Kristel Klaassen

6-mercaptopurine influences TPMT gene transcription in a TPMT gene promoter variable number of tandem repeats-dependent manner

AIM TPMT activity is characterized by a trimodal distribution, namely low, intermediate and high methylator. TPMT gene promoter contains a variable number of GC-rich tandem repeats (VNTRs), namely A, B and C, ranging from three to nine repeats in length in an A(n)B(m)C architecture. We have previously shown that the VNTR architecture in the TPMT gene promoter affects TPMT gene transcription. MATERIALS, METHODS & RESULTS: Here we demonstrate, using reporter assays, that 6-mercaptopurine (6-MP) treatment results in a VNTR architecture-dependent decrease of TPMT gene transcription, mediated by the binding of newly recruited protein complexes to the TPMT gene promoter, upon 6-MP treatment. We also show that acute lymphoblastic leukemia patients undergoing 6-MP treatment display a VNTR architecture-dependent response to 6-MP. CONCLUSION These data suggest that the TPMT gene promoter VNTR architecture can be potentially used as a pharmacogenomic marker to predict toxicity due to 6-MP treatment in acute lymphoblastic leukemia patients.

Molecular and phenotypic characteristics of seven novel mutations causing branched-chain organic acidurias

Specific mitochondrial enzymatic deficiencies in the catabolism of branched‐chain amino acids cause methylmalonic aciduria (MMA), propionic acidemia (PA) and maple syrup urine disease (MSUD). Disease‐causing mutations were identified in nine unrelated branched‐chain organic acidurias (BCOA) patients. We detected eight previously described mutations: p.Asn219Tyr, p.Arg369His p.Val553Glyfs*17 in MUT, p.Thr198Serfs*6 in MMAA, p.Ile144_Leu181del in PCCB, p.Gly288Valfs*11, p.Tyr438Asn in BCKDHA and p.Ala137Val in BCKDHB gene. Interestingly, we identified seven novel genetic variants: p.Leu549Pro, p.Glu564*, p.Leu641Pro in MUT, p.Tyr206Cys in PCCB, p.His194Arg, p.Val298Met in BCKDHA and p.Glu286_Met290del in BCKDHB gene. In silico and/or eukaryotic expression studies confirmed pathogenic effect of all novel genetic variants. Aberrant enzymes p.Leu549Pro MUT, p.Leu641Pro MUT and p.Tyr206Cys PCCB did not show residual activity in activity assays. In addition, activity of MUT enzymes was not rescued in the presence of vitamin B12 precursor in vitro which was in accordance with non‐responsiveness or partial responsiveness of patients to vitamin B12 therapy. Our study brings the first molecular genetic data and detailed phenotypic characteristics for MMA, PA and MSUD patients for Serbia and the whole South‐Eastern European region. Therefore, our study contributes to the better understanding of molecular landscape of BCOA in Europe and to general knowledge on genotype–phenotype correlation for these rare diseases.

Influence of Promoter Polymorphisms of the TNF-α (-308G/A) And IL-6 (-174G/C) Genes On Therapeutic Response To Etanercept In Rheumatoid Arthritis

Summary Background: The study was undertaken to assess the influence of functional -308G/A TNF-α (rs 1800629) and -174G/C IL-6 (rs1800795) promoter polymorphisms on the therapeutic response to etanercept, a TNF-α blocker, in patients with rheumatoid arthritis (RA). Methods: Seventy-three patients suffering from active RA were studied, at baseline and 6 and 12 months after therapy. The therapeutic response was estimated according to the European League Against Rheumatism response criteria. Patients were genotyped for -308G/A TNF-α and -174G/C IL-6 polymorphisms by the PCR-RFLP method, and the influence of genotype on etanercept response was assessed. Results: No difference in the percentage of responders (patients who had DAS28 improvement > 1.2) between patients with the TNF-α-308GG and GA and AA genotype was detected after 6 and 12 months of treatment. After 12 months of treatment the percentage of responders was significantly increased in patients with the IL-6 -174GG genotype compared with those with the GC or CC genotype (p=0.006 by Chi-square test). Evaluation of the patients according to their combined IL-6/TNF-α genotypes showed that patients with the IL-6 -174GG / TNF-α -308GG genotype were more frequent among the responders compared to those with other combined genotypes (p=0.022 by Chisquare test). More precisely, all patients with the combined IL-6 -174GG / TNF-α -308GG genotype were responders after 12 months of etanercept treatment. Conclusions: The study suggests that patients who are genetically low TNF-α and IL-6 producers are the best responders to etanercept therapy.

