Cutaneous manifestations in Costello syndrome: HRAS p.Gly12Ser affects RIN1-mediated integrin trafficking in immortalized epidermal keratinocytes.

Heterozygous germline missense variants in the HRAS gene underlie Costello syndrome (CS). The molecular basis for cutaneous manifestations in CS is largely unknown. We used an immortalized human cell line, HaCaT keratinocytes, stably expressing wild-type or CS-associated (p.Gly12Ser) HRAS and defined RIN1 as quantitatively most prominent, high-affinity effector of active HRAS in these cells. As an exchange factor for RAB5 GTPases, RIN1 is involved in endosomal sorting of cell-adhesion integrins. RIN1-dependent RAB5A activation was strongly increased by HRASGly12Ser, and HRAS-RIN1-ABL1/2 signaling was induced in HRASWT- and HRASGly12Ser-expressing cells. Along with that, HRASGly12Ser expression decreased total integrin levels and enriched ß1 integrin in RAB5- and EEA1-positive early endosomes. The intracellular level of active ß1 integrin was increased in HRASGly12Ser HaCaT keratinocytes due to impaired recycling, whereas RIN1 disruption raised ß1 integrin cell surface distribution. HRASGly12Ser induced co-localization of ß1 integrin with SNX17 and RAB7 in early/sorting and late endosomes, respectively. Thus, by retaining ß1 integrin in intracellular endosomal compartments, HRAS-RIN1 signaling affects the subcellular availability of ß1 integrin. This may interfere with integrin-dependent processes as we detected for HRASGly12Ser cells spreading on fibronectin. We conclude that dysregulation of receptor trafficking and integrin-dependent processes such as cell adhesion are relevant in the pathobiology of CS.

Genetic Variants in ARHGEF6 Cause Congenital Anomalies of the Kidneys and Urinary Tract in Humans, Mice, and Frogs.

BACKGROUND: About 40 disease genes have been described to date for isolated CAKUT, the most common cause of childhood CKD. However, these genes account for only 20% of cases. ARHGEF6, a guanine nucleotide exchange factor that is implicated in biologic processes such as cell migration and focal adhesion, acts downstream of integrin-linked kinase (ILK) and parvin proteins. A genetic variant of ILK that causes murine renal agenesis abrogates the interaction of ILK with a murine focal adhesion protein encoded by Parva , leading to CAKUT in mice with this variant. METHODS: To identify novel genes that, when mutated, result in CAKUT, we performed exome sequencing in an international cohort of 1265 families with CAKUT. We also assessed the effects in vitro of wild-type and mutant ARHGEF6 proteins, and the effects of Arhgef6 deficiency in mouse and frog models. RESULTS: We detected six different hemizygous variants in the gene ARHGEF6 (which is located on the X chromosome in humans) in eight individuals from six families with CAKUT. In kidney cells, overexpression of wild-type ARHGEF6 -but not proband-derived mutant ARHGEF6 -increased active levels of CDC42/RAC1, induced lamellipodia formation, and stimulated PARVA-dependent cell spreading. ARHGEF6-mutant proteins showed loss of interaction with PARVA. Three-dimensional Madin-Darby canine kidney cell cultures expressing ARHGEF6-mutant proteins exhibited reduced lumen formation and polarity defects. Arhgef6 deficiency in mouse and frog models recapitulated features of human CAKUT. CONCLUSIONS: Deleterious variants in ARHGEF6 may cause dysregulation of integrin-parvin-RAC1/CDC42 signaling, thereby leading to X-linked CAKUT.

RIT1 controls actin dynamics via complex formation with RAC1/CDC42 and PAK1.

