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Detection and characterization of seven novel protein S (PROS) gene
lesions: evaluation of reverse transcript-polymerase chain reaction as a
mutation screening strategy
CJ Formstone, AI Wacey, LP Berg, S Rahman, D Bevan, M Rowley, J Voke, F Bernardi, C Legnani and P Simioni
Charter Molecular Genetics Laboratory, Thrombosis Research Institute,
London, UK.
The molecular genetic analysis of protein S deficiency has been hampered by
the complexity of the protein S (PROS) gene and by the existence of a
homologous pseudogene. In an attempt to overcome these problems, a reverse
transcript-polymerase chain reaction (RT-PCR) mutation screening procedure
was developed. However, the application of this mRNA-based strategy to the
detection of gene lesions causing heterozygous type I protein S deficiency
appears limited owing to the high proportion of patients exhibiting absence
of mRNA derived from the mutation-bearing allele ("allelic exclusion").
Nevertheless, this strategy remains extremely effective for rapid mutation
detection in type II/III protein S deficiency. Using the RT-PCR technique,
a G-to-A transition was detected at position +1 of the exon IV donor splice
site, which was associated with type I deficiency and resulted in both exon
skipping and cryptic splice site utilization. No abnormal protein S was
detected in plasma from this patient. A missense mutation (Asn 217 to Ser),
which may interfere with calcium binding, was also detected in exon VIII in
a patient with type III protein S deficiency. A further three PROS gene
lesions were detected in three patients with type I deficiency by direct
sequencing of exon-containing genomic PCR fragments: a single base-pair
(bp) deletion in exon XIV, a 2-bp deletion in exon VIII, and a G0to-A
transition at position -1 of the exon X donor splice site all resulted in
the absence of mRNA expressed from the disease allele. Thus, the RT-PCR
methodology proved effective for further analysis of the resulting protein
S-deficient phenotypes. A missense mutation (Met570 to Thr) in exon XIV of
a further type III- deficient proband was subsequently detected in this
patient's cDNA. No PROS gene abnormalities were found in the remaining four
subjects, three of whom exhibited allelic exclusion. However, the father of
one such patient exhibiting allelic exclusion was subsequently shown to
carry a nonsense mutation (Gly448 to Term) within exon XII.
Volume 86,
Issue 7,
pp. 2632-2641,
10/01/1995
Copyright © 1995 by The American Society of Hematology
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