Early Risk Stratification for Natural Disease Course in Fabry Patients Using Plasma Globotriaosylsphingosine Levels.

BACKGROUND: Fabry disease is a very heterogeneous X-linked lysosomal storage disease. Disease manifestations in the kidneys, heart, and brain vary greatly, even between patients of the same sex and with the same disease classification (classical or nonclassical). A biomarker with a strong association with the development of disease manifestations is needed to determine the need for Fabry-specific treatment and appropriate frequency of follow-up because clinical manifestations of the disorder may take decennia to develop. METHODS: We investigated the levels of plasma lysoGb3 levels over time and its association with disease manifestations and disease course in 237 untreated patients with Fabry disease (median age 42 years, 38% male) using linear mixed-effect models. RESULTS: LysoGb3 levels are stable over time in plasma of untreated patients with Fabry disease. Higher levels of lysoGb3 were associated with steeper decline in eGFR ( P = 0.05) and a faster increase in albuminuria (measured as the urinary albumin-to-creatinine ratio, P < 0.001), left ventricular mass (measured on echocardiography, P < 0.001), left atrial volume index ( P = 0.003), and Fazekas score ( P = 0.003). In addition, regardless of age, higher lysoGb3 levels were associated with higher relative wall thickness ( P < 0.001) and unfavorable functional markers on echocardiography, including septal mitral annular early diastolic velocity (e', P < 0.001) and the ratio of early transmitral velocity (E) to e' (E/e', P = 0.001). CONCLUSIONS: In an individual patient with Fabry disease, the plasma lysoGb3 level reached a specific level in early childhood which, in the absence of Fabry-specific treatment, remained stable throughout life. The level of lysoGb3 in untreated patients was associated with nearly all Fabry-specific disease manifestations, regardless of the sex of the patient.

Development of a Biosimilar of Agalsidase Beta for the Treatment of Fabry Disease: Preclinical Evaluation.

BACKGROUND AND OBJECTIVE: Fabry disease (FD) is a rare lysosomal storage disorder caused by a deficiency of the enzyme α-galactosidase A (aGal A). Since 2001, two different enzyme replacement therapies have been authorized, with agalsidase beta being used in most parts of the Western world. Currently, biosimilars of several expensive enzyme therapies are under development to improve their accessibility for patients. We present the preclinical results of the development of a biosimilar to agalsidase beta. METHODS: Produced in a Chinese hamster ovary (CHO)-cell system, the biosimilar aGal A Biosidus (AGABIO), was compared with agalsidase beta with respect to amino acid sequence, glycosylation, specific α-galactosidase activity, stability in plasma, and effects on cultured human Fabry fibroblasts and Fabry mice. RESULTS: AGABIO had the same amino acid composition and similar glycosylation, enzymatic activity, and stability as compared with agalsidase beta. After uptake in fibroblasts, α-galactosidase A activity increased in a dose-dependent manner, with maximum uptake observed after 24 h, which remained stable until at least 48 h. Both enzymes were localized to lysosomes. Reduction of accumulated globotriaosylceramide (Gb3) and lysoGb3 in cultured Fabry fibroblasts by AGABIO and agalsidase beta showed comparable dose-response curves. In Fabry knockout mice, after a single injection, both enzymes were rapidly cleared from the plasma and showed equal reductions in tissue and plasma sphingolipids. Repeated dose studies in rats did not raise any safety concerns. Anti-drug antibodies from patients with FD treated with agalsidase beta showed equal neutralization activity toward AGABIO. CONCLUSION: These findings support the biosimilarity of AGABIO in comparison with agalsidase beta. The clinical study phase is currently under development.

Sex-specific newborn screening for X-linked adrenoleukodystrophy.

