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The current study examined the association between the carbonic anhydrase VI (CA VI) copy number variations (CNVs) and dental caries experience in adults. In total, 202 of 35–72 years old subjects participating in the Lithuanian National Oral Health Survey (LNOHS) agreed to provide saliva samples; thus, their data were included in the current study. Information about sociodemographic, environmental, and behavioural determinants was acquired via the self-administered World Health Organization (WHO) questionnaire. Fluoride levels in the drinking water were recorded based on information provided by water suppliers. Dental caries experience was recorded by one calibrated examiner using the WHO criteria for recording caries on smooth (including proximal, buccal, and oral) or occlusal surfaces. Caries experience was measured as the total number of decayed (D3), missing (M), filled (F) surfaces. DNA was extracted from saliva samples to examine CA VI CNVs using the QX200 Droplet Digital PCR system. Negative binomial regression and Poisson regression analyses were employed for data analyses. Based on multivariable regression analyses, higher copy number of CA VI were associated with higher caries experience on smooth surfaces (IRR 1.04, 95% CI: 1.005–1.08) and occlusal surfaces (IRR 1.02, 95% CI: 1.003–1.04). Positive associations between higher copy number of CA VI and higher caries experience on smooth and occlusal surfaces were found, suggesting that the CA VI coding gene may be associated with caries development. Future studies are needed to validate our results and to examine the underlying mechanisms of such associations.

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.

Myxoid liposarcoma (MLPS) has different patterns that are often difficult to distinguish from other soft tissue lesions. MLPS is characterized by a reciprocal translocation involving the DNA Damage Inducible Transcript 3 gene (DDIT3) that can be detected using fluorescent in situ hybridization (FISH). Recently, the marker for cancer testis antigen 1b (CTAG1B) was found to be expressed in MLPS. The aim of the present study was to assess the potential use immunohistochemistry (IHC) for CTAG1B expression and DDIT3 rearrangement to diagnose MLPS and distinguish it from similar lesions. Out of 29 cases including MLPS and its mimics, CTAG1B was expressed in 92.86% of cases of MLPS and 20% of its mimics. DDIT3 rearrangement was 100% sensitive and 92.86% specific in distinguishing MLPS from its mimics. The DDIT3 rearrangement was found to be more sensitive but less specific than cytoplasmic expression of CTAG1B marker. DDIT3 polysomy and amplification were detected in some cases. Therefore, both CTAG1B expression and FISH for DDIT3 gene can be used to distinguish MLPS from similar tumors. The use of both immunohistochemistry for CTAG1B in addition to DDIT3 gene rearrangement detection by FISH was more specific than using either of them alone. However, the DDIT3 gene rearrangement alone was the most sensitive test for distinguishing MLPS from its mimics.

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.

Objective: Cardiac septal defects (CSDs), the most common human congenital heart malformations are complex and heterogeneous. Progress in molecular biology has helped to identify many genes responsible for cardiac morphogenesis. However, etiologic factors in familial as well as isolated syndromes are being identified; the root genetic cause still needs to be resolved and its mechanism is yet to be revealed. The objective of this study is to identify DNA copy number variations (CNVs) and their possible association with septal defects. Methods: Multiplex ligation-dependent probe amplification (MLPA) was used to detect DNA copy number in non-syndromic CSDs using the P311-A1 Kit consisting of probes for the key genes, namely, NKX2-5 (NK2 transcription factor related, locus 5), GATA4 (GATA binding protein 4), TBX5 (T-box transcription factor), bone morphogenetic protein 4, and CRELD1 (cysteine rich with EGF-like domains 1). Results: We studied 124 clinically diagnosed CSD subjects, of which 111 (89.5%) had atrial septal defects and 13 (10.5%) had ventricular septal defects. MLPA assay was carried out in all these patients after a thorough clinical and cytogenetic screening. CNVs were identified in 16 (12.9%) cases, of which heterozygous deletions and heterozygous duplications were detected (8 patients each) with apparent phenotypes. Conclusion: MLPA could be a useful assay for the detection of CNVs and to be adopted as the first line of screening in patients with congenital heart diseases.

