MRC-Holland MLPA. Description version 10; 06 April 2018

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1 Description version ; 6 April 8 mix P36-B Y-Chromosome Microdeletions Lot B-5. As compared to version A (Lot A-), all probes f DPY9L, one probe f RBMYCP and one probe f KDM5D have been removed, and one USP9Y probe and 6 reference probes have been replaced. Two probes detecting DDX3Y and 4 reference probes have been added. This SALSA probemix is f basic research and intended f experienced MLPA users only! This probemix is intended to quantify genes 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. Interpretation of results can be complicated. recommends thoughly screening any available literature. 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 (Festa et al., ; PMID: 9485) and are clustered in three specific regions on the long arm of the Y chromosome, designated as azospermia facts (AZF) loci: AZFa (~3.-5. Mb from the p-telomere), AZFb (~ Mb) and AZFc (~ Mb) (Vogt et al., 996; PMID: 88737). Traditionally, these regions are examined by conventional sequence-tagged site PCR analyses. This approach focusses on deletions only, whereas MLPA can also detect duplications and complex rearrangements (Saito et al., 4; PMID: ). Giachini et al. (8; PMID: 88755) found no significant pathogenic effect of AZFc duplications, whereas Lin et al. (7; PMID: 78559) associated these duplications with a significant risk of oligospermia. These inconsistent findings about clinical consequences of e.g. AZF-linked duplications demonstrate the necessity of further research, f which at present, MLPA offers the most suitable technology. The most frequent deletion type is the AZFc region deletion (~8%) followed by AZFa (.5-4%), AZFb (- 5%) and AZFbc (-3%) deletions (Krausz et al., 4; PMID: ). Deletions which are detected as AZFabc are most likely related to abnmal karyotype such as 46,XX male iso (Y) (Lange et al., 9; PMID: ). This probemix contains probes f regions AZFa, b and c. The following genes have been mapped to the AZFa region: RPS4P, ARSEP, USP9Y, DDX3Y, UTY, BPY, VCYB and NLGN4Y. This P36-B probemix contains 6 probes detecting sequences in these genes. The genes RBMYJ, DY, KDM5D, EIFAY and MYJ have been mapped within AZFb. This P36-B probemix includes 5 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. The AZFc region encompasses many genes that are required f nmal spermatogenesis and that are critical f male fertility. AZFc is arranged in repeated sequence blocks, called amplicons, which are ganised into palindromic structures showing nearly identical sequences (Kuroda-Kawaguchi et al., ; PMID: ). The most imptant genes within the AZFc region are DAZ, BPY and CDY. The first and best characterised AZFc deletion spans the whole AZFc region (about 3.5 Mb). In a recent large study (Rozen et al., ; PMID: 333) the frequency of four recurrent partial AZFc deletions was studied in five populations. It was found that partial (interstitial) AZFc deletions are me common among individuals with severe spermatogenic failure than in controls. However, it is imptant to note that certain partial AZFc deletions, such as the.6 Mb gr/gr deletion, are also present in high frequency in fertile men (Machev et al., 4; PMID: 5546; Hucklenbroich et al., 5; PMID: ). This P36-B probemix contains probes detecting sequences in the AZFc region. Four of these probes detect a sequence that is present twice in the AZFc region. Two probes detect three targets within AZFc and one probe (as mentioned above) detects two targets in AZFc and one target in AZFb. Finally, this probemix contains one probe detecting the SRY gene on the Y-chromosome and reference probes that detect several different autosomal chromosomal locations. SALSA MLPA P36-B Y-Chromosome Microdeletions probemix Page of 3

