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Charman et al. Genotype-phenotype associations in Rett syndrome Supplementary data Methods Mutation screening MECP2 mutation screening was carried out for this study on 62 patients. Multiple tissue samples (blood, buccal and hair) were collected from patients, and from their mothers, fathers and siblings over 11 years old where available. DNA extractions from blood were performed at the Department of Medical Genetics, University of Glasgow, UK under NHS medical genetics QC conditions. DNA was extracted from approximately 7ml of EDTA blood by standard kit-based procedures. Buccal brushings were also taken (1 brush per individual). Buccal brushes (Epicentre Technologies; Madison, WI) were used to swab inner cheeks, and left to air dry for a period of 2 hours before packaging under sterile conditions and transportation to the lab. On arrival, brushes were stored at -20˚C before processing. Buccal DNA was extracted using the Qiagen QIAamp DNA Blood mini kit (Qiagen Ltd; Crawley, UK), as per manufacturer’s instructions. Amplification of exonic fragments of MECP2 from genomic DNA was carried out as follows. Exons 2, 3 and 4 were amplified along with splice sites and small amounts of flanking intron. Exon 4 was amplified as 5 separate, overlapping fragments. Primer sequences are given in Supplementary Table ST1 below. Exon 4 fragment ‘a’ (exon 4a) was amplified in some cases using primers exon 4a1F and 4a2R. Amplification was generally carried out for 30 cycles under standard ‘step-down’ conditions (similar to conditions given in Buyse et al. (ref. 35): 10-25l reactions with 20-50ng genomic DNA and employing AB Gene’s (ABgene Ltd; Epsom, UK) PCR MasterMix (final annealing temperatures are available from the authors). S1 Supplementary Table ST1 PCR primers used to screen MECP2 in Glasgow Exon Primer Name Primer Sequence 2 3 4a1 4a2 4b 4c/d 4e Reference 2-For 5’-TAAGCTGGGAAATAGCCTAGTAC-3’ Buyse et al. 200034 2-Rev 5’-TTATATGGCACAGTTTGGCACAG-3’ Buyse et al. 2000 3-For 5’-AGGACATCAAGATCTGAGTGTAT-3’ Buyse et al. 2000 3-Rev 5’-GGTCATTTCAAGCACACCTG-3’ Buyse et al. 2000 4a-For.2 5’-CGCTCTGCCCTATCTCTGA-3’ Buyse et al. 2000 exon 4a1R 5’-GTGGCCGCCTTGGGTCTC-3’ This study exon 4a2 F 5’- AGAGCAGAAACCACCTAAGAAG -3’ This study exon 4a2 R 5’- ACAATCGGATAGAAGACTCCTT -3’ This study 4b-For.3 5’- GGCAGGAAGCGAAAAGCTGAG -3’ Buyse et al. 2000 4b-Rev.3 5’- TGAGTGGTGGTGATGGTGGTGG -3’ Buyse et al. 2000 4c/d-cFor 5’- GGAAAGGACTGAAGACCTGTAAG -3’ Buyse et al. 2000 HZR4 5’- CTCCCTCCCCTCGGTGTTTG -3’ Buyse et al. 2000 4e-For 5’- GGAGAAGATGCCCAGAGGAG -3’ Buyse et al. 2000 4e-Rev 5’- CGGTAAGAAAAACATCCCCAA -3’ Buyse et al. 2000 Sequences of each fragment were ascertained by automated fluorescent sequencing of PCR products in most cases. Exon 2 fragments could only be successfully sequenced in the forward direction due to the presence of mononucleotide tracts close to the R primer. In some cases, PCR products were cloned using T-vector kits and multiple clones were sequenced and aligned using ABI Autoassembler 2.0 (Applied Biosystems; Foster City, CA) to detect variant sites. Exon 4a and 4b fragments were initially screened for the presence of several common mutations that generate or abolish restriction sites. All point mutations and variants observed were confirmed either by two methods (restriction digestion and sequencing), or by sequencing two separate fragments (e.g. exon 4a and 4b), or by sequencing fragments generated in independent PCR reactions in the forward and reverse directions, depending on the identity and location of the mutation/variant. Small insertions and deletions were confirmed by gel electrophoresis of the fragment containing the alteration