Exome sequence analysis in consanguineous Pakistani families inheriting Bardet‐Biedle syndrome determined founder effect of mutation c.299delC (p.Ser100Leufs*24) in BBS9 gene

Abstract Background Bardet‐Biedl syndrome (BBS) is characterized by a heterogeneous phenotypic spectrum of retinopathy, intellectual disability (ID), obesity, polydactyly, and kidney dysfunctions as the major clinical features. Genetic investigations have reported 21 BBS genes, the products of which are mostly located at the centrosome, basal body or the ciliary transition zone. Methods In the present genetic report, we analyzed two apparently unrelated consanguineous BBS families from Dera Ismail Khan (D.I.Khan) district, Pakistan. Genetic mapping was performed using Whole exome sequencing and Sanger sequencing. Results Whole exome sequencing identified a recently reported single base deletion NM_001033604.1:c.299delC in the fourth exon of BBS9 in both families. The identified frameshift mutation is predicted to cause premature truncation of the expressed protein (p.Ser100Leufs*24). This mutation has previously been mapped in a consanguineous Pakistani family; therefore this is the second report of this particular mutation in two additional BBS families originating from different locations. Conclusion We speculate the evolutionary significance of this mutation and assume its strong founder effect in the Khaisoori tribe of D.I.Khan. Based on these findings, we suggest developing a molecular diagnostic test that may be used for premarital and prenatal screening of families at risk of BBS.

The prevalence rate of BBS varies among different ethnicities worldwide. In North America and Europe, BBS affetcs 1 per 140,000-160,000 live births. However, its incidence is extremely high with ratios of 1:18,000, 1:13,500, and 1:3,700 in geographically isolated communities of Newfoundland, Kuwaiti Bedouins and The Faroe Islands, respectively. Nonetheless, the additional contributing factor of elevated BBS incidence in the Kuwaiti population is probably the high ratio of consanguinity (Maria et al., 2016).
This study was aimed at exploring the genetics of BBS phenotypes in two apparently nonrelated consanguineous families of Pashtoon origin, recruited from a small village in Khyber-Pukhtunkhwa (KPK) province of Pakistan. Exome sequence analysis in both families revealed a previously reported single base deletion mutation in BBS9 [c.299delC (p. Ser100Leufs*24)], which suggests its founder effect in the Khaisoori tribe of D.I.Khan city in KPK.

| Family recruitment
Two apparently unrelated consanguineous families of Pashtoon origin exhibiting the BBS phenotype were enrolled in the present genetic study. Pedigree information of both families was procured to explain the consanguineous relationship and degree of consanguinity. Both families were recruited from the Rehmani-Khail village, a remote area in D.I.Khan city in the KPK Province of Pakistan. Prior approval for the study was obtained from the ethical review board of Gomal University, D.I.Khan, Pakistan. Participants or their guardians were briefed on the study scheme, and written consent was obtained.

| Clinical assessment
Details of relevant BBS associated clinical phenotypes were documented using a self-designed questionnaire by comprehensively evaluating the apparent features in all patients. Subsequently, peripheral blood samples from cooperative affected and unaffected participants of the study were collected and DNA was extracted using standard laboratory protocols. Additionally, the peripheral blood samples were subjected to certain biochemical tests, such as lipid profile, liver function tests and renal function test. These spectrophotometric assays were conducted using a chemistry analyzer (Macrolab, Germany). Furthermore, profile photographs of certain patients were obtained to document and publish the patients' phenotype.

| Exome sequencing and mutation analysis
To map the causative gene manifesting BBS in both families, one patient from each family was selected for whole exome sequencing. Sample library preparation was performed with Nextera Rapid Capture Exome Kit (Illumina, USA) and sequenced using the NextSeq550 (Illumina, USA) at the Institute of Human Genetics, Medical University of Graz, Austria. Raw sequence data were aligned to the reference genome using the BaseSpace applications to generate BAM and VCF files. Thereafter, variant filtering was performed using VariantStudio Software (Illumina, USA). Primarily, data were filtered for homozygous, nonsynonymous SNPs and InDel variants with an allele frequency below 1% in the coding regions of known BBS genes.
In addition to this, gene ontology and protein functionbased bioinformatics analysis was performed using PhenIX software (Zemojtel et al., 2014)]. Subsequently, segregation analysis of potentially pathogenic variants in the whole family was done using Sanger DNA sequencing.

| Results
This study focuses on two consanguineous families of Pashtoon origin from the Rehmani-Khail village in D.I.Khan city of Pakistan, exhibiting autosomal recessive BBS. Although, both families ethnically belong to the Rehmani-Khail tribe, they were unable to establish a close relationship with each other or previously published family from Khan, Mohan, et al. (2016) and Khan, Muhammad, et al. (2016). All the BBS children were born from asymptomatic parents with first cousin relationship (Figure 1).

| Clinical outcomes
Patients from both families exhibited postaxial polydactyly of hands and feet, obesity and intellectual disability (ID) as a common phenotype. Gross clinical analysis confirmed diagnosis of BBS syndrome in all cases. All patients from family A showed a mild level of ID, while patient/V from family B had severe ID. Severely reduced visual acuity was only observed in one affected person from family B, along with strabismus of right eye, however this phenotype was not observed in patients from family A. Speech was quite developed in all patients of family A, but patient/V from family B was unable to speak. Analysis of facial photographs did not reveal facial dysmorphology (Figure 1). Patients from both families revealed hexadactyly (central polydactyly) of both feet, however, their hands exhibited syn-hexa-dactyly (see table and figure for details). Furthermore, there were no visceral organ defects observed in either family. The summarized clinical features, along with additional information, are presented in Table 1. We interviewed the family elders from both familes of the current study (Rehmani-khail), and the family described in the previously published study from Khan, Mohan, et al. (2016) and Khan, Muhammad, et al. (2016) (Khanokhail tribe). On the basis of their statements a distant kinship with common ancestors, i.e. the Khaisoori tribe, seems possible.

| Genetic outcomes
Whole exome sequencing identified a recently reported single base deletion c.299delC in the fourth exon of the BBS9 gene in both family A and B. This frameshift mutation is predicted to cause premature truncation of the protein product (p.Ser100Leufs*24), possibly leading to a loss of part of its N-terminal and all of its C terminal domains. In both familes the detected mutation co-segregated with the disease phenotype.
Additionally, exome-wide SNP analysis for HBD mapping revealed both families sharing common haplotype between markers rs201037213 and rs2240350 around the BBS9 locus on chrosomsome 7.

| DISCUSSION
Bardet-Biedl syndrome (BBS) is an autosomal recessive disorder that affects many tissues of the body simultaneously. According to BBS diagnosis criteria, the patients should either have four major features or three major and two minor features for positive diagnosis of BBS. Genetic investigations have reported 21 BBS genes so far, among them BBS1 and BBS10 are highly reported in Europe and North America, while BBS2, BBS4, BBS5, and BBS12 are prevalent in the Middle East and North Africa (Nikkhah et al., 2018). Nonetheless, to date, 18 Pakistani families harboring eight genes have been reported to be involved in BBS (Maria et al., 2016).
In this study, whole exome sequencing in two families revealed a previously reported frameshift mutation p.Ser-100Leufs*24 in BBS9. The identified mutation is predicted to severely truncate the BBS9 protein and completely remove its C-terminus domain. Predictably, the defective mRNA may be removed through mRNA degradation as in an analogous case of protein truncating nonsense mutation p.Q597* reported by Maria et al., (2016).