A NEW TUMOR SUPPRESSOR GENE CANDIDATE REGULATED BY THE NON-CODING RNA PCA3 IN HUMAN PROSTATE CANCER

Prostate cancer is the second leading cause of cancer-related death and the most common non-skin cancer in men in the USA. Considerable advancements in the practice of medicine have allowed a significant improvement in the diagnosis and treatment of this disease and, in recent years, both incidence and mortality rates have been slightly declining. However, it is still estimated that 1 man in 6 will be diagnosed with prostate cancer during his lifetime, and 1 man in 35 will die of the disease. In order to identify novel strategies and effective therapeutic approaches in the fight against prostate cancer, it is imperative to improve our understanding of its complex biology since many aspects of prostate cancer initiation and progression still remain elusive. The study of tumor biomarkers, due to their specific altered expression in tumor versus normal tissue, is a valid tool for elucidating key aspects of cancer biology, and may provide important insights into the molecular mechanisms underlining the tumorigenesis process of prostate cancer. PCA3, is considered the most specific prostate cancer biomarker, however its biological role, until now, remained unknown. PCA3 is a long non-coding RNA (ncRNA) expressed from chromosome 9q21 and its study led us to the discovery of a novel human gene, PC-TSGC, transcribed from the opposite strand and in an antisense orientation to PCA3. With the work presented in this thesis, we demonstrate that PCA3 exerts a negative regulatory role over PC-TSGC, and we propose PC-TSGC to be a new tumor suppressor gene that contrasts the transformation of prostate cells by inhibiting Rho-GTPases signaling pathways. Our findings provide a biological role for PCA3 in prostate cancer and suggest a new mechanism of tumor suppressor gene inactivation mediated by non-coding RNA. Also, the characterization of PCA3 and PC-TSGC led us to propose a new molecular pathway involving both genes in the transformation process of the prostate, thus providing a new piece of the jigsaw puzzle representing the complex biology of prostate cancer.

Analysis of Variation in Clubfoot Candidate Genes

Isolated clubfoot, a common birth defect occurring in more than 135,000 livebirths worldwide each year, is associated with significant health care and financial burdens. Clubfoot is defined by forefoot adduction, hindfoot varus, midfoot cavus and hindfoot equinus. Isolated clubfoot, which is the focus of these studies, is distinct from syndromic clubfoot because there are no other associated malformations. Population, family, twin and segregation analysis studies provide evidence that genetic and environmental factors play an etiologic role in isolated clubfoot. The studies described in this thesis were performed to define the role of genetic variation in isolated clubfoot. Interrogation of a deletion region associated with syndromic clubfoot, suggested that CASP8 and CASP10, two apoptotic genes, play a role in isolated clubfoot. To explore the role of apoptotic genes in clubfoot, SNPs spanning genes involved in the apoptotic pathway in the six chromosomal deletion regions, and limb patterning genes, HOXD and HOXA, were interrogated. SNPs in mitochondrial mediated apoptotic genes and several SNPs in HOXA and HOXD genes were modestly associated with clubfoot with the most significant SNP, rs3801776, located in the basal promoter of HOXA9. Several significant associations were found with SNPs in NFAT2 and TNIP2. Significant gene interactions were detected between SNPs in HOX and apoptotic genes. These findings suggest a model for clubfoot in which variation in one gene is not sufficient to cause the malformation but requires variation several genes to perturb protein expression sufficiently to alter muscle and foot development. These results significantly impact our knowledge base by delineating underlying mechanisms causing clubfoot.

Serum IL-6: a candidate biomarker for intracranial pressure elevation following isolated traumatic brain injury.