RIT1 belongs to the RAS family of small GTPases. Germline and somatic RIT1 mutations have been identified in Noonan syndrome (NS) and cancer, respectively. By using heterologous expression systems and purified recombinant proteins, we identified the p21-activated kinase 1 (PAK1) as novel direct effector of RIT1. We found RIT1 also to directly interact with the RHO GTPases CDC42 and RAC1, both of which are crucial regulators of actin dynamics upstream of PAK1. These interactions are independent of the guanine nucleotide bound to RIT1. Disease-causing RIT1 mutations enhance protein-protein interaction between RIT1 and PAK1, CDC42 or RAC1 and uncouple complex formation from serum and growth factors. We show that the RIT1-PAK1 complex regulates cytoskeletal rearrangements as expression of wild-type RIT1 and its mutant forms resulted in dissolution of stress fibers and reduction of mature paxillin-containing focal adhesions in COS7 cells. This effect was prevented by co-expression of RIT1 with dominant-negative CDC42 or RAC1 and kinase-dead PAK1. By using a transwell migration assay, we show that RIT1 wildtype and the disease-associated variants enhance cell motility. Our work demonstrates a new function for RIT1 in controlling actin dynamics via acting in a signaling module containing PAK1 and RAC1/CDC42, and highlights defects in cell adhesion and migration as possible disease mechanism underlying NS.

Next-generation sequencing of 32 genes associated with hereditary aortopathies and related disorders of connective tissue in a cohort of 199 patients.

PURPOSE: Heritable factors play an important etiologic role in connective tissue disorders (CTD) with vascular involvement, and a genetic diagnosis is getting increasingly important for gene-tailored, personalized patient management. METHODS: We analyzed 32 disease-associated genes by using targeted next-generation sequencing and exome sequencing in a clinically relevant cohort of 199 individuals. We classified and refined sequence variants according to their likelihood for pathogenicity. RESULTS: We identified 1 pathogenic variant (PV; in FBN1 or SMAD3) in 15 patients (7.5%) and ≥1 likely pathogenic variant (LPV; in COL3A1, FBN1, FBN2, LOX, MYH11, SMAD3, TGFBR1, or TGFBR2) in 19 individuals (9.6%), together resulting in 17.1% diagnostic yield. Thirteen PV/LPV were novel. Of PV/LPV-negative patients 47 (23.6%) showed ≥1 variant of uncertain significance (VUS). Twenty-five patients had concomitant variants. In-depth evaluation of reported/calculated variant classes resulted in reclassification of 19.8% of variants. CONCLUSION: Variant classification and refinement are essential for shaping mutational spectra of disease genes, thereby improving clinical sensitivity. Obligate stringent multigene analysis is a powerful tool for identifying genetic causes of clinically related CTDs. Nonetheless, the relatively high rate of PV/LPV/VUS-negative patients underscores the existence of yet unknown disease loci and/or oligogenic/polygenic inheritance.

16p13.11 microdeletion uncovers loss-of-function of a MYH11 missense variant in a patient with megacystis-microcolon-intestinal-hypoperistalsis syndrome.

Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS), a rare condition that affects smooth muscle cells, is caused by biallelic null alleles in MYH11. We report on a girl with MMIHS in addition to growth hormone deficiency, central hypothyroidism and a tonically dilated pupil with accommodation deficit. Sanger sequencing and arrayCGH uncovered the novel heterozygous missense variant c.379C>T in MYH11 and a heterozygous 1.3 Mb deletion in 16q13.11 encompassing MYH11, respectively. Her mother carries the deletion, whereas her father is heterozygous for the c.379C>T p.(Pro127Ser) change. Proline 127 is crucial for the formation of the Adenosine triphosphate binding pocket of the MYH11 motor domain and molecular modeling indicated that p.Pro127Ser alters nucleotide binding properties. Thus, the unusual and complex clinical presentation of the patient results from compound heterozygosity for a 16p13.11 microdeletion including the entire MYH11 gene and a loss-of-function missense variant on the remaining MYH11 allele. In conclusion, we recommend genetic testing both for MYH11 sequence alterations and copy number imbalances in individuals with MMIHS and smooth muscle cell-associated abnormalities in additional organs, that is, multisystemic smooth muscle dysfunction.

