Extending the clinical spectrum of X-linked Tonne-Kalscheuer syndrome (TOKAS): new insights from the fetal perspective.

INTRODUCTION: Tonne-Kalscheuer syndrome (TOKAS) is a recessive X-linked multiple congenital anomaly disorder caused by RLIM variations. Of the 41 patients reported, only 7 antenatal cases were described. METHOD: After the antenatal diagnosis of TOKAS by exome analysis in a family followed for over 35 years because of multiple congenital anomalies in five male fetuses, a call for collaboration was made, resulting in a cohort of 11 previously unpublished cases. RESULTS: We present a TOKAS antenatal cohort, describing 11 new cases in 6 French families. We report a high frequency of diaphragmatic hernia (9 of 11), differences in sex development (10 of 11) and various visceral malformations. We report some recurrent dysmorphic features, but also pontocerebellar hypoplasia, pre-auricular skin tags and olfactory bulb abnormalities previously unreported in the literature. Although no clear genotype-phenotype correlation has yet emerged, we show that a recurrent p.(Arg611Cys) variant accounts for 66% of fetal TOKAS cases. We also report two new likely pathogenic variants in RLIM, outside of the two previously known mutational hotspots. CONCLUSION: Overall, we present the first fetal cohort of TOKAS, describe the clinical features that made it a recognisable syndrome at fetopathological examination, and extend the phenotypical spectrum and the known genotype of this rare disorder.

The detection of a strong episignature for Chung-Jansen syndrome, partially overlapping with Börjeson-Forssman-Lehmann and White-Kernohan syndromes.

Chung-Jansen syndrome is a neurodevelopmental disorder characterized by intellectual disability, behavioral problems, obesity and dysmorphic features. It is caused by pathogenic variants in the PHIP gene that encodes for the Pleckstrin homology domain-interacting protein, which is part of an epigenetic modifier protein complex. Therefore, we hypothesized that PHIP haploinsufficiency may impact genome-wide DNA methylation (DNAm). We assessed the DNAm profiles of affected individuals with pathogenic and likely pathogenic PHIP variants with Infinium Methylation EPIC arrays and report a specific and sensitive DNAm episignature biomarker for Chung-Jansen syndrome. In addition, we observed similarities between the methylation profile of Chung-Jansen syndrome and that of functionally related and clinically partially overlapping genetic disorders, White-Kernohan syndrome (caused by variants in DDB1 gene) and Börjeson-Forssman-Lehmann syndrome (caused by variants in PHF6 gene). Based on these observations we also proceeded to develop a common episignature biomarker for these disorders. These newly defined episignatures can be used as part of a multiclass episignature classifier for screening of affected individuals with rare disorders and interpretation of genetic variants of unknown clinical significance, and provide further insights into the common molecular pathophysiology of the clinically-related Chung-Jansen, Börjeson-Forssman-Lehmann and White-Kernohan syndromes.

Time to diagnosis and determinants of diagnostic delays of people living with a rare disease: results of a Rare Barometer retrospective patient survey.

Timely diagnosis is one of the most serious challenges faced by people living with a rare disease (PLWRD), and this study estimates that in Europe, the average total diagnosis time (TDT) is close to 5 years. We investigated the duration of the TDT for PLWRD in Europe, the difficulties associated with their diagnosis odyssey and the main determinants of diagnosis delays for all rare diseases (RD). We conducted a survey of PLWRD and their families using Rare Barometer, the survey initiative of EURORDIS-Rare Diseases Europe. In geographical Europe, we surveyed 6507 people living with 1675 RD in 41 countries. We then performed a descriptive analysis and ordinal logistic regressions to identify the main determinants of diagnosis delays. Average TDT is 4.7 years. 56% of respondents were diagnosed more than 6 months after a first medical contact. The main determinants of diagnosis delays are symptom onset before 30 years of age, especially during childhood (OR = 3.11; 95% CI: 2.4-4.0) and adolescence (OR = 4.79; 95% CI: 3.7-6.2), being a woman (OR = 1.22; 95% CI:1.1-1.4), living in Northern Europe (OR = 2.15; 95% CI:1.8-2.6) or Western Europe (OR = 1.96; 95% CI:1.6-2.3), the number of healthcare professionals consulted (OR = 5.15; 95% CI:4.1-6.4), misdiagnosis (OR = 2.48; 95% CI:2.1-2.9), referral to a centre of expertise (OR = 1.17; 95% CI:1.0-1.3), unmet needs for psychological support (OR = 1.34; 95% CI:1.2-1.5) and financial support (OR = 1.16; 95% CI:1.0-1.3), having a genetic disease (OR = 1.33; 95% CI:1.1-1.5) and a family history of an RD (OR = 1.36; 95% CI:1.1-1.6). These determinants can inform policies and actions to improve access to diagnosis for all PLWRD.

