Browsing by Subject "Mutation detection"

Now showing 1 - 4 of 4
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    Congenital hypogonadotropic hypogonadism due to GnRH receptor mutations in three brothers reveal sites affecting conformation and coupling
    (Public Library of Science, 2012) Tello, Javier A; Newton, Claire L; Bouligand, Jerome; Guiochon-Mantel, Anne; Millar, Robert P; Young, Jacques
    Congenital hypogonadotropic hypogonadism (CHH) is characterized by low gonadotropins and failure to progress normally through puberty. Mutations in the gene encoding the GnRH receptor ( GNRHR1 ) result in CHH when present as compound heterozygous or homozygous inactivating mutations. This study identifies and characterizes the properties of two novel GNRHR1 mutations in a family in which three brothers display normosmic CHH while their sister was unaffected. Molecular analysis in the proband and the affected brothers revealed two novel non-synonymous missense GNRHR1 mutations, present in a compound heterozygous state, whereas their unaffected parents possessed only one inactivating mutation, demonstrating the autosomal recessive transmission in this kindred and excluding X-linked inheritance equivocally suggested by the initial pedigree analysis. The first mutation at c.845 C>G introduces an Arg substitution for the conserved Pro 282 in transmembrane domain (TMD) 6. The Pro282Arg mutant is unable to bind radiolabeled GnRH analogue. As this conserved residue is important in receptor conformation, it is likely that the mutation perturbs the binding pocket and affects trafficking to the cell surface. The second mutation at c.968 A>G introduces a Cys substitution for Tyr 323 in the functionally crucial N/DPxxY motif in TMD 7. The Tyr323Cys mutant has an increased GnRH binding affinity but reduced receptor expression at the plasma membrane and impaired G protein-coupling. Inositol phosphate accumulation assays demonstrated absent and impaired Gα q/11 signal transduction by Pro282Arg and Tyr323Cys mutants, respectively. Pretreatment with the membrane permeant GnRHR antagonist NBI-42902, which rescues cell surface expression of many GNRHR1 mutants, significantly increased the levels of radioligand binding and intracellular signaling of the Tyr323Cys mutant but not Pro282Arg. Immunocytochemistry confirmed that both mutants are present on the cell membrane albeit at low levels. Together these molecular deficiencies of the two novel GNRHR1 mutations lead to the CHH phenotype when present as a compound heterozygote.
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    A novel angiotensin I-converting enzyme mutation (S333W) impairs N-domain enzymatic cleavage of the anti-fibrotic peptide, AcSDKP
    (Public Library of Science, 2014) Danilov, Sergei M; Wade, Michael S; Schwager, Sylva L; Douglas, Ross G; Nesterovitch, Andrew B; Popova, Isolda A; Hogarth, Kyle D; Bhardwaj, Nakul; Schwartz, David E; Sturrock, Edward D; Garcia, Joe G N
    BACKGROUND: Angiotensin I-converting enzyme (ACE) has two functional N- and C-domain active centers that display differences in the metabolism of biologically-active peptides including the hemoregulatory tetrapeptide, Ac-SDKP, hydrolysed preferentially by the N domain active center. Elevated Ac-SDKP concentrations are associated with reduced tissue fibrosis. RESULTS: We identified a patient of African descent exhibiting unusual blood ACE kinetics with reduced relative hydrolysis of two synthetic ACE substrates (ZPHL/HHL ratio) suggestive of the ACE N domain center inactivation. Inhibition of blood ACE activity by anti-catalytic mAbs and ACE inhibitors and conformational fingerprint of blood ACE suggested overall conformational changes in the ACE molecule and sequencing identified Ser333Trp substitution in the N domain of ACE. In silico analysis demonstrated S333W localized in the S 1 pocket of the active site of the N domain with the bulky Trp adversely affecting binding of ACE substrates due to steric hindrance. Expression of mutant ACE (S333W) in CHO cells confirmed altered kinetic properties of mutant ACE and conformational changes in the N domain. Further, the S333W mutant displayed decreased ability (5-fold) to cleave the physiological substrate AcSDKP compared to wild-type ACE. Conclusions and Significance A novel Ser333Trp ACE mutation results in dramatic changes in ACE kinetic properties and lowered clearance of Ac-SDKP. Individuals with this mutation (likely with significantly increased levels of the hemoregulatory tetrapeptide in blood and tissues), may confer protection against fibrosis.
