A COL17A1 Splice-Altering Mutation Is Prevalent in Inherited Recurrent Corneal Erosions

PurposeCorneal dystrophies are a genetically heterogeneous group of disorders. We previously described a family with an autosomal dominant epithelial recurrent erosion dystrophy (ERED). We aimed to identify the underlying genetic cause of ERED in this family and 3 additional ERED families. We sought to characterize the potential function of the candidate genes using the human and zebrafish cornea.DesignCase series study of 4 white families with a similar ERED. An experimental study was performed on human and zebrafish tissue to examine the putative biological function of candidate genes.ParticipantsFour ERED families, including 28 affected and 17 unaffected individuals.MethodsHumanLinkage-12 arrays (Illumina, San Diego, CA) were used to genotype 17 family members. Next-generation exome sequencing was performed on an uncle–niece pair. Segregation of potential causative mutations was confirmed using Sanger sequencing. Protein expression was determined using immunohistochemistry in human and zebrafish cornea. Gene expression in zebrafish was assessed using whole-mount in situ hybridization. Morpholino-induced transient gene knockdown was performed in zebrafish embryos.Main Outcome MeasuresLinkage microarray, exome analysis, DNA sequence analysis, immunohistochemistry, in situ hybridization, and morpholino-induced genetic knockdown results.ResultsLinkage microarray analysis identified a candidate region on chromosome chr10:12,576,562–112,763,135, and exploration of exome sequencing data identified 8 putative pathogenic variants in this linkage region. Two variants segregated in 06NZ–TRB1 with ERED: COL17A1 c.3156C→T and DNAJC9 c.334G→A. The COL17A1 c.3156C→T variant segregated in all 4 ERED families. We showed biologically relevant expression of these proteins in human cornea. Both proteins are expressed in the cornea of zebrafish embryos and adults. Zebrafish lacking Col17a1a and Dnajc9 during development show no gross corneal phenotype.ConclusionsThe COL17A1 c.3156C→T variant is the likely causative mutation in our recurrent corneal erosion families, and its presence in 4 independent families suggests that it is prevalent in ERED. This same COL17A1 c.3156C→T variant recently was identified in a separate pedigree with ERED. Our study expands the phenotypic spectrum of COL17A1 disease from autosomal recessive epidermolysis bullosa to autosomal dominant ERED and identifies COL17A1 as a key protein in maintaining integrity of the corneal epithelium

Role of G Protein-coupled Receptor Kinase 5 in Desensitisation of the V1b Vasopressin Receptor in Response to Arginine Vasopressin

Arginine vasopressin (AVP) is a hypothalamic nonapeptide which regulates the hypothalamic-pituitary-adrenal axis response to stress by stimulating the secretion of adrenocorticotropin (ACTH) from corticotroph cells of the anterior pituitary. This effect is mediated by binding of AVP to the pituitary vasopressin receptor (V1bR). The V1bR belongs to the G protein-coupled receptor (GPCR) super family. Repeated stimulation of anterior pituitary cells with AVP has been shown to produce a loss of responsiveness to subsequent AVP stimulation. This phenomenon appears to be mediated by desensitisation of the V1bR, and may be due to phosphorylation of the receptor by G protein-coupled receptor kinase 5 (GRK5). The aim of this research was to establish and validate methods that would allow the role of GRK5 in the desensitisation of V1bR to AVP stimulation to be investigated. As no isoform specific inhibitors for GRK5 were available, HEK293 cells transiently transfected with the rat V1bR were used as a model system for this research. This allowed RNA interference (RNAi) to be used to knockdown GRK5 expression. The protocol for RNAi-mediated knockdown of GRK5 was established as part of this research. Protocols for Western blotting and qRT-PCR were also established to allow the RNAi-mediated knockdown of GRK5 protein and mRNA to be measured. Transfection of HEK293 cells with 10nM GRK5-targeting small interfering RNAs (siRNAs) reduced the expression of GRK5 protein to 53.4% ± 3.4% (mean ± SEM) of that seen in untreated control cells at 84 hours after transfection, while GRK5 mRNA levels were reduced to 28.7% ± 1.9% (mean ± SEM) of that of control cells 48 hours after transfection. An experimental protocol was designed in this research that would coordinate the RNAi-mediated knockdown of GRK5 with transient transfection of the HEK293 cells with the rV1bR. Since, activated V1bRs couple to Gq/11 and stimulate the production of inositol phosphates (IPs), the responsiveness of the V1bR can be determined by measuring the accumulation of [H³]-IPs in cells labelled with [H³]-myo-inositol. In the protocol designed, the effect of GRK5 knockdown on V1bR desensitisation is determined by stimulating HEK293 cells expressing the rV1bR (and previously transfected with GRK5-targeting siRNA) with 0nM or 100nM AVP for 0, 5, 15, 30 or 60 minutes, and comparing the accumulation if IPs over time with that of cells that are not transfected with GRK5-targeting siRNA. This protocol can be used in future to investigate the role of GRK5 in V1bR desensitisation, and may be adapted to determine if other GRK isoforms are involved in V1bR desensitisation

