Hana Vinohradská

Targeted metabolomic analysis of plasma samples for the diagnosis of inherited metabolic disorders

Metabolomics has become an important tool in clinical research and diagnosis of human diseases. In this work we focused on the diagnosis of inherited metabolic disorders (IMDs) in plasma samples using a targeted metabolomic approach. The plasma samples were analyzed with the flow injection analysis method. All the experiments were performed on a QTRAP 5500 tandem mass spectrometer (AB SCIEX, U.S.A.) with electrospray ionization. The compounds were measured in a multiple reaction monitoring mode. We analyzed 50 control samples and 34 samples with defects in amino acid metabolism (phenylketonuria, maple syrup urine disease, tyrosinemia I, argininemia, homocystinuria, carbamoyl phosphate synthetase deficiency, ornithine transcarbamylase deficiency, nonketotic hyperglycinemia), organic acidurias (methylmalonic aciduria, propionic aciduria, glutaric aciduria I, 3-hydroxy-3-methylglutaric aciduria, isovaleric aciduria), and mitochondrial defects (medium-chain acyl-coenzyme A dehydrogenase deficiency, carnitine palmitoyltransferase II deficiency). The controls were distinguished from the patient samples by principal component analysis and hierarchical clustering. Approximately 80% of patients were clearly detected by absolute metabolite concentrations, the sum of variance for first two principle components was in the range of 44-55%. Other patient samples were assigned due to the characteristic ratio of metabolites (the sum of variance for first two principle components 77 and 83%). This study has revealed that targeted metabolomic tools with automated and unsupervised processing can be applied for the diagnosis of various IMDs.

The TREC/KREC assay for the diagnosis and monitoring of patients with DiGeorge syndrome.

DiGeorge syndrome (DGS) presents with a wide spectrum of thymic pathologies. Nationwide neonatal screening programs of lymphocyte production using T-cell recombination excision circles (TREC) have repeatedly identified patients with DGS. We tested what proportion of DGS patients could be identified at birth by combined TREC and kappa-deleting element recombination circle (KREC) screening. Furthermore, we followed TREC/KREC levels in peripheral blood (PB) to monitor postnatal changes in lymphocyte production. Methods TREC/KREC copies were assessed by quantitative PCR (qPCR) and were related to the albumin control gene in dry blood spots (DBSs) from control (n = 56), severe immunodeficiency syndrome (SCID, n = 10) and DGS (n = 13) newborns. PB was evaluated in DGS children (n = 32), in diagnostic samples from SCID babies (n = 5) and in 91 controls. Results All but one DGS patient had TREC levels in the normal range at birth, albeit quantitative TREC values were significantly lower in the DGS cohort. One patient had slightly reduced KREC at birth. Postnatal DGS samples revealed reduced TREC numbers in 5 of 32 (16%) patients, whereas KREC copy numbers were similar to controls. Both TREC and KREC levels showed a more pronounced decrease with age in DGS patients than in controls (p<0.0001 for both in a linear model). DGS patients had higher percentages of NK cells at the expense of T cells (p<0.0001). The patients with reduced TREC levels had repeated infections in infancy and developed allergy and/or autoimmunity, but they were not strikingly different from other patients. In 12 DGS patients with paired DBS and blood samples, the TREC/KREC levels were mostly stable or increased and showed similar kinetics in respective patients. Conclusions The combined TREC/KREC approach with correction via control gene identified 1 of 13 (8%) of DiGeorge syndrome patients at birth in our cohort. The majority of patients had TREC/KREC levels in the normal range.

Controlled diet in phenylketonuria and hyperphenylalaninemia may cause serum selenium deficiency in adult patients: the Czech experience

Phenylketonuria is an inherited disorder of metabolism of the amino acid phenylalanine caused by a deficit of the enzyme phenylalanine hydroxylase. It is treated with a low-protein diet containing a low content of phenylalanine to prevent mental affection of the patient. Because of the restricted intake of high-biologic-value protein, patients with phenylketonuria may have lower than normal serum concentrations of pre-albumin, selenium, zinc and iron. The objective of the present study was to assess the compliance of our phenylketonuric (PKU) and hyperphenylalaninemic (HPA) patients; to determine the concentration of serum pre-albumin, selenium, zinc and iron to discover the potential correlation between the amount of proteins in food and their metabolic control. We studied 174 patients of which 113 were children (age 1–18), 60 with PKU and 53 with HPA and 61 were adults (age 18–42), 51 with PKU and 10 with HPA. We did not prove a statistically significant difference in the concentration of serum pre-albumin, zinc and iron among the respective groups. We proved statistically significant difference in serum selenium concentrations of adult PKU and HPA patients (p = 0.006; Mann–Whitney U test). These results suggest that controlled low-protein diet in phenylketonuria and hyperphenylalaninemia may cause serum selenium deficiency in adult patients.

Long-term treatment for hyperphenylalaninemia and phenylketonuria: a risk for nutritional vitamin B12 deficiency?

