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Purifying selection of CCR5-tropic human immunodeficiency virus type 1 variants in AIDS subjects that have developed syncytium-inducing, CXCR4-tropic viruses.

Human immunodeficiency virus type 1 (HIV-1) infection is established by virus variants that use the CCR5 co-receptor for entry (CCR5-tropic or R5 variants), whereas viruses that use CXCR4 as co-receptor (CXCR4-tropic or X4 variants) emerge during disease progression in approximately 50 % of infected subjects. X4 variants may have a higher fitness ex vivo and their detection is usually accompanied by faster T-cell depletion and the onset of AIDS in HIV-1-positive individuals. Here, the relationship between the sequence variation of the HIV-1 env V3-V5 region and positive selective pressure on R5 and X4 variants from infected subjects with CD4 T cell counts below 200 cells microl(-1) was studied. A correlation was found between genetic distance and CD4(+) cell count at late stages of the disease. R5 variants that co-existed with X4 variants were significantly less heterogeneous than R5 variants from subjects without X4 variants (P < 0.0001). Similarly, X4 variants had a significantly higher diversity than R5 variants (P < 0.0001), although residues under positive selection had a similar distribution pattern in both variants. Therefore, both X4 and R5 variants were subjected to high selective pressures from the host. Furthermore, the interaction between X4 and R5 variants within the same subject resulted in a purifying selection on R5 variants, which only survived as a homogeneous virus population. These results indicate that R5 variants from X4 phenotype samples were highly homogeneous and under weakly positive selective pressures. In contrast, R5 variants from R5 phenotype samples were highly heterogeneous and subject to positive selective pressures.

Acquired Immunodeficiency Syndrome↗

Frequency of variants in Mendelian Alzheimer's disease genes within the Alzheimer's Disease Sequencing Project.

BackgroundPrior studies examined variants within presenilin-2 (PSEN2), presenilin-1 (PSEN1), and amyloid precursor protein (APP) genes. However, previously-reported clinically-relevant variants and other predicted damaging missense (DM) variants have not been characterized in a newer release of the Alzheimer's Disease Sequencing Project (ADSP).ObjectiveTo characterize previously-reported clinically-relevant variants and DM variants in PSEN2, PSEN1, APP within the participants from the ADSP.MethodsWe identified rare variants (MAF&#x2009;<&#x2009;1%) in PSEN2, PSEN1, and APP in 14,641 individuals with whole genome sequencing and 16,849 individuals with whole exome sequencing available (Ntotal&#x2009;=&#x2009;31,490). We additionally curated variants from ClinVar, OMIM, and Alzforum and report carriers of variants in clinical databases as well as predicted DM variants in these genes.ResultsWe detected 31 previously-reported clinically-relevant variants with alternate alleles observed within the ADSP: 4 variants in PSEN2, 25 in PSEN1, and 2 in APP. The overall variant carrier rate for the 31 clinically-relevant variants in the ADSP was 0.3%. We observed that 79.5% of the variant carriers were cases compared to 3.9% were controls. In those with AD, the mean age of onset of AD among carriers of these clinically-relevant variants was 19.6&#x2009;&#xb1;&#x2009;1.4 years earlier compared with noncarriers (p&#x2009;=&#x2009;7.8&#x2009;&#xd7;&#x2009;10-57). Additionally, we identified 197 rare variants (MAF&#x2009;<&#x2009;1%) within ADSP participants not reported in known clinical databases.ConclusionsA small proportion of individuals in the ADSP are carriers of a previously-reported clinically-relevant variant allele for AD and these participants have significantly earlier age of AD onset compared to noncarriers.

Humans↗

Systematic functional evaluation of CNGA1 missense variants associated with retinitis pigmentosa.

BACKGROUND: Missense variants are frequently classified as variants of uncertain significance (VUS) according to the guidelines of the American College of Medical Genetics and Genomics and the Association of Molecular Pathology (ACMG/AMP). Consequently, disease relevance remains elusive, impeding molecular genetic diagnostics, patients` and family genetic counseling, and identification of patients eligible for clinical trials. Functional studies are critical for resolving the clinical significance of VUS. CNGA1 encodes the main subunit of the rod cyclic nucleotide-gated (CNG) channel, a vital component of the phototransduction cascade. Variants in CNGA1 are a rare cause of autosomal recessive retinitis pigmentosa and a phase I/II gene augmentation trial (NCT06291935) is currently ongoing highlighting the necessity to differentiate benign from pathogenic variants. METHODS: CNGA1 missense variants compiled from retinal disease patient cohorts, public databases and literature were functionally investigated using a medium-throughput aequorin-based assay and in vitro minigene splice assays for predicted exonic spliceogenic variants. Functional data were correlated with the in silico prediction of five variant effect predictors (VEPs) and applied to support or revise variants' ACMG/AMP classification. RESULTS: Data mining revealed 86 missense CNGA1 variants - including three novel - most of them lacking functional data; 65.1% of the variants were initially classified as VUS. The aequorin-based assay showed that 72.1% of tested variants significantly impaired CNG channel function and were classified as functionally abnormal, while 23.3% were functionally normal and 5% remained functionally uncertain. Correlation of the functional data with in silico predictions identified AlphaMissense and CPT-1 to be the most suitable tools for assessing CNGA1 missense variants. Using in vitro minigene splice assays, two putative missense variants were shown to induce missplicing. Based on the functional findings, 62.1% of the variants initially classified as VUS were re-categorized as likely pathogenic or likely benign. Furthermore, 93.3% of the variants initially classified as likely pathogenic showed an effect on CNGA1 channel function, confirming their disease relevance and supporting their reclassification as pathogenic. CONCLUSION: This study represents the first comprehensive functional assessment of disease-associated CNGA1 missense variants, thus significantly advancing the understanding of their disease relevance and improving molecular genetic diagnostics in patients.

