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Mutation of the XNP/ATR-X Gene in a Family with Severe Mental Retardation, Spastic Paraplegia and Skewed Pattern of X Inactivation: Demonstration that the Mutation is Involved in the Inactivation Bias

1999; Elsevier BV; Volume: 65; Issue: 2 Linguagem: Inglês

10.1086/302499

1537-6605

Anne-Marie Lossi, José M. Millán, Laurent Villard, Carmen Orellana, Carlos Cardoso, F. Prieto, M. Fontés, Francisco Martı́nez,

Sexual Differentiation and Disorders

To the Editor: A family in which severe mental retardation (MR) is segregating with spastic paraplegia (SP) has recently been reported (Martinez et al. Martinez et al., 1998Martinez F Tomas M Millan JM Fernandez A Palau F Prieto F Genetic localisation of mental retardation with spastic diplegia to the pericentromeric region of the X chromosome: X inactivation in female carriers.J Med Genet. 1998; 35: 284-287Crossref PubMed Scopus (16) Google Scholar). The extended pedigree of this family is presented in figure 1. Obligate-carrier females have a totally skewed pattern of X inactivation, detected by amplification of the (CAG)n microsatellite repeat in the androgen-receptor gene and previous digestion of genomic DNA with the methylation-sensitive restriction endonuclease HpaII, as described elsewhere (Martinez et al. Martinez et al., 1998Martinez F Tomas M Millan JM Fernandez A Palau F Prieto F Genetic localisation of mental retardation with spastic diplegia to the pericentromeric region of the X chromosome: X inactivation in female carriers.J Med Genet. 1998; 35: 284-287Crossref PubMed Scopus (16) Google Scholar). Genetic analysis in the family has revealed linkage of the morbid locus to the proximal long arm of the X chromosome, with a maximum LOD score in Xq13.3. Three genes involved in X-linked MR (XLMR) have already been reported in this genomic region. One, encoding oligophrenin-1, is involved in nonsyndromic XLMR (Billuart et al. Billuart et al., 1998Billuart P Bienvenu T Ronce N des Portes V Vinet MC Zemni R Roest-Crollius H et al.Oligophrenin-1 encodes a rhoGAP protein involved in X-linked mental retardation.Nature. 1998; 392: 923-926Crossref PubMed Scopus (367) Google Scholar) and thus does not seem to be a good candidate. The other two are involved in two syndromic XLMR conditions: Menkes syndrome (MIM 309400) and alpha-thalassemia with mental retardation (ATR-X [MIM 301040]) syndrome. The latter is an XLMR condition that associates severe MR, mild alpha-thalassemia, typical facial dysmorphy and a skewed pattern of X inactivation in carrier females (Gibbons et al. Gibbons et al., 1995aGibbons RJ Brueton L Buckle VJ Burn J Clayton-Smith J Davison BCC Gardner RJM et al.Clinical and hematologic aspects of the X-linked alpha-thalassemia/mental retardation syndrome (ATR-X).Am J Med Genet. 1995; 55: 288-299Crossref PubMed Scopus (97) Google Scholara). The clinical characteristics of the reported MR+SP family are close to this description, and thus the presence of an allelic mutation at the ATR-X locus could be hypothesized. In addition to the already reported clinical features in the MR+SP family, hematologic analysis revealed that 3% of the patients' erythrocytes showed HbH inclusions after cresyl-brilliant staining, which reinforced the possibility that a mutation in the XNP/ATR-X gene is present in this family. However, ATR-X syndrome has always been reported to be associated with neonatal hypotonia, which can be severe (Gibbons et al. Gibbons et al., 1995aGibbons RJ Brueton L Buckle VJ Burn J Clayton-Smith J Davison BCC Gardner RJM et al.Clinical and hematologic aspects of the X-linked alpha-thalassemia/mental retardation syndrome (ATR-X).Am J Med Genet. 1995; 55: 288-299Crossref PubMed Scopus (97) Google Scholara). In the case of the family that we studied, affected patients were affected, from birth, with the opposite sign, hypertonia. It was thus interesting to look for a possible new mutation that might lead to a variant of the disorder. The gene involved in the ATR-X syndrome was isolated in Gecz et al., 1994Gecz J Pollard H Consalez G Villard L Stayton C Millasseau P Khrestchatisky M et al.Cloning and expression of the murine homologue of a putative human X-linked nuclear protein gene closely linked to PGK1 (Xq13.3).Hum Mol Genet. 1994; 3: 39-44Crossref PubMed Scopus (40) Google Scholar (by Gecz et al.) and named “XNP.” It has since been shown to be mutated in 13 patients with alpha-thalassemia MR (ATR-X) syndrome (Gibbons et al. Gibbons et al., 1995bGibbons RJ Picketts DJ Villard L Higgs DR Mutations in a putative global transcriptional regulator cause X-linked mental retardation with alpha-thalassemia (ATR-X syndrome).Cell. 1995; 80: 837-845Abstract Full Text PDF PubMed Scopus (471) Google Scholarb). Moreover, mutations in this gene have also been identified in families affected with Juberg-Marsidi syndrome (MIM 309590) and with MR syndromes without alpha-thalassemia (Villard et al. Villard et al., 1996aVillard L Lacombe D Fontès M A point mutation in the XNP gene, associated with an ATR-X phenotype without alpha-thalessemia.Eur J Hum Genet. 