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In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Retinal ganglion cell dendritic pruning has been reported in association with a 50% reduction in Opa1 transcript and protein in retinal and neural tissue, which manifests as visual dysfunction in the heterozygous mutant mouse, B6;C3-Opa1(Q285STOP). Here we report a marked reduction in retinal ganglion cell synaptic connectivity in the absence of soma loss and explore the mechanism and relationship between mitochondrial integrity and synaptic connectivity. We observed decreased levels of postsynaptic density protein 95 in Opa1(+/-) mutant mice consistent with synaptic loss in the inner plexiform layer. Glutamatergic but not γ-aminobutyric acid-ergic synaptic sites were reduced in Opa1(+/-) mice. We observed increased synaptic vesicle number in bipolar cell terminal arbours assessed by immunohistochemistry, electron microscopy and western blot analysis. These changes occur without significant loss of mitochondrial membrane potential in retina and optic nerve. Analysis of biolistically transfected retinal ganglion cells shows the retraction of mitochondria towards the soma, and mitochondrial fragmentation, preceding dendritic loss. These processes cast light on the intimate relationship between normal mitochondrial fusion and fission balances, as influenced by the OPA1 protein, in neural cell connectivity in the mammalian retina.

This study examines the link between divorced nonresident fathers' proximity and children's long-run outcomes, using high-quality data from Norwegian population registers. We follow (from birth to young adulthood) each of 15,992 children born into married households in Norway in the years 1975-1979 whose parents divorced during his or her childhood. We observe the proximity of the child to his or her father in each year following the divorce and link proximity to educational and economic outcomes for the child in young adulthood, controlling for a wide range of observable characteristics of the parents and the child. Our results show that closer proximity to the father following a divorce has, on average, a modest negative association with offspring's outcomes in young adulthood. The negative associations are stronger among children of highly educated fathers. Complementary Norwegian survey data show that highly educated fathers report more post-divorce conflict with their ex-wives as well as more contact with their children (measured in terms of the number of nights that the child spends at the father's house). Consequently, the father's relocation to a more distant location following the divorce may shelter the child from disruptions in the structure of the child's life as they split time between households and/or from post-divorce interparental conflict.

Purpose: To investigate the developmental and ocular expression of Opa3 in a mouse model of 3-methylglutaconicaciduria type III and the effect of mutation on protein localization and mitochondrial morphology., Methods: The B6 C3-Opa3(L122P) mouse carrying a missense mutation in exon 2 (c.365T>C; p.L122P) of Opa3, which displays features of recessive 3-methylglutaconic aciduria type III was studied. The expression of Opa3 was determined with RT-PCR, quantitative PCR, and Western blot, in embryos (embryonic day [E]8 to postnatal day [P]0) and adult tissues, and by ocular immunohistochemistry. Mitochondria were stained using a mitochondrion-selective probe in mouse embryonic fibroblasts from Opa3(-/-) mutants and imaged by electron microscopy of the retinas., Results: The splice variants Opa3a and Opa3b were expressed in the lenses and the retinas in the Opa3(-/-) mice, with the expression of the Opa3a isoform predominant. Opa3 was expressed throughout embryonic development, with high levels of expression in the developing brain, retina, optic nerve, and lens. Opa3 localized to the mitochondria, and the L122P mutant protein did not mislocalize. Neither protein localized to the peroxisome. Opa3(-/-) mice displayed disrupted mitochondrial morphology in the retina. Wild-type Opa3 protein increased as the lenses aged, despite the reduction in Opa3 mRNA occurring as a part of lens differentiation. However, mutant Opa3 mRNA was upregulated in homozygous mutant lenses, suggesting a compensatory increase in expression, which may further increase Opa3 protein levels., Conclusions: Mutant Opa3 protein retains its mitochondrial localization and induces disrupted mitochondrial morphology. Opa3 accumulates in the lens. The results may reflect a slow turnover of Opa3 protein in vivo and may be important in normal lens physiology.

