Multiple Congenital Anomaly Syndrome Research Articles

Novel insights into the phenotypic spectrum and pathogenesis of Hardikar syndrome

PurposeHardikar syndrome (HS, MIM #301068) is a female-specific multiple congenital anomaly syndrome characterized by retinopathy, orofacial clefting, aortic coarctation, biliary dysgenesis, genitourinary malformations, and intestinal malrotation. We previously showed that heterozygous nonsense and frameshift variants in MED12 cause HS. The phenotypic spectrum of disease and the mechanism by which MED12 variants cause disease is unknown. We aim to expand the phenotypic and molecular landscape of HS and elucidate the mechanism by which MED12 variants cause disease. MethodsWe clinically assembled and molecularly characterized a cohort of 11 previously unreported individuals with HS. Additionally, we studied the effect of MED12 deficiency on ciliary biology, hedgehog, and yes-associated protein (YAP) signaling; pathways implicated in diseases with phenotypic overlap with HS. ResultsWe report novel phenotypes associated with HS, including cardiomyopathy, arrhythmia, and vascular anomalies, and expand the molecular landscape of HS to include splice site variants. We additionally demonstrate that MED12 deficiency causes decreased cell ciliation, and impairs hedgehog and YAP signaling. ConclusionOur data support updating HS standard-of-care to include regular cardiac imaging, arrhythmia screening, and vascular imaging. We further propose that dysregulation of ciliogenesis and YAP and hedgehog signaling contributes to the pathogenesis of HS.

RASopathies - what they reveal about RAS/MAPK signaling in skeletal muscle development.

RASopathies are rare developmental genetic syndromes caused by germline pathogenic variants in genes that encode components of the RAS/mitogen-activated protein kinase (MAPK) signal transduction pathway. Although the incidence of each RASopathy syndrome is rare, collectively, they represent one of the largest groups of multiple congenital anomaly syndromes and have severe developmental consequences. Here, we review our understanding of how RAS/MAPK dysregulation in RASopathies impacts skeletal muscle development and the importance of RAS/MAPK pathway regulation for embryonic myogenesis. We also discuss the complex interactions of this pathway with other intracellular signaling pathways in the regulation of skeletal muscle development and growth, and the opportunities that RASopathy animal models provide for exploring the use of pathway inhibitors, typically used for cancer treatment, to correct the unique skeletal myopathy caused by the dysregulation of this pathway.

Prenatal Diagnosis of Fryns Syndrome through Identification of Two Novel Splice Variants in the PIGN Gene-A Case Series.

Fryns syndrome (FS) is a multiple congenital anomaly syndrome with different multisystemic malformations. These include congenital diaphragmatic hernia, pulmonary hypoplasia, and craniofacial dysmorphic features in combination with malformations of the central nervous system such as agenesis of the corpus callosum, cerebellar hypoplasia, and enlarged ventricles. We present a non-consanguineous northern European family with two recurrent cases of FS: a boy with multiple congenital malformations who died at the age of 2.5 months and a female fetus with a complex developmental disorder with similar features in a following pregnancy. Quad whole exome analysis revealed two likely splicing-affecting disease-causing mutations in the PIGN gene: a synonymous mutation c.2619G>A, p.(Leu873=) in the last nucleotide of exon 29 and a 30 bp-deletion c.996_1023+2del (NM_176787.5) protruding into intron 12, with both mutations in trans configuration in the affected patients. Exon skipping resulting from these two variants was confirmed via RNA sequencing. Our molecular and clinical findings identified compound heterozygosity for two novel splice-affecting variants as the underlying pathomechanism for the development of FS in two patients.

