Elucigene Male Factor Infertility Products Guide to Interpretation

Transcription

1 Elucigene Male Factor Infertility Products Guide to Interpretation Manufactured by: Elucigene Diagnostics Citylabs Nelson Street Manchester M13 9NQ For Sales, Customer Service and Technical Support:- T: +44 (0) F: +44 (0) E: E: Elucigene Diagnostics is the trading name of Delta Diagnostics (UK) Limited., a company registered in England and Wales, registration number ANXYAZFGIEN 001 Jun-2015 Page 1 of 28

2 Elucigene Male Factor Infertility Products Guide to Interpretation of Results It is recommended that each laboratory develops its own interpretation and reporting procedures and criteria. Best practice guidelines for QF-PCR have been documented by the UK s Association for Clinical Genetic Science and are available for reference at: The European Andrology Academy (EAA) and European Molecular Genetics Quality Network (EMQN) guidelines for Y chromosome microdeletion testing are discussed in detail in Krausz et al., 2014 (1). Further information about the EAA and EMQN can be found at DISCLAIMER Elucigene Diagnostics does not represent this guide as a comprehensive summary of all possible outcomes using the Male Factor Infertility products and it is intended to be used solely as an aide memoire. It must not be used in any clinical interpretation of the results of the assay. Laboratories must interpret the results of the assay in accordance with their own locally developed procedures. The UK s ACGS QF-PCR Best Practice Guidelines makes recommendations regarding the interpretation of results obtained. The EAA and EMQN guidelines offer guidance on interpretation of Y chromosome microdeletion results. ANXYAZFGIEN 001 Jun-2015 Page 2 of 28

3 General Guidelines for Male Factor Infertility Products 1. The negative control should show no sharp peaks within the read range of 100 to 550bp. 2. The positive control must show the expected results and all peaks must meet the criteria below. 3. The acceptable range for marker peaks is between 50 and 6000 relative fluorescent units (rfu s). Peak heights falling outside this range must not be analysed. 4. Electropherograms of poor quality due to excessive bleed-through between dye colours (also known as pull-up ) or electrophoretic spikes (sharp peaks present in more than one dye) should not be interpreted. The PCR products should be reinjected and re-analysed. 5. Aneuploidy analysis is performed by assessment of peak ratios (A1/A2), where A1 is the peak area of the shorter length fragment and A2 is the peak area of the longer length fragment. The resulting ratio is indicative of locus copy number. ANXYAZFGIEN 001 Jun-2015 Page 3 of 28

4 Interpretation A complete analysis of chromosome copy number status is performed by comparison of peak area ratios. Sex chromosome aneuploidy analysis 1. For STR markers, heterozygous di-allelic (i.e. two alleles) markers should fall within a ratio window of 0.8 to 1.4. However, for two alleles separated by more than 24bp in size a ratio of up to 1.5 is acceptable. Any values falling within this region are referred to as having a ratio of 1:1. If the ratio balance falls out of this window then it may be due to a number of factors, including but not limited to:- Whole chromosome trisomy Partial chromosome trisomy (including sub-microscopic duplications) Stutters causing skewing Preferential amplification of one allele causing skewing Primer site polymorphisms Somatic microsatellite mutations Homozygous markers are uninformative since a ratio cannot be determined. Trisomy is determined by either: Two peaks of uneven height due to one of the peaks representing two alleles which are common to one or both parents. In this case the ratio between the two peaks will be classed as 2:1 or 1:2 such that A1/A2 will give a result in the region of 1.8 to 2.4 when the peak representing the shorter length allele is greater in area than the peak representing the longer length allele, or where A1/A2 will give a result in the region of 0.45 to 0.65 when the peak representing the shorter length allele is smaller in area than the peak representing the longer length allele Three peaks of comparable height present. The ratio of the peaks will be classed as 1:1:1 and their values fall within the normal range of (although for alleles separated by more than 24bp an allele ratio of up to 1.5 is acceptable). If this does not occur then it may be due to one of the factors mentioned in paragraph Peak area ratios that fall between the normal and abnormal ranges are classed as inconclusive 3. The AMEL marker amplifies non-polymorphic sequences on the X (104bp) and Y (110bp) chromosomes and can be used to determine the presence or absence of a Y chromosome and represents the relative amount of X to Y sequence. Note: amplification failure due to mutation of the AMEL-Y sequence has been reported. 4. TAF9L is an invariant paralogous marker with sequences on chromosomes 3 and X. The chromosome 3 specific peak (116bp, representing 2 copies of chromosome 3) can therefore be used as a reference peak to assist in the determination of the number of X chromosomes present (121bp peak). Analysed in combination with Amelogenin and the other sex chromosomes markers, it is particularly useful in the detection of sex chromosome aneuploidy. In a normal female the markers should fall within a ratio window of 0.8 to 1.4. In a normal male the markers will give a ratio 1.8. Examples of results can be found on pages The DXYS218 polymorphic STR marker is present on both the X and Y chromosomes and represents the total number of sex chromosomes. For informative male results it is not possible to determine which allele represents the X or Y chromosome. 6. The informative X-specific marker XHPRT represents the number of X chromosomes. 7. The Y-specific marker, SRY, will give a single peak in males and will not amplify in normal females. ANXYAZFGIEN 001 Jun-2015 Page 4 of 28

