WDR19: an ancient, retrograde, intraflagellar ciliary protein is mutated in autosomal recessive retinitis pigmentosa and in Senior-Loken syndrome.

Autosomal recessive retinitis pigmentosa (arRP) is a clinically and genetically heterogeneous retinal disease that causes blindness. Our purpose was to identify the causal gene, describe the phenotype and delineate the mutation spectrum in a consanguineous Quebec arRP family. We performed Arrayed Primer Extension (APEX) technology to exclude ∼500 arRP mutations in ∼20 genes. Homozygosity mapping [single nucleotide polymorphism (SNP) genotyping] identified 10 novel significant homozygous regions. We performed next generation sequencing and whole exome capture. Sanger sequencing provided cosegregation. We screened another 150 retinitis pigmentosa (RP) and 200 patients with Senior-Løken Syndrome (SLS). We identified a novel missense mutation in WDR19, c.2129T>C which lead to a p.Leu710Ser. We found the same mutation in a second Quebec arRP family. Interestingly, two of seven affected members of the original family developed 'sub-clinical' renal cysts. We hypothesized that more severe WDR19 mutations may lead to severe ciliopathies and found seven WDR19 mutations in five SLS families. We identified a new gene for both arRP and SLS. WDR19 is a ciliary protein associated with the intraflagellar transport machinery. We are currently investigating the full extent of the mutation spectrum. Our findings are crucial in expanding the understanding of childhood blindness and identifying new genes.

Hypomorphic mutations in meckelin (MKS3/TMEM67) cause nephronophthisis with liver fibrosis (NPHP11).

BACKGROUND: Nephronophthisis (NPHP), a rare recessive cystic kidney disease, is the most frequent genetic cause of chronic renal failure in children and young adults. Mutations in nine genes (NPHP1-9) have been identified. NPHP can be associated with retinal degeneration (Senior-Løken syndrome), brainstem and cerebellar anomalies (Joubert syndrome), or liver fibrosis. METHODS: To identify a causative gene for the subset of patients with associated liver fibrosis, the authors performed a genome wide linkage search in a consanguineous family with three affected patients using 50K SNP microarrays and homozygosity mapping. RESULTS: The authors obtained a significant maximum parametric LOD (logarithm of odds) score of Z(max) = 3.72 on chromosome 8q22 and identified a homozygous missense mutation in the gene MKS3/TMEM67. When examining a worldwide cohort of 62 independent patients with NPHP and associated liver fibrosis we identified altogether four novel mutations (p.W290L, p.C615R, p.G821S, and p.G821R) in five of them. Mutations of MKS3/TMEM67, found recently in Meckel-Gruber syndrome (MKS) type 3 and Joubert syndrome (JBTS) type 6, are predominantly truncating mutations. In contrast, the mutations detected here in patients with NPHP and associated liver fibrosis are exclusively missense mutations. This suggests that they may represent hypomorphic alleles, leading to a milder phenotype compared with the more severe MKS or JBTS phenotype. Additionally, mutation analysis for MKS3/TMEM67 in 120 patients with JBTS yielded seven different (four novel) mutations in five patients, four of whom also presented with congenital liver fibrosis. CONCLUSIONS: Hypomorphic MKS3/TMEM67 mutations cause NPHP with liver fibrosis (NPHP11). This is the first report of MKS3 mutations in patients with no vermian agenesis and without neurological signs. Thus NPHP, JBTS, and MKS represent allelic disorders.

Mutational analysis of the RPGRIP1L gene in patients with Joubert syndrome and nephronophthisis.

Joubert syndrome (JS) is an autosomal recessive disorder, consisting of mental retardation, cerebellar vermis aplasia, an irregular breathing pattern, and retinal degeneration. Nephronophthisis (NPHP) is found in 17-27% of these patients, which was designated JS type B. Mutations in four separate genes (AHI1, NPHP1, CEP290/NPHP6, and MKS3) are linked to JS. However, missense mutations in a new ciliary gene (RPGRIP1L) were found in type B patients. We analyzed a cohort of 56 patients with JS type B who were negative for mutations in three (AHI1, NPHP1, and CEP290/NPHP6) of the four genes previously linked to the syndrome. The 26 exons encoding RPGRIP1L were analyzed by means of PCR amplification, CEL I endonuclease digestion, and subsequent sequencing. Using this approach, four different mutations in the RPGRIP1L gene in five different families were identified and three were found to be novel mutations. Additionally, we verified that missense mutations are responsible for JS type B and cluster in exon 15 of the RPGRIP1L gene. Our studies confirm that a T615P mutation represents the most common mutation in the RPGRIP1L gene causing disease in about 8-10% of JS type B patients negative for NPHP1, NPHP6, or AHI1 mutations.

