Lung adenocarcinoma promotion by air pollutants.

A complete understanding of how exposure to environmental substances promotes cancer formation is lacking. More than 70 years ago, tumorigenesis was proposed to occur in a two-step process: an initiating step that induces mutations in healthy cells, followed by a promoter step that triggers cancer development1. Here we propose that environmental particulate matter measuring ≤2.5 µm (PM2.5), known to be associated with lung cancer risk, promotes lung cancer by acting on cells that harbour pre-existing oncogenic mutations in healthy lung tissue. Focusing on EGFR-driven lung cancer, which is more common in never-smokers or light smokers, we found a significant association between PM2.5 levels and the incidence of lung cancer for 32,957 EGFR-driven lung cancer cases in four within-country cohorts. Functional mouse models revealed that air pollutants cause an influx of macrophages into the lung and release of interleukin-1ß. This process results in a progenitor-like cell state within EGFR mutant lung alveolar type II epithelial cells that fuels tumorigenesis. Ultradeep mutational profiling of histologically normal lung tissue from 295 individuals across 3 clinical cohorts revealed oncogenic EGFR and KRAS driver mutations in 18% and 53% of healthy tissue samples, respectively. These findings collectively support a tumour-promoting role for  PM2.5 air pollutants  and provide impetus for public health policy initiatives to address air pollution to reduce disease burden.

Transient genomic instability drives tumorigenesis through accelerated clonal evolution.

Abnormal numerical and structural chromosome content is frequently found in human cancer. To test the role of aneuploidy in tumor initiation and progression, we generated mice with random aneuploidies by transient induction of polo-like kinase 4 (Plk4), a master regulator of centrosome number. Short-term chromosome instability (CIN) from transient Plk4 induction resulted in formation of aggressive T-cell lymphomas in mice with heterozygous inactivation of one p53 allele and accelerated tumor development in the absence of p53. Transient CIN increased the frequency of lymphoma-initiating cells with a specific karyotype profile, including trisomy of chromosomes 4, 5, 14, and 15 occurring early in tumorigenesis. Tumor development in mice with chronic CIN induced by an independent mechanism (through inactivation of the spindle assembly checkpoint) gradually trended toward a similar karyotypic profile, as determined by single-cell whole-genome DNA sequencing. Overall, we show how transient CIN generates cells with random aneuploidies from which ones that acquire a karyotype with specific chromosome gains are sufficient to drive cancer formation, and that distinct CIN mechanisms can lead to similar karyotypic cancer-causing outcomes.

Nuclear chromosome locations dictate segregation error frequencies.

Chromosome segregation errors during cell divisions generate aneuploidies and micronuclei, which can undergo extensive chromosomal rearrangements such as chromothripsis1-5. Selective pressures then shape distinct aneuploidy and rearrangement patterns-for example, in cancer6,7-but it is unknown whether initial biases in segregation errors and micronucleation exist for particular chromosomes. Using single-cell DNA sequencing8 after an error-prone mitosis in untransformed, diploid cell lines and organoids, we show that chromosomes have different segregation error frequencies that result in non-random aneuploidy landscapes. Isolation and sequencing of single micronuclei from these cells showed that mis-segregating chromosomes frequently also preferentially become entrapped in micronuclei. A similar bias was found in naturally occurring micronuclei of two cancer cell lines. We find that segregation error frequencies of individual chromosomes correlate with their location in the interphase nucleus, and show that this is highest for peripheral chromosomes behind spindle poles. Randomization of chromosome positions, Cas9-mediated live tracking and forced repositioning of individual chromosomes showed that a greater distance from the nuclear centre directly increases the propensity to mis-segregate. Accordingly, chromothripsis in cancer genomes9 and aneuploidies in early development10 occur more frequently for larger chromosomes, which are preferentially located near the nuclear periphery. Our findings reveal a direct link between nuclear chromosome positions, segregation error frequencies and micronucleus content, with implications for our understanding of tumour genome evolution and the origins of specific aneuploidies during development.

Systemic complement levels in patients with age-related macular degeneration carrying rare or low-frequency variants in the CFH gene.

