Product Description SALSA MLPA probemix P073-A1 IKBKG To be used with the MLPA General Protocol.

Transcription

1 Version A1, lot A Product Description SALSA probemix P073-A1 IKBKG To be used with the MLPA General Protocol. Catalogue numbers: P R: SALSA probemix P073 IKBKG, 25 reactions. P R: SALSA probemix P073 IKBKG, 50 reactions. P R: SALSA probemix P073 IKBKG, 100 reactions. To be used in combination with SALSA reagent kit EK1 (100 reactions) or EK5 (500 reactions). MLPA reagent kits are available with different fluorescent dyes: EK1-FAM, EK5-FAM (Applied Biosystems sequencers) and EK1-Cy5.0, EK5-Cy5.0 (Beckman sequencers). Precautions and warnings: For professional use only. Always consult the most recent product description AND the MLPA General Protocol before use: 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: This SALSA P073 IKBKG probemix is a research-use-only (RUO) probemix for the detection of copy number status in the chromosome Xq28 region, known to be a cause for Incontinentia Pigmenti and is not intended to be used as a standalone assay for clinical decisions. Due to the high sequence homology, this assay cannot discriminate copy number status between IKBKG and its pseudogene, and the presence of e.g. the common IKBKGexon4_10del mutation always requires confirmation. The assay should be used on blood derived human DNA, free from impurities. Clinical background: Incontinentia pigmenti (IP), or Bloch-Sulzberger Syndrome, is an X-linked dominant disorder that is primarily characterized by skin lesions. Initial clinical manifestations start at birth with a vesiculobullous eruption (Stage I), followed by a verrucous stage (Stage II), a hyperpigmentation stage (Stage III), and finally a hypopigmentation stage (Stage IV) usually continuing throughout life. Other characteristics include ocular abnormalities, central nervous system abnormalities, and teeth defects. IP is easy to diagnose when the classical features are present after birth. However, the diagnosis can be difficult in patients with partial or non-classical clinical features. The IKBKG gene is the only known gene responsible for IP. The absence of a functional IKBKG gene is lethal. Therefore, as an X-linked disorder, IP occurs primarily in females. However, male patients with 47,XXY karyotype or somatic mosaicism have been diagnosed with IKBKG pathogenic variants. The majority of patients (>65%) harbour a 11.7 kb deletion, which is generated by a recombination event between two repeats (MER67B) located in intron 3 and about 6 kb downstream of IKBKG exon 10. This results in the removal of IKBKG exon 4 to 10 (IKBKGexon4_10del). Additionally, other large deletions of different sizes (including parts of neighbouring genes) and single nucleotide substitutions and insertions have been reported (Conte et al., 2014). Notably, over 60% of IP patients are sporadic carrying a de novo IKBKG mutation. Duplication of (part of) the IKBKG gene or deletion of the pseudogene, IKBKGP1, are common polymorphisms throughout the population with a frequency of 1-2% and do not result in any clinical phenotype. Additionally, about 10% of IP patient s parents carry either of these common polymorphisms. These polymorphisms arise due to the structure of this gene region (see Gene Structure) causing novel rearrangements ultimately leading to potential pathogenic or benign variants. Therefore, these polymorphisms are important risk factors for IP in offspring (Fusco et al., 2009). See for more information on IP. Apart from IP, the IKBKG gene is also involved in X-linked hypohidrotic ectodermal dysplasia and immunodeficiency (HED-ID), which is also known as ectodermal dysplasia, anhidrotic, with immunodeficiency (EDA-ID, and HED-ID with osteopetrosis and lymphedema (OL-HED-ID, SALSA Probemix P073 IKBKG Page 1 of 9

