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The term 'endemic parkinsonism' refers to diseases that manifest with a dominant parkinsonian syndrome, which can be typical or atypical, and are present only in a particular geographically defined location or population. Ten phenotypes of endemic parkinsonism are currently known: three in the Western Pacific region; two in the Asian-Oceanic region; one in the Caribbean islands of Guadeloupe and Martinique; and four in Europe. Some of these disease entities seem to be disappearing over time and therefore are probably triggered by unique environmental factors. By contrast, other types persist because they are exclusively genetically determined. Given the geographical clustering and potential overlap in biological and clinical features of these exceptionally interesting diseases, this Review provides a historical reference text and offers current perspectives on each of the 10 phenotypes of endemic parkinsonism. Knowledge obtained from the study of these disease entities supports the hypothesis that both genetic and environmental factors contribute to the development of neurodegenerative diseases, not only in endemic parkinsonism but also in general. At the same time, this understanding suggests useful directions for further research in this area., (© 2023. Springer Nature Limited.)

The deficiency of natural anticoagulants—antithrombin (AT), protein C (PC), and protein S (PS)—is a highly predisposing factor for thrombosis, which is still underdiagnosed at the genetic level. We aimed to establish and evaluate an optimal diagnostic approach based on a high-throughput sequencing platform suitable for testing a small number of genes. A fast, flexible, and efficient method involving automated amplicon library preparation and target sequencing on the Ion Torrent platform was optimized. The cohort consisted of a group of 31 unrelated patients selected for sequencing due to repeatedly low levels of one of the anticoagulant proteins (11 AT-deficient, 13 PC-deficient, and 7 PS-deficient patients). The overall mutation detection rate was 67.7%, highest in PC deficiency (76.9%), and six variants were newly detected—SERPINC1 c.398A > T (p.Gln133Leu), PROC c.450C > A (p.Tyr150Ter), c.715G > C (p.Gly239Arg) and c.866C > G (p.Pro289Arg), and PROS1 c.1468delA (p.Ile490fs) and c.1931T > A (p.Ile644Asn). Our data are consistent with those of previous studies, which mostly used time-consuming Sanger sequencing for genotyping, and the indication criteria for molecular genetic testing were adapted to this process in the past. Our promising results allow for a wider application of the described methodology in clinical practice, which will enable a suitable expansion of the group of indicated patients to include individuals with severe clinical findings of thrombosis at a young age. Moreover, this approach is flexible and applicable to other oligogenic panels.

The short stature homeobox-containing (SHOX) is the most frequently analysed gene in patients classified as short stature patients (ISS) or diagnosed with Leri-Weill dyschondrosteosis (LWD), Langer mesomelic dysplasia (LMD), or Madelung deformity (MD). However, clinical testing of this gene focuses primarily on single nucleotide variants (SNV) in its coding sequences and copy number variants (CNV) overlapping SHOX gene. This review summarizes the clinical impact of variants in noncoding regions of SHOX. RECENT FINDINGS: CNV extending exclusively into the regulatory elements (i.e., not interrupting the coding sequence) are found more frequently in downstream regulatory elements of SHOX. Further, duplications are more frequent than deletions. Interestingly, downstream duplications are more common than deletions in patients with ISS or LWD but no such differences exist for upstream CNV. Moreover, the presence of specific CNVs in the patient population suggests the involvement of additional unknown factors. Some of its intronic variants, notably NM_000451.3(SHOX):c.-9delG and c.-65C>A in the 5'UTR, have unclear clinical roles. However, these intronic SNV may increase the probability that other CNV will arise de novo in the SHOX gene based on homologous recombination or incorrect splicing of mRNA. SUMMARY: This review highlights the clinical impact of noncoding changes in the SHOX gene and the need to apply new technologies and genotype-phenotype correlation in their analysis., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Parkinsonism belongs to the most common neurodegenerative disease. Genetic predisposition could be one of the significant risk factor for disease development. It has been described higher prevalence of parkinsonism in large pedigree from southeastern Moravia region. The study aims were to select accessible subfamily trios from the pedigree suitable for segregation genetic analyses to perform whole exome sequencing (WES) in trio individuals and further to evaluate genetic variants in the each trio. We used IonTorrent platform for WES for five subfamily trios (1-5). Each trio included two affected and one healthy person (as control). Found variants were filtered with respect to MAF < 1% (minor allele frequency), variants effect (based on prediction tools) and disease filter (Parkinsonism responsible genes). Finally, the variants from each trio were assessed with respect to the presence in the patients. There were found no one founder mutation in the subfamilies from the pedigree. Trio 1 shares two variants with trio 2: MC1R :c.322G > A (p.A108T) and MTCL1 :c.1445C > T (p.A482V), trio 3 shares two variants with trio 5: DNAJC6 :c.1817A > C (p.H606P) and HIVEP3 :c.3856C > A (p.R1286W). In trios 4 and 5, there were found two variants in gene CSMD1 :c.3335A > G (p.E1112G) and c.4071C > G (p.I1357M) respectively. As the most potentially damaging, we evaluated the non-shared variant SLC18A2 :c.583G > A (p.G195S). The variant could affect dopamine transport in dopaminergic neurons. The study of the parkinsonism genetic background in isolated Moravian population suggested that there could be significant accumulation of many risk genetic factors. For verification of the variants influence, it would be appropriate to perform a more extensive population study and suitable functional analysis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kolarikova, Vodicka, Vrtel, Stellmachova, Prochazka, Mensikova and Kanovsky.)

