Novel splicing‐site mutation in DCAF17 gene causing Woodhouse‐Sakati syndrome in a large consanguineous family

Abstract Background Woodhouse‐Sakati syndrome is a rare autosomal recessive disease with endocrine and neuroectodermal aberrations with heterogeneous phenotypes and disease course. The most common phenotypes of the disease are progressive sensorineural hearing loss and alopecia, mild‐to‐moderate mental retardation and hypogonadism. The disease results from mutations in the DCAF17 gene. Method Here, we reported a large consanguineous pedigree with multiple affected individuals with Woodhouse‐Sakati syndrome phenotypes. Laboratory tests confirmed the endocrine perturbance in affected individuals. To find out the underlying genetic change, whole‐exome sequencing was carried out. Result Analysis of the exome data identified a splicing‐site deletion NM_025000.3:c.1423‐1_1425delGACA in DCAF17 gene. Sanger sequencing confirmed the co‐segregation of the variant with the disease phenotypes in the family. Conclusion The variant is predicted to cause aberrant splicing, i.e., exon skipping, resulting in the translation of a truncated functionless protein which results in appearance of typical phenotypic features and clinical laboratory findings of Woodhouse‐Sakati syndrome in affected members of the family.


| Study subjects
The studied four-generation consanguineous pedigree has five affected individuals, including two males (IV-3, IV-4) and three females (IV-1, IV-2, and IV-5), segregating autosomal recessive WSS phenotypes ( Figure 1). Approval for this study was obtained from Institutional Review Board, of Khyber Medical University (KMU), Peshawar, and Kohat University of Science and Technology (KUST), Kohat, Pakistan. Informed written consent was obtained from all the participants. Detailed physical and clinical examination of affected members was carried out at a tertiary care hospital.

| Samples collection and DNA extraction
For DNA extraction, peripheral blood samples were collected from all the participants in EDTA containing vacutainer tubes. Extraction of DNA from the whole blood was carried out by following a standard phenol-chloroform protocol, as described somewhere else (Ullah et al., 2017).

| Whole-exome sequencing
DNA sample of an affected individual (IV-1) was submitted to CENTOGENE for whole-exome sequencing (WES). Briefly, exome enrichment was performed using the Twist Human Core Exome plus Kit. Sequencing with a minimum depth of 20× was performed on an Illumina platform for >98% of the targeted bases. The in-house bioinformatics pipeline of CENTOGENE was used for alignment to GRCh37/hg19 genome assembly, variant calling, annotation, and variant filtering. Homozygous variants with a frequency of ≤1% in gnomAD database (https://gnomad.broad insti tute.org/) and exome F I G U R E 1 Pedigree of the family segregating Woodhouse-Sakati syndrome phenotypes in an autosomal recessive manner. Individual who underwent whole-exome sequencing is indicated by arrow sequencing project, (ESP; Exome Variant Server (washington.edu)), and disease causing variants reported HGMD (http://www.hgmd. cf.ac.uk/ac/all.php), in ClinVar (https://www.ncbi.nlm.nih.gov/clinv ar/) or in CentoMD (https://www.cento gene.com/pharm a/mutat ion-datab ase-cento md.html) were considered, focusing on coding exons and exon-intron splicing nucleotides, as described in previous studies. [10][11][12][13][14] For segregation analysis of the pathogenic variant, Sanger sequencing was performed as described earlier. 15

| Clinical findings
A large consanguineous pedigree, including five affected siblings (IV-1, IV-2, IV-3, IV-4, and IV-5), showing WSS phenotypes was studied. Specialized clinicians examination revealed presence of alopecia, intellectual disability, hypogonadism, mild sensory neural deafness, delayed speech, language development, and extrapyramidal features in affected members of the family (Table 1). Hair loss disorder in the affected patients was defined by the presence of sparse, thin, short, dry, and lighter in color scalp hairs that break easily, scanty eyebrows, and absence of axillary and pubic region hair. Nails and teeth are normal. They also display sensory neural      However, the level of thyroxin and TSH was normal. In affected brother IV-3, most laboratory tests showed normal range, such as LH, FSH, thyroxin, TSH, Testosterone except elevated serum cholesterol, and decreased IGF-1 levels ( Table 1).

| Variant identification and sequence analysis
The mutation detected in this study is a deletion mutation at the splicing acceptor site, most probably leading to exon skipping.
However, if the splicing does occur then, due to the deletion of the two nucleotides from the coding region, there will be a frameshift.
In each case, probably a truncated protein will be translated, causing lack/altered residues after 474 amino acids, resulting in a function- atrophy, but further animal-based studies are obligatory to explain these findings. 16 Similarly, the DCAF17 knockout mice generated by gene targeting show that the DCAF17 mice produced a decreased number of sperm, with abnormal shape and significantly low mo-  (III-1 and III-2) and the lower panel shows the homozygous wildtype allele in the unaffected sisters (IV-1, IV-2, IV-3, IV-4, and IV-5) helps to confirm the diagnosis, allow a more accurate prognosis and proper genetic counseling of the patients and their family members.

ACK N OWLED G M ENT
We thank all the individuals for their generous participation in this study.

CO N FLI C T S O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All the data used in this manuscript can be obtained upon request.