Searching for a genetic risk profile in end-stage renal disease.

There is a strong evidence that both, genetic factors and environment, affect the development of end-stage renal disease (ESRD). The factors that initiate renal failure are probably different from those contributing to its progression. Some factors involved in the progression of nephropathy are already identified, like elevated blood pressure, hypercholesterolemia or hyperparathyroidism. Molecular genetic techniques are extensively applied to developing strategies for detection of human renal failure genes. The candidate gene approach for finding an association between polymorphic DNA markers and disease was successfully used in studying genes of the renin-angiotensin system, especially those coding angiotensin I-converting enzyme, angiotensinogen and angiotensin type 1 receptor. Several other genes, for example growth factors and cytokine genes, nitric oxide synthase or genes of the kallikrein-kinin system are being evaluated using this approach. Identification of candidate genes associated with end-stage renal disease will create a need for testing several disease loci simultaneously. Subsets of predictive genetic markers will be developed for routine use in clinical practice. Recognizing genetic changes affecting the initiation and progression of renal failure will have important clinical implications, involving testing individuals who are genetically susceptible but yet unaffected. It will make possible identification of individuals at a higher risk for renal failure and help in the choice of diagnostic tests as well as in planning therapeutic interventions.

[Renin-angiotensin system genes in chronic glomerulonephritis]. / Geny ukladu renina-angiotensyna w przewleklych klebuszkowych zapaleniach nerek.

Glomerulonephritis is a group of diseases with complex etiology, pathogenesis, morphological features and clinical course. The renin-angiotensin system genes are important group of candidate genes involved in pathogenesis of chronic renal diseases. The purpose of our study was to analyze the association of genetic polymorphisms of these genes with glomerular kidney diseases. The study population consisted of 52 patients with immunological glomerular kidney diseases and 50 hemodialyzed patients with end-stage renal failure with glomerulonephritis as primary disease. The control group consisted of 200 healthy subjects. By means of the polymerase chain reaction (PCR) the following polymorphisms were evaluated: insertion/deletion (I/D) polymorphism in intron 16 of the angiotensin-converting enzyme gene (ACE), M235T polymorphism of the angiotensinogen gene (AGT) and A1166C polymorphism of the angiotensin II type 1 receptor gene (AT1R). No significant association was found between the ACE allele and genotype frequencies and the disease. The allele frequency of the M235T polymorphism was different from that observed in the control group, but differences in the genotype distribution were not statistically significant. The CC genotype of the AT1R gene polymorphism was significantly more frequent in patients than controls. This suggests an increased susceptibility to renal diseases in individuals carrying the CC genotype. This relationship is not associated with hypertension. Our results suggest that in the Polish population the AT1R gene polymorphism might be associated with increased susceptibility to chronic renal diseases.

Restriction fragment length polymorphisms in the apolipoprotein B gene in survivors of myocardial infarction.

INTRODUCTION: The aim of this study was to investigate the association between Pvu II and Msp I polymorphisms of the human apolipoprotein B gene and risk of myocardial infarction in 90 survivors of myocardial infarction. Apolipoprotein B is important in the metabolism of lipoproteins and there is an evidence suggesting that this apolipoprotein plays a central role in atherogenesis. Some polymorphisms in the apolipoprotein B gene are associated with peripheral arterial disease, coronary artery disease and risk of myocardial infarction. MATERIAL AND METHODS: DNA was prepared from the whole blood. Samples from patients and control group were digested with Pvu II restriction enzyme. Filters were prepared by Southern blotting technique and hybridized with ApoB probe (LB25-A). Genotypes for Msp I polymorphism were determined with polymerase chain reaction. RESULTS: The frequency of the rarer allele (P2) for Pvu II polymorphism in the apolipoprotein B gene was significantly higher in myocardial infarction group (P = 0.001) compared with healthy individuals. A significant association was also found between P2 allele and the age at which myocardial infarction occurred. CONCLUSION: The results suggest that in Polish population the individuals with P2 allele of the apolipoprotein B gene are at increased risk of developing myocardial infarction. No significant correlation with myocardial infarction event was found for the Msp I polymorphism.

X-linked retinitis pigmentosa in two families with a missense mutation in the RPGR gene and putative change of glycine to valine at codon 60.

