The localization and protein organization of Nebulin within the sarcomere unit of skeletal muscles.

<p>Nebulin is a large myofibrillar protein that binds filamentous actin (F-actin) and may also associate with tropomyosin and tropomodulin while anchoring to the z-line of sarcomeres (A). The largest <i>NEB</i> transcript is predicted to encode a 5907 amino acid long protein consisting of approximately 165 Nebulin motifs (NEBU). The C-terminus contains a 60 amino acid long SH3 domain. A low complexity region (LCR) was identified at both the N and C-terminals of the protein. The position of the three missense variants identified is indicated (K2051R, V2805L and Q2891K) (B).</p

Graphical representation of two-point and multipoint linkage analysis results.

<p>The larger locus identified with using multipoint linkage analysis a maximum LOD_multipoint score of 3.24 (Chr19: 54,949,124–56,765,346) brackets the region identified using two-point linkage analysis (Chr19: 55,358,186–55,848,473) and contains a total of 12 Ensemble-predicted canine genes. Comparison of the linked locus to the human genome reveals shared synteny with a region on chromosome 2. The number and order of genes identified within the canine locus are completely conserved in the syntenic human region.</p

Immunofluorescence localization of Nebulin in in sagittal sections of sucrose embedded human donor eyes.

<p>Positive staining indicating protein expression was noted in the stromal and apical epithelial (EP) cell surface of the cornea (A), the trabecular meshwork (TM) and ciliary cleft structure behind the opening of the iridocorneal angle (marked by a red asterisk) (B). Diffuse staining was observed in the ciliary body (CB) and a stronger signal was detected in the unpigmented epithelial layer of ciliary processes (CP) (C). Significant expression of Nebulin denoted by intense staining was observed in the ciliary muscle (CM) (D). Staining was noted in the apical pigmented epithelium (PE) layer of the iris but not within the iris (E). No staining was noted in the retina [OS = outer segment, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer] (E). Arrows indicate areas where staining was observed.</p

Immunohistochemical localization of Nebulin in sagittal sections of paraffin embedded Basset Hound eyes.

<p>Nebulin (left) and negative control (primary antibody omitted) (right) staining are shown. Positive staining indicating protein expression is noted in the stromal and apical epithelial (EP) cell surface of the cornea (Panel A) as well as the ciliary body (CB) and the unpigmented epithelial layer of ciliary processes (CP) (Panel B). Significant expression of Nebulin is observed in the ciliary muscle (CM) (Panel C). Faint, diffused staining is also noted in close proximity to the pigmented epithelium (PE) layer at the apical surface of the iris (Panel D). Faint to no staining was noted in the optic nerve head (Panel E). Arrows indicate areas where staining was observed.</p

Basset Hound Pedigree used in this study.

<p>The affected Basset 5a in the second generation was duplicated twice (5b and 5c) in order to break two otherwise computationally confounding breeding loops. Genotypes of typed markers within region uncovered following two-point linkage analysis are shown. Shading indicates the transmission pattern of heterozygous parental haplotypes to affected, homozygous offspring. Additional patterns of shading including diagonal lines indicate variation to the same haplotype identified in the other pedigree members. Complete concordance of homozygous haplotype inheritance with the disease phenotype is observed in all affected animals.</p

Sanger sequencing confirmation of the disease segregating variant identified in the linked region.

<p>Candidate region identified using linkage analysis in PACG pedigree animals (left). Sequence chromatograms from a carrier (C), confirms heterozygous genotype whereas affected animals (A) display a homozygous state of variant identified in <i>NEB</i> (g.5588214 A->G) (Right).</p

Whole genome homozygosity mapping results.

<p>A red peak reaching a 1.0 max score of statistical significance depicts a homozygous region on chromosome 19. The region coincides with the 0.49 Mbp locus identified using two-point linkage analysis and is bracketed by recombination spots (indicated in red). The identified haploblock fulfills the zygosity criterion by displaying homozygosity in affected animals (indicated in red) and heterozygosity in unaffected carriers (indicated in green).</p

CEP290 protein analysis in <i>Cep290</i> mouse models.

<p>Representative CEP290 immunodetection in P150 retinas from the three different mouse models. A CEP290-Flag construct was expressed in HEK293-T cells and used as a positive control. Tubulin was used for normalization.</p

A Fisher exact contingency table of genotypes observed in a confirmatory animal cohort for the <i>RIF1</i> variant (g.55723957 C->T).

<p>Forty-four additional unaffected and affected Basset Hounds were selected for confirmatory sequencing of the <i>RIF1</i> variant (g.55723957 C->T). The observed total of individuals and percentage of individuals displaying a specific genotype is shown for each cell respectively. The two-tailed P value is 0.57 (Fisher Exact Probability Test)</p><p>A Fisher exact contingency table of genotypes observed in a confirmatory animal cohort for the <i>RIF1</i> variant (g.55723957 C->T).</p

A Fisher exact contingency table of genotypes observed in a confirmatory animal cohort for the <i>NEB</i> variant (g.55885214 A->G).

<p>Forty-four additional unaffected and affected Basset Hounds were selected for confirmatory sequencing of the <i>NEB</i> variant (g.55885214 A->G). The observed total of individuals and percentage of individuals displaying a specific genotype is shown for each cell respectively.</p><p>The two-tailed P value is 0.00034 (Fisher Exact Probability Test)</p><p>A Fisher exact contingency table of genotypes observed in a confirmatory animal cohort for the <i>NEB</i> variant (g.55885214 A->G).</p