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At least 415 records · Page 23Linked to original sources

PubMed1992

DNA mutations associated with the human butyrylcholinesterase J-variant.

The J-variant of human serum butyrylcholinesterase (BChE) causes both an approximately two-thirds reduction of circulating enzyme molecules and a corresponding decrease in the level of BChE activity present in serum. Since the level of serum BChE activity and the duration of succinylcholine apnea are inversely correlated, this marked decrease in activity makes individuals with the J-variant more susceptible than usual subjects to prolonged apnea from succinylcholine. We reinvestigated the same family in which Garry et al. identified the J-variant phenotype. The atypical, fluoride, and K-variant mutations were also identified in members of the 47-person pedigree. DNA amplification by PCR, followed by direct sequencing of the amplified DNA, led to the finding that the J-variant phenotype of human serum BChE was associated with two DNA point mutations in the coding region. One of these was the mutation previously identified with the K-variant phenotype (GCA----ACA; Ala539----Thr). The other was an adenine-to-thymine transversion at nucleotide 1490, which changed amino acid 497 from glutamic acid to valine (GAA----GTA; Glu497----Val). This latter point mutation was named the J-variant mutation (formal name BCHE*497V). The J-variant mutation has not been identified without the K-variant mutation. The J-variant mutation created an RsaI-enzyme RFLP. Two additional point mutations, located in the noncoding regions of the gene, were also found to be linked with the J-variant and K-variant point mutations on the same allele. These noncoding polymorphic mutations had previously been found linked to the atypical and K-variant point mutations. A summary table shows dibucaine, fluoride, and Hoffmann-La Roche compound Ro 2-0683 inhibition numbers for 119 samples whose DNA has been sequenced. Eighteen BChE genotypes are represented.

Amino Acid Sequence↗

PubMed2025

Identification of pathogenic variants in six Chinese families with keratoconus of autosomal dominant inheritance: pathogenicity analysis and variable phenotype.

PURPOSE: Keratoconus (KC) is a bilateral, asymmetric disease causing corneal thinning, irregular astigmatism, and vision decline, with unclear etiology. This study aims to investigate pathogenic variants of candidate genes in Chinese KC families via whole exome sequencing (WES). METHODS: The Pentacam 3D anterior segment analysis system was applied for keratectasia detection, and the Corvis ST was used for corneal biomechanics measurement. Probands from KC families were screened via WES and further verified in other family members through Sanger sequencing. Additionally, qPCR was used to validate copy number variants and identify pathogenic gene loci. The identified variants were then classified according to the Standards and Guidelines for the Interpretation of Sequence Variants published by the American College of Medical Genetics and Genomics (ACMG). Finally, STRING protein-protein interaction (PPI) networks analysis was performed to investigate interactions among candidate gene-related proteins. RESULTS: Using WES, four heterozygous missense variants were detected in the ZNF469, KRT12, COL8A2, and COL18A1 genes: c.4384G > A: p.Asp1462Asn, c.1229T > G:p.Val410Gly, c.505A > G:p.Ile169Val, and c.1159G > A:p.Gly387Arg. Additionally, a heterozygous frameshift variant was detected in the PMS2 gene: c.1551_1572del:p.Ser517Argfs*71. The affected parents carried the same variants as the probands verified by Sanger sequencing. A copy number variant was detected in the DPP6 gene: seq[GRCh38] dup(7)(q36.2q36.2) chr7:g.153782360_ 153982491dup. According to ACMG guidelines, ZNF469, KRT12, COL8A2, and COL18A1 gene variants are Likely Pathogenic; PMS2 and DPP6 gene variants are Pathogenic. STRING analysis highlights a tightly interconnected network centered on COL8A2, involving COL18A1, FN1, ZNF469, and KRT12. DPP6 was involved in KC via affecting FN1. In four of six autosomal dominant KC (adKC) families, affected parents had the same variants as probands but milder phenotypes. CONCLUSION: In this study, six novel variants in ZNF469, KRT12, COL8A2, COL18A1, PMS2, and DPP6 were linked to adKC. Family phenotypes showed variable expressivity with irregular dominance inheritance. Abnormal KC-related gene protein expression may contribute to corneal structural instability. This study broadened KC genetic screening candidates and suggested genetic testing could aid early KC diagnosis and intervention.

Adult↗

PubMed2026

Automated patch clamp data improve variant classification and penetrance stratification for SCN5A-Brugada syndrome.

