Problems in the regulation of genetic tests in Japan: what can we learn from direct-to-consumer genetic tests?

As one of the countries that have invested greatly in the field of bioscience, Japan is facing difficulties introducing human genetic research to the market. A key issue is how to regulate the quality of genetic testing. Since genetic testing is a part of clinical laboratory tests, the regulatory framework for these tests should cover the regulation of genetic testing. Nevertheless, the quality of clinical laboratory tests has been regulated largely by the authority of medical professionals. The fact that genetic testing can be provided without supervision of medical professionals reveals the necessity for the regulation of quality of genetic testing. While medical geneticists have publicly criticized direct-to-consumer (DTC) genetic testing, a group of industries related to DTC genetic testing have established self-regulatory guidelines on the quality control of genetic analysis, based on the OECD guidelines. This article describes the regulatory framework for clinical laboratory tests including genetic tests, and the gaps in regulation, which are particularly highlighted by the appearance of DTC genetic testing. Furthermore the current initiatives taken by different organizations, especially the self-regulatory initiatives by related industries, will be discussed. To conclude the article, recommendations to improve the situation will be made.

Knowledge and impressions regarding the concept of mutation among Japanese university students.

Although the term mutation is frequently used in genetic counseling, it may carry negative connotations and create misunderstanding. Our objective was to investigate the relationship between the impressions regarding three Japanese terms mutation of gene, change of gene, and lesion of gene as well as to investigate the depth of understanding regarding mutation. A total of 175 university students and auditing students were included and responded to two questionnaires that were Impressions regarding the term in the semantic differential method and Knowledge about the concept of mutation. In factor analysis, three factors (Value, Change Rate, and Intention) were extracted. Participants were divided into three groups depending on their knowledge, and a two-way analysis of variance (Term x Knowledge Group) was conducted on the factor score for each. Results showed that the main effect of the 'Term' was significant for the Value Factor and that interaction was significant for the Change Rate Factor, and that the main effect of Knowledge Group was significant for the Intention Factor. The findings suggest that healthcare professionals should demonstrate an awareness of varying impressions of the different terms used to refer to the identical concepts of mutation. This is of particular importance when communicating with patients and their families.

Filamin C accumulation is a strong but nonspecific immunohistochemical marker of core formation in muscle.

Filamin C is the muscle isoform of a group of large actin-crosslinking proteins. On the one hand, filamin C is associated with the Z-disk of the myofibrillar apparatus and binds to myotilin; on the other hand, it interacts with the sarcoglycan complex at the sarcolemma. Filamin C may be involved in reorganizing the cytoskeleton in response to signalling events and in muscle it may, in addition, fulfill structural functions at the Z-disk. An examination of biopsies from patients with multi-minicore myopathy, central core myopathy and neurogenic target fibers with core-like target formations (TF) revealed strong reactivity of all the cores and target formations with two different anti-filamin C antibodies. In all three conditions, the immunoreactivity in the cores for filamin C was considerably stronger than that for desmin. Only for alphaB-crystallin were comparable levels of immunoreactivity detected. There was no difference in intensity for filamin C between the three pathological conditions. Thus, filamin C along with alphaB-crystallin is a strong and robust, but nonspecific marker of core formation. The reason why filamin C accumulates in cores is unclear at present, but we postulate that it may be critically involved in the chain of events eventually leading to myofibrillar degeneration.

Mutations of the slow muscle alpha-tropomyosin gene, TPM3, are a rare cause of nemaline myopathy.

The alpha-tropomyosin-3 (TPM3) gene was screened in 40 unrelated patients with nemaline myopathy (NM). A single compound heterozygous patient was identified carrying one mutation that converts the stop codon to a serine and a second splicing mutation that is predicted to prevent inclusion of skeletal muscle exon IX. TPM3 mutations are a rare cause of NM, probably accounting for less than 3% of cases. The severity of cases with TPM3 mutations may vary from severe infantile to late childhood onset, slowly progressive forms.

