MRC-Holland
®
Description version 14; 04-02-2014
MLPA
SALSA MLPA probemix P099-C2 GCH1-TH-SGCE
Lot C2-0813. As compared to the previous lot C1-0410, two reference probe have been replaced and the
control fragments have been adjusted (QDX2).
Autosomal dominant dopa-responsive dystonia is characterised by postural and motor disturbances showing
marked diurnal fluctuation (Segawa, M. et al., 1976, Adv Neurol). The disorder is caused by a mutation in
the gene encoding GTP cyclohydrolase I (GCH1). The GTP cyclohydrolase I enzyme is rate-limiting in the
conversion of GTP to BH4, which is a cofactor for tyrosine hydroxylase. Tyrosine hydroxylase is the ratelimiting enzyme for dopamine synthesis. The GCH1 gene has 6 exons, spans ~61 kb of genomic DNA and is
located on chromosome 14q22.2, ~55 Mb from the p-telomere.
The autosomal recessive form of dopa-responsive dystonia is also known as Segawa disease (De Lonlay, P.
et al., 2000, J Inherit Metab Dis). Deficiency of tyrosine hydroxylase (TH) is generally considered as a cause
of this autosomal recessive dystonia. TH is involved in the conversion of tyrosine to dopamine. As the ratelimiting enzyme in the synthesis of catecholamines, tyrosine hydroxylase has a key role in the physiology of
adrenergic neurons. The TH gene is located in the Beckwith-Wiedemann syndrome (BWS) critical region
(Gu, D. et al., 2002, Hum Genet) on chromosome 11p15.5. The TH gene has 14 exons and spans ~8 kb of
genomic DNA, ~2 Mb from the p-telomere.
Mutations in the epsilon-sarcoglycan encoding SGCE gene cause myoclonus-dystonia syndrome. The SGCE
gene has 13 exons, spans ~71 kb of genomic DNA and is located on chromosome 7q21.3, ~94 Mb from the
p-telomere. Please note that exons 10 and 12 are only present in the NM_001099401.1 and
NM_001099400.1 reference sequences, respectively.
Since the phenotypes of the dopa-responsive dystonia diseases are very similar, we combined probes for
TH, GCH1 and SGCE exons in one MLPA probemix. This probemix contains probes for all 6 exons of the
GCH1 gene, 6 out of the 14 exons of the TH gene and an additional flanking probe and all 11 SGCE exons
present in NM_003919.2. For data analysis, 9 reference probes for other human genes located on different
chromosomes are included.
This SALSA® MLPA® probemix is designed to detect deletions/duplications of one or more sequences in the
aforementioned genes in a DNA sample. Heterozygous deletions of recognition sequences should give a 3550% 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 these genes 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).
More information
Website
E-mail
Mail
: www.mlpa.com
: info@mlpa.com (information & technical questions); order@mlpa.com (for orders)
: MRC-Holland bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands
SALSA MLPA probemix P099 GCH1-TH-SGCE
Page 1 of 7
MRC-Holland
®
Description version 14; 04-02-2014
MLPA
References of SALSA® MLPA® probemix P099 GCH1-TH-SGCE
Ling, H. et al., 2011. An intragenic duplication in guanosine triphosphate cyclohydrolase-1 gene in a doparesponsive dystonia family. Mov Disord. 26:905-9
Steinberger, D. et al., 2007. Utility of MLPA in deletion analysis of GCH1 in dopa-responsive dystonia.
Neurogenetics. 8:51-55.
Grϋnewald, A. et al., 2008. Myoclonus-dystonia: significance of large SGCE deletions. Hum Mutat. 29:3312.
Liu, X. et al., 2010. GCH1 mutation and clinical study of Chinese patients with dopa-responsive dystonia.
Mov Disord. 25:447-51.
Wu-Chou, Y. H. et al., 2010. High frequency of multiexonic deletion of the GCH1 gene in a Taiwanese
cohort of dopa-response dystonia. Am J Med Genet B Neuropsychiatr Genet. 153B:903-908.