The heterozygous mutation B6;C3-Opa1(Q285STOP), which models autosomal dominant optic atrophy, leads to a 50% reduction in Opa1 transcript and protein in the mouse retina and neural tissues and is associated with visual dysfunction and structural changes in the murine retina and optic nerve. In this article we use this model to quantify and evaluate the dendritic morphology of retinal ganglion cells. Retinal ganglion cells in Opa1(+/-) mutant mice (n=16) and accompanying age- and sex-matched controls (n=11) (age ranges of <10, 10-15 and >20 months) were labelled DiOlistically with carbocyanine dyes to quantify changes in dendritic tree architecture as a function of age. We observed localized dendritic reduction to sublamina b of the inner plexiform layer without retinal ganglion cell loss, showing dendritic pruning of on- but not off-centre retinal ganglion cells, and this effect was exacerbated with age. The mean dendritic field area was reduced in on-centre retinal ganglion cells of 10- to 15-month-old mice (-24.24%; C(V) =0.68; P<0.05) and >20-month-old mice (-43.22%; C(V) =0.75; P<0.05) compared with age-matched wild-type controls. Similar changes were seen in average total dendritic length in on-centre retinal ganglion cells of 10- to 15-month-old mice (-31.66%; C(V) =0.67; P<0.05) and >20-month-old mice (-49.55%; C(V) =0.63; P<0.05). Sholl analysis showed a marked difference in the dendritic arborization of on-centre retinal ganglion cells in the 10- to 15-month-old group (area under the curve -21.67%; P>0.05) and of the >20-month-old group (area under the curve -42.12%; P<0.05) compared with the control group. There was no detectable change in dendritic morphology in <10-month-old Opa1(+/-) mutant mice compared with wild-type (P>0.05). No significant changes (P>0.05) were seen in off-centre retinal ganglion cells. Finally, there was also no significant change (P>0.05) in the retinal ganglion cell count across all age groups. In conclusion, we show dendritic pruning in on-centre retinal ganglion cells of the Opa1(+/-) mouse model of autosomal dominant optic atrophy from as early as 10 months of age. These results highlight the importance of normal mitochondrial fusion balance, as influenced by the OPA1 protein in maintaining the dendritic morphology of retinal ganglion cells. Dendritic pruning precedes the onset of clinical visual loss and structural changes in the optic nerve in the absence of significant cell loss.

Purpose: To characterize changes in corneal collagen arrangement during mouse postnatal development., Methods: Small-angle X-ray scatter patterns were gathered from the centers of 32 excised mice corneas aged between postnatal days 10 (before eye opening) and 28 (onset of sexual maturity). These were analyzed to produce measurements of the average separation distance between corneal collagen fibrils. Changes in the predominant orientation of corneal collagen and its relative distribution during the same developmental period were determined using wide-angle X-ray scatter data collected at 0.2-mm intervals over the entire cornea and limbal region of each specimen., Results: Collagen interfibrillar spacing decreased in the days leading up to eye opening (61.3 +/- 2.9 nm at day 10 to 45.5 +/- 4.5 nm at day 14), after which it remained constant. However, changes in collagen orientation and distribution occurred throughout the entire developmental period. After eye opening at day 12, collagen alignment gradually increased in the peripheral cornea and limbus. By day 28, an annulus of highly aligned collagen surrounded the cornea., Conclusions: Changes in corneal thickness before and after eye opening are not caused by widespread alterations in the collagen fibrillar array but are more likely caused by expansion and contraction of regions devoid of regularly arranged collagen. The postnatal development of a corneal annulus of collagen, thought to play a role in stabilizing the curvature of the cornea, may be triggered by visual factors.

Purpose: The majority of patients with autosomal dominant optic atrophy (DOA) harbor pathogenic OPA1 mutations and certain missense mutations, mostly within the GTPase domain, have recently been shown to cause multiple mitochondrial DNA (mtDNA) deletions in skeletal muscle. This raises the possibility that the optic neuropathy could be the result of secondary mtDNA defects accumulating within retinal ganglion cells (RGCs). To explore this hypothesis, the authors looked for evidence of mitochondrial dysfunction in a mouse model of DOA and documented the visual and neurologic progression in aging mutant mice., Methods: Visual function was assessed with a rotating optokinetic (OKN) drum at ages 13 and 18 months and neurologic phenotyping was performed using the primary SHIRPA screen at age 13 months, comparing mutant Opa1(+/)(-) mice with wild-type C57Bl/6 mice. The presence of cytochrome c oxidase (COX) deficiency and multiple mtDNA deletions was investigated in gastrocnemius muscle and eye specimens harvested from 2- and 11-month-old Opa1(+/+) and Opa1(+/)(-) mice., Results: At age 13 months, Opa1(+/)(-) mice had a statistically significant reduction in OKN responses compared to C57Bl/6 controls with both 2 degrees and 8 degrees gratings (P < 0.001). At age 18 months, the difference between the two groups was significant for the 8 degrees grating (P = 0.003) but not for the 2 degrees grating (P = 0.082). Opa1(+/)(-) mice did not exhibit any significant neuromuscular deficits and no COX deficient areas or secondary mtDNA deletions were identified in skeletal muscle or the RGC layer. There was also no evidence of significant mtDNA depletion or proliferation in skeletal muscle from Opa1(+/)(-) mice., Conclusions: COX deficiency and mtDNA abnormalities do not contribute to optic nerve dysfunction in pure DOA.

Purpose: To examine retinal ganglion cell (RGC) and axonal abnormalities in an ENU-induced mutant mouse carrying a protein-truncating nonsense mutation in OPA1. Mutations in the OPA1 gene cause autosomal dominant optic atrophy (ADOA) in which loss of RGCs followed by myelin degeneration in the optic nerve leads to progressive decrease in visual acuity., Methods: Ultrastructure of the optic nerve was examined in heterozygous mutants and wild-type littermate controls at 6, 9, and 24 months using electron microscopy. The RGC layer was examined at 6 and 24 months., Results: There was an increase in the number of autophagosomes in the RGC layer in heterozygous mutants compared with wild type at 24 months. Signs of optic nerve degeneration were seen as early as 9 months in Opa1(+/-) mice, with more severe degeneration by 24 months. By 24 months, degeneration of axons was also seen in control mice. Numbers of opaque mitochondria in the Opa1(+/-) mice increased at 6 and 24 months, possibly representing an increase in the density of cristae to fulfill the energy requirements of the axon. In addition, mitochondria with vesiculation of the inner membranes, similar to the mutant mitochondria described in a mouse model of Charcot-Marie-Tooth type 2A, were observed., Conclusions: Mutations in OPA1 cause pathologic changes to optic nerve axons that are similar to, but occur earlier than, age-related degeneration. Increased autophagy is likely to result from an increase in abnormal mitochondria and could be one mechanism contributing to RGC loss and subsequent optic atrophy seen in ADOA.

