False HbA1cValue due to a Rare Variant of Hemoglobin J-Cubujuqui.

BACKGROUND: Hemoglobin (Hb) J-Cubujuqui is a rare Hb variant, and reports about it are very limited. There are no descriptions that it affects the results of glycated Hb. METHODS: In this study, we describe a rare variant discovered during newborn screening. Both high-performance liquid chromatography (HPLC) and capillary electrophoresis for hemoglobin analysis displayed abnormal peaks. The Hb variant was confirmed by Sanger sequencing. RESULTS: The pedigree study shows the variant was inherited from the newborn's father. His fasting blood glucose (FBG) level was 5.5 mmol/L. HbA1c measured by HPLC was falsely low in her father (2.41%), whereas that measured by immunoassay was normal (5.11%). Sanger sequencing revealed a heterozygous mutation (CGT˃AGT) at amino acid position 141 of the α1 gene, corresponding to Hb J-Cubujuqui [α1 141(HC3) Arg→Ser (CGT˃AGT); HBA1:c.424C˃A (or HBA2)]. CONCLUSIONS: This is the first report that Hb J-Cubujuqui interferes with the measurement of HbA1cand prompts clinicians to pay attention to the accuracy of glycated Hb results.

Unexpected HbA1c results in the presence of three rare hemoglobin variants.

Hemoglobin (Hb) variants, characterized by structural abnormalities in the globin chains, are among the most common inherited disorders. It has been shown that Hb variant remains an important cause of erroneous HbA1c results. Thus, it is important to be aware of the extent of the interference of each Hb variant encountered to avoid reporting unreliable results. However, the effects of many types of Hb variants on the measurement of HbA1c remain unclear. Here, we describe three rare Hb variants, Hb J-Tashikuergan [HBA2: c.59 C > A], Hb Pyrgos [HBB: c.251G > A], and Hb Hope [HBB: c.410 G > A], which lead to extremely high values (>25%) determined by Variant II Turbo 2.0. We further investigated their effects on HbA1c measurement by an HPLC system (Bio-Rad D100), a CE system (Sebia Capillarys 3 TERA), a boronate affinity chromatography system (Premier Hb9210), and an immunoassay method (Roche Diagnostics), and found that these Hb variants severely interfered with HbA1c measurement by Variant II Turbo 2.0 and Bio-Rad D100. This study demonstrates that patients with abnormally high HbA1c levels should be highly suspected of carrying Hb variants. When the HbA1c results are considered unreliable, other indicators such as glycated albumin may be a possible alternative to HbA1c in diabetic patients.

Fifteen Cases of Hb J-Meerut: The Rare Association with Hb E and/or HBA1: c.-24C>G (or HBA2) Variants.

Hb J-Meerut [HBA2: c.362C>A (or HBA1)] is a rare, stable, nonpathogenic α-globin gene variant that peaks in the area between the P3 and A0 windows on high performance liquid chromatography (HPLC). Few cases from different ethnic origins have been published but the majority were Asian Indians. Coinheritance with other hemoglobin (Hb) variants are rarer and can change the Hb J-Meerut phenotype making a diagnostic dilemma. In this study, we have reported 15 cases of Hb J-Meerut, discovered during a wide spectrum study of α-globin chain variants in the UK. The diagnosis was confirmed by forward and reverse DNA sequencing of the α1- and α2-globin genes. The average of the Hb J-Meerut expression was 20.9% of total Hb and characterized by a retention time (RT) of 1.9 min. (on average) on HPLC. The median of isoelectric focusing (IEF) was 5.6 mm above Hb A. Among the 15 cases studied, one case coinherited the Hb E (HBB: c.79G>A) mutation in heterozygosity and another case was associated with the Cap +14 (C>G) [HBA1: c.-24C>G (or HBA2)] variant. We noticed that the coinheritance of the Hb E mutation reduced the Hb J-Meerut expression with the formation of a hybrid peak missed on the HPLC chromatograph. We also noticed an increased expression of Hb J-Meerut in the case showing the coinheritance of the HBA2: c.-24C>G (or HBA1) variant.

Hematological and Molecular Findings in the First Case of Hb J-Norfolk [HBA2: c.173G>A (or HBA1] in an Indian Patient.

