Novel sequence variants in the MKKS gene cause Bardet-Biedl syndrome with intra- and inter-familial variable phenotypes.

Abstract

Bardet-Biedl syndrome (BBS) is a multisystem disorder involving retina, kidney, limbs, and nervous system. Additional features associated with BBS include dental anomalies, cardiovascular defects, hearing loss, and speech impairment (Beales et al. 1999; Ullah et al. 2017). Diagnosis of BBS is based on the presence of four out of six primary or three primary and two secondary phenotypes. Variation in the phenotypes within and among different families has been reported (Beales et al. 1999; Khan et al. 2016). So far, mutations have been reported in 21 disease-causing genes (BBS1–21) (Heon et al. 2016; Suspitsin and Imyanitov, 2016). BBS segregates in autosomal recessive manner. In rare cases, triallelic nature of BBS involving three mutated alleles in two genes has been reported as well. Genes causing BBS phenotypes play important roles in ciliogenesis. Defects in the formation or function of cilia result in syndromic form of BBS. Cilia are cell surface structures involved in cell locomotion, signaling, and chemo/mechano/photo-sensation. Due to localization of cilia in different organs including kidney, liver, pancreas, odontoblasts, retinal photoreceptors, and hypothalamic neurons, ciliary defects cause multisystem disorders. Three BBS proteins including BB6/MKKS, BBS10, and BBS12 form chaperonin complex required for BBsome formation. BBsome is composed of eight BBS proteins including BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9, and BBIP10/BBS18. Chaperonin binds to BBS7 and stabilizes it and then interacts with BBS2 and BBS9 to form core of BBsome. Other members bind to BBsome core in order of BBS1, BBS5, BBS8, BBS4, and BBIP10 (Loktev et al. 2008). BBsome participates in the cilia formation. The present study was conducted with the permission of institutional review board of Qauid-i-Azam University Islamabad, Pakistan. Clinical features of affected individuals are presented in Figure 1. Affected members in the two families (A and B) showed previously reported primary features of BBS, which included polydactyly, learning difficulties, retinitis pigmentosa, truncal obesity, and hypogonadism (Khan et al. 2016). In addition, a male affected member of family A showed sparse hairs on scalp and face, and a rare secondary feature of dental anomalies. In the same family, one of the affected members (V-5) had polydactyly in both hands and feet, while the second affected member (V-6) showed this feature only in the right foot. Affected members of family B showed clinodactyly of toes as a secondary feature of BBS (Table S1, Supporting information). DNA from blood was extracted using Sigma GenElute Blood Genomic DNA kit (Sigma Aldrich, St Louis, MO, USA). Homozygosity mapping was performed by genotyping highly polymorphic microsatellite markers which were mapped previously in vicinity of the BBS genes (BBS1–21) ( Fig. S1). This was followed by Sanger sequencing using standard procedure (Ullah et al. 2015), which revealed two novel variants including c.775-775delA in family A and c.287C>T in family B in the MKKS. Mutation effect prediction tools (MutationTaster, SIFT, and Polyphen2) predicted the two novel variants to be deleterious and disease-causing (Table S2). Previously, the MKKS was mapped on chromosome 20p12.2. The gene encodes 570-amino acid long BBS6 protein. It contains three domains including equatorial, intermediate, and apical. The equatorial domain is connected to apical domain by intermediate domain through flexible hinges. The variant (p.Thr259Leufs*21) in MKKS/BBS6, found in family A, is located in the apical domain which is involved in substrate binding. The other variant (p.Ala96Val), found in family B, is located in the most conserved equatorial domain and is highly likely to disrupt its ATPase activity. The BBS6 is a member of chaperonin family, which is involved in the formation of BBsome complex. It is highly likely that sequence variants in the BBS6 would disrupt formation of such a complex leading to buckle cilia formation. We have observed variation in the phenotypes not only in the two families, but also among patients of the same family (Table S1). Position and nature of mutation alter the structure and function of protein differentially. Genetic background and extent of change in the structure and function of protein might be responsible for the intra- and inter-familial variability of phenotypes. In conclusion, we have identified two novel sequence variants in the BBS6 causing BBS phenotypes in two different consanguineous families. This study not only supports the previously reported findings of intra- and inter-familial variations in features of BBS families, but also further extend spectrum of the mutations in the MKKS gene. This study will support genetic testing of BBS patients in Pakistan. We are highly thankful to the family members for their participation in the present research project. MU was supported by Higher Education Commission Islamabad, Pakistan Indigenous PhD fellowship. The authors declare that they have no competing interests. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.