First-trimester use of paroxetine and congenital heart defects: a population-based case-control study.

BACKGROUND: There is a need for case-control studies of the effect of paroxetine on the occurrence of specific heart defects. METHODS: We performed a case-control study with data from a population-based birth defects registry in the Netherlands. All the children born between 1997 and 2006 were selected. Cases were defined as fetuses and children with isolated heart defects, and the controls were fetuses and children with a genetic disorder with no heart defect. We excluded children for whom there was no information on maternal medication use and deceased children and fetuses who were not examined postmortem. First-trimester exposure to paroxetine was compared between cases and controls by calculating adjusted odds ratios (AOR). RESULTS: We included 678 cases with isolated heart defects and 615 controls. The first trimester exposure rate was 1.5% for cases and 1.0% for controls. After excluding mothers who used paroxetine outside the first trimester, or who had used another SSRI, we found no significantly increased risk for heart defects overall (10 exposed cases; AOR, 1.5; 95% confidence interval [CI], 0.5-4.0), but we did find a significantly increased risk for atrium septum defects (three exposed cases; AOR, 5.7; 95% CI, 1.4-23.7). CONCLUSIONS: Our results suggest that the use of paroxetine in early pregnancy is associated with an increased risk of atrium septum defects. The results stress the importance of studying possible teratogenic effects of a drug, preferably in regard to well-specified malformations.

In vitro fertilization with preimplantation genetic screening.

BACKGROUND: Pregnancy rates in women of advanced maternal age undergoing in vitro fertilization (IVF) are disappointingly low. It has been suggested that the use of preimplantation genetic screening of cleavage-stage embryos for aneuploidies may improve the effectiveness of IVF in these women. METHODS: We conducted a multicenter, randomized, double-blind, controlled trial comparing three cycles of IVF with and without preimplantation genetic screening in women 35 through 41 years of age. The primary outcome measure was ongoing pregnancy at 12 weeks of gestation. The secondary outcome measures were biochemical pregnancy, clinical pregnancy, miscarriage, and live birth. RESULTS: Four hundred eight women (206 assigned to preimplantation genetic screening and 202 assigned to the control group) underwent 836 cycles of IVF (434 cycles with and 402 cycles without preimplantation genetic screening). The ongoing-pregnancy rate was significantly lower in the women assigned to preimplantation genetic screening (52 of 206 women [25%]) than in those not assigned to preimplantation genetic screening (74 of 202 women [37%]; rate ratio, 0.69; 95% confidence interval [CI], 0.51 to 0.93). The women assigned to preimplantation genetic screening also had a significantly lower live-birth rate (49 of 206 women [24%] vs. 71 of 202 women [35%]; rate ratio, 0.68; 95% CI, 0.50 to 0.92). CONCLUSIONS: Preimplantation genetic screening did not increase but instead significantly reduced the rates of ongoing pregnancies and live births after IVF in women of advanced maternal age. (Current Controlled Trials number, ISRCTN76355836 [controlled-trials.com].).

Severe myocardial fibrosis caused by a deletion of the 5' end of the lamin A/C gene.

OBJECTIVES: The goal of this study was to identify the underlying gene defect in a family with inherited myocardial fibrosis. BACKGROUND: A large family with an autosomal dominantly inherited form of myocardial fibrosis with a highly malignant clinical outcome has been investigated. Because myocardial fibrosis preceded the clinical and echocardiographic signs, we consider the disease to be a hereditary form of cardiac fibrosis. METHODS: Twenty-five family members were clinically evaluated, and 5 unaffected and 8 affected family members were included in a genome-wide linkage study. RESULTS: The highest logarithm of the odds (LOD) score (LOD = 2.6) was found in the region of the lamin AC (LMNA) gene. The LMNA mutation analysis, both by denaturing gradient gel electrophoresis and sequencing, failed to show a mutation. Subsequent Southern blotting, complementary deoxyribonucleic acid sequencing, and multiplex ligation-dependent probe amplification analysis, however, revealed a deletion of the start codon-containing exon and an adjacent noncoding exon. In vitro studies demonstrated that the deletion results in the formation of nuclear aggregates of lamin, suggesting that the mutant allele is being transcribed. CONCLUSIONS: This novel LMNA deletion causes a distinct, highly malignant cardiomyopathy with early-onset primary cardiac fibrosis likely due to an effect of the shortened mutant protein, which secondarily leads to arrhythmias and end-stage cardiac failure.

Ras/ERK1/2-mediated STAT3 Ser727 phosphorylation by familial medullary thyroid carcinoma-associated RET mutants induces full activation of STAT3 and is required for c-fos promoter activation, cell mitogenicity, and transformation.