2 Gene structure: The IKBKG gene (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma; previously named NEMO, Nuclear factor kappab essential modulator) spans about 17.2 kb of genomic DNA and is located on chromosome Xq28, close to the q-telomere (about Mb from the p- telomere). It contains nine coding exons (exons 2 10) and several alternative noncoding first exons, some of which are located in the neighbouring G6PD gene (see Figure 1). IKBKG is part of a ~36 kb segmental duplication region or low copy repeat (LCR) (Figure 1). LCR1 contains IKBKG and LCR2 contains IKBKG s non-functional truncated pseudogene, IKBKGP1 or pseudonemo, arranged in an opposite orientation. The two LCRs, separated by a 21.7 kb unique sequence, are derived from a duplication event resulting in >99.5% of identity. Figure 1. Schematic representation of the IKBKG locus. Two nearly identical inverted repeat regions are located on chromosome Xq28: LCR1 and LCR2 (horizontal arrows). LCR1 contains, among others, the functional IKBKG gene, while the non-functional pseudogene IKBKGP1 is situated in LCR2. Both regions are separated by a unique ~22 kb sequence. Vertical bars represent exons. The location of target probes in the P073 IKBKG probemix are indicated by vertical arrows, including their length (see also Tables 1 & 2). Probes indicated with an asterisk (*) detect both sequences in LCR1 and LCR2; 4 copies in most females, 2 copies in most males. Figure is not to scale. Exon numbering: The exon numbering used in this P073 IKBKG probemix product description and in the P073 IKBKG Coffalyser.Net analysis sheet is the exon numbering that is also used in IKBKG LRG_70 and G6PD LRG_148 ( These are identical to Genbank NG_ and NG_ , respectively. P073-A1 probemix content: This SALSA MLPA probemix P073 IKBKG contains 38 MLPA probes with amplification products between 136 and 436 nt (Tables 1 & 2), including 13 probes for the IKBKG gene. Ten of these probes also detect sequences in IKBKGP1; hence these probes detect 4 copies in most healthy female individuals. The probemix further contains 7 probes upstream of the IKBKG coding sequence, including 6 probes for G6PD. Also, 9 probes downstream of the IKBKG gene are included, of which 4 are located in the duplicated region and therefore detect 4 copies in most healthy female individuals, and 2 are telomeric of IKBKGP1. Finally, 9 reference probes are included, detecting several locations on the X- chromosome. The identity of the genes detected by the reference probes is available online ( This probemix contains nine quality control fragments generating amplification products between 64 and 105 nt: four DNA Quantity Fragments (Q-fragments), three DNA Denaturation Fragments (D-fragments), and one chromosome X and one chromosome Y-specific fragment (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 (MS-)MLPA General Protocol. MLPA technique: The principles of the MLPA technique (Schouten et al. 2002) are described in the MLPA General Protocol ( 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. Note that duplication of the IKBKG gene and deletion of the pseudogene are relative frequent polymorphisms in the normal population (~2%)! SALSA Probemix P073 IKBKG Page 2 of 9

3 Required specimens: Purified DNA from blood, 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 IP. Reference samples should NOT contain the frequent IKBKG duplication or IKBKGP1 deletion! It is recommended to use reference and 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: cannot provide positive DNA samples. Inclusion of a positive sample in each experiment is recommended. Performance characteristics: In over 65% of IP patients a deletion of IKBKG exon 4-10 is the genetic cause of the disease, which can be identified by this P073 probemix. It is important to emphasize that the probes detecting IKBKG exon 3 to 10 and further downstream sequences cannot discriminate IKBKG and its pseudogene IKBKGP1 (see Figure 1 and Table 2). A reduced signal of these probes might therefore as well reflect a deletion of the pseudogene, and conclusions should not be drawn in the absence of a clinical diagnosis, and conformation with another technique is required. Another known cause of IP are large deletions other than the IKBKGexon4_10 deletion. These deletions vary in size and may extend to neighbouring genes, like G6PD (Fusco et al., 2012). This P073 probemix is able to detect these deletions. In the remaining IP cases, base substitutions and small indels are responsible for the disease, the majority of which will not be detected by this probemix. 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 Use of other non-proprietary software may lead to inconclusive or false results. The analysis method used for P073-A1 is block normalisation. Normalisation of results should be performed within one experiment. For more details on MLPA quality control and data analysis, see the Coffalyser.Net Manual. Interpretation of 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 The majority of IP patients carry the common IKBKGexon4_10 mutation. Probes located in this region will therefore detect 4 copies in reference samples, and a ~25% signal reduction of these probes will be observed in patients (see Figure 2). Note that an exon4_10 deletion in the pseudogene, IKBKGP1, will result in a similar pattern! Individuals carrying IKBKGP1 deletions are, however, without any clinical phenotype. For probes detecting only a single location (outside the LCR regions, see Figure 1 and Table 2), the following cut-off values for the DQ of the probes can be used to interpret MLPA results on female samples: Copy Number status (female) Dosage quotient Normal 0.80 < DQ < 1.20 Homozygous deletion DQ = 0 Heterozygous deletion 0.40 < DQ < 0.65 Heterozygous duplication 1.30 < DQ < 1.65 Heterozygous triplication/ Homozygous duplication 1.75 < DQ < 2.15 Ambiguous copy number All other values SALSA Probemix P073 IKBKG Page 3 of 9