2 Description version ; 6 April 8 Please note that we have no infmation whether not the deletion of individual probe sequences, even the complete deletion of certain genes, has an effect on male fertility. This mix is not intended f diagnostic purposes, but strictly f research use only. This SALSA probemix is designed to detect deletions/duplications of one me sequences in the above mentioned Y chromosomal regions in a DNA sample. F 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 cresponding probe amplification product. Note that a mutation polymphism in the sequence detected by a probe can also cause a reduction in relative peak height, even when not located exactly on the ligation site! In addition, some probe signals are me sensitive to sample purity and small changes in experimental conditions. Therefe, 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. Determination of microdeletions on the Y-chromosome is often perfmed by the analysis of the presence absence of a series of sequence-tagged sites (STSs). Me infmation on Y-chromosome microdeletions and common sequence-tagged sites (STSs) can be found in the EAA/EMQN best practice guidelines f molecular diagnosis of Y-chromosomal microdeletions: state-of-the-art 3 (Krausz et al., 4; PMID: ) at To give an idea about where MLPA probes are located compared to the common STS markers, tables 3, 6 and 7 also include the STS locations. To determine the exact location of the STS, we used the online database which provides regionally targeted catalogues of STSs. SALSA probemixes and reagents are sold by f research purposes and to demonstrate the possibilities of the MLPA technique. They are not CE/FDA certified f use in diagnostic procedures. Purchase of the SALSA test probemixes and reagents includes a limited license to use these products f research purposes. The use of a SALSA probemix and reagents requires a thermocycler with heated lid and sequence type electrophesis equipment. Different fluescent PCR primers are available. The MLPA technique has been first described in Nucleic Acids Research 3, e57 (). Me infmation Website : info@mlpa.com (infmation & technical questions); der@mlpa.com (f ders) Mail : bv; Willem Schoutenstraat, 57 DL Amsterdam, the Netherlands References Krausz C et al. (4). EAA/EMQN best practice guidelines f molecular diagnosis of Y-chromosomal microdeletions: state-of-the-art 3. Andrology. :5-9. Rozen SG et al. (). AZFc deletions and spermatogenic failure: a population-based survey of, Y chromosomes. Am J Hum Genet. 9:89-6. Shahid M et al. (). Association of Y-chromosome subdeletion gr/gr with the prevalence of Y- chromosome haplogroups in infertile patients. Eur J Hum Gen. 9:3-9. Lange J et al. (8). MSY Breakpoint Mapper, a database of sequence-tagged sites useful in defining naturally occurring deletions in the human Y chromosome. Nucleic Acids Res. 36:D89-4. The mapper can be found here: SALSA MLPA P36-B Y-Chromosome Microdeletions probemix Page of 3

3 Description version ; 6 April 8 Data analysis The P36-B Y-Chromosome Microdeletions probemix contains 55 MLPA probes with amplification products between 3 and 57 nt. In addition, it contains 9 control fragments generating an amplification product smaller than nt: four DNA Quantity fragments (Q-fragments) at nt, three DNA Denaturation control fragments (D-fragments) at nt, one X-fragment at nt and one Y-fragment at 5 nt. Me infmation on how to interpret observations on these control fragments can be found in the MLPA protocol. Data generated by this probemix can be nmalised intra-sample by dividing the peak height of each amplification product by the total peak height of only the reference probes in the probemix (block nmalisation). Secondly, inter-sample nmalisation can be achieved by dividing the intra-nmalised probe ratio in a sample by the average intra-nmalised probe ratio of all reference samples. Male reference samples should be used. Please note that this type of nmalisation assumes that no changes occurred in the genomic regions targeted by the reference probes. Data nmalisation should be perfmed within one experiment. Only samples purified by the same method should be compared. Confirmation of most exons deletions and amplifications can be done by e.g. Southern blotting, long range PCR, qpcr FISH. Note that Coffalyser, the MLPA analysis tool developed at, can be downloaded free of charge from our website Many copy number alterations in healthy individuals are described in the database of genomic variants: F example, a duplication of a complete gene might not be pathogenic, while a partial duplication a deletion may result in disease. F some genes, certain in-frame deletions may result in a very mild, no disease. Copy number changes of reference probes are unlikely to be the cause of the condition tested f. Users should always verify the latest scientific literature when interpreting their findings. Recommendations f the analysis of results of the P36-B Y-Chromosome Microdeletions probemix Due to strong homology between large sequence blocks within AZFb and AZFc, some probes included in this probemix detect even 3 target sites (see Table 3). Data interpretation can be complicated and MRC- Holland cannot offer further suppt. We strongly recommend using the P36-B data sheet that is available on request: info@mlpa.com. In this P36-B data sheet, all positions detected by the probes are arranged accding to chromosomal location. Probes detecting two three targets are included two three times, respectively. F each probe, fill in the probe ratio as calculated by Coffalyser.Net. If a probe detects two three target sequences, the probe value needs to be filled in two three times. Due to the fact that some probes target two even three targets, it can be difficult to determine which deleted target is actually causing a decreased signal. Using the data sheet can help to determine the possible deletion boundaries of a certain sample. Please see the Example sheet in the P36-B data sheet f me infmation. 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 included as co-auth. Info/remarks/suggestions f improvement: info@mlpa.com. SALSA MLPA P36-B Y-Chromosome Microdeletions probemix Page 3 of 3

