Factor IXAlabama: a point mutation in a clotting protein results in hemophilia B

Factor IXAlabama is a variant factor IX molecule responsible for a clinically moderate form of hemophilia B. Twenty-five kilobases (kb) of the variant gene, including seven exons coding for the structural protein, were cloned and characterized. The restriction map and the arrangement of coding regions are identical to those of the normal gene. DNA sequence analysis of the coding regions revealed a single base-pair difference between the gene for factor IXAlabama and the normal factor IX gene. An adenine to guanine transition in the first nucleotide of exon d causes the substitution of a glycine codon (GGT) for the normal aspartic acid codon (GAT). This point mutation results in a single amino acid substitution at residue 47 of the zymogen and represents the genetic defect in factor IXAlabama.     

An arginine to cysteine amino acid substitution at a critical thrombin cleavage site in a dysfunctional factor VIII molecule

We have analyzed the factor VIII (FVIII) protein and the nucleotide sequence around two thrombin cleavage sites, at arginine 372 in the FVIII heavy chain and arginine 1689 in the FVIII light chain in a naturally occurring dysfunctional FVIII variant, FVIII Okayama. The patient was a 42-year-old hemophiliac with a FVIII coagulant activity of 0.03 U/mL and a FVIII antigen level of 0.8 U/mL. The patient's FVIII was not thrombin activatable to levels seen in normal plasma. Immunoblotting of partially purified FVIII Okayama and normal FVIII showed that thrombin cleavage of the 92 kilodalton (Kd) heavy chain was impaired in the mutant protein. The patient's genomic DNA was amplified using the polymerase chain reaction with two sets of synthetic oligonucleotide primers spanning amino acid residues 319 to 400 and 1630 to 1720. Sequence analysis of the amplified DNA fragments revealed a cytosine to thymine transition, converting an arginine to a cysteine codon at residue 372. No abnormality was found in the FVIII light chain region analyzed. The patient's hemophilic brother and carrier mother revealed the same mutation. We conclude that the pathogenesis of hemophilia A in this patient is probably due to an arginine to cysteine substitution at a thrombin cleavage site in the FVIII heavy chain.     

A codon 338 nonsense mutation in the factor IX gene in unrelated hemophilia B patients: factor IX338 New York

Hemophilia B is an X-linked recessive bleeding disorder resulting from a deficiency of the coagulation factor IX (FIX) protein activity, a vitamin K-dependent serine protease active in both the intrinsic and extrinsic coagulation systems. DNA analyses of the factor IX gene in two unrelated patients with severe hemophilia B, with a IX coagulant activity less than 1% and undetectable FIX antigen, detected the loss of the second TaqI site in exon h (VIII) in both individuals. Polymerase chain reaction (PCR) amplification of 576 base pairs of exon h (VIII) with cloning and dideoxy sequencing of cloned DNA from one hemophiliac revealed a single C----T transition in codon 338 that changes an arginine residue codon CGA to a nonsense codon TGA. Allele- specific oligonucleotide probe hybridization with a mutant (C----T) and a wild-type allele confirmed the same mutation in amplified genomic DNA of the second hemophilia patient. The C----T transition represents another example of mutation at a CpG dinucleotide. DNA polymorphism analysis of the FIX gene in both individuals revealed each to be on a separate FIX haplotype; therefore, predicting each to be a separate mutation event.     

Hemoglobin Cagliari (beta 60 [E4] Val----Glu): a novel unstable thalassemic hemoglobinopathy

This report describes a patient with thalassemia intermedia-like phenotype born to normal parents in whom globin gene sequencing detected a novel abnormal hemoglobin (Hb) due to a T to A substitution at codon 60 of the beta-globin gene arising as a de novo mutation. Normal sequences were detected at the homologous beta-globin locus. This mutation results in the substitution of a polar (glutamic acid) for a nonpolar (valine) residue near the corner of the heme pocket of the beta-globin chain. The novel variant has been designated Hb Cagliari, from the place of birth of the propositus. Kinetics of globin synthesis performed following splenectomy suggest that this new Hb variant is synthesized at a near normal rate but undergoes rapid breakdown. The extreme lability of the variant explains the clinical and hematologic picture characterized by marked ineffective erythropoiesis, thalassemia-like bone changes, iron overload, high proportion of Hb F in the peripheral blood, reduced beta/alpha-globin chain synthesis ratio in peripheral blood reticulocytes, and absence of the abnormal Hb in peripheral blood at extensive protein structural analysis before splenectomy. This case indicates that a thalassemic hemoglobinopathy should be suspected in the presence of a patient with a thalassemia intermedia-like phenotype born to normal parents, even when protein structural analysis fails to detect an abnormal Hb. DNA sequencing may allow to define the mutation, thus making the proper diagnosis.     

