Limb malformations are paradigmatic of altered gene regulation in human disease. Nail-Patella Syndrome (NPS) is a rare condition characterized mainly by skeletal defects, glomerulonephritis and glaucoma, with variable expressivity. NPS is caused by the haploinsufficiency or loss-of-function of LMX1B, which encodes a transcription factor involved in limb dorsalization, in the renal glomerular filtration barrier and the anterior segment of the eye. The dorsal expression of LMX1B in the developing limbs is under the control of LMX1B autoregulatory modules (LARMs), which are non-coding cis-regulatory elements (CREs) with a limb-specific enhancer activity. Here, we describe the regulatory landscape and report regulatory anomalies at the LMX1B locus in four families, including the deletion of a CRE, two structural variations disrupting the CRE-promoter interaction, and a 5’UTR variant causing an upstream open reading frame (ORF). Molecular mechanisms involving the non-coding genome can have a tissue-specific impact on gene expression, resulting in incomplete forms of the syndrome, and sometimes modifying its classical mode of inheritance. While approximately 95% of individuals with NPS carry pathogenic variants in the coding regions of LMX1B, non-coding alterations explain the remaining cases. This work highlights the importance of genomic diagnosis (gene ORF versus CRE alteration) for precision medicine and genetic counselling in rare diseases.

Background and ObjectivesDevelopmental and epileptic encephalopathies (DEEs) with early burst-suppression EEG (EIDEE-BS) are among the most severe neonatal epileptic syndromes, typically presenting in the first months of life with refractory seizures and profound neurodevelopmental impairment. Although variants in the KCNQ2, STXBP1, and SCN2A genes are recognized as major causes, the full genetic spectrum remains uncertain. We aimed to delineate the electroclinical characteristics, genetic etiologies, and long-term outcomes in a large MRI-negative EIDEE-BS cohort.MethodsWe retrospectively analyzed 110 patients with BS EEG enrolled from a database of 1,540 individuals with suspected genetic epilepsies (2008-2023). Clinical, EEG, and genetic data were systematically collected. Patients were stratified into 4 groups: KCNQ2, STXBP1, "other pathogenic variants," and "without a genetic diagnosis." EEG traces were reviewed independently, and outcomes were assessed through long-term follow-up.ResultsPathogenic or likely pathogenic variants were identified in 62.7% of patients and involved 23 genes, including 2 copy number variants. KCNQ2 (n = 24) and STXBP1 (n = 16) accounted for one-third of diagnoses, whereas SCN2A (n = 3) and KCNT1 (n = 2) were less frequent. In KCNQ2 cases, seizures and BS onset occurred earlier than in STXBP1 cases: mean 2 days vs 6 weeks for seizures and 3 days vs 2 months for BS, respectively. A typical BS pattern (bursts longer than suppressions) strongly correlated with KCNQ2 and STXBP1 variants. Novel associations were found with DPM1, GRIN2A, KCNT2, PIGO, PURA, WWOX, and candidate genes (KMT2E, SNAP25, and SYT1). Most variants were de novo heterozygous; however, recessive and X-linked inheritance patterns were also observed. Mortality was high (25%), primarily from status epilepticus and complications of severe disability. Most patients (72.5%) had persistent seizures at follow-up (a mean of 6.5 years), as well as profound intellectual disabilities, irrespective of genotype.DiscussionThis large series highlights the strong monogenic basis of EIDEE-BS. KCNQ2, STXBP1, and SCN2A were the most commonly affected genes. Early EEG features, particularly BS timing and morphology, can help anticipate the underlying genotype and guide precision therapy, including the early use of sodium channel blockers in selected cases. These findings support recent ILAE reclassification efforts and underscore the importance of comprehensive genomic testing for improved diagnosis and counseling.

The CNTNAP2 gene encodes CASPR2, a transmembrane protein essential for neuronal development and synaptic function. Biallelic pathogenic variants cause Pitt-Hopkins-like syndrome, characterized by intellectual disability, epilepsy, and autistic features. We report two patients with a Pitt-Hopkins-like phenotype carrying compound heterozygous structural variants: an intragenic deletion in trans with a paracentric inversion. Short-read genome sequencing detected both variants, and long-read sequencing refined one breakpoint. Non-reccurent deletions involved exon 3, while CNTNAP2 breakpoints for both inversions were located in intron 1. These findings broaden the mutational spectrum of CNTNAP2 and underscore the value of genome sequencing in identifying complex structural variants.

