Multilocus pathogenic variants in MCM4, RYR1, and G6PD identified by trio-based whole-exome sequencing in a neonate with multisystem symptoms: a case report

Introduction

When neonates present with nonspecific yet concerning signs—such as hypotonia, poor feeding, or growth restriction—it may indicate an underlying genetic etiology (1). However, when the phenotype does not conform to a known syndrome, conventional diagnostic pathways often fail. Whole-exome sequencing (WES), particularly trio-based analysis, provides an opportunity to detect multilocus variation and blended phenotypes (2). The concept of a “blended phenotype” refers to the coexistence of pathogenic variants in two or more disease-associated genes (3), each contributing partially to the clinical presentation. Recent studies have suggested that 4–7% of patients undergoing WES harbor such multilocus pathogenic variants (4,5). This case represents a rare and clinically significant example involving concurrent variants in MCM4, RYR1, and G6PD, expanding the spectrum of neonatal blended phenotypes and emphasizing the importance of genomic testing in atypical clinical scenarios. We present this article in accordance with the CARE reporting checklist (available at https://acr.amegroups.com/article/view/10.21037/acr-2025-131/rc).

Case presentation

A full-term male neonate was delivered at 38+5 weeks of gestation via cesarean section due to fetal distress. The birth weight was 1.73 kg, consistent with small-for-gestational-age status. The mother had gestational diabetes but no other pregnancy complications. The infant experienced perinatal asphyxia and required 37 minutes of resuscitation before stabilization and admission to the neonatal intensive care unit (NICU). At admission, his vital signs were temperature 35.5 ℃, heart rate 130 bpm, respiratory rate 56 breaths/min, and blood pressure 63/38 mmHg. He exhibited generalized hypotonia, weak spontaneous movements, poor feeding, a weak cry, diminished reflexes, and mild chest retractions. Bilateral testes were non-palpable. Laboratory investigations revealed anemia, hyperbilirubinemia, and a positive G6PD enzyme screening. The clinical diagnoses included neonatal pneumonia, transient tachypnea, mild asphyxia, G6PD deficiency, anemia, hyperbilirubinemia, and classification as an infant of a diabetic mother. Both parents were non-consanguineous, with no family history of inherited or metabolic disorders.

He received supportive treatment in the NICU, including oxygen therapy, phototherapy for hyperbilirubinemia, empiric antibiotic therapy, and nutritional support. After clinical improvement, he was discharged in stable condition with scheduled multidisciplinary follow-up.

Given the complex and nonspecific presentation, trio-based WES (trio-WES) was performed with parental consent. The analysis identified compound heterozygous missense variants in MCM4 (c.1829G>A and c.2179G>C; Figure 1), compound heterozygous variants in RYR1 consisting of a canonical splice site mutation (c.11608+1G>A; Figure 2) and a deep intronic variant (c.13660-29G>A; Figure 2), and a hemizygous pathogenic variant in G6PD (c.482G>T, p.Gly161Val; Figure 3). All variants were inherited from the parents, consistent with autosomal recessive and X-linked inheritance patterns, and no pathogenic copy number variants were detected.

Figure 1 Pedigree illustrating the inheritance of MCM4 variants. The proband inherited compound heterozygous missense variants in MCM4 (c.1829G>A from the mother and c.2179G>C from the father). These variants are consistent with autosomal recessive inheritance. Both parents are asymptomatic heterozygous carriers. Red arrows indicate the position of the identified variant.

Figure 2 Pedigree illustrating the inheritance of RYR1 variants. The proband carried a canonical splice site variant (c.11608+1G>A) inherited from the mother and a deep intronic variant (c.13660-29G>A) inherited from the father in the RYR1 gene. These variants are consistent with autosomal recessive inheritance and may be associated with congenital myopathy. Red arrows indicate the position of the identified variant.

Figure 3 Pedigree illustrating the inheritance of the G6PD variant. The proband carried a hemizygous pathogenic variant in G6PD (c.482G>T), inherited from his heterozygous mother. This is consistent with X-linked recessive inheritance. The mother is an asymptomatic carrier. Red arrows indicate the position of the identified variant.

At 18 months of age, the infant showed global developmental delay, mild muscle weakness, and unsteady gait. He could walk with assistance but had limited expressive language. During the 18-month follow-up period, he was hospitalized 14 times for respiratory tract infections. All procedures performed in this study were in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Ethics Committee of Lishui Municipal Central Hospital [No. 2025(I)-209-01]. Written informed consent was obtained from the patient’s parents for publication of this case report and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.

Discussion

This case underscores the diagnostic importance of trio-WES for neonates with complex and nonspecific clinical features. Conventional diagnostic approaches failed to identify the etiology, whereas genomic sequencing revealed multiple rare variants across three disease-associated genes. Recent studies have demonstrated that early genome or exome sequencing in NICUs significantly improves diagnostic yield and guides management in critically ill infants (6,7). Multilocus pathogenic variation has gained increasing attention in patients with atypical or multisystem presentations, occurring in approximately 4–7% of individuals undergoing WES. In our case, the coexistence of pathogenic variants in MCM4, RYR1, and G6PD provided a comprehensive explanation for the neonate’s blended phenotype, encompassing hypotonia, anemia, and growth restriction—findings that could not be attributed to any single variant alone.

