Association of Clinical Characteristics With Familial Hypercholesterolaemia Variants in a Lipid Clinic Setting: A Case-Control Study.

Objective: Familial hypercholesterolaemia (FH) variant positive subjects have over double the cardiovascular risk of low-density-lipoprotein-cholesterol (LDL-C) matched controls. It is desirable to optimise FH variant detection. Methods: We identified 213 subjects with FH gene panel reports (LDLR, APOB, PCSK9, and APOE) based on total cholesterol >310 mg/dL; excluding triglycerides >400 mg/dL, cascade screening, and patients without pre-treatment LDL-C recorded. Demographic, clinical and lipid parameters were recorded. Results: A 31/213 (14.6%) patients had pathogenic or likely pathogenic FH variants. 10/213 (4.7%) had variants of uncertain significance. Compared with patients without FH variants, patients with FH variants were younger (median age, 39 years vs. 48 years), had more tendon xanthomata (25.0% vs. 11.4%), greater proportion of first degree relatives with total cholesterol >95th percentile (40.6% vs. 16.5%), higher LDL-C (median, 271 mg/dL vs. 236 mg/dL), and lower triglycerides (median, 115 mg/dL vs. 159 mg/dL). The Besseling et al. model (c-statistic 0.798) improved FH variant discrimination over Friedewald LDL-C (c-statistic 0.724), however, Dutch Lipid Clinic Network Score (DLCNS) did not (c-statistic 0.665). Sampson LDL-C (c-statistic 0.734) had similar discrimination to Friedewald. Conclusion: Although tendon xanthomata and first degree relatives with high total cholesterol >95th percentile were associated with FH variants, DLCNS or Simon Broome criteria did not improve FH detection over LDL-C. Sampson LDL-C did not significantly improve discrimination over Friedewald. Although lower triglycerides and younger age of presentation are positively associated with presence of FH variants, this information is not commonly used in FH detection algorithms apart from Besseling et al.

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.

Disorders of sex development: insights from targeted gene sequencing of a large international patient cohort.

BACKGROUND: Disorders of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. Clinical management of DSD is often difficult and currently only 13% of patients receive an accurate clinical genetic diagnosis. To address this we have developed a massively parallel sequencing targeted DSD gene panel which allows us to sequence all 64 known diagnostic DSD genes and candidate genes simultaneously. RESULTS: We analyzed DNA from the largest reported international cohort of patients with DSD (278 patients with 46,XY DSD and 48 with 46,XX DSD). Our targeted gene panel compares favorably with other sequencing platforms. We found a total of 28 diagnostic genes that are implicated in DSD, highlighting the genetic spectrum of this disorder. Sequencing revealed 93 previously unreported DSD gene variants. Overall, we identified a likely genetic diagnosis in 43% of patients with 46,XY DSD. In patients with 46,XY disorders of androgen synthesis and action the genetic diagnosis rate reached 60%. Surprisingly, little difference in diagnostic rate was observed between singletons and trios. In many cases our findings are informative as to the likely cause of the DSD, which will facilitate clinical management. CONCLUSIONS: Our massively parallel sequencing targeted DSD gene panel represents an economical means of improving the genetic diagnostic capability for patients affected by DSD. Implementation of this panel in a large cohort of patients has expanded our understanding of the underlying genetic etiology of DSD. The inclusion of research candidate genes also provides an invaluable resource for future identification of novel genes.

Direct analysis of VLDL by TOF-MS allows rapid definition of Apo E genotypes and facilitates characterisation of post translational changes.

BACKGROUND: Apolipoprotein E (Apo E) is a glycoprotein which acts as a ligand facilitating the uptake of lipids. Three common isoforms of Apo E are recognised, E2, E3 and E4. E2 and E4 are associated with altered lipid metabolism and increased cardiovascular risk. We report a novel variant of Apo E (c.382G>A) predicting 110Asp→Asn identified by genotyping, we were prompted to investigate this further as the amino acid substitution produced a prospective N-glycosylation site in this novel variant. METHODS: We present a new rapid approach to genotyping Apo E performed by electrospray TOF-MS, on the same sample analysed by ultracentrifugation. The analysis can be performed in <10min and requires minimal sample volume. Control samples were used to verify the analysis. RESULTS: Spectra showed the expected mass for the E3 isoform at 34,237Da, E2 and E4 isoforms were identifiable by peaks at -53Da and +53Da respectively. Post translational glycosylation of the protein can also be identified. The novel isoform had a mass of 34,237Da without evidence of N-glycosylation. No significant effect on lipid metabolism was identified. CONCLUSION: The electrospray TOF-MS approach potentially provides a rapid alternative method for genotyping Apo E and for the investigation of novel isoforms.

Electrolytes in sick neonates - which sodium is the right answer?

