Dinucleotide Substitution Research Articles

Atypical Droplet Digital Polymerase Chain Reaction Patterns That Indicate Uncommon but Clinically Actionable EGFR Mutations in Lung Cancer.

Droplet digital polymerase chain reaction (ddPCR) is a sensitive method to detect common pathogenic EGFR mutations in non-small cell lung cancer. Although targeted assays have not been specifically designed to detect them, uncommon EGFR mutations have been linked to response to targeted therapy. To describe atypical ddPCR patterns that correspond to uncommon but clinically actionable EGFR mutations. A cohort of 1134 consecutive non-small cell lung cancers that underwent targeted next-generation sequencing was reviewed. Uncommon EGFR mutations involving probe binding sites were evaluated by ddPCR. Two hundred fifty-five of 1134 cancers (22.5%) harbored pathogenic EGFR mutations. One hundred eighty-six of 255 (72.9%) had canonical EGFR exon 19 deletion or exon 21 p.L858R variants designed for detection by ddPCR. An additional 25 of 255 cases (9.8%) had uncommon EGFR mutations within the probe-binding site, including 1 case with concurrent uncommon mutations in both exon 19 and exon 21. These mutations included uncommon EGFR exon 19 deletions (n = 6), EGFR exon 19 substitutions p.L747P (n = 3) and p.L747A (n = 1), dinucleotide substitutions leading to EGFR p.L858R (n = 5), EGFR exon 21 substitutions p.K860I (n = 1) and p.L861Q (n = 9), and EGFR p.[L858R;K860I] (n = 1). Droplet digital polymerase chain reaction generated atypical but reproducible signal for each of these uncommon variants. Droplet digital polymerase chain reaction analysis of uncommon pathogenic EGFR variants can yield unique and reproducible results. Recognition of atypical patterns in EGFR ddPCR testing can prompt confirmatory molecular testing and aid appropriate targeted therapy selection for patients with non-small cell lung cancer.

Coronavirus XBB.1.5 as an Indicator of the Long-Term Continuation of the Covid-19 pandemic. What Next for Vaccination?

The article discusses the properties of the pandemic strain XBB.1.5 S protein in comparison with the 1918 and 2009 pandemic strains hemagglutinin H1. The S-protein XBB.1.5 already contains more than 40 mutations realized by substituting different amino acids through single and dinucleotide substitutions, deletions and the use of predominantly transversions. The variability of H1N1 influenza virus hemagglutinin is associated with single nucleotide substitutions at a constant length. Conditional extrapolation of influenza virus hemagglutinin variability data on coronavirus S-protein sizes suggests that new pandemic strains will emerge in the next 2-3 years, avoiding the immune defense formed by vaccination against the strains preceding them. The inability to create through the adaptive immune system a long-term immunity to pandemic coronaviruses, as well as to other respiratory viruses with a short incubation cycle, puts on the agenda the need to find new vaccine designs that provide a combination of long-term adaptive and trained immunity. The problem in the search for such vaccines is associated with the regulation of the activity of the innate immune system and ensuring the stability of trained immunity.

Abstract 5012: Characterizing the functional significance of PDGFRAK385I and PDGFRAK385L extracellular domain mutations in the newly defined myxoid glioneuronal tumor

