Received: 5 May 2022 Revised: 26 July 2022 Accepted: 13 August 2022 DOI: 10.1002/ajmg.a.63004 ORIGINAL ARTICLE Missense MED12 variants in 22 males with intellectual disability: From nonspecific symptoms to complete syndromes Nuno Maia 1 | Nekane Ibarluzea 2 | Mala Misra-Isrie 3 | Daniel C. Koboldt 4,5 | Isabel Marques 1 | Gabriela Soares 6 | Rosário Santos1 | Carlo L. M. Marcelis7 | Riikka Keski-Filppula8 | Miriam Guitart9 | Elisabeth Gabau Vila9 | April Lehman5,10 | Scott Hickey5,10 | Mari Mori10,11 | Paulien Terhal12 | Irene Valenzuela13 | Amaia Lasa-Aranzasti13 | Anna Maria Cueto-González13 | Brian H. Chhouk14 | Rebecca C. Yeh14 | Jennifer E. Neil14 | Bassam Abu-Libde15 | Tjitske Kleefstra7 | Mariet W. Elting3 | Andrea Császár16 | Judit Kárteszi17 | Beáta Bessenyei18 | Hans van Bokhoven7 | Paula Jorge1 | Johanna M. van Hagen3 | Arjan P. M. de Brouwer7 1 Unidade de Genética Molecular, Centro de Genética Médica Doutor Jacinto de Magalhães (CGM), Centro Hospitalar Universitário do Porto (CHUPorto); Unit for Multidisciplinary Research In Biomedicine (UMIB), Institute of Biomedical Sciences Abel Salazar (ICBAS), and ITR - Laboratory for Integrative and Translational Research in Population Health, University of Porto, Porto, Portugal 2 Biocruces Bizkaia Health Research Institute, Barakaldo, Spain 3 Department of Human Genetics, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands 4 Steve and Cindy Institute for Genomic Medicine, Nationwide Children's Hospital, Columbus, Ohio, USA 5 Department of Pediatrics, The Ohio State University College of Medicine, Columbus, Ohio, USA 6 Unidade de Genética Médica, Centro de Genética Médica Doutor Jacinto de Magalhães (CGM), Centro Hospitalar Universitário do Porto (CHUPorto), Porto, Portugal 7 Department of Human Genetics, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, Nijmegen, The Netherlands 8 Department of Clinical Genetics, Oulu University Hospital, Medical Research Center Oulu and PEDEGO Research Unit, University of Oulu, Oulu, Finland 9  i Innovacio Paediatric Unit, ParcTaulí Hospital Universitari, Institut d'Investigacio  Parc Taulí, I3PTUniversitat Autònoma de Barcelona, Sabadell, Spain 10 Division of Genetic & Genomic Medicine, Nationwide Children's Hospital, Columbus, Ohio, USA 11 Nationwide Children's Hospital, Columbus, Ohio, USA 12 Division Laboratories, Pharmacy and Biomedical Genetics, Wilhelmina Children's Hospital, Utrecht, The Netherlands 13 Department of Clinical and Molecular Genetics, Vall d'Hebron University Hospital and Medicine Genetics Group, Vall d'Hebron Research Institute, Barcelona, Spain 14 Division of Genetics and Genomics and Howard Hughes Medical Institute, Boston Children's Hospital, Boston, Massachusetts, USA 15 Makassed Hospital, Jerusalem, Israel 16 Paediatric Ward, Hospital of Zala County, Zalaegerszeg, Hungary 17 Genetic Counselling, Hospital of Zala County, Zalaegerszeg, Hungary 18 Division of Clinical Genetics, Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary Correspondence Arjan P. M. de Brouwer, Department of Abstract Human Genetics, Donders Institute for Brain, We describe the phenotype of 22 male patients (20 probands) carrying a hemizygous Cognition and Behavior, Radboud University Medical Center, 6500 HB Nijmegen, The missense variant in MED12. The phenotypic spectrum is very broad ranging from Nuno Maia and Nekane Ibarluzea contributed equally to this study. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2022 The Authors. American Journal of Medical Genetics Part A published by Wiley Periodicals LLC. Am J Med Genet. 