Biomedical subjects

Sandya Liyanarachchi

Publications and source records attributed to Sandya Liyanarachchi.

At least 19 recordsLinked to original sources

PubMed2026

Identification of Rare Noncoding Variants in Familial Nonmedullary Thyroid Carcinoma.

BACKGROUND: Familial nonmedullary thyroid carcinoma (FNMTC) occurs when three or more family members are affected by usually papillary thyroid carcinoma (PTC), the most common form of NMTC. While the heritability to NMTC is among the highest of all cancers, the genetic determinants among NMTC families are not well understood. Here, we aim to understand the contribution of rare noncoding germline variants in the etiology of FNMTC. METHODS: We previously reported whole-genome sequencing (WGS) and linkage analysis in 17 PTC families and reported on 41 protein-coding variants in 40 genes that cosegregated with PTC in 11 of the families. Herein, we further leveraged our WGS data to include noncoding variants in our analysis for all 17 families. We hypothesized that most of the pathogenic noncoding variants would be located in theoretical or empirically determined regulatory regions that demonstrate at a minimum, basal thyroid expression, a positive family linkage score, and co-segregation among PTC-affected individuals. To test this hypothesis, we adopted a unique filtering strategy to identify variants that occurred in known DNA elements and transcription factor binding sites, near regions known to impact on gene expression or splicing in thyroid tissue, and/or in characterized thyroid enhancers. We annotated variants using two analyses (ENCODE and transcription factor binding site) within the BasePlayer software. We separately analyzed (1) expression quantitative trait loci, (2) splicing quantitative trait loci, and (3) thyroid enhancers. We then ranked variants according to predicted pathogenicity and performed Sanger sequencing in all individuals of each family. RESULTS: In total, 121 variants were selected based on in-silico prediction and our custom ranking analysis in each pedigree. Of these, 56 variants showed cosegregation among all PTC-affected individuals and were absent from unaffected individuals. This included candidate variants from five of the six PTC families for whom no protein-coding variants were previously found. CONCLUSION: Our data suggest that noncoding variants are important in the etiology of FNMTC and provide a framework for identifying noncoding germline variants using a novel approach. Further studies are needed to functionally characterize these variants to better understand the molecular mechanism of their pathogenicity.

Humans↗

PubMed2007

beta-2-adrenergic receptor gene polymorphism confers susceptibility to Graves disease.

Graves disease (GD) is an autoimmune disorder with genetic predisposition. The polymorphisms 47A-->G (Arg16Gly) and 79C-->G (Gln27Glu) of the adrenergic beta-2 receptor (ADRB2) gene in the 5q32 region affect the functional reaction to adrenergic stimulation, which contributes to the regulation of immunological response. The -367T-->C polymorphism within the 5'-leading regulatory sequence affects ADRB2 transcriptional activity. The aim of the present study was to investigate whether ADRB2 gene variants are associated with susceptibility to GD. All polymorphisms were studied in Polish GD patients (n=300) and healthy control subjects without a family history of autoimmune disorders (n=301). Genotypes were determined by the MassARRAY system (Sequenom, San Diego, CA). Gly16 and Gln27 allele frequencies were 61.4% and 55.2% among healthy controls, almost the same as previously reported in 4441 white participants of a cardiovascular health study. We found a higher risk of GD in Gln27 carriers (CC or CG genotypes) than in Glu27 homozygous (GG genotype) participants (OR=1.99, 95% CI: 1.27-3.12, p=0.003, pcorr=0.03). The frequency of the 79GG protective genotype was significantly smaller in the GD patients without symptoms of Graves ophthalmopathy compared to controls (10% vs 22%, OR=0.41, 95% CI: 0.234-0.706, p=0.0017, pcorr=0.015). We didn't find any association of -367T--> or 47A-->G genotypes/alleles with Graves disease, however, haplotype analysis has shown a significant difference in haplotype distribution between patients and controls (the global p=0.001) with increased -367T/47A/79C haplotype frequency in GD patients compared to controls (34% vs 25%, p=0.00073, pcorr=0.0044). In conclusion, Gln27 carriers (79CC or 79GC genotypes) have increased risk of Graves disease. Our results suggest that ADRB2 plays a role in susceptibility to Graves disease in humans.

