First case report of a successful delivery of a healthy boy by preimplantation genetic testing for Beckwith-Wiedemann syndrome

Abstract

Purpose

To showcase the successful use of ICSI with PGT-M to overcome Beckwith-Wiedemann syndrome (BWS)–related reproductive challenges, resulting in the birth of a healthy baby boy. By targeting the maternally inherited CDKN1C pathogenic gene variant, this report highlights the genetic interventions in BWS reproductive risk management.

Methods

This case report describes a 41-year-old woman seeking fertility assistance after a previous pregnancy revealed a fetal anomaly related to BWS. Families with BWS recurrence face challenges, as maternally inherited CDKN1C pathogenic variants contribute to approximately 40% of genetic alterations, with a potential recurrence risk as high as 50%. Genetic analysis identified a pathogenic variant in the CDKN1C gene of the fetus that was maternally inherited. The pregnancy was terminated due to the fetal anomalies. The couple underwent intra-cytoplasmic sperm injection (ICSI) combined with preimplantation genetic testing for monogenic diseases (PGT-M) and preimplantation genetic testing for aneuploidy (PGT-A).

Results

Two embryos from IVF with low-risk PGT-M and euploid status. One transferred via frozen embryo transfer (FET) in February 2023 resulted in the successful birth of a healthy baby boy. This study reports the first successful delivery of a healthy boy after PGT-M for the CDKN1C gene variant c.79_100delinsGTGACC, contributing to the limited literature on successful outcomes for BWS.

Conclusion

Utilizing PGT-M in combination with IVF can lead to favorable outcomes in managing BWS-associated reproductive challenges, offering insights into potential genetic interventions and successful birth.

Introduction

Beckwith-Wiedemann syndrome (BWS) is a rare imprinting gene disorder, with an estimated prevalence ranging from 1 in 10,000 to 1 in 13,700 live births [1]. This complex syndrome shows a spectrum of clinical characteristics such as neonatal hypoglycemia, macrosomia, macroglossia, and omphalocele and an increased susceptibility to embryonal tumors [2, 3]. BWS is associated with abnormal gene transcription regulation in two imprinted domains on chromosome 11p15.5: imprinting center 1 (IC1) regulates the expression of IGF2 and H19 in domain 1, and imprinting center 2 (IC2) regulates the expression of CDKN1C in domain 2. Differential methylation of the two domains is associated with the expression of specific genes on the paternal and maternal alleles in unaffected individuals [4]. In more than 80% of the individuals with BWS, epigenetic alterations involve loss of methylation of IC2 and gain of methylation of IC1 on the maternal chromosome [5]. Other possible etiologies besides epigenetic alterations include paternal uniparental disomy or maternal heterozygous pathogenic variants in the CDKN1C allele. Usually, clinically relevant pathogenic variants associated with BWS involve those inherited maternally in the CDKN1C gene, as the paternally derived allele is typically silenced due to gene imprinting.

Pathogenic CDKN1C variants are identified in approximately 5% of BWS cases when there is no previous family history. While the reported prevalence likely underestimates the true frequency due to undiagnosed cases with milder phenotypes, families with known family history of BWS may face recurrence risk challenges, particularly when certain genetic mechanisms, such as CDKN1C pathogenic variants and copy number variants involving 11p15, are involved [1]. While the recurrence risk in the majority of the families is less than 1%, maternally inherited CDKN1C pathogenic variants contribute to approximately 40% of genetic alterations in families with BWS recurrence, with a potential recurrence risk as high as 50% and paternally or maternally inherited copy number variants are responsible for approximately 9% of genetic alterations. Preimplantation genetic testing for monogenic disease (PGT-M) has emerged as a strategy for preventing the conception of affected fetuses in hereditary single-gene disorders [6]. PGT-M can be conducted for a familial CDKN1C pathogenic variant and may also be feasible for certain familial genomic variants [1].

Despite the importance of understanding the genetic underpinnings of BWS and the potential applications of PGT-M, the literature is scarce, with only one study reporting a successful pregnancy with a wild-type CDKN1C fetus achieved by PGT-M for a mother carrying a different CDKN1C allele pathogenic variant [7]. Our study, therefore, contributes to the limited literature.

Case report

A 41-year-old woman sought assistance from our fertility center with the goal of conceiving a healthy child. Her previous pregnancy at the age of 40 resulted in a spontaneous conception, but a 20-week gestational scan revealed an anomaly—specifically, exomphalos containing bowel measuring 14.9 mm and increased amniotic fluid level (19.4 cm). All prior assessments, including non-invasive prenatal testing, nuchal translucency, and routine ultrasound scans, failed to detect any abnormalities.