Orbital tissue-derived T lymphocytes from patients with Graves' ophthalmopathy recognize autologous orbital antigens.

Lymphocytic and other mononuclear cell infiltration of the retro-bulbar space is observed in Graves' ophthalmopathy (GO). We investigated the antigenic character of orbital adipose/connective tissue and muscle from 21 euthyroid patients with severe GO after orbital surgery. Orbital tissue proteins were separated and recovered in soluble form by means of an electroelution technique. Twenty-two protein fractions, identified according to their molecular mass ranges, were used as antigens for orbital tissue-derived and peripheral blood T lymphocytes. Seventeen T cell lines from 6 patients were established from in vivo activated orbital T cells using interleukin-2 and anti-CD3 antibodies. T cell proliferation was measured as [3H] thymidine uptake. When screened for their reactivity to autologous adipose/connective tissue proteins, all T cell lines responded significantly to protein fractions 6-10 kDa [stimulation index (SI) = 32.9 +/- 9.8 (mean +/- SE)] and 19-26 kDa (17 +/- 5), but not to tuberculin, which was used as a control. Phenotypic analysis analysis of 10 orbital T cell lines indicated that 6 lines consisted predominantly of CD4+ cells. Incubation of a representative T cell line with allogeneic orbital protein fraction induced a very low response to protein fraction 19-26 kDa, but not to other fractions. Thyroid protein fraction 6-10 kDa also induced the proliferation of orbital T cell lines. Incubation of peripheral blood mononuclear cells with autologous orbital protein fractions gave similar results; positive responses to 6-10 and 19-26 kDa fractions were observed with orbital tissue from 12 of 14 patients (mean SI = 22 +/- 5.9 and 6.3 +/- 1.7, respectively), and positive responses were observed with orbital tissue from 3 of 4 patients to eye muscle fractions 6-10 and 19-26 kDa (13.8 +/- 6.9 and 6 +/- 2, respectively). When proteins from cultured orbital fibroblasts were used as antigens, autologous peripheral blood mononuclear cells from the 7 of the 9 patients tested responded to these 2 fractions (15.2 +/- 6.9 and 6.8 +/- 2.4, respectively), whereas a response to cultured orbital myoblasts was observed with the 19-26 kDa fraction only (SI = 8). Positive responses to abdominal adipose or muscle proteins, as controls, were not found. The demonstration of sensitized, orbital tissue-specific, T lymphocytes in the peripheral blood and orbit from patients with GO provides evidence for a role of cellular immunity in the pathogenesis of this eye disorder.

Retrobulbar adipocytes and humoral immunity in Graves' ophthalmopathy.

As CT and MR-imaging revealed an enlargement of retrobulbar fat tissue in patients with Graves' ophthalmopathy, the role of the retrobulbar adipocytes in the pathogenesis of this disorder remains to be elucidated. To evaluate the in vitro influence of humoral immunity on retrobulbar adipose tissue, the effects of IgG and sera from 56 euthyroid ophthalmopathy patients and 53 controls on both porcine and human (patients' and controls') retrobulbar adipocytes were measured by means of several assays: An enzyme-linked immunosorbent assay was employed to reveal specific binding of antibodies to the adipocytes. Metabolic activity was determined by means of a colorimetric dimethyl thiazolium-diphenyl-tetrazolium bromide test which quantifies the activity of mitochondrial dehydrogenases; cell proliferation was measured by incorporation of [3H]-thymidine in 24 h, and activities of adipocyte specific enzymes, such as membrane-bound lipoprotein lipase and 1-glycerol-3-phosphate-dehydrogenase were determined. By means of these specific enzyme tests no distinctions could be made between patients and controls. Furthermore, a significant difference between patients' (untreated and treated) and controls' IgG to bind to, to activate or to stimulate the proliferation of porcine or human (patients' and controls') retrobulbar adipocytes could not be detected under the employed experimental conditions. The effects of patients' heat-inactivated and non-inactivated sera were indistinguishable from those of the controls. Incubation with autologous sera, however, led to an activation of the retrobulbar adipocytes which was higher than the median caused by the patients' group and that engendered by incubation with autologous IgG.(ABSTRACT TRUNCATED AT 250 WORDS)