Prominent Optic Disc Featured in Inherited Retinopathy.

Background We investigated the relationship between prominent optic disc (POD) and inherited retinal dystrophy (IRD). Patients and Methods A cross-sectional consecutive study was performed in 10 children and 11 adults of 7 non-related families. We performed clinical phenotyping, including a detailed examination, fundus autofluorescence, and colour fundus and OCT imaging. Genetic testing was subsequently performed for all family members presenting retinal pathology. Results In 4 members of a 3-generation family, hyperfluorescent deposits on the surface of POD were related to a p.(L224M) heterozygous mutation in BEST1. In the second family, one member presented deposits located on the surface on hyperaemic OD and a compound p.(R141H);(A195V) mutation in BEST1. In the third family, POD was observed in father and child with early onset cone-rod dystrophy and a novel autosomal recessive p.(W31*) homozygous mutation in ABCA4. In the fourth family, POD with "mulberry-like" deposits and attenuated vessels were observed in a 7-year old girl, with a mutation in USH1A, and with early onset rod-cone dystrophy, associated with hearing loss. In the fifth family, blurry OD with tortuous vessels was observed in 4 consanguineous female carriers and a hemizygous boy with a p.(R200H) mutation in the X-linked retinoschisis RS1. In the sixth family, a mother and her son were both affected with POD and attenuated peripapillary vessels, and presented with a p.(Y836C) heterozygous mutation in TOPORS, thus confirming autosomal dominant RP. In the seventh family, in 3 family members with POD, compound p.(L541P;A1038 V);(G1961E) mutations in ABCA4 confirmed the diagnosis of Stargardt disease. Conclusions A variety of OD findings are found in a genetically heterogeneous group of IRDs. In the presence of POD, an inherited progressive photoreceptor disease should be ruled out.

Potential blindness in children of patients with hereditary bone disease.

Mono- and bi-allelic mutations in the low-density lipoprotein receptor related protein 5 (LRP5) may cause osteopetrosis, autosomal dominant and recessive exudative vitreoretinopathy, juvenile osteoporosis, or persistent hyperplastic primary vitreous (PHPV). We report on a child affected with PHPV and carrying compound mutations. The father carried the splice mutation and suffered from severe bone fragility since childhood. The mother carried the missense mutation without any clinical manifestations. The genetic diagnosis of their child allowed for appropriate treatment in the father and for the detection of osteopenia in the mother. Mono- and bi-allelic mutations in LRP5 may cause osteopetrosis, autosomal dominant and recessive exudative vitreoretinopathy, juvenile osteoporosis, or PHPV. PHPV is a component of persistent fetal vasculature of the eye, characterized by highly variable expressivity and resulting in a wide spectrum of anterior and/or posterior congenital developmental defects, which may lead to blindness. We evaluated a family diagnosed with PHPV in their only child. The child presented photophobia during the first 3 weeks of life, followed by leukocoria at 2 months of age. Molecular resequencing of NDP, FZD4, and LRP5 was performed in the child and segregation of the observed mutations in the parents. At presentation, fundus examination of the child showed a retrolental mass in the right eye. Ultrasonography revealed retinal detachment in both eyes. Thorough familial analysis revealed that the father suffered from many fractures since childhood without specific fragility bone diagnosis, treatment, or management. The mother was asymptomatic. Molecular analysis in the proband identified two mutations: a c.[2091+2T>C] splice mutation and c.[1682C>T] missense mutation. We report the case of a child affected with PHPV and carrying compound heterozygous LRP5 mutations. This genetic diagnosis allowed the clinical diagnosis of the bone problem to be made in the father, resulting in better management of the family. It also enabled preventive treatment to be prescribed for the mother and accurate genetic counseling to be provided.

A Novel Recessive RPGRIP1 Mutation Causing Leber Congenital Amaurosis.

