Download Product description P048-C1-0315 LMNA-MYOT - MRC

MRC-Holland
Description version 17; 01 June 2016
®
MLPA
SALSA MLPA probemix P048-C1 LMNA/MYOT/ZMPSTE24
Lot C1-0315, C1-0112.
Laminopathies have emerged as clinically heterogeneous genetic disorders due to mutations in lamins or
lamin-associated proteins. Lamins are structural protein components of the nuclear lamina, a protein
network underlying the inner nuclear membrane that determines nuclear shape and size. The lamins
constitute a class of intermediate filaments. Three types of lamins, A, B, and C, have been described in
mammalian cells.
Laminopathies regroup at least eight distinct diseases, belonging to the groups of skeletal and/or cardiac
muscular dystrophies, axonal neuropathies, premature ageing syndromes and familial lipodystrophies. These
diseases, such as Emery-Dreifuss muscular dystrophy, Hutchinson-Gilford progeria syndrome and limb-girdle
muscular dystrophy type 1B (LGMD1B), result from alterations in the LMNA gene, encoding type A-lamins.
Pathophysiological mechanisms explaining how mutations in a unique gene could lead to such various
phenotypes are still unknown, but probably involve alterations in cellular mechanical stress responses, in
gene expression, and/or in post-translational maturation of lamin A. One gene that is involved in the posttranslational processing of Lamin A precursor is ZMPSTE24 (also known as FACE-1 in human). Loss of
function of the ZMPSTE24 gene and accumulation of precursor lamin A has been correlated with restrictive
dermopathy (RD).
Mutations in the MYOT gene are associated with LGMD1A and myofibrillar myopathies. Mutations in the
CAV3 gene are associated with LGMD1C.
The LMNA gene (12 exons) spans ~25.4 kb of genomic DNA and is located on 1q22, ~156 Mb from the ptelomere. The ZMPSTE24 gene (10 exons) spans ~36.1 kb of genomic DNA and is located on 1p34.2, ~40.7
Mb from the p-telomere. The MYOT gene (10 exons) spans ~20 kb of genomic DNA and is located on
chromosome 5q31.2, ~137.2 Mb from the p-telomere. The CAV3 gene (2 exons) spans ~13 kb of genomic
DNA and is located on chromosome 3p25.3, ~8.8 Mb from the p-telomere.
The P048-C1 probemix contains probes for all 12 exons of the LMNA gene. Two probes are present for exon
1. This probemix furthermore contains probes for all exons of the ZMPSTE24, MYOT and CAV3 genes.
Finally, 9 reference probes are included in this probemix, detecting several different autosomal chromosomal
locations.
This SALSA® MLPA® probemix is designed to detect deletions/duplications of one or more sequences in the
aforementioned gene(s) in a DNA sample. Heterozygous deletions of recognition sequences should give a
35-50% reduced relative peak height of the amplification product of that probe. Note that a mutation or
polymorphism in the sequence detected by a probe can also cause a reduction in relative peak height, even
when not located exactly on the ligation site! In addition, some probe signals are more sensitive to sample
purity and small changes in experimental conditions. Therefore, deletions and duplications detected by MLPA
should always be confirmed by other methods. Not all deletions and duplications detected by MLPA will be
pathogenic; users should always verify the latest scientific literature when interpreting their findings. We
have no information on what percentage of defects in these genes is caused by deletions/duplications of
complete exons. Finally, note that most defects in this gene are expected to be small (point) mutations
which will not be detected by this SALSA® MLPA® test.
SALSA® MLPA® probemixes and reagents are sold by MRC-Holland for research purposes and to
demonstrate the possibilities of the MLPA technique. They are not CE/FDA certified for use in
diagnostic procedures. Purchase of the SALSA® MLPA® test probemixes and reagents includes a
limited license to use these products for research purposes.
