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Anaesthesia and Genetics of the Ryanodine 1 Receptor

Broman, Marcus Ewert LU (2011) In Lund University Faculty of Medicine Doctoral Dissertation Series 2011:38.
Abstract
The only validated method to characterize the phenotype and to reach a definitive diagnosis

of Malignant Hyperthermia Susceptibility (MHS; OMIM *145600), a pharmacogenetic

disease linked to the Ryanodine 1 receptor gene (RYR1; OMIM *180901), is

an invasive muscle contraction test requiring a muscle biopsy, according to the European

(IVCT) protocol or to the North American (CHCT) protocol.

The series of scientific papers in this thesis tries to reach as far as possible in diagnosing

MHS from peripheral venous blood samples collected at the patients’ local primary

care provider and sent to the laboratory over significant transportation time and distance.

Study I uses a... (More)
The only validated method to characterize the phenotype and to reach a definitive diagnosis

of Malignant Hyperthermia Susceptibility (MHS; OMIM *145600), a pharmacogenetic

disease linked to the Ryanodine 1 receptor gene (RYR1; OMIM *180901), is

an invasive muscle contraction test requiring a muscle biopsy, according to the European

(IVCT) protocol or to the North American (CHCT) protocol.

The series of scientific papers in this thesis tries to reach as far as possible in diagnosing

MHS from peripheral venous blood samples collected at the patients’ local primary

care provider and sent to the laboratory over significant transportation time and distance.

Study I uses a traditional method of direct sequencing of a limited number of central

exons of the RYR1 gene in the Swedish population with an outcome of only one fifth

of the tested probands positive for a known MH causative mutation.

Study II tries to bypass the laborious direct sequencing method of the very large

RYR1 gene by using a method of stabilizing RNA in test tubes for transportation and

synthesizing of RYR1cDNA for direct sequencing.

Study IV implies a recent method based on establishing and analysing High

Resolution Melting (HRM) DNA curves of 131 amplicons of the RYR1 on stable

genomic DNA with an encouraging finding of a sequence variant in 81 % of the tested

patients and similarly reducing the sequencing work by 79 %.

By analysing all the found sequence variants in studies I, II and IV leading to amino

acid changes in the final RYR1 receptor, it seems that the formerly known hotspot

(N-terminal, central and C-terminal) distribution pattern can be seen, but sequence

variants appear also outside of these traditional hotspots.

Therefore coverage of the total coding region of the gene remains necessary.

Another fact is that about half of the found sequence variants leading to amino acid

changes are previously unknown and therefore uncharacterized at functional level.

Study III presents a method of measuring the [Ca2+]i resting level and the increase in

cytoplasmic [Ca2+]i level in prepared B lymphoblastic cell clones from patients carrying

the actual candidate mutations, after influence of a RYR1 agonist. And of combining

the outcome data on cellular level with clinical data.

The series of studies I-IV presented in this thesis cannot give a definitive method

to reach the diagnosis MHS, or even more important; to reach the definitive diagnosis

of MHN, without an IVCT. But it can present advanced techniques to describe the

genetical status of the patients and the impact of new previously unknown candidate

mutations at functional level, and to combine this data with clinical data, based on only

a simple venous blood sample.

Once a MH causative mutation is found in a pedigree this allows for predictive testing

in the family members and the individuals positive for the actual mutation can be

