Molecular diagnosis and clinical onset of Charcot-Marie-Tooth disease in Japan.

To study the genetic background of Japanese Charcot-Marie-Tooth disease (CMT) patients, we analyzed qualitative and quantitative changes in the disease-causing genes mainly by denaturing high performance liquid chromatography and multiplex ligation-dependent probe analysis in 227 patients with demyelinating CMT and 127 patients with axonal CMT. In demyelinating CMT, we identified 53 patients with PMP22 duplication, 10 patients with PMP22 mutations, 20 patients with MPZ mutations, eight patients with NEFL mutations, 19 patients with GJB1 mutations, one patient with EGR2 mutation, five patients with PRX mutations and no mutations in 111 patients. In axonal CMT, we found 14 patients with MFN2 mutations, one patient with GARS mutation, five patients with MPZ mutations, one patient with GDAP1 mutation, six patients with GJB1 mutations and no mutations in 100 patients. Most of the patients carrying PMP22, MPZ, NEFL, PRX and MFN2 mutations showed early onset, whereas half of the patients carrying PMP22 duplication and all patients with GJB1 or MPZ mutations showing axonal phenotype were adult onset. Our data showed that a low prevalence of PMP22 duplication and high frequency of an unknown cause are features of Japanese CMT. Low prevalence of PMP22 duplication is likely associated with the mild symptoms due to genetic and/or epigenetic modifying factors.

Cardiac ion channel gene mutations in sudden infant death syndrome.

Sudden infant death syndrome (SIDS) is multifactorial and may result from the interaction of a number of environmental, genetic, and developmental factors. We studied three major genes causing long QT syndrome in 42 Japanese SIDS victims and found five mutations, KCNQ1-K598R, KCNH2-T895M, SCN5A-F532C, SCN5A-G1084S, and SCN5A-F1705S, in four cases; one case had both KCNH2-T895M and SCN5A-G1084S. All mutations were novel except for SCN5A-F532C, which was previously detected in an arrhythmic patient. Heterologous expression study revealed significant changes in channel properties of KCNH2-T895M, SCN5A-G1084S, and SCN5A-F1705S, but did not in KCNQ1-K598R and SCN5A-F532C. Our data suggests that nearly 10% of SIDS victims in Japan have mutations of the cardiac ion channel genes similar to in other countries.

Periaxin mutation in Japanese patients with Charcot-Marie-Tooth disease.

Periaxin (PRX) plays an important role in the myelination of the peripheral nerve and consequently in the pathogenesis of Charcot-Marie-Tooth disease (CMT). To date, nine nonsense or frameshift PRX mutations have been reported in eight families with CMT. The patients with PRX mutations appeared to show characteristic clinical features with early onset but slow or no progression, a common result of mutations that lead to missing a C-terminal acidic domain. Here, we report a Japanese CMT patient with these characteristic clinical features, who was a compound heterozygote for PRX R1070X and L132FsX153 mutations. We previously reported that three Japanese isolated families also had the homozygous R1070X mutation. To examine the potential founder effect of the R1070X mutation in the Japanese population, we performed haplotype analysis and found that each R1070X allele lay on a different haplotype background in these four families. Therefore, the high frequency of the R1070X mutation among the Japanese population is not likely the consequence of a founder effect, but probably a result of a mutation hot spot.

Sensitive detection of polyalanine expansions in PHOX2B by polymerase chain reaction using bisulfite-converted DNA.

Congenital central hypoventilation syndrome, also known as Ondine's curse, is characterized by idiopathic abnormal control of respiration during sleep. Recent studies indicate that a polyalanine expansion of PHOX2B is relevant to the pathogenesis of this disorder. However, it is difficult to detect the repeated tract because its high GC content inhibits conventional polymerase chain reaction (PCR) amplification. Here, we describe a bisulfite treatment for DNA in which uracil is obtained by deamination of unmethylated cytosine residues. Deamination of DNA permitted direct PCR amplification that yielded a product of 123 bp for the common 20-residue repetitive tract with replacement of C with T by sequencing. It settled allele dropouts accompanied by insufficient amplification of expanded alleles. The defined procedure dramatically improved detection of expansions to 9 of 10 congenital central hypoventilation syndrome patients examined in a previous study. The chemical conversion of DNA before PCR amplification facilitates effective detection of GC-rich polyalanine tracts.

Small heat shock protein 27 mutation in a Japanese patient with distal hereditary motor neuropathy.

