Mutations and altered expression of SERPINF1 in patients with familial otosclerosis

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 disease-causing 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 5'-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 5'-UTR mutations showed that they had reduced expression of SERPINF1-012 All three 5'-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.

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.

Figure 2.

SERPINF1 transcript expression in control and otosclerotic stapes. (A) SERPINF1-001 and SERPINF1-012 transcripts (not to scale) with the identified variants indicated by dashed lines. Open box, 5′-UTR; grey box, coding sequence. The positions of the Taqman® gene expression assays used in RT-qPCR are also indicated. (B) The average read count of SERPINF1 exons is shown in eight otosclerotic and four control stapes subjected to RNA-seq analysis, the read count of the stapes from the proband of family B with a c.601G > A mutation is also shown separately. Data indicate a significant difference in expression between otosclerotic and control stapes of exons 5–8 but not exons 1–4. (*P < 0.05. Error bars indicate standard error of mean. A student’s two-tailed t-test was performed.) (C, D) RT-qPCR data show the average relative expression levels of (C) upstream (exons 3–4) and (D) downstream (exons 6–7) SERPINF1 exons in stapes from: control individuals (n = 5), otosclerosis patients (n = 75) and stapes from one individual with a SERPINF1 mutation (c.441G > C). Expression levels were calculated relative to 18s RNA endogenous control and to levels in controls (calibrator sample). Levels of SERPINF1 are significantly reduced in the otosclerotic stapes and in c.441G > C for both assays, a greater reduction being detected in downstream exons. Error bars indicate 95% confidence intervals (*P < 0.05; ***P < 0.0005; ****P < 0.00005 in a Student’s two-tailed t-test performed on ΔΔCt values compared with control sample).

Figure 3.

Mutations in the 5′-UTR influence translational efficiency of the SERPINF1-012 transcript. (A) Fragments containing the wild-type or mutant 5′-UTRs of the SERPINF1-012 transcript were cloned into the pGL4.10 expression vector and transfected into MG-63 cells. (B) For the luciferase assay wild-type values were set to 1, to which mutant values were normalized. These results are the average of 12 repeats in four independent experiments. The c.440-40_440-38delTCG (3bpDel) and c.441G > C mutations show a significant reduction in translational activity, whereas the c.601G > A mutation displays a significant increase. Error bars indicate standard error of mean (*P < 0.05; **P < 0.01 in a Student’s two-tailed t-test versus control data).

Figure 4

Proposed role of SERPINF1-012 expression in otosclerosis pathophysiology. The alternatively spliced SERPINF1-012 transcript (shown in red) is the major transcript found in healthy stapes bone, whereas SERPINF1-001 (shown in blue) has a lower level of expression. Reduced expression of SERPINF1-012 expression by mutation in familial cases or by other means in sporadic otosclerosis leads to localized stapes bone dysregulation and otosclerosis. Loss of SERPINF1-001 expression due to frameshift and nonsense mutations in osteogenesis imperfecta causes systemic bone dysregulation.