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Results for “mutational profiling”

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PubMed2001

Polyglutamine expansions cause decreased CRE-mediated transcription and early gene expression changes prior to cell death in an inducible cell model of Huntington's disease.

Huntington's disease (HD) is one of 10 known diseases caused by a (CAG)(n) trinucleotide repeat expansion that is translated into an abnormally long polyglutamine tract. We have developed stable inducible neuronal (PC12) cell lines that express huntingtin exon 1 with varying CAG repeat lengths under doxycycline (dox) control. The expression of expanded repeats is associated with aggregate formation, caspase-dependent cell death and decreased neurite outgrowth. Post-mitotic cells expressing mutant alleles were more prone to cell death compared with identical cycling cells. To determine early metabolic changes induced by this mutation in cell models, we studied changes in gene expression after 18 h dox induction, using Affymetrix arrays, cDNA filters and adapter-tagged competitive PCR (ATAC-PCR). At this time point there were low rates of inclusion formation, no evidence of mitochondrial compromise and no excess cell death in the lines expressing expanded compared with wild-type repeats. The expression profiles suggest novel targets for the HD mutation and were compatible with impaired cAMP response element (CRE)-mediated transcription, which we confirmed using CRE-luciferase reporter assays. Reduced CRE-mediated transcription may contribute to the loss of neurite outgrowth and cell death in polyglutamine diseases, as these phenotypes were partially rescued by treating cells with cAMP or forskolin.

Animals↗

PubMed2003

Molecular characterization of human prostate carcinoma cell lines.

BACKGROUND: This study presents a comprehensive survey and characterization of available prostate carcinoma cell lines, most of which have been widely used but are incompletely characterized. METHODS: A total of 21 cell lines were investigated, including three "classical" (DU 145, LNCaP, and PC-3) and 18 "non-classical" lines (1013L, 22Rv1, ALVA-55, ALVA-101, ARCaP, CWR-R1, DuCaP, DuPro-1, LAPC-4, MDA PCa 1, MDA PCa 2a, MDA PCa 2b, NCI-H660, PC-346C, PC-93, PSK-1, UM-SCP-1, and VCaP). Cytogenetics, DNA profiling, expression of basal, luminal, and neuroendocrine differentiation markers, and mutation analyses of the TP53 and androgen receptor (AR) genes were performed. RESULTS: Based on cytogenetics and DNA profiling analyses, out of the 18 "non-classical" lines, six were confirmed to be unique, eight (in four pairs) were confirmed to be related in origin, and four lines were identified as cross-contaminants. Of this latter group, PC-93 was found to be a derivative of HeLa, whereas DuPro-1, ALVA-55, and ALVA-101 were derivatives of PC-3. The 17 genuine prostate cell lines expressed keratin 8 (K8) and K18. Nine showed AR expression, of which five harbored mutations in the AR gene. Prostate-specific antigen and DD3 were exclusively detected in AR expressing cell lines. Seven lines expressed the basal cell marker K5, three of these lines showed co-expression of AR. CONCLUSIONS: This study defines a collection of 17 genuine prostate carcinoma cell lines. This collection, although small, constitutes a variety of different types and stages of prostate cancer, while it also partly reflects the heterogeneous nature of this malignancy.

Blotting, Western↗

PubMed2005

Regulation of type III secretion hierarchy of translocators and effectors in attaching and effacing bacterial pathogens.

Human enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and the mouse pathogen Citrobacter rodentium (CR) belong to the family of attaching and effacing (A/E) bacterial pathogens. They possess the locus of enterocyte effacement (LEE) pathogenicity island, which encodes a type III secretion system. These pathogens secrete a number of proteins into culture media, including type III effector proteins and translocators that are required for the translocation of effectors into host cells. Preliminary evidence indicated that the LEE-encoded SepL and Rorf6/SepD may form a molecular switch that controls the secretion of translocators and effectors in CR. Here, we show that SepL and SepD indeed perform this function in A/E pathogens such as EHEC and EPEC. Their sepL and sepD mutants do not secrete translocators but exhibit enhanced secretion of effectors. We demonstrate that SepL and SepD interact with each other and that both SepL and SepD are localized to the bacterial membranes. Furthermore, we demonstrate that culture media influence the type III secretion profile of EHEC, EPEC, and CR and that low-calcium concentrations inhibit secretion of translocators but promote the secretion of effectors, similar to effects on type III secretion by mutations in sepL and sepD. However, the secretion profile of the sepD and sepL mutants is not affected by these culture conditions. Collectively, our results suggest that SepL and SepD not only are necessary for efficient translocator secretion in A/E pathogens but also control a switch from translocator to effector secretion by sensing certain environmental signals such as low calcium.

