John Old

Rapid detection of alpha-thalassaemia deletions and alpha-globin gene triplication by multiplex polymerase chain reactions.

We describe a sensitive, reliable and reproducible method, based on three multiplex PCR assays, for the rapid detection of seven common α‐thalassaemia deletions and one α‐globin gene triplication. The new assay detects the α0 deletions –u2003–SEA, –u2003(α)20.5, –u2003–MED, –u2003–FIL and –u2003–THAI in the first multiplex PCR, the second multiplex detects the –α3.7 deletion and αααanti3.7 variant, the third multiplex detects the –α4.2 deletion. This simple multiplex method should greatly facilitate the genetic screening and molecular diagnosis of these determinants in populations where α‐thalassaemias are prevalent.

Rapid detection and prenatal diagnosis of β-thalassaemia: studies in Indian and Cypriot populations in the UK

The application of the amplification refractory mutation system (ARMS) to the detection of individual beta-thalassaemia mutations in heterozygous parents and at risk fetuses has been assessed in Indian and Cypriot immigrant populations in the UK. 100 first trimester prenatal diagnoses have been done, entailing the detection of 17 different mutations. The method, which allows the determination of the mutations in both parental and fetal DNA on the same day, should have wide application to the carrier detection and prenatal diagnosis of monogenic diseases with heterogeneous molecular defects.

FIRST-TRIMESTER FETAL DIAGNOSIS FOR HAEMOGLOBINOPATHIES: THREE CASES

Abstract By means of a simplified method for trophoblast biopsy together with restriction endonuclease analysis of fetal DNA, first-trimester diagnoses were successfully made in two fetuses at risk for homozygous β thalassaemia and in one at risk for sickle cell anaemia.

The spectrum of β‐thalassaemia mutations on the Indian subcontinent: the basis for prenatal diagnosis

Summary The β‐thalassaemia mutations in 702 unrelated carriers originating from seven different regions of the Indian subcontinent have been characterized using allele specific priming of the polymerase chain reaction (PCR). It was possible to identify the mutations in 688 (98%) of the individuals studied. Eleven different mutations were identified, of which five common ones accounted for 93·6%; namely the ones at IVS‐1 position 5 (G‐C), codons 8/9 (+G), IVS‐1 position 1 (G‐T), codons 41/42 (‐CTTT) and the 619 bp deletion at the 3’end of the gene. The mutations at IVS‐2 position 1 (G‐A) and codon 30 (G‐C), previously undescribed in Asian Indians, were found in two and six individuals respectively. Some regional variation in the distribution of β‐thalassaemia alleles was noted. These findings should prove useful for the development of a first trimester prenatal diagnosis programme based on direct detection of mutations.

Multicentric origin of hemochromatosis gene (HFE) mutations.

Genetic hemochromatosis (GH) is believed to be a disease restricted to those of European ancestry. In northwestern Europe, >80% of GH patients are homozygous for one mutation, the substitution of tyrosine for cysteine at position 282 (C282Y) in the unprocessed protein. In a proportion of GH patients, two mutations are present, C282Y and H63D. The clinical significance of this second mutation is such that it appears to predispose 1%-2% of compound heterozygotes to expression of the disease. The distribution of the two mutations differ, C282Y being limited to those of northwestern European ancestry and H63D being found at allele frequencies>5%, in Europe, in countries bordering the Mediterranean, in the Middle East, and in the Indian subcontinent. The C282Y mutation occurs on a haplotype that extends </=6 Mb, suggesting that this mutation has arisen during the past 2,000 years. The H63D mutation is older and does not occur on such a large extended haplotype, the haplotype in this case extending </=700 kb. Here we report the finding of the H63D and C282Y mutations on new haplotypes. In Sri Lanka we have found H63D on three new haplotypes and have found C282Y on one new haplotype, demonstrating that these mutations have arisen independently on this island. These results suggest that the HFE gene has been the subject of selection pressure. These selection pressures could be due to infectious diseases, environmental conditions, or other genetic disorders such as anemia.

Analysis of β-globin gene haplotypes in Asian Indians: origin and spread of β-thalassaemia on the Indian subcontinent

Abstractβ-globin gene haplotypes were determined for 196 normal (β-A) and 419 thalassaemia (β-Th) chromosomes of individuals from four different regions of the Indian subcontinent; North-west Pakistan, Gujarat, Punjab and Sindh. Analysis of β-A and β-Th haplotypes and haplotype-mutation associations in each regional group along with a consideration of Indian history provided information about the origin and spread of β-thalassaemia mutations on the Indian subcontinent. The data are consistent with relatively recent and local origins for most β-thalassaemia mutations. The frequencies of particular alleles differ markedly in various regions and these may be useful population markers. Of the high frequency alleles, intervening sequence 1 (IVS-1) nucleotide 5 (G-C) and codons 41/42 (-CTTT) appear to be older as suggested by multiple haplotype associations and a widespread geographical distribution. The microepidemiology of β-thalassaemia in this region reflects considerable ethnic diversity, gene flow from population migration and natural selection by malaria infection.