A novel missense variant in the SDR domain of the WWOX gene leads to complete loss of WWOX protein with early-onset epileptic encephalopathy and severe developmental delay.

The human WWOX (WW domain-containing oxidoreductase) gene, originally known as a tumor suppressor gene, has been shown to be important for brain function and development. In recent years, mutations in WWOX have been associated with a wide phenotypic spectrum of autosomal recessively inherited neurodevelopmental disorders. Whole exome sequencing was completed followed by Sanger sequencing to verify segregation of the identified variants. Functional WWOX analysis was performed in fibroblasts of one patient. Transcription and translation were assessed by quantitative real-time PCR and Western blotting. We report two related patients who presented with early epilepsy refractory to treatment, progressive microcephaly, profound developmental delay, and brain MRI abnormalities. Additionally, one of the patients showed bilateral optic atrophy. Whole exome sequencing revealed homozygosity for a novel missense variant affecting the evolutionary conserved amino acid Gln230 in the catalytic short-chain dehydrogenase/reductase (SDR) domain of WWOX in both girls. Functional studies showed normal levels of WWOX transcripts but absence of WWOX protein. To our knowledge, our patients are the first individuals presenting the more severe end of the phenotypic spectrum of WWOX deficiency, although they were only affected by a single missense variant of WWOX. This could be explained by the functional data indicating an impaired translation or premature degradation of the WWOX protein.

Functional analysis of a duplication (p.E63_D69dup) in the switch II region of HRAS: new aspects of the molecular pathogenesis underlying Costello syndrome.

Costello syndrome is a congenital disorder comprising a characteristic face, severe feeding difficulties, skeletal, cardiac and skin abnormalities, intellectual disability and predisposition to malignancies. It is caused by heterozygous germline HRAS mutations mostly affecting Gly(12) or Gly(13), which impair HRAS-GTPase activity and result in increased downstream signal flow independent of incoming signals. Functional analyses of rarer HRAS mutations identified in individuals with attenuated Costello syndrome phenotypes revealed altered GDP/GTP nucleotide affinities (p.K117R) and inefficient effector binding (p.E37dup). Thus, both phenotypic and functional variability associated with HRAS mutations are evident. Here, we report on a novel heterozygous HRAS germline mutation (c.187_207dup, p.E63_D69dup) in a girl presenting with a phenotype at the milder end of the Costello syndrome spectrum. The p.E63_D69dup mutation impaired co-precipitation of recombinant HRAS with NF1 GTPase-activating protein (GAP) suggesting constitutive HRAS(E63_D69dup) activation due to GAP insensitivity. Indeed, we identified strongly augmented active HRAS(E63_D69dup) that co-precipitated with effectors RAF1, RAL guanine nucleotide dissociation stimulator and phospholipase C1. However, we could not pull down active HRAS(E63_D69dup) using the target protein PIK3CA, indicating a compromised association between active HRAS(E63_D69dup) and PIK3CA. Accordingly, overexpression of HRAS(E63_D69dup) increased steady-state phosphorylation of MEK1/2 and ERK1/2 downstream of RAF, whereas AKT phosphorylation downstream of phosphoinositide 3-kinase (PI3K) was not enhanced. By analyzing signaling dynamics, we found that HRAS(E63_D69dup) has impaired reagibility to stimuli resulting in reduced and disrupted capacity to transduce incoming signals to the RAF-MAPK and PI3K-AKT cascade, respectively. We suggest that disrupted HRAS reagibility, as we demonstrate for the p.E63_D69dup mutation, is a previously unappreciated molecular pathomechanism underlying Costello syndrome.

An attenuated phenotype of Costello syndrome in three unrelated individuals with a HRAS c.179G>A (p.Gly60Asp) mutation correlates with uncommon functional consequences.

