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Abstract Nucleotides across a genome do not mutate at equal frequencies. Instead, specific nucleotide positions can exhibit much higher mutation rates than the genomic average due to their immediate nucleotide neighbors. These “mutational hotspots” can play a prominent role in adaptive evolution, yet we lack knowledge of which short nucleotide sequences drive hotspots. In this work, we employ a combination of experimental evolution with Pseudomonas fluorescens and bioinformatic analysis of various Salmonella species to characterize a short nucleotide motif (≥8 bp) that can drive T:A→G:C mutation rates >1000-fold higher than the baseline T→G rate in bacteria. First, we experimentally confirm previous analysis showing that homopolymeric tracts (≥3) of G with a 3′ T frequently mutate so that the T is replaced with a G, resulting in an extension of the guanine tract, i.e. GGGT → GGGG. We then demonstrate that the potency of this T:A→G:C hotspot is dependent on the nucleotides immediately flanking the GnT sequence. We find that the dinucleotide immediately 5′ to a G4 tract and the dinucleotide immediately 3′ to the T strongly affect the T:A→G:C mutation rate, which ranges from ∼5-fold higher than the typical rate to over 1000-fold higher depending on the flanking elements. GnT motifs are therefore comprised of several modular nucleotide components which each exert a significant, quantifiable effect on the mutation rate. This work advances our ability to accurately identify the position and quantify the mutagenicity of hotspot motifs predicated on short nucleotide sequences.

Abstract Mutation—whilst stochastic—is frequently biased toward certain loci. When combined with selection, this results in highly repeatable and predictable evolutionary outcomes. Immotile variants of the bacterium Pseudomonas fluorescens (SBW25) possess a “mutational hotspot” that facilitates repeated occurrences of an identical de novo single nucleotide polymorphism when re-evolving motility, where ≥95% independent lines fix the mutation ntrB A289C. Identifying hotspots of similar potency in other genes and genomic backgrounds would prove valuable for predictive evolutionary models but to do so we must understand the genomic features that enable such a hotspot to form. Here, we reveal that genomic location, local nucleotide sequence, gene strandedness, and presence of mismatch repair proteins operate in combination to facilitate the formation of this mutational hotspot. Our study therefore provides a framework for utilizing genomic features to predict and identify hotspot positions capable of enforcing near-deterministic evolution.

(CCG) short tandem repeats (STRs) are predominantly enriched in genic regions, mutation hotspots for C to T truncating substitutions, and involved in various neurological and neurodevelopmental disorders. However, intact blocks of this class of STRs are widely overlooked with respect to their link with natural selection. The human neuron-specific gene, DISP2 (dispatched RND transporter family member 2), contains a (CCG) repeat in its 5′ untranslated region. Here, we sequenced this STR in a sample of 448 Iranian individuals, consisting of late-onset neurocognitive disorder (NCD) ( N  = 203) and controls ( N  = 245). We found that the region spanning the (CCG) repeat was highly mutated, resulting in several flanking (CCG) residues. However, an 8-repeat of the (CCG) repeat was predominantly abundant (frequency = 0.92) across the two groups. While the overall distribution of genotypes was not different between the two groups ( p  > 0.05), we detected four genotypes in the NCD group only (2% of the NCD genotypes, Mid- p  = 0.02), consisting of extreme short alleles, 5- and 6-repeats, that were not detected in the control group. The patients harboring those genotypes received the diagnoses of probable Alzheimer’s disease and vascular dementia. We also found six genotypes in the control group only (2.5% of the control genotypes, Mid- p  = 0.01) that consisted of the 8-repeat and extreme long alleles, 9- and 10-repeats, of which the 10-repeat was not detected in the NCD group. The (CCG) repeat specifically expanded in primates. In conclusion, we report an indication of natural selection at a novel hypermutable region in the human genome and divergent alleles and genotypes in late-onset NhCDs and controls. These findings reinforce the hypothesis that a collection of rare alleles and genotypes in a number of genes may unambiguously contribute to the cognition impairment component of late-onset NCDs.

