Czech Probands Research Articles

HIIT system programming and some practical problems of its application examined within the sample of selected Czech probands in Brno

High Intensity Interval Training is a training method based on a combination of periods in which high-intensity load alternate with low-intensity exercise or passive rest, the so-called rest interval or inactive phase. Nowadays it is gaining more popularity among the general population and is applied in modern fitness centers. The authors found that more than 95 % sports centers organizing group lessons in Brno offer some form of HIIT (“Tabata System”, especially). Changing one of the HIIT components will affect the efficiency of the whole system. This effect is demonstrable on the aerobic and anaerobic performance and the composition of body tissues. Our goal within the broad research is to find out what effect changing one variable has on the most widely used HIIT program, and we wanted to examine whether the method is suitable for recreational athletes.The authors have made the first step in the form of pilot research described in this article, trying to design the system and applying the components in it. The experiment involved twenty deliberately selected male probands. They were randomly divided into two intervention groups of ten probands. In both intervention groups, we observed: number of repetitions performed, subjective load assessment (on the Borg scale) and heart rate. The original design of the pilot study included three training units per week for two weeks (a total of six training units). Basic multiple articulated exercises (Burpees and Jump Squats) were selected for both sets in these protocols in order to achieve key intensity for HIIT. There were some limitations of the experiment described in the article.The result of the first pilot study was essential concerning the adequacy of the cycle settings. The authors were forced to stop the piloting after the completion of the first week due to the acute overtraining of the probands. The reason to stop the experiment is attributed to an inadequate frequency of training units in individual weeks, which we reflected in the design of the following pilot study and reduced the number to two. The authors have kept the research questions and present the results of the modified piloting below.It can be assumed that the prolongation of the rest interval has an impact on the ability to perform repeated exercises, heart rate and subjective perception of stress in selected exercises. These results of our pilot research are also related to people's desire to get as much as possible in as little (time) as possible. The HIIT method is (in many aspects) more effective than the continuous method. Its undeniable advantage is time saving, but efficiency is "redeemed" by intensity and demanding character (proved not just in the described experiments). Where is the line between benefit sport and health-threatening sport? What is the "correct" HIIT setting/programming and what causes a change in one of the key variables? Is less sometimes more or more demanding means more effective? Respecting people's demands and desires for performance, mental fitness and physical beauty, with regard to sustainability and health above all, we will seek answers to all these questions. The first step towards finding them is the study carried out.

Hereditary hyperferritinemia-cataract syndrome in three Czech families: molecular genetic testing and clinical implications

Hereditary hyperferritinemia-cataract syndrome (HHCS) is an autosomal dominant disorder manifesting with high serum ferritin levels and the formation of early-onset cataracts, with numerous small opacities, predominantly in the lens cortex. HHCS is caused by mutations in the iron-responsive element of the FTL gene. The aim of this study was to establish a molecular diagnosis in three Czech probands with suspected HHCS. A complex ocular and systemic evaluation, including ferritin and iron measurements, was performed. The 5' untranslated region of FTL was directly sequenced in all available family members, followed by paternity testing in one family. Three different FLT pathogenic variants (c.-161C>T, c.-167C>T, and c.-168G>C) present in the heterozygous state were detected in each of the 3 probands. Two segregated with the disease phenotype within the families, but c.-167C>T occurred de novo (confirmed by paternity testing). Prior to establishing molecular diagnosis, two probands were misdiagnosed with hemochromatosis. One individual, aged 43years, underwent phlebotomy; another, aged 8.5years, was treated with the iron chelator deferasirox, leading to life-threatening acute hyperammonemia, without severe liver injury. Lack of family history does not exclude HHCS, because the pathogenic variant can arise de novo. Noncoding regions are often omitted from diagnostic gene panels, thus evading detection. Careful clinical evaluations and targeted genetic screening are important for avoiding potentially harmful treatments.

