Ian A. Glass

Systematic Localization of Common Disease-Associated Variation in Regulatory DNA

Predictions of Genetic Disease Many genome-wide association studies (GWAS) have identified loci and variants associated with disease, but the ability to predict disease on the basis of these genetic variants remains small. Maurano et al. (p. 1190; see the Perspective by Schadt and Chang; see the cover) characterize the location of GWAS variants in the genome with respect to their proximity to regulatory DNA [marked by deoxyribonuclease I (DNase I) hypersensitive sites] by tissue type, disease, and enrichments in physiologically relevant transcription factor binding sites and networks. They found many noncoding disease associations in regulatory DNA, indicating tissue and developmental-specific regulatory roles for many common genetic variants and thus enabling links to be made between gene regulation and adult-onset disease. Genetic variants that have been associated with diseases are concentrated in regulatory regions of the genome. Genome-wide association studies have identified many noncoding variants associated with common diseases and traits. We show that these variants are concentrated in regulatory DNA marked by deoxyribonuclease I (DNase I) hypersensitive sites (DHSs). Eighty-eight percent of such DHSs are active during fetal development and are enriched in variants associated with gestational exposure–related phenotypes. We identified distant gene targets for hundreds of variant-containing DHSs that may explain phenotype associations. Disease-associated variants systematically perturb transcription factor recognition sequences, frequently alter allelic chromatin states, and form regulatory networks. We also demonstrated tissue-selective enrichment of more weakly disease-associated variants within DHSs and the de novo identification of pathogenic cell types for Crohn’s disease, multiple sclerosis, and an electrocardiogram trait, without prior knowledge of physiological mechanisms. Our results suggest pervasive involvement of regulatory DNA variation in common human disease and provide pathogenic insights into diverse disorders.

The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4

The molecular basis of nephronophthisis, the most frequent genetic cause of renal failure in children and young adults, and its association with retinal degeneration and cerebellar vermis aplasia in Joubert syndrome are poorly understood. Using positional cloning, we here identify mutations in the gene CEP290 as causing nephronophthisis. It encodes a protein with several domains also present in CENPF, a protein involved in chromosome segregation. CEP290 (also known as NPHP6) interacts with and modulates the activity of ATF4, a transcription factor implicated in cAMP-dependent renal cyst formation. NPHP6 is found at centrosomes and in the nucleus of renal epithelial cells in a cell cycle–dependent manner and in connecting cilia of photoreceptors. Abrogation of its function in zebrafish recapitulates the renal, retinal and cerebellar phenotypes of Joubert syndrome. Our findings help establish the link between centrosome function, tissue architecture and transcriptional control in the pathogenesis of cystic kidney disease, retinal degeneration, and central nervous system development.

Transcriptional landscape of the prenatal human brain

The anatomical and functional architecture of the human brain is mainly determined by prenatal transcriptional processes. We describe an anatomically comprehensive atlas of the mid-gestational human brain, including de novo reference atlases, in situ hybridization, ultra-high-resolution magnetic resonance imaging (MRI) and microarray analysis on highly discrete laser-microdissected brain regions. In developing cerebral cortex, transcriptional differences are found between different proliferative and post-mitotic layers, wherein laminar signatures reflect cellular composition and developmental processes. Cytoarchitectural differences between human and mouse have molecular correlates, including species differences in gene expression in subplate, although surprisingly we find minimal differences between the inner and outer subventricular zones even though the outer zone is expanded in humans. Both germinal and post-mitotic cortical layers exhibit fronto-temporal gradients, with particular enrichment in the frontal lobe. Finally, many neurodevelopmental disorder and human-evolution-related genes show patterned expression, potentially underlying unique features of human cortical formation. These data provide a rich, freely-accessible resource for understanding human brain development.

Mutations in the gene encoding the basal body protein RPGRIP1L, a nephrocystin-4 interactor, cause Joubert syndrome.