1996; 4: 316-320PubMed Google Scholara, Villard et al., 1996bVillard L Saugier-Veber P Gecz J Mattéi JF Munnich A Lyonnet S Fontés M XNP mutation in a large family with Juberg-Marsidi syndrome.Nat Genet. 1996; 12: 359-360Crossref PubMed Scopus (73) Google Scholarb, Villard et al., 1996cVillard L Toutain A Lossi AM Gecz J Houdayer C Moraine C Fontés M Splicing mutation in the ATR-X gene can lead to a mental retardation phenotype without alpha-thalassemia.Am J Hum Genet. 1996; 58: 499-505PubMed Google Scholarc). More recently, a new mutation has been found, in Carpenter-Waziri syndrome (Abidi et al. Abidi et al., in pressAbidi F, Carpenter NJ, Villard L, Curtis M, Fontès M, Schwartz CE. The Carpenter-Waziri syndrome results from a mutation in XNP. Am J Med Genet (in press)Google Scholar). The gene is large (probably extending over 300 kb) and consists of 35 exons. It encodes a putative zinc-finger helicase (Villard et al. Villard et al., 1997Villard L Lossi AM Cardoso C Proud V Chiaroni P Colleaux L Schwartz C et al.Determination of the genomic structure of the XNP/ATR-X gene, encoding a potential zinc-finger helicase.Genomics. 1997; 43: 149-155Crossref PubMed Scopus (56) Google Scholar) that is probably involved, by remodeling the structure of the chromatin, in the control of gene expression. This assumption is supported by the finding of an interaction between the XNP protein and the Ezh2 protein (Cardoso et al. Cardoso et al., 1998Cardoso C Timsit S Villard L Khrestchatisky M Fontès M Colleaux L Specific interaction between the XNP/ATR-X gene product and the set domain of the human EZH2 protein.Hum Mol Genet. 1998; 7: 679-684Crossref PubMed Scopus (127) Google Scholar), the human equivalent of Drosophila enhancer of zeste, a chromatinian protein of the Polycomb group. To date, 34 different mutations in 50 pedigrees have been described in the XNP/ATR-X gene, which are located either in the region coding for the three zinc-finger domains of the protein (exons 7 and 8 and the beginning of exon 9) or in the helicase domain, which extends over 3 kb at the COOH terminus of the putative protein. We thus decided to search for mutations in this gene in a patient from the MR+SP family. This was performed by systematic sequencing of the gene. We found one mutation in the gene, changing an arginine from the conserved helicase domain III into a lysine, R1742K (fig. 2). This mutation perfectly segregates with the disorder in the family (fig. 3). Moreover, the mutation was absent from 100 unrelated chromosomes from the same geographical area as that of the family we studied. We performed reverse-transcription PCR (RT-PCR) on RNA from lymphocytes of the patients, to check for any putative effect of the mutation on the mRNA splicing, which has been previously reported to occur in this gene (Villard et al. Villard et al., 1996bVillard L Saugier-Veber P Gecz J Mattéi JF Munnich A Lyonnet S Fontés M XNP mutation in a large family with Juberg-Marsidi syndrome.Nat Genet. 1996; 12: 359-360Crossref PubMed Scopus (73) Google Scholarb) but we failed to find any abnormal-size product (data not shown).Figure 3Detection of 5459G→A mutation in the affected branch of the family. A PCR product obtained by use of two oligonucleotides flanking the mutation is digested with BseRI and electrophoresed. The presence of the mutation destroys an internal BseRI site in the amplification product. Affected males have a single undigested amplified allele (one band); carrier-female DNA harboring both a mutated and a nonmutated allele yields both a digested and a nondigested PCR product (three bands); and noncarrier females have two digested alleles (two bands).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Since this amino acid change (arginine to lysine) is generally considered to be conservative, we used a local structure–prediction program (PEPTIDESTRUCTURE, from the GCG package, which mainly predicts α helices and β sheets). This program has predicted a change in the local structure of the protein (fig. 4), potentially arising because of steric problems. Arginine 1742 is an amino acid that cannot be strictly considered to be evolutionarily conserved, but it is present at the same position in several members of the helicase family of proteins (e.g., in the yeast Rad54 protein). Very interestingly, in exploring another branch of the family for the presence of the mutation, we found two females (individuals III-1 and IV-2 in fig. 1) who share the disease-associated haplotype, spanning both the XNP gene and the X-inactivation center, but who lack the R1742K mutation. We thus can deduce that the mutation arose de novo in the germline of one member of the founding couple (I-1 or I-2 in fig. 1). Furthermore, the fact that females in the nonmutated branch of the family do not exhibit a skewed pattern of X-chromosome inactivation demonstrates that the skewing