followed by sequencing in the forward and reverse directions. Larger deletions were confirmed by S2 gel electrophoresis of amplified fragments (in some cases, larger fragments were amplified for this purpose by combining primers from different amplicons) and sequencing of gel-purified normal and deletion allele products. MECP2 mutations identified were categorised into 3 classes – missense, early truncating and late truncating. The late truncating class included all mutations involving small (1-60bp) deletions and in/dels in the region encoding the C-terminal portion of MECP2, as well as a small number of larger deletions of 100-200bp. All these mutations alter the reading frame and are thus expected to cause premature truncation of the protein at stop codons within the new reading frame. As they are all located in the final exon, they are not expected to lead to nonsense-mediated mRNA decay, and should thus lead to expression of a truncated protein that is presumed to enter the nucleus and be capable of DNA-binding and possibly other aspects of the normal function of MECP2. All nonsense mutations located downstream of the TRD-NLS (amino acids 255-271 of the MECP2e2 isoform) were also included in this class. The early truncating class consisted of nonsense mutations up to and including the TRD-NLS, all larger deletions removing this region and all DNA ‘null’ mutations involving deletions spanning large parts of the gene. The TRD-NLS was chosen as the cut-off, since proteins that do not incorporate it are likely to be excluded from the nucleus and are therefore unlikely to be able to perform the major functions of MECP2 as they are currently understood. Since this effect is probably functionally equivalent to that of large deletions that result in ‘protein null’ phenotypes, the latter mutations were included in this class. The missense mutations were treated as a single group, as their phenotypic effects are not expected to vary consistently with position in the protein’s primary structure. For analyses of the phenotypic effects of the commonly occurring mutations, we have combined R306C and R306H, on the assumption that the phenotype arises from loss of a function provided by R306, and not from separate gains of function. This conclusion is supported by the fact that the substituted residues are of different functional categories, and H306 should have a positive charge (as does R306) in the nuclear environment whereas C306 does not. X chromosome inactivation analysis XCI analysis was performed using the HUMARA test. Briefly, genomic DNA extracted from blood and from buccal samples from female subjects was subjected to PCR amplification at the AR locus with and without prior HpaII digestion and the products were resolved and analysed by ABI GeneScan (Applied Biosystems, Foster City, CA) gel technology. XCI ratios in AR heterozygotes were calculated from peak areas for each allele36 and were expressed as the proportion of the allele with the greater peak area in the sample (i.e. 50-100%). A few additional datapoints were generated by similar analysis of the FMR1 promoter (CGG)n polymorphism S3 according to ref. [37], in patients homozygous at AR. Where available, the XCI ratio was calculated as the mean of replicate experiments performed on blood lymphocyte DNA. In a few cases, only buccal DNA was available, and values are given as means of replicate experiments using these samples. Direction of skewing relative to the MECP2 mutant allele has not yet been ascertained. Results Details of all mutations and mean XCI ratios present in cases analysed in this study are presented below. Table ST2 shows all mutations present in cases analysed in this study, ordered by their position