Bacterial Outer Membrane Proteins↗

PubMedDate not supplied

Microsatellite instability and p53 mutations in hepatocellular carcinoma.

We have studied 27 hepatocellular carcinomas (HCCs) to identify possible relationships between microsatellite instability (MSI), p53 mutations, and HBV infection in hepatocarcinogenesis. MSI was assessed using 19 polymorphic markers and the poly(A) tract BAT-26. All coding regions of p53 were examined for mutations. Tumors were also examined for presence of hepatitis B virus (HBV) DNA sequences; 66.6% of the samples exhibit MSI in at least one microsatellite locus and 44% in two or three loci. None of the tumors examined showed alterations in BAT-26. Moreover, 73.3% of samples with indication of HBV infection showed instability in at least one marker. No association between MSI and pathological profile was found. Five (18.5%) samples harbored mutations in p53, three missense, and two insertions, all in exons 5 and 8 not previously reported. No mutations were detected in codon 249, which has been linked with dietary intake of aflatoxins. Our results support the hypothesis that HCC is a "low" MSI tumor. Only 1/5 samples with MSI in more than two markers harbored a mutation in p53. Although the number of samples is too small to support a statistical significance, this finding may indicate an inverse relationship between p53 mutations and MSI in HCC.

Base Sequence↗

PubMed2002

Diagnosis and confirmation of epidermolytic palmoplantar keratoderma by the identification of mutations in keratin 9 using denaturing high-performance liquid chromatography.

BACKGROUND: Epidermolytic palmoplantar keratoderma (EPPK) is one of a number of disorders characterized by diffuse thickening of palm and sole skin. Although EPPK is not a life-threatening condition, palmoplantar keratoderma can be associated with cancer and heart disease and therefore differential diagnosis is important so that adequate surveillance can be provided for the more serious conditions. Most cases of EPPK are caused by mutations in the gene encoding the palm- and sole-specific keratin 9 (K9), and this provides an option for molecular diagnosis of this condition. OBJECTIVES: To identify the molecular basis of diffuse palmoplantar keratoderma in four British families. METHODS: Denaturing high-performance liquid chromatography (dHPLC) and DNA sequencing were used to screen exon 1 of the k9 gene for sequence variations. RESULTS: The dHPLC profiles obtained from individuals with EPPK differed from control samples, indicating sequence variations within the fragment analysed. The profiles varied between families, suggesting that underlying mutations were different for each family; this was confirmed by DNA sequencing. In three cases previously reported mutations were found that resulted in the change of methionine156 to valine and arginine162 to either tryptophan or glutamine. A novel mutation was identified in a fourth family that changed valine170 to methionine. dHPLC was used to screen control samples for this sequence variation and confirmed that it was not a common polymorphism. CONCLUSIONS: These results confirm the diagnosis of EPPK in these families and underline the usefulness of dHPLC as a method of screening samples for heterozygous mutations.

Chromatography, High Pressure Liquid↗

PubMed2005

Comparative analysis of the mutagenic activity of oxaliplatin and cisplatin in the Hprt gene of CHO cells.

