Metka Ravnik - Academia.edu (original) (raw)

Papers by Metka Ravnik

Research paper thumbnail of Involvement of CFTR Gene Alterations in Obstructive and Nonobstructive Infertility in Men

Genetic Testing, 2001

There have not been many studies concerning CFTR gene alterations in nonobstructive causes of mal... more There have not been many studies concerning CFTR gene alterations in nonobstructive causes of male infertility and subfertility, and in those that have been published, the results reported are not concordant. Therefore, we proposed to determine, in a representative unselected sample of men who were sent for microsurgical epididymal sperm aspiration, if different types of male infertility and impaired fertility were associated with CFTR gene alterations. We screened 80 men with idiopathic azoospermia, 50 men with severe oligozoospermia, 70 men with oligoasthenoteratozoospermia, and 7 men with congenital bilateral absence of the vas deferens (CBAVD), as well as 95 controls from Slovenia, for mutations in 10 CFTR exons that include the majority of the most common cystic fibrosis (CF) disease causing mutations. We also wanted to evaluate the risk for CF in children born after the intracytoplasmic sperm injection (ICSI) method of in vitro fertilization (IVF). No tested individual had mutations in both CFTR alleles. Altogether 13 different nucleotide alterations were identified. The frequencies of both CFTR gene alterations and polymorphisms did not differ significantly between the control group and men with idiopathic nonobstructive azoospermia and subfertility, but were significantly increased in men with CBAVD (DeltaF508, p = 0.039; IVS8-5T, p = 0.006). Our results suggest that CFTR mutations are not associated with errors in spermatogenesis and nonobstructive pathology of urogenital tract in men with any frequency. However, genetic counseling and CFTR mutation screening continue to be recommended for men with obstructive azoospermic conditions and their female partners.

Research paper thumbnail of Sixteen novel mutations identified in COL4A3, COL4A4, and COL4A5 genes in Slovenian families with Alport syndrome and benign familial hematuria

Kidney International, 2007

Research paper thumbnail of Genetics and clinical characteristics of keratoconus

Acta dermatovenerologica Alpina, Pannonica, et Adriatica, 2010

Keratoconus (KC) is a bilateral, non-inflammatory, and progredient corneal ectasia that mostly oc... more Keratoconus (KC) is a bilateral, non-inflammatory, and progredient corneal ectasia that mostly occurs as a sporadic disorder, but it has long been recognized that a significant minority of patients also exhibit a family history. In recent years several candidate genes such as VSX1 and SOD1 have been proposed, and some disease-causing mutations have been identified. Lately research has also focused on collagen genes, especially those that are differentially expressed in KC cornea. Alterations in COL4A3 and COL4A4 genes may be responsible for decreases in collagen types I and III, a feature often detected in KC. To investigate the role of all four genes in 113 Slovenian patients with sporadic or familial keratoconus, DNA extraction, polymerase chain reaction amplification, and sequencing of both genes were performed. No disease-causing mutations were found, but two previously identified single nucleotide polymorphisms were identified (A128A and 627+23G>A) in the VSX1 gene. D326Y in...

Research paper thumbnail of Functional MDR1 polymorphisms (G2677T and C3435T) and TCF4 mutations in colorectal tumors with high microsatellite instability

Cellular & molecular biology letters, 2002

The multidrug resistance 1 (MDRI) gene and transcription factor 4(TCF4) gene are suggested to be ... more The multidrug resistance 1 (MDRI) gene and transcription factor 4(TCF4) gene are suggested to be involved in the WNT signalling pathway, the most important pathway altered in colorectal cancer. Mutations in both genes have been identified and associated with colorectal tumors exhibiting high microsatellite instability (MSI-H). In this study, we report on the distribution of functional polymorphisms in the MDR] gene and somatic frameshift mutations in the TCF4 gene coding mononucleotide repetition in 62 MSI-H colorectal tumors. Somatic frameshift mutations in(of) the TCF4 gene were identified in 24/62 (39%) of the studied MSI-H tumors. The estimated allele frequencies of functional polymorphisms in(of) exon 21 (2677 G>T, Ala893Ser) and exon 26(3435 C>T, Ilel 142I1e) of the MDR] gene were 0.42 and 0.46 in the controls and 0.54 (p=0.035) and 0.60 (p=0.017) in the MSI-H tumors. However, the allele frequency of both functional MDR] polymorphisms did not significantly differ between...

Research paper thumbnail of Genetic analysis of amyotrophic lateral sclerosis in the Slovenian population

Neurobiology of Aging, 2015

Amyotrophic lateral sclerosis (ALS) is a complex fatal neurodegenerative disease characterized by... more Amyotrophic lateral sclerosis (ALS) is a complex fatal neurodegenerative disease characterized by progressive degeneration and loss of upper motor neurons in the cerebral cortex and lower motor neurons in brainstem and spinal cord. We established the frequencies of mutations in 4 major ALS-associated genes, SOD1, TARDBP, FUS, and C9ORF72 in a representative cohort of 85 Slovenian patients with sporadic form of ALS. Pathogenic massive hexanucleotide repeat expansion mutation in C9ORF72 was detected in 5.9% of patients and was the most common cause of the disease. In the remaining 3 genes, we identified 4 changes in 3 patients, p.Val14Met in SOD1, silent mutation p.Arg522Arg in FUS, and p.Gly93Cys in SOD1 together with a novel synonymous variant c.990A>G (p.Leu330Leu) in TARDBP gene, respectively. This study represents the first genetic screening of major causative genes for ALS in a cohort of sporadic ALS patients from Slovenia and is according to our knowledge the first such study in Slavic population. Overall, we genetically characterized 8.2% sporadic ALS patients.