BACKGROUND: Increased intracranial pressure (ICP) is a serious, life-threatening, secondary event following traumatic brain injury (TBI). In many cases, ICP rises in a delayed fashion, reaching a maximal level 48-96 hours after the initial insult. While pressure catheters can be implanted to monitor ICP, there is no clinically proven method for determining a patient's risk for developing this pathology. METHODS: In the present study, we employed antibody array and Luminex-based screening methods to interrogate the levels of inflammatory cytokines in the serum of healthy volunteers and in severe TBI patients (GCS RESULTS: Consistent with previous reports, we observed sustained increases in IL-6 levels in TBI patients irrespective of their ICP status. However, the group of patients who subsequently experienced ICP >or= 25 mm Hg had significantly higher IL-6 levels within the first 17 hours of injury as compared to the patients whose ICP remained 128 pg/ml correctly identified 85% of isolated TBI patients who subsequently developed elevated ICP, and values between these cut-off values correctly identified 75% of all patients whose ICP remained CONCLUSIONS: Our results suggest that serum IL-6 can be used for the differential diagnosis of elevated ICP in isolated TBI.

myogenin: Human candidate gene and knockout mouse effect

The myogenin gene encodes an evolutionarily conserved basic helix-loop-helix transcription factor that regulates the expression of skeletal muscle-specific genes and its homozygous deletion results in mice who die of respiratory failure at birth. The histology of skeletal muscle in the myogenin null mice is reminiscent of that found in some severe congenital myopathy patients, many of whom also die of respiratory complications and provides the rationale that an aberrant human myogenin (myf4) coding region could be associated with some congenital myopathy conditions.^ With PCR, we found similarly sized amplimers for the three exons of the myogenin gene in 37 patient and 40 control samples. In contrast to the GeneBank sequence for human myogenin, we report several differences in flanking and coding regions plus an additional 659 and 498 bps in the first and second introns, respectively, in all patients and controls. We also find a novel (CA)-dinucleotide repeat in the second intron. No causative mutations were detected in the myogenin coding regions of genomic DNA from patients with severe congenital myopathy.^ Severe congenital myopathies in humans are often associated with respiratory complications and pulmonary hypoplasia. We have employed the myogenin null mouse, which lacks normal development of skeletal muscle fibers as a genetically defined severe congenital myopathy mouse model to evaluate the effect of absent fetal breathing movement on pulmonary development.^ Significant differences are observed at embryonic days E14, E17 and E20 of lung:body weight, total DNA and histologically, suggesting that the myogenin null lungs are hypoplastic. RT-PCR, in-situ immunofluorescence and EM reveal pneumocyte type II differentiation in both null and wild lungs as early as E14. However, at E14, myogenin null lungs have decreased BrdU incorporation while E17 through term, augmented cell death is detected in the myogenin null lungs, not seen in wild littermates. Absent mechanical forces appear to impair normal growth, but not maturation, of the developing lungs in myogenin null mouse.^ These investigations provide the basis for delineating the DNA sequence of the myogenin gene and and highlight the importance of skeletal muscle development in utero for normal lung organogenesis. My observation of no mutations within the coding regions of the human myogenin gene in DNA from patients with severe congenital myopathy do not support any association with this condition. ^

Linkage and association of candidate obesity genes in Mexican-Americans from Starr County, Texas

