Preimplantation Genetic Testing for Monogenic Disorders

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1		Roberta Giuliano	--	2096	2023-11-25 16:25:00	\|
2	format correct	Catherine Yang	Meta information modification	2096	2023-11-27 01:39:28	\|

This entry is adapted from the peer-reviewed paper 10.3390/genes14112095

Preimplantation genetic tests have a broad range of applications, conceptually divisible into two main areas: inherited disorders, where alterations can be found in the parents (PGT-M and PGT-SR), and de novo conditions, i.e., not inherited, as in the case of PGT-A. The objective of PGT-M testing is to avoid transferring embryos affected by a specific monogenic disease. This can only be achieved by selecting embryos that either do not carry the mutation or are healthy carriers (in the case of recessive diseases), as may occur in patients with a positive family or personal history for a monogenic condition. This necessitates a preliminary study tailored to each couple, involving family members. In general, PGT-M can be applied to the diagnosis of all hereditary monogenic diseases for which the responsible gene has been identified, one or two index cases are available, and a diagnostic linkage analysis protocol can be developed. Conversely, it is not indicated in cases of large gene deletions/duplications or de novo triplet expansions since the phasing of the at-risk haplotype is not feasible.

preimplantation genetic testing biopsy pgt-m pgt-a pgt-sr

1. Diagnostic Strategies

PGT-M always starts with the biopsy of the TE, from which 5–10 cells are collected. From there, the process can split into either target amplification or whole-genome amplification ^[1]. In the case of target amplification, a multiplex PCR can be performed, identifying both the mutation and genetic markers. With whole-genome amplification, options include multiplex PCR, single nucleotide polymorphism (SNP) array, or NGS. All these techniques are based on the principle of haplotyping, which involves determining the risk-associated haplotype linked to the mutation by observing how chosen informative markers (SNPs or STRs) segregate with the mutation. This approach helps to determine whether the embryo has inherited the risk allele or the wild-type allele. This is a genuine linkage analysis, which overcomes the limitations of working with low DNA quantities. Therefore, during preclinical work-up, the genotyping of SNP markers near the gene of interest in DNA samples from the couple and family members with a known genetic status is required ^[2]^[3].

PGT-M originated from fragment length analysis using STRs, combined with minisequencing for mutation analysis. In parallel, another target technology using SNPs with real-time PCR was developed, enabling the identification of mutations and flanking SNP markers. The workflow involves real-time PCR SNP genotyping, TE biopsy and the lysis of collected cells, multiplex amplification using TaqMan assays for mutations and informative SNPs, real-time PCR amplification, TaqMan genotyping, linkage analysis, and haplotype determination. If CNV assays are added to TaqMan assays in the second step, the simultaneous detection of aneuploidies (PGT-M and PGT-A) is also possible.

Among the advantages of the target protocol is a very low allele drop out (ADO) rate, which increases with WGA instead. Additionally, diagnoses can be obtained quickly (within 4 h of biopsy) due to the short analysis time of real-time PCR compared to NGS. Concurrently, WGA started to develop, allowing the amplification of the entire genome with multiple displacement amplification (MDA), followed by the target amplification of STR markers and fragment length analysis. Following the introduction of WGA, PGT-M approaches diversified greatly. With target amplification, specific loci or gene regions are amplified, whereas, with WGA, the entire genome is amplified. This opens up the possibility to use other techniques, such as SNP array, especially karyomapping, suitable for PGT-M; targeted NGS, enriching specific gene regions linked to particular pathologies; and genome-wide NGS, with an enriched panel (5000 genes) to detect various pathologies ^[4]. The advantages of SNP array are the standardized procedures and reduced time to diagnosis, allowing concurrent PGT-M, PGT-A, and PGT-SR. However, there is a drawback in terms of higher costs and the analysis being limited to inherited pathologies, excluding de novo mutations due to the need for a reference. In particular, SNP genotyping methods are preferable to STRs for indirect analysis, as SNPs are more frequent in the genome compared to STRs and thus more informative in this context. Therefore, whenever possible, SNP analysis should be preferred over STR analysis ^[5]. The diagnostic protocol of the targeted strategy involves the direct identification of the disease-causing mutation post-WGA, along with analyzing at least two informative polymorphic markers, or three markers in cases where the causative mutation is unknown ^[6]. The relevant polymorphic markers are STRs, chosen for their informativeness. An informative single STR is equivalent to three informative SNPs, even though SNPs (biallelic) are more abundant (one SNP every 300–1000 bp), easier to interpret, and suitable for high-throughput analysis ^[3]. Subsequently, a multiplex PCR is set up using pre-made kits, and the heterozygosity of STRs is assessed via capillary electrophoresis. Since markers are chosen based on the chromosome affected by the mutation, those close to the gene of interest allow the discrimination of parental haplotypes. Informative markers (heterozygous) are selected for clinical testing on embryos. High-risk (mutant) and low-risk haplotypes are established during preclinical setup. The diagnostic protocol is optimized for the couple and family members to confirm the presence of the causative variant and define informative linked markers for PGT ^[7]. As a result, PGT-M is a laborious and costly procedure that requires extensive preliminary family study, leading to long waiting lists for couples embarking on this journey. This approach also enables molecular diagnosis while simultaneously testing for DNA contamination, relatedness, and technical artifacts ^[8]^[9].

