Suppression of Hb Bart's to improve tissue oxygenation and fetal development in homozygous alpha-thalassemia.

Intra-uterine reduction of Hb Bart's only reached with exchange transfusions.

Molecular analysis of complex cases of alpha- and beta-thalassemia in Mexican mestizo patients with microcytosis and hypochromia reveals two novel alpha(0) -thalassemia deletions - -(Mex1) and - -(Mex2).

INTRODUCTION: Alpha-thalassemia (α-thal) is a common monogenic disorder worldwide. In mixed ethnic populations, α-thal and beta-thalassemia (ß-thal) can be expected, sometimes giving complex phenotypes, which without molecular analysis are not easily explained. We performed the molecular identification of α- and ß-thal alleles in 51 Mexican patients with microcytosis, hypochromia, and normal or low levels of HbA2 . METHODS: Common deletional alleles (-α(3.7) , -α(4.2) , - -(SEA) , - -(MED) , - -(FIL) , - -(THAI) , -α(20.5) ) and α-triplication were studied by gap-PCR and nondeletional alleles (α(IVSI) ((-5nt)) , α2 (NcoI) , α1 (NcoI) ) by ARMS. ß-thal alleles Cd39 (C>T), IVS1:1 (G>A), IVS1:110 (G>A), and Spanish δß-thal were also investigated. DNA sequencing was performed on HBA2, HBA1, and HBB genes. Negative samples were subjected to MLPA. RESULTS: In 35 subjects, we identified the mutations, -α(3.7) , - -(SEA) , - -(FIL) , α(IVSI) ((-5nt)) , and ααα(anti3.7) and two novel deletion alleles - -(Mex1) (6.8-8.9 kb) and - -(Mex2) (77.6-135.7 kb). Four individuals also had a ß-thal allele (Cd39/IVS1:110). No α-thal alleles were observed in 16 subjects, but three had a ß-thal mutation Cd39, IVS1:110, and Spanish δß-thal. CONCLUSION: α-thal is relatively common in Mexican patients, the combination with ß-thal is sometimes unexpected, and this underlines the importance of performing molecular analysis for both α- and ß-genes defects in patients showing microcytic hypochromic anemia.

Sickle cell anemia and α-thalassemia: a modulating factor in homozygous HbS/S patients in Oman.

We report the general phenotype severity and the hematological presentation in a cohort of 125 sickle cell anemia (SCA) patients with identical homozygous HbS/S genotype and categorized by identical ß(S) haplotype, both with and without alpha thalassemia. No clear general phenotype correlation was found when patients were compared regardless of the haplotype but overall, patients with homozygous alpha thalassemia (α-/α-) had the highest Hb, HCT, RBC and the lowest MCV, MCH and MCHC levels. When patients with identical haplotype were compared, the mildest hematological and clinical conditions were observed in patients of the Asian/Asian haplotype, also known as Arab-Indian haplotype, and carriers of α-thalassemia, suggesting an additional ameliorating effect of alpha thalassemia. In conclusion, our results show that alpha thalassemia improves the hematological conditions but amelioration of the general disease severity is only noticed when compared in cohorts of the same haplotype.

Aberrant glycated haemoglobin (HbA1c) results leading to haemoglobinopathy diagnosis in four Belgian patients.

INTRODUCTION: We report four cases in which haemoglobinopathy screening was triggered following aberrant HbA1c analysis. Either the HbA1c assay was unable to produce a quantifiable result or it showed the presence of an extra fraction and/or the result was discordant with the clinical context. CASE REPORT: In the reported four patients, all from Caucasian, Belgian descent, Hb analysis was performed using cation-exchange high performance liquid chromatography. If necessary, additional Hb electrophoresis was carried out to establish a preliminary (biochemical) diagnosis. Definitive diagnosis was obtained for every sample through DNA-analysis. Three patients were carriers of Hb J-Toronto and one of Hb Stanleyville-II. DISCUSSION: This report underlines the importance of correct interpretation of HbA1c results to avoid mismanagement of (diabetic) patients. Since neither the RBC indices, the clinical context, nor the ethnicity of these patients was suspicious for an underlying haemoglobinopathy, the aberrant HbA1c result was the only indicator for further investigation. Laboratory personnel and clinicians should be aware of the possibility of uncommon, sometimes clinically unsuspected, Hb variants to cause aberrant HbA1c values, even in populations with low prevalence for haemoglobinopathies. Further analysis should be prompted to obtain definitive diagnosis. Alternative methods for monitoring glycaemic control should be used.

