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

Abstract

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.

Keywords: Williams-Beuren syndrome (WBS); blended phenotypes; chronic granulomatous disease (CGD); copy number variation (CNV); dual molecular diagnosis; whole exome sequencing (WES).

Figure 1

Variant filtering strategy. All variants harbored in the exonic regions and splice sites were filtered based on the known IEI-associated genes, variant class, allele frequency and variant functional impact. Five potential causative single nucleotide variants (SNVs) fulfilled the filtering criteria. However, the inheritance pattern and associated features of these genetic disorders did not match the zygosity and clinical symptoms of the patient respectively. IEI, inborn errors of immunity; SNVs, single nucleotide variants; Indels, small insertions or deletions.

Figure 2

Copy number variation (CNV) analysis on chromosome 7q11.23 and assessment of NADPH oxidase activity. (A) The patient was found to have a biallelic deletion of NCF1 localized within the 1.53Mb heterozygous deletion associated with Williams-Beuren syndrome (WBS) (shaded in yellow). The breakpoints of WBS deletion occurred from chromosome 7:73,303,201-74,836,526, affecting 27 genes. Biallelic loss of NCF1 leads to autosomal recessive chronic granulomatous disease (CGD). Aortic stenosis observed in the patient is consistent with the haploinsufficiency of ELN. Ratio of 1.5 indicates one-copy duplication, 1.0 indicates normal copy state, 0.5 indicates one-copy deletion and 0 indicates two-copy deletion. Grey shade represents the 95% confidence interval of expected read ratio. Each red plus symbol indicates the genomic position being probed. Size of genes (black-filled boxes) was approximate and not drawn to scale. (B) Assessment of neutrophil oxidative burst activity using dihydrorhodamine (DHR) flow cytometry assay. Upon stimulation by phorbol 12-myristate 13-acetate (PMA), the patient showed no change in the fluorescence intensity of rhodamine 123 (right panel). This result is consistent with CGD that lacks normal neutrophil oxidative burst activity. The percentage in the histograms indicates the proportion of neutrophils that produced reactive oxygen species (ROS). PMA, phorbol 12-myristate 13-acetate.

Figure 3

Validation of GT-deletion (ΔGT) in NCF1. (A) Coamplification of NCF1 and its pseudogenes at intron 1/exon 2. Overlapping peaks of GTGT-containing NCF1 and ΔGT-containing pseudogenes were observed in the control and parents. The patient only had a product of NCF1 pseudogenes, indicating a ΔGT in NCF1 (NM_000265.7: exon 2: c.75_76delGT: p.Tyr26Hisfs*26). (B) ΔGT gene scan. The fragments of NCF1 and its pseudogenes were depicted by the 198-bp peak and 196-bp peak respectively. A normal ratio of 2:4 was identified in the control and father, reflecting two copies of NCF1 per four copies of pseudogenes. Meanwhile, the mother had a ratio of 1:5, signifying the carrier of ΔGT in NCF1. Only a single peak of NCF1 pseudogenes was observed in the patient. (C) 20-bp gene scan. The 411-bp-peak and 429-bp-peak represented the fragments of NCF1 and its pseudogenes respectively. Results from 20-bp gene scan corroborated the ratios from ΔGT gene scan. The patient was confirmed to have a biallelic loss of NCF1, in which one allele was included in the Williams-Beuren syndrome (WBS) deletion while another allele harbors a ΔGT. bp, base pair.

Figure 4

Familial segregation analysis. The patient was postulated to inherit a paternal allele with de novo 7q11.23 deletion involving NCF1 and a maternal allele with ΔGT-containing NCF1, resulting in Williams-Beuren syndrome (WBS) and NCF1-deficient chronic granulomatous disease (CGD). Both parents were well with no features of WBS and CGD. Square symbol indicates male, half-filled circle indicates female carrier of NCF1 with ΔGT and filled circle indicates female affected by WBS and CGD. The diagram was not drawn to scale. ΔGT, GT-deletion; WBS, Williams-Beuren syndrome; CGD, chronic granulomatous disease.