Wheat powdery mildew is a devastating disease leading to severe yield loss. The powdery mildew resistance gene Pm21 , encoding a nucleotide-binding leucine-rich repeat receptor (NLR) protein, confers broad-spectrum resistance to powdery mildew and has great potential for controlling this disease. In this study, a large-scale mutagenesis was conducted on wheat cultivar (cv.) Yangmai 18 carrying Pm21 . As a result, a total of 113 independent mutant lines susceptible to powdery mildew were obtained, among which, only one lost the whole Pm21 locus and the other 112 harbored one- (107) or two-base (5) mutations in the encoding region of Pm21 . From the 107 susceptible mutants containing one-base change, we found that 25 resulted in premature stop codons leading to truncated proteins and 82 led to amino acid changes involving in 59 functional sites. We determined the mutations per one hundred amino acids (MPHA) indexes of different domains, motifs, and non-domain and non-motif regions of PM21 protein and found that the loss-of-function mutations occurred in a tendentious means. We also observed a new mutation hotspot that was closely linked to RNBS-D motif of the NB-ARC domain and relatively conserved in different NLRs of wheat crops. In addition, we crossed all the susceptible mutants with Yangmai 18 carrying wild-type Pm21 , subsequently phenotyped their F 1 plants and revealed that the variant E44K in the coiled-coil (CC) domain could lead to dominant-negative effect. This study revealed key functional sites of PM21 and their distribution characteristics, which would contribute to understanding the relationship of resistance and structure of Pm21 -encoded NLR.

Purpose: Mutation in the USH2A gene is the most common cause of inherited retinal dystrophy (IRD), including non-syndromic retinitis pigmentosa (RP) and Usher syndrome II (USH2). Gene editing and therapy targeting USH2A, especially the hotspot region, would benefit a large proportion of IRD patients. In this study, we comprehensively analyzed the genetic spectrum of the USH2A gene, aiming to identify global hot spot mutations in USH2A-related IRDs and differences in hot spot regions across continents. Method: A retrospective USH2A-related IRD study was conducted, including our IRD cohort, and reported USH2A studies worldwide. Results: A total of 3972 mutated USH2A alleles of approximately 1935 patients were collected from 33 cohort studies worldwide, containing 102 alleles of 51 patients in our IRD cohort. Mutations in exon 13 were the most common, reaching 18.4% globally and a higher frequency of 22% in America, 19.2% in Europe, and a lower 12% in East Asia. Pathogenic mutations that affected 10 of the 72 exons of USH2A, exon2, exon 13, exon 41-43, exon 50, exon 54, exon 57, exon 61, and exon 63 in total were responsible for half of global USH2A mutant alleles. With base editors including adenine base editor (ABE), cytidine base editor (CBE), and glycosylase base editor (GBE), 76.3% of single nucleotide variations (SNVs) and 58% of all mutations in USH2A are correctable. Meantime, four novel pathogenic mutations were revealed in our IRD cohort, p. (Val1130Cysfs*72), p. (Ala2139fs*14), p. (Gly4139Arg) and p. (Val4166Cysfs*7). Conclusion: In this study, we revealed four novel mutations, expanding the spectrum of USH2A mutations, and importantly presented global hotspot exons and mutations of USH2A as well as the proportion of SNVs that can be restored by different base editors, providing a perspective for exploring high-efficiency and broader-reaching gene editing and gene therapies.

