Mutation of the melastatin-related cation channel, TRPM3, underlies inherited cataract and glaucoma

Abstract

Inherited forms of cataract are a clinically important and genetically heterogeneous cause of visual impairment that usually present at an early age with or without systemic and/or other ocular abnormalities. Here we have identified a new locus for inherited cataract and high-tension glaucoma with variable anterior segment defects, and characterized an underlying mutation in the gene coding for transient receptor potential cation channel, subfamily M, member-3 (TRPM3, melastatin-2). Genome-wide linkage analysis mapped the ocular disease locus to the pericentric region of human chromosome 9. Whole exome and custom-target next-generation sequencing detected a heterozygous A-to-G transition in exon-3 of TRPM3 that co-segregated with disease. As a consequence of alternative splicing this missense mutation was predicted to result in the substitution of isoleucine-to-methionine at codon 65 (c.195A>G; p.I65 M) of TRPM3 transcript variant 9, and at codon 8 (c.24A>G; p.I8 M) of a novel TRPM3 transcript variant expressed in human lens. In both transcript variants the I-to-M substitution was predicted in silico to exert damaging effects on protein function. Furthermore, transient expression studies of a recombinant TRPM3-GFP reporter product predicted that the I-to-M substitution introduced an alternative translation start-site located 89 codons upstream from the native initiator methionine found in eight other TRPM3 transcript variants (1-8). Collectively, these studies have provided the first evidence that TRPM3 is associated with inherited ocular disease in humans, and further provide support for the important role of this cation channel in normal eye development.

Figure 1. Linkage analysis of autosomal dominant cataract and glaucoma co-segregating in a 5-generation family.

(A) Pedigree and haplotype analysis showing segregation of markers across chromosome 9cen, listed in descending order from the short-arm telomere (9p-tel). Affected males (filled squares) and females (filled circles) that were diagnosed with cataract and glaucoma are indicated with a + symbol. (B) Ocular phenotype of affected individual V:4 presenting with high-tension glaucoma and persistent pupillary membrane characterized by fibrous strands extending from the iris border across the pupil. (C) Partial ideogram of chromosome 9 showing cytogenetic location of the ocular disease locus. Note, cytogenetic band regions p11.1, q11 and much of q12 are gene-poor centromeric regions, flanked by gene-rich regions in bands p11.2–p13.3 on the short arm and q13–q21.31 on the long arm.

Figure 2. Mutation analysis of TRPM3.

(A) Sanger sequence trace of the wild-type allele showing translation of isoleucine (I) at codon 65 (ATA) of variant 1 (isoform k), and at codon 8 of a novel lens variant. (B) Sequence trace of the mutant allele showing the heterozygous A-to-G transition (denoted R by the International Union of Pure and Applied Chemistry [IUPAC] code) that is predicted to result in the missense substitution of methionine (ATG) for isoleucine at codon 65 (c.195A>G, p.I65 M) of isoform k, and at codon 8 of a lens abundant isoform (c.24A>G, p.I8 M). (C) Restriction fragment length analysis showing gain of a Fok I site (5′GGATG[9/13]) that co-segregated with affected individuals heterozygous for the A-to-G transition (175 bp).

Figure 3. Schematic showing the gene structure of TRPM3.

(A) Exon organization, splice variants and protein domains. Exons are indicated by numbered boxes (1–28), and codon numbers are shown below each coding exon. Translation start and stop sites are denoted by ATG and asterisks, respectively. Transcript variants are numbered (1–9), and corresponding protein isoforms are indicated by letters (a–h, k). Exons subject to alternative splicing (1, 2, 8, 15 and 17) are indicated by plus and minus symbols below each exon. (−/−) indicates that the exon is absent from both the transcript variant and protein isoform. (+/−) indicates that the exon is present in the transcript variant but is not translated. Exon 2, exon 8 and exon 15 are skipped in transcript variant 9 (isoform k). In transcript variants 1–8 (isoforms a–h) exon 2, exon 3 and all of exon 4 except the last 3 bases (ATG) are non-coding. EST denotes an expressed sequence tag (BM712132) that replaces exon 1 of variant 9, and exon 2 of variants 1–8, joining directly to exon 3 in a novel lens abundant transcript variant (KF987075). Arrows indicate the orientation and exon location of PCR primers used to amplify and sequence the 5′-ends of TRPM3 transcripts. The micro-RNA gene, MIR204, is located in intron 8. The predicted I-to-M substitution (red) identified in the family studied here is located in a putative calmodulin-binding (CaM) motif near the N-terminus of isoform k and the novel lens isoform. The approximate locations of conserved protein domains are indicated: (pfam00520) ion-transport domain; (TRP1–2) transient receptor potential box 1 and 2; (C–C) coiled-coil domain, and (ICF) amino-acid sequence indispensible for channel function . Note that variants 7 and 8 encode short isoforms h and c, respectively, lacking the conserved domains above. These short cytoplasmic isoforms may play a role in regulation of the full-length transmembrane isoforms . (B) Amino acid sequence alignment of the N-terminal calmodulin-binding motif of human TRPM3 isoform-k, a novel human lens isoform, and homologs from other species showing conservation of isoleucine at the site of the predicted methionine substitution (red). Divergent amino-acid residues are shaded grey.

Figure 4. Alternative splicing of TRPM3 transcripts in the human lens.

(A) RT-PCR showing presence of transcripts for variant 9 (exon 1–5, 411 bp), variants 1–8 (exon 2–5, 414 bp) and a novel lens variant, KF987075 (EST - exon 5, 403 bp). (B) Quantitative RT-PCR showing relative copy numbers of TRPM3 variant 9 (2.06E+01, SD 9.46E+00), variants 1–8 combined (2.76E+05, SD 2.15E+04), and lens variant KF987075 (3.17E+04, SD 8.07E+03) standardized against RPL19. TRPM3 primers used for amplification were as follows; RT1F - RT5R for variant 9, RT2F - RT5R for variants 1–8, and ESTF - RT5R for lens variant KF987075 (Table S5).

Figure 5. Transient expression of a TRPM3-GFP fusion product in HEK293T cells.

(A) N-terminal amino-acid sequence of TRPM3 isoform k (NP_001007472), and a lens abundant isoform (KF987075). The sequence coding for amino-acids 60-153 (exon 3) of isoform k (codons 3–96 of the lens isoform) that was inserted into the GFP fusion vector is shaded grey. The p.I65M substitution in isoform k (p.I8M in the lens isoform) is shown in red. Translation initiator methionine residues are shown in bold and underlined. The translation start-site for isoform k is located 57 codons upstream of that for the lens isoform, and 153 codons upstream of that for isoforms 1–8. Italics denote the 17 amino-acid sequence linking the TRPM3 sequence in-frame to GFP (green box). The GFP translation start-site is located in-frame 106 codons downstream from the I-to-M substitution in isoform k and the lens isoform. (:) denotes identical amino-acids, (∧) indicates intron boundaries between exons 1, 3 and 4 (exon 2 is skipped). The CaM binding domain is underlined. (B) Immunoblot analysis of transfected HEK293T cell lysates showing presence of the predicted TRPM3-GFP fusion product (∼39 kDa) that is ∼12 kDa larger than native GFP (27 kDa). (*) indicates non-specific cross-reaction with the HEK293T cell lysate.