Schinzel-Giedion Syndrome with Congenital Megacalycosis in a Turkish Patient: Report of SETBP1 Mutation and Literature Review of the Clinical Features

Schinzel-Giedion syndrome (SGS) is a rare autosomal dominant disorder that results in facial dysmorphism, multiple congenital anomalies, and an increased risk of malignancy. Recently, using exome sequencing, de novo heterozygous mutations in the SETBP1 gene have been identified in patients with SGS. Most affected individuals do not survive after childhood because of the severity of this disorder. Here, we report SETBP1 mutation confirmed by molecular analysis in a case of SGS with congenital megacalycosis

Cleidocranial dysplasia: Clinical, endocrinologic and molecular findings in 15 patients from 11 families

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder characterized by skeletal anomalies such as delayed closure of the cranial sutures, underdeveloped or absent clavicles, multiple dental abnormalities, short stature and osteoporosis. RUNX2, encoding Runt DNA-binding domain protein important in osteoblast differentiation, is the only known gene related to the disease and identified as responsible in 70% of the cases. Our clinical evaluations revealed that short stature present at a rate of 28.6%, osteoporosis at a rate of 57.1% and osteopenia at 21.4%. In this study, RUNX2 sequencing revealed nine different variations in 11 families, eight being pathogenic of which one was novel gross insertion (c.1271_1272ins20) and one other being predicted benign in frame gross deletion (c.241_258del). (C) 2016 Elsevier Masson SAS. All rights reserved.Scientific and Technological Research Institution of Turkey, TUBITAK-ERA NET (CRANIRARE-2) [SBAG-112S398]We sincerely thank the patients and their families for their contribution to this study, which was supported by the Scientific and Technological Research Institution of Turkey, TUBITAK-ERA NET (CRANIRARE-2, grant number: SBAG-112S398)

Overlapping SETBP1 gain-of-function mutations in Schinzel-Giedion syndrome and hematologic malignancies

Schinzel-Giedion syndrome (SGS) is a rare developmental disorder characterized by multiple malformations, severe neurological alterations and increased risk of malignancy. SGS is caused by de novo germline mutations clustering to a 12bp hotspot in exon 4 of SETBP1. Mutations in this hotspot disrupt a degron, a signal for the regulation of protein degradation, and lead to the accumulation of SETBP1 protein. Overlapping SETBP1 hotspot mutations have been observed recurrently as somatic events in leukemia. We collected clinical information of 47 SGS patients (including 26 novel cases) with germline SETBP1 mutations and of four individuals with a milder phenotype caused by de novo germline mutations adjacent to the SETBP1 hotspot. Different mutations within and around the SETBP1 hotspot have varying effects on SETBP1 stability and protein levels in vitro and in in silico modeling. Substitutions in SETBP1 residue I871 result in a weak increase in protein levels and mutations affecting this residue are significantly more frequent in SGS than in leukemia. On the other hand, substitutions in residue D868 lead to the largest increase in protein levels. Individuals with germline mutations affecting D868 have enhanced cell proliferation in vitro and higher incidence of cancer compared to patients with other germline SETBP1 mutations. Our findings substantiate that, despite their overlap, somatic SETBP1 mutations driving malignancy are more disruptive to the degron than germline SETBP1 mutations causing SGS. Additionally, this suggests that the functional threshold for the development of cancer driven by the disruption of the SETBP1 degron is higher than for the alteration in prenatal development in SGS. Drawing on previous studies of somatic SETBP1 mutations in leukemia, our results reveal a genotype-phenotype correlation in germline SETBP1 mutations spanning a molecular, cellular and clinical phenotype

Mutations in SCNM1 cause orofaciodigital syndrome due to minor intron splicing defects affecting primary cilia

Orofaciodigital syndrome (OFD) is a genetically heterogeneous ciliopathy characterized by anomalies of the oral cavity, face, and digits. We describe individuals with OFD from three unrelated families having bi-allelic loss-of-function variants in SCNM1 as the cause of their condition. SCNM1 encodes a protein recently shown to be a component of the human minor spliceosome. However, so far the effect of loss of SCNM1 function on human cells had not been assessed. Using a comparative transcriptome analysis between fibroblasts derived from an OFD-affected individual harboring SCNM1 mutations and control fibroblasts, we identified a set of genes with defective minor intron (U12) processing in the fibroblasts of the affected subject. These results were reproduced in SCNM1 knockout hTERT RPE-1 (RPE-1) cells engineered by CRISPR-Cas9-mediated editing and in SCNM1 siRNA-treated RPE-1 cultures. Notably, expression of TMEM107 and FAM92A encoding primary cilia and basal body proteins, respectively, and that of DERL2, ZC3H8, and C17orf75, were severely reduced in SCNM1-deficient cells. Primary fibroblasts containing SCNM1 mutations, as well as SCNM1 knockout and SCNM1 knockdown RPE-1 cells, were also found with abnormally elongated cilia. Conversely, cilia length and expression of SCNM1-regulated genes were restored in SCNM1-deficient fibroblasts following reintroduction of SCNM1 via retroviral delivery. Additionally, functional analysis in SCNM1-retrotransduced fibroblasts showed that SCNM1 is a positive mediator of Hedgehog (Hh) signaling. Our findings demonstrate that defective U12 intron splicing can lead to a typical ciliopathy such as OFD and reveal that primary cilia length and Hh signaling are regulated by the minor spliceosome through SCNM1 activity.This work was supported by a grant from the Spanish Ministry of Science and Innovation (PID2019-105620RB-I00/AEI/10.13039/501100011033)