The use of a SALSA® MLPA® probemix and reagents requires a thermocycler with heated lid and sequence
type electrophoresis equipment. Different fluorescent PCR primers are available. The MLPA technique has
been first described in Nucleic Acid Research 30, e57 (2002).
SALSA MLPA P048 LMNA/MYOT/ZMPSTE24 probemix
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MRC-Holland
Description version 17; 01 June 2016
®
MLPA
References
Van Spaendonck-Zwarts K et al. (2013). Genetic analysis in 418 index patients with idiopathic dilated
cardiomyopathy: overview of 10 years' experience. Eur J Heart Fail. 15:628–636.
Weterings A et al. (2013). A novel lamin A/C mutation in a Dutch family with premature atherosclerosis.
Atherosclerosis. 229:169-173.
Norton N et al. (2011). Assessment of LMNA copy number variation in 58 probands with dilated
cardiomyopathy. Clin Transl Sci. 4:351–352.
Marsman R et al (2011). A complex double deletion in LMNA underlies progressive cardiac conduction
disease, atrial arrhythmias, and sudden death. Circ Cardiovasc Genet. 4:280-287.
Related SALSA® MLPA® probemixes
P116
P176
P268
P436
P061
SGC: Contains probes for the SGCA, SGCB, SGCD, SGCG and FKRP genes, involved in LGMDs.
CAPN3: Contains probes for the CAPN3 gene, involved in LGMD2A.
DYSF: Contains probes for the DYSF gene, involved in LGMD2B.
ANO5: Contains probes for the ANO5 gene, involved in LGMD2L.
Lissencephaly: Contains probes for the POMT1 gene, involved in LGMD2K.
More information
Website : www.mlpa.com
E-mail
: [email protected] (information & technical questions); [email protected] (for orders)
Mail
: MRC-Holland bv; Willem Schoutenstraat 1, 1057 DL Amsterdam, the Netherlands
Data analysis
The P048-C1 probemix contains 44 MLPA probes with amplification products between 129 and 463 nt. In
addition, it contains 9 control fragments generating an amplification product smaller than 120 nt: four DNA
Quantity fragments (Q-fragments) at 64-70-76-82 nt, three DNA denaturation control fragments (Dfragments) at 88-92-96 nt, one X-fragment at 100 nt and one Y-fragment at 105 nt. More information on
how to interpret observations on these control fragments can be found in the MLPA protocol.
Data generated by this probemix can first be normalised intra-sample by dividing the peak height of each
probe’s amplification product by the total peak height of only the reference probes in this probemix (block
normalisation). Secondly, inter-sample normalisation can be achieved by dividing the intra-normalised probe
ratio in a sample by the average intra-normalised probe ratio of all reference samples. Please note that this
type of normalisation assumes no changes occurred in the genomic regions recognised by the reference
probes.
Data normalisation should be performed within one experiment. Only samples purified by the same method
should be compared. Confirmation of most exons deletions and amplifications can be done by e.g. Southern
blotting, long range PCR, qPCR, FISH.
Note that Coffalyser, the MLPA analysis tool developed at MRC-Holland, can be downloaded free of charge
from our website www.mlpa.com.
Many copy number alterations in healthy individuals are described in the database of genomic variants:
http://dgv.tcag.ca/dgv/app/home. For example, a duplication of a complete gene might not be pathogenic,
while a partial duplication or a deletion may result in disease. For some genes, certain in-frame deletions
may result in a very mild, or no disease. Copy number changes of reference probes are unlikely to be the
cause of the condition tested for. Users should always verify the latest scientific literature when interpreting
their findings.
This probemix was developed at MRC-Holland.
Info/remarks/suggestions for improvement: [email protected].
