Human Molecular Genetics, 2000, Vol. 9, No. 7 1119-1129
© 2000 Oxford University Press
Long-read sequence analysis of the MECP2 gene in Rett syndrome patients: correlation of disease severity with mutation type and location
Institute of Medical Genetics, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK, 1Department of Psychological Medicine, University of Glasgow, Gartnavel Royal Hospital, 1055 Great Western Road, Glasgow, G12 OXH, UK, 2TVW Telethon Institute for Child Health Research, West Perth, WA 6872, Australia, 3Department of Human Genetics, University of Newcastle upon Tyne, 19/20 Claremont Place, Newcastle upon Tyne, NE2 4AA, UK and 4Department of Biological Sciences, University of Warwick, UK
Received 4 January 2000; Revised and Accepted 17 February 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutations in the methyl-CpG-binding protein gene MECP2 at Xq28 cause Rett syndrome (RTT), an X-linked dominant neurodevelopmental disorder characterized by a period of stagnation followed by regression in the development of young girls. Mutations were sought in MECP2 in 48 females with classical sporadic RTT, seven families with possible familial RTT and five sporadic females with features suggestive, but not diagnostic of RTT. Long distance PCR coupled with long-read direct sequencing was employed to sequence the entire MECP2 gene coding region in all cases. Mutations were identified in 44/55 (80%) unrelated classical sporadic and familial RTT patients, but only 1/5 (20%) sporadic cases with suggestive but non-diagnostic features of RTT. Twenty-one different mutations were identified (12 missense, four nonsense and five frame-shift mutations); 14 of these were novel. All missense mutations were located either in the methyl-CpG-binding domain or in the transcription repression domain. Nine recurrent mutations were characterized in a total of 33 unrelated cases (73% of all cases with MECP2 mutations). Significantly milder disease was noted in patients carrying missense mutations as compared with those with truncating mutations (P = 0.0023), and milder disease was associated with late as compared with early truncating mutations (P = 0.0190).
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Rett syndrome (RTT) is an X-linked dominant disorder that occurs almost exclusively in females and has been considered lethal to males in utero (1–4). It has an estimated prevalence of one in 15 000 girls (5–7). RTT is characterized by neurodevelopmental dysfunction in which a period of stagnation is followed by regression of development in young girls, typically between 6 months and 3 years (1,8,9). The skills often lost include purposeful hand use, social contact and language use. After a period of regression lasting some months, affected girls often regain social contact and may begin to make limited developmental progress. Some of those who had not walked previously subsequently do so, but ataxia is usual. Deceleration of head growth, sometimes leading to microcephaly, has been considered to be one of the diagnostic criteria (10). A number of other manifestations are common in affected girls: stereotypical hand movements, seizures, ventilatory irregularities, autonomic (especially vasomotor) dysfunction, growth retardation, scoliosis and underdevelopment of the fourth metacarpal and metatarsal bones (11–15).
The search for the genetic basis of RTT has been problematic. The usually sporadic nature of the condition prevented gene mapping studies by conventional linkage analysis but the possibility of exclusion mapping using rare familial cases was proposed (16,17). Possible mechanisms of familial recurrence include mosaicism, skewed X chromosome inactivation or the existence of unstable premutations. Early studies excluded much of the short arm and pericentromeric region of the X chromosome (18,19). Concordant inheritance for the Xq28 region of the maternal X chromosome was reported in several families (20) and further linkage analyses confirmed this as the most promising candidate region (3,4,21–24). Several groups then identified candidate genes within the Xq28 region and sought evidence for mutation of these genes in females with RTT (25). Recently, mutations have been identified in the methyl-CpG-binding protein gene, MECP2, at Xq28 in RTT patients (26). These have been shown to account for a broader spectrum of disease, including mild intellectual difficulties in a female and neonatal encephalopathy in a male surviving to birth (27).