2023;191A:135–143. wileyonlinelibrary.com/journal/ajmga 135 136 MAIA ET AL. Netherlands. Email: arjan.debrouwer@radboudumc.nl nonspecific intellectual disability (ID) to the three well-known syndromes: Opitz– Kaveggia syndrome, Lujan–Fryns syndrome, or Ohdo syndrome. The identified vari- Present address Gabriela Soares, Consulta de Genética, Centro ants were randomly distributed throughout the gene (p = 0.993, χ2 test), but mostly Hospitalar de Trás-os-Montes e Alto douro, outside the functional domains (p = 0.004; χ2 test). Statistical analyses did not show E.P.E, Vila Real, Portugal. a correlation between the MED12-related phenotypes and the locations of the vari- Funding information ants (p = 0.295; Pearson correlation), nor the protein domain involved (p = 0.422; Foundation for Science and Technology (FCT), Grant/Award Number: UIDB/00215/2020 Pearson correlation). In conclusion, establishing a genotype–phenotype correlation in UIDP/00215/2020 LA/P/0064/2020; Broad MED12-related diseases remains challenging. Therefore, we think that patients with a Institute; National Eye Institute; National Heart, Lung and Blood Institute, Grant/Award causative MED12 variant are currently underdiagnosed due to the broad patients' Number: UM1 HG008900; National Human clinical presentations. Genome Research Institute, Grant/Award Number: R01 HG009141 KEYWORDS intellectual disability, MED12, phenotype, genotype 1 | I N T RO DU CT I O N (Srivastava et al., 2019). Notwithstanding, other nonspecific clinical presentations were also described (Bouazzi et al., 2015; Prontera Intellectual disability (ID) is characterized by impaired intellectual et al., 2016; Yamamoto & Shimojima, 2015), where the three corre- functioning and adaptive behavior arising before the age of sponding missense variants were also randomly distributed along 22 (Schalock et al., 2021). ID is one of the most common neurodeve- MED12. lopmental conditions with a prevalence of about 1%–3% (Patel In the past decade, next-generation sequencing (NGS) has et al., 2018) and X-linked ID (XLID) accounts for about 5%–10% ID in revealed several unique variants in MED12 in male patients with ID, males (Lubs et al., 2012). and there is an urgent need to assess whether the phenotype of those MED12 (OMIM no. *300188) is an X-linked gene that encodes for patients is compatible with either FGS1, Lujan–Fryns or Ohdo syn- the Mediator of RNA polymerase II transcription subunit 12 protein dromes. Nevertheless, the more variants are identified in this gene, that is part of the kinase module of the Mediator complex, together the more difficult is the patients' classification into the different with CDK8, MED13, and Cyclin C, which is involved in the regulation related syndromes due to the high phenotypic variability related to of gene expression through the RNA polymerase II. MED12 comprises MED12 variants (Charzewska et al., 2018). Because of this phenotypic 2177 amino acid (aa) residues and contains three functional domains: variability in the increasing number of MED12 variants that are found Med12 (103–161 aa), LCEWAV (289–757 aa), and PQL-catenin- by NGS, it is essential to gain more knowledge about the different binding (1616–2051 aa) (Polla et al., 2020). Pathogenic variants in the effects of MED12 variants and possible phenotypes. This could result kinase module, including MED12, have been shown to disrupt devel- in more accurate clinical counseling of the involved families, and facili- opmental and oncogenic signaling pathways, such as Wnt, Sonic tate the challenge of distinguishing pathogenic variants from non- Hedgehog, Epidermal Growth Factor, and Notch pathways (Clark pathogenic variants in the MED12 gene. et al., 2015). In this study, we focus on 22 male patients (20 probands) with MED12 germline variants are known to cause syndromic or non- variants in the MED12 gene. We provide an overview of the clinical syndromic forms of XLID. Syndromic forms include (i) Opitz–Kaveggia features and discuss their differences and similarities with the previ- syndrome (also known as FG syndrome 1 [FGS1], OMIM no. 305450) ously reported MED12 mutational spectrum. which is characterized by ID, hypotonia, distinct facial features, rela- tive macrocephaly, broad thumbs and halluces, corpus callosum abnormalities, and gastrointestinal complications (Risheg et al., 2007); 2 | P A T I E N T S A ND M E T H O D S (ii) Lujan–Fryns syndrome (OMIM no. 309520) which partially over- laps with FGS1 but shows several distinctive