Katja Vouk | University of Ljubljana (original) (raw)

Papers by Katja Vouk

Background: Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous d... more Background: Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder caused by mutations in at least two different loci. Prior to performing mutation screening, if DNA samples of sufficient number of family members are available, it is worthwhile to assign the gene involved in disease progression by the genetic linkage analysis.

Archives of Biochemistry and Biophysics, 2000

Mycobacterium belong to the CYP51 family which is evolutionary the most conserved gene family wit... more Mycobacterium belong to the CYP51 family which is evolutionary the most conserved gene family within the cytochrome P450 superfamily. We have characterized a new member of this family, the mouse lanosterol 14␣-demethylase, with the aim to study the in vivo role of this gene in spermatogenesis in mammals. The amino acid sequence of mouse Cyp51 is 96% identical to rat and 91% to human. Comparison of all known CYP51 proteins by the neighbor-joining method suggests that fungal and animal CYP51 genes arose from a common ancestral gene (98.3% probability) and interestingly, that plant and bacterial CYP51 genes share a common progenitor (88.8% probability). This suggests that the first CYP51 gene may have arisen in eukaryotes and has been transferred horizontally from plants to Mycobacterium. The mouse CYP51 gene is ϳ17-kb long and contains 10 exons. Transcription starts at several locations within the CpG island, which is characteristic for the TATA-less housekeeping genes. The mouse 5-untranslated region (800 bp) contains putative cAMP-responsive elements (CRE), sterol regulatory elements (SRE) and GC-boxes at positions similar to human and rat, suggesting an evolutionary conserved mechanism of CYP51 transcriptional regulation in mammals. The mouse Cyp51 gene resides on chromosome 5, region A2, close to the centromere. No signals outside this region were detected as well as no evidence of processed pseudogenes using long PCR was found. This indicates that the mouse genome most likely lacks CYP51 processed pseudogenes.

Chemico-biological Interactions, 2011

17β-Hydroxysteroid dehydrogenases (17β-HSDs) have a dominant role in the local activation and ina... more 17β-Hydroxysteroid dehydrogenases (17β-HSDs) have a dominant role in the local activation and inactivation of estrogens. The reduction of estrone to estradiol is catalyzed by 17β-HSD types 1 and 7, while 17β-HSD type 2 catalyzes the oxidation of estradiol to estrone, thus lowering the concentration of the active hormone. The differential expression of these estrogenic 17β-HSDs may lead to an increased local concentration of the potent estradiol and thus to the development of estrogendependent diseases, such as endometriosis and endometrial cancer. In addition to the estrogenic 17β-HSDs, androgenic 17β-HSD (AKR1C3) and other enzymes of the aromatase (aromatase) and the sulfatase (sulfatase, sulfotransferase) pathways may also be involved in the production of increased levels of estradiol. We have here examined the expression of 17β-HSDs, aromatase, sulfatase and sulfotransferase, and of estrogen receptors (ERs) in 16 specimens of ovarian endometriosis in a comparison with normal endometrium, and in 16 specimens of endometrial cancer and adjacent normal endometrium, by real-time PCR. In the ectopic endometrial specimens, aromatase, 17β-HSD types 1 and 7, AKR1C3, sulfatase and ERβ were significantly up-regulated, while ERα was significantly down-regulated. In the cancerous endometrium specimens, only aromatase and AKR1C3 were upregulated in three and four cases out of 16, respectively; types 1 and 7 17β-HSD, sulfotransferase, ERα and ERβ were significantly down-regulated. Our data thus suggest different mechanisms of excessive estradiol production within the ectopic and the cancerous endometrium, and emphasize a pivotal role of the 17β-HSDs in both of these estrogen-dependent diseases. In ovarian endometriosis, the local estradiol concentrations can be increased due to the up-regulation of 17β-HSD types 1 and 7, while in endometrial cancer, only the up-regulation of AKR1C3 can contribute to the enhanced estrogen action that is associated with the pathogenesis of endometrial adenocarcinomas.

