Gene identification for ocular congenital cranial motor neuron disorders using human sequencing, zebrafish screening, and protein binding microarrays.

Purpose: To functionally evaluate novel human sequence-derived candidate genes and variants for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs). Methods: Through exome and genome sequencing of a genetically unsolved human oCCDD cohort, we previously identified variants in 80 strong candidate genes. Here, we further prioritized a subset of these (43 human genes, 57 zebrafish genes) using a G0 CRISPR/Cas9-based knockout assay in zebrafish and generated F2 germline mutants for seventeen. We tested the functionality of variants of uncertain significance in known and novel candidate transcription factor-encoding genes through protein binding microarrays. Results: We first demonstrated the feasibility of the G0 screen by targeting known oCCDD genes phox2a and mafba. 70-90% of gene-targeted G0 zebrafish embryos recapitulated germline homozygous null-equivalent phenotypes. Using this approach, we then identified three novel candidate oCCDD genes (SEMA3F, OLIG2, and FRMD4B) with putative contributions to human and zebrafish cranial motor development. In addition, protein binding microarrays demonstrated reduced or abolished DNA binding of human variants of uncertain significance in known and novel sequence-derived transcription factors PHOX2A (p.(Trp137Cys)), MAFB (p.(Glu223Lys)), and OLIG2 (p.(Arg156Leu)). Conclusions: This study nominates three strong novel candidate oCCDD genes (SEMA3F, OLIG2, and FRMD4B) and supports the functionality and putative pathogenicity of transcription factor candidate variants PHOX2A p.(Trp137Cys), MAFB p.(Glu223Lys), and OLIG2 p.(Arg156Leu). Our findings support that G0 loss-of-function screening in zebrafish can be coupled with human sequence analysis and protein binding microarrays to aid in prioritizing oCCDD candidate genes/variants.

Chromosomal structural rearrangements implicate long non-coding RNAs in rare germline disorders.

In recent years, there has been increased focus on exploring the role the non-protein-coding genome plays in Mendelian disorders. One class of particular interest is long non-coding RNAs (lncRNAs), which has recently been implicated in the regulation of diverse molecular processes. However, because lncRNAs do not encode protein, there is uncertainty regarding what constitutes a pathogenic lncRNA variant, and thus annotating such elements is challenging. The Developmental Genome Anatomy Project (DGAP) and similar projects recruit individuals with apparently balanced chromosomal abnormalities (BCAs) that disrupt or dysregulate genes in order to annotate the human genome. We hypothesized that rearrangements disrupting lncRNAs could be the underlying genetic etiology for the phenotypes of a subset of these individuals. Thus, we assessed 279 cases with BCAs and selected 191 cases with simple BCAs (breakpoints at only two genomic locations) for further analysis of lncRNA disruptions. From these, we identified 66 cases in which the chromosomal rearrangements directly disrupt lncRNAs. In 30 cases, no genes of any other class aside from lncRNAs are directly disrupted, consistent with the hypothesis that lncRNA disruptions could underly the phenotypes of these individuals. Strikingly, the lncRNAs MEF2C-AS1 and ENSG00000257522 are each disrupted in two unrelated cases. Furthermore, we experimentally tested the lncRNAs TBX2-AS1 and MEF2C-AS1 and found that knockdown of these lncRNAs resulted in decreased expression of the neighboring transcription factors TBX2 and MEF2C, respectively. To showcase the power of this genomic approach for annotating lncRNAs, here we focus on clinical reports and genetic analysis of seven individuals with likely developmental etiologies due to lncRNA disruptions.

Expanding the genetics and phenotypes of ocular congenital cranial dysinnervation disorders.