SALSA MLPA P048 LMNA/MYOT/ZMPSTE24 probemix
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MRC-Holland
®
MLPA
Description version 17; 01 June 2016
Table 1. SALSA MLPA P048-C1 LMNA/MYOT/ZMPSTE24 probemix
Length (nt)
64-70-76-82
88-92-96
100
105
129
136
148
154
160
166
172
179
184
190
196
202
208
214
220
230
238
247
256
262
266
274
280
286
292
301
310
315
321
330
337
346
355 Ж
361
371
382
391
400
409
418
427
436
454
463
SALSA MLPA probe
reference
Chromosomal position
LMNA
ZMPSTE24
MYOT
CAV3
Q-fragments: DNA quantity; only visible with less than 100 ng sample DNA
D-fragments: Low signal of 88 or 96 nt fragment indicates incomplete denaturation
X-fragment: Specific for the X chromosome
Y-fragment: Specific for the Y chromosome
Reference probe 11622-L12379
ZMPSTE24 probe 15689-L21730
ZMPSTE24 probe 15691-L17658
LMNA probe 11552-L12299
LMNA probe 11553-L12300
MYOT probe 11554-L12301
MYOT probe 11555-L12302
ZMPSTE24 probe 15687-L18494
MYOT probe 11556-L12303
MYOT probe 11557-L12304
Reference probe 10138-L10600
MYOT probe 11558-L13504
ZMPSTE24 probe 15684-L18495
MYOT probe 11559-L13505
LMNA probe 05939-L05368
Reference probe 01783-L01347
CAV3 probe 09681-L10061
LMNA probe 02478-L01971
Reference probe 10702-L11284
ZMPSTE24 probe 17350-L17657
Reference probe 08600-L21175
CAV3 probe 17349-L21793
LMNA probe 12287-L13799
LMNA probe 12027-L13939
MYOT probe 11560-L12307
LMNA probe 01916-L01460
ZMPSTE24 probe 16705-L19884
LMNA probe 15694-L20323
Reference probe 05802-L20124
LMNA probe 01918-L21732
LMNA probe 01919-L01463
ZMPSTE24 probe 15692-L17659
ZMPSTE24 probe 16895-SP0379L19286
LMNA probe 13216-L14549
ZMPSTE24 probe 17417-L18992
LMNA probe 01922-L01466
LMNA probe 15693-L17660
Reference probe 15010-L11786
MYOT probe 11561-L21731
MYOT probe 11562-L12309
MYOT probe 11563-L14166
Reference probe 09614-L09909
ZMPSTE24 probe 15695-L17662
Reference probe 10108-L10532
10q25
Exon 7
Exon 10
Exon 5
Exon 9
Exon 4
Exon 1
Exon 2
Exon 8
Exon 5
18q11
Exon 9
Exon 1
Exon 3
Exon 12
13q14
Exon 1
Exon 11
6p12
Exon 8
17p11
Exon 2
Exon 1
Exon 1
Exon 7
Exon 2
Exon 3
Exon 7
15q15
Exon 4
Exon 6
Exon 4
Exon 5
Exon 8
Exon 9
Exon 10a
Exon 3
2p24
Exon 6
Exon 2
Exon 10
20p12
Exon 6
8q22
Ж This probe consists of three parts and has two ligation sites.
Note: Exon numbering used here may differ from literature! Please notify us of any mistakes. The identity
of the genes detected by the reference probes is available on request: [email protected].
