Mutational screening of inverted formin 2 in adult-onset focal segmental glomerulosclerosis or minimal change patients from the Czech Republic.

BACKGROUND: Mutations in INF2 are frequently responsible for focal segmental glomerulosclerosis (FSGS), which is a common cause of end stage renal disease (ESRD); additionally, they are also connected with Charcot-Marie-Tooth neuropathy. INF2 encodes for inverted formin 2. This protein participates in regulation of the dynamics of the actin cytoskeleton, involving not only the polymerisation, but also the depolymerisation of filaments. The present study is the first mutational analysis of INF2 done in the Czech Republic. METHODS: Mutational analysis of INF2 was performed on 109 patients (mean age at onset 41.44 ± 18.91 years) with FSGS or minimal change disease (MCD); and also in 6 patients without renal biopsy who had already developed chronic kidney disease (CKD)/ESRD at the time of diagnosis. We used high resolution melting method (HRM), with subsequent Sanger sequencing, in suspect samples from HRM analysis. The HRM method is an effective method for the screening of large cohorts of patients. RESULTS: Two pathogenic mutations (p.Arg214His and p.Arg218Gln) were detected in INF2. The first (p.Arg214His) was identified in the FSGS patient with a positive family history. The second mutation (p.Arg218Gln) was found in two brothers with ESRD of unknown etiology. The most frequent sequence change was the substitution p.P35P, the incidence of which corresponded with the frequencies available in the ExAC Browser and gnomAD Browser databases. This analysis also detected different exonic and intronic changes that probably did not influence the phenotype of the included patients. CONCLUSIONS: The INF2 mutational screening is useful in familial FSGS cases as well as in patients with an unknown cause for their ESRD, but with a positive family history. INF2 seems to be not only the cause of FSGS, but also of ESRD of unknown etiology. Our study has confirmed that the HRM analysis is a very useful method for the identification of single nucleotide substitutions.

Bilineal inheritance of pathogenic PKD1 and PKD2 variants in a Czech family with autosomal dominant polycystic kidney disease - a case report.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disorder, leading to end stage renal failure and kidney transplantation in its most serious form. The severity of the disease's manifestation depends on the genetic determination of ADPKD. The huge variability of different phenotypes (even within a single family) is not only modulated by the two main ADPKD genes (PKD1 and PKD2) but also by modifier genes and the whole genetic background. CASE PRESENTATION: This is a report of an ADPKD family with co-inheritance of PKD1 and PKD2 pathogenic variants. The proband, with an extremely serious manifestation of ADPKD (the man was diagnosed in early childhood, and with end stage renal disease aged 23), underwent genetic analysis of PKD1 and PKD2, which revealed the presence of pathogenic mutations in both of these genes. The missense PKD2 mutation p.Arg420Gly came from the proband's father, with a mild ADPKD phenotype. The same mutation of the PKD2 gene and similar mild disease presentation were found in the proband's aunt (father's sister) and her son. The nonsense mutation p.Gln2196* within the PKD1 gene was probably inherited from the proband's mother, who died at the age of 45. It was only discovered post mortem, that the real cause of her death was kidney failure as a consequence of untreated ADPKD. Unfortunately, neither the DNA of the proband's mother nor the DNA of any other family members from this side of the pedigree were available for further examination. The proband underwent successful cadaveric kidney transplantation at the age of 24, and this replacement therapy lasted for the next 15 years. CONCLUSIONS: Here, we present a first case of bilineal ADPKD inheritance in the Czech Republic. This report highlights the significant role of modifier genes in genetic determination of ADPKD, especially in connection with seriously deteriorated disease phenotypes. In our case, the modifying role is probably mediated by the PKD2 gene.

Detection of SHOX gene aberrations in routine diagnostic practice and evaluation of phenotype scoring form effectiveness.