DICER1 syndrome is an autosomal dominant inherited syndrome predisposing to various benign and malignant tumors, mainly occurring in children and young adults, requiring broad surveillance starting at birth with repeated irradiating imaging exams and sedations for young patients. It is caused by monoallelic germline pathogenic variants in the DICER1 gene. More than 90% of tumors bear an additional somatic DICER1 missense hotspot mutation, as a second hit, involving 1 of 6 codons clustered in exons 24 and 25. We designed and in vitro validated a drop-off droplet digital PCR (ddPCR) system to scan all DICER1 hotspot codons, allowing for a liquid biopsy test, an alternative to sedation and radiation exposure. Three drop-off ddPCR assays were designed, with 2 TaqMan probes per assay, 1 complementary to the wild-type sequence of the region containing hotspots and another 1 used as a reference. Eight tumor-derived DNAs and 5 synthetic oligonucleotides bearing DICER1 hotspot mutations were tested. All tested mutations were detected, with a limit of detection ranging from 0.07% to 0.31% for codons p. E1705, p. D1709, and p. D1713 in exon 24 and from 0.06% to 0.15% for codons p. G1809, p. D1810, and p. E1813 in exon 25. The high sensitivity of this method is compatible with its use for plasma circulating tumor DNA (ctDNA) analysis for early tumor detection in DICER1 syndrome patients. It may reduce the need for radiation exposure and sedation in surveillance protocols and may also improve patient prognosis. Clinical trials are needed to evaluate ctDNA analysis in these patients.

The advantage of an increasing amount of bioinformatics data on leukemias intrigued us to explore the hot-spot mutation profiles and investigate the implications of those hot-spot mutations in patient survival. We retrieved somatic mutations and their distribution in protein domains through data analysis of The Cancer Genome Atlas and cBioPortal databases. After determining differentially expressed mutant genes related to leukemia, we further conducted principal component analysis and single-factor Cox regression analyses. Moreover, survival analysis was performed for the obtained candidate genes, followed by a multi-factor Cox proportional hazard model method for the impacts of the candidate genes on the survival and prognosis of patients with leukemia. At last, the signaling pathways involved in leukemia were investigated by gene set enrichment analysis. There were 223 somatic missense mutation hot-spots identified with pertinence to leukemia, which were distributed in 41 genes. Differential expression in leukemia was witnessed in 39 genes. We found a close correlation between seven genes and the prognosis of leukemia patients, among which, three genes could significantly influence the survival rate. In addition, among these three genes, CD74 and P2RY8 were highlighted due to close pertinence with survival conditions of leukemia patients. Finally, data suggested that B cell receptor, Hedgehog, and TGF-beta signaling pathways were enriched in low-hazard patients. In conclusion, these data underline the involvement of hot-spot mutations of CD74 and P2RY8 genes in survival status of leukemia patients, highlighting their as novel therapeutic targets or prognostic indicators for leukemia patients.Summary of Graphical Abstract: We identified 223 leukemia-associated somatic missense mutation hotspots concentrated in 41 different genes from 2297 leukemia patients in the TCGA database. Differential analysis of leukemic and normal samples from the TCGA and GTEx databases revealed that 39 of these 41 genes showed significant differential expression in leukemia. These 39 genes were subjected to PCA analysis, univariate Cox analysis, survival analysis, multivariate Cox regression analysis, GSEA pathway enrichment analysis, and then the association with leukemia survival prognosis and related pathways were investigated.

Despite the great utility of mitochondrial DNA (mtDNA) sequence data in population genetics and phylogenetics, key parameters describing the process of mitochondrial mutation (e.g., the rate and spectrum of mutational change) are based on few direct estimates. Furthermore, the variation in the mtDNA mutation process within species or between lineages with contrasting reproductive strategies remains poorly understood. In this study, we directly estimate the mtDNA mutation rate and spectrum using Daphnia pulex mutation-accumulation (MA) lines derived from sexual (cyclically parthenogenetic) and asexual (obligately parthenogenetic) lineages. The nearly complete mitochondrial genome sequences of 82 sexual and 47 asexual MA lines reveal high mtDNA mutation rate of 1.37 × 10−7 and 1.73 × 10−7 per nucleotide per generation, respectively. The Daphnia mtDNA mutation rate is among the highest in eukaryotes, and its spectrum is dominated by insertions and deletions (70%), largely due to the presence of mutational hotspots at homopolymeric nucleotide stretches. Maximum likelihood estimates of the Daphnia mitochondrial effective population size reveal that between five and ten copies of mitochondrial genomes are transmitted per female per generation. Comparison between sexual and asexual lineages reveals no statistically different mutation rates and highly similar mutation spectra.