Aims: Turner syndrome is the only chromosome monosomy that is postnatally compatible with life. The reported incidence of TS is 1 in 2500 liveborn girls. The phenotype of these girls is highly variable, with cardiac abnormalities being life-threatening defects. The aim of the study was to reveal the possible influence of the parental origin of the X chromosome in these patients on a selected phenotype that is associated with Turner syndrome. Selected symptoms and parameters were: a bicuspid aortic valve, aortic coarctation, lymphoedema, pterygium colli, coeliac disease, thyroiditis, otitis media, diabetes mellitus 2, renal abnormalities, spontaneous puberty, and IVF., Methods: The X chromosome haplotype was determined for a group of 45,X patients verified by native FISH. A molecular diagnostic method based on the detection of different lengths of X chromosome-linked STR markers using the Argus X-12 QS kit was used to determine the X haplotype., Results: Our results, analysed by Fisher's exact (factorial) test, suggest independence between the maternal/paternal origin of the inherited X chromosome and the presence of the anomalies that were studied (P=1 to P=0.34)., Conclusion: In the group of 45,X patients, who were precisely selected by means of the native FISH method, no correlation was demonstrated with the parental origin of the X chromosome and the observed symptom.

Parkinson's disease and parkinsonism are relatively common neurodegenerative disorders. This study aimed to assess potential genetic risk factors of haplotypes in genes associated with parkinsonism in a population in which endemic parkinsonism and atypical parkinsonism have recently been found. The genes ADH1C, EIF4G1, FBXO7, GBA, GIGYF2, HTRA2, LRRK2, MAPT, PARK2, PARK7, PINK1 PLA2G6, SNCA, UCHL1, and VPS35 were analyzed in 62 patients (P) and 69 age-matched controls from the researched area (C1). Variants were acquired by high-throughput sequencing using Ion Torrent workflow. As another set of controls, the whole genome sequencing data from 100 healthy non-related individuals from the Czech population were used (C2); the results were also compared with the Genome Project data (C3). We observed shared findings of four intron (rs11564187, rs36220738, rs200829235, and rs3789329) and one exon variant (rs33995883) in the LRRK2 gene in six patients. A comparison of the C1-C3 groups revealed significant differences in haplotype frequencies between ratio of 2.09 for C1, 1.65 for C2, and 6.3 for C3, and odds ratios of 13.15 for C1, 2.58 for C2, and 7.6 for C3 were estimated. The co-occurrence of five variants in the LRRK2 gene (very probably in haplotype) could be an important potential risk factor for the development of parkinsonism, even outside the recently described pedigrees in the researched area where endemic parkinsonism is present.

Background: In Turner syndrome (TS), fluorescent in situ hybridization (FISH) karyotyping offers an alternative to classical karyotyping., Objective: We tested the added value of FISH karyotyping from lymphocytes (mesodermal origin), buccal cells (ectodermal origin), and a rear-tongue smear (endodermal origin) to determine the 45,X cell line fraction and its impact on patient phenotype., Design and Patients: Classical karyotyping and three FISH assays were done in 153 girls and women previously diagnosed with TS in four university hospitals. The 45,X cell line fraction was determined for each method and correlated with the major phenotypic signs., Results: Classical karyotyping identified 45,X/46,XX mosaicism in 77/153 subjects (50%), 45,X monosomy in 52/153 (34%), and other karyotypes in 24/153 (16%). FISH from lymphocytes verified 45,X in 47/52 original cases, whereas 4/52 had 45,X/46,XX and 1/52 45,X/47,XYY mosaicism. The 45,X cell line fraction was higher in FISH from lymphocytes compared to classical karyotyping (median 86.4% vs. 70.0%; p < 0.001), while there was no difference for FISH from buccal or rear-tongue smear cells. The mean 45,X cell line fraction was more abundant in patients with several of the characteristic phenotypic signs compared to patients without them (p < 0.01), but the predictive power was insufficient., Conclusion: FISH analysis confirmed the findings of classical karyotyping; only a few 45,X monosomy cases were reclassified as mosaics. The 45,X cell line fraction did not show clinically meaningful prediction of the phenotype. FISH analysis of buccal or rear-tongue epithelial cells may be a non-inferior, less invasive alternative to classical karyotyping., (© 2022 S. Karger AG, Basel.)

