Download Novel genomic insertion–deletion in MLH1: possible mechanistic

Clin Genet 2005: 68: 234–238
Printed in Singapore. All rights reserved
Copyright # Blackwell Munksgaard 2005
CLINICAL GENETICS
doi: 10.1111/j.1399-0004.2005.00486.x
Short Report
Novel genomic insertion–deletion in
MLH1: possible mechanistic role for nonhomologous end-joining DNA repair
McVety S, Younan R, Li L, Gordon PH, Wong N, Foulkes WD,
Chong G. Novel genomic insertion–deletion in MLH1: possible
mechanistic role for non-homologous end-joining DNA repair.
Clin Genet 2005: 68: 234–238. # Blackwell Munksgaard, 2005
S McVetya, R Younanb, L Lic,
PH Gordond, N Wonga,e,
WD Foulkesa,c,e,f and G Chonga,f
a
Department of Human Genetics,
Department of Surgery, cProgram in
Cancer Genetics, Department of
Oncology and Human Genetics,
d
Department of Surgery, Division of
Colorectal Surgery, Sir Mortimer B. Davis
Jewish General Hospital, eCancer
Prevention Center, and fDepartment of
Diagnostic Medicine, SMBD Jewish
General Hospital, McGill University,
Montreal, Quebec, Canada
b
Hereditary non-polyposis colorectal cancer (HNPCC) is an inherited
cancer syndrome caused by a defect in the mismatch repair pathway.
The majority of HNPCC mutations have been detected in MLH1 and
MSH2. Most reported mutations are substitutions, small insertions and
deletions, but standard methods of mutation analysis do not detect large
rearrangements. It is now established that large deletions, insertions and
rearrangements account for a significant proportion of MLH1 and
MSH2 mutations. We report an unusual rearrangement resulting in the
deletion of exons 6, 7 and 8 of MLH1, with the retention of part of
intron 6 and insertions of two nucleotides each flanking the retained
sequence. The 349-bp-retained sequence is made up of two closely
spaced Alu sequences. The mutation was initially detected by protein
truncation test and cDNA sequencing. Multiplex ligation-dependent
probe amplification confirmed the deletion of three exons. PCR and
sequencing were used to characterize the breakpoint. Despite the high
density of Alu elements in MLH1, there is no homology at the deletion
breakpoints or insertion junctions in this case to suggest that homologous recombination has occurred. We propose a mechanism involving
non-homologous end joining to explain the occurrence of this complex
deletion.
Key words: DNA repair–DNA sequence
analysis–gene rearrangement–hereditary
non-polyposis colorectal neoplasms–
molecular diagnostic techniques–nucleic
acid repetitive sequences
Corresponding author: William Foulkes,
Department of Medical Genetics, Cancer
Prevention Center, Jewish General
Hospital, Room C-107.1, 3755 Cote Ste
Catherine, Montreal, Quebec, Canada
H3T 1E2.
Tel.: þ1 514 340 8222x3965;
fax: þ1 514 340 8600;
e-mail: [email protected]
Received 23 April 2005, revised and
accepted for publication 24 May 2005
Hereditary non-polyposis colorectal cancer is an
inherited cancer syndrome caused by a mutation
in a mismatch repair gene, with the majority of
mutations being detected in MLH1 and MSH2
(1). Somatic inactivation of the remaining copy
leads to cancer. Large rearrangements account
for a significant proportion of germline MLH1
and MSH2 mutations (2–8).
Most large deletions characterized in MLH1
are attributed to homologous recombination
(HR) between Alu repeats (2–4, 6, 7). Alu
sequences are present in MLH1 at an average of
one per 1.5 kb of sequence and it has been suggested that a high density of Alu repeats may
234
promote recombination causing large deletions
(9, 10). However, Gebow et al. (11) showed that
the presence of Alu sequences per se does not
increase the frequency of large deletions, and
suggest that repeating sequences are used preferentially by cells to repair double stranded breaks
(DSBs). Alu sequences may also interact with
other Alus due to homology, bringing sequences
closer together and making recombination in or
near Alu sequences more likely (12).
