Putative Deleterious Mutations Research Articles

Does chromosome 16p12.1-p12.2 harbor a novel gene for essential tremor? (P5-11.012)

<h3>Objective:</h3> To describe a patient with de novo duplication of 16p12.1-p12.2 associated with severe action tremor and developmental delay, and genetic analysis of candidate genes within the duplicated chromosomal segment. <h3>Background:</h3> A family history of essential tremor (ET) can be found in 30–80% of all affected patients, suggesting a strong genetic contribution to its etiology. However, discovery of disease-causing genes has been largely elusive. Candidate genes can be identified using linkage analysis, whole exome or genome sequencing, and genetic association studies. Detection of genomic rearrangements with gene dosage changes in tremor patients provides another unique opportunity to identify additional candidate genes. <h3>Design/Methods:</h3> Case report and genetic analysis of the candidate chromosomal region. Chromosomal microarray analysis was performed in patient and both parents. We designed PCR primers for all coding exons of selected candidate genes. We included 75 unrelated probands from kindreds with familiar ET. Candidate genes were sequenced in all ET controls. <h3>Results:</h3> The proband is a 20-year-old female with significant developmental delay who developed progressive tremor at age of 15 years. Her tremor was a moderate to severe 4–5 Hz postural and action distal tremor, not present at rest, minimally responsive to propranolol. Additional examination revealed diffusely brisk reflexes with positive Hoffman and cross adductor sign, and upgoing toes. MRI brain did not show evidence of hypomyelination. She underwent chromosomal microarray analysis that identified duplications 12p13.33 and 16p12.2-p12.1. Chromosome 12p duplication was also detected in her father. Duplication of 12p was considered nonpathogenic and duplication 16p was <i>de novo</i>. The duplicated segment on chromosome 16p contained eight genes and six were considered possible candidate for tremor: <i>UQCRC2</i>, <i>CDR2</i>, <i>EEF2K</i>, <i>POLR3A</i>, <i>LOG23117</i>, and c16orf65. No putative deleterious mutations have been identified in ET controls. <h3>Conclusions:</h3> We report identification and genetic analysis of six novel candidate genes on chromosome 16p12.1-p12.2 that may be associated with ET. <b>Disclosure:</b> Dr. D’Aguiar Rosa has nothing to disclose. Dr. Yousaf has nothing to disclose. Dr. Hedera has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Alexion. Dr. Hedera has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Abbvie. Dr. Hedera has received personal compensation in the range of $5,000-$9,999 for serving as an Editor, Associate Editor, or Editorial Advisory Board Member for University of Louisville.

Genomic Impact of Whaling in North Atlantic Fin Whales.

It is generally recognized that large-scale whaling in the 19th and 20th century led to a substantial reduction of the size of many cetacean populations, particularly those of the baleen whales (Mysticeti). The impact of these operations on genomic diversity of one of the most hunted whales, the fin whale (Balaenoptera physalus), has remained largely unaddressed because of the paucity of adequate samples and the limitation of applicable techniques. Here, we have examined the effect of whaling on the North Atlantic fin whale based on genomes of 51 individuals from Icelandic waters, representing three temporally separated intervals, 1989, 2009 and 2018 and provide a reference genome for the species. Demographic models suggest a noticeable drop of the effective population size of the North Atlantic fin whale around a century ago. The present results suggest that the genome-wide heterozygosity is not markedly reduced and has remained comparable with other baleen whale species. Similarly, there are no signs of apparent inbreeding, as measured by the proportion of long runs of homozygosity, or of a distinctively increased mutational load, as measured by the amount of putative deleterious mutations. Compared with other baleen whales, the North Atlantic fin whale appears to be less affected by anthropogenic influences than other whales such as the North Atlantic right whale, consistent with the presence of long runs of homozygosity and higher levels of mutational load in an otherwise more heterozygous genome. Thus, genome-wide assessments of other species and populations are essential for future, more specific, conservation efforts.