BACKGROUND: Leber congenital amaurosis is an early-onset childhood severe retinal dystrophy, of significant genetic heterogeneity. RPGRIP1 is ubiquitously expressed, but mutations in RPGRIP1 lead to a retina-restricted phenotype, such as Leber congenital amaurosis and cone-rod dystrophy. PATIENT AND METHODS: We analysed a consanguineous family from Egypt in which one individual, a four-year-old girl, was affected with Leber congenital amaurosis. IROme, a proprietary enrichment system for retinal dystrophy genes, was applied and high throughput sequencing was performed. RESULTS: Severe visual impairment was reported during infancy. The fundus of the affected patient exhibited disc pallor and attenuated vessels. Neurodevelopmental delay and brain atrophy in the CT scan were reported. Genomic sequencing identified a novel homozygous deletion, c.[420delG], in RPGRIP1. This mutation was not detected in 80 ethnically matched controls and has not been reported elsewhere. CONCLUSIONS: Identifying new mutations in Leber congenital amaurosis-related genes and their clinical manifestations can improve our understanding of the disease and could help to stratify the population for potential therapies.

Molecular Analysis of NOTCH2 in Patients with Primary Open-Angle Glaucoma.

BACKGROUND AND PURPOSE: Transgenic mice overexpressing Notch2 in the uvea exhibit a hyperplastic ciliary body leading to increased IOP and glaucoma. The aim of this study was to investigate the possible presence of NOTCH2 variants in patients with primary open-angle glaucoma (POAG). METHODS: We screened DNA samples from 130 patients with POAG for NOTCH2 variants by denaturing high-performance liquid chromatography after PCR amplification and validated our data by direct Sanger sequencing. RESULTS: No mutations were observed in the coding regions of NOTCH2 or in the splice sites. 19 known SNPs (single nucleotide polymorphisms) were detected. An SNP located in intron 24, c.[4005+45A>G], was seen in 28.5% of the patients (37/130 patients). As this SNP is reported to have a minor allele frequency of 7% in the 1000 genomes database, it could be associated with POAG. However, we evaluated its frequency in an ethnic-matched control group of 96 subjects unaffected by POAG and observed a frequency of 29%, indicating that it was not related to POAG. CONCLUSION: NOTCH2 seemed to be a good candidate for POAG as it is expressed in the anterior segment in the human eye. However, mutational analysis did not show any causative mutation. This study also shows that proper ethnic-matched control groups are essential in association studies and that values given in databases are sometimes misleading.

Retinal vessel oxygen saturation in patients suffering from inherited diseases of the retina.

PURPOSE: The aim of this study was to evaluate the oxygen saturation in patients with inherited diseases of the retina. METHODS: Fundus oximetry images were taken using a retinal vessel analyser (IMEDOS Systems UG, Jena, Germany). Retinal vessel oximetry was performed in 53 eyes of 27 patients suffering from inherited retinal diseases and compared to 22 eyes of 11 healthy controls. The oxygen saturation in all four major retinal arterioles (A-SO2) and venules (V-SO2) were measured and their difference (A-V SO2) was calculated. The data were compared within groups and to controls. RESULTS: Based on V-SO2 values, the rod-cone dystrophy group (66.46%; SD, ± 5.09) could well be differentiated from controls 54.02% (SD, ± 3.04), from cone-rod dystrophies 57.56% (SD, ± 5.66), as well as from inherited maculopathies 58.42% (SD, ± 4.74). The mean A-SO2 in the rod-cone dystrophy group was increased to 98.96% (SD, ± 6.06, p<0.014), while in the cone-rod group and in the maculopathy group it was 92.75% (SD, ± 3.75), respectively 94.44% (SD ± 4.85), closer to the normal values (92.68%; SD, ± 3.53, p>0.05). The A-V SO2 difference, as an indirect indicator for retinal oxygen use, was reduced in the rod-cone patients, however only when the controls were taken into account (p=0.01). CONCLUSION: This is to our knowledge the first study which proposes the retinal vessel oximetry to be a sensitive measure for differentiating rod-cone dystrophy patients not only from controls, but also from patients with other inherited retinal dystrophies.

Cone-rod dystrophy caused by a novel homozygous RPE65 mutation in Leber congenital amaurosis.