BACKGROUND AND AIMS: Brugada Syndrome (BrS) is an inherited arrhythmia disorder that causes an elevated risk of sudden cardiac death. Approximately 20% of patients with BrS have rare variants in SCN5A, which encodes the cardiac sodium channel NaV1.5. Genetic workup of BrS is often complicated by SCN5A variants of uncertain significance (VUS) and/or incomplete penetrance. This study deployed an SCN5A-BrS functional assay at cohort scale to facilitate the implementation of genetic and precision medicine. METHODS: All 252 missense and in-frame insertion/deletion SCN5A variants from a previously published large cohort of BrS cases (n = 3335 patients) were analysed using a calibrated high-throughput automated patch-clamp (APC) assay. Variant functional Z-scores were assigned evidence levels ranging from BS3_moderate (normal function) to PS3_strong (loss-of-function), as defined by American College of Medical Genetics and Genomics criteria. Functional evidence was combined with population frequency, hotspot, case counts, protein-length changes, and in silico predictions. Odds ratios of BrS case-control enrichment and penetrance for BrS were calculated from variant frequencies in the BrS cohort and in gnomAD. RESULTS: Most variants (146/252) were functionally abnormal (Z ≤ -2), with 100 having severe loss-of-function (Z ≤ -4). Functional evidence enabled the reclassification of 110 of 225 VUS; 104 to likely pathogenic and 6 to likely benign. SCN5A variants with loss-of-function were mainly localized to the transmembrane domains, especially the regions comprising the central pore. SCN5A variant penetrance was proportional to the severity of loss-of-function; variants with Z ≤ -6 had penetrance of 24.5% (15.9%-37.7% CI) and an odds ratio of 501 for BrS. CONCLUSIONS: This cohort-scale APC dataset stratifies SCN5A variants found in BrS patients into normal function 'bystander' variants that have a low risk of BrS and loss-of-function variants that have a high risk for BrS. Functional data can be integrated with other criteria to reclassify a substantial fraction of VUS. The dataset helps clarify the SCN5A-BrS relationship and will improve the diagnosis and clinical management of BrS probands and their families.

Humans↗

PubMed2025

Novel Protein-Altering Variants in Cleft Genes Transmitted in Families With NSCL±P.

BACKGROUND: Pathogenic protein-altering variants play a role in the etiology of nonsyndromic cleft lip with or without palate (nsCL±P), one of the most common craniofacial anomalies. However, the genetic basis of many cases remains unclear, complicating risk prediction for affected families. PURPOSE: This study utilized whole-genome sequencing (WGS) of 150 case-families with nsCL±P from sub-Saharan Africa to identify pathogenic risk variants. STUDY DESIGN, SETTING, SAMPLE: This study utilized whole-genome sequencing (WGS) of 150 case-families with nsCL±P from sub-Saharan Africa to identify risk variants. PREDICTOR/EXPOSURE/INDEPENDENT VARIABLE: Genetic variants. MAIN OUTCOME VARIABLES: Nonsyndromic cleft lip with or without palate (nsCL±P). ANALYSES: Genomes were sequenced at a mean ×30 coverage, and variants were prioritized using CADD (≥20), REVEL (≥0.5), and ACMG/AMP clinical significance criteria. RESULTS: We identified pathogenic protein-altering variants in CHD7 (p.Arg1345His), LRP2 (p.Asp3245Asn), RYR1 (p.Arg2163Leu, p.Pro2903Thr), SHH (p.Met114Val), and WNT3 (p.Ser112Pro) highlighting the role of hedgehog signaling pathway (FDR=5.32e-12) in nsCL±P. These variants were inherited from unaffected parents suggesting an incomplete penetrance of the variant effect. Although mouse data showed that knockout of these genes produces cleft phenotypes, in vivo studies will help us better understand how the consequences of these variants differ from benign mutations. The presence of these protein-altering variants in unaffected parents-incomplete penetrance, provides additional evidence supporting the trait complexity. CONCLUSIONS AND RELEVANCE: This study identified rare, pathogenic protein-altering variants in genes involved in key developmental pathways in African families affected by nsCL±P. These findings highlight the critical role of the hedgehog signaling pathway and related networks in the etiology of nsCL±P. These findings underscore the importance of whole-genome sequencing in genetically diverse populations to uncover novel risk variants. These findings enhance our understanding of the genetic etiology of nsCL±P, particularly in under-represented African populations and support the multifactorial inheritance and the involvement of developmental pathways, such as hedgehog signaling in the etiology of clefting.