Journal of Steroid Biochemistry and Molecular Biology, 2011

In the search for novel biomarkers of endometriosis, we selected 152 genes from the GeneLogic dat... more In the search for novel biomarkers of endometriosis, we selected 152 genes from the GeneLogic database based on results of genome-wide expression analysis of ovarian endometriosis, plus 20 genes related to estrogen metabolism and action. We then performed low-density array analysis of these 172 genes on 11 ovarian endometriosis samples and 9 control endometrium samples. Principal component analysis of the gene expression levels showed clear separation between the endometriosis and control groups. We identified 78 genes as differentially expressed. Based on Ingenuity pathway analysis, these differentially expressed genes were arranged into groups according to biological function. These analyses revealed that 32 differentially expressed genes are estrogen related, 23 of which have not been reported previously in connection with endometriosis. Functional annotation showed that 25 and 22 genes are associated with the biological terms "secreted" and "extracellular region", respectively. Differential expression of 4 out of 5 genes related to estrogen metabolism and action (ESR1, ESR2, PGR and BGN) was also confirmed by immunohistochemistry. Our study thus reveals differential expression of several genes that have not previously been associated with endometriosis and that encode potential novel biomarkers and drug targets.

Human Mutation, 1999

Using polymerase chain reaction, single-stranded conformational polymorphism (SSCP), TaqI restric... more Using polymerase chain reaction, single-stranded conformational polymorphism (SSCP), TaqI restriction analysis and direct sequencing, exons 1, 7, 8, 9, 12, 13, 14, 18, 22, 23, 24, and 26 of the factor VIII gene were screened for point mutations in 55 Slovenian haemophilia A patients. In eighteen patients eleven different mutations were found; one (in six patients) in exon 26, one (in two patients) in exon 24, two in exon 23, one in intron 23, one in exon 18, one in exon 12, one in exon 8, two (1 + 1 in two patients) in exon 7 and one in exon 1. Of the mutations detected one has recently been reported by us (Q602X), and two are novel; S-1R in exon 1 and IVS23+1G-->A in intron 23.

BMC Medical Genetics, 2006

Background Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous di... more Background Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder caused by mutations in at least two different loci. Prior to performing mutation screening, if DNA samples of sufficient number of family members are available, it is worthwhile to assign the gene involved in disease progression by the genetic linkage analysis. Methods We collected samples from 36 Slovene ADPKD families and performed linkage analysis in 16 of them. Linkage was assessed by the use of microsatellite polymorphic markers, four in the case of PKD1 (KG8, AC2.5, CW3 and CW2) and five for PKD2 (D4S1534, D4S2929, D4S1542, D4S1563 and D4S423). Partial PKD1 mutation screening was undertaken by analysing exons 23 and 31–46 and PKD2 . Results Lod scores indicated linkage to PKD1 in six families and to PKD2 in two families. One family was linked to none and in seven families linkage to both genes was possible. Partial PKD1 mutation screening was performed in 33 patients (including 20 patients from the families where linkage analysis could not be performed). We analysed PKD2 in 2 patients where lod scores indicated linkage to PKD2 and in 7 families where linkage to both genes was possible. We detected six mutations and eight polymorphisms in PKD1 and one mutation and three polymorphisms in PKD2. Conclusion In our study group of ADPKD patients we detected seven mutations: three frameshift, one missense, two nonsense and one putative splicing mutation. Three have been described previously and 4 are novel. Three newly described framesfift mutations in PKD1 seem to be associated with more severe clinical course of ADPKD. Previously described nonsense mutation in PKD2 seems to be associated with cysts in liver and milder clinical course.

