Evaluation of the Patients with the Diagnosis of Pontocerebellar Hypoplasia: A Multicenter National Study.

Pontocerebellar hypoplasia (PCH) is a heterogeneous group of neurodegenerative disorders characterized by hypoplasia and degeneration of the cerebellum and pons. We aimed to identify the clinical, laboratory, and imaging findings of the patients with diagnosed PCH with confirmed genetic analysis. We collected available clinical data, laboratory, and imaging findings in our retrospective multicenter national study of 64 patients with PCH in Turkey. The genetic analysis included the whole-exome sequencing (WES), targeted next-generation sequencing (NGS), or single gene analysis. Sixty-four patients with PCH were 28 female (43.8%) and 36 (56.3%) male. The patients revealed homozygous mutation in 89.1%, consanguinity in 79.7%, pregnancy at term in 85.2%, microcephaly in 91.3%, psychomotor retardation in 98.4%, abnormal neurological findings in 100%, seizure in 63.8%, normal biochemistry and metabolic investigations in 92.2%, and dysmorphic findings in 51.2%. The missense mutation was found to be the most common variant type in all patients with PCH. It was detected as CLP1 (n = 17) was the most common PCH related gene. The homozygous missense variant c.419G > A (p.Arg140His) was identified in all patients with CLP1. Moreover, all patients showed the same homozygous missense variant c.919G > T (p.A307S) in TSEN54 group (n = 6). In Turkey, CLP1 was identified as the most common causative gene with the identical variant c.419G > A; p.Arg140His. The current study supports that genotype data on PCH leads to phenotypic variability over a wide phenotypic spectrum.

DPAGT1-CDG: Report of Two New Pediatric Patients and Brief Review of the Literature.

Introduction: Congenital glycosylation disorders are multisystem diseases with heterogeneous clinical manifestations caused by defects in the synthesis of the glycan moiety of glycoproteins or glycolipids or the binding of glycans to proteins and lipids. DPAGT1 (UDP-GlcNAc: dolichol phosphate N-acetylglucosamine-1-phosphotransferase) is an initiating protein in the biosynthetic pathway of dolichol-linked oligosaccharides required for protein N-glycosylation. Pathogenic variants in DPAGT1 (UDP-GlcNAc: dolichol phosphate N-acetylglucosamine-1-phosphotransferase) gene cause a rare type of congenital glycosylation disorder called DPAGT1-CDG (formerly CDG-Ij) (OMIM #608093). It is a rare autosomal recessive disease or a milder version with congenital myasthenic syndrome known as DPAGT1-CMS. A severe disease course with hypotonia, cataracts, skeletal deformities, resistant epilepsy, intellectual disability, global developmental delay, premature death has been described in most patients with DPAGT1-CDG. Patient Presentation: We describe two patients with variants in the DPAGT1 gene: an 8-month-old boy with a homozygous, missense DPAGT1:c.339T>G (p.Phe113Leu) novel variant and a 13-year-old female patient with compound heterozygous variants, DPAGT1:c.466C>T (p.Arg156Cys, R156C) and DPAGT1:c.161+5G>A. While the 8-month-old patient was diagnosed with congenital cataract at the age of 1 month, had dysmorphic findings, and epilepsy, clinical symptoms in the other patient appeared later but with more prominent muscle weakness, behavioral disorder, dysmorphic findings, and no epilepsy. Discussion: Cholinesterase inhibitor therapy was found to be effective in patients against muscle weakness, supporting DPAGT1 deficiency as the underlying etiology. We started pyridostigmine treatment in our patient with more pronounced muscle weakness, and we saw its benefit. We aimed to present our patients diagnosed with DPAGT1-CDG due to different variants in the same gene and different clinical presentations, treatment and to compare them with other patients in the literature.

The Relationship Between Cardiovascular Disease Risk and Major Depression.

