Coding Sequence Variants Research Articles

Recurrent, low-frequency coding variants contributing to colorectal cancer in the Swedish population.

Genome-wide association studies (GWAS) have identified dozens of common genetic variants associated with risk of colorectal cancer (CRC). However, the majority of CRC heritability remains unclear. In order to discover low-frequency, high-risk CRC susceptibility variants in Swedish population, we genotyped 1 515 CRC patients enriched for familial cases, and 12 108 controls. Case/control association analysis suggested eight novel variants associated with CRC risk (OR 2.0–17.6, p-value < 2.0E-07), comprised of seven coding variants in genes RAB11FIP5, POTEA, COL27A1, MUC5B, PSMA8, MYH7B, and PABPC1L as well as one variant downstream of NEU1 gene. We also confirmed 27 out of 30 risk variants previously reported from GWAS in CRC with a mixed European population background. This study identified rare, coding sequence variants associated with CRC risk through analysis in a relatively homogeneous population. The segregation data suggest a complex mode of inheritance in seemingly dominant pedigrees.

Single molecule real time sequencing in ADTKD-MUC1 allows complete assembly of the VNTR and exact positioning of causative mutations

Recently, the Mucin-1 (MUC1) gene has been identified as a causal gene of autosomal dominant tubulointerstitial kidney disease (ADTKD). Most causative mutations are buried within a GC-rich 60 basepair variable number of tandem repeat (VNTR), which escapes identification by massive parallel sequencing methods due to the complexity of the VNTR. We established long read single molecule real time sequencing (SMRT) targeted to the MUC1-VNTR as an alternative strategy to the snapshot assay. Our approach allows complete VNTR assembly, thereby enabling the detection of all variants residing within the VNTR and simultaneous determination of VNTR length. We present high resolution data on the VNTR architecture for a cohort of snapshot positive (n = 9) and negative (n = 7) ADTKD families. By SMRT sequencing we could confirm the diagnosis in all previously tested cases, reconstruct both VNTR alleles and determine the exact position of the causative variant in eight of nine families. This study demonstrates that precise positioning of the causative mutation(s) and identification of other coding and noncoding sequence variants in ADTKD-MUC1 is feasible. SMRT sequencing could provide a powerful tool to uncover potential factors encoded within the VNTR that associate with intra- and interfamilial phenotype variability of MUC1 related kidney disease.

Uncovering the genetic lesions underlying the most severe form of Hirschsprung disease by whole-genome sequencing.

Hirschsprung disease (HSCR) is a complex birth defect characterized by the lack of ganglion cells along a variable length of the distal intestine. A large proportion of HSCR patients remain genetically unexplained. We applied whole-genome sequencing (WGS) on 9 trios where the probands are sporadically affected with the most severe form of the disorder and harbor no coding sequence variants affecting the function of known HSCR genes. We found de novo protein-altering variants in three intolerant to change genes-CCT2, VASH1, and CYP26A1-for which a plausible link with the enteric nervous system (ENS) exists. De novo single-nucleotide and indel variants were present in introns and non-coding neighboring regions of ENS-related genes, including NRG1 and ERBB4. Joint analysis with those inherited rare variants found under recessive and/or digenic models revealed both patient-unique and shared genetic features where rare variants were found to be enriched in the extracellular matrix-receptor (ECM-receptor) pathway (p = 3.4 × 10-11). Delineation of the genetic profile of each patient might help finding common grounds that could lead to the discovery of shared molecules that could be used as drug targets for the currently ongoing cell therapy effort which aims at providing an alternative to the surgical treatment.

MADS-box genes and crop domestication: the jack of all traits.

MADS-box genes are key regulators of virtually every aspect of plant reproductive development. They play especially prominent roles in flowering time control, inflorescence architecture, floral organ identity determination, and seed development. The developmental and evolutionary importance of MADS-box genes is widely acknowledged. However, their role during flowering plant domestication is less well recognized. Here, we provide an overview illustrating that MADS-box genes have been important targets of selection during crop domestication and improvement. Numerous examples from a diversity of crop plants show that various developmental processes have been shaped by allelic variations in MADS-box genes. We propose that new genomic and genome editing resources provide an excellent starting point for further harnessing the potential of MADS-box genes to improve a variety of reproductive traits in crops. We also suggest that the biophysics of MADS-domain protein-protein and protein-DNA interactions, which is becoming increasingly well characterized, makes them especially suited to exploit coding sequence variations for targeted breeding approaches.

