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PubMed2001

Papillary carcinoma of the thyroid and its variants: a cytohistological correlation.

Fine-needle aspiration cytology (FNAC) is considered highly specific for the diagnosis of papillary carcinoma of the thyroid (PCT). In recent years, several variants of PCT have been described. An attempt was made to gauge the accuracy of classification of variants of PCT on aspirates. Cytology smears from 124 of 150 cases of histologically proven PCT with a prior FNAC were reviewed over a 16-yr period. A diagnosis of papillary carcinoma on FNAC was made in 93 cases. Further subclassification of these cases was done on cytology and tissue sections independently. The variants of PCT classified on FNAC were classical PCT (PCT-CL), 76 cases; Hurthle-cell variant (PCT-HCV), 3 cases; follicular variant (PCT-FV), 6 cases; tall-cell variant (PCT-TCV), 2 cases; high-grade variant (PCT-HG), 2 cases; and 2 cases each which were debatably PCT-CL/PCT-FV and PCT-CL/PCT-HG. Cytology typing was accurate in 65 of the 72 classical variants, while only 7 of the 22 follicular variants were correctly identified on cytology. Two of the 3 high-grade papillary carcinomas (PCT-HG) were identified on FNAC, and the solitary case of tall-cell variant could readily be classified on cytology. In conclusion, identification of the various variants of PCT is possible, though difficulty is encountered in correctly categorizing the follicular variant, which is often mistaken for a follicular neoplasm. Also, identification of the solid variant and the papillary carcinoma with nodular fasciitis-like stroma was a problem on cytology. Another interesting observation in our series is that an admixture of various cell types was seen in the smears and corroborated on histology to be present in focal areas.

Biopsy, Needle↗

PubMed2006

MC1R common variants, CDKN2A and their association with melanoma and breast cancer risk.

We sought to examine the association between MC1R variants and the risk of melanoma and breast cancer in Polish population. We also determined the prevalence of compound heterozygous carriers of MC1R and CDKN2A (A148T) variants. We examined 500 unselected melanoma cases, 511 consecutive invasive breast cancer patients, 800 newborns, 421 healthy adults matched for sex and age with the melanoma cases and 511 healthy women matched for sex and age with the breast cancer cases. A statistically significant association of all 4 MC1R variants with the melanoma risk was found. For the R151C variant p value was 0.000008 and odds ratio 2.9; for the V60L variant p value was 0.007 and OR 1.78; for the R160C p was 0.006 and OR 1.76; for the R163Q p was 0.015 and odds ratio 2.1. None of the compound heterozygotes were significantly over-represented among any of the melanoma cases, the highest OR (4.2) observed in patients harbouring the A148T variant in CDKN2A and the R151C variant in MC1R. Positive association was found between carrying any of the MC1R variants and (i) increased occurrence of melanoma among I degree relatives of the carriers; (ii) increased occurrence of melanoma on UV-non-exposed skin areas. We also observed a tendency of increased risk of multiple melanomas among carriers of MC1R variants. The haplotype analysis demonstrates that MC1R variants do not co-occur in cis, compound carriers have both alleles affected. We found no association with the MC1R variants and breast cancer risk. In conclusion, the results of this population-based study show herein that MC1R variants are associated with increased melanoma risk in the Polish population. The risk of disease seems to be increased additively for patients harbouring also the CDKN2A common variant A148T.

Adult↗

PubMed2004

Identification of rare polymerase variants of hepatitis B virus using a two-stage PCR with peptide nucleic acid clamping.