Humans↗

Differential impacts of exon 1-associated and exon 11-associated variants of the rat mu opioid receptor gene, Oprm1, on buprenorphine- and morphine-induced analgesia and respiratory depression in male rats.

Buprenorphine has long been recognized as a mu opioid agonist with a distinctive and intricate pharmacological profile. It is a partial agonist at the mu opioid receptor, an antagonist at the kappa and delta opioid receptors, and an agonist at the nociception opioid receptor. Similar to other mu agonists such as morphine and fentanyl, buprenorphine can produce side effects, including tolerance, physical dependence, respiratory depression, and addiction. The mu opioid receptor gene, OPRM1, undergoes extensive alternative splicing, generating an array of splice variants or isoforms, which are conserved from rodents to humans. These splice variants can be categorized into 2 main types, exon 1 (E1)-associated variants and exon 11 (E11)-associated variants. E1-associated variants primarily consist of full-length, 7-transmembrane C-terminal variants, whereas E11-associated variants are typically truncated 6-transmembrane variants. Previous studies established that buprenorphine analgesia in mice is dependent on both E1- and E11-associated variants. However, the role of these variants in buprenorphine analgesia and respiratory depression in rats remains unclear. In this study, we used CRISPR/Cas9 technology to develop 2 rat Oprm1 gene-targeting models in which E1- and E11-associated variants were selectively disrupted, aiming to investigate their roles in buprenorphine and morphine's actions. The results showed that both E1- and E11-associated variants are essential for buprenorphine's analgesic and respiratory depressional effects in rats, whereas morphine's effects are solely attributed to the E1-associated variants. These findings provide new and important insights into the distinct contributions of the E1- and E11-associated variants to the pharmacological actions of buprenorphine and morphine. SIGNIFICANCE STATEMENT: Differential dependences of buprenorphine and morphine analgesia and respiratory depression on Oprm1 exon 1- and exon 11-associated variants revealed in rat gene-targeting models provide new and important insights into unique contributions of these variants to buprenorphine and morphine actions.

Animals↗

The mechanism of antigenic drift in influenza viruses: analysis of Hong Kong (H3N2) variants with monoclonal antibodies to the hemagglutinin molecule.

Monoclonal antibodies to the hemagglutinin molecule of the Hong Kong variant, Mem/1/71 (H3N2), influenza virus were used to study antigenic drift in the H3N2 subtype of influenza viruses. Antigenic variants of Mem/1/71 (H3N2) were selected after a single passage of the virus in chick embryos in the presence of monoclonal antibody. The variants showed a marked reduction in the ability to react with the monoclonal antibody used in selection. The monoclonal antibodies could be divided into three groups based on their reactions with the variants, providing evidence for at least three nonoverlapping antigenic areas on the hemagglutinin molecule. Amino acid analysis of tryptic peptides of the hemagglutinin from these variants showed that a single amino acid substitution in the heavy polypeptide chain (HA1) of the hemagglutinin molecule accounted for the reduced antibody interactions, and that variants from each group exhibited sequence changes in different areas of the molecule. Sequence changes were also detected in the HA1 polypeptides of naturally occurring H3N2 variants, but in most cases the changes in the monoclonal antibody selected variants were different from the field strains. Antigenic analysis showed that most of the variants selected with monoclonal antibody could not be distinguished from parental viruses with heterogeneous sera, suggesting that they are probably epidemiologically irrelevant. One variant, however, could be distinguished from parental virus with heterogeneous sera. This variant showed a change in sequence at residue 144 of the HA1 polypeptide from glycine in the parent to aspartic acid in the variant. Similar substitutions have been found in naturally occurring variants at this position. These studies suggest that some amino acid substitutions are more important than other for producing viruses with epidemiological potential. Antigenic analysis of naturally occurring H3N2 strains with monoclonal antibodies established that two variants co-circulated in 1968; Hong Kong/1/68 being distinguishable from Aichi/2/68 in at least two antigenic areas. It would appear that there may have been two separate lineages of H3N2 viruses, Hong Kong/1/68 giving rise to variants in England and Aichi/2/68 to variants in USA and Australia.

