Pathogenic RAB34 variants impair primary cilium assembly and cause a novel oral-facial-digital syndrome.

Oral-facial-digital syndromes (OFDS) are a group of clinically and genetically heterogeneous disorders characterized by defects in the development of the face and oral cavity along with digit anomalies. Pathogenic variants in over 20 genes encoding ciliary proteins have been found to cause OFDS through deleterious structural or functional impacts on primary cilia. We identified by exome sequencing bi-allelic missense variants in a novel disease-causing ciliary gene RAB34 in four individuals from three unrelated families. Affected individuals presented a novel form of OFDS (OFDS-RAB34) accompanied by cardiac, cerebral, skeletal and anorectal defects. RAB34 encodes a member of the Rab GTPase superfamily and was recently identified as a key mediator of ciliary membrane formation. Unlike many genes required for cilium assembly, RAB34 acts selectively in cell types that use the intracellular ciliogenesis pathway, in which nascent cilia begin to form in the cytoplasm. We find that the protein products of these pathogenic variants, which are clustered near the RAB34 C-terminus, exhibit a strong loss of function. Although some variants retain the ability to be recruited to the mother centriole, cells expressing mutant RAB34 exhibit a significant defect in cilium assembly. While many Rab proteins have been previously linked to ciliogenesis, our studies establish RAB34 as the first small GTPase involved in OFDS and reveal the distinct clinical manifestations caused by impairment of intracellular ciliogenesis.

ADGRL1 haploinsufficiency causes a variable spectrum of neurodevelopmental disorders in humans and alters synaptic activity and behavior in a mouse model.

ADGRL1 (latrophilin 1), a well-characterized adhesion G protein-coupled receptor, has been implicated in synaptic development, maturation, and activity. However, the role of ADGRL1 in human disease has been elusive. Here, we describe ten individuals with variable neurodevelopmental features including developmental delay, intellectual disability, attention deficit hyperactivity and autism spectrum disorders, and epilepsy, all heterozygous for variants in ADGRL1. In vitro, human ADGRL1 variants expressed in neuroblastoma cells showed faulty ligand-induced regulation of intracellular Ca2+ influx, consistent with haploinsufficiency. In vivo, Adgrl1 was knocked out in mice and studied on two genetic backgrounds. On a non-permissive background, mice carrying a heterozygous Adgrl1 null allele exhibited neurological and developmental abnormalities, while homozygous mice were non-viable. On a permissive background, knockout animals were also born at sub-Mendelian ratios, but many Adgrl1 null mice survived gestation and reached adulthood. Adgrl1-/- mice demonstrated stereotypic behaviors, sexual dysfunction, bimodal extremes of locomotion, augmented startle reflex, and attenuated pre-pulse inhibition, which responded to risperidone. Ex vivo synaptic preparations displayed increased spontaneous exocytosis of dopamine, acetylcholine, and glutamate, but Adgrl1-/- neurons formed synapses in vitro poorly. Overall, our findings demonstrate that ADGRL1 haploinsufficiency leads to consistent developmental, neurological, and behavioral abnormalities in mice and humans.

TRAPPC6B biallelic variants cause a neurodevelopmental disorder with TRAPP II and trafficking disruptions.