A pilot study about multiple congenital ocular anomalies in trait Comtois horse breed

The Silver gene is at the origin of the special coat color of the Trait Comtois Horse breed, but also predisposes carriers to Multiple Congenital Ocular Anomalies syndrome.
 The objective of this pilot study was to evaluate the awareness and knowledge of Comtois owners about this topic through their response to a questionnaire. After completing it, we offered documentation for them to keep and to refer to when they feel the need.
 The majority of the owners were aware about the gene, but still many are confused with its issues. Genetic testing is not systematically done when ocular problems are noticed, but willingness to pay more attention to genotypes is present in the common spirit. By developing a proper reproduction management and a proper genotyping of the Comtois horse breed population, conservation and enhancement of the breed will be possible. In this regard, Silver homozygous equines should not be discriminated, as they may possess desirable traits and may be able to perform as any other horse in their domain of activity. They should instead be encouraged to be mated with their bay peers, in order to obtain heterozygous foals. Genetic testing campaigns are increasingly offered during competitions and open to all categories of horses.
 This problematic should not divide breeders, but rather motivate them to work altogether to be able to perpetuate the Trait Comtois horse breed, flagship of the Franche-Comté region.

Clinical and molecular analysis of Guangxi patients with Kabuki syndrome and KMT2D mutations

Kabuki syndrome (KS) is a multiple congenital anomaly syndrome that is characterized by postnatal growth deficiency, hypotonia, short stature, mild-to-moderate intellectual disability, skeletal abnormalities, persistence of fetal fingertip pads, and distinct facial appearance. It is mainly caused by pathogenic/likely pathogenic variants in the KMT2D or KDM6A genes. Here, we described the clinical features of nine sporadic KS patients with considerable phenotypic heterogeneity. In addition to intellectual disability and short stature, our patients presented with a high prevalence of motor retardation and recurrent otitis media. We recommended that KS should be strongly considered in patients with motor delay, short stature, intellectual disability, language disorder and facial deformities. Nine KMT2D variants, four of which were novel, were identified by whole-exome sequencing. The variants included five nonsense variants, two frameshift variants, one missense variant, and one non-canonical splice site variant. In addition, we reviewed the mutation types of the pathogenic KMT2D variants in the ClinVar database. We also indicated that effective mRNA analysis, using biological materials from patients, is helpful in classifying the pathogenicity of atypical splice site variants. Pedigree segregation analysis may also provide valuable information for pathogenicity classification of novel missense variants. These findings extended the mutation spectrum of KMT2D and provided new insights into the understanding of genotype-phenotype correlations, which are helpful for accurate genetic counseling and treatment optimization.

Heterozygous rare variants in NR2F2 cause a recognizable multiple congenital anomaly syndrome with developmental delays.

Nuclear receptor subfamily 2 group F member 2 (NR2F2 or COUP-TF2) encodes a transcription factor which is expressed at high levels during mammalian development. Rare heterozygous Mendelian variants in NR2F2 were initially identified in individuals with congenital heart disease (CHD), then subsequently in cohorts of congenital diaphragmatic hernia (CDH) and 46,XX ovotesticular disorders/differences of sexual development (DSD); however, the phenotypic spectrum associated with pathogenic variants in NR2F2 remains poorly characterized. Currently, less than 40 individuals with heterozygous pathogenic variants in NR2F2 have been reported. Here, we review the clinical and molecular details of 17 previously unreported individuals with rare heterozygous NR2F2 variants, the majority of which were de novo. Clinical features were variable, including intrauterine growth restriction (IUGR), CHD, CDH, genital anomalies, DSD, developmental delays, hypotonia, feeding difficulties, failure to thrive, congenital and acquired microcephaly, dysmorphic facial features, renal failure, hearing loss, strabismus, asplenia, and vascular malformations, thus expanding the phenotypic spectrum associated with NR2F2 variants. The variants seen were predicted loss of function, including a nonsense variant inherited from a mildly affected mosaic mother, missense and a large deletion including the NR2F2 gene. Our study presents evidence for rare, heterozygous NR2F2 variants causing a highly variable syndrome of congenital anomalies, commonly associated with heart defects, developmental delays/intellectual disability, dysmorphic features, feeding difficulties, hypotonia, and genital anomalies. Based on the new and previous cases, we provide clinical recommendations for evaluating individuals diagnosed with an NR2F2-associated disorder.

