mix P185-C1 Intersex Lot C1-0611: As compared to the previous version B2 (lot B2-0311), s for CYP21A2 have been removed and s for the CXorf21 gene as well as additional s for NR0B1, NR5A1 and the Y chromosome have been included. The number of reference s has been increased from 6 to 9. The sex-determining region on chromosome Y (SRY) is the most important sex-determining region in humans. As a transcriptional activator, the SRY protein s main function is to initiate male sex determination by regulating a genetic switch in male development. In addition to SRY, the dosage-sensitive sex reversal (DSS) gene, NR0B1 (also known as DAX1) has also been found to influence sex-determination. It is believed that two active copies of DAX1 can override the testis-determining signal, resulting in the development of ovaries and an XY female. In testicular Sertoli and Leydig cells, DAX1 can be up-regulated by WNT4. The SOX9 gene functions as a critical Sertoli cell differentiation factor. Leipoldt M et al. described Cis acting elements upstream of SOX9 where chromosomal aberrations can lead to campomelic dysplasia with or without XY sex reversal (2007, Clin Genet.). NR5A1 encodes the orphan nuclear receptor steroidogenic factor-1, a nuclear receptor transcription factor that plays a key role in regulating adrenal and gonadal development, steroidogenesis and reproduction. Recently, haploinsufficiency of NR5A1 has been described in several 46, XY individuals with mild gonadal dysgenesis and impaired androgenization, but normal adrenal function, suggesting that dosage-sensitive or domain-specific effects of SF1 action are important in human testicular development and function. The P185-C1 mix contains s for the following genes: NR0B1 (DAX1) and CXorf21 on Xp21.2, SOX9 on 17q24.3, SRY and ZFY on Yp11.3, WNT4 on 1p36.12 and NR5A1 on 9q33. Furthermore, s specific for the X and Y-chromosomes are included. In addition, 9 reference s are included in this mix, detecting several different autosomal chromosomal locations. This SALSA mix is designed to detect deletions/duplications of one or more sequences in the aforementioned genes in a DNA sample. Heterozygous deletions of autosomal recognition sequences should give a 35-50% reduced relative peak height of the amplification product of that. Deletions of a s recognition sequence on the X-chromosome will lead to a complete absence of the corresponding amplification product in males, whereas female heterozygotes are recognisable by a 35-50% reduction in relative peak height. Note that a mutation or polymorphism in the sequence detected by a can also cause a reduction in relative peak height, even when not located exactly on the ligation site! In addition, some 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. We have no information on what percentage of defects in these genes is caused by deletions/duplications of complete exons. Finally, note that most defects in these genes are expected to be small (point) mutations, most of which will not be detected by this SALSA test. SALSA mixes 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 mixes and reagents includes a limited license to use these products for research purposes. The use of this SALSA mix 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). Related SALSA mixes P050 CAH: Genes included CYP21A2, CYP21A1P, C4A, C4B and TNXB. P334 Gonadal: Genes included DMRT1, CYP17A1, SRD5A2 and HSD17B3. More information Website : www.mlpa.com E-mail : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 1, 1057 DL Amsterdam, the Netherlands SALSA P185 Intersex mix Page 1 of 7