The MECP2 gene comprises three exons (of which exon 3 is the largest, spanning 1084 bp) and encodes a 486 amino acid protein (28). MECP2 is widely expressed and alternative polyadenylation in the 3'-UTR results in a highly expressed 10.1 kb transcript in the fetal brain and a 5 kb transcript in the adult brain (28,29). MeCP2 contains two functional domains, an 85 amino acid methyl-cytosine-binding domain (MBD) and a 104 amino acid transcriptional repression domain (TRD). The MBD binds to 5-methyl-cytosine residues in symmetrically positioned CpG dinucleotides located in gene promoter regions that are subject to transcriptional silencing after DNA methylation (30,31). The TRD interacts with histone deacetylase and SIN3A, a transcriptional corepressor. Interaction between this transcription repressor complex and chromatin-bound MeCP2 causes deacetylation of core histones resulting in transcriptional repression (32,33).
To provide further insights into the distribution, spectrum and phenotypic consequences of mutations at the MECP2 locus, we investigated 48 females with classical sporadic RTT, seven families with possible familial RTT, and five sporadic females with features suggestive, but not diagnostic, of RTT. To identify mutations in genes with large exons we have developed an approach based on long distance PCR and long-read direct sequencing. We sequenced the entire MECP2 coding region in all cases and identified mutations in 44 out of 55 (80%) unrelated classical sporadic and familial RTT patients, but only one out of five (20%) sporadic cases with suggestive but non-diagnostic features of RTT. Significantly milder disease was noted in cases carrying missense mutations as compared with those with truncating mutations.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Spectrum and distribution of MECP2 mutations in RTT
In total, 21 different mutations were identified in 45 cases with RTT (44 of 55 unrelated classical sporadic and familial RTT patients and one of five sporadic cases with suggestive but non-diagnostic features of RTT) (Figs 1 and 2a); 14 of these mutations were novel. All mutations were confirmed by independent sequencing reactions in both forward and reverse directions. Twelve different missense mutations were present in 20 unrelated cases (Table 1), four different nonsense mutations were present in 18 unrelated cases and five different frame-shifting mutations were present in seven unrelated cases (Table 2). Nine recurrent mutations were characterized in a total of 33 unrelated cases (i.e. 73% of the total with an identified MECP2 mutation): R168X was present in nine cases; R255X in five cases (counting the monozygotic twin-pair as a single mutational event); 806 delG, R270X, T158M, R133C and R306C in three cases each (with an additional case of R133C in a sister pair); and R106W and R306H in two cases each.
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Eleven of the twelve missense mutations were shown to have arisen de novo in sporadic RTT cases, consistent with their pathogenicity. P101T, identified in patient 139, was not present in her mother but her father was unavailable for analysis, and was not found on 70 normal chromosomes sequenced. All missense mutations were clustered within the two functional domains of MeCP2; 13 cases carried substitutions in the MBD and seven cases carried substitutions in the TRD. All missense mutations were located at highly conserved residues by comparison with human, mouse, Xenopus and chicken sequences (Fig. 2b). Three different missense mutations occurred at the same residue (proline 101) within the MBD and two different missense mutations occurred at the same residue (arginine 306) within the TRD.
All truncating mutations were characterized within the third exon, and all except the large deletion in patient 107 and the two smaller deletions in patients 88 and 80 were located downstream of the MBD and within the TRD or the region upstream of it. The 407 del507bp+insGCTTTTAG mutation which was shown to occur de novo, is predicted to result in the premature truncation of the MeCP2 protein at the newly created amino acid residue 138. The de novo 1152 del44bp deletion and the 1157 del41bp deletion, both create identical new stop codons, five and four codons respectively, downstream of these deletions.
Polymorphic variants
Four silent polymorphic variants were characterized: C
A at 375 (I125), CT at 582 (S194) (26), CT at 897 (T299) and CT at 1197 (P399); all were unique except S194, which was characterized on 4/120 chromosomes. Two single examples of putative polymorphic coding variants were identified (out of 120 chromosomes sequenced). A CT transition at nucleotide 686, corresponding to a serine to leucine substitution at residue 229 (S229L) was identified in a normal father and his affected daughter (patient 144) who also carried the nonsense mutation R168X. A GA transition at 1315, corresponding to an alanine to threonine substitution at residue 439 (A439T) was identified in an apparently normal, unaffected mother and her daughter (patient 125) with no characterized MECP2 mutation, but not in an unaffected maternal uncle.