in carrier females is directly linked to the presence of the mutation in this gene. This is the first demonstration of a direct link between a mutation in this gene and the skewed pattern of X inactivation observed in all carrier females, and it further supports the pathogenicity of the R1742K mutation. We can draw, from these data, two main conclusions. First, the analysis of this family extends the phenotype associated with XNP/ATR-X mutations. All patients reported so far were hypotonic (at least in the neonatal period), which made this trait a major criterion of inclusion. In this family, patients are hypertonic and also present spastic paraplegia. This clinical sign has never been observed either in classic ATR-X patients or in related phenotypes such as Juberg-Marsidi syndrome or Carpenter-Waziri syndrome. Spasticity can develop at later stages in ATR-X patients, despite neonatal hypotonia, but the patients reported in this study were affected with spasticity from birth. We have no explanation for this, except that no mutation affecting the helicase III conserved domain has been identified so far. However, it is difficult to draw conclusions based on the analysis of a single family. In contrast, a reexamination of the affected males in this family shows that, although one affected male does not exhibit a markedly dysmorphic phenotype, the two other affected individuals present some facial features in common with the ones observed in ATR-X syndrome: everted lower lip with a “carplike” triangular mouth, hypertelorism, small triangular nose, and broad nasal root. The second point regards the skewed pattern of X inactivation in carrier females. So far, only a close association between a bias in X inactivation and a mutation in the gene has been reported, without any direct proof. In this family, we can observe that the bias in X inactivation occurs only in females carrying the mutation. Other females, who have received the same chromosome but who are not mutated at the XNP locus, do not present a skewed pattern of X inactivation, demonstrating that this phenomenon is directly related to a mutation in the gene. We do not have an explanation for this phenomenon, but we can postulate two hypotheses. First, a mutation in the gene could lead to a selection, during embryogenesis, that favors the cells expressing the normal gene product. The second hypothesis is that the gene, which is involved in chromatin-structure remodeling (Cardoso et al. Cardoso et al., 1998Cardoso C Timsit S Villard L Khrestchatisky M Fontès M Colleaux L Specific interaction between the XNP/ATR-X gene product and the set domain of the human EZH2 protein.Hum Mol Genet. 1998; 7: 679-684Crossref PubMed Scopus (127) Google Scholar), could participate directly or indirectly in one of the processes of X-chromosome inactivation: either spreading or maintenance of the inactive state. This would be lethal in females in whom the mutated allele is expressed, because functional disomy of the X chromosome is known to be a lethal condition. Whatever the truth is, it is really striking to observe that a gene defect that leads to such a strong counterselection in female carriers and that is not restricted to certain cell lineages does not impair male viability. It is, to our knowledge, the first instance in which such a negative selection against the cells expressing an abnormal gene product in females does not imply a male-lethal condition (discussed in Pegoraro et al. Perogaro et al., 1997Perogaro E Whitaker J Mowery-Rushton P Surti U Lanasa M Hoffman EP Familial skewed X inactivation: a molecular trait associated with high spontaneous-abortion rate maps to Xq28.Am J Hum Genet. 1997; 61: 160-170Abstract Full Text PDF PubMed Scopus (106) Google Scholar). Although highly skewed X inactivation has been reported in several other X-linked disorders, it is never systematically present in the obligate-carrier females (Orstavik et al. Orstavik et al., 1998Orstavik KH Orstavik RE Naumova AK D'Adamo P Gedeon A Bolhuis PA Barth PG et al.X-chromosome inactivation in carriers of Barth syndrome.Am J Hum Genet. 1998; 63: 1457-1463Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar; Plenge et al. Plenge et al., 1999Plenge RM Tranebjaerg L Jensen PK Schwartz C Willard HF Evidence that mutations in the X-linked DDP gene cause incompletely penetrant and variable skewed X inactivation.Am J Hum Genet. 1999; 64: 759-767Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). A final point is that the bias in X inactivation is, together with severe MR, the only consistent sign in families. This can certainly be important in a first clinical screening of the patients, before a mutation is sought in the gene. We would like to thank the family members for their cooperation and Dr. Charles Schwartz for helpful discussion. This work was supported by the INSERM program PARMIFR and by the PROGRES network, as well as by Spanish Ministry of Health project FIS98/0170 (Fondo de Investigaciones Sanitarias).