Silent Mutations Research Articles

Nonoptimal Codon Usage Is Critical for Protein Structure and Function of the Master General Amino Acid Control Regulator CPC-1.

Under amino acid starvation conditions, eukaryotic organisms activate a general amino acid control response. In Neurospora crassa, Cross Pathway Control Protein 1 (CPC-1), the ortholog of the Saccharomyces cerevisiae bZIP transcription factor GCN4, functions as the master regulator of the general amino acid control response. Codon usage biases are a universal feature of eukaryotic genomes and are critical for regulation of gene expression. Although codon usage has also been implicated in the regulation of protein structure and function, genetic evidence supporting this conclusion is very limited. Here, we show that Neurospora cpc-1 has a nonoptimal NNU-rich codon usage profile that contrasts with the strong NNC codon preference in the genome. Although substitution of the cpc-1 NNU codons with synonymous NNC codons elevated CPC-1 expression in Neurospora, it altered the CPC-1 degradation rate and abolished its amino acid starvation-induced protein stabilization. The codon-manipulated CPC-1 protein also exhibited different sensitivity to limited protease digestion. Furthermore, CPC-1 functions in rescuing the cell growth of the cpc-1 deletion mutant and activation of the expression of its target genes were impaired by the synonymous codon changes. Together, these results reveal the critical role of codon usage in regulation of CPC-1 expression and function and establish a genetic example of the importance of codon usage in protein folding.IMPORTANCE The general amino acid control response is critical for adaptation of organisms to amino acid starvation conditions. The preference to use certain synonymous codons is a universal feature of all genomes. Synonymous codon changes were previously thought to be silent mutations. In this study, we showed that the Neurospora cpc-1 gene has an unusual codon usage profile compared to other genes in the genome. We found that codon optimization of the cpc-1 gene without changing its amino acid sequence resulted in elevated CPC-1 expression, an altered protein degradation rate, and impaired protein functions due to changes in protein structure. Together, these results reveal the critical role of synonymous codon usage in regulation of CPC-1 expression and function and establish a genetic example of the importance of codon usage in protein structure.

AU richness within the 5′ coding region of the Escherichia coli heat-stable enterotoxin b mRNA affects toxin secretion

The majority of protein secretion in bacteria is mediated by the T2SS pathway. Substrates processed through this pathway are guided by the N-terminal signal sequence within the nascent polypeptide. Recent experimental evidence suggests that in similar secretory pathways, such as the T3SS, information in the 5′ coding region of the mRNA affects secretion and may also participate in mRNA localization. The majority of studies on the effects of AU richness on translation have focussed on the 5′ UTR in mRNAs. To look at the effects of AU richness within the coding region of mRNA on secretion, we have generated several silent mutations within the 5′ coding region of the E. coli heat-stable enterotoxin b (STb). This toxin is a well studied T2SS substrate. The mutations were generated such that AU richness within the 5′ coding region (corresponding to the N-terminal signal sequence) was gradually reduced. Reduction of AU richness within the first 15 codons resulted in reduced secretion of the toxin as the AU/GC ratio was reduced from 2.13 for the WT STb to 1.65 (S-I) and subsequently to 1.30 (S-II). This reduction did not correlate with mRNA accumulation and decreased stability of the transcripts could not account for the reduced secretion observed. Reduction of AU richness beyond the first 15 codons recovered secretion efficiency of the toxin (S-III). To validate the experimental approach, a positive control was used in which a mutation involving the insertion of a positive charge within the hydrophobic domain of the N-terminal signal sequence was constructed. As expected, this mutation abolished secretion of the toxin. In conclusion, reducing AU richness within the 5′coding region in the STb mRNA reduces toxin secretion but other factors, such as formation of hairpins, must also be taken into consideration. This will have implications for both homologous and heterologous expression of STb for biological studies and for toxin production.

