Homozygous Individuals Research Articles

FBXW7 polymorphism asserts susceptibility to colorectal cancer

FBXW7, belonging to the F-Box protein family, is considered a candidate cancer susceptibility gene. Our findings indicate that single nucleotide polymorphisms (SNPs) in the FBXW7 gene are linked to cancer risk, strengthening FBXW7′s role in the pathogenesis of colorectal cancer. Our case-control study comprised of 450 patients diagnosed with colorectal cancer (CRC) and an equal number of 450 healthy subjects. FBXW7 SNPs rs2255137C>T and rs6842544C>T were genotyped using PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) and Single-Stranded Conformation Polymorphism (SSCP) techniques and further cross-checked by direct sequencing. Linkage disequilibrium and haplotype analyses of these SNPs were also assessed. The in-silico approach was used to reveal the functional analysis between the nonsynonymous variation (rs6842544) and CRC followed by its validation at the protein level by western blotting and reverse transcription-PCR. A significant association of colorectal cancer was detected with rs6842544 SNP. However, there was no association between FBXW7 rs2255137 polymorphism and CRC. The homozygous individuals carrying the C variant in FBXW7 rs6842544 showed a slightly higher risk for colorectal cancer (OR = 1.590, 95%CI = 0.39 ∼ 2.89, p = 0.011). The haplotype CC identified in this study seemed to be associated with good prognosis (OR = 1.22, 95% CI = 1.00 ∼ 1.47, p = 0.0013) whereas the TT haplotype was found to reduce the CRC risk (OR = 0.642, 95%CI = 0.48 ∼ 0.84, p = 0.039). In-silico prediction proposed that the variant R133G is responsible for the lower expression of FBXW7. Additionally, the expression profiling of FBXW7 nonsynonymous SNP was significantly lower in primary CRC tissues than in the paired non-cancerous tissues at protein and mRNA levels. The study indicates that the FBXW7 rs6842544 is associated with the risk of development of CRC and could serve as a molecular biological marker to screen high-risk groups for CRC.

Association between ApoE ε4 genotype and attentional function in non-demented, middle-aged, and older adults from rural India.

Several genetic factors have been associated with cognitive decline in aging. Apolipoprotein E (ApoE) ε4 has been widely studied in the risk for pathological cognitive decline, including dementia. However, the association between ApoE ε4 and cognitive functioning in the healthy aging Indian population has been understudied, and the results are ambiguous. This study aims to examine the role of the ApoE genotype with attentional function in aging adults (≥45 years) in a rural Indian population. Cross-sectional (baseline) data (n = 2100) was utilized from an ongoing longitudinal cohort study on aging (Srinivaspura Aging, Neurosenescence, and Cognition study). Participants hailed from villages of Srinivaspura in Karnataka, southern India. Participants were categorized based on ApoE-ε4 status into three categories: No ε4, heterozygous ε4, and homozygous ε4. Attentional function was assessed using the auditory and visual attention subtests from a computerized neurocognitive test battery. Linear regression was performed adjusting for age, gender, and education. In model 1 (unadjusted), we did not find an association between ApoE and attention function. In the partially adjusted model 2 (adjusting for age), ApoE ε4 with age was significantly associated with the attention function. Further, with increasing age, there was a decline in attention among homozygous ε4 individuals. Model 3 (model 2 + gender) found that ApoE ε4, age, and gender explained a significant variance in attention function. In addition, with increasing age, males had poor attention in the homozygous as compared to heterozygous group. Model 4 (model 3+ education) explained a significant variance in attention and also revealed that with increasing age, attention declined in the illiterate and low literacy groups in both homozygous and heterozygous groups among both genders. Although ApoE ε4 alone was not associated, it interacted with age, gender, and education to affect attention function in this rural Indian population. Longitudinal cognitive monitoring will yield insights into understanding whether the ApoE ε4 genotype influences the rate of cognitive decline in this rural, aging population.

Pancreatitis as a Main Consequence of APOC2-Related Hypertriglyceridemia: The Role of Nonsense and Frameshift Variants.

APOC2-related hypertriglyceridemia occurs due to biallelic variants of this gene. Here, genotype-phenotype architecture of all pathogenic APOC2 variants is investigated among heterozygous and homozygous individuals. Clinical heterogeneity of various types of the variants is also described, and pancreatitis in more than half of homozygotes carrying chain-termination variants is highlighted as well. For this study, patients were selected who had a plasma triglyceride level above 250 mg/dL. The coding and intronic regions of the APOC2 gene were amplified using the Sanger sequencing to investigate the presence of variants. The genotypes, lipid profiles, and detailed clinical features were documented for all APOC2-related patients and heterozygous individuals. Pathogenicity of the variants was predicted and categorized using available bioinformatics tools such as MutationTaster and PolyPhen-2 and ACMG criteria. MetaDome and Phyre2 were applied for structural and functional in silico analyses. 40% (12 out of 30) of APOC2 variants were chain-termination (nonsense and frameshift) variants. These types of variants were determined in 60.53% of patients. 55% of these patients showed pancreatitis followed by lipemia retinalis (29%), abdominal pain (24%), hepatosplenomegaly (24%), and xanthomas (18%). The mean age of onset was about 22 years old. In at least 50% of 38 homozygous individuals, the TG level was more than 2000 mg/dL. More than 25% of heterozygous individuals showed at least one symptom. Pancreatitis and a severe form of HTG were found in 5 and 2% of heterozygous individuals, respectively. The main clinical features of APOC2-related hypertriglyceridemia include pancreatitis, lipemia retinalis, abdominal pain, hepatosplenomegaly, and xanthomas. Nonsense and frameshift homozygous variants usually lead to a severe form of hypertriglyceridemia. Pancreatitis is one of the main consequences of these types of mutations; thus, it is important to consider this point when evaluating asymptomatic individuals. Heterozygous individuals may become symptomatic due to the role of unknown modifying agent including environmental genetic factors.