Heterozygous aberrations of SHOX gene have been reported to be responsible for Léri-Weill dyschondrosteosis (LWD) and small portion of idiopathic short stature. The study was established to assess effectiveness of using phenotype 'scoring form' in patients indicated for SHOX gene defect analysis. The submitted study is based on a retrospective group of 352 unrelated patients enrolled as a part of the routine diagnostic practice and analyzed for aberrations affecting the SHOX gene. All participants were scanned for deletion/duplication within the main pseudoautosomal region (PAR1) using the multiplex ligation-dependent probe amplification (MLPA) method. The phenotype 'scoring form' is used in our laboratory practice to preselect patients for subsequent mutation analysis of SHOX gene-coding sequences. The overall detection rate was 11.1% but there was a significant increase in frequency of SHOX gene defect positive with increasing achieved score (P<0.0001). The most frequent aberration was a causal deletion within PAR1. In three probands, MLPA analysis indicated a more complex rearrangement. Madelung deformity or co-occurrence of disproportionate short stature, short forearm and muscular hypertrophy had represented the most potent markers to determine the likelihood of SHOX gene defect detection. We conclude that appliance of phenotype 'scoring form' had saved excessive sample analysis and enabled effective routine diagnostic testing.

[Gitelman´s syndrome as common cause of hypokalemia and hypomagnesemia]. / Gitelmanuv syndrom jako castá prícina hypokalemie a hypomagnezemie.

The Gitelman syndrome (GS) is an autosomal recessive disorder characterized by hypokalemic metabolic alkalosis and presence of hypocalciuria and hypomagnesemia. It is one of the most common congenital "salt-wasting" tubulo-pathies, where the impairment of function of the Na+-Cl- cotransporter (NCCT) in the distal convoluted tubule is primary and hypokalemia secondary. Hypomagnesemia is caused by the impairment of magnesium reabsorption through TRPM6 channel which is located just by NCCT. Clinically, patients suffer from fatigue and hypotension due to loss of salt and water and also have cramps and tetany. In some patients chondrocalcinosis can be identified which leads to protracted pain and repeated aseptic inflammations in the joints. The course of the disease, though, is typically benign, and it rarely leads to structural changes in the kidneys or renal impairment. In the period of 2004-2006 we commenced examination of patients with suspected GS based on clinical and laboratory findings within a grant project in the Czech Republic, and in the following years this methodology was introduced to the common laboratory practice. By the year 2011 we had identified 7 different causal mutations in the gene SLC12A3 (4 of them new) among the Czech population, which is responsible for the origin of this disease. The majority of patients, whose clinical findings indicated the presence of GS, had the mutation actually detected, specifically in heterozygous form; 4 individuals were then homozygous. Most of the identified mutations were missense mutations and the most common type found among the Czech population was the change 1315 G>A within the geneSLC12A3, which causes impairment of glycosylation of the NCCT transporter. Further a great number of single-nucleotide polymorphisms were found that may be involved in clinical manifestation of the disease.Key words: gene mutation - gene sequence - Gitelman´s syndrome - NCC channel - PCR.

Molecular genetic analysis of PKHD1 by next-generation sequencing in Czech families with autosomal recessive polycystic kidney disease.

BACKGROUND: Autosomal recessive polycystic kidney disease (ARPKD) is an early-onset form of polycystic kidney disease that often leads to devastating outcomes for patients. ARPKD is caused by mutations in the PKHD1 gene, an extensive gene that encodes for the ciliary protein fibrocystin/polyductin. Next-generation sequencing is presently the best option for molecular diagnosis of ARPKD. Our aim was to set up the first study of ARPKD patients from the Czech Republic, to determine the composition of their mutations and genotype-phenotype correlations, along with establishment of next-generation sequencing of the PKHD1 gene that could be used for the diagnosis of ARPKD patients. METHODS: Mutational analysis of the PKHD1 gene was performed in 24 families using the amplicon-based next-generation sequencing (NGS) technique. In patients without 2 causal mutations identified by NGS, subsequent MLPA analysis of the PKHD1 gene was carried out. RESULTS: Two underlying mutations were detected in 54% of families (n = 13), one mutation in 13% of families (n = 3), and in 33% of families (n = 8) no mutation could be detected. Overall, seventeen different mutations (5 novel) were detected, including deletion of one exon. The detection rate in our study reached 60% in the entire cohort of patients; but 90% in the group of patients who fulfilled all clinical criteria of ARPKD, and 42% in the group of patients with unknown kidney pathology. The most frequent mutation was T36M, accounting for nearly 21% of all identified mutations. CONCLUSIONS: Next-generation sequencing of the PKHD1 gene is a very useful method of molecular diagnosis in patients with a full clinical picture of ARPKD, and it has a high detection rate. Furthermore, its relatively low costs and rapidity allow the molecular genetic analysis of patients without the full clinical criteria of ARPKD, who might also have mutations in the PKHD1 gene.