Humans↗

PubMed2026

Functional Validation of a Novel Homozygous TTN Splice-Site Variant Reveals Aberrant Splicing in Hypertrophic Cardiomyopathy.

The TTN gene encodes a crucial structural protein within cardiac sarcomeres, and its variants may contribute to hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy; however, phenotype and genotype are different. Whole-exome sequencing (WES) was conducted on a Chinese proband diagnosed with HCM. In silico splicing prediction tools and minigene assays were employed to investigate the impact of the identified variant on mRNA splicing. A literature review was performed to retrieve and analyze previously reported splicing variants in the TTN gene associated with HCM. A 42-year-old male proband presented with nonobstructive HCM and paroxysmal atrial arrhythmias. A novel homozygous TTN variant was found, predicted to cause a 14-base pair deletion at a splice acceptor site. Two asymptomatic offspring were found to carry the heterozygous variant. Based on variant interpretation guidelines, the variant met the PM2_supporting criterion and was classified as a variant of uncertain significance (VUS). The predicted aberrant splicing effect was subsequently confirmed by the minigene splicing assay, demonstrating altered pre-mRNA splicing leading to an in-frame insertion/deletion (p.Arg32498_Glu32504delinsGln). Functional verification confirmed that this mutation conforms to the PM4 criterion, suggesting that it can be reclassified as a tepid VUS (scoring 3 points). The functional result might provide pathogenic evidence. We also reviewed 28 previously reported splicing variants in TTN associated with HCM. Of these, 57.1% (16/28) localized to the I-band region, whereas 21.4% (6/28) were situated in the A-band domain of titin. Notably, 21.4% (6/28) co-occurred with pathogenic variants in other sarcomeric genes (MYH7 or MYBPC3), correlating with more severe clinical phenotypes. Reclassification and reinterpretation of the variants revealed that none met the level of likely pathogenic or higher. The present case contributes a homozygous splice-site variant with experimentally confirmed aberrant splicing and an in-frame protein alteration in titin. The focus of this report is the Mendelian genetic basis of the proband's cardiomyopathy phenotype, and our data provide additional case-level and functional evidence for a possible role of specific TTN splicing defects in HCM.

Humans↗

PubMed2021

Early-Onset Atrial Fibrillation and the Prevalence of Rare Variants in Cardiomyopathy and Arrhythmia Genes.

IMPORTANCE: Early-onset atrial fibrillation (AF) can be the initial manifestation of a more serious underlying inherited cardiomyopathy or arrhythmia syndrome. OBJECTIVE: To examine the results of genetic testing for early-onset AF. DESIGN, SETTING, AND PARTICIPANTS: This prospective, observational cohort study enrolled participants from an academic medical center who had AF diagnosed before 66 years of age and underwent whole genome sequencing through the National Heart, Lung, and Blood Institute's Trans-Omics for Precision Medicine program. Participants were enrolled from November 23, 1999, to June 2, 2015. Data analysis was performed from October 24, 2020, to March 11, 2021. EXPOSURES: Rare variants identified in a panel of 145 genes that are included on cardiomyopathy and arrhythmia panels used by commercial clinical genetic testing laboratories. MAIN OUTCOMES AND MEASURES: Sequencing data were analyzed using an automated process followed by manual review by a panel of independent, blinded reviewers. The primary outcome was classification of rare variants using American College of Medical Genetics and Genomics criteria: benign, likely benign, variant of undetermined significance, likely pathogenic, or pathogenic. Disease-associated variants were defined as pathogenic/likely pathogenic variants in genes associated with autosomal dominant or X-linked dominant disorders. RESULTS: Among 1293 participants (934 [72.2%] male; median [interquartile range] age at enrollment, 56 [48-61] years; median [interquartile range] age at AF diagnosis, 50 [41-56] years), genetic testing identified 131 participants (10.1%) with a disease-associated variant, 812 (62.8%) with a variant of undetermined significance, 92 (7.1%) as heterozygous carriers for an autosomal recessive disorder, and 258 (20.0%) with no suspicious variant. The likelihood of a disease-associated variant was highest in participants with AF diagnosed before the age of 30 years (20 of 119 [16.8%; 95% CI, 10.0%-23.6%]) and lowest after the age of 60 years (8 of 112 [7.1%; 95% CI, 2.4%-11.9%]). Disease-associated variants were more often associated with inherited cardiomyopathy syndromes compared with inherited arrhythmias. The most common genes were TTN (n = 38), MYH7 (n = 18), MYH6 (n = 10), LMNA (n = 9), and KCNQ1 (n = 8). CONCLUSIONS AND RELEVANCE: In this cohort study, genetic testing identified a disease-associated variant in 10% of patients with early-onset AF (the percentage was higher if diagnosed before the age of 30 years and lower if diagnosed after the age of 60 years). Most pathogenic/likely pathogenic variants are in genes associated with cardiomyopathy. These results support the use of genetic testing in early-onset AF.

