Report of a de novo c.2605C > T (p.Pro869Ser) change in the MED13L gene and review of the literature for MED13L-related intellectual disability

Background MED13L-related intellectual disability is a new syndrome that is characterized by intellectual disability (ID), motor developmental delay, speech impairment, hypotonia and facial dysmorphism. Both the MED13L haploinsufficiency mutation and missense mutation were reported to be causative. It has also been reported that patients carrying missense mutations have more frequent epilepsy and show a more severe phenotype. Case presentation We report a child with ID, speech impairment, severe motor developmental delay, facial deformity, hypotonia, muscular atrophy, scoliosis, odontoprisis, abnormal electroencephalogram (EEG), and congenital ureteropelvic junction obstruction (UPJO) combined with high ureter attachment. We used whole-exome sequencing (WES) to detect the genetic aberration of the child and found a de novo mutation, c.2605C > T (p.Pro869Ser), in the MED13L gene. Neither of her parents carried the mutation. Additionally, we review the literature and summarize the phenotypes and features of reported missense mutations. After reviewing the literature, approximately 17 missense mutations in 20 patients have been reported thus far. For 18 patients (including our case) whose clinical manifestations were provided, 100% of the patients had ID or developmental delay (DD). A total of 88.9, 83.3 and 66.7% of the patients had speech impairment, delayed milestones and hypotonia, respectively. A total of 83.3% of the patients exhibited craniofacial deformity or other dysmorphic features. Behavioral difficulties and autistic features were observed in 55.6% of the patients. Cardiac anomalies were seen in only 27.8% of the patients. Of these patients, 44.4% had epileptic seizures. Of the 17 mutations, 2 were located in the N-terminal domain, 8 were located in the C-terminal domain, and 1 was located in an α-helical sequence stretch. One of them was located in the MID domain of the MedPIWI module. Conclusions We report a new patient with a reported missense mutation, c.2605C > T (p.Pro869Ser), who exhibited some infrequent manifestations except common phenotypes, which may broaden the known clinical spectrum. Additionally, by reviewing the literature, we also found that patients with missense mutations have a higher incidence of seizures, MRI abnormalities, autistic features and cardiac anomalies. They also have more severe ID and hypotonia. Our case further demonstrates that Pro869Ser is a hotspot mutation of the MED13L gene.


Background
Mediator complex subunit 13-like gene (MED13L), which is a component of the Mediator complex in HeLa cells [1], was first linked to intellectual disability (ID) and congenital heart disease (CHD) in 2003 by Muncke et al. [2]. The patient reported by Muncke et al. harbored a translocation disrupting MED13L, and three additional missense mutations in MED13L (p.Glu251Gly, p.Arg1872His, and p.Asp2023Gly) were found by mutation screening of dextro-looped transposition of the great arteries (dTGA) patients. Muncke et al. cloned MED13L using a positional cloning approach, and they designated the gene as PROSIT240 due to the protein similarity to the human thyroid hormone receptorassociated protein 240 [2]. Independently, Musante et al. cloned the gene by RT-PCR and 5-prime RACE of human fetal brain and lymphoblastoid cell line cDNA libraries, which they called THRAP2 [3]. A decade later, an increasing number of cases harboring large intragenic or whole gene deletions/duplications of the MED13L gene, chromosomal translocation disrupting the MED13L gene, de novo frameshift variants, nonsense mutations, and splice site mutations were published, exhibiting moderate ID, severe speech impairment, motor developmental delay, facial deformity and/or CHD, and these were recognized as MED13L haploinsufficiency syndrome [4][5][6][7][8][9][10][11]. Several missense mutations in the MED13L gene have also been reported and are supposed to frequently have a more severe phenotype with hypotonia, more frequently epilepsy, severe absent speech, and severely delayed motor function compared to patients with truncating variants [11][12][13][14].
Here, we report another de novo p.Pro869Ser change in the MED13L gene that exhibits ID, speech impairment, severe motor developmental delay, facial deformity, hypotonia, muscular atrophy, hyperlaxity of the joints, scoliosis, odontoprisis, abnormal electroencephalogram (EEG), and congenital ureteropelvic junction obstruction (UPJO) combined with high ureter attachment that has never been reported in MED13L-related syndrome. Our observation of UPJO in this patient further broadens the known clinical spectrum. Additionally, we review the current literature to summarize in detail the missense mutations of the MED13L gene and the clinical characteristics of reported patients with missense mutations of the MED13L gene.

