Manuel L Gonzalez-Garay
- Research Associate Professor, Medicine
- Associate Director, Applied Genomics - Center for Biomedical Informatic / Biostatistics
I am a seasoned expert in bioinformatics, computational biology, and genetics, and I also have extensive training in biostatistics, cell, and molecular biology. I am strongly motivated to contribute to the understanding of genetic disorders. My initial training was in biostatistics, genetics, cell and molecular biology, however during 1997; I redirected my career towards bioinformatics. Since then, I have been working professionally as a bioinformatician for the last eighteen years. In 1998, I joined a drug discovery company, Lexicon Pharmaceuticals as a Scientific Programmer, few months after joining Lexicon, I become engage in a significant number of projects and very soon I got promoted to create my group. During my time at Lexicon, I managed and created databases and software for multiple projects. Some of them were: Omnibank, Lexvision, Lexgene, the lexicon’s mouse knockout database, a target prioritization program, etc. After the burst of the first biotech bubble, I decide to move back to academia and from 2002 to 2009 I worked at Human Genome Sequencing Center (HGSC), Baylor College of Medicine in Houston. During my employment at HGSC, I developed bioinformatics software like Genboree discovery systems and became integrally involved in multiple projects including the Tumor Sequencing Project (TSP), The Cancer Genome Atlas (TCGA) and the ENCODE project. In 2010, I moved to the Institute of Molecular Medicine (UTHealth), where my laboratory was routinely sequencing genomes, exomes and RNAs using multiple sequencing platforms and perform bioinformatics analysis. During that period, I analyzed the whole genome of patients with rare disorders, whole-exome sequences of hundreds of samples. Consequently, I developed the necessary expertise and software to identify genetic variants efficiently. At the IMM, I directed a project in personalized medicine where I analyzed the genetic components of healthy individuals. During 2012, I led a team of clinicians and graduate students to participate in the Boston Children’s Hospital CLARITY challenge with the goal of developing better standards for the interpretation of clinical genomes. Also, I established multiple collaborations at the IMM where I served as the main bioinformatician in a variety of projects directed toward understanding the molecular mechanisms of complex and monogenetic disorders. In 2016, I moved to the University of Arizona where I have been acting as a bioinformatician and biostatistician in multiple projects directed to understand the molecular mechanisms leading to Lipedema, Dercum's disease, Sarcoidosis and other lung disorders.
- Ph.D. Regulatory Biology, 1996
- University of Texas, GSBS
- B. Biology 1988
- Universidad Autónoma de Nuevo León, México
2016-present Associate Professor, Department of Medicine. University of Arizona MS, Tucson, Arizona
2016-present Associate Director for Applied Genomics. Center for Biomedical Informatics and Biostatistics. University of Arizona MS, Tucson, Arizona
2013-present Affiliated Professor, Medical School, Universidad Autónoma de Nuevo Leon (UANL).
2010-2015 Assistant Professor, The Brown Foundation Institute of Molecular Medicine,The University of Texas Health Science Center, Houston, Texas
2002-2009 Senior Scientific Programmer Lead, Bioinformatics Research Laboratory, Human Genome Sequencing Center, Baylor College of Medicine. Houston, TX
2000-2002 Manager of Bioinformatics, Lexicon Genetics Inc. The Woodlands, TX
1999-2000 Bioinformatics Scientist, Lexicon Genetics Inc. The Woodlands, TX
1998-1999 Scientific Programmer, Lexicon Genetics Inc. The Woodlands, TX
1996-1998 Postdoctoral Fellow, University of Texas, MS, Dept. of Integrative Biology, Houston, TX
1988-1991 Research Assistant, University of Texas, Medical School, Dept. of Pharmacology, Houston
- Ph.D. Cell and Regulatory Biology
- The University of Texas UTHealth Graduate School of Biomedical Sciences, Houston, Texas, United States
- Analysis of the mechanisms that maintain coordinate production of alpha and beta- tubulin in mammalian cells
- B.S. Biology
- Universidad Autónoma de Nuevo Leon, Monterrey, Nuevo Leon, Mexico
- Detection of DNA sequences from Human Papillomavirus type 16 inCervical Cancer
- Research Associate Professor & Associate Director of Applied Genomics, University of Arizona, Tucson, Arizona (2016 - Ongoing)
- Assistant Professor, UThealth, Institute of Molecular Medicine (IMM) (2010 - 2015)
- Senior Scientific Programmer Lead, Human Genome Sequencing Center, Baylor College of Medicine (2002 - 2009)
- Sr. Manager of Bioinformatics, Lexicon Pharmaceuticals (1998 - 2002)
- Postdoctoral Fellow, University of Texas (1996 - 1998)
Rare Genetic disordersLymphedemaLipedemaDercum's DiseasePulmonary hypertension (PH)Pulmonary Fibrosis
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- Gonzalez-Garay, M. L. (2015). Introduction to Isoform Sequencing Using Pacific Biosciences Technology (Iso-Seq). In Transcriptomics and Gene Regulation Volume 9 of the series Translational Bioinformatics(pp 141-160). Springer Netherlands: Springer. doi:10.1007/978-94-017-7450-5_6More infoAlternative RNA splicing is a known phenomenon, but we still do not have a complete catalog of isoforms that explain variability in the human transcriptome. We have made significant progress in developing methods to study variability of the transcriptome, but we are far away of having a complete picture of the transcriptome. The initial methods to study gene expression were based on cloning of cDNAs and Sanger sequencing. The strategy was labor-intensive and expensive. With the development of microarrays, different methods based on exon arrays and tiling arrays provided valuable information about RNA expression. However, the microarray presented significant limitations. Most of the limitations became apparent by 2005, but it was not until 2008 that an alternative method to study the transcriptome was developed. RNA Sequencing using next-generation sequencing (RNA-Seq) quickly became the technology of choice for gene expression profiling. Recently, the precision and sensitivity of RNA-Seq have come into question, especially for transcriptome reconstruction. This chapter will describe a relatively new method, “Isoform Sequencing” (Iso-Seq). Iso-Seq was developed by Pacific Biosciences (PacBio), and it is capable of identifying new isoforms with extraordinary precision due to its long-read technology. The technique to create libraries is straightforward, and the PacBio RS II instrument generates the information in hours. The bioinformatics analysis is performed using the freely available SMRT® Portal software. The SMRT® Portal is easy to use and capable of performing all the steps necessary to analyze the raw data and to generate high-quality full-length isoforms. For the universal acceptance of the Iso-Seq method, the capacity of the SMRT® Cells needs to improve at least 10- to 100-fold to make the system affordable and attractive to users.