We here report a case of a 23-year-old female from Mumbai, Maharashtra, India who was detected to carry the α chain variant Hb J-Norfolk [HBA2: c.173G>A (or HBA1]. She had no clinical symptoms and was referred to us for routine investigations and screening. An abnormal peak was detected on both high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) with a fast-moving band on cellulose acetate electrophoresis. There is no detailed study on the HPLC and CE pattern of this hemoglobin (Hb) variant, and therefore, this study will help in detecting and avoiding missing these variants during routine investigations and population screening. This is the first report of this variant in the Indian population.

First Report of a Chinese Family Carrying a Double Heterozygosity for Hb Q-Thailand and Hb J-Bangkok.

The double heterozygosity for α and ß chain variants leads to the formation of abnormal heterodimer hybrids, which could render laboratory diagnostics in a routine setting difficult. The following is the first report of a double heterozygosity for Hb Q-Thailand [α74(EF3)Asp→His; HBA1: c.223G>C] with α+-thalassemia (α+-thal) and Hb J-Bangkok [ß56(D7)Gly→Asp; HBB: c.170G>A] found in a Chinese family. Both subjects were healthy with normal or borderline hematological parameters. Hemoglobin (Hb) analyses showed a novel variant, Hb Q-Thailand and Hb J-Bangkok. Family studies helped in the initial recognition and in making presumptive diagnoses, but definitive diagnoses of these cases with complex α and ß chain variants could only be obtained after DNA analysis.

A Mosaic Expression of a Hb J-Amiens (HBB: c.54G > T; p.Lys18Asn) and its Interference with Hb A1c Analysis.

We report the case of a 56-year-old Caucasian woman in whom hemoglobinopathy screening was triggered following an aberrant Hb A1c analysis. Preliminary diagnosis of the hemoglobin (Hb) variant was obtained through cation exchange high performance liquid chromatography (HPLC) and gel electrophoresis. DNA analysis confirmed the presence of Hb J-Amiens [ß17(A14)Lys→Asn; HBB: c.[54G > C or 54G > T)]. However, an unbalanced ratio between wild type and mutant signal after direct sequencing and a lower than expected percentage of this Hb variant led to the suggestion of a mosaic expression. Furthermore, different methods [capillary zone electrophoresis (CZE), cation exchange HPLC and boronate affinity] were tested to study the possible interference of this variant with Hb A1c measurements. These investigations showed a clinically relevant difference between the methods tested. Hb A1c analysis may lead to the discovery of new Hb variants or mosaicism for previously described Hb variants. This may have genetic consequences for the offspring of carriers and brings about the question of partner testing.

Asociación de la hemoglobina Groene Hart con la hemoglobina J-París-I: primer caso en España / Association between hemoglobin Groene Hart and hemoglobin J-Paris-I: First case in Spain

Fundamento y objetivo: Las talasemias son las enfermedades monogénicas más frecuentes a nivel mundial. Representan un grave problema sanitario en las regiones de mayor incidencia. Las α-talasemias se deben a un déficit de síntesis de las cadenas α de la hemoglobina (Hb). La Hb Groene Hart es una variante hiperinestable. En este trabajo se presentan 24 casos pertenecientes a 17 familias afectadas por la Hb Groene Hart, uno de ellos asociado con Hb J-París-I. Pacientes y método: Veinticuatro pacientes de 17 familias no relacionadas fueron incluidos en este estudio. La caracterización se realizó mediante secuenciación. Resultados: La secuenciación del gen α1 mostró la mutación CCT→TCT (Pro→Ser) en el codón 119 (Hb Groene Hart). En un paciente se asoció con la Hb J-París-I. Conclusiones: En la Hb Groene Hart se encuentra afectado un residuo clave para preservar la estabilidad de las cadenas α de globina. La presencia de esta variante es elevada en población española e inmigrante. La aparición de formas graves de la enfermedad podría ser evitada incorporando esta mutación al cribado de las mutaciones α-talasemia no deleción (AU)

