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.

De novo variants in FRYL are associated with developmental delay, intellectual disability, and dysmorphic features.

FRY-like transcription coactivator (FRYL) belongs to a Furry protein family that is evolutionarily conserved from yeast to humans. The functions of FRYL in mammals are largely unknown, and variants in FRYL have not previously been associated with a Mendelian disease. Here, we report fourteen individuals with heterozygous variants in FRYL who present with developmental delay, intellectual disability, dysmorphic features, and other congenital anomalies in multiple systems. The variants are confirmed de novo in all individuals except one. Human genetic data suggest that FRYL is intolerant to loss of function (LoF). We find that the fly FRYL ortholog, furry (fry), is expressed in multiple tissues, including the central nervous system where it is present in neurons but not in glia. Homozygous fry LoF mutation is lethal at various developmental stages, and loss of fry in mutant clones causes defects in wings and compound eyes. We next modeled four out of the five missense variants found in affected individuals using fry knockin alleles. One variant behaves as a severe LoF variant, whereas two others behave as partial LoF variants. One variant does not cause any observable defect in flies, and the corresponding human variant is not confirmed to be de novo, suggesting that this is a variant of uncertain significance. In summary, our findings support that fry is required for proper development in flies and that the LoF variants in FRYL cause a dominant disorder with developmental and neurological symptoms due to haploinsufficiency.

Allelic strengths of encephalopathy-associated UBA5 variants correlate between in vivo and in vitro assays.

Protein UFMylation downstream of the E1 enzyme UBA5 plays essential roles in development and endoplasmic reticulum stress. Variants in the UBA5 gene are associated with developmental and epileptic encephalopathy 44 (DEE44), an autosomal recessive disorder characterized by early-onset encephalopathy, movement abnormalities, global developmental delay, intellectual disability, and seizures. DEE44 is caused by at least 12 different missense variants described as loss of function (LoF), but the relationships between genotypes and molecular or clinical phenotypes remain to be established. We developed a humanized UBA5 fly model and biochemical activity assays in order to describe in vivo and in vitro genotype-phenotype relationships across the UBA5 allelic series. In vivo, we observed a broad spectrum of phenotypes in viability, developmental timing, lifespan, locomotor activity, and bang sensitivity. A range of functional effects was also observed in vitro across comprehensive biochemical assays for protein stability, ATP binding, UFM1 activation, and UFM1 transthiolation. Importantly, there is a strong correlation between in vivo and in vitro phenotypes, establishing a classification of LoF variants into mild, intermediate, and severe allelic strengths. By systemically evaluating UBA5 variants across in vivo and in vitro platforms, this study provides a foundation for more basic and translational UBA5 research, as well as a basis for evaluating current and future individuals afflicted with this rare disease.

Although rare diseases only impact a small fraction of the population, they still affect hundreds of millions of people around the world. Many of these conditions are caused by variations in inherited genetic material, which nowadays can be readily detected using advanced sequencing technologies. However, establishing a connection between these genetic changes and the disease they cause often requires further in-depth study. One such rare inherited disorder is developmental and epileptic encephalopathy type 44 (DEE44), which is caused by genetic variations within the gene for UBA5 (short for ubiquitin-like modifier activating enzyme 5). For DEE44 to occur, both copies of the gene for UBA5, known as alleles, must contain one or more detrimental variation. Although all these variations prevent UBA5 from working correctly, the level of disruption they cause, known as allelic strength, varies between them. However, it remained unclear whether the severity of the DEE44 disease directly corresponds with the allelic strength of these variants. To answer this question, Pan et al. tested how different genetic variants found in patients with DEE44 affected the behavior and health of fruit flies. These results were then compared against in vitro biochemical assays testing how alleles containing these variants impacted the function of UBA5. When the fly gene for the enzyme was replaced with the human gene containing variations associated with DEE44, flies exhibited changes in their survival rates, developmental progress, lifespan, and neurological well-being. However, not all of the variants caused ill effects. Using this information, the patient variants were classified into three categories based on the severity of their effect: mild, intermediate, and severe. Biochemical assays supported this classification and revealed that the variants that caused more severe symptoms tended to inhibit the activity of UBA5 more significantly. Pan et al. further analyzed the nature of the variants in the patients and showed that most patients typically carried one mild and one strong variant, although some individuals did have two intermediate variants. Notably, no patients carried two severe variants. This indicates that DEE44 is the result of UBA5 only partially losing its ability to work correctly. The study by Pan et al. provides a framework for assessing the impact of genetic variants associated with DEE44, aiding the diagnosis and treatment of the disorder. However, further research involving more patients, more detailed clinical data, and testing other newly identified DEE44-causing variants is needed to solidify the correlation between allelic strength and disease severity.