Transmission of Mycobacterium tuberculosis presenting unusually high discordance between genotypic and phenotypic resistance to rifampicin in an endemic tuberculosis setting

BackgroundSince the implementation of the Xpert MTB/RIF in Sao Paulo, Brazil, numerous Mycobacterium tuberculosis isolates presenting “rifampicin-resistant genotype with rifampicin-susceptible phenotype” were observed. ObjectiveTo evaluate the prevalence, rpoB mutations and transmission of M. tuberculosis resistant to rifampicin on Xpert MTB/RIF but susceptible on BACTEC MGIT system, in Sao Paulo state. MethodsPatients’ isolates with this pattern of rifampicin discordance, collected from 2014 to 2017, had their rpoB predominant rifampicin-resistance-determining region sequenced and were genotyped by IS6110 restriction fragment-length polymorphism. FindingsThe prevalence of rifampicin-discordant M. tuberculosis with genotypic resistance was 55.1% (156/283). Among the sequenced and genotyped isolates, 75.5% (111/147) were in clusters, largely associated with the type of rpoB mutation. Most isolates (98.6%; 72/73) harbouring the predominant mutation, His445Asn, were pooled into the two largest clusters, SP2ga (42/72; 58.3%) and SP5o (12/72; 16.7%). Ranking second, isolates carrying the silent mutation Phe433Phe were mostly (92.3%; 24/26) gathered into four groups of the family SP25. ConclusionThese findings suggest that this unusual high rifampicin discrepancy proportion was greatly influenced by few actively circulating clusters. Further studies on many of the rpoB mutations identified in our setting are needed to elucidate their association with phenotypic rifampicin resistance.

SUPT5H Post-Transcriptional Silencing Modulates PIN1 Expression, Inhibits Tumorigenicity, and Induces Apoptosis of Human Breast Cancer Cells.

Breast cancer (BrCa) is one of the most common cancers and a highly heterogenous disease, both at the pathological and molecular levels. A common element for the progression of cancer is the presence of aberrant transcription. Targeting the misregulation of transcription may serve as a tool for cancer therapeutics. SUPT5H (Suppressor of Ty 5 homolog) is a highly conserved RNA polymerase II-associated transcription elongation and processivity factor. However, few studies have examined the relationship between SUPT5H and cancer. Yeast two-hybrid and colocalization by immunofluorescence were performed to investigate protein-protein interaction. Colony formation assay, CTG assay, and crystal violet assays were performed for cell viability, clonogenicity, and cell proliferation study. Data mining was performed for expression analysis of SUPT5H in breast cancers. Flow cytometry was performed for the assessment of cell cycle and apoptosis. The Transwell chambers were employed for the migration and invasion assays. Quantitative real-time polymerase chain reaction (qRT PCR) and Western blotting were performed to measure the mRNA and protein levels of SUPT5H and other markers related to viability, migration, cell cycle, and apoptosis. Silent mutations were generated for rescue experiments. The biological function of SUPT5H was investigated through siRNA depletion of SUPT5H mRNA in vitro. We showed that SUPT5H is upregulated in breast cancer tissue as compared with the adjacent normal tissue in breast cancer patients. In human breast cancer cells, the levels of SUPT5H and PIN1 are positively correlated with each other. Our biochemical analysis showed that PIN1 interacts with SUPT5H through WW domain, that was required to promote SUPT5H protein stability. Depletion of SUPT5H by siRNA technology reduced the tumorigenic and metastatic properties, promoted s-phase cell cycle arrest and apoptosis of MDA-MB-231 cells. Moreover, depletion of SUPT5H abrogated MAPK molecules thereby regulates the oncogenic behavior of breast cancer cells. Our findings demonstrated an essential role of SUPT5H in BrCa tumorigenicity by regulating the expression levels of genes that control proliferation, migration, cell cycle, and apoptosis of breast cancer MDA-MB-231 cells.

Development of an improved dual-promoter-based reverse genetics system for emerging Senecavirus A

Senecavirus A (SVA), a recently emerging picornavirus, poses a great threat to the swine industry because it causes swine idiopathic vesicular disease and epidemic transient neonatal losses. Thus far, the progress in SVA viral pathogenesis studies and vaccine development remains sluggish, and an available and convenient reverse genetics system would undoubtedly promote relevant research. Herein, we established an improved universal dual-promoter reverse genetics system with an SVA-specific hammerhead ribozyme and hepatitis delta virus ribozyme at both terminals of the viral genome; this system could be applied to rescue all SVA strains by both eukaryotic and prokaryotic RNA polymerase systems. The genome of the clone-derived Chinese field strain CH/HeN-2018 was assembled into the universal vector pcDNA-rSVAuni through the Gibson assembly technique. Moreover, two silent mutations, G6848C and C7163 G, were separately engineered into the full-length cDNA clone with one step site-directed mutagenesis to create a KpnI restriction enzyme site, which served as a unique genetic marker. The viruses, designated rCH/HeN-2018-T7, rCH/HeN-2018-CMV, rCH/HeN-2018-6484 m and rCH/HeN-2018-7163 m, were successfully rescued through both CMV- and T7-dependent pathways, and their biological properties were further evaluated. The results showed that all four viruses grew rapidly in PK-15 cells and exhibited viral titers and growth kinetics similar to those of parental wtCH/HeN-2018. The established reverse genetics system is easily operated and can be applied to rescue all SVA strains in a short time, which will be helpful for studying SVA biology, including viral pathogenesis, antiviral therapies and vaccine development.