Males with X-linked adrenoleukodystrophy (ALD) are at high risk for developing adrenal insufficiency and/or progressive leukodystrophy (cerebral ALD) at an early age. Pathogenic variants in ABCD1 result in elevated levels of very long-chain fatty acids (VLCFA), including C26:0-lysophosphatidylcholine (C26:0-LPC). Newborn screening for ALD enables prospective monitoring and timely therapeutic intervention, thereby preventing irreversible damage and saving lives. The Dutch Health Council recommended to screen only male newborns for ALD without identifying untreatable conditions associated with elevated C26:0-LPC, like Zellweger spectrum disorders and single peroxisomal enzyme defects. Here, we present the results of the SCAN (Screening for ALD in the Netherlands) study which is the first sex-specific newborn screening program worldwide. Males with ALD are identified based on elevated C26:0-LPC levels, the presence of one X-chromosome and a variant in ABCD1, in heel prick dried bloodspots. Screening of 71 208 newborns resulted in the identification of four boys with ALD who, following referral to the pediatric neurologist and confirmation of the diagnosis, enrolled in a long-term follow-up program. The results of this pilot show the feasibility of employing a boys-only screening algorithm that identifies males with ALD without identifying untreatable conditions. This approach will be of interest to countries that are considering ALD newborn screening but are reluctant to identify girls with ALD because for girls there is no direct health benefit. We also analyzed whether gestational age, sex, birth weight and age at heel prick blood sampling affect C26:0-LPC concentrations and demonstrate that these covariates have a minimal effect.

Skin biomarkers predict development of atopic dermatitis in infancy.

BACKGROUND: There is currently no insight into biomarkers that can predict the onset of pediatric atopic dermatitis (AD). METHODS: Nested in a prospective birth cohort study that examined the occurrence of physician-diagnosed AD in 300 children, 44 random children with onset of AD in the first year of life were matched on sex and season of birth with 44 children who did not develop AD. Natural moisturizing factor (NMF), corneocyte surface protrusions, cytokines, free sphingoid bases (SBs) of different chain lengths and their ceramides were analyzed from tape strips collected at 2 months of age before onset of AD using liquid chromatography, atomic force microscopy, multiplex immunoassay, and liquid chromatography mass spectrometry, respectively. RESULTS: Significant alterations were observed for four lipid markers, with phytosphingosine ([P]) levels being significantly lower in children who developed AD compared with children who did not (median 240 pmol/mg vs. 540 pmol/mg, p < 0.001). The two groups of children differed in the relative amounts of SB of different chain lengths (C17, C18 and C20). Thymus- and activation-regulated chemokine (TARC/CCL17) was slightly higher in children who developed AD, whereas NMF and corneocyte surface texture were similar. AD severity assessed by the eczema area and severity index (EASI) at disease onset was 4.2 (2.0;7.2). [P] had the highest prediction accuracy among the biomarkers (75.6%), whereas the combination of 5 lipid ratios gave an accuracy of 89.4%. CONCLUSION: This study showed that levels and SB chain length were altered in infants who later developed AD, and that TARC/CCL17 levels were higher.

Mind the B2: Life-Threatening Neonatal Complications of a Strict Vegan Diet during Pregnancy.

An increasing number of women of reproductive age follow vegan diets. Because vegan diets are deficient in a number of essential nutrients, guidelines address the necessity of supplementations such as iron, zinc, and vitamin B12. However, the risk of riboflavin (vitamin B2) deficiency is not properly addressed. We report a case of a male neonate with a life-threatening hypoglycaemia and lactic acidosis due to severe riboflavin deficiency. The mother followed a strict vegan diet with intermittent use of supplements (folic acid, vitamin B12, vitamin D, omega 3). This case highlights the importance of adequate counselling of all pregnant women adhering to vegan diets to ensure sufficient intake of essential nutrients and vitamins, including riboflavin.

Children with atopic dermatitis show increased activity of ß-glucocerebrosidase and stratum corneum levels of glucosylcholesterol that are strongly related to the local cytokine milieu.

