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.

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.

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.

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.

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 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.

Identification of a novel mutation in the ß-globin gene 3' untranslated region (HBB: c.*+118A > G) in Spain.

The 3' untranslated region (3'UTR) region is well known to be associated with mRNA stability because of its associations with 3' end processing, polyadenylation and mRNA capping. Mutations located in this area cause a ß-thalassemia (ß-thal) phenotype compatible with ß(+)-thal. Two brothers, 49- and 41-years-old, were diagnosed with ß-thal intermedia (ß-TI) at 2 years of age. The ß-globin gene from the promoter region to the 3'UTR was sequenced and both brothers were diagnosed to be compound heterozygotes for the 3'UTR +1592 (A > G) (HBB: c.*+118A > G) and codon 39 (C > T) (HBB: c.118C > T) mutations. Their mother was a carrier of the nonsense codon 39 mutation and her hematological data suggested ß-thal trait; their father was a carrier of the 3'UTR +1592 mutation, though he did not have hematological parameters associated with ß-thal. The adenine at position +1592 or +118 bases downstream of the termination codon is highly conserved among primates and placental mammals, as it is located between the polyadenylation A signal (PAS) and the polyadenylation A cleavage (PAC) sites. Given its location, it is likely that this mutation would interfere with mRNA maturation; however, the clinical data of the heterozygous carriers show virtually no significant alterations. Therefore, we suggest that the impact on cleavage-stimulation factor (CstF) recognition of the mRNA sequence would be minimal and not significantly alter polyadenylation. Although the mechanism is not known, and because the carrier has no ß-thal minor, the mRNA is stable enough that the synthesis of the ß-globin chain is unaffected.

Heterozygosity for deletion of hypersensitive site 3 in the human locus control region has an unexpected minor effect on red cell phenotype.

The locus control region (LCR) is a genetic region that regulates the expression of the ß-globin locus (HBB locus). This region is composed of several DNase I hypersensitive sites (HSs) in which the regulatory functions of the LCR may reside. To date, some individuals bearing deletions of several HSs or even the complete LCR have been described. Although the globin genes of the HBB locus are intact, most of these patients suffer thalassemia due to the reduced expression of such genes. The LCR and the HSs forming it have been thoroughly studied in different genetic models. However, seemingly contradictory results are often obtained. Here, we describe the first deletion found in humans exclusively affecting the HS3 element of the LCR. The adult carrying this deletion shows very mild hematological modifications, indicating that HS3 deletion does not severely impair the ß-gene expression. Our results also reveal limitations of the murine models when studying the native mouse genes for understanding human diseases like thalassemias.

Erythrocytosis in a child due to Hb Andrew-Minneapolis [ß144(HC1)Lys→Asn (AAG>AAT or AAC)] associated with a Spanish (δß)(0)-thalassemia.

We report a rare association of δß-thalassemia (δß-thal) and a hemoglobin (Hb) variant with high oxygen affinity in a Spanish newborn. The proband had no Hb A and showed microcytosis and hypochromia; the peripheral blood smear was compatible with a thalassemia trait. Molecular studies revealed that the proband had a Spanish (δß)(0)-thal (inherited from his father) and also carried a de novo variant (Hb Andrew-Minneapolis) because from the point of hematology, his mother was quite normal. The hemoglobinopathies with high affinity for oxygen constitute an infrequent cause of secondary congenital erythrocytosis. The degree of erythrocytosis and the resulting clinical manifestations are highly variable, depending on the degree of altered oxygen affinity and the presence of thalassemic genes. Thus, when these variants are associated with ß(0)- or δß-thal, as in our case, the proportion of abnormal Hb is ∼100.0%, which may cause polycythemia, hyperviscosity, and iron deficiency. This type of association is very rare and few have been described, especially in children, as they would normally be detected in adults as the increased packed cell volume (PCV) also increases blood viscosity and causes the typical symptoms (cephalalgia, drowsiness, dizziness). The association of a high oxygen affinity Hb and a δß-thal presents a greater degree of erythrocytosis than when this same variant is associated with a ß(0)-thal, mainly because the Hb F percentage is usually greater in the δß-thal, and Hb F normally shows a greater affinity for oxygen and a reduced P(50), although one must always take into account the degree of oxygen affinity of the Hb variant. Familial erythrocytosis and an abnormal electrophoresis finding are indicative of a high affinity Hb. However, the absence of these findings does not reject the possibility of hemoglobinopathies, and in these cases, functional and molecular studies would be justified and should be mandatory for the differential diagnosis of erythrocytosis.