Investigation of glucose-6-phosphate dehydrogenase (G6PD) deficiency prevalence in a Plasmodium vivax-endemic area in the Republic of Korea (ROK)

BackgroundGlucose-6-phosphate dehydrogenase (G6PD) deficiency is the most prevalent inborn disorder. This X-chromosome-linked recessive disease affects more than 400 million people globally, and is associated with haemolytic anaemia after medication with the anti-latent malaria drug, primaquine. To prevent malaria, the Republic of Korea (ROK) Army administers malaria chemoprophylaxis. Due to the previously low G6PD deficiency prevalence in the ROK, prior to primaquine administration, testing for G6PD deficiency was not mandatory. In this study, to evaluate the risk from malaria chemoprophylaxis in the ROK, G6PD deficiency prevalence was investigated.MethodsBlood specimens from 1632 soldiers entering training camp for the 3rd Infantry of the ROK Army were collected. CareStart™ Biosensor for G6PD and haemoglobin (Hb) was used to detect G6PD levels. G6PD variants using the DiaPlexC G6PD Genotyping kit (Asian type) and full-length sequencing were examined.ResultsOf 1632 blood specimens tested, none was observed to be G6PD deficient. The median value of all tested samples was 7.582 U/g Hb. An investigation of 170 G6PD DNA variants was analysed and categorized as partially low normal [n = 131, 30–80% (2.27–6.05 U/g Hb) of the median value], high [n = 3, > 150% (> 11.373 U/g Hb) of the median value], or normal [n = 36, 80–150% (6.05–11.373 U/g Hb) of the median value], and none was amplified by the DiaPlexC kit. Five silent mutations (C→T) in 131 partially low normal specimens were found at the 1311th nucleotide position by sequence analysis. Another 8 silent mutations (T93C) were also detected in 131 partially low normal specimens. Thus, it is inferred that these silent mutations could be related to G6PD activity.ConclusionsThis G6PD deficiency prevalence study, conducted among participants from the 3rd Infantry of the ROK Army, provided crucial evidence for the safety of malaria chemoprophylaxis. This study showed that the prevalence of G6PD deficiency among 1632 young soldiers was wholly absent. Although G6PD phenotypic mutations were not detected, many silent mutations (C1311T and T93C) were observed. Thus, it is inferred that malaria chemoprophylaxis is relatively safe against G6PD deficiency-mediated haemolytic anaemia. However, given the number of individuals whose G6PD were at the partially low normal range and the frequent detection of G6PD deficiency-related mutations, consistent monitoring of G6PD deficiency is needed.

Role of oqxA and oqxB Genes in the Development of Multidrug Resistant Phenotype among Clinical Klebsiella pneumoniae Isolates from Various Cases

This study investigated the prevalence of oqxA and oqxB genes and their effective roles in the development of multidrug resistant (MDR) phenotype among clinical isolates of Klebsiella pneumoniae. Out of 150 clinical samples, 50 (33%) isolates were recognized as K. pneumoniae according to the morphological and biochemical properties. The minimum inhibitory concentrations (MICs) assay revealed that the resistance values of the isolates were 43 (86%) against ceftriaxone (4- ≥64 µg/ml), 42 (84%) against ceftazidime (16- ≥64 µg/ml), 41 (82%) against cefepime (≥16 µg/ml), 21 (42%) against ertapenem (≥8 µg/ml), 18 (36%) against imipenem (4- ≥16 µg/ml), 15 (30%) against ciprofloxacin (≥4 µg/ml), 11 (22%) against levofloxacin (≥8 µg/ml), 45 (90%) against nitrofurantoin (128- ≥512 µg/ml), 36 (72%) against trimethoprime-sulfamethoxazole (≥320 µg/ml), and 4 (8%) against tigecycline (≥8 µg/ml). Genotype detection revealed that oqxA was found in 48 (96%) of K. pneumoniae isolates, whereas oqxB was found in 6 (12%) isolates. The MDR phenotype was observed in 40 (80%) isolates, of which 38 (95%) were harbored oqxA and/or oqxB genes. DNA sequencing of oqxA revealed the presence of three silent mutations. The phylogenetic tree of oqxA variants showed a significant deviation of these variants from K. pneumoniae species. The high prevalence of oqxA among K. pneumoniae isolates may contribute to the reduction of their susceptibility to multiple antimicrobial agents.

