[show abstract][hide abstract] ABSTRACT: Purpose: Mutations at some retinitis pigmentosa (RP) loci are associated with variable penetrance and expressivity, exacerbating diagnostic challenges. The purpose of this study was to dissect the genetic underpinnings of non-syndromic RP with variable age of onset in a large Mexican family. Methods: We ascertained members of a large, multigenerational pedigree using a complete ophthalmic examination. We performed whole-exome sequencing on two affected first cousins, an obligate carrier, and a married-in spouse. Confirmatory sequencing of candidate variants was performed in the entire pedigree, as well as genotyping and mRNA studies to investigate expression changes in the causal locus. Results: We identified a 14-bp deletion in PRPF31, a gene implicated previously in autosomal dominant (ad) RP. The mutation segregated with the phenotype of all ten affected females, but was also present in six asymptomatics (two females and four males). Studies in patient cells showed that the penetrance/expressivity of the PRPF31 deletion allele was concordant with the expression levels of wild-type message. However, neither the known PRPF31 modulators nor cis-eQTLs within 1Mb of the locus could account for the variable expression of message or the clinical phenotype. Conclusions: We have identified a novel 14-bp deletion in PRPF31 as the genetic driver of adRP in a large Mexican family that exhibits both non-penetrance and variable expressivity, a known property of this locus. However, our studies intimate the presence of additional loci that can modify PRPF31 expression.
[show abstract][hide abstract] ABSTRACT: Ciliopathies are characterized by a pattern of multisystem involvement that is consistent with the developmental role of the primary cilium. Within this biological module, mutations in genes that encode components of the cilium and its anchoring structure, the basal body, are the major contributors to both disease causality and modification. However, despite rapid advances in this field, the majority of the genes that drive ciliopathies and the mechanisms that govern the pronounced phenotypic variability of this group of disorders remain poorly understood. Here, we show that mutations in CSPP1, which encodes a core centrosomal protein, are disease causing on the basis of the independent identification of two homozygous truncating mutations in three consanguineous families (one Arab and two Hutterite) affected by variable ciliopathy phenotypes ranging from Joubert syndrome to the more severe Meckel-Gruber syndrome with perinatal lethality and occipital encephalocele. Consistent with the recently described role of CSPP1 in ciliogenesis, we show that mutant fibroblasts from one affected individual have severely impaired ciliogenesis with concomitant defects in sonic hedgehog (SHH) signaling. Our results expand the list of centrosomal proteins implicated in human ciliopathies.
The American Journal of Human Genetics 12/2013; · 11.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rare single-gene disorders cause chronic disease. However, half of the 6000 recessive single gene causes of disease are still unknown. Because recessive disease genes can illuminate, at least in part, disease pathomechanism, their identification offers direct opportunities for improved clinical management and potentially treatment. Rare diseases comprise the majority of chronic kidney disease (CKD) in children but are notoriously difficult to diagnose. Whole-exome resequencing facilitates identification of recessive disease genes. However, its utility is impeded by the large number of genetic variants detected. We here overcome this limitation by combining homozygosity mapping with whole-exome resequencing in 10 sib pairs with a nephronophthisis-related ciliopathy, which represents the most frequent genetic cause of CKD in the first three decades of life. In 7 of 10 sibships with a histologic or ultrasonographic diagnosis of nephronophthisis-related ciliopathy, we detect the causative gene. In six sibships, we identify mutations of known nephronophthisis-related ciliopathy genes, while in two additional sibships we found mutations in the known CKD-causing genes SLC4A1 and AGXT as phenocopies of nephronophthisis-related ciliopathy. Thus, whole-exome resequencing establishes an efficient, noninvasive approach towards early detection and causation-based diagnosis of rare kidney diseases. This approach can be extended to other rare recessive disorders, thereby providing accurate diagnosis and facilitating the study of disease mechanisms.Kidney International advance online publication, 20 November 2013; doi:10.1038/ki.2013.450.