Introduction: Cardiovascular risk in depression has been investigated in small clinical samples and population-based studies revealing inconclusive results. However, cardiovascular risk in drug-naive depressed patients has not been tested extensively. Methods: Body mass index-based Framingham Cardiovascular Risk Scores and soluble intercellular adhesion molecule-1 (sICAM-1) levels were used to assess the risk of cardiovascular disease in drug-naive depressed patients and healthy volunteers. Conclusion: There were no significant differences in Framingham Cardiovascular Risk Scores and individually assessed risk variables between patients and healthy controls (HC). Both groups were comparable in terms of sICAM-1. Results: The widely recognized association between cardiovascular risk and major depression might be more prominent in older depressed patients and patients with recurring episodes.

Associations Between Blood Levels of NLRP3 Inflammasome Components and Obsessive Compulsive Disorder.

Introduction: Even though the effect of inflammation on pathogenesis of obsessive compulsive disorder (OCD) is known, information regarding the underlying mechanisms are yet to be revealed. The NLRP3 inflammasome complex is an important component of the innate immune system that initiates and mediates inflammatory response to a variety of stimuli. This study aims to inquire into a possible association between NLRP3 inflammasome complex and OCD. Methods: This case-control study included 103 participants (51 cases with OCD and 52 healthy controls). All participants were evaluated with the Yale Brown Obsessive Compulsive Scale, Hamilton Depression Scale, and Hewitt Multidimensional Perfectionism Scale. RNA and proteins were extracted from peripheral blood mononuclear cells. Expression of NLRP3 inflammasome components were determined using quantitative real-time polymerase chain reaction (PCR) and Western blotting. Levels of Serum IL-1beta and IL-18 cytokine were determined by ELISA. Results: NEK7 and CASP1 mRNA levels were significantly higher in OCD patients, compared to controls. Pro-caspase-1 protein levels were elevated, as well. Regression analysis showed that NEK7 mRNA and pro-caspase-1 protein levels can differentiate OCD and healthy control groups. Conclusion: Our results provide insight into the molecular alterations that could explain the inflammation-OCD association.

Bi-allelic variants in the ESAM tight-junction gene cause a neurodevelopmental disorder associated with fetal intracranial hemorrhage.

The blood-brain barrier (BBB) is an essential gatekeeper for the central nervous system and incidence of neurodevelopmental disorders (NDDs) is higher in infants with a history of intracerebral hemorrhage (ICH). We discovered a rare disease trait in thirteen individuals, including four fetuses, from eight unrelated families associated with homozygous loss-of-function variant alleles of ESAM which encodes an endothelial cell adhesion molecule. The c.115del (p.Arg39Glyfs∗33) variant, identified in six individuals from four independent families of Southeastern Anatolia, severely impaired the in vitro tubulogenic process of endothelial colony-forming cells, recapitulating previous evidence in null mice, and caused lack of ESAM expression in the capillary endothelial cells of damaged brain. Affected individuals with bi-allelic ESAM variants showed profound global developmental delay/unspecified intellectual disability, epilepsy, absent or severely delayed speech, varying degrees of spasticity, ventriculomegaly, and ICH/cerebral calcifications, the latter being also observed in the fetuses. Phenotypic traits observed in individuals with bi-allelic ESAM variants overlap very closely with other known conditions characterized by endothelial dysfunction due to mutation of genes encoding tight junction molecules. Our findings emphasize the role of brain endothelial dysfunction in NDDs and contribute to the expansion of an emerging group of diseases that we propose to rename as "tightjunctionopathies."

An in silico approach to the identification of diagnostic and prognostic markers in low-grade gliomas.

