Severe coagulation factor V deficiency caused by a 4 bp deletion in the factor V gene

Factor V (FV) deficiency (parahaemophilia) is an autosomal recessive bleeding disorder with an incidence of 1:10⁶. We have studied a young girl with very mild bleeding symptoms and undetectable levels of plasma factor V antigen and activity (<0.3% and <1.6% of normal, respectively). Both parents showed plasma levels of factor V activity of about 50% of normal. Sequence analysis of the 5'- and 3'-untranslated, coding and adjacent regions of the factor V gene revealed the presence of a 4 bp deletion in exon 13. Subsequent screening of members of the family for the mutation showed that both parents were heterozygous for the mutation, that one healthy sister carried only normal alleles, and that the patient was homozygous for the mutated allele. The mutation introduced a frameshift and a novel premature stop codon in codon 1303, and would predict the synthesis of a truncated factor V molecule that lacks part of the B domain and the complete light chain. However, no factor V heavy chain could be detected in the plasma of the patient. Furthermore, factor V activity could not be detected in the patients' platelets. This is the first reported mutation in the factor V gene that predicts a type I quantitative factor V deficiency. Surprisingly, the patient, who is homozygous for the mutation, so far has only a very mild bleeding tendency.     

A novel two base pair deletion in the factor V gene associated with severe factor V deficiency

We studied a family in which the proband, a 13-year-old boy, had unmeasurable plasma levels of coagulation factor V antigen and activity. Clinical symptoms were severe, with several episodes of haemorrhages in the mucosal tracts (gastrointestinal, nose and urinary) and recurrent haemarthroses that caused permanent arthropathy. Sequence analysis of the factor V gene demonstrated the presence of a novel 2 base pair (bp) homozygous deletion in exon 13 at positions 2833-2834. This mutation, present in the heterozygous state in the asymptomatic mother and absent in the healthy brother, introduced a frameshift and a premature stop at codon 900. This would predict the synthesis of a truncated factor V molecule, lacking part of the B domain and the complete light chain. Because of the existence of a surveillance mechanism that selectively recognizes and degrades mRNA molecules carrying premature termination codons, we analysed the relative abundance of mutant vs. wild-type mRNA molecules in the platelets of the heterozygous proband's mother. The mutant mRNA was significantly reduced in amount (mutant/wild-type ratio 0.35). This is the first reported mutation in the factor V gene causing severe factor V deficiency, the effect of which was quantitatively analysed at mRNA level.     

Nonsense mutation in exon V of the factor XI gene does not abolish platelet factor XI expression

Factor XI (FXI) is a plasma glycoprotein produced by the liver that plays an essential role in blood coagulation. Individuals deficient in this protein are prone to bleeding following trauma or surgery. Well-washed platelets possess FXI-like activity and FXI antigen that differs in molecular weight from plasma FXI and is associated with FXI mRNA from platelets in which exon V is absent. Some individuals deficient in plasma FXI produce the platelet form of the protein. The molecular basis for the presence of platelet FXI in plasma FXI-deficient patients is unknown. In the current study, to help determine the mechanism for this differential expression, the FXI genotype was determined for three plasma FXI-deficient individuals who express platelet FXI. All three individuals had a nonsense mutation encoding a stop codon in exon V of the FXI gene that would result in a severely truncated polypeptide. An exon V nonsense mutation in the FXI gene combined with the absence of exon V in platelet FXI mRNA provides a plausible mechanism for the differential expression of platelet FXI in plasma FXI-deficient patients.     