Expectations, needs and mid-term outcomes in people accessing to secondary findings from ES: 1st French mixed study (FIND Study).

Generation and subsequently accessibility of secondary findings (SF) in diagnostic practice is a subject of debate around the world and particularly in Europe. The French FIND study has been set up to assess patient/parent expectations regarding SF from exome sequencing (ES) and to collect their real-life experience until 1 year after the delivery of results. 340 patients who had ES for undiagnosed developmental disorders were included in this multicenter mixed study (quantitative N = 340; qualitative N = 26). Three groups of actionable SF were rendered: predisposition to late-onset actionable diseases; genetic counseling; pharmacogenomics. Participants expressed strong interest in obtaining SF and a high satisfaction level when a SF is reported. The medical actionability of the SF reinforced parents' sense of taking action for their child and was seen as an opportunity. While we observed no serious psychological concerns, we showed that these results could have psychological consequences, in particular for late-onset actionable diseases SF, within families already dealing with rare diseases. This study shows that participants remain in favor of accessing SF despite the potential psychological, care, and lifestyle impacts, which are difficult to anticipate. The establishment of a management protocol, including the support of a multidisciplinary team, would be necessary if national policy allows the reporting of these data.

DNA methylation episignature and comparative epigenomic profiling for Pitt-Hopkins syndrome caused by TCF4 variants.

Pitt-Hopkins syndrome (PTHS) is a neurodevelopmental disorder caused by pathogenic variants in TCF4, leading to intellectual disability, specific morphological features, and autonomic nervous system dysfunction. Epigenetic dysregulation has been implicated in PTHS, prompting the investigation of a DNA methylation (DNAm) "episignature" specific to PTHS for diagnostic purposes and variant reclassification and functional insights into the molecular pathophysiology of this disorder. A cohort of 67 individuals with genetically confirmed PTHS and three individuals with intellectual disability and a variant of uncertain significance (VUS) in TCF4 were studied. The DNAm episignature was developed with an Infinium Methylation EPIC BeadChip array analysis using peripheral blood cells. Support vector machine (SVM) modeling and clustering methods were employed to generate a DNAm classifier for PTHS. Validation was extended to an additional cohort of 11 individuals with PTHS. The episignature was assessed in relation to other neurodevelopmental disorders and its specificity was examined. A specific DNAm episignature for PTHS was established. The classifier exhibited high sensitivity for TCF4 haploinsufficiency and missense variants in the basic-helix-loop-helix domain. Notably, seven individuals with TCF4 variants exhibited negative episignatures, suggesting complexities related to mosaicism, genetic factors, and environmental influences. The episignature displayed degrees of overlap with other related disorders and biological pathways. This study defines a DNAm episignature for TCF4-related PTHS, enabling improved diagnostic accuracy and VUS reclassification. The finding that some cases scored negatively underscores the potential for multiple or nested episignatures and emphasizes the need for continued investigation to enhance specificity and coverage across PTHS-related variants.

FOXG1 variants can be associated with milder phenotypes than congenital Rett syndrome with unassisted walking and language development.

Since 2008, FOXG1 haploinsufficiency has been linked to a severe neurodevelopmental phenotype resembling Rett syndrome but with earlier onset. Most patients are unable to sit, walk, or speak. For years, FOXG1 sequencing was only prescribed in such severe cases, limiting insight into the full clinical spectrum associated with this gene. Next-generation sequencing (NGS) now enables unbiased diagnostics. Through the European Reference Network for Rare Malformation Syndromes, Intellectual and Other Neurodevelopmental Disorders, we gathered data from patients with heterozygous FOXG1 variants presenting a mild phenotype, defined as able to speak and walk independently. We also reviewed data from three previously reported patients meeting our criteria. We identified five new patients with pathogenic FOXG1 missense variants, primarily in the forkhead domain, showing varying nonspecific intellectual disability and developmental delay. These features are not typical of congenital Rett syndrome and were rarely associated with microcephaly and epilepsy. Our findings are consistent with a previous genotype-phenotype analysis by Mitter et al. suggesting the delineation of five different FOXG1 genotype groups. Milder phenotypes were associated with missense variants in the forkhead domain. This information may facilitate prognostic assessments in children carrying a FOXG1 variant and improve the interpretation of new variants identified with genomic sequencing.

Penetrance, variable expressivity and monogenic neurodevelopmental disorders.