Low-grade gliomas (LGG) are central nervous system Grade I tumors, and as they progress they are becoming one of the deadliest brain tumors. There is still great need for timely and accurate diagnosis and prognosis of LGG. Herein, we aimed to identify diagnostic and prognostic biomarkers associated with LGG, by employing diverse computational approaches. For this purpose, differential gene expression analysis on high-throughput transcriptomics data of LGG versus corresponding healthy brain tissue, derived from TCGA and GTEx, respectively, was performed. Weighted gene co-expression network analysis of the detected differentially expressed genes was carried out in order to identify modules of co-expressed genes significantly correlated with LGG clinical traits. The genes comprising these modules were further used to construct gene co-expression and protein-protein interaction networks. Based on the network analyses, we derived a consensus of eighteen hub genes, namely, CD74, CD86, CDC25A, CYBB, HLA-DMA, ITGB2, KIF11, KIFC1, LAPTM5, LMNB1, MKI67, NCKAP1L, NUSAP1, SLC7A7, TBXAS1, TOP2A, TYROBP, and WDFY4. All detected hub genes were up-regulated in LGG, and were also associated with unfavorable prognosis in LGG patients. The findings of this study could be applicable in the clinical setting for diagnosing and monitoring LGG.

Whole Genome Analysis of Dizygotic Twins With Autism Reveals Prevalent Transposon Insertion Within Neuronal Regulatory Elements: Potential Implications for Disease Etiology and Clinical Assessment.

Transposable elements (TEs) have been implicated in autism spectrum disorder (ASD). However, our understanding of their roles is far from complete. Herein, we explored de novo TE insertions (dnTEIs) and de novo variants (DNVs) across the genomes of dizygotic twins with ASD and their parents. The neuronal regulatory elements had a tendency to harbor dnTEIs that were shared between twins, but ASD-risk genes had dnTEIs that were unique to each twin. The dnTEIs were 4.6-fold enriched in enhancers that are active in embryonic stem cell (ESC)-neurons (p < 0.001), but DNVs were 1.5-fold enriched in active enhancers of astrocytes (p = 0.0051). Our findings suggest that dnTEIs and DNVs play a role in ASD etiology by disrupting enhancers of neurons and astrocytes.

StemnesScoRe: an R package to estimate the stemness of glioma cancer cells at single-cell resolution.

Background/aim: Glioblastoma is the most heterogeneous and the most difficult-to-treat type of brain tumor and one of the deadliest among all cancers. The high plasticity of glioma cancer stem cells and the resistance they develop against multiple modalities of therapy, along with their high heterogeneity, are the main challenges faced during treatment of glioblastoma. Therefore, a better understanding of the stemness characteristics of glioblastoma cells is needed. With the development of various single-cell technologies and increasing applications of machine learning, indices based on transcriptomic and/or epigenomic data have been developed to quantitatively measure cellular states and stemness. In this study, we aimed to develop a glioma-specific stemness score model using scATAC-seq data for the first time. Materials and methods: We first applied three powerful machine-learning algorithms, i.e. random forest, gradient boosting, and extreme gradient boosting, to glioblastoma scRNA-seq data to discover the most important genes associated with cellular states. We then identified promoter and enhancer regions associated with these genes. After downloading the scATAC-seq peaks and their read counts for each patient, we identified the overlapping regions between the single-cell peaks and the peaks of genes obtained through machine-learning algorithms. Then we calculated read counts that were mapped to these overlapping regions. We finally developed a model capable of estimating the stemness score for each glioma cell using overlapping regions and the importance of genes predictive of glioblastoma cellular states. We also created an R package, accessible to all researchers regardless of their coding proficiency. Results: Our results showed that mesenchymal-like stem cells display higher stemness scores compared to neural-progenitor-, oligodendrocyte-progenitor-, and astrocyte-like cells. Conclusion: scATAC-seq can be used to assess heterogeneity in glioblastoma and identify cells with high stemness characteristics. The package is publicly available at https://github.com/Necla/StemnesScoRe and includes documentation with implementation of a real-data experiment.

Could lysosomal acid lipase enzyme activity be used for clinical follow-up in cryptogenic cirrhosis?