Emergence of lamivudine-resistant variants, with amino acid substitutions in the Tyr-Met-Asp-Asp (YMDD) motif of hepatitis B virus (HBV) reverse transcriptase, is a serious problem in antiviral therapy. Presence of YMDD motif variants in patients who had never been treated with lamivudine has been reported recently. However, no analysis of nucleotide and amino acid sequences of these variants has been performed. In the present study, using polymerase chain reaction (PCR) with peptide nucleic acid (PNA) clamping, we detected many new variants, such as Tyr-Arg-Asp-Asp (YRDD), Tyr-Met-Asp-Asn (YMDN). Many of them had stop codon(s) in overlapping HBs gene. Although the biological activity of these HBV polymerase variants remains to be determined, our results showed that numerous quasispecies are created during virus replication. A typical lamivudine-resistant Tyr-Val-Asp-Asp (YVDD) variant was detected in only one of 62 (1.6%) anti-HBe patients with HBV infection before administration of lamivudine. This variant did not have the L528M mutation, which is often associated with YVDD variants, and lamivudine therapy in this patient suppressed HBV replication. Thus, care should be taken when interpreting the results of detection of YMDD variants, especially when the sensitivity of the assay is very high. Amplification of rare variants by PCR with PNA seems a useful tool to examine the emergence of drug-resistant variants as well as naturally occurring mutants, such as the hepatitis B e antigen (HBeAg) stop codon and vaccine escape mutants. Examination of rare variants should enhance the understanding of the mechanism for emergence of drug-resistant HBV variants and help in developing strategies for new antiviral drugs.

Adult↗

PubMed1995

Agrin mRNA variants are differentially regulated in developing chick embryo spinal cord and sensory ganglia.

Agrin is a synapse-organizing protein synthesized and externalized by motor neurons in the spinal cord, which organizes the postsynaptic apparatus of the developing neuromuscular junction. Agrin mRNA in the nervous system consists of several alternatively spliced variants. Splicing of agrin gene transcripts at the major site of variability results in four variants: encoding 8 (B8) or 11 (B11) amino acid inserts, both (B19), or predominant variant (B0) without inserts. The insert-containing variants are neuron specific and encode agrin proteins with greater synapse-organizing activity than the B0 variant. Here, we report the localization and developmental regulation of agrin mRNA variants in chick embryo spinal cord and dorsal root ganglia. In situ hybridization using antisense oligodeoxynucleotide (ODN) probes specific for the B8 and B11 sequences shows that the neuron-specific variants are concentrated in ventrolateral cells of the chick embryo spinal cord, presumably motor neurons, beginning at embryonic day 4 (E4). By E14, the insert-containing mRNAs are found almost exclusively in presumptive motor neurons. These variants are also found in dorsal root ganglia and sympathetic ganglia, but not in non-neural tissues. Analysis by polymerase chain reaction showed that the B11 and B19 mRNA variants appeared in spinal cord at E4, whereas the B8 variant was first seen at E14. During development, B11 decreased and disappeared by E20, whereas B8 increased from E14 to E20. A similar time course was seen in dorsal root ganglia. The greatest acetylcholine receptor-aggregating activity in the spinal cord was seen from E6 to E10, coincident with the highest proportion of B11-containing transcripts and with the peak of synaptogenesis in limb muscles. These data provide the first evidence linking appearance of the neuron-specific agrin mRNA variants with expression of the functional protein. The B11 and B19 variants appeared in E2 (stage 15) neural tubes cultured for 2 days with or without notochord and trunk tissues, indicating that there is no peripheral signal required to induce these agrin mRNA variants in developing motor neurons.

Agrin↗

PubMed2026

Novel splice site variants in GBA1 are associated with Gaucher disease and genotype-phenotype correlations.

BACKGROUND: Variants in GBA1 are associated with neurodegenerative disease. This study aimed to explore pathogenic GBA1 variants. METHODS: Four patients with progressive myoclonic epilepsy (PME) and extremely low β-glucosidase levels were recruited. Whole-exome sequencing and long-range PCR were performed to identify GBA1 variants. Bioinformatic analyses were used to predict the impact of the identified variants. A literature review was performed to explore the genotype-phenotype correlations. GBA1 expression data across different brain regions and developmental stages were analyzed using the BrainSpan database. RT-PCR was performed to verify the splicing effects. RESULTS: Compound heterozygous GBA1 variants were identified in four patients. Five distinct variants were detected, including two novel splice site variants (c.308-2A>G and c.762-2A>C) and three previously reported variants. All identified variants were rare or absent in gnomAD. Splice site variants c.308-2A>G and c.762-2A>C were predicted to cause aberrant splicing. Minigene-based splicing assays coupled with RT-PCR and Sanger sequencing confirmed that both variants cause complete exon skipping (exon 4 and exon 7, respectively). All patients presented with PME onset in childhood/adolescence, intellectual regression, low β-glucosidase, and diffuse brain atrophy and were subsequently diagnosed with Gaucher disease type 3. GBA1 expression in the brain showed two distinct peaks: one in infancy and another after five years of age. The onset age of PME aligned with the second GBA1 expression peak (after five years of age). CONCLUSION: This study identified compound heterozygous GBA1 variants, including two novel candidate pathogenic splice site variants, in Gaucher disease type 3 patients, expanding the known mutational spectrum.

