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The objective of this study was to compare the CYP 21A2 genetic profiles of couples with unexplained fertility problems (UFP) with genetic profiles of healthy controls (HCs). Furthermore, we analyzed associations between mutations in theCYP21A2gene and various clinical and laboratory parameters. Allele-specific polymerase chain reaction (PCR) was used in 638 probands with UFP and 200 HCs. Statistic analysis with χ2was used to study the association of mutations with infertility. The effect of mutations on particular clinical and laboratory parameters was assessed with the analysis of variance (ANOVA) test. With regard to theCYP21A2gene, 0.6% of probands with UFP and 0.5% of HCs were positive for the c.290-13A/C>G mutation; 0.6% of probands with UFP and 1.5% of HCs were positive for the p.I172N mutation; there were no probands with UFP positive for the p.P30L mutation, whereas 0.5% of HCs were; and 0.2% of probands with UFP and 0.5% of HCs were found to have the p.V281L mutation. We found a significant association between c.290-13A/C>G mutation and the frequency of significant hormone deviations (χ2= 6.997,p= 0.008). Similar association was also observed between the c.29013A/C>G mutation and the frequency of polycystic ovary syndrome (PCOS) (χ2= 16.775,p= 0.000). Our findings indicate that no significant difference in the prevalence of CYP 21A2 mutations can be found in probands with UFP when compared with HCs without infertility history. The results also imply the significant association of the c.290-13A/ C>G mutation in theCYP21A2gene, not only with the frequency of PCOS, but also with the frequency of significant hormone deviations.

Background Copy number variations (CNSs) of large genomic regions are an important mechanism implicated in the development of head and neck cancer, however, for most changes their exact role is not well understood. The aim of this study was to find possible associations between gains/losses of genomic regions and clinically distinct subgroups of head and neck cancer patients. Results Array comparative genomic hybridization (aCGH) analysis was performed on DNA samples in 64 patients with cancer in oral cavity, oropharynx or hypopharynx. Overlapping genomic regions created from gains and losses were used for statistical analysis. Following regions were overrepresented: in tumors with stage I or II a gain of 2.98 Mb on 6p21.2-p11 and a gain of 7.4 Mb on 8q11.1-q11.23; in tumors with grade I histology a gain of 1.1 Mb on 8q24.13, a loss of a large part of p arm of chromosome 3, a loss of a 1.24 Mb on 6q14.3, and a loss of terminal 32 Mb region of 8p23.3; in cases with affected lymph nodes a gain of 0.75 Mb on 3q24, and a gain of 0.9 Mb on 3q26.32-q26.33; in cases with unaffected lymph nodes a gain of 1.1 Mb on 8q23.3, in patients not treated with surgery a gain of 12.2 Mb on 7q21.3-q22.3 and a gain of 0.33 Mb on 20q11.22. Conclusions Our study identified several genomic regions of interest which appear to be associated with various clinically distinct subgroups of head and neck cancer. They represent a potentially important source of biomarkers useful for the clinical management of head and neck cancer. In particular, the PIK3CA and AGTR1 genes could be singled out to predict the lymph node involvement.

Background The inherited JAK2 46/1 haplotype is strongly associated with the development of myeloproliferative neoplasms (MPNs), and its increased frequency has also been reported in splanchnic venous thrombosis (SVT). In the present study, the role of the JAK2 46/1 haplotype in non-splanchnic venous thrombosis (non-SVT) was investigated. Methods and Results We genotyped 438 patients with non-SVT, 226 patients with MPNs and 459 healthy controls for three single nucleotide polymorphisms (SNPs) which tag the JAK2 46/1 haplotype (rs12342421 G > C, rs12343867 T > C and rs10974944 C > G). We found statistically significant association of the rs12342421 GC + CC genotypes (OR = 1.40; p = 0.023) and the rs12343867 TC + CC genotypes (OR = 1.83; p = 7.02x10- 5) with non-SVT. We also found that the CC haplotype of these two SNPs was associated with an increased risk of the disease (OR = 1.68; p = 0.009). Stratification analysis indicated that the observed association of the JAK2 46/1 haplotype with non-SVT was probably largely free of confounding effect of thrombophilic risk factors. In addition, we established a strong association of SNPs rs12342421 and rs10974944 and their CG haplotype with MPNs and with JAK2 V617F-positive MPNs. Conclusions This study provides statistical evidence that SNPs rs12342421 and rs12343867 are associated with an increased risk of non-SVT. Consistently, haplotypes of the SNPs were also associated with non-SVT risk, suggesting that inherited genetic variation in the JAK2 gene may play a role in the pathogenesis of non-SVT. Furthermore, the reported associations of the JAK2 46/1 haplotype with MPNs as well as with the occurrence of the JAK2 V617F mutation in MPNs were confirmed.