BACKGROUND: Cholesterol ester storage disease (CESD) is one of the rare causes that should be kept in mind in the etiology of cirrhosis. Recent studies detected that significantly reduced lysosomal acid lipase deficiency enzyme (LAL) in patients with cryptogenic cirrhosis (CC). Moreover, studies have evaluated that LAL activity is as effective as scoring systems in assessing the severity of cirrhosis. In this study, we aimed to investigate the CESD with LAL level and mutation analysis of LIPA gene in patients diagnosed with CC and to compare LAL activities between patients with CC and healthy volunteers. METHODS: Laboratory parameters and cirrhosis stage (CHILD and MELD) were recorded for the patient group included in the study. In addition, blood samples were taken from each case included in the study for LAL activity determination and LIPA gene analysis. RESULTS: A statistically significant decrease in LAL activity was found in patients diagnosed with CC compared to the healthy group. LIPA gene analysis did not detect CESD in any patient group. Correlation analysis showed a positive correlation between LAL activity and white blood cell and platelet counts in both healthy volunteers and CC patient groups. In the univariate and multivariate logistic regression analysis of the parameters associated with the MELD of ≥10 in patients with CC, significant relationship was found between the MELD of ≥10 and the LAL activity. DISCUSSION: In our study, LAL activity was significantly lower in CC patients than in the normal population. LAL activity level appears to be a parameter that can be used to assess the severity of cirrhosis.

Novel insights into PORCN mutations, associated phenotypes and pathophysiological aspects.

BACKGROUND: Goltz syndrome (GS) is a X-linked disorder defined by defects of mesodermal- and ectodermal-derived structures and caused by PORCN mutations. Features include striated skin-pigmentation, ocular and skeletal malformations and supernumerary or hypoplastic nipples. Generally, GS is associated with in utero lethality in males and most of the reported male patients show mosaicism (only three non-mosaic surviving males have been described so far). Also, precise descriptions of neurological deficits in GS are rare and less severe phenotypes might not only be caused by mosaicism but also by less pathogenic mutations suggesting the need of a molecular genetics and functional work-up of these rare variants. RESULTS: We report two cases: one girl suffering from typical skin and skeletal abnormalities, developmental delay, microcephaly, thin corpus callosum, periventricular gliosis and drug-resistant epilepsy caused by a PORCN nonsense-mutation (c.283C > T, p.Arg95Ter). Presence of these combined neurological features indicates that CNS-vulnerability might be a guiding symptom in the diagnosis of GS patients. The other patient is a boy with a supernumerary nipple and skeletal anomalies but also, developmental delay, microcephaly, cerebral atrophy with delayed myelination and drug-resistant epilepsy as predominant features. Skin abnormalities were not observed. Genotyping revealed a novel PORCN missense-mutation (c.847G > C, p.Asp283His) absent in the Genome Aggregation Database (gnomAD) but also identified in his asymptomatic mother. Given that non-random X-chromosome inactivation was excluded in the mother, fibroblasts of the index had been analyzed for PORCN protein-abundance and -distribution, vulnerability against additional ER-stress burden as well as for protein secretion revealing changes. CONCLUSIONS: Our combined findings may suggest incomplete penetrance for the p.Asp283His variant and provide novel insights into the molecular etiology of GS by adding impaired ER-function and altered protein secretion to the list of pathophysiological processes resulting in the clinical manifestation of GS.

High diagnostic rate of trio exome sequencing in consanguineous families with neurogenetic diseases.