2. PGT-M Limitations

One of the main limitations of PGT-M is the genetic variability and complexity inherent to monogenic diseases themselves. Hereditary genetic diseases can be caused by different mutations on the same gene or mutations on different genes that lead to similar phenotypes. This genetic variability can make the accurate identification of specific disease-causing mutations challenging. Moreover, certain genetic diseases can be influenced by environmental or epigenetic factors, further complicating the association between genotype and phenotype. The challenge in addressing genetic variability in monogenic disorders is that a preimplantation diagnostic test should accurately identify the presence or absence of specific mutations in embryonic cells. The current clinical practice in PGT centers does not involve the preimplantation diagnosis of VUS. However, the array of mutations and the heterogeneity of phenotypes can impact the efficacy of such tests. Furthermore, the detection of Variants of Uncertain Significance (VUS) can further complicate result interpretation, generating uncertainty about which mutations actually represent a concrete risk in terms of developing the disease. Dealing with genetic variability and the complexity of monogenic disorders requires a combination of advanced molecular, diagnostic, and bioinformatic approaches. Identifying specific genetic variants responsible for a disease requires a deep understanding of its genetic basis and the metabolic pathways involved. Additionally, collaboration between clinical geneticists, molecular biologists, and bioinformaticians is crucial in interpreting genetic information correctly and identifying relevant mutations for PGT-M.

Another major technical limitation of PGT-M protocols is the small quantity of input samples, which is why WGA is performed following tubing and cell lysis in order to obtain a sufficient quantity of genomic DNA for one or more molecular analyses ^[9]^[10]. As mentioned earlier, the gold standard for PGT-M involves an indirect analysis because the risk of allele drop out (ADO), amplification failure (AF), and contamination is very high when working with embryos, as only a few cells are available for the examination. Moreover, determining the haplotype through SNP array also requires a sample from at least a first-degree relative of the carrier partner. If unavailable, this leads to reduced SNP informativeness. Conversely, this limitation can be mitigated with a targeted protocol, although it is more time-intensive compared to NGS, which provides a greater quantity of information in less time.

Certain mutations (e.g., small deletions, duplications, or complex chromosomal rearrangements) cannot be detected and diagnosed using current analytical strategies. This has led to the introduction of new screening approaches capable of analyzing genetic markers present throughout the genome (genome-wide), thereby overcoming these limitations. The use of WGA has made it possible to apply genome-wide analysis methods to samples consisting of single or few cells ^[11]^[12]. An example is karyomapping, which is based on SNP array technology and determines an individual’s genotype by analyzing thousands of SNPs distributed across the genome. Each SNP is biallelic, presenting one of two possible nucleotides on each chromosome. Individually, they are less informative than STRs, which possess high allelic heterogeneity. However, a set of four SNP markers is sufficient to determine the genotypes of the parents and the index case ^[13]. Karyomapping is based on linkage analysis: by comparing the SNPs associated with the disease-causing mutation in the index case and parental chromosomes with those present in embryo cells, it is possible to identify the presence or absence of the mutation-carrying allele. Therefore, karyomapping marks a transition from a family- or disease-specific diagnostic approach to a “genome-wide” approach, applicable, in principle, to any monogenic alteration with informative SNPs. Moreover, this technique overcomes the issue of failed allele amplification in single-cell cases, distinguishing key SNPs in the embryo sample from non-key SNPs. This does not completely eliminate genotyping errors but significantly reduces their occurrence and, in most cases, leads to the identification of a set of key SNP markers with consistent results.

