Detection of large deletions in the factor viii (f8) and von Willebrand factor (vwf) genes using multiplex ligation-dependent probe amplification (MLPA)

O’Brien, Michelle (2013) Detection of large deletions in the factor viii (f8) and von Willebrand factor (vwf) genes using multiplex ligation-dependent probe amplification (MLPA). Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Haemophilia A and von Willebrand Disease (VWD) are disorders which can cause mild to severe bleeding in an individual. In many cases, knowledge of the molecular basis of these disorders is required to enable appropriate genetic counselling within families, carrier diagnosis and, in some cases, prenatal diagnosis. The current ‘gold standard’ for diagnosing patients with Haemophilia A and VWD disorders is by direct sequencing of genomic DNA to identify mutations. However this method may not reveal the presence of larger heterozygous deletions or duplications of F8 and VWF due to masking by an unaffected allele. Therefore the genetic basis of disease in a proportion of cases may remain unresolved. Multiplex ligation-dependent probe amplification (MLPA) is a method based on gene dosage that can be applied to the detection of large gene deletions or duplications in Haemophilia and VWD, aiding effective genetic diagnosis. This project developed and applied MLPA to investigate the molecular basis of disease in a selected cohort of patients diagnosed with Haemophilia A, type 1 and type 3 VWD, where standard direct sequencing protocols of F8 and VWF had failed to reveal causative mutations, and to assess its application in the genetic diagnosis of these disorders. This extended previous work on the molecular basis of Haemophilia A and VWD carried out in the Molecular Diagnostics Centre (MDC) at Central Manchester NHS Foundation Trust. MLPA confirmed the presence of large deletions in seven Haemophilia A index patients, two type 1 VWD patients, seven type 3 VWD patients and one possible duplication in a female haemophiliac. The presence of a masked gene abnormality was also confirmed in two carrier females. This demonstrates that MLPA is a useful diagnostic tool for detecting or confirming larger scale mutations not suited to DNA sequence analysis. MLPA is an important addition to the range of molecular diagnostic tools for the investigation of these haemostatic disorders and may be utilised when conventional mutation screening has failed to identify a causative mutation.