Robert Petrovič

Adult onset cerebral form of X-linked adrenoleukodystrophy with dementia of frontal lobe type with new L160P mutation in ABCD1 gene

BACKGROUND X-linked adrenoleukodystrophy is a genetically determined disorder that causes varying degrees of malfunction of the adrenal cortex and central nervous system and is characterized by abnormally high levels of very long chain fatty acid in tissues and body fluids. The gene ABCD1, responsible for X-ALD, has been mapped on chromosome Xq28. More than 500 different mutations have been reported but no correlation between genotype and phenotype has been found. OBJECTIVES To investigate the occurrence of known or new mutations in the ABCD1 gene in patients with clinically and biochemical proven adrenoleukodystrophy. PATIENT AND METHODS A 37-year-old patient with history of one-year progression of personality and behavioral changes such as, fluctuation of apathy and euphoria, perseveration, bizarre affect, and general disengagement, preliminarily assessed as adrenoleukodystrophy has undergone a clinical, biochemical and genetic examination in order to confirm the diagnosis and discover a possible mutation. RESULTS The clinical examination has shown signs of the severe prefrontal syndrome, and a neurological examination disclosed deliberation signs and a spastic quadruparesis predominantly on the lower extremities. MRIs showed confluent hyperintensive lesions in T2 and FLAIR images in both hemispheres with severe progression over 6 to 12 months. Clinical findings referred to adrenoleukodystrophy, consecutively performed genetic analyses showed missense mutation at the codon 479 (T>C) in exon 1 of ABCD 1 gene, predicting the substitution L160P in ALD protein. The same mutation has also been found in patients mother. CONCLUSION We examined a patient with progressive development of early onset frontal lobe type dementia and upper motor neuron signs in which neuroimaging methods and biochemical tests refer to adrenoleukodystrophy. Genetic tests revealed a new mutation at position L160P in ALD protein.

Frequency of selected single nucleotide polymorphisms influencing the warfarin pharmacogenetics in Slovak population

Warfarin represents the most commonly prescribed oral anticoagulants, which functions as an antagonist of vitamin K, an essential factor of blood coagulation cascade. Warfarin has a narrow therapeutic index. An insufficient dose can cause failure of antithrombotic effect, and an overdose increases a risk of bleeding. It is known that variability in two genes (CYP2C9 and VKORC1) has a significant effect on individual response to warfarin dose. These polymorphisms influence more than one‐third of known warfarin dose effect. Pharmacogenetics of warfarin is less affected by polymorphisms in the other genes such as CYP4F2, CYP2C19, and GGCX.

Frequencies of polymorphisms in CYP2C9 and VKORC1 genes influencing warfarin metabolism in Slovak population: implication for clinical practice.

OBJECTIVES The study was aimed at establishing an effective molecular-genetic method for detecting polymorphisms in genes CYP2C9 and VKORC1, which affect the pharmacogenetics of warfarin, and at determining their prevalence in Slovak population. BACKGROUND Warfarin, derivative of coumarin, belongs to the most commonly prescribed oral anticoagulants with narrow therapeutic index. An insufficient dose of warfarin can result in failure to produce the antithrombotic effect, whereas an overdose increases the risk of bleeding. It was proven that genetic variability in two genes, CYP2C9 a VKORC1, has a significant influence on the individuals response to the dosage of warfarin. METHODS In a control group of 112 randomly selected individuals, we tested the frequency of selected single nucleotide polymorphisms including CYP2C9*2 (430C>T), CYP2C9*3 (1075A>C), VKORC1*2 (1173C>T) by allele-specific Real-Time PCR and VKORC1*2 (-1639G>A) by using PCR-RFLP. RESULTS Due to the combination of frequent alleles CYP2C9*2, CYP2C9*3 and VKORC1*2 in Slovak population we determine that 25% of population need a standard 5-mg daily dose of warfarin, while 44%, 23%, and 8% need 4 mg, 3 mg and 2 mg of warfarin per day. CONCLUSION Slovak population is in Hardy-Weinberg equilibrium and frequencies of SNPs were in accordance with other published results in European populations (Tab. 5. Fig. 3, Ref. 51).

ERVW-1 gene polymorphisms related to preeclampsia.

