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. 2024 Mar 21;12(3):e2409. doi: 10.1002/mgg3.2409

Preimplantation genetic testing as a means of preventing hereditary congenital myasthenic syndrome caused by RAPSN

Zhiping Zhang 1, Xueluo Zhang 1, Huiqin Xue 1, Liming Chu 2, Lina Hu 2, Xingyu Bi 1, Pengfei Zhu 1, Dongdong Zhang 1, Jiayao Chen 1, Xiangrong Cui 1, Lingyin Kong 2, Bo Liang 3, Xueqing Wu 1,✉
PMCID: PMC10955331  PMID: 38511267

Abstract

Background

Congenital myasthenic syndrome is a heterogeneous group of inherited neuromuscular transmission disorders. Variants in RAPSN are a common cause of CMS, accounting for approximately 14%–27% of all CMS cases. Whether preimplantation genetic testing for monogenic disease (PGT‐M) could be used to prevent the potential birth of CMS‐affected children is unclear.

Methods

Application of WES (whole‐exome sequencing) for carrier testing and guidance for the PGT‐M in the absence of a genetically characterized index patient as well as assisted reproductive technology were employed to prevent the occurrence of birth defects in subsequent pregnancy. The clinical phenotypes of stillborn fetuses were also assessed.

Results

The family carried two likely pathogenic variants in RAPSN(NM_005055.5): c.133G>A (p.V45M) and c.280G>A (p.E94K). And the potential birth of CMS‐affected child was successfully prevented, allowing the family to have offspring devoid of disease‐associated variants and exhibiting a normal phenotype.

Conclusion

This report constitutes the first documented case of achieving a CMS‐free offspring through PGT‐M in a CMS‐affected family. By broadening the known variant spectrum of RAPSN in the Chinese population, our findings underscore the feasibility and effectiveness of PGT‐M for preventing CMS, offering valuable insights for similarly affected families.

Keywords: congenital myasthenic syndrome, haplotype analysis, PGT‐M, RAPSN, WES

1. INTRODUCTION

Congenital myasthenic syndromes (CMS, OMIM#: 616326) constitute a group of rare inherited muscular disorders caused by genetic defects that affect the signal transmission to the neuromuscular junction (NMJ; Costain et al., 2016). The incidence rate varies with race and is approximately 0.1–1/100,000 (Pattrakornkul et al., 2020). It is a clinically heterogeneous disease characterized by muscle weakness and fatigue, with most patients showing symptoms of difficulty feeding, suffocation, ptosis, and generalized weakness of the face, neck, limbs, and trunk starting in the neonatal or early childhood periods (Finsterer, 2019). Some patients may even experience scoliosis and respiratory failure. Symptoms tend to exacerbate during fever, infection, pregnancy, or excitation. In rare cases, onset occurs during adolescence or adulthood with milder clinical symptoms (Finsterer, 2019). CMS can be classified into presynaptic, synaptic cleft, postsynaptic, glycosylation defect, and other rare subtypes based on the location and function of the defective proteins at the NMJ, and postsynaptic membrane protein defects accounting for the largest proportion (Müller et al., 2007). To date, pathogenic variants in over 30 genes that encode proteins important for neuromuscular transmission have been identified, including 9 presynaptic, 4 synaptic cleft, 12 postsynaptic, 5 glycosylation‐related, and 2 other genes (Bestue‐Cardiel & Natera‐de Benito, 2017; Finsterer, 2019; Wang et al., 2022). The most frequently mutated genes in CMS are CHAT, COLQ, RAPSN, CHRNE, DOK7, and GFPT1 (Aharoni et al., 2017; Cossins et al., 2020).