One sequence difference from the published GenBank sequence (X99686) was also identified; in all chromosomes sequenced, the nucleotide at position 869 was A rather than G, corresponding to a predicted glutamic acid residue at amino acid 290 rather than glycine.
Familial and sporadic, classical and non-classical RTT
Members of seven families with potentially more than one case of RTT were analysed. Family 1: patients 6 and 10, reported as cases 1 and 2 by Clarke et al. (34), and their clinically unaffected mother carried the missense mutation R133C. Family 2: patient 53 had classical RTT and carried the de novo missense mutation S134C. Her younger sister had an episode of developmental stagnation in infancy but then made good developmental progress and does not have RTT nor an MECP2 mutation. Family 3: two sisters (patients 22 and 26) fulfilled the diagnostic criteria but had less severe developmental disturbance than is usual in classical RTT. Neither carried an MECP2 mutation. Family 4: a classically affected girl (patient 4) carried the nonsense mutation R294X. Her aunt was developmentally delayed but was less severely retarded than in classical RTT and did not carry the R294X mutation. Family 5: two second cousins (patients 65 and 67) with classical RTT but with some unusual features were related through unaffected males (35). Only patient 67 carried an MECP2 mutation (P225R), which was absent in other family members. Family 6: both members of a pair of monozygotic twins (patients 14 and 20) had classical RTT but exhibited a marked difference in severity of developmental disturbance, whilst harbouring the same nonsense mutation R255X. Genotyping with the highly polymorphic microsatellite markers D4S43, D6S250, D7S636, D15S945 and D16S665 was consistent with monozygosity. The parents were not available for analysis. Family 7: two mildly affected sisters had features diagnostic of RTT. One was tested (patient 72) and found to be negative for an MECP2 mutation.
Thirty-nine of the 48 (81%) classical sporadic RTT patients carried MECP2 mutations. Blood DNA samples from the mothers of 29 of these patients were tested for the mutation and all were negative. Only one MECP2 mutation was identified in the five (20%) sporadic RTT-like patients, significantly fewer than in the classical sporadic RTT cases (P = 0.01, Fisher’s Exact test). The non-classical group of girls had some features of RTT but met only three of the major diagnostic criteria (Table 3).
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Milder disease associated with missense mutations
Mean phenotypic severity scores observed in patients with different types of mutation are summarized in Table 4. Patients carrying a truncating (i.e. nonsense or frameshift) mutation were found to rank significantly higher for the combined score, and therefore suffered more severe disease overall, than patients with missense mutations (P = 0.0023, corrected P = 0.0138) or those with no characterized MECP2 mutation (P = 0.0009, corrected P = 0.0054). Two sporadic patients (patients 88 and 80) with extremely late truncating mutations (1152 del44bp and 1157 del41bp), also representing obvious outliers in terms of their clinical presentation, had been excluded prior to this analysis. Some component severity scores were also found to yield P values <0.05, but most failed to reach statistical significance when corrected for multiple testing. The only exceptions were provided by the smaller severity of speech scores of patients carrying a missense mutation (P = 0.0069, corrected P = 0.0414) and the significantly milder manual impairment exhibited by patients in whom an MECP2 mutation has not yet been found (P = 0.0002, corrected P = 0.0012). Within the group of patients carrying truncating mutations, a significant difference in combined severity score (P = 0.0190) was observed between early (upstream of the TRD) and late (within or downstream of the TRD) truncating mutations (Table 5). There was no evidence of a difference in severity between cases harbouring missense mutations in the MBD and the TRD.
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Other genotype–phenotype correlations
Patient 107, with an ~500 bp deletion within exon 3, had classical severe RTT. At 14 months, she spoke three words and was unable to walk. She has had frequent absence and grand mal seizures from the age of 4 years and is now profoundly delayed developmentally and has a phenotype score of nine (the maximum observed). In contrast, the two cases with small deletions towards the 3' end of the gene (patients 80 and 88) are mildly affected with phenotype scores of three (the minimum observed).