Adolescent↗

PubMed2006

Prognostic DNA methylation biomarkers in ovarian cancer.

PURPOSE: Aberrant DNA methylation, now recognized as a contributing factor to neoplasia, often shows definitive gene/sequence preferences unique to specific cancer types. Correspondingly, distinct combinations of methylated loci can function as biomarkers for numerous clinical correlates of ovarian and other cancers. EXPERIMENTAL DESIGN: We used a microarray approach to identify methylated loci prognostic for reduced progression-free survival (PFS) in advanced ovarian cancer patients. Two data set classification algorithms, Significance Analysis of Microarray and Prediction Analysis of Microarray, successfully identified 220 candidate PFS-discriminatory methylated loci. Of those, 112 were found capable of predicting PFS with 95% accuracy, by Prediction Analysis of Microarray, using an independent set of 40 advanced ovarian tumors (from 20 short-PFS and 20 long-PFS patients, respectively). Additionally, we showed the use of these predictive loci using two bioinformatics machine-learning algorithms, Support Vector Machine and Multilayer Perceptron. CONCLUSION: In this report, we show that highly prognostic DNA methylation biomarkers can be successfully identified and characterized, using previously unused, rigorous classifying algorithms. Such ovarian cancer biomarkers represent a promising approach for the assessment and management of this devastating disease.

Adenocarcinoma↗

PubMed2006

Genome-wide analysis of core promoter elements from conserved human and mouse orthologous pairs.

BACKGROUND: The canonical core promoter elements consist of the TATA box, initiator (Inr), downstream core promoter element (DPE), TFIIB recognition element (BRE) and the newly-discovered motif 10 element (MTE). The motifs for these core promoter elements are highly degenerate, which tends to lead to a high false discovery rate when attempting to detect them in promoter sequences. RESULTS: In this study, we have performed the first analysis of these core promoter elements in orthologous mouse and human promoters with experimentally-supported transcription start sites. We have identified these various elements using a combination of positional weight matrices (PWMs) and the degree of conservation of orthologous mouse and human sequences--a procedure that significantly reduces the false positive rate of motif discovery. Our analysis of 9,010 orthologous mouse-human promoter pairs revealed two combinations of three-way synergistic effects, TATA-Inr-MTE and BRE-Inr-MTE. The former has previously been putatively identified in human, but the latter represents a novel synergistic relationship. CONCLUSION: Our results demonstrate that DNA sequence conservation can greatly improve the identification of functional core promoter elements in the human genome. The data also underscores the importance of synergistic occurrence of two or more core promoter elements. Furthermore, the sequence data and results presented here can help build better computational models for predicting the transcription start sites in the promoter regions, which remains one of the most challenging problems.

Animals↗

PubMed2006

Combinatorial analysis of transcription factor partners reveals recruitment of c-MYC to estrogen receptor-alpha responsive promoters.

In breast cancer and normal estrogen target tissues, estrogen receptor-alpha (ERalpha) signaling results in the establishment of spatiotemporal patterns of gene expression. Whereas primary target gene regulation by ERalpha involves recruitment of coregulatory proteins, coactivators, or corepressors, activation of these downstream promoters by receptor signaling may also involve partnership of ERalpha with other transcription factors. By using an integrated, genome-wide approach that involves ChIP-chip and computational modeling, we uncovered 13 ERalpha-responsive promoters containing both ERalpha and c-MYC binding elements located within close proximity (13-214 bp) to each other. Estrogen stimulation enhanced the c-MYC-ERalpha interaction and facilitated the association of ERalpha, c-MYC, and the coactivator TRRAP with these estrogen-responsive promoters, resulting in chromatin remodeling and increased transcription. These results suggest that ERalpha and c-MYC physically interact to stabilize the ERalpha-coactivator complex, thereby permitting other signal transduction pathways to fine-tune estrogen-mediated signaling networks.

