The CD6 protein has been shown to play important roles in T cell costimulation and adhesion. Rece... more The CD6 protein has been shown to play important roles in T cell costimulation and adhesion. Recently, variably spliced isoforms of CD6 mRNA have been identified in both human and murine T cells. Here we report on the genomic organization of the human CD6 gene, its chromosomal localization, and the characterization of novel isoforms. Human CD6 is encoded by at least 13 exons. The amino terminal signal sequence, extracellular region, and transmembrane domain are encoded by seven exons, while the cytoplasmic domain of CD6 is encoded by six exons. Each of the three extracellular scavenger receptor cysteine-rich domains is encoded by a separate exon. Fluorescence in situ hybridization studies and screening of a chromosome-specific YAC (yeast artificial chromosome) library revealed that the gene encoding CD6 is located on chromosome 11 at 11q13 in close proximity to the gene encoding the related molecule CD5 and within 600 kb of CD20. Analysis of mRNA transcripts encoding CD6 isolated fr...
ABSTRACTThe sequencing of human virus genomes from wastewater samples is an efficient method for ... more ABSTRACTThe sequencing of human virus genomes from wastewater samples is an efficient method for tracking viral transmission and evolution at the community level. However, this requires the recovery of viral nucleic acids of high quality. We developed a reusable tangential-flow filtration system to concentrate and purify viruses from wastewater for whole-genome sequencing. A pilot study was conducted with 94 wastewater samples from four local sewersheds, from which viral nucleic acids were extracted, and the whole genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was sequenced using the ARTIC V4.0 primers. Our method yielded a high probability (0.9) of recovering complete or near-complete SARS-CoV-2 genomes (>90% coverage at 10× depth) from wastewater when the COVID-19 incidence rate exceeded 33 cases per 100 000 people. The relative abundances of sequenced SARS-CoV-2 variants followed the trends observed from patient-derived samples. We also identified SARS-...
Traditional analysis of genomic data from bulk sequencing experiments seek to group and compare s... more Traditional analysis of genomic data from bulk sequencing experiments seek to group and compare sample cohorts into biologically meaningful groups. To accomplish this task, large scale databases of patient-derived samples, like that of TCGA, have been established, giving the ability to interrogate multiple data modalities per tumor. We have developed a computational strategy employing multimodal integration paired with spectral clustering and modern dimension reduction techniques such as PHATE to provide a more robust method for cancer sub-type classification. Using this integrated approach, we have examined 514 Head and Neck Squamous Carcinoma (HNSC) tumor samples from TCGA across gene-expression, DNA-methylation, and microbiome data modalities. We show that these approaches, primarily developed for single-cell sequencing can be efficiently applied to bulk tumor sequencing data. Our multimodal analysis captures the dynamic heterogeneity, identifies new and refines subtypes of HNSC,...
5374 Irinotecan (CPT-11) interacts synergistically with a wide variety of anticancer agents, incl... more 5374 Irinotecan (CPT-11) interacts synergistically with a wide variety of anticancer agents, including thymidylate synthetase inhibitors, taxane derivatives, platinum compounds and anthracyclines, as well as immunotherapeutic agents. The wide variety of known mechanisms of action of these synergistic agents and the schedule-dependency of synergy suggest that synergistic interactions with CPT-11 are associated with a CPT-11-induced change in a general cellular process, such as the apoptotic response. Study of time kinetic changes in expression of apoptosis-associated genes studied by cDNA microarray analysis in HL60 cells following two-hour in vitro exposure to 0.3 μM SN-38, the active metabolite of CPT-11, revealed downregulation of the anti-apoptotic transcriptome survivin, with a nadir at six hours. In order to determine whether survivin downregulation was related to altered transcription regulation, expression profiles of factors controlling the NFkB transcription complex, which ...
