Loss of TBC1D2B causes a progressive neurological disorder with gingival overgrowth.

Biallelic loss-of-function variants in TBC1D2B have been reported in five subjects with cognitive impairment and seizures with or without gingival overgrowth. TBC1D2B belongs to the family of Tre2-Bub2-Cdc16 (TBC)-domain containing RAB-specific GTPase activating proteins (TBC/RABGAPs). Here, we report five new subjects with biallelic TBC1D2B variants, including two siblings, and delineate the molecular and clinical features in the ten subjects known to date. One of the newly reported subjects was compound heterozygous for the TBC1D2B variants c.2584C>T; p.(Arg862Cys) and c.2758C>T; p.(Arg920*). In subject-derived fibroblasts, TBC1D2B mRNA level was similar to control cells, while the TBC1D2B protein amount was reduced by about half. In one of two siblings with a novel c.360+1G>T splice site variant, TBC1D2B transcript analysis revealed aberrantly spliced mRNAs and a drastically reduced TBC1D2B mRNA level in leukocytes. The molecular spectrum included 12 different TBC1D2B variants: seven nonsense, three frameshifts, one splice site, and one missense variant. Out of ten subjects, three had fibrous dysplasia of the mandible, two of which were diagnosed as cherubism. Most subjects developed gingival overgrowth. Half of the subjects had developmental delay. Seizures occurred in 80% of the subjects. Six subjects showed a progressive disease with mental deterioration. Brain imaging revealed cerebral and/or cerebellar atrophy with or without lateral ventricle dilatation. The TBC1D2B disorder is a progressive neurological disease with gingival overgrowth and abnormal mandible morphology. As TBC1D2B has been shown to positively regulate autophagy, defects in autophagy and the endolysosomal system could be associated with neuronal dysfunction and the neurodegenerative disease in the affected individuals.

Craniofacial dysmorphism, skeletal anomalies, and impaired intellectual development syndrome-1 in two new patients with the same homozygous TMCO1 variant and review of the literature.

Craniofacial dysmorphism, skeletal anomalies, and impaired intellectual development syndrome-1 (CFSMR1; OMIM#213980) is a rare autosomal recessive disorder characterized by the clinical triad of developmental delay and/or intellectual disability, a typical facial gestalt with brachycephaly, highly-arched bushy eyebrows, synophrys, hypertelorism, wide nasal bridge, and short nose, as well as multiple vertebrae and rib malformations, such as bifid and fused ribs and abnormal vertebral segmentation and fusion. Biallelic loss-of-function variants in TMCO1 cause CFSMR1. We report on two unrelated Egyptian patients with a phenotype suggestive of CFSMR. Single whole-exome sequencing in patient 1 and Sanger sequencing of TMCO1 in patient 2 revealed the same homozygous TMCO1 nonsense variant c.187C > T/p.(Arg63*) in both affected individuals; patients' healthy parents were heterozygous carriers of the variant. Congenital hearing loss in patients 1 and 2 is an occasional finding in individuals affected by CFSMR. Camptodactyly and syndactyly, which were noted in patient 2, have not or rarely been reported in CFSMR. Review of the literature revealed a total of 30 individuals with the clinically recognizable and unique phenotype of CFSMR1, including the patients reported here, who all carried biallelic TMCO1 variants. Six different TMCO1 variants have been reported in the 30 patients from 14 families, comprising three nonsense, two 2-bp deletions, and a splice donor site variant. All disease-associated TMCO1 variants likely represent null alleles resulting in absence of the encoded protein. TMCO1 has been proposed to act as a Ca2+ channel, while other data revealed TMCO1 as a mitochondrial protein and a component of the translocon at the endoplasmic reticulum, a cellular machinery important for the biogenesis of multi-pass membrane proteins. RAB5IF/C20orf24 has recently been identified as causative gene for craniofacial dysmorphism, skeletal anomalies, and impaired intellectual development syndrome-2 (CFSMR2; OMIM#616994). Heterodimerization of RAB5IF/C20orf24 and TMCO1 and their interdependence may suggest a pathophysiological role of ER-mitochondria interaction underlying CFSMR.

INPP4A-related genetic and phenotypic spectrum and functional relevance of subcellular targeting of INPP4A isoforms.

