Protocol for the Sequence Analysis of Ryanodine Receptor Subtype 1 Gene Transcripts from Human Leukocytes

Kraev, Natasha; Loke, Julian; Kraev, Alexander; MacLennan, David H.

doi:10.1097/00000542-200308000-00010

Cited by 29 publications

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“…34 Using RNA from purified or transformed lymphocytes, others have successfully amplified mRNA encoding beta-myosin heavy chain (MYH7), one of the proteins mutated in HCM, 14 as well as genes involved in cystic fibrosis (CFTR), 35 Duchenne muscular dystrophy (DMD), 36 and malignant hyperthermia (RYR1). 37 Differences in gene expression patterns between purified or transformed lymphocytes and unfractionated whole blood cells 38 may actually favor the detection of certain transcripts. 14,15 On the other hand, using whole blood RNA, we have successfully amplified mRNA from multiple genes not previously accessible to RNA sequencing, including those linked to LQTS/Brugada syndrome (SCN5A), ARVD (PKP2), dilated cardiomyopathy (lamin A/C, LMNA) and Barth Syndrome (tafazzin, TAZ; our unpublished data).…”

Section: Applicability Of Whole Blood Rna Testing In Other Disordersmentioning

confidence: 99%

Whole blood RNA offers a rapid, comprehensive approach to genetic diagnosis of cardiovascular diseases

Miller

You

Myerburg

et al. 2007

Genetics in Medicine

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Purpose: Long QT Syndrome, Marfan Syndrome, hypertrophic and dilated cardiomyopathy are caused by mutations in large, multi-exon genes that are principally expressed in cardiovascular tissues. Genetic testing for these disorders is labor-intensive and expensive. We sought to develop a more rapid, comprehensive, and cost-effective approach.Methods: Paired whole blood samples were collected into tubes with or without an RNA-preserving solution, and harvested for whole blood RNA or leukocyte DNA, respectively. Large overlapping cDNA fragments from KCNQ1 and KCNH2 (Long QT Syndrome), MYBPC3 (hypertrophic and dilated cardiomyopathy), or FBN1 (Marfan Syndrome) were amplified from RNA and directly sequenced. Variants were confirmed in leukocyte DNA. Results: All 4 transcripts were amplified and sequenced from whole blood mRNA. Six known and 2 novel mutations were first identified from RNA of 10 probands, and later confirmed in genomic DNA, at considerable savings in time and cost. In one patient with MFS, RNA sequencing directly identified a splicing mutation. Results from RNA and DNA were concordant for single nucleotide polymorphisms at the same loci. Conclusion: Taking advantage of new whole blood RNA stabilization methods, we have designed a cost-effective, comprehensive method for mutation detection that should significantly facilitate clinical genetic testing in four lethal cardiovascular disorders. Genet Med 2007:9(1):23-33.

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Section: Applicability Of Whole Blood Rna Testing In Other Disordersmentioning

confidence: 99%

Whole blood RNA offers a rapid, comprehensive approach to genetic diagnosis of cardiovascular diseases

Miller

You

Myerburg

et al. 2007

Genetics in Medicine

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“…25,26 Ribonucleic acid (RNA) isolation from blood leukocytes as well as complementary DNA (cDNA) synthesis and polymerase chain reaction (PCR) amplification of the RYR1 transcript were performed as described previously, 27 with minor modifications. Sequencing reactions were run at the DNA Sequencing and Synthesis Facility of The Centre for Applied Genomics, Toronto, Canada.…”

Section: Transcript Sequencingmentioning

confidence: 99%

Ryanodine receptor type 1 gene mutations found in the Canadian malignant hyperthermia population