Monoallelic loss-of-function BMP2 variants result in BMP2-related skeletal dysplasia spectrum

PurposeBone morphogenic proteins (BMPs) regulate gene expression that is related to many critical developmental processes, including osteogenesis for which they are named. In addition, BMP2 is widely expressed in cells of mesenchymal origin, including bone, cartilage, skeletal and cardiac muscle, and adipose tissue. It also participates in neurodevelopment by inducing differentiation of neural stem cells. In humans, BMP2 variants result in a multiple congenital anomaly syndrome through a haploinsufficiency mechanism. We sought to expand the phenotypic spectrum and highlight phenotypes of patients harboring monoallelic missense variants in BMP2. MethodsWe used retrospective chart review to examine phenotypes from an international cohort of 18 individuals and compared these with published cases. Patient-derived missense variants were modeled in zebrafish to examine their effect on the ability of bmp2b to promote embryonic ventralization. ResultsThe presented cases recapitulated existing descriptions of BMP2-related disorders, including craniofacial, cardiac, and skeletal anomalies and exhibit a wide phenotypic spectrum. We also identified patients with neural tube defects, structural brain anomalies, and endocrinopathies. Missense variants modeled in zebrafish resulted in loss of protein function. ConclusionWe use this expansion of reported phenotypes to suggest multidisciplinary medical monitoring and management of patients with BMP2-related skeletal dysplasia spectrum.

ALDH1A2-related disorder: A new genetic syndrome due to alteration of the retinoic acid pathway.

Aldehyde Dehydrogenase 1, Family Member A2 (ALDH1A2) is essential for the synthesis of retinoic acid from vitamin A. Studies in model organisms demonstrate a critical role for ALDH1A2 in embryonic development, yet few pathogenic variants are linked to congenital anomalies in humans. We present three siblings with multiple congenital anomaly syndrome linked to biallelic sequence variants in ALDH1A2. The major congenital malformations affecting these children include tetralogy of Fallot, absent thymus, diaphragmatic eventration, and talipes equinovarus. Upper airway anomalies, hypocalcemia, and dysmorphic features are newly reported in this manuscript. In vitro functional validation of variants indicated that substitutions reduced the expression of the enzyme. Our clinical and functional data adds to a recent report of biallelic ALDH1A2 pathogenic variants in two families with a similar constellation of congenital malformations. These findings provide further evidence for an autosomal recessive ALDH1A2-deficient recognizable malformation syndrome involving the diaphragm, cardiac and musculoskeletal systems.

Establishment of a human induced pluripotent stem cell line, KMUGMCi002-A, from a patient bearing a heterozygous c.6362_6364del mutation in the NIPBL gene leading Cornelia de Lange syndrome (CdLS)

Cornelia de Lange syndrome (CdLS) is a multiple congenital anomalies syndrome caused by mutations in the cohesion complex. The mutations in NIPBL, one of cohesion regulatory proteins, are the most frequent cause of CdLS. The peripheral blood mononuclear cells (PBMCs) from a patient carrying a heterozygous 3 bp deletion in Exon 37 of the NIPBL gene were reprogrammed using the CytoTune-iPS2.0 Sendai Reprogramming Kit. The deleted mutation in NIPBL will cause the abnormal truncated protein, which is known to associated with CdLS. The established human induced pluripotent cell (hiPSC) line will enable proper in vitro disease modelling of CdLS.Resource Table:

Expanding the novel MAPKAPK5-related developmental disorder's genotype-phenotype correlation: Patient report and 19months of follow-up.

This study aimed to widen the knowledge of a recently identified, autosomal‐recessive, multiple congenital anomalies syndrome to date observed in only other three children. This is the second report of biallelic mutations in MAPKAPK5 whose impairment during human development has been associated with neurological, cardiac, and facial anomalies combined with fingers and toes malformations. Through the affected patients' genetic and phenotypic features overlap, this report confirms MAPKAPK5 as causative gene and adds unique neurodevelopmental characterization. Moreover, based on the complex congenital genitourinary anomalies reported and MAPKAPK5 literature review, we also propose kidney and external genitalia involvement as a key syndromic feature whose expressivity may be more severe in males.