Heterozygous germline mutations in the proto-oncogene HRAS cause Costello syndrome (CS), an intellectual disability condition with severe failure to thrive, cardiac abnormalities, predisposition to tumors, and neurologic abnormalities. More than 80% of patients share the HRAS mutation c.34G>A (p.Gly12Ser) associated with the typical, relatively homogeneous phenotype. Rarer mutations occurred in individuals with an attenuated phenotype and less characteristic facial features. Most pathogenic HRAS alterations affect hydrolytic HRAS activity resulting in constitutive activation. "Gain-of-function" and "hyperactivation" concerning downstream pathways are widely used to explain the molecular basis and dysregulation of the RAS-MAPK pathway is the biologic mechanism shared amongst rasopathies. Panel testing for rasopathies identified a novel HRAS mutation (c.179G>A; p.Gly60Asp) in three individuals with attenuated features of Costello syndrome. De novo paternal origin occurred in two, transmission from a heterozygous mother in the third. Individuals showed subtle facial features; curly hair and relative macrocephaly were seen in three; atrial tachycardia and learning difficulties in two, and pulmonic valve dysplasia and mildly thickened left ventricle in one. None had severe failure to thrive, intellectual disability or cancer, underscoring the need to consider HRAS mutations in individuals with an unspecific rasopathy phenotype. Functional studies revealed strongly increased HRAS(Gly60Asp) binding to RAF1, but not to other signaling effectors. Hyperactivation of the MAPK downstream signaling pathways was absent. Our results indicate that an increase in the proportion of activated RAS downstream signaling components does not entirely explain the molecular basis of CS. We conclude that the phenotypic variability in CS recapitulates variable qualities of molecular dysfunction.

RASopathy-associated CBL germline mutations cause aberrant ubiquitylation and trafficking of EGFR.

Noonan syndrome, a congenital disorder comprising a characteristic face, short stature, heart defects, learning difficulties, and a predisposition to malignancies, is caused by heterozygous germline mutations in genes encoding components of RAS-MAPK signaling pathways. Mutations in the CBL tumor suppressor gene have been reported in patients with a Noonan syndrome-like phenotype. CBL encodes a multivalent adaptor protein with ubiquitin ligase activity, which promotes ubiquitylation and vesicle-mediated internalization and degradation of the epidermal growth factor (EGF) receptor (EGFR). We investigated the functional consequences of disease-associated CBL amino acid changes p.K382E, p.D390Y, and p.R420Q on ligand-induced EGFR trafficking. Expression of CBL(K382E), CBL(D390Y), or CBL(R420Q) in COS-7 cells resulted in increased levels of surface EGFR and reduced amounts of intracellular EGFR; both consequences indicate ineffective EGFR internalization. Accordingly, receptor-mediated uptake of EGF was decreased. Furthermore, the p.K382E, p.D390Y, and p.R420Q lesions impaired CBL-mediated EGFR ubiquitylation and degradation. Together, these data indicate that pathogenic CBL mutations severely affect vesicle-based EGFR trafficking. Since we detected enhanced ERK phosphorylation in cells expressing mutant CBL, we conclude that aberrant EGFR trafficking contributes to augmented RAS-MAPK signaling, the common trait of Noonan syndrome and related RASopathies. Thus, our data suggest that EGFR trafficking is a novel disease-relevant regulatory level in the RASopathy network.

Dysregulation of Rho GTPases in the αPix/Arhgef6 mouse model of X-linked intellectual disability is paralleled by impaired structural and synaptic plasticity and cognitive deficits.