Objective The C216T mutation in the TERT promoter (TERTp) is a rarely reported genetic alteration in papillary thyroid carcinoma (PTC). Its clinical significance remains unclear. This study aimed to compare the impact of the C216T and hot spot mutations (C228T and C250T) of TERTp on the clinicopathologic characteristics and the expression of S100A10, a member of the S100 protein family, in PTC. Methods In this retrospective study, a cohort comprising 8 PTC cases with the C216T mutation, 12 cases with the hot spot mutations, and 120 cases with the wildtype genotype was established. The influence of TERTp mutations on the clinicopathologic profiles of PTC was assessed. Results The C216T mutation was mutually exclusive with the hot spot mutations and its frequency (0.19%) fell between that of C228T (0.68%) and C250T (0.06%). Compared to PTC cases with the wildtype genotype, cases with C216T mutations did not exhibit significant differences in clinicopathologic characteristics and S100A10 expression levels. In contrast, the hot spot mutations were positively associated with extrathyroidal extension ( p  = 0.001), ATA recurrence risk ( p  < 0.001), AJCC staging ( p  < 0.001), and increased expression of S100A10 ( p  = 0.005). Furthermore, a significant correlation was found between S100A10 expression and extrathyroidal extension ( p  = 0.005), lymph node metastasis ( p  = 0.013), and ATA recurrence risk ( p  = 0.023). Conclusion The C216T mutation did not induce the aggressiveness of PTC as the hot spot mutations did. Furthermore, the hot spot mutations were closely associated with the increased expression of S100A10. The latter may contribute to the pro-invasive effect of the hot spot mutations on PTC.

Background Multi‐cancer early detection (MCED) through a single blood test significantly advances cancer diagnosis. However, most MCED tests rely on a single type of biomarkers, leading to limited sensitivity, particularly for early‐stage cancers. We previously developed SPOT‐MAS, a multimodal ctDNA‐based assay analyzing methylation and fragmentomic profiles to detect five common cancers. Despite its potential, SPOT‐MAS exhibited moderate sensitivities for early‐stage cancers. This study investigated whether integrating hotspot mutations into SPOT‐MAS could enhance its detection rates. Method A targeted amplicon sequencing approach was developed to profile 700 hotspot mutations in cell‐free DNA and integrated into the SPOT‐MAS assay, creating a single‐blood draw workflow. This workflow, namely SPOT‐MAS Plus was retrospectively validated in a cohort of 255 non‐metastatic cancer patients (breast, colorectal, gastric, liver, and lung) and 304 healthy individuals. Results Hotspot mutations were detected in 131 of 255 (51.4%) cancer patients, with the highest rates in liver cancer (96.5%), followed by colorectal (59.3%) and lung cancer (53.7%). Lower detection rates were found for cancers with low tumor mutational burden, such as breast (31.3%) and gastric (41.9%) cancers. In contrast, SPOT‐MAS demonstrated higher sensitivities for these cancers (51.6% for breast and 62.9% for gastric). The combination of hotspot mutations with SPOT‐MAS predictions improved early‐stage cancer detection, achieving an overall sensitivity of 78.5% at a specificity of 97.7%. Enhanced sensitivities were observed for colorectal (81.36%) and lung cancer (82.9%). Conclusion The integration of genetic and epigenetic alterations into a multimodal assay significantly enhances the early detection of various cancers. Further validation in larger cohorts is necessary to support broader clinical applications.