1997 — 2014 |
Layman, Lawrence C |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R29Activity Code Description: Undocumented code - click on the grant title for more information. |
Genetics of Delayed Puberty @ Georgia Regents University
DESCRIPTION (provided by applicant): Reproductive dysfunction manifested as infertility, early embryonic loss, or congenital anomalies afflicts 10- 15% of reproductive aged individuals and results in high economic and psychological costs. Many infertile males and females display impaired function of the hypothalamic-pituitary-gonadal (HPG) axis, but the precise etiology is often not known. Coordination of the HPG axis is dependent upon appropriate migration and development of hypothalamic gonadotropin releasing hormone (GnRH) neurons followed by proper GnRH synthesis and pulsatile release. Idiopathic hypogonadotropic hypogonadism (IHH) represents a naturally occurring human model of impaired GnRH action which provides insight into hypothalamic-pituitary function in reproduction. Kallmann syndrome (KS), a specific subtype of IHH, includes anosmia, as well as other neurologic deficits and congenital anomalies. Although mutations in KAL1, FGFR1, and GNRHR genes account for 20-30% of cases and digenic disease has been reported in a few families, the molecular basis for most IHH/KS cases is not well understood. Recently, we identified mutations in the chromodomain helicase DNA binding protein-7 (CHD7) and nasal embryonic LHRH factor (NELF) genes in sporadic IHH/KS patients. However, the precise phenotypic findings and segregation patterns within families for either of these genes are not known. Our overlying hypothesis is that CHD7 and NELF mutations cause autosomal dominant IHH/KS by the disruption of GnRH and olfactory neuron migration and/or by impaired expression of reproductive genes within the hypothalamus. We will test this hypothesis with the following Specific Aims: Aim 1) We will test the hypothesis that NELF or CHD7 mutations cause autosomal dominant IHH/KS. We will genotype individuals from families with mutations in either of these genes to characterize the segregation patterns and define the phenotype. To exclude digenic disease, affected individuals will be genotyped for mutations in more than one gene. Aim 2) We will test the hypothesis that both CHD7 and NELF have: A) an early embryologic function to regulate GnRH and olfactory neuron migration and; B) overexpression of human mutations will interfere with GnRH and olfactory neuron migration. In Sub Aim 2A, we will knockdown NELF and CHD7 expression in migratory neurons (GnRH neuronal cells and human olfactory neuroblasts) and chd7 expression in zebrafish to determine the effect upon neuron migration and the expression of key genes involved in neuron migration. In Sub Aim 2B, we will determine if overexpression of human NELF and CHD7 mutations in these neuronal cell lines and zebrafish affects migration and the expression of key genes involved in neuron migration. Demonstration that NELF and CHD7 mutations cause autosomal dominant disease will directly lead to improved genetic counseling. Characterization of the basic function of NELF and CHD7 will provide novel information related to the developmental control of GnRH neuron migration and reproduction. Our findings also have the potential to define more common, but less severe causes of IHH/KS and other forms of infertility.
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1 |
2002 — 2005 |
Layman, Lawrence C |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core--Bioassay @ Medical College of Georgia (McG)
DESCRIPTION (provided by applicant): The primary role of the Bioassay Core is to provide services related to biochemical, genotyping and microarray analyses for the four proposed projects. It will serve all projects in very different but complementary roles. All projects will use the Biochemical Assay services which include various biochemical assays from plasma and urine for the SNS (projects 1-3), RAAS (projects 1-3), and ES (projects 1-4). Projects 1-3 will use Genotyping Assay services for polymorphisms related to candidate genes that have been associated with BP or EH via the SNS (projects 1-3), RAAS (projects 1 and 2) and ES (projects 1 and 3). Project 4 will use the Microarray Assay services related to the gene expression analyses using DNA microarray technology. This will allow the study of gene-gene interactions, the characterization of signaling and regulatory pathways, as well as the identification of new candidate genes that can be added to projects 1-3 in future applications. Dr. Lawrence Layman will be the Core B Leader...Dr. Jennifer Pollock will be a Core co-Leader...Dr. Anita Kulharya will also be a Core co-Leader...
