Neil Van de Water

Mutations within the protein Z-dependent protease inhibitor gene are associated with venous thromboembolic disease: a new form of thrombophilia

Protein Z‐dependent protease inhibitor (ZPI) is a serpin that inhibits the activated coagulation factors X and XI. The precise physiological significance of ZPI in the control of haemostasis is unknown although a deficiency of ZPI may be predicted to alter this balance. The coding region of the ZPI gene was screened for mutations using denaturing high‐performance liquid chromatography. 16 mutations/polymorphisms within the coding region of ZPI were identified including two mutations, which generated stop codons at residues R67 and W303. We observed nonsense mutations within the ZPI gene in 4·4% of thrombosis patients (n = 250) compared with 0·8% of controls (n = 250). The difference in distribution of stop codon mutations between thrombosis patients and controls was significant (P = 0·02) with an odds ratio of 5·7 (95% confidence interval, 1·25–26·0). Our results suggest an association between ZPI deficiency and venous thrombosis and we propose that ZPI deficiency is potentially a new form of thrombophilia.

Factor IX gene mutations in haemophilia B: a New Zealand population-based study.

Summary. Haemophilia B (Christmas disease) is an X‐linked bleeding disorder resulting from an inherited deficiency of coagulation factor IX activity. Due to the heterogeneity of mutations within the factor IX gene there is a marked clinical variability in disease severity. By applying techniques of mutational analysis and direct sequencing of PCR products it is now potentially possible to determine the pathogenic gene defect in entire haemophilia B populations. We report here characterization of the factor IX gene defect in all the haemophilia B patients in New Zealand as part of a nationwide approach towards providing efficient and cost‐effective haemophilia B genetic counselling services for these families. Twenty‐six different mutations were identified in 32 unrelated haemophilia B families. Three defects at nucleotide positions +8,6659 and 17696 are novel mutations which have not been reported by other laboratories. A PCR‐based diagnostic screening test for direct mutational analysis could be performed in most cases; 17 of the 26 mutations altered a restriction enzyme recognition sequence and, with the exception of the total gene deletion, base changes not affecting a restriction enzyme site could be detected by allele‐specific PCR.

POSSIBLE MISDIAGNOSIS USING THE XBA I POLYMORPHISM FOR PRENATAL DIAGNOSIS OF HAEMOPHILIA A

Summary. A mother of two haemophilia A sons presented at 7 weeks pregnancy for a prenatal diagnosis. Sourthern blot analysis of haemophilia DNA at the factor VII intron 22 Xba I restriction fragment lenght polymorphism (RFLP) site revealed an Xba I haplotype of A‐B+C‐. This haplotype has been alluded to but not reported before, and when a 1.6kb Bst XI fragment of p482.6 is used as a probe the resulting band pattern is similar to that of females heterozygous at site A.

Hb Feilding [β12(A9)Thr → Pro; HBB: c.37A>C]: A Novel Unstable β-Globin Chain Variant

Abstract We report here a patient heterozygous for a previously unreported β chain variant. A 72-year-old Caucasian female was found to have an abnormal hemoglobin (Hb) as an incidental finding following Hb A1C analysis. There was no family history of anemia or hemoglobinopathy. Her full blood count revealed a mild normochromic anemia with Hb 11.1 g/dL (range 11.5–15.0), mean corpuscular volume (MCV) 93.0 fL (range 80.0–100.0) and mean corpuscular Hb (MCH) 30.0 pg (range 27.0–32.0). Isopropanol stability tests and a variant Hb on high performance liquid chromatography (HPLC) comprizing 37.0% of the total Hb suggested an unstable Hb variant. Sanger sequencing of the β-globin gene revealed a single base substitution, HBB: c.37A>C, causing the missense mutation β12(A9)Thr → Pro in exon 1 of the HBB gene. This mutation changes the threonine residue at position 12(A9) to a proline in the β-globin chain. We propose that this variant be called Hb Feilding after the town where the proband lived. Three dimensional modeling suggested that the disruption of the Hb structure was due to the introduction of a proline at helix A9 which caused distortion of the helical structure and resulted in reduced solubility.

