{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,27]],"date-time":"2026-06-27T06:57:35Z","timestamp":1782543455463,"version":"3.54.5"},"reference-count":37,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2025,5,9]],"date-time":"2025-05-09T00:00:00Z","timestamp":1746748800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001350","name":"Ministry of Health, Singapore","doi-asserted-by":"publisher","award":["MOH-000644-01"],"award-info":[{"award-number":["MOH-000644-01"]}],"id":[{"id":"10.13039\/501100001350","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Combined preimplantation genetic testing of aneuploidy (PGT-A) and monogenic disease (PGT-M) can be achieved through PCR-based whole genome amplification (WGA) and next-generation sequencing (NGS). However, pathogenic variant detection is usually achieved indirectly through single nucleotide polymorphism haplotyping, as direct detection of pathogenic variants is not always possible. We evaluated whether isothermal WGA was suitable for combined PGT-A and PGT-M that also permitted direct detection of repeat expansions and large deletions, in addition to indirect linkage analysis using microsatellite markers. Five-cell replicates from selected cell lines were subjected to isothermal or PCR-based WGA, followed by NGS-based PGT-A and direct and indirect PGT-M of Huntington\u2019s disease and spinal muscular atrophy. Both WGA methods accurately detected aneuploidy and large (10 Mb) segmental imbalances. However, isothermal WGA produced higher genotyping accuracy compared with PCR-based WGA for all analysed microsatellite markers (93.5% vs. 75.6%), as well as at the HTT CAG repeat locus (100% vs. 7.7%) and the SMN1\/2 locus (100% vs. 71.8%). These results demonstrate that isothermal WGA is potentially ideal for combined PGT-A and PGT-M that permits both direct and indirect detection of pathogenic variants including repeat expansions and gene deletions.<\/jats:p>","DOI":"10.3390\/ijms26104532","type":"journal-article","created":{"date-parts":[[2025,5,9]],"date-time":"2025-05-09T06:18:51Z","timestamp":1746771531000},"page":"4532","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["A Strategy Potentially Suitable for Combined Preimplantation Genetic Testing of Aneuploidy and Monogenic Disease That Permits Direct Detection of Pathogenic Variants Including Repeat Expansions and Gene Deletions"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6070-2333","authenticated-orcid":false,"given":"Vivienne J.","family":"Tan","sequence":"first","affiliation":[{"name":"Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, NUHS Tower Block, Level 12, 1E Kent Ridge Road, Singapore 119228, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ying","family":"Liang","sequence":"additional","affiliation":[{"name":"Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, NUHS Tower Block, Level 12, 1E Kent Ridge Road, Singapore 119228, Singapore"},{"name":"Reproductive Medical Center, Shijiazhuang Obstetrics and Gynaecology Hospital, The Fourth Hospital of Shijiazhuang, Shijiazhuang 050011, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2772-2583","authenticated-orcid":false,"given":"Arnold S.","family":"Tan","sequence":"additional","affiliation":[{"name":"Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, NUHS Tower Block, Level 12, 1E Kent Ridge Road, Singapore 119228, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simin","family":"Wong","sequence":"additional","affiliation":[{"name":"Preimplantation Genetic Diagnosis Centre, Department of Obstetrics and Gynaecology, National University Hospital, Singapore 119074, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-7678-2779","authenticated-orcid":false,"given":"Nur","family":"Asherah","sequence":"additional","affiliation":[{"name":"Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, NUHS Tower Block, Level 12, 1E Kent Ridge Road, Singapore 119228, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pengyian","family":"Chua","sequence":"additional","affiliation":[{"name":"Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, NUHS Tower Block, Level 12, 1E Kent Ridge Road, Singapore 119228, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4323-3635","authenticated-orcid":false,"given":"Caroline G.","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117596, Singapore"},{"name":"Cellular and Molecular Research, National Cancer Centre Singapore, Singapore 168583, Singapore"},{"name":"Duke-NUS Medical School, Singapore 169857, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mahesh