Diagnostics of Primary Immunodeficiencies through Next-Generation Sequencing

Abstract

BackgroundRecently, a growing number of novel genetic defects underlying primary immunodeficiencies (PIDs) have been identified, increasing the number of PID up to more than 250 well-defined forms. Next-generation sequencing (NGS) technologies and proper filtering strategies greatly contributed to this rapid evolution, providing the possibility to rapidly and simultaneously analyze large numbers of genes or the whole exome.ObjectiveTo evaluate the role of targeted NGS and whole exome sequencing (WES) in the diagnosis of a case series, characterized by complex or atypical clinical features suggesting a PID, difficult to diagnose using the current diagnostic procedures.MethodsWe retrospectively analyzed genetic variants identified through targeted NGS or WES in 45 patients with complex PID of unknown etiology.ResultsForty-seven variants were identified using targeted NGS, while 5 were identified using WES. Newly identified genetic variants were classified into four groups: (I) variations associated with a well-defined PID, (II) variations associated with atypical features of a well-defined PID, (III) functionally relevant variations potentially involved in the immunological features, and (IV) non-diagnostic genotype, in whom the link with phenotype is missing. We reached a conclusive genetic diagnosis in 7/45 patients (~16%). Among them, four patients presented with a typical well-defined PID. In the remaining three cases, mutations were associated with unexpected clinical features, expanding the phenotypic spectrum of typical PIDs. In addition, we identified 31 variants in 10 patients with complex phenotype, individually not causative per se of the disorder.ConclusionNGS technologies represent a cost-effective and rapid first-line genetic approach for the evaluation of complex PIDs. WES, despite a moderate higher cost compared to targeted, is emerging as a valuable tool to reach in a timely manner, a PID diagnosis with a considerable potential to draw genotype–phenotype correlation. Nevertheless, a large fraction of patients still remains without a conclusive diagnosis. In these patients, the sum of non-diagnostic variants might be proven informative in future studies with larger cohorts of patients.

Clinical challenges in the management of patients with B cell immunodeficiencies

“…In the past, clinical considerations in addition to measurements of various T cell types in blood have been used to separate the heterogeneous group of CVIDs from combined immune deficiencies (CIDs), but time has proved this combination to be insufficient. Whole exome screening (WES) has shown that many patients presenting initially with primary antibody failure turn out to have significant disease‐causing mutations in unsuspected genes . This supports the suggestion from Oksenhendler's group that those patients with mild T cell defects, unexplained opportunistic infections, a positive family history for a PID and probably those presenting in childhood should always be tested for a CID .…”

Hermansky–Pudlak syndrome: Mutation update

“…Next‐generation sequencing of different cohorts of subjects with primary immunodeficiency or hemophagocytic lymphohistiocytosis (HLH) reported least eight AP3B1 variants (Table S4) in a heterozygous state with or without a variant in another (synergistic) gene. These findings suggest that heterozygous AP3B1 variants may contribute to an immunologic phenotype (Chi et al, 2018; Gallo et al, 2016; Gao, Zhu, Huang, & Zhou, 2015; Miao et al, 2019; Mukda et al, 2017; Tesi et al, 2015; Xu et al, 2017). These variants have not been reported in HPS subjects but were included in this report because they may cause HPS when occurring in a homozygous or compound heterozygous state.…”

“…Individuals with HPS‐2 that present to an immunologist with (severe) immunodeficiency may escape diagnosis due to emphasis on their immunodeficiency, other mild manifestations (e.g., hypopigmentation, ocular findings, and bleeding diathesis) may be overlooked, there may be unfamiliarity with HPS‐2, and costs and lack of availability of AP3B1 genetic testing may provide obstacles to diagnosis. However, the recent significant number of heterozygous AP3B1 variants identified by next‐generation sequencing in cohorts with immunodeficiency disorders (Chi et al, 2018; Gallo et al, 2016; Gao et al, 2015; Miao et al, 2019; Mukda et al, 2017; Tesi et al, 2015; Xu et al, 2017) emphasizes the importance of including AP3B1 in immunodeficiency‐related gene panels and may result in the diagnosis of additional HPS‐2 cases.…”

“…Heterozygous HPS1 , HPS4 , and DTNBP1 variants were reported in familial (Stearman et al, 2019) or sporadic (Deng et al, 2018) pulmonary fibrosis cases (Tables 3, 6, and 9). Heterozygous AP3B1 variants have been reported in cases with primary immunodeficiency (Chi et al, 2018; Gallo et al, 2016) or HLH (Gao et al, 2015; Miao et al, 2019; Mukda et al, 2017; Tesi et al, 2015; Xu et al, 2017; Table 4), and heterozygous variants in AP3D1 , HPS3 , and HPS4 were reported in cases with autism spectrum disorder or schizophrenia (Fromer et al, 2014; Iossifov et al, 2014; Takata et al, 2018).…”

Clinical efficacy of a next‐generation sequencing gene panel for primary immunodeficiency diagnostics

“…The most widely used technique within routine clinical care remains NGS gene panels due to economic, data handling, and the result turnaround time advantages . NGS gene panels can provide a comprehensive method for diagnostics in PID, with reported diagnostic rates between 15% and 70%, depending on the PID population and phenotypic criteria of patients assessed …”