The molecular pathology of hemolytic disease of the fetus and newborn (HDFN) is determined by different RHD , RHCE , and KEL genotypes and by blood group incompatibility between the mother and fetus that is caused by erythrocyte antigen presence/absence on the cell surface. In the Czech Republic, clinically significant antierythrocyte alloantibodies include anti-D, anti-K, anti C/c, and anti-E. Deletion of the RHD gene and then three single nucleotide polymorphisms in the RHCE and KEL genes (rs676785, rs609320, and rs8176058) are the most common. The aim of this study is to develop effective and precise monitoring of fetal genotypes from maternal plasma of these polymorphisms using droplet digital (dd)PCR. Fifty-three plasma DNA samples (from 10 to 18 weeks of gestation) were analyzed (10 RHD , 33 RHCE , and 10 KEL ). The ddPCR methodology was validated on the basis of the already elaborated and established method of minisequencing and real-time PCR and with newborn phenotype confirmation. The results of ddPCR were in 100% agreement with minisequencing and real-time PCR and also with newborn phenotype. ddPCR can fully replace the reliable but more time-consuming method of minisequencing and real-time PCR RHD examination. Accurate and rapid noninvasive fetal genotyping minimizes the possibility of HDFN developing.

Objectives: The primary aim of the study was to evaluate the prevalence of fetal heart tumors in a single tertiary referral center over a period of 15 years. The secondary aim was to confirm the presence of tuberous sclerosis complex (TSC) through the evaluation of germline mutation in TSC1 / TSC2 and assess the outcomes in affected fetuses and newborns. Methods: A retrospective study was conducted between 2003 and 2017. Fetal echocardiography was performed in the second trimester of pregnancy in the study population. The identification of heart tumors and further follow-up were performed by a pediatric cardiologist. Molecular genetic analysis was conducted on fetuses and children in cases where TSC was suspected. Results: In total, 39,018 fetuses were examined between 2003 and 2017. Heart tumors were detected in nine fetuses and were diagnosed as rhabdomyoma in all cases. We identified mutations in one of the TSC1 or TSC2 genes in all cases with multiple rhabdomyomas (8/9). In all born children (5/9), the genetically confirmed diagnosis of TSC was established, and clinically pathological deposits in the brain were found. Conclusion: Fetal heart tumors are usually represented by rhabdomyomas having a good cardiac prognosis. However, rhabdomyoma is usually the first symptom of TSC with a subsequent brain disorder and impaired neurological development.

Ovarian cancer (OC) is the deadliest gynecologic malignancy with a substantial proportion of hereditary cases and a frequent association with breast cancer (BC). Genetic testing facilitates treatment and preventive strategies reducing OC mortality in mutation carriers. However, the prevalence of germline mutations varies among populations and many rarely mutated OC predisposition genes remain to be identified. We aimed to analyze 219 genes in 1333 Czech OC patients and 2278 population-matched controls using next-generation sequencing. We revealed germline mutations in 18 OC/BC predisposition genes in 32.0% of patients and in 2.5% of controls. Mutations in BRCA1/BRCA2 , RAD51C/RAD51D , BARD1 , and mismatch repair genes conferred high OC risk (OR > 5). Mutations in BRIP1 and NBN were associated with moderate risk (both OR = 3.5). BRCA1/2 mutations dominated in almost all clinicopathological subgroups including sporadic borderline tumors of ovary (BTO). Analysis of remaining 201 genes revealed somatic mosaics in PPM1D and germline mutations in SHPRH and NAT1 associating with a high/moderate OC risk significantly; however, further studies are warranted to delineate their contribution to OC development in other populations. Our findings demonstrate the high proportion of patients with hereditary OC in Slavic population justifying genetic testing in all patients with OC, including BTO.

