Transgenic Overexpression of Myocilin Leads to Variable Ocular Anterior Segment and Retinal Alterations Associated with Extracellular Matrix Abnormalities in Adult Zebrafish.

Myocilin is an enigmatic glaucoma-associated glycoprotein whose biological role remains incompletely understood. To gain novel insight into its normal function, we used transposon-mediated transgenesis to generate the first zebrafish line stably overexpressing myocilin [Tg(actb1:myoc-2A-mCherry)]. qPCR showed an approximately four-fold increased myocilin expression in transgenic zebrafish embryos (144 hpf). Adult (13 months old) transgenic animals displayed variable and age-dependent ocular anterior segment alterations. Almost 60% of two-year-old male, but not female, transgenic zebrafish developed enlarged eyes with severe asymmetrical and variable abnormalities in the anterior segment, characterized by corneal limbus hypertrophy, and thickening of the cornea, iris, annular ligament and lens capsule. The most severe phenotype presented small or absent ocular anterior chamber and pupils, due to iris overgrowth along with dysplastic retinal growth and optic nerve hypertrophy. Immunohistochemistry revealed increased presence of myocilin in most altered ocular tissues of adult transgenic animals, as well as signs of retinal gliosis and expanded ganglion cells and nerve fibers. The preliminary results indicate that these cells contributed to retinal dysplasia. Visual impairment was demonstrated in all old male transgenic zebrafish. Transcriptomic analysis of the abnormal transgenic eyes identified disrupted expression of genes involved in lens, muscular and extracellular matrix activities, among other processes. In summary, the developed transgenic zebrafish provides a new tool to investigate this puzzling protein and provides evidence for the role of zebrafish myocilin in ocular anterior segment and retinal biology, through the influence of extracellular matrix organization and cellular proliferation.

RNA and protein expression analysis of HLA-DQB1*03:01:01:21Q allele: A null allele renamed as HLA-DQB1*03:01:01:21N.

The characterization of the expression profile of HLA questionable alleles (Q) is clinically relevant in allogeneic hematopoietic stem cell transplantation (HSTC) because an aberrant expression of these alleles could lead to transplantation-related complications. HLA-DQB1*03:01:01:21Q shows a substitution at the donor splice site of intron 3 that potentially could affect the expression of this allele. In order to determine their expression profile at RNA and protein level, we analyzed the presence of the HLA-DQ7 molecule by complement-dependent cytotoxicity test (CDC) and flow cytometry, and their RNA processing by cDNA analyses and sequencing by Sanger methods. Our results reveal that HLA-DQ7 is not detectable by serological methods, this is confirmed by cDNA methods demonstrating the absence of specific HLA-DQB1*03:01:01:21Q mRNA, probably due to an intron 3 retention that creates a premature TGA stop codon, leading to mRNA degradation via nonsense-mediated decay (NMD). These findings demonstrate that the HLA-DQB1*03:01:01:21Q allele is nonexpressed, thus it has been renamed as DQB1*03:01:01:21N.

Null cyp1b1 Activity in Zebrafish Leads to Variable Craniofacial Defects Associated with Altered Expression of Extracellular Matrix and Lipid Metabolism Genes.

CYP1B1 loss of function (LoF) is the main known genetic alteration present in recessive primary congenital glaucoma (PCG), an infrequent disease characterized by delayed embryonic development of the ocular iridocorneal angle; however, the underlying molecular mechanisms are poorly understood. To model CYP1B1 LoF underlying PCG, we developed a cyp1b1 knockout (KO) zebrafish line using CRISPR/Cas9 genome editing. This line carries the c.535_667del frameshift mutation that results in the 72% mRNA reduction with the residual mRNA predicted to produce an inactive truncated protein (p.(His179Glyfs*6)). Microphthalmia and jaw maldevelopment were observed in 23% of F0 somatic mosaic mutant larvae (144 hpf). These early phenotypes were not detected in cyp1b1-KO F3 larvae (144 hpf), but 27% of adult (four months) zebrafish exhibited uni- or bilateral craniofacial alterations, indicating the existence of incomplete penetrance and variable expressivity. These phenotypes increased to 86% in the adult offspring of inbred progenitors with craniofacial defects. No glaucoma-related phenotypes were observed in cyp1b1 mutants. Transcriptomic analyses of the offspring (seven dpf) of cyp1b1-KO progenitors with adult-onset craniofacial defects revealed functionally enriched differentially expressed genes related to extracellular matrix and cell adhesion, cell growth and proliferation, lipid metabolism (retinoids, steroids and fatty acids and oxidation-reduction processes that include several cytochrome P450 genes) and inflammation. In summary, this study shows the complexity of the phenotypes and molecular pathways associated with cyp1b1 LoF, with species dependency, and provides evidence for the dysregulation of extracellular matrix gene expression as one of the mechanisms underlying the pathogenicity associated with cyp1b1 disruption.