Amino Acid Sequence↗

RBM20 Truncating Variants and Human Cardiomyopathy.

IMPORTANCE: Genetic diagnosis has become increasingly important to guide clinical decision-making for patients with dilated cardiomyopathy (DCM). Pathogenic or likely pathogenic (P/LP) missense variants in the gene RBM20 cause a highly penetrant arrhythmogenic DCM, but the role of RBM20 truncating variants (RBM20tvs) is unclear. OBJECTIVE: To assess the contribution of RBM20 variants to arrhythmogenic DCM. DESIGN, SETTING, AND PARTICIPANTS: In this cohort study, participants in the genome-first UK Biobank (UKB) and All of Us populations were evaluated to assess the etiologic fraction, natural history and penetrance of RBM20 variants. Retrospective data were collected from an international cohort of patients with DCM and RBM20 variants identified at centers of excellence for genetic heart disease and compared based on time to event. Study dates are not disclosed because the institutional review board did not authorize the sharing of this information. EXPOSURES: RBM20 variants were compared to known P/LP variants and variants of uncertain significance in RBM20 as well as titin truncating variants (TTNtvs). MAIN OUTCOMES AND MEASURES: Major ventricular arrhythmias, end-stage heart failure, and heart failure hospitalization as measured by medical record review (retrospective cohort) and diagnostic codes (UKB). RESULTS: Two main cohorts were studied for this project. In UK Biobank, a cohort of participants with RBM20tvs, RBM20 synonymous variants, and TTNtvs was studied. Of these 4249 participants, 1869 (44%) were male. The mean (SD) age at enrollment was 56 (8.2) years. In the RBM20 registry, of 179 patients, 105 (58.6%) were male, and the mean (SD) age at enrollment was 43.8 (19.1) years. A validation cohort from the All of Us biobank was also used. This consisted of 7002 participants, 4342 of whom (62.0%) were male, and the mean (SD) age was 52.7 (16.7) years. The etiologic fraction of RBM20 variants in arrhythmogenic DCM was 0.53 (95% CI, 0.32-0.67; P&#x2009;<&#x2009;.001). In genome-first biobanks, lifetime incidence of cardiomyopathy, heart failure, or major ventricular arrhythmia diagnosis was lower in participants with RBM20 variants than in those with TTNtvs (hazard ratio, 0.55; 95% CI, 0.36-0.84; P&#x2009;<&#x2009;.001). Patients with RBM20tvs and DCM presented to referral centers later in life than those with P/LP RBM20 and DCM (mean [SD], 53 [10] vs 34 [18] years; P&#x2009;<&#x2009;.001) and were less likely to have a family history of sudden cardiac arrest (2 of 10 [20%] vs 11 of 17 [65%]; P&#x2009;=&#x2009;.046) or cardiomyopathy (2 of 10 [20%] vs 14 of 18 [78%]; P&#x2009;<&#x2009;.001). There was no significant difference in age- and sex-adjusted incident major heart failure or arrhythmia events between patients with RBM20tv and DCM or those with P/LP RBM20 and DCM, though sex-adjusted lifetime hazard was reduced in those with RBM20tv and DCM (hazard ratio, 0.13; 95% CI, 0.03-0.56; P&#x2009;=&#x2009;.01). CONCLUSIONS AND RELEVANCE: This study found that RBM20 variants contributed to arrhythmogenic DCM phenotypes but conferred reduced lifetime disease penetrance compared to TTNtvs and milder disease severity alone than P/LP RBM20 variants. Their potential for additive interactions with other damaging variants should be considered in patients with DCM and their families.

Humans↗

Response to mivacurium in patients carrying the k variant in the butyrylcholinesterase gene.

BACKGROUND: Mivacurium is hydrolyzed by the butyrylcholinesterase enzyme, and patients with hereditary changes of the enzyme often have prolonged duration of action of mivacurium. In this study, the authors investigated the significance of the most commonly occurring variant, the Kalow (K) variant, established using DNA analysis, for the response to mivacurium. METHODS: A total of 58 patients carrying either the wild-type butyrylcholinesterase or different combinations of the atypical (A) variant and the K variant were included. Patients who were homozygous for the A variant were given 0.03 mg/kg mivacurium. All other patients received 0.2 mg/kg mivacurium. The neuromuscular block was measured using train-of-four nerve stimulation and mechanomyography. Genotyping was performed with complete nucleotide sequencing. RESULTS: Heterozygosity of the K variant prolonged the time to train-of-four 0.70 from 26.6 to 34.5 min (30%; not significant) as compared with the wild type. Heterozygosity of the K variant linked to the A variant prolonged the corresponding time from 32 to 42.7 min (33%; P = 0.03) as compared with patients who were heterozygous for solely an A allele. For eight patients who were homozygous for both the A and K variants, the time to 25% recovery was 78-89 min as compared with 44-57 min in patients who were homozygous for the A variant or had only one linked K variant. CONCLUSION: The K variant prolongs the duration of action of mivacurium. The current results indicate that the effect is modest when the K variant occurs heterozygously with the wild type or the A variant but is marked in patients who are homozygous for both the A and K variants.

