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International audience; Adaptation to changing environmental conditions represents a challenge to parthenogenetic organisms, and until now, how phenotypic variants are generated in clones in response to the selection pressure of their environment remains poorly known. The obligatory parthenogenetic root-knot nematode species Meloidogyne incognita has a worldwide distribution and is the most devastating plant-parasitic nematode. Despite its asexual reproduction, this species exhibits an unexpected capacity of adaptation to environmental constraints, for example, resistant hosts. Here, we used a genomewide comparative hybridization strategy to evaluate variations in gene copy numbers between genotypes of M. incognita resulting from two parallel experimental evolution assays on a susceptible vs. resistant host plant. We detected gene copy number variations (CNVs) associated with the ability of the nematodes to overcome resistance of the host plant, and this genetic variation may reflect an adaptive response to host resistance in this parthenogenetic species. The CNV distribution throughout the nematode genome is not random and suggests the occurrence of genomic regions more prone to undergo duplications and losses in response to the selection pressure of the host resistance. Furthermore, our analysis revealed an outstanding level of gene loss events in nematode genotypes that have overcome the resistance. Overall, our results support the view that gene loss could be a common class of adaptive genetic mechanism in response to a challenging new biotic environment in clonal animals.

This study aims to investigate the relationship between the TSHR, BRAF, and PIK3CA gene copy number variations (CNVs) and thyroid nodules by analyzing gene CNVs, and to explore the interaction between iodine status and the above genes CNVs in the occurrence of thyroid nodules. Three hundred and ninety-five subjects were selected from 3 regions with different iodine status in Shanxi Province of China, including 192 patients with thyroid nodules and 203 healthy controls. The basic information about subjects had been obtained through a questionnaire. B ultrasound was utilized to check thyroid nodules. Blood and urine samples were harvested to detect the thyroid function and urinary iodine concentration. Real-time quantitative polymerase chains reaction (RT-PCR) served to detect CNVs in DNA from human blood. There was an association between TSHR gene CNV and thyroid nodules (χ2 = 8.403, P = 0.004). The prevalence of BRAF and PIK3CA gene CNVs was not statistically significant between the case group and the control group. Differences in the TSHR gene CNV rates for cases of the 3 areas were statistically significant (χ2 = 10.072, P = 0.007). No statistical difference in the prevalence rates of the 3 genes CNVs between diverse characteristics of thyroid nodules was observed. UIC > 300 μg/L (OR = 1.74, 95% CI: 1.02–2.96, P = 0.041) and TSHR gene CNV (OR = 3.53, 95% CI: 1.40–8.92, P = 0.008) were risk factors for thyroid nodules. There was no significant interaction between the UIC and the examined genes CNVs. TSHR gene CNV and high urinary iodine levels can increase the risk of thyroid nodules. But the interactions between the 3 above genes CNVs and iodine nutrition were not found in the occurrence of thyroid nodules.

Copy number variations (CNVs) are gains and losses of genomic sequence of more than 50 bp between two individuals of a species. Also, CNV is considered to be one of the main elements affecting the phenotypic diversity and evolutionary adaptation of animals. ORMDL sphingolipid biosynthesis regulator 1 (ORMDL1) is a protein-coding gene associated with diseases and development. In our study, the polymorphism of ORMDL1 gene copy numbers in four Chinese sheep breeds (abbreviated CK, HU, STH, and LTH) was detected. In addition, we analyzed the transcriptional expression level of ORMDL1 gene in different tissues of sheep and examined the association of ORMDL1 CNV with growth traits. The statistical analysis revealed that ORMDL1 CNV was remarkably correlated with body height, heart girth, and circumference of cannon bone in HU sheep (P

Leishmania infantum is one of the causative agents of visceral leishmaniasis (VL), a widespread, life-threatening disease. This parasite is responsible for the majority of human VL cases in Brazil, the Middle East, China, Central Asia and the Mediterranean basin. Its main reservoir are domestic dogs which, similar to human patients, may develop severe visceral disease and die if not treated. The drug allopurinol is used for the long-term maintenance of dogs with canine leishmaniasis. Following our report of allopurinol resistance in treated relapsed dogs, we investigated the mechanisms and markers of resistance to this drug. Whole genome sequencing (WGS) of clinical resistant and susceptible strains, and laboratory induced resistant parasites, was carried out in order to detect genetic changes associated with resistance. Significant gene copy number variation (CNV) was found between resistant and susceptible isolates at several loci, including a locus on chromosome 30 containing the genes LinJ.30.3550 through LinJ.30.3580. A reduction in copy number for LinJ.30.3560, encoding the S-adenosylmethionine synthetase (METK) gene, was found in two resistant clinical isolates and four induced resistant clonal strains. Using quantitative real time PCR, this reduction in METK copy number was also found in three additional resistant clinical isolates. Furthermore, inhibition of S-adenosylmethionine synthetase encoded by the METK gene in allopurinol susceptible strains resulted in increased allopurinol resistance, confirming its role in resistance to allopurinol. In conclusion, this study identified genetic changes associated with L. infantum resistance to allopurinol and the reduction in METK copy number identified may serve as a marker for resistance in dogs, and reduced protein activity correlated with increased allopurinol resistance., Graphical abstract Distribution of relative quantity values (RQ) of gene LinJ.30.3560 from the 20 L. infantum clinical strains in relation to their allopurinol IC50.Image 1, Highlights • Allopurinol resistance was previously described in L. infantum isolated from dogs. • This study aimed at defining the genetic differences between susceptible and resistant strains. • Gene and chromosome copy numbers differed between susceptible and resistant L. infantum strains. • Decrease in METK gene copies was associated with increased allopurinol resistance. • Inhibition of the enzyme encoded by METK increased allopurinol resistance.

Gene copy number variations (CNVs) involved in phenotypic variations have already been shown in plants, but genomewide testing of CNVs for adaptive variation was not doable until recent technological developments. Thus, reports of the genomic architecture of adaptation involving CNVs remain scarce to date. Here, we investigated F1 progenies of an intraprovenance cross (north-north cross, 58th parallel) and an interprovenances cross (north-south cross, 58th/49th parallels) for CNVs using comparative genomic hybridization on arrays of probes targeting gene sequences in balsam poplar (Populus balsamifera L.), a widespread North American forest tree. A total of 1,721 genes were found in varying copy numbers over the set of 19,823 tested genes. These gene CNVs presented an estimated average size of 8.3 kb and were distributed over poplar's 19 chromosomes including 22 hotspot regions. Gene CNVs number was higher for the interprovenance progeny in accordance with an expected higher genetic diversity related to the composite origin of this family. Regression analyses between gene CNVs and seven adaptive trait variations resulted in 23 significant links; among these adaptive gene CNVs, 30% were located in hotspots. One-to-five gene CNVs were found related to each of the measured adaptive traits and annotated for both biotic and abiotic stress responses. These annotations can be related to the occurrence of a higher pathogenic pressure in the southern parts of balsam poplar's distribution, and higher photosynthetic assimilation rates and water-use efficiency at high latitudes. Overall, our findings suggest that gene CNVs typically having higher mutation rates than SNPs may in fact represent efficient adaptive variations against fast-evolving pathogens.

During the last few years multiplex real-time or quantitative polymerase chain reaction PCR (qPCR) has become the method of choice for multiplex gene expression changes and gene copy number variations (CNVs) analysis. However, such determinations require the use of different fluorescent labels for the different amplified sequences, which increases significantly the costs of the analysis and limits the applicability of the technique for simultaneous amplification of many targets of interest in a single reaction. In this regard, the use of the coupling between gel electrophoresis (GE) separation with inductively coupled plasma mass spectrometry (ICP-MS) detection allows the label-free determination of multiplex PCR-amplified sequences (amplicons) by monitoring the P present in the DNA backbone. The quantitative dimension is obtained since under optimal and controlled multiplex PCR conditions the peak areas of the separated amplicons are directly proportional to the amount of DNA template in the original sample. Moreover, the calibration of the GE-ICP-MS system with a DNA ladder permits direct estimation of the size (bp) of the PCR products. The suitability of the proposed multiplex strategy has been evaluated addressing two different situations: determination of CNVs and gene expression changes in human ovarian cancer cells. In the first case, the results obtained for the simultaneous quantitation of CNVs of four genes (HER2, CCNE1, GSTM1, ACTB) on DNA obtained from OVCAR-3 cells were in accordance with the literature data, and also with the results obtained by conventional simplex qPCR. In the second case, multiplex gene expression changes of BAX, ERCC1 and CTR1 genes, using ACTB as constitutive gene, on A2780cis respect to A2780 cells, resistant and sensitive to cisplatin, respectively, provided the same information as single reaction reverse transcription (RT)-qPCR.

