A BBS1 SVA F retrotransposon insertion is a frequent cause of Bardet-Biedl syndrome

Abstract

Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinitis pigmentosa, obesity, polydactyly, cognitive impairment and renal failure. Pathogenic variants in 24 genes account for the molecular basis of >80% of cases. Toward saturated discovery of the mutational basis of the disorder, we carefully explored our cohorts and identified a hominid-specific SINE-R/VNTR/Alu type F (SVA-F) insertion in exon 13 of BBS1 in eight families. In six families, the repeat insertion was found in trans with c.1169 T > G, p.Met390Arg and in two families the insertion was found in addition to other recessive BBS loci. Whole genome sequencing, de novo assembly and SNP array analysis were performed to characterize the genomic event. This insertion is extremely rare in the general population (found in 8 alleles of 8 BBS cases but not in >10 800 control individuals from gnomAD-SV) and due to a founder effect. Its 2435 bp sequence contains hallmarks of LINE1 mediated retrotransposition. Functional studies with patient-derived cell lines confirmed that the BBS1 SVA-F is deleterious as evidenced by a significant depletion of both mRNA and protein levels. Such findings highlight the importance of dedicated bioinformatics pipelines to identify all types of variation.

Keywords: BBS1; Bardet-Biedl syndrome; Mobile element insertion; SVA F; founder effect.

Figure 1.. Pedigree of BBS families and variant analysis

(A) 8 families carrying the SVA F insertion in exon 13 of BBS1. (B) Next generation sequencing data displayed from the Integrative Genomics Viewer surrounding the BBS1 locus from A.II-3 and one control individual. (C) Magnified view of the incorrectly called deletion insertion with the sorting and coloring of the reads according to the “insert size”. The sequence reveals all reads ending at the exact same position with a “AG” deletion and a “T” insertion (c.1215_1216delinsT). (D) Region of interest with the “Show soft-clipped bases” turned off revealing multiple reads with aberrant alignments on their right side and corresponding to an SVA F insertion. The spacing between the reads on the left (including the 3’ end of the SVA F with the poly(A) tail) and the reads on the right (including the 5’ end of the SVA F) corresponds to the TSD. (E) Segregation analysis using Sanger sequencing for M1 (left) and M2 (right).

Figure 2.. Schematic of the inserted SVA F element in exon 13 of BBS1.

(A) Canonical SVA F structure composed of 6 parts: (1) hexameric CCCTCT repeats (2) Alu-like domain, (3) a Variable Number Tandem Repeats (VNTR) domain, (4) a Short Interspersed Nucleotide Elements (SINE-R) domain, (5) a poly(A) tail and (6) the target site duplication (TSD) at both extremities. Numbers above the scheme indicate the size of each element. (B) Schematic of both BBS1 alleles in A.II-3 with primer positioning and amplification products highlighting each contribution to the resolution of the full-length sequence.

Figure 3.. Quantification of BBS1 mRNA and protein levels in A.II-3

(A) Reduced BBS1 mRNA expression by RT-qPCR (A.II-3) compared to control or homozygous p.Met390Arg patient. The major fraction of BBS1 expression likely corresponds to the p.Met390Arg allele. Normalization was performed using both GAPDH and HPRT as reference genes. Mean of both results are shown. Controls are in gray (n=3), homozygous p.Met390Arg in black (n=2), and patient A.II-3 (p.Met390Arg and SVA F) in white (n=2). Bars show the mean of 2 independent experiments +/− SEM (n=2, t-test *: p<0.05, ns: not significant). (B) Western blot analysis of BBS1 protein level. No truncated version of BBS1 could be detected. Half reduced BBS1 protein level in A.II-3 compared to control. Bars show mean of 3 independent experiments +/− SEM (n=3, t-test *: p<0.05, ns: not significant). M: molecular weight ladder (kDa).