Research paper thumbnail of The role of P-glycoprotein ( MDR1 ) polymorphisms and mutations in colorectal cancer

Pfl?gers Archiv European Journal of Physiology, 2001

To better understand physiological function of P-glycoprotein (P-gp), encoded by MDR1 gene, and i... more To better understand physiological function of P-glycoprotein (P-gp), encoded by MDR1 gene, and its role in cancer, we analyzed tumor and corresponding normal tissue from 400 patients with previously non treated colorectal cancer for germline and somatic alterations in MDR1 gene and compared the results to histopathological data and microsatellite instability status of tumors. We have identified naturally occurring mutations in the MDRI gene associated with colorectal cancers with high microsatellite instability (MSI-H) suggesting that tumor cells with MDR1 mutations are selected for during development of MSI-H cancers and that MDR1 plays an important role in tumor initiation and progression in at least a proportion of MSI-H cancers. We found that in all MSI-H tumors with MDR1 mutations, both, the coding and promoter regions were mutated. These results and results from others suggest that alterations in MDR1 promoter are important for P-gp function and that screening for naturally occurring mutations in the promoter region of MDR1 is important in some of the human cancers. We have identified also 12 different germline polymorphisms and at least two of them were significantly associated with increased lymphoid infiltration in tumors suggesting physiological function for P-gp in immune response in addition to protection from xenobotics.

Research paper thumbnail of Evidence that hereditary pancreatitis is genetically heterogeneous disorder

Pfl?gers Archiv European Journal of Physiology, 2000

Hereditary pancreatitis (HP) is an autosomal dominant disorder characterized by recurrent acute a... more Hereditary pancreatitis (HP) is an autosomal dominant disorder characterized by recurrent acute attacks of severe abdominal pain with an onset in early childhood. Many HP patients progress to complicated chronic pancreatitis and/or pancreatic cancer. Initially, a single mutation R117H in the cationic trypsinogen gene was detected in all affected members of five unrelated HP families. Further studies identified a second mutation (N21L) in two HP families without the R117H mutation. Before the association between cationic trypsinogen and HP was found, we detected a cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation (L327R) in all affected individuals of a family with HP. We therefore performed a mutational analysis for R117H and N21L in cationic trypsinogen in this and three additional unrelated families with HP. The R117H mutation was detected in all 9 affected members of three HP families and in 3 asymptomatic but at-risk relatives. However, neither the R117H nor the N21L mutation in the cationic trypsinogen were found in the HP family with the L327R alteration in CFTR. The L327R allele segregates with the disease within this HP family and was not detected on 360 unrelated Caucasian non-CF chromosomes. Although close to 800 different mutations have been detected in the CF gene of cystic fibrosis patients, L327R is a new alteration, not yet reported in connection with CF. The results of this study indicate that the CFTR gene may playa role in the etiology of minority of cases with HP and suggest that hereditary pancreatitis is genetically heterogeneous disease. Keywords hereditary pancreatitis • cationic trypsinogen gene mutations • cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations • genetic heterogeneity

Research paper thumbnail of Evaluation of microsatellite markers for efficient assessment of high microsatellite instabile colorectal tumors

Pfl?gers Archiv European Journal of Physiology, 2000

Two hundred thirty randomly collected primary colorectal tumors were initially screened for micro... more Two hundred thirty randomly collected primary colorectal tumors were initially screened for microsatellite instability (MSI) with three highly informative micro satellite markers (BAT26, D2S123 and D5S346), Forty one (17.8%) tumors showed alterations in at least one marker, In further MSI analysis of these 41 MSI tumors with additional 9 markers, 21 tumors (9,6% of 230 analyzed) exhibited MSI at more than 40% and the rest 20 (8,7% of 230 analyzed) tumors exhibited MSI at 8%-20% tested markers, These results support classification of MSI tumors into high MSI tumors (more than 40% unstable loci) and low MSI tumors (less than 20% unstable loci). Based on our results the combination of BAT26 and two out of four other highly informative markers (D2S123, D5S346, BAT25 or BAT40) is recommended for rapid and reliable assessment of high MSI tumors, Keywords microsatelIite instability • high MSI tumors • hereditary non-polyposis • colorectal cancer • sporadic tumors

Research paper thumbnail of Screening methods for cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in non-human primates

Pfl?gers Archiv European Journal of Physiology, 2000

We report here a comparison of isotopic and non-isotopic conformation analysis approach, for scre... more We report here a comparison of isotopic and non-isotopic conformation analysis approach, for screening genomic DNA for coding variations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. A large pool of non-human primates was tested in order to detect naturally occuring CFfR carriers, for future testing of gene therapy of cystic fibrosis. We screened 25 of 27 CFTR exons in over 1,000 animals. We have detected numerous missense mutations and single nucleotide polymorphisms. We found that both methods are highly efficient for detection of variations in DNA sequence, but the non-radioactive approach is faster, less expensive and in some cases more sensitive.

