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1.  Correction: Hearing Loss in a Mouse Model of 22q11.2 Deletion Syndrome 
PLoS ONE  2014;9(1):10.1371/annotation/f10a686b-b6e2-4630-8c6f-7a91191ae732.
doi:10.1371/annotation/f10a686b-b6e2-4630-8c6f-7a91191ae732
PMCID: PMC3879389
2.  Deregulated FGF and homeotic gene expression underlies cerebellar vermis hypoplasia in CHARGE syndrome 
eLife  2013;2:e01305.
Mutations in CHD7 are the major cause of CHARGE syndrome, an autosomal dominant disorder with an estimated prevalence of 1/15,000. We have little understanding of the disruptions in the developmental programme that underpin brain defects associated with this syndrome. Using mouse models, we show that Chd7 haploinsufficiency results in reduced Fgf8 expression in the isthmus organiser (IsO), an embryonic signalling centre that directs early cerebellar development. Consistent with this observation, Chd7 and Fgf8 loss-of-function alleles interact during cerebellar development. CHD7 associates with Otx2 and Gbx2 regulatory elements and altered expression of these homeobox genes implicates CHD7 in the maintenance of cerebellar identity during embryogenesis. Finally, we report cerebellar vermis hypoplasia in 35% of CHARGE syndrome patients with a proven CHD7 mutation. These observations provide key insights into the molecular aetiology of cerebellar defects in CHARGE syndrome and link reduced FGF signalling to cerebellar vermis hypoplasia in a human syndrome.
DOI: http://dx.doi.org/10.7554/eLife.01305.001
eLife digest
CHARGE syndrome is a rare genetic condition that causes various developmental abnormalities, including heart defects, deafness and neurological defects. In most cases, it is caused by mutations in a human gene called CHD7. CHD7 is known to control the expression of other genes during embryonic development, but the molecular mechanisms by which mutations in CHD7 lead to the neural defects found in CHARGE syndrome are unclear.
During embryonic development, the neural tube—the precursor to the nervous system—is divided into segments, which give rise to different neural structures. The r1 segment, for example, forms the cerebellum, and the secretion of a protein called FGF8 (short for fibroblast growth factor 8) by a nearby structure called the isthmus organiser has an important role in this process. Since a reduction in FGF8 causes defects similar to those found in CHARGE syndrome, Yu et al. decided to investigate if the FGF signalling pathway was involved in this syndrome.
Mice should have two working copies of the Chd7 gene, and mice that lack one of these suffer from symptoms similar to those of humans with CHARGE syndrome. Yu et al. examined the embryos of these mice and found that the isthmus organiser produced less FGF8. Embryos with no working copies of the gene completely lost the r1 segment. The loss of this segment appeared to be caused by changes in the expression of homeobox genes (the genes that determine the identity of brain segments).
Embryos that did not have any working copies of the Chd7 gene died early in development, which made further studies impossible. However, embryos that had one working copy of the Chd7 gene survived, and Yu et al. took advantage of this to study the effects of reduced FGF8 expression on these mice. These experiments showed that mice with just one working copy of the Fgf8 gene and one working copy of the Chd7 gene had a small cerebellar vermis. This part of the cerebellum is known to be very sensitive to changes in FGF8 signalling. Yu et al. then used an MRI scanner to look at the cerebellar vermis in patients with CHARGE syndrome, and found that more than half of the patients had abnormal cerebella.
In addition to confirming that studies on mouse embryos can provide insights into human disease, the work of Yu et al. add defects in the cerebellar vermis to the list of developmental abnormalities associated with CHARGE syndrome. The next step will be to test if any mutations in the human FGF8 gene can contribute to cerebellar defects in CHARGE syndrome, and to investigate if any other developmental defects in CHARGE syndrome are associated with abnormal FGF8 levels.
DOI: http://dx.doi.org/10.7554/eLife.01305.002
doi:10.7554/eLife.01305
PMCID: PMC3870572  PMID: 24368733
cerebellum; CHARGE syndrome; CHD7; FGF8; OTX2; GBX2; Human; Mouse
3.  Hearing Loss in a Mouse Model of 22q11.2 Deletion Syndrome 
PLoS ONE  2013;8(11):e80104.
