Hemostasis Reference Laboratory

DNA Hemophilia A Mutation Evaluation

• Transaction code: 3250-02
• CPT codes (suggested): 83890, 83898 (x33), 83908 (x2), 83904 (x34), 83909, 83892, 83894, 83912

Severe patients that are negative for the DNA Factor VIII inversion or patients that have moderate or mild hemophilia can be evaluated for mutations. Through our current screening methods, a mutation is found ~90-95% of the time. A reference for the DNA FVIII mutation database can be found at: HAMSTeRS.

If the specific mutation cannot be identified, intragenic linkage is often useful to trace the affected allele through the family tree by following 5 polymorphic sites. Linkage studies also help identify the de novo origin of mutations in families with an isolated occurrence (eg: maternal grandparents). Intragenic linkage may be useful in 80-90% of families where key family members (patient and both parents of potential carrier) are available.

Method
For approximately half of the severe hemophilia A patients (and moderate cases with FVIII levels of ~1%), the DNA Factor VIII Inversion will be negative. For these patients, along with the moderate and mild patients, the Hemophilia A Mutation Evaluation will be performed to identify the mutation. A MDE (mutation detection enhancement) screen will be performed initially and if needed, will be followed by a sequence screen. Once identified, the genotype will be tested by confirmation analysis.

Confirmation is performed one of two ways. If the mutation creates/removes a restriction enzyme digestion site, the testing will be performed by RFLP. If a restriction enzyme digestion site is not present, testing will be done by sequencing the exon in which the mutation is located.

Test Details

• Turnaround Time: FVIII inversion testing takes 1-2 weeks. If the mutation is unknown an evaluation will be performed, which may take an additional month. If the mutation is identified, screening for family members takes 1-2 weeks.
• Sample Requirements: 5-10mL whole blood drawn in EDTA and shipped at 4°C or ambient temperature is acceptable. Refer to the sample requirements page for more information on shipping.
• Analysis of amniocytes can be performed on cultured amniocytes. Two T-25 flasks of cells cultured to confluency will be accepted. Puget Sound Blood Center does not have facilities to culture amniocytes.