Adult↗

Effects of ethnicity on the distribution of clinically relevant endothelial nitric oxide variants.

Polymorphisms in the endothelial nitric oxide synthase (eNOS) gene have been associated inconsistently with cardiovascular diseases. A maldistribution of eNOS variants among ethnic groups may explain interethnic differences in nitric oxide (NO)-mediated vasodilation and response to drugs. To test this possibility, we examined the distribution of genetic variants of three clinically relevant eNOS polymorphisms (T-786C in the promoter, the variable number of tandem repeats (VNTR) in intron 4, and the Glu298Asp variant in exon 7) in 305 ethnically well-characterized DNA samples (100 Caucasians, 100 African-Americans, and 105 Asians). We estimated the haplotype frequency, and evaluated associations between these variants. The Asp298 variant was more common in Caucasians (34.5%) than in African-Americans (15.5%) or Asians (8.6%)(P < 0.0001). The C-786 variant was also more common in Caucasians (42.0%) than in African-Americans (17.5%) or Asians (13.8%) (P < 0.0001). The 4a variant in intron 4 was more common in African-Americans (26.5%) than in Caucasians (16.0%) or Asians (12.9%) (P < 0.0001). The most common predicted haplotype in the three groups combined only wild-type variants. Asians had the highest frequency of this haplotype (77% in Asians v. 46% in the other groups). In Caucasians, the Asp298 and C-786variants were associated, and this haplotype was predicted to have a frequency of 24%. In African-Americans, the second most common haplotype included the variant 4a and wild-type variants; the Asp298 and 4a variants were associated negatively in this group. The C-786 and 4a variants were associated in Asians (P < 0.0001). The marked interethnic differences that we found in the distribution of eNOS variants, in the estimated haplotype frequency, and in the association between variants may help us to understand how the combination of these genetic variants may influence cardiovascular diseases.

Alleles↗

No phenotypic resistance observed for most group-3 and -4 variants in Mycobacterium tuberculosis genes related to bedaquiline, clofazimine, delamanid, and pretomanid in a Central and West African context.

The interpretation of genetic variants' association (or not) with phenotypic resistance to newly introduced and repurposed antituberculosis drugs remains challenging, as many mutations detected by whole-genome sequencing (WGS) are classified as of uncertain significance (group 3) or not associated with resistance-interim (group 4) by the World Health Organization (WHO) mutation catalog v2. We evaluated the phenotypic impact of such variants on minimum inhibitory concentrations (MICs) for bedaquiline (BDQ), clofazimine (CFZ), delamanid (DLM), and pretomanid (PA) in Mycobacterium tuberculosis complex isolates from the multi-country DIAMA cohort in sub-Saharan Africa (SSA), which recruited RR/RS-TB patients na&#xef;ve to these drugs. Among 1,475 isolates with available WGS data, 163 variants met eligibility criteria; due to viable strain unavailability, 89 isolates carrying 29 unique BDQ/CFZ-related and 60 unique DLM/PA-related variants were tested for MIC determination using broth microdilution. Additional structural modeling was performed to explore potential effects of amino-acid substitutions on protein stability. Among BDQ/CFZ-related variants, MICs above the critical concentrations (CCs) were consistently associated with mmpR5 variants, whereas variants in atpE, pepQ, and Rv1979c were not. DLM/PA variants (ddn, fbiA-D, and fgd1) were frequently detected as non-fixed populations, yet rarely yielding MIC values above the CC. Predicted structural destabilization showed no consistent association with MIC values or variant fixation status. Under the conditions tested, phenotypic resistance was not detected for most group 3 and 4 variants detected by WGS. Our data provide evidence from SSA to support improved interpretation of resistance-associated mutations for new and repurposed antituberculosis drugs.IMPORTANCEWhole-genome sequencing increasingly detects Mycobacterium tuberculosis complex mutations classified by the World Health Organization (WHO) mutation catalog v2 as group 3 variants of uncertain significance or group 4 variants not associated with resistance-interim, limiting reliable prediction of resistance to new and repurposed antituberculosis drugs. By generating minimum inhibitory concentration (MIC) data for such variants identified in a multi-country sub-Saharan African cohort, this study provides phenotypic evidence to support future refinement and expansion of the WHO mutation catalog v2. Notably, mmpR5 variants associated with elevated bedaquiline/clofazimine MICs were identified in eight isolates, suggesting that some patients in this cohort may have harbored pre-existing resistance-associated variants yet remained potentially eligible for bedaquiline-containing regimens. These findings contribute to improving the interpretation of genomic resistance data and strengthening surveillance of resistance to bedaquiline, clofazimine, delamanid, and pretomanid.