22q11.2 Deletion Syndrome (22q11DS) arises from an interstitial chromosomal microdeletion encompassing at least 30 genes. This disorder is one of the most significant known cytogenetic risk factors for schizophrenia, and can also cause heart abnormalities, cognitive deficits, hearing difficulties, and a variety of other medical problems. The Df1/+ hemizygous knockout mouse, a model for human 22q11DS, recapitulates many of the deficits observed in the human syndrome including heart defects, impaired memory, and abnormal auditory sensorimotor gating. Here we show that Df1/+ mice, like human 22q11DS patients, have substantial rates of hearing loss arising from chronic middle ear infection. Auditory brainstem response (ABR) measurements revealed significant elevation of click-response thresholds in 48% of Df1/+ mice, often in only one ear. Anatomical and histological analysis of the middle ear demonstrated no gross structural abnormalities, but frequent signs of otitis media (OM, chronic inflammation of the middle ear), including excessive effusion and thickened mucosa. In mice for which both in vivo ABR thresholds and post mortem middle-ear histology were obtained, the severity of signs of OM correlated directly with the level of hearing impairment. These results suggest that abnormal auditory sensorimotor gating previously reported in mouse models of 22q11DS could arise from abnormalities in auditory processing. Furthermore, the findings indicate that Df1/+ mice are an excellent model for increased risk of OM in human 22q11DS patients. Given the frequently monaural nature of OM in Df1/+ mice, these animals could also be a powerful tool for investigating the interplay between genetic and environmental causes of OM.
doi:10.1371/journal.pone.0080104
PMCID: PMC3828191  PMID: 24244619
4.  Novel exomphalos genetic mouse model: The importance of accurate phenotypic classification 
Journal of Pediatric Surgery  2013;48(10):2036-2042.
Background
Rodent models of abdominal wall defects (AWD) may provide insight into the pathophysiology of these conditions including gut dysfunction in gastroschisis, or pulmonary hypoplasia in exomphalos. Previously, a Scribble mutant mouse model (circletail) was reported to exhibit gastroschisis. We further characterise this AWD in Scribble knockout mice.
Method
Homozygous Scrib knockout mice were obtained from heterozygote matings. Fetuses were collected at E17.5–18.5 with intact amniotic membranes. Three mutants and two control fetuses were imaged by in amnio micro-MRI. Remaining fetuses were dissected, photographed and gut length/weight measured. Ileal specimens were stained for interstitial cells of Cajal (ICC), imaged using confocal microscopy and ICC quantified.
Results
127 fetuses were collected, 15 (12%) exhibited AWD. Microdissection revealed 3 mutants had characteristic exomphalos phenotype with membrane-covered gut/liver herniation into the umbilical cord. A further 12 exhibited extensive AWD, with eviscerated abdominal organs and thin covering membrane (intact or ruptured). Micro-MRI confirmed these phenotypes. Gut was shorter and heavier in AWD group compared to controls but morphology/number of ICC was not different.
Discussion
The Scribble knockout fetus exhibits exomphalos (intact and ruptured), in contrast to the original published phenotype of gastroschisis. Detailed dissection of fetuses is essential ensuring accurate phenotyping and result reporting.
doi:10.1016/j.jpedsurg.2013.04.010
PMCID: PMC4030649  PMID: 24094954
Exomphalos; Gastroschisis; Abdominal wall defect; Rodent model; Scrib; In amnio micro-MRI; Interstitial cells of Cajal
5.  Mutations in CCDC39 and CCDC40 are the major cause of primary ciliary dyskinesia with axonemal disorganisation and absent inner dynein arms 
Human mutation  2013;34(3):462-472.
Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder caused by cilia and sperm dysmotility. About 12% of cases show perturbed 9+2 microtubule cilia structure and inner dynein arm (IDA) loss, historically termed ‘radial spoke defect’. We sequenced CCDC39 and CCDC40 in 54 ‘radial spoke defect’ families, as these are the two genes identified so far to cause this defect. We discovered biallelic mutations in a remarkable 69% (37/54) of families, including identification of 25 (19 novel) mutant alleles (12 in CCDC39 and 13 in CCDC40). All the mutations were nonsense, splice and frameshift predicting early protein truncation, which suggests this defect is caused by ‘null’ alleles conferring complete protein loss. Most families (73%; 27/37) had homozygous mutations, including families from outbred populations. A major putative hotspot mutation was identified, CCDC40 c.248delC, as well as several other possible hotspot mutations. Together, these findings highlight the key role of CCDC39 and CCDC40 in PCD with axonemal disorganisation and IDA loss, and these genes represent major candidates for genetic testing in families affected by this ciliary phenotype. We show that radial spoke structures are largely intact in these patients and propose this ciliary ultrastructural abnormality be referred to as ‘IDA and nexin-dynein regulatory complex (N-DRC) defect’, rather than ‘radial spoke defect’.
