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Keywords:

  • factor X deficiency;
  • acceptor splicing site;
  • polypyrimidine tract;
  • spliceosomes

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. Acknowledgements
  6. References

We investigated the molecular defect underlying congenital factor X (FX) deficiency in a Japanese patient. A novel three-base-pair deletion was identified within intron D of the FX gene. An allele-specific restriction analysis showed the propositus was homozygous and her two daughters were heterozygous for the deletion. It was not detected in 53 unrelated Japanese (106 alleles), indicating a probable cause of the FX deficiency. The deletion resides within a polypyrimidine tract of the acceptor splicing site where U2 snRNP binds to form spliceosomes. The defect could alter the formation of spliceosomes, resulting in incorrect splicing and decreased FX production.

Factor X (FX) is a vitamin K dependent plasma protein required for the intrinsic and extrinsic pathways of blood coagulation ( Davie et al, 1991 ). It is synthesized in the liver as a single chain polypeptide of 488 amino acids, and circulates in the plasma as disulphide-linked light and heavy chains after removal of a signal peptide and a tripeptide, Arg140-Lys141-Arg142, during processing ( Jackson & Nemerson, 1980). The FX gene consists of eight exons spanning approximately 27 kilobases in length on chromosome 13q32, and the sequence of the cDNA and exon/intron boundary have been determined ( Leytus et al, 1984 ; Fung et al, 1985 ; Royle et al, 1986 ). Congenital deficiency of functional FX, one of the rarest coagulation disorders, was first described by Graham & Barrow (1957), and its genetic abnormality was first identified by Reddy et al (1989 ). Subsequently, 17 mutations have been reported in the literature (HGMD; http://www.cf.ac.uk/uwcm/mg/hgmd0.htm, accessed on 27 February 1998). Here we describe a novel mutation of the FX gene observed in the FX-deficient patient.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. Acknowledgements
  6. References

Patient's profile

A 56-year-old Japanese female with life-long history of frequent subcutaneous haemorrhage, epistaxis and hypermenorrhea was referred to our department. She had suffered prolonged bleeding after a tooth extraction and two vaginal deliveries at age 23 and 30. At the age of 45 she was admitted to our hospital due to hyperthyroidism. Laboratory examinations showed a prolonged prothrombin time (26.9 s v 10.2 s, control) and activated partial thromboplastin time (62.8 s v 31.2 s, control). The activity of coagulation FX was 2.5% (PT-derived method) and 4.5% (with Russell's viper venom). The FX antigen was < 5% (Laurell's method), and an inhibitor for FX was not detected using the cross-mixing test. Consanguinity was noted for her parents. Her two daughters showed decreased FX activity (first daughter 50%, second daughter 69%). At that time the patient was diagnosed with congenital FX deficiency. Prior to the study, informed consent was obtained from the family members entering this study.

Western blotting

Citrated plasma was diluted 1:50 with phosphate-buffered saline (pH 7.4), and 10 μl of the samples were boiled for 5 min. The samples were subjected to SDS-PAGE through a 4–15% gradient gel, then transferred onto a nitrocellulose membrane. The membrane was incubated with rabbit anti-human factor X antibody (Nordic Immunol Laboratory, Netherlands), then with biotinylated anti-rabbit IgG (1:3000, Vector Laboratories Inc., Burlingame, Calif.). FX antigen was visualized by using streptavidin alkaline phosphatase conjugate (1:5000, Amersham), nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.

Polymerase chain reaction (PCR) and DNA sequencing

PCR primers for all exons and exon/intron boundaries of the FX gene were synthesized according to the published information with some modifications ( James et al, 1991 ). After PCR, the amplified fragments were subcloned into pGEM-T vectors (Promega Co., Madison, Wis.) for preparing single-stranded DNA for nucleotide sequencing.

Allele-specific restriction enzyme analysis (ASRA)

Primers encompassing the mutation site were as follows: FXPst-F; 5′-GATGTAGCTGGCACCCTTGG-3′ (distance from 5′-end of primer to exon 5 was 79 bp according to sequence data; Fung, 1988) and FXPst-R; 5′-GCTCAGTCCTGTCCTCTTGG-3′ (142 bp to exon 5). After PCR, the amplified fragments were digested with an endonuclease PstI at 37°C for 90 min, then the digests were analysed by electrophoresis.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. Acknowledgements
  6. References