The identified variants have distinct but interrelated functional implications. The MCM4 gene encodes a DNA helicase essential for genomic stability, and biallelic mutations have been linked to immunodeficiency-54 and growth failure. In our patient, intrauterine growth restriction and postnatal weakness likely reflect MCM4-related replication dysfunction, although no overt immunodeficiency has yet appeared—consistent with reports describing delayed or incomplete immune phenotypes (8). However, the patient’s recurrent respiratory infections (occurring 14 times over 18 months of follow-up) may suggest subtle immunologic abnormalities. A previous study on MCM4-related disorders has described variable immune phenotypes, including reduced natural killer cell cytotoxicity, impaired lymphocyte proliferation, recurrent respiratory or viral infections, and adrenal insufficiency (9). These findings imply that MCM4 dysfunction may result in a continuum of immune dysregulation ranging from subclinical susceptibility to overt immunodeficiency. The RYR1 variants, including a canonical splice-site mutation and a deep intronic variant (c.13660-29G>A), may jointly disrupt calcium channel function and contribute to subclinical congenital myopathy. Deep intronic RYR1 variants have been shown to alter pre-mRNA splicing and cause severe fetal or neonatal myopathies (10). Furthermore, recent structure-based reclassification efforts have refined RYR1 variant pathogenicity and its association with malignant hyperthermia susceptibility (11). Meanwhile, the G6PD variant (c.482G>T, p.Gly161Val) accounts for the neonatal anemia and hyperbilirubinemia observed and has lifelong implications for oxidative stress management.

We hypothesize that these variants may act synergistically to exacerbate muscular and metabolic vulnerability, with MCM4 and RYR1 defects jointly impairing energy homeostasis and tissue resilience. Such blended or synergistic mechanisms among multiple disease genes have been increasingly recognized in atypical neonatal phenotypes (12). Moreover, maternal gestational diabetes may have modified the clinical phenotype, worsening intrauterine growth restriction and postnatal adaptation.

This case highlights the clinical value of comprehensive genomic testing in unexplained neonatal disorders and underscores the need for functional validation—such as RNA or protein-level assays—to confirm the pathogenicity of novel variants. Long-term follow-up evaluating neurodevelopmental, immunologic, and metabolic outcomes remains essential to define the full spectrum of multilocus pathogenic variation.

Conclusions

To our knowledge, this is the first reported case involving concurrent variants in MCM4, RYR1, and G6PD. It expands the known phenotypic spectrum of multilocus pathogenic variation and reinforces the clinical utility of trio-WES in diagnosing complex neonatal conditions.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the CARE reporting checklist. Available at https://acr.amegroups.com/article/view/10.21037/acr-2025-131/rc

Peer Review File: Available at https://acr.amegroups.com/article/view/10.21037/acr-2025-131/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://acr.amegroups.com/article/view/10.21037/acr-2025-131/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in this study were in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Ethics Committee of Lishui Municipal Central Hospital [No. 2025(I)-209-01]. Written informed consent was obtained from the patient’s parents for publication of this case report and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Han M, Shi W, Chen X, et al. Genetic Manifestations and Phenotype Spectrum in Infants With Feeding Difficulty. Mol Genet Genomic Med 2024;12:e70001. [Crossref] [PubMed]
2. Wright CF, FitzPatrick DR, Firth HV. Paediatric genomics: diagnosing rare disease in children. Nat Rev Genet 2018;19:253-68. [Crossref] [PubMed]
3. Retterer K, Juusola J, Cho MT, et al. Clinical application of whole-exome sequencing across clinical indications. Genet Med 2016;18:696-704. [Crossref] [PubMed]
4. Posey JE, Harel T, Liu P, et al. Resolution of Disease Phenotypes Resulting from Multilocus Genomic Variation. N Engl J Med 2017;376:21-31. [Crossref] [PubMed]
5. Yang Y, Muzny DM, Xia F, et al. Molecular findings among patients referred for clinical whole-exome sequencing. JAMA 2014;312:1870-9. [Crossref] [PubMed]
6. Zhang R, Cui X, Zhang Y, et al. Whole-exome sequencing as the first-tier test for patients in neonatal intensive care unit: a Chinese single-center study. BMC Pediatr 2024;24:351. [Crossref] [PubMed]
7. Marom D, Mory A, Reytan-Miron S, et al. National Rapid Genome Sequencing in Neonatal Intensive Care. JAMA Netw Open 2024;7:e240146. [Crossref] [PubMed]
8. Gineau L, Cognet C, Kara N, et al. Partial MCM4 deficiency in patients with growth retardation, adrenal insufficiency, and natural killer cell deficiency. J Clin Invest 2012;122:821-32. [Crossref] [PubMed]
9. Hughes CR, Guasti L, Meimaridou E, et al. MCM4 mutation causes adrenal failure, short stature, and natural killer cell deficiency in humans. J Clin Invest 2012;122:814-20. [Crossref] [PubMed]
10. Hou W, Huang G, Wei H, et al. A novel homozygous intronic variant affecting splicing in the RYR1 gene contributes to fetal hydrops. Genes Dis 2024;11:101375. [Crossref] [PubMed]
11. van den Bersselaar LR, Schiemann AH, Yang CY, et al. Pathogenicity assessment of seven RYR1 variants in patients with confirmed susceptibility to malignant hyperthermia in the Netherlands. Br J Anaesth 2026;136:317-25. [Crossref] [PubMed]
12. Bozkurt-Yozgatli T, Pehlivan D, Gibbs RA, et al. Multilocus pathogenic variants contribute to intrafamilial clinical heterogeneity: a retrospective study of sibling pairs with neurodevelopmental disorders. BMC Med Genomics 2024;17:85. [Crossref] [PubMed]