Gene-Environment Interaction of Interleukin 10 Gene Polymorphism, rs1800896, with Lifestyle on Cardiovascular Risk in Type 2 Diabetes

Abstract Background: Gene–environment interactions play a major role in the phenotypic expression of complex disease traits such as those for cardiovascular diseases. Aims: This study aimed to determine the gene–environment interactions that underpin the relationship between interleukin-10 (IL-10) single-nucleotide polymorphism (1082 G/A [rs1800896]) with lifestyle on cardiovascular disease risk in adult Nigerians with type 2 diabetes mellitus (DM). Setting and Design: This case–control study involved patients with type 2 DM with high cardiovascular risk, determined by the Framingham’s classification, and age, sex, and diabetes-duration matched subjects with low and intermediate cardiovascular risks. Subjects and Methods: The genotypes were detected by polymerase chain reaction (PCR) followed by allelic discrimination using the Applied Biosystems 7900HT Fast Real-Time PCR System. One-way analysis of variance and logistic regression were employed for analysis. Odds ratios (ORs) with 95% confidence intervals (CI) were calculated to estimate the risk caused by the polymorphism. P <0.05 was considered significant. Results: The odds for cardiovascular risk decreased progressively in individuals with the GG, GA, and AA genotypes (OR = 0.80, 95% CI = 0.49–1.28, P = 0.345 and OR = 0.46, 95% CI = 0.24–0.88, P = 0.018 for GA and AA genotypes, respectively. A significantly higher proportion of homozygous (AA) individuals were in the low cardiovascular risk group (54.2%, P = 0.018). Compared with the whole study population, individuals with the AA genotype had consistently lower odds for cardiovascular risk in subpopulations like alcohol users (OR = 0.25 [0.11–0.55], P = 0.001), but the odds were higher among smokers (OR = 1.80 [1.14–2.90], P = 0.017) and those with sedentary lifestyles (OR = 2.46, 95% CI = 1.14–5.33, P = 0.024). Conclusions: The homozygous mutant genotype AA of the IL-10 gene 1082 G/A had a protective effect on cardiovascular risk in type 2 DM. However, this protection was absent in those leading a sedentary lifestyle.

High Population Frequency of GNRHR p.Q106R in Malta: An Evaluation of Fertility and Hormone Profiles in Heterozygotes.

The gonadotropin-releasing hormone receptor variant GNRHR p.Q106R (rs104893836) in homozygosity, compound heterozygosity, or single heterozygosity is often reported as the causative variant in idiopathic hypogonadotropic hypogonadism (IHH) patients with GnRH deficiency. Genotyping of a Maltese newborn cord-blood collection yielded a minor allele frequency (MAF) 10 times higher (MAF = 0.029; n = 493) than that of the global population (MAF = 0.003). To determine whether GNRHR p.Q106R in heterozygosity influences profiles of endogenous hormones belonging to the hypothalamic-pituitary axis and the onset of puberty and fertility in adult men (n = 739) and women (n = 239). Analysis of questionnaire data relating to puberty and fertility, genotyping of the GNRHR p.Q106R variant, and hormone profiling of a highly phenotyped Maltese adult cohort from the Maltese Acute Myocardial Infarction Study. Out of 978 adults, 43 GNRHR p.Q106R heterozygotes (26 men and 17 women) were identified. Hormone levels and fertility for all heterozygotes are within normal parameters except for TSH, which was lower in men 50 years or older. Hormone data and baseline fertility characteristics of GNRHR p.Q106R heterozygotes are comparable to those of homozygous wild-type individuals who have no reproductive problems. The heterozygous genotype alone does not impair the levels of investigated gonadotropins and sex steroid hormones or affect fertility. GNRHR p.Q106R heterozygotes who exhibit IHH characteristics must have at least another variant, probably in a different IHH gene, that drives pathogenicity. We also conclude that GNRHR p.Q106R is likely a founder variant due to its overrepresentation and prevalence in the island population of Malta.

Spleen function is reduced in individuals with NR5A1 variants with or without a difference of sex development: a cross-sectional study.

