MOLECULAR GENETICS OF HAEMOPHILIA A IN SINGAPORE
FONG LOONG KWAN IAN
FONG LOONG KWAN IAN
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Abstract
As the DNA polymorphisms of haemophilia A are influenced by ethnic variations, an initial population study is required in the major ethnic groups in Singapore. This could help to lead to the development of a more sophisticated genetic diagnosis programme through linkage analysis to be established in the future. Our study involved the use of indirect polymorphism-based genetic markers and direct mutation detection techniques to study 344 normals and 46 affected families. Our main objective is to determine suitable DNA polymorphisms which reveals maximum informativity in the three major ethnic groups, namely Chinese, Indian and Malay, in Singapore for linkage studies in their respective family members.
Four Four intragenic bi-allelic polymorphisms (RFLP) namely G:A - intron 7, BcII - intron 18, HindlII - intron 19, and XbaI - intron 22, and two multi-allelic dinucleotide repeat polymorphisms in introns 13 (JCA13) and 22 (JCA22) were studied. The results obtained from intragenic polymorphism studies using BcII, HindIII, and XbaI RFLPs, showed high informativity in our Singapore population. The former two RFLPs, were found to be most informative in the Indian ethnic group with heterozygosity rates of 46% for both RFLPs. In contrast, the XbaI RFLP showed to be the most informative for the Chinese ethnic group with a heterozygosity rate of 48%. As for the dinucleotide repeats in intron 13 and 22, the highest heterozygosity rates of 59% and 55% respectively were found in the Malay ethnic group In addition, the allele sizes of intron 13 repeats were found to be larger when compared to the Caucasian population. A new allele of 30 (CA)n repeat was found in intron 13, and three new alleles of 31, 30 and 28 (GT)n(AG)n repeat were also found in intron 22. Intron 7, G:A polymorphism was not found to be useful for pedigree analysis of our population because of its insignificant heterozygosity rate of 3.1%. For the pedigree analysis of the 46 affected families, the best combination of genetic markers was determined to the HindIII, XbaI, JCA13 and JCA22. This combination contributed to 80.4% (37/46) informativity in the affected families. Carrier status was ascertained in 14 out of the 15 females at rick from 12 informative cases with family history.
As for the direct mutation detection, the PCR-heteroduplex technique was used to analyse almost the entire coding region of factor VIII gene (9 kb) for point mutations, small deletions and insertions. Ten out of 16 genetic defects detected were novel and this included two missense mutations, five frameshift mutations, two silent mutations and one polymorphism. Pedigree analysis for 1 out of 9 families which were uninformative from the DNA polymorphism analysis, was eventually made possible through direct analysis of the causative of both indeirect and direct molecular analysis, 82.6% (38/46) of the affected families in this study were found to be informative. In conclusion, there is definitely a need of a more exhaustive study in this field as to enhance the level of standard and sophistication of carrier and pedigree analysis to an ideal 100% informativity in Singapore in the near future.
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Date
1996
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