BACKGROUND: Atopic dermatitis (AD) is characterized by immune dysregulations and an impaired skin barrier, including abnormalities in lipid organization. In the stratum corneum (SC), ß-glucocerebrosidase (GBA) mediates transformation of glucosylceramide (GlcCER) into ceramide (CER) and cholesterol into glucosylcholesterol (GlcChol). Alteration in GBA activity might contribute to skin barrier defects in AD. OBJECTIVES: To investigate GBA activity in the SC of children with AD before and after topical corticosteroid therapy and to compare it with healthy controls; to determine SC levels of GlcCER- and CER-containing hydroxysphingosine base (GlcCER[H] and CER[H], respectively) and GlcChol; and to relate them to disease severity, skin barrier function and the local cytokine milieu. METHODS: Lipid markers and cytokines of innate, T helper 1 and T helper 2 immunity were determined in SC collected from healthy children and from clinically unaffected skin of children with AD, before and after 6 weeks of therapy with topical corticosteroids. AD severity was assessed by Scoring Atopic Dermatitis and skin barrier function by transepidermal water loss (TEWL). RESULTS: Baseline GBA activity and GlcChol levels were increased in children with AD but declined after therapy. CER[H] levels and the CER[H] to GlcCER[H] ratio were increased in AD. GBA activity and GlcChol correlated with TEWL and levels of multiple cytokines, especially interleukin-1α and interleukin-18. GlcChol was strongly associated with disease severity. CONCLUSIONS: We show increased GBA activity and levels of GlcChol in AD. Our data suggest an important role of inflammation in disturbed lipid processing. GBA activity or GlcChol might be useful biomarkers in the monitoring of therapeutic responses in AD. What is already known about this topic? Patients with atopic dermatitis (AD) have a reduced skin barrier, mainly caused by altered lipid organization. The mechanisms underlying these lipid anomalies are not fully understood but likely reflect both genetic abnormalities in AD skin and the local cutaneous inflammatory environment. What does this study add? We show increased activity of the ceramide-generating enzyme ß-glucocerebrosidase in AD. Activity of this enzyme was correlated with the local cytokine milieu and declined after local corticosteroid therapy. We show that glucosylcholesterol levels in the stratum corneum are increased in AD. The function of glucosylcholesterol and the physiological consequences of increased levels are not clear yet; however, its levels were strongly correlated with skin barrier function: high transepidermal water loss strongly correlated with high levels of glucosylcholesterol. What is the translational message? Correction of cutaneous inflammation largely restores alterations in lipid metabolism in the stratum corneum of infants with AD.

An improved functional assay in blood spot to diagnose Barth syndrome using the monolysocardiolipin/cardiolipin ratio.

Barth syndrome is an X-linked disorder characterized by cardiomyopathy, skeletal myopathy, and neutropenia, caused by deleterious variants in TAFAZZIN. This gene encodes a phospholipid-lysophospholipid transacylase that is required for the remodeling of the mitochondrial phospholipid cardiolipin (CL). Biochemically, individuals with Barth syndrome have a deficiency of mature CL and accumulation of the remodeling intermediate monolysocardiolipin (MLCL). Diagnosis typically relies on mass spectrometric measurement of CL and MLCL in cells or tissues, and we previously described a method in blood spot that uses a specific MLCL/CL ratio as diagnostic biomarker. Here, we describe the evolution of our blood spot assay that is based on the implementation of reversed phase-UHPLC separation followed by full scan high resolution mass spectrometry. In addition to the MLCL/CL ratio, our improved method also generates a complete CL spectrum allowing the interrogation of the CL fatty acid composition, which considerably enhances the diagnostic reliability. This addition negates the need for a confirmatory test in lymphocytes thereby providing a shorter turn-around-time while achieving a more certain test result. As one of the few laboratories that offer this assay, we also evaluated the diagnostic yield and performance from 2006 to 2021 encompassing the use of both the original and improved assay. In this period, we performed 796 diagnostic analyses of which 117 (15%) were characteristic of Barth syndrome. In total, we diagnosed 93 unique individuals with Barth syndrome, including three females, which together amounts to about 40% of all reported individuals with Barth syndrome in the world.

