Download Product description P018-G1 SHOX-v03 - MRC

MRC-Holland
Product Description version G1-03; Issued 23 February 2017
®
MLPA
Product Description SALSA® MLPA® probemix
P018-G1 SHOX
To be used with the MLPA General Protocol.
Version G1. One SHOX area probe has been replaced (CNE4) and four new probes have been included for
the upstream SHOX enhancer sequences CNE-2, CNE-3 and CNE-5 and the downstream CNE7 enhancer
sequence. For complete product history see page 10.
Catalogue numbers:
• P018-025R: SALSA® MLPA® probe mix P018 SHOX, 25 reactions.
• P018-050R: SALSA® MLPA® probe mix P018 SHOX, 50 reactions.
• P018-100R: SALSA® MLPA® probe mix P018 SHOX, 100 reactions.
To be used in combination with a SALSA® MLPA® reagent kit, available for various number of reactions.
MLPA reagent kits are either provided with FAM or Cy5.0 dye-labelled PCR primer, suitable for Applied
Biosystems and Beckman capillary sequencers, respectively (see www.mlpa.com).
Certificate of Analysis: Information regarding storage conditions, quality tests, and a sample
electropherogram from the current sales lot is available at www.mlpa.com.
Precautions and warnings: For professional use only. Always consult the most recent product description
AND the MLPA General Protocol before use: www.mlpa.com. It is the responsibility of the user to be aware
of the latest scientific knowledge of the application before drawing any conclusions from findings generated
with this product.
Intended use: The SALSA® MLPA® probemix P018 SHOX is an in vitro diagnostic (IVD)1 or research use
only (RUO) assay for the detection of deletions or duplications in the human short stature homeobox (SHOX)
gene and its regulatory regions located on Xp22.33/Yp11.32 as a cause for disorders associated with short
stature, including Leri-Weill dyschondrosteosis (LWD), Langer mesomelic dysplasia (LMD), and Idiopathic
short stature (ISS). This assay can be used with human DNA derived from peripheral blood and buccal swab.
In the majority of patients, defects in the SHOX gene region are deletions, but point mutations can occur
which will not be detected by MLPA. It is therefore recommended to use this SALSA® MLPA® probemix in
combination with sequence analysis of the SHOX coding region. Copy number changes detected by only a
single probe always require validation by another method. This probemix is not intended to be used as a
standalone assay for clinical decisions. The results of this test should be interpreted by a clinical molecular
geneticist or equivalent.
1
Please note that this probemix is for In Vitro Diagnostic use (IVD) in the countries specified at the end of this
product description. In all other countries, the product is for Research Use Only (RUO).
Clinical background: SHOX is located in the pseudoautosomal region 1 (PAR1) on the short arm of the X
and Y chromosomes. Located upstream and downstream of SHOX are highly conserved non-coding elements
(CNEs), important SHOX enhancer sequences. Mutations in SHOX or its regulatory regions cause a range of
disorders associated with short stature, including LWD, LMD, and ISS, as SHOX is a known transcription
factor highly expressed in tissues responsible for bone development (Benito-Sanz et al. 2012b).
LWD is a dominant skeletal disorder characterised by short stature, mesomelic shortening of the limbs, and
the characteristic Madelung deformity. LMD is a more severe form of LWD and is a result of mutations in
both SHOX alleles (Bertorelli et al. 2007, Campos-Barros et al. 2007, Shears et al. 2002, Zinn et al. 2002).
ISS classifies individuals with a height below the third centile when in the absence of an identified cause.
Heterozygous mutations of SHOX and/or its regulatory elements are detected in approximately 60% of LWD
patients and approximately 5-15% of ISS cases. Homozygous or compound heterozygous mutations of
SHOX and/or its downstream enhancers are detected in 75% of LMD patients (Benito-Sanz et al. 2006,
Benito-Sanz et al. 2012a, Chen et al. 2009, Huber et al. 2006).
SALSA MLPA Probemix P018 SHOX
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Product Description version G1-03; Issued 23 February 2017
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MLPA
In individuals with a SHOX related disorder, 70-80% of mutations are due to a large deletion, 2-6% is from
a partial deletion, and 20-25% are from point mutations (Binder 2011, Caliebe et al. 2012). Duplications
have also been reported in LWD and ISS patients (Benito-Sanz et al. 2011b). The MLPA technique can
detect most deletions and duplications and therefore complements sequence analysis of SHOX.
More information is available on http://www.ncbi.nlm.nih.gov/books/NBK1215/.
Gene structure and Transcript variants: SHOX spans 35 kb of the pseudoautosomal region 1 (PAR1)
located on Xp22.33/Yp11.32. The Genbank chromosomal sequence is NG_009385.1. Two major transcript
variants have been described: http://www.ncbi.nlm.nih.gov/gene/6473. SHOX transcript variant 1
(NM_000451.3, 3757 nt, coding sequence 692-1570) represents the longer transcript and encodes the
longer active isoform (SHOXa). The ATG translation start site is located in exon 2 and the termination site is
located in exon 6. The second transcript variant NM_006883.2 (1951 nt, coding sequence 692-1369)
contains an alternate 3' terminal exon compared to transcript variant 1 and encodes a shorter isoform
(SHOXb) with a different C-terminus compared to isoform SHOXa. Several regulatory sequences located
outside of SHOX and affecting SHOX transcription have been described (e.g. Benito-Sanz et al. 2012b,
Durand et al. 2010, Fukami et al. 2006, Sandbacka et al. 2011).