Data analysis
The P099-C2 GCH1-TH-SGCE probemix contains 36 MLPA probes with amplification products between 130
and 418 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 height of each
probe’s amplification product by the total peak height of only the reference probes in the probemix (block
normalisation). Secondly, inter-sample normalisation can be achieved by dividing the intra-normalized 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: info@mlpa.com.
SALSA MLPA probemix P099 GCH1-TH-SGCE
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MRC-Holland
®
MLPA
Description version 14; 04-02-2014
Table 1. SALSA MLPA P099-C2 GCH1-TH-SGCE probemix
Length
(nt)
64-70-76-82
88-92-96
100
105
130
136
142
148 ¬
153 #
159
165
171
178
185
191
202
210
218
229
239
246 ¥ #
256
265
274
283
290
298
310 *
319
328
337
346 *
355 #
364
371 ¥
382
391
401
409
418
SALSA MLPA probe
reference
Chromosomal position
TH
GCH1
SGCE
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 00797-L00463
Reference probe 11992-L12820
GCH1 probe 04619-L03894
IGF2AS probe 02218-L01712
SGCE probe 07004-L07150
Reference probe 02597-L02068
TH probe 03143-L02611
SGCE probe 07892-L07706
GCH1 probe 03139-L02607
TH probe 03144-L03587
SGCE probe 07005-L07151
GCH1 probe 03140-L02608
TH probe 03145-L02613
SGCE probe 03382-L05789
GCH1 probe 03141-L02609
TH probe 03146-L14800
SGCE probe 07003-L26246
GCH1 probe 03682-L03586
TH probe 03147-L02615
SGCE probe 07009-L07153
GCH1 probe 03686-L03101
SGCE probe 03384-L05790
TH probe 03148-L05945
Reference probe 13275-L14608
SGCE probe 03385-L02778
Reference probe 09571-L10025
SGCE probe 07002-L06608
Reference probe 14980-L16716
SGCE probe 03386-L02779
GCH1 probe 15131-L16901
SGCE probe 19656-L26248
SGCE probe 07891-L07705
Reference probe 01795-L01358
SGCE probe 07931-L06613
Reference probe 02669-L02136
Reference probe 12930-L14081
5q31
8p11
Exon 1
11p15
Exon 3
5q35
Exon 1
Exon 9
Exon 2
Exon 3
Exon 4
Exon 3
Exon 4
Exon 1
Exon 5
Exon 8
Exon 2
Exon 1
Exon 12
Exon 13 (11)
Exon 6
Exon 5
Exon 14
1p21
Exon 7
22q13
Exon 1
6q22
Exon 11 (10)
Exon 4
Exon 6
Exon 8
13q14
Exon 8
11q22
2p23
* New in version C2 (from lot C2-0813 onwards).
¥ Changed in version C2 (from lot C2-0813 onwards). Small change in length, no change in sequence
detected.
¬ Flanking probe. Included to facilitate determination of the extend of a deletion / duplication. Copy number
alterations of flanking and reference probes are unlikely to be related to the condition being tested.
# The following probes have a validated SNP on their ligation site: 246 nt probe rs11548284; 153 nt probe
rs200979763; 355 nt probe rs148126317. In case of an apparent deletion it is recommended to sequence
the target site.
The SGCE exon numbering has changed. From description version 08 onwards, we have adopted the
NCBI exon numbering that is present in the NM_ sequences for the SGCE gene. This exon numbering used
here may differ from literature! The exon numbering used in previous versions of this product description
can be found between brackets in Table 1 & 2.
SALSA MLPA probemix P099 GCH1-TH-SGCE
Page 3 of 7
MRC-Holland
®
MLPA
Description version 14; 04-02-2014
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: info@mlpa.com.
