Mecp2 deficiency decreases bone formation and reduces bone volume in a rodent model of Rett syndrome.

Rett syndrome (RTT), a neurological disorder characterized by neurological impairment and a high frequency of osteopenia which often manifests early in childhood, most often is caused by inactivating mutations in the X-linked gene encoding a regulator of epigenetic gene expression, methyl CpG binding protein, MeCP2. Clinical data show that, along with neurological defects, females with RTT frequently have marked decreases in bone mineral density (BMD) beyond that expected from disuse atrophy. To investigate the relationship between loss of Mecp2 and reduced BMD, we used a Mecp2 null mouse model, Mecp2 (-/yBIRD), for our histological and biochemical studies. Mecp2 (-/yBIRD) mice have significantly shorter femurs and an overall reduced skeletal size compared to wild-type mice by post-natal day 60 (P60). Histological and histomorphometric studies identified growth plate abnormalities as well as decreased cortical and trabecular bone in P21 and especially in P60 Mecp2 (-/yBIRD) mice. Dynamic histomorphometry revealed decreased mineral apposition rates (MAR) in Mecp2 null femoral trabecular bone as well as in calvarial bone samples. While changes in MAR of cortical bone were not significant, loss of Mecp2 significantly reduced cortical, trabecular and calvarial bone volume compared with age-matched wild-type animals. These differences indicate that Mecp2 deficiency leads to osteoblast dysfunction, which translates into reduced osteoid deposition accounting for the reduced bone volume phenotype. While individual variations were observed in OPG and Rankl concentrations, molar ratios of OPG:Rankl at P21 and P60 were comparable between wild-type and Mecp2 (-/yBIRD) mice and showed a consistent excess of OPG. In tibial sections, TRAP staining demonstrated equivalent osteoclast number per bone surface measurements between wild-type and null animals. Our work with a Mecp2 null mouse model suggests epigenetic regulation of bone in the Mecp2 (-/yBIRD) mice which is associated with decreased osteoblast activity rather than increased osteoclastic bone loss.

Chromosome 15q11-13 duplication syndrome brain reveals epigenetic alterations in gene expression not predicted from copy number.

BACKGROUND: Chromosome 15q11-13 contains a cluster of imprinted genes essential for normal mammalian neurodevelopment. Deficiencies in paternal or maternal 15q11-13 alleles result in Prader-Willi or Angelman syndromes, respectively, and maternal duplications lead to a distinct condition that often includes autism. Overexpression of maternally expressed imprinted genes is predicted to cause 15q11-13-associated autism, but a link between gene dosage and expression has not been experimentally determined in brain. METHODS: Postmortem brain tissue was obtained from a male with 15q11-13 hexasomy and a female with 15q11-13 tetrasomy. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to measure 10 15q11-13 transcripts in maternal 15q11-13 duplication, Prader-Willi syndrome, and control brain samples. Southern blot, bisulfite sequencing and fluorescence in situ hybridisation were used to investigate epigenetic mechanisms of gene regulation. RESULTS: Gene expression and DNA methylation correlated with parental gene dosage in the male 15q11-13 duplication sample with severe cognitive impairment and seizures. Strikingly, the female with autism and milder Prader-Willi-like characteristics demonstrated unexpected deficiencies in the paternally expressed transcripts SNRPN, NDN, HBII85, and HBII52 and unchanged levels of maternally expressed UBE3A compared to controls. Paternal expression abnormalities in the female duplication sample were consistent with elevated DNA methylation of the 15q11-13 imprinting control region (ICR). Expression of non-imprinted 15q11-13 GABA receptor subunit genes was significantly reduced specifically in the female 15q11-13 duplication brain without detectable GABRB3 methylation differences. CONCLUSION: Our findings suggest that genetic copy number changes combined with additional genetic or environmental influences on epigenetic mechanisms impact outcome and clinical heterogeneity of 15q11-13 duplication syndromes.

Supernumerary tricentric derivative chromosome 15 in two boys with intractable epilepsy: another mechanism for partial hexasomy.

Rearrangements of chromosome 15q, including isodicentric 15 chromosomes and interstitial duplications and triplications, have been previously reported in association with autism spectrum disorders. We have identified two boys with exceptionally large der(15) chromosomes that are tricentric and contain four copies of the proximal long arm, including the Prader Willi/Angelman critical region, and leading to hexasomy of the involved segment. Biallelic inheritance of maternal alleles and methylation analysis indicate that the markers are maternally derived. Clinical assessment of the boys indicated severe cognitive impairment associated with marked delays in gross and fine motor skills. Social and language deficits were present in both, although the severity of the mental retardation precluded diagnosis of autism (both were considered to have pervasive developmental disorder-not otherwise specified). Neurologic manifestations included infantile spasms evolving into intractable early-onset myoclonic seizures, psychomotor regression, and profound diffuse hypotonia. These patients represent the most severe end of the spectrum of phenotypes associated with segmental aneuploidy for chromosome 15q11-q13.