Kidney International 11/2013; · 7.92 Impact Factor
[show abstract][hide abstract] ABSTRACT: The motive forces for ciliary movement are generated by large multiprotein complexes referred to as outer dynein arms (ODAs), which are preassembled in the cytoplasm prior to transport to the ciliary axonemal compartment. In humans, defects in structural components, docking complexes, or cytoplasmic assembly factors can cause primary ciliary dyskinesia (PCD), a disorder characterized by chronic airway disease and defects in laterality. By using combined high resolution copy-number variant and mutation analysis, we identified ARMC4 mutations in twelve PCD individuals whose cells showed reduced numbers of ODAs and severely impaired ciliary beating. Transient suppression in zebrafish and analysis of an ENU mouse mutant confirmed in both model organisms that ARMC4 is critical for left-right patterning. We demonstrate that ARMC4 is an axonemal protein that is necessary for proper targeting and anchoring of ODAs.
The American Journal of Human Genetics 07/2013; · 11.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: White matter hyperintensities (WMHs) of the brain are important markers of aging and small-vessel disease. WMHs are rare in healthy children and, when observed, often occur with comorbid neuroinflammatory or vasculitic processes. Here, we describe a complex 4 kb deletion in 2q36.3 that segregates with early childhood communication disorders and WMH in 15 unrelated families predominantly from Southeast Asia. The premature brain aging phenotype with punctate and multifocal WMHs was observed in ∼70% of young carrier parents who underwent brain MRI. The complex deletion removes the penultimate exon 3 of TM4SF20, a gene encoding a transmembrane protein of unknown function. Minigene analysis showed that the resultant net loss of an exon introduces a premature stop codon, which, in turn, leads to the generation of a stable protein that fails to target to the plasma membrane and accumulates in the cytoplasm. Finally, we report this deletion to be enriched in individuals of Vietnamese Kinh descent, with an allele frequency of about 1%, embedded in an ancestral haplotype. Our data point to a constellation of early language delay and WMH phenotypes, driven by a likely toxic mechanism of TM4SF20 truncation, and highlight the importance of understanding and managing population-specific low-frequency pathogenic alleles.
The American Journal of Human Genetics 06/2013; · 11.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: Skeletal dysplasias are challenging to diagnose because of their phenotypic variability, genetic heterogeneity, and diverse inheritance patterns. We conducted WES of a Turkish male with a suspected X-linked skeletal dysplasia of unknown etiology as well as his unaffected mother and maternal uncle. Bioinformatic filtering of variants implicated in skeletal system development revealed a novel hemizygous mutation, c.341-(11_9)delAAT, in an intron of TRAPPC2, the causative locus of spondyloepiphyseal dysplasia tarda (SEDT). We show that this deletion leads to the loss of wild-type TRAPPC2 and the generation of two functionally impaired mRNAs in patient cells. These consequences are predicted to disrupt function of SEDLIN/TRAPPC2. The clinical and research data were returned, with appropriate caveats, to the patient and informed his disease status and reproductive choices. Our findings expand the allelic repertoire of SEDT and demonstrate how prior filtering of the morbid human genome informed by inheritance pattern and phenotype, when combined with appropriate functional tests in patient-derived cells, can expedite discovery, overcome issues of missing data and help interpret variants of unknown significance. Finally, this example demonstrates how the return of a clinically confirmed mutational finding, supported by research allele pathogenicity data, can assist individuals with inherited disorders with life choices.
[show abstract][hide abstract] ABSTRACT: Seminoma is a subclass of human testicular germ cell tumors (TGCT), the most frequently observed cancer in young men with a rising incidence. Here we describe the identification of a novel gene predisposing specifically to seminoma formation in a vertebrate model organism. Zebrafish carrying a heterozygous nonsense mutation in Leucine-Rich Repeat Containing protein 50 (lrrc50 also called dnaaf1), associated previously with ciliary function, are found to be highly susceptible to the formation of seminomas. Genotyping of these zebrafish tumors shows loss of heterozygosity (LOH) of the wild-type lrrc50 allele in 44.4% of tumor samples, correlating with tumor progression. In humans we identified heterozygous germline LRRC50 mutations in two different pedigrees with a family history of seminomas, resulting in a nonsense Arg488* change and a missense Thr590Met change, which show reduced expression of the wild-type allele in seminomas. Zebrafish in vivo complementation studies indicate the Thr590Met to be a loss-of-function mutation. Moreover, we show that a pathogenic Gln307Glu change is significantly enriched in individuals with seminoma tumors (13% of our cohort). Together, our study introduces an animal model for seminoma and suggests LRRC50 to be a novel tumor suppressor implicated in human seminoma pathogenesis.
