Download SALSA MLPA probemix P222-A2 LCA mix-2 - MRC

MRC-Holland
Description version 11; 17-07-2015
®
MLPA
SALSA MLPA probemix P222-A2 LCA mix-2
Lot A2-0511. As compared to lot A1-0607, new control fragments (QDX2-fragments, 88 and 96 nt), one Xfragment (100 nt) and one Y-fragment (105 nt) have been added.
Leber congenital amaurosis (LCA) is the most common inherited cause of blindness in childhood and is
characterized by a severe retinal dystrophy before the age of one year. Eight genes have been identified
that together account for approximately 45% of all LCA patients. This SALSA® MLPA® P222 LCA probemix-2
contains probes for 3 of these genes: GUCY2D, RDH12 and RPGRIP1. In addition, this probemix contains 2
probes for the CEP290 gene of which one detects the wild type sequence of the c.2991+1655A>G mutation.
Mutations in the GUCY2D gene that encodes retinal guanylate cyclase-1 (retGC1) account for 10%-20% of
all LCA cases. These mutations disrupt synthesis of cGMP in photoreceptor cells, a key second messenger
required for function of these cells. The GUCY2D gene (20 exons) spans ~18 kb of genomic DNA and is
located on chromosome 17p13.1, ~8 Mb from the p-telomere. The P222 probemix contains probes for 9
different GUCY2D exons.
RDH12 encodes a photoreceptor-specific retinal dehydrogenase. Retinoids are indispensable light-sensitive
elements of vision and also serve as essential modulators of cellular differentiation and proliferation in
diverse cell types. RDH12 belongs to a family of dual-specificity retinol dehydrogenases that metabolize both
all-trans- and cis-retinols (Haeseleer et al., J Biol Chem, 2002). The RDH12 gene (9 exons) spans ~33 kb of
genomic DNA and is located on chromosome 14q24.1, ~68 Mb from the p-telomere. The P222 probemix
contains one probe for exons 3 to 9.
RPGRIP1 (RPGR-interacting protein 1) is a key component of cone and rod photoreceptor cells, where it
interacts with RPGR (retinitis pigmentosa GTPase regulator). Mutations in RPGRIP1 have been detected in
4.5% of LCA patients. The RPGRIP1 gene (24 exons) spans ~63.3 kb of genomic DNA and is located on
chromosome 14q11, ~21 Mb from the p-telomere. The P222 probemix contains probes for 18 different
RPGRIP1 exons.
The CEP290 protein is localized to the connecting cilium of photoreceptors. The gene is located at
chromosome 12q21.33, ~88 Mb from the p-telomere and spans ~93 kb of genomic DNA. Its mutation
c.2991+1655AG seem to be an important cause of LCA and may explain up to 21% of LCA cases. (den
Hollander et al., Am J Hum Genet, 2006). The P222 probemix contains 2 probes for the CEP290 gene. One
probe is specific for the wild type sequence of the c.2991+1655AG mutation and the other probe detects a
sequence at a short distance from the mutation specific probe.
This SALSA® MLPA® probemix is designed to detect deletions/duplications of one or more sequences of the
aforementioned genes in a DNA sample. Heterozygous deletions of recognition sequences should give a 3550% reduced relative peak area 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 area, 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 probemix P222 LCA mix-2
Page 1 of 6
MRC-Holland
Description version 11; 17-07-2015
®
MLPA
Related SALSA® MLPA® probemixes
P221 LCA mix-1: Contains probes for the AIPL1, CRB1, CRX and RPE65 genes.
More information
Website : www.mlpa.com
E-mail
: [email protected] (information & technical questions); [email protected] (for orders)
Mail
: MRC-Holland bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands
Data analysis
The P222-A2 LCA mix-2 probemix contains 42 MLPA probes with amplification products between 124 and
427 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
(D-fragments) 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 area of each
probe’s amplification product by the total area 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://projects.tcag.ca/variation. 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 by N. Laddach & J.P. Schouten at MRC-Holland. In case the results obtained
with this probemix lead to a scientific publication, it would be very much appreciated if the probemix
designer could be made a co-author.
Info/remarks/suggestions for improvement: [email protected].