CPAMD8 loss-of-function underlies non-dominant congenital glaucoma with variable anterior segment dysgenesis and abnormal extracellular matrix.

Abnormal development of the ocular anterior segment may lead to a spectrum of clinical phenotypes ranging from primary congenital glaucoma (PCG) to variable anterior segment dysgenesis (ASD). The main objective of this study was to identify the genetic alterations underlying recessive congenital glaucoma with ASD (CG-ASD). Next-generation DNA sequencing identified rare biallelic CPAMD8 variants in four patients with CG-ASD and in one case with PCG. CPAMD8 is a gene of unknown function and recently associated with ASD. Bioinformatic and in vitro functional evaluation of the variants using quantitative reverse transcription PCR and minigene analysis supported a loss-of-function pathogenic mechanism. Optical and electron microscopy of the trabeculectomy specimen from one of the CG-ASD cases revealed an abnormal anterior chamber angle, with altered extracellular matrix, and apoptotic trabecular meshwork cells. The CPAMD8 protein was immunodetected in adult human ocular fluids and anterior segment tissues involved in glaucoma and ASD (i.e., aqueous humor, non-pigmented ciliary epithelium, and iris muscles), as well as in periocular mesenchyme-like cells of zebrafish embryos. CRISPR/Cas9 disruption of this gene in F0 zebrafish embryos (96 hpf) resulted in varying degrees of gross developmental abnormalities, including microphthalmia, pharyngeal maldevelopment, and pericardial and periocular edemas. Optical and electron microscopy examination of these embryos showed iridocorneal angle hypoplasia (characterized by altered iris stroma cells, reduced anterior chamber, and collagen disorganized corneal stroma extracellular matrix), recapitulating some patients' features. Our data support the notion that CPAMD8 loss-of-function underlies a spectrum of recessive CG-ASD phenotypes associated with extracellular matrix disorganization and provide new insights into the normal and disease roles of this gene.

Role of FOXC2 and PITX2 rare variants associated with mild functional alterations as modifier factors in congenital glaucoma.

Congenital glaucoma (CG) is a severe and inherited childhood optical neuropathy that leads to irreversible visual loss and blindness in children. CG pathogenesis remains largely unexplained in most patients. Herein we have extended our previous studies to evaluate the role of FOXC2 and PITX2 variants in CG. Variants of the proximal promoter and transcribed sequence of these two genes were analyzed by Sanger sequencing in a cohort of 133 CG families. To investigate possible oligogenic inheritance involving FOXC2 or PITX2 and CYP1B1, we also analyzed FOXC2 and PITX2 variants in a group of 25 CG cases who were known to carry CYP1B1 glaucoma-associated genotypes. The functional effect of three identified variants was assessed by transactivation luciferase reporter assays, protein stability and subcellular localization analyses. We found eight probands (6.0%) who carried four rare FOXC2 variants in the heterozygous state. In addition, we found an elevated frequency (8%) of heterozygous and rare PITX2 variants in the group of CG cases who were known to carry CYP1B1 glaucoma-associated genotypes, and one of these PITX2 variants arose de novo. To the best of our knowledge, two of the identified variants (FOXC2: c.1183C>A, p.(H395N); and PITX2: c.535C>A, p.(P179T)) have not been previously identified. Examination of the genotype-phenotype correlation in this group suggests that the presence of the infrequent PITX2 variants increase the severity of the phenotype. Transactivation reporter analyses showed partial functional alteration of three identified amino acid substitutions (FOXC2: p.(C498R) and p.(H395N); PITX2: p.(P179T)). In summary, the increased frequency in PCG patients of rare FOXC2 and PITX2 variants with mild functional alterations, suggests they play a role as putative modifier factors in this disease further supporting that CG is not a simple monogenic disease and provides novel insights into the complex pathological mechanisms that underlie CG.