Research paper thumbnail of Cystic fibrosis gene mutations detected in hereditary pancreatitis

Pflügers Archiv European Journal of Physiology, 1996

To determine the potential role of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)... more To determine the potential role of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene in the etiology of Hereditary Pancreatits (HP), we analyzed this gene in two HP families. PCR-SSCP analysis was employed, followed by direct sequencing of DNA samples showing alterations by SSCP. A new alteration not yet detected in connection with CF was detected in one HP family. The alteration is in exon 7 and causes an amino acid change from Leu to Arg at position 327 (L327R). L327R segregates with the disease within the family. We discovered another novel alteration (Vll90P) in an HP patient of the second family. These results indicate that CFTR gene mutations may be involved in the etiology of HP and represent a challenge for further study of the role of (CTFR) in other digestive diseases.

Research paper thumbnail of Genomic DNA sequence of Rhesus (M. mulatta) cystic fibrosis (CFTR) gene

Mammalian Genome, 1998

Cystic fibrosis is a common human genetic disease caused by mutations in CFTR, a gene that codes ... more Cystic fibrosis is a common human genetic disease caused by mutations in CFTR, a gene that codes for a chloride channel that is regulated by phosphorylation and cytosolic nucleotides. As part of a program to discover natural animal models for human genetic diseases, we have determined the genomic sequence of CFTR in the Rhesus monkey, Macaca mulatta. The coding region of rhesus CFTR is 98.3% identical to human CFTR at the nucleotide level and 98.2% identical and 99.7% similar at the amino acid level. Partial sequences of flanking introns (5582 base pair positions analyzed) revealed 91.1% identity with human introns. Relative to rhesus intronic sequence, the human sequences had 27 insertions and 22 deletions. Primer sequences for amplification of rhesus genomic CFTR sequences are provided. The accession number is AF013753 (all 27 exons and some flanking intronic sequence).

Research paper thumbnail of Characterization and mapping of three new mammalian ATP-binding transporter genes from an EST database

Mammalian Genome, 1995

Analysis of the human expressed sequence tag (EST) database identified four clones that contain s... more Analysis of the human expressed sequence tag (EST) database identified four clones that contain sequences of previously uncharacterized genes, members of the ATP-binding cassette (ABC) superfamily. Two new ABC genes (EST20237, 31252) are located at Chromosome (Chr) lq42 and lq25 respectively in humans, as determined by FISH; at locations distinct from previously mapped genes of this superfamily. Two additional clones, EST 600 and EST 1596, were found to represent different ATP-binding domains of the same gene, ABC2. This gene was localized to 9q34 in humans by FISH and to the proximal region of Chr 2 in mice by linkage analysis. All genes display extensive diversity in sequence and expression pattern. We present several approaches to characterizing EST clones and demonstrate that the analysis of EST clones from different tissues is a powerful approach to identify new members of important gene families. Some drawbacks of using EST databases, including chimerism of cDNA clones, are discussed.

Research paper thumbnail of Incidence of germline hMLH1 and hMSH2 mutations (HNPCC patients) among newly diagnosed colorectal cancers in a Slovenian population

Journal of Medical Genetics, 2000

The Ministry of Science and Technology of the Republic of Slovenia supported this study (Project ... more The Ministry of Science and Technology of the Republic of Slovenia supported this study (Project J3-7919-0381). The scope of the study was agreed by the Medical Ethical Commission of the Republic of Slovenia in October 1995. We are grateful to the patients who participated in the study. We thank Rastko Golouh and Anton Cerar for patients' tissue samples. We also thank Tomaz Krašovec and Aleš Novak for excellent technical assistance.

Research paper thumbnail of Screening for CF mutations in adult cystic fibrosis patients with a directed and optimized SSCP strategy

Human Mutation, 1994

Twenty adolescent and adult cystic fibrosis (CF) patients have been studied for the presence of m... more Twenty adolescent and adult cystic fibrosis (CF) patients have been studied for the presence of mutations in the CFTR gene. Mutations other than deltaF508 have been detected by comparison to the single-stranded conformation polymorphism (SSCP) pattern of known mutations in eight exons, in which 80% of the more common mutations are present. Each mutation was confirmed by direct sequencing. For each of the analyzed exons, optimal SSCP conditions have been determined that allow all available known mutations in that exon to be distinguished from each other. This approach allowed mutations to be defined in 75% of the non deltaF508 alleles and 92% of all CF alleles in this cohort.

Research paper thumbnail of Single-stranded conformation polymorphism analysis of the CFTR gene in slovenian cystic fibrosis patients: Detection of mutations and sequence variations

Human Mutation, 1993

Cystic fibrosis (CF) mutations have been identified in Slovenian CF patients using single-strande... more Cystic fibrosis (CF) mutations have been identified in Slovenian CF patients using single-stranded conformation polymorphism (SSCP) analysis. The entire coding region and all of the splice junction sites were screened in 24 patients. By varying the electrophoretic conditions and composition of the gel, 16 different nucleotide changes have been observed in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Three newly described mutations and four previously reported mutations were found. In addition two new polymorphisms have been identified. Of 35 non-delta F508 chromosomes examined, mutations were detected on 25.7%, raising the proportion of Slovenian CF alleles characterized to 67.5%. Because of the high sensitivity of the SSCP technique most of the remaining uncharacterized CF mutations probably lie in large introns, promoter sequences, or putative regulatory regions not yet analyzed.