Mutational mechanisms
Sixteen different single base-pair substitutions were found within the MECP2 coding region; nine were CT transitions in CpG dinucleotides (Tables 1 and 2) and eight of these CpG sites manifested multiple independent mutations (present in a total of 30 patients). CT transitions were noted in four of the five CGA (Arg) codons in the MECP2 gene, the exception being codon 453, just 34 codons upstream of the normal termination codon. In the other four CGA codons, CT transitions causing nonsense mutations were found with decreasing frequency in a 5' to 3' direction: codons 168 (nine cases), 255 (five cases), 270 (three cases) and 294 (one case).
Five intragenic deletions of between 1 and 507 bp were characterized and three patients possessed identical deletions of a single base (806 delG), a lesion that has been reported once before (27). Two of the other deletions (1152 del44bp and 1157 del41bp) occurred at approximately the same location, which may represent a second deletion hotspot in the MECP2 gene.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A number of techniques have been developed to scan for DNA sequence variants, including denaturing gradient gel electrophoresis, chemical cleavage, heteroduplex analysis, single strand conformation polymorphism analysis, hybridization to oligonucleotide arrays and DNA sequencing (36). Advantages of DNA sequencing include its ease of use, its automation with fluorescence-based protocols, and its ability to provide complete information about the location and nature of the sequence variant(s). Disadvantages include labour intensity and, particularly with dye-labelled terminators, difficulty in accurately identifying heterozygous sites because of the variability in fluorescence signal and the inconsistency of base calling at these sites (37). We have developed an approach based on coupling long-distance PCR to long-read direct sequencing to allow rapid scanning of genes with compact genomic structures and/or large exons, such as exon 3 of MECP2. The use of dye-labelled primers generates sequence with better signal-to-noise ratios and more even peaks than with dye-labelled terminators (37). Using this approach, we produced clear sequencing runs well in excess of 650 bp, significantly greater than the 400 bp runs achieved by Thomas et al. (38) using terminator cycle sequencing of long distance PCR products at the PKD1 locus. This may explain in part why we detected MECP2 mutations in 80% of RTT patients, as compared with ~50% characterized by Wan et al. (27), who also used fluorescent-dye terminator cycle sequencing.
We failed to identify mutations in 11 (20%) cases with classical RTT. However, the promoter region was not screened for point mutations, nor were large DNA rearrangements or deletions sought by Southern, pulsed-field gel electrophoresis or FISH analysis. Gross gene deletions and rearrangements have been commonly observed in patients with other X-linked diseases such as Duchenne muscular dystrophy and haemophilia A. Furthermore, the highly conserved portions of the 8.5 kb 3'-UTR were not screened for mutations; these regions have been suggested to be evolutionarily conserved and important for post-transcriptional regulation of MECP2 (29). Therefore, although locus heterogeneity remains a theoretical possibility in RTT, any other gene(s) involved would account for only a very small proportion of classical RTT cases.
From studies of both sporadic and familial, classical and non-classical RTT-like cases, we have shown that the classical cases are likely to carry a coding region MECP2 mutation. It remains possible that the non-classical cases in which a coding region mutation was not identified, carry a different type of MECP2 lesion or may involve one of the other members of the MBD protein family. Therefore, although there is the possibility of wide phenotypic variability associated with an MECP2 mutation (27), presumably through varying patterns of X inactivation, the presentation of a classic RTT phenotype in any family member is likely to indicate the presence of an MECP2 mutation.
All missense mutations except P101T were shown to have arisen de novo, consistent with their pathogenicity. Since no other disease causing variants were detected in the patient carrying P101T and that this change was not identified on 70 normal chromosomes sequenced, and since two other disease-causing missense mutations were identified at amino acid residue P101 (P101H and P101L), we speculate that the threonine substitution is also pathogenic. All 12 missense mutations occurred either in the MBD or in the TRD. Within these domains, the substitutions were sometimes clustered, probably either for structural/functional reasons (residues 101, 133/134) or as a consequence of hypermutability (residue 306). No phenotypic difference was found between cases harbouring missense mutations in one or the other functional domain. Given the process of random X inactivation in females and the wide range of phenotypes observed with the same MECP2 mutation both within a family and in unrelated individuals, this is not surprising. However, we did observe milder disease in cases carrying missense mutations as compared with those with truncating mutations. This could reflect some residual function of the mutant MeCP2 proteins.