Molecular diagnostics of the HBB gene in an Omani cohort using bench-top DNA Ion Torrent PGM technology.

Hemoglobinopathies, such as sickle cell disease (SCD) and beta-thalassemia major (TM), are severe diseases and the most common autosomal recessive condition worldwide and in particular in Oman. Early screening and diagnosis of carriers are the key for primary prevention. Once a country-wide population screening program is mandated by law, a sequencing technology that can rapidly confirm or identify disease-causing mutations for a large number of patients in a short period of time will be necessary. While Sanger sequencing is the standard protocol for molecular diagnosis, next generation sequencing starts to become available to reference laboratories. Using the Ion Torrent PGM sequencer, we have analyzed a cohort of 297 unrelated Omani cases and reliably identified mutations in the beta-globin (HBB) gene. Our model study has shown that Ion Torrent PGM can rapidly sequence such a small gene in a large number of samples using a barcoded uni-directional or bi-directional sequence methodology, reducing cost, workload and providing accurate diagnosis. Based on our results we believe that the Ion Torrent PGM sequencing platform, able to analyze hundreds of patients simultaneously for a single disease gene can be a valid molecular screening alternative to ABI sequencing in the diagnosis of hemoglobinopathies and other genetic disorders in the near future.

State of the art and new developments in molecular diagnostics for hemoglobinopathies in multiethnic societies.

For detecting carriers of thalassemia traits, the basic part of diagnostics consists of measurement of the hematological indices followed by mostly automatic separation and measurement of the Hb fractions, while direct Hb separation either on high pressure liquid chromatography or capillary electrophoresis is sufficient to putatively identify carriers of the common Hb variants like HbS, C, E, D, and O-Arab. A putative positive result is reported together with an advice for parents, partner, or family analysis. For couples, presumed at-risk confirmation at the DNA level is essential. In general, this part of diagnostics is done in specialized centers provided with sufficient experience and the technical tools needed to combine hematological and biochemical interpretation with identification of the mutations at the molecular level. State-of-the-art tools are usually available in centers that also provide prenatal diagnosis and should consist of gap-PCR for the common deletions, direct DNA sequencing for all kind of point-mutations and the capacity to uncover novel or rare mutations or disease mechanisms. New developments are MLPA for large and eventually unknown deletion defects and microarray technology for fine mapping and primer design for breakpoint analysis. Gap-PCR primers designed in the region flanking the deletion breakpoints can subsequently be used to facilitate carrier detection of uncommon deletions in family members or isolated populations in laboratories where no microarray technology or MLPA is available.

Hb Haaglanden: a new nonsickling ß7Glu>Val variant. Consequences for basic diagnostics, screening, and risk assessment when dealing with HbS-like variants.

INTRODUCTION: To report a new hemoglobin variant undistinguishable from the common HbS on HPLC. To show the efficiency of the simplest confirmation method for HbS and to discuss the implications that may occur if HbS-like variants are wrongly reported as HbS. METHODS: Basic hematology, separation and measurement of the Hb fractions, 'sickle test,' and molecular analysis. RESULTS: The abnormal Hb fractions were eluting in the HbS window on HPLC, sickle test was however negative, and DNA sequencing of the beta globin gene revealed an unclassified variant HBBc.23A>T, p.Glu8Val in heterozygous form. CONCLUSIONS: Although the amino acid substitution of this new variant is identical to that of HbS and shifted of a single amino acid position, no polymerization occurs in vitro. The sickle test is a valid method to confirm or exclude HbS trait in individual cases. Whenever the case is part of a possible couple at risk, then one has to use full DNA analysis in both partners not to miss hidden concomitant defects important for genetic risk predictions.

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.

Iron depletion: an ameliorating factor for sickle cell disease?

We report some observations from our laboratory practice that might be important for the treatment of sickle cell disease (SCD). We describe data from two cases indicating that iron depletion might have a beneficial effect diminishing the formation of HbS in favor of HbF, possibly reducing the severity of the disease. We believe that it would be worthwhile to monitor the course of the disease comparing cases with identical genotypes with and without iron depletion, and we advise to consider chelation therapy to reduce iron overload in patients with SCD.