Myozenin: an alpha-actinin- and gamma-filamin-binding protein of skeletal muscle Z lines.

To better understand the structure and function of Z lines, we used sarcomeric isoforms of alpha-actinin and gamma-filamin to screen a human skeletal muscle cDNA library for interacting proteins by using the yeast two-hybrid system. Here we describe myozenin (MYOZ), an alpha-actinin- and gamma-filamin-binding Z line protein expressed predominantly in skeletal muscle. Myozenin is predicted to be a 32-kDa, globular protein with a central glycine-rich domain flanked by alpha-helical regions with no strong homologies to any known genes. The MYOZ gene has six exons and maps to human chromosome 10q22.1-q22.2. Northern blot analysis demonstrated that this transcript is expressed primarily in skeletal muscle with significantly lower levels of expression in several other tissues. Antimyozenin antisera stain skeletal muscle in a sarcomeric pattern indistinguishable from that seen by using antibodies for alpha-actinin, and immunogold electron microscopy confirms localization specifically to Z lines. Thus, myozenin is a skeletal muscle Z line protein that may be a good candidate gene for limb-girdle muscular dystrophy or other neuromuscular disorders.

Gene structure of the P100 serine-protease component of the human Ra-reactive factor.

The Ra-reactive factor (RaRF) is a complement dependent anti-microbial factor that reacts with numerous microorganisms such as viruses, bacteria, fungi and protozoa. It is a complex of a mannan-binding lectin (MBL) and the serine protease, P100 (MASPI). P100 activates the C4 component of the complement system and its domain organization is similar to C1r and C1s. In this study, determination was made of the structure of the human P100 gene which was found longer than 67 kbp and to be comprised of 16 exons. Its non-protease region consisted of 10 exons, as in the case of C1r and C1s, and the introns were found present in the boundary separating two CUB domains, an EGF-like domain and two CCP domains and each CUB and CCP domain contained extra internal introns. The serine protease region was comprised of 6 exons in contrast to C1r and C1s, either of which consists of a single exon. The exon-intron structure was found to reflect the evolution of these molecules and P100 to have derived earlier in the stage of evolution than C1r or C1s.

[Frequent missense mutations of fibroblast growth factor receptor (FGFR) gene families in craniofacial syndromes in Japanese patients].

Craniofacial syndromes, including Crouzon syndrome, Pfeiffer syndrome, Jackson-Weiss syndrome, Apert syndrome and achondroplasia, have been indicated that syndromes were associated with mutations of fibroblast growth factor receptor (FGFR) gene families. In this report, seven Japanese patients with craniofacial syndromes, three Crouzon syndromes and four achondroplasias, were analyzed on FGFR2 and FGFR3 genes by non RI-SSCP (single strand conformation polymorphisms) and direct sequencing. Missense mutations of the FGFR3 exon 10, at codon 380 in two sporadic cases and codon 375 in two familial cases, were detected in all cases of achondroplasia. Mutations of the FGFR2 were noted in Crouzon and Apert syndromes. One of three Crouzon syndromes has a missense mutation at codon 342 on exon 9. Highly frequent mutations were clustered within some localized regions of the FGFR genes in craniofacial syndromes. Alterations in these receptors due to missense mutations would thus appear closely involved in pathogenesis of craniofacial syndrome. The non RI-SSCP and direct sequencing of the FGFR genes, shown in this report, may be an appropriate approach for diagnosis of these syndromes with extensive clinical application.

[Nucleotide sequences at intron 6 and exon 7 junction of fibroblast growth factor receptor 2 and rapid mutational analysis in Apert syndrome].

