Altered HIV-1 mRNA Splicing Due to Drug-Resistance-Associated Mutations in Exon 2/2b

The underlying molecular mechanism and their general effect on the replication capacity of HIV 1 drug-resistance-associated mutations is often poorly understood. To elucidate the effect of two such mutations located in a region with a high density of spicing regulatory elements on the HIV-1-splicing outcome, bioinformatic predictions were combined with transfection and infection experiments. Results show that the previously described R263K drug-resistance-associated integrase mutation has additionally a severe effect on the ESE2b splicing regulatory element (SRE) in exon 2b, which causes loss of SD2b recognition. This was confirmed by an R263R silent mutation with a similar predicted effect on the exon 2b SRE. In contrast, a V260I mutation and its silent counterpart with a lower effect on ESS2b did not exhibit any differences in the splicing pattern. Since HIV-1 highly relies on a balanced splicing reaction, changes in the splicing outcome can contribute to changes in viral replication and might add to the effect of escape mutations toward antiviral drugs. Thus, a classification of mutations purely addressing proteins is insufficient.

Indonesian Genomic Landscape of Pathogenic Mutation of APC, KRAS, TP53, PIK3CA, and MLH1 in Colorectal Cancer

Background : Knowing colorectal cancer’s heterogeneity and dynamic features, recognizing its biological behaviour requires detailed identification of mutated genes involved. Colorectal cancer (CRC) requires several mutated genes to occur and those are dissimilar in each person hence essential to be discovered in specific population. Until recently, there is no known study describing genomic landscape of CRC in Indonesian population. This study aims to describe profile of pathogenic mutation of APC, TP53, PIK3CA, KRAS, and MLH1 in CRC patients treated at 3 different hospitals in Jakarta. Methods : This is a descriptive study conducted on CRC patients who underwent neoadjuvant, surgical, and adjuvant therapy at RSCM, RSKJ, and MRCCC in 2017-2018. DNA analysis was performed using next-generation sequencing and aligned against GRCh38. Pathogenic variant was identified using ACMG classification and FATHMM score. Data related to behaviour and survival were collected from medical records. Results : There were total 22 subjects in which APC, TP53, and PIKCA were mutated. KRAS mutation occurred in 64%, while MLH1 in 45%. Five types of mutation were identified, including nonsense, missense, frameshift, splice-site, and silent mutation. There are 4 groups of co-occurring mutations, which are APC, TP53, PIK3CA (triple mutation/TM) alone; TM+KRAS; TM+MLH1; and TM+KRAS+MLH1, presenting different nature and survival. Conclusion : Indonesia having various ethnicities with diverse diet and lifestyle has distinct profile of pathogenic mutation presenting mostly with locally-advanced stage with various outcome and survival rate.

Construction of an Influenza D Virus with an Eight-Segmented Genome

Influenza D virus (IDV) may cause the bovine respiratory disease complex, which is the most common and costly disease affecting the cattle industry. Previously, we revealed that eight segments could be actively packaged in its single virion, suggesting that IDV with the seven-segmented genome shows an agnostic genome packaging mechanism. Herein, we engineered an eight-segmented recombinant IDV in which the NS1 or NS2 genes were separated from NS segment into independent segments (NS1 or NS2 segments, respectively), leading to monocistronic translation of each NS protein. We constructed two plasmids: one for the viral RNA (vRNA)-synthesis of the NS1 segment with a silent mutation at the splicing acceptor site, which controls NS2 transcription in the NS segment; and another for the RNA synthesis of the NS2 segment, with deletion of the intron in the NS segment. These plasmids and six other vRNA-synthesis plasmids were used to fabricate an infectious eight-segmented IDV via reverse genetics. This system enables analysis of the functions of NS1 or NS2. We tested the requirement of the N-terminal overlapping region (NOR) in these proteins for viral infectivity. We rescued a virus with NOR-deleted NS2 protein, which displayed a growth rate equivalent to that of the eight-segmented virus with intact NS2. Thus, the NOR may not influence viral growth. In contrast, a virus with NOR-deleted NS1 protein could not be rescued. These results indicate that the eight-segmented rescue system of IDV may provide an alternative method to analyze viral proteins at the molecular level.

