Does size matter? Two new deletions in the HBB gene cause ß0-thalassemia.

Most ß-thalassemias are caused by mutations involving one or a limited number of nucleotides within the gene or its adjacent regions. They can be substitutions or deletions; in these cases, the loss ranges from a single nucleotide to even the entire HBB gene, so we wonder if the phenotype is due to the size of the deletion or the location of the mutation. To clarify this, we present two new deletions in the ß-globin gene that cause ß0-thalassemia. The hematological parameters were determined with an automated cell counter; the Hb A2 and Hb F levels were measured by performance liquid chromatography. Hemoglobins were analyzed by capillary zone electrophoresis (Sebia Capillarys Flex system) and ion-exchange HPLC (BioRad Variant II ß-thalassemia Short Program). Molecular characterization was performed by automatic Sanger sequencing. The screening of common α-thalassemia point mutations and deletions in the world (21 in total) were carried out using multiplex PCR followed by reverse-hybridization with a commercial Alpha-Globin StripAssay kit. We have characterized two new mutations-(1) 1-bp deletion [CD61/62(-G)] [HBB:c.186_187delG], (2) 105-bp deletion [IVS-2-nt767-CD111] [HBB:c.316-84_333del]-and we have described, for first time in Spain, the 25-bp deletion [ß nts 252 - 276 deleted] [HBB:c.93-22_95del] mutation. These mutations were classified as pathogenic by UniProt Variants confirmed according to the American College of Medical Genetics and Genomics guidelines. These mutations present a phenotype compatible with ß0-thalassemia, supported by hematological parameters that correlate the degree of reduction in the synthesis of the ß-globin chain. Identification of this type of mutation is important for genetic counselling of partners where both are carriers, so that they are aware of the genetic risk of having affected children, allowing them to take an informed decision about their reproductive choices.

ß-Thalassemia Intermedia: Interaction of α-Globin Gene Triplication With ß-thalassemia Heterozygous in Spain.

Objectives: To verify with hematimetric data that the diagnosis and clinical grade of ß-TI can be established when a triplication of alpha genes (αααanti 3.7) and heterozygous ß-thalassemia coexist. Materials and Methods: Retrospective study in which 73 patients of Caucasian origin participated, who simultaneously showed a triplication or quadruplication of genes α and ß-thalassemia.Screening for the most frequent α-thalassemia mutations as well as gene triplication (αααanti 3.7) was carried out by multiplex PCR followed by reverse hybridization with a commercial Alpha-Globin StripAssay kit and confirmed by MLPA (Multiplex ligation-dependent probe amplification). The molecular diagnosis of ß-thalassemia was carried out by automatic sequencing according to the Sanger method. Results: The genotypes have been classified into three groups according to the number of α globin genes and the severity of the alteration in the ß globin gene. All had a mutation in the HBB gene (ß0-thalassemia, ß+-thalassemia severe, and ß+-thalassemia mild). Group I patients who have coherent 6 α genes and groups II and III with 5 α globin genes. In group III, the patients were carriers of mutations affecting the ß and δ globin genes. The most significant hematological parameters were hemoglobin levels, MCV, RDW, and the percentage of Hb F. Conclusions: In group I, regardless of the distribution of the 6 α globin genes, homozygous triplication (ααα/ααα) or heterozygous quadruplication (αααα/αα), the association with heterozygous ß-thalassemia results in severe to moderate anemia that may or may not require transfusion therapy, is the severity of the HBB gene mutation that would determine the clinical variation. Group II patients phenotypically behaved like mild thalassemia intermedia, except for one case that presented thalassemic trait because it also presented an associated α-thalassemia (ααα/-α3.7). Finally, group III patients behaved as a thalassemic trait since all were carriers of mutations that increase the overexpression of γ genes.

Genetic variation patterns of ß-thalassemia in Western Andalusia (Spain) reveal a structure of specific mutations within the Iberian Peninsula.

