Monoclonal antibody analysis of ocular basement membranes during development

As a first step in a study of the role(s) of basement membranes in ocular morphogenesis, we have produced a variety of monoclonal antibodies against native lens capsule from adult chicks, and have used these reagents to stain histological sections of ocular tissues from 4 1/2- to 18-day-old chicken embryos. Four different patterns of immunofluorescence were observed in sections of corneas of 18-day-old chicken embryos stained with these antibodies. The antibodies in group 1 stained the basement membranes of both the corneal epithelium and the endothelium (as well as Descemet's membrane). Those in groups 2 and 3 stained only the epithelial or endothelial basement membranes, respectively. The group 4 antibody stained the corneal stroma as well as Bowman's membrane and Descemet's membrane. The antibodies in group 1 could be further subdivided into groups 1a and 1b on the basis of temporal differences in the onset of staining in corneas from 4 1/2- to 7-day-old embryos. Thus, this series of monoclonal antibodies appears to recognize at least five different antigenic determinants. When these antibodies were used to stain sections of eyes at different stages of development, we found that the characteristic differential staining of some basement membranes was maintained throughout development, while the staining properties of others changed. This indicates that many of the ocular basement membranes may differ from one another in composition or conformation, and that at least some of them may undergo developmental changes. We also noticed a similarity in the pattern of fluorescence associated with the basement membranes of the limbal blood vessels and the corneal endothelium that is consistent with the hypothesis that the corneal endothelium is derived from the early periocular vascular endothelium. Our observations of developing corneas also revealed that the antigen recognized by the group 4 antibody may be produced by both the corneal epithelium and the stromal fibroblasts. The suitability of monoclonal antibodies for probing basement membrane heterogeneity is discussed.     

Altered expression of aquaporins in bullous keratopathy and Fuchs' dystrophy corneas.

Corneas with edema-related diseases lose transparency, which causes significant vision loss. This study analyzed seven aquaporins (AQPs) in normal corneas, pseudophakic/aphakic bullous keratopathy (PBK/ABK) corneas, Fuchs' dystrophy corneas, keratoconus corneas, post-cataract surgery (PCS) corneas, and normal organ-cultured corneas. RNA levels for AQP1, AQP4, and beta2-microglobulin were measured by RT-PCR. AQP1 antibody localized to stromal cells of all corneas. PBK/ABK and Fuchs' dystrophy corneas had decreased endothelial cell staining compared with normal. AQP1 mRNA was found in whole corneas and cultured stromal fibroblasts but not in isolated epithelial cells. AQP3 staining was found in basal epithelial cells of the normal, Fuchs' dystrophy, and keratoconus corneas but throughout the entire epithelium of PBK/ABK corneas. AQP4 antibody localized to endothelial cells of all corneas and in stromal cells of PBK/ABK corneas. AQP4 mRNA was identified in whole human corneas. AQP5 was found in epithelial cells of all corneas. AQP0, AQP2, and AQP9 were not found in any corneas. Normal AQP distributions were found in PCS and organ-cultured corneas, although they showed signs of swelling. Our study demonstrates that AQP abnormalities are found in PBK/ABK corneas (decreased AQP1, increased AQP3 and AQP4) and Fuchs' dystrophy corneas (decreased AQP1). Although both have vision-disrupting corneal edema, the mechanisms of fluid accumulation may be different in each disease.     

Mutation analysis and embryonic expression of the HLXB9 Currarino syndrome gene

The HLXB9 homeobox gene was recently identified as a locus for autosomal dominant Currarino syndrome, also known as hereditary sacral agenesis (HSA). This gene specifies a 403-amino acid protein containing a homeodomain preceded by a very highly conserved 82-amino acid domain of unknown function; the remainder of the protein is not well conserved. Here we report an extensive mutation survey that has identified mutations in the HLXB9 gene in 20 of 21 patients tested with familial Currarino syndrome. Mutations were also detected in two of seven sporadic Currarino syndrome patients; the remainder could be explained by undetected mosaicism for an HLXB9 mutation or by genetic heterogeneity in the sporadic patients. Of the mutations identified in the 22 index patients, 19 were intragenic and included 11 mutations that could lead to the introduction of a premature termination codon. The other eight mutations were missense mutations that were significantly clustered in the homeodomain, resulting, in each patient, in nonconservative substitution of a highly conserved amino acid. All of the intragenic mutations were associated with comparable phenotypes. The only genotype-phenotype correlation appeared to be the occurrence of developmental delay in the case of three patients with microdeletions. HLXB9 expression was analyzed during early human development in a period spanning Carnegie stages 12-21. Signal was detected in the basal plate of the spinal cord and hindbrain and in the pharynx, esophagus, stomach, and pancreas. Significant spatial and temporal expression differences were evident when compared with expression of the mouse Hlxb9 gene, which may partly explain the significant human-mouse differences in mutant phenotype.     

