Karyomegalic interstitial nephritis and DNA damage-induced polyploidy in fan1 nuclease-defective knock-in mice

The Fan1 endonuclease is required for repair of DNA interstrand cross-links (ICLs). Mutations in human Fan1 cause karyomegalic interstitial nephritis (KIN), but it is unclear whether defective ICL repair is responsible or whether Fan1 nuclease activity is relevant. We show that Fan1 nuclease-defective (Fan1nd/nd) mice develop a mild form of KIN. The karyomegalic nuclei from Fan1nd/nd kidneys are polyploid, and fibroblasts from Fan1nd/nd mice become polyploid upon ICL induction, suggesting that defective ICL repair causes karyomegaly. Thus, Fan1 nuclease activity promotes ICL repair in a manner that controls ploidy, a role that we show is not shared by the Fanconi anemia pathway or the Slx4–Slx1 nuclease also involved in ICL repair

MSH3 polymorphisms and protein levels affect CAG repeat instability in huntington's disease mice

Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)~100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of DNA repair genes may have prognostic implications for various repeat-associated diseases

Absence of MutSβ leads to the formation of slipped-DNA for CTG/CAG contractions at primate replication forks

Typically disease-causing CAG/CTG repeats expand, but rare affected families can display high levels of contraction of the expanded repeat amongst offspring. Understanding instability is important since arresting expansions or enhancing contractions could be clinically beneficial. The MutSβ mismatch repair complex is required for CAG/CTG expansions in mice and patients. Oddly, by unknown mechanisms MutSβ-deficient mice incur contractions instead of expansions. Replication using CTG or CAG as the lagging strand template is known to cause contractions or expansions respectively; however, the interplay between replication and repair leading to this instability remains unclear. Towards understanding how repeat contractions may arise, we performed in vitro SV40-mediated replication of repeat-containing plasmids in the presence or absence of mismatch repair. Specifically, we separated repair from replication: Replication mediated by MutSβ- and MutSα-deficient human cells or cell extracts produced slipped-DNA heteroduplexes in the contraction- but not expansion-biased replication direction. Replication in the presence of MutSβ disfavoured the retention of replication products harbouring slipped-DNA heteroduplexes. Post-replication repair of slipped-DNAs by MutSβ-proficient extracts eliminated slipped-DNAs. Thus, a MutSβ-deficiency likely enhances repeat contractions because MutSβ protects against contractions by repairing template strand slip-outs. Replication deficient in LigaseI or PCNA-interaction mutant LigaseI revealed slipped-DNA formation at lagging strands. Our results reveal that distinct mechanisms lead to expansions or contractions and support inhibition of MutSβ as a therapeutic strategy to enhance the contraction of expanded repeats

CDKL5 kinase controls transcription‐coupled responses to DNA damage

Mutations in the gene encoding the CDKL5 kinase are among the most common genetic causes of childhood epilepsy and can also give rise to the severe neurodevelopmental condition CDD (CDKL5 deficiency disorder). Despite its importance for human health, the phosphorylation targets and cellular roles of CDKL5 are poorly understood, especially in the cell nucleus. Here, we report that CDKL5 is recruited to sites of DNA damage in actively transcribed regions of the nucleus. A quantitative phosphoproteomic screen for nuclear CDKL5 substrates reveals a network of transcriptional regulators including Elongin A (ELOA), phosphorylated on a specific CDKL5 consensus motif. Recruitment of CDKL5 and ELOA to damaged DNA, and subsequent phosphorylation of ELOA, requires both active transcription and the synthesis of poly(ADP‐ribose) (PAR), to which CDKL5 can bind. Critically, CDKL5 kinase activity is essential for the transcriptional silencing of genes induced by DNA double‐strand breaks. Thus, CDKL5 is a DNA damage‐sensing, PAR‐controlled transcriptional modulator, a finding with implications for understanding the molecular basis of CDKL5‐related diseases

Incidence and phenotypes of childhood-onset genetic epilepsies:a prospective population-based national cohort