features, such as a tall 2.1 | Patient cohort stature, hypernasal voice, and hyperextensible digits (Schwartz et al., 2007) and (iii) Ohdo syndrome (OMIM no. 300895) which We report a series of male patients with ID who harbor a variant shows particular features, as ptosis and blepharophimosis (Vulto-van in MED12. The patients were gathered from an international col- Silfhout et al., 2013). These MED12-related syndromes have been laboration through Genematcher (Au et al., 2015; Sobreira assigned to different regions of the protein: variants in leucine-rich et al., 2015). In total, we have managed to recruit 22 male patients and serine-rich region (LS, 500–1616 aa) have been associated with from 10 different hospitals/research institutes. Of these, 20 are FGS1 and Lujan syndrome, while variants in LS and PQL-catenin- index male patients. All reported patients were evaluated by their binding domain have been associated with Ohdo syndrome respective clinical geneticists and DNA sequencing studies (either MAIA ET AL. 137 targeted or exome sequencing) were performed for diagnostic affected amino acid residue, clinical features, and syndrome purposes. The severity of ID was determined with different tests: classification. WISC-III, WISC-IV, and WAIS-III, by checking developmental mile- stones, or by performing a complete neuropsychological evalua- tion depending on the age of the individuals. Written informed 3 | RE SU LT S consent has been obtained from all the legal representatives of the participants to publish clinical data and photographs of the Here, we describe 22 male patients (20 probands) carrying a missense included patients. variant in the MED12 gene. Most of the probands were found through exome sequencing (11/20) and the other patients by single gene test- ing (5/20), targeted ID (2/20), or X-linked ID (1/20) panels (Table 1 2.2 | Statistical analyses and Table S1). Individuals, who had undergone single gene testing, reported with a phenotype that fitted a number of the characteristics For the purposes of this study, statistical analyses, namely chi-square of the MED12-related phenotypes. In total, 13 of these variants were (χ2) and Pearson's chi-squared test (Pearson's χ2), have been per- maternally inherited and 4 occurred de novo. The mothers of three formed by using SPSS Statistics 26.0 (IBM-SPSS, Chicago, Illinois). A remaining patients were unavailable for testing. Patient 11 also had a p-value of <0.05 was considered statistically significant. The variables maternally-inherited pathogenic variant c.913C > T (p.Arg305*) in tested included variant exon location, protein domain involved, RPS6KA3 pointing to a diagnosis of Coffin–Lowry syndrome. TABLE 1 Molecular data of MED12 patients Classification Genomic Protein Mother Diagnostic Variant classification (syndrome) Patient (NM_005120.2) (NP_005111.2) Domain carrier methodology (ACMG/AMP guidelines) Opitz-Kaveggia 1 c.2881C > T p.(Arg961Trp) No Yes Exome sequencing P 2 c.2881C > T p.(Arg961Trp) No No Targeted variant P analysis 3 c.3020A > G p.(Asn1007Ser) No Yes Single gene testing LP 4 c.3469A > G p.(Asn1157Asp) No No Single gene testing VUS 5 c.3883C > T p.(Arg1295Cys) No Yes Single gene testing P Lujan–Fryns 6 c.67G > A p.(Asp23Asn) No NT Exome sequencing VUS 7 c.4879C > T p.(Arg1627Cys) PQL Yes Targeted ID panel LP Ohdo 8 c.1054 T > G p.(Cys352Gly) LCEWAV Yes Exome sequencing LP 9 c.3064A > G p.(Met1022Val) No No Exome sequencing LP 10 c.4147G > A p.(Ala1383Thr) No No Exome sequencing LP 11 c.5965C > T p.(Arg1989Cys) PQL Yes Exome sequencing LP Unclassified or 12 c.745C > G p.(Leu249Val) No Yes Trio exome VUS unknown sequencing 13 c.887G > A p.(Arg296Gln) LCEWAV Yes Exome sequencing LP 14 c.1546C > T p.(Arg516Cys) LCEWAV Yes Trio exome LP sequencing 15 c.1862G > A p.(Arg621Gln) LCEWAV Yes Single gene testing LP 16 c.2639 T > G p.(Met880Arg) No No Exome sequencing LP 17 c.3797G > A p.(Arg1266His) No Yes Single gene testing B 18 c.3884G > A p.(Arg1295His) No NT Unknown P 19 c.3946C > G p.(Gln1316Glu) No Yes Exome sequencing VUS 20 c.5009C > T p.