Apert syndrome, acrocephalosyndactyly Type I, is an autosomal dominant craniosynostosis comprising acrocephaly, facial dysmorphism and severe syndactyly of the hands and feet. Missense mutations at codons 252 and 253 at 5'-end on exon 7 of fibroblast growth factor receptor (FGFR) 2 have been identified in a large number of patients with Apert syndrome. In this study, nucleotide sequences on the intron 6 were determined by vector ligation-PCR and direct sequencing. Five DNA samples from sporadic Apert syndrome were examined by non-RI SSCP and direct sequencing using a primer pair of intron 6 and exon 7. All cases of the syndrome showed abnormal banding pattern in the SSCP and missense mutations from Ser to Trp at codon 252 of the FGFR2 gene. The non-RI SSCP and direct sequencing of the FGFR2 exon 7 from genomic DNAs may be a useful and rapid molecular means for clinical diagnosis of Apert syndrome.

Mutations of the fibroblast growth factor receptor-3 gene in one familial and six sporadic cases of achondroplasia in Japanese patients.

Achondroplasia, the most common cause of chondrodysplasia in man, is characterized by short-limbed dwarfism, macrocephaly, and dysplasia of metaphyses of the tubular bones. Recently, mutations in the gene encoding fibroblast growth factor receptor-3 (FGFR-3) have been found in patients with achondroplasia. All mutations so far reported had occurred at codon 380, resulting in the substitution of an arginine for a glycine in the transmembrane domain of the predicted protein. We have examined the transmembrane domain of the FGFR-3 gene in seven Japanese patients with achondroplasia. Of the six cases that were sporadic, all carried a mutation in codon 380; the single familial case bore a novel mutation of a G-to-T transition at codon 375, which resulted in substitution of a cysteine for a glycine.

[Molecular diagnosis of a kindred with novel mutation of methylmalonyl-CoA mutase gene using non-RI SSCP].

A deficiency of methylmalonyl-CoA mutase (MCM) results in methylmalonic acidemia, which is inherited as an autosomal recessive disease and is characterized by accumulation of precursors and abnormal derivatives of methylmalonyl-CoA in body fluids. Abnormal splicing with 13 base pairs (bp) insertion at MCM exons 2 and 3 junction in MCM transcripts and a homozygous point mutation, g to a transition, on 5 bp downstream exon 2 were detected in a proband with methylmalonic acidemia. The parents in the kindred were heterozygous carriers of the g to a transition in MCM intron 2. Non-RI single strand conformation polymorphisms (SSCP) was conducted to devise for analysis of this MCM mutation. This non-RI SSCP is considered to be useful diagnostic means with high potential for extended clinical application.

Localization of the genes for the 100-kDa complement-activating components of Ra-reactive factor (CRARF and Crarf) to human 3q27-q28 and mouse 16B2-B3.

Human and mouse genes for the complement-activating component (P100) of Ra-reactive factor, a novel bactericidal factor (CRARF and Crarf), were mapped to R-banded metaphase chromosomes by fluorescence in situ hybridization with human and mouse P100 cDNA 2.7 and 2.0 kb long, respectively. The localization of fluorescent signals showed that CRARF and Crarf mapped to human 3q27-q28 and mouse 16B2-B3, respectively. This evidence is consistent with the previous assumption that the distal portion of the long arm of human chromosome 3 is homologous to the proximal portion of mouse chromosome 16.

Asphyxiating thoracic dystrophy: surgical correction and 2-year follow-up in a girl.

A 15-month-old girl under mechanical ventilation with asphyxiating thoracic dystrophy underwent surgical thoracic expansion according to the procedure of Todd et al. (1986). Now aged 4 years, she is free from respiratory distress, is of normal intelligence, and leads an active life.

Metacarpophalangeal pattern profile analysis in 14 Japanese children with Sotos syndrome.