Opa3 mRNA is expressed in all tissues examined to date, but currently the function of the OPA3 protein is unknown. Intriguingly, various mutations in the OPA3 gene lead to two similar diseases in humans: autosomal dominant inherited optic atrophy and cataract (ADOAC) and a metabolic condition; type 3-methylglutaconic aciduria (MGA). Early onset bilateral optic atrophy is a common characteristic of both disorders; retinal ganglion cells are lost and visual acuity is impaired from an early age. In order to investigate the function of the OPA3 protein, we have generated a novel ENU-induced mutant mouse carrying a missense mutation in the OPA3 gene. The heterozygous mutation in exon 2, causes an amino acid change p.L122P (c.365T>C), which is predicted to alter tertiary protein structure. In the heterozygous state, the mice appear uncompromised however; in the homozygous state mice display some of the features of MGA. Visual function is severely reduced, consistent with significant loss of retinal ganglion cells and degeneration of axons in the optic nerve. In the homozygous optic nerve, there was evidence of increased mitochondrial activity, as demonstrated by the increased presence of mitochondrial marker Cytochrome C Oxidase (COX) histochemistry. Mice homozygous for the opa3(L122P) mutation also display a severe multi-systemic disease characterized by reduced lifespan (majority dying before 4 months), decreased weight, dilated cardiomyopathy, extrapyramidal dysfunction and gross neuro-muscular defects. All of these defects are synonymous with the phenotypic characteristics of Type III MGA found in humans. This model will be of major importance for future studies of the specific function of the OPA3 gene.

This study is a comparative study of the relationship between corneal structure, morphology, and function in a range of mammalian species. X-ray scattering patterns were gathered at regular spatial intervals over the excised cornea (and in most cases also the scleral rim) of humans, marmosets, horses, cows, pigs, rabbits, and mice. All patterns were analyzed to produce quantitative information regarding the predominant orientation of fibrillar collagen throughout the tissue. The predominant direction of corneal collagen varies between mammals. This variation is not related to the size, shape, or thickness of the cornea or the frequency with which the animal blinks. A relationship does, however, appear to exist between corneal collagen arrangement and visual acuity. An excess of collagen directed toward one or both sets of opposing rectus muscles is a feature of animals that have an intermediate to high level of visual acuity. There is a significant variation in the arrangement of corneal collagen between different mammalian species. This finding may be related to differences in the frequency of action and the forces generated by the various extraocular muscles during eye movement and image fixation.

OPA1 is a ubiquitously expressed, nuclear dynamin-related GTPase, targeted to the inner mitochondrial membrane, which plays a role in mitochondrial fusion. Mutations in the OPA1 gene on chromosome 3q28-qter are associated with autosomal dominant optic atrophy (ADOA), the most common inherited optic neuropathy, in which retinal ganglion cells (RGCs) are lost and visual acuity is impaired from an early age. We have generated a novel ENU-induced mutant mouse carrying a protein-truncating nonsense mutation in opa1 in order to explore the pathophysiology of ADOA. The heterozygous mutation, B6; C3-Opa1(Q285STOP), located in exon 8 immediately before the central dynamin-GTPase, leads to approximately 50% reduction in opa1 protein in retina and all tissues on western analysis. The homozygous mutation is embryonic lethal by 13.5 days post coitum, demonstrating the importance of Opa1 during early development. Fibroblasts taken from adult heterozygous mutant mice show an apparent alteration in morphology, with an increase in mitochondrial fission and fragmentation. Heterozygous mutants show a slow onset of degeneration in the optic nerve electron microscopy. Furthermore, they demonstrate a functional reduction in visual function on testing with the optokinetic drum and the circadian running wheel. These findings indicate that the opa1 GTPase contains crucial information required for the survival of RGCs and that Opa1 is essential for early embryonic survival. The Opa1 +/- mice described here provide a means to directly investigate the cellular pathophysiology of OPA1 ADOA.