Indonesian Genomic Landscape of Pathogenic Mutation of APC, KRAS, TP53, PIK3CA, and MLH1 in Colorectal Cancer

Background : Knowing colorectal cancer’s heterogeneity and dynamic features, recognizing its biological behaviour requires detailed identification of mutated genes involved. Colorectal cancer (CRC) requires several mutated genes to occur and those are dissimilar in each person hence essential to be discovered in specific population. Until recently, there is no known study describing genomic landscape of CRC in Indonesian population. This study aims to describe profile of pathogenic mutation of APC, TP53, PIK3CA, KRAS, and MLH1 in CRC patients treated at 3 different hospitals in Jakarta. Methods : This is a descriptive study conducted on CRC patients who underwent neoadjuvant, surgical, and adjuvant therapy at RSCM, RSKJ, and MRCCC in 2017-2018. DNA analysis was performed using next-generation sequencing and aligned against GRCh38. Pathogenic variant was identified using ACMG classification and FATHMM score. Data related to behaviour and survival were collected from medical records. Results : There were total 22 subjects in which APC, TP53, and PIKCA were mutated. KRAS mutation occurred in 64%, while MLH1 in 45%. Five types of mutation were identified, including nonsense, missense, frameshift, splice-site, and silent mutation. There are 4 groups of co-occurring mutations, which are APC, TP53, PIK3CA (triple mutation/TM) alone; TM+KRAS; TM+MLH1; and TM+KRAS+MLH1, presenting different nature and survival. Conclusion : Indonesia having various ethnicities with diverse diet and lifestyle has distinct profile of pathogenic mutation presenting mostly with locally-advanced stage with various outcome and survival rate.

Reduced pathogenicity of velogenic NDV strain AF22420-I via site-directed mutagenesis of V gene

Newcastle disease virus (NDV), an avian paramyxovirus, has the potential to be used as an anti-cancer therapeutic vaccine due to its oncolytic and immunostimulatory activities. The virus can be categorised into three pathotypes: lentogenic, mesogenic, and velogenic; of the three pathotypes, the lentogenic strains such as the La Sota are the preferred pathotype for vaccine development due to their low virulence to birds. On the other hand, the translation of the virus to clinic of the velogenic strain AF2240-I is hindered by its virulence towards birds although it exhibits strong oncolysis with significant outcomes both in vitro and in vivo. This study aims to reduce the pathogenicity of AF2240-I yet retaining the anti-cancer properties of the virus. To achieve this, the V protein that acts as an interferon antagonist was chosen to be mutated. It is a non-structural protein that does not interfere with the binding and infection of the virus; hence, mutation of this virulence factor was deducted to be able to reduce harm to the avian species but retain its anti-cancer properties as much as possible. The V protein, which was produced from the insertion of an additional G into a conserved editing site of the P gene, was mutated by substituting the G nucleotide at position 411 from the start of P gene to a T nucleotide. This mutation will produce a premature stop codon from the V mRNA, resulting in a truncated V protein; but only causes a silent mutation in the P protein. The recombinant virus was recovered by the use of BHK cells stably expressing the phage T7 RNA polymerase. The pathogenicity of the mutated virus was determined in 9- to 11-day-old embryonated SPF chicken eggs. The mean death time (MDT) was determined to be 73.6 hours at the minimal lethal dose of 10-7, resembling to that of a mesogenic strain. The virulence of the mutated virus has been successfully reduced where it could be potentially used as the vector for the development of recombinant oncolytic virus for cancer treatment.