Obesity and related chronic diseases represent a tremendous public health burden among Mexican Americans, a young and rapidly-expanding population. This study investigated the impact of variation within eight candidate obesity genes, which include leptin (LEP), leptin receptor (LEPR), neuropeptide Y (NPY), NPYY1 receptor (NPYY1), glucagon-like peptide-1 (GLP-1), GLP-1 receptor (GLP1R), beta-3 adrenergic receptor (β3AR), and uncoupling protein (UCP1), on variation in human obesity status and/or quantitative traits related to obesity in Mexican Americans from Starr County, Texas. The Trp64Arg polymorphism within β3AR was typed in 820 random individuals and 240 pedigrees (N = 2,044). The Arg allele frequency was significantly greater in obese versus non-obese individuals (0.20 versus 0. 15, respectively). In addition, within the random sample, the Arg allele was associated with significantly greater body weight (p = 0.031) and body mass index (BMI, p = 0.008) than the Trp allele. In the family sample, the Trp64Arg locus was also linked to percent fat (p = 0.045) but not to body weight or BMI. No linkage between obesity, diabetes, hypertension, or gallbladder disease and the Trp64Arg mutation was observed in families using affected sib pair linkage analysis or the transmission disequilibrium test. Microsatellite markers proximate to the remaining seven genes were typed in 302 individuals from 59 families. Sib pair linkage analysis provided evidence for linkage between obesity and NPY within affected sibling pairs (p = 0.042; n = 170 pairs). NPY was also linked to weight (p = 0.020), abdominal circumference (p = 0.031), hip circumference (p = 0.012), DBP (p ≤ 0.005), and a composite measure of body mass/fat (p ≤ 0.048) in all sibling pairs (n = 545 pairs). Additionally, LEP was linked to waist/hip ratio (p ≤ 0.009), total cholesterol (p ≤ 0.030), and HDL cholesterol (p ≤ 0.026), and LEPR was linked to fasting blood glucose (p ≤ 0.018) and DBP (p ≤ 0.003). Subsequent to the linkage analyses, the NPY gene was sequenced and eight variant sites identified. Two variant sites (-880I/D and 69I/D) were typed in a random sample of 914 individuals. The 880I/D variant was significantly associated with waist/hip ratio (p = 0.035) in the entire sample (N = 914) and with BMI (p = 0. 031), abdominal circumference (p = 0.044), and waist/hip ratio (p = 0.041) in a non-obese subsample (BW < 30 kg/m2, n = 594). The 69I/D variant was a rare mutation observed in only one pedigree and was not associated with obesity or body size/mass within this pedigree. Results of this study indicate that variation at or near β3AR, LEP, LEPR, and NPY may exert effects which increase obesity susceptibility and influence obesity-related measures in this population. ^

The localization and identification of candidate tumor suppressor genes involved in prostate cancer

Frequent loss of heterozygosity (LOH) at specific chromosomal regions are highly associated with the inactivation of tumor suppressor genes (TSGs) (Weinberg, 1991; Bishop, 1989). Chromosome 8p is the most frequently reported site of LOH (∼60%) in prostate cancer (PC), suggesting that there may be inactivated TSG(s) involved in PC on chromosome 8p. (Bergerheim et. al., 1991; Kagan et. al., 1995). In order to identify the smallest common regions of frequent LOH (SCLs) on chromosome 8, we screened 52 PC patient/tumor samples with 39 polymorphic markers in successive screenings. In the course of refining the SCLs, we identified 3 tumors with >6 Mb homozygous deletions (HZDs) at 8p22 and 8p21, suggesting the presence of candidate TSGs at both loci. These HZDs spanned the two SCLs at 8p22 (46%) and 8p21 (45%). The SCLs were narrowed to 3.2 cM at 8p22 and less than 3 cM at 8p21. ^ In order to identify candidate TSGs within the SCLs on 8p, two approaches were used. In the candidate gene approach, thirty genes that mapped to the SCLs were evaluated for expression in normal prostate and in PC cell lines. One of the candidate genes, Clusterin, showed decreased expression in 4/7 (57%) prostate cancer cell lines by Northern blot analysis. Clusterin will be further examined as a candidate TSG. ^ The second approach involved utilizing subtractive hybridization and hybrid affinity capture to generate pools of expressed sequence tags (ESTs) enriched for genes that are downregulated or deleted in PC and that map to specific regions of interest. We took advantage of a prostate cancer cell line (PC3) with a known HZD of a candidate TSG, CTNNA1 on 5q31, to develop and validate a model system. We then developed subtracted libraries enriched for 8p22 and 8p21 ESTs by this method, using two cell lines, MDAPCa-2b and PC3. The ESTs were cloned, and 40 were sequenced and evaluated for expression in normal prostate and PC cell lines. Three ESTs from the subtracted libraries, C2, C17 and F12, showed decreased expression in 29–57% of the prostate tumor cell lines studied, and will be further examined as candidate TSGs. ^

