Inherited metabolic diseases are rare in the general population but can cause significant harm to affected families and society due to their often untreatable nature (Ferreira and van Karnebeek, 2019). Citrullinemia type 1, with an incidence rate of about 1/22,000, is characterized primarily by high blood ammonia levels (Posset et al., 2020). Symptoms range from mild to severe and can include increased intracranial pressure, heightened neuromuscular tension, spasticity, seizures, loss of consciousness, and even death due to elevated blood ammonia and other toxic substances (Kölker et al., 2015). Citrullinemia type 1 can lead to intellectual disability or death despite the availability of effective therapies (Häberle et al., 2019). Preimplantation genetic testing (PGT) offers hope for affected families by preventing intergenerational inheritance (Cho et al., 2014). High-risk families can use PGT to prevent genetic diseases in offspring, making gene diagnosis crucial throughout the process.

Since the American College of Medical Genetics and Genomics (ACMG) issued standards for classifying unreported gene variants in 2015, a unified standard has been established (Richards et al., 2015). However, the efficacy of these standards in successfully blocking the transmission of inherited metabolic diseases requires further clinical support (Chen, 2023). Mutations in ASS1 can cause citrullinemia type 1 (Xiong and Chen, 2022), and functional tests showed that mutation in ASS1 affects its expression (Liu et al., 2023). In this research, we present a family case of citrullinemia type 1 involving previously unreported mutation sites in ASS1, classified as pathogenic or likely pathogenic according to ACMG guidelines. Preimplantation genetic testing for monogenic disease (PGT-M) was performed, followed by monitoring related conditions until birth.

Citrullinemia type 1 symptoms vary widely, and without early diagnosis and treatment, the prognosis for children with severe symptoms is very poor (Häberle et al., 2019). Therefore, PGT and prenatal diagnosis are critically important. Our data provide further clinical evidence for preventing citrullinemia type 1 in a family, including observing allele dropout (ADO) during testing and gene recombination during generational transmission.

Current guidelines and consensus recommendations primarily address medical interventions for variants classified as pathogenic or likely pathogenic. However, the potential for diagnostic errors in identifying such variants and the risk of misdiagnosis in PGT remain (Chen, 2023). In this case, the ASS1.847G>A variant, originating from the male partner, is a pathogenic missense mutation. Previous reports suggest that this mutation may affect the folding of specific protein domains, although no affected patients have been documented. The evidence for this variant is classified as PM3_Very strong + PM1+PM2_Supporting + PP3 according to ACMG guidelines (Gao et al., 2003).

The c.1127 + 1G>T variant, originating from the female partner, is classified as likely pathogenic. It is a previously unreported alternative splicing variant categorized as potentially causative. The evidence type for this variant is PVS1+PM2_Supporting according to ACMG guidelines (Engel, Höhne, and Häberle 209). This splicing variant occurs at the donor site with a SpliceAI score of 0.99. Given the ACMG rating of likely pathogenic and the clinical diagnosis of the related disease corresponding to this gene variant (PP3), this variant could be considered pathogenic. As the variant is classified as pathogenic, we did not further perform functional studies to assess protein expression abnormalities caused by this splicing site, such as exon skipping, intron inclusion, or cryptic splice site usage. However, these functional validations are crucial for understanding the disease’s pathogenesis.

Determining the embryonic genotype based on haplotypes is a commonly used technique in genetic diagnosis of embryos (Harton et al., 2011). Establishing haplotypes through family members clarifies the haplotype where the genetic variant is located, effectively reducing the risk associated with allele dropout during embryonic genetic testing. However, diagnostic errors due to genetic recombination remain possible, underscoring the importance of prenatal diagnosis (Wilton et al., 2009). This case includes a detailed description of citrullinemia type 1 occurrence, diagnosis, tracing, prevention, verification, and follow-up in a family. We observed ADO and recombination, which can complicate embryonic diagnosis, but haplotype analysis combined with Sanger sequencing can increase PGT success rates.

ADO is a phenomenon observed during PCR, potentially leading to genetic diagnostic errors (Blais et al., 2015), ADO occurrence may be related to high CG content (Wenzel et al., 2009), and the amount of PCR amplification template and analysis methods (Hedell et al., 2015), Despite mitigating high-risk factors, ADO cannot be completely avoided (Blais et al., 2015). This is more likely to occur in embryonic testing, where linkage analysis can reduce misdiagnosis rates caused by ADO (Rechitsky et al., 1998). However, genetic recombination during haplotype determination can affect diagnosis. In this study, we observed two recombination events in the tested region over three generations, possibly due to this region being a recombination hotspot. Our data support varying recombination regions across different embryos. The necessity of simultaneous CNV testing during PGT-M is debated (Yang et al., 2022), but our data indicate that concurrent CNV testing can effectively reduce embryo aneuploidy rates.

Amniocentesis verification confirmed the fetal genotype, underscoring the importance of prenatal diagnosis in accurately assessing fetal genetic risks. Unlike PCR, prenatal diagnosis does not carry ADO-related risks and involves cells from multiple embryonic layers, offering more accurate evaluations than trophoblast cell sampling during the embryonic stage (Kahraman et al., 2020; Greco et al., 2023).

Although our research suggests that clinical intervention through PGT methods could prevent the birth of children with citrullinemia type 1, our study has several limitations. Firstly, expanding the sample size of citrullinemia type 1 patients is necessary to further validate the effectiveness of PGT methods in preventing the transmission of ASS1 gene mutations. Establishing the relationship between genetic variants and phenotypic outcomes is crucial for PGT. While the ACMG guidelines offer valuable references, many variants lack clear causal relationships with phenotypes, emphasizing the necessity for further research and clinical data. Furthermore, our study did not include functional studies on the two variants identified, which are essential for understanding changes in protein function and the underlying pathogenic mechanisms. Additionally, there is a need for further research on the accuracy and detection methods of PGT. Based on the above problems, future research will involve conducting scientific experiments to verify our findings and enhance the understanding of PGT’s potential in managing citrullinemia type 1 Engel et al., 2009.

Preimplantation Genetic Testing (PGT) targeting likely pathogenic and pathogenic variants in the ASS1 gene, as rated by ACMG, allows the birth of healthy infants free from citrullinemia type 1. Additionally, the establishment of single haplotypes and Sanger sequencing can reduce the misdiagnosis rate caused by allele dropout (ADO) and genetic recombination.