HNRNPC haploinsufficiency affects alternative splicing of intellectual disability-associated genes and causes a neurodevelopmental disorder.

Heterogeneous nuclear ribonucleoprotein C (HNRNPC) is an essential, ubiquitously abundant protein involved in mRNA processing. Genetic variants in other members of the HNRNP family have been associated with neurodevelopmental disorders. Here, we describe 13 individuals with global developmental delay, intellectual disability, behavioral abnormalities, and subtle facial dysmorphology with heterozygous HNRNPC germline variants. Five of them bear an identical in-frame deletion of nine amino acids in the extreme C terminus. To study the effect of this recurrent variant as well as HNRNPC haploinsufficiency, we used induced pluripotent stem cells (iPSCs) and fibroblasts obtained from affected individuals. While protein localization and oligomerization were unaffected by the recurrent C-terminal deletion variant, total HNRNPC levels were decreased. Previously, reduced HNRNPC levels have been associated with changes in alternative splicing. Therefore, we performed a meta-analysis on published RNA-seq datasets of three different cell lines to identify a ubiquitous HNRNPC-dependent signature of alternative spliced exons. The identified signature was not only confirmed in fibroblasts obtained from an affected individual but also showed a significant enrichment for genes associated with intellectual disability. Hence, we assessed the effect of decreased and increased levels of HNRNPC on neuronal arborization and neuronal migration and found that either condition affects neuronal function. Taken together, our data indicate that HNRNPC haploinsufficiency affects alternative splicing of multiple intellectual disability-associated genes and that the developing brain is sensitive to aberrant levels of HNRNPC. Hence, our data strongly support the inclusion of HNRNPC to the family of HNRNP-related neurodevelopmental disorders.

Tle4 controls both developmental acquisition and early post-natal maturation of corticothalamic projection neuron identity.

Identities of distinct neuron subtypes are specified during embryonic development, then maintained during post-natal maturation. In cerebral cortex, mechanisms controlling early acquisition of neuron-subtype identities have become increasingly understood. However, mechanisms controlling neuron-subtype identity stability during post-natal maturation are largely unexplored. We identify that Tle4 is required for both early acquisition and post-natal stability of corticothalamic neuron-subtype identity. Embryonically, Tle4 promotes acquisition of corticothalamic identity and blocks emergence of core characteristics of subcerebral/corticospinal projection neuron identity, including gene expression and connectivity. During the first post-natal week, when corticothalamic innervation is ongoing, Tle4 is required to stabilize corticothalamic neuron identity, limiting interference from differentiation programs of developmentally related neuron classes. We identify a deacetylation-based epigenetic mechanism by which TLE4 controls Fezf2 expression level by corticothalamic neurons. This contributes to distinction of cortical output subtypes and ensures identity stability for appropriate maturation of corticothalamic neurons.

De novo missense variants in phosphatidylinositol kinase PIP5KIγ underlie a neurodevelopmental syndrome associated with altered phosphoinositide signaling.

