SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions Lot A

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1 SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions Lot A This SALSA MLPA probemix is for basic research and intended for experienced MLPA users only! This probemix enables you to quantify genes or chromosomal regions in which the occurrence of copy number changes is not yet wellestablished and the relationship between genotype and phenotype is not yet clear. Since it will not provide you with clear cut answers, interpretation of results can be complicated. recommends thoroughly screening any available literature. SALSA probemixes and reagents are sold by 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 test probemixes and reagents includes a limited license to use these products for research purposes. Microdeletions of the Y chromosome are the second most frequent genetic cause of spermatogenetic failure in infertile men, after Klinefelter syndrome. These microdeletions are caused by intrachromosomal recombination events between large homologous repetitive sequence blocks and are clustered in three specific regions on the long arm of the Y chromosome, known as azospermia factors (AZF) loci: AZFa (~ Mb from the p-telomere), AZFb (~ Mb) and AZFc (~ Mb). The AZFc region contains many gene families required for normal spermatogenesis and is critical for male fertility. This region is most frequently involved in Yq deletions (up to 79%). AZFc is arranged in repeated sequence blocks called amplicons organised into palindromic structure showing nearly identical sequences. The most important genes within the AZFc region are DAZ, BPY2 and CDY. The first and best characterised AZFc deletion spans the whole AZFc region (about 3.5 Mb). Partial (interstitial) AZFc deletions are found in higher frequency in individuals with severe spermatogenic failure. In a recent large study (Rozen et al, 2012; PMID ) the frequency of four recurrent partial AZFc deletions was studied in 5 populations. Please note however that certain partial AZFc deletions, such as the 1.6 Mb gr/gr deletion, are also present in high frequency in fertile man. This P360-A1 probemix contains 13 probes detecting sequences within the AZFc region. Five of these probes detect a sequence that is present twice in the AZFc region. Two probes detect three targets within AZFc and one probe detects two targets in AZFc and one target in AZFb. Deletions in the AZFb region account for about 9% of the Yq microdeletions, further 6% is involved in AZFb + AZFc deletions. The RBMY1J, DY2, KDM5D, EIF1AY and MY1J genes have been mapped within AZFb. This P360-A1 probemix includes 16 probes detecting AZFb, including three probes that detect two targets within AZFb and one probe which detects a sequence that is present once in AZFb and twice in AZFc. Microdeletions in the AZFa region account for about 3% of Yq microdeletions. The USP9Y, DDX3Y, UTY and BPY2 genes have been mapped in AZFa. These genes have homologous copies on the X chromosome. This P360-A1 probemix contains 14 probes detecting AZFa sequences. More information on Y chromosome microdeletions can be found at Furthermore, two probes are present for the autosomal DPY19L2 gene, which was shown to be homozygously deleted in patients with globozoospermia. (Ray et al., 2011; Am J Hum Genet. 88:351-61). The DPY19L2 gene (22 exons) spans ~109.7 kb of genomic DNA and is located on 12q14, 64 Mb from the p-telomere. Finally, 8 reference probes are included in this probemix, detecting several different autosomal chromosomal locations. Please note that we have no information whether or not the deletion of individual probe sequences, or even the complete deletion of certain genes, has an effect on male fertility. This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the above mentioned Y chromosomal regions in a DNA sample. For probes that detect a sequence which is present in a single copy on the Y-chromosome, deletion will lead to a complete absence of the corresponding probe amplification product. 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 SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions Page 1 of 12

2 Description version 06; 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. More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands The use of a SALSA 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). Data analysis The P360-A1 Y-Chromosome Microdeletions probemix contains 53 MLPA probes with amplification products between 136 and 500 nt. In addition, it contains 9 control fragments generating an amplification product smaller than 120 nt: four DNA Quantity fragments (Q-fragments) at nt, three DNA denaturation control fragments (D-fragments) at 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. Male reference samples should be used. 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 or FISH. Note that Coffalyser, the MLPA analysis tool developed at, can be downloaded free of charge from our website Many copy number alterations in healthy individuals are described in the database of genomic variants: 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. Recommendations for the analysis of results of the P360-A1 Y-Chromosome Microdeletions probemix: Due to high homology between large sequence blocks within AZFb and AZFc some probes included in this probemix detect 2 or even 3 target sites (see Table 3). In case the probe detects two target sites, a deletion of one single target site is expected to result in a ~50% decrease in signal. In case the probe detects three target sites, a deletion of one single target site is expected to result in ~33% decrease in signal. For data interpretation, we strongly recommend to create a sheet compared to Table 4 (see appendix) wherein all positions detected by each probe are included. The value of the normalised results is copied to every location. This probemix was developed by N. Laddach at. 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: info@mlpa.com. SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions Page 2 of 12

