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Purpose:We report the diagnostic yield of whole-exome sequencing (WES) in 3,040 consecutive cases at a single clinical laboratory.Methods: WES was performed for many different clinical indications and included the proband plus two or more family members in 76% of cases. Results:The overall diagnostic yield of WES was 28.8%. The diagnostic yield was 23.6% in proband-only cases and 31.0% when three family members were analyzed. The highest yield was for patients who had disorders involving hearing (55%, N = 11), vision (47%, N = 60), the skeletal muscle system (40%, N = 43), the skeletal system (39%, N = 54), multiple congenital anomalies (36%, N = 729), skin (32%, N = 31), the central nervous system (31%, N = 1,082), and the cardiovascular system (28%, N = 54). Of 2,091 cases in which secondary findings were analyzed for 56

SummaryThe glycerophospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE) account for greater than 50% of the total phospholipid species in eukaryotic membranes and thus play major roles in the structure and function of those membranes. In most eukaryotic cells, PC and PE are synthesized by an aminoalcoholphosphotransferase reaction, which uses sn-1,2-diradylglycerol and either CDP-choline or CDP-ethanolamine, respectively. This is the last step in a biosynthetic pathway known as the Kennedy pathway, so named after Eugene Kennedy who elucidated it over 50 years ago. This review will cover various aspects of the Kennedy pathway including: each of the biosynthetic steps, the functions and roles of the phospholipid products PC and PE, and how the Kennedy pathway has the potential of being a chemotherapeutic target against cancer and various infectious diseases. IUBMBIUBMB Life, 62(6): [414][415][416][417][418][419][420][421][422][423][424][425][426][427][428] 2010

The Kennedy Pathway. See The Kennedy Pathway–De Novo Synthesis of Phosphatidylethanolamine and Phosphatidylcholine by Federica Gibellini and Terry K. Smith, pp. 414–428.

Purpose:Germ-line testing for panels of cancer genes using next-generation sequencing is becoming more common in clinical care. We report our experience as a clinical laboratory testing both well-established, high-risk cancer genes (e.g., BRCA1/2, MLH1, MSH2) as well as more recently identified cancer genes (e.g., PALB2, BRIP1), many of which have increased but less well-defined penetrance.Genet Med 18 8, 823–832.Methods:Clinical genetic testing was performed on over 10,000 consecutive cases referred for evaluation of germ-line cancer genes, and results were analyzed for frequency of pathogenic or likely pathogenic variants, and were stratified by testing panel, gene, and clinical history.Genet Med 18 8, 823–832.Results:Overall, a molecular diagnosis was made in 9.0% of patients tested, with the highest yield in the Lynch syndrome/colorectal cancer panel. In patients with breast, ovarian, or colon/stomach cancer, positive yields were 9.7, 13.4, and 14.8%, respectively. Approximately half of the pathogenic variants identified in patients with breast or ovarian cancer were in genes other than BRCA1/2.Genet Med 18 8, 823–832.Conclusion:The high frequency of positive results in a wide range of cancer genes, including those of high penetrance and with clinical care guidelines, underscores both the genetic heterogeneity of hereditary cancer and the usefulness of multigene panels over genetic tests of one or two genes.Genet Med 18 8, 823–832.

A gene expression signature of tumor proliferation rate in mantle cell lymphoma (MCL) is an overriding molecular predictor of the length of survival following diagnosis. Many strongly proliferative MCL tumors have exceptionally high cyclin D1 mRNA levels and preferentially express short cyclin D1 mRNA isoforms. We demonstrate here that these short mRNAs are cyclin D1a isoforms with truncated 3UTRs, not alternatively spliced cyclin D1b mRNA isoforms. Among 15 MCL tumors with truncated cyclin D1 mRNAs, 7 had genomic deletions in the CCND1 3UTR region. In 3 others, CCND1 contained point mutations that created premature polyadenylation signals, giving rise to 1.5-kb mRNAs lacking most of the 3UTR. Both types of genomic alteration created transcripts lacking mRNA destabilization elements present in the wild-type cyclin D1a mRNA. Premature polyadenylation due to a 3UTR mutation also was present in the Z-138 MCL cell line, which expressed both truncated and full-length cyclin D1a mRNAs. In these cells, the half-life of the short cyclin D1a mRNA was much longer than that of the full-length mRNA. We conclude that alterations of CCND1 3UTR IntroductionMantle cell lymphoma (MCL) comprises about 6% of all nonHodgkin lymphoma and is considered incurable with standard chemotherapy. 1,2 Median survival is approximately 3 years, but survival ranges from less than one year to Ͼ 6 years. The hallmark genetic feature of MCL is the t(11;14) translocation that leads to misexpression of cyclin D1 in the malignant cells. [3][4][5] The t(11;14) is not unique to MCL and occurs also in multiple myeloma. 6 Cyclin D1 is a member of the D-type cyclins that regulate the transition from G 0 /G 1 phase to S phase of the cell cycle. 7 Cyclin D1 is not normally expressed at high levels in lymphoid cells, and its expression from the t(11;14) translocated allele is driven by enhancer elements in the immunoglobulin heavy chain locus. Most t(11;14) translocations take place at the 5Ј end of the cyclin D1 locus, but translocations at the 3Јend of the gene also have been described in some cases. 8 CCND1 has 5 exons, which can be alternatively spliced to create 2 major isoforms, cyclin D1a and D1b. The cyclin D1a isoform is 4.5 kb in length, with a coding region of only 882 bp. The majority of this mRNA consists of 3ЈUTR sequences containing mRNA destabilizing elements. The cyclin D1b isoform lacks exon 5 but retains intron 4, which contains a translation stop codon after 99 bp and a polyadenylation signal less then 300 bp 3Ј from this stop codon. The 1.7-kb cyclin D1b mRNA is found in most tumors and cell lines that express cyclin D1 and encodes a 274 amino acid protein that differs at the C terminus from the 294 amino acid protein encoded by the cyclin D1a mRNA. [9][10][11][12][13] In contrast to cyclin D1a, cyclin D1b is potently transforming in experimental models. 13,14 The relative abundance of the cyclin D1b isoform is reportedly affected by a G/A single nucleotide polymorphism at the last base of exon 4 (position 870, codon 241), which is the...