The Shiga Toxin Receptor Globotriaosylceramide as Therapeutic Target in Shiga Toxin E. coli Mediated HUS.

In 90% of the cases, childhood hemolytic uremic syndrome (HUS) is caused by an infection with the Shiga toxin (Stx) producing E. coli bacteria (STEC-HUS). Stx preferentially binds to its receptor, the glycosphingolipid, globotriaosylceramide (Gb3), present on the surface of human kidney cells and various organs. In this study, the glycosphingolipid pathway in endothelial cells was explored as therapeutic target for STEC-HUS. Primary human glomerular microvascular endothelial cells (HGMVECs) and human blood outgrowth endothelial cells (BOECs) in quiescent and activated state were pre-incubated with Eliglustat (Cerdelga®; glucosylceramide synthase inhibitor) or Agalsidase alpha (Replagal®; human cell derived alpha-galactosidase) in combination with various concentrations of Stx2a. Preincubation of endothelial cells with Agalsidase resulted in an increase of α-galactosidase activity in the cell, but had no effect on the binding of Stx to the cell surface when compared to control cells. However, the incubation of both types of endothelial cells incubated with or without the pro-inflammatory cytokine TNFα in combination with Eliglustat resulted in significant decrease of Stx binding to the cell surface, a decrease in protein synthesis by Stx2a, and diminished cellular Gb3 levels as compared to control cells. In conclusion, inhibition of the synthesis of Gb3 may be a potential future therapeutic target to protect against (further) endothelial damage caused by Stx.

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.

Sex differences in the association of sphingolipids with age in Dutch and South-Asian Surinamese living in Amsterdam, the Netherlands.

BACKGROUND: Men have a higher risk for cardiovascular disease (CVD) early in life, while women have a higher risk later in life. The sex-related differences in CVD risk, especially by age, could be related to sphingolipid metabolism. We compared plasma sphingolipid concentrations and its increase by age in men and women. METHODS: Plasma concentrations of 13 types of sphingolipids were measured by liquid chromatography-tandem mass spectrometry in a random subsample of 328 men and 372 women of Dutch and South-Asian Surinamese ethnic origin, participating in the HELIUS study. Sphingolipid concentrations were compared between men and women by age group (18-39, 40-55, and 56-70 years). Multiple linear regression was used to determine sex differences in age trends in sphingolipids stratified by ethnicity. Analyses were performed without adjustment and adjusted for body mass index (BMI) and waist circumference. RESULTS: At age 18-39 years, sphingolipid concentrations were lower in women than those in men, but at age 56-70 years this was reversed. At higher age, women showed higher concentrations than men. In line, we observed a more rapid increase of sphingolipid concentrations by age in women than in men. The observed sex differences were not explained by BMI or waist circumference. Patterns of sex differences were similar across ethnic groups, although the strength of associations differed. CONCLUSIONS: Mean sphingolipid concentrations increase more rapidly with age in women than in men. Therefore, plasma lipid concentrations of sphingolipids, although lower in women than in men at younger age, are higher in women than in men at older age.

Fumarase Deficiency: A Case With a New Pathogenic Mutation and a Review of the Literature.

Fumarase deficiency (FD) is a rare and severe autosomal disorder, caused by inactivity of the enzyme fumarase, due to biallelic mutations of the fumarase hydratase (FH) gene. Several pathogenic mutations have been published. The article describes an infant with failure to thrive, microcephaly, axial hypotonia, and developmental retardation with increased excretion of fumarate, no activity of fumarase and a homozygous mutation of the FH gene, which was until recently only known as a variant of unknown significance. Carriers of pathogenic mutations in the FH gene are at risk for developing renal cell carcinoma and should therefore be screened. Both parents were healthy carriers of the mutation and had decreased levels of enzyme activity. In addition, the article presents an overview and analysis of all cases of FD reported thus far in the literature.