Expression of two type 2N von Willebrand disease mutations identified in exon 18 of von Willebrand factor gene

Type 2N von Willebrand disease (VWD) is characterized by a markedly decreased affinity of von Willebrand factor (VWF) for factor VIII (FVIII). The FVIII binding site has been localized within the first 272 amino acid residues of mature VWF, encoded by exons 18-23. Two substitutions in exon 18 of VWF gene, inducing candidate mutations Y795C and C804F were identified in the heterozygous state in two French patients who also displayed the frequent R854Q mutation in exon 20. Expression studies in Cos-7 cells showed that these abnormalities, which implicate cysteine residues, induced secretion, multimerization and FVIII binding defects of corresponding recombinant VWF. Results from transfection experiments with R854Q, performed to reproduce the hybrid VWF present in patient plasma, were in agreement with those obtained for patient's plasma VWF. These findings confirm the importance of the VWF D' domain in FVIII binding. In addition, this work shows that exon 18 should preferentially be sequenced in type 2N VWD patients when the frequent R854Q mutation in exon 20 has been excluded or detected in the heterozygous state.     

Homozygous factor V splice site mutation associated with severe factor V deficiency

We investigated a family whose proband has a severe bleeding disorder and factor V antigenic and functional levels of 8% and less than 1% of control values, respectively. Molecular analysis of the factor V gene revealed a novel homozygous mutation in the last nucleotide of exon 10. 1701G>T causes activation of a cryptic exonic splice site in exon 10, which encodes part of the factor V heavy chain (A2 domain). This leads to the deletion of 35 nucleotides and results in a frameshift with a premature stop codon at amino acid position 498. The G1701 and corresponding Gln509 are conserved in murine, bovine, and porcine factor V and in human factor VIII. Few factor V deficiency mutations have been identified as yet. Several are present in the heterozygous form in combination with factor V Leiden (Arg506Gln). This is the first reported homozygous splice site mutation in a patient with factor V deficiency.     

Coagulation factor XI gene analysis in three factor XI deficient Austrian patients

Abstract: Objectives: Hereditary factor XI deficiency is a rare bleeding disorder with worldwide distribution. In Austrian patients only one mutation leading to congenital factor XI deficiency has been reported. In the present study, we identified the molecular basis of factor XI deficiency in three Austrian patients. Methods: Patients attended hospital for other reasons than bleeding disorders. Routine laboratory tests revealed prolonged APTTs due to decreased factor XI levels. We performed automated fluorescent sequencing of the promotor region, exons 1–15 and the flanking intronic regions of the factor XI gene. The mutations found were confirmed by restriction enzyme analysis or sequencing of the non‐coding strand. Results: Fluorescent sequencing revealed two novel mutations, the nonsense mutation Gln116X (443C>T) in exon 5 and a deletion of Ile¹⁹⁷ and Asp ¹⁹⁸ (687_692delTCGACA) in exon 7. Furthermore, we detected a heterozygous A>G exchange at the third nucleotide of IVS6 (IVS 6 +3A>G), which had already been reported in a FXI deficient individual of French Basque origin. Conclusion: While the IVS 6 +3A>G decreases the calculated splice consensus score from 0.98 in the wild type to 0.56 in the altered sequence and therefore interferes with the consensus splice sequence, the complete loss of the two amino acids Ile¹⁹⁷ and Asp¹⁹⁸ is expected to interfere with the steric structure and hence the functions of the third apple domain. The Gln116X leads to a premature termination codon resulting in a lack of the light as well as parts of the heavy chain of the FXI protein, most likely resulting in rapid degradation of the truncated mRNA.     

Identification of two novel mutations in non-Jewish factor XI deficiency

Summary. We have studied two heterozygous unrelated CRM non-Jewish FXI-deficient patients. Neither of the patients carries a previously described mutation. Their FXI genes were screened by SSCP analysis following PCR amplification of each exon and the flanking intronic sequences. DNA fragments showing aberrant mobility were cloned and sequenced. The following mutations were identified: in case 1, a T to G transition in exon 12 results in the substitution of Phe-442 by Val (FXI-F442V); in case 2 a C to A transition in exon 5 results in the substitution of Cys-128 by a nonsense codon (FXI-C128X). The missense mutation results in a substitution within the protease domain of FXI. Molecular modelling locates this residue in a structurally conserved region of the protease domain and the amino acid substitution may therefore interfere with either chain folding and subsequent secretion or the stability of the protein in plasma. We conclude that the mutations which we have identified are responsible for the inherited abnormality in these patients.     