5 Y chromosome microdeletion analysis Elucigene Male Factor Infertility The Y chromosome microdeletion primers in the Elucigene Male Factor Infertility assay recognize wild type sequence in the AZFa (sy84 and sy86), AZFb (sy127 and sy134) and AZFc (sy254 and sy255) microdeletion regions of chromosome Y (Figure 1). The use of these markers is in accordance with the Best Practice guidelines laid down by the European Academy of Andrology (EAA) and the European Molecular Genetics Quality Network (EMQN) The presence of a peak at these loci denotes the presence of wild type sequence while the loss of a peak is indicative of a microdeletion affecting that particular region (Figure 1 and Table 1). 1. The sy84 and sy86 markers will each give single peaks in normal males. Loss of both peaks is indicative of AZFa region microdeletion*. 2. The sy127 and sy134 markers will each give single peaks in normal males. Loss of both peaks is indicative of AZFb region microdeletion. 3. The sy254 and sy255 markers will each give single peaks in normal males. Loss of both peaks is indicative of AZFc region microdeletion*. 4. The ZFX/ZFY marker represents sequences on both the X and Y chromosomes. As the amplicon generated from both sequences cannot be distinguished by size, this marker will give a single peak regardless of sample gender. This marker serves only as an amplification control. Note- Microdeletions in the AZFa region usually leads to the loss of both sy84 and sy86 markers however on rare occasions loss of the sy84 marker alone has been described. This is often due to the presence of the rs SNP which affects sy84 primer binding. Therefore sequencing of the sy84 primer binding sites should be carried out to eliminate the possibility of a false positive in these incidences (See Figure 6 for an example). Primers for the detection of this polymorphism can be found in Wu et al.,2011 (3). Note- Microdeletion of the AZFc invariably leads to the loss of both sy254 and sy255 markers. Loss of only one of these markers should be treated as a false positive. Examples of the detection of loss of each of the AZF microdeletion regions with the Elucigene Male Factor Infertility kit are shown in the Examples section (pages 9-25). ANXYAZFGIEN 001 Jun-2015 Page 5 of 28

6 Figure 1: Representation of chromosome Y showing the relative positions of the AZF microdeletion regions and sequence-tagged site (STS) markers used by the Male Factor Infertility assay (Krausz et al., 2014). Repetitive sequences thought to underlie the origin of microdeletions affecting the AZFb and AZFc regions are shown by coloured arrows. Table 1: Predicted STS marker pattern for AZF region microdeletions analysed using the Elucigene Male Factor Infertility assay. ANXYAZFGIEN 001 Jun-2015 Page 6 of 28