PURPOSE: Incomplete penetrance is observed for most monogenic diseases. However, for neurodevelopmental disorders, the interpretation of single and multi-nucleotide variants (SNV/MNVs) is usually based on the paradigm of complete penetrance. METHOD: From 2020 to 2022, we proposed a collaboration study with the French molecular diagnosis for intellectual disability network. The aim was to recruit families for whom the index case, diagnosed with a neurodevelopmental disorder, was carrying a pathogenic or likely pathogenic variant for an OMIM morbid gene and inherited from an asymptomatic parent. Grandparents were analyzed when available for segregation study. RESULTS: We identified 12 patients affected by a monogenic neurodevelopmental disorder caused by likely pathogenic or pathogenic variant (SNV/MNV) inherited from an asymptomatic parent. These genes were usually associated with de novo variants. The patients carried different variants (1 splice-site variant, 4 nonsense and 7 frameshift) in 11 genes: CAMTA1, MBD5, KMT2C, KMT2E, ZMIZ1, MN1, NDUFB11, CUL3, MED13, ARID2 and RERE. Grandparents have been tested in 6 families, and each time the variant was confirmed de novo in the healthy carrier parent. CONCLUSION: Incomplete penetrance for SNV and MNV in neurodevelopmental disorders might be more frequent than previously thought. This point is crucial to consider for interpretation of variants, family investigation, genetic counseling, and prenatal diagnosis. Molecular mechanisms underlying this incomplete penetrance still need to be identified.

DISP1 deficiency: Monoallelic and biallelic variants cause a spectrum of midline craniofacial malformations.

PURPOSE: DISP1 encodes a transmembrane protein that regulates the secretion of the morphogen, Sonic hedgehog, a deficiency of which is a major cause of holoprosencephaly (HPE). This disorder covers a spectrum of brain and midline craniofacial malformations. The objective of the present study was to better delineate the clinical phenotypes associated with division transporter dispatched-1 (DISP1) variants. METHODS: This study was based on the identification of at least 1 pathogenic variant of the DISP1 gene in individuals for whom detailed clinical data were available. RESULTS: A total of 23 DISP1 variants were identified in heterozygous, compound heterozygous or homozygous states in 25 individuals with midline craniofacial defects. Most cases were minor forms of HPE, with craniofacial features such as orofacial cleft, solitary median maxillary central incisor, and congenital nasal pyriform aperture stenosis. These individuals had either monoallelic loss-of-function variants or biallelic missense variants in DISP1. In individuals with severe HPE, the DISP1 variants were commonly found associated with a variant in another HPE-linked gene (ie, oligogenic inheritance). CONCLUSION: The genetic findings we have acquired demonstrate a significant involvement of DISP1 variants in the phenotypic spectrum of midline defects. This underlines its importance as a crucial element in the efficient secretion of Sonic hedgehog. We also demonstrated that the very rare solitary median maxillary central incisor and congenital nasal pyriform aperture stenosis combination is part of the DISP1-related phenotype. The present study highlights the clinical risks to be flagged up during genetic counseling after the discovery of a pathogenic DISP1 variant.

A recurrent missense variant in the E3 ubiquitin ligase substrate recognition subunit FEM1B causes a rare syndromic neurodevelopmental disorder.

PURPOSE: Fem1 homolog B (FEM1B) acts as a substrate recognition subunit for ubiquitin ligase complexes belonging to the CULLIN 2-based E3 family. Several biological functions have been proposed for FEM1B, including a structurally resolved function as a sensor for redox cell status by controlling mitochondrial activity, but its implication in human disease remains elusive. METHODS: To understand the involvement of FEM1B in human disease, we made use of Matchmaker exchange platforms to identify individuals with de novo variants in FEM1B and performed their clinical evaluation. We performed functional validation using primary neuronal cultures and in utero electroporation assays, as well as experiments on patient's cells. RESULTS: Five individuals with a recurrent de novo missense variant in FEM1B were identified: NM_015322.5:c.377G>A NP_056137.1:p.(Arg126Gln) (FEM1BR126Q). Affected individuals shared a severe neurodevelopmental disorder with behavioral phenotypes and a variable set of malformations, including brain anomalies, clubfeet, skeletal abnormalities, and facial dysmorphism. Overexpression of the FEM1BR126Q variant but not FEM1B wild-type protein, during mouse brain development, resulted in delayed neuronal migration of the target cells. In addition, the individuals' cells exhibited signs of oxidative stress and induction of type I interferon signaling. CONCLUSION: Overall, our data indicate that p.(Arg126Gln) induces aberrant FEM1B activation, resulting in a gain-of-function mechanism associated with a severe syndromic developmental disorder in humans.

