Haplotype analysis at the FRAXA locus in Thai subjects.

The prevalence of fragile X syndrome (FXS) is approximately 7% in Thai boys with developmental delay of unknown cause. To determine if FXS might have a specific haplotype association, we analyzed 125 unrelated control subjects and 25 unrelated FXS patients using 3 microsatellites, DXS548, FRAXAC1 and FRAXE, and two single nucleotide polymorphisms, ATL1 and IVS10. FRAXAC1 and DXS548 are located approximately 7 kb and approximately 150 kb proximal to the CGG-FMR1 whereas ATL1, IVS10 and FRAXE are located approximately 5.6 kb, approximately 24.5 kb and approximately 600 kb distal to the CGG-FMR1. We found 40 haplotypes in the control group and 14 haplotypes in the FXS group. Of 14 haplotypes in the FXS group, 6 haplotypes were not found in the control group suggesting possible new mutations or admixture of immigrant haplotypes. We observed that most diverse haplotypes came from different FRAXE alleles. For this reason, we analyzed haplotypes composed from the remaining markers alone (DXS548-FRAXAC1-ATL1-IVS10). We found 2 major haplotypes (20-18-G-T and 20-19-A-C) with no significant haplotype differences between the control group (67/125 of 20-18-G-T and 25/125 of 20-19-A-C) and FXS group (16/25 of 20-18-G-T and 6/25 of 20-19-A-C). The other haplotypes found were 33/125 in the control group and 3/25 in the FXS group. The two major haplotypes associated FXS in Thai subjects were the two most common haplotypes in the normal Thai subjects. We could not prove, therefore, that there were founder effects at the FRAXA locus in Thailand. We could not, however, exclude it completely. These findings apparently contrast with most other reports on FXS founder effects in various ethnic groups.

Prenatal diagnosis of beta-thalassaemia by reverse dot-blot hybridization.

Thalassaemia is the most common genetic disease and is a public health problem of Thailand. Prevention and control of beta-thalassaemia diseases need accurate diagnosis of carriers and proper genetic counselling. Prenatal diagnosis is needed to prevent birth of the thalassaemic offspring in the couple at risk. This can be performed in the first trimester of pregnancy by DNA analysis using the polymerase chain reaction (PCR). Since there are more than 20 mutations causing beta-thalassaemia in Thailand, the point mutation detection by reverse dot-blot allele-specific oligonucleotide (ASO) hybridization was developed using two sets of ASO probes. The first battery of ASO probes has been designed to detect 10 common beta-globin gene mutations including codon 26, G->A (Hb E): codons 41/42, -TCTT; codon 17, A->T; IVS 2 nt 654, C->T; IVS 1 nt 1, G->T; IVS 1 nt 5. G->C; codon 19, A->G (Hb Malay); codon 35, C->A; codons 71/72, +A and -28 ATA, A->G. The second set of ASO probes detect 14 uncommon beta-thalassaemia mutations. We applied this reverse dot-blot hybridization technique to perform prenatal diagnosis in 105 pregnancies at risk of having severe beta-thalassaemia diseases. 36 fetuses (34 per cent) were found to be affected with homozygous beta-thalassaemia or beta-thalassaemia/Hb E disease in which one was twin pregnancy. The others included 31 fetuses with heterozygous beta-thalassaemia, 22 heterozygous Hb E, 1 homozygous Hb E and 16 normal fetuses. The common set of ASO probes detected about 95 per cent of cases which suggests that prenatal diagnosis for beta-thalassaemia disease can be easily carried out by this approach.

A novel 105 basepair deletion causing beta(0)-thalassemia in members of a Thai family.

We identified and characterized a novel beta(0)-thalassemia mutation due to partial deletion of the 5' end beta-globin gene including the mRNA cap site and a part of exon 1. The deletion was precisely 105 basepair (bp) in length extending from position -24 or -25 to +80 or +81 relative to the beta-globin gene mRNA cap site. This mutation was detected in three individuals from a family originating in the area of southern Thailand. The propositus was a 39-year-old female and noted to be heterozygous for beta-thalassemia with hemoglobin (Hb) level of 10.1 g/dl, MCV 70 fl, MCH 23.1 pg, HbA2 6.3%, and HbF 2.4%. Her son was 9 years of age and was also heterozygous for the mutation, having Hb level of 10.8 g/dl, MCV 58 fl, MCH 19.0 pg, HbA2 5.6%, and HbF 4.3%. Her 6-year-old daughter was affected, having a genotype of this mutation and a G-C transition at IVS 1 nt 5. Although the deletion does not include the beta-globin gene promoter sequences, the individuals heterozygous for this mutation have an elevated HbA2 level slightly higher than observed in most carriers of beta-thalassemia caused by point mutations.