Cell adhesion molecules in stromal corneal dystrophies

The aim of the present study was to investigate the expression pattern of different cell adhesion molecules in corneal stromal dystrophies. Fifteen corneal buttons from patients diagnosed with three different types of stromal corneal dystrophies and healthy corneas were investigated. Paraffin embedded sections were stained immunohistochemically with monoclonal antibodies against human intercellular adhesion molecule-1 (ICAM-1), endothelial selectin (E-selectin) and endothelial cadherin (E-cadherin) using the avidin-biotin-peroxidase-complex technique. The sections were compared to normal eye bank controls. In corneas from granular dystrophy patients ICAM-1 was expressed focally in epithelial cells and in keratocytes, and expressed diffusely in endothelial cells. In corneas from macular dystrophy patients diffuse epithelial staining was observed and the stromal and endothelial expression was found to be similar to that of granular dystrophy. In lattice dystrophy, only the epithelial cells and endothelium were intensively positive for ICAM-1. E-selectin was not present on any layer of the corneal specimens. E-cadherin was observed only in the epithelium of all three types of corneal dystrophies. Normal corneas did not express any of the investigated adhesion molecules. We found different expression patterns of adhesion molecules in corneas from stromal dystrophies. Our results suggest that adhesion molecules may be involved in the pathogenesis of corneal stromal dystrophies.     

Using Caveolin-1 epithelial immunostaining patterns to stratify human breast cancer patients and to predict the Caveolin-1 (P132L) mutation

Caveolin-1 (Cav-1) mutations, such as P132L, are associated with ER-positive human breast cancers. However, no immuno-histochemical methods have yet been described to predict the presence of Cav-1 mutations in human breast cancer. Since the P132L mutation acts in a dominant-negative fashion and causes the mis-localization Cav-1 in cultured cells in vitro, we hypothesized that patients carrying this mutation would show a similar Cav-1 staining pattern in vivo. Indeed, while performing histological analysis of Cav-1 immunostaining on human breast cancer samples, we noted the emergence of two distinct epithelial staining patterns: 1) punctate peri-nuclear “Golgi-like” localization; or 2) diffuse cytoplasmic staining. The punctate peri-nuclear staining pattern was associated with ER-alpha positivity and was present mainly in well-differentiated samples. In striking contrast, the diffuse staining pattern was present in poorly differentiated samples, and was not associated with ER-status. DNA sequence analysis revealed that only well-differentiated samples with a punctate staining pattern harbored the Cav-1 P132L mutation. Thus, immunostaining of Cav-1 can be used as a first step to stratify human breast patients and to predict the presence of Cav-1 mutations. As such, the punctate Cav-1 immunostaining pattern can now be used as a screening tool to select patients for Cav-1 mutational analysis.     

Intragenic telSMN mutations: frequency, distribution, evidence of a founder effect, and modification of the spinal muscular atrophy phenotype by cenSMN copy number.

The autosomal recessive neuromuscular disorder proximal spinal muscular atrophy (SMA) is caused by the loss or mutation of the survival motor neuron (SMN) gene, which exists in two nearly identical copies, telomeric SMN (telSMN) and centromeric SMN (cenSMN). Exon 7 of the telSMN gene is homozygously absent in approximately 95% of SMA patients, whereas loss of cenSMN does not cause SMA. We searched for other telSMN mutations among 23 SMA compound heterozygotes, using heteroduplex analysis. We identified telSMN mutations in 11 of these unrelated SMA-like individuals who carry a single copy of telSMN: these include two frameshift mutations (800ins11 and 542delGT) and three missense mutations (A2G, S262I, and T274I). The telSMN mutations identified to date cluster at the 3' end, in a region containing sites for SMN oligomerization and binding of Sm proteins. Interestingly, the novel A2G missense mutation occurs outside this conserved carboxy-terminal domain, closely upstream of an SIP1 (SMN-interacting protein 1) binding site. In three patients, the A2G mutation was found to be on the same allele as a rare polymorphism in the 5' UTR, providing evidence for a founder chromosome; Ag1-CA marker data also support evidence of an ancestral origin for the 800ins11 and 542delGT mutations. We note that telSMN missense mutations are associated with milder disease in our patients and that the severe type I SMA phenotype caused by frameshift mutations can be ameliorated by an increase in cenSMN gene copy number.     