Exon numbering: The exon numbering used in this P018-G1 SHOX product description and in the P018-G1
SHOX Coffalyser.Net analysis sheet is the exon numbering from the Genbank NG_009385.1 reference
sequence. This exon numbering is different from the SHOX exon numbering in many articles where exon 7 is
referred to as exon 6b.
P018-G1 probemix content: This SALSA® MLPA® probemix P018 SHOX contains 48 MLPA probes with
amplification products between 124 and 503 nt: 26 probes located in the SHOX + Xp22 areas (including
SHOX and its regulatory regions); 13 probes elsewhere on the X-chromosome and 9 reference probes
detecting autosomal chromosomes (table 1 and table 2).
One probe is present for each exon of human SHOX. In addition, one probe is included detecting the area
just before the SHOX promoter region. Furthermore, several probes are present for the regions that have
been identified to be SHOX regulatory regions, located upstream and downstream of SHOX. Finally, several
probes for the X-chromosome are included in this probemix that can be used to characterise larger deletions
and to distinguish SHOX deletions from a Turner syndrome karyotype.
This probemix contains ten quality control fragments generating amplification products between 64 and 118
nt: four DNA Quantity Fragments (Q-Fragments), three DNA Denaturation Fragments (D-Fragments), one
chromosome X and two chromosome Y-specific fragments (Table 1). The Q-fragments are only visible when
less than 100 ng sample DNA is used. Low signal of the 88 or 96 nt fragment indicates incomplete DNA
denaturation. More information on how to interpret observations on these control fragments can be found in
the MLPA General Protocol.
MLPA technique: The principles of the MLPA technique (Schouten et al. 2002) are described in the MLPA
General Protocol (www.mlpa.com).
MLPA technique validation: Internal validation of the MLPA technique using 16 DNA samples from
healthy individuals of the same sex is required, in particular, when using MLPA for the first time, or when
changing the sample handling procedure, DNA extraction method or instruments used. This validation
experiment should result in a standard deviation <0.10 for all probes over the experiment.
Required specimens: Purified DNA from peripheral blood or buccal swab, free from impurities known to
affect MLPA reactions. For more information please refer to the section on DNA sample treatment found in
the MLPA General Protocol.
Reference samples: Reference DNA samples should be derived from the same tissue type, handled using
the same procedure, and prepared using the same DNA extraction method as the patient samples.
Reference samples should be derived from unrelated individuals who are from families without a history of
short stature. All reference samples should be of the same sex. It is recommended to use reference and
SALSA MLPA Probemix P018 SHOX
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MLPA
Product Description version G1-03; Issued 23 February 2017
patient samples of the same sex to facilitate interpretation. More information regarding the selection and use
of reference samples can be found in the MLPA General Protocol.
Positive control DNA samples: MRC-Holland cannot provide positive DNA samples. Inclusion of a positive
sample in each experiment is recommended.
Performance characteristics: In individuals with a SHOX related disorder, 70-80% of all mutations are
due to a large deletion and should be detected by this MLPA probemix (Binder 2011, Caliebe et al. 2012).
The analytical sensitivity and specificity for the detection of deletions/duplications in SHOX and its
surrounding enhancer regions (based on a 2006-2014 literature review) is very high and can be considered
>99%.
Analytical performance can be compromised by: SNPs or other polymorphisms (e.g. indels) in the DNA
target sequence, impurities in the DNA sample, incomplete DNA denaturation, the use of insufficient or too
much sample DNA, the use of insufficient or unsuitable reference samples, problems with capillary
electrophoresis or a poor data normalisation procedure and other technical errors. The MLPA General
Protocol contains technical guidelines and information on data evaluation/normalisation.
Data analysis: Coffalyser.Net software must be used for data analysis in combination with the appropriate
lot-specific MLPA Coffalyser sheet. For both, the latest version should be used which are freely downloadable
at www.mlpa.com. Use of other non-proprietary software may lead to inconclusive or false results. For more
details on MLPA quality control and data analysis, see the Coffalyser.Net Manual.
Interpretation of results: The expected results for SHOX-specific MLPA probes are allele copy numbers of
2 (normal), 0 (homozygous deletion), 1 (heterozygous deletion), 3 (heterozygous duplication), and 4
(heterozygous triplication/homozygous duplication). Please see table 3 for examples of potential results.
The standard deviation of all probes in the reference samples should be <0.10 and the dosage quotient
(DQ) of the reference probes in the patient samples should be between 0.80 and 1.20. When these criteria
are fulfilled, the following cut-off values for the DQ of the probes can be used to interpret MLPA results:
Copy Number status
Normal
Homozygous deletion
Heterozygous deletion
Heterozygous duplication
Heterozygous triplication/Homozygous duplication
Ambiguous copy number
-
-
-
-
-
Dosage quotient
0.80 < DQ < 1.20
DQ = 0
0.40 < DQ < 0.65
1.30 < DQ < 1.65
1.75 < DQ < 2.15
All other values
Arranging probes according to chromosomal location facilitates interpretation of the results and may
reveal more subtle changes such as those observed in mosaic cases. Analysis of parental samples may
be necessary for correct interpretation of complex results.