Molecular Human Reproduction, 2005

The cAMP-responsive element modulator (CREM) gene plays a pivotal role in the mouse spermatogenes... more The cAMP-responsive element modulator (CREM) gene plays a pivotal role in the mouse spermatogenesis, but its role in the human infertility has not been fully established. We performed a mutation screening in 13 Slovenian men with round spermatid arrest and in six controls. Eleven genetic changes have been identified in the human CREM gene, three novel single-nucleotide polymorphisms [within the promoters P1, P3 and intervening sequence 1 (IVS1)], one insertion (IVS2) and one non-sense mutation (exon ␥). Some infertile patients seem to accumulate potentially harmful genetic changes. We identified a patient with no CREM immunoreactive protein that was homozygous for the nucleotide changes in all promoters, IVS 1, 2, 6, and was heterozygous for the mutation in exon ␥. Interestingly, insertion in IVS2 (IVS2-58_55insT) results in a four-fold decrease in binding of nuclear proteins. Computer predictions suggested the presence of a potential novel CREM promoter, however, random amplification of cDNA ends from the human testis cDNA library was not successful in confirming a novel transcription start site of the CREM gene. Screening of a larger number of patients and controls is required to elucidate whether the observed combinations of genetic changes in the CREM gene can explain some forms of male infertility.

Human Heredity, 2000

A novel missense mutation was detected in the L12 region of keratin 5 (K5) in a Slovene family di... more A novel missense mutation was detected in the L12 region of keratin 5 (K5) in a Slovene family diagnosed with a Weber-Cockayne variant of epidermolysis bullosa simplex (EBS). Direct sequencing identified a heterozygous GAC to GAA substitution altering codon 328 of K5 from Asp to Glu in all affected family members, while no mutation was observed either in the healthy individual or the 50 unrelated control samples. Asp 328 of K5 (position 12 in the L12 domain) is remarkably conserved among all type II keratins. K5 L12:D12E is the third mutation found to affect this residue in K5-related EBS, indicating the importance of Asp 328 for K5 structure and the dramatic effect that fine changes can have on keratin intermediate filament integrity.

Pflugers Archiv-european Journal of Physiology, 2000

More than 800 mutations have been indentified in the CFTR gene. This vast mutation diversity make... more More than 800 mutations have been indentified in the CFTR gene. This vast mutation diversity makes the search for molecular defects in cystic fibrosis difficult. Out of 100 Slovenian CF families, we have screened 30, using DGGE and SSCP as mutation detection techniques, while the remaining 70 have been studied previously. Together our and the previous studies have been able to indentify 18 CF mutations which cover 77.6% of the CF alleles in those families. The relative frequency of ΔF508 is 62.7% which is significantly higher than the average reported for the Mediterranean South European region (51.6%). At the same time, significant differences in mutation frequencies were found for the G542X, R1162X, W1282X, N1303K and 3905insT mutations. Several, otherwise rare mutations have been detected, such as: I148T, Q552X, 457TAT→G, R1006H, 2907delTT, 3667ins4, A559T and G576A. An interesting fact is that A559T was so far found mostly in CF patients of African-American origin. These results imply that a high heterogeneity of CF mutations occurs within the small population of Slovenia, consisting only of 2 million inhabitants. In view of the spectrum and frequencies of detected mutations, Slovenian population expresses characteristics of Mediterranean and central European countries, and at the same time shows also distinctive differences and unique region specific CF mutations (Q685X, D192G, S4X).

Human Mutation, 1999

Using polymerase chain reaction, single-stranded conformational polymorphism (SSCP), TaqI restric... more Using polymerase chain reaction, single-stranded conformational polymorphism (SSCP), TaqI restriction analysis and direct sequencing, exons 1, 7, 8, 9, 12, 13, 14, 18, 22, 23, 24, and 26 of the factor VIII gene were screened for point mutations in 55 Slovenian haemophilia A patients. In eighteen patients eleven different mutations were found; one (in six patients) in exon 26, one (in two patients) in exon 24, two in exon 23, one in intron 23, one in exon 18, one in exon 12, one in exon 8, two (1 + 1 in two patients) in exon 7 and one in exon 1. Of the mutations detected one has recently been reported by us (Q602X), and two are novel; S-1R in exon 1 and IVS23+1G-->A in intron 23.

