Hb G-Waimanalo [A1] [α64(E13)Asp→Asn; HBA1: c.193 G > A] with Decreased Oxygen Affinity.

A clinically asymptomatic 12-year-old girl showed microcytosis in routine examination. Cation exchange high performance liquid chromatography (HPLC), revealed two additional peaks eluting after Hb A and DNA sequencing uncovered a novel heterozygous mutation at codon 64 of the α1-globin gene. The hemoglobin (Hb) variant was annotated as Hb G-Waimanalo [A1]. Further analyses demonstrated a decreased oxygen affinity Hb compared to the normal Hb configuration.

A new α1-globin mutation, Hb Brugg [α20(B1)His→Gln].

A 2½-year-old male child and a 23-year-old woman with no clinical symptoms were investigated during routine consultations. Cation exchange high performance liquid chromatography (HPLC) revealed an additional peak eluting before Hb A. DNA sequencing showed a novel heterozygous mutation at codon 20 of the α1-globin gene. The hemoglobin (Hb) variant was named Hb Brugg. Analysis of oxygen affinity Hb and Hb stability did not show any changes compared to normal Hb constellation.

Unraveling the hydroxypropionaldehyde (HPA) system: an active antimicrobial agent against human pathogens.

The hydroxypropionaldehyde (HPA) system is a natural defense system synthesized by the probiotic bacterium Lactobacillus reuteri. To elucidate which of the molecules composing the HPA system (3-hydroxypropionaldehyde (3-HPA), reuterin (HPA dimer), and HPA hydrate) is responsible for the potent antimicrobial activity in biological systems, a combination of biochemical, genetic, and proteomic assays was used. The HPA system reacts with sulfhydryl-containing compounds such as cysteine and reduced glutathione (GSH) in solution. In situ, GSH knock-out Escherichia coli is significantly more susceptible to HPA-mediated cell death than E. coli wild type; GSH supplementation protects either bacteria from HPA attack. Proteomic analysis of HPA-treated bacteria ( Haemophilus influenzae ) revealed induction of redox- and heat shock-related proteins. A new antimicrobial mechanism of HPA is proposed, whereby the activity of HPA leads to depletion of free SH- groups in GSH and proteins through the action of 3-hydroxypropionaldehyde, causing an imbalance of the cellular redox status, ultimately resulting in cell death.

Hb Alperton [beta135(H13)Ala-->Val] shows decreased oxygen affinity.

A 14-year-old male child presented with microcytosis, a known alpha(+)-thalassemia (alpha-thal) heterozygote and a hemizygous glucose-6-phosphate dehydrogenase (G6PD) deficiency. Furthermore, cation exchange high performance liquid chromatography (HPLC) revealed an additional peak eluting slightly before Hb A. The peak area of the variant was equal to that of Hb A, suggesting a beta-globin variant. Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis confirmed the mutation at the protein level. The variant was detectable by isoelectric focusing (IEF) or by reversed phase HPLC. DNA sequencing revealed a heterozygous mutation at codon 135 of the beta gene, already described as Hb Alperton. Hb Alperton showed decreased oxygen affinity. Neither biochemical nor clinical characteristics for Hb Alperton have been reported so far.

A new alpha-globin variant with increased oxygen affinity in a Swiss family: Hb Frauenfeld [alpha 138(H21)Ser-->Phe, TCC>TTC (alpha 2)].

A new alpha-globin mutation [alpha 138(H21)Ser-->Phe] was found in a 55-year-old male proband with an erythrocytosis known since his youth. Cation exchange high performance liquid chromatography (HPLC) revealed an additional peak eluting slightly before Hb A indicating the presence of a variant. The peak area of the variant was approximately one-third that of Hb A suggesting an alpha-globin variant. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis confirmed the mutation at the protein level. The variant is also detectable with isoelectric focusing and reversed phase HPLC. DNA analysis revealed a heterozygous sequence mutation at codon 138 of the alpha2 gene. A C>T transition at the second nucleotide of the codon indicated a Ser-->Phe exchange. The variant showed increased oxygen affinity and was named Hb Frauenfeld.

THE Hb S/beta+ -thalassemia phenotype demonstrates that the IVS-I (-2) (A>C) mutation is a mild beta-thalassemia allele.

We report a family in which two siblings are compound heterozygotes for Hb S [beta6(A3)GluVal] and a rare beta-globin mutation [IVS-I (-2) (A>C)]. Both patients had significant levels of Hb A, indicating that the IVS-I (-2) mutation is a relatively mild beta(+)-thalassemia (beta(+)-thal) allele. This mutation, in compound heterozygosity with Hb S, does not necessarily lead to a mild clinical course.

Mass spectrometry: a tool for enhanced detection of hemoglobin variants.