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    Transmission of HIV-1 CTL escape variants provides HLA-mismatched recipients with a survival advantage
    (Public Library of Science, 2008) Chopera, Denis R; Woodman, Zenda; Mlisana, Koleka; Mlotshwa, Mandla; Martin, Darren P; Seoighe, Cathal; Treurnicht, Florette; Rosa, Debra Assis de; Hide, Winston; Karim, Salim Abdool
    Author Summary Following infection with HIV, it is well established that a person's genetic makeup is a major determinant of how quickly they will progress to AIDS. Particularly important is the class I Human leukocyte antigen (HLA) gene that is responsible for alerting the immune system to HIV's presence. One of the reasons our immune systems are unable to beat HIV is that the virus can mutate to forms that our HLA genes no longer recognise. However, some people have versions of the HLA gene (for example HLA-B*57 and HLA-B*5801) that are known to force HIV to tolerate mutations that damage its ability to reproduce. Slower HIV reproduction is thought to be one reason that HLA-B*57 and HLA-B*5801 positive people progress to AIDS more slowly than most other HIV infected persons. We report here on a study of HLA-B*57 and HLA-B*5801 negative women in which better control of disease tended to be associated with their being infected with viruses carrying mutations that have been previously shown to reduce replication. These mutations characterise viruses found infecting HLA-B*57 and HLA-B*5801 positive people. This indicates for the first time that HLA-B*57 or HLA-B*5801 negative people that are infected by such reproductively compromised viruses may also experience better survival prospects.
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    Virologic failure of protease inhibitor-based second-line antiretroviral therapy without resistance in a large HIV treatment program in South Africa
    (Public Library of Science, 2012) Levison, Julie H; Orrell, Catherine; Gallien, Sébastien; Kuritzkes, Daniel R; Fu, Naishin; Losina, Elena; Freedberg, Kenneth A; Wood, Robin
    BACKGROUND: We investigated the prevalence of wild-type virus (no major drug resistance) and drug resistance mutations at second-line antiretroviral treatment (ART) failure in a large HIV treatment program in South Africa. Methodology/ Principal FINDINGS: HIV-infected patients ≥15 years of age who had failed protease inhibitor (PI)-based second-line ART (2 consecutive HIV RNA tests >1000 copies/ml on lopinavir/ritonavir, didanosine, and zidovudine) were identified retrospectively. Patients with virologic failure were continued on second-line ART. Genotypic testing for drug resistance was performed on frozen plasma samples obtained closest to and after the date of laboratory confirmed second-line ART failure. Of 322 HIV-infected patients on second-line ART, 43 were adults with confirmed virologic failure, and 33 had available plasma for viral sequencing. HIV-1 RNA subtype C predominated (n = 32, 97%). Mean duration on ART (SD) prior to initiation of second-line ART was 23 (17) months, and time from second-line ART initiation to failure was 10 (9) months. Plasma samples were obtained 7(9) months from confirmed failure. At second-line failure, 22 patients (67%) had wild-type virus. There was no major resistance to PIs found. Eleven of 33 patients had a second plasma sample taken 8 (5.5) months after the first. Median HIV-1 RNA and the genotypic resistance profile were unchanged. Conclusions/ Significance Most patients who failed second-line ART had wild-type virus. We did not observe evolution of resistance despite continuation of PI-based ART after failure. Interventions that successfully improve adherence could allow patients to continue to benefit from second-line ART therapy even after initial failure.