Copy number variations (CNVs) recently have been recognized as another important genetic variability followed single nucleotide polymorphisms (SNPs). The guanylate binding protein 2 (GBP2) gene plays an important role in cell proliferation. This study was performed to determine the presence of GBP2 CNV (relative to Angus cattle) in 466 individuals representing six main cattle breeds from China, identify its relationship with growth, and explore the biological effects of gene expression. There were two CNV regions in the GBP2 gene, for three types, CNV1 loss type (relative to Angus cattle) was more frequent in XN than other breeds, and CNV2 loss type (relative to Angus cattle) was more frequent in XN and CDM than other breeds. Though the GBP2 gene copy number presented no correlation with the transcriptional expression of JX (P .05), but the transcriptional expression in heart is higher than other tissues, and the copy number in muscles and fat of JX is higher than others breeds. Statistical analysis revealed that the GBP2 gene CNV1 and CNV2 were significantly associated with growth traits (P .05). In conclusion, this research established the correlations between CNVs of GBP2 gene and growth traits in different cattle breeds, and our results suggested that the CNVs in GBP2 gene may be considered markers for the molecular breeding of Chinese beef cattle.

Gene copy number variation (CNV) has been associated with phenotypic variability in animals and plants, but a genomewide understanding of their impacts on phenotypes is largely restricted to human and agricultural systems. As such, CNVs have rarely been considered in investigations of the genomic architecture of adaptation in wild species. Here, we report on the genetic mapping of gene CNVs in white spruce, which lacks a contiguous assembly of its large genome (~20 Gb), and their relationships with adaptive phenotypic variation. We detected 3,911 gene CNVs including de novo structural variations using comparative genome hybridization on arrays (aCGH) in a large progeny set. We inferred the heterozygosity at CNV loci within parents by comparing haploid and diploid tissues and genetically mapped 82 gene CNVs. Our analysis showed that CNVs were distributed over 10 linkage groups and identified four CNV hotspots that we predict to occur in other species of the Pinaceae. Significant relationships were found between 29 of the gene CNVs and adaptive traits based on regression analyses with timings of bud set and bud flush, and height growth, suggesting a role for CNVs in climate adaptation. The importance of CNVs in adaptive evolution of white spruce was also indicated by functional gene annotations and the clustering of 31% of the mapped adaptive gene CNVs in CNV hotspots. Taken together, these results illustrate the feasibility of studying CNVs in undomesticated species and represent a major step towards a better understanding of the roles of CNVs in adaptive evolution.

Endometriosis is a common chronic and polygenic disease among women. Considering the importance of the relationship between endometriosis and the increasing risk of being diagnosed with various cancers among women with endometriosis, it has been demonstrated that genetic factors are clearly involved in half of these cases. The amplification/overexpression of growth factors as a dominant mechanism in developing cancer is of high importance in this progress. HER-2 is one of the main growth factors and contributors in tumorigenesis. The evident role of this biomarker in identifying and curing prevalent cancers among women is an indicator of its possible role in the cancer development process after being diagnosed with endometriosis. Thus, according to the distinctive genetic model of Iran's population, the aim of this study was to explore the relationship between HER-2 gene copy number variations and the risk of endometriosis in Iranian women. In a population-based case-control study conducted in Tehran, in-person interviews were completed for 100 women aged 15–49 years and an equal number of controls frequency-matched to cases by age. All cases were newly diagnosed with endometriosis and all controls were with no laparoscopic evidence of disease between Dec 2014 and Aug 2015. We extracted the genomic DNA from peripheral blood and then HER-2 copy number was detected by real-time PCR. OR and 95% CI were calculated with Mann–Whitney test (P

Chinese Hamster Ovary (CHO) cells are aneuploid in nature. The genome of recombinant protein producing CHO cell lines continuously undergoes changes in its structure and organization. We analyzed nine cell lines, including parental cell lines, using a comparative genomic hybridization (CGH) array focused on gene-containing regions. The comparison of CGH with copy-number estimates from sequencing data showed good correlation. Hierarchical clustering of the gene copy number variation data from CGH data revealed the lineage relationships between the cell lines. On analyzing the clones of a clonal population, some regions with altered genomic copy number status were identified indicating genomic changes during passaging. A CGH array is thus an effective tool in quantifying genomic alterations in industrial cell lines and can provide insights into the changes in the genomic structure during cell line derivation and long term culture. Biotechnol. Bioeng. 2017;114: 1903-1908. © 2017 Wiley Periodicals, Inc.

To analyze copy number variations of HER-2/neu, c-MYC and CCNE1 oncogenes and their protein expression in endometrioid endometrial carcinomas in relation to the degree of tumor progression and presence of a family history of cancer in cancer patients.The study was conducted on endometrial cancer (EC) samples from 68 patients with I-II FIGO stages of disease. Copy number analysis of HER-2/neu, c-MYC and CCNE1 genes was performed by quantitative PCR. Protein expression was analyzed using immunohistochemistry.Assessment of copy number variations of HER-2/neu, c-MYC and CCNE1 genes revealed their amplification in the tumors of 18.8, 25.0 and 14.3% of EC patients, respectively. High expression of corresponding proteins was detected in 14.6, 23.5 and 65.6% of patients, respectively. It was established that HER-2/neu gene amplification is more common in the group of tumors of low differentiation grade than in moderate grade EC (35.7 and 5.5% of cases, respectively, p0.05). Also, high expression of c-Myc protein was more frequently observed in low differentiated tumors compared to the moderately differentiated EC (36.6 and 13.2% of cases, respectively, p0.05). Expression of HER-2/neu and cyclin E proteins was found to be dependent on the depth of tumor invasion into the myometrium. High expression of HER-2/neu protein was observed in 25.0 and 4.1% of EC patients with tumor invasion½ and½ of the myometrium, respectively, and cyclin E - in 86.7 and 46.6% of cases, respectively, p0.05. It was shown that among patients with a family history of cancer, a larger proportion of cases with high expression of c-Myc protein was observed compared to the group of patients with sporadic tumors (43.8 and 17.3%, respectively; p0.05).Amplification of HER-2/neu gene, along with high expression of c-Myc, HER-2/neu and cyclin E proteins, are associated with such indices of tumor progression as a low differentiation grade and deep myometrial invasion, suggesting the potential possibility of including these markers in the panel for determining the molecular EC subtype associated with an aggressive course of the disease. In a certain category of EC patients, there is a relationship between a family history of cancer and high expression of c-Myc protein.

The CCND1 locus is located in 11q13 and encodes the G1-S regulatory protein, cyclin D1. Cyclin D1 is frequently amplified in various types of cancers, and is an attractive potential therapeutic target. Multiplex ligation-dependent probe amplification (MLPA) is a new, high-resolution method for the detection of amplification of numerous genes including CCND1 in small amounts of DNA fragments derived from formalin-fixed, paraffin-embedded material in a single reaction. This approach is, however, based on PCR and averages many different cells, so validation by morphological methods such as fluorescence in situ hybridization (FISH) is theoretically mandatory. Here we describe detection of CCND1 gene copy number variations by commercially available MLPA kits and FISH using a bacterial artificial chromosome (BAC) probe.

ObjectiveComplement-mediated vasculopathy of muscle and skin are clinical features of juvenile dermatomyositis (JDM). We assess gene copy-number variations (CNVs) for complement C4 and its isotypes, C4A and C4B, in genetic risks and pathogenesis of JDM.MethodsThe study population included 105 patients with JDM and 500 healthy European Americans. Gene copy-numbers (GCNs) for total C4, C4A, C4B and HLA-DRB1 genotypes were determined by Southern blots and qPCRs. Processed activation product C4d bound to erythrocytes (E-C4d) was measured by flow cytometry. Global gene-expression microarrays were performed in 19 patients with JDM and seven controls using PAXgene-blood RNA. Differential expression levels for selected genes were validated by qPCR.ResultsSignificantly lower GCNs and differences in distribution of GCN groups for total C4 and C4A were observed in JDM versus controls. Lower GCN of C4A in JDM remained among HLA DR3-positive subjects (p=0.015). Homozygous or heterozygous C4A-deficiency was present in 40.0% of patients with JDM compared with 18.2% of controls (OR=3.00 (1.87 to 4.79), p=8.2×10−6). Patients with JDM had higher levels of E-C4d than controls (p=0.004). In JDM, C4A-deficient subjects had higher levels of E-C4d (p=0.0003) and higher frequency of elevated levels of multiple serum muscle enzymes at diagnosis (p=0.0025). Microarray profiling of blood RNA revealed upregulation of type I interferon-stimulated genes and lower abundance of transcripts for T-cell and chemokine function genes in JDM, but this was less prominent among C4A-deficient or DR3-positive patients.ConclusionsComplement C4A deficiency appears to be an important factor for the genetic risk and pathogenesis of JDM, particularly in patients with a DR3-positive background.