Mycobacterium tuberculosis↗

The macrophage scavenger receptor CD163: endocytic properties of cytoplasmic tail variants.

CD163 is the monocyte/macrophage-specific receptor for haptoglobin-hemoglobin (Hp-Hb) complexes. The cytoplasmic tail of human CD163 exists as a short tail variant and two long tail variants. Reverse transcriptase-polymerase chain reaction analysis indicated that all three CD163 variants are substantially expressed in blood, liver, and spleen, and the short tail variant is the predominant mRNA species. Using cell transfectants in which cDNA encoding the CD163 variants was inserted at the same site in the genome, we evaluated the expression and endocytic properties of the tail variants. Ligand uptake analysis showed that cells expressing the CD163 short tail variant exhibited a higher capacity for ligand endocytosis than cells expressing the CD163 long tail variants. The difference in endocytic activity was explained by confocal microscopic analysis, showing marked deviations in subcellular distribution. Surface expression was far most pronounced for the CD163 short tail variant, whereas the long tail variants were most abundant in the Golgi region/endosomes. Mutational change of a putative signal for endocytosis (Tyr-Arg-Glu-Met), present in a common part of the cytoplasmic tail of the variants, almost completely inactivated the endocytic activity of the short tail variant. In conclusion, the three physiological tail variants of CD163 may contribute to Hp-Hb endocytosis by means of the common ligand-binding region and endocytic signal. However, the high mRNA expression level and relatively high endocytic capacity of the short tail variant suggest that it accounts for the majority of Hp-Hb uptake from the circulation, whereas the long tail variants may have yet-unknown intracellular roles.

Amino Acid Sequence↗

HPLC retention time as a diagnostic tool for hemoglobin variants and hemoglobinopathies: a study of 60000 samples in a clinical diagnostic laboratory.

BACKGROUND: Previous evaluations of HPLC as a tool for detection of hemoglobin variants have done so within newborn-screening programs and/or by use of stored samples. We describe a 32-month prospective study in a clinical diagnostic laboratory in which we evaluated the imprecision of HPLC retention times and determined the retention times for hemoglobin variants seen in a multiethnic setting. METHODS: We analyzed all samples on the Bio-Rad Variant II HPLC system. For normal hemoglobin fractions and hemoglobin variants, we recorded and analyzed their retention times, their proportion of the total hemoglobin (%), and the peak characteristics. We compared the imprecision of retention time with the imprecision of retention time normalized to the retention time of hemoglobin A0 (Hb A0) and to the retention time of Hb A2. Alkaline and acid hemoglobin electrophoresis, and in certain cases globin chain electrophoresis, isoelectric focusing, and DNA analysis, were performed to document the identities of the hemoglobin variants. RESULTS: The mean (SD) imprecision (CV) of the retention time was 1.0 (0.7)% with no statistical difference compared with the imprecision for normalized retention times. Among 60,293 samples tested, we encountered 34 unique hemoglobin variants and 2 tetramers. Eighteen variants and 2 tetramers could be identified solely by retention time and 3 variants by retention time and proportion of total hemoglobin. Four variants could be identified by retention time and peak characteristics and eight variants by retention time and electrophoretic mobility. One variant (Hb New York) was missed on HPLC. Retention time on HPLC was superior to electrophoresis for the differentiation and identification of six members of the Hb J family, four members of the Hb D family, and three variants with electrophoretic mobilities identical or similar to that of Hb C. Six variants with electrophoretic mobilities identical or similar to that of Hb S could be differentiated and identified by retention time and proportion of total hemoglobin. HPLC detected two variants (Hb Ty Gard and Hb Twin Peaks) missed on electrophoresis. CONCLUSIONS: The retention time on HPLC is reliable, reproducible, and in many cases superior to conventional hemoglobin electrophoresis for the detection and identification of hemoglobin variants. Confirmatory testing by electrophoresis can be eliminated in the majority of cases by use of retention time, proportion of total hemoglobin, and peak characteristics of HPLC.

Chromatography, High Pressure Liquid↗

Role of common sequence variants in insulin secretion in familial type 2 diabetic kindreds: the sulfonylurea receptor, glucokinase, and hepatocyte nuclear factor 1alpha genes.

