A rare male patient with classic Rett syndrome caused by MeCP2_e1 mutation

Rett syndrome (RTT) is a severe neurodevelopmental disorder typically affecting females. It is mainly caused by loss‐of‐function mutations that affect the coding sequence of exon 3 or 4 of methyl‐CpG‐binding protein 2 (MECP2). Severe neonatal encephalopathy resulting in death before the age of 2 years is the most common phenotype observed in males affected by a pathogenic MECP2 variant. Mutations in MECP2 exon 1 affecting the MeCP2_e1 isoform are relatively rare causes of RTT in females, and only one case of a male patient with MECP2‐related severe neonatal encephalopathy caused by a mutation in MECP2 exon 1 has been reported. This is the first reported case of a male with classic RTT caused by a 5‐bp duplication in the open‐reading frame of MECP2 exon 1 (NM_001110792.1:c.23_27dup) that introduced a premature stop codon [p.(Ser10Argfs*36)] in the MeCP2_e1 isoform, which has been reported in one female patient with classic RTT. Therefore, both males and females displaying at least some type of MeCP2_e1 mutation may exhibit the classic RTT phenotype.

     

Rett综合征MECP2基因突变分析

目的 分析我国典型的Rett综合征患儿甲基化CpG结合蛋白－2基因（methyl-CpG-binding protein 2,MECP2）突变。方法 使用PCR扩增、单链构象多态性分析、PCR产物克隆和DNA测序的方法。检测分析了26例Rett综合征患儿、其父母和其中2例患儿的妹妹MECP2基因3个外显子的基因突变。结果 26例Rett综合征患儿中发现14例有9种类型MECP2基因的杂合性突变，突变均位于第3外显子。其中7例有3种错义突变：C473T（T158M)4例,C674G(P225R)1例，C916T（R306C）2例；4例有3种无义突变：C502T（R168X）2例，C763T（R255X）1例，C880T（R294X）1例；2种由于缺失导致的突变：1例为1152del 44bp和1例1158－1167／1171－1186del 26bp；1鲍由于碱基插入导致的移码突变：874insA。1158－1167／1171－1186del 26bp和874insA突变为首次报千，突变均为新生突变。此外，新发现了一种源于你亲的错义变异1141G（P381A）。结论 我国Rett综合征患儿存在MECP2基因突变，典型的Rett综合征MECP2基因突变率大于50％。     

MECP2 deletions and genotype-phenotype correlation in Rett syndrome

Rett syndrome is a neurodevelopmental disorder that represents one of the most common genetic causes of mental retardation in girls. MECP2 point mutations in exons 2-4 account for about 80% of classic Rett cases and for a lower percentage of variant patients. We investigated the genetic cause in 77 mutation-negative Rett patients (33 classic, 31 variant, and 13 Rett-like cases) by searching missed MECP2 defects. DHPLC analysis of exon 1 and MLPA analysis allowed us to identify the defect in 17 Rett patients: one exon 1 point mutation (c.47_57del) in a classic case and 16 MECP2 large deletions (15/33 classic and 1/31 variant cases). One identical intragenic MECP2 deletion, probably due to gonadal mosaicism, was found in two sisters with discordant phenotype: one classic and one "highly functioning" preserved speech variant. This result indicates that other epigenetic or genetic factors, beside MECP2, may contribute to phenotype modulation. Three out of 16 MECP2 deletions extend to the adjacent centromeric IRAK1 gene. A putative involvement of the hemizygosity of this gene in the ossification process is discussed. Finally, results reported here clearly indicate that MECP2 large deletions are a common cause of classic Rett, and MLPA analysis is mandatory in MECP2-negative patients, especially in those more severely affected (P = 0.044).     

Clinical profiles of four patients with Rett syndrome carrying a novel exon 1 mutation or genomic rearrangement in the MECP2 gene

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the X-linked MECP2 gene encoding methyl CpG binding protein 2 (MeCP2). Recently, a new isoform of MeCP2 including exon 1 was identified. This new isoform is more abundantly expressed in brain than the isoform including exons 2-4. Very little is known about the phenotypes associated with mutations in exon 1 of MECP2 since only a limited number of RTT patients carrying such mutations have been identified so far. In this study, we screened a cohort of 20 girls with RTT for exon 1 mutations by sequencing and multiplex ligation-dependent probe amplification (MLPA). We identified one girl with a novel exon 1 mutation (c.30delCinsGA) by sequencing and three with genomic rearrangements by MLPA. Comparison of the phenotypes showed that the girls carrying a mutation or rearrangement encompassing exon 1 were more severely affected than the girls with rearrangements not affecting exon 1.     