The apparent excess of 5' CT transitions in the five CGA (Arg) codons in the MECP2 gene mirrors the combined data from the two previous studies (26,27) and could either reflect variable site-specific methylation in the germline or a decrease in the likelihood of clinical sequelae arising from nonsense mutations toward the C-terminal of the MECP2 gene product. A decrease in severity was noted in cases harbouring truncating mutations within or downstream of the TRD as compared with cases with truncating mutations N-terminal to this domain. Two cases (patients 80 and 88), with similar frameshift deletions towards the 3' end of the gene which were predicted to result in MeCP2 proteins truncated by ~20%, were both associated with very mild RTT.
Wan et al. (27) described a patient, presenting with incomplete diagnostic features of RTT and highly localized skin findings resembling incontinentia pigmenti, who carried a frameshift mutation that was predicted to truncate the MeCP2 protein at codon 138. They suggested the possibility that since the truncated product was missing half of the MBD, it would no longer be able to bind to methyl-CpG and might cause a more complex phenotype. In this study, we identified a patient (patient 107) with a large deletion that was also predicted to result in the premature truncation of the MeCP2 protein at amino acid 138. However, this patient did not have any signs of incontinentia pigmenti and presented with classical severe RTT, supporting the alternative explanation suggested by Wan et al. (27) that their patient was also mosaic for a postzygotic mutation in the IP2 gene.
Sixty-nine percent (31/45) of unrelated RTT cases with MECP2 mutations exhibited CT transitions in CpG dinucleotides, reflecting the inherent hypermutability of this doublet (39). Of the 201 genes listed in the Human Gene Mutation Database (40) with more than 15 different single base-pair substitutions, only four exhibit a higher proportion of CpG mutations than the MECP2 gene. This high proportion of CpG mutations does not, however, explain the very high mutation rate in RTT.
The recurrent deletion 806 delG in the MECP2 gene that has now been identified in four unrelated cases (27 and this study) involves a single base which is flanked by an interrupted direct repeat that could have served to template the deletion through slipped mispairing (41). The breakpoints of the almost concurrent 41 and 44 bp deletions were flanked by both direct and indirect complementary repeats, which again could have mediated the deletions via slipped mispairing. Direct repeats were also noted flanking the breakpoints of the two other MECP2 gene deletions identified.
The pathogenetic process linking MECP2 gene mutations to the RTT phenotype is unclear. We speculate that decreased MeCP2 activity will lead to derepression of transcription at multiple loci and to a generally increased level of synthesis of a broader range of proteins than is usual in any particular cell type. RTT may arise where cells are metabolically disturbed in this way and where their functions cannot be readily substituted by their neighbours, as in the developing brain, once the early neuronal plasticity and substitutability are lost. Were such a cell to be lost or become dysfunctional in fetal life, its functions might be taken over by its neighbours, but such plasticity is progressively reduced as the infant matures into a child. The characteristic features of RTT could therefore be the result of processes that are only apparent at the level of whole organ development and function. Attempts to identify a simple, linear sequence of alterations to the transcription of specific loci, linking MECP2 mutations to the RTT phenotype in a causal chain, would then be fruitless. This model would satisfactorily account for many features of RTT such as regression in infancy but would be difficult to investigate experimentally. It is possible that some mutations cause RTT through mechanisms compatible with our model whereas others, perhaps including particular missense mutations, could lead to specific ‘toxic’ consequences for gene transcription.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Patients
The blood samples studied were obtained over more than a decade from a variety of sources, including 12 samples courtesy of the UK Rett Syndrome Association Genetic Bank established by M.H., and samples taken for combined metabolic and genetic studies of RTT as approved by the relevant Local Research Ethics Committees (in Newcastle upon Tyne and South Glamorgan). In total, we investigated 48 females with classical sporadic RTT, seven families with possible familial RTT and five sporadic females with some features of RTT but in whom the diagnosis was clinically uncertain: patient 84, gradual developmental progress from late infancy and no regression; patient 111, developmental delay noted before 4 months, seizures from 6 months, marked regression at 5 years coinciding with worsening seizures and some dysmorphic features; patient 123, seizures from neonatal period and no clear regression; patient 125, salaam spasms from 6 months, eyelid myoclonia from 18 months, no regression and development of some hand skills; patient 131, infantile spasms from 3 months and no clear regression (Table 3).