An amniocentesis was conducted at the Feto Maternal Center, followed by a comprehensive genetic analysis involving polymerase chain reaction (PCR), karyotyping, array CGH, and whole exome sequencing (WES). While PCR, karyotyping, and array CGH showed normal results, WES identified a heterozygous pathogenic variant (c.79_100delinsGTCGACC) in exon 1 of the CDKN1C gene, resulting in the loss of five amino acids. This pathogenic variant indicated a likelihood of the fetus being affected by BWS due to the maternally inherited CDKN1C allele. This is attributed to the clinical relevance of maternally inherited pathogenic variants in this syndrome [5]. Unfortunately, due to the fetal anomalies, the pregnancy was terminated at 27 weeks.

Further genetic analysis, including segregation analysis of the mother’s and father’s blood, confirmed that the mother was a carrier of the pathogenic variant c.79_100delinsGTGGACC in the CDKN1C gene, while the father did not carry the pathogenic variant. This raised the possibility that the mother’s pathogenic variant could be de novo or paternally inherited, as she was unaffected and none of her family members had any sign or symptom of BWS.

The couple received comprehensive counseling from an experienced Geneticist and the clinic’s IVF expert team. Luckily, the amniocentesis fetal sample from the previous affected pregnancy was successfully preserved at the Feto Maternal Center and subsequently transferred to the genetics laboratory. The analysis of both the couple’s DNA samples and the amniocentesis sample from the terminated pregnancy confirmed the feasibility of PGT-M for the couple. Seven CDKN1C gene-linked polymorphic markers were analyzed for informativity, out of which four were fully informative and the c.79_100delinsGTGACC variant was identified through fluorescent PCR, genotyping, and fragment analysis, revealing an expected variant allele compared to the normal wild-type. Consequently, the recommendation was made for intra-cytoplasmic sperm injection (ICSI) combined with PGT-M and preimplantation genetic testing for aneuploidy (PGT-A). This suggestion took into account the 50% probability of the fetus inheriting the BWS pathogenic variant from the mother, coupled with her advanced maternal age.

The entire IVF process, including ovarian stimulation, egg retrieval, fertilization, embryo culture, blastocyst biopsy, vitrification, survival of thawed embryos, potential misdiagnosis inherent to PGT, and the likelihood of pregnancy, was thoroughly explained to the couple. The couple decided to proceed with ICSI and PGT to achieve a healthy offspring.

The patient underwent controlled ovarian stimulation on four cycles using the GnRH antagonist protocol. Transvaginal oocyte retrieval and ICSI were successfully performed. A total of 57 mature eggs were collected, out of which 41 fertilized normally. Trophectoderm biopsy was conducted on 17 blastocysts, followed by vitrification for future use. The treatment outcomes are summarized in Table 1. The Gardner embryo grading system was employed for embryo scoring [8].

Table 1.

Summary of cycle outcomes

Treatment number	Eggs collected	Eggs mature	Eggs fertilized	Embryos biopsied	Embryo No	Embryo scoring	PGT-M	PGT-A	Gender	Recommend for transfer
1	20	14	12	5	2	5BC	Low-risk	Complex aneuploid	Male	No
					4	5BB	Low-risk	Aneuploid	Male	No
					6	5BA	At-risk	Aneuploid	Male	No
					9	5AA	Low-risk	Complex aneuploid	Male	No
					12	5BC	At-risk	Euploid	Female	No
2	19	12	9	2	7	5AA	At-risk	Euploid	Female	No
					9	5AA	Low-risk	Aneuploid	Female	No
3	25	19	11	7	1	5AA	At-risk	Aneuploid	Male	No
					2	5BB	At-risk	Complex aneuploid	Female	No
					4	5AA	At-risk	Euploid	Female	No
					7	5BB	Low-risk	Complex aneuploid	Male	No
					9	5AA	Non-informative	Complex aneuploid	Female	No
					11	5AA	Low-risk	Euploid	Male	Yes*
					14	5AA	At-risk	Aneuploid	Male	No
4	19	12	9	3	2	5AA	At-risk	Aneuploid	Female	Np
					9	5AB	Low-risk	Euploid	Female	Yes
					12	5AB	At-risk	Euploid	Male	No
Total	83	57	41	17

^*Embryo transferred

For PGT-M, whole genome amplification was performed. Polymorphic markers linked to the CDKN1C gene (D11S922 at the 5′ end, D11S1318 and D11S3702 intergenically, and D11S4088 at the 3′ end) underwent PCR amplification. Subsequent to amplification, capillary electrophoresis on an automated sequencer (Thermo Fisher) facilitated fragment analysis of the PCR products. PGT-A was also conducted, covering all 24 chromosomes through next-generation sequencing.