BACKGROUND: The aim of this study was to describe an unexpected phenotype in a family with Leber congenital amaurosis (LCA) due to a retinal pigment epithelium-specific protein 65 kDa (RPE65) homozygous mutation. HISTORY AND SIGNS: We analyzed a family from Yemen in which 3 individuals were affected with LCA. Linkage analysis using markers flanking the known LCA genes was done, followed by direct sequencing of RPE65. THERAPY AND OUTCOME: Severe visual impairment and night blindness were observed during infancy. We observed photophobia only in the 8-year-old patient. The youngest affected had bilateral hyperopia of +3.50 and visual acuity of 1/60. The oldest two had visual acuity limited to hand movements in the right eye (OD) and counting fingers in the left eye (OS) for the oldest and of 5/60 OD, 6/60 OS for the other. They showed disc pallor, attenuated vessels, white flecks in the retina mid-periphery and bull's eye maculopathy. ERGs of the oldest child were completely unresponsive. Genomic sequencing identified a novel homozygous missense mutation, IVS2-3C>G, in the second RPE65 intron. CONCLUSIONS: We identified a novel LCA-related homozygous RPE65 mutation associated with a severe clinical presentation including an early and severe cone dysfunction. This is in contrast with the presentation associated with other RPE65 mutations predominantly causing rod-cone dystrophy with residual visual function.

ERK1/2 pathway is activated in degenerated Rpe65-deficient mice.

The MAPK family is composed of three majors kinases, JNK, p38 and ERK1/2, and is implicated in many degenerative processes, including retinal cell death. The purpose of our study was to evaluate the activation of ERK1/2 kinase, and its potential role in Müller cell gliosis, during photoreceptor cell death in Rpe65(-/-) mice. We assayed ERK1/2 mRNA and protein levels, and evaluated ERK1/2 phosphorylation involved in kinase activation, in 2, 4 and 6 month-old Rpe65(-/-) mice and in age-matched wild-type controls. No differences in ERK1/2 expression were detected between Rpe65(-/-) and wild-type mice, however, ERK1/2 phosphorylation was dramatically increased in the knock out mice at 4 and 6 months-of-age. Phosphorylated ERK1/2 co-localized with GFAP in the ganglion cell layer, and correlated with an increase in GFAP protein expression and retinal cell death. Accumulation of cFOS protein in the ganglion cell layer occurred concomitant with pERK1/2 activation. Müller cell proliferation was not observed. ERK1/2 activation did not occur in 2 month-old Rpe65(-/-) or in the Rpe65(-/-)/Gnat1(-/-) mice, in which no degeneration was evident. The observed activation ERK1/2 and GFAP, both markers of Müller cell gliosis, in the absence of Müller cell proliferation, is consistent with the activation of atypical gliosis occurring during the slow process of degeneration in Rpe65(-/-) mice. As Müller cell gliosis is activated in many neuronal and retinal degenerative diseases, further studies will be needed to determine whether atypical gliosis in Rpe65(-/-) mice contributes to, or protects against, the pathogenesis occurring in this model of Leber congenital amaurosis.

The gene mutated in thiamine-responsive anaemia with diabetes and deafness (TRMA) encodes a functional thiamine transporter.

Thiamine-responsive megaloblastic anaemia with diabetes and deafness (TRMA; MIM 249270) is an autosomal recessive disease thought to be due to a defect in thiamine (vitamin B1) transport. Pharmacological doses of thiamine correct the anaemia, and in some cases improve the diabetes, although progressive sensorineural deafness is irreversible. Previous studies localized the TRMA gene to a 4-cM region on chromosome 1q23.3 (ref. 5), and fine-mapping has recently narrowed that region further. We have previously demonstrated that fibroblasts from people with TRMA lack high-affinity thiamine transport. Expression of a gene encoding a known yeast thiamine transporter, THI10 (refs 8-10), in TRMA mutant cells prevents apoptotic cell death in thiamine-depleted medium. On the basis of these studies, we hypothesized that a defective thiamine transporter causes TRMA. We undertook a candidate gene approach to identify putative thiamine transporters in the 1q23.3 critical region. Here we present evidence that the gene SLC19A2 (for solute carrier family 19 (thiamine transporter), member 2) encodes the first known mammalian thiamine transporter, which we designate thiamine transporter-1 (THTR-1).