- Gonzalez-Garay, M. L., Gonzalez-Guerrero, J. F., & Barrera-Saldana, H. A. (1988). Detection of the Human Papillomavirus Genome in Cervical Cancer. In Cell Function and Disease(pp 333-341). Springer US: Springer. doi:10.1007/978-1-4613-0813-3_29More infoCervical cancer is one of the primary neoplasias responsible for deaths of women in the world. In 1975, the world incidence of this sickness was approximately five hundred thousand cases, a value comparable to the number of women in which breast cancer was detected in the same year (1).
- Gonzalez-Garay, M. L. (2016). Biomechanisms of Comorbidity: Reviewing Integrative Analyses of Multi-omics Datasets and Electronic Health Records.. Yearb Med Inform, 10(1), 194-206. doi:10.15265/IY-2016-040More infoObjectives: Disease comorbidity is a pervasive phenomenon impacting patients’ health outcomes, disease management, and clinical decisions. This review presents past, current and future research directions leveraging both phenotypic and molecular information to uncover disease similarity underpinning the biology and etiology of disease comorbidity. Methods: We retrieved ~130 publications and retained 59, ranging from 2006 to 2015, that comprise a minimum number of five diseases and at least one type of biomolecule. We surveyed their methods, disease similarity metrics, and calculation of comorbidities in the electronic health records, if present. Results: Among the surveyed studies, 44% generated or validated disease similarity metrics in context of comorbidity, with 60% being published in the last two years. As inputs, 87% of studies utilized intragenic loci and proteins while 13% employed RNA (mRNA, LncRNA or miRNA). Network modeling was predominantly used (35%) followed by statistics (28%) to impute similarity between these biomolecules and diseases. Studies with large numbers of biomolecules and diseases used network models or naïve overlap of disease-molecule associations, while machine learning, statistics, and information retrieval were utilized in smaller and moderate sized studies. Multiscale computations comprising shared function, network topology, and phenotypes were performed exclusively on proteins. Conclusion: This review highlighted the growing methods for identifying the molecular mechanisms underpinning comorbidities that leverage multiscale molecular information and patterns from electronic health records. The survey unveiled that intergenic polymorphisms have been overlooked for similarity imputation compared to their intragenic counterparts, offering new opportunities to bridge the mechanistic and similarity gaps of comorbidity.
- Gonzalez-Garay, M. L. (2016). Robust extracellular pH modulation by Candida albicans during growth in carboxylic acids. mBio, 7(6), Published online 2016 Nov 15. doi:10.1128/mBio.01646-16More infoThe opportunistic fungal pathogen Candida albicans thrives within diverse niches in the mammalian host. Among the adaptations that underlie this fitness is an ability to utilize a wide array of nutrients, especially sources of carbon that are disfavored by many other fungi; this contributes to its ability to survive interactions with the phagocytes that serve as key barriers against disseminated infections. We have reported that C. albicans generates ammonia as a byproduct of amino acid catabolism to neutralize the acidic phagolysosome and promote hyphal morphogenesis in a manner dependent on the Stp2 transcription factor. Here, we report that this species rapidly neutralizes acidic environments when utilizing carboxylic acids like pyruvate, α-ketoglutarate (αKG), or lactate as the primary carbon source. Unlike in cells growing in amino acid-rich medium, this does not result in ammonia release, does not induce hyphal differentiation, and is genetically distinct. While transcript profiling revealed significant similarities in gene expression in cells grown on either carboxylic or amino acids, genetic screens for mutants that fail to neutralize αKG medium identified a nonoverlapping set of genes, including CWT1, encoding a transcription factor responsive to cell wall and nitrosative stresses. Strains lacking CWT1 exhibit retarded αKG-mediated neutralization in vitro, exist in a more acidic phagolysosome, and are more susceptible to macrophage killing, while double cwt1Δ stp2Δ mutants are more impaired than either single mutant. Together, our observations indicate that C. albicans has evolved multiple ways to modulate the pH of host-relevant environments to promote its fitness as a pathogen.IMPORTANCE The fungal pathogen Candida albicans is a ubiquitous and usually benign constituent of the human microbial ecosystem. In individuals with weakened immune systems, this organism can cause potentially life-threatening infections and is one of the most common causes of hospital-acquired infections. Understanding the interactions between C. albicans and immune phagocytic cells, such as macrophages and neutrophils, will define the mechanisms of pathogenesis in this species. One such adaptation is an ability to make use of nonstandard nutrients that we predict are plentiful in certain niches within the host, including within these phagocytic cells. We show here that the metabolism of certain organic acids enables C. albicans to neutralize acidic environments, such as those within macrophages. This phenomenon is distinct in several significant ways from previous reports of similar processes, indicating that C. albicans has evolved multiple mechanisms to combat the harmful acidity of phagocytic cells.
- Gonzalez-Garay, M. L., Aldrich, M. B., Rasmussen, J. C., Guilliod, R., Lapinski, P. E., King, P. D., & Sevick-Muraca, E. M. (2016). A novel mutation in CELSR1 is associated with hereditary lymphedema. Vascular cell, 8, 1.More infoBiological evidence reported in the literature supports the role of CELSR1 as being essential for valvular function in murine lymphatics. Yet thus far, there have been no variants in CELSR1 associated with lymphatic dysfunction in humans.
- Kin, K., Chen, X., Gonzalez-Garay, M., & Fakhouri, W. D. (2016). The effect of non-coding DNA variations on P53 and cMYC competitive inhibition at cis-overlapping motifs. Human molecular genetics, 25(8), 1517-27.More infoNon-coding DNA variations play a critical role in increasing the risk for development of common complex diseases, and account for the majority of SNPs highly associated with cancer. However, it remains a challenge to identify etiologic variants and to predict their pathological effects on target gene expression for clinical purposes. Cis-overlapping motifs (COMs) are elements of enhancer regions that impact gene expression by enabling competitive binding and switching between transcription factors. Mutations within COMs are especially important when the involved transcription factors have opposing effects on gene regulation, like P53 tumor suppressor and cMYC proto-oncogene. In this study, genome-wide analysis of ChIP-seq data from human cancer and mouse embryonic cells identified a significant number of putative regulatory elements with signals for both P53 and cMYC. Each co-occupied element contains, on average, two COMs, and one common SNP every two COMs. Gene ontology of predicted target genes for COMs showed that the majority are involved in DNA damage, apoptosis, cell cycle regulation, and RNA processing. EMSA results showed that both cMYC and P53 bind to cis-overlapping motifs within a ChIP-seq co-occupied region in Chr12. In vitro functional analysis of selected co-occupied elements verified enhancer activity, and also showed that the occurrence of SNPs within three COMs significantly altered enhancer activity. We identified a list of COM-associated functional SNPs that are in close proximity to SNPs associated with common diseases in large population studies. These results suggest a potential molecular mechanism to identify etiologic regulatory mutations associated with common diseases.
