A novel compound heterozygous mutation in ttc8 identified in a japanese patient

ABSTRACT Bardet–Biedl syndrome (BBS), characterized by rod-cone dystrophy, postaxial polydactyly, central obesity, hypogonadism, renal abnormalities, and mental retardation, is a rare

autosomal recessive disorder. To date, 21 causative genes have been reported. Here we describe a Japanese BBS patient with a novel compound heterozygous mutation in _TTC8_. To the best of

our knowledge, this is the first description of a BBS patient with a mutation in the _TTC8_ gene in Japan. 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. 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. 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. HGV DATABASE The relevant data from this Data Report are hosted at the Human Genome Variation Database at https://doi.org/10.6084/m9.figshare.hgv.2528;

https://doi.org/10.6084/m9.figshare.hgv.2531 REFERENCES * Forsythe, E., Kenny, J., Bacchelli, C. & Beales, P. L. Managing Bardet-Biedl Syndrome-now and in the future. _Front. Pediatr._

6, 23 (2018). Article  Google Scholar  * Dilan, T. L. et al. Bardet-Biedl syndrome-8 (BBS8) protein is crucial for the development of outer segments in photoreceptor neurons. _Hum. Mol.

Genet._ 15, 283–294 (2018). Article  Google Scholar  * Beales, P. L., Elcioglu, N., Woolf, A. S., Parker, D. & Flinter, F. A. New criteria for improved diagnosis of Bardet-Biedl

syndrome: results of a population survey. _J. Med. Genet._ 36, 437–446 (1999). CAS  PubMed  PubMed Central  Google Scholar  * Ansley, S. J. et al. Basal body dysfunction is a likely cause of

pleiotropic Bardet-Biedl syndrome. _Nature_ 425, 628–633 (2003). Article  CAS  Google Scholar  * Hug, N., Longman, D. & Cáceres, J. F. Mechanism and regulation of the nonsense-mediated

decay pathway. _Nucleic Acids Res._ 44, 1483–1495 (2016). Article  Google Scholar  * Bin, J. et al. BBS7 and TTC8 (BBS8) mutations play a minor role in the mutational load of Bardet-Biedl

syndrome in a multiethnic population. _Hum. Mutat._ 30, E-737–E746 (2009). Article  Google Scholar  * Stoetzel, C. et al. BBS8 is rarely mutated in a cohort of 128 Bardet-Biedl syndrome

families. _J. Hum. Genet._ 51, 81–84 (2006). Article  Google Scholar  * Hirano, M. et al. The first nationwide survey and genetic analysis of Bardet-Biedl syndrome in Japan. _PLoS ONE_ 10,

e0136317 (2015). Article  Google Scholar  * Kurata, K. et al. Clinical characteristics of a Japanese patient with Bardet-Biedl syndrome caused by BBS 10 mutations. _Jpn J. Ophthalmol._ 62,

458–466 (2018). Article  CAS  Google Scholar  * Smaoui, N. et al. Screening of the eight BBS genes in Tunisian families: no evidence of triallelism. _Invest. Ophthalmol. Vis. Sci._ 47,

3487–3495 (2006). Article  Google Scholar  * Harville, H. M. et al. Identification of 11 novel mutations in eight BBS genes by high-resolution homozygosity mapping. _J. Med. Genet._ 47,

262–267 (2010). Article  CAS  Google Scholar  * Janssen, S. et al. Mutation analysis in Bardet-Biedl syndrome by DNA pooling and massively parallel resequencing in 105 individuals. _Hum.

Genet._ 129, 79–90 (2011). Article  CAS  Google Scholar  * Redin, C. et al. Targeted high-throughput sequencing for diagnosis of genetically heterogeneous diseases: efficient mutation

detection in Bardet-Biedl and Alström syndromes. _J. Med. Genet._ 49, 502–512 (2012). Article  CAS  Google Scholar  * M’hamdi, O. et al. Clinical and genetic characterization of Bardet-Biedl

syndrome in Tunisia: defining a strategy for molecular diagnosis. _Clin. Genet._ 85, 172–177 (2014). Article  Google Scholar  * Ullah, A. et al. Sequence variants in four genes underlying

Bardet-Biedl syndrome in consanguineous families. _Mol. Vis._ 23, 482–494 (2017). CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS We thank E. Suga, M.

Morita, Y. Hasegawa, S. Tanaka, and S. Ishino for their technical assistance. We thank Editage (www.editage.jp) for the English language editing. This research was supported by the Project

Promoting Clinical Trials for the Development of New Drugs and Medical Devices (Japan Medical Association) from the Japan Agency for Medical Research and Development, AMED. AUTHOR

INFORMATION AUTHORS AND AFFILIATIONS * Department of Ophthalmology, Osaka University Graduate School of Medicine, Osaka, Japan Shigeru Sato, Takeshi Morimoto, Takashi Fujikado & Kohji

Nishida * Department of Applied Visual Science, Osaka University Graduate School of Medicine, Osaka, Japan Takeshi Morimoto & Takashi Fujikado * Department of Medical Innovation, Osaka

University Hospital, Osaka, Japan Kikuko Hotta Authors * Shigeru Sato View author publications You can also search for this author inPubMed Google Scholar * Takeshi Morimoto View author

publications You can also search for this author inPubMed Google Scholar * Kikuko Hotta View author publications You can also search for this author inPubMed Google Scholar * Takashi

Fujikado View author publications You can also search for this author inPubMed Google Scholar * Kohji Nishida View author publications You can also search for this author inPubMed Google

Scholar CORRESPONDING AUTHOR Correspondence to Shigeru Sato. ETHICS DECLARATIONS CONFLICT OF INTEREST The authors declare that they have no conflict of interest. ADDITIONAL INFORMATION

PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. RIGHTS AND PERMISSIONS OPEN ACCESS This article is

licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give

appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in

this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative

Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a

copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Sato, S., Morimoto, T., Hotta, K. _et al._ A novel

compound heterozygous mutation in _TTC8_ identified in a Japanese patient. _Hum Genome Var_ 6, 14 (2019). https://doi.org/10.1038/s41439-019-0045-y Download citation * Received: 24 November

2018 * Revised: 24 December 2018 * Accepted: 10 January 2019 * Published: 12 March 2019 * DOI: https://doi.org/10.1038/s41439-019-0045-y SHARE THIS ARTICLE Anyone you share the following

link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature

SharedIt content-sharing initiative