Background and aims COX16 is a nuclear-encoded assembly factor essential for mitochondrial cytochrome c oxidase (complex IV) biogenesis. Only two patients with COX16-related disease have previously been reported. Methods We describe two siblings born to consanguineous parents who presented with neonatal hypotonia, respiratory failure, lactic acidosis and early death. Neuroimaging revealed diffuse white matter abnormalities; neither had cardiac involvement. Muscle and fibroblast studies demonstrated isolated complex IV deficiency. Whole-exome sequencing identified a novel homozygous intronic COX16 variant (c.70-11_70-8del), extremely rare in population databases and observed only in the heterozygous state and predicted to have minimal splicing impact. Results Reverse transcription-PCR and long-read complementary DNA sequencing confirmed complete exon 2 skipping in patient fibroblasts. Blue native polyacrylamide gel electrophoresis showed defective complex IV assembly. Conclusions These findings establish the pathogenicity of a splice-altering COX16 variant and expand the genetic and phenotypic spectrum of COX16 -related mitochondrial disease. Our report underscores the importance of transcript-level assays when in silico predictions are inconclusive and highlights the usefulness of integrated genomic and functional approaches.

BACKGROUND AND OBJECTIVES:Developmental and epileptic encephalopathies (DEEs) with early burst-suppression EEG (EIDEE-BS) are among the most severe neonatal epileptic syndromes, typically presenting in the first months of life with refractory seizures and profound neurodevelopmental impairment. Although variants in the KCNQ2, STXBP1, and SCN2A genes are recognized as major causes, the full genetic spectrum remains uncertain. We aimed to delineate the electroclinical characteristics, genetic etiologies, and long-term outcomes in a large MRI-negative EIDEE-BS cohort. METHODS:We retrospectively analyzed 110 patients with BS EEG enrolled from a database of 1,540 individuals with suspected genetic epilepsies (2008-2023). Clinical, EEG, and genetic data were systematically collected. Patients were stratified into 4 groups: KCNQ2, STXBP1, "other pathogenic variants," and "without a genetic diagnosis." EEG traces were reviewed independently, and outcomes were assessed through long-term follow-up. RESULTS:Pathogenic or likely pathogenic variants were identified in 62.7% of patients and involved 23 genes, including 2 copy number variants. KCNQ2 (n = 24) and STXBP1 (n = 16) accounted for one-third of diagnoses, whereas SCN2A (n = 3) and KCNT1 (n = 2) were less frequent. In KCNQ2 cases, seizures and BS onset occurred earlier than in STXBP1 cases: mean 2 days vs 6 weeks for seizures and 3 days vs 2 months for BS, respectively. A typical BS pattern (bursts longer than suppressions) strongly correlated with KCNQ2 and STXBP1 variants. Novel associations were found with DPM1, GRIN2A, KCNT2, PIGO, PURA, WWOX, and candidate genes (KMT2E, SNAP25, and SYT1). Most variants were de novo heterozygous; however, recessive and X-linked inheritance patterns were also observed. Mortality was high (25%), primarily from status epilepticus and complications of severe disability. Most patients (72.5%) had persistent seizures at follow-up (a mean of 6.5 years), as well as profound intellectual disabilities, irrespective of genotype. DISCUSSION:This large series highlights the strong monogenic basis of EIDEE-BS. KCNQ2, STXBP1, and SCN2A were the most commonly affected genes. Early EEG features, particularly BS timing and morphology, can help anticipate the underlying genotype and guide precision therapy, including the early use of sodium channel blockers in selected cases. These findings support recent ILAE reclassification efforts and underscore the importance of comprehensive genomic testing for improved diagnosis and counseling.