(Ser1670Phe) PQL Yes Targeted XLID VUS panel 21 c.5009C > T p.(Ser1670Phe) PQL Yes Targeted XLID VUS panel 22 c.6371C > T p.(Ala2124Val) No NT Targeted ID panel VUS Abbreviations: ACMG, American College of Medical Genetics; AMP, Association for Molecular Pathology; B, Benign; ID, Intellectual disability; LCEWAV, Mediator complex, subunit Med12 LCEWAV-domain; LP, Likely pathogenic; NA, not assessed; P, Pathogenic; PQL, Mediator complex, subunit Med12, catenin-binding domain; VUS, Variant of uncertain significance; XLID, X-linked intellectual disability. 138 MAIA ET AL. The majority of the variants are located outside currently recog- in 11/22 patients and hearing impairment in 5/22 patients. One nized functional domains (13/20; p = 0.004, χ2 test), despite the vari- patient had dysplasia of his semicircular canals. This could be an ants being randomly located among the 45 MED12 exons (p = 0.993, underreported feature in individuals with a MED12 variant as a χ2 test). Arginine showed to be the most frequently mutated amino T2-weighted MRI specifically directed at finding this aberration needs acid (9/20; p = 0.013, χ2 test) (Tables 2 and 3), which is in line with to be performed. We have collected two uncle/nephew families: previous studies (Khan & Vihinen, 2007). Patients 1 and 2, and Patients 20 and 21, respectively. The phenotype The phenotypic spectrum of our cohort is very broad, in terms of is relatively consistent among the two individual family members. The clinical features and of severity, which is expected as MED12 is a gene main noticeable difference is vision impairment (Table 4), which associated with a large phenotypic variability (Table 4 and Table S1). occurs in one case the younger (Patient 1), and the other case the Our patients present from borderline to severe ID. While autism is older family member (Patient 20). rare (5/22 patients), behavioral problems are common (15/22), rang- The presence of facial dysmorphic features is also diverse ing from problems with concentration to aggressivity. Delay or (Table 4, Figure 1, and Table S1). They range from absence of dys- absence of speech is reported in 20/22 patients. MRI was performed morphisms (β) to multiple congenital anomalies such as severe prena- in 19/22 patients, 12 of which showed structural brain abnormalities, tal craniosynostosis, tall prominent forehead, hypertelorism, down- such as corpus callosum agenesis. Eyes/vision involvement is present slanting palpebral fissures, high narrow palate, maxillary hypoplasia, low-set ears, and a cloverleaf skull (Patient 1). Most of the patients described in this cohort could not be classi- T A B L E 2 Statistical results of the identified variants in all fied into any of the reported syndromes. However, some patients individuals with a MED12 variant except for individual 11 who also has a pathogenic variant in RPS6KA3 were classified as FGS1 (5/22), Lujan–Fryns syndrome (2/22), or Ohdo syndrome (4/22). One patient (Patient 11) also has a maternally χ2 test inherited pathogenic variant c.913C > T (p.Arg305*) in RPS6KA3 Null hypothesis p-value pointing to a diagnosis of Coffin–Lowry syndrome in addition to The variants are randomly distributed over the different 0.993 Ohdo–FG spectrum. No significant correlation was observed between exons the variants' genomic localization and MED12-related syndromes The variants affect randomly the different protein domains 0.004 (Tables 2 and 3; exon: p = 0.295; protein domain: p = 0.422 Pearson's The wild-type amino acid residues are equally affected 0.013 χ2). However, a statistically significant correlation was observed between the affected domain and cardiac defects (p = 0.010; Pear- Note: A χ2 test was calculated to determine if there was correlation between the different phenotype–genotype characteristics. Depicted in son's χ2). Furthermore, statistical significance was observed in the cor- bold are the correlations with a p < 0.05. relation analysis between hearing impairment and cardiac defects T A B L E 3 