Oxaliplatin is a platinum-derived antitumor drug that is active against cisplatin-resistant tumors and has lower overall toxicity than does cisplatin. DNA adduct formation is believed to mediate the cytotoxic activity of both compounds; however, the adducts may also be responsible for mutagenic and secondary tumorigenic activities. In this study, we have compared the mutagenicity of oxaliplatin and cisplatin in the Hprt gene of CHO-K1 cells. Both drugs produced dose-related increases in mutant frequency. For 1-hr treatments, oxaliplatin was less mutagenic than cisplatin at equimolar doses, while similar mutant frequencies were induced at equitoxic doses. Sequencing of mutant Hprt genes indicated that the mutation spectra of both oxaliplatin and cisplatin were significantly different from the spontaneous mutation spectrum (P = 0.014 and P = 0.008, respectively). A significant difference was also observed between the spectra of oxaliplatin- and cisplatin-induced mutations (P = 0.033). Although G:C-->T:A transversion was the most common mutation produced by both compounds, oxaliplatin produced higher frequencies of A:T-->T:A transversion than did cisplatin, most commonly at nucleotide 307, and higher frequencies of small deletions/insertions. Also, cisplatin induced tandem base-pair substitutions, mainly at positions 135/136, and a higher frequency of G:C-->A:T transition than did oxaliplatin. These results provide the first evidence that oxaliplatin is mutagenic and that the profiles of cisplatin- and oxaliplatin-induced mutations display not only similarities but also distinctive features relating to the type and sequence-context preference for mutation. Environ.

Animals↗

PubMed2004

Augmentin (amoxicillin/clavulanate) in the treatment of community-acquired respiratory tract infection: a review of the continuing development of an innovative antimicrobial agent.

Amoxicillin/clavulanate (Augmentin) is a broad-spectrum antibacterial that has been available for clinical use in a wide range of indications for over 20 years and is now used primarily in the treatment of community-acquired respiratory tract infections. Amoxicillin/clavulanate was developed to provide a potent broad spectrum of antibacterial activity, coverage of beta-lactamase-producing pathogens and a favourable pharmacokinetic/pharmacodynamic (PK/PD) profile. These factors have contributed to the high bacteriological and clinical efficacy of amoxicillin/clavulanate in respiratory tract infection over more than 20 years. This is against a background of increasing prevalence of antimicrobial resistance, notably the continued spread of beta-lactamase-mediated resistance in Haemophilus influenzae and Moraxella catarrhalis, and penicillin, macrolide and quinolone resistance in Streptococcus pneumoniae. The low propensity of amoxicillin/clavulanate to select resistance mutations as well as a favourable PK/PD profile predictive of high bacteriological efficacy may account for the longevity of this combination in clinical use. However, in certain defined geographical areas, the emergence of S. pneumoniae strains with elevated penicillin MICs has been observed. In order to meet the need to treat drug-resistant S. pneumoniae, two new high-dose amoxicillin/clavulanate formulations have been developed. A pharmacokinetically enhanced tablet dosage form of amoxicillin/clavulanate 2000/125 mg twice daily (available as Augmentin XR in the USA), has been developed for use in adult respiratory tract infection due to drug-resistant pathogens, such as S. pneumoniae with reduced susceptibility to penicillin, as well as beta-lactamase-producing H. influenzae and M. catarrhalis. Amoxicillin/clavulanate 90/6.4 mg/kg/day in two divided doses (Augmentin ES-600) is for paediatric use in persistent or recurrent acute otitis media where there are risk factors for the involvement of beta-lactamase-producing strains or S. pneumoniae with reduced penicillin susceptibility. In addition to high efficacy, amoxicillin/clavulanate has a well known safety and tolerance profile of the two new high-dose formulations are not significantly different from those of conventional formulations. Amoxicillin/clavulanate is included in guidelines and recommendations for the treatment of bacterial sinusitis, acute otitis media, community-acquired pneumonia and acute exacerbations of chronic bronchitis. Amoxicillin/clavulanate continues to be an important agent in the treatment of community-acquired respiratory tract infections, both now and in the future.

Amoxicillin-Potassium Clavulanate Combination↗

PubMed1973

Mutation in Neurospora crasa affecting some of the extracellular enzymes and several growth characteristics.

A mutant of Neurospora crassa, strain T9, which shows reduced activity of glucoamylase, was isolated. The mutant glucoamylase was not altered in pH optimum, or thermostability, but altered in elution profile of Bio-Gel filtration. The T9 mutation led to other distinct phenotypic effects: a poor growth response, resistance to the effect of sorbose, a high activity of extracellular acid phosphatase, and an increased sensitivity to high osmolarity of the growth medium. The discussion was based on the idea that the T9 mutation occurred at a gene governing the synthesis of cell wall precursors and resulted in an alteration in various characteristics related to extracellular enzymes and growth.