A.","family":"Choolani","sequence":"additional","affiliation":[{"name":"Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119228, Singapore"},{"name":"Department of Obstetrics and Gynaecology, National University Hospital, Singapore 119074, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8587-0440","authenticated-orcid":false,"given":"Truong","family":"Dang","sequence":"additional","affiliation":[{"name":"Department of Anatomy, Vietnam Military Medical University, Hanoi 10000, Vietnam"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1872-5937","authenticated-orcid":false,"given":"Samuel S.","family":"Chong","sequence":"additional","affiliation":[{"name":"Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, NUHS Tower Block, Level 12, 1E Kent Ridge Road, Singapore 119228, Singapore"},{"name":"Preimplantation Genetic Diagnosis Centre, Department of Obstetrics and Gynaecology, National University Hospital, Singapore 119074, Singapore"},{"name":"Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119228, Singapore"},{"name":"Molecular Diagnosis Centre, Department of Laboratory Medicine, National University Hospital, Singapore 119074, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,5,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"768","DOI":"10.1038\/344768a0","article-title":"Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification","volume":"344","author":"Handyside","year":"1990","journal-title":"Nature"},{"key":"ref_2","first-page":"1786","article-title":"The international glossary on infertility and fertility care, 2017","volume":"32","author":"Adamson","year":"2017","journal-title":"Fertil. Steril."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"ESHRE PGT-M Working Group, Carvalho, F., Moutou, C., Dimitriadou, E., Dreesen, J., Gim\u00e9nez, C., Goossens, V., Kakourou, G., Vermeulen, N., and Zuccarello, D. (2020). ESHRE PGT Consortium good practice recommendations for the detection of monogenic disorders\u2020. Hum. Reprod. Open, 2020, hoaa018.","DOI":"10.1093\/hropen\/hoaa018"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"577","DOI":"10.1038\/nm.1924","article-title":"Chromosome instability is common in human cleavage-stage embryos","volume":"15","author":"Vanneste","year":"2009","journal-title":"Nat. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"928","DOI":"10.1016\/j.fertnstert.2019.01.017","article-title":"Role of aneuploidy screening in preimplantation genetic testing for monogenic diseases in young women","volume":"111","author":"Hou","year":"2019","journal-title":"Fertil. Steril."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1327","DOI":"10.1007\/s10897-016-9975-4","article-title":"Preimplantation genetic diagnosis (PGD) for monogenic disorders: The value of concurrent aneuploidy screening","volume":"25","author":"Goldman","year":"2016","journal-title":"J. Genet. Couns."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Xiao, M., Shi, H., Rao, J., Xi, Y., Zhang, S., Wu, J., Zhu, S., Zhou, J., Xu, H., and Lei, C. (2022). Combined preimplantation genetic testing for genetic kidney disease: Genetic risk identification, assisted reproductive cycle, and pregnancy outcome analysis. Front. Med., 9.","DOI":"10.3389\/fmed.2022.936578"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"651","DOI":"10.1136\/jmg.2009.069971","article-title":"Karyomapping: A universal method for genome wide analysis of genetic disease based on mapping crossovers between parental haplotypes","volume":"47","author":"Handyside","year":"2009","journal-title":"J. Med. Genet."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1390","DOI":"10.1038\/s41436-018-0351-7","article-title":"Haploseek: A 24-hour all-in-one method for preimplantation genetic diagnosis (PGD) of monogenic disease and aneuploidy","volume":"21","author":"Backenroth","year":"2019","journal-title":"Genet. Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"103647","DOI":"10.1016\/j.ejmg.2019.04.004","article-title":"Validation of concurrent preimplantation genetic testing for polygenic and monogenic disorders, structural rearrangements, and whole and segmental chromosome aneuploidy with a single universal platform","volume":"62","author":"Treff","year":"2019","journal-title":"Eur. J. Med. Genet."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1608","DOI":"10.1093\/humrep\/dez106","article-title":"Multi-centre evaluation of a comprehensive preimplantation genetic test through haplotyping-by-sequencing","volume":"34","author":"Masset","year":"2019","journal-title":"Hum. Reprod."