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M Ravnik-Glavac

Publications and source records attributed to M Ravnik-Glavac.

At least 19 recordsLinked to original sources
PubMed2003

Genotyping microarray (gene chip) for the ABCR (ABCA4) gene.

Genetic variation in the ABCR (ABCA4) gene has been associated with five distinct retinal phenotypes, including Stargardt disease/fundus flavimaculatus (STGD/FFM), cone-rod dystrophy (CRD), and age-related macular degeneration (AMD). Comparative genetic analyses of ABCR variation and diagnostics have been complicated by substantial allelic heterogeneity and by differences in screening methods. To overcome these limitations, we designed a genotyping microarray (gene chip) for ABCR that includes all approximately 400 disease-associated and other variants currently described, enabling simultaneous detection of all known ABCR variants. The ABCR genotyping microarray (the ABCR400 chip) was constructed by the arrayed primer extension (APEX) technology. Each sequence change in ABCR was included on the chip by synthesis and application of sequence-specific oligonucleotides. We validated the chip by screening 136 confirmed STGD patients and 96 healthy controls, each of whom we had analyzed previously by single strand conformation polymorphism (SSCP) technology and/or heteroduplex analysis. The microarray was >98% effective in determining the existing genetic variation and was comparable to direct sequencing in that it yielded many sequence changes undetected by SSCP. In STGD patient cohorts, the efficiency of the array to detect disease-associated alleles was between 54% and 78%, depending on the ethnic composition and degree of clinical and molecular characterization of a cohort. In addition, chip analysis suggested a high carrier frequency (up to 1:10) of ABCR variants in the general population. The ABCR genotyping microarray is a robust, cost-effective, and comprehensive screening tool for variation in one gene in which mutations are responsible for a substantial fraction of retinal disease. The ABCR chip is a prototype for the next generation of screening and diagnostic tools in ophthalmic genetics, bridging clinical and scientific research.

ATP-Binding Cassette Transporters↗
PubMed2001

Causes of microsatellite instability in colorectal tumors: implications for hereditary non-polyposis colorectal cancer screening.

Microsatellite instability (MSI) analysis was performed using a "reference panel" of microsatellite markers in 345 unselected primary colorectal cancers (CRC). Thirty-five (10%) tumors were classified as high MSI (MSI-H). We identified 6 (17%) MSI-H tumors with germline mutations in mismatch repair (MMR) genes (tumors from patients with hereditary non-polyposis colorectal cancer (HNPCC) syndrome) and 29 (83%) MSI-H tumors without germline MMR mutations (sporadic MSI-H tumors). Hypermethylation of the hMLH1 promoter was found in 26/29 (90%) sporadic MSI-H tumors but only in 1/6 (17%) HNPCC tumors (P<.001). Somatic alterations were identified in both MMR genes in HNPCC tumors but mainly in the hMSH2 gene in sporadic MSI-H tumors. LOH at MMR loci was detected in 3/6 (50%) HNPCC tumors and in 4/26 (15%) informative sporadic MSI-H tumors. These results together indicate different mode of inactivation of MMR genes in sporadic MSI-H tumors versus MSI-H tumors in HNPCC patients. We therefore propose that MSI analysis of newly diagnosed primary CRC followed by methylation analysis of hMLH1 promoter in MSI-H tumors and mutational analysis of MMR genes in MSI-H tumors lacking hMLH1 promoter methylation might be an efficient molecular genetic approach for HNPCC screening.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Involvement of CFTR gene alterations in obstructive and nonobstructive infertility in men.

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.

Cystic Fibrosis↗
PubMed2000

Screening methods for cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in non-human primates.

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 CFTR 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.

Alleles↗
PubMed2000

Evaluation of microsatellite markers for efficient assessment of high microsatellite instabile colorectal tumors.

Two hundred thirty randomly collected primary colorectal tumors were initially screened for microsatellite instability (MSI) with three highly informative microsatellite 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.

Colorectal Neoplasms↗
PubMed2000

Evidence that hereditary pancreatitis is genetically heterogeneous disorder.

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 play a role in the etiology of minority of cases with HP and suggest that hereditary pancreatitis is genetically heterogeneous disease.

Chromosome Mapping↗
PubMed2000

Study of mutant and polyvariant mutant CFTR genes in patients with congenital absence of the vas deferens.

Congenital bilateral absence of the vas deferens (CBAVD) is a form of male infertility in which mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been identified. Cystic fibrosis patients have mutations in both alleles of the gene while most CBAVD patients have mutations in only one allele. Frequently, the second CFTR allele is not mutant but polyvariant. We have studied CFTR gene mutations and polyvariant allele (TG)m(T)n in intron 8 in 37 patients with CBAVD. Ten (27%) compound heterozygotes and 16 (43.3%) heterozygotes have been detected, while no alteration have been found in the coding and intronic bounderies regions in 11 (29.7%) patients. DeltaF508 was the most frequent mutation present in 15/74 (20.3%) alleles. Additional mutant alleles include: R117H (6.8%), R75Q (5.4%), D1270N (2.7%) and G576A (2.7%). We identified in total 14 different mutations and mutant variants, of which one E804V has not been reported previously. Of 37 alleles with not detectable alteration in the coding or splice site regions the (TG)12(T)5 allele has been found in 14 (37.8%) genes. If we consider that (TG)12(T)5 is believed per se to be a highly penetrant pathogenic allele connected with CBAVD, we have detected a disease causing alterations in 49/74 (66.2%) CFTR alleles of CBAVD patients. These results are extremely important for genetic counseling of couples indicated for in vitro fertilization.