Novel mutations of PKD genes in the Czech population with autosomal dominant polycystic kidney disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disorder caused by mutation in either one of two genes, PKD1 and PKD2. High structural and sequence complexity of PKD genes makes the mutational diagnostics of ADPKD challenging. The present study is the first detailed analysis of both PKD genes in a cohort of Czech patients with ADPKD using High Resolution Melting analysis (HRM) and Multiplex Ligation-dependent Probe Amplification (MLPA). METHODS: The mutational analysis of PKD genes was performed in a set of 56 unrelated patients. For mutational screening of the PKD1 gene, the long-range PCR (LR-PCR) strategy followed by nested PCR was used. Resulting PCR fragments were analyzed by HRM; the positive cases were reanalyzed and confirmed by direct sequencing. Negative samples were further examined for sequence changes in the PKD2 gene by the method of HRM and for large rearrangements of both PKD1 and PKD2 genes by MLPA. RESULTS: Screening of the PKD1 gene revealed 36 different likely pathogenic germline sequence changes in 37 unrelated families/individuals. Twenty-five of these sequence changes were described for the first time. Moreover, a novel large deletion was found within the PKD1 gene in one patient. Via the mutational analysis of the PKD2 gene, two additional likely pathogenic mutations were detected. CONCLUSIONS: Probable pathogenic mutation was detected in 71% of screened patients. Determination of PKD mutations and their type and localization within corresponding genes could help to assess clinical prognosis of ADPKD patients and has major benefit for prenatal and/or presymptomatic or preimplantational diagnostics in affected families as well.

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

BACKGROUND: Autosomal 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 PRESENTATION: Here 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. CONCLUSION: We 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.

Improvement of Diagnostic Yield by an Additional Amplicon Module to Hybridization-Based Next-Generation Sequencing Panels.

Many approaches aimed at improving next-generation sequencing output for clinical purposes exist. However, sequencing gaps or misalignments for regions that are difficult to cover because of their low complexity or high homology still exist. Our aim was to improve the yield of sequencing data. A hybridization-based next-generation sequencing library was pooled with custom add-on amplicon-based libraries processed by the same commercial test and run in parallel and sequenced simultaneously. Formulas and steps for proper amplicon pooling (250 to 7000 bp) and final library merging are presented. The novel strategy was tested on selected archetypal situations: diagnostics of a gene with many pseudogenes, a genomic region surrounded by Alu repeats, simple one-time addition of an extra gene, and mosaicism detection. The sequence of all supplemented genomic regions was traced with reasonable coverage at the background of a hybridization captured library. The flexible add-on module expands the possibilities of routine diagnostics. The technical solution makes it possible to mix amplicons that differ significantly in size and process them in one tube simultaneously with samples of the hybridization-based panel. The proposed approach reduces turnaround time and increases diagnostic yield.

Ambulatory blood pressure and hypertension control in children with autosomal recessive polycystic kidney disease: clinical experience from two central European tertiary centres.

OBJECTIVE: : Arterial hypertension is a common complication in patients with autosomal recessive polycystic kidney disease (ARPKD), occurring in 33-75% of children when measured by office blood pressure (OBP). Ambulatory blood pressure monitoring (ABPM) is a superior tool for investigating blood pressure relative to OBP. The aim of our study was to investigate the prevalence and control of hypertension in children with ARPKD based on ABPM. METHODS: This retrospective study evaluated 36 children with ARPKD and at least one ABPM performed in two our tertiary paediatric nephrology centres and 29 children with at least two ABPM. Ambulatory hypertension was defined as mean daytime or night-time BP at least 95th percentile or use of antihypertensives and controlled hypertension as normal ambulatory BP in children on antihypertensive drugs. RESULTS: The first ABPM study revealed ambulatory hypertension in 94% of children. Untreated or uncontrolled ambulatory hypertension was diagnosed in 67% and controlled hypertension in only 28%. Masked hypertension was found in 5.5% and white-coat hypertension in 14%. The last ABPM study revealed ambulatory hypertension in 86% (all 86% hypertensive children on drugs, i.e. no untreated hypertension), the prevalence of controlled hypertension increased to 59%. Masked hypertension was detected in 8.3% and white-coat hypertension in 10%. Ambulatory blood pressure correlated neither with kidney length nor with glomerular filtration rate. Echocardiography demonstrated left ventricular hypertrophy (LVH) in 27% of children at the time of their first ABPM. CONCLUSION: The prevalence of ambulatory hypertension is very high in children with ARPKD, while the control of hypertension improves over time.