Occurrence of common and rare δ-globin gene defects in two multiethnic populations: thirteen new mutations and the significance of δ-globin gene defects in ß-thalassemia diagnostics.

INTRODUCTION: The aim of this review is to study the frequency of common and the occurrence of rare and novel mutations of the delta-globin gene and of Hb Lepore defects that might interfere with thalassemia diagnostics and to report the rationale of HbA2 estimation in the presence of delta- or alpha-gene mutations. METHODS: A total of 135 cases suspected to have a delta-globin gene defect collected in a diagnostic center in the USA and in a reference laboratory in the Netherlands were characterized by molecular analysis. RESULTS: Hb B2 was found at a frequency of at least 0.5% in the USA and 0.87% in the Netherlands. Known variants such as Hb A2-Babinga, Hb A2-Sphakia, Hb A2-Fitzroy, Hb A2-Flatbush, Hb A2-NYU, Hb A2-Grovetown, HbA2-Yialousa, Hb A2-Indonesia and several delta-thalassemia mutations were found together with 13 new mutations and two new polymorphisms, while Hb Lepores were regularly observed. CONCLUSION: HbA2 mutations either structurally stable and visible or undetectable because of a thalassemia effect or instability are clinically asymptomatic but may compromise the diagnosis of beta-thalassemia minor. Stable mutations result in two HbA2 fractions of about half of the expected value. Expression defects are undetectable as a protein fraction but reduce the amount of HbA2 by half.

Newborn screening for hemoglobinopathies using capillary electrophoresis technology: Testing the Capillarys Neonat Fast Hb device.

OBJECTIVES: To diagnose hemoglobinopathies in newborns by separating and measuring the Hb fractions on high throughput capillary electrophoresis. To test and validate the Capillarys Neonat Fast Hb device (Sebia) on fresh and dry blood samples. DESIGN AND METHODS: The Hb fractions in 1.600 cord blood samples from the multi ethnic Dutch population were separated and measured. Further, the sensitivity, specificity and reproducibility of the device in detecting abnormalities and measuring the Hb fractions were estimated. RESULTS: The apparatus separated all significant Hb fractions that should be detected during newborn screening (NBS) with 100% sensitivity. The reproducibility of the migrations guaranteed putative specificity for the few relevant frequent variants observed (HbS, C, and E). The estimation of the HbA and F fractions proved reliable using a well-designed integration mode. DISCUSSION: Due to the limited number of samples no cases with sickle cell disease or ß-thalassemia major were found in this cohort. However, the heterozygous state for the common variants associated with these diseases was clearly recognizable. The measurements were sufficiently precise to recognize sickle cell disease, ß-thalassemia major and intermedia and to identify carriers including possible ß-thalassemia. Therefore, Capillarys Neonat Fast Hb (Sebia) can be considered as a valid instrument for NBS of the Hemoglobinopathies on fresh and dry blood samples.

Evaluating five dedicated automatic devices for haemoglobinopathy diagnostics in multi-ethnic populations.

We have tested five haemoglobin (Hb) separation apparatuses, dedicated to haemoglobinopathy diagnostics. These are the four high performance liquid chromatography devices: VARIANT II, HA 8160, G7, Ultra(2) and the Capillary Electrophoresis apparatus from Sebia. In the first place, we focussed on the capacity of all apparatuses to detect the most common structural variants relevant for public health, these being HbS, HbC, HbE, HbD-Punjab and HbO-Arab. We then compared how the high HbA(2)beta-thalassaemia carriers were identified. All apparatuses were able to identify carriers of these traits with the expected sensitivity and specificity. With the primary goal of a high degree of conformity in basic diagnostics of haemoglobinopathies, we present the interpretation and the significance of the results on all apparatuses, and we comment on the unavoidable problems and solutions.

Segmental duplications involving the alpha-globin gene cluster are causing beta-thalassemia intermedia phenotypes in beta-thalassemia heterozygous patients.