Background and objective: Thalassemias are the most frequent monogenic disorder around the world. α-thalassemias are due to a deficiency of synthesis in the alpha-globin chain of the hemoglobin (Hb). Hb Groene Hart is a hyperunstable variant. In this work, we have studied 24 cases affected by Hb Groene Hart, one of them associated with Hb J-Paris-I. Patients and methods: Twenty-four patients from 17 unrelated families were included in this study. The characterization was done by sequencing. Results: α1 gene sequencing showed the mutation CCT→TCT (Pro→Ser) at codon 119 (Hb Groene Hart) in all patients. In one case, there was an association with Hb J-Paris-I. Conclusions: In the Hb Groene Hart, the residue 119 of alpha-globin chain is affected. This amino acid has a key role in preserving the stability of alpha-globin chain. It is also remarkable the presence of this variant in both the immigrant and native population. Thus, the identification of Hb Groene Hart carriers should be considered in the screening of α-thalassemia in Spain, as it is done in Northern Africa (AU)

[Association between hemoglobin Groene Hart and hemoglobin J-Paris-I: first case in Spain]. / Asociación de la hemoglobina Groene Hart con la hemoglobina J-París-I: primer caso en España.

BACKGROUND AND OBJECTIVE: Thalassemias are the most frequent monogenic disorder around the world. α-thalassemias are due to a deficiency of synthesis in the alpha-globin chain of the hemoglobin (Hb). Hb Groene Hart is a hyperunstable variant. In this work, we have studied 24 cases affected by Hb Groene Hart, one of them associated with Hb J-Paris-I. PATIENTS AND METHODS: Twenty-four patients from 17 unrelated families were included in this study. The characterization was done by sequencing. RESULTS: α1 gene sequencing showed the mutation CCT→TCT (Pro→Ser) at codon 119 (Hb Groene Hart) in all patients. In one case, there was an association with Hb J-Paris-I. CONCLUSIONS: In the Hb Groene Hart, the residue 119 of alpha-globin chain is affected. This amino acid has a key role in preserving the stability of alpha-globin chain. It is also remarkable the presence of this variant in both the immigrant and native population. Thus, the identification of Hb Groene Hart carriers should be considered in the screening of α-thalassemia in Spain, as it is done in Northern Africa.

[Analysis of clinical phenotypes of compound heterozygotes of Hb J-Bangkok and ß-thalassemia].

OBJECTIVE: To analyze hematological characteristics of compound heterozygotes of Hb J-Bangkok and ß-thalassemia, and to explore the influence of Hb J-Bangkok on the phenotype of ß-thalassemia. METHODS: Peripheral blood samples from a patient carrying Hb J-Bangkok and a ß-thalassemia mutation, her family members and three sporadic Hb J-Bangkok carriers were collected. RBC analysis and hemoglobin electrophoresis were performed. Genotypes of α- and ß-globin genes were analyzed. RESULTS: The father of the proband and the three sporadic cases were single carriers of Hb J-Bangkok. All of them were asymptomatic and have normal hematological parameters except for an abnormal hemoglobin band detected on hemoglobin electrophoresis. The proband was a compound heterozygote for Hb J-Bangkok and ß-thalassemia mutation IVS-Ⅱ-654. She presented typical ß-thalassemia trait, featuring hypochromic microcytic anemia and increased Hb A2 level. An abnormal hemoglobin band was also detected. CONCLUSION: Carriers of Hb J-Bangkok alone are asymptomatic. Co-existence of Hb J-Bangkok and ß-thalassemia may not aggravate the phenotype. Therefore, couples with one carrying Hb J-Bangkok and another carrying a ß-thalassemia mutation do not require prenatal diagnosis.

Report of haemoglobin J-Toronto and alpha thalassemia in a family from North of Iran.

We report of an Iranian family with history of a rare haemoglobin variant, Haemoglobin J associated with alpha thalassemia, discovered while performing premarital thalassemia screening. In the present study we report the first case of haemoglobin J-Toronto [alpha 5 (A3) Ala > Asp] on -globin gene, found in a 16-year-old female from Mazandaran Province, North of Iran. Further investigation characterized the same mutation for mother and brother of the proband, whilst mother was also a carrier of an alpha thalassemia gene mutation (-alpha3.7). Haemoglobin J-Toronto was previously just reported from Canada and has not been found in any part of Iran.

Isolated Hb Providence ß82Asn and ß82Asp fractions are more stable than native HbA(0) under oxidative stress conditions.

