Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-09-07
2004-03-23
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
Reexamination Certificate
active
06709817
ABSTRACT:
The work herein was supported by grants from the United States Government. The Government may have certain rights in the invention.
FIELD OF THE INVENTION
The invention relates to detection of mutations in a methyl-CpG-binding domain-containing protein. More particularly it relates to detecting mutations in MECP2, MECP1, MBD1, MBD2, MBD3, and MBD4. It further relates to detection and treatment of neurodevelopmental disease.
BACKGROUND OF THE INVENTION
Rett syndrome (herein used interchangeably with the term “RTT”), first described by Andreas Rett (1966), is a progressive neurodevelopmental disorder and one of the most common causes of mental retardation in females, with an incidence of 1 in 10-15,000 (Hagberg, 1985). Patients with classic Rett syndrome 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 (Hagberg et al., 1983). After initial regression, patients stabilize and usually survive into adulthood. Since Rett syndrome occurs almost exclusively in females, it was proposed that RTT is caused by an X-linked dominant mutation with lethality in hemizygous males (Hagberg et al., 1983, Zoghbi 1988, Zoghbi et al., 1990, Ellison et al., 1992 and Schanen et al., 1997). Other hypotheses—such as an autosomal dominant mutation with sex-limited expression or two mutations, one autosomal and one X-linked—remained theoretical possibilities (Behler et al., 1990 and Migeon et al., 1995). Although most cases are sporadic, there have been a few familial occurrences of Rett syndrome with evidence for inheritance through the maternal germline. Further support for the X-linked inheritance model came from three families in which a non-random pattern of X-inactivation was confirmed in the obligate carrier females (Zoghbi et al., 1990, Schanen et al., 1997 and Sirianni et al., 1998). In two of these families, a male sibling with a severe neonatal encephalopathy died within a few months of birth (Schanen et al., 1998). Because of the very few familial cases, investigators favoring the X-linkage hypothesis pursued exclusion mapping on the X-chromosome to define the smallest region shared amongst affected kindred cases (Ellison et al., 1992, Schanen et al., 1997, Sirianni et al., 1998, Schanen et al., 1998, Archidiacono et al., 1991 and Curtis et al., 1993). These analyses eventually mapped the RTT gene telomeric to DXS998 in a 10 Mb gene-rich region in distal Xq.
In pursuit of the Rett gene, a systematic mutational analysis of genes located in Xq28 in Rett syndrome patients was performed. This region harbors a number of good candidate genes. Several were selected for mutation analysis because of their known function and expression patterns, but recently were excluded (Amir et al., 1999, incorporated by reference herein). The gene encoding methyl-CpG-binding protein 2 (MECP2), which maps to Xq28 between L1CAM and the RCP/GCP loci and undergoes X-inactivation was then analyzed (D'Esposito et al., 1996). MeCP2 is an abundant chromosome-binding protein that selectively binds 5-methyl cytosine residues in symmetrically positioned CpG dinucleotides in mammalian genomes (Lewis et al., 1992). These residues are preferentially located in the promoter regions of genes that are subject to transcriptional silencing after DNA methylation. Recent studies established that MeCP2 is the molecular link between DNA methylation and transcriptional silencing by histone deacetylation (Nan et al., 1998 and Jones et al., 1988). It contains at least two functional domains: an 85 amino acid (aa) methyl-CpG-binding domain (MBD), essential for its binding to 5-methyl cytosine (Nan et al., 1993), and a 104 aa transcriptional repression domain (TRD) that interacts with histone deacetylase and the transcriptional corepressor Sin3A. Interactions between this transcription repressor complex and chromatin-bound MeCP2 leads to deacetylation of core histones, which in turn leads to transcriptional repression (Nan et al., 1998 and Jones et al., 1988). Furthermore, this complex can inhibit transcription from a promoter at a distance (Nan et al., 1997). The surprising discovery of the present invention regards mutations in Rett syndrome of a member of a family of genes encoding methyl-CpG-binding domain proteins. This discovery facilitates development of a test for early diagnosis and prenatal detection of neurodevelopmental diseases. More importantly, the finding that epigenetic regulation plays a role in the pathogenesis of Rett syndrome provides opportunities for therapy.
SUMMARY OF THE INVENTION
In one embodiment of the present invention there is a method of screening a vertebrate for neurodevelopmental disease comprising the step of detecting a mutation in the nucleic acid sequence of a gene encoding a methyl-CpG-binding domain containing protein. In a specific embodiment, the neurodevelopmental disease is selected from the group consisting of Rett syndrome, autism, non-syndromic mental retardation, idiopathic neonatal encephalopathy, idiopathic infantile spasms, idiopathic cerebral palsy, Angelman syndrome, and schizophrenia.
In a specific embodiment said mutation is found in the sequences selected from the group consisting of a regulatory sequence, an exon, an intron, an exon/intron junction, and a 3′ untranslated region.
A further embodiment of the present invention is the method wherein said mutation is detected by sequencing, a probe, electrophoretic mobility, nucleic acid hybridization, fluorescent in situ hybridization, nucleic acid-chip technology, polymerase chain reaction or reverse transcription-polymerase chain reaction.
Another embodiment of the present invention is a method of screening a vertebrate for neurodevelopmental disease comprising the step of detecting an alteration in the amino acid sequence of a methyl-CpG-binding domain containing protein. In a specific embodiment of the present invention said alteration is detected by electrophoresis, through chromosomal binding pattern analysis, by the methylation pattern of genomic DNA, by measuring upregulation of expression of a target gene, by measuring increased production of a protein encoded by a target gene, by measuring increased production of a protein encoded by a target gene wherein said protein is secreted from the cell, by antibodies, by amino acid sequencing, and by determining the molecular weight.
Another embodiment of the present invention is the method of screening a vertebrate for neurodevelopmental disease comprising the step of detecting a mutation in a nucleic acid sequence or in the corresponding amino acid sequence of a protein wherein said protein is present in a MECP2/complex and said mutation disrupts function of a protein present in said MECP2/complex. A specific embodiment of the present invention is the method wherein said nucleic acid sequence or corresponding amino acid sequence is selected from the group consisting of Sin3A, HDAC1, HDAC2, and RbAp48.
An additional embodiment of the present invention is a method of screening a vertebrate for neurodevelopmental disease comprising the step of detecting a mutation in a first gene involved in regulation of expression of a second gene encoding a methyl-CpG-binding domain containing protein. Said first gene may encode a transcription factor or a gene associated with X-inactivation. In a further embodiment the gene associated with X-inactivation is MECP2.
In another embodiment said gene involved in regulation of expression is associated with localization patterns of RNAs transcribed from said gene encoding a methyl-CpG-binding domain containing protein wherein said RNAs vary in length.
In an additional embodiment is the method of treating a vertebrate with a neurodevelopmental disease wherein a mutation in a first gene encoding a methyl-CpG-binding domain containing protein causes upregulation of expression of said second gene comprising the step of administering into said vertebrate a therapeutically effective amount
Amir Ruthie
Francke Uta
Van den Veyver Ignatia B.
Zoghbi Huda Y.
Baylor College of Medicine
Fulbright & Jaworski LLP
McKelvey Terry
LandOfFree
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