Mutations in the ARHGEF6 gene, encoding the guanine nucleotide exchange factor αPIX/Cool-2 for the Rho GTPases Rac1 and Cdc42, cause X-linked intellectual disability (ID) in humans. We show here that αPix/Arhgef6 is primarily expressed in neuropil regions of the hippocampus. To study the role of αPix/Arhgef6 in neuronal development and plasticity and gain insight into the pathogenic mechanisms underlying ID, we generated αPix/Arhgef6-deficient mice. Gross brain structure in these mice appeared to be normal; however, analysis of Golgi-Cox-stained pyramidal neurons revealed an increase in both dendritic length and spine density in the hippocampus, accompanied by an overall loss in spine synapses. Early-phase long-term potentiation was reduced and long-term depression was increased in the CA1 hippocampal area of αPix/Arhgef6-deficient animals. Knockout animals exhibited impaired spatial and complex learning and less behavioral control in mildly stressful situations, suggesting that this model mimics the human ID phenotype. The structural and electrophysiological alterations in the hippocampus were accompanied by a significant reduction in active Rac1 and Cdc42, but not RhoA. In conclusion, we suggest that imbalance in activity of different Rho GTPases may underlie altered neuronal connectivity and impaired synaptic function and cognition in αPix/Arhgef6 knockout mice.

Reelin and the Cdc42/Rac1 guanine nucleotide exchange factor αPIX/Arhgef6 promote dendritic Golgi translocation in hippocampal neurons.

In the cerebral cortex of reeler mutant mice lacking reelin expression, neurons are malpositioned and display misoriented apical dendrites. Neuronal migration defects in reeler have been studied in great detail, but how misorientation of apical dendrites is related to reelin deficiency is poorly understood. In wild-type mice, the Golgi apparatus transiently translocates into the developing apical dendrite of radially migrating neurons. This dendritic Golgi translocation has recently been shown to be promoted by reelin. However, the underlying signalling mechanisms are largely unknown. Here, we show that the Cdc42/Rac1 guanine nucleotide exchange factor αPIX/Arhgef6 promoted translocation of Golgi cisternae into developing dendrites of hippocampal neurons. Reelin treatment further increased the αPIX-dependent effect. In turn, overexpression of exchange activity-deficient αPIX or dominant-negative (dn) Cdc42 or dn-Rac1 impaired dendritic Golgi positioning, an effect that was not compensated by reelin treatment. Together, these data suggest that αPIX may promote dendritic Golgi translocation, as a downstream component of a reelin-modulated signalling pathway. Finally, we found that reelin promoted the translocation of the Golgi apparatus into the dendrite that was most proximal to the reelin source. The distribution of reelin may thus contribute to the selection of the process that becomes the apical dendrite.

WDR11, a WD protein that interacts with transcription factor EMX1, is mutated in idiopathic hypogonadotropic hypogonadism and Kallmann syndrome.

By defining the chromosomal breakpoint of a balanced t(10;12) translocation from a subject with Kallmann syndrome and scanning genes in its vicinity in unrelated hypogonadal subjects, we have identified WDR11 as a gene involved in human puberty. We found six patients with a total of five different heterozygous WDR11 missense mutations, including three alterations (A435T, R448Q, and H690Q) in WD domains important for ß propeller formation and protein-protein interaction. In addition, we discovered that WDR11 interacts with EMX1, a homeodomain transcription factor involved in the development of olfactory neurons, and that missense alterations reduce or abolish this interaction. Our findings suggest that impaired pubertal development in these patients results from a deficiency of productive WDR11 protein interaction.

Duplication of Glu37 in the switch I region of HRAS impairs effector/GAP binding and underlies Costello syndrome by promoting enhanced growth factor-dependent MAPK and AKT activation.

