Bell et al. (1) reported mutations
in the hCHK2 gene in families with classical Li-Fraumeni
syndrome (LFS) and in Li-Fraumeni-like (LFL) families. We have
screened genomic DNA for mutations in this gene in one individual from
each of 11 LFS families and 25 LFL families, using primers designed
with reference to GenBank accession number AL117330. Five of these
individuals had what at first appeared to be a single base pair
deletion at nucleotide 1422 (1422delT); four out of 80 normal control
individuals were also found to have a similar sequence variant. In view
of the presence of other sequence variants in all samples, we realized that we may have been amplifying at least two homologous fragments of
the gene that include exons 11 through 14 and that have very similar
but not identical sequences to the gene.
Database searches using the NCBI BLAST server (2, 3)
revealed six homologous fragments that are located on chromosomes 7, 10, 15, 16, 22, and X and that between them encompass exons 10 through
14 of the gene and share 95 to 98% homology. By subcloning polymerase
chain reaction (PCR) products of a sample from a normal control
individual who had the delT variant and subsequently sequencing the
separate alleles, we found that the delT was in a homologous fragment
which had exactly the same sequence as the copy on chromosome 15 except
for the delT (4). There was also evidence of two
other copies that are not in the database. The sequence data in the
BLAST "htgs" database (corresponding to unfinished sequences from
the Human Genome Project), which includes the chromosome 15 homologous
fragment, are unconfirmed and subject to change, so we verified the
presence of this fragment in a chromosome 15 somatic cell hybrid
(5). Because about 5% of the normal controls screened had
the sequence variant, we conclude that this variant is a polymorphism
in the homologous fragment present on chromosome 15.
Bell et al. (1) also reported the 1422delT
variant in a sample from an individual within an LFL family. The
individual had multiple colonic polyps, colorectal cancer, and
bilateral ocular melanomas, which are not among the typical cancers
seen in LFS families. The high level of homology in the introns between the original copy and the homologous fragment (92.5%) suggests that
Bell et al. may also have been amplifying the homologous fragment and that these cancers may not be attributable to mutations in
the hCHK2 gene. The presence of several homologous fragments of the gene has implications for mutation screening in this region of
the gene using genomic DNA.
Nayanta Sodha
Royal Marsden NHS Trust
Downs Road
Sutton
Surrey SM2 5PT, UK
Richard Williams
Jonathan Mangion
Sarah L. Bullock
Martin R. Yuille
Rosalind A. Eeles*
Institute of Cancer Research
Sections of Cancer, Genetics,
and Pediatrics and
Department of
Hematology
and Cytogenetics
Cotswold Road
Sutton
Surrey SM2
5NG, UK
* Also
Royal Marsden NHS Trust
REFERENCES
-
D. W. Bell,
et al.,
Science
286,
2528
(1999)
[Abstract/Free Full Text]
.
-
http://www.ncbi.nlm.nih.gov/BLAST/
-
S. F. Altschul,
et al.,
Nucleic Acids Res.
25,
3389
(1997)
[Abstract/Free Full Text]
.
-
GenBank accession number ACO18963.
-
D. P. Kelsall,
et al.,
Ann. Hum. Genet.
59,
233
(1995)
[Web of Science] [Medline]
.
5 June 2000; accepted 29 June 2000
Response: Sodha et al. note that
recently deposited human genome sequence data reveal duplication of the
3' terminal exons and introns of the hCHK2 gene on
chromosomes 7, 10, 15, 16, and X. Further mapping studies show
additional 3' gene fragments on chromosomes 2, 13, and Y. These partial
gene sequences are highly conserved with that of the parental gene on
chromosome 22, but they are not expressed. PCR-based analysis using
genomic DNA templates leads to variable amplification of these
hCHK2-related sequences, which can be distinguished
from each other by the presence of nucleotide variations.
We have reexamined case DF593, an archival case of LFS variant for
which only genomic DNA was available, and confirm that the 1422delT
mutation is indeed present on the chromosome 15 sequence. Sodha
et al. have now shown that mutation to be a rare
polymorphism in the population. In contrast, the other two reported LFS
cases with germ-line mutations in hCHK2 were identified
using both reverse transcriptase-PCR analysis of the hCHK2
transcript and genomic DNA sequencing. Examination of the sequence
variants surrounding the 1100delC mutation, another focus of our report
(1), confirms that this mutation is present in the
functional hCHK2 gene on chromosome 22. The T470C mutation
within the forkhead homology-associated (FHA) domain is also present in
the transcribed gene, and is located within exon 3, which is not
duplicated.
We agree with Sodha et al. that the discovery of partial 3'
duplications of the hCHK2 gene complicates mutation analysis
based on genomic DNA. Sequence variants identified in these 3' exons may originate from nonfunctional alleles, while genuine mutations present within the hCHK2 transcript may not be readily
confirmed by genomic DNA analysis. In fact, our initial attempts at
detecting loss of heterozygosity (LOH) for the 1100delC mutation within a formalin-fixed tumor specimen were complicated by the
co-amplification of the derivative hCHK2 chromosomal
fragments. Analysis of the chromosome 22-derived sequences, however,
now shows the 1100delC mutation in 11 of 13 independent clones, which
suggests LOH.
The multiple duplications of highly conserved hCHK2 genomic
fragments are highly unusual, but similar genetic events may become apparent as the full human genome sequence is made available.
Daphne W. Bell
Doke C. R. Wahrer
Massachusetts General Hospital
Cancer Center
and Harvard
Medical School
Charlestown, MA 02129, USA
Jennifer M. Varley
Cancer Research Campaign
Department of Cancer Genetics
Paterson Institute for
Cancer Research
Manchester M20 4BX, UK
Daniel A. Haber
Massachusetts General Hospital
Cancer Center
and Harvard Medical School
REFERENCES
-
D. W. Bell,
et al.,
Science
286,
2528
(1999)
.
21 June 2000; accepted 29 June 2000