Kerato-epithelin mutations in four 5q31-linked corneal dystrophies.

Granular dystrophy Groenouw type I (CDGG1), Reis-Bücklers (CDRB), lattice type I (CDL1) and Avellino (ACD) are four 5q31-linked human autosomal dominant corneal dystrophies. Clinically, they show progressive opacification of the cornea leading to severe visual handicap. The nature of the deposits remains unknown in spite of amyloid aetiology ascribed to the last two. We generated a YAC contig of the linked region and, following cDNA selection, recovered the beta ig-h3 gene. In six affected families we identified missense mutations. All detected mutations occurred at the CpG dinucleotide of two arginine codons: R555W in one CDGG1, R555Q in one CDRB, R124C in two CDL1 and R124H in two ACD families. This suggests, as the last two diseases are characterized by amyloid deposits, that R124 mutated kerato-epithelin (the product of beta ig-h3) forms amyloidogenic intermediates that precipitate in the cornea. Our data establish a common molecular origin for the 5q31-linked corneal dystrophies.

The human pseudoautosomal GM-CSF receptor alpha subunit gene is autosomal in mouse.

The gene encoding the granulocyte macrophage colony stimulating factor receptor alpha subunit (CSF2RA) has previously been mapped to the pseudoautosomal region of the human sex chromosomes. In contrast, we report that the murine locus, Csf2ra, maps to an autosome in the laboratory mouse. By in situ hybridization and genetic mapping, Csf2ra maps at telomeric band D2 of mouse chromosome 19. This first instance of a pseudoautosomal locus in human being autosomal in mouse, indicates incomplete conservation between the human and mouse X chromosomes and suggests that the genetic content of the pseudoautosomal region may differ between species of eutherian mammals due to chromosomal rearrangements.

A single EFEMP1 mutation associated with both Malattia Leventinese and Doyne honeycomb retinal dystrophy.

Malattia Leventinese (ML) and Doyne honeycomb retinal dystrophy (DHRD) refer to two autosomal dominant diseases characterized by yellow-white deposits known as drusen that accumulate beneath the retinal pigment epithelium (RPE). Both loci were mapped to chromosome 2p16-21 (refs 5,6) and this genetic interval has been subsequently narrowed. The importance of these diseases is due in large part to their close phenotypic similarity to age-related macular degeneration (AMD), a disorder with a strong genetic component that accounts for approximately 50% of registered blindness in the Western world. Just as in ML and DHRD, the early hallmark of AMD is the presence of drusen. Here we use a combination of positional and candidate gene methods to identify a single non-conservative mutation (Arg345Trp) in the gene EFEMP1 (for EGF-containing fibrillin-like extracellular matrix protein 1) in all families studied. This change was not present in 477 control individuals or in 494 patients with age-related macular degeneration. Identification of this mutation may aid in the development of an animal model for drusen, as well as in the identification of other genes involved in human macular degeneration.

Mutations of keratinocyte transglutaminase in lamellar ichthyosis.

Lamellar ichthyosis is a severe congenital skin disorder characterized by generalized large scales and variable redness. Affected individuals in three families exhibited drastically reduced keratinocyte transglutaminase (TGK) activity. In two of these families, expression of TGK transcripts was diminished or abnormal and no TGK protein was detected. Homozygous or compound heterozygous mutations of the TGK gene were identified in all families. These data suggest that defects in TGK cause lamellar ichthyosis and that intact cross-linkage of cornified cell envelopes is required for epidermal tissue homeostasis.

New VMD2 gene mutations identified in patients affected by Best vitelliform macular dystrophy.

