The Mediator is an evolutionarily conserved, multi-subunit complex that regulates multiple steps of transcription. Mediator activity is regulated by the reversible association of a four-subunit module comprising CDK8 or CDK19 kinases, together with cyclin C, MED12 or MED12L, and MED13 or MED13L. Mutations in MED12, MED13, and MED13L were previously identified in syndromic developmental disorders with overlapping phenotypes. Here, we report CDK8 mutations (located at 13q12.13) that cause a phenotypically related disorder. Using whole-exome or whole-genome sequencing, and by international collaboration, we identified eight different heterozygous missense CDK8 substitutions, including 10 shown to have arisen de novo, in 12 unrelated subjects; a recurrent mutation, c.185C>T (p.Ser62Leu), was present in five individuals. All predicted substitutions localize to the ATP-binding pocket of the kinase domain. Affected individuals have overlapping phenotypes characterized by hypotonia, mild to moderate intellectual disability, behavioral disorders, and variable facial dysmorphism. Congenital heart disease occurred in six subjects; additional features present in multiple individuals included agenesis of the corpus callosum, ano-rectal malformations, seizures, and hearing or visual impairments. To evaluate the functional impact of the mutations, we measured phosphorylation at STAT1-Ser727, a known CDK8 substrate, in a CDK8 and CDK19 CRISPR double-knockout cell line transfected with wild-type (WT) or mutant CDK8 constructs. These experiments demonstrated a reduction in STAT1 phosphorylation by all mutants, in most cases to a similar extent as in a kinase-dead control. We conclude that missense mutations in CDK8 cause a developmental disorder that has phenotypic similarity to syndromes associated with mutations in other subunits of the Mediator kinase module, indicating probable overlap in pathogenic mechanisms.

Mutations in the GDAP1 gene cause different forms of Charcot-Marie-Tooth (CMT) disease, and the primary clinical expression of this disease is markedly variable in the dominant inheritance form (CMT type 2K; CMT2K), in which carriers of the GDAP1 p.R120W mutation can display a wide range of clinical severity. We investigated the JPH1 gene as a genetic modifier of clinical expression variability because junctophilin-1 (JPH1) is a good positional and functional candidate. We demonstrated that the JPH1-GDAP1 cluster forms a paralogon and is conserved in vertebrates. Moreover, both proteins play a role in Ca(2+) homeostasis, and we demonstrated that JPH1 is able to restore the store-operated Ca(2+) entry (SOCE) activity in GDAP1-silenced cells. After the mutational screening of JPH1 in a series of 24 CMT2K subjects who harbour the GDAP1 p.R120W mutation, we characterized the JPH1 p.R213P mutation in one patient with a more severe clinical picture. JPH1(p.R213P) cannot rescue the SOCE response in GDAP1-silenced cells. We observed that JPH1 colocalizes with STIM1, which is the activator of SOCE, in endoplasmic reticulum-plasma membrane puncta structures during Ca(2+) release in a GDAP1-dependent manner. However, when GDAP1(p.R120W) is expressed, JPH1 seems to be retained in mitochondria. We also established that the combination of GDAP1(p.R120W) and JPH1(p.R213P) dramatically reduces SOCE activity, mimicking the effect observed in GDAP1 knock-down cells. In summary, we conclude that JPH1 and GDAP1 share a common pathway and depend on each other; therefore, JPH1 can contribute to the phenotypical consequences of GDAP1 mutations.

Purpose Enrichment of heterozygous missense and truncating SMAD6 variants was previously reported in nonsyndromic sagittal and metopic synostosis, and interaction of SMAD6 variants with a common polymorphism nearBMP2 (rs1884302) was proposed to contribute to inconsistent penetrance. We determined the occurrence of SMAD6 variants in all types of craniosynostosis, evaluated the impact of different missense variants on SMAD6 function, and tested independently whether rs1884302 genotype significantly modifies the phenotype. Methods We performed resequencing of SMAD6 in 795 unsolved patients with any type of craniosynostosis and genotyped rs1884302 in SMAD6-positive individuals and relatives. We examined the inhibitory activity and stability of SMAD6 missense variants. Results We found 18 (2.3%) different rare damaging SMAD6 variants, with the highest prevalence in metopic synostosis (5.8%) and an 18.3-fold enrichment of loss-of-function variants comparedwith gnomAD data (P < 10⁻⁷). Combined with eight additional variants, ≥20/26 were transmitted from an unaffected parent but rs1884302 genotype did not predict phenotype. Conclusion Pathogenic SMAD6 variants substantially increase the risk of both nonsyndromic and syndromic presentations of craniosynostosis, especially metopic synostosis. Functional analysis is important to evaluate missense variants. Genotyping of rs1884302 is not clinically useful. Mechanisms to explain the remarkable diversity of phenotypes associated with SMAD6 variants remain obscure.

Next-generation sequencing is a powerful tool for the discovery of genes related to neurodevelopmental disorders (NDDs). Here, we report the identification of a distinct syndrome due to de novo or inherited heterozygous mutations in Tousled-like kinase 2 (TLK2) in 38 unrelated individuals and two affected mothers, using whole-exome and whole-genome sequencing technologies, matchmaker databases, and international collaborations. Affected individuals had a consistent phenotype, characterized by mild-borderline neurodevelopmental delay (86%), behavioral disorders (68%), severe gastro-intestinal problems (63%), and facial dysmorphism including blepharophimosis (82%), telecanthus (74%), prominent nasal bridge (68%), broad nasal tip (66%), thin vermilion of the upper lip (62%), and upslanting palpebral fissures (55%). Analysis of cell lines from three affected individuals showed that mutations act through a loss-of-function mechanism in at least two case subjects. Genotype-phenotype analysis and comparison of computationally modeled faces showed that phenotypes of these and other individuals with loss-of-function variants significantly overlapped with phenotypes of individuals with other variant types (missense and C-terminal truncating). This suggests that haploinsufficiency of TLK2 is the most likely underlying disease mechanism, leading to a consistent neurodevelopmental phenotype. This work illustrates the power of international data sharing, by the identification of 40 individuals from 26 different centers in 7 different countries, allowing the identification, clinical delineation, and genotype-phenotype evaluation of a distinct NDD caused by mutations in TLK2.