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

Background Bardet Biedl Syndrome (BBS) is a rare condition of multi-organ dysfunction with characteristic clinical features of retinal degeneration, truncal obesity, postaxial polydactyly, genital anomaly, intellectual disability and renal dysfunction. It is a hetero-genetic disorder and nineteen BBS genes have been discovered so far. Methods Whole genome SNP genotyping was performed by using CytoScan® 750 K array (Affymetrix). Subsequently, the segregation of the disease locus in the whole family was carried out by genotyping STS markers within the homozygous interval. Finally, the mutation analysis was performed by Sanger DNA sequencing. Results In the present molecular study a consanguineous Pakistani family, with autosomal recessive BBS, was analyzed. The clinical analysis of affected individuals presented with synpolydactyly, obesity, intellectual disability, renal abnormality and retinitis pigmentosa. The presented phenotype was consistent with the major features of BBS syndrome. Homozygosity mapping identified a common homozygous interval within the known BBS9 locus. Sequence analysis of BBS9/PTHB1 gene revealed a single base deletion of c.299delC (p.Ser100Leufs*24) in exon 4. This frame-shift mutation presumably leads to a 122 amino acid truncated protein with complete loss of its C-terminal PTHB1 domain in combination with a partial loss of the N-terminal PTHB1 domain as well. BBS9/PTHB1 gene mutations have been shown to be associated with BBS syndrome and to the best of our knowledge this study reports the first Pakistani family linked to the BBS9 gene. Conclusion Our molecular findings expand the mutational spectrum of BBS9 gene and also explain the genetic heterogeneity of Pakistan families with BBS syndrome. The growing number of mutations in BBS genes in combination with a detailed phenotypical description of patients will be helpful for genotype-phenotype correlation, targeted genetic diagnosis, prenatal screening and carrier testing of familial and non-familial BBS patients.


Background
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].
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.

From sampling to DNA isolation
The consanguineous family for the present molecular investigation was ascertained from the rural area of D.I.Khan in the Khyber-Pakhtoonkhwa Province of Pakistan. The study was approved by the ethical review board of Gomal Centre of Biochemistry and Biotechnology, Gomal University D.I.Khan, Pakistan. Moreover, informed written consent was obtained from all individuals participating in the study. The family was visited at its residence and blood samples were collected. The patients were examined in detail for all apparent phenotypic features to document the major as well as the minor BBS symptoms. The parents were also counseled in detail on prenatal, perinatal, and neonatal medical problems. Subsiquently, the DNA was isolated from the peripheral blood sample by using standard Phenol-Chloroform method.

From genome to gene analysis
To investigate the underlying disease gene mutation, genome-wide SNP genotyping was performed by using Affymetrix CytoScan 750 K SNP microarray. The SNP data were analyzed using the Chromosome Analysis Suite (ChAS) version 1.2 (Affymetrix) to identify the homozygous by descend region (HBD). The perfect segregation of identified disease associated HBD region in the complete family was determined by genotyping STR markers D7S493 (36.08 cM), D7S516 (42.93 cM), D7S484 (53.73 cM), and D7S510 (59.8 cM) {Rutgers map B37 [22]} within the homozygous region. For statistical significance of linkage, two point and multi point LOD scores were calculated by using Superlink and GeneHunter software packages plugged-in the EasyLinkage plus v5.02 software. The analysis was performed assuming a disease allele frequency of 0.001 with full penetrance at zero recombination fraction [23].
The primers for protein coding exons of BBS9 gene were designed using the online version of Primer 3 plus software (version 0.4.0) [24]. The sequence reactions were performed with BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystem, USA) and the sequencing products were analyzed by the Genetic analyzer 3130xl (Life Technologies, USA). The sequence data were then aligned with the BBS9 reference sequence via blast like alignment tool (BLAT) tool package of UCSC Genome Browser [25].

Results
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).

Clinical findings
The clinical examination of both BBS patients demonstrated bilateral postaxial synpolydactyly of hands and feet, mild progressive retinal degeneration, intellectual disability, renal dysfunction, and obesity as a major BBS features, while mild hearing loss was also noticed as a minor feature in both affected individuals. The pattern of postaxial synpolydactyly in the female showed hepta-dactyly of left hand and foot but the right hand and foot had six digits, while the affected male had only hepta-dactyly of the right hand. The detailed clinical description of both affected individuals is shown in Table 1.

Genetic findings Whole genome SNP genotyping and linkage analysis
Genome-wide homozygosity mapping revealed a 18.45 Mb (HG19: chr7: 21,596,876-39,320,676) long HBD region between markers rs56356943 to rs12112084, on the short arm of chromosome 7. Segregation mapping by STR markers in the complete family, through STR markers, confirmed the inheritance of the homozygous region with disease etiology. Linkage analysis generated a suggestive two point and multipoint LOD scores of 0.92 and 1.26 respectively at same marker D7S484 (53.73 cM) with zero recombination fraction. The identified homozygous interval harbored the BBS9 gene which is reported to be associated with BBS.

Sequence analysis of the BBS9 gene
Sanger DNA sequencing of the BBS9 gene revealed a homozygous deletion of cytosine nucleotide in exon 4 (c.299delC). At protein level the reported deletion is predicted to result in a frame-shift mutation p.Ser100-Leufs*24 (UniProtKB accession: Q3SYG4). The predicted premature stop codon presumably disrupts the open reading frame and results in a truncated polypeptide

Discussion
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 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 multigene 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 In summary, our data represent the first BBS9 gene mutation being reported in a Pakistani family associated with Bardet Biedl syndrome. In addition to cardinal features of BBS, slight clinical variations were also observed in Pakistani siblings. It is assumed that the current study will contribute to the elucidation of the genetic heterogeneity of BBS patients and that the screening of additional BBS cases in future might detect the mutational hotspot and thus would be helpful in designing a multigene panel for routine diagnosis.

Conclusion
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. , heterozygous mutation in a carrier (III-2) and wild-type sequence in an normal individual (IV-6). c-The protein truncating mutation is depicting a loss of complete PTHB1 C-terminus domain along with some part of N-terminus, leaving behind a 122 amino acid truncated protein