Abstract 1318: APOBEC/ADAR aberrations are potentially implicated in certain pancancer hypermutation patterns

Abstract Background: Known endogenous mutators include certain APOBEC family members, whose mRNA levels have been implicated in various cancers. Other important nucleic acid deaminases include ADAR family of proteins, with some recent evidence supporting their potential mutator activity. Some external mutators have also been reported to act through endogenous ones. Although the ADAR and particularly APOBEC mutations are infrequent among cancer genomes, their potential contributions in cancer mutagenesis might not be underestimated. Methods: We assessed 10,294 exomes from 32 TCGA cancer types for mutations among fourteen APOBEC/ADAR family members. The mean number of single nucleotide variants (SNVs) and insertions/deletions (indels) was compared among those exomes with nonsilent APOBEC/ADAR mutations and those with wild-type APOBEC/ADAR or silent mutations. Multivariate analyses identified the relative impacts of APOBEC/ADAR mutations and mRNA levels as well as DNA repair/replication defects, as determined by mutations in KEGG pathway genes, on main variant classes across pancancer exomes. Results: Preliminary analysis identified higher mean number of both SNVs and indels among those exomes with any of the fourteen APOBEC/ADAR genes mutated compared to those with wild-type ones. Further analyses showed higher mean number of certain indel variants among those exomes with nonsilent APOBEC1 (A1; 6.1 fold), A3B (5.1 fold), A3C (20.5 fold), or ADAR (2.2 fold) mutations, and higher mean number of T>N SNVs (2.8 fold) among those with nonsilent ADARB1 mutations compared to silent ones, suggesting their true implication in cancer hypermutation. Besides DNA repair/replication defects, multivariate analyses revealed that C>G variants were determined mostly by APOBEC (A3B, A3A, A3G) mRNA levels, while other SNV classes determined mainly by various APOBEC/ADAR mutations. Indels were found to be determined mainly by mutations in A1, A3B, ADAR, and ADARB1, with a small impact of A1 mRNA level. The impacts of DNA repair/replication defects were more prominent on indels as well as C>G SNVs. Conclusions: These results provide a landscape of cancer variants as correlated with endogenous mutators and DNA repair defects. They propose the implication of certain APOBEC/ADAR mutations in most SNV classes as well as indels, in contrast to C>G SNVs mostly determined by APOBEC overexpression. These potentially reflect the gain/ loss of function impacts of various mutations and/or oligomerization capacities of both families. Further analyses linking APOBEC/ADAR mutations and mRNA levels with distinct mutational signatures are shown in the accompanying abstract “Distinct pancancer mutational signatures are determined by APOBEC/ADAR aberrations”. Citation Format: Ahmadreza Niavarani, Milad Bagheri, Joseph CF Ng, Franca Fraternali, Reza Malekzadeh. APOBEC/ADAR aberrations are potentially implicated in certain pancancer hypermutation patterns [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1318.

Determination of genetic changes of Rev-erb beta and Rev-erb alpha genes in Type 2 diabetes mellitus by next-generation sequencing.