Costello syndrome (CS) is a developmental disorder characterized by postnatal reduced growth, facial dysmorphism, cardiac defects, mental retardation and skin and musculo-skeletal defects. CS is caused by HRAS germline mutations. In the majority of cases, mutations affect Gly(12) and Gly(13) and are associated with a relatively homogeneous phenotype. The same amino acid substitutions are well known as somatic mutations in human tumors and promote constitutive HRAS activation by impairing its GTPase activity. In a small number of cases with mild phenotype, a second class of substitutions involving codons 117 and 146 and affecting GTP/GDP binding has been described. Here, we report on the identification and functional characterization of two different three-nucleotide duplications resulting in a duplication of glutamate 37 (p.E37dup) associated with a homogeneous phenotype reminiscent of CS. Ectopic expression of HRAS(E37dup) in COS-7 cells resulted in enhanced growth factor-dependent stimulation of the MEK-ERK and phosphoinositide 3-kinase (PI3K)-AKT signaling pathways. Recombinant HRAS(E37dup) was characterized by slightly increased GTP/GDP dissociation, lower intrinsic GTPase activity and complete resistance to neurofibromin 1 GTPase-activating protein (GAP) stimulation due to dramatically reduced binding. Co-precipitation of GTP-bound HRAS(E37dup) by various effector proteins, however, was inefficient because of drastically diminished binding affinities. Thus, although HRAS(E37dup) is predominantly present in the active, GTP-bound state, it promotes only a weak hyperactivation of downstream signaling pathways. These findings provide evidence that the mildly enhanced signal flux through the MAPK and PI3K-AKT cascades promoted by these disease-causing germline HRAS alleles results from a balancing effect between a profound GAP insensitivity and inefficient binding to effector proteins.

A novel HRAS substitution (c.266C>G; p.S89C) resulting in decreased downstream signaling suggests a new dimension of RAS pathway dysregulation in human development.

Costello syndrome is caused by HRAS germline mutations affecting Gly(12) or Gly(13) in >90% of cases and these are associated with a relatively homogeneous phenotype. Rarer mutations in other HRAS codons were reported in patients with an attenuated or mild phenotype. Disease-associated HRAS missense mutations result in constitutive HRAS activation and increased RAF-MEK-ERK and PI3K-AKT signal flow. Here we report on a novel heterozygous HRAS germline alteration, c.266C>G (p.S89C), in a girl presenting with severe fetal hydrops and pleural effusion, followed by a more benign postnatal course. A sibling with the same mutation and fetal polyhydramnios showed a Dandy-Walker malformation; his postnatal course was complicated by severe feeding difficulties. Their apparently asymptomatic father is heterozygous for the c.266C>G change. By functional analyses we identified reduced levels of active HRAS(S89C) and diminished MEK, ERK and AKT phosphorylation in cells overexpressing HRAS(S89C) , which represent novel consequences of disease-associated HRAS mutations. Given our patients' difficult neonatal course and presence of this change in their asymptomatic father, we hypothesize that its harmful consequences may be time limited, with the late fetal stage being most sensitive. Alternatively, the phenotype may develop only in the presence of an additional as-yet-unknown genetic modifier. While the pathogenicity of the HRAS c.266C>G change remains unproven, our data may illustrate wide functional and phenotypic variability of germline HRAS mutations.

A novel HRAS c.466C>T p.(Phe156Leu) variant in two patients with attenuated features of Costello syndrome.

Costello syndrome (CS) is caused by heterozygous HRAS germline mutations. Most patients share the HRAS variant p.Gly12Ser that is associated with a typical, homogeneous phenotype. Rarer pathogenic HRAS variants (e.g., p.Thr56Ile) were identified in individuals with attenuated CS phenotypes. The obvious phenotypical variability reflects different dysfunctional consequences of distinct HRAS variants. We report on two boys with the novel de novo HRAS variant c.466 C > T p.(Phe156Leu). Both had severe feeding difficulties, airway obstruction and developmental delay, which are typical findings in CS. They showed subtle facial and dermatologic features consistent with attenuated CS. They significantly differed in their musculoskeletal, cardiovascular and endocrinologic manifestations underscoring the clinical variability of individuals with identical, in particular rarer pathogenic HRAS variants. Functional studies revealed enhanced effector-binding, increased downstream signaling activation and impaired growth factor-induced signaling dynamics in cells expressing HRASPhe156Leu. Our data further illustrate the molecular and phenotypic variability of CS.

The focal adhesion protein ß-parvin controls cardiomyocyte shape and sarcomere assembly in response to mechanical load.

