X-linked intellectual disability (XLID) constitutes a significant subset of inherited neurodevelopmental disorders, resulting from pathogenic variants in genes located on the X-chromosome, and it accounts for approximately 10% of inherited intellectual disability cases (1). Over 140 XLID-associated genes have been identified to date, including DDX3X, USP9X, KDM6A, KDM5C, SMC1A, HDAC8, PCDH19, MECP2, FMR1, and CDKL5. Males are predominantly and severely affected due to hemizygosity for X-linked genes. Female carriers, benefiting from random X-chromosome inactivation (XCI), are typically asymptomatic or exhibit milder phenotypes. However, de novo pathogenic variants combined with skewed XCI (SXCI), where the X chromosome carrying the wild-type allele is predominantly inactivated, can disrupt this protective mechanism, leading to full phenotypic expression in females (2, 3). Recently, several X-linked disorders that disproportionately affect females have been described, illustrating diverse inheritance modes such as semi-dominant (e.g., DDX3X), co-existing recessive and dominant patterns (e.g., USP9X), and unique phenotypes resulting from cellular mosaicism (e.g., PCDH19). These conditions highlight the expanding spectrum of X-linked disorders with female-predominant presentation, complex inheritance patterns, and notable sex differences (4).

The NONO gene (OMIM *300084), located at Xq13.1, encodes a multifunctional nuclear protein belonging to the Drosophila Behavior Human Splicing (DBHS) family. NONO plays crucial roles in transcriptional regulation, RNA processing (including splicing, stability, and nuclear retention), and DNA repair (5). NONO is also essential for neurodevelopment: studies in mouse models demonstrate that its deficiency impairs TET1 chromatin association and neuronal differentiation (6), and disrupts cortical neuronal migration and postnatal maturation (7). These mechanisms likely underlie the synaptic and cognitive deficits observed in humans with NONO-related disorders. Pathogenic variants in NONO cause MRXS34 (OMIM #300967), characterized by intellectual disability/global developmental delay, distinctive craniofacial dysmorphism (e.g., relative macrocephaly, long face, micrognathia), cardiac defects (notably left ventricular non-compaction—LVNC, septal defects), and structural brain abnormalities. Since its initial description in 2015, all 27 reported postnatal MRXS34 cases have been male (8–12). A single female fetus with a NONO missense variant was prenatally diagnosed but not molecularly confirmed postnatally.

Here, we present the first molecularly confirmed female patient with MRXS34 harboring a de novo NONO frameshift variant. Extreme skewing of XCI and the significantly reduced expression NONO gene confirmed by RNA-Seq, leading to the characteristic neurodevelopmental and cardiac phenotype. This case expands the known genotype-phenotype spectrum of MRXS34, provides evidence for the role of non-random XCI in female expression of XLID disorders, and offers critical insights for the diagnosis of X-linked recessive conditions in females.

This study presents the first molecularly confirmed female patient with MRXS34, resulting from a de novo NONO frameshift variant (c.994del, p.Gln322Lysfs*31) and extreme skewing of XCI (99% paternal inactivation). This mechanism effectively created a functional hemizygous state for the mutant allele, overriding the typical protective effect of random XCI in female carriers of X-linked recessive disorders (2, 3). Her phenotype—global developmental delay, relative macrocephaly, hypotonia, PFO, tricuspid regurgitation, and white matter abnormalities—aligns remarkably well with the core features observed in affected males (8–11), although her cardiac manifestations (PFO, resolving TR) were milder than the prevalent LVNC in males. This case critically expands the clinical spectrum of MRXS34 and provides a paradigm for understanding female expression in XLID disorders.