Variants in ZFX are associated with an X-linked neurodevelopmental disorder with recurrent facial gestalt.

Pathogenic variants in multiple genes on the X chromosome have been implicated in syndromic and non-syndromic intellectual disability disorders. ZFX on Xp22.11 encodes a transcription factor that has been linked to diverse processes including oncogenesis and development, but germline variants have not been characterized in association with disease. Here, we present clinical and molecular characterization of 18 individuals with germline ZFX variants. Exome or genome sequencing revealed 11 variants in 18 subjects (14 males and 4 females) from 16 unrelated families. Four missense variants were identified in 11 subjects, with seven truncation variants in the remaining individuals. Clinical findings included developmental delay/intellectual disability, behavioral abnormalities, hypotonia, and congenital anomalies. Overlapping and recurrent facial features were identified in all subjects, including thickening and medial broadening of eyebrows, variations in the shape of the face, external eye abnormalities, smooth and/or long philtrum, and ear abnormalities. Hyperparathyroidism was found in four families with missense variants, and enrichment of different tumor types was observed. In molecular studies, DNA-binding domain variants elicited differential expression of a small set of target genes relative to wild-type ZFX in cultured cells, suggesting a gain or loss of transcriptional activity. Additionally, a zebrafish model of ZFX loss displayed an altered behavioral phenotype, providing additional evidence for the functional significance of ZFX. Our clinical and experimental data support that variants in ZFX are associated with an X-linked intellectual disability syndrome characterized by a recurrent facial gestalt, neurocognitive and behavioral abnormalities, and an increased risk for congenital anomalies and hyperparathyroidism.

Expansion of the neurodevelopmental phenotype of individuals with EEF1A2 variants and genotype-phenotype study.

Translation elongation factor eEF1A2 constitutes the alpha subunit of the elongation factor-1 complex, responsible for the enzymatic binding of aminoacyl-tRNA to the ribosome. Since 2012, 21 pathogenic missense variants affecting EEF1A2 have been described in 42 individuals with a severe neurodevelopmental phenotype including epileptic encephalopathy and moderate to profound intellectual disability (ID), with neurological regression in some patients. Through international collaborative call, we collected 26 patients with EEF1A2 variants and compared them to the literature. Our cohort shows a significantly milder phenotype. 83% of the patients are walking (vs. 29% in the literature), and 84% of the patients have language skills (vs. 15%). Three of our patients do not have ID. Epilepsy is present in 63% (vs. 93%). Neurological examination shows a less severe phenotype with significantly less hypotonia (58% vs. 96%), and pyramidal signs (24% vs. 68%). Cognitive regression was noted in 4% (vs. 56% in the literature). Among individuals over 10 years, 56% disclosed neurocognitive regression, with a mean age of onset at 2 years. We describe 8 novel missense variants of EEF1A2. Modeling of the different amino-acid sites shows that the variants associated with a severe phenotype, and the majority of those associated with a moderate phenotype, cluster within the switch II region of the protein and thus may affect GTP exchange. In contrast, variants associated with milder phenotypes may impact secondary functions such as actin binding. We report the largest cohort of individuals with EEF1A2 variants thus far, allowing us to expand the phenotype spectrum and reveal genotype-phenotype correlations.

Allelic heterogeneity in a patient with postzygotic MTOR-related hypomelanosis of Ito with neurodevelopmental abnormalities.

A case of mosaic MTOR-associated hemimegalencephaly and hypomelanosis of Ito, died at 33 probably because of sudden unexpected death in epilepsy. Assessment of the variant allele fraction (VAF) in different tissues postmortem showed high variability not correlated with clinical features, representing the most detailed assessment of VAFs in different tissues to date.

Early prenatal diagnosis of causative homozygous variants in ASCC1 in a fetus with cystic hygroma and additional homozygous variants of unknown significance associated with a neurological phenotype not visible in early gestation: Dual diagnosis or not?