Variation in exon 1 coding region and promoter of MECP2 in Rett syndrome and controls

Mutations in MECP2 are a cause of Rett syndrome. Recently, a new isoform of MeCP2 was described, which has an alternative N-terminus, transcribed from exon 1. We screened exon 1 and the promoter region of MECP2 in 97 mutation-negative Rett syndrome cases. We found two sequence variants, but there was no evidence that they are pathogenic. Mutations in exon 1 and the promoter of MECP2 are not a common cause of Rett syndrome.     

Real-time quantitative PCR as a routine method for screening large rearrangements in Rett syndrome: Report of one case of MECP2 deletion and one case of MECP2 duplication

Mutations in the X-linked methyl-CpG-binding protein 2 gene (MECP2) are found in 70-80% of cases of classical Rett syndrome (RTT) and in about 50% of cases of preserved speech variant (PSV). This high percentage of MECP2 mutations, especially in classical RTT cases, suggests that another major RTT locus is unlikely. Missed mutations may be due to the limited sensitivity of the methodology used for mutation scanning and/or the presence of intronic mutations. In a double-copy gene, such as MECP2 in females, current methodologies (e.g., DGGE, SSCP, DHPLC, direct sequencing) are prone to miss gross rearrangements. Three previous reports during 2001-2003 have shown the presence of large deletions in a fraction of MECP2-negative classical RTT patients. We developed a reliable, single tube, quantitative PCR assay for rapid determination of MECP2 gene dosage. This method involves a multiplex reaction using a FAM labeled TaqMan probe with a TAMRA quencher derived from MECP2 exon 4 and two primers derived from the same exon and RNAaseP as an internal reference. The copy number of the MECP2 gene was determined by the comparative threshold cycle method (ddCt). Each sample was run in quadruplicate. We validated this assay through the analysis of 30 healthy controls (15 female and 15 male) and we then applied this method to eight classical RTT and six PSV patients, all negative for MECP2 mutations. We identified gross rearrangements in two patients: a deletion in a classical RTT patient and a duplication in a PSV patient. Our results confirm that a fraction of MECP2-negative RTT cases have MECP2 gross rearrangements and we propose real-time quantitative PCR as a simple and reliable method for routine screening of MECP2 in addition to DHPLC analysis.     

Spectrum and distribution of MECP2 mutations in 424 Rett syndrome patients: a molecular update

Mutations in the MECP2 (Methyl-CpG-binding protein) gene have been reported to cause Rett syndrome (RTT), an X-linked progressive encephalopathy. Recent studies have identified large gene rearrangements that escape the common PCR-based mutation screening strategy and mutations in a novel MeCP2 isoform (named MECP2B). We have collected the results of MECP2 mutational analysis concerning 424 RTT patients conducted in eight laboratories in France. In total, 121 different MECP2 mutations were identified. R168X (11.5%) is the most common of MECP2 mutations, followed by R270X (9%), R255X (8.7%), T158 M (8.3%) and R306C (6.8%). Only eight mutations had relative frequency>3%. Large and complex rearrangements not previously detected using only a PCR-based strategy represent 5.8% of MECP2 mutations. On the contrary, mutation in exon 1 appears to be rare (less than 0.5%). These data demonstrate the high allelic heterogeneity of RTT in France and suggest that routine mutation screening in MECP2 should include quantitative analysis of the MECP2 gene. This study represents an important instrument for molecular diagnosis strategy and genetic counseling in RTT families.     

Detection of heterozygous deletions and duplications in the MECP2 gene in Rett syndrome by Robust Dosage PCR (RD-PCR)

Fifty to eighty percent of Rett syndrome (RTT) cases have point mutations in the gene encoding methyl-CpG-binding protein-2 (MECP2). A fraction of MECP2 negative classical RTT patients has large heterozygous deletions. Robust Dosage PCR (RD-PCR) assays were developed as a rapid, convenient and accurate method to detect large heterozygous deletions and duplications. A blinded analysis was performed for 65 RTT cases from Portugal by RD-PCR in the coding exons 2-4 of the MECP2 gene. Neither the patients with point mutations nor the non-classical RTT patients without point mutation had a deletion or duplication. One of remaining eight female patients with classical RTT without point mutation had a heterozygous deletion. This is the first report of a deletion spanning the entire MECP2 gene. The deletion was confirmed by Southern blotting analysis and the deletion junction was localized 37 kb upstream from exon 1 and 18 kb downstream from exon 4. No duplications were detected. Our results suggest that RD-PCR is an accurate and convenient molecular diagnostic method.     