Factor XII Tenri, a novel cross-reacting material negative factor XII deficiency, occurs through a proteasome-mediated degradation.

A homozygous cross-reacting material negative factor XII-deficient patient with 3% antigen and activity levels of factor XII was screened for the identification of a mutation at the genomic level. Low-ionic strength single-stranded conformation polymorphism (SSCP) analysis and sequence analysis showed that the proband's gene for factor XII had an A-->G substitution at nucleotide position 7832 in exon 3, resulting in a Tyr34 to Cys substitution in the NH2-terminal type II domain of factor XII. We designated this mutation as factor XII Tenri. Mutagenic polymerase chain reaction (PCR), followed by KpnI digestion, showed a homozygous mutation in the proband's gene and heterozygous mutations in his parents and sister. Immunoprecipitation and Western blot analyses of plasma samples from the factor XII Tenri family indicated that the proband had a trace amount of variant factor XII with an apparent molecular mass of 115 kD, which was converted to the normal 80-kD form after reduction, suggesting that factor XII Tenri was secreted as a disulfide-linked heterodimer with a approximately 35-kD protein, which we identified as alpha1-microglobulin by immunoblotting. Pulse-chase experiments using baby hamster kidney (BHK) cells showed that Tenri-type factor XII was extensively degraded intracellularly, but the addition of cystine resulted in increased secretion of the mutant. Using membrane-permeable inhibitors, we observed that the degradation occurred in the pre-Golgi, nonlysosomal compartment and a proteasome appeared to play a major role in this process. On the basis of these in vitro results, we speculate that the majority of the factor XII Tenri is degraded intracellularly through a quality control mechanism in the endoplasmic reticulum (ER), and a small amount of factor XII Tenri that formed a disulfide-linked heterodimer with alpha1-microglobulin is secreted into the blood stream.     

Two novel mutations (Pro864His, Val867Glu) causing type 2A von Willebrand disease and affecting a single restriction site in exon 28

We detected two transversions in two unrelated Italian patients with type 2A von Willebrand disease (VWD): a C to A at nucleotide 8821 and a T to A at nucleotide 8830, resulting in the missense mutations Pro864His and Val867Glu respectively. Both mutations were in the heterozygous form and abolished the BstXI restriction site in exon 28 of the VWF gene. In both mutations plasma VWF multimer pattern improved by antiproteases. Moreover, DDAVP normalized plasma VWF multimers in the Pro864His patient, especially when protease inhibitors were present. These new mutations appear to be of the 2A VWD subtype due to the increased susceptibility to proteases.     

Compound heterozygosity for two novel mutations (1203insG/Y1456X) in the von Willebrand factor gene causing type 3 von Willebrand disease

A 23-year-old Chinese woman with severe von Willebrand factor (VWF) deficiency and her parents were investigated by PCR/direct sequencing of the VWF gene. The patient was found to be compound heterozygous for two novel null mutations. The first was a microinsertion in exon 8 (1203insG) that introduced a frameshift at codon 298 leading to premature translational termination at codon 302. The second was a C to A transversion in exon 28 which resulted in the replacement of tyrosine 562 by a stop codon (Y1456X). The failure to amplify VWF cDNA from the patient by semi-nested PCR is consistent with the induction of nonsense-mediated mRNA decay.     

