Relationship between neonatal screening results by HPLC and the number of α-thalassaemia gene mutations; consequences for the cut-off value.

OBJECTIVES: To evaluate the relationship between FAST peak percentage by adapted Bio-Rad Vnbs analysis using the valley-to-valley integration and genotypes with the aim to improve differentiation between severe α-thalassaemia forms (HbH disease) and the milder disease types. METHOD: DNA analysis for α-thalassaemia was performed on 91 dried blood spot samples presenting normal and elevated FAST peak levels, selected during three years of Dutch national newborn screening. RESULTS: Significant differences were found between samples with and without α-thalassaemia mutations, regardless of the genetic profiles. No significant difference was demonstrated between HPLC in -α/αα and -α/-α, between -α/-α and - -/αα and between - -/αα and - -/-α genotypes. CONCLUSION: This study confirms that the percentage HbBart's, as depicted by the FAST peak, is only a relative indication for the number of α genes affected in α-thalassaemia. Based on the data obtained using the modified Bio-Rad Vnbs software, we adopted a cut-off value of 22.5% to discriminate between possible severe α-thalassaemia or HbH disease and other α-thalassaemia phenotypes. Retrospectively, if this cut-off value was utilized during this initial three-year period of neonatal screening, the positive predictive value would have been 0.030 instead of 0.014.

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.

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 (- -/-alpha) and 36 (14%) patients had the non-deletional type (- -/alpha(ND)alpha). 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 mug/l and in adults, was 192 +/- 180 mug/l in females and 363 +/- 303 mug/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.

Cholelithiasis and Gilbert's syndrome in homozygous beta-thalassaemia.

Cholelithiasis has been reported with a variable incidence in homozygous beta-thalassaemia, the reasons for which have only partially been defined. Disease-associated factors or specific modifier genes may be implicated. We assessed the prevalence of cholelithiasis and the effect of co-inherited Gilbert's syndrome genotype on its development in 261 thalassaemia major (TM) and 35 thalassaemia intermedia (TI) patients. Cholelithiasis was found in 20.3% of TM and in 57.1% of TI patients. Its incidence was higher (P < 0.05) in patients homozygous for the (TA7) motif in the promoter of the UGT1-A1 gene, the genotype associated with Gilbert's syndrome, which seems to be a risk factor for the development of gallstones in TM and TI patients.

Hyperbilirubinemia, glucose-6-phosphate-dehydrogenase deficiency and Gilbert's syndrome.

The pathogenesis of neonatal hyperbilirubinemia has not yet been completely defined in normal and glucose-6-phosphate-dehydrogenase (G6PD)-deficient newborns. The recent identification of a variant promoter in the gene encoding for the bilirubin uridine-diphosphoglucuronosyl-transferase (UGT-1 A) associated with Gilbert's syndrome, allowed us to explore whether the presence of this variant promoter is a risk factor for the development of neonatal hyperbilirubinemia in normal newborns and in association with G6PD deficiency. We found that the variant (TA)7/(TA)7 promoter shows no statistically significant difference in normal or G6PD-deficient newborns developing severe hyperbilirubinemia and in control subjects from the same population. This finding indicates that the variant promoter of UGT-1 A does not contribute to the development of hyperbilirubinemia in the newborn.

Heterozygous beta-thalassemia with thalassemia intermedia phenotype.

In this study we investigated the molecular bases of the beta-thalassemia intermedia phenotype in six patients belonging to two unrelated families of Sardinian descent. Sequence analysis of the beta globin gene from these patients detected, as the sole abnormality, the heterozygosity for the codon 39 nonsense mutation. The A gamma and Ggamma promoters as well as the HS2 and HS3 core sequences of the beta globin LCR from these patients, did not show any non-polymorphic nucleotide variation from the consensus sequence. One of the parents was heterozygous for codon 39 nonsense mutation but showed the beta-thalassemia carrier phenotype; the other was hematologically normal and had an entirely normal beta globin gene sequence. In both families, other members showed the typical hematological phenotype, clinically silent, of heterozygous beta thalassemia. To explain the thalassemia intermedia phenotype, we postulated the presence of an unknown molecular defect interacting with the beta globin gene mutation. Haplotype analysis excluded that this postulated defect lies in the beta globin gene cluster.

Hyperbilirubinaemia in heterozygous beta-thalassaemia is related to co-inherited Gilbert's syndrome.

