Identification of Genomic Alterations in Thai Patients With Colorectal Cancer Using Next-Generation Sequencing-Based Multigene Cancer Panel.

Introduction Colorectal cancer (CRC) is one of the leading causes of death and illness in the general population. Although the incidence of CRC is steadily decreasing worldwide, it is being diagnosed more in individuals under 50 years of age. Multiple disease-causing variants have been reported to be involved in the development of CRC. This study aimed to investigate the molecular and clinical characteristics of Thai patients with CRC. Methods NGS-based multigene cancer panel testing was performed on 21 unrelated patients. Target enrichment was performed using a custom-designed Ion AmpliSeq on-demand panel. Thirty-six genes associated with CRC and other cancer were analyzed for variant detection. Results Sixteen variants (five nonsense, eight missense, two deletions, and one duplication) in nine genes were identified in 12 patients. Eight (66.7%) patients harboring disease-causing deleterious variants in genes APC, ATM, BRCA2, MSH2, and MUTYH. One of the eight patients also carried additional heterozygous variants in genes ATM, BMPR1A, and MUTYH. In addition, four patients carried variants of uncertain significance in genes APC, MLH1, MSH2, STK11, and TP53. Among all detected genes, APC was the most frequent causative gene observed in CRC patients, which is consistent with previous reports. Conclusion This study demonstrated the comprehensive molecular and clinical characterization of CRC patients. These findings showed the benefits of using multigene cancer panel sequencing for pathogenic gene detection and showed the prevalence of genetic aberrations in Thai patients with CRC.

Shrimp Parvovirus Circular DNA Fragments Arise From Both Endogenous Viral Elements and the Infecting Virus.

Some insects use endogenous reverse transcriptase (RT) to make variable viral copy DNA (vcDNA) fragments from viral RNA in linear (lvcDNA) and circular (cvcDNA) forms. The latter form is easy to extract selectively. The vcDNA produces small interfering RNA (siRNA) variants that inhibit viral replication via the RNA interference (RNAi) pathway. The vcDNA is also autonomously inserted into the host genome as endogenous viral elements (EVE) that can also result in RNAi. We hypothesized that similar mechanisms occurred in shrimp. We used the insect methods to extract circular viral copy DNA (cvcDNA) from the giant tiger shrimp (Penaeus monodon) infected with a virus originally named infectious hypodermal and hematopoietic necrosis virus (IHHNV). Simultaneous injection of the extracted cvcDNA plus IHHNV into whiteleg shrimp (Penaeus vannamei) resulted in a significant reduction in IHHNV replication when compared to shrimp injected with IHHNV only. Next generation sequencing (NGS) revealed that the extract contained a mixture of two general IHHNV-cvcDNA types. One showed 98 to 99% sequence identity to GenBank record AF218266 from an extant type of infectious IHHNV. The other type showed 98% sequence identity to GenBank record DQ228358, an EVE formerly called non-infectious IHHNV. The startling discovery that EVE could also give rise to cvcDNA revealed that cvcDNA provided an easy means to identify and characterize EVE in shrimp and perhaps other organisms. These studies open the way for identification, characterization and use of protective cvcDNA as a potential shrimp vaccine and as a tool to identify, characterize and select naturally protective EVE to improve shrimp tolerance to homologous viruses in breeding programs.

Homologues of Piwi control transposable elements and development of male germline in Penaeus monodon.