Physiological and pathological cardiac stress induced by exercise and hypertension, respectively, increase the hemodynamic load for the heart and trigger specific hypertrophic signals in cardiomyocytes leading to adaptive or maladaptive cardiac hypertrophy responses involving a mechanosensitive remodeling of the contractile cytoskeleton. Integrins sense load and have been implicated in cardiac hypertrophy, but how they discriminate between the two types of cardiac stress and translate mechanical loads into specific cytoskeletal signaling pathways is not clear. Here, we report that the focal adhesion protein ß-parvin is highly expressed in cardiomyocytes and facilitates the formation of cell protrusions, the serial assembly of newly synthesized sarcomeres, and the hypertrophic growth of neonatal rat ventricular cardiomyocytes (NRVCs) in vitro. In addition, physiological mechanical loading of NRVCs by either the application of cyclic, uni-axial stretch, or culture on physiologically stiff substrates promotes NRVC elongation in a ß-parvin-dependent manner, which is achieved by binding of ß-parvin to α/ß-PIX, which in turn activates Rac1. Importantly, loss-of-function studies in mice also revealed that ß-parvin is essential for the exercise-induced cardiac hypertrophy response in vivo. Our results identify ß-parvin as a novel mechano-responsive signaling hub in hypertrophic cardiomyocytes that drives cell elongation in response to physiological mechanical loads.

Expanding the clinical spectrum of COL2A1 related disorders by a mass like phenotype.

MASS phenotype is a connective tissue disorder clinically overlapping with Marfan syndrome and caused by pathogenic variants in FBN1. We report four patients from three families presenting with a MASS-like phenotype consisting of tall stature, arachnodactyly, spinal deformations, dural ectasia, pectus and/or feet deformations, osteoarthritis, and/or high arched palate. Gene panel sequencing was negative for FBN1 variants. However, it revealed likely pathogenic missense variants in three individuals [c.3936G > T p.(Lys1312Asn), c.193G > A p.(Asp65Asn)] and a missense variant of unknown significance in the fourth patient [c.4013G > A p.(Ser1338Asn)] in propeptide coding regions of COL2A1. Pathogenic COL2A1 variants are associated with type II collagenopathies comprising a remarkable clinical variablility. Main features include skeletal dysplasia, ocular anomalies, and auditory defects. A MASS-like phenotype has not been associated with COL2A1 variants before. Thus, the identification of likely pathogenic COL2A1 variants in our patients expands the phenotypic spectrum of type II collagenopathies and suggests that a MASS-like phenotype can be assigned to various hereditary disorders of connective tissue. We compare the phenotypes of our patients with related disorders of connective tissue and discuss possible pathomechanisms and genotype-phenotype correlations for the identified COL2A1 variants. Our data recommend COL2A1 sequencing in FBN1-negative patients suggestive for MASS/Marfan-like phenotype (without aortopathy).

Mutations in CHD7, encoding a chromatin-remodeling protein, cause idiopathic hypogonadotropic hypogonadism and Kallmann syndrome.

CHARGE syndrome and Kallmann syndrome (KS) are two distinct developmental disorders sharing overlapping features of impaired olfaction and hypogonadism. KS is a genetically heterogeneous disorder consisting of idiopathic hypogonadotropic hypogonadism (IHH) and anosmia, and is most commonly due to KAL1 or FGFR1 mutations. CHARGE syndrome, a multisystem autosomal-dominant disorder, is caused by CHD7 mutations. We hypothesized that CHD7 would be involved in the pathogenesis of IHH and KS (IHH/KS) without the CHARGE phenotype and that IHH/KS represents a milder allelic variant of CHARGE syndrome. Mutation screening of the 37 protein-coding exons of CHD7 was performed in 101 IHH/KS patients without a CHARGE phenotype. In an additional 96 IHH/KS patients, exons 6-10, encoding the conserved chromodomains, were sequenced. RT-PCR, SIFT, protein-structure analysis, and in situ hybridization were performed for additional supportive evidence. Seven heterozygous mutations, two splice and five missense, which were absent in > or = 180 controls, were identified in three sporadic KS and four sporadic normosmic IHH patients. Three mutations affect chromodomains critical for proper CHD7 function in chromatin remodeling and transcriptional regulation, whereas the other four affect conserved residues, suggesting that they are deleterious. CHD7's role is further corroborated by specific expression in IHH/KS-relevant tissues and appropriate developmental expression. Sporadic CHD7 mutations occur in 6% of IHH/KS patients. CHD7 represents the first identified chromatin-remodeling protein with a role in human puberty and the second gene to cause both normosmic IHH and KS in humans. Our findings indicate that both normosmic IHH and KS are mild allelic variants of CHARGE syndrome and are caused by CHD7 mutations.