Goniodysgenesis variability and activity of CYP1B1 genotypes in primary congenital glaucoma.

Mutations in the CYP1B1 gene are currently the main known genetic cause of primary congenital glaucoma (PCG), a leading cause of blindness in children. Here, we analyze for the first time the CYP1B1 genotype activity and the microscopic and clinical phenotypes in human PCG. Surgical pieces from trabeculectomy from patients with PCG (n = 5) and sclerocorneal rims (n = 3) from cadaver donors were processed for transmission electron microscopy. Patients were classified into three groups depending on goniodysgenesis severity, which was influenced by CYP1B1 enzymatic activity. The main histological changes observed in the outflow pathway of patients with PCG and mutations in CYP1B1 were: i) underdeveloped collector channels and the Schlemm's canal; ii) abnormal insertion of the ciliary muscle; iii) death of the trabecular endothelial cells. Our findings could be useful in improving treatment strategy of PCG associated with CYP1B1 mutations.

Functional characterization of eight rare missense CYP1B1 variants involved in congenital glaucoma and their association with null genotypes.

PURPOSE: To evaluate the function of eight missense CYP1B1 single nucleotide variants (SNVs) previously identified in patients with primary congenital glaucoma (PCG). METHODS: The eight variants were obtained by site-directed mutagenesis and transiently expressed in human embryonic kidney 293-T (HEK-293T) cells. The catalytic activity, protein stability and subcellular localization of the different recombinant CYP1B1 variants were assessed in this cell line. RESULTS: Six of the mutant CYP1B1 proteins (p.L89P, p.A106D, p.R390S, p.P437L, p.C470Y and S485F) showed catalytic activity values ranging from 0% to 4% of those of the wild-type protein and were considered null variants. The activity values of the two remaining variants (p.F123L and p.A237E) were close to 20% of that of the wild-type enzyme and were classified as hypomorphic variants. Reduced protein stability contributed partially to the decreased catalytic activity of two of the mutant enzymes (p.L89P and p.A106D). None of the CYP1B1 variants showed intracellular aggregation and they all displayed a normal subcellular localization in the endoplasmic reticulum, suggesting that they had folded into a wild-type-like structure. The enzymatic activity associated with the different genotypes in which these CYP1B1 variants were present was estimated to range from 0% to 10% of that of the wild-type genotype. CONCLUSION: These results confirm the pathogenicity of the analysed missense CYP1B1 variants and further support the concept that either absent or very low CYP1B1 activity levels are the primary molecular defect involved in PCG pathogenesis.

Clinical Variability of Primary Congenital Glaucoma in a Spanish Family With Cyp1b1 Gene Mutations.

BACKGROUND: Primary congenital glaucoma (PCG) is a rare disease. In around a third of Spanish patients, the disease is attributed to mutations in the CYP1B1 gene inherited in an autosomal recessive manner. Such mutations are the main known genetic cause of PCG. CASE REPORT: We describe the case of a family with 3 of 7 siblings diagnosed with PCG. In a genetic study of the CYP1B1 gene, 2 null mutations were identified in the affected siblings (R355fsX69/T404fsX38). Two of the 4 healthy siblings were heterozygous for mutation R355fsX69 and the remaining 2 had no mutations. The healthy parents were found to be heterozygous for mutations T404fsX38 (mother) and R355fsX69 (father). High variation in the expression of PCG was observed, especially in terms of disease onset and severity: Patient 1 in the eldest affected sibling, PCG was diagnosed at 8 years of age and is presently stable after 60 months of medical treatment; patient 2 the second affected child underwent surgery at 7 days of age. Today, at 104 months, she has undergone 2 operations on the right eye and 3 on the left eye; patient 3 the youngest sibling, the disease also manifested at birth and the boy underwent surgery at 4 days. Currently he is 84 months old, he has required 7 operations for glaucoma, 3 in the right eye and 4 in the left. CONCLUSIONS: This clinical case reveals the etiological relationship between CYP1B1 mutations and PCG. In addition, it indicates a highly variable clinical picture associated with a single disease genotype mainly affecting disease onset and progression.