Animals↗

PubMed2005

The role of microRNA genes in papillary thyroid carcinoma.

Apart from alterations in the RET/PTC-RAS-BRAF pathway, comparatively little is known about the genetics of papillary thyroid carcinoma (PTC). We show that numerous microRNAs (miRNAs) are transcriptionally up-regulated in PTC tumors compared with unaffected thyroid tissue. A set of five miRNAs, including the three most up-regulated ones (miR-221, -222, and -146), distinguished unequivocally between PTC and normal thyroid. Additionally, miR-221 was up-regulated in unaffected thyroid tissue in several PTC patients, presumably an early event in carcinogenesis. Tumors in which the up-regulation (11- to 19-fold) of miR-221, -222, and -146 was strongest showed dramatic loss of KIT transcript and Kit protein. In 5 of 10 such cases, this down expression was associated with germline single-nucleotide changes in the two recognition sequences in KIT for these miRNAs. We conclude that up-regulation of several miRs and regulation of KIT are involved in PTC pathogenesis, and that sequence changes in genes targeted by miRNAs can contribute to their regulation.

Base Sequence↗

PubMed2005

Hypermethylation of 18S and 28S ribosomal DNAs predicts progression-free survival in patients with ovarian cancer.

PURPOSE: Repetitive ribosomal DNA (rDNA) genes are GC-rich clusters in the human genome. The aim of the study was to determine the methylation status of two rDNA subunits, the 18S and 28S genes, in ovarian tumors and to correlate methylation levels with clinicopathologic features in a cohort of ovarian cancer patients. EXPERIMENTAL DESIGN: 18S and 28S rDNA methylation was examined by quantitative methylation-specific PCR in 74 late-stage ovarian cancers, 9 histologically uninvolved, and 11 normal ovarian surface epithelial samples. In addition, methylation and gene expression levels of 18S and 28S rDNAs in two ovarian cancer cell lines were examined by reverse transcription-PCR before and after treatment with the demethylating drug 5'-aza-2'-deoxycytidine. RESULTS: The methylation level (amount of methylated rDNA/beta-actin) of 18S and 28S rDNAs was significantly higher (P < 0.05) in tumors than in normal ovarian surface epithelial samples. Methylation of 18S and 28S rDNA was highly correlated (R2= 0.842). Multivariate analysis by Cox regression found that rDNA hypermethylation [hazard ratio (HR), 0.25; P < 0.01], but not age (HR, 1.29; P = 0.291) and stage (HR, 1.09; P = 0.709), was independently associated with longer progression-free survival. In ovarian cancer cell lines, methylation levels of rDNA correlated with gene down-regulation and 5'-aza-2'-deoxycytidine treatment resulted in a moderate increase in 18S and 28S rDNA gene expressions. CONCLUSION: This is the first report of rDNA hypermethylation in ovarian tumors. Furthermore, rDNA methylation levels were higher in patients with long progression-free survival versus patients with short survival. Thus, rDNA methylation as a prognostic marker in ovarian cancer warrants further investigation.

Adult↗

PubMed2005

p53 codon 72 and MDM2 SNP309 polymorphisms and age of colorectal cancer onset in Lynch syndrome.