Phosphoinositides (PIs) are membrane phospholipids produced through the local activity of PI kinases and phosphatases that selectively add or remove phosphate groups from the inositol head group. PIs control membrane composition and play key roles in many cellular processes including actin dynamics, endosomal trafficking, autophagy, and nuclear functions. Mutations in phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] phosphatases cause a broad spectrum of neurodevelopmental disorders such as Lowe and Joubert syndromes and congenital muscular dystrophy with cataracts and intellectual disability, which are thus associated with increased levels of PI(4,5)P2. Here, we describe a neurodevelopmental disorder associated with an increase in the production of PI(4,5)P2 and with PI-signaling dysfunction. We identified three de novo heterozygous missense variants in PIP5K1C, which encodes an isoform of the phosphatidylinositol 4-phosphate 5-kinase (PIP5KIγ), in nine unrelated children exhibiting intellectual disability, developmental delay, acquired microcephaly, seizures, visual abnormalities, and dysmorphic features. We provide evidence that the PIP5K1C variants result in an increase of the endosomal PI(4,5)P2 pool, giving rise to ectopic recruitment of filamentous actin at early endosomes (EEs) that in turn causes dysfunction in EE trafficking. In addition, we generated an in vivo zebrafish model that recapitulates the disorder we describe with developmental defects affecting the forebrain, including the eyes, as well as craniofacial abnormalities, further demonstrating the pathogenic effect of the PIP5K1C variants.

Allelic strengths of encephalopathy-associated UBA5 variants correlate between in vivo and in vitro assays.

Protein UFMylation downstream of the E1 enzyme UBA5 plays essential roles in development and ER stress. Variants in the UBA5 gene are associated with developmental and epileptic encephalopathy 44 (DEE44), an autosomal recessive disorder characterized by early-onset encephalopathy, movement abnormalities, global developmental delay, intellectual disability, and seizures. DEE44 is caused by at least twelve different missense variants described as loss of function (LoF), but the relationships between genotypes and molecular or clinical phenotypes remains to be established. We developed a humanized UBA5 fly model and biochemical activity assays in order to describe in vivo and in vitro genotype-phenotype relationships across the UBA5 allelic series. In vivo, we observed a broad spectrum of phenotypes in viability, developmental timing, lifespan, locomotor activity, and bang sensitivity. A range of functional effects was also observed in vitro across comprehensive biochemical assays for protein stability, ATP binding, UFM1 activation, and UFM1 transthiolation. Importantly, there is a strong correlation between in vivo and in vitro phenotypes, establishing a classification of LoF variants into mild, intermediate, and severe allelic strengths. By systemically evaluating UBA5 variants across in vivo and in vitro platforms, this study provides a foundation for more basic and translational UBA5 research, as well as a basis for evaluating current and future individuals afflicted with this rare disease.

A de novo missense variant in EZH1 associated with developmental delay exhibits functional deficits in Drosophila melanogaster.

EZH1, a polycomb repressive complex-2 component, is involved in a myriad of cellular processes. EZH1 represses transcription of downstream target genes through histone 3 lysine27 (H3K27) trimethylation (H3K27me3). Genetic variants in histone modifiers have been associated with developmental disorders, while EZH1 has not yet been linked to any human disease. However, the paralog EZH2 is associated with Weaver syndrome. Here we report a previously undiagnosed individual with a novel neurodevelopmental phenotype identified to have a de novo missense variant in EZH1 through exome sequencing. The individual presented in infancy with neurodevelopmental delay and hypotonia and was later noted to have proximal muscle weakness. The variant, p.A678G, is in the SET domain, known for its methyltransferase activity, and an analogous somatic or germline mutation in EZH2 has been reported in patients with B-cell lymphoma or Weaver syndrome, respectively. Human EZH1/2 are homologous to fly Enhancer of zeste (E(z)), an essential gene in Drosophila, and the affected residue (p.A678 in humans, p.A691 in flies) is conserved. To further study this variant, we obtained null alleles and generated transgenic flies expressing wildtype [E(z)WT] and the variant [E(z)A691G]. When expressed ubiquitously the variant rescues null-lethality similar to the wildtype. Overexpression of E(z)WT induces homeotic patterning defects but notably the E(z)A691G variant leads to dramatically stronger morphological phenotypes. We also note a dramatic loss of H3K27me2 and a corresponding increase in H3K27me3 in flies expressing E(z)A691G, suggesting this acts as a gain-of-function allele. In conclusion, here we present a novel EZH1 de novo variant associated with a neurodevelopmental disorder. Furthermore, we found that this variant has a functional impact in Drosophila.

De novo MCM6 variants in neurodevelopmental disorders: a recognizable phenotype related to zinc binding residues.