Null CYP1B1 genotypes in primary congenital and nondominant juvenile glaucoma.

PURPOSE: To assess the mutation spectrum, enzymatic activity, and phenotypic features associated with CYP1B1 genotypes in primary congenital glaucoma (PCG) and nondominant juvenile glaucoma (ndJG). DESIGN: CYP1B1 genotyping, segregation analysis, and functional evaluation of mutations in a cohort of patients. PARTICIPANTS: A total of 177 probands clinically diagnosed with PCG (161) or ndJG (16). METHODS: Automatic DNA sequencing of the promoter (-1 to -867) and the 3 CYP1B1 exons. CYP1B1 enzymatic activity was evaluated using an ethoxyresorufin O-deethylation assay in transfected HEK-293T cells. MAIN OUTCOME MEASURES: Screening and functional evaluation of CYP1B1 mutations. Glaucoma diagnosis based on slit-lamp examination, measurement of intraocular pressure, gonioscopy, and fundus examination. RESULTS: Thirty-one different mutations were identified in 56 PCG and 7 ndJG index cases. To the best of our knowledge, 3 of the identified mutations were novel (-337G>T, F123L, and I399_P400del). Approximately 56% of all mutation carriers were compound heterozygotes, 25% were homozygotes, and both groups inherited glaucoma as an autosomal recessive trait. Nineteen percent of carriers were heterozygotes and showed non-Mendelian segregation. In vitro and inferred functional analysis showed that no less than approximately 74% of the recessive genotypes result in null enzymatic activity. We detected variable expressivity in relation to age of onset and a possible case of incomplete penetrance in 3 of 6 families (50%), with more than 1 affected child or more than 1 subject carrying 2 CYP1B1 mutant alleles. Altogether, these data support that PCG is not a simple monogenic disease. In addition, most patients with PCG carrying null or putative null genotypes showed severe bilateral phenotypes featured by early disease onset, frequently at birth. The mean number of trabeculectomies per eye was significantly higher in carriers than in noncarriers. CONCLUSIONS: This is the largest analysis of CYP1B1 mutations performed in European patients with PCG to date. Our data show that null CYP1B1 genotypes, and therefore complete absence of CYP1B1 activity, frequently lead to severe phenotypes. Our results support that CYP1B1 glaucoma is not a simple monogenic disease and that CYP1B1 activity levels could influence the phenotype.

Role of CYP1B1 gene polymorphisms in bladder cancer susceptibility.

PURPOSE: CYP1B1 activates procarcinogens in some human tissues, including the urinary tract. Changes related to genetic polymorphisms are a known risk factor for cancer. We analyzed the association between CYP1B1 sequence variations and bladder cancer. MATERIALS AND METHODS: Sequence variations in the coding region (exons 2 and 3) and the neighboring introns of CYP1B1 were analyzed by direct polymerase chain reaction and DNA sequencing in 208 unrelated patients with bladder cancer and 208 healthy controls. RESULTS: We identified 6 known single nucleotide polymorphisms organized into 2 linkage disequilibrium blocks. The Ala/Ala and Leu/Val genotypes of the Ala119Ser and Leu432Val polymorphisms were significantly more common in patients than in controls (55.3% vs 42.8% and 54.8% vs 42.3%, respectively). The strongest individual single nucleotide polymorphism risk was found under an over dominant model for Leu432Val (OR 1.65, CI 95% 1.12-2.44). The 2 susceptibility single nucleotide polymorphisms were predicted to be structured into 4 haplotypes and more than 10 diplotypes. No individual haplotype was associated with bladder cancer but the diplotype Ala-Leu/Ala-Val was significantly overrepresented in cases compared to controls (31.73% vs 17.31%, OR 2.22, 95% CI 1.36-3.62, p = 0.001). The OR was approximately 1.6 for the individual genotypes Ala/Ala and Leu/Val, which increased to 2.2 for the Ala-Leu/Ala-Val diplotype. A risk occupation had a modifying effect, increasing the crude OR of the combined genotype Ala/Ala + Leu/Val from 2.2 to 8.3. CONCLUSIONS: This study provides strong evidence for the role of common CYP1B1 variants as risk factors for bladder cancer, which increases with occupational exposure.

GSTT1, GSTM1, and CYP1B1 gene polymorphisms and susceptibility to sporadic renal cell cancer.

