Two sisters with Rett syndrome and non-identical paternally-derived microdeletions in the MECP2 gene
© Springer Science+Business Media B.V. 2008
Received: 11 June 2008
Accepted: 27 August 2008
Published: 20 September 2008
The unique case of two sisters with symptoms of RTT and two quite distinct, novel, and apparently de novo microdeletions of the MECP2 gene is described. One sister possessed an 18 base-pair (bp) deletion (c.1155_1172del18) within the deletion hotspot region of exon 4, whereas the other sister exhibited a 43 bp deletion at a different location in the same exon (c.1448_1461del14+29). Although these lesions occurred on the same paternally-derived X chromosome, this is probably due to chance co-occurrence owing to the relatively high mutation rate of the MECP2 gene rather than to a constitutional mutator phenotype.
KeywordsRett syndrome MECP2 gene Familial non-identical mutations
Rett syndrome (RTT; RTT MIM#312750) is an X-linked dominant neurological disorder of development which affects 1 in 10,000–15,000 females (Percy 2002; Chahrour and Zoghbi 2007). Classic RTT has a characteristic presentation with subtle abnormalities in development from birth, a period of stagnation, followed by regression with loss of hand and social skills and development of hand stereotypies, deceleration in head growth, severe learning difficulties and gait dyspraxia (Weaving et al. 2005; Williamson and Christodoulou 2006).
Mutations in the MECP2 gene (MIM# 300005) were first reported in RTT by Amir et al. (1999) and are identifiable in ~80% of classic RTT cases, although less frequently in atypical RTT (Huppke and Gärtner 2005). The MECP2 gene contains four exons and encodes methyl-CpG-binding protein-2 (MeCP2), a multifunctional protein with at least four different functional domains: (i) a methyl-CpG-binding domain, (ii) an arginine-glycine repeat RNA-binding domain, (iii) a transcriptional repression domain and (iv) an RNA splicing factor binding region (WW group II binding domain) [Fan and Hutnick 2005]. Two distinct isoforms have been reported; MECP2A is encoded by exons 2–4 whereas MECP2B is encoded by exons 1, 3 and 4 (Mnatzakanian et al. 2004). To date, more than 200 different nucleotide substitutions have been reported in the MECP2 gene as a cause of RTT (RettBASE, http://mecp2.chw.edu.au; Miltenberger-Miltenyi and Laccone 2003; Williamson and Christodoulou 2006). In addition, ~10% of RTT patients possess microdeletions which cluster within a ~150 bp exon 4 hotspot region, between nucleotides 1050 and 1200 (Williamson and Christodoulou 2006; Bienvenu and Chelly 2006; Philippe et al. 2006).
The vast majority of RTT cases are sporadic (>99%), but some familial cases have been described which are usually caused by either maternal or paternal germline mosaicism (Evans et al. 2006; Venâncio et al. 2007) or skewing of X-inactivation towards the wild-type MECP2 allele in the asymptomatic carrier mother (Villard et al. 2001; Knudsen et al. 2006; Dayer et al. 2007). In such cases, affected relatives invariably possess the same molecular lesion. Here, we describe the unique case of two sisters with symptoms of RTT who were found to have two quite distinct, and apparently de novo, microdeletions on the same paternally-derived MECP2 allele.
Case 2, now 4 years of age, weighed 3.24 kg at term. The pregnancy was largely uncomplicated, although occasional periods of fetal inactivity were noted by the mother (now aged 47 years; father 51 years). She was noted to be quite floppy during feeding but was able to sit at 9 months, and walked unsteadily from 18 months. Developmental progression was slow, with no recognisable speech, although she did babble. She did not develop imaginative play, and engaged in frequent hand-wringing and clapping. On assessment at age 2 years 4 months, she was markedly hypotonic but non-dysmorphic. Height and weight were on the 25th and 75th centiles, respectively, but head circumference was on the 9th centile, and had stalled at 48 cm by 3 years 4 months (~2nd centile). Further developmental regression had been noted from the age of 2 years 6 months, with loss of stacking skills and spoon handling ability. She did not have seizures. MRI brain scan was normal but an EEG has not yet been carried out. The affected girls have two apparently normal sisters.
Materials and methods
Polymerase chain reaction (PCR) amplification of the four exons of the MECP2 gene was performed using oligonucleotide primers and amplification conditions described by Cheadle et al. (2000). PCR products and plasmid clones were sequenced using Thermosequenase (Amersham Pharmacia, Little Chalfont, UK) according to the manufacturers’ instructions. PCR-amplified genomic fragments were then sequenced in both directions using the primers used for PCR amplification.