Abstract In 2021, the World Health Organization (WHO) approved new diagnostic guidelines that stratify central nervous system (CNS) tumors based on molecular, transcriptomic, and epigenetic analysis instead of relying solely on phenotypic characterization and added twenty-two new CNS tumor entities based on these guidelines. Our focus is on one of these newly defined entities—myxoid glioneuronal tumors—a slow growing, mucinous glial tumor defined by a pathognomonic dinucleotide substitution in the extracellular domain of PDGFRA p.K385I/L. PDGFRA p.K385I/L remains a variant of unknown significance, however, constitutive activation of PDGFRA is well established as a pharmacologically actionable, oncogenic driver present in a variety of tumor types. Here we report the functional characterization of PDGFRAK385I and PDGFRAK385L by assessing transforming potential using Ba/F3 cells and immortalized human astrocytes. Our results demonstrate that both p.K385I and K385L substitutions are oncogenic, driving cytokine-independent Ba/F3 cell growth and anchorage-independent colony formation. Western blot analysis of downstream cellular signaling pathways in the absence of PDGF-ligand further confirm the activating nature of these mutations, showing robust phosphorylation of PDGFRA and constitutive phosphorylation of downstream signaling effectors ERK1/2, SHP2, and STAT3. To assess whether PDGFRAK385I and PDGFRAK385L are pharmacologically actionable, we tested multiple tyrosine kinase inhibitors with known activity against PDGFRA aberrant cancers using our mutant-transformed PDGFRA Ba/F3 cells. We found that the PDGFRAK385I and PDGFRAK385L mutants were sensitive to inhibitors axitinib, AZD-3229, and avapritinib at therapeutically relevant concentrations. Further, these mutants showed resistance to imatinib, nilotinib, and crenolanib underscoring the importance of pharmacogenomic investigation. No appreciable difference in sensitivity or resistance profiles was observed between the two mutants. In summary, our findings indicate that these previously uncharacterized p.K385I/L extracellular domain mutations in PDGFRA are oncogenic and represent a pharmacologically actionable target that could be harnessed to increase therapeutic options in the setting of myxoid glioneuronal tumors. Citation Format: Catherine Z. Beach, Romel Somwar, Matthew D. Wood, Monika A. Davare. Characterizing the functional significance of PDGFRAK385I and PDGFRAK385L extracellular domain mutations in the newly defined myxoid glioneuronal tumor. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5012.

Myxoid glioneuronal tumor: Histopathologic, neuroradiologic, and molecular features in a single center series

BackgroundMyxoid glioneuronal tumor (MGT) is a benign glioneuronal neoplasm recently introduced in the World Health Organization (WHO) classification of the central nervous system (CNS) tumors. MGTs are typically located in the septum pellucidum, foramen of Monro or periventricular white matter of the lateral ventricle. They were previously diagnosed as dysembryoplastic neuroepithelial tumors (DNT), showing histological features almost indistinguishable from classical cortical DNT. Despite that, MGTs have been associated with a specific dinucleotide substitution at codon 385 in the platelet-derived growth factor receptor alpha (PDGFRA) gene, replacing a lysine residue with either leucine or isoleucine (p. LysK385Leu/Iso). This genetic variation has never been described in any other CNS tumor. Materials and methodsThirty-one consecutive tumors, previously diagnosed as DNTs at the Meyer Children's Hospital IRCCS between January 2010 and June 2021 were collected for a comprehensive study of their clinical, imaging, pathological features, and molecular profile. ResultsIn six out of the thirty-one tumors we had previously diagnosed as DNTs, we identified the recurrent dinucleotide mutation in the PDGFRA. All six tumors were typically located within the periventricular white matter of the lateral ventricle and in the septum pellucidum. We then renamed these lesions as MGT, according to the latest WHO CNS classification. In all patients we observed an indolent clinical course, without recurrence. ConclusionMGT represent a rare but distinct group of neoplasm with a typical molecular profiling, a characteristic localization, and a relative indolent clinical course.