A set of diagnostic criteria was adapted from Trevarthen et al. (10) and Hagberg (6). Classical cases were defined as fulfilling all five major diagnostic criteria or four out of five major and two out of six minor criteria. Major diagnostic criteria were (i) normal pre- and perinatal period and apparently normal development for the first 5–6 months; (ii) developmental stagnation and regression with onset between 6 months and 3 years; (iii) documented evidence of deceleration of growth in occipito-frontal circumference; (iv) acquisition of hand movement stereotypies; and (v) marked developmental/cognitive delay. Minor (supportive) diagnostic criteria were (i) growth retardation; (ii) vasomotor (autonomic) dysfunction and atrophy of the feet; (iii) abnormalities of the EEG; (iv) scoliosis; (v) ventilatory irregularities; and (vi) presence of shortened 4th metatarsal or metacarpal bones (15).
In addition, a phenotypic score of clinical severity was derived by assessing: (a) hand use, where a score of 1 indicates the ability to use a spoon/finger feed/hold a cup or bottle, 2 indicates the ability to grasp or hold briefly, and 3 denotes no purposeful use; (b) speech, where 1 indicates the ability to understand and say some words, 2 denotes the ability to say a few words, possibly with meaning, and 3 denotes no speech; (c) walking, where 1 indicates walking independently, 2 denotes walking with aid (walking frame or hands held), and 3 denotes no ability to walk. The scores allocated represented the highest level of functioning achieved after the period of regression. Higher scores (maximum score 9) indicate more severe disease.
PCR amplification
Genomic DNA was prepared from peripheral blood lymphocytes by standard methods. PCR and sequencing primers were designed with the aid of the Oligo Analysis software package using parameters that we have previously defined (42). PCR primers were synthesized by Oswel DNA Services (Southampton University, Southampton, UK). Exons 1, 2 and 3, the flanking intronic splice site sequences and the 5'-UTR of MECP2 were amplified as three non-overlapping fragments of 174, 454 and 1340 bp, respectively.
Exon 1: JC1F, 5'-AGGCTCCATAAAAATACAGACT-3'
and JC2R, 5'-GGCCAAACCAGGACATATAC-3';
exon 2: JC3F, 5'-TGCCTCTGCTCACTTGTTCT-3'
and JC4R, 5'-TGCCCTGTAGAGATAGGAGTT-3';
exon 3: JC5F, 5'-ATCCGCTCTGCCCTATCTCT-3'
and JC6R, 5'-CCCCAATGCTCCAACTACTC-3'.
Standard PCR (exons 1 and 2) was carried out in 50 µl reaction volumes containing 100 ng genomic DNA, 25 pmol primers, 0.2 mM dNTP, 5 µl reaction buffer (100 mM Tris pH 8.3, 500 mM KCl, 15 mM MgCl2, 0.01% gelatin), and 1 U AmpliTaq Gold Polymerase (Cetus). Cycling parameters were 94°C for 10 min, followed by 32 cycles of 57°C for 1 min, 72°C for 1 min, 94°C for 30 s, and a final step of 72°C for 10 min. Long PCR (exon 3) was carried out in 50 µl reaction volumes containing 100 ng genomic DNA, 4 pmol primers, 0.35 mM dNTP, 5 µl Boehringer Mannheim reaction buffer 3 [20 mM Tris–HCl, 100 mM KCl, 22.5 mM MgCl2, 1 mM dithiothreitol, 0.1 mM EDTA, 0.5% (v/v) Tween-20, 0.5% (v/v) Nonidet P-40, 50% (v/v) glycerol] and 2 U Expand Long Template polymerase. Cycling parameters were 95°C for 2 min, followed by 30 cycles of 60°C for 30 s, 68°C for 2 min and 94°C for 30 s. The elongation step was increased by 20 s for each cycle after cycle number 10. Exon 3 PCR products were purified using the Qiagen PCR purification kit.