Expression cloning of a candidate prostate cancer oncogene

Genetic analysis, both karyotyping and comparative genomic hybridization, of prostate cancer cell lines and specimens have revealed multiple areas of concordant increases in DNA content. An increase of DNA in specific regions of the genome in cancer is often associated with the amplification of oncogenes. Based on these observations we have hypothesized that oncogenes are involved in the initiation or progression of prostate cancer. An expression cloning approach was utilized to identify candidate oncogenes in prostate cancer. ^ A full-length, unidirectional cDNA expression library was constructed from DU145 prostate cancer cells. The cDNA library was screened using CP12, a rat prostate epithelial cell line. In soft agarose assays, CP12 (parental or vector transfected) do not form colonies. However, upon the introduction of a number of known oncogenes CP12 becomes anchorage independent in soft agarose. Based on this in-vitro phenotypic shift, a DU145 cDNA library was stably transfected into CP12, and selected for anchorage independence. Two hundred fifty nine anchorage independent clones were isolated. Some colonies contained more than one insert, bringing the candidate oncogene pool to approximately 400. Seven inserts were sequenced at random. Using the sequences obtained, GenBank was screened, and matches were found with p53, PARG1, a mitochondrial ATPase, RNF6, and three unknown genes that mapped to Unigene clusters. As the pool of cDNA inserts appeared promising, overexpressed genes were further selected. From 259 clones, 17 clones were overexpressed more than 6-fold in DU145 compared to Normal Prostate. From the 17 clones, 12 cDNA inserts were strongly expressed in DU145 and were isolated for sequencing. ^ Two of the sequences, 1G6 and 3E9, were identical. Expression of 1G6/2G9/3E9 was tested by RT-PCR. 1G6/2G9/3E9 was not expressed in normal prostate, but was expressed in all prostate cancer cell lines tested as well as six prostate cancer samples. When retransfected into CP12, 1G6/2G9/3E9 induced the formation of foci and anchorage independent colonies. Thus, functional and expression data suggest that 1G6/2G9/3E9 may be a prostate cancer oncogene. ^

Positional candidate cloning of the RP10 form of retinitis pigmentosa

Retinitis pigmentosa (RP) is a genetically heterogeneous group of retinal degenerations that affects over one million people worldwide. To date, 11 autosomal dominant, 13 autosomal recessive, and 5 X-linked forms of retinitis pigmentosa have been identified through linkage analysis, but the disease-causing genes and mutations have been found for only half of these loci. My research uses a positional candidate cloning approach to identify the gene and mutations responsible for one type of autosomal dominant retinitis pigmentosa, RP10. The premise is that identifying the genes and mutations responsible for disease will provide insight into disease mechanisms and provide treatment options. Previous research mapped the RP10 locus to a 5cM region on chromosome 7q31 between markers D7S686 and D7S530. Linkage and fine-point haplotype analysis was used to reduce and refine the RP10 disease interval to a 4cM region located between D7S2471 and a new marker located 45,000bp telomeric of D7S461. In order to identify genes located in the RP10 interval, an extensive EST map was created of this region. Five EST clusters from this map were analyzed to determine if mutations in these genes cause the RP10 form of retinitis pigmentosa. The genomic structure of a known metabotrophic glutamate receptor, GRMS8, was determined first. DNA sequencing of GRM8 in RP10 family members did not identify any disease-causing mutations. Four other EST clusters (A170, A173, A189, and A258) were characterized and determined to be part of the same gene, UBNL1 (ubinuclein-like 1). The full-length mRNA sequence and genomic structure of UBNL1 was determined and then screened in patients. No disease-causing mutations were identified in any of the RP10 family members tested. Recent data made available with the release of the public and Celera genome assemblies indicates that UBNL1 is outside of the RP10 disease region. Despite this complication, characterization of UBNL1 is still important in the understanding of normal visual processes and it is possible that mutations in UBNL1 could cause other forms of retinopathy. The EST map and list of RP10 candidates will continue to aid others in the search for the RP10 gene and mutations. ^

