Download Identification of a factor IX point mutation using SSCP analysis and

Nucleic Acids Research, Vol. 18, No. 18 5575
Identification of a factor IX point mutation using SSCP
analysis and direct sequencing
Daniel B.Demers*, Shannon J.Odelberg1 and L.McA. Fisher2
Department of Pathology, PO Box 662, MCV Station, Medical College of Virginia, Virginia
Commonwealth University, Richmond, VA 23298, department of Human Genetics, 501 Wintrobe
Building, University of Utah, Salt Lake City, UT 84132 and department of Pathology, PO Box 597,
MCV Station, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298,
USA
Submitted May 29, 1990
Hemophilia B is an X-linked recessive disorder characterized by
reduced or functionally defective factor IX. Severity of the disease
depends on the functional significance of the genetic alteration.
We report here a C - T transition at nucleotide 20518 (numbering
by (1)) of the factor IX gene of an Idaho patient with severe
hemophilia B (factor IX activity < 1%).
A molecular defect was localized to exon VI by single-strand
conformation polymorphism (SSCP) analysis (2). To obtain
sequence data the polymerase chain reaction (PCR, (3)) was used
to symmetrically amplify a 250 bp fragment encompassing all
of exon VI including both intron—exon splice junctions. The PCR
product was subsequently used as template for asymmetric
amplification followed by direct sequencing using a Sequenase
Version 2.0 kit (US Biochemicals). Sequence analysis revealed
a C —T transition at nucleotide 20518 predicting an Arg to Trp
replacement at amino acid 180 of the protein (Figure 1). This
mutation would alter the conserved carboxy-terminal cleavage
site of the activation peptide (Arg18O-Val181) thereby resulting
in a protein unable to participate in the clotting cascade (4). We
designate this variant FIX 180 (CGG—TGG). Although it cannot
as yet be classified as a hemophilia B^ variant (CRM status and
ox brain prothrombin time unknown), it is likely to represent
the same mutation responsible for the Arg180—Trp substitution
in factor IX BM Nagoya (5) and factor TX BM Deventer (4, 5,
6). It is similar to factor IX BM Hilo (180 (CGG—CAG)) in that
it involves a base substitution in codon 180 and results in severe
hemophilia B (4).
ACKNOWLEDGEMENTS
We thank M.H.Ford and B.Baty of the Comprehensive
Hemophilia Program at the University of Utah for providing us
with blood samples and patient information.
* To whom correspondence should be addressed
REFERENCES
1. Yoshitake,S., Schach.B.G., FosterAC., Davie.E.W. and KurachiJC. (1985)
Biochemistry 24, 3736-3750.
2. Orita.M., Suzuki.Y., Sekiya.T. and Hayashi.K. (1989) Genomics 5,
874-879.
3. Saiki.R.K., Gelfand.D.H., Stoffd.S., Scharf.S.J., Higuchi.R., Hom.G.T.,
Mullis.K.B. and Erlich.H.A. (1988) Science 239, 487-491.
4. Huang,M.-N., Kasper.C.K., Roberts.H.R., Stafford.D.W. and High.K.A.
(1989) Blood 73, 718-721.
5. Suehiro.K., Kawabata,S., Miyata.T., Takeya.H., TakamatsuJ., Ogata.K.,
Kamiya.T., Saito.H., Niho.Y. and Iwanaga.S. (1989) / . Biol. Otem. 264,
21257-21265.
6. Bertina.R.M. and van der Lindcn.I.K. (1982) Thromb. Haemostas. 47,
136-140.
NORMAL
G
A
PATIENT
T
C
G
A
T
C
Val 182
T
Val 182
Val 181
T
Val 181
Arg 180
Thr 179
Phe 178
Figure 1. Part of the nucleotide sequence for exon VI of the human factor IX
gene along with translation products and their corresponding codon number. The
normal sequence is shown on the left and that of the patient on the right. Arrow
indicates the nucleotide difference between the normal and mutant sequence. The
affected codon is marked with an asterisk.