Jitka Štekrová

SHOX gene defects and selected dysmorphic signs in patients of idiopathic short stature and Léri–Weill dyschondrosteosis

The aim of the study was to analyze frequency of SHOX gene defects and selected dysmorphic signs in patients of both idiopathic short stature (ISS) and Léri-Weill dyschondrosteosis (LWD), all derived from the Czech population. Overall, 98 subjects were analyzed in the study. Inclusion criteria were the presence of short stature (-2.0 SD), in combination with at least one of the selected dysmorphic signs for the ISS+ group; and the presence of Madelung deformity, without positive karyotyping for the LWD+ group. Each proband was analyzed by use of P018 MLPA kit, which covers SHOX and its regulatory sequences. Additionally, mutational analysis was done of the coding portions of the SHOX. Both extent and breakpoint localizations in the deletions/duplications found were quite variable. Some PAR1 rearrangements were detected, without obvious phenotypic association. In the ISS+ group, MLPA analysis detected four PAR1 deletions associated with a SHOX gene defect, PAR1 duplication with an ambiguous effect, and two SHOX mutations (13.7%). In the LWD+ group, MLPA analysis detected nine deletions in PAR1 region, with a deleterious effect on SHOX, first reported case of isolated SHOX enhancer duplication, and SHOX mutation (68.8%). In both ISS+ and LWD+ groups were positivity associated with a disproportionately short stature; in the ISS+ group, in combination with muscular hypertrophy. It seems that small PAR1 rearrangements might be quite frequent in the population. Our study suggests disproportionateness, especially in combination with muscular hypertrophy, as relevant indicators of ISS to be the effect of SHOX defect.

Autosomal dominant polycystic kidney disease in a family with mosaicism and hypomorphic allele

BackgroundAutosomal dominant polycystic kidney disease (ADPKD) is the most common form of inherited kidney disease that results in renal failure. ADPKD is a systemic disorder with cysts and connective tissue abnormalities involving many organs. ADPKD caused by mutations in PKD1 gene is significantly more severe than the cases caused by PKD2 gene mutations. The large intra-familial variability of ADPKD highlights a role for genetic background.Case presentationHere we report a case of ADPKD family initially appearing unlinked to the PKD1 or PKD2 loci and the influence of mosaicism and hypomorphic allele on the variability of the clinical course of the disease. A grandmother with the PKD1 gene mutation in mosaicism (p.Val1105ArgfsX4) and with mild clinical course of ADPKD (end stage renal failure at the age of 77) seemed to have ADPKD because of PKD2 gene mutation. On the other hand, her grandson had a severe clinical course (end stage renal disease at the age of 45) in spite of the early treatment of mild hypertension. There was found by mutational analysis of PKD genes that the severe clinical course was caused by PKD1 gene frameshifting mutation inherited from his father and mildly affected grandmother in combination with inherited hypomorphic PKD1 allele with described missense mutation (p.Thr2250Met) from his clinically healthy mother. The sister with two cysts and with PKD1 hypomorphic allele became the kidney donor to her severely affected brother.ConclusionWe present the first case of ADPKD with the influence of mosaicism and hypomorphic allele of the PKD1 gene on clinical course of ADPKD in one family. Moreover, this report illustrates the role of molecular genetic testing in assessing young related kidney donors for patients with ADPKD.

Novel APC mutations in Czech and Slovak FAP families: clinical and genetic aspects