4 Description version ; 6 April 8 Table. SALSA MLPA P36-B Y-Chromosome Microdeletions probemix reference Chromosomal position target-specific Q-fragments: DNA quantity; only visible with less than ng sample DNA D-fragments: Low signal of nt fragment indicates incomplete denaturation X-fragment: Specific f the X chromosome 5 Y-fragment: Specific f the Y chromosome Number of sequences detected 3 * Reference probe 966-L674 4p3 36 EIFAY probe 734-L57 Yq.3 4 PPPRBP probe 733-L4796 Yq.3 48 * Reference probe 57-L8 3q 54 * Reference probe 46-L64 p34 6 CDYB probe 536-L7486 Yq. 66 BPY probe 739-L38 Yq.3 7 * Reference probe 9-L579 9q34 78 BPY probe 74-L45 Yq.3 84 VCYB probe 538-L7485 Yq. 9 Reference probe 6378-L5844 6p5 96 Reference probe 87-L85 5q * Reference probe 559-L694 6q3 8 KDM5D probe 747-L53 Yq.3 5 UTY probe 8-L334 Yq. CDYA probe 673-L865 Yq. 7 RPS4P probe 539-L867 Yq. 34 * DDX3Y probe 86-L6 Yq. 4 CDYA probe 545-L8543 Yq KDM5D probe 754-L8544 Yq. 5 ARSEP probe 88-L8545 Yq. 56 * USP9Y probe 8-L66 Yq. 63 RBMYJ probe 757-L8748 Yq.3 67 DAZ probe 758-L89 Yq.3 74 KDM5D probe 39-L8749 Yq.3 79 SRY probe 3-L875 Yp.3 84 DAZ probe 738-L463 Yq.3 9 CDYB probe 759-L875 Yq. 95 * Reference probe 3796-L977 q 3 DAZ probe 76-L875 Yq.3 38 * DDX3Y probe 36-L8753 Yq. 35 RPS4P probe 393-L8553 Yq. 38 UTY probe 39-L893 Yq. 336 USP9Y probe 86-L8756 Yq. 34 BPY probe 543-L893 Yq. 35 UTY probe 88-L93 Yq. 356 VCYB probe 394-L869 Yq. 364 CDYB probe 546-L8757 Yq.3 37 USP9Y probe 544-L8758 Yq. 378 BPY probe 768-L8759 Yq * Reference probe 558-L3858 p3 39 RBMYDP probe 54-L67 Yq.3 4 HSFY probe 547-L863 Yq. 45 VCYB probe 85-L863 Yq. 4 HSFY probe 77-L555 Yq. 48 BPY probe 773-L556 Yq.3 47 RBMYJ probe 774-L89 Yq HSFY probe 74-L863 Yq. 445 KDM5D probe 776-L559 Yq. 454 * Reference probe 874-L853 8q4 463 NLGN4Y probe 853-L65 Yq. 47 * Reference probe 979-L36 p4 SALSA MLPA P36-B Y-Chromosome Microdeletions probemix Page 4 of 3

5 Description version ; 6 April 8 reference Chromosomal position target-specific Number of sequences detected 486 BPY probe 548-L7487 Yq EIFAY probe 549-L857 Yq.3 57 * Reference probe 4883-L896 4q * New in version B (from lot B-5 onwards). Changed in version B (from lot B-5 onwards). Change in length, no change in sequence detected. Based on the UCSC Genome Browser. Table. Reference sequences and gene synonyms Gene name accding to HUGO location NCBI reference sequence Alias gene names ARSEP Yq. NG_ VCYB Yq. NM_888. BPY, VCY, VCY, VCYB BPY Yq. NM_4678. BPYA, VCYA, VCY CDYB Yq. NM_ CDYA Yq. NM_485. CDY CDYB Yq. NM_7. CDY DAZ Yq. NM_363.3 PDP678, MGC644 EIFAY Yq. NM_ HSFY Yq. NM_5584. HSFY, HSFL KDM5D Yq. NM_ JARIDD NLGN4Y Yq. NM_ KIAA95 PPPRBP Yq. NG_ RPS4P Yq. NC_4.9 - RBMYJ Yq. NM_67. - RBMYDP Yq. NC_4.9 RBM SRY Yp.3 NM_34. TDF USP9Y Yq. NM_ DFFRY UTY Yq. 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 P36-B Y-Chromosome Microdeletions probemix Page 5 of 3

