year-old woman, had a 2-year-old asymptomatic
daughter with the HCCAA-causing mutation and
requested DNA analysis for her seeond pregnancy.
DNA from ehorionie villi, sampied between the
9th and 10th week of gestation, was analysed for
the presenee of the 630-bp
Alu/
marker for HCCAA
(20). The fetus did not have the 630-bp
Alul
marker and therefore has not inherited the disease
(Fig. lb).
Southern blot analysis of the
Alul
RFLP is an
accurate, although time-consurning diagnostic
procedure for HCCAA.
Polymerase chain reaction based analysis
To further simplify the diagnosis of HCCAA, a
rapid polymerase chain reaction (PCR)- based
procedure has been devised recently (18). This
method is based on the same principle as the
Southern blot analysis, i.e., the
Alul
RFLP due to
a loss of an
Alu/
restfiction site in HCCAApatients
(16, 18). A 275-bp genomic segment encoding
exon 2 of the cystatio C gene is amplified by PCR
(Fig. 2). Digestion of this segment with
Alul
results in the appearance of 4 DNA fragments of
29, 70, 176, and 205 bp, respectively, in carriers
5'-(GGGAATTC)TGTAAGAAACTGACAGAACG -3'
... AGTGAAGTGCCTGTGTGTGTAAGAAACTGACAGAACGTGCTGTCCCTGCCTCCTGCT
56
60
(Gln)
IleValAlaGlyValAsnTyrPheLeuAspValGluLeuGlyArg
CTTTCACATGTGTAGATCGT~GGGGTGAACTACTTCTTGGACGTGG~GGGCCGA
(A)
80
90
ThrThrCysThrLysThrGlnProAsnLeuAspAsnCysProPheHisAspGlnProHis
ACCACGTGTACCAAGACCCAGCCCAACTTGGACAACTGCCCCTTCCATGACCAGCCACAT
93
LeuLysArg
CTGAAAAGGGTATGTGCCTTATATGGGTCCAGGGCCAGTCATACACTGCAGAGGGGTGTG
TGTGTGTGTGTGTGTGTGTGATGCACATGTTCTGCAGGGTACGTGTGCATGTGCCTG ...
3 ' -
CTACGTGTACAAGACGTCCC(TTCGAAGG) -5'
LEGEND FOR FIG. 2
Prirnary structure of the genornic
DNA
segment arnplified in the PCR-based diagnostic proeecture for
HCCAA.
The
nucleotide sequence of exon 2 of the normal human cystatin Cgene and its flanking intron sequences is shown with
the deduced arnino acid sequence given above (13). The sequences ofthe primers u
sed
in the PCR are shown at their
sites of specific hybridization (18). The
Alul
restfiction sites within the genarnie region are underlined. The arnino
acid substituted in the cystatin
C
from
HCCAA
patients and the corresponding base substitution are in parentheses.
Klinisk kemi i Norden 2, /993
19