False positive results: Please note that abnormalities detected by a single probe (or multiple consecutive
probes) still have a considerable chance of being a false positive result. Incomplete DNA denaturation,
due to salt contamination, can lead to a decreased probe signal, in particular for probes located within a
CpG island in or near the SHOX gene. The use of an additional purification step or an alternative DNA
extraction method may resolve such cases.
False positive duplication results: Contamination of DNA samples with cDNA or PCR amplicons of
individual exons can lead to false positive duplication results (Varga et al. 2012). Analysis of an
independently collected secondary DNA sample can exclude these kinds of contamination artefacts.
Copy number variation in healthy individuals has been found using this probemix. Normal copy number
variation in healthy individuals is described in the database of genomic variants:
http://dgv.tcag.ca/dgv/app/home. Users should always verify the latest updates of the database and
scientific literature when interpreting their findings.
Not all abnormalities detected by MLPA are pathogenic. In some genes, intragenic deletions are known
that result in very mild, or no disease (Schwartz et al. 2007). For many genes, more than one transcript
variant exists. Copy number changes of exons that are not present in all transcript variants may not
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Product Description version G1-03; Issued 23 February 2017
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MLPA
have clinical significance. Duplications that include the first or last exon of a gene (e.g. exons 1-3) might
in some cases not result in inactivation of that gene copy.
Copy number changes detected by reference probes are unlikely to have any relation to the condition
tested for.
Notes SHOX results:
- Short stature and skeletal deformities due to SHOX defects are pseudoautosomal dominant disorders.
Therefore, a heterozygous mutation of SHOX is expected to result in these phenotypes.
- A recurrent 47.5 kb deletion downstream of SHOX has been described by Benito-Sanz et al. (2012b).
This 47.5 kb deletion is covered by three probes in this P018-G1 probemix (Table 2).
- Complete or partial duplications found within SHOX or its surrounding regulatory regions have been
found in LWD and ISS patients (Benito-Sanz et al. 2011b).
- Deletion of the SHOX intron 6 probes, which are located downstream of the stop codon, may not affect
SHOX gene function.
- Please note that single exon deletions have a considerable chance of being a false positive result, either
due to non-pathogenic copy number variants (Benito-Sanz et al. 2011a), mutations within the probe
binding site (Barca-Tierno et al. 2011) or due to impurities in the DNA.
- Not all copy number changes detected by the “SHOX-area” probes will affect SHOX gene function.
Analysis of family members may be required for correct interpretation of results.
Limitations of the procedure:
− MLPA cannot detect any changes that lie outside the target sequence of the probes and will not detect
most inversions or translocations. Even when MLPA did not detect any aberrations, the possibility
remains that biological changes in that gene or chromosomal region do exist but remain undetected.
− Sequence changes (e.g. SNPs, point mutations, small indels) in the target sequence detected by a probe
can cause false positive results. Mutations/SNPs (at least up to 20 nt from the probe ligation site) can
reduce the probe signal by preventing ligation of the probe oligonucleotides or by destabilising the
binding of a probe oligonucleotide to the sample DNA.
Confirmation of results: Copy number changes detected by only a single probe always require
confirmation by another method. An apparent deletion detected by a single probe can be due to e.g. a
mutation/polymorphism that prevents ligation or destabilises the binding of probe oligonucleotides to the
DNA sample. Sequence analysis can establish whether mutations or polymorphisms are present in the probe
target sequence. The finding of a heterozygous mutation or polymorphism indicates that two different alleles
of the sequence are present in the sample DNA and that a false positive MLPA result was obtained.
Copy number changes detected by one or more than one consecutive probe should be confirmed by another
independent technique such as long range PCR, qPCR, array CGH or Southern blotting, whenever possible.
Deletions/duplications of more than 50 kb in length can often be confirmed by FISH.
SHOX mutation database: https://grenada.lumc.nl/LOVD2/MR/home.php?select_db=SHOX. We strongly
encourage users to deposit positive results in the X-chromosome gene database. Recommendations for the
nomenclature to describe deletions/duplications of one or more exons can be found on
www.hgvs.org/mutnomen/. Please report copy number changes detected by the reference probes, false
positive results due to SNPs and unusual results (e.g. a duplication of SHOX exons 3 and 5, but not exon 4)
to MRC-Holland: [email protected].