Age-related macular degeneration (AMD) is a major cause of vision loss among the elderly in the Western world. Genetic variants in the complement factor H (CFH) gene are associated with AMD, but the functional consequences of many of these variants are currently unknown. In this study, we aimed to determine the effect of 64 rare and low-frequency variants in the CFH gene on systemic levels of factor H (FH) and complement activation marker C3bBbP using plasma samples of 252 carriers and 159 non-carriers. Individuals carrying a heterozygous nonsense, frameshift or missense variant in CFH presented with significantly decreased FH levels and significantly increased C3bBbP levels in plasma compared to non-carrier controls. FH and C3bBbP plasma levels were relatively stable over time in samples collected during follow-up visits. Decreased FH and increased C3bBbP concentrations were observed in carriers compared to non-carriers of CFH variants among different AMD stages, with the exception of C3bBbP levels in advanced AMD stages, which were equally high in carriers and non-carriers. In AMD families, FH levels were decreased in carriers compared to non-carriers, but C3bBbP levels did not differ. Rare variants in the CFH gene can lead to reduced FH levels or reduced FH function as measured by increased C3bBbP levels. The effects of individual variants in the CFH gene reported in this study will improve the interpretation of rare and low-frequency variants observed in AMD patients in clinical practice.

Common haplotypes at the CFH locus and low-frequency variants in CFHR2 and CFHR5 associate with systemic FHR concentrations and age-related macular degeneration.

Age-related macular degeneration (AMD) is the principal cause of blindness in the elderly population. A strong effect on AMD risk has been reported for genetic variants at the CFH locus, encompassing complement factor H (CFH) and the complement-factor-H-related (CFHR) genes, but the underlying mechanisms are not fully understood. We aimed to dissect the role of factor H (FH) and FH-related (FHR) proteins in AMD in a cohort of 202 controls and 216 individuals with AMD. We detected elevated systemic levels of FHR-1 (p = 1.84 × 10-6), FHR-2 (p = 1.47 × 10-4), FHR-3 (p = 1.05 × 10-5) and FHR-4A (p = 1.22 × 10-2) in AMD, whereas FH concentrations remained unchanged. Common AMD genetic variants and haplotypes at the CFH locus strongly associated with FHR protein concentrations (e.g., FH p.Tyr402His and FHR-2 concentrations, p = 3.68 × 10-17), whereas the association with FH concentrations was limited. Furthermore, in an International AMD Genomics Consortium cohort of 17,596 controls and 15,894 individuals with AMD, we found that low-frequency and rare protein-altering CFHR2 and CFHR5 variants associated with AMD independently of all previously reported genome-wide association study (GWAS) signals (p = 5.03 × 10-3 and p = 2.81 × 10-6, respectively). Low-frequency variants in CFHR2 and CFHR5 led to reduced or absent FHR-2 and FHR-5 concentrations (e.g., p.Cys72Tyr in CFHR2 and FHR-2, p = 2.46 × 10-16). Finally, we showed localization of FHR-2 and FHR-5 in the choriocapillaris and in drusen. Our study identifies FHR proteins as key proteins in the AMD disease mechanism. Consequently, therapies that modulate FHR proteins might be effective for treating or preventing progression of AMD. Such therapies could target specific individuals with AMD on the basis of their genotypes at the CFH locus.

The H3.3K27M oncohistone affects replication stress outcome and provokes genomic instability in pediatric glioma.

While comprehensive molecular profiling of histone H3.3 mutant pediatric high-grade glioma has revealed extensive dysregulation of the chromatin landscape, the exact mechanisms driving tumor formation remain poorly understood. Since H3.3 mutant gliomas also exhibit high levels of copy number alterations, we set out to address if the H3.3K27M oncohistone leads to destabilization of the genome. Hereto, we established a cell culture model allowing inducible H3.3K27M expression and observed an increase in mitotic abnormalities. We also found enhanced interaction of DNA replication factors with H3.3K27M during mitosis, indicating replication defects. Further functional analyses revealed increased genomic instability upon replication stress, as represented by mitotic bulky and ultrafine DNA bridges. This co-occurred with suboptimal 53BP1 nuclear body formation after mitosis in vitro, and in human glioma. Finally, we observed a decrease in ultrafine DNA bridges following deletion of the K27M mutant H3F3A allele in primary high-grade glioma cells. Together, our data uncover a role for H3.3 in DNA replication under stress conditions that is altered by the K27M mutation, promoting genomic instability and potentially glioma development.

Effect of rare coding variants in the CFI gene on Factor I expression levels.

