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Fulltext Revealing Chronic Granulomatous Disease in a Patient With Williams-Beuren Syndrome Using Whole Exome Sequencing

Ripen AM, Chiow MY, Rama Rao PR, Mohamad SB

Front Immunol, 2021;12:778133.
PMID: 34804071 DOI: 10.3389/fimmu.2021.778133

Blended phenotypes exhibited by a patient may present a challenge to the establishment of diagnosis. In this study, we report a seven-year-old Murut girl with unusual features of Williams-Beuren syndrome (WBS), including recurrent infections and skin abscesses. Considering the possibility of a second genetic disorder, a mutation screening for genes associated with inborn errors of immunity (IEI) was conducted using whole exome sequencing (WES). Analysis of copy number variations (CNVs) from the exome data revealed a 1.53Mb heterozygous deletion on chromosome 7q11.23, corresponding to the known WBS. We also identified a biallelic loss of NCF1, which indicated autosomal recessive chronic granulomatous disease (CGD). Dihydrorhodamine (DHR) flow cytometric assay demonstrated abnormally low neutrophil oxidative burst activity. Coamplification of NCF1 and its pseudogenes identified a GT-deletion (ΔGT) at the start of exon 2 in NCF1 (NM_000265.7: c.75_76delGT: p.Tyr26Hisfs*26). Estimation of NCF1-to-NCF1 pseudogenes ratio using ΔGT and 20-bp gene scans affirmed nil copies of NCF1 in the patient. While the father had a normal ratio of 2:4, the mother had a ratio of 1:5, implicating the carrier of ΔGT-containing NCF1. Discovery of a 7q11.23 deletion involving one NCF1 allele and a ΔGT in the second NCF1 allele explained the coexistence of WBS and CGD in our patient. This study highlights the capability of WES to establish a molecular diagnosis for a case with blended phenotypes, enabling the provision of appropriate prophylactic treatment.
Fulltext Whole exome sequencing identifies compound heterozygous variants of CR2 gene in monozygotic twin patients with common variable immunodeficiency

Mat Ripen A, Ghani H, Chear CT, Chiow MY, Syed Yahya SNH, Kassim A, et al.

SAGE Open Med, 2020;8:2050312120922652.
PMID: 32547748 DOI: 10.1177/2050312120922652

Objectives: A pair of female Malay monozygotic twins who presented with recurrent upper respiratory tract infections, hepatosplenomegaly, bronchiectasis and bicytopenia were recruited in this study. Both patients were suspected with primary immunodeficiency diseases. However, the definite diagnosis was not clear due to complex disease phenotypes. The objective of this study was to identify the causative gene mutation in these patients.
Methods: Lymphocyte subset enumeration test and whole exome sequencing were performed.
Results: We identified a compound heterozygous CR2 mutation (c.1916G>A and c.2012G>A) in both patients. These variants were then confirmed using Sanger sequencing.
Conclusion: Whole exome sequencing analysis of the monozygotic twins revealed compound heterozygous missense mutations in CR2.
Fulltext Visual inspection reveals a novel pathogenic mutation in PKD1 missed by the variant caller in whole‑exome sequencing

Koay BT, Chiow MY, Ismail J, Fahmy NK, Yee SY, Mustafa N, et al.

Mol Med Rep, 2022 Dec;26(6).
PMID: 36281931 DOI: 10.3892/mmr.2022.12882

Autosomal dominant polycystic kidney disease (ADPKD) is the most common type of inherited cystic kidney disease. The feasibility of whole‑exome sequencing (WES) to obtain molecular diagnosis of ADPKD is still in question as previous studies showed conflicting results. Utilizing WES on a patient with ADPKD, standard bioinformatics pipeline demonstrated no pathogenic variant in the genes of interest. By visualizing read alignments using the Integrative Genomics Viewer, a region with atypical alignment of numerous soft‑clipped reads at exon 45 of polycystin 1, transient receptor potential channel interacting (PKD1) gene was demonstrated. A total of four visual inspection steps were outlined to assess the origin of these soft‑clipped reads as strand bias during capture, poor mapping, sequencing error or DNA template contamination. Following assessment, the atypical alignment at PKD1 was hypothesized to be caused by an insertion/deletion mutation. Sanger sequencing confirmed the presence of a novel 20‑bp insertion in PKD1 (NM_001009944.3; c.12143_12144insTCCCCGCAGTCTTCCCCGCA; p.Val4048LeufsTer157), which introduced a premature stop codon and was predicted to be pathogenic. The present study demonstrated that WES could be utilized as a molecular diagnostic tool for ADPKD. Furthermore, visual inspection of read alignments was key in identifying the pathogenic variant. The proposed visual inspection steps may be incorporated into a typical WES data analysis workflow to improve the diagnostic yield.
A single-center pilot study in Malaysia on the clinical utility of whole-exome sequencing for inborn errors of immunity

Ripen AM, Chear CT, Baharin MF, Nallusamy R, Chan KC, Kassim A, et al.

Clin Exp Immunol, 2021 Nov;206(2):119-128.
PMID: 34060650 DOI: 10.1111/cei.13626

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.