PURPOSE: The Arg/Pro polymorphism in codon 72 of p53 was recently associated with age of onset of colorectal cancer in Lynch syndrome. A novel polymorphism in the promoter region of MDM2 was associated with age of cancer onset in Li-Fraumeni syndrome. We studied the influence of both polymorphisms on age of onset in Lynch syndrome and of the p53 polymorphism also in sporadic colorectal cancer. EXPERIMENTAL DESIGN: We genotyped p53 codon 72 in 193 individuals with Lynch syndrome mutations, 93 patients with sporadic microsatellite unstable colorectal cancer, and 93 patients with sporadic microsatellite stable colorectal cancer from Finland and 323 Finnish controls. We genotyped 30 colorectal cancer patients with Lynch syndrome mutations from Ohio and 118 U.S. controls. We genotyped SNP309 of MDM2 in the Lynch syndrome groups. We used chi2 test, Kaplan-Meier statistics, and Cox regression model to analyze the data. RESULTS: Allele frequencies of both polymorphisms were similar in subjects and controls from both populations and showed Hardy-Weinberg equilibrium. Neither polymorphism was associated with age of colorectal cancer onset in any of the subject groups. CONCLUSIONS: This study failed to show any role of the p53 polymorphism on age of colorectal cancer onset in Lynch syndrome and sporadic colorectal cancer. The polymorphism in the MDM2 promoter had no affect on age of onset in Lynch syndrome. Accurate information about age of onset is important in clinical practice, especially in high-risk conditions. As association studies are vulnerable to biologically insignificant variation, both positive and negative findings need to be reported to enable unbiased assessment of the significance of putative risk variants.

Adult↗

PubMed2005

TWIST2 demonstrates differential methylation in immunoglobulin variable heavy chain mutated and unmutated chronic lymphocytic leukemia.

PURPOSE: Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease for which natural history can be predicted based on the presence or absence of immunoglobulin (Ig) variable heavy chain (V(H)) gene mutations. Herein we report selective epigenetic silencing of the transcription factor TWIST2 (DERMO1) in Ig V(H) mutated CLL and describe a semiquantitative assay to study promoter methylation of this gene in primary tumor cells. MATERIALS AND METHODS: TWIST2 promoter methylation was identified by restriction landmark genome scanning. Southern blot (SB), bisulfite sequencing, and combined bisulfite restriction analysis (COBRA), and quantitative SB-COBRA was performed to study methylation of the TWIST2 promoter. Reverse transcription polymerase chain reaction assays were used to study TWIST2 expression in CLL cells. RESULTS: Following identification and confirmation of TWIST2 methylation in CLL patients, we demonstrated that expression of this transcription factor is related to the degree of promoter methylation. Expression of TWIST2 in a CLL cell line in which the promoter is methylated was increased following decitabine treatment. We next studied 53 patients by COBRA and demonstrated that 72% of patient samples with mutated Ig V(H) show TWIST2 methylation, while only 16% of patient samples with unmutated Ig V(H) were methylated (P < .001). In a subset of patients, methylation of TWIST2 correlated with mRNA expression. CONCLUSION: TWIST2 is differentially methylated in CLL cells relative to Ig V(H) mutational status and can be quantitatively monitored by SB-COBRA. Based on the known role of TWIST2 in silencing p53 function in other malignancies, future studies should focus on the role of TWIST2 in CLL and related lymphoproliferative diseases.

Antimetabolites, Antineoplastic↗

PubMed2005

Changes in gene expression associated with loss of function of the NSDHL sterol dehydrogenase in mouse embryonic fibroblasts.

Seven human disorders of postsqualene cholesterol biosynthesis have been described. One of these, congenital hemidysplasia with ichthyosiform nevus and limb defects (CHILD) syndrome, results from mutations in the X-linked gene NADH sterol dehydrogenase-like (NSDHL) encoding a sterol dehydrogenase. A series of mutant alleles of the murine Nsdhl gene are carried by bare patches (Bpa) mice, with Bpa(1H) representing a null allele. Heterozygous Bpa(1H) females display skin and skeletal abnormalities in a distribution reflecting random X inactivation, whereas hemizygous male embryos die before embryonic day 10.5. To investigate the molecular basis of defects associated with perturbations in cholesterol biosynthesis, microarray analysis was performed comparing gene expression in embryonic fibroblasts expressing the Bpa(1H) allele versus wild-type (wt) cells. Labeled cDNAs from cells grown in normal serum or lipid-depleted serum (LDS) were hybridized to microarrays containing 22,000 mouse genes. Among 44 genes that showed higher expression in the Bpa(1H) versus wt cells grown in LDS, 11 function in cholesterol biosynthesis, 7 are involved in fatty acid synthesis, 3 (Srebp2, Insig1, and Orf11) encode sterol-regulatory proteins, and 2 (Ldlr and StarD4) are lipid transporters. Of the 21 remaining genes, 16 are known genes, some of which have been implicated previously in cholesterol homeostasis or lipid-mediated signaling, and 5 are uncharacterized cDNA clones.