Adult↗

PubMed2000

Variant estrogen receptor alpha mRNAs in human breast cancer specimens.

A panel of human breast cancer specimens was examined for single base change mutations by DNA sequencing and for larger deletions using a PCR-based assay. In the cancer specimens examined, no sequencing variants were detected other than a previously characterized polymorphism. Although most of the specimens contained estrogen receptor (ER) variants at a low level, 2 of 118 specimens exhibited variants which, after amplification, constituted most of the amplified ER cDNA. One specimen contained a single variant form, and there was little evidence of the wild-type ER mRNA by PCR, Northern blotting or immunocytochemistry. The second specimen, despite the presence of a normal-sized mRNA by Northern blotting and normal immunocytochemical staining for ER, contained at least 5 different variant forms as well as the wild-type ER. All but 1 of the variant forms were processing variants, and 3 of these processing variants have not been described before. One variant, although lacking exons 2-4, has break points in exons 1 and 5 that do not correspond to intron-exon boundaries. This variant might reflect more widespread damage to the genome in this breast cancer specimen. The low level of occurrence of variants suggests that ER variant forms, at least in the coding region, do not contribute generally to the progression of breast cancer.

Blotting, Northern↗

PubMed2026

Additional PAX3 Variants for Dominant Blue Eyes in Cats.

Since the recent publication of the first feline variant of the PAX3 (Paired Box 3) gene associated with blue eyes and minimal white spotting (DBE, Dominant Blue Eyes), three further variants of PAX3 have been reported in felines. However, these four variants do not account for all the different feline lines that exhibit DBE. Whole-genome sequencing using short-read and long-read technologies identified a large complex structural variant involving two deletions and an inversion in a purebred line of British shorthair and longhair cats. All 39 British cats with a BDE phenotype were heterozygous for the variant, that was absent in 47 non-DBE cats and 16 DBE cats from other breeding lines. The purebred British shorthair founder female named Nadeya was also heterozygous for the variant, and the segregation of the variant is consistent with dominant inheritance. We named this NC_058375.1:g.[205097674_207411974del;207411975_211290497inv;211290498_211387174del] variant the DBENAD (Nadeya Dominant Blue Eyes) allele. In addition, a candidate gene approach identified a nonsense variant, XM_019838731.3:c.784C>T, in the fifth exon of PAX3 in a second breeding line originating from a female named Marusya. The segregation of this nonsense variant is consistent with dominant inheritance, and a perfect genotype-phenotype correlation was observed in the 16 cats from this line, so we propose that this sixth variant of PAX3 represents the DBEMARU (Marusya Dominant Blue Eyes) allele in the domestic cat. Our study has identified two further variants of the PAX3 gene associated with DBE in domestic cats, which will help to improve genotyping of the breeding stock.

Animals↗

PubMed2006

Tensin2 variant 3 is associated with aggressive tumor behavior in human hepatocellular carcinoma.

Tensins are a new family of proteins that act as an important link among extracellular matrix, actin cytoskeleton, and signal transduction and have been implicated in human cancers. Tensin2 was initially identified in a search for new tensin family members that share extensive sequence homology with tensin1. Tensin2 was highly expressed in liver tissues. A recent study reported that one of the splicing variants of tensin2, variant 3, promotes cell migration. In the present study, we aimed to elucidate the role of variant 3 in hepatocarcinogenesis by assessing the expression of variant 3 mRNA in hepatocellular carcinoma (HCC) tissue and ectopically expressing variant 3 in HCC cell lines. Analysis of variant 3 expression in human HCC tissue revealed it was overexpressed in 46% (23/50) of tumor tissues as compared with the corresponding nontumorous livers. High expression of variant 3 was significantly associated with venous invasion (P = .037), tumor microsatellite formation (P = .022), and tumor nonencapsulation (P = .049). Our ectopic expression study showed that variant 3 significantly promoted the cell growth and motility of HCC cells. The clonal transfectants of variant 3 were more closely packed and resulted in a higher saturation density than in the control vector transfectants. Variant 3 expression also enhanced the proliferation rate in culture and in vivo tumorigenicity in nude mice. In conclusion, we reveal a novel role for variant 3 in the progression of HCC and suggest the feasibility of elevated variant 3 expression as a tumor progression marker for HCC.

