Protocol for the sequence analysis of ryanodine receptor subtype 1 gene transcripts from human leukocytes.
BACKGROUND
The search for novel mutations in the ryanodine receptor subtype 1 (RYR1) gene causing malignant hyperthermia and central core disease is hampered by the fact that the gene contains 106 exons. Searching for novel mutations in complementary DNA (cDNA) requires an invasive muscle biopsy. Accordingly, an alternate source of RYR1 cDNA was sought for sequence analysis.


METHODS
Leukocytes were isolated from human blood and used for extraction of RNA and reverse transcription of messenger RNA into cDNA. A detailed protocol was developed in which overlapping fragments of RYR1 cDNA were amplified by polymerase chain reaction in a series of steps and used for double-strand sequencing.


RESULTS
The sequences of full-length leukocyte RYR1 cDNA obtained from four human blood samples were shown to be identical to the sequence of a human muscle RYR1 cDNA. The incidence of aberrant splicing was more pronounced in the blood-derived cDNAs, but this could be minimized by adequate sample preparation. Protocols to sequence alternatively spliced products were also developed. Several silent nucleotide polymorphisms were detected, and minor revisions were made to the RYR1 sequence.


CONCLUSIONS
Because there are no differences in RYR1 transcript structure between muscle and leukocytes, aside from those that may be ascribed to RNA splicing aberrations during processing, leukocytes seem to be an adequate substitute tissue for screening the RYR1 gene for previously undiscovered mutations in families with malignant hyperthermia or central core disease.
Kraev
Natasha
N
Department of Anaesthesia, University of Toronto, Ontario, Canada.
Loke
Julian C P
JC
Kraev
Alexander
A
MacLennan
David H
DH
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007962:Leukocytes / Q000378:metabolism*; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D019837:Ryanodine Receptor Calcium Release Channel