Acid Phosphatase↗

PubMed2005

Distinct genomic profiles in hereditary breast tumors identified by array-based comparative genomic hybridization.

Mutations in BRCA1 and BRCA2 account for a significant proportion of hereditary breast cancers. Earlier studies have shown that inherited and sporadic tumors progress along different somatic genetic pathways and that global gene expression profiles distinguish between these groups. To determine whether genomic profiles similarly discriminate among BRCA1, BRCA2, and sporadic tumors, we established DNA copy number profiles using comparative genomic hybridization to BAC-clone microarrays providing <1 Mb resolution. Tumor DNA was obtained from BRCA1 (n = 14) and BRCA2 (n = 12) mutation carriers, as well as sporadic cases (n = 26). Overall, BRCA1 tumors had a higher frequency of copy number alterations than sporadic breast cancers (P = 0.00078). In particular, frequent losses on 4p, 4q, and 5q in BRCA1 tumors and frequent gains on 7p and 17q24 in BRCA2 tumors distinguish these from sporadic tumors. Distinct amplicons at 3q27.1-q27.3 were identified in BRCA1 tumors and at 17q23.3-q24.2 in BRCA2 tumors. A homozygous deletion on 5q12.1 was found in a BRCA1 tumor. Using a set of 169 BAC clones that detect significantly (P < 0.001) different frequencies of copy number changes in inherited and sporadic tumors, these could be discriminated into separate groups using hierarchical clustering. By comparing DNA copy number and RNA expression for genes in these regions, several candidate genes affected by up- or down-regulation were identified. Moreover, using support vector machines, we correctly classified BRCA1 and BRCA2 tumors (P < 0.0000004 and 0.00005, respectively). Further validation may prove this tumor classifier to be useful for selecting familial breast cancer cases for further mutation screening, particularly, as these data can be obtained using archival tissue.

Breast Neoplasms↗

PubMed2006

[Prophylactic oophorectomy among carriers of BRCA1/2 mutations--demographic and pathologic data].

BACKGROUND: During the past few years, the genes BRCAI and BRCA2 were cloned. Mutations in each gene are responsible for the syndrome of familial breast and ovarian carcinoma. Among women who carry such a mutation, there is a 56%-80% life-time risk of developing breast cancer and a 16%-60% risk of developing ovarian cancer. Recently, it has been proven that prophylactic mastectomy and/or oophorectomy might reduce such risks of developing cancer. Neither of these treatments offers full protection and furthermore, compliance of carriers is partial, considering the physical and mental consequences of such treatment. GOALS AND METHODS: This study describes the sociodemographic profile of 30 healthy carriers of BRCA1/2 mutations that underwent prophylactic salpingo-oophorectomy. We also examined the pathological specimens and point out the ratio of significant pathological findings, especially the presence of cancer. The women are being followed-up at Hadassah Medical Center, Jerusalem. RESULTS: Pathological examination of the ovaries and fallopian tubes of 30 healthy carriers of BRCA1/2 mutations who underwent prophylactic salpingo-oophorectomy revealed tumor in three cases (10%). Two tumors were in the ovaries and one in the fallopian tube. One of these tumors was in an advanced stage and two were small and confined to the organ. CONCLUSIONS: Based on the above results, we noted that salpingo-oophorectomy, despite being quite a radical preventive method, might offer protection for the carriers against life-threatening silently-developing cancer. We found cancer in 10% (3) of the women, and in two of these cases, prophylactic salpingo-oophorectomy became the definitive treatment for a small occult tumor.

BRCA1 Protein↗

PubMed1999

A common mutation of cholesteryl ester transfer protein gene in dialysis patients.

