De Novo Missense Variants in FBXW11 Cause Diverse Developmental Phenotypes Including Brain, Eye, and Digit Anomalies.

The identification of genetic variants implicated in human developmental disorders has been revolutionized by second-generation sequencing combined with international pooling of cases. Here, we describe seven individuals who have diverse yet overlapping developmental anomalies, and who all have de novo missense FBXW11 variants identified by whole exome or whole genome sequencing and not reported in the gnomAD database. Their phenotypes include striking neurodevelopmental, digital, jaw, and eye anomalies, and in one individual, features resembling Noonan syndrome, a condition caused by dysregulated RAS signaling. FBXW11 encodes an F-box protein, part of the Skp1-cullin-F-box (SCF) ubiquitin ligase complex, involved in ubiquitination and proteasomal degradation and thus fundamental to many protein regulatory processes. FBXW11 targets include ß-catenin and GLI transcription factors, key mediators of Wnt and Hh signaling, respectively, critical to digital, neurological, and eye development. Structural analyses indicate affected residues cluster at the surface of the loops of the substrate-binding domain of FBXW11, and the variants are predicted to destabilize the protein and/or its interactions. In situ hybridization studies on human and zebrafish embryonic tissues demonstrate FBXW11 is expressed in the developing eye, brain, mandibular processes, and limb buds or pectoral fins. Knockdown of the zebrafish FBXW11 orthologs fbxw11a and fbxw11b resulted in embryos with smaller, misshapen, and underdeveloped eyes and abnormal jaw and pectoral fin development. Our findings support the role of FBXW11 in multiple developmental processes, including those involving the brain, eye, digits, and jaw.

Expanding the KIF4A-associated phenotype.

Kinesin super family (KIF) genes encode motor kinesins, a family of evolutionary conserved proteins, involved in intracellular trafficking of various cargoes. These proteins are critical for various physiological processes including neuron function and survival, ciliary function and ciliogenesis, and cell-cycle progression. Recent evidence suggests that alterations in motor kinesin genes can lead to a variety of human diseases, including monogenic disorders. Neuropathies, impaired higher brain functions, structural brain abnormalities and multiple congenital anomalies (i.e., renal, urogenital, and limb anomalies) can result from pathogenic variants in many KIF genes. We expand the phenotype associated with KIF4A variants from developmental delay and intellectual disability with or without epilepsy to a congenital anomaly phenotype with hydrocephalus and various brain anomalies at the more severe end of phenotypic manifestations. Additional anomalies of the kidneys and urinary tract, congenital lymphedema, eye, and dental anomalies seem to be variably associated and overlap with clinical signs observed in other kinesinopathies. Caution still applies to missense variants, but hopefully, future work will further establish genotype-phenotype correlations in a larger number of patients and functional studies may give further insights into the complex function of KIF4A.

Mutations in ARMC9, which Encodes a Basal Body Protein, Cause Joubert Syndrome in Humans and Ciliopathy Phenotypes in Zebrafish.

Joubert syndrome (JS) is a recessive neurodevelopmental disorder characterized by hypotonia, ataxia, abnormal eye movements, and variable cognitive impairment. It is defined by a distinctive brain malformation known as the "molar tooth sign" on axial MRI. Subsets of affected individuals have malformations such as coloboma, polydactyly, and encephalocele, as well as progressive retinal dystrophy, fibrocystic kidney disease, and liver fibrosis. More than 35 genes have been associated with JS, but in a subset of families the genetic cause remains unknown. All of the gene products localize in and around the primary cilium, making JS a canonical ciliopathy. Ciliopathies are unified by their overlapping clinical features and underlying mechanisms involving ciliary dysfunction. In this work, we identify biallelic rare, predicted-deleterious ARMC9 variants (stop-gain, missense, splice-site, and single-exon deletion) in 11 individuals with JS from 8 families, accounting for approximately 1% of the disorder. The associated phenotypes range from isolated neurological involvement to JS with retinal dystrophy, additional brain abnormalities (e.g., heterotopia, Dandy-Walker malformation), pituitary insufficiency, and/or synpolydactyly. We show that ARMC9 localizes to the basal body of the cilium and is upregulated during ciliogenesis. Typical ciliopathy phenotypes (curved body shape, retinal dystrophy, coloboma, and decreased cilia) in a CRISPR/Cas9-engineered zebrafish mutant model provide additional support for ARMC9 as a ciliopathy-associated gene. Identifying ARMC9 mutations as a cause of JS takes us one step closer to a full genetic understanding of this important disorder and enables future functional work to define the central biological mechanisms underlying JS and other ciliopathies.