from 5’- to 3’- in the gene (amino acid position is given relative to the initiator methionine of the MECP2e2 isoform, as used in most previous reports). Mutations found in Glasgow as part of this study are listed separately. References for mutations reported in other studies are given where appropriate. In addition to the data in the Table, a further 9 patients without identified mutations in MECP2 have been included in previously published datasets (four in ref. 22; three in ref. 23; one in ref. 38; one in both ref. 25 and ref. 24 [in both of these papers, the patient was reported as carrying p.R168X, but further analysis indicated that no mutation could be confirmed]). ## indicates patients reported previously as ‘No mutation’ in whom mutations were later discovered. Mutations in bold are those common mutations that were analysed singly (see text). The large deletions were found using either the MLPA procedure31 or a quantitative PCR procedure30. Note that several cases had two separate mutations. In these cases, the mutation predicted to have the more severe effect (early truncating or missense, in comparison with late truncating, for example, or bigger deletion vs. smaller deletion/indel) was used in the analysis. All cases with more than one mutation will be described in other publications. For those mutations observed in more than one case, their proportion in the mutation-positive set is given (%). The bold line below p.R270X represents the split between the Early truncating mutation category and the Late truncating category for nonsense/frameshifting mutations. CR indicates cases with classic Rett syndrome together with a small number of cases who have a typical but incomplete classic profile, usually due to their age (incCR). RnonC indicates atypical forms of Rett syndrome. * indicates case (female) was mosaic for mutation. ** indicates a small-medium truncating deletion or in/del mutation in the region of MECP2 encoding the C-terminal portion of the protein. Y indicates that the mutation is novel and reported for the first time here. Informative XCI ratio mean values (expressed as %, in the range 50100%) are given for each informative case for each mutation. ‡ indicates buccal sample only was available for XCI ratio testing. XCI ratio values in bold are moderately skewed (75%) and those in bold italic are extremely skewed (>90%). 7 S4 Supplementary Table ST2 Mutation (DNA) Mutation (Protein) All CR All Glasgow Glasgow % of cases Novel No. previously XCI ratio RnonC CR RnonC with this study reported (%) mutation (and ref.) 1 2 0 3.7 N/A 2 [23] 53, 58, 65, 1 [25]## 85, 95 0 81 g.Large deletion, multiple exons Null 4 c.91delG p.V31fsX30 1 c.139C>T p.Q47X 1 0 83 c.302C>T p.P101L 1 0 85 c.316C>T p.R106W 3 1 1 0 g.IVS3 -3C>G Null 0 1 0 0 c.382C>T p.Q128X 1 c.397C>T p.R133C 4 c.423C>G p.Y141X 1 0 c.455C>G p.P152R 6 5 4.4 c.473C>T p.T158M 13 8 9.6 c.481_987del507 + 481_482ins8 p.G161-G329delfsX8 1 0 c.495_1163del669 p.S166-P388del223 1 c.502C>T p.R168X 14 2 4 0 c.502C>T + 1136_1142del7 p.R168X + other 0 1 0 0 c.622C>T p.Q208X 1 0 c.730C>T p.Q244X 2 1 c.748_749insC p.R250fsX257 1 0 c.753delC p.G252fsX287 2 2 c.763C>T p.R255X 7 c.784C>T p.Q262X 1 0 c.792_804del13 p.I264fsX283 1 1 c.806delG p.G269fsX287 1 0 c.808C>T p.R270X 9 6 c.808C>T* p.R270X* 0 1* 0 0 c.880C>T p.R294X 3 1 1 0 c.905C>G p.P302R 2 1 1.5 c.905C>T p.P302L 2 0 1.5 c.910A>C p.K304Q 1 c.916C>T p.R306C 8 c.917G>A p.R306H 2 1 c.1054_1259del206** p.K352fsX366 1 1 Y 96 c.1101_1201del101** p.H367fsX369 1 1 Y 66 c.1116_1201del86** p.H372fsX374 1 0 c.1126_1159del34ins39** p.P376fsX400 1 1 c.1152_1192del41** p.P385fsX390 1 1 c.1152_1195del44** p.P385fsX389 1 0 c.1154_1197del44** p.P385fsX402 1 0 c.1157_1196del40** p.L386fsX394 