3-Hydroxysteroid Dehydrogenases↗

PubMed2005

Allelic variation in gene expression in thyroid tissue.

Heritable allelic variation in gene expression may contribute to sporadic and familial disease, but is relatively unexplored. Papillary thyroid cancer (PTC) is characterized by strong heritability but predisposing genes have not been detected. Here we tested the hypothesis that inherited variation in allelic expression occurs in thyroid tissue and so might contribute to disease. We studied genes with a role in thyroid function, signaling, and/or tumorigenesis (PRKAR1A, DUOX1, IP3RI, ARGBP2, TPO, TG, and PEG10). We screened thyroid tissues from controls and patients with thyroid disease and lymphoblastoid cell lines from 40 healthy individuals. We demonstrated the robustness of the technique, a fluorescent dideoxy terminator-based method distinguishing the mRNA products of alleles in individuals who are heterozygous for a polymorphism in the transcript. We confirmed that the PEG10 gene transcript was derived only from one allele because of genetic imprinting. Three other genes, DUOX1, TPO, and ARGBP2 showed allelic variation in thyroid tissue and/or lymphoblastoid cells. Benign and malignant thyroid tissue from the same patient always gave concordant results. DUOX1 variation in expression was seen in 3 of 13 patients with PTC and 6 of 27 patients with benign disease. Further studies are needed to determine the significance of these and other allelic variations.

Alleles↗

PubMed2004

Identifying estrogen receptor alpha target genes using integrated computational genomics and chromatin immunoprecipitation microarray.

The estrogen receptor alpha (ERalpha) regulates gene expression by either direct binding to estrogen response elements or indirect tethering to other transcription factors on promoter targets. To identify these promoter sequences, we conducted a genome-wide screening with a novel microarray technique called ChIP-on-chip. A set of 70 candidate ERalpha loci were identified and the corresponding promoter sequences were analyzed by statistical pattern recognition and comparative genomics approaches. We found mouse counterparts for 63 of these loci and classified 42 (67%) as direct ERalpha targets using classification and regression tree (CART) statistical model, which involves position weight matrix and human-mouse sequence similarity scores as model parameters. The remaining genes were considered to be indirect targets. To validate this computational prediction, we conducted an additional ChIP-on-chip assay that identified acetylated chromatin components in active ERalpha promoters. Of the 27 loci upregulated in an ERalpha-positive breast cancer cell line, 20 having mouse counterparts were correctly predicted by CART. This integrated approach, therefore, sets a paradigm in which the iterative process of model refinement and experimental verification will continue until an accurate prediction of promoter target sequences is derived.

Animals↗

PubMed2004

Role of cancer-associated stromal fibroblasts in metastatic colon cancer to the liver and their expression profiles.