doi:10.1002/humu.22261
PMCID: PMC3630464  PMID: 23255504
primary ciliary dyskinesia; cilia; CCDC39; CCDC40; radial spoke; dynein regulatory complex; nexin link
6.  An Ift80 mouse model of short rib polydactyly syndromes shows defects in hedgehog signalling without loss or malformation of cilia 
Human Molecular Genetics  2011;20(7):1306-1314.
IFT80, a protein component of intraflagellar transport (IFT) complex B, is required for the formation, maintenance and functionality of cilia. Mutations in IFT80 cause Jeune asphyxiating thoracic dystrophy (JATD) and short rib polydactyly (SRP) type III. Both diseases are autosomal recessive chondrodysplasias and share clinical and radiological similarities, including shortening of the long bones and constriction of the thoracic cage. A murine Ift80 gene-trap line was used to investigate the role of Ift80 during development. The homozygote appears hypomorphic rather than a true null due to low level wild-type transcript production by alternative splicing around the gene-trap cassette. Hypomorphic levels of Ift80 result in embryonic lethality highlighting a key role for Ift80 in development. In rare cases, gene-trap homozygotes survive to postnatal stages and phenocopy both JATD and SRP type III by exhibiting growth retardation, shortening of the long bones, constriction of the ribcage and polydactyly. Mouse embryonic fibroblasts made from this line showed a significant reduction in hedgehog pathway activation in response to Hedgehog analog treatment. This defective signalling was not accompanied by the loss or malformation of cilia as seen in some knockout models of other IFT component genes. Phenotypes indicative of defects in cilia structure or function such as situs inversus, cystic renal disease and retinal degeneration were not observed in this line. These data suggest that there is an absolute requirement for Ift80 in hedgehog signalling, but low level expression permits ciliogenesis indicating separate but linked roles for this protein in formation and function.
doi:10.1093/hmg/ddr013
PMCID: PMC3049354  PMID: 21227999
7.  Expression of Fraser syndrome genes in normal and polycystic murine kidneys 
Background
Fraser syndrome (FS) features renal agenesis and cystic kidneys. Mutations of FRAS1 (Fraser syndrome 1) and FREM2 (FRAS1-related extracellular matrix protein 2) cause FS. They code for basement membrane proteins expressed in metanephric epithelia where they mediate epithelial/mesenchymal signalling. Little is known about whether and where these molecules are expressed in more mature kidneys.
Methods
In healthy and congenital polycystic kidney (cpk) mouse kidneys we sought Frem2 expression using a LacZ reporter gene and quantified Fras family transcripts. Fras1 immunohistochemistry was undertaken in cystic kidneys from cpk mice and PCK (Pkhd1 mutant) rats (models of autosomal recessive polycystic kidney disease) and in wild-type metanephroi rendered cystic by dexamethasone.
Results
Nascent nephrons transiently expressed Frem2 in both tubule and podocyte epithelia. Maturing and adult collecting ducts also expressed Frem2. Frem2 was expressed in cpk cystic epithelia although Frem2 haploinsufficiency did not significantly modify cystogenesis in vivo. Fras1 transcripts were significantly upregulated, and Frem3 downregulated, in polycystic kidneys versus the non-cystic kidneys of littermates. Fras1 was immunodetected in cpk, PCK and dexamethasone-induced cyst epithelia.
Conclusions
These descriptive results are consistent with the hypothesis that Fras family molecules play diverse roles in kidney epithelia. In future, this should be tested by conditional deletion of FS genes in nephron segments and collecting ducts.
doi:10.1007/s00467-012-2100-5
PMCID: PMC3337421  PMID: 21993971
Basement membrane; Cyst; Development; Fras1; Frem2; LacZ; Reporter gene; Medicine & Public Health; Pediatrics
8.  Endogenous Retinoic Acid Activity in Principal Cells and Intercalated Cells of Mouse Collecting Duct System 
PLoS ONE  2011;6(2):e16770.
