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.

Hb Amsterdam-A1 [α32(B13)Met→Ile; HBA1: c.99G>A]: A Hyperunstable Variant Due to a New Mutation on the α1 Gene.

Patients with hyperunstable α chain variants usually present with a thalassemic, rather than hemolytic, phenotype. Electrophoretic, ion exchange and reverse phase separations usually fail to detect the variant and when DNA sequencing identifies a 'silent' substitution it is usually presumed to be hyperunstable. We report the identification of such a variant, α32(B13)Met→Ile; HBA1: c.99G>A, arising from a new mutation on the α1 gene. The hemoglobin (Hb) was unequivocally detected by the isopropanol stability test and confirmed as hyperunstable by mass spectrometry (MS) of the precipitate and lysate, which showed proportions of 55% and 2.5% of α chains, respectively. The instability appears to be driven by perturbation of globin-heme, and possibly α1ß1 subunit, interactions.

The development of a peptide SRM-based tandem mass spectrometry assay for prenatal screening of Down syndrome.

Two new biomarkers, serum amyloid-P (SAP) and plasma C1-inhibitor protein are elevated in the maternal circulation of mothers carrying Down syndrome foetuses. Much emphasis of late\ has been put on the lack of translational tests being developed following the identification of new biomarkers. We have created a single-reaction-monitoring (SRM) tandem mass spectrometry-based assay for the quantitation of these biomarkers and compared these results with an in-house developed immunofluorescence-based technique (IF). This MS-based assay is a rapid 5 min test and a simple "one pot reaction," requiring only 5µl of plasma. To evaluate the potential of SRM-based quantitation in a clinical setting, SAP and C1-inhibitor were quantitated in 38 normal and Down syndrome affected pregnancies. Plasma SAP levels in the Down's group were significantly raised at 10-14 weeks (p<0.0015) and 14-20 weeks (p<0.0001). Plasma C1-inhibitor levels were also observed significantly elevated in the Down's group (10-14 weeks, p<0.0193, 14-20 weeks, p<0.0001). Analysis using the IF technique did not show any significant elevation of plasma SAP levels or C1-inhibitor levels. This rapid and sensitive assay demonstrates the potential of multiplexed tandem MS-based quantitation of proteins in chemical pathology labs and in a more cost-effective, accurate manner than conventionally used antibody methods.

Identification of new biomarkers for Down's syndrome in maternal plasma.

Using ProteinChip Technology (SELDI TOF MS), the maternal plasma of 53 chromosomally-normal control and 28 Down's syndrome affected pregnancies was profiled between 10 and 20 weeks' gestation. Preliminary studies demonstrated two distinct phases of changes in protein expression, the first at 10-14 weeks and second at 14-20 weeks. Using this data, analysis of the 10-14 weeks' plasma samples (Down's syndrome n=13, control n=20) showed the presence of a protein of mass 100.3 kDa that was elevated in the Down's syndrome group compared to the controls (p<0.002). This protein was further isolated using SAX Q-spin columns and identified using QTOF MS and Western blotting as being plasma protease C1-inhibitor. Analysis of the 14-20 week cohort demonstrated changes in protein expression of three additional proteins. Two of these proteins were found to be up-regulated (serum amyloid P-component, p<0.004 and transthyretin, p<0.006) and complement C3-α chain was observed to be down-regulated (p<0.0005). The identification of these biomarkers in maternal plasma and their potential to improve current Down's syndrome screening are discussed.

Implementing prenatal diagnosis based on cell-free fetal DNA: accurate identification of factors affecting fetal DNA yield.

OBJECTIVE: Cell-free fetal DNA is a source of fetal genetic material that can be used for non-invasive prenatal diagnosis. Usually constituting less than 10% of the total cell free DNA in maternal plasma, the majority is maternal in origin. Optimizing conditions for maximizing yield of cell-free fetal DNA will be crucial for effective implementation of testing. We explore factors influencing yield of fetal DNA from maternal blood samples, including assessment of collection tubes containing cell-stabilizing agents, storage temperature, interval to sample processing and DNA extraction method used. METHODS: Microfluidic digital PCR was performed to precisely quantify male (fetal) DNA, total DNA and long DNA fragments (indicative of maternal cellular DNA). Real-time qPCR was used to assay for the presence of male SRY signal in samples. RESULTS: Total cell-free DNA quantity increased significantly with time in samples stored in K(3)EDTA tubes, but only minimally in cell stabilizing tubes. This increase was solely due to the presence of additional long fragment DNA, with no change in quantity of fetal or short DNA, resulting in a significant decrease in proportion of cell-free fetal DNA over time. Storage at 4 °C did not prevent these changes. CONCLUSION: When samples can be processed within eight hours of blood draw, K(3)EDTA tubes can be used. Prolonged transfer times in K(3)EDTA tubes should be avoided as the proportion of fetal DNA present decreases significantly; in these situations the use of cell stabilising tubes is preferable. The DNA extraction kit used may influence success rate of diagnostic tests.

2D DIGE analysis of maternal plasma for potential biomarkers of Down Syndrome.

BACKGROUND: Prenatal screening for Down Syndrome (DS) would benefit from an increased number of biomarkers to improve sensitivity and specificity. Improving sensitivity and specificity would decrease the need for potentially risky invasive diagnostic procedures. RESULTS: We have performed an in depth two-dimensional difference gel electrophoresis (2D DIGE) study to identify potential biomarkers. We have used maternal plasma samples obtained from first and second trimesters from mothers carrying DS affected fetuses compared with mothers carrying normal fetuses. Plasma samples were albumin/IgG depleted and expanded pH ranges of pH 4.5 - 5.5, pH 5.3 - 6.5 and pH 6 - 9 were used for two-dimensional gel electrophoresis (2DE). We found no differentially expressed proteins in the first trimester between the two groups. Significant up-regulation of ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4, complement proteins C1s subcomponent, C4-A, C5, and C9 and kininogen 1 were detected in the second trimester in maternal plasma samples where a DS affected fetus was being carried. However, ceruloplasmin could not be confirmed as being consistently up-regulated in DS affected pregnancies by Western blotting. CONCLUSIONS: Despite the in depth 2DE approach used in this study the results underline the deficiencies of gel-based proteomics for detection of plasma biomarkers. Gel-free approaches may be more productive to increase the number of plasma biomarkers for DS for non-invasive prenatal screening and diagnosis.

A functional mutation in the LDLR promoter (-139C>G) in a patient with familial hypercholesterolemia.

A novel sequence change in repeat 3 of the promoter of the low-density lipoprotein receptor (LDLR) gene, -139C>G, has been identified in a patient with familial hypercholesterolemia (FH). LDLR -139G has been passed to one offspring who also shows an FH phenotype. Transient transfection studies using luciferase gene reporter assays revealed a considerable reduction (74+/-1.4% SEM) in reporter gene expression from the -139G variant sequence compared to the wild-type sequence, strongly suggesting that this change is the basis for FH in these patients. Analysis using electrophoretic mobility shift assay demonstrated the loss of Sp1 binding to the variant sequence in vitro, explaining the reduction of transcription.