Teresa L. Johnson-Pais

Mechanism of calcium gating in small-conductance calcium-activated potassium channels

The slow afterhyperpolarization that follows an action potential is generated by the activation of small-conductance calcium-activated potassium channels (SK channels). The slow afterhyperpolarization limits the firing frequency of repetitive action potentials (spike-frequency adaption) and is essential for normal neurotransmission. SK channels are voltage-independent and activated by submicromolar concentrations of intracellular calcium. They are high-affinity calcium sensors that transduce fluctuations in intracellular calcium concentrations into changes in membrane potential. Here we study the mechanism of calcium gating and find that SK channels are not gated by calcium binding directly to the channel α-subunits. Instead, the functional SK channels are heteromeric complexes with calmodulin, which is constitutively associated with the α-subunits in a calcium-independent manner. Our data support a model in which calcium gating of SK channels is mediated by binding of calcium to calmodulin and subsequent conformational alterations in the channel protein.

Three novel mutations in SQSTM1 identified in familial Paget's disease of bone.

Mutations in Sequestosome 1 (SQSTM1) have been shown to segregate with familial Pagets disease of bone (PDB). We examined the coding sequence of SQSTM1 in five PDB pedigrees and found three novel mutations clustered around the C‐terminal ubiquitin associated domain. Disruptions of the C‐terminal domain of SQSTM1 seem to be a leading cause of familial PDB.

l-2-Hydroxyglutarate: An Epigenetic Modifier and Putative Oncometabolite in Renal Cancer

UNLABELLED Through unbiased metabolomics, we identified elevations of the metabolite 2-hydroxyglutarate (2HG) in renal cell carcinoma (RCC). 2HG can inhibit 2-oxoglutaratre (2-OG)-dependent dioxygenases that mediate epigenetic events, including DNA and histone demethylation. 2HG accumulation, specifically the d enantiomer, can result from gain-of-function mutations of isocitrate dehydrogenase (IDH1, IDH2) found in several different tumors. In contrast, kidney tumors demonstrate elevations of the l enantiomer of 2HG (l-2HG). High-2HG tumors demonstrate reduced DNA levels of 5-hydroxymethylcytosine (5hmC), consistent with 2HG-mediated inhibition of ten-eleven translocation (TET) enzymes, which convert 5-methylcytosine (5mC) to 5hmC. l-2HG elevation is mediated in part by reduced expression of l-2HG dehydrogenase (L2HGDH). L2HGDH reconstitution in RCC cells lowers l-2HG and promotes 5hmC accumulation. In addition, L2HGDH expression in RCC cells reduces histone methylation and suppresses in vitro tumor phenotypes. Our report identifies l-2HG as an epigenetic modifier and putative oncometabolite in kidney cancer. SIGNIFICANCE Here, we report elevations of the putative oncometabolite l-2HG in the most common subtype of kidney cancer and describe a novel mechanism for the regulation of DNA 5hmC levels. Our findings provide new insight into the metabolic basis for the epigenetic landscape of renal cancer.

Single and Multigenic Analysis of the Association between Variants in 12 Steroid Hormone Metabolism Genes and Risk of Prostate Cancer

To estimate the prostate cancer risk conferred by individual single nucleotide polymorphisms (SNPs), SNP-SNP interactions, and/or cumulative SNP effects, we evaluated the association between prostate cancer risk and the genetic variants of 12 key genes within the steroid hormone pathway (CYP17, HSD17B3, ESR1, SRD5A2, HSD3B1, HSD3B2, CYP19, CYP1A1, CYP1B1, CYP3A4, CYP27B1, and CYP24A1). A total of 116 tagged SNPs covering the group of genes were analyzed in 2,452 samples (886 cases and 1,566 controls) in three ethnic/racial groups. Several SNPs within CYP19 were significantly associated with prostate cancer in all three ethnicities (P = 0.001-0.009). Genetic variants within HSD3B2 and CYP24A1 conferred increased risk of prostate cancer in non-Hispanic or Hispanic Caucasians. A significant gene-dosage effect for increasing numbers of potential high-risk genotypes was found in non-Hispanic and Hispanic Caucasians. Higher-order interactions showed a seven-SNP interaction involving HSD17B3, CYP19, and CYP24A1 in Hispanic Caucasians (P = 0.001). In African Americans, a 10-locus model, with SNPs located within SRD5A2, HSD17B3, CYP17, CYP27B1, CYP19, and CYP24A1, showed a significant interaction (P = 0.014). In non-Hispanic Caucasians, an interaction of four SNPs in HSD3B2, HSD17B3, and CYP19 was found (P < 0.001). These data are consistent with a polygenic model of prostate cancer, indicating that multiple interacting genes of the steroid hormone pathway confer increased risk of prostate cancer. (Cancer Epidemiol Biomarkers Prev 2009;18(6):1869–80)