Tandem Substitutions in Somatic Hypermutation

Upon antigen recognition, activation-induced cytosine deaminase initiates affinity maturation of the B-cell receptor by somatic hypermutation (SHM) through error-prone DNA repair pathways. SHM typically creates single nucleotide substitutions, but tandem substitutions may also occur. While tandem substitutions have been described in mice and other species, the incidence of this phenomenon and its underlying mechanism in humans is currently unknown.We investigated incidence and sequence context of tandem substitutions by massive parallel sequencing of V(D)J repertoires in healthy human donors generated by unbiased ARTISAN PCR. Selection of unique, clonally unrelated, antigen-experienced sequences carrying up to 5% mutations yielded 13.532 VDJ, 7.952 VJ-kappa and 7.598 VJ-lambda. Comparison to the closest germline allele allowed for identification of a total of 122.878 single nucleotide substitutions (SNS), 10.735 tandem dinucleotide substitutions (TDNS) and 2.615 longer contiguous substitutions. After correcting for expected clusters of adjacent SNS, tandem substitutions comprised 5,7% of all AID-induced mutations. The mutation of more than one nucleotide in a single event, was shown to overcome amino acid codon redundancy and may therefore enhance the adaptive immune response. Clustering of such mutations around AID hotspots and their overall distribution indicates that tandem substitutions are an integral part of the SHM spectrum.In the majority of tandem substitutions, the mutated sequence may be identified in the directly adjacent reference sequence context. Tandem substitutions in humans therefore represent single nucleotide juxtalocations. Such juxtalocations appear to be favored in polydipyramidine stretches. These observations could be confirmed in patients with MSH2/6 deficiency, but were absent in a VDJ library from an UNG-deficient patient, indicating a strict dependence on abasic sites as an instigating mechanism.Together, these findings delineate a model where tandem substitutions are predominantly generated by translesion synthesis across an apyramidinic site that is typically created by UNG. During replication, apyrimidinic sites transiently adapt an extruded configuration, causing skipping of the extruded base. Consequent strand decontraction leads to the juxtalocation, after which exonucleases repair the apyramidinic site and any directly adjacent mismatched base pairs. The mismatch repair pathway appears to account for the remainder of tandem substitutions.Our study shows that a significant portion of mutations acquired during SHM are caused by tandem substitutions, and that this mechanism may enhance affinity maturation and expedite the adaptive immune response by overcoming amino acid codon degeneracies or mutating two adjacent amino acid residues simultaneously.Figure legend. Corrected incidence of tandem dinucleotide substitutions in healthy donors. (A) Dinucleotide substitutions from unique IGHV, IGKV and IGLV sequences and corrected after in silico predictions of dinucleotide substitutions that did not occur in tandem. Burgundy cells represent sequence inversions, light and dark purple cells represent juxtalocations of the 5‘ and 3‘ base in the pair (as seen from the non-transcribed strand), respectively. For unshaded cells, juxtalocation could not be assessed due to one or more nucleotides in the reference sequence matching the mutated sequence. (B) Relative contribution of sequence inversions and juxtalocations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

C. elegans genome-wide analysis reveals DNA repair pathways that act cooperatively to preserve genome integrity upon ionizing radiation.

Ionizing radiation (IR) is widely used in cancer therapy and accidental or environmental exposure is a major concern. However, little is known about the genome-wide effects IR exerts on germ cells and the relative contribution of DNA repair pathways for mending IR-induced lesions. Here, using C. elegans as a model system and using primary sequencing data from our recent high-level overview of the mutagenic consequences of 11 genotoxic agents, we investigate in detail the genome-wide mutagenic consequences of exposing wild-type and 43 DNA repair and damage response defective C. elegans strains to a Caesium (Cs-137) source, emitting γ-rays. Cs-137 radiation induced single nucleotide variants (SNVs) at a rate of ~1 base substitution per 3 Gy, affecting all nucleotides equally. In nucleotide excision repair mutants, this frequency increased 2-fold concurrently with increased dinucleotide substitutions. As observed for DNA damage induced by bulky DNA adducts, small deletions were increased in translesion polymerase mutants, while base changes decreased. Structural variants (SVs) were augmented with dose, but did not arise with significantly higher frequency in any DNA repair mutants tested. Moreover, 6% of all mutations occurred in clusters, but clustering was not significantly altered in any DNA repair mutant background. Our data is relevant for better understanding how DNA repair pathways modulate IR-induced lesions.

Identification of a Novel Cis-Acting Regulator of HIV-1 Genome Packaging.

Human immunodeficiency virus type 1 (HIV-1) uptakes homo-dimerized viral RNA genome into its own particle. A cis-acting viral RNA segment responsible for this event, termed packaging signal (psi), is located at the 5′-end of the viral genome. Although the psi segment exhibits nucleotide variation in nature, its effects on the psi function largely remain unknown. Here we show that a psi sequence from an HIV-1 regional variant, subtype D, has a lower packaging ability compared with that from another regional variant, HIV-1 subtype B, despite maintaining similar genome dimerization activities. A series of molecular genetic investigations narrowed down the responsible element of the selective attenuation to the two sequential nucleotides at positions 226 and 227 in the psi segment. Molecular dynamics simulations predicted that the dinucleotide substitution alters structural dynamics, fold, and hydrogen-bond networks primarily of the psi-SL2 element that contains the binding interface of viral nucleocapsid protein for the genome packaging. In contrast, such structural changes were minimal within the SL1 element involved in genome dimerization. These results suggest that the psi 226/227 dinucleotide pair functions as a cis-acting regulator to control the psi structure to selectively tune the efficiency of packaging, but not dimerization of highly variable HIV-1 genomes.