Cloning of PCR amplified fragments
Exon 4 of the MECP2 gene was PCR amplified from DNA derived from both sisters and their parents as described above. PCR fragments were cloned into pGEM-T (Promega, Southampton, UK). Four clones from each individual were then sequenced in both directions using vector-based primers pGEMT5B (5′ CAGTCACGACGTTGTAAAACG 3′) and pGEMT3B (5′ ATGACCATGATTACGCCAAGC 3′) using BigDye v3.1 (Applied Biosystems, Warrington, UK) and analysed on an ABI 3100 sequencer (Applied Biosystems, Warrington, UK).
The rare c.1373G>A variant serendipitously provided a marker for the paternal MECP2 allele in this family. The sequencing of cloned exon 4-containing fragments in the two affected sisters demonstrated that both microdeletions had occurred in cis to the paternal c.1373G>A variant. We may therefore conclude that both microdeletions occurred independently on the same paternal allele. This is consistent with the observations of Trappe et al. (2001) that most de novo MECP2 gene lesions in RTT arise on the paternally derived X chromosome.
Complexity analysis (Gusev et al. 1999) was used to examine the potential role of the local DNA sequence environment in mediating the two microdeletions in the MECP2 gene. Several repetitive sequence elements were identified which could have mediated the two mutational events. Deletion 1155_1172del18, which occurred within the deletion hotspot region, could have been mediated by two CCACC direct repeats via slipped mispairing (Fig. 2a). However, the 1448_1461del14+29 deletion appears to be more complex and may have originated via either one of two distinct deletions both of which could have been mediated by direct repeats. These repeats, GAG and CAA (Fig. 2b), appear to be capable of forming slipped structures (Fig. 2c). Such slipped structures can adopt non-B DNA conformations which are known to be susceptible to double strand breaks and hence are also highly mutagenic (Wang and Vasquez 2006).
The vast majority of cases of Rett syndrome (RTT) are female and sporadic. However, a number of familial cases of RTT, exhibiting recurrent RTT within a sibship, have been reported and have generally been explained either by parental gonadal mosaicism or by a clinically unaffected carrier mother with skewed X-inactivation; in such cases, affected relatives have invariably possessed the same molecular lesion (Villard et al. 2000; Mari et al. 2005; Archer et al. 2007; Hardwick et al. 2007; Venâncio et al. 2007). As far as we are aware, the independent occurrence of two non-identical MECP2 gene lesions in the same family, as reported here, is unprecedented. The question therefore arises as to whether the X chromosome upon which the microdeletions arose could somehow be predisposed to mutation. Although both microdeletions occurred on the same paternally inherited chromosome and within the WW group II binding domain, only c.1155_1172del18 occurred within the deletion hotspot region in exon 4. Moreover, the two micro-deletions appear to have arisen via entirely different mutational mechanisms. Finally, inspection of the sequence of the affected MECP2 allele failed to identify any private sequence characteristics that could account for a particular predisposition to deletional mutagenesis on this paternally-derived chromosome. We therefore conclude that, in all likelihood, this unique occurrence of two non-identical MECP2 gene lesions within a sibship probably represents a chance event.
This case is reminiscent of our previous report of a family with three individuals displaying clinical evidence of neurofibromatosis type 1 (NF1) who were found to possess three different heritable and pathological mutations in their NF1 genes (Upadhyaya et al. 2003). Although several possible explanations for this unique finding were considered, including the possibility that the NF1 alleles segregating in the family might be unstable, on balance we concluded that it probably represented chance co-occurrence (Upadhyaya et al. 2003). These cases are not altogether without precedent. Indeed, other such examples of independently segregating non-identical pathological gene lesions have involved the NF1 gene in a second quite unrelated family with NF1 (Klose et al. 1999), the RB1 gene in a family with retinoblastoma (Munier et al. 1993) and the APC gene in a family with adenomatous polyposis coli (Davidson et al. 2002). Examples of this phenomenon have also been reported in the X-linked recessive conditions haemophilia A (Tizzano et al. 2005) and Duchenne/Becker muscular dystrophy (Laing et al. 1992; Mostacciuolo et al. 1994; Morandi et al. 1995; Zatz et al. 1998). Irrespective of whether these cases represent examples of mutationally unstable alleles or simply chance co-occurrence owing to the relatively high mutation rates exhibited by some of these genes, such reports should serve as a stark reminder that it is unwise to extrapolate from the detection of a single mutation in a specific individual to the assumption of an identical molecular genetic aetiology in other clinically affected members of the same family.