Sequence variation in melanocortin-1-receptor and tyrosinase-related protein 1 genes and their relationship with melanin-based plumage trait expression in Lesser Kestrel (Falco naumanni) males

Lesser kestrel males display inter-individual variation in melanin-based plumage traits, like ventral plumage colouration and breast/underwing spottiness. We explored whether such plumage variability was associated with single-nucleotide genetic polymorphisms of melanocortin-1-receptor (MC1R) and tyrosinase-related protein 1 (TYRP1), two genes involved in the melanogenesis pathway. No statistically significant associations between single-nucleotide mutations and male plumage traits emerged, though in some cases very rare (< 2%) homozygous mutated individuals displayed extreme plumage phenotypes. Hence, large inter-individual male Lesser Kestrel plumage variation, which is consistent between years and partly age related, was only marginally related to untranslated region and coding sequence variation of MC1R and TYRP1.

VaDiR: an integrated approach to Variant Detection in RNA.

BackgroundAdvances in next-generation DNA sequencing technologies are now enabling detailed characterization of sequence variations in cancer genomes. With whole-genome sequencing, variations in coding and non-coding sequences can be discovered. But the cost associated with it is currently limiting its general use in research. Whole-exome sequencing is used to characterize sequence variations in coding regions, but the cost associated with capture reagents and biases in capture rate limit its full use in research. Additional limitations include uncertainty in assigning the functional significance of the mutations when these mutations are observed in the non-coding region or in genes that are not expressed in cancer tissue.ResultsWe investigated the feasibility of uncovering mutations from expressed genes using RNA sequencing datasets with a method called Variant Detection in RNA(VaDiR) that integrates 3 variant callers, namely: SNPiR, RVBoost, and MuTect2. The combination of all 3 methods, which we called Tier 1 variants, produced the highest precision with true positive mutations from RNA-seq that could be validated at the DNA level. We also found that the integration of Tier 1 variants with those called by MuTect2 and SNPiR produced the highest recall with acceptable precision. Finally, we observed a higher rate of mutation discovery in genes that are expressed at higher levels.ConclusionsOur method, VaDiR, provides a possibility of uncovering mutations from RNA sequencing datasets that could be useful in further functional analysis. In addition, our approach allows orthogonal validation of DNA-based mutation discovery by providing complementary sequence variation analysis from paired RNA/DNA sequencing datasets.

FOXP2 variation in great ape populations offers insight into the evolution of communication skills

The gene coding for the forkhead box protein P2 (FOXP2) is associated with human language disorders. Evolutionary changes in this gene are hypothesized to have contributed to the emergence of speech and language in the human lineage. Although FOXP2 is highly conserved across most mammals, humans differ at two functional amino acid substitutions from chimpanzees, bonobos and gorillas, with an additional fixed substitution found in orangutans. However, FOXP2 has been characterized in only a small number of apes and no publication to date has examined the degree of natural variation in large samples of unrelated great apes. Here, we analyzed the genetic variation in the FOXP2 coding sequence in 63 chimpanzees, 11 bonobos, 48 gorillas, 37 orangutans and 2 gibbons and observed undescribed variation in great apes. We identified two variable polyglutamine microsatellites in chimpanzees and orangutans and found three nonsynonymous single nucleotide polymorphisms, one in chimpanzees, one in gorillas and one in orangutans with derived allele frequencies of 0.01, 0.26 and 0.29, respectively. Structural and functional protein modeling indicate a biochemical effect of the substitution in orangutans, and because of its presence solely in the Sumatran orangutan species, the mutation may be associated with reported population differences in vocalizations.

Transcriptome Signatures of Selection, Drift, Introgression, and Gene Duplication in the Evolution of an Extremophile Endemic Plant.