Variants in RAPSN (NC_000011.9), encoding RAPSYN, are a common cause of CMS and account for 14%–27% of all cases (Abicht et al., 2012; Müller et al., 2007; Natera‐de Benito et al., 2016; Parr et al., 2014). RAPSYN functions at the postsynaptic membrane to aggregate and anchor acetylcholine receptors (AChRs; Estephan et al., 2018). RAPSN variants can lead to loss or destabilization of AChR clusters, causing receptor deficiency and impaired neuromuscular transmission. RAPSN variants typically manifest as either severe, early onset or mild, late‐onset phenotypes (Burke et al., 2003). The N88K and ‐38A/G variants are hotspot RAPSN variants, with homozygous N88K variants usually leading to milder symptoms (Müller et al., 2004).

Preimplantation genetic testing for monogenic diseases (PGT‐M) offers a valuable tool for couples carrying pathogenic variants. By selecting euploid embryos without these variants for transfer, PGT‐M prevents the transmission of monogenic diseases and ultimately results in the birth of a healthy child (De Rycke & Berckmoes, 2020; Du et al., 2023). One significant advantage of PGT‐M is the avoidance of terminating an affected pregnancy, which can lead to complications (Hu et al., 2021). Currently, PGT‐M effectively prevents the transmission of numerous monogenic diseases (Chen et al., 2016). The safety and efficacy of this approach are supported by the successful birth of tens of thousands of unaffected offspring, demonstrating no apparent adverse effects.

Next‐generation sequencing (NGS) has revolutionized genetic testing prior to embryo transfer. Considering that 85% of pathogenic human variants are found in exons (Choi et al., 2009), whole‐exome sequencing (WES) based on NGS enables accurate detection of most exonic variants and copy number variations within its coverage range. WES streamlines the efficient and rapid identification of pathogenic genes and variants, facilitating the diagnosis of genetic diseases (Choi et al., 2009). Haplotype analysis not only distinguishes chromosomes carrying pathogenic variants from their homologous counterparts but also circumvents misdiagnosis due to allele dropout (ADO), making it a crucial component of PGT‐M (Altarescu et al., 2008, 2013). The integration of NGS‐based haplotyping analysis with single‐nucleotide polymorphism (SNP) haplotyping analysis can further enhance the accuracy of prenatal diagnosis and reduce misdiagnosis rates (Chen et al., 2016, 2017). In this study, couples carrying the RAPSN variant have a 50% chance of having offspring who are carriers of the variant and a 25% chance of having affected children (compound heterozygotes). The couple opted for PGT‐M to conceive, ultimately resulting in the birth of a healthy male infant without the pathogenic variant. This marks the first instance of using PGT‐M to prevent the intergenerational transmission of CMS caused by RAPSN, holding significant clinical implications for the application of PGT‐M in avoiding the transmission of similar monogenic diseases within families.

2. METHODS

2.1. Family participating in PGT‐M

In this study, we explored the genetic underpinnings of adverse pregnancy outcomes in a specific family. This family, having a documented history of reproductive challenges, sought intervention through assisted reproductive technology (ART) at the Center of Reproductive Medicine, Children's Hospital of Shanxi, and Women Health Center of Shanxi. This study received approval from the Ethics Committee of Affiliated Children's Hospital of Shanxi & Women Health Center of Shanxi Medicine University, China (ID: IRB‐KY‐2020‐013 [017]). The participants provided their written informed consent to participate in this study.