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

e17531 Background: More than 90% of malignant vulvar tumors and almost 5% of all gynecological cancers are squamous cell carcinoma. 1/3 of patients with vulvar cancer relapse after initial treatment, and the 2-year overall survival is less than 15%. Development of more effective treatments requires understanding of the molecular pathogenesis of vulvar cancer. The aim of this study was the screening of potential prognostic tumor markers in vulvar cancer based on relative copy number analysis (CNV) of MYC, PTEN, CCND1, PIK3CA, TP53, CDKN2A, MDM2, MCL1, AEPX1, NFKBIA, MTAP, BIRC2, SYNE1, KMT2C genes. Methods: The study was performed on 300 patients aged 29-80 years diagnosed with vulvar squamous cell carcinoma with lymph nodes metastases (n = 150) and without metastases (n = 150). The material for the study was DNA from FFPE-blocks of tumor and non-tumor vulvar tissues. The RT-qPCR method was used to evaluate the relative copy number of MYC, PTEN, CCND1, PIK3CA, TP53, CDKN2A, MDM2, MCL1, AEPX1, NFKBIA, MTAP, BIRC2, SYNE1, KMT2C, GAPDH and B2M genes (the last two were reference genes). Differences were assessed using the Mann-Whitney test (Bonferroni correction was used). Results: All genes showed a change in the dose in vulva tumor cells relative to normal ones: amplifications for the MYC, MCL1, AEPX1, NFKBIA, CCND1, PIK3CA, TP53, MDM2, BIRC2, SYNE1, KMT2C genes, respectively, in 46%, 36%, 28%, 40%, 56%, 43%, 62%, 39%, 57%, 35%, 44% of cases, and a decrease in copy number for PTEN, MTAP, CDKN2A genes, respectively, in 34%, 28%, 35% of cases. At the same time, a statistically significant increase in the CNV of the CCND1 gene (p < 0.05) by 1.7 times and a decrease in the PTEN gene (p < 0.05) by 1.9 times were observed in the group of vulvar squamous cell carcinoma patients with lymph nodes metastases. No statistically significant change in the CNV of other genes was found. Conclusions: Thus, the identified aberrant copy numbers of CCND1 and PTEN genes can serve as prognostic markers for vulvar cancer patients.

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.

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 (χTSHR 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.

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.

Recent studies showed that genetic lost or gain in the genome can predispose cells toward malignancy. Hepatocellular carcinoma (HCC) is the most common type of liver cancer which occurs predominantly in patients with underlying chronic liver disease and cirrhosis. Prognosis of HCC is strongly connected with diagnostic delay. To date, no ideal screening modality has been developed for HCC. Recent findings demonstrated that Copy number variation (CNVs) can lead to activation of oncogenes and inactivation of tumor suppressor genes in cancers. In this study, CNV profile of 361 HCC samples was evaluated to reveal the potent - chromosomal regions involved in the disease. The obtained data showed that the chr1q and chr8p were two hotspot regions for gene amplifications and deletions in studied samples respectively. In this research, YY1AP1 (Yin Yang-1 Associated Protein 1) on chr1q22 was the most amplified gene in HCC samples and showed the positive correlation with tumor grade. Deletion of CHMP7 (Charged Multivesicular Body Protein 7) on chr8p21.3 was another frequently observed CNV among HCC patients. Both genes were interacted with variety of well-known oncogenes and tumor suppressor genes including YY1 (Yin Yang 1), CCND1 (Cyclin D1), HDAC1 (Histone deacetylase 1), VHL (von Hippel-Lindau tumor suppressor), MAD2L2 (Mitotic Arrest Deficient 2 Like 2), CEBPA (CCAAT/enhancer-binding protein alpha), CHMP4A, CHMP5, CHMP2A, CHMP3 and ENSG00000249884 (RNF103-CHMP3 gene), all of them are well-known in carcinogenesis. Although this study was based on in silico evaluations, our findings can open a new window for researchers of HCC to focus on such candidate genes during experimental assays.