Plants on serpentine soils provide extreme examples of adaptation to environment, and thus offer excellent models for the study of evolution at the molecular and genomic level. Serpentine outcrops are derived from ultramafic rock and have extremely low levels of essential plant nutrients (e.g., N, P, K, and Ca), as well as toxic levels of heavy metals (e.g., Ni, Cr, and Co) and low moisture availability. These outcrops provide habitat to a number of endemic plant species, including the annual mustard Caulanthus amplexicaulis var. barbarae (Cab) (Brassicaceae). Its sister taxon, C. amplexicaulis var. amplexicaulis (Caa), is intolerant to serpentine soils. Here, we assembled and annotated comprehensive reference transcriptomes of both Caa and Cab for use in protein coding sequence comparisons. A set of 29,443 reciprocal best Blast hit (RBH) orthologs between Caa and Cab was compared with identify coding sequence variants, revealing a high genome-wide dN/dS ratio between the two taxa (mean = 0.346). We show that elevated dN/dS likely results from the composite effects of genetic drift, positive selection, and the relaxation of negative selection. Further, analysis of paralogs within each taxon revealed the signature of a period of elevated gene duplication (∼10 Ma) that is shared with other species of the tribe Thelypodieae, and may have played a role in the striking morphological and ecological diversity of this tribe. In addition, distribution of the synonymous substitution rate, dS, is strongly bimodal, indicating a history of reticulate evolution that may have contributed to serpentine adaptation.

Genetic Control of Serum Marinobufagenin in the Spontaneously Hypertensive Rat and the Relationship to Blood Pressure.

BackgroundWe have investigated serum levels of immunoreactive marinobufagenin (MBG) in 16‐ to 20‐week‐old spontaneously hypertensive rats (SHRs)‐A3 and in the normotensive Wistar‐Kyoto (WKY) rat strain in the absence of salt loading, and we have investigated the genetic control of serum MBG.Methods and ResultsWe genotyped the F2 progeny of an SHR‐A3×WKY intercross using a genome‐wide panel of 253 single‐nucleotide polymorphism markers that were dimorphic between SHR‐A3 and WKY and measured serum MBG by ELISA. Serum MBG levels were lower in SHR‐A3 than WKY rats (0.39±0.07 and 1.27±0.40 nmol/L, respectively), suggesting that MBG may not play a role in the markedly divergent blood pressure measured by telemetry in rats of these 2 strains (SHR‐A3 and WKY, 198.3±4.43 and 116.8±1.51 mm Hg, respectively). The strain difference in serum MBG was investigated to determine whether genomic regions influencing MBG might be identified by genetic mapping. Quantitative trait locus mapping indicated a single locus influencing serum MBG in the region of chromosome 6q12. Homozygosity of WKY alleles at this locus was associated with increased serum MBG levels. We surveyed whole genome sequences from our SHR‐A3 and WKY lines, seeking coding sequence variation between SHR‐A3 and WKY within the mapped locus that might explain the inherited strain difference in serum MBG.ConclusionsWe identified amino acid substitution in the sterol transport protein Abcg5, present in SHR‐A3, but absent in WKY, that is a potential mechanism influencing MBG levels.

Next generation sequencing reveals the potential functions of the genes involved in controlling several important commercial traits in peach (Prunus persica (L). Batsch) fruits

ABSTRACTNext-generation sequencing techniques provide a new insight into the genetic bases of the most important commercial traits in fruit trees. The genomic deep sequencing results of peach cultivar ‘Xiaobaitao’ and its bud variation type ‘Jinliuzaohong’ are reported here. The numbers of detected Single nucleotide polymorphisms (SNPs) in wild type and in mutant were 591,485 and 593,484, with that of InDel as 113,934 and 115,065, respectively. Interestingly, the numbers of SNPs and InDels that differentiate ‘Jinliuzaohong’ from ‘Xiaobaitao’ were 50,217 and 19,368, respectively. The number of genes with non-synonymous mutation was 887, which involved in 109 pathways. The authors found dozens of genes involved in the important commercial traits of peach fruit which are of variations in Coding sequence (CDS) regions, including 3 NAC family genes, i.e. NAC66, NAC68, and NAC78. The relevant commercial traits of peach fruit include colour coverage, maturity date, fruit size, and sugar content. A phylogenetic tree of homological NAC proteins indicated relationship among the species. Genome sequence data suggested that a series of genes and several genetic pathways were likely involved in the formation of the important traits in peach fruits. The findings provide new evidence for the functions of the candidate genes controlling peach fruit traits which were proposed previously by researchers using QTL analysis.