BackgroundGermline mutations in the adenomatous polyposis gene (APC) result in familial adenomatous polyposis (FAP). FAP is an autosomal dominantly inherited disorder predisposing to colorectal cancer. Typical FAP is characterized by hundreds to thousands of colorectal adenomatous polyps and by several extracolonic manifestations. An attenuated form of polyposis (AFAP) is characterized by less than 100 adenomas and later onset of the disease.MethodsHere, we analyzed the APC gene for germline mutations in 59 Czech and 15 Slovak FAP patients. In addition, 50 apparently APC mutation negative Czech probands and 3 probands of Slovak origin were screened for large deletions encompassing the APC gene. Mutation screening was performed using denaturing gradient gel electrophoresis and/or protein truncation test. DNA fragments showing an aberrant electrophoretic banding pattern were sequenced. Screening for large deletions was performed by multiplex ligation dependent probe amplification. The extent of deletions was analyzed using following microsatellite markers: D5S299, D5S82, D5S134 and D5S346.ResultsIn the set of Czech and Slovak patients, we identified 46 germline mutations among 74 unrelated probands. Total mutation capture is 62,2% including large deletions. Thirty seven mutations were detected in 49 patients presenting a classical FAP phenotype (75,5%) and 9 mutations in 25 patients with attenuated FAP (36%). We report 20 novel germline APC mutations and 3 large deletions (6%) encompassing the whole-gene deletions and/or exon 14 deletion. In the patients with novel mutations, correlations of the mutation localization are discussed in context of the classical and/or attenuated phenotype of the disease.ConclusionThe results of the molecular genetic testing are used both in the establishment of the predictive diagnosis and in the clinical management of patients. In some cases this study has also shown the difficulty to classify clinically between the classical and the attenuated form of FAP according to the established criteria. Interfamilial and/or intrafamilial phenotype variability was also confirmed in some cases which did not fit well with predicted genotype-phenotype correlation. All these findings have to be taken into consideration both in the genetic counselling and in the patient care.

Influence of the Alpha-Adducin and ACE Gene Polymorphism on the Progression of Autosomal-Dominant Polycystic Kidney Disease

Background: A significant phenotypical variability is observed in autosomal dominant polycystic kidney disease (ADPKD). The variability can not be fully explained by the genetic heterogenity of the disease. We examined the influence of the ACE I/D polymorphism, adducin Trp460Gly polymorphism and the association of both polymorphisms on the progression of ADPKD towards end-stage renal failure (ESRF). Methods: 320 ADPKD patients (pts) were analyzed, 220 pts (113 males, 107 females) with ESRF before 63 years of age, with a subgroup (rapid progressors) of 20 pts (12 males, 8 females) with ESRF before 40 years of age, 52 pts (23 males, 29 females) with ESRF later than 63 years of age (slow progressors), 48 ADPKD pts (18 males, 30 females) with mean age ±50 years with serum creatinine <110 µmol/l (slow progressors) and 200 genetically unrelated healthy Czech subjects. DNA samples from collected blood were genotyped for the ACE I/D polymorphism and the Trp460Gly of α-adducin gene polymorphism. Results: The α-adducin genotypes showed no differences among the groups of slow progressors (74% Gly/Gly, 22.9% Gly/Trp and 3.1% Trp/Trp), pts with ESRF before 63 years of age (67.7% Gly/Gly, 30.5% Gly/Trp and 1.8% Trp/Trp) and rapid progressors (75% Gly/Gly, 25% Gly/Trp). The ACE genotypes did not differ among the groups of slow progressors (27.1% I/I, 44.8% I/D and 28.1% D/D), pts with ESRF before 63 years of age (23.6% I/I, 51.4% I/D and 25% D/D) and rapid progressors (20% I/I, 55% I/D and 25% D/D). The distribution did not differ from the control group. The ages of ESRF according to different genotypes did not significantly differ. We observed a significant tendency to better prognosis in Trp allele carriers for I/I genotype in comparison with Gly/Gly homozygous subjects. Conclusion: The ACE and α-adducin polymorphisms do not play a significant role in the progression of ADPKD to ESRF.

Influence of VEGF polymorphism on progression of autosomal dominant polycystic kidney disease.