The novel duplication HRAS c.186_206dup p.(Glu62_Arg68dup): clinical and functional aspects.

Specific activating missense HRAS variants cause Costello syndrome (CS), a RASopathy with recognizable facial features. The majority of these dominant disease causing variants affect the glycine residues in position 12 or 13. A clinically suspected CS diagnosis can be confirmed through identification of a dominant pathogenic HRAS variant. A novel HRAS variant predicting p.(Glu62_Arg68dup) was identified in an individual with hypertrophic cardiomyopathy, Chiari 1 malformation and ectodermal findings consistent with a RASopathy. Functional studies showed that the p.Glu62_Arg68dup alteration affects HRAS interaction with effector protein PIK3CA (catalytic subunit of phosphoinositide 3-kinase) and the regulator neurofibromin 1 (NF1) GTPase-activating protein (GAP). HRASGlu62_Arg68dup binding with effectors rapidly accelerated fibrosarcoma (RAF1), RAL guanine nucleotide dissociation stimulator (RALGDS) and phospholipase C1 (PLCE1) was enhanced. Accordingly, p.Glu62_Arg68dup increased steady-state phosphorylation of MEK1/2 and ERK1/2 downstream of RAF1, whereas AKT phosphorylation downstream of PI3K was not significantly affected. Growth factor stimulation revealed that expression of HRASGlu62_Arg68dup abolished the HRAS' capacity to modulate downstream signaling. Our data underscore that different qualities of dysregulated HRAS-dependent signaling dynamics determine the clinical severity in CS.

Exome sequencing in 38 patients with intracranial aneurysms and subarachnoid hemorrhage.

OBJECTIVE: Genetic risk factors for unruptured intracranial aneurysms (UIA) and aneurysmal subarachnoid hemorrhage (aSAH) are poorly understood. We aimed to verify recently reported risk genes and to identify novel sequence variants involved in the etiology of UIA/aSAH. METHODS: We performed exome sequencing (ES) in 35 unrelated individuals and 3 family members, each with a history of UIA and/or aSAH. We searched for sequence variants with minor allele frequency (MAF) ≤ 5% in the reported risk genes ADAMTS15, ANGPTL6, ARHGEF17, LOXL2, PCNT, RNF213, THSD1 and TMEM132B. To identify novel putative risk genes we looked for unknown (MAF = 0) variants shared by the three relatives. RESULTS: We identified 20 variants with MAF ≤ 5% in 18 individuals: 9 variants in PCNT (9 patients), 4 in RNF213 (3 patients), 3 in THSD1 (6 patients), 2 in ANGPTL6 (3 patients), 1 in ADAMTS15 (1 patient) and 1 in TMEM132B (1 patient). In the affected family, prioritization of shared sequence variants yielded five novel putative risk genes. Based on predicted pathogenicity of identified variants, population genetics data and a high functional relevance for vascular biology, EDIL3 was selected as top candidate and screened in additional 37 individuals with UIA and/or aSAH: a further very rare EDIL3 sequence variant in two unrelated sporadic patients was identified. CONCLUSIONS: Our data support a role of sequence variants in PCNT, RNF213 and THSD1 as susceptibility factors for cerebrovascular disease. The documented function in vascular wall integrity, the crucial localization of affected amino acids and gene/variant association tests suggest EDIL3 as a further valid candidate disease gene for UIA/aSAH.