Results of targeted next-generation sequencing in children with cystic kidney diseases often change the clinical diagnosis.

Cystic kidney diseases are a very heterogeneous group of chronic kidney diseases. The diagnosis is usually based on clinical and ultrasound characteristics and the final diagnosis is often difficult to be made. Next-generation sequencing (NGS) may help the clinicians to find the correct final diagnosis. The aim of our study was to test the diagnostic yield of NGS and its ability to improve the diagnosis precision in a heterogeneous group of children with cystic kidney diseases. Next-generation sequencing of genes responsible for the formation of cystic kidneys was performed in 31 unrelated patients with various clinically diagnosed cystic kidney diseases gathered at the Department of Pediatrics of Motol University Hospital in Prague between 2013 and 2018. The underlying pathogenic variants were detected in 71% of patients (n = 22), no or only one (in case of autosomal recessive inheritance) pathogenic variant was found in 29% of patients (n = 9). The result of NGS correlated with the clinical diagnosis made before the NGS in 55% of patients (n = 17), in the remaining 14 children (45%) the result of NGS revealed another type of cystic kidney disease that was suspected clinically before or did not find causal mutation in suspected genes. The most common unexpected findings were variants in nephronophthisis (NPHP) genes in children with clinically suspected autosomal recessive polycystic kidney disease (ARPKD, n = 4). Overall, 24 pathogenic or probably pathogenic variants were detected in the PKHD1 gene, 8 variants in the TMEM67 gene, 4 variants in the PKD1 gene, 2 variants in the HNF1B gene and 2 variants in BBS1 and NPHP1 genes, respectively. NGS is a valuable tool in the diagnostics of various forms of cystic kidney diseases. Its results changed the clinically based diagnoses in 16% (n = 5) of the children.

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

BACKGROUND: Autosomal 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. METHODS: The 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. RESULTS: In 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. CONCLUSION: Twenty 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.

Genotype-phenotype correlation in children with autosomal dominant polycystic kidney disease.

Adults with autosomal dominant polycystic kidney disease (ADPKD) and PKD1 mutations have a more severe disease than do patients with PKD2 mutations. The aim of this study was to compare phenotypes between children with mutations in the PKD1/PKD2 genes. Fifty PKD1 children and ten PKD2 children were investigated. Their mean age was similar (8.6 +/- 5.4 years and 8.9 +/- 5.6 years). Renal ultrasound was performed, and office blood pressure (BP), ambulatory BP, creatinine clearance and proteinuria were measured. The PKD1 children had, in comparison with those with PKD2, significantly greater total of renal cysts (13.3 +/- 12.5 vs 3.0 +/- 2.1, P = 0.004), larger kidneys [right/left kidney length 0.89 +/- 1.22 standard deviation score (SDS) vs 0.17 +/- 1.03 SDS, P = 0.045, and 1.19 +/- 1.42 SDS vs 0.12 +/- 1.09 SDS, P = 0.014, successively] and higher ambulatory day-time and night-time systolic BP (day-time/night-time BP index 0.93 +/- 0.10 vs 0.86 +/- 0.05, P = 0.021 and 0.94 +/- 0.07 vs 0.89 +/- 0.04, P = 0.037, successively). There were no significant differences in office BP, creatinine clearance or proteinuria. Prenatal renal cysts (14%), hypertension defined by ambulatory BP (27%) and enlarged kidneys (32%) were observed only in the PKD1 children. This is the first study on genotype-phenotype correlation in children with ADPKD. PKD1 children have more and larger renal cysts, larger kidneys and higher ambulatory BP than do PKD2 children. Renal cysts and enlarged kidneys detected prenatally are highly specific for children with PKD1.