Abstract 3406: Hereditary risks of male breast cancer in a multi-gene panel testing cohort

Abstract While the population-based risk for breast cancer in males remains relatively low (1:1000), inherited predisposition can significantly raise this to as high as 10%, in men who carry a mutation in the BRCA2 gene. Lifetime breast cancer risks of 1-2% have also been reported in men who carry a mutation in BRCA1. These risks, as well as elevated risks of prostate, pancreatic, and melanoma cancers, are important to discuss in families diagnosed with Hereditary Breast and Ovarian Cancer Syndrome, which is often viewed as being clinically relevant only to the women in an affected family. However, beyond the BRCA1/2 genes, limited data is available on hereditary predisposition to male breast cancer. We analyzed clinical histories and molecular results from multi-gene panel testing for hereditary cancer predisposition in a cohort of 715 men affected by breast cancer. A total of 708 male breast cancer patients were eligible for inclusion in the final analysis. Molecular testing included analysis of 5 to 59 genes for DNA coding sequence and copy number variants by next-generation sequencing and microarray. Genetic variants identified were classified according to a 5-tier system using previously validated algorithms1,2. Only those variants classified as pathogenic or likely pathogenic were included in the analyses as positive for a mutation. Overall, a mutation was detected in 18% of these men. Four subjects carried a mutation in two different breast cancer predisposition genes. BRCA2 and CHEK2 were the most frequently mutated genes. The risk of breast cancer was significantly elevated compared to public controls in individuals with a mutation in BRCA2 (odds ratio (OR)=13.9; p=1.92x10-16), CHEK2 (OR=3.8; p=6.24x10-24) and PALB2 (OR=6.6, p=0.01). Average age was similar amongst men with (63.5±2.7 years) and without (62.3±1.2 years) mutations, as were clinical and family histories of additional cancers. The high overall diagnostic yield suggests the utility of testing all male breast cancer patient regardless of age or family history by multigene panel testing, and provides data to support risk-based counseling and medical recommendations for screening and/or prevention in male mutation carriers. 1. 1. LaDuca et al. Genet Med. 2014 Nov;16(11):830-7 2. 2. Richards et al. Genet Med. 2015 May;17(5):405-24 Citation Format: Elizabeth C. Chao, Mary Pritzlaff, Summerour Pia, Rachel McFarland, Shuwei Li, Jill Dolinsky, David Goldgar, Hermela Shimelis, Fergus Couch, LaDuca Holly. Hereditary risks of male breast cancer in a multi-gene panel testing cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3406. doi:10.1158/1538-7445.AM2017-3406

Rapid and Parallel Adaptive Evolution of the Visual System of Neotropical Midas Cichlid Fishes.

Midas cichlid fish are a Central American species flock containing 13 described species that has been dated to only a few thousand years old, a historical timescale infrequently associated with speciation. Their radiation involved the colonization of several clear water crater lakes from two turbid great lakes. Therefore, Midas cichlids have been subjected to widely varying photic conditions during their radiation. Being a primary signal relay for information from the environment to the organism, the visual system is under continuing selective pressure and a prime organ system for accumulating adaptive changes during speciation, particularly in the case of dramatic shifts in photic conditions. Here, we characterize the full visual system of Midas cichlids at organismal and genetic levels, to determine what types of adaptive changes evolved within the short time span of their radiation. We show that Midas cichlids have a diverse visual system with unexpectedly high intra- and interspecific variation in color vision sensitivity and lens transmittance. Midas cichlid populations in the clear crater lakes have convergently evolved visual sensitivities shifted toward shorter wavelengths compared with the ancestral populations from the turbid great lakes. This divergence in sensitivity is driven by changes in chromophore usage, differential opsin expression, opsin coexpression, and to a lesser degree by opsin coding sequence variation. The visual system of Midas cichlids has the evolutionary capacity to rapidly integrate multiple adaptations to changing light environments. Our data may indicate that, in early stages of divergence, changes in opsin regulation could precede changes in opsin coding sequence evolution.

Abstract P1-05-01: Landscape of somatic mutations in inflammatory breast cancer whole-genome sequences