The cancer microenvironment and interaction between cancer and stromal cells play critical roles in tumor development and progression. The molecular features of cancer stroma are less well understood than those of cancer cells. Cancer-associated stromal fibroblasts are the predominant component of stroma associated with colon cancer and its functions remain unclear. Fibroblast cell cultures were established from metastatic colon cancer in liver, liver away from the metastatic lesions, and skin from three patients with metastatic colorectal cancer. We generated expression profiles of cancer-associated fibroblasts using oligochip arrays and compared them to those of uninvolved fibroblasts. The conditioned media from the cancer-associated fibroblast cultures enhanced proliferation of colon cancer cell line HCT116 to a greater extent than cultures from uninvolved fibroblasts. In microarray expression analysis, cancer-associated fibroblasts clustered tightly into one group and skin fibroblasts into another. Approximately 170 of 22,000 genes were up-regulated in cancer-associated fibroblasts (fold change > 2, P < 0.05) as compared to skin fibroblasts, including many genes encoding cell adhesion molecules, growth factors, and COX2. By immunohistochemistry in-vivo, we confirmed COX2 and TGFB2 expression in cancer-associated fibroblasts in metastatic colon cancer. The distinct molecular expression profiles of cancer-associated fibroblasts in colon cancer metastasis support the notion that these fibroblasts form a favorable microenvironment for cancer cells.

Cell Line, Tumor↗

PubMed2004

Papillary and follicular thyroid carcinomas show distinctly different microarray expression profiles and can be distinguished by a minimum of five genes.

PURPOSE: We have previously conducted independent microarray expression analyses of the two most common types of nonmedullary thyroid carcinoma, namely papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC). In this study, we sought to combine our data sets to shed light on the similarities and differences between these tumor types. MATERIALS AND METHODS: Microarray data from six PTCs, nine FTCs, and 13 normal thyroid samples were normalized to remove interlaboratory variability and then analyzed by unsupervised clustering, t test, and by comparison of absolute and change calls. Expression changes in four genes not previously implicated in thyroid carcinogenesis were verified by reverse transcriptase polymerase chain reaction on these same samples, together with eight additional FTC tumors. RESULTS: PTCs showed two distinct groups of genes that were either over- or underexpressed compared with normal thyroid, whereas the predominant changes in FTCs were of decreased expression. Five genes could collectively distinguish the two tumor types. PTCs showed overexpression of CITED1, claudin-10 (CLDN10), and insulin-like growth factor binding protein 6 (IGFBP6) but showed no change in expression of caveolin-1 (CAV1) or -2 (CAV2); conversely, FTCs did not express CLDN10 and had decreased expression of IGFBP6 and/or CAV1 and CAV2. CONCLUSION: PTC and FTC show distinctive microarray expression profiles, suggesting that either they have different molecular origins or they diverge distinctly from a common origin. Furthermore, if verified in a larger series of tumors, these genes could, in combination with known tumor-specific chromosome translocations, form the basis of a valuable diagnostic tool.

Adenocarcinoma, Follicular↗

PubMed2004

Epigenetic profiling in chronic lymphocytic leukemia reveals novel methylation targets.

CpG island methylation is an epigenetic alteration that contributes to tumorigenesis by transcriptional inactivation of genes. Little is known about the overall levels of CpG island methylation in chronic lymphocytic leukemia (CLL). To provide a baseline estimate of global aberrant methylation and identify target sequences for additional investigation, we performed Restriction Landmark Genomic Scanning on 10 CLL samples. Two methylation-sensitive landmark enzymes were used (NotI and AscI), allowing assessment of over 3000 CpG islands in each sample. Tumor-derived Restriction Landmark Genomic Scanning profiles were compared with profiles from CD19-selected B cells from normal volunteers and matched normal neutrophils from 4 CLL patients. We found 2.5-8.1% (mean 4.8%) of the CpG islands in CLL samples were aberrantly methylated compared with controls, and the methylation events had a nonrandom distribution (P < 0.0001). Furthermore, we identified 193 aberrantly methylated sequences, of which 93% have CpG island characteristics and 90% have homology to genes or expressed sequences. One such gene, the G protein-coupled metabotropic glutamate receptor 7 (GRM7), possibly inhibits cyclic AMP signaling in the induction of apoptosis. Bisulfite sequencing of GRM7 confirmed extensive CpG island methylation, and treatment with 5-aza-2'-deoxycytidine (decitabine) resulted in up-regulated expression of several genes in vitro with concurrent cellular depletion of DNMT1 protein. Our dual-enzyme global methylation study shows that CLL is characterized by widespread nonrandom CpG island methylation similar to other tumors and provides a panel of novel methylation targets that can be used in larger studies designed to assess impact on disease progression and survival.