Consanguineous marriages have a prevalence rate of 24% in Turkey. These carry an increased risk of autosomal recessive genetic conditions, leading to severe disability or premature death, with a significant health and economic burden. A definitive molecular diagnosis could not be achieved in these children previously, as infrastructures and access to sophisticated diagnostic options were limited. We studied the cause of neurogenetic disease in 246 children from 190 consanguineous families recruited in three Turkish hospitals between 2016 and 2020. All patients underwent deep phenotyping and trio whole exome sequencing, and data were integrated in advanced international bioinformatics platforms. We detected causative variants in 119 known disease genes in 72% of families. Due to overlapping phenotypes 52% of the confirmed genetic diagnoses would have been missed on targeted diagnostic gene panels. Likely pathogenic variants in 27 novel genes in 14% of the families increased the diagnostic yield to 86%. Eighty-two per cent of causative variants (141/172) were homozygous, 11 of which were detected in genes previously only associated with autosomal dominant inheritance. Eight families carried two pathogenic variants in different disease genes. De novo (9.3%), X-linked recessive (5.2%) and compound heterozygous (3.5%) variants were less frequent compared to non-consanguineous populations. This cohort provided a unique opportunity to better understand the genetic characteristics of neurogenetic diseases in a consanguineous population. Contrary to what may be expected, causative variants were often not on the longest run of homozygosity and the diagnostic yield was lower in families with the highest degree of consanguinity, due to the high number of homozygous variants in these patients. Pathway analysis highlighted that protein synthesis/degradation defects and metabolic diseases are the most common pathways underlying paediatric neurogenetic disease. In our cohort 164 families (86%) received a diagnosis, enabling prevention of transmission and targeted treatments in 24 patients (10%). We generated an important body of genomic data with lasting impacts on the health and wellbeing of consanguineous families and economic benefit for the healthcare system in Turkey and elsewhere. We demonstrate that an untargeted next generation sequencing approach is far superior to a more targeted gene panel approach, and can be performed without specialized bioinformatics knowledge by clinicians using established pipelines in populations with high rates of consanguinity.

Bi-allelic variants in SPATA5L1 lead to intellectual disability, spastic-dystonic cerebral palsy, epilepsy, and hearing loss.

Spermatogenesis-associated 5 like 1 (SPATA5L1) represents an orphan gene encoding a protein of unknown function. We report 28 bi-allelic variants in SPATA5L1 associated with sensorineural hearing loss in 47 individuals from 28 (26 unrelated) families. In addition, 25/47 affected individuals (53%) presented with microcephaly, developmental delay/intellectual disability, cerebral palsy, and/or epilepsy. Modeling indicated damaging effect of variants on the protein, largely via destabilizing effects on protein domains. Brain imaging revealed diminished cerebral volume, thin corpus callosum, and periventricular leukomalacia, and quantitative volumetry demonstrated significantly diminished white matter volumes in several individuals. Immunofluorescent imaging in rat hippocampal neurons revealed localization of Spata5l1 in neuronal and glial cell nuclei and more prominent expression in neurons. In the rodent inner ear, Spata5l1 is expressed in the neurosensory hair cells and inner ear supporting cells. Transcriptomic analysis performed with fibroblasts from affected individuals was able to distinguish affected from controls by principal components. Analysis of differentially expressed genes and networks suggested a role for SPATA5L1 in cell surface adhesion receptor function, intracellular focal adhesions, and DNA replication and mitosis. Collectively, our results indicate that bi-allelic SPATA5L1 variants lead to a human disease characterized by sensorineural hearing loss (SNHL) with or without a nonprogressive mixed neurodevelopmental phenotype.

Alternative splicing and gene co-expression network-based analysis of dizygotic twins with autism-spectrum disorder and their parents.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with high heritability, however, understanding the complexity of the underlying genetic basis has proven to be a challenging task. We hypothesized that dissecting the aberrations in alternative splicing (AS) and their effects on expression networks might provide insight. Therefore, we performed AS and co-expression analyses of total RNA isolated from Peripheral Blood Mononuclear Cells (PBMCs) of two pairs of dizygotic (DZ) twins with non-syndromic autism and their parents. We identified 183 differential AS events in 146 genes, seven of them being Simons Foundation Autism Research Initiative (SFARI) Category 1-3 genes, three of which had previously been reported to be alternatively spliced in ASD post-mortem brains. Gene co-expression analysis identified 7 modules with 513 genes, 5 of which were SFARI Category 1 or Category 2 genes. Among differentially AS genes within the modules, ZNF322 and NR4A1 could be potentially interesting targets for further investigations.

