[Show abstract][Hide abstract] ABSTRACT: Wilson's disease is an autosomal recessive disorder caused by more than 500 mutations in ATP7B gene presenting considerably clinical manifestations heterogeneity even in patients with a particular mutation. Previous findings suggested a potential role of additional genetic modifiers and environment factors on phenotypic expression among the affected patients. We conducted clinical and genetic investigations to perform genotype-phenotype correlation in two large families living in a socio-culturally isolated community with the highest prevalence of Wilson's disease ever reported of 1∶1130. Sequencing of ATP7B gene in seven affected individuals and 43 family members identified a common compound heterozygous genotype, H1069Q/M769H-fs, in five symptomatic and two asymptomatic patients and detected the presence of two out of seven identified single nucleotide polymorphisms in all affected patients. Symptomatic patients had similar clinical phenotype and age at onset (18±1 years) showing dysarthria and dysphagia as common clinical features at the time of diagnosis. Moreover, all symptomatic patients presented Kayser-Fleischer rings and lack of dystonia accompanied by unfavourable clinical outcomes. Our findings add value for understanding of genotype-phenotype correlations in Wilson's disease based on a multifamily study in an isolated population with high extent of genetic and environmental homogeneity as opposed to majority of reports. We observed an equal influence of presumed other genetic modifiers and environmental factors on clinical presentation and age at onset of Wilson's disease in patients with a particular genotype. These data provide valuable inferences that could be applied for predicting clinical management in asymptomatic patients in such communities.
[Show abstract][Hide abstract] ABSTRACT: Since the Middle Ages, Romanian population lived in three distinct provinces Wallachia, Moldavia and Transylvania until the 19th century. Over the centuries, their territories were repeatedly invaded by different peoples and subjected to external political influences resulting in demographic changes that could affected the genetic structure of populations. We performed mitochondrial DNA analysis in order to visualize the relationships between Romanian and other populations Europe based on HVSI and HVS II mtDNA sequence data using Sanger sequencing. We presented here a large scale mtDNA analysis of 612 Romanians from these historical provinces of Romania. Multidimensional scaling (MDS) plot was constructed from the pairwise Fst values. The results showed that present day Romanians in all provinces share their maternal ancestry with both eastern/central European and Balkan populations. The three populations of Romania analyzed here exhibit slightly different mtDNA lineage compositions, mainly consisting of the haplogroups H, U, J, T, K, N and W, with significant frequency differences corresponding for H, U and W haplogroups. H haplogroup accounts for 47% in Moldavia, 35 % in Wallachia and 33 % in Transylvania. Overall, this study provides a first comprehensive analysis of mtDNA genome variation in Romania revealing the existence of different degrees of provincial differences of haplogroup frequencies.
[Show abstract][Hide abstract] ABSTRACT: The CCR5-delta 32 mutation in human chemokine receptor gene can be considered a rare example of a beneficial mutation. The mutation gives its homozygous carriers complete resistance against HIV infection and has been proposed to provide protection against different lethal epidemics. Although the origin of the mutation indicates prehistoric times, repeated waves of lethal epidemics over medieval centuries strongly modified the European gene pool via bottleneck effect and seem to increase the frequency of CCR5-delta
32 up to 20% as a result of selective advantage. In order to investigate if the lethal epidemics of the medieval times selected the allele we analyzed the presence of this mutation in aDNA isolated from the skeletons discovered in Piaţa Universităţii archeological site and compared them to the frequency in a control group from local modern population. This archaeological site is one of the biggest medieval cemeteries from Romania providing the opportunity to sample the same population at different points during the medieval period and beyond. The comparisons couldn’t reveal strikingly different frequencies of the CCR5-delta 32 allele. Our preliminary results based on 18 aDNA of 150 samples and 74 of 2500 modern DNA indicate that the frequency of the allele in medieval Romanian population is not statistically significant when compared to contemporary one suggesting that lethal epidemics had little effect on its present day frequency. We can conclude based on preliminary data that increase in the allelic frequency of the mutant CCR5-delta 32 allele
has been positive selected before the medieval centuries.