Abstract Goal: Inflammatory breast cancer (IBC) is a rare, aggressive form of breast cancer that is characterized by a highly metastatic phenotype. Numerous previous attempts failed to identify, recurrent, IBC-specific gene expression or DNA copy number alterations. We performed whole genome sequencing (WGS) of IBC biopsies obtained before any therapy to define a comprehensive genomic landscape of this disease. Methods: Illumina paired-end whole genome sequencing (WGS) of 20 IBC (n=9 ER+, n=11 ER-) and matched normal samples were performed with median coverage of 60X and 40X for cancer and normal, the percentages of mapped reads were 99.3% and 99.2%, respectively. We identified germ-line and somatic variants, indels as well as large scale structural variants, using GATK Haplotype Caller, MuTect and CREST, respectively. We performed the same analysis on WGS data from 23, age, race and ER and HER2 matched, non-IBC (n=12 ER+, n=11 ER-) from the TCGA for comparison. Variants in both coding and noncoding sequences were categorized by FunSeq to identify potential drivers. Mutation clustering in each gene, as well as significantly mutated non-coding regulatory modules, were identified using LARVA. DeconstructSigs were used to decompose the mutational spectrum of each cancer into 30 validated, mutational signatures provided by COSMIC. Contributions of each validated signature to mutations in IBC vs. non-IBC were compared using Welch's t-test. Results: We identified 118,818 somatic variants in the IBC samples (median: 3,856; minimum: 1,109; maximum: 24,815) including 1,060 variants (~0.9%) in coding regions. 5,287 somatic indels and 5,959 large scale structural variants were detected including 1,028 insertions and 1,857 deletions. Recurrent, non-synonymous mutations were detected in the coding region of GRIN2A gene in 3/20 IBC samples (15%), (previously reported as a potential driver mutation in 1.7% of breast cancers). Other significant mutations in coding regions included GRHL1, PIK3R2, ESR1, FLG2 and etc. Three DNase I hypersensitive sites (DHSs) in non-coding regions were altered in 20% (4/20) IBC samples vs. fewer than 8.7% (2/23) in non-IBC. Mutational frequency of GATA3 is 80.0% vs. 47.8% (p=0.03), and PTEN is 45.0% vs. 73.9% (p=0.05), in IBC vs. non-IBC samples when including both coding and non-coding variants. Contributions of mutational signature 9, that is associated with polymerase η , were significantly higher in IBC cohort than non-IBC cohort (p-value=0.056). Conclusion: This is the first whole genome sequencing analysis of IBC and comparison with the results from non-IBC. We identified promising candidate drivers in the coding sequence and in non-coding regulatory modules of expressed genes. We also identified mutational signature 9, and mutations in several DHS as significantly more frequent alterations in IBC compared to non-IBC. Citation Format: Li X, Krishnamurthy S, Kumar S, Reddy S, Woodward W, Reuben J, Hatzis C, Ueno NT, Gerstein M, Pusztai L. Landscape of somatic mutations in inflammatory breast cancer whole-genome sequences [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-01.

Variations in endothelin receptor B subtype 2 (EDNRB2) coding sequences and mRNA expression levels in 4 Muscovy duck plumage colour phenotypes

ABSTRACT1. Endothelin receptor B subtype 2 (EDNRB2) is a paralog of EDNRB, which encodes a 7-transmembrane G-protein coupled receptor. Previous studies reported that EDNRB was essential for melanoblast migration in mammals and ducks.2. Muscovy ducks have different plumage colour phenotypes. Variations in EDNRB2 coding sequences (CDSs) and mRNA expression levels were investigated in 4 different Muscovy duck plumage colour phenotypes, including black, black mutant, silver and white head.3. The EDNRB2 gene from Muscovy duck was cloned; it had a length of 6435 bp and encoded 437 amino acids. The coding region was screened and potential single nucleotide polymorphisms were identified. Eight mutations were obtained, including one missense variant (c.64C > T) and 7 synonymous substitutions. The substitutions were associated with plumage colour phenotypes.4. The EDNRB2 mRNA expression levels were compared between feather pulp from black birds and black mutant birds. The results indicated that EDNRB2 transcripts in feather pulp were significantly higher in black feathers than in white feathers.5. The results determined the variation of EDNRB2 CDS and mRNA expression in Muscovy ducks of various plumage colours.

Mycobacterium tuberculosis Complex Exhibits Lineage-Specific Variations Affecting Protein Ductility and Epitope Recognition.