3 Table 1. SALSA MLPA P360-A1 Y-Chromosome Microdeletions probemix Length (nt) SALSA MLPA probe Chromosomal position reference target-specific 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 Number of sequences detected (according to UCSC Genome Browser) 136 EIF1AY probe L12517 Yq PPP1R12BP1 probe L14796 Yq RBMY2CP probe L12599 Yq / Yq Reference probe L p CDY2B probe L17486 Yq BPY2 probe L13811 Yq BPY2 probe L14251 Yq VCY1B probe L17485 Yq Reference probe L p Reference probe L q KDM5D probe L13987 Yq KDM5D probe L12530 Yq UTY probe L13342 Yq CDY2A probe L18625 Yq RPS24P1 probe L18627 Yq * CDY2A probe L18626 Yq11.221/Yq11.223/Yq KDM5D probe L12537 Yq ARSEP probe L17483 Yq KDM5D probe L13817 Yq RBMY1J probe L13818 Yq DAZ2 probe L12541 Yq / Yq Reference probe L q SRY probe L15420 Yp DAZ2 probe L12543 Yq / Yq CDY2B probe L14252 Yq / Yq Reference probe L q DAZ2 probe L12544 Yq / Yq DPY19L2 probe L q UTY probe L20831 Yq RPS24P1 probe L20832 Yq USP9Y probe L12621 Yq Reference probe L q VCY1B probe L15069 Yq UTY probe L19232 Yq VCY1B probe L18629 Yq CDY1B probe L12554 Yq / Yq USP9Y probe L12618 Yq * BPY2 probe L14716 Yq (2x)/ Yq11.23 (1x) DPY19L2 probe L q RBMY2DP probe L12617 Yq HSFY1 probe L18630 Yq (2x) VCY1B probe L18631 Yq HSFY1 probe L12555 Yq BPY2 probe L12556 Yq RBMY1J probe L12557 Yq HSFY1 probe L18632 Yq (2x) 2 SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions Page 3 of 12

4 Length (nt) SALSA MLPA probe Chromosomal position reference target-specific Number of sequences detected (according to UCSC Genome Browser) 445 KDM5D probe L12559 Yq USP9Y probe L12631 Yq NLGN4Y probe L12650 Yq Reference probe L p * BPY2 probe L19234 Yq11.221/Yq11.223/ Yq EIF1AY probe L12560 Yq Reference probe L q12 1 Detects 2 sequences (UCSC Genome Browser). * Detects 3 sequences (UCSC Genome Browser). The signal of this probe is relatively low. Table 2. Reference sequences and gene synonyms Gene name according to HUGO location NCBI reference sequence Alias gene names ARSEP Yq11.2 NG_ VCY1B Yq11.2 NM_ BPY1, VCY1, VCY, VCYB BPY2 Yq11.2 *NM_ BPY2A, VCY2A, VCY2 CDY1B Yq11.2 NM_ CDY2A Yq11.2 NM_ CDY2 CDY2B Yq11.2 NM_ CDY DAZ2 Yq11.2 *NM_ pdp1678, MGC EIF1AY Yq11.2 NM_ HSFY1 Yq11.2 *NM_ HSFY, HSF2L KDM5D Yq11.2 NM_ JARID1D NLGN4Y Yq11.2 *NM_ KIAA0951 PPP1R12BP1 Yq11.2 NG_ RPS24P1 Yq11.2 NC_ RBMY1J Yq11.2 NM_ RBMY2CP Yq11.2 NC_ RBMY2 RBMY2DP Yq11.2 NC_ RBM SRY Yp11.3 *NM_ TDF USP9Y Yq11.2 *NM_ DFFRY UTY Yq11.2 NM_ DPY19L2 12q14.2 NM_ *These sequences are reference standards in the NCBI RefSeqGene project. Note: Please notify us of any mistakes. The identity of the genes detected by the reference probes and the complete probe sequences are available on request: info@mlpa.com. SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions Page 4 of 12

