The common restriction fragment length polymorphisms (RFLPs) associated with the FIX gene: 5' BamH I, Dde I, BamH I (2), Taq I and 3' Hha I were absent or of low incidence in Southern Chinese and are therefore not useful for linkage analysis. No deletion was detected amongst seven consecutive unrelated haemophilia B patients, but one had an insertion of a 15 kb Pvu II fragment containing exon d. Using an alternate strategy of polymerase chain reaction (PCR) amplification and direct sequencing, the molecular defect in the other six patients was defined. The four novel mutations characterized were: nucleotide (nt) 6410 G----C (Gly12----Ala); nt 31261 delta T (stop codon 31 bp downstream); nt 31260 C----G (Thr380----Ser) and nt 31122 C----A (Ala34----Asp). Two patients had the same mutation at nt 6365, G----A (Arg-4----Gln), identical to one previously described in other ethnic groups, suggesting that this is a hotspot for mutation. Each of the mutations was found to affect an enzyme recognition site and could thus be identified by direct visualization of abnormal restriction fragments in amplified genomic DNA. This allows rapid and accurate DNA diagnosis of haemophilia B in an ethnic group which otherwise shows little or no polymorphism for the common RFLP sites.

Three XbaI restriction fragment length polymorphisms (RFLPs) can be detected using the factor VIII-intron 22 probe (p482.6) in a XbaI-KpnI double digest of genomic DNA. The XbaI (A) site had been reported by Wion et al (1986) to be in intron 22, while the two additional sites. XbaI (B) and XbaI (C), are shown here to be X-linked and close to the XbaI (A) site. The frequencies of heterozygosity for these three sites are 0.49, 0.18 and 0.30 respectively. In 75 females the observed heterozygosity rate for the XbaI (A) site is 0.41 and this increased to 0.57 with the two additional sites. Care should be exercised when interpreting the XbaI RFLPs, since the 1.4 kb XbaI/KpnI fragment and the 4.8 kb XbaI fragment are associated with both positive XbaI (A) and XbaI (B) sites. By the combined use of the multiple XbaI polymorphisms with the BclI site in intron 18, the carrier detection rate would increase to 67%. Four prenatal diagnoses had been performed using the multiple XbaI polymorphisms.

The molecular basis of haemophilia B is heterogeneous and many mutations of the Factor IX (FIX) gene have been characterised. Using the allele-specific arrayed primer extension (AS-APEX) technology, we have designed a FIX array to simultaneously analyse 69 mutations found in British, Thai and Chinese patients. This technology overcomes the problem of multiple reverse dot-blot analysis and has a 100% accuracy in the detection of both affected subjects and carriers in families with known mutations. In seven unknown mutations from Thailand, the array could detect the specific mutation in five and in the remainders the normal primer at specific spots failed to extend due to a mutation a few nucleotides upstream, thus allowing their identification. Hence this FIX array can detect 53% of the 2891 mutation entries in the FIX database. Each of the microarray slide can be used for three different test samples and would be useful for carrier testing for common mutations and prenatal diagnosis. It is simpler and more cost effective than genome sequencing and would be particularly useful in laboratories with limited technical capabilities.

Allelic frequencies ofRFLPs at loci closely linked to the HD gene, D4S95, D4S91, D4S141, and D4S90, were determined in 13 Huntington's disease (HD) patients from nine Chinese families and 129 normal subjects. These were similar for non-HD and HD chromosomes and the HD gene in Chinese is associated with multiple haplotypes. Hence the HD gene probably arose independently in the background haplotypes of the Chinese population. The heterozygosity rates for the two most useful RFLP sites are 0-659 for D4S95-AccI VNTR and 0-494 for D4S141-HindIII.(CAG)n repeat numbers ranged from 12 to 27 in 174 normal chromosomes. In 52 meiotic recombinations, the (CAG)n repeats were stably inherited in normal families. In HD families, 12 of 13 HD patients had expanded (CAG)n repeats of 40 to 58. Additionally, 10 asymptomatic family members had expanded (CAG)n repeats and the inheritance ofthe expanded repeat was unstable in these families.