Molecular defect of PKD1 gene resulting in abnormal RNA processing in a Thai family.

Autosomal dominant polycystic kidney disease (ADPKD) is a common human autosomal disorder caused mainly by mutations of the PKD1 gene. In analysis of PKD1 transcripts by long RT-PCR and nested PCR procedures, we observed PKD1-cDNA fragments from three ADPKD siblings from the same family with a size approximately 250 base pairs (bp) shorter than normal. Further investigations showed that the PKD1 transcripts from these patients had been abnormally processed, the nucleotide sequence of exon 43 containing 291 nt was missing from the transcripts, which would result in an abnormal polycystin-1 with an in-frame deletion of 97 amino acids. This splicing defect did not result from a mutation that disrupted the splice donor or acceptor sites adjacent to exon 43 or the branch sites in flanking introns but was most likely due to 20-bp deletion observed in intron 43. The intronic deletion was present in 8 affected members but absent in 11 unaffected members, corresponding with the results of genetic linkage analysis using 5 polymorphic markers in the PKD1 region. Molecular diagnosis of PKD1 in this family could, therefore, be carried out by genomic DNA amplification to directly detect the PKD1 intronic deletion.

Detection and typing of human papilloma virus DNAs in normal cervix, intraepithelial neoplasia and cervical cancer in Bangkok.

We detected and typed HPV-DNA by polymerase chain reaction (PCR) in cervico-vaginal lavages of 102 women with normal cervical cytology, 57 patients with cervical intraepithelial neoplasia (CIN), and 23 cervical cancer patients. HPV-DNA detection and typing by in situ hybridization were also performed in cervical biopsies from CIN lesions and cancers. Five percent of women with normal cervical cytology, 46% of CIN, and 61% of cervical cancer were positive for HPV-DNA. Of CIN cases with positive HPV-DNA, 69, 15, 8, 4 and 4% were HPV-16, -33, -18, -11 and -16/33 respectively. Of cervical cancer cases with positive HPV-DNA, 86% were HPV-16, 7% were HPV-16/33, 7% were HPV-18/31. HPV typing was performed in biopsies from 37 CIN and 18 cervical cancers by in situ hybridization. By this method, 38% of CIN were HPV-DNA positive, of which 71% were HPV-16 and 7% were each of HPV-11, -18, -31 and -33. Thirty-nine percent of cervical cancers were positive, of which 71% and 29% were HPV-16 and HPV-16/18 respectively.

Rapid detection and identification of dengue viruses by polymerase chain reaction (PCR).

A polymerase chain reaction (PCR) method using sets of newly designed primers for rapid detection and simultaneous identification of dengue virus serotypes was developed and tested. The test is based on two sets of primers specific within the envelope (E) and non-structural (NS1) regions of the dengue-virus genome. Two sets of universal primers that bind to two target sequences which are shared by all the four serotypes of the virus within the E and NS1 regions are used. The resulting products are further amplified by another pair of inner or nested universal primers, which also bind to another set of shared sequences within the E and NS1 regions, respectively. The nested PCR of both the E and NS1 regions can detect dengue virus of all the four serotypes at a sensitivity of 1 plaque forming unit (pfu) or less. For the identification of serotypes, a mixture of four pairs of serotype-specific primers, specific to the E region, was used. The primers have been designed to bind to serotype specific sequences within the regions flanked by the outer universal primers, and giving the amplified products of different sizes, each corresponds to one particular serotype (405 bp for Den1, 346 bp for Den2, 196 bp for Den3, and 143 bp for Den4). A protocol has been developed and successfully applied to detect dengue virus in cell-culture supernatants and patients sera. The technique is simple and rapid, capable of not only detecting the dengue virus but also identifying the serotypes of the virus in clinical specimens.

Heterogeneity of beta thalassaemia in Thailand.

Beta thalassaemia in Thailand is heterogeneous. Clinical, genetical, haematological and globin chain biosynthetic studies were performed in seven beta-thalassaemia families. The results showed different gene combinations. These were alpha-thalassaemia/homozygous beta 0-thalassaemia, questionable double heterozygosity between a beta-thalassaemia and a silent beta-thalassaemia genes with low Hb F of unexplained cause, silent beta-thalassaemia/beta+ - or beta 0-thalassaemia, high Hb A2 high Hb F-beta-thalassaemia - a new mutant, mild beta+-thalassaemia/Hb E, and beta 0-thalassaemia/heterocellular HPFH or delta beta-thalassaemia associated with alpha-thalassaemia. Most of variability of clinical and haematological findings in these families is due to heterogeneity of the beta-thalassaemia and related genes.