The advent of whole-genome sequencing has provided an unprecedented detail about the evolution and genetic significance of species-specific variations across the whole Mycobacterium tuberculosis Complex. However, little attention has been focused on understanding the functional roles of these variations in the protein coding sequences. In this work, we compare the coding sequences from 74 sequenced mycobacterial species including M. africanum, M. bovis, M. canettii, M. caprae, M. orygis, and M. tuberculosis. Results show that albeit protein variations affect all functional classes, those proteins involved in lipid and intermediary metabolism and respiration have accumulated mutations during evolution. To understand the impact of these mutations on protein functionality, we explored their implications on protein ductility/disorder, a yet unexplored feature of mycobacterial proteomes. In agreement with previous studies, we found that a Gly71Ile substitution in the PhoPR virulence system severely affects the ductility of its nearby region in M. africanum and animal-adapted species. In the same line of evidence, the SmtB transcriptional regulator shows amino acid variations specific to the Beijing lineage, which affects the flexibility of the N-terminal trans-activation domain. Furthermore, despite the fact that MTBC epitopes are evolutionary hyperconserved, we identify strain- and lineage-specific amino acid mutations affecting previously known T-cell epitopes such as EsxH and FbpA (Ag85A). Interestingly, in silico studies reveal that these variations result in differential interaction of epitopes with the main HLA haplogroups.

Most ApoL1 Is Secreted by the Liver.

Two coding sequence variants in the APOL1 gene (G1 and G2) explain much of the increased risk for FSGS, HIV-associated nephropathy, and hypertension-attributed ESRD among people of recent African ancestry. The ApoL1 protein is expressed in a wide variety of cell tissues. It has been assumed that the majority of circulating ApoL1 is produced by the liver, but this has not been shown. Using mass spectrometry, we genotyped and quantified the circulating ApoL1 in two liver transplant recipients whose native APOL1 genotype differed from the genotype of the deceased donors, allowing us to differentiate liver- from nonliver-produced ApoL1. Our findings confirm that the liver is indeed the main source of circulating ApoL1. However, the liver is not the sole source of circulating ApoL1, because we found that residual amounts of native ApoL1 continued to circulate in the blood, even after the liver transplant.

Pathogenic Relevance of Germ Line TET2 Alterations

Several familial mutations predisposing to the development of leukemia have been identified, mostly in association with childhood or adolescent presentations of myeloid neoplasms or bone marrow failure states. However, many of these genetic syndromes have variable penetrance and latency. Thus, late presentations of familial diseases are possible as with DDX41 mutations associated with adult myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Growing evidence exists to support the notion that germ line (GL) alterations may play a greater than previously known role in otherwise typical spontaneous adult myeloid neoplasms. These alterations may include known predisposition leukemia genes (i.e.,. ETV6, RUNX1, CEBPa, and DDX41), heterozygous alterations known to generate recessive disorders, or new unidentified genes. The search for such predisposition genes is difficult but we hypothesized that genes frequently damaged by somatic mutations may also be affected by GL alterations, a similar principle as seen for CEBPa or TP53. Further, that a collection of rare variants (MAF<1%) within a gene could be associated with risk of MDS according to the allelic burden (AB) theory. To develop proof-of-principle of this theory we focused on the gene TET2. In comparisons of the AB of rare TET2 GL variants of patients vs. ethnically-matched controls (selected according to ethnic indicator SNPs), MDS patients examined by whole exome sequencing (WES, N=368) had more rare coding-sequence variants than controls (p<.001). On average, patients had 3 TET2 rare variants, while controls had only 1.5 variants. This suggests that the burden of TET2 rare variants could increase risks of myeloid neoplasms. This finding is in agreement with several other observations; 1.) TET2 is frequently mutated and extremely polymorphic; 2.) it is often associated with biallelic mutations, of which some may be a combination of GL and somatic lesions; and 3.) GL alterations in TET2 may predispose to acquisition of additional lesions.We analyzed a cohort of 4159 patients with myeloid neoplasms using WES and targeted deep next generation sequencing (NGS). Extensive scoring algorithms, along with mutational databases were used to detect GL variants. Serial tumor samples were examined to discriminate GL from somatic defects. We identified 2518 TET2 SNPs in 1475 patients. Multiple SNPs were seen in the same patient, 28% of patients had 2 SNPs, 11% had 3 and 6% had 4 or more SNPs. Of those 52% were found in proximity to the catalytic domain, and 10% were homozygous. TET2 SNPs irrespective of their population frequency were found in 21% of MDS, 27% of MDS/MPN, 31% of MPN, and 33% of AML patients.We focused our analysis on the top 10 canonical SNPs with the highest odds ratio (OR) when comparing frequencies in our population to that of healthy controls derived from the ExAC base of 60,000 individuals. The SNPs with the highest OR was present in 2% of patients (p.Val1718Leu) vs. 0.5% of controls. Combinations of SNPs (biallelic configuration) were then investigated; SNPs p.Leu1721Trp and p.Pro363Leu co-occurred in 8% of patients, and p.Val218Met, p.Leu34Phe and p.His1778Arg co-occurred in 3%. We also queried whether GL TET2 SNPs create a haplotype (i.e., increased the risk for acquisition of somatic TET2 mutations) or are mutually exclusive. Overall, 32% of cases with any TET2 SNP also acquired a TET2 somatic mutation vs the WT form for these (68%). Conversely, among the somatic mutant carriers, for example SNP p.Met1701Ile, SNP p.Tyr864His and SNP p.Gly355Asp had an OR vs. controls of 1.1. In particular, 55% of all TET2 mutants had SNP p.Leu1721Trp in a biallelic fashion vs. 46% of somatic WT cases. This suggests a predisposition haplotype. We also examined the relationship between unrelated somatic mutations and "top OR" TET2 SNPs. No correlation was seen between SNPs and other somatic mutations.In sum, several GL TET2 variants or their combination may constitute complex, likely low penetrance predisposing factors for myeloid neoplasm that also interact with somatic lesions and lead to cancer decades later. DisclosuresMeggendorfer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Maciejewski:Celgene: Consultancy, Honoraria, Speakers Bureau; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau.