References Benko S et al., 2011. Disruption of a long distance regulatory region upstream of SOX9 in isolated disorders of sex development. J Med Genet. 48:825-30. Kim GJ et al., 2015. Copy number variation of two separate regulatory regions upstream of SOX9 causes isolated 46,XY or 46,XX disorder of sex development. J Med Genet. 52: 240-247. Kon M and Fukami M, 2015. Submicroscopic copy-number variations associated with 46,XY disorders of sex development. Molecular and Cellular Pediatrics 2:7. Data analysis The P185-C1 Intersex mix contains 45 MLPA s with amplification products between 130 and 494 nt. In addition, it contains 10 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 two Y-fragment at 105 nt and 118 nt. More information on how to interpret observations on these control fragments can be found in the MLPA protocol. The 105, 118, 214, 240 and 351 nt Y chromosome specific s will only generate a signal on male DNA samples. Data generated by this mix can be normalised intra-sample by dividing the peak height of each amplification product by the total peak height of only the reference s in the mix (block normalisation). Secondly, inter-sample normalisation can be achieved by dividing the intra-normalised ratio in a sample by the average intra-normalised ratio of all reference samples. Please note that this type of normalisation assumes that no changes occurred in the genomic regions targeted by the reference s. It is strongly recommended to use reference and patient samples of the same sex to minimize variation, as intersex comparison makes analysis more difficult. Sex determination can also be done by visual examination of the electropherogram. 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, 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://dgv.tcag.ca/dgv/app/home. 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 s are unlikely to be the cause of the condition tested for. Users should always verify the latest scientific literature when interpreting their findings. This mix was developed by A.O.H. Nygren at. In case the results obtained with this mix lead to a scientific publication, it would be very much appreciated if the mix designer could be included as a co-author. Info / remarks / suggestions for improvement: info@mlpa.com. SALSA P185 Intersex mix Page 2 of 7
Table 1. P185-C1 Intersex mix Chromosomal position reference SOX9 chr X/Y NR0B1 WNT4 NR5A1 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 118 * ZFY S0135-L16766 Yp11.31 130 Reference 00797-L00463 5q31 136 ± SOX9 07156-L06795 Exon 1 142 Reference 02956-L02388 7q31 148 NR0B1 06048-L05503 Exon 1 154 Reference 15163-L16938 3q27 165 ± SOX9 06051-L05506 Exon 1 171 ± NR5A1 16876-L20237 Exon 2 178 Reference 14329-L15998 6p22 187 ± WNT4 06054-L19826 Exon 1 200 SOX9 16990-L19999 upstream 206 * PHEX 07621-L08286 Xp22.11 214 * UTY 11812-L12607 Yq11.221 221 SOX9 09604-L12188 upstream 228 COL4A5 05874-L05274 Xq22.3 234 NR0B1 06049-L12569 Exon 1 240 UTY 01071-L00464 Yq11.221 247 Reference 02869-L02336 1p21 256 ± SOX9 06052-L05507 Exon 2 265 Reference 14385-L11820 13q22 274 WNT4 07159-L06798 Exon 4 283 ± NR5A1 16992-L20001 Exon 3 292 WNT4 06055-L05510 Exon 2 301 SOX9 11501-L09981 upstream 310 NR0B1 06050-L05505 Exon 2 319 WNT4 16991-L20000 Exon 5 328 * NR5A1 16993-SP0424-L20002 Exon 5 337 ± SOX9 06053-L05508 Exon 3 343 ± NR5A1 09673-L12239 Exon 1 351 SRY 01191-L19672 Yp11.31 364 Reference 04357-L03777 7q21 373 * SOX9 16994-SP0425-L20003 upstream 382 SOX9 09607-L09980 upstream 390 * NR5A1 16995-SP0426-L20004 Exon 6 400 WNT4 07158-L06797 Exon 3 408 ± NR5A1 09675-L10047 Exon 4 418 NR5A1 16996-L12240 Exon 7 427 GJB1 06188-L02097 Xq13.1 436 PQBP1 07886-L07700 Xp11.23 445 Reference 08793-L08817 10q21 454 * NR0B1 14797-L16506 NR0B1 region 463 * CXorf21 14798-L16507 NR0B1 region 474 * CXorf21 14799-L16508 NR0B1 region 481 * NR0B1 14800-L16509 NR0B1 region 494 Reference 06676-L15676 11p15 * New in version C1 (from lot 0611 onwards). Changed in version C1 (from lot 0611 onwards). Small change in length, no change in sequence detected. ± These s are located within, or close to, a very strong CpG island. A low signal of these s can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. SALSA P185 Intersex mix Page 3 of 7