Large genomic rearrangements in MECP2

In 1999, mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2) were first reported in patients with Rett syndrome (RTT). The MECP2 gene is located at Xq28 and consists of 4 exons. About 80-90 % of the classic RTT patients harbor mutations in the coding region of MECP2, while the molecular cause is unknown in the remaining 10-20%. Several groups have searched for large rearrangements within the MECP2 and the results indicate that a fraction of MECP2-negative RTT cases has large deletions of the MECP2. In this study we have used the Multiplex Ligation-dependent Probe Amplification (MLPA) technique to screen 45 RTT patients, who have previously been tested negative for mutations in the coding region of MECP2. The MECP2-MLPA is a semi-quantitative multiplex PCR approach. It determines the relative number of copies of each MECP2 exon. With this approach we detected seven RTT patients with genomic deletions and further characterized the deletions using real time quantitative PCR (qPCR) and long-range PCR. The seven patients were given a severity score and their X chromosome inactivation profiles were determined in order to identify a possible genotype-phenotype correlation. The results from this study indicate that large deletions in MECP2 cause classic RTT.     

Skewed X chromosome inactivation failed to explain the normal phenotype of a carrier female with MECP2 mutation resulting in Rett syndrome

Mutations in the X-linked MECP2 gene cause Rett syndrome, a neurodevelopmental disorder that exclusively affects girls. Females with the MECP2 mutations exhibit a broad spectrum of clinical presentations ranging from classical Rett syndrome to asymptomatic carriers, which can be explained by differences in X chromosome inactivation (XCI). Here, we report a family with a girl with Rett syndrome in whom a novel missense mutation in the MECP2 gene was transmitted through the maternal germ line. The carrier mother was asymptomatic and presented non-random XCI in the peripheral blood cells, which resulted in the X chromosome harboring the mutant allele that was predominantly active. Thus, the presence of non-random XCI in the peripheral blood cells did not provide an explanation for the normal phenotype of the carrier mother. This result suggests that mechanisms other than XCI may contribute to the phenotypic heterogeneity associated with MECP2 mutations.     

MECP2 gene mutation analysis in Chinese patients with Rett syndrome

Rett syndrome (RTT) is a progressive neurodevelopmental disorder that affects almost exclusively girls. Mutations in the X-linked methyl-CpG-binding protein 2 gene (MECP2) have been found to be a cause. In order to study the spectrum of MECP2 mutations in Chinese patients, we employed PCR and sequencing of the coding region of MECP2 gene in 31 Chinese cases of classical sporadic RTT. Mutations in MECP2 were found in about 55%. Twelve different mutations in exon 3 were identified in 17 of these 31 patients; two of these are novel. A novel missense variant was detected in the C-terminal region in a patient and her father who was normal. In addition, there was a single nucleotide variant in the 3'UTR.     

Somatic mosaicism for a MECP2 mutation associated with classic Rett syndrome in a boy

Rett syndrome is a severe neurodevelopmental disorder that arises from mutations in the X-linked MECP2 gene. It is almost exclusively seen in girls due to the predominant occurrence of the mutations on the paternal X-chromosome, and also the early postnatal lethal effect of the disease causing mutations in hemizygous boys. We identified a boy with features of classic Rett syndrome who is mosaic for the truncating MECP2 mutation R270X. Chromosome analysis showed normal karyotype. These results indicate that a MECP2 mutation associated with Rett syndrome in females could lead to a similar phenotype in males as a result of somatic mosaicism.     

Five novel frameshift mutations in exon 3 and 4 of the MECP2 gene identified in Rett patients: Consequences for the molecular diagnosis strategy

Rett syndrome (RTT) is a severe progressive neurological disorder that affects almost exclusively females. The gene responsible for this disorder, MECP2, was recently identified by candidate gene strategy. Mutations were detected in 70-85% of RTT cases. We report here five novel frameshift mutations (named 345delC, 895del202, 989ins18del8, 996insAG and 1124del53) in exon 3 and 4 of the MECP2 gene. To avoid the missing of few small deletions in RTT patients using classical mutation screening approaches, we suggest that screening of the mutations in the MECP2 gene in RTT girls should include at least a large PCR to amplify exon 4 entirely.     