Hypomorphic Recessive Variants in SUFU Impair the Sonic Hedgehog Pathway and Cause Joubert Syndrome with Cranio-facial and Skeletal Defects.

The Sonic Hedgehog (SHH) pathway is a key signaling pathway orchestrating embryonic development, mainly of the CNS and limbs. In vertebrates, SHH signaling is mediated by the primary cilium, and genetic defects affecting either SHH pathway members or ciliary proteins cause a spectrum of developmental disorders. SUFU is the main negative regulator of the SHH pathway and is essential during development. Indeed, Sufu knock-out is lethal in mice, and recessive pathogenic variants of this gene have never been reported in humans. Through whole-exome sequencing in subjects with Joubert syndrome, we identified four children from two unrelated families carrying homozygous missense variants in SUFU. The children presented congenital ataxia and cerebellar vermis hypoplasia with elongated superior cerebellar peduncles (mild "molar tooth sign"), typical cranio-facial dysmorphisms (hypertelorism, depressed nasal bridge, frontal bossing), and postaxial polydactyly. Two siblings also showed polymicrogyria. Molecular dynamics simulation predicted random movements of the mutated residues, with loss of the native enveloping movement of the binding site around its ligand GLI3. Functional studies on cellular models and fibroblasts showed that both variants significantly reduced SUFU stability and its capacity to bind GLI3 and promote its cleavage into the repressor form GLI3R. In turn, this impaired SUFU-mediated repression of the SHH pathway, as shown by altered expression levels of several target genes. We demonstrate that germline hypomorphic variants of SUFU cause deregulation of SHH signaling, resulting in recessive developmental defects of the CNS and limbs which share features with both SHH-related disorders and ciliopathies.

Mandibulofacial Dysostosis with Microcephaly: Mutation and Database Update.

Mandibulofacial dysostosis with microcephaly (MFDM) is a multiple malformation syndrome comprising microcephaly, craniofacial anomalies, hearing loss, dysmorphic features, and, in some cases, esophageal atresia. Haploinsufficiency of a spliceosomal GTPase, U5-116 kDa/EFTUD2, is responsible. Here, we review the molecular basis of MFDM in the 69 individuals described to date, and report mutations in 38 new individuals, bringing the total number of reported individuals to 107 individuals from 94 kindreds. Pathogenic EFTUD2 variants comprise 76 distinct mutations and seven microdeletions. Among point mutations, missense substitutions are infrequent (14 out of 76; 18%) relative to stop-gain (29 out of 76; 38%), and splicing (33 out of 76; 43%) mutations. Where known, mutation origin was de novo in 48 out of 64 individuals (75%), dominantly inherited in 12 out of 64 (19%), and due to proven germline mosaicism in four out of 64 (6%). Highly penetrant clinical features include, microcephaly, first and second arch craniofacial malformations, and hearing loss; esophageal atresia is present in an estimated ∼27%. Microcephaly is virtually universal in childhood, with some adults exhibiting late "catch-up" growth and normocephaly at maturity. Occasionally reported anomalies, include vestibular and ossicular malformations, reduced mouth opening, atrophy of cerebral white matter, structural brain malformations, and epibulbar dermoid. All reported EFTUD2 mutations can be found in the EFTUD2 mutation database (http://databases.lovd.nl/shared/genes/EFTUD2).

Novel mutations including deletions of the entire OFD1 gene in 30 families with type 1 orofaciodigital syndrome: a study of the extensive clinical variability.

OFD1, now recognized as a ciliopathy, is characterized by malformations of the face, oral cavity and digits, and is transmitted as an X-linked condition with lethality in males. Mutations in OFD1 also cause X-linked Joubert syndrome (JBTS10) and Simpson-Golabi-Behmel syndrome type 2 (SGBS2). We have studied 55 sporadic and six familial cases of suspected OFD1. Comprehensive mutation analysis in OFD1 revealed mutations in 37 female patients from 30 families; 22 mutations have not been previously described including two heterozygous deletions spanning OFD1 and neighbouring genes. Analysis of clinical findings in patients with mutations revealed that oral features are the most reliable diagnostic criteria. A first, detailed evaluation of brain MRIs from seven patients with cognitive defects illustrated extensive variability with the complete brain phenotype consisting of complete agenesis of the corpus callosum, large single or multiple interhemispheric cysts, striking cortical infolding of gyri, ventriculomegaly, mild molar tooth malformation and moderate to severe cerebellar vermis hypoplasia. Although the OFD1 gene apparently escapes X-inactivation, skewed inactivation was observed in seven of 14 patients. The direction of skewing did not correlate with disease severity, reinforcing the hypothesis that additional factors contribute to the extensive intrafamilial variability.