The extent of structural variants (SVs), in particular copy number variations (CNVs) in plant and animal genomes remains unknown, mainly due to the lack of population-scale truly high-quality genomes. Here, we de novo assembled and annotated 31 gold-standard reference genomes from varieties representing all major cultivated rice subtypes, and accurately detected 107,251 non-redundant SVs affecting 644.91Mbp. A position-resolved pan-genome comprising 66,636 genes enabled our discovery that more than 38% of protein-coding genes in cultivated rice harbor CNVs, a far greater proportion than previous estimate. Illustrating functional consequences of these variations, CNVs of Awn3-1, OsVIL1 and OsMADS18, as well as a 345kb inversion likely contributed to major events in rice evolution, domestication and environmental adaptation. Beyond fully resolving the SVs of cultivated rice and comprehensively cataloguing CNVs among protein-coding genes, our study suggests that SVs-affected genes likely contribute to many mechanisms underlying domestication and phenotypic variation in rice.

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.

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.

Background Human SLE is characterised by fluctuating serum levels of complement proteins. There are frequent copy number variations (CNVs) of complement C4A and C4B genes among different individuals. Previously, we demonstrated that C4A deficiency is a strong genetic risk factor for SLE. Objectives To investigate how CNVs of C4 contribute to the great variability of C4 serum levels and how deficiencies of C4A or C4B modulate the clinical presentations, including organ damage, of SLE. Methods Our study population included 499 patients from Hong Kong, who fulfilled ≥4 of the 2013 ACR/SLICC criteria for SLE. Among them 93% were women, the mean age of SLE onset was 32.8±13.0 years, and SLE duration was 14.4±7.6 years. Gene copy numbers (GCNs) of total C4 (C4T), C4A and C4B were determined by real-time PCRs. Serial serum levels over the past 5 years for C4 and C3 of each patient were retrieved through the laboratory data registry system. Serum C4 and C3 levels are shown as mg/100 ml (unit). Clinical manifestations and organ damage of SLE were correlated with CNVs of C4 genes and serum levels. Continuous data between groups were compared by t-tests and categorical data by χ2 analyses. Logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals for effects of C4 CNVs on cumulative clinical manifestations of SLE and accrued organ damage, adjusted for durations of disease. Results Serum levels for C4 varied from 1–84 units (Median: 17) and for C3 from 8–314 units (Median: 86). There was a very strong correlation between C4 and C3 protein levels (R=0.70, p=5.3×10–75). The GCN of C4T varied between 2 and 9 with a median of 4 copies (54%), followed by 2 and 3 copies (21%). Each additional gene copy correlated to an increase of 4 and 6 units for the mean and maximum serum C4 levels, respectively. A higher GCN of C4T (≥3 vs Conclusions CNVs of C4 genes confer great variability of serum C4 levels among SLE patients. While C4A deficiency contributes to genetic predisposition of SLE, persistently low levels of serum C4 among patients were strongly correlated with low GCN of total C4 and C4B deficiency. Elucidating C4-CNVs may have prognostic significance of SLE as high GCNs of C4B and C4A appeared to correlate with organ damage and cardiovascular disease, respectively. Disclosure of Interest None declared

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.

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 ligation-dependent 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.

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.

To explore the association of TLR7 gene copy number variations (CNVs) with the susceptibility of ankylosing spondylitis (AS).The case control study was performed in 649 Chinese Han patients with AS and 628 healthy controls. The copy numbers of TLR7 gene (2 fragments) were measured by AccuCopyTM methods. Chi-square and logistic regression models were performed to investigate the association of TLR7 gene CNVs with AS. Odds ratio (ORs) and 95% confidence intervals (CIs) was calculated to estimate AS risk and the Bonferroni correction was applied owing to multiple testing.The logistic regression analysis showed that one copy was significantly associated with AS susceptibility after Bonferroni correction (for the TLR7_1 fragment: OR=1.458, 95%CI(1.098,1.936), p=0.009; for the TLR7_2 fragment: OR=1.451, 95%CI (1.093,1.927), p=0.010), and this association still exists after adjustment of age and sex (for the TLR7_1 fragment: adjusted OR=2.066, 95%CI (1.318,3.238), p=0.002; for the TLR7_2 fragment: adjusted OR=2.061, 95%CI (1.315,3.230), p=0.002). However, logistic regression analysis stratified by gender showed a higher OR in males (for the TLR7_1 fragment: OR(95%CI)=7.987(3.756,16.983); for the TLR7_2 fragment: OR(95%CI)=7.947(3.738,16.897)) than in females (for the TLR7_1 fragment: OR(95%CI)=0.204(0.080,0.524); for the TLR7_2 fragment: OR(95%CI)=0.204(0.080,0.524)).We conclude that the lower copy number (=1) of TLR7 gene confers a risk factor for AS susceptibility in males but protective factor in females.