e16504 Background: A proof of principle array-based comparative genomic hybridization (aCGH) anal... more e16504 Background: A proof of principle array-based comparative genomic hybridization (aCGH) analysis was performed in archival formalin-fixed and paraffin-embedded (FFPE) stage I ovarian cancers (EOC) to determine if frequent (>40%) copy number aberrations (CNAs) can be detected in DNA repair genes including the Fanconi anemia complementation group (FANC) and RAD51 families compared with the rest of the genome. Methods: Tumor DNA was isolated from 22 serous cancers from the GOG-175 virtual tissue bank. RPCI 19K BAC arrays were hybridized (GeneTAC HybStation) and scanned (Gene Pix 4200AL Laser Scanner). Spot fluorescence values were quantified using ImaGene, vetted for quality and loess corrected with adjustments for chip-specific spatial effects. The genome was segmented to identify regions with common copy number means (DNAcopy software). Posterior aberration probabilities for the regions were obtained using CGHcall and data was visualized and annotated using iGenomicViewer in ...
The hallmark of Hodgkin’s Lymphoma (HL) is the presence of large Hodgkin’s and Reed/Sternberg (HR... more The hallmark of Hodgkin’s Lymphoma (HL) is the presence of large Hodgkin’s and Reed/Sternberg (HRS) cells that comprise only ~1–3% of the cellular infiltrate. Given the paucity of genomic analyses of these cells, we sought to characterize their DNA copy number alterations (CNA) by array comparative genomic hybridization (aCGH) using DNA isolated from laser capture microdissected (LCM) CD30+ HRS cells. Primary paraffin-embedded diagnostic samples were obtained from 27 patients (pts), including 15 pts with chemotherapy responsive and 12 pts with primary refractory HL. From each sample, 150 HRS cells were isolated by LCM from 5-μm thick tissue sections, amplified using whole genome amplification (WGA), and hybridized to a minimal tiling 19K whole genome bacterial artificial chromosome (BAC) array (BAC center to center distance of 165 kb). To define thresholds for calling gains and loss and to control for WGA noise, 1.05 ng DNA (DNA equivalent of 150 cells) was obtained from six normal ...
Chemotherapy or targeted cancer therapies have greatly improved the treatment outcome of patients... more Chemotherapy or targeted cancer therapies have greatly improved the treatment outcome of patients with leukemia; however, many will ultimately die because of disease relapse and development of drug resistance. Leukemias are cancers of the blood cells that result from alteration of the normal physiological constraints that regulate hematopoietic stem cells (HSCs). General characteristics of leukemia stem cells (LSCs) such as self-renewal, self-protection and proliferative quiescence represent inherent mechanisms that at least partially explain drug resistance and recurrence in post-therapy leukemia patients. Acute myeloid leukemia (AML) is a heterogeneous disease, both biologically and clinically, in which a number of distinct genetic abnormalities have been described. Several recent studies suggest that this heterogeneity extends to LSCs and can vary between patient subgroups, and even within individual patients. Moreover, the complexity of AML is further complicated by the existenc...
17 Some hematologic malignancies arise from a few genomic events such as simple translocations (i... more 17 Some hematologic malignancies arise from a few genomic events such as simple translocations (i.e., breakage and removal of a large segment of DNA from one chromosome and attachment of the segment to a different chromosome). Solid tumors, however, reflect the consequences of accumulated genetic and epigenetic (i.e., changes “on” the gene, for example coupling to the DNA, rather than changes to the DNA sequence itself) events arising over a period of many years. In solid tumors, this process is facilitated by genomic instability resulting from environmental agents, such as smoking, and from defects in genes whose role involves the maintenance of genomic integrity.1 During the past several years, new technologies have dramatically increased our understanding of cancer at its fundamental genomic level. For the most part such technologies have revealed both point mutations and ever smaller and ever more abundant amplifications (increases in the number of copies of any particular piece of DNA), deletions (loss of pieces of DNA from chromosomes), and translocations, along with genomic heterogeneity at the cellular level.2,3 Genomic sampling approaches first detected numerous events. This knowledge has been dramatically enhanced and augmented since 2006 by direct DNA sequencing of entire cancer genomes by The Cancer Genome Anatomy Project in the United States and by The Cancer Genome Project in the United Kingdom.4,5 These studies have revealed genomic alterations far more extensive than long believed, and have conclusively established genomic heterogeneity within and among tumors.2,6 Moving from research into practice, clinical utilization of such genomic data becomes a question of what do physicians need to know about a particular case—and not physicians must know everything they can possibly learn. The cost-benefit ratio associated with obtaining genomic data also must be considered. In other words, while a multitude of genomic events have educated researchers about the enormous diversity and complexity of cancer, only a finite number of specific events currently have been found to be clinically valuable. These include the diagnostic BCR-ABL translocation in chronic myelogenous leukemia that underlies responsiveness to Gleevec® and amplification of ERBB2 in breast cancer that is associated with responsiveness to Herceptin®.
