Fluorescent PCR Based Gene Dose Assessment for Detection of Deletion Mutations in the FIX Gene Among Carriers of Hemophilia B.

Abstract 3486

Poster Board III-423

Haemophilia B (HB), an X linked inherited disorder is caused by heterogeneous mutations in the F9 gene. Approximately 3% of hemophilia B patients have major deletions in the F9 gene. Gross and small deletions in the F9 gene in HB affected males are easily detected by PCR but detecting the carrier state of females in the family is challenging due to the presence of the normal allele. Different methods like linkage analysis, real time PCR and MLPA have been used to assess the carrier status in this situation. Linkage analysis is limited by the availability of informative markers and adequate number of family members. Real time PCR involves standardisation and preparation of calibration curves for each run. Although MLPA is a better alternative, it can be time consuming and involves multiple steps. We have therefore developed a fluorescent PCR based gene dosage approach which is simple, rapid and cost-effective for determining the carrier status of females in families with deletions in the F9 gene. 200ng of DNA extracted by standard protocols was used for amplification with primers designed to amplify a 160bp product from exon h in the F9 gene. One of the primers was fluorescently labelled. Amplification was carried out using these primers for 20 cycles only and the amplified product was subjected to capillary electrophoresis on an ABI 310 genetic analyser. A 230 bp fragment in the albumin gene was used as the control. Analysis was done using Genescan and Genotyper software. The ratio between the peak heights of the exon h in the F9 and control genes in the patient samples were normalised to a normal control. Five families with deletional HB were analysed (in toto deletion-1; Ex g-h – 1; Ex g-poly A-1; Ex h-poly A-2). The ratios in the probands and the family members are presented in the table. Out of eight females analysed, 6 were carriers and 2 were normal. The mean ratio in the carriers was 0.49±0.08 and 0.75±0.05 in the normal. Deletions are not uncommon in HB and deletions involving the exon g and h constitute a major group. Among 212 families with HB assessed at our centre, we have identified large deletions in 8 families (3.7%). It is interesting to note that all except one of these deletional mutations involved exon h. This method confirmed the presence of these deletions in the males and helped us to identify the carrier status of the females in the family.

Identification of carrier status of females with deletions in F9 gene by gene dosage

Subject ID .	Peak height of Exh in F9 .	Peak height of albumin .	Normalised Ratio .	Interpretation .
HB5	284	442	0.59	Carrier
HB6	305	489	0.57	Carrier
HB22	188	372	0.47	Carrier
HB63	85	165	0.48	Carrier
HB129	247	295	0.78	Normal
HB238	94	326	0.4	Carrier
HB280	372	679	0.77	Normal
HB384	202	670	0.4	Carrier

Subject ID .	Peak height of Exh in F9 .	Peak height of albumin .	Normalised Ratio .	Interpretation .
HB5	284	442	0.59	Carrier
HB6	305	489	0.57	Carrier
HB22	188	372	0.47	Carrier
HB63	85	165	0.48	Carrier
HB129	247	295	0.78	Normal
HB238	94	326	0.4	Carrier
HB280	372	679	0.77	Normal
HB384	202	670	0.4	Carrier

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