Molecular defect in coagulation factor XFriuli results from a substitution of serine for proline at position 343

Our previous findings suggested that coagulation factor XFriuli could be functionally defective owing to a point mutation in the portion of the factor X gene coding for the fully activated heavy chain. To verify the existence of this postulated change, we analyzed all eight exons of the normal and Friuli factor X gene. Each exon was amplified from genomic DNA using the polymerase chain reaction and cloned into the plasmid pUC19. The amplified DNA inserts were subjected to direct sequencing by the dideoxy chain termination method with forward and reverse oligonucleotide sequencing primers. A point mutation (C to T transition at nucleotide position 19,297) that results in coding for serine (TCC) in place of proline (CCC) at amino acid position 343 was found. This substitution involves a highly conserved proline residue oriented spatially close to both the cleavage site of the zymogen and the active site of the enzyme and explains the previous observations of discrete biochemical and functional differences between factor XFriuli and normal factor X. The mutation abolished an HgiCI restriction site present in the normal factor X gene, and this change constitutes the basis for a convenient method for screening individuals carrying this molecular defect. Proline343 is in conserved region 5 of the serine protease superfamily to which factor X belongs and is part of a 14-residue L*****P******C motif that occurs in at least 16 other enzymes. Computer analysis suggests that the motif may be an essential aspect of conformational features important to functional properties of factor X as well as other serine proteases.     

A new β chain hemoglobin variant with increased oxygen affinity: Hb Santa Giusta Sardegna [β93(F9)Cys→Trp; HBB c.282T>G

During a screening program for the identification of β-thalassemia (β-thal) carriers in Sardinia, Italy, we identified two subjects with increased hemoglobin (Hb) levels and an abnormal Hb variant. The same variant was detected in a family member. DNA sequencing revealed a TGT > TGG mutation at codon 93 of the β-globin gene. Structural analysis demonstrated that the cystine residue at position 93 of the β chain was substituted by tryptophan. Since this amino acid substitution had not yet been reported, we designated this variant Hb Santa Giusta Sardegna for the place of birth of the subjects. This amino acid substitution occurs at the tyrosine pocket of the β chain as well as at the α1β2/α2β1 contact of the quaternary structure of the molecule. The presence of this Hb in the hemolysate causes an increased oxygen affinity, a slightly reduced Bohr effect and a reduced heme-heme interaction (n(50), Hill's constant) in comparison with those of Hb A.     

A factor XI deficiency associated with a nonsense mutation (Trp501stop) in the catalytic domain

We identified a novel mutation in an asymptomatic 65-year-old Japanese man with severe factor XI deficiency. Sequence analysis after polymerase chain reaction single-stranded conformation polymorphism (PCR-SSCP) analysis of his factor XI gene revealed a G-->A transition in codon 501 of exon 13, resulting in a substitution of Trp501 (TGG) by a stop codon (TAG) in the catalytic domain. This mutation abolished a FokI restriction site. The PCR product from normal subjects was digested with FokI and yielded two fragments, one of 223 bp and one of 47 bp. The PCR product from the patient gave a single 270-bp fragment, demonstrating possible homozygosity.     

Direct characterization of factor VIII in plasma: detection of a mutation altering a thrombin cleavage site (arginine-372----histidine).

An immunoadsorbent method has been developed for the direct analysis of normal and variant plasma factor VIII. Using this method, the molecular defect responsible for mild hemophilia A has been identified for a patient whose plasma factor VIII activity is 0.05 unit/ml, even though the factor VIII antigen content is 3.25 units/ml. Although the variant factor VIII has an apparently normal molecular mass and chain composition, the 92-kDa heavy chain accumulates when the variant protein is incubated with thrombin and the 44-kDa heavy chain fragment cannot be detected. In contrast, thrombin cleavage of the 80-kDa light chain to the 72-kDa fragment is normal. As these data indicate a loss of factor VIII cleavage by thrombin at arginine-372, the genetic defect was determined by polymerase-chain-reaction amplification of exon 8 of the factor VIII gene and direct sequencing of the amplified product. A single-base substitution (guanine----adenine) was identified that produces an arginine to histidine substitution at amino acid residue 372. These data identify the molecular basis of an abnormal factor VIII, "factor VIII-Kumamoto," that lacks procoagulant function because of impaired thrombin activation.     

