Andrew P. Fellowes

Molecular Mechanisms of Hypo- and Afibrinogenemia

Abstract: Point mutations responsible for hypo‐ and afibrinogenemia are yielding new insights into amino acid side chains involved in the molecular processing, assembly, secretion, and domain stability of fibrinogen. Reverse phase chromatography, isoelectric focussing, electrospray mass spectrometry, and tryptic peptide mass mapping have shown that chains with heterozygous mutations of γ284 Gly→Arg, Bβ316 Asp→Tyr and γ371 Thr→Ile are absent from plasma fibrinogen. The nonexpression of these mutations appears to result from perturbation of the five‐stranded β sheet of the D domain. We propose that this is due to retention of the variant in the endoplasmic reticulum and that in turn this leads to hypofibrinogenemia. Other mutations effect intracellular proteolysis and chain assembly. For example the mutation, Aα20 Val→Asp, makes the protein a substrate for furin, which removes the first 19 residues of the Aα chain as the mature molecule transits the trans golgi complex. Transient expression of γ153 Cys→Arg chains together with Aα and Bβ chains suggests this mutation might perturb chain assembly, and the incorporation of mutations of Bβ353 Leu→Arg or Bβ400 Gly→Asp into intracellular fibrinogen precludes its subsequent export from host cells expressing fibrinogen genes. The graded severity of the hypo‐ and afibrinogenemias associated with homozygous Aα chain truncations suggest the absolute minimal requirement for molecular assembly is the formation of the C terminal disulfide ring of the coiled coil.

Familial hypofibrinogenaemia associated with heterozygous substitution of a conserved arginine residue; Bβ255 Arg→His (Fibrinogen Merivale)

Sequencing of all three fibrinogen genes from an individual with hypofibrinogenaemia led to the identification of two new point mutations in the Bbeta gene. Family studies showed the mutations Bbeta255 Arg-->His (Fibrinogen Merivale) and Bbeta148 Lys-->Asn (Fibrinogen Merivale II) were on different alleles and that only the Bbeta255 Arg-->His mutation segregated with hypofibrinogenaemia. Three simple heterozygotes for this mutation had mean fibrinogen concentrations of 1.4 mg/ml, while heterozygotes for the Bbeta148 Lys-->Asn mutation had normal fibrinogen concentrations. ESI MS analysis of endoproteinase Asp-N digests of Bbeta chains showed that the Bbeta255 Arg-->His substitution was not expressed in plasma, confirming it as the cause of the hypofibrinogenaemia. The Bbeta148 Lys-->Asn chains, on the other hand, were equally expressed with wild-type Bbeta chains in simple heterozygotes. Genotype analysis failed to detect either substitution in 182 healthy controls. Arg(255) is located in the first strand of the five-stranded sheet that forms the main feature of the betaD domain and appears to form an essential H bond with Gly(414). Both the Arg and Gly are absolutely conserved, not only in all known Bbeta chains, but also in all homologous alphaE and gamma chains and in all fibrinogen-related proteins. Protein instability from loss of this contact could easily explain the association of this mutation with hypofibrinogenaemia.

Albumin Rugby Park: a truncated albumin variant caused by a G → C splice-site mutation in nitro 13

Three members of a family were found to be heterozygous for a fast albumin variant (Albumin Rugby Park) that made up only 8% of total serum albumin. Isoelectric focussing indicated an increased negative charge on the C-terminal CNBr peptide and C-terminal sequence analysis of the native protein showed an aberrant sequence of -Ser-Phe. Sequence analysis of PCR-amplified DNA indicated a G-->C mutation at position 1 of the 13th intron and this was confirmed by restriction digestion. The replacement of the obligate GT sequence by CT at the exon/intron boundary prevents splicing of the 13th intron and translation continues for 21 nucleotides until a stop codon is reached. The new protein lacks the 14 amino acids coded for in the 14th exon (GKKLVAASQAALGH), but these are replaced by 7 new residues (LLQFSSF), giving a truncated albumin of 578 residues.

Identification and characterization of five new fibrinogen gene polymorphisms.