Gross rearrangements in the MECP2 gene in three patients with Rett syndrome: implications for routine diagnosis of Rett syndrome

Since 1999, many laboratories have confirmed that mutations in the MECP2 gene are the primary cause of Rett syndrome (RTT or RS) and identified mutations in 70 to 90% of the sporadically affected girls. Most of the screenings are PCR-based and restricted to the coding part of the gene and therefore prone to miss gross rearrangements. By Southern blot analysis we identified large deletions (>1 kb) in three female patients with classical, severe Rett syndrome. Further characterization by semi-quantitative PCR and amplification of junction fragments confirmed the presence of a 7.6-kb deletion in one patient and an 8.1-kb deletion in the other patient, both including exon 3 and the coding part of exon 4. The exact nature of the rearrangement in the third patient remained elusive. These results underline the importance of screening for major genomic rearrangements in Rett syndrome. Hum Mutat 22:116-120, 2003.     

De novo MECP2 frameshift mutation in a boy with moderate mental retardation,obesity and gynaecomastia

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the MECP2 gene, with apparent lethality in male embryos. However, recent studies indicate that mutations in the MECP2 gene can cause congenital encephalopathy, an Angelman-like phenotype and even nonspecific mental retardation in males. We report on a 10-year-old boy with moderate mental retardation, hypotonia, obesity and gynaecomastia and a de novo 2-bp deletion in the MECP2 gene that resulted in a frameshift and premature stop codon. As some of the clinical features were suggestive of the Prader-Willi syndrome, it might be worthwhile screening for MECP2 mutations in patients with an atypical Prader-Willi phenotype but without the characteristic abnormalities on chromosome 15q. This report contributes to the phenotypic knowledge of male patients with MECP2 mutations. Moreover, this is the first reported male case of a de novo MECP2 mutation.     

Effects of MECP2 mutation type,location and X-inactivation in modulating Rett syndrome phenotype

Rett syndrome (RTT) is a clinically defined disorder that describes a subset of patients with mutations in the X-linked MECP2 gene. However, there is a high degree of variability in the clinical phenotypes produced by mutations in MECP2, even amongst classical RTT patients. In a large-scale screening project, this variability has been examined by looking at the effects of mutation type, functional domain affected and X-inactivation. Mutations have been identified in 60% of RTT patients in this study (25% of whom were atypical), including 23 novel mutations and polymorphisms. More mutations were found in classical patients (63%) compared to atypical patients (44%). All of the pathogenic mutations were de novo in patients for whom parent DNA was available for screening. A composite phenotype score was developed, based on the recommendations for reporting clinical features in RTT of an international collaborative group. This score proved useful for summarising phenotypic severity, but did not correlate with mutation type, domain affected or X-inactivation, probably due to complex interactions between all three. Other correlations suggested that truncating mutations and mutations affecting the methyl-CpG-binding domain tend to lead to a more severe phenotype. Skewed X-inactivation was found in a large proportion (43%) of our patients, particularly in those with truncating mutations and mutations affecting the MBD. It is therefore likely that X-inactivation does modulate the phenotype in RTT.     

p.R270X MECP2 mutation and mortality in Rett syndrome

Among cases in the Australian Rett Syndrome Database, the nonsense mutation p.R270X is one of the most commonly occurring single pathogenic MECP2 mutations. In two recent published reports of the MECP2 mutational spectrum the p.R270X appeared to be under represented. We hypothesised that increased mortality arising from this mutation may underlie this apparent discrepancy. We investigated our hypothesis in two independent study groups from Australia and the UK with prospective data collections (total n=524). Only females with Rett syndrome and an identified MECP2 mutation were included. Significant differences in survival were detected among Rett syndrome cases grouped for the eight most frequent mutations (log-rank chi(2) (7)=15.71, P=0.03). Moreover, survival among cases with p.R270X, when compared with survival among cases with all the other mutations was reduced (log-rank chi(2) (2)=6.94, P=0.01). Our observation of a reduced survival associated with the p.R270X mutation offers an explanation for the under representation of p.R270X in older subjects with Rett syndrome.     

Novel de novo nonsense mutation of MECP2 in a patient with Rett syndrome

Because of the recent identification of several mutations of methyl-CpG-binding protein 2 (MECP2) in patients with Rett syndrome (RTT), a patient with suspected RTT from an autism clinic was screened for mutations. She was found to have a novel heterozygous nonsense mutation, 129C>T (Q19X), which leads to the most severely truncated MECP2 protein reported to date. Sequencing of parental DNA revealed the mutation was de novo. The patient was not affected with microcephaly or hyperventilation, but had other features of Rett syndrome including severe mental retardation and symptoms of autistic disorder. Moderately skewed X-chromosome inactivation (XCI) may have contributed to her relatively mild phenotype.