Objective: To determine the incremental costs of pneumonia occurring during hospitalization for stroke., Methods: We reviewed hospital records of all Medicare patients admitted for ischemic or hemorrhagic stroke to 29 hospitals in a large metropolitan area, 1991 through 1997, excluding those who died or had do not resuscitate orders written within 3 days of admission. Hospital costs of patients with stroke were determined using Medicare Provider Analysis and Review data after adjustment for baseline factors affecting cost and propensity for pneumonia. Secondary analyses examined the risk-adjusted relationship of pneumonia to discharge disposition., Results: Pneumonia occurred in 5.6% (635/11,286) of patients with stroke, and was more common among patients admitted from nursing homes and those with greater severity of illness (p < 0.001). Mean adjusted costs of hospitalization for patients with stroke with pneumonia were $21,043 (95% CI $19,698 to 22,387) and were $6,206 (95% CI $6,150 to 6,262) for patients without pneumonia, resulting in an incremental cost of $14,836 (95% CI $14,436 to 15,236). Patients with pneumonia were over 70% more likely to be discharged with requirements for extended care (adjusted OR 1.73, 95% CI 1.32 to 2.26)., Conclusion: Extrapolated to the over 500,000 similar patients hospitalized for stroke in the United States, the annual cost of pneumonia as a complication after acute stroke is approximately $459 million.

Purpose: Autosomal dominant optic atrophy (ADOA) is characterized by primary degeneration of retinal ganglion cells and atrophy of the optic nerve. The OPA1 gene encodes a 960-amino-acid protein. In the current study the temporal and spatial localization of OPA1 were examined in developing and adult murine ocular tissues and the adult human eye. Because the Bst/+ mouse has been postulated as a model of ADOA, the mOPA1 expression in the Bst/+ retina was also examined., Methods: A polyclonal antibody generated against a C-terminal peptide of OPA1 was used to assess by immunohistochemistry the expression of mOPA1 in the wild-type embryonic and postnatal mouse ocular tissues and the Bst/+ retina. Western blot analyses of total proteins from a panel of adult human tissues were used to examine the expression of human OPA1, and spatial localization was assessed by immunohistochemistry., Results: The ocular expression of mOPA1 begins at E15 in the inner retina in a location corresponding to that of the subsequently developing ganglion cell layer (GCL) and peaks between postnatal day (P)0 and P1 in the retina and the optic nerve. There is a sharp decline in mOPA1 expression after P2, but it is expressed at a basal level until at least P12 in the GCL, inner plexiform layer (IPL), and inner nuclear layer (INL) of the retina as well as in the optic nerve. In the adult Bst/+ retina, mOPA1 is strongly expressed in the GCL and IPL and weakly in the INL. In the adult human eye, OPA1 is expressed in the GCL, IPL, INL, and outer plexiform layer (OPL) of the retina and in the optic nerve, where it is observed only in the myelinated region., Conclusions: OPA1 is not restricted to the GCL of the mammalian retina, and its expression extends into the IPL, INL, and OPL. OPA1 is distinctly expressed in the myelinated region beyond the lamina cribrosa in the human optic nerve, whereas its expression is weaker in the mouse optic nerve. In the Bst/+ mouse retina, despite the structural defects, mOPA1 expression is comparable to that observed in the wild-type adult mouse retina. These observations suggest a wider role for OPA1 than previously anticipated.

The case of a 12-year-old girl with Jaffé-Lichtenstein-Uehlinger syndrome is presented. A bone scan pattern exhibits clinical symptoms, X ray images and histological findings are described in a case of polyostotic form of the fibrous dysplasia. A biopsy with histology was performed in consideration of bone scan findings and a confirmed final diagnosis.

Objective: To characterize the clinical phenotype, with emphasis on electrophysiology and blood flow measurements, of a family with dominant optic nerve atrophy and an identified mutation in the OPA1 gene., Methods: Seven family members were examined. Ophthalmological evaluation included testing of visual acuity, ophthalmolscopy, kinetic perimetry, color vision testing, full-field electroretinography (ERG), multifocal electroretinography (MERG), and multifocal visual evoked potential (MVEP). Retrobulbar arterial blood flow and retinal capillary perfusion was measured in three patients using scanning laser Doppler flowmetry (SLDF) and color Doppler imaging techniques. PCR-SSCP and DNA sequencing determined the presence of a mutation in exon 18 of the OPA1 gene., Results: The clinical characteristics varied considerably in the family. The ERG and the MERG demonstrated normal retinal function, while the MVEP was abnormal in all examined patients. Retinal and optic nerve head capillary perfusion was significantly decreased in the three patients examined with SLDF. Retrobulbar blood flow velocities were significantly decreased in the central retinal and ophthalmic arteries. In all seven examined subjects, a microdeletion (1756-1767del(12 bp)) in the OPA1 gene was identified., Conclusion: Patients with a mutation in the OPA1 gene have a very variable phenotype. MVEP and blood flow measurements are two new objective methods for an easier detection of this specific genetic optic nerve atrophy.

