Bardet–Biedl syndrome (BBS) is a rare autosomal recessive disorder characterized by rod-cone dystrophy, postaxial polydactyly, central obesity, hypogonadism, renal abnormalities, and mental retardation. BBS is often complicated by strabismus/cataracts/astigmatism, diabetes mellitus, Hirschsprung disease, heart disease, and/or liver fibrosis. To date, 21 causative genes have been reported, comprising ~80% of BBS genetic abnormalities1,2. The remaining 20% of genetic abnormalities among BBS patients are not yet known. In the present study, we performed whole-exome sequencing (WES) of a classical BBS patient.

The patient was diagnosed with BBS at 8 years of age, in accordance with criteria reported previously3. Primary and secondary signs of BBS in this patient are listed in Table 1. When the patient first visited Osaka University Hospital at 17 years of age, his best-corrected visual acuity (BCVA) was 0.07 in the right eye and 0.2 in the left eye. At 28 years of age, his BCVA was 0.01 in the right eye and 0.04 in the left eye; he exhibited bilateral diffuse retinal degeneration, including macular atrophy, attenuated retinal vessels, and optic nerve head pallor with little pigmentary dispersion. His parents were not consanguineous. His mother showed no sign of BBS or rod-cone dystrophy. His father did not have symptoms of BBS.

Table 1 Primary and secondary signs of BBS in this patient
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All experimental procedures were approved by the Ethics Committee at Osaka University (No. 719–2, Osaka, Japan) and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from the patient (at the time of the report, a 28-year-old male) and his 61-year-old mother. Both individuals underwent ophthalmologic examinations: BCVA in decimal units, slit-lamp biomicroscopy, fundoscopy, visual field testing with Goldmann perimetry, optical coherence tomography (SSOCT; DRI OCT1, Topcon Corp., Tokyo, Japan), and fundus autofluorescence (Optos, Optos KK, Tokyo, Japan). Genomic DNA was extracted from blood samples using NucleoSpin Blood XL (Macherey-nagel, Düren, Germany). DNA libraries were constructed using SureSelectXT Human All Exon Kit V6 and SureSelectXT Reagent Kit (Agilent, Santa Clara, CA, USA) and then subjected to 100 bp paired-end sequencing on an Illumina HiSeq2500 Platform (Illumina, San Diego, CA, USA). Sequence reads were aligned to the reference human genome (UCSC hg19) in BWA (http://www.bio-bwa.sourceforge.net/) to align short reads after adaptor sequences were removed by Cutadapt (https://cutadapt.readthedocs.io/en/stable/). SAM tools (Version 0.1.17; http://www.samtools.sourceforge.net/) were used for sequence data conversion, sorting, and indexing. To exclude duplicate reads, Picard (http://picard.sourceforge.net) was used. Variants were determined using GATK (http://www.broadinstitute.org/gatk/). ANNOVAR (http://www.openbioinformatics.org/annovar/) was used to annotate the resulting genetic variants. Rare variants (minor allele frequency < 0.05) were selected using the Exome Sequencing Project, 1000 Genomes Project, and Human Genetic Variation databases; possible pathogenic variants, such as nonsynonymous, nonsense, and frameshift mutations, were extracted from among the retinal degenerative disease-related genes registered in the Ret.Net.TM database.

Ten candidate pathogenic rare variants in genes related to retinal degenerative diseases were detected in this patient. All were heterozygous variants; however, two novel nonsense (NM_001288781.1 [TTC8_v001]: c.226 C > T, p.Q76X) and frameshift (NM_001288781.1 [TTC8_v001]: c.309_310insTA, p.T103fs) mutations were located in the TTC8 gene (also known as BBS8). Both mutations were validated by direct sequencing of PCR products (Applied Biosystems 3730 DNA Analyzer; Thermo Fisher Scientific K.K., Tokyo, Japan). The primer sets used for PCR were as follows: c.226 C > T, 5′-TGGGTTTTAGGCAGCTTGGA-3′ and 5′-ACCATAAGGCAGAACAGAAACCA-3′; c.308_309insAT, 5′-TAGGCCCTGGAACGTCTTTG-3′ and 5′- ACCATAAGGCAGAACAGAAACCA-3′. This mutation is likely to be pathogenic, because the TTC8 gene has been reported as a causative gene for BBS84. The nonsense mutation was located in exon 3 of the TTC8 gene, thus producing a truncated protein without tetratricopeptide repeats 11 and 15, which are involved in pilus formation and twitching mobility. The frameshift mutation in exon 5 (c.309_310insTA) generates a premature stop codon in exon 6, which also produces TTC8 lacking normal tetratricopeptide repeats 11 and 15. The premature stop codon is located before the last exon; notably, a mRNA transcribed from a gene with a truncating mutation often undergoes nonsense-mediated mRNA decay before translation5. Thus, transcripts with nonsense and frameshift mutations are likely to be rapidly degraded to reduce the translation of the truncated TTC8 protein. Therefore, this compound heterozygous patient would not have a functional TTC8 protein to support the formation of the BBSome, leading to the development of BBS. His mother exhibited the heterozygous nonsense mutation, but no frameshift mutation. Although the genetic and clinical data were not available from his father, this patient’s BBS was determined to result from a compound heterozygous TTC8 gene mutation.

BBS patients with mutations in the TTC8 gene comprise only 2.8% of all BSS patients6,7. In Japan, the genetics of four BBS families have been reported: BBS2, BBS5, and BBS7 homozygotes, as well as a BBS10 compound heterozygote8,9. To the best of our knowledge, this is the first BBS patient with a mutation in the TTC8 gene in Japan. Thus far, 16 families with the TTC8 genetic abnormality have been reported (Table 2)4,7,10,11,12,13,14,15. Most of these families have homozygous mutations; only our patient and a Hispanic family were compound heterozygotes. Although full clinical information was not available for some cases, most of the cases in these 16 families exhibit classical BBS without obvious differences in phenotypes.

Table 2 List of variants and phenotype reported in patients of BBS8
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In summary, we identified a novel compound heterozygous mutation in a Japanese BBS patient by WES. Our findings suggest that WES may be a useful tool for genetic diagnosis and characterization of BBS.