OBJECTIVE: We have demonstrated high heritability of insulin secretion measured as acute insulin response to glucose times insulin sensitivity (disposition index). Furthermore, we showed that obese normoglycemic family members of a type 2 diabetic proband failed to compensate for the insulin resistance of obesity by increasing insulin secretion. In this study, we tested the primary hypotheses that previously described variants in the pancreatic sulfonylurea receptor gene (SUR1 or ABCC8), glucokinase (GCK) gene, or hepatocyte nuclear factor 1alpha (TCF1 or HNF1alpha) gene contribute to the inherited deficiencies of insulin secretion and beta-cell compensation to insulin resistance, as well as the secondary hypotheses that these variants altered insulin sensitivity. RESEARCH DESIGN AND METHODS: We typed 124 nondiabetic members of 26 familial type 2 diabetic kindreds who had undergone tolbutamide-modified intravenous glucose tolerance tests for two variants of the ABCC8 (sulfonylurea) gene, two variants of the GCK gene, and one common amino acid variant in the TCF1 (HNF1alpha) gene. All family members were classified as normal or having impaired glucose tolerance based on oral glucose tolerance testing. We used minimal model analysis to calculate the insulin sensitivity index (S1) and glucose effectiveness (SG), and acute insulin response to glucose was calculated as the mean insulin excursion above baseline during the first 10 min after the glucose bolus. Disposition index (DI), a measure of beta-cell compensation for insulin sensitivity, was calculated as insulin sensitivity times acute insulin response. Effects of polymorphisms were determined using mixed effects models that incorporated family membership and by a likelihood analysis that accounted for family structure through polygenic inheritance. RESULTS: An intronic variant of the ABCC8 gene just upstream of exon 16 was a significant determinant of both DI and an analogous index based on acute insulin response to tolbutamide. Surprisingly, heterozygous individuals showed the lowest indexes, whereas the DI in the two homozygous states did not differ significantly. Neither the exon 18 variant nor the variants in the GCK and TCF1 genes were significant in this model. However, combined genotypes of ABCC8 exon 16 and 18 variants again significantly predicted both indexes of glucose and tolbutamide-stimulated insulin secretion. Unexpectedly, a variant in the 3' untranslated region of the GCK gene interacted significantly with BMI to predict insulin sensitivity. CONCLUSIONS: The exon 16 variant of the ABCC8 gene reduced beta-cell compensation to the decreased insulin sensitivity in the heterozygous state. This may explain the observation from several groups of an association of the ABCC8 variants in diabetes and is consistent with other studies showing a role of ABCC8 variants in pancreatic beta-cell function. However, our study focused on individuals from relatively few families. Ascertainment bias, family structure, and other interacting genes might have influenced our unexpected result. Additional studies are needed to replicate our observed deficit in beta-cell compensation in individuals heterozygous for ABCC8 variants. Likewise, the role of the GCK 3' variant in the reduced insulin sensitivity of obesity will require further study.

ATP-Binding Cassette Transporters↗

Different phenotypic variants of the mouse B cell tumor A20/2J are selected by antigen- and mitogen-triggered cytotoxicity of L3T4-positive, I-A-restricted T cell clones.

The L3T4+, Lyt-2-, cloned BALB/c T cell lines 5.9.24 and 5.8.6 are cytotoxic for the BALB/c B cell tumor line A20/2J. The T cell cytotoxicity against A20/2J cells could be triggered either by the specific antigen ovalbumin (OVA), which is recognized by the T cell clones in association with I-Ad determinants, or by the T cell mitogens Con A and rabbit anti-mouse brain (RaMBr) antiserum. Repeated exposure of A20/2J cells to 5.9.24 and 5.8.6 T cell cytotoxicity selected variant cell lines that had developed resistance to cytotoxicity. The variant lines could be classified into four different variant phenotypes of which three were stably maintained in vitro. The type of variant obtained appeared to be related to the nature of the ligand used to trigger T cell cytotoxicity during selection. Cytotoxicity triggered by the antigen OVA generated type 1 variants that expressed abnormally low levels of I-Ad determinants at the cell surface. Type 1 variants were resistant to OVA-triggered 5.9.24 T cell cytotoxicity, but were fully susceptible to cytotoxicity triggered by Con A or RaMBr antiserum. RaMBr-triggered cytotoxicity generated two unique types of variant cell lines: type 3 variants that were deficient in cell surface Fc receptors and resistant to 5.9.24 cytotoxicity only when triggered by RaMBr antiserum, and type 4 variants that were resistant to cytotoxicity triggered by all three ligands. One type 4 variant, the IC-1 cell line, appeared to be resistant to soluble cytotoxic factors released by 5.9.24 T cells after activation by antigen. All of these variant lines retained sensitivity to cytotoxicity by classic Lyt-2+ cytotoxic T lymphocytes (CTL), a finding that indicates that L3T4a+ T cells and Lyt-2+ CTL use different molecules to attack their target cells. The variant phenotypes were inherited by clones derived from the original cell lines. Because the variants were generated without mutagenesis, they are thought to have been derived by the immunoselection of pre-existing variant cells that arose spontaneously in the parental A20/2J cell line. It is postulated that inheritable variation of A20/2J cells may represent changes that normally occur during B cell differentiation in response to T cell signals. The variant A20/2J cell lines described here provide material for the investigation of B cell surface structures that may regulate T-B cell interactions.

Animals↗

Identification of Rare Noncoding Variants in Familial Nonmedullary Thyroid Carcinoma.