BACKGROUND: In patients on maintenance hemodialysis, a decreased concentration of high-density lipoprotein cholesterol (HDL-C) is an apparent independent risk factor for vascular disease (VD). A common missense mutation of cholesteryl ester transfer protein (CETP) gene, D442G (Asp442 to Gly), increases HDL-C levels, but the mutation may also diminish the activity of reverse cholesterol transport. METHODS: We compared the genotype distribution of the D442G polymorphism and postprandial serum lipid levels between patients with and without VD in 414 hemodialysis patients. RESULTS: Serum levels of total cholesterol and HDL-C did not differ in patients with the mutation [group M (+)] and without the mutation [group M (-)] and in patients with and without VD. However, patients with below median HDL-C levels (< 45 mg/dl) had a significantly higher prevalence of VD than those with above median HDL levels (26.0 vs. 15.2%, P < 0.01). Moreover, in this low-HDL-C subgroup, group M (+) patients had a significantly higher prevalence of VD than group M (-) patients (54.5 vs. 24.4%, P < 0.05). In the subgroup, group M (+) patients with VD had higher levels of total cholesterol and a higher atherogenic index than those without VD, whereas group M (-) patients with VD had lower levels of total cholesterol and a lower atherogenic index than those without VD. CONCLUSIONS: The D442G mutation may be a risk factor for atherosclerotic complications in dialysis patients with HDL-C levels below 45 mg/dl. Atherogenic lipid profiles may promote atherosclerosis in the patients with the mutation, but not in those with no mutation.

Adult↗

PubMed2000

Lipoprotein lipase genetic variation and gender-specific ischemic cerebrovascular disease risk.

The lipoprotein lipase gene is highly polymorphic, and several common mutations have been associated with an increased atherogenic lipid profile. One of these, Asn291Ser, was also found to be associated with increased ischemic heart disease in women. More recently, this same mutation, present in approximately 5% of the population, was also associated with increased ischemic cerebrovascular disease risk. The magnitude of this increase is less than or equal to that attributed to other modifiable risk factors, such as hypertension, smoking, and hypercholesterolemia. A prudent diet and lifestyle should therefore attenuate the higher genetic predisposition to atherosclerosis observed in women carrying this mutation.

Arteriosclerosis↗

PubMed1998

Heterogeneous expression of fusidic acid resistance in Staphylococcus aureus with plasmid or chromosomally encoded fusidic acid resistance genes.

Fusidic acid resistance expression in a methicillin susceptible Staphylococcus aureus strain (WBG1576), which carries fusidic acid resistance on plasmid pUB101, and a prevalent Western Australian methicillin-fusidic acid resistant strain (WBG8287) were compared. WBG8287 carries fusidic acid resistance on the chromosome and its plasmid content has no effect on the levels of this resistance. WBG1576 and WBG8287 exhibited similar heterogeneous populations in respect to fusidic acid resistance levels in population analyses. A high-level fusidic acid resistant mutant of WBG1576 (BE8) had alterations in Smal chromosomal profiles, but not in plasmid size or resistance expression. Mutations causing increased fusidic acid resistance in WBG1576 are chromosomally located. A high-level fusidic acid resistant mutant of WBG8287 (BE3) had no alterations in Smal chromosomal profiles, or plasmid content and resistances. Comparison of resistance levels to kanamycin and spectinomycin, between high-level resistant colonies of WBG8287 and WBG8287, indicate that mutations in the chromosomal gene fusA, which encodes elongation factor-G, are probably the cause of the increased resistance levels observed in these mutant strains.

Anti-Bacterial Agents↗

PubMed1999

Distinct molecular pathogeneses of early-onset breast cancers in BRCA1 and BRCA2 mutation carriers: a population-based study.