[show abstract][hide abstract] ABSTRACT: Intraflagellar transport (IFT) depends on two evolutionarily conserved modules, subcomplexes A (IFT-A) and B (IFT-B), to drive ciliary assembly and maintenance. All six IFT-A components and their motor protein, DYNC2H1, have been linked to human skeletal ciliopa-thies, including asphyxiating thoracic dystrophy (ATD; also known as Jeune syndrome), Sensenbrenner syndrome, and Mainzer-Saldino syndrome (MZSDS). Conversely, the 14 subunits in the IFT-B module, with the exception of IFT80, have unknown roles in human disease. To identify additional IFT-B components defective in ciliopathies, we independently performed different mutation analyses: candidate-based sequencing of all IFT-B-encoding genes in 1,467 individuals with a nephronophthisis-related ciliopathy or whole-exome resequencing in 63 individuals with ATD. We thereby detected biallelic mutations in the IFT-B-encoding gene IFT172 in 12 fam-ilies. All affected individuals displayed abnormalities of the thorax and/or long bones, as well as renal, hepatic, or retinal involvement, consistent with the diagnosis of ATD or MZSDS. Additionally, cerebellar aplasia or hypoplasia characteristic of Joubert syndrome was present in 2 out of 12 families. Fibroblasts from affected individuals showed disturbed ciliary composition, suggesting alteration of ciliary transport and signaling. Knockdown of ift172 in zebrafish recapitulated the human phenotype and demonstrated a genetic interaction between ift172 and ift80. In summary, we have identified defects in IFT172 as a cause of complex ATD and MZSDS. Our findings link the group of skeletal ciliopathies to an additional IFT-B component, IFT172, similar to what has been shown for IFT-A. Cilia are hair-like structures that project from the surface of most mammalian cells and are involved in diverse signaling pathways. Mutations in genes encoding ciliary
The American Journal of Human Genetics 01/2013; · 11.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.(1) These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.
[show abstract][hide abstract] ABSTRACT: Cilia are evolutionarily conserved microtubule-based organelles that are crucial for diverse biological functions, including motility, cell signaling and sensory perception. In humans, alterations in the formation and function of cilia manifest clinically as ciliopathies, a growing class of pleiotropic genetic disorders. Despite the substantial progress that has been made in identifying genes that cause ciliopathies, therapies for these disorders are not yet available to patients. Although mice with a hypomorphic mutation in the intraflagellar transport protein IFT88 (Ift88(Tg737Rpw) mice, also known as ORPK mice) have been well studied, the relevance of IFT88 mutations to human pathology is unknown. We show that a mutation in IFT88 causes a hitherto unknown human ciliopathy. In vivo complementation assays in zebrafish and mIMCD3 cells show the pathogenicity of this newly discovered allele. We further show that ORPK mice are functionally anosmic as a result of the loss of cilia on their olfactory sensory neurons (OSNs). Notably, adenoviral-mediated expression of IFT88 in mature, fully differentiated OSNs of ORPK mice is sufficient to restore ciliary structures and rescue olfactory function. These studies are the first to use in vivo therapeutic treatment to reestablish cilia in a mammalian ciliopathy. More broadly, our studies indicate that gene therapy is a viable option for cellular and functional rescue of the complex ciliary organelle in established differentiated cells.
[show abstract][hide abstract] ABSTRACT: The last decade has witnessed an explosion in the identification of genes, mutations in which appear sufficient to cause clinical phenotypes in humans. This is especially true for disorders of ciliary dysfunction in which an excess of 50 causal loci are now known; this discovery was driven partly by an improved understanding of the protein composition of the cilium and the co-occurrence of clinical phenotypes associated with ciliary dysfunction. Despite this progress, the fundamental challenge of predicting phenotype and or clinical progression based on single locus information remains unsolved. Here, we explore how the combinatorial knowledge of allele quality and quantity, an improved understanding of the biological composition of the primary cilium, and the expanded appreciation of the subcellular roles of this organelle can be synthesized to generate improved models that can explain both causality but also variable penetrance and expressivity.