In-silico analysis unravels the structural and functional consequences of non-synonymous SNPs in the human IL-10 gene

BackgroundInterleukin-10 (IL-10) is an anti-inflammatory cytokine that affects different immune cells. It is also associated with the stimulation of the T and B cells for the production of antibodies. Several genetic polymorphisms in the IL-10 gene have been reported to cause or aggravate certain diseases like inflammatory bowel disease, rheumatoid arthritis, systemic sclerosis, asthma, etc. However, the disease susceptibility and abnormal function of the mutated IL-10 variants remain obscure.ResultsIn this study, we used seven bioinformatics tools (SIFT, PROVEAN, PMut, PANTHER, PolyPhen-2, PHD-SNP, and SNPs&GO) to predict the disease susceptible non-synonymous SNPs (nsSNPs) of IL-10. Nine nsSNPs of IL-10 were predicted to be potentially deleterious: R42G, R45Q, F48L, E72G, M95T, A98D, R125S, Y155C, and I168T. Except two, all of the putative deleterious mutations are found in the highly conserved region of IL-10 protein structure, thus affecting the protein's stability. The 3-D structure of mutant proteins was modeled by project HOPE, and the protein–protein interactions were assessed with STRING. The predicted nsSNPs: R42Q, R45Q, F48L, E72G, and I168T are situated in the binding site region of the IL-10R1 receptor. Disruption of binding affinity with its receptor leads to deregulation of the JAK-STAT pathway and results in enhanced inflammation that imbalance in cellular signaling. Finally, Kaplan–Meier Plotter analysis displayed that deregulation of IL-10 expression affects gastric and ovarian cancer patients' survival rate. Thus, IL-10 could be useful as a potential prognostic marker gene for some cancers.ConclusionThis study has determined the deleterious nsSNPs of IL-10 that might contribute to the malfunction of IL-10 protein and ultimately lead to the IL-10 associated diseases.

The patterns of deleterious mutations during the domestication of soybean

Globally, soybean is a major protein and oil crop. Enhancing our understanding of the soybean domestication and improvement process helps boost genomics-assisted breeding efforts. Here we present a genome-wide variation map of 10.6 million single-nucleotide polymorphisms and 1.4 million indels for 781 soybean individuals which includes 418 domesticated (Glycine max), 345 wild (Glycine soja), and 18 natural hybrid (G. max/G. soja) accessions. We describe the enhanced detection of 183 domestication-selective sweeps and the patterns of putative deleterious mutations during domestication and improvement. This predominantly selfing species shows 7.1% reduction of overall deleterious mutations in domesticated soybean relative to wild soybean and a further 1.4% reduction from landrace to improved accessions. The detected domestication-selective sweeps also show reduced levels of deleterious alleles. Importantly, genotype imputation with this resource increases the mapping resolution of genome-wide association studies for seed protein and oil traits in a soybean diversity panel.

Adaptive and maladaptive genetic diversity in small populations: Insights from the Brook Charr (Salvelinus fontinalis) case study.

Investigating the relative importance of neutral versus selective processes governing the accumulation of genetic variants is a key goal in both evolutionary and conservation biology. This is particularly true in the context of small populations, where genetic drift can counteract the effect of selection. Using Brook Charr (Salvelinus fontinalis) from Québec, Canada, as a case study, we investigated the importance of demographic versus selective processes governing the accumulation of both adaptive and maladaptive mutations in closed versus open and connected populations to assess gene flow effect. This was achieved by using 14,779 high-quality filtered SNPs genotyped among 1,416 fish representing 50 populations from three life history types: lacustrine (closed populations), riverine and anadromous (connected populations). Using the PROVEAN algorithm, we observed a considerable accumulation of putative deleterious mutations across populations. The absence of correlation between the occurrence of putatively beneficial or deleterious mutations and local recombination rate supports the hypothesis that genetic drift might be the main driver of the accumulation of such variants. However, despite a lower genetic diversity observed in lacustrine than in riverine or anadromous populations, lacustrine populations do not exhibit more deleterious mutations than the two other history types, suggesting that the negative effect of genetic drift in lacustrine populations may be mitigated by that of relaxed purifying selection. Moreover, we also identified genomic regions associated with anadromy, as well as an overrepresentation of transposable elements associated with variation in environmental variables, thus supporting the importance of transposable elements in adaptation.

