Maria Franchina

Deep sequencing of uveal melanoma identifies a recurrent mutation in PLCB4.

Next generation sequencing of uveal melanoma (UM) samples has identified a number of recurrent oncogenic or loss-of-function mutations in key driver genes including: GNAQ, GNA11, EIF1AX, SF3B1 and BAP1. To search for additional driver mutations in this tumor type we carried out whole-genome or whole-exome sequencing of 28 tumors or primary cell lines. These samples have a low mutation burden, with a mean of 10.6 protein changing mutations per sample (range 0 to 53). As expected for these sun-shielded melanomas the mutation spectrum was not consistent with an ultraviolet radiation signature, instead, a BRCA mutation signature predominated. In addition to mutations in the known UM driver genes, we found a recurrent mutation in PLCB4 (c.G1888T, p.D630Y, NM_000933), which was validated using Sanger sequencing. The identical mutation was also found in published UM sequence data (1 of 56 tumors), supporting its role as a novel driver mutation in UM. PLCB4 p.D630Y mutations are mutually exclusive with mutations in GNA11 and GNAQ, consistent with PLCB4 being the canonical downstream target of the former gene products. Taken together these data suggest that the PLCB4 hotspot mutation is similarly a gain-of-function mutation leading to activation of the same signaling pathway, promoting UM tumorigenesis.

Meta-analysis of human methylation data for evidence of sex-specific autosomal patterns

BackgroundSeveral individual studies have suggested that autosomal CpG methylation differs by sex both in terms of individual CpG sites and global autosomal CpG methylation. However, these findings have been inconsistent and plagued by spurious associations due to the cross reactivity of CpG probes on commercial microarrays. We collectively analysed 76 published studies (n = 6,795) for sex-associated differences in both autosomal and sex chromosome CpG sites.ResultsOverall autosomal methylation profiles varied substantially by study, and we encountered substantial batch effects. We accounted for these by conducting random effects meta-analysis for individual autosomal CpG methylation associations. After excluding non-specific probes, we found 184 autosomal CpG sites differentially methylated by sex after correction for multiple testing. In line with previous studies, average beta differences were small. Many of the most significantly associated CpG probes were new. Of note was differential CpG methylation in the promoters of genes thought to be involved in spermatogenesis and male fertility, such as SLC9A2, SPESP1, CRISP2, and NUPL1. Pathway analysis revealed overrepresentation of genes differentially methylated by sex in several broad Gene Ontology biological processes, including RNA splicing and DNA repair.ConclusionsThis study represents a comprehensive analysis of sex-specific methylation patterns. We demonstrate the existence of sex-specific methylation profiles and report a large number of novel DNA methylation differences in autosomal CpG sites between sexes.

Hypomethylation of the IL17RC Promoter in Peripheral Blood Leukocytes Is Not A Hallmark of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a leading cause of visual impairment worldwide. Aberrant DNA methylation within the promoter of IL17RC in peripheral blood mononuclear cells has recently been reported in AMD. To validate this association, we examined DNA methylation of the IL17RC promoter in peripheral blood. First, we used Illumina Human Methylation450 Bead Arrays, a widely accepted platform for measuring global DNA methylation. Second, methylation status at multiple sites within the IL17RC promoter was determined by bisulfite pyrosequencing in two cohorts. Third, a methylation-sensitive quantitative PCR-based assay was performed on a subset of samples. In contrast to previous findings, we did not find evidence of differential methylation between AMD cases and age-matched controls. We conclude that hypomethylation within the IL17RC gene promoter in peripheral blood is not suitable for use as a clinical biomarker of AMD. This study highlights the need for considerable replication of epigenetic association studies prior to clinical application.

Evidence that general genomic hypomethylation and focal hypermethylation are two independent molecular events of non-Hodgkin's lymphoma.

