Falsely high HbA1c value due to a novel α1-globin gene mutation: Hb shantou [α127(H10)Lys > Glu; HBA1: c.382 A > G]

Abstract

Hemoglobin A_1c (HbA_1c) is a widely utilized biomarker for the diagnosis and management of diabetes mellitus. Here, we describe an α1-globin chain hemoglobin variant and investigate its effect on HbA_1c measurement. A 26-year-old pregnant woman was suspected to harbor a hemoglobin variant following HbA_1c measurement during a routine prenatal examination using D10 (Bio-Rad). An oral glucose tolerance test (OGTT) was performed using an AU5800 clinical chemistry system (Beckman Coulter). HbA_1c was reanalyzed using VII-T 2.0 (Bio-Rad), Capillarys 2 Flex Piercing (C2FP, HbA_1c program, Sebia), Premier Hb9210 (Trinity Biotech), and Cobas c501 (Cobas Tina-quant Hemoglobin A_1c Gen.3). Glycated albumin (GA) level was also quantified using an enzymic method GA Kit (Lucica GA-L, Japan). Hemoglobin analysis was performed using high performance liquid chromatography on the Bio-Rad Variant II (β-thalassemia short program) and capillary electrophoresis (Capillarys 2 Flex Piercing, Hb program). Sanger sequencing of α and β genes was also conducted. HbA_1c was initially measured at 16.0% (151?mmol/mol) using the D10 (Bio-Rad). Her OGTT result was normal. Subsequently, HbA_1c values determined by VII-T 2.0, C2FP, Premier Hb9210, and Cobas c501 were 4.8% (29?mmol/mol), 4.9% (30?mmol/mol), 4.6% (27?mmol/mol), and 4.8% (29?mmol/mol), respectively. The glycated albumin level was 12.3% (reference: 10.8?～?17.1%). Hemoglobin analyzed using CE and HPLC revealed an abnormal hemoglobin. By Sanger sequencing, we identified a transition mutation in the α1 gene Hb Shantou [α127(H10)Lys?>?Glu; HBA1: c.382 A?>?G]. Clinically silent variants may interfere with HbA_1c determination by common methods.     

Molecular and hematological characteristics of a novel form of α-globin gene triplication: The hemoglobin St.Luke's-Thailand [α95(G2)Pro→Arg] or Hb St. Luke's [A2] HBA2

ObjectivesThis study aims to report a hitherto un-described α-globin gene triplicated allele with a novel hemoglobin (Hb) variant on α2-globin gene, Hb St.Luke's-Thailand [α95(G2)Pro-Arg].Designs and methodsA study was done on a 1.5-year-old Thai boy who showed pallor and jaundice after birth. Hb analysis was performed by HPLC and capillary electrophoresis. cDNAs of the two α-globin genes were analyzed by sequencing and novel diagnostic methods based on PCR-RFLP and multiplex PCR assays were developed.ResultsHb analysis by both HPLC and capillary electrophoresis revealed unknown variants for Hb A (amounting to 9.0 and 9.4%) and Hb A₂ (0.7 and 0.6%). cDNA analysis of α-globin genes revealed a normal α1-globin gene sequence and a CCG (Pro) to CGG (Arg) mutation at codon 95 of the α2-globin gene leading to the Hb St.Luke's-Thailand or Hb St. Luke's [A2] HBA2: c.287C>G. DNA analysis of the patient and his mother identified the in cis α-globin gene triplication. This novel orientation of α-globin gene could be confirmed by PCR-NlaIV restriction digestion or a multiplex allele specific PCR assay developed.ConclusionsA relatively lower percentage of the Hb St.Luke's-Thailand due to a HBA2 gene mutation could be explained by a linked anti-3.7 α-globin gene triplication. Clinical and hematological features, effect of α-globin gene triplication on the phenotypic expression of Hb St.Luke's-Thailand and diagnostics using combined HPLC, capillary electrophoresis and molecular techniques were presented.     

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

BackgroundIn 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.ObjectivesWe present a new mutation in CD16 of the HBA1 gene, where the change AAG>TAG generates a stop codon.MethodsA 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 β-thalassemia). Molecular characterization was performed by automatic sequencing of alpha globin genes.ResultsThe 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].ConclusionIn 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 (−/αα^T or -α/−α^T).     

Additional molecular and clinical evidence open the way to definitive IARC classification of the BRCA1 c.5332G > A (p.Asp1778Asn) variant

ObjectivesIn silico splicing analysis, a mini-gene assay and splicing data, obtained using RNA from blood samples, have shown that the BRCA1 c.5332G > A variant induces exon 21 skipping. However, despite these evidences, up to date, this variant is unclassified.The aim of this study is to provide further molecular and clinical evidence for the BRCA1 c.5332G > A variant in a patient with high grade serous ovarian carcinoma (HGSOC) to allow a definitive classification of this variant.Design and methodThe effect of the BRCA1 c.5332G > A variant on RNA splicing was evaluated by amplifying regions of BRCA1 from the cDNA of the patient. Loss of heterozygosity (LOH) in tumor tissue was also investigated.ResultsThe c.5332G > A allele causes significantly aberrant splicing of the BRCA1 exon 21. Evaluation of the c.5332A allele in tumor tissue highlights a possible loss of heterozygosity, supporting her pathogenic effect.ConclusionsOur results regarding the c.5332G > A variant confirm that it contributed to predisposition and onset of ovarian carcinoma in the patient. We propose to classify this variant as ‘likely-pathogenic’ (class IV).