Tuberous sclerosis complex (TSC) is a rare autosomal dominant disorder causing benign tumors in the brain and other vital organs. The genes implicated in disease development are TSC1 and TSC2. Here, we have performed mutational analysis followed by a genotype-phenotype correlation study based on the clinical characteristics of the affected individuals. Twenty unrelated probands or families from Greece have been analyzed, of whom 13 had definite TSC, whereas another 7 had a possible TSC diagnosis. Using direct sequencing, we have identified pathogenic mutations in 13 patients/families (6 in TSC1 and 7 in TSC2), 5 of which were novel. The mutation identification rate for patients with definite TSC was 85%, but only 29% for the ones with a possible TSC diagnosis. Multiplex ligation-dependent probe amplification (MLPA) did not reveal any genomic rearrangements in TSC1 and TSC2 in the samples with no mutations identified. In general, TSC2 disease was more severe than TSC1, with more subependymal giant cell astrocytomas and angiomyolipomas, higher incidence of pharmacoresistant epileptic seizures, and more severe neuropsychiatric disorders. To our knowledge, this is the first comprehensive TSC1 and TSC2 mutational analysis carried out in TSC patients in Greece.

Background: Presence of multiple cardiac rhabdomyomas is one of the major features of Tuberous sclerosis (TSC), but isolated progressing single giant rhabdomyoma is very rare and not typical of TSC., Case Report: This report presents family without obvious history of TSC with occurrence of giant mediastinal rhabdomyoma affecting the haemodynamics in male foetus, without other TSC symptoms. Girl from the next gravidity had prenatally detected multiple rhabdomyomas and small subcortical tuber of brain detected after birth. DNA analysis found novel c.4861A>T TSC2 variant and large deletion in TSC2 in tumour tissue from male foetus. The novel TSC2 variant was also present in the girl and her healthy father, in silico analysis suggested its functional effect on TSC2. Brain MRI of the father detected mild TSC specific abnormality., Conclusion: We suggest the novel TSC2 mutation is a cause of mild TSC in this family and has reduced expression. The clinical and molecular findings in this family also emphasize that TSC diagnosis should be also evaluated in case of single giant foetal cardiac rhabdomyoma.

Background: A higher prevalence of parkinsonism was recently identified in southeastern Moravia (Czech Republic). Further research confirmed 3 large pedigrees with familial autosomal-dominant parkinsonism spanning 5 generations., Methods: This case report concerns a patient belonging to one of these 3 pedigrees, in whom motor and oculomotor symptoms were accompanied by frontal-type dementia, who finally developed a clinical phenotype of progressive supranuclear palsy. Molecular genetic examinations were performed due to the positive family history., Results: No previously described causal mutation was found. After filtering against common variants (minor allele frequency (MAF) < 0.01), 2 noncoding and 1 synonymous rare mutation potentially associable with parkinsonism were identified: GIGYF2-GRB10 Interacting GYF Protein 2, PARK11 (c.*2030G > A, rs115669549); VPS35 gene-vacuolar protein sorting 35, PARK17 (c.102 + 33G > A, rs192115886); and FBXO7-F-box only protein 7 gene, PARK15 (c.540A > G, rs41311141)., Conclusion: As to the changes in the FBXO7 and VPS35 genes (despite phylogenetic conservation in primates), probably neither the FBXO7 nor the VPS35 variants will be direct causal mutations. Both described variants, and possibly the influence of their combination, could increase the risk of the disease., Competing Interests: The authors have no conflicts of interest to disclose.