Changes in the DNA methylation profile, including general genomic hypomethylation and regional hypermethylation, have been shown to coexist in many neoplastic tissues. However, the relationship between, and significance of, these different forms of DNA methylation dysregulation in disease onset, progression, or maintenance remains unclear. Previously, our work has shown that the CpG dinucleotide-rich gene Myf-3 is hypermethylated in most cases of malignant lymphoproliferative disease (LPD). However, it is unknown whether malignant transformation of lymphoid cells is associated with general genomic hypomethylation and whether regional hypermethylation is restricted to CpG islands. The relationship between the status of general genomic methylation and the methylation of CpG and non-CpG islands can be clearly investigated in DNA from tumors of patients suffering malignant LPD, as monoclonalilty of malignant cells in LPD can be readily confirmed. In this study, the relationships between the methylation status of a region of the PAX7 paired box, which is not contained within a CpG island, general genomic hypomethylation, and the methylation status of Myf-3 was examined in 24 cases of LPD. Results revealed that hypermethylation of the PAX7 paired box is strongly associated with hypermethylation of Mvf-3, indicating the abnormal hypermethylating activity in malignant lymphoid cells does not specifically target CpG islands. Further, general genomic hypomethylation was shown to be associated with malignant LPD but not with regional hypermethylation, indicating that the mechanisms responsible for the generation of each of these disturbed DNA methylation phenotypes act independently as one of a number of permissive but not essential steps in the malignant transformation of lymphoid cells.

Swimming goggle wear is not associated with an increased prevalence of glaucoma.

Background/aims Previous studies have demonstrated a small but significant transient increase in intraocular pressure (IOP) in individuals wearing certain types of swimming goggles. These findings suggested that wearing goggles could represent a significant risk factor for developing and/or worsening of glaucoma in people who swim regularly. The aim of this study was to determine if glaucoma prevalence is increased among adult swimmers. Methods A comprehensive ocular examination was performed on 231 members of local swimming clubs and 118 non-swimmers. IOP was measured using iCARE tonometry and visual field testing was performed using Humphrey SITA fast 24–2. Retinal nerve fibre layer thickness was assessed using spectral domain optical coherence tomography. Results Based on measurements of IOP and visual fields, we did not detect any new cases of glaucoma in our cohort of frequent swimmers. Similarly, we found no difference in the thickness of the retinal nerve fibre layer between swimmers and non-swimmers; the mean right global thickness (GT) was 94.0 μm (IQR 88.0, 100.3) vs 93.0 μm (IQR 89.0, 101.0), respectively (p=0.976), and the median left GT was 93.7 μm (IQR 88.0, 101) in both groups (p=0.799). Conclusions These findings suggest that frequently wearing swim goggles does not lead to an increased risk of glaucoma over time in adults.

DNA methylation landscape of ocular tissue relative to matched peripheral blood

Epigenetic variation is implicated in a range of non-communicable diseases, including those of the eye. However, investigating the role of epigenetic variation in central tissues, such as eye or brain, remains problematic and peripheral tissues are often used as surrogates. In this study, matched human blood and eye tissue (n = 8) were obtained post-mortem and DNA methylation profiling performed on blood, neurosensory retina, retinal pigment epithelium (RPE)/choroid and optic nerve tissue using the Illumina Infinium HumanMethylation450 platform. Unsupervised clustering and principal components analysis revealed tissue of origin as the main driver of methylation variation. Despite this, there was a strong correlation of methylation profiles between tissues with >255,000 CpG sites found to have similar methylation levels. An additional ~16,000 show similarity across ocular tissues only. A small proportion of probes showing inter-individual variation in blood co-varied with eye tissues within individuals, however much of this variation may be genetically driven. An improved understanding of the epigenetic landscape of the eye will have important implications for understanding eye disease. Despite a generally high correlation irrespective of origin, tissue type is the major driver of methylation variation, with only limited covariation between blood and any specific ocular tissue.

Allele-specific variation in the gene copy number of human cytosine 5-methyltransferase.

Previously, we have identified two alternate allelic forms of cytosine 5-methyltransferase, 5-MT I and 5-MT II, specified by polymorphic fragments of 1.5 and 1.1 kb, respectively. In the presence study, a 0.8-kb genomic probe was prepared which was confirmed to be included within the polymorphic fragments. The 0.8-kb probe hybridised with greater intensity to the 1.1-kb fragment than the 1.5-kb fragment. Densitometric analysis indicated that there is 1 copy of 5-MT associated with 5-MT I, whereas there may be 1–4 copies of the gene associated with the 5-MT II allele. Segregation studies demonstrated that the multiple copies of 5-MT II are inherited in a Mendelian fashion. These results allow novel approaches to investigating the underlying mechanisms of cytosine methylation and gene duplication.

Myopia and skin cancer are inversely correlated: results of the Busselton Healthy Ageing Study

TO THE EDITOR: Kerry Breen has hit the nail squarely on the head.1 The Medical Board of Australia should heed Peter Ustinov’s advice, “Don’t just do something, stand there!” And Oliver Cromwell’s plea, “think it possible you may be mistaken”. And mine: “Please think again!” It is easier (more effective and cost-effective) to move the tail of the bell curve to the right than to shift the bulk of it to the right. While every doctor’s performance could possibly be improved, there is little point and dubious costeffectiveness in improving doctors on the right of the bell curve from “good” or “very good” to “a little bit better”, when the point of the exercise is to improve the game of poor performers. Alison Reid, former Medical Director of the New South Wales Medical Board, has suggested that the focus could readily be directed at doctors about whom there are a number of complaints, and at those in the groups known to be at risk: those with an impairment, the aged, and the professionally and geographically isolated.2 It is among these groups that the evidence shows that poor performance is most likely to be found. It would seem to me that assessment of doctors in these groups, in their practices, as is done by the Royal Australasian College of Physicians in its clinical audit program,3 would be more productive and cost-effective than wholesale assessment of all practising doctors.

Novel Nucleotide Substitutions within the Coding Region of DNMT2 Are in Strong Linkage Disequilibrium in Caucasians and Japanese

Investigations into mechanims by which cytosine methylation may be genetically controlled have led to the identification of single nucleotide polymorphisms within the coding region of DNMT2 that are conserved in different ethnic groups. The DNMT2 I allele includes a G at nucleotide position 104 of exon 2 and a C at position 50 of exon 4. The alternative allele, DNMT2 II, includes an A and T, respectively, at these positions. G was never found in the absence of C and vice versa and A was never found in the absence of T and vice versa. The gene products of DNMT2 I and DNMT2 II differ by the inclusion of a histidine or tyrosine residue at the position specified by codon 101. This amino acid substitution alters the amino acid composition of a conserved methylating enzyme motif shown to be involved in S-adenosylmethionine binding in M.HhaI, a bacterial methyltransferase that is almost identical to DNMT2 in size and structure. Demonstration of strong linkage disequilibrium between the nucleotide substitutions associated with each DNMT2 allele provides valuable tools for the investigation of molecular genetic mechanisms of evolution and speciation.

Ferritin light chain gene mutation in a large Australian family with hereditary hyperferritinemia-cataract syndrome

ABSTRACT Background: Hereditary hyperferritinemia-cataract syndrome (HHCS) is an autosomal dominant Mendelian disorder characterized by early onset cataracts and elevated levels of serum ferritin in the absence of iron overload. Numerous mutations associated with the development of HHCS have been reported in the 5′ non-coding region of the ferritin light chain (FTL) gene in family studies. We present an FTL mutation in an Australian family with 10 HHCS-affected members spanning three generations. Materials and methods: Blood and saliva samples were collected from affected and unaffected family members and DNA was extracted using commercially available kits (Qiagen). The complete sequencing of the iron-responsive element (IRE) of the FTL gene was analyzed using bi-directional genomic sequencing. Results: A heterozygous single nucleotide substitution (c.-167 C>T) was identified in the proband and five affected family members (logarithm of the odds score [Z] = 3.61, recombination distance [θ = 0]). All affected individuals had previously been found to have high ferritin levels and early onset cataracts. Conclusion: This is the first Australian report of the c.-167 C>T mutation in a large family with multiple affected individuals. This finding raises the possibility that identification of HHCS mutations may be an effective means of disease detection and may aid in facilitating appropriate genetic counseling.