[Show abstract][Hide abstract] ABSTRACT: Ancient DNA population studies may yield interesting results in cases where are indications from archaeology and history that a population demographic modification has taken place and significantly reduce the genetic diversity via bottleneck effect. We analyzed mtDNA variation using aDNA samples from Piata Universității archaeological site and modern DNA to study nature and extent of temporal changes in genetic variation in Bucharest region during 16th-19th centuries. The archaeological site of Piata Universității cemetery unearthed about 676 graves with 900 skeletons. We have analyzed by now 18 aDNA samples out
of a total of 150 dental pieces recovered from the skeletons excavated from the cemetery and 74 out of 250 modern DNA samples collected. Standard contamination precautions and authentication criteria were applied. Hypervariable regions I and II of ancient mtDNA were amplified and sequenced using twelve overlapping fragments, each with a length of approximately 100 base pairs. The majority of the ancient and modern mtDNA samples analyzed by now falls into the common West Eurasian mitochondrial haplogroups.
In conclusion, to fully assess the dynamics of the historical population composition by comparing genotypes in a temporal context we have to complete the comparative analysis of all aDNA and modern DNA samples. Moreover, in order to reveal possible genetic data changes caused by a possible population bottleneck corresponding to the waves of lethal epidemics, in which an almost one-third of the population was lost, we will also investigate a set of 17 fast evolving short tandem repeat loci (STR).
[Show abstract][Hide abstract] ABSTRACT: Moldova has a rich historical and cultural heritage, which may be reflected in the current genetic makeup of its population. To date, no comprehensive studies exist about the population genetic structure of modern Moldavians. To bridge this gap with respect to paternal lineages, we analyzed 37 binary and 17 multiallelic (STRs) polymorphisms on the non-recombining portion of the Y chromosome in 125 Moldavian males. In addition, 53 Ukrainians from eastern Moldova and 54 Romanians from the neighboring eastern Romania were typed using the same set of markers. In Moldavians, 19 Y chromosome haplogroups were identified, the most common being I-M423 (20.8%), R-M17* (17.6%), R-M458 (12.8%), E-v13 (8.8%), R-M269* and R-M412* (both 7.2%). In Romanians, 14 haplogroups were found including I-M423 (40.7%), R-M17* (16.7%), R-M405 (7.4%), E-v13 and R-M412* (both 5.6%). In Ukrainians, 13 haplogroups were identified including R-M17 (34.0%), I-M423 (20.8%), R-M269* (9.4%), N-M178, R-M458 and R-M73 (each 5.7%). Our results show that a significant majority of the Moldavian paternal gene pool belongs to eastern/central European and Balkan/eastern Mediterranean Y lineages. Phylogenetic and AMOVA analyses based on Y-STR loci also revealed that Moldavians are close to both eastern/central European and Balkan-Carpathian populations. The data correlate well with historical accounts and geographical location of the region and thus allow to hypothesize that extant Moldavian paternal genetic lineages arose from extensive recent admixture between genetically autochthonous populations of the Balkan-Carpathian zone and neighboring Slavic groups.