Breast cancers arising in women with and without a germline mutation in the BRCA1 or BRCA2 gene display different histological features, which suggests unique mechanisms of molecular pathogenesis: We used a molecular pathological analysis to define the genetic abnormalities relevant to these specific pathogeneses. Tumor material was studied from 40 women with breast cancer diagnosed before 40 years of age, sampled from a population-based study and stratified by BRCA1 and BRCA2 germline mutation status. Cases were not selected for family history or ethnic origin, and none were known to be genetically related. Thus, germline mutation itself is likely to impact on the molecular pathogenesis of these tumors, with no substantial influence due to modifying genetic or environmental factors. Breast cancers occurring in BRCA1 mutation carriers had significantly higher levels of p53 expression, including the preinvasive (carcinoma in situ) stage of disease, compared with cancers occurring in BRCA2 mutation carriers or women with no detectable germline mutation. These cancers also had a higher proliferation rate as measured by Ki-67 antibody. Expression of the prognostic factors c-erbB-2, cyclin D1, and estrogen receptor was significantly less common in BRCA1 mutation carriers. Lower levels of cyclin D1 were also found in cancers from BRCA2 mutation carriers compared with non-mutation carriers. Direct p53 mutation analysis revealed mutations in 18% of all of the early-onset breast cancers within the study and included rare insertion and deletional mutations in cancers from BRCA1 mutation carriers. Our data indicate that a BRCA1 breast cancer phenotype may be recognized by an exceptionally high proliferation rate and early and frequent p53 overexpression but infrequent selection for overexpression of several other prognostic factor proteins known to be involved in breast oncogenesis. In contrast, breast cancers arising in BRCA2 mutation carriers have a more heterogeneous phenotypic profile.

Age of Onset↗

PubMedDate not supplied

Gas chromatographic-mass spectrometric urinary metabolome analysis to study mutations of inborn errors of metabolism.

Urine contains numerous metabolites, and can provide evidence for the screening or molecular diagnosis of many inborn errors of metabolism (IEMs). The metabolomic analysis of urine by the combined use of urease pretreatment, stable-isotope dilution, and capillary gas chromatography/mass spectrometry offers reliable and quantitative data for the simultaneous screening or molecular diagnosis of more than 130 IEMs. Those IEMs include hyperammonemias and lactic acidemias, and the IEMs of amino acids, pyrimidines, purines, carbohydrates, and others including primary hyperoxalurias, hereditary fructose intolerance, propionic acidemia, and methylmalonic acidemia. Metabolite analysis is comprehensive for mutant genotypes. Enzyme dysfunction-either by the abnormal structure of an enzyme/apoenzyme, the reduced quantity of a normal enzyme/apoenzyme, or the lack of a coenzyme-is involved. Enzyme dysfunction-either by an abnormal regulatory gene, abnormal sub-cellular localization, or by abnormal post-transcriptional or post-translational modification-is included. Mutations-either known or unknown, common or uncommon-are involved. If the urine metabolome approach can accurately observe quantitative abnormality for hundreds of metabolites, reflecting 100 different disease-causing reactions in a body, then it is possible to simultaneously detect different mutant genotypes of far more than tens of thousands.

DNA Mutational Analysis↗

PubMed2003

Gene discovery using a human vestibular schwannoma cDNA library constructed from a patient with neurofibromatosis type 2 (NF2).

BACKGROUND: Despite a strong association of schwannomin/merlin gene mutations with vestibular schwannoma formation, the regulatory mechanisms and biologic pathways involved are still largely unknown. The hypothesis of this study is that the genesis and growth characteristics of neurofibromatosis type 2 (NF2)-associated vestibular schwannomas are determined by genetic alterations that vary in gene transcript expression; this transcript expression includes oncogenic gene products that may be identified by construction and sequencing of a cDNA library from NF2-associated vestibular schwannoma. METHODS: Approximately 3 mL of fresh tumor was obtained during resection of a 4-cm vestibular schwannoma from a patient with NF2. Poly(A)(+) mRNA was isolated, synthesized into double-stranded cDNA, and unidirectionally inserted into Uni-Zap XR (Stratagene, La Jolla, CA) bacteriophage vectors. Bacteriophage vectors containing cDNA inserts were processed into phagemids according to Uni-Zap XR protocol, and inserted vectors were sequenced and analyzed using BLAST software (National Institutes of Health, Bethesda, MD) with GenBank, EMBL, DDBJ, and PBD databases. RESULTS: The cDNA library contained 2.4 million primary plaques. Inserts averaged 1.8 kilobases (kb) in length, with a range of 0.8 to 3.0 kb. BLAST multidatabase comparison of the sequence data obtained from 50 randomly selected clones yielded identification of 13 sequences representing known human genes and 17 sequences representing cloned sequences with unknown function. Three clones represented sequences not previously described in vestibular schwannomas but strongly implicated in oncogenesis within other tissues. CONCLUSIONS: These data have implications for understanding the molecular mechanisms of vestibular schwannoma tumor biology. Identified genes may provide future diagnostic/prognostic markers and therapeutic targets.

