Skip to main content
SpringerLink
Log in

Genotyping single-sperm cells by universal MARSALA enables the acquisition of linkage information for combined pre-implantation genetic diagnosis and genome screening

  • Technological Innovations
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

This paper aims to investigate the feasibility of performing pre-implantation genetic diagnosis (PGD) and pre-implantation genetic screening (PGS) simultaneously by a universal strategy without the requirement of genotyping relevant affected family members or lengthy preliminary work on linkage analysis.

Methods

By utilizing a universal Mutated Allele Revealed by Sequencing with Aneuploidy and Linkage Analyses (MARSALA) strategy based on low depth whole genome sequencing (~3x), not involving specific primers' design nor the enrichment of SNP markers for haplotype construction. Single-sperm cells and trephectoderm cells from in vitro fertilized embryos from a couple carrying HBB mutations were genotyped. Haplotypes of paternal alleles were constructed and investigated in embryos, and the chromosome copy number profiles were simultaneously analyzed.

Results

The universal MARSALA strategy allows the selection of a euploid embryo free of disease mutations for in uterus transfer and successful pregnancy. A follow-up amniocentesis was performed at 17 weeks of gestation to confirm the PGD/PGS results.

Conclusion

We present the first successful PGD procedure based on genotyping multiple single-sperm cells to obtain SNP linkage information. Our improved PGD/PGS procedure does not require genotyping the proband or relevant family members and therefore can be applicable to a wider population of patients when conducting PGD for monogenic disorders.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Genes and human disease: Human Genetic Programmes. 2015. http://www.who.int/genomics/public/geneticdiseases/en/index2.html

  2. Peltonen L, Perola M, Naukkarinen J, Palotie A. Lessons from studying monogenic disease for common disease. Hum Mol Genet. 2006;15(suppl 1):R67–74.

    Article  PubMed  CAS  Google Scholar 

  3. Handyside AH, Pattinson JK, Penketh RJ, Delhanty JD, Winston RM, Tuddenham EG. Biopsy of human preimplantation embryos and sexing by DNA amplification. Lancet. 1989;333(8634):347–9.

    Article  Google Scholar 

  4. Hellani A, Coskun S, Benkhalifa M, Tbakhi A, Sakati N, Al-Odaib A, et al. Multiple displacement amplification on single cell and possible PGD applications. Mol Hum Reprod. 2004;10(11):847–52.

    Article  PubMed  CAS  Google Scholar 

  5. Wells D, Sherlock JK. Strategies for preimplantation genetic diagnosis of single gene disorders by DNA amplification. Prenat Diagn. 1998;18(13):1389–401.

    Article  PubMed  CAS  Google Scholar 

  6. Natesan SA, Bladon AJ, Coskun S, Qubbaj W, Prates R, Munne S, et al. Genome-wide karyomapping accurately identifies the inheritance of single-gene defects in human preimplantation embryos in vitro. Genet Med. 2014;16(11):838–45.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Thornhill AR, Handyside AH, Ottolini C, Natesan SA, Taylor J, Sage K, et al. Karyomapping—a comprehensive means of simultaneous monogenic and cytogenetic PGD: comparison with standard approaches in real time for Marfan syndrome. J Assist Reprod Genet. 2015;32(3):347–56.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Natesan SA, Handyside AH, Ottolini C, Natesan SA, Taylor J, Sage K, et al. Live birth after PGD with confirmation by a comprehensive approach (karyomapping) for simultaneous detection of monogenic and chromosomal disorders. Reprod BioMed Online. 2014;29(5):600–5.

    Article  PubMed  Google Scholar 

  9. Wilton L, Thornhill A, Traeger-Synodinos J, Sermon KD, Harper JC. The causes of misdiagnosis and adverse outcomes in PGD. Hum Reprod. 2009;24(5):1221–8.

    Article  PubMed  CAS  Google Scholar 

  10. Wang, L, Cram DS, Shen J, Wang X, Zhang J, Song Z, Xu G, Li N, Fan J, Wang S, Luo Y, Wang J, Yu L, Liu J, Yao Y, Validation of copy number variation sequencing for detecting chromosome imbalances in human preimplantation embryos. Biology of reproduction, 2014: p. biolreprod. 114.120576.