2.2. Gene mutation detection

Peripheral blood was collected from the couple. Genomic DNA was extracted using the TIANamp Blood DNA Kit (#DP348‐03, TIANGEN, Beijing, China) according to the manufacturer's instructions. The IDT xGen Exome Research Panel v2 capture probes were employed for hybridization with the gDNA library in a liquid phase, enriching DNA fragments in the target region to construct a comprehensive exome library. The xGen Exome Research Panel v2 comprises 415,115 probes, spanning a 34 Mb target region (encompassing 19,433 genes) of the human genome and 39 Mb of probe space (the genomic regions covered by the probes). Detailed information on the specific probe coverage areas can be found on the IDT official website (https://www.idtdna.com/pages/products/next‐generation‐sequencing/workflow/xgen‐ngs‐hybridization‐capture/pre‐designed‐hyb‐cap‐panels/exome‐hyb‐panel‐v2). Libraries were subjected to high‐throughput sequencing on the Illumina NovaSeq 6000 platform (San Diego, CA) using 150 bp paired‐end reads. Reads not meeting quality control criteria were removed. The remaining reads were aligned to the UCSC hg19 (https://genome.ucsc.edu/) reference genome using BWA (Burrows–Wheeler aligner). Variant analysis was performed using GATK v3.7 (Genome Analysis Toolkit). Variants were classified as pathogenic, likely pathogenic, variants of uncertain significance, likely benign or benign based on ACMG (American College of Medical Genetics and Genomics) standards and guidelines. Selected variants were validated by Sanger sequencing.

2.3. Embryo biopsy, single‐cell whole‐genome amplification and sequencing, and CNV detection

The couple opted for PGT‐M for their next pregnancy. Ovulation was induced using the ultra‐long protocol and in vitro fertilization was performed by intracytoplasmic sperm injection (ICSI). Embryos were cultured sequentially to Day 5. Trophectoderm biopsies were obtained from blastocysts and subjected to multiple annealing and looping‐based amplification cycles (MALBAC) for whole‐genome amplification (WGA) according to the ChromSwift™ Kit (XK‐028, Yikon Genomics, Su Zhou, China). WGA products were fragmented and used to construct libraries for sequencing on the Illumina NextSeq 550 platform. Data were analyzed for CNVs and SNP haplotyping and selected variants were validated by Sanger sequencing.

CNV analysis was performed as follows: raw reads were stripped of duplicates and aligned in 1 Mb bins to the reference genome. Counts across the whole genome were normalized using GC content and a reference dataset. A 50% increase in reads was expected when copy number (CN) increased from 2 to 3 per bin, while a 50% decrease was expected when CN decreased from 2 to 1 per bin. CNVs ≥4 Mb were reported using the circular binary segmentation algorithm (Olshen et al., 2004; Pfundt et al., 2017). CNVs across the 22 pairs of autosomes were visualized for each bin using R programming language.

2.4. Haplotyping analysis

Haplotype analysis of family members was conducted utilizing the Illumina iScan microarray scanner (Infinium Asian Screening Array‐24 v1.0 Kit). A total of 15 informative SNP markers located upstream and 15 informative SNP markers located downstream of the RAPSN gene (located at 11p11.2) were selected for haplotype analysis. The selection of informative SNP markers was performed based on stringent criteria: each SNP had one parental allele that displayed heterozygosity while the other parental allele exhibited homozygosity. Subsequent to the construction of SNP‐based haplotypes, we employed linkage analysis to effectively distinguish the haplotype carrying the pathogenic variant from the normal haplotype.

2.5. Frozen embryo transfer and prenatal diagnosis

Embryos devoid of pathogenic variants and exhibiting normal chromosomal ploidy, as determined by linkage analysis of SNPs flanking the variant site and validation of the variant and chromosomal status, were chosen for FET in a subsequent cycle. At 20 weeks of gestation, amniocentesis was performed to confirm the normal chromosomal ploidy and genotype of the fetus.

3. RESULTS

3.1. Basic clinical characteristics and family history

The couple was in good health. The wife initially became pregnant in 2009, giving birth to a healthy female infant at term. However, in 2013 and 2017, she underwent term cesarean sections, with both male infants unfortunately passing away due to developmental abnormalities. The affected infants exhibited pulmonary dysfunction and skeletal abnormalities (Figure 1a).

FIGURE 1.

FIGURE 1

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Pedigree diagram and linkage analysis identified for the disease‐carrying allele (a) Pedigree diagram and the RAPSN variants identified in the family. (b) The Sanger‐sequencing results of c.133G>A and c.280G>A within RAPSN in various family members. Family members I‐1/2 and III‐2/3 were not subjected to genetic testing for the RAPSN gene due to the unavailability of the corresponding samples. (c) The pedigree haplotypes were constructed based on informative SNPs that flank RAPSN. The haplotypes that were distinguished by slashes indicated the pathogenic alleles. Informative SNPs were shown on the left, with numerical annotations representing the distance from RAPSN, measured in kilobases (kb).