The precise role of STAT3 Ser(727) phosphorylation in RET-mediated cell transformation and oncogenesis is not well understood. In this study, we have shown that familial medullary thyroid carcinoma (FMTC) mutants RET(Y791F) and RET(S891A) induced, in addition to Tyr(705) phosphorylation, constitutive STAT3 Ser(727) phosphorylation. Using inhibitors and dominant negative constructs, we have demonstrated that RET(Y791F) and RET(S891A) induce STAT3 Ser(727) phosphorylation via a canonical Ras/ERK1/2 pathway and that integration of the Ras/ERK1/2/ELK-1 and STAT3 pathways was required for up-regulation of the c-fos promoter by FMTC-RET. Moreover, inhibition of ERK1/2 had a more severe effect on cell proliferation and cell phenotype in HEK293 cells expressing RET(S891A) compared with control and RET(WT)-transfected cells. The transforming activity of RET(Y791F) and RET(S891A) in NIH-3T3 cells was also inhibited by U0126, indicating a role of the ERK1/2 pathway in RET-mediated transformation. To investigate the biological significance of Ras/ERK1/2-induced STAT3 Ser(727) phosphorylation for cell proliferation and transformation, N-Ras-transformed NIH-3T3 cells were employed. These cells displayed elevated levels of activated ERK1/2 and Ser(727)-phosphorylated STAT3, which were inhibited by treatment with U0126. Importantly, overexpression of STAT3, in which the Ser(727) was mutated into Ala (STAT3(S727A)), rescued the transformed phenotype of N-Ras-transformed cells. Immunohistochemistry in tumor samples from FMTC patients showed strong nuclear staining of phosphorylated ERK1/2 and Ser(727) STAT3. These data show that FMTC-RET mutants activate a Ras/ERK1/2/STAT3 Ser(727) pathway, which plays an important role in cell mitogenicity and transformation.

FISH and array-CGH analysis of a complex chromosome 3 aberration suggests that loss of CNTN4 and CRBN contributes to mental retardation in 3pter deletions.

Imbalances of 3p telomeric sequences cause 3p- and trisomy 3p syndrome, respectively, showing distinct, but also shared clinical features. No causative genes have been identified in trisomy 3p patients, but for the 3p- syndrome, there is growing evidence that monosomy for one or more of four genes at 3pter, CHL1, CNTN4, CRBN, and MEGAP/srGAP3, may play a causative role. We describe here an analysis of a complex chromosome 3p aberration in a severely mentally retarded patient that revealed two adjacent segments with different copy number gains and a distal deletion. The deletion in this patient included the loci for CHL1, CNTN4, and CRBN, and narrowed the critical segment associated with the 3p- syndrome to 1.5 Mb, including the loci for CNTN4 and CRBN. We speculate that the deletion contributes more to this patient's phenotype than the gains that were observed. We suggest that 3p- syndrome associated features are primarily caused by loss of CNTN4 and CRBN, with loss of CHL1 probably having an additional detrimental effect on the cognitive functioning of the present patient.

Functional analysis of lung tumor suppressor activity at 3p21.3.

The early and frequent occurrence of deletions at 3p21.3 in lung cancer has led to the consideration of this chromosomal region as a lung cancer (LUCA) critical region with tumor suppressor activity. We covered this 19 genes-containing region with overlapping P1 artificial chromosomes (PACs), in which genes are likely accompanied by their own promoters or other regulatory sequences. With these PACs we transfected cells from a small cell lung cancer (SCLC) cell line which readily caused tumors in nude mice. Per PAC we selected two cell clones with a low number of PAC copies integrated at a single genomic site. The selected clones were s.c. injected into nude mice to investigate whether the integrated genes suppressed the tumor-inducing capacity of the original SCLC cell line. We could demonstrate PAC-specific gene expression in the transfected cells. All of the PAC integration sites were different. It appeared that introduction of a PAC or even an empty PAC vector causes some chromosomal instability, which in principle may either promote or inhibit cell growth. However, both cell clones with integration of the same PAC from the centromeric part of the LUCA region in different genomic sites were the sole pair of clones that caused smaller tumors than did the original SCLC cell line. This suggests that rather than the induced chromosomal instability, the DNA sequence of that PAC, which in addition to two protein-encoding genes contains at least one potential miRNA gene, is responsible for the tumor suppressor activity.

Quality aspects of prenatal cytogenetic diagnosis: determining the effect of various factors involved in handling amniotic fluid and chorionic villus material for cytogenetic diagnosis.

OBJECTIVES: To investigate the effect of factors involved in cell culturing and slide preparation of amniotic fluid (AF) and chorionic villus biopsies (CVB) for prenatal cytogenetic diagnosis. METHODS: The effect on the outcome of our standard AF cell culture procedure of volume and appearance of the submitted AF specimen, gynaecologist performing the amniocentesis, week of gestation in which the specimen was taken and culture medium was retrospectively investigated. In a prospective study controlled experimental variation was introduced in composition of fixative, relative humidity, temperature and airflow during slide preparation from primary CVB and AF in situ cultures. For evaluation, analysis of regression or variance was used. RESULTS: Provided that at least 0.8 mL AF per culture dish was admitted, none of the investigated factors appeared as critical resulting in unacceptable variation in outcome. Variation in appearance of the AF had a relatively major impact: bloody or brown AF resulted in a 3 days longer culture time. To a limited degree, metaphase quality of AF and CVB cells was affected by composition of fixative, relative humidity, ambient temperature and airflow during slide preparation. CONCLUSION: Current prenatal cytogenetic practice as described here appears in general to be robust and reliable. The investigated conditions are not critical within the investigated range. Expensive measures for fine control of these conditions are, therefore, not required.