Background: Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous d... more Background: Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder caused by mutations in at least two different loci. Prior to performing mutation screening, if DNA samples of sufficient number of family members are available, it is worthwhile to assign the gene involved in disease progression by the genetic linkage analysis.

Archives of Biochemistry and Biophysics, 2000

Mycobacterium belong to the CYP51 family which is evolutionary the most conserved gene family wit... more Mycobacterium belong to the CYP51 family which is evolutionary the most conserved gene family within the cytochrome P450 superfamily. We have characterized a new member of this family, the mouse lanosterol 14␣-demethylase, with the aim to study the in vivo role of this gene in spermatogenesis in mammals. The amino acid sequence of mouse Cyp51 is 96% identical to rat and 91% to human. Comparison of all known CYP51 proteins by the neighbor-joining method suggests that fungal and animal CYP51 genes arose from a common ancestral gene (98.3% probability) and interestingly, that plant and bacterial CYP51 genes share a common progenitor (88.8% probability). This suggests that the first CYP51 gene may have arisen in eukaryotes and has been transferred horizontally from plants to Mycobacterium. The mouse CYP51 gene is ϳ17-kb long and contains 10 exons. Transcription starts at several locations within the CpG island, which is characteristic for the TATA-less housekeeping genes. The mouse 5-untranslated region (800 bp) contains putative cAMP-responsive elements (CRE), sterol regulatory elements (SRE) and GC-boxes at positions similar to human and rat, suggesting an evolutionary conserved mechanism of CYP51 transcriptional regulation in mammals. The mouse Cyp51 gene resides on chromosome 5, region A2, close to the centromere. No signals outside this region were detected as well as no evidence of processed pseudogenes using long PCR was found. This indicates that the mouse genome most likely lacks CYP51 processed pseudogenes.

Chemico-biological Interactions, 2011

17β-Hydroxysteroid dehydrogenases (17β-HSDs) have a dominant role in the local activation and ina... more 17β-Hydroxysteroid dehydrogenases (17β-HSDs) have a dominant role in the local activation and inactivation of estrogens. The reduction of estrone to estradiol is catalyzed by 17β-HSD types 1 and 7, while 17β-HSD type 2 catalyzes the oxidation of estradiol to estrone, thus lowering the concentration of the active hormone. The differential expression of these estrogenic 17β-HSDs may lead to an increased local concentration of the potent estradiol and thus to the development of estrogendependent diseases, such as endometriosis and endometrial cancer. In addition to the estrogenic 17β-HSDs, androgenic 17β-HSD (AKR1C3) and other enzymes of the aromatase (aromatase) and the sulfatase (sulfatase, sulfotransferase) pathways may also be involved in the production of increased levels of estradiol. We have here examined the expression of 17β-HSDs, aromatase, sulfatase and sulfotransferase, and of estrogen receptors (ERs) in 16 specimens of ovarian endometriosis in a comparison with normal endometrium, and in 16 specimens of endometrial cancer and adjacent normal endometrium, by real-time PCR. In the ectopic endometrial specimens, aromatase, 17β-HSD types 1 and 7, AKR1C3, sulfatase and ERβ were significantly up-regulated, while ERα was significantly down-regulated. In the cancerous endometrium specimens, only aromatase and AKR1C3 were upregulated in three and four cases out of 16, respectively; types 1 and 7 17β-HSD, sulfotransferase, ERα and ERβ were significantly down-regulated. Our data thus suggest different mechanisms of excessive estradiol production within the ectopic and the cancerous endometrium, and emphasize a pivotal role of the 17β-HSDs in both of these estrogen-dependent diseases. In ovarian endometriosis, the local estradiol concentrations can be increased due to the up-regulation of 17β-HSD types 1 and 7, while in endometrial cancer, only the up-regulation of AKR1C3 can contribute to the enhanced estrogen action that is associated with the pathogenesis of endometrial adenocarcinomas.