BACKGROUND: Analyses of the genomic variation in the western Mediterranean population are being used to reveal its evolutionary history and to understand the molecular basis of particular diseases. AIM: To observe the ß-thalassemia mutational spectrum in western Andalusia, Spain, in the context of the Mediterranean. In addition, associations between disease and neutral gene variants within the ß-globin gene (HBB) were also evaluated. SUBJECTS AND METHODS: This study included 63 unrelated individuals diagnosed with ß-thalassemia. In addition, 97 unrelated, healthy subjects of the same territory were also analysed as proxies of the normal genetic background. Allele associations and population genetic structure analyses were performed using different methodologies. RESULTS: Data have revealed a rather restricted spectrum of ß-thalassemia mutations in the analysed sample. Although the detected variants fit well with the Mediterranean pattern, certain singularities support a structure of some specific ß-thalassemia alleles. The IVSI-1 (G > A) shows a strong regionalisation. The spatial correlogram revealed a typically narrow wave structure, presumably linked to genetic isolation and genetic drift. CONCLUSIONS: The long history of endemic malaria in the study territory, the rather high consanguinity rates among its autochthonous population, and other demographic features have been used here to understand the western Andalusian ß-thalassemia molecular portrait.

Hb Maruchi [α165 (E14) Ala>Pro; HBA1: c.196G>C]: A new thalassemia hemoglobinopathy related to the alpha1 globin gene.

OBJECTIVE: To describe a new mutation causing alpha thalassemia and its mechanism of action. DESIGN AND METHODS: The propositus was a 37-year-old man who presented maintained microcytosis without iron deficiency. Molecular characterization was undertaken using automatic sequencing after testing negative for the most frequent α-globin mutations by multiplex PCR followed by reverse-hybridization. RESULTS: The mutation is a single base substitution at codon 65 of the α1 globin gene [α65(E14) Ala>Pro; HBA1: c.196G>C] and leads to the substitution of a proline residue in the E helix. The resulting hemoglobin variant has been named Hb Maruchi. This new variant cannot be separated from Hb A by electrophoretic and chromatographic techniques. CONCLUSIONS: The substitution α65(E14) Ala>Pro; HBA1: c.196G>C causes a α-thalassemia silent associated with a very mild phenotype. The diagnosis of this type of mutation is important because it may cause alpha thalassemia if inherited with other clinically relevant HBA1/HBA2 variants.

Why is the novel Hb Miguel Servet visualised by CE-HPLC newborn and not by the CE-HPLC ß-thalassaemia programme?

Screening of haemoglobinopathies is indicated for the detection of sickle cell anaemia; thus, neonates can benefit from early and adequate treatment that prevents neurological damage, reduces morbidity and mortality associated with the disease. These types of programmes sometimes lead to unexpected findings. We present a new haemoglobin (Hb) variant (Hb Miguel Servet) detected by newborn screening. During neonatal screening of haemoglobinopathies by cation-exchange high-performance liquid chromatography (CE-HPLC) newborn, an Hb variant was detected. An analysis at 8 months of age using capillary zone electrophoresis (CZE) confirmed the presence of this new Hb. The molecular characterisation was performed by automatic sequencing of the α and ß globin genes in an ABI PRISM 3100 Genetic Analyzer. Hb analysis by CE-HPLC ß-thalassaemia short programmedid not indicate the presence of abnormal Hbs. By CZE showed a peak in the zone 12 zone comprising 3.3% of the total Hb. A new analysis by CE-HPLC on a Tosoh G8-2 (Horiba) shown a peak, in the region of HbA1b, did not interfere with the quantification of HbA1c. Sequencing of the ß gene revealed the substitution of a guanine for a thymine (GGT >TGT) in codon 69 of the second exon, resulting in substitution of cysteine for the amino acid glutamine (HBB:c.208G>T). Hb Miguel Servet is a ß-chain globin variant detected by CE-HPLC newborn (BioRad), by CZE and by CE-HPLC-CE Tosoh G8-2 (Horiba), but no by CE-HPLC-CE ß-thalassaemia short programme (BioRad). In fact, for all the techniques that are visualised, what would be detected would be the glutathione variant of Hb (Miguel Servet).