The minichromosome maintenance (MCM) complex acts as a DNA helicase during DNA replication, and thereby regulates cell cycle progression and proliferation. In addition, MCM-complex components localize to centrosomes and play an independent role in ciliogenesis. Pathogenic variants in genes coding for MCM components and other DNA replication factors have been linked to growth and developmental disorders as Meier-Gorlin syndrome and Seckel syndrome. Trio exome/genome sequencing identified the same de novo MCM6 missense variant p.(Cys158Tyr) in two unrelated individuals that presented with overlapping phenotypes consisting of intra-uterine growth retardation, short stature, congenital microcephaly, endocrine features, developmental delay and urogenital anomalies. The identified variant affects a zinc binding cysteine in the MCM6 zinc finger signature. This domain, and specifically cysteine residues, are essential for MCM-complex dimerization and the induction of helicase activity, suggesting a deleterious effect of this variant on DNA replication. Fibroblasts derived from the two affected individuals showed defects both in ciliogenesis and cell proliferation. We additionally traced three unrelated individuals with de novo MCM6 variants in the oligonucleotide binding (OB)-fold domain, presenting with variable (neuro)developmental features including autism spectrum disorder, developmental delay, and epilepsy. Taken together, our findings implicate de novo MCM6 variants in neurodevelopmental disorders. The clinical features and functional defects related to the zinc binding residue resemble those observed in syndromes related to other MCM components and DNA replication factors, while de novo OB-fold domain missense variants may be associated with more variable neurodevelopmental phenotypes. These data encourage consideration of MCM6 variants in the diagnostic arsenal of NDD.

A Pooled Electronic Consultation Program to Improve Access to Genetics Specialists.

Innovative service delivery models are needed to increase access to genetics specialists. Electronic consultation (e-Consult) programs can connect clinicians with specialists. At Massachusetts General Hospital, an e-Consult service was created to address genomics-related questions. In its first year, the e-Consult service triaged 153 requests and completed 122 in an average of 3.2 days. Of the 95 e-Consults with actionable recommendations, there was documentation that most ordering clinicians followed through (82%). A variety of providers used the service, although the majority (77%) were generalists. E-Consult models should be considered as one way to increase access to genetics care.

A de novo missense variant in EZH1 associated with developmental delay exhibits functional deficits in Drosophila melanogaster.

EZH1 ( Enhancer of Zeste, homolog 1) , a Polycomb Repressive Complex-2 (PRC2) component, is involved in a myriad of cellular processes through modifying histone 3 lysine27 (H3K27) residues. EZH1 represses transcription of downstream target genes through H3K27 trimethylation (H3K27me3). Genetic mutations in histone modifiers have been associated with developmental disorders, while EZH1 has not yet been linked to any human disease. However, the paralog EZH2 is associated with Weaver syndrome. Here we report a previously undiagnosed individual with a novel neurodevelopmental phenotype identified to have a de novo variant in EZH1 , p.Ala678Gly, through exome sequencing. The individual presented in infancy with neurodevelopmental delay and hypotonia and was later noted to have proximal muscle weakness. The variant, p.A678G, is in the SET domain, known for its methyltransferase activity, and was the best candidate variant found in the exome. Human EZH1 / 2 are homologous to fly Enhancer of zeste E(z) , an essential gene in flies, and the residue (A678 in humans, A691 in Drosophila ) is conserved. To further study this variant, we obtained Drosophila null alleles and generated transgenic flies expressing wild-type (E(z) WT ) and the variant (E(z) A691G ) . The E(z) A691G variant led to hyper H3K27me3 while the E(z) WT did not, suggesting this is as a gain-of-function allele. When expressed under the tubulin promotor in vivo the variant rescued null-lethality similar to wild-type but the E(z) A691G flies exhibit bang sensitivity and shortened lifespan. In conclusion, here we present a novel EZH1 de novo variant associated with a neurodevelopmental disorder. Furthermore, we found that this variant has a functional impact in Drosophila . Biochemically this allele leads to increased H3K27me3 suggesting gain-of-function, but when expressed in adult flies the E(z) A691G has some characteristics of partial loss-of-function which may suggest it is a more complex allele in vivo .

The recurrent de novo c.2011C>T missense variant in MTSS2 causes syndromic intellectual disability.