Background
Retinoic acid is the bioactive derivative of vitamin A, which plays an indispensible role in kidney development by activating retinoic acid receptors. Although the location, concentration and roles of endogenous retinoic acid in post-natal kidneys are poorly defined, there is accumulating evidence linking post-natal vitamin A deficiency to impaired renal concentrating and acidifying capacity associated with increased susceptibility to urolithiasis, renal inflammation and scarring. The aim of this study is to examine the presence and the detailed localization of endogenous retinoic acid activity in neonatal, young and adult mouse kidneys, to establish a fundamental ground for further research into potential target genes, as well as physiological and pathophysiological roles of endogenous retinoic acid in the post-natal kidneys.
Methodology/Principal Findings
RARE-hsp68-lacZ transgenic mice were employed as a reporter for endogenous retinoic acid activity that was determined by X-gal assay and immunostaining of the reporter gene product, β-galactosidase. Double immunostaining was performed for β-galactosidase and markers of kidney tubules to localize retinoic acid activity. Distinct pattern of retinoic acid activity was observed in kidneys, which is higher in neonatal and 1- to 3-week-old mice than that in 5- and 8-week-old mice. The activity was present specifically in the principal cells and the intercalated cells of the collecting duct system in all age groups, but was absent from the glomeruli, proximal tubules, thin limbs of Henle's loop and distal tubules.
Conclusions/Significance
Endogenous retinoic acid activity exists in principal cells and intercalated cells of the mouse collecting duct system after birth and persists into adulthood. This observation provides novel insights into potential roles for endogenous retinoic acid beyond nephrogenesis and warrants further studies to investigate target genes and functions of endogenous retinoic acid in the kidney after birth, particularly in the collecting duct system.
doi:10.1371/journal.pone.0016770
PMCID: PMC3033902  PMID: 21326615
9.  Hes1 expression is reduced in Tbx1 null cells and is required for the development of structures affected in 22q11 deletion syndrome 
Developmental Biology  2010;340(2):369-380.
22q11 deletion syndrome (22q11DS) is characterised by aberrant development of the pharyngeal apparatus and the heart with haploinsufficiency of the transcription factor TBX1 being considered the major underlying cause of the disease. Tbx1 mutations in mouse phenocopy the disorder. In order to identify the transcriptional dysregulation in Tbx1-expressing lineages we optimised fluorescent-activated cell sorting of β-galactosidase expressing cells (FACS-Gal) to compare the expression profile of Df1/Tbx1lacZ (effectively Tbx1 null) and Tbx1 heterozygous cells isolated from mouse embryos. Hes1, a major effector of Notch signalling, was identified as downregulated in Tbx1−/− mutants. Hes1 mutant mice exhibited a partially penetrant range of 22q11DS-like defects including pharyngeal arch artery (PAA), outflow tract, craniofacial and thymic abnormalities. Similar to Tbx1 mice, conditional mutagenesis revealed that Hes1 expression in embryonic pharyngeal ectoderm contributes to thymus and pharyngeal arch artery development. These results suggest that Hes1 acts downstream of Tbx1 in the morphogenesis of pharyngeal-derived structures.
doi:10.1016/j.ydbio.2010.01.020
PMCID: PMC2877781  PMID: 20122914
Tbx1; Hes1; 22q11; Pharyngeal arch artery; Thymus; Mouse; Microarray; FACS
10.  Great vessel development requires biallelic expression of Chd7 and Tbx1 in pharyngeal ectoderm in mice  
The Journal of Clinical Investigation  2009;119(11):3301-3310.