NR5A1 is a key regulator of sex differentiation and has been implicated in spleen development through transcription activation of TLX1. Concerns exist about hypo- or asplenism in individuals who have a difference of sex development (DSD) due to an NR5A1 disease-causing variant. We aimed to assess spleen anatomy and function in a clinical cohort of such individuals and in their asymptomatic family member carriers. Cross-sectional assessment in 22 patients with a DSD or primary ovarian insufficiency and 5 asymptomatic carriers from 18 families, harboring 14 different NR5A1 variants. Spleen anatomy was assessed by ultrasound, spleen function by peripheral blood cell count, white blood cell differentiation, percentage of nonswitched memory B cells, specific pneumococcal antibody response, % pitted red blood cells, and Howell-Jolly bodies. Patients and asymptomatic heterozygous individuals had significantly decreased nonswitched memory B cells compared to healthy controls, but higher than asplenic patients. Thrombocytosis and spleen hypoplasia were present in 50% of heterozygous individuals. Four out of 5 individuals homozygous for the previously described p.(Arg103Gln) variant had asplenia. Individuals harboring a heterozygous NR5A1 variant that may cause DSD have a considerable risk for functional hyposplenism, irrespective of their gonadal phenotype. Splenic function should be assessed in these individuals, and if affected or unknown, prophylaxis is recommended to prevent invasive encapsulated bacterial infections. The splenic phenotype associated with NR5A1 variants is more severe in homozygous individuals and is, at least for the p.(Arg103Gln) variant, associated with asplenism.

Genome-Wide Association Analysis Identifies the PMEL Gene Affecting Coat Color and Birth Weight in Simmental × Holstein.

Coat color and birth weight, as easily selected traits in cattle, play important roles in cattle breeding. Therefore, we carried out a genome-wide association study on birth weight and coat color to identify loci or potential linkage regions in 233 Simmental × Holstein crossbred beef cattle. The results revealed that nine SNPs were significantly associated with coat color (rs137169378, rs110022687, rs136002689, Hypotrichosis_PMel17, PMEL_1, rs134930689, rs383170073, rs109924971, and rs109146332), and these were in RNF41, ZC3H10, ERBB3, PMEL, and OR10A7 on BTA5. Interestingly, rs137169378, rs110022687, rs136002689, Hypotrichosis_PMel17, and PMEL_1 showed strong linkage disequilibrium (r2 > 0.8) and were significantly associated with coat color. Notably, Hypotrichosis_PMel17 and PMEL_1 were located in the gene PMEL (p = 2.22 × 10-18). Among the five significant SNPs associated with coat color, the birth weight of heterozygous individuals (AB) was greater than that of homozygous individuals (AA). Notably, the birth weight of heterozygous individuals with Hypotrichosis_PMel17 and PMEL_1 genotypes was significantly greater than that of homozygous individuals (0.01 < p < 0.05). Interestingly, the two loci were homozygous in black/white individuals and heterozygous in gray/white individuals, and the birth weight of heterozygous brown/white individuals (43.82 ± 5.25 kg) was greater than that of homozygous individuals (42.58 ± 3.09 kg). The birth weight of calves with the parental color (41.95 ± 3.53 kg) was significantly lower than that of calves with a non-parental color (43.54 ± 4.78 kg) (p < 0.05), and the birth weight of gray/white individuals (49.40 ± 7.11 kg) was the highest. Overall, PMEL appears to be a candidate gene affecting coat color in cattle, and coat color may have a selective effect on birth weight. This study provides a foundation for the breeding of beef cattle through GWAS for coat color and birth weight.

The contribution of a novel PHEX gene mutation to X-linked hypophosphatemic rickets: a case report and an analysis of the gene mutation dosage effect in a rat model.

A Chinese family was identified to have two patients with rickets, an adult female and a male child (proband), both exhibiting signs related to X-linked hypophosphatemic rickets (XLH). Gene sequencing analysis revealed a deletion of adenine at position 1985 (c.1985delA) in the PHEX-encoding gene. To investigate the relationship between this mutation and the pathogenicity of XLH, as well as analyze the effects of different dosages of PHEX gene mutations on clinical phenotypes, we developed a rat model carrying the PHEX deletion mutation. The CRISPR/Cas9 gene editing technology was employed to construct the rat model with the PHEX gene mutation (c.1985delA). Through reproductive procedures, five genotypes of rats were obtained: female wild type (X/X), female heterozygous (-/X), female homozygous wild type (-/-), male wild type (X/Y), and male hemizygous (-/Y). The rats with different genotypes underwent analysis of growth, serum biochemical parameters, and bone microstructure. The results demonstrated the successful generation of a stable rat model inheriting the PHEX gene mutation. Compared to the wild-type rats, the mutant rats displayed delayed growth, shorter femurs, and significantly reduced bone mass. Among the female rats, the homozygous individuals exhibited the smallest body size, decreased bone mass, shortest femur length, and severe deformities. Moreover, the mutant rats showed significantly lower blood phosphorus concentration, elevated levels of FGF23 and alkaline phosphatase, and increased expression of phosphorus regulators. In conclusion, the XLH rat model with the PHEX gene mutation dosage demonstrated its impact on growth and development, serum biochemical parameters, and femoral morphology.