A novel variant of transthyretin (Glu89Lys) associated with familial amyloidotic polyneuropathy.

We detected a point mutation in the transthyretin (TTR) gene associated with familial amyloidotic polyneuropathy (FAP) in a 57-year old male presenting with sensorimotor polyneuropathy, severe autonomic dysfunction and cardiomyopathy using a non-isotopic RNase cleavage assay (NIRCA). NIRCA suggested that the mutation site was near either amino acid position 58 of mature TTR or the 3' end of exon 3. Direct DNA sequencing showed both a normal GAG (Glu) and a variant AAG (Lys) codon at amino acid position 89 of mature TTR, which has not been previously reported. The site of this mutation is near the 3' end of exon 3, consistent with the result of NIRCA. This mutation was also confirmed by polymerase chain reaction-induced mutation restriction analysis (PCR-IMRA).     

Isolation and characterization of the factor X Friuli variant

Factor X Friuli was isolated from plasma by immunoaffinity and ion exchange chromatography and compared with normal factor X purified by the same method. Similar molecular weights were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the intact or activated factor X molecules including their respective heavy and light chains. These data indicated that there were no gross structural differences between the normal and variant proteins. Immunochemical assays employing either polyclonal or 46 monoclonal antibodies (MoAbs) did not reveal any structural deviations. Two- dimensional peptide maps indicated that while the light chains of normal and Friuli factor X were very similar, the heavy chains of the native and activated molecules contained a limited number of differences. These data suggested that the defect in factor X Friuli may be a point mutation which lies within the activated heavy chain defined by the 195-424 amino acid sequence. Activation of factor X Friuli in purified systems showed that Russell's viper venom cleaved the molecule at 70% of the normal rate, while the rate of proteolysis of the variant protein was reduced 98% and 75% when incubated with the extrinsic and intrinsic activation complexes, respectively. These data support the clinical laboratory findings and the hypothesis that the defect associated with the Friuli variant may reflect an abnormal interaction between factor X Friuli and the nonproteolytic cofactors of the extrinsic and intrinsic factor X activation complexes. Fluorescence polarization studies suggested that a bound dansylated inhibitor of factor Xa was not oriented to the same extent within the active site of the variant enzyme relative to normal factor Xa until the addition of phospholipid and factor Va. Activated factor X Friuli generated thrombin from prothrombin in a purified system, but at one third the normal rate that was attributed to the Kcat suggesting a secondary effect of this defect.     

Proteolytic events that regulate factor V activity in whole plasma from normal and activated protein C (APC)-resistant individuals during clotting: an insight into the APC-resistance assay

Human factor V is activated to factor Va by alpha-thrombin after cleavages at Arg709, Arg1018, and Arg1545. Factor Va is inactivated by activated protein C (APC) in the presence of a membrane surface after three sequential cleavages of the heavy chain. Cleavage at Arg506 provides for efficient exposure of the inactivating cleavages at Arg306 and Arg679. Membrane-bound factor V is also inactivated by APC after cleavage at Arg306. Resistance to APC is associated with a single nucleotide change in the factor V gene (G1691-->A) corresponding to a single amino acid substitution in the factor V molecule: Arg506-->Gln (factor V Leiden). The consequence of this mutation is a delay in factor Va inactivation. Thus, the success of the APC-resistance assay is based on the fortuitous activation of factor V during the assay. Plasmas from normal individuals (1691 GG) and individuals homozygous for the factor V mutation (1691 AA) were diluted in a buffer containing 5 mmol/L CaCl2, phospholipid vesicles (10 micromol/L), and APC. APC, at concentrations < or = 5.5 nmol/L, prevented clot formation in normal plasma, whereas under similar conditions, a clot was observed in plasma from APC-resistant individuals. Gel electrophoresis analyses of factor V fragments showed that membrane-bound factor V is primarily cleaved at Arg306 in both plasmas. However, whereas in normal plasma production of factor Va heavy chain is counterbalanced by fast degradation after cleavage at Arg506/Arg306, in the APC-resistant individuals' plasma, early generation and accumulation of the heavy chain portion of factor Va occurs as a consequence of delayed cleavage at Arg306. At elevated APC concentrations (>5.5 nmol/L), no clot formation was observed in either plasma from normal or APC-resistant individuals. Our data show that resistance to APC in patients with the Arg506-->Gln mutation is due to the inefficient degradation (inactivation) of factor Va heavy chain by APC.     

