array:24 [
  "pii" => "S0021755721001376"
  "issn" => "00217557"
  "doi" => "10.1016/j.jped.2021.09.003"
  "estado" => "S300"
  "fechaPublicacion" => "2022-07-01"
  "aid" => "1031"
  "copyright" => "Sociedade Brasileira de Pediatria"
  "copyrightAnyo" => "2021"
  "documento" => "article"
  "crossmark" => 1
  "licencia" => "http://creativecommons.org/licenses/by-nc-nd/4.0/"
  "subdocumento" => "fla"
  "cita" => "J Pediatr (Rio J). 2022;98:383-9"
  "abierto" => array:3 [
    "ES" => true
    "ES2" => true
    "LATM" => true
  ]
  "gratuito" => true
  "lecturas" => array:1 [
    "total" => 0
  ]
  "itemSiguiente" => array:19 [
    "pii" => "S002175572100142X"
    "issn" => "00217557"
    "doi" => "10.1016/j.jped.2021.09.006"
    "estado" => "S300"
    "fechaPublicacion" => "2022-07-01"
    "aid" => "1036"
    "copyright" => "Sociedade Brasileira de Pediatria"
    "documento" => "article"
    "crossmark" => 1
    "licencia" => "http://creativecommons.org/licenses/by-nc-nd/4.0/"
    "subdocumento" => "fla"
    "cita" => "J Pediatr (Rio J). 2022;98:390-8"
    "abierto" => array:3 [
      "ES" => true
      "ES2" => true
      "LATM" => true
    ]
    "gratuito" => true
    "lecturas" => array:1 [
      "total" => 0
    ]
    "en" => array:11 [
      "idiomaDefecto" => true
      "cabecera" => "<span class="elsevierStyleTextfn">Original article</span>"
      "titulo" => "Biochemical markers and anthropometric profile of children enrolled in public daycare centers"
      "tienePdf" => "en"
      "tieneTextoCompleto" => "en"
      "tieneResumen" => "en"
      "paginas" => array:1 [
        0 => array:2 [
          "paginaInicial" => "390"
          "paginaFinal" => "398"
        ]
      ]
      "contieneResumen" => array:1 [
        "en" => true
      ]
      "contieneTextoCompleto" => array:1 [
        "en" => true
      ]
      "contienePdf" => array:1 [
        "en" => true
      ]
      "autores" => array:1 [
        0 => array:2 [
          "autoresLista" => "Luiz Felipe de Paiva Louren&#231;&#227;o, Nara Cristina de Paula, Maria Ang&#233;lica Cardoso, Paula Ribeiro Santos, Izabela Regina Cardoso de Oliveira, Fernando Luiz Affonso Fonseca, Glaucia Luciano da Veiga, Beatriz da Costa Aguiar Alves, Miriam Monteiro de Castro Graciano, Stela M&#225;rcia Pereira-Dourado"
          "autores" => array:10 [
            0 => array:2 [
              "nombre" => "Luiz Felipe de Paiva"
              "apellidos" => "Louren&#231;&#227;o"
            ]
            1 => array:2 [
              "nombre" => "Nara Cristina"
              "apellidos" => "de Paula"
            ]
            2 => array:2 [
              "nombre" => "Maria Ang&#233;lica"
              "apellidos" => "Cardoso"
            ]
            3 => array:2 [
              "nombre" => "Paula Ribeiro"
              "apellidos" => "Santos"
            ]
            4 => array:2 [
              "nombre" => "Izabela Regina Cardoso"
              "apellidos" => "de Oliveira"
            ]
            5 => array:2 [
              "nombre" => "Fernando Luiz Affonso"
              "apellidos" => "Fonseca"
            ]
            6 => array:2 [
              "nombre" => "Glaucia Luciano"
              "apellidos" => "da Veiga"
            ]
            7 => array:2 [
              "nombre" => "Beatriz da Costa Aguiar"
              "apellidos" => "Alves"
            ]
            8 => array:2 [
              "nombre" => "Miriam Monteiro de Castro"
              "apellidos" => "Graciano"
            ]
            9 => array:2 [
              "nombre" => "Stela M&#225;rcia"
              "apellidos" => "Pereira-Dourado"
            ]
          ]
        ]
      ]
    ]
    "idiomaDefecto" => "en"
    "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S002175572100142X?idApp=UINPBA000049"
    "url" => "/00217557/0000009800000004/v2_202207140636/S002175572100142X/v2_202207140636/en/main.assets"
  ]
  "itemAnterior" => array:19 [
    "pii" => "S0021755721001340"
    "issn" => "00217557"
    "doi" => "10.1016/j.jped.2021.09.001"
    "estado" => "S300"
    "fechaPublicacion" => "2022-07-01"
    "aid" => "1028"
    "copyright" => "Sociedade Brasileira de Pediatria"
    "documento" => "article"
    "crossmark" => 1
    "licencia" => "http://creativecommons.org/licenses/by-nc-nd/4.0/"
    "subdocumento" => "fla"
    "cita" => "J Pediatr &#40;Rio J&#41;. 2022;98:376-82"
    "abierto" => array:3 [
      "ES" => true
      "ES2" => true
      "LATM" => true
    ]
    "gratuito" => true
    "lecturas" => array:1 [
      "total" => 0
    ]
    "en" => array:12 [
      "idiomaDefecto" => true
      "cabecera" => "<span class="elsevierStyleTextfn">Original article</span>"
      "titulo" => "Skin-to-skin contact and deaths in newborns weighing up to 1800 grams&#58; a cohort study"
      "tienePdf" => "en"
      "tieneTextoCompleto" => "en"
      "tieneResumen" => "en"
      "paginas" => array:1 [
        0 => array:2 [
          "paginaInicial" => "376"
          "paginaFinal" => "382"
        ]
      ]
      "contieneResumen" => array:1 [
        "en" => true
      ]
      "contieneTextoCompleto" => array:1 [
        "en" => true
      ]
      "contienePdf" => array:1 [
        "en" => true
      ]
      "resumenGrafico" => array:2 [
        "original" => 0
        "multimedia" => array:8 [
          "identificador" => "fig0001"
          "etiqueta" => "Figure 1"
          "tipo" => "MULTIMEDIAFIGURA"
          "mostrarFloat" => true
          "mostrarDisplay" => false
          "figura" => array:1 [
            0 => array:4 [
              "imagen" => "gr1.jpeg"
              "Alto" => 2350
              "Ancho" => 2667
              "Tamanyo" => 317662
            ]
          ]
          "detalles" => array:1 [
            0 => array:3 [
              "identificador" => "alt0001"
              "detalle" => "Figure "
              "rol" => "short"
            ]
          ]
          "descripcion" => array:1 [
            "en" => "<p id="spara001" class="elsevierStyleSimplePara elsevierViewall">Multivariate analysis &#40;classification tree&#41; of factors associated with the relationship between permanence in SSC and neonatal death&#46;</p>"
          ]
        ]
      ]
      "autores" => array:1 [
        0 => array:2 [
          "autoresLista" => "Marivanda J&#46;F&#46; Goudard, Zeni C&#46; Lamy, S&#233;rgio T&#46;M&#46; Marba, Milady C&#46;V&#46; Cavalcante, Alcione M&#46; dos Santos, Vivian M&#46;G&#46;de O&#46; Azevedo, Roberta Costa, Carolina N&#46;M&#46; Guimar&#227;es, Fernando Lamy-Filho"
          "autores" => array:9 [
            0 => array:2 [
              "nombre" => "Marivanda J&#46;F&#46;"
              "apellidos" => "Goudard"
            ]
            1 => array:2 [
              "nombre" => "Zeni C&#46;"
              "apellidos" => "Lamy"
            ]
            2 => array:2 [
              "nombre" => "S&#233;rgio T&#46;M&#46;"
              "apellidos" => "Marba"
            ]
            3 => array:2 [
              "nombre" => "Milady C&#46;V&#46;"
              "apellidos" => "Cavalcante"
            ]
            4 => array:2 [
              "nombre" => "Alcione M&#46;"
              "apellidos" => "dos Santos"
            ]
            5 => array:2 [
              "nombre" => "Vivian M&#46;G&#46;de O&#46;"
              "apellidos" => "Azevedo"
            ]
            6 => array:2 [
              "nombre" => "Roberta"
              "apellidos" => "Costa"
            ]
            7 => array:2 [
              "nombre" => "Carolina N&#46;M&#46;"
              "apellidos" => "Guimar&#227;es"
            ]
            8 => array:2 [
              "nombre" => "Fernando"
              "apellidos" => "Lamy-Filho"
            ]
          ]
        ]
      ]
    ]
    "idiomaDefecto" => "en"
    "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0021755721001340?idApp=UINPBA000049"
    "url" => "/00217557/0000009800000004/v2_202207140636/S0021755721001340/v2_202207140636/en/main.assets"
  ]
  "en" => array:20 [
    "idiomaDefecto" => true
    "cabecera" => "<span class="elsevierStyleTextfn">Original article</span>"
    "titulo" => "Possible association between a polymorphism of <span class="elsevierStyleItalic">EPAS1</span> gene and persistent pulmonary hypertension of the newborn&#58; a case-control study"
    "tieneTextoCompleto" => true
    "paginas" => array:1 [
      0 => array:2 [
        "paginaInicial" => "383"
        "paginaFinal" => "389"
      ]
    ]
    "autores" => array:1 [
      0 => array:4 [
        "autoresLista" => "Narongsak Nakwan, Surakameth Mahasirimongkol, Nusara Satproedprai, Tassamonwan Chaiyasung, Punna Kunhapan, Cheep Charoenlap, Kamonnut Singkhamanan, Chariyawan Charalsawadi"
        "autores" => array:8 [
          0 => array:3 [
            "nombre" => "Narongsak"
            "apellidos" => "Nakwan"
            "referencia" => array:2 [
              0 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">a</span>"
                "identificador" => "aff0001"
              ]
              1 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">b</span>"
                "identificador" => "aff0002"
              ]
            ]
          ]
          1 => array:3 [
            "nombre" => "Surakameth"
            "apellidos" => "Mahasirimongkol"
            "referencia" => array:1 [
              0 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">c</span>"
                "identificador" => "aff0003"
              ]
            ]
          ]
          2 => array:3 [
            "nombre" => "Nusara"
            "apellidos" => "Satproedprai"
            "referencia" => array:1 [
              0 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">c</span>"
                "identificador" => "aff0003"
              ]
            ]
          ]
          3 => array:3 [
            "nombre" => "Tassamonwan"
            "apellidos" => "Chaiyasung"
            "referencia" => array:1 [
              0 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">c</span>"
                "identificador" => "aff0003"
              ]
            ]
          ]
          4 => array:3 [
            "nombre" => "Punna"
            "apellidos" => "Kunhapan"
            "referencia" => array:1 [
              0 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">c</span>"
                "identificador" => "aff0003"
              ]
            ]
          ]
          5 => array:3 [
            "nombre" => "Cheep"
            "apellidos" => "Charoenlap"
            "referencia" => array:1 [
              0 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">d</span>"
                "identificador" => "aff0004"
              ]
            ]
          ]
          6 => array:3 [
            "nombre" => "Kamonnut"
            "apellidos" => "Singkhamanan"
            "referencia" => array:1 [
              0 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">a</span>"
                "identificador" => "aff0001"
              ]
            ]
          ]
          7 => array:4 [
            "nombre" => "Chariyawan"
            "apellidos" => "Charalsawadi"
            "email" => array:1 [
              0 => "cchariya@medicine.psu.ac.th"
            ]
            "referencia" => array:2 [
              0 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">e</span>"
                "identificador" => "aff0005"
              ]
              1 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">&#42;</span>"
                "identificador" => "cor0001"
              ]
            ]
          ]
        ]
        "afiliaciones" => array:5 [
          0 => array:3 [
            "entidad" => "Prince of Songkla University&#44; Faculty of Medicine&#44; Department of Biomedical Sciences&#44; Hat Yai&#44; Thailand"
            "etiqueta" => "a"
            "identificador" => "aff0001"
          ]
          1 => array:3 [
            "entidad" => "Hat Yai Hospital&#44; Hat Yai Medical Education Center&#44; Department of Pediatrics&#44; Hat Yai&#44; Thailand"
            "etiqueta" => "b"
            "identificador" => "aff0002"
          ]
          2 => array:3 [
            "entidad" => "Ministry of Public Heath&#44; Department of Medical Sciences&#44; Division of Genomic Medicine and Innovation Support&#44; Medical Genetics Center&#44; Nonthaburi&#44; Thailand"
            "etiqueta" => "c"
            "identificador" => "aff0003"
          ]
          3 => array:3 [
            "entidad" => "Hat Yai Hospital&#44; Hat Yai Medical Education Center&#44; Department of Anatomical Pathology&#44; Hat Yai&#44; Thailand"
            "etiqueta" => "d"
            "identificador" => "aff0004"
          ]
          4 => array:3 [
            "entidad" => "Prince of Songkla University&#44; Faculty of Medicine&#44; Department of Pathology&#44; Hat Yai&#44; Thailand"
            "etiqueta" => "e"
            "identificador" => "aff0005"
          ]
        ]
        "correspondencia" => array:1 [
          0 => array:3 [
            "identificador" => "cor0001"
            "etiqueta" => "&#8270;"
            "correspondencia" => "Corresponding author&#46;"
          ]
        ]
      ]
    ]
    "resumenGrafico" => array:2 [
      "original" => 0
      "multimedia" => array:8 [
        "identificador" => "fig0001"
        "etiqueta" => "Figure 1"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "gr1.jpeg"
            "Alto" => 1710
            "Ancho" => 2751
            "Tamanyo" => 257819
          ]
        ]
        "detalles" => array:1 [
          0 => array:3 [
            "identificador" => "alt0003"
            "detalle" => "Fig&#46; "
            "rol" => "short"
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="spara001" class="elsevierStyleSimplePara elsevierViewall">A flow chart illustrating subject selection and study workflow&#46;</p>"
        ]
      ]
    ]
    "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0001" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cesectitle0007">Introduction</span><p id="para0005" class="elsevierStylePara elsevierViewall">Persistent pulmonary hypertension of the newborn &#40;PPHN&#41; is the most serious complication of several neonatal respiratory diseases&#44; leading to adverse outcomes such as asphyxia&#44; chronic lung disease&#44; neurodevelopmental sequelae&#44; and death&#44; despite the use of modern therapeutic modalities for treating PPHN such as high-frequency oscillatory ventilation&#44; inhaled nitric oxide&#44; and extracorporeal membrane oxygenation&#46;<a class="elsevierStyleCrossRef" href="#bib0001"><span class="elsevierStyleSup">1</span></a><span class="elsevierStyleSup">&#44;</span><a class="elsevierStyleCrossRef" href="#bib0002"><span class="elsevierStyleSup">2</span></a> Although the primary etiologies of PPHN are various factors such as chronic hypoxia&#44; meconium aspiration syndrome&#44; birth asphyxia&#44; sepsis&#44; infection&#44; and respiratory distress syndrome&#44;<a class="elsevierStyleCrossRef" href="#bib0001"><span class="elsevierStyleSup">1</span></a><span class="elsevierStyleSup">&#44;</span><a class="elsevierStyleCrossRef" href="#bib0003"><span class="elsevierStyleSup">3</span></a><span class="elsevierStyleSup">&#44;</span><a class="elsevierStyleCrossRef" href="#bib0004"><span class="elsevierStyleSup">4</span></a> a genetic contribution to PPHN has been increasingly recognized&#46; Previous studies have shown that SNPs in the <span class="elsevierStyleItalic">CRHR1</span> &#40;rs4458044&#41;&#44; <span class="elsevierStyleItalic">CPS1</span> &#40;rs41272673&#44; rs4399666&#44; rs2287599&#44; rs192759073&#44; rs1047883&#44; and rs2229589&#41;&#44; <span class="elsevierStyleItalic">EDN1</span> &#40;rs2070699&#41;&#44; and <span class="elsevierStyleItalic">NOTCH3</span> &#40;rs1044008&#41; genes are susceptibility loci associated with PPHN&#46;<a class="elsevierStyleCrossRefs" href="#bib0005"><span class="elsevierStyleSup">5&#8211;9</span></a> It has also recently been reported that mutations in a few genes such as <span class="elsevierStyleItalic">TTLL3</span> and <span class="elsevierStyleItalic">ITGAM</span><a class="elsevierStyleCrossRef" href="#bib0010"><span class="elsevierStyleSup">10</span></a> were associated with PPHN&#46; <span class="elsevierStyleItalic">TBX4</span> variant has also been found in relation to neonatal and pediatric pulmonary hypertension&#46;<a class="elsevierStyleCrossRef" href="#bib0011"><span class="elsevierStyleSup">11</span></a></p><p id="para0006" class="elsevierStylePara elsevierViewall">In this study&#44; the authors aimed to determine any associations between SNPs of previous PPHN candidate genes and to perform a validation of any genetic associations found to elucidate a possible biological involvement of the protein&#40;s&#41; encoded from the gene&#40;s&#41; in the etiology of PPHN&#46;</p></span><span id="sec0002" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cesectitle0008">Materials and methods</span><span id="sec0003" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cesectitle0009">Study population</span><p id="para0007" class="elsevierStylePara elsevierViewall">The authors recruited 45 patients with PPHN who were hospitalized at Hat Yai hospital&#44; one of the biggest hospitals in Southern Thailand where the prevalence of PPHN is relatively high&#44; from 2&#46;6 to 2&#46;8 per 1000 live births&#44; and constituting &#8764;30&#37; of the all-cause mortality rate&#46;<a class="elsevierStyleCrossRef" href="#bib0001"><span class="elsevierStyleSup">1</span></a><span class="elsevierStyleSup">&#44;</span><a class="elsevierStyleCrossRef" href="#bib0012"><span class="elsevierStyleSup">12</span></a> Retrospective data of PPHN infants discharged from the hospital between January 2013 and March 2019&#44; born to mothers with a gestational age of &#8805; 35 weeks and birth weight of &#8805; 2000&#160;g&#44; were retrieved from the hospital computer data system&#46; The authors recruited 20 patients from the retrospective data&#46; The authors also recruited additional PPHN neonates who were hospitalized at the hospital between April 2019 and November 2019 &#40;<a class="elsevierStyleCrossRef" href="#fig0001">Figure 1</a>&#41;&#46; The authors excluded patients with congenital structural cyanotic heart diseases and other congenital disorders such as congenital diaphragmatic hernia and birth defects due to chromosomal disorders&#46; For a healthy control group&#44; the authors recruited 59 neonates who were born at the hospital between April 2019 and November 2019 and 235 adults who participated in the Thai SNP database project of Ramathibodi Hospital&#44; Bangkok&#44; Thailand&#46; A summary of subjects involving in the study is shown in <a class="elsevierStyleCrossRef" href="#fig0001">Figure&#160;1</a> and baseline characteristics