- Amir RE, Van den Veyver I, Wan M, Tran CQ, Francke U, Zoghbi HY (1999) Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet 23:185–188. doi:10.1038/13810PubMedView ArticleGoogle Scholar
- Archer H, Evans J, Leonard H, Colvin L, Ravine D, Christodoulou J, Williamson S, Charman T, Bailey MES, Sampson J, de Klerk N, Clarke A (2007) Correlation between clinical severity in patients with Rett syndrome with a p.R168X or p.T158M MECP2 mutation, and the direction and degree of skewing of X-chromosome inactivation. J Med Genet 44:148–152PubMedPubMed CentralView ArticleGoogle Scholar
- Bienvenu T, Chelly J (2006) Molecular genetics of Rett syndrome: when DNA methylation goes unrecognized. Nat Rev Genet 7:415–426. doi:10.1038/nrg1878PubMedView ArticleGoogle Scholar
- Chahrour M, Zoghbi HY (2007) The story of Rett syndrome: from clinic to neurobiology. Neuron 56:422–437. doi:10.1016/j.neuron.2007.10.001PubMedView ArticleGoogle Scholar
- Cheadle JP, Gill H, Fleming N, Maynard J, Kerr A, Leonard H et al (2000) Long-read sequence analysis of the MECP2 gene in Rett syndrome patients: correlation of disease severity with mutation type and location. Hum Mol Genet 9:1119–1129. doi:10.1093/hmg/9.7.1119PubMedView ArticleGoogle Scholar
- Davidson S, Leshanski L, Rennert G, Eidelman S, Amikam D (2002) Maternal mosaicism for a second mutational event—a novel deletion—in a familial adenomatous polyposis family harbouring a new germ-line mutation in the alternatively spliced exon 9 region of APC. Hum Mutat 19:83–84. doi:10.1002/humu.9006PubMedView ArticleGoogle Scholar
- Dayer AG, Bottani A, Bouchardy I, Fluss J, Antonarakis SE, Haenggeli CA et al (2007) MECP2 mutant allele in a boy with Rett syndrome and his unaffected heterozygous mother. Brain Dev 29:47–50. doi:10.1016/j.braindev.2006.06.001PubMedView ArticleGoogle Scholar
- Evans JC, Archer HL, Whatley SD, Clarke A (2006) Germline mosaicism for a MECP2 mutation in a man with two Rett daughters. Clin Genet 70:336–338. doi:10.1111/j.1399-0004.2006.00691.xPubMedView ArticleGoogle Scholar
- Fan G, Hutnick L (2005) Methyl-CpG binding proteins in the nervous system. Cell Res 15:255–261. doi:10.1038/sj.cr.7290294PubMedView ArticleGoogle Scholar
- Gusev VD, Nemytikova LA, Chuzhanova NA (1999) On the complexity measures of genetic sequences. Bioinformatics 15:994–999. doi:10.1093/bioinformatics/15.12.994PubMedView ArticleGoogle Scholar
- Hardwick SA, Reuter K, Williamson SL, Vasudevan V, Donald J, Slater K et al (2007) Delineation of large deletions of the MECP2 gene in Rett syndrome patients, including a familial case with a male proband. Eur J Hum Genet 15:1218–1229. doi:10.1038/sj.ejhg.5201911PubMedView ArticleGoogle Scholar
- Huppke P, Gärtner J (2005) Molecular diagnosis of Rett syndrome. J Child Neurol 20:732–736PubMedView ArticleGoogle Scholar
- Klose A, Peters H, Hoffmeyer S, Buske A, Luder A, Hess D et al (1999) Two independent mutations in a family with neurofibromatosis type 1 (NF1). Am J Med Genet 83:6–12. doi:10.1002/(SICI)1096-8628(19990305)83:1<6::AID-AJMG3>3.0.CO;2-EPubMedView ArticleGoogle Scholar
- Knudsen GP, Neilson TC, Pedersen J, Kerr A, Schwartz M, Hulten M et al (2006) Increased skewing of X chromosome inactivation in Rett syndrome patients and their mothers. Eur J Hum Genet 14:1189–1194. doi:10.1038/sj.ejhg.5201682PubMedView ArticleGoogle Scholar
- Laing NG, Layton MG, Johnsen RD, Chandler DC, Mears ME, Goldblatt J et al (1992) Two distinct mutations in a single dystrophin gene: chance occurrence or premutation? Am J Med Genet 42:688–692. doi:10.1002/ajmg.1320420512PubMedView ArticleGoogle Scholar
- Mari F, Caselli R, Russo S, Cogliati F, Ariani F, Longo I et al (2005) Germline mosaicism in Rett syndrome identified by prenatal diagnosis. Clin Genet 67:258–260. doi:10.1111/j.1399-0004.2005.00397.xPubMedView ArticleGoogle Scholar
- Miltenberger-Miltenyi G, Laccone F (2003) Mutations and polymorphisms in the human methyl CpG-binding protein MECP2. Hum Mutat 22:107–115. doi:10.1002/humu.10243PubMedView ArticleGoogle Scholar