We describe two cases of simple heterozygosity for the common beta degrees -thalassemia mutation beta 39 (C-->T), both presenting with a thalassemia intermedia phenotype. In both cases synergic effect deriving from membrane defects or red cell enzyme deficiencies were excluded. In one case a triplication of the alpha-globin genes was found which did not justify the severity of the transfusion-dependent phenotype. Multiplex ligation-dependent probe amplification (MLPA) analysis of the alpha-globin gene cluster revealed two new rearrangements, consisting of a full duplication of the alpha-globin genes locus including the upstream regulatory element. In one case the duplication was in the presence of the common anti-alpha(3.7) triplication in trans, resulting in a total of 7 active alpha-globin genes. In the other case the duplicated allele and the normal allele in trans resulted into a total of 6 active alpha-globin genes. We report the clinical and hematological data and the molecular analysis and discuss the occurrence of alpha-globin genes duplication defects in cases of beta-thalassemia heterozygotes with thalassemia intermedia phenotypes.

A new deletion defect leading to alpha-thalassaemia in a large Dutch Caucasian family.

alpha-thalassaemia is a common inherited haemoglobin disorder that can cause only mild symptoms in carriers and is often either not diagnosed or mistaken for iron deficiency anaemia in the Netherlands. Although considered rare in North-Europeans, we also regularly observe common and rare defects in this population. It is important to be alert for the mild symptoms of these carriers because compound heterozygous and homozygous combinations can result in intermediate, severe or fatal disease in the progeny of healthy carriers. Using a new technical application, a novel alpha degrees -thalassaemia deletion was recently detected in our laboratories in a propositus of a large Dutch Caucasian family. We report the phenotypic and molecular study of this new form of alpha(o)-thalassaemia (called--(OH)alpha-thalassaemia deletion), which was observed in 10 of the 19 individuals studied in the index family. Our results indicate that the frequency of these unsuspected alpha(o)-thalassaemia defects is probably underestimated in the Netherlands.

[Prevention of hereditary haemoglobinopathies in The Netherlands]. / Preventie van erfelijke hemoglobinopathieën in Nederland.

Haemoglobinopathies are defects that interfere with the synthesis of haemoglobin. Parents who are both healthy carriers (heterozygotes) may produce severely affected homozygous or compound heterozygous children who can be treated only symptomatically. Offering Dutch couples at risk the possibility of primary prevention is a matter of good healthcare and its organisation deserves priority. The prevention of haemoglobinopathy is based on the provision of information to potential carriers, carrier diagnostics and counselling, services that can in principle be provided by existing public health care institutions. Three moments at which the prevention of recessive diseases can be offered are the phase of preconception, the phase of early pregnancy (prospective primary prevention) and the postnatal phase at the time of a following pregnancy (retrospective primary prevention). By providing basic information and requesting laboratory tests for the diagnosis ofhaemoglobinopathy, the general practitioner plays a crucial role in primary prevention.

Nine unknown rearrangements in 16p13.3 and 11p15.4 causing alpha- and beta-thalassaemia characterised by high resolution multiplex ligation-dependent probe amplification.

BACKGROUND: Approximately 80% of the alpha- and 10% of the beta-thalassaemias are caused by genomic deletions involving the alpha- and beta-globin gene clusters on chromosomes 16p13.3 and 11p15.5, respectively. Gap-PCR, Southern blot analysis, and fluorescent in situ hybridisation are commonly used to identify these deletions; however, many deletions go undetected using conventional techniques. METHODS: Patient samples for which no abnormalities had been found using conventional DNA techniques were analysed by a three colour multiplex ligation-dependent probe amplification assay. Two sets of 35 and 50 probes, covering a region of 700 kb of the alpha- and 500 kb of the beta-globin gene cluster, respectively, were designed to detect rearrangements in the alpha- and beta-globin gene clusters. RESULTS: In 19 out of 38 patient samples, we found 11 different alpha-thalassaemia deletions, six of which were not previously described. Two novel deletions leaving the alpha-globin gene cluster intact were found to cause a complete downregulation of the downstream alpha-genes. Similarly, 31 out of 51 patient samples were found to carry 10 different deletions involving the beta-globin gene cluster, three of which were not previously described. One involves the deletion of the locus control region leaving the beta-globin gene cluster intact. CONCLUSIONS: These deletions, which are not easily detected by conventional techniques, may have clinical implications during pregnancy ranging from mild to life threatening microcytic haemolytic anaemia in neonates. The approach as described here is a rapid and sensitive method for high resolution analysis of the globin gene clusters and for any region of the genome.