PURPOSE: The mutations responsible for Best vitelliform macular dystrophy (BVMD) are found in a gene called VMD2. The VMD2 gene encodes a transmembrane protein named bestrophin-1 (hBest1) which is a Ca(2+)-sensitive chloride channel. This study was performed to identify disease-specific mutations in 27 patients with BVMD. Because this disease is characterised by an alteration in Cl(-) channel function, patch clamp analysis was used to test the hypothesis that one of the VMD2 mutated variants causes the disease. METHODS: Direct sequencing analysis of the 11 VMD2 exons was performed to detect new abnormal sequences. The mutant of hBest1 was expressed in HEK-293 cells and the associated Cl(-) current was examined using whole-cell patch clamp analysis. RESULTS: Six new VMD2 mutations were identified, located exclusively in exons four, six and eight. One of these mutations (Q293H) was particularly severe. Patch clamp analysis of human embryonic kidney cells expressing the Q293H mutant showed that this mutant channel is non-functional. Furthermore, the Q293H mutant inhibited the function of wild-type bestrophin-1 channels in a dominant negative manner. CONCLUSIONS: This study provides further support for the idea that mutations in VMD2 are a necessary factor for Best disease. However, because variable expressivity of VMD2 was observed in a family with the Q293H mutation, it is also clear that a disease-linked mutation in VMD2 is not sufficient to produce BVMD. The finding that the Q293H mutant does not form functional channels in the membrane could be explained either by disruption of channel conductance or gating mechanisms or by improper trafficking of the protein to the plasma membrane.

Preserved functional and structural integrity of the papillomacular area correlates with better visual acuity in retinitis pigmentosa.

PurposeLinking multifocal electroretinography (mfERG) and optical coherence tomography (OCT) findings with visual acuity in retinitis pigmentosa (RP) patients.DesignProspective, cross-sectional, nonintervention study.SubjectsPatients with typical RP and age-matched controls, who underwent SD-OCT (spectral domain OCT) and mfERG, were included.MethodsMfERG responses were averaged in three zones (zone 1 (0°-3°), zone 2 (3°-8°), and zone 3 (8°-15°)). Baseline-to-trough- (N1) and trough-to-peak amplitudes (N1P1) of the mfERG were compared with corresponding areas of the OCT. The papillomacular area (PMA) was analyzed separately. Correlations between best-corrected visual acuity (BCVA, logMAR) and each parameter were determined.Main outcome measuresComparing structural (OCT) and functional (mfERG) measures with the BCVA.ResultsIn RP patients, the N1 and N1P1 responses showed positive association with the central retinal thickness outside zone 1 (P≤0.002), while the central N1 and the N1P1 responses in zones 1, 2, and 3-with the BCVA (P≤0.007). The integrity of the IS/OS line on OCT showed also a positive association with the BCVA (P<0.001). Isolated analysis of the PMA strengthened further the structure-function association with the BCVA (P≤0.037). Interactions between the BCVA and the OCT, respectively, the mfERG parameters were more pronounced in the RP subgroup without macular edema (P≤0.020).ConclusionIn RP patients, preserved structure-function of PMA, measured by mfERG amplitude and OCT retinal thickness, correlated well with the remaining BCVA. The subgroup analyses revealed stronger links between the examined parameters, in the RP subgroup without appearance of macular edema.

Rearrangements of minisatellites in the human telomerase reverse transcriptase gene are not correlated with its expression in colon carcinomas.

Telomerase activation is crucial in human carcinogenesis. The limiting component of telomerase, the catalytic subunit (hTERT), is undetectable in normal somatic cells but present in most tumor cells, including the earliest stages of colon carcinoma. The mechanisms involved in the differential expression in normal and tumor cells are not understood. In normal cells hTERT expression is shut down by a repressor, and upregulation could be a consequence of cis-acting changes in the hTERT gene, making it resistant to repression. We have identified a polymorphic and a monomorphic minisatellite in the second intron of the hTERT gene, and polymorphic one in intron 6. The polymorphic minisatellite in intron 2 contains binding sites for c-Myc, which has been shown to upregulate hTERT transcription. Screening colon carcinoma DNAs for rearrangements of hTERT minisatellites we detected no changes in 33 samples from tumors, most of which express hTERT. This indicates that size rearrangements of the hTERT minisatellites are not required for telomerase expression in colon carcinomas. Minor changes and one LOH were seen in five tumors.