Quantification of encephalic transcripts of the APOE allele specific gene of European and African ancestry

AbstractBackgroundThe APOE gene is identified as a major risk factor for late onset Alzheimer’s disease and has three alleles, ε2, ε3 and ε4, related to two non‐synonymous substitutions. The presence of the ε4 allele confers an increased risk for the disease in a dose dependent manner (odds ratio 12.9 for homozygous individuals and between 3.2 and 4.2 for heterozygous individuals). The most prevalent form of Alzheimer’s disease has a multifactorial etiology, where several genetic and environmental factors influence its pathology. Factors such as age and sex are relevant in determining the risk for APOE, recent studies point out that the ancestry around the APOE gene is also relevant to the risk for the disease. Individuals of African‐American ancestry have a reduced risk of developing the disease compared to European and Asian whites. These regions may act in a cis‐regulatory manner in modulating gene expression. The Brazilian population results from miscegenation between indigenous, African and European populations, offering an opportunity to study the effects of different ancestries on the risk of Alzheimer’s disease, fill the lack of genetic information about admixed groups and characterize the differential patterns of APOE’s expression in Brazilian population.MethodThis project aims to quantitatively characterize the role of different local ancestry in the different APOE genotypes using 36 post‐mortem brain tissue, with distinct genotypes and local ancestries combinations (33,34,44, European‐European, African‐African, European‐African, African‐European) and associating them with the expression of this gene through molecular marker genotyping and RT‐qPCR techniques. It is expected that the mRNA expression is differentially increased when European local ancestry is shown to be present, compared to non‐European ancestry and may explain, in part, the difference in risks observed in populations of different ancestries.Resultongoing researchConclusionongoing research

NTHL1 is a recessive cancer susceptibility gene

In search of novel breast cancer (BC) risk variants, we performed a whole-exome sequencing and variant analysis of 69 Finnish BC patients as well as analysed loss-of-function variants identified in DNA repair genes in the Finns from the Genome Aggregation Database. Additionally, we carried out a validation study of SERPINA3 c.918-1G>C, recently suggested for BC predisposition. We estimated the frequencies of 41 rare candidate variants in 38 genes by genotyping them in 2482–4101 BC patients and in 1273–3985 controls. We further evaluated all coding variants in the candidate genes in a dataset of 18,786 BC patients and 182,927 controls from FinnGen. None of the variants associated significantly with cancer risk in the primary BC series; however, in the FinnGen data, NTHL1 c.244C>T p.(Gln82Ter) associated with BC with a high risk for homozygous (OR = 44.7 [95% CI 6.90–290], P = 6.7 × 10–5) and a low risk for heterozygous women (OR = 1.39 [1.18–1.64], P = 7.8 × 10–5). Furthermore, the results suggested a high risk of colorectal, urinary tract, and basal-cell skin cancer for homozygous individuals, supporting NTHL1 as a recessive multi-tumour susceptibility gene. No significant association with BC risk was detected for SERPINA3 or any other evaluated gene.

Phenotypes Associated with HFE p.C282Y Homozygosity, the Main Hereditary Hemochromatosis Genotype, in Four Large Genetic Cohorts