BACKGROUND: Familial nonmedullary thyroid carcinoma (FNMTC) occurs when three or more family members are affected by usually papillary thyroid carcinoma (PTC), the most common form of NMTC. While the heritability to NMTC is among the highest of all cancers, the genetic determinants among NMTC families are not well understood. Here, we aim to understand the contribution of rare noncoding germline variants in the etiology of FNMTC. METHODS: We previously reported whole-genome sequencing (WGS) and linkage analysis in 17 PTC families and reported on 41 protein-coding variants in 40 genes that cosegregated with PTC in 11 of the families. Herein, we further leveraged our WGS data to include noncoding variants in our analysis for all 17 families. We hypothesized that most of the pathogenic noncoding variants would be located in theoretical or empirically determined regulatory regions that demonstrate at a minimum, basal thyroid expression, a positive family linkage score, and co-segregation among PTC-affected individuals. To test this hypothesis, we adopted a unique filtering strategy to identify variants that occurred in known DNA elements and transcription factor binding sites, near regions known to impact on gene expression or splicing in thyroid tissue, and/or in characterized thyroid enhancers. We annotated variants using two analyses (ENCODE and transcription factor binding site) within the BasePlayer software. We separately analyzed (1) expression quantitative trait loci, (2) splicing quantitative trait loci, and (3) thyroid enhancers. We then ranked variants according to predicted pathogenicity and performed Sanger sequencing in all individuals of each family. RESULTS: In total, 121 variants were selected based on in-silico prediction and our custom ranking analysis in each pedigree. Of these, 56 variants showed cosegregation among all PTC-affected individuals and were absent from unaffected individuals. This included candidate variants from five of the six PTC families for whom no protein-coding variants were previously found. CONCLUSION: Our data suggest that noncoding variants are important in the etiology of FNMTC and provide a framework for identifying noncoding germline variants using a novel approach. Further studies are needed to functionally characterize these variants to better understand the molecular mechanism of their pathogenicity.

Humans↗

Multiple isoelectric variants of copper, zinc-superoxide dismutase from rat liver.

Isoelectric variants of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) have been reported to exist in various organs including rat liver. To elucidate the biochemical characteristics of the variants, rat liver Cu,Zn-SOD was purified and isolated into eight variants, i.e., pI 5.15, 4.88, 4.80, 4.75, 4.70, 4.65, 4.60, and 4.50. The pI 4.88 variant had the highest specific activity (4245 U/mg protein) and the highest yield (45% of original activity). The descending order of specific activity for the other variants was pI 4.80, 4.75, 5.15, 4.70, 4.65, 4.60, and 4.50. The specific activity correlated well with metal content. The specific activity for most variants was 5-9 times greater when determined at pH 10.0 than at pH 7.8. However, three preparations of pI 4.80 and 4.70 variants had 13.9-16.3 times greater specific activity at pH 10.0 versus 7.8, while one of the pI 4.60 variants was only 3.5 times greater. The rate of Coomasie brilliant blue G-250 binding was lowest with pI 4.88 followed by pIs 4.80 and 4.75. To evaluate the mechanisms which might produce these variants, the pI 4.88 variant was incubated with xanthine-xanthine oxidase or a mixture of rat liver microsome, NADPH, and sodium azide, and a shift to variants pI 4.80 and pI 4.75 was found. The shift was greatly inhibited by the presence of mannitol or by the omitting of azide, respectively. The existence of these variants was also confirmed by other methods: (i) direct application of rat liver 105,000g supernatant to an isoelectric focusing, and (ii) extraction of SOD from acetone powder prepared from rat liver homogenate. Results indicate that several variants most likely arise in tissue as a result of activated oxygen radical modification of variant pI 4.88.

Animals↗

Glycosylation study of the major genetic variants of human alpha1-acid glycoprotein and of their pharmacokinetics in the rat.

Human alpha1-acid glycoprotein (AAG) is a mixture of at least two genetic variants, the A variant and the F1 and/or S variant or variants, which are encoded by two different genes. AAG is also an extensively glycosylated protein which possesses five N-linked glycans exhibiting substantial heterogeneity in their structures. The first objective of this study was to investigate the glycosylation of the two major gene products of AAG, i.e. the A variant and a mixture of the F1 and S variants (F1*S). To this end, we combined a chromatographic method for the fractionation of the AAG variants with a lectin-binding assay to characterise the glycosylation of purified glycoproteins. Secondly, because the oligosaccharides can influence the disposition of AAG, a kinetic study of the AAG variants was carried out in the rat. After intravenous administration of whole human AAG, the separation and quantification of the AAG variants in plasma was performed by application of specific methods by isoelectric focusing and immunonephelometry. The binding studies carried out on a panel of lectins showed significant differences in the lectin-binding characteristics of the separated F1*S and A variants, accounting for differences in the degree of branching of their glycan chains and substitution with sialic acid and fucose. The plasma concentration-time profiles of the F1*S and A variants were biphasic, and only small differences were observed between the variants for their initial and terminal half-lives, clearance and distribution volume. This indicates that the structural differences between the two AAG gene products do not affect their pharmacokinetics in the rat. Specific drug transport roles have been previously demonstrated for the F1*S and A variants, calling for further investigations into their effects on the disposition of drugs they bind in plasma. The present study shows that such investigations are possible without being complicated by kinetic differences between these variants.