Estimating the predictive power of silent mutations on cancer classification and prognosis

In recent years it has been shown that silent mutations, in and out of the coding region, can affect gene expression and may be related to tumorigenesis and cancer cell fitness. However, the predictive ability of these mutations for cancer type diagnosis and prognosis has not been evaluated yet. In the current study, based on the analysis of 9,915 cancer genomes and approximately three million mutations, we provide a comprehensive quantitative evaluation of the predictive power of various types of silent and non-silent mutations over cancer classification and prognosis. The results indicate that silent-mutation models outperform the equivalent null models in classifying all examined cancer types and in estimating the probability of survival 10 years after the initial diagnosis. Additionally, combining both non-silent and silent mutations achieved the best classification results for 68% of the cancer types and the best survival estimation results for up to nine years after the diagnosis. Thus, silent mutations hold considerable predictive power over both cancer classification and prognosis, most likely due to their effect on gene expression. It is highly advised that silent mutations are integrated in cancer research in order to unravel the full genomic landscape of cancer and its ramifications on cancer fitness.

Cross-sectional genomic perspective of epidemic waves of SARS-CoV-2: a pan India study

COVID-19 has posed unforeseen circumstances and throttled major economies worldwide. India has witnessed two waves affecting around 31 million people representing 16% of the cases globally. To date, the epidemic waves have not been comprehensively investigated to understand pandemic progress in India. In the present study, we aim for a cross-sectional analysis since its first incidence up to 26th July 2021. We have performed the pan Indian evolutionary study using 20,086 high-quality complete genomes of SARS-CoV-2. Based on the number of cases reported and mutation rates, we could divide the Indian epidemic into seven different phases. First, three phases constituting the pre-first wave had a very less average mutation rate (<11), which increased in the first wave to 17 and then doubled in the second wave (~34). In accordance with the mutation rate, variants of concern (alpha, beta, gamma and delta) and interest (eta and kappa) also started appearing in the first wave (1.5% of the genomes), which dominated the second (~96% of genomes) and post-second wave (100% of genomes) phases. Whole genome-based phylogeny could demarcate the post-first wave isolates from previous ones by the point of diversification leading to incidences of VOCs and VOIs in India. Nation-wide mutational analysis depicted more than 0.5 million events with four major mutations in ~97% of the total 20,086 genomes in the study. These included two mutations in coding (spike (D614G) and NSP 12b (P314L) of RNA dependent RNA polymerase), one silent mutation (NSP3 F106F) and one extragenic mutation (5 UTR 241). Large scale genome-wide mutational analysis is crucial in expanding knowledge on evolution of deadly variants of SARS-CoV-2 and timely management of the pandemic.

Analisis Mutasi Gen Pancreatic Duodenal Homeobox 1 (PDX-1) Berdasarkan pada Kadar Glukosa Darah Kucing Domestik (Felis catus) Post Ovariohisterektomy (OH)

Ovariohisterectomy (OH) pada kucing domestik menyebabkan beberapa efek samping, diantaranya yaitu obesitas. Obesitas berhubungan sangat erat dengan penyakit diabetes. Gen glukoregulator yang mungkin akan menjadi penyebab resistensi insulin jika mengalami mutasi gen adalah gen PDX-1. Resistensi insulin tersebut akan berpengaruh sangat erat dengan glukosa darah dalam tubuh. Tujuan penelitian adalah melihat hubungan antara polimorfisme gen PDX-1 dengan kadar glukosa darah pada kucing domestik yang mengalami OH. Metode yang digunakan pada penelitian ini adalah amplifikasi invitro PCR gen PDX-1 dengan primer Forward ‘CCG AGC TGT CAA AGC TAG CG’ dan Reverse ‘GAA CCG GTA GAG GGT CTT GG’ dan dilanjutkan dengan sekuensing terhadap produk PCR. Hasil penelitian menunjukkan bahwa terdapat perbedaan mendasar pada urutan basa dalam sekuen gen PDX-1 pada c.435G>A, c.927A>T, dan c.584A>G. Perbedaan mendasar pada urutan gen PDX-1 merupakan silent mutation.