The gene for the human mineralocorticoid receptor (MLR) was previously localized to chromosome 4.... more The gene for the human mineralocorticoid receptor (MLR) was previously localized to chromosome 4. Here, we have localized this gene to 4q31.2 by in situ hybridization. This precise mapping of MLR will assist in the linkage analysis and genetic characterization of pseudohypoaldosteronism, an autosomal recessive disorder which likely results from a defect in the MLR gene.
Cold Spring Harbor Symposia on Quantitative Biology, 1986
Human chromosome 11 is clearly a model autosome encoding genes and characteristics associated wit... more Human chromosome 11 is clearly a model autosome encoding genes and characteristics associated with both normal and abnormal growth and development, and several significant disorders. A fine-structure molecular, genetic, and physical map of this chromosome would add considerably to our knowledge of the organization and control of human genes and to an understanding of normal and abnormal human biology.
STK15/Aurora-A is a serine/threonine kinase essential for chromosome segregation and cytokinesis,... more STK15/Aurora-A is a serine/threonine kinase essential for chromosome segregation and cytokinesis, and is considered to be a cancer susceptibility gene in mice and humans. Two coding single nucleotide polymorphisms in Aurora-A, 91T>A [phenylalanine/isoleucine (F/I)] and 169G>A [valine/isoleucine (V/I)], create four haplotypes, 91T-169G, 91A-169G, 91T-169A, and 91A-169A. We evaluated the association between these coding single nucleotide polymorphisms and esophageal cancer risk by genotyping 197 esophageal cancer cases and 146 controls. Haplotype 91A-169A (I31/I57) was observed to be statistically more frequent in cancer cases (odds ratio, 3.1452; 95% confidence interval, 1.0258-9.6435). Functional differences among the four isoforms were then analyzed to reveal the source of the cancer risk. Kinase activity levels of I31/I57 and F31/I57 were reduced to 15% and 40% compared with I31/V57 in vivo and in vitro. We considered the differences between the kinase activities and divided...
American Journal of Respiratory Cell and Molecular Biology, 1997
A partial cDNA (pAM1) encoding a major airway mucin glycoprotein with novel tandem repetitive seq... more A partial cDNA (pAM1) encoding a major airway mucin glycoprotein with novel tandem repetitive sequence has recently been cloned (Shankar, V., M. S. Gilmore, R. C. Elkins, and G. P. Sachdev. 1994. Biochem. J. 300:295-298). In this article, we report additional new sequence derived by 3'-rapid amplification of cDNA ends technique. The sequence corresponds to a stop codon, 3'-untranslated region of 458 bp, a polyadenylation signal, and poly A+ tail, and represents the extreme carboxy terminus of MUC8. A plasmid construct (pAM3) in pBluescript was generated by in-frame ligation of pAM1 to the 479-bp 3'UTR of MUC8. A 5'-end 325-bp fragment of this cDNA subcloned into the protein fusion and expression vector pET28b(+) was used to generate fusion protein under the control of T7 promoter. The purified fusion protein as well as synthetic peptide corresponding to the MUC8 repeat sequence (TSCPRPLQEGTPGS) were used to raise polyclonal antibodies in rabbits. The antiserum to the fusion protein and to the synthetic peptide reacted with the deglycosylated major tracheobronchial mucin. Immunohistochemical studies using the above antibodies localized the MUC8 protein product to submucosal glands in human tracheal epithelium. Furthermore, the gene from which this cDNA is derived, was mapped to chromosome 12 using DNA from a panel of human-mouse somatic cell hybrids. Fluorescence in situ hybridization was used to assign the regional localization to 12q24.3. Since the eight known human mucin genes map to other chromosomes, we have named this gene MUC8, in accordance with mucin gene nomenclature.