OBJECTIVE: This study describes the ophthalmic findings in two unrelated white families with X-linked retinitis pigmentosa (XLRP) caused by a missense mutation in the retinitis pigmentosa GTPase regulator (RPGR) gene. DESIGN: Genetic screening and clinical correlation. PARTICIPANTS: Thirty-six families with XLRP seen by the authors were screened for a possible mutation in the RPGR gene to identify three affected hemizygotes with retinitis pigmentosa and four heterozygote carriers in one family and one hemizygote and one carrier in a second family. INTERVENTION: All nine patients underwent a routine ocular examination, including slit-lamp biomicroscopy and a dilated fundus examination. Goldmann visual field kinetic perimetry, static threshold perimetry, and electroretinography also were obtained. The DNA screening was performed on the three affected male patients and four obligate carriers examined from one family and the two examined patients, plus an additional male and obligate carrier, from the second family to determine the presence of any causative mutation in the RPGR gene. MAIN OUTCOME MEASURES: Findings on fundus examination, static threshold and kinetic perimetry, and electroretinography testing were the main outcome measures. RESULTS: A G-->T nucleotide change at position 238 in exon 3 of the RPGR gene resulting in a putative substitute of Gly-->Val at codon 60 was shown to segregate with RP in affected males and the carrier state in female heterozygotes in these two families. The ophthalmologic findings in hemizygotes as well as the carriers in this family were within the spectrum of findings characteristically noted in XLRP families. A tapetal-like reflex was not observed in any of the five female carriers. Psychophysical and electrophysiologic testing on the carriers indicated that cone and rod functions were impaired equivalently. When present in the carriers, visual field restriction was most apparent in, or limited to, the superotemporal quadrant, which corresponded to the retinal pigmentary changes that tended to occur in the inferonasal retina. CONCLUSIONS: A mutation in exon 3 of the RPGR gene, which would result in a putative glycine to valine substitution at codon 60, is associated with a severe clinical phenotype in male patients and a patchy retinopathy without a tapetal-like reflex in carrier females. In these families, heterozygote carriers showed equivalent impairment of their cone and rod function.

Phenotype of an X-linked retinitis pigmentosa family with a novel splice defect in the RPGR gene.

PURPOSE: To assess the clinical phenotype in a Swedish family with X-linked retinitis pigmentosa (XLRP) resulting from a novel splice defect in the RPGR gene. METHODS: RPGR mutation analysis was performed in one family with XLRP, and several individuals from the family were examined clinically. RESULTS: The causative mutation in the family was demonstrated to be a single base-pair change at the splice donor site in intron 7 that resulted in skipping of the complete exon 7 in the mature RPGR transcript. The aberrant mRNA is predicted to produce an RPGR protein with an in-frame deletion of 53 amino acids, corresponding to an RCC1-homology repeat. Clinical studies that included ophthalmological examination and full-field electroretinography showed that this splice mutation resulted in a comparatively less severe form of RP. CONCLUSIONS: Correlation of a causative RPGR genotype with clinical findings in hemizygotes and carrier heterozygotes is an important step toward predictive diagnosis and should assist in the development of gene-based therapies in the future.

A new 2-base pair deletion in the RPGR gene in a black family with X-linked retinitis pigmentosa.

OBJECTIVE: To report the genetic and ophthalmic findings in a black family with X-linked retinitis pigmentosa resulting from a newly identified mutation in the RPGR (retinitis pigmentosa GTPase regulator) gene. PATIENTS: Four affected hemizygotes with retinitis pigmentosa and 2 obligate carriers were examined. Two unaffected family members, 1 woman and her unaffected son, were also examined. METHODS: Patients underwent a routine ocular examination including slitlamp examination and a dilated fundus examination. Certain patients also underwent testing with Goldmann visual field kinetic perimetry and electroretinography. DNA screening from affected male patients, 2 obligate carriers, and 2 unaffected family members was performed to determine the presence of any mutation in the RPGR gene. RESULTS: A 2-base pair deletion in exon 13 of the RPGR gene that creates a frameshift was found to segregate with the retinal disease in affected males and the carrier state in female heterozygotes in this family. The ophthalmic findings in hemizygotes and carriers were within the spectrum of findings characteristically noted in families with X-linked retinitis pigmentosa. In 2 obligate carriers, a tapetal-like reflex was not clinically apparent. CONCLUSIONS: The described mutation is the first RPGR gene mutation reported in a black family. A 2-base pair deletion in exon 13 segregates with a clinical phenotype of X-linked retinitis pigmentosa.