BACKGROUND: More than 900 hemoglobin (Hb) variants are currently known. Common techniques used in Hb analysis are electrophoretic and chromatographic assays. In our laboratory, we routinely apply chromatographic methods. To ascertain whether Hb variants are missed with our procedures, we additionally analyzed all samples with mass spectrometry (MS). METHODS: Database evaluation was performed using all entries made in the Hb variant database HbVar, and possible Hb variants were calculated based on DNA variations. During a 5-year period, we analyzed 2105 lysates with cation-exchange HPLC (PolyCAT A column) and reversed-phase HPLC and additionally with electrospray ionization or MALDI-TOF MS. Globin chains were identified by their molecular masses. RESULTS: Database evaluation revealed that 43.2% of all possible Hbalpha- and beta-chain variants were found to date (considering only single-point mutations). Currently, 68.2% of the possible charge difference variants and only 28.7% of the neutral variants are found. Among 2105 Hb samples we identified 4 samples with Hb variants that were detected only with the MS method; 2 were new Hb variants (Hb Zurich-Hottingen and Hb Zurich-Langstrasse). With cation-exchange HPLC, 1 sample was found to be a beta-thalassemia and was identified by MS to be a beta-variant (Hb Malay). More common variants, such as Hb C, Hb D, and Hb E, and thalassemias could not be detected with the MS method. CONCLUSIONS: Application of MS improves the sensitivity of Hb analysis. The combination of MS with electrophoretic and chromatographic methods is optimal for the detection of Hb variants.

Analysis of minor hemoglobins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

BACKGROUND: Hemoglobin (Hb) heterogeneity arises mainly from posttranslational modifications of the globin chains, and cation-exchange chromatography reveals falsely increased concentrations of some minor Hbs in the presence of abnormal Hbs. Here we describe a method for identification of the globin chains and their posttranslational modifications contained in the Hb fractions. METHODS: We used cation-exchange HPLC (PolyCAT A column) for separation of Hb fractions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for analysis of the separated globin chains. Globin chains were identified by their molecular masses. Posttranslational modifications of globin chains were identified by digestion of the proteins with endoproteinase V8 before MALDI-TOF MS of the resulting peptides. RESULTS: Analysis of the HbA2 fractions of patients with HbS revealed 4 different globin chains. We found, in addition to the expected alpha- and delta-chains, the carbamylated alpha- and the betaS-chains. Additionally, we analyzed HbH, Hb Barts, HbA 1b, pre-HbA 1c, HbA 1c, HbF1, HbF, HbA 1d3a, HbA 1d3b, HbA2, and HbC1 fractions from control and pathologic blood samples. We identified several posttranslational modifications of the globin chains, such as pyruvatization, glycation, acetylation, carbamylation, and acetaldehyde adduct formation. CONCLUSIONS: The native and posttranslationally modified globin chains in minor and major Hbs are unambiguously identified by MALDI-TOF MS. A minor Hb containing the carbamylated alpha- and the betaS-chain elutes at the same time as normal HbA2 (alpha2delta2) and thus leads to falsely increased HbA2 values in patients with HbS when blood is analyzed with PolyCAT A chromatography.

Severe neurological impairment in hereditary methaemoglobinaemia type 2.

UNLABELLED: Recessive congenital methaemoglobinaemia (RCM) due to NADH-cytochrome b5 reductase (cytb5r) deficiency is a very rare disorder. We report on two unrelated patients (4 and 2.5 years old) with RCM type 2. Developmental delay was obvious at the age of 4 months. On follow-up, both children showed severe tetraspastic cerebral palsy, profound cognitive impairment, strabismus, impressive secondary microcephaly and failure to thrive. One novel mutation in the DIA1gene was identified. Prenatal diagnosis was successfully done in both families by mutation analysis in chorionic villi or measurement of cytb5r in fetal amniotic cells. CONCLUSION: Due to the severity of this disease and its 25% recurrence risk, prenatal diagnosis should be made available to all affected families.

Mutagenesis of the HMGB (high-mobility group B) protein Cmb1 (cytosine-mismatch binding 1) of Schizosaccharomyces pombe: effects on recognition of DNA mismatches and damage.

Cmb1 (cytosine-mismatch binding 1) is a high-mobility group (HMG) protein of Schizosaccharomyces pombe, which consists of 223 amino acids and has a single HMG domain at the C-terminal end. We have created several mutant and deletion forms of the Cmb1 protein and studied the effects on general DNA binding and specific binding to DNA mismatches and damaged DNA. Cmb1Delta41 (i.e. Cmb1 from which the 41 N-terminal amino acids have been deleted) bound specifically to cytosine-containing mismatches, to the cisplatin-induced intrastrand cross-links cis -GG and cis -AG and to an O (6)-methylguanine lesion. DNA binding was not affected when the 45 N-terminal amino acids were deleted, but was abolished in the absence of the 50 N-terminal amino acids, and was reduced when Cmb1 was truncated by between five and eleven C-terminal amino acids. Cmb1, both with and without the C-terminal truncations, retained its DNA binding affinity after heating at 95 degrees C. The cmb1 gene was induced when S. pombe cells were treated with cisplatin. Mitotic mutation rates were increased in a S. pombe cmb1 null mutant and in a cmb1-(1-212) mutant, which encodes a Cmb1 protein lacking the 11 C-terminal amino acids. We conclude that mutation avoidance by Cmb1 is distinct from Msh2-dependent mismatch repair, but related to nucleotide excision repair.