We had previously sequenced the exomes of individuals from four families, where at least one member was affected with MBS. We identified a few candidate genes (1-3) per family that had variants segregating with MBS. Notably, we had not identified candidate genes in common across two or more of the four families studied. Thus, it became clear that interrogating more homogeneous subgroups of affected individuals based on detailed clinical phenotyping was required given the complexity and heterogeneity of this disorder to identify common genetic and potential environmental factors. In light of this, we established a new NHGRI protocol in 2014 (14-HG-0055, ClinicalTrials.gov ID: NCT02055248; PI: Irini Manoli) dedicated to defining the phenotypes and the genetic factors associated with Moebius syndrome and other congenital facial weakness disorders. This protocol has been partially funded by a competitive UO1 grant awarded in January 2014, 1U01HD079068-03 (coPIs: Jabs, Engle, Manoli, Brooks, Pierpaoli) and a 2-year research grant from the Moebius Syndrome Foundation, awarded in January 2017. As part of the protocol, the affected individual(s) from each family undergoes standardized multisystem phenotyping at the NIH Clinical Center. We have enrolled 72 patients and 104 family members as well as 31 healthy controls for brain imaging data analysis, for a total of 207 subjects. To date, 143/207 participants have visited the NIH CC for full clinical evaluation. Initially, we prioritized families with more than one affected individual, since we believed these would provide a better chance of identifying a causative germline mutation. Since 2017, we have focused on individuals with MBS, including a subset with mirror movements. Under the collaborative agreement between NIH, MSSM and BCH, each of the three teams in the UO1 grant (NIH: Manoli/Brooks/Pierpaoli; MSSM Jabs; BCH: Engle) analyzes the clinical and genetic data. To facilitate this, we have created a REDCap database to enable sharing of the clinical phenotype information. The detailed phenotype information helps inform and direct the genomic analyses that are currently in progress. To date, we have sequenced the exomes of 148 individuals. We have identified more than 130 variants in 44 genes segregating with affection/disease status and thus, of interest for follow-up analyses. We worked with the NHGRI Secondary Genomic Findings Service (SGFS) to screen the sequence data of 73 individuals, who opted to learn secondary findings during informed consent, and found no medically actionable/reportable results to report. The Gabriela Miller Kids First Pediatric Research Program awarded resources (Dr. Engle, BCH; X01HL132377) for whole genome sequencing at the Baylor Sequencing Core of 200 individuals and their family members with MBS or hereditary congenital facial paresis (HCFP). Data generated by this funding are being analyzed in collaboration with Daniel MacArthur at the Broad Institute. Our preliminary analyses have identified non-coding variations in a genomic region that is common to several families that meet a specific subset of diagnostic criteria. Analysis of the ENCODE chromatin state data for a neuroblastoma cell line supports a potential regulatory role for this region and we are currently performing functional assays to determine its relevance in our studies. We also assessed four individuals with Carey-Fineman-Ziter syndrome enrolled through John Carey (University of Utah) and Elizabeth Engle (BCH) and in collaboration with Stephen Robertson (University of Otago, New Zealand) and Eric Olson (University of Texas Southwestern) identified myomaker (MYMK) as the causative gene and validated the functional relevance of MYMK mutations in human myoblasts and a zebrafish model system. A manuscript describing the MYMK results was published at Nature Communications. Additionally, we have identified compound heterozygous mutations in STAC3, the gene causing Native-American myopathy, in two non-Native American siblings with a CFZS-like phenotype. Brain imaging studies in patients enrolled in the clinical protocol (10 MBS, 5 HCFP and 15 controls) were analyzed by diffusion tensor-based morphometry (DTBM), a novel approach developed by co-PI Carlo Pierpaoli (NIBIB). This approach enables the identification of morphological changes of individual white matter pathways in subjects with facial palsy, as compared to healthy age and gender matched control subjects. We identified volumetric reduction in the brainstem (posterior pons, pontine tegmentum), indicating atrophy or hypoplasia of the brainstem at this level. However, HCFP did not show hypoplasia in this region. 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, including transcranial magnetic stimulation (TMS), have been performed in a subset of MBS subjects (N=7) with mirror movements (characterized as simultaneous, contralateral, involuntary movements that accompany voluntary movements on the other side) to understand the underlying pathophysiology. 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 in patients with bilateral or unilateral mirror movements. 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 interesting endophenotype may guide further analysis of their WES/WGS findings. We have obtained autopsy material of two adults with classic Moebius syndrome and a young child with Moebius syndrome and severe intellectual disability, short stature, club feet, history of ileal atresia and severe gastrointestinal dysmotility. Postmortem brain imaging and high-resolution DTI tractography findings are correlated with detailed histopathology of motor neurons in the affected regions. Small areas of calcification were identified at the predicted location of Cn VI with aberrant dorsoventral tracts near the expected Cn VI tract, that are undergoing further histopathological characterization and cross-validation between the different specimens. Blood- and brainstem-derived DNA from 2 of the 3 autopsied probands and blood DNA from their parents were used for whole genome sequencing to investigate the role of somatic mutations. We are currently analyzing these data to identify likely candidates for follow-up functional studies. We believe this collaborative approach of bringing together expert investigators from multiple sites to combine valuable resources/data, will maximize 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 516 individuals, 204 affecteds, from 169 families, as well as rich phenotypic and genotypic data that have been deposited in dbGaP (Accession ID phs001383.v1.p1.) for further exploration by other investigators interested in conditions involving facial palsy.

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Telegrafi, Aida; Webb, Bryn D; Robbins, Sarah M et al. (2017) Identification of STAC3 variants in non-Native American families with overlapping features of Carey-Fineman-Ziter syndrome and Moebius syndrome. Am J Med Genet A 173:2763-2771
Di Gioia, Silvio Alessandro; Connors, Samantha; Matsunami, Norisada et al. (2017) A defect in myoblast fusion underlies Carey-Fineman-Ziter syndrome. Nat Commun 8:16077