Evaluation of candidate genes for susceptibility to spina bifida

Neural tube defects (NTDs) are the most common severely disabling birth defects in the United States, with a frequency of approximately 1–2 of every 1,000 births. This text includes the identification and evaluation of candidate susceptibility genes that confer risk for the development of neural tube defects (NTDs). The project focused on isolated meningomyelocele, also termed spina bifida (SB). ^ Spina bifida is a complex disease with multifactorial inheritance, therefore the subject population (consisting of North American Caucasians and Hispanics of Mexicali-American descent) was composed of 459 simplex SB families who were tested for genetic associations utilizing the transmission disequilibrium test (TDT), a nonparametric linkage technique. Three categories of candidate genes were studied, including (1) human equivalents of genes determined in mouse models to cause NTDs, (2) HOX and PAX genes, and (3) the MTHFR gene involved in the metabolic pathway of folate. ^ The C677T variant of the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene was the first mutation in this gene to be implicated as a risk factor for NTDs. Our evaluation of the MTHFR gene provides evidence that maternal C677T homozygosity is a risk factor for upper level spina bifida defects in Hispanics [OR = 2.3, P = 0.02]. This observed risk factor is of great importance due to the high prevalence of this homozygous genotype in the Hispanic population. Additionally, maternal C677T/A1298C compound heterozygosity is a risk factor for upper level spina bifida defects in non-Hispanic whites [OR = 3.6, P = 0.03]. ^ For TDT analysis, our total population of 1128 subjects were genotyped for 54 markers from within and/or flanking the 20 candidate genes/gene regions of interest. Significant TDT findings were obtained for 3 of the 54 analyzed markers: d20s101 flanking the PAX1 gene (P = 0.019), d1s228 within the PAX7 gene (P = 0.011), and d2s110 within the PAX8 gene (P = 0.013). These results were followed-up by testing the genes directly for mutations utilizing single-strand conformational analysis (SSCA) and direct sequencing. Multiple variations were detected in each of these PAX genes; however, these variations were not passed from parent to child in phase with the positively transmitted alleles. Therefore, these variations do not contribute to the susceptibility of spina bifida, but rather are previously unreported single nucleotide polymorphisms. ^

Familial aggregation, candidate genes and genome scans: Analyzing the role of genetics in stroke

The genetic etiology of stroke likely reflects the influence of multiple loci with small effects, each modulating different pathophysiological processes. This research project utilized three analytical strategies to address the paucity of information related to the identification and characterization of genetic variation associated with stroke in the general population. ^ First, the general contribution of familial factors to stroke susceptibility was evaluated in a population-based sample of unrelated individuals. Increased risk of subclinical cerebral infarction was observed among individuals with a positive parental history of stroke. This association did not appear to be mediated by established stroke risk factors, specifically blood pressure levels or hypertension status. ^ The need to identify specific gene variation associated with stroke in the general population was addressed by evaluating seven candidate gene polymorphisms in a population-based sample of unrelated individuals. Three polymorphisms were significantly associated with increased subclinical cerebral infarction or incident clinical ischemic stroke risk. These relationships include the G-protein β3 subunit 825C/T polymorphism and clinical stroke in Whites, the lipoprotein lipase S/X447 polymorphism and subclinical and clinical stroke in men, and the angiotensin I-converting enzyme Ins/Del polymorphism and subclinical stroke in White men. These associations did not appear to be obfuscated by the stroke risk factors adjusted for in the analysis models specifically blood pressure levels or anti-hypertensive medication use. ^ The final research strategy considered, on a genome-wide scale, the idea that genetic variation may contribute to the occurrence of hypertension or stroke through a common etiologic pathway. Genomic regions were identified for which significant evidence of heterogeneity was observed among hypertensive sibpairs stratified by family history of stroke information. Regions identified on chromosome 15 in African Americans, and chromosome 13 in Whites and African Americans, suggest the presence of genes influencing hypertension and stroke susceptibility. ^ Insight into the role of genetics in stroke is useful for the potential early identification of individuals at increased risk for stroke and improved understanding of the etiology of the disease. The ultimate goal of these endeavors is to guide the development of therapeutic intervention and informed prevention to provide a lasting and positive impact on public health. ^