Current opinion in genetics & development 05/2012; 22(3):290-303. · 8.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: Otocephaly or dysgnathia complex is characterised by mandibular hypoplasia/agenesis, ear anomalies, microstomia, and microglossia; the molecular basis of this developmental defect is largely unknown in humans.
This study reports a large family in which two cousins with micro/anophthalmia each gave birth to at least one child with otocephaly, suggesting a genetic relationship between anophthalmia and otocephaly. OTX2, a known microphthalmia locus, was screened in this family and a frameshifting mutation was found. The study subsequently identified in one unrelated otocephalic patient a sporadic OTX2 mutation. Because OTX2 mutations may not be sufficient to cause otocephaly, the study assayed the potential of otx2 to modify craniofacial phenotypes in the context of known otocephaly gene suppression in vivo. It was found that otx2 can interact genetically with pgap1, prrx1, and msx1 to exacerbate mandibular and midline defects during zebrafish development. However, sequencing of these loci in the OTX2-positive families did not unearth likely pathogenic lesions, suggesting further genetic heterogeneity and complexity.
Identification of OTX2 involvement in otocephaly/dysgnathia in humans, even if loss of function mutations at this locus does not sufficiently explain the complex anatomical defects of these patients, suggests the requirement for a second genetic hit. Consistent with this notion, trans suppression of otx2 and other developmentally related genes recapitulate aspects of the otocephaly phenotype in zebrafish. This study highlights the combined utility of genetics and functional approaches to dissect both the regulatory pathways that govern craniofacial development and the genetics of this disease group.
Journal of Medical Genetics 05/2012; 49(6):373-9. · 5.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: Neighboring genes are often coordinately expressed within cis-regulatory modules, but evidence that nonparalogous genes share functions in mammals is lacking. Here, we report that mutation of either TMEM138 or TMEM216 causes a phenotypically indistinguishable human ciliopathy, Joubert syndrome. Despite a lack of sequence homology, the genes are aligned in a head-to-tail configuration and joined by chromosomal rearrangement at the amphibian-to-reptile evolutionary transition. Expression of the two genes is mediated by a conserved regulatory element in the noncoding intergenic region. Coordinated expression is important for their interdependent cellular role in vesicular transport to primary cilia. Hence, during vertebrate evolution of genes involved in ciliogenesis, nonparalogous genes were arranged to a functional gene cluster with shared regulatory elements.
[show abstract][hide abstract] ABSTRACT: Joubert syndrome related disorders (JSRDs) have broad but variable phenotypic overlap with other ciliopathies. The molecular etiology of this overlap is unclear but probably arises from disrupting common functional module components within primary cilia. To identify additional module elements associated with JSRDs, we performed homozygosity mapping followed by next-generation sequencing (NGS) and uncovered mutations in TMEM237 (previously known as ALS2CR4). We show that loss of the mammalian TMEM237, which localizes to the ciliary transition zone (TZ), results in defective ciliogenesis and deregulation of Wnt signaling. Furthermore, disruption of Danio rerio (zebrafish) tmem237 expression produces gastrulation defects consistent with ciliary dysfunction, and Caenorhabditis elegans jbts-14 genetically interacts with nphp-4, encoding another TZ protein, to control basal body-TZ anchoring to the membrane and ciliogenesis. Both mammalian and C. elegans TMEM237/JBTS-14 require RPGRIP1L/MKS5 for proper TZ localization, and we demonstrate additional functional interactions between C. elegans JBTS-14 and MKS-2/TMEM216, MKSR-1/B9D1, and MKSR-2/B9D2. Collectively, our findings integrate TMEM237/JBTS-14 in a complex interaction network of TZ-associated proteins and reveal a growing contribution of a TZ functional module to the spectrum of ciliopathy phenotypes.