The nuclear receptors Rev-erb alpha and Rev-erb beta are transcription factors that regulate the function of genes in glucose and lipid metabolism, and they also form a link between circadian rhythm and metabolism. We evaluated the variations in Rev-erb alpha and Rev-erb beta genes together with biochemical parameters as risk factors in type 2 diabetic (T2DM) patients. Molecular analyses of Rev-erb alpha and Rev-erb beta genes were performed on genomic DNA by using next-generation sequencing in 42 T2DM patients (21 obese and 21 non-obese) and 66 healthy controls. We found 26 rare mutations in the study groups, including 13 missense mutations, 9 silent mutations, 3 5'UTR variations, and a 3'UTR variation, of which 9 were novel variations (5 missense and 3 silent and 1 5'UTR). Six common variations were also found in the Rev-erb genes; Rev-erb beta Chr3:24003765 A>G, Rev-erb beta rs924403442 (Chr3:24006717) G>T, Rev-erb alpha Chr17:38253751T>C, Rev-erb alpha rs72836608 C>A, Rev-erb alpha rs2314339 C>T and Rev-erb alpha rs2102928 C>T. Of these, Rev-erb beta Chr3:24003765 A>G was a novel missense mutation (p.Q197R), while others were identified as intronic variants. T2DM patients with Rev-erb beta rs924403442 T allele had lower body surface area (BSA) than noncarriers (GG genotype) (p=0.039). Rev-erb alpha rs72836608 A allele and Rev-erb alpha rs2314339 CC genotype were associated with decreased serum HDL-cholesterol levels in T2DM patients (p=0.025 and p=0.027, respectively). In our study, different effects of Rev-erbs polymorphisms were found according to gender and presence of obesity. Rev-erb alpha rs72836608 (C>A) and rs2314339 (C>T) and Rev-erb alpha rs2102928 (C>T) were associated with low HDL-C levels in male T2DM patients. In female patients, Rev-erb alpha rs2102928 (C>T) was associated with high microalbuminuria and Rev-erb beta rs9244403442 G>T was associated with low HDL and high BSA values. In addition, Rev-erb alpha Chr17: 38,253,751 (T>C), rs72836608 (C>A), and rs2314339 (C>T) and Rev-erb beta Chr3:24003765 (A>G) were associated with increased serum GGT levels in obese T2DM patients. In non-obese patients, Rev-erbs SNPs had no effect on serum GGT levels. Our findings indicate that variations in the Rev-erb alpha and Rev-erb beta genes can affect metabolic changes in T2DM and these effects may vary depending on gender and obesity.

Short Communication: Identification of the exon 1 myostatin gene polymorphism and its association with slaughtered weight in Indonesian Kampung and Broiler Chicken

Abstract. Suhartati L, Khaerunnisa L, Gunawan A, Rukmiasih, Darwati S, Sumantri C, Rizqan. 2020. Short Communication: Identification of the exon 1 myostatin gene polymorphism and its association with slaughtered weight in Indonesian Kampung and Broiler Chicken. Biodiversitas 21: 3893-3897. Myostatin is widely known as a negative regulator of skeletal muscle development by controlling hypertrophy and hyperplasia. Silent mutation in exon 1 (c.234G>A) was then thought to be associated with chicken growth performances. This study was designed to analyze polymorphisms of c.234G>A mutation of the myostatin gene and its effect on slaughtered weight in Indonesian Kampung and meat-type chicken. The SatI restriction enzyme was used for genotype determination through the PCR-RFLP technique. The effect of genotype on the slaughtered weight was analyzed using the General Linear Model (GLM) procedure. Genotyping was performed on 138 chicken from Kampung (n=67) and meat type (cobb Broiler) (n=71) chicken. The amplification product contained 226 bp nucleotides. This myostatin gene was polymorphic in both chicken population, produced 2 alleles (G and A), and 3 genotypes (GG, GA, and AA). The A allele had a higher frequency than the G allele in all populations. In this study, genotype of AA and GA had higher live weight, carcass weight, breast weight, thigh weight, drumsticks weight, wings weight, breast muscle weight, thighs muscle weight, drumsticks muscle weight than GG genotype of 12 weeks-old Kampung chicken. The results suggested that the c.234G>A mutation in exon 1 could be potentially recommended as a genetic marker for chicken slaughtered weight traits.

Polymorphism of CD1B Gene and Its Association with Yolk Immunoglobulin (IgY) Concentration and Newcastle Disease Antibody Titer in IPB-D1 Chicken

The CD1B gene has an important role in the immune system of poultry by mediating antibody induction. The study aimed to identify the CD1B gene polymorphism and its association with the concentration of IgY and ND antibody titers in IPB-D1 chicken. As many as 111 of IPB-D1 chickens at 21 weeks old were used in this study. Polymorphism identification of the CD1B gene was made using the PCR Sequencing method, while the IgY and ND antibody titers were done using the ELISA and HI test, respectively. The associations of gene polymorphism with IgY and ND antibody titers were analyzed using the General Linear Model (GLM) procedure and Duncan`s Multiple Range test. The results show that there are 4 SNPs in exon 3, i.e., c.550 G>A, c.562 T>A, c.588 A>G, and c.612 C>G. All the SNPs are missense, silent mutations, and polymorphic. The c.550 G>A and c.562 T>A SNPs were in Hardy Weinberg’s equilibrium and heterozygosity (0.054-0.252) condition, while the c.588 A>G and c.612 C>G SNPs were not in equilibrium and their heterozygosity was low (0.072-0.252). The combination of 4 SNPs generated 8 haplotypes, i.e., haplotypes 1, 2, 3, 4, 5, 6, 7, and 8. Haplotypes 1, 2, and 8 had high frequencies (17.6%-23.5%). The c.588 A>G and c.612 C>G mutations were significantly associated (p A were significantly associated (p G and c.612 C>G mutations had higher IgY concentration and ND antibody titers (p<0.05) compared to the other haplotypes. In conclusion, this study has identified the CD1B gene as a polymorphic and is associated with IgY concentration and ND antibody titers in IPB-D1 chicken.

