Harshwardhan Thaker

Cell type-specific over-expression of chromosome 21 genes in fibroblasts and fetal hearts with trisomy 21.

BackgroundDown syndrome (DS) is caused by trisomy 21 (+21), but the aberrations in gene expression resulting from this chromosomal aneuploidy are not yet completely understood.MethodsWe used oligonucleotide microarrays to survey mRNA expression in early- and late-passage control and +21 fibroblasts and mid-gestation fetal hearts. We supplemented this analysis with northern blotting, western blotting, real-time RT-PCR, and immunohistochemistry.ResultsWe found chromosome 21 genes consistently over-represented among the genes over-expressed in the +21 samples. However, these sets of over-expressed genes differed across the three cell/tissue types. The chromosome 21 gene MX1 was strongly over-expressed (mean 16-fold) in senescent +21 fibroblasts, a result verified by northern and western blotting. MX1 is an interferon target gene, and its mRNA was induced by interferons present in +21 fibroblast conditioned medium, suggesting an autocrine loop for its over-expression. By immunohistochemistry the p78MX1 protein was induced in lesional tissue of alopecia areata, an autoimmune disorder associated with DS. We found strong over-expression of the purine biosynthesis gene GART (mean 3-fold) in fetal hearts with +21 and verified this result by northern blotting and real-time RT-PCR.ConclusionDifferent subsets of chromosome 21 genes are over-expressed in different cell types with +21, and for some genes this over-expression is non-linear (>1.5X). Hyperactive interferon signaling is a candidate pathway for cell senescence and autoimmune disorders in DS, and abnormal purine metabolism should be investigated for a potential role in cardiac defects.

Complete hydatidiform mole retaining a chromosome 11 of maternal origin: molecular genetic analysis of a case

Hydatidiform moles are pregnancies characterized by abnormal development of both embryonic and extraembryonic tissues and are associated with the misexpression of imprinted genes. The vast majority of complete hydatidiform moles are diploid and androgenetic, whereas partial hydatidiform moles are triploid, with an extra set of chromosomes of paternal origin. Here, we present an unusual complete mole that showed strong expression of two imprinted, maternally transcribed genes, CDKN1C (encoding p57KIP2) and PHLDA2 (TSSC3/IPL), both part of a large imprinted gene domain on chromosome 11. Using microsatellite genotyping and fluorescent in situ hybridization, we show that this paradoxical gene expression was due to retention of a maternal copy of chromosome 11 in addition to the two paternal copies normally present in complete moles. These findings demonstrate that, despite being predominantly androgenetic, some complete moles contain small amounts of DNA of maternal origin. Furthermore, these results provide an explanation for rare false negatives that can arise when p57KIP2 is used as a diagnostic marker for complete moles.

A novel mutation of STK11/LKB1 gene leads to the loss of cell growth inhibition in head and neck squamous cell carcinoma

To investigate whether genetic alteration of the STK11 (serine/threonine kinase 11)/LKB1 tumor-suppressor gene is involved in the carcinogenesis of head and neck squamous cell carcinoma (HNSCC), the entire encoding exons and flanking intronic sequences of the STK11/LKB1 gene were analysed with direct genomic sequencing of 15 HNSCC specimens. A novel missense mutation with presumed loss of heterozygosity (LOH) and 10 polymorphisms were identified in these samples. The novel mutation of STK11/LKB1 at nucleotide position 613 G → A, which causes the amino-acid substitution from alanine to threonine at residue 205 within the catalytic kinase domain, was identified in cell line RPMI 2650. To further determine whether this point mutation affects the gene function, constructs of the wild type and A205T mutant of the STK11/LKB1 gene expression vectors were created and transfected into RPMI 2650 cells. Our results showed that the reintroduction of the wild-type but not the mutant STK11/LKB1 construct into RPMI 2650 cells induced suppression of the cell growth. The mutation also affected the kinase activity of the Stk11/Lkb1 protein. This led us to conclude that the A205T point mutation of the STK11/LKB1 gene produces functionally inactive proteins. This is the first described mutation of the STK11/LKB1 gene in HNSCC. While the mutation frequency of the STK11/LKB1 gene in HNSCC remains to be determined in future studies, our data strongly suggests that STK11/LKB1 is involved in the carcinogenesis of HNSCC.

Placental biomarkers of phthalate effects on mRNA transcription: application in epidemiologic research

BackgroundCYP19 and PPARγ are two genes expressed in the placental trophoblast that are important to placental function and are disrupted by phthalate exposure in other cell types. Measurement of the mRNA of these two genes in human placental tissue by quantitative real-time polymerase chain reaction (qPCR) offers a source of potential biomarkers for use in epidemiologic research. We report on methodologic challenges to be considered in study design.MethodsWe anonymously collected 10 full-term placentas and, for each, sampled placental villi at 12 sites in the chorionic plate representing the inner (closer to the cord insertion site) and outer regions. Each sample was analyzed for the expression of two candidate genes, aromatase (CYP19) and peroxisome proliferator activated receptor protein gamma (PPARγ) and three potential internal controls: cyclophilin (CYC), 18S rRNA (18S), and total RNA. Between and within placenta variability was estimated using variance component analysis. Associations of expression levels with sampling characteristics were estimated using mixed effects models.ResultsWe identified large within-placenta variability in both transcripts (>90% of total variance) that was minimized to <20% of total variance by using 18S as an internal control and by modelling the means by inner and outer regions. 18S rRNA was the most appropriate internal control based on within and between placenta variability estimates and low correlations of 18S mRNA with target gene mRNA. Gene expression did not differ significantly by delivery method. We observed decreases in the expression of both transcripts over the 25 minute period after delivery (CYP19 p-value for trend = 0.009 and PPARγ (p-value for trend = 0.002). Using histologic methods, we confirmed that our samples were comprised predominantly of villous tissue of the fetal placenta with minimal contamination of maternally derived cell types.ConclusionqPCR-derived biomarkers of placental CYP19 and PPARγ gene expression show high within-placental variability. Sampling scheme, selection of an appropriate internal control and the timing of sample collection relative to delivery can be optimized to minimize within-placenta and other sources of underlying, non-etiologic variability.