We have previously shown that hydrogen peroxide (H(2)O(2)) triggers irreversible oxidation of amino acids exclusive to the ß-chains of purified human hemoglobin (HbAo). However, it is not clear, whether α- or ß-subunit Hb variants exhibit different oxidative resistance to H(2)O(2) when compared to their native HbAo. Hb Providence contains two ß-subunit variants with single amino acid mutations at ßLys82→Asp (ßK82D) and at ßLys82→Asn (ßK82N) positions and binds oxygen at lower affinity than wild type HbA. We have separated Hb Providence into its 3 component fractions, and contrasted oxidative reactions of its ß-mutant fractions with HbAo. Relative to HbAo, both ßK82N and ßK82D fractions showed similar autoxidation kinetics and similar initial oxidation reaction rates with H(2)O(2). However, a more profound pattern of changes was seen in HbAo than in the two Providence fractions. The structural changes in HbAo include a collapse of ß-subunits, and α-α dimer formation in the presence of excess H(2)O(2). Mass spectrometric and amino acid analysis revealed that ßCys93 and ßCys112 were oxidized in the HbAo fraction, consistent with oxidative pathways driven by a ferrylHb and its protein radical. These amino acids were oxidized at a lesser extent in ßK82D fraction. While the 3 isolated components of Hb Providence exhibited similar ligand binding and oxidation reaction kinetics, the variant fractions were more effective in consuming H(2)O(2) and safely internalizing radicals through the ferric/ferryl pseudoperoxidase cycle.

Falsely low HbA1c value due to a rare variant of hemoglobin J-Baltimore.

HbA1c values are heavily relied on to assess glycemic control. Hemoglobin variants may interfere with measurements of HbA1c resulting in falsely low values. We present the first report of a rare variant of Hb in a patient with type 2 diabetes, which lead to a falsely low HbA1c.

Ion mobility augments the utility of mass spectrometry in the identification of human hemoglobin variants.

The global dispersion of hemoglobin variants through population migration has precipitated a need for their identification. A particularly effective mass spectrometry (MS)-based procedure involves analysis of the intact globin chains in diluted blood to detect the variant through mass anomalies, followed by location of the variant amino acid residue by direct analysis of the enzymatically digested globins. Here we demonstrate the use of ion mobility separation in combination with this MS procedure to reduce mass spectral complexity. In one example, the doubly charged tryptic peptide from a low abundance variant (4%) occurred at the same m/z value as a singly and a doubly charged interfering ion. In another example, the singly charged tryptic peptide from an alpha-chain variant (26%) occurred at the same m/z value as a doubly charged interfering ion. Ion mobility was used to separate the variant ions from the interfering ions, thus allowing the variant peptides to be observed and sequenced by tandem mass spectrometry.

Hb J-Cape Town [alpha92(FG4)Arg-->Gln (alpha1), CGG-->CAG] in Southern Italy found in a patient with erythrocytosis.

A high oxygen affinity hemoglobin (Hb) variant, Hb J-Cape Town [alpha92(FG4)Arg-->Gln (alpha1), CGG-->CAG] was identified in a 30-year-old woman patient from Cosenza (Southern Italy) who had previously been diagnosed with juvenile polycythemia in other hospitals. The occurrence of the variant Hb was assessed by both cation exchange chromatography and liquid chromatography-mass spectrometry (LC-MS) analyses. A detailed structural and functional characterization of the variant was performed at both the protein and DNA level. Structural investigation of the Hb variant by mass spectrometric methodologies and peptide sequencing identified the amino acid replacement as Arg-->Gln at alpha92. The corresponding DNA mutation CGGCAG was assigned to codon 92 of the alpha1 gene by DNA sequencing. These findings highlight the importance of investigating the hypothesis of a high affinity variant in the presence of a polycythemia so as to avoid unnecessary bone marrow examination or radioactive treatment. This report represents the first observation of the Hb J-Cape Town variant in Italy.

Hb J Meerut, a fast-moving hemoglobin: a study of seven cases from India and a review of literature.

Fast-moving hemoglobins (FMH) are rare abnormal hemoglobin variants which migrate anodal to hemoglobin A on alkaline agarose gel electrophoresis. Only five cases of Hb J Meerut, an alpha variant FMH, have been reported so far. We here report seven cases.