are presented in <a class="elsevierStyleCrossRef" href="#sec0015">supplementary&#160;Table 1</a>&#46; This study was approved by the Research Ethics Committee of the Hat Yai Hospital &#40;EC No&#46; 24&#47;2562&#41;&#46;</p><elsevierMultimedia ident="fig0001"></elsevierMultimedia></span><span id="sec0004" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cesectitle0010">Study definitions</span><p id="para0008" class="elsevierStylePara elsevierViewall">The diagnostic criteria for PPHN were presentation shortly after birth with refractory hypoxemia plus one of the three following conditions&#58; 1&#41; documented pulmonary hypertension&#44; as defined by echocardiographic evidence of elevated pulmonary pressure &#40;right to left or bidirectional shunt&#41; and&#47;or 2&#41; a pre-to-postductal partial pressure of oxygen gradient equal to or greater than 20 mm Hg&#44; and&#47;or 3&#41; a pulse oximetry oxygen saturation &#40;SpO<span class="elsevierStyleInf">2</span>&#41; gradient equal to or greater than 10&#37;&#46;<a class="elsevierStyleCrossRef" href="#bib0013"><span class="elsevierStyleSup">13</span></a></p></span><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cesectitle0011">Genomic DNA extraction</span><p id="para0009" class="elsevierStylePara elsevierViewall">Genomic DNA was extracted from a 200 &#956;L sample of peripheral blood using a Qiagen DNA Mini kit &#40;Qiagen&#44; Hilden&#44; Germany&#41;&#46; The quantity and quality of the DNA samples were evaluated using an Eppendorf BioPhotometer plus &#40;Eppendorf AG&#44; Hamburg&#44; Germany&#41; and automated electrophoresis &#40;Agilent 2200 TapeStation system&#44; Santa Clara&#44; California&#44; United States&#41;&#46;</p></span><span id="sec0006" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cesectitle0012">Genotyping and quality control</span><p id="para0010" class="elsevierStylePara elsevierViewall">A total of 659&#44;184 single nucleotide polymorphism &#40;SNPs&#41; probes specific to several regions across the genome were fabricated on an Illumina Asian Screening Array-24 v1&#46;0 BeadChip Array&#46; SNP genotyping using an Illumina Infinium HTS Assay platform &#40;Illumina&#44; San Diego&#44; California&#41; was carried out and 612&#44;128 &#40;93&#37;&#41; SNPs were successfully genotyped&#46; For quality control&#44; the authors applied a low call rate &#60; 98&#37;&#44; a Hardy&#8211;Weinberg equilibrium calculations <span class="elsevierStyleItalic">p</span>-value &#60; 1&#160;&#215;&#160;10<span class="elsevierStyleSup">&#8722;8</span>&#44; and minor allele frequency &#40;MAF&#41; of each SNP &#60;0&#46;05 to exclude data with low quality&#46; After the quality control exclusion&#44; 498&#44;160 autosomal SNPs were left for the genome-wide association study &#40;GWAS&#41; analysis&#46; The authors selected the SNPs in the genes related to PPHN and pulmonary arterial hypertension &#40;PAH&#41;&#46; Finally&#44; 285 SNPs of 11 candidate genes &#40;<span class="elsevierStyleItalic">BMPR2&#44; EPAS1&#44; PDE3A&#44; VEGFA&#44; ENG&#44; NOTCH3&#44; SOD3&#44; CPS1&#44; ABCA3&#44; ACVRL1&#44;</span> and <span class="elsevierStyleItalic">SMAD9</span>&#41; were analyzed&#46;</p></span><span id="sec0007" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cesectitle0013">Immunohistochemistry &#40;IHC&#41; detection of HIF-2&#945; in umbilicus and lung tissues</span><p id="para0011" class="elsevierStylePara elsevierViewall">For the validation study&#44; the authors evaluated the expression of hypoxia-inducible factor 2-alpha &#40;HIF-2&#945;&#41;&#44; a protein encoded from the <span class="elsevierStyleItalic">EPAS1</span> gene&#44; in the pulmonary vascular endothelium of lung tissues obtained from deceased PPHN patients &#40;<span class="elsevierStyleItalic">n</span>&#160;&#61;&#160;3&#41; compared to normal adult lung tissue &#40;<span class="elsevierStyleItalic">n</span>&#160;&#61;&#160;3&#41;&#46; In addition&#44; the authors carried out IHC staining of HIF-2&#945; in the umbilicuses of 8 healthy newborns to evaluate the expression of the protein in the umbilical artery and vein&#46; Paraffin-embedded umbilical and lung tissues were stained with hematoxylin and eosin to examine histopathology of the tissues&#46; Then&#44; IHC using anti-HIF-2&#945; antibody &#40;ab199&#44; Abcam&#44; UK&#41; with dilution 1&#58;100 v&#47;v was performed on VENTANA BenchMark ULTRA platform &#40;Ventana Medical Systems Inc&#46;&#44; USA&#41; to examine for HIF-2&#945; expression&#46;</p></span><span id="sec0008" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cesectitle0014">Statistical analysis</span><p id="para0012" class="elsevierStylePara elsevierViewall">The authors used FastLmmC &#40;v2&#46;02&#46;20121124&#41; and the R package for GWAS &#40;v&#46; 3&#46;3&#46;3&#41; for statistical analysis&#46; Regional plots were generated using LocusZoom v&#46;0&#46;4&#46;8&#46; The allele and genotype frequencies of the candidate genes were calculated by the genotypes of the subjects&#46; The chi-square test was used to compare the allele and genotype frequencies between the groups&#46; When comparing the genotype frequency of each significant SNP between groups&#44; the authors used the Cochrane-Armitage trend test between all genotypes&#46; Odds ratio &#40;OR&#41; and 95&#37; confidence intervals &#40;CI&#41; were calculated to indicate the degree of association between groups&#46;</p></span></span><span id="sec0009" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cesectitle0015">Results</span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cesectitle0016">Association of SNPs in previously reported candidate genes and PPHN&#47; PAH</span><p id="para0013" class="elsevierStylePara elsevierViewall">The authors analyzed 285 SNPs in 11 candidate genes&#44; including <span class="elsevierStyleItalic">BMPR2&#44; EPAS1&#44; PDE3A&#44; VEGFA&#44; ENG&#44; NOTCH3&#44; SOD3&#44; CPS1&#44; ABCA3&#44; ACVRL1&#44;</span> and <span class="elsevierStyleItalic">SMAD9&#44;</span> that were found to be associated with the pathogenesis of PPHN&#47;PAH in previous studies&#46; A <span class="elsevierStyleItalic">p</span>-value of &#60; 0&#46;05 was considered significant&#46; The authors found that 17 SNPs in 6 candidate genes&#58; <span class="elsevierStyleItalic">BMPR2&#44; EPAS1&#44; PDE3A&#44; VEGFA&#44; ENG&#44;</span> and <span class="elsevierStyleItalic">NOTCH3&#44;</span> were significantly associated with PPHN &#40;<a class="elsevierStyleCrossRef" href="#tbl0001">Table 1</a>&#41;&#46; Of these&#44; rs17034984 in the <span class="elsevierStyleItalic">EPAS1</span> gene was the only one to remain significant after the Bonferroni correction &#40;P&#160;&#60;&#160;0&#46;00018&#41;&#46;</p><elsevierMultimedia ident="tbl0001"></elsevierMultimedia></span><span id="sec0011" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cesectitle0017">Genotype distribution&#44; allele frequency&#44; and fit inheritance model of significant SNPs in candidate genes &#40;P&#160;&#60;&#160;0&#46;05&#41; associated with PPHN&#47;PAH</span><p id="para0014" class="elsevierStylePara elsevierViewall">The genotype and allele frequencies of candidate gene polymorphisms are summarized in <a class="elsevierStyleCrossRef" href="#sec0015">Supplementary&#160;Table 2</a>&#46; Various models of inheritance &#40;codominant&#44; dominant&#44; and recessive&#41; were tested and genetic effects were calculated for each genotype compared to the reference genotype in each model&#46; The authors found that the frequency of the TC&#47;TT genotype of rs17034984 in the <span class="elsevierStyleItalic">EPAS1</span> gene with the dominant model was significant in the patients with PPHN &#40;OR&#160;&#61;&#160;5&#46;38&#44; 95&#37; CI&#58; 2&#46;15&#8211;13&#46;49&#41;&#46; In addition&#44; the allele frequency of the minor allele T of rs17034984 in the <span class="elsevierStyleItalic">EPAS1</span> gene showed a significant difference compared with the control subjects &#40;OR&#160;&#61;&#160;4&#46;89&#44; 95&#37; CI&#58; 2&#46;03&#8211;11&#46;82&#41;&#46;</p></span><span id="sec0012" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cesectitle0018">HIF-2&#945; immunohistochemistry staining of the vascular endothelium of umbilicus and lung tissues</span><p id="para0015" class="elsevierStylePara elsevierViewall">HIF-2&#945; protein was expressed in lung tissues from 2 infants with PPHN&#46; Localization of HIF-2&#945; was seen in the nuclei of endothelial cells of the pulmonary arteries &#40;<a class="elsevierStyleCrossRef" href="#fig0002">Figure 2</a>A&#41;&#46; However&#44; there was one infant with PPHN in whom HIF-2&#945; protein expression was not detected &#40;<a class="elsevierStyleCrossRef" href="#fig0002">Figure 2</a>B&#41;&#46; The HIF-2&#945; protein was also expressed in the endothelial cells of the umbilical arteries and veins from the 8 normal newborns &#40;<a class="elsevierStyleCrossRef" href="#fig0002">Figure 2</a>C&#41;&#46; The HIF-2&#945; protein was not expressed in normal adult lung tissues and endothelial cells of the pulmonary vessels &#40;<a class="elsevierStyleCrossRef" href="#fig0002">Figure 2</a>D&#41;&#46; Determination of protein expression was scored by two pathologists &#40;intensity of staining&#58; 0&#44; no staining&#59; 1&#44; weak&#59; 2&#44; moderate&#59; and 3&#44; strong&#41;&#46; In addition&#44; semiquantitative analysis of the IHC images was performed using ImageJ Fiji software &#40;version 2&#46;1&#46;0&#47;1&#46;53C&#41;&#44; following an integrated protocol&#46;<a class="elsevierStyleCrossRef" href="#bib0014"><span class="elsevierStyleSup">14</span></a> Expression of HIF-2&#945; in different tissues were shown in <a class="elsevierStyleCrossRef" href="#tbl0002">Table 2</a>&#46;</p><elsevierMultimedia ident="fig0002"></elsevierMultimedia><elsevierMultimedia ident="tbl0002"></elsevierMultimedia></span></span><span id="sec0013" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cesectitle0019">Discussion</span><p id="para0016" class="elsevierStylePara elsevierViewall">PPHN is the most serious respiratory disorder in Thailand and has a high mortality rate&#46; Its high prevalence in the study&#39;s institution led the authors to hypothesize whether genetic factors play an important role in the development of PPHN&#46; Using SNP analysis&#44; the authors found that rs17034984 in the <span class="elsevierStyleItalic">EPAS1</span> gene showed a significant association with PPHN in the studied population&#46; In addition&#44; the authors found that the T allele of rs17034984 in the <span class="elsevierStyleItalic">EPAS1</span> gene is a possible predisposing factor for PPHN and the TC&#47;TT genotype with the dominant model could be a predictor of PPHN&#46;</p><p id="para0017" class="elsevierStylePara elsevierViewall">The rs17034984 SNP is located in intron 1 of the <span class="elsevierStyleItalic">EPAS1</span> gene&#46; The <span class="elsevierStyleItalic">EPAS1</span> gene&#44; located on the short arm of chromosome 2 &#40;2p21&#41;&#44; encodes endothelial PAS domain-containing protein 1&#44; which is also known as hypoxia-inducible factor 2 alpha &#40;HIF-2&#945;&#41; protein&#46;<a class="elsevierStyleCrossRef" href="#bib0015"><span class="elsevierStyleSup">15</span></a> This protein is a subunit of the HIF protein complex&#44; which plays a crucial role in the body&#39;s ability to adapt to changing oxygen levels&#46; HIF-2&#945; is a transcription factor involved in the induction of oxygen-regulated genes&#46; It regulates erythropoiesis and physiological responses to hypoxia through the HIFs signaling pathway&#46;<a class="elsevierStyleCrossRef" href="#bib0016"><span class="elsevierStyleSup">16</span></a> In hypoxic conditions&#44; the HIFs protein is translocated to the nucleus to bind the hypoxic response element &#40;HRE&#41; of target gene promoters and subsequently initiate gene transcription&#46; When oxygen levels rise&#44; HIFs are hydroxylated by prolyl hydroxylase domain proteins &#40;PHD&#41;&#44; leading to interaction with other proteins&#44; and with the addition to ubiquitin results in HIFs degradation&#46;<a class="elsevierStyleCrossRef" href="#bib0015"><span class="elsevierStyleSup">15</span></a> Previous studies reported that the <span class="elsevierStyleItalic">EPAS1</span> gene polymorphisms were associated with adaptation to hypobaric hypoxia in the Tibetan population&#46;<a class="elsevierStyleCrossRef" href="#bib0017"><span class="elsevierStyleSup">17</span></a><span class="elsevierStyleSup">&#44;</span><a class="elsevierStyleCrossRef" href="#bib0018"><span class="elsevierStyleSup">18</span></a> In addition&#44; HIF-2&#945; regulates the vascular endothelial growth factor &#40;VEGF&#41; expression and seems to be implicated in the development of blood vessels and the tubular system of the lung&#46; An animal model study also showed that the gain-of-function of HIF-2&#945; was related to the development of pulmonary vascular remodeling&#46;<a class="elsevierStyleCrossRef" href="#bib0019"><span class="elsevierStyleSup">19</span></a> Another study found that chronic hypoxia could induce HIF-2&#945; stability&#44; leading to increased arginase expression and dysregulating normal vascular nitric oxide homeostasis&#46;<a class="elsevierStyleCrossRef" href="#bib0020"><span class="elsevierStyleSup">20</span></a> In addition&#44; endothelial HIF-2&#945; was a major mediator in the development of hypoxic-induced pulmonary hypertension&#44; and its functions could induce pulmonary hypertension through an increased expression of endothelin-1&#44; encoded from the <span class="elsevierStyleItalic">EDN1</span> gene and a concomitant decrease in vasodilatory apelin receptor signaling&#46;<a class="elsevierStyleCrossRef" href="#bib0021"><span class="elsevierStyleSup">21</span></a> Gene network analysis showed that <span class="elsevierStyleItalic">EPAS1</span> gene is co-expressed with <span class="elsevierStyleItalic">EDN1</span> gene&#46; The Gene network of 11 candidate genes in the present study study &#40;<span class="elsevierStyleItalic">BMPR2&#44; EPAS1&#44; PDE3A&#44; VEGFA&#44; ENG&#44; NOTCH3&#44; SOD3&#44; CPS1&#44; ABCA3&#44; ACVRL1&#44;</span> and <span class="elsevierStyleItalic">SMAD9&#41;</span> and other genes that had been shown to be associated with PPHN is shown in <a class="elsevierStyleCrossRef" href="#sec0015">Supplementary&#160;Figure 1</a>&#46;</p><p id="para0018" class="elsevierStylePara elsevierViewall">In the present study&#44; 10 SNPs of the <span class="elsevierStyleItalic">EPAS1</span> gene showed a significant difference between patients with PPHN and the controls &#40;<a class="elsevierStyleCrossRef" href="#tbl0001">Table 1</a>&#41;&#59; however&#44; rs17034984 was the only SNP that remained associated with PPHN after the Bonferroni correction&#46; The T allele on rs17034984 was a risk factor for PPHN and the TC&#47;TT genotype with the dominant model was significantly higher in patients with PPHN&#46; However&#44; to date&#44; there have been few studies regarding HIF-2&#945; in neonates&#46; One study reported that HIF-2&#945; was highly expressed in the lungs of neonates with a congenital diaphragmatic hernia and was possibly correlated with PPHN in neonates&#46;<a class="elsevierStyleCrossRef" href="#bib0022"><span class="elsevierStyleSup">22</span></a> Taking these findings together&#44; the authors believe that a genetic variant of the <span class="elsevierStyleItalic">EPAS1</span> gene is possibly associated with PPHN&#46;</p><p id="para0019" class="elsevierStylePara elsevierViewall">To demonstrate that expression of the HIF-2&#945; protein &#40;i&#46;e&#46;&#44; expression of the <span class="elsevierStyleItalic">EPAS1</span> gene&#41; in pulmonary vascular endothelial cells is involved in PPHN pathophysiology&#44; the authors carried out IHC staining in pulmonary vascular endothelial cells and other tissues&#46; The authors found that HIF-2&#945; protein was expressed in the nucleus of pulmonary vascular endothelial cells of some PPHN patients&#44; whereas there was no HIF-2&#945; protein expression in normal adult lung tissues&#46; Additionally&#44; the authors observed HIF-2&#945; protein expression in fetal umbilical vascular endothelial cells&#44; possibly as a result of exposure to a hypoxic environment during physiological development&#46; During <span class="elsevierStyleItalic">in utero</span> development&#44; expression of both HIF-1&#945; and HIF-2&#945; are relatively high in the fetal lungs due to fetal exposure to a hypoxic environment&#46;<a class="elsevierStyleCrossRefs" href="#bib0023"><span class="elsevierStyleSup">23&#8211;25</span></a> HIF-1&#945; expression is predominant in respiratory epithelial cells&#44; while HIF-2&#945; is predominant in vascular endothelial cells and type II pneumocytes&#46;<a class="elsevierStyleCrossRef" href="#bib0026"><span class="elsevierStyleSup">26</span></a> Due to a limited number of PPHN lung samples&#44; the authors could not perform statistical analysis to make a solid conclusion that HIF-2&#945; was significantly higher expressed in neonatal PPHN lungs&#46; However&#44; the present study&#39;s results suggest that more studies are needed to further explicate the role&#40;s&#41; of HIF-2&#945; in the pathophysiology of PPHN&#46;</p><p id="para0020" class="elsevierStylePara elsevierViewall">There are some limitations to the present study&#46; First&#44; the number of neonates with PPHN recruited into this study was relatively small&#46; Second&#44; the number of SNP probes fabricated onto the microarray might not cover all SNPs associated with the pathogenesis of PPHN&#46; Third&#44; although the present study carried out a preliminary validation study&#44; using IHC staining to detect HIF-2&#945; protein expression in lung tissues from neonates with PPHN&#44; the authors could not compare the study&#39;s findings with normal lung tissues from neonates because autopsies of deceased neonates were not routinely performed in the study&#39;s center&#46; Instead&#44; the authors carried out IHC staining to detect HIF-2&#945; protein expression in normal adult lung tissues&#46; Concerning potential bias in adult control subject selection due to unknown medical history in their neonatal period&#44; this concern was deemed minimal&#46; Since the prevalence of PPHN is &#8764;3 per 1000 livebirth&#44; which means the vast majority of individuals do not have PPHN&#46;</p><p id="para0021" class="elsevierStylePara elsevierViewall">In conclusion&#44; in this study&#44; the authors explored novel candidate SNPs that might be involved in the pathophysiology of PPHN&#46; The authors&#8217; analysis revealed that rs17034984 in the <span class="elsevierStyleItalic">EPAS1</span> gene is a possible genetic susceptibility locus associated with PPHN&#46; In combination with IHC staining to detect HIF-2&#945; protein expression&#44; the authors found that HIF-2&#945;&#44; encoded by the <span class="elsevierStyleItalic">EPAS1</span> gene&#44; might be a factor associated with PPHN via pulmonary vascular remodeling related to different oxygen levels in fetuses&#46; However&#44; further studies are needed to confirm the authors&#8217; findings and to further explore the pathophysiological mechanism&#40;s&#41; underlying this association&#46;</p></span><span id="sec0014" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cesectitle0020">Funding</span><p id="para0022" class="elsevierStylePara elsevierViewall">Graduate School and Faculty of Medicine Foundation&#44; Prince of Songkla University&#44; and Medical Genetics Center&#44; Division of Genomic Medicine and Innovation Support&#44; Department of Medical Sciences&#44; Ministry of Public Health&#44; Thailand&#46;</p></span></span>"