Trichothiodystrophy-associated MPLKIP maintains DBR1 levels for proper lariat debranching and ectodermal differentiation

The brittle hair syndrome Trichothiodystrophy (TTD) is characterized by variable clinical features, including photosensitivity, ichthyosis, growth retardation, microcephaly, intellectual disability, hypogonadism, and anaemia. TTD-associated mutations typically cause unstable mutant proteins involved in various steps of gene expression, severely reducing steady-state mutant protein levels. However, to date, no such link to instability of gene-expression factors for TTD-associated mutations in MPLKIP/TTDN1 has been established. Here, we present seven additional TTD individuals with MPLKIP mutations from five consanguineous families, with a newly identified MPLKIP variant in one family. By mass spectrometry-based interaction proteomics, we demonstrate that MPLKIP interacts with core splicing factors and the lariat debranching protein DBR1. MPLKIP-deficient primary fibroblasts have reduced steady-state DBR1 protein levels. Using Human Skin Equivalents (HSEs), we observed impaired keratinocyte differentiation associated with compromised splicing and eventually, an imbalanced proteome affecting skin development and, interestingly, also the immune system. Our data show that MPLKIP, through its DBR1 stabilizing role, is implicated in mRNA splicing, which is of particular importance in highly differentiated tissue.</p

A novel ICK mutation causes ciliary disruption and lethal endocrine-cerebro-osteodysplasia syndrome

Background: Endocrine-cerebro-osteodysplasia (ECO) syndrome [MIM:612651] caused by a recessive mutation (p.R272Q) in Intestinal cell kinase (ICK) shows significant clinical overlap with ciliary disorders. Similarities are strongest between ECO syndrome, the Majewski and Mohr-Majewski short-rib thoracic dysplasia (SRTD) with polydactyly syndromes, and hydrolethalus syndrome. In this study, we present a novel homozygous ICK mutation in a fetus with ECO syndrome and compare the effect of this mutation with the previously reported ICK variant on ciliogenesis and cilium morphology. Results: Through homozygosity mapping and whole-exome sequencing, we identified a second variant (c.358G\u3eT; p.G120C) in ICK in a Turkish fetus presenting with ECO syndrome. In vitro studies of wild-type and mutant mRFP-ICK (p.G120C and p.R272Q) revealed that, in contrast to the wild-type protein that localizes along the ciliary axoneme and/or is present in the ciliary base, mutant proteins rather enrich in the ciliary tip. In addition, immunocytochemistry revealed a decreased number of cilia in ICK p.R272Q-affected cells. Conclusions: Through identification of a novel ICK mutation, we confirm that disruption of ICK causes ECO syndrome, which clinically overlaps with the spectrum of ciliopathies. Expression of ICK-mutated proteins result in an abnormal ciliary localization compared to wild-type protein. Primary fibroblasts derived from an individual with ECO syndrome display ciliogenesis defects. In aggregate, our findings are consistent with recent reports that show that ICK regulates ciliary biology in vitro and in mice, confirming that ECO syndrome is a severe ciliopathy

IL11 is elevated in systemic sclerosis and IL11-dependent ERK signalling underlies TGF beta-mediated activation of dermal fibroblasts

Objectives Interleukin 11 (IL11) is highly upregulated in skin and lung fibroblasts from patients with systemic sclerosis (SSc). Here we tested whether IL11 is mechanistically linked with activation of human dermal fibroblasts (HDFs) from patients with SSc or controls. Methods We measured serum IL11 levels in volunteers and patients with early diffuse SSc and manipulated IL11 signalling in HDFs using gain- and loss-of-function approaches that we combined with molecular and cellular phenotyping. Results In patients with SSc, serum IL11 levels are elevated as compared with healthy controls. All transforming growth factor beta (TGFβ) isoforms induced IL11 secretion from HDFs, which highly express IL11 receptor α-subunit and the glycoprotein 130 (gp130) co-receptor, suggestive of an autocrine loop of IL11 activity in HDFs. IL11 stimulated ERK activation in HDFs and resulted in HDF-to-myofibroblast transformation and extracellular matrix secretion. The pro-fibrotic action of IL11 in HDFs appeared unrelated to STAT3 activity, independent of TGFβ upregulation and was not associated with phosphorylation of SMAD2/3. Inhibition of IL11 signalling using either a neutralizing antibody against IL11 or siRNA against IL11RA reduced TGFβ-induced HDF proliferation, matrix production and cell migration, which was phenocopied by pharmacological inhibition of ERK. Conclusions These data reveal that autocrine IL11-dependent ERK activity alone or downstream of TGFβ stimulation promotes fibrosis phenotypes in dermal fibroblasts and suggest IL11 as a potential therapeutic target in SSc