Identification of a point mutation in growth factor repeat C of the low density lipoprotein-receptor gene in a patient with homozygous familial hypercholesterolemia that affects ligand binding and intracellular movement of receptors.

The coding region of the low density lipoprotein (LDL)-receptor gene from a patient (MM) with homozygous familial hypercholesterolemia (FH) has been sequenced from six overlapping 500-base-pair amplified fragments of the cDNA from cultured skin fibroblasts. Two separate single nucleotide base changes from the normal sequence were detected. The first involved substitution of guanine for adenine in the third position of the codon for amino acid residue Cys-27 and did not affect the protein sequence. The second mutation was substitution of thymine for cytosine in the DNA for the codon for amino acid residue 664, changing the codon from CCG (proline) to CTG (leucine) and introducing a new site for the restriction enzyme PstI. MM is a true homozygote with two identical genes, and the mutation cosegregated with clinically diagnosed FH in his family in which first cousin marriages occurred frequently. The amino acid change occurs in the center of growth factor repeat C in the epidermal growth factor precursor-homology domain of the protein, a region of highly conserved sequence between bovine and human LDL receptors, and results in slowed, but complete, maturation of the precursor to the mature form of the receptor and, despite its remoteness from the ligand-binding domain, in impaired binding of LDL. LDL receptors in MM's skin fibroblasts bind less LDL than normal and with reduced affinity. Thus this naturally occurring single point mutation affects both intracellular transport of the protein and ligand binding and occurs in growth factor-like repeat C, a region that has not previously been found to influence LDL binding.     

Hb Belfast [beta15(A12)Trp-->Arg]: definition of the clinical and hematological phenotype

We report the sixth occurrence of Hb Belfast [beta15(A12)Trp-->Arg], a mild, unstable beta chain variant, in a large family wherein nine subjects were affected. DNA analysis showed a TUG-->AGG mutation at codon 15 of the beta-globin gene, confirming a Trp-->Arg amino acid substitution. The oxygen affinity of the isolated variant was increased. The clinical phenotype is silent or very mild, the only clinical finding being an intermittent moderate jaundice.     

Factor VII R110C: a novel missense mutation (Arg110Cys) in the second epidermal growth factor-like domain causing factor VII deficiency in members of a Japanese family.

This report describes the findings of a genetic analysis of the factor VII (FVII) gene in a Japanese, male patient with FVII deficiency. The proband showed FVII activity level of 25% and FVII antigen level of 28% of the normal value, but he had no severe bleeding episodes. We identified the mutation by direct sequencing of polymerase chain reaction products representing all exons except 1b and their flanking intronic regions of his FVII gene. We detected a single point mutation, a C-->T substitution at nucleotide position 7863 in exon 5, which results in an amino acid replacement of Arg (CGC) to Cys (TGC) at codon 110 in the second epidermal growth factor-like domain. Homozygosity was confirmed in the propositus by loss of a site for the restriction endonuclease Eco47III. Furthermore, his parents, who had moderately reduced levels of factor VII activity and antigen, carried this mutation site as a heterozygote. Although the Arg11O residue is located distal to the tissue factor (TF) in the soluble TF-FVIIa crystal structure, we infer that the replacement of the positively charged and larger Arg residue with a neutral Cys residue may be likely to impair proper folding, resulting in destabilization of the protein structure.     

Hb Zoetermeer: a new mutation on the alpha2 gene inducing an Ala-->Ser substitution at codon 21 is possibly associated with a mild thalassemic phenotype

A 52-year-old Dutch male was referred to our laboratory for hemoglobinopathy analysis because of persistent microcytic hypochromic parameters and moderate erythrocytosis in the absence of iron deficiency. The hemoglobin (Hb) pattern was normal and breakpoint polymerase chain reaction (PCR) excluded the six common deletion defects of the alpha gene cluster. Direct sequencing revealed a GCT-->TCT transversion at codon 21 of the alpha2 gene generating an Ala-->Ser single amino acid substitution. The hematological parameters observed in the presence of this mutation are consistent with a compensated heterozygous alpha(+)-thalassemia (thal). However, the neutral mutation and the external position of the residue do not explain an association with this phenotype. Nevertheless, we cannot exclude that the mutation could induce the observed hematological abnormalities and could eventually be considered as a mutation associated with a mild alpha-thalassemic phenotype.     

A novel variant of transthyretin (prealbumin), Thr119 to Met, associated with increased thyroxine binding.