Protein-protein interaction analyses have uncovered a ciliary and basal body protein network that, when disrupted, can result in nephronophthisis (NPHP), Leber congenital amaurosis, Senior-Løken syndrome (SLSN) or Joubert syndrome (JBTS). However, details of the molecular mechanisms underlying these disorders remain poorly understood. RPGRIP1-like protein (RPGRIP1L) is a homolog of RPGRIP1 (RPGR-interacting protein 1), a ciliary protein defective in Leber congenital amaurosis. We show that RPGRIP1L interacts with nephrocystin-4 and that mutations in the gene encoding nephrocystin-4 (NPHP4) that are known to cause SLSN disrupt this interaction. RPGRIP1L is ubiquitously expressed, and its protein product localizes to basal bodies. Therefore, we analyzed RPGRIP1L as a candidate gene for JBTS and identified loss-of-function mutations in three families with typical JBTS, including the characteristic mid-hindbrain malformation. This work identifies RPGRIP1L as a gene responsible for JBTS and establishes a central role for cilia and basal bodies in the pathophysiology of this disorder.

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.

CC2D2A Is Mutated in joubert Syndrome and Interacts with the Ciliopathy-Associated Basal Body Protein CEP290

Joubert syndrome and related disorders (JSRD) are primarily autosomal-recessive conditions characterized by hypotonia, ataxia, abnormal eye movements, and intellectual disability with a distinctive mid-hindbrain malformation. Variable features include retinal dystrophy, cystic kidney disease, and liver fibrosis. JSRD are included in the rapidly expanding group of disorders called ciliopathies, because all six gene products implicated in JSRD (NPHP1, AHI1, CEP290, RPGRIP1L, TMEM67, and ARL13B) function in the primary cilium/basal body organelle. By using homozygosity mapping in consanguineous families, we identify loss-of-function mutations in CC2D2A in JSRD patients with and without retinal, kidney, and liver disease. CC2D2A is expressed in all fetal and adult tissues tested. In ciliated cells, we observe localization of recombinant CC2D2A at the basal body and colocalization with CEP290, whose cognate gene is mutated in multiple hereditary ciliopathies. In addition, the proteins can physically interact in vitro, as shown by yeast two-hybrid and GST pull-down experiments. A nonsense mutation in the zebrafish CC2D2A ortholog (sentinel) results in pronephric cysts, a hallmark of ciliary dysfunction analogous to human cystic kidney disease. Knockdown of cep290 function in sentinel fish results in a synergistic pronephric cyst phenotype, revealing a genetic interaction between CC2D2A and CEP290 and implicating CC2D2A in cilium/basal body function. These observations extend the genetic spectrum of JSRD and provide a model system for studying extragenic modifiers in JSRD and other ciliopathies.

Joubert syndrome (and related disorders) (OMIM 213300)

Joubert syndrome (JS) and related disorders are characterized by the ‘molar tooth sign’ (cerebellar vermis hypoplasia and brainstem anomalies) on MRI, hypotonia, developmental delay, ataxia, irregular breathing pattern and abnormal eye movements. Combinations of additional features such as polydactyly, ocular coloboma, retinal dystrophy, renal disease, hepatic fibrosis, encephalocele, and other brain malformations define clinical sub-types. Recent identification of the NPHP1, AHI1, and CEP290 genes has started to reveal the molecular basis of JS, which may implicate the primary cilium in these disorders. Additional genes remain to be identified.

CEP41 is mutated in Joubert syndrome and is required for tubulin glutamylation at the cilium

Tubulin glutamylation is a post-translational modification that occurs predominantly in the ciliary axoneme and has been suggested to be important for ciliary function. However, its relationship to disorders of the primary cilium, termed ciliopathies, has not been explored. Here we mapped a new locus for Joubert syndrome (JBTS), which we have designated as JBTS15, and identified causative mutations in CEP41, which encodes a 41-kDa centrosomal protein. We show that CEP41 is localized to the basal body and primary cilia, and regulates ciliary entry of TTLL6, an evolutionarily conserved polyglutamylase enzyme. Depletion of CEP41 causes ciliopathy-related phenotypes in zebrafish and mice and results in glutamylation defects in the ciliary axoneme. Our data identify CEP41 mutations as a cause of JBTS and implicate tubulin post-translational modification in the pathogenesis of human ciliary dysfunction.