The objective of this study was to analyze the methylenetetrahydrofolate reductases (MTHFRs) C677T and A1298C genotype distributions in couples with unexplained fertility problems (UFP) and healthy controls, and to analyze the genotype and haplotype distribution in spontaneously aborted embryonic tissues (SAET) using allele specific polymerase chain reaction (PCR) in 200 probands with UFP, 353 samples of SAET and 222 healthy controls. The analysis revealed a significant overall representation of the 677T allele in male probands from couples with UFP (p = 0.036). The combined genotype distribution for both MTHFR polymorphisms was also significantly altered (χ2 21.73, p

Several techniques can be used to diagnose Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP), but no technique combines simplicity with high sensitivity. Multiplex ligation-dependent probe amplification (MLPA) was applied to develop an efficient and sensitive test for the detection of duplication/deletion of the peripheral myelin protein 22 (PMP22) gene. The study sample included 70 probands that had each been previously analysed by fluorescence in situ hibridization (FISH) and the restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) assay, both of which detect a unique recombination fragment uniquely present in most patients with the duplication. A total of nine duplications and 19 deletions were detected in the 70 probands using MLPA, and there was 100% concordance between MPLA and FISH. A single duplication was missed by the RFLP-PCR assay, which accords with the lower sensitivity of this method. It is concluded that the MLPA allows accurate detection of PMP22 gene duplications/deletions and could be used for the molecular diagnosis of these two neuropathies.

Aim: To determine whether four potential genetic factors (polymorphisms in genes for alpha-adducin, beta-adducin, the G-protein beta-3 subunit and nitric oxide synthase) are important for the development of essential hypertension (EH) in Slovenian children and young adults with EH. Methods: Both a nuclear families approach and case-control study have been performed. Genotyping of common polymorphisms in these genes using polymerase chain reaction was carried out in 104 nuclear families (an affected child, both parents) and in 200 control patients. Results: Using the transmission disequilibrium test, no statistically significant differences were found between the frequencies of transmitted and non-transmitted alleles in nuclear families for all four investigated polymorphisms. In addition, the distributions of genotypes and alleles for the four polymorphisms did not differ significantly between our children and 200 healthy control patients. The allele frequencies of all polymorphisms were concordant with those observed in some other Caucasian populations. Conclusion: We found no association between the investigated gene variants and EH, so we conclude that they do not confer a significantly increased risk of the development of EH in the Slovenian population of hypertensive children.

The C825T polymorphism in the GNB3 gene encoding a beta3 subunit from heterotrimeric G-proteins correlates strongly with the variation in activity of the G-proteins. It has so far been associated with a variety of medical conditions, but has not been tested for association with vesico-ureteric reflux (VUR). Primary VUR is a condition of genetic origin that appears to be inherited in an autosomal dominant mode, but with reduced penetrance. The constitutional change in G-protein-mediated cell signaling associated with the C825T polymorphism might be one of the factors that participate in the development of VUR by modifying the effect of still unknown mutated gene(s). A significant difference in genotype frequencies (chi(2) = 7.38, P = 0.025, df = 2) was observed between patients with primary VUR (33 CC homozygotes, 40 CT heterozygotes, 12 TT homozygotes) and healthy controls with no medical record of reflux (114 CC homozygotes, 88 CT heterozygotes, 18 TT homozygotes). This result suggests that the C825T polymorphism of the GNB3 gene might be associated with the development of VUR.

In this paper we present the case of a girl at the age of 32 months with dysmorphic features, including general muscular hypotonia, developmental delay and mental retardation. The cytogenetic analysis revealed de novo partial duplication of Xp: 46,X,dup(X)(p11.23-->p22.33: :p11.23-->p22.33). To characterize the duplication, X painting, Kallman (KAL), yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs) covering Xp11.23-->Xp22.33 region were used. Selective inactivation of the abnormal X chromosome using HpaII digestion of the AR gene was evident. After BrdU incorporation the abnormal X was late-replicating in all lymphocytes examined. There was one peculiar exception observed: the break-point region was consistently early replicating. The replicating pattern of this region corresponded to the active X chromosome. Methylation pattern of late replicating X chromosome was studied also using antibodies against 5-methylcytosine. The pattern corresponded to the normally inactive X chromosome, with the exception of the previously observed break-point region which revealed an early replicating pattern with strong fluorescent signal, similar to the pattern of the active X chromosome. The observed phenomenon could lead to the abnormal phenotype of the patient, with some normally inactive genes of the break-point region escaping the inactivation process. The abnormal clinical findings could also be due to tissue-dependent differences in the inactivation pattern.