Spectrum of mutations in the RPGR gene that are identified in 20% of families with X-linked retinitis pigmentosa.

The RPGR (retinitis pigmentosa GTPase regulator) gene for RP3, the most frequent genetic subtype of X-linked retinitis pigmentosa (XLRP), has been shown to be mutated in 10%-15% of European XLRP patients. We have examined the RPGR gene for mutations in a cohort of 80 affected males from apparently unrelated XLRP families, by direct sequencing of the PCR-amplified products from the genomic DNA. Fifteen different putative disease-causing mutations were identified in 17 of the 80 families; these include four nonsense mutations, one missense mutation, six microdeletions, and four intronic-sequence substitutions resulting in splice defects. Most of the mutations were detected in the conserved N-terminal region of the RPGR protein, containing tandem repeats homologous to those present in the RCC-1 protein (a guanine nucleotide-exchange factor for Ran-GTPase). Our results indicate that mutations either in as yet uncharacterized sequences of the RPGR gene or in another gene located in its vicinity may be a more frequent cause of XLRP. The reported studies will be beneficial in establishing genotype-phenotype correlations and should lead to further investigations seeking to understand the mechanism of disease pathogenesis.

Analysis of the RPGR gene in 11 pedigrees with the retinitis pigmentosa type 3 genotype: paucity of mutations in the coding region but splice defects in two families.

X-linked retinitis pigmentosa (XLRP) is a severe form of inherited progressive retinal degeneration. The RP3 (retinitis pigmentosa type 3) locus at Xp21.1 is believed to account for the disease in the majority of XLRP families. Linkage analysis and identification of patients with chromosomal deletion have refined the location of the RP3 locus and recently have led to the cloning of the RPGR (retinitis pigmentosa GTPase regulator) gene, which has been shown to be mutated in 10%-15% of XLRP patients. In order to systematically characterize the RPGR mutations, we identified 11 retinitis pigmentosa type III (RP3) families by haplotype analysis. Sequence analysis of the PCR-amplified genomic DNA from patients representing these RP3 families did not reveal any causative mutation in RPGR exons 2-19, spanning >98% of the coding region. In patients from two families, we identified transition mutations in the intron region near splice sites (IVS10+3 and IVS13-8). RNA analysis showed that both splice-site mutations resulted in the generation of aberrant RPGR transcripts. Our results support the hypothesis that mutations in the reported RPGR gene are not a common defect in the RP3 subtype of XLRP and that a majority of causative mutations may reside either in as yet unidentified RPGR exons or in another nearby gene at Xp21.1.

Disease expression in X-linked retinitis pigmentosa caused by a putative null mutation in the RPGR gene.

PURPOSE: To determine the disease expression in X-linked retinitis pigmentosa (XLRP) caused by a putative null mutation in the RPGR (retinitis pigmentosa GTPase regulator) gene. METHODS: In a family with XLRP, haplotype analysis was performed with polymorphic microsatellite markers from the Xp chromosomal region, and genomic polymerase chain reaction sequencing was used to identify sequence variations in the RPGR gene. Hemizygotes and heterozygotes were evaluated clinically and with visual function tests. Optical coherence tomography (OCT) was performed on heterozygotes. Postmortem donor retinas from a heterozygote were examined by microscopy and immunocytochemistry. RESULTS: X-linked inheritance was confirmed by haplotype analysis using Xp markers. Sequence analysis of the RPGR gene identified a single base pair change, a G-->T transversion, that converts codon 52 GGA (Gly) to TGA (stop codon); the mutation segregates with the disease. A hemizygote in the third decade of life had barely measurable rod function and severely impaired cone function that diminished further over a 7-year interval. Heterozygotes varied in degree of disease expression from mild to severe. Perimetry showed loci with normal rod and cone sensitivity interspersed with loci having either equal rod and cone dysfunction or rod > cone dysfunction. Electroretinographic photoreceptor responses had equal reductions in rod and cone maximal amplitude. OCT cross sectional reflectance images of retinal regions with severe dysfunction showed reduced thickness of the retina and retinal pigment epithelium-choriocapillaris (RPE-CC) complex and increased reflections posteriorly. Regions with mild dysfunction showed similar OCT findings but with preserved retinal thickness. Retinal histopathology in a heterozygote revealed loss of photoreceptors throughout, with retention of only a few islands of cones with tiny or absent outer segments and rods lacking outer segments. CONCLUSIONS: This RPGR gene mutation, in its mildest expression in heterozygotes, causes a relatively equal disturbance of rod and cone photoreceptor function. Detectable structural change by OCT at the level of the RPE-CC can be present in patches of retina with minimal functional disturbance. More advanced disease stages in heterozygotes show greater rod than cone dysfunction, and the end stage in hemizygotes and heterozygotes is that of typical RP, with only barely detectable cone function from residual cones in a thinned retina with abnormal RPE and choriocapillaris.

