Rapid mutation detection by the Transgenomic wave analyser DHPLC identifies MYOC mutations in patients with ocular hypertension and/or open angle glaucoma

Cobb, Caroline; Scott, Gordon; Swingler, Robert; Wilson, Sheri L.; Ellis, James; MacEwen, C J; McLean, W.H. Irwin

doi:10.1136/bjo.86.2.191

Cited by 16 publications

(11 citation statements)

References 16 publications

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“…Ion-pair reversed-phase dHPLC identifies mutations by detecting heteroduplex formation between mismatched nucleotides in PCR-amplified DNA (40). Using the WAVE Nucleic Acid Fragment Analysis System (Transgenomics, Inc., Omaha, NE), we analyzed selected regions of each candidate TSG for the presence of mutations (41,42). Amplicons encoding the following functional domains were selected for dHPLC analysis: the kinase domain of CKI⑀; the PDZ2 domain of hDlg; and the NH 2 -terminal region of the EDD HECT domain.…”

Section: Introductionmentioning

confidence: 99%

Somatic Mutations and Altered Expression of the Candidate Tumor Suppressors CSNK1ε , DLG1 , and EDD/hHYD in Mammary Ductal Carcinoma

Fuja

Lin²,

Osann³

et al. 2004

Cancer Research

121

111

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We report somatic mutations in three genes (CSNK1⑀, encoding the Ser/Thr kinase casein kinase I ⑀; DLG1, encoding a membrane-associated putative scaffolding protein; and EDD/hHYD, encoding a progestin induced putative ubiquitin-protein ligase) in mammary ductal carcinoma. These genes were suspected of playing a role in cancer because loss-offunction mutations in their Drosophila homologues cause excess tissue growth. Using DNA from 82 laser-microdissected tumor samples, followed by microsatellite analysis, denaturing HPLC and direct sequencing, we found multiple somatic point mutations in all three genes, and these mutations showed significant association with loss of heterozygosity of closely linked polymorphic microsatellite markers. For CSNK1⑀ and DLG1, most of the mutations affected highly conserved residues, some were found repetitively in different patients, and no synonymous mutations were found, indicating that the observed mutations were selected in tumors and may be functionally significant. Immunohistochemical reactivity of each protein was reduced in poorly differentiated tumors, and there was a positive association between altered protein reactivity, loss of heterozygosity, and somatic mutations. There was a statistically significant association of hDlg staining with p53 and Ki67 reactivity, whereas CSK1⑀ and EDD/hHYD staining levels were associated with progesterone receptor status. The results provide strong indications for a role of all three genes in mammary ductal carcinoma. They also justify additional studies of the functional significance of the changes, as well as a search for additional changes in these and other genes identified from studies on model systems.

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Section: Introductionmentioning

confidence: 99%

Somatic Mutations and Altered Expression of the Candidate Tumor Suppressors CSNK1ε , DLG1 , and EDD/hHYD in Mammary Ductal Carcinoma

Fuja

Lin²,

Osann³

et al. 2004

Cancer Research

121

111

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“…Information on MYOC variants in European POAG patients is presently limited (Cobb et al, 2002;Hulsman et al, 2002a;Michels-Rautenstrauss et al, 2002). In the present work, we investigated the genetic variation of the coding region of MYOC in 237 unrelated French POAG patients and 108 control subjects, using denaturing high performance liquid chromatography (DHPLC).…”

Section: Introductionmentioning

confidence: 99%

Myocilin analysis by DHPLC in French POAG patients: Increased prevalence of Q368X mutation

et al. 2003

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Primary open-angle glaucoma (POAG) is a prevalent optic neuropathy with complex genetics. A small number of patients carry a mutation in the coding region of the myocilin (MYOC) gene. The nature and the frequency of these mutations, however, vary substantially, notably with the age at onset and the ethnic origin of the patients. Here, we showed that denaturing high performance liquid chromatography (DHPLC) is an appropriate method for screening carriers of MYOC mutations. We have applied the method to a group of 237 POAG patients and 108 control subjects from France. Mutations were found in 17 (7.5%) patients and in none of the controls. A single mutation, Q368X (c.1102C>T), accounted for the majority (12/17) of these mutations, corresponding to a frequency of 5% among POAG patients, the highest ever reported for this mutation. Furthermore, analysis of allelic associations at closely linked microsatellite markers indicated that most, if not all, patients inherited Q368X from a same ancestor. Five other patients carried four distinct mutations, including N480K (c.1440C>A) (2 cases), I499F (c.1495A>T), G367R (c.1099G>A) and T438I (c.1313C>T), which is reported here for the first time. Altogether, MYOC mutations in French patients were associated with a significantly increased intraocular pressure at diagnosis. In addition, the age at diagnosis of patients with a mutation other than Q368X was significantly younger than that of Q368X carriers or of patients with a normal MYOC. Based on these observations, a screening strategy of MYOC mutations in French POAG patients is briefly outlined.