Statistical results of the identified variants in all individuals with a MED12 variant except for individual 11 who also has a pathogenic variant in RPS6KA3 Pearson's χ2 test Syndromea Intellectual Autism Facial Eyes/vision Hearing Cardiac disabilityb dysmorphismsc involvement impairment defects Exond 0.295 0.568 0.288 0.162 0.872 0.330 0.184 Domain 0.422 0.242 0.242 0.059 0.129 0.410 0.010 Syndromea 0.798 0.523 0.138 0.059 0.238 0.904 Intellectual 0.703 0.145 0.355 0.296 0.319 disabilityb Autism 0.407 0.058 0.329 0.439 Facial 0.286 0.293 0.454 dysmorphismsc Eye involvement 0.548 0.291 Hearing 0.034 impairment Cardiac defects Note: A Pearson correlation was calculated to determine if there was correlation between the different phenotype–genotype characteristics. Depicted in bold are the correlations with a p < 0.05. a MED12-related phenotypes: FGS1; LFS; Ohdo; nonspecific; no clinical information. b Intellectual disability: developmental delay; borderline; mild; moderate; severe; profound; no information. c Facial dysmorphisms: number of clinical features. d Domains: (i) Mediator complex, subunit Med12; (ii) LCEWAV-domain; (iii) catenin-binding domain; and (iv) outside important domains. MAIA ET AL. TABLE 4 Summary of the clinical data of MED12 patients Neurodevelopment Dysmorphic features Other features Classification Behavioral Speech MRI Face Mouth Extremities Eyes/vision Hearing Cardiac (Syndrome) Patient ID ASD disturbances impairment findings shape Eyes Nose and chin Ears abnormalities impairment Impairment defects Opitz-Kaveggia 1 Severe NA NA + + + + + + + + 2 Moderate + + + + + + + + + 3 Mild + + + + + + + + + NA 4 Mild + + + + + + + + + NA 5 Moderate + + + + + + + + + Lujan–Fryns 6 Borderline + + + + + + + + + 7 + + + + + NA + NA Ohdo 8 Mild + + + + + + + + + + 9 + + + + + + + + + + + + 10 + NA + + + + + + + + 11 Severe NA NA + + + + + + + NA Unclassified or 12 DD + + + NA + + + + + + NA + unknown 13 DD + + + + + + + + + 14 Moderate + + + 15 Moderate NA NA + NA + + + + + + 16 Severe NA + + + + + + 17 Mild NA + NA + + + + + + 18 Mild NA + NA + + + + + + NA 19 Mild + + + + + + + + + + + + 20 Borderline NA + + + + + + + + NA NA 21 NA + NA + NA + + + NA NA 22 Severe + + + + + + Abbreviations: ASD, autism spectrum disorder; DD, developmental delay; ID, intellectual disability; MRI, magnetic resonance imaging; NA, not assessed. 139 140 MAIA ET AL. F I G U R E 1 Clinical aspects of patients with a MED12 variant. Clearly visible are the different phenotypes. For instance, Patient 21 has a completely normal face, whereas Patient 1 presented with multiple congenital anomalies such as severe prenatal craniosynostosis, tall prominent forehead, hypertelorism, down-slanting palpebrae, high narrow palate, maxillary hypoplasia, low-set ears, and a cloverleaf skull. (p = 0.034; Pearson's χ2). Taken together, these data suggest that variability, even intrafamilial (Martinez et al., 2017; Patil et al., 2017). when a patient presents hearing impairment, the clinician should also Schwartz et al. (2007) described “the original Lujan syndrome family” look for hidden cardiac defects. carrying the variant c.3020A > G (p.Asn1007Ser) that is the same vari- ant as found in our Patient 3, who is classified as FGS1. The variant c.3883C > T (p.Arg1295Cys) identified in Patient 5, was previously 4 | DISCUSSION reported in patients classified as FGS1 (Graham et al., 1999; Hu et al., 2016). Interestingly, a different substitution affecting the same In this study, we describe a cohort of 22 male patients carrying mis- amino acid residue p.