Research paper thumbnail of DHPLC screening of cystic fibrosis gene mutations

Human Mutation, 2002

Denaturing high performance liquid chromatography (DHPLC) using ion-pairing reverse phase chromat... more Denaturing high performance liquid chromatography (DHPLC) using ion-pairing reverse phase chromatography (IPRPC) columns is a technique for the screening of gene mutations. In order to evaluate the potential utility of this assay method in a clinical laboratory setting, we subjected the PCR products of 73 CF patients known to bear CFTR mutations to this analytic technique. We used thermal denaturation profile parameters specified by the MELT program tool, made available by Stanford University. Using this strategy, we determined an initial analytic sensitivity of 90.4% for any of 73 known CFTR mutations. Most of the mutations not detected by DHPLC under these conditions are alpha-substitutions. This information may eventually help to improve the MELT algorithm. Increasing column denaturation temperatures for one or two degrees above those recommended by the MELT program allowed 100% detection of CFTR mutations tested. By comparing DHPLC methodology used in this study with the recently reported study based on Wavemaker 3.4.4 software (Transgenomic, Omaha, NE) [Le Marechal et al., 2001) and with previous SSCP analysis of CFTR mutations [Ravnik-Glavac et al., 1994] we emphasized differences and similarities in order to refine the DHPLC system and discuss the relationship to the alternative approaches. We conclude that the DHPLC method, under optimized conditions, is highly accurate, rapid, and efficient in detecting mutations in the CFTR gene and may find high utility in screening individuals for CFTR mutations. Hum Mutat 19:374-383, 2002. Published 2002 Wiley-Liss, Inc.

Research paper thumbnail of Identification of a rare cystic fibrosis mutation (S4X) in a Slovenian population

Human Molecular Genetics, 1993

Research paper thumbnail of A Novel Q562X Mutation Identified in the hMLH1 Gene in a Slovenian Patient with Hereditary Nonpolyposis Colorectal Cancer

Human Heredity, 1999

Microsatellite instability W Hereditary nonpolyposis colorectal cancer W HNPCC W hMLH1 gene W SSC... more Microsatellite instability W Hereditary nonpolyposis colorectal cancer W HNPCC W hMLH1 gene W SSCA/HA analysis W Nonsense mutation W Slovenian origin Description of the Mutation

Research paper thumbnail of Analysis of 14 cystic fibrosis mutations in five South European populations

Human Genetics, 1991

We have analysed five Southern European populations (Albanian, Greek, Italian, Spanish and Yugosl... more We have analysed five Southern European populations (Albanian, Greek, Italian, Spanish and Yugoslavian) for 14 cystic fibrosis (CF) mutations. The most frequent mutations, apart from delta F508, were G542X (6.04%), R1162X (3.61%) and N1303K (3.24%). Each of the other analysed mutations were present at a frequency of less than 1% (R347P, R334W, S549RA, S549I, G551D, R553X and W1282X), and four mutations (D110H, delta I507, S549RT, and S1255X) were not found in this sample. The data presented here allows the use of mutation analysis in 69.5% of Spanish, 58% of Greek, and 56.5% of Italian CF cases.

Research paper thumbnail of Characterization of a novel 21-kb deletion, CFTRdele2,3(21 kb), in the CFTR gene: a cystic fibrosis mutation of Slavic origin common in Central and East Europe

Human Genetics, 2000

We report a large genomic deletion of the cystic fibrosis transmembrane conductance regulator (CF... more We report a large genomic deletion of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, viz., a deletion that is frequently observed in Central and Eastern Europe. The mutation, termed CFTRdele2,3(21 kb), deletes 21,080 bp spanning introns 1-3 of the CFTR gene. Transcript analyses have revealed that this deletion results in the loss of exons 2 and 3 in epithelial CFTR mRNA, thereby producing a premature termination signal within exon 4. In order to develop a simple polymerase chain reaction assay for this allele, we defined the end-points of the deletion at the DNA sequence level. We next screened for this mutation in a representative set of European and European-derived populations. Some 197 CF patients, including seven homozygotes, bearing this mutation have been identified during the course of our study. Clinical evaluation of CFTRdele2,3(21 kb) homozygotes and a comparison of compound heterozygotes for deltaF508/CFTRdele2,3(21 kb) with pairwise-matched deltaF508 homozygotes indicate that this deletion represents a severe mutation associated with pancreatic insufficiency and early age at diagnosis. Current data show that the mutation is particularly common in Czech (6.4% of all CF chromosomes), Russian (5.2%), Belorussian (3.3%), Austrian (2.6%), German (1.5%), Polish (1.5%), Slovenian (1.5%), Ukrainian (1.2%), and Slovak patients (1.1%). It has also been found in Lithuania, Latvia, Macedonia and Greece and has sporadically been observed in Canada, USA, France, Spain, Turkey, and UK, but not in CF patients from Bulgaria, Croatia, Romania or Serbia. Haplotype analysis has identified the same extragenic CF-haplotype XV-2c/KM. 19 "A" and the same infrequent intragenic microsatellite haplotype 16-33-13 (IVS8CA-IVS 17bTA-IVS 17bCA) in all examined CFTRdele2,3(21 kb) chromosomes, suggesting a common origin for this deletion. We conclude that the 21-kb deletion is a frequent and severe CF mutation in populations of Eastern- and Western-Slavic descent.