3.2. Mutation detection and parental haplotype construction

Whole‐exome sequencing was performed on the peripheral blood of the couple, revealing heterozygous RAPSN variants in both partners. The husband (II‐2) carried the c.133G>A (p.V45M) heterozygous variant; however, further validation from his parents (I‐1, I‐2) was not available to determine the origin of the variant. The wife (II‐3) carried the RAPSN c.280G>A (p.E94K) heterozygous variant, which was inherited from her mother (I‐4). Their healthy daughter (III‐1) was also a carrier of this variant (Figure 1a,b). Both RAPSN variants carried by the couple were classified as likely pathogenic. Based on the clinical phenotypes of the two deceased infants and the pathogenic variants carried by their parents, it was inferred that both infants had CMS. Combining the variant carrier status of the family, we successfully constructed the corresponding haplotypes (Figure 1c).

3.3. Single‐cell WGA and aneuploidy detection

For the PGT‐M cycle of this pedigree, three blastocysts were subjected to single‐cell WGA of their trophectoderm cells (TE cells) using the MALBAC method. DNA amplification was successful for all blastocysts. Chromosomal ploidy analysis of the embryos revealed that all three blastocysts were euploid, with no detected duplications or deletions larger than 4 Mb (Figure 2a).

FIGURE 2.

FIGURE 2

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Results of CNV, haplotype, and variants in embryos. (a) Embryonic and prenatal amniocentesis samples subjected to CNV analysis. (b) Haplotype analysis conducted on embryonic and prenatal amniocentesis samples. The haplotypes that were distinguished by slashes indicated the pathogenic alleles. Informative SNPs were shown on the left, with numerical annotations representing the distance from RAPSN, measured in kilobases (kb). (c) Sanger sequencing of two variants (c.280G>A and c.133G>A) in RAPSN across embryonic and prenatal amniocentesis samples.

3.4. Haplotype analysis of three blastocysts

The WGA products of the three blastocysts from this pedigree were subjected to SNP haplotype analysis and Sanger sequencing targeting the mutation site to determine whether the blastocysts carried the RAPSN variants (Figure 2b,c). Blastocyst 1 carried the c.133G>A (p.V45M) homozygous variant from the father (II‐2) and the c.280G>A (p.E94K) heterozygous variant from the mother (II‐3), resulting in compound heterozygosity. In contrast, blastocysts 2 and 3 did not carry either of these RAPSN variants (Figure 2b,c).

3.5. Embryo selection of non‐RAPSN variant carriers and results of frozen embryo transfer

Combining the results of chromosomal ploidy analysis and haplotype analysis, the couple from this pedigree obtained two chromosomally normal embryos that did not carry the RAPSN variants. Blastocyst 2 was chosen for frozen embryo transfer (FET) and underwent amniocentesis in December 2022, confirming the PGT‐M results (Figure 2a–c). At 37+4 weeks of gestation, a male infant was delivered by cesarean section, weighing 3100 g and measuring 50 cm in length. Further physical examination revealed normal respiration, heart rate, and muscle tone, with an Apgar score of 9/10 (assessing activity, pulse, grimace, appearance, and respiration).

4. DISCUSSION

In this study, both parents possess heterozygous pathogenic variant genes (c.133G>A and c.280G>A), presenting their descendants with a 25% probability of disease manifestation, a 50% likelihood of being a pathogenic variant carrier, and a 25% chance of normalcy. To prevent the transmission of CMS within this family, we employed the MALBAC technique for single‐cell WGA and sequencing. By selecting one embryo that did not carry pathogenic variants from either the maternal or paternal source, we successfully achieved the birth of a healthy offspring, effectively interrupting the transmission of CMS in this family. This represents the first reported case within a CMS family where the utilization of PGT‐M resulted in the generation of phenotype‐normal embryos free from pathogenic variants, highlighting the clinical feasibility and effectiveness of PGT‐M as a valuable tool in CMS prevention and interception.