Polyploidy or whole-genome duplication is recurrent in plant evolution, yet only a small fraction of whole-genome duplications has led to successful speciation. A major challenge in the establishment of nascent polyploids is sustained karyotype instability, which compromises fitness. The three putative diploid progenitors of bread wheat, with AA, SS (S ∼ B), and DD genomes occurred sympatrically, and their cross-fertilization in different combinations may have resulted in fertile allotetraploids with various genomic constitutions. However, only SSAA or closely related genome combinations have led to the speciation of tetraploid wheats like Triticum turgidum and Triticum timopheevii. We analyzed early generations of four newly synthesized allotetraploid wheats with genome compositions S(sh)S(sh)A(m)A(m), S(l)S(l)AA, S(b)S(b)DD, and AADD by combined fluorescence and genomic in situ hybridization-based karyotyping. Results of karyotype analyses showed that although S(sh)S(sh)A(m)A(m) and S(l)S(l)AA are characterized by immediate and persistent karyotype stability, massive aneuploidy and extensive chromosome restructuring are associated with S(b)S(b)DD and AADD in which parental subgenomes showed markedly different propensities for chromosome gain/loss and rearrangements. Although compensating aneuploidy and reciprocal translocation between homeologs prevailed, reproductive fitness was substantially compromised due to chromosome instability. Strikingly, localized genomic changes in repetitive DNA and copy-number variations in gene homologs occurred in both chromosome stable lines, S(sh)S(sh)A(m)A(m) and S(l)S(l)AA. Our data demonstrated that immediate and persistent karyotype stability is intrinsic to newly formed allotetraploid wheat with genome combinations analogous to natural tetraploid wheats. This property, coupled with rapid gene copy-number variations, may have laid the foundation of tetraploid wheat establishment.

Map of parent provenances for the four pedigrees. (DOCX 240Â kb)

The design and evaluation of analytical methods that permit quantitative analysis of specific DNA sequences is exponentially increasing. For this purpose, highly sensitive methodologies usually based on labeling protocols with fluorescent dyes or nanoparticles are often explored. Here, the possibility of label-free signal amplification using end-point polymerase chain reaction (PCR) are exploited using on-column agarose gel electrophoresis as separation and inductively coupled plasma-mass spectrometry (ICP-MS) for the detection of phosphorus in amplified DNA sequences. The calibration of the separation system with a DNA ladder permits direct estimation of the size of the amplified gene fragment after PCR. With this knowledge, and considering the compound-independent quantification capabilities exhibited by ICP-MS for phosphorus (it is only dependent on the number of P atoms per molecule), the correlation of the P-peak area of the amplified gene fragment, with respect to the gene copy numbers (in the starting DNA), is then established. Such a relationship would permit the determination of copy number variations (CNVs) in genomic DNA using ICP-MS measurements. The method detection limit, in terms of the required amount of starting DNA, is ∼6 ng (or 1000 cells if 100% extraction efficiency is expected). The suitability of the proposed label-free amplification strategy is applied to CNVs monitoring in cells exposed to a chemical agent capable of deletion induction, such as cisplatin.

Copy number variations (CNVs) have been recently recognized as another important genetic variability complementary to single nucleotide polymorphisms (SNPs). Compelling evidence has indicated that CNVs are responsible for phenotypic traits by changing the copy numbers of functional genes. Myosin heavy chain 3 (MYH3) gene is a critical regulatory factor in skeletal muscle development, and has been detected in the CNVs region by comparative genomic hybridization (CGH) array. This study was conducted to validate and detect the distribution of MYH3 copy numbers (relative to Angus cattle) in four Chinese cattle breeds (NY, QC, LX, and CY), and further to investigate the associations of the copy number changes with its transcriptional expression and cattle growth traits. Substantial genetic differences of MYH3 copy numbers were identified between NY and the other three breeds (P