Quantitative-fluorescence polymerase chain reaction (QF-PCR) was used to detect common fetal aneuploidies in pregnancies with increased (maternal age) or high risk (increased nuchal translucency, abnormal fetal ultrasonography, positive biochemical hormone test, or positive family history) for fetal aneuploidy.The QF-PCR testing was performed on 642 prenatal samples (73.3% amniotic fluids, 26.7% chorionic villus). DNA from prenatal samples were analyzed using an in-house-developed QF-PCR method with 20 micro-satellite markers located on the chromosomes 13, 18, 21, X and Y. Karyotyping of the 392 samples was done and both results were compared.634/642 samples were successfully analyzed. In 7.1% of 634 cases numerical chromosome abnormalities were detected. Results of QF-PCR and karyotyping were compared in 392 cases. In the group, with increased risk of fetal trisomy the specificity and sensitivity of QF-PCR method was 100%. Among cases with high risk for fetal aneuploidy, sensitivity was 100% (86.6%-100%); however, the specificity was lower, 91.1% to 100%, depending on the referral reason.In women, at advanced age QF-PCR can be used alone without karyotyping. In cases with higher risk, especially those with abnormal ultrasound findings, analysis performed only with QF-PCR is not a sufficient diagnostic method.

Background/Aims: The association between venous thrombosis outside the splanchnic area as well arterial thromboembolism and the JAK2 V617F mutation, an important marker for chronic myeloproliferative neoplasms (MPN), is not completely clear. Methods: Four hundred forty-four patients with venous thrombosis of the lower/upper limbs and/or pulmonary embolism and 60 patients with ischemic stroke were screened for the JAK2 V617F mutation, factor V Leiden, and factor II G20210A. Results: The JAK2 V617F mutation was detected in 1.4% of patients with venous thrombosis and in 3.3% of patients with ischemic stroke. Because 6 out of 2,430 control individuals with no medical history of venous thrombosis, stroke, or MPN were positive for the JAK2 V617F mutation, a significant association was observed (OR 5.53, CI 1.77–17.2, p = 0.0053 for venous thrombosis; OR 13.9, CI 2.75–70.5, p = 0.0145 for stroke). Conclusion: We provide evidence of the association between the JAK2 V617F mutation and different forms of thrombosis. This association is comparable with the association between inherited risk factors (factor V Leiden and factor II G20210A) and thrombotic events, but with a much lower prevalence of the mutation. Finally, the JAK2 V617F mutation is not absent from the general population despite being considered somatic and an acquired genetic variation.

We report a 13-month-old male infant with an apparently normal karyotype, severe growth and developmental delay, ichthyosis, hypogonadism, limb shortness, hypoplasia of the corpus callosum and a round, flat face and thin upper lip as a consequence of a subtelomeric del/dup event of the X chromosome. The recombinant X chromosome (rec(X)), derived from crossing-over within the inversion, was identified in a family, in which the mother is a carrier of pericentric inversion of one X chromosome and pericentric inversion of the heterochromatic region of chromosome 9. The inv(X) chromosome was also analysed in her sister and daughter. The rec(X) had a duplication of the segment Xq27.3→Xqter and deletion of the Xp22.31→Xpter and was interpreted as Xqter-Xq27.3::Xp22.31-Xqter. The rec (X) was characterised by FISH using a number of BAC probes. There are only three published reports of chromosome rearrangements resulting in a similar subtelomeric duplication of Xq in males. The proband’s phenotype corresponds to descriptions of contiguous gene syndromes due to deletion of the STS, SHOX, ARSE and KAL genes. Despite the loss of the ARSE gene there was no evidence of chondrodysplasia punctata. Additional conditions associated with duplication of the Xq28 segment, such as severe growth retardation and developmental delay, a peculiar head shape, atrophy of the cerebral hemispheres and hypoplasia of the cerebellum and corpus callosum, were observed. Conclusion:Fluorescent in situ hybridisation techniques using subtelomeric DNA probes are essential tools for detection of such complex submicroscopic chromosomal rearrangements as the dup/del event of the X chromosome described in our patient.

Partial trisomy of the long arm of chromosome 10 is a well-defined but rare syndrome. Clinical features of this chromosomopathy are a distinctive dysmorphic appearance, developmental delay, growth retardation, and in some cases, abnormalities of the extremities and renal, cardiac and ocular anomalies. This report describes a neonate with symmetric growth retardation and multiple dysmorphic features, in whom chromosomal analysis revealed a partial trisomy of chromosome 10q with a monosomy of the 13q34 region. The phenotype shares many common features with previously published cases. In addition to the typical features, our case also shows renal hypoplasia with early renal insufficiency and some genital anomalies.

