Mutations in Mediator Complex Genes CDK8, MED12, MED13, and MEDL13 Mediate Overlapping Developmental Syndromes

To identify genes that are probably causing a rare genetic disorder, medical geneticists could initially only rely on karyotyping hoping to find a de novo translocation, inversion, or deletion. Such a chromosomal rearrangement was thought to either disrupt a gene or its chromosomal context, i.e., a topologically associated domain [Poot and Haaf, 2015; Lupiáñez et al., 2016]. In this way, translocations and an inversion involving chromosomal band 7q35 produced disruptions of CNTNAP2 in patients with Gilles de la Tourette syndrome or autism [Verkerk et al., 2003; Bakkaloglu et al., 2008; Poot et al., 2010]. An apparently balanced translocation t(6;7)(q16.2;p15.3) disrupted a topologically associated domain affecting the expression of TWIST1 in a patient with Saethre Chotzen syndrome [Krebs et al., 1997]. A de novo balanced translocation t(12,17)(q24.1;q21) that interrupted the MED13L gene was found in a 7-year-old girl with postnatal microcephaly; developmental delay (DD), in particular delayed motor development and ataxia; mental retardation, with nearly absent speech; a ventricular septal defect, open foramen ovale, and coarctation of the aorta [Muncke et al., 2003]. This complex set of phenotypes suggests that MED13L is involved in regulation of development at a very early stage. This inference was supported by findings in a patient with a de novo balanced translocation t(12;19)(q24;q12) that interrupted only the MED13L gene [Utami et al., 2014]. This patient showed DD, moderate intellectual disability, very poor speech, absence seizures, cerebral atrophy, and dysmorphic facial features, including a cleft palate, glossoptosis, retrognathia, hypertelorism, flat philtrum, broad nasal bridge, bulbous nose, strabismus, and hirsutism. In addition, multiple limb contractures, camptodactyly, and foot deformities were seen. Cultured cells from this patient showed decreased mRNA and protein levels of MED13L compared to controls, which is in agreement with haploinsufficiency of this gene. While the findings from these 2 translocation carriers suggested a MED13L haploinsufficiency syndrome, the associated phenotypes appeared rather varied, and a molecular mechanism for this was not clear. Haploinsufficiency syndromes arise if the gene, of which one copy is lost, encodes a protein that is part of a signaling pathway, a protein complex, or a receptor [Veitia, 2010; Poot et al., 2011; Birchler and Veitia, 2012]. MED13L is indeed part of a protein complex, the Mediator complex (MC), which regulates transcription of mRNAs by RNA polymerase II [Conaway et al., 2005; Asadollahi et al., 2017; Harper and Taatjes, 2018].

Karyotyping detects genome rearrangements in approximately 9.5% of the children with DD, whereas genome wide copy number profiling finds a probably pathogenic copy number variation (CNV) in around 19% of the referrals [Hochstenbach et al., 2009]. In a girl with DD, a perimembranous ventricular septal defect, and mild hypotonia, a heterozygous 1-Mb de novo triplication involving the MED13L gene as well as several nonprotein-coding RNA genes and the MAP1LC3B2 gene was identified [Asadollahi et al., 2013]. In 2 unrelated patients with cardiac anomalies, an inherited heterozygous duplication or a de novo deletion, both encompassing, among other genes, MED13L was found [Adegbola et al., 2015]. While these cases share MED13L CNVs as a common denominator, they do not rigorously prove that this gene is responsible for the patient's phenotype. High-resolution arrays allowed detection of losses or duplications of a single gene or even an exon [Boone et al., 2010]. Applying this technology, van Haelst et al. [2015] found a patient with delayed motor and speech development, small eyelids, and mild retrognathia, but no cardiac anomalies, who carried a de novo deletion of exons 6-20 of MED13L. A second patient with DD, hypertonia of the extremities, a slightly asymmetric face with short, upslanted palpebral fissures, a bulbous nasal tip and protruding tongue, without cardiac or pulmonary anomalies had a de novo mutation in the splice acceptor site of exon 5 of MED13L. This resulted in an in-frame deletion of 15 amino acids in the middle of the MED13L protein. In 2 unrelated girls with intellectual delay, facial dysmorphisms and cardiac anomalies, one patient carried a 17-kb deletion encompassing exon 2 of MED13L and the other a 115-kb deletion encompassing exons 3 and 4 [Asadollahi et al., 2013]. In a cohort of 41 patients, a 7-year-old girl with delayed psychomotor development and nonsyndromic intellectual disability, a de novo heterozygous truncating mutation in the MED13L gene was identified [Hamdan et al., 2014]. Finally, among 106 patients with DD, an 18-year-old man with a de novo heterozygous truncating mutation in MED13L was found [Redin et al., 2014]. Reviewing patients with intragenic CNVs of MED13L, Abegdola et al. [2015] reported 5 deletions, 3 duplications, and 1 truncating mutation. In contrast, 18 patients with a nucleotide variant in MED13L have been reported [Asadollahi et al., 2017]. The preponderance of heterozygous intragenic CNVs points towards a possible pathogenic mechanism of MED13L inactivation. Intragenic deletions hint at possible interactions with other proteins as has been suggested for CNTNAP2 and TCF4 [Bedeschi et al., 2017; Poot, 2019].