EP404: Evidence supporting the pathogenicity of the NADSYN1 c.1717G>A (p.Ala573Thr) variant in individuals referred for clinical testing

Vertebral, cardiac, renal, and limb defects syndrome 3 (VCRL3; OMIM #618845) is an autosomal recessive multiple congenital anomaly syndrome caused by pathogenic variants in the NADSYN1 gene. NADSYN1 encodes a nicotinamide adenine dinucleotide (NAD) synthetase that is part of the de novo NAD synthesis pathway. Other genes involved in this pathway include HAAO and KYNU. Genetic variants in any of these genes result in reduced de novo NAD synthesis and cause congenital anomalies of the heart, kidney, and vertebrae. Of note, presence of these three features is also considered clinical evidence for vertebral defects, anal atresia, cardiac defects, tracheo-esophageal fistula, renal anomalies, and limb abnormalities (VACTERL) association, which is often a diagnosis of exclusion (ie, other genetic conditions with overlapping features must be ruled out before assigning this diagnosis). The recommended approach for genetic testing in possible VACTERL patients is to either pursue a comprehensive testing option, such as exome or genome sequencing, or a panel approach with the most likely candidates. The NADSYN1 gene is included on a VACTERL Association and Related Disorders panel and several patients were noted to be homozygous for the NADSYN1 c.1717G>A (p.Ala573Thr) variant, thus providing additional evidence for both the pathogenicity and high carrier frequency of this change. To date, we have identified five patients who are homozygous for the NADSYN1 (NM_018161.4) c.1717G>A (p.Ala573Thr) variant. One patient was identified through targeted testing, one through testing with the VACTERL Association and Related Disorders panel, and three through exome sequencing. The first four cases were from pregnancies evaluated for the differential diagnosis of VACTERL association via panel testing or exome sequencing. All four fetuses were reported to have cardiac malformation and segmental vertebral anomalies. Parental data was available for three of the four fetuses, and all parents tested were heterozygous for the NADSYN1 c.1717G>A (p.Ala573Thr) variant. All cases denied consanguinity. In contrast to the severe and early onset features of the other four patients identified, the targeted testing case was an adult patient who previously had the NADSYN1 c.1717G>A variant identified in a research study. This individual was noted to have cardiac defects, vertebral defects, and short stature. This variant is relatively common in non-Finnish Europeans with an allele frequency of 0.15% in the gnomAD v2 dataset. Five patients from four families with VCRL3 have been described in the literature. Of these patients, three had the c.1717G>A variant: two homozygous siblings and one compound heterozygote with a loss of function variant on the opposite allele. Parental sequencing data confirmed the biallelic variants in all reported patients. In addition, yeast model functional studies performed by Szot and colleagues demonstrated that the p.Ala573Thr change diminished NAD synthetase activity and reduced yeast viability, consistent with a loss of function mechanism. Taken together, although the NADSYN1 c.1717G>A (p.Ala573Thr) has a relatively high minor allele frequency, there is sufficient evidence for this variant to be classified as pathogenic using ACMG/AMP variant classifications and internal variant interpretation guidelines. The NADSYN1 c.1717G>A (p.Ala573Thr) variant should be interpreted as pathogenic given the 5 additional homozygous patients and parental segregation data presented here. Additionally, the diagnosis of VCRL3 in an adult patient suggests that although penetrance for NAD deficiency is complete, variable expressivity is observed and could be explained by other genetic or non-genetic factors modulating disease severity. Given the high population frequency of this variant, genetic testing for VACTERL association should always include evaluation of NADSYN1 to consider VCRL3 in the differential diagnosis.