Towards the identification of a tumor suppressor gene on chromosome 3p12: Physical mapping and candidate gene screening

Renal cell carcinoma (RCC) is the most common malignant tumor of the kidney. Characterization of RCC tumors indicates that the most frequent genetic event associated with the initiation of tumor formation involves a loss of heterozygosity or cytogenetic aberration on the short arm of human chromosome 3. A tumor suppressor locus Nonpapillary Renal Carcinoma-1 (NRC-1, OMIM ID 604442) has been previously mapped to a 5–7 cM region on chromosome 3p12 and shown to induce rapid tumor cell death in vivo, as demonstrated by functional complementation experiments. ^ To identify the gene that accounts for the tumor suppressor activities of NRC-1, fine-scale physical mapping was conducted with a novel real-time quantitative PCR based method developed in this study. As a result, NRC-1 was mapped within a 4.6-Mb region defined by two unique sequences within UniGene clusters Hs.41407 and Hs.371835 (78,545Kb–83,172Kb in the NCBI build 31 physical map). The involvement of a putative tumor suppressor gene Robo1/Dutt1 was excluded as a candidate for NRC-1. Furthermore, a transcript map containing eleven candidate genes was established for the 4.6-Mb region. Analyses of gene expression patterns with real-time quantitative RT-PCR assays showed that one of the eleven candidate genes in the interval (TSGc28) is down-regulated in 15 out of 20 tumor samples compared with matched normal samples. Three exons of this gene have been identified by RACE experiments, although additional exon(s) seem to exist. Further gene characterization and functional studies are required to confirm the gene as a true tumor suppressor gene. ^ To study the cellular functions of NRC-1, gene expression profiles of three tumor suppressive microcell hybrids, each containing a functional copy of NRC-1, were compared with those of the corresponding parental tumor cell lines using 16K oligonucleotide microarrays. Differentially expressed genes were identified. Analyses based on the Gene Ontology showed that introduction of NRC-1 into tumor cell lines activates genes in multiple cellular pathways, including cell cycle, signal transduction, cytokines and stress response. NRC-1 is likely to induce cell growth arrest indirectly through WEE1. ^

Candidate gene identification following linkage: Search for hypertension susceptibility genes

Following up genetic linkage studies to identify the underlying susceptibility gene(s) for complex disease traits is an arduous yet biologically and clinically important task. Complex traits, such as hypertension, are considered polygenic with many genes influencing risk, each with small effects. Chromosome 2 has been consistently identified as a genomic region with genetic linkage evidence suggesting that one or more loci contribute to blood pressure levels and hypertension status. Using combined positional candidate gene methods, the Family Blood Pressure Program has concentrated efforts in investigating this region of chromosome 2 in an effort to identify underlying candidate hypertension susceptibility gene(s). Initial informatics efforts identified the boundaries of the region and the known genes within it. A total of 82 polymorphic sites in eight positional candidate genes were genotyped in a large hypothesis-generating sample consisting of 1640 African Americans, 1339 whites, and 1616 Mexican Americans. To adjust for multiple comparisons, resampling-based false discovery adjustment was applied, extending traditional resampling methods to sibship samples. Following this adjustment for multiple comparisons, SLC4A5, a sodium bicarbonate transporter, was identified as a primary candidate gene for hypertension. Polymorphisms in SLC4A5 were subsequently genotyped and analyzed for validation in two populations of African Americans (N = 461; N = 778) and two of whites (N = 550; N = 967). Again, SNPs within SLC4A5 were significantly associated with blood pressure levels and hypertension status. While not identifying a single causal DNA sequence variation that is significantly associated with blood pressure levels and hypertension status across all samples, the results further implicate SLC4A5 as a candidate hypertension susceptibility gene, validating previous evidence for one or more genes on chromosome 2 that influence hypertension related phenotypes in the population-at-large. The methodology and results reported provide a case study of one approach for following up the results of genetic linkage analyses to identify genes influencing complex traits. ^