Characteristics of mutational signatures of unknown etiology.

Although not all somatic mutations are cancer drivers, their mutational signatures, i.e. the patterns of genomic alterations at a genome-wide scale, provide insights into past exposure to mutagens, DNA damage and repair processes. Computational deconvolution of somatic mutation patterns and expert curation pan-cancer studies have identified a number of mutational signatures associated with point mutations, dinucleotide substitutions, insertions and deletions, and rearrangements, and have established etiologies for a subset of these signatures. However, the mechanisms underlying nearly one-third of all mutational signatures are not yet understood. The signatures with established etiology and those with hitherto unknown origin appear to have some differences in strand bias, GC content and nucleotide context diversity. It is possible that some of the hitherto ‘unknown’ signatures predominantly occur outside gene regions. While nucleotide contexts might be adequate to establish etiologies of some mutational signatures, in other cases additional features, such as broader (epi)genomic contexts, including chromatin, replication timing, processivity and local mutational patterns, may help fully understand the underlying DNA damage and repair processes. Nonetheless, remarkable progress in characterization of mutational signatures has provided fundamental insights into the biology of cancer, informed disease etiology and opened up new opportunities for cancer prevention, risk management, and therapeutic decision making.

Recurrent gene mutations detected in canine mast cell tumours by next generation sequencing.

Genetic causes of canine mast cell tumours (MCTs), except for mutations in the KIT gene detected in some MCTs, are generally unknown. We used whole exome sequencing to reveal mutation spectra in canine MCTs. We detected somatic mutations in 87 genes including 10 genes recognized as human cancer drivers. Besides KIT, 14 other genes were recurrently mutated. Subsequently, we performed next generation sequencing of a panel of 50 selected genes in additional MCT samples. In this group, the most frequently altered gene was GNB1 showing a recurrent dinucleotide substitution at position of Gly116 in 30% of the MCT samples (n = 6/20) and Ile80 substitution accompanied by a splice region mutation in one case. We extended the study by analysis of the above mentioned GNB1 regions in additional MCT samples by Sanger sequencing, and assessed the overall prevalence of GNB1 mutations to 17.3% (n = 14/81), which is similar to the prevalence of KIT alterations. Our results indicate that GNB1 mutations are probably involved in canine MCT pathogenesis in both cutaneous and subcutaneous MCT cases. As opposed to KIT alterations, the presence of GNB1 mutations did not negatively affect survival times, and our data even showed a trend towards positive prognosis. If our results are confirmed in a larger number of MCTs, an extension of molecular testing of canine MCTs by GNB1 analysis would help to refine the molecular stratification of MCTs, and become useful for targeted treatment strategies.

Biochemical reconstitution of UV-induced mutational processes.

We reconstituted two biochemical processes that may contribute to UV-induced mutagenesis in vitro and analysed the mutational profiles in the products. One process is translesion synthesis (TLS) by DNA polymerases (Pol) δ, η and ζ, which creates C>T transitions at pyrimidine dimers by incorporating two dAMPs opposite of the dimers. The other process involves spontaneous deamination of cytosine, producing uracil in pyrimidine dimers, followed by monomerization of the dimers by secondary UV irradiation, and DNA synthesis by Pol δ. The mutational spectrum resulting from deamination without translesion synthesis is similar to a mutational signature found in melanomas, suggesting that cytosine deamination encountered by the replicative polymerase has a prominent role in melanoma development. However, CC>TT dinucleotide substitution, which is also commonly observed in melanomas, was produced almost exclusively by TLS. We propose that both TLS-dependent and deamination-dependent mutational processes are likely involved in UV-induced melanoma development.