Introduction Hereditary hemochromatosis (HH) is the most common known recessive hereditary disease in people of northern European descent. HH is most commonly caused by the HFE-C282Y homozygosity. Disease penetrance is highly variable (1-20%). We have examined the association between C282Y homozygosity and multiple phenotypes, both case-control and quantitative, across 4 international cohorts (deCODE genetics, UK Biobank, Denmark (Copenhagen Hospital Biobank / Danish Blood Donor Study) and Intermountain Healthcare) in an effort to shed a new light on the pathophysiology of this common hereditary disease. Methods We preformed a phenome wide association study meta-analyses for C282Y homozygosity across the 4 beforementioned cohorts, comprising around 970.000 individuals, of those 5928 were homozygous for the C282Y mutation (746 in deCODE/Iceland MAF 6.71%, 2865 in UK Biobank MAF 7.94%, 1703 in CHB MAF 5.6% and 614 in Intermountain Healthcare MAF 6.19%). The analyses comprised both case-control phenotypes (approximately 7000), cases being defined by ICD-10 codes, and continuous quantitative phenotypes (approximately 5500), both results from blood tests and proteome data from deCODE and UK Biobank. Results C282Y homozygotes had a higher prevalence of the following liver, hematological and rheumatic diseases compared to a control group with no copy of the C282Y variant. Diagnosed fibrosis and cirrhosis of liver (odds ratio 4.30, 95% confidence interval 3.35 to 5.51, p=1.77x10 -30), liver cell carcinoma (9.04, 6.01 to 13.58, p=3.24x10 -26) and fatty liver disease (1.75, 1.45 to 2.11, p=5.42x10 -09) were strikingly more common in C282Y homozygotes. Unsurprisingly C282Y homozygotes also had higher liver parameters. Notably C282Y homozygotes had a higher prevalence of diagnosed polycythemia vera (7.24, 4.93 to 10.62, p=4.37x10 -24) yet no association was found with clonal hematopoiesis (1.09, 0.88 to 1.34, p=4.47x10 -01). Strong association was found between C282Y homozygosity and increased hemoglobin (0.41 standard deviations, 95% confidence interval 0.38 to 0.45, p=6.84x10 -142), MCH (0.98, 0.93 to 1.03, p=&amp;lt;1.00x10 -300) and MCV (0.82, 0.78 to 0.86, p=&amp;lt;1.00x10 -300). Red blood cell count (RBC) was decreased (-0.27, -0.30 to -0.23, p=1.06x10 -50), while reticulocyte count (0.30, 0.26 to 0.34, p=1.42x10 -54) and percentage (0.36, 0.32 to 0.40, p=7.91x10 -79) were increased. Erythropoietin (EPO) measurements were markedly decreased in C282Y homozygotes (-0.31, -0.40 to -0.22, p=1.77x10 -11). Homozygous individuals had an increased risk of being diagnosed with osteoarthritis of hip (1.83, 1.63 to 2.07, p=4.30x10 -23) and were more likely to undergo total hip replacement (2.00, 1.69 to 2.29, p=5.81x10 -18). In contrast to these risk enhancing effects C282Y homozygotes had a lower prevalence of cardiovascular disease (CVD). C282Y homozygotes had decreased risk of coronary artery disease (CAD) (0.79, 0.72 to 0.86, p=4.26x10 -07) and angina pectoris (0.77, 0.69 to 0.86, p=4.28x10 -06) and were less likely to undergo coronary artery bypass graft (0.60, 0.49 to 0.74, p=2.39x10 -06). Pure hypercholesterolemia (0.76, 0.69 to 0.83, p=2.52x10 -09) and disorders of lipoprotein metabolism (0.75, 0.66 to 0.85, p=8.41x10 -06) was less commonly diagnosed. C282Y homozygotes were less likely to take cholesterol lowering statin drugs (0.74, 0.67 to 0.81, p=1.45x10 -09). Unsurprisingly with regard to these results strong association was found with lower cholesterol measurements in C282Y homozygotes, the effects being most pronounced on LDL cholesterol (-0.38, -0.42 to -0.35, p=1.61x10 -99). Total cholesterol was also markedly lowered (-0.35, -0.38 to -0.31, p=1.38x10 -91), as well as non HDL cholesterol (-0.34, -0.37 to -0.30, p=2.36x10 -81). No significant association was found with HDL cholesterol and triglyceride measurements. Conclusions C282Y homozygosity was associated with significantly increased risk of liver disease, liver cell carcinoma and osteoarthritis of hip. Homozygous individuals had increased risk of non-clonal primary polycythemia, association with polycythemia vera likely due to misdiagnosis or early diagnosis exacerbated by significantly increased hemoglobin. C282Y homozygotes had a significant decrease in LDL, non-HDL and total cholesterol measurements and were protected against cardiovascular disease.

Genetic Variants Associated with High Plasma Levels of Factor VIII and Von Willebrand Factor in a Family with Thrombotic Episodes

Background: High levels of factor VIII (FVIII) and von Willebrand factor (vWF) have been indicated as independent risk factors for venous thromboembolism. However, the genetic factors responsible for their increase remain still poorly known. In a woman referred to our Thrombosis unit following a venous thrombotic event, very high levels of FVIII and vWF were found at the acute episode and in subsequent measurements. High levels of FVIII and vWF were also detected in other family members, suggesting a possible genetic cause (Figure 1). Aim: To identify variants/genes associated with high levels of FVIII and vWF. Methods: In a large Italian family with high levels of FVIII/vWF and thrombotic episodes, whole-exome sequencing was performed in 12 family members (SureSelect-Agilent). Data were analyzed according to the guidelines of the Broad Institute (https://software.broadinstitute.org/gatk/best-practices/). Variants were annotated (Annovar) and filtered (KGGseq) according to an autosomal dominant inheritance model of high levels of FVIII or vWF. RNA levels were evaluated in PBMCs by qPCR (Thermo Fisher). Results: 20 variants, spread over a 8300-Kb region on chromosome 5, were identified in 12 genes, including the low frequency rs13158382 located upstream of the MIR143/145 genes. Since variants in the promoter of MIR143/145 have been reported to affect their expression and reduced levels of miR-145 have been associated with high vWF, the potential correlation between rs13158382 and vWF levels was evaluated. Lower levels of both miRNAs and higher level of vWF mRNA were measured in family members carrying the variant (n=3) compared to family members without the variant (n=3). Analysis of genetic and expression data from of a larger cohort of individuals from the 1000 Genomes and GEUVADIS project confirmed a statistically significant reduction (p=0.023) of miR-143 in heterozygous subjects (n=35) compared to homozygous wild-type individuals (n=386). Since low levels of pri-miRNA-143/145 were measured in heterozygous family members and structural changes of pre-miR-143 with rs13158382 was predicted by miRVaS tool, the reduction of mature miRNAs might result from a lowered synthesis of pri-miRNAs and/or from an aberrant processing of pre-miRNA. Conclusion: This family-based study revealed a new genetic variant potentially involved in vWF increase by reducing the expression of miR-143/145.