SALSA MLPA P048 LMNA/MYOT/ZMPSTE24 probemix
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MRC-Holland
®
MLPA
Description version 17; 01 June 2016
Table 2. P048 probes arranged according to chromosomal location
Table 2a. ZMPSTE24 gene
Length SALSA MLPA ZMPSTE24
(nt)
probe
exon
start codon
208
179
310
346
355 Ж
454
136
262
371
148
15684-L18495
15687-L18494
16705-L19884
15692-L17659
16895-SP0379L19286
15695-L17662
15689-L21730
17350-L17657
17417-L18992
15691-L17658
Exon
Exon
Exon
Exon
1
2
3
4
Exon 5
Exon 6
Exon 7
Exon 8
Exon 9
Exon 10
Ligation site
NM_005857.3
212-214 (exon 1)
Partial sequence (24 nt
adjacent to ligation site)
187-188
375-376
523-524
653-654
16 nt before exon 5;
700-701
900-901
1011-1012
1215-1216
1356-1357
2878-2879
GGTGCACGCTGA-AGGAGCCGGCGG
ACCACCGGAGTT-AGGACAGATCAT
CTCTGGAGACTT-TCTGGACGGTTC
ATAATACTTTTG-TGATAGAAGAAA
TTCTTGTGGTAA-31 nt spanning
oligo-ATGAAAGATGCA
CACACCTCTGCC-TGAGGGAAAGCT
CAGCAATGCTTA-TTTTTATGGCTT
TGTACTAGGCCA-TGAACTGGGGCA
TTATGATAGCCA-ACCCACTCTTAT
AAAGTGGGATCA-ACTGTACGCCTT
Distance to
next probe
2.6
6.9
0.7
1.5
kb
kb
kb
kb
2.0 kb
9.4
4.6
5.0
3.0
kb
kb
kb
kb
stop codon
1637-1639 (exon 10)
Ж This probe consists of three parts and has two ligation sites.
The NM_005857.3 sequence is the reference standard in the NCBI RefSeqGene project.
Table 2b. LMNA gene
Length SALSA MLPA
(nt)
probe
280
286
301
391
330
154
337
315
361
160
382
247
220
12287-L13799
12027-L13939
01916-L01460
15693-L17660
01918-L21732
11552-L12299
01919-L01463
15694-L20323
13216-L14549
11553-L12300
01922-L01466
02478-L01971
05939-L05368
LMNA
exon
start codon
Ligation site
NM_170707.2
250-252 (exon 1)
Partial sequence (24 nt
adjacent to ligation site)
Exon 1
Exon 1
Exon 2
Exon 3
Exon 4
Exon 5
Exon 6
Exon 7
Exon 8
Exon 9
Exon 10a
Exon 11
Exon 12
534-535
589-590
639-640
781-780 reverse
936-937
1067-1066 reverse
1335-1336
1515-1516
161 nt before exon 8
1831-1830 reverse
1893-1894
2094-2095
9 nt before exon 12
CTTGACTCAGTA-GCCAAGGAGCGC
GTGAGGAGTTTA-AGGAGCTGAAAG
CTGATAGCTGCT-CAGGCTCGGCTG
CTTCTTGGCCTC-ACCTAGGGCTGC
CTGGTGGAGATT-GACAATGGGAAG
ACTGCCTGGCAT-TGTCCAGCTGGA
TACCAGGAGCTT-CTGGACATCAAG
CGCAAACTGGAG-TCCACTGAGAGC
TGTGTCCACAGA-TCATGGCTATTA
GATGAGAGCCGT-ACGCAGGCTGTT
CGCTCAGTGACT-GTGGTTGAGGAC
TCCTCTGGCTCT-TCTGCCTCCAGT
CTCCTCTGTTTT-CTCTCTTAGAGC
start codon
2242-2244 (exon 12)
Distance to
next probe
0.1
15.4
3.8
0.4
0.3
0.9
0.3
0.4
0.4
0.5
0.9
0.4
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
The NM_170707.2 sequence represents transcript variant 1 and is a reference standard in the NCBI
RefSeqGene project.
Table 2c. CAV3 gene
Length SALSA MLPA
(nt)
probe
238
274
09681-L10061
17349-L21793
CAV3
exon
start codon
Ligation site
NM_033337.2
78-80 (exon 1)
Partial sequence (24 nt
adjacent to ligation site)
Exon 1
Exon 2
142-143
435-436
CTGCAAGGAGAT-TGACCTGGTGAA
GCATCAGCCACA-TCTACTCACTCT
stop codon
531-533 (exon 2)
Distance to
next probe
11.8 kb
The NM_033337.2 sequence represents transcript variant 1 and is a reference standard in the NCBI
RefSeqGene project.