5 Table 3. P360 probes arranged according to chromosomal location Table 3a. Y chromosome Length (nt) SALSA MLPA probe HG19 AZF probe start locus Partial sequence (24 nt adjacent to ligation site) Distance to next probe 276 SRY probe L GCACTGAAAGCT-GTAACTCTAAGT kb 319 RPS24P1 probe L AZFa TTACAGAAGGTA-TGTCCTTGCACT 28.2 kb 228 RPS24P1 probe L AZFa TCCCTAGTGCTA-CTGCCTCACTTA kb 247 ARSEP probe L AZFa ACCTTCCCAGCA-AGCCGCCTTGAA kb 366 USP9Y probe L AZFa ATCATGTGGCAT-TACCTCATTTGC kb sy85 STS marker USP9Y probe L AZFa GAAAGGCAAGGA-CTTTACCTTAAA kb 454 USP9Y probe L AZFa CTGTTCCTGATA-GAAGGAATCATG kb G34990 STS marker UTY probe L AZFa TGCATTATTGCA-GTACTTTCTTCA 54.7 kb UTY probe L AZFa ATTGGTCCAGGA-GATTGTGAATGG kb UTY probe L AZFa AGGATCCTGGAT-ATTCCACTACCA kb 342 VCY1B probe L AZFa TCCCTTTCTACA-CTTAGATCTCTG kb 184 VCY1B probe L AZFa GCATATTGAGTA-GATCATTCCTAG kb 402 VCY1B probe L AZFa CACGTTCGCTCA-GTTCTCACTGAT kb 355 VCY1B probe L AZFa GGAGTAGACCAA-GAGAGGAATATA kb 463 NLGN4Y probe L AZFa TTCTGCGTGGCA-TCACAGTCTTCC kb 160 CDY2B probe L AZFb TTCCAGCCAGGA-CACATCTGGAAA 5.1 kb 288 CDY2B probe L AZFb CTTCTGGCTGAA-CTGCGGCACCAA kb 223 CDY2A probe L AZFb GGCTGTTAATGA-ATTCGTTAATGC 0.1 kb 234 * CDY2A probe L AZFb ATTTTCTGTTAA-CCTTAGTGTAAA kb 288 CDY2B probe L AZFb CTTCTGGCTGAA-CTGCGGCACCAA 97.1 kb 396 HSFY1 probe L AZFb ATTTGATGATGA-AGATTTAGCAGA 84.4 kb 436 HSFY1 probe L AZFb AAAGAACACATA-CCAATATAGCTG 21.1 kb 409 HSFY1 probe L AZFb CTGGACTATGGA-TGCAACTTCCGA 44.0 kb 436 HSFY1 probe L AZFb AAAGAACACATA-CCAATATAGCTG 84.4 kb 396 HSFY1 probe L AZFb ATTTGATGATGA-AGATTTAGCAGA kb 241 KDM5D probe L AZFb CCAACAAAGTCT-TACAATTATACT 16.1 kb 202 KDM5D probe L AZFb CAATTTTGTCCA-TACTTTTGGTTA kb 445 KDM5D probe L AZFb CTGATTGGAGCA-CTCAGCCTAAAC 77.9 kb 208 KDM5D probe L AZFb GACCAGGTTCAT-GCCAATATATTT 19.6 kb 252 KDM5D probe L AZFb GATCTGAAGTTA-CTGATGAATCTG kb sy127 STS marker EIF1AY probe L AZFb ACTTTCTAAATG-TTCTTGAATGTA 24.1 kb 136 EIF1AY probe L AZFb CCTGATTCTCCA-ATGGCTTCATAG kb sy133 STS marker sy142 STS marker sy1258 STS marker sy1161 STS marker ٨ RBMY1J probe L AZFb TGGCAAATCCAT-AATATTACAACA 6.4 kb 259 ٨ RBMY1J probe L AZFb TACAACCAGAGA-TAATGTAAATAG kb sy1197 STS marker sy1192 STS marker ٨ BPY2 probe L AZFc TTTACATGGTAA-ATTGATGTGCTT 0.5 kb 166 ٨ BPY2 probe L AZFc TAGGAGAAAATA-ACAAAATAATGA 1.7 kb 178 ٨ BPY2 probe L AZFc CACAGAAATATA-TACACTGTTTGA 42.6 kb sy1191 STS marker *@٨ BPY2 probe L AZFc TCTTTGTATTCA-TGCCAAGAAACG 49.2 kb 373 *@٨ BPY2 probe L AZFc TCATATGTCTGA-AGTCAGAACTTG kb sy254 STS marker copy sy254 STS marker copy sy1291 & sy1189 STS markers SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions Page 5 of 12