[Show abstract][Hide abstract] ABSTRACT: Non-melanoma skin cancer is one of the most common of all cancers and the incidence has increased in the last years as a result of many factors including increased tanning, life style and possible global climate change. Inflammation plays an important role in cancer development and is frequently evaluated by serum C-reactive protein (CRP) levels. PTGS2 -765C allele coding for COX-2 has been found to be associated with lower plasma levels of CRP. The objectives of this study are: evaluation of the association between PTGS2 -765G>C polymorphism and the occurrence of non-melanoma skin cancer, the relationship between this polymorphism and cyclooxygenase-2 activity in skin tissue, as well as the correlation with serum CRP levels in patients with non-melanoma skin cancer. We used PCR-RFLP technique to explore -765G>C PTGS2 gene polymorphism, colorimetric analysis for cyclooxygenase-2 activity in skin tissue and immunoturbidimetric assay for CRP serum levels in 174 patients with non-melanoma skin cancer [54 patients with basal cell carcinoma (BCC) and 120 patients with squamous cell carcinoma (SCC)] and 80 healthy subjects. PTGS2 -765G>C polymorphism failed to show an association with non-melanoma skin cancer risk. We observed a significant increase in COX-2 activity in SCC and BCC patients compared to control tissue (0.58 ± 0.11 and 0.63 ± 0.09 U/mg protein, respectively vs. 0.16 ± 0.01 U/mg protein). BCC and SCC intra-group analysis showed lower COX-2 activity in C-allele carriers versus non-carriers (p < 0.001 and p < 0.0001, respectively). In BCC and SCC patients with GG genotype, CRP level is significantly increased compared to control group (p < 0.0001 and p < 0.0001, respectively). Intra-group comparison of CRP levels showed significantly lower CRP levels in patients carrying C-allele compared to GG homozygotes in BCC (p = 0.0001) and SCC patients (p < 0.0001). PTGS2 -765G>C polymorphism failed to show an association with non-melanoma skin cancer risk. Regarding prognostic indicators, no consistent association emerged between PTGS2 -765G>C polymorphism and COX-2 activity or CRP levels.
No preview · Article · Dec 2011 · Archives for Dermatological Research
[Show abstract][Hide abstract] ABSTRACT: About 10% of cases of male infertility are due to the presence of microdeletions within the long arm of the Y chromosome (Yq). Despite the large literature covering this critical issue, very little is known about the pathogenic mechanism leading to spermatogenesis disruption in patients carrying these microdeletions. In order to identify the presence of specific molecular pathways leading to spermatogenic damage, testicular gene expression profiling was carried out by employing a microarray assay in 16 patients carrying an AZFc microdeletion or affected by idiopathic infertility. Hierarchical clustering was performed pooling the data set from 26 experiments (16 patients, 10 replicates).
An intriguing and unexpected finding is that all the samples showing the AZFc deletion cluster together irrespectively of their testicular phenotypes. This cluster, including also four patients affected by idiopathic infertility, showed a downregulation of several genes related to spermatogenesis that are mainly involved in testicular mRNA storage. Interestingly, the four idiopathic patients present in the cluster showed no testicular expression of DAZ despite the absence of AZFc deletion in the peripheral blood.
Our expression profiles analysis indicates that several forms of infertility can be triggered by a common pathogenic mechanism that is likely related to alterations in testicular mRNA storage. Our data suggest that a lack of testicular DAZ gene expression may be the trigger of such mechanism. Furthermore, the presence of AZFc deletions in mosaic or the loss of function of AZFc genes in absence of Yq deletion can perhaps explain these findings. Finally, based on our data, it is intriguing to hypothesize that DAZ gene dysfunctions can account for a larger number of previously thought "idiopathic" infertility cases and investigation of such testicular gene dysfunction can be important to reveal the molecular determinant of infertility than are undetected when only testing Yq deletions in peripheral blood.
[Show abstract][Hide abstract] ABSTRACT: Mutations in the X-linked androgen receptor (AR) gene cause androgen insensitivity syndrome (AIS), resulting in an impaired embryonic sex differentiation in 46,XY genetic men. Complete androgen insensitivity (CAIS) produces a female external phenotype, whereas cases with partial androgen insensitivity (PAIS) have various ambiguities of the genitalia. Mild androgen insensitivity (MAIS) is characterized by undermasculinization and gynecomastia. Here we describe a 2-month-old 46,XY female patient, with all of the characteristics of CAIS. Defects in testosterone (T) and dihydrotestosterone (DHT) synthesis were excluded. Sequencing of the AR gene showed the presence in exon 6 of a T to C transition in the second base of codon 790, nucleotide position 2369, causing a novel missense Leu790Pro mutation in the ligand-binding domain of the AR protein. The identification of a novel AR mutation in a girl with CAIS provides significant information due to the importance of missense mutations in the ligand-binding domain of the AR, which are able to induce functional abnormalities in the androgen binding capability, stabilization of active conformation, or interaction with coactivators.