Table 2. P099 probes arranged according to chromosomal location
Table 2a. GCH1 gene
Length
(nt)
SALSA MLPA
probe
142
256
178
202
364
229
283
04619-L03894
03682-L03586
03139-L02607
03140-L02608
15131-L16901
03141-L02609
03686-L03101
GCH1
exon
Ligation site
NM_000161.2
start codon
162-164 (ex 1)
Exon 1
Exon 1
Exon 2
Exon 3
Exon 4
Exon 5
Exon 6
188 nt before exon 1
360-361
569-570
626-627
61 nt before exon 4
751-752
904-905
Partial sequence (24 nt adjacent Distance to
to ligation site)
next probe
CGCCCTTTTCCT-TCCCTCCCTGCG
ATAACGAGCTGA-ACCTCCCTAACC
AAGGACATAGAC-ATGTTTTCCATG
GTCCATATTGGT-TATCTTCCTAAC
GTTTTATGAGGA-AGGCTTATCAAT
CACGGAAGCCTT-GCGGCCTGCTGG
CCTGACTCTCAT-TAGGAGCTGAGC
0.6
37.1
5.6
12.6
1.4
1.8
kb
kb
kb
kb
kb
kb
stop codon
912-914 (ex 6)
The NM_000161.2 sequence represents transcript variant 1 and is a reference standard in the NCBI
RefSeqGene project.
Table 2b. TH gene
Length
(nt)
SALSA MLPA
probe
TH
exon
Ligation site
NM_199292.2
165
185
210
239
265
298
03143-L02611
03144-L03587
03145-L02613
03146-L14800
03147-L02615
03148-L05945
start codon
20-22 (ex 1)
Exon 1
Exon 3
Exon 4
Exon 8
Exon 12
Exon 14
59-60
224-225
470-471
877-878
1251-1252
1467-1468
148 ¬
02218-L01712
IGF2AS gene
stop codon
1604-1606 (ex 14)
Partial sequence (24 nt adjacent Distance to
to ligation site)
next probe
AGGCCAAGGGCT-TCCGCAGGGCCG
TCATTGGGCGCA-GGCAGAGCCTCA
GGCCCGCCCAGA-GGCCGCGAGCTG
CTGGAGGCCTTT-GCTTTGCTGGAG
CGGGCTGTGTAA-GCAGAACGGGGA
CTCACGCATCCA-GCGCCCCTTCTC
1.9
1.2
1.7
1.2
1.4
20.2
kb
kb
kb
kb
kb
kb
GGGTTTCTTCCT-TACACCCGGACG
The NM_199292.2 sequence represents transcript variant 1 and is a reference standard in the NCBI
RefSeqGene project.
Table 2c. SGCE gene
Length
(nt)
SALSA MLPA
probe
218
337
246 ¥ #
153 #
191
290
371 ¥
319
401
382
171
03382-L05789
07002-L06608
07003-L26246
07004-L07150
07005-L07151
03384-L05790
19656-L26248
03385-L02778
07931-L06613
07891-L07705
07892-L07706
No probe
Exon 10
355 #
03386-L02779
Exon 11 (10)
No probe
Exon 12
07009-L07153
Exon 13 (11)
274
SGCE
exon
Ligation site
NM_003919.2
start codon
112-114 (ex 1)
Exon 1
Exon 1
Exon 2
Exon 3
Exon 4
Exon 5
Exon 6
Exon 7
Exon 8
Exon 8
Exon 9
158-159
564 nt after exon 1
257-256 reverse
405-404 reverse
206 nt after exon 4
671-672
797-798
1048-1049
96 nt before exon 8
1174-1175
1339-1340
Partial sequence (24 nt adjacent Distance to
to ligation site)
next probe
CTGTGCTTGGAC-GGGACAGGGTCG
GAAATCTCGGTT-TTCTTCGGTTTT
ATGGGTATACAT-TCCGATCGGAGT
CGAAGCCATCCA-GGTCGGTCTGGG
AACTCAGATAGG-AAGCCAAACAGA
TCTTGGCGCAGT-GAAAAATGTGTG
GGTTGGTGCAGA-TGTCCCGTTTTC
AAAGCAGAGACT-ATTACACGGATT
GTGACAATGTCA-GCATTTCCACAT
TGCAAACACCAG-AGTAAGTGTCTT
ATGATAGCACAA-ACATGCCATTGA
0.6
25.6
1.5
5.2
4.3
15.4
2.7
0.9
0.1
0.9
10.1
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
Only present in NM_001099401.1
1392-1391 reverse
GTCTGCTGTTGG-GGAATCTGAGTC
3.2 kb
Only present in NM_001099400.1
1418-1417 reverse
TTCTTCAGGGAT-ACCATTTACCTG
stop codon
1423-1425 (ex 13)
¥ Changed in version C2 (from lot C2-0813 onwards). Small change in length, no change in sequence
detected.
