Diagnosing, discarding, or de-VUSsing: A practical guide to (un)targeted metabolomics as variant-transcending functional tests.

PURPOSE: For patients with inherited metabolic disorders (IMDs), any diagnostic delay should be avoided because early initiation of personalized treatment could prevent irreversible health damage. To improve diagnostic interpretation of genetic data, gene function tests can be valuable assets. For IMDs, variant-transcending functional tests are readily available through (un)targeted metabolomics assays. To support the application of metabolomics for this purpose, we developed a gene-based guide to select functional tests to either confirm or exclude an IMD diagnosis. METHODS: Using information from a diagnostic IMD exome panel, Kyoto Encyclopedia of Genes and Genomes, and Inborn Errors of Metabolism Knowledgebase, we compiled a guide for metabolomics-based gene function tests. From our practical experience with this guide, we retrospectively selected illustrative cases for whom combined metabolomic/genomic testing improved diagnostic success and evaluated the effect hereof on clinical management. RESULTS: The guide contains 2047 metabolism-associated genes for which a validated or putative variant-transcending gene function test is available. We present 16 patients for whom metabolomic testing either confirmed or ruled out the presence of a second pathogenic variant, validated or ruled out pathogenicity of variants of uncertain significance, or identified a diagnosis initially missed by genetic analysis. CONCLUSION: Metabolomics-based gene function tests provide additional value in the diagnostic trajectory of patients with suspected IMD by enhancing and accelerating diagnostic success.

High childhood serum triglyceride concentrations associate with hepatocellular adenoma development in patients with glycogen storage disease type Ia.

Background & Aims: Glycogen storage disease type Ia (GSDIa) is an inborn error of carbohydrate metabolism caused by pathogenic variants in the glucose-6-phosphatase catalytic subunit 1 (G6PC1) gene and is associated with hepatocellular adenoma (HCA) formation. Data on risk factors for HCA occurrence in GSDIa are scarce. We investigated HCA development in relation to sex, G6PC1 genotype, and serum triglyceride concentration (TG). Methods: An observational study of patients with genetically confirmed GSDIa ≥12 years was performed. Patients were categorised for sex; presence of 2, 1, or 0 predicted severe G6PC1 variant (PSV); and median TG during childhood (<12 years; stratified for above/below 5.65 mmol/L, i.e. 500 mg/dl). Results: Fifty-three patients (23 females) were included, of which 26 patients developed HCA at a median (IQR) age of 21 (17-25) years. At the age of 25 years, 48% of females and 30% of males had developed HCA (log-rank p = 0.045). Two-thirds of patients with GSDIa carried 2 PSVs, 20% carried 1, and 13% carried none. Neither the number of PSVs nor any specific G6PC1 variants were associated with HCA occurrence. Childhood TG was 3.4 (3.0-4.2) mmol/L in males vs. 5.6 (4.0-7.9) mmol/L in females (p = 0.026). Childhood TG >5.65 mmol/L was associated with HCA development at younger age, compared with patients with childhood TG <5.65 mmol/L (18 vs. 33 years; log-rank p = 0.001). Cox regression analysis including TG, sex, and TG-sex interaction correction revealed childhood TG >5.65 mmol/L as an independent risk factor for HCA development (hazard ratio [HR] 6.0; 95% CI 1.2-29.8; p = 0.028). Conclusions: In patients with GSDIa, high childhood TG was associated with an increased risk of HCA, and earlier onset of HCA development, independent of sex-associated hypertriglyceridaemia, and G6PC1 genotype. Lay summary: Glycogen storage disease type Ia (GSDIa) is a rare, inherited metabolic disease that can be complicated by liver tumours (hepatocellular adenomas), which in turn may cause bleeding or progress to liver cancer. Risk factors associated with hepatocellular adenoma formation in patients with GSDIa are largely unknown. In our study, we found that high serum triglyceride concentrations during childhood, but not specific genetic variants, were associated with increased risk of hepatocellular adenoma diagnosis later in life.

Monitoring phenylalanine concentrations in the follow-up of phenylketonuria patients: An inventory of pre-analytical and analytical variation.

BACKGROUND: Reliable measurement of phenylalanine (Phe) is a prerequisite for adequate follow-up of phenylketonuria (PKU) patients. However, previous studies have raised concerns on the intercomparability of plasma and dried blood spot (DBS) Phe results. In this study, we made an inventory of differences in (pre-)analytical methodology used for Phe determination across Dutch laboratories, and compared DBS and plasma results. METHODS: Through an online questionnaire, we assessed (pre-)analytical Phe measurement procedures of seven Dutch metabolic laboratories. To investigate the difference between plasma and DBS Phe, participating laboratories received simultaneously collected plasma-DBS sets from 23 PKU patients. In parallel, 40 sample sets of DBS spotted from either venous blood or capillary fingerprick were analyzed. RESULTS: Our data show that there is no consistency on standard operating procedures for Phe measurement. The association of DBS to plasma Phe concentration exhibits substantial inter-laboratory variation, ranging from a mean difference of -15.5% to +30.6% between plasma and DBS Phe concentrations. In addition, we found a mean difference of +5.8% in Phe concentration between capillary DBS and DBS prepared from venous blood. CONCLUSIONS: The results of our study point to substantial (pre-)analytical variation in Phe measurements, implicating that bloodspot Phe results should be interpreted with caution, especially when no correction factor is applied. To minimize variation, we advocate pre-analytical standardization and analytical harmonization of Phe measurements, including consensus on application of a correction factor to adjust DBS Phe to plasma concentrations.

[Iron supplementation in iron deficiency anaemia]. / Suppletie van ijzer bij ijzergebreksanemie.

