A single-center pilot study in Malaysia on the clinical utility of whole-exome sequencing for inborn errors of immunity

Abstract

Primary immunodeficiency diseases refer to inborn errors of immunity (IEI) that affect the normal development and function of the immune system. The phenotypical and genetic heterogeneity of IEI have made their diagnosis challenging. Hence, whole-exome sequencing (WES) was employed in this pilot study to identify the genetic etiology of 30 pediatric patients clinically diagnosed with IEI. The potential causative variants identified by WES were validated using Sanger sequencing. Genetic diagnosis was attained in 46.7% (14 of 30) of the patients and categorized into autoinflammatory disorders (n = 3), diseases of immune dysregulation (n = 3), defects in intrinsic and innate immunity (n = 3), predominantly antibody deficiencies (n = 2), combined immunodeficiencies with associated and syndromic features (n = 2) and immunodeficiencies affecting cellular and humoral immunity (n = 1). Of the 15 genetic variants identified, two were novel variants. Genetic findings differed from the provisional clinical diagnoses in seven cases (50.0%). This study showed that WES enhances the capacity to diagnose IEI, allowing more patients to receive appropriate therapy and disease management.

Keywords: bioinformatics analysis; genetic diagnosis; genetic variant; inborn errors of immunity; whole-exome sequencing.

FIGURE 1

Overview of the diagnostic workflow using whole‐exome sequencing (WES) of 30 pediatric patients aged 18 years and below. Patients with clinical suspicion of X‐linked agammaglobulinemia (XLA) and chronic granulomatous disease (CGD) were excluded based on their abnormal Bruton’s tyrosine kinase (BTK) protein expression and abnormal neutrophil oxidative burst, respectively. Patient clinical history was collected along with peripheral venous blood samples and written informed consent. Laboratory testing including lymphocyte subset enumeration and quantitation of serum immunoglobulins and complement were performed. Purified genomic DNA from patients was subjected to WES that utilized the Illumina paired‐end sequencing approach. Bioinformatics processing of the sequencing data and variant filtering were performed following the workflows previously described [21]. Lastly, causative variants with functional impact relating to the clinical and immunological features of individual patient were validated using Sanger sequencing. Disease inheritance was confirmed by sequencing the parents’ DNA when available. For patients with negative findings, the exome data were reanalyzed based on the updated International Union of Immunological Societies (IUIS) gene list and latest published reports

FIGURE 2

Clinical presentations and laboratory findings of 30 patients. (a) Inborn errors of immunity (IEI) patients were more likely to experience respiratory tract infections, particularly lower respiratory tract infections (LRTI), including pneumonia, bronchiectasis, tuberculosis and bronchiolitis. (b) Skin abscesses were more commonly seen than internal abscesses. (c) Most patients contracted infections caused by bacteria while only a minority had fungal and viral infections. Infections induced by Mycobacterium sp. and Staphylococcus sp. were common among the IEI patients

FIGURE 3

Duration from the age of onset to age recruited for whole‐exome sequencing (WES). A median duration of 4 years was observed from the onset of symptoms to the recruitment for WES

FIGURE 4

Genetic variants uncovered by whole‐exome sequencing (WES) in 14 patients. (a) Of the 15 variants identified, 10 were missense single nucleotide variants (SNVs), whereas two were stopgain SNVs. Two splice site mutations and a frameshift deletion were also detected by WES. (b) Most of the variants detected led to autosomal dominant disorders (64.3%). Familial segregation examined by Sanger sequencing of six patients showed three patients had de‐novo mutations and the other three had familial mutations. (c) One compound heterozygous mutation induced by a missense SNV and a frameshift deletion was detected