Widening the spectrum of deletions and molecular mechanisms underlying alpha-thalassemia.

Inherited deletions of α-globin genes and/or their upstream regulatory elements (MCSs) give rise to α-thalassemia, an autosomal recessive microcytic hypochromic anemia. In this study, multiplex ligation-dependent probe amplification performed with commercial and synthetic engineered probes, Gap-PCR, and DNA sequencing were used to characterize lesions in the sub-telomeric region of the short arm of chromosome 16, possibly explaining the α-thalassemia/HbH disease phenotype in ten patients. We have found six different deletions, in heterozygosity, ranging from approximately 3.3 to 323 kb, two of them not previously described. The deletions fall into two categories: one includes deletions which totally remove the α-globin gene cluster, whereas the other includes deletions removing only the distal regulatory elements and keeping the α-globin genes structurally intact. An indel was observed in one patient involving the loss of the MCS-R2 and the insertion of 39 bp originated from a complex rearrangement spanning the deletion breakpoints. Finally, in another case, no α-globin gene cluster deletion was found and the patient revealed to be a very unusual case of acquired α-thalassemia-myelodysplastic syndrome. This study further illustrates the diversity of genomic lesions and underlying molecular mechanisms leading to α-thalassemia.

Novel large deletions in the human alpha-globin gene cluster: Clarifying the HS-40 long-range regulatory role in the native chromosome environment.

Globin genes, which encode the protein subunits of hemoglobin (Hb), are organized in two different gene clusters and present a coordinated and differential pattern of expression during development. Concerning the human alpha-globin gene cluster (located at chromosome region 16p13.3), four upstream highly conserved elements known as multispecies conserved sequences (MCS-R1-4) or DNase I hypersensitive sites (HSs) are implicated in the long-range regulation of downstream gene expression. However, only the absence of the MCS-R2 site (HS-40) has proven to drastically downregulate the expression of those genes, and consequently, it has been regarded as the major and crucial distal regulatory element. In this study, Multiplex Ligation-dependent Probe Amplification was used to screen for deletions in the telomeric region of the short arm of chromosome 16, in an attempt to explain the alpha-thalassemia or the HbH disease present in a group of Portuguese patients. We report four novel and five uncommon deletions that remove the alpha-globin distal regulatory elements and/or the complete alpha-globin gene cluster. Interestingly, one of them occurred de novo and removes all HSs except HS-10, while other eliminates only the HS-40 site, the latter being replaced by the insertion of a 39 nucleotide orphan sequence. Our results demonstrate that HS-10 alone does not significantly enhance the alpha-globin gene expression. The absence of HS-40 in homozygosity, found in a patient with Hb H disease, strongly downregulates the expression of alpha-globin genes but it is not associated with a complete absence of alpha-globin chain production. The study of naturally occurring deletions in this region is of great interest to understand the role of each upstream regulatory element in the native human erythroid environment.

Mutational spectrum of delta-globin gene in the Portuguese population.

The phenotype of increased Hb A2 typical of beta-thalassaemia (beta-thal) carriers can be reduced to normal or borderline values because of the co-inheritance of a delta-globin gene (HBD, MIM #142000) mutation, which may lead to misinterpretation of diagnostic results. To know the spectrum of delta-globin mutations in the Portuguese population we performed a mutational analysis of the delta-globin gene in a group of 51 Portuguese beta-thal carriers presenting microcytosis, hypochromia and a normal/borderline Hb A2 level and in another group of 15 individuals suspected to have delta-globin structural abnormalities. The heterozygosity for the beta(+)IVS-I-6T-->C (HBB:c. 92+6T>C) mutation was the main cause for the mentioned atypical beta-thal carrier phenotype. Furthermore, eight individuals were double heterozygous for one common beta-thal mutation and the delta(+)Cd27G-->T mutation (Hb A2-Yialousa; HBD:c.82G>T). One of them also presented a novel delta-globin gene promoter mutation,-80G-->A (HBD:c.-130G>A), responsible for about 25% decrease of the promoter activity in transient expression assays. One the other hand, in the other group of 15 individuals suspected to have delta-globin structural abnormalities observed by biochemical methods, some known Hb A2 variants were identified - Hb A2' (HBD:c.49G>C), Hb A2-Babinga (HBD:c.410G>A), and Hb A2-Wrens (HBD:c.295G>A), and the novel Hb A2-Fogo [delta64(E8)(Gly-->Ser); (HBD:c.193G>A)]. This novel Hb A2 variant was observed segregating in linkage with Hb E (HBB:c.79G>A) in a three generation family. In conclusion, six different delta-globin mutations were found, being two of them new molecular defects. All delta-alleles identified were found linked to the expected beta-globin cluster haplotype. All mutations caused a low Hb A2 level and through this could lead to misdiagnosis when inherited together with a beta-thal allele.