Nondeletional α-Thalassemia: Two New Mutations on the α2 Gene.

About 10.0% of α-thalassemia (α-thal) cases are due to point mutations, small deletions, or insertions of one or more bases on the α genes that can alter mRNA processing at the transcription, translation, or post-translation level; these cases are called nondeletional α-thalassemias (α-thal). Most occur within the domain of the α2 gene without changes in the expression of the α1 gene. We present two new frameshift mutations on the HBA2 gene, associated with a nondeletional α-thal phenotype. The probands were referred to our clinic because of persistent microcytosis and hypochromia. The molecular characterization was performed by automatic sequencing of the α-globin genes. Two new mutations were detected on the HBA2 gene; HBA2: c.85delG, p.(Ala29fs*21), and HBA2: c.268_280delCACAAGCTTCGGG, p.(His90Trpfs*9). These new mutations cause a change of the reading frame, the first on codon 28 and the second from codons 89 to 93. In the first mutation, the result is an altered amino acid sequence and a premature termination codon at position 87, while the elimination of 13 bp generates a protein of 95 residues and in this case, the premature termination codon is at position 96. These types of mutation are among the most damaging changes to the coding of a protein. Not only do they lead to changes in the length of the polypeptide, but they also vary the chemical composition, which would result in a nonfunctional protein. The importance of identifying these new mutations lies in their possible association with α0-thal, which could lead to a severe thalassemia.

C>A substitution in NT 46 of the 3' UTR region (the α complex protected region) of the alpha-1 globin gene: a non-deletional mutation or polymorphism?

AIMS: Untranslated regions (UTRs) play an important role in post-transcriptional regulation of gene expression, including by modulating messenger RNA (mRNA) transport out of the nucleus, translation efficiency, subcellular localisation and stability. Any mutation in this region could alter the stability of mRNA and thereby affect protein synthesis. We analysed if a mutation located in the α complex protected region of the α1 globin gene could cause non-deletional α-thalassaemia by affecting post-transcriptional stability (mRNA stability). METHODS: A total of 14 patients without anaemia, normal or slight microcytosis and hypochromia (medium concentration haemoglobin [MCH] <27 pg) were studied. Haemoglobin subtypes were screened using capillary zone electrophoresis and ion-exchange high-performance liquid chromatography (VARIANT II ß-Thalassaemia Short Program). The most common α-globin mutations were identified by multiplex PCR (Alpha-Globin StripAssay kit) and the molecular characterisation by automatic sequencing of alpha globin genes. RESULTS: All of them shown a novel transversion mutation in nt 778 (C>A), which is located in the 3' UTR in the α complex protected region [HBA1: c.*+46C>A]. CONCLUSIONS: This mutation is in the αRNAmin binding site, so a single nucleotide substitution in this region can decrease mRNA stability by potentially compromising the binding of α-complex protein to αRNAmin, favouring the decay of α-globin mRNA via erythroid cell-enriched endoribonuclease cleavage. In this case, it is a non-deletional α-thalassaemia. However, in silico and empirical studies predicted that it could be a silent polymorphism. Functional studies should be carried out to confirm whether it is a pathological mutation or a silent polymorphism.

Characterization of deletional and non-deletional alpha globin variants in a large cohort from Spain between 2009 and 2014.

