Takahito Wada

Parent-of-Origin Specific Histone Acetylation and Reactivation of a Key Imprinted Gene Locus in Prader-Willi Syndrome

To examine the chromatin basis of imprinting in chromosome 15q11-q13, we have investigated the status of histone acetylation of the SNURF-SNRPN locus, which is a key imprinted gene locus in Prader-Willi syndrome (PWS). Chromatin immunoprecipitation (ChIP) studies revealed that the unmethylated CpG island of the active, paternally derived allele of SNURF-SNRPN was associated with acetylated histones, whereas the methylated maternally derived, inactive allele was specifically hypoacetylated. The body of the SNURF-SNRPN gene was associated with acetylated histones on both alleles. Furthermore, treatment of PWS cells with the DNA methyltransferase inhibitor 5-azadeoxycytidine (5-aza-dC) induced demethylation of the SNURF-SNRPN CpG island and restoration of gene expression on the maternal allele. The reactivation was associated with increased H4 acetylation but not with H3 acetylation at the SNURF-SNRPN CpG island. These findings indicate that (1) a significant role for histone deacetylation in gene silencing is associated with imprinting in 15q11-q13 and (2) silenced genes in PWS can be reactivated by drug treatment.

Wide clinical variability in a family with a CACNA1A T666m mutation: hemiplegic migraine, coma, and progressive ataxia.

We report a Japanese family carrying a T666M missense mutation of CACNA1A. Affected members demonstrated a strikingly wide clinical spectrum including migraine, hemiplegia, coma, and progressive cerebellar ataxia. Despite such variability of the clinical features, they demonstrated similar magnetic resonance imaging findings demonstrating cerebellar atrophy predominantly of the cerebellar vermis. These magnetic resonance images appeared not to correlate with clinical severity. Our findings should indicate that a T666M mutation of CACNA1A may be associated with more variable clinical features and that paroxysmal hemiplegic migraine attacks and progressive cerebellar atrophy should have distinct mechanisms of pathogenesis.

Germline mosaicism of a novel mutation in lysosome‐associated membrane protein‐2 deficiency (Danon disease)

We identified a family with lysosome‐associated membrane protein‐2 deficiency (Danon disease) associated with a novel 883 ins‐T mutation in the lysosome‐associated membrane protein‐2 gene located at Xq24. Although the affected son and daughter carried the same mutation, it was not detected in their mothers peripheral blood or buccal cells; this indicated germline mosaicism. This is the first molecular evidence for germline mosaicism in Danon disease and has important implications for genetic counseling.

Molecular genetic study of Japanese patients with X-linked α-thalassemia/mental retardation syndrome (ATR-X)

X-linked alpha-thalassemia/mental retardation syndrome (ATR-X) is one of the many known X-linked mental retardation syndromes. Mutations in the ATR-X gene (ATRX) that encodes a putative global transcription factor have been identified in patients with ATR-X as well as those with other forms of X-linked mental retardation syndrome. To better understand the genetic basis of ATR-X, we investigated nine patients with the ATR-X phenotype from eight independent Japanese families for mutations in ATRX. We identified seven missense mutations, including six novel mutations, all of which were located either in the N-terminal region corresponding to the putative zinc finger domain (N179S, P190L, V194I, and R246C) or in the C-terminal region corresponding to the helicase domain (V1552F, L1645S, and Y1847C). R246C was found in two independent patients. Furthermore, we investigated the origin of the mutations in seven mothers. Five mothers were found to be carriers, and two were not, indicating de novo origin of the mutations. When we compared clinical manifestations with respective mutations, we could not find apparent phenotype-genotype correlation. Therefore, the putative zinc finger domain and the helicase domains may have similar functional significance for the function of ATRX.

A novel P gene missense mutation in a Japanese patient with oculocutaneous albinism type II (OCA2).

BACKGROUNDnOculocutaneous albinism type II (OCA2) is an autosomal recessively inherited disorder, characterized by white hair and skin, and loss of pigment in the eyes. Mutations in P gene have been shown to result in OCA2. So far, two cases have been reported from Japan.nnnOBJECTIVEnWe had an opportunity to examine a case of albinism, and screened the mutations of tyrosinase and P gene.nnnMETHODSnGenomic DNA was prepared from peripheral leukocytes. All of the exons and flanking introns of tyrosinase and P gene were PCR-direct-sequenced.nnnRESULTSnAlthough no mutations were found in tyrosinase, we found two missense substitutions, A481T and Q799H in P gene. The A481T has previously been shown to result in partial function of the P protein.nnnCONCLUSIONnThe Q799H mutation is not a common polymorphism among normal Japanese, seems most likely to be a pathological OCA2 mutation among Japanese with this form of albinism.