Abstract: It is clear that plasma fibrinogen levels are strongly influenced by genetic factors. To date 14 polymorphic sites have been identified within the fibrinogen gene cluster, mainly by restriction fragment length polymorphism (RFLP) and single‐stranded conformation polymorphism (SSCP) analyses. Since elevated plasma fibrinogen is an independent risk factor for cardiovascular disease, these and other polymorphisms are of practical interest in defining haplotypes that correlate with fibrinogen levels. Here, DNA sequencing of fibrinogen genes from four patients led to the identification of 17 variations from the published sequence. Nine of these occurred in all chromosomes sequenced and were considered to be errors in the published data. Of the remaining eight, five represented novel variations, three having been previously described. The population frequency of the five novel variations, together with six known polymorphisms, was estimated by genotyping 50 normal individuals at each locus. The five new variations were all found at polymorphic frequencies in this group. Two of these new polymorphisms, Bβ intron 2 and Bβ codon 159, belong to the Bβ linkage group defined by Behague et al., 1 since their rare alleles occurred in complete concordance with the rare alleles of BβMnl I and BβBcl I. Calculation of pairwise linkage disequilibrium coefficients showed that the three remaining novel polymorphisms, AαDde I, BβHinf I, and γ intron 9 exhibited linkage equilibrium with respect to all other loci examined, including nearby polymorphisms that are themselves in strong linkage disequilibrium. This data indicates that these polymorphisms occur randomly with respect to background haplotype, and suggests that they are mutational hot spots.

Electrospray ionization mass spectrometry identification of fibrinogen Banks Peninsula (γ280Tyr→Cys) : a new variant with defective polymerization

Fibrinogen Banks Peninsula was identified in the mother of a patient referred for investigation following recurrent epistaxis. Coagulation tests revealed prolonged thrombin and reptilase times and a decreased functional fibrinogen level. Thrombin‐catalysed release of fibrinopeptides A and B was normal, and no abnormalities were detected by DNA sequencing of the regions encoding the thrombin cleavage sites in the Aα and Bβ genes. Reducing SDS‐PAGE and reverse‐phase HPLC analysis of purified fibrinogen chains were normal, as was electrospray ionization mass spectrometry (ESI‐MS) analysis of isolated Aα and Bβ chains. However ESI‐MS revealed a mass of 48 345 D for the isolated γ chains, 31 D less than the measured mass of control chains (48 376 D). Since normal and abnormal γ chains were not resolved, this implies a 60–62 D mass decrease in 50% of the molecules. A 60 D decrease was confirmed when DNA sequencing indicated heterozygosity for a mutation of Tyr→Cys at codon 280 of the γ chain gene. Fibrin monomer polymerization revealed a delayed lag phase and reduced final turbidity and although factor XIIIa crosslinking of fibrinogen was normal, it is likely that this delay is due to impaired D:D self association. Recent crystallographic studies show residues γ280 and γ275 make contact across the D:D interface, suggesting a similar mechanism for the polymerization defects in fibrinogens Banks Peninsula and Tokyo II (γ275Arg→Cys).

Apolipoprotein B-32: a new truncated mutant of human apolipoprotein B capable of forming particles in the low density lipoprotein range

We have identified a new species of apolipoprotein (apo) B in an individual with heterozygous hypobetalipoproteinemia. The new apo B (apo B-32) is the result of a single point mutation (1450 Gln----Stop) in the apo B gene that prevents full length translation. Apo B-32 is predicted to contain the 1449 amino-terminal amino acids of apo B-100 and is associated with a markedly decreased low density lipoprotein (LDL) cholesterol level. The density distribution of apo B-32 in the plasma lipoproteins makes it unique amongst other truncated apo B species. Normally, apo B-100 is found in both very low density lipoprotein (VLDL) and LDL particles. However, the majority of the apo B-32 protein was found in the high density lipoprotein (HDL) and lipoprotein-deplete (d greater than 1.21 g/ml) fractions, suggesting that it was mainly assembled into abnormally dense lipoprotein particles. A small amount of apo B-32 was also found in the LDL, making it the shortest known apo B variant capable of forming particles in this density range. Apo B-32 was undetected in VLDL. The apo B-32 mutation further defines the minimum length of the apo B protein that is required for the assembly of LDL.