Background: Significant phenotypical variability is observed in autosomal dominant polycystic kidney disease (ADPKD). Dysregulation of vascular endothelial growth factor (VEGF) expression in the kidney has been demonstrated in a wide range of renal diseases. The aim of the present study was to assess the influence of the –2578 C/A and the –1154 G/A polymorphisms in the regulatory region of the VEGF gene upon the progression of ADPKD toward end-stage renal disease (ESRD). Methods: The study was performed on 283 ADPKD patients (145 males, 138 females, mean age 51.7 ± 10.3 years) who had reached ESRD. Patients were divided into three groups: (1) ESRD development later than in 63 years (slow progressors, n = 47), (2) ESRD development before 45 years (rapid progressors, n = 69), and (3) ESRD development between 45 and 63 years (intermediate progressors, n = 167). Genetically unrelated healthy Czech individuals were analyzed as a control group (n = 311, 153 males, 158 females, mean age 44.6 ± 9.2 years). DNA samples were genotyped for the –2578 C/A and for the –1154 G/A polymorphisms of the VEGF gene promoter. The serum levels of VEGF were established in 111 healthy Czech individuals from the control group. Results: The VEGF –2578 C/A and –1154 G/A genotype distribution showed no differences among the groups of slow, rapid and intermediate progressors. The age of ESRD with regard to different genotypes was not significantly different in all ADPKD patients. However, the AA genotype of the –2578 C/A polymorphism was associated with a significantly higher age of ESRD than other genotypes in rapid progressors (42.7 vs. 40.5 years, p = 0.01). The CG haplotype was found significantly more frequent in ADPKD rapid progressors than in slow progressors (p = 0.047). Serum levels of VEGF did not significantly differ in the control group, according to different genotypes of both polymorphisms. Conclusion: To conclude, AA genotype of the –2578 C/A polymorphism was related to better prognosis of the disease in a limited group of ADPKD patients. Classical genetic recessive and dominant model did not find significant influence of separate VEGF polymorphisms on the progression of ADPKD. Accordingly, CG haplotype was associated with earlier onset of ESRD in ADPKD patients.

Influence of the endothelial nitric oxide synthase polymorphism on the progression of autosomal dominant polycystic kidney disease and IgA nephropathy.

Background: The reason of variability of clinical course and progression to end-stage renal failure (ESRF) of two widespread chronic nephropathies–-autosomal dominant polycystic kidney disease (ADPKD) and IgA nephropathy (IGAN) is not clear. The endothelial dysfunction is considered in the number of factors possibly influencing the prognosis of these nephropathies. Our study tried to verify the hypothesis that endothelial nitric oxide synthase (ecNOS) gene polymorphisms in intron 4 could have some relevance to the progression of ADPKD and/or IgA nephropathy. Methods: We examined 128 Czech patients with ADPKD (62 males, 66 females) and 93 patients with IGAN (51 males, 42 females). As a control group we used 100 genetically unrelated healthy subjects (50 men, 50 women, mean age 51.2 ± 8.2). The genomic DNA was amplified by polymerase chain reaction (PCR) and the products were separated on 1.5% agarose gel and visualized by ultraviolet transillumination. We compared homozygous subjects for ecNOSb allele with homozygous and heterozygous subjects for ecNOSa allele. Results: The frequencies of ecNOSa/b + a/a and ecNOSb/b genotypes were 19% (19/100) and 81% (81/100) in the control group. The frequencies of ecNOSa/b + a/a and ecNOSb/b genotypes in ADPKD patients were: 26.6% (8/30) and 73.4% (22/30) in ADPKD patients with normal renal function, 30% (9/30) and 70% (21/30) in ADPKD with ESRF, 35.2% (18/51) and 64.8% (33/51) in young ADPKD patients, 60% (12/20) and 40% (8/20) in ADPKD patients with ESRF later than in 62 years. In IGAN the frequencies of ecNOSa/b + a/a and ecNOSb/b genotypes were 24% (12/50) and 76% (38/50) in IgA with normal renal function and 20.9 % (9/43) and 79.1% (38/43) in IgA with ESRF. Conclusion: Both in ADPKD and IGAN groups there was no significant difference in the frequencies of ecNOS genotypes between patients with normal renal function and age matched patients with ESRF and between patients with normal renal function and control group. The frequency of ecNOS a allele was significantly higher in a number limited group ADPKD patients with ESRF later than in 62 years (Chi-square test p < 0.05). This higher frequency of a allele among ADPKD patients with later onset of ESRF could suggest the trend of positive influence of a allele in ADPKD patients.