Comparison of the Diagnostic Performance of C26:0-Lysophosphatidylcholine and Very Long-Chain Fatty Acids Analysis for Peroxisomal Disorders.

Peroxisomes are subcellular organelles that are involved in various important physiological processes such as the oxidation of fatty acids and the biosynthesis of bile acids and plasmalogens. The gold standard in the diagnostic work-up for patients with peroxisomal disorders is the analysis of very long-chain fatty acid (VLCFA) levels in plasma. Alternatively, C26:0-lysophosphatidylcholine (C26:0-LPC) can be measured in dried blood spots (DBS) using liquid chromatography tandem mass spectrometry (LC-MS/MS); a fast and easy method but not yet widely used. Currently, little is known about the correlation of C26:0-LPC in DBS and C26:0-LPC in plasma, and how C26:0-LPC analysis compares to VLCFA analysis in diagnostic performance. We investigated the correlation between C26:0-LPC levels measured in DBS and plasma prepared from the same blood sample. For this analysis we included 43 controls and 38 adrenoleukodystrophy (ALD) (21 males and 17 females) and 33 Zellweger spectrum disorder (ZSD) patients. In combined control and patient samples there was a strong positive correlation between DBS C26:0-LPC and plasma C26:0-LPC, with a Spearman's rank correlation coefficient of r (114) = 0.962, p < 0.001. These data show that both plasma and DBS are suitable to determine blood C26:0-LPC levels and that there is a strong correlation between C26:0-LPC levels in both matrices. Following this, we investigated how VLCFA and C26:0-LPC analysis compare in diagnostic performance for 67 controls, 26 ALD males, 19 ALD females, and 35 ZSD patients. For C26:0-LPC, all ALD and ZSD samples had C26:0-LPC levels above the upper limit of the reference range. For C26:0, one out of 67 controls had C26:0 levels above the upper reference range. For 1 out of 26 (1/26) ALD males, 1/19 ALD females and 3/35 ZSD patients, the C26:0 concentration was within the reference range. The C26:0/C22:0 ratio was within the reference range for 0/26 ALD males, 1/19 ALD females and 2/35 ZSD patients. Overall, these data demonstrate that C26:0-LPC analysis has a superior diagnostic performance compared to VLCFA analysis (C26:0 and C26:0/C22:0 ratio) in all patient groups. Based on our results we recommend implementation of C26:0-LPC analysis in DBS and/or plasma in the diagnostic work-up for peroxisomal disorders.

Contributions of amino acid, acylcarnitine and sphingolipid profiles to type 2 diabetes risk among South-Asian Surinamese and Dutch adults.

INTRODUCTION: People of South Asian origin are at high risk of type 2 diabetes (T2D), but the underpinning mechanisms are not fully understood. We determined ethnic differences in acylcarnitine, amino acid and sphingolipid concentrations and determined the associations with T2D. RESEARCH DESIGN AND METHODS: Associations between these metabolites and incident T2D among Dutch and South-Asian Surinamese were determined in participants from the Healthy Life in an Urban Setting (HELIUS) study (Amsterdam, the Netherlands) using Prentice-weighted Cox regression. The HELIUS study includes 95 incident T2D cases and a representative subcohort of 700 people from a cohort of 5977 participants with a mean follow-up of 4 years. RESULTS: Concentrations of acylcarnitines were comparable between both ethnic groups. Amino acid and lactosylceramide concentrations were higher among South-Asian Surinamese than Dutch (eg, isoleucine 65.7 (SD 16.3) vs 60.7 (SD 15.6) µmol/L). Ceramide concentrations were lower among South-Asian Surinamese than Dutch (eg, Cer d18:1 8.48 (SD 2.04) vs 9.08 (SD 2.29) µmol/L). Metabolic dysregulation preceded T2D without evidence for a multiplicative interaction by ethnicity. Most amino acids and (dihydro)ceramides were associated with increased risk (eg, Cer d18:1 HR 2.38, 95% CI 1.81 to 3.12) while acylcarnitines, glycine, glutamine and lactosylceramides were associated with decreased risk for T2D (eg, LacCer d18:2 HR 0.56, 95% CI 0.42 to 0.77). CONCLUSIONS: Overall, these data suggest that the disturbances underlying amino acid and sphingolipid metabolism may be predictive of T2D risk in populations of both South Asian and European background. These observations may be used as starting point to unravel the underlying metabolic disturbances.