Aortic arch artery patterning defects account for approximately 20% of congenital cardiovascular malformations and are observed frequently in velocardiofacial syndrome (VCFS). In the current study, we screened for chromosome rearrangements in patients suspected of VCFS, but who lacked a 22q11 deletion or TBX1 mutation. One individual displayed hemizygous CHD7, which encodes a chromodomain protein. CHD7 haploinsufficiency is the major cause of coloboma, heart defect, atresia choanae, retarded growth and development, genital hypoplasia, and ear anomalies/deafness (CHARGE) syndrome, but this patient lacked the major diagnostic features of coloboma and choanal atresia. Because a subset of CHARGE cases also display 22q11 deletions, we explored the embryological relationship between CHARGE and VCSF using mouse models. The hallmark of Tbx1 haploinsufficiency is hypo/aplasia of the fourth pharyngeal arch artery (PAA) at E10.5. Identical malformations were observed in Chd7 heterozygotes, with resulting aortic arch interruption at later stages. Other than Tbx1, Chd7 is the only gene reported to affect fourth PAA development by haploinsufficiency. Moreover, Tbx1+/–;Chd7+/– double heterozygotes demonstrated a synergistic interaction during fourth PAA, thymus, and ear morphogenesis. We could not rescue PAA morphogenesis by restoring neural crest Chd7 expression. Rather, biallelic expression of Chd7 and Tbx1 in the pharyngeal ectoderm was required for normal PAA development.
doi:10.1172/JCI37561
PMCID: PMC2769172  PMID: 19855134
11.  Tbx1 Regulates the BMP-Smad1 Pathway in a Transcription Independent Manner 
PLoS ONE  2009;4(6):e6049.
Tbx1 is a T-box transcription factor implicated in DiGeorge syndrome. The molecular function of Tbx1 is unclear although it can transactivate reporters with T-box binding elements. We discovered that Tbx1 binds Smad1 and suppresses the Bmp4/Smad1 signaling. Tbx1 interferes with Smad1 to Smad4 binding, and a mutation of Tbx1 that abolishes transactivation, does not affect Smad1 binding nor does affect the ability to suppress Smad1 activity. In addition, a disease-associated mutation of TBX1 that does not prevent transactivation, prevents the TBX1-SMAD1 interaction. Expression of Tbx1 in transgenic mice generates phenotypes similar to those associated with loss of a Bmp receptor. One phenotype could be rescued by transgenic Smad1 expression. Our data indicate that Tbx1 interferes with Bmp/Smad1 signaling and provide strong evidence that a T-box transcription factor has functions unrelated to transactivation.
doi:10.1371/journal.pone.0006049
PMCID: PMC2698216  PMID: 19557177
12.  Fras1, a basement membrane-associated protein mutated in Fraser syndrome, mediates both the initiation of the mammalian kidney and the integrity of renal glomeruli 
Human Molecular Genetics  2008;17(24):3953-3964.
FRAS1 is mutated in some individuals with Fraser syndrome (FS) and the encoded protein is expressed in embryonic epidermal cells, localizing in their basement membrane (BM). Syndactyly and cryptophthalmos in FS are sequelae of skin fragility but the bases for associated kidney malformations are unclear. We demonstrate that Fras1 is expressed in the branching ureteric bud (UB), and that renal agenesis occurs in homozygous Fras1 null mutant blebbed (bl) mice on a C57BL6J background. In vivo, the bl/bl bud fails to invade metanephric mesenchyme which undergoes involution, events replicated in organ culture. The expression of glial cell line-derived neurotrophic factor and growth-differentiation factor 11 was defective in bl/bl renal primordia in vivo, whereas, in culture, the addition of either growth factor restored bud invasion into the mesenchyme. Mutant primordia also showed deficient expression of Hoxd11 and Six2 transcription factors, whereas the activity of bone morphogenetic protein 4, an anti-branching molecule, was upregulated. In wild types, Fras1 was also expressed by nascent nephrons. Foetal glomerular podocytes expressed Fras1 transcripts and Fras1 immunolocalized in a glomerular BM-like pattern. On a mixed background, bl mutants, and also compound mutants for bl and my, another bleb strain, sometimes survive into adulthood. These mice have two kidneys, which contain subsets of glomeruli with perturbed nephrin, podocin, integrin α3 and fibronectin expression. Thus, Fras1 protein coats branching UB epithelia and is strikingly upregulated in the nephron lineage after mesenchymal/epithelial transition. Fras1 deficiency causes defective interactions between the bud and mesenchyme, correlating with disturbed expression of key nephrogenic molecules. Furthermore, Fras1 may also be required for the formation of normal glomeruli.
doi:10.1093/hmg/ddn297
PMCID: PMC2638576  PMID: 18787044
13.  Hyperdynamic Plasticity of Chromatin Proteins in Pluripotent Embryonic Stem Cells 
Developmental cell  2006;10(1):105-116.
