Frederike L. Harms

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.

Delineating SPTAN1 associated phenotypes: from isolated epilepsy to encephalopathy with progressive brain atrophy

&NA; De novo in‐frame deletions and duplications in the SPTAN1 gene, encoding the non‐erythrocyte &agr;II spectrin, have been associated with severe West syndrome with hypomyelination and pontocerebellar atrophy. We aimed at comprehensively delineating the phenotypic spectrum associated with SPTAN1 mutations. Using different molecular genetic techniques, we identified 20 patients with a pathogenic or likely pathogenic SPTAN1 variant and reviewed their clinical, genetic and imaging data. SPTAN1 de novo alterations included seven unique missense variants and nine in‐frame deletions/duplications of which 12 were novel. The recurrent three‐amino acid duplication p.(Asp2303_Leu2305dup) occurred in five patients. Our patient cohort exhibited a broad spectrum of neurodevelopmental phenotypes, comprising six patients with mild to moderate intellectual disability, with or without epilepsy and behavioural disorders, and 14 patients with infantile epileptic encephalopathy, of which 13 had severe neurodevelopmental impairment and four died in early childhood. Imaging studies suggested that the severity of neurological impairment and epilepsy correlates with that of structural abnormalities as well as the mutation type and location. Out of seven patients harbouring mutations outside the &agr;/&bgr; spectrin heterodimerization domain, four had normal brain imaging and three exhibited moderately progressive brain and/or cerebellar atrophy. Twelve of 13 patients with mutations located within the spectrin heterodimer contact site exhibited severe and progressive brain, brainstem and cerebellar atrophy, with hypomyelination in most. We used fibroblasts from five patients to study spectrin aggregate formation by Triton‐X extraction and immunocytochemistry followed by fluorescence microscopy. &agr;II/&bgr;II aggregates and &agr;II spectrin in the insoluble protein fraction were observed in fibroblasts derived from patients with the mutations p.(Glu2207del), p.(Asp2303_Leu2305dup) and p.(Arg2308_Met2309dup), all falling in the nucleation site of the &agr;/&bgr; spectrin heterodimer region. Molecular modelling of the seven SPTAN1 amino acid changes provided preliminary evidence for structural alterations of the A‐, B‐ and/or C‐helices within each of the mutated spectrin repeats. We conclude that SPTAN1‐related disorders comprise a wide spectrum of neurodevelopmental phenotypes ranging from mild to severe and progressive. Spectrin aggregate formation in fibroblasts with mutations in the &agr;/&bgr; heterodimerization domain seems to be associated with a severe neurodegenerative course and suggests that the amino acid stretch from Asp2303 to Met2309 in the &agr;20 repeat is important for &agr;/&bgr; spectrin heterodimer formation and/or &agr;II spectrin function.

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.

αPIX Is a Trafficking Regulator that Balances Recycling and Degradation of the Epidermal Growth Factor Receptor.

Endosomal sorting is an essential control mechanism for signaling through the epidermal growth factor receptor (EGFR). We report here that the guanine nucleotide exchange factor αPIX, which modulates the activity of Rho-GTPases, is a potent bimodal regulator of EGFR trafficking. αPIX interacts with the E3 ubiquitin ligase c-Cbl, an enzyme that attaches ubiquitin to EGFR, thereby labelling this tyrosine kinase receptor for lysosomal degradation. We show that EGF stimulation induces αPIX::c-Cbl complex formation. Simultaneously, αPIX and c-Cbl protein levels decrease, which depends on both αPIX binding to c-Cbl and c-Cbl ubiquitin ligase activity. Through interaction αPIX sequesters c-Cbl from EGFR and this results in reduced EGFR ubiquitination and decreased EGFR degradation upon EGF treatment. However, quantitatively more decisive for cellular EGFR distribution than impaired EGFR degradation is a strong stimulating effect of αPIX on EGFR recycling to the cell surface. This function depends on the GIT binding domain of αPIX but not on interaction with c-Cbl or αPIX exchange activity. In summary, our data demonstrate a previously unappreciated function of αPIX as a strong promoter of EGFR recycling. We suggest that the novel recycling regulator αPIX and the degradation factor c-Cbl closely cooperate in the regulation of EGFR trafficking: uncomplexed αPIX and c-Cbl mediate a positive and a negative feedback on EGFR signaling, respectively; αPIX::c-Cbl complex formation, however, results in mutual inhibition, which may reflect a stable condition in the homeostasis of EGF-induced signal flow.

Mutations in EBF3 disturb transcriptional profiles and underlie a novel syndrome of intellectual disability, ataxia and facial dysmorphism

From a GeneMatcher-enabled international collaboration, we identified ten individuals with intellectual disability, speech delay, ataxia and facial dysmorphism and a mutation in EBF3, encoding a transcription factor required for neuronal differentiation. Structural assessments, transactivation assays, in situ fractionation, RNA-seq and ChlP-seq experiments collectively show that the mutations are deleterious and impair EBF3 transcriptional regulation. These findings demonstrate that EBF3-mediated dysregulation of gene expression has profound effects on neuronal development in humans.

Phenotypic variability in patients with interstitial 6q21‐q22 microdeletion and Acro–Cardio–Facial syndrome

Deletions of 6q are known to be associated with variable clinical phenotypes including facial dysmorphism, hand malformations, heart defects, microcephaly, intellectual disability, epilepsy, and other neurodevelopmental and neuropsychiatric conditions. Here, we report a 7‐year‐old boy evaluated for facial dysmorphism, trigonocephaly, microcephaly, global developmental delay, and behavioral abnormalities. Molecular karyotyping revealed a 13‐Mb deletion within 6q21‐q22.31, (chr6:105,771,520‐119,130,805; hg19, GRch37) comprising 81 genes. Review of 15 cases with interstitial 6q21‐q22.3 deletion from the literature showed that facial dysmorphism, intellectual disability, and corpus callosum abnormalities are the most consistent clinical features in these individuals. Deleted genes and breakpoints in the 6q21‐q22 region of the patient reported here are similar to two earlier reported cases with the clinical diagnosis of Acro–Cardio–Facial syndrome. However, the present case lacks characteristic clinical findings of Acro–Cardio–Facial syndrome. We discuss, the considerable phenotypic variability seen in individuals with 6q21‐q22 microdeletion and emphasize the need for further scrutiny into the hypothesis of Acro–Cardio–Facial syndrome being a microdeletion syndrome.

Coinheritance of biallelic SLURP1 and SLC39A4 mutations cause a severe genodermatosis with skin peeling and hair loss all over the body.

Next-generation sequencing (NGS), especially multi-gene panels and whole-exome sequencing (WES), is a tool for identifying the cause of monogenic disorders and has played a role in uncovering the genetic cause of previously uncharacterized genodermatoses.1 By the application of NGS, the concept of apparently novel or atypical clinical presentations has been challenged by the finding of two or more genetic diagnoses in affected individuals. Approximately 5% of cases in which WES was informative had dual or multiple molecular diagnoses.2 This article is protected by copyright. All rights reserved.