Cowden syndrome (CS) and Bannayan-Riley-Ruvalcaba syndrome are allelic, defined by germline PTEN mutations, and collectively referred to as PTEN hamartoma tumor syndrome. To date, there are no existing criteria based on large prospective patient cohorts to select patients for PTEN mutation testing. To address these issues, we conducted a multicenter prospective study in which 3042 probands satisfying relaxed CS clinical criteria were accrued. PTEN mutation scanning, including promoter and large deletion analysis, was performed for all subjects. Pathogenic mutations were identified in 290 individuals (9.5%). To evaluate clinical phenotype and PTEN genotype against protein expression, we performed immunoblotting (PTEN, P-AKT1, P-MAPK1/2) for a patient subset (n = 423). In order to obtain an individualized estimation of pretest probability of germline PTEN mutation, we developed an optimized clinical practice model to identify adult and pediatric patients. For adults, a semiquantitative score-the Cleveland Clinic (CC) score-resulted in a well-calibrated estimation of pretest probability of PTEN status. Overall, decreased PTEN protein expression correlated with PTEN mutation status; decreasing PTEN protein expression correlated with increasing CC score (p < 0.001), but not with the National Comprehensive Cancer Network (NCCN) criteria (p = 0.11). For pediatric patients, we identified highly sensitive criteria to guide PTEN mutation testing, with phenotypic features distinct from the adult setting. Our model improved sensitivity and positive predictive value for germline PTEN mutation relative to the NCCN 2010 criteria in both cohorts. We present the first evidence-based clinical practice model to select patients for genetics referral and PTEN mutation testing, further supported biologically by protein correlation.

We have combined genetic and biochemical approaches to analyze the function of the RNA-binding protein Nova-1, the paraneoplastic opsoclonus-myoclonus ataxia (POMA) antigen. Nova-1 null mice die postnatally from a motor deficit associated with apoptotic death of spinal and brainstem neurons. Nova-1 null mice show specific splicing defects in two inhibitory receptor pre-mRNAs, glycine alpha2 exon 3A (GlyRalpha2 E3A) and GABA(A) exon gamma2L. Nova protein in brain extracts specifically bound to a previously identified GlyRalpha2 intronic (UCAUY)3 Nova target sequence, and Nova-1 acted directly on this element to increase E3A splicing in cotransfection assays. We conclude that Nova-1 binds RNA in a sequence-specific manner to regulate neuronal pre-mRNA alternative splicing; the defect in splicing in Nova-1 null mice provides a model for understanding the motor dysfunction in POMA.

Paraneoplastic opsoclonus myoclonus ataxia (POMA) is a neurologic disorder thought to be mediated by an autoimmune attack against onconeural disease antigens that are expressed by gynecologic or lung tumors and by neurons. One POMA disease antigen, termed Nova-1, has been identified as a neuron-specific KH-type RNA-binding protein.Nova-1 expression is restricted to specific regions of the central nervous system, primarily the hindbrain and ventral spinal cord, which correlate with the predominantly motor symptoms in POMA. However, POMA antisera recognize antigens that are widely expressed in both caudal and rostral regions of the central nervous system, and some patients develop cognitive symptoms. We have used POMA antisera to clone a cDNA encoding a second POMA disease antigen termed Nova-2. Nova-2 is closely related to Nova-1, and is expressed at high levels in neurons during development and in adulthood, and at lower levels in the adult lung. In the postnatal mouse brain, Nova-2 is expressed in a pattern that is largely reciprocal with Nova-1, including high levels of Nova-2 expression in the neocortex and hippocampus. Functional characterization of Nova-2 in RNA selection and nitrocellulose filter-binding assays reveals that Nova-2 binds RNA with high affinity and with sequence specificity that differs from Nova-1. Our results demonstrate that the immune response in POMA targets a family of highly related sequencespecific neuronal RNA-binding proteins. The expression pattern of the Nova-2 protein is likely to underlie the development of cognitive deficits in some POMA patients.The paraneoplastic neurologic diseases (PNDs) are an unusual group of diseases at the intersection of neurobiology, immunology, and oncology. Patients with PNDs harbor systemic tumors and develop immune responses against onconeural antigens that are expressed both by their tumors and by neurons (for review, see refs. 1 and 2). The presence of high-titer antibodies in patient sera has allowed the identification and characterization of PND antigens and has provided a unique approach toward understanding the expression and function of these proteins in discrete regions of the brain. For

The Nova paraneoplastic antigens are neuron-specific RNA binding proteins that participate in the control of alternative splicing. We have used the yeast two-hybrid system to isolate Nova interacting proteins and identify an RNA binding protein that is closely related to the polypyrimidine tract-binding protein (PTB). The expression of this protein, brPTB, is enriched in the brain, where it is expressed in glia and neurons. brPTB interacts with Nova proteins in cell lines and colocalizes with Nova within neuronal nuclei. We previously found that Nova binds to a pyrimidine-rich RNA element present upstream of an alternatively spliced exon, E3A, in glycine receptor ␣2 (GlyR␣2) pre-mRNA, and this binding is implicated in Novadependent regulation of splicing. Cotransfection assays with a GlyR␣2 minigene demonstrate that brPTB antagonizes the action of Nova to increase utilization of GlyR␣2 E3A. brPTB binds to a 90-nt GlyR␣2 RNA adjacent to the Nova binding site, but with an affinity that is more than 10-fold lower than Nova. When a putative binding site for brPTB on the GlyR␣2 RNA is mutated, binding is abolished and the inhibitory effect on Nova-dependent exon selection disappears. These results suggest that brPTB is a tissuerestricted RNA binding protein that interacts with and inhibits the ability of Nova to activate exon selection in neurons.N eurons make extensive use of alternative splicing to regulate functional differences in proteins. A wide variety of neurotransmitter receptor activities are regulated by alternative splicing, including NR1 N-methyl-D-aspartate (NMDA) receptor subcellular localization (1) and interaction with neurofilaments (2), the physiology of the glutamate (3) and NMDA (4) receptors, and the ability of agrin to induce clustering of acetylcholine receptors (5). Moreover, several neurologic diseases such as spinal muscular atrophy, amyotrophic lateral sclerosis, and paraneoplastic opsoclonus-myoclonus ataxia (POMA) have been associated with defects in proteins involved in generating the splicing machinery or in the accurate splicing of target .Since the discovery of tissue-specific splicing of the calcitonin͞ calcitonin gene-related peptide (CGRP) transcript in neurons, there has been an extensive search for cis-acting RNA elements and trans-acting RNA binding proteins that mediate neuronspecific splicing. The first example of cis-acting regulatory elements in neuronal pre-mRNAs identified was in calcitonin͞ CGRP pre-mRNA (9), and a number of specific sequences have been identified that are responsible for calcitonin͞CGRP tissuespecific processing (10, 11). Subsequent work identified regulatory sequences near other neuron-specific exons such as the N1 exon of src (12) and a 24-nt exon of the ␥-aminobutyric acid type A receptor ␥2 subunit (13).The identification of trans-acting factors that regulate neuronal splicing has been a greater challenge. Two general mechanisms might account for the way such factors could mediate regulation of neuronal splicing. Brain-specific variants in splicing ...