Construction and analysis of dual selectable microcell hybrids and their use in the identification of suppressor gene loci.

Cancer can be considered as a class of genetic diseases which arises as a result of mutations in oncogenes and tumour suppressor genes. These mutations alter the function, morphology, and behaviour of the affected cells, ultimately leading to tumour initiation, progression, and metastasis. These phenotypic changes also impact upon the ability of tumour cells to either respond to chemotherapy with cell death, or evade chemotherapeutic induced cell death through drug-resistant pathways. To identify cancer-related genes which contribute to cell survival during chemotherapy, hybrids between normal human fibroblasts and murine melanoma cells (B78) were generated and screened for the ability to survive short term exposure to the chemotherapeutic agent, PALA (N(phosphonoacetyl)- scL-aspartate). Whereas PALA induced growth arrest in normal fibroblasts, B78 responded to PALA with apoptosis (active cell death). It was hypothesized that the introduction of normal human chromosomes from the fibroblasts into B78 would result in an alteration in some of the hybrids' responses to the drug; in this instance, survival via growth arrest as opposed to apoptosis. Those human chromosomes which were associated with improved PALA survival were theorized to express genes responsible for cellular survival during chemotherapeutic treatment. The hypothesis was first tested using whole cell hybrids constructed between human fibroblasts and B78. Whole cell hybrids showed improved survival during PALA exposure, as opposed to B78, and showed morphological features of growth arrest. To identify individual human chromosomes involved in this response, a panel of dual selectable microcell hybrids was constructed and screened. These hybrids contained one or more normal human chromosomes in a B78 background. A drug selectable marker permitted the selective retention, or removal of the tagged human chromosome, as desired. The property of double selection was required to show that phenotypic changes were always due to the presence or removal of the tagged chromosome, rather than due to clonal variation. A pharmacological screen (using PALA) of the microcell hybrid panel resulted in the identification of human chromosome 3, which was consistently associated with improved survival and growth arrest during drug exposure. To attempt to identify genes which were involved in drug survival, a molecular strategy, known as differential display reverse transcriptase PCR (DDRT-PCR) was employed. This strategy was used to identify those cDNAs derived from differentially expressed mRNAs during PALA exposure in hybrids that showed improved survival during PALA exposure, as compared to B78 and the hybrids that died readily in the presence of PALA. Several candidate cDNAs were identified, cloned and sequenced. Characterization of these cDNA's may ultimately lead to the identity of new survival genes and an improved understanding of cancer cell drug responses

Identification of chromosomes implicated in suppression of apoptosis in somatic cell hybrids

In vitro exposure of tumorigenic cell lines to the chemotherapeutic agent PALA (N-(phosphonoacetyl)-L-aspartate) usually results in cell death (shown here to be apoptosis), followed by clonal growth of rare survivors. On the other hand, normal diploid cells respond to PALA by arresting in G1 and G2 of the cell cycle. It was previously suggested that growth control mechanisms might exist to prevent cells from entering S phase under toxic conditions and that genes involved in such mechanisms were mutated or deleted in tumor cells. Interestingly, the tumor suppressor gene p53, a putative G1 control gene, was shown to mediate PALA-induced growth arrest. However, growth arrest occurs in cells that lack wild-type p53, suggesting that other genes are involved as well. To identify these genes, we have generated whole cell hybrids between mouse melanoma and normal human fibroblast cells. At early passage, a whole cell hybrid (BHF12) responds to PALA with growth arrest, while at later passage, the same hybrid undergoes apoptosis. To determine which human chromosomes are required for the PALA-induced growth arrest phenotype, we isolated subclones of the hybrid and tested them for their PALA response. FISH (fluorescence in situ hybridization) and PCR (polymerase chain reaction) amplification have been used to identify the human chromosome content of BHF12 and its subclones. Several human chromosomes, in addition to chromosome 17 (the location of p53), are consistently associated with the growth arrest phenotype. These findings provide evidence that one or more genes in addition to p53 may be involved in the suppression of a drug-induced apoptosis pathway and may lie on one or more of these chromosomes.Key words: apoptosis, growth arrest, N-(phosphonoacetyl)-L-aspartate, somatic cell hybrids. </jats:p

Data sharing to improve concordance in variant interpretation across laboratories: results from the Canadian Open Genetics Repository

BackgroundThis study aimed to identify and resolve discordant variant interpretations across clinical molecular genetic laboratories through the Canadian Open Genetics Repository (COGR), an online collaborative effort for variant sharing and interpretation.MethodsLaboratories uploaded variant data to the Franklin Genoox platform. Reports were issued to each laboratory, summarising variants where conflicting classifications with another laboratory were noted. Laboratories could then reassess variants to resolve discordances. Discordance was calculated using a five-tier model (pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), likely benign (LB), benign (B)), a three-tier model (LP/P are positive, VUS are inconclusive, LB/B are negative) and a two-tier model (LP/P are clinically actionable, VUS/LB/B are not). We compared the COGR classifications to automated classifications generated by Franklin.ResultsTwelve laboratories submitted classifications for 44 510 unique variants. 2419 variants (5.4%) were classified by two or more laboratories. From baseline to after reassessment, the number of discordant variants decreased from 833 (34.4% of variants reported by two or more laboratories) to 723 (29.9%) based on the five-tier model, 403 (16.7%) to 279 (11.5%) based on the three-tier model and 77 (3.2%) to 37 (1.5%) based on the two-tier model. Compared with the COGR classification, the automated Franklin classifications had 94.5% sensitivity and 96.6% specificity for identifying actionable (P or LP) variants.ConclusionsThe COGR provides a standardised mechanism for laboratories to identify discordant variant interpretations and reduce discordance in genetic test result delivery. Such quality assurance programmes are important as genetic testing is implemented more widely in clinical care.</jats:sec

A large data resource of genomic copy number variation across neurodevelopmental disorders

AbstractCopy number variations (CNVs) are implicated across many neurodevelopmental disorders (NDDs) and contribute to their shared genetic etiology. Multiple studies have attempted to identify shared etiology among NDDs, but this is the first genome-wide CNV analysis across autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), schizophrenia (SCZ), and obsessive-compulsive disorder (OCD) at once. Using microarray (Affymetrix CytoScan HD), we genotyped 2,691 subjects diagnosed with an NDD (204 SCZ, 1,838 ASD, 427 ADHD and 222 OCD) and 1,769 family members, mainly parents. We identified rare CNVs, defined as those found in &lt;0.1% of 10,851 population control samples. We found clinically relevant CNVs (broadly defined) in 284 (10.5%) of total subjects, including 22 (10.8%) among subjects with SCZ, 209 (11.4%) with ASD, 40 (9.4%) with ADHD, and 13 (5.6%) with OCD. Among all NDD subjects, we identified 17 (0.63%) with aneuploidies and 115 (4.3%) with known genomic disorder variants. We searched further for genes impacted by different CNVs in multiple disorders. Examples of NDD-associated genes linked across more than one disorder (listed in order of occurrence frequency) are NRXN1, SEH1L, LDLRAD4, GNAL, GNG13, MKRN1, DCTN2, KNDC1, PCMTD2, KIF5A, SYNM, and long non-coding RNAs: AK127244 and PTCHD1-AS. We demonstrated that CNVs impacting the same genes could potentially contribute to the etiology of multiple NDDs. The CNVs identified will serve as a useful resource for both research and diagnostic laboratories for prioritization of variants.</jats:p