Genetic Variability of the Factor VIII Gene in the Normal Population

Genetic Variability of the Factor VIII Gene in the Normal Population

H. Singer, R. Ahmed, V. Ivaskevicius, M. Watzka, J. Schröder, O. El-Maarri, E. Seifried, R. Schwaab, P. Hanfland

and J. Oldenburg

Introduction

The factor VIII protein (FVIII) is involved in two hereditary conditions. Deficiency of FVIII leads to hemophilia A while increased levels of > 150% are known to repre- sent a risk factor for thromboembolic complications (odds ratio about 5). The mechanisms that lead to inherited increased levels of the FVIII protein are still unknown. In order to analyze the genetic variability of the F8 gene in the normal population we screened 560 alleles from male and female blood donors for the pre- sence of sequence variations by DHPLC.

Materials and Methods

The genomic DNA of 330 healthy blood donors has been amplified by PCR and screened using denaturing high performance liquid chromatography (DHPLC) (Fig. 1). The abnormal peaks obtained by DHPLC have been further sequenced by automated sequencing. From the underpresentation of missense mutations in the B-domain in hemophilic patients it was concluded that the B-domain is functio- nally less important. Furthermore, the successful treatment of hemophiliacs with a recombinant B-domain deleted FVIII also demonstrates that the B-domain might

I. Scharrer/W. Schramm (Ed.)

34^thHemophilia Symposium Hamburg 2003

” Springer Medizin Verlag Heidelberg 2005

330 blood donors Blood Donor – DNA Bank

560 alleles allow the detection of most polymorphisms with frequencies of l ess than 1%

560 alleles

230 f emales 100 males

Fig. 1. Screening strategy for

polymorphism detection.

be dispensable for the FVIII clotting function. However, in the 560 alleles of blood donors only 6 exhibited a SNP beside the known SNPs in codons 1241 and 1269 within the 3 kb encoding the B-domain, thus indicating a genetically highly con- served region.

Results and Discussion

In so far 29 of 33 analyzed fragments of the F8 gene we found 11 polymorphisms (Table 1, Fig. 2). The already reported frequent SNPs in the B-domain, Asp1241Glu and Ser1269Ser, showed frequencies of 27 and 7%, respectively, thus corresponding Genetic Variability of the Factor VIII Gene in the Normal Population 349

Table 1. Polymorphisms within the human factor VIII gene. red color indicates those with amino acid exchange.

Exon codon nt exchange aa exchange frequency

1 15 nt 102 C>T Asp>Asp 2/560

1 21 nt 120 C>T Leu>Leu 1/560

9 408 nt 1279 A>G Lys>Glu 1/560

IVS 9 – IVS 9-5 T>C – 1/560

14 794 nt 2439 G>A Leu>Leu 1/560

14 831 nt 2550 G>T Leu>Leu 1/560

14 981 nt 2998 C>T Pro>Ser 2/560

14 1038 nt 3170 A>C Glu>Ala 1/560

14 1241 nt 3780 C>G Asp>Glu 151/560

14 1269 nt 3864 A>C Ser>Ser 39/560

14 1667 nt 5058 C>T Thr>Thr 1/560

mature protein FVIII- gene

cDNA 7 kb

26 exons 186 kb

Exon 1 14 22 26

NH

A1 A2 B A3 C1 C2 COOH 2332 aa 300 kDa Fig. 2. Localization

of polymorphism

within the

FVIII gene and

FVIII protein.

very well to those reported in the literature. Beside these known polymorphisms only 9 more rare SNPs were found, that together altered 11 of 560 analyzed alleles.

While 5 SNPs (codon 15, 21, 794, 831, and 1667) showed neutral amino acid ex- changes, 3 SNPs (coden 408 Lys>Glu, codon 981 Pro>Ser, and codon 1038 Glu>Ala) led to an exchange of amino acids and one alters a splice site consensus sequence (intron 9).

Conclusion

Since SNPs are very rare within the F8 gene, the hereditary increased FVIII activity levels in the normal population cannot be explained by polymorphic variants of the FVIII protein but instead should be due to other mechanism regulating the FVIII activity.

350 H. Singer et al.