Utility of multiplex ligation-dependent probe amplification (MLPA) for hemophilia mutation screening.

Hemophilia A (HA) and B (HB) are estimated to affect 1 in 5,000 male births in the United States each year.[1] Inheritance of mutations in the Factor VIII (F8) gene or Factor IX (F9) gene causes these bleeding disorders. Identification of mutations causing a patient’s hemophilia can lead to better understanding of risk of complications [2], as well as aid in carrier detection in family members [3]. Mutation screening for HA has involved testing for inversions of introns 1 and 22 of F8, as approximately 45% of severe HA patients carry an inversion as their causative mutation [2], and sequencing of the coding regions of F8 to identify point mutations, deletions, or splice-site mutations. Similarly, mutation screening for HB has involved sequencing of the coding regions of F9. However, a subset of patients presenting with hemophilia do not to have a detectable mutation with these methods.[4] Duplication of part of F8 or F9, for example, may not be detected. Also, female family members heterozygous for a large F8 or F9 deletion may not be identified as carriers using these methods, as dosage of the genes is not determined. Recently, Multiplex LigationDependent Probe Amplification (MLPA®, MRC Holland, Amsterdam, Netherlands) has been successfully used to identify large deletions and duplications within F8 and F9.[5-7] This assay quantitatively compares copy numbers of a set of DNA sequences in a patient sample to those in a control sample to screen for the presence of deletions or duplications.[8] The assessment of how this or similar technologies will fit into currently-used mutation screening protocols should be critically evaluated.