The hemoglobinopathies are a group of disorders passed down through families (inherited) in which there is abnormal production or structure of the hemoglobin molecule. They are among the most common inherited diseases around the world. Those that produce abnormal hemoglobin are called structural hemoglobinopathies while thalassemia is another type of disorder that is caused by a defect in the gene production of the globin chains. In a study ambispective comprising 1623 patients, 153 subjects showed an abnormal hemoglobin and 1470 with hypochromic and microcytic anemia, and of these 1470, 23 patients were studied for simultaneously α-thalassemias and structural hemoglobinopathies. Among the α-thalassaemia cases, 1282 cases (87.2%) were deletional α-thalassemia, 172 cases (11.7%) were non-deletional α-thalassemia, and 16 cases (1.1%) were deletional and non-deletional α-thalassamias simultaneously. Thus, approximately 12% of the cases were non-deletional α-thalassaemia. Clinical diagnosis, only 19 severe cases (1 hydrops fetalis and 18 instances of Hb H disease), 1200 thalassamias traits, and 160 thalassaemia silent carriers were recorded within the α-thalassaemia. Regarding structural hemoglobinopathies, there were only 2 cases of hemoglobinopathies with low oxygen affinity and 1 case of hemoglobin M; the remaining 150 were silent hemoglobinopathies. Non-deletional α-thalassaemia represented 12% of all α-thalassemias in our region; the most common deletion in our area was the 3.7-kb deletions, followed by Asian --(SEA) and --(FIL). The alterations responsible for non-deletional α-thalassaemia are most represented by the Hph and Hb Groene Hart and, in the case of structural hemoglobinopathies, Hb Le Lamentin and Hb J-Paris.

Novel nonsense mutation in the α1-globin gene [HBA1:C.49A>T] is responsible for non-deletion α-thalassemia.

BACKGROUND: In the α-thalassemia one of the less frequent mechanisms is the nonsense mutations, which generate the substitution of a triplet that encodes an amino acid for a stop codon and, therefore, protein synthesis stops prematurely. At present, 9 mutations of this type have been documented, 6 that affect the HBA2 gene and 3 that affect the HBA1 gene. OBJECTIVES: We present a new mutation in CD16 of the HBA1 gene, where the change AAG>TAG generates a stop codon. METHODS: A 48-year-old woman from Madrid, was studied because she had maintained microcytosis without iron deficiency. Hb A2 and Hb F levels were measured by ion exchange HPLC (VARIANT II). Hemoglobin was studied by capillary zone electrophoresis and ion exchange HPLC (short program of ß-thalassemia). Molecular characterization was performed by automatic sequencing of alpha globin genes. RESULTS: The propositus presented no abnormal hemoglobins and Hb A2 and Hb F levels were within normal limits. The molecular characterization identified the new transversion mutation HBA1: c.49 A>T, which resulted in an amino acid change of Lys > Stop at codon 16 of exon 1 in the state heterozygous [α116 (A14) Lys>Stop; HBA1: c.49A>T]. CONCLUSION: In this new nonsense mutation, short genetic products may suffer nonsense-mediated degradation, whereas the abnormal protein will be eliminated through the proteolytic pathway mediated by ubiquitin. Regardless, the phenotype is mild. The most severe end of the clinical spectrum will probably occur when a mutation is inherited together with a mutation that results in suppression of two genes (-/ααT or -α/-αT).

Hb Palencia: a novel δßδ-type two-way fusion variant with ß-globin-like expression levels.

AIMS: Fusion proteins of unequal recombination events at the ß-globin locus have pathological effect. The haemoglobin (Hb) variants of type Lepore are fusion proteins characterised by ß-like globin chains with a δ-globin (HBD) N-terminus and a ß-globin (HBB) C-terminus, whereas reciprocal products of underlying crossover events hold a HBB N-terminus and HBD C-terminus instead. Finally, Hb Parchman contains a ß-like globin chain with a central HBB fragment and HBD-derived N-termini and C-termini, whereas reciprocal hybrid proteins are as yet unknown. METHODS: The propositus was an 80-year-old Caucasian man, whose HbA1c quantification by HPLC (Variant II turbo) for exclusion of type-2 diabetes revealed an abnormal peak. Haemoglobins were analysed by ion-exchange HPLC (Variant II) and capillary electrophoresis (Sebia Capillarys Flex) and DNA by automatic Sanger sequencing of δ-globin and ß-globin genes. RESULTS: Sequencing showed an HBB-HBD-HBB hybrid gene, with HBD-derived central codons 9-31, and HBB-derived UTRs and complementary coding regions. The corresponding new hybrid haemoglobin (Hb Palencia) is represented at ≈40%, similar to HbA. CONCLUSION: Hb Palencia contains the first globin variant with internal HBD sequences and HBB-derived N-terminal and C-terminal and regulatory sequences. Relative quantity of the new ßδß-type variant suggests transcriptional control by HBB elements and a half-life similar to normal HBB.

