Homozygosity mapping identified a novel protein truncating mutation (p.Ser100Leufs*24) of the BBS9 gene in a consanguineous Pakistani family with Bardet Biedl syndrome

Bardet Beidl syndrome (BBS) is a multi-symptomatic ciliopathy. The major clinical manifestations of this disorder are cone-rod dystrophy, truncal obesity, postaxial polydactyly, genital anomaly, learning disability and renal dysfunction [1]. Clinically BBS is a heterogeneous disorder with variable inter- and intra-familial features [2], therefore its diagnosis is established by the presence of four or three of the major features and two minor features like speech delay, brachydactyly/syndactyly, cardiovascular anomalies etc. [3, 4]. The incidence rate of BBS varies among the different ethnicities, and occur with the ratio of 1: 156,000 in Tunisia [5], 1: 140,000–160,000 in North America to Europe, 1: 17,000 in Kuwait, and 1: 18,000 in Newfoundland [6, 7].

Genetically BBS is segregated in an autosomal recessive fashion with high degree of locus and allele heterogeneity. The combinatorial approach of SNP microarray (for homozygosity mapping) with targeted exome sequencing (for mutation analysis) has speed-up the discovery of causative gene and its underlying pathogenic mutation [8, 9]. Genetic analysis of BBS patients have identified nineteen disease responsible genes (BBS1-19), most of which encode BBSome protein (BBS1, BBS2, BBS4, BBS5, BBS7, BBS8/TTC8, BBS9/PTHB1, BBS17/LZTFL1 and BBS18/BBIP1) while some produce basal body interacting protein (BSS13/MKS1, BBS14/EP290/NPHP6, BBS15/WDPCP and BBS16/SDCCAG8), chaperonin complex protein (MKKS, BBS10 and BBS12), E3 ubiquitin ligase (BBS11/TRIM32), and a GTPase protein complex (BBS3/ARL6 and BBS19/IFT27) [10, 11]. Physiologically BBSome is of prime importance in ciliogenesis due to its functional role in membrane trafficking in primary cilium [12]. The mutational spectrum of BBS genes is variable among different populations. A high proportion of European and Caucasian families are reported to have mutation in BBS1 and BBS10 gene with the ratio of 23.2 % and 20 % respectively [13, 14], while in the Arab population most of the mutations are found in BBS1, BBS2, BBS3, BBS4 and BBS8 [10, 15‐17] with a total prevalence rate of 67 %. Although no prevalence data of the BBS genes have been estimated yet in the Pakistani population however most of the mutations occur in BBS1 [18], BBS3/ARL6 [19], BBS10 [19, 20], and BBS12 [21].

In the present linkage study, we have ascertained a Saraiki origin BBS family from the Kirikhaisor village of D.I.Khan in the KPK province of Pakistan. It was a consanguineous family with two affected siblings presenting with BBS features. Homozygosity mapping in combination with Sanger sequencing of a candidate gene identified a novel single base deletion mutation in BBS9 gene [(c.299delC) & (p.Ser100Leufs*24)]. This frame shift mutation presumably leads to truncated nonfunctional protein. These data present the first report of the BBS9 gene mutation in a Pakistani family and thus increase the mutational spectrum of Bardet Beidl syndrome in this ethnic region.

In this study, four individuals from a multi-generation family were enrolled for molecular investigation of an underlying disease gene mutation. The recruited individuals consist of both affected siblings (IV-4 and IV-5), one unaffected brother (IV-6) and an asymptomatic carrier mother (III-2). Pedigree analysis revealed an autosomal recessive mode of disease segregation (see Fig. 1).

Ciliopathies are a distinct group of genetic disorders which are caused by primary ciliary dysfunction with pleiotropic effect. Bardet Biedl syndrome is a rare autosomal recessive ciliopathic disorder characterized by retinal degeneration, intellectual disability, obesity, renal complications and polydactyly as predominant phenotypes. It is a heterogenetic disorder with 19 causative genes, whose protein products together constitute centrosome and/or basal body which is physiologically involved in ciliary function [26, 27]. Here in this report, we discovered a novel single base deletion in the 4th exon of BBS9 gene (c.299delC), which presumably leads to truncated protein with complete loss of the C terminal PTHB1 domain (UniProtKB accession:Q3SYG4). The presence of the premature stop codon will likely lead to degradation of the resulting mRNA through Nonsense-mediated mRNA Decay.

BBS9 protein is the component of BBSome protein complex, a central entity of ciliogenesis, which is localized in the primary cilia and peri-centriolar region. Ten BBSome subunits (from BBS1 to 10) have been discovered, and BBS9 has a possible role in associating other subunits [12]. STRING protein interaction database (URL: www.​string-db.​org) predicted strong interaction of BBS9 with BBS5 protein, which suggests that BBS5 could be involved in the assembly of BBSome subunits through BBS9 interaction. Hence, loss of function mutation in BBS9 may affect the integrity of BBSome proteins complex due to a structural abnormality in cilia. This structural aberration could be the reason for disassembly of BBSome subunits due to defective BBS9-BBS5 protein interaction which might be required for proper organization. The knockdown studies in zebrafish and mouse have clearly demonstrated the significant role of BBS9 in cilia biogenesis [28]. Although extensive work has been performed for the genetic screening of BBS genes, but the actual function of most BBS proteins are yet to be discovered.

The mutational spectrum of BBS9 gene has reported 12 missense/non-sense mutations, 2 splice site changes, 2 small deletions, 2 small insertions and 4 gross deletions in BBS phenotype (HGMD database; URL: http://​www.​hgmd.​cf.​ac.​uk/​ac/​index.​php), where as one complex rearrangement in BBS9 is associated with Wilms tumor (HGMD database; [29]). The clinical spectrum of both patients reported in this study is consistent with previous reports, except for a few novel phenotypic variations. These clinical variations included digestion problems (constipation) in both affected (IV-4 & IV-5), irregular menstrual cycle (IV-5), strabismus (IV-4), and synpolydactyly of internal digits (IV-4 & IV-5).

The molecular genetics testing of any inherited disorder either involves multi-gene panel or the most implicated genes. In case of BBS, the high cost of multi-gene panels (BBS1- BBS19) and weakly established genotype-phenotype correlations favor the screening of highly prevalent BBS1 and BBS10 genes. Hence, keeping in view the genetic counseling of BBS patients, the large number of reported BBS genes and unclear phenotypic association data, genetic screening of the most implicated BBS1 and BBS10 is considered as cost effective. Nevertheless, prevalences of supposedly rare BBS genes such as BBS9 might vary in different ethicities and therefore prioritization of genes have to be adapted accordingly in a diagnostic setup.

Here in this study we report the first Pakistani family segregating a novel deletion mutation in BBS9 gene (c.299delC). The mutation shifts the reading frame and presumably results in a truncated protein with loss of functional PTHB1 domains (p.Ser100Leufs*24). The current finding has increased the mutational spectrum of the Bardet Biedl Syndrome mutation in the Pakistani population and thereby will increase our knowledge to understand the potential role of the BBS9 protein in BBSome protein complex.