MTSS2, also known as MTSS1L, binds to plasma membranes and modulates their bending. MTSS2 is highly expressed in the central nervous system (CNS) and appears to be involved in activity-dependent synaptic plasticity. Variants in MTSS2 have not yet been associated with a human phenotype in OMIM. Here we report five individuals with the same heterozygous de novo variant in MTSS2 (GenBank: NM_138383.2: c.2011C>T [p.Arg671Trp]) identified by exome sequencing. The individuals present with global developmental delay, mild intellectual disability, ophthalmological anomalies, microcephaly or relative microcephaly, and shared mild facial dysmorphisms. Immunoblots of fibroblasts from two affected individuals revealed that the variant does not significantly alter MTSS2 levels. We modeled the variant in Drosophila and showed that the fly ortholog missing-in-metastasis (mim) was widely expressed in most neurons and a subset of glia of the CNS. Loss of mim led to a reduction in lifespan, impaired locomotor behavior, and reduced synaptic transmission in adult flies. Expression of the human MTSS2 reference cDNA rescued the mim loss-of-function (LoF) phenotypes, whereas the c.2011C>T variant had decreased rescue ability compared to the reference, suggesting it is a partial LoF allele. However, elevated expression of the variant, but not the reference MTSS2 cDNA, led to similar defects as observed by mim LoF, suggesting that the variant is toxic and may act as a dominant-negative allele when expressed in flies. In summary, our findings support that mim is important for appropriate neural function, and that the MTSS2 c.2011C>T variant causes a syndromic form of intellectual disability.

Gain-of-function mutations in ALPK1 cause an NF-κB-mediated autoinflammatory disease: functional assessment, clinical phenotyping and disease course of patients with ROSAH syndrome.

OBJECTIVES: To test the hypothesis that ROSAH (retinal dystrophy, optic nerve oedema, splenomegaly, anhidrosis and headache) syndrome, caused by dominant mutation in ALPK1, is an autoinflammatory disease. METHODS: This cohort study systematically evaluated 27 patients with ROSAH syndrome for inflammatory features and investigated the effect of ALPK1 mutations on immune signalling. Clinical, immunologic and radiographical examinations were performed, and 10 patients were empirically initiated on anticytokine therapy and monitored. Exome sequencing was used to identify a new pathogenic variant. Cytokine profiling, transcriptomics, immunoblotting and knock-in mice were used to assess the impact of ALPK1 mutations on protein function and immune signalling. RESULTS: The majority of the cohort carried the p.Thr237Met mutation but we also identified a new ROSAH-associated mutation, p.Tyr254Cys.Nearly all patients exhibited at least one feature consistent with inflammation including recurrent fever, headaches with meningeal enhancement and premature basal ganglia/brainstem mineralisation on MRI, deforming arthritis and AA amyloidosis. However, there was significant phenotypic variation, even within families and some adults lacked functional visual deficits. While anti-TNF and anti-IL-1 therapies suppressed systemic inflammation and improved quality of life, anti-IL-6 (tocilizumab) was the only anticytokine therapy that improved intraocular inflammation (two of two patients).Patients' primary samples and in vitro assays with mutated ALPK1 constructs showed immune activation with increased NF-κB signalling, STAT1 phosphorylation and interferon gene expression signature. Knock-in mice with the Alpk1 T237M mutation exhibited subclinical inflammation.Clinical features not conventionally attributed to inflammation were also common in the cohort and included short dental roots, enamel defects and decreased salivary flow. CONCLUSION: ROSAH syndrome is an autoinflammatory disease caused by gain-of-function mutations in ALPK1 and some features of disease are amenable to immunomodulatory therapy.

Heterozygous variants in PRPF8 are associated with neurodevelopmental disorders.

The pre-mRNA-processing factor 8, encoded by PRPF8, is a scaffolding component of a spliceosome complex involved in the removal of introns from mRNA precursors. Previously, heterozygous pathogenic variants in PRPF8 have been associated with autosomal dominant retinitis pigmentosa. More recently, PRPF8 was suggested as a candidate gene for autism spectrum disorder due to the enrichment of sequence variants in this gene in individuals with neurodevelopmental disorders. We report 14 individuals with various forms of neurodevelopmental conditions, found to have heterozygous, predominantly de novo, missense, and loss-of-function variants in PRPF8. These individuals have clinical features that may represent a new neurodevelopmental syndrome.