Cell-permeable peptide inhibitors of JNK: novel blockers of beta-cell death.

Stress conditions and proinflammatory cytokines activate the c-Jun NH2-terminal kinase (JNK), a member of the stress-activated group of mitogen-activated protein kinases (MAPKs). We recently demonstrated that inhibition of JNK signaling with the use of the islet-brain (IB) 1 and 2 proteins prevented interleukin (IL)-1beta-induced pancreatic beta-cell death. Bioactive cell-permeable peptide inhibitors of JNK were engineered by linking the minimal 20-amino acid inhibitory domains of the IB proteins to the 10-amino acid HIV-TAT sequence that rapidly translocates inside cells. Kinase assays indicate that the inhibitors block activation of the transcription factor c-Jun by JNK. Addition of the peptides to the insulin-secreting betaTC-3 cell line results in a marked inhibition of IL-1beta-induced c-jun and c-fos expression. The peptides protect betaTC-3 cells against apoptosis induced by IL-1beta. All-D retro-inverso peptides penetrate cells as efficiently as the L-enantiomers, decrease c-Jun activation by JNK, and remain highly stable inside cells. These latter peptides confer full protection against IL-1beta-induced apoptosis for up to 2 weeks of continual treatment with IL-1beta. These data establish these bioactive cell-permeable peptides as potent pharmacological compounds that decrease intracellular JNK signaling and confer long-term protection to pancreatic beta-cells from IL-1beta-induced apoptosis.

Morphological, clinical and genetic aspects in a family with a novel LAMP-2 gene mutation (Danon disease).

A family with several cases of severe cardiomyopathy and moderate myopathy is described, affecting two brothers and their cousin as well as their mothers. One boy died of sudden cardiac arrest at 17 years of age. The two brothers were treated with an implantable defibrillator and their mother died suddenly at 40 years of age. Muscle biopsy in males showed vacuolar myopathy in two cases, and no abnormality on standard staining in the third case. Cardiac biopsies showed hypertrophic and vacuolated fibres. Complete absence of LAMP-2 was demonstrated by immunohistochemistry on the vacuolated skeletal and cardiac muscle, but also on the morphologically normal skeletal muscle. Sequencing of LAMP-2 gene showed a novel S157X mutation in exon 4. Danon disease is a rare and potentially lethal cause of hypertrophic cardiomyopathy. Diagnosis can be made by immunohistochemistry performed on cardiac or muscle biopsy, and confirmed by genetic analysis, which also allows for easy family screening and counselling.

Linkage of the steroid sulfatase gene to the sex-reversed mutation in the mouse.

Dosage studies and the inheritance pattern of the gene for steroid sulfatase (Sts) in the mouse have previously provided indirect evidence for a functional Y-linked allele which recombines obligatorily with its X-linked allele in male meiosis. In this study, we have investigated the linkage relationship of Sts and the sex-reversed mutation (Sxr), a gene which is known to reside in the pairing region of the Y chromosome. The results clearly demonstrate that Sxr and Sts are linked in a region of obligatory recombination and Sts maps proximal to Sxr with most recombinants occurring proximal to the two genes.

Molecular and clinical characteristics in 32 families affected with familial adenomatous polyposis.

Germ-line mutations in the 5' half of the Adenomatous Polyposis Coli (APC) gene are found in about 80% of the patients affected with familial adenomatous polyposis (FAP). The vast majority of these are nonsense or frameshift mutations which result in the loss of the carboxyl terminus of the APC protein. Using an in vivo assay in yeast, we have identified pathogenic germ-line mutations in 26 of 32 (81%) unrelated Swiss families affected with FAP. Nine mutations were novel and eight families were shown to harbor two recurrent mutations. Correlations were attempted between the location of APC germ-line mutations and clinical manifestations of the disease.