Reviews and feature articleExome and genome sequencing for inborn errors of immunity
Section snippets
Generating NGS data
NGS, also known as deep sequencing, massive parallel sequencing, or second-generation sequencing, is a sequencing method in which hundreds of millions of small DNA fragments are sequenced in parallel (Fig 1). NGS can be used to sequence entire genomes (WGS) or a targeted panel of genes, ranging from a small number of genes (eg, all genes known to cause PIDs, hereafter referred to as a gene panel) to the whole exome (WES).7, 8, 9, 10 The technical process of NGS is summarized in Fig 1, and the
Filtering and selecting appropriate variants
NGS identifies between 20,000 and 50,000 high-quality variants per exome, depending on the kit used and some of the criteria for data processing.11, 12, 22 The variants/calls are analyzed and selected according to criteria both at the variant level (allele frequency [AF], variant annotation, and potential functional effect) and the gene level (gene expression, gene function, and gene population genetics). An allele with an AF of greater than 1% is regarded as common; the remainder are rare or
Testing a genetic hypothesis
The experimenter who does not know what he is looking for will not understand what he sees. This is true in human genetics, particularly for unbiased, genome-wide, so-called “hypothesis-generating” approaches based on NGS. These approaches are unbiased from a physiologic point of view and might generate immunologic hypotheses, but first, they must be designed and interpreted in light of a genetic hypothesis. A thorough knowledge of the clinical and cellular phenotype, its prevalence in the
Validating genetic findings experimentally
Experimental validation of the causal relationship between genotype and phenotype is crucial. NGS can easily lead to a false-positive result being associated with disease if the genetic hypothesis is flawed, population genetics is neglected, or experimental validation is insufficient (eg, the recent report of a presumed novel disease-causing mutation in 7 patients with multiple sclerosis from 2 multiplex families).44, 45 Only in rare cases of phenotypic and genetic homogeneity can a strong
Discoveries of PID-causing mutations by using NGS
The introduction of NGS in research settings has resulted in an exponential increase in the number of disease-causing genotypes identified for PIDs. AR disorders remain 4 times more common than AD disorders because they are easier to identify by using classic molecular methods and NGS (Fig 3 and Table III). Since 2010, the number of AD conditions identified has been steadily increasing because NGS has made it possible to decipher disease-causing mutations in small pedigrees and in multiple
The thin line between diagnostic and research settings
NGS is increasingly being used for the molecular diagnosis of PIDs.83 Limited resources have invited some to question the benefits of providing a molecular diagnosis to patients with PIDs.84 However, a molecular diagnosis is a definitive diagnosis. Second, in case of a strong genotype-phenotype correlation, molecular diagnosis offers prognostic information. Third, genetic analysis allows for identifying potentially fatal PIDs before onset of symptoms, enabling timely intervention (eg,
Conclusion
The advent of NGS has revolutionized gene detection for both research and diagnostic purposes. NGS-based gene panel sequencing, WES, and WGS are most useful in the field of PIDs. WES is presently the most cost-effective approach for PID research and diagnosis, but WGS provides more uniform coverage. The scientific or diagnostic challenge is the selection of 1 or 2 candidate variants among thousands of NGS calls, a task truly resembling finding a needle in a haystack. We stress the importance of
References (89)
- et al.
Discovery of single-gene inborn errors of immunity by next generation sequencing
Curr Opin Immunol
(2014) - et al.
Characterizing natural variation using next-generation sequencing technologies
Trends Genet
(2009) - et al.
IL-7 receptor deficient SCID with a unique intronic mutation and post-transplant autoimmunity due to chronic GVHD
Clin Immunol
(2007) - et al.
Exome sequencing identifies biallelic MSH3 germline mutations as a recessive subtype of colorectal adenomatous polyposis
Am J Hum Genet
(2016) - et al.
Genomic signatures of selective pressures and introgression from archaic hominins at human innate immunity genes
Am J Hum Genet
(2016) - et al.
Rare-variant association analysis: study designs and statistical tests
Am J Hum Genet
(2014) - et al.
Whole-exome-sequencing-based discovery of human FADD deficiency
Am J Hum Genet
(2010) - et al.
A new case of Fas-associated death domain protein deficiency and update on treatment outcomes
J Allergy Clin Immunol
(2015) - et al.
Nuclear receptor NR1H3 in familial multiple sclerosis
Neuron
(2016) - et al.