PURPOSE: To identify genetic etiologies and genotype/phenotype associations for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs). METHODS: We coupled phenotyping with exome or genome sequencing of 467 probands (550 affected and 1108 total individuals) with genetically unsolved oCCDDs, integrating analyses of pedigrees, human and animal model phenotypes, and de novo variants to identify rare candidate single nucleotide variants, insertion/deletions, and structural variants disrupting protein-coding regions. Prioritized variants were classified for pathogenicity and evaluated for genotype/phenotype correlations. RESULTS: Analyses elucidated phenotypic subgroups, identified pathogenic/likely pathogenic variant(s) in 43/467 probands (9.2%), and prioritized variants of uncertain significance in 70/467 additional probands (15.0%). These included known and novel variants in established oCCDD genes, genes associated with syndromes that sometimes include oCCDDs (e.g., MYH10, KIF21B, TGFBR2, TUBB6), genes that fit the syndromic component of the phenotype but had no prior oCCDD association (e.g., CDK13, TGFB2), genes with no reported association with oCCDDs or the syndromic phenotypes (e.g., TUBA4A, KIF5C, CTNNA1, KLB, FGF21), and genes associated with oCCDD phenocopies that had resulted in misdiagnoses. CONCLUSION: This study suggests that unsolved oCCDDs are clinically and genetically heterogeneous disorders often overlapping other Mendelian conditions and nominates many candidates for future replication and functional studies.

Rare germline disorders implicate long non-coding RNAs disrupted by chromosomal structural rearrangements.

In recent years, there has been increased focus on exploring the role the non-protein-coding genome plays in Mendelian disorders. One class of particular interest is long non-coding RNAs (lncRNAs), which has recently been implicated in the regulation of diverse molecular processes. However, because lncRNAs do not encode protein, there is uncertainty regarding what constitutes a pathogenic lncRNA variant, and thus annotating such elements is challenging. The Developmental Genome Anatomy Project (DGAP) and similar projects recruit individuals with apparently balanced chromosomal abnormalities (BCAs) that disrupt or dysregulate genes in order to annotate the human genome. We hypothesized that rearrangements disrupting lncRNAs could be the underlying genetic etiology for the phenotypes of a subset of these individuals. Thus, we assessed 279 cases with BCAs and selected 191 cases with simple BCAs (breakpoints at only two genomic locations) for further analysis of lncRNA disruptions. From these, we identified 66 cases in which the chromosomal rearrangements directly disrupt lncRNAs. Strikingly, the lncRNAs MEF2C-AS1 and ENSG00000257522 are each disrupted in two unrelated cases. Furthermore, in 30 cases, no genes of any other class aside from lncRNAs are directly disrupted, consistent with the hypothesis that lncRNA disruptions could underly the phenotypes of these individuals. To showcase the power of this genomic approach for annotating lncRNAs, here we focus on clinical reports and genetic analysis of two individuals with BCAs and additionally highlight six individuals with likely developmental etiologies due to lncRNA disruptions.

Fetal Head and Neck Imaging.

Prenatal MRI plays an essential role in the evaluation of the head and neck. This article overviews technical considerations and both isolated and syndromic anomalies of the fetal calvarium, globes and orbits, ears, maxilla, mandible, and neck.

Pediatric cerebrospinal fluid rhinorrhea caused by low flow vascular anomaly of the temporal bone: A case series.

OBJECTIVE: Atraumatic cerebrospinal fluid (CSF) rhinorrhea is uncommon in children and necessitates a multi-disciplinary evaluation for an etiology. Underlying osseous abnormality due to extensive or multifocal low flow vascular anomaly should be considered as a potential cause of spontaneous CSF leak. Treatment of multifocal low flow vascular anomalies may include medical and surgical approaches. In this series, we seek to determine the presenting signs and symptoms and medical and surgical treatment options for multifocal or extensive low flow vascular anomalies. METHODS: A retrospective case series at a quaternary care children's hospital was compiled. All children with CSF rhinorrhea diagnosed and treated for multifocal low flow vascular anomalies at our institution were included. A total of four patients were identified. RESULTS: All four patients had delay in initial diagnosis of underlying cause of meningitis and CSF rhinorrhea. Average age at diagnosis of multifocal low flow vascular anomaly was 7 years. This was on average 4 years after initial presentation for medical attention. Treatment approach was multidisciplinary and included medical management with sirolimus and bisphosphonates as well as surgical approaches to the skull base (lateral and anterior) to prevent CSF egress. CONCLUSION: Consideration of multifocal low flow vascular anomaly should be included in any pediatric patient presenting with CSF rhinorrhea.