SALSA MLPA probemix P222 LCA mix-2
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MRC-Holland
®
MLPA
Description version 11; 17-07-2015
Table 1. SALSA MLPA P222-A2 LCA mix-2 probemix
Length
(nt)
64-70-76-82
88-92-96
100
105
124
130 ~
139
143
148
154±
159
166
172
177
184
190
195 ‡
202
208
214
220
230
238
247
256
261
266
274
283
292
301
310
319
328
337
346
355
364
370
378
382
391
400 *
408
418
427
SALSA MLPA probe
reference
Chromosomal position
GUCY2D
RDH12
RPGRIP1
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 04235-L08770
8p22
GUCY2D probe 07036-L06647
Exon 3
RPGRIP1 probe 07023-L06634
Exon 10
RDH12 probe 08890-L09782
Exon 5
RPGRIP1 probe 07018-L06629
Exon 2
GUCY2D probe 07038-L06649
Exon 7
GUCY2D probe 07040-L06651
Exon 12
Reference probe 01588-L06028
17p13
RPGRIP1 probe 07032-L06643
Exon 22
RPGRIP1 probe 07021-L06632
Exon 6
RPGRIP1 probe 07024-L06635
Exon 11
Reference probe 03574-L02400
7q31
RPGRIP1 probe 07033-L08019
Exon 23
GUCY2D probe 07035-L06646
Exon 2
RPGRIP1 probe 07019-L06630
Exon 3
RPGRIP1 probe 07025-L06636
Exon 13
RPGRIP1 probe 07029-L06640
Exon 19
RDH12 probe 08889-L08851
Exon 4
RPGRIP1 probe 07030-L06641
Exon 20
GUCY2D probe 07037-L06648
Exon 5
GUCY2D probe 07039-L08018
Exon 10
RPGRIP1 probe 07031-L06642
Exon 21
Reference probe 00830-L00345
17q21
CEP290 probe 08887-L08849
GUCY2D probe 07041-L06652
Exon 14
RPGRIP1 probe 07017-L06628
Exon 1
RPGRIP1 probe 07022-L06633
Exon 8
RPGRIP1 probe 07028-L06639
Exon 18
RDH12 probe 08892-L08854
Exon 7
RPGRIP1 probe 07020-L06631
Exon 4
GUCY2D probe 07042-L06653
Exon 17
RPGRIP1 probe 07027-L06638
Exon 16
RPGRIP1 probe 07034-L06645
Exon 24
RDH12 probe 08893-L08855
Exon 8
RPGRIP1 probe 08141-L06637
Exon 14
Reference probe 05921-L05366
17q11
GUCY2D probe 07043-L06654
Exon 20
RDH12 probe 08891-L08853
Exon 6
CEP290 probe 08886-L08848
RDH12 probe 08894-L08856
Exon 9
RDH12 probe 08888-L08850
Exon 3
Reference probe 00680-L00121
7q34
CEP290
Intron 26
Intron 26
~ This probe has been found duplicated in healthy individuals. Please disregard.
‡ More variable. This probe has a low signal and will be replaced in the next lot.
* This probe detects the common allele of the intronic polymorphism c.2991+1655AG. The presence of the rare allele will result in a
lower signal.
± This probe is located within, or close to, a very strong CpG island. A low signal of this probe can be due to incomplete sample DNA
denaturation, e.g. due to the presence of salt in the sample DNA.
Note
The RDH12 and RPGRIP1 exon numbering has changed. From description version 06 onwards, we have adopted the NCBI
exon numbering that is present in the latest NM_ sequences for the RDH12 and RPGRIP1 gene. This exon numbering might be
different as compared to literature! The exon numbering used in previous versions of this product description can be found between
brackets in Table 2.
SALSA MLPA probemix P222 LCA mix-2
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MRC-Holland
®
MLPA
Description version 11; 17-07-2015
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].