- Laskowski, T. J., Van Caeneghem, Y., Pourebrahim, R., Ma, C., Ni, Z., Garate, Z., Crane, A. M., Li, X. S., Liao, W., Gonzalez-Garay, M., Segovia, J. C., Paschon, D. E., Rebar, E. J., Holmes, M. C., Kaufman, D., Vandekerckhove, B., & Davis, B. R. (2016). Gene Correction of iPSCs from a Wiskott-Aldrich Syndrome Patient Normalizes the Lymphoid Developmental and Functional Defects. Stem cell reports, 7(2), 139-48.More infoWiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disease caused by mutations in the gene encoding the WAS protein (WASp). Here, induced pluripotent stem cells (iPSCs) were derived from a WAS patient (WAS-iPSC) and the endogenous chromosomal WAS locus was targeted with a wtWAS-2A-eGFP transgene using zinc finger nucleases (ZFNs) to generate corrected WAS-iPSC (cWAS-iPSC). WASp and GFP were first expressed in the earliest CD34(+)CD43(+)CD45(-) hematopoietic precursor cells and later in all hematopoietic lineages examined. Whereas differentiation to non-lymphoid lineages was readily obtained from WAS-iPSCs, in vitro T lymphopoiesis from WAS-iPSC was deficient with few CD4(+)CD8(+) double-positive and mature CD3(+) T cells obtained. T cell differentiation was restored for cWAS-iPSCs. Similarly, defects in natural killer cell differentiation and function were restored on targeted correction of the WAS locus. These results demonstrate that the defects exhibited by WAS-iPSC-derived lymphoid cells were fully corrected and suggests the potential therapeutic use of gene-corrected WAS-iPSCs.
- Mamenko, M., Dhande, I., Tomilin, V., Zaika, O., Boukelmoune, N., Zhu, Y., Gonzalez-Garay, M. L., Pochynyuk, O., & Doris, P. A. (2016). Defective Store-Operated Calcium Entry Causes Partial Nephrogenic Diabetes Insipidus. Journal of the American Society of Nephrology : JASN, 27(7), 2035-48.More infoStore-operated calcium entry (SOCE) is the mechanism by which extracellular signals elicit prolonged intracellular calcium elevation to drive changes in fundamental cellular processes. Here, we investigated the role of SOCE in the regulation of renal water reabsorption, using the inbred rat strain SHR-A3 as an animal model with disrupted SOCE. We found that SHR-A3, but not SHR-B2, have a novel truncating mutation in the gene encoding stromal interaction molecule 1 (STIM1), the endoplasmic reticulum calcium (Ca(2+)) sensor that triggers SOCE. Balance studies revealed increased urine volume, hypertonic plasma, polydipsia, and impaired urinary concentrating ability accompanied by elevated circulating arginine vasopressin (AVP) levels in SHR-A3 compared with SHR-B2. Isolated, split-open collecting ducts (CD) from SHR-A3 displayed decreased basal intracellular Ca(2+) levels and a major defect in SOCE. Consequently, AVP failed to induce the sustained intracellular Ca(2+) mobilization that requires SOCE in CD cells from SHR-A3. This effect decreased the abundance of aquaporin 2 and enhanced its intracellular retention, suggesting impaired sensitivity of the CD to AVP in SHR-A3. Stim1 knockdown in cultured mpkCCDc14 cells reduced SOCE and basal intracellular Ca(2+) levels and prevented AVP-induced translocation of aquaporin 2, further suggesting the effects in SHR-A3 result from the expression of truncated STIM1. Overall, these results identify a novel mechanism of nephrogenic diabetes insipidus and uncover a role of SOCE in renal water handling.
- Messina-Baas, O., Gonzalez-Garay, M. L., González-Huerta, L. M., Toral-López, J., & Cuevas-Covarrubias, S. A. (2016). Whole Exome Sequencing Reveals a Mutation in CRYBB2 in a Large Mexican Family with Autosomal Dominant Pulverulent Cataract. Molecular syndromology, 7(2), 87-92.More infoCongenital cataract, an important cause of reversible blindness, is due to several causes including Mendelian inheritance. Thirty percent of cataracts are hereditary with participation of the gamma crystallin genes. Clinical and genetic heterogeneity is observed in patients with gene mutations and congenital cataract; about 40 genetic loci have been associated with hereditary cataract. In this study, we identified the underlying genetic cause of an autosomal dominant pulverulent cataract (ADPC) in a large Mexican family. Twenty-one affected patients and 20 healthy members of a family with ADPC were included. Genomic DNA was analyzed by whole exome sequencing in the proband, a normal daughter, and in an affected son, whereas DNA Sanger sequencing was performed in all members of the family. After the bioinformatics analysis, all samples were genotyped using Sanger sequencing to eliminate variants that do not cosegregate with the cataract. We observed a perfect cosegregation of a nonsense mutation c.475C>T (p.Q155*) in exon 6 of the CRYBB2 gene with ADPC. We calculated a logarithm of the odds score of 5.5. This mutation was not detected in healthy members of the family and in 100 normal controls. This is the first Mexican family with ADPC associated with a p.Q155* mutation. Interestingly, this specific mutation in the CRYBB2 gene seems to be exclusively associated with pulverulent/cerulean cataract (with some clinical variability) independent of the population's genetic background.
- Crane, A. M., Kramer, P., Bui, J. H., Chung, W. J., Li, X. S., Gonzalez-Garay, M. L., Hawkins, F., Liao, W., Mora, D., Choi, S., Wang, J., Sun, H. C., Paschon, D. E., Guschin, D. Y., Gregory, P. D., Kotton, D. N., Holmes, M. C., Sorscher, E. J., & Davis, B. R. (2015). Targeted correction and restored function of the CFTR gene in cystic fibrosis induced pluripotent stem cells. Stem cell reports, 4(4), 569-77.More infoRecently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources-potentially useful for the development of new therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc-finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR). We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other. The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells.