Speech, Phonological Awareness and Literacy in New Zealand Children with Down Syndrome

Children with Down syndrome (DS) are reported to experience difficulty with spoken and written language which can persist through the lifespan. However, little is known about the spoken and written language profiles of children with DS in the New Zealand social and education environment, and a thorough investigation of these profiles has yet to be conducted. The few controlled interventions to remediate language deficits in children with DS that are reported in the literature typically focus on remediation of a single language domain, with the effectiveness of interventions which integrate spoken and written language goals yet to be explored for this population. The experiments reported in this thesis aim to address these areas of need. The following questions are asked 1) What are the phonological awareness, speech, language and literacy skills of New Zealand children with DS? 2) What are the home and school literacy environments of New Zealand children with DS and how do they support written language development? and 3) What are the immediate and longer term effects of an integrated phonological awareness intervention on enhancing aspects of spoken and written language development in young children with DS? These questions will be addressed through the following chapters. The first experiment (presented in Chapter 2) was conducted in two parts. Part 1 consisted of the screening of the early developing phonological awareness, letter knowledge, and decoding skills of 77 primary school children with DS and revealed considerable variability between participants on all measures. Although some children were able to demonstrate mastery of the phoneme identity and letter knowledge skills, floor effects were also apparent. Data were analysed by age group (5 - 8 years and 9 -14 years) which revealed increased performance with maturation, with older children outperforming their younger peers on all measures. Approximately one quarter of all children were unable to decode any words, 6.6% demonstrated decoding skills at a level expected for 7 - 8 year old children and one child demonstrated decoding skills at an age equivalent level. Significant relationships between decoding skills and letter knowledge were found to exist. In Part 2 of the experiment, 27 children with DS who participated in the screening study took part in an in-depth investigation into their speech, phonological awareness, reading accuracy and comprehension and narrative language skills. Results of the speech assessments revealed the participants’ speech was qualitatively and quantitatively similar to the speech of younger children with typical development, but that elements of disorder were also evident. Results of the phonological awareness measures indicated participants were more successful with blending than with segmentation at both sentence and syllable level. Rhyme generation scores were particularly low. Reading accuracy scores were in advance of reading comprehension, with strong relationships demonstrated between reading accuracy and phonological awareness and letter knowledge. Those children who were better readers also had better language skills, producing longer sentences and using a greater number of different words in their narratives. The production of more advanced narrative structures was restricted to better readers. In the second experiment (presented in Chapter 3), the home literacy environment of 85 primary school aged children with DS was investigated. Parents of participants completed a questionnaire which explored the frequency and duration of literacy interactions, other ways parents support and facilitate literacy, parents’ priorities for their children at school, and the child’s literacy skills. Results revealed that the homes of participants were generally rich in literacy resources, and that parents and children read together regularly, although many children were reported to take a passive role duding joint story reading. Many parents also reported actively teaching their child letter names and sounds and encouraging literacy development in other ways such as language games, computer use, television viewing and library access. Writing at home was much less frequent than reading, and the allocation of written homework was much less common than reading homework. In the third experiment (presented in Chapter 4), the school literacy environment of 87 primary school aged children with DS (identified in the second experiment) was explored. In a parallel survey to the one described in Chapter 3, the teachers of participants completed a questionnaire which explored the frequency and duration of literacy interactions, the role of the child during literacy interactions, the child’s literacy skills, and other ways literacy is supported. The results of the questionnaire revealed nearly all children took part in regular reading instruction in the classroom although the amount of time reportedly dedicated to reading instruction was extremely variable amongst respondents. The average amount of time spent on reading instruction was consistent with that reported nationally and in advance of the international average for Year 5 children. Reading instruction was typically given in small groups or in a one on one setting and included