Familial tauopathy with P364S MAPT mutation: clinical course, neuropathology and ultrastructure of neuronal tau inclusions.

AIMS: This report presents the clinical course, neuropathology and ultrastructure of neuronal tau inclusions of four Slovene relatives with P364S MAPT mutation. METHODS: The clinical history of three out of four P364S MAPT mutation carriers was taken. After formalin fixation, thorough sampling of the central nervous system was followed by paraffin embedding, H&E, Gallyas, Bielschowsky and immunostaining with AT8, anti-3R, anti-4R tau, anti-amyloid-ß, anti-TDP43 and anti-alpha-synuclein antibodies. The distribution and density of different types of neuronal tau inclusions were semiquantitatively assessed. In addition, the ultrastructure of neuronal tau inclusions was analysed. RESULTS: Macroscopic examination of the brains was unremarkable. Microscopically, neuronal tau inclusions of almost all known types were widespread and distributed fairly uniformly in all cases. Pick bodies and swollen neurones were found in only one family member. Mutant tau was composed of 3R and 4R isoforms, with a slight predominance of 3R tau. Composite neuronal tau inclusion (CNTI), found in all four relatives, was a hallmark of the P364S MAPT mutation. CNTI showed compartmental differences in H&E and Gallyas staining, tau isoforms immunolabelling and ultrastructure, displaying fuzzy fibrils in the core and paired twisted tubules at the periphery. CONCLUSIONS: P364S MAPT mutation is characterized clinically by a variable combination of frontotemporal dementia, parkinsonism and motor neurone disease of short duration, and neuropathologically by a widespread uniform distribution of all known neuronal tau inclusions in one family member. Two-compartment CNTI is a unique characteristic of the P364S MAPT mutation.

Hepatic expression of miR-122, miR-126, miR-136 and miR-181a and their correlation to histopathological and clinical characteristics of patients with hepatitis C.

It has been shown for hepatitis C virus (HCV) infection that host miRNAs contribute to the replication of the viral RNA genome. However, the clinical impact of these and many other cellular miRNAs on HCV in humans is still largely unclear. We therefore analysed the expression of miR-122, miR-126, miR-181a and miR-136 in HCV-infected patients. The study included liver biopsies of 65 patients infected with HCV of different genotypes (gt 1, gt 1a, gt 1b, gt 3 and gt 4) and nine noninfected individuals. Expression analysis of miRNAs was performed by qPCR, and they were analysed for differences between patient gender and age, genotypes, stage of fibrosis, grade of inflammation, serum level of liver enzymes, serum viral load, the presence of steatosis and mode of transmission. Different target prediction algorithms were used to search for targets of analyzed miRNAs. Statistical analysis revealed significant up-regulation of miR-136 and down-regulation of miR-126 and miR-181a in patients infected with HCV of different genotypes compared with noninfected individuals. The same expression pattern was observed in different stages and grades of liver disease. miR-122 was up-regulated in women relative to men and associated to portal inflammation, miR-122 and miR-126 correlated with serum HCV load and miR-136 and miR-122 correlated with the presence of steatosis. miR-126 and miR-136 were differentially expressed between different modes of HCV transmission. There were approximately 2000 different targets predicted for all four miRNAs and each of the analyzed miRNAs could be involved in more than a 100 different biochemical pathways. miR-122, miR-126, miR-136 and miR-181a have been shown to be involved in HCV infection with different genotypes. Their expression has been associated with the gender, stage and grade of liver disease, mode of transmission, serum HCV load and the presence of steatosis. Numerous target genes and biochemical pathways are predicted for each of the analyzed miRNAs. All these results suggest their role in HCV-infected liver disease.

MicroRNA miR-1 is up-regulated in remote myocardium in patients with myocardial infarction.