Mutations in the cilia gene ARL13B lead to the classical form of Joubert syndrome.

Joubert syndrome (JS) and related disorders are a group of autosomal-recessive conditions sharing the "molar tooth sign" on axial brain MRI, together with cerebellar vermis hypoplasia, ataxia, and psychomotor delay. JS is suggested to be a disorder of cilia function and is part of a spectrum of disorders involving retinal, renal, digital, oral, hepatic, and cerebral organs. We identified mutations in ARL13B in two families with the classical form of JS. ARL13B belongs to the Ras GTPase family, and in other species is required for ciliogenesis, body axis formation, and renal function. The encoded Arl13b protein was expressed in developing murine cerebellum and localized to the cilia in primary neurons. Overexpression of human wild-type but not patient mutant ARL13B rescued the Arl13b scorpion zebrafish mutant. Thus, ARL13B has an evolutionarily conserved role mediating cilia function in multiple organs.

Postzygotic inactivating mutations of RHOA cause a mosaic neuroectodermal syndrome.

Hypopigmentation along Blaschko's lines is a hallmark of a poorly defined group of mosaic syndromes whose genetic causes are unknown. Here we show that postzygotic inactivating mutations of RHOA cause a neuroectodermal syndrome combining linear hypopigmentation, alopecia, apparently asymptomatic leukoencephalopathy, and facial, ocular, dental and acral anomalies. Our findings pave the way toward elucidating the etiology of pigmentary mosaicism and highlight the role of RHOA in human development and disease.

Flores hominid: new species or microcephalic dwarf?

The proposed new hominid "Homo floresiensis" is based on specimens from cave deposits on the Indonesian island Flores. The primary evidence, dated at approximately 18,000 y, is a skull and partial skeleton of a very small but dentally adult individual (LB1). Incomplete specimens are attributed to eight additional individuals. Stone tools at the site are also attributed to H. floresiensis. The discoverers interpreted H. floresiensis as an insular dwarf derived from Homo erectus, but others see LB1 as a small-bodied microcephalic Homo sapiens. Study of virtual endocasts, including LB1 and a European microcephalic, purportedly excluded microcephaly, but reconsideration reveals several problems. The cranial capacity of LB1 ( approximately 400 cc) is smaller than in any other known hominid < 3.5 Ma and is far too small to derive from Homo erectus by normal dwarfing. By contrast, some associated tools were generated with a prepared-core technique previously unknown for H. erectus, including bladelets otherwise associated exclusively with H. sapiens. The single European microcephalic skull used in comparing virtual endocasts was particularly unsuitable. The specimen was a cast, not the original skull (traced to Stuttgart), from a 10-year-old child with massive pathology. Moreover, the calotte does not fit well with the rest of the cast, probably being a later addition of unknown history. Consideration of various forms of human microcephaly and of two adult specimens indicates that LB1 could well be a microcephalic Homo sapiens. This is the most likely explanation for the incongruous association of a small-brained recent hominid with advanced stone tools.

Two Hundred Thirty-Six Children With Developmental Hydrocephalus: Causes and Clinical Consequences.

Few systematic assessments of developmental forms of hydrocephalus exist. We reviewed magnetic resonance images (MRIs) and clinical records of patients with infancy-onset hydrocephalus. Among 411 infants, 236 had hydrocephalus with no recognizable extrinsic cause. These children were assigned to 1 of 5 subtypes and compared on the basis of clinical characteristics and developmental and surgical outcomes. At an average age of 5.3 years, 72% of children were walking independently and 87% could eat by mouth; in addition, 18% had epilepsy. Distinct patterns of associated malformations and syndromes were observed within each subtype. On average, children with aqueductal obstruction, cysts, and encephaloceles had worse clinical outcomes than those with other forms of developmental hydrocephalus. Overall, 53% of surgically treated patients experienced at least 1 shunt failure, but hydrocephalus associated with posterior fossa crowding required fewer shunt revisions. We conclude that each subtype of developmental hydrocephalus is associated with distinct clinical characteristics, syndromology, and outcomes, suggesting differences in underlying mechanisms.

AHI1 gene mutations cause specific forms of Joubert syndrome-related disorders.