Androgen Receptor Length Polymorphism Associated with Prostate Cancer Risk in Hispanic Men

PURPOSE The transcriptional activation domain of the androgen receptor gene includes a CAG repeat length polymorphism. A smaller number of repeats is reported to increase the risk of prostate cancer. We investigated the association of CAG repeat length and the risk of prostate cancer in a case-control study of Hispanic men. MATERIALS AND METHODS To estimate the magnitude of the association of repeat length with prostate cancer risk, samples from 82 white patients of Hispanic origin (Hispanic) with prostate cancer and 145 Hispanic controls were genotyped. To determine the allelic distribution of repeats by race/ethnicity we genotyped 132 black men, 163 white men of nonHispanic origin (white) and 175 Hispanic men with no family history of prostate cancer, and performed pairwise comparison. RESULTS In the case-control study of Hispanic men with a repeat length of 18 or less versus greater than 18 we found an approximately 3-fold increased risk of prostate cancer (OR 2.7, 95% CI 1.21 to 6.01, t test p = 0.013, age adjusted OR 3.03, 95% CI 1.27 to 7.26). The distribution of alleles differed significantly by race/ethnicity. The mean prevalence of short CAG repeat alleles plus or minus SD was higher in black than in white men (19.8 +/- 3.2 versus 21.8 +/- 2.7, t test p <0.0001) and lower in Hispanic men than in other white men (22.7 +/- 3.3, t test p = 0.014). CONCLUSIONS To our knowledge, our study represents the first case-control study of the androgen receptor gene in a Hispanic population and provides evidence of the increased prostate cancer risk associated with short CAG repeats. Our results suggest that short CAG repeats are associated with an increased prostate cancer risk in Hispanic men.

Identification of viral infections in the prostate and evaluation of their association with cancer

BackgroundSeveral viruses with known oncogenic potential infect prostate tissue, among these are the polyomaviruses BKV, JCV, and SV40; human papillomaviruses (HPVs), and human cytomegalovirus (HCMV) infections. Recently, the Xenotropic Murine Leukemia Virus-related gammaretrovirus (XMRV) was identified in prostate tissue with a high prevalence observed in prostate cancer (PC) patients homozygous for the glutamine variant of the RNASEL protein (462Q/Q). Association studies with the R462Q allele and non-XMRV viruses have not been reported. We assessed associations between prostate cancer, prostate viral infections, and the RNASEL 462Q allele in Mexican cancer patients and controls.Methods130 subjects (55 prostate cancer cases and 75 controls) were enrolled in the study. DNA and RNA isolated from prostate tissues were screened for the presence of viral genomes. Genotyping of the RNASEL R462Q variant was performed by Taqman method.ResultsR/R, R/Q, and Q/Q frequencies for R462Q were 0.62, 0.38, and 0.0 for PC cases and 0.69, 0.24, and 0.07 for controls, respectively. HPV sequences were detected in 11 (20.0%) cases and 4 (5.3%) controls. XMRV and HCMV infections were detected in one and six control samples, respectively. The risk of PC was significantly increased (Odds Ratio = 3.98; 95% CI: 1.17-13.56, p = 0.027) by infection of the prostatic tissue with HPV. BKV, JCV, and SV40 sequences were not detected in any of the tissue samples examined.ConclusionsWe report a positive association between PC and HPV infection. The 462Q/Q RNASEL genotype was not represented in our PC cases; thus, its interaction with prostate viral infections and cancer could not be evaluated.

pRB induces Sp1 activity by relieving inhibition mediated by MDM2.

pRB activates transcription by a poorly understood mechanism that involves relieving negative regulation of the promoter specificity factor Sp1. We show here that MDM2 inhibits Sp1-mediated transcription, that MDM2 binds directly to Sp1 in vitro as well as in vivo, and that MDM2 inhibits the DNA-binding activity of Sp1. Forced expression of pRB relieves MDM2-mediated repression, and interaction of pRB with the MDM2-Sp1 complex releases Sp1 and restores DNA binding. These results suggest a model in which the opposing activities of MDM2 and pRB regulate Sp1 DNA-binding and transcriptional activity.

