Ruthie E. Amir

Bio: Ruthie E. Amir is an academic researcher from Baylor College of Medicine. The author has contributed to research in topics: Rett syndrome & MECP2. The author has an hindex of 9, co-authored 10 publications receiving 5446 citations.

Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2.

Abstract: Rett syndrome (RTT, MIM 312750) is a progressive neurodevelopmental disorder and one of the most common causes of mental retardation in females, with an incidence of 1 in 10,000-15,000 (ref. 2). Patients with classic RTT appear to develop normally until 6-18 months of age, then gradually lose speech and purposeful hand use, and develop microcephaly, seizures, autism, ataxia, intermittent hyperventilation and stereotypic hand movements. After initial regression, the condition stabilizes and patients usually survive into adulthood. As RTT occurs almost exclusively in females, it has been proposed that RTT is caused by an X-linked dominant mutation with lethality in hemizygous males. Previous exclusion mapping studies using RTT families mapped the locus to Xq28 (refs 6,9,10,11). Using a systematic gene screening approach, we have identified mutations in the gene (MECP2 ) encoding X-linked methyl-CpG-binding protein 2 (MeCP2) as the cause of some cases of RTT. MeCP2 selectively binds CpG dinucleotides in the mammalian genome and mediates transcriptional repression through interaction with histone deacetylase and the corepressor SIN3A (refs 12,13). In 5 of 21 sporadic patients, we found 3 de novo missense mutations in the region encoding the highly conserved methyl-binding domain (MBD) as well as a de novo frameshift and a de novo nonsense mutation, both of which disrupt the transcription repression domain (TRD). In two affected half-sisters of a RTT family, we found segregation of an additional missense mutation not detected in their obligate carrier mother. This suggests that the mother is a germline mosaic for this mutation. Our study reports the first disease-causing mutations in RTT and points to abnormal epigenetic regulation as the mechanism underlying the pathogenesis of RTT.

Rett syndrome and beyond: recurrent spontaneous and familial MECP2 mutations at CpG hotspots.

Abstract: Rett syndrome (RTT) is a neurodevelopmental disorder characterized by loss of acquired skills after a period of normal development in infant girls. The responsible gene, encoding methyl-CpG binding protein 2 (MeCP2), was recently discovered. Here we explore the spectrum of phenotypes resulting from MECP2 mutations. Both nonsense (R168X and R255X) and missense (R106W and R306C) mutations have been found, with multiple recurrences. R168X mutations were identified in six unrelated sporadic cases, as well as in two affected sisters and their normal mother. The missense mutations were de novo and affect conserved domains of MeCP2. All of the nucleotide substitutions involve C→T transitions at CpG hotspots. A single nucleotide deletion, at codon 137, that creates a L138X stop codon within the methyl-binding domain was found in an individual with features of RTT and incontinentia pigmenti. An 806delG deletion causing a V288X stop in the transcription-repression domain was identified in a woman with motor-coordination problems, mild learning disability, and skewed X inactivation; in her sister and daughter, who were affected with classic RTT; and in her hemizygous son, who died from congenital encephalopathy. Thus, some males with RTT-causing MECP2 mutations may survive to birth, and female heterozygotes with favorably skewed X-inactivation patterns may have little or no involvement. Therefore, MECP2 mutations are not limited to RTT and may be implicated in a much broader phenotypic spectrum.

Influence of mutation type and X chromosome inactivation on Rett syndrome phenotypes

Abstract: We screened 71 sporadic and 7 familial Rett syndrome (RTT) patients for MECP2 mutations by direct sequencing and determined the pattern of X chromosome inactivation (XCI) in 39 RTT patients. We identified 23 different disease-causing MECP2 mutations in 54 of 71 (76%) sporadic patients and in 2 of 7 (29%) familial cases. We compared electrophysiological findings, cerebrospinal fluid neurochemistry, and 13 clinical characteristics between patients carrying missense mutations and those carrying truncating mutations. Thirty-one of 34 patients (91%) with classic RTT had random XCI. Nonrandom XCI was associated with milder phenotypes, including a mitigated classic RTT caused by a rare early truncating mutation. Patients with truncating mutations have a higher incidence of awake respiratory dysfunction and lower levels of cerebrospinal fluid homovanillic acid. Scoliosis is more common in patients with missense mutations. These data indicate that different MECP2 mutations have similar phenotypic consequences, and random XCI plays an important role in producing the full phenotypic spectrum of classic RTT. The association of early truncating mutations with nonrandom XCI, along with the fact that chimeric mice lacking methyl-CpG-binding protein 2 (MeCP2) function die during embryogenesis, supports the notion that RTT is caused by partial loss of MeCP2 function.

Rett syndrome: methyl-CpG-binding protein 2 mutations and phenotype-genotype correlations.

