We can now routinely identify coding variants within individual human genomes. A pressing challenge is to determine which variants disrupt the function of disease-associated genes. Both experimental and computational methods exist to predict pathogenicity of human genetic variation. However, a systematic performance comparison between them has been lacking. Therefore, we developed and exploited a panel of 26 yeast-based functional complementation assays to measure the impact of 179 variants (101 disease-and 78 non-disease-associated variants) from 22 human disease genes. Using the resulting reference standard, we show that experimental functional assays in a 1-billion-year diverged model organism can identify pathogenic alleles with significantly higher precision and specificity than current computational methods.

As a natural defense mechanism of the immune system, a group of white blood cells known as B cells produce antibodies to neutralize and clear pathogens and toxins from circulation. In order to increase the antibody binding strength, B cells undergo two types of genomic alterations: somatic hypermutation and class switch recombination (CSR). CSR is accomplished by the B cell-specific activation-induced cytidine deaminase protein (AID) which allows activated B cells to produce antibodies of different isotypes, which have distinct effector functions. Current research concerning the nature of antibody diversification examines the role of specific proteins in the CSR process. To test the role of these genes in CSR, I cloned the cDNAs of these genes. Through polymerase chain reaction (PCR) and PCR clean-up, the PCR products were ligated into an expression vector that harbours a drug-resistant gene. A variety of primers including RNF20 'UTR, RNF40 'UTR, RNF40 clone and RNF40 clone targeted cDNA samples through gradient PCR. Although electrophoresis analyses show inconsistency with PCR products, conclusions can be drawn about the efficiency of primers, Phusion, and cDNA samples. Successful PCR products will be expressed in the CH12F3-2 B cell line that undergoes CSR at high rates and assess whether the CSR process is affected. I will also knockdown RNF20 and RNF40 by siRNA. If the hypothesis is correct, RNF20 and RNF40 will be discovered to be critical factors in the CSR process in B cells, and therefore in establishing an effective antibody response that is critical in protective immunity En tant qu'un mécanisme de défense naturel du système immunitaire, un groupe de globules blancs appelés lymphocytes B produisent des anticorps afin de neutraliser et élimi-ner les agents pathogènes et les toxines de la circulation. Manuscript edited by Siddharth Nath and Sujay NagarajCathy Tie CharaCTerizinG rnF20 and rnF40 in The ClaSS SwiTChinG OF B CellS entreprises (RSE). La RSE se réalise grâce à l'activation induite des protéines cytidine désaminase (AID) spécifique aux cellules B, qui permet aux cellules B activées de produire des anticorps de différents isotypes détenant des fonctions effectrices différentes. La recherche actuelle concernant la nature diversifiée d'anticorps examine le rôle de protéines spécifiques dans le processus RSE. Afin de tester le rôle de ces gènes dans RSE, j'ai cloné l'ADNc dans ces gènes. Par réaction en chaîne par polymérase (PCR) et la purification PCR, les produits obtenus par PCR ont subi une ligature dans un vecteur d'expression qui abrite un gène résistant aux médicaments. Une variété d'amorces dont RNF20 'UTR, RNF40'UTR, le clone RNF40 et le clone RNF40 ciblaient des échantillons d'ADNc par un gradient PCR. Bien que les analyses d'électrophorèse montrent une certaine incompatibilité avec les produits de la PCR, certaines conclusions au sujet de l'efficacité des échantillons d'amorces, de Physion et de l'ADNc peuvent en être tirées. Les produits de la PCR retenus seront exprimés dans la ligné...