Table 2. P185 s arranged according to chromosomal location Table 2a. NR0B1 and other chromosome X s Gene / exon next 228 05874-L05274 COL4A5 CTTGCTTCAACT-GCATTGGAACTG 37365 kb 427 06188-L02097 GJB1 ACAAGGTCCACA-TCTCAGGGACAC 21716 kb 436 07886-L07700 PQBP1 GCTCTCCCCACA-TGACCCCAACTC 18119 kb 474 14799-L16508 463 14798-L16507 CXorf21 CXorf21 19.4 kb before CXorf21; 522 kb after last DMD exon NM_025159.2; 132 nt after exon 1 reverse 481 14800-L16509 NR0B1 126.3 kb upstream NR0B1 454 14797-L16506 NR0B1 17.8 kb upstream NR0B1 148 06048-L05503 234 06049-L12569 310 06050-L05505 start codon NR0B1 Exon 1 NR0B1 Exon 1 NR0B1 Exon 2 stop codon 16-18 (ex1) 10 nt before exon 1, 26 nt before ATG 942-943 in NM_000475.4 1303-1304 in NM_000475.4 1426-1428 (ex2) TGCCATCACAAT-AATTAGTCGTCT TCTGATATGGTA-AGCTAGAAAAGC TTAGCTCAAATC-AAAGCTCCTAAC CCACATTTTCTG-TAGCTAATTTAT GCCACTGGGCAG-AACTGGGCTACG TTCGAGACTGTG-GAAGTCTCGGAG TCATCGAACTTA-ATAGTACCCTTT 19.7 kb 141.9 kb 108.6 kb 17.7 kb 1.0 kb 3.7 kb 8126 kb 206 07621-L08286 PHEX CTCGCAAGTATT-TAGCACAGTCTG Flanking. Included to facilitate the determination of the extent of a deletion/duplication. Copy number alterations of flanking and reference s are unlikely to be related to the condition tested. The NM_000475.4 sequence is a reference standard in the NCBI RefSeqGene project. Note: NR0B1 (DAX1) region duplication results in 46XY females. Table 2b. Chromosome Y s Gene next 351 01191-L19672 SRY 639-640 in NM_003140.1 CAACAGGTTGTA-CAGGGATGACTG 174.1 kb 118 S0135-L16766 ZFY TCATAGAGGAGG-ATGTTCAGTGCT 11087.3 kb 105 S0369-L12720 UTY TCGACAACCACA-TGGTGCTCTGAC 124.5 kb 240 01071-L00464 UTY CTTCGGTAGCTT-AAGTCTTTGCCT 124.6 kb 214 11812-L12607 UTY AGGATCCTGGAT-ATTCCACTACCA The NM_003140.1 sequence is a reference standard in the NCBI RefSeqGene project. Table 2c. WNT4 WNT4 exon NM_030761.4 next start codon 105-107 (ex1) 187 06054-L19826 exon 1 175-174 reverse CTTACAGCCAGT-TGCTCGCGGCGG 13.1 kb 292 06055-L05510 exon 2 243-244 GCGAGAAACTCA-AGGGCCTGATCC 8.3 kb 400 07158-L06797 exon 3 510-511 AGCTGGAGAAGT-GCGGCTGTGACA 0.2 kb 274 07159-L06798 exon 4 630-631 TGCGGGAGAGAA-GCAAGGGGGCCT 2.8 kb 319 16991-L20000 exon 5 2777-2778 GACATGCATGGA-TCAAGACCTTGC stop codon 1158-1160 (ex5) The NM_030761.4 sequence is a reference standard in the NCBI RefSeqGene project. SALSA P185 Intersex mix Page 4 of 7
Table 2d. NR5A1 NR5A1 exon NM_004959.4 next exon start codon 188-190 (ex1) 343 09673-L12239 Exon 1 170-171 GCTGCTTCCGCT-TCGTAAGTGAGG 3.9 kb 171 16876-L20237 Exon 2 192-191 reverse CGTACGAATAGT-CCATGCCCGCGG 0.2 kb 283 16992-L20001 Exon 3 323-324 AGAACAACAAGC-ACTACACGTGCA 3.1 kb 408 09675-L10047 Exon 4 1004-1005 CCGACCAGACCT-TCATCTCCATCG 7.0 kb 328 Ж 16993-SP0424-L20002 Exon 5 1081-1082; 1107- ATGACGCTGCTG-26nt spanning 1108 oligo -GTTCGACCACAT 2.0 kb 390 Ж 16995-SP0426-L20004 Exon 6 1319-1320; 21nt TCATCCTCTTCA-27nt spanning after exon 6 oligo -GAGAGGTGGAGA 8.2 kb 418 16996-L12240 Exon 7 1498-1499 AAGGAGTACCTG-TACCACAAGCAC stop codon 1571-1573 (ex7) Ж This consists of three parts and has two ligation sites. The NM_004959.4 sequence is a reference standard in the NCBI RefSeqGene project. Table 2e. SOX9 SOX9 exon NM_000346.3 next 221 09604-L12188 1500 kb before exon 1 CGTGTCAGAAAT-ATCCAGTGAGAA 