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
Abstract:BACKGROUND
Preimplantation genetic diagnosis (PGD) of Huntington disease (HD) generally employs linkage analysis of flanking microsatellite markers to complement direct mutation testing, as well as for exclusion testing. Thus far, only 10 linked markers have been developed for use in HD PGD, with a maximum of 3 markers coamplified successfully. We aimed to develop a single-tube multiplex PCR panel of highly polymorphic markers to simplify HD PGD.
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“…The TP-PCR assay, in parallel with multiplex-PCR of a highly polymorphic tridecaplex (13-plex) microsatellite marker panel 32 (Fig. 2) will allow the haplotype phase of the disease/mutant allele to be determined during the PGT-M itself, which is especially useful when there are no family members available for a priori haplotype phasing of the disease allele in the affected spouse.Figure 2Schematic illustration of the combined PGT-M strategy.…”
Section: Resultsmentioning
confidence: 99%
“…This ADO risk can be reduced, but not entirely eliminated, by testing directly on blastomeres instead of first subjecting them to WGA which is known to increase ADO rates (Harton et al ., 2011), and by testing trophectoderm samples instead of blastomeres. Nonetheless, ADO is best overcome by parallel analysis of a highly polymorphic multi-microsatellite panel of closely linked markers 32 to establish haplotype phase of the disease/mutant allele. For example, haplotype analysis provided a definitive diagnosis when ADO of the affected spouse’s expanded HTT allele was observed in embryo 18 of the clinical IVF PGT-M case (Figs 5 and 6).…”
Section: Discussionmentioning
confidence: 99%
“…Tridecaplex microsatellite marker PCR analysis was separately performed on genomic DNA or single cell WGA products according to conditions as previously described 32 .…”
Section: Methodsmentioning
confidence: 99%
“…We describe a versatile strategy for HD PGT-M involving the use of TP-PCR for robust detection of the HTT CAG repeat expansion 30 , in combination with multiplex-PCR analysis of 13 closely linked microsatellite markers to establish disease haplotype phase 32 . We also report the first clinical application of this strategy for IVF PGT-M of HD in an at-risk couple for which family members of the affected spouse were unavailable for development of a standalone linkage-based PGT-M.…”
Huntington disease (HD) is a lethal neurodegenerative disorder caused by expansion of a CAG repeat within the huntingtin (HTT) gene. Disease prevention can be facilitated by preimplantation genetic testing for this monogenic disorder (PGT-M). We developed a strategy for HD PGT-M, involving whole genome amplification (WGA) followed by combined triplet-primed PCR (TP-PCR) for HTT CAG repeat expansion detection and multi-microsatellite marker genotyping for disease haplotype phasing. The strategy was validated and tested pre-clinically in a simulated PGT-M case before clinical application in five cycles of a PGT-M case. The assay reliably and correctly diagnosed all embryos, even where allele dropout (ADO) occurred at the HTT CAG repeat locus or at one or more linked markers. Ten of the 27 embryos analyzed were diagnosed as unaffected. Four embryo transfers were performed, two of which involved fresh cycle double embryo transfers and two were frozen-thawed single embryo transfers. Pregnancies were achieved from each of the frozen-thawed single embryo transfers and confirmed to be unaffected by amniocentesis, culminating in live births at term. This strategy enhances diagnostic confidence for PGT-M of HD and can also be employed in situations where disease haplotype phase cannot be established prior to the start of PGT-M.
“…The TP-PCR assay, in parallel with multiplex-PCR of a highly polymorphic tridecaplex (13-plex) microsatellite marker panel 32 (Fig. 2) will allow the haplotype phase of the disease/mutant allele to be determined during the PGT-M itself, which is especially useful when there are no family members available for a priori haplotype phasing of the disease allele in the affected spouse.Figure 2Schematic illustration of the combined PGT-M strategy.…”
Section: Resultsmentioning
confidence: 99%
“…This ADO risk can be reduced, but not entirely eliminated, by testing directly on blastomeres instead of first subjecting them to WGA which is known to increase ADO rates (Harton et al ., 2011), and by testing trophectoderm samples instead of blastomeres. Nonetheless, ADO is best overcome by parallel analysis of a highly polymorphic multi-microsatellite panel of closely linked markers 32 to establish haplotype phase of the disease/mutant allele. For example, haplotype analysis provided a definitive diagnosis when ADO of the affected spouse’s expanded HTT allele was observed in embryo 18 of the clinical IVF PGT-M case (Figs 5 and 6).…”
Section: Discussionmentioning
confidence: 99%
“…Tridecaplex microsatellite marker PCR analysis was separately performed on genomic DNA or single cell WGA products according to conditions as previously described 32 .…”
Section: Methodsmentioning
confidence: 99%
“…We describe a versatile strategy for HD PGT-M involving the use of TP-PCR for robust detection of the HTT CAG repeat expansion 30 , in combination with multiplex-PCR analysis of 13 closely linked microsatellite markers to establish disease haplotype phase 32 . We also report the first clinical application of this strategy for IVF PGT-M of HD in an at-risk couple for which family members of the affected spouse were unavailable for development of a standalone linkage-based PGT-M.…”
Huntington disease (HD) is a lethal neurodegenerative disorder caused by expansion of a CAG repeat within the huntingtin (HTT) gene. Disease prevention can be facilitated by preimplantation genetic testing for this monogenic disorder (PGT-M). We developed a strategy for HD PGT-M, involving whole genome amplification (WGA) followed by combined triplet-primed PCR (TP-PCR) for HTT CAG repeat expansion detection and multi-microsatellite marker genotyping for disease haplotype phasing. The strategy was validated and tested pre-clinically in a simulated PGT-M case before clinical application in five cycles of a PGT-M case. The assay reliably and correctly diagnosed all embryos, even where allele dropout (ADO) occurred at the HTT CAG repeat locus or at one or more linked markers. Ten of the 27 embryos analyzed were diagnosed as unaffected. Four embryo transfers were performed, two of which involved fresh cycle double embryo transfers and two were frozen-thawed single embryo transfers. Pregnancies were achieved from each of the frozen-thawed single embryo transfers and confirmed to be unaffected by amniocentesis, culminating in live births at term. This strategy enhances diagnostic confidence for PGT-M of HD and can also be employed in situations where disease haplotype phase cannot be established prior to the start of PGT-M.