Functional Architecture of Deleterious Genetic Variants in the Genome of a Wrangel Island Mammoth.

Woolly mammoths were among the most abundant cold-adapted species during the Pleistocene. Their once-large populations went extinct in two waves, an end-Pleistocene extinction of continental populations followed by the mid-Holocene extinction of relict populations on St. Paul Island ∼5,600 years ago and Wrangel Island ∼4,000 years ago. Wrangel Island mammoths experienced an episode of rapid demographic decline coincident with their isolation, leading to a small population, reduced genetic diversity, and the fixation of putatively deleterious alleles, but the functional consequences of these processes are unclear. Here, we show that a Wrangel Island mammoth genome had many putative deleterious mutations that are predicted to cause diverse behavioral and developmental defects. Resurrection and functional characterization of several genes from the Wrangel Island mammoth carrying putatively deleterious substitutions identified both loss and gain of function mutations in genes associated with developmental defects (HYLS1), oligozoospermia and reduced male fertility (NKD1), diabetes (NEUROG3), and the ability to detect floral scents (OR5A1). These data suggest that at least one Wrangel Island mammoth may have suffered adverse consequences from reduced population size and isolation.

Deleterious Mutation Burden and Its Association with Complex Traits in Sorghum (Sorghum bicolor).

Sorghum (Sorghum bicolor L.) is a major food cereal for millions of people worldwide. The sorghum genome, like other species, accumulates deleterious mutations, likely impacting its fitness. The lack of recombination, drift, and the coupling with favorable loci impede the removal of deleterious mutations from the genome by selection. To study how deleterious variants impact phenotypes, we identified putative deleterious mutations among ∼5.5 M segregating variants of 229 diverse biomass sorghum lines. We provide the whole-genome estimate of the deleterious burden in sorghum, showing that ∼33% of nonsynonymous substitutions are putatively deleterious. The pattern of mutation burden varies appreciably among racial groups. Across racial groups, the mutation burden correlated negatively with biomass, plant height, specific leaf area (SLA), and tissue starch content (TSC), suggesting that deleterious burden decreases trait fitness. Putatively deleterious variants explain roughly one-half of the genetic variance. However, there is only moderate improvement in total heritable variance explained for biomass (7.6%) and plant height (average of 3.1% across all stages). There is no advantage in total heritable variance for SLA and TSC. The contribution of putatively deleterious variants to phenotypic diversity therefore appears to be dependent on the genetic architecture of traits. Overall, these results suggest that incorporating putatively deleterious variants into genomic models slightly improves prediction accuracy because of extensive linkage. Knowledge of deleterious variants could be leveraged for sorghum breeding through either genome editing and/or conventional breeding that focuses on the selection of progeny with fewer deleterious alleles.

Targeted massively parallel sequencing of a panel of putative breast cancer susceptibility genes in a large cohort of multiple-case breast and ovarian cancer families

IntroductionGene panel testing for breast cancer susceptibility has become relatively cheap and accessible. However, the breast cancer risks associated with mutations in many genes included in these panels are unknown.MethodsWe...

Mutations in the RAS Signaling, B-Cell Development, TP53/RB1, and JAK Signaling Pathways Are Common in High Risk B-Precursor Childhood Acute Lymphoblastic Leukemia (ALL): A Report From the Children's Oncology Group (COG) High-Risk (HR) ALL TARGET Project.