0.4 kb 200 16990-L19999 1500 kb before exon1 CAGTCTAGGGCT-GCTGTTTTATTG 492.5 kb 382 09607-L09980 1007 kb before exon 1 AAGGGCCATCAT-TCTGGACAGTGC 523.8 kb 301 11501-L09981 483 kb before exon 1 CCTGCAGGACAA-ATCTGATCACTG 242.5 kb 373 Ж 16994-SP0425-L20003 241 kb before exon 1 TTCTGGCAGCTA-31nt spanning oligo -GGTCCTCCCTTA 240.8 kb start codon 373-375 (ex1) 165 06051-L05506 Exon 1 35-36 AACTGACTGGAA-ACTTCAGTGGCG 0.7 kb 136 07156-L06795 Exon 1 760-759 reverse GGCGTTGTGCAA-GTGCGGGTACTG 1.1 kb 256 06052-L05507 Exon 2 985-986 ACGCCATCTTCA-AGGCGCTGCAGG 2.5 kb 337 06053-L05508 Exon 3 2954-2955 AGGCAGATGCCT-GCTCGCTCTGTC stop codon 1900-1902 (ex3) Ж This consists of three parts and has two ligation sites. Flanking. Included to facilitate the determination of the extent of a deletion/duplication. Copy number alterations of flanking and reference s are unlikely to be related to the condition tested. The NM_000346.3 sequence is a reference standard in the NCBI RefSeqGene project. Note: Exon numbering used here may differ from literature! The identity of the genes detected by the reference s and complete sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA P185 Intersex mix Page 5 of 7
mix P185-C1 Intersex sample pictures 40000 177.79 170.54 35000 86.37 96.52 140.07 128.26 153.36 186.04 199.22 220.64 326.37 319.34 30000 25000 91.26 105.59 134.04 145.52 163.17 264.95 292.35 255.71 246.86 273.16 282.17 301.80 335.93 342.78 406.86 399.67 417.03 446.11 494.22 205.33 372.57 20000 115.06 233.36 350.82 362.35 390.01 382.12 15000 10000 100.77 213.68 226.98 239.15 309.84 426.97 437.11 463.38 454.69 474.26 482.68 D ye Signal 5000 0 50 100 150 200 250 300 350 400 450 500 Size Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with mix P185-C1 Intersex (lot C1-0611). 55000 145.45 50000 205.38 45000 40000 35000 30000 25000 20000 96.48 86.32 100.73 91.23 153.35 140.00 128.23 133.99 163.18 170.49 177.79 186.03 199.25 220.67 233.37 227.00 246.87 265.00 255.73 273.21 282.20 292.41 309.89 301.84 326.40 319.38 335.97 342.81 372.61 362.37 389.99 382.15 406.81 399.66 416.98 426.92 437.03 446.07 463.36 454.66 474.23 482.68 494.20 15000 10000 D ye Signal 5000 0 100 150 200 250 300 350 400 450 500 Size Figure 2. Capillary electrophoresis pattern of a sample of approximately 50 ng human female control DNA analysed with mix P185-C1 Intersex (lot C1-0611). SALSA P185 Intersex mix Page 6 of 7
Implemented Changes compared to the previous product description versions Version 08 07 May 2015 (54) - New references added on page 2. - Electropherogram pictures using the old MLPA buffer removed. - Various minor textual changes. Version 07 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 06 (48) - Product description adapted to a new product version (version number changed, lot number added, changes in Table 1 and Table 2, new picture included). - Various minor textual changes. - Remark on RefSeqGene standard added below Table 2. - Reference added on page 2. Version 05 (46) - Product description adapted to a new product version (version number changed, lot number added, small changes in Table 1 and Table 2, new picture included). - Various minor layout changes. - Warning added in table 1, 328 nt (09677-L12238) and 418 nt (09606-L09979). Version 04 (46) - Table 1 layout improved. - Warning added in Table 1 and 2, 265 nt 09536-L04181 and 172 nt 01974-L01507. - s of the s targeting the NR0B1 gene updated according to new version of the NM_reference sequence. - Small changes of lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Data analysis method has been modified. - Various minor textual changes on page 1. - Various minor layout changes. SALSA P185 Intersex mix Page 7 of 7