  11. Korenberg J, Chen XN, Schipper R, Sun Z, Gonsky R, Gerwehr S, et al. Down syndrome phenotypes: the consequences of chromosomal imbalance. Proc Natl Acad Sci. 1994;91(11):4997–5001.

    Article  PubMed  CAS  Google Scholar 

  12. Treff NR, Northrop LE, Kasabwala K, Su J, Levy B, Scott RT Jr. Single nucleotide polymorphism microarray–based concurrent screening of 24-chromosome aneuploidy and unbalanced translocations in preimplantation human embryos. Fertil and Steril. 2011;95(5):1606–12. e2.

    Article  CAS  Google Scholar 

  13. Yin X, Tan K, Vajta G, Jiang H, Tan Y, Zhang C, et al. Massively parallel sequencing for chromosomal abnormality testing in trophectoderm cells of human blastocysts. Biol Reprod. 2013;88(3):69.

    Article  PubMed  CAS  Google Scholar 

  14. Yang Z, Tan K, Vajta G, Jiang H, Tan Y, Zhang C, et al. Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study. Mol Cytogenet. 2012;5(1):1–8.

    Article  CAS  Google Scholar 

  15. Schoolcraft W, Surrey E, Minjarez D, Gustofson RL, Scott RT Jr, Katz-Jaffe MG. Comprehensive chromosome screening (CCS) with vitrification results in improved clinical outcome in women > 35 years: a randomized control trial. Fertil Steril. 2012;98(3):S1.

    Article  Google Scholar 

  16. Forman EJ, Hong KH, Ferry KM, Tao X, Taylor D, Levy B, et al. In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial. Fert Steril. 2013;100(1):100–7. e1.

    Article  Google Scholar 

  17. Scott RT Jr, Upham KM, Forman EJ, Hong KH, Scott KL, Taylor D, et al. Blastocyst biopsy with comprehensive chromosome screening and fresh embryo transfer significantly increases in vitro fertilization implantation and delivery rates: a randomized controlled trial. Fertil Steril. 2013;100(3):697–703.

    Article  PubMed  Google Scholar 

  18. Yan L, Huang L, Xu L, Huang J, Ma F, Zhu X, et al. Live births after simultaneous avoidance of monogenic diseases and chromosome abnormality by next-generation sequencing with linkage analyses. Proc Natl Acad Sci U S A. 2015;112(52):15964–9.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Ren Y, Zhi X, Zhu X, Huang J, Lian Y, Li R, et al. Clinical applications of MARSALA for preimplantation genetic diagnosis of spinal muscular atrophy. J Genet Genomics. 2016;43(9):541–7.

    Article  PubMed  Google Scholar 

  20. Jiao Z, Zhou C, Li J, Shu Y, Liang X, Zhang M, et al. Birth of healthy children after preimplantation diagnosis of β-thalassemia by whole-genome amplification. Prenat Diagn. 2003;23(8):646–51.

    Article  PubMed  CAS  Google Scholar 

  21. Zong C, Lu S, Chapman AR, Xie XS. Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Science. 2012;338(6114):1622–6.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Zhang L, Cui X, Schmitt K, Hubert R, Navidi W, Arnheim N. Whole genome amplification from a single cell: implications for genetic analysis. Proc Natl Acad Sci. 1992;89(13):5847–51.

    Article  PubMed  CAS  Google Scholar 

  23. Lu S, Zong C, Fan W, Yang M, Li J, Chapman AR, et al. Probing meiotic recombination and aneuploidy of single sperm cells by whole-genome sequencing. Science. 2012;338(6114):1627–30.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Zhou X, Ren L, Meng Q, Li Y, Yu Y, Yu J. The next-generation sequencing technology and application. Protein Cell. 2010;1(6):520–36.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Wang J, Fan HC, Behr B, Quake SR. Genome-wide single-cell analysis of recombination activity and de novo mutation rates in human sperm. Cell. 2012;150(2):402–12.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Navin N, Kendall J, Troge J, Andrews P, Rodgers L, McIndoo J, et al. Tumour evolution inferred by single-cell sequencing. Nature. 2011;472(7341):90–4.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Hou Y, Song L, Zhu P, Zhang B, Tao Y, Xu X, et al. Single-cell exome sequencing and monoclonal evolution of a JAK2-negative myeloproliferative neoplasm. Cell. 2012;148(5):873–85.

