Williams-Beuren syndrome (WBS) (OMIM ID: 194050) is a contiguous gene deletion syndrome inherited in autosomal dominant pattern. Most cases occurred sporadically with an estimated prevalence of 1/7,500 to 1/20,000 live births (1, 2). Patients with WBS typically exhibited distinct facial appearance, cardiovascular abnormalities and developmental delay (3). Other features including idiopathic infantile hypercalcemia, gastrointestinal problems, musculoskeletal defects, hypothyroidism and hernias were noted in some patients (4). This genetic disorder is caused by a heterozygous deletion of 1.5-1.8Mb on chromosome 7q11.23 that encodes approximately 26 to 28 genes (5). Phenotypic variability between WBS patients may complicate the establishment of diagnosis based on clinical symptoms. Hence, a comprehensive genetic testing is necessary to confirm the diagnosis, particularly in cases with unusual manifestations.

Our study involved a seven-year-old patient with blended phenotypes. The patient manifested typical WBS features such as aortic stenosis, inguinal hernia, subclinical hypothyroidism and learning difficulty with developmental delay. Nevertheless, she also had recurrent infections, diarrhea and skin abscesses since the age of one year. As WBS patients are not commonly associated with immune disorders, we suspected the possibility of a primary immunodeficiency. Primary immunodeficiencies, recently termed as inborn errors of immunity (IEI) are characterized by increased susceptibility to infections, autoimmune diseases, autoinflammatory disorders, allergies and malignancies (6). These disorders are caused by monogenic mutations in at least 430 genes (6, 7). Diagnosing IEI based on the clinical and immunological abnormalities can be challenging owing to their phenotypic and genetic heterogeneity (8). Since the application of next-generation sequencing (NGS), the molecular diagnostics of IEI was revolutionized, facilitating the continuous discovery of IEI-associated genes (6, 9).

Previously, we utilized whole exome sequencing (WES) to diagnose patients suspected with IEI and obtained molecular diagnoses in 46.7% of the cohort (10). Among the three NGS approaches, whole genome sequencing (WGS) has the highest diagnostic capacity compared to targeted gene panel and WES. Still, WGS is minimally used in the clinical settings because of its high cost and large data output. Although a targeted gene panel generates more manageable data, it restricts the identification of novel disease-causing genes. In light of the affordable cost and computational requirements, WES is reasonable for both diagnostic and research purposes of IEI (11). The likelihood of encountering incidental findings and variants of uncertain significance increases as the NGS coverage expands. Thus, NGS data should be interpreted meticulously to ensure accurate reporting of deleterious mutations. Herein, we identified the genetic etiology responsible for the unexpected features of WBS in our patient using WES.

In this study, we reported a case with dual molecular diagnosis of WBS and chronic granulomatous disease (CGD). Repeated infections and formation of skin abscesses in our patient raised the suspicion of an IEI. We performed initial screening for germline short variants associated with IEI using WES, but detected no causative variants matching the clinical presentation. Further analysis involving CNV detection from exome data uncovered a biallelic loss of NCF1, suggesting CGD. This finding was corroborated by DHR assay that showed remarkably low neutrophil oxidative burst activity. DHR assay is commonly used to diagnose CGD owing to its quantitative measurement of NADPH oxidase activity and high sensitivity (22). However, abnormal DHR oxidation in complete myeloperoxidase deficiency and Rac2 deficiency may lead to false-positive CGD diagnosis (23, 24). Thus, genetic analysis is useful to confirm the diagnosis of CGD.

Oxidative burst activity of NADPH oxidase, which involves ROS production is essential for the elimination of invading pathogens (25). Defects in one of the five subunits of NADPH oxidase will impair the release of ROS, resulting in CGD (26). Due to ineffective clearance of microbes, CGD patients are predisposed to recurrent infections and excessive inflammation (22). This IEI was estimated to affect 1/200,000 to 1/250,000 newborns, with majority of the cases caused by X-linked CYBB mutations (27). Monogenic defects in CYBA, CYBC1, NCF1, NCF2 and NCF4 led to the less common form of CGD inherited in autosomal recessive pattern (28, 29).

Mutation detection in NCF1 can be tricky due to the presence of two pseudogenes that share at least 98% of DNA sequence similarity with the gene (30). Prominent differences, including GTGT at exon 2 and single 20-bp stretch at intron 2 distinguished NCF1 from its pseudogenes that contain a ΔGT and duplicated 20-bp stretch respectively (31). Taking these two features into account, Dekker et al. had established a reliable gene scan method to determine the ratio of NCF1 and its pseudogenes (21). The ΔGT and 20-bp gene scans revealed NCF1 deficiency caused by a ΔGT in our patient as opposed to a normal ratio of 2:4 and a carrier ratio of 1:5 in the father and mother respectively. Deletion of GT in NCF1 led to a frameshift followed by premature termination at amino acid residue 51, generating a truncated p47^phox. Unusually high incidence of ΔGT in unrelated patients was thought to arise from unequal crossing-over between NCF1 and its highly homologous pseudogenes (31, 32). Clinical symptoms of WBS and CGD in the patient can be explained by a WBS deletion involving one NCF1 allele and a ΔGT in the second NCF1 allele. As the mother was found to be the carrier of ∆GT-containing NCF1, we hypothesized that the patient inherited the de novo 7q11.23 deletion including NCF1 from her father ( Figure 4 ). The genetic diagnosis has enabled our patient to be treated with continuous prophylactic antibiotics and antifungal to prevent severe infections.