Alternative Mutations and Isoform Dysregulation in MYD88 in Waldenstrom's Macroglobulinemia

BackgroundMutations in MYD88 are highly recurring in Waldenstrom's Macroglobulinemia (WM) patients and are important for establishing the diagnosis of WM. The most common mutation in MYD88 is c.978T>C resulting a proline substitution for leucine at amino acid position 265 (p.Leu265Pro). Both allele specific PCR (AS-PCR) and clinical diagnostic next generation sequencing (NGS) panels are used to detect mutated MYD88, though they differ in sensitivity and scope. In this study we screened 734 patients with WM by AS-PCR for MYD88 c.978T>C MYD88 followed by Sanger sequencing to clarify negative results for non-MYD88 p.Leu265Pro mutations and compared the findings to clinical NGS panel data from the same biopsy when available. We also investigated MYD88 isoform dysregulation and isoform specific effects of the observed mutations that may impact mutated MYD88 regulation which has not been previously studied in WM.MethodsDNA from CD19-selected bone marrow mononuclear cells (BMMC) of 734 WM patients were used for the MYD88 c.978T>C AS-PCR assay previously described by us (Xu et al, Blood 2013). For patients wild-type for MYD88 c.978T>C by AS-PCR, Sanger sequencing of the open reading frame of MYD88 was performed for both DNA and RNA simultaneously isolated from CD19-selected BMMC. DNA was also used to validate the presence of c.978T>C by Sanger. Findings were compared to 222/734 (30.2%) patients who also underwent illumina miSeq based targeted next generation sequencing on a clinical diagnostic platform using unselected BMMC. NGS isoform specific expression estimates were calculated using Salmon for 77 WM patients and 34 healthy donors (Hunter et al, Blood 2016).Results688/734 (93.7%) WM patients tested positive for the c.978T>C mutation. To confirm these results, Sanger sequencing at the DNA level covering the c.978T>C mutation was performed in 361/688 (52.5%) patients confirming the presence of the mutation in all cases. These Sanger studies revealed that one patient had two somatic mutations in addition to c.978T>C. Of the 46/734 (6.3%) that were wild-type by AS-PCR, 18 had cDNA available to screen for alternative MYD88 mutations. Of these, 13/18 (72.2%) were confirmed to be truly wild type for MYD88, and 5/18 (27.8%) harbored alternative MYD88 mutations making up 0.7% of the study population. Taken together 693/734 (94.4%) of patients were found to harbor somatic MYD88 mutations. Of the 222 patients form whom matching NGS panel data was available, the finding between the NGS and AS-PCR studies were largely concordant. The only discrepancies observed were 69 (31.1%) cases where targeted NGS gave false negative results for c.978T>C but was detected by AS-PCR. Of the four patients with alternative MYD88 mutations, one patient had a dinucleotide substitution that also resulted in p.Leu265Pro but tested as wild-type by AS-PCR, two patients each had one previously documented mutation (either pVal217Phe or p.Ser243Asn) and one patient had a mutation that was synonymous at the protein level (p.Phe277Phe). The patient with the two novel mutations in addition to c.978T>C had a mutation in the polypyrimidine track leading to the final exon and one resulting in p.Gly259Gly in the primary transcript but presents as a highly disruptive p.Val199Glu in the shorter regulatory isoforms. This is similar to c.978T>C which presents as p.L265P in the primary transcripts but acts as a stop loss in the shorter isoforms. We therefore looked for evidence of isoform level dysregulation in MYD88 using RNASeq and found highly significant and distinctive MYD88 isoform signatures for MYD88 mutant, MYD88 wild-type and healthy donor samples (Figure 1).ConclusionsUsing CD19-selected BMMC, MYD88 c.978T>C (p.Leu265Pro) was found in 93.7% of 734 patients, while non-c.978T>C mutations were present in <1% of WM population. Discordance with targeted NGS is common, with a false negative rate of 31%. Distinct isoform usage patterns and isoform specific mutational effects among MYD88 mutated patients was observed, highly suggestive of a functional role for MYD88 splicing in WM pathogenesis. [Display omitted] DisclosuresCastillo:Millennium: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Genentech: Consultancy; Beigene: Consultancy, Research Funding. Treon:Johnson & Johnson: Consultancy; Janssen: Consultancy, Other: Travel, Accommodations, Expenses; BMS: Research Funding; Pharmacyclics: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding. Hunter:Pharmacyclics: Consultancy.

In-depth characterization of the cisplatin mutational signature in human cell lines and in esophageal and liver tumors.