SALSA MLPA Probemix P018 SHOX
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MLPA
Product Description version G1-03; Issued 23 February 2017
Table 1. SALSA® MLPA® P018-G1 SHOX probemix
Length
(nt)
SALSA MLPA probe
Chromosomal position(a)
reference
Outside PAR1
SHOX region / PAR1
64-70-76-82 Q-fragments (Only visible with <100 ng sample DNA)
88-92-96
D-fragments (Low signal of 88 or 96 fragment indicates incomplete denaturation)
100
X-fragment (X chromosome specific)
105
Y-fragment (Y chromosome specific)
118
Y-fragment (Y chromosome specific)
124 *
Reference probe 15370-L13762
7q11
130 *
SHOX AREA probe 18885-L24430
Xp22-PAR1
136
SHOX AREA probe 05642-L05096
Xp22-PAR1
142
IL3RA probe 13597-L15055
Xp22-PAR1
148
SHOX AREA probe 05648-L06218
Xp22-PAR1
154
SHOX AREA probe 13821-L14642
Xp22-PAR1
160
Reference probe 04966-L04696
1p22
166
SHOX probe 01145-L00702
Exon 1 Xp22-PAR1
172 *
SHOX AREA probe 18886-L24431
Xp22-PAR1
178
SHOX AREA probe 05649-L20176
Xp22-PAR1
185
SHOX AREA probe 06293-L20177
Xp22-PAR1
191
Reference probe 06057-L05512
4p16
199
SHOX AREA probe 13296-L20175
Xp22-PAR1
204
SHOX probe 01146-L06220
Exon 2 Xp22-PAR1
211
PPP2R3B probe 09333-L10292
Xp22-PAR1
219
Reference probe 03247-L02684
13q14
226
SHOX probe 09336-L20178
Intron 6 Xp22-PAR1
231
SHOX probe 09337-L00911
Exon 6 Xp22-PAR1
238
ANOS1 (KAL1) probe 06402-L09795
Xp22.31
245
SHOX probe 01147-L00802
Exon 3 Xp22-PAR1
254
ARSF probe 16846-L20647
Xp22.33
261
Reference probe 00587-L20649
18q21
266
SHOX probe 01341-L20651
4 kb before SHOX-PAR1
274
FANCB probe 03906-L03066
Xp22.2
283
NLGN4X probe 05587-L04577
Xp22.31
290
SHOX AREA probe 06291-L06222
Xp22-PAR1
300
SHOX probe 01148-L15501
Exon 4 Xp22-PAR1
310
ASMT probe 01153-L00712
Xp22-PAR1
318
SHOX AREA probe 05645-L05099
Xp22-PAR1
328
PRKX probe 16898-L19768
Xp22.33
337 ∆
SHOX probe 01149-L19676
Exon 5 Xp22-PAR1
346
Reference probe 06560-L06118
1q32
355
VAMP7 probe 01156-L00659
Xq28-PAR2
364 *
SHOX AREA probe 18889-L25087
Xp22-PAR1
379 ¥
SHOX AREA probe 14697-L24245
Xp22-PAR1
387 ¥
CSF2RA probe 10251-L24246
Xp22-PAR1
395 ¥
SHOX probe 09338-L24247
Intron 6 Xp22-PAR1
403
CRLF2 probe 13911-L19678
Xp22-PAR1
412
Reference probe 09793-L12593
15q21
420 ¥
AIFM1 probe 00820-L25090
Xq26.1
427 *
SHOX AREA probe 18891-L25088
Xp22-PAR1
439 ¥
SHOX AREA probe 05646-L24249
Xp22-PAR1
445 ¥
SHOX AREA probe 09335-L24250
Xp22-PAR1
456 ¥
ZBED1 probe 16858-L25227
Xp22-PAR1
466 ¥
SHOX AREA probe 13297-L24253
Xp22-PAR1
474
Reference probe 09888-L10301
16p13
481 *
SHOX AREA probe 18893-L25091
Xp22-PAR1
503 *
Reference probe 09870-L19465
2p16
(a) The exon numbering used in this P018-G1 SHOX product description and in the P018-G1 SHOX Coffalyser.Net
analysis sheet is the exon numbering from the Genbank NG_009385.1 reference sequence. This exon numbering is
SALSA MLPA Probemix P018 SHOX
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Product Description version G1-03; Issued 23 February 2017
different from the SHOX exon numbering in many articles where exon 7 is referred to as exon 6b.
*
New in version G1 (from lot G1-0513 onwards).
¥
Changed in version G1 (from lot G1-0513 onwards). Small change in length, but no change in sequence detected.
∆
This probe has been reported to be more variable.
Table 2. Xp/Yp probes arranged according to chromosomal location
Length
(nt)
SALSA MLPA
probe
Gene
Exon(a)
Ligation site(b)
Partial sequence(c) (24 nt
adjacent to ligation site)
p-telomere
------------------------------------------ Start of PAR1 -----------------------------------------211 ¬
09333-L10292 PPP2R3B gene
PAR1
CGTCCGAGTTCC-ACTCGCGCTACA
364
18889-L25087
SHOX AREA
CNE-5
GAAATGTTAACA-GCTCCCCGAGCT
130
18885-L24430
SHOX AREA
CNE-3
ATGGCAGAGCAT-TTGTACCCCTGG
427
18891-L25088
SHOX AREA
CNE-2
TACACCGTTATG-CGGATGCTCGTT
SHOX AREA
266
01341-L20651
Upstream of SHOX GCCTGGAACAGA-ACTTCCGCGGGG
LOC159015
SHOX
start codon