Background/aims: Patients with autosomal dominant optic atrophy (ADOA) are genetically heterogeneous, but all have disc pallor. A degree of cupping in ADOA can make the distinction from normal tension glaucoma (NTG) clinically difficult. This study aimed to clarify the features of the optic nerve of patients with ADOA at the OPA1 locus., Methods: 29 patients (58 eyes), from 12 families, were identified in a prospective observational study of patients with ADOA examined by a single observer between 1995 and 1998, in whom genetic analysis showed either evidence for linkage to chromosome 3q28 or mutations in the ADOA gene, OPA1. All of the patients had disc and fundal photographs available for retrospective analysis. Clinical data collected included disc appearance, intraocular pressure, Snellen visual acuity, Hardy-Rand-Rittler colour vision plates, and Humphrey 30-2 visual fields., Results: Mean age at time of examination was 37 years and mean visual acuity was 6/24. Disc morphology showed temporal disc pallor in 30 eyes (52%) and total disc pallor in 28 eyes (48%). At least one disc showed a cup to disc ratio of more than 0.5 in 18 patients (28 discs, 48%). The temporal neuroretinal rim always showed pallor and shallow shelving (or saucerisation) was seen in 46 eyes (79%). Only 12 discs (21%) had deep excavation and baring of blood vessels. All of the patients had normal intraocular pressure and no family history of glaucoma. There was a temporal grey, pigmentary crescent in 12 patients (18 eyes, 31%) and peripapillary atrophy in 20 patients (40 eyes, 69%), but disc margin haemorrhages were not seen. There was no maculopathy or retinopathy., Conclusion: The optic disc morphology, described for the first time in this genetically homogeneous population of patients with OPA1 ADOA, shows a distinctive absence of a healthy neuroretinal rim and shallow saucerisation of the optic disc cup, with frequent peripapillary atrophy.

The primary inherited optic neuropathies are a heterogeneous group of disorders that result in loss of retinal ganglion cells, leading to the clinical appearance of optic atrophy. They affect between 1:10,000 to 1:50,000 people. The main clinical features are a reduction in visual acuity, colour vision abnormalities, centro-caecal visual field defects and pallor of the optic nerve head. Electrophysiological testing shows a normal flash electroretinogram, absent or delayed pattern visually evoked potentials suggestive of a conduction deficit and N95 waveform reduction on the pattern electroretinogram, consistent with a primary ganglion cell pathology. The primary inherited optic neuropathies may be sporadic or familial. The mode of inheritance may be autosomal dominant, autosomal recessive, X-linked recessive or mitochondrial. Within each of these groups, the phenotypic characteristics vary in such features as the mode and age of onset, the severity of the visual loss, the colour deficit and the overall prognosis. A number of different genes (most as yet unidentified) in both nuclear and mitochondrial genomes, underlie these disorders. The elucidation of the role of the encoded proteins will improve our understanding of basic mechanisms of ganglion cell development, physiology and metabolism and further our understanding of the pathophysiology of optic nerve disease. It will also improve diagnosis, counselling and management of patients, and eventually lead to the development of new therapeutic modalities.

Purpose: To characterize the spectrum of mutations in the OPA1 gene in a large international panel of patients with autosomal dominant optic atrophy (adOA), to improve understanding of the range of functional deficits attributable to sequence variants in this gene, and to assess any genotype-phenotype correlations., Methods: All 28 coding exons of OPA1, intron-exon splice sites, 273 bp 5' to exon 1, and two intronic regions with putative function were screened in 94 apparently unrelated white patients of European origin with adOA by single-strand conformational polymorphism (SSCP)-heteroduplex analysis and direct sequencing. Clinical data were collated, and putative mutations were tested for segregation in the respective families by SSCP analysis or direct sequencing and in 100 control chromosomes. Further characterization of selected splice site mutations was performed by RT-PCR of patient leukocyte RNA. Staining of mitochondria in leukocytes of patients and control subjects was undertaken to assess gross differences in morphology and cellular distribution., Results: Twenty different mutations were detected, of which 14 were novel disease mutations (missense, nonsense, deletion-frameshift, and splice site alterations) and six were known mutations. Mutations were found in 44 (47%) of the 94 families included in the study. Ten new polymorphisms in the OPA1 gene were also identified. Mutations occur throughout the gene, with three clusters emerging: in the mitochondrial leader, in the highly conserved guanosine triphosphate (GTP)-binding domain, and in the -COOH terminus. Examination of leukocyte mitochondria from two unrelated patients with splice site mutations in OPA1 revealed no abnormalities of morphology or cellular distribution when compared with control individuals., Conclusions: This study describes 14 novel mutations in the OPA1 gene in patients with adOA, bringing the total number so far reported to 54. It is likely that many cases of adOA are due to mutations outside the coding region of OPA1 or to large-scale rearrangements. Evaluation of the mutation spectrum indicates more than one pathophysiological mechanism for adOA. Preliminary data suggests that phenotype-genotype correlation is complex, implying a role for other genetic modifying or environmental factors. No evidence was found of pathologic changes in leukocyte mitochondria of patients with adOA.