To explore the association of β-defensin gene copy number variations (CNVs) with ankylosing spondylitis (AS).In this study, 405 unrelated Chinese Han patients with AS and 401 unrelated healthy controls were enrolled. The copy numbers of DEFB4 gene (2 fragments) were measured by AccuCopy™ methods. The association of DEFB4 gene CNVs with AS susceptibility was analyzed by chi-square and logistic regression models. Besides, P values, odds ratio, and 95% confidence intervals (CIs) were used to estimate the effects of risk.The range of DEFB4_1 CN was 0-7 and the range of DEFB4_2 CN was 1-8 both in patients and controls. P values of χ(2) trend test for the association of DEFB4_1 and DEFB4_2 with AS were 0.607 and 0.005, respectively. The results of DEFB4_2, compared with the individual having median 3 copies, those carrying ≤ 2-copies [OR = 0.68, 95%CI: (0.46, 0.99), P = 0.049; adjusted OR = 0.69, 95%CI(0.47, 1.03), P = 0.067.]; and those carrying ≥ 4-copies [OR = 0.62, 95%CI: (0.45, 0.86), P = 0.004; adjusted OR = 0.64, 95%CI: (0.46, 0.88), P = 0.006], were significantly associated with decreasing risk of AS. Univariate analysis showed that both DEFB4_1 and DEFB4_2 were associated with Bath AS Disease Activity Index or BASDAI. After adjusted by age, sex, and disease duration, the results changed little, which demonstrated that high copies may be linked with decrease in the risk of disease severity [OR = 0.71, 95%CI: (0.56, 0.90), P = 0.005; OR = 0.75, 95%CI: (0.60, 0.94), P = 0.013, respectively].The CNs of DEFB4 gene may be associated with AS and involved in disease progression.

CYP2D6 and SULT1A1 occasionally show copy number variations (CNVs), with a larger number generally indicating greater enzymic activity. However, those variations are difficult to calculate using standard methods. With digital PCR, a recently introduced method for CNV analysis, DNA molecules are subjected to limited dilution and separated into nano-scale droplets prior to a PCR assay. Absolute quantitation of copy number can then be performed with high accuracy and sensitivity by determining the number of droplets showing an amplified signal for the target gene. This is the first report of analyses of CYP2D6 and SULT1A1 CNVs using a digital PCR method with blood sample from Japanese subject. Primers and probes were synthesized for the target and reference genes, and copy number calculation was performed using a QX200 Droplet Digital PCR System. Our results showed that the copy numbers in CYP2D6*5 hetero, non-CNV, and CYP2D6xN subjects were 1, 2, and 3 to 4, respectively. In addition, in non-CNV and multiplication subjects, the number of copies for SULT1A1 was 2 and 3 to 6, respectively. We found that the present digital PCR method was useful as well as accurate. In the future, a combined genotyping, allele distinction, and copy number calculation technique will be helpful for analysis of enzymic activity.

KIR (Killer Immunoglobulin‐like Receptor) variants influence immune responses and are genetic factors in disease susceptibility. Using sequence‐specific priming PCR, we have previously described the diversity of KIR genes in term of presence/absence in northeastern Thais (NETs). To provide additional resolution beyond conventional methods, quantitative PCR was applied to determine KIR copy number profiles. Novel expanded and contracted KIR copy number profiles were identified at cumulatively high frequencies. These all comprise haplotypes with duplication (6·9%) or deletion (2·7%) of KIR3DL1/S1 along with adjacent genes. Five expanded KIR profiles comprised haplotypes with duplications of KIR2DP1, 2DL1, 3DP1, 2DL4, 3DL1/S1 and 2DS1/4, whereas two contracted profiles contained only a single copy of KIR3DP1, 3DL1/S1 and 2DL4. Using a KIR haplotype prediction program (KIR Haplotype Identifier), 14% of NET haplotypes carried atypical haplotypes based on the gene copy number data.

Multiplex ligation-dependent probe amplification (MLPA) is based on simultaneous multiplex PCR of specific probes that hybridize to multiple different target DNA regions. The method can identify copy number changes, gross gene rearrangements, methylation patterns or even point mutations. MLPA has been a reliable approach to identify copy number changes in the clinical and research settings and is widely used for the screening and investigation of copy number variations and genomic aberrations of interest in various diseases. In this chapter the analysis of the copy number changes in the RB1 gene locus by MLPA is described.

Objective At the molecular and cellular levels, heart development entails the precise orchestration of genetic events such as the interplay of master transcriptional regulators, signaling pathways, and chromatin remodeling. Recent studies among patients with congenital heart disease (CHD) have shown the importance of recurrent copy number variations (CNVs) in a significant number of patients. Recurrent CNVs that span several genes may affect other important organs, besides the heart. Because CHD may be the first presenting symptom in such patients, the analysis of recurrent CNVs in the genomic regions containing genes associated with CHD in patients referring to cardiology clinics may lead to an early diagnosis and the treatment of extracardiac symptoms in these patients. In this study, we aimed to screen CNVs of genomic regions including GATA4, NKX2-5, TBX5, BMP4, and CRELD1 genes and to analyse the 22q11.2 chromosomal region in apparently nonsyndromic patients with cardiac septal defects. Methods Genomic regions including GATA4, NKX2-5, TBX5, BMP4, and CRELD1 genes and the 22q11.2 chromosomal region were analyzed in apparently nonsyndromic 45 patients with cardiac septal defects using the MLPA P-311 A2 Congenital Heart Disease kit. Multiplex ligationdependent probe amplification (MLPA) is an established technique for the detection of known CNVs. MLPA is substantially less expensive than array CGH and is relatively simple to use for clinicians without specific expertise in genomic technology; thus, MLPA could be used as a first-tier screening assay. Results We screened 45 patients with cardiac septal defects for CNVs using the MLPA P-311 A2 kit. We identified three CNVs (n=3/45, 6.66%) and three 22q11 deletions. The CNVs were confirmed using fluorescence in situ hybridization. Conclusion Our study confirmed that the analysis of recurrent CNVs using the MLPA assay within pediatric cardiology clinics can led to an early syndrome diagnosis in nonsyndromic patients with CHD.

Background: Cervical cancer is the second most common gynecologic cancer worldwide. Human papillomavirus (HPV) infection is a leading etiological factor in cervical carcinoma.The aim of this study was to compare HPV positivity, EGFR and TOP2A gene copy number variations and cervical cytologic findings. Materials and Methods: The study group comprised 100 female volunteers between 21-64 years old. Cytologic analysis was performed using the liquid-based cytology technique. HPV DNA testing was performed in all cases. Copy number variations that belong to EGFR and TOP2A genes evaluated by using FISH analysis. Results: Cytologic analysis of the cervical samples revealed abnormal findings in 13 of the 100 study subjects. ASCUS , LSIL, HSIL were determined in 8, 2 and 3 cases respectively as the result of cytologic analysis on all cases. Forty-one (41%) of the 100 women were HPV-positive. Chi-square analysis confirmed that HPV-positive women showed significantly more abnormal cytology (P = 0.035). EGFR deletion, TOP2A deletion and both EGFR and TOP2A deletion were determined in 1, 8 and 1 cases respectively. We found no statistical difference in abnormal cytologic findings between subjects with these gene deletions and subjects with normal gene copy numbers (P > 0.05 for both). No cases of amplification were determined for either gene. Conclusion: As a result, HPV positivity and the determination of changes that may occur in the gene copy number in patients with abnormal cytology can be an important tool on account of prognosis. Research with more patients may be suggested.