Thalassaemia in Sri Lanka: implications for the future health burden of Asian populations

BACKGROUNDnThalassaemias pose an increasing problem for the Indian subcontinent and many Asian countries. We analysed the different types of thalassaemia in the Sri Lankan population, surveyed gene frequencies in schoolchildren, and estimated the burden of disease and requirements for its control.nnnMETHODSnWe analysed blood samples from patients attending clinics in nine hospitals and defined the different types of beta thalassaemia by high-performance liquid chromatography (HPLC) and DNA analysis. The range of mutations was obtained by analysis of beta-globin genes. Capillary blood was obtained from schoolchildren from different parts of the island and analysed by HPLC to provide an approximate assessment of the carrier frequency of beta thalassaemia and haemoglobin E (HbE). To estimate the frequency of alpha thalassaemia the alpha-globin genotypes were also analysed when it was possible.nnnFINDINGSnBlood samples were obtained from 703 patients with beta thalassaemia and from 1600 schoolchildren. The thalassaemia mutations were unevenly spread. Although 23 different beta-thalassaemia mutations were found, three accounted for the thalassaemia phenotype in about 70% of the patients, most whom are homozygotes or compound heterozygotes for IVS1-5 (G-->C) or IVS1-1 (G-->A). The third common mutation, codon 26 (G-->A), which produces HbE, interacts with one or other of these mutations to produce HbE/beta thalassaemia; this comprises 13.0-30.9% of cases in the main centres. Samples from 472 patients were analysed to determine the alpha-globin genotype. Overall, 15.5% patients were carriers for deletion forms of alpha+ thalassaemia. Average gene frequencies showed that there will be more than 2000 patients requiring treatment at any one time, in the future, of whom those with HbE/beta thalassaemia will account for about 40%.nnnINTERPRETATIONnIn Sri Lanka, interactions of the two common beta-thalassaemia alleles will nearly always result in a transfusion-dependent disorder. However, about 40% of patients will have HbE/beta thalassaemia, which has a variable course. The management of these disorders could require about 5% of the total health budget. We need to learn more about the natural history and appropriate management of HbE/beta thalassaemia if resources are to be used effectively.

Association of thalassaemia intermedia with a beta-globin gene haplotype

We have identified 14 Asian patients with homozygous β° thalassaemia who had a mild clinical disorder related to an augmented production of haemoglobin F. None of their parents had an elevated level of Hb F. Restriction fragment length polymorphism analysis of the β‐globin cluster of these patients and a control group of Asian thalassaemia major patients showed that 6/14 of the thalassaemia intermedia patients were homozgyous for a particular 5′β‐globin haplotype (−+−++), in contrast to 1/42 of the thalassaemia major patients. Furthermore, the —+—++β haplotype is also associated with amelioration of disease severity in β thalassaemia in an Italian population. This β haplotype is linked to a DNA sequence variation 5′ (at position — 158) to the cγ globin gene which can be detected by the presence (+) of an Xmn I restriction enzyme site. The possible role of the Xmn I‐γ polymorphism in relation to this variant HPFH is discussed. We conclude that much of the observed clinical variability of β thalassaemia can now be explained by the inheritance of β thalassaemia chromosomes with different propensities for fetal haemoglobin production.

Informed choice in genetic screening for thalassaemia during pregnancy: audit from a national confidential inquiry.

Abstract Objective: National audit of informed choice in antenatal screening for thalassaemia. Design: Audit from the UK Confidential Enquiry into Counselling for Genetic Disorders. Setting: Thalassaemia module of the UK Confidential Enquiry into Counselling for Genetic Disorders. Subjects: 138 of 156 couples who had had a pregnancy affected by a major β thalassaemia from 1990 to 1994. Main outcome measures: How and when genetic risk was identified for each couple, and whether and when prenatal diagnosis was offered. Results: Risk was detected by screening before or during the first pregnancy in 49% (68/138) of couples and by diagnosis of an affected child in 28% (38/138) of couples. Prenatal diagnosis was offered in 69% (274/400) of pregnancies, ranging from 94% (122/130) for British Cypriots to 54% (80/149) for British Pakistanis and from 90% in the south east of England to 39% in the West Midlands. Uptake of prenatal diagnosis was 80% (216/274), ranging from 98% (117/120) among British Cypriots in either the first or second trimester to 73% (35/48) among British Pakistanis in the first trimester and 39% (11/28) in the second trimester. A demonstrable service failure occurred in 28% (110/400) of pregnancies, including 110 of 126 where prenatal diagnosis was not offered and 48 of 93 that ended with an affected liveborn infant. Conclusion: Although antenatal screening and counselling for haemoglobin disorders are standard practices in the United Kingdom, they are delivered inadequately and inequitably. An explicit national policy is needed, aiming to make prenatal diagnosis in the first trimester available to all couples and including ongoing national audit.

Rare β-thalassaemia mutations in Asian Indians

Five β‐thalassaemia mutations hitherto undescribed in Asian Indians were identified in β‐thalassaemia carriers originating from the Indian subcontinent by direct sequencing of their β‐globin genes which were amplified by the polymerase chain reaction (PCR). A T‐G substitution at IVS 2 position 837, which probably creates an alternative acceptor splice site and a T insertion in codon 88, resulting in a shift in the reading frame with a premature stop codon, are new β‐thalassaemia mutations. The others were framshift codon 5 (‐CT), IVS 1 position 110 (G‐A) and IVS‐1 minus 1 (G‐A) which have been described previously in other populations. These results complete the characterization of the β‐thalassaemia mutations in 708 carriers of Asian Indian origin and will enable a comprehensive programme of carrier screening and prenatal diagnosis of β‐thalassaemia in this population.