Altered Levels of Sphingosine, Sphinganine and Their Ceramides in Atopic Dermatitis Are Related to Skin Barrier Function, Disease Severity and Local Cytokine Milieu.

Dysfunctional skin barrier plays a key role in the pathophysiology of atopic dermatitis (AD), a common inflammatory skin disease. Altered composition of ceramides is regarded as a major cause of skin barrier dysfunction, however it is not clear whether these changes are intrinsic or initiated by inflammation and aberrant immune response in AD. This study investigated the levels of free sphingoid bases (SBs) sphingosine and sphinganine and their ceramides and glucosylceramide in the stratum corneum (SC) and related them to skin barrier function, disease severity and local cytokine milieu. Ceramides were measured in healthy skin, and lesional and non-lesional skin of AD patients by a novel method based on deacylation of ceramides which were subsequently determined as corresponding sphingoid bases by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The cytokine levels were determined by multiplex immunoassay. Atopic skin showed increased levels of most investigated markers, predominantly in lesional skin. The largest difference in respect to healthy skin was found for glucosylceramide with respective median values of 0.23 (IQR 0.18-0.61), 0.56 (IQR 0.32-0.76) and 19.32 (IQR 7.86-27.62) pmol/g protein for healthy, non-lesional and lesional skin. The levels of investigated ceramide markers were correlated with disease severity (scoring atopic dermatitis, SCORAD) and skin barrier function (trans-epidermal water loss, TEWL) and furthermore with cytokines involved in innate, Th-1, and Th-2 immune response. Interestingly, the strongest association with SCORAD was found for sphinganine/sphingosine ratio (r = -0.69, p < 0.001; non-lesional skin), emphasizing the importance of SBs in AD. The highest correlation with TEWL was found for glucosylceramide (r2 = 0.60, p < 0.001), which was investigated for the first time in AD. Findings that the changes in SBs and ceramide levels were predominant in lesional skin and their association with disease severity and cytokine levels suggest an immune-system driven effect. a novel analysis method demonstrates a robust and simple approach that might facilitate wider use of lipid biomarkers in the clinics e.g., to monitor (immune) therapy or dissect disease endotypes.

Biochemical and imaging parameters in acid sphingomyelinase deficiency: Potential utility as biomarkers.

Acid Sphingomyelinase Deficiency (ASMD), or Niemann-Pick type A/B disease, is a rare lipid storage disorder leading to accumulation of sphingomyelin and its precursors primarily in macrophages. The disease has a broad phenotypic spectrum ranging from a fatal infantile form with severe neurological involvement (the infantile neurovisceral type) to a primarily visceral form with different degrees of pulmonary, liver, spleen and skeletal involvement (the chronic visceral type). With the upcoming possibility of treatment with enzyme replacement therapy, the need for biomarkers that predict or reflect disease progression has increased. Biomarkers should be validated for their use as surrogate markers of clinically relevant endpoints. In this review, clinically important endpoints as well as biochemical and imaging markers of ASMD are discussed and potential new biomarkers are identified. We suggest as the most promising biomarkers that may function as surrogate endpoints in the future: diffusion capacity measured by spirometry, spleen volume, platelet count, low-density lipoprotein cholesterol, liver fibrosis measured with a fibroscan, lysosphingomyelin and walked distance in six minutes. Currently, no biomarkers have been validated. Several plasma markers of lipid-laden cells, fibrosis or inflammation are of high potential as biomarkers and deserve further study. Based upon current guidelines for biomarkers, recommendations for the validation process are provided.

