Detection of mutations in the CYP21A2 gene: genotype-phenotype correlation in Slovenian couples with conceiving problems.

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 the CYP21A2 gene 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 χ2 was 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 the CYP21A2 gene, 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 the CYP21A2 gene, not only with the frequency of PCOS, but also with the frequency of significant hormone deviations.

MTHFR C677T and A1298C Genotypes and Haplotypes in Slovenian Couples with Unexplained Infertility Problems and in Embryonic Tissues from Spontaneous Abortions.

The objective of this study was to analyze the methylenetetrahydrofolate reductases ( MTHFR s) 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 (χ (2) 21.73, p <0.001) although female probands made no contribution (χ (2) 1.33, p = 0.72). The overall representation of the 677T allele was more pronounced in SAET (0.5 vs. 0.351 in controls, p <0.001) regardless of the karyotype status (aneuploidy vs. normal karyotype). In addition, the frequencies of the CA and CC haplotypes were significantly lower than in the control group ( p = 0.021 and p = 0.001, respectively), whereas the frequency of the TC haplotype was significantly higher than in controls ( p <0.0001). The presented findings indicate that only male probands contribute to the association of MTHFR mutations with fertility problems in grown adults and demonstrate a high prevalence of mutated MTHFR genotypes in SAET.

Human telomerase gene amplification and high-risk human papillomavirus infection in women with cervical intra-epithelial neoplasia.

This study was designed to investigate whether a correlation exists between amplification of the human telomerase gene (human telomerase RNA component [TERC]) and high-risk human papillomavirus (HR-HPV) infection in 101 women with cervical intra-epithelial neoplasia (CIN). Eight patients (7.9%) had CIN 1, 24 (23.8%) had CIN 2 and 69 (68.3%) had CIN 3. TERC was amplified in 31.7% of all CIN patients. The difference in frequency of TERC amplification between patients with low-grade CIN (CIN 1) and those with high-grade CIN (CIN 2 and CIN 3) was not significant. HR-HPV infection was detected in 88.1% of all CIN cases and was significantly more frequent in patients with CIN 2 and CIN 3 than in patients with CIN 1. There was no significant difference in the frequency of HR-HPV infection between groups of patients with and without TERC amplification. In conclusion, this study found no correlation between TERC amplification and HR-HPV infection in patients with CIN.

Molecular diagnosis of PMP22 gene duplications and deletions: comparison of different methods.

Several techniques can be used to diagnose Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuro pathy 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.

Evidence for two transferrin loci in the Salmo trutta genome.

To determine the organization of transferrin (TF) locus in the Salmo trutta genome, partial DNA and cDNA sequencing, fluorescent in situ hybridization (FISH) and Salmo salar BAC analysis were performed. TF expression levels and copy number prediction were assessed using real-time PCR. In addition to two previously reported DNA TF variant sequences of S. trutta and Salmo marmoratus (TF1), two novel variant sequences (TF2) were revealed in both species. Variant-specific sequence tags, characterizing two variants for each TF type (TF1 and TF2), were identified in genomic clones from each of the F1 hybrids between S. trutta and S. marmoratus. These clearly documented double heterozygote status at the TF loci. The real-time PCR data showed that each of the two TF types (TF1 and TF2) existed in one copy only and that the transcription of TF2 was considerably lower compared with TF1. Using FISH, hybridization signals were observed on two medium-sized acrocentric chromosomes of S. trutta karyotype. A TF type-specific PCR followed by a restriction analysis revealed the presence of two TF loci in the majority of analysed BAC clones. It was concluded that the TF gene is duplicated in the genome of S. trutta, and that the two TF loci are located adjacent to one another on the same chromosome. The differing transcription levels of TF1 and TF2 appear to depend on the corresponding promoter activity, which at least for TF2 seems to vary between different Salmo congeners.

Early renal insufficiency in a neonate with de novo partial trisomy of chromosome 10q.

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.

Partial Xp duplication in a girl with dysmorphic features: the change in replication pattern of late-replicating dupX chromosome.

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.

Hypomethylation of alphoid DNA and classical satellite DNA on chromosome 1, 9, 16 and Y in extraembryonic tissue.

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.

A case of insertional translocation resulting in partial trisomy 16p.

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.

DNA methylation of the extraembryonic tissues: an in situ study on human metaphase chromosomes.