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

BACKGROUND: Germline 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. METHODS: Here, 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. RESULTS: In 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. CONCLUSION: The 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.

PKD1 and PKD2 mutations in Slovenian families with autosomal dominant polycystic kidney disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder caused by mutations in at least two different loci. Prior to performing mutation screening, if DNA samples of sufficient number of family members are available, it is worthwhile to assign the gene involved in disease progression by the genetic linkage analysis. METHODS: We collected samples from 36 Slovene ADPKD families and performed linkage analysis in 16 of them. Linkage was assessed by the use of microsatellite polymorphic markers, four in the case of PKD1 (KG8, AC2.5, CW3 and CW2) and five for PKD2 (D4S1534, D4S2929, D4S1542, D4S1563 and D4S423). Partial PKD1 mutation screening was undertaken by analysing exons 23 and 31-46 and PKD2 . RESULTS: Lod scores indicated linkage to PKD1 in six families and to PKD2 in two families. One family was linked to none and in seven families linkage to both genes was possible. Partial PKD1 mutation screening was performed in 33 patients (including 20 patients from the families where linkage analysis could not be performed). We analysed PKD2 in 2 patients where lod scores indicated linkage to PKD2 and in 7 families where linkage to both genes was possible. We detected six mutations and eight polymorphisms in PKD1 and one mutation and three polymorphisms in PKD2. CONCLUSION: In our study group of ADPKD patients we detected seven mutations: three frameshift, one missense, two nonsense and one putative splicing mutation. Three have been described previously and 4 are novel. Three newly described framesfift mutations in PKD1 seem to be associated with more severe clinical course of ADPKD. Previously described nonsense mutation in PKD2 seems to be associated with cysts in liver and milder clinical course.

Hereditary Renal Hypouricemia Type 1 and Autosomal Dominant Polycystic Kidney Disease.

BACKGROUND: Renal hypouricemia (RHUC) is a heterogeneous inherited disorder characterized by impaired tubular uric acid (UA) transport with severe complications, such as acute kidney injury. Type 1 is caused by a mutation in the SLC22A12 gene (URAT1) and type 2 in the SLC2A9 gene (GLUT9). In this article, the authors present a coexpression functional characterization of variants responsible for RHUC type 1 in a Czech family with polycystic kidney disease (PKD). METHODS: The serum UA concentration in the proband was 1.1 mg/dL and was expressed as an increase in the fractional excretion of UA (43%). The URAT1 allelic variants were prepared and their urate uptake and subcellular localization were studied by Xenopus oocytes expression system. Linkage analysis using a panel of 4 CA-repeat flanking markers for chromosome 16 and a panel of 3 CA-repeat flanking markers for chromosome 4 was performed, which carry the PKD1 and PKD2 genes, respectively. RESULTS: Coexpression results showed that p.G366R and p.R477H suppressed urate transport by wild types. Colocalization studies showed an accumulation of URAT1 in the endoplasmic reticulum of the p.G366R variant and mainly retention of wild-type protein by variants p.G366R and p.R477H. CONCLUSIONS: The findings suggest that not only loss-of-function mutation of URAT1 but also the dominant-negative effect cause RHUC through loss of UA absorption, partly due to protein misfolding caused by accumulation of URAT1 protein in the endoplasmic reticulum.

APC germline mutations identified in Czech patients with familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is an autosomal dominantly inherited predisposition to colorectal cancer, which is caused by germline mutations in the adenomatous polyposis coli (APC) gene. The APC mutations have been investigated in 46 Czech unrelated FAP families and 9 suspected FAP families using DGGE analysis and direct DNA sequencing. We found 25 germline APC mutations and identified 11 which were not previously reported. Of the identified mutations, 10 were 1 to 5 bp deletions, four were 1 bp insertions and six were nonsense, all leading to the formation of premature stop codon. In addition, we detected two mutations in the splice-donor region of APC intron 11, one missense and two samesense mutations. Phenotypes of patients with known and novel types of mutations are presented and discussed.

Synchronous gastric and sebaceous cancers, a rare manifestation of MLH1-related Muir-Torre syndrome.