Factor I (FI) is one of the main inhibitors of complement activity, and numerous rare coding variants have been reported in patients with age-related macular degeneration, atypical hemolytic uremic syndrome and C3 glomerulopathy. Since many of these variants are of unknown clinical significance, this study aimed to determine the effect of rare coding variants in the complement factor I (CFI) gene on FI expression. We measured FI levels in plasma samples of carriers of rare coding variants and in vitro in the supernatants of epithelial cells expressing recombinant FI. FI levels were measured in 177 plasma samples of 155 individuals, carrying 24 different rare coding variants in CFI. In carriers of the variants p.Gly119Arg, p.Leu131Arg, p.Gly188Ala and c.772G>A (r.685_773del), significantly reduced FI plasma levels were detected. Furthermore, recombinant FI expression levels were determined for 126 rare coding variants. Of these variants 68 (54%) resulted in significantly reduced FI expression in supernatant compared to wildtype (WT). The recombinant protein expression levels correlated significantly with the FI level in plasma of carriers of CFI variants. In this study, we performed the most comprehensive FI expression level analysis of rare coding variants in CFI to date. More than half of CFI variants lead to reduced FI expression, which might impair complement regulation in vivo. Our study will aid the interpretation of rare coding CFI variants identified in clinical practice, which is in particular important in light of patient inclusion in ongoing clinical trials for CFI gene supplementation in AMD.

Analysis of hemopexin plasma levels in patients with age-related macular degeneration.

Purpose: A protein quantitative trait locus (pQTL) analysis recently revealed a strong association between hemopexin (HPX) levels and genetic variants at the complement factor H (CFH) locus. In this study, we aimed to determine HPX plasma levels in patients with age-related macular degeneration (AMD) and to compare them with those in controls. We also investigated whether genetic variants at the CFH locus are associated with HPX plasma levels. Methods: HPX levels were quantified in 200 advanced AMD cases and 200 controls using an enzyme-linked immunosorbent assay and compared between the two groups. Furthermore, HPX levels were analyzed per genotype group of three HPX-associated variants (rs61818956, rs10494745, and rs10801582) and four AMD-associated variants (rs794362 [proxy for rs187328863], rs570618, rs10922109, and rs61818924 [proxy for rs61818925]) at the CFH locus. Results: HPX levels were similar in the control group compared with the AMD group. The three variants at the CFH locus, which were previously associated with the HPX levels, showed no association with the HPX levels in our data set. No significant differences in HPX levels were detected between the different genotype groups of AMD-associated variants at the CFH locus. Conclusions: In this study, HPX levels were not associated with AMD or AMD-associated variants at the CFH locus. The finding of a previous pQTL study that variants at the CFH locus were associated with HPX levels was also not confirmed in this study.

Chromosomal Instability Characterizes Pediatric Medulloblastoma but Is Not Tolerated in the Developing Cerebellum.

Medulloblastoma is a pediatric brain malignancy that consists of four transcriptional subgroups. Structural and numerical aneuploidy are common in all subgroups, although they are particularly profound in Group 3 and Group 4 medulloblastoma and in a subtype of SHH medulloblastoma termed SHHα. This suggests that chromosomal instability (CIN), the process leading to aneuploidy, is an important player in medulloblastoma pathophysiology. However, it is not known if there is ongoing CIN in medulloblastoma or if CIN affects the developing cerebellum and promotes tumor formation. To investigate this, we performed karyotyping of single medulloblastoma cells and demonstrated the presence of distinct tumor cell clones harboring unique copy number alterations, which is suggestive of ongoing CIN. We also found enrichment for processes related to DNA replication, repair, and mitosis in both SHH medulloblastoma and in the highly proliferative compartment of the presumed tumor cell lineage-of-origin, the latter also being sensitive to genotoxic stress. However, when challenging these tumor cells-of-origin with genetic lesions inducing CIN using transgenic mouse modeling, we found no evidence for large chromosomal aberrations in the cerebellum or for medulloblastoma formation. We therefore conclude that without a background of specific genetic mutations, CIN is not tolerated in the developing cerebellum in vivo and, thus, by itself is not sufficient to initiate medulloblastoma.

Development of a Genotype Assay for Age-Related Macular Degeneration: The EYE-RISK Consortium.

PURPOSE: To develop a genotype assay to assess associations with common and rare age-related macular degeneration (AMD) risk variants, to calculate an overall genetic risk score (GRS), and to identify potential misdiagnoses with inherited macular dystrophies that mimic AMD. DESIGN: Case-control study. PARTICIPANTS: Individuals (n = 4740) from 5 European cohorts. METHODS: We designed single-molecule molecular inversion probes for target selection and used next generation sequencing to sequence 87 single nucleotide polymorphisms (SNPs), coding and splice-site regions of 10 AMD-(related) genes (ARMS2, C3, C9, CD46, CFB, CFH, CFI, HTRA1, TIMP3, and SLC16A8), and 3 genes that cause inherited macular dystrophies (ABCA4, CTNNA1, and PRPH2). Genetic risk scores for common AMD risk variants were calculated based on effect size and genotype of 52 AMD-associated variants. Frequency of rare variants was compared between late AMD patients and control individuals with logistic regression analysis. MAIN OUTCOME MEASURES: Genetic risk score, association of genetic variants with AMD, and genotype-phenotype correlations. RESULTS: We observed high concordance rates between our platform and other genotyping platforms for the 69 successfully genotyped SNPs (>96%) and for the rare variants (>99%). We observed a higher GRS for patients with late AMD compared with patients with early/intermediate AMD (P < 0.001) and individuals without AMD (P < 0.001). A higher proportion of pathogenic variants in the CFH (odds ratio [OR] = 2.88; P = 0.006), CFI (OR = 4.45; P = 0.005), and C3 (OR = 6.56; P = 0.0003) genes was observed in late AMD patients compared with control individuals. In 9 patients, we identified pathogenic variants in the PRPH2, ABCA4, and CTNNA1 genes, which allowed reclassification of these patients as having inherited macular dystrophy. CONCLUSIONS: This study reports a genotype assay for common and rare AMD genetic variants, which can identify individuals at intermediate to high genetic risk of late AMD and enables differential diagnosis of AMD-mimicking dystrophies. Our study supports sequencing of CFH, CFI, and C3 genes because they harbor rare high-risk variants. Carriers of these variants could be amendable for new treatments for AMD that currently are under development.

Functional analyses of rare genetic variants in complement component C9 identified in patients with age-related macular degeneration.

Age-related macular degeneration (AMD) is a progressive disease of the central retina and the leading cause of irreversible vision loss in the western world. The involvement of abnormal complement activation in AMD has been suggested by association of variants in genes encoding complement proteins with disease development. A low-frequency variant (p.P167S) in the complement component C9 (C9) gene was recently shown to be highly associated with AMD; however, its functional outcome remains largely unexplored. In this study, we reveal five novel rare genetic variants (p.M45L, p.F62S, p.G126R, p.T170I and p.A529T) in C9 in AMD patients, and evaluate their functional effects in vitro together with the previously identified (p.R118W and p.P167S) C9 variants. Our results demonstrate that the concentration of C9 is significantly elevated in patients' sera carrying the p.M45L, p.F62S, p.P167S and p.A529T variants compared with non-carrier controls. However, no difference can be observed in soluble terminal complement complex levels between the carrier and non-carrier groups. Comparing the polymerization of the C9 variants we reveal that the p.P167S mutant spontaneously aggregates, while the other mutant proteins (except for C9 p.A529T) fail to polymerize in the presence of zinc. Altered polymerization of the p.F62S and p.P167S proteins associated with decreased lysis of sheep erythrocytes and adult retinal pigment epithelial-19 cells by carriers' sera. Our data suggest that the analyzed C9 variants affect only the secretion and polymerization of C9, without influencing its classical lytic activity. Future studies need to be performed to understand the implications of the altered polymerization of C9 in AMD pathology.

Association of rs10490924 in ARMS2/HTRA1 with age-related macular degeneration in the Pakistani population.

Age-related macular degeneration (AMD) is a disease of the elderly in which central vision is lost because of degenerative changes of the macula. The current study investigated the association of single-nucleotide polymorphisms (SNPs) with AMD in the Pakistani population. Four SNPs were analyzed in this study: rs1061170 in the CFH, rs429608 near CFB, rs2230199 in the C3, and rs10490924 in ARMS2/HTRA1. This case-control association study was conducted on 300 AMD patients (125 wet AMD and 175 dry AMD) and 200 unaffected age- and gender-matched control individuals. The association of the SNP genotypes and allele frequency distributions were compared between patients and healthy controls, keeping age, gender, and smoking status as covariates. A significant genotype and variant allele association was found of rs10490924 in ARMS2/HTRA1 with wet AMD, while the SNPs in CFH, CFB, and C3 were not associated with AMD in the current Pakistani cohort. The lack of association of CFH, CFB, and C3 may be attributed to limited sample size. This study demonstrates that genetic causative factors of AMD differ among populations and supports the need for genetic association studies among cohorts from various populations to increase our global understanding of the disease pathogenesis.

Whole-Exome Sequencing in Age-Related Macular Degeneration Identifies Rare Variants in COL8A1, a Component of Bruch's Membrane.

PURPOSE: Genome-wide association studies and targeted sequencing studies of candidate genes have identified common and rare variants that are associated with age-related macular degeneration (AMD). Whole-exome sequencing (WES) studies allow a more comprehensive analysis of rare coding variants across all genes of the genome and will contribute to a better understanding of the underlying disease mechanisms. To date, the number of WES studies in AMD case-control cohorts remains scarce and sample sizes are limited. To scrutinize the role of rare protein-altering variants in AMD cause, we performed the largest WES study in AMD to date in a large European cohort consisting of 1125 AMD patients and 1361 control participants. DESIGN: Genome-wide case-control association study of WES data. PARTICIPANTS: One thousand one hundred twenty-five AMD patients and 1361 control participants. METHODS: A single variant association test of WES data was performed to detect variants that are associated individually with AMD. The cumulative effect of multiple rare variants with 1 gene was analyzed using a gene-based CMC burden test. Immunohistochemistry was performed to determine the localization of the Col8a1 protein in mouse eyes. MAIN OUTCOME MEASURES: Genetic variants associated with AMD. RESULTS: We detected significantly more rare protein-altering variants in the COL8A1 gene in patients (22/2250 alleles [1.0%]) than in control participants (11/2722 alleles [0.4%]; P = 7.07×10-5). The association of rare variants in the COL8A1 gene is independent of the common intergenic variant (rs140647181) near the COL8A1 gene previously associated with AMD. We demonstrated that the Col8a1 protein localizes at Bruch's membrane. CONCLUSIONS: This study supported a role for protein-altering variants in the COL8A1 gene in AMD pathogenesis. We demonstrated the presence of Col8a1 in Bruch's membrane, further supporting the role of COL8A1 variants in AMD pathogenesis. Protein-altering variants in COL8A1 may alter the integrity of Bruch's membrane, contributing to the accumulation of drusen and the development of AMD.

Genetic screening for macular dystrophies in patients clinically diagnosed with dry age-related macular degeneration.

It can be clinically challenging to distinguish dry age-related macular degeneration (AMD) from AMD-mimicking dystrophies, and sometimes misdiagnosis occurs. With upcoming therapies for dry AMD it is important to exclude patients with a different retinal disease from clinical trials. In this study we evaluated the occurrence of AMD-mimicking dystrophies in an AMD cohort. Whole-exome sequencing (WES) was performed in 218 patients with intermediate AMD or geographic atrophy secondary to AMD and 133 control individuals. WES data was analyzed for rare variants in 19 genes associated with autosomal dominant and recessive macular dystrophies mimicking AMD. In three (1.4%) of 218 cases we identified a pathogenic heterozygous variant (PRPH2 c.424C > T; p.R142W) causal for autosomal dominant central areolar choroidal dystrophy (CACD). Phenotypically, these patients all presented with geographic atrophy. In 12 (5.5%) of 218 cases we identified a heterozygous variant of unknown clinical significance, but predicted to be highly deleterious, in genes previously associated with autosomal dominant macular dystrophies. The distinction between AMD and AMD-mimicking dystrophies, such as CACD, can be challenging based on fundus examination alone. Genetic screening for genes associated with macular dystrophies, especially PRPH2, can be beneficial to help identify AMD-mimicking dystrophies.

How to count chromosomes in a cell: An overview of current and novel technologies.

Aneuploidy, an aberrant number of chromosomes in a cell, is a feature of several syndromes associated with cognitive and developmental defects. In addition, aneuploidy is considered a hallmark of cancer cells and has been suggested to play a role in neurodegenerative disease. To better understand the relationship between aneuploidy and disease, various methods to measure the chromosome numbers in cells have been developed, each with their own advantages and limitations. While some methods rely on dividing cells and thus bias aneuploidy rates to that population, other, more unbiased methods can only detect the average aneuploidy rates in a cell population, cloaking cell-to-cell heterogeneity. Furthermore, some techniques are more prone to technical artefacts, which can result in over- or underestimation of aneuploidy rates. In this review, we provide an overview of several "traditional" karyotyping methods as well as the latest high throughput next generation sequencing karyotyping protocols with their respective advantages and disadvantages.

Chronic central serous chorioretinopathy is associated with genetic variants implicated in age-related macular degeneration.

PURPOSE: In this study, single nucleotide polymorphisms (SNPs) at 19 loci, previously associated with age-related macular degeneration (AMD), were systematically tested for association in patients with chronic central serous chorioretinopathy (cCSC). In addition, we evaluated the effect of detailed phenotyping on these genetic associations. DESIGN: Case-control study. PARTICIPANTS: We included 292 cCSC patients, 1147 AMD patients, and 1311 control individuals. METHODS: We genotyped SNPs at 19 AMD-associated loci and 6 additional SNPs at the complement factor H (CFH) locus. Phenotyping of all patients was based on fundoscopy, spectral-domain optical coherence tomography, fluorescein angiography (FA), and indocyanine green angiography. MAIN OUTCOME MEASURES: We measured the allele frequencies of 25 AMD-associated SNPs and CFH haplotype frequencies in patients with cCSC and the effect of phenotypic subdivision of cCSC on genetic associations. RESULTS: One SNP in ARMS2 (rs10490924) was significant after Bonferroni correction (Punadjusted=0.002; odds ratio [OR]=0.64). The SNPs at 3 other AMD loci (CFH, TNFRSF10A, ADAMTS9) showed a trend toward association with typical cCSC. Further analysis of the CFH locus identified 2 SNPs that significantly conferred increased risk for cCSC and 1 that was protective. The CFH-H3 haplotype was also found to be protective (P=0.01; OR=0.54). Using multimodal imaging, 197 patients were classified as having typical cCSC, 52 patients had unilateral abnormalities on FA that were otherwise typical of cCSC, and 43 patients had a clinical picture that could be compatible with cCSC, but with features that could also indicate other macular diseases. Significant differences of the minor allele frequencies of the tested SNPs were observed between these 3 phenotypic subgroups. CONCLUSIONS: Chronic CSC is associated with genetic variants in ARMS2 and CFH, indicating a genetic and pathophysiologic overlap between cCSC and AMD. Intriguingly, alleles in ARMS2 and CFH that confer risk of AMD may be protective for cCSC, and alleles in CFH that are protective for AMD confer risk for cCSC. Significant differences in allele frequencies were found among the phenotypic subgroups for several SNPs, illustrating the importance of correct clinical classification.

CINcere Modelling: What Have Mouse Models for Chromosome Instability Taught Us?

Chromosomal instability (CIN) is a process leading to errors in chromosome segregation and results in aneuploidy, a state in which cells have an abnormal number of chromosomes. CIN is a hallmark of cancer, and furthermore linked to ageing and age-related diseases such as Alzheimer's. Various mouse models have been developed to explore the role of CIN in ageing and cancer. While these models reveal only a modest contribution of CIN to the initiation of cancer, they also clearly show that CIN is a powerful accelerator of cancer in a predisposed background. Other than cancer, CIN also appears to provoke premature ageing in some of the CIN models. In this review, we discuss the phenotypes of the various available mouse models, what we have learnt so far, and importantly, also which questions still need to be addressed.

Identification of novel CYP1B1 gene mutations in patients with primary congenital and primary open-angle glaucoma.

BACKGROUND: CYP1B1 is the most commonly mutated gene in primary congenital glaucoma (PCG), and mutations have also been identified in primary open-angle glaucoma (POAG). This study was undertaken to describe mutations in CYP1B1 in patients and families with PCG and POAG from Pakistan. DESIGN: Case-control series. PARTICIPANTS: Forty families, 190 sporadic POAG cases and 140 controls from Pakistan. METHODS: Patients and healthy individuals of one consanguineous Pakistani family were genotyped with high-resolution single nucleotide polymorphism microarrays. Homozygosity mapping was performed using HomozygosityMapper. Direct sequencing of CYP1B1 gene was performed in probands of the families, sporadic POAG cases and control individuals. MAIN OUTCOME MEASURES: Mutations in the CYP1B1 gene in PCG and POAG patients. RESULTS: Homozygosity mapping in a consanguineous Pakistani family revealed one 11-Mb homozygous region encompassing the CYP1B1 gene. A homozygous CYP1B1 missense mutation (p.Arg390His) was identified in this family. Sequence analysis of CYP1B1 in 39 additional families revealed one known and three novel homozygous mutations in PCG (p.Ala288Pro, p.Asp242Ala, p.Arg355* and p.Arg290Profs*37). In POAG, one novel heterozygous missense mutation (p.Asp316Val) was identified in one family and a previously reported mutation (p.Glu229Lys) was identified in three families. Analysis of CYP1B1 in a panel of 190 sporadic POAG patients revealed three novel heterozygous variants (p.Thr234Lys, p.Ala287Pro and p.Gln362*) and three previously reported heterozygous variants (p.Gly61Glu, p.Glu229Lys and p.Arg368His). The p.Glu229Lys variant was significantly associated with POAG (P = 0.03; odds ratio 2.49). CONCLUSIONS: This study confirms that CYP1B1 mutations are associated with POAG and PCG in the Pakistani population.

Association of known common genetic variants with primary open angle, primary angle closure, and pseudoexfoliation glaucoma in Pakistani cohorts.

PURPOSE: Despite the different etiology of primary open angle glaucoma (POAG), primary angle closure glaucoma (PACG), and pseudoexfoliative glaucoma (PEXG), several studies have suggested that these forms of glaucoma have overlapping genetic risk factors. Therefore, the aim of this study was to evaluate the role of genetic variants recently associated with POAG in different types of glaucoma in Pakistani POAG, PACG, and PEXG patient cohorts. METHODS: Six variants in CDKN2B-AS1 (rs4977756), CDKN2B (rs1063192), ATOH7 (rs1900004), CAV1 (rs4236601), TMCO1 (rs4656461), and SIX1 (rs10483727) were genotyped using TaqMan assays. A total of 513 unrelated patients with glaucoma (268 with POAG, 125 with PACG, and 120 with PEXG) and 233 healthy controls were included in the study. Genotypic and allelic associations were analyzed with a chi-square test. RESULTS: The frequency of the G allele of TMCO1 rs4656461 was significantly lower in the patients with POAG (p=0.003; OR [odds ratio]=0.57), PACG (p=0.009; OR=0.52), and PEXG (p=0.01; OR=0.54) compared to the control individuals. The T allele of ATOH7 rs1900004 was observed less frequently in the patients with PACG (p=0.03; OR=0.69) compared to the control individuals. The A allele of CAV1 rs4236601 was found more frequently in the patients with POAG (p=0.008; OR=1.49) compared to the control individuals. This study demonstrates that the TMCO1 rs4656461 variant is associated with POAG, PACG and PEXG in the Pakistani population. Our study was unable to confirm previous associations reported for variants in CDKN2B-AS1, CDKN2B, and SIX1 with any type of glaucoma. CONCLUSIONS: In conclusion, we found consistent evidence of the significant association of three common variants in TMCO1, ATOH7, and CAV1.

The role of APOBEC3B in lung tumor evolution and targeted cancer therapy resistance.

In this study, the impact of the apolipoprotein B mRNA-editing catalytic subunit-like (APOBEC) enzyme APOBEC3B (A3B) on epidermal growth factor receptor (EGFR)-driven lung cancer was assessed. A3B expression in EGFR mutant (EGFRmut) non-small-cell lung cancer (NSCLC) mouse models constrained tumorigenesis, while A3B expression in tumors treated with EGFR-targeted cancer therapy was associated with treatment resistance. Analyses of human NSCLC models treated with EGFR-targeted therapy showed upregulation of A3B and revealed therapy-induced activation of nuclear factor kappa B (NF-κB) as an inducer of A3B expression. Significantly reduced viability was observed with A3B deficiency, and A3B was required for the enrichment of APOBEC mutation signatures, in targeted therapy-treated human NSCLC preclinical models. Upregulation of A3B was confirmed in patients with NSCLC treated with EGFR-targeted therapy. This study uncovers the multifaceted roles of A3B in NSCLC and identifies A3B as a potential target for more durable responses to targeted cancer therapy.