BACKGROUND: Recent advances in sequencing technologies have enhanced patient diagnosis; however, causal pathogenic variants remain unidentified for a significant number of patients due to limited understanding of certain variants, regulatory sequences, or sequencing challenges, such as complex rearrangements. Investigating the epigenetic landscape has become essential to improve the diagnostic yield. Diseases caused by pathogenic variants in epigenetic regulators, often associated with growth abnormalities, intellectual disability, and facial dysmorphism, are prime models for studying episignatures. Among them, Snijders Blok-Campeau syndrome (ORPHA:599082), caused by pathogenic variants in the CHD3 gene, remains largely understudied. METHODS: A European cohort of 23 patients displaying typical Snijders Blok-Campeau syndrome traits and carrying pathogenic/likely pathogenic CHD3 variants was analysed using the Illumina EPIC array, identifying 270 differentially methylated positions distinguishing patients from 62 healthy matched controls. A subset of these regions serves as diagnostic tools for complex cases or variants of uncertain significance and helps uncover deregulated pathways linked to this syndrome. Four patients carrying pathogenic/likely pathogenic variants but with atypical clinical presentation, as well as 10 patients with variants of uncertain significance, were analysed as the testing set. RESULTS: Comparing methylomes of patients carrying pathogenic variants in CHD3, CHD7 (CHARGE syndrome, ORPHA:138), and CHD8 (Intellectual developmental disorder with autism and macrocephaly, ORPHA:642675) genes allows us to identify distinct subgroups with unique methylation profiles. This CHD3 DNA methylation signature aids in reclassifying variants and diagnosing atypical cases. CONCLUSIONS: Our findings advance the field of epigenetic signatures in rare diseases. We have opened new avenues for further investigation into subtypes defined by methylome assays (such as in the context of chromatinopathies), which could refine the phenotype spectrum and help predict patient outcomes.

Pathogenic variants in the nuclear gene NDUFAF8 are a rare cause of mitochondrial complex I deficiency with only three cases described to date. We report here a new case of NDUFAF8 deficiency confirming the phenotype of NDUFAF8-induced complex I biochemical defect, Leigh syndrome and premature death. As a mitochondrial DNA variant in a gene encoding a complex I subunit was also identified in this patient, we discuss the molecular heterogeneity of Leigh syndrome and the need to explore the mitochondrial and nuclear genome to ensure a reliable diagnosis.

The major spliceosome contains five small nuclear RNAs (snRNAs; U1, U2, U4, U5 and U6) essential for splicing. Variants in RNU4-2, encoding U4, cause a neurodevelopmental disorder called ReNU syndrome. We investigated de novo variants in 50 snRNA-encoding genes in a French cohort of 23,649 individuals with rare disorders and gathered additional cases through international collaborations. Altogether, we identified 145 previously unreported probands with (likely) pathogenic variants in RNU4-2 and 21 individuals with de novo and/or recurrent variants in RNU5B-1 and RNU5A-1, encoding U5. Pathogenic variants typically arose de novo on the maternal allele and cluster in regions critical for splicing. RNU4-2 variants mainly localize to two structures, the stem III and T-loop/quasi-pseudoknot, which position the U6 ACAGAGA box for 5' splice site recognition and associate with different phenotypic severity. RNU4-2 variants result in specific defects in alternative 5' splice site usage and methylation patterns (episignatures) that correlate with variant location and clinical severity. This study establishes RNU5B-1 as a neurodevelopmental disorder gene, suggests RNU5A-1 as a strong candidate and highlights the role of de novo variants in snRNAs.

The GenIDA project aims to improve the understanding and management of rare genetic forms of intellectual disability by fostering collaboration among patients, caregivers, healthcare professionals, and research professionals. Clinical data is provided by patients' families via a structured questionnaire to identify medically relevant insights and better understand the natural history of rare diseases. This study focused on MED13L syndrome, analyzing data from 41 patients in the GenIDA database and comparing it with 102 cases from the scientific literature and 6 new descriptions of patients from our medical center.The GenIDA series confirmed the key features of MED13L syndrome, including global developmental delay, poor speech, intellectual disability, and cardiac defects (OMIM #616789), at frequencies similar to those reported in the literature. The GenIDA series identified a higher prevalence of visual impairment (76%) and highlighted under-recognized musculoskeletal issues, such as foot deformities, which had previously received little attention. This study highlights the value of family-reported data in describing the full phenotype of rare syndromes. A comprehensive review of published cases showed that patients with missense variants have more severe impairments, including increased cardiac defects, global developmental delay, and a higher incidence of epilepsy, than patients with premature truncated variants.These findings highlight the importance of family involvement in rare disease research and the need for further studies to explore genotype-phenotype correlations to improve patient care and outcomes.

Structural variants (SVs) significantly contribute to human disease, but their complexity often makes accurate characterization difficult with conventional methods. Advances in long-read sequencing (LRS) offer potential by spanning kilobases and directly resolving SVs. In this study, we examined two individuals with unresolved SVs. LRS on both DNA and cDNA provided single-base resolution of all breakpoint junctions, revealing detailed rearrangement structures and underlying mechanisms. Transcriptomic analyses identified abnormal fusion transcripts and clarified their functional consequences, including haploinsufficiency and potential dominant-negative effects. In one case, a triplication affecting the ZMYM2 gene was precisely mapped, revealing a truncated variant that may escape nonsense-mediated decay. In the second case, a highly complex reciprocal translocation involving RERE and FHAD1 disrupted RERE expression, with Hi-C data showing minimal impact on enhancer-promoter interactions. Due to their complexity, these SVs were not fully resolved by standard methods. By integrating LRS with transcriptomic and chromosomal conformation analyses, we provided a comprehensive understanding of SV formation and its pathogenic impact. Our findings emphasize the need for advanced genomic approaches to resolve complex SVs, enhance diagnostic accuracy, and inform clinical management.