Development and validation of a multivariable prediction rule for detecting a severe acquired ADAMTS13 activity deficiency in patients with thrombotic microangiopathies.

BACKGROUND: Thrombotic microangiopathies (TMAs) are a group of diseases that have different aetiologies and treatments, but a clinical differential diagnosis remains difficult. Among TMAs, thrombotic thrombocytopenic purpura (TTP) is characterised by a severe ADAMTS13 functional deficiency. However, assays exploring ADAMTS13 activity are limited to some specialised laboratories. Our objective was to develop and validate a diagnostic method for TTP in adult patients with TMA. METHODS: We generated a multivariable model (four predictors) on a cohort of 174 TMA patients in order to predict an ADAMTS13 activity deficiency (AUC of 0.927). The multivariable model was simplified into a binary rule to facilitate the interpretation of the predictions. There were two scenarios for a patient: (1) Predicted ADAMTS13 deficiency; if the patient met four conditions simultaneously (platelets ≤44×109/L, creatinine ≤2 mg/dL (≤176.84 µmol/L) for males or ≤1.9 mg/dL (≤168 µmol/L) for females, age ≤68 years and no history of haematopoietic stem cell transplant [HSCT]); or (2) Predicted "normal" activity; if any of the above conditions are not met. This rule was validated on a second cohort of 86 patients and performed with sensitivity of 87.7% and specificity of 92.7%. RESULTS AND CONCLUSIONS: This could lead to the earlier confirmation or rapid exclusion of TTP when ADAMTS13 testing is not avalilable, facilitating a more suitable therapy based on the aetiology of the TMA.

Hb Moncloa: A new variant of haemoglobin that interferes in the quantification of Hb A1c.

BACKGROUND: In most routine laboratories in Spain, the commonly used method for evaluating HbA1c is ion-exchange high performance liquid chromatography (HPLC). The presence of a variant of Hb may interfere with the quantification of HbA1c. AIMS: Here, we report a novel haemoglobin variant, named Hb Moncloa, which was found during a routine health check at the Hospital Clínico San Carlos in Moncloa (Madrid), Spain. METHODS AND RESULTS: Molecular characterization of ß gene identified a novel transversion mutation [ß80(EF4)Asn>Ser; HBB:c.242A>G]. CONCLUSIONS: When there is no correlation between clinical, glycemic status and glycated haemoglobin of the patient, the chromatogram of HbA1c should be carefully checked to detect the possible presence of variants that cause interference in their measurement.

Phenotype of mutations in the promoter region of the ß-globin gene.

BACKGROUND: ß+-Thalassaemia is characterised by reduced production of ß chains, which decrease can be caused by mutations in the promoter region (CACCC or TATA box), and is classified as mild or silent depending on the extent of ß-globin chain reduction. In both cases, homozygotes or compound heterozygotes for these mutations usually have thalassaemia intermedia. Frequently the diagnosis is made in adulthood or even in old age. A total of 37 alterations in the promoter region have been described so far. AIMS: In this report we describe the mutations found in the promoter region of the ß-globin gene in a single hospital in Madrid. METHODS: Between 1998 and 2015, more than 9000 blood samples were analysed for full blood count and underwent haemoglobin electrophoresis and high performance liquid chromatography. Genetic analysis of the ß and Gγ-globin genes was carried out by automatic sequencing and, in the case of α genes, by multiplex PCR. RESULTS: 35 samples showed mutation in the promoter region of the ß-globin gene, with a total of six different mutations identified: one in the distal CACCC box, two in the proximal CACCC box, three in the ATA box. CONCLUSIONS: Any alterations in the proximal CACCC and TATA boxes lead to a moderate decrease in synthesis of the ß-globin chain, which has been demonstrated in cases of thalassaemia intermedia that have presented in the second decade of life with a moderate clinical course.