Human cornea proteome: identification and quantitation of the proteins of the three main layers including epithelium, stroma, and endothelium

Diseases of the cornea are common and refer to conditions like infections, injuries and genetic defects. Morphologically, many corneal diseases affect only certain layers of the cornea and separate analysis of the individual layers is therefore of interest to explore the basic molecular mechanisms involved in corneal health and disease. In this study, the three main layers including, the epithelium, stroma and endothelium of healthy human corneas were isolated. Prior to analysis by LC-MS/MS the proteins from the different layers were either (i) separated by SDS-PAGE followed by in-gel trypsinization, (ii) in-solution digested without prior protein separation or, (iii) in-solution digested followed by cation exchange chromatography. A total of 3250 unique Swiss-Prot annotated proteins were identified in human corneas, 2737 in the epithelium, 1679 in the stroma, and 880 in the endothelial layer. Of these, 1787 proteins have not previously been identified in the human cornea by mass spectrometry. In total, 771 proteins were quantified, 157 based on in-solution digestion and 770 based on SDS-PAGE separation followed by in-gel digestion of excised gel pieces. Protein analysis showed that many of the identified proteins are plasma proteins involved in defense responses.     

Cranioectodermal Dysplasia, Sensenbrenner Syndrome, Is a Ciliopathy Caused by Mutations in the IFT122 Gene

Cranioectodermal dysplasia (CED) is a disorder characterized by craniofacial, skeletal, and ectodermal abnormalities. Most cases reported to date are sporadic, but a few familial cases support an autosomal-recessive inheritance pattern. Aiming at the elucidation of the genetic basis of CED, we collected 13 patients with CED symptoms from 12 independent families. In one family with consanguineous parents two siblings were affected, permitting linkage analysis and homozygosity mapping. This revealed a single region of homozygosity with a significant LOD score (3.57) on chromosome 3q21-3q24. By sequencing candidate genes from this interval we found a homozygous missense mutation in the IFT122 (WDR10) gene that cosegregated with the disease. Examination of IFT122 in our patient cohort revealed one additional homozygous missense change in the patient from a second consanguineous family. In addition, we found compound heterozygosity for a donor splice-site change and a missense change in one sporadic patient. All mutations were absent in 340 control chromosomes. Because IFT122 plays an important role in the assembly and maintenance of eukaryotic cilia, we investigated patient fibroblasts and found significantly reduced frequency and length of primary cilia as compared to controls. Furthermore, we transiently knocked down ift122 in zebrafish embryos and observed the typical phenotype found in other models of ciliopathies. Because not all of our patients harbored mutations in IFT122, CED seems to be genetically heterogeneous. Still, by identifying CED as a ciliary disorder, our study suggests that the causative mutations in the unresolved cases most likely affect primary cilia function too.     

Mutations in the WFS1 gene that cause low-frequency sensorineural hearing loss are small non-inactivating mutations

Hereditary hearing impairment is an extremely heterogeneous trait, with more than 70 identified loci. Only two of these loci are associated with an auditory phenotype that predominantly affects the low frequencies (DFNA1 and DFNA6/14). In this study, we have completed mutation screening of the WFS1 gene in eight autosomal dominant families and twelve sporadic cases in which affected persons have low-frequency sensorineural hearing impairment (LFSNHI). Mutations in this gene are known to be responsible for Wolfram syndrome or DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness), which is an autosomal recessive trait. We have identified seven missense mutations and a single amino acid deletion affecting conserved amino acids in six families and one sporadic case, indicating that mutations in WFS1 are a major cause of inherited but not sporadic low-frequency hearing impairment. Among the ten WFS1 mutations reported in LFSNHI, none is expected to lead to premature protein truncation, and nine cluster in the C-terminal protein domain. In contrast, 64% of the Wolfram syndrome mutations are inactivating. Our results indicate that only non-inactivating mutations in WFS1 are responsible for non-syndromic low-frequency hearing impairment.     

Germ-line mutational analysis of the TSC2 gene in 90 tuberous-sclerosis patients.

