Hb Kirikiriroa [α57(E6)Gly→Cys; HBA1: c.172G>T]: A Novel Unstable α-Globin Variant with Oxidized Derivatives Interfering with Hb A1c.

We report the identification of a novel hemoglobin (Hb) variant [α57(E6)Gly→Cys; HBA1: c.172G>T], to be referred to as Hb Kirikiriroa. The variant was detected in five subjects from two families, with familial relationship established between the families following diagnosis. A persistently elevated Hb A1c over a 1-year period prompted hemoglobinopathy screening in an adolescent male of New Zealand (NZ) European descent (case 1). Capillary electrophoresis (CE) revealed the variant was negatively charged and susceptible to oxidation, with multiple abnormal peaks detected (0.4-5.1% total Hb). Hb A1c analysis by cation exchange high performance liquid chromatography (HPLC) was the first indication of the variant in a pregnant female of NZ European descent (case 2). Cases 1 and 2 had normal complete blood counts. Isopropanol stability testing provided evidence the variant was unstable. We herein describe the characterization of Hb Kirikiriroa and clinical significance of the variant for interference with Hb A1c analysis by CE and cation exchange HPLC.

Hb Westport [ß121 (GH4) Glu>Asp; HBB:c.366A>C]: A novel ß-globin variant interfering with HbA1c measurement.

OBJECTIVES: To describe a novel ß-globin variant that interferes with HbA1c analysis by cation exchange HPLC. DESIGN AND METHODS: Diabetes screening by HbA1c measurement was assessed using cation exchange HPLC and an immunoassay point-of-care analyzer. Routine hemoglobinopathy screening was performed including CBC, HbF and HbA2 measurement by cation exchange HPLC and capillary electrophoresis (CE). Further variant characterization was undertaken by ESI TOF mass spectrometry and DNA sequencing. RESULTS: Discordant HbA1c results were obtained for our subject, with elevated HbA1c of 52 mmol/mol measured by cation exchange HPLC and a normal level of 34 mmol/mol by immunoassay. Abnormal HbA1c peak shape prompted hemoglobinopathy screening to investigate potential variant interference. Cation exchange HPLC (using ß-thalassemia program) and CE results were apparently normal, with HbF and HbA2 detected within reference intervals. ESI TOF mass spectrometry revealed the presence of a variant ß-globin chain. A novel missense variant was confirmed at codon 121 of the ß-globin gene [ß121 (GH4) Glu>Asp; HBB: c.366A>C], which we have named Hb Westport. CONCLUSIONS: Hb Westport is a novel ß-globin variant that interferes with HbA1c measurement by Bio-Rad D-100 cation exchange HPLC, giving a falsely elevated result. This was clinically significant for our subject because the erroneously elevated HbA1c value was above the diabetes diagnostic threshold. Alternative methods for diabetes assessment should be considered in subjects with Hb Westport.

Hb Tacoma: G>T or G>C, and Does It Matter?

Hb Tacoma [ß30(B12)Arg→Ser] is a missense variant that is caused by either an AGG>AGT or AGG>AGC substitution at codon 30 of the HBB gene. Currently, the latter is classified as a rare cause of ß0-thalassemia (ß0-thal). We propose that HBB: c.93G>C has been incorrectly assigned as ß0-thal and discuss whether HBB: c.93G>T or HBB: c.93G>C should be classified as ß+-thal instead, or as ß-globin variants without thalassemic effect. We present several subjects who are heterozygous for Hb Tacoma, one with HBB: c.93G>T and two with HBB: c.93G>C, to support our conclusions.

Hb Waikato [α127(H10)Lys→Gln; HBA1: c.382A>C]: A Novel High Oxygen Affinity Variant.

We report the identification of a novel, high oxygen affinity hemoglobin (Hb) variant [α127(H10)Lys→Gln; HBA1: c.382A>C]. The variant was detected in an adolescent male (proband) of Syrian descent by cation exchange high performance liquid chromatography (HPLC), during Hb A1c analysis. A complete blood count (CBC) showed elevated red blood cells (RBCs) (6.08 × 1012/L), Hb (16.1 g/dL) and packed cell volume (PCV) (0.48 L/L). Capillary electrophoresis (CE) revealed the variant was more negatively charged and represented 18.2% of total Hb. Isopropanol stability was normal. Cyanosis in the subject prompted investigation of oxygen affinity, with a reduced p50 of 20.8 mm Hg and a left shifted oxygen dissociation curve demonstrating increased oxygen affinity. We propose the novel variant be named Hb Waikato, which reflects the Hospital Laboratory where the variant was discovered and region where the proband was born and herein describe characterization.

Novel α0-Thalassemia Deletion Identified in an Indian Infant with Hb H Disease.

We report the identification of a large deletion of the α-globin gene cluster, which removed both HBA2 and HBA1 and included the region from HBZ to HBQ1 on chromosome 16 (16p13.3). The α0-thalassemia (α0-thal) deletion was discovered in an Indian family residing in New Zealand. The proband was a 3-month-old female, who presented with a Hb H disease of unknown molecular origin. Routine hematology showed marked hypochromic microcytic anemia, with numerous Hb H inclusion bodies. In the absence of iron deficiency, there was a strong clinical suspicion of α-thal. On initial screening using a multiplex gap polymerase chain reaction (gap-PCR), only the common rightward deletion (-α3.7) was detected. Investigation of the proband's mother and father revealed the mother was heterozygous for the -α3.7 deletion, while none of the seven most common pathogenic α-thal deletions were detected in the father. Multiplex ligation-dependent probe amplification (MLPA) was employed to detect the presence of a novel α0-thal deletion in both the proband and her father. For the proband, the α0-thal deletion in combination with the -α3.7 deletion, eliminated three copies of HBA consistent with a clinical diagnosis of Hb H disease.

Hb Manitoba [α102(G9)Ser→Arg] in Pasifika: Tongan Case Report.

Hb Manitoba [α102(G9)Ser→Arg] results from an AGC>CGC or AGC>AGA substitution at codon 102 of the HBA1 or HBA2 genes. The variant is mildly unstable but carriers typically have normal clinical presentation and hematological profile. Hb Manitoba has not been reported in Pasifika of Tongan, Samoan or New Zealand (NZ) Maori descent before. The cases presented here support the findings from existing literature but include results from alternative methodology including capillary zone electrophoresis (CZE), which may slightly underestimate the true variant percentage. The subject of our case report, a Tongan male with microcytic indices, was shown to be heterozygous for Hb Manitoba III (HBA2: c.309C>A) coinherited with the -α3.7 (rightward) deletion.