Comprehensive Mutation Analysis for Congenital Muscular Dystrophy: A Clinical PCR-Based Enrichment and Next-Generation Sequencing Panel

The congenital muscular dystrophies (CMDs) comprise a heterogeneous group of heritable muscle disorders with often difficult to interpret muscle pathology, making them challenging to diagnose. Serial Sanger sequencing of suspected CMD genes, while the current molecular diagnostic method of choice, can be slow and expensive. A comprehensive panel test for simultaneous screening of mutations in all known CMD-associated genes would be a more effective diagnostic strategy. Thus, the CMDs are a model disorder group for development and validation of next-generation sequencing (NGS) strategies for diagnostic and clinical care applications. Using a highly multiplexed PCR-based target enrichment method (RainDance) in conjunction with NGS, we performed mutation detection in all CMD genes of 26 samples and compared the results with Sanger sequencing. The RainDance NGS panel showed great consistency in coverage depth, on-target efficiency, versatility of mutation detection, and genotype concordance with Sanger sequencing, demonstrating the test''s appropriateness for clinical use. Compared to single tests, a higher diagnostic yield was observed by panel implementation. The panel''s limitation is the amplification failure of select gene-specific exons which require Sanger sequencing for test completion. Successful validation and application of the CMD NGS panel to improve the diagnostic yield in a clinical laboratory was shown.     

Determination of common genetic variants in cytidine deaminase (CDA) gene in Indian ethnic population

Cytidine deaminase (CDA) is the major enzyme involved in metabolism of gemcitabine, a pyrimidine analog widely used for chemotherapy of solid tumors. While only low amounts of administered gemcitabine undergo intracellular phosphorylation into active forms and involve in antineoplastic activities, majority of it is rapidly inactivated by CDA and excreted to avoid drug toxicity. Knowledge of the genetic polymorphisms mildly effecting cellular activity of the enzyme CDA is therefore crucial to understanding drug-induced toxicities associated with gemcitabine. Functional significance and allele frequencies for common SNPs including 79A>C (*2) and 208G>A (*3) have been reported in various ethnic populations including Caucasian, African, Korean and Japanese. However, such studies have not been reported in any Indian sub-population. In the present study, conventional polymerase chain reaction (PCR) based amplification using gene specific primers and Sanger sequencing were performed to identify CDA variants in 50 healthy individuals from Indian sub-population. Established common variant 79A>C known to reduce CDA activity was observed at a frequency of 0.14 in the study cohort. In addition to other known variants, one novel variant, c.325−209T>C was detected at a frequency of 0.06. Genetic variants in CDA gene and their frequencies established in our study hold value in pharmacogenetics.     

Mir1-CP,a novel defense cysteine protease accumulates in maize vascular tissues in response to herbivory

When lepidopteran larvae feed on the insect-resistant maize genotype Mp708 there is a rapid accumulation of a defensive cysteine protease, Maize insect resistance 1-cysteine protease (Mir1-CP), at the feeding site. Silver-enhanced immunolocalization visualized with both light and transmission electron microscopy was used to determine the location of Mir1-CP in the maize leaf. The results indicated that Mir1-CP is localized predominantly in the phloem of minor and intermediate veins. After 24 h of larval feeding, Mir1-CP increased in abundance in the vascular parenchyma cells and in the thick-walled sieve element (TSE); it was also found localized to the bundle sheath and mesophyll cells. In situ hybridization of mRNA encoding Mir1-CP indicated that the primary sites of Mir1-CP synthesis in the whorl are the vascular parenchyma and bundle sheath cells. In addition to the phloem, Mir1-CP was also found in the metaxylem of the leaf and root. After 24 h of foliar feeding, the amount of Mir1-CP in the root xylem increased and it appeared to move from xylem parenchyma into the root metaxylem elements. The accumulation of Mir1-CP in maize vascular elements suggests Mir1-CP may move through these tissues to defend against insect herbivores.