Study on characteristics of gene mutation G6PD in patient of enzyme G6PD deficiency

G6PD deficiency is the most common genetic enzyme pathology in humans. The disease is caused by a gene mutation G6PD-the majority of cases are asymptomatic when exposed to oxidizing agents (drugs, chemicals, or foods). The disease is temporarily stopped at symptomatic treatment, so early detection is to help improve the quality of life for the patient, to prevent possible complications. The study was conducted to detect mutations in all regions of the gene G6PD. Through the study of 262 boys, 38 girls in 25 provinces in the northern region of Vietnam of 5 ethnic groups showed the results finding 11 deviated mutations that affect the coding region of the G6PD gene is Viangchan (c.871G> A) (24.33%), Kaipping (c.1388G> A) (22.67%), Canton (c.1376G> T) (18.67%), Union (c.1360C> T) (13.33%), Gaohe (cc95A > G) (6.67%), QuingYuang (c.392G> T) (4.33%), Chiniese-5 (c.1024C> T) (4.33%), Orissa (c.131C> G) (1.33%) Chatham (c.1003G> A) (1%) and Taiwan2 (c.1330G> A) (0.33%); 78/297 cases of silent mutation (c.1311C> T) associated with most of the above mutations. Discovered 2 mutant forms Orisa and Taiwan 2 that have never appeared in Vietnam. These mutations are spread evenly among the Kinh, Muong, Tay, Nung and Thai ethnic groups, of which the Kinh mainly carry the Viangchan mutation, the remaining Muong are the Union and the Tay, Nung Thai are most of Kaiping mutation

Polymorphisms in coding and non-coding regions of rabbit (Oryctolagus cuniculus) myogenin (MyoG) gene

In animal breeding, selection based on growth is very often used, as this trait affects the profitability of animal production. Identification of polymorphisms within the genes affecting the growth process seems to be very important. Therefore, we decided to analyse rabbit myogenin (<em>MyoG</em> gene) for potential polymorphic sites and their association with growth and carcass traits in Termond White (TER), Belgian Giant Grey (BGG) and crossbred New Zealand White×Belgian Giant Grey (NZW×BGG) rabbits. We found three single nucleotide polymorphisms (SNPs) – in 5’ upstream sequence g.68679476 C&gt;T, in exon 1 – silent mutation g.68680096 T&gt;C and g.68680097 G&gt;A resulting in change of GTG triplet (valine) into ATG triplet (methionine). Association analysis showed that GG genotype weaning weight was statistically higher compared to GA in TER population (<em>P</em>=0.005), and that the hind parts for GG genotypes were heavier compared to those of GA (<em>P</em>=0.024), but association analysis of dissectible parts showed this was caused by higher bone weight (<em>P</em>=0.015). For g.68679476 C&gt;T in NZW×BGG population, the CC genotypes for fore (678±35) and hind part (615±29) weights were heavier compared to CT (588±16 and 549±13, respectively); moreover, association analysis of dissectible parts showed that weight of dissectible meat in hind part. Unfortunately, we did not find similar associations for other analysed breeds. For g.68679476 C&gt;T in NZWxBGG musculus longissimus lumborum pH leg after 24 h chilling (pH24L) were statistically lower for CC genotypes compared to CT (<em>P</em>=0.027). For g.68680097 G&gt;A in Termond White population L* value on the hind leg after 24 h chilling (L*24H) was higher for GA genotypes compared to GG (<em>P</em>=0.03), while for g.68679476 C&gt;T for musculus longissimus lumborum L* value after 24 h (L*24L) CC genotypes had higher value compared to CT (<em>P</em>=0.016) in BGG population. Moreover, in BGG population CT genotypes had higher weaning weight compared to CC (<em>P</em>=0.018). Our results show that SNPs within the <em>MyoG </em>gene may influence growth traits in some rabbit breeds, but the evolutionary conserved sequence may not be favourable for changes within coding sequences. For a better understanding thereof, additional analysis is required.