Endothelial Nitric Oxide Synthase Affects the Progression of Autosomal Dominant Polycystic Kidney Disease

Background/Aim: The phenotypic variability of autosomal dominant polycystic kidney disease (ADPKD) cannot be explained only by various mutations of two known genes (PKD1 and PKD2), but the influence of other unknown factors should also be considered. Impairment of endothelial function has been observed in ADPKD patients. Our study tried to verify the hypothesis that endothelial nitric oxide synthase (ecNOS) gene polymorphisms in intron 4 could have some relevance to the progression of ADPKD. Methods: We examined 155 Czech patients with ADPKD (80 males, 75 females; mean age 43.4 ± 14.7 years) and 100 genetically unrelated healthy subjects (50 men, 50 women; mean age 51.2 ± 8.2 years). The genomic DNA was amplified by polymerase chain reaction and the products were separated on 1.5% agarose gel and visualised by ultraviolet transillumination. We compared subjects homozygous for the ecNOSb allele with subjects homozygous and heterozygous for the ecNOSa allele. Results: The frequencies of ecNOSb/b, ecNOSa/b and ecNOSa/a were 81% (81/100), 17% (17/100) and 2% (2/100), respectively, in the control group, 60% (30/50), 32% (16/50) and 8% (4/50), respectively, in ADPKD patients with end-stage renal failure (ESRF), and 68.5% (72/105), 28.6% (30/105) and 2.9% (3/105), respectively, in ADPKD patients with normal renal function. The two-tailed t test revealed that the frequency of the ecNOSa allele (ecNOSa/b and ecNOSa/a) in dialysed patients was significantly higher than in ADPKD patients with normal renal function (p < 0.05). Conclusion: The a allele of the ecNOS gene polymorphism showed a significantly higher incidence among patients with ESRF caused by ADPKD. The a allele might have a negative influence on the progression of ADPKD.

Influence of Endothelin-1 Gene Polymorphisms on the Progression of Autosomal Dominant Polycystic Kidney Disease

Background/Aim:A significant phenotypical variability is observed in autosomal dominant polycystic kidney disease (ADPKD). The variability cannot be fully explained by the genetic heterogeneity of the disease. Endothelin-1 (ET-1) has been suggested to be a major promoting factor in renal diseases. The role of the ET-1 gene locus (EDN1) in the renal function in the general nondiabetic population was evaluated. We examined the influence of three single-nucleotide polymorphisms of the ET-1 gene (EDN1) – K198N, 3A/4A, and T-1370G – on the progression of ADPKD towards end-stage renal disease (ESRD). Methods: Two hundred and five ADPKD patients (113 males and 92 females) who had reached ESRD were analyzed. The patients were divided into three groups: (1) 48 patients (23 males and 25 females) with ESRD later than 63 years of age (slow progressors), (2) 74 patients (41 males and 33 females) with ESRD before 45 years of age (rapid progressors), and (3) 83 patients (49 males and 34 females) with ESRD between 45 and 63 years old. DNA samples from collected blood were genotyped for three single-nucleotide polymorphisms of EDN1: K198N, 3A/4A, and T-1370G. Haplotype analysis was also done in 200 healthy individuals. We compared the frequencies of the different genotypes between the groups of slow and rapid progressors and the ages at the time of ESRD regarding the EDN1 genotypes. Results: The EDN1 genotype distribution showed no differences among the groups of slow progressors, rapid progressors, the ADPKD group with ESRD between 45 and 63 years old, and the control group. Comparing the ages of ESRD of all patients, we did not find significant differences with regard to the different genotypes. Furthermore, we compared the combinations of the different haplotypes and the ages at the time of ESRD. We found no differences in ages at the time of ESRD in patients with different haplotypes in the endothelin promoter (T-1370G) in combination with 3A/4A or K198N polymorphisms. Comparing the ages at the time of ESRD in patients with different 3A/4A and K198N haplotypes, we found a significantly lower age at the time of ESRD (47.1 ± 8.7 years) in the carriers of the 4A allele in combination with the 198N allele (4A/4A, 3A/4A + 198KN,NN) than in the carriers of the 4A allele homozygous for the K198 allele (52.9 ± 10.9 years; 4A/4A, 3A/4A + 198KK; t test: p < 0.01) and in the carriers of the 198N allele homozygous for the 3A allele (53 ± 11.2 years; 3A/3A + 198KN,NN; t test: p < 0.05). Conclusions: We excluded an effect of K198N, 3A/4A, and T-1370G polymorphisms of EDN1 on the progression of ADPKD. However, a deleterious effect of the combination of 4A and 198N alleles of EDN1 was observed in APKDK individuals.

