First study of the C2491t nonsense mutation frequency in Moroccan healthy population

First Study of the C2491t Nonsense Mutation Frequency in Moroccan Healthy Population

K. Hamzi_&B. Diakité_&W. Hmimech_&S. Nadifi

Received: 13 May 2013 / Accepted: 4 June 2013

#Springer Science+Business Media New York 2013

Abstract The mutation FV Leiden (G1691A) is the most common mutation worldwide with a variable allelic frequen- cy between countries. More frequent in the European and Caucasian populations and rare or absent in African native population, the FVL was studied in the Moroccan population (They-They et al. Ann Hum Biol 37(6):767–777,2010) and is totally absent as reported previously by (Mathonnet et al.

Thromb Haemost 88(6):1073–1074, 2002). Here, another mutation in FV (Q773Term) was detected in a Moroccan patient, which took our interest for this study and establishes the first epidemiological database for future associated stud- ies concerning neurovascular diseases.

Keywords First Study . C2491t Nonsense Mutation . Moroccan Health

Introduction

Human coagulation factor V (FV) is a single-chain glyco- protein that plays an important role in maintaining hemostat- ic balance (Jenny et al.1994). It circulates in the blood as an inactive procoagulant enzyme. In its active form, FV forms an essential part of the prothrombinase complex that cata- lyzes the conversion of prothrombin to thrombin by factor Xa in the presence of calcium and a phospholipid membrane.

Activated protein C (APC) inactivates FV through cleavage of the active cofactor at R306, R506, and R679 and requires FV as a cofactor in the APC-mediated inactivation of factor V III (Tuddenham and Cooper 1994). The gene for

coagulation FV has been mapped to chromosome 1q23 and spans more than 80 kb (Rosing et al.1997). It consists of 25 exons and the messenger RNA encodes a leader peptide of 28 amino acids and a mature protein of 2,196 amino acids.

Roughly, the heavy chain is encoded by exons 1 to 12 and the light chain by exons 14 to 25 (Rosing et al. 1997).

The entire B domain is encoded by exon 13, which contains two tandem repeats of 17 amino acids and 31 tandem repeats of nine amino acids that are absent in the B domain of FV III (Rosing et al.1997). The mutation FV Leiden (G1691A) is the most common mutation world- wide with a variable allelic frequency between countries (Hoekema et al. 1997). More frequent in the European and Caucasian population and rare or absent in African native population, the FVL was studied in the Moroccan population (They-They et al.2010) and is totally absent as reported previously by Mathonnet et al. (2002).

Another mutation in FV (Q773Term) was detected in a Moroccan patient (Mathonnet et al. 2002), which took our interest for this study and establishes the first epidemiological database for future associated studies concerning neurovascular diseases.

Patients and Methods

The study group consisted of 194 unrelated healthy indi- vidual residents in Casablanca representing the major Moroccan population. Since Casablanca is the industrial capital of Morocco, there are a lot of migration events from all countries that the entire major ethnical groups can be found here. Informed consent was obtained from all con- trol individuals.

Their DNA was extracted by phenol chloroform proto- col (van Wijk et al. 2001) used to establish the allelic K. Hamzi (*)

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Laboratory of Human Genetics and Molecular Pathology, Medical School, University Hassan II Casablanca, Casablanca, Morocco e-mail: khalil.hamzi@gmail.com

J Mol Neurosci

DOI 10.1007/s12031-013-0045-1

frequencies of the Q773Term mutation (C2491T nonsense mutation in exon 13). PCR and enzymatic restriction were done as described by (van Wijk et al.2001). This mutation was confirmed by HphI digestion and was named FV Casablanca mutation. The 605-bp PCR product normally contains one recognition site for HphI, producing frag- ments of 458 and 147 bp, which were abolished by this base change and thus results in an uncut PCR product from the mutant allele.

Results

194 unrelated healthy individual residents in Casablanca representing the major Moroccan population, 106 women and 88 males, middle age is 38.30±2.01. Clinical character- istics of our populations are summarized in Table1.

In the population studied, the allelic frequency T is about 15.46 vs 84.54 % (T allele), 3.6 % mutant homozygote vs 72.7 % wild homozygote and 23.7 % heterozygote (Table2).

Discussion

FV plays an important role in the procoagulant pathway as well as in the protein C anticoagulant pathway. Deficiency of FV was first described by Owren (1947). It is a rare autosomal dominant bleeding disorder with an estimated frequency of one in one million. The recently published complete nucleotide sequence of the FV gene has facilitat- ed the molecular characterization underlying FV activity variation and has identified mutations in the FV gene.

Little is known about mutations and molecular basis un- derlying FV deficiency due to its low frequency in the population combined with the complexity of the gene itself. Indeed and before the detection of FV deficiency mutation, many cases of stroke and general thrombosis

events was reported with FV activity deficiency but with no molecular basis. (Petiot et al.1991) reported a case of thalamic stroke associated with congenital FV deficiency (Reich et al.1976; Norbert E et al.1976) described cases of thrombophlebitis and pulmonary diseases as complication of FV deficiency (Goodnough et al.1983). As most cases of FV deficiency were detected in the 1970s and 1980s, no molecular tools were available to detect mutation and genetic defects. Generally, incidence of thromboembolic events in FV deficiency is between 5 and 8.7 % and thrombotic complication seems to appear preferentially in young patients (Goodnough et al. 1983). Many studies showed the presence of R506Q mutation (FVL) and proved the absence of this mutation in the African and Moroccan population (Rosing et al. 1997; They-They et al.2010; Mathonnet et al.2002). However, Q773Term mutation was detected in a young Moroccan patient, re- ported by van Wijk et al. (2001). Q773Term is a nonsense mutation in exon 13 where a C→T substitution at nt2491 predicted a stop codon at residue 773 instead of the wild- type glutamine (van Wijk et al. 2001). Mutations in exon 13 occur frequently in the FV gene and because the B domain is relatively unimportant for FV procoagulant function, “ribosomal frame shifting” may thereby reflect one of the modulating factors involved in the phenotypic differences observed in these patients. However, mutations in exon 13 can still lead to a severe phenotypic expression of the disease.

In the Moroccan population, the presence of Q773Term mutation will lead to exploration, for a second time, of the association with cardiovascular and neurovascular diseases (stroke, MI…) and confirm the effect of our novel mutation as an important genetic factor in interaction with other ge- netic factors.

The allelic frequency found in our population (15.46 %) is relatively high; however, as our study is the first in the world concerning the Q773Term mutation, we didn’t have any data to compare with and analyze the mutation evolution.

Table 1 Clinical characteristics

of our studied population Parameters Frequency (%)

Age 194

<45 127 (65.5)

≥45 67 (34.5)

Sex 194

Female 106 (54.6)

Male 88 (45.4)

Ethnicity 194 Arabic 141 (72.7) Berber 53 (27.3)

Table 2 Allelic distribution of C2491T nonsense mutation with Hardy Weinberg equilibrium

Frequency (%) Pvalue Chi²

Genotype N=194 0.39 1.84

CC 141 (72.7)

CT 46 (23.7)

TT 7 (3.6)

Allele N=388 0.80 0.07

C 328 (84.54)

T 60 (15.46)

J Mol Neurosci

Acknowledgments The authors would like to acknowledge the Acad- emy Hassan II of Sciences and Technology for the funding of this study.

Authors are deeply gratiful to Dr Yaya and Dr Dehbi for collaboration with healthy sampling. Many thanks to Dr Van Wirk for precious help.

Conflict of Interest None declared.

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