The periosteum contributes to bone repair and maintenance of cortical bone mass. In contrast to the understanding of bone development within the epiphyseal growth plate, factors that regulate periosteal osteogenesis have not been studied as intensively. Osteofibrous dysplasia (OFD) is a congenital disorder of osteogenesis and is typically sporadic and characterized by radiolucent lesions affecting the cortical bone immediately under the periosteum of the tibia and fibula. We identified germline mutations in MET, encoding a receptor tyrosine kinase, that segregate with an autosomal-dominant form of OFD in three families and a mutation in a fourth affected subject from a simplex family and with bilateral disease. Mutations identified in all families with dominant inheritance and in the one simplex subject with bilateral disease abolished the splice inclusion of exon 14 in MET transcripts, which resulted in a MET receptor (MET(Δ14)) lacking a cytoplasmic juxtamembrane domain. Splice exclusion of this domain occurs during normal embryonic development, and forced induction of this exon-exclusion event retarded osteoblastic differentiation in vitro and inhibited bone-matrix mineralization. In an additional subject with unilateral OFD, we identified a somatic MET mutation, also affecting exon 14, that substituted a tyrosine residue critical for MET receptor turnover and, as in the case of the MET(Δ14) mutations, had a stabilizing effect on the mature protein. Taken together, these data show that aberrant MET regulation via the juxtamembrane domain subverts core MET receptor functions that regulate osteogenesis within cortical diaphyseal bone.

Frontometaphyseal dysplasia (FMD) is a progressive sclerosing skeletal dysplasia affecting the long bones and skull. The cause of FMD in some individuals is gain-of-function mutations in FLNA, although how these mutations result in a hyperostotic phenotype remains unknown. Approximately one half of individuals with FMD have no identified mutation in FLNA and are phenotypically very similar to individuals with FLNA mutations, except for an increased tendency to form keloid scars. Using whole-exome sequencing and targeted Sanger sequencing in 19 FMD-affected individuals with no identifiable FLNA mutation, we identified mutations in two genes-MAP3K7, encoding transforming growth factor β (TGF-β)-activated kinase (TAK1), and TAB2, encoding TAK1-associated binding protein 2 (TAB2). Four mutations were found in MAP3K7, including one highly recurrent (n = 15) de novo mutation (c.1454C>T [ p.Pro485Leu]) proximal to the coiled-coil domain of TAK1 and three missense mutations affecting the kinase domain (c.208G>C [p.Glu70Gln], c.299T>A [p.Val100Glu], and c.502G>C [p.Gly168Arg]). Notably, the subjects with the latter three mutations had a milder FMD phenotype. An additional de novo mutation was found in TAB2 (c.1705G>A, p.Glu569Lys). The recurrent mutation does not destabilize TAK1, or impair its ability to homodimerize or bind TAB2, but it does increase TAK1 autophosphorylation and alter the activity of more than one signaling pathway regulated by the TAK1 kinase complex. These findings show that dysregulation of the TAK1 complex produces a close phenocopy of FMD caused by FLNA mutations. Furthermore, they suggest that the pathogenesis of some of the filaminopathies caused by FLNA mutations might be mediated by misregulation of signaling coordinated through the TAK1 signaling complex.

Robinow syndrome (RS) is a phenotypically and genetically heterogeneous condition that can be caused by mutations in genes encoding components of the non-canonical Wnt signaling pathway. In contrast, germline mutations that act to increase canonical Wnt signaling lead to distinctive osteosclerotic phenotypes. Here, we identified de novo frameshift mutations in DVL1, a mediator of both canonical and non-canonical Wnt signaling, as the cause of RS-OS, an RS subtype involving osteosclerosis, in three unrelated individuals. The mutations all delete the DVL1 C terminus and replace it, in each instance, with a novel, highly basic sequence. We showed the presence of mutant transcript in fibroblasts from one individual with RS-OS and demonstrated unimpaired protein stability with transfected GFP-tagged constructs bearing a frameshift mutation. In vitro TOPFlash assays, in apparent contradiction to the osteosclerotic phenotype, revealed that the mutant allele was less active than the wild-type allele in the canonical Wnt signaling pathway. However, when the mutant and wild-type alleles were co-expressed, canonical Wnt activity was 2-fold higher than that in the wild-type construct alone. This work establishes that DVL1 mutations cause a specific RS subtype, RS-OS, and that the osteosclerosis associated with this subtype might be the result of an interaction between the wild-type and mutant alleles and thus lead to elevated canonical Wnt signaling.