An Sp1/Sp3 site polymorphism associated with hypermethylation of the candidate tumor suppressor gene RIL in cancer

Gene silencing due to promoter methylation is an alternative to mutations and deletions, which inactivate tumor suppressor genes (TSG) in cancer. We identified RIL by Methylated CpG Island Amplification technique as a novel aberrantly methylated gene. RIL is expressed in normal tissues and maps to the 5q31 region, frequently deleted in leukemias. We found methylation of RIL in 55/80 (69%) cancer cell lines, with highest methylation in leukemia and colon. We also observed methylation in 46/80 (58%) primary tumors, whereas normal tissues showed substantially lower degrees of methylation. RIL expression was lost in 13/16 cancer cell lines and was restored by demethylating agent. Screening of 38 cell lines and 13 primary cancers by SSCP revealed no mutations in RIL, suggesting that methylation and LOH are the primary inactivation mechanisms. Stable transfection of RIL into colorectal cancer cells resulted in reduction in cell growth, clonogenicity, and increased apoptosis upon UVC treatment, suggesting that RIL is a good candidate TSG. ^ In searching for a cause of RIL hypermethylation, we identified a 12-bp polymorphic sequence around the transcription start site of the gene that creates a long allele containing 3CTC repeat. Evolutionary studies suggested that the long allele appeared late in evolution due to insertion. Using bisulfite sequencing, in cancers heterozygous for RIL, we found that the short allele is 4.4-fold more methylated than the long allele (P = 0.003). EMSA results suggested binding of factor(s) to the inserted region of the long allele, but not to the short. EMSA mutagenesis and competition studies, as well as supershifts using nuclear extracts or recombinant Sp1 strongly indicated that those DNA binding proteins are Sp1 and Sp3. Transient transfections of RIL allele-specific expression constructs showed less than 2-fold differences in luciferase activity, suggesting no major effects of the additional Sp1 site on transcription. However, stable transfection resulted in 3-fold lower levels of transcription from the short allele 60 days post-transfection, consistent with the concept that the polymorphic Sp1 site protects against time-dependent silencing. Thus, an insertional polymorphism in the RIL promoter creates an additional Sp1/Sp3 site, which appears to protect it from silencing and methylation in cancer. ^

Alpha C protein of group B Streptococcus as a potential vaccine candidate

Group B Streptococcus (GBS) is a leading cause of life-threatening infection in neonates and young infants, pregnant women, and non-pregnant adults with underlying medical conditions. Immunization has theoretical potential to prevent significant morbidity and mortality from GBS disease. Alpha C protein (α C), found in 70% of non-type III capsule polysaccharide group B Streptococcus, elicits antibodies protective against α C-expressing strains in experimental animals and is an appealing carrier for a GBS conjugate vaccine. We determined whether natural exposure to α C elicits antibodies in women and if high maternal α C-specific serum antibody at delivery is associated with protection against neonatal disease. An ELISA was designed to measure α C-specific IgM and IgG in human sera. A case-control design (1:3 ratio) was used to match α C-expressing GBS colonized and non-colonized women by age and compare quantified serum α C-specific IgM and IgG. Sera also were analyzed from bacteremic neonates and their mothers and from women with invasive GBS disease. Antibody concentrations were compared using t-tests on log-transformed data. Geometric mean concentrations of α C-specific IgM and IgG were similar in sera from 58 α C strain colonized and 174 age-matched non-colonized women (IgG 245 and 313 ng/ml; IgM 257 and 229 ng/ml, respectively). Delivery sera from mothers of 42 neonates with GBS α C sepsis had similar concentrations of α C-specific IgM (245 ng/ml) and IgG (371 ng/ml), but acute sera from 13 women with invasive α C-expressing GBS infection had significantly higher concentrations (IgM 383 and IgG 476 ng/ml [p=0.036 and 0.038, respectively]). Convalescent sera from 5 of these women 16-49 days later had high α C-specific IgM and IgG concentrations (1355 and 4173 ng/ml, respectively). In vitro killing of α C-expressing GBS correlated with total α C-specific antibody concentration. Invasive disease but not colonization elicits α C-specific IgM and IgG in adults. Whether α C-specific IgG induced by vaccine would protect against disease in neonates merits further investigation. ^