Genetic characterization of GSD I in Serbian population revealed unexpectedly high incidence of GSD Ib and 3 novel SLC37A4 variants: SKAKICet al.

Glycogen storage disease (GSD) type I is inborn metabolic disease characterized by accumulation of glycogen in multiple organs. We analyzed 38 patients with clinical suspicion of GSD I using Sanger and next‐generation sequencing (NGS). We identified 28 GSD Ib and 5 GSD Ia patients. In 5 patients, GSD III, VI, IX, cholesteryl‐ester storage disease and Shwachman‐Diamond syndrome diagnoses were set using NGS. Incidences for GSD Ia and GSD Ib were estimated at 1:172 746 and 1:60 461 live‐births, respectively. Two variants were identified in G6PC gene: c.247C>T (p.Arg83Cys) and c.518T>C (p.Leu173Pro). In SLC37A4 gene, 6 variants were detected. Three previously reported variants c.81T>A (p.Asn27Lys), c.162C>A (p.Ser54Arg) and c.1042_1043delCT (p.Leu348Valfs*53) accounted for 87% of all analyzed alleles. Computational, transcription studies and/or clinical presentation in patients confirmed pathogenic effect of 3 novel variants: c.248G>A (p.Gly83Glu), c.404G>A (p.Gly135Asp) and c.785G>A (p.Ser263Glyfs*33 or p.Gly262Asp). In the cohort, hepatomegaly, hypoglycemia and failure to thrive were the most frequent presenting signs of GSD Ia, while hepatomegaly and recurrent bacterial infections were clinical hallmarks of GSD Ib. All GSD Ib patients developed neutropenia while 20.6% developed inflammatory bowel disease. Our study revealed the highest worldwide incidence of GSD Ib. Furthermore, description of 3 novel variants will facilitate medical genetic practice.

Functional Characterization of Novel Phenylalanine Hydroxylase p.Gln226Lys Mutation Revealed Its Non-responsiveness to Tetrahydrobiopterin Treatment in Hepatoma Cellular Model

Treatment with tetrahydrobiopterin (BH4) is the latest therapeutic option approved for patients with phenylketonuria (PKU)—one of the most frequent inborn metabolic diseases. PKU or phenylalanine hydroxylase (PAH) deficiency is caused by mutations in the PAH gene. Given that some PAH mutations are responsive to BH4 treatment while others are non-responsive, for every novel mutation that is discovered it is essential to confirm its pathogenic effect and to assess its responsiveness to a BH4 treatment in vitro, before the drug is administered to patients. We found a c.676C>A (p.Gln226Lys) mutation in the PAH gene in two unrelated patients with PKU. The corresponding aberrant protein has never been functionally characterized in vitro and its response to BH4 treatment is unknown. Computational analyses proposed that glutamine at position 226 is an important, evolutionary conserved amino acid while the substitution with lysine probably disturbs tertiary protein structure and impacts posttranslational PAH modifications. Using hepatoma cellular model, we demonstrated that the amount of mutant p.Gln226Lys PAH detected by Western blot was only 1.2% in comparison to wild-type PAH. The addition of sepiapterin, intracellular precursor of BH4, did not increase PAH protein yield thus marking p.Gln226Lys as BH4-non-responsive mutation. Therefore, computational, experimental, and clinical data were all in accordance showing that p.Gln226Lys is a severe pathogenic PAH mutation. Its non-responsiveness to BH4 treatment in hepatoma cellular model should be considered when deciding treatment options for PKU patients carrying this mutation. Consequently, our study will facilitate clinical genetic practice, particularly genotype-based stratification of PKU treatment.