The X-linked NONO gene encodes Non-Pou Domain-Containing Octamer-Binding Protein, a multifunctional member of the DBHS family involved in transcriptional regulation, RNA splicing and DNA repair. Pathogenic variants in NONO cause Intellectual Developmental Disorder, X-linked Syndromic (MIM #300967), characterised by intellectual disability, neurodevelopmental delay, cardiomyopathy, such as left ventricular non-compaction (LVNC), and congenital heart defects such as including atrial septal defect (ASD), ventricular septal defect (VSD), patent ductus arteriosus (PDA), and patent foramen ovale (PFO). This study reports three new patients with pathogenic hemizygous frameshift variants in NONO identified with exome sequencing, broadening the clinical presentation. The patients present with neurodevelopmental delay, macrocephaly, agenesis or hypoplasia of the corpus callosum and LVNC, confirming previous findings. These findings contribute to the understanding of the phenotypic diversity in patients with NONO pathogenic variants and highlight the need for further investigation of genotype-phenotype correlations, particularly with regard to early cardiac development, and prenatal presentations.

Background SATB2-associated syndrome (SAS) results from various mutations of the SATB2 gene and associates a neurodevelopmental disorder including major speech delay, intellectual disability, and behavioral problems with dental anomalies, sometimes a cleft palate, risk of osteoporosis, and facial dysmorphism. The principal objective of this study was to describe the oral phenotype of young children with SATB2-associated syndrome, especially in terms of orofacial malformation of Robin Sequence (RS) spectrum (bifid uvula, cleft palate, or RS, dental malformation, feeding and communication, with data from a national cohort. The secondary objective was to determine whether feeding and communication disorders were more severe when associated with an orofacial malformation of RS spectrum. Methods We conducted a retrospective cross-sectional study among the largest possible cohort of patients with a mutation of the SATB2 gene in France. A questionnaire completed by the referring physicians and by telephone with parents enabled us to collect the following clinical information: (1) orofacial morphology, feeding difficulties, and pharyngeal functioning from birth to 3 years, (2) communication and language from 0 to 6 years, (3) speech development at the last examination. Results The study included 40 patients. Early and persistent feeding difficulties were found in 55% of the children. Communication was abnormal from the first months of life, with poor babbling in 85% of them. A major language delay was described in all patients; 65% had a vocabulary of 10 words or less. An anomaly of RS spectrum was found in half the cases, and dental malformations were described in 90%. Feeding difficulties and language delay were greater in the group with one or more orofacial malformations than the group with none. Conclusion This study confirmed the severity of oral involvement, affecting feeding and speech simultaneously, in individuals with SAS. It raises the question of why the oral phenotype involving feeding and speech is more severe in the presence of cleft palate or RS. We recommend close monitoring of prelanguage communication in infants with apparently isolated cleft palate or RS and the search for SATB2 impairment when a cleft palate or RS is found, especially in the prenatal period.

OBJECTIVE:Fibroblast growth factor 12 (FGF12), a member of the fibroblast homologous factor family, plays a key role in the modulation of voltage-gated sodium (Nav) channels. Pathogenic variants in the FGF12 gene leading to a gain-of-function mechanism and partial duplication encompassing the FGF12 gene leading to a loss-of-function mechanism are associated with developmental and epileptic encephalopathy (DEE), characterized by developmental delay, intellectual disability, ataxia, and drug-resistant epilepsy. We report two patients with DEE harboring de novo recurrent intragenic duplications of FGF12 identified by long-read sequencing (LRS). METHODS:We applied LRS to the DNA and cDNA of patients with FGF12 duplication to fully characterize the DNA's structural organization and its transcriptional consequences. Additionally, we reanalyzed electroencephalographic (EEG) data from patients at different timepoints to identify phenotypical specificities and refine the electroclinical spectrum. RESULTS:These duplications, spanning approximately 536 kbp, were mediated by nonallelic homologous recombination between L1PA2 elements (LINE-1 Primate-specific subfamily A, number 2). cDNA analysis revealed aberrant transcripts, one predicted to encode an elongated FGF12 protein and another leading to premature termination. Both patients shared overlapping clinical features, including postepilepsy onset regression, global developmental delay, and ataxia. EEG studies revealed a marked early encephalopathic pattern with disorganized and high-voltage slow background activity with multifocal spikes at onset evolving later into subcontinuous generalized spike and wave activation. SIGNIFICANCE:Our findings are consistent with previous reports linking structural variants to functional disruption, suggesting impaired Nav channel activity due to a shift in inactivation to hyperpolarized potential, leading to a loss-of-function effect. These findings underscore the utility of LRS for DNA and cDNA analysis in resolving structural variants and expanding the electroclinical spectrum of patients with FGF12 duplications.