4 Figure 2. Comparative analysis using Coffalyser.Net software of P073-A1 IKBKG probemix on DNA from 3 female reference samples (left) and 2 female IP patients (right). Since the MLPA probes for the most part of the IKBKG gene detect 4 copies in the reference samples, a ~25% reduction is observed in the IP patients carrying the common IKBKGexon4_10 deletion (shaded box). Note that a similar pattern will be obtained when the common IKBKGP1 deletion is present. IKBKGP1 deletions do not result in any abnormal phenotype and conclusions should not be drawn in the absence of clinical data! 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 (e.g. due to salt contamination) can lead to a decreased probe signal, in particular for probes located within a CpG island in or near the IKBKG 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. Normal copy number variation in healthy individuals is described in the database of genomic variants: Users should always consult the latest update 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 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. SALSA Probemix P073 IKBKG Page 4 of 9

5 Limitations of the procedure: - Due to the homology between the two repeat regions, this assay cannot discriminate IKBKG from its pseudogene (see Figure 1, Table 2). - In most populations, a significant portion of IP cases (20-25%) is caused by small (point) mutations and insertions, most of which will not be detected using SALSA probemix P073 IKBKG. - Low level mosaic cases are likely not detectable using this probemix. - MLPA cannot detect any changes that lie outside the target sequence of the probes and will not detect copy number neutral 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 (even when >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: Confirmation of the IKBKGexon4_10 deletion can be done by long range PCR, as described in Bardaro et al. (2004) and Steffann et al. (2004). 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. IKBKG mutation database: We strongly encourage users to deposit positive results in an available IKBKG database. Recommendations for the nomenclature to describe deletions/duplications of one or more exons can be found on Please report copy number changes detected by the reference probes, false positive results due to SNPs and unusual results (e.g., a duplication of IKBKG exons 6 and 8, but not exon 7) to : info@mlpa.com. SALSA Probemix P073 IKBKG Page 5 of 9

6 Table 1. SALSA probemix P073-A1 IKBKG probemix Length Chromosomal position (a) SALSA MLPA probe IKBKG (nt) reference IKBKG IKBKGP1 upstream 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 100 X-fragment: Specific for the X chromosome 105 Y-fragment: Specific for the Y chromosome IKBKG downstream 136 Reference probe L14743 Xp ± IKBKG probe L26986 Exon CTAG1A probe L28165 CTAG1A down 154 G6PD probe L26975 G6PD exon Reference probe L20191 Xp IKBKG probe L27679 Exon 3 Exon IKBKG probe L27608 Exon 8 Exon Reference probe L22348 Xp CTAG1A probe L28742 CTAG1A up 190 ± IKBKG probe L28766 Exon G6PD probe L26988 G6PD exon IKBKG probe L28765 Exon 4 Exon FAM223B probe L28743 FAM223B down 220 IKBKG probe L27680 Exon Reference probe L17416 Xq CTAG1A probe L28744 CTAG1A/B exon IKBKG probe L27604 Exon 9 Exon CTAG1A probe L26978 CTAG1A down 265 IKBKG probe L28745 Exon 10 Exon ± G6PD probe L27692 G6PD exon FAM3A probe L26980 FAM3A exon Reference probe L07377 Xp ± G6PD probe L27996 G6PD exon FAM223B probe L28746 FAM223B up 319 IKBKG probe L14030 Exon 10 Exon ± G6PD probe L26981 G6PD exon CTAG2 probe L26982 CTAG2 up 346 Reference probe L26139 Xq IKBKG probe L27606 Exon 7 Exon Reference probe L03190 Xq IKBKG probe L27607 Exon 10 Exon Ж CTAG1A probe SP0906-L26983 CTAG1A down 391 IKBKG probe L14035 Exon 6 Exon G6PD probe L26984 G6PD exon Reference probe L21770 Xq CTAG2 probe L26985 CTAG2 down 426 Ж IKBKG probe SP0108-L28339 Exon 5 Exon Reference probe L20735 Xq13 Probes marked in GREY detect sequences in both LCR1 and LCR2, see also Figure 1. Hence, each probe detects 4 copies in most healthy females and 2 copies in most healthy males! (a) The exon numbering used in this P073-A1 IKBKG product description and in the P073-A1 IKBKG Coffalyser.Net analysis sheet is the exon numbering from the Genbank NG_ reference sequence for IKBKG and the NG_ reference sequence for G6PD. ± 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. Ж This probe consists of three parts and has two ligation sites. SALSA Probemix P073 IKBKG Page 6 of 9