PURPOSE: To estimate the prevalence and importance of GSTT1, GSTM1, and CYP1B1 genotypes in renal cell carcinoma (RCC), and to identify their value as a prognostic factor. MATERIALS AND METHODS: Cross-sectional study of a group of patients diagnosed with RCC (n = 133) and a control group (n = 208) with benign conditions and no history of tumor. Controls were selected by cumulative samples and mixed pairing. All subjects pertained to the catchment area for our hospital. Sociodemographic variables, anatomical pathology features, and presence of GSTT1, GSTM1, and CYP1B1 polymorphisms by multiplex PCR and sequencing techniques. RESULTS: There were no differences in the genotype distribution of the GSTT1 and GSTM1 genes between cases and controls. In the case of CYP1B1, the GG genotype (Ala119) was more prevalent in patients with RCC (OR = 2.08; 95% CI: 1.32-2.28) and may be implicated in 34.3% (95% CI: 16.3-52.2) of RCCs. In patients with GSTT1 deletion, TNM stages III to IV were more common (39.1%); whereas in Val432 homozygous patients in CYP1B1, Fuhrman grades 3 to 4 (54.6%) were more common. Because this was a cross-sectional study, longitudinal studies are needed in the future to confirm these data. CONCLUSIONS: No relationship between GSTT1 and GSTM1 genotypes and RCC risk was observed. Homozygous subjects with Ala119 in CYP1B1 had twice the risk of RCC as homozygous for Ser119 or heterozygotes. Patients with GSTT1 deletion had tumors of more advanced stages, and those with Val432 polymorphism in CYP1B1 had tumors of higher Fuhrman grade.

WDR36 and P53 gene variants and susceptibility to primary open-angle glaucoma: analysis of gene-gene interactions.

PURPOSE: To investigate the role of WDR36 and P53 sequence variations in POAG susceptibility. METHODS: The authors performed a case-control genetic association study in 268 unrelated Spanish patients (POAG1) and 380 control subjects matched for sex, age, and ethnicity. WDR36 sequence variations were screened by either direct DNA sequencing or denaturing high-performance liquid chromatography. P53 polymorphisms p.R72P and c.97-147ins16bp were analyzed by single-nucleotide polymorphism (SNP) genotyping and PCR, respectively. Positive SNP and haplotype associations were reanalyzed in a second sample of 211 patients and in combined cases (n = 479). RESULTS: The authors identified almost 50 WDR36 sequence variations, of which approximately two-thirds were rare and one-third were polymorphisms. Approximately half the variants were novel. Eight patients (2.9%) carried rare mutations that were not identified in the control group (P = 0.001). Six Tag SNPs were expected to be structured in three common haplotypes. Haplotype H2 was consistently associated with the disease (P = 0.0024 in combined cases). According to a dominant model, genotypes containing allele P of the P53 p.R72P SNP slightly increased glaucoma risk. Glaucoma susceptibility associated with different WDR36 genotypes also increased significantly in combination with the P53 RP risk genotype, indicating the existence of a genetic interaction. For instance, the OR of the H2 diplotype estimated for POAG1 and combined cases rose approximately 1.6 times in the two-locus genotype H2/RP. CONCLUSIONS: Rare WDR36 variants and the P53 p.R72P polymorphism behaved as moderate glaucoma risk factors in Spanish patients. The authors provide evidence for a genetic interaction between WDR36 and P53 variants in POAG susceptibility, although this finding must be confirmed in other populations.

Interaction of recombinant myocilin with the matricellular protein SPARC: functional implications.