7 Extension Analysis using Elucigene MFI-Yplus In the event of detection of a microdeletion it is strongly recommended that extension analysis of flanking markers be undertaken to better map the breakpoint as outlined in the EAA and EMQN guidelines (1, 2), this can be carried out using the Elucigene MFI-Yplus assay. Such additional screening is deemed appropriate as partial microdeletions affecting the AZF regions typically have a better prognosis than the classical full microdeletions and as such can effect treatment. The presence of a peak at these loci denotes the presence of wild type sequence while the loss of a peak is indicative of a microdeletion affecting that particular region (Figure 2 and Examples section). 1. The sy82 (flanking), sy83, sy1182 and sy88 (flanking) markers each give single peaks in normal males. Loss of sy83 and sy1182 is observed in the event of a classic complete deletion of the AZFa region. Loss of one of these markers indicates a partial deletion of the AZFa region. 2. The sy105 (flanking), sy121, sy143 and sy153 (flanking) markers each give single peaks in normal males. Loss of sy121 and sy143 is observed in the event of a classic complete deletion of the AZFb region. Loss of one of these markers indicates a partial deletion of the AZFb region. Note- samples exhibiting complete deletion of the AZFb region may also show loss of the sy1191 and sy1291 markers. This is due to overlap of the AZFb and AZFc microdeletion regions and does not indicate an AZFc microdeletion. 3. The sy1191 and sy1291 markers each give single peaks in normal males. Loss of sy1291 is observed in the event of a gr/gr deletion, a small microdeletion affecting the AZFc region. Note- Loss of markers sy1191 and sy1291 may also be accompanied by loss or a reduction in peak amplitude of the AZFb region flanking marker sy153. This is due to overlap of the AZFb and AZFc microdeletion regions as well as the fact that sy153 is a multicopy marker. This result does not indicate an AZFb microdeletion. 4. The sy160 marker will give a single peak in a normal male. Loss of this marker indicates a terminal deletion of Yq. This in combination with an AZFc deletion may result in a more severe phenotype. Examples of the extension analysis of each of the AZF microdeletion regions with the Elucigene MFI-Yplus kit are shown in the Examples section (pages 9-25). ANXYAZFGIEN 001 Jun-2015 Page 7 of 28

8 Figure 2: Predicted STS marker pattern for selected AZF microdeletions undergoing extension analysis using the Elucigene MFI-Yplus assay (Adapted from Krausz et al.,2014). *= sy153 is a multicopy STS marker, the relative position with relation to the other markers is for the first copy, additional copies are located distally. ANXYAZFGIEN 001 Jun-2015 Page 8 of 28

9 Examples 1. AZFa Region Microdeletion Loss of peaks corresponding to sy84 and sy86 demonstrate a microdeletion affecting the AZFa region of the Y chromosome (see red arrows). All other Y chromosome microdeletion peaks (sy127, sy134, sy254 and sy255) are present suggesting that the Y microdeletion is confined to the AZFa region alone. The ZFX/ZFY peak confirms that PCR amplification conditions were correct (Figure 3a). Figure 3a. AZFa deletion (Male Factor Infertility) ANXYAZFGIEN 001 Jun-2015 Page 9 of 28

10 MFI-Yplus analysis of this sample shows loss of the peaks corresponding to sy1182 and sy83 again demonstrating a microdeletion affecting the AZFa region (see red arrows). All other peaks are present including the AZFa region flanking markers sy82 and sy88 suggesting that this is a classical full AZFa region microdeletion (Figure 3b). Figure 3b. AZFa deletion (MFI-Yplus) ANXYAZFGIEN 001 Jun-2015 Page 10 of 28

11 2. AZFb Region Microdeletion Loss of peaks corresponding to sy127 and sy134 demonstrate a microdeletion affecting the AZFb region of the Y chromosome (see red arrows). All other Y chromosome microdeletion peaks (sy84, sy86, sy254 and sy255) are present suggesting that the microdeletion is confined to the AZFb region alone. The ZFX/ZFY peak confirms that PCR amplification conditions were correct (Figure 4a). Figure 4a. AZFb deletion (Male Factor Infertility) ANXYAZFGIEN 001 Jun-2015 Page 11 of 28

12 MFI-Yplus analysis of this sample shows loss of the peaks corresponding to sy121 and sy143 again demonstrating a microdeletion affecting the AZFb region (see red arrows). Peaks corresponding to the AZFb flanking regions sy105 and sy153 are present indicating that this is a classical full AZFb region microdeletion (Figure 4b). Loss of the sy1191 and sy1291 markers are also observed, this is due to the overlapping nature of the AZFb and AZFc microdeletion regions and is not indicative of an AZFc microdeletion. All other peaks are present. Figure 4b. AZFb deletion (MFI-Yplus) ANXYAZFGIEN 001 Jun-2015 Page 12 of 28