NONO, a core member of the DBHS protein family (alongside SFPQ and PSPC1), functions as a multifunctional nuclear scaffold protein (13). Its domains (RRM1, RRM2, NOPS, Coiled-coil) facilitate interactions with DNA, RNA, and other proteins, enabling roles in transcriptional regulation, RNA processing (splicing, nuclear retention), DNA repair, and paraspeckle formation (5). NONO is crucial for neurodevelopment: knockout mice exhibit cerebellar defects, impaired synaptic function (particularly involving GABAergic transmission via genes like GABRA2), spatial memory deficits, and anxiety-like behaviors, mirroring the human intellectual disability phenotype (8). In cardiac development, NONO deficiency disrupts cardiomyocyte differentiation and promotes fibroblast proliferation, leading to LVNC and other structural defects (14). Its recent identification as a key candidate for X-linked contributions to congenital heart disease (CHD) gender differences further underscores its systemic importance (15). Additional roles in circadian rhythm regulation, metabolic gene coordination, DNA damage response via R-loop modulation, and myogenesis, highlight the pleiotropic effects of NONO dysfunction, explaining the multi-system involvement in MRXS34 (neurodevelopmental, cardiac, potential musculoskeletal) (16–18). The truncating variant reported here (p.Gln322Lysfs*31), located within the essential NOPS dimerization domain, is predicted to undergo NMD, resulting in haploinsufficiency, consistent with the observed significant reduction in NONO transcript levels.

The phenotypic spectrum of MRXS34, now encompassing both sexes, is characterized by near-universal neurodevelopmental delay (GDD/ID), distinctive craniofacial features (macrocephaly, long face, micrognathia), and a high prevalence of cardiac defects (91.3%, LVNC being hallmark) (Table 1). Structural brain anomalies, particularly corpus callosum dysgenesis (85%), are highly characteristic. While our proband lacked overt callosal abnormalities, she exhibited white matter signal changes, suggesting a broader spectrum of neurological involvement. Hypotonia and feeding difficulties are common. Seizures occur but are less frequent (∼7%). Skeletal issues (scoliosis, joint laxity) and ophthalmological problems (strabismus) are also reported (19). Prenatal cases uniformly present with severe, often complex, cardiac malformations, frequently associated with other anomalies and poor outcomes (11, 16–20). Compared to typical severe male presentations, our female patient had relatively milder cardiac involvement (PFO vs. LVNC), potentially reflecting partial functional compensation from the residual wild-type allele expression on the extremely inactivated paternal X chromosome, particularly relevant in cardiac tissue. Alternatively, tissue-specific XCI patterns or modifier genes might attenuate cardiac severity in females, as suggested by studies on X-linked congenital heart disease contributions. However, the degree of neurodevelopmental impairment was substantial and comparable to males, suggesting the developing nervous system is exquisitely sensitive to NONO dosage.

The predominance of LOF variants (85.7%) scattered across NONO gene, underscores haploinsufficiency as the primary disease mechanism (Figure 4). Most LOF variants trigger NMD, leading to absent or severely reduced NONO protein, as demonstrated in patient fibroblasts (8). Compensatory upregulation of other DBHS proteins (SFPQ, PSPC1) occurs but is insufficient to rescue the phenotype (20) or potential structural disruption (e.g., disulfide bond formation interference) (13). The genotype-phenotype correlation remains complex; recurrent variants like p.Arg365* and p.Ala377 = (splicing defect) cause variable severity, likely influenced by genetic background and modifying factors (8, 21). ur novel variant (p.Gln322Lysfs*31) fits the LOF paradigm, causing NMD and protein truncation upstream of the critical coiled-coil domain. This case emphasizes that NONO screening is warranted not only in males with ID + cardiac defects (especially LVNC) +/- corpus callosum anomalies but also in females presenting with compatible phenotypes, necessitating adjunct XCI analysis.

