Uses of Next-Generation Sequencing Technologies for the Diagnosis of Primary Immunodeficiencies

Abstract

Primary immunodeficiencies (PIDs) are genetic disorders impairing host immunity, leading to life-threatening infections, autoimmunity, and/or malignancies. Genomic technologies have been critical for expediting the discovery of novel genetic defects underlying PIDs, expanding our knowledge of the complex clinical phenotypes associated with PIDs, and in shifting paradigms of PID pathogenesis. Once considered Mendelian, monogenic, and completely penetrant disorders, genomic studies have redefined PIDs as a heterogeneous group of diseases found in the global population that may arise through multigenic defects, non-germline transmission, and with variable penetrance. This review examines the uses of next-generation DNA sequencing (NGS) in the diagnosis of PIDs. While whole genome sequencing identifies variants throughout the genome, whole exome sequencing sequences only the protein-coding regions within a genome, and targeted gene panels sequence only a specific cohort of genes. The advantages and limitations of each sequencing approach are compared. The complexities of variant interpretation and variant validation remain the major challenge in wide-spread implementation of these technologies. Lastly, the roles of NGS in newborn screening and precision therapeutics for individuals with PID are also addressed.

Targeted NGS Platforms for Genetic Screening and Gene Discovery in Primary Immunodeficiencies

“…Although the prioritization methods applied in this study follows all common assumptions for a correct data analysis, the identification of novel variants currently under investigation represents a challenge and their validation needs the essential support of further in-depth experimental studies (6)(7)(8)63). The integration of clinical, immunological, biochemical and molecular data might favor a revised PIDs classification of patients with similar phenotype due to a different genetic cause, or patients with different phenotypes but with the same genetic cause.…”

“…Phenotypic and genotypic heterogeneity of PIDs make genetic diagnosis often complex and delayed. Indeed, more than one genotype might cause similar clinical phenotypes, but identical genotypes will not often produce the same phenotype and finally clinical penetrance may be different (6)(7)(8)(9). The characterization of PIDassociated genes is expected to significantly contribute to define the molecular events governing immune system development and will provide new insights into the pathogenesis of PIDs.…”

“…A better understanding of the genetic and immune defects of patients is critical to develop therapeutic strategies aimed at changing the clinical course of the disease and to guarantee an appropriate genetic counseling allowing the identification of PID patients before the onset of the disease (11)(12)(13). The application of Next Generation Sequencing (NGS) to PIDs has been a revolution and it has accelerated the discovery and identification of novel disease-causing genes and the genetic diagnosis of patients with monogenic inborn errors of immunity (7,8,(14)(15)(16). Targeted gene-panel sequencing (17)(18)(19)(20)(21), whole exome sequencing (WES) (22,23) or whole genome sequencing (WGS) (24) approaches can rapidly identify candidate gene variants in an increasing number of genetically undefined diseases (17,24) and are widely used in several laboratories for the diagnosis of PIDs (10).…”

“…These tools increase the amount of data analysis that can identify causative genes in both clinically defined and atypical diseases. Nonetheless, delay in diagnosis can be caused by the huge amount of data retrieved from whole sequencing, increased costs sustained by clinical laboratories and the requirement of trained personnel to validate variants (7,8,22). An increased depth of the sequencing coverage is generally obtained using targeted gene panels, in favor of a high accuracy, amelioration of sensitivity and management of datasets, reducing the time of analysis, the costs and the interpretation of results, thus accelerating the diagnosis for the majority of PIDs (14,(16)(17)(18).…”

“…An increased depth of the sequencing coverage is generally obtained using targeted gene panels, in favor of a high accuracy, amelioration of sensitivity and management of datasets, reducing the time of analysis, the costs and the interpretation of results, thus accelerating the diagnosis for the majority of PIDs (14,(16)(17)(18). On the other hand, the usefulness of targeted exome sequencing approach for the identification of PID patients has been demonstrated, with accurate detection of point mutations and exonic deletions in patients with either known or unknown genetic diagnosis (7,8).…”