Patients with defective NADPH oxidase are increasingly susceptible to infections caused by catalase-positive microorganisms (27). This is because microbes with catalase are able to break down both host and microbial hydrogen peroxide and escape from the oxidative killing. On the contrary, catalase-negative microorganisms seldom caused infections in CGD patients as they were killed by their self-produced hydrogen peroxide. Interestingly, we detected the growth of catalase-negative Streptococcus sp. in our patient in addition to catalase-positive bacteria, i.e., Escherichia coli and Salmonella typhi. The pathogenicity of Streptococci in CGD can be explained by some strains that produced no hydrogen peroxide (33, 34). Hence, the possibility of getting infected by catalase-negative microbes should be taken into consideration despite being rare in CGD patients.

Misalignment of segmental duplications on chromosome 7q11.23 mediates nonallelic homologous recombination during meiosis, resulting in WBS deletion (35). Cases with a 1.5Mb deletion are more common than those with a larger deletion of 1.8Mb (36). Diagnosing WBS using fluorescence in situ hybridization with an ELN-targeting probe hampers the detection of deletion size (37). This limitation was then overcome by chromosomal microarray and multiplex ligation-dependent probe amplification (38, 39). Alternatively, we used read depth data from WES to call CNVs, revealing a heterozygous deletion of 1.53Mb on chromosome 7q11.23. The deletion breakpoints were predicted within NSUN5 and GTF2IRD2, affecting 27 genes, including ELN and NCF1. Phenotypic consequences were identified in some of the deleted genes. As reported by Curran et al., haploinsufficiency of ELN was responsible for the pathogenesis of aortic stenosis (40). This heart abnormality was noted in our patient, whom had single copy of ELN. Arterial stiffening, possibly due to elastin insufficiency, may increase the risk for cardiovascular events (41, 42). Contrastingly, hemizygosity for NCF1 generated lower oxidative stress, serving as a protective factor for hypertension (43). Biallelic pathogenic mutations in NCF1 will lead to autosomal recessive CGD (20). Stenton et al. recently revealed a causal link between homozygous mutations in DNAJC30 and autosomal recessive Leber’s hereditary optic neuropathy in humans (44). This feature has yet to be reported in WBS patients. Understanding the potential impacts of genetic variants help to predict the disease progression and tailor a proper therapeutic treatment.

Dual molecular diagnosis caused by two disease loci within a patient was less widely recognized before the adoption of NGS. According to a retrospective analysis of exome by Smith et al., patients with clinically complex phenotypes were more likely to have multiple genetic diagnoses (45). This finding suggests the need for reevaluation of existing NGS data and additional laboratory screening in patients with phenotypic complexity. Our study described the first case from Southeast Asia, whom was diagnosed with WBS and NCF1-deficient CGD using WES. Until now, only six patients were reported with similar dual diagnosis (46–50). Despite NCF1 hemizygosity conferred a lower risk for hypertension, Stasia et al. reported one NCF1-deficient patient suffering from high blood pressure (49). Hence, regular medical evaluation is crucial to detect the unexpected medical conditions and initiate a timely regimen.

There are several limitations in this study. Firstly, WES impedes the detection of true positive variants in genes with pseudogenes and highly repetitive sequence due to poor mapping in these regions. Although WES may not be an ideal approach for mutation screening in NCF1, it is useful for the identification of exonic and splicing variants in other CGD genes. Additional genetic testing, particularly WGS is required to detect disease-causing deep intronic variants that will be missed by WES. Secondly, uneven coverage resulted from amplification bias in WES may reduce the sensitivity and accuracy of CNV detection. Wide coverage of WGS made it more superior to detect CNVs that affect both coding and noncoding regions. Chromosomal microarray and multiplex ligation-dependent probe amplification can be used to validate CNVs identified from NGS data.

Our study revealed a case with dual diagnosis of WBS and NCF1-deficient CGD. Blended phenotypes of two genetic conditions can get misinterpreted as atypical presentation of a single disease, resulting in misdiagnosis or underdiagnosis. The use of NGS has facilitated the elucidation of multiple molecular diagnoses caused by multilocus genomic variations. Still, NGS has a low variant detection rate in genes with homologous pseudogenes, which can be complemented by Sanger sequencing. This study demonstrated the capacity of WES to detect both germline short variants and CNVs that may provide insights into pathogenesis and optimal treatment options.

The dataset presented in this study can be found in online repository. The name of the repository and accession number are as follows: https://www.ncbi.nlm.nih.gov/, PRJNA763322.

The studies involving human participants were reviewed and approved by Medical Research and Ethics Committee, Ministry of Health Malaysia. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

AMR and SBM designed the study framework and supervised the study. MYC performed the experiments and data analysis. MYC and AMR drafted the manuscript. RRPR recruited the patient, provided clinical treatments and contributed critical views to the study. All co-authors critically reviewed and approved the final version of the manuscript.

This study was funded by a grant from the Ministry of Health Malaysia (NMRR-16-892-31023) awarded to AMR.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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