Research paper thumbnail of Involvement of CFTR Gene Alterations in Obstructive and Nonobstructive Infertility in Men

Genetic Testing, 2001

There have not been many studies concerning CFTR gene alterations in nonobstructive causes of mal... more There have not been many studies concerning CFTR gene alterations in nonobstructive causes of male infertility and subfertility, and in those that have been published, the results reported are not concordant. Therefore, we proposed to determine, in a representative unselected sample of men who were sent for microsurgical epididymal sperm aspiration, if different types of male infertility and impaired fertility were associated with CFTR gene alterations. We screened 80 men with idiopathic azoospermia, 50 men with severe oligozoospermia, 70 men with oligoasthenoteratozoospermia, and 7 men with congenital bilateral absence of the vas deferens (CBAVD), as well as 95 controls from Slovenia, for mutations in 10 CFTR exons that include the majority of the most common cystic fibrosis (CF) disease causing mutations. We also wanted to evaluate the risk for CF in children born after the intracytoplasmic sperm injection (ICSI) method of in vitro fertilization (IVF). No tested individual had mutations in both CFTR alleles. Altogether 13 different nucleotide alterations were identified. The frequencies of both CFTR gene alterations and polymorphisms did not differ significantly between the control group and men with idiopathic nonobstructive azoospermia and subfertility, but were significantly increased in men with CBAVD (DeltaF508, p = 0.039; IVS8-5T, p = 0.006). Our results suggest that CFTR mutations are not associated with errors in spermatogenesis and nonobstructive pathology of urogenital tract in men with any frequency. However, genetic counseling and CFTR mutation screening continue to be recommended for men with obstructive azoospermic conditions and their female partners.

Research paper thumbnail of Sixteen novel mutations identified in COL4A3, COL4A4, and COL4A5 genes in Slovenian families with Alport syndrome and benign familial hematuria

Kidney International, 2007

Research paper thumbnail of Genetics and clinical characteristics of keratoconus

Acta dermatovenerologica Alpina, Pannonica, et Adriatica, 2010

Keratoconus (KC) is a bilateral, non-inflammatory, and progredient corneal ectasia that mostly oc... more Keratoconus (KC) is a bilateral, non-inflammatory, and progredient corneal ectasia that mostly occurs as a sporadic disorder, but it has long been recognized that a significant minority of patients also exhibit a family history. In recent years several candidate genes such as VSX1 and SOD1 have been proposed, and some disease-causing mutations have been identified. Lately research has also focused on collagen genes, especially those that are differentially expressed in KC cornea. Alterations in COL4A3 and COL4A4 genes may be responsible for decreases in collagen types I and III, a feature often detected in KC. To investigate the role of all four genes in 113 Slovenian patients with sporadic or familial keratoconus, DNA extraction, polymerase chain reaction amplification, and sequencing of both genes were performed. No disease-causing mutations were found, but two previously identified single nucleotide polymorphisms were identified (A128A and 627+23G>A) in the VSX1 gene. D326Y in...

Research paper thumbnail of Functional MDR1 polymorphisms (G2677T and C3435T) and TCF4 mutations in colorectal tumors with high microsatellite instability

Cellular & molecular biology letters, 2002

The multidrug resistance 1 (MDRI) gene and transcription factor 4(TCF4) gene are suggested to be ... more The multidrug resistance 1 (MDRI) gene and transcription factor 4(TCF4) gene are suggested to be involved in the WNT signalling pathway, the most important pathway altered in colorectal cancer. Mutations in both genes have been identified and associated with colorectal tumors exhibiting high microsatellite instability (MSI-H). In this study, we report on the distribution of functional polymorphisms in the MDR] gene and somatic frameshift mutations in the TCF4 gene coding mononucleotide repetition in 62 MSI-H colorectal tumors. Somatic frameshift mutations in(of) the TCF4 gene were identified in 24/62 (39%) of the studied MSI-H tumors. The estimated allele frequencies of functional polymorphisms in(of) exon 21 (2677 G>T, Ala893Ser) and exon 26(3435 C>T, Ilel 142I1e) of the MDR] gene were 0.42 and 0.46 in the controls and 0.54 (p=0.035) and 0.60 (p=0.017) in the MSI-H tumors. However, the allele frequency of both functional MDR] polymorphisms did not significantly differ between...

Research paper thumbnail of Genetic analysis of amyotrophic lateral sclerosis in the Slovenian population

Neurobiology of Aging, 2015

Amyotrophic lateral sclerosis (ALS) is a complex fatal neurodegenerative disease characterized by... more Amyotrophic lateral sclerosis (ALS) is a complex fatal neurodegenerative disease characterized by progressive degeneration and loss of upper motor neurons in the cerebral cortex and lower motor neurons in brainstem and spinal cord. We established the frequencies of mutations in 4 major ALS-associated genes, SOD1, TARDBP, FUS, and C9ORF72 in a representative cohort of 85 Slovenian patients with sporadic form of ALS. Pathogenic massive hexanucleotide repeat expansion mutation in C9ORF72 was detected in 5.9% of patients and was the most common cause of the disease. In the remaining 3 genes, we identified 4 changes in 3 patients, p.Val14Met in SOD1, silent mutation p.Arg522Arg in FUS, and p.Gly93Cys in SOD1 together with a novel synonymous variant c.990A>G (p.Leu330Leu) in TARDBP gene, respectively. This study represents the first genetic screening of major causative genes for ALS in a cohort of sporadic ALS patients from Slovenia and is according to our knowledge the first such study in Slavic population. Overall, we genetically characterized 8.2% sporadic ALS patients.

