Mutagenic Potential of Telomeric Repeats and the Role of Werner Syndrome Helicase Protein in Facilitating Telomeric DNA Replication

Chromosome termini form nucleoprotein structures called telomeres that consist of tandem repeats of TTAGGG DNA sequences (mammals) and telomeric proteins. Telomeres play a critical role in cell survival and genomic stability. Biochemical studies showed that the G-rich strand of telomeres can fold into secondary DNA structures called G-quadruplexes (G4-DNA), which are thought to impact telomere length regulation and telomeric DNA stability. G4 DNA structures are capable of interfering with DNA synthesis by blocking DNA polymerases in vitro and are proposed to hinder replication in vivo. We cloned telomeric repeats into reporter cassettes on shuttle vectors and replicated them in normal human somatic cells to determine if telomeric repeats induce mutations and deletions due to their ability to fold into G4 DNA structures. We demonstrated for the first time that G-rich telomeric repeats, in spite of their G4 DNA forming ability are stable upon replication in normal human cells. In contrast, ciliate telomeric sequences that form more stable G4 DNA than human telomeric sequences, induce more mutations. Stochastic telomere loss is seen in the premature aging disorder Werner Syndrome, which is caused by loss of the RecQ helicase protein WRN. We hypothesized that WRN deficiency leads to replication fork stalling and collapse due to G4 DNA formed by telomeric repeats resulting in deletions of DNA sequence. Shuttle vectors with a telomeric or control sequence were replicated in U2OS cells deficient or proficient for WRN. Replication of shuttle vectors in normal cells did not influence shuttle vector mutant frequencies, while WRN depleted cells exhibited elevated mutant frequencies for both telomeric and control vectors but the increase was significantly higher for the telomeric vector. We demonstrated that WRN is involved in suppressing mutagenesis in shuttle vectors with telomeric sequences. We are also testing DNA synthesis in plasmids through regions of single stranded DNA containing telomere repeats in WRN proficient and deficient cells. Public health significance: Shortened telomeres are associated with age related diseases such as heart disease, cancer and premature aging disorders. These assays will help us investigate factors that cause accelerated telomere loss with the goal of preventing or delaying disease

DNAH6 and Its Interactions with PCD Genes in Heterotaxy and Primary Ciliary Dyskinesia.

Heterotaxy, a birth defect involving left-right patterning defects, and primary ciliary dyskinesia (PCD), a sinopulmonary disease with dyskinetic/immotile cilia in the airway are seemingly disparate diseases. However, they have an overlapping genetic etiology involving mutations in cilia genes, a reflection of the common requirement for motile cilia in left-right patterning and airway clearance. While PCD is a monogenic recessive disorder, heterotaxy has a more complex, largely non-monogenic etiology. In this study, we show mutations in the novel dynein gene DNAH6 can cause heterotaxy and ciliary dysfunction similar to PCD. We provide the first evidence that trans-heterozygous interactions between DNAH6 and other PCD genes potentially can cause heterotaxy. DNAH6 was initially identified as a candidate heterotaxy/PCD gene by filtering exome-sequencing data from 25 heterotaxy patients stratified by whether they have airway motile cilia defects. dnah6 morpholino knockdown in zebrafish disrupted motile cilia in Kupffer\u27s vesicle required for left-right patterning and caused heterotaxy with abnormal cardiac/gut looping. Similarly DNAH6 shRNA knockdown disrupted motile cilia in human and mouse respiratory epithelia. Notably a heterotaxy patient harboring heterozygous DNAH6 mutation was identified to also carry a rare heterozygous PCD-causing DNAI1 mutation, suggesting a DNAH6/DNAI1 trans-heterozygous interaction. Furthermore, sequencing of 149 additional heterotaxy patients showed 5 of 6 patients with heterozygous DNAH6 mutations also had heterozygous mutations in DNAH5 or other PCD genes. We functionally assayed for DNAH6/DNAH5 and DNAH6/DNAI1 trans-heterozygous interactions using subthreshold double-morpholino knockdown in zebrafish and showed this caused heterotaxy. Similarly, subthreshold siRNA knockdown of Dnah6 in heterozygous Dnah5 or Dnai1 mutant mouse respiratory epithelia disrupted motile cilia function. Together, these findings support an oligogenic disease model with broad relevance for further interrogating the genetic etiology of human ciliopathies