- Mnatzakanian GN, Lohi H, Munteanu I, Alfred SE, Yamada T, MacLeod PJ et al (2004) A previously unidentified MECP2 open reading frame defines a new protein isoform relevant to Rett syndrome. Nat Genet 36:339–341. doi:10.1038/ng1327PubMedView ArticleGoogle Scholar
- Morandi L, Mora M, Tedeschi S, Di Blasi C, Curcio C, De Leonardis P et al (1995) DMD and BMD in the same family due to distinct mutations. Am J Med Genet 59:501–505. doi:10.1002/ajmg.1320590418PubMedView ArticleGoogle Scholar
- Mostacciuolo ML, Miorin M, Vitiello L, Rampazzo A, Fanin M, Angelini C et al (1994) Occurrence of two different intragenic deletions in two male relatives affected with Duchenne muscular dystrophy. Am J Med Genet 50:84–86. doi:10.1002/ajmg.1320500118PubMedView ArticleGoogle Scholar
- Munier FL, Wang MX, Spence MA, Thonney F, Balmer A, Pescia G et al (1993) Pseudo low penetrance in retinoblastoma. Fortuitous familial aggregation of sporadic cases caused by independently derived mutations in two large pedigrees. Arch Ophthalmol 111:1507–1511PubMedView ArticleGoogle Scholar
- Percy AK (2002) Rett syndrome. Current status and new vistas. Neurol Clin 20:1125–1141. doi:10.1016/S0733-8619(02)00022-1PubMedView ArticleGoogle Scholar
- Philippe C, Villard L, De Roux N, Raynaud M, Bonnefond JP, Pasquier L et al (2006) Spectrum and distribution of MECP2 mutations in 424 Rett syndrome patients: a molecular update. Eur J Med Genet 49:9–18. doi:10.1016/j.ejmg.2005.04.003PubMedView ArticleGoogle Scholar
- Tizzano EF, Venceslá A, Baena M, Cornet M, Calvo MT, Lucía JF et al (2005) First report of two independent point factor VIII mutations in a family with haemophilia A: a word of caution for carrier diagnosis. Thromb Haemost 94:675–677PubMedGoogle Scholar
- Trappe R, Laccone F, Cobilanschi J, Meins M, Huppke P, Hanefeld F et al (2001) MECP2 mutations in sporadic cases of Rett syndrome are almost exclusively of paternal origin. Am J Hum Genet 68:1093–1101. doi:10.1086/320109PubMedPubMed CentralView ArticleGoogle Scholar
- Upadhyaya M, Majounie E, Thompson P, Han S, Consoli C, Krawczak M et al (2003) Three different pathological lesions in the NF1 gene originating de novo in a family with neurofibromatosis type 1. Hum Genet 112:12–17. doi:10.1007/s00439-002-0840-1PubMedView ArticleGoogle Scholar
- Venâncio M, Santos M, Pereira SA, Maciel P, Saraiva JM (2007) An explanation for another familial case of Rett syndrome: maternal germline mosaicism. Eur J Hum Genet 15:902–904. doi:10.1038/sj.ejhg.5201835PubMedView ArticleGoogle Scholar
- Villard L, Kpebe A, Cardoso C, Chelly PJ, Tardieu PM, Fontes M (2000) Two affected boys in a Rett syndrome family: clinical and molecular findings. Neurology 55:1188–1193PubMedView ArticleGoogle Scholar
- Villard L, Lévy N, Xiang F, Kpebe A, Labelle V, Chevillard C, Zhang Z, Schwartz CE, Tardieu M, Chelly J, Anvret M, Fontès M (2001) Segregation of a totally skewed pattern of X chromosome inactivation in four familial cases of Rett syndrome without MECP2 mutation: implications for the disease. J Med Genet 38:435–442PubMedPubMed CentralView ArticleGoogle Scholar
- Wang G, Vasquez KM (2006) Non-B DNA structure-induced genetic instability. Mutat Res 598:103–119. doi:10.1016/j.mrfmmm.2006.01.019PubMedView ArticleGoogle Scholar
- Weaving LS, Ellaway CJ, Gécz J, Christodoulou J (2005) Rett syndrome: clinical review and genetic update. J Med Genet 42:1–7. doi:10.1136/jmg.2004.027730PubMedPubMed CentralView ArticleGoogle Scholar
- Williamson SL, Christodoulou J (2006) Rett syndrome: new clinical and molecular insights. Eur J Hum Genet 14:896–903. doi:10.1038/sj.ejhg.5201580PubMedView ArticleGoogle Scholar
- Zatz M, Sumita D, Campiotto S, Canovas M, Cerqueira A, Vainzof M et al (1998) Paternal inheritance or different mutations in maternally related patients occur in about 3% of Duchenne familial cases. Am J Med Genet 78:361–365. doi:10.1002/(SICI)1096-8628(19980724)78:4<361::AID-AJMG11>3.0.CO;2-GPubMedView ArticleGoogle Scholar