Humans↗

PubMed2000

Identification of the F17 fimbrial subunit- and adhesin-encoding (f17A and f17G) gene variants in necrotoxigenic Escherichia coli from cattle, pigs and humans.

Putative colonization factors of the F17 family of fimbrial adhesins have been identified in necrotoxigenic Escherichia coli Type 1 and Type 2 (NTEC1 and NTEC2) from calves, pigs, and humans. The f17A and f17G gene variants, coding respectively for the major subunit and for the adhesin of the F17 fimbriae, were typed in 70 E. coli carrying f17-related sequences (15 NTEC1, 51 NTEC2, and four non-NTEC) by colony hybridisation with gene probes derived from the different f17A gene variants (a, b, c, and d) and by PCRs specific for each f17A and f17G (I and II) gene variants. Typing of f17A genes was not possible by colony hybridisation, as most 70 E. coli were positive with more than one gene probe. On the other hand, the PCRs allowed the typing of the f17A gene in 37 E. coli and of the f17G gene in all 70 E. coli. The f17Ab gene variant was detected in 13 NTEC2; the f17Ac, in all 15 NTEC1, six NTEC2 and two non-NTEC; and the f17Ad, in one non-NTEC. Seven additional NTEC2 were positive with the PCRs for two variants: f17Ab and f17Ac in three of them; f17Ac and f17Ad in four of them. Either these seven NTEC2 harbour two variants or the variant present can be detected by two PCRs. The remaining 25 NTEC2 and one non-NTEC tested negative with the PCRs for the four f17A gene variants, suggesting the existence of other variant(s). In contrast, all 70 E. coli were positive with the PCR for the f17GII gene variant and none with the PCR for the f17GI gene variant. The f17-related sequences were present on the CNF2/Vir plasmids in 27 out of the 46 NTEC2 from which plasmid DNA could be extracted: all but one of those positive for the f17Ab gene variant and various proportions of those positive for other variants. In contrast, no plasmid carried f17-related sequences in NTEC1 and non-NTEC.

Adhesins, Escherichia coli↗

PubMed2000

Variants of the long control region of human papillomavirus type 16.

Expression of the human papillomavirus (HPV) E6 and E7 oncogenes is regulated on the transcriptional level by specific protein-binding sites contained in the viral long control region (LCR). Sequence changes within the LCR region may have an impact on the transcription of viral oncogenes, possibly resulting in differences in the oncogenic potential of the virus. The present study was designed to determine the sequence variability of the LCR of HPV 16 and to assess whether certain LCR variants do correlate with the clinical outcome of the disease of the uterine cervix. The entire LCR segment of HPV 16 was analysed from 37 cervical biopsy specimens derived from 28 women included in the Kuopio long-term prospective follow-up study. The LCR sequence was identical with the reference sequence in six HPV 16 isolates. Overall, 14 different HPV 16 LCR variants were identified. One of the variants showed sequence variation typical of the Asian-American variant lineage of HPV 16, and all the other variants appeared to belong to the European variant group. The European variants exhibited low genetic diversity, and only five of these LCR variants contained nucleotide changes involving known or proposed binding sites for transcription factors. The variants with changes at nucleotide positions 7193 and 7521 was the most prevalent, accounting for almost 37% of infections. This variant (7193; 7521) has been previously demonstrated to have similar transcriptional activity compared with the reference isolate by Veress and colleagues J Gen Virol 1999, 80, 1035-1043. The reference isolate, variant (7193; 7521) and variants with changes within transcription factor binding sites accounted for most of the infections, and no significant differences were found in the comparison of the distribution of these different LCR isolates in cases where the disease showed progression to severe cervical intraepithelial neoplasia (CIN) or carcinoma in situ (CIS). Notably, both the reference isolate and variant (7193; 7521) were also closely associated with infections showing more aggressive behaviour. According to the present findings, in European HPV 16 isolates, intratype genetic variation of the LCR region does not seem to be commonly responsible for differences in the pathogenicity of the virus and thereby for a risk of progressive infections.

