GLI1 inactivation is associated with developmental phenotypes overlapping with Ellis–van Creveld syndrome

Peer reviewe

EVC-EVC2 complex stability and ciliary targeting are regulated by modification with ubiquitin and SUMO

Ellis van Creveld syndrome and Weyers acrofacial dysostosis are two rare genetic diseases affecting skeletal development. They are both ciliopathies, as they are due to malfunction of primary cilia, microtubule-based plasma membrane protrusions that function as cellular antennae and are required for Hedgehog signaling, a key pathway during skeletal morphogenesis. These ciliopathies are caused by mutations affecting the EVC-EVC2 complex, a transmembrane protein heterodimer that regulates Hedgehog signaling from inside primary cilia. Despite the importance of this complex, the mechanisms underlying its stability, targeting and function are poorly understood. To address this, we characterized the endogenous EVC protein interactome in control and Evc-null cells. This proteomic screen confirmed EVC’s main known interactors (EVC2, IQCE, EFCAB7), while revealing new ones, including USP7, a deubiquitinating enzyme involved in Hedgehog signaling. We therefore looked at EVC-EVC2 complex ubiquitination. Such ubiquitination exists but is independent of USP7 (and of USP48, also involved in Hh signaling). We did find, however, that monoubiquitination of EVC-EVC2 cytosolic tails greatly reduces their protein levels. On the other hand, modification of EVC-EVC2 cytosolic tails with the small ubiquitin-related modifier SUMO3 has a different effect, enhancing complex accumulation at the EvC zone, immediately distal to the ciliary transition zone, possibly via increased binding to the EFCAB7-IQCE complex. Lastly, we find that EvC zone targeting of EVC-EVC2 depends on two separate EFCAB7-binding motifs within EVC2’s Weyers-deleted peptide. Only one of these motifs had been characterized previously, so we have mapped the second herein. Altogether, our data shed light on EVC-EVC2 complex regulatory mechanisms, with implications for ciliopathies

Heterozygous pathogenic variants in GLI1 are a common finding in isolated postaxial polydactyly A/B

ECEMC Working Group on Polydactyly: et al.Postaxial polydactyly (PAP) is a frequent limb malformation consisting in the duplication of the fifth digit of the hand or foot. Morphologically, this condition is divided into type A and B, with PAP‐B corresponding to a more rudimentary extra‐digit. Recently, biallelic truncating variants in the transcription factor GLI1 were reported to be associated with a recessive disorder, which in addition to PAP‐A, may include syndromic features. Moreover, two heterozygous subjects carrying only one inactive copy of GLI1 were also identified with PAP. Herein, we aimed to determine the level of involvement of GLI1 in isolated PAP, a condition previously established to be autosomal dominantly inherited with incomplete penetrance. We analyzed the coding region of GLI1 in 95 independent probands with nonsyndromic PAP and found 11.57% of these subjects with single heterozygous pathogenic variants in this gene. The detected variants lead to premature termination codons or result in amino acid changes in the DNA‐binding domain of GLI1 that diminish its transactivation activity. Family segregation analysis of these variants was consistent with dominant inheritance with incomplete penetrance. We conclude that heterozygous changes in GLI1 underlie a significant proportion of sporadic or familial cases of isolated PAP‐A/B.This study was supported by a grant from the Spanish Ministry of Economy and Competitiveness (SAF2016–75434‐R) to V.L. R‐P and by funding from Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Science, Innovation and Universities and the Fundación 1000 sobre Defectos Congénitos to E. B‐S.Peer reviewe

La inactivación de GLI1 causa alteraciones del desarrollo solapantes con el Síndrome de Ellis-vanCreveld

Resumen del trabajo presentado a la XI Reunión Anual CIBERER, celebrada en Castelldefels, Barcelona del 12 al 14 de marzo de 2018.Los factores transcripcionales GLI1, GLI2 y GLI3 actúan en el desarrollo embrionario como mediadores de los morfógenos Hedgehog (Hh). En consecuencia, variantes deletéreas en GLI2 y GLI3 han sido reportadas como responsables de defectos congénitos. Sin embargo, hasta la fecha no se habían descrito mutaciones en GLI1, por lo que el papel de este gen en el desarrollo humano permanecía desconocido. Aquí presentamos 8 pacientes de tres familias distintas con mutaciones en GLI1 y características clínicas similares a las del Síndrome de Ellis-van Creveld (EvC), una enfermedad causada por la disminución de la actividad de la vía de Hh. Dos familias portaban mutaciones de fin de mensaje en el último exón del gen y la tercera un codón de parada en la región N-terminal. El análisis de fibroblastos de uno de los pacientes con mutaciones en el último exón demostró que las células de este paciente sintetizan la proteína truncada correspondiente e inducen su expresión en respuesta a un agonista químico de Hh. Sin embargo, ensayos “in vivo” y en cultivo celular revelaron que la actividad transcripcional de la proteína mutante se encontraba drásticamente disminuida. Consistentemente, las células del paciente presentaron menor expresión de la diana de Hh, PTCH1. Este trabajo muestra que la inactivación de GLI1 da lugar a alteraciones del desarrollo que varían desde polidactilia postaxial aislada hasta fenotipos solapantes con EvC, a la vez que pone de manifiesto que todos los miembros de la triada GLI son necesarios en el desarrollo humano.Peer reviewe

Common atrium/atrioventricular canal defect and postaxial polydactyly: A mild clinical subtype of Ellis-van Creveld syndrome caused by hypomorphic mutations in the EVC gene