Phenotypes in three Swedish families with X-linked retinitis pigmentosa caused by different mutations in the RPGR gene.

PURPOSE: To assess the clinical phenotypes in three Swedish families with X-linked retinitis pigmentosa caused by different mutations in the RPGR gene. METHODS: Three families from different parts of Sweden, including nine patients with retinitis pigmentosa and six female carriers of X-linked retinitis pigmentosa, were examined clinically. Ophthalmologic examination included kinetic perimetry with a Goldmann perimeter using standardized objects I4e and V4e, dark adaptation final thresholds with a Goldmann-Weeker adaptometer, and full-field electroretinograms. RESULTS: The clinical findings in the patients demonstrated a severe form of retinitis pigmentosa with visual handicap early in life. Patients with a microdeletion of exons 8 through 10 of the RPGR gene had a more severe phenotype compared to the patients with single base-pair mutations in the introns 10 and 13 of the RPGR gene, resulting in splicing defects. Furthermore, heterozygous carriers in these families displayed a wide spectrum of clinical features, from minor symptoms to severe visual disability. CONCLUSION: These three families show a variable clinical phenotype resulting from different mutations in the RPGR gene. A microdeletion spanning at least parts of exons 8 through 10 seems to result in a severe phenotype compared to the splice defects. Heterozygous carriers of X-linked retinitis pigmentosa with these specific RPGR genotypes also show a variability of the phenotype; carriers with the microdeletion may be severely visually handicapped.

Construction of human embryonic cDNA libraries: HD, PKD1 and BRCA1 are transcribed widely during embryogenesis.

We used the polymerase chain reaction (PCR) to construct cDNA libraries from small amounts of tissue and to screen cDNA libraries efficiently for the presence of given sequences. To isolate genes expressed in early human development, we constructed both oligo dT-primed and random hexamer-primed cDNA libraries from ten different tissues of human embryos aged 53 to 78 days post conception. Given the small amount of RNA available, it was necessary to amplify the resultant cDNA using PCR to generate sufficient amounts of cDNA for library construction. As a result of using PCR followed by sizing to eliminate smaller synthesized fragments, the size of the synthesized product was > or = 650 base pairs and the average initial complexity of the given libraries was 10(6). We screened these cDNA libraries efficiently using PCR. Primers corresponding to a given gene were used to amplify DNA from phages encompassing a cDNA library. Successful amplification of the appropriate-sized fragment demonstrated that the DNA in question was transcribed in a given tissue. We demonstrated that HD (huntingtin, the protein transcribed from the Huntington disease locus), PKD1 (the most common gene responsible for familial polycystic kidney disease) and BRCA1 (a gene responsible for familial breast cancer) are synthesized nearly ubiquitously (including during embryogenesis). Thus, these human embryonic cDNA libraries constitute a unique resource to study early human development.

Sequence-tagged sites (STSs) for a set of mapped markers on chromosome 21.

Sequence tagged sites (STSs) have been proposed as a "common language" for comparing physical and genetic maps of the human genome produced by a variety of techniques. We have produced 44 STSs from 38 mapped loci on human chromosome 21. The STSs represent most of the loci designated as genetic reference or ordered physical framework markers, along with a number of others chosen to span all regions of 21q. Of the STSs, 12 are from gene segments, including 4 from exons of the APP gene encoding the amyloid beta protein precursor, and 32 mark anonymous DNA loci. These STSs make each of the corresponding loci readily accessible to the research community without the need for exchange of clones. These sites also represent multiple start points for the isolation of YAC clones that should permit overlapping the entire chromosome 21 long arm as cloned DNA.