Background Understanding the genetic basis of adaptive evolution is one of the major goals in evolutionary biology. Recently, it has been revealed that gene copy number variations (GCNVs) constitute significant proportions of genomic diversities within natural populations. However, it has been unclear whether GCNVs are under positive selection and contribute to adaptive evolution. Parallel evolution refers to adaptive evolution of the same trait in related but independent lineages, and three-spined stickleback (Gasterosteus aculeatus) is a well-known model organism. Through identification of genetic variations under parallel selection, i.e., variations shared among related but independent lineages, evidence of positive selection is obtained. In this study, we investigated whole-genome resequencing data from the marine and freshwater groups of three-spined sticklebacks from diverse areas along the Pacific and Atlantic Ocean coastlines, and searched for GCNVs under parallel selection. Results We identified 24 GCNVs that showed significant differences in the numbers of mapped reads between the two groups, and this number was significantly larger than that expected by chance. The derived group, i.e., freshwater group, was typically characterized by larger gene-copy numbers, which implied that gene duplications or multiplications helped with adaptation to the freshwater environment. Some of the identified GCNVs were those of multigenic family genes, which is consistent with the theory that fatal effects due to copy-number changes of multigenic family genes tend to be less than those of single-copy genes. Conclusion The identification of GCNVs that were likely under parallel selection suggests that contribution of GCNVs should be considered in studies on adaptive evolution. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-735) contains supplementary material, which is available to authorized users.

Background: Characterization of genomic alterations that drive patient disease development is critical in cancer management. The genomic alteration and population characteristics between the primary tumors and metastases in patients with non-small cell lung cancer (NSCLC) is still unclear. Here we evaluated the frequency and distribution of all classes of genomic alterations in both primary and metastatic NSCLC by comprehensive genomic profiling approach. Methods: FFPE tumor and matched blood samples of 1135 Chinese NSCLC patients (female:male is 670: 465), including 954 primary tumors and 181 metastases, were collected for next-generation sequencing (NGS) based panel assay. Gene copy number variations (CNV) including gene amplifications and deletions, short variants, and gene fusions were assessed. Results: On average, 0.65 gene copy number variations were detected in the 181 metastatic lesions, which is higher than 0.43 CNVs per sample in the primary lesions (p value is 0.003). No significant difference was found in short variants (2.48 vs 2.31), fusions (0.27 vs 0.20) and germline mutations (0.26 vs 0.31) between the primary and metastatic NSCLC. In the top-ranked genes with copy number alterations, EGFR amplifications were detected in 11% of the primary and 15% in metastases. MET amplifications, CDKN2A/B deletions, and PMS2 deletions were more frequently found in metastatic lesions: MET (7.7% vs 1.7%, p=0.0001), CDKN2A (5.5% vs 1.5%, p=0.002), CDKN2B (5.0% vs 1.9%, p=0.0007), and PMS2 (3.9% vs 1.2%, p=0.016). Conclusions: Copy number variations are common driver mutations in NSCLC. We reported here that the prevalence of copy number variations between primary and metastatic lung cancers was different, including driver genes MET, CDKN2A/B, and PMS2. No difference was observed in terms of frequency on short variants or gene fusions in NSCLC. Citation Format: Yunfei Shi, Naiquan Mao, Xiao Ding, Jianji Guo, Gang Guo, Zheng Chen, Weiguang Gu, Lei Dai, Shiwang Wen, Hui Jia, Ziqiang Tian, Junping Shi, Taiyan Guo, Jicheng Yao, Fei Pang, Gungwei Chirn, Ming Yao. Comparison of gene copy number variations (CNV) in primary and metastatic lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3391.

The goal of this study was CGH array profiling of breast cancer from Malian women in order to define differences with those from USA. CGH array was performed in 28 samples, 17 with a triple negative phenotype. The profiles were compared to those of 106 tumors from USA. 6 chromosomal regions (6p21, 9q34, 11q13, 12q24, 17q25 and 22q12.1-22q13.1) were identified with a significant higher rate of copy number alterations. These regions contain several genes of interest including BCR. FISH and IHC confirmed that BCR was amplified and overexpressed particularly in triple negative tumors. Finally, 5 regions presented a high level of amplification in two or more samples, including 2 regions located between 9p22.3-9p23 and 9p23-9p24.1. This study confirms that breast cancers from African women present biological differences with those from USA. Larger studies are needed to go further in the identification of therapeutic targets that would be specific to African women.

