Genetics of Moebius syndrome and other congenital facial weakness disorders

National Human Genome Research Institute

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Abstract

In 2014, we established a NHGRI protocol (14-HG-0055, ClinicalTrials.gov ID: NCT02055248; PI: Eirini Manoli) dedicated to defining the phenotypes and the genetic factors associated with MBS and other congenital facial weakness disorders. This protocol was partially funded by a 2014 UO1 grant 1U01HD079068-03 (coPIs: Jabs, Engle, Manoli, Brooks, Pierpaoli) and a 2-year research grant from the Moebius Syndrome Foundation, awarded in January 2017. Under this protocol, affected individual(s) underwent a standardized multisystem phenotyping at the NIH Clinical Research Center (NIH CRC). We enrolled 80 probands and 96 family members as well as 31 healthy controls for brain imaging data analysis, for a total of 207 subjects. Detailed clinical evaluations of 143/207 participants have been completed at the NIH CRC. As of June 2019, the clinical protocol is open only for data analysis. Analysis of the clinical and genetic data is performed by each of the three UO1 teams. We created a database to share clinical phenotypes across the three sites. We have sequenced the exomes of 148 individuals, including 37 proband-parent trios and the genomes of 18 individuals (including 5 trios). We have also genotyped 150 individuals, including 27 family trios. By jointly analyzing 148 exomes sequenced at the NIH and 142 whole genomes sequenced by our collaborator (Engle, BCH), we identified 445,980 variants (SNPs/Indels), including some in the coding region of several candidate genes for facial palsy. We prioritized candidate variants for follow-up based on allele frequencies and segregation with affection/disease status. We submitted our top candidates to GeneMatcher and are assessing genotype and phenotype data to further prioritize candidates for functional analysis. No recurrent variants have been observed in more than one family in subjects with sporadic Moebius syndrome. To investigate the role of somatic mutations, whole genome sequencing was performed from blood-derived DNA from proband/parent trios, as well as for brainstem-derived DNA from two unrelated autopsies of MBS probands. We obtained autopsy material from three individuals, two with classic and one with complex Moebius syndrome. Two full-body research autopsies were performed at the NIH CRC. Postmortem brain imaging and high-resolution DTI and tractography findings were correlated with detailed histopathology of motor neurons in the affected regions. Small areas of calcification (not observed in brain MRI or CT) were identified at the predicted location of Cn VI, with gliosis in the region of Cn VII. Aberrant dorsoventral tracts were documented near the expected Cn VI tract. The case of complex Moebius was a younger child with Moebius syndrome associated with severe intellectual disability, short stature, club feet, history of ileal atresia and severe gastrointestinal dysmotility/recurrent obstructions requiring multiple surgeries. The Gabriella Miller Kids First Pediatric Research Program awarded resources (Dr. Engle, BCH) for whole genome sequencing at the Baylor Sequencing Core of 142 individuals and their family members with MBS or hereditary congenital facial paresis (HCFP). Our preliminary analyses identified six non-coding single nucleotide variants in a conserved cis-regulatory element in seven families and two overlapping tandem duplications in two families with hereditary congenital facial palsy (HCFP1), for a total of 34 mutation-positive individuals. Analysis of the ENCODE chromatin state data for a neuroblastoma cell line supported a potential regulatory role for this region and follow-up functional studies suggests the presence of a cis-acting silencer operating in conjunction with nearby enhancers to regulate cell-type specific expression of GATA2. The mutated cis-regulatory element no longer binds NR2F1 (COUP-TF1) or functions as a silencer, resulting in a disruption of the developmental switch of neuronal progenitors from the inner ear efferent neurons (IEEs) to facial branchial motor neurons in rhombomere 4 early in embryonic development. Two mouse models related to this work were developed by the Boston and Mt Sinai groups and phenotyping efforts are ongoing. We are currently completing additional experiments suggested by the reviewers of our submission to Nature Genetics. We also assessed four individuals with Carey-Fineman-Ziter syndrome enrolled through J. Carey (University of Utah) and E. Engle (BCH) with variants in a novel gene (myomaker, MYMK) and in collaboration with S. Robertson (University of Otago, NZ) and E. Olson (University of Texas Southwestern) confirmed the functional relevance of these mutations in human myoblasts and the zebrafish model system. We have published the results for MYMK. Brain imaging studies in patients enrolled in the clinical protocol (21 MBS, 9 CFW and 15 controls) were analyzed by tensor-based morphometry (TBM), developed by co-PI Carlo Pierpaoli (NIBIB). TBM enables the identification of morphological changes in individual white matter pathways in subjects with facial palsy. We identified a small region of highly significant volumetric reduction in brainstem structures important for the initiation and coordination of conjugate horizontal gaze, indicating atrophy or hypoplasia of the brainstem at this level. However, HCFP or subjects with undefined myopathies causing facial weakness and ophthalmoplegia did not show hypoplasia in this region. Moreover, the size of this volumetric abnormality correlated with a clinical severity score reflecting the disease burden for the subjects. This study was published in Brain Communications. Functional MRI studies are underway with collaborators at NIMH (Japee Shruti and Leslie Ungerleider) to understand differences in emotion processing in MBS subjects compared to controls. Findings to date suggest there are deficits in fearful emotion processing in subjects with MBS, that are accompanied by reduced connectivity in CNS regions that process expressions such as pSTS (posterior superior temporal sulcus) and amygdala. Lastly, fMRI, DTI and electrophysiology studies, have been performed for seven MBS subjects with mirror movements (characterized as simultaneous, contralateral, involuntary movements that accompany voluntary movements on the other side). TMS and fMRI studies identified three patient groups with bilateral, unilateral and no mirror movements. Ipsilateral motor evoked and somatosensory evoked potentials, and decreased interhemispheric inhibition were observed on both sides in patients with bilateral mirror movement. DTI findings showed decreased or absent pyramidal decussation at the level of the medulla. We conclude that a certain portion of the motor and sensory pathways do not decussate in Moebius syndrome subjects with mirror movement, suggesting abnormalities in neuronal guidance outside the cranial nerve VI and VII region in the brainstem. This collaborative approach across multiple investigators/sites enables the use of joint resources and maximizes our capacity to identify germline or somatic genetic causes of the various syndromes associated with facial palsy. Moreover, this collaboration has allowed the generation of a patient registry/clinical database in REDCap, including a total of 584 individuals, 210 affecteds, from 203 families, including 17 multiplex families, as well as rich phenotypic and genotypic data in 487 individuals that has been deposited in dbGaP for use by other investigators. Several clinical manuscripts are currently in preparation from the deep phenotyping effort at the NIH CRC with colleagues from different principles (dental/craniofacial, audiology, neuropsychology, socioehavioral), while we collectively work on a paper summarizing the clinical phenotype and WES/WGS for the entire project.