Mutations in MTMR13, a new pseudophosphatase homologue of MTMR2 and Sbf1, in two families with an autosomal recessive demyelinating form of Charcot-Marie-Tooth disease associated with early-onset glaucoma

Abstract

Charcot-Marie-Tooth disease (CMT) with autosomal recessive (AR) inheritance is a heterogeneous group of inherited motor and sensory neuropathies. In some families from Japan and Brazil, a demyelinating CMT, mainly characterized by the presence of myelin outfoldings on nerve biopsies, cosegregated as an autosomal recessive trait with early-onset glaucoma. We identified two such large consanguineous families from Tunisia and Morocco with ages at onset ranging from 2 to 15 years. We mapped this syndrome to chromosome 11p15, in a 4.6-cM region overlapping the locus for an isolated demyelinating ARCMT (CMT4B2). In these two families, we identified two different nonsense mutations in the myotubularin-related 13 gene, MTMR13. The MTMR protein family includes proteins with a phosphoinositide phosphatase activity, as well as proteins in which key catalytic residues are missing and that are thus called "pseudophosphatases." MTM1, the first identified member of this family, and MTMR2 are responsible for X-linked myotubular myopathy and Charcot-Marie-Tooth disease type 4B1, an isolated peripheral neuropathy with myelin outfoldings, respectively. Both encode active phosphatases. It is striking to note that mutations in MTMR13 also cause peripheral neuropathy with myelin outfoldings, although it belongs to a pseudophosphatase subgroup, since its closest homologue is MTMR5/Sbf1. This is the first human disease caused by mutation in a pseudophosphatase, emphasizing the important function of these putatively inactive enzymes. MTMR13 may be important for the development of both the peripheral nerves and the trabeculum meshwork, which permits the outflow of the aqueous humor. Both of these tissues have the same embryonic origin.

Figure 1

Haplotype reconstruction of 14 markers in the Tunisian (A) and Moroccan (B) families with demyelinating ARCMT and glaucoma. Deduced haplotypes for individuals for whom DNA was not available are bracketed. Microsatellite markers are ordered, from telomere (top) to centromere (bottom), according to the Généthon genetic map. Boxes indicate the haplotype segregating with the disease, and shaded areas indicate the common region of homozygosity. Recombination events are indicated by arrows.

Figure 2

Physical map of 11p15 and genomic structure of MTMR13. A, Physical map of the critical region, including the position of the linkage markers (top) and STS (bottom) tested. Physical distances (Ensembl Genome Browser; GenBank) are indicated in megabases. The marker closest to the gene is in bold. B, Location of the two ESTs, LOC19611/LOC283105 and KIAA1766, from which the coding sequence of MTMR13 (top) and intron-exon structure of MTMR13 (bottom) was constructed. This region was covered by four different genomic clones from contig NT_028309.7. Clone AC080023.6 contains only the first exon of MTMR13. AC100763.2 includes exons 2 and 3 and partially overlaps with clone AC011092.4, which extended from exons 3 to 17. The remaining portion of the gene is found in clone AC026250.16.

Figure 3

RT-PCR analysis of MTMR13 expression in several human tissues. A, RT-PCR performed using primers amplifying a fragment corresponding to the first 1,250 bp of the MTMR13 ORF, starting from the ATG. A similar expression pattern was demonstrated for the remaining four overlapping fragments into which the ORF was divided (not shown). B, G3PDH was used as positive control for the quality of RNA and cDNA synthesis. −MMLV is a control reaction in which the MMLV reverse transcriptase was omitted. RT corresponds to the negative control of the PCR performed by omitting the cDNA thereafter.

Figure 4

Chromatograms of the mutation site sequence (arrow) are presented for both families. A, Family TUN-RE: a healthy homozygous sib (V11), a heterozygous sib (V17), and an affected sib (V6); N:C/T (forward strand sequence). B, Family RBT-HAD: a heterozygous sib (IV5) and two affected sibs (IV10 and IV14); N:A/G (reverse strand sequence).

Figure 5

Phylogenetic relationship of MTMR13 within the myotubularin family. The radial distance tree of the human, D. melanogaster, and S. cerevisiae protein is shown, illustrating the six subgroups, each defined by one protein in D. melanogaster. MTMR13 belongs to the same subgroup as MTMR5 (or Sbf1), its closest homologue (60% identity). MTMR2 belongs to the same subgroup as MTM1 and MTMR1 (70% identity with the latter). MTMR11 was also called “3-PAP” (Nandurkar and Huysmans 2002). While submitting this article, another MTMR was published that is not included in this analysis (CRAα/β) (Wishart and Dixon 2002a). The scale represents the percentage of divergence. Human genetic disorders associated with mutations in myotubularin protein are indicated: CMT4B1 (CMT type 4B1), CMT4B2 (CMT type 4B2 with early-onset glaucoma), and XLMTM (X-linked myotubular myopathy). The tree was generated using sequences encompassing the PTPc/DSPc homology domain and the SET interacting domain. Hs, Homo sapiens; Dm, D. melanogaster; Sc, S. cerevisiae.

Figure 6

Domain structure of MTMR13 and homologous proteins. Top, Scaled representations of Hs MTMR2, Hs MTMR5 (Sbf1), and the three orthologous MTMR13 proteins in human (Hs MTMR13), D. melanogaster (Dm CG6939), and yeast (Sc MTM). The main protein domains are depicted. The DENN (differentially expressed in neoplastic versus normal cells) domain is formed by three subdomains that are always present together: uDENN, DENN, and dDENN (Levivier et al. 2001), all depicted as “DENN” here. In Dm CG6939, the first DENN subdomain is missing, suggesting incomplete prediction of the N-terminus of the protein. The GRAM domain is found in glucosyltransferase, rab-like GTPase activators and myotubularins and has no known function. The PTPc/DSPc homology domain (catalytic domain of tyrosine and dual-specificity phosphatases) is found in all members. However, the consensus sequence of the catalytic pocket, present in Hs MTMR2 and Sc MTM, is degenerated in Hs MTMR13. SET interacting domain (SID) was defined on the basis of interaction between Hs MTMR5 and proteins with SET (suvar 3–9, enhancer-of-zeste, trithorax) domains and is present in all members. The PH domain (pleckstrin homology) is a phosphoinositide-binding domain. Additional protein domains include a coiled-coil domain (amino acids 593–627) and a PDZ-binding domain (amino acids 640–643) at the C-terminus of Hs MTMR2, a protein kinase C conserved region in Dm CG6939 (C1 domain/diacyglycerol and phrobol ester binding; amino acids 1546–1593), and the RID (Rac1-induced recruitment domain), the boundaries of which have not been defined (around amino acid 283 in Hs MTMR2), and which is responsible for the plasma membrane recruitment of MTMR2 and other active and inactive homologues upon Rac 1 activation (Laporte et al. 2002). Bottom, Sequence alignment of the PTPc/DSPc homology domain in Hs MTMR13 and homologous proteins. Names of the proteins are on the left, and the sequence intervals are on the right. Dm CG9115 is the D. melanogaster orthologue of Hs MTMR2 and Hs MTM1. Shaded amino acids are conserved in all the depicted proteins. The underlined sequence HCSDGWDRT is conserved 100% in all active myotubularins from yeast to human and contains the compulsory consensus sequence CysX₅Arg for the phosphatase activity. The catalytic cysteine and arginine are indicated by stars.