    "textoCompletoSecciones" => array:1 [
      "secciones" => array:9 [
        0 => array:3 [
          "identificador" => "xres1747029"
          "titulo" => "Abstract"
          "secciones" => array:4 [
            0 => array:2 [
              "identificador" => "abss0001"
              "titulo" => "Objective"
            ]
            1 => array:2 [
              "identificador" => "abss0002"
              "titulo" => "Methods"
            ]
            2 => array:2 [
              "identificador" => "abss0003"
              "titulo" => "Results"
            ]
            3 => array:2 [
              "identificador" => "abss0004"
              "titulo" => "Conclusions"
            ]
          ]
        ]
        1 => array:2 [
          "identificador" => "xpalclavsec1539160"
          "titulo" => "Keywords"
        ]
        2 => array:2 [
          "identificador" => "sec0001"
          "titulo" => "Introduction"
        ]
        3 => array:3 [
          "identificador" => "sec0002"
          "titulo" => "Materials and methods"
          "secciones" => array:6 [
            0 => array:2 [
              "identificador" => "sec0003"
              "titulo" => "Study population"
            ]
            1 => array:2 [
              "identificador" => "sec0004"
              "titulo" => "Study definitions"
            ]
            2 => array:2 [
              "identificador" => "sec0005"
              "titulo" => "Genomic DNA extraction"
            ]
            3 => array:2 [
              "identificador" => "sec0006"
              "titulo" => "Genotyping and quality control"
            ]
            4 => array:2 [
              "identificador" => "sec0007"
              "titulo" => "Immunohistochemistry &#40;IHC&#41; detection of HIF-2&#945; in umbilicus and lung tissues"
            ]
            5 => array:2 [
              "identificador" => "sec0008"
              "titulo" => "Statistical analysis"
            ]
          ]
        ]
        4 => array:3 [
          "identificador" => "sec0009"
          "titulo" => "Results"
          "secciones" => array:3 [
            0 => array:2 [
              "identificador" => "sec0010"
              "titulo" => "Association of SNPs in previously reported candidate genes and PPHN&#47; PAH"
            ]
            1 => array:2 [
              "identificador" => "sec0011"
              "titulo" => "Genotype distribution&#44; allele frequency&#44; and fit inheritance model of significant SNPs in candidate genes &#40;P&#160;&#60;&#160;0&#46;05&#41; associated with PPHN&#47;PAH"
            ]
            2 => array:2 [
              "identificador" => "sec0012"
              "titulo" => "HIF-2&#945; immunohistochemistry staining of the vascular endothelium of umbilicus and lung tissues"
            ]
          ]
        ]
        5 => array:2 [
          "identificador" => "sec0013"
          "titulo" => "Discussion"
        ]
        6 => array:2 [
          "identificador" => "sec0014"
          "titulo" => "Funding"
        ]
        7 => array:2 [
          "identificador" => "xack616947"
          "titulo" => "Acknowledgments"
        ]
        8 => array:1 [
          "titulo" => "References"
        ]
      ]
    ]
    "pdfFichero" => "main.pdf"
    "tienePdf" => true
    "fechaRecibido" => "2021-07-30"
    "fechaAceptado" => "2021-09-22"
    "PalabrasClave" => array:1 [
      "en" => array:1 [
        0 => array:4 [
          "clase" => "keyword"
          "titulo" => "Keywords"
          "identificador" => "xpalclavsec1539160"
          "palabras" => array:5 [
            0 => "PPHN"
            1 => "Polymorphism"
            2 => "SNP"
            3 => "<span class="elsevierStyleItalic">EPAS1</span> gene"
            4 => "Association study"
          ]
        ]
      ]
    ]
    "tieneResumen" => true
    "resumen" => array:1 [
      "en" => array:3 [
        "titulo" => "Abstract"
        "resumen" => "<span id="abss0001" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cesectitle0002">Objective</span><p id="spara006" class="elsevierStyleSimplePara elsevierViewall">To explore possible genes related to the development of persistent pulmonary hypertension of the newborn &#40;PPHN&#41;&#46;</p></span> <span id="abss0002" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cesectitle0003">Methods</span><p id="spara007" class="elsevierStyleSimplePara elsevierViewall">The authors identified 285 single nucleotide polymorphisms &#40;SNPs&#41; of 11 candidate genes &#40;<span class="elsevierStyleItalic">BMPR2&#44; EPAS1&#44; PDE3A&#44; VEGFA&#44; ENG&#44; NOTCH3&#44; SOD3&#44; CPS1&#44; ABCA3&#44; ACVRL1&#44;</span> and <span class="elsevierStyleItalic">SMAD9</span>&#41;&#44; using an Illumina Asian Screening Array-24 v1&#46;0 BeadChip Array&#46; The FastLmmC and R package was used for statistical analyses&#46; The chi-square test and Cochrane-Armitage trend test were used to compare the allele and genotype frequencies between the groups and to test the genetic models&#44; respectively&#46;</p></span> <span id="abss0003" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cesectitle0004">Results</span><p id="spara008" class="elsevierStyleSimplePara elsevierViewall">A total of 45 PPHN infants and 294 control subjects were analyzed&#46; The most common cause of PPHN was meconium aspiration syndrome&#46; Among the 285 SNPs&#44; 17 SNPs from 6 candidate genes &#40;<span class="elsevierStyleItalic">BMPR2&#44; EPAS1&#44; PDE3A&#44; VEGFA&#44; ENG&#44;</span> and <span class="elsevierStyleItalic">NOTCH3</span>&#41; were significantly associated with PPHN &#40;<span class="elsevierStyleItalic">P</span> &#60; 0&#46;05&#41;&#46; After using the Bonferroni correction &#40;<span class="elsevierStyleItalic">P</span>&#160;&#60;&#160;0&#46;00018&#41;&#44; only the rs17034984 SNP located in intron 1 of the <span class="elsevierStyleItalic">EPAS1</span> gene remained significantly different between the PPHN and control subjects &#40;<span class="elsevierStyleItalic">P</span>&#160;&#61;&#160;0&#46;00014&#41;&#46; The frequency of the TC&#47;TT genotype of rs17034984 in the gene with the dominant model was significant in the patients with PPHN &#40;OR&#160;&#61;&#160;5&#46;38&#44; 95&#37; CI&#58; 2&#46;15&#8211;13&#46;49&#41;&#46; The T allele frequency of rs17034984 in the gene showed a significant difference compared with the control subjects &#40;OR&#160;&#61;&#160;4&#46;89&#44; 95&#37; CI&#58; 2&#46;03&#8211;11&#46;82&#41;&#46;</p></span> <span id="abss0004" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cesectitle0005">Conclusions</span><p id="spara009" class="elsevierStyleSimplePara elsevierViewall">The present study suggests that the rs17034984 variant of <span class="elsevierStyleItalic">EPAS1</span> gene is associated with PPHN&#46;</p></span>"
        "secciones" => array:4 [
          0 => array:2 [
            "identificador" => "abss0001"
            "titulo" => "Objective"
          ]
          1 => array:2 [
            "identificador" => "abss0002"
            "titulo" => "Methods"
          ]
          2 => array:2 [
            "identificador" => "abss0003"
            "titulo" => "Results"
          ]
          3 => array:2 [
            "identificador" => "abss0004"
            "titulo" => "Conclusions"
          ]
        ]
      ]
    ]
    "apendice" => array:1 [
      0 => array:1 [
        "seccion" => array:1 [
          0 => array:4 [
            "apendice" => "<p id="para0023a" class="elsevierStylePara elsevierViewall"><elsevierMultimedia ident="ecom0001"></elsevierMultimedia></p>"
            "etiqueta" => "Appendix"
            "titulo" => "Supplementary materials"
            "identificador" => "sec0016"
          ]
        ]
      ]
    ]
    "multimedia" => array:5 [
      0 => array:8 [
        "identificador" => "fig0001"
        "etiqueta" => "Figure 1"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "gr1.jpeg"
            "Alto" => 1710
            "Ancho" => 2751
            "Tamanyo" => 257819
          ]
        ]
        "detalles" => array:1 [
          0 => array:3 [
            "identificador" => "alt0003"
            "detalle" => "Fig&#46; "
            "rol" => "short"
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="spara001" class="elsevierStyleSimplePara elsevierViewall">A flow chart illustrating subject selection and study workflow&#46;</p>"
        ]
      ]
      1 => array:8 [
        "identificador" => "fig0002"
        "etiqueta" => "Figure 2"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "gr2.jpeg"
            "Alto" => 1879
            "Ancho" => 2500
            "Tamanyo" => 402266
          ]
        ]
        "detalles" => array:1 [
          0 => array:3 [
            "identificador" => "alt0004"
            "detalle" => "Fig&#46; "
            "rol" => "short"
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="spara002" class="elsevierStyleSimplePara elsevierViewall">A&#44; Positive HIF-2&#945; was detected in the nucleuses of few endothelial cells &#40;arrow&#41; of bronchial vessels in PPHN patients&#59; B&#44; Endothelial cells of bronchial arterioles stained negatively for HIF-2&#945;&#59; C&#44; Positive HIF-2&#945; was detected in the nucleuses of many endothelial cells &#40;arrow&#41; of umbilical cord vessels in healthy newborns&#59; D&#44; Immunostaining of HIF-2&#945; stained negatively in adult bronchial vessels&#46; Scale bar&#160;&#61;&#160;20 &#956;m&#46;</p>"
        ]
      ]
      2 => array:8 [
        "identificador" => "tbl0001"
        "etiqueta" => "Table 1"
        "tipo" => "MULTIMEDIATABLA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "detalles" => array:1 [
          0 => array:3 [
            "identificador" => "alt0001"
            "detalle" => "Table "
            "rol" => "short"
          ]
        ]
        "tabla" => array:1 [
          "tablatextoimagen" => array:1 [
            0 => array:1 [
              "tabla" => array:1 [
                0 => """
                  <table border="0" frame="\n
                  \t\t\t\t\tvoid\n
                  \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><a name="en0001"></a><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Gene&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><a name="en0002"></a><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Gene name&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><a name="en0003"></a><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Chromosome position&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><a name="en0004"></a><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Total SNPs tested&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><a name="en0005"></a><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="" valign="top" scope="col" style="border-bottom: 2px solid black">Significant SNPs&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><a name="en0006"></a><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="" valign="top" scope="col" style="border-bottom: 2px solid black">Position&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><a name="en0007"></a><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="" valign="top" scope="col" style="border-bottom: 2px solid black"><span class="elsevierStyleItalic">p</span>-value&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><a name="en0008"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="top"><span class="elsevierStyleItalic">BMPR2</span>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0009"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="top"><span class="elsevierStyleItalic">Bone morphogenetic protein receptor type2</span>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0010"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="top">2q33&#46;1&#8211;33&#46;2&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0011"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="top">14&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0012"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">rs79171078&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0013"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Intron&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0014"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;037&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0015"></a><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowgroup " rowspan="10" align="left" valign="top"><span class="elsevierStyleItalic">EPAS1</span></td><a name="en0016"></a><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowgroup " rowspan="10" align="left" valign="top"><span class="elsevierStyleItalic">Endothelial PAS domain protein 1</span></td><a name="en0017"></a><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowgroup " rowspan="10" align="left" valign="top">2p21</td><a name="en0018"></a><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowgroup " rowspan="10" align="left" valign="top">41</td><a name="en0019"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">rs17034984&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0020"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Intron&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0021"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;00014&#42;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0026"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">rs77238140&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0027"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Intron&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0028"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;008&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0033"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">rs2121267&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0034"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Intron&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0035"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;008&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0040"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">rs6726454&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0041"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Intron&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0042"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;014&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0047"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">rs4953344&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0048"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Intron&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0049"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;021&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0054"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">rs2346176&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0055"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Intron&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0056"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;024&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0061"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">rs1374749&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0062"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Intron&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0063"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;032&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0068"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">rs1868089&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0069"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Intron&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0070"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;038&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0075"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">rs6758592&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0076"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Intron&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0077"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;038&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0082"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">rs4953345&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0083"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Intron&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0084"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;045&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0085"></a><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowgroup " rowspan="3" align="left" valign="top"><span class="elsevierStyleItalic">PDE3A</span></td><a name="en0086"></a><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowgroup " rowspan="3" align="left" valign="top"><span class="elsevierStyleItalic">Phosphodiesterase 3A</span></td><a name="en0087"></a><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowgroup " rowspan="3" align="left" valign="top">12p12&#46;2</td><a name="en0088"></a><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowgroup " rowspan="3" align="left" valign="top">132</td><a name="en0089"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">rs10770682&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0090"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Intron&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0091"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;013&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0096"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">rs57238599&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0097"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">3&#180; untranslated region UTR