The CD6 protein has been shown to play important roles in T cell costimulation and adhesion. Rece... more The CD6 protein has been shown to play important roles in T cell costimulation and adhesion. Recently, variably spliced isoforms of CD6 mRNA have been identified in both human and murine T cells. Here we report on the genomic organization of the human CD6 gene, its chromosomal localization, and the characterization of novel isoforms. Human CD6 is encoded by at least 13 exons. The amino terminal signal sequence, extracellular region, and transmembrane domain are encoded by seven exons, while the cytoplasmic domain of CD6 is encoded by six exons. Each of the three extracellular scavenger receptor cysteine-rich domains is encoded by a separate exon. Fluorescence in situ hybridization studies and screening of a chromosome-specific YAC (yeast artificial chromosome) library revealed that the gene encoding CD6 is located on chromosome 11 at 11q13 in close proximity to the gene encoding the related molecule CD5 and within 600 kb of CD20. Analysis of mRNA transcripts encoding CD6 isolated fr...
ABSTRACTThe sequencing of human virus genomes from wastewater samples is an efficient method for ... more ABSTRACTThe sequencing of human virus genomes from wastewater samples is an efficient method for tracking viral transmission and evolution at the community level. However, this requires the recovery of viral nucleic acids of high quality. We developed a reusable tangential-flow filtration system to concentrate and purify viruses from wastewater for whole-genome sequencing. A pilot study was conducted with 94 wastewater samples from four local sewersheds, from which viral nucleic acids were extracted, and the whole genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was sequenced using the ARTIC V4.0 primers. Our method yielded a high probability (0.9) of recovering complete or near-complete SARS-CoV-2 genomes (>90% coverage at 10× depth) from wastewater when the COVID-19 incidence rate exceeded 33 cases per 100 000 people. The relative abundances of sequenced SARS-CoV-2 variants followed the trends observed from patient-derived samples. We also identified SARS-...
Traditional analysis of genomic data from bulk sequencing experiments seek to group and compare s... more Traditional analysis of genomic data from bulk sequencing experiments seek to group and compare sample cohorts into biologically meaningful groups. To accomplish this task, large scale databases of patient-derived samples, like that of TCGA, have been established, giving the ability to interrogate multiple data modalities per tumor. We have developed a computational strategy employing multimodal integration paired with spectral clustering and modern dimension reduction techniques such as PHATE to provide a more robust method for cancer sub-type classification. Using this integrated approach, we have examined 514 Head and Neck Squamous Carcinoma (HNSC) tumor samples from TCGA across gene-expression, DNA-methylation, and microbiome data modalities. We show that these approaches, primarily developed for single-cell sequencing can be efficiently applied to bulk tumor sequencing data. Our multimodal analysis captures the dynamic heterogeneity, identifies new and refines subtypes of HNSC,...
5374 Irinotecan (CPT-11) interacts synergistically with a wide variety of anticancer agents, incl... more 5374 Irinotecan (CPT-11) interacts synergistically with a wide variety of anticancer agents, including thymidylate synthetase inhibitors, taxane derivatives, platinum compounds and anthracyclines, as well as immunotherapeutic agents. The wide variety of known mechanisms of action of these synergistic agents and the schedule-dependency of synergy suggest that synergistic interactions with CPT-11 are associated with a CPT-11-induced change in a general cellular process, such as the apoptotic response. Study of time kinetic changes in expression of apoptosis-associated genes studied by cDNA microarray analysis in HL60 cells following two-hour in vitro exposure to 0.3 μM SN-38, the active metabolite of CPT-11, revealed downregulation of the anti-apoptotic transcriptome survivin, with a nadir at six hours. In order to determine whether survivin downregulation was related to altered transcription regulation, expression profiles of factors controlling the NFkB transcription complex, which ...
e16504 Background: A proof of principle array-based comparative genomic hybridization (aCGH) anal... more e16504 Background: A proof of principle array-based comparative genomic hybridization (aCGH) analysis was performed in archival formalin-fixed and paraffin-embedded (FFPE) stage I ovarian cancers (EOC) to determine if frequent (>40%) copy number aberrations (CNAs) can be detected in DNA repair genes including the Fanconi anemia complementation group (FANC) and RAD51 families compared with the rest of the genome. Methods: Tumor DNA was isolated from 22 serous cancers from the GOG-175 virtual tissue bank. RPCI 19K BAC arrays were hybridized (GeneTAC HybStation) and scanned (Gene Pix 4200AL Laser Scanner). Spot fluorescence values were quantified using ImaGene, vetted for quality and loess corrected with adjustments for chip-specific spatial effects. The genome was segmented to identify regions with common copy number means (DNAcopy software). Posterior aberration probabilities for the regions were obtained using CGHcall and data was visualized and annotated using iGenomicViewer in ...