Different selection dynamics of S and RdRp between SARS-CoV-2 genomes with and without the dominant mutations.

SARS-CoV-2 is a betacoronavirus responsible for the COVID-19 pandemic that has affected millions of people worldwide. Pharmaceutical research against COVID-19 and the most frequently used tests for SARS-CoV-2 both depend on the genomic and peptide sequences of the virus for their robustness. Therefore, understanding the mutation rates and content of the virus is critical. Two key proteins for SARS-CoV-2 infection and replication are the S protein, responsible for viral entry into the cells, and RdRp, the RNA polymerase responsible for replicating the viral genome. Due to their roles in the viral cycle, these proteins are crucial for the fitness and infectiousness of the virus. Our previous findings had shown that the two most frequently observed mutations in the SARS-CoV-2 genome, 14408C>T in the RdRp coding region, and 23403A>G in the S gene, are correlated with higher mutation density over time. In this study, we further detail the selection dynamics and the mutation rates of SARS-CoV-2 genes, comparing them between isolates carrying both mutations, and isolates carrying neither. We find that the S gene and the RdRp coding region show the highest variance between the genotypes, and their selection dynamics contrast each other over time. The S gene displays higher tolerance for positive selection in mutant isolates early during the appearance of the double mutant genotype, and undergoes increasing negative selection over time, whereas the RdRp region in the mutant isolates shows strong negative selection throughout the pandemic.

Current mutatome of SARS-CoV-2 in Turkey reveals mutations of interest.

As the underlying pathogen for the COVID-19 pandemic that has affected tens of millions of lives worldwide, SARS-CoV-2 and its mutations are among the most urgent research topics worldwide. Mutations in the virus genome can complicate attempts at accurate testing or developing a working treatment for the disease. Furthermore, because the virus uses its own proteins to replicate its genome, rather than host proteins, mutations in the replication proteins can have cascading effects on the mutation load of the virus genome. Due to the global, rapidly developing nature of the COVID-19 pandemic, local demographics of the virus can be difficult to accurately analyze and track, disproportionate to the importance of such information. Here, we analyzed available, high-quality genome data of SARS-CoV-2 isolates from Turkey and identified their mutations, in comparison to the reference genome, to understand how the local mutatome compares to the global genomes. Our results indicate that viral genomes in Turkey has one of the highest mutation loads and certain mutations are remarkably frequent compared to global genomes. We also made the data on Turkey isolates available on an online database to facilitate further research on SARS-CoV-2 mutations in Turkey.

Autosomal recessive variants in TUBGCP2 alter the γ-tubulin ring complex leading to neurodevelopmental disease.

Microtubules help building the cytoskeleton of neurons and other cells. Several components of the gamma-tubulin (γ-tubulin) complex have been previously reported in human neurodevelopmental diseases. We describe two siblings from a consanguineous Turkish family with dysmorphic features, developmental delay, brain malformation, and epilepsy carrying a homozygous mutation (p.Glu311Lys) in TUBGCP2 encoding the γ-tubulin complex 2 (GCP2) protein. This variant is predicted to disrupt the electrostatic interaction of GCP2 with GCP3. In primary fibroblasts carrying the variant, we observed a faint delocalization of γ-tubulin during the cell cycle but normal GCP2 protein levels. Through mass spectrometry, we observed dysregulation of multiple proteins involved in the assembly and organization of the cytoskeleton and the extracellular matrix, controlling cellular adhesion and of proteins crucial for neuronal homeostasis including axon guidance. In summary, our functional and proteomic studies link TUBGCP2 and the γ-tubulin complex to the development of the central nervous system in humans.

Mutations and Copy Number Alterations in IDH Wild-Type Glioblastomas Are Shaped by Different Oncogenic Mechanisms.