166
204
245
300 #
337 ∆
231
01145-L00702
01146-L06220
01147-L00802
01148-L15501
01149-L19676
09337-L00911
Exon
Exon
Exon
Exon
Exon
Exon
1
2
3
4
5
6
stop codon
226
09336-L20178
SHOX Intron 6
Distance to
next probe
227.0 kb
91.6
61.7
56.3
63.5
4.7 kb
NM_000451.3
692-694 (Ex 2)
99-100
920-921
1032-1033
1198-1199
1261-1262
1506-1507
TTTCTACTGCAA-ACAGAAATGGGA
ACCACGTAGACA-ATGACAAGGAGA
CGGGCAGACCAA-GCTGAAACAGAG
CAGAACCGGAGA-GCCAAGTGCCGC
ACAGCCAACCAC-CTAGACGCCTGC
AAGCAACAGCAA-GAATTCCAGCAT
6.7
3.6
6.2
0.2
3.5
6.4
¬
x ¬
06402-L09795
ANOS1 (KAL1)
gene
FANCB gene
AIFM1 gene
kb
kb
kb
kb
kb
kb
TGGCTTCACGAG-TTCAGCCCATTG
6.4 kb
1568-1570 (Ex 6)
6.4 kb after Ex 6;
8 kb before Ex 7
1.4 kb before Ex 7
6.2 kb after CNE2
CNE3
CNE4
CNE5
CNE7
2 kb before CNE8
5.4 kb after CNE8
6 kb before CNE9
CNE9
CNE9
395
09338-L24247 SHOX Intron 6
TCCCACATTCTT-GGAATCACAATG
56.9
136
05642-L05096
SHOX AREA
GCAGCAGTGAAA-GTGAGCATTCCC
19.8
154
13821-L14642
SHOX AREA
GATGGCTGATAA-TTACTCCGTATG
19.4
172
18886-L24431
SHOX AREA
GCCTCCGATACA-GTTTACGGCTTC
37.4
199
13296-L20175
SHOX AREA
GGAAAACCACGT-TCCTATCGATCC
29.6
481 ^
18893-L25091
SHOX AREA
CAGACCAGGTCT-CCTGTTTCATGT
28.1
318 ^
05645-L05099
SHOX AREA
TGTTCCCACCGT-AAAACTCACTCC
8.4
439 ^
05646-L24249
SHOX AREA
TGCATGTCTGCT-TTTTGAATGGCC
10.7
466
13297-L24253
SHOX AREA
TACAGCAAATGA-TACGTATAAATT
6.3
290
06291-L06222
SHOX AREA
CTTGAAAGGGCA-GGAACTCTAATT
0.4
185
06293-L20177
SHOX AREA
TAATTGATGAGA-TGCAGAAGCCAG
15.4
148
05648-L06218
Xp22-PAR1
Downstream
TGGTGCTGAAAT-GAGGAAGCCCTG
48.7
178
05649-L20176
Xp22-PAR1
Downstream
TGAGGAGGTACC-TCAAAGCTAAAC
64.4
445
09335-L24250
Xp22-PAR1
Downstream
GAAATTCAGTTT-TAATAACACAGA
66.0
379
14697-L24245
Xp22-PAR1
Downstream
CTCTGGTGAGAT-GCCATCTAGAGA
338.0
403 ¬
13911-L19678
CRLF2 gene
PAR1-Xp22PAR
GAATGCCAGCAA-ATACTCCAGGAC
73.9
387 ¬
10251-L24246
CSF2RA gene
PAR1-Xp22PAR
GACAAGCCTTCT-GCTCTGTGAGTT
69.8
142 ¬
13597-L15055
IL3RA gene
PAR1-Xp22PAR
TGCACAGATAAG-TTTGTCGTCTTT
280.7
310 ¬
01153-L00712
ASMT gene
PAR1-Xp22PAR
GACATCCCAGAA-GTGGTGTGGACG
706.4
456 ¬
16858-L25227
ZBED1 gene
PAR1-Xp22PAR
TCGTCAAGAGCA-ACACGGAGCAGA
593.8
------------------------------------------------- End of PAR1 ------------------------------------------------254 x ¬
16846-L20647
ARSF gene
Xp22.33
CATCCATATAAT-TATGGGTTTGAC
536.9
328 x ¬
16898-L19768
PRKX gene
Xp22.33
CGATTAGGAAAC-ATGAAGGTCAGT
2606.5
283 x ¬
05587-L04577
NLGN4X gene
Xp22.31
GACGGCTTGGGT-GATGCACGAAAT
2311.9
238 x
kb
kb
kb
kb
Xp22.31
GTTTCCTGAAGC-GTGTGCCCACAA
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
6304.2 kb
274
03906-L03066
Xp22.2
TCTCATCAGAAT-TCTCCCTATAAA
114321.4 kb
420 x ¬
00820-L25090
Xq26.1
TATTGGTCTTGT-GGACAGTAGTTT
25732.3 kb
-------------------------------------------------Start of PAR2 ------------------------------------------------355 + ¬
01156-L00659
VAMP7 gene
PAR2
TGTGGGAAAAGT-GTTTCCATTCTG
98 kb
SALSA MLPA Probemix P018 SHOX
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Product Description version G1-03; Issued 23 February 2017
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MLPA
q-telomere
(a) The exon numbering used in this P018-G1 SHOX product description and in the P018-G1 SHOX Coffalyser.Net
analysis sheet is the exon numbering from the Genbank NG_009385.1 reference sequence. This exon numbering is
different from the SHOX exon numbering in many articles where exon 7 is referred to as exon 6b.
(b) Ligation sites of the P018 SHOX MLPA probes are indicated according to Refseq sequence NM_000451.3 containing 7
exons.
(c) Only partial probe sequences are shown. Complete probe sequences are available at www.mlpa.com. Please notify
us of any mistakes: [email protected].
∆
This probe has been reported to be more variable.
A founder SHOX point mutation in the Spanish Gypsy population, c.508G>C (p.A170P), results in a strongly reduced
probe signal of the 300 nt exon 4 probe (Barca-Tierno et al. 2011).
