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American Journal of Medical Genetics 71:238–239 (1997)
Letter to the Editor
Questions and Problems in Direct Predictive
Testing for Huntington’s Disease
To the Editor:
Huntington’s disease (HD) affects about 1 in 10,000
individuals in most European countries. The gene responsible for HD was discovered in 1993, and the mutation mechanism found to be the expansion of a CAG
repeat in the 58-translated region of the gene. Since the
first description of a polymerase chain reaction (PCR)
method to detect the mutation in HD gene was published, several workers have had difficulties reproducing the results [Huntington’s Disease Collaborative
Consortium, 1993]. The other problem with the initial
method was that it described the polymerase chain reaction test with primers flanking a region of a polymorphic CCG repeat, as well as the CAG repeat at the 58
end of the gene. Therefore, a PCR test has been developed that is easy to use and does not amplify the CCG
region [Warner et al., 1993]. We use this modified procedure to test for the presence of a CAG repeat amplification in DNAs from patients with HD and their relatives. Although a false-negative result was reported using this method, we have not had any problems with
the detection of CAG repeats among Hungarian patients until now [Tóth et al., 1996]. Here we report on
a family where several questions were raised during
the test.
A young woman (II) asked for direct predictive testing since her mother had had HD. On repeated PCR
testing, the patient appeared to possess a single allele
in the normal range. In order to check for the homozygosity of the patient, DNA was extracted from the peripheral blood of her parents. We found that the
mother (I) of the patient had one HD and one normal
allele. The latter was equal in size to her daughter’s
allele. The father (IV) had a single allele in the normal
range, but its size was smaller than the patient’s allele
(Fig. 1). Because the father and his daughter also had
a single allele that was not equal in size, we thought
that they both had an allele which was refractory to
PCR amplification at the primer-annealing temperature normally used for the test (65°C in our protocol).
This could be a polymorphism at one of the primer-
*Correspondence to: Tamás Tóth, M.D., I. Department of Obstetrics and Gynaecology, Semmelweis University, Baross u. 27.,
Budapest, H-1088, Hungary. E-mail:
Received 9 July 1996; Accepted 3 February 1997
© 1997 Wiley-Liss, Inc.
annealing sites. To check this possibility, PCR amplifications were repeated with reduced annealing temperatures, as reported earlier [Cross et al., 1994]. On
lowering the temperature to 60°C, a single allele was
detected in both cases, and the amount of mispriming
prevented a further lowering of the temperature. The
second alternative was non-paternity. To confirm this
possibility, we chose the D1S80 VNTR on chromosome
1 for PCR amplification, which is known to be highly
polymorphic. The results indicated that paternity could
be excluded.
Meanwhile, the sister (III) of the patient also visited
us for predictive testing. One normal and one HD allele
were found and those were consistent with her parents’
alleles. Similar results were found on checking her
D1S80 alleles.
Because of the high heterozygosity observed for the
CAG repeats, it was strange that two relatives in this
family were homozygous for this repeat. Therefore, it
was strongly suggested that a non-amplificated allele
is present, but non-paternity was proved, which explained the results. This case suggests the problems
that might be found during tests where a single allele
in the normal size range is seen. We think that nonpaternity is much more frequent in the investigated
families than a polymorphism at the primer annealing
Fig. 1. EA: expanded alleles; NA: normal alleles. I—mother, II—
patient, III—sister of the patient, IV—father.
Letter to the Editor
sites, therefore, one should take this possibility into
consideration first.
Cross G, Pitt T, Sharif A, Bates G, Lehrach H (1994): False-negative result
for Huntington’s disease mutation. Lancet 343:1232.
Huntington’s Disease Collaborative Consortium (1993): A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. Cell 72:971–83.
Tóth T, Németi M, Papp Z (1996): Presymptomatic diagnosis of Huntington’s disease with polymerase chain reaction. Orv Hetil 137:451–454.
Warner JP, Barron LH, Brock DJH (1993): A new polymerase chain reaction (PCR) assay for the trinucleotide repeat that is unstable and expanded on Huntington’s disease chromosomes. Mol Cell Probes 7:235–
Tamás Tóth*
Csaba Papp
Margit Németi
Zoltán Papp
I. Department of Obstetrics and Gynaecology
Semmelweis University
Budapest, Hungary
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