A case of Gross E esophageal atresia discovered following a unique clinical course

The patient was a 15 months-old boy who had been diagnosed CHARGE syndrome, which is a multiple congenital anomaly syndrome caused by mutations in the CHD7 gene. Mechanical ventilation management was initiated 2 hours after birth for dysphagia and respiratory failure, and tracheotomy was performed 3 months after birth for dysphagia and failed extubation. He was repeatedly hospitalized due to pneuomoniae. Approximately 1 year after birth, the boy had two consecutive episodes of sudden ventilatory insufficiency while replacing the tracheotomy cannula. A bronchoscopic examination under general anesthesia revealed a tracheoesophageal fistula directly below the tracheostomy. The patient was diagnosed with Gross E esophageal atresia, and we speculated that the cannula migrated to the esophagus via the fistula during tracheostomy cannula replacement. Gross E esophageal atresia is a rare disease. Its diagnosis is often delayed, and it is discovered by recurrent pneumonia in many cases. A tracheoesophageal fistula may also be found in children with deformities of the respiratory system. Furthermore, tracheoesophageal fistulae are often found in the neck. Therefore, when sudden ventilatory insufficiency occurs in a child with a tracheostomy after replacing the tracheostomy cannula, caution must be exercised since the cannula may have migrated to the esophagus via a fistula. J. Med. Invest. 69 : 141-144, February, 2022.

Metabolomics in Clinical Practice: Improving Diagnosis and Informing Management.

Metabolomics is the study of small molecules to simultaneously identify multiple low molecular weight molecules in a system. Broadly speaking, metabolomics can be subdivided into targeted and untargeted types of analysis, each type having advantages and drawbacks. Targeted metabolomics can quantify analytes but only looks for known or expected analytes related to particular disease(s), whereas untargeted metabolomics is typically nonquantitative but can detect thousands of analytes from an agnostic or nonhypothesis driven perspective, allowing for novel discoveries. One application of metabolomics is the study of inborn errors of metabolism (IEM). The biochemical hallmark of IEMs is decreased concentrations of analytes distal to the enzymatic defect and buildup of analytes proximal to the defect. Metabolomics can detect these changes with one test and is effective in screening for and diagnosis of IEMs. Metabolomics has also been used to study many nonmetabolic diseases such as autism spectrum disorder, various cancers, and multiple congenital anomalies syndromes. Metabolomics has led to the discovery of many novel biomarkers of disease. Recent publications demonstrate how metabolomics can be useful clinically in the diagnosis and management of patients, as well as for research and clinical discovery. Metabolomics has proved to be a useful tool clinically for screening and diagnostic purposes and from a research perspective for the detection of novel biomarkers. In the future, metabolomics will likely become a routine part of the evaluation for many diseases as either a supplementary test or it may simply replace historical analyses that require several individual tests and sample types.

BRANCHIO-OTO-RENAL (BOR) SYNDROME

Introduction. This work is devoted to the problem of embryology of the ears and kidneys. First ear and kidney abnormalities were reported in 1946 by Edith Potter’s association of crumpled and flattened ears with bilateral kidney agenesis. Ear malformations are associated with an increased frequency of clinically significant structural renal anomalies compared with the general population. These include specific multiple congenital anomaly syndromes, Townes-Brocks syndrome, branchio-oto-renal syndrome.The link can be explained by structural and functional similarities between tissues in the inner ear and in the kidney. Also, toxins that accumulate in kidney failure can damage nerves, including those in the inner ear.Goal. To study the causes, clinical manifestations of Branchio-oto-renal (BOR) syndrome. Materials and Methods. Review of modern and foreign literary sources; methods - description, analysis, abstracting.Results and discussion. Mutations in three genes, EYA1, SIX1, and SIX5, have been reported in people with BOR/BO syndrome. About 40 percent of people with this condition have a mutation in the EYA1 gene. SIX1 gene mutations are a much less common cause of the disorder. SIX5 gene mutations have been found in a small number of people with BOR syndrome, although researchers question whether mutations in this gene cause the condition. Some affected individuals originally reported to have SIX5 gene mutations were later found to have EYA1 gene mutations as well, and researchers suspect that the EYA1 gene mutations may be the actual cause of the condition in these people.The proteins produced from the EYA1, SIX1, and SIX5 genes play important roles in development before birth. The EYA1 protein interacts with several other proteins, including SIX1 and SIX5, to regulate the activity of genes involved in many aspects of embryonic development. Research suggests that these protein interactions are essential for the normal formation of many organs and tissues, including the second branchial arch, ears, and kidneys. Mutations in the EYA1, SIX1, or SIX5 gene may disrupt the proteins’ ability to interact with one another and regulate gene activity.Conclusions. The link between ear anomalies and kidney function can be explained by structural and functional similarities between tissues in the inner ear and in the kidney. Additionally, toxins that accumulate in kidney failure can damage nerves, including those in the inner ear.