The extreme XCI skewing (99:1) observed here is the key determinant enabling full phenotypic expression in our female patient. This mechanism parallels other X-linked disorders like Incontinentia Pigmenti or X-linked Lissencephaly in females (21). Skewed XCI is increasingly recognized as a diagnostic clue in unsolved neurodevelopmental cases, guiding re-evaluation of X-linked genes. Such skewing is common and influences disease severity in conditions like Fabry disease and various neurodevelopmental disorders (22). Additionally, XCI is often incomplete, with many genes “escaping” silencing, which creates natural differences in gene expression between males and females and contributes to individual variation (23). Crucially, testing for XCI skewing in undiagnosed patients—especially females with neurodevelopmental issues—is a powerful diagnostic tool, as it can reveal hidden disease-causing mutations on the X chromosome, significantly improving diagnostic rates (24). Studies on tissue-specific XCI landscapes further illustrate how inactivation patterns can modulate phenotypic severity across different organ systems (25, 26). Previously reported asymptomatic female carriers likely had random or favorable (wild-type active) XCI. The single prenatally diagnosed female fetus with a NONO VUS (p.Arg142His) highlights the diagnostic challenge and the need for functional studies and postnatal follow-up in such cases (27). Our patient's de novo frameshift variant (c.994del), coupled with 99% paternal X-chromosome inactivation, resulted in predominant mutant allele expression, overcoming typical X-linked protective mechanisms and manifesting as characteristic neurodevelopmental deficits alongside mild cardiac anomalies (patent foramen ovale). This parallels the pathophysiology of X-linked disorders like Incontinentia Pigmenti, emphasizing the diagnostic necessity of X-inactivation analysis in female evaluations (3). While the patient's cardiac phenotype was milder than typical male cases (possibly due to partial maternal X-chromosome compensation), the comparable severity of neurodevelopmental impairment suggests heightened nervous system sensitivity to NONO gene dosage effects. This case represents the first molecularly confirmed live-born female MRXS34 patient globally, contrasting with the single prior Chinese report (a male with p.Arg153* variant and asymptomatic female carriers) (9). By demonstrating how extreme XCI skewing can unmask X-linked recessive phenotypes in females, this case redefines the diagnostic paradigm for gender differences in X-linked disorders and significantly expands the clinical understanding of MRXS34.

For couples with a family history or previous affected child, genetic counseling should be offered, noting a 50% transmission risk to male offspring (who will be affected) and female offspring (who will typically be asymptomatic carriers). Prenatal diagnosis (via CVS/amniocentesis with WES or NONO-specific testing) and preimplantation genetic testing (PGT) are viable options, with prenatal ultrasound focusing on relative macrocephaly, cardiac defects (particularly LVNC, septal defects, and Ebstein anomaly), and corpus callosum abnormalities. Neonates/infants with suspected MRXS34 require immediate cardiac and respiratory evaluation due to risks of heart failure or pulmonary hypertension, alongside essential brain MRI and echocardiography (28). Early intervention for hypotonia, feeding difficulties (including potential NG/G-tube placement), and gastroesophageal reflux is crucial, as is prompt initiation of multidisciplinary rehabilitation (physical, occupational, and speech therapy). Long-term management involves regular monitoring of growth, nutrition, cardiac and neurological function, vision, hearing, and orthopedic status (e.g., scoliosis), with developmental assessments (e.g., Gesell, Bayley) guiding tailored educational and therapeutic support; anti-seizure medications may be necessary if epilepsy develops. Prognosis varies based on cardiac, neurological, and systemic involvement severity, with prenatal cases often having poor outcomes. While postnatal survival into adulthood is documented, significant neurodevelopmental disability is universal. The use of mouse nerve growth factor in our patient's rehabilitation regimen was based on its neurotrophic properties, which may support synaptic plasticity and neuronal survival in neurodevelopmental disorders; however, specific evidence in MRXS34 remains limited and warrants further investigation.

This study reports the first molecularly confirmed female case of MRXS34, caused by a de novo NONO frameshift variant (c.994del, p.Gln322Lysfs*31) and extreme skewing of X-chromosome inactivation (99%). This case definitively demonstrates that non-random XCI can unmask X-linked recessive disorders like MRXS34 in females, leading to phenotypes closely resembling those seen in affected males, albeit potentially milder in some systems (e.g., cardiac). Our findings significantly expand the clinical and genetic understanding of MRXS34, emphasizing the critical role of XCI analysis in the diagnostic workup of females with intellectual disability and compatible features. This case underscores the importance of including X-linked genes in the differential diagnosis for females with neurodevelopmental disorders and highlights NONO screening combined with XCI studies as a crucial diagnostic strategy for such patients. Future research should focus on elucidating tissue-specific effects of NONO haploinsufficiency, modifiers of XCI skewing, and potential therapeutic avenues targeting downstream pathways.