Haemoglobinopathies that occur with decreased HbA2 levels: a gene mutation set involving the δ gene at a Spanish centre.

AIMS: Haemoglobin A2 (HbA2) consists of two globin chains, α and ß. Alterations in any of these genes influences the level of HbA2. Here, we present cases of structural Hb variants and thalassaemias which present either alone or together and reduce the level of HbA2 at varying degrees. Furthermore, we present a novel structural mutation in the δ globin gene, called Hb A2-Madrid. METHODS: The levels of HbA2 and HbF and the different haemoglobin variants were measured and analysed by ion exchange high performance liquid chromatography (HPLC, VARIANT II), the types of haemoglobins were determined by capillary zone electrophoresis (CZE) (Sebia) and the globin chains were determined by reversed-phase HPLC. Genetic analysis was performed by automatic sequencing of the α and δ genes as well as by multiple PCRs for the α globin genes. RESULTS: In α thalassaemia (n=94), the HbA2 levels ranged from 1.39% to 2.43%. Among individuals with δ thalassaemia (n=5), the HbA2 level of those with δ+ thalassaemia was 1.77%, and that of those with δ0 thalassaemia was 1.70%. Among the individuals with 뫧 thalassaemia (n=13), those who were homozygous lacked HbA2. All structural haemoglobinopathies (n=97) were heterozygous; the α chain variants (n=84) presented with an HbA2 level of 1.76%, while the δ chain variants (n=13) presented with a level of 1.75%. CONCLUSION: HbA2 is an essential parameter in the diagnostics of haemoglobinopathies. HPLC-EC and CZE allow the quantification of HbA2. Here, we show that quantification of HbA2 is critical for the identification of α, δ and ßδ thalassaemias. Structural variants are discovered by HPLC. Molecular genetics is required for the proper identification of the mutations. Only with this knowledge is genetic counselling possible.

Hemoglobin Le Lamentin in the province of Albacete, Spain: Discovery of 32 cases.

OBJECTIVES: Hemoglobin Le Lamentin (α20(B1)His→Gln) is a ubiquitous variant that has been previously described in a small number of isolated patients. We report the incidental observation of Hb Le Lamentin in a large population from the province of Albacete, in southeastern Spain. Our study investigates possible reasons for the elevated number of carriers of this variant and its implications for the management of diabetes in our region. DESIGN & METHODS: The subjects are 32 diabetic patients whose hemoglobin displayed an unusual peak while they were being tested for glycated hemoglobin at the laboratory of the University General Hospital of Albacete over a 3-year period. Measurements were made by high performance liquid chromatography using a Variant™ II Turbo Kit-2.0, and subsequently the samples of the 32 patients with anomalous peaks were sent to the Hospital Clínico San Carlos (Madrid, Spain) for molecular characterization of any Hb variants. RESULTS: Molecular studies revealed 31 out of 32 patients heterozygous for Le Lamentin, and in one of them, Hb City of Hope was associated with Hb Le Lamentin. The remaining patient was homozygous for the Le Lamentin mutation. Additionally, most patients were native to the northeastern half of the province of Albacete and were unrelated. CONCLUSIONS: Our study describes the largest finding to date of hemoglobin Le Lamentin in a sample of patients. The fact that our region has been perpetually depopulated, with a population that has remained stable in small localities over the centuries, may have favored the survival of the mutation. Since the presence of this variant underestimates the true value of glycated hemoglobin measured by HPLC, it is necessary to systematically review chromatograms.

