Fig. 2. Identification of the R328W mutation in transcript and genomic DNA derived from leukocytes. (A ) The C982T transition was first identified by direct sequencing of the RYR1  transcript and then of the corresponding fragment from genomic DNA. The four chromatographs, from top  to bottom , correspond to malignant hyperthermia–susceptible (MHS) transcript, normal transcript, malignant hyperthermia–susceptible genomic DNA, and normal genomic DNA. The arrow  indicates the position of the C982T heterozygous mutation. (B ) The R328W mutation was detected by Aci  I restriction endonuclease digestion of the 395-bp polymerase chain reaction–amplified fragment from genomic DNA, as described in the Materials and Methods section, from individuals within the pedigree shown in figure 1. Lane 1 , before Aci  I digestion; lane 2 , Aci  I digestion of the normal allele generates 173-, 137-, and 85-bp fragments; lanes 3–5 , loss of one Aci  I restriction site in one allele in malignant hyperthermia–susceptible individuals is detected by the appearance of a 258-bp fragment and a reduction in intensity of the 173- and 85-bp fragments; lane M , a 50-bp marker ladder. (C ) Alignment of the human RyR1 protein sequence with those in rabbit, mouse, pig, bullfrog, and fish in the region flanking the mutated amino acid residue shows evolutionary conservation of Arg328.