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A missense mutation in TTC8/BBS8 affecting mRNA splicing in patients with non-syndromic retinitis pigmentosa

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Abstract

Splicing disruption is one type of mutation mechanism for disease-predisposing alleles. To date, less than 30 mutations in TTC8/BBS8 have been reported; however, mutations affecting the splice site are rare. Generally missense mutations are assumed to alter protein function; however, reports have shown that mutations in protein coding exons can disrupt splicing by altering exonic splicing silencer or enhancer motifs. Hence, a missense mutation c.1347G > C (p.Q449H) involving final base of the exon 13 in the TTC8, previously identified by us to be linked with non-syndromic autosomal recessive retinitis pigmentosa (arRP), in an Indian family, that might deleteriously affect splicing has been functionally characterized. RNA was isolated, cDNA prepared and amplified using region-specific primers. PCR products were purified and sequenced bi-directionally by Sanger sequencing. Effect of mutation (c.1347G > C) on mRNA splicing has been predicted using bioinformatics tools. We reported that missense mutation (c.1347G > C) at the last base of exon 13 of TTC8 disrupted the canonical donor splice-site resulting in aberrant RNA splicing. A cryptic donor splice-site got activated 77 bases downstream of the authentic splice donor site in intron 13, resulting in the retention of 77 bases of intron 13, and a frameshift leading to pre-mature termination codon in exon 14 at codon 486. Further, duplication of exon 15 and fusion of its duplicated copy occurred with exon 13. The binding site for SC35 protein, normally involved in splicing, also got disrupted (as predicted by SpliceAid2 software), hence, leading to alternative splicing. Our findings strongly suggest that a missense mutation c.1347G > C in TTC8 disrupted the splice donor site causing retention of 77 bases of intron 13, resulting in a frameshift and subsequently introduced a pre-mature termination codon into exon 14, hence creating an altered mRNA transcript. These findings emphasize the significance of examining missense mutations especially in TTC8, to determine their pathogenic role through alternative splicing. Present findings also reiterate the notion that mutations in the TTC8/BBS8 cause phenotypic heterogeneity and does not always follow Mendelian genetics in this ciliopathy.

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Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

The authors are grateful to all the family members for their participation in this study and their kind cooperation. Research grants under the Department of Science and Technology-Promotion of University Research and Scientific Excellence (DST-PURSE) scheme from DST and University Grants Commission-University with Potential for Excellence (UGC-UPE) scheme from UGC, Government of India, to the Guru Nanak Dev University (GNDU) are also duly acknowledged. This paper is dedicated to the fond memories of our respected teacher Dr. Daljit Singh Ji.

Funding

This work was supported in part by a grant sanctioned from the Department of Biotechnology, Government of India, BT/IN/German/13/VK/2010 IND 10/036 to VV under the framework of Indo-German bilateral cooperation for research. The authors are also thankful to the DST-PURSE scheme from DST and UGC-UPE scheme from UGC, Government of India, for providing research grants to GNDU. The authors are also thankful to the Indian Council of Medical Research for providing a Senior Research Fellowship to Dr. Shiwali Goyal and for providing a research grant (No.3/1/2(4)/OPH/2016/NCD-II) to Dr. Vanita.

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  1. Department of Human Genetics, Guru Nanak Dev University, Amritsar, Punjab, 143005, India

    Shiwali Goyal & Vanita Vanita

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  1. Shiwali Goyal
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  2. Vanita Vanita
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VV: conceptualization; SG: data curation; SG: formal analysis; VV: funding acquisition; VV: investigation; VV: methodology; VV: project administration; VV: resources; VV: software; VV: supervision; SG: validation; SG: visualization; SG: roles/writing—original draft; VV: writing—review & editing.

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Correspondence to Vanita Vanita.

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Communicated by Shuhua Xu.

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Goyal, S., Vanita, V. A missense mutation in TTC8/BBS8 affecting mRNA splicing in patients with non-syndromic retinitis pigmentosa. Mol Genet Genomics 297, 1439–1449 (2022). https://doi.org/10.1007/s00438-022-01933-y

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