Normal tension glaucoma (NTG) is a major form of glaucoma, associated with intraocular pressures that are within the statistically normal range of the population. OPA1, the gene responsible for autosomal dominant optic atrophy represents an excellent candidate gene for NTG, as the clinical phenotypes are similar and OPA1 is expressed in the retina and optic nerve. Eighty-three well-characterized NTG patients were screened for mutations in OPA1 by heteroduplex analysis and bi-directional sequencing. Sequences found to be altered in NTG subjects were examined for variations in 100 population controls. A second cohort of 80 NTG patients and 86 population controls was subsequently screened to determine whether the initial findings could be replicated. A single nucleotide polymorphism (SNP) on intervening sequence (IVS) 8 (IVS8 + 4 C/T) was found to be strongly associated with the occurrence of NTG in both cohorts (chi(2)=7.97, P=0.005 in the first cohort, chi(2)=9.93, P=0.002 in the second cohort; odds ratio 3.1 (95% CI: 1.8-5.6). A second SNP (IVS8 + 32 T/C) appeared to be associated with disease in the first cohort (chi(2)=4.71, P=0.030), but this finding could not be replicated in the second cohort. In the combined cohort, the compound at-risk genotype IVS8 + 4 C/T, + 32 T/C was strongly associated with the occurrence of NTG (chi(2)=22.04, P=0.00001 after correcting for testing four genotypes). These results indicate that polymorphisms in the OPA1 gene are associated with NTG and may be a marker for the disease.

In humans, low peak bone mass is a significant risk factor for osteoporosis. We report that LRP5, encoding the low-density lipoprotein receptor-related protein 5, affects bone mass accrual during growth. Mutations in LRP5 cause the autosomal recessive disorder osteoporosis-pseudoglioma syndrome (OPPG). We find that OPPG carriers have reduced bone mass when compared to age- and gender-matched controls. We demonstrate LRP5 expression by osteoblasts in situ and show that LRP5 can transduce Wnt signaling in vitro via the canonical pathway. We further show that a mutant-secreted form of LRP5 can reduce bone thickness in mouse calvarial explant cultures. These data indicate that Wnt-mediated signaling via LRP5 affects bone accrual during growth and is important for the establishment of peak bone mass.

Dominant optic atrophy (DOA) is a hereditary optic neuropathy characterised by decreased visual acuity, colour vision deficits, centro-coecal scotoma and optic nerve pallor. The gene OPA1, encoding a dynamin-related GTPase, has recently been identified within the genetic linkage interval for the major locus for DOA on chromosome 3q28 and shown to harbour genetic aberrations segregating with disease in DOA families. The prevalence of the disorder in Denmark is reported to be the highest of any geographical location, suggestive of a founder effect. In order to establish the genetic basis of disease in a sample of 33 apparently unrelated Danish families, we screened DNA from affected members for OPA1 gene mutations by heteroduplex analysis and direct sequencing. A novel identical mutation in exon 28 (2826delT) was associated with DOA in 14 pedigrees and led to a frameshift and abnormal OPA1 protein -COOH terminus. Haplotype analysis of a region of approximately 1 Mb flanking the OPA1 gene using eight polymorphic markers revealed a common haplotype shared by all 14 patients; this haplotype was markedly over-represented compared with ethnically matched controls. Statistical analysis confirmed significant linkage disequilibrium with DOA over approximately 600 kb encompassing the disease mutation. We have therefore demonstrated that the relatively high frequency of DOA in Denmark is attributable to a founder mutation responsible for approximately 42% of the examined families and suggest that presymptomatic screening for the (2826delT) mutation may facilitate diagnosis and genetic counselling in a significant proportion of DOA patients of Danish ancestry.

Supplement with keto acids/amino acids (KA) and erythropoietin can independently improve the metabolic sequels of chronic renal insufficiency. Our study was designed to establish whether a supplementation with keto acids/amino acids (KA) exerts additional beneficial metabolic effects in patients with chronic renal insufficiency (CRF) treated with a low-protein diet (LPD) and recombinant human erythropoietin (EPO). In a prospective randomized controlled trial over a period of 12 months, we evaluated a total of 38 patients (20 M/18 F) aged 32-68 years with a creatinine clearance (CCr) of 20-36 ml/min. All patients were receiving EPO (40 U/kg twice a week s.c.) and a low-protein diet (0.6 g protein/kg/day and 145 kJ/kg/day). The diet of 20 patients (Group I) was supplemented with KA at a dosage of 100 mg/kg/day while 18 patients (Group II) received no supplementation. During the study period, the glomerular filtration rate slightly decreased (CCr from 28.2 +/- 3.4 to 26.4 +/- 4.1 ml/min and 29.6 +/- 4.8 to 23.4 +/- 4.4 ml/min in groups I and II, respectively and Cin); this however was more marked in Group II (Group I vs. Group II, p < 0.01). The serum levels of urea also declined (p < 0.01), more pronouncedly in Group I (p < 0.025). In Group I, there was a significant rise in the levels of leucine (p < 0.01), isoleucine (p < 0.01), valine (p < 0.02) and albumin (p < 0.01) and a decrease in protein-uria (p < 0.01). Analysis of the lipid spectrum revealed a mild yet significant decrease in total cholesterol and LDL-cholesterol (p < 0.02), more pronounced in Group I. In Group I, there was a decrease in plasma triglycerides (from 4.2 +/- 0.8 down to values a low as 2.2 +/- 0.6 mmol/L; p < 0.01) whereas HDL-cholesterol levels increased (from 0.9 +/- 0.1 to 1.2 +/- 0.1 mmol/L, p < 0.01). A further remarkable finding was a reduction in the serum concentration of free radicals (p < 0.01). We conclude that a KA supplementation in patients with CRF receiving LPD and EPO potentiates the beneficial effects on metabolism of proteins, amino acids and surprisingly, also lipids. Long-term co-administration of KA, EPO and LPD was also associated with a delay in progression of renal insufficiency and a reduction in proteinuria. Thus, concomitant administration of KA and EPO during a low-protein diet presents an effective treatment modality in the conservative management of CRF.