OBJECTIVES Identification of genetic association between the gene ERVW-1 and preeclampsia. BACKGROUND Preeclampsia is a multifactorial disease affecting women during pregnancy and it is one of the main causes of perinatal and maternal morbidity and mortality. The pathophysiology of preeclampsia is very complex and several aspects of the disease have not been elucidated yet. Abnormal placentation frequently occurs during severe preeclampsia. Protein syncytin 1, a product of the ERVW-1 gene, plays a crucial role in the syncytiotrophoblast differentiation and optimal placentation. The syncytin 1 expression is disturbed during preeclampsia. The main focus of this study was the analysis of the ERVW-1 regulatory regions and identification of DNA polymorphisms associated with preeclamptic cases in Slovak population. METHODS Regulatory region of gene ERVW-1 was analyzed by sequencing to identify genetic variants. RESULTS We identified four DNA variants, namely rs4727276, rs148592540, rs569899772 and rs555416193, in samples of Slovak population. CONCLUSION No relation between polymorphisms and preeclampsia was observed, indicating that further investigations with a larger sampling are still required. However, our work represents new original approach in genetic differential diagnosis of preeclampsia with possible useful findings in the future (Tab. 3, Fig. 1, Ref. 34).

Peroxisomal D-bifunctional protein deficiency: First case reports from Slovakia

D-bifunctional protein deficiency (#OMIM 261515) is a rare autosomal recessive hereditary metabolic disorder causing severe clinical and biochemical abnormalities that are usually fatal in the course of the first years of life. This disease is classified as single enzyme peroxisomal disorder affecting the β-oxidation pathway in this compartment. In this paper we present a full overview of the clinical presentation, magnetic resonance imaging, biochemical and molecular data of two Slovak D-bifunctional protein deficient patients. In the clinical presentation of both patients severe generalized hypotonia, depression of neonatal reflexes, craniofacial dysmorphism and seizures dominated starting from the second day of life. In both patients, who died up to two years of life, we found elevated plasma levels of very long chain fatty acids and we identified the presence of causative mutations in the HSD17B4 gene. In the first case, we found the homozygous mutation c.46G>A, which is responsible for a defect in the dehydrogenase domain. In the second patient, the heterozygous mutations c.1369A>G and c.1516C>T were present and functionally they are related to the hydratase domain of the protein. This combination of mutations in the second patient is very rare and has not been reported until now. The presence of mutations was examined in all family members, and the resulting data were successfully utilized for prenatal diagnosis.

GAI - distinct genotype and phenotype characteristics in reported Slovak patients.

OBJECTIVES The clinical, biochemical and genetic findings in two Slovak patients with glutaric aciduria type I (GAI) are presented. BACKGROUND GAI is a rare autosomal recessive neuro-metabolic disorder caused by deficiency of glutaryl-CoA dehydrogenase, which is involved in the catabolic pathways of lysine, hydroxylysine and tryptophan. This enzymatic defect gives rise to elevated levels of glutaric acid (GA), 3-hydroxyglutaric acid (3-OH-GA) and glutarylcarnitine (C5DC) in body fluids. METHODS Biochemical and molecular-genetic tests were performed. Urinary organic acids were analysed by Gas Chromatography/Mass Spectrometry (GC/MS) and the entire coding region of the GCDH gene, including flanking parts, was sequenced. RESULTS We found the presence of typical metabolic profile and novel causal pathogenic variants in both GAI patients. CONCLUSION We present the first report of two Slovak patients with GAI, which differed in the clinical and biochemical phenotype significantly. They were diagnosed by two distinct approaches - selective and newborn screening. Their diagnosis was complexly confirmed by biochemical and later on molecular-genetic examinations. Though we agreed with a thesis that early diagnostics might positively influenced patients health outcome, contradictory facts should be considered. Supposed extremely low prevalence of GAI patients in the general population and/or the existence of asymptomatic individuals with a questionable benefit of the applied therapeutic intervention for them lead to doubts whether the inclusion of disease into the newborn screening programme is justified well enough (Tab. 1, Fig. 3, Ref. 41).

