MRC-Holland
Description version 07; 07-05-2008
®
MLPA
SALSA MLPA KIT P025 CANAVAN DISEASE
Lot 0407. As compared to the previous lot 0204, one extra ASPA exon 1 probe is included.
CANAVAN DISEASE is a disease that is characterised by a spongy degeneration of the central nervous
system, resulting in blindness, megalocephaly, severe mental defects, atonia of neck muscles and
hyperextension of legs and flexion of arms. Although average life expectancy of Canavan patients is only 18
months, several congenital, infantile and late-onset forms of Canavan disease have been reported.
The cause of Canavan disease is a defect in the ASPA gene on chromosome 17pter-p13, coding for
aspartoacylase. Diagnosis can be performed by measuring aspartoacylase activity in cultured fibroblasts.
DNA methods detecting the gene defect are desirable for prenatal diagnosis as aspartoacylase activity is low
or undetectable in direct or cultured normal chorionic villi and in normal cultured amniocytes. The great
majority of Canavan patients are of Jewish ancestry and carry one or more of the three common point
mutations. In non-Jewish patients, a variety of mutations have been found, including deletions of one or
more ASPA exons. An inframe deletion of exon 4 appears to be a founder mutation in patients of Turkish
origin.
The ASPA gene spans 29 Kb of genomic sequence and consists of 6 exons. This P025 probemix contains
probes for each ASPA exon. In addition, it contains 3 other probes for the 17p13 region and 14 reference
probes for other regions. Two probes are present for ASPA exon 1.
This SALSA MLPA kit is designed to detect deletions/duplications of one or more exons of the ASPA gene.
Heterozygote deletions of probe recognition sequences should give a 35-50% reduced relative peak area of
the amplification product of that probe. However, mutations and/or polymorphisms very close to the probe
ligation site may also result in a reduced relative peak area. Therefore, apparent deletions detected by a
single probe always require confirmation by other methods. We have no information on what percentage of
defects in these genes is caused by deletions/duplications of complete exons. Please note that most defects
in these genes are expected to be small (point) mutations, most of which will not be detected by this MLPA
test.
SALSA® MLPA® kits are sold by MRC-Holland for research purposes and to demonstrate the
possibilities of the MLPA technique. This kit is not CE/FDA certified for use in diagnostic
procedures. SALSA MLPA kits are supplied with all necessary buffers and enzymes. Purchase of
the SALSA MLPA test kits includes a limited license to use these products for research purposes.
The use of this MLPA kit 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).
References of SALSA MLPA kit P025 Canavan
Zeng BJ. Et al. (2006). Rapid detection of three large novel deletions of the aspartoacylase gene in nonJewish patients with Canavan disease. Mol Genet Metab. 2006 Sep-Oct;89(1-2):156-63. Epub 2006 Jul 18.
More information
Website
E-mail
Fax
Mail
: www.mlpa.com
:info@mlpa.com
:+31-20-6891149
:MRC-Holland bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands
SALSA MLPA kit P025 Canavan
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MRC-Holland
Description version 07; 07-05-2008
®
MLPA
Data analysis
The P025 Canavan probemix contains 24 different MLPA probes with amplification products between 148
and 355 bp. In addition, it contains 5 control fragments generating an amplification product smaller than
120 nt and one synthetic ligation-dependent control fragment at 92 nt. More information on how to interpret
observations on these control fragments can be found in the MLPA protocol.
When only small numbers of samples are tested, visual comparison of peak profiles should be sufficient to
easily identify exon deletions. Comparison of results should preferably 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 blots or long range PCR.
Note that Coffalyser, the MLPA analysis tool developed at MRC-Holland, can be downloaded free of charge
from our website www.mlpa.com .
This probemix was developed by A. Errami & 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 first probemix
designer could be made a coauthor.
Info/remarks/suggestions for improvement: info@mlpa.com.
SALSA MLPA kit P025 Canavan
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MRC-Holland
®
MLPA
Description version 07; 07-05-2008
SALSA MLPA P025 Canavan probemix
Length
(nt)
SALSA MLPA probe
64-70-76-82
92
148
157
166
175
184‡
193
202‡
211
220
229‡
238
247±
256
265¥
274
283
292
301
310
319
328
337
346‡
355
Chromosomal position
Reference
Gene
Q-fragments: DNA quantity; only visible with less than 100 ng sample DNA
Synthetic control fragment
TRPV1 probe 1472-L0946
Reference probe 1063-L0061
Reference probe 1286-L0847
Reference probe 1301-L0857
ASPA probe 7937-L7718
Reference probe 0976-L0563
Reference probe 1245-L0793
Reference probe 1220-L0689
Reference probe 1119-L0559
Reference probe 1461-L0926
Reference probe 0523-L0363
ASPA probe 1324-L0870
Reference probe 1055-L0628
ASPA probe 1325-L7456
Reference probe 0978-L0565
ASPA probe 1326-L0873
GEMIN4 probe 1079-L0646
ASPA probe 1327-L0872
Reference probe 0440-L0366
ASPA probe 1328-L0871
Reference probe 1043-L0619
ASPA probe 1659-L1241
GARNL4 probe 0689-L8392
Reference probe 0781-L0284
17p13
14q13
9p22
5q35
Exon 1
11p13
14q24
10p15
10q22
17p12
6p21
Exon 1
17q21
Exon 5
15q25
Exon 4
17p13
Exon 3
17q24
Exon 2
8q13
Exon 6
17p13
17q21
‡ New from lot 0407 onwards.
± Less reliable than most other probes.
¥ Small change in length and or amplification characteristics as compared to the previous lot. No change in
sequence detected.
17p probes arranged according to chromosomal location
Length
(nt)
SALSA
MLPA probe
P-telomere
292
346
184†
247
319
301
283
265
337
148
1079-L0646
0689-L8392
7937-L7718
1324-L0870
1328-L0871
1327-L0872
1326-L0873
1325-L0874
1659-L1241
1472-L0946
Exon
exon
exon
exon
exon
exon
exon
exon
1
1
2
3
4
5
6
Distance to next
probe
597
2252
478
0.1
5.4
1.7
5.7
5.1
4.5
68
Kb
Kb
Kb
Kb
Kb
Kb
Kb
Kb
Kb
Kb
† New from lot 0407 onwards.
Note: Exon numbering might be different as compared to literature! Please notify us of any mistakes. The
identity of the genes detected by the reference probes is available on request: info@mlpa.com.
SALSA MLPA kit P025 Canavan
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MRC-Holland
Description version 07; 07-05-2008
®
MLPA
SALSA MLPA kit P025 Canavan sample picture
Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA
analyzed with SALSA MLPA kit P025 Canavan (lot 0407).
SALSA MLPA kit P025 Canavan
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MRC-Holland b.v.
Description version 07; 07-05-2008
Homozygous deletion ASPA exon 1-6 (old lot)
1
5
3
2
6
2
6
4
Normal sample
3
5
1
SALSA MLPA kit P025 Canavan
4
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MRC-Holland b.v.
Description version 07; 07-05-2008
Heterozygous ASPA deletion exon 1-6 (old lot)
5
1
4
3
Normal sample
SALSA MLPA kit P025 Canavan
6
2
6
3
5
1
2
4
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