Pre-eclampsia is a pregnancy-specific hypertensive disorder that affects 2–8 % of pregnancies. This disorder can lead to seizure, multi-organ failure and maternal death. The best approach to prevent pre-eclampsia-associated adverse outcomes is to be able to prevent pre-eclampsia as early as possible. Unfortunately, current diagnostic methods are ineffective at predicting the risk of pre-eclampsia during early pregnancy. In humans, low levels of a group of placenta-derived Pregnancy Specific Glycoproteins (PSGs) have been associated with intrauterine growth retardation and pre-eclampsia and there is a significant enrichment of cases with deletions in the PSG gene locus in pre-eclampsia patients. Based on these observations, we hypothesize that genomic variations at human PSG locus of maternal and/or fetal genomes may confer increased risks of pre-eclampsia. To test this hypothesis, we have recruited 90 normal control and 30 pre-eclamptic women for the analysis of fetal PSG copy number variations (CNVs).

Aims and Background Copy number variations (CNVs) contribute to genome variability and their pathogenic role is becoming evident in an increasing number of human disorders. Commercial assays for routine diagnosis of CNVs are available only for a fraction of known genomic rearrangements. Thus, it is important to develop flexible and cost-effective methods that can be adapted to the detection of CNVs of interest, both in research and clinical settings. Methods We describe a new multiplex PCR-based method for CNV analysis that exploits automated microfluidic capillary electrophoresis through lab-on-a-chip technology (LOC-CNV). We tested the reproducibility of the method and compared the results obtained by LOC-CNV with those obtained using previously validated semiquantitative assays such as multiplex ligation-dependent probe amplification (MLPA) and nonfluorescent multiplex PCR coupled to HPLC (NFMP-HPLC). Results The results obtained by LOC-CNV in control individuals and carriers of pathogenic MLH1 or BRCA1 genomic rearrangements (losses or gains) were concordant with those obtained by previously validated methods, indicating that LOC-CNV is a reliable method for the detection of genomic rearrangements. Conclusion Because of its advantages with respect to time, costs, easy adaptation of previously developed multiplex assays and flexibility in novel assay design, LOC-CNV may represent a practical option to evaluate relative copy number changes in genomic targets of interest, including those identified in genome-wide analyses.

Leishmania tropica is one of the main causative agents of cutaneous leishmaniasis (CL). Population structures of L. tropica appear to be genetically highly diverse. However, the relationship between L. tropica strains genomic diversity, protein coding gene evolution and biogeography are still poorly understood. In this study, we sequenced the genomes of three new clinical L. tropica isolates, two derived from a recent outbreak of CL in camps hosting Syrian refugees in Lebanon and one historical isolate from Azerbaijan to further refine comparative genome analyses. In silico multilocus microsatellite typing (MLMT) was performed to integrate the current diversity of genome sequence data in the wider available MLMT genetic population framework. Single nucleotide polymorphism (SNPs), gene copy number variations (CNVs) and chromosome ploidy were investigated across the available 18 L. tropica genomes with a main focus on protein coding genes. MLMT divided the strains in three populations that broadly correlated with their geographical distribution but not populations defined by SNPs. Unique SNPs profiles divided the 18 strains into five populations based on principal component analysis. Gene ontology enrichment analysis of the protein coding genes with population specific SNPs profiles revealed various biological processes, including iron acquisition, sterols synthesis and drug resistance. This study further highlights the complex links between L. tropica important genomic heterogeneity and the parasite broad geographic distribution. Unique sequence features in protein coding genes identified in distinct populations reveal potential novel markers that could be exploited for the development of more accurate typing schemes to further improve our knowledge of the evolution and epidemiology of the parasite as well as highlighting protein variants of potential functional importance underlying L. tropica specific biology., Author summary Human cutaneous leishmaniasis (CL) is a parasitic infection transmitted through the bite of an infected sandfly vector. Members of the Leishmania tropica complex are one of the main causative agents of CL in the Old World. Despite having varying phenotypical manifestations and degrees of severity, the genomic differences underlying the observed phenotypic diversity are not well understood. CL outbreaks in Lebanon, Syria and in Azerbaijan and neighbouring countries are evidence that leishmaniasis is still an important public health concern. Here, we explored unique genetic features in L. tropica genomes by comparing the gene variants and gene copy number variations, as well as chromosome ploidy, of two recent isolates collected during a CL outbreak in Lebanon and one historical isolate from Azerbaijan with 16 published L. tropica genomes. In silico MLMT was also performed to integrate the current diversity of genome sequence data in the wider geography-related genetic populations. This study further highlights the distinct population structure and genomic heterogeneity of the L. tropica species complex in the context of the parasite broad geographic distribution. Unique sequence features identified in distinct populations could be exploited for the development of more accurate typing schemes and highlight proteins of potential functional importance to gain new insights into L. tropica specific biology.

Molecular analyses have become mandatory for treatment choices in patients with advanced non-small cell lung cancers (NSCLC). Among them, HER2 gene mutation, HER2 gene amplification, and HER2 protein expression consist in potential targets of various treatments. Tumor heterogeneity and overlapping of molecular alterations may cause dilemmas in treatment choices but to date there are few that reported about HER2 with discrepant data. We led a retrospective study evaluating HER2 protein expression and HER2 gene/chromosome 17 copy number variations across different tumor areas and samples from patients with advanced NSCLC harboring HER2 gene mutations and other oncogenic mutations. Among patients with HER2-mutated (10 patients) and nonmutated lung adenocarcinomas (10 patients), we observed frequent heterogeneous HER2 protein expression with no correlation with HER2 gene copy number variations. HER2 gene amplification was observed in 6 patients (3 HER2-mutated and 3 HER2-nonmutated), but with intrasample heterogeneity in 2 cases and intersample heterogeneity in another case. Our small case series emphasizes the potential overlapping and spatial heterogeneity of HER2 alterations in NSCLC, which must be taken into account as a limitation in building predictive strategies accompanying the development of anti-HER2 therapeutic strategies in patients with advanced NSCLC.

Malaria is among the most devastating and widespread tropical parasitic diseases in which most prevalent in developing countries. Antimalarial drug resistance is the ability of a parasite strain to survive and/or to multiply despite the administration and absorption of medicine given in doses equal to or higher than those usually recommended. Among the factors which facilitate the emergence of resistance to existing antimalarial drugs: the parasite mutation rate, the overall parasite load, the strength of drug selected, the treatment compliance, poor adherence to malaria treatment guideline, improper dosing, poor pharmacokinetic properties, fake drugs lead to inadequate drug exposure on parasites, and poor-quality antimalarial may aid and abet resistance. Malaria vaccines can be categorized into three categories: pre-erythrocytic, blood-stage, and transmission-blocking vaccines. Molecular markers of antimalarial drug resistance are used to screen for the emergence of resistance and assess its spread. It provides information about the parasite genetics associated with resistance, either single nucleotide polymorphisms or gene copy number variations which are associated with decreased susceptibility of parasites to antimalarial drugs. Glucose transporter PfHT1, kinases (Plasmodium kinome), food vacuole, apicoplast, cysteine proteases, and aminopeptidases are the novel targets for the development of new antimalarial drugs. Therefore, this review summarizes the antimalarial drug resistance and novel targets of antimalarial drugs.

Background. MYC (v-myc avian myelocytomatosis viral oncogene homolog) is one of the most frequently amplified genes in lung tumors. For the analysis of gene copy number variations, dPCR (digital PCR) is an appropriate tool. The aim of our study was the assessment of dPCR for the detection of MYC copy number variations (CNV) in lung tissue considering clinicopathological parameters. Material and Methods. MYC status was analyzed with dPCR as well as qPCR (quantitative PCR) using gDNA (genomic DNA) from tumor and adjacent nontumor tissue samples of lung cancer patients. The performance of MYC was estimated based on the AUC (area under curve). Results. The results of the MYC amplification correlated significantly between dPCR and qPCR (rS=0.81, P<0.0001). The MYC copy number revealed by dPCR showed statistically significant differences between tumor and adjacent nontumor tissues. For discrimination, a sensitivity of 43% and a specificity of 99% were calculated, representing 55 true-positive and one false-positive tests. No statistically significant differences could be observed for age, sex, and smoking status or the clinicopathological parameters (histological subtype, grade, and stage). Conclusion. The results of the study show that dPCR is an accurate and reliable method for the determination of MYC copy numbers. The application is characterized by high specificity and moderate sensitivity. MYC amplification is a common event in lung cancer patients, and it is indicated that the determination of the MYC status might be useful in clinical diagnostics.