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"15964","DOI":"10.1073\/pnas.1523297113","article-title":"Live births after simultaneous avoidance of monogenic diseases and chromosome abnormality by next-generation sequencing with linkage analyses","volume":"112","author":"Yan","year":"2015","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"e22267","DOI":"10.1002\/jcla.22267","article-title":"The performance of MALBAC and MDA methods in the identification of concurrent mutations and aneuploidy screening to diagnose beta-thalassaemia disorders at the single- and multiple-cell levels","volume":"32","author":"Liu","year":"2018","journal-title":"J. Clin. Lab. Anal."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41598-023-45292-z","article-title":"SHaploseek is a sequencing-only, high-resolution method for comprehensive preimplantation genetic testing","volume":"13","author":"Backenroth","year":"2023","journal-title":"Sci. Rep."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"365","DOI":"10.1007\/s10815-020-02006-y","article-title":"Development and clinical application of a preimplantation genetic testing for monogenic disease (PGT-M) for beta thalassemia in Vietnam","volume":"38","author":"Mai","year":"2020","journal-title":"J. Assist. Reprod. Genet."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"549","DOI":"10.1007\/s10815-020-01732-7","article-title":"Eleven healthy live births: A result of simultaneous preimplantation genetic testing of \u03b1- and \u03b2-double thalassemia and aneuploidy screening","volume":"37","author":"Chen","year":"2020","journal-title":"J. Assist. Reprod. Genet."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"407","DOI":"10.1038\/s10038-018-0411-5","article-title":"Systematic assessment of the performance of whole-genome amplification for SNP\/CNV detection and \u03b2-thalassemia genotyping","volume":"63","author":"He","year":"2018","journal-title":"J. Hum. Genet."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1016\/j.jgg.2015.03.001","article-title":"The performance of whole genome amplification methods and next-generation sequencing for pre-implantation genetic diagnosis of chromosomal abnormalities","volume":"42","author":"Li","year":"2015","journal-title":"J. Genet. Genom."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Satirapod, C., Sukprasert, M., Panthan, B., Charoenyingwattana, A., Chitayanan, P., Chantratita, W., Choktanasiri, W., Trachoo, O., and Hongeng, S. (2019). Clinical utility of combined preimplantation genetic testing methods in couples at risk of passing on beta thalassemia\/hemoglobin E disease: A retrospective review from a single center. PLoS ONE, 14.","DOI":"10.1371\/journal.pone.0225457"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1016\/j.ejogrb.2024.04.033","article-title":"Impact of double trophectoderm biopsy on reproductive outcomes following single euploid blastocyst transfer","volume":"298","author":"Theodorou","year":"2024","journal-title":"Eur. J. Obstet. Gynecol. Reprod. Biol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1007\/s10815-020-02009-9","article-title":"Third-generation sequencing: Any future opportunities for PGT?","volume":"38","author":"Liu","year":"2021","journal-title":"J. Assist. Reprod. Genet."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1089\/gtmb.2021.0268","article-title":"Assessment of multiple annealing and looping-based amplification cycle-based whole-genome amplification for short tandem repeat genotyping of low copy number-DNA","volume":"26","author":"Liao","year":"2022","journal-title":"Genet. Test. Mol. Biomark."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Mir Pardo, P., Mart\u00ednez-Conejero, J.A., Mart\u00edn, J., Sim\u00f3n, C., and Cervero, A. (2020). Combined preimplantation genetic testing for autosomal dominant polycystic kidney disease: Consequences for embryos available for transfer. Genes, 11.","DOI":"10.3390\/genes11060692"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"94","DOI":"10.1186\/s13039-015-0197-5","article-title":"Emergence of clonal chromosomal alterations during the mesenchymal stromal cell cultivation","volume":"8","author":"Borgonovo","year":"2015","journal-title":"Mol. Cytogenet."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Nikitina, V., Astrelina, T., Nugis, V., Ostashkin, A., Karaseva, T., Dobrovolskaya, E., Usupzhanova, D., Suchkova, Y., Lomonosova, E., and Rodin, S. (2018). Clonal chromosomal and genomic instability during human multipotent mesenchymal stromal cells long-term culture. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0192445"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1093\/humrep\/deq231","article-title":"ESHRE PGD consortium best practice guidelines for amplification-based PGD","volume":"26","author":"Harton","year":"2011","journal-title":"Hum. Reprod."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e21","DOI":"10.1016\/j.rbmo.2017.10.051","article-title":"Validation of two whole genome amplification methods for PGD on monogenetic diseases and aneuploidy screening","volume":"36","author":"Chow","year":"2018","journal-title":"Reprod. Biomed. Online"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1096","DOI":"10.1373\/clinchem.2016.255711","article-title":"Identification of novel microsatellite markers <1 Mb from the HTT CAG repeat and development of a single-tube tridecaplex PCR panel of highly polymorphic markers for preimplantation genetic diagnosis of Huntington disease","volume":"62","author":"Zhao","year":"2016","journal-title":"Clin. Chem."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Zhao, M., Lian, M., Cheah, F.S., Tan, A.S., Agarwal, A., and Chong, S.S. (2019). Identification of novel microsatellite markers flanking the SMN1 and SMN2 duplicated region and inclusion into a single-tube tridecaplex panel for haplotype-based preimplantation genetic testing of spinal muscular atrophy. Front. Genet., 10.","DOI":"10.3389\/fgene.2019.01105"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2914","DOI":"10.1002\/elps.201500146","article-title":"Identification of novel microsatellite markers <1 Mb from the HBB gene and development of a single-tube pentadecaplex PCR panel of highly polymorphic markers for preimplantation genetic diagnosis of beta-thalassemia","volume":"36","author":"Chen","year":"2015","journal-title":"Electrophoresis"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1473","DOI":"10.1111\/jth.13685","article-title":"Single-tube tetradecaplex panel of highly polymorphic microsatellite markers <1 Mb from F8 for simplified preimplantation genetic diagnosis of hemophilia A","volume":"15","author":"Zhao","year":"2017","journal-title":"J. Thromb. Haemost."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"869","DOI":"10.1038\/gim.2015.185","article-title":"Identification of microsatellite markers <1 Mb from the FMR1 CGG repeat and development of a single-tube tetradecaplex PCR panel of highly polymorphic markers for preimplantation genetic diagnosis of fragile X syndrome","volume":"18","author":"Chen","year":"2016","journal-title":"Genet. Med."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1127","DOI":"10.1373\/clinchem.2017.271528","article-title":"Single-tube dodecaplex PCR panel of polymorphic microsatellite markers closely linked to the DMPK CTG repeat for preimplantation genetic diagnosis of myotonic dystrophy type 1","volume":"63","author":"Lian","year":"2017","journal-title":"Clin. Chem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2285","DOI":"10.1016\/j.gim.2022.08.008","article-title":"Segmental aneuploidies with 1 Mb resolution in human preimplantation blastocysts","volume":"24","author":"Xie","year":"2022","journal-title":"Genet. Med."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Zhang, J., Qin, M., Ma, M., Li, H., Wang, N., Zhu, X., Yan, L., Qiao, J., and Yan, Z. (2024). Assessing the necessity of screening \u22645 Mb segmental aneuploidy in routine PGT for aneuploidies. Reprod. Biomed. Online, 49.","DOI":"10.1016\/j.rbmo.2024.103991"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"395","DOI":"10.1016\/j.jmoldx.2025.02.002","article-title":"A Novel and comprehensive whole-genome sequencing\u2013based preimplantation genetic testing approach for different genetic conditions","volume":"27","author":"Li","year":"2025","journal-title":"J. Mol. Diagn."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"348","DOI":"10.1159\/000444714","article-title":"Enhanced detection and sizing of the HTT CAG repeat expansion in Huntington disease using an improved triplet-primed PCR assay","volume":"16","author":"Zhao","year":"2016","journal-title":"Neurodegener. Dis."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/10\/4532\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:30:02Z","timestamp":1760031002000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/10\/4532"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,5,9]]},"references-count":37,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2025,5]]}},"alternative-id":["ijms26104532"],"URL":"https:\/\/doi.org\/10.3390\/ijms26104532","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,5,9]]}}}