Robert Lindeman

Polymerase chain reaction (PCR) mutagenesis enabling rapid non‐radioactive detection of common β‐thalassaemia mutations in Mediterraneans

Summary The IVS‐1–110 (G→A) and IVS‐1–1 (G→A) mutations occur in approximately 33% and 9% respectively of β‐thalassaemia alleles in Mediterraneans (Kazazian & Boehm, 1988). They are generally detected in polymerase chain reaction (PCR)‐amplified material by allele‐specific oligonucleotide (ASO) hybridization patterns. In this study, artificial base substitutions in amplified material have been created to distinguish normal from mutant alleles on the basis of restriction enzyme digestion patterns. Invariant target sites provide an internal control for restriction enzyme activity. Mutagenesis was achieved by 3’base mismatches in primers selected to anneal immediately adjacent to target sites. Digestion of PCR products from normal and thalassaemic alleles with the restriction enzymes MboI (IVS‐1–110) and Hinfl (IVS‐1–1) produced different fragments on electrophoresis. The above strategy was validated by allele‐specific oligonucleotide probing. Identification of the three commonest mutations in this population (IVS‐1–110, codon 39 and IVS‐1–1), which account for approximately 69% of thalassaemic alleles (Kazazian & Boehm, 1988), was subsequently undertaken in seven chorion villus biopsies.

Australian β0‐thalassaemia: a high haemoglobin A2β0‐thalassaemia due to a 12 kb deletion commencing 5′to the β‐globin gene

Summary A large novel deletional β0‐thalassaemia mutation associated with unusually high levels of haemoglobin A2 in heterozygotes is described in an Australian family. The deletion was characterized by restriction enzyme analysis followed by PCR amplification and sequencing of the breakpoint region. Australian β0‐thalassaemia extends from 835 basepairs (bp) 5’to the cap site of the β‐globin gene downstream for 12.023 kb. This deletion, similar to previously described deletional β0‐thalassaemias associated with high Hb A2, removes sequences 5’to the β‐globin gene promoter and emphasizes the functional importance of the 5’β‐globin region in eliciting the unusually high Hb A2 phenotype.

Complex phenotypes in the haemoglobinopathies: recommendations on screening and DNA testing

&NA; This document considers a number of scenarios involving complex haemoglobinopathies and provides 28 recommendations at both the clinical and laboratory levels on how these should be managed.

DETECTION AND CHARACTERIZATION OF THE Hb LEPORE (BOSTON) DEFECT BY THE POLYMERASE CHAIN REACTION

venous thrombosis has been reported both in patients with latent myeloproliferative disorders and in heterozygotes for protein C deficiency, yet in our case it seems reasonable to identify the myeloproliferative disorder as the triggering event. Therefore in situations such as congenital protein C deficiency, where a high thrombotic risk is not necessarily expected (Miletich et al, 1987), an extensive screening for concomitant congenital or acquired thrombogenic factors is mandatory in the case of major thrombotic events, as recently stressed by reports of families with the presence of traits for either antithrombin 111 defect, protein S defect or lstituto di Semeiotica Medica, Universith Cattolica del LUCIANA TEOFILI Sacro Cuore, BIANCA ROCCA Roma. Italy GIOVANNI NICOLETTI VALERIO DE STEFANO

Cytogenetic microdeletions in the Prader-Willi syndrome assist in the understanding of genomic imprinting

The PWS is a rare multisystem genetic disorder associated with mild mental retardation, obesity, hypotonia, hypogonadism and hypopigmentation. The major clinical problem is obesity which results from uncontrollable eating in affected individuals. We have investigated cytogenetically and with DNA mapping 27 PWS patients. 59% demonstrated a cytogenetic deletion whilst 74% were deleted on DNA testing. In two families where the proband was cytogenetically normal it was possible to confirm that the paternal PWCR allele was absent because of the inheritance of a novel DNA band. A third family in which the PWS proband was cytogenetically deleted was shown to have a paternal deletion on the basis of restriction fragment length polymorphism patterns. The above results confirm the hypothesis that genomic imprinting is involved in pathogenesis of PWS. It is proposed that deletion of a part of the paternal PWCR produces PWS whilst loss in the maternal allele leads to Angelman’s syndrome, a genetic disorder with a different clinical phenotype. Mechanism(s) involved in genomic imprinting in PWS include deletions or uniparental disomy in those cases which are non-deletional (Nicholls et al; 1989 Nature; 342:281).