Journal of Steroid Biochemistry and Molecular Biology, 2011

In the search for novel biomarkers of endometriosis, we selected 152 genes from the GeneLogic dat... more In the search for novel biomarkers of endometriosis, we selected 152 genes from the GeneLogic database based on results of genome-wide expression analysis of ovarian endometriosis, plus 20 genes related to estrogen metabolism and action. We then performed low-density array analysis of these 172 genes on 11 ovarian endometriosis samples and 9 control endometrium samples. Principal component analysis of the gene expression levels showed clear separation between the endometriosis and control groups. We identified 78 genes as differentially expressed. Based on Ingenuity pathway analysis, these differentially expressed genes were arranged into groups according to biological function. These analyses revealed that 32 differentially expressed genes are estrogen related, 23 of which have not been reported previously in connection with endometriosis. Functional annotation showed that 25 and 22 genes are associated with the biological terms "secreted" and "extracellular region", respectively. Differential expression of 4 out of 5 genes related to estrogen metabolism and action (ESR1, ESR2, PGR and BGN) was also confirmed by immunohistochemistry. Our study thus reveals differential expression of several genes that have not previously been associated with endometriosis and that encode potential novel biomarkers and drug targets.

Human Mutation, 1999

Using polymerase chain reaction, single-stranded conformational polymorphism (SSCP), TaqI restric... more Using polymerase chain reaction, single-stranded conformational polymorphism (SSCP), TaqI restriction analysis and direct sequencing, exons 1, 7, 8, 9, 12, 13, 14, 18, 22, 23, 24, and 26 of the factor VIII gene were screened for point mutations in 55 Slovenian haemophilia A patients. In eighteen patients eleven different mutations were found; one (in six patients) in exon 26, one (in two patients) in exon 24, two in exon 23, one in intron 23, one in exon 18, one in exon 12, one in exon 8, two (1 + 1 in two patients) in exon 7 and one in exon 1. Of the mutations detected one has recently been reported by us (Q602X), and two are novel; S-1R in exon 1 and IVS23+1G-->A in intron 23.

BMC Medical Genetics, 2006

Background Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous di... more Background Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder caused by mutations in at least two different loci. Prior to performing mutation screening, if DNA samples of sufficient number of family members are available, it is worthwhile to assign the gene involved in disease progression by the genetic linkage analysis. Methods We collected samples from 36 Slovene ADPKD families and performed linkage analysis in 16 of them. Linkage was assessed by the use of microsatellite polymorphic markers, four in the case of PKD1 (KG8, AC2.5, CW3 and CW2) and five for PKD2 (D4S1534, D4S2929, D4S1542, D4S1563 and D4S423). Partial PKD1 mutation screening was undertaken by analysing exons 23 and 31–46 and PKD2 . Results Lod scores indicated linkage to PKD1 in six families and to PKD2 in two families. One family was linked to none and in seven families linkage to both genes was possible. Partial PKD1 mutation screening was performed in 33 patients (including 20 patients from the families where linkage analysis could not be performed). We analysed PKD2 in 2 patients where lod scores indicated linkage to PKD2 and in 7 families where linkage to both genes was possible. We detected six mutations and eight polymorphisms in PKD1 and one mutation and three polymorphisms in PKD2. Conclusion In our study group of ADPKD patients we detected seven mutations: three frameshift, one missense, two nonsense and one putative splicing mutation. Three have been described previously and 4 are novel. Three newly described framesfift mutations in PKD1 seem to be associated with more severe clinical course of ADPKD. Previously described nonsense mutation in PKD2 seems to be associated with cysts in liver and milder clinical course.