Purpose: To review treatment strategies in neovascular age-related macular degeneration (ARMD)., Methods: Medline and Embase search., Results: Age-related macular degeneration (ARMD) is the commonest cause of blindness in the developed world in individuals over 50 years of age. ARMD may lead to loss of vision by atrophy of the retinal pigment epithelium or by the development of choroidal neovascular membranes (CNVM) under the macula, which leak serous fluid and blood and ultimately cause a blinding disciform scar. Treatment options currently being investigated fall into three main approaches: elimination of the CNVM from the subfoveal area (by laser or surgery), modification of the CNVM (by laser, radiotherapy or chemotherapeutic agents) or lastly prevention of the formation of CNVM (by laser prophylaxis, diet or gene targeting). Whilst almost no therapy restores normal visual acuity, any significant visual improvement over the natural history may be regarded as beneficial., Conclusions: Both the current and immediate future potential therapies for choroidal neovascularisation in ARMD require considerable advances to be made before they will make any impact on blindness caused by ARMD. Of the current treatments none are curative and the treatment benefits are small. There is an urgent need for new therapies.

Purpose: To compare visual and pupil afferent function in dominant optic atrophy (DOA)., Methods: Patients with DOA who belonged to families showing evidence of linkage to the locus on chromosome 3q28-qter were recruited from the Moorfields Genetic Register. Patients and healthy control subjects underwent visual and pupil perimetry using a modified automated perimeter (Octopus 1-2-3; Interzeag, Schlieren, Switzerland). Five stimulus locations were tested: fixation, and at 17 degrees eccentricity along the 45 degrees and 135 degrees meridians in all four quadrants. The visual deficit (difference in decibels between the patient's luminance threshold and that in age-matched healthy control subjects) was compared directly with the pupil deficit (difference in decibels between the stimulus intensity giving the patient's pupil response and that giving an equivalent pupil response in healthy control subjects) at each test location., Results: Visual deficits and pupil afferent deficits were found at all five locations. The visual deficits were significantly greater than the pupil deficits at the four peripheral locations (median difference = 6.3 dB, P: < 0.001). At fixation, the difference was not significant (median difference = 2.3 dB, P: = 0.407)., Conclusions: Pupil function appears less affected than visual function at four of five locations tested. This result provides evidence that the retinotectal fibers serving the pupil light reflex are less susceptible to damage from the OPA1 genetic defect than the retinogeniculate fibers serving vision.

Autosomal dominant optic atrophy (ADOA) is the most prevalent hereditary optic neuropathy resulting in progressive loss of visual acuity, centrocoecal scotoma and bilateral temporal atrophy of the optic nerve with an onset within the first two decades of life. The predominant locus for this disorder (OPA1; MIM 165500) has been mapped to a 1.4-cM interval on chromosome 3q28-q29 flanked by markers D3S3669 and D3S3562 (ref. 3). We established a PAC contig covering the entire OPA1 candidate region of approximately 1 Mb and a sequence skimming approach allowed us to identify a gene encoding a polypeptide of 960 amino acids with homology to dynamin-related GTPases. The gene comprises 28 coding exons and spans more than 40 kb of genomic sequence. Upon sequence analysis, we identified mutations in seven independent families with ADOA. The mutations include missense and nonsense alterations, deletions and insertions, which all segregate with the disease in these families. Because most mutations probably represent null alleles, dominant inheritance of the disease may result from haploinsufficiency of OPA1. OPA1 is widely expressed and is most abundant in the retina. The presence of consensus signal peptide sequences suggests that the product of the gene OPA1 is targeted to mitochondria and may exert its function in mitochondrial biogenesis and stabilization of mitochondrial membrane integrity.

Measurements of the intraorbital optic nerve were made using high-resolution coronal MRI in 10 adults with autosomal dominant optic atrophy. Comparisons were made with previous studies of 10 normal adult subjects. The cross-sectional diameters of the optic nerve and the perineural subarachnoid space were measured and a ratio of there diameters at anterior, mid and posterior positions along the optic nerve was determined. We found a statistically significant difference in the mean optic nerve: sheath ratio between the control group and patients with autosomal dominant optic atrophy. At anterior, mid and posterior locations along the optic nerve it is significantly smaller in patients with optic atrophy. We have demonstrated that the loss of ganglion cells, previously documented in dominant optic atrophy, is associated with a significant loss of optic nerve tissue and thinning of the nerve along its length.