Laboratory parameters provided by Advia 2120 analyser identify structural haemoglobinopathy carriers and discriminate between Hb S trait and Hb C trait.

BACKGROUND: Haemoglobinopathies have spread owing to human migration, and the number of people needing diagnosis and management of these conditions is increasing. Clinicians need to accurately identify carriers and provide adequate genetic counselling in order to prevent the occurrence of homozygous or compound heterozygous offspring. OBJECTIVES: To identify red blood cell (RBC) laboratory parameters that discriminate between structural haemoglobinopathy carriers and healthy subjects, and to compare RBC laboratory indices between HbAS and HbAC individuals. METHODS: Samples of 500 variant Hb carriers (355 HbAS, 104 HbAC, 19 HbAD, 7 HbAE, 7 HbAO-Arab, 4 α-chain variants and 4 Hb Lepore) and 251 normal controls were run on an Advia 2120 analyser (Siemens). Classic haematological parameters and RBC populations were assessed in all subjects. A multivariable binary logistic regression model was created to predict the probability of a subject carrying any structural haemoglobinopathy. HbAS (n=355, 71%) and HbAC (n=104, 20.8%) subjects were compared. RESULTS: A clinical prediction rule was developed by assigning one point to each of the most efficient variables: mean corpuscular volume (MCV) <88.4 fL, RBC distribution width >13.4%, percentage of microcytic RBCs (%MICRO) >0.7% and the ratio of microcytic RBCs to hypochromic RBCs >0.8. A score of 0, 1, 2, 3 or 4, resulted in a probability of 9.6%, 36.3%, 66.7%, 85.2% or 98.3%, respectively. Among the most frequent variant Hb, HbAC subjects had lower values of parameters related to cell size (MCV, %MICRO) and higher values of parameters related to haemoglobin concentration (MCHC, %HYPER) than HbAS subjects. Coexistence of α-thalassaemia in both HbAS and HbAC individuals resulted in decreased Hb, MCV, MCH and MCHC. CONCLUSIONS: Structural haemoglobinopathy should be investigated in subjects belonging to ethnic groups with high prevalence of variant Hb and with a score of 3 or 4. Erythrocytes of HbAC subjects are smaller and denser than those of HbAS subjects.

HB Puerta del Sol [HBA1:c.148A>C], HB Valdecilla [HBA2:c.3G>T], HB Gran Vía [HBA2:c.98T>G], HB Macarena [HBA2:c.358C>T] and HB El Retiro [HBA2:c.364_366dupGTG]: description of five new hemoglobinopathies.

BACKGROUND: Structural hemoglobinopathies do not usually have a clinical impact, but they can interfere with the analytical determination of some parameters, such as the glycated hemoglobin in diabetic patients. Thalassemias represent a serious health problem in areas where their incidence is high. The defects in the post-translational modifications produce hyper-unstable hemoglobin that is not detected by most of electrophoretic or chromatographic methods that are available so far. METHODS: We studied seven patients who belong to six unrelated families. The first two families were studied because they had peak abnormal hemoglobin (Hb) during routine analytical assays. The other four families were studied because they had microcytosis and hypochromia with normal HbA2 and HbF without iron deficiency. HbA2 and F quantification and abnormal Hb separation were performed by chromatographic and electrophoretic methods. The molecular characterization was performed using specific sequencing. RESULTS: The Hb Puerta del Sol presents electrophoretic mobility and elution in HPLC that is different from HbA and similar to HbS. The electrophoretic and chromatographic profiles of the four other variants are normal and do not show any anomalies, and their identification was only possible with sequencing. CONCLUSIONS: Some variants, such as Hb Valdecilla, Hb Gran Vía, Hb Macarena and Hb El Retiro, have significant clinical impact when they are associated with other forms of α-thalassemia, which could lead to more serious forms of this group of pathologies as for HbH disease. Therefore, it is important to maintain an adequate program for screening these diseases in countries where the prevalence is high to prevent the occurrence of severe forms.