High‐throughput DNA analyses in cancers allow the detection of molecular abnormalities that can guide patients' treatment. The collection of a tumour DNA is mainly performed on a biopsy, an invasive procedure for the patients. Another less invasive method consists in using a fine needle. We showed that fine‐needle biopsies are as suitable as a classical biopsy for DNA analysis., High‐throughput molecular profiling of solid tumours using core needle biopsies (CNB) allows the identification of actionable molecular alterations, with around 70% success rate. Although several studies have demonstrated the utility of small biopsy specimens for molecular testing, there remains debate as to the sensitivity of the less invasive fine‐needle aspiration (FNA) compared to CNB to detect molecular alterations. We aimed to prospectively evaluate the potential of FNA to detect such alterations in various tumour types as compared to CNB in cancer patients included in the SHIVA02 trial. An in‐house amplicon‐based targeted sequencing panel (Illumina TSCA 99.3 kb panel covering 87 genes) was used to identify pathogenic variants and gene copy number variations (CNV) in concomitant CNB and FNA samples obtained from 61 patients enrolled in the SHIVA02 trial (NCT03084757). The main tumour types analysed were breast (38%), colon (15%), pancreas (11%), followed by cervix and stomach (7% each). We report 123 molecular alterations (85 variants, 23 amplifications and 15 homozygous deletions) among which 98 (80%) were concordant between CNB and FNA. The remaining discordances were mainly related to deletions status, yet undetected alterations were not exclusively specific to FNA. Comparative analysis of molecular alterations in CNB and FNA showed high concordance in terms of variants as well as CNVs identified. We conclude FNA could therefore be used in routine diagnostics workflow and clinical trials for tumour molecular profiling with the advantages of being minimally invasive and preserve tissue material needed for diagnostic, prognostic or theranostic purposes.

Background: In addition to protein tyrosine kinases, accumulating evidence has shown that protein tyrosine phosphatases (PTPs) are suitable therapeutic targets in cancer. PRL-3 is a PTP member that has been well studied in many malignant tumours. The goal of the present study was to elucidate the role of PRL-3 in hepatocellular carcinoma (HCC), which remains largely unknown. Methods: Bioinformatic and immunohistochemical analyses were performed to analyse PRL-3 expression in HCC tissue samples and determine its clinical relevance. PRL-3 gene copy number variations were evaluated by bioinformatic analysis and quantitative-genomic polymerase chain reaction. The biological functions of PRL-3 were investigated in vivo and vitro. Gene microarray assays, RT-qPCR, western blotting and luciferase experiments were performed to identify the downstream effectors of PRL-3 that mediate its functions in HCC. Results: PRL-3 expression was upregulated in HCC samples from public databases and in cohort samples from our centre. High PRL-3 expression was associated with poor prognosis. Copy number gains and amplification of chromosome 8q24.3 in HCC were determined to be positively correlated with the PRL-3 overexpression. PRL-3 overexpression promoted HCC cell proliferation, migration and adhesion, while its loss had the opposite effects. Further study showed that focal adhesion kinase (FAK) was co-amplified and co-expressed with PRL-3 in HCC. Interestingly, PRL-3 also promoted the phosphorylation of FAK, which subsequently mediated the oncogenic functions of PRL-3 in HCC cells. Moreover, TGFB1 was identified as a downstream molecule of PRL-3. TGF-β signalling was shown to mediate the PRL-3-induced activation of FAK. Furthermore, the p38 and PI3K/AKT pathways were observed to mediate the PRL-3-induced expression of TGFB1 and the subsequent activation of FAK, while the activation of FAK in turn stimulated activation of the p38 and PI3K/AKT pathways, forming a PRL-3-triggered AKT/p38/TGFB1/FAK positive feedback loop. Conclusion: Collectively, our findings indicate that the PTP PRL-3 plays a crucial role in the progression of HCC and provides an example of how co-amplified genes work together in HCC.

Hepatocellular carcinoma (HCC) is a common malignant tumor worldwide, but effective immunotherapy is still limited for those affected. Therefore, there is an urgent need to explore the specific mechanisms governing tumor immunity to improve the survival rate for those diagnosed with HCC. In the present study, we performed a new immune stratification of HCC samples into two subclasses (A and B) from The Cancer Genome Atlas and the International Cancer Genome Consortium databases, and comprehensive multi-omic analyses of major histocompatibility complex genes, gene copy-number variations, somatic mutations, DNA methylation, and non-coding RNAs. Subclass A was found to have a higher survival rate compared with subclass B, and there were significant immunological differences between the two clusters. Based on these differences, we identified DRD1 and MYCN as key hub genes in the immune-phenotype gene expression regulatory network. These results provide novel ideas and evidence for HCC regulatory mechanisms that may improve immunotherapy for this cancer.

Phytoplankton account for >45% of primary production on Earth, and hence have an enormous impact on aquatic food webs and on the entire Earth System. Their members are found across many of the major clades of the tree of life, including prokaryotes (cyanobacteria) and multiple eukaryotic lineages. Phytoplankton communities are generally studied by PCR amplification of rRNA marker genes from the DNA extracted from environmental samples and high-throughput sequencing of the amplicons. However, our appreciation of phytoplankton abundance or biomass is limited by PCR-amplification biases, rRNA gene copy number variations across taxa and the fact that rRNA do not provide insights into metabolic traits such as photosynthesis (i.e., it is sometimes difficult to distinguish an rRNA sequence of a phytoplanktonic species from a phylogenetically close non-photosynthetic species). In addition, rRNA marker genes fail to capture both cyanobacteria and photosynthetic eukaryotes simultaneously, thus hampering assessment of the entire phytoplankton community. Here, we propose to use the core single-copy photosynthetic gene psbO extracted from metagenomes to circumvent these limitations: the method is PCR-free, and the gene is present in both prokaryotes and eukaryotes, mainly in one copy per genome. We applied and validated this new strategy with the Tara Oceans datasets, and showed in most cases improved correlations with flow cytometry and microscopy than when based on rRNA genes. Furthermore, we revealed unexpected features of the ecology of these organisms, such as the high abundance of picocyanobacterial aggregates and symbionts in the ocean, and the decrease in relative abundance of phototrophs towards the larger size classes of marine communities of dinoflagellates. To facilitate the incorporation of psbO in future molecular-based surveys, we released a curated database including >20,000 unique sequences. Overall, psbO appears to be a promising new gene marker for improving molecular-based evaluations of phytoplankton communities.

Conventional plasmid-based gene expression tends to introduce genetic instability and gene copy number variations that lead to degenerated production. The limited number of auxotrophic markers in Yarrowia lipolytica also restricts our ability to perform iterative genetic modifications and manipulate long gene clusters. To overcome these limitations, we combined the high recombination efficiency of the Cre-loxP system and the high integration rate of 26s rDNA, and developed a versatile framework to iteratively integrate multicopy metabolic pathways in Y. lipolytica. We demonstrated the efficient genome integration of a plant-derived flavonoid pathway at random sites with multiple copies. Transient expression of Cre recombinase enabled efficient marker removal and allowed for the next round of genome integration. Investigating the recombination events demonstrated that the iterative integration is happening at sufficiently high rates (more than 80%) without disrupting the previous integration. Both the flavonoid precursor pathway and the plant-derived cytochrome c P450 enzymes were functionally integrated to improve flavonoid and hydroxylated flavonoid production. The engineered strains produced 71.2 mg/L naringenin, 54.2 mg/L eriodyctiol, and 48.1 mg/L taxifolin. The reported work provides a versatile platform to iteratively integrate functional gene clusters at high copy numbers. This work may streamline and expand our capability to build efficient microbial cell factories for high-value natural products and commodity chemical production in Y. lipolytica.

Recent findings implicate the complement C4 gene in gray matter loss in schizophrenia. In a large cohort of patients with first-episode psychosis (FEP), we aimed to (1) characterize the frequency of C4 gene copy number variations (CNVs) and HERV-K Ins/Del events as compared to that in healthy controls (HCs) and (2) evaluate whether C4 gene structural variants influence baseline clinical symptoms and treatment response to amisulpride. A total of 271 FEP subjects and 221 HCs were genotyped for C4 CNV and HERV-Ins/Del (C4A and C4B isoforms; C4-HERV structural forms [C4AL, C4AS, C4BL, C4BS] variations using droplet digital PCR. Overall, the gene frequencies of both C4 isoforms and C4-HERV structural forms did not significantly differ between groups. At the genotype level, we found that the C4 AL-AL-BL-BL genotype (AL-BL haplotype) was significantly more frequent in FEP as compared to HC. Apart from a marginal observation concerning the C4 AL-AL-BL-BL genotype (AL-BL haplotype), possibly reflecting a relationship with schizophrenia, we did not find any correlation between C4 genetic and clinical characteristics or treatment response in FEP.