Molecular Analysis of 6-pyruvoyltetrahydropterin Synthase Gene in Atypical Phenylketonuric Egyptian Patients

Background Hyperphenylalaninemia (HPA) combined with neurological signs due to impaired catecholamine, dopamine and serotonin synthesis. Symptoms may appears in first week of life but most seen in age of 4 months. Atypical PKU disease caused mainly by deficiency in 6-pyruvoyltetrahydropterin synthase (PTPS) involved in synthesis of BH4. Clinical symptoms may include poor sucking, impaired tone, ataxia, and seizures. The purpose of this study was to analyze the genotype-phenotype relation among BH4 deficient patients because of PTPS mutations in different state of Egypt. Methods Suspected PKU patients loaded with phenylalanine/Kuvan, and the level of phe and phe/tyrosine ratio determined using tandem mass spectrometry by dried blood spots. Blood samples of 13 unrelated Egyptian patients were collected for total RNA extraction, amplification of PTPS gene by PCR followed with sequencing by Sanger method and finally mutations were recorded for genetic analysis. Results The mean value of phe in 13 patients decreased after loaded of phenylalanine from 482.5μmol/L to 270.63 μmol/L as well as phe/tyrosine ratio was decreased from 13.4 to 6.36 after 24hour of treatment with Kuvan. Sanger sequencing of PTPS gene of those patient showed 21 SNPs and Indels mutations. The most repeated mutation is a novel 23 base pair homozygous deletion in 12/13; c.200C&gt;T in four patients, a novel c.86A&gt;T in two patients and three different mutations located once in three different patients (novel c.22C&gt;T; novel c.273G&gt;A and 405T&gt;C) among patients. On amino acid predicted sequences 4 different types of mutations on protein level were presented, 1 deletion mutation in seven amino acid and 3 different missense mutations in addition to 2 silent mutations among 13 patients. Conclusion Patients were the first case of clinical diagnosis as hyperphenylalaninemia (HPA) undergoing genetic diagnosis for PTPS deficiency in Egypt. The sever HPA patients with severe nervous system damage mainly accompanied with deletion mutations and should pay more attention to the BH4 deficiency. While mild HPA is associated with base substitution mutations with mainly transition mutations (7/9; 78%). Next-generation sequencing technique can increase the mutation detection rate when the hereditary diseases are highly suspected in clinic.

Synonymous Mutation in DKC1 Causes Telomerase RNA Insufficiency Manifesting as Familial Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is the most common of short telomere phenotypes. Familial clustering of IPF is common, but the genetic basis remains unknown in more than one-half of cases. We identified a 65-year-old man with familial IPF, short telomere length, and low telomerase RNA levels. He was diagnosed with a short telomere syndrome after developing hematologic complications post-lung transplantation, but no mutations were identified in a clinical testing pipeline. What is the molecular basis underlying the familial IPF and low telomerase RNA levels in this patient? We analyzed whole-genome sequence data and performed functional molecular studies on cells derived from the patient and his family. We identified a previously unreported synonymous variant c.942G>A p.K314K in DKC1, the gene encoding the dyskerin ribonucleoprotein, which is required for telomerase RNA biogenesis. The mutation created a competing de novo exonic splicing enhancer, and the misspliced product was degraded by nonsense-mediated decay causing an overall dyskerin deficiency in mutation carriers. In silico tools identified other rare silent DKC1 variants that warrant functional evaluation if found in patients with short telomere-mediated disease. Our data point to silent mutation in telomere maintenance genes as a mechanism of familial pulmonary fibrosis. In contrast to DKC1 missense mutations, which primarily manifest in children as dyskeratosis congenita, hypomorphic mutations affecting dyskerin levels likely have a predilection to presenting in adults as pulmonary fibrosis.