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0.925 |
2002 — 2006 |
Layman, Lawrence C |
K24Activity Code Description: To provide support for the clinicians to allow them protected time to devote to patient-oriented research and to act as mentors for beginning clinical investigators. |
Genotype /Phenotype Correlations in Infertility @ Medical College of Georgia (McG)
DESCRIPTION (provided by applicant): Although infertility affects 10-15% of all individuals attempting to have children, little is known about the molecular basis of human puberty and fertility. The long-term goal of this laboratory is to elucidate the mechanisms underlying the development of normal puberty and reproductive capability by utilizing patients with infertility who possess gene mutations. The applicant will study two groups of infertile patients: those with idiopathic hypogonadotropic hypogonadism (LHH) and those with normal puberty who have ovulation disorders or sperm abnormalities. Patients with IHH constitute a severe reproductive-deficient phenotype with absent puberty, low serum gonadotropins, and infertility. Most infertility patients have normal puberty, and constitute men with sperm abnormalities (azoospermia, oligospermia. and/or asthenospermia) or women with ovulation disorders. The applicant?s overlying hypothesis is that identification of the genetic mutations in these groups will lead to a better understanding of: 1) which forms of IHH are hereditary; 2) whether FSH is necessary for normal sperm concentration and fertility in men, follicular development beyond the antral stage in women, and for normal androgens in both men and women; and 3) whether gene mutations affect the function of the encoded proteins. These hypotheses will be addressed by the following specific aims: Specific Aim 1: To test candidate genes for linkage and/or mutations in IHH patients; Specific Aim 2: To screen infertility patients for FSH beta mutations, specifically those with abnormal semen analyses and those with ovulation disorders, likely to possess FSH-beta mutations; Specific Aim 3: To create the mutants, express them in vitro, and determine their effects upon the encoded protein. The elucidation and analysis of gene mutations in infertile patients will be important to determine the genetic basis of some forms of infertility and to determine the underlying mechanisms of puberty and reproduction.
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0.925 |
2003 — 2007 |
Layman, Lawrence C |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Genetics of Delayed Puberty. @ Medical College of Georgia (McG)
DESCRIPTION (provided by applicant): The interaction of hypothalamic gonadotropin releasing hormone (GnRH) with pituitary gonadotrophs to produce follicle stimulating hormone (FSH) and luteinizing hormone (LH) represents the principle orchestrating event of normal gonadal function and reproduction in mammals. When gonadotropins are deficient, absent puberty and infertility result in the clinical disorder idiopathic hypogonadotropic hypogonadism (IHH). Although initially only mutations in the X-linked KAL1 and NROB 1 (AHC) genes were identified in IHH patients, studies from our laboratory were instrumental in documenting mutations in the GNRHR gene, the first autosomal gene shown to be causative in IHH. We have also described mutations in the FSHB gene in families with isolated FSH deficiency, and were the first to perform functional studies demonstrating that the FSHB mutants impaired FSH production in vitro. In addition, we presented evidence suggesting that FSH also plays an important role in LH-mediated ovarian androgen production. Although KAL1 and GNRHR comprise about 20% of the mutations described to date, the molecular basis for most IHH patients remains unknown. The long-term goal of our laboratory is to advance our understanding of normal puberty and reproduction by determining the molecular basis of IHH. Our overlying hypothesis is that the genes that regulate gonadotropin production and/or secretion are important in normal puberty and possess mutations in IHH patients. We will test these hypotheses by the following specific aims: Specific Aim 1: We hypothesize that chromosomal abnormalities, such as translocations and inversions, identified in IHH patients will provide clues to the location of causative genes etiologic in the pathophysiology of IHH. We will identify the locations of possible candidate genes by determining the prevalence of chromosomal abnormalities in IHH patients. Candidate genes located in the breakpoint regions will then be identified. Specific Aim 2: We hypothesize that genes suspected to regulate GnRH and/or gonadotropin function will possess mutations in IHH patients and not controls. We will test candidate genes, determined by their possible effects upon GnRH and gonadotropin function or by their location in chromosomal breakpoints regions, for association and for mutations in patients with IHH vs. fertile controls. Specific Aim 3: We hypothesize that gene mutations causing IHH will decrease the expression of the transcript or the amount or function of the encoded protein. We will create the mutant genes, express them in appropriate cell lines in vitro, and determine their effects upon transcription and protein function. Specific Aim 4: We hypothesize that patients with more severe mutations (i.e., nonsense mutations and deletions) will have more severe disease than those with less severe mutations. We also hypothesize that the severity of the phenotype may vary for different causative genes. We will perform clinical endocrinologic studies on patients with gene mutations in order to make genotype/phenotype correlations.