Direct DNA sequencing
Manual PCR product sequencing was performed for exons 1 (primer JC1F) and 2 (primers JC3F and JC4R) using the ThermoSequenase cycle sequencing kit (Amersham Pharmacia Biotech, Uppsala, Sweden) at a 60°C annealing temperature, according to the manufacturer’s specifications. Sequencing reactions were loaded onto 6% polyacrylamide gels, which were run for 1.5 h at 65 W, dried and exposed to film. Automated PCR product sequencing for exon 3 was performed on an MWG RoboAmp 4200 robotic workstation using two overlapping bi-directional sequencing reactions:
JC5F to JC10R (5'-GGGCTTCACCACTTCCTTGAC-3')
and JC9F (5'-TCCACCCAGGTCATGGTGATC-3') to
JC11R (5'-TGTCAGAGCCCTACCCATAAG-3')
which generated long, high quality sequencing reads of 666 and 821 bp, respectively, with the ThermoSequenase fluorescence cycle sequencing kit (Amersham) at a 54°C annealing temperature. Sequencing primers were synthesized by MWG Biotech with forward primers labelled with IRD-700 and reverse with IRD-800. Sequencing reactions were loaded onto 4.75% polyacrylamide gels (Rapid Gel XL; Amersham USB) and run at 2800 V, 40 mA and 55 W at 45°C on a Li-Cor 4200 DNA sequencer. Every patient was sequenced for all three fragments, and mutations were easily detected by eye and confirmed on both strands.
Statistical analysis
Mean phenotypic severity scores of groups of patients classified by mutation type were tested for significant pairwise differences using a resampling test. In terms of their validity, such randomization procedures are known to be superior to parametric tests when the underlying distribution functions are unknown or when sampling has not been random (43). By way of illustration of the methodology, let x1,..,xn be the severity scores observed in the first patient group, and let xn+1,...,xm be the severity scores in the second group. The two mean scores to compare were thus
For a given permutation 
of integers 1...m, define
An empirical P-value to test {µ1/µ2} against the null hypothesis {µ1 = µ2} was obtained by evaluating the difference between µ1() and µ2() for a large number of randomly chosen permutations . In the present study, a two-sided test was required and P was thus set equal to the relative frequency of permutations , among 10 000 replicates, for which 
µ1() µ2() 
µ1 – µ2. Multiple testing was corrected for by means of a Bonferroni approach. To this end, P-values were multiplied by the number of pairwise comparisons (i.e. six) performed for each phenotypic measure.
Mutation nomenclature
Nucleotides were numbered from the first base of the translation iniation ATG codon (GenBank accession no. X99686) in accord with Wan et al. (27). Mutations were described as recommended by the Ad Hoc Committee on Mutation Nomenclature (44) and Antonarakis et al. (45).
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ACKNOWLEDGEMENTS |
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We would like to thank the patients with RTT syndrome and their families, UK Rett Syndrome Association (UKRSA), Mrs Emer Parker, Mrs Meinir Cheadle, Mr Edward Ball, members of the Institute of Medical Genetics DNA Diagnostic laboratory, Cardiff, Dr John Wilson (formerly of The Hospital for Sick Children, Great Ormond Street, London), Dr Athel Hockey (Disability Services Commission, Perth, Australia), Dr James Manson (Women’s and Children’s Hospital, Adelaide, Australia), Mr Mark Davis (Neurogenetics laboratory, Royal Perth Hospital, Perth, Australia), Dr David Shortland (Department of Paediatrics, Poole Hospital, Dorset) and Dr Gillian McCarthy (MacKeith Centre, Royal Alexandra Hospital for Sick Children, UK) for their support of this project. This work was supported by a research grant from The Patrick Berthoud Charitable Trust to M.H. and UKRSA.
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FOOTNOTES |
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+ These authors contributed equally to this work
§ To whom correspondence should be addressed. Tel: +44 29 20744035; Fax: +44 29 20747603; Email: clarkeaj@cardiff.ac.uk
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REFERENCES |
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