Here, we report an unusual rearrangement that
results in the deletion of exons 6, 7 and 8 of
MLH1. A short sequence in intron 6 is preserved
and insertions of two nucleotides each flank the
Novel genomic insertion–deletion in MLH1
(b)
Ex6
Ex7
Ex6
Ex7
Ex8
Control DNA
Dosage quotient
Patient DNA
Ex8
1.200
1.100
1.000
0.900
0.800
0.700
0.600
0.500
0.400
0.300
0.200
0.100
0.000
C5q31
C10p11
MLH1 EX1
MSH2 EX1
MLH1 EX2
MSH2 EX2
MLH1 EX3
MSH2 EX3
MLH1 EX4
MSH2 EX4
C11p13
MLH1 EX5
MSH2 EX5
MLH1 EX6
MSH2 EX6
MLH1 EX7
MSH2 EX7
MLH1 EX8
MSH2 EX8
MLH1 EX9
C17q21
MSH2 EX9
MLH1 EX10
MSH2 EX10
MLH1 EX11
MSH2 EX11
MLH1 EX12
MSH2 EX12
MLH1 EX13
MSH2 EX13
C4q25
MLH1 EX14
MSH2 EX14
MLH1 EX15
MSH2 EX15
MLH1 EX16
MSH2 EX16
MLH1 EX17
MLH1 EX18
MLH1 EX19
C10p14
C11p12
(a)
Amplicons
(c)
AluSp
TG
Mutant sequence
Normal intron 5
(nucleotides 14123–14176)
Normal intron 6
(nucleotides 16260–16313)
(d)
AluSp
CA
Mutant sequence
Normal intron 6
(nucleotides 16598–16640)
Normal intron 8
(nucleotides 19257–19299)
Fig. 1. Detection and characterization of the deletion of exons 6, 7 and 8 of MLH1. (a) The MLPA output encompassing the
exons 5–9 from a control and the patient samples are shown. The fragment peaks corresponding to exons 6, 7 and 8 of MLH1
are indicated. Exon deletions in the patient sample are apparent by a reduction in the peak height. (b) Peak heights of each
fragment of the patient sample were compared with those of the control sample to obtain a dosage quotient representing the
gene dosage of each exon, as described by Taylor et al. (7). The expected dosage quotient is 1 for normal copy exons and 0.5
for deleted exons. Bars for control probes are yellow and for the deleted exons are red. (c) Sequencing output is shown for the
patient. The wildtype sequence aligned below the output is taken RefSeq NC_000003.9. Nucleotides are numbered from A of
the start codon. Underlined bases are present in the mutant sequence. Sequencing with primer MLH1 IVS5 388F
(50 -GCAAAGAATACTTGCCCTCTTGACTTAAA-30 ) shows normal intron 5 sequence joined to normal intron 6 sequence,
with the insertion of a TG. d) Sequencing with primer MLH1 IVS8 1065R (50 -TAGCTGGGTGTGGTGACAGG-30 ) reveals
normal intron 6 sequence joined to normal intron 8 sequence, with the insertion of a CA.
retained sequence. The 349-bp sequence from
intron 6 that is retained is made up of two closely
spaced Alu sequences.
the cDNA relevant exons of the genomic DNA,
using methods described elsewhere (7, 14, 15).
The deletion breakpoint was characterized by
PCR and sequencing of the genomic DNA. For
details, please contact the correspondence
author.
Case and methods
The mutation was detected in a male diagnosed
with CRC at 36 years of age. The subject met the
Bethesda guidelines for genetic testing (13).
Informed consent was obtained.