both ‘top-down’ and bottom up’ strategies. Children were more likely to be assigned reading homework compared to written homework, with writing activities and instruction reported to be particularly challenging. In the fourth experiment (reported in Chapter 5), the effectiveness of an experimental integrated phonological awareness intervention was evaluated for ten children with DS, who ranged in age from 4;04 to 5;05 (M = 4;11, SD = 4.08 months). The study employed a multiple single-subject design to evaluate the effect of the intervention on participants’ trained and untrained speech measures, and examined the development of letter knowledge and phonological awareness skills. The 18 week intervention included the following three components; 1. parent implemented print referencing during joint story reading, 2. speech goals integrated with letter knowledge and phoneme awareness activities conducted by the speech-language therapist (SLT) in a play based format, and 3. letter knowledge and phoneme awareness activities conducted by the computer specialist (CS) adapted for presentation on a computer. The intervention was implemented by the SLT and CS at an early intervention centre during two 20 minute sessions per week, in two 6 week therapy blocks separated by a 6 week break (i.e. 8 hours total). The parents implemented the print referencing component in four 10 minute sessions per week across the 18 week intervention period (approximately 12 hours total). Results of the intervention revealed all ten children made statistically significant gains on their trained and untrained speech targets with some children demonstrating transfer to other phonemes in the same sound class. Six children demonstrated gains in letter knowledge and nine children achieved higher scores on phonological awareness measures at post-intervention, however all phonological awareness scores were below chance. The findings demonstrated that dedicating some intervention time to facilitating the participants’ letter knowledge and phonological awareness was not at the expense of speech gains. The fifth experiment (presented in Chapter 6) comprises a re-evaluation of the speech, phonological awareness, and letter knowledge, and an evaluation of the decoding and spelling development in children with DS who had previously participated in an integrated phonological awareness intervention (see Chapter 5), after they had subsequently received two terms (approximately 20 weeks) of formal schooling. Speech accuracy was higher at follow-up than at post-intervention on standardised speech measures and individual speech targets for the group as a whole, with eight of the ten participants demonstrating increased scores on their individual speech targets. Group scores on both letter knowledge measures were higher at follow-up than at post-intervention, with nine participants maintaining or improving on post-intervention performance. The majority of participants exhibited higher phonological awareness scores at follow-up on both the phoneme level assessments, with above chance scores achieved by five participants on one of the tasks, however, scores on the rhyme matching task demonstrated no evidence of growth. Some transfer of phonological awareness and letter knowledge was evident, with five children able to decode some words on the single word reading test and three children able to represent phonemes correctly in the experimental spelling task. The emergence of these early literacy skills highlighted the need for ongoing monitoring of children’s ability to transfer their improved phonological awareness and letter knowledge to decoding and spelling performance. In the sixth experiment (presented in Chapter 7) the long term effects of the integrated phonological awareness intervention was evaluated for one boy with DS aged 5;2 at the start of the intervention. The study monitored Ben’s speech and literacy development up to the age of 8;0 (34 months post pre-school intervention) which included two years of formal schooling. Ben demonstrated sustained growth on all measures with evidence of a growing ability to transfer letter-sound knowledge and phoneme-grapheme correspondences to the reading and spelling process. The results indicated an intervention which is provided early and which simultaneously targets speech, letter knowledge and phonological awareness goals provides a promising alternative to conventional therapy, and that integrating spoken and written therapy goals for children with DS can be effective in facilitating development in both domains. This thesis provides evidence that the spoken and written language abilities of New Zealand children with DS exhibit a pattern of delay and disorder that is largely consistent with those of children with DS from other countries reported in the literature. The home and school literacy environments of children in New Zealand with DS are rich in literacy resources and are, for the most part, supportive of their literacy development. The immediate and longer term results of the integrated phonological awareness intervention suggest that it is possible to achieve significant and sustained gains in speech, letter knowledge and phonological awareness which may contribute to the remediation of the persistent and compromised spoken and written language profile characteristic of individuals with DS