Capecitabine-based treatment of a patient with a novel DPYD genotype and complete dihydropyrimidine dehydrogenase deficiency.

Fluoropyrimidines are frequently used anti-cancer drugs. It is known that patients with reduced activity of dihydropyrimidine dehydrogenase (DPD), the key metabolic enzyme in fluoropyrimidine inactivation, are at increased risk of developing severe fluoropyrimidine-related toxicity. Upfront screening for DPD deficiency and dose reduction in patients with partial DPD deficiency is recommended and improves patient safety. For patients with complete DPD deficiency, fluoropyrimidine-treatment has generally been discouraged. During routine pretreatment screening, we identified a 59-year-old patient with a sigmoid adenocarcinoma who proved to have a complete DPD deficiency. Genetic analyses showed that this complete absence of DPD activity was likely to be caused by a novel DPYD genotype, consisting of a combination of amplification of exons 17 and 18 of DPYD and heterozygosity for DPYD*2A. Despite absence of DPD activity, the patient was treated with capecitabine-based chemotherapy, but capecitabine dose was drastically reduced to 150 mg once every 5 days (0.8% of original dose). Pharmacokinetic analyses showed that the area under the concentration-time curve (AUC) and half-life of 5-fluorouracil were respectively tenfold and fourfold higher than control values of patients receiving capecitabine 850 mg/m2 . When extrapolating from the dosing schedule of once every 5 days to twice daily, the AUC of 5-fluorouracil was comparable to controls. Treatment was tolerated well for eight cycles by the patient without occurrence of capecitabine-related toxicity. This case report demonstrates that a more comprehensive genotyping and phenotyping approach, combined with pharmacokinetically-guided dose administration, enables save fluoropyrimidine-treatment with adequate drug exposure in completely DPD deficient patients.

Catecholamines profiles at diagnosis: Increased diagnostic sensitivity and correlation with biological and clinical features in neuroblastoma patients.

INTRODUCTION: Neuroblastoma (NBL) accounts for 10% of the paediatric malignancies and is responsible for 15% of the paediatric cancer-related deaths. Vanillylmandelic acid (VMA) and homovanillic acid (HVA) are most commonly analysed in urine of NBL patients. However, their diagnostic sensitivity is suboptimal (82%). Therefore, we performed in-depth analysis of the diagnostic sensitivity of a panel of urinary catecholamine metabolites. PATIENTS AND METHODS: Retrospective study of a panel of 8 urinary catecholamine metabolites (VMA, HVA, 3-methoxytyramine [3MT], dopamine, epinephrine, metanephrine, norepinephrine and normetanephrine [NMN]) from 301 NBL patients at diagnosis. Special attention was given to subgroups, metaiodobenzylguanidine (MIBG) non-avid tumours and VMA/HVA negative patients. RESULTS: Elevated catecholamine metabolites, especially 3MT, correlated with nine out of 12 NBL characteristics such as stage, age, MYCN amplification, loss of heterozygosity for 1p and bone-marrow invasion. The combination of the classical markers VMA and HVA had a diagnostic sensitivity of 84%. NMN was the most sensitive single diagnostic metabolite with overall sensitivity of 89%. When all 8 metabolites were combined, a diagnostic sensitivity of 95% was achieved. Among the VMA and HVA negative patients, were also 29% with stage 4 disease, which usually had elevation of other catecholamine metabolites (93%). Diagnostic sensitivity for patients with MIBG non-avid tumour was improved from 33% (VMA and/or HVA) to 89% by measuring the panel. CONCLUSIONS: Our study demonstrates that analysis of a urinary catecholamine metabolite panel, comprising 8 metabolites, ensures the highest sensitivity to diagnose NBL patients.