Incomplete partition type II in its various manifestations: isolated, in association with EVA, syndromic, and beyond; a multicentre international study.

PURPOSE: Incomplete partition type II (IP-II) is characterized by specific histological features and radiological appearance. It may occur in isolation or in association with an enlarged vestibular aqueduct (EVA). Among those with IP-II and EVA, a subset has a diagnosis of Pendred syndrome. This study aimed to explore the prevalence of isolated IP-II, IP-II with EVA, and cases with a genetic or syndromic basis in our cohort. METHODS: From a large, multicentre database of dysplastic cochleae (446 patients, 892 temporal bones), those with imaging features of IP-II were examined in detail, including whether there was a genetic or syndromic association. RESULTS: A total of 78 patients with IP-II were identified. Among these, 55 patients had bilateral IP-II and EVA (only 12 with typical Mondini triad), 8 with bilateral IP-II and normal VA, 2 with bilateral IP-II and unilateral EVA, and 13 with unilateral IP-II (9 with unilateral EVA). Among the group with bilateral IP-II and bilateral EVA in whom genetic analysis was available, 14 out of 29 (48%) had SLC26A4 mutations and a diagnosis of Pendred syndrome, 1 had a FOXI1 mutation, and a few other genetic abnormalities; none had KCNJ10 pathogenic variants. CONCLUSION: Bilateral IP-II-bilateral EVA may be seen in the context of Pendred syndrome (SLC26A4 or FOXI1 mutations) but, in the majority of our cohort, no genetic abnormalities were found, suggesting the possibility of unknown genetic associations. IP-II in isolation (without EVA) is favored to be genetic when bilateral, although the cause is often unknown.

Expanding the genetics and phenotypes of ocular congenital cranial dysinnervation disorders.

Purpose: To identify genetic etiologies and genotype/phenotype associations for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs). Methods: We coupled phenotyping with exome or genome sequencing of 467 pedigrees with genetically unsolved oCCDDs, integrating analyses of pedigrees, human and animal model phenotypes, and de novo variants to identify rare candidate single nucleotide variants, insertion/deletions, and structural variants disrupting protein-coding regions. Prioritized variants were classified for pathogenicity and evaluated for genotype/phenotype correlations. Results: Analyses elucidated phenotypic subgroups, identified pathogenic/likely pathogenic variant(s) in 43/467 probands (9.2%), and prioritized variants of uncertain significance in 70/467 additional probands (15.0%). These included known and novel variants in established oCCDD genes, genes associated with syndromes that sometimes include oCCDDs (e.g., MYH10, KIF21B, TGFBR2, TUBB6), genes that fit the syndromic component of the phenotype but had no prior oCCDD association (e.g., CDK13, TGFB2), genes with no reported association with oCCDDs or the syndromic phenotypes (e.g., TUBA4A, KIF5C, CTNNA1, KLB, FGF21), and genes associated with oCCDD phenocopies that had resulted in misdiagnoses. Conclusion: This study suggests that unsolved oCCDDs are clinically and genetically heterogeneous disorders often overlapping other Mendelian conditions and nominates many candidates for future replication and functional studies.

A recurrent missense variant in the E3 ubiquitin ligase substrate recognition subunit FEM1B causes a rare syndromic neurodevelopmental disorder.