The DNA damage checkpoints provide an anti-cancer barrier in diverse tumour types, however this concept has remained unexplored in prostate cancer (CaP). Furthermore, targeting DNA repair defects by PARP1 inhibitors (PARPi) as a cancer treatment strategy is emerging yet requires suitable predictive biomarkers. To address these issues, we performed immunohistochemical analysis of multiple markers of DNA damage signalling, oxidative stress, DNA repair and cell cycle control pathways during progression of human prostate disease from benign hyperplasia, through intraepithelial neoplasia to CaP, complemented by genetic analyses of TMPRSS2-ERG rearrangement and NQO1, an anti-oxidant factor and p53 protector. The DNA damage checkpoint barrier (γH2AX, pATM, p53) mechanism was activated during CaP tumorigenesis, albeit less and with delayed culmination compared to other cancers, possibly reflecting lower replication stress (slow proliferation despite cases of Rb loss and cyclin D1 overexpression) and progressive loss of ATM activator NKX3.1. Oxidative stress (8-oxoguanine lesions) and NQO1 increased during disease progression. NQO1 genotypes of 390 men did not indicate predisposition to CaP, yet loss of NQO1 in CaP suggested potential progression-opposing tumour suppressor role. TMPRSS2-ERG rearrangement and PTEN loss, events sensitizing to PARPi, occurred frequently along with heterogeneous loss of DNA repair factors 53BP1, JMJD1C and Rev7 (all studied here for the first time in CaP) whose defects may cause resistance to PARPi. Overall, our results reveal an unorthodox DNA damage checkpoint barrier scenario in CaP tumorigenesis, and provide novel insights into oxidative stress and DNA repair, with implications for biomarker guidance of future targeted therapy of CaP., (Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Background: An epidemiological study conducted over four years revealed increased prevalence of neurodegenerative parkinsonism in a small, isolated region (10 villages, with a combined population of 8664, with approx. 2927 over 50 years of age) of south-eastern Moravia, Czech Republic. The aim of this study was to obtain more detailed information on the medical history of the relatives of individuals with confirmed parkinsonism in an isolated rural population in south-eastern Moravia, Czech Republic., Methods: We did detailed genealogical research on the families of all inhabitants with confirmed parkinsonism and compiled the pedigrees. These were modified on the basis of information from a consecutive door-to-door survey and local municipal and church registers., Results: In the first stage, three large pedigrees with a familial occurrence of parkinsonism were found; two originated in one of the region's villages. In the second stage, these two pedigrees were combined into one large family tree., Conclusions: The high prevalence of parkinsonism in the researched area is caused by the familial aggregation of parkinsonism that was found in two large family trees. This is probably the result of the genetic isolation of the regional population due to the very low migration rate of its inhabitants to neighboring regions in the last two centuries.

Background: The clinical importance of assessing the fetal KEL genotype is to exclude 'K'-positive fetuses (genotype KEL1/KEL2) in 'K'-alloimmunized pregnant women (genotype KEL2/KEL2). Noninvasive assessment of the fetal KEL genotype is not yet available in the Czech Republic., Objective: The aim of this study was to assess the fetal KEL1/KEL2 genotype from cell-free fetal DNA in the plasma of KEL2/KEL2 pregnant women., Methods: The fetal genotype was assessed by minisequencing (a dilution series including control samples). A total of 138 pregnant women (between the 8th and 23rd gestational week) were tested by minisequencing. The fetal genotype was further verified by analysis of a buccal swab from the newborn., Results: Minisequencing proved to be a reliable method. In 2.2% (3/138) of the examined women, plasma sample testing failed; 94.8% (128/135) had the KEL2/KEL2 genotype, and a total of 3.1% of fetuses (4/128) had the KEL1/KEL2 genotype. Sensitivity and specificity reached 100% (p < 0.0001)., Conclusion: Minisequencing is a reliable method for the assessment of the fetal KEL1 allele from the plasma of KEL2/KEL2 pregnant women., (© 2015 S. Karger AG, Basel.)

Introduction: ADAM33 is the candidate gene most commonly associated with asthma and airway hyperreactivity (AHR)., Aim: The aim of this study was to determine whether level of AHR is associated with certain alleles or haplotypes of the ADAM33 gene in asthmatic children., Methods: One hundred and nine asthmatic children and 46 controls from the general population were examined with spirometry before and after histamine and methacholine inhalation. All subjects were genotyped for single-nucleotide polymorphisms (SNPs) of the ADAM33 gene. Haplotypes were determined according to genotypes of the patient's parents., Results: We found the three most frequent ADAM33 haplotypes (a1-3) were associated with the highest level of AHR to methacholine and histamine in 66% of asthmatic children. The paternally transmitted GGGCTTTCGCA haplotype was seen in 73.3% asthmatic children with serious AHR to methacholine challenge (paternal and maternal origin of haplotype 73.3% to 37.5, P=0.046) Significant differences in the relative frequency of paternal haplotypes with high levels of AHR to histamine were found (P=0.013)., Conclusion: ADAM33 haplotypes (a1, a2, a3) are associated with severity of AHR and are significantly more often transmitted in the paternal line.