13 3. AZFc Region Microdeletion Loss of peaks corresponding to sy254 and sy255 (see red arrows) demonstrate a microdeletion affecting the AZFc region of chromosome Y. All other Y chromosome microdeletion peaks (sy84, sy86, sy127 and sy134) are present suggesting that the microdeletion is confined to the AZFc region alone. The ZFX/ZFY peak confirms that PCR amplification conditions were correct (Figure 5a). Figure 5a. AZFc deletion (Male Factor Infertility) ANXYAZFGIEN 001 Jun-2015 Page 13 of 28

14 MFI-Yplus analysis of this sample shows loss of the peaks corresponding to sy1191 and sy1291 again demonstrating a microdeletion affecting the AZFc region (see red arrows). The loss of the sy1191 peak suggests that this is a classic full AZFc microdeletion rather than a gr/gr subdeletion (Figure 5b). Loss of the AZFb flanking marker sy153 is also observed, this is due to the overlapping nature of the AZFb and AZFc microdeletion regions and is not indicative of an AZFb microdeletion. Figure 5b. AZFc deletion (MFI-Yplus) ANXYAZFGIEN 001 Jun-2015 Page 14 of 28

15 4. Primer Binding Site Polymorphism (PSP) Partial or complete allelic drop-out as a result of a primer binding site polymorphism is seen in the example below. This sample carries an AZFc microdeletion with associated loss of peaks for sy254 and sy255 (see black arrows). The sample also exhibits loss of the AZFa marker sy84 (see red arrow Figure 6). Sequence analysis confirmed the presence of the rs SNP in the sy84 primer binding region, this polymorphism has been shown to interfere with SY84 primer binding, potentially leading to a false positive (3). Note: Primer binding site polymorphism can often be distinguished by repeating the assay at a significantly reduced annealing temperature (e.g. 4 degrees lower). This allows more permissive annealing of the primer and may result in the restoration of the expected profile/peak size. If this is the case, it is recommended that the marker is not used to assess microdeletion status for that patient as amplification may be incomplete and an alternative should be used (1,2,3). Figure 6. Primer binding site polymorphism affecting sy84 in an AZFc deletion sample (Male Factor Infertility) ANXYAZFGIEN 001 Jun-2015 Page 15 of 28

16 5. Small insertions/deletions Small insertions and/or deletions can lead to aberrant amplicon migration and shifting of the resulting peak out of its assigned bin. An example is shown below in which a small deletion (approximately 19bp) affecting the SY86 marker amplicon (Black arrow shows the expected position) leads to a shift in the resulting peak into the neighbouring SY134 bin (see red arrow Figure 7). Care should be taken when analyzing data in which loss of a peak is accompanied by the appearance of an aberrant peak elsewhere on the trace. The clinical significance of these small insertions/deletions are as yet unknown. Figure 7. Small deletion affecting migration and sizing of the sy86 amplicon (Male Factor Infertility) ANXYAZFGIEN 001 Jun-2015 Page 16 of 28

17 Sex Chromosome Anueploidy 6. Klinefelter Syndrome The most common form of Klinefelter syndrome is caused by the presence of an extra X chromosome in a male resulting in a 47,XXY karyotype (see Figure 8). This is suggested by the 2:1 ratio of the AMEL X and Y specific peaks, the 1:1 ratio of the TAF9L chromosome 3 and X specific peaks, the presence of an SRY peak, and the presence of two X specific HPRT peaks (See Figure 13 for a more detailed explanation). Figure 8. Example of classic 47,XXY Klinefelter profile (Male Factor Infertility) ANXYAZFGIEN 001 Jun-2015 Page 17 of 28

18 Examples Figure 9. Shoulder peak at sy134 (Male Factor Infertility) A defined shoulder peak is often observed at the sy134 marker during Male Factor Infertility analysis. The presence of this peak is not clinically relevant and should not interfere with interpretation of the data (Figure 9). AN000GIEN 001 Jul-2015 Page 18 of 28