Congenital thrombocytopenia and radio-ulnar synostosis: a new familial syndrome.

The association of bone marrow failure and skeletal defects has been frequently noted, however, the genetic basis for most of these syndromes remains unclear. We describe a previously uncharacterized autosomal dominant syndrome of amegakaryocytic thrombocytopenia associated with radial-ulnar synostosis. The clinical features of this syndrome appear to be distinct from other similar conditions, including Fanconi's anaemia and thrombocytopenia-absent radii (TAR). The physical findings at diagnosis and clinical management of each case are detailed, as well as a discussion of this disorder in the context of other syndromes in which marrow failure and skeletal defects are prominent features. We also review recent developments in molecular genetics that may provide important clues to the underlying aetiology of this condition.

Branchial cleft anomaly, congenital heart disease, and biliary atresia: Goldenhar complex or Lambert syndrome?

The features of Goldenhar complex have been well-described and classically include branchial arch abnormalities, epibulbar dermoid and vertebral abnormalities. We have identified an infant with these features in association with complex congenital heart disease and intrahepatic biliary atresia. Although Lambert described an autosomal recessive disorder with an association of biliary atresia and branchial arch abnormalities, none of those cases had epibulbar dermoid. Diagnostic considerations in this case include inclusion of biliary atresia as a new feature in the expanding spectrum of the Goldenhar complex, versus Lambert syndrome with epibulbar dermoid.

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

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.

Molecular approaches to the Rett syndrome gene.

Rett syndrome is a neurodevelopmental disorder affecting 1 in 10,000 to 15,000 females worldwide. Apparently normal at birth, girls with Rett syndrome undergo developmental regression and acquire a neurologic and behavioral profile that has been used to define diagnostic criteria for the disorder. Neurochemical and anatomic alterations indicate that Rett syndrome appears to result from an arrest of normal neuronal maturation. Although Rett syndrome generally occurs sporadically, rare familial recurrences indicate a genetic basis for the disorder. Data from familial recurrences are consistent with an X-linked dominant locus causing the classic phenotype in female patients and a distinct, more severe phenotype in hemizygous male patients. Exclusion mapping data from rare kindreds with recurrent Rett syndrome localize the gene to the distal long arm of the X chromosome (Xq27.3-Xqter).

Identification of a new polymorphism in the 3'-untranslated region of the human serotonin receptor 2C (5-HT2C) gene.

We identified a polymorphism (2831T > G) in the 3'-untranslated region of 5-HT2C receptor gene, approximately 100 kb from a previously reported coding sequence polymorphism, 796G > C (C23S). Allele frequencies were 0.90 (T) and 0.10 (G) and cosegregation analysis of the alleles at the two loci demonstrated frequencies of 0.82 (GT), 0.08 (CT), 0.10 (GG), and 0 (CC). The increased informativity gained by analysis of both polymorphisms will prove useful for future studies of this gene in X-linked neuropsychiatric disorders.

Exclusion of the gastrin-releasing peptide receptor (GRPR) locus as a candidate gene for Rett syndrome.

The gene for the gastrin-releasing peptide receptor (GRPR) has been mapped to a candidate region for Rett syndrome (RTT) on the short arm of the X chromosome. The recent report of a translocation that disrupted the gene in an individual with mental retardation and autistic behavior prompted us to examine GRPR as a possible locus for RTT. Genomic polymerase chain reaction amplification of exons followed by single-strand conformation analysis screening in 25 unrelated RTT-affected individuals and by direct sequencing in 12 others has failed to detect any mutation. No gross structural rearrangements were found by Southern analysis of DNA from six unrelated RTT-affected individuals. A high-frequency biallelic polymorphism caused by two single nucleotide substitutions in exon 2 was discovered. The allele frequencies were identical in the RTT population as compared to 100 normal control X chromosomes. This polymorphism will enable future evaluation of the GRPR locus as a candidate for other X-linked mental retardation or neurobehavioral syndromes.

Neonatal encephalopathy in two boys in families with recurrent Rett syndrome.

Rett syndrome (RTT) has been described in its classic form only in females. Although the majority of cases are sporadic, familial cases give valuable insight into the genetic basis and phenotypic variability of the disorder. The exclusive occurrence of classic Rett syndrome in females led to the hypothesis that the Rett syndrome locus is likely to be X-linked and mutations are lethal in hemizygous males. We identified two boys in families with recurrent Rett syndrome who had encephalopathies with neonatal onset and who may represent the phenotype of males harboring Rett syndrome mutations. The difference in severity of disease in these males and their female relatives supports the location of Rett syndrome locus on the X-chromosome.