SALSA MLPA probemix P099 GCH1-TH-SGCE
Page 4 of 7
MRC-Holland
®
Description version 14; 04-02-2014
MLPA
¬ Flanking probe. Included to facilitate determination of the extent of a deletion / duplication. Copy number
alterations of flanking and reference probes are unlikely to be related to the condition tested.
# The following probes have a validated SNP on their ligation site: 246 nt probe rs11548284; 153 nt probe
rs200979763; 355 nt probe rs148126317. In case of an apparent deletion it is recommended to sequence
the target site.
The NM_003919.2 sequence represents transcript variant 2 and is a reference standard in the NCBI
RefSeqGene project.
The SGCE exon numbering has changed. From description version 08 onwards, we have adopted the
NCBI exon numbering that is present in the NM_003919.2 sequence for the SGCE gene. This exon
numbering used here may differ from literature! The exon numbering used in previous versions of this
product description can be found between brackets in Table 1 & 2.
Note: Exon numbering used here may differ from literature! Please notify us of any mistakes. Complete
probe sequences are available on request: info@mlpa.com.
SALSA MLPA probemix P099 GCH1-TH-SGCE
Page 5 of 7
MRC-Holland
®
MLPA
Description version 14; 04-02-2014
SALSA MLPA probemix P099-C2 GCH1-TH-SGCE sample picture
100000
128.23
90000
96.39
86.00
80000
246.86
70000
170.94
163.88
91.14
60000
105.51
140.26
157.60
134.14
152.16
145.74
217.86
210.13
371.14
202.09
177.92
228.65
238.90 254.83
50000
288.93
264.06
317.52
364.08
297.77
327.65
40000
409.70
185.02
191.22
310.15
281.81
274.58
100.66
345.60
336.52
354.85
419.03
380.36
401.00
391.08
30000
D ye S ign a l
20000
10000
0
50
100
150
200
250
Size (nt)
300
350
400
450
Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA
analysed with SALSA MLPA probemix P099-C2 GCH1-TH-SGCE (lot C2-0813).
Implemented Changes – compared to the previous product description version(s).
Version 14 (53)
- Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new
picture included).
- Warning added on SNPs being present on the ligation site of the 246 nt 153 nt and 355 nt probes
Version 13 (48)
- Warning about 210nt probe removed from product description. Non-specific peak formation by this
probe is prevented by new PCR primer mix and One-tube protocol.
Version 12 (48)
- Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added.
Version 11 (48)
-
Small correction of chromosomal locations in Table 1 and 2.
Various minor textual changes.
Various minor layout changes.
Remark on RefSeqGene standard and transcript variant added below Table 2.
Version 10 (46)
- Warning added in Table 1, 210 nt probe 03145-L02613.
- New reference added on page 2.
- Minor textual and layout changes on page 1 and 2.
Version 09 (46)
- Extra information added regarding the exon numbering of SGCE. Exons 10 and 12 are present in other
transcript variants and no probes are included for these exons in this probemix.
Version 08 (45)
- 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).
SALSA MLPA probemix P099 GCH1-TH-SGCE
Page 6 of 7
MRC-Holland
®
Description version 14; 04-02-2014
MLPA
- Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the
amplification products.
- Sentence “when only small numbers of samples are tested, visual comparison of peak profiles should be
sufficient” removed from data analysis section
- Ligation sites updated according to new version of the NM_reference sequence.
SALSA MLPA probemix P099 GCH1-TH-SGCE
Page 7 of 7