Epilepsy is common in early childhood. In this age group it is associated with high rates of therapy-resistance, and with cognitive, motor, and behavioural comorbidity. A large number of genes, with wide ranging functions, are implicated in its aetiology, especially in those with therapy-resistant seizures. Identifying the more common single-gene epilepsies will aid in targeting resources, the prioritization of diagnostic testing and development of precision therapy. Previous studies of genetic testing in epilepsy have not been prospective and population-based. Therefore, the population-incidence of common genetic epilepsies remains unknown. The objective of this study was to describe the incidence and phenotypic spectrum of the most common single-gene epilepsies in young children, and to calculate what proportion are amenable to precision therapy. This was a prospective national epidemiological cohort study. All children presenting with epilepsy before 36 months of age were eligible. Children presenting with recurrent prolonged (>10 min) febrile seizures; febrile or afebrile status epilepticus (>30 min); or with clusters of two or more febrile or afebrile seizures within a 24-h period were also eligible. Participants were recruited from all 20 regional paediatric departments and four tertiary children’s hospitals in Scotland over a 3-year period. DNA samples were tested on a custom-designed 104-gene epilepsy panel. Detailed clinical information was systematically gathered at initial presentation and during follow-up. Clinical and genetic data were reviewed by a multidisciplinary team of clinicians and genetic scientists. The pathogenic significance of the genetic variants was assessed in accordance with the guidelines of UK Association of Clinical Genetic Science (ACGS). Of the 343 patients who met inclusion criteria, 333 completed genetic testing, and 80/333 (24%) had a diagnostic genetic finding. The overall estimated annual incidence of single-gene epilepsies in this well-defined population was 1 per 2120 live births (47.2/100 000; 95% confidence interval 36.9–57.5). PRRT2 was the most common single-gene epilepsy with an incidence of 1 per 9970 live births (10.0/100 000; 95% confidence interval 5.26–14.8) followed by SCN1A: 1 per 12 200 (8.26/100 000; 95% confidence interval 3.93–12.6); KCNQ2: 1 per 17 000 (5.89/100 000; 95% confidence interval 2.24–9.56) and SLC2A1: 1 per 24 300 (4.13/100 000; 95% confidence interval 1.07–7.19). Presentation before the age of 6 months, and presentation with afebrile focal seizures were significantly associated with genetic diagnosis. Single-gene disorders accounted for a quarter of the seizure disorders in this cohort. Genetic testing is recommended to identify children who may benefit from precision treatment and should be mainstream practice in early childhood onset epilepsy

Diabetes Ther

INTRODUCTION: Type 2 diabetes represents a significant public health issue, with increasing prevalence in developing countries while adherence to insulin treatment remains a challenge. No studies have evaluated the relationship between adherence to insulin, diabetes-related distress, and trust in physician among persons with diabetes. Our objectives were to evaluate treatment adherence to insulin, emotional distress (using the Problem Areas in Diabetes Questionnaire, PAID), trust in physician, and to examine associations between them among Lebanese patients with diabetes. METHODS: This cross-sectional study, conducted in all districts of Lebanon between August 2016 and April 2017, enrolled 135 adult patients. RESULTS: The mean percentage score of adherence to insulin was 79.7 +/- 19.94. A significantly higher mean adherence score was found in non-sedentary (81.96) compared to sedentary patients (67.41) (p = 0.017), with no difference between gender, employment, rural vs non-rural residence, or familial history of diabetes. In addition, no significant relationship was seen between adherence score and education level, smoking, or alcohol intake. A significant positive association was found between trust in physician and adherence scores, whereas a significant but negative one was found between PAID and adherence scores. The results of linear regressions showed that a secondary level of education (beta = - 13.48) significantly decreased the trust in physician score, whereas the total number of oral antidiabetics (beta = 0.93) increased it. Having a sedentary lifestyle (beta = - 12.73) and smoking < 3 waterpipes/week compared to no smoking (beta = - 16.82) significantly decreased the adherence score. Female gender (beta = 10.46), smoking < 3 waterpipes (beta = 27.42) and 3 + waterpipes/week (beta = 17.95) significantly increased the PAID score. CONCLUSION: Trust in physician is associated with an increased adherence and with decreased diabetes-related distress. This distress was also associated with poor adherence in our study

Kaitlyn Slean - Bachelor of Music - Senior Recital

Rustiques / Eugene Bozza (1905-1991) -- Andante et Scherzo / Joseph Edouard Barat (1882-1963) -- Concerto for Trumpet / Aleksandra Pakhmutova (b. 1929)Music, Moores School o

Role of DNA Mismatch Repair in Disease-associated Trinucleotide Repeat Instability

Trinucleotide repeat (TNR) tract expansions in specific genes are known to cause numerous neurological and neuromuscular diseases. The mechanism of DNA instability is believed to involve the formation of slipped-repeat structures, and ongoing expansion requires the presence of functional mismatch repair (MMR) proteins. It is known that MMR efficiency is affected by the structure of the DNA lesion, leading to outcomes such as G-T mismatches being repaired more readily than C-C mismatches. It has also been demonstrated that the structure of slipped TNRs can affect their repair, with CAG slip-outs being repaired with higher efficiency than CTG slip-outs. Here I will show that the length of the slip-out, the number of slip-outs, and the slip-out junction all affect repair; additionally, I show that their formation/processing can occur during DNA replication. While isolated short slip-outs are efficiently repaired (much more efficiently than long slip-outs), clustered slip-outs are poorly repaired. The repair of short TNR slip-outs involves the MMR protein MutSβ, but not MutSα. MutSβ is known to contribute to TNR instability, and my results indicate a potential mechanism: attempts to repair short slip-outs meet with interference due to the presence of numerous adjacent slip-outs, and this leads to errors in the repair process causing mutagenesis. Expansion products can also occur during the processing of longer slip-outs. Long CAG and CTG slip-outs can vary widely at the slip-out junction, and I show that these variations can lead to differences in correct repair, even occasionally leading to repair of the wrong strand and thus the retention of excess repeats. Finally, I show that the processing of slipped-repeats by MMR proteins can occur during DNA replication. Altogether, this thesis reveals the role of mismatch repair proteins in instability during the repair and replication of trinucleotide repeat tracts, and how features of DNA structures in the repetitive tracts affect their processing.Ph