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

SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions Lot A1-1011. 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 (~13.1-15.2 Mb from the p-telomere), AZFb (~18.5-22.8 Mb) and AZFc (~23.3-25.5 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; PMID23103232) 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 http://www.unileipzig.de/~eaa/guidelines_2004.pdf 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

Description version 06; 23-07-2013 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 : www.mlpa.com E-mail : 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 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 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 www.mlpa.com. Many copy number alterations in healthy individuals are described in the database of genomic variants: http://projects.tcag.ca/variation. 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

Table 1. SALSA MLPA P360-A1 Y-Chromosome Microdeletions probemix Length (nt) SALSA MLPA probe Chromosomal position reference target-specific 64-70-76-82 Q-fragments: DNA quantity; only visible with less than 100 ng sample DNA 88-92-96 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 11734-L12517 Yq11.222 1 142 PPP1R12BP1 probe 12733-L14796 Yq11.23 1 147 RBMY2CP probe 11804-L12599 Yq11.223 / Yq11.23 2 153 Reference probe 12212-L13140 3p21 1 160 CDY2B probe 15236-L17486 Yq11.221 1 166 BPY2 probe 11739-L13811 Yq11.223 1 178 BPY2 probe 11740-L14251 Yq11.223 1 184 VCY1B probe 15238-L17485 Yq11.221 1 190 Reference probe 06378-L05844 6p25 1 196 Reference probe 08170-L08050 5q22 1 202 KDM5D probe 11744-L13987 Yq11.222 1 208 KDM5D probe 11747-L12530 Yq11.222 1 215 UTY probe 11812-L13342 Yq11.21 1 223 CDY2A probe 11749-L18625 Yq11.221 1 228 RPS24P1 probe 15239-L18627 Yq11.21 1 234 * CDY2A probe 15245-L18626 Yq11.221/Yq11.223/Yq11.23 3 241 KDM5D probe 11754-L12537 Yq11.222 1 247 ARSEP probe 11818-L17483 Yq11.21 1 252 KDM5D probe 11756-L13817 Yq11.223 1 259 RBMY1J probe 11757-L13818 Yq11.223 1 265 DAZ2 probe 11758-L12541 Yq11.223 / Yq11.23 2 271 Reference probe 14361-L00694 4q35 1 276 SRY probe 01023-L15420 Yp11.31 1 283 DAZ2 probe 12738-L12543 Yq11.223 / Yq11.23 2 288 CDY2B probe 11759-L14252 Yq11.221 / Yq11.222 2 292 Reference probe 08790-L11322 10q21 1 301 DAZ2 probe 11761-L12544 Yq11.223 / Yq11.23 2 307 DPY19L2 probe 17170-L20718 12q14.2 1 312 UTY probe 11824-L20831 Yq11.21 1 319 RPS24P1 probe 11825-L20832 Yq11.21 1 327 USP9Y probe 11826-L12621 Yq11.21 1 333 Reference probe 01790-L10676 13q14 1 342 VCY1B probe 15243-L15069 Yq11.221 1 347 UTY probe 11828-L19232 Yq11.21 1 355 VCY1B probe 15240-L18629 Yq11.221 1 361 CDY1B probe 15246-L12554 Yq11.223 / Yq11.23 2 366 USP9Y probe 15244-L12618 Yq11.21 1 373 * BPY2 probe 11768-L14716 Yq11.223 (2x)/ Yq11.23 (1x) 3 380 DPY19L2 probe 17171-L20390 12q14.2 1 389 RBMY2DP probe 15241-L12617 Yq11.23 1 396 HSFY1 probe 15247-L18630 Yq11.222 (2x) 2 402 VCY1B probe 11852-L18631 Yq11.221 1 409 HSFY1 probe 11772-L12555 Yq11.222 1 418 BPY2 probe 11773-L12556 Yq11.223 1 427 RBMY1J probe 11774-L12557 Yq11.223 1 436 HSFY1 probe 12740-L18632 Yq11.222 (2x) 2 SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions Page 3 of 12