- Guo, L., Milburn, M. V., Ryals, J. A., Lonergan, S. C., Mitchell, M. W., Wulff, J. E., Alexander, D. C., Evans, A. M., Bridgewater, B., Miller, L., Gonzalez-Garay, M. L., & Caskey, C. T. (2015). Plasma metabolomic profiles enhance precision medicine for volunteers of normal health. Proceedings of the National Academy of Sciences of the United States of America, 112(35), E4901-10.More infoPrecision medicine, taking account of human individuality in genes, environment, and lifestyle for early disease diagnosis and individualized therapy, has shown great promise to transform medical care. Nontargeted metabolomics, with the ability to detect broad classes of biochemicals, can provide a comprehensive functional phenotype integrating clinical phenotypes with genetic and nongenetic factors. To test the application of metabolomics in individual diagnosis, we conducted a metabolomics analysis on plasma samples collected from 80 volunteers of normal health with complete medical records and three-generation pedigrees. Using a broad-spectrum metabolomics platform consisting of liquid chromatography and GC coupled with MS, we profiled nearly 600 metabolites covering 72 biochemical pathways in all major branches of biosynthesis, catabolism, gut microbiome activities, and xenobiotics. Statistical analysis revealed a considerable range of variation and potential metabolic abnormalities across the individuals in this cohort. Examination of the convergence of metabolomics profiles with whole-exon sequences (WESs) provided an effective approach to assess and interpret clinical significance of genetic mutations, as shown in a number of cases, including fructose intolerance, xanthinuria, and carnitine deficiency. Metabolic abnormalities consistent with early indications of diabetes, liver dysfunction, and disruption of gut microbiome homeostasis were identified in several volunteers. Additionally, diverse metabolic responses to medications among the volunteers may assist to identify therapeutic effects and sensitivity to toxicity. The results of this study demonstrate that metabolomics could be an effective approach to complement next generation sequencing (NGS) for disease risk analysis, disease monitoring, and drug management in our goal toward precision care.
- Agollah, G. D., Gonzalez-Garay, M. L., Rasmussen, J. C., Tan, I., Aldrich, M. B., Darne, C., Fife, C. E., Guilliod, R., Maus, E. A., King, P. D., & Sevick-Muraca, E. M. (2014). Evidence for SH2 domain-containing 5'-inositol phosphatase-2 (SHIP2) contributing to a lymphatic dysfunction. PloS one, 9(11), e112548.More infoThe lymphatic vasculature plays a critical role in a number of disease conditions of increasing prevalence, such as autoimmune disorders, obesity, blood vascular diseases, and cancer metastases. Yet, unlike the blood vasculature, the tools available to interrogate the molecular basis of lymphatic dysfunction/disease have been lacking. More recently, investigators have reported that dysregulation of the PI3K pathway is involved in syndromic human diseases that involve abnormal lymphatic vasculatures, but there have been few compelling results that show the direct association of this molecular pathway with lymphatic dysfunction in humans. Using near-infrared fluorescence lymphatic imaging (NIRFLI) to phenotype and next generation sequencing (NGS) for unbiased genetic discovery in a family with non-syndromic lymphatic disease, we discovered a rare, novel mutation in INPPL1 that encodes the protein SHIP2, which is a negative regulator of the PI3K pathway, to be associated with lymphatic dysfunction in the family. In vitro interrogation shows that SHIP2 is directly associated with impairment of normal lymphatic endothelial cell (LEC) behavior and that SHIP2 associates with receptors that are associated in lymphedema, implicating its direct involvement in the lymphatic vasculature.
- Braun, M. C., Herring, S. M., Gokul, N., Monita, M., Bell, R., Zhu, Y., Gonzalez-Garay, M. L., Wenderfer, S. E., & Doris, P. A. (2014). Hypertensive renal injury is associated with gene variation affecting immune signaling. Circulation. Cardiovascular genetics, 7(6), 903-10.More infoThe spontaneously hypertensive rat (SHR) strain exists in lines that contrast strongly in susceptibility to renal injury in hypertension. These inbred lines share common ancestry, and only 13% of their genomes arise from different ancestors.
- Brownstein, C. A., Beggs, A. H., Homer, N., Merriman, B., Yu, T. W., Flannery, K. C., DeChene, E. T., Towne, M. C., Savage, S. K., Price, E. N., Holm, I. A., Luquette, L. J., Lyon, E., Majzoub, J., Neupert, P., McCallie, D., Szolovits, P., Willard, H. F., Mendelsohn, N. J., , Temme, R., et al. (2014). An international effort towards developing standards for best practices in analysis, interpretation and reporting of clinical genome sequencing results in the CLARITY Challenge. Genome biology, 15(3), R53.More infoThere is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance.
- Caskey, C. T., Gonzalez-Garay, M. L., Pereira, S., & McGuire, A. L. (2014). Adult genetic risk screening. Annual review of medicine, 65, 1-17.More infoRecent advances in genetic analysis especially DNA sequencing technology open a new strategy for adult disease prevention by genetic screening. Physicians presently treat disease pathology with less emphasis on disease risk prevention/reduction. Genetic screening has reduced the incidence of untreatable childhood genetic diseases and improved the care of newborns. The opportunity exists to expand screening programs and reduce the incidence of adult onset diseases via genetic risk identification and disease intervention. This article outlines the approach, challenges, and benefits of such screening for adult genetic disease risks.
- Gonzalez-Garay, M. L. (2014). The road from next-generation sequencing to personalized medicine. Personalized medicine, 11(5), 523-544.More infoMoving from a traditional medical model of treating pathologies to an individualized predictive and preventive model of personalized medicine promises to reduce the healthcare cost on an overburdened and overwhelmed system. Next-generation sequencing (NGS) has the potential to accelerate the early detection of disorders and the identification of pharmacogenetics markers to customize treatments. This review explains the historical facts that led to the development of NGS along with the strengths and weakness of NGS, with a special emphasis on the analytical aspects used to process NGS data. There are solutions to all the steps necessary for performing NGS in the clinical context where the majority of them are very efficient, but there are some crucial steps in the process that need immediate attention.