RAPSYN, encoded by the RAPSN, is a postsynaptic protein involved in the aggregation and anchoring of acetylcholine receptors (AChR) at the postsynaptic membrane (Natera‐de Benito et al., 2016). Variants in the RAPSN lead to defects in endplate AChR or reduced stability of AChR clusters, resulting in receptor deficiency and impaired neuromuscular transmission (Cossins et al., 2006). RAPSYN is composed of four distinct functional domains: an N‐terminal myristoylation domain mediating membrane interactions, seven tetratricopeptide repeats (TPR) involved in self‐aggregation and binding to the cytoplasmic region of muscle‐specific kinase MuSK, a coiled‐coil domain interacting with the cytoplasmic loop of AChR subunits, and a C‐terminal RING‐H2 domain that binds to cytoskeletal proteins (such as dystroglycan) to link the RAPSYN‐AChR complex to the cytoskeleton (Cossins et al., 2006). Variants located in different domains of RAPSYN can result in varying degrees of protein dysfunction (Table 1, Figure 3). Homozygous or compound heterozygous variants in RAPSN are known to cause two distinct clinical phenotypes: a severe early‐onset form and a milder late‐onset form. The early‐onset phenotype is more common, typically presenting with contractures, joint fusion, facial dysmorphism, hypotonia, bilateral ptosis, and respiratory distress, either at birth or before 2 years of age. The late‐onset phenotype, on the other hand, is rare and usually manifests in early adulthood or adolescence, characterized by bilateral ptosis and distal muscle weakness (Müller et al., 2007; Natera‐de Benito et al., 2016). In this CMS family, the two deceased infants exhibited pulmonary dysfunction and skeletal anomalies, suggesting a more severe phenotype. The parental carriers in this family were found to harbor heterozygous variants in RAPSN, specifically c.133G>A (p.V45M) and c.280G>A (p.E94K). However, due to the lack of genetic testing on the deceased infants, the definitive genotype could not be determined. Previous reports have shown that the c.133G>A and c.280G>A mutations are often found as compound heterozygous variants in conjunction with other variants, leading to CMS (Table 1). For instance, a patient with compound heterozygosity for c.264C>A and c.280G>A variants presented with hypotonia, poor sucking, subjective diplopia, and neck muscle weakness at birth (Natera‐de Benito et al., 2016). Another patient harboring compound heterozygosity for c.133G>A and c.484G>A variants exhibited symptom onset at 16 months and presented with normal cognition, bilateral ptosis, mild facial muscle weakness, and mild proximal limb weakness (Maselli et al., 2007).

TABLE 1.

Known RAPSN variants and their corresponding clinical manifestations.