DNA, Viral↗

PubMed1985

Receptor-binding and down-regulatory properties of 22000-Mr human growth hormone and its natural 20000-Mr variant on IM-9 human lymphocytes.

Our earlier binding studies of the 22000- and 20000-Mr variants of human growth hormone (somatotropin) to pregnant-rabbit liver and mammary receptors [Closset, Smal, Gomez & Hennen (1983) Biochem. J. 214, 885-892] suggested that the 20000-Mr variant was a lower-affinity analogue of the 22000-Mr molecule. Since the receptor population in these tissues is not fully characterized, we have now investigated the binding of both variants to the well-characterized and highly specific human-growth-hormone receptor of the human lymphocyte IM-9 cell line. The maximum bindability of radioiodinated 22000- and 22000-Mr to IM-9 cells was 60 and 45% respectively. Both hormone variants have essentially the same binding characteristics: slow association (equilibrium reached in 8-10h at 30 degrees C), poor reversibility ('tight binding'), linear Scatchard plot, same specificity as shown by lack of competition by bovine, porcine or equine growth hormones or human growth hormone-(32-46)-(missing in the 20000-Mr variant),-(1-134)- and -(141-191)-peptides. Both unlabelled hormones inhibit binding of both tracers completely, with the 20000-Mr variant being only half as potent as the 22000-Mr one. The apparent affinity is 2.8 X 10(9)M-1 for the 22000-Mr variant and 1.6 X 10(9)M-1 for the 20000-Mr variant. This decreased affinity of the 20000-Mr variant appears to be due to a lower association rate constant. Concentrations (5 ng/ml) of the two variants that occupy about 15% of the total sites induce a marked down-regulation of the receptors after 18h incubation, but the 20000-Mr variant (50% decrease) has a smaller effect than the 22000-Mr variant (75% decrease). Thus the only consequence of the residues-32-46 deletion in the 20000-Mr variant is a lower association rate and affinity for the IM-9 lymphocyte human-growth-hormone receptor. The close binding characteristics of the two forms suggest that the known differences in their insulin-like effects cannot be explained by differences in the nature of their interaction with the human-growth-hormone receptor.

Binding Sites↗

PubMed2000

The butyrylcholinesterase K-variant shows similar cellular protein turnover and quaternary interaction to the wild-type enzyme.

A recent study has linked the butyrylcholinesterase (BChE) K-variant and the apolipoprotein epsilon4 isoform to late-onset Alzheimer's disease. These findings have been controversial and have led us to examine the differences between wild-type and K-variant BChE in enzyme activity, protein stability, and quaternary structure. J-variant BChE (E497V/A539T) was also studied because it is associated with the K-variant mutation. The K-variant mutation (A539T) is located in the C-terminal tetramerization domain. Wild-type, K-variant, and J-variant BChE were expressed in Chinese hamster ovary cells and purified. The purified enzymes had similar binding affinity (Km) values and catalytic rates for butyrylthiocholine and benzoylcholine. In pulse-chase studies the K-variant, J-variant, and wildtype BChE were degraded rapidly within the cell, with a half-time of approximately 1.5 h. Less than 5% of the intracellular BChE was exported. The C-terminal peptide containing the K-variant mutation interacted with itself as strongly as did the wild-type peptide in the yeast two-hybrid system. Both K-variant and wild-type BChE assembled into tetramers in the presence of poly-L-proline or the proline-rich attachment domain of the collagen tail. The native K-variant BChE in serum showed the same proportion of tetramers as the native serum wild-type BChE. We conclude that the K-variant BChE is similar to wild-type BChE in enzyme activity, protein turnover, and tetramer formation.