Adult↗

PubMed2003

Drug resistance patterns of recombinant herpes simplex virus DNA polymerase mutants generated with a set of overlapping cosmids and plasmids.

Herpes simplex virus (HSV) DNA polymerase (Pol) mutations can confer resistance to all currently available antiherpetic drugs. However, discrimination between mutations responsible for drug resistance and those that are part of viral polymorphism can be difficult with current methodologies. A new system is reported for rapid generation of recombinant HSV type 1 (HSV-1) DNA Pol mutants based on transfection of a set of overlapping viral cosmids and plasmids. With this approach, twenty HSV-1 recombinants with single or dual mutations within the DNA pol gene were successfully generated and subsequently evaluated for their susceptibilities to acyclovir (ACV), foscarnet (FOS), cidofovir (CDV), and adefovir (ADV). Mutations within DNA Pol conserved regions II (A719T and S724N), VI (L778M, D780N, and L782I), and I (F891C) were shown to induce cross-resistance to ACV, FOS, and ADV, with two of these mutations (S724N and L778M) also conferring significant reduction in CDV susceptibility. Mutant F891C was associated with the highest levels of resistance towards ACV and FOS and was strongly impaired in its replication capacity. One mutation (D907V) lying outside of the conserved regions was also associated with this ACV-, FOS-, and ADV-resistant phenotype. Some mutations (K522E and Y577H) within the delta-region C were lethal, whereas others (P561S and V573M) induced no resistance to any of the drugs tested. Recombinants harboring mutations within conserved regions V (N961K) and VII (Y941H) were resistant to ACV but susceptible to FOS. Finally, mutations within conserved region III were associated with various susceptibility profiles. This new system allows a rapid and accurate evaluation of the functional role of various DNA Pol mutations, which should translate into improved management of drug-resistant HSV infections.

Animals↗

PubMed2005

Genotyping microarray (disease chip) for Leber congenital amaurosis: detection of modifier alleles.

PURPOSE: Leber congenital amaurosis (LCA) is an early-onset inherited disorder of childhood blindness characterized by visual impairment noted soon after birth. Variants in at least six genes (AIPL1, CRB1, CRX, GUCY2D, RPE65, and RPGRIP1) have been associated with a diagnosis consistent with LCA or early-onset retinitis pigmentosa (RP). Genetically heterogeneous inheritance complicates the analyses of LCA cases, especially in patients without a family history of the disorder, and conventional methods are of limited value. METHODS: To overcome these limitations, arrayed primer extension (APEX) technology was used to design a genotyping microarray for early-onset, severe retinal degenerations that includes all of the >300 disease-associated variants currently described in eight genes (in addition to the six just listed, the early-onset RP genes LRAT and MERTK were added). The resultant LCA array allows simultaneous detection of all known disease-associated alleles in any patient with early-onset RP. The array was validated by screening 93 confirmed patients with LCA who had known mutations. Subsequently, 205 novel LCA cases were screened on the array, followed by segregation analyses in families, if applicable. RESULTS: The microarray was >99% effective in determining the existing genetic variation and yielded at least one disease-associated allele in approximately one third of the novel patients. More than two (expected) variants were discovered in a substantial fraction (22/300) of the patients, suggesting a modifier effect from more than one gene. In support of the latter hypothesis, the third allele segregated with a more severe disease phenotype in at least five families. CONCLUSIONS: The LCA genotyping microarray is a robust and cost-effective screening tool, representing the prototype of a disease chip for genotyping patients with a genetically heterogeneous condition. Simultaneous screening for all known LCA-associated variants in large LCA cohorts allows systematic detection and analysis of genetic variation, facilitating prospective diagnosis and ultimately predicting disease progression.

Alleles↗