Kraeva

Riazi

Loke

et al. 2011

Can J Anesth/J Can Anesth

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Purpose Malignant hyperthermia (MH) is an autosomal dominant pharmacogenetic disorder that is manifested on exposure of susceptible individuals to halogenated anesthetics or succinylcholine. Since MH is associated primarily with mutations in the ryanodine receptor type 1 (RYR1) gene, the purpose of this study was to determine the distribution and frequency of MH causative RyR1 mutations in the Canadian MH susceptible (MHS) population. Methods In this study, we screened a representative cohort of 36 unrelated Canadian MHS individuals for RYR1 mutations by sequencing complete RYR1 transcripts and selected regions of CACNA1S transcripts. We then analyzed the correlation between caffeine-halothane contracture test (CHCT) results and RYR1 genotypes within MH families. Results Eighty-six percent of patients had at least one RyR1 mutation (31 out of 36), five of which were unrelated individuals who were double-variant carriers. Fifteen of the 27 mutations identified in RYR1 were novel. Eight novel mutations, involving highly conserved amino acid residues, were predicted to be causal. Two of the mutations co-segregated with the MHS phenotype within two large independent families (a total of 79 individuals). Fourteen percent of MHS individuals (five out of 36) carried neither RYR1 nor known CACNA1S mutations. Conclusions The distribution and frequency of MH causative RyR1 mutations in the Canadian MHS population are close to those of European MHS populations. Novel mutations described in this study will contribute to the worldwide pool of MH-associated mutations in the RYR1 gene, ultimately increasing the value of MH genetic diagnostic testing. RésuméObjectif L'hyperthermie maligne (HM) est une maladie pharmacoge´ne´tique he´re´ditaire dominante autosomique qui se manifeste lors de l'exposition des personnes susceptibles a`des anesthe´siques haloge´ne´s ou à la succinylcholine. E´tant donne´que l'HM est principalement associe´e aux mutations au niveau du ge`ne des re´cepteurs de ryanodine de type 1 (RYR1), l'objectif de cette e´tude e´tait de de´terminer la distribution et la fre´quence des mutations RyR1 causant une HM chez la population canadienne susceptible a`l'HM (SHM). Méthode Dans cette e´tude, nous avons examine´une cohorte repre´sentative de 36 personnes canadiennes SHM This article is accompanied by an editorial. Please see Can J Anesth 2011; 58: 6.

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“…16 Sequencing reactions were performed at the DNA Sequencing and Synthesis Facility of The Centre for Applied Genomics (Toronto, ON, Canada). Raw sequence data analysis (contig building and sequence comparison with the reference RYR1 sequence, GenBank accession NC 000019) was carried out using Sequencher 4.10.1 (Gene Codes, Ann Arbor, MA, USA).…”

Section: Genetic Testingmentioning

confidence: 99%

Analysis of histomorphology in malignant hyperthermia-susceptible patients

Orlov

Keith

Rosen

et al. 2013

Can J Anesth/J Can Anesth

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Background Malignant hyperthermia (MH) is a potentially lethal disorder of skeletal muscle triggered by anesthetic agents. A histomorphological examination of diseased muscle may provide insight into MH pathophysiology, but it is not a routine part of standardof-care practice for the identification of MH-susceptibility. In this study, we investigated muscle histomorphology in a large cohort of MH-susceptible (MHS) patients and examined its relationship to genotype and phenotype.Methods All consenting patients who were identified as MHS based on a caffeine-halothane contracture test (CHCT) performed during 1992-2011 were retrospectively identified and recruited for this study. Results of the histomorphological examination, which is a routine part of our centre-specific practice, were reviewed. Patient demographics, MH proband status, histological features, CHCTs, and genetic results for MH-causative mutations were summarized. Results Seven of the 399 patients classified as MHS had histological characteristics consistent with central core disease, and one patient was a carrier of Duchenne's muscular dystrophy. Eighty-six (22%) patients had histological abnormalities, and five (6%) of these had evidence of ''frank'' myopathy. No histologic abnormalities were consistent among the MHS patients; however, a higher proportion of MH probands had abnormal histomorphology compared with the general MHS population, and patients with evidence of ''frank'' myopathy showed similarities in clinical history, biochemistry, CHCT, and genetic testing. Author contributions David Orlov and Sheila Riazi participated in the analysis and interpretation of data. David Orlov wrote the original draft. David Orlov, Julia Keith, Derek Rosen, Sidney Croul, and Sheila Riazi were involved with the critical revision of the original manuscript. Julia Keith, Derek Rosen, Sidney Croul, Natalia Kraeva, and Sheila Riazi were involved with the acquisition of data. Julia Keith and Sidney Croul were involved with the analysis and interpretation of the histomorphological results. Derek Rosen, Natalia Kraeva, and Sheila Riazi were involved with the conception and design of the study.

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Protocol for the Sequence Analysis of Ryanodine Receptor Subtype 1 Gene Transcripts from Human Leukocytes

Cited by 29 publications

References 0 publications

Whole blood RNA offers a rapid, comprehensive approach to genetic diagnosis of cardiovascular diseases

Whole blood RNA offers a rapid, comprehensive approach to genetic diagnosis of cardiovascular diseases

Ryanodine receptor type 1 gene mutations found in the Canadian malignant hyperthermia population

Analysis of histomorphology in malignant hyperthermia-susceptible patients

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