Highly conserved transport protein particle (TRAPP) complexes regulate subcellular trafficking pathways. Accurate protein trafficking has been increasingly recognized to be critically important for normal development, particularly in the nervous system. Variants in most TRAPP complex subunits have been found to lead to neurodevelopmental disorders with diverse but overlapping phenotypes. We expand on limited prior reports on TRAPPC6B with detailed clinical and neuroradiologic assessments, and studies on mechanisms of disease, and new types of variants. We describe 29 additional patients from 18 independent families with biallelic variants in TRAPPC6B. We identified seven homozygous nonsense (n = 12 patients) and eight canonical splice-site variants (n = 17 patients). In addition, we identified one patient with compound heterozygous splice-site/missense variants with a milder phenotype and one patient with homozygous missense variants. Patients displayed non-progressive microcephaly, global developmental delay/intellectual disability, epilepsy and absent expressive language. Movement disorders including stereotypies, spasticity and dystonia were also observed. Brain imaging revealed reductions in cortex, cerebellum and corpus callosum size with frequent white matter hyperintensity. Volumetric measurements indicated globally diminished volume rather than specific regional losses. We identified a reduced rate of trafficking into the Golgi apparatus and Golgi fragmentation in patient-derived fibroblasts that was rescued by wild-type TRAPPC6B. Molecular studies revealed a weakened interaction between mutant TRAPPC6B (c.454C>T, p.Q152*) and its TRAPP binding partner TRAPPC3. Patient-derived fibroblasts from the TRAPPC6B (c.454C>T, p.Q152*) variant displayed reduced levels of TRAPPC6B as well as other TRAPP II complex-specific members (TRAPPC9 and TRAPPC10). Interestingly, the levels of the TRAPPC6B homologue TRAPPC6A were found to be elevated. Moreover, co-immunoprecipitation experiments showed that TRAPPC6A co-precipitates equally with TRAPP II and TRAPP III, while TRAPPC6B co-precipitates significantly more with TRAPP II, suggesting enrichment of the protein in the TRAPP II complex. This implies that variants in TRAPPC6B may preferentially affect TRAPP II functions compared to TRAPP III functions. Finally, we assessed phenotypes in a Drosophila TRAPPC6B-deficiency model. Neuronal TRAPPC6B knockdown impaired locomotion and led to wing posture defects, supporting a role for TRAPPC6B in neuromotor function. Our findings confirm the association of damaging biallelic TRAPPC6B variants with microcephaly, intellectual disability, language impairments, and epilepsy. A subset of patients also exhibited dystonia and/or spasticity with impaired ambulation. These features overlap with disorders arising from pathogenic variants in other TRAPP subunits, particularly components of the TRAPP II complex. These findings suggest that TRAPPC6B is essential for brain development and function, and TRAPP II complex activity may be particularly relevant for mediating this function.

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.

Differential alternative splicing analysis links variation in ZRSR2 to a novel type of oral-facial-digital syndrome.

PURPOSE: Oral-facial-digital (OFD) syndromes are genetically heterogeneous developmental disorders, caused by pathogenic variants in genes involved in primary cilia formation and function. We identified a previously undescribed type of OFD with brain anomalies, ranging from alobar holoprosencephaly to pituitary anomalies, in 6 unrelated families. METHODS: Exome sequencing of affected probands was supplemented with alternative splicing analysis in patient and control lymphoblastoid and fibroblast cell lines, and primary cilia structure analysis in patient fibroblasts. RESULTS: In 1 family with 2 affected males, we identified a germline variant in the last exon of ZRSR2, NM_005089.4:c.1211_1212del NP_005080.1:p.(Gly404GlufsTer23), whereas 7 affected males from 5 unrelated families were hemizygous for the ZRSR2 variant NM_005089.4:c.1207_1208del NP_005080.1:p.(Arg403GlyfsTer24), either occurring de novo or inherited in an X-linked recessive pattern. ZRSR2, located on chromosome Xp22.2, encodes a splicing factor of the minor spliceosome complex, which recognizes minor introns, representing 0.35% of human introns. Patient samples showed significant enrichment of minor intron retention. Among differentially spliced targets are ciliopathy-related genes, such as TMEM107 and CIBAR1. Primary fibroblasts containing the NM_005089.4:c.1207_1208del ZRSR2 variant had abnormally elongated cilia, confirming an association between defective U12-type intron splicing, OFD and abnormal primary cilia formation. CONCLUSION: We introduce a novel type of OFD associated with elongated cilia and differential splicing of minor intron-containing genes due to germline variation in ZRSR2.

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.

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.

Discovery of pathogenic variants in EFEMP2 and RAG1 and undetectable fetal phenotype: A challenge of prenatal exome sequencing.