7 Table 2. P073 IKBKG 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) MV Location (GRCh38/hg38) SALSA Probemix P073 IKBKG Page 7 of 9 Distance to next probe L26980 FAM3A Exon 1 NM_ ; AGATCGTGAGCC-AACGGGTTCCTG kb G6PD stop codon (exon 14); NM_ L26975 G6PD Exon TGACATTCCTTG-TCACCAGCAACA kb L26984 G6PD Exon ATGCATCTCAGA-GGTGCAGGCCAA kb L26988 G6PD Exon GCAAGGCCAGGT-AGAAGAGGCGGT kb 301 ± L27996 G6PD Exon 3 10 nt after exon 3 TGGTAAGTGTGT-CCCACCACTGCC kb IKBKG start codon (exon 2); NM_ ± L26986 IKBKG Exon GAGGGGAAGAGA-CTGGGATTGATA kb 328 ± L26981 G6PD Exon CGGGAAGAGCTT-TTCCAGGGCGAT kb 274 ± L27692 G6PD Exon GTCCGCCAGGCT-CTGCAGGCCCGC kb G6PD start codon (exon 1); NM_ ± L28766 IKBKG Exon CCGGCAGCAGAT-CAGGACGTACTG kb L27680 IKBKG Exon nt before exon 3 AGGGAGGACCAA-TACCGAGCATCT kb L27679 IKBKG Exon CTGAAGAGGCAG-AAGGAGCAGGCT kb L28765 IKBKG Exon reverse GGCATTCCTTAG-TGGCAGCCTCCA kb 426 Ж SP0108- L28339 IKBKG Exon nt and 132 nt after exon 5 GCCTTCTGAGGA-24 nt spanning oligo-gtgggcccattc kb L14035 IKBKG Exon 6 18 nt before exon 6 GTTTCTCCAGCA-AAAGCTCCCTTT kb L27606 IKBKG Exon reverse TCCTTCAGCTTA-TCGATCACCTCC kb L27608 IKBKG Exon CAGCAAACTGAA-GGCCAGCTGTCA kb L27604 IKBKG Exon AAGGATCGAGGA-CATGAGGAAGCG kb L27607 IKBKG Exon CTTCTGCTGTCC-CAAGTGCCAGTA kb L14030 IKBKG Exon CTGTCTGCTCGA-ACCACTTGCCTC kb L28745 IKBKG Exon nt after exon 10 TAGCCTTGCCAA-GGAGGAGGTGGG kb IKBKG stop codon (exon 10); NM_ L28746 FAM223B Up 1.2 kb before FAM223B AAACCAAAACGG-ATTCCTCTTCCT kb L28743 FAM223B Down 3.3 kb after FAM223B GCACCAAGACTA-TGGACTGAAGTG kb L28742 CTAG1A Up 5.6 kb before CTAG1A GGACTGTGTGGT-GCTACCTGAGTG kb L28744 CTAG1A 105 nt after exon 2 AGGCTGCTAGAA-ACCTGGGAACAC kb 382 Ж SP0906- L26983 CTAG1A Down 5.2 kb and 5.2 kb after CTAG1A AAAGGACAGGCA-30 nt spanning oligo-attgaggagttt kb L28165 CTAG1A Down 15.8 kb after CTAG1A TTCAGGTAAGTA-AAACAACCAAGT kb L26978 CTAG1A Down 21 kb after CTAG1A CCTGAACCTAAA-TAGCATCACAAT kb L28744 CTAG1B 105 nt after exon 2 AGGCTGCTAGAA-ACCTGGGAACAC kb L28742 CTAG1B Up 5.6 kb before CTAG1B GGACTGTGTGGT-GCTACCTGAGTG kb L28743 FAM223A Down 3.3 kb after FAM223A GCACCAAGACTA-TGGACTGAAGTG kb L28746 FAM223A Up 1.2 kb before FAM223A AAACCAAAACGG-ATTCCTCTTCCT kb IKBKGP1; NG_ L28745 Exon nt after NG_ TAGCCTTGCCAA-GGAGGAGGTGGG kb L14030 Exon CTGTCTGCTCGA-ACCACTTGCCTC kb L27607 Exon CTTCTGCTGTCC-CAAGTGCCAGTA kb L27604 Exon AAGGATCGAGGA-CATGAGGAAGCG kb L27608 Exon CAGCAAACTGAA-GGCCAGCTGTCA kb L27606 Exon TCCTTCAGCTTA-TCGATCACCTCC kb L14035 Exon GTTTCTCCAGCA-AAAGCTCCCTTT kb 426 Ж SP and GCCTTCTGAGGA-24 nt spanning Exon 5 L oligo-gtgggcccattc kb L28765 Exon reverse GGCATTCCTTAG-TGGCAGCCTCCA kb L27679 Exon CTGAAGAGGCAG-AAGGAGCAGGCT kb IKBKGP1; NG_ L26985 CTAG2 Up 2.7 kb after exon 3 GGGCAGAAGGAG-AACCTGCTCTCT kb L26982 CTAG2 Down 2.6 kb before exon 1 GAGATGTGAGAA-GATGTCTACGAA NOTE: Probes marked in GREY detect sequences in both LCR1 and LCR2, see also Figure 1. Hence, each probe detects 4 copies in most healthy females and 2 copies in most healthy males! Probes marked in ITALIC detect sequences that are ~25% reduced in peak height when the IKBKGexon4_10 deletion is present.