Length (nt) SALSA MLPA probe Chromosomal position reference target-specific Number of sequences detected (according to UCSC Genome Browser) 445 KDM5D probe 11776-L12559 Yq11.222 1 454 USP9Y probe 11836-L12631 Yq11.21 1 463 NLGN4Y probe 11853-L12650 Yq11.221 1 472 Reference probe 14160-L20657 2p25 1 486 * BPY2 probe 15248-L19234 Yq11.221/Yq11.223/ Yq11.23 3 490 EIF1AY probe 15249-L12560 Yq11.222 1 500 Reference probe 12462-L19605 22q12 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_000880.3 - VCY1B Yq11.2 NM_181880.1 BPY1, VCY1, VCY, VCYB BPY2 Yq11.2 *NM_004678.2 BPY2A, VCY2A, VCY2 CDY1B Yq11.2 NM_001003894.1 - CDY2A Yq11.2 NM_004825.2 CDY2 CDY2B Yq11.2 NM_001001722.1 CDY DAZ2 Yq11.2 *NM_020363.3 pdp1678, MGC126442 EIF1AY Yq11.2 NM_004681.2 - HSFY1 Yq11.2 *NM_152584.1 HSFY, HSF2L KDM5D Yq11.2 NM_004653.4 JARID1D NLGN4Y Yq11.2 *NM_014893.4 KIAA0951 PPP1R12BP1 Yq11.2 NG_004755.2 - RPS24P1 Yq11.2 NC_000024.9 - RBMY1J Yq11.2 NM_001006117.2 - RBMY2CP Yq11.2 NC_000024.9 RBMY2 RBMY2DP Yq11.2 NC_000024.9 RBM SRY Yp11.3 *NM_003140.1 TDF USP9Y Yq11.2 *NM_004654.3 DFFRY UTY Yq11.2 NM_007125.3 - DPY19L2 12q14.2 NM_173812.4 - *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