“…Notably, exclusion protocols (that is, not transferring all embryos with the same haplotype as the affected parent) should be considered in some cases. They have the advantage that the affected parent (usually with AD or late-onset neurodegenerative disorders such as Huntington’s disease) can choose to not know their genotype status, whereas a disadvantage is that the number of transferable embryos will be apparently reduced, inevitably affecting the fertility outcome (such as the implantation rate and livebirth rate) [54].…”
Preimplantation genetic diagnosis (PGD) has become a crucial approach in helping carriers of inherited disorders to give birth to healthy offspring. In this study, we review PGD methodologies and explore the use of amplification refractory mutation system quantitative polymerase chain reaction (ARMS-qPCR) and/or linkage analysis for PGD in neurodegenerative diseases that are clinically relevant with typical features, such as late onset, and which are severely debilitating. A total of 13 oocyte retrieval cycles were conducted in 10 cases with various neurodegenerative diseases. Among the 59 embryos analyzed, 49.2% (29/59) were unaffected and 50.8% (30/59) were affected. Of the 12 embryo transfer cycles, three resulted in pregnancy, and all pregnancies were delivered. The implantation rate and livebirth rate were 23.1% (3/13) per oocyte retrieval cycle and 25.0% (3/12) per embryo transfer cycle. Allele dropout (ADO) was noted in two embryos that were classified as unaffected by ARMS-qPCR but were evidenced as affected after prenatal diagnosis, rendering the false negative rate as 6.3% (2/32). Four among the 13 cycles underwent PGD by ARMS-qPCR coupled with linkage analysis, and all were correctly diagnosed. We conclude that PGD by ARMS-qPCR and/or linkage analysis is a feasible strategy, whereas ADO is a concern when ARMS-qPCR is used as the sole technology in PGD, especially in autosomal dominant diseases.
Preimplantation genetic testing for the monogenic disorder (PGT-M) spinal muscular atrophy (SMA) is significantly improved by supplementation of SMN1 deletion detection with marker-based linkage analysis. To expand the availability of informative markers for PGT-M of SMA, we identified novel non-duplicated and highly polymorphic microsatellite markers closely flanking the SMN1 and SMN2 duplicated region. Six of the novel markers within 0.5 Mb of the 1.7 Mb duplicated region containing SMN1 and SMN2 (SMA6863, SMA6873, SMA6877, SMA7093, SMA7115, and SMA7120) and seven established markers (D5S1417, D5S1413, D5S1370, D5S1408, D5S610, D5S1999, and D5S637), all with predicted high heterozygosity values, were selected and optimized in a tridecaplex PCR panel, and their polymorphism indices were determined in two populations. Observed marker heterozygosities in the Chinese and Caucasian populations ranged from 0.54 to 0.86, and 98.4% of genotyped individuals (185 of 188) were heterozygous for ≥2 markers on either side of SMN1. The marker panel was evaluated for disease haplotype phasing using single cells from two parent–child trios after whole-genome amplification, and applied to a clinical IVF (in vitro fertilization) PGT-M cycle in an at-risk couple, in parallel with SMN1 deletion detection. Both direct and indirect test methods determined that none of five tested embryos were at risk for SMA, with haplotype analysis further identifying one embryo as unaffected and four as carriers. Fresh transfer of the unaffected embryo did not lead to implantation, but subsequent frozen-thaw transfer of a carrier embryo produced a pregnancy, with fetal genotype confirmed by amniocentesis, and a live birth at term.
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