Metacarpophalangeal pattern profile (MCPP) was analyzed in 14 Japanese children (mean age 6.7 years old) with Sotos syndrome. The patients were divided into 2 groups based on age; group 1 (n = 8): 6 years or over; group 2 (n = 6): less than 6 years. The mean values of standard deviation of the 14 patients with obviously large hand were all above 1.4. The MCPP in group 1 showed (1) two major peaks in metacarpal and proximal phalangeal areas, (2) a small peak in middle phalangeal area, and (3) relatively short distal phalangeal bones compared with the metacarpal and proximal phalangeal bones. The MCPP in group 2 was similar to that in group 1, but an additional peak was observed in distal phalangeal area. The MCPP of Japanese patients showed a quite similar pattern to that of Caucasian patients, and we conclude the method can also be a useful tool in the diagnosis of the Japanese patients. In correlation study, 8 of the 14 patients had a significant positive correlation, but 2 patients in group 2, less than 3 years, had no positive correlation. We suggest the method is not applicable to young patients less than 3 years.

A 100-kDa protein in the C4-activating component of Ra-reactive factor is a new serine protease having module organization similar to C1r and C1s.

Ra-reactive factor (RaRF), a C-dependent bactericidal factor in mice, is composed of one polysaccharide-binding component and one C4/C2-activating component. The former is an oligomer of 28-kDa protein corresponding to the mannose-binding protein of mice. The 100-kDa protein, P100, has been shown to be present in the C4/C2-activating component. This protein generates 29- and 70-kDa polypeptide chains when reduced. In this study, we determined the nucleotide sequence of cDNA coding for P100. cDNAs were prepared by reverse transcription PCR and cassette-ligation-mediated PCR on mRNA from BALB/c mouse liver, using primers synthesized by reference to the sequence determined in a previous study. The results of cDNA sequencing indicate that the precursor protein of P100 containing a 24-residue signal peptide consists of 704 amino acid residues. Taking the results of the previous electrophoretic study into consideration, it is thought that the cleavage of mature P100 protein generates a 29-kDa chain of 251 residues and a 70-kDa chain of 429 residues. Although homology in the amino acid sequence of P100 with that of human C1r and C1s subcomponents of C was less than 40%, a striking similarity in domain organization was found among these proteins, indicating that P100 is a new C4-activating serine protease structurally similar to C1r and C1s. Northern hybridization showed that the liver was the primary site of the expression of the P100 gene.

A new member of the C1s family of complement proteins found in a bactericidal factor, Ra-reactive factor, in human serum.

The Ra-reactive factor (RaRF) found in vertebrate sera activates the C4 and C2 components of complement. The C4/C2-activating component of mouse RaRF has been found to contain a 100-kDa serine protease called P100. In the present study, we cloned a cDNA with cDNA of mouse RaRF P100 as a probe from a human liver cDNA library. An open reading frame of 2097 nucleotides encoding a protein of 699 residues was found in the cloned cDNA of 4489 nucleotides. This protein exhibits 87.4% amino acid homology with mouse P100, and 36.4% and 37.1% homologies with that of the C1r and C1s subcomponents of human complement, respectively. The characteristic nodules and domain of C1r and C1s were highly conserved in this protein. This indicates that the P100, together with the C1r and C1s, forms a unique protein family having the same module/domain constitution.

Cytogenetic and molecular study of Angelman syndrome.

Six patients, including two sibs, with Angelman syndrome (AS; three females and three males, aged 11 to 18 years) were studied cytogenetically. Molecular analysis was also performed. Using high-resolution banding technique, we detected a microdeletion in the proximal region of chromosome 15q in four cases. The deleted segment was heterogenous between these patients, and the common deleted region appeared to be 15q11.2. Four patients with deleted 15q were all sporadic cases, whereas in the sib cases we could not detect a visible deletion in the long arm of chromosome 15. However, there was no clinical difference between sporadic cases and sib cases. Densitometric analysis of autoradiographic bands of Southern hybridization using two DNA segments, pML34 and pTD3-21, as probes demonstrated that two patients had only one copy for each of the probes. In the remaining four patients, including the sibs, two copies of each sequence were retained. The probes used here detect a molecular deletion in most Prader-Willi syndrome patients. Thus the segment causing AS is localized adjacent to the critical segment of Prader-Willi syndrome. There seemed to be heterogeneity for the molecular deletion within AS individuals.