Research paper thumbnail of The role of P-glycoprotein ( MDR1 ) polymorphisms and mutations in colorectal cancer

Pfl?gers Archiv European Journal of Physiology, 2001

To better understand physiological function of P-glycoprotein (P-gp), encoded by MDR1 gene, and i... more To better understand physiological function of P-glycoprotein (P-gp), encoded by MDR1 gene, and its role in cancer, we analyzed tumor and corresponding normal tissue from 400 patients with previously non treated colorectal cancer for germline and somatic alterations in MDR1 gene and compared the results to histopathological data and microsatellite instability status of tumors. We have identified naturally occurring mutations in the MDRI gene associated with colorectal cancers with high microsatellite instability (MSI-H) suggesting that tumor cells with MDR1 mutations are selected for during development of MSI-H cancers and that MDR1 plays an important role in tumor initiation and progression in at least a proportion of MSI-H cancers. We found that in all MSI-H tumors with MDR1 mutations, both, the coding and promoter regions were mutated. These results and results from others suggest that alterations in MDR1 promoter are important for P-gp function and that screening for naturally occurring mutations in the promoter region of MDR1 is important in some of the human cancers. We have identified also 12 different germline polymorphisms and at least two of them were significantly associated with increased lymphoid infiltration in tumors suggesting physiological function for P-gp in immune response in addition to protection from xenobotics.

Research paper thumbnail of Evidence that hereditary pancreatitis is genetically heterogeneous disorder

Pfl?gers Archiv European Journal of Physiology, 2000

Hereditary pancreatitis (HP) is an autosomal dominant disorder characterized by recurrent acute a... more Hereditary pancreatitis (HP) is an autosomal dominant disorder characterized by recurrent acute attacks of severe abdominal pain with an onset in early childhood. Many HP patients progress to complicated chronic pancreatitis and/or pancreatic cancer. Initially, a single mutation R117H in the cationic trypsinogen gene was detected in all affected members of five unrelated HP families. Further studies identified a second mutation (N21L) in two HP families without the R117H mutation. Before the association between cationic trypsinogen and HP was found, we detected a cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation (L327R) in all affected individuals of a family with HP. We therefore performed a mutational analysis for R117H and N21L in cationic trypsinogen in this and three additional unrelated families with HP. The R117H mutation was detected in all 9 affected members of three HP families and in 3 asymptomatic but at-risk relatives. However, neither the R117H nor the N21L mutation in the cationic trypsinogen were found in the HP family with the L327R alteration in CFTR. The L327R allele segregates with the disease within this HP family and was not detected on 360 unrelated Caucasian non-CF chromosomes. Although close to 800 different mutations have been detected in the CF gene of cystic fibrosis patients, L327R is a new alteration, not yet reported in connection with CF. The results of this study indicate that the CFTR gene may playa role in the etiology of minority of cases with HP and suggest that hereditary pancreatitis is genetically heterogeneous disease. Keywords hereditary pancreatitis • cationic trypsinogen gene mutations • cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations • genetic heterogeneity

Research paper thumbnail of Evaluation of microsatellite markers for efficient assessment of high microsatellite instabile colorectal tumors

Pfl?gers Archiv European Journal of Physiology, 2000

Two hundred thirty randomly collected primary colorectal tumors were initially screened for micro... more Two hundred thirty randomly collected primary colorectal tumors were initially screened for microsatellite instability (MSI) with three highly informative micro satellite markers (BAT26, D2S123 and D5S346), Forty one (17.8%) tumors showed alterations in at least one marker, In further MSI analysis of these 41 MSI tumors with additional 9 markers, 21 tumors (9,6% of 230 analyzed) exhibited MSI at more than 40% and the rest 20 (8,7% of 230 analyzed) tumors exhibited MSI at 8%-20% tested markers, These results support classification of MSI tumors into high MSI tumors (more than 40% unstable loci) and low MSI tumors (less than 20% unstable loci). Based on our results the combination of BAT26 and two out of four other highly informative markers (D2S123, D5S346, BAT25 or BAT40) is recommended for rapid and reliable assessment of high MSI tumors, Keywords microsatelIite instability • high MSI tumors • hereditary non-polyposis • colorectal cancer • sporadic tumors

Research paper thumbnail of Screening methods for cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in non-human primates

Pfl?gers Archiv European Journal of Physiology, 2000

We report here a comparison of isotopic and non-isotopic conformation analysis approach, for scre... more We report here a comparison of isotopic and non-isotopic conformation analysis approach, for screening genomic DNA for coding variations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. A large pool of non-human primates was tested in order to detect naturally occuring CFfR carriers, for future testing of gene therapy of cystic fibrosis. We screened 25 of 27 CFTR exons in over 1,000 animals. We have detected numerous missense mutations and single nucleotide polymorphisms. We found that both methods are highly efficient for detection of variations in DNA sequence, but the non-radioactive approach is faster, less expensive and in some cases more sensitive.