Abstract 85▪▪This icon denotes an abstract that is clinically relevant.Despite the overall favorable treatment outcome for childhood ALL, about 20% of patients still experience relapse. Moreover, many ALL cases lack sentinel chromosomal alterations, and the genetic factors contributing to leukemogenesis in these cases are poorly understood. Genome-wide profiling of DNA copy number alterations (CNA) coupled with focused candidate gene resequencing has identified novel genetic alterations that contribute to leukemogenesis and are associated with treatment outcome in ALL. However, large-scale analysis of somatic sequence mutations in ALL has not yet been performed. As part of the COG HR-ALL TARGET (Therapeutically Applicable Research to Generate Effective Targets) Project, 125 genes (selected based on recurrent CNAs, gene expression profiles and known cancer genes) were sequenced in 187 HR B-precursor ALL patients enrolled in COG P9906. The entire coding region and UTRs of each gene were sequenced and more than 98% of the targeted nucleotides have high quality sequence coverage. We found that somatic mutations are frequent in genes that encode for proteins involved in signal transduction, B-cell development and p53/RB signaling. A notable finding was the presence of somatic mutations resulting in constitutive activation of RAS signaling in at least 39% of the cohort. Seventy-three cases have at least one mutation in NRAS (30), KRAS (28), PTPN11 (9), FLT3 (7) and NF1 (6), including 7 patients with multiple mutations (KRAS and NRAS (3), FLT3 and NF1 (1), PTPN11 and FLT3 (1), PTPN11 and FLT3 (1), PTPN11 and NRAS (1), PTPN11 and KRAS (1)). There was no association between RAS pathway mutations and event-free survival or cumulative incidence of relapse in this HR patient cohort. Notably, RAS pathway mutations were uncommon in ALL cases with TCF3-PBX1 (1/22 cases) or MLL translocations (2/18 cases), but occurred frequently in cases lacking known sentinel cytogenetic lesions (68/145 cases, 47%, p<0.0001). Sequence mutations that are known or predicted to impair normal B-cell development were observed in at least 14% of the cohort (PAX5 (21), IKZF1 (7)), with confirmatory germline sequencing underway to clarify whether lymphoblast sequence alterations present in other B-cell development genes including BLK, ETV6, IKZF3, TCF3, RAG1, and BCL11A are somatic or germline. Sequence mutations disrupting TP53/RB1 signaling ((TP53 (10), RB1 (4), CDKN2A (4)) occurred in 10% of cases. Activating sequence mutations in JAK family members (JAK2 (16) and JAK1 (3)) were present in 10% of the cases. Of the 103 cases (55%) with at least one sequence mutation in these four (RAS signaling, B-cell development, p53/RB, and JAK) pathways, 30 have somatic mutations in multiple pathways. When both CNAs and sequence mutations are considered, 94% of the cases have lesions in at least one of the pathways and 31% of the cases have somatic alterations in all three of the B-cell development, RAS, and p53/RB1 signaling pathways, suggesting that aberrations in multiple pathways may be central to development of HR-ALL. Our results contrast sharply with known genetic alterations in T-cell ALL, where RAS pathway alterations are relatively uncommon (<10% of cases). PTEN somatic mutations and deletions occur in approximately 25% of cases of T-cell ALL, but we found no PTEN alterations in this cohort, suggesting differential activation of RAS and PI-3K signaling pathways in T-cell and HR B-precursor ALL. Validation of putative deleterious mutations in other sequenced genes (e.g., TBL1XR1, BLK, and CREBBP) is ongoing. These results from the TARGET COG HR-ALL Pilot Project confirm and extend prior knowledge of genetics of this subtype of ALL and point the way to new potential therapeutic strategies for this patient population. Disclosures:No relevant conflicts of interest to declare.

RNAmutants: a web server to explore the mutational landscape of RNA secondary structures

The history and mechanism of molecular evolution in DNA have been greatly elucidated by contributions from genetics, probability theory and bioinformatics—indeed, mathematical developments such as Kimura's neutral theory, Kingman's coalescent theory and efficient software such as BLAST, ClustalW, Phylip, etc., provide the foundation for modern population genetics. In contrast to DNA, the function of most noncoding RNA depends on tertiary structure, experimentally known to be largely determined by secondary structure, for which dynamic programming can efficiently compute the minimum free energy secondary structure. For this reason, understanding the effect of pointwise mutations in RNA secondary structure could reveal fundamental properties of structural RNA molecules and improve our understanding of molecular evolution of RNA. The web server RNAmutants provides several efficient tools to compute the ensemble of low-energy secondary structures for all k-mutants of a given RNA sequence, where k is bounded by a user-specified upper bound. As we have previously shown, these tools can be used to predict putative deleterious mutations and to analyze regulatory sequences from the hepatitis C and human immunodeficiency genomes. Web server is available at http://bioinformatics.bc.edu/clotelab/RNAmutants/, and downloadable binaries at http://rnamutants.csail.mit.edu/.