A homozygous splice variant in ATP5PO, disrupts mitochondrial complex V function and causes Leigh syndrome in two unrelated families.

Mitochondrial complex V plays an important role in oxidative phosphorylation by catalyzing the generation of ATP. Most complex V subunits are nuclear encoded and not yet associated with recognized Mendelian disorders. Using exome sequencing, we identified a rare homozygous splice variant (c.87+3A>G) in ATP5PO, the complex V subunit which encodes the oligomycin sensitivity conferring protein, in three individuals from two unrelated families, with clinical suspicion of a mitochondrial disorder. These individuals had a similar, severe infantile and often lethal multi-systemic disorder that included hypotonia, developmental delay, hypertrophic cardiomyopathy, progressive epileptic encephalopathy, progressive cerebral atrophy, and white matter abnormalities on brain MRI consistent with Leigh syndrome. cDNA studies showed a predominant shortened transcript with skipping of exon 2 and low levels of the normal full-length transcript. Fibroblasts from the affected individuals demonstrated decreased ATP5PO protein, defective assembly of complex V with markedly reduced amounts of peripheral stalk proteins, and complex V hydrolytic activity. Further, expression of human ATP5PO cDNA without exon 2 (hATP5PO-∆ex2) in yeast cells deleted for yATP5 (ATP5PO homolog) was unable to rescue growth on media which requires oxidative phosphorylation when compared to the wild type construct (hATP5PO-WT), indicating that exon 2 deletion leads to a non-functional protein. Collectively, our findings support the pathogenicity of the ATP5PO c.87+3A>G variant, which significantly reduces but does not eliminate complex V activity. These data along with the recent report of an affected individual with ATP5PO variants, add to the evidence that rare biallelic variants in ATP5PO result in defective complex V assembly, function and are associated with Leigh syndrome.

TLE4 Is a Critical Mediator of Osteoblast and Runx2-Dependent Bone Development.

Healthy bone homeostasis hinges upon a delicate balance and regulation of multiple processes that contribute to bone development and metabolism. While examining hematopoietic regulation by Tle4, we have uncovered a previously unappreciated role of Tle4 on bone calcification using a novel Tle4 null mouse model. Given the significance of osteoblasts in both hematopoiesis and bone development, this study investigated how loss of Tle4 affects osteoblast function. We used dynamic bone formation parameters and microCT to characterize the adverse effects of Tle4 loss on bone development. We further demonstrated loss of Tle4 impacts expression of several key osteoblastogenic genes, including Runx2, Oc, and Ap, pointing toward a potential novel mechanism for Tle4-dependent regulation of mammalian bone development in collaboration with the RUNX family members.

Clinical, neuroimaging, and molecular spectrum of TECPR2-associated hereditary sensory and autonomic neuropathy with intellectual disability.

Bi-allelic TECPR2 variants have been associated with a complex syndrome with features of both a neurodevelopmental and neurodegenerative disorder. Here, we provide a comprehensive clinical description and variant interpretation framework for this genetic locus. Through international collaboration, we identified 17 individuals from 15 families with bi-allelic TECPR2-variants. We systemically reviewed clinical and molecular data from this cohort and 11 cases previously reported. Phenotypes were standardized using Human Phenotype Ontology terms. A cross-sectional analysis revealed global developmental delay/intellectual disability, muscular hypotonia, ataxia, hyporeflexia, respiratory infections, and central/nocturnal hypopnea as core manifestations. A review of brain magnetic resonance imaging scans demonstrated a thin corpus callosum in 52%. We evaluated 17 distinct variants. Missense variants in TECPR2 are predominantly located in the N- and C-terminal regions containing ß-propeller repeats. Despite constituting nearly half of disease-associated TECPR2 variants, classifying missense variants as (likely) pathogenic according to ACMG criteria remains challenging. We estimate a pathogenic variant carrier frequency of 1/1221 in the general and 1/155 in the Jewish Ashkenazi populations. Based on clinical, neuroimaging, and genetic data, we provide recommendations for variant reporting, clinical assessment, and surveillance/treatment of individuals with TECPR2-associated disorder. This sets the stage for future prospective natural history studies.