Iron deficiency anaemia is a common problem. The majority of patients are treated with oral iron supplements. The current recommended dosage for oral supplementation of 200 mg ferrous fumarate 3x per day however, is based on a single small study of poor quality. There is no consensus concerning parenteral dosing. In recent years, new insights have been gained regarding both the dosage of oral supplementation and the indication for parenteral supplementation. Oral therapy is preferred. In principle, 100 mg ferrous fumarate once a day is sufficient for the treatment of symptom-free patients with anaemia. In cases of severe anaemia, or in patients with symptoms, 200 mg/day should be prescribed. If side effects appear, it can be dosed every other day. Where oral therapy does not show effectiveness, the anaemia is severe, or rapid increase of haemoglobin is indicated, parenteral supplementation should be chosen. Parenteral supplementation is more effective than oral supplementation in specific conditions, such as dialysis-dependent renal insufficiency, heart failure or active IBD.

Iron refractory iron deficiency anemia: a heterogeneous disease that is not always iron refractory.

TMPRSS6 variants that affect protein function result in impaired matriptase-2 function and consequently uninhibited hepcidin production, leading to iron refractory iron deficiency anemia (IRIDA). This disease is characterized by microcytic, hypochromic anemia and serum hepcidin values that are inappropriately high for body iron levels. Much is still unknown about its pathophysiology, genotype-phenotype correlation, and optimal clinical management. We describe 14 different TMPRSS6 variants, of which 9 are novel, in 21 phenotypically affected IRIDA patients from 20 families living in the Netherlands; 16 out of 21 patients were female. In 7 out of 21 cases DNA sequencing and multiplex ligation dependent probe amplification demonstrated only heterozygous TMPRSS6 variants. The age at presentation, disease severity, and response to iron supplementation were highly variable, even for patients and relatives with similar TMPRSS6 genotypes. Mono-allelic IRIDA patients had a milder phenotype with respect to hemoglobin and MCV and presented significantly later in life with anemia than bi-allelic patients. Transferrin saturation (TSAT)/hepcidin ratios were lower in IRIDA probands than in healthy relatives. Most patients required parenteral iron. Genotype alone was not predictive for the response to oral iron. We conclude that IRIDA is a genotypically and phenotypically heterogeneous disease. The high proportion of female patients and the discrepancy between phenotypes of probands and relatives with the same genotype, suggest a complex interplay between genetic and acquired factors in the pathogenesis of IRIDA. In the absence of inflammation, the TSAT/hepcidin ratio is a promising diagnostic tool, even after iron supplementation has been given. Am. J. Hematol. 91:E482-E490, 2016. © 2016 Wiley Periodicals, Inc.

JTT-705: is there still future for a CETP inhibitor after torcetrapib?

BACKGROUND: Despite reduction in low-density lipoprotein cholesterol, there is still a considerable amount of residual atherosclerosis-related disease. Epidemiological and pathophysiological data strongly favour increasing plasma high-density lipoprotein (HDL) cholesterol levels as antiatherogenic therapy, for example with cholesteryl ester transfer inhibition (CETP). However, negative Phase III studies on clinical end points with the CETP inhibitor torcetrapib challenge the future perspectives of other CETP inhibitors such as JTT-705. OBJECTIVE: Is there potential for CETP inhibition with JTT-705 after torcetrapib's collapse? METHODS: Search of articles in Pubmed citing JTT-705, torcetrapib and anacetrapib, or citing effects of pharmacological HDL-cholesterol raising or CETP inhibition. RESULTS/CONCLUSION: There is possibly a future for HDL-cholesterol raising therapies. Phase III clinical studies with either JTT-705 or anacetrapib will determine whether CETP inhibition is beneficial.

Fluid retention and vascular effects of rosiglitazone in obese, insulin-resistant, nondiabetic subjects.

OBJECTIVE: The use of thiazolidinedione (TZD) derivatives is associated with fluid retention, especially when combined with insulin. Because TZDs improve the metabolic effect of insulin, they may also reverse the blunted vascular response to insulin. We hypothesize that improvement of the action of insulin on vascular tone or permeability is the key mechanism of TZD-related fluid retention. RESEARCH DESIGN AND METHODS: In a randomized, double-blind, placebo-controlled, cross-over study in 18 obese, nondiabetic subjects with features of the metabolic syndrome, we investigated the effects of a 12-week treatment with 4 mg rosiglitazone twice a day on glucose disposal, hemodynamics (including forearm vasoconstrictor response to nitric oxide [NO]), synthase inhibition by N-monomethyl-L-arginine-acetate (L-NMMA), vascular permeability (transcapillary escape rate of albumin), and plasma volume during a hyperinsulinemic-euglycemic clamp (120 min, 120 mU/m(2) per min). RESULTS: As expected, rosiglitazone increased the glucose infusion rate during clamping. However, neither vascular permeability nor forearm blood flow response to hyperinsulinemia or L-NMMA was affected by rosiglitazone. Compared with placebo, rosiglitazone decreased diastolic blood pressure by 5 mmHg (95% CI 2.35-6.87, P = 0.0005) and increased plasma volume by 255 ml/1.73 m(2) (80-430, P = 0.007). Interestingly, the positive effect of rosiglitazone on glucose disposal correlated with change in foot volume (R(2) = 0.53, P = 0.001). CONCLUSIONS: Rosiglitazone improved insulin sensitivity but had no effect on NO-dependent vasodilatation in the forearm or vascular permeability in obese, insulin-resistant, nondiabetic subjects. As such, TZD-related fluid retention was not caused by improvement of the vascular actions of insulin. Nonetheless, rosiglitazone-induced improvement in insulin sensitivity appears to be correlated to edema formation.