BACKGROUND: Exome sequencing in prenatal context confronts with pathogenic variants associated with phenotypes that are not detectable prenatally. MATERIALS AND METHODS: A consanguineous couple was referred at 24 weeks of gestation for prenatal genetic investigations after ultrasonography findings including decreased fetal movements, hypoplastic male external genitalia, retrognathia, prefrontal edema, anomalies of the great vessels with pulmonary atresia and dilated tortuous aorta. RESULT: Prenatal trio exome sequencing identified two homozygous likely pathogenic variants, i.e. a missense in EFEMP2 involved in cutis laxa and a nonsense in RAG1 involved in several types of severe combined immunodeficiency. DISCUSSION: The fetal ultrasonographic phenotype was partially compatible with previously reported prenatal presentations secondary to EFEMP2 biallelic variants, but prenatal presentations have never been reported for RAG1 related disorders because the RAG1 phenotype is undetectable during pregnancy. CONCLUSION: Both EFEMP2 and RAG1 variants were disclosed to the couple because the EFEMP2 variant was considered causative for the fetal ultrasonographic phenotype and the RAG1 variant was considered a finding of strong interest for genetic counselling and monitoring of future pregnancies following the American College of Medical Genetics and Genomics recommendations about the discovery of incidental findings in fetal exome sequencing in prenatal diagnosis.

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.

Multiple molecular diagnoses in the field of intellectual disability and congenital anomalies: 3.5% of all positive cases.

PURPOSE: Wide access to clinical exome/genome sequencing (ES/GS) enables the identification of multiple molecular diagnoses (MMDs), being a long-standing but underestimated concept, defined by two or more causal loci implicated in the phenotype of an individual with a rare disease. Only few series report MMDs rates (1.8% to 7.1%). This study highlights the increasing role of MMDs in a large cohort of individuals addressed for congenital anomalies/intellectual disability (CA/ID). METHODS: From 2014 to 2021, our diagnostic laboratory rendered 880/2658 positive ES diagnoses for CA/ID aetiology. Exhaustive search on MMDs from ES data was performed prospectively (January 2019 to December 2021) and retrospectively (March 2014 to December 2018). RESULTS: MMDs were identified in 31/880 individuals (3.5%), responsible for distinct (9/31) or overlapping (22/31) phenotypes, and potential MMDs in 39/880 additional individuals (4.4%). CONCLUSION: MMDs are frequent in CA/ID and remain a strong challenge. Reanalysis of positive ES data appears essential when phenotypes are partially explained by the initial diagnosis or atypically enriched overtime. Up-to-date clinical data, clinical expertise from the referring physician, strong interactions between clinicians and biologists, and increasing gene discoveries and improved ES bioinformatics tools appear all the more fundamental to enhance chances of identifying MMDs. It is essential to provide appropriate patient care and genetic counselling.

New insights into CC2D2A-related Joubert syndrome.

PURPOSE: In this study, we describe the phenotype and genotype of the largest cohort of patients with Joubert syndrome (JS) carrying pathogenic variants on one of the most frequent causative genes, CC2D2A. METHODS: We selected 53 patients with pathogenic variants on CC2D2A, compiled and analysed their clinical, neuroimaging and genetic information and compared it to previous literature. RESULTS: Developmental delay (motor and language) was nearly constant but patients had normal intellectual efficiency in 74% of cases (20/27 patients) and 68% followed mainstream schooling despite learning difficulties. Epilepsy was found in only 13% of cases. Only three patients had kidney cysts, only three had genuine retinal dystrophy and no subject had liver fibrosis or polydactyly. Brain MRIs showed typical signs of JS with rare additional features. Genotype-phenotype correlation findings demonstrate a homozygous truncating variant p.Arg950* linked to a more severe phenotype. CONCLUSION: This study contradicts previous literature stating an association between CC2D2A-related JS and ventriculomegaly. Our study implies that CC2D2A-related JS is linked to positive neurodevelopmental outcome and low rate of other organ defects except for homozygous pathogenic variant p.Arg950*. This information will help modulate patient follow-up and provide families with accurate genetic counselling.

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.

Characterization of Vps13b-mutant mice reveals neuroanatomical and behavioral phenotypes with females less affected.