Ninety patients with tuberous-sclerosis complex (TSC) were tested for subtle mutations in the TSC2 gene, by means of single-strand conformational analysis (SSCA) of genomic DNA. Patients included 56 sporadic cases and 34 familial probands. For all patients, SSCA was performed for each of the 41 exons of the TSC2 gene. We identified 32 SSCA changes, 22 disease-causing mutations, and 10 polymorphic variants. Interestingly, we detected mutations at a much higher frequency in the sporadic cases (32%) than in the multiplex families (9%). Among the eight families for which linkage to the TSC2 region had been determined, only one mutation was found. Mutations were distributed equally across the gene; they included 5 deletions, 3 insertions, 10 missense mutations, 2 nonsense mutations, and 2 tandem duplications. We did not detect an increase in mutations either in the GTPase-activating protein (GAP)-related domains of TSC2 or in the activating domains that have been identified in rat tuberin. We did not detect any mutations in the exons (25 and 31) that are spliced out in the isoforms. There was no evidence for correspondence between variability of phenotype and type of mutation (missense versus early termination). Diagnostic testing will be difficult because of the genetic heterogeneity of TSC (which has at least two causative genes: TSC1 and TSC2), the large size of the TSC2 gene, and the variety of mutations. More than half of the mutations that we identified (missense, small in-frame deletion, and tandem duplication) are not amenable to the mutation-detection methods, such as protein-truncation testing, that are commonly employed for genes that encode proteins with tumor-suppressor function.     

Evaluation of acute corneal damage induced by 222-nm and 254-nm ultraviolet light in Sprague–Dawley rats

Two hundred twenty-two nanometres ultraviolet (UV) light produced by a krypton–chlorine excimer lamp is harmful to bacterial cells but not skin. However, the effects of 222-nm UV light exposure to the eye are not fully known. We evaluated acute corneal damage induced by 222- and 254-nm UV light in albino rats. Under deep anaesthesia, 6-week-old Sprague–Dawley albino rats were exposed to UV light. The exposure levels of corneal radiation were 30, 150, and 600?mJ/cm². Epithelial defects were detected by staining with fluorescein. Superficial punctate keratitis developed in corneas exposed to more than 150?mJ/cm² of UV light, and erosion was observed in corneas exposed to 600?mJ/cm² of UV light. Haematoxylin and eosin staining also showed corneal epithelial defects in eyes exposed to 254-nm UV light. However, no damage developed in corneas exposed to 222-nm UV light. Cyclobutane pyrimidine dimer-positive cells were observed only in normal corneas and those exposed to 254-nm UV light. Although some epithelial cells were stained weakly in normal corneas, squamous epithelial cells were stained moderately, and the epithelial layer that was detached from the cornea exposed to 600?mJ/cm² of light was stained intensely in corneas exposed to 254-nm UV light. In the current study, no corneal damage was induced by 222-nm UV light, which suggested that 222-nm UV light may not harm rat eyes within the energy range and may be useful for sterilising or preventing infection in the future.     

Whole-exome sequencing identified compound heterozygous variants in MMKS in a Chinese pedigree with Bardet-Biedl syndrome

Bardet-Biedl syndrome(BBS) is a genetically heterogeneous disorder characterized by retinal dystrophy, polydactyly, obesity,developmental delay, and renal defects. At least 21 candidate BBS-associated genes(BBS1-19, NPHP1, and IFT172) have previously been identified, and all of them play important roles in ciliary function. Here, we collected a BBS pedigree with four members and performed whole-exome sequencing on the proband. The variants were analyzed and evaluated to confirm their pathogenicity. We found compound heterozygous variants(c.1192CT, p.Q398* and c.1175CT, p.T392M) in MKKS in both the siblings, and these were likely to be pathogenic variants. We also found a missense variant(c.2029GC, p.E677Q) in NPHP1 and a missense variant(c.2470CT, p.R824C) in BBS9 in the proband only, which are variants of uncertain significance. The compound heterozygous variants were probably responsible for the BBS phenotype in this Chinese pedigree and the missense mutations in NPHP1 and BBS9 might contribute to the mutation load.     

Infection-Triggered Familial or Recurrent Cases of Acute Necrotizing Encephalopathy Caused by Mutations in a Component of the Nuclear Pore, RANBP2

Acute necrotizing encephalopathy (ANE) is a rapidly progressive encephalopathy that can occur in otherwise healthy children after common viral infections such as influenza and parainfluenza. Most ANE is sporadic and nonrecurrent (isolated ANE). However, we identified a 7 Mb interval containing a susceptibility locus (ANE1) in a family segregating recurrent ANE as an incompletely penetrant, autosomal-dominant trait. We now report that all affected individuals and obligate carriers in this family are heterozygous for a missense mutation (c.1880C→T, p.Thr585Met) in the gene encoding the nuclear pore protein Ran Binding Protein 2 (RANBP2). To determine whether this mutation is the susceptibility allele, we screened controls and other patients with ANE who are unrelated to the index family. Patients from 9 of 15 additional kindreds with familial or recurrent ANE had the identical mutation. It arose de novo in two families and independently in several other families. Two other patients with familial ANE had different RANBP2 missense mutations that altered conserved residues. None of the three RANBP2 missense mutations were found in 19 patients with isolated ANE or in unaffected controls. We conclude that missense mutations in RANBP2 are susceptibility alleles for familial and recurrent cases of ANE.     