Variant&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0098"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;002&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0103"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">rs6487091&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0104"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Intron&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0105"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;042&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0106"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="top"><span class="elsevierStyleItalic">VEGFA</span>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0107"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="top"><span class="elsevierStyleItalic">Vascular endothelial growth factor A</span>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0108"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="top">6p21&#46;1&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0109"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="top">25&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0110"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">rs3025012&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0111"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Intron&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0112"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;021&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0113"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="top"><span class="elsevierStyleItalic">ENG</span>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0114"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="top"><span class="elsevierStyleItalic">Endoglin</span>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0115"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="top">9q34&#46;11&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0116"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="top">9&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0117"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">rs34116890&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0118"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Intron&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0119"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;018&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0120"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="top"><span class="elsevierStyleItalic">NOTCH3</span>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0121"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="top"><span class="elsevierStyleItalic">NOTCH receptor 3</span>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0122"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="top">19p13&#46;12&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0123"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="top">17&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0124"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">rs10404382&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0125"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Intron&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0126"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;043&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr></tbody></table>
                  """
              ]
            ]
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="spara003" class="elsevierStyleSimplePara elsevierViewall">Candidate genes related to the pathogenesis of PPHN &#40;17 SNPs from 6 candidate genes&#41;&#46;</p>"
        ]
      ]
      3 => array:8 [
        "identificador" => "tbl0002"
        "etiqueta" => "Table 2"
        "tipo" => "MULTIMEDIATABLA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "detalles" => array:1 [
          0 => array:3 [
            "identificador" => "alt0002"
            "detalle" => "Table "
            "rol" => "short"
          ]
        ]
        "tabla" => array:2 [
          "leyenda" => "<p id="spara005" class="elsevierStyleSimplePara elsevierViewall">Note&#58; intensity of staining&#58; 0&#44; no staining&#59; 1&#44; weak&#59; 2&#44; moderate&#59; and 3&#44; strong&#46;</p>"
          "tablatextoimagen" => array:1 [
            0 => array:1 [
              "tabla" => array:1 [
                0 => """
                  <table border="0" frame="\n
                  \t\t\t\t\tvoid\n
                  \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><a name="en0127"></a><th class="td-with-role" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t ; entry_with_role_rowgroup " rowspan="2" align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Tissue</th><a name="en0128"></a><th class="td-with-role" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t ; entry_with_role_colgroup " colspan="2" align="center" valign="top" scope="col" style="border-bottom: 2px solid black">Level of HIF-2&#945; protein</th></tr><tr title="table-row"><a name="en0130"></a><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="" valign="top" scope="col" style="border-bottom: 2px solid black">Manual scoring&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><a name="en0131"></a><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="" valign="top" scope="col" style="border-bottom: 2px solid black">IHC staining intensity normalized by nucleus &#40;image analysis&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><a name="en0132"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">PPHN lung &#40;case 1&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0133"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">1&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0134"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;009&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0135"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">PPHN lung &#40;case 2&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0136"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">1&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0137"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;010&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0138"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">PPHN lung &#40;case 3&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0139"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0140"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;001&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0141"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Normal adult lung &#40;n&#160;&#61;&#160;3&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0142"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0143"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;002&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0144"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Umbilical cord &#40;case 1&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0145"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">2&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0146"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;026&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0147"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Umbilical cord &#40;case 2&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0148"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">2&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0149"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;038&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0150"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Umbilical cord &#40;case 3&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0151"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">2&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0152"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;036&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0153"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Umbilical cord &#40;case 4&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0154"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">2&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0155"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;033&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0156"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Umbilical cord &#40;case 5&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0157"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">2&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0158"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;029&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0159"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Umbilical cord &#40;case 6&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0160"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">2&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0161"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;085&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0162"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Umbilical cord &#40;case 7&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0163"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">2&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0164"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;042&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><a name="en0165"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">Umbilical cord &#40;case 8&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0166"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">2&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><a name="en0167"></a><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="top">0&#46;039&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr></tbody></table>
                  """
              ]
            ]
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="spara004" class="elsevierStyleSimplePara elsevierViewall">Expression of HIF-2&#945; protein in different tissues&#46;</p>"
        ]
      ]
      4 => array:6 [
        "identificador" => "ecom0001"
        "tipo" => "MULTIMEDIAECOMPONENTE"
        "mostrarFloat" => false
        "mostrarDisplay" => true
        "detalles" => array:1 [
          0 => array:3 [
            "identificador" => "alt0005"
            "detalle" => "Image&#44; application "
            "rol" => "short"
          ]
        ]
        "Ecomponente" => array:2 [
          "fichero" => "mmc1.docx"
          "ficheroTamanyo" => 1633679
        ]
      ]
    ]
    "bibliografia" => array:2 [
      "titulo" => "References"
      "seccion" => array:1 [
        0 => array:2 [
          "identificador" => "cebibsec1"
          "bibliografiaReferencia" => array:26 [
            0 => array:3 [
              "identificador" => "bib0001"
              "etiqueta" => "1"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "An Asian multicenter retrospective study on persistent pulmonary hypertension of the newborn&#58; incidence&#44; etiology&#44; diagnosis&#44; treatment and outcome"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "N Nakwan"
                            1 => "S Jain"
                            2 => "K Kumar"
                            3 => "S Hosono"
                            4 => "M Hammoud"
                            5 => "YY Elsayed"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1080/14767058.2018.1536740"
                      "Revista" => array:6 [
                        "tituloSerie" => "J Matern Fetal Neonatal Med"
                        "fecha" => "2020"
                        "volumen" => "33"
                        "paginaInicial" => "2032"
                        "paginaFinal" => "2037"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/30318951"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            1 => array:3 [
              "identificador" => "bib0002"
              "etiqueta" => "2"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Persistent pulmonary hypertension of the newborn&#58; advances in diagnosis and treatment"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:2 [
                            0 => "A Jain"
                            1 => "PJ&#46; McNamara"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:1 [
                      "Revista" => array:5 [
                        "tituloSerie" => "Semin Fetal Neonat Med"
                        "fecha" => "2015"
                        "volumen" => "20"
                        "paginaInicial" => "262"
                        "paginaFinal" => "271"
                      ]
                    ]
                  ]
                ]
              ]
            ]
            2 => array:3 [
              "identificador" => "bib0003"
              "etiqueta" => "3"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Persistent pulmonary hypertension of non-cardiac causes in a neonatal intensive care unit"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:3 [
                            0 => "G Rocha"
                            1 => "MJ Baptista"
                            2 => "H&#46; Guimar&#227;es"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:1 [
                      "Revista" => array:5 [
                        "tituloSerie" => "Pulm Med"
                        "fecha" => "2012"
                        "volumen" => "2012"
                        "paginaInicial" => "818"
                        "paginaFinal" => "971"
                      ]
                    ]
                  ]
                ]
              ]
            ]
            3 => array:3 [
              "identificador" => "bib0004"
              "etiqueta" => "4"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Persistent pulmonary hypertension of the newborn in late preterm and term infants in California"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:6 [
                            0 => "MA Steurer"
                            1 => "LL Jelliffe-Pawlowski"
                            2 => "RJ Baer"
                            3 => "JC Partridge"
                            4 => "EE Rogers"
                            5 => "RL&#46; Keller"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:1 [
                      "Revista" => array:3 [
                        "tituloSerie" => "Pediatrics"
                        "fecha" => "2017"
                        "volumen" => "139"
                      ]
                    ]
                  ]
                ]
              ]
            ]
            4 => array:3 [
              "identificador" => "bib0005"
              "etiqueta" => "5"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Neonatal pulmonary hypertension&#8211;urea-cycle intermediates&#44; nitric oxide production&#44; and carbamoyl-phosphate synthetase function"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "DL Pearson"
                            1 => "S Dawling"
                            2 => "WF Walsh"
                            3 => "JL Haines"
                            4 => "BW Christman"
                            5 => "A Bazyk"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1056/NEJM200106143442404"
                      "Revista" => array:6 [
                        "tituloSerie" => "N Engl J Med"
                        "fecha" => "2001"
                        "volumen" => "344"
                        "paginaInicial" => "1832"
                        "paginaFinal" => "1838"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/11407344"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            5 => array:3 [
              "identificador" => "bib0006"
              "etiqueta" => "6"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Variations in CRHR1 are associated with persistent pulmonary hypertension of the newborn"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:6 [
                            0 => "HM Byers"
                            1 => "JM Dagle"
                            2 => "JM Klein"
                            3 => "KK Ryckman"
                            4 => "EL McDonald"
                            5 => "JC&#46; Murray"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1038/pr.2011.24"
                      "Revista" => array:6 [
                        "tituloSerie" => "Pediatr Res"
                        "fecha" => "2012"
                        "volumen" => "71"
                        "paginaInicial" => "162"
                        "paginaFinal" => "167"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/22258127"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            6 => array:3 [
              "identificador" => "bib0007"
              "etiqueta" => "7"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "EDN1 gene variant is associated with neonatal persistent pulmonary hypertension"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "M Mei"
                            1 => "G Cheng"
                            2 => "B Sun"
                            3 => "L Yang"
                            4 => "H Wang"
                            5 => "J Sun"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1038/srep29877"
                      "Revista" => array:5 [
                        "tituloSerie" => "Sci Rep"
                        "fecha" => "2016"
                        "volumen" => "6"
                        "paginaInicial" => "29877"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/27425626"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            7 => array:3 [
              "identificador" => "bib0008"
              "etiqueta" => "8"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Polymorphisms in urea cycle enzyme genes are associated with persistent pulmonary hypertension of the newborn"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:6 [
                            0 => "DC Kaluarachchi"
                            1 => "CJ Smith"
                            2 => "JM Klein"
                            3 => "JC Murray"
                            4 => "JM Dagle"
                            5 => "KK&#46; Ryckman"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1038/pr.2017.143"
                      "Revista" => array:6 [
                        "tituloSerie" => "Pediatr Res"
                        "fecha" => "2018"
                        "volumen" => "83"
                        "paginaInicial" => "142"
                        "paginaFinal" => "147"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/28609431"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            8 => array:3 [
              "identificador" => "bib0009"