Schnyder corneal dystrophy and associated phenotypes caused by novel and recurrent mutations in the UBIAD1 gene

BackgroundThe purpose of this study was to identify the genetic cause and describe the clinical phenotype of Schnyder corneal dystrophy (SCD) in six unrelated probands.MethodsWe identified two white Czech, two white British and two South Asian families with a clinical diagnosis of SCD. Ophthalmic assessment included spectral domain optical coherence tomography (SD-OCT) of one individual with advanced disease, and SD-OCT and confocal microscopy of a child with early stages of disease. UBIAD1 coding exons were amplified and Sanger sequenced in each proband. A fasting serum lipid profile was measured in three probands. Paternity testing was performed in one family.ResultsA novel heterozygous c.527G>A; p.(Gly176Glu) mutation in UBIAD1 was identified in one Czech proband. In the second Czech proband, aged 6 years when first examined, a previously described de novo heterozygous c.289G>A; p.(Ala97Thr) mutation was found. Two probands of South Asian descent carried a known c.305G>A; p.(Asn102Ser) mutation in the heterozygous state. Previously reported heterozygous c.361C>T; p.(Leu121Phe) and c.308C>T; p.(Thr103Ile) mutations were found in two white British families. Although crystalline deposits were present in all probands the affected area was small in some individuals. Corneal arcus and stromal haze were the most prominent phenotypical feature in two probands. In the Czech probands, SD-OCT confirmed accumulation of reflective material in the anterior stroma. Crystalline deposits were visualized by confocal microscopy. Mild dyslipidemia was found in all three individuals tested.ConclusionAlthough de novo occurrence of mutations in UBIAD1 is extremely rare, SCD should be considered in the differential diagnosis of bilateral corneal haze and/or crystal deposition, especially in children.

Autosomal recessive limb-girdle muscular dystrophies in the Czech Republic.

BackgroundAutosomal recessive limb-girdle muscular dystrophies (LGMD2) include a number of disorders with heterogeneous etiology that cause predominantly weakness and wasting of the shoulder and pelvic girdle muscles. In this study, we determined the frequency of LGMD subtypes within a cohort of Czech LGMD2 patients using mutational analysis of the CAPN3, FKRP, SGCA, and ANO5 genes.MethodsPCR-sequencing analysis; sequence capture and targeted resequencing.ResultsMutations of the CAPN3 gene are the most common cause of LGMD2, and mutations in this gene were identified in 71 patients in a set of 218 Czech probands with a suspicion of LGMD2. Totally, we detected 37 different mutations of which 12 have been described only in Czech LGMD2A patients. The mutation c.550delA is the most frequent among our LGMD2A probands and was detected in 47.1% of CAPN3 mutant alleles. The frequency of particular forms of LGMD2 was 32.6% for LGMD2A (71 probands), 4.1% for LGMD2I (9 probands), 2.8% for LGMD2D (6 probands), and 1.4% for LGMD2L (3 probands).Further, we present the first results of a new approach established in the Czech Republic for diagnosis of neuromuscular diseases: sequence capture and targeted resequencing. Using this approach, we identified patients with mutations in the DYSF and SGCB genes.ConclusionsWe characterised a cohort of Czech LGMD2 patients on the basis of mutation analysis of genes associated with the most common forms of LGMD2 in the European population and subsequently compared the occurrence of particular forms of LGMD2 among countries on the basis of our results and published studies.

Macular corneal dystrophy and associated corneal thinning.

To identify the molecular genetic cause of macular corneal dystrophy (MCD) in four probands, and characterize phenotypic similarities between MCD and keratoconus. We performed ophthalmological examination, Scheimpflug imaging (Pentacam, Oculus Inc.), histopathological examination of excised corneal buttons, and direct sequencing of the CHST6 coding region. Pentacam measurements were taken in six eyes of three probands. All showed diffuse corneal thinning with paracentral steepening of the anterior corneal surface that was graded as keratoconus by the integrated software, but without associated ectasia of the posterior corneal surface or regional thinning. Homozygous or compound heterozygous CHST6 mutations were identified in all cases, including two novel mutations, c.13C>T; p.(Arg5Cys) and c.289C>T; p.(Arg97Cys). Localized elevation of the anterior corneal curvature can occur in MCD in the absence of other features of keratoconus. The identification of a further two Czech probands with the compound allele c.[484C>G; 599T>G] supports the enrichment of this allele in the study population.