The American Journal of Human Genetics 12/2011; 89(6):713-30. · 11.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: KIF7, the human ortholog of Drosophila Costal2, is a key component of the Hedgehog signaling pathway. Here we report mutations in KIF7 in individuals with hydrolethalus and acrocallosal syndromes, two multiple malformation disorders with overlapping features that include polydactyly, brain abnormalities and cleft palate. Consistent with a role of KIF7 in Hedgehog signaling, we show deregulation of most GLI transcription factor targets and impaired GLI3 processing in tissues from individuals with KIF7 mutations. KIF7 is also a likely contributor of alleles across the ciliopathy spectrum, as sequencing of a diverse cohort identified several missense mutations detrimental to protein function. In addition, in vivo genetic interaction studies indicated that knockdown of KIF7 could exacerbate the phenotype induced by knockdown of other ciliopathy transcripts. Our data show the role of KIF7 in human primary cilia, especially in the Hedgehog pathway through the regulation of GLI targets, and expand the clinical spectrum of ciliopathies.
[show abstract][hide abstract] ABSTRACT: Ciliary dysfunction leads to a broad range of overlapping phenotypes, collectively termed ciliopathies. This grouping is underscored by genetic overlap, where causal genes can also contribute modifier alleles to clinically distinct disorders. Here we show that mutations in TTC21B, which encodes the retrograde intraflagellar transport protein IFT139, cause both isolated nephronophthisis and syndromic Jeune asphyxiating thoracic dystrophy. Moreover, although resequencing of TTC21B in a large, clinically diverse ciliopathy cohort and matched controls showed a similar frequency of rare changes, in vivo and in vitro evaluations showed a significant enrichment of pathogenic alleles in cases (P < 0.003), suggesting that TTC21B contributes pathogenic alleles to ∼5% of ciliopathy cases. Our data illustrate how genetic lesions can be both causally associated with diverse ciliopathies and interact in trans with other disease-causing genes and highlight how saturated resequencing followed by functional analysis of all variants informs the genetic architecture of inherited disorders.
[show abstract][hide abstract] ABSTRACT: Primary ciliary dyskinesia (PCD) is an inherited disorder characterized by recurrent infections of the upper and lower respiratory tract, reduced fertility in males and situs inversus in about 50% of affected individuals (Kartagener syndrome). It is caused by motility defects in the respiratory cilia that are responsible for airway clearance, the flagella that propel sperm cells and the nodal monocilia that determine left-right asymmetry. Recessive mutations that cause PCD have been identified in genes encoding components of the outer dynein arms, radial spokes and cytoplasmic pre-assembly factors of axonemal dyneins, but these mutations account for only about 50% of cases of PCD. We exploited the unique properties of dog populations to positionally clone a new PCD gene, CCDC39. We found that loss-of-function mutations in the human ortholog underlie a substantial fraction of PCD cases with axonemal disorganization and abnormal ciliary beating. Functional analyses indicated that CCDC39 localizes to ciliary axonemes and is essential for assembly of inner dynein arms and the dynein regulatory complex.
[show abstract][hide abstract] ABSTRACT: Bardet-Biedl syndrome (BBS) is a rare, primarily autosomal-recessive ciliopathy. The phenotype of this pleiotropic disease includes retinitis pigmentosa, postaxial polydactyly, truncal obesity, learning disabilities, hypogonadism and renal anomalies, among others. To date, mutations in 15 genes (BBS1-BBS14, SDCCAG8) have been described to cause BBS. The broad genetic locus heterogeneity renders mutation screening time-consuming and expensive. We applied a strategy of DNA pooling and subsequent massively parallel resequencing (MPR) to screen individuals affected with BBS from 105 families for mutations in 12 known BBS genes. DNA was pooled in 5 pools of 21 individuals each. All 132 coding exons of BBS1-BBS12 were amplified by conventional PCR. Subsequent MPR was performed on an Illumina Genome Analyzer II™ platform. Following mutation identification, the mutation carrier was assigned by CEL I endonuclease heteroduplex screening and confirmed by Sanger sequencing. In 29 out of 105 individuals (28%), both mutated alleles were identified in 10 different BBS genes. A total of 35 different disease-causing mutations were confirmed, of which 18 mutations were novel. In 12 additional families, a total of 12 different single heterozygous changes of uncertain pathogenicity were found. Thus, DNA pooling combined with MPR offers a valuable strategy for mutation analysis of large patient cohorts, especially in genetically heterogeneous diseases such as BBS.
Human Genetics 10/2010; 129(1):79-90. · 4.63 Impact Factor