Maria Carla Sollaino

Amelioration of Sardinian β0 thalassemia by genetic modifiers

Sardinian beta-thalassemia patients all are homozygotes for the same null allele in the beta-globin gene, but the clinical manifestations are extremely variable in severity. Previous studies have shown that the coinheritance of alpha-thalassemia or the presence of genetic variants that sustain fetal hemoglobin production has a strong impact on ameliorating the clinical phenotype. Here we evaluate the contribution of variants in the BCL11A, and HBS1L-MYB genes, implicated in the regulation of fetal hemoglobin, and of alpha-thalassemia coinheritance in 50 thalassemia intermedia and 75 thalassemia major patients. We confirm that alpha-thalassemia and allele C of single nucleotide polymorphism rs-11886868 in BCL11A were selectively represented in thalassemia intermedia patients. Moreover, allele G at single nucleotide polymorphism rs9389268 in the HBS1L-MYB locus was significantly more frequent in the thalassemia intermedia patients. This trio of genetic factors can account for 75% of the variation differences in phenotype severity.

Association of α globin gene quadruplication and heterozygous β thalassemia in patients with thalassemia intermedia

The degree of the globin chain imbalance is the pathogenetic clue to the clinical phenotype of thalassemia syndromes. This paper reports a duplication of the α globin gene locus in a group of hetereozygous β-thalassemia patients with the unexplained phenotype of thalassemia intermedia. Ten patients with thalassemia intermedia with variable severity and apparent simple heterozygosis for β0 39 C>T nonsense mutation were submitted to clinical, hematologic and molecular studies. The presence of an unknown molecular defect (silent β-thalassemia) unlinked to the β cluster interacting with the heterozygous β thalassemia, was previously postulated in these families. Analysis of the α globin gene cluster with PCR-based methods (MLPA, GAP-PCR, digestion with restriction enzymes) detected complex rearrangements in the α cluster. A duplication of the α globin gene locus, including the upstream regulatory region, was present in all the patients, associated in some of them with deletion or non-deletion α thalassemia. The variability of the clinical phenotype correlates with the degree of the globin chain imbalance. The presence of α globin cluster duplication should be considered in patients heterozygote for β-thalassemia with thalassemia intermedia phenotype and in the carriers of suspected silent β thalassemia.

Clinical and molecular analysis of haemoglobin H disease in Sardinia: haematological, obstetric and cardiac aspects in patients with different genotypes

In this study, 251 Sardinian patients (187 adults and 64 children) with haemoglobin (Hb) H disease were evaluated. Two‐hundred and sixteen patients (86%) had the deletional type (‐ ‐/‐α) and 36 (14%) patients had the non‐deletional type (‐ ‐/αNDα). A clear genotype–phenotype correlation was found, with the non‐deletional type more severe than the deletional type. Diagnosis of Hb H disease was incidental in about 60% of cases. Aplastic crises due to B19 parvovirus infection were found in five patients (2·1%), while 23 patients (9·6%) experienced one or more haemolytic crises. Nineteen patients with Hb H received sporadic red blood cell transfusions and three patients were repeatedly transfused. Forty‐seven of 61 married women (77%) had 82 pregnancies. In children, mean serum ferritin was 87 ±92 μg/l and in adults, was 192 ± 180 μg/l in females and 363 ± 303 μg/l in males. For the 98 male patients, a significant correlation was found between ferritin values and age (r2 = 0·33, P < 0·0001). In the Sardinian population, Hb H disease needs regular monitoring for early detection and treatment of possible complications, such as worsening of anaemia that may require red cell transfusion, cholelithiasis and iron overload.

A genetic score for the prediction of beta-thalassemia severity.

Clinical and hematologic characteristics of beta(β)-thalassemia are determined by several factors resulting in a wide spectrum of severity. Phenotype modulators are: HBB mutations, HBA defects and fetal hemoglobin production modulators (HBG2:g.−158C>T polymorphism, HBS1L-MYB intergenic region and the BCL11A). We characterized 54 genetic variants at these five loci robustly associated with the amelioration of beta-thalassemia phenotype, to build a predictive score of severity using a representative cohort of 890 β-thalassemic patients. Using Cox proportional hazard analysis on a training set, we assessed the effect of these loci on the age at which patient started regular transfusions, built a Thalassemia Severity Score, and validated it on a testing set. Discriminatory power of the model was high (C-index=0.705; R2=0.343) and the validation conducted on the testing set confirmed its predictive accuracy with transfusion-free survival probability (P<0.001) and with transfusion dependency status (Area Under the Receiver Operating Characteristic Curve=0.774; P<0.001). Finally, an automatized on-line calculation of the score was made available at http://tss.unica.it. Besides the accurate assessment of genetic predictors effect, the present results could be helpful in the management of patients, both as a predictive score for screening and a standardized scale of severity to overcome the major-intermedia dichotomy and support clinical decisions.