Diaphragmatic Hernia as a Prenatal Feature of Glycosylphosphatidylinositol Biosynthesis Defects and the Overlap With Fryns Syndrome - Literature Review.

Fryns syndrome is an autosomal recessive multiple congenital anomaly syndrome, with diaphragmatic defects and secondary lung hypoplasia as cardinal features. Despite it was reported first in 1979, its exact etiology has not been established to date. With this review, we would like to draw attention to the prenatal presentation of multiple congenital anomalies syndromes, resulting from defects in the synthesis of glycosylphosphatidylinositol anchors, to be considered in a prenatal assessment of fetuses with DH and Fryns-like phenotype.

Genome-wide copy number variations in a large cohort of bantu African children

BackgroundCopy number variations (CNVs) account for a substantial proportion of inter-individual genomic variation. However, a majority of genomic variation studies have focused on single-nucleotide variations (SNVs), with limited genome-wide analysis of CNVs in large cohorts, especially in populations that are under-represented in genetic studies including people of African descent.MethodsWe carried out a genome-wide copy number analysis in > 3400 healthy Bantu Africans from Tanzania. Signal intensity data from high density (> 2.5 million probes) genotyping arrays were used for CNV calling with three algorithms including PennCNV, DNAcopy and VanillaICE. Stringent quality metrics and filtering criteria were applied to obtain high confidence CNVs.ResultsWe identified over 400,000 CNVs larger than 1 kilobase (kb), for an average of 120 CNVs (SE = 2.57) per individual. We detected 866 large CNVs (≥ 300 kb), some of which overlapped genomic regions previously associated with multiple congenital anomaly syndromes, including Prader-Willi/Angelman syndrome (Type1) and 22q11.2 deletion syndrome. Furthermore, several of the common CNVs seen in our cohort (≥ 5%) overlap genes previously associated with developmental disorders.ConclusionsThese findings may help refine the phenotypic outcomes and penetrance of variations affecting genes and genomic regions previously implicated in diseases. Our study provides one of the largest datasets of CNVs from individuals of African ancestry, enabling improved clinical evaluation and disease association of CNVs observed in research and clinical studies in African populations.

Biallelic hypomorphic variants in ALDH1A2 cause a novel lethal human multiple congenital anomaly syndrome encompassing diaphragmatic, pulmonary, and cardiovascular defects.

This study shows a causal association between ALDH1A2 variants and a novel, severe multiple congenital anomaly syndrome in humans that is neonatally lethal due to associated pulmonary hypoplasia and respiratory failure. In two families, exome sequencing identified compound heterozygous missense variants in ALDH1A2. ALDH1A2 is involved in the conversion of retinol (vitamin A) into retinoic acid (RA), which is an essential regulator of diaphragm and cardiovascular formation during embryogenesis. Reduced RA causes cardiovascular, diaphragmatic, and associated pulmonary defects in several animal models, matching the phenotype observed in our patients. In silico protein modeling showed probable impairment of ALDH1A2 for three of the four substitutions. In vitro studies show a reduction of RA. Few pathogenic variants in genes encoding components of the retinoic signaling pathway have been described to date, likely due to embryonic lethality. Thus, this study contributes significantly to knowledge of the role of this pathway in human diaphragm and cardiovascular development and disease. Some clinical features in our patients are also observed in Fryns syndrome (MIM# 229850), syndromic microphthalmia 9 (MIM# 601186), and DiGeorge syndrome (MIM# 188400). Patients with similar clinical features who are genetically undiagnosed should be tested for recessive ALDH1A2-deficient malformation syndrome.