Ion Torrent sequencing for conducting genome-wide scans for mutation mapping analysis

Mutation mapping in mice can be readily accomplished by genome wide segregation analysis of polymorphic DNA markers. In this study, we showed the efficacy of Ion Torrent next generation sequencing for conducting genome-wide scans to map and identify a mutation causing congenital heart disease in a mouse mutant, Bishu, recovered from a mouse mutagenesis screen. The Bishu mutant line generated in a C57BL/6J (B6) background was intercrossed with another inbred strain, C57BL/10J (B10), and the resulting B6/B10 hybrid offspring were intercrossed to generate mutants used for the mapping analysis. For each mutant sample, a panel of 123 B6/B10 polymorphic SNPs distributed throughout the mouse genome was PCR amplified, bar coded, and then pooled to generate a single library used for Ion Torrent sequencing. Sequencing carried out using the 314 chip yielded > 600,000 usable reads. These were aligned and mapped using a custom bioinformatics pipeline. Each SNP was sequenced to a depth > 500x, allowing accurate automated calling of the B6/B10 genotypes. This analysis mapped the mutation in Bishu to an interval on the proximal region of mouse chromosome 4. This was confirmed by parallel capillary sequencing of the 123 polymorphic SNPs. Further analysis of genes in the map interval identified a splicing mutation in Dnaic1 (c.204+1G > A), an intermediate chain dynein, as the disease causing mutation in Bishu. Overall, our experience shows Ion Torrent amplicon sequencing is high throughput and cost effective for conducting genome-wide mapping analysis and is easily scalable for other high volume genotyping analyses

Prickle1 mutation causes planar cell polarity and directional cell migration defects associated with cardiac outflow tract anomalies and other structural birth defects

Planar cell polarity (PCP) is controlled by a conserved pathway that regulates directional cell behavior. Here, we show that mutant mice harboring a newly described mutation termed Beetlejuice (Bj) in Prickle1 (Pk1), a PCP component, exhibit developmental phenotypes involving cell polarity defects, including skeletal, cochlear and congenital cardiac anomalies. Bj mutants die neonatally with cardiac outflow tract (OFT) malalignment. This is associated with OFT shortening due to loss of polarized cell orientation and failure of second heart field cell intercalation mediating OFT lengthening. OFT myocardialization was disrupted with cardiomyocytes failing to align with the direction of cell invasion into the outflow cushions. The expression of genes mediating Wnt signaling was altered. Also noted were shortened but widened bile ducts and disruption in canonical Wnt signaling. Using an in vitro wound closure assay, we showed Bj mutant fibroblasts cannot establish polarized cell morphology or engage in directional cell migration, and their actin cytoskeleton failed to align with the direction of wound closure. Unexpectedly, Pk1 mutants exhibited primary and motile cilia defects. Given Bj mutant phenotypes are reminiscent of ciliopathies, these findings suggest Pk1 may also regulate ciliogenesis. Together these findings show Pk1 plays an essential role in regulating cell polarity and directional cell migration during development

Novel Jbts17 mutant mouse model of Joubert syndrome with cilia transition zone defects and cerebellar and other ciliopathy related anomalies

Recent studies identified a previously uncharacterized gene C5ORF42 (JBTS17) as a major cause of Joubert syndrome (JBTS), a ciliopathy associated with cerebellar abnormalities and other birth defects. Here we report the first Jbts17 mutant mouse model, Heart Under Glass (Hug), recovered from a forward genetic screen. Exome sequencing identified Hug as a S235P missense mutation in the mouse homolog of JBTS17 (2410089e03rik). Hug mutants exhibit multiple birth defects typical of ciliopathies, including skeletal dysplasia, polydactyly, craniofacial anomalies, kidney cysts and eye defects. Some Hug mutants exhibit congenital heart defects ranging from mild pulmonary stenosis to severe pulmonary atresia. Immunostaining showed JBTS17 is localized in the cilia transition zone. Fibroblasts from Hug mutant mice and a JBTS patient with a JBTS17 mutation showed ciliogenesis defects. Significantly, Hug mutant fibroblasts showed loss of not only JBTS17, but also NPHP1 and CEP290 from the cilia transition zone. Hug mutants exhibited reduced ciliation in the cerebellum. This was associated with reduction in cerebellar foliation. Using a fibroblast wound-healing assay, we showed Hug mutant cells cannot establish cell polarity required for directional cell migration. However, stereocilia patterning was grossly normal in the cochlea, indicating planar cell polarity is not markedly affected. Overall, we showed the JBTS pathophysiology is replicated in the Hug mutant mice harboring a Jbts17 mutation. Our findings demonstrate JBTS17 is a cilia transition zone component that acts upstream of other Joubert syndrome associated transition zone proteins NPHP1 and CEP290, indicating its importance in the pathogenesis of Joubert syndrome