6 Length (nt) SALSA MLPA probe HG19 AZF probe start locus Partial sequence (24 nt adjacent to ligation site) Distance to next probe DAZ2 probe L AZFc GTTCAGCTGGCA-AGCTAGCTGTGC 48.3 kb RBMY2CP probe L AZFc TTGCAATGCCAA-TGCAATCCCACA 49.7 kb DAZ2 probe L AZFc AGTATATTCCCA-TTCCTAATAATG kb DAZ2 probe L AZFc CAGTGCTTCTGA-ATGATTTTCAGT kb CDY1B probe L AZFc CCTTACTGCTTA-AGGCCGTATTTC kb 234 *@ CDY2A probe L AZFc ATTTTCTGTTAA-CCTTAGTGTAAA kb sy1206 STS marker copy *@ BPY2 probe L AZFc TCTTTGTATTCA-TGCCAAGAAACG 49.2 kb 373 *@ BPY2 probe L AZFc TCATATGTCTGA-AGTCAGAACTTG kb sy254 STS marker copy sy254 STS marker copy * BPY2 probe L AZFc TCATATGTCTGA-AGTCAGAACTTG 49.2 kb 486 * BPY2 probe L AZFc TCTTTGTATTCA-TGCCAAGAAACG kb sy1206 STS marker copy * CDY2A probe L AZFc ATTTTCTGTTAA-CCTTAGTGTAAA kb 361 CDY1B probe L AZFc CCTTACTGCTTA-AGGCCGTATTTC kb 265 DAZ2 probe L AZFc CAGTGCTTCTGA-ATGATTTTCAGT kb 301 DAZ2 probe L AZFc AGTATATTCCCA-TTCCTAATAATG 49.7 kb 147 RBMY2CP probe L AZFc TTGCAATGCCAA-TGCAATCCCACA 48.3 kb 283 DAZ2 probe L AZFc GTTCAGCTGGCA-AGCTAGCTGTGC 41.1 kb sy1201 STS marker PPP1R12BP1 probe AZFc L AGCATTTGGAGA-TGCTCCAGAAGA 88.4 kb 389 RBMY2DP probe L AZFc CACTGAATGGAA-AAGTACAGCTGG Parts of the Y chromosome can have an inverted orientation! The sy255 STS marker detects 4 sequences, close to the sy254 locations. The locations of the gr/gr and b1/b3 deletions are based on Shahid et al (PMID ). These probes detect 2 sequences (UCSC Genome Browser). Deletion of a single target site is expected to result in an ~50% decrease in signal. * These probes detect 3 sequences (UCSC Genome Browser). Deletion of a single target site is expected to result in an ~33% decrease in signal. + The 105 nt Y probe detects a sequence located 52 kb after the 312 nt probe and 250 kb before the 215 nt probe. The signal of this probe is relatively These probes are expected to be deleted in DNA samples with the gr/gr deletion, resulting in a 50% decreased signal for the 147, 265, 283, 301 and 361 nt probes, as well as a 67% decreased signal for the 373 and 486 nt probes and a 33% decreased signal for the 234 nt probe. The gr/gr deletion is also frequently present in DNA samples of fertile man. ٨ These probes are expected to be deleted in DNA samples with the b1/b3 deletion, resulting in the signals for the 166, 178, 259, 418 and 427 probes being absent and the probes for the 373 and 486 nt probes being 33% reduced in signal. Table 3b. Partial AZFc deletions as described in Rozen et al (2012); PMID AZFc deletion gr/gr STS markers deleted sy1291, sy1189, sy254 (2 copies deleted) STS markers not deleted sy1191, sy254 (2 copies remaining) Deletion size (Mb) Frequency (fertile + non-fertile males) Effect on severe spermatogenic failure 1.6 Mb 1: fold increase in SSF risk b2/b3 sy1192, sy1191 sy142, sy Mb 1:90 Not a risk factor for SSF b1/b3 sy1192, sy1191, sy1291, sy1189, sy254 (2 copies deleted) sy142, sy254 (2 copies remaining) 1.6 Mb 1: fold increase in SSF risk b2/b4 sy1192, sy1191, sy1291, sy1189, sy254 (all copies deleted) sy142, sy Mb 1:2320 (Frequency of each deletion type is population dependent) 145 fold increase in SSF risk SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions Page 6 of 12

7 Table 3c. DPY19L2 Length (nt) SALSA MLPA probe DPY19L2 exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (ex 1) L20390 Intron 3 841nt before exon 4 AACACGAGTAAG-TACTAAATTACA 90.7 kb L20718 Intron nt before exon 20 AAGATGATCTGC-GGGAAGAAAACA stop codon (ex 22) The NM_ sequence is a reference standard in the NCBI RefSeqGene project. Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions Page 7 of 12