Biallelic truncating variants in MAPKAPK5 cause a new developmental disorder involving neurological, cardiac, and facial anomalies combined with synpolydactyly

This study aimed to identify the genetic cause of a new multiple congenital anomalies syndrome observed in three individuals from two unrelated families. Clinical assessment was conducted prenatally and at different postnatal stages. Genetic studies included exome sequencing (ES) combined with single-nucleotide polymorphism (SNP) array based homozygosity mapping and trio ES. Dermal fibroblasts were used for functional assays. A clinically recognizable syndrome characterized by severe developmental delay, variable brain anomalies, congenital heart defects, dysmorphic facial features, and a distinctive type of synpolydactyly with an additional hypoplastic digit between the fourth and fifth digits of hands and/or feet was identified. Additional features included eye abnormalities, hearing impairment, and electroencephalogram anomalies. ES detected different homozygous truncating variants in MAPKAPK5 in both families. Patient-derived cells showed no expression of MAPKAPK5 protein isoforms and reduced levels of the MAPKAPK5-interacting protein ERK3. F-actin recovery after latrunculin B treatment was found to be less efficient in patient-derived fibroblasts than in control cells, supporting a role of MAPKAPK5 in F-actin polymerization. Our data indicate that loss-of-function variants in MAPKAPK5 result in a severe developmental disorder and reveal a major role of this gene in human brain, heart, and limb development.

De novo loss-of-function variants in X-linked MED12 are associated with Hardikar syndrome in females

Hardikar syndrome (MIM 612726) is a rare multiple congenital anomaly syndrome characterized by facial clefting, pigmentary retinopathy, biliary anomalies, and intestinal malrotation, but with preserved cognition. Only four patients have been reported previously, and none had a molecular diagnosis. Our objective was to identify the genetic basis of Hardikar syndrome (HS) and expand the phenotypic spectrum of this disorder. We performed exome sequencing on two previously reported and five unpublished female patients with a clinical diagnosis of HS. X-chromosome inactivation (XCI) studies were also performed. We report clinical features of HS with previously undescribed phenotypes, including a fatal unprovoked intracranial hemorrhage at age 21. We additionally report the discovery of de novo pathogenic nonsense and frameshift variants in MED12 in these seven individuals and evidence of extremely skewed XCI in all patients with informative testing. Pathogenic missense variants in the X-chromosome gene MED12 have previously been associated with Opitz-Kaveggia syndrome, Lujan syndrome, Ohdo syndrome, and nonsyndromic intellectual disability, primarily in males. We propose a fifth, female-specific phenotype for MED12, and suggest that nonsense and frameshift loss-of-function MED12 variants in females cause HS. This expands the MED12-associated phenotype in females beyond intellectual disability.

Ras/MAPK dysregulation in development causes a skeletal myopathy in an activating BrafL597V mouse model for cardio-facio-cutaneous syndrome.

Cardio-facio-cutaneous (CFC) syndrome is a human multiple congenital anomaly syndrome that is caused by activating heterozygous mutations in either BRAF, MEK1, or MEK2, three protein kinases of the Ras/mitogen-activated protein kinase (MAPK) pathway. CFC belongs to a group of syndromes known as RASopathies. Skeletal muscle hypotonia is a ubiquitous phenotype of RASopathies, especially in CFC syndrome. To better understand the underlying mechanisms for the skeletal myopathy in CFC, a mouse model with an activating BrafL597V allele was utilized. The activating BrafL597V allele resulted in phenotypic alterations in skeletal muscle characterized by a reduction in fiber size which leads to a reduction in muscle size which are functionally weaker. MAPK pathway activation caused inhibition of myofiber differentiation during embryonic myogenesis and global transcriptional dysregulation of developmental pathways. Inhibition in differentiation can be rescued by MEK inhibition. A skeletal myopathy was identified in the CFC BrafL597V mouse validating the use of models to study the effect of Ras/MAPK dysregulation on skeletal myogenesis. RASopathies present a novel opportunity to identify new paradigms of myogenesis and further our understanding of Ras in development. Rescue of the phenotype by inhibitors may help advance the development of therapeutic options for RASopathy patients.