19 Figure 10. Appearance of the sy1291 peak (MFI-Yplus) The sy1291 peak is noticeably wider than the other peaks in the MFI-Yplus panel, furthermore splitting of the peak at the top may be observed, this will be particularly noticeable when higher DNA input amounts are used. These features are likely the consequence of enzyme slippage due to a poly A tract located in the sy1291 PCR amplicon and do not interfere with interpretation of the MFI-Yplus assay (Figure 10). AN000GIEN 001 Jul-2015 Page 19 of 28

20 Interpretation of the TAF9L marker in conjunction with Amelogenin and SRY The TAF9L marker has been included as a quantitative marker to assist in determining the number of X chromosomes present. TAF9L is a paralogous marker with sequences on both chromosome 3 and chromosome X. The peak resulting from amplification of the sequence on chromosome 3 is expected to represent a normal diploid chromosome complement except in the case of triploidy (or other rare aneuploidy). Using this peak as a reference, it is possible to compare it with that amplified from the sequence on the X chromosome and thereby determine the number of X chromosomes present. Note: All sex chromosome marker information should be assessed together when drawing conclusions over the copy numbers of the sex chromosome markers. AN000GIEN 001 Jul-2015 Page 20 of 28

21 Figure 11. Normal XY male (Male Factor Infertility) Amelogenin: 1:1 ratio consistent with equal number of X and Y Presence of SRY consistent with presence of Y chromosome. SRY is similar in height to AM X/AM Y and TAF X peaks TAF9L: 2:1 ratio consistent with 2 copies of chromosome 3 and 1 copy of X chromosome Y chromosome microdeletion markers present consistent with presence of Y chromosome ZFX/ZFY amplification control marker present consistent with correct PCR conditions Conclusion: Result is consistent with XY sex chromosome complement. AN000GIEN 001 Jul-2015 Page 21 of 28

22 Figure 12. Normal XX female (Male Factor Infertility) Amelogenin: 1 peak only, similar in size to TAF9L chromosome 3 peak consistent with 2 copies of chromosome X and a lack of chromosome Y. No SRY peak consistent with a lack of a Y chromosome. TAF9L: 1:1 ratio consistent with 2 copies of chromosome 3 and 2 copies of X chromosome Y chromosome microdeletion markers absent consistent with no Y chromosome present ZFX/ZFY amplification control marker present consistent with correct PCR conditions Conclusion: Result is consistent with XX sex chromosome complement AN000GIEN 001 Jul-2015 Page 22 of 28

23 Figure 13. XXY Male (Male Factor Infertility) Amelogenin: 2:1 ratio - consistent with presence of 2 copies of X chromosome and 1 copy of Y chromosome Presence of SRY consistent with presence of Y chromosome. SRY is similar in height to AM Y peak TAF9L: 1:1 ratio consistent with 2 copies of chromosome 3 and 2 copies of X chromosome Y chromosome microdeletion markers present consistent with presence of Y chromosome ZFX/ZFY amplification control marker present consistent with correct PCR conditions Conclusion: Result is consistent with XXY sex chromosome complement AN000GIEN 001 Jul-2015 Page 23 of 28

24 Interpretation of Y chromosome microdeletion extension marker analysis by MFI-Yplus Figure 14. Normal XY male (MFI-Yplus) This sample has previously tested as wild type using the Elucigene Male Factor Infertility assay All diagnostic peaks are present confirming that none of the tested for microdeletions are present in this sample. Note: Samples testing as wild type using the Elucigene Male Factor Infertility assay would not normally be subjected to further testing with the MFI-Yplus assay. AN000GIEN 001 Jul-2015 Page 24 of 28

25 Figure 15. Normal XX female (MFI-Yplus) This sample has previously tested as wild type female using the Elucigene Male Factor Infertility assay Female samples would not normally be tested using either Elucigene Male Factor Infertility or MFI-Yplus No diagnostic peaks are present confirming that no Y chromosome is present in this sample. Note: Female samples would not normally be subjected to further testing with the MFI-Yplus assay. AN000GIEN 001 Jul-2015 Page 25 of 28