The hallmark of Hodgkin’s Lymphoma (HL) is the presence of large Hodgkin’s and Reed/Sternberg (HR... more The hallmark of Hodgkin’s Lymphoma (HL) is the presence of large Hodgkin’s and Reed/Sternberg (HRS) cells that comprise only ~1–3% of the cellular infiltrate. Given the paucity of genomic analyses of these cells, we sought to characterize their DNA copy number alterations (CNA) by array comparative genomic hybridization (aCGH) using DNA isolated from laser capture microdissected (LCM) CD30+ HRS cells. Primary paraffin-embedded diagnostic samples were obtained from 27 patients (pts), including 15 pts with chemotherapy responsive and 12 pts with primary refractory HL. From each sample, 150 HRS cells were isolated by LCM from 5-μm thick tissue sections, amplified using whole genome amplification (WGA), and hybridized to a minimal tiling 19K whole genome bacterial artificial chromosome (BAC) array (BAC center to center distance of 165 kb). To define thresholds for calling gains and loss and to control for WGA noise, 1.05 ng DNA (DNA equivalent of 150 cells) was obtained from six normal ...
Chemotherapy or targeted cancer therapies have greatly improved the treatment outcome of patients... more Chemotherapy or targeted cancer therapies have greatly improved the treatment outcome of patients with leukemia; however, many will ultimately die because of disease relapse and development of drug resistance. Leukemias are cancers of the blood cells that result from alteration of the normal physiological constraints that regulate hematopoietic stem cells (HSCs). General characteristics of leukemia stem cells (LSCs) such as self-renewal, self-protection and proliferative quiescence represent inherent mechanisms that at least partially explain drug resistance and recurrence in post-therapy leukemia patients. Acute myeloid leukemia (AML) is a heterogeneous disease, both biologically and clinically, in which a number of distinct genetic abnormalities have been described. Several recent studies suggest that this heterogeneity extends to LSCs and can vary between patient subgroups, and even within individual patients. Moreover, the complexity of AML is further complicated by the existenc...
17 Some hematologic malignancies arise from a few genomic events such as simple translocations (i... more 17 Some hematologic malignancies arise from a few genomic events such as simple translocations (i.e., breakage and removal of a large segment of DNA from one chromosome and attachment of the segment to a different chromosome). Solid tumors, however, reflect the consequences of accumulated genetic and epigenetic (i.e., changes “on” the gene, for example coupling to the DNA, rather than changes to the DNA sequence itself) events arising over a period of many years. In solid tumors, this process is facilitated by genomic instability resulting from environmental agents, such as smoking, and from defects in genes whose role involves the maintenance of genomic integrity.1 During the past several years, new technologies have dramatically increased our understanding of cancer at its fundamental genomic level. For the most part such technologies have revealed both point mutations and ever smaller and ever more abundant amplifications (increases in the number of copies of any particular piece of DNA), deletions (loss of pieces of DNA from chromosomes), and translocations, along with genomic heterogeneity at the cellular level.2,3 Genomic sampling approaches first detected numerous events. This knowledge has been dramatically enhanced and augmented since 2006 by direct DNA sequencing of entire cancer genomes by The Cancer Genome Anatomy Project in the United States and by The Cancer Genome Project in the United Kingdom.4,5 These studies have revealed genomic alterations far more extensive than long believed, and have conclusively established genomic heterogeneity within and among tumors.2,6 Moving from research into practice, clinical utilization of such genomic data becomes a question of what do physicians need to know about a particular case—and not physicians must know everything they can possibly learn. The cost-benefit ratio associated with obtaining genomic data also must be considered. In other words, while a multitude of genomic events have educated researchers about the enormous diversity and complexity of cancer, only a finite number of specific events currently have been found to be clinically valuable. These include the diagnostic BCR-ABL translocation in chronic myelogenous leukemia that underlies responsiveness to Gleevec® and amplification of ERBB2 in breast cancer that is associated with responsiveness to Herceptin®.