Full-text · Article · Aug 2008 · Asian Journal of Andrology
[Show abstract][Hide abstract] ABSTRACT: Microarray is a recently developed simultaneous analysis of expression patterns of thousand of genes. The aim of this research was to evaluate the expression profile of human healthy dental pulp in order to find the presence of genes activated and encoding for proteins involved in the physiological process of human dental pulp. We report data obtained by analyzing expression profiles of human tooth pulp from single subjects, using an approach based on the amplification of the total RNA.
Experiments were performed on a high-density array able to analyse about 21,000 oligonucleotide sequences of about 70 bases in duplicate, using an approach based on the amplification of the total RNA from the pulp of a single tooth. Obtained data were analyzed using the S.A.M. system (Significance Analysis of Microarray) and genes were merged according to their molecular functions and biological process by the Onto-Express software.
The microarray analysis revealed 362 genes with specific pulp expression. Genes showing significant high expression were classified in genes involved in tooth development, protoncogenes, genes of collagen, DNAse, Metallopeptidases and Growth factors.
We report a microarray analysis, carried out by extraction of total RNA from specimens of healthy human dental pulp tissue. This approach represents a powerful tool in the study of human normal and pathological pulp, allowing minimization of the genetic variability due to the pooling of samples from different individuals.
Full-text · Article · Feb 2008 · Clinical and investigative medicine. Medecine clinique et experimentale
[Show abstract][Hide abstract] ABSTRACT: Male infertility represents one of the clearest examples of a complex disease with a substantial genetic basis. Numerous male mouse models, mutation screening and association studies reported over the last few years reveal the high prevalence of genetic causes of spermatogenic impairment, accounting for 10-15% of severe male infertility, including chromosomal aberrations and single gene mutations. Natural selection prevents the transmission of mutations causing infertility, but this protective mechanism may be overcome by assisted reproduction techniques. Consequently, the identification of genetic factors is important for appropriate management of the infertile couple. However, a large proportion of infertile males are diagnosed as idiopathic, reflecting poor understanding of the basic mechanisms regulating spermatogenesis and sperm function. Furthermore, the molecular mechanisms underlying spermatogenic damage in cases of genetic infertility (for example Yq microdeletions) are not known. These problems can be addressed only by large scale association studies and testicular or spermatozoal expression studies in well-defined alterations of spermatogenesis. It is conceivable that these studies will have important diagnostic and therapeutic implications in the future. This review discusses the genetic causes of male infertility known to date, the genetic polymorphisms possibly associated with male infertility, and reports novel results of global gene expression profiling of normal human testis by microarray technology.
[Show abstract][Hide abstract] ABSTRACT: The area between the Dniester and the eastern Carpathian mountain range is at a geographical crossroads between eastern Europe and the Balkans. Little is known about the genetics of the population of this region. We performed an analysis of 12 binary autosomal markers in samples from six Dniester-Carpathian populations: two Moldavian, one Romanian, one Ukrainian and two Gagauz populations. The results were compared with gene frequency data from culturally and linguistically related populations from Southeast Europe and Central Asia. Small genetic differences were found among southeastern European populations (in particular those of the Dniester-Carpathian region). The observed homogeneity suggests either a very recent common ancestry of all southeastern European populations or strong gene flow between them. Despite this low level of differentiation, tree reconstruction and principle component analyses allowed a distinction between Balkan-Carpathian (Macedonians, Romanians, Moldavians, Ukrainians and Gagauzes) and eastern Mediterranean (Turks, Greeks and Albanians) population groups. The genetic affinities among Dniester-Carpathian and southeastern European populations do not reflect their linguistic relationships. The results indicate that the ethnic and genetic differentiations occurred in these regions to a considerable extent independently of each other. In particular, Gagauzes, a Turkic-speaking population, show closer affinities to their geographical neighbors than to other Turkic populations.