Heat shock protein 27 (HSP27) belongs to a family of small heat shock proteins that play significant roles in the cellular stress response and are also involved in the control of protein-protein interactions as chaperons. Mutation in HSP27 has been identified as the cause of axonal Charcot-Marie-Tooth disease (CMT) and distal hereditary motor neuropathy (HMN). Heat shock protein 22 (HSP22) is a molecular counterpart of HSP27, and its mutation is another cause of distal HMN. We screened the mutation of HSP27 and HSP22 in 68 Japanese patients with axonal CMT or unclassified CMT and six Japanese patients with distal HMN. We detected a heterozygous P182S mutation of HSP27 in a patient with distal HMN, but we found no mutations in HSP22. Mutation in HSP27 may impair the formation of the stable neurofilament network that is indispensable for the maintenance of peripheral nerves.

[A pedigree of Charcot-Marie-Tooth disease type 4F (Periaxin mutation)].

We report a 51-year-old man genetically diagnosed as Charcot-Marie-Tooth disease type 4F. The patient was the first child of healthy, consanguineous parents. He had two sisters and one of them showed similar but milder symptoms. He had gait disturbance since childhood. Then he noticed muscle weakness of his hands at the age of early forties, and more difficulties in gait at the age of late forties. On examination at age 51, he showed absence of all deep tendon reflexes, weakness of the hand and distal leg muscles, pes cavus and decreased sensitivity to touch and vibration in the lower extremities. Electrophysiological studies of the median nerve showed delayed motor nerve conduction velocity and undetectable sensory nerve action potentials. The histology of his sural nerve revealed moderate loss of large myelinated fibers and the diameters of residual fibers shifted to small shown as size-frequency histogram. Many fibers are thinly myelinated and some of the Schwann cells looked as wrapping around the myelinate fibers with their processes. On gene analyses, we identified an Arg 1070 Stop homozygous mutation in the Periaxin, known to be a causative gene for CMT type 4F. Based on these observations, we emphasized that broad genetic analyses are necessary for diagnosis of CMT disease, including so far unidentified mutations among the Japanese populations.

Neonatal hyperbilirubinemia and the bilirubin uridine diphosphate-glucuronosyltransferase gene: the common -3263T > G mutation of phenobarbital response enhancer module is not associated with the neonatal hyperbilirubinemia in Japanese.

BACKGROUND: Neonatal hyperbilirubinemia is frequent and severe in Japanese newborns. Previously, it has been reported that half of the Japanese neonates with severe hyperbilirubinemia carried the 211G > A (p.G71R) mutation of the bilirubin uridine diphosphate-glucuronosyltransferase (UGT1A1) gene causing Gilbert syndrome. Recently, it was reported that the -3263T > G mutation in the phenobarbital response enhancer module in UGT1A1 was associated with the majority of cases of Gilbert syndrome. The gene frequency of the -3263T > G mutation was determined and the relation with neonatal hyperbilirubinemia in Japanese was studied. METHODS: UGT1A1 in 119 neonates born at Yamagata University Hospital, Yamagata, Japan, and 26 subjects who had undergone phototherapy due to severe hyperbilirubinemia at four other hospitals were studied. The gene frequency of -3263T > G mutation in Japanese, Korean, Chinese and German healthy adult controls was also determined. Hyperbilirubinemia was assessed with a Jaundice Meter and UGT1A1 was analyzed by sequence determination or restriction enzyme method. RESULTS: The gene frequency of the -3263T > G mutation was 0.26 in Japanese subjects and was similar to the prevalence in Korean, Chinese and German populations. However, there was no significant increase in the gene frequency of the mutation in the neonates who required phototherapy for hyperbilirubinemia compared to that in the neonates without severe hyperbilirubinemia. In addition, neonates with or without the mutation did not show a significant change in the level of bilirubin and the mutation also did not show a synergic effect with the 211G > A mutation on the level of bilirubin. CONCLUSION: The -3263T > G mutation is not likely to be associated with the neonatal hyperbilirubinemia in Japanese.

Mitochondrial GTPase mitofusin 2 mutation in Charcot-Marie-Tooth neuropathy type 2A.

Charcot-Marie-Tooth disease (CMT) has been classified into two types, CMT1 and CMT2, demyelinating and axonal forms, respectively. CMT2 has been further subdivided into eight groups by linkage studies. CMT2A is linked to chromosome 1p35-p36 and mutation in the kinesin family member 1B-beta (KIF1B) gene had been reported in one pedigree. However, no mutation in KIF1B was detected in other pedigrees with CMT2A and the mutations in the mitochondrial fusion protein mitofusin 2 (MFN2) gene were recently detected in those pedigrees. MFN2, a mitochondrial transmembrane GTPase, regulates the mitochondrial network architecture by fusion of mitochondria. We studied MFN2 in 81 Japanese patients with axonal or unclassified CMT and detected seven mutations in seven unrelated patients. Six of them were novel and one of them was a de novo mutation. Most mutations locate within or immediately upstream of the GTPase domain or within two coiled-coil domains, which are critical for the functioning or mitochondrial targeting of MFN2. Formation of a mitochondrial network would be required to maintain the functional peripheral nerve axon.