Adult↗

PubMed1998

Melanocortin 1 receptor variants in an Irish population.

The identification of an association between variants in the human melanocortin 1 receptor (MC1R) gene and red hair and fair skin, as well as the relation between variants of this gene and coat color in animals, suggests that the MC1R is an integral control point in the normal pigmentation phenotype. In order to further define the contribution of MC1R variants to pigmentation in a normal population, we have looked for alterations in this gene in series of individuals from a general Irish population, in whom there is a preponderance of individuals with fair skin type. Seventy-five per cent contained a variant in the MC1R gene, with 30% containing two variants. The Arg151Cys, Arg160Trp, and Asp294His variants were significantly associated with red hair (p = 0.0015, p < 0.001, and p < 0.005, respectively). Importantly, no individuals harboring two of these three variants did not have red hair, although some red-haired individuals only showed one alteration. The same three variants were also over-represented in individuals with light skin type as assessed using a modified Fitzpatrick scale. Despite these associations many subjects with dark hair/darker skin type harbored MC1R variants, but there was no evidence of any particular association of variants with the darker phenotype. The Asp294His variant was similarly associated with red hair in a Dutch population, but was infrequent in red-headed subjects from Sweden. The Asp294His variant was also significantly associated with nonmelanoma skin cancer in a U.K. population. The results show that the Arg151Cys, Arg160Trp, and Asp294His variants are of key significance in determining the pigmentary phenotype and response to ultraviolet radiation, and suggest that in many cases the red-haired component and in some cases fair skin type are inherited as a Mendelian recessive.

Adult↗

PubMed1964

POSSIBLE ROLE OF INTERFERON IN DETERMINING THE ONCOGENIC EFFECT OF POLYOMA VIRUS VARIANTS.

1. Infection of newborn C(3)Hf/Bi mice with both the highly oncogenic S variant and the poorly oncogenic M variant of polyoma virus caused significantly fewer tumors than infection with the S variant alone 2. Infection of newborn C(3)Hf/Bi mice at birth with either M or S variant caused resistance to infection with a heterologous agent, encephalomyocarditis virus, but the M variant caused a somewhat greater degree of resistance. 3. Extracts of tissues of animals infected at birth with the M variant had measurable levels of interferon, reaching a peak on the 5th day after infection with the virus. Extracts of animals infected with the S variant showed no such activity. 4. The growth curves of the two variants in newborn C(3)Hf/Bi mice showed that the S variant grew to a 1000-fold greater titer than did the M variant at 5 days after infection. Samples tested at 10 days showed a 10-fold difference. 5. These findings suggested that the difference between the variants in oncogenic potential might have been due to the greater interferon production induced by infection with the M variant than by infection with the S variant.

Animals↗

PubMed2000

Allelic variants of human cytochrome P450 1A1 (CYP1A1): effect of T461N and I462V substitutions on steroid hydroxylase specificity.