PIWI belongs to the Argonaute protein family, which is a major protein component in RNA silencing pathway. Piwi proteins play roles in the control of transposons and germline development. They have been widely studied in vertebrates and flies, while very little is known in crustacean so far. We have previously identified and characterized a cDNA encoding Piwi protein (PmPiwi1) in the black tiger shrimp Penaeus monodon. In this study, a cDNA encoding another Piwi protein namely PmPiwi2 was identified by rapid amplification of cDNA ends (RACEs). PmPiwi2 was expressed solely in shrimp testis and ovary, indicating its potential role in germ cell development. Similar to PmPiwi1, PmPiwi2 also plays a part in the control of transposons as PmPiwi2-knockdown shrimp showed a significant increase in the expression of gypsy2 retrotransposon and mariner element in the testis. In addition, a reduction of sperm numbers in the spermatophore of PmPiwi2-knockdown shrimp suggests that PmPiwi2 is required for spermatogenesis similar to PmPiwi1. This study further demonstrated that apoptotic cell death was strongly detected in spermatogonia and spermatocyte cells of both PmPiwi-knockdown shrimp and thus, could be the cause of reduced sperm count. Investigation of sperm morphology showed a remarkably high proportion of abnormal sperms in the spermatophore of the PmPiwi1-knockdown shrimp, while PmPiwi2-knockdown shrimp had comparable percentage of abnormal sperms to the control shrimp. Consistently, the expression of KIFC1, a gene that is necessary for spermiogenesis was significantly reduced upon PmPiwi1 silencing, but not in the PmPiwi2-knockdown shrimp. Our results suggested that while both PmPiwis are required for the development of spermatid, only PmPiwi1 is possibly involved in the final stage of sperm maturation.

Functional characterization of a cDNA encoding Piwi protein in Penaeus monodon and its potential roles in controlling transposon expression and spermatogenesis.

Piwi proteins comprise a subfamily of Argonaute that plays a major role in germline development by association with a distinct class of small RNAs called Piwi interacting RNA (piRNA). Although the functions of Piwi in the development of germline cells as well as transposon regulation were reported in a number of mammalians and insects, developmental expression and function of Piwi subfamily in crustaceans is poorly known. This study is aimed at cloning and characterization of a Piwi cDNA in the black tiger shrimp, Penaeus monodon. The cDNA encoding a Piwi protein of P. monodon (PmPiwi1) was obtained by rapid amplification of cDNA ends (RACE). The PmPiwi1 coding cDNA contains 2811 nt encoding a putative protein of 936 amino acids, and was specifically expressed in testis and ovary, suggesting its possible function in gametogenesis. RNAi experiment showed that suppression of PmPiwi1 expression led to a significant up-regulation of retrotransposon gypsy2 and DNA element transposon mariner in shrimp testis. Investigation of the function of PmPiwi1 in spermatogenesis by sperm count showed significantly lower number of sperms in the spermatophore sac of PmPiwi1-knockdown shrimp compared with that in the control shrimp. Our study thus reported for the first time the cDNA encoding a Piwi protein in the shrimp P. monodon. Its roles in controlling transposons and spermatogenesis as implied by the results in this study will be important for understanding sperm development and could be useful for the improvement of reproduction in male shrimp in the future.

A novel gonad-specific Argonaute 4 serves as a defense against transposons in the black tiger shrimp Penaeus monodon.

Argonaute is a key protein of the small-RNA guided gene regulation process. The Argonaute family is generally divided into two subfamilies; AGO and PIWI. In this study, a cDNA encoding a novel type of Argonaute (PmAgo4) in the black tiger shrimp Penaeus monodon was identified and characterized. PmAgo4 cDNA contained an open reading frame of 2433 nucleotides that can be translated into a deduced amino acid with the conserved PAZ and PIWI domains. PmAgo4 was phylogenetically clustered with the AGO subfamily while exhibited a gonad-specific expression pattern similar to that of proteins in the PIWI subfamily. The expression of PmAgo4 did not change significantly in response to either double-stranded RNA or yellow head virus injection suggesting that PmAgo4 may not be the main AGO proteins that play a role in dsRNA-mediated gene silencing or antiviral defense. Interestingly, PmAgo4 appeared to participate in the control of transposons since the activation of both DNA transposon and retrotransposon was detected in the testis of PmAgo4-knockdown shrimp. Our study thus provided the first evidence for an unusual type of the AGO proteins that was predominantly expressed in shrimp gonad and implication of its role in protecting the shrimp genome against an invasion of transposons.