Sudden infant death syndrome is not associated with the mutation of PHOX2B gene, a major causative gene of congenital central hypoventilation syndrome.

Sudden infant death syndrome (SIDS) is a major cause of infant death, but its etiology is unknown. There are several independent risk factors for SIDS, and prone sleeping is a major risk factor. SIDS is probably based on a compromise in arousal response to breathing or blood pressure during sleep. Congenital central hypoventilation syndrome (CCHS or Ondine's curse) is a disorder characterized by an idiopathic failure of the autonomic control of breathing and has been regarded as one of the compromised conditions in SIDS. Recently, mutations of the PHOX2B gene have been detected in half to two-thirds of CCHS patients. We therefore analyzed the PHOX2B gene in 23 cases of SIDS and did not find any mutations, except for three polymorphic nucleotidic substitutions. The mutation of PHOX2B is thus not likely associated with SIDS.

Periaxin mutation causes early-onset but slow-progressive Charcot-Marie-Tooth disease.

Periaxin (PRX) plays a significant role in the myelination of the peripheral nerve. To date, seven non-sense or frameshift PRX mutations have been reported in six pedigrees with Dejerine-Sottas neuropathy or severe Charcot-Marie-Tooth neuropathy (CMT). We detected a PRX mutation in three patients in the screening of 66 Japanese demyelinating CMT patients who were negative for the gene mutation causing dominant or X-linked demyelinating CMT. Three unrelated patients were homozygous for a novel R1070X mutation and presented early-onset but slowly progressive distal motor and sensory neuropathies. Mutations lacking the carboxyl-terminal acidic domain may show loss-of-function effects and cause severe demyelinating CMT.

Molecular analysis of congenital central hypoventilation syndrome.

Congenital central hypoventilation syndrome (CCHS or Ondine's curse; OMIM 209880) is a disorder characterized by an idiopathic failure of the automatic control of breathing. CCHS is frequently complicated with neurocristopathies such as Hirschsprung's disease (HSCR). The genes involved in the RET-GDNF signaling and/or EDN3-EDNRB signaling pathways have been analyzed as candidates for CCHS; however, only a few patients have mutations of the RET, EDN3, and GDNF genes. Recently, mutations of the PHOX2B gene, especially polyalanine expansions, have been detected in two thirds of patients. We studied the RET, GDNF, GFRA1, PHOX2A, PHOX2B, HASH-1, EDN1, EDN3, EDNRB, and BDNF genes in seven patients with isolated CCHS and three patients with HSCR. We detected polyalanine expansions and a novel frameshift mutation of the PHOX2B gene in four patients and one patient, respectively. We also found several mutations of the RET, GFRA1, PHOX2A, and HASH-1 genes in patients with or without mutations of the PHOX2B gene. Our study confirmed the prominent role of mutations in the PHOX2B gene in the pathogenesis of CCHS. Mutations of the RET, GFRA1, PHOX2A, and HASH-1 genes may also be involved in the pathogenesis of CCHS. To make clear the pathogenesis of CCHS, the analysis of more cases and further candidates concerned with the development of the autonomic nervous system is required.

Screening of the early growth response 2 gene in Japanese patients with Charcot-Marie-Tooth disease type 1.

Charcot-Marie-Tooth disease type 1 (CMT1) is a heterogeneous disorder. Most CMT1 patients are associated with a duplication of 17p11.2-p12 (CMT1A duplication), but a small number of patients have mutations of peripheral myelin protein 22 (PMP22), myelin protein zero (MPZ), connexin 32 (Cx32) and early growth response 2 (EGR2) genes. In our previous study, we identified the responsible mutations in 72 of 128 Japanese CMT1 patients as CMT1A duplication in 40, PMP22 mutation in 6, MPZ mutation in 12 and Cx32 mutation in 14 patients. A total of 56 Japanese CMT1 patients with no identified mutations were screened for EGR2 mutation by denaturing gradient gel electrophoresis (DGGE). We detected a heterozygous Asp383Tyr mutation of EGR2 in one patient with severe CMT1, Dejerine-Sottas syndrome. EGR2 mutation is rare cause of CMT1 in Japan as in other nations. We were unable to identify the responsible mutation in 55 of 128 CMT1 patients and need further analysis to identify their candidate genes.