The gene for the human mineralocorticoid receptor (MLR) was previously localized to chromosome 4.... more The gene for the human mineralocorticoid receptor (MLR) was previously localized to chromosome 4. Here, we have localized this gene to 4q31.2 by in situ hybridization. This precise mapping of MLR will assist in the linkage analysis and genetic characterization of pseudohypoaldosteronism, an autosomal recessive disorder which likely results from a defect in the MLR gene.
Cold Spring Harbor Symposia on Quantitative Biology, 1986
Human chromosome 11 is clearly a model autosome encoding genes and characteristics associated wit... more Human chromosome 11 is clearly a model autosome encoding genes and characteristics associated with both normal and abnormal growth and development, and several significant disorders. A fine-structure molecular, genetic, and physical map of this chromosome would add considerably to our knowledge of the organization and control of human genes and to an understanding of normal and abnormal human biology.
STK15/Aurora-A is a serine/threonine kinase essential for chromosome segregation and cytokinesis,... more STK15/Aurora-A is a serine/threonine kinase essential for chromosome segregation and cytokinesis, and is considered to be a cancer susceptibility gene in mice and humans. Two coding single nucleotide polymorphisms in Aurora-A, 91T>A [phenylalanine/isoleucine (F/I)] and 169G>A [valine/isoleucine (V/I)], create four haplotypes, 91T-169G, 91A-169G, 91T-169A, and 91A-169A. We evaluated the association between these coding single nucleotide polymorphisms and esophageal cancer risk by genotyping 197 esophageal cancer cases and 146 controls. Haplotype 91A-169A (I31/I57) was observed to be statistically more frequent in cancer cases (odds ratio, 3.1452; 95% confidence interval, 1.0258-9.6435). Functional differences among the four isoforms were then analyzed to reveal the source of the cancer risk. Kinase activity levels of I31/I57 and F31/I57 were reduced to 15% and 40% compared with I31/V57 in vivo and in vitro. We considered the differences between the kinase activities and divided...
American Journal of Respiratory Cell and Molecular Biology, 1997
A partial cDNA (pAM1) encoding a major airway mucin glycoprotein with novel tandem repetitive seq... more A partial cDNA (pAM1) encoding a major airway mucin glycoprotein with novel tandem repetitive sequence has recently been cloned (Shankar, V., M. S. Gilmore, R. C. Elkins, and G. P. Sachdev. 1994. Biochem. J. 300:295-298). In this article, we report additional new sequence derived by 3'-rapid amplification of cDNA ends technique. The sequence corresponds to a stop codon, 3'-untranslated region of 458 bp, a polyadenylation signal, and poly A+ tail, and represents the extreme carboxy terminus of MUC8. A plasmid construct (pAM3) in pBluescript was generated by in-frame ligation of pAM1 to the 479-bp 3'UTR of MUC8. A 5'-end 325-bp fragment of this cDNA subcloned into the protein fusion and expression vector pET28b(+) was used to generate fusion protein under the control of T7 promoter. The purified fusion protein as well as synthetic peptide corresponding to the MUC8 repeat sequence (TSCPRPLQEGTPGS) were used to raise polyclonal antibodies in rabbits. The antiserum to the fusion protein and to the synthetic peptide reacted with the deglycosylated major tracheobronchial mucin. Immunohistochemical studies using the above antibodies localized the MUC8 protein product to submucosal glands in human tracheal epithelium. Furthermore, the gene from which this cDNA is derived, was mapped to chromosome 12 using DNA from a panel of human-mouse somatic cell hybrids. Fluorescence in situ hybridization was used to assign the regional localization to 12q24.3. Since the eight known human mucin genes map to other chromosomes, we have named this gene MUC8, in accordance with mucin gene nomenclature.
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Papers by Norma Nowak