- Gonzalez-Garay, M. L., Cranford, S. M., Braun, M. C., & Doris, P. A. (2014). Diversity in the preimmune immunoglobulin repertoire of SHR lines susceptible and resistant to end-organ injury. Genes and immunity, 15(8), 528-33.More infoWe used next-generation sequencing to identify immunoglobulin heavy chain (IGH) genetic variation in two closely related hypertensive rat lines that differ in susceptibility to end-organ disease (SHR-A3 and SHR-B2). The two SHR lines differ extensively at the IGH locus from the rat reference genome sequence and from each other, creating 306 sequence unique IGH genes. Compared with IGH genes mapped in the rat reference genome sequence, 98 are null gene alleles (31 are null in both SHR lines, 45 are null in SHR-A3 only and 23 are null in SHR-B2 only). Of the 306 divergent gene sequences, 126 result in amino acid substitution and, among these, SHR-A3 and SHR-B2 differ from one another at the amino acid level in 96 segments. Twelve pseudogenes in the rat reference genome sequence had changes displacing the stop codon and creating probable functional genes in either or both SHR-A3 and SHR-B2. A further five alleles that encoded functional rat reference genome sequence genes or open reading frames were converted to pseudogenes in either or both SHR-A3 and SHR-B2. These studies reveal that the preimmune immunoglobulin repertoire is highly divergent among SHR lines differing in end-organ injury susceptibility and this may modify immune mechanisms in hypertensive renal injury.
- Burrows, P. E., Gonzalez-Garay, M. L., Rasmussen, J. C., Aldrich, M. B., Guilliod, R., Maus, E. A., Fife, C. E., Kwon, S., Lapinski, P. E., King, P. D., & Sevick-Muraca, E. M. (2013). Lymphatic abnormalities are associated with RASA1 gene mutations in mouse and man. Proceedings of the National Academy of Sciences of the United States of America, 110(21), 8621-6.More infoMutations in gene RASA1 have been historically associated with capillary malformation-arteriovenous malformation, but sporadic reports of lymphatic involvement have yet to be investigated in detail. To investigate the impact of RASA1 mutations in the lymphatic system, we performed investigational near-infrared fluorescence lymphatic imaging and confirmatory radiographic lymphangiography in a Parkes-Weber syndrome (PKWS) patient with suspected RASA1 mutations and correlated the lymphatic abnormalities against that imaged in an inducible Rasa1 knockout mouse. Whole-exome sequencing (WES) analysis and validation by Sanger sequencing of DNA from the patient and unaffected biological parents enabled us to identify an early-frameshift deletion in RASA1 that was shared with the father, who possessed a capillary stain but otherwise no overt disease phenotype. Abnormal lymphatic vasculature was imaged in both affected and unaffected legs of the PKWS subject that transported injected indocyanine green dye to the inguinal lymph node and drained atypically into the abdomen and into dermal lymphocele-like vesicles on the groin. Dermal lymphatic hyperplasia and dilated vessels were observed in Rasa1-deficient mice, with subsequent development of chylous ascites. WES analyses did not identify potential gene modifiers that could explain the variability of penetrance between father and son. Nonetheless, we conclude that the RASA1 mutation is responsible for the aberrant lymphatic architecture and functional abnormalities, as visualized in the PKWS subject and in the animal model. Our unique method to combine investigatory near-infrared fluorescence lymphatic imaging and WES for accurate phenoptyping and unbiased genotyping allows the study of molecular mechanisms of lymphatic involvement of hemovascular disorders.
- Gonzalez-Garay, M. L., McGuire, A. L., Pereira, S., & Caskey, C. T. (2013). Personalized genomic disease risk of volunteers. Proceedings of the National Academy of Sciences of the United States of America, 110(42), 16957-62.More infoNext-generation sequencing (NGS) is commonly used for researching the causes of genetic disorders. However, its usefulness in clinical practice for medical diagnosis is in early development. In this report, we demonstrate the value of NGS for genetic risk assessment and evaluate the limitations and barriers for the adoption of this technology into medical practice. We performed whole exome sequencing (WES) on 81 volunteers, and for each volunteer, we requested personal medical histories, constructed a three-generation pedigree, and required their participation in a comprehensive educational program. We limited our clinical reporting to disease risks based on only rare damaging mutations and known pathogenic variations in genes previously reported to be associated with human disorders. We identified 271 recessive risk alleles (214 genes), 126 dominant risk alleles (101 genes), and 3 X-recessive risk alleles (3 genes). We linked personal disease histories with causative disease genes in 18 volunteers. Furthermore, by incorporating family histories into our genetic analyses, we identified an additional five heritable diseases. Traditional genetic counseling and disease education were provided in verbal and written reports to all volunteers. Our report demonstrates that when genome results are carefully interpreted and integrated with an individual's medical records and pedigree data, NGS is a valuable diagnostic tool for genetic disease risk.
- Network, T. C. (2013). Corrigendum: Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature, 494(7438), 506.
- Schaaf, C. P., Gonzalez-Garay, M. L., Xia, F., Potocki, L., Gripp, K. W., Zhang, B., Peters, B. A., McElwain, M. A., Drmanac, R., Beaudet, A. L., Caskey, C. T., & Yang, Y. (2013). Truncating mutations of MAGEL2 cause Prader-Willi phenotypes and autism. Nature genetics, 45(11), 1405-8.More infoPrader-Willi syndrome (PWS) is caused by the absence of paternally expressed, maternally silenced genes at 15q11-q13. We report four individuals with truncating mutations on the paternal allele of MAGEL2, a gene within the PWS domain. The first subject was ascertained by whole-genome sequencing analysis for PWS features. Three additional subjects were identified by reviewing the results of exome sequencing of 1,248 cases in a clinical laboratory. All four subjects had autism spectrum disorder (ASD), intellectual disability and a varying degree of clinical and behavioral features of PWS. These findings suggest that MAGEL2 is a new gene causing complex ASD and that MAGEL2 loss of function can contribute to several aspects of the PWS phenotype.
- Ding, L., Getz, G., Wheeler, D. A., Mardis, E. R., McLellan, M. D., Cibulskis, K., Sougnez, C., Greulich, H., Muzny, D. M., Morgan, M. B., Fulton, L., Fulton, R. S., Zhang, Q., Wendl, M. C., Lawrence, M. S., Larson, D. E., Chen, K., Dooling, D. J., Sabo, A., , Hawes, A. C., et al. (2008). Somatic mutations affect key pathways in lung adenocarcinoma. Nature, 455(7216), 1069-75.More infoDetermining the genetic basis of cancer requires comprehensive analyses of large collections of histopathologically well-classified primary tumours. Here we report the results of a collaborative study to discover somatic mutations in 188 human lung adenocarcinomas. DNA sequencing of 623 genes with known or potential relationships to cancer revealed more than 1,000 somatic mutations across the samples. Our analysis identified 26 genes that are mutated at significantly high frequencies and thus are probably involved in carcinogenesis. The frequently mutated genes include tyrosine kinases, among them the EGFR homologue ERBB4; multiple ephrin receptor genes, notably EPHA3; vascular endothelial growth factor receptor KDR; and NTRK genes. These data provide evidence of somatic mutations in primary lung adenocarcinoma for several tumour suppressor genes involved in other cancers--including NF1, APC, RB1 and ATM--and for sequence changes in PTPRD as well as the frequently deleted gene LRP1B. The observed mutational profiles correlate with clinical features, smoking status and DNA repair defects. These results are reinforced by data integration including single nucleotide polymorphism array and gene expression array. Our findings shed further light on several important signalling pathways involved in lung adenocarcinoma, and suggest new molecular targets for treatment.
