To determine the spectrum of mutations of the COL4A5 gene encoding type IV collagen among Japanese Alport syndrome (AS) patients, 60 unrelated patients (47 males and 13 females) from all over the country were recruited. Screening for mutations in all the exons (1 to 51) of the COL4A5 gene was carried out by PCR-SSCP analysis. A mobility shift was observed in 22 of 60 patients, and their genomic DNA were analyzed by the direct sequence method and using cloned ssDNA. Nine of these had missense mutations in the collagenous domain (in exons 39, 37, 31, 29, 28, 27, 21, 20, 19). Eight of these mutations were observed in a codon of glycine residue. Two were altered to arginine, two to valine, two to glutamic acid and two to aspartic acid. The other missense mutation was a change from isoleucine to serine in a interruption region. Five patients had small size base deletions and one had a 4 bp insertion resulting in frameshift (in exons 49, 41, 19, 14, 13). Three had a splice site mutation (in exons 49, 47, 27). One had a nonsense mutation (in exon 17). These mutations seemed to be pathogenic, but the phenotype, which includes extrarenal manifestations, can vary with respect to both expression and severity. The remaining mutations were three silent ones (in exons 19, 39, 46). In addition, major gene rearrangement seemed to be rare in Japanese AS patients.

Samples from 916 members of various ethnic groups from malaria-endemic southern Shan State, Myanmar, were analyzed for 3-thalassemia (3-thal), 3-thalassemia (3-thal), abnormal hemoglobin variants, and glucose-6-phosphate dehydrogenase (G6PD) deficiency. Of these subjects, 530 (57.9%) were found to have at least one of these red cell genetic disorders. The overall frequencies for the various red cell genetic disorders were as follows: 3-thal, 37.5% (343/916); hemoglobin E (Hb-E), 20.3% (186/916); G6PD-Mahidol, 17.5% (160/916); and 3-thal, 0.3% (3/916). The frequencies of combined disorders were 6.9% (63/ 916) for 3-thal/Hb-E, 5.7% (52/916) for 3-thal/G6PD-Mahidol, 2.8% (26/916) for Hb-E/G6PD-Mahidol, 1.1% (10/916) for 3-thal/Hb-E/G6PD-Mahidol, and 0.1% (1/916) for 3-thal/3-thal/G6PD-Mahidol. Of the various ethnic and non-ethnic groups, the Bamar population showed the highest frequencies of 3-thal (56.9%, 177/311), Hb-E (28.3%, 88/311), and G6PD-Mahidol (21.2%, 66/311) (all duplicated and triplicated cases were included). In addition, 2 new mutations, an 3 gene triplication (/333(anti3.7); 0.2%, 2/916) and Hb-Neapolis (0.1%, 1/916), were detected. Our results showed that race was the dominant factor affecting the frequencies of red cell genetic disorders in malaria-endemic areas of Myanmar.

A novel compound heterozygous mutation of 317 CGC-->TGC with 142 GCT-->ACT in human red cell band 4.2 deficiency is described. A proband and his son suffered from compensated haemolysis with nearly complete deficiency of red cell band 4.2. Their red cell morphology exhibited microspherocytosis resembling classic hereditary spherocytosis (HS). Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed band 4.2 to be nearly missing (< 1% of normal controls) with the presence of 74 kD and 72 kD isoforms in trace amounts. Other family members (daughters older and younger than the son) exhibited nearly normal amounts of 72kD as a wild form of band 4.2 on SDS-PAGE with the presence of the 74kD isoform in a trace amount. The proband and his son demonstrated two compound heterozygous mutations in trans: i.e. nucleotide (nt) 949 CGC-->TGC (codon 317 Arg-->Cys) in exon 7 and nt 424 GCT--ACT (codon 142 Ala-->Thr) in exon 3 of the band 4.2 gene. The two daughters demonstrated only the mutation of nt 949 CGC-->TGC in exon 7 in heterozygous states, but no 142 mutation. Therefore the proband and his son were compound heterozygotes of these two mutations in trans. It is interesting to note that the 74 kD isoform of band 4.2 protein existed in a trace amount in the two daughters in spite of the absence of the 142 Ala-->Thr mutation. In addition, even in the presence of the 142 mutation in one allele in the proband and his son, their red cell morphology demonstrated classic HS with microspherocytosis, although a homozygous state of the 142 mutation known as the Nippon type of band 4.2 deficiency exhibits ovalostomatocytosis.

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DNA sequence analysis of the alpha 5(IV) collagen chain gene (COL4A5) was carried out between exon 47 and 51, which encode the noncollagenous (NC) domain, in eight Japanese families with Alport's syndrome. In one family with X-linked inheritance of the disease, a point mutation (G to C) was found at the 3' end of exon 49 in the COL4A5. This mutation converted the codon of a conserved methionine-1601 to the codon for isoleucine, and also altered the normal splicing process. The polymerase chain reaction (PCR) product amplified between exons 47 and 51 of cDNA in the affected male (hemizygote) of this family contained four fragments with various molecular weights, whereas that of a normal control contained one with the expected molecular weight. Sequence analysis of the PCR fragments of the male patient revealed various types of alternative splicing between the exons, reflecting the various sizes of PCR fragments. The PCR amplified product of the cDNA of the affected female (heterozygote), on the other hand, contained a fragment with the same molecular weight as the normal control. Sequence analysis of the PCR fragments of her cDNA revealed normal splicing and no point mutation at the 3' end of exon 49. These findings indicate that this point mutation at the consensus sequence not only converted the codon but also altered the splicing between these exons encoding the NC domain of the COL4A5. Resulting in missense of the alpha 5(IV) chain, changing a large portion of the carboxyl terminal crosslinking NC domain, this mutation can alter the normal structure of the type IV collagen network.(ABSTRACT TRUNCATED AT 250 WORDS)

We describe a silent and unstable hemoglobin variant, Hb Toranomon, which was discovered by high performance liquid chromatography in an apparently healthy Japanese male during a Hb A1c assay. Isoelectrofocusing of his hemolysate and chromatographic separation of globin on a CM-cellulose column revealed no abnormality. The isopropanol precipitation test gave a positive result. Amino acid analysis of all peptides isolated from the tryptic digests of the aminoethylated and non-aminoethylated beta chain (beta A + beta X) by reversed phase high performance liquid chromatography indicated a substitution of Cys-->Trp at position 112 of the beta chain, which was confirmed by protein sequencing. cDNA sequencing showed the nucleotide sequence of the beta 112 codon to be TGG instead of TGT, confirming the amino acid substitution described above. The globin biosynthesis ratio (beta/alpha) was 1.00.

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A new 'silent' abnormal hemoglobin, Hb Okayama IB2 (NA 2) His + Gln], happened to be discovered in a diabetic Japanese female living in Okayama Prefecture, Japan, in the course of glyco-Hb measurement of the blood samples of diabetic patients. This variant did not differ from Hb A by conventional electrophoretic tests. Only the isoelectric focusing on PAG plate for the determination of glyco-Hb and the cation exchanger chromatography were successful in the separation of this abnormal variant from Hb A and glyco-Hb. Functional study of the whole blood demonstrated a slight increase of oxygen affinity. Hb Okayama 682 (NA 2) His + Gln]Silent Hb variant