Tetrahydrobiopterin deficiency among Serbian patients presenting with hyperphenylalaninemia

Abstract Hyperphenylalaninemia (HPA) [phenylketonuria (PKU) and tetrahydrobiopterin (BH4) deficiencies] is rare inborn metabolic disease characterized by elevated phenylalanine level in body fluids. In Serbia, 62 HPA patients have been identified through newborn screening since 1983. However, pterin pattern analysis is not performed. We present a patient initially diagnosed and treated as classical PKU. At 3 years of age, during infection with H1N1 influenza A virus, the patient first developed a neurologic crisis with encephalopathy and dystonic movements. We suspected that the patient is the first case of BH4 deficiency identified in Serbia. Genetic analyses showed that the patient does not have disease-causing variants of the PAH gene and carries a p.Asp136Val mutation in homozygous state in the PTS gene. For patients with treatable rare diseases, like PKU and BH4 deficiencies, correct diagnosis is crucial for the implementation of optimal treatment. If biochemical tests needed for differential diagnosis are not available, our experience emphasizes the necessity of immediate genetic testing after newborn screening.

Importance of TLR9-IL23-IL17 axis in inflammatory bowel disease development: Gene expression profiling study

BACKGROUND AND AIMS Mucosal gene expression have not been fully enlightened in inflammatory bowel disease (IBD). Aim of this study was to define IL23A, IL17A, IL17F and TLR9 expression in different IBD phenotypes. METHODS Evaluation of mRNA levels was performed in paired non-inflamed and inflamed mucosal biopsies of newly diagnosed 50 Crohns disease (CD) and 54 ulcerative colitis (UC) patients by quantitative real-time PCR analysis. RESULTS IL17A and IL17F expression levels were significantly increased in inflamed IBD mucosa. Inflamed CD ileal and UC mucosa showed increased IL23A, while only inflamed CD ileal samples showed increased TLR9 mRNA level. Correlation between analysed mRNAs levels and endoscopic and clinical disease activity were found in UC, but only with clinical activity in CD. CONCLUSION Both CD and UC presented expression of Th17-associated genes. Nevertheless, expression profiles between different disease forms varies which should be taken into account for future research and therapeutics strategies.

Functional Analysis of an (A)γ-Globin Gene Promoter Variant (HBG1: g.-225_-222delAGCA) Underlines Its Role in Increasing Fetal Hemoglobin Levels Under Erythropoietic Stress.

Abstract Hereditary persistence of fetal hemoglobin (HPFH) is a condition characterized by persistent γ-globin gene expression and synthesis of high levels of fetal hemoglobin (Hb F; α2γ2) during adult life. It is usually caused by promoter variants or large deletions affecting the human fetal globin (HBG1 and HBG2) genes. Some of these HPFH-causing variants, such as HBG2: g.-158 C > T, exert their effect only under conditions of erythropoietic stress, typical for β-thalassemia (β-thal) patients. Namely, the presence of HBG2: g.-158 C > T favors a higher Hb F response, while it has little effect in healthy individuals. We analyzed a previously reported deletion residing in the promoter region of the HBG1 gene (HBG1: g.-225_-222delAGCA), both in normal conditions and under conditions of erythropoietic stress. Our results indicate that this deletion is responsible for decreased HBG1 gene expression. Specifically, this deletion was shown to result in drastically reduced reporter gene expression in K562 cells, compared to the wild-type sequence but only under conditions of erythropoietic stress, mimicked by introduction of erythropoietin (EPO) into the cell culture. Also, electrophoretic mobility shift analysis showed that the HBG1: g.-225_-222delAGCA deletion creates additional transcriptional factors’ binding sites, which, we propose, bind a transcriptional repressor, thus decreasing the HBG1 gene promoter activity. These results are consistent with in silico analysis, which indicated that this deletion creates a binding site for GATA1, known to be a repressor of the γ-globin gene expression. These data confirm the regulatory role of the HBG1: g.-225_-222 region that exerts its effect under conditions of erythropoietic stress characteristic for β-thal patients.