Gordon syndrome (GS), or distal arthrogryposis type 3, is a rare, autosomal-dominant disorder characterized by cleft palate and congenital contractures of the hands and feet. Exome sequencing of five GS-affected families identified mutations in piezo-type mechanosensitive ion channel component 2 (PIEZO2) in each family. Sanger sequencing revealed PIEZO2 mutations in five of seven additional families studied (for a total of 10/12 [83%] individuals), and nine families had an identical c.8057G>A (p.Arg2686His) mutation. The phenotype of GS overlaps with distal arthrogryposis type 5 (DA5) and Marden-Walker syndrome (MWS). Using molecular inversion probes for targeted sequencing to screen PIEZO2, we found mutations in 24/29 (82%) DA5-affected families and one of two MWS-affected families. The presence of cleft palate was significantly associated with c.8057G>A (Fisher's exact test, adjusted p value < 0.0001). Collectively, although GS, DA5, and MWS have traditionally been considered separate disorders, our findings indicate that they are etiologically related and perhaps represent variable expressivity of the same condition.

We report 15 individuals with de novo pathogenic variants in WDR26. Eleven of the individuals carry loss-of-function mutations, and four harbor missense substitutions. These 15 individuals comprise ten females and five males, and all have intellectual disability with delayed speech, a history of febrile and/or non-febrile seizures, and a wide-based, spastic, and/or stiff-legged gait. These subjects share a set of common facial features that include a prominent maxilla and upper lip that readily reveal the upper gingiva, widely spaced teeth, and a broad nasal tip. Together, these features comprise a recognizable facial phenotype. We compared these features with those of chromosome 1q41q42 microdeletion syndrome, which typically contains WDR26, and noted that clinical features are consistent between the two subsets, suggesting that haploinsufficiency of WDR26 contributes to the pathology of 1q41q42 microdeletion syndrome. Consistent with this, WDR26 loss-of-function single-nucleotide mutations identified in these subjects lead to nonsense-mediated decay with subsequent reduction of RNA expression and protein levels. We derived a structural model of WDR26 and note that missense variants identified in these individuals localize to highly conserved residues of this WD-40-repeat-containing protein. Given that WDR26 mutations have been identified in ∼1 in 2,000 of subjects in our clinical cohorts and that WDR26 might be poorly annotated in exome variant-interpretation pipelines, we would anticipate that this disorder could be more common than currently appreciated.

Netrin-1 is a secreted protein that was first identified 20 years ago as an axon guidance molecule that regulates midline crossing in the CNS. It plays critical roles in various tissues throughout development and is implicated in tumorigenesis and inflammation in adulthood. Despite extensive studies, no inherited human disease has been directly associated with mutations in NTN1, the gene coding for netrin-1. Here, we have identified 3 mutations in exon 7 of NTN1 in 2 unrelated families and 1 sporadic case with isolated congenital mirror movements (CMM), a disorder characterized by involuntary movements of one hand that mirror intentional movements of the opposite hand. Given the diverse roles of netrin-1, the absence of manifestations other than CMM in NTN1 mutation carriers was unexpected. Using multimodal approaches, we discovered that the anatomy of the corticospinal tract (CST) is abnormal in patients with NTN1-mutant CMM. When expressed in HEK293 or stable HeLa cells, the 3 mutated netrin-1 proteins were almost exclusively detected in the intracellular compartment, contrary to WT netrin-1, which is detected in both intracellular and extracellular compartments. Since netrin-1 is a diffusible extracellular cue, the pathophysiology likely involves its loss of function and subsequent disruption of axon guidance, resulting in abnormal decussation of the CST.