^
The 481, 318 and 439 probes are within the recurrent ~47.5 kb deletion region described by Benito-Sanz et al.
(2012b).
x
X-chromosome, outside PAR region. Gives 50% reduced signal in males as compared to females.
+
The VAMP7 probe at 355 nt is located very close to the X and Y q-telomere in PAR2.
¬
Flanking probe. Included to help determine the extent of a deletion/duplication. Copy number alterations of flanking
and reference probes are unlikely to be related to the condition tested.
#
CNE = Conserved Non-coding DNA Element. Locations of the upstream regulatory regions (CNE-2, CNE-3 and CNE-5)
are based on Durand et al. (2010) and Benito-Sanz et al. (2012b). Locations of the downstream regulatory regions
(CNE2 through CNE9) are based on Benito-Sanz et al. (2012b), Fukami et al. (2006), and Sabherwal et al. (2007).
SALSA MLPA Probemix P018 SHOX
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MRC-Holland
®
MLPA
Product Description version G1-03; Issued 23 February 2017
Table 3. Examples of probe signal ratios from potential results
RATIO: when
compared to
Type of test sample
Normal FEMALE DNA
Normal MALE DNA
FEMALE DNA with a
deletion that includes
one or more exons of
the SHOX gene
MALE DNA with a
deletion that includes
one or more exons of
the SHOX gene
FEMALE or MALE DNA
with a deletion in the
area upstream or
downstream of SHOX,
but not including
SHOX gene probes
FEMALE sample with
Turner syndrome
(45,X)
MALE sample with
Klinefelter syndrome
(47,XXY)
FEMALE sample with
Triple X-syndrome
(47,XXX).
Probes
All PAR1/PAR2 probes, incl.
SHOX AREA, Xp22
X-chromosome probes
OUTSIDE PAR1/2 region
105 & 118 nt Y-chromosome
probes outside PAR1 + PAR2
All PAR1/PAR2 probes, incl.
SHOX AREA, Xp22
X-chromosome probes
OUTSIDE PAR1/2 region
105 & 118 nt Y-chromosome
probes outside PAR1 + PAR2
One or more SHOX probes +
possibly one or more SHOX
AREA and/or Xp22 probes
Deletions extending outside
PAR1: one or more of the 6
chromosome X probes
(marked x in Table 2) also
show a lower copy number
One or more SHOX probes +
possibly one or more SHOX
AREA and/or Xp22 probes
Deletions extending outside
PAR1: one or more of the 6
chromosome X probes
(marked x in Table 2) also
show a lower copy number
One or more probes
upstream or downstream of
SHOX, indicated with SHOX
AREA in table 2
All PAR1/PAR2 probes, incl.
SHOX AREA, Xp22
X-chromosome probes
OUTSIDE PAR1/2 region
105 & 118 nt Y-chromosome
probes outside PAR1 + PAR2
All PAR1/PAR2 probes, incl.
SHOX AREA, Xp22.
X-chromosome probes
OUTSIDE PAR1/2 region
105 & 118 nt Y-chromosome
probes outside PAR1 + PAR2
All PAR1/PAR2 probes, incl.
SHOX AREA, Xp22.
X-chromosome probes
OUTSIDE PAR1/2 region
105 & 118 nt Y-chromosome
probes outside PAR1 + PAR2
SALSA MLPA Probemix P018 SHOX
Explanation
Male
ref
Female
ref
1
1
2
1
0
-
1
1
1
0.5
1
∞
0.5
0.5
Heterozygous deletion in essential SHOX area in PAR1.
PAR1 region is located on X and Y.
This typically causes LWD or short stature.
1
0.5
Heterozygous deletion outside PAR1.
Sequences present on only X.
0.5
0.5
Heterozygous deletion in essential SHOX area in PAR1.
PAR1 region is located on X and Y.
This typically causes LWD or short stature.
0
0
0.5
0.5
0.5
0.5
1
0.5
0
-
1.5
1.5
2
1
1
∞
1.5
1.5
3
1.5
0
-
PAR1 & PAR2 regions are located on X and Y:
normal females: 2 copies; normal males: 2 copies.
Sequences present on only X:
normal females: 2 copies; normal males: 1 copy.
Sequences present on only Y:
normal females: 0 copies; normal males: 1 copy.
PAR1 & PAR2 regions are located on X and Y:
normal females: 2 copies; normal males: 2 copies.
Sequences present on only X:
normal females: 2 copies; normal males: 1 copy.
Sequences present on only Y:
normal females: 0 copies; normal males: 1 copy.
Heterozygous deletion outside PAR1.
Sequences present on only X.
Heterozygous deletion.
PAR1 region is located on X and Y.
Deletions in this region have been associated
with LWD & ISS (Benito-Sanz et al. 2005, BenitoSanz et al. 2012a, Benito-Sanz et al. 2012b, Chen et al.
2009). Not all deletions detected by these probes
will result in LWD or ISS!
PAR1 & PAR2 regions are located on X and Y.
Only one X is present in this case.
Probe sequences located on only X.
Only one X is present in this case.
Probe sequences located on only Y.
No Y present in this case.
PAR1 & PAR2 regions are located on X and Y.
Three instead of the normal two copies present.
Probe sequences present on only X.
Two Xs are present in this case.
Probe sequences present on only Y.
One Y present in this case.
PAR1 & PAR2 regions are located on X and Y.