6 Description version ; 6 April 8 Table 3. P36 probes accding to chromosomal location F clarity, the location of the most commonly used STS markers has also been indicated. HG38 probe start Partial sequence (4 nt adjacent to ligation site) Distance to next probe (kb) 79 SRY probe 3-L GCACTGAAAGCT-GTAACTCTAAGT AZFa locus: 35 RPS4P probe 393-L TTACAGAAGGTA-TGTCCTTGCACT 8. sy8 STS marker RPS4P probe 539-L TCCCTAGTGCTA-CTGCCTCACTTA 7.6 sy64 STS marker ARSEP probe 88-L ACCTTCCCAGCA-AGCCGCCTTGAA 54.4 sy86 STS marker USP9Y probe 544-L ATCATGTGGCAT-TACCTCATTTGC 38.8 sy85 STS marker USP9Y probe 86-L GAAAGGCAAGGA-CTTTACCTTAAA 3.9 sy84 STS marker USP9Y probe 8-L TGGAGAAGGCAA-ACTTAGTCCACC 8.8 sy34 STS marker 7968 sy36 STS marker sy74 STS marker DDX3Y probe 86-L6 95 AAGCAAAGAACA-TGTCAGTGACTA. 38 DDX3Y probe 36-L AGCCTTCACTCT-TGTTATTGCTTA 33. sy65 STS marker 3497 sy8 STS marker 3 35 UTY probe 88-L TGCATTATTGCA-GTACTTTCTTCA UTY probe 39-L ATTGGTCCAGGA-GATTGTGAATGG 3.5 sy88 STS marker UTY probe 8-L AGGATCCTGGAT-ATTCCACTACCA BPY probe 543-L TCCCTTTCTACA-CTTAGATCTCTG VCYB probe 538-L GCATATTGAGTA-GATCATTCCTAG VCYB probe 85-L CACGTTCGCTCA-GTTCTCACTGAT VCYB probe 394-L GGAGTAGACCAA-GAGAGGAATATA NLGN4Y probe 853-L TTCTGCGTGGCA-TCACAGTCTTCC 37.7 sy5 STS marker AZFb locus: 6 CDYB probe 536-L TTCCAGCCAGGA-CACATCTGGAAA 5. 9 Ħ CDYB probe 759-L CTTCTGGCTGAA-CTGCGGCACCAA 49.7 CDYA probe 673-L GGCTGTTAATGA-ATTCGTTAATGC. 4 ⱷ CDYA probe 545-L ATTTTCTGTTAA-CCTTAGTGTAAA.8 sy4 STS marker Ħ CDYB probe 759-L CTTCTGGCTGAA-CTGCGGCACCAA 97.7 sy4 STS marker Ħ HSFY probe 547-L ATTTGATGATGA-AGATTTAGCAGA 84.4 sy967 STS marker Ħ HSFY probe 74-L AAAGAACACATA-CCAATATAGCTG. sy39 STS marker HSFY probe 77-L CTGGACTATGGA-TGCAACTTCCGA Ħ HSFY probe 74-L AAAGAACACATA-CCAATATAGCTG 84.4 sy967 STS marker Ħ HSFY probe 547-L ATTTGATGATGA-AGATTTAGCAGA sy STS marker 8899 sy399 STS marker KDM5D probe 754-L CCAACAAAGTCT-TACAATTATACT KDM5D probe 776-L CTGATTGGAGCA-CTCAGCCTAAAC KDM5D probe 747-L GACCAGGTTCAT-GCCAATATATTT. 74 KDM5D probe 39-L GATCTGAAGTTA-CTGATGAATCTG 39.7 sy7 STS marker EIFAY probe 549-L ACTTTCTAAATG-TTCTTGAATGTA SALSA MLPA P36-B Y-Chromosome Microdeletions probemix Page 6 of 3