A novel troponin I rule-out value below the upper reference limit for acute myocardial infarction.

OBJECTIVE: To determine cut-off values for a recently introduced high sensitive cardiac troponin assay (hs-cTnI) which provide similar sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) for acute myocardial infarction (AMI) as known cut-off values for an hs-cTnT assay. METHODS: A prospective observational study was performed. Hs-cTnT (Roche) and hs-cTnI (Abbott) were measured in consecutive patients with symptoms suggestive of AMI. Representative measurements (obtained at least 3 h after chest pain has started) and serial measurements with a time delay between 2.5 h and 4.5 h were used to determine cut-off levels. Two independent clinicians adjudicated the final diagnosis. RESULTS: 1490 patients were included in the study of whom 114 (8%) received a final diagnosis of AMI. Receiver operating characteristics analysis showed no statistically significant differences in the areas under the curve between the two assays. Cut-off values for representative hs-TnI were found to be as follows: rule-out: 10 ng/L (sensitivity: 98.2%; 95% CI 95.7% to 100.0% and NPV: 99.8%; 99.5% to 100.0%); rule-in: 70 ng/L (specificity: 90.8%; 89.3% to 92.4% and PPV: 39.7%; 36.1% to 43.3%). For serial measurements we found a Δ rule-out cut-off value of 20 ng/L (sensitivity: 94.9%; 88.0% to 100.0% and NPV: 98.7%; 96.9% to 100.0%) and Δ rule-in cut-off values of 100 ng/L (specificity: 92.7%; 87.9% to 95.8% and PPV: 57.6%; 39.4% to 74.0%) and 300% (specificity: 93.8%; 90.4% to 97.2% and PPV: 61.3%; 51.1% to 71.5%). CONCLUSIONS: Cut-off values for hs-cTnI measurements are determined which allow a similar diagnostic classification as compared with hs-cTnT. Importantly, for a rule-out paradigm this cut-off value is unmistakably lower than the upper reference limit.

PAK2 is an effector of TSC1/2 signaling independent of mTOR and a potential therapeutic target for Tuberous Sclerosis Complex.

Tuberous sclerosis complex (TSC) is caused by inactivating mutations in either TSC1 or TSC2 and is characterized by uncontrolled mTORC1 activation. Drugs that reduce mTOR activity are only partially successful in the treatment of TSC, suggesting that mTOR-independent pathways play a role in disease development. Here, kinome profiles of wild-type and Tsc2(-/-) mouse embryonic fibroblasts (MEFs) were generated, revealing a prominent role for PAK2 in signal transduction downstream of TSC1/2. Further investigation showed that the effect of the TSC1/2 complex on PAK2 is mediated through RHEB, but is independent of mTOR and p21RAC. We also demonstrated that PAK2 over-activation is likely responsible for the migratory and cell cycle abnormalities observed in Tsc2(-/-) MEFs. Finally, we detected high levels of PAK2 activation in giant cells in the brains of TSC patients. These results show that PAK2 is a direct effector of TSC1-TSC2-RHEB signaling and a new target for rational drug therapy in TSC.

Sarcoidosis-related hypercalcaemia due to production of parathyroid hormone-related peptide.

Hypercalcaemia is frequently observed in patients with sarcoidosis. This is classically attributed to ectopic production of 1,25 dihydroxy vitamin D by sarcoid granulomas. We present a case of sarcoidosis-related hypercalcaemia with normal vitamin D levels. In this patient, production of parathyroid hormone-related peptide (PTHrp) was the cause for sarcoidosis-induced hypercalcaemia. As such, plasma PTHrp levels were increased and bone marrow granulomas stained positively for PTHrp expression. Medium-dose prednisolone treatment improved symptoms of sarcoidosis and normalised serum calcium, and PTHrp concentrations. Thus, production of PTHrp may be the cause for hypercalcaemia in some patients with sarcoidosis.