Excess exogenous retinoic acid (RA) has been well documented to have teratogenic effects in the limb and craniofacial skeleton. Malformations that have been observed in this context include craniosynostosis, a common developmental defect of the skull that occurs in 1 in 2500 individuals and results from premature fusion of the cranial sutures. Despite these observations, a physiological role for RA during suture formation has not been demonstrated. Here, we present evidence that genetically based alterations in RA signaling interfere with human development. We have identified human null and hypomorphic mutations in the gene encoding the RA-degrading enzyme CYP26B1 that lead to skeletal and craniofacial anomalies, including fusions of long bones, calvarial bone hypoplasia, and craniosynostosis. Analyses of murine embryos exposed to a chemical inhibitor of Cyp26 enzymes and zebrafish lines with mutations in cyp26b1 suggest that the endochondral bone fusions are due to unrestricted chondrogenesis at the presumptive sites of joint formation within cartilaginous templates, whereas craniosynostosis is induced by a defect in osteoblastic differentiation. Ultrastructural analysis, in situ expression studies, and in vitro quantitative RT-PCR experiments of cellular markers of osseous differentiation indicate that the most likely cause for these phenomena is aberrant osteoblast-osteocyte transitioning. This work reveals a physiological role for RA in partitioning skeletal elements and in the maintenance of cranial suture patency.

Cantú syndrome is a rare disorder characterized by congenital hypertrichosis, neonatal macrosomia, a distinct osteochondrodysplasia, and cardiomegaly. Using an exome-sequencing approach applied to one proband-parent trio and three unrelated single cases, we identified heterozygous mutations in ABCC9 in all probands. With the inclusion of the remaining cohort of ten individuals with Cantú syndrome, a total of eleven mutations in ABCC9 were found. The de novo occurrence in all six simplex cases in our cohort substantiates the presence of a dominant disease mechanism. All mutations were missense, and several mutations affect Arg1154. This mutation hot spot lies within the second type 1 transmembrane region of this ATP-binding cassette transporter protein, which may suggest an activating mutation. ABCC9 encodes the sulfonylurea receptor (SUR) that forms ATP-sensitive potassium channels (K(ATP) channels) originally shown in cardiac, skeletal, and smooth muscle. Previously, loss-of-function mutations in this gene have been associated with idiopathic dilated cardiomyopathy type 10 (CMD10). These findings identify the genetic basis of Cantú syndrome and suggest that this is a new member of the potassium channelopathies.

The family of Tre2-Bub2-Cdc16 (TBC)-domain containing GTPase activating proteins (RABGAPs) is not only known as key regulatorof RAB GTPase activity but also has GAP-independent functions. Rab GTPases are implicated in membrane trafficking pathways, such as vesicular trafficking. We report biallelic loss-of-function variants in TBC1D2B, encoding a member of the TBC/RABGAP family with yet unknown function, as the underlying cause of cognitive impairment, seizures, and/or gingival overgrowth in three individuals from unrelated families. TBC1D2B messenger RNA amount was drastically reduced, and the protein was absent in fibroblasts of two patients. In immunofluorescence analysis, ectopically expressed TBC1D2B colocalized with vesicles positive for RAB5, a small GTPase orchestrating early endocytic vesicle trafficking. In two independent TBC1D2B CRISPR/Cas9 knockout HeLa cell lines that serve as cellular model of TBC1D2B deficiency, epidermal growth factor internalization was significantly reduced compared with the parental HeLa cell line suggesting a role of TBC1D2B in early endocytosis. Serum deprivation of TBC1D2B-deficient HeLa cell lines caused a decrease in cell viability and an increase in apoptosis. Our data reveal that loss of TBC1D2B causes a neurodevelopmental disorder with gingival overgrowth, possibly by deficits in vesicle trafficking and/or cell survival.