Type I inositol polyphosphate-4-phosphatase (INPP4A) belongs to the group of phosphoinositide phosphatases controlling proliferation, apoptosis, and endosome function by hydrolyzing phosphatidylinositol 3,4-bisphosphate. INPP4A produces multiple transcripts encoding shorter and longer INPP4A isoforms with hydrophilic or hydrophobic C-terminus. Biallelic INPP4A truncating variants cause a spectrum of neurodevelopmental disorders ranging from moderate intellectual disability to postnatal microcephaly with developmental and epileptic encephalopathy and (ponto)cerebellar hypoplasia. We report a girl with the novel homozygous INPP4A variant NM_001134224.2:c.2840del/p.(Gly947Glufs*12) (isoform d). She presented with postnatal microcephaly, global developmental delay, visual impairment, myoclonic seizures, and pontocerebellar hypoplasia and died at the age of 27 months. The level of mutant INPP4A mRNAs in proband-derived leukocytes was comparable to controls suggesting production of C-terminally altered INPP4A isoforms. We transiently expressed eGFP-tagged INPP4A isoform a (NM_004027.3) wildtype and p.(Gly908Glufs*12) mutant [p.(Gly947Glufs*12) according to NM_001134224.2] as well as INPP4A isoform b (NM_001566.2) wildtype and p.(Asp915Alafs*2) mutant, previously reported in family members with moderate intellectual disability, in HeLa cells and determined their subcellular distributions. While INPP4A isoform a was preferentially found in perinuclear clusters co-localizing with the GTPase Rab5, isoform b showed a net-like distribution, possibly localizing near and/or on microtubules. Quantification of intracellular localization patterns of the two INPP4A mutants revealed significant differences compared with the respective wildtype and similarity with each other. Our data suggests an important non-redundant function of INPP4A isoforms with hydrophobic or hydrophilic C-terminus in the brain.

Biallelic loss-of-function variants in TBC1D2B cause a neurodevelopmental disorder with seizures and gingival overgrowth.

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.

Biallelic variants in SMAD6 are associated with a complex cardiovascular phenotype.

Rare heterozygous variants in SMAD6 have been identified as a significant genetic contributor to bicuspid aortic valve-associated thoracic aortic aneurysm on one hand and non-syndromic midline craniosynostosis on the other. In this study, we report two individuals with biallelic missense variants in SMAD6 and a complex cardiac phenotype. Trio exome sequencing in Proband 1, a male who had aortic isthmus stenosis, revealed the homozygous SMAD6 variant p.(Ile466Thr). He also had mild intellectual disability and radio-ulnar synostosis. Proband 2 is a female who presented with a more severe cardiac phenotype with a dysplastic and stenotic pulmonary valve and dilated cardiomyopathy. In addition, she had vascular anomalies, including a stenotic left main coronary artery requiring a bypass procedure, narrowing of the proximal left pulmonary artery and a venous anomaly in the brain. Proband 2 has compound heterozygous SMAD6 missense variants, p.(Phe357Ile) and p.(Ser483Pro). Absence of these SMAD6 variants in the general population and high pathogenicity prediction scores suggest that these variants caused the probands' phenotypes. This is further corroborated by cardiovascular anomalies and appendicular skeletal defects in Smad6-deficient mice. SMAD6 acts as an inhibitory SMAD and preferentially inhibits bone morphogenetic protein (BMP)-induced signaling. Our data suggest that biallelic variants in SMAD6 may affect the inhibitory activity of SMAD6 and cause enhanced BMP signaling underlying the cardiovascular anomalies and possibly other clinical features in the two probands.

A homozygous ATAD1 mutation impairs postsynaptic AMPA receptor trafficking and causes a lethal encephalopathy.

Members of the AAA+ superfamily of ATPases are involved in the unfolding of proteins and disassembly of protein complexes and aggregates. ATAD1 encoding the ATPase family, AAA+ domain containing 1-protein Thorase plays an important role in the function and integrity of mitochondria and peroxisomes. Postsynaptically, Thorase controls the internalization of excitatory, glutamatergic AMPA receptors by disassembling complexes between the AMPA receptor-binding protein, GRIP1, and the AMPA receptor subunit GluA2. Using whole-exome sequencing, we identified a homozygous frameshift mutation in the last exon of ATAD1 [c.1070_1071delAT; p.(His357Argfs*15)] in three siblings who presented with a severe, lethal encephalopathy associated with stiffness and arthrogryposis. Biochemical and cellular analyses show that the C-terminal end of Thorase mutant gained a novel function that strongly impacts its oligomeric state, reduces stability or expression of a set of Golgi, peroxisomal and mitochondrial proteins and affects disassembly of GluA2 and Thorase oligomer complexes. Atad1-/- neurons expressing Thorase mutantHis357Argfs*15 display reduced amount of GluA2 at the cell surface suggesting that the Thorase mutant may inhibit the recycling back and/or reinsertion of AMPA receptors to the plasma membrane. Taken together, our molecular and functional analyses identify an activating ATAD1 mutation as a new cause of severe encephalopathy and congenital stiffness.