A novel TC deletion resulting in Pro(260)-->Stop in the human LCAT gene is associated with a dominant effect on HDL-cholesterol

Human lecithin:cholesterol acyltransferase (LCAT) plays a key role in the biogenesis of circulating high-density lipoprotein-cholesterol (HDL-C) and reverse cholesterol efflux. We investigated the molecular defect in the LCAT gene in a family with low levels of HDL-C. The proband, a 53-year-old woman from Oklahoma City, had a HDL-C level of 0.21 mmol/l. The LCAT activity in the proband was 5 nmol/ml/h and cholesterol esterification rate was 54.2 nmol/ml/h, consistent with LCAT deficiency. Analysis of polymerase chain reaction (PCR) amplified subgenomic fragments of LCAT DNA on polyacrylamide gels revealed heteroduplex bands in the proband and three other affected individuals in exon 6. DNA sequence analyses of the proband's LCAT gene identified a 2 base pair deletion (TC) (base pairs 4544-4545, corresponding to amino acid 255) in the heteroduplex allele, thereby converting Pro(260) to a premature stop codon and a predicted truncated protein of 260 amino acids. This is approximately 60% of the length of the normal translated protein. The heterozygous individuals also revealed significant reduction in apolipoprotein A-1 levels compared with the unaffected family members (n=4). The marked reduction in HDL-C in the proband and sibling suggests a dominant effect of this mutation on HDL-C levels. Furthermore, because the deletion results in a heterozygous allele that can be detected by a simple PCR reaction and polyacrylamide gel-size fractionation, it may be possible to rapidly screen susceptible individuals for the presence of this mutation.     

Two novel factor V null mutations associated with activated protein C resistance phenotype/genotype discrepancy

Activated protein C (APC) resistance phenotype/genotype discrepancy is a very rare event. The objective of this study was to characterize the molecular mechanisms in two cases of APC phenotype/genotype discrepancy. An approach using direct sequencing of each exon and splicing junctions of the factor V gene showed that two novel factor V null mutations combined with heterozygous factor V Leiden mutation were responsible for this discrepancy. Our results suggest the necessity to use both phenotypic and genotypic analyses in some cases to determine an accurate diagnosis.     

Factor XIIIA subunit deficiency due to a homozygous 13-base pair deletion in exon 3 of the A subunit gene

We investigated the molecular basis of factor XIII_A subunit deficiency in a Greek family. Each of the 15 exons of the A subunit gene were individually amplified by polymerase chain reaction, using previously reported oligoprimers. The proband with severe deficiency was found to have a homozygous 13-base pair deletion in the 3′ half of exon 3. The deleted sequence, extending from codons 82–86, results in a frameshift and generates a downstream termination codon in exon 4. Single strand conformation polymorphism (SSCP) analysis detected no additional mutations in the coding or consensus splice sequences of the A subunit gene. Both parents of the proband were heterozygous for the defect. Only one previous microdeletion (AG dinucleotide) has been reported in the A subunit gene, and was located at the intron B-exon 3 boundary. Further studies are necessary to determine whether this region of the gene is a “hot spot” for microdeletion mutations. © 1996 Wiley-Liss, Inc.     

Clinical follow up of mexican women with early onset of breast cancer and mutations in the BRCA1 and BRCA2 genes

OBJECTIVE: This study describes the presence of mutations in BRCA1 and BRCA2 genes in a group of Mexican women and the clinical evolution of early onset breast cancer (EOBC). MATERIAL AND METHODS: A prospective hospital-based study was performed in a sample of 22 women with EOBC (7 in clinical stage IIA, 8 in IIB, and 7 in IIIA). The patients attended a tertiary care hospital in northeastern Mexico in 1997 and were followed up over a 5-year period. Molecular analysis included: 1) a mutation screening by heteroduplex analysis (HA) of BRCA1 and BRCA2 genes and 2) a sequence analysis. RESULTS: Of 22 patients, 14 (63.6%) showed a variant band detected by heteroduplex analysis of the BRCA1 and BRCA2 genes: 8 polymorphisms, 4 mutations of uncertain significance, and 2 novel truncated protein mutations, one in BRCAI (exon 11, 3587delT) and the other in the BRCA2 gene (exon 11, 2664InsA). CONCLUSIONS: These findings support future studies to determine the significance and impact of the genetic factor in this Mexican women population.     