New mutations in the PKD1 gene in Czech population with autosomal dominant polycystic kidney disease

BackgroundAutosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disease. The disease is caused by mutations of the PKD1 (affecting roughly 85% of ADPKD patients) and PKD2 (affecting roughly 14% of ADPKD patients) genes, although in several ADPKD families, the PKD1 and/or PKD2 linkage was not found. Mutation analysis of the PKD1 gene is complicated by the presence of highly homologous genomic duplications of the first two thirds of the gene.MethodsThe direct detection of mutations in the non-duplicated region of the PKD1 gene was performed in 90 unrelated individuals, consisting of 58 patients with end-stage renal failure (manifesting before their 50th year of life) and 32 individuals from families where the disease was clearly linked to the PKD1 gene. Mutation screening was performed using denaturing gradient gel electrophoresis (DGGE). DNA fragments showing an aberrant electrophoretic banding pattern were sequenced.ResultsIn the non-duplicated region of the PKD1 gene, 19 different likely pathogenic germline sequence changes were identified in 19 unrelated families/individuals. Fifteen likely pathogenic sequence changes are unique for the Czech population. The following probable mutations were identified: 9 nonsense mutations, 6 likely pathogenic missense mutations, 2 frameshifting mutations, one in-frame deletion and probable splice site mutation. In the non-duplicated region of the PKD1 gene, 16 different polymorphisms or unclassified variants were detected.ConclusionTwenty probable mutations of the PKD1 gene in 90 Czech individuals (fifteen new probable mutations) were detected. The establishment of localization and the type of causal mutations and their genotype - phenotype correlation in ADPKD families will improve DNA diagnosis and could help in the assessment of the clinical prognosis of ADPKD patients.

The Influence of the Endothelin-Converting Enzyme-1 Gene Polymorphism on the Progression of Autosomal Dominant Polycystic Kidney Disease

Background. A significant phenotypical variability is observed in autosomal dominant polycystic kidney disease (ADPKD), the most common renal hereditary disease. Endothelin-1 (ET-1) has been suggested to be an important disease-promoting factor of the kidney. Endothelin-converting enzyme-1 (ECE-1) is the main protease responsible for ET-1 generation by cleavage of its functionally inactive precursor. We examined the influence of the ECE-1b C-338A polymorphism on the progression of ADPKD toward end-stage renal disease (ESRD). The A allele was suggested to be associated with higher plasma level of ET-1. Methods. 200 ADPKD patients (107 males, 93 females) who had reached ESRD were analyzed. Patients were divided into three groups: (1) 47 patients (23 males, 24 females) with ESRD later than in 63 yr (slow progressors); (2) 71 patients (38 males, 33 females) with ESRD before 45 yr (rapid progressors); and (3) 82 patients (46 males, 36 females) with ESRD between 45–63 yr. Moreover, we analyzed 160 genetically unrelated healthy Czech subjects as the control group (82 males, 78 females, mean age 51.4 ± 8.2 yr). DNA samples from collected blood were genotyped for ECE-1b C-338A polymorphism using described polymerase chain reaction (PCR) followed by restriction enzyme digestion. We compared the frequencies of different genotypes between the groups of slow and rapid progressors and the ages of ESRD with regard to different genotypes. Results. The ECE-1b C-338A genotype distribution showed no differences among the groups of slow progressors, rapid progressors, ADPKD group with ESRD between 45–63 yr and control group. Comparing the ages of ESRD of all patients, we did not find significant differences in the ages with regard to different genotypes: CC (51.5 ± 10.1 yr), AC (51.6 ± 11.4 yr), AA (48.2 ± 5.9 yr). There was a tendency to lower age of ESRD in AA homozygotes in comparison with other genotypes (t-test, p = 0.12). We found no influence of gender. Conclusion. We excluded the effect of ECE-1b C-338A polymorphism on the progression of ADPKD. We could observe a mild tendency toward faster decline of renal function in AA homozygous individuals.