7 Description version ; 6 April 8 HG38 probe start Partial sequence (4 nt adjacent to ligation site) Distance to next probe (kb) 36 EIFAY probe 734-L CCTGATTCTCCA-ATGGCTTCATAG 77.9 sy33 STS marker sy34 STS marker sy4 STS marker 8378 sy43 STS marker sy3 STS marker RBMYJ probe 774-L TGGCAAATCCAT-AATATTACAACA RBMYJ probe 757-L TACAACCAGAGA-TAATGTAAATAG 44. sy99 STS marker sy97 STS marker AZFc locus: sy9 STS marker BPY probe 773-L TTTACATGGTAA-ATTGATGTGCTT.5 66 BPY probe 739-L TAGGAGAAAATA-ACAAAATAATGA.7 78 BPY probe 74-L CACAGAAATATA-TACACTGTTTGA 4.6 sy9 STS marker ⱷ BPY probe 548-L TCTTTGTATTCA-TGCCAAGAAACG ⱷ BPY probe 768-L TCATATGTCTGA-AGTCAGAACTTG 45.6 sy53 STS marker copy sy54 STS marker copy 3746 sy54 STS marker copy sy37 STS marker copy 3797 sy89 STS marker sy9 STS marker Ħ DAZ probe 738-L GTTCAGCTGGCA-AGCTAGCTGTGC Ħ DAZ probe 76-L AGTATATTCCCA-TTCCTAATAATG 4. sy5 STS marker copy Ħ DAZ probe 758-L CAGTGCTTCTGA-ATGATTTTCAGT 96. sy858 STS marker copy sy74 STS marker copy Ħ CDYB probe 546-L CCTTACTGCTTA-AGGCCGTATTTC ⱷ CDYA probe 545-L ATTTTCTGTTAA-CCTTAGTGTAAA 3.3 sy6 STS marker ⱷ BPY probe 548-L TCTTTGTATTCA-TGCCAAGAAACG ⱷ BPY probe 768-L TCATATGTCTGA-AGTCAGAACTTG 76.7 sy53 STS marker copy 4547 sy37 STS marker copy sy54 STS marker copy sy54 STS marker copy ⱷ BPY probe 768-L TCATATGTCTGA-AGTCAGAACTTG ⱷ BPY probe 548-L TCTTTGTATTCA-TGCCAAGAAACG 3.3 sy6 STS marker copy sy55 STS marker copy ⱷ CDYA probe 545-L ATTTTCTGTTAA-CCTTAGTGTAAA Ħ CDYB probe 546-L CCTTACTGCTTA-AGGCCGTATTTC Ħ DAZ probe 758-L CAGTGCTTCTGA-ATGATTTTCAGT 4.6 sy5 STS marker copy Ħ DAZ probe 76-L AGTATATTCCCA-TTCCTAATAATG Ħ DAZ probe 738-L GTTCAGCTGGCA-AGCTAGCTGTGC 4. sy STS marker PPPRBP probe 733- L AGCATTTGGAGA-TGCTCCAGAAGA RBMYDP probe 54-L CACTGAATGGAA-AAGTACAGCTGG sy55 STS marker copy sy55 STS marker copy Ħ These probes detect sequences (f a detailed explanation, please see table 4a). ⱷ These probes detect 3 sequences (f a detailed explanation, please see table 4b). + The 5 nt Y probe detects a sequence located 5 kb after the 38 nt probe and 5 kb befe the 5 nt probe. SALSA MLPA P36-B Y-Chromosome Microdeletions probemix Page 7 of 3