Abstract Purpose: The objective of the study was to determine the incidence of vitamin B12 deficiency in patients under long-term treatment for phenylketonuria (PKU) and hyperphenylalaninemia (HPA), as well as its associations with B12 vitamin parameters (holotranscobalamin – active vitamin B12, serum folate, total plasma homocysteine, and plasma methylmalonic acid concentration). Patients and methods: The group consisted of 51 PKU (n=29) and HPA (n=22) patients aged 3–48 years (28 children, 23 adults). Results: A significant difference in serum folate levels was discovered between adult HPA patients and PKU patients (p=0.004, Mann-Whitney U-test). A significant difference in plasma homocysteine concentrations within the normal levels (p=0.032, χ2-test) was detected between adult HPA and PKU patients. In the group of adults, we also found significant differences in serum holotranscobalamin concentrations regarding both concentration levels and the proportion of patients with concentrations within the normal levels (p=0.031, Mann-Whitney U-test; p=0.006, χ2-test). Conclusion: We have proven that adult patients with PKU and HPA are at risk of vitamin B12 nutritional deficiency. The most effective parameter for these adults is the monitoring of holotranscobalamin in the serum.

Neonatal screening in the Czech Republic: increased prevalence of selected diseases in low birthweight neonates

Neonates with low birthweight (LBW) represent a vulnerable population. This retrospective study analyzed the birth frequency of diseases detected by neonatal screening (NBS) in normal and LBW neonates in the Czech Republic. Between years 2002 and 2016, the number of screened disorders in the Czech Republic gradually increased from two to 13. Prevalence of screened diseases was calculated for cohorts ranging from 777,100 to 1,277,283 neonates stratified by birthweight. Odds ratio of the association of LBW with each disease was calculated and statistical significance was evaluated using the chi-square test or Fisher’s exact test, as appropriate. Three diseases were associated with higher risk of prevalence in LBW neonates, namely congenital hypothyroidism (OR 2.50, CI 1.92; 3.25), cystic fibrosis (OR 2.44, CI 1.51; 3.94), and long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) (OR 7.74, CI 2.18; 27.42).Conclusion: Although the underlying mechanisms are not well understood, results can be hypothesized that LBW (respectively prematurity) may lead to the secondary and often transitory hypothyroidism while cystic fibrosis and LCHADD may manifest already prenatally and result into preterm birth and LBW.What is Known:• The percentage of low birthweight (LBW) neonates in the Czech Republic has been increasing.• Previously published studies reported positive association between LBW and congenital hypothyroidism and cystic fibrosis.What is New:• The association between LCHADD and LBW has not yet been described.• LBW can be the first manifestation of cystic fibrosis and LCHADD.

PO-0136 Increased Risk Of Vitamin B12 Nutritional Deficiency In Long-term Treated Patients With Phenylketonuria And Hyperfenylalaninemia

The aim of this study was to assess the prevalence of nutritional deficiency of vitamin B12 in long-term treated patients with phenylketonuria (PKU) and hyperfenylalaninemia (HPA), together with parameters of vitamin B12 and metabolic control. Methods In 51 patients aged 3–48 years (28 children, 23 adults) was examined levels of active vitamin B12 in serum, folate concentration in blood, plasma homocysteine and methylmalonic acid concentrations in urine. Results We found a statistically significant difference between the levels of folate in the blood among patients with PKU and HPA (p = 0.046, Mann Whitney U test). This difference was also statistically significant for adults with HPA and PKU (p = 0.004, Mann Whitney U test). There was a statistically significant difference in the proportion of normal homocysteine concentrations in plasma in the overall evaluation of both groups (p = 0.023, chi -square test). This difference was also statistically significant in adults with HPA and PKU (p = 0.032, chi -square test). In the group of adults we detected a statistically significant difference in the concentrations of active vitamin B12 in the blood as in the evaluation of the concentration and the proportion of patients with normal levels (p = 0.031, Mann Whitney U test, p = 0.006, chi -square test). Conclusions In the analysed group of patients we demonstrated that our patients are at risk of vitamin B12 nutritional deficiency and the risk increases with age.

1037 Determination of Prealbumin, Selenium, Zinc and Iron Concentration in Serum for Monitoring the Nutrition Status of Phenylketonuric and Hyperphenylalaninemic Patients

Background and Aims Phenylketonuria is an inherited disorder of metabolism of the amino acid phenylalanine caused by a deficit of the enzyme phenylalaninhydroxylase. It is treated with a low-protein diet containing a low content of phenylalanine to prevent mental affection of the patient. The objective of the present study was to assess the compliance of our phenylketonuric (PKU) and hyperphenylalaninemic (HPA) patients; to determine the concentration of serum pre-albumin and trace elements to discover the potential correlation between the amount of proteins in food and their metabolic control. Methods The prospective study contained altogether 174 patients, of which 113 were children, 60 with PKU and 53 with HPA and 61 were adults, 51 with PKU and 10 with HPA. Results We did not prove a statistically significant difference in the levels of serum pre-albumin, zinc and iron among the respective groups. We proved statistically significant difference in the level of serum selenium among PKU and HPA patients in adulthood (p=0.006, Mann-Whitney U test). Conclusion The therapeutic restrictive diet for PKU and HPA makes the patient liable to the risk of nutritional deficit.