Analysis of beta-thalassemia mutations and beta-locus control region hypersensitive sites 2, 3 and 4 in southern Thailand.

beta-Thalassemia mutations in 221 chromosomes of unrelated southern Thai patients were analyzed. Using dot blot hybridization of PCR amplified DNA with 15 allele specific oligonucleotide probes for beta-thalassemia mutations 196/221 (89%) of the alleles were characterized. Ten mutations were identified, of which six [codon 41/42 (TTCTTT-TT), IVS1 nt5(G-C), codon 19 (AAC-AGC), codon 17 (AAG-TAG), IVS1 nt1(G-T), -28 TATA (A-G)], accounted for 85%. Among the 25 uncharacterized alleles, 15 were analyzed by automated fluorescent DNA sequencing of the whole beta-globin gene with normal results in 7 alleles. Four mutations, previously described were detected in 8 alleles. They were a G-A at IVS1 nt1 in one heterozygote, a G-T at IVS1 nt1 in one heterozygote, codon 15 (TGG-TAG) in two heterozygotes and poly A(AATAAA-AATAGA) in two homozygotes. The polyadenylation mutations, previously demonstrated in the Malaysian population have been first detected in Thailand. It is remarkable that the IVS1 nt1 (G-A) mutation, previously reported in the Mediterranean population has been found only in the south of Thailand. This mutation was probably imported from Portugal. In former times the Portuguese had settled in Phuket in southern Thailand. In order to find a causative mutation in the rest of 7 true unknowns we performed direct DNA sequencing of the core fragments of the beta-Locus Control Region Hypersensitive Sites (LCR HS) 2,3 and 4 in these 7 samples. DNA sequencing of HS2 and HS3 fragments showed normal results. The heterozygote A/G was present in the palindromic sequence of the LCR HS4 (TGGGGACCCCA) in 6 beta-thalassemia samples. The same heterozygote A/G was found in 5/12 normal subjects. The allele frequency of A (0.79) is obviously higher than that of G (0.21). This could be due to the stability of the palindromic structure. When an A is in the middle of the palindromic sequence, the hairpin structure is formed. In contrast the hairpin structure disappears when a G is in the middle of the palindromic sequence. This structure is not further symmetric and may not be so stable as the hairpin structure. beta-Thalassemia mutations in southern Thailand are very heterogeneous and their distribution is different from other parts of the country.

Mitochondrial genome analysis in Kearns-Sayre syndrome.

We analysed the mitochondrial genome of one patient with chronic and progressive bilateral ophthalmoplegia. This patient also had abnormal EKG showing cardiac conduction defects and pigmentary retinopathy, suggestive of the Kearns-Sayre syndrome. On muscle biopsy, with Gomori trichrome stain, the fibers showed an increase in red-staining material in the intermyofibrillary network and the subsarcolemmal region. On electron microscopy, aggregations of abnormal mitochondria were demonstrated, confirming the diagnosis of mitochondrial myopathy. Analysis of mitochondrial DNA (mtDNA) from the patient and her mother showed no deleted mtDNA.

The spectrum of beta-thalassemia mutations in southern Thailand.

Beta-thalassemia mutations in 282 alleles of 253 unrelated individuals originating from various provinces in the south of Thailand were characterized by dot blot hybridization, specific PCR-amplification and direct DNA sequencing. It was possible to characterize the mutations in 274 (97.2%) of alleles studied. Twelve different point mutations and two different large deletions of the beta-globin gene were identified. Seven common mutations, namely 4 bp deletion at codons 41/42. IVS1 position 5 (G-C), codon 19 (AAC-AGC), codon 17 (AAG-TAG), IVS1 position 1 (G-T), position -28 (A-G) and 3.5 kb deletion, accounted for about 91.5%. The mutations at mRNA cap site + 1 (A-C) and IVS1 position 1 (G-A), previously undescribed in Thailand, were found in 1 and 2 individuals, respectively. A novel mutation of 105 bp deletion at the 5' end of beta-globin gene was detected in a family originating from this area. The knowledge from this study should be useful for planning of genetic counseling and prenatal diagnosis programs for patients with beta-thalassemia in the south of Thailand.