Molecular Human Reproduction, 2005

The cAMP-responsive element modulator (CREM) gene plays a pivotal role in the mouse spermatogenes... more The cAMP-responsive element modulator (CREM) gene plays a pivotal role in the mouse spermatogenesis, but its role in the human infertility has not been fully established. We performed a mutation screening in 13 Slovenian men with round spermatid arrest and in six controls. Eleven genetic changes have been identified in the human CREM gene, three novel single-nucleotide polymorphisms [within the promoters P1, P3 and intervening sequence 1 (IVS1)], one insertion (IVS2) and one non-sense mutation (exon ␥). Some infertile patients seem to accumulate potentially harmful genetic changes. We identified a patient with no CREM immunoreactive protein that was homozygous for the nucleotide changes in all promoters, IVS 1, 2, 6, and was heterozygous for the mutation in exon ␥. Interestingly, insertion in IVS2 (IVS2-58_55insT) results in a four-fold decrease in binding of nuclear proteins. Computer predictions suggested the presence of a potential novel CREM promoter, however, random amplification of cDNA ends from the human testis cDNA library was not successful in confirming a novel transcription start site of the CREM gene. Screening of a larger number of patients and controls is required to elucidate whether the observed combinations of genetic changes in the CREM gene can explain some forms of male infertility.

Human Heredity, 2000

A novel missense mutation was detected in the L12 region of keratin 5 (K5) in a Slovene family di... more A novel missense mutation was detected in the L12 region of keratin 5 (K5) in a Slovene family diagnosed with a Weber-Cockayne variant of epidermolysis bullosa simplex (EBS). Direct sequencing identified a heterozygous GAC to GAA substitution altering codon 328 of K5 from Asp to Glu in all affected family members, while no mutation was observed either in the healthy individual or the 50 unrelated control samples. Asp 328 of K5 (position 12 in the L12 domain) is remarkably conserved among all type II keratins. K5 L12:D12E is the third mutation found to affect this residue in K5-related EBS, indicating the importance of Asp 328 for K5 structure and the dramatic effect that fine changes can have on keratin intermediate filament integrity.

Pflugers Archiv-european Journal of Physiology, 2000

More than 800 mutations have been indentified in the CFTR gene. This vast mutation diversity make... more More than 800 mutations have been indentified in the CFTR gene. This vast mutation diversity makes the search for molecular defects in cystic fibrosis difficult. Out of 100 Slovenian CF families, we have screened 30, using DGGE and SSCP as mutation detection techniques, while the remaining 70 have been studied previously. Together our and the previous studies have been able to indentify 18 CF mutations which cover 77.6% of the CF alleles in those families. The relative frequency of ΔF508 is 62.7% which is significantly higher than the average reported for the Mediterranean South European region (51.6%). At the same time, significant differences in mutation frequencies were found for the G542X, R1162X, W1282X, N1303K and 3905insT mutations. Several, otherwise rare mutations have been detected, such as: I148T, Q552X, 457TAT→G, R1006H, 2907delTT, 3667ins4, A559T and G576A. An interesting fact is that A559T was so far found mostly in CF patients of African-American origin. These results imply that a high heterogeneity of CF mutations occurs within the small population of Slovenia, consisting only of 2 million inhabitants. In view of the spectrum and frequencies of detected mutations, Slovenian population expresses characteristics of Mediterranean and central European countries, and at the same time shows also distinctive differences and unique region specific CF mutations (Q685X, D192G, S4X).

Human Mutation, 1999

Using polymerase chain reaction, single-stranded conformational polymorphism (SSCP), TaqI restric... more Using polymerase chain reaction, single-stranded conformational polymorphism (SSCP), TaqI restriction analysis and direct sequencing, exons 1, 7, 8, 9, 12, 13, 14, 18, 22, 23, 24, and 26 of the factor VIII gene were screened for point mutations in 55 Slovenian haemophilia A patients. In eighteen patients eleven different mutations were found; one (in six patients) in exon 26, one (in two patients) in exon 24, two in exon 23, one in intron 23, one in exon 18, one in exon 12, one in exon 8, two (1 + 1 in two patients) in exon 7 and one in exon 1. Of the mutations detected one has recently been reported by us (Q602X), and two are novel; S-1R in exon 1 and IVS23+1G-->A in intron 23.