High-resolution DNA melt-curve analysis for cost-effective mass screening of pairwise species interactions

Ecological studies of pairwise interactions are constrained by the methods available for rapid species identification of the interacting organisms. The resolution of data required to characterize species interaction networks at multiple spatio-temporal scales can be intensive, and therefore laborious and costly to collect. We explore the utility of high-resolution DNA melt-curve analysis (HRM) as a rapid species identification method. An approach was developed to identify organisms at the pairwise interaction level, with particular application to cryptic species interactions that are traditionally difficult to study. Here, we selected a challenging application; to identify the presence/absence of pathogenic fungi (Sporothrix inflata, Ophiostoma nigrocarpum and Ophiostoma galeiforme) transported by bark beetle vectors (Hylastes ater and Hylurgus ligniperda). The technique was able to distinguish between different species of DNA within a single, pooled sample. In test applications, HRM was effective in the mass screening and identification of pathogenic fungal species carried by many individual bark beetle vectors (n\ua0=\ua0455 beetles screened) across large geographic scales. For two of the fungal species, there was no difference in the frequency of association with either of their vectors, but for the third fungal species there was a shift in vector-pathogen associations across locations. This technique allows rapid, mass screening and characterization of species interactions at a fraction of the time and cost of traditional methods. It is anticipated that this method can be readily applied to explore other cryptic species interactions, or other studies requiring rapid generation of large data sets and/or high-throughput efficiency

Impact of <i>TCF4</i> Repeat Number on Resolution of Corneal Edema after Descemet’s Stripping Only in Fuchs Dystrophy: A Pilot Study

Purpose: To investigate whether Fuchs endothelial corneal dystrophy (FECD) genotype, specifically transcription factor 4 (TCF4) CTG triplet repeat “load” predicts time to clearance following Descemet’s Stripping Only (DSO). Methods: This prospective, interventional trial was conducted on consecutive FECD patients undergoing DSO. Genetic analysis using patients’ saliva was performed to assess the extent of CTG expansion using short tandem repeat analysis, corroborated gel electrophoresis and Sanger sequencing. Polymerase chain reaction and bidirectional Sanger sequencing was undertaken. Partial least square regression and logistic regression modelling was used to evaluate the predictive power of TCF4 repeats on corneal clearance. Results: Of 11 eyes of 11 patients, 8 showed complete corneal clearance. For these 8 patients, mean TCF4 allele repeat was 24.8 (SD: 23.7, range: 11–63) and 63.4 (SD: 30.3; range: 11–97), respectively. In total, 9/11 (81.8%) had expanded CTG repeats (>40) in one allele. In cases with an allele repeat ≥80, there was a significantly increased risk of corneal non-clearance (odds ratio 18.2, p = 0.009). Conclusion: Whilst it was not possible to predict time to corneal clearance based on CTG repeats, there is a significant correlation between allele repeats and achievement of corneal clearance

Geometric morphometrics and molecular systematics of Xanthocnemis sobrina (McLachlan, 1873) (Odonata: Coenagrionidae) and comparison to its congeners

