Mutations and altered expression of SERPINF1 in patients with familial otosclerosis

Human Molecular Genetics, 2016, Vol. 25, No. 12 2393–2403 doi: 10.1093/hmg/ddw106 Advance Access Publication Date: 7 April 2016 Original Article ORIGINAL ARTICLE Mutations and altered expression of SERPINF1 in patients with familial otosclerosis 1 UCL Ear Institute, University College London, London WC1X 8EE, UK, 2Royal National Throat Nose and Ear Hospital, London WC1X 8EE, UK, 3Department of ENT Surgery, The Princess Margaret Hospital, Windsor SL4 3SJ, UK and 4Wellcome Trust Sanger Institute, Hinxton CB10 1SA, UK *To whom correspondence should be addressed. Tel: þ44 2076798935; Email: Abstract Otosclerosis is a relatively common heterogenous condition, characterized by abnormal bone remodelling in the otic capsule leading to fixation of the stapedial footplate and an associated conductive hearing loss. Although familial linkage and candidate gene association studies have been performed in recent years, little progress has been made in identifying diseasecausing genes. Here, we used whole-exome sequencing in four families exhibiting dominantly inherited otosclerosis to identify 23 candidate variants (reduced to 9 after segregation analysis) for further investigation in a secondary cohort of 84 familial cases. Multiple mutations were found in the SERPINF1 (Serpin Peptidase Inhibitor, Clade F) gene which encodes PEDF (pigment epithelium-derived factor), a potent inhibitor of angiogenesis and known regulator of bone density. Six rare heterozygous SERPINF1 variants were found in seven patients in our familial otosclerosis cohort; three are missense mutations predicted to be deleterious to protein function. The other three variants are all located in the 50 -untranslated region (UTR) of an alternative spliced transcript SERPINF1-012. RNA-seq analysis demonstrated that this is the major SERPINF1 transcript in human stapes bone. Analysis of stapes from two patients with the 50 -UTR mutations showed that they had reduced expression of SERPINF1-012. All three 50 -UTR mutations are predicted to occur within transcription factor binding sites and reporter gene assays confirmed that they affect gene expression levels. Furthermore, RT-qPCR analysis of stapes bone cDNA showed that SERPINF1-012 expression is reduced in otosclerosis patients with and without SERPINF1 mutations, suggesting that it may be a common pathogenic pathway in the disease. Introduction Otosclerosis is one of the most common causes of hearing impairment among young adults. It is characterized by abnormal bone homeostasis within the otic capsule (1). The remodelling process often involves the stapedio-vestibular joint, and can lead to fixation of the stapedial footplate and a conductive hearing loss that result in clinical otosclerosis. Histological otosclerosis is seen more frequently than clinical otosclerosis; however in these cases, the stapes is not fixed and the diagnosis can only be made post-mortem (2). The age of onset is variable, although hearing loss in clinical otosclerosis typically begins in the third decade and progressively becomes more severe. † Present address: Wolfson Centre for Age-Related Diseases, King’s College London, London SE1 1UL, UK. Received: February 24, 2016. Revised: March 24, 2016. Accepted: March 25, 2016 C The Author 2016. Published by Oxford University Press. V This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 2393 Joanna L. Ziff1, Michael Crompton1, Harry R.F. Powell2, Jeremy A. Lavy2, Christopher P. Aldren3, Karen P. Steel4,†, Shakeel R. Saeed1,2 and Sally J. Dawson1,* 2394 | Human Molecular Genetics, 2016, Vol. 25, No. 12 population but one that often occurs in multiple members of the same family consistent with a monogenic type of inheritance. Hence, a WES approach in these families may provide a powerful discovery tool to reveal the genetic causes and molecular pathways disrupted in otosclerosis. However, because of the frequency of the disease the causal variants are unlikely to be identified by WES alone as frequency filters will have to be set higher than in rare disorders. In this work, we used a combination of WES, RNAseq and functional analysis to identify disease-causing genes in familial otosclerosis (Fig. 1A). In one family, we detected a rare heterozygous missense variant, c.601G > A, in SERPINF1 (Serpin Peptidase Inhibitor, Clade F; Fig. 1B). Five additional rare variants were identified in SERPINF1 from a cohort of 84 familial cases of otosclerosis. We analysed the effect of these mutations and identified SERPINF1 as the first disease-causing gene in otosclerosis. Results WES in four families identifies candidate variants for otosclerosis We sequenced the exomes of 10 individuals from four families with familial otosclerosis, three of European ancestry and one of mixed European and Caribbean ancestry in which the otosclerosis has been inherited via the European ancestral line. Exome capture was performed with an Agilent SureSelect Human All Exon 50 Mb Kit and subject to massively parallel sequencing. An average of 16.5 Gb of sequence was generated per individual as paired-end 100 bp reads. Reads were mapped to the reference sequence (GRCh37_hs37d5) with 96.16% of the bases mapping at >10 coverage. The mean depth of coverage was 178.5-fold with an average of 89 656 variants Figure 1. Familial otosclerosis and the next-generation sequencing approach for discovering causal variants. (A) Schematic presenting the workflow implementing a combination of whole-exome sequencing, RNA-seq and functional analysis to identify disease-causing variants in familial otosclerosis. (B) Pedigree of Family B shows an autosomal dominant inheritance pattern, consistent with familial otosclerosis. A rare heterozygous missense variant, c.601G > A, was identified in SERPINF1. Open symbol, clinically unaffected; black symbol, confirmed otosclerosis; light grey symbol, other hearing loss; arrow, proband. Hearing loss is bilateral in 70–85% of cases and is usually asymmetrical, developing initially in one ear before progressing to the other (3). Patients choose to undergo surgery to replace all or part of the affected stapes with a prosthetic device which ameliorates the hearing loss to varying degrees. Alternatively, the condition can be managed with hearing-aids (4). The clinically relevant form of otosclerosis is relatively common in individuals of Indian and European extraction with a reported frequency of 0.3–0.4% in white Europeans; it is much rarer in African, Native American and other Asian populations (4–7). In most individuals, otosclerosis appears to be sporadic and is considered a complex condition with both environmental and genetic factors contributing (7–9). However, otosclerosis also occurs with a strong familial inherita (...truncated)