Pteridine reductase (PTR1) is essential for salvage of pterins by parasitic trypanosomatids and is a target for the development of improved therapies. To identify inhibitors of Leishmania major and Trypanosoma cruzi PTR1, we combined a rapid-screening strategy using a folate-based library with structure-based design. Assays were carried out against folate-dependent enzymes including PTR1, dihydrofolate reductase (DHFR), and thymidylate synthase. Affinity profiling determined selectivity and specificity of a series of quinoxaline and 2,4-diaminopteridine derivatives, and nine compounds showed greater activity against parasite enzymes compared with human enzymes. Compound 6a displayed a Ki of 100 nM toward LmPTR1, and the crystal structure of the LmPTR1:NADPH:6a ternary complex revealed a substrate-like binding mode distinct from that previously observed for similar compounds. A second round of design, synthesis, and assay produced a compound (6b) with a significantly improved Ki (37 nM) against LmPTR1, and the structure of this complex was also determined. Biological evaluation of selected inhibitors was performed against the extracellular forms of T. cruzi and L. major, both wild-type and overexpressing PTR1 lines, as a model for PTR1-driven antifolate drug resistance and the intracellular form of T. cruzi. An additive profile was observed when PTR1 inhibitors were used in combination with known DHFR inhibitors, and a reduction in toxicity of treatment was observed with respect to administration of a DHFR inhibitor alone. The successful combination of antifolates targeting two enzymes indicates high potential for such an approach in the development of previously undescribed antiparasitic drugs.antitrypanosomatid agents ͉ antifolates ͉ drug discovery P rotozoan parasites of the order Kinetoplastida are the causal agents of serious human diseases, including African sleeping sickness, Chagas' disease, and leishmaniasis. There is an urgent need for new, more effective drugs targeting these neglected diseases, because those in current use are toxic, expensive, and often difficult to administer. The problem is compounded by an increase in drug resistance and lack of progress in drug development. Only a single new effective treatment has been developed in the last 25 years, Miltefosine (hexadecylphosphocholine), recently approved in India (1).Enzymes involved in the provision and use of reduced folate cofactors such as dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are valued drug targets for the treatment of bacterial infections (2), cancer (3), and certain parasitic diseases, notably malaria (4). DHFR catalyzes the two-step reduction of folate to tetrahydrofolate, which is then transformed to N 5 ,N 10 -methylene tetrahydrofolate and is used by TS as a methyl donor and reducing agent in the conversion of 2Ј-deoxyuridine-5Ј-monophosphate to 2Ј-deoxythymidine-5Ј-monophosphate. Inhibition of DHFR or TS reduces the cellular pool of 2Ј-deoxythymidine-5Ј-monophosphate, impairing DNA replication and resulting ...

SummaryThe protozoan Trypanosoma brucei has a functional pteridine reductase (TbPTR1), an NADPH-dependent short-chain reductase that participates in the salvage of pterins, which are essential for parasite growth. PTR1 displays broad-spectrum activity with pterins and folates, provides a metabolic bypass for inhibition of the trypanosomatid dihydrofolate reductase and therefore compromises the use of antifolates for treatment of trypanosomiasis. Catalytic properties of recombinant TbPTR1 and inhibition by the archetypal antifolate methotrexate have been characterized and the crystal structure of the ternary complex with cofactor NADP + and the inhibitor determined at 2.2 Å resolution. This enzyme shares 50% amino acid sequence identity with Leishmania major PTR1 (LmPTR1) and comparisons show that the architecture of the cofactor binding site, and the catalytic centre are highly conserved, as are most interactions with the inhibitor. However, specific amino acid differences, in particular the placement of Trp221 at the side of the active site, and adjustment of the b6-a6 loop and a6 helix at one side of the substrate-binding cleft significantly reduce the size of the substrate binding site of TbPTR1 and alter the chemical properties compared with LmPTR1. A reactive Cys168, within the active site cleft, in conjunction with the C-terminus carboxyl group and His267 of a partner subunit forms a triad similar to the catalytic component of cysteine proteases. TbPTR1 therefore offers novel structural features to exploit in the search for inhibitors of therapeutic value against African trypanosomiasis.