8 Description version ; 6 April 8 Table 4. Expected probe signals and ratios Table 4a. Expected probe signals and ratios f probes detecting sequences PROBES detecting sequences (Ħ) Remaining signal Decrease of signal Expected ratio Both targets present % % Deletion of target site; target remaining 5% 5%.5 Deletion of target sites; no target remaining % % Table 4b. Expected probe signals and ratios f probes detecting 3 sequences PROBES detecting 3 sequences (ⱷ) Remaining signal Decrease of signal Expected ratio All 3 targets present % Deletion of target site; targets remaining 66% 33%.66 Deletion of target sites; target remaining 33% 66%.33 Deletion of all 3 target sites; no target remaining % Table 5. Partial AZFc deletions as described by Rozen et al. & Shahid et al. AZFc deletion STS markers deleted STS markers not deleted Deletion size Frequency Risk increase f SSF 3 b/b3 sy9 sy4 sy9 sy9.8 Mb :9 No increase sy9 b/b3 sy9 sy89 sy4 sy.6 Mb :994.5 sy9 sy54: copies deleted sy54: copies remaining b/b4 sy9 sy9 sy89 sy9 sy54: all 4 copies deleted sy4 sy sy54: copies remaining 3.5 Mb :3 45 sy4 gr/gr sy9 sy6 sy89 sy9.6 Mb :4.9 sy54: copies deleted sy54: copies remaining. Rozen et al., ; PMID: 333; Shahid et al., ; PMID: Frequency in the total male population. The exact frequency of each deletion type varies per population. SSF: Severe Spermatogenic Failure. SALSA MLPA P36-B Y-Chromosome Microdeletions probemix Page 8 of 3

9 Description version ; 6 April 8 Table 6. Expected probe numbers in samples with partial AZFc deletions Nmal HG38 copy probe start number left: expected remaining copies per deletion type (based on table 5) right: graphical representation of expected extent of the deletion b/b3 b/b3 b/b4 gr/gr* sy9 STS marker BPY probe 773-L BPY probe 739-L BPY probe 74-L sy9 STS marker ⱷ BPY probe 548-L ⱷ BPY probe 768-L sy53 STS marker copy sy54 STS marker copy sy54 STS marker copy sy37 STS marker copy 3797 sy89 STS marker sy9 STS marker Ħ DAZ probe 738-L Ħ DAZ probe 76-L sy5 STS marker copy Ħ DAZ probe 758-L sy858 STS marker copy sy74 STS marker copy Ħ CDYB probe 546-L ⱷ CDYA probe 545-L sy6 STS marker copy ⱷ BPY probe 548-L ⱷ BPY probe 768-L sy53 STS marker copy 4547 sy37 STS marker copy sy54 STS marker copy sy54 STS marker copy ⱷ BPY probe 768-L ⱷ BPY probe 548-L sy6 STS marker copy sy55 STS marker copy SALSA MLPA P36-B Y-Chromosome Microdeletions probemix Page 9 of

10 Description version ; 6 April 8 Nmal HG38 copy probe start number 4 ⱷ CDYA probe 545-L Ħ CDYB probe 546-L Ħ DAZ probe 758-L sy5 STS marker copy Ħ DAZ probe 76-L Ħ DAZ probe 738-L left: expected remaining copies per deletion type (based on table 5) right: graphical representation of expected extent of the deletion b/b3 b/b3 b/b4 gr/gr* 3 sy STS marker PPPRBP probe 733- L RBMYDP probe 54- L Ħ These probes detect sequences (f a detailed explanation, please see table 4a). ⱷ These probes detect 3 sequences (f a detailed explanation, please see table 4b). * The location of the gr/gr deletion has been confirmed by testing several samples carrying this specific deletion. Please find the nmalised results of one example in table 7. Solid line: represents the area that is certainly absent in this deletion type. question mark: represents an area of which it is not clear if it is absent present in this deletion type. (No empirical values are available.) Dotted line: represents an area that is also missing in this deletion type, but of which the exact location cannot be established with certainty because there are two identical repeat areas flanking the solid line, either of which could be the deleted area. + An STS marker with a plus sign is expected to be present in this deletion type based on the results in table 5. - An STS marker with a minus sign is expected to be absent in this deletion type based on the results in table 5. SALSA MLPA P36-B Y-Chromosome Microdeletions probemix Page of 3