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).The identification of novel PSG CNV-disease relationships will provide not only a better understanding of the pathology of pre-eclampsia but also a novel opportunity to identify patients with a high risk of developing early-onset pre-eclampsia, which has a five- to tenfold higher risk of life-threatening maternal complications and fetal demise as compared to late-onset pre-eclampsia patients.

Background: Metastatic hepatocellular carcinoma (HCC) is a highly aggressive tumor with limited treatment options. While sorafenib has recently been shown to provide a survival advantage in patients with advanced HCC, the overall outcomes such as time to progression (TTP) and overall survival (OS) ought to be further improved. To that end, several targeted agents aimed at amplified oncogenes such as HER2 and FGFR2 have recently been developed. In this study, we aimed to identify genetic markers in the form of copy number variations (CNVs) that influence clinical outcomes post-sorafenib treatment in advanced HCC patients. Methods: We surveyed 38 metastatic HCC patients who were treated with sorafenib for the presence of CNVs using the NanoString nCounter assay. Results: The median TTP and OS for all patients were 2.7 months (95% confidence interval [CI]: 2.0-3.3 months) and 13.4 months (95% CI: 8.4-18.4 months), respectively. Several genes previously implicated in liver cancer were amplified, including CCND1 (n = 4), CDKN1A (n = 2), KRAS (n = 2), MDM2 (n = 1), and ERBB2 (n = 1). However, we found no correlations between CNVs and survival in our sorafenib-treated patients. Conclusions: The clinical features and biomarkers that account for sensitivity to sorafenib in HCC are complicated and remain unclear. Further investigation to identify predictive biomarkers and therapeutic strategies, including combining sorafenib with other target agents, are warranted.

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).

The rationale of this work is based on recent evidences suggesting that: 1) both qualitative and quantitative β-lactoglobulin (β-LG) polymorphism may be found in bovine milk; 2) quantitative polymorphisms are often the result of expression gradients in multiple copies of a gene; 3) the β-LG gene is duplicated in the dog and bovine genome; 4) mammary genes are highly conserved across Mammalia. Thus, an investigation was conducted on ovine β-LG polymorphism checking phenotypic evidence for copy-number variants of β-LG in sheep. To the purpose, 206 milk samples were collected, during a small-scale survey within sheep farms breeding Southern Italian breeds. PAGIF screening of the samples revealed that approximately 50% individuals exhibited β-LG polymorphism and 4 different quantitative patterns, which were characterized in detail by a proteomic approach relying on combined chromatographic and mass spectrometric techniques. The expected figures based on the expression gradient models were compared with well-established α-globin gene arrangements in sheep. The different phenotypes suggest the presence of both duplicate and triplicate BLG haplotypes. The occurrence of a triplicate haplotype was supported by population data. The current study supports the helpfulness of up-to-date proteomics for inferring copy number polymorphisms through the characterization of the phenotypic expression.

Copy number variations (CNVs) are one of the primary sources of variation found in the human genome. They involve both the deletion and multi plication of DNA segments [1]. In the human genome, there are thousands of copy number variable regions that range from 100 bp to several Mb pairs [2]. The human genome project revealed that many regions of the genome have CNVs and that some are more common than others [1,3]. According to the Database of Genomic Variants [3], there are 66,741 catalogued CNVs from a total of 15,963 CNV loci (accessed in December, 2010). CNVs have been increasingly implicated in a number of diseases including cancer, developmental diseases, mental illness, autoimmune diseases and infectious diseases [4,5]. The importance of CNVs in genes controlling drug metabolism has been appreciated for many years. A partial list of those affected include UDP-glucuronosyltransferase 2B17 and 2B28 genes (UGT2B17 and UGT2B28) [6], sulfotransferase 1A1 (SULTA1) [7], glutathione S-transferase M1 and T1 genes (GSTT1 and GSTM1) [8], CYP2A6 [9] and CYP2D6 [10]. In fact, UGT2B28, UGT2B17, CYP2A6, GSTM1 and GSTT1 are among the most commonly deleted genes in the human genome [6].