Case no. Age at onset Delayed motor development Ptosis Ophthalmoparesis Facial weakness Bulbar weakness Cervical weakness Proximal/distal weakness Episodic worsening Other Variants PMID
1 Birth Y + N + + ++ ++/+ Y Strabismus c.‐27C>G,p.N88K 19620612 (Milone et al., 2009)
2 Birth Y + N ++ + +++ +/+ Y c.1177_1178delAA,p.N88K 19620612
3 Birth Y ++ N ++ ++ ++ ++/+ N p.L14P, p.N88K 19620612
4 Birth Y ++ N +++ ++ ++ ++/+ ND p.N88K, p.N88K 19620612
5 <1 N Y E E N +++ N/N Y p.N88K, p.N88K 19620612
6 Birth ND ND ND ND ND ND ND/ND ND p.R242W*, p.N88K 19620612
7 Birth Y E N E E Y Y/Y Y p.N88K, p.N88K 19620612
8 Birth Y Y N Y Y ND ++/+ Y Strabismus p.L326P*, p.N88K 19620612
9 2 N Y N ND ND Y Y/Y ND Strabismus p.N88K, p.N88K 19620612
10 3/12 N Y E Y + Y Y/N Y p.E147K, p.N88K 19620612
11 Birth N N E + N N +/+ Y p.C97X, p.N88K 19620612
12 Birth Y ++ N N N N N/N Y Strabismus p.N88K, p.N88K 19620612
13 Birth Y + N + + N +/N Y c.553_554insGTTCT, p.N88K 19620612
14 Birth Y E ND ND Y Y Y/Y Y p.R151P, p.N88K 19620612
15 3/12 Y + N ++ N Y Y/N Y p.N88K, p.N88K 19620612
16 Birth N ++ N +++ Y + +/+ N c.‐38A/G, c.‐38A/G 19620612
17 Birth N E N N E N N/N Y p.E333X, p.N88K 19620612
18 Birth N Y E Y N N +/N ND p.A142D, p.N88K 19620612
19 2 N +++ N ++ Y + N/N N c.‐38A/G, c.‐38A/G 19620612
20 2/12 N ++ N +++ Y + N/N N c.‐38A/G, c.‐38A/G 19620612
21 Birth N ++ N +++ Y N +/N N c.‐38A/G, c.‐38A/G 19620612
22 <2 N ++ N ++ Y N N/N N c.‐38A/G, c.‐38A/G 19620612
23 9/12 N ++ N ++ Y N N/N N c.‐38A/G, c.‐38A/G 19620612
24 Birth N ++ N +++ Y N N/N N c.‐38A/G, c.‐38A/G 19620612
25 1/12 N ++ N ++ Y N N/N N c.‐38A/G, c.‐38A/G 19620612
26 Birth Y Y N ND ND ND ND/ND ND Strabismus p.V50_S55del, p.N88K 19620612
27 Birth N ++ N ++ +++ ++ ++/++ Y c.1177_1178delAA, p.N88K 19620612
28 Birth Y +++ + Y N ND +/ND ND p.V165M, p.N88K 19620612
29 Birth ND ND N ND ND ND ND/ND ND c.966+1GT>AG, p.N88K 19620612
30 9/12 Y ND ND N E +++ Y/ND Y p.V45M, p.N88K 19620612
31 Birth Y Y Y + N N N/+ Y p.L14P, p.N88K 19620612
32 Birth Y Y N N N N +++/N Y Strabismus p.E147K, p.N88K 19620612
33 Birth Y +++ + ++ In infancy N N/N Y p.N88K, p.N88K 19620612
34 Birth Y + ++ ++ +++ + ++/+ Y p.A142D, p.N88K 19620612
35 Birth Y + N E E + +/+ Y Strabismus c.‐27C>G, p.N88K 19620612
36 Birth Y Y N ND ND ND ND/ND Y p.Q325X, p.N88K 19620612
37 Birth Y E N ++ ++ +++ Y/+ Y p.K373del, p.N88K 19620612
38 5 N N N N N N +/N N p.N88K, p.N88K 19620612
39 Birth N + + + + +++ +++/+ N p.R164H*, p.N88K 19620612
40 Y Y A N + Y ND +/N ND V45M, E162K 17594401 (Maselli et al., 2007)
41 Birth Y Y N Y Y ND Y/ND ND p.N88K, p.N88K 36308527 (Krenn et al., 2023)
42 Infancy Y Y N Y Y ND ND/ND ND c.193‐15C>A, p.N88K 36308527
43 N/A Y Y N Y Y ND ND/ND ND c.193‐15C>A, p.N88K 36308527
44 Birth Y ND ND Y ND Y Y/ND ND Micrognathia, Deformity of the joint, Death c.1177‐1178delAA 18179903 (Vogt et al., 2008)
45 1 N Y N Y N N N/N ND Mandibular prognathism, and Facial dysmorphism c.‐38A/G, p.224 insT 22326364 (Leshinsky‐Silver et al., 2012)
46 3/12 N Y N Y N N N/N Y c.‐38A/G, c.673–676 insACT 22326364
47 Infancy ND ND ND ND ND ND ND ND Skin edema and pleural effusions p.K373del 33897756 (Zhou et al., 2021)
48 Infancy ND ND ND ND ND ND ND ND Skin edema and pleural effusions p.Val50Glufs*114, p.G123D 33897756
49 Infancy ND ND ND ND ND ND ND ND Skin thickening at level of fetal skull and ascites p.K373del, p.L63P 33897756
50 Birth ND N N Y N Y Y/N Y Respiratory distress, hypotonia, poor sucking, and diplopia p.N88K, p.N88K 26782015 (Natera‐de Benito et al., 2016)
51 Birth ND N N N N Y N/N Y Respiratory distress, hypotonia, poor sucking, and diplopia p.N88K, p.N88K 26782015
52 Birth ND Y Y Y N Y N/N Y Respiratory distress, hypotonia, poor sucking, and diplopia p.N88K, p.L290P 26782015
53 Birth ND N N N N N N/N Y Hypotonia, poor sucking, and diplopia p.N88K, p.Thr396Pfs*12 26782015
54 Birth ND N N N N Y N/N Y Hypotonia, poor sucking, and diplopia p.N88K, p.E94K 26782015
55 Birth ND Y N N N Y Y/N Y Respiratory distress, hypotonia, poor sucking, and diplopia p.N88K, p.Thr396Pfs*12 26782015
56 Birth ND Y N N Y Y Y/N Y Hypotonia, poor sucking, anddiplopia p.N88K, p.Thr396Pfs*12 26782015
57 Birth ND Y N Y Y N Y/N Y Hypotonia, poor sucking, anddiplopia p.N88K, p.Thr396Pfs*12 26782015
58 Birth ND N N Y N Y N/N Y Respiratory distress, hypotonia, and poor sucking p.N88K, p.Gln120Serfs*8 26782015
59 Birth ND Y N N N Y Y/N Y Respiratory distress, hypotonia, and poor sucking p.N88K, p.V165M 26782015
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Abbreviations: +, mild weakness; ++, moderate weakness; +++, severe weakness; A, asymmetry.; E, episodic; N, no; ND, no data; Y, weakness present but the extent is unknown.