Clinical expression of Ellis‐van Creveld syndrome (EvC) is variable and mild phenotypes have been described, including patients with mostly cardiac and limb involvement. Whether these cases are part of the EvC phenotypic spectrum or separate conditions is disputed. Herein, we describe a family with vertical transmission of atrioventricular canal defect (AVCD), common atrium, and postaxial polydactyly. Targeted sequencing of EVC, EVC2, WDR35, DYNC2LI1, and DYNC2H1 identified different compound heterozygosity in EVC genotypes in the two affected members, consisting of a nonsense (p.Arg622Ter) and a missense (p.Arg663Pro) variant in the father, and the same nonsense variant and a noncanonical splice‐site in‐frame change (c.1316–7A>G) in the daughter. Complementary DNA sequencing, immunoblot, and immunofluorescence experiments using patient‐derived fibroblasts and Evc–/– mouse embryonic fibroblasts showed that p.Arg622Ter is a loss‐of‐function mutation, whereas p.Arg663Pro and the splice‐site change c.1316–7A>G are hypomorphic variants resulting in proteins that retain, in part, the ability to complex with EVC2. Our molecular and functional data demonstrate that at least in some cases the condition characterized as “common atrium/AVCD with postaxial polydactyly” is a mild form of EvC due to hypomorphic EVC mutations, further supporting the occurrence of genotype‐phenotype correlations in this syndrome.This study was supported by funding from the Italian Ministry of Health (RC‐2019) to Alessandro De Luca, Fondazione Bambino Gesù (Vite Coraggiose) to Marco Tartaglia, and the Spanish Ministry of Science, Innovation and Universities to Victor L. Ruiz‐Perez (SAF2016‐75434‐R (AEI/FEDER, UE) and PID2019‐105620RB‐I00/AEI/10.13039/501100011033)

Germline and Mosaic Variants in PRKACA and PRKACB Cause a Multiple Congenital Malformation Syndrome

PRKACA and PRKACB code for two catalytic subunits (Cα and Cβ) of cAMP-dependent protein kinase (PKA), a pleiotropic holoenzyme that regulates numerous fundamental biological processes such as metabolism, development, memory, and immune response. We report seven unrelated individuals presenting with a multiple congenital malformation syndrome in whom we identified heterozygous germline or mosaic missense variants in PRKACA or PRKACB. Three affected individuals were found with the same PRKACA variant, and the other four had different PRKACB mutations. In most cases, the mutations arose de novo, and two individuals had offspring with the same condition. Nearly all affected individuals and their affected offspring shared an atrioventricular septal defect or a common atrium along with postaxial polydactyly. Additional features included skeletal abnormalities and ectodermal defects of variable severity in five individuals, cognitive deficit in two individuals, and various unusual tumors in one individual. We investigated the structural and functional consequences of the variants identified in PRKACA and PRKACB through the use of several computational and experimental approaches, and we found that they lead to PKA holoenzymes which are more sensitive to activation by cAMP than are the wild-type proteins. Furthermore, expression of PRKACA or PRKACB variants detected in the affected individuals inhibited hedgehog signaling in NIH 3T3 fibroblasts, thereby providing an underlying mechanism for the developmental defects observed in these cases. Our findings highlight the importance of both Cα and Cβ subunits of PKA during human development.This work was partially supported by funding from the Spanish Ministry of Science, Innovation and Universities (SAF2016-75434-R [AEI/FEDER, UE] and PID2019-105620RB-I00/AEI/10.13039/501100011033) to V.L.R.-P. S.S.T. was supported by NIH grant R03TR002947, E.M.F.M. by Kassel graduate school “Clocks”, and A.D.L. by the Italian Ministry of Health (RC-2019). The University of Antwerp supported G.M. and W.V.H. with Methusalem funding (FFB190208) and S.P. with a predoctoral grant. E.B. was supported by The Research Foundation Flanders with a postdoctoral grant (12A3814N). The study was also funded by a National Health and Medical Research Council Program Grant (1091593) to I.E.S., a Practitioner Fellowship (1006110) to I.E.S., a Senior Research Fellowship (1102971) to M.B., and an R.D. Wright Career Development Fellowship (1063799) to M.S.H. B.S.S. and G.L. were supported by Throne Holst Foundation UiO (2019-2021) and Strategic PhD fund by UiO/IMB

Heterozygous pathogenic variants in GLI1 are a common finding in isolated postaxial polydactyly A/B

Postaxial polydactyly (PAP) is a frequent limb malformation consisting in the duplication of the fifth digit of the hand or foot. Morphologically, this condition is divided into type A and B, with PAP-B corresponding to a more rudimentary extra-digit. Recently, biallelic truncating variants in the transcription factor GLI1 were reported to be associated with a recessive disorder, which in addition to PAP-A, may include syndromic features. Moreover, two heterozygous subjects carrying only one inactive copy of GLI1 were also identified with PAP. Herein, we aimed to determine the level of involvement of GLI1 in isolated PAP, a condition previously established to be autosomal dominantly inherited with incomplete penetrance. We analyzed the coding region of GLI1 in 95 independent probands with nonsyndromic PAP and found 11.57% of these subjects with single heterozygous pathogenic variants in this gene. The detected variants lead to premature termination codons or result in amino acid changes in the DNA-binding domain of GLI1 that diminish its transactivation activity. Family segregation analysis of these variants was consistent with dominant inheritance with incomplete penetrance. We conclude that heterozygous changes in GLI1 underlie a significant proportion of sporadic or familial cases of isolated PAP-A/B