Steroid hydroxylation specificities were determined for the wild-type and the two allelic variants of the polymorphic human cytochrome P450 1A1 (CYP1A1) that were associated with amino acid exchanges near the active site of the enzyme. All three variants were expressed in insect cells using recombinant baculoviruses. Each variant protein was spectrally and enzymatically active, as judged by the ability of the prepared microsomes to catalyse O-dealkylation of ethoxyresorufin and pentoxyresorufin in cumene hydroperoxide-mediated reactions. With progesterone and testosterone as substrate, all variants of CYP1A1 exhibited high, but different steroid hydroxylation activities (8-40 pmol hydroxysteroid/min/pmol CYP1A1, i.e. approximately 800-4000 pmol/min/mg microsomal protein). All three variants exclusively catalysed 6beta-hydroxylation of both steroids. In addition, towards progesterone as substrate, all variants also catalysed 16alpha-hydroxylations with approximately half of the rate of 6beta-hydroxylation activity. With progesterone as substrate for hydroxylation in 6beta position, CYP1A1 T461N had the lowest catalytic efficiency (Vmax/Km) followed by the CYP1A1 I462V variant and the wild-type enzyme. For 16alpha-hydroxylation of progesterone, the catalytic efficiencies of the three variants are not statistically significantly different. With testosterone as substrate the CYP1A1 1462V variant catalysed 6beta-hydroxylation with an efficiency considered not significantly different compared to the wild-type, although both the apparent Km and Vmax were significantly decreased. In contrast, the CYP1A1 T461N variant exhibited significantly decreased catalytic efficiencies compared to both the 1462V variant and the wild-type enzyme. These results indicate that all three naturally occurring allelic variants of human CYP1A1 hydroxylate steroid hormones with varying efficiencies in a stereo- and regioselective manner, whereby the CYP1A1 T461N variant exhibited the lowest catalytic efficiency.

Alleles↗

PubMed1995

Prevalence of an immunological LH beta-subunit variant in a UK population of healthy women and women with polycystic ovary syndrome.

OBJECTIVE: An immunological LH beta-subunit variant has been described, which is undetectable using monoclonal antibodies directed to the intact LH molecule alone. Subjects have been found homozygous or heterozygous for nucleotide mutations within codons 8 and 15 in the LH beta-subunit gene. The prevalence of the variant LH beta-subunit has been estimated in a healthy UK population of women of reproductive age and in women with polycystic ovary syndrome (PCOS). The relationship of the variant molecule to the clinical and hormonal parameters of the subjects has been evaluated. DESIGN: The control and PCOS subjects were screened for the presence of the mutation by using a ratio of two immunofluorometric assays using monoclonal antibodies (Mab). One assay, not detecting the LH variant, uses a Mab directed to the intact LH molecule and a beta-specific Mab. The other assay, detecting both the variant and wild-type LH, uses two beta-subunit specific Mabs. The mutations in the LH beta-subunit gene were confirmed by restriction fragment length polymorphism. The relationship of the presence of the variant to the clinical and hormonal parameters was assessed by ANOVA. PATIENTS: Two hundred and twelve normal ovulatory women, of whom 66 (31%) were obese (body mass index > 25) and 146 (69%) non-obese, and 153 women with PCOS, 115 (75%) obese and 38 (25%) non-obese participated in the study. RESULTS: The variant LH was detected in 31 (15%) controls and 32 (21%) PCOS subjects (P = 0.124) using specific Mab. Obese PCOS had a higher incidence of the heterozygous LH variant compared to obese controls (odds ratio 2.5, P = 0.03), and compared to non-obese PCOS (odds ratio 6.3, P = 0.01). The previously described two mutations in codon 8 and codon 15 were present in all subjects detected to be mutant hetero of homo-zygous by RFLP. There was no relationship between the presence of the variant LH and the clinical and hormonal parameter in the PCOS subjects; however, in the controls the presence of the variant LH was associated with a higher serum total testosterone (P = 0.046), oestradiol (P = 0.03) and SHBG (P = 0.002). CONCLUSIONS: The results of this study show that the variant LH beta-subunit is a common polymorphism occurring in 15% of a healthy UK population of women. The prevalence was not higher in women with PCOS, though it was over represented in obese women with PCOS. The presence of the variant did not alter the clinical or hormonal expression of the disorder in women with PCOS. Its presence in the controls was however associated with higher serum oestradiol and probably secondary elevation of SHBG and testosterone, suggesting that the variant form of LH may be associated with subtle changes in the function of the hypothalamic-pituitary-gonadal axis.

Adult↗

PubMed1963

Effect of heat on spores of rough and smooth variants of Bacillus stearothermophilus.