DNA methylation level and pattern of human metaphase chromosomes from extraembryonic tissues (chorionic villi and placental fibroblasts) were analysed in situ. The DNA methylation global level of these tissues was studied by comparing them with the one observed in fetal fibroblasts and adult lymphocytes. In order to assess the tissue specificity and significance of the observed differences, chromosomal preparations were then treated in parallel. They were first stained with distamycin A/DAPI and pictured, then treated with immunofluorescent staining using monoclonal antibodies raised against 5-methylcytosine. Compared with metaphases from lymphocytes or placental and fetal fibroblasts, distamycin-A/DAPI stained metaphases and constitutive heterochromatic regions with very similar intensities. In contrast, in chorionic villi, the immunofluorescent intensities revealing the presence of 5-methylcytosine was much duller than in the other tissues. In addition, in both chorionic villi and placental fibroblasts, large differences were observed between various chromosome structures within individual metaphases. In particular, the secondary constriction of chromosome 9, the distal segment of chromosome Y and the short arms of acrocentric chromosomes exhibited a much lower staining than the one observed for the secondary constrictions of chromosome 1 and 16 of the same metaphases. Because all these structures are known to be deeply methylated in other somatic tissues, this suggests that in extraembryonic tissues DNA methylation level remained hypomethylated and the pattern is under precise control.

New sites of methylcytosine-rich DNA detected on metaphase chromosomes.

In situ immunofluorescence detection of antibodies against 5-methylcytosine on metaphase chromosomes prepared by a new procedure allows the display of new 5-methylcytosine-rich sites as compared to previously published methods. In short-term culture lymphocytes, the immunofluorescent signals give a recurrent pattern in which four types of binding sites can be distinguished. Type I sites are the secondary constrictions and a few juxtacentromeric regions, type II sites correspond to T-bands. Both types I and II sites emit a strong fluorescence. Type III sites form an R-band pattern and emit a weaker fluorescence. Type IV sites are the short arms of acrocentrics, they emit strong but polymorphic signals. The results obtained from control experiments suggest that the pattern observed is rather the expression of an uneven distribution of 5-methylcytosine-rich sites than a consequence of the various treatments used. In a lymphoblastoid cell line known to have a reduced 5-methylcytosine content, it was possible to demonstrate a heterogeneous hypomethylation among chromosome structures, principally involving type I sites. The method opens the possibility of studying in situ on chromosomes, regional variations of methylation in pathological conditions.

Detection of methylcytosine-rich heterochromatin on banded chromosomes. Application to cells with various status of DNA methylation.

A procedure including incorporation of 5-bromodeoxyuridine (BrdU) in DNA and a thermal denaturation step was developed to obtain both R-banding and efficient binding of anti-5-methylcytosine antibodies on metaphase chromosomes. BrdU incorporation improved the efficiency of antibody binding disclosed by immunofluorescence staining. This method allowed semiquantitative analysis of the antibody binding sites on straightforward characterized metaphase chromosomes and was applied to normal human lymphocytes and lymphoblastoid cell lines for which DNA methylation status had been previously analyzed. A correlation was established between level of DNA methylation and the semiquantitative estimate of antibody fixation. This procedure can be used to study DNA methylation on metaphase chromosomes in transformed and cancerous cell lines.

[Characterization of chromosomal rearrangements by in situ hybridization in glioblastoma]. / Caractérisation par hybridation in situ des remaniements chromosomiques dans un glioblastome.

In a case of glioblastoma, the following karyotype was determined: 47, X, - Y, + der(1) t(1;9)(p21;p23), t(1;9)(p21;p23), + 3, + 7, der(9) t(Y;9)(q11;p21), - 13, t(13;16)(p13,p11), del(14)(q11q22). Classical satellite DNAs are mainly located in chromosomes 1, 9, 15, 16 and Y. Because, most of these chromosomes were implicated in the rearrangements, a detailed cytogenetic study was undertaken. This study included in situ hybridization of the satellite and alphoid DNAs of chromosomes 1, 9, 16 and Y combined with various chromosome banding methods (DA-DAPI, quinacrine mustard and R-banding). The data obtained, demonstrated that the breakpoints were always located outside the areas containing the satellite and alphoid DNAs. The situation observed here differs from that reported in breast cancers for which a high proportion of the breakpoints occur within these areas. These findings suggest that in glioblastoma, chromosome rearrangements result from different mechanisms than those implicated in breast cancers. Thus, in cancers, chromosomal instabilities may result from several mechanisms.

