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.
- Department of Clinical Sciences, Lund
- Bodelsson, Mikael, Supervisor
- Islander, Gunilla, Supervisor
- Muller, Clemenz, Supervisor, External person
|Award date||2011 May 6|
|Publication status||Published - 2011|
Place: Skåne University Hospital, Lund, Lecture Hall 4
Name: Brandom, Barbara
Affiliation: Department of Anaesthesiology, Childrens Hospital of Pittsburgh, USA
- General Anaesthesia
- Ryanodine 1 Receptor
- Malignant Hyperthermia