Longitudinal and logistic analysis of endocrine hormone profiles, physical maturation, and candidate genes related to pubertal events in children

Children who experience early pubertal development have an increased risk of developing cancer (breast, ovarian, and testicular), osteoporosis, insulin resistance, and obesity as adults. Early pubertal development has been associated with depression, aggressiveness, and increased sexual prowess. Possible explanations for the decline in age of pubertal onset include genetics, exposure to environmental toxins, better nutrition, and a reduction in childhood infections. In this study we (1) evaluated the association between 415 single nucleotide polymorphisms (SNPs) from hormonal pathways and early puberty, defined as menarche prior to age 12 in females and Tanner Stage 2 development prior to age 11 in males, and (2) measured endocrine hormone trajectories (estradiol, testosterone, and DHEAS) in relation to age, race, and Tanner Stage in a cohort of children from Project HeartBeat! At the end of the 4-year study, 193 females had onset of menarche and 121 males had pubertal staging at age 11. African American females had a younger mean age at menarche than Non-Hispanic White females. African American females and males had a lower mean age at each pubertal stage (1-5) than Non-Hispanic White females and males. African American females had higher mean BMI measures at each pubertal stage than Non-Hispanic White females. Of the 415 SNPs evaluated in females, 22 SNPs were associated with early menarche, when adjusted for race ( p<0.05), but none remained significant after adjusting for multiple testing by False Discovery Rate (p<0.00017). In males, 17 SNPs were associated with early pubertal development when adjusted for race (p<0.05), but none remained significant when adjusted for multiple testing (p<0.00017). ^ There were 4955 hormone measurements taken during the 4-year study period from 632 African American and Non-Hispanic White males and females. On average, African American females started and ended the pubertal process at a younger age than Non-Hispanic White females. The mean age of Tanner Stage 2 breast development in African American and Non-Hispanic White females was 9.7 (S.D.=0.8) and 10.2 (S.D.=1.1) years, respectively. There was a significant difference by race in mean age for each pubertal stage, except Tanner Stage 1 for pubic hair development. Both Estradiol and DHEAS levels in females varied significantly with age, but not by race. Estradiol and DHEAS levels increased from Tanner Stage 1 to Tanner Stage 5.^ African American males had a lower mean age at each Tanner Stage of development than Non-Hispanic White males. The mean age of Tanner Stage 2 genital development in African American and Non-Hispanic White males was 10.5 (S.D.=1.1) and 10.8 (S.D.=1.1) years, respectively, but this difference was not significant (p=0.11). Testosterone levels varied significantly with age and race. Non-Hispanic White males had higher levels of testosterone than African American males from Tanner Stage 1-4. Testosterone levels increased for both races from Tanner Stage 1 to Tanner Stage 5. Testosterone levels had the steepest increase from ages 11-15 for both races. DHEAS levels in males varied significantly with age, but not by race. DHEAS levels had the steepest increase from ages 14-17. ^ In conclusion, African American males and females experience pubertal onset at a younger age than Non-Hispanic White males and females, but in this study, we could not find a specific gene that explained the observed variation in age of pubertal onset. Future studies with larger study populations may provide a better understanding of the contribution of genes in early pubertal onset.^