The vacuolar protein sorting-associated protein 13B (VPS13B) is a large and highly conserved protein. Disruption of VPS13B causes the autosomal recessive Cohen syndrome, a rare disorder characterized by microcephaly and intellectual disability among other features, including developmental delay, hypotonia, and friendly-personality. However, the underlying mechanisms by which VPS13B disruption leads to brain dysfunction still remain unexplained. To gain insights into the neuropathogenesis of Cohen syndrome, we systematically characterized brain changes in Vps13b-mutant mice and compared murine findings to 235 previously published and 17 new patients diagnosed with VPS13B-related Cohen syndrome. We showed that Vps13b is differentially expressed across brain regions with the highest expression in the cerebellum, the hippocampus and the cortex with postnatal peak. Half of the Vps13b-/- mice die during the first week of life. The remaining mice have a normal lifespan and display the core phenotypes of the human disease, including microcephaly, growth delay, hypotonia, altered memory, and enhanced sociability. Systematic 2D and 3D brain histo-morphological analyses reveal specific structural changes in the brain starting after birth. The dentate gyrus is the brain region with the most prominent reduction in size, while the motor cortex is specifically thinner in layer VI. The fornix, the fasciculus retroflexus, and the cingulate cortex remain unaffected. Interestingly, these neuroanatomical changes implicate an increase of neuronal death during infantile stages with no progression in adulthood suggesting that VPS13B promotes neuronal survival early in life. Importantly, whilst both sexes were affected, some neuroanatomical and behavioral phenotypes were less pronounced or even absent in females. We evaluate sex differences in Cohen patients and conclude that females are less affected both in mice and patients. Our findings provide new insights about the neurobiology of VPS13B and highlight previously unreported brain phenotypes while defining Cohen syndrome as a likely new entity of non-progressive infantile neurodegeneration.

Retrotransposon insertion as a novel mutational cause of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder resulting from biallelic alterations of the SMN1 gene: deletion, gene conversion or, in rare cases, intragenic variants. The disease severity is mainly influenced by the copy number of SMN2, a nearly identical gene, which produces only low amounts of full-length (FL) mRNA. Here we describe the first example of retrotransposon insertion as a pathogenic SMN1 mutational event. The 50-year-old patient is clinically affected by SMA type III with a diagnostic odyssey spanning nearly 30 years. Despite a mild disease course, he carries a single SMN2 copy. Using Exome Sequencing and Sanger sequencing, we characterized a SINE-VNTR-Alu (SVA) type F retrotransposon inserted in SMN1 intron 7. Using RT-PCR and RNASeq experiments on lymphoblastoid cell lines, we documented the dramatic decrease of FL transcript production in the patient compared to subjects with the same SMN1 and SMN2 copy number, thus validating the pathogenicity of this SVA insertion. We described the mutant FL-SMN1-SVA transcript characterized by exon extension and showed that it is subject to degradation by nonsense-mediated mRNA decay. The stability of the SMN-SVA protein may explain the mild course of the disease. This observation exemplifies the role of retrotransposons in human genetic disorders.

Bi-allelic Variants in the GPI Transamidase Subunit PIGK Cause a Neurodevelopmental Syndrome with Hypotonia, Cerebellar Atrophy, and Epilepsy.

Glycosylphosphatidylinositol (GPI)-anchored proteins are critical for embryogenesis, neurogenesis, and cell signaling. Variants in several genes participating in GPI biosynthesis and processing lead to decreased cell surface presence of GPI-anchored proteins (GPI-APs) and cause inherited GPI deficiency disorders (IGDs). In this report, we describe 12 individuals from nine unrelated families with 10 different bi-allelic PIGK variants. PIGK encodes a component of the GPI transamidase complex, which attaches the GPI anchor to proteins. Clinical features found in most individuals include global developmental delay and/or intellectual disability, hypotonia, cerebellar ataxia, cerebellar atrophy, and facial dysmorphisms. The majority of the individuals have epilepsy. Two individuals have slightly decreased levels of serum alkaline phosphatase, while eight do not. Flow cytometric analysis of blood and fibroblasts from affected individuals showed decreased cell surface presence of GPI-APs. The overexpression of wild-type (WT) PIGK in fibroblasts rescued the levels of cell surface GPI-APs. In a knockout cell line, transfection with WT PIGK also rescued the GPI-AP levels, but transfection with the two tested mutant variants did not. Our study not only expands the clinical and known genetic spectrum of IGDs, but it also expands the genetic differential diagnosis for cerebellar atrophy. Given the fact that cerebellar atrophy is seen in other IGDs, flow cytometry for GPI-APs should be considered in the work-ups of individuals presenting this feature.

Evaluation of DNA Methylation Episignatures for Diagnosis and Phenotype Correlations in 42 Mendelian Neurodevelopmental Disorders.

