MED12-Related Disease in a Chinese Girl: Clinical Characteristics and Underlying Mechanism

Abstract

The RNA polymerase II transcription subunit 12 homolog (MED12) is a member of the mediator complex, which plays a critical role in RNA transcription. Mutations in MED12 cause X-linked intellectual disability and other anomalies collectively grouped as MED12-related disorders. While MED12 mutations have been most commonly reported in male patients, we present the case of a 1-year-old girl with clinical characteristics similar to MED12-related disorders. To explore the clinical characteristics of the condition and its possible pathogenesis, we analyzed the patient's clinical data; genetic testing by whole-exome sequencing revealed a de novo heterozygous mutation (c.1249-1G > C) in MED12. Further cDNA experiments revealed that the patient had an abnormal splicing at the skipping of exon9, which may have produced a truncated protein. qPCR showed decreased MED12 gene expression level in the patient, and an X-chromosome inactivation test confirmed a skewed inactivation of the X-chromosome. The lymphoblast transcription levels of the genes involved in the Gli3-dependent sonic hedgehog (SHH) signaling pathway, namely, CREB5, BMP4, and NEUROG2, were found to be significantly elevated compared with those of her parents and sex- and age-matched controls. Our results support the view that MED12 mutations may dysregulate the SHH signaling pathway, which may have accounted for the aberrant craniofacial morphology of our patient.

Keywords: MED12; SHH signaling pathway; X-chromosome inactivation; craniofacial morphology; intellectual disability; mutation.

Figure 1

(A–C) The clinical characteristics of the patient at age 1 were consistent with the phenotype of MED12-related disease: (A) long forehead, low ear position, prominent nasal bridge, short philtrum, and repaired cleft lip and palate. (B) The hypotonia and the overextension joints of the thumb and toe. (C) Magnetic resonance imaging (T1- and T2-weighted and T2-fluid attenuated inversion recovery) showed that the corpus callosum was thin. (D) The sequences of genomic DNA in the patient’s nuclear family with the detection of a novel heterozygous splicing variant of MED12 c.1249-1G > C in a girl patient. (E) PCR results of cDNA from the patient’s nuclear family. The PCR products of the patient appeared to be two bands: one band like her parents’ and the other truncated band. (F) The result of the sequences from the gel purified products of the parents and the patient’s normal band. (G) The result of the sequence from the gel purified product of the patient’s truncated band showed the patient had an exon9 skip when compared with the normal sequence of MED12 gene.

Figure 2

(A) The relative expression of MED12 was investigated with RT-qPCR in the patient’s nuclear family, as well as in a sex- and age-matched control group. The patient exhibited significantly lower expression of the MED12 gene c.1249-1G > C relative to that of her parents. The discrepancy in the levels of MED12 was not related to age or sex according to the results of the control group. (B) The transcription levels of the sonic hedgehog (SHH)-signaling genes (BMP4, CREB5, and NEUROG2) were investigated with RT-qPCR in the patient’s nuclear family, as well as in a sex- and age-matched control group. The expressions of BMP4, CREB5, and NEUROG2 were all significantly enhanced in our patient relative to that in her parents and a sex- and age-matched control group ( Supplementary Figure 1 ), suggesting the hyperacitvated output of GLI3-dependent SHH signaling. Each dataset for the transcription levels in (A) and (B) was generated from triplicate studies presented as mean ± SEM. Statistical analyses of the qPCR data were performed to compare the means of the samples according to the 2^-ΔΔCtmethod. Asterisks denote statistically significant differences (Student t test, **P < 0.05, ***P < 0.01).

Figure 3

PCR products of the androgen receptor (AR) with and without HpaII digestion of the patient’s nuclear family. The peak represents the amplified AR allele. The PCR products of the patient derived from the undigested DNA yielded two peaks because of the different numbers of CAG repeats in the two alleles (275 bp from the father and 285 bp from the mother). However, after HpaII was digested, one peak (275 bp) appeared hypermethylated. The results revealed a skewed X-inactivation pattern in the patient with the paternal allele highly inactivated.