Three instead of the normal two copies present.
Probe sequences present on only X.
Three Xs are present in this case.
Probe sequences located on only Y.
No Y present in this case.
Page 8 of 11
MRC-Holland
Product Description version G1-03; Issued 23 February 2017
®
MLPA
Related SALSA® MLPA® probe mixes
P329 CRLF2CSF2RA-IL3RA
P216 GHD
P217 IGF1R
P262 GHI
P026 Sotos
P384 Human Height
Probes for these PAR1 genes. Deletions of the IL3RA and CSF2RA genes, resulting in
CRLF2 overexpression, are found in Acute Lymphoblastic Leukaemia samples.
Probes for GH1, PROP1, POU1F1, GHRHR, HESX1, LHX3 and LHX4.
Probes for IGF1R and IGFBP3.
Probes for IGF1, GHR, JAK2 and STAT5B.
Probes for NSD1.
Probes for HHIP, HMGA2, ADAMTSL3, NPPC and IHH.
References
Barca-Tierno V et al. (2011). Identification of a Gypsy SHOX mutation (p.A170P) in Leri-Weill dyschondrosteosis and
Langer mesomelic dysplasia. Eur J Hum Genet. 19:1218-1225.
Benito-Sanz S et al. (2005). A novel class of Pseudoautosomal region 1 deletions downstream of SHOX is associated
with Leri-Weill dyschondrosteosis. Am J Hum Genet. 77:533-544.
Benito-Sanz S et al. (2006). PAR1 deletions downstream of SHOX are the most frequent defect in a Spanish cohort
of Leri-Weill dyschondrosteosis (LWD) probands. Hum Mutat. 27:1062.
Benito-Sanz S et al. (2011a). A non-pathogenic pseudoautosomal region 1 copy number variant downstream of
SHOX. Am J Med Genet A. 155A:935-937.
Benito-Sanz S et al. (2011b). Clinical and molecular evaluation of SHOX/PAR1 duplications in Leri-Weill
dyschondrosteosis (LWD) and idiopathic short stature (ISS). J Clin Endocrinol Metab. 96:E404-412.
Benito-Sanz S et al. (2012a). Identification of the first PAR1 deletion encompassing upstream SHOX enhancers in a
family with idiopathic short stature. Eur J Hum Genet. 20:125-127.
Benito-Sanz S et al. (2012b). Identification of the first recurrent PAR1 deletion in Leri-Weill dyschondrosteosis and
idiopathic short stature reveals the presence of a novel SHOX enhancer. J Med Genet. 49:442-450.
Bertorelli R et al. (2007). The homozygous deletion of the 3' enhancer of the SHOX gene causes Langer mesomelic
dysplasia. Clin Genet. 72:490-491.
Binder G (2011). Short stature due to SHOX deficiency: genotype, phenotype, and therapy. Horm Res Paediatr.
75:81-89.
Caliebe J et al. (2012). IGF1, IGF1R and SHOX mutation analysis in short children born small for gestational age and
short children with normal birth size (idiopathic short stature). Horm Res Paediatr. 77:250-260.
Campos-Barros A et al. (2007). Compound heterozygosity of SHOX-encompassing and downstream PAR1 deletions
results in Langer mesomelic dysplasia (LMD). Am J Med Genet A. 143A:933-938.
Chen J et al. (2009). Enhancer deletions of the SHOX gene as a frequent cause of short stature: the essential role of
a 250 kb downstream regulatory domain. J Med Genet. 46:834-839.
Durand C et al. (2010). Enhancer elements upstream of the SHOX gene are active in the developing limb. Eur J Hum
Genet. 18:527-532.
Fukami M et al. (2006). Transactivation function of an approximately 800-bp evolutionarily conserved sequence at
the SHOX 3' region: implication for the downstream enhancer. Am J Hum Genet. 78:167-170.
Huber C et al. (2006). High incidence of SHOX anomalies in individuals with short stature. J Med Genet. 43:735-739.
Sabherwal N et al. (2007). Long-range conserved non-coding SHOX sequences regulate expression in developing
chicken limb and are associated with short stature phenotypes in human patients. Hum Mol Genet. 16:210-222.
Sandbacka M et al. (2011). Evaluation of SHOX copy number variations in patients with Mullerian aplasia. Orphanet
J Rare Dis. 6:53.
Schouten JP et al. (2002). Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe
amplification. Nucleic Acids Res. 30:e57.
Schwartz M et al. (2007). Deletion of exon 16 of the dystrophin gene is not associated with disease. Hum Mutat.
28:205.
Shears DJ et al. (2002). Pseudodominant inheritance of Langer mesomelic dysplasia caused by a SHOX homeobox
missense mutation. Am J Med Genet. 110:153-157.
Varga RE et al. (2012). MLPA-based evidence for sequence gain: pitfalls in confirmation and necessity for exclusion
of false positives. Anal Biochem. 421:799-801.
Zinn AR et al. (2002). Complete SHOX deficiency causes Langer mesomelic dysplasia. Am J Med Genet. 110:158163.
Selected publications using SALSA® MLPA® Probemix P018 SHOX
Albuisson J et al. (2012). Clinical utility gene card for: Leri-Weill dyschondrosteosis (LWD) and Langer mesomelic
dysplasia (LMD). Eur J Hum Genet. 20.