Phytophthora infestans, the causal agent of the Irish Potato Famine in the 1840s, is one of the most destructive crop pathogens that threaten global food security. Host resistance (R) genes may help to control the disease, but recognition by through the gene products can be evaded by newly emerging isolates. Such isolates are dangerous as they may cause disease outbreaks under favorable conditions. However, our lack of knowledge about adaptation in these isolates jeopardizes an apt response to resistance breakdown. Here we performed genome and transcriptome sequencing of HB1501 and HN1602, two field isolates from distinct Chinese geographic regions. We found extensive polymorphisms in these isolates, including gene copy number variations, nucleotide polymorphisms, and gene expression changes. Effector encoding genes, which contribute to virulence, show distinct expression landscapes in P. infestans isolates HB1501 and HN1602. In particular, polymorphisms at multiple effectors required for recognition (Avr loci) enabled these isolates to overcome corresponding R gene based resistance. Although the isolates evolved multiple strategies to evade recognition, we experimentally verified that several R genes such as R8, RB, and Rpi-vnt1.1 remain effective against these isolates and are valuable to potato breeding in the future. In summary, rapid characterization of the adaptation in emerging field isolates through genomic tools inform rational agricultural management to prevent potential future epidemics.

Structural variations (SVs) and gene copy number variations (gCNVs) have contributed to crop evolution, domestication, and improvement. Here, we assembled 31 high-quality genomes of genetically diverse rice accessions. Coupling with two existing assemblies, we developed pan-genome-scale genomic resources including a graph-based genome, providing access to rice genomic variations. Specifically, we discovered 171,072 SVs and 25,549 gCNVs and used an Oryza glaberrima assembly to infer the derived states of SVs in the Oryza sativa population. Our analyses of SV formation mechanisms, impacts on gene expression, and distributions among subpopulations illustrate the utility of these resources for understanding how SVs and gCNVs shaped rice environmental adaptation and domestication. Our graph-based genome enabled genome-wide association study (GWAS)-based identification of phenotype-associated genetic variations undetectable when using only SNPs and a single reference assembly. Our work provides rich population-scale resources paired with easy-to-access tools to facilitate rice breeding as well as plant functional genomics and evolutionary biology research.

Genetic polymorphism of cytochrome P450s results in clinically significant modifications in patients' drug metabolizing capacities. CYP2D6 has a crucial role in the elimination of several clinically important drugs (antiarrhythmics, beta-adrenergic blockers, psychopharmacons and analgesics); however, the prediction of the phenotypic appearance of CYP2D6 is a challenge. Since single nucleotide polymorphisms and gene copy number variations (gene deletion and multiplication) frequently occur in CYP2D6 gene, CYP2D6 activity particularly depends on the genetic factors. Microsomal CYP2D6 activities (dextromethorphan O -demethylation) and CYP2D6 genotypes for the most frequent allelic variants ( CYP2D6*3 , *4 , *5 , *6 , *10 , *41 and duplication) were determined in 128 human liver samples derived from Hungarian organ donors. Substantial inter-individual variations were observed in CYP2D6 metabolic activities that were successfully predicted from the CYP2D6 genotypes in 67% of the donors. The underestimation of CYP2D6 phenotypes in 12.5% of the donors was assumed to be originated from the overlapping ranges of CYP2D6 activity among similar diplotypes or from the presence of − 1584C > G in the promoter region evoking increased transcription of the wild-type CYP2D6 allele. In an appreciable number of donors (20.3%), the genotype-based CYP2D6 phenotype prediction was overestimated because of the rare CYP2D6 allelic variants which were not included in our genotyping platform or some external factors that could alter CYP2D6 activity (medication with CYP2D6 substrate/inhibitor) and hepatic function (Augmentin therapy, chronic alcohol consumption). In conclusion, CYP2D6 genotyping for the most frequent allelic variants was able to reliably predict CYP2D6 phenotypes in most donors; however, the external factors modifying the phenotypic appearance of CYP2D6 had to be taken into account. Personalized medication strategy should include monitoring of CYP2D6 genotype in a more comprehensive manner and should take external factors into consideration for an appropriate prediction of CYP2D6 metabolizing capacity.

BACKGROUND Approximately 1% of normal tension glaucoma (NTG) cases are caused by TANK-binding kinase 1 (TBK1) gene duplications and triplications. However, the precise borders and orientation of these TBK1 gene copy number variations (CNVs) on chromosome 12 are unknown. METHODS We determined the exact borders of TBK1 CNVs and the orientation of duplicated or triplicated DNA segments in 5 NTG patients with different TBK1 mutations using whole-genome sequencing. RESULTS Tandemly duplicated chromosome segments spanning the TBK1 gene were detected in 4 NTG patients, each with unique borders. Four of 5 CNVs had borders located within interspersed repetitive DNA sequences (Alu and long interspersed nuclear element-L1 elements), suggesting that mismatched homologous recombinations likely generated these CNVs. A fifth NTG patient had a complex rearrangement including triplication of a chromosome segment spanning the TBK1 gene. CONCLUSIONS No specific mutation hotspots for TBK1 CNVs were detected, however, interspersed repetitive sequences (ie, Alu elements) were identified at the borders of TBK1 CNVs, which suggest that mismatch of these elements during meiosis may be the mechanism that generated TBK1 gene dosage mutations.

Summary Cryptorchidism is the most common congenital disorder in boys, but the cause for most cases remains unknown. Patients with Noonan Syndrome are characterized by a typical face, growth retardation, congenital heart defects, learning disabilities and cryptorchidism. Copy number variations of Ras/MAPK pathway genes are unusual in patients with several clinical features of Noonan Syndrome; however, they have not been studied in patients with only one feature of this condition, such as cryptorchidism. Our aim was to determine whether patients with isolated cryptorchidism exhibit Ras/MAPK pathway gene copy number variations (CNVs). Fifty-nine patients with isolated cryptorchidism and negative for mutations in genes associated with Noonan Syndrome were recruited. Determination of Ras/MAPK pathway gene CNVs was performed by Comparative Genome Hybridization array. A CNV was identified in two individuals, a ~175 kb microduplication at 3p25.2, partially including RAF1. A similar RAF1 microduplication has been observed in a patient with testicular aplasia. This suggests that some patients with isolated cryptorchidism may harbor Ras/MAPK pathway gene CNVs.

Background IDH-mutant glioblastoma is classified by the 2016 CNS WHO as a group with good prognosis. However, the actual number of cases examined in the literature is relatively small. We hypothesize that IDH-mutant glioblastoma is not a uniform group and should be further stratified. Methods We conducted methylation profiles and estimated copy number variations of 57 IDH-mutant glioblastomas. Results Our results showed that 59.6% and 40.4% of tumors belonged to glioma-CpG island methylator phenotype (G-CIMP)-high and G-CIMP-low methylation subgroups, respectively. G-CIMP-low subgroup was associated with significantly worse overall survival (OS) as compared to G-CIMP-high (P = .005). CDKN2A deletion (42.1%) was the most common gene copy number variation, and was significantly associated with G-CIMP-low subgroup (P = .004). Other frequent copy number changes included mesenchymal–epithelial transition (MET) (5.3%), CCND2 (19.3%), PDGFRA (14.0%), CDK4 (12.3%), and EGFR (12.3%) amplification. Both CDKN2A deletion (P = .036) and MET amplification (P < .001) were associated with poor OS in IDH-mutant glioblastomas. Combined epigenetic signature and gene copy number variations separated IDH-mutant glioblastomas into Group 1 (G-CIMP-high), Group 2 (G-CIMP-low without CDKN2A nor MET alteration), and Group 3 (G-CIMP-low with CDKN2A and/or MET alteration). Survival analysis revealed Groups 1 and 2 exhibited a favorable OS (median survival: 619 d [20.6 mo] and 655 d [21.8 mo], respectively). Group 3 exhibited a significant shorter OS (median survival: 252 d [8.4 mo]). Multivariable analysis confirmed the independent prognostic significance of our Groups. Conclusions IDH-mutant glioblastomas should be stratified for risk with combined epigenetic signature and CDKN2A/MET status and some cases have poor outcome.