PURPOSE: Fem1 homolog B (FEM1B) acts as a substrate recognition subunit for ubiquitin ligase complexes belonging to the CULLIN 2-based E3 family. Several biological functions have been proposed for FEM1B, including a structurally resolved function as a sensor for redox cell status by controlling mitochondrial activity, but its implication in human disease remains elusive. METHODS: To understand the involvement of FEM1B in human disease, we made use of Matchmaker exchange platforms to identify individuals with de novo variants in FEM1B and performed their clinical evaluation. We performed functional validation using primary neuronal cultures and in utero electroporation assays, as well as experiments on patient's cells. RESULTS: Five individuals with a recurrent de novo missense variant in FEM1B were identified: NM_015322.5:c.377G>A NP_056137.1:p.(Arg126Gln) (FEM1BR126Q). Affected individuals shared a severe neurodevelopmental disorder with behavioral phenotypes and a variable set of malformations, including brain anomalies, clubfeet, skeletal abnormalities, and facial dysmorphism. Overexpression of the FEM1BR126Q variant but not FEM1B wild-type protein, during mouse brain development, resulted in delayed neuronal migration of the target cells. In addition, the individuals' cells exhibited signs of oxidative stress and induction of type I interferon signaling. CONCLUSION: Overall, our data indicate that p.(Arg126Gln) induces aberrant FEM1B activation, resulting in a gain-of-function mechanism associated with a severe syndromic developmental disorder in humans.

Single-institution Series of Hirayama Disease in North America.

STUDY DESIGN: A retrospective chart review. OBJECTIVE: The aims of this study were to review pathophysiology, workup, and treatment for Hirayama disease (HD); and to assess outcomes from a single institution. SUMMARY OF BACKGROUND DATA: HD is a rare, painless, cervical myelopathy with distal upper extremity weakness, muscle wasting, and spinal cord atrophy. Disease progression-a consequence of repeat flexion injury-occurs up to 5 years from the initial diagnosis. METHODS: Single-institution review of pediatric HD patients from 2010 to 2020. RESULTS: Patients (n=10 male, n=2 female) presented in the second decade (14-20 y) with painless progressive distal upper extremity weakness and atrophy without sensory loss. Electromyography (n=12) demonstrated denervation in C7-T1 myotomes and flexion/extension magnetic resonance imaging showed focal cord atrophy and anterior displacement of the posterior dura with epidural enhancement in flexion. Treatment included observation and external orthoses (n=9) and anterior cervical discectomy with fusion (n=3). One of the 9 patients managed conservatively experienced further deterioration; no patient who underwent anterior cervical discectomy with fusion progressed. CONCLUSIONS: Patients with HD require a multidisciplinary approach to diagnosis and treatment to preserve function. Treatment is preventive and aims to minimize flexion injury by inhibiting motion across involved joints. First-line management is avoidance of neck flexion and use of rigid orthosis; in cases of failed conservative management and/or rapid clinical deterioration, surgical fixation can be offered.

Non-Syndromic Sensorineural Hearing Loss in Children.

Pediatric hearing loss is common with significant consequences in terms of language, communication, social and emotional development, and academic advancement. Radiological imaging provides useful information regarding hearing loss etiology, prognosis, therapeutic options, and potential surgical pitfalls. This review provides an overview of temporal bone imaging protocols, an outline of the classification of inner ear anomalies associated with sensorineural hearing loss and illustrates some of the more frequently encountered and/or important causes of non-syndromic hearing loss.

Conductive Hearing Loss in Children.

A variety of congenital and acquired disorders result in pediatric conductive hearing loss. Malformations of the external auditory canal are invariably associated with malformations of the middle ear space and ossicles. Isolated ossicular malformations are uncommon. Syndromes associated with external and middle ear malformations are frequently associated with abnormal development of first and second pharyngeal arch derivatives. Chronic inflammatory disorders include cholesteatoma, cholesterol granuloma, and tympanosclerosis.

Syndromic Hearing Loss in Children.

Pattern recognition of specific temporal bone radiological phenotypes, in association with abnormalities in other organ systems, is critical in the diagnosis and management of syndromic causes of hearing loss. Several recent publications have demonstrated the presence of specific radiological appearances, allowing precise genetic and/or syndromic diagnosis, in the right clinical context. This review article aims to provide an extensive but practical guide to the radiologist dealing with syndromic causes of hearing loss.