1 1 3.0 60, 75, 77 0 5 2 0 6.7 2 1 [22,25] 1 [22;25##] 1 1 3 [22] 1 [25] 51, 52, 64, 65, 66‡, 68, 77 57‡, 58, 60, 63, 82 51, 56, 62, 68, 68, 69, 74, 78, 80 80 Y 0 11.9 2 [24] 1 [25] 50, 57, 63, 64, 73, 74, 78‡ 1 [25] 62‡ 1.5 79, 98 1.5 64 5.9 1 [22,25] 1[22,24,25] 1 [25] 59, 68, 72, 72, 77, 91 54 Y 6.7 1 [22] 3.0 63, 67, 70, 70, 74, 75, 91 51 1 [22] 0 2 S5 6 1 7.4 1 [22; 25##] 1.5 54, 58, 64, 66, 66, 67, 72, 72, 84 54, 63 Y 52‡ 1 [22, 25] 67 50 Y 88 c.1157_1197del41** p.L386fsX389 3 c.1157_1200del44** p.L386fsX388 4 1 c.1157_1200del44**, +c.862G>A 1 0 c.1157_1202del46** p.L386fsX388, +p.V288M p.L386fsX392 1 0 c.1158_1201del44** p.P387fsX388 1 c.1159_1202del44**, +1098_1100del3 0 1 0 0 83 c.1163_1206del44** p.P387fsX389 +p.H367del p.P388fsX486 0 1 0 0 72 c.1164_1207del44** p.P389fsX388 2 0 c.1224_1267del44 [=c.1160_1203del44**?] p.S409fsX481? 1 0 c.1398_1428del31**? 1 0 p.M466fsX470 Totals 1 1 0 3.0 1 [22] 58, 59 3.0 1 [25] 60, 61, 71 74 0 N=116 N=19 N=51 1.5 N=1 77.3% 55 6 Novel Mutation screening A summary of the results for Set A (patients screened specifically for this study in Glasgow) is set out here: of the 62 RS patients, both classic (n=57) and atypical (n=5), specifically screened for this study, mutations were identified in 52, including two cases with large deletions of the region of MECP2 containing exons 3 and 4, identified using the MLPA assay. Ten patients had no identifiable mutations in exons 2, 3 or 4 by sequencing, seven of whom were screened using the MLPA assay or by qPCR - no copy number anomalies were found in these patients. Of the 57 classic RS patients in Set A, 51 (89.5%) were found to have MECP2 mutations; 2 of the 6 classic cases without identified mutations remain to be checked by appropriate dosagesensitive methods. One of the 6 atypical Rett patients was found to have a mutation (p.R306C). The following statistics are presented for the entire dataset reported in this paper (set A and set B): 77% of cases with identified mutations carried a ‘recurrent’ mutation (observed in more than 1 case in the dataset). 19% of classic CR cases (22/116) and 16% of atypical cases (3/19) with identified mutations in our dataset carried small-medium truncating deletions or in/dels in the region encoding the C-terminal portion of MECP2 (indicated by ** in Table ST2). 85% of classic CR cases with mutations carried ‘recurrent’ mutations, including the C-terminal region deletions and in/dels. XCI ratio analysis Of the 135 patients with identified mutations, we were able to generate or gather existing XCI ratio data for 110 (81%). Of these, only 85 were informative, the rest being homozygous at the AR and FMR1 repeat loci genotyped or for whom the assay failed. We also generated or gathered XCI ratio data for 39 cases with no identified mutation, of which 33 were informative (data not shown). S6 N=85 References 35 Buyse IM, Fang P, Hoon KT, Amir RE, Zoghbi HY, Roa BB. Diagnostic testing for Rett syndrome by DHPLC and direct sequencing analysis of the MECP2 gene: Identification of several novel mutations and polymorphisms. Am J Hum Genet 2000; 67: 1428-36 36 Pegoraro E, Schimke RN, Arahara K, et al. Detection of new paternal dystrophin gene mutations in isolated cases of dystrophinopathy in females. Am J Hum Genet 1994; 54: 989-1003 37 Carrel L, Willard HF. An assay for X inactivation based on differential methylation at the fragile X locus, FMR1. Am J Med Genet 1996; 64: 27-30 38 Borg I, Freude K, Kübart S, Hoffmann K, Menzel C, Laccone F, Firth H, Ferguson-Smith MA, Tommerup N, Ropers H-H, Sargan D, Kalscheuer VM. Disruption of Netrin G1 by a balanced chromosome translocation in a girl with Rett syndrome. Eur J Hum Genet; AOP doi:10.1038/sj.ejhg.5201429 S7