              "etiqueta" => "9"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Identification of genetic factors underlying persistent pulmonary hypertension of newborns in a cohort of Chinese neonates"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "X Liu"
                            1 => "M Mei"
                            2 => "X Chen"
                            3 => "Y Lu"
                            4 => "X Dong"
                            5 => "L Hu"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1186/s12931-019-1148-1"
                      "Revista" => array:5 [
                        "tituloSerie" => "Respir Res"
                        "fecha" => "2019"
                        "volumen" => "20"
                        "paginaInicial" => "174"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/31382961"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            9 => array:3 [
              "identificador" => "bib0010"
              "etiqueta" => "10"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Frequent mutation of hypoxia-related genes in persistent pulmonary hypertension of the newborn"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "M Wang"
                            1 => "D Zhuang"
                            2 => "M Mei"
                            3 => "H Ma"
                            4 => "Z Li"
                            5 => "F He"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1186/s12931-020-1314-5"
                      "Revista" => array:5 [
                        "tituloSerie" => "Respir Res"
                        "fecha" => "2020"
                        "volumen" => "21"
                        "paginaInicial" => "53"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/32054482"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            10 => array:3 [
              "identificador" => "bib0011"
              "etiqueta" => "11"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Phenotype characterisation of <span class="elsevierStyleItalic">TBX4</span> mutation and deletion carriers with neonatal and paediatric pulmonary hypertension"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "C Galambos"
                            1 => "MP Mullen"
                            2 => "JT Shieh"
                            3 => "N Schwerk"
                            4 => "MJ Kielt"
                            5 => "N Ullmann"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:1 [
                      "Revista" => array:3 [
                        "tituloSerie" => "Eur Respir J"
                        "fecha" => "2019"
                        "volumen" => "54"
                      ]
                    ]
                  ]
                ]
              ]
            ]
            11 => array:3 [
              "identificador" => "bib0012"
              "etiqueta" => "12"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Acute kidney injury and pneumothorax are risk factors for mortality in persistent pulmonary hypertension of the newborn in Thai neonates"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:2 [
                            0 => "N Nakwan"
                            1 => "S&#46; Pithaklimnuwong"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.3109/14767058.2015.1060213"
                      "Revista" => array:6 [
                        "tituloSerie" => "J Matern Fetal Neonatal Med"
                        "fecha" => "2016"
                        "volumen" => "29"
                        "paginaInicial" => "1741"
                        "paginaFinal" => "1746"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/26217981"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            12 => array:3 [
              "identificador" => "bib0013"
              "etiqueta" => "13"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "The practical challenges of diagnosis and treatment options in persistent pulmonary hypertension of the newborn&#58; a developing country&#39;s perspective"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:1 [
                            0 => "N&#46; Nakwan"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1055/s-0038-1660462"
                      "Revista" => array:6 [
                        "tituloSerie" => "Am J Perinatol"
                        "fecha" => "2018"
                        "volumen" => "35"
                        "paginaInicial" => "1366"
                        "paginaFinal" => "1375"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/29920641"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            13 => array:3 [
              "identificador" => "bib0014"
              "etiqueta" => "14"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Semi-quantitative determination of protein expression using immunohistochemistry staining and analysis&#58; an integrated protocol"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:2 [
                            0 => "AR Crowe"
                            1 => "W&#46; Yue"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.21769/BioProtoc.3465"
                      "Revista" => array:5 [
                        "tituloSerie" => "Bio-protocol"
                        "fecha" => "2019"
                        "volumen" => "9"
                        "paginaInicial" => "e3465"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/31867411"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            14 => array:3 [
              "identificador" => "bib0015"
              "etiqueta" => "15"
              "referencia" => array:1 [
                0 => array:1 [
                  "referenciaCompleta" => "Reference SNP &#40;rs&#41; Report&#58; rs17034984&#46; &#91;Cited 2020 August 19&#93;&#46; Available from&#58; <a target="_blank" href="https://www.ncbi.nlm.nih.gov/snp/rs17034984">https&#58;&#47;&#47;www&#46;ncbi&#46;nlm&#46;nih&#46;gov&#47;snp&#47;rs17034984</a>"
                ]
              ]
            ]
            15 => array:3 [
              "identificador" => "bib0016"
              "etiqueta" => "16"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "HIF and the lung&#58; role of hypoxia-inducible factor in pulmonary development and disease"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:2 [
                            0 => "LA Shimoda"
                            1 => "GL&#46; Semenza"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1164/rccm.201009-1393PP"
                      "Revista" => array:6 [
                        "tituloSerie" => "Am J Respir Crit Care Med"
                        "fecha" => "2011"
                        "volumen" => "183"
                        "paginaInicial" => "152"
                        "paginaFinal" => "156"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/21242594"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            16 => array:3 [
              "identificador" => "bib0017"
              "etiqueta" => "17"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Natural selection on <span class="elsevierStyleItalic">EPAS1</span> &#40;HIF2alpha&#41; associated with low hemoglobin concentration in Tibetan highlanders"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "CM Beall"
                            1 => "GL Cavalleri"
                            2 => "L Deng"
                            3 => "RC Elston"
                            4 => "Y Gao"
                            5 => "J Knight"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1073/pnas.1002443107"
                      "Revista" => array:6 [
                        "tituloSerie" => "Proc Natl Acad Sci U S A"
                        "fecha" => "2010"
                        "volumen" => "107"
                        "paginaInicial" => "11459"
                        "paginaFinal" => "11464"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/20534544"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            17 => array:3 [
              "identificador" => "bib0018"
              "etiqueta" => "18"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Genetic evidence for high-altitude adaptation in Tibet"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "TS Simonson"
                            1 => "Y Yang"
                            2 => "CD Huff"
                            3 => "H un"
                            4 => "G Qin"
                            5 => "DJ Witherspoon"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1126/science.1189406"
                      "Revista" => array:6 [
                        "tituloSerie" => "Science"
                        "fecha" => "2010"
                        "volumen" => "329"
                        "paginaInicial" => "72"
                        "paginaFinal" => "75"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/20466884"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            18 => array:3 [
              "identificador" => "bib0019"
              "etiqueta" => "19"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Increased prevalence of EPAS1 variant in cattle with high-altitude pulmonary hypertension"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "JH Newman"
                            1 => "TN Holt"
                            2 => "JD Cogan"
                            3 => "B Womack"
                            4 => "JA Phillips 3rd"
                            5 => "C Li"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1038/ncomms7863"
                      "Revista" => array:5 [
                        "tituloSerie" => "Nat Commun"
                        "fecha" => "2015"
                        "volumen" => "6"
                        "paginaInicial" => "6863"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/25873470"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            19 => array:3 [
              "identificador" => "bib0020"
              "etiqueta" => "20"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "HIF2&#945;-arginase axis is essential for the development of pulmonary hypertension"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "AS Cowburn"
                            1 => "A Crosby"
                            2 => "D Macias"
                            3 => "C Branco"
                            4 => "RD Cola&#231;o"
                            5 => "M Southwood"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1073/pnas.1602978113"
                      "Revista" => array:6 [
                        "tituloSerie" => "Proc Natl Acad Sci USA"
                        "fecha" => "2016"
                        "volumen" => "113"
                        "paginaInicial" => "8801"
                        "paginaFinal" => "8806"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/27432976"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            20 => array:3 [
              "identificador" => "bib0021"
              "etiqueta" => "21"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "The endothelial prolyl-4-hydroxylase domain 2&#47;hypoxia-inducible factor 2 axis regulates pulmonary artery pressure in mice"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "PP Kapitsinou"
                            1 => "G Rajendran"
                            2 => "L Astleford"
                            3 => "M Michael"
                            4 => "MP Schonfeld"
                            5 => "T Fields"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1128/MCB.01055-15"
                      "Revista" => array:6 [
                        "tituloSerie" => "Mol Cell Biol"
                        "fecha" => "2016"
                        "volumen" => "36"
                        "paginaInicial" => "1584"
                        "paginaFinal" => "1594"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/26976644"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            21 => array:3 [
              "identificador" => "bib0022"
              "etiqueta" => "22"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Hypoxia inducible factor 2&#945; &#40;HIF2&#945;&#47;EPAS1&#41; is associated with development of pulmonary hypertension in severe congenital diaphragmatic hernia patients"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:6 [
                            0 => "Y Huang"
                            1 => "A Boerema-de Munck"
                            2 => "M Buscop-van Kempen"
                            3 => "R de Krijger"
                            4 => "D Tibboel"
                            5 => "RJ&#46; Rottier"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:1 [
                      "Revista" => array:3 [
                        "tituloSerie" => "Pulm Circ"
                        "fecha" => "2018"
                        "volumen" => "8"
                      ]
                    ]
                  ]
                ]
              ]
            ]
            22 => array:3 [
              "identificador" => "bib0023"
              "etiqueta" => "23"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Hypoxia-inducible factors in the first trimester human lung"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:5 [
                            0 => "F Groenman"
                            1 => "M Rutter"
                            2 => "I Caniggia"
                            3 => "D Tibboel"
                            4 => "M&#46; Post"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1369/jhc.6A7129.2006"
                      "Revista" => array:6 [
                        "tituloSerie" => "J Histochem Cytochem"
                        "fecha" => "2007"
                        "volumen" => "55"
                        "paginaInicial" => "355"
                        "paginaFinal" => "363"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/17189520"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            23 => array:3 [
              "identificador" => "bib0024"
              "etiqueta" => "24"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Hypoxia-inducible factors HIF-1alpha and HIF-2alpha are decreased in an experimental model of severe respiratory distress syndrome in preterm lambs"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:5 [
                            0 => "TR Grover"
                            1 => "TM Asikainen"
                            2 => "JP Kinsella"
                            3 => "SH Abman"
                            4 => "CW&#46; White"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1152/ajplung.00372.2006"
                      "Revista" => array:6 [
                        "tituloSerie" => "Am J Physiol Lung Cell Mol Physiol"
                        "fecha" => "2007"
                        "volumen" => "292"
                        "paginaInicial" => "L1345"
                        "paginaFinal" => "L1351"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/17307811"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            24 => array:3 [
              "identificador" => "bib0025"
              "etiqueta" => "25"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Effect of preterm birth on hypoxia-inducible factors and vascular endothelial growth factor in primate lungs"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:4 [
                            0 => "TM Asikainen"
                            1 => "A Ahmad"
                            2 => "BK Schneider"
                            3 => "CW White"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1002/ppul.20321"
                      "Revista" => array:6 [
                        "tituloSerie" => "Pediatr Pulmonol"
                        "fecha" => "2005"
                        "volumen" => "40"
                        "paginaInicial" => "538"
                        "paginaFinal" => "546"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/16231377"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            25 => array:3 [
              "identificador" => "bib0026"
              "etiqueta" => "26"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Hypoxia-inducible factor 2&#945; plays a critical role in the formation of alveoli and surfactant"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "Y Huang"
                            1 => "MB Kempen"
                            2 => "AB Munck"
                            3 => "S Swagemakers"
                            4 => "S Driegen"
                            5 => "P Mahavadi"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1165/rcmb.2011-0024OC"
                      "Revista" => array:6 [
                        "tituloSerie" => "Am J Respir Cell Mol Biol"
                        "fecha" => "2012"
                        "volumen" => "46"
                        "paginaInicial" => "224"
                        "paginaFinal" => "232"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/22298531"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
          ]
        ]
      ]
    ]
    "agradecimientos" => array:1 [
      0 => array:4 [
        "identificador" => "xack616947"
        "titulo" => "Acknowledgments"
        "texto" => "<p id="para0025" class="elsevierStylePara elsevierViewall">The authors thank Dr&#46; Jakris Eu-ahsunthornwattana of the Department of Community Medicine&#44; Faculty of Medicine Ramathibodi Hospital&#44; Mahidol University for allowing the authors to use the Thai SNP database&#46; The authors thank Mr&#46; David Patterson of the Office of International Relations&#44; Faculty of Medicine&#44; Prince of Songkla University for editing the manuscript&#46;</p>"
        "vista" => "all"
      ]
    ]
  ]
  "idiomaDefecto" => "en"
  "url" => "/00217557/0000009800000004/v2_202207140636/S0021755721001376/v2_202207140636/en/main.assets"
  "Apartado" => array:4 [
    "identificador" => "10179"
    "tipo" => "SECCION"
    "en" => array:2 [
      "titulo" => "Original articles"
      "idiomaDefecto" => true
    ]
    "idiomaDefecto" => "en"
  ]
  "PDF" => "https://static.elsevier.es/multimedia/00217557/0000009800000004/v2_202207140636/S0021755721001376/v2_202207140636/en/main.pdf?idApp=UINPBA000049&text.app=https://jped.elsevier.es/"
  "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0021755721001376?idApp=UINPBA000049"
]
Share
Journal Information
Vol. 98. Issue 4.
Pages 383-389 (July - August 2022)
Share
Share
Download PDF
More article options
Visits
3140
Vol. 98. Issue 4.
Pages 383-389 (July - August 2022)
Original article
Open Access
Possible association between a polymorphism of EPAS1 gene and persistent pulmonary hypertension of the newborn: a case-control study
Visits
3140
Narongsak Nakwana,b, Surakameth Mahasirimongkolc, Nusara Satproedpraic, Tassamonwan Chaiyasungc, Punna Kunhapanc, Cheep Charoenlapd, Kamonnut Singkhamanana, Chariyawan Charalsawadie,
Corresponding author
cchariya@medicine.psu.ac.th