- Gonzalez-Garay, M. L. (2008). Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature, 455(7216), 1061-8.More infoHuman cancer cells typically harbour multiple chromosomal aberrations, nucleotide substitutions and epigenetic modifications that drive malignant transformation. The Cancer Genome Atlas (TCGA) pilot project aims to assess the value of large-scale multi-dimensional analysis of these molecular characteristics in human cancer and to provide the data rapidly to the research community. Here we report the interim integrative analysis of DNA copy number, gene expression and DNA methylation aberrations in 206 glioblastomas--the most common type of adult brain cancer--and nucleotide sequence aberrations in 91 of the 206 glioblastomas. This analysis provides new insights into the roles of ERBB2, NF1 and TP53, uncovers frequent mutations of the phosphatidylinositol-3-OH kinase regulatory subunit gene PIK3R1, and provides a network view of the pathways altered in the development of glioblastoma. Furthermore, integration of mutation, DNA methylation and clinical treatment data reveals a link between MGMT promoter methylation and a hypermutator phenotype consequent to mismatch repair deficiency in treated glioblastomas, an observation with potential clinical implications. Together, these findings establish the feasibility and power of TCGA, demonstrating that it can rapidly expand knowledge of the molecular basis of cancer.
- , S. U., Sodergren, E., Weinstock, G. M., Davidson, E. H., Cameron, R. A., Gibbs, R. A., Angerer, R. C., Angerer, L. M., Arnone, M. I., Burgess, D. R., Burke, R. D., Coffman, J. A., Dean, M., Elphick, M. R., Ettensohn, C. A., Foltz, K. R., Hamdoun, A., Hynes, R. O., Klein, W. H., , Marzluff, W., et al. (2006). The genome of the sea urchin Strongylocentrotus purpuratus. Science (New York, N.Y.), 314(5801), 941-52.More infoWe report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes.
- Muzny, D. M., Scherer, S. E., Kaul, R., Wang, J., Yu, J., Sudbrak, R., Buhay, C. J., Chen, R., Cree, A., Ding, Y., Dugan-Rocha, S., Gill, R., Gunaratne, P., Harris, R. A., Hawes, A. C., Hernandez, J., Hodgson, A. V., Hume, J., Jackson, A., , Khan, Z. M., et al. (2006). The DNA sequence, annotation and analysis of human chromosome 3. Nature, 440(7088), 1194-8.More infoAfter the completion of a draft human genome sequence, the International Human Genome Sequencing Consortium has proceeded to finish and annotate each of the 24 chromosomes comprising the human genome. Here we describe the sequencing and analysis of human chromosome 3, one of the largest human chromosomes. Chromosome 3 comprises just four contigs, one of which currently represents the longest unbroken stretch of finished DNA sequence known so far. The chromosome is remarkable in having the lowest rate of segmental duplication in the genome. It also includes a chemokine receptor gene cluster as well as numerous loci involved in multiple human cancers such as the gene encoding FHIT, which contains the most common constitutive fragile site in the genome, FRA3B. Using genomic sequence from chimpanzee and rhesus macaque, we were able to characterize the breakpoints defining a large pericentric inversion that occurred some time after the split of Homininae from Ponginae, and propose an evolutionary history of the inversion.
- Scherer, S. E., Muzny, D. M., Buhay, C. J., Chen, R., Cree, A., Ding, Y., Dugan-Rocha, S., Gill, R., Gunaratne, P., Harris, R. A., Hawes, A. C., Hernandez, J., Hodgson, A. V., Hume, J., Jackson, A., Khan, Z. M., Kovar-Smith, C., Lewis, L. R., Lozado, R. J., , Metzker, M. L., et al. (2006). The finished DNA sequence of human chromosome 12. Nature, 440(7082), 346-51.More infoHuman chromosome 12 contains more than 1,400 coding genes and 487 loci that have been directly implicated in human disease. The q arm of chromosome 12 contains one of the largest blocks of linkage disequilibrium found in the human genome. Here we present the finished sequence of human chromosome 12, which has been finished to high quality and spans approximately 132 megabases, representing approximately 4.5% of the human genome. Alignment of the human chromosome 12 sequence across vertebrates reveals the origin of individual segments in chicken, and a unique history of rearrangement through rodent and primate lineages. The rate of base substitutions in recent evolutionary history shows an overall slowing in hominids compared with primates and rodents.
- Gibbs, R. A., Weinstock, G. M., Metzker, M. L., Muzny, D. M., Sodergren, E. J., Scherer, S., Scott, G., Steffen, D., Worley, K. C., Burch, P. E., Okwuonu, G., Hines, S., Lewis, L., DeRamo, C., Delgado, O., Dugan-Rocha, S., Miner, G., Morgan, M., Hawes, A., , Gill, R., et al. (2004). Genome sequence of the Brown Norway rat yields insights into mammalian evolution. Nature, 428(6982), 493-521.More infoThe laboratory rat (Rattus norvegicus) is an indispensable tool in experimental medicine and drug development, having made inestimable contributions to human health. We report here the genome sequence of the Brown Norway (BN) rat strain. The sequence represents a high-quality 'draft' covering over 90% of the genome. The BN rat sequence is the third complete mammalian genome to be deciphered, and three-way comparisons with the human and mouse genomes resolve details of mammalian evolution. This first comprehensive analysis includes genes and proteins and their relation to human disease, repeated sequences, comparative genome-wide studies of mammalian orthologous chromosomal regions and rearrangement breakpoints, reconstruction of ancestral karyotypes and the events leading to existing species, rates of variation, and lineage-specific and lineage-independent evolutionary events such as expansion of gene families, orthology relations and protein evolution.