SALSA MLPA P048 LMNA/MYOT/ZMPSTE24 probemix
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MRC-Holland
®
MLPA
Description version 17; 01 June 2016
Table 2d. MYOT gene
Length SALSA MLPA
(nt)
probe
172
418
214
166
190
409
292
184
202
427
11555-L12302
11562-L12309
11559-L13505
11554-L12301
11557-L12304
11561-L21731
11560-L12307
11556-L12303
11558-L13504
11563-L14166
MYOT
exon
start codon
Ligation site
NM_006790.2
310-312 (exon 2)
Partial sequence (24 nt
adjacent to ligation site)
Exon 1
Exon 2
Exon 3
Exon 4
Exon 5
Exon 6
Exon 7
Exon 8
Exon 9
Exon 10
13 nt after exon 1
164-165
726-727
889-890
957-956 reverse
1063-1064
1163-1164
1431-1432
1611-1612
1686-1687
AAGTCCCTTCTT-TATTCCTACTAC
TCAGGATCTCAA-CAAGGAAGAGCA
ACTGCAAATGCT-AAGCCCATACCA
TAGGACCACAGA-ATGCAGCTGCTG
AGTCGCGCATGT-TCTGAGTTGTGT
ACCCACCACGTT-TCATTCAAGTGC
GTCATGGTATCT-AAATGGAAGAAC
CAGATCTCGGCT-ATACCTCCACCA
GTGACTACATGT-AACACAAGATTA
GTTCGACCAACA-TTCAGCAAATAT
stop codon
1804-1806 (exon 10)
Distance to
next probe
2.5
5.4
1.7
3.2
1.2
1.4
2.7
0.8
0.3
kb
kb
kb
kb
kb
kb
kb
kb
kb
The NM_006790.2 sequence represents transcript variant 1 and is a reference standard in the NCBI
RefSeqGene project.
Note: Exon numbering used here may differ from literature! Complete probe sequences are available on
request: [email protected]. Please notify us of any mistakes: [email protected].
SALSA MLPA probemix P048-C1 LMNA/MYOT/ZMPSTE24 sample
picture
Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA
analysed with SALSA MLPA probemix P048-C1 LMNA/MYOT/ZMPSTE24 (lot C1-0315).
SALSA MLPA P048 LMNA/MYOT/ZMPSTE24 probemix
Page 5 of 6
MRC-Holland
Description version 17; 01 June 2016
®
MLPA
Implemented Changes – compared to the previous product description versions.
Version 17 – 01 June 2016 (55)
- Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new
picture included).
- Warning on probe located in CpG island removed.
- Various minor textual changes.
- New references added on page 2.
Version 16 – 12 August 2015 (54)
- Various minor textual changes.
- Figure(s) based on the use of old MLPA buffer (replaced in December 2012) removed.
- “Peak area” replaced with “peak height”.
Version 15 (48)
- Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added.
Version 14 (48)
- Product description adapted to a new product version (version number changed, lot number added,
small changes in Table 1 and Table 2, new picture included).
- Various minor textual changes on page 1.
Version 13 (48)
- Warning about salt sensitivity of 337 nt probe added.
- Various minor textual changes.
Version 12 (46)
- Product description adapted to a new version (lot number added, small changes in Table 1 and Table 2,
new pictures included).
Version 11 (46)
- Ligation sites of the probes targeting the CAV3 gene updated according to new version of the
NM_reference sequence.
- Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the
amplification products.
- Warning added in Table 1, 178 nt probe 08416-L08305 and 364 nt probe 01921-L01465.
- Data analysis method has been modified.
- Various minor textual changes on page 1.
SALSA MLPA P048 LMNA/MYOT/ZMPSTE24 probemix
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