A new Rett syndrome family consistent with X-linked inheritance expands the X chromosome exclusion map.

Although familial recurrences of Rett syndrome (RTT) comprise only approximately 1% of the reported cases, it is these cases that hold the key for the understanding of the genetic basis of the disorder. Families in which RTT occurs in mother and daughter, aunt and niece, and half sisters are consistent with dominant inheritance and variable expressivity of the phenotype. Recurrence of RTT in sisters is likely due to germ-line mosaicism in one of the parents, rather than to recessive inheritance. The exclusive occurrence of classic RTT in females led to the hypothesis that it is X-linked and may be lethal in males. In an X-linked dominant disorder, unaffected obligate-carrier females would be expected to show nonrandom or skewed inactivation of the X chromosome bearing the mutant allele. We investigated the X chromosome inactivation (XCI) patterns in the female members of a newly identified family with recurrence of RTT in a maternal aunt and a niece. Skewing of XCI is present in the obligate carrier in this family, supporting the hypothesis that RTT is an X-linked disorder. However, evaluation of the XCI pattern in the mother of affected half sisters shows random XCI, suggesting germ-line mosaicism as the cause of repeated transmission in this family. To determine which regions of the X chromosome were inherited concordantly/discordantly by the probands, we genotyped the individuals in the aunt-niece family and two previously reported pairs of half sisters. These combined exclusion-mapping data allow us to exclude the RTT locus from the interval between DXS1053 in Xp22.2 and DXS1222 in Xq22.3. This represents an extension of the previous exclusion map.

Is Rett syndrome caused by a triplet repeat expansion?

Rett syndrome is usually sporadic, but rare pedigrees with nonpenetrance in obligate carriers and possible anticipation suggest that it could be caused by a triplet repeat expansion (TRE). Rett probands and controls were systematically screened for expansions of any of the 10 possible triplet repeats by using a modified Repeat Expansion Detection (RED) assay that had been shown to detect expanded disease alleles in myotonic dystrophy and Huntington disease. No significant expansions were found in 26 sporadic and six familial Rett probands. Our results exclude the possibility that Rett syndrome is caused by a large TRE. We cannot exclude, however, causation by a small TRE that is masked by the background of longer polymorphic repeats in the normal population.

Trinucleotide repeats in the human genome: size distributions for all possible triplets and detection of expanded disease alleles in a group of Huntington disease individuals by the repeat expansion detection method.

Using a modified Repeat Expansion Detection (RED) assay, that was optimized for individual oligonucleotides, unrelated individuals were systematically screened for maximal repeat sizes of each of the ten possible trinucleotide repeats. Cloned trinucleotide repeats were generated and used as standards for the detectability of single copy trinucleotide repeat fragments. When the size distributions of trinucleotide repeats were compared to previously reported data, significant differences were found for the CTT repeat, which corresponds to the expanded GAA repeat in Friedreich ataxia, as well as for ATT, CCT and GTT repeats. Since 30-35% of normal individuals have CTG/CAG trinucleotide repeat sizes of 180 bp or more, we investigated the question whether small-scale CTG/CAG repeat expansions are detectable on a population basis by using the RED technique. We blindly screened 20 HD probands with CAG expansions of the HD gene, ranging in size between 120 and 174 bp, and found that a shift to larger CAG size ranges is clearly detectable when comparing the distribution of maximal repeat sizes in the disease group to a control group. Our study, therefore, demonstrates that the application of the RED assay to a population of probands and a population of controls allows the detection of small-scale CTG/CAG repeat expansions in the size range of the expanded HD gene and present in a single allele. We also provide standards and control data for the detection of other trinucleotide repeat expansions.

Isolation and characterization of microtubule-associated protein 2 (MAP2) kinase from rat brain.

Microtubule-associated protein 2 (MAP2) kinase has been isolated and characterized from rat brain. The enzyme has an apparent M(r) of approximately 42,000 and its pI is 4.9. MAP2 was the preferred substrate, but it also phosphorylated myelin basic protein (MBP), histone V-S, tubulin and the PC12 protein substrate pp250. The enzyme is distinct from protein kinase C, cAMP-dependent kinase and the calcium/calmodulin-dependent kinases, as specific inhibitors of these kinases did not affect MAP2 phosphorylation. The addition of the relatively non-specific protein kinase inhibitor H7 (20 microM) had a modest inhibitory effect. The enzyme was active in both 5 mM Mn2+ and Mg2+, and displayed Kms for MAP2, MBP, and ATP of 56 nM, 254 nM, and 4 microM, respectively. This enzyme, which represents a low abundance protein in whole brain, is analogous to the MAP2 kinase observed in growth factor-stimulated cell lines.