PURPOSE: Myocilin is an extracellular glycoprotein with unknown function that is associated with glaucoma. Calpain II cleaves recombinant myocilin within the linker region of the protein, releasing the C-terminal olfactomedin domain from the N-terminal domain. The authors previously reported that myocilin interacts with the C-terminal region of hevin, a secretory glycoprotein belonging to the SPARC family of matricellular proteins. This study aims to investigate the interaction of myocilin with SPARC. METHODS: Protein-protein interactions were evaluated by the yeast two-hybrid system. The positive interactions were confirmed by solid-phase binding assays using Ni-chelating HPLC purified recombinant proteins and coexpression of recombinant proteins in HEK-293T cells. Coexpression of myocilin, SPARC, and hevin in ocular tissues was identified by immunoflorescence microscopy, Western blot, and array-based gene profiling. RESULTS: Yeast two-hybrid analyses showed that myocilin interacted with the highly conserved C-terminal extracellular calcium binding (EC) domain within SPARC and hevin. Solid-phase binding assays confirmed these interactions and showed that both myocilin and its C-terminal olfactomedin fragment interacted noncovalently with SPARC and a peptide containing the EC domain of SPARC. Full-length myocilin interacted with higher affinity with SPARC and its EC domain than the myocilin C-terminal fragment. Coexpression of the two recombinant proteins in HEK-293T cells also indicated their intracellular interaction. CONCLUSIONS: Recombinant myocilin and SPARC interact through their C-terminal domains. The data suggest that the proteolytic processing of myocilin modulates this interaction as well as the interactions of myocilin with other extracellular matrix and matricellular proteins, further supporting a functional role for this proteolytic cleavage.

Polymorphic deletions of the GSTT1 and GSTM1 genes and susceptibility to bladder cancer.

UNLABELLED: What's known on the subject? and What does the study add? Bladder cancer susceptibility may be determined by genetic differences in the activity of glutathione S-transferases, enzymes that regulate the conversion of exogenous carcinogens to excretable hydrophilic metabolites by glutathione conjugation. The discrepancy of results regarding the association of common genetic polymorphisms and complex diseases such as cancer has raised scepticism in this area of research. Although the evidence generally supports the implication of GSTM1 and GSTT1 polymorphisms in bladder cancer, there is still some debate, with some studies in favour and some against. This study shows a greater risk of bladder cancer in individuals with GSTM1 null genotype, particularly women. This relationship is less evident with GSTT1 null genotypes. Null genotypes in both genes appear to be synergistic, particularly among smokers, and to increase the predisposition to more aggressive tumours. Nevertheless, the role of GSTM1 and GSTT1 polymorphisms in predisposition to bladder cancer should be viewed with caution, due to the multifactorial genetic origin of this condition and the need for long-term longitudinal studies to confirm these results. OBJECTIVE: To estimate the prevalence and importance of GSTT1 and GSTM1 genotypes (implicated in glutathione S-transferase activity) in bladder cancer, to determine whether smoking and occupational factors influence this relationship, and to identify the value of GSTT1 and GSTM1 genotypes as prognostic factors. PATIENTS AND METHODS: A cross-sectional study was conducted with a group of patients with bladder carcinoma and a control group with benign conditions and no history of tumours. The controls were selected and paired as subjects were recruited. Sociodemographic variables, smoking, professional occupation, histological features and the presence of GSTT1 and GSTM1 polymorphisms by multiplex PCR techniques were assessed. RESULTS: GSTM1 genotypes were investigated in 201 patients and 193 controls and GSTT1 genotypes in 190 patients and 163 controls. In the patients group, GSTT1 null genotype was observed in 22.1% (not significant) and GSTM1 null genotype in 54.2% (P=0.008) (odds ratio, OR, 1.7); when considered together, 15.5% (P<0.05; OR, 3.5) of patients had both null genotypes. In the multivariate analysis, the presence of GSTM1 null genotype remained in the model (OR, 2.1) in addition to smoking and age. Subjects with bladder tumour and GSTM1 null genotype were younger than patients without gene deletion (P=0.049). Women with GSTM1 null genotype presented a higher OR than men (P=0.024). When stratified by smoking habit, smokers with both null genotypes showed an OR of 4.7. The percentage of patients with G3 tumours was higher in patients with GSTT1 null genotype (P=0.013) and in patients with both null genotypes (P=0.002). A higher percentage of infiltrating tumours was also observed in patients with both null genotypes (P=0.035). CONCLUSIONS: The data obtained in the present study suggest a higher risk of bladder cancer in individuals with the GSTM1 null genotype. This risk is twofold higher when GSTM1 and GSTT1 null genotypes are both present and is also higher in smokers. A greater predisposition for more aggressive tumours appears to exist, particularly when both null genotypes are combined. Longer-term longitudinal studies are needed to confirm these results.

CYP1B1 mutations in Spanish patients with primary congenital glaucoma: phenotypic and functional variability.