Clinical and genetic characterization of a progressive RBL2-associated neurodevelopmental disorder

Retinoblastoma (RB) proteins are highly conserved transcriptional regulators that play important roles during development by regulating cell-cycle gene expression. RBL2 dysfunction has been linked to a severe neurodevelopmental disorder. However, to date, clinical features have only been described in six individuals carrying five biallelic predicted loss of function (pLOF) variants. To define the phenotypic effects of RBL2 mutations in detail, we identified and clinically characterized a cohort of 35 patients from 20 families carrying pLOF variants in RBL2, including fifteen new variants that substantially broaden the molecular spectrum. The clinical presentation of affected individuals is characterized by a range of neurological and developmental abnormalities. Global developmental delay and intellectual disability were uniformly observed, ranging from moderate to profound and involving lack of acquisition of key motor and speech milestones in most patients. Disrupted sleep was also evident in some patients. Frequent features included postnatal microcephaly, infantile hypotonia, aggressive behaviour, stereotypic movements, seizures, and non-specific dysmorphic features. Neuroimaging features included cerebral atrophy, white matter volume loss, corpus callosum hypoplasia and cerebellar atrophy. In parallel, we used the fruit fly, Drosophila melanogaster, to investigate how disruption of the conserved RBL2 orthologue Rbf impacts nervous system function and development. We found that Drosophila Rbf LOF mutants recapitulate several features of patients harbouring RBL2 variants, including developmental delay, alterations in head and brain morphology, locomotor defects, and perturbed sleep. Surprisingly, in addition to its known role in controlling tissue growth during development, we found that continued Rbf expression is also required in fully differentiated post-mitotic neurons for normal locomotion in Drosophila, and that adult-stage neuronal re-expression of Rbf is sufficient to rescue Rbf mutant locomotor defects. Taken together, our study provides a clinical and experimental basis to understand genotype-phenotype correlations in an RBL2-linked neurodevelopmental disorder, and suggests that restoring RBL2 expression through gene therapy approaches may ameliorate some symptoms caused by RBL2 pLOF

Variant characterisation and clinical profile in a large cohort of patients with Ellis-van Creveld syndrome and a family with Weyers acrofacial dysostosis

[Background]: Ellis-van Creveld syndrome (EvC) is a recessive disorder characterised by acromesomelic limb shortening, postaxial polydactyly, nail-teeth dysplasia and congenital cardiac defects, primarily caused by pathogenic variants in EVC or EVC2. Weyers acrofacial dysostosis (WAD) is an ultra-rare dominant condition allelic to EvC. The present work aimed to enhance current knowledge on the clinical manifestations of EvC and WAD and broaden their mutational spectrum.[Methods]: We conducted molecular studies in 46 individuals from 43 unrelated families with a preliminary clinical diagnosis of EvC and 3 affected individuals from a family with WAD and retrospectively analysed clinical data. The deleterious effect of selected variants of uncertain significance was evaluated by cellular assays.[Main results]: We identified pathogenic variants in EVC/EVC2 in affected individuals from 41 of the 43 families with EvC. Patients from each of the two remaining families were found with a homozygous splicing variant in WDR35 and a de novo heterozygous frameshift variant in GLI3, respectively. The phenotype of these patients showed a remarkable overlap with EvC. A novel EVC2 C-terminal truncating variant was identified in the family with WAD. Deep phenotyping of the cohort recapitulated ‘classical EvC findings’ in the literature and highlighted findings previously undescribed or rarely described as part of EvC.[Conclusions]: This study presents the largest cohort of living patients with EvC to date, contributing to better understanding of the full clinical spectrum of EvC. We also provide comprehensive information on the EVC/EVC2 mutational landscape and add GLI3 to the list of genes associated with EvC-like phenotypes.This work was funded by the Spanish Ministry of Economy and Competitiveness (SAF2010-17901, SAF2013-43365-R, SAF2016-75434-R, PID2019-105620RB-I00/AEI/10.13039/501100011033) and FEDER funds through ISCIII grant PI20/01053 and PMP21/00063/Instituto de Salud Carlos III.Peer reviewe