MicroRNAs are small regulatory RNA molecules that mediate regulation of gene expression, thus affecting a variety of physiological, developmental and pathological conditions. They are believed to be new promising therapeutic targets. In recent studies two muscle-specific microRNAs were discovered to contribute to heart diseases and development: miR-1 and miR-133, but there is little data on their expression patterns in human myocardial infarction. We performed simultaneous expression analysis of miR-1, miR-133a, miR-133b in samples of infarcted tissue and remote myocardium from twenty- four patients with acute myocardial infarction. MicroRNA expression was analysed using quantitative real-time PCR and compared to the expression patterns in myocardium of eight healthy adults who died in accidents. We found ~3.8-fold miR-1 up-regulation in remote myocardium when compared to infarcted tissue or healthy adult hearts. As miR-1 has been shown in animal models and clinical studies to contribute to arrhythmogenesis by regulating pacemaker channel genes, our finding of miR-1 up-regulation in patients with myocardial infarction indicates that it might be responsible for the higher risk for arrhythmias in these patients. In addition, miR-133a/b down-regulation in infarcted tissue and remote myocardium was observed, indicating miR-133a/b involvement in the heart response to myocardial infarction. We conclude that miR-1 and miR-133 seem to be important regulators of heart adaptation after ischaemic stress.

Pattern electroretinography of larger stimulus field size and spectral-domain optical coherence tomography in patients with Stargardt disease.

AIMS: To investigate the importance of a larger stimulus field for pattern electroretinography (PERG) in evaluating macular function in Stargardt disease, and to determine the relationship between PERG and spectral-domain optical coherence tomography (SD-OCT). METHODS: In this prospective cross-sectional study, PERG from standard (12 degrees x 16 degrees ) and larger (24 degrees x 32 degrees ) stimulus fields and SD-OCT were recorded in 18 patients with genetically confirmed Stargardt disease, and in 18 control subjects. RESULTS: A PERG P50 response to the larger stimulus field was detectable in 86% of eyes, with a mean P50 amplitude of 2.3 microV, compared with 22% and 1.0 muV for the standard stimulus field. The specificity and sensitivity of PERG to the standard stimulus field were greater than for the larger field. For both PERG P50 and N95, the differences in their amplitudes between the standard and larger stimulus fields correlated significantly with visual acuity and SD-OCT parameters. CONCLUSION: The higher sensitivity and specificity of PERG to the standard stimulus field provide detection of early maculopathy in Stargardt disease, while PERG with the larger stimulus field allows for longer follow-up. The PERG amplitude for the larger stimulus field correlated with severity of transverse photoreceptor loss in SD-OCT. These methods are complementary for evaluation of progression of photoreceptor damage in patients with Stargardt disease.

Sixteen novel mutations identified in COL4A3, COL4A4, and COL4A5 genes in Slovenian families with Alport syndrome and benign familial hematuria.

Alport syndrome (ATS) and benign familial hematuria (BFH) are type IV collagen inherited disorders. Mutations in COL4A5 are generally believed to cause X-linked ATS, whereas mutations in COL4A3 and COL4A4 genes can be associated with the autosomal-recessive and -dominant type of ATS or BFH. In view of the wide spectrum of phenotypes, an exact diagnosis is sometimes difficult to achieve. This study involved screening each exon with boundary intronic sequences of COL4A3, COL4A4, and COL4A5 genes by optimized polymerase chain reaction-single-stranded conformational polymorphism analysis in 17 families with ATS and in 40 families diagnosed as having BFH. Twelve different mutations were found in the COL4A5 gene in ATS patients, comprising nine missense mutations, a splice site mutation, a mutation causing frameshift, and a nonsense mutation. One of the missense mutations (p.G624D) was present not only in one family with ATS but also in five families with suspected BFH. Three heterozygous mutations in the COL4A3 gene (two missense and one frameshift) and four heterozygous mutations in COL4A4 (two splice site, one in-frame deletion, and one missense) were identified in patients with BFH. Sixteen mutations are to the best of our knowledge new and private.

Polymorphisms in multidrug resistance 1 (MDR1) gene are associated with refractory Crohn disease and ulcerative colitis.