Background: STAT6 has an important role in the IL-4 / IL-13 signalling pathway. Genome - wide association studies have shown that particular polymorphism (SNP) or haplotype variants of STAT6 as well as epigenetic gene modifications are associated with IgE level and asthma in childhood., Methods: A review of the available literature was performed to map out the function and signalling pathway of STAT6, studies of STAT6 SNPs association with susceptibility to asthma and atopy, covering the years 1997 - 2012 were summarized, and the value of epigenetic and epistatic influences on STAT6 and their relevance to the development of the studied phenotype (atopy or asthma) were determined., Results: There are 2 SNPs (rs71802646 and rs320411) with clinical association and proven functional effect on STAT6 expression. The effect of STAT6 SNPs cumulates in haplotypes and more potently during interaction with SNPs in the genes from the signalling pathway (IL4, IL4Ra, and IL13). Expression of STAT6 is also influenced by DNA methylation. Atopy is traditionally believed to be maternally inherited but there is one report about paternally overtransmitted STAT6 haplotype (TCA haplotype, built from rs324011, rs3024974 and rs4559 SNPs)., Conclusions: STAT6 polymorphisms and their combinations have an important influence on IgE level and development of asthma. However, the interaction between SNPs in the IL-4 / IL-13 signalling pathway is of greater impact. Hypermethylation of the STAT6 promoter is also significant in the regulation of STAT6 expression and this fact opens possibilities for targeting therapy in asthma.

Background: Some promising treatments for Huntington's disease (HD) may require pre-clinical testing in large animals. Minipig is a suitable species because of its large gyrencephalic brain and long lifespan., Objective: To generate HD transgenic (TgHD) minipigs encoding huntingtin (HTT)1-548 under the control of human HTT promoter., Methods: Transgenesis was achieved by lentiviral infection of porcine embryos. PCR assessment of gene transfer, observations of behavior, and postmortem biochemical and immunohistochemical studies were conducted., Results: One copy of the human HTT transgene encoding 124 glutamines integrated into chromosome 1 q24-q25 and successful germ line transmission occurred through successive generations (F0, F1, F2 and F3 generations). No developmental or gross motor deficits were noted up to 40 months of age. Mutant HTT mRNA and protein fragment were detected in brain and peripheral tissues. No aggregate formation in brain up to 16 months was seen by AGERA and filter retardation or by immunostaining. DARPP32 labeling in WT and TgHD minipig neostriatum was patchy. Analysis of 16 month old sibling pairs showed reduced intensity of DARPP32 immunoreactivity in neostriatal TgHD neurons compared to those of WT. Compared to WT, TgHD boars by one year had reduced fertility and fewer spermatozoa per ejaculate. In vitro analysis revealed a significant decline in the number of WT minipig oocytes penetrated by TgHD spermatozoa., Conclusions: The findings demonstrate successful establishment of a transgenic model of HD in minipig that should be valuable for testing long term safety of HD therapeutics. The emergence of HD-like phenotypes in the TgHD minipigs will require more study.

Background: Giant angiofibromas in patients with tuberous sclerosis complex (TSC) are rare., Objective: We sought to report two patients who had TSC with unusually large and disfiguring facial angiofibromas and to identify underlying histopathologic changes that may possibly explain the clinical features., Methods: We performed a clinicopathologic, immunohistochemical, and molecular biologic study using 42 lesional specimens and peripheral blood from one of the two patients. The immunohistochemical investigations were mainly focused on the vascular moiety of the lesions. TSC1 and TSC2 alterations were studied using multiplex ligation-dependent probe amplification for large deletion/duplication mutations, whereas screening for small mutations was performed using polymerase chain reaction amplification of individual coding exons and exon-intron junctions of both genes followed by an analysis on denaturation gradient gel electrophoresis., Results: Histopathologic examination revealed, in addition to findings typical of angiofibroma, several unusual features including multinucleated giant cells containing multiple intracytoplasmic vacuoles, Touton-like cells, emperipolesis, pagetoid dyskeratosis, vacuolar alteration at the dermoepidermal junction, Civatte bodies, and melanophages in the subjacent dermis. Numerous dilated lymphatic vessels were detected indicating localized lymphostasis, probably caused by secondary lymphedema. The lymphatic nature of the vessels was confirmed by immunohistochemical study. Genetic testing for TSC1 and TSC2 gene mutations revealed a substitution on position c.2251C>T resulting in a nonsense mutation R751X in fragment 20.2., Limitations: Histopathologic specimens and peripheral blood were available from only one patient., Conclusion: Localized lymphedema may contribute to the formation of large disfiguring angiofibromas in patients with TSC., (Copyright © 2012 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.)