INTRODUCTION: Hypoproteinaemia leads to spuriously high-sodium values when measured by indirect ion-selective electrodes (ISE) as used in main laboratory analysers compared with direct ISE employed in point-of-care analysers (POCT). The authors, therefore, investigated the occurrence of hypoalbuminaemia and its effect on measured sodium from POCT and the main laboratory analyser of neonatal intensive-care samples. METHOD: Sodium, in paired retrospective samples, measured by the main laboratory and neonatal unit blood-gas (POCT) analysers were compared. RESULTS: Hypoalbuminaemia (<30 g/l) was present in 1400/2420 paired results. Sodium was higher when measured by laboratory analyser, the difference increased with decreasing albumin; sodium (laboratory - POCT)=7.6 (±1.1)-0.22 (±0.04)×albumin. A difference >3 mmol/l was present in 31% and consequently underestimated (9.4%) hyponatraemia and overestimated (3.8%) hypernatraemia. CONCLUSION: Hypoalbuminaemia is common in sick neonates and monitoring electrolytes using POCT and laboratory analysers frequently yield significantly different results with consequent misclassification. In these patients, measurement of electrolytes by direct ISE (blood-gas analyser) may be more accurate.

A mass-spectroscopic method for measuring des-Leu albumin--a novel marker for chronic pancreatitis.

OBJECTIVES: Chronic pancreatitis is a progressive inflammatory disease leading to pancreatic insufficiency. The diagnosis of chronic pancreatitis is challenging, especially in early disease and the current tests have low sensitivity, may be invasive or have limited availability. We previously identified a truncated form of albumin lacking the C-terminal leucine, des-Leu albumin, which was present at high concentration in pancreatitis. We have developed a liquid-chromatography tandem-mass spectrometry (LC-MS/MS) method for measuring this peptide and make some preliminary observations on patient samples. METHODS: Serum samples from patients with established pancreatitis and controls were obtained. Diluted serum samples or prepared standards were digested with trypsin. Aliquots of the digest were separated on a reversed-phase column using water:acetonitrile:formic acid mobile-phase with tandem-mass spectrometry detection. Percentage composition of des-Leu albumin was determined from a response curve. RESULTS: The C-terminal peptide, LVAASQAALG- of des-Leu albumin was identified by m/z 901→725, wild type albumin by m/z 1014→825. Additional fragments were monitored as internal reference for digestion and sample integrity. Inter-assay imprecision was estimated at 10%. The percentage composition of des-Leu albumin segregated with the diagnosis of established pancreatitis with median levels of des-Leu albumin of 68% in patients compared to 5% in controls. CONCLUSIONS: Des-Leu albumin is a promising novel biomarker for chronic pancreatitis. It allowed clear discrimination of patients with pancreatitis from controls and its long half-life may facilitate monitoring of disease activity. The method described could readily be undertaken in modern clinical chemistry laboratories and will form the basis for further study.

BCS1L gene mutation presenting with GRACILE-like syndrome and complex III deficiency.

The clinical presentation of a neonate with GRACILE-like syndrome, complex III deficiency and BCS1L mutations is discussed. This case is compared and contrasted with the original Finnish reports of GRACILE syndrome and other cases with a similar phenotype. This case confirms the pathogenicity of the BCS1L gene mutation c.166C>T, and provides support for the pathogenicity of a sequence variation, c.-588T>A, previously reported.

ß37Trp→Cys mutation leads to multiple new hemoglobin species in red cells.

OBJECTIVES: To determine the cause of an unusual hemoglobin (Hb) pattern detected during HbA(1)c monitoring. DESIGN AND METHOD: Hemolysate was analysed by ESI MS, and individual components purified by reverse phase HPLC. Peptide mapping was used to pinpoint the substitution and DNA sequencing to confirm the mutation. RESULTS: ESI MS of lysate showed three novel ß chains with mass changes of -83, -51 and +222 Da. Peptide mapping and DNA sequencing indicated a ß37Trp→Cys substitution. Reverse phase chromatography showed three new ß globins eluting ahead of ß(A) CONCLUSION: The new Hbs result from an initial ß37Trp→Cys mutation (-83 Da) followed by oxidation to cysteine sulfinic acid (+32 Da) and the formation of a glutathione adduct (+305 Da). Despite the hydrophobicity change and the critical location of the side chain on the α1ß2 interface, there was no evidence of molecular instability or altered oxygen affinity, and no clear phenotype apart from discordant HbA(1)c.

How paraproteins can affect laboratory assays: spurious results and biological effects.

The presence of large amounts of monoclonal paraprotein in the serum can lead to a range of spurious laboratory results. These may result from analytical interference of the M-protein through chemical or immunological means, or from pre-analytical interference; the different modes of interference are explored in this review along with suggested methods of prevention or detection.