Corresponding author.
a Prince of Songkla University, Faculty of Medicine, Department of Biomedical Sciences, Hat Yai, Thailand
b Hat Yai Hospital, Hat Yai Medical Education Center, Department of Pediatrics, Hat Yai, Thailand
c Ministry of Public Heath, Department of Medical Sciences, Division of Genomic Medicine and Innovation Support, Medical Genetics Center, Nonthaburi, Thailand
d Hat Yai Hospital, Hat Yai Medical Education Center, Department of Anatomical Pathology, Hat Yai, Thailand
e Prince of Songkla University, Faculty of Medicine, Department of Pathology, Hat Yai, Thailand
This item has received

Under a Creative Commons license
Article information
Abstract
Full Text
Bibliography
Download PDF
Statistics
Figures (2)
Additional material (1)
Abstract
Objective

To explore possible genes related to the development of persistent pulmonary hypertension of the newborn (PPHN).

Methods

The authors identified 285 single nucleotide polymorphisms (SNPs) of 11 candidate genes (BMPR2, EPAS1, PDE3A, VEGFA, ENG, NOTCH3, SOD3, CPS1, ABCA3, ACVRL1, and SMAD9), using an Illumina Asian Screening Array-24 v1.0 BeadChip Array. The FastLmmC and R package was used for statistical analyses. The chi-square test and Cochrane-Armitage trend test were used to compare the allele and genotype frequencies between the groups and to test the genetic models, respectively.

Results

A total of 45 PPHN infants and 294 control subjects were analyzed. The most common cause of PPHN was meconium aspiration syndrome. Among the 285 SNPs, 17 SNPs from 6 candidate genes (BMPR2, EPAS1, PDE3A, VEGFA, ENG, and NOTCH3) were significantly associated with PPHN (P < 0.05). After using the Bonferroni correction (P < 0.00018), only the rs17034984 SNP located in intron 1 of the EPAS1 gene remained significantly different between the PPHN and control subjects (P = 0.00014). The frequency of the TC/TT genotype of rs17034984 in the gene with the dominant model was significant in the patients with PPHN (OR = 5.38, 95% CI: 2.15–13.49). The T allele frequency of rs17034984 in the gene showed a significant difference compared with the control subjects (OR = 4.89, 95% CI: 2.03–11.82).