Alleles↗
PubMed2000

Animal model in the study of colorectal carcinogenesis.

Experimental animal models of neoplastic diseases are important in understanding etiological and pathophysiological processes also in humans. In order to investigate whether the mechanism of genomic instability is associated with chemically induced colorectal tumorigenesis in rat we performed the following study: One hundred and fifty Wistar rats (males 220-280 g and females 140-180 g) were used in the study. Colorectal tumors were induced by means of 15 s.c. applications (20 mg/kg) of 1,2-dimethylhydrazine (DMH). On autopsy, all intestinal lesions were assessed by histological criteria used in human pathology. Forty five tumors were found in the large intestine--30 of these in males and 15 in females, i.e. in 27% of all animals. In four animals multiple primary tumors were found. Histologically 24 tumours were adenocarcinomas, 14 signet-cell carcinoma and 7 adenomas. DNA was extracted from rat neoplastic lesions and adjoining microscopically normal tissues from the same slide and amplified by PCR, using 10 different microsatellite markers from chromosomes 1, 3, 5, 7 and 8. PCR amplicon were analyzed for microsatellite instability with non-isotopic method. In 13 adenocarcinomas (29%) microsatellite instability was found at a minimum of 1 locus. Seven tumors (15.5%) showed microsatellite instability at multiple loci. The results of our experiment suggest that genomic instability is an important molecular event in the pathophysiology of DMH induced colorectal carcinogenesis in rats.

1,2-Dimethylhydrazine↗
PubMed1998

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

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).

Amino Acid Sequence↗
PubMed1996

Cystic fibrosis gene mutations detected in hereditary pancreatitis.

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 (V1190P) 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.

Autoradiography↗
PubMed1996

Genetic changes in the origin and development of renal cell carcinoma (RCC).

64 kidney tumours of clear cell histopathology were analysed with non-isotopic SSCP and HA for the presence of VHL gene defects. All positive cases were further characterised by direct sequencing. In 30 tumours (48%) mutations were identified in the coding region of the VHL gene. Other tumours were examined for methylation changes in 5' CpG islands in exon 1 Bisulphite genomic sequencing which gives positive signal for methylated cytosines, was used in this analysis and in 7 tumours hypermethylation of 5' CpG islands was found. These findings suggest that VHL gene mutations together with methylation associated inactivation of the VHL gene are important events that predispose to renal cell tumorigenesis.

Base Sequence↗
PubMed1995

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

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) 1q42 and 1q25 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.

ATP-Binding Cassette Transporters↗
PubMed1994

Screening for CF mutations in adult cystic fibrosis patients with a directed and optimized SSCP strategy.

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.

Adolescent↗
PubMed1994

Polymorphisms in the 3' untranslated region of the I kappa B/MAD-3 (NFKBI) gene located on chromosome 14.

The NF-kappa B transcription factor regulates the expression of a number of genes, including immune function and growth control loci, and several viruses. For example, the long terminal repeat of the human immunodeficiency virus contains NF-kappa B binding sites. NF-kappa B activity in the nucleus is regulated by a cellular inhibitory protein I kappa B. To analyze the potential role of these genes in genetic disease we have mapped the NF-kappa B (NFKB2) and I kappa B/MAD-3 (NFKBI) loci in a panel of somatic cell hybrids to chromosomes 4 and 14, respectively. Amplification of the 3' untranslated region of NFKBI allows the detection of three independent polymorphisms within 410 bp. In combination these polymorphisms were informative in 27 of 36 CEPH families and allowed the gene to be placed onto the linkage map of chromosome 14, between the D14S32 and D14S42 markers.

Base Sequence↗
PubMed1994

Sensitivity of single-strand conformation polymorphism and heteroduplex method for mutation detection in the cystic fibrosis gene.

The gene responsible for cystic fibrosis (CF) contains 27 coding exons and more than 300 independent mutations have been identified. An efficient and optimized strategy is required to identify additional mutations and/or to screen patient samples for the presence of known mutations. We have tested several different conditions for performing single-stranded conformation polymorphism (SSCP) analysis in order to determine the efficiency of the method and to identify the optimum conditions for mutation detection. Each exon and corresponding exon boundaries were amplified. A panel of 134 known CF mutations were used to test the efficiency of detection of mutations. The SSCP conditions were varied by altering the percentage and cross-linking of the acrylamide, employing MDE (an acrylamide substitute), and by adding sucrose and glycerol. The presence of heteroduplexes could be detected on most gels and in some cases contributed to the ability to distinguish certain mutations. Each analysis condition detected 75-98% of the mutations, and all of the mutations could be detected by at least one condition. Therefore, an optimized SSCP analysis can be used to efficiently screen for mutations in a large gene.

Base Sequence↗
PubMed1993

Single-stranded conformation polymorphism analysis of the CFTR gene in Slovenian cystic fibrosis patients: detection of mutations and sequence variations.

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.

Adult↗