Animals↗

PubMed1983

Rat serum albumin synthesis in variant rat hepatoma cells.

Variant subclones of the rat hepatoma cell line FU5-5 have been isolated that are altered in their production of rat serum albumin. Three of these variants, isolated in a random screening, have been categorized as high, intermediate, and low producers. They secrete albumin into the culture medium at different rates: 16, 1.7, and 0.3 microgram/mg cell protein/48 h. A fourth variant, isolated on the basis of altered morphology, secretes no detectable albumin. Unlike the albumin-producing variants, this null variant is also deficient in the level and inducibility of tyrosine aminotransferase activity. Albumin biosynthesis as determined in pulse-labeling experiments is affected similarly in the four variants, yielding albumin synthetic rates of 0.24, 0.035, 0.006, and less than 0.002% of total protein synthesis. The translatable albumin messenger RNA content in these variants was measured using a rabbit reticulocyte lysate system. The null variant contains no detectable mRNA, and the three quantitative variants contain levels of translatable albumin messenger RNA corresponding to 0.07, 0.03, and 0.005% of total stimulated polypeptide synthesis. The highest producing variant contains less translatable albumin mRNA than expected on the basis of cellular biosynthetic measurements, suggesting a translation efficiency difference in this clone. Cell hybrids constructed by fusing the high-producing clone and the null variant produce little or no albumin. This extinction indicates that the null variant contains a diffusible regulatory factor capable of decreasing albumin gene expression. The relatively stable and discrete heritable phenotypic changes exhibited by these clones may serve as a model for similar changes that occur during hepatic differentiation.

Animals↗

PubMed2004

Liver transplant donor candidates: associations between vascular and biliary anatomic variants.

Our objective was to investigate the coexistence of vascular and biliary anatomic variants, the latter of which are known to increase the risk of biliary complications in living liver donor transplantation. A total of 108 consecutive liver donor candidates were examined by magnetic resonance (MR) imaging that included 2 MR cholangiography methods, T2-weighted MR cholangiography and mangofodipir-enhanced T1-weighted three-dimensional (3D) MR cholangiography, as well as gadolinium-enhanced MR angiography and venography of the liver. Images were interpreted by at least 2 investigators in consensus for definition of hepatic arterial, portal venous, and biliary anatomy. A subset of 51 subjects underwent laparotomy for right hepatectomy. Of the 108 subjects examined, 50 (46%) demonstrated normal hepatic artery, portal vein, and biliary anatomy. Variants of the hepatic artery were found in 27 of 108 (25%) subjects, of the portal vein in 12 of 108 (11%) subjects, and of the bile ducts in 30 of 108 (28%) subjects. Of the 27 subjects with hepatic arterial variants, 8 (30%) also had variant biliary anatomy. The association between hepatic arterial variants and biliary variants was not statistically significant (P >.5). However, of the 12 subjects with portal vein variants, 7 (58%) had biliary variants, and in 6 of 7 cases, the right posterior hepatic duct was anomalous. By chi-square analysis, the association between portal venous and biliary variants was significant (P =.012). In conclusion, over half of subjects with portal vein variants were found to have anomalous biliary anatomy, which always involved the hepatic ducts of the right lobe. The association between portal venous and biliary variants is statistically significant, while there is no significant association between hepatic arterial and biliary variants.

Biliary Tract↗

PubMed1995

Human mammary carcinomas express homologues of rat metastasis-associated variants of CD44.