Factor VIII gene inversions in severe hemophilia a patients

&NA; The mutations causing hemophilia A are very heterogeneous with the exception of a large inversion involving intron 22 in the factor VIII (FVIII) gene which appears to be the underlying defect in approximately 45% of all severely affected patients (FVIII ˇ‐ 1%). In these patients it is thought that the factor VIII gene is disrupted within intron 22 due to inappropriate recombination of FVIIIA with one of 2 homologous regions upstream of the factor VIII gene resulting in a large (∼500 kb) inversion. The inversion can be detected by Southern blot analysis and greatly enhances the accuracy of genetic counselling services available to families with severe hemophilia A. We report here the presence of this mutation in a study of 27 unrelated families with severe hemophilia. The factor VIII inversion was identified in 12 of 27 (44%) severe hemophilia A patients and has been successfully used for direct carrier analysis and prenatal diagnosis.

A +8 (C→T) Mutation within the 5′ Untranslated Region of β-Globin Down-Regulates the mRNA Transcription

The 5′ untranslated region (5′UTR) of β-globin has been well characterized and is often used as a model for eukaryotic transcription/translation, but there are still questions regarding the mechanism of translational control. Mutations affecting the Cap site at  + 1 and at positions +10, +22, +33 and +40–43 have been described, and it is thought that the initiator element required for transcription stretches from −2 to +7 relative to the Cap site with a downstream element situated from +10 to +15. The influence on initiation or translation of sequences between +7 and +10 is unknown. We report here a family with β-thalassemia (β-thal) who have a + 8 (C→T) mutation. Molecular studies indicate that this mutation leads to a reduction in mRNA levels and we discuss the implications of a C→T change at this position on the transcription/translation process.

Efficient identification of somatic mutations in acute myeloid leukaemia using whole exome sequencing of fingernail derived DNA as germline control

Recent advances in next-generation sequencing have made it possible to perform genome wide identification of somatic mutation in cancers. Most studies focus on identifying somatic mutations in the protein coding portion of the genome using whole exome sequencing (WES). Every human genome has around 4 million single nucleotide polymorphisms (SNPs). A sizeable fraction of these germline SNPs is very rare and will not be found in the databases. Thus, in order to unambiguously identify somatic mutation, it is absolutely necessary to know the germline SNPs of the patient. While a blood sample can serve as source of germline DNA from patients with solid tumours, obtaining germline DNA from patients with haematological malignancies is very difficult. Tumor cells often infiltrate the skin, and their DNA can be found in saliva and buccal swab samples. The DNA in the tips of nails stems from keratinocytes that have undergone keratinization several months ago. DNA was successfully extracted from nail clippings of 5 probands for WES. We were able to identify somatic mutations in one tumor exome by using the nail exome as germline reference. Our results demonstrate that nail DNA is a reliable source of germline DNA in the setting of hematological malignancies.

Evaluation of an immunochromatographic strip test for alpha-thalassaemia screening

Hb H inclusion test (HbH‐i) commonly used for α‐thalassaemia screening is not standardised and is labour‐intensive. This study evaluated a strip test based on immunochromatographic detection of Hb Barts (ICT) for use as a routine screening test for α‐thalassaemia screening in the clinical laboratory setting.

Novel α-Globin Splice Site Mutation (HBA2: c.96-5C>A) in Combination with Three-Gene Deletion Hb H Disease

Abstract The choice of acceptor splice site during exon-exon splicing by the spliceosome is determined by a variety of factors. We report here a family with a novel acceptor splice site variant within intron 1 of the α-globin gene that provides some in vivo insight into the rules governing RNA splicing in homo sapiens. A 2-year-old female with Hb H disease, was found to have not only three α-globin genes deleted (– –FIL/–α3.7) but also a HBA2: c.96-5C>A variant on her remaining α-globin gene. The HBA2: c.96-5C>A variant was in cis with –α3.7 and mRNA studies indicate that this variant creates a new acceptor splice site which is used in approximately 35.0% of α-globin mRNA transcripts. The reduced levels of normal mRNA transcript predicts a more severe Hb H disease than expected for the three-gene deletion Hb H disease with a phenotype similar to nondeletional Hb H disease. We propose that this variant be called Hb Beach Haven (HBA2: c.96-5C>A).