Cisplatin reacts with DNA and thereby likely generates a characteristic pattern of somatic mutations, called a mutational signature. Despite widespread use of cisplatin in cancer treatment and its role in contributing to secondary malignancies, its mutational signature has not been delineated. We hypothesize that cisplatin's mutational signature can serve as a biomarker to identify cisplatin mutagenesis in suspected secondary malignancies. Knowledge of which tissues are at risk of developing cisplatin-induced secondary malignancies could lead to guidelines for noninvasive monitoring for secondary malignancies after cisplatin chemotherapy. We performed whole genome sequencing of 10 independent clones of cisplatin-exposed MCF-10A and HepG2 cells and delineated the patterns of single and dinucleotide mutations in terms of flanking sequence, transcription strand bias, and other characteristics. We used the mSigAct signature presence test and nonnegative matrix factorization to search for cisplatin mutagenesis in hepatocellular carcinomas and esophageal adenocarcinomas. All clones showed highly consistent patterns of single and dinucleotide substitutions. The proportion of dinucleotide substitutions was high: 8.1% of single nucleotide substitutions were part of dinucleotide substitutions, presumably due to cisplatin's propensity to form intra- and interstrand crosslinks between purine bases in DNA. We identified likely cisplatin exposure in nine hepatocellular carcinomas and three esophageal adenocarcinomas. All hepatocellular carcinomas for which clinical data were available and all esophageal cancers indeed had histories of cisplatin treatment. We experimentally delineated the single and dinucleotide mutational signature of cisplatin. This signature enabled us to detect previous cisplatin exposure in human hepatocellular carcinomas and esophageal adenocarcinomas with high confidence.

Saturation mutagenesis reveals manifold determinants of exon definition.

To illuminate the extent and roles of exonic sequences in the splicing of human RNA transcripts, we conducted saturation mutagenesis of a 51-nt internal exon in a three-exon minigene. All possible single and tandem dinucleotide substitutions were surveyed. Using high-throughput genetics, 5560 minigene molecules were assayed for splicing in human HEK293 cells. Up to 70% of mutations produced substantial (greater than twofold) phenotypes of either increased or decreased splicing. Of all predicted secondary structural elements, only a single 15-nt stem–loop showed a strong correlation with splicing, acting negatively. The in vitro formation of exon-protein complexes between the mutant molecules and proteins associated with spliceosome formation (U2AF35, U2AF65, U1A, and U1-70K) correlated with splicing efficiencies, suggesting exon definition as the step affected by most mutations. The measured relative binding affinities of dozens of human RNA binding protein domains as reported in the CISBP-RNA database were found to correlate either positively or negatively with splicing efficiency, more than could fit on the 51-nt test exon simultaneously. The large number of these functional protein binding correlations point to a dynamic and heterogeneous population of pre-mRNA molecules, each responding to a particular collection of binding proteins.

A Model Study of In Silico Proficiency Testing for Clinical Next-Generation Sequencing.

-Most current proficiency testing challenges for next-generation sequencing assays are methods-based proficiency testing surveys that use DNA from characterized reference samples to test both the wet-bench and bioinformatics/dry-bench aspects of the tests. Methods-based proficiency testing surveys are limited by the number and types of mutations that either are naturally present or can be introduced into a single DNA sample. -To address these limitations by exploring a model of in silico proficiency testing in which sequence data from a single well-characterized specimen are manipulated electronically. -DNA from the College of American Pathologists reference genome was enriched using the Illumina TruSeq and Life Technologies AmpliSeq panels and sequenced on the MiSeq and Ion Torrent platforms, respectively. The resulting data were mutagenized in silico and 26 variants, including single-nucleotide variants, deletions, and dinucleotide substitutions, were added at variant allele fractions (VAFs) from 10% to 50%. Participating clinical laboratories downloaded these files and analyzed them using their clinical bioinformatics pipelines. -Laboratories using the AmpliSeq/Ion Torrent and/or the TruSeq/MiSeq participated in the 2 surveys. On average, laboratories identified 24.6 of 26 variants (95%) overall and 21.4 of 22 variants (97%) with VAFs greater than 15%. No false-positive calls were reported. The most frequently missed variants were single-nucleotide variants with VAFs less than 15%. Across both challenges, reported VAF concordance was excellent, with less than 1% median absolute difference between the simulated VAF and mean reported VAF. -The results indicate that in silico proficiency testing is a feasible approach for methods-based proficiency testing, and demonstrate that the sensitivity and specificity of current next-generation sequencing bioinformatics across clinical laboratories are high.