A group of patients with prealbumin associated hyperthyroxinemia possess a common single base substitution in the fourth exon of their transthyretin gene. This cytosine to thymine substitution occurs in the codon for residue 119 and results in the predicted replacement of a threonine residue with a methionine at this position. A new NcoI restriction endonuclease cleavage site is created by the point mutation and can be detected by a rapid and simple assay based on the polymerase chain reaction. This variant transthyretin is inherited in an autosomal dominant manner and is apparently not amyloidogenic but is associated with increased thyroxine binding. As healthy heterozygous individuals have normal serum thyroxine concentrations, the hyperthyroxinemia sometimes found may not be primarily due to the variant.     

Hb Belfast [β15(A12)Trp→Arg]: Definition of the Clinical and Hematological Phenotype

We report the sixth occurrence of Hb Belfast [β15(A12)Trp→Arg], a mild, unstable β chain variant, in a large family wherein nine subjects were affected. DNA analysis showed a TGG→AGG mutation at codon 15 of the β‐globin gene, confirming a Trp→Arg amino acid substitution. The oxygen affinity of the isolated variant was increased. The clinical phenotype is silent or very mild, the only clinical finding being an intermittent moderate jaundice.     

The "Normandy" variant of von Willebrand disease: characterization of a point mutation in the von Willebrand factor gene

We previously reported a functional defect of von Willebrand factor (vWF) in a new variant of von Willebrand disease (vWD) tentatively named vWD "Normandy." The present work has attempted to characterize the molecular abnormality of this vWF that fails to bind factor VIII (FVIII). The immunopurified vWF from normal and patient's plasma were digested by trypsin and the resulting peptides were compared. The electrophoresis of "vWF Normandy" showed a shift in the band corresponding to a polypeptide from amino acid 1 to 272. Consequently, we performed the molecular analysis of the portion of the vWF gene of this patient encoding this amino acid sequence. Exons 18-24 were amplified by the use of polymerase chain reaction and their nucleotide sequences corresponding to 1.8 kb were determined. Our analysis showed a point mutation C to T at codon 791, resulting in the substitution of Methionine for Threonine at position 28 of the mature vWF subunit. Because this nucleotide substitution destroyed a Mae II restriction site, this mutation was conveniently sought in various individual DNAs. The patterns obtained were consistent with the homozygous and heterozygous state of this mutation in the patient and in her son, respectively, and with its absence in 28 normal individuals. We conclude that Threonine at position 28 in plasma vWF may be crucial for the conformation and FVIII-binding capacity of its cystine-rich N-terminal domain.     

Hb Zoetermeer: A New Mutation on the α2 Gene Inducing an Ala→Ser Substitution at Codon 21 is Possibly Associated with a Mild Thalassemic Phenotype

A 52-year-old Dutch male was referred to our laboratory for hemoglobinopathy analysis because of persistent microcytic hypochromic parameters and moderate erythrocytosis in the absence of iron deficiency. The hemoglobin (Hb) pattern was normal and breakpoint polymerase chain reaction (PCR) excluded the six common deletion defects of the α gene cluster. Direct sequencing revealed a GCT→TCT transversion at codon 21 of the α2 gene generating an Ala→Ser single amino acid substitution. The hematological parameters observed in the presence of this mutation are consistent with a compensated heterozygous α⁺-thalassemia (thal). However, the neutral mutation and the external position of the residue do not explain an association with this phenotype. Nevertheless, we cannot exclude that the mutation could induce the observed hematological abnormalities and could eventually be considered as a mutation associated with a mild α-thalassemic phenotype.     

Hb Youngstown [β101(G3)Glu → Ala; HBB: c.305A > C]: An Unstable Hemoglobin Variant Causing Severe Hemolytic Anemia

Hb Youngstown is a rare hemoglobin (Hb) variant caused by substitution of glutamic acid with alanine at amino acid residue 101 of the β-globin chain as a result of an A?>?C transversion on the β-globin gene nucleotide sequences [β101(G3)Glu?→?Ala; HBB: c.305A?>?C]. We now report three patients from two different families, one from South Africa and the other from Costa Rica, who are heterozygous for this Hb variant. All three carriers had marked hemolysis, consistent with Hb Youngstown being a highly unstable variant. The substitution of glutamic acid, a large and negatively charged amino acid, with alanine, a small and non polar amino acid, in the interface of the α1- and β2-globin subunits might interfere with the transition between the oxy- and deoxyHb, and lead to Hb instability and hemolytic anemia.