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Introduction GATA1 mutation plays an important role in initiating transient abnormal myelopoiesis (TAM) and in the clonal evolution towards acute megakaryoblastic leukaemia (AMKL) associated with Down syndrome (DS). This study aimed to develop and validate the clinical utility of a complementary DNA (cDNA) analysis in parallel with the conventional genomic DNA (gDNA) Sanger sequencing (Ss), as an initial screening test for GATA1 mutations. Methods GATA1 mutations were evaluated using both gDNA and cDNA in 14 DS patients using Ss and fragment analysis (FA), respectively. Results The detection sensitivity of conventional gDNA sequencing was limited in low blast percentage TAM (LBP-TAM); however, cDNA-based Ss readily detected all the pathognomonic GATA1 mutations. The cDNA-based FA readily detected GATA1 frameshift mutation with a reliable sensitivity ranging from 0.005% to 0.01% of clonal cells. Conclusions GATA1 mutations are heterogeneous; therefore, we would like to propose a dual cDNA and gDNA analysis as a standard diagnostic approach, especially for LBP-TAM. cDNA-based FA promises an excellent sensitivity for detecting frameshift GATA1 mutations in the longitudinal clonal evolution towards AMKL without using a patient specific primer.

Myoclonus-dystonia syndrome (MDS) is a rare autosomal-dominant movement disorder characterized by brief, frequently alcohol-responsive myoclonic jerks that begin in childhood or early adolescence, caused by mutations in the e-sarcoglycan gene (SGCE). The patient was a 6-year-old boy. At 2 years 8 months, he had abnormal movement when he ran due to dystonia of his left leg. At 3 years 5 months, he exhibited dystonia and myoclonic movement of his arms when eating. Myoclonus was likely to develop when he felt anxiety or exhaustion. Genomic DNA showed a heterozygous mutation in SGCE (c.109 + 1 G > T). His father and uncle with the same mutation also experienced milder dystonia or myoclonic movements. SGCE mutation can cause a broad range of clinical symptoms between and within families. We should consider MDS as a differential diagnosis for patients with paroxysmal walking abnormalities and/or myoclonic movements.

Summary Purpose: De novo STXBP1 mutations have been found in individuals with early infantile epileptic encephalopathy with suppression-burst pattern (EIEE). Our aim was to delineate the clinical spectrum of subjects with STXBP1 mutations, and to examine their biologic aspects. Methods: STXBP1 was analyzed in 29 and 54 cases of cryptogenic EIEE and West syndrome, respectively, as a second cohort. RNA splicing was analyzed in lymphoblastoid cells from a subject harboring a c.663 + 5G>A mutation. Expression of STXBP1 protein with missense mutations was examined in neuroblastoma2A cells. Results: A total of seven novel STXBP1 mutations were found in nine EIEE cases, but not in West syndrome. The mutations include two frameshift mutations, three nonsense mutations, a splicing mutation, and a recurrent missense mutation in three unrelated cases. Including our previous data, 10 of 14 individuals (71%) with STXBP1 aberrations had the onset of spasms after 1 month, suggesting relatively later onset of epileptic spasms. Nonsense-mediated mRNA decay associated with abnormal splicing was demonstrated. Transient expression revealed that STXBP1 proteins with missense mutations resulted in degradation in neuroblastoma2A cells. Discussion: Collectively, STXBP1 aberrations can account for about one-third individuals with EIEE (14 of 43). These genetic and biologic data clearly showed that haploinsufficiency of STXBP1 is the important cause for cryptogenic EIEE.

Monosomy 1p36 is a common subtelomeric microdeletion syndrome, characterized by craniofacial dysmorphisms, developmental delay, mental retardation, hypotonia, epilepsy, cardiovascular complications, and hearing impairment; deleted regions have been mapped within 10.0 Mb from the telomere in most documented cases. We report on a girl with a 10.5-11.1 Mb terminal deletion of 1p36 shown by fluorescence in situ hybridization (FISH). She had three distinct structural abnormalities: bilateral perisylvian polymicrogyria, periventricular nodular heterotopia, and left ventricular noncompaction. She died in early infancy with intractable epilepsy, progressive congestive heart failure and pulmonary hypertension. To date, this is the first case with monosomy 1p36, complicated by this combination of manifestations; she is also the first who had possibly a simple terminal deletion of 1p36 and died in early infancy. An atypically large deletion in this patient might be the basis for the development of these features and the severe clinical course.