AHI1 mutations cause both retinal dystrophy and renal cystic disease in Joubert syndrome

Background: Joubert syndrome (JS) is an autosomal recessive disorder characterised by hypotonia, ataxia, mental retardation, altered respiratory pattern, abnormal eye movements, and a brain malformation known as the molar tooth sign (MTS) on cranial MRI. Four genetic loci have been mapped, with two genes identified (AHI1 and NPHP1). Methods: We screened a cohort of 117 JS subjects for AHI1 mutations by a combination of haplotype analysis and sequencing of the gene, and for the homozygous NPHP1 deletion by sequencing and marker analysis. Results: We identified a total of 15 novel AHI1 mutations in 13 families, including nonsense, missense, splice site, and insertion mutations, with some clustering in the WD40 domains. Eight families were consanguineous, but no single founder mutation was apparent. In addition to the MTS, retinal dystrophy was present in 11 of 12 informative families; however, no subjects exhibited variable features of JS such as polydactyly, encephalocele, colobomas, or liver fibrosis. In contrast to previous reports, we identified two families with affected siblings who developed renal disease consistent with nephronophthisis (NPH) in their 20s. In addition, two individuals with classic NPH were found to have homozygous NPHP1 deletions. Conclusions: Overall, 11% of subjects had AHI1 mutations, while ∼2% had the NPHP1 deletion, representing a total of less than 15% in a large JS cohort. Some preliminary genotype-phenotype correlations are possible, notably the association of renal impairment, specifically NPH, in those with NPHP1 deletions. Subjects with AHI1 mutations may be at risk of developing both retinal dystrophy and progressive kidney disease.

Mutations in 3 genes (MKS3, CC2D2A and RPGRIP1L) cause COACH syndrome (Joubert syndrome with congenital hepatic fibrosis)

Objective To identify genetic causes of COACH syndrome Background COACH syndrome is a rare autosomal recessive disorder characterised by Cerebellar vermis hypoplasia, Oligophrenia (developmental delay/mental retardation), Ataxia, Coloboma, and Hepatic fibrosis. The vermis hypoplasia falls in a spectrum of mid-hindbrain malformation called the molar tooth sign (MTS), making COACH a Joubert syndrome related disorder (JSRD). Methods In a cohort of 251 families with JSRD, 26 subjects in 23 families met criteria for COACH syndrome, defined as JSRD plus clinically apparent liver disease. Diagnostic criteria for JSRD were clinical findings (intellectual impairment, hypotonia, ataxia) plus supportive brain imaging findings (MTS or cerebellar vermis hypoplasia). MKS3/TMEM67 was sequenced in all subjects for whom DNA was available. In COACH subjects without MKS3 mutations, CC2D2A, RPGRIP1L and CEP290 were also sequenced. Results 19/23 families (83%) with COACH syndrome carried MKS3 mutations, compared to 2/209 (1%) with JSRD but no liver disease. Two other families with COACH carried CC2D2A mutations, one family carried RPGRIP1L mutations, and one lacked mutations in MKS3, CC2D2A, RPGRIP1L and CEP290. Liver biopsies from three subjects, each with mutations in one of the three genes, revealed changes within the congenital hepatic fibrosis/ductal plate malformation spectrum. In JSRD with and without liver disease, MKS3 mutations account for 21/232 families (9%). Conclusions Mutations in MKS3 are responsible for the majority of COACH syndrome, with minor contributions from CC2D2A and RPGRIP1L; therefore, MKS3 should be the first gene tested in patients with JSRD plus liver disease and/or coloboma, followed by CC2D2A and RPGRIP1L.