Immunocytochemical localization of aquaporin-1 in bovine corneal endothelial cells and keratocytes

For immunocytochemistry, cultured bovine corneal endothelial cells (CBCEC) and bovine corneal cryosections were utilized. Preparations were fixed, permeabilized, and incubated with primary rabbit anti-rat aquaporin 1 (AQP1) antibody followed by rhodamine-conjugated secondary antibody, and were counter-stained with Sytox nuclear acid stain. Confocal microscopy of CBCEC in the x, y, and z planes showed rhodamine fluorescence, indicating the presence of AQP1 antibody localized to the apical and basolateral domains of the plasma membrane, but not to the membranes of intracellular compartments or other subcellular locations. Preabsorption with control antigenic peptide yielded no positive staining. Similar results were obtained using freshly dissected bovine corneas; in addition, these images showed AQP1 distributed to the plasma membranes of keratocytes. No AQP1 staining was seen in corneal epithelium, and no staining was observed in CBCEC layers exposed to AQP3, AQP4, and AQP5 antibodies.     

Mutations in FAM111B Cause Hereditary Fibrosing Poikiloderma with Tendon Contracture,Myopathy, and Pulmonary Fibrosis

Congenital poikiloderma is characterized by a combination of mottled pigmentation, telangiectasia, and epidermal atrophy in the first few months of life. We have previously described a South African European-descent family affected by a rare autosomal-dominant form of hereditary fibrosing poikiloderma accompanied by tendon contracture, myopathy, and pulmonary fibrosis. Here, we report the identification of causative mutations in FAM111B by whole-exome sequencing. In total, three FAM111B missense mutations were identified in five kindreds of different ethnic backgrounds. The mutation segregated with the disease in one large pedigree, and mutations were de novo in two other pedigrees. All three mutations were absent from public databases and were not observed on Sanger sequencing of 388 ethnically matched control subjects. The three single-nucleotide mutations code for amino acid changes that are clustered within a putative trypsin-like cysteine/serine peptidase domain of FAM111B. These findings provide evidence of the involvement of FAM111B in congenital poikiloderma and multisystem fibrosis.     

Niemann-Pick C1 disease: correlations between NPC1 mutations, levels of NPC1 protein, and phenotypes emphasize the functional significance of the putative sterol-sensing domain and of the cysteine-rich luminal loop

To obtain more information of the functional domains of the NPC1 protein, the mutational spectrum and the level of immunoreactive protein were investigated in skin fibroblasts from 30 unrelated patients with Niemann-Pick C1 disease. Nine of them were characterized by mild alterations of cellular cholesterol transport (the "variant" biochemical phenotype). The mutations showed a wide distribution to nearly all NPC1 domains, with a cluster (11/32) in a conserved NPC1 cysteine-rich luminal loop. Homozygous mutations in 14 patients and a phenotypically defined allele, combined with a new mutation, in a further 10 patients allowed genotype/phenotype correlations. Premature-termination-codon mutations, the three missense mutations in the sterol-sensing domain (SSD), and A1054T in the cysteine-rich luminal loop all occurred in patients with infantile neurological onset and "classic" (severe) cholesterol-trafficking alterations. By western blot, NPC1 protein was undetectable in the SSD missense mutations studied (L724P and Q775P) and essentially was absent in the A1054T missense allele. Our results thus enhance the functional significance of the SSD and demonstrate a correlation between the absence of NPC1 protein and the most severe neurological form. In the remaining missense mutations studied, corresponding to other disease presentations (including two adults with nonneurological disease), NPC1 protein was present in significant amounts of normal size, without clear-cut correlation with either the clinical phenotype or the "classic"/"variant" biochemical phenotype. Missense mutations in the cysteine-rich luminal loop resulted in a wide array of clinical and biochemical phenotypes. Remarkably, all five mutant alleles (I943M, V950M, G986S, G992R, and the recurrent P1007A) definitively correlated with the "variant" phenotype clustered within this loop, providing new insight on the functional complexity of the latter domain.