Confirmation of Single Exon Deletions in MLH1 and MSH2 Using Quantitative Polymerase Chain Reaction

Vaughn, Cecily P.; Lyon, Elaine; Samowitz, Wade S.

doi:10.2353/jmoldx.2008.080021

Cited by 15 publications

(5 citation statements)

References 19 publications

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“…Ratios obtained from gene single‐copy deletions range from 0.4 to 0.7, whereas ratios obtained from the presence of two copies range from 0.8 to 1.2. Ratios obtained from gene duplications (3 copies vs. the normal 2 copies) range from 1.3 to 1.7 [Vaughn et al, 2008].…”

Section: Methodsmentioning

confidence: 99%

Chromosomal loss of 3q26.3‐3q26.32, involving a partial neuroligin 1 deletion, identified by genomic microarray in a child with microcephaly, seizure disorder, and severe intellectual disability

Millson

LaGrave

Willis

et al. 2011

American J of Med Genetics Pt A

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Neuroligin 1 (NLGN1) is one of five members of the neuroligin gene family and may represent a candidate gene for neurological disorders, as members of this family are involved in formation and remodeling of central nervous system synapses. NLGN1 is expressed predominantly in the central nervous system, where it dimerizes and then binds with β-neurexin to form a functional synapse. Mutations in neurexin 1 (NRXN1) as well as two other members of the neuroligin family, NLGN3 and NLGN4, have been associated with autism and mutations in NLGN4 have also been associated with intellectual disability, seizures, and EEG abnormalities. Genomic microarray is recommended for the detection of chromosomal gains or losses in patients with intellectual disability and multiple congenital anomalies. Results of uncertain significance are not uncommon. Parental studies can provide additional information by demonstrating that the imbalance is either de novo or inherited, and therefore is more or less likely to be causative of the clinical phenotype. However, the possibility that even inherited deletions and duplications may play a role in the phenotype of the proband cannot be excluded as many copy number variants associated with neurodevelopmental conditions show incomplete penetrance and may be inherited from an unaffected parent. Here, we report on a patient with a 2.2 Mb deletion at 3q26.3-3q26.32-encompassing the terminal end of NLGN1 and the entire NAALADL2 gene-detected by genomic microarray, and confirmed by FISH and real-time quantitative PCR. The same size deletion was subsequently found in her healthy, asymptomatic, adult mother.

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Section: Methodsmentioning

confidence: 99%

Chromosomal loss of 3q26.3‐3q26.32, involving a partial neuroligin 1 deletion, identified by genomic microarray in a child with microcephaly, seizure disorder, and severe intellectual disability

Millson

LaGrave

Willis

et al. 2011

American J of Med Genetics Pt A

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“…In patient 7 we detected a homozygous deletion of PMS2 exons 14-15. Because this deletion was homozygous in the patient, we were able to use quantitative PCR [Vaughn et al, 2008] to confirm the deletion. This was done by amplifying exons 14 and 15 in PMS2 and PMS2CL (two alleles remaining in the patient) and comparing to the amplification of exon 11 in both genes (four alleles) (data not shown).…”

Section: Discussionmentioning

confidence: 99%

Avoidance of pseudogene interference in the detection of 3′ deletions in PMS2

et al. 2011

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Lynch syndrome is characterized by mutations in the mismatch repair genes MLH1, MSH2, MSH6, and PMS2. In PMS2, detection of mutations is confounded by numerous pseudogenes. Detection of 3' deletions is particularly complicated by the pseudogene PMS2CL, which has strong similarity to PMS2 exons 9 and 11-15, due to extensive gene conversion. A newly designed multiplex ligation-dependent probe amplification (MLPA) kit incorporates probes for variants found in both PMS2 and PMS2CL. This provides detection of deletions, but does not allow localization of deletions to the gene or pseudogene. To address this, we have developed a methodology incorporating reference samples with known copy numbers of variants, and paired MLPA results with sequencing of PMS2 and PMS2CL. We tested a subset of clinically indicated samples for which mutations were either unidentified or not fully characterized using existing methods. We identified eight unrelated patients with deletions encompassing exons 9-15, 11-15, 13-15, 14-15, and 15. By incorporating specific, characterized reference samples and sequencing the gene and pseudogene it is possible to identify deletions in this region of PMS2 and provide clinically relevant results. This methodology represents a significant advance in the diagnosis of patients with Lynch syndrome caused by PMS2 mutations.

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“…Additionally, all single exon deletions were confirmed using quantitative real-time PCR as previously described [Vaughn et al, 2008].…”

Section: Multiplex Ligation-dependent Probe Amplificationmentioning

confidence: 99%

Clinical analysis ofPMS2: mutation detection and avoidance of pseudogenes

et al. 2010

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Germline mutation detection in PMS2, one of four mismatch repair genes associated with Lynch syndrome, is greatly complicated by the presence of numerous pseudogenes. We used a modification of a long-range PCR method to evaluate PMS2 in 145 clinical samples. This modification avoids potential interference from the pseudogene PMS2CL by utilizing a long-range product spanning exons 11-15, with the forward primer anchored in exon 10, an exon not shared by PMS2CL. Large deletions were identified by MLPA. Pathogenic PMS2 mutations were identified in 22 of 59 patients whose tumors showed isolated loss of PMS2 by immunohistochemistry (IHC), the IHC profile most commonly associated with a germline PMS2 mutation. Three additional patients with pathogenic mutations were identified from 53 samples without IHC data. Thirty-seven percent of the identified mutations were large deletions encompassing one or more exons. In 27 patients whose tumors showed absence of either another protein or combination of proteins, no pathogenic mutations were identified. We conclude that modified long-range PCR can be used to preferentially amplify the PMS2 gene and avoid pseudogene interference, thus providing a clinically useful germline analysis of PMS2. Our data also support the use of IHC screening to direct germline testing of PMS2.

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Confirmation of Single Exon Deletions in MLH1 and MSH2 Using Quantitative Polymerase Chain Reaction

Cited by 15 publications

References 19 publications

Chromosomal loss of 3q26.3‐3q26.32, involving a partial neuroligin 1 deletion, identified by genomic microarray in a child with microcephaly, seizure disorder, and severe intellectual disability

Chromosomal loss of 3q26.3‐3q26.32, involving a partial neuroligin 1 deletion, identified by genomic microarray in a child with microcephaly, seizure disorder, and severe intellectual disability

Avoidance of pseudogene interference in the detection of 3′ deletions in PMS2

Clinical analysis ofPMS2: mutation detection and avoidance of pseudogenes

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