Summary
Differentiation of embryonic stem (ES) cells from a pluripotent to a committed state involves global changes in genome expression patterns. Gene activity is critically determined by chromatin structure and interactions of chromatin binding proteins. Here, we show that major architectural chromatin proteins are hyperdynamic and bind loosely to chromatin in ES cells. Upon differentiation, the hyperdynamic proteins become immobilized on chromatin. Hyperdynamic binding is a property of pluripotent cells, but not of undifferentiated cells that are already lineage committed. ES cells lacking the nucleosome assembly factor HirA exhibit elevated levels of unbound histones, and formation of embryoid bodies is accelerated. In contrast, ES cells, in which the dynamic exchange of H1 is restricted, display differentiation arrest. We suggest that hyperdynamic binding of structural chromatin proteins is a functionally important hallmark of pluripotent ES cells that contributes to the maintenance of plasticity in undifferentiated ES cells and to establishing higher-order chromatin structure.
doi:10.1016/j.devcel.2005.10.017
PMCID: PMC1868458  PMID: 16399082
14.  Targeted Mutagenesis of the Hira Gene Results in Gastrulation Defects and Patterning Abnormalities of Mesoendodermal Derivatives Prior to Early Embryonic Lethality 
Molecular and Cellular Biology  2002;22(7):2318-2328.
The Hira gene encodes a nuclear WD40 domain protein homologous to the yeast transcriptional corepressors Hir1p and Hir2p. Using targeted mutagenesis we demonstrate that Hira is essential for murine embryogenesis. Analysis of inbred 129Sv embryos carrying the null mutation revealed an initial requirement during gastrulation, with many mutant embryos having a distorted primitive streak. Mutant embryos recovered at later stages have a range of malformations with axial and paraxial mesendoderm being particularly affected, a finding consistent with the disruption of gastrulation seen earlier in development. This phenotype could be partially rescued by a CD1 genetic background, although the homozygous mutation was always lethal by embryonic day 11, with death probably resulting from abnormal placentation and failure of cardiac morphogenesis.
doi:10.1128/MCB.22.7.2318-2328.2002
PMCID: PMC133693  PMID: 11884616
15.  Combined exome and whole-genome sequencing identifies mutations in ARMC4 as a cause of primary ciliary dyskinesia with defects in the outer dynein arm 
Journal of Medical Genetics  2013;51(1):61-67.
Background
Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous ciliopathy disorder affecting cilia and sperm motility. A range of ultrastructural defects of the axoneme underlie the disease, which is characterised by chronic respiratory symptoms and obstructive lung disease, infertility and body axis laterality defects. We applied a next-generation sequencing approach to identify the gene responsible for this phenotype in two consanguineous families.
Methods and results
Data from whole-exome sequencing in a consanguineous Turkish family, and whole-genome sequencing in the obligate carrier parents of a consanguineous Pakistani family was combined to identify homozygous loss-of-function mutations in ARMC4, segregating in all five affected individuals from both families. Both families carried nonsense mutations within the highly conserved armadillo repeat region of ARMC4: c.2675C>A; pSer892* and c.1972G>T; p.Glu658*. A deficiency of ARMC4 protein was seen in patient's respiratory cilia accompanied by loss of the distal outer dynein arm motors responsible for generating ciliary beating, giving rise to cilia immotility. ARMC4 gene expression is upregulated during ciliogenesis, and we found a predicted interaction with the outer dynein arm protein DNAI2, mutations in which also cause PCD.
Conclusions
We report the first use of whole-genome sequencing to identify gene mutations causing PCD. Loss-of-function mutations in ARMC4 cause PCD with situs inversus and cilia immotility, associated with a loss of the distal outer (but not inner) dynein arms. This addition of ARMC4 to the list of genes associated with ciliary outer dynein arm defects expands our understanding of the complexities of PCD genetics.
doi:10.1136/jmedgenet-2013-101938
PMCID: PMC3888613  PMID: 24203976
Clinical Genetics; Developmental; Genetics; Molecular Genetics; Other Respiratory Medicine
16.  TTC21B contributes both causal and modifying alleles across the ciliopathy spectrum 
Nature genetics  2011;43(3):189-196.