- Barlow, S. B., Gonzalez-Garay, M. L., & Cabral, F. (2002). Paclitaxel-dependent mutants have severely reduced microtubule assembly and reduced tubulin synthesis. Journal of cell science, 115(Pt 17), 3469-78.More infoA subset of mutant cell lines selected for resistance to the antitumor drug paclitaxel are unable to progress normally through mitosis unless the drug is present in the growth medium. Without paclitaxel the cells form defective spindles, undergo aberrant mitoses, fail to complete cell division and eventually die. Analysis of these drug-dependent cells revealed a low amount of microtubule polymer and less tubulin production than wild-type cells. Ribonuclease protection experiments indicated that the decreased tubulin protein was due to decreased tubulin mRNA. Enhancing microtubule assembly by treating the cells with paclitaxel, restored tubulin to levels comparable with those of paclitaxel-treated wild-type cells, which demonstrated that the drug-dependent cells do not have a permanent impairment in their capacity to synthesize tubulin. Paclitaxel-resistant (but not dependent) cells have a smaller reduction in microtubule polymer with little or no decrease in tubulin production, whereas colcemidresistant cells have increased microtubule assembly but also exhibit little or no change in tubulin production. Finally, a mutant cell line producing an unstable beta-tubulin protein has normal growth as well as normal synthesis and polymerization of tubulin, despite an approximately 30% decrease in steady state tubulin content. These studies establish a lower limit of tubulin assembly needed for cell survival and indicate that tubulin assembly must fall below this point to trigger a significant decrease in tubulin synthesis.
- Gonzalez-Garay, M. L., Chang, L., Blade, K., Menick, D. R., & Cabral, F. (1999). A beta-tubulin leucine cluster involved in microtubule assembly and paclitaxel resistance. The Journal of biological chemistry, 274(34), 23875-82.More infoAnalysis of beta-tubulin alleles from nine paclitaxel-resistant Chinese hamster ovary cell lines revealed an unexpected cluster of mutations affecting Leu-215, Leu-217, and Leu-228. Six of the mutant alleles encode a His, Arg, or Phe substitution at Leu-215; another mutant allele has an Arg substitution at Leu-217; and the final two mutant alleles have substitutions of His or Phe at Leu-228. Using plasmids that allow tetracycline regulated expression, the L215H, L217R, and L228F mutations were introduced into a hemagglutinin antigen-tagged beta-tubulin cDNA and transfected into wild-type Chinese hamster ovary cells. In all three cases, low to moderate expression of the transfected mutant gene conferred paclitaxel resistance. Higher levels of expression caused disruption of microtubule assembly, cell cycle arrest at mitosis, and failure to proliferate. Consistent with reduced microtubule stability, cells expressing mutant hemagglutinin beta-tubulin had fewer acetylated microtubules than nonexpressing cells in the same population. These data, together with previous studies showing that the paclitaxel-resistant mutant cell lines have less stable microtubules, indicate that the leucine cluster represents an important structural motif for microtubule assembly.
- Yu, X. C., Margolin, W., Gonzalez-Garay, M. L., & Cabral, F. (1999). Vinblastine induces an interaction between FtsZ and tubulin in mammalian cells. Journal of cell science, 112 ( Pt 14), 2301-11.More infoThe Escherichia coli cell division protein FtsZ was expressed in Chinese hamster ovary cells, where it formed a striking array of dots that were independent of the mammalian cytoskeleton. Although FtsZ appears to be a bacterial homolog of tubulin, its expression had no detectable effects on the microtubule network or cell growth. However, treatment of the cells with vinblastine at concentrations that caused microtubule disassembly rapidly induced a network of FtsZ filaments that grew from and connected the dots, suggesting that the dots are an active storage form of FtsZ. Cells producing FtsZ also exhibited vinblastine- and calcium-resistant tubulin polymers that colocalized with the FtsZ network. The FtsZ polymers could be selectively disassembled, indicating that the two proteins were not copolymerized. The vinblastine effects were readily reversible by washing out the drug or by treating the cells with the vinblastine competitor, maytansine. These results demonstrate that FtsZ assembly can occur in the absence of bacterial chaperones or cofactors, that FtsZ and tubulin do not copolymerize, and that tubulin-vinblastine complexes have an enhanced ability to interact with FtsZ.
- Boggs, B. A., Gonzalez-Garay, M. L., & Cabral, F. (1996). Significant divergence in nucleotide sequences for beta-tubulin from different laboratory strains of Chinese hamster ovary cells. DNA sequence : the journal of DNA sequencing and mapping, 6(3), 171-4.More infoThe nucleotide sequences for isotype 1 beta-tubulin cDNAs cloned from different laboratory strains of Chinese hamster ovary (CHO) cells were compared and found to contain an unexpected number of sequence differences in both translated and untranslated regions of the gene. The results indicate significant changes in the DNA, but not protein, sequence while the cells have been in culture and reveal sequences in the 5' and 3' untranslated regions that have resisted these changes.
- Gonzalez-Garay, M. L., & Cabral, F. (1996). alpha-Tubulin limits its own synthesis: evidence for a mechanism involving translational repression. The Journal of cell biology, 135(6 Pt 1), 1525-34.More infoA Chinese hamster alpha-tubulin cDNA was modified to encode an 11-amino acid carboxyl-terminal extension containing the immunodominant epitope from influenza hemagglutinin antigen (to create HA alpha 1-tubulin) and was cloned into a vector for expression in mammalian cells. 12 stable CHO cell lines expressing this HA alpha 1-tubulin were isolated and characterized. HA alpha 1-tubulin incorporated into all classes of microtubules, assembled to the same extent as the endogenous tubulin, and did not perturb the growth of the cells in which it was expressed. However, overexpression of HA alpha 1-tubulin strongly repressed the synthesis of endogenous alpha-tubulin while having little or no effect on the synthesis of beta-tubulin. Treatment of transfected cells with sodium butyrate to induce even greater expression of HA alpha 1-tubulin led to a further decrease in synthesis of endogenous alpha-tubulin that was fully reversible upon removal of the inducer. Decreased synthesis of alpha-tubulin in transfected cells did not result from decreased levels of alpha-tubulin mRNA, as demonstrated by ribonuclease protection assays. On the other hand, colchicine, a drug previously shown to destabilize the tubulin message, caused a clear reduction in both protein synthesis and mRNA levels for transfected HA alpha 1-tubulin and endogenous alpha-tubulin, thus indicating that the decreased alpha-tubulin synthesis observed as a result of HA alpha 1-tubulin overexpression is distinct from the previously described autoregulation of tubulin. The results are consistent with a mechanism in which free alpha-tubulin inhibits the translation of its own message as a way of ensuring stoichiometric synthesis of alpha- and beta-tubulin.