11 Description version ; 6 April 8 Table 7. Nmalised results of a positive sample carrying a gr/gr deletion HG38 probe start Remaining robe ratios with a gr/gr deletion Empirical value The extent of the gr/gr deletion Expected remaining probe value, see also Table 6 47 RBMYJ probe 774-L ~ 63 RBMYJ probe 757-L ~ sy99 STS marker sy97 STS marker AZFc locus: sy9 STS marker BPY probe 773-L ~ 66 BPY probe 739-L ~ 78 BPY probe 74-L ~ sy9 STS marker ⱷ BPY probe 548-L ⱷ BPY probe 768-L sy53 STS marker copy sy54 STS marker copy 3746 sy54 STS marker copy sy37 STS marker copy 3797 sy89 STS marker sy9 STS marker Ħ DAZ probe 738-L Ħ DAZ probe 76-L sy5 STS marker copy Ħ DAZ probe 758-L sy858 STS marker copy sy74 STS marker copy Ħ CDYB probe 546-L ⱷ CDYA probe 545-L sy6 STS marker copy ⱷ BPY probe 548-L ⱷ BPY probe 768-L sy53 STS marker copy 4547 sy37 STS marker copy sy54 STS marker copy sy54 STS marker copy ⱷ BPY probe 768-L ⱷ BPY probe 548-L sy6 STS marker copy sy55 STS marker copy ⱷ CDYA probe 545-L Ħ CDYB probe 546-L Ħ DAZ probe 758-L sy5 STS marker copy Ħ DAZ probe 76-L Ħ DAZ probe 738-L sy STS marker PPPRBP probe 733-L ~ + 39 RBMYDP probe 54-L ~ +. Data courtesy of David J. Bunyan, Salisbury NHS Foundation Trust, UK. This data is based on a sample analysed with MLPA probemix P36-B Y-Chromosome Microdeletions. Solid line: represents the area that is certainly absent in this deletion type. Dotted line: represents an area that is also missing in this deletion type, but of which the exact location cannot be established with certainty because there are two identical repeat areas flanking the solid line, either of which could be the deleted area. SALSA MLPA P36-B Y-Chromosome Microdeletions probemix Page of 3

12 Description version ; 6 April 8 mix P36-B Y-Chromosome Microdeletions sample picture Figure. Capillary electrophesis pattern of a sample of approximately 5 ng human male control DNA analysed with mix P36-B Y-Chromosome Microdeletions (lot B-5). SALSA MLPA P36-B Y-Chromosome Microdeletions probemix Page of 3

13 Description version ; 6 April 8 Implemented Changes compared to the previous product description version Version 6 April 8 (55) - RBMYCP has been removed from table. - Various min textual changes. Version 9 4 January 8 (55) - Table 3 has been revised: the Y-fragment (5 nt) has been removed, and a remark has been added below the table. Version 8 9 July 6 (55) - Various textual and graphical changes have been made throughout the document to improve readability. - References to literature have been added updated throughout the document. - Symbols have been changed in the tables. - Table 3 (previously table 3a) has been revised, and the Y-fragment (5 nt) has been included. - Table 4 with detailed infmation about remaining peak signals and expected ratios f probes detecting two three targets has been added. - Table 5 (previously table 3b) has been revised. - Table 6 (previously table 3c) has been revised, and infmation on the b/b3 deletion has been crected. - Table 7 with the nmalized results f a sample with a gr/gr deletion has been added. Version 7 9 November 5 (55) - Product description adapted to a new product version (version number changed, lot number added, small changes in Table and Table 3, new pictures included). - Updated link f Database of Genomic Variants. - Peak area replaced with peak height. - Crected text on data analysis. - Updated link f EAA/EMQN best practical guidelines f molecular diagnosis of Y-chromosomal microdeletions: state-of-the-art 3. - Manufacturer s address adjusted. - Additional STS markers added to Table 3. - Content of Table 3c changed. - Appendix deleted. Version 6 (5) - Basic research box added on page. Version 5 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December ) added. Version 4 (48) - Location of STS markers and the presumed locations of the gr/gr and b/b3 deletions included in Table 3a. Textual change of page. Version 3 (48) - Change of probe position 546-L554 in Table 4. Version (48) - Various min textual and layout changes. - Remark on RefSeqGene standard added below Table 3b. - Small changes of probe lengths in Table and 3 in der to better reflect the true lengths of the amplification products. Version - Not applicable, new document. SALSA MLPA P36-B Y-Chromosome Microdeletions probemix Page 3 of 3