Clinical and molecular pathology of the β+ IVSI-6C thalassaemia in Malta

Abstract

CLINICAL AND MOLECULAR PATHOLOGY OF THE β+ IVSI-6C THALASSAEMIA IN MALTA The main objectives of this study were to characterise the β -thalassaemia mutations present in Malta, evaluate treatment protocols, study the correlation between the genotypical and phenotypical pictures, evaluate the criteria for the proper identification of thalassaemia heterozygotes and investigate the causes for the bone disease in thalassaemia homozygotes. Data on the molecular defects leading to β -thalassaemia were obtained from 28 homozygotes out of the known 29 subjects. Four different mutations were encountered, with the β+IVSI-6(T->C) accounting for 71.4% of all β-thal alle1es [β+IVSI-110(G->A) = 12.5%; β °IVSII-1 (G->A) = 10.7%; β °Codon 39(C->T) = 5.4%]. The β+IVSI-6 (C) allele was present on both haplotype VI and VII while the β °Codon 39(T)and the β+IVSI-110(A) were associated with haplotypes I and IX respectively. The β °IVSII-1(A) mutation was found within haplotype III except in one case that had an unusual VI/Ill hybrid haplotype. The VI/Ill hybrid haplotype was characterised by a low HbF (7.7%) in contrast to the other 5 cases that had a high HbF (-60%). Data collected prospectively on the β+IVSI-6C homozygote children, indicated that the disease presented as a moderate to severe condition needing regular blood transfusions (mean = 70ml/kg/year) for normal growth and development. Splenectomy had little effect on the blood transfusion requirement of the β+IVSI-6C homozygotes. On the other hand, the adult 13+ rvSI-6C homozygous condition was characterised by a mild disease (mean Hb= 8.2g/dl) with only occasional transfusions. Intra-allelic heterogeneity in the level of HbF was observed among the β+IVSI-6C homozygotes and these could be divided into two groups, one with a relatively high HbF (mean=15.0%) and one with a low HbF (4.5%). Statistically significant (p<0.05) gender difference in the level of HbF was also observed with female patients having in general, a higher HbF level. The high incidence of a relatively mild mutation and the presence of iron deficiency amongst the population, posed problems in the proper identification of β-thalassaemia heterozygotes. Indeed 37% (N=19) of obligate β+IVSI-6C heterozygotes had an MCV<80fl and a HbA2 between 3.0 and 3.5%. In an attempt to improve in the identification of the β+IVSI-6C heterozygotes, while keeping the cost of population testing to a minimum, a new approach in the identification of individuals at risk was evaluated with a computed index (10 X RBC3 x HbA²1Hb3) The exclusion of iron deficiency was further considered with a revised discriminant/cut-off value for serum ferritin, which was quite higher than that employed so far. A definite diagnosis amongst those individuals deemed at risk would be obtained by DNA analysis for the prevalent mutation within the population. Osteopenia as documented by a low bone mineral density using DEXA, was present in the majority of the homozygote subjects and was apparent as early as 5 years of age, despite a seemingly adequate hypertransfusion regime. In an attempt to elucidate possible causes for the observed osteopenia, the level of bone biochemical markers for bone formation (serum procollagen I carboxyterminal propeptide and osteocalcin) and for bone turnover (urinary deoxypyridinoline crosslinks and serum tartrate resistant acid phosphatase) were measured amongst the thalassaemia patients. The low serum osteocalcin level accompanied by normal levels for urinary deoxypyridinoline crosslinks and serum procollagen I carboxyterminal propeptide indicated that lack of proper mineralisation could result in the osteopenia observed within this group of patients. Nutritional deficiencies associated with low body weight might be possible causes of the lack of mineralisation among the patients with "mild" alleles.