Table 2. P222 probes arranged according to chromosomal location
Table 2a. GUCY2D gene
Length
(nt)
SALSA
MLPA probe
GUCY2D
Exon
start codon
Ligation site
NM_000180.3
75-77 (ex 2)
Partial sequence (24 nt
adjacent to ligation site)
202
130 ~
247
07035-L06646
07036-L06647
07037-L06648
Exon 2
Exon 3
Exon 5
CAGCCTGCTCCG-TCTGTGTTCGCA
TGCACTCGGTGC-TGCTGGGTGGCG
CTCCTGTCACTG-TCCCTTCAGGAC
0.9 kb
3.2 kb
0.9 kb
154±
07038-L06649
Exon 7
CCTGGGCCACCT-AGCAGGTAGAGG
4.6 kb
256
159
283
337
382
07039-L08018
07040-L06651
07041-L06652
07042-L06653
07043-L06654
Exon
Exon
Exon
Exon
Exon
13 nt before ex 2
822-823
9 nt before ex 5
1 nt before ex 7
reverse
2103-2104
2411-2412
2688-2689
3154-3155
3483-3484
TAGTGGATGGCA-GATTCGTACTCA
GCACCTGTCGAG-TGTATCCTCCTG
GGACACCAGTGG-AGCCCGAGTACT
CACTGTGGGGAT-TCTCCGTGCTCT
TGGGGTGAACTG-CTCCTTGGCAGG
1.4
0.9
1.1
4.2
10
12
14
17
20
Distance to
next probe
kb
kb
kb
kb
stop codon
3384-3386 (ex 19)
~ This probe has been found duplicated in healthy individuals. Please disregard.
± This probe is located within, or close to, a very strong CpG island. A low signal of this probe can be due to
incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA.
The NM_000180.3 sequence is a reference standard in the NCBI RefSeqGene project.
Table 2b. RDH12 gene
Length
(nt)
SALSA
MLPA probe
418
230
143
391
319
364
408
08888-L08850
08889-L08851
08890-L09782
08891-L08853
08892-L08854
08893-L08855
08894-L08856
RDH12
Exon
start codon
Exon
Exon
Exon
Exon
Exon
Exon
Exon
3
4
5
6
7
8
9
Ligation site
NM_152443.2
325-327 (ex 3)
Partial sequence (24 nt
adjacent to ligation site)
255-256
420-421
581-582
80 nt before ex 6
29 nt before ex 7
74 nt before ex 8
1629-1630
GCCAGACCAGGA-ACCTGAGCCAGA
GGAGTGTGTAGA-ACAAATGTGCAG
AATCCGAGTGGA-TACAAAGAACTC
TGAACAAAGGGA-AAGGGCAATTAT
GTCATTCCACTT-TCAATCTTCCCT
TCACTTGTGTAT-TTTGCTGCAGGA
GCTGAGGCAAGA-AGAGCACCATCA
Distance to
next probe
1.9
0.7
0.8
1.0
2.2
5.1
kb
kb
kb
kb
kb
kb
stop codon
1273-1275 (ex 9)
The NM_152443.2 sequence 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 on any mistakes: [email protected]
SALSA MLPA probemix P222 LCA mix-2
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MRC-Holland
®
MLPA
Description version 11; 17-07-2015
Table 2c. RPGRIP1 gene
Length
(nt)
SALSA
MLPA probe
RPGRIP1
Exon
start codon
Ligation site
NM_020366.3
1-3 (ex 1)
Partial sequence (24 nt
adjacent to ligation site)
292
148
208
328
07017-L06628
07018-L06629
07019-L06630
07020-L06631
No probe
07021-L06632
No probe
07022-L06633
No probe
07023-L06634
07024-L06635
No probe
07025-L06636
08141-L06637
No probe
07027-L06638
No probe
07028-L06639
07029-L06640
07030-L06641
07031-L06642
07032-L06643
07033-L08019
07034-L06645
Exon 1
Exon 2
Exon 3
Exon 4
Exon 5
Exon 6
Exon 7
Exon 8
Exon 9
Exon 10
Exon 11
Exon 12
Exon 13
Exon 14
Exon 15
Exon 16
Exon 17
Exon 18
Exon 19
Exon 20
Exon 21
Exon 22
Exon 23
Exon 24
31-32
122-123
3 nt before exon 3
556-557
CTACATCAGGAG-ACTTGCCAGTTA
CTTGAGCAGGAT-GAACCGGGAGGA
TCTATTTGTCCA-CAGGCTGAGGAC
CAAATGAAAACA-GAGGTGAAGTAG
6.7
6.2
1.6
5.2
4 nt before exon 6
GCGCCTTTCTCT-GCAGAGCTTCCA
4.2 kb
5.8 kb
995-996
GCTCAGGGCAGA-GCTGAAGGAAGA
1178-1179
1349-1350
TGACAGCTCCAG-TCAGCCCCACTG
CAACATACTTCA-GAAGCATAAACA
2.3 kb
1.9 kb
1751-1752
1977-1978
AAGCCATGACCT-TCCAACATCTGG
TATTCCTTCTAT-GACTTTGAAACC
2.8 kb