Ciliary dysfunction leads to a broad range of overlapping phenotypes, termed collectively as ciliopathies. This grouping is underscored by genetic overlap, where causal genes can also contribute modifying alleles to clinically distinct disorders. Here we show that mutations in TTC21B/IFT139, encoding a retrograde intraflagellar transport (IFT) protein, cause both isolated nephronophthisis (NPHP) and syndromic Jeune Asphyxiating Thoracic Dystrophy (JATD). Moreover, although systematic medical resequencing of a large, clinically diverse ciliopathy cohort and matched controls showed a similar frequency of rare changes, in vivo and in vitro evaluations unmasked a significant enrichment of pathogenic alleles in cases, suggesting that TTC21B contributes pathogenic alleles to ∼5% of ciliopathy patients. Our data illustrate how genetic lesions can be both causally associated with diverse ciliopathies, as well as 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 disorders.
doi:10.1038/ng.756
PMCID: PMC3071301  PMID: 21258341
17.  Distinct factors control histone variant H3.3 localization at specific genomic regions 
Cell  2010;140(5):678-691.
Summary
The incorporation of histone H3 variants has been implicated in the epigenetic memory of cellular state. Using genome editing with zinc finger nucleases to tag endogenous H3.3, we report genome-wide profiles of H3 variants in mammalian embryonic stem (ES) cells and neuronal precursor cells. Genome-wide patterns of H3.3 are dependent on amino acid sequence, and change with cellular differentiation at developmentally regulated loci. The H3.3 chaperone Hira is required for H3.3 enrichment at active and repressed genes. Strikingly, Hira is not essential for localization of H3.3 at telomeres and many transcription factor binding sites. Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3.3 in a Hira-independent manner. Atrx is required for Hira-independent localization of H3.3 at telomeres, and for the repression of telomeric RNA. Our data demonstrate that multiple and distinct factors are responsible for H3.3 localization at specific genomic locations in mammalian cells.
doi:10.1016/j.cell.2010.01.003
PMCID: PMC2885838  PMID: 20211137
18.  Exome sequencing identifies DYNC2H1 mutations as a common cause of asphyxiating thoracic dystrophy (Jeune syndrome) without major polydactyly, renal or retinal involvement 
Journal of Medical Genetics  2013;50(5):309-323.
Background
Jeune asphyxiating thoracic dystrophy (JATD) is a rare, often lethal, recessively inherited chondrodysplasia characterised by shortened ribs and long bones, sometimes accompanied by polydactyly, and renal, liver and retinal disease. Mutations in intraflagellar transport (IFT) genes cause JATD, including the IFT dynein-2 motor subunit gene DYNC2H1. Genetic heterogeneity and the large DYNC2H1 gene size have hindered JATD genetic diagnosis.
Aims and methods
To determine the contribution to JATD we screened DYNC2H1 in 71 JATD patients JATD patients combining SNP mapping, Sanger sequencing and exome sequencing.
Results and conclusions
We detected 34 DYNC2H1 mutations in 29/71 (41%) patients from 19/57 families (33%), showing it as a major cause of JATD especially in Northern European patients. This included 13 early protein termination mutations (nonsense/frameshift, deletion, splice site) but no patients carried these in combination, suggesting the human phenotype is at least partly hypomorphic. In addition, 21 missense mutations were distributed across DYNC2H1 and these showed some clustering to functional domains, especially the ATP motor domain. DYNC2H1 patients largely lacked significant extra-skeletal involvement, demonstrating an important genotype–phenotype correlation in JATD. Significant variability exists in the course and severity of the thoracic phenotype, both between affected siblings with identical DYNC2H1 alleles and among individuals with different alleles, which suggests the DYNC2H1 phenotype might be subject to modifier alleles, non-genetic or epigenetic factors. Assessment of fibroblasts from patients showed accumulation of anterograde IFT proteins in the ciliary tips, confirming defects similar to patients with other retrograde IFT machinery mutations, which may be of undervalued potential for diagnostic purposes.
doi:10.1136/jmedgenet-2012-101284
PMCID: PMC3627132  PMID: 23456818
Clinical Genetics; Molecular Genetics; Developmental; Diagnostics; Genetic Screening/Counselling

Results 1-18 (18)