Genome editing approaches with CRISPR/Cas9: the association of NOX4 expression in breast cancer patients and effectiveness evaluation of different strategies of CRISPR/Cas9 to knockout Nox4 in cancer cells

BackgroundThe increasing prevalence of cancer detection necessitated practical strategies to deliver highly accurate, beneficial, and dependable processed information together with experimental results. We deleted the cancer biomarker NOX4 using three novel genetic knockout (KO) methods. Homology-directed repair (HDR), Dual allele HITI (Du-HITI) and CRISPR-excision were utilized in this study.MethodsThe predictive value of the NOX4 expression profile was assessed using a combined hazard ratio (HR) with a 95% confidence interval (CI). With a 95% confidence interval, a pooled odd ratio (OR) was used to calculate the relationship between NOX4 expression patterns and cancer metastasis. There were 1060 tumor patients in all sixteen research that made up this meta-analysis. To stop the NOX4 from being transcribed, we employed three different CRISPR/Cas9-mediated knockdown methods. The expression of RNA was assessed using RT-PCR. We employed the CCK-8 assay, colony formation assays, and the invasion transwell test for our experiments measuring cell proliferation and invasion. Using a sphere-formation test, the stemness was determined. Luciferase reporter tests were carried out to verify molecular adhesion. Utilizing RT-qPCR, MTT, and a colony formation assay, the functional effects of NOX4 genetic mutation in CRISPR-excision, CRISPR-HDR, and CRISPR du-HITI knockdown cell lines of breast cancer were verified.ResultsThere were 1060 malignant tumors in the 16 studies that made up this meta-analysis. In the meta-analysis, higher NOX4 expression was linked to both a shorter overall survival rate (HR = 1.93, 95% CI 1.49–2.49, P < 0.001) and a higher percentage of lymph node metastases (OR = 3.22, 95% CI 2.18–4.29, P < 0.001). In breast carcinoma cells, it was discovered that NOX4 was overexpressed, and this increase was linked to a poor prognosis. The gain and loss-of-function assays showed enhanced NOX4 breast carcinoma cell proliferation, sphere-forming capacity, and tumor development. To activate transcription, the transcriptional factor E2F1 also attaches to the promoter region of the Nanog gene. The treatment group (NOX4 ablation) had substantially more significant levels of proapoptotic gene expression than the control group (P < 0.01). Additionally, compared to control cells, mutant cells expressed fewer antiapoptotic genes (P < 0.001). The du-HITI technique incorporated a reporter and a transcription termination marker into the two target alleles. Both donor vector preparation and cell selection were substantially simpler using this approach than with “CRISPR HDR” or “CRISPR excision.“ Furthermore, single-cell knockouts for both genotypes were created when this method was applied in the initial transfection experiment.ConclusionsThe NOX4 Knockout cell lines generated in this research may be used for additional analytical studies to reveal the entire spectrum of NOX4 activities. The du-HITI method described in this study was easy to employ and could produce homozygous individuals who were knockout for a specific protein of interest.

Extracranial carotid artery atherosclerotic plaque and APOE polymorphisms: a systematic review and meta-analysis.

Carotid atherosclerotic plaque is an important independent risk factor for stroke. Apolipoprotein E (APOE) influences cholesterol levels and certain isoforms are associated with increased carotid atherosclerosis, though the exact association between APOE and carotid plaque is uncertain. The study aimed to evaluate the association between APOE and carotid plaque. A systematic review was performed to retrieve all studies which examined the association between carotid plaque and APOE. This study was conducted in accordance with the PRISMA guidelines. Independent readers extracted the relevant data from each study including the type of imaging assessment, plaque definition, frequency of APOE E4 carrier status and type of genotyping. Meta-analyses with an assessment of study heterogeneity and publication bias were performed. Results were presented in a forest plot and summarized using a random-effects model. After screening 838 studies, 17 studies were included for systematic review. A meta-analysis of 5 published studies showed a significant association between ε4 homozygosity and carotid plaque [odds ratio (OR), 1.53; 95% CI, 1.16, 2.02; p = .003]. Additionally, there was a significant association between patients possessing at least one ε4 allele, heterozygotes or homozygotes, and carotid plaque (OR, 1.25; 95% CI, 1.03, 1.52; p = .03). Lastly, there was no association between ε4 heterozygosity and carotid plaque (OR, 1.08; 95% CI, 0.93, 1.26; p = .30). APOE ε4 allele is significantly associated with extracranial carotid atherosclerotic plaque, especially for homozygous individuals.