Abstract: Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder that manifests in females, typically after the first year of life. It is a leading cause of mental retardation and autistic behavior in girls and women; a hallmark of the disease is incessant hand movements in the form of wringing, twisting, or clapping. It was recently discovered that RTT is caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene. MECP2 assists in the transcriptional silencing process via DNA methylation; we hypothesize that disruption of this gene alters the normal developmental expression of various other genes, some of which must account for the peculiar neurologic phenotype of RTT. Molecular studies have identified MECP2 mutations in up to 80% of classic RTT patients; mutation type has some effect on the phenotypic manifestation of RTT, but the pattern of X inactivation seems to determine phenotypic severity. Favorable (skewed) X inactivation can so spare a patient from the effects of mutant MECP2 that they display only the mildest learning disability or no phenotype at all. The unmitigated impact of mutant MECP2 can be inferred from the few males who have been born into RTT kindreds with such severe neonatal encephalopathy that they did not survive their second year. MECP2 mutations thus manifest in a far broader array of phenotypes than classic RTT. This discovery should prove helpful in diagnosing cases of mild learning disability or severe neonatal encephalopathies of unknown cause and also should provide insight into the pathogenesis of RTT.

Diagnostic Testing for Rett Syndrome by DHPLC and Direct Sequencing Analysis of the MECP2 Gene: Identification of Several Novel Mutations and Polymorphisms

Abstract: Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder affecting 1/10,000–15,000 girls. The disease-causing gene was identified as MECP2 on chromosome Xq28, and mutations have been found in ∼80% of patients diagnosed with RTT. Numerous mutations have been identified in de novo and rare familial cases, and they occur primarily in the methyl-CpG–binding and transcriptional-repression domains of MeCP2. Our first diagnostic strategy used bidirectional sequencing of the entire MECP2 coding region. Subsequently, we implemented a two-tiered strategy that used denaturing high-performance liquid chromatography (DHPLC) for initial screening of nucleotide variants, followed by confirmatory sequencing analysis. If a definite mutation was not identified, then the entire MECP2 coding region was sequenced, to reduce the risk of false negatives. Collectively, we tested 228 unrelated female patients with a diagnosis of possible (209) or classic (19) RTT and found MECP2 mutations in 83 (40%) of 209 and 16 (84%) of 19 of the patients, respectively. Thirty-two different mutations were identified (8 missense, 9 nonsense, 1 splice site, and 14 frameshifts), of which 12 are novel and 9 recurrent in unrelated patients. Seven unclassified variants and eight polymorphisms were detected in 228 probands. Interestingly, we found that T203M, previously reported as a missense mutation in an autistic patient, is actually a benign polymorphism, according to parental analysis performed in a second case identified in this study. These findings highlight the complexities of missense variant interpretation and emphasize the importance of parental DNA analysis for establishing an etiologic relation between a possible mutation and disease. Overall, we found a 98.8% concordance rate between DHPLC and sequence analyses. One mutation initially missed by the DHPLC screening was identified by sequencing. Modified conditions subsequently enabled its detection, underscoring the need for multiple optimized conditions for DHPLC analysis. We conclude that this two-tiered approach provides a sensitive, robust, and efficient strategy for RTT molecular diagnosis.

DNA methylation patterns and epigenetic memory

Abstract: The character of a cell is defined by its constituent proteins, which are the result of specific patterns of gene expression. Crucial determinants of gene expression patterns are DNA-binding transcription factors that choose genes for transcriptional activation or repression by recognizing the sequence of DNA bases in their promoter regions. Interaction of these factors with their cognate sequences triggers a chain of events, often involving changes in the structure of chromatin, that leads to the assembly of an active transcription complex (e.g., Cosma et al. 1999). But the types of transcription factors present in a cell are not alone sufficient to define its spectrum of gene activity, as the transcriptional potential of a genome can become restricted in a stable manner during development. The constraints imposed by developmental history probably account for the very low efficiency of cloning animals from the nuclei of differentiated cells (Rideout et al. 2001; Wakayama and Yanagimachi 2001). A “transcription factors only” model would predict that the gene expression pattern of a differentiated nucleus would be completely reversible upon exposure to a new spectrum of factors. Although many aspects of expression can be reprogrammed in this way (Gurdon 1999), some marks of differentiation are evidently so stable that immersion in an alien cytoplasm cannot erase the memory. The genomic sequence of a differentiated cell is thought to be identical in most cases to that of the zygote from which it is descended (mammalian B and T cells being an obvious exception). This means that the marks of developmental history are unlikely to be caused by widespread somatic mutation. Processes less irrevocable than mutation fall under the umbrella term “epigenetic” mechanisms. A current definition of epigenetics is: “The study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence” (Russo et al. 1996). There are two epigenetic systems that affect animal development and fulfill the criterion of heritability: DNA methylation and the Polycomb-trithorax group (Pc-G/trx) protein complexes. (Histone modification has some attributes of an epigenetic process, but the issue of heritability has yet to be resolved.) This review concerns DNA methylation, focusing on the generation, inheritance, and biological significance of genomic methylation patterns in the development of mammals. Data will be discussed favoring the notion that DNA methylation may only affect genes that are already silenced by other mechanisms in the embryo. Embryonic transcription, on the other hand, may cause the exclusion of the DNA methylation machinery. The heritability of methylation states and the secondary nature of the decision to invite or exclude methylation support the idea that DNA methylation is adapted for a specific cellular memory function in development. Indeed, the possibility will be discussed that DNA methylation and Pc-G/trx may represent alternative systems of epigenetic memory that have been interchanged over evolutionary time. Animal DNA methylation has been the subject of several recent reviews (Bird and Wolffe 1999; Bestor 2000; Hsieh 2000; Costello and Plass 2001; Jones and Takai 2001). For recent reviews of plant and fungal DNA methylation, see Finnegan et al. (2000), Martienssen and Colot (2001), and Matzke et al. (2001).

Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals

Abstract: Cells of a multicellular organism are genetically homogeneous but structurally and functionally heterogeneous owing to the differential expression of genes. Many of these differences in gene expression arise during development and are subsequently retained through mitosis. Stable alterations of this kind are said to be 'epigenetic', because they are heritable in the short term but do not involve mutations of the DNA itself. Research over the past few years has focused on two molecular mechanisms that mediate epigenetic phenomena: DNA methylation and histone modifications. Here, we review advances in the understanding of the mechanism and role of DNA methylation in biological processes. Epigenetic effects by means of DNA methylation have an important role in development but can also arise stochastically as animals age. Identification of proteins that mediate these effects has provided insight into this complex process and diseases that occur when it is perturbed. External influences on epigenetic processes are seen in the effects of diet on long-term diseases such as cancer. Thus, epigenetic mechanisms seem to allow an organism to respond to the environment through changes in gene expression. The extent to which environmental effects can provoke epigenetic responses represents an exciting area of future research.

Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2.

Abstract: Rett syndrome (RTT, MIM 312750) is a progressive neurodevelopmental disorder and one of the most common causes of mental retardation in females, with an incidence of 1 in 10,000-15,000 (ref. 2). Patients with classic RTT appear to develop normally until 6-18 months of age, then gradually lose speech and purposeful hand use, and develop microcephaly, seizures, autism, ataxia, intermittent hyperventilation and stereotypic hand movements. After initial regression, the condition stabilizes and patients usually survive into adulthood. As RTT occurs almost exclusively in females, it has been proposed that RTT is caused by an X-linked dominant mutation with lethality in hemizygous males. Previous exclusion mapping studies using RTT families mapped the locus to Xq28 (refs 6,9,10,11). Using a systematic gene screening approach, we have identified mutations in the gene (MECP2 ) encoding X-linked methyl-CpG-binding protein 2 (MeCP2) as the cause of some cases of RTT. MeCP2 selectively binds CpG dinucleotides in the mammalian genome and mediates transcriptional repression through interaction with histone deacetylase and the corepressor SIN3A (refs 12,13). In 5 of 21 sporadic patients, we found 3 de novo missense mutations in the region encoding the highly conserved methyl-binding domain (MBD) as well as a de novo frameshift and a de novo nonsense mutation, both of which disrupt the transcription repression domain (TRD). In two affected half-sisters of a RTT family, we found segregation of an additional missense mutation not detected in their obligate carrier mother. This suggests that the mother is a germline mosaic for this mutation. Our study reports the first disease-causing mutations in RTT and points to abnormal epigenetic regulation as the mechanism underlying the pathogenesis of RTT.

Epigenetics in human disease and prospects for epigenetic therapy

Abstract: Epigenetic mechanisms, which involve DNA and histone modifications, result in the heritable silencing of genes without a change in their coding sequence. The study of human disease has focused on genetic mechanisms, but disruption of the balance of epigenetic networks can cause several major pathologies, including cancer, syndromes involving chromosomal instabilities, and mental retardation. The development of new diagnostic tools might reveal other diseases that are caused by epigenetic alterations. Great potential lies in the development of ‘epigenetic therapies’ — several inhibitors of enzymes controlling epigenetic modifications, specifically DNA methyltransferases and histone deacetylases, have shown promising anti-tumorigenic effects for some malignancies.

Strong Association of De Novo Copy Number Mutations with Autism

Abstract: We tested the hypothesis that de novo copy number variation (CNV) is associated with autism spectrum disorders (ASDs). We performed comparative genomic hybridization (CGH) on the genomic DNA of patients and unaffected subjects to detect copy number variants not present in their respective parents. Candidate genomic regions were validated by higher-resolution CGH, paternity testing, cytogenetics, fluorescence in situ hybridization, and microsatellite genotyping. Confirmed de novo CNVs were significantly associated with autism (P = 0.0005). Such CNVs were identified in 12 out of 118 (10%) of patients with sporadic autism, in 2 out of 77 (3%) of patients with an affected first-degree relative, and in 2 out of 196 (1%) of controls. Most de novo CNVs were smaller than microscopic resolution. Affected genomic regions were highly heterogeneous and included mutations of single genes. These findings establish de novo germline mutation as a more significant risk factor for ASD than previously recognized.