Following PGT-M, the results showed that seven embryos were classified at low risk, which means that the embryos did not inherit the parental “at-risk haplotype” but the one linked to the wild-type allele. Additionally, following PGT-A, six embryos were classified as euploid. Only two embryos exhibited both a low-risk PGT-M result and euploid status after PGT-A. Consequently, these two embryos were potential candidates for future transfer. In February 2023, the patient underwent a frozen embryo transfer (FET) of an excellent quality euploid blastocyst at low risk of carrying BWS (Fig. 1). The procedure resulted in a successful and healthy pregnancy, leading to the live birth of a healthy baby boy. Non-invasive prenatal testing (NIPT) was performed during pregnancy yielding normal results, and although amniocentesis was also recommended, the couple opted out of it.

Fig. 1.

Embryo transferred in February 2023 leading to the live birth of a healthy boy

Discussion

Our study presents the first successful birth of a healthy male baby with a wild-type CDKN1C gene following PGT-M for the c.79_100delinsGTGACC variant. As per our knowledge, there is only one published case report of PGT-M on BWS by Yang et al. (2022) using PGT-M for a different variant (c.187 T > C/WT, p.Tyr63His), underscoring the genetic diversity in BWS [7]. In both our report and the study by Yang et al. (2022), patients underwent stimulation using the GnRH antagonist protocol. Additionally, multiple stimulation cycles were required for both patients to achieve a low-risk, euploid embryo. In order to assess the risk of each embryo carrying the specific pathogenic variant, both studies utilized analysis of the familial variant by detecting polymorphic markers of informative short tandem repeats (STRs) previously defined by fluorescent PCR. Therefore, our case contributes to the limited literature on successful PGT-M outcomes for BWS, offering valuable insights into preventing affected pregnancies in families with a significant risk of BWS recurrence.

The decision to conduct multiple IVF cycles was driven by the patient’s advanced maternal age (41 years old), which posed challenges in obtaining both low-risk and euploid embryos simultaneously. Despite obtaining one low-risk euploid embryo in the third cycle, an additional cycle was pursued as a precautionary measure, in case the first transfer did not yield desired results, and also to provide the option for a second future child if desired by the patient.

The preservation of the amniocentesis fetal sample was crucial for making PGT-M feasible for the couple. This emphasizes the importance of retaining products of conception following the termination of a pregnancy or miscarriage, especially when a genetic disorder is suspected. Without access to such specimens, conducting PGT-M would be significantly challenging and could hinder the establishment of informative linkage markers.

The male unaffected euploid embryo was prioritized to be transferred over the female. Our decision to transfer a male embryo is an approach to protect the future reproductive attempts of the offspring in case of the small but present risk of misdiagnosis by PGT-M. In such a case, the reproductive risks would be more significant for the female embryo if it was affected in comparison to the male. As carrier mothers of the CDKN1C pathogenic variant have a 50% chance of passing the gene to their children, our choice reflects a strategic measure to minimize the potential for inherited BWS in subsequent generations in case of PGT-M misdiagnosis, emphasizing the importance of personalized genetic counseling, informed choices for couples and forward-looking decision-making in assisted reproductive technologies.

This successful treatment allowed us to navigate the complexities of this rare syndrome, potentially offering valuable insights into its genetic mechanisms, recurrence risks, and how PGT-M and PGT-A can help select healthy embryos with unaffected CDKN1C genes. However, as this is a single case report and available literature is scarce, it is advisable to interpret these findings cautiously. Despite the successful outcome, several questions remain for future research. The genetic diversity in BWS cases requires further investigation into the functional significance of different CDKN1C variants and their impact on disease severity. Additionally, comprehensive follow-up studies are required to investigate the long-term health outcomes and developmental potential of children born through PGT-M for BWS.

Conclusion

In conclusion, our case report sheds light on the successful management of reproductive risk for BWS through PGT-M, leading to the birth of a healthy baby boy. This study underscores the importance of personalized genetic counseling, emphasizes the potential role of PGT-M in preventing BWS recurrence, and contributes valuable insights to the limited body of literature on the genetic underpinnings and management of this rare syndrome.

Data availability

Data regarding any of the subjects in the study has not been previously published. Data will be made available to the editors of the journal for review or query upon request.

Declarations

Ethical approval

Ethical approval was sought from the Dubai Scientific Research Ethics Committee (DSREC), which confirmed that approval was unnecessary for a single case report as it did not constitute research. However, the couple had already provided their consent.

Conflict of interest

The authors declare no competing interests.