Genetic association studies often examine features independently, potentially missing subpopulations with multiple phenotypes that share a single cause. We describe an approach that aggregates phenotypes on the basis of patterns described by Mendelian diseases. We mapped the clinical features of 1204 Mendelian diseases into phenotypes captured from the electronic health record (EHR) and summarized this evidence as phenotype risk scores (PheRSs). In an initial validation, PheRS distinguished cases and controls of five Mendelian diseases. Applying PheRS to 21,701 genotyped individuals uncovered 18 associations between rare variants and phenotypes consistent with Mendelian diseases. In 16 patients, the rare genetic variants were associated with severe outcomes such as organ transplants. PheRS can augment rare-variant interpretation and may identify subsets of patients with distinct genetic causes for common diseases.

In membranes from COS-7 cells expressing rPLD, addition of myristoylated ADP-ribosylation factor (ARF) and RhoA in vitro stimulated PLD activity. The effect of ARF was greater than that of RhoA, although the concentrations for half-maximal stimulation (0.08 -0.2 M) were similar. Membranes isolated from cells expressing rPLD plus L71ARF3 and/or V14RhoA also showed higher PLD activity but no synergism between the two G proteins. Addition of phorbol ester and protein kinase C ␣ (PKC␣) also stimulated PLD activity in membranes from COS-7 cells expressing rPLD, but it had no effect on the activity in control (vector) membranes and did not enhance the effects of constitutively active ARF or Rho. The stimulation by PKC␣ did not require ATP and was not increased by addition of this nucleotide. No synergism between ARF and Rho and between these and PKC␣ on PLD activity was observed when these were added to membranes from cells expressing rPLD. Oleate inhibited the PLD activity of membranes from both control and rPLD-expressing cells.In summary, these results indicate that in vitro, rPLD is stimulated by ARF, RhoA, and PKC␣ and inhibited by oleate. However, in intact COS-7 cells, ARF activates endogenous PLD but not rPLD, whereas the reverse is true for RhoA. In addition, the effects of phorbol ester are much greater in the intact cells. It is concluded that the regulation of rPLD in intact COS-7 cells differs significantly from that seen in vitro; possible reasons for this are discussed.

Ketamine abusers develop severe lower urinary tract symptoms. The major aims of the present study were to elucidate ketamine-induced ulcerative cystitis and bladder apoptosis in association with oxidative stress mediated by mitochondria and the endoplasmic reticulum (ER). Sprague-Dawley rats were distributed into three different groups, which received normal saline or ketamine for a period of 14 or 28 days, respectively. Double-labeled immunofluorescence experiments were performed to investigate tight junction proteins for urothelial barrier functions. A TUNEL assay was performed to evaluate the distribution of apoptotic cells. Western blot analysis was carried out to examine the expressions of urothelial tight junction proteins, ER stress markers, and apoptosis-associated proteins. Antioxidant enzymes, including SOD and catalase, were investigated by real-time PCR and immunofluorescence experiments. Ketamine-treated rats were found to display bladder hyperactivity. This bladder dysfunction was accompanied by disruptions of epithelial cadherin- and tight junction-associated proteins as well as increases in the expressions of apoptosis-associated proteins, which displayed features of mitochondria-dependent apoptotic signals and ER stress markers. Meanwhile, expressions of mitochondria respiratory subunit enzymes were significantly increased in ketamine-treated bladders. Conversely, mRNA expressions of the antioxidant enzymes Mn-SOD (SOD2), Cu/Zn-SOD (SOD1), and catalase were decreased after 28 days of ketamine treatment. These results demonstrate that ketamine enhanced the generation of oxidative stress mediated by mitochondria- and ER-dependent pathways and consequently contributed to bladder apoptosis and urothelial lining defects. Such oxidative stress-enhanced bladder cell apoptosis and urothelial barrier defects are potential factors that may play a crucial role in bladder overactivity and ulceration.

Rat brain phospholipase D1 (rPLD1) belongs to a superfamily defined by the highly conserved catalytic motif (H(X)K(X) 4 D, denoted HKD. RPLD1 contains two HKD domains, located in the N-and C-terminal regions. Deletion mutants of rPLD1 that contained only an N-or C-terminal HKD domain exhibited no catalytic activity when expressed in COS 7 cells. However, when N-terminal fragments containing one of the HKD domains were cotransfected with a C-terminal fragment containing the other HKD domain, PLD activity was restored. Furthermore, immunoprecipitation assays showed that the N-and C-terminal halves of rPLD1 were physically associated when expressed in COS 7 cells. In addition, deletion of 168 amino acids from the N terminus of rPLD1 significantly enhanced basal PLD activity while inhibiting the response to phorbol ester. Likewise, the coexpression of this truncated N-terminal half with the C-terminal half resulted in increased PLD activity. In summary, this study provides direct evidence that the enzymatic activity of rPLD1 requires the presence of the HKD domains in both the N-and C-terminal regions of the molecule. More importantly, the two halves of rPLD1 can associate, and this may be essential to bring the two HKD domains together to form an active catalytic center. These findings provide new insights into the catalytic mechanism of enzymes of the PLD superfamily. Phospholipase D (PLD)1 catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline (1). It also carrys out a phosphatidyl transfer reaction which is used as a specific measure of PLD activity (2). PLD activity has been detected in almost all organisms and is involved in a variety of signal transduction cascades (3, 4). PLD activity has been shown to be regulated by small G proteins, PKC, proteintyrosine kinases, and intracellular Ca 2ϩ. It was first cloned from plant (5) followed by yeast (6). To date, two types of mammalian PLD genes, termed PLD1 and PLD2, have been cloned (7-16). PLD1 has a low basal activity and responds to PKC and small G protein of the ADP-ribosylation factor and Rho families. On the other hand, PLD2 is constitutively active and shows little response to stimuli.Data base searches using PLD1 and other sequences reveal that PLD belongs to a superfamily (17-19) with four highly conserved regions (17). The most prominent conserved sequences reside in regions I and IV and contain the invariant motif, HXK(X) 4 D, denoted HKD. The HKD motif is found in other enzymes (17-19), including phosphatidyltransferases, poxvirus envelope proteins, a Yersinia murine toxin, and several endonucleases, including Nuc (20). Mutation of the HKD motifs residing in region I or IV renders human PLD1 and mouse PLD2 inactive (21). Studies of Nuc also suggest that the histidine in the HKD domain is directly involved in the catalytic reaction by forming a phosphoenzyme intermediate (22). However, the mechanism(s) by which the two HKD domains are organized spatially to form an active catalytic center is not clear. Vaccinia virus protein VP...