Splice variants of CD44 expressed in a metastasizing cell line derived from a rat pancreatic adenocarcinoma have been shown recently to confer metastatic potential onto non-metastasizing rat pancreatic carcinoma and sarcoma cell lines. Homologues of these variants have also been detected in a variety of human malignancies. Using antibodies raised against a bacterially expressed fusion protein containing variant CD44 sequences, we have explored the expression of variant CD44 glycoproteins on tumors of the female breast. The material examined included normal tissue, hyperplastic lesions, 103 primary invasive mammary carcinomas, 10 in situ carcinomas, 12 local recurrences and 18 lymph node metastases. Using a polyclonal serum directed against several variant CD44 epitopes, normal mammary epithelia as well as ductal hyperplasias were negative for these splice variants, while the variant CD44 epitopes were detectable in all but six of the primary invasive carcinomas. From the reaction with various monoclonal antibodies and polyclonal sera specific for individual epitopes it is obvious that the tumors predominantly express CD44 variants encoded by exons v5 to v7. Interestingly, all investigated lymph node metastases reacted positively with the variant-specific antibodies, in contrast to primary tumors which reacted in 54% to 86% of the cases, depending on the antibody used. Statistical analysis revealed a significant correlation between expression of variant exons v3/v4 and v6 and increased tumor grade (p = 0.001 and p < 0.05, respectively; Fisher's exact test). Exon v6 is carried by the variants which confer metastatic capability in the rat. These results indicate that the expression of the CD44 variants is upregulated in mammary carcinomas and is closely linked to tumor anaplasia.

Animals↗

PubMed1992

Selection of tumor cell variants for resistance to tumor necrosis factor also induces a form of pleiotropic drug resistance.

This study has addressed the question of whether there may be some common mechanism underlying the induction or expression of acquired cytokine and drug resistance in a tumor cell line. This study employed the tumor-necrosis-factor(TNF)-sensitive U937 tumor cell line as a model system to determine if selection of a tumor cell variant for cytokine resistance would also result in drug resistance and vice versa. Variants were selected by culturing in the presence of purified recombinant TNF or a mixed-lymphokine-containing supernatant derived from concanavalin-A-stimulated peripheral blood lymphocytes. The resulting variants were resistant not only to TNF, but also to certain chemotherapeutic drugs. The variants were most resistant to colchicine and the Vinca alkaloids, requiring drug concentrations 50- to 5000-fold higher to mediate levels of cytotoxicity comparable to that seen with the parental U937. The variants were moderately resistant to cycloheximide, actinomycin D, and mitomycin C. In contrast, these lines were relatively sensitive to doxorubicin or daunomycin. This phenomenon was not unique to U937 cells since we obtained a similar pattern of drug resistance by selecting TNF-resistant variants of the WEHI-164 tumor cell line. The cytokine-selected U937 variants were still lysed by NK cells, although they were somewhat less sensitive than the parental U937. Both variants were relatively resistant to lysis by activated macrophages, probably because of their TNF resistance. In an alternative selection procedure, U937 variants were derived by culturing in the presence of increasing concentrations of colchicine. The resulting variants were relatively resistant to TNF, providing further support for the existence of some common mechanism operating in induction or expression of acquired cytokine and drug resistance. The resistance mechanism apparently does not involve the P glycoprotein since the cytokine-selected U937 variants do not overexpress the mdr gene. This study has demonstrated that selection of TNF-resistant variants results in coexpression of a unique form of drug resistance that is characterized by resistance to microtubule-active drugs but not to the anthracycline antibiotics and is not associated with overexpression of the mdr gene.

Animals↗

PubMed2004

The G972R variant of the insulin receptor substrate-1 (IRS-1) gene is associated with insulin resistance in "uncomplicated" obese subjects evaluated by hyperinsulinemic-euglycemic clamp.

Several association studies have indicated the insulin receptor substrate-1 (IRS-1) gene G972R variant as a genetic risk factor for insulin resistance, particularly in presence of obesity. A few studies have also suggested a possible effect of the G972R variant on insulin secretion. The aim of this study was to evaluate the role of the IRS-1 gene G972R variant in 61 subjects with "uncomplicated" obesity [i.e. without diabetes, hypertension, dyslipidemia, coronary artery disease (CAD)], studied by hyperinsulinemic-euglycemic clamp. The presence of the G972R variant, detected in real-time with LightCycler hybridisation probes, was related to the indexes of insulin sensitivity. Furthermore, the possible role of this variant on insulin secretion was studied by means of insulin release indexes derived from oral tolerance test (OGTT). Twenty-four point five percent (24.5%) (no.=15) of the obese subjects proved to be carriers of the G972R variant. M index (p<0.05), non-oxidative glucose (p<0.01), insulin clearance (p<0.03) and insulin sensitivity index (ISI) (p<0.005) were all significantly reduced in G972R carriers compared to non-carriers, indicating a significant reduction in insulin sensitivity in carriers of the variant. A logistic regression analysis confirmed the independent association between the G972R variant and reduced insulin sensitivity (p<0.03). The interaction between obesity and the G972R variant was also independently associated with a reduced insulin sensitivity (p<0.005), suggesting that obesity and G972R variant were more than additive in predicting insulin resistance. The analysis of insulin release indexes did not show any significant differences. Our results demonstrate the association of the G972R variant of the IRS-1 gene with reduced insulin sensitivity in obese subjects, and indicate a possible interaction between the IRS-1 variant and obesity in worsening of insulin sensitivity.