The mammalian neocortex has undergone remarkable changes through evolution. A consequence of such rapid evolutionary events could be a trade-off that has rendered the brain susceptible to certain neurodevelopmental and neuropsychiatric conditions. We analyzed the exomes of 65 patients with the structural brain malformation periventricular nodular heterotopia (PH). De novo coding variants were observed in excess in genes defining a transcriptomic signature of basal radial glia, a cell type linked to brain evolution. In addition, we located two variants in human isoforms of two genes that have no ortholog in mice. Modulating the levels of one of these isoforms for the gene PLEKHG6 demonstrated its role in regulating neuroprogenitor differentiation and neuronal migration via RhoA, with phenotypic recapitulation of PH in human cerebral organoids. This suggests that this PLEKHG6 isoform is an example of a primate-specific genomic element supporting brain development.

Multinucleate cellular syncytial formation is a hallmark of skeletal muscle differentiation. Myomaker, encoded by Mymk (Tmem8c), is a well-conserved plasma membrane protein required for myoblast fusion to form multinucleated myotubes in mouse, chick, and zebrafish. Here, we report that autosomal recessive mutations in MYMK (OMIM 615345) cause Carey-Fineman-Ziter syndrome in humans (CFZS; OMIM 254940) by reducing but not eliminating MYMK function. We characterize MYMK-CFZS as a congenital myopathy with marked facial weakness and additional clinical and pathologic features that distinguish it from other congenital neuromuscular syndromes. We show that a heterologous cell fusion assay in vitro and allelic complementation experiments in mymk knockdown and mymkinsT/insT zebrafish in vivo can differentiate between MYMK wild type, hypomorphic and null alleles. Collectively, these data establish that MYMK activity is necessary for normal muscle development and maintenance in humans, and expand the spectrum of congenital myopathies to include cell-cell fusion deficits.

Multicentric carpotarsal osteolysis (MCTO) is a rare skeletal dysplasia characterized by aggressive osteolysis, particularly affecting the carpal and tarsal bones, and is frequently associated with progressive renal failure. Using exome capture and next-generation sequencing in five unrelated simplex cases of MCTO, we identified previously unreported missense mutations clustering within a 51 base pair region of the single exon of MAFB, validated by Sanger sequencing. A further six unrelated simplex cases with MCTO were also heterozygous for previously unreported mutations within this same region, as were affected members of two families with autosomal-dominant MCTO. MAFB encodes a transcription factor that negatively regulates RANKL-induced osteoclastogenesis and is essential for normal renal development. Identification of this gene paves the way for development of novel therapeutic approaches for this crippling disease and provides insight into normal bone and kidney development.

Periventricular neuronal heterotopia (PH) is one of the most common forms of cortical malformation in the human cortex. We show that human neuronal progenitor cells (hNPCs) derived from PH patients with a DCHS1 or FAT4 mutation as well as isogenic lines had altered migratory dynamics when grafted in the mouse brain. The affected migration was linked to altered autophagy as observed in vivo with an electron microscopic analysis of grafted hNPCs, a Western blot analysis of cortical organoids, and time-lapse imaging of hNPCs in the presence of bafilomycin A1. We further show that deficits in autophagy resulted in the accumulation of paxillin, a focal adhesion protein involved in cell migration. Strikingly, a single-cell RNA-seq analysis of hNPCs revealed similar expression levels of autophagy-related genes. Bolstering AMPK-dependent autophagy by metformin, an FDA-approved drug, promoted migration of PH patients-derived hNPCs. Our data indicate that transcription-independent homeostatic modifications in autophagy contributed to the defective migratory behavior of hNPCs in vivo and suggest that modulating autophagy in hNPCs might rescue neuronal migration deficits in some forms of PH.