Research paper thumbnail of Cystic fibrosis gene mutations detected in hereditary pancreatitis

Pflügers Archiv European Journal of Physiology, 1996

To determine the potential role of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)... more To determine the potential role of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene in the etiology of Hereditary Pancreatits (HP), we analyzed this gene in two HP families. PCR-SSCP analysis was employed, followed by direct sequencing of DNA samples showing alterations by SSCP. A new alteration not yet detected in connection with CF was detected in one HP family. The alteration is in exon 7 and causes an amino acid change from Leu to Arg at position 327 (L327R). L327R segregates with the disease within the family. We discovered another novel alteration (Vll90P) in an HP patient of the second family. These results indicate that CFTR gene mutations may be involved in the etiology of HP and represent a challenge for further study of the role of (CTFR) in other digestive diseases.

Research paper thumbnail of Genomic DNA sequence of Rhesus (M. mulatta) cystic fibrosis (CFTR) gene

Mammalian Genome, 1998

Cystic fibrosis is a common human genetic disease caused by mutations in CFTR, a gene that codes ... more Cystic fibrosis is a common human genetic disease caused by mutations in CFTR, a gene that codes for a chloride channel that is regulated by phosphorylation and cytosolic nucleotides. As part of a program to discover natural animal models for human genetic diseases, we have determined the genomic sequence of CFTR in the Rhesus monkey, Macaca mulatta. The coding region of rhesus CFTR is 98.3% identical to human CFTR at the nucleotide level and 98.2% identical and 99.7% similar at the amino acid level. Partial sequences of flanking introns (5582 base pair positions analyzed) revealed 91.1% identity with human introns. Relative to rhesus intronic sequence, the human sequences had 27 insertions and 22 deletions. Primer sequences for amplification of rhesus genomic CFTR sequences are provided. The accession number is AF013753 (all 27 exons and some flanking intronic sequence).

Research paper thumbnail of Characterization and mapping of three new mammalian ATP-binding transporter genes from an EST database

Mammalian Genome, 1995

Analysis of the human expressed sequence tag (EST) database identified four clones that contain s... more Analysis of the human expressed sequence tag (EST) database identified four clones that contain sequences of previously uncharacterized genes, members of the ATP-binding cassette (ABC) superfamily. Two new ABC genes (EST20237, 31252) are located at Chromosome (Chr) lq42 and lq25 respectively in humans, as determined by FISH; at locations distinct from previously mapped genes of this superfamily. Two additional clones, EST 600 and EST 1596, were found to represent different ATP-binding domains of the same gene, ABC2. This gene was localized to 9q34 in humans by FISH and to the proximal region of Chr 2 in mice by linkage analysis. All genes display extensive diversity in sequence and expression pattern. We present several approaches to characterizing EST clones and demonstrate that the analysis of EST clones from different tissues is a powerful approach to identify new members of important gene families. Some drawbacks of using EST databases, including chimerism of cDNA clones, are discussed.

Research paper thumbnail of Incidence of germline hMLH1 and hMSH2 mutations (HNPCC patients) among newly diagnosed colorectal cancers in a Slovenian population

Journal of Medical Genetics, 2000

The Ministry of Science and Technology of the Republic of Slovenia supported this study (Project ... more The Ministry of Science and Technology of the Republic of Slovenia supported this study (Project J3-7919-0381). The scope of the study was agreed by the Medical Ethical Commission of the Republic of Slovenia in October 1995. We are grateful to the patients who participated in the study. We thank Rastko Golouh and Anton Cerar for patients' tissue samples. We also thank Tomaz Krašovec and Aleš Novak for excellent technical assistance.

Research paper thumbnail of Screening for CF mutations in adult cystic fibrosis patients with a directed and optimized SSCP strategy

Human Mutation, 1994

Twenty adolescent and adult cystic fibrosis (CF) patients have been studied for the presence of m... more Twenty adolescent and adult cystic fibrosis (CF) patients have been studied for the presence of mutations in the CFTR gene. Mutations other than deltaF508 have been detected by comparison to the single-stranded conformation polymorphism (SSCP) pattern of known mutations in eight exons, in which 80% of the more common mutations are present. Each mutation was confirmed by direct sequencing. For each of the analyzed exons, optimal SSCP conditions have been determined that allow all available known mutations in that exon to be distinguished from each other. This approach allowed mutations to be defined in 75% of the non deltaF508 alleles and 92% of all CF alleles in this cohort.

Research paper thumbnail of Single-stranded conformation polymorphism analysis of the CFTR gene in slovenian cystic fibrosis patients: Detection of mutations and sequence variations

Human Mutation, 1993

Cystic fibrosis (CF) mutations have been identified in Slovenian CF patients using single-strande... more Cystic fibrosis (CF) mutations have been identified in Slovenian CF patients using single-stranded conformation polymorphism (SSCP) analysis. The entire coding region and all of the splice junction sites were screened in 24 patients. By varying the electrophoretic conditions and composition of the gel, 16 different nucleotide changes have been observed in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Three newly described mutations and four previously reported mutations were found. In addition two new polymorphisms have been identified. Of 35 non-delta F508 chromosomes examined, mutations were detected on 25.7%, raising the proportion of Slovenian CF alleles characterized to 67.5%. Because of the high sensitivity of the SSCP technique most of the remaining uncharacterized CF mutations probably lie in large introns, promoter sequences, or putative regulatory regions not yet analyzed.