26 Glossary of Terms ANEUPLOIDY Definition: any deviation from the standard chromosome complement. Effect: A deviation from the expected 1:1 ratio at informative loci. AZFa Definition: Azoospermia factor a region (on chromosome Y) Effect: Male Factor Infertility: Microdeletion of this region leads to a loss of peaks at markers sy84 and sy86 MFI-Yplus: Classical full AZFa microdeletions result in the loss of peaks at markers sy83 and sy1182 Note: AZFa deletions typically result in loss of both sy84 and sy86. Loss of the sy84 marker alone has been reported and is often associated with the presence of the rs SNP (3). In cases when only one marker is deleted it is highly recommended that sequencing of the primer binding region should be carried out to rule out the presence of polymorphisms. AZFb Definition: Azoospermia factor b region (on chromosome Y) Effect: Male Factor Infertility: Microdeletion of this region leads to a loss of peaks at markers sy127 and/or sy134 MFI-Yplus: Classical full AZFb microdeletions result in the loss of peaks at markers sy121 and sy143 AZFc Definition: Azoospermia factor c region (on chromosome Y) Effect: Male Factor Infertility: Microdeletion of this region leads to a loss of peaks at markers sy254 and sy255 MFI-Yplus: Relevant markers are sy1191 and sy1291 Note: AZFc deletions always lead to the loss of both sy254 and sy255. Loss of only one marker should be regarded as a false positive GR/GR MICRODELETION Definition: green/red microdeletion, named after the probes used to first describe this microdeletion, Effect: Partial AZFc microdeletion (1.6Mb), associated with variable clinical phenotype including subfertility. Effect appears to be dependent on genetic background. Characterized by loss of the sy1291 marker on the MFI-Yplus assay. AN000GIEN 001 Jul-2015 Page 26 of 28

27 PRIMER BINDING SITE POLYMORPHISM Definition: a polymorphism on the template DNA strand where a PCR primer anneals. Effect: Allelic drop-out. In the case of complete drop-out, the profile shows apparent homozygosity for an individual marker. In the case of a trisomic chromosome, the profile may show apparent disomy. Partial drop-out is evident as an additional peak at a reduced height which can result in skewed, inconclusive or apparent 1:2/2:1 allele ratios. Note: Primer site polymorphisms can be distinguished by repeating the assay at a significantly reduced annealing temperature (e.g. 4 degrees lower). This allows more permissive annealing of the primer and may result in the restoration of the expected profile/peak size. If this is the case, it is recommended that the marker is not used to assess chromosome status as amplification may be incomplete. TRISOMY Definition: Three copies of an individual chromosome. Effect: All informative markers for an individual chromosome demonstrate a deviation from the expected normal 1:1 ratio resulting in a 2:1/1:2 ratio for diallelic markers or a 1:1:1 ratio where markers demonstrate three alleles. Y CHROMOSOME MICRODELETION Definition: Microdeletions affecting the AZF(a-c) regions of chromosome Y. These microdeletions are associated with male infertility. Effect: Microdeletions affecting one or more of these regions lead to the loss of peaks at the sy84, sy86 (AZFa), sy127, sy134 (AZFb), sy254 and sy255 (AZFc) loci (Elucigene Male Factor Infertility) AN000GIEN 001 Jul-2015 Page 27 of 28

28 References: 1. Krausz C, Hoefsloot L, Simoni M, Tuttelmann F, European Academy of Andrology, European Molecular Genetics Quality Network. EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions: state-of-the art Andrology Jan:2(1): Simoni M, Bakker E, Krausz C. EAA/EMQN best practice guidelines for molecular diagnosis of y-chromosomal microdeletions. State of the art International Journal of Andrology : Wu Q, Chen G, Yan T, Wang H, Liu Y, Li Z, Duan S, Sun F, Feng Y and Shi H. Prevalent false positives of azoospermia factor a (AZFa) microdeletions caused by single-nucleotide polymorphism rs in s Y84 screening of male infertility. Asian Journal of Andrology : ELUCIGENE is a trademark of Delta Diagnostics (UK) Ltd. Copyright 2015 Delta Diagnostics (UK) Ltd. AN000GIEN 001 Jul-2015 Page 28 of 28