Nucleotide Diversity and Molecular Evolution of the ALK Gene in Cultivated Rice and its Wild Relatives

Gelatinization temperature (GT), an important parameter for rice cooking quality, is mainly regulated by the ALK gene encoding starch synthase IIa. Here, we reported the nucleotide diversity of the ALK gene in 122 cultivated accessions and 199 wild rice accessions that were collected around the Pearl River Basin in China. A total of 93 single nucleotide ploymorphisms (SNPs) were identified, with an average of one SNP per 40 bp. Tajima D statistics revealed that the DNA sequences covering the last exon have probably evolved under balancing selection. Based on two functional SNPs (an A to G substitution at 4198 bp and a GC to TT dinucleotide substitution at 4330/4331 bp), three haplotypes, G/GC, G/TT, and A/GC, were identified in both wild and cultivated accessions, with the G/GC haplotype being predominant. Interestingly, the A/GC haplotype was exclusively found in the wild accessions from Guangdong province, while the G/TT haplotype was only present in the wild accessions from Jiangxi province and Hainan Island. This suggests that the G/TT and A/GC variants may have arisen independently and undergone balancing selection on separate haplotypes in multiple populations. Our result supports earlier hypothesis that cultivated rice was independently domesticated from multiple domestication events in China. Our study aids in the understanding of the domestication process that led to the improvement of rice grain quality.

CAPN1, CAST, and DGAT1 genetic effects on preweaning performance, carcass quality traits, and residual variance of tenderness in a beef cattle population selected for haplotype and allele equalization1,2,3,4

Genetic marker effects and type of inheritance are estimated with poor precision when minor marker allele frequencies are low. A stable composite population (MARC III) was subjected to marker-assisted selection for multiple years to equalize specific marker frequencies to 1) estimate effect size and mode of inheritance for previously reported SNP on targeted beef carcass quality traits (n=254), 2) estimate pleiotropic effects of previously reported SNP on nontarget performance traits (n=542 or 254), and 3) evaluate tenderness SNP specific residual variance for LM tenderness. Three haplotypes within μ-calpain (CAPN1), a SNP in calpastatin (CAST), and a dinucleotide substitution in diacylglycerol O-acyltransferase 1 (DGAT1) were successfully selected to equalize their frequencies. Traits evaluated were birth BW, weaning BW, yearling BW, final BW, dressing percent, HCW, fat thickness, LM area, USDA marbling score, yield grade, LM slice shear force (SSF), and visible and near-infrared (VISNIR)-predicted SSF. While the CAPN1 genotype effect on SSF was not significant (P=0.12), the direction and size of CAPN1 contrasts were consistent with previous research. Effects on SSF between divergent CAPN1 haplotypes (1.153 kg) and the additive effect of CAST (0.902 kg) were large, and animals homozygous for tender alleles at both CAPN1 and CAST would have 4.11 kg lower SSF (27.5% of the mean) than animals homozygous tough for both markers. Furthermore, the interaction between CAPN1 and CAST for SSF was not significant (P=0.40). There were significant effects for DGAT1 on adjusted fat thickness (P=0.02) and VISNIR-predicted SSF (P<0.001) with additive and dominance modes of inheritance (P<0.05) for both traits. Furthermore, CAST genotype specific residual variance models fit significantly better (P<0.001) than single residual variance models for SSF, with the tougher genotypes having progressively larger residual (and hence phenotypic) variances. Therefore, risk of a tough steak from the undesired CAST genotype is increased through both an increase in mean and an increase in variation. This work is supportive of the importance of CAPN1 and CAST for mean tenderness in beef, confirms an effect of CAST on beef LM tenderness variation, and identifies an effect of DGAT1 on subcutaneous fat thickness.