1.1 kb
2485-2486
TGTACCGCTTCT-TCACCTTTTCTG
2.5 kb
2925-2926
3131-3132
3273-3274
3379-3380
3599-3600
3654-3655
3783-3784
GAGGTTCCCATT-GAAGCTGGCCAG
GAAGTTCTCAGA-GACTAACAGCTT
TCTGAAGTCAGT-GAAGCACAAACT
TCTCCCTGGCCT-TCTACCCAGAGG
GCTGAATGGACA-AGATCCTGATCA
CCTCTGGATGAA-GAAAAGAAAGAA
ACCCCAATAGGA-AGGCTGAAGGTT
1.8
4.4
8.4
2.1
3.0
3.0
stop codon
3859-3861 (ex 24)
177
301
139
184
214
370
346
310
220
238
261
172
195 ‡
355
Distance to
next probe
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
‡ Low signal. This probe is less reliable and will be replaced in the next lot.
The NM_020366.3 sequence is a reference standard in the NCBI RefSeqGene project.
Table 2d. CEP290 gene
Length
(nt)
SALSA
MLPA probe
CEP290
Exon
start codon
400 *
08886-L08848
Intron 26
274
08887-L08849
Intron 26
stop codon
Ligation site
NM_025114.3
345-347 (ex 2)
1655 nt after exon
26
1791 nt after exon
26 reverse
Partial sequence (24 nt
adjacent to ligation site)
TGTAATTGTGAA-TATCTCATACCT
Distance to
next probe
0.1 kb
ACAGGGTAGGAT-TCATGTTTAGAA
7782-7784 (ex 54)
* This probe detects the common allele of the intronic polymorphism c.2991+1655AG. The presence of the rare allele
will result in a lower signal.
The NM_025114.3 sequence 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 on any mistakes: [email protected]
SALSA MLPA probemix P222 LCA mix-2
Page 5 of 6
MRC-Holland
®
MLPA
Description version 11; 17-07-2015
SALSA MLPA probemix P222-A2 LCA mix-2 sample picture
35000
188.78
121.25
259.45
213.09
30000
308.45
247.24
207.53
25000
96.22
20000
146.37
219.89
354.12
319.86
200.32
105.41
391.11
336.29
282.46
182.36
127.92
377.70
345.24
266.35
151.61
15000
299.71
273.89
255.32
289.79
137.14
90.99
85.67
229.72
237.23
175.97
170.35
165.55
326.03
407.10
417.73
369.26
382.36
141.50
363.19
426.69
157.41
D ye S ign al
10000
400.64
194.39
100.55
5000
0
100
150
200
250
Size (nt)
300
350
400
450
Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA
analysed with SALSA MLPA probemix P222-A2 LCA mix-2 (lot A2-0511).
Implemented Changes – compared to the previous product description version(s).
Version 11 (48)
- Electropherogram picture of the old buffer (introduced Dec. 2012) removed.
Version 10 (48)
- Warning added in Table 1 and Table 2, 154 nt probe 07038-L06649.
Version 09 (48)
- Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added.
Version 08 (48)
- Minor textual changes.
Version 07 (46)
- Remark on RefSeqGene standard added below Table 2.
- Minor textual changes.
Version 06 (46)
- Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new
picture included).
- Warning added in Table 1, 408 nt RDH12 probe 08894-L08856.
- Various minor textual changes on page 1 and 2.
- Exon numbering of the RDH12 and RPGRIP1 gene has been changed in Tables 1 and 2.
- Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the
amplification products.
Version 05 (44)
- Various minor textual changes on page 1.
- Minor changes in the data analysis section on page 2.
- Sentence “when only small numbers of samples are tested, visual comparison of peak profiles should be
sufficient” removed from data analysis section.
- Tables have been numbered.
- Various minor layout changes in Table 1 and 2.
SALSA MLPA probemix P222 LCA mix-2
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