Detection of lymphocytes and granulocytes expressing the mutant WASP message in carriers of Wiskott-Aldrich syndrome

Wiskott‐Aldrich syndrome (WAS) is an X‐linked recessive disease caused by mutations in the recently identified WAS protein gene (WASP). In some X‐linked genetic disorders skewed X‐inactivation has been observed in all cell populations or some specific cell lineages of female carriers. Recently, female carriers of WAS were also revealed to present skewed X‐inactivation patterns at the haemopoietic stem cell level. However, it is not clear if all haematological cells expressing the mutant WASP allele are eliminated in WAS carriers. By reverse transcription PCR methods, we studied 14 WAS carriers from 10 different families to assess whether blood cells expressing the mutant WASP message were present in their peripheral blood. The mutations of each WAS patient were known and carrier diagnosis of their female family members was performed using specific mutation analysis. We detected circulating lymphocytes and granulocytes expressing the mutant WASP message in most of the WAS carriers, nevertheless they showed skewed X‐chromosomal inactivation patterns. Interestingly, the presence of blood cells expressing the mutant WASP message seemed to correlate to the WASP genotype and the age of the carriers.

Phenotypic variability in a family with a mitochondrial DNA T8993C mutation

Two patients are described in a family with a mitochondrial DNA T8993C point mutation. Patient 1, the proband, was a 4-year-old male, and his clinical features were consistent with those of Leigh syndrome, including lactic acidosis, motor development delay, and symmetric basal ganglia lesions on magnetic resonance imaging (MRI). His mental development was delayed mildly, but he has not demonstrated neurologic deterioration. Patient 2 was his maternal aunt. She developed her first neurologic sign at 18 months of age, thereafter her development ceased and regressed. She had lost her head control and become bedridden by 4 years of age and died at 20 years of age, demonstrating a more severe clinical course than that of Patient 1. Analysis of mitochondrial DNA from peripheral leukocytes of Patient 1 revealed a T8993C mutation of 99%. Patient 2 was demonstrated to have the same mutation at high abundance (99%) in the frozen myocardium and in the formaldehyde preserved spinal cord, with only 18% mutant mitochondrial DNA present in the formaldehyde preserved sciatic nerve. The mother of Patient 1, who was phenotypically normal (sister of Patient 2), had 35% mutant mitochondrial DNA in peripheral leukocytes. The authors findings suggest that T8993C phenotypes are highly variable and that the proportion of the mutant mitochondrial DNA may vary among tissues and not correlate well with clinical severity.

Non-chromosome 15 marker chromosome in a Prader-Willi syndrome patient with uniparental disomy.

Correspondence : Shinji Saitoh MD, Department of Pediatrics, Hokkaido University Graduate School of Medicine, N-15, W-7, Kita-ku, Sapporo 060-8638, Japan. Email: ss11@med.hokudai.ac.jp Received 2 September 2004; revised 12 December 2004; accepted 21 January 2005. Prader-Willi syndrome (PWS) is characterized by neonatal hypotonia, mild-to-moderate mental retardation, hypogonadism, facial dysmorphism, and hyperphagia with subsequent obesity. PWS is caused by the lack of contribution of paternally expressed imprinted genes in chromosome 15q11-q13. 1

Molecular Genetic Study on Angelman Syndrome Patients without a Chromosomal Deletion

Purpose: Angelman syndrome (AS) is a ncurobehavioral disorder characterized by severe mental retardation, easily cvoked laughter, ataxic gait, and epilepsy. Epilepsy associated with AS is characterized by early childhood onset gencralized seizures with profound EEG abnormalities. Therefore, AS is a good human model for genetic epilepsy syndromes. Approximately 70% of AS cases are caused by maternal deletions of chromosomc 15q I I‐qI3; whereas, 30% are not associated with a chromosomal dcletion. Thcse non‐deletion AS patients are caused by paternal uniparental disomy (UPD), imprinting mutation (IM), or loss‐or‐function mutations of the UBE3A gene, cach of which predisposes different recurrence risk. To elucidate molecular etiology of non‐dclction AS patients, we investigated 34 AS patients without a chromosomal deletion.