MOLECULAR CHARACTERISTICS, PHENOTYPIC DIVERSITY AND GENOTYPE-ESTIMATED THERAPEUTIC RESPONSIVENESS OF SERBIAN PATIENTS WITH PHENYLKETONURIA MOLEKULARNE KARAKTERISTIKE, FENOTIPSKA RAZNOLIKOST I PROCENA ODGOVORA NA TERAPIJU ZASNOVANA NA GENOTIPU KOD SRPSKIH PACIJENATA SA FENILKETONURIJOM

Summary Phenylketonuria (PKU) is a rare, inherited metabolic disease which is transmitted in an autosomal recessive pattern. PKU is caused by mutations in the gene encoding the phenylalanine hydroxylase (PAH) enzyme. This review cites the most prominent methods for the detection of mutations in the PAH gene. Since the image of PKU transcends »simple« monogenic disease, the known data on non-coding PAH gene variants and their role and PKU modifier genes have been further reviewed. It has been shown that there is a significant correlation between mutant PAH genotypes and PKU phenotypes. However, genotype-phenotype correlation inconsistencies have also been found. This review discusses the possible causes of phenotypic inconsistencies, such as oversight of more than two mutations present in the patient’s PAH genotype, pitfalls of patient phenotypic classification (plasma phenylalanine concentration and phenylalanine tolerance), the inter-allelic complementation (positive and negative) phenomenon. A new therapeutic approach, tetrahydrobiopterin (BH4) supplementation therapy, is an important innovation in the course of PKU patients’ treatment. How - ever, in countries where the BH4-loading test and BH4-supplementation therapy are not available, a genotype-based estimation of responsiveness to the therapy is a valuable approach. It enables BH4-potential benefit estimation, which provides vital information both for the patient and for the population. An optimal molecular diagnostics algorithm, established according to the published mutation frequencies in Serbian PKU patients, has been suggested. In the future, the molecular-genetic algorithm for PKU could be expanded to include a variety of transcriptional regulatory elements located in noncoding PAH gene regions and yet to be discovered modifier genes. Kratak sadržaj Fenilketonurija (PKU) jeste retko metaboličko oboljenje koje se nasleđuje autozomalno recesivno. Uzrok PKU su mutacije u genu koji kodira za enzim fenilalanin-hidroksilazu (PAH). U ovom revijskom radu su opisane najznačajnije metode za detekciju mutacija u PAH genu. Budući da slika o PKU prevazilazi »jednostavno« monogensko oboljenje, prikazani su i podaci o varijantama u nekodirajućim regionima PAH gena, kao i genima modifikatorima za PKU. Iako postoji značajna korelacija između PAH geno tipa i PKU fenotipa, uočene su i neusaglašenosti. U ovom revijskom radu su izneti mogući uzroci za neusaglašenost između genotipa i fenotipa, kao što su: propust u detekciji više od dve mutacije prisutne u PAH genotipu pacijenta, neusaglašenost u fenotipskoj klasifikaciji pacijenata (koncentracija fenilalanina u serumu pre terapije i tolerancija fenilalanina), fenomen interalelske komplemen - tacije (pozitivne i negativne). Novi terapijski pristup, te rapija tetrahidrobio pterinom (BH4), predstavlja važnu inova ciju u lečenju pacijenata sa PKU. Međutim, u zemljama u kojima test opte re će nja sa BH4 i terapija sa BH4 nisu dostupni, procena odgovora na terapiju koja je zasnovana na ge no tipu predstavlja koristan pristup. Ovakav pristup omo gućava procenu potencijalne koristi od terapije sa BH4 što je bitna informacija, kako za pacijenta, tako i na nivou populacije. Predložen je i optimalni algoritam za molekularnu dijagnostiku, uspostavljen prema objavljenim učestalostima mutacija kod pacijenata sa PKU u Srbiji. U budućnosti, molekularno-genetički algoritam za PKU bi mogao da bude proširen tako da uključi i različite transkripcione regulatorne elemente u nekodirajućim regionima PAH gena, kao i gene modifikatore koji tek treba da budu otkriveni.