Full-text · Article · Feb 2007 · Journal of Human Genetics
[Show abstract][Hide abstract] ABSTRACT: Deletions of the Y chromosome are a significant cause of spermatogenic failure. Three major deletion intervals have been defined and termed AZFa, AZFb and AZFc. Here, we report an unusual case of a proximal AZFb deletion that includes the Y chromosome palindromic sequence P4 and a novel heat shock factor (HSFY). This deletion neither include the genes EIF1AY, RPS4Y2 nor copies of the RBMY1 genes. The individual presented with idiopathic azoospermia. We propose that deletions of the testis-specific HSFY gene family may be a cause of unexplained cases of idiopathic male infertility. This deletion would not have been detected using current protocols for Y chromosome microdeletion screens, therefore we recommend that current screening protocols be extended to include this region and other palindrome sequences that contain genes expressed specifically in the testis.
Full-text · Article · May 2005 · Molecular Human Reproduction
[Show abstract][Hide abstract] ABSTRACT: About 30% of couple infertilities are of male origin, some of them caused by genetic abnormalities of the Y chromosome. Deletions in AZF region can cause severe spermatogenic defects ranging from non-obstructive azoospermia to oligospermia. The intracytoplasmatic sperm injection technique (ICSI) is rapidly becoming a versatile procedure for human assisted reproduction in case of male infertility. The use of ICSI allows Y chromosome defects to be passed from father. The goal of our study is to evaluate the frequency of microdeletions in the long arm of Y chromosome, within the AZF regions, in these cases of infertilities, using molecular genetics techniques. Thirty infertile men with azoospermia or oligozoospermia, determined by spermogram, were studied after exclusion of patients with endocrine or obstructive causes of infertility. Peripheral blood DNA was extracted from each patient, then amplified by multiplex PCR with STS genomic markers from the Y chromosome AZF zones. Each case was checked by multiplex PCR through coamplification with the SRY marker. Three men with microdeletions of the long arm of the Y chromosome were diagnosed among the 30 patients, corresponding to a proportion of 10%. The relatively high proportion of microdeletions found in our population suggest the need for strict patient selection to avoid unnecessary screening for long arm Y chromosome microdeletions. The molecular diagnostics was performed according to the current European Academy of Andrology laboratory guidelines for molecular diagnosis of Y chromosomal microdeletions.
Full-text · Article · Jan 2003 · Journal of Cellular and Molecular Medicine
[Show abstract][Hide abstract] ABSTRACT: Institutul de Genetică al Universităţii din Bucuresti, Aleea Portocalilor Nr.1-3 Sector 6 Bucureşti Introducere Prionii sunt particule infecţioase care cauzează un grup de maladii neurodegenerative caracterizate prin degenerarea sistemului nervos central. Sunt lipsiţi de orice tip de acid nucleic şi pot induce o serie de maladii atât la om cât şi la animale. La om, cele mai cunoscute sunt: maladia Creutzfeld-Jacob (CJD), sindromul Gerstmann-Straussler-Scheinker (GSS), insomnia fatală familială (FFI), maladia Kuru şi sindromul Alpers (întâlnit numai la copii), iar la animale: scrapie (la oi şi capre), encefalopatia spongiformă bovină (BSE) sau "boala vacilor nebune", encefalopatia spongiformă a felinelor, a cervidelor, etc. Procesul care declanşează boala este reprezentat de conversia unei proteine normale, sintetizată în mod natural în creierul tuturor mamiferelor (PrP c), într-una mutantă, anormală (PrP Sc). In funcţie de factorul care induce conversia PrP c în PrP Sc , natura acestor afecţiuni poate fi genetică (în cazul unei mutaţii punctiforme a genei pentru PrP, transmisă autozomal dominant), infecţioasă (ca urmare a consumului alimentelor contaminate, a folosirii unor instrumente chirurgicale nesterile, injectării unor hormoni derivaţi din hipofiza prelevată de la cadavre), sau sporadică (datorată unor mutaţii spontane ale genei PrP, care intensifică rata conversiei proteinei