Conclusions

The present study suggests that the rs17034984 variant of EPAS1 gene is associated with PPHN.

Keywords:
PPHN
Polymorphism
SNP
EPAS1 gene
Association study
Full Text
Introduction

Persistent pulmonary hypertension of the newborn (PPHN) is the most serious complication of several neonatal respiratory diseases, leading to adverse outcomes such as asphyxia, chronic lung disease, neurodevelopmental sequelae, and death, despite the use of modern therapeutic modalities for treating PPHN such as high-frequency oscillatory ventilation, inhaled nitric oxide, and extracorporeal membrane oxygenation.1,2 Although the primary etiologies of PPHN are various factors such as chronic hypoxia, meconium aspiration syndrome, birth asphyxia, sepsis, infection, and respiratory distress syndrome,1,3,4 a genetic contribution to PPHN has been increasingly recognized. Previous studies have shown that SNPs in the CRHR1 (rs4458044), CPS1 (rs41272673, rs4399666, rs2287599, rs192759073, rs1047883, and rs2229589), EDN1 (rs2070699), and NOTCH3 (rs1044008) genes are susceptibility loci associated with PPHN.5–9 It has also recently been reported that mutations in a few genes such as TTLL3 and ITGAM10 were associated with PPHN. TBX4 variant has also been found in relation to neonatal and pediatric pulmonary hypertension.11

In this study, the authors aimed to determine any associations between SNPs of previous PPHN candidate genes and to perform a validation of any genetic associations found to elucidate a possible biological involvement of the protein(s) encoded from the gene(s) in the etiology of PPHN.

Materials and methodsStudy population

The authors recruited 45 patients with PPHN who were hospitalized at Hat Yai hospital, one of the biggest hospitals in Southern Thailand where the prevalence of PPHN is relatively high, from 2.6 to 2.8 per 1000 live births, and constituting ∼30% of the all-cause mortality rate.1,12 Retrospective data of PPHN infants discharged from the hospital between January 2013 and March 2019, born to mothers with a gestational age of ≥ 35 weeks and birth weight of ≥ 2000 g, were retrieved from the hospital computer data system. The authors recruited 20 patients from the retrospective data. The authors also recruited additional PPHN neonates who were hospitalized at the hospital between April 2019 and November 2019 (Figure 1). The authors excluded patients with congenital structural cyanotic heart diseases and other congenital disorders such as congenital diaphragmatic hernia and birth defects due to chromosomal disorders. For a healthy control group, the authors recruited 59 neonates who were born at the hospital between April 2019 and November 2019 and 235 adults who participated in the Thai SNP database project of Ramathibodi Hospital, Bangkok, Thailand. A summary of subjects involving in the study is shown in Figure 1 and baseline characteristics are presented in supplementary Table 1. This study was approved by the Research Ethics Committee of the Hat Yai Hospital (EC No. 24/2562).

Figure 1.

A flow chart illustrating subject selection and study workflow.

(0.25MB).
Study definitions

The diagnostic criteria for PPHN were presentation shortly after birth with refractory hypoxemia plus one of the three following conditions: 1) documented pulmonary hypertension, as defined by echocardiographic evidence of elevated pulmonary pressure (right to left or bidirectional shunt) and/or 2) a pre-to-postductal partial pressure of oxygen gradient equal to or greater than 20 mm Hg, and/or 3) a pulse oximetry oxygen saturation (SpO2) gradient equal to or greater than 10%.13

Genomic DNA extraction

Genomic DNA was extracted from a 200 μL sample of peripheral blood using a Qiagen DNA Mini kit (Qiagen, Hilden, Germany). The quantity and quality of the DNA samples were evaluated using an Eppendorf BioPhotometer plus (Eppendorf AG, Hamburg, Germany) and automated electrophoresis (Agilent 2200 TapeStation system, Santa Clara, California, United States).

Genotyping and quality control

A total of 659,184 single nucleotide polymorphism (SNPs) probes specific to several regions across the genome were fabricated on an Illumina Asian Screening Array-24 v1.0 BeadChip Array. SNP genotyping using an Illumina Infinium HTS Assay platform (Illumina, San Diego, California) was carried out and 612,128 (93%) SNPs were successfully genotyped. For quality control, the authors applied a low call rate < 98%, a Hardy–Weinberg equilibrium calculations p-value < 1 × 10−8, and minor allele frequency (MAF) of each SNP <0.05 to exclude data with low quality. After the quality control exclusion, 498,160 autosomal SNPs were left for the genome-wide association study (GWAS) analysis. The authors selected the SNPs in the genes related to PPHN and pulmonary arterial hypertension (PAH). Finally, 285 SNPs of 11 candidate genes (BMPR2, EPAS1, PDE3A, VEGFA, ENG, NOTCH3, SOD3, CPS1, ABCA3, ACVRL1, and SMAD9) were analyzed.

Immunohistochemistry (IHC) detection of HIF-2α in umbilicus and lung tissues

For the validation study, the authors evaluated the expression of hypoxia-inducible factor 2-alpha (HIF-2α), a protein encoded from the EPAS1 gene, in the pulmonary vascular endothelium of lung tissues obtained from deceased PPHN patients (n = 3) compared to normal adult lung tissue (n = 3). In addition, the authors carried out IHC staining of HIF-2α in the umbilicuses of 8 healthy newborns to evaluate the expression of the protein in the umbilical artery and vein. Paraffin-embedded umbilical and lung tissues were stained with hematoxylin and eosin to examine histopathology of the tissues. Then, IHC using anti-HIF-2α antibody (ab199, Abcam, UK) with dilution 1:100 v/v was performed on VENTANA BenchMark ULTRA platform (Ventana Medical Systems Inc., USA) to examine for HIF-2α expression.

Statistical analysis

The authors used FastLmmC (v2.02.20121124) and the R package for GWAS (v. 3.3.3) for statistical analysis. Regional plots were generated using LocusZoom v.0.4.8. The allele and genotype frequencies of the candidate genes were calculated by the genotypes of the subjects. The chi-square test was used to compare the allele and genotype frequencies between the groups. When comparing the genotype frequency of each significant SNP between groups, the authors used the Cochrane-Armitage trend test between all genotypes. Odds ratio (OR) and 95% confidence intervals (CI) were calculated to indicate the degree of association between groups.

ResultsAssociation of SNPs in previously reported candidate genes and PPHN/ PAH

The authors analyzed 285 SNPs in 11 candidate genes, including BMPR2, EPAS1, PDE3A, VEGFA, ENG, NOTCH3, SOD3, CPS1, ABCA3, ACVRL1, and SMAD9, that were found to be associated with the pathogenesis of PPHN/PAH in previous studies. A p-value of < 0.05 was considered significant. The authors found that 17 SNPs in 6 candidate genes: BMPR2, EPAS1, PDE3A, VEGFA, ENG, and NOTCH3, were significantly associated with PPHN (Table 1). Of these, rs17034984 in the EPAS1 gene was the only one to remain significant after the Bonferroni correction (P < 0.00018).

Table 1.

Candidate genes related to the pathogenesis of PPHN (17 SNPs from 6 candidate genes).

Gene  Gene name  Chromosome position  Total SNPs tested  Significant SNPs  Position  p-value 
BMPR2  Bone morphogenetic protein receptor type2  2q33.1–33.2  14  rs79171078  Intron  0.037 
EPAS1Endothelial PAS domain protein 12p2141rs17034984  Intron  0.00014* 
rs77238140  Intron  0.008 
rs2121267  Intron  0.008 
rs6726454  Intron  0.014 
rs4953344  Intron  0.021 
rs2346176  Intron  0.024 
rs1374749  Intron  0.032 
rs1868089  Intron  0.038 
rs6758592  Intron  0.038 
rs4953345  Intron  0.045 
PDE3APhosphodiesterase 3A12p12.2132rs10770682  Intron  0.013 
rs57238599  3´ untranslated region UTR Variant  0.002 
rs6487091  Intron  0.042 
VEGFA  Vascular endothelial growth factor A  6p21.1  25  rs3025012  Intron  0.021 
ENG  Endoglin  9q34.11  rs34116890  Intron  0.018 
NOTCH3  NOTCH receptor 3  19p13.12  17  rs10404382  Intron  0.043 
Genotype distribution, allele frequency, and fit inheritance model of significant SNPs in candidate genes (P < 0.05) associated with PPHN/PAH

The genotype and allele frequencies of candidate gene polymorphisms are summarized in Supplementary Table 2. Various models of inheritance (codominant, dominant, and recessive) were tested and genetic effects were calculated for each genotype compared to the reference genotype in each model. The authors found that the frequency of the TC/TT genotype of rs17034984 in the EPAS1 gene with the dominant model was significant in the patients with PPHN (OR = 5.38, 95% CI: 2.15–13.49). In addition, the allele frequency of the minor allele T of rs17034984 in the EPAS1 gene showed a significant difference compared with the control subjects (OR = 4.89, 95% CI: 2.03–11.82).

HIF-2α immunohistochemistry staining of the vascular endothelium of umbilicus and lung tissues

HIF-2α protein was expressed in lung tissues from 2 infants with PPHN. Localization of HIF-2α was seen in the nuclei of endothelial cells of the pulmonary arteries (Figure 2A). However, there was one infant with PPHN in whom HIF-2α protein expression was not detected (Figure 2B). The HIF-2α protein was also expressed in the endothelial cells of the umbilical arteries and veins from the 8 normal newborns (Figure 2C). The HIF-2α protein was not expressed in normal adult lung tissues and endothelial cells of the pulmonary vessels (Figure 2D). Determination of protein expression was scored by two pathologists (intensity of staining: 0, no staining; 1, weak; 2, moderate; and 3, strong). In addition, semiquantitative analysis of the IHC images was performed using ImageJ Fiji software (version 2.1.0/1.53C), following an integrated protocol.14 Expression of HIF-2α in different tissues were shown in Table 2.

Figure 2.

A, Positive HIF-2α was detected in the nucleuses of few endothelial cells (arrow) of bronchial vessels in PPHN patients; B, Endothelial cells of bronchial arterioles stained negatively for HIF-2α; C, Positive HIF-2α was detected in the nucleuses of many endothelial cells (arrow) of umbilical cord vessels in healthy newborns; D, Immunostaining of HIF-2α stained negatively in adult bronchial vessels. Scale bar = 20 μm.

(0.38MB).
Table 2.

Expression of HIF-2α protein in different tissues.

TissueLevel of HIF-2α protein
Manual scoring  IHC staining intensity normalized by nucleus (image analysis) 
PPHN lung (case 1)  0.009 
PPHN lung (case 2)  0.010 
PPHN lung (case 3)  0.001 
Normal adult lung (n = 3)  0.002 
Umbilical cord (case 1)  0.026 
Umbilical cord (case 2)  0.038 
Umbilical cord (case 3)  0.036 
Umbilical cord (case 4)  0.033 
Umbilical cord (case 5)  0.029 
Umbilical cord (case 6)  0.085 
Umbilical cord (case 7)  0.042 
Umbilical cord (case 8)  0.039 

Note: intensity of staining: 0, no staining; 1, weak; 2, moderate; and 3, strong.

Discussion

PPHN is the most serious respiratory disorder in Thailand and has a high mortality rate. Its high prevalence in the study's institution led the authors to hypothesize whether genetic factors play an important role in the development of PPHN. Using SNP analysis, the authors found that rs17034984 in the EPAS1 gene showed a significant association with PPHN in the studied population. In addition, the authors found that the T allele of rs17034984 in the EPAS1 gene is a possible predisposing factor for PPHN and the TC/TT genotype with the dominant model could be a predictor of PPHN.

