Genetic Evaluation of the Patients with Clinically Diagnosed Inborn Errors of Immunity by Whole Exome Sequencing: Results from a Specialized Research Center for Immunodeficiency in Türkiye

Molecular diagnosis of inborn errors of immunity (IEI) plays a critical role in determining patients’ long-term prognosis, treatment options, and genetic counseling. Over the past decade, the broader utilization of next-generation sequencing (NGS) techniques in both research and clinical settings has facilitated the evaluation of a significant proportion of patients for gene variants associated with IEI. In addition to its role in diagnosing known gene defects, the application of high-throughput techniques such as targeted, exome, and genome sequencing has led to the identification of novel disease-causing genes. However, the results obtained from these different methods can vary depending on disease phenotypes or patient characteristics. In this study, we conducted whole-exome sequencing (WES) in a sizable cohort of IEI patients, consisting of 303 individuals from 21 different clinical immunology centers in Türkiye. Our analysis resulted in likely genetic diagnoses for 41.1% of the patients (122 out of 297), revealing 52 novel variants and uncovering potential new IEI genes in six patients. The significance of understanding outcomes across various IEI cohorts cannot be overstated, and we believe that our findings will make a valuable contribution to the existing literature and foster collaborative research between clinicians and basic science researchers. Supplementary Information The online version contains supplementary material available at 10.1007/s10875-024-01759-w.

The LoF variant in a particular exon is frequent in the general population if: The 99th percentile of the allele frequency distribution of the previously reported LoF variants (GnomAD) in the exon is higher than the predefined threshold of 0.1%.
The Truncated/Altered region is critical for the protein function if: For the nonsense/frameshift mutations: The altered region is the region between the variant position and the last base of the coding sequence (excluding the stop codon).
For the splice mutations: If the ORF is preserved, we consider the nearby (upstream for splice donor, downstream for splice acceptor) skipped exon as the altered region.Otherwise, it is the region between the variant position and the last base of the coding sequence.
For the deletion mutations: If the ORF is preserved, we consider only the skipped region of the protein.Otherwise, it is the region between the variant position and the last base of the coding sequence.
For the altered region to be critical for the protein, we require at least 3 pathogenic ClinVar variants with a minimum of 2 stars (multiple submitters) or higher.
Expected to undergo NMD (nonsense-mediated decay) if: The variant is not present in the 3' most coding exon or the 3' most 50 bp of the penultimate coding exon.
The transcript has a multi-coding exon.

PS1
The same amino acid change as a previously established pathogenic variant regardless of the nucleotide change.
Evidence code strength is modified based on the Clinvar review status: 1-star: PS1 (Supporting) active site of an enzyme) without benign variation.

Detection of a mutational hotspot
A hotspot region is defined as the largest pathogenic variant dense region between two benign variations and meeting the following conditions: Presence of a minimum of 5 pathogenic and 0 benign variants reported in the ClinVar.
Pathogenic variant count per base pair of the region is greater than the predefined threshold of 0.2.
Domains without benign variation in the ClinVar (with a minimum of 1 star).
At least 3 pathogenic variants in the ClinVar (with a minimum of 1 star).

PM2
Absent from controls (or at extremely low frequency if recessive) in the Exome Sequencing Project, the 1000 Genomes Project, or the Exome Aggregation Consortium databases.
Maximum allele frequency of the variant is less than the predefined threshold.

PM4
The protein length changes as a result of in-frame deletions/insertions in a non-repeat region or stop-loss variants.

PM5
A novel missense change at an amino acid residue where a different missense change was previously observed as a pathogenic variant.

PP2
A missense variant in a gene that has a low rate of benign missense variation and in which missense variants are a common mechanism of disease.
This rule only applies to missense variants.The following thresholds are applied to determine if a missense variant is a common mechanism of disease: Minimum missense pathogenic variant count in the transcript: 5 Missense/total pathogenic variant count ratio threshold: 70% Missense pathogenic/benign count threshold: 0.65

PP3
Multiple lines of computational evidence support a deleterious effect on the gene or the gene product (conservation, evolutionary, splicing impact, etc.).
PP3 is assigned if one the following criteria is met (Ionnadis et al. 2016 (opens new window)): The REVEL score is greater than 0.75 The REVEL score is greater than 0.5 and MetalR is damaging The REVEL score is not available and MetalR is damaging

PP5
A reputable source recently reported the variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.

BA1
The allele frequency is >5% in the Exome Sequencing Project, the 1000 Genomes Project, or the Exome Aggregation Consortium databases.
Maximum allele frequency of the variant is greater than 5% Variants on the BA1 exception list provided by Clingen are excluded.Clingen Exception List(opens new window)

BS1
The allele frequency is greater than the expected frequency for the disorder.
Maximum allele frequency of the variant is less than the predefined BS1 threshold

BS2
Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age.
A variant was observed (at least 2 times) in the control cohort of the Gnomad (v2.1.1)database in a homozygous state.

BP1
A Missense variant in a gene for which primarily truncating variants are known to cause disease.
At least 5 pathogenic variants are reported in the ClinVar database.
All pathogenic variants in the ClinVar database are truncating.

BP3
In-frame deletions/insertions in a repetitive region without a known function.
BP3 is assigned if the variant is in a repetitive region and the repetitive region meets the following criteria.
Do not overlap with a functional UniProt domain.
Do not have a pathogenic variant reported in the ClinVar.

BP4
Multiple lines of computational evidence suggest no impact on the gene or the gene product (conservation, evolutionary, splicing impact, etc.).
BP4 is assigned if one the following criteria is met (Ionnadis et al. 2016 (opens new window)): The REVEL score is less than 0.5 The REVEL score is less than 0.75 and MetalR is benign The REVEL score is not available and MetalR is benign

BP6
A Reputable source recently reported the variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.
Evidence code strength is modified based on ClinVar review status: 1-2-star: BP6 (Supporting) mutational hotspot and/or critical and well-established functional domain (e.g., an Annotation Dependent Depletion ACMG: American College of Medical Genetics and Genomics VUS: Variant of Uncertain Significance AIHA: Autoimmune hemolytic anemia HSM: Hepatosplenomegaly IBD: Inflammatory bowel disease ITP: Immune thrombocytopenic purpura DNT: Double negative Total number od reads (million) Average read depth Total number od reads (million)

Table 1 .
Pathogenicity of the variantsLoss of function is a mechanism of the disease if (either of the below is required):At least 3 pathogenic LoF variants from the ClinVar (with a minimum of 1 star) need to be presentThe LOEUF score of the transcript is smaller than 0.35 as suggested by GNOMADThe transcript is biologically relevant if it is present in: CADD: Combined Annotation Dependent Depletion ACMG: American College of Medical Genetics and Genomics VUS: Variant of Uncertain Significance *Number of homozygous individuals in Gnomad 4.1 #Allele frequencies in Turkish VariomeACMG-based pathogenicity: Our data analysis application "Seq, Genomize" uses ACMG 2015 criteria with minor modifications as explained below:

Table 2 :
Detailed evaluation of possible disease-causing monoallelic variants