A consanguineous couple was referred at 10 weeks of gestation (WG) for prenatal genetic investigations due to isolated cystic hygroma. Prenatal trio exome sequencing identified causative homozygous truncating variants in ASCC1 previously implicated in spinal muscular atrophy with congenital bone fractures. Prenatal manifestations in ASCC1 can usually include hydramnios, fetal hypo-/akinesia, arthrogryposis, contractures and limb deformities, hydrops fetalis and cystic hygroma. An additional truncating variant was identified in CSPP1 associated with Joubert syndrome. Presentations in CSPP1 include cerebellar and brainstem malformations with vermis hypoplasia and molar tooth sign, difficult to visualize in early gestation. A second pregnancy was marked by the recurrence of isolated increased nuchal translucency at 10 + 2 WG. Sanger prenatal diagnosis targeted on ASCC1 and CSPP1 variants showed the presence of the homozygous familial ASCC1 variant. In this case, prenatal exome sequencing analysis is subject to a partial ASCC1 phenotype and an undetectable CSPP1 phenotype at 10 weeks of gestation. As CSPP1 contribution is unclear or speculative to a potentially later in pregnancy or postnatal phenotype, it is mentioned as a variant of uncertain significance. The detection of pathogenic or likely pathogenic variants involved in severe disorders but without phenotype-genotype correlation because the pregnancy is in the early stages or due to prenatally undetectable phenotypes, will encourage the clinical community to define future practices in molecular prenatal reporting.

Further description of two individuals with de novo p.(Glu127Lys) missense variant in the ASCL1 gene.

Achaete-Scute Family basic-helix-loop-helix (bHLH) Transcription Factor 1 (ASCL1) is a proneural transcription factor involved in neuron development in the central and peripheral nervous system. While initially suspected to contribute to congenital central hypoventilation syndrome-1 (CCHS) with or without Hirschsprung disease (HSCR) in three individuals, its implication was ruled out by the presence, in one of the individuals, of a Paired-like homeobox 2B (PHOX2B) heterozygous polyalanine expansion variant, known to cause CCHS. We report two additional unrelated individuals sharing the same sporadic ASCL1 p.(Glu127Lys) missense variant in the bHLH domain and a common phenotype with short-segment HSCR, signs of dysautonomia, and developmental delay. One has also mild CCHS without polyalanine expansion in PHOX2B, compatible with the diagnosis of Haddad syndrome. Furthermore, missense variants with homologous position in the same bHLH domain in other genes are known to cause human diseases. The description of additional individuals carrying the same variant and similar phenotype, as well as targeted functional studies, would be interesting to further evaluate the role of ASCL1 in neurocristopathies.

A syndromic neurodevelopmental disorder caused by rare variants in PPFIA3.

PPFIA3 encodes the protein-tyrosine phosphatase, receptor-type, F-polypeptide-interacting-protein-alpha-3 (PPFIA3), which is a member of the LAR-protein-tyrosine phosphatase-interacting-protein (liprin) family involved in synapse formation and function, synaptic vesicle transport, and presynaptic active zone assembly. The protein structure and function are evolutionarily well conserved, but human diseases related to PPFIA3 dysfunction are not yet reported in OMIM. Here, we report 20 individuals with rare PPFIA3 variants (19 heterozygous and 1 compound heterozygous) presenting with developmental delay, intellectual disability, hypotonia, dysmorphisms, microcephaly or macrocephaly, autistic features, and epilepsy with reduced penetrance. Seventeen unique PPFIA3 variants were detected in 18 families. To determine the pathogenicity of PPFIA3 variants in vivo, we generated transgenic fruit flies producing either human wild-type (WT) PPFIA3 or five missense variants using GAL4-UAS targeted gene expression systems. In the fly overexpression assays, we found that the PPFIA3 variants in the region encoding the N-terminal coiled-coil domain exhibited stronger phenotypes compared to those affecting the C-terminal region. In the loss-of-function fly assay, we show that the homozygous loss of fly Liprin-α leads to embryonic lethality. This lethality is partially rescued by the expression of human PPFIA3 WT, suggesting human PPFIA3 function is partially conserved in the fly. However, two of the tested variants failed to rescue the lethality at the larval stage and one variant failed to rescue lethality at the adult stage. Altogether, the human and fruit fly data reveal that the rare PPFIA3 variants are dominant-negative loss-of-function alleles that perturb multiple developmental processes and synapse formation.

Lessons from two series by physicians and caregivers' self-reported data in DDX3X-related disorders.

INTRODUCTION AND METHODS: We report two series of individuals with DDX3X variations, one (48 individuals) from physicians and one (44 individuals) from caregivers. RESULTS: These two series include several symptoms in common, with fairly similar distribution, which suggests that caregivers' data are close to physicians' data. For example, both series identified early childhood symptoms that were not previously described: feeding difficulties, mean walking age, and age at first words. DISCUSSION: Each of the two datasets provides complementary knowledge. We confirmed that symptoms are similar to those in the literature and provides more details on feeding difficulties. Caregivers considered that the symptom attention-deficit/hyperactivity disorder were most worrisome. Both series also reported sleep disturbance. Recently, anxiety has been reported in individuals with DDX3X variants. We strongly suggest that attention-deficit/hyperactivity disorder, anxiety, and sleep disorders need to be treated.