Adult↗

PubMed1990

The effect of chemotherapy on the in vivo frequency of glycophorin A 'null' variant erythrocytes.

A human in vivo somatic cell assay based on the enumeration of variant erythrocytes lacking expression of an allelic form of the cell-surface sialoglycoprotein, glycophorin A, was applied to the study of blood samples from patients obtained prior to, during, and following chemotherapy for malignant disease in order to determine the effect of mutagenic chemical agents on the frequency of variant cells. In 22 patients assayed prior to therapy, the mean variant cell frequency was 11.9 per million, which was not significantly different from that observed in healthy controls. In an initial cross-sectional survey, blood samples were obtained at various times during and after therapy from 30 patients diagnosed with a variety of malignancies who were treated with one or more known mutagenic agents including adriamycin, bleomycin, cis-platinum, cyclophosphamide, dacarbazine, etoposide, lomustine, mechlorethamine, melphalan, mitomycin C, and procarbazine. Significant elevations in the mean frequency of variant cells over pre-therapy and normal levels were observed in samples obtained during and after therapy. In a time-series study, 14 breast cancer patients treated with CAF (cyclophosphamide, adriamycin, 5-fluorouracil), CMF (cyclophosphamide, methotrexate, 5-fluorouracil), or VMF (vinblastine, methotrexate, 5-fluorouracil) adjuvant chemotherapy were sampled repeatedly during and after therapy. For the CAF and CMF patients an increase in the frequency of variant cells was observed with a lag in the appearance of induced variants after initiation of therapy; variant frequencies gradually increased during therapy reaching a maximum at or shortly after the end of therapy, then declined to near pre-therapy levels within 6 months. The maximum level of induced variants ranged from 2- to 7-fold over pre-therapy or normal levels depending on the combination of agents used. The breast cancer patients treated with both adriamycin and cyclophosphamide showed consistent elevations in the frequency of variant cells; patients treated only with cyclophosphamide showed lower and more variable elevations. The data demonstrate that mutagenic chemotherapy agents induce elevated levels of glycophorin A variant erythrocytes consistent with the hypothesis that variant cells result from somatic mutation. The elevations in variant cells were transient, suggesting that these agents primarily affect the rapidly cycling committed erythroid cell population.

Adolescent↗

PubMed2005

Identification of a dysfunctional missense single nucleotide variant of human adenylyl cyclase VI.

Genetic variants have been described for a range of G protein-coupled receptors (as well as for G proteins) linked to adenylyl cyclase. Furthermore, expression of these variants resulted in alterations in receptor-mediated activation of adenylyl cyclase, as well as alterations in more "downstream" effector pathways mediated by cyclic adenosine monophosphate. However, the identification of dysfunctional variants of adenylyl cyclase has been far more limited. Screening a region of the molecule that we recently demonstrated to be critical in regulation of enzyme activity, we have identified a missense single-nucleotide variant at amino acid 674 of human adenylyl cyclase isoform VI. In a population of 286 healthy white subjects, this variant has an allelic frequency of 3.1% (although 0/90 nonwhite subjects had this variant). Expression of this variant of adenylyl cyclase VI (whether expressed as the S674 human adenylyl cyclase VI [ADCY6] or the S686 ADCY6 rat analog) is characterized by a significant decrease in stimulated adenylyl cyclase activity (forskolin-stimulated activity of the S674 human ADCY6 variant was decreased to 56% +/- 6% of the activity of the A674 variant [mean +/- SEM]; n = 9; P = .004). Furthermore, subjects with the S674 variant demonstrated a significantly higher lymphocyte count (2.68 +/- 4.13 x 10(3)/mm3 versus 1.90 +/- 0.72 x 10(3)/mm3, P = .019). Paralleling this phenotype, expression of the variant was associated with attenuation of the forskolin-mediated reduction in cell growth rate to 64% +/- 5% of the effect seen with expression of the wild-type ADCY6 (n = 4; P = .001). In summary, these data demonstrate an unappreciated variant of adenylyl cyclase isoform VI that has a functional impact on both enzyme activity and cyclic adenosine monophosphate-mediated regulation of cell growth.