Pathogenic variants in ARID1B are one of the most frequent causes of intellectual disability (ID) as determined by large-scale exome sequencing studies. Most studies published thus far describe clinically diagnosed Coffin-Siris patients (ARID1B-CSS) and it is unclear whether these data are representative for patients identified through sequencing of unbiased ID cohorts (ARID1B-ID). We therefore sought to determine genotypic and phenotypic differences between ARID1B-ID and ARID1B-CSS. In parallel, we investigated the effect of different methods of phenotype reporting.

Distal arthrogryposis (DA) syndromes are the most common of the heritable congenital-contracture disorders, and ~50% of cases are caused by mutations in genes that encode contractile proteins of skeletal myofibers. DA type 5D (DA5D) is a rare, autosomal-recessive DA previously defined by us and is characterized by congenital contractures of the hands and feet, along with distinctive facial features, including ptosis. We used linkage analysis and whole-genome sequencing of a multiplex consanguineous family to identify in endothelin-converting enzyme-like 1 (ECEL1) mutations that result in DA5D. Evaluation of a total of seven families affected by DA5D revealed in five families ECEL1 mutations that explain ~70% of cases overall. ECEL1 encodes a neuronal endopeptidase and is expressed in the brain and peripheral nerves. Mice deficient in Ecel1 exhibit perturbed terminal branching of motor neurons to the endplate of skeletal muscles, resulting in poor formation of the neuromuscular junction. Our results distinguish a second developmental pathway that causes congenital-contracture syndromes.

Genitopatellar syndrome (GPS) is a skeletal dysplasia with cerebral and genital anomalies for which the molecular basis has not yet been determined. By exome sequencing, we found de novo heterozygous truncating mutations in KAT6B (lysine acetyltransferase 6B, formerly known as MYST4 and MORF) in three subjects; then by Sanger sequencing of KAT6B, we found similar mutations in three additional subjects. The mutant transcripts do not undergo nonsense-mediated decay in cells from subjects with GPS. In addition, human pathological analyses and mouse expression studies point to systemic roles of KAT6B in controlling organismal growth and development. Myst4 (the mouse orthologous gene) is expressed in mouse tissues corresponding to those affected by GPS. Phenotypic differences and similarities between GPS, the Say-Barber-Biesecker variant of Ohdo syndrome (caused by different mutations of KAT6B), and Rubinstein-Taybi syndrome (caused by mutations in other histone acetyltransferases) are discussed. Together, the data support an epigenetic dysregulation of the limb, brain, and genital developmental programs.

Progeroid disorders overlapping with De Barsy syndrome (DBS) are collectively denoted as autosomal-recessive cutis laxa type 3 (ARCL3). They are caused by biallelic mutations in PYCR1 or ALDH18A1, encoding pyrroline-5-carboxylate reductase 1 and pyrroline-5-carboxylate synthase (P5CS), respectively, which both operate in the mitochondrial proline cycle. We report here on eight unrelated individuals born to non-consanguineous families clinically diagnosed with DBS or wrinkly skin syndrome. We found three heterozygous mutations in ALDH18A1 leading to amino acid substitutions of the same highly conserved residue, Arg138 in P5CS. A de novo origin was confirmed in all six probands for whom parental DNA was available. Using fibroblasts from affected individuals and heterologous overexpression, we found that the P5CS-p.Arg138Trp protein was stable and able to interact with wild-type P5CS but showed an altered sub-mitochondrial distribution. A reduced size upon native gel electrophoresis indicated an alteration of the structure or composition of P5CS mutant complex. Furthermore, we found that the mutant cells had a reduced P5CS enzymatic activity leading to a delayed proline accumulation. In summary, recurrent de novo mutations, affecting the highly conserved residue Arg138 of P5CS, cause an autosomal-dominant form of cutis laxa with progeroid features. Our data provide insights into the etiology of cutis laxa diseases and will have immediate impact on diagnostics and genetic counseling.