Little is known about the mutational processes that shape the genetic landscape of gliomas. Numerous mutational processes leave marks on the genome in the form of mutations, copy number alterations, rearrangements or their combinations. To explore gliomagenesis, we hypothesized that gliomas with different underlying oncogenic mechanisms would have differences in the burden of various forms of these genomic alterations. This was an analysis on adult diffuse gliomas, but IDH-mutant gliomas as well as diffuse midline gliomas H3-K27M were excluded to search for the possible presence of new entities among the very heterogenous group of IDH-WT glioblastomas. The cohort was divided into two molecular subsets: (1) Molecularly-defined GBM (mGBM) as those that carried molecular features of glioblastomas (including TERT promoter mutations, 7/10 pattern, or EGFR-amplification), and (2) those who did not (others). Whole exome sequencing was performed for 37 primary tumors and matched blood samples as well as 8 recurrences. Single nucleotide variations (SNV), short insertion or deletions (indels) and copy number alterations (CNA) were quantified using 5 quantitative metrics (SNV burden, indel burden, copy number alteration frequency-wGII, chromosomal arm event ratio-CAER, copy number amplitude) as well as 4 parameters that explored underlying oncogenic mechanisms (chromothripsis, double minutes, microsatellite instability and mutational signatures). Findings were validated in the TCGA pan-glioma cohort. mGBM and "Others" differed significantly in their SNV (only in the TCGA cohort) and CNA metrics but not indel burden. SNV burden increased with increasing age at diagnosis and at recurrences and was driven by mismatch repair deficiency. On the contrary, indel and CNA metrics remained stable over increasing age at diagnosis and with recurrences. Copy number alteration frequency (wGII) correlated significantly with chromothripsis while CAER and CN amplitude correlated significantly with the presence of double minutes, suggesting separate underlying mechanisms for different forms of CNA.

Mutations of SARS-CoV-2 nsp14 exhibit strong association with increased genome-wide mutation load.

SARS-CoV-2 is a betacoronavirus responsible for COVID-19, a pandemic with global impact that first emerged in late 2019. Since then, the viral genome has shown considerable variance as the disease spread across the world, in part due to the zoonotic origins of the virus and the human host adaptation process. As a virus with an RNA genome that codes for its own genomic replication proteins, mutations in these proteins can significantly impact the variance rate of the genome, affecting both the survival and infection rate of the virus, and attempts at combating the disease. In this study, we analyzed the mutation densities of viral isolates carrying frequently observed mutations for four proteins in the RNA synthesis complex over time in comparison to wildtype isolates. Our observations suggest mutations in nsp14, an error-correcting exonuclease protein, have the strongest association with increased mutation load without selective pressure and across the genome, compared to nsp7, nsp8 and nsp12, which form the core polymerase complex. We propose nsp14 as a priority research target for understanding genomic variance rate in SARS-CoV-2 isolates and nsp14 mutations as potential predictors for high mutability strains.

Mutation density changes in SARS-CoV-2 are related to the pandemic stage but to a lesser extent in the dominant strain with mutations in spike and RdRp.

Since its emergence in Wuhan, China in late 2019, the origin and evolution of SARS-CoV-2 have been among the most debated issues related to COVID-19. Throughout its spread around the world, the viral genome continued acquiring new mutations and some of them became widespread. Among them, 14408 C>T and 23403 A>G mutations in RdRp and S, respectively, became dominant in Europe and the US, which led to debates regarding their effects on the mutability and transmissibility of the virus. In this study, we aimed to investigate possible differences between time-dependent variation of mutation densities (MDe) of viral strains that carry these two mutations and those that do not. Our analyses at the genome and gene level led to two important findings: First, time-dependent changes in the average MDe of circulating SARS-CoV-2 genomes showed different characteristics before and after the beginning of April, when daily new case numbers started levelling off. Second, this pattern was much delayed or even non-existent for the "mutant" (MT) strain that harbored both 14408 C>T and 23403 A>G mutations. Although these differences were not limited to a few hotspots, it is intriguing that the MDe increase is most evident in two critical genes, S and Orf1ab, which are also the genes that harbor the defining mutations of the MT genotype. The nature of these unexpected relationships warrants further research.