Research paper thumbnail of DHPLC screening of cystic fibrosis gene mutations

Human Mutation, 2002

Denaturing high performance liquid chromatography (DHPLC) using ion-pairing reverse phase chromat... more Denaturing high performance liquid chromatography (DHPLC) using ion-pairing reverse phase chromatography (IPRPC) columns is a technique for the screening of gene mutations. In order to evaluate the potential utility of this assay method in a clinical laboratory setting, we subjected the PCR products of 73 CF patients known to bear CFTR mutations to this analytic technique. We used thermal denaturation profile parameters specified by the MELT program tool, made available by Stanford University. Using this strategy, we determined an initial analytic sensitivity of 90.4% for any of 73 known CFTR mutations. Most of the mutations not detected by DHPLC under these conditions are alpha-substitutions. This information may eventually help to improve the MELT algorithm. Increasing column denaturation temperatures for one or two degrees above those recommended by the MELT program allowed 100% detection of CFTR mutations tested. By comparing DHPLC methodology used in this study with the recently reported study based on Wavemaker 3.4.4 software (Transgenomic, Omaha, NE) [Le Marechal et al., 2001) and with previous SSCP analysis of CFTR mutations [Ravnik-Glavac et al., 1994] we emphasized differences and similarities in order to refine the DHPLC system and discuss the relationship to the alternative approaches. We conclude that the DHPLC method, under optimized conditions, is highly accurate, rapid, and efficient in detecting mutations in the CFTR gene and may find high utility in screening individuals for CFTR mutations. Hum Mutat 19:374-383, 2002. Published 2002 Wiley-Liss, Inc.

Research paper thumbnail of Identification of a rare cystic fibrosis mutation (S4X) in a Slovenian population

Human Molecular Genetics, 1993

Research paper thumbnail of A Novel Q562X Mutation Identified in the hMLH1 Gene in a Slovenian Patient with Hereditary Nonpolyposis Colorectal Cancer

Human Heredity, 1999

Microsatellite instability W Hereditary nonpolyposis colorectal cancer W HNPCC W hMLH1 gene W SSC... more Microsatellite instability W Hereditary nonpolyposis colorectal cancer W HNPCC W hMLH1 gene W SSCA/HA analysis W Nonsense mutation W Slovenian origin Description of the Mutation

Research paper thumbnail of Analysis of 14 cystic fibrosis mutations in five South European populations

Human Genetics, 1991

We have analysed five Southern European populations (Albanian, Greek, Italian, Spanish and Yugosl... more We have analysed five Southern European populations (Albanian, Greek, Italian, Spanish and Yugoslavian) for 14 cystic fibrosis (CF) mutations. The most frequent mutations, apart from delta F508, were G542X (6.04%), R1162X (3.61%) and N1303K (3.24%). Each of the other analysed mutations were present at a frequency of less than 1% (R347P, R334W, S549RA, S549I, G551D, R553X and W1282X), and four mutations (D110H, delta I507, S549RT, and S1255X) were not found in this sample. The data presented here allows the use of mutation analysis in 69.5% of Spanish, 58% of Greek, and 56.5% of Italian CF cases.

Research paper thumbnail of Characterization of a novel 21-kb deletion, CFTRdele2,3(21 kb), in the CFTR gene: a cystic fibrosis mutation of Slavic origin common in Central and East Europe

Human Genetics, 2000

We report a large genomic deletion of the cystic fibrosis transmembrane conductance regulator (CF... more We report a large genomic deletion of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, viz., a deletion that is frequently observed in Central and Eastern Europe. The mutation, termed CFTRdele2,3(21 kb), deletes 21,080 bp spanning introns 1-3 of the CFTR gene. Transcript analyses have revealed that this deletion results in the loss of exons 2 and 3 in epithelial CFTR mRNA, thereby producing a premature termination signal within exon 4. In order to develop a simple polymerase chain reaction assay for this allele, we defined the end-points of the deletion at the DNA sequence level. We next screened for this mutation in a representative set of European and European-derived populations. Some 197 CF patients, including seven homozygotes, bearing this mutation have been identified during the course of our study. Clinical evaluation of CFTRdele2,3(21 kb) homozygotes and a comparison of compound heterozygotes for deltaF508/CFTRdele2,3(21 kb) with pairwise-matched deltaF508 homozygotes indicate that this deletion represents a severe mutation associated with pancreatic insufficiency and early age at diagnosis. Current data show that the mutation is particularly common in Czech (6.4% of all CF chromosomes), Russian (5.2%), Belorussian (3.3%), Austrian (2.6%), German (1.5%), Polish (1.5%), Slovenian (1.5%), Ukrainian (1.2%), and Slovak patients (1.1%). It has also been found in Lithuania, Latvia, Macedonia and Greece and has sporadically been observed in Canada, USA, France, Spain, Turkey, and UK, but not in CF patients from Bulgaria, Croatia, Romania or Serbia. Haplotype analysis has identified the same extragenic CF-haplotype XV-2c/KM. 19 "A" and the same infrequent intragenic microsatellite haplotype 16-33-13 (IVS8CA-IVS 17bTA-IVS 17bCA) in all examined CFTRdele2,3(21 kb) chromosomes, suggesting a common origin for this deletion. We conclude that the 21-kb deletion is a frequent and severe CF mutation in populations of Eastern- and Western-Slavic descent.