We used coding and noncoding polymorphisms evenly spaced across the ABCB1/MDR1 gene to perform association analysis in Slovenian patients with inflammatory bowel diseases and to obtain haplotype structure and patterns of linkage disequilibrium (LD) in the MDR1 gene. A disease association study was performed in 307 IBD patients, including 144 patients with ulcerative colitis (UC) and 163 patients with Crohn's disease (CD), and 355 healthy controls. Here we report an association between MDR1 alleles, polymorphisms and haplotypes and refractory CD patients, who do not respond to standard therapy, including patients who develop fistulas. We also report an association with UC and MDR1 polymorphisms in a Slovenian population. Haplotypes significantly associated with diseases were defined by single-nucleotide polymorphisms (SNPs) in exons 12 (1236 C>A), 21(A893S), and 26 (3435 C>T). In addition, two intronic SNPs in LD with the disease haplotype, one in intron 13 (rs2235035) and another in intron 16 (rs1922242), were significantly associated with refractory Crohn (P=0.026, odds ratio (OR) 2.7 and P=0.025, OR 2.8, respectively), as well as with UC (P=0.006, OR 1.8 and P=0.026, OR 1.9, respectively). Our results suggest that MDR1 is a potential target for therapy in refractory CD patients and in patients with UC.

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.

Mutation detection 2001: Sixth International Symposium on Mutations in the Human Genome, May 3-7, 2001, Bled, Slovenia.

Mutation Detection 2001, an international symposium on human mutations and mutation detection methodologies, was held in Bled, Republic of Slovenia, on May 3-7, 2001. The event was sponsored by the Human Genome Organization (HUGO) and several other co-sponsors. It provided an important forum for not only defining the state-of-the-art in mutation detection methodologies but also a valuable chance for international collaboration. A special issue of Human Mutation with articles derived from the symposium is scheduled for publication in early 2002. Meeting highlights are described at http://www.mutations2001.bled.si.

The role of P-glycoprotein (MDR1) polymorphisms and mutations in colorectal cancer.

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.

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.

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.

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.

VHL alterations in human clear cell renal cell carcinoma: association with advanced tumor stage and a novel hot spot mutation.

To elucidate the role of somatic alterations for renal cancer etiology and prognosis, we analyzed 227 sporadic renal epithelial tumors for mutations and hypermethylations in the von Hippel-Lindau tumor suppressor gene VHL. Tumors were classified according to the recommendations of the Union Internationale Contre le Cancer (UICC) and the American Joint Committee on Cancer (AJCC). Somatic VHL mutations were identified by PCR, single-strand conformation polymorphism analysis, and sequencing, and hypermethylations were identified by restriction enzyme digestion and Southern blotting. Frequencies of VHL alterations were established, and an association with tumor type or tumor type and tumor stage was evaluated. VHL mutations and hypermethylations were identified in 45% of clear cell renal cell carcinomas (CCRCCs) and occasionally (3 of 28) in papillary (chromophilic) renal cell carcinomas (RCCs). Lack of VHL mutations and hypermethylations in chromophobe RCCs and oncocytomas was statistically significant (P = 0.0001 and P = 0.0004, respectively). RCCs carrying VHL alterations showed, in nine cases (12%), mutations at a hot spot involving a thymine repeat (ATT.TTT) in exon 2. Tumor staging was critical to the VHL mutation/hypermethylation detection rate in CCRCCs shown by separate evaluation of patients from medical centers in Munich, Heidelberg, and Mainz. The spectrum of pT1, pT2, and pT3 CCRCCs and the VHL mutation/hypermethylation detection rate varied among these three groups. Altogether, VHL alterations were significantly associated with pT3 CCRCCs (P = 0.009). This is the first evidence of frequent somatic VHL mutations at a particular site within exon 2 and an association of VHL mutations/hypermethylations with a standard prognostic factor.

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.

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.