3. Karyomapping Work

SNP array is a highly dense array spotted with 300,000 SNPs. Firstly, the parents and a reference individual need to be genotyped—collectively called a trio—across hundreds of thousands of sites distributed throughout the genome. This is because karyomapping is based on genome-wide linkage analysis, targeting all 300,000 available platform SNPs. The first step identifies a set of informative SNP markers (heterozygous for one parent and homozygous for the other) for each of the four parental chromosomes. Subsequently, the allele phase for each informative SNP locus is determined, and the linkage of the parental risk alleles with the corresponding chromosomes is established. A linkage is defined with respect to the reference individual’s genotype, usually an individual with a known disease state, such as an affected child or fetus from a previous pregnancy. The goal is to determine the parental origin of each chromosome in the embryo with regard to the reference genotype.

The first publication concerning karyomapping technology dates back to 2010 and was authored by Professor Handyside, the father of PGD, and colleagues ^[13]. This publication summarizes the key characteristics and execution of PGT-M karyomapping technology. Firstly, it is a genome-wide linkage analysis, a distinct advantage from certain viewpoints as it is performed on an extensive, non-target set of markers—specifically, all 300,000 available on the platform. Another distinguishing feature is its applicability at the single-cell level, necessitating a whole-genome amplification step.

Furthermore, unlike other methods, no patient-, family-, or disease-specific setup is required. In 2014, Natesan et al., 2014, ^[14] conducted a concordance analysis, assessing karyomapping’s accuracy against the gold standard at that time—direct mutation analysis plus linkage analysis with flanking STR markers. This study involved 218 embryo samples from 44 PGD cycles, achieving an extremely high concordance rate of 97.7%. Discrepancies mainly arose in consanguineous families where, without direct mutation analysis, distinguishing the inheritance of the four alleles solely from haplotypes became more challenging. Another advantage is that, as no preclinical work-up is necessary, waiting times for setup, the potentially necessary acquisition of locus-specific probes, and validation on parents and family members are significantly reduced ^[14]. The WGA technology used in SNP array is multiple displacement amplification (MDA). This involves isothermal whole-genome amplification utilizing random hexamer molecules that bind to denatured DNA strands, continuously extending on the nascent strand, assisted by a phi polymerase. This generates large fragments and maintains high sequencing quality due to minimal biases. This feature is unique to karyomapping, as it is not shared by other techniques like NGS ^[15].

In conclusion, one of karyomapping’s advantages is the substantial reduction in laboratory workload and waiting times for couples due to the absence of the need for preclinical work-up. Additionally, it enables the simultaneous analysis of PGT-M and PGT-A since both SNP genotyping and chromosome copy number information are obtained from raw data. Disadvantages include the high costs of equipment and consumables, the underlying algorithm not providing an all-in-one solution for molecular and chromosomal diagnosis, being limited to inherited chromosomal or monogenic abnormalities only, and the necessity of pertinent familial samples for haplotyping. Lastly, direct mutation analysis is not included in this approach.

4. Future Perspectives for PGT-M

Improving diagnostic capabilities and accessibility for PGT-M is a key direction for the future. This includes addressing challenges associated with de novo mutations or mutations that are still difficult to identify, such as repeat expansions. New technologies like haplotyping-by-sequencing offer the potential to obtain both genetic and chromosomal information in a single workflow. However, it is important to note that this approach still requires family members for phasing, and, currently, it may not be suitable for the detection of de novo mutations.