Homozygous deletion of the major alpha-globin regulatory element (MCS-R2) responsible for a severe case of hemoglobin H disease

To the editor: Alpha-thalassemia commonly results from deletions or point mutations in one or both alpha-globin genes, located on chromosome 16p13.3. Rarely, alpha-thalassemia is caused by deletions in a region, located 30 to 70 kb upstream of the alpha-globin genes, containing 4 remote,

Complexity of the alpha-globin genotypes identified with thalassemia screening in Sardinia.

α-Thalassemia commonly results from deletions or point mutations in one or both α-globin genes located on chromosome 16p13.3 giving rise to complex and variable genotypes and phenotypes. Rarely, unusual non-deletion defects or atypical deletions down-regulate the expression of the α-globin gene. In the last decade of the program for β-thalassemia carrier screening and genetic counseling in Sardinia, the association of new techniques of molecular biology such as gene sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) to conventional methods has allowed to better define several thalassemic genotypes and the complex variability of the α-cluster with its flanking regions, with a high frequency of different genotypes and compound heterozygosity for two α mutations even in the same family. The exact molecular definition of the genotypes resulting from the interactions among the large number of α-thalassemia determinants and with β-thalassemia, is important for a correct correlation of genotype-phenotype and to prevent underdiagnosis of carrier status which could hamper the effectiveness of a screening program particularly in those regions where a high frequency of hemoglobinopathies is present.

The Problem of Borderline Hemoglobin A2 Levels in the Screening for β-Thalassemia Carriers in Sardinia

Background: The increase in HbA2 is the most important parameter for the identification of thalassemia carriers. However, in routine screening for hemoglobinopathies, some cases are difficult to classify because the level of HbA2 is not typically elevated. In this work, we report the results of a molecular investigation on a cohort of subjects with borderline HbA2. Methods: All subjects with a β-thalassemia carrier partner and a borderline percentage level of HbA2 were investigated for the presence of a pathological mutation in the β-globin gene. All negative subjects were screened for both the KLF1 mutation and the presence of ααα/ or αααα/ alleles. The subjects with reduced MCV and/or MCH were also screened for deletional and nondeletional α-globin gene defects. Results: Various β-globin mutations and KLF1 gene defects are the most common genetic determinants responsible for this phenotype in our population. Conclusion: KLF1 mutations are important in a screening program for hemoglobinopathies. An increase in HbF in association with borderline HbA2 levels is a useful but not exclusive marker that suggests the investigation of this gene. On the basis of our findings, we are able to suggest the molecular procedure to use in a population characterized by a high prevalence of thalassemia carriers.

First Detection of Hb Taybe [α38(C3) or α39(C4) Thr→0 (α1)] in An Italian Child

Hb Taybe [α38(C3) or α39(C4) Thr→0 (α1)] is an unstable hemoglobin (Hb) variant caused by a deletion of a threonine residue at codon 39 of the α1-globin chain. Usually asymptomatic or with minimal hematological abnormalities in the heterozygous state, Hb Taybe becomes clinically evident in compound heterozygosity with α-thalassemia (α-thal) or in homozygous patients. To date, Hb Taybe has been described in Israeli-Arab and Greek individuals. We report, for the first time, a patient with chronic hemolytic anemia due to the presence of Hb Taybe in trans to the α2 initiation codon mutation ATG>ACG in an Italian child. Hb Taybe was not evident at Hb analysis with cellulose acetate electrophoresis and high performance liquid chromatography (HPLC). Globin biosynthetic studies revealed an α/β-globin ratio in the range of β-thal trait. Consequently, an investigation of the α- and β-globin genes was requested in order to avoid missing any rare globin chain variant and to offer accurate genetic counseling.