Insulin-like growth factor (IGF) dysregulation and gene copy number variations (CNV) are hallmarks of adrenocortical carcinoma (ACC). The contribution of IGF CNVs in adrenal carcinogenesis has not been studied previously. In addition, studies demonstrating an association between SLC12A7 gene amplifications and enhanced metastatic behavior in ACC, as well as reported IGF-SLC12A7 signaling interactions in other cancers, suggest a potential IGF-SLC12A7 signaling circuitry in ACC. Here we investigate the potential complicity of IGF-SLC12A7 signaling in ACC.Insulin-like growth factor CNVs were determined by whole-exome sequencing analysis in an exploratory cohort of ACC. Quantitative polymerase chain reaction methods determined IGF1 and IGF2 expression levels and were evaluated for correlation with SLC12A7 expression and tumor characteristics. Insulin-like growth factor CNVs and expression patterns were compared with The Cancer Genome Atlas. In vitro studies determined the relationship of IGF and SLC12A7 co-expression in 2 ACC cell lines, SW-13 and NCI-H295R. Immunohistochemistry assessed IGF1 receptor (IGF1R) activation.The IGF1 gene was amplified in 9 of 19 ACC samples, similar to findings in The Cancer Genome Atlas database. The IGF1 overexpression was observed in 5 samples and was associated with SLC12A7 overexpression and non-functional, early-stage tumors (p0.05). In contrast, IGF2 overexpression was associated with larger tumors (p0.05). In vitro IGF treatment of ACC cell lines did not stimulate SLC12A7 expression, and endogenous overexpression and silencing of SLC12A7 significantly altered IGF1 and IGF1R expression without impacting other IGFs. The IGF1R activation was associated with IGF1 overexpression in ACC tumor samples.These findings indicate that IGF1 overexpression, caused in part by gene amplifications, is correlated with SLC12A7 overexpression in non-functional, early-stage ACCs, suggesting a potentially targeted IGF1-SLC12A7 therapeutic opportunity for these tumors.

Fungi are a diverse kingdom of organisms capable of thriving in various niches across the world including those in close association with multicellular eukaryotes. Fungal pathogens that contribute to human disease reside both within the host as commensal organisms of the microbiota and the environment. Their niche of origin dictates how infection initiates but also places specific selective pressures on the fungal pathogen that contributes to its genome organization and genetic repertoire. Recent efforts to catalogue genomic variation among major human fungal pathogens have unveiled evolutionary themes that shape the fungal genome. Mechanisms ranging from large scale changes such as aneuploidy and ploidy cycling as well as more targeted mutations like base substitutions and gene copy number variations contribute to the evolution of these species, which are often under multiple competing selective pressures with their host, environment, and other microbes. Here, we provide an overview of the major selective pressures and mechanisms acting to evolve the genome of clinically important fungal pathogens of humans.

The study sought to identify genetic aberrations driving oral squamous cell carcinoma (OSCC) development among users of shammah, an Arabian preparation of smokeless tobacco. Twenty archival OSCC samples, 15 of which with a history of shammah exposure, were whole-exome sequenced at an average depth of 127×. Somatic mutations were identified using a novel, matched controls-independent filtration algorithm. CODEX and Exomedepth coupled with a novel, Database of Genomic Variant-based filter were employed to call somatic gene-copy number variations. Significantly mutated genes were identified with Oncodrive FM and the Youn and Simon's method. Candidate driver genes were nominated based on Gene Set Enrichment Analysis. The observed mutational spectrum was similar to that reported by the TCGA project. In addition to confirming known genes of OSCC (TP53, CDKNA2, CASP8, PIK3CA, HRAS, FAT1, TP63, CCND1 and FADD) the analysis identified several candidate novel driver events including mutations of NOTCH3, CSMD3, CRB1, CLTCL1, OSMR and TRPM2, amplification of the proto-oncogenes FOSL1, RELA, TRAF6, MDM2, FRS2 and BAG1, and deletion of the recently described tumor suppressor SMARCC1. Analysis also revealed significantly altered pathways not previously implicated in OSCC including Oncostatin-M signalling pathway, AP-1 and C-MYB transcription networks and endocytosis. There was a trend for higher number of mutations, amplifications and driver events in samples with history of shammah exposure particularly those that tested EBV positive, suggesting an interaction between tobacco exposure and EBV. The work provides further evidence for the genetic heterogeneity of oral cancer and suggests shammah-associated OSCC is characterized by extensive amplification of oncogenes.

Background Targeted therapies including monoclonal antibodies and small molecule inhibitors have dramatically changed the treatment of cancer over past 10 years. Their therapeutic advantages are more tumor specific and with less side effects. For precisely tailoring available targeted therapies to each individual or a subset of cancer patients, next-generation sequencing (NGS) has been utilized as a promising diagnosis tool with its advantages of accuracy, sensitivity, and high throughput. Methods We developed and validated a NGS-based cancer genomic diagnosis targeting 115 prognosis and therapeutics relevant genes on multiple specimen including blood, tumor tissue, and body fluid from 10 patients with different cancer types. The sequencing data was then analyzed by the clinical-applicable analytical pipelines developed in house. Results We have assessed analytical sensitivity, specificity, and accuracy of the NGS-based molecular diagnosis. Also, our developed analytical pipelines were capable of detecting base substitutions, indels, and gene copy number variations (CNVs). For instance, several actionable mutations of EGFR, PIK3CA,TP53, and KRAS have been detected for indicating drug susceptibility and resistance in the cases of lung cancer. Conclusion Our study has shown that NGS-based molecular diagnosis is more sensitive and comprehensive to detect genomic alterations in cancer, and supports a direct clinical use for guiding targeted therapy.

Leishmania amazonensis is one of the causative agents of the different forms of cutaneous leishmaniasis present in Latin America. This species has been isolated from humans and animals (canine/feline) in some endemic regions of Colombia. Therefore, L. amazonensis is of great relevance at the clinical and epidemiological levels in medicine and veterinary science. Until now, very few genomes from this species are available. Here, we report the complete genome sequence of a laboratory-adapted L. amazonensis strain isolated from a human patient with clinical manifestation of cutaneous leishmaniasis in Colombia. The genome sequence not only allowed inter and intra-species comparative analyses to be performed with the sequenced genomes of L. amazonensis strains from different geographical regions, but also increased our knowledge about the genomic behavior of this L. amazonensis Colombian strain. This isolate was also characterized in terms of single nucleotide polymorphisms, chromosome and gene copy number variations (CNVs). The results revealed moderate aneuploidy, CNVs in genes involved in the virulence, growth, and survival of the parasite, and in the distributions of the multicopy genes on some chromosomes, as well as a high level of heterozygosity. The data confirmed previous reports that identified unique variations in L. amazonensis, suggesting aneuploidy may not have a high fitness cost and may allow the rapid generation of diversity in Leishmania parasites growing normally. © 2019 Elsevier B.V.

Cytomegalovirus (CMV) is a member of the herpesviridae family and remains latent in the host cells after primary infection. The virus reactivates in the immunosuppressed host and is a leading cause of morbidity following allogeneic hematopoietic stem cell transplant (allo-HSCT). Natural killer (NK) cells provide the first-line defence against viruses and a subset of NK cells expressing NKG2C have been shown to play a role in the immune surveillance of human CMV. Deletion of the NKG2C gene has been reported as a risk factor for viral infections including CMV and HIV, but current data on the influence of NKG2C gene-copy number variations in the donor graft on the risk of CMV reactivation after allo-HSCT is limited. Following cord blood transplantation (CBT), where the T-cell compartment is functionally naïve, the NKG2C genotype may have an even more pronounced impact on the risk of CMV reactivation. Therefore, we studied NKG2C copy number in the donor graft and the risk of CMV reactivation after double umbilical cord blood transplantation (DUCBT) in 100 CMV seropositive DUCBT recipients and their corresponding cord blood grafts (n=200). The gene encoding NKG2C is present at different copy numbers in the genomes of different individuals, with possible genotypes including 2 copies (wt/wt), 1 copy due to heterozygous deletion (wt/del) or 0 copies due to homozygous deletion (del/del). Among the donor CB units, approximately 2/3 had both copies of the gene (wt/wt), 1/3 had only one copy (wt/del) and only a minority of units had 0 copies with both alleles deleted (del/del). In the setting of DUCBT, the combined graft may contain 0 to 4 functional copies of NKG2C gene. In our cohort, patients whose combined grafts contained only 1 or 2 NKG2C gene copies had a significantly higher probability of CMV reactivation than patients whose combined grafts had 3 or 4 NKG2C copies. The 6-month cumulative incidence of CMV reactivation for the 4 groups was 100%, 92.9%, 60.5% and 55.9% respectively (p=0.005). No patient received a graft with zero gene copies. For the rest of the analysis we divided the patients into two groups, namely the 84 patients who received a combined graft with 3 or 4 NKG2C copies and the 16 patients who received two units with 1 or 2 NKG2C copies. The 6-month cumulative incidence of CMV reactivation for the two groups was 58.4% and 93.7% respectively (p=0.0003). In a multivariate analysis, receiving a combined graft with low NKG2C copy number (1 or 2 copies) (HR=2.72, CI=1.59-4.64; p Disclosures Oran: ASTEX: Research Funding; AROG pharmaceuticals: Research Funding; Celgene: Consultancy, Research Funding. Champlin:Sanofi: Research Funding; Otsuka: Research Funding. Shpall:Affirmed GmbH: Research Funding.

