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This study describes a new molecular condition in the α2-globin gene (HBA2) found in six unrelated families from Southern Italy (Campania and Sicily). This new double mutant form of haemoglobin is called Hb Southern Italy and originated from the coexistence of two known mutations occurring in the same globin gene, HBA2 26 GA (Hb Caserta) and HBA2 130 GC (Hb Sun Prairie). Hb Sun Prairie was originally observed in Indian patients in either the homozygous state, with severe hemolytic anemia, and in the heterozygous state with microcytosis, or in asymptomatic cases as an α-thalassemia carrier phenotype. Hb Caserta was observed for the first time in a Casertian family (South Italy) that displayed a slowmigrating haemoglobin upon investigation. We report the clinical phenotype and molecular study of this new double mutant form of haemoglobin in heterozygous and homozygous subjects, as well as in association with α° delectional thalassemia.
α-Thalassemia is the most common human single gene disorder worldwide (Bernini & Harteveld, 1998; Weatherall et al, 2001). In Southern Italy, β-thalassemia is the most common form of thalassemia, though α-thalassemia is also prevalent. In Sicily, α-thalassemia does not constitute a public or clinical health problem because α+-thalassemia is the predominant form (Fichera et al, 1997; Guida et al, 2006). However, an accurate diagnosis is important in order to provide genetic counseling for the prevention of severe forms of α-thalassemia that range from mortal Hb Bart’s hydrops fetalis to severe HbH disease. Moreover, a proper diagnosis is also needed to differentiate between the phenotypes of heterozygous α-thalassemia and cases that phenotypically resemble α-thalassemia, such as co-inherited δ and β-thalassemia (Giambona et al, 2006).
The Sun Prairie haemoglobin variant was first reported in the homozygous state (Harkness et al, 1990) in a young Indian patient with severe hemolytic anemia, microytosis and hypochromia. Subsequently, the same abnormal hemoglobin was described in the heterozygous state in an Asian Indian family with an α thalassemia carrier phenotype (Ho et al, 1996).
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- Materials and methods
Hb variants with more than one point mutation in the same polypeptide chain are rare; so far 29 such variants have been described in the beta chain, while none has been identified in the alpha chain (database of Human Hemoglobin Variants http://globin.cse.psu.edu; Blackwell et al, 1972).
Hb Southern Italy is the first variant haemoglobin of HBA2 that originated from an unusual combination of two known molecular defects, GCGACG at codon 26 that was previously found in Hb Caserta, and GCTCCT at codon 130, already described in Hb Sun Prairie.
Hb Caserta was described for the first time in a family from Caserta (Southern Italy) as a slow-migrating form of haemoglobin, and then as a variant haemoglobin with reduced haematological parameters (MCV and MCH) and normal HbA2. More recently, Hb Caserta was described to be in association with an α-thalassemia-like phenotype, and it was suggested that there was a decreased production of normal mRNA due to the activation of a cryptic splicing site at codons 25–27 (unpublished observations). These reports did not mention any other alterations (mutations or polymorphisms) in HBA2, either because they were absent or because HBA2 was not completely analyzed by sequencing methods. The results presented here suggest that when new or rare mutations are observed and little information is available, entire HBA1, HBA2 and HBB genes should be completely analyzed using sequencing method.
In Hb Caserta, a conservative replacement of an alanine residue with threonine occurs within the B helix, buried in the tertiary structure of the α-globin chain and not involved in the α/β contacts or in the heme globin linkage. Taking these factors into consideration, it may be suggested that this mutation should not significantly affect Hb function.
Hb Sun Prairie was also associated with another molecular defect in a patient of Indian origin (Sarkar et al, 2005). The authors described Hb Sun Prairie in association with a CT transition in the 5′ untranslated region (UTR) of the same HBA2 gene. Molecular modelling studies did not display large distortions in the Hb molecule, suggesting that the severe clinical picture of the patients carrying these mutations might have been related to the co-inheritance of the mutation in the 5′-UTR of the same HBA2 gene containing the Sun Prairie substitution. The authors then suggested that the co-occurrence of these two defects would lead to an additional depression of HBA2 expression.
We have also investigated a 1-year-old girl of Philippine origin who displayed thalassemia with splenomegaly and hepatomegaly. Her Hb level was lower than 83 g/l and she was regularly treated with blood transfusions. Molecular analysis of the proband and her parents showed that she had inherited Hb Sun Prairie from her father and a deletional α0-allele from her mother (--SEA). Sequencing analysis of the propositus and her father did not indicate the presence of any further mutations, either at the 5′UTR or at codon 26, and the polymorphic site HBA2 +861 was also absent.
Our molecular analysis provided evidences that the mutation at codon 130 present in the Hb Sun Prairie variant (identified in the Indian population and in Eastern Indians) and in the Hb Southern Italy double variant, has different origins. In the new Hb Southern Italy variant, mutation at codon 130 is always associated with mutation at codon 26 and with the polymorphic site HBA2 +861.
Two possible mechanisms may have contributed to the formation of Hb Southern Italy allele. First, a crossover between a gene encoding Hb Caserta and a gene encoding Hb Sun Prairie. The Mediterranean basin was the crossroads of the world for thousands of years and was traversed by several races, each of which spread their ideas and cultures. The geographical position of Sicily and Campania made them a natural seaport point on these travels. Large numbers of Greeks, Byzantines, Saracens, and Normans came to Campania to settle, and not just to rule and exploit the region. Each of these people contributed to the gene pool and left an imprint on the genetic composition of the Campanian people.
Alternatively, the variant allele might have been originated by the occurrence of two separate point mutational events, perhaps sequentially, although the specific family in which one of the point mutations existed in a parent and both in a child, could not be found.
The founder effect would explain the unexpected frequency of this rare type of mutation in a restricted area like South of Italy allele.
The phenotypes of both homozygote and heterozygote subjects for Hb Sun Prairie described by other authors were very similar to those of our patients carrying the Hb Southern Italy variant in the homozygous and heterozygous states. Both groups of patients showed α-thalassemia-like carrier phenotypes with microcytosis and normal HbA2 levels in the heterozygous state, while chronic hemolytic anemia was observed in homozygotes. However, the instability of the Hb Southern Italy variant, which is rapidly catabolized and therefore not detected by routine analysis of hemolysate, was slightly higher than Hb Sun Prairie. The variant alpha globin from Hb Sun Prairie, but not that from Hb Southern Italy could be detected by liquid chromatography mass spectrometry analysis (LC-MS) (data not shown), which suggests that the mutation at codon 26 might somehow contribute to the instability of Hb Southern Italy. However, the reduction of HBA2 expression and the consequent severity of the clinical picture in patients with the Hb Southern Italy variant should essentially be associated with the mutation in codon 130.
It is important to note that, although, in the homozygous state Hb Southern Italy presents as a severe anemia, as in the Hb Sun Prairie homozygous state, this does not necessarily require transfusion. However, the combination of Hb Southern Italy with an α0-thalassemia deletional determinant leads to the presentation of a severe HbH disease, which is blood transfusion-dependent.