PURPOSE: To analyze the contributions of cytochrome P4501B1 (CYP1B1) mutations to primary congenital glaucoma (PCG) in Spanish patients. METHODS: We analyzed, by polymerase chain reaction (PCR) DNA sequencing, the presence of promoter (-1 to -867) and exon CYP1B1 mutations in 38 unrelated Spanish probands affected by PCG. Functional analysis of nine identified mutations was performed measuring ethoxyresorufin O-deethylation activity and CYP1B1 stability in transiently transfected human embryonic kidney 293T (HEK-293-T) cells. RESULTS: We found a total of 16 different mutations in 13 (34.2%) index cases. The identified mutations included nine missense and three nonsense nucleotide changes, three small deletions, and a short duplication. Eleven probands were compound heterozygotes and two were heterozygotes. Six of the identified mutations were novel (A106D, E173X, F261L, E262X, W341X, and P513_K514del). Mutations T404fsX30 and R355fsX69 were the most prevalent among index cases and were detected in six (23.0%) and three (11.5%) patients, respectively. Functional analysis showed that the three nonsense mutants assayed (E173X, E262X, and W341X) and F261L were null alleles. Of the remaining mutants, four (P52L, G61E, Y81N, and E229K) showed catalytic activities ranging from 20% to 40% of wild-type CYP1B1 and high protein instability. Mutation P400S showed normal catalytic activity and moderate instability. These five mutants were classified as hypomorphic alleles. Patients carrying two null alleles showed severe phenotypes featured by very early PCG onset usually at birth or in the first month of life (0.6+/-0.9 months). Incomplete penetrance was detected in patients carrying hypomorphic alleles. CONCLUSIONS: Our data indicate that approximately one-third of Spanish patients with PCG carry loss-of-function CYP1B1 and show that null alleles are associated with the most severe phenotypes. Hypomorphic alleles may contribute to some cases of incomplete penetrance.

Implications of mismatch repair genes hMLH1 and hMSH2 in patients with sporadic renal cell carcinoma.

OBJECTIVES: To analyse the implications of DNA mismatch repair genes hMLH1 and hMSH2 in sporadic renal cell carcinoma (RCC). MATERIALS AND METHODS: Specimens of tumour and healthy renal tissue were collected from 89 patients treated for sporadic RCC. Another 95 blood samples taken from individuals with no history of cancer were also analysed. After DNA extraction and PCR amplification, microsatellite instability (MSI) was determined using the Bethesda microsatellite panel, two exonic microsatellites of the TGFbRII and BAX genes, and the microsatellite D3S1611. The promoter methylation status of hMLH1 was investigated using the HpaII and MspI restriction enzymes. In addition, a sequencing analysis of complete coding region of hMLH1 and hMSH2 genes was performed. RESULTS: MSI and promoter hypermethylation of hMLH1 were not detected. Interestingly, loss of heterozygosity (LOH) was common among patients with RCC, particularly in microsatellite D3S1611 (34.9%). Mutations were identified in eight patients: K618A and V716M in gene hMLH1; and I145V, G322D, and the novel mutation P349A, in gene hMSH2. The mutations also appeared in healthy renal tissue and therefore, were considered as germline DNA sequence variations. There were G322D and K618A changes in >1% of the healthy control subjects, suggesting that they are DNA polymorphisms. CONCLUSIONS: Our data show that loss of function of both hMLH1 and hMSH2 is not involved in sporadic RCC, either by promoter methylation or mutation in their exons. However, LOH indicated that chromosomal instability affecting large fragments of DNA was the main genetic alteration we detected associated with RCC.

Expression and purification of functional recombinant human pigment epithelium-derived factor (PEDF) secreted by the yeast Pichia pastoris.