Background: ADAM33 and STAT6 belong to the candidate genes that have been commonly associated with asthma, bronchial hyperresponsiveness or IgE levels. Our objective was to assess the association of 11 SNPs of the ADAM33 and 6 of the STAT6 and their haplotypes with IgE levels and asthma. We also evaluated the possible role of parental origin of haplotypes on IgE levels., Methods: We enrolled 109 children with asthma and 45 healthy controls. Genotyping was performed by TaqMan probes and confirmed by sequencing. Haplotype construction was based on the knowledge of parental genotypes and also inferred by using the EM algorithm and Bayes' theorem., Results: None of the SNPs were associated with elevated IgE level or asthma. We found that the most frequent STAT6 haplotype ATTCAA (built from rs324012, rs324011, rs841718, rs3024974, rs3024974, rs4559 SNPs, respectively) was associated with elevated total IgE levels (P=0.01) and this haplotype was predominantly transmitted paternally (P<0.001). We compared our results with those of studies performed on German and Australian Caucasian populations and found that rs324011, rs3024974 and rs4559 SNPs in STAT6 should have a major effect on IgE levels. Therefore, we suggest the TCA haplotype alone (built from rs324011, rs3024974 and rs4559 SNPs, respectively) in STAT6 is associated with total IgE elevation., Conclusions: The influence of paternal origin of the STAT6 haplotype on IgE levels is surprising but the exact role of possible paternal imprinting in STAT6 regulation should be investigated and confirmed in future studies.

NQO1 participates in cellular defense against oxidative stress and regulates apoptosis via p53- and NFκB-mediated pathways. We have previously found that homozygous missense variant NQO1*2 (rs1800566) predicts poor survival among breast cancer patients, particularly after anthracycline-based adjuvant chemotherapy. Here, we investigated NQO1 and NFκB protein expression and global gene expression profiles in breast tumors with correlation to tumor characteristics and survival after adjuvant chemotherapy. We used immunohistochemical analysis of tissue microarrays to study NQO1 and NFκB expression in two series of tumors: 1000 breast tumors unselected for treatment and 113 from a clinical trial comparing chemotherapy regimens after anthracycline treatment in advanced breast cancer. We used gene expression arrays to define genes co-expressed with NQO1 and NFκB. NQO1 and nuclear NFκB were expressed in 83% and 11% of breast tumors, and correlated inversely (P = 0.012). NQO1 protein expression was associated with estrogen receptor (ER) expression (P = 0.011), whereas 34.5% of NFκB-nuclear/activated tumors were ER negative (P = 0.001). NQO1 protein expression and NFκB activation showed only trends, but no statistical significance for patient survival or outcome after anthracycline treatment. Gene expression analysis highlighted 193 genes that significantly correlated with both NQO1 and NFκB in opposite directions, consistent with the expression patterns of the two proteins. Inverse correlation was found with genes related to oxidation/reduction, lipid biosynthesis and steroid metabolism, immune response, lymphocyte activation, Jak-STAT signaling and apoptosis. The inverse relationship between NQO1 protein expression and NFκB activation, underlined also by inverse patterns of association with ER and gene expression profiles of tumors, suggests that NQO1-NFκB interaction in breast cancer is different from several other tissue types, possibly due to estrogen receptor signaling in breast cancer. Neither NQO1 nor NFκB protein expression appear as significant prognostic or predictive markers in breast cancer.

Aim: The aim of this study was to evaluate TSPY (testis specific protein on the Y chromosome) gene and 5'UTR (UnTranslated Region) polymorphisms in men with impaired fertility compared to fertile controls., Methods: We analyzed 72 infertile men and 31 fertile controls usingconventional sequencing analysis to find crucial SNPs (single nucleotide polymorphism) and other changes., Results: The most remarkable changes were found in the 1(st) exon only. In one half of the both infertile men and fertile controls, the most frequent finding was 26 SNPs with a similar pattern. In the other half we found highly relevant changes, generating a stop codon in the first third of exon 1. Early termination cut down the protein by 78.5%. This kind of change was not found in the fertile controls. No correlation was found between the spermiogram and the changes leading to the stop codon. The distribution of men with deletions, insertion and higher gene copy number was not statistically different., Conclusion: The changes found in exon 1 in infertile men could fundamentally affect the process of spermatogenesis. These findings could significantly enhance our understanding of the molecular-genetic causes of male infertility.