Pathogenic heterozygous variants in CHD4 cause Sifrim-Hitz-Weiss syndrome, a neurodevelopmental disorder associated with brain anomalies, heart defects, macrocephaly, hypogonadism, and additional features with variable expressivity. Most individuals have non-recurrent missense variants, complicating variant interpretation. A few were reported with truncating variants, and their role in disease is unclear. DNA methylation episignatures have emerged as highly accurate diagnostic biomarkers in a growing number of rare diseases. We aimed to study evidence for the existence of a CHD4-related DNA methylation episignature. We collected blood DNA samples and/or clinical information from 39 individuals with CHD4 variants, including missense and truncating variants. Genomic DNA methylation analysis was performed on 28 samples. We identified a sensitive and specific DNA methylation episignature in samples with pathogenic missense variants within the ATPase/helicase domain. The same episignature was observed in a family with variable expressivity, a de novo variant near the PHD domain, variants of uncertain significance within the ATPase/helicase domain, and a sample with compound heterozygous variants. DNA methylation data revealed higher percentages of shared probes with BAFopathies, CHD8, and the terminal ADNP variants encoding a protein known to form the ChAHP complex with CHD4. Truncating variants, as well as a sample with a recurrent pathogenic missense variant, exhibited DNA methylation profiles distinct from the ATPase/helicase domain episignature. These DNA methylation differences, together with the distinct clinical features observed in those individuals, provide preliminary evidence for clinical and molecular sub-types in the CHD4-related disorder.

Heterozygous pathogenic variants in the Mediator complex subunit 13-like gene located in the locus 12q21.21 (MED13L) are associated with intellectual disability, developmental delay, and distinctive facial features. While nonsense and frameshift variants typically cause haploinsufficiency, resulting in a well-characterized clinical presentation, missense variants have been associated with a broader range of phenotypes, including epilepsy and severe motor delay. In this study, we investigated five pathogenic missense variants in MED13L-c.2597C>T p.Pro866Leu, c.2605C>T p.Pro869Ser, c.3392G>A p.Cys1131Tyr, c.5695G>A p.Gly1899Arg, and c.6485C>T p.Thr2162Met-associated with different clinical severities. We identified significant reductions in protein stability across these variants, with some exhibiting aberrant cytoplasmic localization, suggesting disruptions in structural integrity and function. In particular, exon 15 variants (p.Pro866Leu and p.Pro869Ser) correlated with severe phenotypes, including epilepsy and severe motor impairment, whereas p.Gly1899Arg and p.Thr2162Met were associated with milder manifestations. 3D protein modeling suggested that these missense variants may disrupt MED13L's interaction with the CDK8 kinase module, leading to functional deficits. Our findings highlight different pathogenic mechanisms, ranging from protein instability to altered molecular interactions, that contribute to the clinical variability observed in MED13L-related disorders.

Recent advances in the understanding of infantile developmental epileptic encephalopathies (IDEE) have revealed the association of biallelic pathogenic variants in UGDH. In this study, we report two novel combinations identified by exome sequencing: p.(Arg135Trp) with p.(Arg65*) and p.(Arg102Trp) with p.(Arg65*). Both combinations share a common pathogenic nonsense variant, with the missense variants strategically located in the NAD-binding domain of the UGDH protein, predicted in structural models to create new interactions with the central domain. The first patient exhibited the typical UGDH-related disease phenotype and progressive microcephaly, a rarely reported feature. In contrast, the second patient presented an atypical phenotype, including absence of seizure, severe intellectual disability, ataxic gait, and abnormal eye movements. This comprehensive analysis extends the phenotypic spectrum of UGDH syndrome beyond early infantile intractable encephalopathy to include intellectual disability without epilepsy.