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 01023-L15420 2655484 GCACTGAAAGCT-GTAACTCTAAGT 11671.5 kb 319 RPS24P1 probe 11825-L20832 14326976 AZFa TTACAGAAGGTA-TGTCCTTGCACT 28.2 kb 228 RPS24P1 probe 15239-L18627 14355188 AZFa TCCCTAGTGCTA-CTGCCTCACTTA 127.7 kb 247 ARSEP probe 11818-L17483 14482862 AZFa ACCTTCCCAGCA-AGCCGCCTTGAA 145.6 kb 366 USP9Y probe 15244-L12618 14628475 AZFa ATCATGTGGCAT-TACCTCATTTGC 138.8 kb sy85 STS marker 14638000 327 USP9Y probe 11826-L12621 14767317 AZFa GAAAGGCAAGGA-CTTTACCTTAAA 160.8 kb 454 USP9Y probe 11836-L12631 14928146 AZFa CTGTTCCTGATA-GAAGGAATCATG 431.9 kb G34990 STS marker 15032000 347 UTY probe 11828-L19232 15360054 AZFa TGCATTATTGCA-GTACTTTCTTCA 54.7 kb 312 + UTY probe 11824-L20831 15414769 AZFa ATTGGTCCAGGA-GATTGTGAATGG 301.5 kb 215 + UTY probe 11812-L13342 15716249 AZFa AGGATCCTGGAT-ATTCCACTACCA 159.0 kb 342 VCY1B probe 15243-L15069 15875243 AZFa TCCCTTTCTACA-CTTAGATCTCTG 161.4 kb 184 VCY1B probe 15238-L17485 16036660 AZFa GCATATTGAGTA-GATCATTCCTAG 153.7 kb 402 VCY1B probe 11852-L18631 16190399 AZFa CACGTTCGCTCA-GTTCTCACTGAT 159.0 kb 355 VCY1B probe 15240-L18629 16349354 AZFa GGAGTAGACCAA-GAGAGGAATATA 209.5 kb 463 NLGN4Y probe 11853-L12650 16558890 AZFa TTCTGCGTGGCA-TCACAGTCTTCC 3007.7 kb 160 CDY2B probe 15236-L17486 19566622 AZFb TTCCAGCCAGGA-CACATCTGGAAA 5.1 kb 288 CDY2B probe 11759-L14252 19571702 AZFb CTTCTGGCTGAA-CTGCGGCACCAA 492.7 kb 223 CDY2A probe 11749-L18625 20064415 AZFb GGCTGTTAATGA-ATTCGTTAATGC 0.1 kb 234 * CDY2A probe 15245-L18626 20064544 AZFb ATTTTCTGTTAA-CCTTAGTGTAAA 543.5 kb 288 CDY2B probe 11759-L14252 20607999 AZFb CTTCTGGCTGAA-CTGCGGCACCAA 97.1 kb 396 HSFY1 probe 15247-L18630 20705073 AZFb ATTTGATGATGA-AGATTTAGCAGA 84.4 kb 436 HSFY1 probe 12740-L18632 20789494 AZFb AAAGAACACATA-CCAATATAGCTG 21.1 kb 409 HSFY1 probe 11772-L12555 20810589 AZFb CTGGACTATGGA-TGCAACTTCCGA 44.0 kb 436 HSFY1 probe 12740-L18632 20854606 AZFb AAAGAACACATA-CCAATATAGCTG 84.4 kb 396 HSFY1 probe 15247-L18630 20939031 AZFb ATTTGATGATGA-AGATTTAGCAGA 555.3 kb 241 KDM5D probe 11754-L12537 21494326 AZFb CCAACAAAGTCT-TACAATTATACT 16.1 kb 202 KDM5D probe 11744-L13987 21510472 AZFb CAATTTTGTCCA-TACTTTTGGTTA 571.7 kb 445 KDM5D probe 11776-L12559 22082204 AZFb CTGATTGGAGCA-CTCAGCCTAAAC 77.9 kb 208 KDM5D probe 11747-L12530 22160145 AZFb GACCAGGTTCAT-GCCAATATATTT 19.6 kb 252 KDM5D probe 11756-L13817 22179719 AZFb GATCTGAAGTTA-CTGATGAATCTG 465.4 kb sy127 STS marker 22570000 490 EIF1AY probe 15249-L12560 22645153 AZFb ACTTTCTAAATG-TTCTTGAATGTA 24.1 kb 136 EIF1AY probe 11734-L12517 22669283 AZFb CCTGATTCTCCA-ATGGCTTCATAG 1756.1 kb sy133 STS marker 23499000 sy142 STS marker 23978000 sy1258 STS marker 24071000 sy1161 STS marker 24239000 427 ٨ RBMY1J probe 11774-L12557 24425431 AZFb TGGCAAATCCAT-AATATTACAACA 6.4 kb 259 ٨ RBMY1J probe 11757-L13818 24431853 AZFb TACAACCAGAGA-TAATGTAAATAG 441.1 kb sy1197 STS marker 24524000 sy1192 STS marker 24872000 418 ٨ BPY2 probe 11773-L12556 24873002 AZFc TTTACATGGTAA-ATTGATGTGCTT 0.5 kb 166 ٨ BPY2 probe 11739-L13811 24873540 AZFc TAGGAGAAAATA-ACAAAATAATGA 1.7 kb 178 ٨ BPY2 probe 11740-L14251 24875225 AZFc CACAGAAATATA-TACACTGTTTGA 42.6 kb sy1191 STS marker 24876000 486 *@٨ BPY2 probe 15248-L19234 24917874 AZFc TCTTTGTATTCA-TGCCAAGAAACG 49.2 kb 373 *@٨ BPY2 probe 11768-L14716 24967068 AZFc TCATATGTCTGA-AGTCAGAACTTG 555.1 kb sy254 STS marker copy 1 25316000 sy254 STS marker copy 2 25373000 sy1291 & sy1189 STS markers 25505000 SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions Page 5 of 12