Two atypical forms of HbH disease in Sardinia

Hemoglobin H disease is usually caused by deletion or inactivation of three α-globin genes, leaving only one α-globin gene intact and active.1 The most frequent defects responsible for HbH disease in Sardinia are the coinheritance of the --Med deletion in one chromosome and the -α3.7 Kb deletion or, less frequently, the α2 initiation codon mutation ATG>ACG (α2NcoI) in the other chromosome.2,3 HbH disease due to deletions including the major upstream regulatory element (MCS-R2) and leaving intact both α-globin genes have also been described.4,5 We report here two new α0 deletions, both located on the short arm of chromosome 16, responsible for HbH disease in two different Sardinian families. These unusual deletions were respectively associated with the common α2NcoI mutation and -α3.7 deletion in trans. Table 1 shows the hematologic and molecular data of the probands and their family members. All patients had severe microcytic anemia (Hb 2.6–9.5 g/dL, MCV 52.0–75.7 fl). Jaundice, spleen enlargement, sporadic hemolytic and aplastic crisis due to B19 parvovirus infection requiring red blood cell transfusions were detected in patients II-1 and II-2 of Family A. A mild thalassemia-like facies was present only in II-1 of the same family. Molecular screening for the most common α-globin gene deletion and non-deletion defects revealed the apparent homozygosity for the α2NcoI mutation in the proband of Family A and in her sister, and the apparent homozygosity for the -α3.7 deletion in the proband of Family B. In spite of that, the α2NcoI mutation was present only in the father of the Family A proband and the -α3.7 deletion was present only in the mother of the Family B proband. Table 1. Hematologic data and genotype of the patients and their parents. In Family A, MLPA analysis, made using MLPA kit (HBA140-B3 MCR-Holland), revealed a deletion of at least 7535 bp beginning in the region between α1-pseudo-globin gene and α2-globin gene and extending to 2.4 kb downstream of α1-globin gene in the proband, in her sister and in their mother. Sequencing analysis of an ~500 bp breakpoint fragment, obtained using specific primers around MLPA deleted probes, allowed us to define the exact deletion breakpoint at position 161276/7 (5′) and 170485/6 (3′). This deletion removed a region of 9209 nt involving both α-globin genes and part of the first exon of the θ gene. In addition, an insertion of six nucleotides (ATTAGT) at position 161216 before the 5′ breakpoint was detected. No orphan sequence was found. The 5′ breakpoint is shifted 2 nt up and the 3′ breakpoint is shifted 1032 nt down, as compared to the breakpoints of the α0 thalassemia deletion found in a recently reported Dutch family.6 In Family B, MLPA analysis revealed a larger deletion which removes all the MLPA probes specific for the sub-telomeric region, including an α-globin gene cluster with all regulatory elements, in the proband and in her father. CGH-array analysis with oligonucleotides (8x60K Agilent Technologies) and SNP genotyping allowed us to define the 3′ breakpoint between the 4th exon of the NME4 gene and the IVSII of the DECR2 gene (389660 and 395647 coordinates) (Figure 1). Figure 1. Schematic representation of the short arm of chromosome 16 (16p13.3) and of the α0 deletions in the two families. The α-globin regulatory region (MCS-R 1 to 4) is indicated as black dots. Black bars represent deleted DNA regions. White ... The greater severity of the α2NcoI non-deletion defect as compared to the -α3.7 deletion in trans to α0 deletions, could be the reason for the different phenotypes in the HbH patients of the two families.2 The different size of alpha0 deletions and the loss of genes located in the deleted region in Family B do not seem to interfere in the determination of specific phenotype. Several large deletions involving the α-globin gene cluster have been recently described.7–10 Although these deletions also remove other genes, affected heterozygotes appear phenotypically normal, apart from α-thalassemia carrier phenotype; however, an HbH patient with a telomeric deletion of ~285 kb associated with the common -α3.7 deletion in trans presented scoliosis, the severity of which remains unexplained.8 A region on chromosome 16p for which haploinsufficiency leads to mental retardation typical of ATR16 has been narrowed down to a region ~0.9 and 1.5–1.7 Mb from telomere. Alu-family repeats, frequent in the genome and particularly common in and around the α-globin gene cluster, facilitate DNA strand exchanges during replication and non-homologous recombinations which are a frequent cause of α0 deletions.9–11 In addition to the common conventional molecular techniques, recent alternative methods, such as MLPA and CGH, become essential for a correct α-globin genotype definition. The exact identification of uncommon and unknown alpha deletion defects, although rare, allows appropriate genetic counselling to be offered to couples at risk for HbH disease or hemoglobin Bart’s hydrops fetalis syndrome, especially in Sardinia were small isolated communities at risk can still be found.

Red cell pyruvate kinase deficiency in Southern Sardinia.

Pyruvate kinase (PK) deficiency is the most frequent red cell enzymatic defect responsible for hereditary non-spherocytic hemolytic anemia. The clinical picture is quite variable and the reasons of this variability have been only partially clarified. We report the clinical description and the extended molecular analysis in 3 PK deficient patients with clinical phenotype of variable severity. We studied the clinical and hematological aspects of 3 patients and analyzed the following genes: pyruvate kinase-R, glucose-6-phosphate-dehydrogenase, α-globin, uridindiphosphoglucuronil transferase and HFE. One patient (A) with a severe clinical picture resulted homozygote for exon 8 nt994A substitution, the other 2 (brothers) were compound heterozygotes for exon 8 nt994A and exon 11 nt1456T mutation. One of the two brothers with a more severe phenotype coinherited also had G6PD deficiency, while both had microcytosis due to the homozygosity for the non-deletional form of α-thalassemia ATG→ACG substitution at the initiation codon of the alpha2 globin gene. Our results suggest that extended molecular analysis is useful for studying how several interacting gene mutations contribute to the clinical variability of pyruvate kinase deficiency.