Cytogenomic identification and long-read single molecule real-time (SMRT) sequencing of a Bardet-Biedl Syndrome 9 (BBS9) deletion

Abstract

Bardet-Biedl syndrome (BBS) is a recessive disorder characterized by heterogeneous clinical manifestations, including truncal obesity, rod-cone dystrophy, renal anomalies, postaxial polydactyly, and variable developmental delays. At least 20 genes have been implicated in BBS, and all are involved in primary cilia function. We report a 1-year-old male child from Guyana with obesity, postaxial polydactyly on his right foot, hypotonia, ophthalmologic abnormalities, and developmental delay, which together indicated a clinical diagnosis of BBS. Clinical chromosomal microarray (CMA) testing and high-throughput BBS gene panel sequencing detected a homozygous 7p14.3 deletion of exons 1-4 of BBS9 that was encompassed by a 17.5 Mb region of homozygosity at chromosome 7p14.2-p21.1. The precise breakpoints of the deletion were delineated to a 72.8 kb region in the proband and carrier parents by third-generation long-read single molecule real-time (SMRT) sequencing (Pacific Biosciences), which suggested non-homologous end joining as a likely mechanism of formation. Long-read SMRT sequencing of the deletion breakpoints also determined that the aberration included the neighboring RP9 gene implicated in retinitis pigmentosa; however, the clinical significance of this was considered uncertain given the paucity of reported cases with unambiguous RP9 mutations. Taken together, our study characterized a BBS9 deletion, and the identification of this shared haplotype in the parents suggests that this pathogenic aberration may be a BBS founder mutation in the Guyanese population. Importantly, this informative case also highlights the utility of long-read SMRT sequencing to map nucleotide breakpoints of clinically relevant structural variants.

Fig. 1

Clinical and genetic diagnosis of Bardet–Biedl syndrome. Clinical evaluation of a 14-month-old proband revealed features suggestive of BBS, including low-set ears and a right ear pit (arrow) (a), and postaxial polydactyly on the right foot (arrow) (b). Analysis of the patient specimen on the Agilent 4 × 180 CGH + SNP array (c, left panel) detected a single region of homozygosity at chromosome 7p14.2–p21.1. A homozygous deletion (indicated by an asterisk) of the 5′ region of BBS9 was nested within the region of homozygosity at 7p14.3. Analysis of the proband DNA with the higher-resolution Affymetrix CytoScan HD array (c, right panel) confirmed these results and further refined the deletion breakpoints

Fig. 2

Deletion breakpoint identification. The chromosome 7p14.3 genomic region is illustrated with tracks for the proband chromosomal microarray (CMA) results, genomic PCR mapping amplicon locations (1–9; green: amplified; red: did not amplify), and copy number variants detected among healthy individuals in the Database of Genomic Variants (DGV; blue: duplication; red: deletion) (a). Unambiguous breakpoint mapping was performed by long-read single molecule real-time (SMRT) sequencing (PacBio) of long-range PCR products that amplified across the deleted interval in the proband (b). These SMRT sequencing data were also aligned to a modified human genome reference that excluded the identified 72.8 kb deletion (chr7:33130616–33203409) (c), confirming that there were no other sequence alterations at the breakpoint locations. The precise deletion breakpoints were subsequently confirmed in the proband and both carrier parents by Sanger sequencing of the long-range PCR amplicons (d)