The mutation was identified by protein truncation test (PTT), multiplex ligation-dependent
probe amplification (MLPA) and sequencing of
Results
PTT analysis detected a truncated MLH1 product in the patient sample. Sequencing of the
cDNA revealed the deletion of exons 6, 7 and 8
of MLH1. Sequencing of the genomic DNA
235
McVety et al.
elements, and a mechanism involving HR
between branches of a hairpin loop stabilized by
the inverted repeats was invoked (11, 18, 19).
However, here, the retained sequence is not
inverted, and there is no homology between the
deletion breakpoints or insertion junctions.
REPEATMASKER
(http://www.repeatmasker.org)
identified the retained intron 6 sequence as two
closely spaced inverted Alu sequences, both interrupted by the deletion. The positions of these Alu
elements relative to the breakpoints in this mutation are shown in Fig. 2.
Several mechanisms could have caused this
insertion–deletion. Because the inserted sequence
is composed of Alu sequences, the insertion may
be due to retrotransposition. Retrotransposition
events are often associated with large deletions
(2, 20–22). However, recent Alu insertions are
usually flanked by short direct repeats (23), and
there are no direct repeats flanking the insertion
in this case to suggest that retrotransposition has
occurred. It seems more likely that the mutation
was introduced by non-homologous end joining
(NHEJ) during the repair of DSBs that occurred
between exons 5 and 9. NHEJ is the preferred
mechanism for repairing DSBs in mammalian
revealed no abnormalities, which suggested that
the deletion was genomic. MLPA detected a
decrease in the copy number of exons 6, 7 and 8
of MLH1, which confirmed the genomic deletion
of these exons (Fig. 1a,b).
Sequencing of the mutant PCR products using
primers in intron 5 revealed a duplication resulting in the insertion of a TG in intron 5 followed
by 142 bp of normal intron 6 sequence (Fig. 1c).
The mutation, g.14141–16278del2139dupTG,
removes exon 6.
Sequencing with reverse primers in intron 8
revealed the insertion of a CA in intron 8
followed by 28 bp of normal intron 6 sequence
(Fig. 1d). The mutation, g.16628–19288del2661
insCA, removes exons 7 and 8.
Discussion
Studies of deletions occurring in model systems
(11, 16, 17) and of disease-causing mutations (18,
19) have revealed a class of deletions coupled
with the insertion of an inverted sequence derived
from the deleted region. In most cases, the deletions were flanked by inverted repeating
5
AluSp
6
7
AluSq AluSp
8
9
AluY
DSB formation
5
6
AluSp
7
AluSq
Alu AluSp
Alu
8
AluY
9
Processing of ends by endogenous
nucleases and polymerases
5
tg
ca
AluY
9
NHEJ
5
tg
ca
9
AluY
624-bp
ivs 5
349-bp
ivs 6
1595-bp
ivs 8
Fig. 2. A pathway involving non-homologous end joining (NHEJ) explains the occurrence of the complex deletion of exons 6,
7 and 8 of MLH1. Grey open boxes represent exons. Straight lines represent introns. Arrows represent repeating elements and
indicate the element’s orientation. In the first step, double stranded breaks are introduced in introns 5 and 8. The free DNA
ends are modified by endogenous polymerases and exonucleases, represented by slanted arrows. This results in the loss of
exons 6, 7 and 8, and the addition of a TG and a CA. In the final step, the modified ends are joined by NHEJ.
236
Novel genomic insertion–deletion in MLH1
cells (17, 24, 25). It is not uncommon for large
deletions to occur during NHEJ (26), and insertions of one or more bases are commonly seen at
the repair junctions (26–29). The deletions are
attributed to exonucleases, which act on the free
ends of the DNA they are joined (19, 30). The
free ends are also subjected to the actions of
polymerases (22, 28). Larger insertions may be
due to incorporation of oligonucleotides present
in the cell during NHEJ (27, 28). In the present
case, the insert appears to be derived from the
deleted region. The steps necessary to create the
mutation by NHEJ are shown in Fig. 2. First,
two DSBs are introduced in the DNA flanking
the retained sequence in intron 6. DSBs may be
caused by a variety of factors, including ionizing
radiation or certain mutagens. The free ends of
the DNA are exposed to the actions of exonucleases and polymerases, resulting in the degradation of the DNA and the addition of novel
bases at the breakpoints. Finally, the free ends
are joined by NHEJ.