Elsahy-Waters syndrome is caused by biallelic mutations in CDH11.

Elsahy-Waters syndrome (EWS), also known as branchial-skeletal-genital syndrome, is a distinct dysmorphology syndrome characterized by facial asymmetry, broad forehead, marked hypertelorism with proptosis, short and broad nose, midface hypoplasia, intellectual disability, and hypospadias. We have recently published a homozygous potential loss of function variant in CDH11 in a boy with a striking resemblance to EWS. More recently, another homozygous truncating variant in CDH11 was reported in two siblings with suspected EWS. Here, we describe in detail the clinical phenotype of the original CDH11-related patient with EWS as well as a previously unreported EWS-affected girl who was also found to have a novel homozygous truncating variant in CDH11, which confirms that EWS is caused by biallelic CDH11 loss of function mutations. Clinical features in the four CDH11 mutation-positive individuals confirm the established core phenotype of EWS. Additionally, we identify upper eyelid coloboma as a new, though infrequent clinical feature. The pathomechanism underlying EWS remains unclear, although the limited phenotypic data on the Cdh11-/- mouse suggest that this is a potentially helpful model to explore the craniofacial and brain development in EWS-affected individuals.

The novel RAF1 mutation p.(Gly361Ala) located outside the kinase domain of the CR3 region in two patients with Noonan syndrome, including one with a rare brain tumor.

Noonan syndrome is characterized by typical craniofacial dysmorphism, postnatal growth retardation, congenital heart defect, and learning difficulties and belongs to the RASopathies, a group of neurodevelopmental disorders caused by germline mutations in genes encoding components of the RAS-MAPK pathway. Mutations in the RAF1 gene are associated with Noonan syndrome, with a high prevalence of hypertrophic cardiomyopathy (HCM). RAF1 mutations cluster in exons encoding the conserved region 2 (CR2), the kinase activation segment of the CR3 domain, and the C-terminus. We present two boys with Noonan syndrome and the identical de novo RAF1 missense variant c.1082G>C/p.(Gly361Ala) affecting the CR3, but located outside the kinase activation segment. The p.(Gly361Ala) mutation has been identified as a RAF1 allele conferring resistance to RAF inhibitors. This amino acid change favors a RAF1 conformation that allows for enhanced RAF dimerization and increased intrinsic kinase activity. Both patients with Noonan syndrome showed typical craniofacial dysmorphism, macrocephaly, and short stature. One individual developed HCM and was diagnosed with a disseminated oligodendroglial-like leptomeningeal tumor (DOLT) of childhood at the age of 9 years. While there is a well-established association of NS with malignant tumors, especially childhood hemato-oncological diseases, brain tumors have rarely been reported in Noonan syndrome. Our data demonstrate that mutation scanning of the entire coding region of genes associated with Noonan syndrome is mandatory not to miss rare variants located outside the known mutational hotspots.

Mannose receptor induces T-cell tolerance via inhibition of CD45 and up-regulation of CTLA-4.

The mannose receptor (MR) is an endocytic receptor involved in serum homeostasis and antigen presentation. Here, we identify the MR as a direct regulator of CD8(+) T-cell activity. We demonstrate that MR expression on dendritic cells (DCs) impaired T-cell cytotoxicity in vitro and in vivo. This regulatory effect of the MR was mediated by a direct interaction with CD45 on the T cell, inhibiting its phosphatase activity, which resulted in up-regulation of cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) and the induction of T-cell tolerance. Inhibition of CD45 prevented expression of B-cell lymphoma 6 (Bcl-6), a transcriptional inhibitor that directly bound the CTLA-4 promoter and regulated its activity. These data demonstrate that endocytic receptors expressed on DCs contribute to the regulation of T-cell functionality.