The reasons why heterozygotes for beta-thalassaemia have considerable variation in serum bilirubin levels are unknown. High levels of bilirubin could be related to the co-inherited Gilbert's syndrome, determined either by mutations of the coding region or by variation in the A(TA)nTAA motif of the promoter of the bilirubin UDP-glucuronosyltransferase gene (UGT-1). We sequenced the coding and the promoter region of UGT-1A or characterized the A(TA)nTAA motif of the promoter by denaturing gel electrophoresis of radioactive amplified products. The results were correlated with bilirubin levels in 49 beta-thalassaemia heterozygotes for codon 39 (CAG --> TAG) nonsense mutation. 21 normal individuals and 32 unrelated patients with Gilbert's syndrome served as controls. The coding sequence region of the UGT-1A was normal. Five beta-thalassaemia heterozygotes, who were homozygous for the extra (TA) bases in the A(TA)nTAA element of the promoter of UGT-1A, the configuration present in homozygosity in Gilbert's syndrome, had higher bilirubin levels compared to those with the (TA)6/(TA)7 or (TA)6/(TA)6 configurations. In the group of 32 patients with Gilbert's syndrome, 31 of whom had the (TA)7/(TA)7 configuration, we detected 14 heterozygotes for beta-thalassaemia, a figure much higher than predicted on the basis of the carrier rate. Homozygosity for the (TA)7 motif, the typical promoter configuration of Gilbert's syndrome, is one of the factors determining hyperbilirubinaemia in heterozygous beta-thalassaemia.

Hb Puttelange [beta 140(H19)Ala-->Val] in an Italian man with polycythemia.

Hb Puttelange [beta 140(H18)Ala-->Val] was found in a 51-year-old Italian man who had mild polycythemia. The variant eluted from ion exchange high performance liquid chromatography at a position between Hb A and Hb A2. It comprised approximately 34% of the total hemoglobin, was weakly unstable and exhibited an increased oxygen affinity. Amplification of the beta-globin exons and nucleotide sequencing revealed a heterozygosity for a GCC-->GTC mutation in codon 140 corresponding to an Ala-->Val replacement. This substitution accounts for the altered functional properties, probably by producing indirect perturbation of the 2 3-diphosphoglycerate pocket through the nearby lysine residue at beta 82(EF6).

Evaluation of an automatic HPLC analyser for thalassemia and haemoglobin variants screening.

In this paper the authors report the evolution of a new automatic HPLC analyser for screening haemoglobinopathies. HbA(2) and F determinations are accurate and reproducible. The analysis time is short (6.5 min) and there is a good separation between the HbA(2) values of beta-thalassemia carriers from normals and alpha-thalassemia carriers, with no overlap between these groups. In addition, the system is also able to detect and quantitate most of the haemoglobin variants, particularly those (HbS, HbC, HbE and Hb Lepore) able to interact with beta-thalassemia and could make haemoglobin electrophoresis unnecessary in all samples. The ease of operation and the limited technical work make this system especially suitable for laboratories with a high workload and allow the cost of screening to be reduced.

Genotype of subjects with borderline hemoglobin A2 levels: implication for beta-thalassemia carrier screening.

In this study, we have defined by molecular analysis, the alpha, beta, and delta globin genotype in a group of individuals with normal or thal-like red cell indices but borderline hemoglobin (Hb)A2 levels, who were identified in a program for beta-thal carrier screening. In 37 of 125 individuals with borderline HbA2 levels, we detected a molecular defect in the beta, in both the delta and the beta, or in the alpha globin gene. Specifically seven of these subjects were carriers of the -101 C T mutation, ten of the IVSI nt6 T C mutation, 16 were double heterozygotes for delta and beta thal, and two had the triple alpha globin gene and two the single alpha globin gene deletion. From these results, we may conclude that subjects with borderline HbA2, particularly when they marry a typical beta-thal carrier, should be extensively investigated in order not to miss heterozygous beta-thalassemia.

Normal individuals with high Hb A2 levels.

Increased haemoglobin (Hb) A2 levels associated with reduced mean corpuscular volume (MCV) and Hb content per cell (MCH) are the most typical features of heterozygous beta thalassaemia. However, double heterozygotes for alpha and beta thalassaemia may have normal MCV and MCH but Hb A2 always in the carrier range. In this report we describe two Sardinian families who have increased Hb A2 levels, normal red blood cell indices and normal globin chain synthesis and in whom DNA sequence analysis of beta and delta globin genes did not reveal any abnormality. Our findings demonstrate the existence of a genetic trait not resulting from a defect of the beta globin gene cluster, transmitted in a dominant manner and manifested as isolated increase of Hb A2.