Genetic syndromes frequently present with overlapping clinical features and inconclusive or ambiguous genetic findings which can confound accurate diagnosis and clinical management. An expanding number of genetic syndromes have been shown to have unique genomic DNA methylation patterns (called "episignatures"). Peripheral blood episignatures can be used for diagnostic testing as well as for the interpretation of ambiguous genetic test results. We present here an approach to episignature mapping in 42 genetic syndromes, which has allowed the identification of 34 robust disease-specific episignatures. We examine emerging patterns of overlap, as well as similarities and hierarchical relationships across these episignatures, to highlight their key features as they are related to genetic heterogeneity, dosage effect, unaffected carrier status, and incomplete penetrance. We demonstrate the necessity of multiclass modeling for accurate genetic variant classification and show how disease classification using a single episignature at a time can sometimes lead to classification errors in closely related episignatures. We demonstrate the utility of this tool in resolving ambiguous clinical cases and identification of previously undiagnosed cases through mass screening of a large cohort of subjects with developmental delays and congenital anomalies. This study more than doubles the number of published syndromes with DNA methylation episignatures and, most significantly, opens new avenues for accurate diagnosis and clinical assessment in individuals affected by these disorders.

Heterozygous pathogenic variants in POMC are not responsible for monogenic obesity: Implication for MC4R agonist use.

PURPOSE: Recessive deficiency of proopiomelanocortin (POMC) causes childhood-onset severe obesity. Cases can now benefit from the melanocortin 4 receptor agonist setmelanotide. Furthermore, a phase 3 clinical trial is evaluating setmelanotide in heterozygotes for POMC. We performed a large-scale genetic analysis to assess the effect of heterozygous, pathogenic POMC variants on obesity. METHODS: A genetic analysis was performed in a family including 2 cousins with childhood-onset obesity. We analyzed the obesity status of heterozygotes for pathogenic POMC variants in the Human Gene Mutation Database. The association between heterozygous pathogenic POMC variants and obesity risk was assessed using 190,000 exome samples from UK Biobank. RESULTS: The 2 cousins carried a compound heterozygous pathogenic variant in POMC. Six siblings were heterozygotes; only 1 of them had obesity. In Human Gene Mutation Database, we identified 60 heterozygotes for pathogenic POMC variants, of whom 14 had obesity. In UK Biobank, heterozygous pathogenic POMC variants were not associated with obesity risk, but they modestly increased body mass index levels. CONCLUSION: Heterozygous pathogenic POMC variants do not contribute to monogenic obesity, but they slightly increase body mass index. Setmelanotide use in patients with obesity, which would only be based on the presence of a heterozygous POMC variant, can be questioned.

YWHAE loss of function causes a rare neurodevelopmental disease with brain abnormalities in human and mouse.

PURPOSE: Miller-Dieker syndrome is caused by a multiple gene deletion, including PAFAH1B1 and YWHAE. Although deletion of PAFAH1B1 causes lissencephaly unambiguously, deletion of YWHAE alone has not clearly been linked to a human disorder. METHODS: Cases with YWHAE variants were collected through international data sharing networks. To address the specific impact of YWHAE loss of function, we phenotyped a mouse knockout of Ywhae. RESULTS: We report a series of 10 individuals with heterozygous loss-of-function YWHAE variants (3 single-nucleotide variants and 7 deletions <1 Mb encompassing YWHAE but not PAFAH1B1), including 8 new cases and 2 follow-ups, added with 5 cases (copy number variants) from literature review. Although, until now, only 1 intragenic deletion has been described in YWHAE, we report 4 new variants specifically in YWHAE (3 splice variants and 1 intragenic deletion). The most frequent manifestations are developmental delay, delayed speech, seizures, and brain malformations, including corpus callosum hypoplasia, delayed myelination, and ventricular dilatation. Individuals with variants affecting YWHAE alone have milder features than those with larger deletions. Neuroanatomical studies in Ywhae-/- mice revealed brain structural defects, including thin cerebral cortex, corpus callosum dysgenesis, and hydrocephalus paralleling those seen in humans. CONCLUSION: This study further demonstrates that YWHAE loss-of-function variants cause a neurodevelopmental disease with brain abnormalities.

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing.

PURPOSE: Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the "ClinVar low-hanging fruit" reanalysis, reasons for the failure of previous analyses, and lessons learned. METHODS: Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. RESULTS: We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). CONCLUSION: The "ClinVar low-hanging fruit" analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock.