Genome-wide association study of brain amyloid deposition as measured by Pittsburgh Compound-B (PiB)-PET imaging.

Deposition of amyloid plaques in the brain is one of the two main pathological hallmarks of Alzheimer's disease (AD). Amyloid positron emission tomography (PET) is a neuroimaging tool that selectively detects in vivo amyloid deposition in the brain and is a reliable endophenotype for AD that complements cerebrospinal fluid biomarkers with regional information. We measured in vivo amyloid deposition in the brains of ~1000 subjects from three collaborative AD centers and ADNI using 11C-labeled Pittsburgh Compound-B (PiB)-PET imaging followed by meta-analysis of genome-wide association studies, first to our knowledge for PiB-PET, to identify novel genetic loci for this endophenotype. The APOE region showed the most significant association where several SNPs surpassed the genome-wide significant threshold, with APOE*4 being most significant (P-meta = 9.09E-30; ß = 0.18). Interestingly, after conditioning on APOE*4, 14 SNPs remained significant at P < 0.05 in the APOE region that were not in linkage disequilibrium with APOE*4. Outside the APOE region, the meta-analysis revealed 15 non-APOE loci with P < 1E-05 on nine chromosomes, with two most significant SNPs on chromosomes 8 (P-meta = 4.87E-07) and 3 (P-meta = 9.69E-07). Functional analyses of these SNPs indicate their potential relevance with AD pathogenesis. Top 15 non-APOE SNPs along with APOE*4 explained 25-35% of the amyloid variance in different datasets, of which 14-17% was explained by APOE*4 alone. In conclusion, we have identified novel signals in APOE and non-APOE regions that affect amyloid deposition in the brain. Our data also highlights the presence of yet to be discovered variants that may be responsible for the unexplained genetic variance of amyloid deposition.

Rare coding variants in the phospholipase D3 gene confer risk for Alzheimer's disease.

Genome-wide association studies (GWAS) have identified several risk variants for late-onset Alzheimer's disease (LOAD). These common variants have replicable but small effects on LOAD risk and generally do not have obvious functional effects. Low-frequency coding variants, not detected by GWAS, are predicted to include functional variants with larger effects on risk. To identify low-frequency coding variants with large effects on LOAD risk, we carried out whole-exome sequencing (WES) in 14 large LOAD families and follow-up analyses of the candidate variants in several large LOAD case-control data sets. A rare variant in PLD3 (phospholipase D3; Val232Met) segregated with disease status in two independent families and doubled risk for Alzheimer's disease in seven independent case-control series with a total of more than 11,000 cases and controls of European descent. Gene-based burden analyses in 4,387 cases and controls of European descent and 302 African American cases and controls, with complete sequence data for PLD3, reveal that several variants in this gene increase risk for Alzheimer's disease in both populations. PLD3 is highly expressed in brain regions that are vulnerable to Alzheimer's disease pathology, including hippocampus and cortex, and is expressed at significantly lower levels in neurons from Alzheimer's disease brains compared to control brains. Overexpression of PLD3 leads to a significant decrease in intracellular amyloid-ß precursor protein (APP) and extracellular Aß42 and Aß40 (the 42- and 40-residue isoforms of the amyloid-ß peptide), and knockdown of PLD3 leads to a significant increase in extracellular Aß42 and Aß40. Together, our genetic and functional data indicate that carriers of PLD3 coding variants have a twofold increased risk for LOAD and that PLD3 influences APP processing. This study provides an example of how densely affected families may help to identify rare variants with large effects on risk for disease or other complex traits.

Evaluation of the use of PEEK material in implant-supported fixed restorations by finite element analysis.

AIMS: The purpose of this study is to compare the stresses occurring in the peri-implant bones, implants, crowns, abutments, and screws after loading through finite element analysis by using the poly-ether-ether-ketone (PEEK) materials, which are alternative to titanium abutment and metal supported restorations and to try to reduce the level of neck resorption. MATERIALS AND METHODS: In our study, three-dimensional modeling of 2 PEEK and titanium abutments, metal-ceramic, and monolithic PEEK upper central dental restorations were made on four titanium implants (Biohorizons® Implant Systems Ins., Birmingham, AL, USA) with diameters of 3.8 mm and 10.5 mm and four groups were obtained. Then, a stress analysis of the finite element was performed by applying a 178 N oblique force of 45° to the long axis of the tooth 2 mm below the incisal edge of the model's palatal surface. RESULTS: It has been observed that the PEEK material reduces the stresses caused by the force applied on itself during all tests. In all groups, PEEK abutments and PEEK crowns have reduced stress on the abutment. The most significant difference is observed in the stresses on the crowns and screws. When the stresses on the crown are examined, the use of PEEK crown reduces the stresses on itself and the use of PEEK abutment increases the stresses on the crown. CONCLUSIONS: The stress on the implant system can be changed through the usage of different prosthetic materials.

Multiple signals at the extended 8p23 locus are associated with susceptibility to systemic lupus erythematosus.

BACKGROUND: A major systemic lupus erythematosus (SLE) susceptibility locus lies within a common inversion polymorphism region (encompassing 3.8 - 4.5 Mb) located at 8p23. Initially implicated genes included FAM167A-BLK and XKR6, of which BLK received major attention due to its known role in B-cell biology. Recently, additional SLE risk carried in non-inverted background was also reported. OBJECTIVE AND METHODS: In this case -control study, we further investigated the 'extended' 8p23 locus (~ 4 Mb) where we observed multiple SLE signals and assessed these signals for their relation to the inversion affecting this region. The study involved a North American discovery data set (~ 1200 subjects) and a replication data set (> 10 000 subjects) comprising European-descent individuals. RESULTS: Meta-analysis of 8p23 SNPs, with p < 0.05 in both data sets, identified 51 genome-wide significant SNPs (p < 5.0 × 10-8). While most of these SNPs were related to previously implicated signals (XKR6-FAM167A-BLK subregion), our results also revealed two 'new' SLE signals, including SGK223-CLDN23-MFHAS1 (6.06 × 10-9 ≤ meta p ≤ 4.88 × 10-8) and CTSB (meta p = 4.87 × 10-8) subregions that are located > 2 Mb upstream and ~ 0.3 Mb downstream from previously reported signals. Functional assessment of relevant SNPs indicated putative cis-effects on the expression of various genes at 8p23. Additional analyses in discovery sample, where the inversion genotypes were inferred, replicated the association of non-inverted status with SLE risk and suggested that a number of SLE risk alleles are predominantly carried in non-inverted background. CONCLUSIONS: Our results implicate multiple (known+novel) SLE signals/genes at the extended 8p23 locus, beyond previously reported signals/genes, and suggest that this broad locus contributes to SLE risk through the effects of multiple genes/pathways.

Antiproliferative and antiapoptotic effect of thymoquinone on cancer cells in vitro.

OBJECTIVES: Nigella sativa oil and thymoquinone were comparatively tested in vitro for their effects on human cancer cell lines (glioma,T98; prostate, LnCaP) as well as mouse embryonic fibroblast cell lines (3T3), and for the induction of apoptosis. METHODS: Individual cell lines were treated with thymoquinone and N. sativa oil for 24 and 48 hr. Survival rate with MTT, apoptosis with flow cytometry and caspase-9 mRNA enzyme levels with RT-PCR were determined in vitro. RESULTS: Application of respective concentrations of N. sativa oil (excluding 100 µg/mL for 48 hr) did not change the number of tested cell lines, however, treatment with thymoquinone reduced the number of all cells significantly. Thymoquinone also exerted its apoptosis inducing effect through the activation of caspase-9. CONCLUSION: Differing with the type of cancer cells, thymoquinone posseses a strong contentration and time dependent survival reducing effect on cancer cells via apoptosis (Fig. 6, Ref. 22). Text in PDF www.elis.sk.

A replication study of 49 Type 2 diabetes risk variants in a Punjabi Pakistani population.

AIM: The burden of Type 2 diabetes is alarmingly high in South Asia, a region that has many genetically diverse ethnic populations. Genome-wide association studies (GWAS) conducted largely in European populations have identified a number of loci predisposing to Type 2 diabetes risk, however, the relevance of such genetic loci in many South Asian sub-ethnicities remains elusive. The aim of this study was to replicate 49 single nucleotide polymorphisms (SNPs) previously identified through GWAS in Punjabis living in Pakistan. METHODS: We examined the association of 49 SNPs in 853 Type 2 diabetes cases and 1945 controls using additive logistic regression models after adjusting for age and gender. RESULTS: Of the 49 SNPs investigated, eight showed a nominal association (P < 0.05) that also remained significant after controlling for the false discovery rate. The most significant association was found for rs7903146 at the TCF7L2 locus. For a per unit increase in the risk score comprising of all the 49 SNPs, the odds ratio in association with Type 2 diabetes risk was 1.16 (95% CI 1.13-1.19, P < 2.0E-16). CONCLUSION: These results suggest that some Type 2 diabetes susceptibility loci are shared between Europeans and Punjabis living in Pakistan.

Genetic link of type 1 diabetes susceptibility loci with rheumatoid arthritis in Pakistani patients.

Rheumatoid arthritis (RA) and type 1 diabetes (T1D) are two autoimmune disorders that have been reported to co-occur in the same subjects or in different subjects from the same family. This suggests the sharing of disease susceptibility loci between RA and T1D. This study was aimed to find out such susceptibility loci that are common in both T1D and RA in Pakistani population. A total of 366 Pakistanis comprising related and unrelated RA cases and controls were recruited. Blood samples were collected from all patients followed by DNA isolation. Thirty-one single-nucleotide polymorphisms (SNPs) previously reported to be associated with T1D were genotyped in RA cases and controls using TaqMan SNP genotyping assays. Data was analyzed using FamCC software. We have identified seven SNP associations that survived multiple testing corrections using false discovery rate: SKAP2/rs7804356 (p = 2.47E-04), GLIS3/rs7020673 (p = 2.86E-04), GSDMB/rs2290400 (p = 23.48E-04), BACH2/rs11755527 (p = 9.16E-04), C6orf173/ rs9388489 (p = 3.11E-03), PRKCQ/DKFZp667F0711/ rs947474 (p = 4.53E-03), and DLK1/ rs941576 (p = 9.51E-03). Our results support the presence of overlapping loci between RA and T1D in Pakistani patients.

Genetic contribution of SCARB1 variants to lipid traits in African Blacks: a candidate gene association study.

BACKGROUND: High-density lipoprotein cholesterol (HDL-C) exerts many anti-atherogenic properties including its role in reverse cholesterol transport (RCT). Scavenger receptor class B member 1 (SCARB1) plays a key role in RCT by selective uptake of HDL cholesteryl esters. We aimed to explore the genetic contribution of SCARB1 to affecting lipid levels in African Blacks from Nigeria. METHODS: We resequenced 13 exons and exon-intron boundaries of SCARB1 in 95 individuals with extreme HDL-C levels using Sanger method. Then, we genotyped 147 selected variants (78 sequence variants, 69 HapMap tagSNPs, and 2 previously reported relevant variants) in the entire sample of 788 African Blacks using either the iPLEX Gold or TaqMan methods. A total of 137 successfully genotyped variants were further evaluated for association with major lipid traits. RESULTS: The initial gene-based analysis demonstrated evidence of association with HDL-C and apolipoprotein A-I (ApoA-I). The follow-up single-site analysis revealed nominal evidence of novel associations of nine common variants with HDL-C and/or ApoA-I (P < 0.05). The strongest association was between rs11057851 and HDL-C (P = 0.0043), which remained significant after controlling for multiple testing using false discovery rate. Rare variant association testing revealed a group of 23 rare variants (frequencies ≤ 1%) associated with HDL-C (P = 0.0478). Haplotype analysis identified four SCARB1 regions associated with HDL-C (global P < 0.05). CONCLUSIONS: To our knowledge, this is the first report of a comprehensive association study of SCARB1 variations with lipid traits in an African Black population. Our results showed the consistent association of SCARB1 variants with HDL-C across various association analyses, supporting the role of SCARB1 in lipoprotein-lipid regulatory mechanism.

Lupus nephritis susceptibility loci in women with systemic lupus erythematosus.

Lupus nephritis is a manifestation of SLE resulting from glomerular immune complex deposition and inflammation. Lupus nephritis demonstrates familial aggregation and accounts for significant morbidity and mortality. We completed a meta-analysis of three genome-wide association studies of SLE to identify lupus nephritis-predisposing loci. Through genotyping and imputation, >1.6 million markers were assessed in 2000 unrelated women of European descent with SLE (588 patients with lupus nephritis and 1412 patients with lupus without nephritis). Tests of association were computed using logistic regression adjusting for population substructure. The strongest evidence for association was observed outside the MHC and included markers localized to 4q11-q13 (PDGFRA, GSX2; P=4.5×10(-7)), 16p12 (SLC5A11; P=5.1×10(-7)), 6p22 (ID4; P=7.4×10(-7)), and 8q24.12 (HAS2, SNTB1; P=1.1×10(-6)). Both HLA-DR2 and HLA-DR3, two well established lupus susceptibility loci, showed evidence of association with lupus nephritis (P=0.06 and P=3.7×10(-5), respectively). Within the class I region, rs9263871 (C6orf15-HCG22) had the strongest evidence of association with lupus nephritis independent of HLA-DR2 and HLA-DR3 (P=8.5×10(-6)). Consistent with a functional role in lupus nephritis, intra-renal mRNA levels of PDGFRA and associated pathway members showed significant enrichment in patients with lupus nephritis (n=32) compared with controls (n=15). Results from this large-scale genome-wide investigation of lupus nephritis provide evidence of multiple biologically relevant lupus nephritis susceptibility loci.

Lipoprotein lipase gene sequencing and plasma lipid profile.

Lipoprotein lipase (LPL) plays a crucial role in lipid metabolism by hydrolyzing triglyceride (TG)-rich particles and affecting HDL cholesterol (HDL-C) levels. In this study, the entire LPL gene plus flanking regions were resequenced in individuals with extreme HDL-C/TG levels (n = 95), selected from a population-based sample of 623 US non-Hispanic White (NHW) individuals. A total of 176 sequencing variants were identified, including 28 novel variants. A subset of 64 variants [common tag single nucleotide polymorphisms (tagSNP) and selected rare variants] were genotyped in the total sample, followed by association analyses with major lipid traits. A gene-based association test including all genotyped variants revealed significant association with HDL-C (P = 0.024) and TG (P = 0.006). Our single-site analysis revealed seven independent signals (P < 0.05; r² < 0.40) with either HDL-C or TG. The most significant association was for the SNP rs295 exerting opposite effects on TG and HDL-C levels with P values of 7.5.10⁻4 and 0.002, respectively. Our work highlights some common variants and haplotypes in LPL with significant associations with lipid traits; however, the analysis of rare variants using burden tests and SKAT-O method revealed negligible effects on lipid traits. Comprehensive resequencing of LPL in larger samples is warranted to further test the role of rare variants in affecting plasma lipid levels.

Differential genetic associations for systemic lupus erythematosus based on anti-dsDNA autoantibody production.

Systemic lupus erythematosus (SLE) is a clinically heterogeneous, systemic autoimmune disease characterized by autoantibody formation. Previously published genome-wide association studies (GWAS) have investigated SLE as a single phenotype. Therefore, we conducted a GWAS to identify genetic factors associated with anti-dsDNA autoantibody production, a SLE-related autoantibody with diagnostic and clinical importance. Using two independent datasets, over 400,000 single nucleotide polymorphisms (SNPs) were studied in a total of 1,717 SLE cases and 4,813 healthy controls. Anti-dsDNA autoantibody positive (anti-dsDNA +, n = 811) and anti-dsDNA autoantibody negative (anti-dsDNA -, n = 906) SLE cases were compared to healthy controls and to each other to identify SNPs associated specifically with these SLE subtypes. SNPs in the previously identified SLE susceptibility loci STAT4, IRF5, ITGAM, and the major histocompatibility complex were strongly associated with anti-dsDNA + SLE. Far fewer and weaker associations were observed for anti-dsDNA - SLE. For example, rs7574865 in STAT4 had an OR for anti-dsDNA + SLE of 1.77 (95% CI 1.57-1.99, p = 2.0E-20) compared to an OR for anti-dsDNA - SLE of 1.26 (95% CI 1.12-1.41, p = 2.4E-04), with p(heterogeneity)<0.0005. SNPs in the SLE susceptibility loci BANK1, KIAA1542, and UBE2L3 showed evidence of association with anti-dsDNA + SLE and were not associated with anti-dsDNA - SLE. In conclusion, we identified differential genetic associations with SLE based on anti-dsDNA autoantibody production. Many previously identified SLE susceptibility loci may confer disease risk through their role in autoantibody production and be more accurately described as autoantibody propensity loci. Lack of strong SNP associations may suggest that other types of genetic variation or non-genetic factors such as environmental exposures have a greater impact on susceptibility to anti-dsDNA - SLE.

Genome-wide association analysis of age-at-onset in Alzheimer's disease.

The risk of Alzheimer's disease (AD) is strongly determined by genetic factors and recent genome-wide association studies (GWAS) have identified several genes for the disease risk. In addition to the disease risk, age-at-onset (AAO) of AD has also strong genetic component with an estimated heritability of 42%. Identification of AAO genes may help to understand the biological mechanisms that regulate the onset of the disease. Here we report the first GWAS focused on identifying genes for the AAO of AD. We performed a genome-wide meta-analysis on three samples comprising a total of 2222 AD cases. A total of ~2.5 million directly genotyped or imputed single-nucleotide polymorphisms (SNPs) were analyzed in relation to AAO of AD. As expected, the most significant associations were observed in the apolipoprotein E (APOE) region on chromosome 19 where several SNPs surpassed the conservative genome-wide significant threshold (P<5E-08). The most significant SNP outside the APOE region was located in the DCHS2 gene on chromosome 4q31.3 (rs1466662; P=4.95E-07). There were 19 additional significant SNPs in this region at P<1E-04 and the DCHS2 gene is expressed in the cerebral cortex and thus is a potential candidate for affecting AAO in AD. These findings need to be confirmed in additional well-powered samples.

Genome-wide association study of Alzheimer's disease with psychotic symptoms.

Psychotic symptoms occur in ~40% of subjects with Alzheimer's disease (AD) and are associated with more rapid cognitive decline and increased functional deficits. They show heritability up to 61% and have been proposed as a marker for a disease subtype suitable for gene mapping efforts. We undertook a combined analysis of three genome-wide association studies (GWASs) to identify loci that (1) increase susceptibility to an AD and subsequent psychotic symptoms; or (2) modify risk of psychotic symptoms in the presence of neurodegeneration caused by AD. In all, 1299 AD cases with psychosis (AD+P), 735 AD cases without psychosis (AD-P) and 5659 controls were drawn from Genetic and Environmental Risk in AD Consortium 1 (GERAD1), the National Institute on Aging Late-Onset Alzheimer's Disease (NIA-LOAD) family study and the University of Pittsburgh Alzheimer Disease Research Center (ADRC) GWASs. Unobserved genotypes were imputed to provide data on >1.8 million single-nucleotide polymorphisms (SNPs). Analyses in each data set were completed comparing (1) AD+P to AD-P cases, and (2) AD+P cases with controls (GERAD1, ADRC only). Aside from the apolipoprotein E (APOE) locus, the strongest evidence for association was observed in an intergenic region on chromosome 4 (rs753129; 'AD+PvAD-P' P=2.85 × 10(-7); 'AD+PvControls' P=1.11 × 10(-4)). SNPs upstream of SLC2A9 (rs6834555, P=3.0 × 10(-7)) and within VSNL1 (rs4038131, P=5.9 × 10(-7)) showed strongest evidence for association with AD+P when compared with controls. These findings warrant further investigation in larger, appropriately powered samples in which the presence of psychotic symptoms in AD has been well characterized.

Genetic Aspects of Conjunctival Melanoma: A Review.

Conjunctival melanoma (CM) is a rare but aggressive cancer. Over the past decade, molecular studies using rapidly advancing technologies have increasingly improved our understanding of CM genetics. CMs are mainly characterized by dysregulated MAPK and PI3K/AKT/mTOR pathways, driven by commonly mutated (BRAF, NRAS, NF1) or less commonly mutated (KIT, PTEN) genes. Another group of genes frequently mutated in CMs include TERT and ATRX, with known roles in telomere maintenance and chromatin remodeling/epigenetic regulation. Uveal melanoma-related genes (BAP1, SF3B1, GNAQ/11) can also be mutated in CMs, albeit infrequently. Additional CM-related mutated genes have increasingly been identified using more comprehensive genetic analyses, awaiting further confirmation in additional/larger studies. As a tumor arising in a partly sun-exposed mucosal tissue, CM exhibits a distinct genomic profile, including the frequent presence of an ultraviolet (UV) signature (and high mutational load) and also the common occurrence of large structural variations (distributed across the genome) in addition to specific gene mutations. The knowledge gained from CM genetic studies to date has led to new therapeutic avenues, including the use of targeted and/or immuno-therapies with promising outcomes in several cases. Accordingly, the implementation of tumor genetic testing into the routine clinical care of CM patients holds promise to further improve and personalize their treatments. Likewise, a growing knowledge of poor prognosis-associated genetic changes in CMs (NRAS, TERT, and uveal melanoma signature mutations and chromosome 10q deletions) may ultimately guide future strategies for prognostic testing to further improve clinical outcomes (by tailoring surveillance and considering prophylactic treatments in patients with high-risk primary tumors).

Investigating Vitreous Cytokines in Choroidal Melanoma.

Due to the close relationship between the vitreous and posterior eye layers, the microenvironment of these layers can affect the composition of the vitreous. Molecular analysis of the vitreous may therefore provide important insights into the pathogenesis of chorioretinal diseases. In this study, vitreous cytokines (n = 41) were evaluated to gain further insights into the tumor microenvironment in uveal melanoma (UM) arising from the choroid (CM). Cytokine levels were measured using a bead-based multiplex immunoassay panel in vitreous samples obtained from 32 eyes, including 18 with CM and 14 controls. Median fluorescence intensity values were extracted and used as relative quantification of the cytokine abundance. Vitreous cytokine levels were compared between the CM and non-CM groups and between different prognostic categories within the CM group (classified as having low or high metastatic risk using tumor biopsy-based gene expression profiling). Correlations between vitreous cytokine levels and tumor dimensions were also evaluated. Our analysis revealed twenty-six vitreous cytokines significantly upregulated in CM-affected eyes compared to the control eyes. Within the CM group, six vitreous cytokines showed altered levels (five upregulated and one downregulated) in eyes with high- vs. low-risk tumors. Levels of these six plus several other cytokines showed correlations with the tumor dimensions. In conclusion, our study has uncovered several UM-relevant vitreous cytokines, worthy of follow-up in larger studies as potential candidates for liquid biopsy-based biomarker development and/or new therapeutic targeting.

Intralesional or intraorbital rituximab injection for the management of biopsy-proven idiopathic orbital inflammation involving the lacrimal gland.

OBJECTIVE: To evaluate the efficacy of intralesional rituximab injection for the management of idiopathic orbital inflammation (IOI) involving the lacrimal gland, which is the most common subtype. METHOD: Eighteen consecutive patients with biopsy-proven IOI involving the lacrimal gland were included. Rituximab (50 mg/5 mL) was injected intralesionally at monthly intervals. RESULTS: Clinically, all patients presented with upper eyelid swelling and ptosis. Most patients (56%) had periocular pain and a palpable superotemporal mass. Biopsies showed chronic inflammation without fibrosis in 14 patients (78%) and chronic inflammation and fibrosis in 4 patients (22%). Intralesional rituximab was injected once in 1 patient (6%) because of complete response after the first injection, twice in 11 patients (61%), and 3 times in 6 patients (33%) because of partial response after 2 injections. After a mean follow-up of 33 months (median, 33 months; range, 11-59 months), 16 patients (89%) showed a clinical response, including 14 patients (78%) a complete response (i.e., disappearance of all lesions) and 2 patients (11%) with a partial response (i.e., ≤30% decrease in lesion diameter). Two patients (11%) did not respond after 3 injections and were placed on systemic corticosteroid and methotrexate therapies. Two patients (11%) with a complete response developed subsequent recurrence 12 and 49 months after their last injections. Both were treated with 2 additional rituximab injections, 1 month apart, and showed complete response when examined 27 and 11 months after treatment, respectively. CONCLUSION: Intralesional rituximab injection may be an effective treatment for IOI involving the lacrimal gland, achieving a 78% complete response rate in this series. Local treatment with rituximab has the potential to avoid the ocular and systemic side effects of corticosteroid and systemic immunosuppressive treatment.

Aqueous Humor-Derived MYD88 L265P Mutation Analysis in Vitreoretinal Lymphoma: A Potential Less Invasive Method for Diagnosis and Treatment Response Assessment.

PURPOSE: To investigate whether MYD88 L265P mutation, which is frequently present in vitreoretinal lymphoma, can be detected in aqueous humor, a specimen that can be obtained in a clinic setting, potentially mitigating the need for more invasive vitrectomy procedures, and whether this approach can be used to monitor treatment response. DESIGN: Observational case series. SUBJECTS: Patients who were diagnosed with biopsy-confirmed or clinically diagnosed vitreoretinal lymphoma or biopsy-confirmed vitritis. METHODS: We evaluated aqueous humor-derived (AHD) MYD88 L265P mutation during vitreous biopsy or at the initial presentation in the clinic if vitreous biopsy was not feasible. Demographic or clinical features of patients were retrospectively reviewed. Aqueous humor-derived MYD88 L265P mutation was re-evaluated after patients completed a course of intravitreal methotrexate and rituximab injection therapy. The NM_002468.4: c.794T>C (p.L265P) mutation in the MYD88 gene was evaluated in AHD cellular and cell-free DNA using allele-specific polymerase chain reaction. MAIN OUTCOME MEASURES: Detection of AHD MYD88 L265P mutation at the initial diagnosis and to monitor the treatment response. RESULTS: Aqueous humor from 18 eyes of 14 patients with biopsy-confirmed or clinically diagnosed vitreoretinal lymphoma and 3 eyes of 3 patients with biopsy-confirmed vitritis were evaluated. Aqueous humor-derived MYD88 L265P mutation was detected in cell-based and cell-free DNA from 15 (83%) of 18 eyes with biopsy-confirmed or clinically diagnosed vitreoretinal lymphoma but not identified in any of the 3 eyes with vitritis. The mutation was less readily detectable in cellular DNA (10 of 18) compared with cell-free DNA (15 of 18). Furthermore, aqueous sampling after intravitreal methotrexate and rituximab injection therapy revealed absence of this mutation after complete response in 7 eyes. The mutation was detected in 1 eye that developed recurrence in a posttreatment window of 6 months. After a mean of follow-up of 9 months, there was no clinical evidence of vitreoretinal lymphoma recurrence in the 7 eyes with no detectable AHD MYD88 L265P mutation. CONCLUSIONS: This investigational study suggests that AHD MYD88 L265P can be detected in eyes with lymphoma and may thus serve as a surrogate, less invasive biopsy in the diagnosis and follow-up of vitreoretinal lymphoma, particularly when cell-free DNA is evaluated.

First Report of Phytophthora cryptogea on Potato Tubers in Turkey.

Potato (Solanum tuberosum L.) is an important agricultural crop in Turkey, with 150,000 ha and a production of about 4 million tons per year. There are numerous pathogens that limit potato production. In 2011, a new disease was observed on the potato cultivar Ramos in Erzincan Province in Turkey. Disease symptoms were similar to pink rot. Infected tubers had a rubbery texture and when cut, infected areas were cream colored. When the infected area was exposed to air for 10 min, the color turned to salmon pink and after 15 to 20 min it turned black. A funguslike organism was isolated from diseased tubers on carrot agar. The colonies were uniform and there were no petaloid patterns. Soil extract solution was prepared using 1,000 ml of distilled water and 15 g of field soil according to Jeffers and Aldwinckle (2) and used for sporangia production. Agar discs from 5-day-old colonies were placed in soil extract solution and incubated under continuous fluorescent light at room temperature. Sporangia began forming after 24 h and were abundant after 48 h. Oogonia were not observed on corn meal agar plates supplemented by beta-cytosterol. Sporangia were nonpapillate and noncaducous, mostly ovoid, but some were obpyriform. The mean size of the sporangia was 43 ± 4 × 30 ± 2 µm. The Oomycete was identified as Phytophthora cryptogea Pethybr. & Laff., according to its morphological characters (1). Isolate identity was confirmed by sequence analysis of the ribosomal DNA internal transcribed spacer using primers ITS1 and ITS4. The ITS sequence was a 99% match to P. cryptogea strains (GenBank Accession Nos. AF087475.1, AF228101.1, GU111626.1, AY995400.1, GU111624.1, AY995400.1, and GU111624.1). The isolate from Turkey was deposited in GenBank as Accession No. JQ071495. Pathogenicity tests were conducted on potato tubers (cv. Ramos). Tubers were surface disinfected with 0.5% NaOCl for 5 min., then washed twice with sterile water. When dried, 20 tubers were wounded with a shallow cut made parallel to the surface by a sterile knife. An agar disc from a 1-week-old colony was placed on the artificial wound on each of 10 potatoes. For controls, agar discs without fungal mycelia were placed on the wounds of the other 10 potatoes. Each inoculated and uninoculated potato was incubated for 5 days at 24°C in the dark and in separate plastic cups containing moistened filter paper at the bottom. Tubers were then cut open and examined for disease. Cream colored lesions were first observed; after 10 min lesions turned salmon pink and after 20 min they turned black. Cream colored lesions were not observed inside uninoculated potatoes. To the best of our knowledge, this is the first report of P. cryptogea causing pink rot of potato in Turkey. Pink rot disease of potato is commonly caused by P. erytroseptica, but P. cryptogea can cause similar symptoms (3) and severe tuber rot. This pathogen could cause important losses of potato in Turkey, especially during storage of tubers. References: (1) M. E. Gallegly and C. Hong, Phytophthora: Identifying Species by Morphology and DNA Fingerprints. APS Press, St. Paul, MN, 2008. (2) S. N. Jeffers and H. S. Aldwinckle, Phytopathology 77:1475, 1987. (3) E. C. Rowe and A. F. Schimitthenner, Plant Dis. Rep. 61:807, 1977.

First Report of Phaeoacremonium scolyti Associated with Esca and Petri Diseases of Grapevine in Turkey.

Grapevine (Vitis vinifera L.) is a widely planted and economically important crop for production of raisin grapes, table grapes, and wine grapes in Turkey. Esca and petri diseases are two of the most important and destructive diseases of young and old vines worldwide (1). During the summers of 2009 and 2010, a survey was carried out in 63 vineyards in six locations of Ankara Province. Root and trunk samples were collected from 4- to 15-year-old grapevines showing esca and petri disease symptoms, including reduced trunk diameters, shortened internodes, stunted growth, chlorotic or necrotic leaves, and brown-to-black spots or streaks in the xylem vessels in cross or longitudinal sections of the rootstock trunk (1,3). Small pieces of internal tissues were surface disinfested in 1% sodium hypochlorite for 2 min, washed twice with sterile distilled water, and plated onto potato dextrose agar (PDA) amended with tetracycline hydrochloride (0.1 g liter-1). Plates were incubated at 25°C in the dark for 14 to 21 days. Phaeoacremonium spp. were consistently isolated from necrotic tissues. Single-conidial isolates of these Phaeoacremonium spp. were grown on PDA, malt extract agar (MEA), and oatmeal agar (OA) in the dark at 25°C for 2 to 3 weeks until colonies produced spores (3). Of these, Phaeoacremonium aleophilum was the most prevalent species, however, one isolate identified as P. scolyti was described by L. Mostert et al. (3). Conidiophores were mostly short and usually unbranched, subcylindrical to navicular, and often consisting of an elongate-ampuliform phialide. Phialides were terminal or lateral and pale brown to hyaline. Type II phialide were predominant. Type I phialide were 4 to 6 µm (average), type II phialide were 7 to 14 µm (average), and type III phialide were 14 to 20 µm (average). Conidia were hyaline, oblong-ellipsoidal, occasionally reniform or allantoid, 2.5 to 5 µm long (average), and 1 to 1.5 µm wide (average). Colony colors were reddish gray on PDA, pinkish white on MEA, and grayish pink on OA. Identity was confirmed by ß-tubulin sequence analysis using primers T1 and Bt2b (2). Additionally, the ß-tubulin gene fragment (primers T1 and Bt2b) of this isolate was sequenced (GenBank Accession No. JF909894). The sequence showed high similarity (99%) with the sequence of P. scolyti (GenBank Accession No. EU260415). Pathogenicity tests were completed using five, 3-month-old rooted cuttings of cv. Sultana. A hole approximately 3 mm in diameter was drilled on the crown 2 cm aboveground level from the bark to the pith and filled with a 30-µl spore suspension (106 spores/ml) harvested from 21-day-old cultures grown on PDA at 25°C in the dark. Five control plants were used. Controls were inoculated with sterile distilled water. Filled holes were sealed with a sheet of Parafilm. The plants were incubated for 3 months in a controlled environment facility at 25°C. After 3 months, the fungus was reisolated from black discoloration of vascular tissue and pith tissue of the crown area of all inoculated cuttings, completing Koch's postulates. The black discoloration was more compact near the point of inoculation. Control plants were asymptomatic and P. scolyti was not recovered from control plants. To our knowledge, this is the first report of the presence of P. scolyti causing esca and petri disease of grapevine in Turkey. References: (1) A. Eskalen et al. Plant Dis. 91:1100, 2007. (2) S. Essakhi et al. Persoonia 21:119, 2008. (3) L. Mostert et al. Stud. Mycol. 54:1, 2006.