Identification of a Novel Tandem Duplication in Exon 1 of the TNFRSF11A Gene in Two Unrelated Patients With Familial Expansile Osteolysis

Familial expansile osteolysis (FEO) is a rare autosomal dominant disorder characterized by striking focal expansile osteolytic bone lesions and generalized osteopenia, often accompanied by characteristic early hearing loss and dental disease. The TNFRSF11A gene encodes the receptor activator of nuclear factor‐κB (RANK), which has been demonstrated to be essential in bone remodeling and osteoclast differentiation. Identical insertional mutations in the first exon of RANK have been identified in all published FEO kindreds. The mutation is an 18 base pair tandem duplication in the sequence coding for the signal peptide of RANK, which causes an increase in NF‐κB signaling. We report the identification and mutational analysis of two unrelated FEO patients. One had no family history of FEO, but presented with bilateral hearing loss at an early age, deterioration of teeth, and severe pain and swelling in the distal tibia before the age of 20. The second patient had a family history of FEO and exhibited an extensive expansile tibial lesion and lesions in one humerus and a phalanx. She also had early hearing loss and dental disease. Mutational analysis of the TNFRSF11A gene in our patients demonstrated an 18 base pair tandem duplication, one base proximal to the duplications previously reported. This novel mutation results in addition of the same six amino acids to the RANK signal peptide that has been observed previously. Further analysis of the exon 1 sequence demonstrated that it has the ability to form a stable secondary structure that may facilitate the generation of tandem duplications.

Molecular characterization of a patient with central nervous system dysmyelination and cryptic unbalanced translocation between chromosomes 4q and 18q.

We report on a 12‐year‐old boy who presented with delayed development and CNS dysmyelination. Genetic studies showed a normal 46,XY karyotype by routine cytogenetic analysis, and 46,XY.ish del(18)(q23)(D18Z1+, MBP−) by FISH using a locus‐specific probe for the MBP gene (18q23). Though the patient appeared to have normal chromosome 18s by repeated high resolution banding analysis, his clinical features were suggestive of a deletion of 18q. These included hearing loss secondary to stenosis of the external auditory canals, abnormal facial features, and foot deformities. FISH studies with genomic probes from 18q22.3 to 18qter confirmed a cryptic deletion which encompassed the MBP gene. In an attempt to further characterize the deletion, whole genome screening was conducted using array based comparative genomic hybridization (array CGH) analysis. The array CGH data not only confirmed a cryptic deletion in the 18q22.3 to 18qter region of approximately 7 Mb, it also showed a previously undetected 3.7 Mb gain of 4q material. FISH studies demonstrated that the gained 4q material was translocated distal to the 18qter deletion breakpoint. The 18q deletion contains, in addition to MBP, other known genes including CYB5, ZNF236, GALR1, and NFATC1, while the gained 4q material includes the genes FACL1 and 2, KLKB1, F11 and MTNR1A. The use of these combined methodologies has resulted in the first reported case in which array CGH has been used to characterize a congenital chromosomal abnormality, highlighting the need for innovative molecular cytogenetic techniques in the diagnosis of patients with idiopathic neurological abnormalities.

Association of RNASEL Variants with Prostate Cancer Risk in Hispanic Caucasians and African Americans

Purpose: The RNASEL gene at 1q25 has been identified as a hereditary prostate cancer susceptibility gene, but to date, no study has investigated the role of RNASEL variants in Hispanic Caucasian men with prostate cancer. Experimental Design: Two RNASEL common variants, located at amino acids 462 and 541, were genotyped in non-Hispanic Caucasian, Hispanic Caucasian, and African American prostate cancer cases and controls. Results: The RNASEL 462 AA genotype was found to increase prostate cancer risk over 4-fold in Hispanic Caucasians [odds ratio (OR), 4.43; 95% confidence interval (95% CI), 1.68-11.68; P = 0.003] and over 10-fold in African Americans (OR, 10.41; 95% CI, 2.62-41.40; P = 0.001) when compared with the GG genotype. Analysis of the RNASEL 541 variant showed that Hispanic Caucasian patients with the GG genotype had a statistically significant increase in their risk for developing prostate cancer when compared with the TT and GT genotypes (OR, 1.91; 95% CI, 1.16-3.14; P = 0.01). A common G-T haplotype for the combination of the RNASEL 462 and 541 variants was found to occur more frequently in controls compared with cases in African Americans (P = 0.04) but not in non-Hispanic Caucasians or Hispanic Caucasians. Conclusions: This is the first study that investigates the association of prostate cancer risk with RNASEL variants in Hispanic men. Our data support the role of RNASEL as a predisposition gene for prostate cancer and showed a significant association between the RNASEL 462 variant and prostate cancer risk in African Americans and Hispanic Caucasians.