prionice, cât şi unor factori necunoscuţi). Maladiile prionice reprezintă un grup heterogen de disfuncţii neurodegenerative cunoscute ca encefalopatii spongiforme transmisibile (TSE), ce au în comun leziuni cerebrale, produse prin degenerescenţa vacuolară a neuronilor. Post-mortem se constată un aspect spongios al creierului, aspect datorat prezenţei vacuolelor care se găsesc în special la nivelul substanţei cenusii, corespunzător corpilor neuronali. Perioada de latenţă a acestor maladii este variabilă (luni sau chiar zeci de ani). Odată instalată, boala evoluează spre debilitate mintală, demenţă, pierderea controlului motor, imobilizare etc. Interesul crescut faţă de aceste afecţiuni este datorat unor particularităţi legate de structura, modul de transmitere şi patologia indusă, intra sau interspecific, de către prion. Toate aceste maladii sunt asociate cu alterarea conformaţională determinată de conversia formei normale a proteinei prionice, sensibilă la protează (PrP C), la o formă rezistentă la protează (PrP Sc), marker specific TSE-urilor; această modificare are loc doar după ce proteina prionică a fost ancorată la nivelul membranei celulare. Atunci când la nivelul genei care codifică proteina prionică normală, au loc mutaţii punctiforme, se realizează sinteza unei izoforme anormale, denumită, după numele bolii la care a fost studiat "PrP scrapie" sau PrP Sc . Aceasta proteină scrapie mai este cunoscută si sub numele de prion. Conceptul de prion a fost introdus de Prusiner în urma studierii scrapiei la oi (encefalopatia spongiformă a ovinelor). Prionii reprezintă o categorie de agenţi 91 POMPILIA APOSTOL, FLORINA RAICU, GABRIELA BORDEIANU, IONELA MOANŢĂ, D. CIMPONERIU, L.O. POPA . infecţioşi, considerându-se ca sunt un mister din punct de vedere biologic, deoarece, în structura lor intră doar o moleculă proteică. Fiind lipsiţi de genom, diferă de toate categoriile de agenţi infecţioşi cunoscute până în prezent, prin faptul că nu conţin nici un tip de acid nucleic (ADN sau ARN). Este interesant de relatat faptul că prionii rezistă la acţiunea multor factori fizico-chimici, printre care amintim: formol 10% (timp de 28 de luni), caldură (rezistă la fierbere timp de 3 ore), factori inhibitori ai acizilor nucleici, precum şi la acţiunea radiaţiilor UV (activitate infecţioasă 100%). Prin analogie, forma normală a acestei proteine, prezentă în condiţii normale în celulă, a fost desemnată PrP C (proteina prionică ceulară). Figura 1. Reprezentarea activitatii infecţioase a prionului si a organismelor cu grad diferit de organizare a ADN, sub actiunea UV.
[Show abstract][Hide abstract] ABSTRACT: A method called variable-stringency PCR was designed to identify the sex of an unknown origin human sample. The reaction amplified a specific 281/285 bp product from male DNA, and some random DNA products from both male and female samples by using specific primers for the alphoid region of the Y chromosome. The amplification of Y chromosome alphoid DNA is a very robust reaction, not very sensitive to the quality and source of the DNA sample. The variable-stringency profile of the amplification reaction consisted in a succession of high-stringency / low-stringency / high-stringency stages, in all cases the stringency being determined by the annealing temperature in the PCR reaction. A blind test was performed, with samples of unknown sex, and the sex identification was successful in all cases. Résumé. Une méthode appelée PCR en conditions de stringence variable a été développée, pour identifier le sexe d'un individu inconnu, dont on analyse un échantillon d'ADN. La méthode consiste en l'amplification d'une série de fragments de 281-285 paires de bases quand on utilise de l'ADN provenant d'un home, alors qu'une série de fragments aléatoires sont amplifies également chez les deux sexes. On a utilisé des amorces spécifiques pour la région alphoid du chromosome Y, cette réaction étant très robuste et peu sensible au qualité de l'ADN. La réaction d'amplification consiste en une succession de trois stages haute stringence/faible stringence/haute stringence, stringence détermineé par la température d'alignement d'amorces.