Another possibility is that two deletions
occurred independently resulting in two closely
spaced deletions. The model presented here,
which predicts that both breakpoints were
formed in one NHEJ event, is more likely than
a ‘two deletion’ model because it is more parsimonious. A similar case of a discontinuous mutation in TP53 has been described (31). Our model,
solely implicating NHEJ, is less able to explain
the likely repair process in that case, because the
first breakpoint removes the entire TP53 gene
and appears to be the result of nonhomologous
recombination (NHR), whereas the second breakpoint removes intergenic sequence and part of the
ATP1B2 gene and appears to be due to HR
between Alu sequences (31). Interestingly, a TG
was inserted at the NHR breakpoint in that case
as well. It seems more probable that this deletion
is the result of two distinct events.
Acknowledgements
We thank the Cancer Research Society and the Judy Steinberg
Trust for their financial support of this project.
References
1. de la Chapelle A. Genetic predisposition to colorectal cancer. Nat Rev Cancer 2004: 4: 769–780.
2. Marshall B, Isidro G, Boavida MG. Insertion of a short
Alu sequence into the hMSH2 gene following a double
cross over next to sequences with chi homology. Gene
1996: 174: 175–179.
3. Mauillon JL, Michel P, Limacher JM et al. Identification of
novel germline hMLH1 mutations including a 22 kb Alumediated deletion in patients with familial colorectal cancer. Cancer Res 1996: 56: 5728–5733.
4. Viel A, Petronzelli F, Della Puppa L et al. Different molecular mechanisms underlie genomic deletions in the MLH1
gene. Hum Mutat 2002: 20: 368–374.
5. Wagner A, van der Klift H, Franken P et al. A 10-Mb
paracentric inversion of chromosome arm 2p inactivates
MSH2 and is responsible for hereditary nonpolyposis colorectal cancer in a North-American kindred. Genes
Chromosomes Cancer 2002: 35: 49–57.
6. Wang Y, Friedl W, Lamberti C et al. Hereditary nonpolyposis colorectal cancer: frequent occurrence of large genomic deletions in MSH2 and MLH1 genes. Int J Cancer
2003: 103: 636–641.
7. Taylor CF, Charlton RS, Burn J, Sheridan E, Taylor GR.
Genomic deletions in MSH2 or MLH1 are a frequent cause
of hereditary non-polyposis colorectal cancer: identification
of novel and recurrent deletions by MLPA. Hum Mutat
2003: 22: 428–433.
8. Charbonnier F, Olschwang S, Wang Q et al. MSH2 in
contrast to MLH1 and MSH6 is frequently inactivated by
exonic and promoter rearrangements in hereditary nonpolyposis colorectal cancer. Cancer Res 2002: 62: 848–853.
9. Deininger PL, Batzer MA. Mammalian retroelements.
Genome Res 2002: 12: 1455–1465.
10. Kolomietz E, Meyn MS, Pandita A, Squire JA. The role of
Alu repeat clusters as mediators of recurrent chromosomal
aberrations in tumors. Genes Chromosomes Cancer 2002:
35: 97–112.
11. Gebow D, Miselis N, Liber HL. Homologous and nonhomologous recombination resulting in deletion: effects of
p53 status, microhomology, and repetitive DNA length and
orientation. Mol Cell Biol 2000: 20: 4028–4035.
12. Chen SJ, Chen Z, Font MP, d’Auriol L, Larsen CJ, Berger
R. Structural alterations of the BCR and ABL genes in Ph1
positive acute leukemias with rearrangements in the BCR
gene first intron: further evidence implicating Alu sequences
in the chromosome translocation. Nucleic Acids Res 1989:
17: 7631–7642.