Spores of variants of Bacillus stearothermophilus were subjected to activating and lethal temperatures. Spore suspensions which were incubated longer contained a higher percentage of spores of the rough variant. The effect of sublethal heat on spore suspensions containing mixed variants (rough and smooth) was difficult to measure at sublethal temperatures (110 C), since the rough variant was not as heat-resistant. While the rough variant was activated in a shorter time, the smooth variant was not activated; when the smooth variant was activated, the rough was killed. A higher percentage of the smooth variant was forced into dormancy after being held at 50 C for 30 hr than the rough variant. When mixed populations were subjected to a lethal temperature (120 C), the curves only reflected the smooth variant. Since the curves which represented the smooth variant or mixtures containing the smooth variant were not linear, this was thought to be due to activation overbalancing the lethal effect. This research emphasized the importance of variants in explaining differences in spore resistance among spore suspensions of the same strain.

Bacillus↗

PubMed2002

Identification of Anaplasma phagocytophila (formerly Ehrlichia phagocytophila) variants in blood from sheep in Norway.

A total of 41 blood samples were collected from 40 Anaplasma phagocytophila-infected sheep in 11 sheep flocks from four different counties of southern Norway. The presence and nature of the Anaplasma species were identified by microscopic detection of morulae, PCR, reverse line blot hybridization, and 16S rRNA gene sequencing. A. phagocytophila was identified in all of the samples, and sequencing of the 16S rRNA gene revealed the presence of four variants of A. phagocytophila. Two of these variants have been described before, but two were newly identified 16S rRNA variants of this species. A. phagocytophila variant 1 was found in nine flocks, A. phagocytophila variant 2 was found in four flocks, the A. phagocytophila prototype was found in two flocks, and A. phagocytophila variant 5 was found in one flock. In two flocks, some sheep were infected with A. phagocytophila variant 1, whereas others were infected with A. phagocytophila variant 2, and in three animals a double infection with two variants was registered. Analyses of the blood samples revealed that blood from sheep infected with A. phagocytophila variant 2 contained nearly twice as many neutrophils and eight times as many Anaplasma-infected neutrophils as blood from sheep infected with the A. phagocytophila variant 1. Furthermore, only 43% of the A. phagocytophila variant 2-infected sheep displayed antibody responses in an immune fluorescence assay, whereas 93% of the sheep with the A. phagocytophila variant 1-infected sheep were seropositive.

Anaplasma↗

PubMed2005

Genetic, functional, and histopathological evaluation of two C-terminal BRCA1 missense variants.

BACKGROUND: The vast majority of BRCA1 missense sequence variants remain uncharacterized for their possible effect on protein expression and function, and therefore are unclassified in terms of their pathogenicity. BRCA1 plays diverse cellular roles and it is unlikely that any single functional assay will accurately reflect the total cellular implications of missense mutations in this gene. OBJECTIVE: To elucidate the effect of two BRCA1 variants, 5236G>C (G1706A) and 5242C>A (A1708E) on BRCA1 function, and to survey the relative usefulness of several assays to direct the characterisation of other unclassified variants in BRCA genes. METHODS AND RESULTS: Data from a range of bioinformatic, genetic, and histopathological analyses, and in vitro functional assays indicated that the 1708E variant was associated with the disruption of different cellular functions of BRCA1. In transient transfection experiments in T47D and 293T cells, the 1708E product was mislocalised to the cytoplasm and induced centrosome amplification in 293T cells. The 1708E variant also failed to transactivate transcription of reporter constructs in mammalian transcriptional transactivation assays. In contrast, the 1706A variant displayed a phenotype comparable to wildtype BRCA1 in these assays. Consistent with functional data, tumours from 1708E carriers showed typical BRCA1 pathology, while tumour material from 1706A carriers displayed few histopathological features associated with BRCA1 related tumours. CONCLUSIONS: A comprehensive range of genetic, bioinformatic, and functional analyses have been combined for the characterisation of BRCA1 unclassified sequence variants. Consistent with the functional analyses, the combined odds of causality calculated for the 1706A variant after multifactorial likelihood analysis (1:142) indicates a definitive classification of this variant as "benign". In contrast, functional assays of the 1708E variant indicate that it is pathogenic, possibly through subcellular mislocalisation. However, the combined odds of 262:1 in favour of causality of this variant does not meet the minimal ratio of 1000:1 for classification as pathogenic, and A1708E remains formally designated as unclassified. Our findings highlight the importance of comprehensive genetic information, together with detailed functional analysis for the definitive categorisation of unclassified sequence variants. This combination of analyses may have direct application to the characterisation of other unclassified variants in BRCA1 and BRCA2.