ALLELIC DISTRIBUTION OF GENES FOR APOLIPOPROTEIN E AND MTHFR IN PATIENTS WITH ALZHEIMER’S DISEASE AND THEIR EPISTATIC INTERACTION

APOEε3 and APOEε4 whereof the latter increases the risk of disease 4-15 fold in a dose-dependent manner whilst APOEε2 appears to be protective. The biological mechanisms underlying the modified risk of disease in APOEε2 and APOEε4 carriers are not known. We previously showed that APOEε4carriers exhibit a prominent plasma apolipoprotein E (apoE) deficiency caused by a specific reduction of the apoE4 isoform. This apoE deficiency was not observed in cerebrospinal fluid. In cognitively intact APOEε3/ε4 carriers an increased relative ratio of plasma apoE4 to apoE3 correlated to glucose hypometabolism and gray matter volume reductions in brain areas most often affected in AD. Hence, we speculate that peripheral apoE levels are linked to processes driving the risk of developing AD brain pathology. In order to determine the cause of the observed phenotype of plasma apoE deficiency in APOEε4-carriers we aimed to investigate the expression of different APOE alleles in liver biopsies from individuals with an APOEε3/ε4 versus an APOEε2/ε3 genotype. Methods: Liver biopsies from liver explants were received from n1⁄43 APOEε3/ε4 and n1⁄43 APOEε2/ε3 carriers who had undergone liver transplantation at the Karolinska University Hospital in Sweden. Total RNA ( 80% in DV200 score) was isolated according to routine laboratory methods and used for RNA sequencing employing the high-throughput Illumina platform. Paired-end sequencing reads were aligned to the human reference genome (hg19), using TopHat, and gene counts for expression determined using HTseq-count. Differential expression between the genotype groups was calculated using DEseq2. Results: Preliminary analyses revealed differential expression of n1⁄4624 genes between individuals with an APOEε3/ε4 versus an APOEε2/ε3 genotype. Conclusions: In total n1⁄4624 genes are differentially expressed in livers from APOEε3/ε4 versus APOEε2/ε3 carriers. Further analyses will reveal the identity of the differentially expressed genes and whether there is a specific difference in the expression of APOE alleles that could explain the observed APOEε4 related plasma apoE deficiency

A novel mutation in the PEX12 gene causing a peroxisomal biogenesis disorder.

The peroxisomal biogenesis disorders are autosomal recessive diseases morphologically characterised by lacking peroxisomes, biochemically by generalised deficiency of peroxisomal constituent and clinically manifested by serious health problems. Genes involved in the peroxisomal biogenesis are defined as the PEX genes encoding proteins called the peroxins. These peroxins are required for function in assembly of the peroxisomal membrane or in import of the enzymes into the peroxisomes. In this study we present a full overview of the clinical presentation, biochemical and molecular data of patient with Zellweger syndrome from Slovakia. We investigated biochemical metabolites using gas chromatography/mass spectrometry. The presence of causal ins/del mutations we identified by a Sanger sequencing and RFLP. We reported that the patient was a compound heterozygote for mutations in the gene PEX12: a 2-bp insertion (c.767_768dupAT) and a 2-bp deletion (c.887_888delTC). The first one mentioned is a novel mutation, which has not been reported before. Both mutations create a frameshift of the open reading frame which result a premature STOP codon and generate a complete loss of the C-terminal RING finger domain that is crucial for the correct import of proteins into peroxisomes. We found causal mutations responsible for a severe phenotype, and moreover we noted a novel mutation c.767_768dupAT that has not been reported before. The presence of mutations was studied in all family members, and the resulting data were successfully utilized for prenatal diagnosis.

Tumour suppressor gene and protein TP53/P53 in normal endometrium and endometrial carcinoma

Endometrial carcinoma (ECa) is the most common neoplasia of the female genital system. There are two basic types of ECa, endometrioid (estrogen related, indolent) and non-endometrioid (estrogen unrelated, aggressive). The tp53 is a tumour suppressor gene (17pl3) encoding a DNA-binding phosphoprotein P53, 53kDa, a transcription factor that mediates the cell’s response to various kinds of genotoxic stress by preventing cell division and/or inducing DNA excise repair or apoptosis. Tp53 alterations results in an aberrant P53 with a longer half-life that accumulates in the cell. A total of 40 archived formalin-fixed and paraffin-embedded human biopsy tissue specimens with normal proliferative endometrium, endometrioid grade G1 and G3 and serous (SC) subtype of ECa were evaluated by immunohistochemistry for P53 expression/accumulation in nuclei of endometrial-epithelial cells; and by molecular biology methods for the exons 5-8 tp53 DNA sequence alterations. The expression/accumulation of tp53/P53 was related only to aggressive (mainly SC) types of ECa. Tp53 mutations (substitutions) were detected in SC only. There is no expression/accumulation of tp53/P53 in normal endometrium. The tp53/P53 expression/accumulation and tp53 mutations presence are associated with aggressive type of ECa. Evaluation of tp53/P53 in ECa could be a relevant component useful in research and clinical practice.