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“…However, since all patients were recruited in a single centre, similar studies in other parts of the UK would be necessary to confirm that this is not a localized effect. A small study of affected sib-pairs in Scotland identified mutations in four families, of which three were Q368X (Cobb et al 2002). Likewise, we cannot exclude the possibility that our cohort differs clinically or demographically from those studied elsewhere.…”

Section: Discussionmentioning

confidence: 85%

“…All mutations were verified by analyzing a duplicate tube of stored blood. Exons 1 and 2 were analyzed by denaturing high performance liquid chromatography (dHPLC) using primers described by Cobb et al (2002). PCR was conducted essentially as described above, except that HotStar Taq polymerase and buffer (Qiagen) were used and the annealing temperature was 52°C.…”

Section: Methodsmentioning

confidence: 99%

“…These mutations were then found in 4.4% of 227 American POAG patients with an affected first-degree relative and 2.9% of 103 patients that were unselected for family history. Additional mutations have since been identified in other glaucoma families of diverse origin, while population-based studies have confirmed that MYOC mutations are involved in a significant proportion of all POAG cases and are not confined to those with a strong family history or juvenile onset (Adam et al 1997;Alward et al 1998;Cobb et al 2002;Faucher et al 2002;Fingert et al 1999;Hulsman et al 2002;Mataftsi et al 2001;Suzuki et al 1997). The majority of mutations are clustered in a single exon that encodes an evolutionarily conserved domain with homology to the frog olfactomedin protein (Fingert et al 1999).…”

Section: Introductionmentioning

confidence: 99%

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Low prevalence of MYOC mutations in UK primary open-angle glaucoma patients limits the utility of genetic testing

et al. 2004

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Primary open angle glaucoma (POAG) affects 1% of people over age 40. Early detection and treatment can prevent blindness, but the disease is often asymptomatic until a late stage. Positive family history is an important risk factor and previous studies indicate that approximately 5% of POAG results from mutations in the myocilin ( MYOC) gene, raising the possibility of identifying individuals genetically predisposed to glaucoma. We collected DNA samples from 426 unselected UK POAG patients and analyzed them for MYOC mutations. The Q368X mutation was found in six patients (1.4%). No other mutations were identified, suggesting that amongst patients unselected for family history, the prevalence of MYOC mutations in the UK is lower than in other populations. Genetic and glaucoma screening was offered to first-degree relatives of these six probands (group 1) and of age/sex-matched mutation-negative controls (group 2). Of 11 group-1 relatives, three carried Q368X, one of whom already had glaucoma. Notably, of the 13 relatives in both groups who were mutation negative, one was already being treated for ocular hypertension. We therefore caution against changing glaucoma surveillance regimens in such individuals and suggest that routine untargeted genetic testing for MYOC mutations in patients with POAG would be of limited value until additional significant genetic risk factors are identified.

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Rapid mutation detection by the Transgenomic wave analyser DHPLC identifies MYOC mutations in patients with ocular hypertension and/or open angle glaucoma

Cited by 16 publications

References 16 publications

Somatic Mutations and Altered Expression of the Candidate Tumor Suppressors CSNK1ε , DLG1 , and EDD/hHYD in Mammary Ductal Carcinoma

Somatic Mutations and Altered Expression of the Candidate Tumor Suppressors CSNK1ε , DLG1 , and EDD/hHYD in Mammary Ductal Carcinoma

Myocilin analysis by DHPLC in French POAG patients: Increased prevalence of Q368X mutation

Low prevalence of MYOC mutations in UK primary open-angle glaucoma patients limits the utility of genetic testing

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