Adult↗

PubMed2004

Acute myeloid leukemia with complex karyotypes and abnormal chromosome 21: Amplification discloses overexpression of APP, ETS2, and ERG genes.

Molecular mechanisms of leukemogenesis have been successfully unraveled by studying genes involved in simple rearrangements including balanced translocations and inversions. In contrast, little is known about genes altered in complex karyotypic abnormalities. We studied acute myeloid leukemia (AML) patients with complex karyotypes and abnormal chromosome 21. High-resolution bacterial artificial chromosome (BAC) array-based comparative genomic hybridization disclosed amplification predominantly in the 25- to 30-megabase (MB) region that harbors the APP gene (26.3 MB) and at position 38.7-39.1 MB that harbors the transcription factors ERG and ETS2. Using oligonucleotide arrays, APP was by far the most overexpressed gene (mean fold change 19.74, P = 0.0003) compared to a control group of AML with normal cytogenetics; ERG and ETS2 also ranked among the most highly expressed chromosome 21 genes. Overexpression of APP and ETS2 correlated with genomic amplification, but high APP expression occurred even in a subset of AML patients with normal cytogenetics (10 of 64, 16%). APP encodes a glycoprotein of unknown function previously implicated in Alzheimer's disease, but not in AML. We hypothesize that APP and the transcription factors ERG and ETS2 are altered by yet unknown molecular mechanisms involved in leukemogenesis. Our results highlight the value of molecularly dissecting leukemic cells with complex karyotypes.

Acute Disease↗

PubMed2003

A high performance test of differential gene expression for oligonucleotide arrays.

Logit-t employs a logit-transformation for normalization followed by statistical testing at the probe-level. Using four publicly-available datasets, together providing 2,710 known positive incidences of differential expression and 2,913,813 known negative incidences, performance of statistical tests were: Logit-t provided 75% positive-predictive value, compared with 5% for Affymetrix Microarray Suite 5, 6% for dChip perfect match (PM)-only, and 9% for Robust Multi-array Analysis at the p < 0.01 threshold. Logit-t provided 70% sensitivity, Microarray Suite 5 provided 46%, dChip provided 53% and Robust Multi-array Analysis provided 63%.

Algorithms↗

PubMed2003

Amnionless, essential for mouse gastrulation, is mutated in recessive hereditary megaloblastic anemia.

The amnionless gene, Amn, on mouse chromosome 12 encodes a type I transmembrane protein that is expressed in the extraembryonic visceral layer during gastrulation. Mice homozygous with respect to the amn mutation generated by a transgene insertion have no amnion. The embryos are severely compromised, surviving to the tenth day of gestation but seem to lack the mesodermal layers that normally produce the trunk. The Amn protein has one transmembrane domain separating a larger, N-terminal extracellular region and a smaller, C-terminal cytoplasmic region. The extracellular region harbors a cysteine-rich domain resembling those occurring in Chordin, found in Xenopus laevis embryos, and Sog, found in Drosophila melanogaster. As these cysteine-rich domains bind bone morphogenetic proteins (Bmps), it has been speculated that the cysteine-rich domain in Amn also binds Bmps. We show that homozygous mutations affecting exons 1-4 of human AMN lead to selective malabsorption of vitamin B12 (a phenotype associated with megaloblastic anemia 1, MGA1; OMIM 261100; refs. 5,6) in otherwise normal individuals, suggesting that the 5' end of AMN is dispensable for embryonic development but necessary for absorption of vitamin B12. When the 5' end of AMN is truncated by mutations, translation is initiated from alternative downstream start codons.

Amnion↗