FIGURE 3.

FIGURE 3

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Schematic diagram of the main domains of RAPSYN with known pathogenic variants. The two variants we identified were marked in red.

Currently, the main treatment approach for CMS primarily involves pharmacotherapy, including cholinergic agonists (such as pyridostigmine and 3,4‐diaminopyridine), adrenergic agonists (such as ephedrine and salbutamol), and long‐lived open‐channel blockers of the acetylcholine receptor ion channel (such as fluoxetine and quinidine; Engel et al., 2015). While some CMS patients can achieve a normal quality of life and have a normal lifespan with timely and standardized treatment, others may experience relapses or a progression of symptoms years later (Iyadurai, 2020). Due to the incurable nature of CMS and the challenges in its management, preventing the birth of affected children represents a crucial measure.

PGT‐M offers a crucial means of preventing the intergenerational transmission of genetic diseases by selectively implanting embryos that do not carry disease‐causing variants into the maternal uterus (De Rycke & Berckmoes, 2020). It currently represents a paramount approach in the prevention and reduction of birth defects, serving as an effective primary preventive measure for monogenic diseases. In the application of PGT‐M, patients undergo a process that includes genetic counseling, variant detection, risk assessment, PGT, assisted reproductive technologies, and prenatal diagnostic validation, thereby increasing the likelihood of having healthy offspring. Furthermore, the genetic risk assessment also benefits at‐risk relatives within the same pedigree and their subsequent generations (Xu et al., 2022). Moreover, it is noteworthy that in PGT‐M, ADO, a potential complication in the Sanger sequencing process, can be effectively avoided through haplotype analysis. The results of the Sanger sequencing for c.133G>A, as depicted in Figure 2c, suggest a homozygous state. When combined with the findings from haplotype analysis, we hypothesize that this observation can be attributed to ADO occurring during the detection process.