Disorders of neuronal mispositioning during brain development are phenotypically heterogeneous and their genetic causes remain largely unknown. Here, we report biallelic variants in a Hippo signaling factor-MOB2-in a patient with one such disorder, periventricular nodular heterotopia (PH). Genetic and cellular analysis of both variants confirmed them to be loss-of-function with enhanced sensitivity to transcript degradation via nonsense mediated decay (NMD) or increased protein turnover via the proteasome. Knockdown of Mob2 within the developing mouse cortex demonstrated its role in neuronal positioning. Cilia positioning and number within migrating neurons was also impaired with comparable defects detected following a reduction in levels of an upstream modulator of Mob2 function, Dchs1, a previously identified locus associated with PH. Moreover, reduced Mob2 expression increased phosphorylation of Filamin A, an actin cross-linking protein frequently mutated in cases of this disorder. These results reveal a key role for Mob2 in correct neuronal positioning within the developing cortex and outline a new candidate locus for PH development.

Spondylocarpotarsal synostosis syndrome (SCTS) is characterized by intervertebral fusions and fusion of the carpal and tarsal bones. Biallelic mutations in FLNB cause this condition in some families, whereas monoallelic variants in MYH3, encoding embryonic heavy chain myosin 3, have been implicated in dominantly inherited forms of the disorder. Here, five individuals without FLNB mutations from three families were hypothesized to be affected by recessive SCTS on account of sibling recurrence of the phenotype. Initial whole-exome sequencing (WES) showed that all five were heterozygous for one of two independent splice-site variants in MYH3. Despite evidence indicating that three of the five individuals shared two allelic haplotypes encompassing MYH3, no second variant could be located in the WES datasets. Subsequent genome sequencing of these three individuals demonstrated a variant altering a 5' UTR splice donor site (rs557849165 in MYH3) not represented by exome-capture platforms. When the cohort was expanded to 16 SCTS-affected individuals without FLNB mutations, nine had truncating mutations transmitted by unaffected parents, and six inherited the rs557849165 variant in trans, an observation at odds with the population allele frequency for this variant. The rs557849165 variant disrupts splicing in the 5' UTR but is still permissive of MYH3 translational initiation, albeit with reduced efficiency. Although some MYH3 variants cause dominant SCTS, these data indicate that others (notably truncating variants) do not, except in the context of compound heterozygosity for a second hypomorphic allele. These observations make genetic diagnosis challenging in the context of simplex presentations of the disorder.

Missense mutations in filamin B (FLNB) are associated with the autosomal dominant atelosteogenesis (AO) and the Larsen group of skeletal malformation disorders. These mutations cluster in particular FLNB protein domains and act in a presumptive gain-of-function mechanism. In contrast the loss-of-function disorder, spondylocarpotarsal synostosis syndrome, is characterised by the complete absence of FLNB. One cluster of AO missense mutations is found within the second of two calponin homology (CH) domains that create a functional actin-binding domain (ABD). This N-terminal ABD is required for filamin F-actin crosslinking activity, a crucial aspect of filamin's role of integrating cell-signalling events with cellular scaffolding and mechanoprotection. This study characterises the wild type FLNB ABD and investigates the effects of two disease-associated mutations on the structure and function of the FLNB ABD that could explain a gain-of-function mechanism for the AO diseases. We have determined high-resolution X-ray crystal structures of the human filamin B wild type ABD, plus W148R and M202V mutants. All three structures display the classic compact monomeric conformation for the ABD with the CH1 and CH2 domains in close contact. The conservation of tertiary structure in the presence of these mutations shows that the compact ABD conformation is stable to the sequence substitutions. In solution the mutant ABDs display reduced melting temperatures (by 6-7 degrees C) as determined by differential scanning fluorimetry. Characterisation of the wild type and mutant ABD F-actin binding activities via co-sedimentation assays shows that the mutant FLNB ABDs have increased F-actin binding affinities, with dissociation constants of 2.0 microM (W148R) and 0.56 microM (M202V), compared to the wild type ABD K(d) of 7.0 microM. The increased F-actin binding affinity of the mutants presents a biochemical mechanism that differentiates the autosomal dominant gain-of-function FLNB disorders from those that arise through the complete loss of FLNB protein.