Coagulation Factor XII ( F12) Haploinsufficiency Is Protective Against Venous Thromboembolism in 415,921 Individuals

Background: Coagulation factor XII (FXII) has drawn significant attention as a potential drug target due to the observation that severe congenital deficiency in FXII is not associated with bleeding, while deletion or inhibition of FXII in pre-clinical models consistently protects against thrombosis. However, the contribution of FXII to thrombosis in humans remains controversial, with early reports suggesting that FXII deficiency may even be associated with an increased risk of venous thromboembolism (VTE). Meanwhile, epidemiologic studies of FXII deficiency have yielded conflicting results and are fraught with methodological challenges in part because FXII-deficient patients have no known phenotype and do not typically present to medical attention. To overcome these limitations, we performed the first large-scale analysis of differential thrombotic risk associated with germline loss of function in the gene encoding FXII ( F12) across over 400,000 individuals. Aims: To determine the impact of rare F12 loss-of-function variants on the risk of VTE in a large-scale whole exome sequencing dataset. Methods: The UK Biobank (UKBB) is a population-scale registry dataset containing paired clinical and genomics data for nearly 500,000 individuals. We identified individuals in the UKBB with rare (MAF &amp;lt;1%) variants in F12 that were predicted in silico to result in altered protein function. Qualifying variants included missense variants with an in silico functional impact score of ≥0.9 and high-confidence loss-of-function (HCLOF) variants such as frameshift, nonsense, and essential splice site mutations. We used Firth's logistic regression to assess the association between qualifying F12 variants in a single-gene collapsing analysis and the risk of VTE after adjusting for age, sex, and ancestry as well as several known contributory factors. Differences in the levels of 1,472 plasma proteins between carriers and non-carriers of qualifying F12 variants were evaluated in a subset of 48,892 UKBB participants using the Olink Explore 1536 proteomics platform. Results: Data from 415,921 UKBB participants were included in our analysis. In total, 1,598 individuals carried qualifying variants in F12, of which 99.8% were heterozygous. The presence of rare, function-altering variants in F12 was significantly associated with protection against VTE (OR=0.74, 95% CI: 0.55-0.97, P=0.029) ( Figure 1). This finding remained unchanged when we restricted the analysis to HCLOF variants (OR=0.74, 95% CI: 0.55-0.97, P=0.031). Further, we replicated several known risk factors for VTE, including female sex (P=0.0015), older age (P&amp;lt;1 x 10 -16), higher BMI (P&amp;lt;1 x 10 -16), non-O blood group (P&amp;lt;1 x 10 -16), higher CRP (P&amp;lt;1 x 10 -16), and a history of smoking (P&amp;lt;1 x 10 -16), suggesting that clinical annotation for the dataset is reliable. As expected, the presence of F12 variants was not associated with increased risk of hemorrhage (OR=0.90, P=0.44). Because FXII is also involved in pathways of innate immunity, we interrogated the relationship between carriage of rare F12 variants and sepsis and found no increased risk (OR=0.95, P=0.73). Next, we compared the circulating plasma levels of 1,472 proteins between participants with and without qualifying F12 variants ( Figure 2). We determined that plasma concentrations of three proteins are significantly elevated in participants with a variant F12 allele. Of these, heme binding protein 1 (HEBP1) levels demonstrated the most striking difference (fold-change=7.08, P=1.439 x 10 -43). However, germline loss-of-function variants in the HEBP1 gene are not associated with decreased risk of VTE (OR=1.109, P=0.88), suggesting that HEPB1 levels are regulated by FXII and do not directly influence VTE risk. Conclusions: Operating at the level of germline genetics, our data are the first to demonstrate that FXII loss-of-function is protective against VTE in humans. Because F12 variant carriers in the UKBB dataset predominantly possess only a single defective allele, it is possible that larger effect sizes would be observed among homozygous or compound heterozygous individuals. Additionally, F12 loss-of-function does not appear to increase the risk of hemorrhage or severe infection. Our findings suggest that therapeutic blockade of FXII in humans is likely to be safe and effective.

Genetic Variants Identified by Whole Exome Sequencing in a Large Italian Family with High Plasma Levels of Factor VIII and Von Willebrand Factor.

High plasma levels of factor VIII (FVIII) and von Willebrand factor (VWF) have been indicated as independent risk factors for venous thromboembolism. However, the genetic factors responsible for their increase remain poorly known. In a large Italian family with high FVIII/VWF levels and thrombotic episodes, whole exome sequencing (WES) was performed on 12 family members to identify variants/genes involved in FVIII/VWF increase. Twenty variants spread over a 8300 Kb region on chromosome 5 were identified in 12 genes, including the low frequency rs13158382, located upstream of the MIR143/145 genes, which might affect miR-143/145 transcription or processing. The expression of miR-143/145 and VWF mRNA were evaluated in the peripheral blood mononuclear cells of six family members. Members with the variant (n = 3) showed lower levels of both miRNAs and higher levels of VWF mRNA compared to members without the variant (n = 3). An analysis of genetic and expression data from a larger cohort of individuals from the 1000 Genomes and GEUVADIS project confirmed a statistically significant reduction (p-value = 0.023) in miR-143 in heterozygous (n = 35) compared to homozygous wild-type individuals (n = 386). This family-based study identified a new genetic variant potentially involved in VWF increase by affecting miR-143/145 expression.

Sorting Mosquito Larvae with a COPAS Machine.