Mutations in the human SIX3 gene in holoprosencephaly are loss of function

Holoprosencephaly (HPE) is the most common developmental anomaly of the human forebrain; however, the genetics of this heterogeneous and etiologically complex malformation is incompletely understood. Heterozygous mutations in SIX3, a transcription factor gene expressed in the anterior forebrain and eyes during early vertebrate development, have been frequently detected in human HPE cases. However, only a few mutations have been investigated with limited functional studies that would confirm a role in HPE pathogenesis. Here, we report the development of a set of robust and sensitive assays of human SIX3 function in zebrafish and apply these to the analysis of a total of 46 distinct mutations (19 previously published and 27 novel) located throughout the entire SIX3 gene. We can now confirm that 89% of these putative deleterious mutations are significant loss-of-function alleles. Since disease-associated single point mutations in the Groucho-binding eh1-like motif decreases the function in all assays, we can also confirm that this interaction is essential for human SIX3 co-repressor activity; we infer, in turn, that this function is important in HPE causation. We also unexpectedly detected truncated versions with partial function, yet missing a SIX3-encoded homeodomain. Our data indicate that SIX3 is a frequent target in the pathogenesis of HPE and demonstrate how this can inform the genetic counseling of families.

S12.18 A genetic system to study mitochondrial DNA mutations and their propagation in mice

The number of mutations in mitochondrial DNA (mtDNA) thought to causemetabolic disorders in patients is growing. At present, we lack the technology to introduce these mutations into the mtDNA of model organisms for detailed study. In this study, random mtDNA mutations were generated using the transgenic mtDNA mutator mouse, which expresses an engineered mitochondrial polymerase defective in its proofreading function. Female lines were then established to transmit and segregate the resulting mutations. We observed the selective loss of nonsense mutations in the protein coding genes, consistent with strong purifying selection by the female germline. The selection was evident two generations after the founder mtDNA mutator mouse, eliminating these nonsense mutations before they were detectable by Sanger sequencing methods. Curiously, the tRNA and rRNA genes do not show the same rapid selection against mutations despite strong evolutionary sequence conservation. Despite this purifying selection, putative deleterious mutations were identified and propagated in the mouse lines. Also, mutations analogous to human pathogenic mtDNA mutations were identified. These results will aid in the generation of more accurate models of inheritance of disease-causing mtDNA mutations and allow for the generation of laboratory mouse models of mtDNA mutations known in human disease.

Mutation and expression analysis of human BUB1 and BUB1B in aneuploid breast cancer cell lines

Genetic instability is a hallmark feature of breast, colorectal and other types of cancers. One type characterized by chromosomal instability is thought to be important in the pathogenesis of many solid tumors displaying aneuploidy. Two related protein kinases and homologues of the yeast checkpoint genes, hBUB1 and hBUB1B, have been implicated in the pathogenesis of colorectal cancers. Mutations in hBUB1 have demonstrated a dominant negative effect by disrupting the mitotic checkpoint when transfected into euploid colon cancer cell lines. In Brca2 deficient murine cells, Bub1 mutants potentiate growth and cellular transformation. This would suggest that aneuploidy in solid tumors including breast, could be the result of defects in mitotic checkpoint genes and may be responsible for a chromosomal instability phenotype contributing to tumor progression. We conducted mutational analysis of 19 aneuploid breast cancer cell lines. No mutations were found but we identified nine sequence variations including five previously unreported sequence variants in hBUB1B, two of which affect restrictions sites. None of these nucleotide changes predict significant changes in the predicted protein structure. Expression analysis by Northern blot of breast cell lines showed variable expression of hBUB1 and hBUB1B genes. This suggest that while regulation of expression of these genes may be important in cancer, the lack of putative deleterious mutations in the coding sequence does not support a frequent role for mutant hBUB1 and hBUB1B alleles in the pathogenesis of breast cancer.