Adenylyl Cyclases↗

PubMed2005

Variants of the 5'-untranslated region of human NCF2: expression and translational efficiency.

The NCF2 gene encodes p67(phox), an essential component of the multi-protein NADPH oxidase enzyme in phagocytic leukocytes, as well as in certain non-phagocytic cells. In humans, the NCF2 gene is expressed as multiple NCF2 variants that differ in the 5'-untranslated region (5'-UTR). Previously, we reported the presence of four NCF2 5'-UTR mRNA variants (designated as NCF2 exon 1, intron 1a, intron 1b and intron 1c). As each of the gene variants encodes an identical p67(phox) protein, the functional significance of these message variants was not apparent. In this study, we investigated the relative expression levels and tissue-specificity of NCF2 5'-UTR variant mRNAs and their translation efficiency and stability. NCF2 5'-UTR variant transcripts were differentially expressed in various cell lines and human tissues. In vitro translation assays indicated that the NCF2 5'-UTR variants also differed in their effects on the translation of a luciferase reporter mRNA and NCF2 mRNA. Notably, NCF2 intron 1 5'-UTR variants, which are the predominantly expressed variants found in vivo, strongly inhibited translation when compared to the NCF2 exon 1 5'-UTR variant. In contrast, RNA decay assays demonstrated that there was no significant difference between stability of NCF2 intron 1 transcripts and the exon 1 5'-UTR variant in HL-60, MonoMac 6, and U937 cells. Moreover, expression of the variant transcripts remained unchanged after neutrophil phagocytosis, and was similar in normal neutrophils and neutrophils from a patient with X-linked chronic granulomatous disease. These studies suggest that expression of p67(phox) is regulated through mechanisms that include modulation of transcription and translation.

5' Untranslated Regions↗

PubMed2001

Quantitative mRNA analysis of five C-terminal splice variants of the human 5-HT4 receptor in the central nervous system by TaqMan real time RT-PCR.

5-HT4 receptors mediate several physiological effects of 5-HT, particularly in the central nervous system (CNS), heart and gut. Recently, several C-terminal splice variants of the human 5-HT4 (h5-HT4) receptor have been described, namely h5-HT4(a), h5-HT4(b), h5-HT4(c), h5-HT4(d) and h5-HT4(g). Previous tissue distribution data suggest some degree of specificity in the mRNA expression patterns of the different h5-HT4 receptor splice variants. However, comparison of the mRNA expression profiles of these splice variants is difficult due to the non-quantitative methods used, and in addition, there is very limited data on the expression of each splice variant in human CNS subregions. In the present study we used a single technique, TaqMan real time quantitative RT-PCR, to investigate the mRNA distribution of 5-HT4 receptor C-terminal splice variants in multiple human CNS and peripheral tissues. Using a primer/probe set that amplified all 5-HT4 splice variants (5-HT4pan), the highest CNS expression of 5-HT4 receptor mRNA was observed in basal ganglia, amygdala and hippocampus, consistent with previous studies. h5-HT4(a), h5-HT4(b), h5-HT4(c) and h5-HT4(g) were predominantly expressed in various CNS tissues, compared to most peripheral tissues, but there were differences in expression levels and distribution patterns of each variant. The distribution profile and expression levels observed for the 5-HT4(b) splice variant were virtually identical to that obtained with the 5-HT4pan primer/probe set, whilst the other splice variants were expressed at much lower levels and with different expression patterns obtained with both 5-HT4(b) and 5-HT4pan primer/probe sets. Highest levels of 5-HT4(g) were observed in the hypothalamus and cortex, whilst the 5-HT4(a) variant was highest in the amygdala. 5-HT4(c) expression was highest in the pituitary gland whilst 5-HT4(d) mRNA was only detected in the small intestine at very low levels and not in the CNS. In conclusion, we have shown quantitative differences in the mRNA distribution profiles of the 5-HT4 receptor C-terminal splice variants in human CNS subregions as well as peripheral tissues. In addition, our data suggests that the h5-HT4(b) variant is the most predominant form of the 5-HT4 receptor in humans.

Alternative Splicing↗