Association of Mitochondrial DNA Variants and Cognitive Impairment of Phenylketonuria Patients / POVEZANOST VARIJANTI U MITOHONDRIJALNOJ DNK I KOGNITIVNOG FENOTIPA KOD PACIJENATA SA FENILKETONURIJOM

Summary Background: Phenylketonuria (PKU) is a metabolic disorder caused by phenylalanine hydroxylase gene (PAH) mutations. If left untreated, PKU patients develop severe mental retardation potentially due to neurodegeneration. This is the first study that investigates presence of mitochondrial DNA variants in PKU patients, m.10398A, reportedly associated with neurodegenerative diseases and m.10410T. Methods: We analyzed 64 PKU patients and 50 healthy controls from Serbian population. PKU patients were categorized into groups according to time of diagnosis and compliance to low-phenylalanine diet. The IQ was determined according to age-appropriate scales. Results: We detected m.10398A and m.10410T variants by direct sequencing. Frequency of m.10398A was similar in patients and healthy controls (82.81% and 82.00% respectively) suggesting their identical ethnic background. No variation was detected for m.10410. In group with late diagnosis and poorly controlled diet, no statistically significant difference in average IQ was found between patients with m.10398A and m.10398G. The same was shown for PKU patients with higher IQ, diagnosed at neonatal screening and treated with low-phenylalanine diet. However, when patients carrying p.L48S, a PAH mutation with inconsistent effect, were excluded from the study, presence of m.10398A variant was associated with lower IQ. Conclusions: This study emphasizes the importance of neonatal screening and good control of low-phenylalanine diet in PKU patients. Statistical analysis did not indicate clear impact of mitochondrial DNA variant m.10398A on IQ of PKU patients, except when PAH genotype was also considered. Studies in larger cohorts will elucidate the association between PAH gene mutations, mitochondrial DNA variants and complex PKU cognitive phenotype. Kratak sadržaj Uvod: Fenilketonurija (PKU) je metaboličko oboljenje uzro- kovano mutacijama u genu za fenilalanin hidroksilazu (PAH). Ukoliko se ne leče, pacijenti sa fenilketonurijom razvijaju tešku mentálnu retardaciju, koja može biti i posledica neu- rodegeneracije. Ovo je prva študija koja istražuje prisustvo mitohondrijalnih DNK varijanti kod pacijenata sa fenilketonurijom, m.10398A, za koju je pokazana povezanost sa neurodegenerativnim oboljenjima, kao i m.10410T. Metode: U ovoj študiji je analizirano 64 pacijenta sa fenilke- tonurijom i 50 zdravih kontrola iz srpske populacije. Pacijenti su podeljeni u grupe prema uzrastu u kome je postav- ljena dijagnoza i odnosu prema dijeti sa niskim sadržajem fenilalanina. IQ je odreden pomoču odgovarajučih uzrasnih skala. Rezultati: Mitohondrijalne DNK varijante, m.10398A i m.10410T, su detektovane sekvenciranjem. Učestalost m.10398A je bila jednaka kod pacijenata i zdravih kontro- la (82,81% i 82,00%), što ukazuje na isto etničko poreklo ovih ispitanika. U slučaju m.10410, nisu detektovane različite varijante. U grupi pacijenata kojima je kasno postavlje- na dijagnoza i koji su neadekvatno lečeni, nije pronadena statistička značajnost u srednjoj vrednosti IQ izmedu paci- jenata sa m.10398A i m.10398G. Isto je pokazano i za pacijente sa visim IQ koji su otkriveni prilikom neonatalnog skrininga i koji su pravilno lečeni. Medutim, kada su iz ove grupe isključeni pacijenti koji nose p.L48S mutaciju u PAH genu, koja ima nekonzistentan uticaj na fenotip, prisustvo m.10398A alela je povezano sa nižim IQ. Zaključak: Ova študija je potvrdila važnost neonatalnog skrininga i pravilnog sprovodenja dijete kod pacijenata sa fenilketonurijom. Štatistické analize nisu jasno utvrdile uti- caj mitohondrijalne DNK varijante, m.10398A, na IQ ovih pacijenata, osim kad je i PAH genotip uključen u analizu. Študije u večim grupama če rasvetliti povezanost izmedu mutacija u PAH genu, mitohondrijalnih DNKvarijanti i slo- ženog kognitivnog fenotipa kod pacijenata sa PKU.