Molecular defects in haemophilia B: detection by direct restriction enzyme analysis

Abstract

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.

Structural Integrity of the γ-Carboxyglutamic Acid Domain of Human Blood Coagulation Factor IXa Is Required for Its Binding to Cofactor VIIIa

“…This suggests a critical role for glycine (or serine) at this position. Two other mutations have been reported in F.IX affecting amino acid 12 (6,46). In F.IX Hong Kong1 , an alanine substitution results in a molecule with 3% normal activity.…”

Single nucleotide polymorphisms of the factor IX gene for linkage analysis in the southern Chinese population

“…The frequencies of these SNPs reported in the literature are summarized in Table II and are population specific. Although most SNP sites studied are polymorphic in the Caucasian and Black populations, and together can be used for up to 89% of the families at risk (Peake et al, 1993), they are not as useful in Chinese and Asian populations (Chan et al, 1991;Graham et al, 1991). On the other hand, the present described site, as well as the 5 H MseI and 3 H HhaI sites, are significantly polymorphic in the southern Chinese, giving heterozygous rates of 0´193 to 0´444 respectively.…”

“…The mother of one haemophiliac infant had the promoter region (nt 2124 to 282), all eight exons and their immediate 5 H and 3 H splice junctions, as well as the poly-A region (nt 32410±32920) of her FIX gene amplified by polymerase chain reaction (PCR). Each amplified DNA fragment was purified from 5% polyacrylamide gel and direct sequencing performed using the primary (amplification) primers and T7 DNA polymerase, as described previously (Chan et al, 1991). Sequencing was performed in both the sense and antisense strands.…”

“…Restriction fragment length polymorphism (RFLP) can be used to link the affected gene in 89% of Caucasian families using six intragenic sites (Peake et al, 1993). However, Oriental populations lack heterozygosity for these sites and diagnosis by this approach had been limited until the discovery of the 3 H HhaI site (Winship et al, 1989), with a heterozygosity rate of 0´28 in the Chinese population (Chan et al, 1991), and the 5 H MseI site , with a heterozygosity rate of 0´42 in the Thai population. We now report an additional T/C single nucleotide polymorphism (SNP) at nucleotide (nt) 32770 in the poly-A, 3 H untranslated region (3 H UTR) of the FIX gene.…”

A new hemophilia B mutation in the propeptide region of the FIX gene

“…The molecular basis of the disease is heterogenous and many mutations have been described [2]. While a number of restriction fragment length polymorphisms (RFLPs) can be used for linkage of the affected gene in Caucasians [3], in Orientals, who lack heterozygosity for these RFLP sites [3,4], prenatal diagnosis is mainly performed by direct detection of the defect.…”