Association of GHR Polymorphisms with Milk Production in Buffaloes.

Simple SummaryThe present study reported two missense mutations in the buffalo GHR gene: A novel (c.380G>A) and (c.836T>A) which was described in previous studies. These two single nucleotide polymorphisms (SNPs) were found to be associated with milk yield, fat %, protein %, and 305 day-milk, fat and protein yield, with higher performance for AA haplotype animals. Therefore, selection of buffaloes with AA haplotype would more likely improve milk production traits. Consequently, this would allow breeders to take more precise selection decisions, leading to significantly higher productivity and profitability within the Egyptian buffalo herds.For its role in the mediation of growth hormone (GH) galactopoietic effect, growth hormone receptor (GHR) was considered a functional candidate gene for milk performance in cattle. However, its genetic variation and potential effect have not been investigated in Egyptian buffaloes. This study aimed to screen GHR for polymorphisms and study their associations with milk traits in Egyptian buffaloes. Polymerase chain reaction, single-strand conformation polymorphism, and sequencing were used to identify mutations in 4 exons (E4–E6 and E8) of the GHR gene in 400 Egyptian buffaloes. No polymorphisms were found in E4, while 2 SNPs (c.380G>A/p.Arg127Lys and c.387C>T/p.Gly129) in E5, one silent mutation (c.435A>G/p.Pro145) in E6, and another missense mutation (c.836T>A/p.Phe279Tyr) in E8 were detected. The c.380G>A SNP in the extracellular domain was associated with milk yield, fat %, protein %, and 305-day milk, fat and protein yield, with higher levels in animals carrying the mutant A allele. The c.836T>A SNP in the transmembrane domain was associated with milk yield, fat %, protein %, and 305-day milk, fat and protein yield, with higher milk yield and lower fat %, protein %, fat and protein yield in the mutant A allele-animals. Interestingly, animals with the two mutant AA alleles produced higher milk yield, fat %, protein %, fat and protein yield, accompanied with upregulated expressions of GHR, GH, insulin-like growth factor 1 (IGF1), prolactin (PRL), prolactin receptor (PRLR), β-casein (encoded by CSN2 gene), and diacylglycerol acyltransferase-1 (DGAT1) genes and proteins in milk somatic cells. Therefore, selection of Egyptian buffaloes with mutant AA haplotypes for the novel c.380G>A SNP and the well-known c.836T>A SNP could improve milk yield and quality in buffaloes.

Human genes differ by their UV sensitivity estimated through analysis of UV-induced silent mutations in melanoma.

We hypothesized that human genes differ by their sensitivity to ultraviolet (UV) exposure. We used somatic mutations detected by genome-wide screens in melanoma and reported in the Catalog Of Somatic Mutations In Cancer. As a measure of UV sensitivity, we used the number of silent mutations generated by C>T transitions in pyrimidine dimers of a given transcript divided by the number of potential sites for this type of mutations in the transcript. We found that human genes varied by UV sensitivity by two orders of magnitude. We noted that the melanoma-associated tumor suppressor gene CDKN2A was among the top five most UV-sensitive genes in the human genome. Melanoma driver genes have a higher UV-sensitivity compared with other genes in the human genome. The difference was more prominent for tumor suppressors compared with oncogene. The results of this study suggest that differential sensitivity of human transcripts to UV light may explain melanoma specificity of some driver genes. Practical significance of the study relates to the fact that differences in UV sensitivity among human genes need to be taken into consideration whereas predicting melanoma-associated genes by the number of somatic mutations detected in a given gene.

Structural and functional conservation of the programmed −1 ribosomal frameshift signal of SARS coronavirus 2 (SARS-CoV-2)

Approximately 17 years after the severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, the world is currently facing the COVID-19 pandemic caused by SARS corona virus 2 (SARS-CoV-2). According to the most optimistic projections, it will take more than a year to develop a vaccine, so the best short-term strategy may lie in identifying virus-specific targets for small molecule–based interventions. All coronaviruses utilize a molecular mechanism called programmed −1 ribosomal frameshift (−1 PRF) to control the relative expression of their proteins. Previous analyses of SARS-CoV have revealed that it employs a structurally unique three-stemmed mRNA pseudoknot that stimulates high −1 PRF rates and that it also harbors a −1 PRF attenuation element. Altering −1 PRF activity impairs virus replication, suggesting that this activity may be therapeutically targeted. Here, we comparatively analyzed the SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar −1 PRF rates and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablate −1 PRF activity. We noted that the upstream attenuator hairpin activity is also functionally retained in both viruses, despite differences in the primary sequence in this region. Small-angle X-ray scattering analyses indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 have the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit −1 PRF was similarly effective against −1 PRF in SARS-CoV-2, suggesting that such frameshift inhibitors may be promising lead compounds to combat the current COVID-19 pandemic.