EphA3 is a member of Eph receptors, which is involved in tumorigenesis. The expression and clinical significance of EphA3 in colorectal cancer (CRC) have not been fully investigated. Four colon cancer cell lines and a set of CRC tissues were examined for EphA3 expression. The methylation status of a CpG island within the EphA3 promoter, the presence of four somatic EPHA3 mutations, and EPHA3 gene copy number variations were also analyzed in colon cancer cell lines. EphA3 expression was lost in all colon cancer cell lines examined. EphA3 expression was lower in tumor tissues when compared with normal intestinal tissues (P

Personalized drug therapy aims to optimize the efficacy of pharmacological treatments by considering genetic, pathophysiological, dietary, and environmental factors as well as comedications and compliance. A multitude of associations between the specific genetic constitution of the patient and drug pharmacokinetics and pharmacodynamics has been identified in the last decades that encompass mainly common single nucleotide variants (SNVs) and gene copy number variations (CNVs) of importance for the function of genes encoding drug-metabolizing enzymes and transporters involved in drug absorption, distribution, metabolism, and excretion (ADME). In addition, genetic variation in factors encoding the major histocompatibility complex have been helpful to predict immune-mediated drug toxicity. This knowledge has been translated into clinical applications through the implementation of pharmacogenomic biomarkers. Over 230 of such markers that can, to a certain extent, predict drug efficacy or the likelihood of adverse drug reactions (ADRs) are recognized by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) and are included in the drug labels of the respective medication. They are of particular value in cancer therapy to provide information of use to avoid ADRs and lack of response.

Background Gene copy number variations (CNVs) have been reported to be frequent in renal cell carcinoma (RCC), with potential prognostic value for some. However, their clinical utility, especially to guide treatment of metastatic disease remains to be established. Our objectives were to assess CNVs on a panel of selected genes and determine their clinical relevance in patients who underwent treatment of metastatic RCC. Patients and Methods The genetic assessment was performed on frozen tissue samples of clear cell metastatic RCC using quantitative multiplex polymerase chain reaction of short fluorescent fragment method to detect CNVs on a panel of 14 genes of interest. The comparison of the electropherogram obtained from both tumor and normal renal adjacent tissue allowed for CNV identification. The clinical, biologic, and survival characteristics were assessed for their associations with the most frequent CNVs. Results Fifty patients with clear cell metastatic RCC were included. The CNV rate was 21.4%. The loss of CDKN2A and PLG was associated with a higher tumor stage ( P PLG and ALDOB was associated with a higher Fuhrman grade ( P ALDOB was also associated with a worse Heng prognostic score (95% vs. 66%; P = .029) and lower 24-month survival rate (18% vs. 58%; P = .012). The loss of both ALDOB and PLG was frequent (32%) and was associated with a higher tumor stage and grade ( P Conclusion As expected, we showed that several CNVs were associated with clinical relevance, especially those located on CDKN2A , PLG , and ALDOB , in a homogeneous cohort of patients with clear cell metastatic RCC.

International audience; While gene copy-number variations play major roles in long-term evolution, their early dynamics remains largely unknown. However, examples of their role in short-term adaptation are accumulating: identical repetitions of a locus (homogeneous duplications) can provide a quantitative advantage, while the association of differing alleles (heterogeneous duplications) allows carrying two functions simultaneously. Such duplications often result from rearrangements of sometimes relatively large chromosome fragments, and even when adaptive, they can be associated with dele-terious side effects that should, however, be reduced by subsequent evolution. Here, we took advantage of the unique model provided by the malaria mosquito Anopheles gambiae s.l. to investigate the early evolution of several duplications, heterogeneous and homogeneous, segregating in natural populations from West Africa. These duplications encompass ~200 kb and 11 genes, including the adaptive insecticide resistance ace-1 locus. Through the survey of several populations from three countries over 3-4 years, we showed that an internal deletion of all coamplified genes except ace-1 is currently spreading in West Africa and introgressing from An. gambiae s.s. to An. coluzzii. Both observations provide evidences of its selection, most likely due to reducing the gene-dosage disturbances caused by the excessive copies of the nona-daptive genes. Our study thus provides a unique example of the early adaptive tra-jectory of duplications and underlines the role of the environmental conditions (insecticide treatment practices and species ecology). It also emphasizes the striking diversity of adaptive responses in these mosquitoes and reveals a worrisome process of resistance/cost trade-off evolution that could impact the control of malaria vectors in Africa.

The importance and impact of gene copy number variations (CNVs) as a source of polymorphism in the human and other genomes is being increasingly recognized. Less information is available about CNVs in forest tree species, mainly due to the relative lack of genomic resources. In this study, several methods—quantitative polymerase chain reaction, comparative high-resolution melting curve analysis (C-HRM), and digital polymerase chain reaction (dPCR)—were used to investigate CNV of the Scots pine thaumatin-like protein gene (PsTLP). The obtained results were supported by transcriptome analysis of a single Pinus sylvestris individual and publically available pine genome sequences. Although estimations of gene copy number (CN) varied, depending on the region of the PsTLP gene investigated and the endogenous control utilized, our results revealed the existence of copy number variations of the PsTLP gene between Scots pine individuals. Of 23 individuals analyzed, two had an increased calculated relative CN regardless of the analyzed gene region and endogenous control used, while several samples had increased copy numbers of regions of the PsTLP gene. C-HRM results were highly correlated with qPCR data (R 2 TLP3′ = 0.88; R 2 TLPc = 0.92), but interpretation of gene CN from C-HRM results proved to be difficult. The results from selected samples analyzed by digital PCR also were highly correlated with qPCR results (R 2 = 0.90).

While gene copy number variations (CNVs) are abundant in the human genome, and often are associated with disease consequences, the mutagenic pathways and environmental exposures that cause these large structural mutations are understudied relative to conventional nucleotide substitutions in DNA. The members of the environmental mutagenesis community are currently seeking to remedy this deficiency, and there is a renewed interest in the development of mutagenicity assays to identify and characterize compounds that may induce de novo CNVs in humans. To achieve this goal, it is critically important to acknowledge that CNVs exist in two very distinct classes: nonrecurrent and recurrent CNVs. The goal of this commentary is to emphasize the deep contrasts that exist between the proposed pathways that lead to these two mutation classes. Nonrecurrent de novo CNVs originate primarily in mitotic cells through replication-dependent DNA repair pathways that involve microhomologies ( 10,000 bp), without an associated paternal age effect. Given the biological differences between the two CNV classes, it is our belief that nonrecurrent and recurrent CN mutagens will probably differ substantially in their modes of action. Therefore, each CNV class may require their own uniquely designed assays, so that we as a field may succeed in uncovering the broadest possible spectrum of environmental CN mutagens.

To the Editor: Serum amyloid A (SAA), including SAA1 and SAA2 , is not only an acute-phase reactant but also an apolipoprotein involved in the pathogenesis of atherosclerosis. A moderate increase in the concentration of SAA is thought to be an indicator of chronic systemic inflammation that plays an important role in the initiation and progression of cardiovascular disease (CVD) (1). However, prospective studies have shown conflicting results regarding moderately increased concentrations of SAA as a risk factor of CVD (2). It is also noteworthy that the reported physiological SAA concentrations varied substantially from 0.1 to 40 mg/L, which reflects the heterogeneity of SAA baseline concentrations (1). This heterogeneity could be a major obstacle for the applicability of SAA as a prognostic factor of CVD in clinical practice. Twin studies suggest that there is a substantial genetic contribution to SAA baseline concentrations, with heritability estimates of 59% (95% CI: 49–67%) (3). Recently, like other types of genetic variation, the impact of submicroscopic gene copy number variations (CNVs) on human diseases has been investigated and found to be associated with susceptibility or resistance to many diseases (4). Because the aim of this study was to identify the association …