(Arg1295His) (c.3884G > A), carried by Patient sense MED12 variants, most of which could not be classified into one 18, was also reported in patients with a mild phenotype (Callier of the three known syndromes. The phenotypic spectrum presented et al., 2013; Srivastava et al., 2019). Finally, two siblings carrying the by the patients is very broad, with different levels of severity. In fact, variant c.4147G > A (p.Ala1383Thr) previously described as Ohdo trying to establish a genotype–phenotype correlation in MED12- syndrome (Langley et al., 2015), shared clinical features with our related diseases remains challenging despite the fact that more than Patient 10, who carried the same variant, except for the cleft palate, 80 MED12 variants in both males and females have been identified hearing impairment, and cardiac defects. This indicates that the (Figure 2). MED12 phenotype can vary quite a bit even in patients with the same Eight of the variants carried by the patients reported herein are variants, which can be the result of the presence genetic modifiers of described in the literature, and the majority are related to different the phenotype in the patient's genome or certain stochastic effects. phenotypes. For instance, the variant c.887G > A (p.Arg296Gln) car- Arginine is the most mutated residue among the missense vari- ried by the Patient 13, is described as associated with clinical ants described herein. This amino acid is the only one containing CpG MAIA ET AL. 141 F I G U R E 2 MED12 protein structure and reported ID-causing variants. (a) Variants found in this study. (b) Variants reported in females by Polla et al., 2020. (c) All pathogenic and likely pathogenic variants were reported to ClinVar as of September 19, 2021. The latter include the variants by Polla et al. M, Med12 domain. Blue dots represent the missense variants; in red the nonsense variants are given; pink dots indicate the splice site variants; and orange dots signify frameshift variants. Gene diagrams were created using lollipops v1.5.3 using UniProt ID Q93074. nucleotides in the first and second codon position (CGA, CGG, CGT, mechanism underlying the disease. For example, the data suggest that and CGC), conferring a high mutability resulting from the spontaneous when a patient shows hearing impairment, the clinician should also deamination either to TG or CA dinucleotides (Shen & Ji, 2015). Argi- look for hidden cardiac defects. Taken together, understanding the nine positive charge and structure contribute for general protein sta- molecular and phenotypic spectrum associated with a given gene bility. The replacement of an arginine residue, especially by neutral or results in a decrease of misdiagnosed patients, especially (i) in patients negatively charged residues, occurs in seven out of the nine variants. that might go underdiagnosed due to presenting clinical features not This could impair protein stability by disruption of electrostatic or sur- typically associated with these genes, or (ii) when patients carry mis- face charge–charge interactions (Strickler et al., 2006). Furthermore, classified variants, for instance by in silico predictions. DNA-binding proteins such as transcription factors may use arginine to recognize and bind specific DNA sites (Lin & Guo, 2019; Zhou & AUTHOR CONTRIBU TIONS Pang, 2018), and the loss of this residue can compromise this protein Nuno Maia and Nekane Ibarluzea were involved in the design of the function. study, the acquisition of data, the analysis and interpretation of data, In this cohort, no protein truncating variant was identified in con- and in drafting the article. Mala Misra-Isrie was involved in the con- trast with the study of Polla et al. (2020) in which 18 female patients ception and design of the study and in the analysis and interpreta- with ID carried a de novo MED12 variant. Seven of these variants tion of data. Daniel C. Koboldt was involved in the analysis and were protein truncating: two splice site changes and five nonsense interpretation of data, and in drafting the article. Isabel Marques, variants. In this study, females carrying protein truncating variants Gabriela Soares, Rosário Santos, Carlo L.M. Marcelis, Riikka Keski- clearly showed a more severe phenotype than those with missense Filppula, Miriam Guitart, Elisbeth Gabau Vila, April Lehman, Scott variants (Polla et al., 2020). This observation led us to propose that Hickey, Mari Mori, Paulien Terhal, Irene Valenzuela, Amaia Lasa-Ara- MED12 is intolerant to