Next-generation sequencing targeted disease panel in rod-cone retinal dystrophies in Maori and Polynesian reveals novel changes and a common founder mutation.

IMPORTANCE: This study identifies unique genetic variation observed in a cohort of Maori and Polynesian patients with rod-cone retinal dystrophies using a targeted next-generation sequencing retinal disease gene panel. BACKGROUND: With over 250 retinal disease genes identified, genetic diagnosis is still only possible in 60-70% of individuals and even less within unique ethnic groups. DESIGN: Prospective genetic testing in patients with rod-cone retinal dystrophies identified from the New Zealand Inherited Retinal Disease Database, PARTICIPANTS: Sixteen patients of Maori and Polynesian ancestry. METHODS: Next-generation sequencing of a targeted retinal gene panel. Sanger sequencing for a novel PDE6B mutation in subsequent Maori patients. MAIN OUTCOME MEASURES: Genetic diagnosis, genotype-phenotype correlation. RESULTS: Thirteen unique pathogenic variants were identified in 9 of 16 (56.25%) patients in 10 different genes. A definitive genetic diagnosis was made in 7/16 patients (43.7%). Six changes were novel and not in public databases of human variation. In four patients, a homozygous, novel pathogenic variant (c.2197G > C, p.(Ala 733Pro)) in PDE6B was identified and also present in a further five similarly affected Maori patients. CONCLUSIONS AND RELEVANCE: Over half of the Maori and Polynesian patients with inherited rod-cone diseases have no pathogenic variant(s) detected with a targeted retinal next-generation sequencing strategy, which is supportive of novel genetic mechanisms in this population. A novel PDE6B founder variant is likely to account for 16% of recessive inherited retinal dystrophy in Maori. Careful characterization of the clinical presentation permits identification of further Maori patients with a similar phenotype and simplifies the diagnostic algorithm.

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

PURPOSE: Corneal 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. DESIGN: Case 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. PARTICIPANTS: Four ERED families, including 28 affected and 17 unaffected individuals. METHODS: HumanLinkage-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 MEASURES: Linkage microarray, exome analysis, DNA sequence analysis, immunohistochemistry, in situ hybridization, and morpholino-induced genetic knockdown results. RESULTS: Linkage 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. CONCLUSIONS: The 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.

ADAMTSL4 assessment in ectopia lentis reveals a recurrent founder mutation in Polynesians.

BACKGROUND: To clinically characterize a cohort of patients with ectopia lentis (EL), or Marfanoid features in whom a definite genetic diagnosis of Marfan syndrome (MFS) had been excluded (atypical MFS), and to evaluate the contribution of mutations in ADAMTSL4 (OMIM * 610113), and P3H2 (LEPREL1; OMIM * 610341) to disease in this population. MATERIALS AND METHODS: Subjects underwent comprehensive ophthalmic examination, including keratometry. Mutational analysis of ADAMTSL4 and P3H2 was undertaken using PCR, high resolution melting analysis, and sequencing. The frequency of c.2237G>A; p.(Arg746His) was determined in an unaffected Polynesian cohort. Haplotype analysis used tagged single nucleotide polymorphic markers. RESULTS: Mutational analysis of ADAMTSL4 identified two pathogenic variants in ADAMTSL4 in 11/31 (35%) probands, consistent with the autosomal recessive EL phenotype. A recurrent, rare missense variant in ADAMTSL4, c.2237G>A; p.(Arg746His), was present in 10 probands -(8 homozygotes), predominantly of Polynesian descent, and all shared the same haplotype. p.(Arg746His) affects the Thrombospondin1 (TSP1) domain of the protein and is predicted to be pathogenic. No pathogenic variants in P3H2 were identified. CONCLUSION: A recurrent pathogenic ADAMTSL4 variant is a major cause of early onset autosomal recessive EL in a Cook Island Maori population and associated with a common haplotype, suggesting a founder effect. Children presenting under the age of 5 years, particularly of Cook Island or New Zealand Maori descent, with isolated ectopia lentis, should in the first instance be tested for this single variant.

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 = 455 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.