- Gonzalez-Garay, M. L., & Cabral, F. (1995). Overexpression of an epitope-tagged beta-tubulin in Chinese hamster ovary cells causes an increase in endogenous alpha-tubulin synthesis. Cell motility and the cytoskeleton, 31(4), 259-72.More infoA Chinese hamster beta-tubulin cDNA, engineered to express a 9 amino acid epitope from the influenza hemagglutinin antigen (HA), was transfected into Chinese hamster ovary (CHO) cells. The recombinant protein (HA beta 1-tubulin) appeared to behave normally by the following criteria: immunofluorescence indicated that HA beta 1-tubulin incorporated into all classes of interphase and spindle microtubules as well as microtubule organizing centers. The sensitivity of the cells expressing HA beta 1-tubulin to Colcemid and taxol was unchanged. A 210 kD microtubule associated protein (MAP) remained associated with microtubules that incorporate HA beta 1-tubulin. The synthesis of both endogenous beta-tubulin and HA beta 1-tubulin was repressed by colchicine. The HA beta 1-tubulin incorporated into microtubules to the same extent as the endogenous beta-tubulin, and the overall extent of microtubule assembly in transfected cells was unchanged. Finally, transfected cells had normal growth rates and morphologies. When effects on endogenous tubulin production were measured, it was found that expression of the HA beta 1-tubulin reduced the synthesis of endogenous wild-type beta-tubulin but increased the synthesis of alpha-tubulin. At steady state, a small increase in total tubulin consistent with the increased synthesis of alpha-tubulin was found. The results indicate that expression of excess exogenous beta-tubulin perturbs the synthesis of endogenous alpha-tubulin in a manner that is not easily explained by current models of tubulin regulation. The changes in tubulin synthesis along with degradation of excess tubulin subunits may reflect mechanisms that exist to ensure coordinate levels of alpha- and beta-tubulin for assembly.
- Barlow, S., Gonzalez-Garay, M. L., West, R. R., Olmsted, J. B., & Cabral, F. (1994). Stable expression of heterologous microtubule-associated proteins (MAPs) in Chinese hamster ovary cells: evidence for differing roles of MAPs in microtubule organization. The Journal of cell biology, 126(4), 1017-29.More infoTo study the effects of microtubule-associated proteins (MAPs) on in vivo microtubule assembly, cDNAs containing the complete coding sequences of a Drosophila 205-kD heat stable MAP, human MAP 4, and human tau were stably transfected into CHO cells. Constitutive expression of the transfected genes was low in most cases and had no obvious effects on the viability of the transfected cell lines. High levels of expression, as judged by Western blots, immunofluorescence, and Northern blots, could be induced by treating cells with sodium butyrate. High levels of MAPs were maintained for at least 24-48 h after removal of the sodium butyrate. Immunofluorescence analysis indicated that all three MAPs bound to cellular microtubules, but only the transfected tau caused a rearrangement of microtubules into bundles. Despite high levels of expression of these exogenous MAPs and the bundling of microtubules in cells expressing tau, transfected cells had normal levels of assembled and unassembled tubulin. With the exception of the tau-induced bundles, microtubules in transfected cells showed the same sensitivity as control cells to microtubule depolymerization by Colcemid. Further, all three MAPs were ineffective in reversing the taxol-dependent phenotype of a CHO mutant cell line. The absence of a quantitative effect of any of these heterologous proteins on the assembly of tubulin suggests that these MAPs may have different roles in vivo from those inferred previously from in vitro experiments.
- Liu, W., Dotson, D. G., Lin, X., Mullen, J. J., Gonzalez-Garay, M. L., Lu, Q., & Putkey, J. A. (1994). The presence but not the sequence of the N-terminal peptide in cardiac TnC is important for function. FEBS letters, 347(2-3), 152-6.More infoThe most diverged region of the primary amino acid sequence between cardiac (cTnC) and fast skeletal troponin C is the N-terminal ten amino acids. We report here that major changes in the primary sequence of this region in cTnC had a minimal effect on the ability of the mutant proteins to recover maximal activity in TnC-extracted cardiac and fast skeletal muscle myofibrils. However, deletion of the N-terminal nine amino acids resulted in a 60% decrease in maximal Ca(2+)-dependent ATPase activity with only a small change in the pCa50 of activation. Deletion of the N-terminal peptide did not appear to appreciably affect the Ca(2+)-binding properties of cTnC, but it did alter the interaction with hydrophobic fluorescent probes. Thus, the presence but not the sequence, of the N-terminal extension is important for the maximal activity of cTnC. The N-terminal helix may function in a relatively non-specific manner to prevent unfavorable interactions between domains in cTnC or between cTnC and other troponin subunits.
- Barrera Saldaña, H. A., Rojas Martínez, A., Rivera Pérez, J. A., Vázquez Alemán, R. M., & González Garay, M. L. (1992). [The molecular diagnosis of hereditary diseases. In memoriam Dr. Eduardo Aguirre Pequeño]. Gaceta medica de Mexico, 128(6), 613-20; discussion 620-1.More infoAccordingly, we have established in our unit a DNA diagnosis laboratory and have started molecular genetics and epidemiological studies of several inherited diseases. We have started with cystic fibrosis, muscular dystrophy and hemophilia A. We practice the molecular diagnosis with both, Southern transfer and the polymerase chain reaction, using either direct (detection of mutations) or indirect (restriction fragment length polymorphisms) approaches. With the studies we have so far carried out, we have been able to provide genetic counseling and gained valuable information on the type and frequency of mutation associated to these diseases in our region.
- González-Garay, M. L., Barrera-Saldaña, H. A., Avilés, L. B., Alvarez-Salas, L. M., & Gariglio, P. (1992). Prevalence in two mexican cities of human papillomavirus DNA sequences in cervical cancer. Revista de investigacion clinica; organo del Hospital de Enfermedades de la Nutricion, 44(4), 491-9.More infoIn Mexico, about 30% of all malignant tumors in women are uterine cervix carcinomas. It is one of their main causes of death. We have previously shown that in Mexico City, 31% (5/16) of the analyzed tumoral samples contained HPV-16 DNA sequences. We have now extended this observation in Mexico City and included the city of Monterrey and found that the prevalence of HPV-16 is similar in both: 26% (6/23) for Monterrey and 29% (4/14) for Mexico City. HPV-18 was detected in only 10% (1/10) and 7% (1/14) of the tumors in these two populations when assayed with an HPV-18 specific probe. In both cities, the majority of the samples analyzed (including samples from the four stages of severity of the disease) contained integrated papillomavirus DNA sequences. Our results suggest that the mexican population contains a rather low proportion of HPV-16 and HPV-18 sequences in uterine-cervix carcinoma.