Western blotting showed a trace amount of FX antigen in the propositus plasma, we diagnosed the patient as cross-reacting material negative FX deficiency (data not shown). Molecular abnormality was analysed by PCR amplification followed by DNA sequencing. A novel three-base-pair deletion was identified within the polypyrimidine tract at the acceptor splicing site of the FX intron D (Fig 1). This deletion created a new PstI restriction site, which was analysed by ASRA. It was suggested that the propositus was homozygous and her two daughters were heterozygous for this deletion (Fig 2), although another explanation of hemizygosity (one FX gene missing and one three-base-pair deleted gene) of the propositus was not completely ruled out (her parents were deceased).

image

Figure 1. Fig 1. Autoradiogram illustrating the DNA sequence analysis of the propositus and control subject. Nucleotide sequence around intron D/exon 5 junction of the factor X gene was shown. A three-base deletion, CTT, was observed in an acceptor splicing site of the intron D of the patient's factor X gene. This deletion resides within the polypyrimidine tract, and creates a new PstI restriction site in the affected allele (right panel: side-lined).

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image

Figure 2. . Positions of molecular weight markers are shown on the left, and corresponding fragment sizes on the right.

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Fifty-three unrelated Japanese were also analysed for the presence of this deletion, but no individual showed the same digestion pattern (data not shown). These results suggest that the deletion was a possible cause of congenital FX deficiency. 17 known mutations, consisting of 16 missense/nonsense mutations and one small deletion resulting in a frame shift have been reported ( Reddy et al, 1989 ), but our case is unique in that it appears to be a splicing site mutation.

In eukaryotes, almost all of the nuclear genes coding for proteins are split into coding (exons) and non-coding (introns) sequences. The latter are removed by a highly accurate cleavage/ligation reaction, known as splicing, before the mRNA is transported to the cytoplasm for translation ( Padgett et al, 1986 ). This process is critically dependent upon the sequences around the exon/intron boundaries; invariable GT and AG dinucleotides are present at the 5′ and 3′ exon/intron junctions, respectively ( Shapiro & Senapathy, 1987). Other than these short sequence motifs, extensive consensus sequences spanning vertebrate 5′ and 3′ splicing sites (ss) have been drawn up. One characteristic of 3′-ss is the existence of a long tract of pyrimidines (polypyrimidine tract) and ‘branch-site’. The length and location of the polypyrimidine tract and ‘branch-site’ is considered to be important for binding to U2 snRNP (small nuclear ribonucleoprotein particle) which is a key component in the formation of spliceosomes, the machinery for mRNA splicing ( Padgett et al, 1984 , 1986).

The present mutation resides within the polypyrimidine tract and therefore it may influence the formation of spliceosomes and accurate intron elimination. It is therefore of importance to know the resultant mRNA sequence. For this purpose we performed reverse transcription (RT)-PCR using ectopic RNAs derived from the patient's peripheral blood mononuclear cells. Primers prepared for nested RT-PCR were as follows: nFXf-1; 5′-GGAAGAGACCTGCTCATACGA-3′ (positions 174–194 in exon 2 according to GenBank, M57285), nFXf-2; 5′-GAGGCCCGCGAGGTCTTTGAGG-3′ (196–217 in exon 2), nFXf-3; 5′-GACGGCCTCGGGGAATACACC-3′ (307–327 in exon 4), nFXr-1; 5′-CCCTACCCTCACCTTGAATCT-3′ (847–867 in exon 7), nFXr-2; 5′-GTGGGCTGCCGTTAGGATGTAG-3′ (807–828 in exon 7), nFXr-3; 5′-AGGCTGCGTCTGGTTGAAGTC-3′ (655–675 in exon 6). The combination of primers for nested RT-PCR were as follows: (nFXf-1/nFXr-1)(nFXf-2/nFXr-2), (nFXf-2/nFXr-2)(nFXf-3/nFXr-3) or (nFXf-3/nFXr-2)(nFXf-3/nFXr-3). Any combination of nested primers, however, failed to amplify corresponding fragments in the patient, whereas combination of (nFXf-2/nFXr-2)(nFXf-3/nFXr-3) did amplify successfully in normal control (data not shown). The patient did not agree to donate a liver specimen. Although we could not ascertain the effect of the mutation on mRNA splicing due to limited sources for RT-PCR, the present results indicated a strong possibility of this being the causative mutation. (The nucleotide sequence data reported here will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases, AB005892.)

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. Acknowledgements
  6. References

This work was supported in part by a grant No. 10670933 for scientific research (to T.H.) from the Japanese Ministry of Education, Science, and Culture.

References

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. Acknowledgements
  6. References
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