Mutations in MED12 cause X-linked Ohdo syndrome

Abstract

Ohdo syndrome comprises a heterogeneous group of disorders characterized by intellectual disability (ID) and typical facial features, including blepharophimosis. Clinically, these blepharophimosis-ID syndromes have been classified in five distinct subgroups, including the Maat-Kievit-Brunner (MKB) type, which, in contrast to the others, is characterized by X-linked inheritance and facial coarsening at older age. We performed exome sequencing in two families, each with two affected males with Ohdo syndrome MKB type. In the two families, MED12 missense mutations (c.3443G>A [p.Arg1148His] or c.3493T>C [p.Ser1165Pro]) segregating with the phenotype were identified. Upon subsequent analysis of an additional cohort of nine simplex male individuals with Ohdo syndrome, one additional de novo missense change (c.5185C>A [p.His1729Asn]) in MED12 was detected. The occurrence of three different hemizygous missense mutations in three unrelated families affected by Ohdo syndrome MKB type shows that mutations in MED12 are the underlying cause of this X-linked form of Ohdo syndrome. Together with the recently described KAT6B mutations resulting in Ohdo syndrome Say/Barber/Biesecker/Young/Simpson type, our findings point to aberrant chromatin modification as being central to the pathogenesis of Ohdo syndrome.

Figure 1

Overview of Clinical and Genetic Data of Families Affected by Ohdo Syndrome MKB Type and MED12 Mutations (A) Photographs of individuals with MED12 mutations: family 1 proband (III-1) in infancy (Aa and Ac) and at age 4 years (Ab) and family 1 uncle (II-3) at age 4 years (Ad) and 18 years (Ae and Af); family 2 proband (II-1) at ages 1 year (Ag), 9 years (Ah), and 15 years (Ai) and family 2 brother (II-2) at ages 1 month (Aj), 7 years (Ak), and 13 years (Al); and family 3 proband (II-1) at ages 3 years (Am), 10 years (An), and 16 years (Ao). Beside the photographs, the electropherograms show the mutation in bold of an affected individual (top) and a control (bottom), and the pedigrees show results of segregation analysis. Mutant alleles are represented by a plus sign (+), and wild-type alleles are represented by a minus sign (–). (B) Schematic overview of MED12, including the PQL domain, which is involved in β-catenin and GLI3 binding, shows previously published amino acid changes leading to Opitz-Kaveggia syndrome and Lujan-Fryns syndrome and the presently identified amino acid changes leading to Ohdo syndrome MKB type.

Figure 2

Ohdo Syndrome Mutations in MED12 Disrupt Its Gene-Repression Function (A) HEK293 cells were transfected with control (siCNTL) or MED12-specific (siMED12) siRNAs. Where indicated (FLAG-MED12), MED12-knockdown cells were transfected with FLAG-tagged siRNA-resistant WT, p.Arg1148His (R1148H), or p.Ser1165Pro (S1165P) MED12 expression plasmids. RNA-expression levels of CHRM4, SNAP25, or SYN1 were determined by quantitative RT-PCR. mRNA levels are expressed relative to mRNA levels in control siRNA-transfected cells. Data represent the mean ± the SEM of at least three independent experiments performed in duplicate. Asterisks denote statistically significant differences compared to WT FLAG-MED12 (Student’s t test, ^∗p < 0.05, ^∗∗p < 0.01). FLAG-tagged MED12 mutants p.Arg1148His and p.Ser1165Pro were unable to repress CHRM4, SNAP25, and SYN1 expression. (B) Nuclear extracts from a representative transient expression assay were resolved by SDS-PAGE and processed by immunoblot analysis with antibodies specific to MED12, the FLAG epitope on FLAG-MED12 derivatives, or the TFIIEβ that was used as an internal loading control. Representative immunoblots show that siMED12 significantly diminished expression of MED12. Cotransfection with FLAG-MED12 restored MED12 expression for the WT and both mutants. For the doublet bands, the band with the higher molecular mass was previously shown to represent full-length MED12. The relative levels of ectopically expressed FLAG-MED12 WT and mutant proteins averaged over four independent experiments are given below the immunoblots and were calculated first by normalization of FLAG-MED12 immunoblot signals to internal-control TFIIEβ signals (within the linear range of detection; Figure S2) and subsequent division of the normalized expression levels of FLAG-MED12 mutants by those of FLAG-MED12 WT. Immunoblot signals were quantified with ImagQuant TL software. No statistically significant differences in expression levels were observed (Student’s t test; p values are given above the bars). (C) Nuclear extracts from untransfected (CNTL) or transfected HEK293 cells transiently expressing FLAG epitope-tagged WT, p.Arg1148His, or p.Ser1165Pro MED12 derivatives were subjected to immunoprecipitation (IP) with IgG or antibodies specific to MED30, as indicated. Mediator immunoprecipitates were resolved by SDS-PAGE and processed by immunoblot analysis with antibodies specific to the FLAG epitope or the indicated Mediator subunits. Input corresponds to 10% of the nuclear extracts subjected to IP. FLAG-tagged MED12 mutants p.Arg1148His and p.Ser1165Pro were incorporated into Mediator comparably to WT MED12. (D) HA-tagged G9a was expressed without or with FLAG-tagged MED12 WT, p.Arg1148His, or p.Ser1165Pro derivatives in HEK293 cells prior to the processing of nuclear extracts by IP with antibodies specific to the HA epitope. Immunoprecipitates were resolved by SDS-PAGE and processed by immunoblot analysis with FLAG- or HA-specific antibodies, as indicated. Input corresponds to 10% of the nuclear extracts subjected to IP. FLAG-tagged MED12 mutants p.Arg1148His and p.Ser1165Pro bound to G9a comparably to WT MED12.