Mutations in a gene encoding a novel SH3/TPR domain protein cause autosomal recessive Charcot-Marie-Tooth type 4C neuropathy

Abstract

Charcot-Marie-Tooth disease type 4C (CMT4C) is a childhood-onset demyelinating form of hereditary motor and sensory neuropathy associated with an early-onset scoliosis and a distinct Schwann cell pathology. CMT4C is inherited as an autosomal recessive trait and has been mapped to a 13-cM linkage interval on chromosome 5q23-q33. By homozygosity mapping and allele-sharing analysis, we refined the CMT4C locus to a suggestive critical region of 1.7 Mb. We subsequently identified mutations in an uncharacterized transcript, KIAA1985, in 12 families with autosomal recessive neuropathy. We observed eight distinct protein-truncating mutations and three nonconservative missense mutations affecting amino acids conserved through evolution. In all families, we identified a mutation on each disease allele, either in the homozygous or in the compound heterozygous state. The CMT4C gene is strongly expressed in neural tissues, including peripheral nerve tissue. The translated protein defines a new protein family of unknown function with putative orthologues in vertebrates. Comparative sequence alignments indicate that members of this protein family contain multiple SH3 and TPR domains that are likely involved in the formation of protein complexes.

Figure 1

Homozygosity mapping in CMT4C-affected families (CMT-133 and M149) with chromosome 5q32 markers. A subset of markers used in this study is given in cen-qter orientation. The physical map positions are according to the UCSC November 2002 freeze. The informativity of each marker is shown as a calculated heterozygosity rate for microsatellite markers (“D5S” locus numbers and plain numerals) and as a minor allele frequency for SNPs (“rs” numbers). Values determined in the Turkish population are marked by an asterisk (*). Haplotypes are shown for affected and healthy offspring. Paternal haplotypes are shown at left; maternal haplotypes are at right. Informative alleles in the affected individuals are underlined. Regions of homozygosity and shared haplotypes are boxed. The recombination events suggested from regions of homozygosity in patient M149.V.1 support a 5.6-Mb critical interval flanked by markers D5S1360 and D5S636 (single arrowheads). Further refinement was inferred through identical haplotypes shared by patients CMT-133.IV.2 and M149.V.1 between markers 91948 and rs2276982 (double arrowheads). This narrowed the critical genetic region to a suggestive interval of 1.7 Mb.

Figure 2

Transcript map of the CMT4C region on 5q32, genomic organization of the CMT4C gene, and alternative splicing of its transcripts. A, Partial physical and transcript map of the 1.7-Mb CMT4C linkage region delimited by the centromeric marker 91948 and telomeric marker rs2276982. Known genes (shown in bold text) and predicted genes (shown in plain text) are indicated by arrows in the direction of transcription. B, Genomic structure of the CMT4C gene (KIAA1985). The gene covers 62 kb of genomic sequence and consists of ⩾18 variably spliced exons. Exons are indicated by vertical hatches and are numbered. Empty bars represent alternatively spliced sequences. The sizes of introns are shown relative to each other. C, Alternative splice products of KIAA1985. The transcript encoding the putative full-length protein is given above the products. Coding regions of exons are drawn to scale. Use of exon 6s (instead of 6), presence of exon 8A, and retention of intron 10 predict shorter translation products. An asterisk (*) indicates predicted translation-initiation site; a double asterisk (**) indicates predicted translation-termination signal.

Figure 3

KIAA1985 mutations in four families with CMT4C. Arrowheads in the electropherograms indicate the disease-causing mutations. A, Homozygous c.26delG mutation in family M2045, resulting in the R9fsX13 frameshift mutation. The wild-type sequence is shown in the lower chromatogram. B, Compound heterozygote c.2829T→G and c.2860C→T base substitutions in family M983, resulting in Y943X and R954X nonsense mutations. C, Compound heterozygote c.1972C→T and c.2860C→T mutations in family H1351, resulting in the R658C missense and the R954X nonsense mutations. D, Homozygous acceptor splice site IVS5-2A→G mutation in family CMT-189. The wild-type sequence is shown in the lower chromatogram.