Induced pluripotent stem cells: a novel frontier in the study of human primary immunodeficiencies
J Allergy Clin Immunol
(2011)
Human TRAF3 adaptor molecule deficiency leads to impaired Toll-like receptor 3 response and susceptibility to herpes simplex encephalitis
Immunity
Immunological loss-of-function due to genetic gain-of-function in humans: autosomal dominance of the third kind
Curr Opin Immunol
Hypomorphic mutation in TTC7A causes combined immunodeficiency with mild structural intestinal defects
Blood
A homozygous mutation of RTEL1 in a child presenting with an apparently isolated natural killer cell deficiency
J Allergy Clin Immunol
Characterization of Crohn disease in X-linked inhibitor of apoptosis-deficient male patients and female symptomatic carriers
J Allergy Clin Immunol
A novel Rab27a mutation binds melanophilin, but not Munc13-4, causing immunodeficiency without albinism
J Allergy Clin Immunol
Inborn errors of human STAT1: allelic heterogeneity governs the diversity of immunological and infectious phenotypes
Curr Opin Immunol
Early-onset lymphoproliferation and autoimmunity caused by germline STAT3 gain-of-function mutations
Blood
RNA sequencing reveals the consequences of a novel insertion in dedicator of cytokinesis-8
J Allergy Clin Immunol
Massively parallel sequencing reveals maternal somatic IL2RG mosaicism in an X-linked severe combined immunodeficiency family
J Allergy Clin Immunol
Is it necessary to identify molecular defects in primary immunodeficiency disease?
J Allergy Clin Immunol
Targeted gene editing restores regulated CD40L function in X-linked hyper-IgM syndrome
Blood
Gene therapy: myth or reality?
C R Biol
ACMG recommendations for reporting of incidental findings in clinical exome and genome sequencing
Genet Med
The 2015 IUIS Phenotypic Classification for Primary Immunodeficiencies
J Clin Immunol
A phenotypic approach for IUIS PID classification and diagnosis: guidelines for clinicians at the bedside
J Clin Immunol
Next generation sequencing data analysis in primary immunodeficiency disorders—future directions
J Clin Immunol
Contribution of high-throughput DNA sequencing to the study of primary immunodeficiencies
Eur J Immunol
Novel primary immunodeficiency candidate genes predicted by the human gene connectome
Front Immunol
Massively parallel genetics
Genetics
Sequencing studies in human genetics: design and interpretation
Nat Rev Genet
Exome sequencing as a tool for Mendelian disease gene discovery
Nat Rev Genet
Whole-genome sequencing is more powerful than whole-exome sequencing for detecting exome variants
Proc Natl Acad Sci U S A
Analysis of protein-coding genetic variation in 60,706 humans
Nature
Sequencing depth and coverage: key considerations in genomic analyses
Nat Rev Genet
Copy number variation detection and genotyping from exome sequence data
Genome Res
Genome-wide in situ exon capture for selective resequencing
Nat Genet
Intronic point mutation in the IL2RG gene causing X-linked severe combined immunodeficiency
Hum Mol Genet
Hypomorphic mutation of ZAP70 in human results in a late onset immunodeficiency and no autoimmunity
Eur J Immunol
New insights into the performance of human whole-exome capture platforms
Nucleic Acids Res
Sources of PCR-induced distortions in high-throughput sequencing data sets
Nucleic Acids Res
Performance comparison of exome DNA sequencing technologies
Nat Biotechnol
The Human Gene Mutation Database: building a comprehensive mutation repository for clinical and molecular genetics, diagnostic testing and personalized genomic medicine
Hum Genet
Characterization of greater Middle Eastern genetic variation for enhanced disease gene discovery
Nat Genet
Cited by (154)
Human germline gain-of-function in STAT6: from severe allergic disease to lymphoma and beyond
2024, Trends in ImmunologyApproach to Diagnosing Inborn Errors of Immunity
2023, Rheumatic Disease Clinics of North AmericaDeciphering actin remodelling in immune cells through the prism of actin-related inborn errors of immunity
2023, European Journal of Cell BiologyLymphocyte disturbance and functional assessment of the [Asp521Asn] ZAP70 mutation
2023, Clinical ImmunologyHow to: Diagnose inborn errors of intrinsic and innate immunity to viral, bacterial, mycobacterial, and fungal infections
2022, Clinical Microbiology and Infection
I.M. is funded by a KOF mandate of the KU Leuven and by the Jeffrey Modell Foundation. B.B. is funded by a Research Mandate of the FWO Vlaanderen. X.B. is funded by a research grant from the Research Council of the Catholic University of Leuven. The Laboratory of Human Genetics of Infectious Diseases (J.-L.C., A.B., B.B., and Y.I.) was supported in part by grants from the Institut National de la Santé et de la Recherche Médicale (INSERM), University Paris Descartes, the Rockefeller University, the St. Giles Foundation, the European Research Council (grant no. ERC-2010-AdG-268777; to L.A.), the French National Research Agency (ANR) under the “Investments for the future” program (grant no. ANR-10-IAHU-01), and the National Institute of Allergy and Infectious Diseases (grant no. R37AI095983).
Disclosure of potential conflict of interest: I. Meyts has received travel support from Octapharma and Gilead Sciences and has received payment from the Jeffrey Modell Foundation. B. Bosch has received a grant from Research Foundation Flanders (FWO). A. Bolze is employed by Helix and owns stock in Illumina. L. Abel has received research support from the European Research Council and the French National Research Agency. J.-L. Casanova has received grants from Biogen Idec. The rest of the authors declare that they have no relevant conflicts of interest.