Establishment of a luciferase-based method for measuring cancer cell adhesion and proliferation.

BACKGROUND: Methods measuring cell proliferation and adhesion are widely used but each hold limitations. We, therefore, introduce novel methods for measuring cell proliferation and adhesion based on CRISPR-modified cancer cell lines secreting luciferase to the growth media. MATERIALS AND METHODS: Using CRISPR genome editing, we generated stable luciferase-secreting LS174T, HCT 116, Caco-2, and PANC-1 cell lines. The modified cells were seeded, and luciferase activity was measured in the media and compared to Coulter counter cell counts and iCELLigence impedance assay to evaluate the value of the secreted luciferase activities as a measurement for adhesion and proliferation. RESULTS: Our results demonstrate that luciferase secreted into the media can be used quantifying cell proliferation and adhesion. The adhesion luciferase assay and the iCELLigence impedance assay showed similar results with increased significant difference observed in the luciferase assays. The luciferase proliferation assay showed increased growth following increased serum concentrations in all cell lines vs. only two cell lines in the iCELLigence impedance assay. CONCLUSIONS: Our results show that the luciferase adhesion and proliferation assays are reliable methods for measuring adhesion and proliferation. The luciferase assays have advantages over existing assays as they are highly sensitive, easy to perform, non-invasive and suitable as high-throughput measurements.

Investigation of Genetic Alterations in Congenital Heart Diseases in Prenatal Period.

The prenatal diagnosis of congenital heart disease (CHD) is important because of mortality risk. The onset of CHD varies, and depending on the malformation type, the risk of aneuploidy is changed. To identify possible genetic alterations in CHD, G-banding, chromosomal microarray or if needed DNA mutation analysis and direct sequence analysis should be planned. In present study, to identify genetic alterations, cell culture, karyotype analysis, and single nucleotide polymorphism, array analyses were conducted on a total 950 samples. Interventional prenatal genetic examination was performed on 23 (2, 4%, 23/950) fetal CHD cases. Chromosomal abnormalities were detected in 5 out of 23 cases (21, 7%). Detected chromosomal abnormalities were 10q23.2 deletion, trisomy 18, 8p22.3-p23.2 deletion, 8q21.3-q24.3 duplication, 11q24.2q24.5 (9 Mb) deletion, and 8p22p12 (16.8 Mb) deletion. Our study highlights the importance of genetic testing in CHD.

Clinical Features of Aberrations Chromosome 22q: A Pilot Study.

Objective A significant number of genetic variations have been identified in chromosome 22, using molecular genetic techniques. Various genomic disorders on chromosome 22, including cat's eye syndrome caused by extra copies of the proximal region of the 22q chromosome, are now well-defined. Our aim in the study was to show phenotypic variability associated with rearrangements of the 22q chromosomal region. Methods We focused our study on clinical aspects of these disorders, including genetic testing, genotype-phenotype correlation, and potential treatments. A total of 998 patients were referred for genetic analysis (Karyotyping, MLPA, array-CGH) during January 2015 to February 2020 because of intellectual deficiency, behavior issues, and/or multiple congenital abnormalities in several genetics departments. Informed consent was obtained from all the patients and/or their parents. Results 22q11.21 or 22q13.33 microdeletions and 22q11.22-q11.23 microduplication were identified in 31 patients out of referrals. The 22q aberrations were detected in 31/998 patients, giving a prevalence of 3.1%. In this study, 18 patients with 22q11.2 (LCR22A-H) deletion, three patients with 22q13.31 deletion, 9 patients with 22q11.2 duplication and one patient with 22q13.31 duplication were identified. We report on the clinical and molecular characterization of 31 individuals with distal deletions and duplications of chromosome 22q. Conclusions The current study demonstrated in the largest postnatal case series reporting the whole spectrum of atypical phenotypic and genotypic variations at 22q. We believe that when all the phenotypic differences are taken into account, various anomalies including developmental delay and intellectual disability might be considered as an indication to search for aberrations of 22q along with congenital heart diseases.

Investigation the Relationship of Autism Spectrum Disorder and FOXP2, GRIN2B, KATNAL2, GABRA4 Genes.

INTRODUCTION: Autism spectrum disorder is a genetically and phenotypically heterogeneous group. Genetic studies carried out to date have suggested that both common and rare genetic variants play a role in the etiology of this disorder. In our study, we aimed to investigate the effect of FOXP2, GRIN2B, KATNAL2 and GABRA4 gene variants in the pathogenesis of autism spectrum disorder. METHOD: In our prospectively planned study, all exons and exon-intron junctions of FOXP2, GRIN2B, KATNAL2 and GABRA4 genes were screened by next generation sequencing analysis in 96 patients who diagnosed with autism spectrum disorder. RESULTS: In our study, the average age was 10.1 and the male/female ratio was 75/21. Pathogenic or likely pathogenic variants were not detected in FOXP2, GRIN2B, KATNAL2 and GABRA4 genes, however, 69 intronic variants of unknown clinical significance were detected in 50 cases (52%). Among those, 26 were in the GABRA4 gene, 22 in the FOXP2 gene, 13 in the KATNAL2 gene, and 8 in the GRIN2B gene. Twenty three of these 69 variants were novel that were not previously reported in the literature. CONCLUSION: In our study, we could not identify a relationship between the autism spectrum disorder and FOXP2, GRIN2B, KATNAL2 and GABRA4 genes. Identifying genetic risk factors that play a role in the etiopathogenesis of autism spectrum disorder will contribute significantly to understanding the molecular mechanisms of the disease and the development of new treatment strategies. In this context, comprehensive molecular genetic studies such as whole exome or whole genome sequencing are required with higher number of cases in different populations.

Chromosomal Microarray Analysis in Turkish Patients with Unexplained Developmental Delay and Intellectual Developmental Disorders.

INTRODUCTION: Aneuploids, copy number variations (CNVs), and single nucleotide variants in specific genes are the main genetic causes of developmental delay (DD) and intellectual disability disorder (IDD). These genetic changes can be detected using chromosome analysis, chromosomal microarray (CMA), and next-generation DNA sequencing techniques. Therefore; In this study, we aimed to investigate the importance of CMA in determining the genomic etiology of unexplained DD and IDD in 123 patients. METHOD: For 123 patients, chromosome analysis, DNA fragment analysis and microarray were performed. Conventional G-band karyotype analysis from peripheral blood was performed as part of the initial screening tests. FMR1 gene CGG repeat number and methylation analysis were carried out to exclude fragile X syndrome. RESULTS: CMA analysis was performed in 123 unexplained IDD/DD patients with normal karyotypes and fragile X screening, which were evaluated by conventional cytogenetics. Forty-four CNVs were detected in 39 (39/123=31.7%) patients. Twelve CNV variant of unknown significance (VUS) (9.75%) patients and 7 CNV benign (5.69%) patients were reported. In 6 patients, one or more pathogenic CNVs were determined. Therefore, the diagnostic efficiency of CMA was found to be 31.7% (39/123). CONCLUSION: Today, genetic analysis is still not part of the routine in the evaluation of IDD patients who present to psychiatry clinics. A genetic diagnosis from CMA can eliminate genetic question marks and thus alter the clinical management of patients. Approximately one-third of the positive CMA findings are clinically intervenable. However, the emergence of CNVs as important risk factors for multiple disorders increases the need for individuals with comorbid neurodevelopmental conditions to be the priority where the CMA test is recommended.

Genetic screening results of individuals with high risk BRCA-related breast/ovarian cancer in Trakya region of Turkey.

PURPOSE: Pathogenic/likely pathogenic (P/LP) germline variations in BRCA1 and BRCA2 genes are responsible for the majority of hereditary breast and ovarian cancers. This study presents the BRCA1/BRCA2 sequencing and deletion duplication analyses results of of 493 participants (485 women, 8 men) selected based on the National Comprehensive Cancer Network (NCCN) guidelines. METHODS: Next generation sequencing (NGS) and multiplex ligation-dependent probe amplification methods (MLPA) were used to define germline BRCA1/BRCA2 positivity. RESULTS: Overall, the P/LP frequency of the participants was 17.8%. Five of the likely pathogenic variants were novel. The 5266dupC pathogenic variation, which is a founder mutation in the Ashkenazi Jewish population, was the most common variation among the patients, with a frequency of 5.47%. The pathogenic/likely pathogenic variation frequency was significantly higher (p=0.01) among clinically diagnosed familial cancer patisents than those participants without personal history of cancer but enrolled for BRCA1 testing due to familial risk. BRCA1/BRCA mutation positivity was significantly higher (p=0.000) among those who had at least one first- or second-degree relative with breast/ovarian cancer from patients who had no family history. BRCA1/BRCA2 mutation positivity was 69.23% between the patients who had personal history of both breast and ovarian cancer. CONCLUSION: Based on our findings, we suggest that sequencing all of the coding regions of the BRCA1/BRCA2 genes using NGS is a feasible approach for individuals who are at risk of developing BRCA-related cancer according to NCCN guidelines. The 5266dupC pathogenic variation, as the most common pathogenic variation in the Trakya region of Turkey, should be included if a targeted mutatin screening is planned.