Muir-Torre syndrome (MTS), a rare variant of the hereditary non polyposis colorectal cancer syndrome, is an autosomal dominant genodermatosis characterised by coincidence of sebaceous gland neoplasms (sebaceous adenoma, epithelioma, or carcinoma) and at least one internal malignancy. The underlying cause of MTS is a germline mutation in DNA mismatch repair genes MSH2, MLH1 and MSH6. We report the case of a 52-year-old caucasian woman with the development of metachronous colon cancer at the age of 38 years, uterine cancer at the age of 43 years, and unique occurrence of synchronous gastric and sebaceous carcinomas related to germline point mutation c. 2194A>T in the last exon of MLH1 gene, resulting in truncated protein in C-terminal region p. Lys732X due to premature stop codon. This mutation, not previously reported in MTS, disrupts the function of MutL complexes presumably by preventing the interaction with PMS1/PMS2 and impairing the endonuclease active site. This case points out the importance of sebaceous neoplasia, especially sebaceous adenocarcinoma, as cutaneous markers of MTS for timely implementation of cancer screening programs.

Gitelman syndrome as a cause of psychomotor retardation in a toddler.

INTRODUCTION: Gitelman syndrome (GS) is a very rare autosomal recessive tubulopathy due to loss-of-function or mutation in solute carrier family12, member 3 gene (SLC12A3 gene) encoding thiazide-sensitive NaCl co-transporter in the distal convoluted tubule, leading to hypokalemia, metabolic alkalosis, hypomagnesemia, hypocalciuria and low-to-normal blood pressure. Clinical signs are mostly secondary to chronic hypokalemia and include dizziness, fatigue, constipation and weakness. Patients can also present with muscle cramps, tetany, fatigue and convulsions due to severe metabolic alkalosis or hypomagnesemia. Manifestations of GS are rarely apparent before the age of five, and the syndrome is usually diagnosed during adolescence or adulthood. Here we describe a case of GS presenting in infancy with hypokalemia and psychomotor retardation. CASE REPORT: We present an 18-month-old boy who presented with psychomotor retardation and failure to thrive. Investigations revealed hypokalemia at 2.7 mmol/L, metabolic alkalosis, hypocalciuria and normal serum magnesium level. The diagnoses of Barter syndrome (BS) and Gitelman syndrome (GS) were considered. Genetic studies confirmed the diagnosis of GS and three different mutations of in SLC12A3 gene were detected. Two mutations (c.2576T>C and c.2929C>Ty) were considered as causal ones, with the patient´s parents being the heterozygous carriers. Oral potassium supplementation resulted in normalisation of the hypokalemia and psychomotor improvement. CONCLUSION: We report a rare case of psychomotor retardation occurring at an early age in genetically confirmed GS. In spite of being a rare disorder, GS has to be considered in children with developmental delay and muscle weakness. With adequate treatment, GS patients have an excellent prognosis.

Novel mutations of the APC gene and genetic consequences of splicing mutations in the Czech FAP families.

Familial adenomatous polyposis (FAP) is an autosomal dominant syndrome with almost 100 % risk of colorectal cancer. The typical FAP is characterized by hundreds to thousands of colorectal adenomatous polyps and by extracolonic manifestations, later onset and lower number of polyps in colon is characteristic of an attenuated form (AFAP). We analyzed the APC gene for germline mutations in 90 FAP/AFAP patients. Mutation screening was performed using Denaturing Gradient Gel Electrophoresis. DNA fragments showing an aberrant electrophoretic banding pattern were sequenced. APC-mutation-negative probands were screened for large deletions of the APC gene using multiplex ligation dependent probe amplification. Analysis of mRNA variants followed in probands with possible splicing mutation by PCR amplification of target site flanking exons and sequencing the normal and aberrant products. We identified 30 germline variants among 36 unrelated probands including large deletions. Eleven APC variants detected last two years have not been reported yet. At all, fifteen of them are expected to cause errors in mRNA splicing. Analysis of mRNA in ten of these patients revealed exon skipping in seven cases, exonisation of intron in one of these as well, change of the amount of alternatively spliced product in one case, and no effect was found in three cases. In two of the patients, the biopsy of colon mucosa and polyp enabled us to examine the effect of the mutation on splicing pattern in colon cells directly. The comparison of alternative and standard transcript amount showed similar transcription pattern of exon 14 in control colon mucosa tissue (9 samples) as in 51 blood control samples.