An absolute procedure to test the growth potential of medium and the influence of decreased oxygen tension in primary amniotic fluid cell cultures.

OBJECTIVE: For prenatal cytogenetic diagnosis, cell cultures should be maximally successful. When introducing a change in conditions, e.g. a new batch of medium, the growth potential of a culture is usually compared under both the new condition and the one already in use. Such a relative test is in principle subject to drift and may over time increasingly lead to rejection of new adequate conditions, c.q. good batches of medium. We therefore wanted to design an absolute test to assess the quality of a new condition for amniotic fluid (AF) in situ cell culturing. METHODS: We tested batches of medium under sub-optimal (stress) conditions, expecting that differences in growth potential would thereby be more readily observed. In our stress test, we diluted the culture medium to the extent of achieving a 50% growth reduction. Thresholds for rejecting a new condition were empirically determined, based on the acceptance of a less than 1% probability of false rejection of a good condition. RESULTS: Testing three cultures per patient for ten patients, i.e. 30 cultures in total, in a medium diluted to 30% of the original concentration, showed that a minimal number of 23 successful cultures and an average number of three or more colonies per culture appeared as thresholds meeting our rejection criteria. Testing five different media resulted in the rejection of one. Using the same stress test to evaluate the effect of culturing under decreased oxygen tension showed that 2.5 and 5% oxygen tension caused a larger colony size. CONCLUSION: We designed a sensitive absolute test to assess the quality of culturing conditions for cells to be used in prenatal diagnosis in general and in particular to test the growth potential of different batches of culture medium.

Analysis of a new homozygous deletion in the tumor suppressor region at 3p12.3 reveals two novel intronic noncoding RNA genes.

Homozygous deletions or loss of heterozygosity (LOH) at human chromosome band 3p12 are consistent features of lung and other malignancies, suggesting the presence of a tumor suppressor gene(s) (TSG) at this location. Only one gene has been cloned thus far from the overlapping region deleted in lung and breast cancer cell lines U2020, NCI H2198, and HCC38. It is DUTT1 (Deleted in U Twenty Twenty), also known as ROBO1, FLJ21882, and SAX3, according to HUGO. DUTT1, the human ortholog of the fly gene ROBO, has homology with NCAM proteins. Extensive analyses of DUTT1 in lung cancer have not revealed any mutations, suggesting that another gene(s) at this location could be of importance in lung cancer initiation and progression. Here, we report the discovery of a new, small, homozygous deletion in the small cell lung cancer (SCLC) cell line GLC20, nested in the overlapping, critical region. The deletion was delineated using several polymorphic markers and three overlapping P1 phage clones. Fiber-FISH experiments revealed the deletion was approximately 130 kb. Comparative genomic sequence analysis uncovered short sequence elements highly conserved among mammalian genomes and the chicken genome. The discovery of two EST clusters within the deleted region led to the isolation of two noncoding RNA (ncRNA) genes. These were subsequently found differentially expressed in various tumors when compared to their normal tissues. The ncRNA and other highly conserved sequence elements in the deleted region may represent miRNA targets of importance in cancer initiation or progression.

MUTYH and the mismatch repair system: partners in crime?

Biallelic germline mutations of MUTYH-a gene encoding a base excision repair protein-are associated with an increased susceptibility of colorectal cancer. Whether monoallelic MUTYH mutations also increase cancer risk is not yet clear, although there is some evidence suggesting a slight increase of risk. As the MUTYH protein interacts with the mismatch repair (MMR) system, we hypothesised that the combination of a monoallelic MUTYH mutation with an MMR gene mutation increases cancer risk. We therefore investigated the prevalence of monoallelic MUTYH mutations in carriers of a germline MMR mutation: 40 carriers of a truncating mutation (group I) and 36 of a missense mutation (group II). These patients had been diagnosed with either colorectal or endometrial cancer. We compared their MUTYH mutation frequencies with those observed in a group of 134 Dutch colorectal and endometrial cancer patients without an MMR gene mutation (0.7%) and those reported for Caucasian controls (1.5%). In group I one monoallelic MUTYH mutation was found (2.5%). In group II five monoallelic germline MUTYH mutations were found (14%), four of them in MSH6 missense mutation carriers (20%). Of all patients with an MMR gene mutation, only those with a missense mutation showed a significantly higher frequency of (monoallelic) MUTYH mutations than the Dutch cancer patients without MMR gene mutations (P = 0.002) and the published controls (P = 0.001). These results warrant further study to test the hypothesis of mutations in MMR genes (in particular MSH6) and MUTYH acting together to increase cancer risk.