Hemoglobin Cagliari (beta 60 [E4] Val----Glu): a novel unstable thalassemic hemoglobinopathy.

This report describes a patient with thalassemia intermedia-like phenotype born to normal parents in whom globin gene sequencing detected a novel abnormal hemoglobin (Hb) due to a T to A substitution at codon 60 of the beta-globin gene arising as a de novo mutation. Normal sequences were detected at the homologous beta-globin locus. This mutation results in the substitution of a polar (glutamic acid) for a nonpolar (valine) residue near the corner of the heme pocket of the beta-globin chain. The novel variant has been designated Hb Cagliari, from the place of birth of the propositus. Kinetics of globin synthesis performed following splenectomy suggest that this new Hb variant is synthesized at a near normal rate but undergoes rapid breakdown. The extreme lability of the variant explains the clinical and hematologic picture characterized by marked ineffective erythropoiesis, thalassemia-like bone changes, iron overload, high proportion of Hb F in the peripheral blood, reduced beta/alpha-globin chain synthesis ratio in peripheral blood reticulocytes, and absence of the abnormal Hb in peripheral blood at extensive protein structural analysis before splenectomy. This case indicates that a thalassemic hemoglobinopathy should be suspected in the presence of a patient with a thalassemia intermedia-like phenotype born to normal parents, even when protein structural analysis fails to detect an abnormal Hb. DNA sequencing may allow to define the mutation, thus making the proper diagnosis.

The effect of the beta thalassemia mutation on the clinical severity of the sickle beta thalassemia syndrome.

In this study, we have defined the beta thalassemia mutation and characterized the beta globin haplotype and the alpha globin gene arrangement in a group of patients of Sicilian descent with beta (s)/beta thalassemia. We found that those patients carrying a beta(+) thalassemia mutation associated with a moderate reduction of beta chain synthesis (beta(+) IVS-1 nt 6) have normal or reduced Hb levels and mild to moderate clinical manifestations, as defined by the number of hospital admission and sickle cell crises per year. Those patients carrying a beta(+) thalassemia mutation associated with a severe reduction of beta chain synthesis (beta(+) IVS-1 nt 110) have a disease of moderate severity. In those carrying a beta(0) thalassemia gene the disease was clinically very heterogeneous, ranging in severity from mild to severe with no difference related to the type of mutation [beta(0) 39, beta(0) IVS-1 nt 1, beta(0) IVS-2 nt 1, beta(0) 6 (-1bp)]. In this last group of patients part of the clinical variability may be attributed to the HbF levels, which were higher in those with mild to moderate clinical severity.

Molecular characterization of a normal Hb A2 beta-thalassaemia determinant in a Sardinian family.

In this study we have carried out haplotype analysis at the beta-globin gene cluster and defined the beta-thalassaemia mutations in a large Sardinian family, ascertained through a proband with thalassaemia major, in which several members were carriers of a beta-thalassaemia allele characterized by microcytosis, hypochromia and normal Hb A2 levels (type 2 normal Hb A2 heterozygous beta-thalassemia). The proband was a compound heterozygote for the beta zero 39 and the beta + IVS-2, nt 745 mutations and all the beta-thalassaemia heterozygotes with normal Hb A2 showed the beta + IVS-2, nt 745 mutation, always associated with haplotype VII. Because of the consistent association of a specific beta-thalassaemia mutation and normal Hb A2 levels, we postulate that this beta-thalassaemia chromosome carries a delta gene (delta-thalassaemia) which is unable to increase the delta-globin output in response to beta-thalassaemia.

Alpha thalassaemia in Sardinian newborns.

In this study we describe the correlation between the haematological parameters (red cell indices and Hb Bart's levels) and the alpha-globin genotype in Sardinian newborns. Increased Hb Bart's levels at birth always indicates alpha-thalassaemia, either of the deletion or non-deletion variety. Infants with two alpha-globin genes deleted (- alpha/- alpha and --/ alpha alpha genotypes) had microcytosis, low MCH and Hb Bart's in the 2.0-7.1% range. A minority (38.9%) of infants with the (- alpha/ alpha alpha) globin genotype had detectable Hb Bart's, in the 0.78-2.5% range, frequently associated with minimal microcytosis while the remainder (61.1%) were completely silent. Infants carriers of a non-deletion type of alpha-thalassaemia showed Hb Bart's levels within the range found in the (- alpha / alpha alpha) genotype. The association of heterozygous beta 0-thalassemia seems to have no effect on the expression of any of these alpha-thalassaemia lesions at birth.