Animals↗

Genomic diversity of human papillomavirus-16, 18, 31, and 35 isolates in a Mexican population and relationship to European, African, and Native American variants.

Cervical cancer, mainly caused by infection with human papillomaviruses (HPVs), is a major public health problem in Mexico. During a study of the prevalence of HPV types in northeastern Mexico, we identified, as expected from worldwide comparisons, HPV-16, 18, 31, and 35 as highly prevalent. It is well known that the genomes of HPV types differ geographically because of evolution linked to ethnic groups separated in prehistoric times. As HPV intra-type variation results in pathogenic differences, we analyzed genomic sequences of Mexican variants of these four HPV types. Among 112 HPV-16 samples, 14 contained European and 98 American Indian (AA) variants. This ratio is unexpected as people of European ethnicity predominate in this part of Mexico. Among 15 HPV-18 samples, 13 contained European and 2 African variants, the latter possibly due to migration of Africans to the Caribbean coast of Mexico. We constructed phylogenetic trees of HPV-31 and 35 variants, which have never been studied. Forty-six HPV-31 isolates from Mexico, Europe, Africa, and the United States (US) contained a total of 35 nucleotide exchanges in a 428-bp segment, with maximal distances between any two variants of 16 bp (3.7%), similar to those between HPV-16 variants. The HPV-31 variants formed two branches, one apparently the European, the other one an African branch. The European branch contained 13 of 29 Mexican isolates, the African branch 16 Mexican isolates. These may represent the HPV-31 variants of American Indians, as a 55% prevalence of African variants in Mexico seems incomprehensible. Twenty-seven HPV-35 samples from Mexico, Europe, Africa, and the US contained 11 mutations in a 893-bp segment with maximal distances between any two variants of only 5 mutations (0.6%), including a characteristic 16-bp insertion/deletion. These HPV-35 variants formed several phylogenetic clusters rather than two- or three-branched trees as HPV-16, 18, and 31. An HPV-35 variant typical for American Indians was not identifiable. Our research suggests type specific patterns of evolution and spread of HPV-16, 18, 31, and 35 both before and after the worldwide migrations of the last four centuries. The high prevalence of highly carcinogenic HPV-16 AA variants, and the extensive diversity of HPV-18, 31, and 35 variants with unknown pathogenic properties raise the possibility that HPV intra-type variation contributes to the high cervical cancer burden in Mexico.

Cohort Studies↗

Rapid detection of lamivudine-resistant hepatitis B virus polymerase gene variants.

The amino acid substitution from methionine to valine or isoleucine at the YMDD (tyrosine, methionine, aspartate, aspartate) motif of the HBV polymerase gene is the main mutation responsible for resistance to lamivudine treatment. Detection of emerging HBV variants by direct sequencing of the HBV genome is excessively time-consuming for studying large numbers of clinical samples. The aim of the study was to analyse the emergence of lamivudine-resistant HBV genotypes by means of restriction fragment length polymorphism (RFLP) of the PCR product generated from a fragment of domain C of the polymerase gene, in clinical samples from patients receiving treatment. The results with this method were compared with those obtained with a direct sequencing technique. In total, 139 serum samples were studied from 37 patients with chronic hepatitis B, obtained at pre-treatment and at 9, 12, 18 and 24 months of treatment. Variants were detected by cleavage of the products of the three PCRs with the following enzymes: FokI (identifies the normal variant, YMDD, and the mutant variant YVDD), SspI (identifies the mutant variant, YIDD) and Alw44I (identifies the variant, YVDD). The digested fragments were separated by electrophoresis in 3% agarose gel. Of the 139 serum samples analysed, the wild-type YMDD sequence was detected in 106 (76%), the YVDD variant in 20 (15%) and the YIDD variant in 13 (9%) cases. The non-mutated variant, YMDD, was detected in all the pre-treatment samples. After 9 months of treatment the mutant variant was detected in four of 37 serum samples analysed (11%) (two YVDD and two YIDD). At 12 months, 12 of the 37 serum samples (32%) showed mutations in the YMDD sequence (seven YVDD and five YIDD). Among the 16 serum samples obtained at 18 months, nine had the YMDD variant (56%) (seven YVDD and two YIDD). At 24 months, variants in the YMDD sequence were detected in eight of the 12 patients treated (66%) (four YVDD and four YIDD). HBV genotypes were confirmed by direct sequencing, with consistent results. In 45% of cases, the emergence of HBV variants was not associated with ALT breakthrough. The PCR-RFLP assay used in this study, in perfect concordance with direct sequencing, is an accurate method for genotyping lamivudine-resistant HBV variants. Since it is a rapid low-cost technique, PCR- RFLP is suitable for large-scale screening of these polymorphisms in clinical samples.

Antiviral Agents↗