Figure 4

Expression analysis of KIAA1985. A, Human adult multiple-tissue northern blot. Upper panel, Membrane probed with a radioactively labeled cDNA fragment corresponding to KIAA1985 exons 15–17. Two strong signals at ∼7.5 kb and ∼4.5 kb are seen in brain and spinal cord, whereas striated muscle (tongue) gives only faint signals. All other tissues are negative for KIAA1985 expression by northern blotting. Comparable results were obtained when using a 5′ probe (exons 1–6). Lower panel, Membrane hybridized with radioactively labeled β-actin cDNA fragment for an internal control. B, Expression analysis by RT-PCR. Upper panel, Amplification of a 535-bp KIAA1985 fragment corresponding to exons 15–17. Lower panel, β2-microglobulin (β-2M)–specific primers were used in control reactions. KIAA1985 expression levels in sciatic nerve and spinal cord seem similar. Weak signals are obtained on skeletal muscle mRNA. Genomic DNA was used as a control to test for specific amplification from cDNA targets. (MW = DNA size standard) C, Expression of different KIAA1985 splice variants in human brain, spinal cord, and sciatic nerve. RT-PCR with primers positioned in exons 6 and 11 yielded a 694-bp product from transcripts encoding the longest continuous ORF (arrowheads). The additional bands arise from alternative usage of exons 6s or 8A or retention of intron 10, which would be predicted to result in truncated forms of the KIAA1985 protein (also see fig. 2). The intact mRNA is the dominant transcript in sciatic nerve, whereas spinal cord and brain predominantly express alternative splice products.

Figure 5

Protein prediction and sequence conservation among the KIAA1985 protein family. A, Full-length protein of 1,288 amino acids, showing domains as predicted by SMART (blue = SH3 domains; orange = TPR motifs). The sites of mutations detected in this study are indicated. B, Multiple protein sequence alignment generated with the program ClustalW, using translation from genome assemblies and expressed sequences. The amino acid numbering is according to the human KIAA1985 protein. Sequence comparison shows that the three KIAA1985 missense mutations (R529Q, E657K, and R658C) observed in patients with CMT4C affect amino acids that are identical in KIAA1985 and FLJ20356 and their putative orthologues. (Hs = Homo sapiens; Mm = Mus musculus; Rn = Rattus norvegicus; Gg = Gallus gallus; Dr = Danio rerio; Fr = Fugu rubripes.) Upper panel, Sequence of the first TPR motif. Residues generating the TPR consensus sequence are shown on gray background. Sequence conservation outside these consensus residues (shown on black background) is believed to correlate with functional specialization of the TPR motif (Blatch and Lässle 1999). Lower panel, Residues from one of the interdomain regions. Conserved amino acids are given on a black background. C, Average distance tree of KIAA1985 (residues 1000–1100) aligned with homologous sequences. The average distance tree, with percentage identities, was generated by the program Jalview on the basis of a multiple-sequence alignment generated with the program ClustalW. (Ss = Sus scrofa; Bt = Bos taurus; Xl = Xenopus laevis.) The following GenBank database entries were used for generating the multiple-sequence alignments: KIAA1985 [accession number AY341075], Bt.KIAA1985 [accession numbers BI680206, BF652287, BU239452, AW656867, and BI976766], Ss.KIAA1985 [accession numbers BF442400, BI186309, BI183894, and BI345340], Mm.KIAA1985/D430044G18Rik [accession number AK052534], Rn.KIAA1985 [accession numbers BF567582 and XM_225887], Gg.KIAA1985 [accession numbers BU239452 and BU355692], FLJ20356 [accession number AK000363], Mm.FLJ20356 [accession numbers AK028482 and BC024909], Rn.FLJ20356 [accession numbers XM_223527 and XM_223528], Gg.FLJ20356 [accession numbers BU232586, BU471861, and BU474979], Xl.FLJ20356 [accession number BJ062830]). D. rerio and F. rubripes orthologues were retrieved by translated BLAST searches on unfinished genomes, with “human protein sequence” as a query (Ensembl).

Figure 6

Electron micrographs. A, Patient AC70.II.1. A thinly remyelinated axon is surrounded by several layers of basal membranes (arrow) that occasionally contain remnants of Schwann cell cytoplasm (9,000×). B, Patient CMT-133.IV.2. Thin Schwann cell processes (arrow) connect isolated unmyelinated axons and show supernumerary extensions (10,000×).

Figure A

KIAA1985 cDNA sequence encoding the longest potential reading frame (GenBank accession number AY341075). Bases appearing in bold text and italics establish the exon-exon boundaries. The translation start site and the stop codon are in bold text and underlined.

Figure A

KIAA1985 cDNA sequence encoding the longest potential reading frame (GenBank accession number AY341075). Bases appearing in bold text and italics establish the exon-exon boundaries. The translation start site and the stop codon are in bold text and underlined.

Figure B

Amino acid sequence of KIAA1985 encoded by the longest ORF. Blue denotes SH3 domains; orange denotes TPR motifs.