Diagnosis of a point mutation in the LDL receptor gene

Direct sequencing demonstrates a mutation in
exon 9. First, exon 9 is amplified by PCR (P1 and
P2 = primers 1 and 2). The mutation in codon
408, GTG (valine) to GTA (methionine), produces
a recognition site (N) for NlaIII (GATC)
that is not normally present. This results in two
fragments of 126 and 96 base pairs (bp) instead
of the usual 222 bp fragment. Thus, affected individuals
(1 and 3 in the pedigree) have two
smaller fragments of 126 and 96 kb (2) in addition
to the 222 kb fragment. Sequence analysis
of the patient (individual 1 in the pedigree)
demonstrates the mutation by the presence of
an additional adenine (A) next to the normal
guanine (3). With this knowledge about the
mutation, the latter can be indirectly demonstrated
within a family by the additional recognition
site for a restriction enzyme.