OBJECTIVE: Joubert syndrome (JS) is a recessively inherited developmental brain disorder with several identified causative chromosomal loci. It is characterized by hypoplasia of the cerebellar vermis and a particular midbrain-hindbrain "molar tooth" sign, a finding shared by a group of Joubert syndrome-related disorders (JSRDs), with wide phenotypic variability. The frequency of mutations in the first positionally cloned gene, AHI1, is unknown. METHODS: We searched for mutations in the AHI1 gene among a cohort of 137 families with JSRD and radiographically proven molar tooth sign. RESULTS: We identified 15 deleterious mutations in 10 families with pure JS or JS plus retinal and/or additional central nervous system abnormalities. Mutations among families with JSRD including kidney or liver involvement were not detected. Transheterozygous mutations were identified in the majority of those without history of consanguinity. Most mutations were truncating or splicing errors, with only one missense mutation in the highly conserved WD40 repeat domain that led to disease of similar severity. INTERPRETATION: AHI1 mutations are a frequent cause of disease in patients with specific forms of JSRD.

Human malformations of the midbrain and hindbrain: review and proposed classification scheme.

Although a great deal of interest in the genetics and etiology of cerebral, particularly forebrain, malformations has been generated in the past decade, relatively little is known about the basis of congenital malformations of the structures of the posterior fossa, namely the midbrain, cerebellum, pons, and medulla. In this review, we present a classification scheme for malformations of the midbrain and hindbrain based on their embryologic derivation, highlight four of the conditions associated with such abnormalities, and describe the genetics, prognosis, and recurrence risks for each. We describe several disorders in addition to Joubert syndrome with the distinctive radiologic sign known as the "molar tooth sign," comprised of midbrain and hindbrain malformations. We discuss Dandy-Walker malformation, its classical definition, and the surprisingly good outcome in the absence of other brain malformations. We consider the heterogeneous entity of cerebellar vermis hypoplasia and describe the recently identified gene associated with an X-linked form of this condition. Finally, the pontocerebellar hypoplasias are discussed in the context of their generally progressive degenerative and severe course, and the differential diagnosis is emphasized. We anticipate that as imaging technologies improve, differentiation of the various disorders should aid in efforts to identify the causative genes.

Molar tooth sign of the midbrain-hindbrain junction: occurrence in multiple distinct syndromes.

The Molar Tooth Sign (MTS) is defined by an abnormally deep interpeduncular fossa; elongated, thick, and mal-oriented superior cerebellar peduncles; and absent or hypoplastic cerebellar vermis that together give the appearance of a "molar tooth" on axial brain MRI through the junction of the midbrain and hindbrain (isthmus region). It was first described in Joubert syndrome (JS) where it is present in the vast majority of patients with this diagnosis. We previously showed that the MTS is a component of several other syndromes, including Dekaban-Arima (DAS), Senior-Löken, and COACH (cerebellar vermis hypoplasia (CVH), oligophrenia, ataxia, coloboma, and hepatic fibrosis). Here we present evidence that the MTS is seen together with polymicrogyria, Váradi-Papp syndrome (Orofaciodigital VI (OFD VI)), and a new syndrome with encephalocele and cortical renal cysts. We also present a new patient with COACH syndrome plus the MTS. We propose that the MTS is found in multiple distinct clinical syndromes that may share common developmental mechanisms. Proper classification of patients with these variants of the MTS will be essential for localization and identification of mutant genes.

The NPHP1 gene deletion associated with juvenile nephronophthisis is present in a subset of individuals with Joubert syndrome.

Joubert syndrome (JS) is an autosomal recessive multisystem disease characterized by cerebellar vermis hypoplasia with prominent superior cerebellar peduncles (the "molar tooth sign" [MTS] on axial magnetic resonance imaging), mental retardation, hypotonia, irregular breathing pattern, and eye-movement abnormalities. Some individuals with JS have retinal dystrophy and/or progressive renal failure characterized by nephronophthisis (NPHP). Thus far, no mutations in the known NPHP genes, particularly the homozygous deletion of NPHP1 at 2q13, have been identified in subjects with JS. A cohort of 25 subjects with JS and either renal and/or retinal complications and 2 subjects with only juvenile NPHP were screened for mutations in the NPHP1 gene by standard methods. Two siblings affected with a mild form of JS were found to have a homozygous deletion of the NPHP1 gene identical, by mapping, to that in subjects with NPHP alone. A control subject with NPHP and with a homozygous NPHP1 deletion was also identified, retrospectively, as having a mild MTS and borderline intelligence. The NPHP1 deletion represents the first molecular defect associated with JS in a subset of mildly affected subjects. Cerebellar malformations consistent with the MTS may be relatively common in patients with juvenile NPHP without classic symptoms of JS.