Pigment epithelium-derived factor (PEDF) combines neurotrophic, neuroprotective, anti-angiogenic, anti-tumor and neural stem cell self-renewal properties in a single molecule, making this protein a valuable potential therapeutic agent. We herein analyzed the expression of human recombinant full-length PEDF, and its N- and C-terminal regions (amino acids 1-243 and 195-418, respectively) in three mammalian cell lines (HEK-293T, COS-1, and 26HCMsv), and in the yeast Pichia pastoris. The highest production of recombinant PEDF was achieved in P. pastoris which secreted approximately 30 microg of full-length rPEDF, and 47 microg of C-terminal/ml of culture medium. Full-length rPEDF was purified by one-step Ni-chelating high-performance liquid chromatography, recovering almost 70% of secreted rPEDF with a purity of 98.6%. The C-terminal region of PEDF was isolated by low-pressure liquid chromatography, recovering around 4% of the recombinant molecule with a purity of 98%. The N-terminal region of PEDF was not secreted by any expression system assayed. The two isolated recombinant PEDF polypeptides inhibited in vitro endothelial cell migration, and full-length rPEDF also increased cerebellar granule cell survival, thus demonstrating their biological activity. These polypeptides can be used to investigate the therapeutic role of PEDF in cancer, neurodegenerative and ocular diseases, and stem cell-based therapies.

MYOC gene mutations in Spanish patients with autosomal dominant primary open-angle glaucoma: a founder effect in southeast Spain.

PURPOSE: Primary open angle glaucoma (POAG) is a genetically heterogeneous disease resulting in optic disc cupping and visual impairment. It can be inherited as either a complex or a monogenic trait. Autosomal dominant POAG is the most frequent type of monogenic glaucoma. In this study, we investigated the role of myocilin MYOC in Spanish patients with autosomal dominant POAG. METHODS: We retrospectively analyzed the MYOC gene by PCR-DNA sequencing in five Southeast Spanish families and one Colombian family of Hispanic origin affected by autosomal dominant juvenile-onset open angle glaucoma (JOAG). We also analyzed two families with adult-onset POAG (AOAG). RESULTS: MYOC mutations D380A and P370L segregated with the disease in the five JOAG Spanish families and the Colombian family, respectively. Neither MYOC mutations nor cytochrome P4501B1 CYP1B1 mutations were detected in the AOAG families. The disease showed an insidious onset in D380A carriers, making early diagnosis difficult. A delay in diagnosis resulted in severe visual impairment. Topical medications were effective in controlling intraocular pressure (IOP) in D380A carriers, but 72.2% of them required surgery for long-term IOP control. Conversely, only 30% of AOAG patients required surgery. Mutation P370L was associated with a severe phenotype unresponsive to medical treatment. Analysis of the four MYOC-linked polymorphic microsatellite markers in the JOAG Spanish families revealed a common disease haplotype, indicating that the D380A mutation was inherited from the same founder. CONCLUSIONS: This is the first evidence of a founder effect for a MYOC mutation in Spanish JOAG patients. Analysis of the MYOC gene in Spanish patients with JOAG is useful to identify at-risk individuals thus help prevent visual impairment through early treatment.

New perspectives in aqueous humor secretion and in glaucoma: the ciliary body as a multifunctional neuroendocrine gland.

The discovery in the human ocular ciliary body of glaucoma-associated genes (i.e., MYOC, CYP1B1), neuroendocrine processing enzymes, neuroendocrine peptides, steroid-converting enzymes, glutamate transporters, glutamate-metabolizing enzymes, and anti-angiogenic factors requires a reevaluation of its function on aqueous humor secretion, intraocular pressure and its role in glaucoma. The ciliary body should be considered as a multifunctional and interactive tissue. The intrinsic hypotensive and/or hypertensive biological activities of many of the endocrine peptides released by the ciliary epithelium are best explained within the context of a neuroendocrine system, linking the inflow and the outflow of aqueous humor. This interpretation is consistent with physiological and genetic studies indicating that changes altering the inflow affects intraocular pressure. In the proposed endocrine system, regulatory peptides secreted by the ciliary epithelium may subserve multiple functions in the following: inflow and outflow pathways of aqueous humor, ciliary blood flow, the immune privilege status of the anterior segment and the diurnal circadian rhythms of aqueous humor secretion and intraocular pressure. These previously unsuspected and challenging functions of the ciliary epithelium should be considered when assessing the multifactorial events which lead to the pathophysiology of glaucoma affecting the outflow pathways of aqueous humor. This review highlights published, and ongoing studies on authors' labs supporting neuroendocrine, steroidogenic and glutamatergic features of the ciliary epithelium and the endocrine communication between the inflow and outflow pathways of aqueous humor. We also discuss how glaucoma-associated genes expressed in the ciliary body and their mutant proteins could influence intraocular pressure, contributing to the development of glaucoma.