DGAT1 polymorphism in Egyptian Zaraibi goat breed and their association with milk yield and composition

BackgroundThe triacylglycerols in milk fat determine the physical and functional properties of dairy products rich in milk fat. Therefore, understanding the variability of genes related to fat synthesis is important for food production. We investigated the effect of diacylglycerol O-acyltransferase 1 (DGAT1) gene polymorphism on milk production parameters of the Zaraibi goat. Milk components were estimated by infrared spectroscopy. Moreover, Restriction Fragment Length Polymorphisms (RFLP) were used to detect genetic variants in DGAT1 genes. The amplified products were sequenced and aligned to the caprine reference sequence of this gene.ResultsTwo alleles (T and C) were identified in Zaraibi goats. The T allele resulted in one silent mutation while the C allele specified two-point mutations: one was located within a non-coding region (T703C) and the other (T713C) causing a Ile → Thr substitution in the deduced amino acid sequence. Moreover, the DGAT1 polymorphism significantly (p < 0.05) affected total solid content of milk, wherein harboring CC genotype had significantly higher amount of total solid than those with TC genotype. Milk contents and yields did not differ significantly between goats with CC and TC genotype.ConclusionThese results advance our understanding of the genetic architecture of Zaraibi milk composition and will help to improve the management and breeding program of the Egyptian dairy goat.Graphical

Abstract IA24: Molecular correlates of T cell-inflamed and non-T cell-inflamed tumors

Abstract IA24: Molecular correlates of T cell-inflamed and non-T cell-inflamed tumors

Rapid Evaluation of CRISPR Guides and Donors for Engineering Mice.

Although the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) technique has dramatically lowered the cost and increased the speed of generating genetically engineered mice, success depends on using guide RNAs and donor DNAs which direct efficient knock-out (KO) or knock-in (KI). By Sanger sequencing DNA from blastocysts previously injected with the same CRISPR components intended to produce the engineered mice, one can test the effectiveness of different guide RNAs and donor DNAs. We describe in detail here a simple, rapid (three days), inexpensive protocol, for amplifying DNA from blastocysts to determine the results of CRISPR point mutation KIs. Using it, we show that (1) the rate of KI seen in blastocysts is similar to that seen in mice for a given guide RNA/donor DNA pair, (2) a donor complementary to the variable portion of a guide integrated in a more all-or-none fashion, (3) donor DNAs can be used simultaneously to integrate two different mutations into the same locus, and (4) by placing silent mutations about every 6 to 10 bp between the Cas9 cut site and the desired mutation(s), the desired mutation(s) can be incorporated into genomic DNA over 30 bp away from the cut at the same high efficiency as close to the cut.

Mutation identification of unreported and reported mutation profile in exon 7 of N-Acetylgalactosamine-6-Sulfates (GALNS) gene of mucopolysaccharidosis type IV A (MPS IVA) patients in Indonesia

Mucopolysaccharidosis type IVA (MPS IVA), is an autosomal recessive genetic disorder because of N-acetylgalactosamine-6-sulfate deficiency which causes keratan sulfate and chondroitin sulfate to not degrade in lysosome. MPS IVA was caused of GALNS gene which located in chromosome 16q24.3 with the most frequent mutation occurrence in exon 7. There are no report or publication about MPS IVA or GALNS gene mutation in Indonesia, therefore this research is aimed to analyze the mutation profile in exon 7 of the GALNS gene in MPS IVA patients in Indonesia. The DNA from blood samples of four patients and three control samples from RSUPN Cipto Mangunkusumo were analyzed. Amplification by polymerase chain reaction was done after designing the primer. Furthermore, electrophoresis and sequencing analysis has been performed. The result shows that there is silent mutation c.708C>T and nonsense mutation c.751C>T. The silent mutation is categorized as a benign variant, while the nonsense mutation is categorized as a pathogenic variant because it may affect protein features and cause neuromuscular disorder. The silent and nonsense mutation that were found were already reported by Laradi and Morrone, but has not been reported in Indonesia. Further experiment was needed to find other mutation in other genes.