8 ± 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. Ж This probe consists of three parts and has two ligation sites. (a) The exon numbering used in this P073-A1 IKBKG product description and in the P073-A1 IKBKG Coffalyser.Net analysis sheet is the exon numbering from the Genbank NG_ reference sequence for IKBKG and the NG_ reference sequence for G6PD. (b) Ligation sites of the P073 IKBKG MLPA probes are indicated according to reference standard in the RefSeqGene project: NM_ for IKBKG, NM_ for G6PD. For IKBKGP1, NG_ reference sequence is used. (c) Only partial probe sequences are shown. Complete probe sequences are available at Please notify us of any mistakes: info@mlpa.com. P073 Product history Version Sold from Modification A1 January 2016 New product Implemented changes in the product description Version January 2016 (02) - Not applicable, new document. More information: bv; Willem Schoutenstraat DL, Amsterdam, The Netherlands info@mlpa.com (information & technical questions); order@mlpa.com (orders) Phone References Bardaro et al., Two cases of misinterpretation of molecular results in incontinentia pigmenti, and a PCR-based method to discriminate NEMO/IKKgamma gene deletion. Hum Mutat. 21: Conte et al., Insight into IKBKG/NEMO Locus: Report of New Mutations and Complex Genomic Rearrangements Leading to Incontinentia Pigmenti Disease. Hum Mutat. 35: Fusco et al., Microdeletion/duplication at the Xq28 IP locus causes a de novo IKBKG/NEMO/IKKgamma exon4_10 deletion in families with Incontinentia Pigmenti. Hum Mut. 30: Fusco et al., Genomic architecture at the Incontinentia Pigmenti locus favours de novo pathological alleles through different mechanisms. Hum Mol Genet. 21: Schouten et al., Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res. 30:e57. Schwartz et al., Deletion of exon 16 of the dystrophin gene is not associated with disease. Hum Mutat. 28:205. Steffann et al., A novel PCR approach for prenatal detection of the common NEMO rearrangement in incontinentia pigmenti. Prenat Diagn. 24: Varga et al., MLPA-based evidence for sequence gain: pitfalls in confirmation and necessity for exclusion of false positives. Anal Biochem. 421: SALSA Probemix P073 IKBKG Page 8 of 9

9 SALSA MLPA probemix P073-A1 IKBKG sample pictures Figure 3. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P073-A1 IKBKG (lot A1-1015). Figure 4. Capillary electrophoresis pattern of a sample of approximately 50 ng human female control DNA analysed with SALSA MLPA probemix P073-A1 IKBKG (lot A1-1015). SALSA Probemix P073 IKBKG Page 9 of 9