8 SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions sample pictures D ye Sign al Size (nt) Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA probemix P360-A1 Y-Chromosome Microdeletions (lot A1-1011). Implemented Changes compared to the previous product description version Version 06 (50) - Basic research box added on page 1. Version 05 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 04 (48) - Location of STS markers and the presumed locations of the gr/gr and b1/b3 deletions included in Table 3a. Textual change of page 1. Version 03 (48) - Change of probe position L12554 in Table 4. Version 02 (48) - Various minor textual and layout changes. - Remark on RefSeqGene standard added below Table 3b. - Small changes of probe lengths in Table 1 and 3 in order to better reflect the true lengths of the amplification products. Version 01 - Not applicable, new document. SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions Page 8 of 12

9 Appendix In this appendix the results of three positive test samples, analysed with a preliminary version of the SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions are described. Probe combination of this preliminary version differs slightly from the final probemix. On the following pages you will also find the accordant capillary electrophoresis patterns of human male control DNA and the three positive test samples mentioned above. Table 4. Normalised results of positive test samples AZF region detected target sites in case of more than 1 site (2A=2A, 3A=3A=3A) Probe ID Length Position on Y- Chromosome sample 1 sample 2 sample 3 values AZFb AZFc deletion values AZFc deletion values AZFc deletion SRY probe L AZFa RPS24P1 probe L AZFa RPS24P1 probe L AZFa ARSEP1 probe L AZFa USP9Y probe L AZFa USP9Y probe L AZFa USP9Y probe L AZFa UTY probe L AZFa UTY probe L AZFa UTY probe L AZFa BPY1 probe L AZFa VCY1B probe L AZFa VCY1B probe L AZFa VCY1B probe L AZFa NLGN4Y probe L AZFb CDY2B probe L AZFb 2A CDY2B probe L AZFb CDY2A probe L del AZFb 3A CDY2A probe L del AZFb 2A CDY2B probe L del AZFb 2B HSFY1 probe L del AZFb 2C HSFY1 probe L del AZFb HSFY1 probe L del AZFb 2C HSFY1 probe L del AZFb 2B HSFY1 probe L del AZFb KDM5D probe L del AZFb KDM5D probe L del AZFb KDM5D probe L del SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions Page 9 of 12

10 AZFb KDM5D probe L del AZFb KDM5D probe L del AZFb EIF1AY probe L del AZFb EIF1AY probe L del AZFb RBMY1J probe L del AZFb RBMY1J probe L del AZFc BPY2 probe L del 0 del 0.97 AZFc BPY2 probe L del 0 del 1.01 AZFc BPY2 probe L del 0 del 0.99 AZFc 3B BPY2 probe L del 0 del 0.68 AZFc 3C BPY2 probe L del 0 del 0.75 AZFc 2D DAZ2 probe L del 0.5 del 0.52 AZFc 2E RBMY2CP probe L del 0.55 del 0.58 AZFc 2F DAZ2 probe L del 0.45 del 0.47 AZFc 2G DAZ2 probe L del 0 del 0.54 AZFc 2H CDY1B probe L del 0 del 0.5 AZFc 3A CDY2A probe L del 0.35 del 0.67 AZFc 3B BPY2 probe L del 0 del 0.68 AZFc 3C BPY2 probe L del 0 del 0.75 AZFc 3C BPY2 probe L del 0 del 0.75 del AZFc 3B BPY2 probe L del 0 del 0.68 del AZFc 3A CDY2A probe L del 0.67 del AZFc 2H CDY1B probe L del 0.5 del AZFc 2G DAZ2 probe L del 0.54 del AZFc 2F DAZ2 probe L del AZFc 2E RBMY2CP probe L del AZFc 2D DAZ2 probe L del AZFc PPP1R12BP1 probe L AZFc RBMY2DP probe L SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions Page 10 of 12

11 Sample pictures of human male control DNA and test samples Figure 3. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with a preliminary version of the SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions. Figure 4. Capillary electrophoresis pattern of sample 1 showing an AZFa-AZFc deletion. Sample DNA was analysed with a preliminary version of the SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions. SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions Page 11 of 12

12 Figure 5. Capillary electrophoresis pattern of sample 2 showing an AZFc deletion. Sample DNA was analysed with a preliminary version of the SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions. Figure 6. Capillary electrophoresis pattern of sample 3 showing an AZFc deletion. Sample DNA was analysed with with the preliminary version of a SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions. SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions Page 12 of 12