Molecular Diagnosis And Prenatal Diagnosis Of Hemophilia A

Background:Hemophilia A（HA, OMIM#306700） is the most common X-linked recessive bleeding disease, with a worldwide incidence of about1:5000in male births. It is caused by mutation of F8gene, which has26exons spanning186kb. The severity of HA is defined by the activity of coagulation factor VIII（FVIII:C） and it is classified into three clinical phenotypes:severe （FVIII:C<0.01IU/mL）; moderate （FVIII:C0.01-0.05IU/mL）; and mild （FVIII:C>0.05IU/mL）. Almost one half of patients with severe HA have large DNA inversions that disrupt either intron22（inv22） or intron l（invl） of F8gene. Lacking of effective method for curing this disease, detecting the carrier and prenatal diagnosis of HA are vital means to stop the disease-caused allele transmission and cut down the morbidity of HA.Objective:The purpose of the research is to detect the F8gene defect in13probands of unrelated Chinese HA family from the Hunan Jiahui genetic hospital, and perform genetic diagnosis on the females and prenatal disgnosis on the high-risk foetus in HA families.Methods:Intron22inversions were identified by IS-PCR（inverse shifting-polymerase chain reaction）, non-inversion mutations of F8gene were identified by direct sequencing. HA with mutation negative was further investigated by the MLPA（Multiplex ligation-dependent probe amplification, MLPA） method.Results:The intron22inversion（distal）（inv22-1） was identified on6families by IS-PCR, six different mutations were identified in non-inversion patients by direct sequencing and MLPA including five novel mutations that were not reported previous. The five novel mutations were c.4875₄876insA, c.6267delG, c.994delT, IVS20-1G>A, duplication of exons2-22.10of18females were identified carring the same mutation as the probands in their families. Three female carriers, one female normal and one male normal fetus were detected in the five high-risk foetus.Conclusions:IS-PCR approach provides a rapid and robust tool for inv22genotyping for severe HA, and MLPA is a highly sensitive and accurate detection method for large deletion/duplication. Combining IS-PCR, direct sequencing and MLPA can efficiently detect HA, also with carried detecting and prenatal diagnosis.