Human complement C4 is complex with multiple layers of diversity. This study aims to elucidate the copy-number variations (CNVs) of C4A and C4B in disease risk of SLE, and compare the basis of race-specific C4A-deficiency in East-Asians (EA) and Europeans. Our study-populations included (a) 999 EA-SLE patients and 1,347 healthy subjects; and (b) 232 European SLE patients and 500 healthy subjects. Variations in gene copy-numbers (GCNs) for total C4, C4A, C4B, long and short genes were determined and validated by independent genotyping technologies. Genomic regions with C4B96 were investigated to determine the basis of the most basic C4B protein that is concurrent with C4A-deficiency. In EA, strong protective effects for high GCNs of total C4 and C4A against SLE were notable; low and medium GCNs of total C4 and C4A, and the absence of short genes were risk factors for SLE. Homozygous C4A-deficiency was infrequent but had an odds-ratio (OR) of 12.4 (p=0.0015) in SLE. Low serum complement levels were strongly associated with low GCNs of total C4 (OR=3.27, p=7.0×10−7) and C4B (OR=2.55, p=2.5×10−5). Patients with low complement had high frequencies of anti-dsDNA (OR=4.96, p=9.7×10−17), hemolytic anemia (OR=3.89, p=3.6×10−10) and renal disease (OR=2.18, p=8.5×10−6). The monomodular-short haplotype with C4A-deficiency and in linkage-disequilibrium with HLA-DRB1*0301 prevalent in Europeans was scarce in EA. Instead, most EA-subjects with C4A-deficiency shared a recombinant haplotype with bimodular-LS encoding C4B1 and C4B96, which was linked to HLA-DRB1*1501. DNA-sequencing revealed the E920K polymorphism for C4B96. In conclusion, C4 CNVs and C4A-deficiency are important in the risk and manifestations of East-Asian and European SLE.

Background Many patients with enlarged vestibular aqueduct (EVA) have either only one allelic mutant of the SLC26A4 gene or lack any detectable mutation. In this study, multiplex ligation-dependent probe amplification (MLPA) was used to screen for copy number variations (CNVs) of SLC26A4 and to reveal the pathogenic mechanisms of non-syndromic EVA (NSEVA). Methods Between January 2003 and March 2010, 923 Chinese patients (481 males, 442 females) with NSEVA were recruited. Among these, 68 patients (7.4%) were found to carry only one mutant allele of SLC26A4 and 39 patients (4.2%) lacked any detectable mutation in SLC26A4; these 107 patients without double mutant alleles were assigned to the patient group. Possible copy number variations in SLC26A4 were detected by SALSA MLPA. Results Using GeneMapper, no significant difference was observed between the groups, as compared with the standard probe provided in the assay. The results of the capillary electrophoresis showed no significant difference between the patients and controls. Conclusion Our results suggest that CNVs and the exon deletion in SLC26A4 are not important factors in NSEVA. However, it would be premature to conclude that CNVs have no role in EVA. Genome-wide studies to explore CNVs within non-coding regions of the SLC26A4 gene and neighboring regions are warranted, to elucidate their roles in NSEVA etiology.

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.

GATA4mutations are found in patients with different isolated congenital heart defects (CHDs), mostly cardiac septal defects and tetralogy of Fallot. In addition,GATA4is supposed to be the responsible gene for the CHDs in the chromosomal 8p23 deletion syndrome, which is recognized as a malformation syndrome with clinical symptoms of facial anomalies, microcephaly, mental retardation, and congenital heart defects. Thus far, no study has been carried out to investigate the role ofGATA4copy number variations (CNVs) in non-syndromic CHDs. To explore the possible occurrence ofGATA4gene CNVs in isolated CHDs, we analyzed by multiplex ligation-dependent probe amplification (MLPA) a cohort of 161 non-syndromic patients with cardiac anomalies previously associated withGATA4gene mutations. The patients were mutation-negative forGATA4,NKX2.5, andFOG2genes after screening with denaturing high performance liquid chromatography. MLPA analysis revealed that normalized MLPA signals were all found within the normal range values for all exons in all patients, excluding a major contribution ofGATA4gene CNVs in CHD pathogenesis.