Association in cis of the mutations +20 (C>T) in the 5' untranslated region and IVS-II-745 (C>G) on the ß-globin gene.

In the 5' untranslated region (5'UTR), which is transcribed but not translated and is involved in posttranscriptional regulation of the gene promoting and stabilizing the mRNA translation, several mutations associated with mild ß-thalassemia (ß-thal) have been described. One of these, the +20 (C>T) mutation, is described in the HbVar database as a mutation responsible for ß(+)-thal. In heterozygote cases, it gives rise to a phenotype of ß-thal minor and ß-thal intermediate (ß-TI) when the mutation is associated with ß(+) IVS-II-745 (C>G). To clarify if this mutation is responsible for ß(+)-thal, we studied nine cases where we found an association of the +20 and IVS-II-745 mutations. All patients were carriers of four α genes. Three patients carried ß-thal major (ß-TM), two were compound heterozygotes for IVS-II-745 and codon 8 (-AA) or codon 39 (C>T), and the third was homozygous for IVS-II-745; all had the +20 mutation in the 5'UTR. The remaining patients showed the mutation IVS-II-745 associated with a replacement of C>T at nucleotide (nt) +20 of the 5'UTR. Contrary to reports in the HbVar database, the +20 mutation should be considered as an innocuous single nt polymorphism associated with the IVS-II-745 mutation in cis.

Study of three families with Hb Agrinio [α29(B10)Leu→Pro, CTG>CCG (α2)] in the Spanish population: three homozygous cases.

Most α-thalassemia (α-thal) mechanisms are deletions of one or both α-globin genes and less than 5.0-10.0% are point mutations. Hb Agrinio [α29(B10)Leu→Pro, CTG>CCG (α2)] is a hyperunstable α chain structural variant in which the thalassemic phenotype is determined by a post translational precipitation of the structurally anomalous chain in erythroid precursors. This study involved 14 cases with Hb Agrinio from three families. Selective sequencing of the α2 gene showed a CTG(Leu)>CCG(Pro) mutation at codon 29. The mutation was found in a heterozygous state in 11 cases and in a homozygous state in three cases. These are the first cases with Hb Agrinio described in Spain. In all cases where a leucine is exchanged for a proline, an unstable hemoglobin (Hb) will occur both in the α and the ß chain. Some of these are as unstable as Hb Agrinio and their presence is difficult to detect except by DNA sequencing.

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.

Hb Extremadura [ß64(E8)Gly→Ser;ß133(H11)Val→Leu]: a new molecular analysis.

Unstable hemoglobin (Hb) variants account for 9.5% of structural hemoglobinopathies. The majority of these unstable variants are the result of gene point mutations resulting in the substitution of a single amino acid by another. The presence of two mutations in the same allele is infrequent: of the 781 variants of the ß-globin cluster described, only 32 are due to two point mutations (4.1%). Hb Extremadura is a structural variant that is included within the so-called unstable Hb anomalies. It was first described in 1989, employing the most up-to-date techniques available at that time, reversed phase high performance liquid chromatography (HPLC) to separate the abnormal chain (ß(X)) digesting it with trypsin and analysis of the fragments with an automatic analyzer.

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).

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.

[Prenatal diagnosis of hemoglobinopathies and thalassemias]. / Diagnóstico prenatal de hemoglobinopatías y talasemias.

BACKGROUND AND OBJECTIVE: Thalassemias are the most common single-gene disorders in the world population and the most important health problem in several countries. The best program of prevention of new births is prenatal diagnosis. Here we gather the experience from 1996 of the Spanish Group of Erythropathology related to the prenatal diagnosis of hemoglobinopathies in Spain. SUBJECT AND METHOD: 36 couples carriers of beta-thalassemia or Hb S were studied. Fetal material was obtained by amniocentesis and BCV. The genotype was determined by molecular biology technologies. RESULTS: We observed 3 spontaneous abortions (8.3%), 6 interruptions of pregnancy (16.7%) and a case of maternal contamination (2.8%). Prenatal diagnosis could be completed in 97.2% of the cases (35). CONCLUSIONS: In our experience, with regards to hemoglobinopathies, it is essential to provide a good genetic advice in order to identify the molecular alteration in the progenitors before the first pregnancy. This would allow a prenatal diagnosis during the first quarter and, in case of a positive result, to perform an early interruption of the pregnancy without risks.

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).