Further Genetic and Clinical Insights of Posterior Polymorphous Corneal Dystrophy 3

Posterior polymorphous corneal dystrophy (PPCD) is a very rare disorder characterized by primary changes of the posterior corneal layers. Sequence variants in 3 genes are associated with the development of PPCD, including ZEB1 that is responsible for PPCD3. Evidence suggests at least 1 more gene remains to be identified. To determine the molecular genetic cause of PPCD3. We performed extensive ophthalmological examination, including rotating Scheimpflug imaging technology and specular microscopy, and direct sequencing of the ZEB1 coding region. Comprehensive review of published PPCD3-causing variants was undertaken. Ophthalmology department of a university hospital. Four Czech probands. Results of ophthalmological examination and direct sequencing of the ZEB1 coding region. The following 2 novel frameshift mutations within ZEB1 were identified: c.2617dup in exon 8 in a 22-year-old woman, considered to be most likely de novo in origin, and c.698dup in exon 6 in a 20-year-old man. The first patient had mild changes consistent with PPCD and bilateral best-corrected visual acuity of 1.00. The corneal phenotype of the patient in the second case was more severe, with best-corrected visual acuity of 0.40 OD and 0.05 OS. Corneas of both probands were abnormally steep (keratometry readings, flat ≥ 47.4 diopters [D] and steep ≥ 49.2 D) with increased pachymetry values but no pattern indicative of keratoconus. Specular microscopy in both patients revealed reduced endothelial cell density (range, 1055/mm² to 1655/mm²). Both probands had a history of surgery for inguinal hernia; the male patient also reported hydrocele. Nucleotide changes within the coding region of ZEB1 underlie the pathogenesis of PPCD in 4 of 23 Czech probands (17%). The cumulative de novo ZEB1 mutation rate is at least 14%. Possible involvement of ZEB1 sequence variants not readily identified by direct sequencing of coding regions needs to be further investigated. Our findings also have implications for patient counseling.

Genotype‐phenotype correlation in two patients with posterior polymorphous corneal dystrophy 3

AbstractPurpose To determine the molecular genetic cause of posterior polymorphous corneal dystrophy (PPCD) in four Czech probands.Methods Extensive ophthalmological examination including Pentacam and specular microscopy, and direct sequencing of the ZEB1 coding region were performed.Results Two novel frameshift mutations within ZEB1 were identified; c.2617dup in exon 8 in a 21‐year‐old female, considered to be most likely de novo in origin, and c.698dup in exon 6 in a 21‐year‐old male. The first case had mild changes consistent with a PPCD diagnosis and best corrected visual acuity (BCVA) bilaterally was 1.0. The corneal phenotype of the second case was more severe with a BCVA of 0.4 in the right and 0.05 in the left eye. Corneas of both probands were abnormally steep (keratometry readings K1 ≥ 47.4 D and K2 45 ≥ 49.2 D) with increased pachymetry values, but no pattern indicative of keratoconus. Specular microscopy in both patients revealed reduced endothelial cell density (range 1055‐ 655 per mm2). Both probands had a history of surgery for inguinal hernia; the male patient also reported the presence of hydrocele.Conclusion Nucleotide changes within the coding region of ZEB1 underlie the pathogenesis of PPCD in 4 out of 23 (17.4%) Czech probands.

The molecular basis of familial hypercholesterolemia in the Czech Republic: Spectrum of LDLR mutations and genotype–phenotype correlations