The successful implementation of PGT‐M in our study's CMS family was largely attributed to the key role of SNP linkage analysis‐based haplotyping. SNP linkage analysis offers several advantages, including (1) high precision and reliability, which helps overcome misdiagnosis resulting from allelic dropout observed in direct sequencing (Li et al., 2018); (2) high efficiency with a relatively short experimental process; (3) the capability to simultaneously detect aneuploidies, allowing for the distinction between monosomy and trisomy; and (4) mitigating the potential harm caused by failed prenatal diagnosis to the female psychological well‐being (Xu et al., 2022). However, PGT‐M also has certain limitations. For instance, haplotype analysis requires clear family information, and it may not detect de novo variants. Allelic recombination, although rare, can lead to changes in the SNPs used for haplotype analysis, thereby affecting the accuracy of SNP linkage analysis. To minimize the risk of misdiagnosis resulting from recombination, combining SNP linkage analysis with prenatal diagnosis through chorionic villus sampling can be employed (de Massy, 2013).

5. CONCLUSION

In summary, this study successfully identified pathogenic variants in RAPSN in a family without available affected samples, using whole‐exome sequencing. Furthermore, by employing PGT‐M, the potential birth of CMS‐affected child was successfully prevented, allowing the family to have offspring devoid of disease‐associated variants and exhibiting a normal phenotype. These findings provide valuable insights for families carrying similar monogenic disease‐related variants, offering a reference for their reproductive decisions.

AUTHOR CONTRIBUTIONS

Zhiping Zhang, Xueluo Zhang, and Huiqin Xue collected clinical data and samples and performed DNA extraction and gene mutation detection. Xingyu Bi, Pengfei Zhu, Dongdong Zhang, Jiayao Chen, and Xiangrong Cui performed embryo biopsy, single‐cell whole‐genome amplification and sequencing, and CNV detection. Zhiping Zhang, Xueluo Zhang, Huiqin Xue, and Xueqing Wu performed haplotyping analysis, frozen embryo transfer, and prenatal diagnosis. Zhiping Zhang, Liming Chu, and Lina Hu collected the literature and wrote the manuscript. Zhiping Zhang, Liming Chu, Lina Hu, Lingyin Kong, Bo Liang, and Xueqing Wu were responsible for the study design and guiding of the study implementation and revised the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING INFORMATION

Innovation plan of Medical Science and Technology of “Four in a batch” (2020TD19).

CONFLICT OF INTEREST STATEMENT

Authors LC, LH, and LK were employed by Basecare Medical Device Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

ETHICS STATEMENT

All procedures were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and its later amendments. Informed consent was obtained from all participants before being included in the study. This study was approved by the Ethics Committee of Affiliated Children's Hospital of Shanxi & Women Health Center of Shanxi Medicine University, China (ID:IRB‐KY‐2020‐013 [017]).

ACKNOWLEDGMENTS

The authors thank the family members for participating in this study. This study was funded by the Innovation Plan of Medical Science and Technology of “Four in a batch” (2020TD19).

Zhang, Z. , Zhang, X. , Xue, H. , Chu, L. , Hu, L. , Bi, X. , Zhu, P. , Zhang, D. , Chen, J. , Cui, X. , Kong, L. , Liang, B. , & Wu, X. (2024). Preimplantation genetic testing as a means of preventing hereditary congenital myasthenic syndrome caused by RAPSN . Molecular Genetics & Genomic Medicine, 12, e2409. 10.1002/mgg3.2409

DATA AVAILABILITY STATEMENT

Raw data for this study are available upon request from the corresponding authors.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Raw data for this study are available upon request from the corresponding authors.

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