Global genetic analysis in mice unveils central role for cilia in congenital heart disease.

Congenital heart disease (CHD) is the most prevalent birth defect, affecting nearly 1% of live births; the incidence of CHD is up to tenfold higher in human fetuses. A genetic contribution is strongly suggested by the association of CHD with chromosome abnormalities and high recurrence risk. Here we report findings from a recessive forward genetic screen in fetal mice, showing that cilia and cilia-transduced cell signalling have important roles in the pathogenesis of CHD. The cilium is an evolutionarily conserved organelle projecting from the cell surface with essential roles in diverse cellular processes. Using echocardiography, we ultrasound scanned 87,355 chemically mutagenized C57BL/6J fetal mice and recovered 218 CHD mouse models. Whole-exome sequencing identified 91 recessive CHD mutations in 61 genes. This included 34 cilia-related genes, 16 genes involved in cilia-transduced cell signalling, and 10 genes regulating vesicular trafficking, a pathway important for ciliogenesis and cell signalling. Surprisingly, many CHD genes encoded interacting proteins, suggesting that an interactome protein network may provide a larger genomic context for CHD pathogenesis. These findings provide novel insights into the potential Mendelian genetic contribution to CHD in the fetal population, a segment of the human population not well studied. We note that the pathways identified show overlap with CHD candidate genes recovered in CHD patients, suggesting that they may have relevance to the more complex genetics of CHD overall. These CHD mouse models and \u3e8,000 incidental mutations have been sperm archived, creating a rich public resource for human disease modelling. Nature 2015 May 28; 521(7553):520-4

<i>DNAH6</i> and PCD gene mutations identified in heterotaxy patients.

<p><i>DNAH6</i> and PCD gene mutations identified in heterotaxy patients.</p

<i>Dnah6</i> genetically interacts with <i>Dnai1</i> and <i>Dnah5</i> to cause heterotaxy and PCD.

<p><b>(A,B)</b> Embryos injected with subthreshold dose of <i>dnah6</i> and <i>dnai1</i> MO show increased heart looping defects compared with Ctrl MO injections (n = 177, p-value = 3.8x10^-8), or single injection of either <i>dnai1</i> (p = 9.29x10^-9) or <i>dnah6</i> (p = 2.04x10^-8) MO at the same MO dose (A). Similar results were observed with subthreshold <i>dnah5/dnah6</i> double MO knockdown (n = 82; p = 1.74x10^-5, Bonferroni corrected)<b>. (C,D)</b> Reciliating mouse airway epithelia from wildtype (+/+) and heterozygous (+/-) <i>Dnai1</i> knockout (C) or <i>Dnah5</i> mutant (D) mice show robust ciliation and ciliary motion (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005821#pgen.1005821.s017" target="_blank">S5 Movie</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005821#pgen.1005821.s018" target="_blank">S6 Movie</a>). 30nM <i>Dnah6</i> siRNA had no effect on ciliation or cilia motility in wildtype airway epithelia, but in heterozygous <i>Dnai1 or Dnah5</i> mutant airway, ciliation was reduced and ciliary motion was dyskinetic (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005821#pgen.1005821.s017" target="_blank">S5 Movie</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005821#pgen.1005821.s018" target="_blank">S6 Movie</a>). With 50nM siRNA, little or no cilia was seen in wildtype and heterozygous <i>Dnai1</i> or <i>Dnah5</i> mutant mouse airway.</p