HCCAA has been shown to be eaused by a T–
>A point mutation in the codon for leucine at
position 68 in thecystatioC genewith aconcornitant
glutarnine for leucine exchange in the cystatin C
molecule (16-18). Sequence analysis has shown
that the 5' end of the structural gene for cystatin C,
isolated froman HCCAApatient, is intact indicating
that the cystatin C amyloid is probably eaused by
post-tfanslational modification (17).
Molecular diagnosis of HCCAA
Diagnosis ofHCCAA has earlier been based upon
immunohistochernical studies ofbiopsy specimens
(9-11) and measurements of cystatin C in the CSF,
as HCCAA is characterized by an abnormally low
level of cystatio C in the CSF (12).
Since the HCCAA-causing mutation abolishes a
normally occurring
Alul
restfiction site in exon 2
1
2 3
4
of the cystatio C gene, analysis of this
Alul
restfiction fragment length polymorphism (RFLP)
enables simple and accurate molecular diagnosis
ofHCCAA (16, 18).
Southern blot analysis
A full-length cystatin C cDNA (19) and the
restfiction enzyme
Alul
have been used to identify
the RFLP, which deteets a loss ofan
Alu/
restfiction
site in the cystatin C gene in HCCAA patients and
a corresponding increase in 600-bp fragment to
630-bp fragment (16). The patients heterozygous
for the HCCAA-causing mutation also display the
600-bp
Alul
fragment which represents the normal
cystatin C gene (Fig. l).
This 630-bp
Alul
marker has been used for the
diagnosis of patients, asymptomatic affected
individuals and for prenatal diagnosis, which has
been performed in one farnily. The patient, an 18-
5 6
o
c:
1
8
l
630
.,...._
&DO Il,
l
•
a)
b)
LEGEND FOR FIG. l
Hybridization of cystatin C cDNA probe to
Alul
digested DNA from two HCCAA families. (a) Family origrnating
in the south of Iceland. (b) Prenatal diagnosis in an HCCAA family . Patients are shown as black circles (females)
andsquares (males). The 630-bp fragment is the marker for HCCAA (arrow). Unaffected individuals, including a
fetus (arrow), have only the 600-bp
Alu/
fragment (lanes 2,4, 5, 6 and 8) whereas patients also have the 630-bp
Alu/
fragment (lanes l, 3, 7 and 9).
18
Klinisk kemi i Norden 2, 1993