assigned MHS without an IVCT, whereas the individuals negative for the mutation

must go through IVC testing in order to confirm the MHN status. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • professor Brandom, Barbara, Department of Anaesthesiology, Childrens Hospital of Pittsburgh, USA
organization
publishing date
type
Thesis
publication status
published
subject
keywords
General Anaesthesia, Ryanodine 1 Receptor, Malignant Hyperthermia, Genetics
in
Lund University Faculty of Medicine Doctoral Dissertation Series
volume
2011:38
pages
110 pages
publisher
Department of Clinical Sciences, Lund University
defense location
Skåne University Hospital, Lund, Lecture Hall 4
defense date
2011-05-06 13:00
ISSN
1652-8220
ISBN
978-91-86671-86-0
language
English
LU publication?
yes
id
24474597-d4ce-4b02-8e5e-337da835201b (old id 1988274)
date added to LUP
2011-06-28 13:44:03
date last changed
2016-09-19 08:44:49
@phdthesis{24474597-d4ce-4b02-8e5e-337da835201b,
  abstract     = {The only validated method to characterize the phenotype and to reach a definitive diagnosis<br/><br>
of Malignant Hyperthermia Susceptibility (MHS; OMIM *145600), a pharmacogenetic<br/><br>
disease linked to the Ryanodine 1 receptor gene (RYR1; OMIM *180901), is<br/><br>
an invasive muscle contraction test requiring a muscle biopsy, according to the European<br/><br>
(IVCT) protocol or to the North American (CHCT) protocol.<br/><br>
The series of scientific papers in this thesis tries to reach as far as possible in diagnosing<br/><br>
MHS from peripheral venous blood samples collected at the patients’ local primary<br/><br>
care provider and sent to the laboratory over significant transportation time and distance.<br/><br>
Study I uses a traditional method of direct sequencing of a limited number of central<br/><br>
exons of the RYR1 gene in the Swedish population with an outcome of only one fifth<br/><br>
of the tested probands positive for a known MH causative mutation.<br/><br>
Study II tries to bypass the laborious direct sequencing method of the very large<br/><br>
RYR1 gene by using a method of stabilizing RNA in test tubes for transportation and<br/><br>
synthesizing of RYR1cDNA for direct sequencing.<br/><br>
Study IV implies a recent method based on establishing and analysing High<br/><br>
Resolution Melting (HRM) DNA curves of 131 amplicons of the RYR1 on stable<br/><br>
genomic DNA with an encouraging finding of a sequence variant in 81 % of the tested<br/><br>
patients and similarly reducing the sequencing work by 79 %.<br/><br>
By analysing all the found sequence variants in studies I, II and IV leading to amino<br/><br>
acid changes in the final RYR1 receptor, it seems that the formerly known hotspot<br/><br>
(N-terminal, central and C-terminal) distribution pattern can be seen, but sequence<br/><br>
variants appear also outside of these traditional hotspots.<br/><br>
Therefore coverage of the total coding region of the gene remains necessary.<br/><br>
Another fact is that about half of the found sequence variants leading to amino acid<br/><br>
changes are previously unknown and therefore uncharacterized at functional level.<br/><br>
Study III presents a method of measuring the [Ca2+]i resting level and the increase in<br/><br>
cytoplasmic [Ca2+]i level in prepared B lymphoblastic cell clones from patients carrying<br/><br>
the actual candidate mutations, after influence of a RYR1 agonist. And of combining<br/><br>
the outcome data on cellular level with clinical data.<br/><br>
The series of studies I-IV presented in this thesis cannot give a definitive method<br/><br>
to reach the diagnosis MHS, or even more important; to reach the definitive diagnosis<br/><br>
of MHN, without an IVCT. But it can present advanced techniques to describe the<br/><br>
genetical status of the patients and the impact of new previously unknown candidate<br/><br>
mutations at functional level, and to combine this data with clinical data, based on only<br/><br>
a simple venous blood sample.<br/><br>
Once a MH causative mutation is found in a pedigree this allows for predictive testing<br/><br>
in the family members and the individuals positive for the actual mutation can be<br/><br>
assigned MHS without an IVCT, whereas the individuals negative for the mutation<br/><br>
must go through IVC testing in order to confirm the MHN status.},
  author       = {Broman, Marcus Ewert},
  isbn         = {978-91-86671-86-0},
  issn         = {1652-8220},
  keyword      = {General Anaesthesia,Ryanodine 1 Receptor,Malignant Hyperthermia,Genetics},
  language     = {eng},
  pages        = {110},
  publisher    = {Department of Clinical Sciences, Lund University},
  school       = {Lund University},
  series       = {Lund University Faculty of Medicine Doctoral Dissertation Series},
  title        = {Anaesthesia and Genetics of the Ryanodine 1 Receptor},
  volume       = {2011:38},
  year         = {2011},
}