Barroso E et al. (2010). Identification of the first de novo PAR1 deletion downstream of SHOX in an individual
diagnosed with Leri-Weill dyschondrosteosis (LWD). Eur J Med Genet. 53:204-207.
Fukami M et al. (2008). Identification and characterization of cryptic SHOX intragenic deletions in three Japanese
patients with Leri-Weill dyschondrosteosis. J Hum Genet. 53:454-459.
SALSA MLPA Probemix P018 SHOX
Page 9 of 11
MRC-Holland
Product Description version G1-03; Issued 23 February 2017
®
MLPA
Funari MF et al. (2008). Cryptic intragenic deletion of the SHOX gene in a family with Leri-Weill dyschondrosteosis
detected by Multiplex Ligation-Dependent Probe Amplification (MLPA). Arq Bras Endocrinol Metabol. 52:1382-1387.
Funari MF et al. (2010). Usefulness of MLPA in the detection of SHOX deletions. Eur J Med Genet. 53:234-238.
Gatta V et al. (2007). Identification and characterization of different SHOX gene deletions in patients with Leri-Weill
dyschondrosteosys by MLPA assay. J Hum Genet. 52:21-27.
Gatta V et al. (2014). Spectrum of phenotypic anomalies in four families with deletion of the SHOX enhancer region.
BMC Med Genet. 15:87.
Gervasini C et al. (2010). SHOX duplications found in some cases with type I Mayer-Rokitansky-Kuster-Hauser
syndrome. Genet Med. 12:634-640.
Hirschfeldova K et al. (2012). SHOX gene defects and selected dysmorphic signs in patients of idiopathic short
stature and Leri-Weill dyschondrosteosis. Gene. 491:123-127.
Iughetti L et al. (2010). Unexpected phenotype in a boy with trisomy of the SHOX gene. J Pediatr Endocrinol Metab.
23:159-169.
Kant SG et al. (2013). Phenotypic characterization of patients with deletions in the 3'-flanking SHOX region. PeerJ.
1:e35.
Rodriguez FA et al. (2013). Clinical and molecular characterization of Chilean patients with Leri-Weill
dyschondrosteosis. J Pediatr Endocrinol Metab. 26:729-734.
Sandoval GT et al. (2014). SHOX gene and conserved noncoding element deletions/duplications in Colombian
patients with idiopathic short stature. Molecular genetics & genomic medicine. 2:95-102.
Stuppia L et al. (2010). Different approaches in the molecular analysis of the SHOX gene dysfunctions. J Endocrinol
Invest. 33:30-33.
van Steensel MA et al. (2008). Contiguous gene syndrome due to a maternally inherited 8.41 Mb distal deletion of
chromosome band Xp22.3 in a boy with short stature, ichthyosis, epilepsy, mental retardation, cerebral cortical
heterotopias and Dandy-Walker malformation. Am J Med Genet A. 146A:2944-2949.
Wolters B et al. (2013). Short stature before puberty: which children should be screened for SHOX deficiency? Horm
Res Paediatr. 80:273-280.
P018 Product history
Version
Modification
G1
One SHOX area probe has been replaced (CNE4) and four new probes have been included for the
upstream SHOX enhancer sequences CNE-2, CNE-3 and CNE-5 and the downstream CNE7
enhancer sequence.
Three new probes near the PAR1 boundary have been included. One probe (GPR143) has been
removed. The 88 and 96 nt control fragments have been replaced (QDX2).
Six probes located on chromosome X and six reference probes have been replaced.
One target and six reference probes have been replaced.
Several reference probes replaced. Variable probes in the SHOX downstream region removed.
Extra control fragments added.
Many probes outside the SHOX gene have been added.
First release.
F1
E1
D1
C1
B1
A1
Implemented changes in the product description
Version G1-03 – 23 February 2017 (03)
- KAL1 gene name changed to ANOS1 in Table 1 & 2.
- Added remark below Table 1 & 2 for the 337 nt SHOX exon 5 probe.
Version G1-02 – 25 August 2016 (03)
- All reference samples should be of the same sex added to the Reference samples section.
- Ligation sites and remarks updated in Table 2.
- Product description adapted to a new template.
Version G1-01 – 07 July 2015 (02)
- Product description restructured and adapted to a new template.
Version 20 – 03 December 2013 (01)
-
Complete product description rewritten. Names of certain regulatory regions changed. P.3 rewritten;
corrected error (Most defects in SHOX are point mutations into deletions); edited Publication list.
Table 2: corrections in the names of regulatory regions detected by certain probes, denoted SHOX area
extended. Table Interpretation of results and Table 3 – Change in relative probe signals as compared to
normal male or female reference DNA samples added. Thanks to Karen Heath for the corrections.
Version 19 - 03 December 2013 (01)
- Product description completely rewritten.
SALSA MLPA Probemix P018 SHOX
Page 10 of 11
MRC-Holland
Product Description version G1-03; Issued 23 February 2017
®
MLPA
- Product description adapted to a new product version (version number changed, lot number added,
changes in Table 1 and Table 2, new picture included).
More information: www.mlpa.com; www.mlpa.eu
MRC-Holland bv; Willem Schoutenstraat 1
1057 DL, Amsterdam, The Netherlands
E-mail
Phone
[email protected] (information & technical questions); [email protected] (orders)
+31 888 657 200
SALSA MLPA Probemix P018 SHOX
Page 11 of 11