13. Umar A, Boland CR, Terdiman JP et al. Revised Bethesda
Guidelines for hereditary nonpolyposis colorectal cancer
(Lynch syndrome) and microsatellite instability. J Natl
Cancer Inst 2004: 96: 261–268.
14. Luce MC, Marra G, Chauhan DP et al. In vitro transcription/translation assay for the screening of hMLH1 and
hMSH2
mutations
in
familial
colon
cancer.
Gastroenterology 1995: 109: 1368–1374.
15. Thiffault I, Hamel N, Pal T et al. Germline truncating
mutations in both MSH2 and BRCA2 in a single kindred.
Br J Cancer 2004: 90: 483–491.
16. Rebuzzini P, Khoriauli L, Azzalin CM, Magnani E,
Mondello C, Giulotto E New mammalian cellular systems
to study mutations introduced at the break site by nonhomologous end-joining. DNA Repair (Amst) 2005: 4:
546–555.
17. Honma M, Izumi M, Sakuraba M et al. Deletion, rearrangement, and gene conversion; genetic consequences of chromosomal double-strand breaks in human cells. Environ
Mol Mutagen 2003: 42: 288–298.
18. Chuzhanova N, Abeysinghe SS, Krawczak M, Cooper DN.
Translocation and gross deletion breakpoints in human
inherited disease and cancer II: potential involvement of
repetitive sequence elements in secondary structure
formation between DNA ends. Hum Mutat 2003: 22:
245–251.
237
McVety et al.
19. Zucman-Rossi J, Legoix P, Victor JM, Lopez B, Thomas G.
Chromosome translocation based on illegitimate recombination in human tumors. Proc Natl Acad Sci USA 1998: 95:
11786–11791.
20. Ricci V, Regis S, Di Duca M, Filocamo M. An
Alu-mediated rearrangement as cause of exon skipping in
Hunter disease. Hum Genet 2003: 112: 419–425.
21. Van de Water N, Williams R, Ockelford P, Browett P. A
20.7 kb deletion within the factor VIII gene associated with
LINE-1 element insertion. Thromb Haemost 1998: 79:
938–942.
22. Wurtele H, Little KC, Chartrand P. Illegitimate DNA integration in mammalian cells. Gene Ther 2003: 10: 1791–1799.
23. Batzer MA, Deininger PL. Alu repeats and human genomic
diversity. Nat Rev Genet 2002: 3: 370–379.
24. Lees-Miller SP, Meek K. Repair of DNA double strand
breaks by non-homologous end joining. Biochimie 2003:
85: 1161–1173.
25. Valerie K, Povirk LF. Regulation and mechanisms of mammalian double-strand break repair. Oncogene 2003: 22:
5792–5812.
238
26. Roth DB, Porter TN, Wilson JH. Mechanisms of nonhomologous recombination in mammalian cells. Mol Cell
Biol 1985: 5: 2599–2607.
27. Little KC, Chartrand P. Genomic DNA is captured and
amplified during double-strand break (DSB) repair in
human cells. Oncogene 2004: 23: 4166–4172.
28. Roth DB, Chang XB, Wilson JH. Comparison of filler
DNA at immune, nonimmune, and oncogenic rearrangements suggests multiple mechanisms of formation. Mol Cell
Biol 1989: 9: 3049–3057.
29. Roth DB, Proctor GN, Stewart LK, Wilson JH.
Oligonucleotide capture during end joining in mammalian
cells. Nucleic Acids Res 1991: 19: 7201–7205.
30. Bannister LA, Waldman BC, Waldman AS. Modulation of
error-prone double-strand break repair in mammalian
chromosomes by DNA mismatch repair protein Mlh1.
DNA Repair (Amst) 2004: 3: 465–474.
31. Bougeard G, Brugieres L, Chompret A et al. Screening for
TP53 rearrangements in families with the Li-Fraumeni syndrome reveals a complete deletion of the TP53 gene.
Oncogene 2003: 22: 840–846.