    Article  PubMed  CAS  Google Scholar 

  28. Xu X, Zhou YQ, Luo GX, Liao C, Zhou M, Chen PY, et al. The prevalence and spectrum of α and β thalassaemia in Guangdong Province: implications for the future health burden and population screening. J Clin Pathol. 2004;57(5):517–22.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Huang L, Ma F, Chapman A, Lu S, Xie XS. Single-cell whole-genome amplification and sequencing: methodology and applications. Annu Rev Genomics Hum Genet. 2015;16:79–102.

    Article  PubMed  CAS  Google Scholar 

  30. Zamani Esteki M, Dimitriadou E, Mateiu L, Melotte C, Van der Aa N, Kumar P, et al. Concurrent whole-genome haplotyping and copy-number profiling of single cells. Am J Hum Genet. 2015;96(6):894–912.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Sermon K. Novel technologies emerging for preimplantation genetic diagnosis and preimplantation genetic testing for aneuploidy, expert review of molecular diagnostics. Expert Rev Mol Diagn. 2017;17(1):71–82.

    Article  PubMed  CAS  Google Scholar 

  32. Shang W, Zhang Y, Shu M, Wang W, Li R, Chen F, et al. Comprehensive chromosomal and mitochondrial copy number profiling in human IVF embryos. Reprod BioMed Online. 2017;36:67–74. https://doi.org/10.1016/j.rbmo.2017.10.110.

    Article  PubMed  CAS  Google Scholar 

Download references

Funding

The work was supported by grants from the National Natural Science Foundation of China (81370765), Guangdong Provincial Key Laboratory of Reproductive Medicine (2012A06140003), Guangzhou Science and Technology Foundation (201300000097), National Key Technologies Research and Development program (2016YFC0900100 to L.H.), Beijing Municipal Science & Technology Commission Grants (D1511000024150002 to X.S.X.), the Pilot Construction of Reproductive Clinical Research and Transformation Center of Guangzhou (155700011/201508020006), and the funding from Beijing Advanced Innovation Center for Genomics at Peking University.

Author information

Author notes

  1. Haitao Wu, Xiaoting Shen and Lei Huang contributed equally to this work.

Authors and Affiliations

  1. Reproductive Medicine Center, Jiangmen Central Hospital, Affiliated Jiangmen Hospital of Sun Yat-Sen University, Jiangmen, Guangdong, 529030, China

    Haitao Wu

  2. Reproductive Medicine Center, First Affiliated Hospital of Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong, 510080, China

    Haitao Wu, Xiaoting Shen, Yanhong Zeng, Baomin Lu, Yiping Zhong, Benyu Miao, Yanwen Xu, Yali Wang, Yubin Li & Canquan Zhou

  3. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, 01238, USA

    Lei Huang & X. Sunney Xie

  4. Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women’s Hospital, Boston, MA, 02115, USA

    Lei Huang

  5. Yikon Genomics Co., Ltd., 1698 Wangyuan Road, Building #26, Fengxian District, Shanghai, 201400, China

    Yumei Gao, Lin Shao & Sijia Lu

  6. Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing, 100871, China

    Luoxing Xiong & X. Sunney Xie

  7. Beijing Advanced Innovation Center for Genomics, Peking University, Beijing, 100871, China

    Luoxing Xiong & X. Sunney Xie

  8. Peking-Tsinghua Center for Life Sciences (CLS), Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, 100871, China

    Luoxing Xiong

Authors
  1. Haitao Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaoting Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lei Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yanhong Zeng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yumei Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lin Shao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Baomin Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yiping Zhong
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Benyu Miao
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yanwen Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Yali Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Yubin Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Luoxing Xiong
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Sijia Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. X. Sunney Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Canquan Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Canquan Zhou.

Ethics declarations

Conflict of interest

X.S.X. and S.L. are cofounders of Yikon Genomics Company, Ltd.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wu, H., Shen, X., Huang, L. et al. Genotyping single-sperm cells by universal MARSALA enables the acquisition of linkage information for combined pre-implantation genetic diagnosis and genome screening. J Assist Reprod Genet 35, 1071–1078 (2018). https://doi.org/10.1007/s10815-018-1158-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10815-018-1158-9

Keywords

Search

Navigation