Length (nt) SALSA MLPA probe HG19 AZF probe start locus Partial sequence (24 nt adjacent to ligation site) Distance to next probe 283 @ DAZ2 probe 12738-L12543 25522145 AZFc GTTCAGCTGGCA-AGCTAGCTGTGC 48.3 kb 147 @ RBMY2CP probe 11804-L12599 25570491 AZFc TTGCAATGCCAA-TGCAATCCCACA 49.7 kb 301 @ DAZ2 probe 11761-L12544 25620137 AZFc AGTATATTCCCA-TTCCTAATAATG 242.0 kb 265 @ DAZ2 probe 11758-L12541 25862163 AZFc CAGTGCTTCTGA-ATGATTTTCAGT 196.1 kb 361 @ CDY1B probe 15246-L12554 26058233 AZFc CCTTACTGCTTA-AGGCCGTATTTC 193.1 kb 234 *@ CDY2A probe 15245-L18626 26251332 AZFc ATTTTCTGTTAA-CCTTAGTGTAAA 300.3 kb sy1206 STS marker copy 1 26527000 486 *@ BPY2 probe 15248-L19234 26551617 AZFc TCTTTGTATTCA-TGCCAAGAAACG 49.2 kb 373 *@ BPY2 probe 11768-L14716 26600816 AZFc TCATATGTCTGA-AGTCAGAACTTG 760.7 kb sy254 STS marker copy 3 26952000 sy254 STS marker copy 4 26987000 373 * BPY2 probe 11768-L14716 27361515 AZFc TCATATGTCTGA-AGTCAGAACTTG 49.2 kb 486 * BPY2 probe 15248-L19234 27410721 AZFc TCTTTGTATTCA-TGCCAAGAAACG 300.3 kb sy1206 STS marker copy 2 27436000 234 * CDY2A probe 15245-L18626 27711031 AZFc ATTTTCTGTTAA-CCTTAGTGTAAA 193.1 kb 361 CDY1B probe 15246-L12554 27904086 AZFc CCTTACTGCTTA-AGGCCGTATTTC 196.0 kb 265 DAZ2 probe 11758-L12541 28100127 AZFc CAGTGCTTCTGA-ATGATTTTCAGT 242.6 kb 301 DAZ2 probe 11761-L12544 28342689 AZFc AGTATATTCCCA-TTCCTAATAATG 49.7 kb 147 RBMY2CP probe 11804-L12599 28392357 AZFc TTGCAATGCCAA-TGCAATCCCACA 48.3 kb 283 DAZ2 probe 12738-L12543 28440706 AZFc GTTCAGCTGGCA-AGCTAGCTGTGC 41.1 kb sy1201 STS marker 28458000 142 PPP1R12BP1 probe 12733- AZFc L14796 28481778 AGCATTTGGAGA-TGCTCCAGAAGA 88.4 kb 389 RBMY2DP probe 15241-L12617 28570146 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 (PMID20823911). 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 low. @ 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); PMID23103232 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 1:41 1.9 fold increase in SSF risk b2/b3 sy1192, sy1191 sy142, sy1291 1.8 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 1:994 2.5 fold increase in SSF risk b2/b4 sy1192, sy1191, sy1291, sy1189, sy254 (all copies deleted) sy142, sy1201 3.5 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

Table 3c. DPY19L2 Length (nt) SALSA MLPA probe DPY19L2 exon Ligation site NM_173812.4 Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon 182-184 (ex 1) 380 17171-L20390 Intron 3 841nt before exon 4 AACACGAGTAAG-TACTAAATTACA 90.7 kb 307 17170-L20718 Intron 19 776nt before exon 20 AAGATGATCTGC-GGGAAGAAAACA stop codon 2456-2458 (ex 22) The NM_173812.4 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

SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions sample pictures 25000 96.51 195.43 183.63 245.88 306.75 20000 15000 86.38 105.57 91.21 133.98 151.55 145.11 158.69 165.46 189.16 177.45 201.61 214.69 233.67 226.99 221.89 240.46 252.27 263.42 258.08 275.99 270.29 281.69 287.64 318.78 312.86 325.85 346.86 365.84 360.04 379.16 395.78 388.77 402.26 478.64 486.28 10000 100.74 139.77 207.74 292.94 299.73 340.98 332.63 353.59 372.37 409.62 427.32 416.98 436.16 445.74 464.13 454.41 500.91 5000 492.25 D ye Sign al 0 50 100 150 200 250 300 350 400 450 500 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 15246-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

