Campbell R. Sheen

The molecular aetiology of haemophilia A in a New Zealand patient group

Summary.  The genetic basis of haemophilia A (HA) is well‐established, and many haematology services are supported by molecular biology laboratories that offer factor VIII genetic testing for HA patients. This report describes the results from factor VIII gene (F8) analysis of a New Zealand cohort of 45 proband HA patients. We screened all proband HA patients attending local clinics to determine the molecular basis of disease in each case. We also aimed to evaluate the significance of founder effect in this population and to explain an unusual case of HA in a female patient. HA patients were screened for the common F8 gene inversion mutations using previously described PCR‐based techniques, and for single base substitution mutations using denaturing high performance liquid chromatography and DNA sequencing. Analysis of microsatellite markers located within or near F8 was used to determine identity by descent and trace inheritance patterns of disease alleles. X‐chromosome inactivation (XCI) patterns were detected using methylation specific PCR. Pathogenic F8 gene mutations were detected in all 45 HA patients in this cohort and non‐random XCI was confirmed in a female haemophiliac. We report nine novel F8 mutations, including two splicing mutations, a five nucleotide deletion and a large deletion at the 5′ end of the gene. The molecular aetiology of HA was similar to that described in other studies but the distribution of mutations was unusual due to founder effects, with almost a quarter of all probands being descended from just three individuals.

Fibrinogen Montreal: A novel missense mutation (Aα D496N) associated with hypofibrinogenaemia

Fibrinogen Montreal: A novel missense mutation (Aα D496N) associated with hypofibrinogenaemia -

Fibrinogen Darlinghurst: Hypofibrinogenaemia caused by a W253G mutation in the gamma chain in a patient with both bleeding and thrombotic complications

Fibrinogen Darlinghurst: Hypofibrinogenaemia caused by a W253G mutation in the gamma chain in a patient with both bleeding and thrombotic complications -

Hb MANAWATU [α37(C2)Pro→Leu]: A NEW MILDLY UNSTABLE MUTATION AT AN INVARIANT PROLINE RESIDUE

Hb Manawatu [a37(C2)Pro!Leu] was detected in a 28-year-old female of British descent. Stability tests gave the first indication of the presence of an abnormal Hb, the isopropanol test giving a strong precipitate by 5 mins. Her MCV and MCH were low at 74.0 fL and 24.0 pg, respectively, while other red cell indices were within the normal range (Hb 12.2 g=L, RBC 5.2610=L, PCV 0.38 L=L, MCHC 31.7 g=dL). Hb F (measured by alkaline resistance) and Hb A2 levels were normal at <1% and 2.7%, respectively. No Hb H bodies were detected and there was no indication of hemolysis, as evinced by a normal haptoglobin level of 1.4 g=L and a normal reticulocyte count (57610=L). Cellulose acetate electrophoresis at pH 8.6 was normal, as was the elution profile from cation exchange high performance liquid chromatography (HPLC) at pH 6.5. Electrospray ionization mass spectrometry (ESI=MS), however, showed that some 20% of the a chain material had a mass of 15,144 Da compared to 15,126 Da for the normal a chain (Fig. 1). The fact that the new signal was not resolved from the Na adduct of a at þ22 Da meant that the actual mass increase associated with the putative substitution might be somewhat less than the measured þ18 Da. This was confirmed by examination of the re-dissolved (1% HCOOH) isopropanol precipitate which showed that the unstable Hb had a chains of mass 15,142 Da, 16 Da higher than normal (Fig. 1).

Hb Taradale [β82(EF6)Lys→Arg]: A Novel Mutation at a 2,3-Diphosphoglycerate Binding Site

Hb Taradale [β82(EF6)Lys→Arg] was initially detected as a split Hb A0 peak on Hb A1c monitoring. Red cell parameters, hemoglobin (Hb) electrophoresis and stability tests were normal. Mass spectrometry (ms) clearly identified a variant β chain with a mass increase of 28 Da and peptide mapping located the mutation site to peptide βT-9. DNA sequencing confirmed the presence of a novel β82(EF6)Lys→Arg mutation. This conservative substitution at a 2,3-diphosphoglycerate (2,3-DPG) binding site did not, however, appear to affect the P50 for oxygen binding.

Hb Amsterdam-A1 [α32(B13)Met→Ile; HBA1: c.99G>A]: A Hyperunstable Variant Due to a New Mutation on the α1 Gene

Abstract Patients with hyperunstable α chain variants usually present with a thalassemic, rather than hemolytic, phenotype. Electrophoretic, ion exchange and reverse phase separations usually fail to detect the variant and when DNA sequencing identifies a ‘silent’ substitution it is usually presumed to be hyperunstable. We report the identification of such a variant, α32(B13)Met→Ile; HBA1: c.99G>A, arising from a new mutation on the α1 gene. The hemoglobin (Hb) was unequivocally detected by the isopropanol stability test and confirmed as hyperunstable by mass spectrometry (MS) of the precipitate and lysate, which showed proportions of 55% and 2.5% of α chains, respectively. The instability appears to be driven by perturbation of globin-heme, and possibly α1β1 subunit, interactions.

Genotyping the factor VIII intron 22 inversion locus using fluorescent in situ hybridization.

The factor VIII intron 22 inversion is the most common cause of hemophilia A, accounting for approximately 40% of all severe cases of the disease. Southern hybridization and multiplex long distance PCR are the most commonly used techniques to detect the inversion in a diagnostic setting, although both have significant limitations. Here we describe our experience establishing a multicolor fluorescent in situ hybridization (FISH) based assay as an alternative to existing methods for genetic diagnosis of the inversion. Our assay was designed to apply three differentially labelled BAC DNA probes that when hybridized to interphase nuclei would exhibit signal patterns that are consistent with the normal or the inversion locus. When the FISH assay was applied to five normal and five inversion male samples, the correct genotype was assignable with p<0.001 for all samples. When applied to carrier female samples the assay could not assign a genotype to all female samples, probably due to a lower proportion of informative nuclei in female samples caused by the added complexity of a second X chromosome. Despite this complication, these pilot findings show that the assay performs favourably compared to the commonly used methods.

Hb Lusaka [α131(H14)Ser→Phe (α1)]: A New Variant Found in a Woman Heterozygous for Hb S [β6(A3)Glu→Val]

Hb Lusaka [a131(H14)Ser!Phe] was detected in association with Hb S [b6(A3)Glu!Val] in a 40-year-old Zambian woman now living in Auckland, New Zealand. Her red cell indices were within the normal range with the following values: Hb 12.2 g dL, PCV 0.37 L L, MCV 80 fL, MCH 27 pg; her Hb F level was also normal at <2.0%. Hb A2, as measured by ion exchange chromatography (VARIANT ; Bio-Rad Laboratories, Hercules, CA, USA), appeared elevated at 4.4%, but with hindsight, this elevation was probably due to co-migration with Hb S1c. The red cells appeared normal and the reticulocyte count (84 10 L), and isopropanol stability were also normal. Cellulose acetate electrophoresis (pH 8.6) indicated an abnormal band running in the Hb S position, and this was quantified at 40% of the total by cation exchange high performance liquid chromatography (HPLC). Electrospray ionization mass spectrometry of whole lysate (1), however, showed two abnormal chains; an abnormal b chain with a mass decrease of 30 Da, and an abnormal a chain with a mass increase of 60 Da (Fig. 1). These variant chains were present at levels of 47 and 20%, respectively, compared to the total b and a chains. The electrophoretically abnormal component of the lysate was purified [Fig. 1 (panel c)] by chromatography on DEAE-Sephadex, and tryptic peptide mapping (1)