CTNNB1 and APC Mutations in Sinonasal Myxoma : Expanding the Spectrum of Tumors Driven By WNT/ß-catenin Pathway.

Sinonasal myxoma (SNM) is a rare, benign mesenchymal neoplasm with distinct clinicopathologic features and aberrant nuclear localization of ß-catenin by immunohistochemistry. The molecular underpinnings have been linked to that of a "myxoid variant" of desmoid fibromatosis. Herein, we describe a series of 8 cases of SNM and propose clinical and biologic differences compared with desmoid fibromatosis. Our patient cohort is comprised of 5 males and 3 females (age range: 10 mo to 12 y), 6 of whom are aged less than or equal to 24 months. All presented with facial swelling, reflecting lesions involving the maxillary bone, and all underwent resection. All tumors were variably cellular and comprised of bland spindled to stellate cells in a profusely myxoid background with diffuse nuclear ß-catenin expression. All cases of SNM were analyzed by next-generation sequencing using the Oncopanel assay. Three cases failed sequencing, 2 of 5 successful cases exhibited exon 3 CTNNB1 alterations involving the ubiquitin recognition motif, and 3 had adenomatous polyposis coli ( APC ) deletions. One patient had APC germline testing which was negative. No germline testing was available for the remaining 7 patients. Follow-up data over a range of 1 month to 23 years was available for 7 of the 8 SNMs. One case patient had local recurrence, and all were alive without evidence of disease. This is in contrast to the high recurrence rate typically seen in desmoid fibromatosis, particularly after resection. Our findings expand the spectrum of tumors with underlying WNT/ß-catenin pathway and highlight the histologic, clinical, and genetic differences of SNM compared with desmoid fibromatosis. APC deletion raises the possibility of underlying germline alteration and familial adenomatous polyposis.

Noncoding variants alter GATA2 expression in rhombomere 4 motor neurons and cause dominant hereditary congenital facial paresis.

Hereditary congenital facial paresis type 1 (HCFP1) is an autosomal dominant disorder of absent or limited facial movement that maps to chromosome 3q21-q22 and is hypothesized to result from facial branchial motor neuron (FBMN) maldevelopment. In the present study, we report that HCFP1 results from heterozygous duplications within a neuron-specific GATA2 regulatory region that includes two enhancers and one silencer, and from noncoding single-nucleotide variants (SNVs) within the silencer. Some SNVs impair binding of NR2F1 to the silencer in vitro and in vivo and attenuate in vivo enhancer reporter expression in FBMNs. Gata2 and its effector Gata3 are essential for inner-ear efferent neuron (IEE) but not FBMN development. A humanized HCFP1 mouse model extends Gata2 expression, favors the formation of IEEs over FBMNs and is rescued by conditional loss of Gata3. These findings highlight the importance of temporal gene regulation in development and of noncoding variation in rare mendelian disease.

A multi-disciplinary team approach to pediatric malignant mandibular tumors.

OBJECTIVE: Mandibular tumors in the pediatric population are rare. These malignancies are variable in their histology, and combined with their rarity, has made it difficult to describe their clinical course, and treatment guidelines. The aim of this paper is to describe the experience of Boston Children's Hospital, a pediatric tertiary referral center, with treating malignant mandibular malignancies, as well as provide multi-disciplinary team approach in managing this clinical entity. METHODS: A retrospective search was performed for mandibular malignancies in pediatric patients between 1995 and 2020 via the pathological database at Boston Children's Hospital. Only patients with malignant solid mandibular neoplasms were included, leaving 15 patients for final analysis. RESULTS: The median age at presentation was 10.1 ± 10.3 years. Nine of 15 patients (60%) presented with jaw mass which was the most common clinical presentation. The most commonly identified histological diagnosis was rhabdomayosarcoma and osteosarcoma (n = 4, 26% each). A mandibulectomy was performed in 12 (80%) cases. Reconstruction of the mandible was performed using a fibular free flap in 6 (40%) cases, and a plate in 3 (20%) cases. Mean follow-up was 4.6 ± 4.9 years. CONCLUSION: Malignant tumors most commonly present with a jaw mass, however asymptomatic and incidental presentations follow closely and pathologies can vary greatly. Surgical resection and reconstruction is often indicated, multidisciplinary tumor board review is required to determine when children are best treated with neo-/adjuvant treatment with chemo- and radiotherapy.