Downregulation of a GPCR by β-Arrestin2-Mediated Switch from an Endosomal to a TGN Recycling Pathway

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone involved in nutrient homeostasis. GIP receptor (GIPR) is constitutively internalized and returned to the plasma membrane, atypical behavior for a G-protein-coupled receptor (GPCR). GIP promotes GIPR downregulation from the plasmamembrane by inhibiting recycling without affecting internalization. This transient desensitization is achieved by altered intracellular trafficking of activated GIPR. GIP stimulation induces a switch in GIPR recycling from a rapid endosomal to a slow trans-Golgi network (TGN) pathway. GPCR kinases and β-arrestin2 are required for this switch in recycling. A coding sequence variant of GIPR, which hasbeen associated with metabolic alterations, has altered post-activation trafficking characterized byenhanced downregulation and prolonged desensitization. Downregulation of the variant requires β-arrestin2targeting to the TGN but is independent ofGPCR kinases. The single amino acid substitution in the variant biases the receptor to promote GIP-stimulated β-arrestin2 recruitment without receptor phosphorylation, thereby enhancing downregulation.

Coupled enhancer and coding sequence evolution of a homeobox gene shaped leaf diversity.

Here we investigate mechanisms underlying the diversification of biological forms using crucifer leaf shape as an example. We show that evolution of an enhancer element in the homeobox gene REDUCED COMPLEXITY (RCO) altered leaf shape by changing gene expression from the distal leaf blade to its base. A single amino acid substitution evolved together with this regulatory change, which reduced RCO protein stability, preventing pleiotropic effects caused by its altered gene expression. We detected hallmarks of positive selection in these evolved regulatory and coding sequence variants and showed that modulating RCO activity can improve plant physiological performance. Therefore, interplay between enhancer and coding sequence evolution created a potentially adaptive path for morphological evolution.

Multiple Transcript Properties Related to Translation Affect mRNA Degradation Rates in Saccharomyces cerevisiae.

Degradation of mRNA contributes to variation in transcript abundance. Studies of individual mRNAs have shown that both cis and trans factors affect mRNA degradation rates. However, the factors underlying transcriptome-wide variation in mRNA degradation rates are poorly understood. We investigated the contribution of different transcript properties to transcriptome-wide degradation rate variation in the budding yeast, Saccharomyces cerevisiae, using multiple regression analysis. We find that multiple transcript properties are significantly associated with variation in mRNA degradation rates, and that a model incorporating these properties explains ∼50% of the genome-wide variance. Predictors of mRNA degradation rates include transcript length, ribosome density, biased codon usage, and GC content of the third position in codons. To experimentally validate these factors, we studied individual transcripts expressed from identical promoters. We find that decreasing ribosome density by mutating the first translational start site of a transcript increases its degradation rate. Using coding sequence variants of green fluorescent protein (GFP) that differ only at synonymous sites, we show that increased GC content of the third position of codons results in decreased rates of mRNA degradation. Thus, in steady-state conditions, a large fraction of genome-wide variation in mRNA degradation rates is determined by inherent properties of transcripts, many of which are related to translation, rather than specific regulatory mechanisms.