Case presentation
The patient is a girl who was born at term after 38 weeks of pregnancy with a birth weight of 2500 g, without a history of asphyxia at birth. She is one of a fraternal pair of twins as the second pregnancy of healthy, nonconsanguineous parents. Her older sister and twin brother are healthy. She is 4 years and 5 months old now, weighs 11 kg (<P3) and is 100 cm tall (between P3 and P10). Her head circumference was 45 cm (<P3). She had hypotonia since birth and presented muscular atrophy of the extremities as well as hyperlaxity of the joints. She had scoliosis and had spontaneous fracture of the distal right femur at 1 year old. Abnormal facial deformity includes frontal bossing, low-set ears, hypertelorism, epicanthus, depressed nasal bridge, bulbous nasal tip, cupid-bow upper lip combined with open mouth appearance and micrognathia (Fig. 1). From 3 years and 4 months old, she had unconscious frequent odontoprisis. Her developmental milestones were severely delayed, as she raised her head at 1 year. At the last evaluation, the girl was 4 years and 5 months old, and she could not yet sit or stand without support, let alone walk. Her speech was also severely delayed; she can speak single words such as "Ma, Pa" with ambiguous pronunciation, and she can understand simple instructions. She also exhibits autistic features. She had no clinically observed seizures but had abnormal EEG showing spike and slow wave colligation and multi-spike and slow waves in the bilateral occipital and posterior temporal regions, as well as rapid rhythm distribution in the occipital area (Fig. 2). Magnetic Resonance Imaging (MRI) at 5 months old showed enlarged bilateral lateral ventricles. Echocardiography found patent ductus arteriosus that was closed at 2 years and 10 months, and mild aortic coarctation, mild aortic regurgitation and slight tricuspid regurgitation Fig. 1 Photograph of the patient. The picture shows microcephaly, frontal bossing, low-set ears, hypertelorism, epicanthus, depressed nasal bridge, bulbous nasal tip, cupid-bow upper lip combined with open mouth appearance, micrognathia, muscular atrophy appeared. UPJO combined with high ureter attachment of the right was discovered in this girl due to uronephrosis, and she underwent surgery at 6 months old. Now she still has a mild right kidney seeper.
Whole-exome sequencing (WES) found a de novo mutation, c.2605C > T (p.Pro869Ser), in the MED13L gene. Neither of her parents had the mutation. The region of the mutation is an important part of the protein, with highly conserved amino acid sequences in different species. This mutation is predicted to be disease causing by Mutation Taster (http://www.mutationtaster.org/) and is predicted to be damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00) by Polyphen 2 (http://genetics. bwh.harvard.edu/pph2/) and damaging with a score of 0.000 by SIFT (cutoff = 0.05) (http://sift.jcvi.org/www/ SIFT_BLink_submit.html).
By reviewing the literature, approximately 17 missense mutations in 20 patients have been reported thus far. Together with our case, a total of 21 patients with MEDL13L missense mutations are summarized in Table 1. Among them, 18 patients (including our case) were provided with clinical manifestations. One hundred percent of the patients have ID or DD. A total of 88.9, 83.3 and 66.7% of the patients had speech impairment, delayed milestones and hypotonia, respectively. A total of 83.3% of the patients exhibited craniofacial deformity or other dysmorphic features, and the most common features were low-set ears, hypertelorism, depressed nasal bridge, bulbous nasal tip, cupid-bow upper lip and open mouth appearance. Behavioral difficulties, such as self-harm and autistic features, were seen in 55.6% of the patients. Cardiac anomalies are seen in only 27.8% of the patients, and there is no complex CHD. Of the patients with missense mutations, 44.4% have epileptic seizures, and even one patient with c.2579A > G(Asp860Gly) has intractable seizures. In 12 patients who received MRI examination, 8 (66.7%) had abnormalities, and most of these anomalies were nonspecific. Of the 17 mutations, 2 were located in the N-terminal domain, 8 were located in the C-terminal domain, and 1 was located in an α-helical sequence

Target capture and sequencing
After obtaining informed consent from her parents, peripheral blood of the proband and her parents was sent to Guangzhou Jiajian Medical Testing Co., Ltd. Genomic DNA was extracted from peripheral blood using the Solpure Blood DNA Kit (Magen) according to the manufacturer's instructions. The genomic DNA of the proband and her parents was then fragmented by the Q800R Sonicator (Qsonica) to generate 300-500 bp insert fragments. The paired-end libraries were prepared following the Illumina library preparation protocol. Customdesigned NimbleGen SeqCap probes (Roche NimbleGen, Madison, Wis) were used for in-solution hybridization to enrich target sequences. Enriched DNA samples were indexed and sequenced on a NextSeq500 sequencer (Illumina, San Diego, Calif) with 100,150 cycles of single end reads, according to the manufacturer's protocols.