Influence of DGAT1 K232A Polymorphism on Milk Fat Percentage and Fatty Acid Profiles in Romanian Holstein Cattle

Milk and dairy products are considered the main sources of saturated fatty acids, which are a valuable source of nutrients in the human diet. Fat composition can be adjusted through guided nutrition of dairy animals but also through selective breeding. Recently, a dinucleotide substitution located in the exon 8 of the gene coding for acyl CoA: diacylglycerol acyltransferase 1 (DGAT1), that alters the amino acid sequence from a lysine to an alanine (p.Lys232Ala) in the mature protein, was shown to have a strong effect on milk fat content in some cattle breeds. Therefore, the objectives of this work were to study the occurrence of the DGAT1 p.Lys232Ala polymorphism in Romanian Holstein cattle and Romanian Buffalo breeds and to further investigate its possible influence on fat percentage and fatty acid profiles. The results obtained in this study show that in Romanian Holstein cattle the K allele is associated with increased fat percentage and higher levels of C16:0 and C18:0 fatty acids. The ratio of saturated fatty acids versus unsaturated fatty acids (SFA/UFA) was also higher in KK homozygous individuals, whereas the fractions of C14:0, unsaturated C18 decreased. The DGAT1 p.Lys232Ala polymorphism revealed a high genetic variance for fat percentage, unsaturated C18, C16:0, and SFA/UFA. Although the effect of this polymorphism was not so evident for short chain fatty acids such as C4:0–C8:0, it was significant for C14:0 fatty acids. We concluded that selective breeding of carriers of the A allele in Romanian Holsteins can contribute to improvement in unsaturated fatty acids content of milk. However, in buffalo, the lack of the A allele makes selection inapplicable because only the K allele, associated with higher saturated fatty acids contents in milk, was identified.

BRAF/NRAS Wild-Type Melanomas Have a High Mutation Load Correlating with Histologic and Molecular Signatures of UV Damage

The mutation load in melanoma is generally high compared with other tumor types due to extensive UV damage. Translation of exome sequencing data into clinically relevant information is therefore challenging. This study sought to characterize mutations identified in primary cutaneous melanomas and correlate these with clinicopathologic features. DNA was extracted from 34 fresh-frozen primary cutaneous melanomas and matched peripheral blood. Tumor histopathology was reviewed by two dermatopathologists. Exome sequencing was conducted and mutation rates were correlated with age, sex, tumor site, and histopathologic variables. Differences in mutations between categories of solar elastosis, pigmentation, and BRAF/NRAS mutational status were investigated. The average mutation rate was 12 per megabase, similar to published results in metastases. The average mutation rate in severely sun damaged (SSD) skin was 21 per Mb compared with 3.8 per Mb in non-SSD skin (P=0.001). BRAF/NRAS wild-type (WT) tumors had a higher average mutation rate compared with BRAF/NRAS-mutant tumors (27 vs. 5.6 mutations per Mb; P=0.0001). Tandem CC>TT/GG>AA mutations comprised 70% of all dinucleotide substitutions and were more common in tumors arising in SSD skin (P=0.0008) and in BRAF/NRAS WT tumors (P=0.0007). Targetable and potentially targetable mutations in WT tumors, including NF1, KIT, and NOTCH1, were spread over various signaling pathways. Melanomas arising in SSD skin have higher mutation loads and contain a spectrum of molecular subtypes compared with BRAF- and NRAS-mutant tumors indicating multigene screening approaches and combination therapies may be required for management of these patients.

Detection of BRAF V600 Mutations in Metastatic Melanoma: Comparison of the Cobas 4800 and Sanger Sequencing Assays

Detection of BRAF V600 Mutations in Metastatic Melanoma: Comparison of the Cobas 4800 and Sanger Sequencing Assays

Use of ChIP-Seq data for the design of a multiple promoter-alignment method

We address the challenge of regulatory sequence alignment with a new method, Pro-Coffee, a multiple aligner specifically designed for homologous promoter regions. Pro-Coffee uses a dinucleotide substitution matrix estimated on alignments of functional binding sites from TRANSFAC. We designed a validation framework using several thousand families of orthologous promoters. This dataset was used to evaluate the accuracy for predicting true human orthologs among their paralogs. We found that whereas other methods achieve on average 73.5% accuracy, and 77.6% when trained on that same dataset, the figure goes up to 80.4% for Pro-Coffee. We then applied a novel validation procedure based on multi-species ChIP-seq data. Trained and untrained methods were tested for their capacity to correctly align experimentally detected binding sites. Whereas the average number of correctly aligned sites for two transcription factors is 284 for default methods and 316 for trained methods, Pro-Coffee achieves 331, 16.5% above the default average. We find a high correlation between a method's performance when classifying orthologs and its ability to correctly align proven binding sites. Not only has this interesting biological consequences, it also allows us to conclude that any method that is trained on the ortholog data set will result in functionally more informative alignments.