Rapid mutation screening in type 2A von Willebrand's disease using universal heteroduplex generators

Patients with type 2A von Willebrand's disease (VWD) commonly have missense mutations in the A2 domain of the von Willebrand factor (VWF) protein. This domain is encoded by the 3' region of VWF gene exon 28 and the large majority of patients have heterozygous mutations clustered in the sequence between codons 742 and 909. We describe a DNA-based diagnostic technique which enables at least 10 previously described mutations to be rapidly identified. The method involves polymerase chain reaction (PCR) amplification of two exon 28 gene segments between codons 717–788 and 803–893, respectively. Each fragment is then hybridized with a synthetic complementary DNA molecule of similar size, termed a Universal Heteroduplex Generator (UHG). The UHG contains base deletions contiguous to the sites of known mutations and, following hybridization, allele-specific heteroduplexes are generated which can be detected by simple polyacrylamide gel electrophoresis and ethidium bromide staining. A small panel of UHG molecules covering the 3' region of exon 28 should enable the large majority of type 2A VWD patients to be rapidly diagnosed by genotype.     

Two novel mutations 685del 1 and D129G in the low-density lipoprotein receptor gene in a compound heterozygote Chinese family with familial hypercholesterolemia

DNA sequencing analysis was used to scan the genes in a Chinese family with clinically diagnosed autosomal genetic hypercholesterolemia. Two mutations were identified in exon 4 of the low-density lipoprotein receptor gene, which is the possible molecular mechanism of etiology of the family. The proband's extremely high level of serum cholesterol and the related manifestations suggested that he was a familial hypercholesterolemia homozygote and that his parents were in a relatively milder condition. DNA sequencing revealed that the proband had an abnormal pattern of exon 4 of the low-density lipoprotein receptor gene due to a heterozygosity (A/G) at nucleotide 386 and a heterozygous single-base deletion (A) at 685. Nucleotide 386 is the second base of codon 129, and A-->G mutation (D129G) changed this codon from Asp(GAC) to Gly(GGC). The single-base deletion of A at 685 (685del 1) is a frameshift mutation. It changes the phase of triplets, so that all codons are misread after this site of mutation; consequently, the protein expressed by the gene must be abnormal in structure and function. DNA analysis of the other family members showed that the 2 mutations should be respectively located in different alleles of the proband. Both of the 2 mutations have not been reported previously.     

Single base mutation in the pro alpha 2(I) collagen gene that causes efficient splicing of RNA from exon 27 to exon 29 and synthesis of a shortened but in-frame pro alpha 2(I) chain.

Previous observations demonstrated that a lethal variant of osteogenesis imperfecta had two altered alleles for pro alpha 2(I) chains of type I procollagen. One mutation produced a nonfunctioning allele in that there was synthesis of mRNA but no detectable synthesis of pro alpha 2(I) chains from the allele. The mutation in the other allele caused synthesis of shortened pro alpha 2(I) chains that lacked most or all of the 18 amino acids encoded by exon 28. Subclones of the pro alpha 2(I) gene were prepared from the proband's DNA and the DNA sequence was determined for a 582-base-pair (bp) region that extended from the last 30 bp of intervening sequence 26 to the first 26 bp of intervening sequence 29. Data from six independent subclones demonstrated that all had the same sequence as a previously isolated normal clone for the pro alpha 2(I) gene except that four subclones had a single base mutation at the 3' end of intervening sequence 27. The mutation was a substitution of guanine for adenine that changed the universal consensus sequence for the 3' splicing site of RNA from -AG- to -GG-. S1 nuclease experiments demonstrated that about half the pro alpha 2(I) mRNA in the proband's fibroblasts was abnormally spliced and that the major species of abnormal pro alpha 2(I) mRNA was completely spliced from the last codon of exon 27 to the first codon of exon 29. The mutation is apparently unique among RNA splicing mutations of mammalian systems in producing a shortened polypeptide chain that is in-frame in terms of coding sequences, that is used in the subunit assembly of a protein, and that contributes to a lethal phenotype.