The association of tuberous sclerosis complex (TSC) and autosomal dominant polycystic kidney disease (ADPKD), termed TSC2/PKD1 contiguous gene syndrome, is a result of molecular defect demonstrating by deletion disrupting TSC2 and PKD1. Dermatopathology of this syndrome has never been addressed. We report 2 sporadic cases form of TSC2/PKD1 contiguous gene syndrome, with emphasis on dermatopathologic findings. Both patients presented with a typical phenotype of TSC and early-onset renal polycystic requiring kidney transplantation in one of the patients. Of a total of 13 cutaneous lesions studied, there were 7 facial angiofibromas, 2 shagreen patches, 1 periungual fibroma, 1 hypopigmented macule, 1 epidermoid cyst, and 1 intradermal melanocytic nevus. The histological features were basically similar to those occurring in TSC, but some unusual features were identified. In both patients, deletions in the region of TSC2 and PKD1 were revealed performing by multiplex ligation probe amplification test. It is concluded that the histopathological features of skin lesions in this syndrome are similar to those encountered in TSC. Clinical awareness and appropriate molecular investigation of TSC2/PKD1 contiguous gene syndrome is necessary in all patients with a typical phenotype of TSC in infancy, adolescence, or adult age, because of severity of the renal alterations.

Osteosarcoma is the most common primary bone cancer. Mutations of the RB gene represent the most frequent molecular defect in this malignancy. A major consequence of this alteration is that the activity of the key cell cycle regulator E2F1 is unleashed from the inhibitory effects of pRb. Studies in animal models and in human cancers have shown that deregulated E2F1 overexpression possesses either "oncogenic" or "oncosuppressor" properties, depending on the cellular context. To address this issue in osteosarcomas, we examined the status of E2F1 relative to cell proliferation and apoptosis in a clinical setting of human primary osteosarcomas and in E2F1-inducible osteosarcoma cell line models that are wild-type and deficient for p53. Collectively, our data demonstrated that high E2F1 levels exerted a growth-suppressing effect that relied on the integrity of the DNA damage response network. Surprisingly, induction of p73, an established E2F1 target, was also DNA damage response-dependent. Furthermore, a global proteome analysis associated with bioinformatics revealed novel E2F1-regulated genes and potential E2F1-driven signaling networks that could provide useful targets in challenging this aggressive neoplasm by innovative therapies.

NQO1 guards against oxidative stress and carcinogenesis and stabilizes p53. We find that a homozygous common missense variant (NQO1(*)2, rs1800566(T), NM&#95;000903.2:c.558C>T) that disables NQO1 strongly predicts poor survival among two independent series of women with breast cancer (P = 0.002, N = 1,005; P = 0.005, N = 1,162), an effect particularly evident after anthracycline-based adjuvant chemotherapy with epirubicin (P = 7.52 x 10(-6)) and in p53-aberrant tumors (P = 6.15 x 10(-5)). Survival after metastasis was reduced among NQO1(*)2 homozygotes, further implicating NQO1 deficiency in cancer progression and treatment resistance. Consistently, response to epirubicin was impaired in NQO1(*)2-homozygous breast carcinoma cells in vitro, reflecting both p53-linked and p53-independent roles of NQO1. We propose a model of defective anthracycline response in NQO1-deficient breast tumors, along with increased genomic instability promoted by elevated reactive oxygen species (ROS), and suggest that the NQO1 genotype is a prognostic and predictive marker for breast cancer.

Background: Cell-free fetal (cff) DNA analysis by short tandem repeats (STR) has the advantage of better recognizing the different genotypes. However, quantitative examination by quantitative fluorescent (QF) polymerase chain reaction (PCR) by STRs is limited to only a rough approximation. This project focuses on a more precise calculation of the relative cff DNA amount tested in the STRs' loci., Methods: The cff DNA was analyzed in 363 samples from 258 pregnant women with physiological fetuses in different stages of pregnancy (from 4-37 gestational weeks) separately in three STRs [D21S1435, D21S1446 and PentaD (pD)] and also by gonosomal sequences amelogenin gene, X/Y-linked/testis specific protein, Y-linked (AMELX/Y/TSPY). Seventeen samples of cff DNA from fetuses with Down syndrome (DS) were compared. We optimized the refined quantitative fluorescent (RQF) PCR for STRs in a particular locus., Results and Conclusions: The cff DNA detection rate was 74% in at least one of the STRs. The efficiency decreased from shorter to longer PCR fragments. All three STR and gonosomal loci proved an increase in cff DNA during pregnancy. The stutter variability rate is greatest in short STR fragments and decreases as the STR fragments increase in length. Results showed that DS samples had a significantly higher amount of cff DNA., (2008 John Wiley & Sons, Ltd)