γ371 Thr→Ile substitution in the fibrinogen γD domain causes hypofibrinogenaemia

Abstract Six members of a family with hypofibrinogenaemia had fibrinogen concentrations ranging from 0.5 to 1.1 mg/ml and, after sequencing the entire coding region and the intron exon boundaries of all three fibrinogen genes, a single heterozygous ACT→ATT mutation was identified in the γ gene. This novel mutation was not detected in normal family members or unrelated controls. The γ371 Thr→Ile substitution occurs at a conserved threonine in the γD domain, but molecules containing the new isoleucine were not present in circulating fibrinogen. The evidence for this was that purified γ chains had a normal mass of 48 375 Da compared to a control of 48 374 Da, and tryptic peptide maps were entirely normal. The mutation predicts a mass increase of 12 Da in peptide T-36, but on mass mapping only the normal [M+2H] ion was detected, at 948 m/z. There was no new signal at 954 m/z that would indicate expression of variant chains. Also the normal 948 m/z signal was at the same intensity in digests from the proposita and controls. Crystal structures show a hydrogen bond from the threonine hydroxyl to the main chain and this case suggests this bond is critical in maintaining the structure of the γD domain.

Albumin Hawkes Bay; a low level variant caused by loss of a sulphydryl group at position 177

A slow migrating minor albumin component, representing 5% of total circulating albumin, was detected by routine serum protein electrophoresis and immunofixation. After treatment with 5 mM dithiothreitol the abnormal component was found to migrate normally suggesting the attachment of some component to the free thiol at position 34. However, purification and analysis by SDS-PAGE showed that the abnormal component had a slightly lower apparent molecular weight than normal albumin. Limited tryptic cleavage indicated the abnormal site to be in the N-terminal third of the molecule. HPLC analysis of tryptic peptides from this domain showed the presence of a new peptide of sequence Ala-Ala-Phe-Leu- Leu-Pro-Lys, indicating either a point mutation of 177 Cys-->Phe or the deletion of residues 166-177. DNA sequencing of PCR-amplified DNA confirmed the former Cys-->Phe substitution by indicating a point mutation of C to A at nucleotide position 5185. It appears that the aberrant electrophoretic mobility of the variant might be due to a gross conformational change associated with the formation of a new disulphide bond between Cys-168 and Cys-124.

Novel sequence insertion in a Mâori patient with transfusion-dependent β-thalassaemia

Although β‐thalassaemia is common throughout the world, it has not been previously described in Polynesia. We report a novel sequence insertion where homozygosity for the defect results in transfusion‐dependent anaemia. The repeated 45 base pair (bp) insertion causes duplication of the start codon and consequent transcription from the original initiation code would be predicted to lead to the production of an irrelevant seven‐residue peptide, while residual translation from the novel initiation site would result in diminished yields of β‐globin and consequent clinical β+‐thalassaemia.

Concurrence of hereditary spherocytosis and alpha thalassaemia

We report a man of MaorilIndian descent with both hereditary spherocytosis and alpha thalassaemia. Alpha thalassaemia is not infrequent in Polynesians and is most often due to the deletion -a3.’ 111.’ Heterozygotes for this deletion usually have mild microcytosis without anaemia.2 In the Indian population the -a3.’ I deletion is seen.’ The propositus was aged three when first investigated for anaemia and found to have spherocytosis with haemolysis of moderate severity (Table 1). At age ten her spleen and gall bladder (containing stones) were removed because of recurrent abdominal pain. The spleen weighed 455g (adult normal 50-2508). Her symptoms disappeared and her haemoglobin rose to levels in the upper normal range without evidence of significant haemolysis. Examination of her father’s blood revealed spherocytes with an increased osmotic fragility comparable to his daughter’s. However, in contrast to his daughter, his haemoglobin level was normal and his spleen was not enlarged. An additional feature was microcytosis of the red cells with an occasional cell containing haemoglobin H inclusion bodies. DNA studies (Figure 1) showed he was heterozygous for a 3.7 kb deletion of one of his alpha globin genes. This man had been adopted and it was not possible to study his biological parents. He had identified himself as being Maori but had recently been informed that his father was Maori and mother Indian. Investigation of the child’s mother showed no haematological disorder (Table 1) and no alpha gene deletion was detected in either mother or daughter (Figure 1). There are no other children in the family. Combined hereditary spherocytosis and thalassaemia has been reported only rarely. Cohen3 in 1959 described an American Negro family segregating for hereditary spherocytosis (mild), sickle cell disease and a form of thalassaemia which appears to have been alpha thalassaemia. No family member had combined hereditary spherocytosis and thalassaemia alone, though one had all three defects. A further six reports document the combination of hereditary spherocytosis and beta thalassaemia, but without a consistent conclusion regarding the interaction of the two &I Ba Bg Ba 3 4