PMC3447225_01
PMC3447225_02
Upper lip of the patient at age 3 6/12 yrs. Note the moustache with dark pigmented hair over the upper lip and the scars of the cleft lip operations.
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fig1
Except the moustache with dark pigmented hair all over the upper lip (Figure 1) and the scars of the cleft lip operations, physical examination was normal. 
PMC3447225_03
Results of CYP21A2 sequencing of DNA of the patient. (a) panel shows part of the exon 3 sequence with the R132C mutation indicated. (b) panel shows part of the exon 8 sequence with the R339H mutation indicated. Both mutations are present in a heterozygous state.
CRIM.GENETICS2011-913020.002.jpg
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fig2
The obtained sequences were compared to the normal sequence, and in the patient 2 mutations were found: R132C in exon 3 and R339H in exon 8 (Figure 2). The observed mutations were confirmed by resequencing (R132C) or by restriction analysis (R339H).
Results of restriction analysis. Lane 1: molecular weight markers; lane 2: normal control; lane 3: the mother of the patient: lane 4: the patient. The wild-type sequence has a ApaI site resulting in two fragments of 152 bp and 144 bp (are not separated on the gel). The mutation destroys this site, leaving the PCR product of 296 bp uncut.
CRIM.GENETICS2011-913020.003.jpg
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fig3
Restriction of the PCR product from the patient confirmed the heterozygosity, showing both 296 (mutant) + 144 + 152 (wild-type) (Figure 3).