A MED13L mutation is suspected in patients with a syndrome consisting of DD, in particular speech impairment, a bulbous nasal tip, macroglossia, macrostomia, or an open mouth [Tørring et al., 2019]. Carriers of a MED13L mutation affecting CNV harbor the so-called MED13L haploinsufficiency syndrome, which in all cases consists of moderate to severe intellectual delay and facial dysmorphic traits [Assodolahi et al., 2017]. In the majority of these patients, severe speech delay and muscle hypotonia are also noted [Assodolahi et al., 2017]. In roughly 20-50% of the patients, further features include abnormal MRI findings of myelination, an abnormal corpus callosum, ataxia and coordination problems, autism spectrum disorder, seizures and/or abnormal EEG, and variable congenital heart defects. In most patients, facial anomalies include a broad forehead, low-set ears, bitemporal narrowing, upslanting palpebral fissures, depressed or flat nasal bridge, a bulbous nose, and an abnormal chin. In 30% of the cases, macroglossia and horizontal eyebrows were observed. These features are key in distinguishing MED13L haploinsufficiency syndrome from the 1p36 deletion and Kleefstra syndromes. The facial gestalt of MED13L haploinsufficiency syndrome also bears resemblance with 22q11.2 deletion syndrome (also known as DiGeorge or velacardiofacial syndrome) [Assodolahi et al., 2013; Cafiero et al., 2015].

Since MED13L, together with CDK8, cyclin C, MED12, MED12L and MED13, forms the regulatory subunit of MC, it is tempting to suggest that either perturbation of interactions of MED13L with these proteins or mutations in them will result in comparable syndromes. Patients with protein-altering mutations in the MED13 gene showed intellectual disability and/or DD, including speech delay or speech-related disorders [Snijders-Blok et al., 2018]. In a cohort of 13 patients, 2 or more showed autism spectrum disorder, attention deficit hyperactivity disorder, optic nerve abnormalities, Duane anomaly, hypotonia, mild congenital heart abnormalities, and dysmorphisms [Snijders-Blok et al., 2018]. In 12 unrelated patients presenting with hypotonia, mild to moderate intellectual disability, behavioral disorders, and variable facial dysmorphism, CDK8 mutations were found [Calpena et al., 2019]. In several patients, congenital heart disease, agenesis of the corpus callosum, anorectal malformations, seizures, and hearing or visual impairments were detected. In a cohort of 7 patients with intellectual disability and/or DD, including speech impairment and features such as autism spectrum disorder, aggressive behavior, corpus callosum abnormality, and facial dysmorphologies, mutations affecting the MED12L gene were found [Nizon et al., 2019]. CNVs were detected in 3 patients, while 4 others harbored single nucleotide variants. Based on their phenotypic presentation, patients with MED13, MED13L, MED12L, or CDK8 mutations are difficult to distinguish from the MED12-related Opitz-Kaveggia, Lujan-Fryns, and Ohdo syndrome [Adegbola et al., 2015; Caro Llopis et al., 2016; Calpena et al., 2019; Srivastava et al., 2019]. Rather, these syndromes appear to form a continuum, which has been subsumed under the term of “transcriptomopathies” [Yuan et al., 2015; Caro Llopis et al., 2016].

It appears that patients with mutations affecting any proteins forming the regulatory component of MC present with a syndrome with variable expressivity, in particular with respect to congenital heart defects. Functional studies may help to elucidate the effects of genetic alterations of MC. Analysis of cells from 2 individuals with MED12L mutations revealed a modest yet significant alteration of MED12L mRNA synthesis rates, akin to findings with an intragenic deletion in the CNTNAP2 gene [Lee et al., 2015; Nizon et al., 2019]. In cells with missense mutations in CDK8, STAT1 phosphorylation is reduced, in most cases to a similar extent as in a kinase-dead control [Calpena et al., 2019]. MED12 mutations in patients with Opitz-Kaveggia or Lujan syndromes disrupt the GLI3-dependent sonic hedgehog (SHH) signaling pathway under the control of MC [Zhou et al., 2012]. In lymphoblast cell lines from these patients, expression levels of multiple SHH/GLI3 target genes such as ASCL1, BMP4, CREB5, and NEUROG2 were significantly elevated [Zhou et al., 2012]. In patients with X chromosome-linked intellectual delay, different MED12 mutations caused distinct patterns of altered expression of the SHH/GLI3 target genes CREB5, BMP4, and NEUROG2 [Srivastava et al., 2019]. GLI3 is a zinc finger transcription factor with a dual function as transcriptional activator and repressor of the SHH system, which is known as a regulator of cranial suture development [Tanimoto et al., 2012; Poot, 2019]. The studies discussed above implicate the regulatory components of MC as a core regulatory mechanism in much more complex developmental syndromes. Assembly of MC cannot only be disrupted by mutations in its component genes, but in mouse liver, it is also sensitive to the nutrition state [Youn et al., 2019]. Thus, during fasting, genetically insulin-resistant and obese mice displayed loss of the kinase module of MC [Youn et al., 2019]. It is not known to what extent this may lead in pregnant women to offspring with DD and other phenotypes of the MED12-MED13L-CDK8 continuum. Given the phenotypic overlap between chromosome 1p36 deletion syndrome and 22q11 deletion syndrome with the MED12-MED13L-CDK8 syndrome continuum, it may also be worthwhile to study the expression levels of the genes in these deletions in patients with MED12, MED13, MED13L, or CDK8 mutations. Following a path from karyotyping, via genome wide aneuploidy screening and exome sequencing, we saw individual patients becoming manifestations of syndromes, which merged in an MC-based continuum at the center of a complex and only partly understood regulatory mechanism of development.

Martin Poot