Spectrum of beta thalassemia mutations and HbF levels in the heterozygous Moroccan population.

A comprehensive hematological and molecular analysis of 57 beta thalassemic heterozygotes, 28 homozygotes, 18 double heterozygotes, 3 compound heterozygotes beta thal/beta S and one compound heterozygote beta thal/Hb Newcastle, in 46 Moroccan families with at least one beta thalassemia patient is reported. Six major mutations: beta(0)39 (C-->T), beta(0)FsCD8(-AA), beta(+)IVS1,nt6 (T-->C) and beta(0)IVS1,nt1 (G-->A), beta(0)FsCD6 (-A) and beta(+)-29 (A-->G) cap site account for 75% of the 86 independent beta thal chromosomes studied. For the first time, an extensive mutation/haplotype study has been performed on the Moroccan population, and data are consistent with the geographical location of the country and historical links with both the Mediterranean and the Sub-Saharan Africa communities. Despite the heterogeneous spectrum of mutations, good genetic counseling can be offered to the carrier population. This study focuses on the analysis of fetal hemoglobin levels in beta thalassemic heterozygotes and its correlation with beta globin cluster polymorphic markers in this population. Fetal hemoglobin levels in heterozygotes vary from trace quantities to 17.9% (2.38 g/dl) of total hemoglobin in the adult. No statistically significant correlation was found, either between genders and HbF levels, or between the mutation and the HbF level, with the exception of mutation beta(0)FSCD6(-A). We have examined the alpha globin genotype and the beta globin genotype of heterozygotes, namely, the extended haplotype, which includes the XmnI site at -158 bp of the Ggamma gene and the microsatellite (AT)(x)T(y) at -540 bp of the beta globin gene. In this sample, we confirm the existence of linkage disequilibrium between the C-->T variation at -158 bp of Ggamma globin gene (XmnI(+)) and Orkin's haplotypes III, IV, or IX (the 5' subhaplotype class A). At 5' beta globin gene, we observe exclusively the allele (AT)(7)T(7). In the beta thalassemic heterozygotes studied, no correlation of those genetic markers with HbF levels is observed.

The beta-thalassemia mutation/haplotype distribution in the moroccan population.

The present study compiles the results of our own research and of a prior study on beta-thalassemia (thal) in Morocco, comprising a total of 187 beta-thalassemic chromosomes. Six major mutations: (beta0) codon 39 (C --> T), (beta+) IVS-I-6 (T --> C), (beta0) frameshift codon (FSC) 6 (-A), (beta0) FSC 8 (-AA), (beta0) IVS-I-1 (G --> A) and (beta+) -29 (A --> G) account for 75.7% of the independent chromosomes studied. A regional predominance was observed (Gharb and West regions) for the (beta+) IVS-I-6 (T --> C) mutation. Despite an observed heterogeneity of molecular anomalies, a direct method of diagnosis of the prevalent mutations is feasible in this population. The distributions of mutations and haplotypes are in conformity with the geographical location of Morocco and the historical links with both the Mediterranean communities that have successively interspersed with the Berbers, the Phoenicians, the Carthaginians, the Romans, the Arabs, the population of the Iberian Peninsula and, to a lesser degree, the Vandals and the Byzantines and permanently, with the Sub-Saharan Africans. In the adult population, the levels of fetal hemoglobin (Hb) in heterozygotes vary from trace quantities to 2.38 g/dL of total Hb. With the exception of the (beta0) codon 39 (C --> T) nonsense mutation, no statistically significant correlation was found, neither between mutation and Hb F levels, nor gender and Hb F levels in heterozygotes. The genetic markers for Hb F increase, located within cis active sites such as the XmnI site at -158 bp of the Ggamma-globin gene and the AT(X)T(Y) repeat region at -540 bp of the beta-globin gene, were assessed. The polymorphism XmnI shows linkage disequilibrium with haplotypes III, IV and IX, as previously observed in the Algerian, Sicilian and Portuguese beta-thal populations. Contrary to what has previously been reported for a population of beta-thal carriers of European descent, this sample does not show a statistically significant correlation between Hb F levels and the presence of the genetic markers XmnI restriction site at -158 bp of the Ggamma-globin gene and AT(X)T(Y) alleles at 5' of the beta-globin gene.