A 56-year-old Japanese woman with mandibuloacral dysplasia and type A lipodystrophy is described. Mutation analysis identified a homozygous missense mutation (1585G > A) in exon 9 of the LMNA gene that replaces well-conserved residue alanine at position 529 to threonine (A529T). The woman showed, in addition to the usual clinical manifestations of the disorder, severe progressive skeletal changes: osteoporotic changes with multiple fractures; osteolysis of the right radius; and destructive changes of the vertebrae, leading to compression of the cervical spinal cord and paraplegia. Laboratory findings included markedly reduced bone mineral density; significantly increased urine N-telopeptide of collagen type I, an osteoclast marker; and normal serum bone specific alkaline phosphatase, an osteoblast marker. Regular follow up of adult patients with the disorder is desirable, including skeletal radiography, estimates of bone mineral density, and biochemical markers of bone turnover. Treatment with bisphosphonates to inhibit osteoclast activity is likely to be beneficial.

We report a Japanese woman with 46,XX,dup(16)(p13.11p13.3), who closely resembled the phenotype of X-linked alpha-thalassemia/mental retardation syndrome (ATR-X, MIM # 301040). Although she never had alpha-thalassemia, she showed characteristic clinical features including severe mental retardation, characteristic facies and behavior. ATR-X is caused by mutations of the ATRX gene. Although the function of ATRX protein has remained unclarified, it is thought to be involved in the regulation of several genes. The only target gene identified so far is the alpha-globin gene at 16p13.3. Clinical similarity among patients with ATR-X and dup(16)(p13.11p13) may indicate that some target genes regulated by ATRX reside in the duplicated region between 16p13.11 and 16p13.3, and that these genes are abnormally upregulated in ATR-X differently from the alpha-globin gene.

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.

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. Methods: Thirty sporadic AS patients, and 4 familial AS patients (2 families of 2 sibs) were enrolled to the study. The diagnosis of AS was based on Williams’ criteria (Williams et al., Am J Med Genet 1995, 56: 237). Genomic DNA was extracted from peripheral blood by a standard procedure. DNA mcthylation tcst at SNRPN locus and genotyping using 7 highly informative PCR-based polymorphisms within 15q I I - q I3 were carried out to identify UPD and IM. When both UPD and IM were ruled out, the patients were classified :LS non-UPD, non-IM. For thcsc non-UPD, non-1M paticnts, UBE3A mutations were screened by PCR-SSCP analysis using 10 sets ofprimcrs covering all coding exons. Results: Among 30 sporadic patients, I UPD and 3 IM patients were identified, and the remaining 26 patients were classified as non-UPD, non-IM. Among 4 familial patients, 2 sibs from I family were detected as IM, whcrcas 2 sibs from another family were classified as non-UPD, non-IM. No UBE3A mutations were identified within 26 sporadic and 2 familial non-UPD, non-IM patients. Conclusion: Threc molecular classes were identified for noindeletion AS patients. Therefore, the underlying genetic mechanism was dcmonstratcd to be complex for AS patients without a chromosomal deletion. Combination of the DNA methylation test and PCR-based polymorphisms was sufficient to detect UPD and IM patients. Because recurrence risk is low for UPD and high lor IM, systematic molecular investigation including the DNA methylation test and PCR-based polymorphisms should bc donc for non-delction AS paticnts for genetic counscling purpose. A majority of non-deletion patients were classified as noii-UPD, non-1M. Although, approximate 30% of non-UPD, nonIM patients arc rcportcd to have UBE3A mutations, no such mutations were identified in our study. An underlying molecular mechanism was not rcvealcd for this group of patients, and therefore, assessment of recurrence risk was difficult. Further investigation is necessary for noii-UPD, non-1M paticnts.