Hypomethylation of classical satellite DNA and chromosome instability in lymphoblastoid cell lines.

To determine possible relationships between DNA hypomethylation and chromosome instability, human lymphoblastoid cell lines from different genetic constitutions were studied with regard to 1) uncoiling and rearrangements, which preferentially affect the heterochromatic segments of chromosomes 1 and 16; 2) the methylation status of the tandemly repetitive sequences (classical satellite and alphoid DNAs) from chromosomes 1 and 16, and of the L1Hs interspersed repetitive sequences. The methylation status largely varied from cell line to cell line, but for a given cell line, the degree of methylation was similar for all the repetitive DNAs studied. Two cell lines, one obtained from a Fanconi anemia patient and the other from an ataxia telangiectasia patient were found to be heavily hypomethylated. The heterochromatic segments of their chromosomes 1 and 16 were more frequently elongated and rearranged than those from other cell lines, which were found to be less hypomethylated. Thus, in these lymphoblastoid cell lines, alterations characterized by uncoiling and rearrangements of heterochromatic segments from chromosomes 1 and 16 seem to correlate with the hypomethylation of their repetitive DNAs. Two-color in situ hybridizations demonstrated that these elongations and rearrangements involved only classical satellite-DNA-containing heterochromatin. This specificity may be related to the excess of breakages affecting the chromosomes carrying these structures in a variety of pathological conditions.

Two-color FISH characterization of i(1q) and der(1;16) in human breast cancer cells.

Two-color fluorescent in situ hybridizations using probes for alphoid (alpha) and classical satellite (CS) DNAs from chromosomes 1 and 16 were performed to characterize i(1q), der(1;16), and complex rearrangements observed in breast cancer cells from fresh tumors and established cell lines. Six of seven i(1q) occurred after breakage in the alpha 1 containing region and one of seven was dicentric, with breakage in 1p11.2. The five der(1;16)(q10;p10) studied appeared to result from a variety of breakpoints involving alpha 1, alpha 16, CS1, and CS16 DNAs. All had conserved alpha 16 DNA, suggesting a segregation of the der(1;16) leading to a loss of 16q and a gain of 1q in most cases. One complex rearrangement of chromosome 1 also appeared to involve chromosome 16, suggesting that a der(1;16) occurred first, followed by another rearrangement. Both the apparent preferential involvement of constitutive heterochromatin harboring alpha and CS DNAs and the variety of breakpoints spanning along heterochromatin suggest that the important consequence of the rearrangement is not the breakage per se but the resulting imbalance.

Specific induction of uncoiling and recombination by azacytidine in classical satellite-containing constitutive heterochromatin.

Azacytidine (ACR) is known to induce uncoiling and somatic association involving the constitutive heterochromatin of human chromosomes 1, 9, 15, and 16 and the Y. These regions are composed of alphoid and classical satellite DNA sequences. Using specific probes for chromosomes 1 and 16, we have performed two-color fluorescence in situ hybridization on human lymphocytes cultured in the presence of ACR. We demonstrate that for these two chromosomes (1) uncoiling and association specifically occur in classical satellite-containing regions at the first cell generation, (2) breakages also affect these regions, and (3) somatic recombinations occur between these regions and lead to translocations at the next cell generation. These results suggest that changes in methylation of repetitive DNA sequences are related to chromosomal instability occurring during cell transformation and tumorigenesis.

A t(X;15)(q23;q25) with Xq reactivation in a lymphoblastoid cell line from Fanconi anemia.

A t(X:15)(q23;q25) was detected during cytogenetic investigation of a lymphoblastoid cell line established from a female patient with Fanconi anemia. The translocation was apparently balanced at passage 300 and unbalanced at passage 13. A chromatid exchange between both the normal and the der(15), between the centromere and band 15q25, may explain these results. Replication studies, following BrdU incorporation, indicate that the segment Xq23----qter from the der(15) is early replicating whereas segment Xpter----q23 from the der(X) is late replicating. Since the normal X was early replicating, it is concluded that the segment of the long arm of chromosome X, separated from its inactivation center by the translocation, was reactivated. This interpretation is confirmed by the methylation patterns of the hypoxanthine phosphoribosyltransferase gene (HPRT), mapped on Xq26, which corresponds to that of an active gene, whereas that of phosphoglycerate kinase (PGK1), which remained on the der(X), corresponds to that of an inactive gene. This is the first example of reactivation of a segment of the X chromosome following a structural rearrangement in somatic cells.