The rs17034984 SNP is located in intron 1 of the EPAS1 gene. The EPAS1 gene, located on the short arm of chromosome 2 (2p21), encodes endothelial PAS domain-containing protein 1, which is also known as hypoxia-inducible factor 2 alpha (HIF-2α) protein.15 This protein is a subunit of the HIF protein complex, which plays a crucial role in the body's ability to adapt to changing oxygen levels. HIF-2α is a transcription factor involved in the induction of oxygen-regulated genes. It regulates erythropoiesis and physiological responses to hypoxia through the HIFs signaling pathway.16 In hypoxic conditions, the HIFs protein is translocated to the nucleus to bind the hypoxic response element (HRE) of target gene promoters and subsequently initiate gene transcription. When oxygen levels rise, HIFs are hydroxylated by prolyl hydroxylase domain proteins (PHD), leading to interaction with other proteins, and with the addition to ubiquitin results in HIFs degradation.15 Previous studies reported that the EPAS1 gene polymorphisms were associated with adaptation to hypobaric hypoxia in the Tibetan population.17,18 In addition, HIF-2α regulates the vascular endothelial growth factor (VEGF) expression and seems to be implicated in the development of blood vessels and the tubular system of the lung. An animal model study also showed that the gain-of-function of HIF-2α was related to the development of pulmonary vascular remodeling.19 Another study found that chronic hypoxia could induce HIF-2α stability, leading to increased arginase expression and dysregulating normal vascular nitric oxide homeostasis.20 In addition, endothelial HIF-2α was a major mediator in the development of hypoxic-induced pulmonary hypertension, and its functions could induce pulmonary hypertension through an increased expression of endothelin-1, encoded from the EDN1 gene and a concomitant decrease in vasodilatory apelin receptor signaling.21 Gene network analysis showed that EPAS1 gene is co-expressed with EDN1 gene. The Gene network of 11 candidate genes in the present study study (BMPR2, EPAS1, PDE3A, VEGFA, ENG, NOTCH3, SOD3, CPS1, ABCA3, ACVRL1, and SMAD9) and other genes that had been shown to be associated with PPHN is shown in Supplementary Figure 1.

In the present study, 10 SNPs of the EPAS1 gene showed a significant difference between patients with PPHN and the controls (Table 1); however, rs17034984 was the only SNP that remained associated with PPHN after the Bonferroni correction. The T allele on rs17034984 was a risk factor for PPHN and the TC/TT genotype with the dominant model was significantly higher in patients with PPHN. However, to date, there have been few studies regarding HIF-2α in neonates. One study reported that HIF-2α was highly expressed in the lungs of neonates with a congenital diaphragmatic hernia and was possibly correlated with PPHN in neonates.22 Taking these findings together, the authors believe that a genetic variant of the EPAS1 gene is possibly associated with PPHN.

To demonstrate that expression of the HIF-2α protein (i.e., expression of the EPAS1 gene) in pulmonary vascular endothelial cells is involved in PPHN pathophysiology, the authors carried out IHC staining in pulmonary vascular endothelial cells and other tissues. The authors found that HIF-2α protein was expressed in the nucleus of pulmonary vascular endothelial cells of some PPHN patients, whereas there was no HIF-2α protein expression in normal adult lung tissues. Additionally, the authors observed HIF-2α protein expression in fetal umbilical vascular endothelial cells, possibly as a result of exposure to a hypoxic environment during physiological development. During in utero development, expression of both HIF-1α and HIF-2α are relatively high in the fetal lungs due to fetal exposure to a hypoxic environment.23–25 HIF-1α expression is predominant in respiratory epithelial cells, while HIF-2α is predominant in vascular endothelial cells and type II pneumocytes.26 Due to a limited number of PPHN lung samples, the authors could not perform statistical analysis to make a solid conclusion that HIF-2α was significantly higher expressed in neonatal PPHN lungs. However, the present study's results suggest that more studies are needed to further explicate the role(s) of HIF-2α in the pathophysiology of PPHN.

There are some limitations to the present study. First, the number of neonates with PPHN recruited into this study was relatively small. Second, the number of SNP probes fabricated onto the microarray might not cover all SNPs associated with the pathogenesis of PPHN. Third, although the present study carried out a preliminary validation study, using IHC staining to detect HIF-2α protein expression in lung tissues from neonates with PPHN, the authors could not compare the study's findings with normal lung tissues from neonates because autopsies of deceased neonates were not routinely performed in the study's center. Instead, the authors carried out IHC staining to detect HIF-2α protein expression in normal adult lung tissues. Concerning potential bias in adult control subject selection due to unknown medical history in their neonatal period, this concern was deemed minimal. Since the prevalence of PPHN is ∼3 per 1000 livebirth, which means the vast majority of individuals do not have PPHN.

In conclusion, in this study, the authors explored novel candidate SNPs that might be involved in the pathophysiology of PPHN. The authors’ analysis revealed that rs17034984 in the EPAS1 gene is a possible genetic susceptibility locus associated with PPHN. In combination with IHC staining to detect HIF-2α protein expression, the authors found that HIF-2α, encoded by the EPAS1 gene, might be a factor associated with PPHN via pulmonary vascular remodeling related to different oxygen levels in fetuses. However, further studies are needed to confirm the authors’ findings and to further explore the pathophysiological mechanism(s) underlying this association.

Funding

Graduate School and Faculty of Medicine Foundation, Prince of Songkla University, and Medical Genetics Center, Division of Genomic Medicine and Innovation Support, Department of Medical Sciences, Ministry of Public Health, Thailand.

Acknowledgments

The authors thank Dr. Jakris Eu-ahsunthornwattana of the Department of Community Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University for allowing the authors to use the Thai SNP database. The authors thank Mr. David Patterson of the Office of International Relations, Faculty of Medicine, Prince of Songkla University for editing the manuscript.

References
[1]
N Nakwan, S Jain, K Kumar, S Hosono, M Hammoud, YY Elsayed, et al.
An Asian multicenter retrospective study on persistent pulmonary hypertension of the newborn: incidence, etiology, diagnosis, treatment and outcome.
J Matern Fetal Neonatal Med, 33 (2020), pp. 2032-2037
[2]
A Jain, PJ. McNamara.
Persistent pulmonary hypertension of the newborn: advances in diagnosis and treatment.
Semin Fetal Neonat Med, 20 (2015), pp. 262-271
[3]
G Rocha, MJ Baptista, H. Guimarães.
Persistent pulmonary hypertension of non-cardiac causes in a neonatal intensive care unit.
Pulm Med, 2012 (2012), pp. 818-971
[4]
MA Steurer, LL Jelliffe-Pawlowski, RJ Baer, JC Partridge, EE Rogers, RL. Keller.
Persistent pulmonary hypertension of the newborn in late preterm and term infants in California.
Pediatrics, 139 (2017),
[5]
DL Pearson, S Dawling, WF Walsh, JL Haines, BW Christman, A Bazyk, et al.
Neonatal pulmonary hypertension–urea-cycle intermediates, nitric oxide production, and carbamoyl-phosphate synthetase function.
N Engl J Med, 344 (2001), pp. 1832-1838
[6]
HM Byers, JM Dagle, JM Klein, KK Ryckman, EL McDonald, JC. Murray.
Variations in CRHR1 are associated with persistent pulmonary hypertension of the newborn.
Pediatr Res, 71 (2012), pp. 162-167
[7]
M Mei, G Cheng, B Sun, L Yang, H Wang, J Sun, et al.
EDN1 gene variant is associated with neonatal persistent pulmonary hypertension.
Sci Rep, 6 (2016), pp. 29877
[8]
DC Kaluarachchi, CJ Smith, JM Klein, JC Murray, JM Dagle, KK. Ryckman.
Polymorphisms in urea cycle enzyme genes are associated with persistent pulmonary hypertension of the newborn.
Pediatr Res, 83 (2018), pp. 142-147
[9]
X Liu, M Mei, X Chen, Y Lu, X Dong, L Hu, et al.
Identification of genetic factors underlying persistent pulmonary hypertension of newborns in a cohort of Chinese neonates.
Respir Res, 20 (2019), pp. 174
[10]
M Wang, D Zhuang, M Mei, H Ma, Z Li, F He, et al.
Frequent mutation of hypoxia-related genes in persistent pulmonary hypertension of the newborn.
[11]
C Galambos, MP Mullen, JT Shieh, N Schwerk, MJ Kielt, N Ullmann, et al.
Phenotype characterisation of TBX4 mutation and deletion carriers with neonatal and paediatric pulmonary hypertension.
Eur Respir J, 54 (2019),
[12]
N Nakwan, S. Pithaklimnuwong.
Acute kidney injury and pneumothorax are risk factors for mortality in persistent pulmonary hypertension of the newborn in Thai neonates.
J Matern Fetal Neonatal Med, 29 (2016), pp. 1741-1746
[13]
N. Nakwan.
The practical challenges of diagnosis and treatment options in persistent pulmonary hypertension of the newborn: a developing country's perspective.
Am J Perinatol, 35 (2018), pp. 1366-1375
[14]
AR Crowe, W. Yue.
Semi-quantitative determination of protein expression using immunohistochemistry staining and analysis: an integrated protocol.
Bio-protocol, 9 (2019), pp. e3465
[15]
Reference SNP (rs) Report: rs17034984. [Cited 2020 August 19]. Available from: https://www.ncbi.nlm.nih.gov/snp/rs17034984
[16]
LA Shimoda, GL. Semenza.
HIF and the lung: role of hypoxia-inducible factor in pulmonary development and disease.
Am J Respir Crit Care Med, 183 (2011), pp. 152-156
[17]
CM Beall, GL Cavalleri, L Deng, RC Elston, Y Gao, J Knight, et al.
Natural selection on EPAS1 (HIF2alpha) associated with low hemoglobin concentration in Tibetan highlanders.
Proc Natl Acad Sci U S A, 107 (2010), pp. 11459-11464
[18]
TS Simonson, Y Yang, CD Huff, H un, G Qin, DJ Witherspoon, et al.
Genetic evidence for high-altitude adaptation in Tibet.
Science, 329 (2010), pp. 72-75
[19]
JH Newman, TN Holt, JD Cogan, B Womack, JA Phillips 3rd, C Li, et al.
Increased prevalence of EPAS1 variant in cattle with high-altitude pulmonary hypertension.
Nat Commun, 6 (2015), pp. 6863
[20]
AS Cowburn, A Crosby, D Macias, C Branco, RD Colaço, M Southwood, et al.
HIF2α-arginase axis is essential for the development of pulmonary hypertension.
Proc Natl Acad Sci USA, 113 (2016), pp. 8801-8806
[21]
PP Kapitsinou, G Rajendran, L Astleford, M Michael, MP Schonfeld, T Fields, et al.
The endothelial prolyl-4-hydroxylase domain 2/hypoxia-inducible factor 2 axis regulates pulmonary artery pressure in mice.
Mol Cell Biol, 36 (2016), pp. 1584-1594
[22]
Y Huang, A Boerema-de Munck, M Buscop-van Kempen, R de Krijger, D Tibboel, RJ. Rottier.
Hypoxia inducible factor 2α (HIF2α/EPAS1) is associated with development of pulmonary hypertension in severe congenital diaphragmatic hernia patients.
Pulm Circ, 8 (2018),
[23]
F Groenman, M Rutter, I Caniggia, D Tibboel, M. Post.
Hypoxia-inducible factors in the first trimester human lung.
J Histochem Cytochem, 55 (2007), pp. 355-363
[24]
TR Grover, TM Asikainen, JP Kinsella, SH Abman, CW. White.
Hypoxia-inducible factors HIF-1alpha and HIF-2alpha are decreased in an experimental model of severe respiratory distress syndrome in preterm lambs.
Am J Physiol Lung Cell Mol Physiol, 292 (2007), pp. L1345-L1351
[25]
TM Asikainen, A Ahmad, BK Schneider, CW White.
Effect of preterm birth on hypoxia-inducible factors and vascular endothelial growth factor in primate lungs.
Pediatr Pulmonol, 40 (2005), pp. 538-546
[26]
Y Huang, MB Kempen, AB Munck, S Swagemakers, S Driegen, P Mahavadi, et al.
Hypoxia-inducible factor 2α plays a critical role in the formation of alveoli and surfactant.
Am J Respir Cell Mol Biol, 46 (2012), pp. 224-232
Copyright © 2021. Sociedade Brasileira de Pediatria
Download PDF
Idiomas
Jornal de Pediatria (English Edition)
Article options
Tools
Supplemental materials
en pt
Taxa de publicaçao Publication fee
Os artigos submetidos a partir de 1º de setembro de 2018, que forem aceitos para publicação no Jornal de Pediatria, estarão sujeitos a uma taxa para que tenham sua publicação garantida. O artigo aceito somente será publicado após a comprovação do pagamento da taxa de publicação. Ao submeterem o manuscrito a este jornal, os autores concordam com esses termos. A submissão dos manuscritos continua gratuita. Para mais informações, contate assessoria@jped.com.br. Articles submitted as of September 1, 2018, which are accepted for publication in the Jornal de Pediatria, will be subject to a fee to have their publication guaranteed. The accepted article will only be published after proof of the publication fee payment. By submitting the manuscript to this journal, the authors agree to these terms. Manuscript submission remains free of charge. For more information, contact assessoria@jped.com.br.
Cookies policy Política de cookies
To improve our services and products, we use "cookies" (own or third parties authorized) to show advertising related to client preferences through the analyses of navigation customer behavior. Continuing navigation will be considered as acceptance of this use. You can change the settings or obtain more information by clicking here. Utilizamos cookies próprios e de terceiros para melhorar nossos serviços e mostrar publicidade relacionada às suas preferências, analisando seus hábitos de navegação. Se continuar a navegar, consideramos que aceita o seu uso. Você pode alterar a configuração ou obter mais informações aqui.