The taxonomy of the damselfly genus Xanthocnemis is revised, with particular focus on populations inhabiting the North Island of New Zealand. Earlier studies revealed two species: X. sobrina, restricted to cool, shaded streams in kauri forests and other forested areas, and X. zealandica, a common species throughout New Zealand except the Chatham and subantarctic islands. A field study encompassing aquatic habitats throughout the whole North Island was carried out to establish the relationship between morphological variation (body size and various morphological traits over the entire body) observed by previous researchers with ecological conditions and/or geographical location. The main aim was to propose reliable diagnostic features that could be used in future studies. Morphological and molecular variation was assessed. Morphological examination included assigning landmarks for all body parts corresponding to the external morphological features that are usually used in Odonata taxonomy. Molecular analysis targeted fragments of the 28S and 16S rRNA genes. Congruence was sought between both types of data, statistical support for two morphological types previously described as different species and a maximum likelihood phylogenetic tree in conjunction with a pairwise genetic distance matrix constructed from the DNA sequences obtained from the sampled specimens. Geometric morphometrics revealed statistically significant differentiation between specimens identified as X. zealandica and X. sobrina for four traits: (1) dorsal view of the head for both sexes as well as male appendages from (2) dorsal, (3) ventral and (4) lateral views. Wings appeared different when analysed for males only. Molecular analysis, however, grouped all specimens into a single undifferentiated cluster with very low mean pairwise distance (96% and similar to 93% pairwise identity with X. tuanuii sequences obtained from the Chatham Island specimens. A careful investigation of the thin plate spline deformations generated for the geometric morphometric landmarks showed that the significant variations in the appendages of the Xanthocnemis specimens appeared to be the result of size, rather than shape, differences. Therefore, X. sobrina is proposed as a synonym of X. zealandica. Recently Amaya-Perilla et al. (2014) synonymised X. sinclairi with X. zealandica and confirmed the status of the Chatham Island X. tuanuii as a distinct species. It is therefore proposed that the genus Xanthocnemis consists of two species only: zealandica occurring all over the North, South and Stewart Islands, and tuanuii, endemic to Chatham and Pitt islands. Considering several statistical tests involving body measurements and ecological variables recorded during the field study, as well as various discussion points from similar studies of other species of Odonata, two alternative hypotheses are proposed for future testing. The first hypothesis synonymises X. sobrina with X.zealandica and suggests a possible explanation for the evolution of the two morphological traits that have previously been considered diagnostic for these species. The second hypothesis suggests that as typical X. sobrina were not sampled during this study this could represent a species that is now extinct, unless future studies prove it otherwise

Circular replication-associated protein encoding DNA viruses identified in the faecal matter of various animals in New Zealand

In recent years, innovations in molecular techniques and sequencing technologies have resulted in a rapid expansion in the number of known viral sequences, in particular those with circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA genomes. CRESS DNA viruses are present in the virome of many ecosystems and are known to infect a wide range of organisms. A large number of the recently identified CRESS DNA viruses cannot be classified into any known viral families, indicating that the current view of CRESS DNA viral sequence space is greatly underestimated. Animal faecal matter has proven to be a particularly useful source for sampling CRESS DNA viruses in an ecosystem, as it is cost-effective and non-invasive. In this study a viral metagenomic approach was used to explore the diversity of CRESS DNA viruses present in the faeces of domesticated and wild animals in New Zealand. Thirty-eight complete CRESS DNA viral genomes and two circular molecules (that may be defective molecules or single components of multicomponent genomes) were identified from forty-nine individual animal faecal samples. Based on shared genome organisations and sequence similarities, eighteen of the isolates were classified as gemycircularviruses and twelve isolates were classified as smacoviruses. The remaining eight isolates lack significant sequence similarity with any members of known CRESS DNA virus groups. This research adds significantly to our knowledge of CRESS DNA viral diversity in New Zealand, emphasising the prevalence of CRESS DNA viruses in nature, and reinforcing the suggestion that a large proportion of CRESS DNA viruses are yet to be identified. (C) 2016 Elsevier B.V. All rights reserved