Guidelines for magnetic resonance imaging in pediatric head and neck pathologies: a multicentre international consensus paper.

The use of standardized imaging protocols is paramount in order to facilitate comparable, reproducible images and, consequently, to optimize patient care. Standardized MR protocols are lacking when studying head and neck pathologies in the pediatric population. We propose an international, multicenter consensus paper focused on providing the best combination of acquisition time/technical requirements and image quality. Distinct protocols for different regions of the head and neck and, in some cases, for specific pathologies or clinical indications are recommended. This white paper is endorsed by several international scientific societies and it is the result of discussion, in consensus, among experts in pediatric head and neck imaging.

TUBB3 Arg262His causes a recognizable syndrome including CFEOM3, facial palsy, joint contractures, and early-onset peripheral neuropathy.

Microtubules are formed from heterodimers of alpha- and beta-tubulin, each of which has multiple isoforms encoded by separate genes. Pathogenic missense variants in multiple different tubulin isoforms cause brain malformations. Missense mutations in TUBB3, which encodes the neuron-specific beta-tubulin isotype, can cause congenital fibrosis of the extraocular muscles type 3 (CFEOM3) and/or malformations of cortical development, with distinct genotype-phenotype correlations. Here, we report fourteen individuals from thirteen unrelated families, each of whom harbors the identical NM_006086.4 (TUBB3):c.785G>A (p.Arg262His) variant resulting in a phenotype we refer to as the TUBB3 R262H syndrome. The affected individuals present at birth with ptosis, ophthalmoplegia, exotropia, facial weakness, facial dysmorphisms, and, in most cases, distal congenital joint contractures, and subsequently develop intellectual disabilities, gait disorders with proximal joint contractures, Kallmann syndrome (hypogonadotropic hypogonadism and anosmia), and a progressive peripheral neuropathy during the first decade of life. Subsets may also have vocal cord paralysis, auditory dysfunction, cyclic vomiting, and/or tachycardia at rest. All fourteen subjects share a recognizable set of brain malformations, including hypoplasia of the corpus callosum and anterior commissure, basal ganglia malformations, absent olfactory bulbs and sulci, and subtle cerebellar malformations. While similar, individuals with the TUBB3 R262H syndrome can be distinguished from individuals with the TUBB3 E410K syndrome by the presence of congenital and acquired joint contractures, an earlier onset peripheral neuropathy, impaired gait, and basal ganglia malformations.

Neuroimaging in Kabuki syndrome and another KMT2D-related disorder.

Recognition of distinct phenotypic features is an important component of genetic diagnosis. Although CHARGE syndrome, Kabuki syndrome, and a recently delineated KMT2D Ex 38/39 allelic disorder exhibit significant overlap, differences on neuroimaging may help distinguish these conditions and guide genetic testing and variant interpretation. We present an infant clinically diagnosed with CHARGE syndrome but subsequently found to have a de novo missense variant in exon 38 of KMT2D, the gene implicated in both Kabuki syndrome and a distinct KMT2D allelic disorder. We compare her brain and inner ear morphology to a retrospective cohort of 21 patients with classic Kabuki syndrome and to typical CHARGE syndrome findings described in the literature. Thirteen of the 21 Kabuki syndrome patients had temporal bone imaging (5/13 CT, 12/13 MRI) and/or brain MRI (12/13) which revealed findings distinct from both CHARGE syndrome and the KMT2D allelic disorder. Our findings further elucidate the spectrum of inner ear dysmorphology distinguishing Kabuki syndrome and the KMT2D allelic disorder from CHARGE syndrome, suggesting that these three disorders may be differentiated at least in part by their inner ear anomalies.