References

Harton, G.L.; Magli, M.C.; Lundin, K.; Montag, M.; Lemmen, J.; Harper, J.C.; European Society for Human Reproduction and Embryology (ESHRE) PGD Consortium/Embryology Special Interest Group. ESHRE PGD Consortium/Embryology Special Interest Group--best practice guidelines for polar body and embryo biopsy for preimplantation genetic diagnosis/screening (PGD/PGS). Hum. Reprod. 2011, 26, 41–46.
Jeffreys, A.J.; Wilson, V.e.; Thein, S.L. Hypervariable ‘minisatellite’ regions in human DNA. Nature 1985, 314, 67–73.
ESHRE PGT-M Working Group; Carvalho, F.; Moutou, C.; Dimitriadou, E.; Dreesen, J.; Giménez, C.; Goossens, V.; Kakourou, G.; Vermeulen, N.; Zuccarello, D.; et al. ESHRE PGT Consortium good practice recommendations for the detection of monogenic disorders. Hum. Reprod. Open 2020, 2020, hoaa018.
Treff, N.R.; Fedick, A.; Tao, X.; Devkota, B.; Taylor, D.; Scott, R.T., Jr. Evaluation of targeted next-generation sequencing-based preimplantation genetic diagnosis of monogenic disease. Fertil. Steril. 2013, 99, 1377–1384.e6.
Diagnosi Genetica Preimpianto—PGT Raccomandazioni SIGU 2017 per la Pratica Clinica. Documento Redatto dal Tavolo Tecnico istituito nell’ambito del GdL SIGU di Citogenetica-Citogenomica. 9 agosto 2017. Available online: https://sigu.net/wp-content/uploads/2020/11/2043-2017_08_09_Raccomandazioni-PGT2017-ApprovatoCDSIGU.pdf (accessed on 8 October 2023).
Renwick, P.J.; Trussler, J.; Ostad-Saffari, E.; Fassihi, H.; Black, C.; Braude, P.; Ogilvie, C.M.; Abbs, S. Proof of principle and first cases using preimplantation genetic haplotyping--a paradigm shift for embryo diagnosis. Reprod. Biomed. Online. 2006, 13, 110–119.
Fiorentino, F.; Biricik, A.; Nuccitelli, A.; De Palma, R.; Kahraman, S.; Iacobelli, M.; Trengia, V.; Caserta, D.; Bonu, M.A.; Borini, A.; et al. Strategies and clinical outcome of 250 cycles of Preimplantation Genetic Diagnosis for single gene disorders. Hum. Reprod. 2006, 21, 670–684.
Huang, L.; Ma, F.; Chapman, A.; Lu, S.; Xie, X.S. Single-Cell Whole-Genome Amplification and Sequencing: Methodology and Applications. Annu. Rev. Genomics Hum. Genet. 2015, 16, 79–102.
Harton, G.L.; De Rycke, M.; Fiorentino, F.; Moutou, C.; SenGupta, S.; Traeger-Synodinos, J.; Harper, J.C. ESHRE PGD consortium best practice guidelines for amplification- based PGD. Hum. Reprod. 2011, 26, 33–40.
Vanneste, E.; Melotte, C.; Voet, T.; Robberecht, C.; Debrock, S.; Pexsters, A.; Staessen, C.; Tomassetti, C.; Legius, E.; D’Hooghe, T.; et al. PGD for a complex chromosomal rearrangement by array comparative genomic hybridization. Hum. Reprod. 2011, 26, 941–949.
Handyside, A.H.; Robinson, M.D.; Simpson, R.J.; Omar, M.B.; Shaw, M.A.; Grudzinskas, J.G.; Rutherford, A. Isothermal whole genome amplification from single and small numbers of cells: A new era for preimplantation genetic diagnosis of inherited disease. Mol. Hum. Reprod. 2004, 10, 767–772.
Hellani, A.; Coskun, S.; Benkhalifa, M.; Tbakhi, A.; Sakati, N.; Al-Odaib, A.; Ozand, P. Multiple displacement amplification on single cell and possible PGD applications. Mol. Hum. Reprod. 2004, 10, 847–852.
Handyside, A.H.; Harton, G.L.; Mariani, B.; Thornhill, A.R.; Affara, N.; Shaw, M.A.; Griffin, D.K. Karyomapping: A universal method for genome wide analysis of genetic disease based on mapping crossovers between parental haplotypes. J. Med. Genet. 2010, 47, 651–658.
Natesan, S.A.; Bladon, A.J.; Coskun, S.; Qubbaj, W.; Prates, R.; Munne, S.; Coonen, E.; Dreesen, J.C.; Stevens, S.J.; Paulussen, A.D.; et al. Genome-wide karyomapping accurately identifies the inheritance of single-gene defects in human preimplantation embryos in vitro. Genet. Med. 2014, 16, 838–845.
Volozonoka, L.; Miskova, A.; Gailite, L. Whole Genome Amplification in Preimplantation Genetic Testing in the Era of Massively Parallel Sequencing. Int. J. Mol. Sci. 2022, 23, 4819.

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Subjects: Genetics & Heredity

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Roberta Giuliano

Anna Maione

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Ugo Sorrentino

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Update Date: 27 Nov 2023

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