Autosomal dominant optic atrophy (OPA1) maps to Chromosome (Chr) 3q28, and the disease interval has been refined to within 1.4 cM, flanked by the markers D3S3669 and D3S3562. HRY, the human homolog of the Drosophila segmentation gene, hairy, maps by in situ hybridization to the chromosomal region 3q28-q29. We screened for mutations in HRY in 36 patients from 18 pedigrees with dominant optic atrophy and a group of normal control individuals. Heteroduplex mutation analysis and direct sequencing of all four coding exons and one upstream putative untranslated exon were performed. No disease-associated sequence alterations were identified. A polymorphism in the untranslated region of exon 2 was found, with four alleles. PCR amplification of this part of exon 2 in four of the pedigrees affected by autosomal dominant optic atrophy mapping to chromosome 3q, followed by haplotype analysis, showed recombination between HRY and OPA1 in one pedigree. This allows us to genetically position HRY in relation to known microsatellite markers in the region, placing HRY telomeric to marker D3S3562 and centromeric to D3S1305. This is outside the published critical disease interval for dominant optic atrophy. We have, therefore, excluded HRY as the gene for dominant optic atrophy by sequence analysis, mapped it genetically, and identified a polymorphism in our population.

Objective: To assess phenotypic variation of affected individuals from British families with autosomal dominant optic atrophy., Design: Eighty-seven patients from 21 families showing evidence of linkage to chromosome 3q were identified via the Genetic Clinic of Moorfields Eye Hospital, London, England. Genetic linkage analysis was carried out with markers from chromosome 3q28-qter. Patients underwent clinical examination and psychophysical and electrophysiological testing., Results: Best-corrected visual acuity ranged from 20/20 (6/6 m) to light perception. Although visual acuity was not significantly worse in older patients in the group (chi2=3.20, df=4, P>.50), it did deteriorate with age in one third of the families. Subtle or temporal pallor of the optic disc occurred in 96 (55%) of 174 eyes and total atrophy in 76 (44%). Tritanopia was found in 6 (7.5%) of 80 patients; 65 (81.2%) had a mixed color deficit. A cecocentral scotoma was found in the vast majority. Peripheral motion detection threshold was elevated in areas of visual field with raised mean surround sensitivity but not elsewhere. Pattern visual evoked potentials were of reduced amplitude and delayed. Pattern electroretinograms showed a reduced N95 component in keeping with primary ganglion cell dysfunction., Conclusions: There is wide intrafamilial and interfamilial phenotypic variation in autosomal dominant optic atrophy, with visual function in some, but not all, families deteriorating with age. There is evidence of degeneration of the ganglion cell layer predominantly from central retina, but this is not the exclusive result of either parvocellular or magnocellular cell loss.

Pattern and flash visual evoked cortical potentials (PVEP, FVEP), and pattern electroretinograms, (PERG) were recorded in 13 affected individuals from 8 families with DOA. These were selected as representative from 87 affected members of 21 pedigrees with DOA who were examined, and who underwent genetic linkage analysis. Linkage to the OPA1 locus on chromosome 3q 28-qter was demonstrated in all families. VA ranged from 6/9 to HM: visual fields showed a variable centro-caecal defect; SLO (when performed) showed diffuse nerve fibre loss; MRI (when performed) showed small intra-orbital optic nerves. In 9/13 patients the PVEP was absent in one or both eyes. Most recordable PVEPs were of abnormal latency, but the delays were not marked (peak times 116-135 msec); amplitudes were low or subnormal. PERG fell within the normal range in 9 eyes of 7 patients. 14 eyes showed an abnormal N95:P50 ratio in keeping with ganglion cell dysfunction. Some severely affected eyes showed P50 component involvement, but in no eye was the PERG extinguished. Significant interocular asymmetries in at least one electrophysiological measure were present in 6/13 patients. Colour contrast thresholds were significantly elevated for all three colour confusion axes, with tritan being most affected.

Dominant optic atrophy, a hereditary optic neuropathy causing decreased visual acuity, colour vision deficits, a centro-caecal scotoma and optic nerve pallor, has been mapped to a genetic interval of 1.4 cM between loci D3S3669 and D3S3562 on chromosome 3q28-qter. In order to further refine the critical disease interval, and to test the power of haplotype analysis and linkage disequilibrium mapping, we identified a total of 38 families with dominant optic atrophy, unrelated on the basis of genealogy, from a data base of genetic eye disease families originating from the British Isles. They were studied with 12 highly polymorphic microsatellite markers spanning a region of 12 cM around the dominant optic atrophy locus (OPA1). Allelic frequency analysis [chi-squared test, likelihood ratio test (LRT) and P values] and haplotype parsimony analysis showed evidence of a founder effect in 36 of the 38 pedigrees. Six markers (D3S3669, D3S1523, D3S3642, D3S2305, D3S3590 and D3S3562), spanning 1.4 cM across the disease-associated region, demonstrated significant linkage disequilibrium by LRT (P < 0.05). A peak LRT value of 10.86 (P < 0.0005, lambda = 0.4) occurred at D3S3669. On linkage disequilibrium multipoint analysis the maximum lod score of 8.01 is achieved at D3S1523, and 95% confidence intervals suggest that OPA1 lies within ca. 400 kb of D3S1523.