Transgenic mosquitoes are widely used in mosquito research. To distinguish transgenic individuals from wild types, genes for fluorescent proteins are the most commonly used genetic markers in transgenic constructs, offering all the advantages of visual selection. Although manual selection under a fluorescence binocular microscope is perfect for the selection of first-generation transgenics, managing established fluorescent lines can be facilitated by complex object parametric analyzer and sorter (COPAS) sorting, which we describe in this protocol. COPAS sorting allows researchers to purify large mosquito larval populations containing only homozygous transgenic individuals, only heterozygotes, or a mix of homozygous, wild types, and heterozygotes in desired proportions. Sorting large populations of a single sex is also possible. Finally, especially when several transgenes of different fluorescence colors are inserted in the same docking site (a recombination site previously inserted in the mosquito genome, which can be used to insert new transgenes into the same locus), they can be maintained together in a single mosquito population to save insectarium space and labor. COPAS sorting can then be used to extract the desired genotype when needed and to readjust transgene frequencies every few generations in case drift is observed.

Techniques for Identifying and Sorting Transgenic Mosquito Larvae.

Transgenic mosquitoes are used in many aspects of mosquito research. First, they can help answer biological questions to advance scientific knowledge-for example, in the fields of mosquito-pathogen interactions, insect immunity, or olfaction. Second, transgenic technologies may be used to develop much needed novel vector control strategies, such as mosquitoes that are unable to transmit disease or transgenes that sterilize mosquito females to suppress vector populations. Here, we introduce how researchers use various selection markers to screen for transgenic mosquito larvae following a transgenesis experiment. Common procedures include using a binocular fluorescence microscope for initial screening. For higher-throughput screening, a flow cytometer known as Complex Object Parametric Analyzer and Sorter (COPAS) can be used to stabilize transgenic lines through the purification of homozygous individuals or to manage transgene frequency in established transgenic lines. In particular, COPAS sorting allows the production of mosquito larval cultures composed of a mixture of genotypes (control and genetically modified larvae) with the goal of raising both groups of mosquitoes under the same environmental conditions in preparation for a controlled phenotype assessment. It can also be used to produce large populations of male mosquitoes, which should facilitate the development of mosquito control intervention strategies similar to the sterile insect technique (SIT), which aims to release large numbers of sterile males that will mate with and sterilize wild females to suppress mosquito populations. Finally, the utilization of a puromycin resistance marker cassette to screen for transgenic Anopheles larvae is also introduced.

High Prevalence of a c.5979dupA Variant in the Dysferlin Gene (DYSF) in Individuals from a Semiarid Region of Brazil.

Dysferlinopathies represent a group of limb girdle or distal muscular dystrophies with an autosomal-recessive inheritance pattern resulting from the presence of pathogenic variants in the dysferlin gene (DYSF). In this work, we describe a population from a small city in Brazil carrying the c.5979dupA pathogenic variant of DYSF responsible for limb girdle muscular dystrophy type 2R and distal muscular dystrophy. Genotyping analyses were performed by qPCR using customized probe complementary to the region with the duplication under analysis in the DYSF. A total of 104 individuals were examined. c.5979dupA was identified in 48 (46.15%) individuals. Twenty-three (22%) were homozygotes, among whom 13 (56.5%) were female. A total of 91.3% (21) of homozygous individuals had a positive family history, and seven (30.4%) reported consanguineous marriages. Twenty-five (24%) individuals were heterozygous (25.8±16 years) for the same variant, among whom 15 (60%) were female. The mean CK level was 697 IU for homozygotes, 140.5 IU for heterozygotes and 176 IU for wild-type homo-zygotes. The weakness distribution pattern showed 17.3% of individuals with a proximal pattern, 13% with a distal pattern and 69.6% with a mixed pattern. Fatigue was present in 15 homozygotes and one heterozygote. The high prevalence of this variant in individuals from this small community can be explained by a possible founder effect associated with historical, geographical and cultural aspects.

Distortion of Mendelian segregation across the Angus cattle genome uncovering regions affecting reproduction

Nowadays, the availability of genotyped trios (sire-dam-offspring) in the livestock industry enables the implementation of the transmission ratio distortion (TRD) approach to discover deleterious alleles in the genome. Various biological mechanisms at different stages of the reproductive cycle such as gametogenesis, embryo development and postnatal viability can induce signals of TRD (i.e., deviation from Mendelian inheritance expectations). In this study, TRD was evaluated using both SNP-by-SNP and sliding windows of 2-, 4-, 7-, 10- and 20-SNP across 92,942 autosomal SNPs for 258,140 genotyped Angus cattle including 7,486 sires, 72,688 dams and 205,966 offspring. Transmission ratio distortion was characterized using allelic (specific- and unspecific-parent TRD) and genotypic parameterizations (additive- and dominance-TRD). Across the Angus autosomal chromosomes, 851 regions were clearly found with decisive evidence for TRD. Among these findings, 19 haplotypes with recessive patterns (potential lethality for homozygote individuals) and 52 regions with allelic patterns exhibiting complete or quasi-complete absence for homozygous individuals in addition to under-representation (potentially reduced viability) of the carrier (heterozygous) offspring were found. In addition, 64 (12) and 20 (4) regions showed significant influence on the trait heifer pregnancy at p-value < 0.05 (after chromosome-wise false discovery rate) and 0.01, respectively, reducing the pregnancy rate up to 15%, thus, supporting the biological importance of TRD phenomenon in reproduction.