Introduction
Box 1 General Recommendations for the Analysis of NGS Data for PIDs
WES is Superior to Targeted Sequencing as a First-Tier Diagnostic Approach
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WES facilitates analysis of (almost) all protein coding regions of the genome instead of a selected gene panel.
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WES in silico gene panels can be more easily updated than targeted sequencing panels as these leverage the existing exome data, while actual sequencing panels require a more laborious procedure and acquisition of new data.
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The diagnostic yield for PIDs using next-generation sequencing was on average 29%, and 38% for WES alone.
Analysis of WES Data: Prioritization and Strategies
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Variants called from WES data are subjected to standardized variant filtering for non-synonymous, rare variants impacting exons and splice sites.
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To prioritize variants exome-wide, a detailed description of the patient phenotype and pedigree is helpful. The phenotype-genotype analysis requires judgement both at the variant and gene level. Metrics for the variant level include variant effect predictions, nucleotide alteration, and amino acid conservation. For the gene level, these include constraint against loss-of-function, functional/pathway annotations, and phenotypes of animal models such as knockout mice [12, 13].
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In sporadic cases of PIDs, a trio analysis can be performed by sequencing of both the patient and parents, allowing the exploration of de novo variants [14, 15]. In familial PID cases, variants from affected and/or unaffected family members can be overlapped to find candidate variants [3]. These approaches can drastically decrease the number of possible candidate variants.
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Functional testing in PID patients is minimally invasive and often required to demonstrate a possible functional defect for variants of uncertain significance (VUS).
Opportunities to improve the Genetic Diagnosis of PID Patients
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There remains a high potential to find new genes associated with PIDs, also indicated by the low average yield seen for NGS-based methods in PIDs.
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Challenges in the diagnosis of PIDs include extremely rare or heterogeneous phenotypes, complex mutational mechanisms, or incomplete penetrance, which is in part due to the requirement of exposure to a specific pathogen to cause an overt phenotype.
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WGS starts to be used as a method to explore non-coding variation and its relevance in PIDs and has recently demonstrated its potential to diagnose patients with non-coding mutations [18].
Genetics of PIDs
Practical Guide for WES Analysis
Yield of WES for PID Diagnostics
Yield (N (%)) | ||||||||||
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Authors | Year | Selection | Country | Approach | Genes (N) | N | Diagnostic | Whole exome | Total | Ref |
Bisgin et al. | 2018 | PID | Turkey | Targeted gene panel | 60 | 37 | 17 (46) | 17 (46) | [47] | |
Erman et al. | 2017 | SCID | Turkey | Targeted gene panel | 356 | 19 | 6 (33) | 6 (33) | [38] | |
Rae et al. | 2018 | PID | UK | Targeted gene panel | 242 | 27 | 13 (46) | 13 (46) | [48] | |
Moens et al. | 2014 | Patients known disease-causing mutation or with agammaglobulinemia without BTK mutations | Sweden + Poland | Targeted gene panel | 179 | 15 | 6 (40) | 6 (40) | [49] | |
Stoddard et al. | 2014 | PID | USA | Targeted gene panel | 173 | 120 | 18 (15) | 18 (15) | [50] | |
Nijman et al. | 2014 | CID, ALPS, granulopenia, HLH/XLP | The Netherlands | Targeted gene panel | 170 | 26 | 4 (15) | 4 (15) | [51] | |
Al-Mousa et al. | 2016 | Suspected PID | Saudi Arabia | Targeted gene panel | 162 | 139 | 35 (25) | 35 (25) | [39] | |
Yu et al. | 2016 | SCID | USA | Targeted gene panel | 46 | 20 | 14 (70) | 14 (70) | [34] | |
Kojima et al. | 2016 | PID | Japan | Targeted gene panel | 349 | 59 | 8 (14) | 8 (14) | [52] | |
Gallo et al. | 2016 | Suspected PID | Italy | Targeted gene panel + WES | 571 | 45 | 7 (16) | 7 (16) | [41] | |
Abolhassani et al. | 2018 | CID | Iran | Targeted gene panel + WES | 200 | 243 | 189 (79) | 189 (79) | [33] | |
Suspitsin et al. | 2020 | Pediatric PID patients | Russia | Targeted gene panel | 344 | 333 | 69 (21) | 69 (21) | [53] | |
Batlle-Maso et al. | 2020 | Autoinflammatory diseases | Spain | WES | 4813 | 22 | 5 (23) | 5 (23) | [42] | |
Abolhassani et al. | 2019 | Primary antibody deficiency (CVID, agammaglobulinemia, HIGM, IGAD) | Iran | Targeted gene panel + WES | 378 | 126 | 86 (68) | 86 (68) | [37] | |
Arts et al. | 2019 | PID | The Netherlands, Saudi Arabia, Finland | WES | 302 | 254 | 62 (24) | 10 (4) | 72 (28) | [2] |
Simon et al. | 2020 | PID | Israel | WES | ? | 106 | 74 (70) | 74 (70) | [36] | |
Stray-Pedersen et al. | 2017 | PID | USA + Norway | WES | 475 | 278 | 110 (40) | 110 (40) | [35] | |
Okano et al. | 2020 | PID patients with severe symptoms with negative previous genetic targeted screening | Japan | WES | 430 | 136 | 36 (26.5) | 36 (26.5) | [54] | |
Maffucci et al. | 2016 | CVID | USA | WES | 269 | 50 | 15 (30) | 15 (30) | [55] | |
Rudilla et al. | 2019 | PID | Spain | WES | 260 | 61 | 12 (20) | 7 (11) | 19 (31) | [8] |
Borghesi et al. | 2020 | Pediatric PID patients with sepsis | Switzerland | WES | 240 | 176 | 35 (20) | 35 (20) | [56] | |
DeValles-Ibanez et al. | 2018 | Pediatric CVID patients | Spain | WES | 16 | 36 | 5–8 (15–24) | 5–8 (15–24) | [57] | |
Mukda et al. | 2017 | HLH | Thailand | WES | 12 | 25 | 12 (48) | 12 (48) | [43] | |
Thaventhiran et al. | 2020 | PID | UK | WGS | NA | 886 | 91 (10.3) | 91 (10.3) | [18] |