BackgroundFamilial hypercholesterolemia (FH), a major risk for coronary heart disease, is predominantly associated with mutations in the genes encoding the low-density lipoprotein receptor (LDLR) and its ligand apolipoprotein B (APOB). ResultsIn this study, we characterize the spectrum of mutations causing FH in 2239 Czech probands suspected to have FH. In this set, we found 265 patients (11.8%) with the APOB mutation p.(Arg3527Gln) and 535 patients (23.9%) with a LDLR mutation. In 535 probands carrying the LDLR mutation, 127 unique allelic variants were detected: 70.1% of these variants were DNA substitutions, 16.5% small DNA rearrangements, and 13.4% large DNA rearrangements. Fifty five variants were novel, not described in other FH populations. For lipid profile analyses, FH probands were divided into groups [patients with the LDLR mutation (LDLR+), with the APOB mutation (APOB+), and without a detected mutation (LDLR−/APOB−)], and each group into subgroups according to gender. The statistical analysis of lipid profiles was performed in 1722 probands adjusted for age in which biochemical data were obtained without FH treatment (480 LDLR+ patients, 222 APOB+ patients, and 1020 LDLR−/APOB− patients). Significant gradients in i) total cholesterol (LDLR+ patients > APOB+ patients = LDLR−/APOB− patients) ii) LDL cholesterol (LDLR+ patients > APOB+ patients = LDLR−/APOB− patients in men and LDLR+patients > APOB+ patients >LDLR−/APOB− patients in women), iii) triglycerides (LDLR−/APOB− patients > LDLR+ patients > APOB+ patients), and iv) HDL cholesterol (APOB+ patients > LDLR−/APOB− patients = LDLR+ patients) were shown. ConclusionOur study presents a large set of Czech patients with FH diagnosis in which DNA diagnostics was performed and which allowed statistical analysis of clinical and biochemical data.

Glucokinase diabetes in 103 families from a country-based study in the Czech Republic: geographically restricted distribution of two prevalent GCK mutations

Glucokinase diabetes, also called GCK-MODY or maturity-onset diabetes of the young type 2 (MODY2), is caused by heterozygous mutations in the gene encoding glucokinase (GCK). The aim of study was to investigate the current prevalence of GCK mutations in a large cohort of Czech patients with typical clinical appearance of GCK-MODY. In addition, we reanalyzed the negative results obtained previously by screening using the denaturing high-performance liquid chromatography (dHPLC). We studied 140 unrelated Czech probands with clinical picture of GCK-MODY who were referred to our center from the whole of the Czech Republic between the years 1999-2009 by direct sequencing of GCK gene. A mutation in GCK was identified in 103 of 140 probands (74%). We identified 46 different GCK mutations of which 13 were novel. Several mutations were detected in multiple families: p.Glu40Lys (20 families), p.Gly318Arg (12), p.Leu315His (7) and p.Val33Ala (six families). Direct sequencing detected a GCK mutations in 9 of 20 previously dHPLC-negative samples; the sensitivity of the dHPLC screening was calculated as 84%. The study shows a relatively high proportion of GCK mutations among individuals with GCK-like phenotype, confirming the effectiveness of carefully applied clinical criteria prior to genetic testing. In the Czech MODY registry, GCK-MODY represents the biggest subgroup of MODY (35%). We report several prevalent GCK mutations with a likely founder effect in the Czech population. Furthermore, our results provide ground for a possible recommendation to reinspect all negative results previously obtained by screening using dHPLC.

Identification of CYP21A2 mutant alleles in Czech patients with 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) is comprised of a group of autosomal recessive disorders caused by an enzymatic deficiency which impairs the biosynthesis of cortisol and, in most of the severe cases, also the biosynthesis of aldosterone. Approximately 90-95% of all the CAH cases are due to mutations in the steroid 21-hydroxylase gene (CYP21A2). In this study, the molecular genetic analysis of CYP21A2 was performed in 267 Czech probands suspected of 21-hydroxylase deficiency (21OHD). 21OHD was confirmed in 241 probands (2 mutations were detected). In 26 probands, a mutation was found only in 1 CYP21A2 allele. A set of 30 different mutant alleles was determined. We describe i) mutated CYP21A2 alleles carrying novel point mutations (p.Thr168Asn, p.Ser169X and p.Pro386Arg), ii) mutated CYP21A2 alleles carrying the novel chimeric gene designated as CH-7, which was detected in 21.4% of the mutant alleles, iii) an unusual genotype with a combination of the CYP21A2 duplication, 2 point mutations and the CYP21A2 large-scale gene conversion on the second allele, and (iv) a detailed analysis of the chimeric CYP21A1P/CYP21A2 genes. In conclusion, our genotyping approach allowed for the accurate identification of the CYP21A2 gene mutations in 21OHD patients and their families and provided some useful information on diagnosis and genetic counselling.