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 01023-L15420 276 2715484 0.95 1.02 0.98 AZFa RPS24P1 probe 11825-L20832 319 12836976 0.98 1 1.02 AZFa RPS24P1 probe 15239-L18627 228 12865189 1.02 0.97 1.03 AZFa ARSEP1 probe 11818-L17483 247 12992870 1 0.96 1.02 AZFa USP9Y probe 15244-L12618 366 13138483 0.99 1 0.99 AZFa USP9Y probe 11826-L12621 327 13276711 1.04 1.06 1.01 AZFa USP9Y probe 11836-L12631 454 13437540 1.01 0.99 1 AZFa UTY probe 11828-L19232 347 13869448 1.06 1.06 0.99 AZFa UTY probe 11824-L20831 312 13924163 1.02 0.98 1 AZFa UTY probe 11812-L13342 215 14225643 1.03 0.99 1.01 AZFa BPY1 probe 15243-L15069 342 14384640 1.08 1.03 1.05 AZFa VCY1B probe 15238-L17485 184 14546054 1 0.96 1 AZFa VCY1B probe 11852-L18631 402 14699793 1.06 1.03 1.02 AZFa VCY1B probe 15240-L18629 355 14858748 0.99 0.99 0.98 AZFa NLGN4Y probe 11853-L12650 463 15068284 0.96 1.02 1.02 AZFb CDY2B probe 15236-L17486 160 18076016 0.87 1.01 0.99 AZFb 2A CDY2B probe 11759-L14252 288 18081096 0.44 0.94 1.01 AZFb CDY2A probe 11749-L18625 223 18573809 0 del 0.97 0.99 AZFb 3A CDY2A probe 15245-L18626 234 18573938 0.34 del 0.35 0.67 AZFb 2A CDY2B probe 11759-L14252 288 19067391 0.44 del 0.94 1.01 AZFb 2B HSFY1 probe 15247-L18630 396 19164461 0 del 0.88 0.98 AZFb 2C HSFY1 probe 12740-L18632 436 19248882 0 del 0.91 0.96 AZFb HSFY1 probe 11772-L12555 409 19269977 0 del 0.94 1.01 AZFb 2C HSFY1 probe 12740-L18632 436 19313994 0 del 0.91 0.96 AZFb 2B HSFY1 probe 15247-L18630 396 19398417 0 del 0.88 0.98 AZFb KDM5D probe 11754-L12537 241 19953714 0 del 1.05 1 AZFb KDM5D probe 11744-L13987 202 19969860 0 del 0.99 0.94 AZFb KDM5D probe 11776-L12559 445 20541592 0 del 0.94 1.04 SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions Page 9 of 12

AZFb KDM5D probe 11747-L12530 208 20619533 0 del 0.92 0.92 AZFb KDM5D probe 11756-L13817 252 20639107 0 del 0.95 0.98 AZFb EIF1AY probe 15249-L12560 490 21054541 0 del 1 0.99 AZFb EIF1AY probe 11734-L12517 136 21078671 0 del 0.98 0.98 AZFb RBMY1J probe 11774-L12557 427 22834819 0 del 0.9 0.96 AZFb RBMY1J probe 11757-L13818 259 22841241 0 del 0.92 0.98 AZFc BPY2 probe 11773-L12556 418 23282390 0 del 0 del 0.97 AZFc BPY2 probe 11739-L13811 166 23282929 0 del 0 del 1.01 AZFc BPY2 probe 11740-L14251 178 23284612 0 del 0 del 0.99 AZFc 3B BPY2 probe 15248-L19234 486 23327263 0 del 0 del 0.68 AZFc 3C BPY2 probe 11768-L14716 373 23376456 0 del 0 del 0.75 AZFc 2D DAZ2 probe 12738-L12543 283 23931533 0.51 del 0.5 del 0.52 AZFc 2E RBMY2CP probe 11804-L12599 147 23979879 0.57 del 0.55 del 0.58 AZFc 2F DAZ2 probe 11761-L12544 301 24029525 0.48 del 0.45 del 0.47 AZFc 2G DAZ2 probe 11758-L12541 265 24271551 0.48 del 0 del 0.54 AZFc 2H CDY1B probe 15246-L12554 361 24467621 0.49 del 0 del 0.5 AZFc 3A CDY2A probe 15245-L18626 234 24660720 0.34 del 0.35 del 0.67 AZFc 3B BPY2 probe 15248-L19234 486 24961006 0 del 0 del 0.68 AZFc 3C BPY2 probe 11768-L14716 373 25010203 0 del 0 del 0.75 AZFc 3C BPY2 probe 11768-L14716 373 25770903 0 del 0 del 0.75 del AZFc 3B BPY2 probe 15248-L19234 486 25820108 0 del 0 del 0.68 del AZFc 3A CDY2A probe 15245-L18626 234 26120421 0.34 0.35 del 0.67 del AZFc 2H CDY1B probe 15246-L12554 361 2631474 0.49 0 del 0.5 del AZFc 2G DAZ2 probe 11758-L12541 265 26509515 0.48 0 del 0.54 del AZFc 2F DAZ2 probe 11761-L12544 301 26752077 0.48 0.45 0.47 del AZFc 2E RBMY2CP probe 11804-L12599 147 26801745 0.57 0.55 0.58 del AZFc 2D DAZ2 probe 12738-L12543 283 26850094 0.51 0.5 0.52 del AZFc PPP1R12BP1 probe 12733-L14796 142 26891166 0.94 0.96 1 AZFc RBMY2DP probe 15241-L12617 389 28570146 0.94 0.93 1 SALSA MLPA probemix P360-A1 Y-Chromosome Microdeletions Page 10 of 12

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

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