Variant annotation and interpretation
Primary data came in fastq form after image analysis, and base calling was conducted using the Illumina Pipeline. The data were filtered to generate 'clean reads' by removing adaptors and low-quality reads (Q20). Sequencing reads were mapped to the reference human genome version hg19 (2009-02 release, http://genome.ucsc.edu/). Nucleotide changes observed in aligned reads were called and reviewed by using NextGENe software (Soft-Genetics, State College, Pa). In addition to the detection of deleterious mutations and novel single nucleotide variants, a coverage-based algorithm developed in-house, eCNVscan, was used to detect large exonic deletions and duplications. The normalized coverage depth of each exon of a test sample was compared with the mean coverage of the same exon in the reference file to detect copy number variants (CNVs). Sequence variants were annotated using population and literature databases including 1000 Genomes, dbSNP, GnomAD, Clinvar, HGMD and OMIM. Some online software programs were used to analyze the structure of the protein, predict the conservation domain and function domain and perform the multiple sequence alignment. Variant interpretation was performed according to the American College of Medical Genetics (ACMG) guidelines [20].

Discussion and conclusion
In this study, we report a new patient with a previously reported missense mutation but with some new clinical manifestations. In addition to ID, speech impairment, motor developmental delay and hypotonia, she also exhibits congenital UPJO combined with high ureter attachment on the right, odontoprisis, appendicular muscular atrophy, scoliosis, and spontaneous facture of femur. Until 4 y 5 m, she had no clinically observed seizures, but EEG revealed spike and slow wave colligation and multi-spike and slow waves in the bilateral occipital and posterior temporal regions, as well as rapid rhythm distribution in the occipital area. However, it is not known whether epilepsy will occur in the future. The two patients P28 and P35 reported by Smol et al. with the same mutation as our case both had seizures, but the ages at first examination were 12 y and 24 y, respectively. Therefore, seizures were not observed, which may be due to the limited follow-up time. P28 did not exhibit hypotonia, but P35 and our patient have severe hypotonia. Whereas our patient exhibits autistic features, both P28 and P35 have no autism or behavioral difficulty [12]. Three patients with the same mutation did not have exactly the same clinical manifestations, demonstrating the clinical heterogeneity of patients with MED13L missense mutations. Additionally, urinary system abnormality was not a common manifestation in patients with MED13L missense mutations. However, our case had congenital UPJO, P28 with the same mutation as our case reported by Smol [11,12]. The incidence of ID and hypotonia was similar to the overall incidence, but the phenotypes were much more serious. The incidence of cardiac anomalies was slightly higher than the overall incidence (27.8% vs 19%) [11].
Of the 17 mutations, 2 (11.8%) were located in the Nterminal domain, 8 (47%) were located in the highly conserved C-terminal domain, 1 of them (Asp860Gly) was located in an α-helical sequence stretch spanning residues Val858-Met864, and replacement of Asp860 by a flexible glycine decreased the helix stability, thereby affecting the secondary structure of MED13L [14]. Another mutation, Arg1416His, was located in the MID domain of the MedPIWI module. MedPIWI is the core globular domain of the Med13 protein. Med13 is a member of the CDK8 subcomplex of the Mediator transcriptional coactivator complex. The MedPIWI module in Med13 is predicted to bind double-stranded nucleic acids, triggering the experimentally observed conformational switch in the CDK8 subcomplex, which regulates the Mediator complex (Fig. 3). By analysis with SIFT, Polyphen2 and Mutation Taster, all the mutations except Prol1761Leu were predicted to be pathogenic by at least two of the prediction software programs. The mutation Prol1761Leu was predicted to be tolerated, benign, and polymorphic by SIFT, Polyphen2 and Mutation Taster, respectively. However, the mRNA expression levels of MED13L are significantly decreased, as revealed by quantitative RT-PCR, and are supposed to be pathogenic [19]. Therefore, in regard to a missense mutation that was predicted to be benign by software, while the clinical manifestations are highly coincident with MED13L-related disorder, researchers or clinicians should carry out further functional experiments to define the pathogenicity of the mutation.
In this paper, we describe a new patient with MED13L missense mutation who exhibited some infrequent manifestations except common phenotypes, which may broaden the known clinical spectrum. Additionally, we review the literature to summarize patients' phenotypes and features of all reported missense mutations. We also found that patients with missense mutations have a higher incidence of seizures, MRI abnormalities, autistic features and cardiac anomalies. They also have more severe ID and hypotonia, which is consistent with the literature [12]. We need more functional experiments to demonstrate why patients carrying missense mutations have more severe phenotypes. Our case further demonstrates that Pro869Ser is a hotspot mutation of the MED13L gene. Fig. 3 The locations of the reported 17 missense mutations. Most of them located in exon 15-31. 2 (11.8%) were located in the N-terminal domain, 8 (47%) were located in the highly conserved C-terminal domain, 1 of them (Asp860Gly) was located in an α-helical sequence stretch spanning residues Val858-Met864, Another mutation, Arg1416His, was located in the MID domain of the MedPIWI module. MedPIWI is the core globular domain of the Med13 protein