Adult↗

PubMed2025

Personalizing CA125 Levels Using Tumor Marker Variants: A Case-Control Analysis of Diagnostic Performance for Pancreatic Cancer.

BACKGROUND: Cancer antigen 125 (CA125) is widely recognized as a useful biomarker for the surveillance of patients with ovarian and other cancers. Prior genome-wide association studies have identified variants that influence CA125 levels. We evaluated the utility of stratifying CA125 levels by such variants and evaluated diagnostic performance in control subjects and patients with pancreatic ductal adenocarcinoma (PDAC). METHODS: We measured CA125 levels in 807 control subjects and 450 patients with PDAC and genotyped 10 variants involving four genes (GAL3ST2, MSLN, D2HGDH, and MUC16). We compared CA125 levels in controls by variant and generated variant-defined CA125 cutoffs and then classified cases and controls into functional groups based on their variant profile. We used this variant classification to evaluate the diagnostic performance of CA125 in patients with PDAC. RESULTS: Six variants associated with CA125 levels were used to group controls into one of four groups. Mean CA125 levels in the highest variant group were approximately fourfold higher than in the lowest group. African Americans were more likely to have a variant group associated with low CA125 levels. After setting diagnostic cutoffs by variant group, the diagnostic sensitivity of CA125 for PDAC was 20.2% at 98% specificity (areas under the ROC curve, 0.702), not significantly different from a uniform CA125 diagnostic cutoff (areas under the ROC curve, 0.700). CONCLUSIONS: Gene variants can be used to generate personalized CA125 reference ranges. This approach did not significantly improve CA125's diagnostic performance for pancreatic cancer, but it merits evaluation in other diagnostic settings, such as detecting ovarian cancer. IMPACT: Gene variants can be used to personalize CA125 levels.

Humans↗

PubMed2006

Real-time RT-PCR quantification of human telomerase reverse transcriptase splice variants in tumor cell lines and non-small cell lung cancer.

BACKGROUND: We developed and validated a real-time reverse transcription (RT)-PCR for the quantification of 4 individual human telomerase reverse transcriptase (TERT) splice variants (alpha+beta+, alpha-beta+, alpha+beta-, alpha-beta-) in tumor cell lines and non-small cell lung cancer (NSCLC). METHODS: We used in silico designed primers and a common TaqMan probe for highly specific amplification of each TERT splice variant, PCR transcript-specific DNA external standards as calibrators, and the MCF-7 cell line for the development and validation of the method. We then quantified TERT splice variants in 6 tumor cell lines and telomerase activity and TERT splice variant expression in cancerous and paired noncancerous tissue samples from 28 NSCLC patients. RESULTS: In most tumor cell lines, we observed little variation in the proportion of TERT splice variants. The alpha+beta- splice variant showed the highest expression and alpha-beta+ and alpha-beta- the lowest. Quantification of the 4 TERT splice variants in NSCLC and surrounding nonneoplastic tissues showed the highest expression percentage for the alpha+beta- variant in both NSCLC and adjacent nonneoplastic tissue samples, followed by alpha+beta+, with the alpha-beta+ and alpha-beta- splice variants having the lowest expression. In the NSCLC tumors, the alpha+beta+ variant had higher expression than other splice variants, and its expression correlated with telomerase activity, overall survival, and disease-free survival. CONCLUSIONS: Real-time RT-PCR quantification is a specific, sensitive, and rapid method that can elucidate the biological role of TERT splice variants in tumor development and progression. Our results suggest that the expression of the TERT alpha+beta+ splice variant may be an independent negative prognostic factor for NSCLC patients.

Carcinoma, Non-Small-Cell Lung↗