haploinsufficiency. Based on this assumption, nzasti, Anna Ma Cueto-González, Brian H. Chhouk, Rebecca C. Yeh, and taking into account other X-linked genes, such as OFD1, SMC1A, Jennifer E. Neil, Bassam Abu-Libde, Tjitske Kleefstra, Mariet and NSDHL, among others (Migeon, 2020), we hypothesize that pro- W. Elting, Andrea Császár, Judit Kárteszi, and Beáta Bessenyei were tein truncating variants drastically compromise the protein function, involved in the analysis and interpretation of data. Hans van Bokho- causing severe or lethal outcomes in males. Unfortunately, X- ven, Paula Jorge, and Johanna M. van Hagen were involved in the chromosome inactivation (XCI) cannot be used as an additional crite- conception and design of the study. Arjan P. M. de Brouwer was rion as we show that while a skewed XCI pattern was demonstrated involved in the conception and design of the study, the acquisition in 3 mothers, 11 showed a random XCI pattern in blood cells of data, the analysis and interpretation of data, and in drafting the (Table S1). A higher number of patients, as well as the study of other article. relevant tissues, could contribute to disclose the mechanism underly- ing XCI skewing in MED12 variants carriers. ACKNOWLEDG MENTS This study, by describing the genotype and phenotype of MED12 The authors are very grateful for the indispensable contribution that patients, gives new insights into the molecular and clinical diversity the patients and their families made for the article. For Patient 12, we associated with this gene. It highlights the importance of a detailed would like to acknowledge Muna Al Saffar, R. Sean Hill, Alisa Mo, clinical characterization for the establishment of accurate genotype– Ramzi Nasir, and Joan Stoler for their contribution to the clinical and phenotype correlations, and for disclosing the pathophysiological genetic characterization. 142 MAIA ET AL. FUND ING INFORMATION Clark, A. D., Oldenbroek, M., & Boyer, T. G. (2015). Mediator kinase mod- Unit for Multidisciplinary Research in Biomedicine (UMIB) and ITR- ule and human tumorigenesis. Critical Reviews in Biochemistry and Molecular Biology, 50(5), 393–426. https://doi.org/10.3109/ Laboratory for Integrative and Translational Research in Population 10409238.2015.1064854 Health, supported by national funds through the Foundation for Sci- Graham, J. M., Jr., Superneau, D., Rogers, R. C., Corning, K., ence and Technology (FCT) Portugal (grant numbers Schwartz, C. E., & Dykens, E. M. (1999). Clinical and behavioral charac- UIDB/00215/2020 UIDP/00215/2020 LA/P/0064/2020, respec- teristics in FG syndrome. American Journal of Medical Genetics, 85(5), 470–475. https://www.ncbi.nlm.nih.gov/pubmed/10405444 tively). The sequencing and analysis of Patient 12 was provided by the Hu, H., Haas, S. A., Chelly, J., Van Esch, H., Raynaud, M., de Brouwer, A. P., Broad Institute of MIT and Harvard Center for Mendelian Genomics Weinert, S., Froyen, G., Frints, S. G., Laumonnier, F., Zemojtel, T., (Broad CMG), which are funded by the National Human Genome Love, M. I., Richard, H., Emde, A. K., Bienek, M., Jensen, C., Research Institute, the National Eye Institute, and the National Heart, Hambrock, M., Fischer, U., Langnick, C., … Kalscheuer, V. M. (2016). X- exome sequencing of 405 unresolved families identifies seven novel Lung and Blood Institute grant UM1 HG008900 and in part by intellectual disability genes. Molecular Psychiatry, 21(1), 133–148. National Human Genome Research Institute grant R01 HG009141. https://doi.org/10.1038/mp.2014.193 Khan, S., & Vihinen, M. (2007). Spectrum of disease-causing mutations in CONF LICT OF IN TE RE ST protein secondary structures. BMC Structural Biology, 7, 56. https:// doi.org/10.1186/1472-6807-7-56 The author declares that there is no conflict of interest that could be Langley, K. G., Brown, J., Gerber, R. J., Fox, J., Friez, M. J., Lyons, M., & perceived as prejudicing the impartiality of the research reported. Schrier Vergano, S. A. (2015). 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