Autosomal inheritance of diabetes in two families characterized by obesity and a novel H241Q mutation inNEUROD1

The aim of the study was to search for mutations in the NEUROD1 and IPF-1 genes in patients with clinical characteristics of maturity-onset diabetes of the young (MODY) but with no mutations in the HNF-4A (MODY1), GCK (MODY2) and TCF1 (MODY3) genes. We studied 30 unrelated Czech probands with a clinical diagnosis of MODY (median age at testing, 18 yr; median age at the recognition of hyperglycaemia, 16 yr). The promoter, exons and exon/intron boundaries of the NEUROD1 and IPF-1 genes were examined by polymerase chain reaction-denaturing high performance liquid chromatography and direct sequencing. While no mutations were found in the IPF-1 gene, a novel H241Q substitution of NEUROD1 gene was identified in two unrelated families. In the first proband, the H241Q mutation led to early diagnosed (20 yr) hyperglycaemia followed by development of diabetic microvascular complications by the age of 32 yr. The second proband suffered from slowly progressing hyperglycaemia detected at the age of 30 yr. Affected members of both families were obese. The overall prevalence of the variant among the general population was 4 of 13 568 chromosomes. We report a novel disease-associated variant in NEUROD1 identified among a set of MODYX families. The variant seems to precipitate type-2-like diabetes in excessively obese individuals.

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.

Hypertrophic Cardiomyopathy

Our research is a pilot study that specializes in the molecular genetic investigation of the TNNT2 gene in Czech patients with HCM/FHC disease. This study was initiated with exons 9 and 11 of TNNT2 because of their crucial role in the binding ability of cardiac troponin T to alpha-tropomyosin, and continued with analyses in other regions of the gene. Hundred and eighty-one Czech probands with HCM/FHC were enrolled in this study. The study group consisted of 24 families with FHC and probands without FHC history but with HCM diagnosis. The clinical diagnosis was based on echocardiography. DNA was isolated from peripheral blood lymphocytes and subsequently analyzed by the polymerase chain reaction (PCR), followed by DNA sequencing analyses, which were cross-sequenced. The DeltaGlu160 mutation was observed in a sequence of the TNNT2 gene in a patient with the severe form of hypertrophic cardiomyopathy. No sequence alteration was found in exons 9 and 11 of the TNNT2 gene found in the rest of the DNA samples. The DeltaGlu160 mutation was observed in patients with severe forms of hypertrophic cardiomyopathy. This region is responsible for binding troponin T to alpha-tropomyosin. This mutation may lead to functional and structural effects on the troponin T protein. Mutations in this region are reported relatively rarely and therefore it was unique to observe the DeltaGlu160 mutation in our study.

Is a Pro12Ala polymorphism of the PPARgamma2 gene related to obesity and type 2 diabetes mellitus in the Czech population?

The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor subfamily of transcription factors. PPARgamma2 plays a key role in regulation of adipocyte differentiation and energy homeostasis. Recent studies provide evidence that the Pro12Ala polymorphism is linked to obesity and type 2 diabetes mellitus, but the results are controversial and depend on ethnicity. The aim of this study was to determine allele frequencies and to study the influence of the polymorphism on biochemical and anthropometric parameters in a Czech healthy adult population, in type 2 diabetics, and in a group of obese women. The frequency of the Pro12Ala PPARgamma2 gene polymorphism in Czech probands is similar to other central European populations. Frequency of the Pro12Ala substitution tends to be higher in obese women and diabetics compared with controls. The fasting insulin levels in the 12Ala carriers were significantly lower within the group of diabetics even after adjustment for age, BMI, and the length of diabetes duration. In obese women, higher WHR was found in subjects with the 12Ala allele. This study indicates that the substitution Pro12Ala is not associated with a decreased obesity or diabetes risk in the Czech population. However, the present data show that fasting insulin concentrations are lower in diabetics with the 12Ala allele than in those without it. This finding provides evidence that the polymorphism may influence glucose homeostasis.