A 77-year-old woman presented to the outpatient hematology clinic in August 2001 with leukocytosis, recurrent bacterial infections, sweating and weight loss. Bone marrow biopsy showed 80% infiltration with lymphoid cells having a prolymphocytic morphology. Flow-cytometric immunophenotype analysis showed that over 80% of the cells were positive for CD2, CD3, CD4, CD5 and CD7 antigens and negative for terminal deoxynucleotidyl transferase and CD1a antigens. T cell prolymphocytic leukemia (T-PLL) was diagnosed on the basis of these findings. The diagnosis was later confirmed by cytogenetic analysis and fluorescence in situ hibridization. The patient had the following karyotype: 46,X,der(X)t(X;3) (q28;p25) t(X;16)(p14;q12), der(3) t(X;3)(q28;p25), der(6) t (X;6) (p14;q25), (8) (q10), del(11) (q14q23), der(13) t (5;13) (q34;p11), der(13) t(13;14)(q22;q11), inv(14)(q11q32), der (16) t(X;16)(q28;q12), r(17)(p13q21), der(20) t(17;20) (q21; q13),22p+. The cytogenetic rearrangements der(6)t(X;6) (p14;q25), der(13)t(13;14)(q22;q11),t(5;13)(q34;p11), r(17) (p13q21) and t(17;20)(q21;q13) have not been described previously in the literature in patients with T-PLL.

This report concerns the case of a boy with partial trisomy 16p resulting from the insertional translocation of the short arm of chromosome 16 into the long arm of chromosome 1 in his father. He was referred for genetic testing because of mental retardation, short stature, microcephaly, seizures and multiple dysmorphic features. Chromosome analysis performed in the child demonstrated the presence of additional material in the long arm of chromosome 1. Paternal high resolution chromosome analysis and fluorescence in situ hybridisation revealed the following karyotype: 46,XY,ins(1;16)(q42;p13.1p13.3), while the karyotype of the boy is 46,XY,der(1),ins(1;16)(q42;p13.1p13.3)pat. This is the first reported case of partial trisomy 16p due to paternal insertional translocation.

The pattern of DNA methylation can be analyzed on methaphase chromosomes with fluorescein labeled antibodies against 5-methylcytosine. In human extraembryonic tissue lower overall intensity of immunofluorescence in centromeric chromosomal regions correspond to hypomethylation of the DNA when compared with normal human lymphocytes. Pericentromeric regions on chromosomes 1,9,16 and heterochromatin on chromosome Y, which reveal lower levels of immunofluorescence, are rich in classical satellite DNA type II and III. In our experiment methylation-sensitive restriction enzymes, alphoid and classical satellite DNA probes specific for chromosomes 1,9,16 and Y were used. Southern blot analysis on cells from extraembryonic tissue revealed different extent of hypomethylation in different chromosomal regions. Our results confirm overall and sequence-specific hypomethylation of DNA in cells from extraembryonic tissue in comparison with somatic cells.

Objectives Essential hypertension (EH) has a multifactorial origin and is thought to arise from an interaction between susceptibility genes and environmental factors. A number of candidate genes have been proposed. In our study, the role of polymorphisms in α-adducin (ADD1) and G-protein β-3 subunit (GNB3) genes in EH was investigated. Design and Methods Ninety-three children and young adults, aged from 4 to 27 years, with both parents, were included in our study, giving 93 nuclear families. Two polymorphisms were detected using the polymerase chain reaction technique (PCR). Investigating the C825T polymorphism of GNB3 the C allele was identified by the presence of 152 and 116 bp signals and the T allele was present when a non-digested 268 bp long band was detected. The allele-specific polymerase chain reaction (PCR) was used to study the G460W polymorphism of ADD1. For statistical analysis the transmission disequilibrium test (TDT) was used. By using non-transmitted alleles as the control population, problems of population admixture and mismatched controls are avoided. Results The following genotype frequencies for G460W polymorphism in ADD1gene were observed: 67 (72.0%) were GG homozygotes, 23 (24.7%) were GW heterozygotes and 3 (3.3%) were WW homozygotes. Using the TDT test, 44 nuclear families were used. There were 63 transmitted G alleles, 25 transmitted W alleles, 60 non-transmitted G alleles and 28 non-transmitted W alleles. The observed difference was not statistically significant (χ2 = 0.24, P = 0.62).The following genotype frequencies for the C825T polymorphism in GNB3 gene were found: 50 (53.8%) were CC homozygotes, 35 (37.6%) were CT heterozygotes and 8 (8.6%) were TT homozygotes. 73 nuclear families were used. There were 95 transmitted C alleles, 51 transmitted T alleles, 84 non-transmitted C alleles and 62 non-transmitted T alleles. The observed difference again was not statistically significant (χ2 = 1.75, P = 0.19). Conclusions Our study found no association of the polymorphisms in ADD1 and GNB3 genes with EH, respectively. However, our sample size was rather small. It seems very likely that many genes with small effects (such as the gene variants above) account for the heritability of EH.