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0.925 |
2018 — 2021 |
Layman, Lawrence C |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Genetics of Mullerian Development
ABSTRACT Uterovaginal developmental abnormalities occur in ~7-10% of women and impair reproductive function. Congenital absence of the uterus/vagina, known as Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome, represents the most drastic Mullerian anomaly. Other problems such as unilateral renal agenesis, skeletal, cardiac, deafness, and emotional stress may occur in MRKH. There is a genetic predisposition, and autosomal dominant (AD) and genetic heterogeneity are likely. Many candidate chromosomal regions and genes have been studied, but there is only convincing evidence for mutations in 2 genes (WNT4 and HNF1B) as determined by in vitro analyses and family studies. A critical barrier to diagnose and counsel MRKH women is the lack of understanding of its pathogenesis. We found a new gene?ZNHIT3?by whole exome sequencing (WES), supported by a de novo heterozygous frameshift mutation, two intragenic exon 5 deletions, three whole gene deletions, and two 17q12 deletions including ZNHIT3. ZNHIT3 is zinc finger protein important for post- transcriptional modification, which we showed is expressed in the human uterus, kidney, and heart. Our goal is to understand the molecular basis and pathogenesis of urogenital anomalies by identifying genetic causes of MRKH. Genes and mechanisms identified as a result of this proposal support our translational strategy to identify and understand pathways important in human Mullerian development. Our central hypothesis is that MRKH is AD, and is caused by intragenic mutations and copy number variants (CNVs). Our expected outcomes include the identification of genes causing MRKH and associated anomalies. The impact of our findings include: providing the basis for improved diagnosis, genetic counseling, and prevention of MRKH; and increasing our understanding of normal Mullerian developmental pathways, which are relevant to more common, less severe anomalies. Aim 1: We hypothesize that point mutations, splicing mutations, and CNVs will be present in ZNHIT3, perhaps a hotspot for CNVs and rearrangements. We will perform RT-PCR and Sanger DNA sequencing on MRKH patient lymphoblast RNA to identify multiple types of mutations in one assay. We will determine in vitro effects of human ZNHIT3 variants and generate a Znhit3 knockout mouse model. Aim 2: To test the hypothesis that MRKH is AD, we will perform WES on 43 quads and 26 trios and filter with standard methods, and use Cartagenia, PhenoDB, and pVAAST to determine MRKH inheritance. We will perform whole genome sequencing (WGS) to identify variants that would be missed by WES in well characterized quad families. Identified variants will be confirmed by Sanger sequencing. Aim 3: Perform a comprehensive analysis of MRKH families to determine the frequency and spectrum of variants in known and suspected MRKH genes by RT-PCR from lymphoblast RNA and Sanger sequencing. Our strong preliminary data, large family cohort, innovative approach, and interdisciplinary research team will instigate significant advances in human Mullerian development as outlined by the Fertility & Infertility Branch of the NICHD.
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0.957 |