Moderate alcohol consumption as risk factor for adverse brain outcomes and cognitive decline: longitudinal cohort study.

Objectives To investigate whether moderate alcohol consumption has a favourable or adverse association or no association with brain structure and function.Design Observational cohort study with weekly alcohol intake and cognitive performance measured repeatedly over 30 years (1985-2015). Multimodal magnetic resonance imaging (MRI) was performed at study endpoint (2012-15).Setting Community dwelling adults enrolled in the Whitehall II cohort based in the UK (the Whitehall II imaging substudy).Participants 550 men and women with mean age 43.0 (SD 5.4) at study baseline, none were "alcohol dependent" according to the CAGE screening questionnaire, and all safe to undergo MRI of the brain at follow-up. Twenty three were excluded because of incomplete or poor quality imaging data or gross structural abnormality (such as a brain cyst) or incomplete alcohol use, sociodemographic, health, or cognitive data.Main outcome measures Structural brain measures included hippocampal atrophy, grey matter density, and white matter microstructure. Functional measures included cognitive decline over the study and cross sectional cognitive performance at the time of scanning.Results Higher alcohol consumption over the 30 year follow-up was associated with increased odds of hippocampal atrophy in a dose dependent fashion. While those consuming over 30 units a week were at the highest risk compared with abstainers (odds ratio 5.8, 95% confidence interval 1.8 to 18.6; P≤0.001), even those drinking moderately (14-21 units/week) had three times the odds of right sided hippocampal atrophy (3.4, 1.4 to 8.1; P=0.007). There was no protective effect of light drinking (1-<7 units/week) over abstinence. Higher alcohol use was also associated with differences in corpus callosum microstructure and faster decline in lexical fluency. No association was found with cross sectional cognitive performance or longitudinal changes in semantic fluency or word recall.Conclusions Alcohol consumption, even at moderate levels, is associated with adverse brain outcomes including hippocampal atrophy. These results support the recent reduction in alcohol guidance in the UK and question the current limits recommended in the US

A hypomorphic PALB2 allele gives rise to an unusual form of FA-N associated with lymphoid tumour development

Patients with biallelic truncating mutations in PALB2 have a severe form of Fanconi anaemia (FA-N), with a predisposition for developing embryonal-type tumours in infancy. Here we describe two unusual patients from a single family, carrying biallelic PALB2 mutations, one truncating, c.1676_1677delAAinsG;(p.Gln559ArgfsTer2), and the second, c.2586+1G>A; p.Thr839_Lys862del resulting in an in frame skip of exon 6 (24 amino acids). Strikingly, the affected individuals did not exhibit the severe developmental defects typical of FA-N patients and initially presented with B cell non-Hodgkin lymphoma. The expressed p.Thr839_Lys862del mutant PALB2 protein retained the ability to interact with BRCA2, previously unreported in FA-N patients. There was also a large increased chromosomal radiosensitivity following irradiation in G2 and increased sensitivity to mitomycin C. Although patient cells were unable to form Rad51 foci following exposure to either DNA damaging agent, U2OS cells, in which the mutant PALB2 with in frame skip of exon 6 was induced, did show recruitment of Rad51 to foci following damage. We conclude that a very mild form of FA-N exists arising from a hypomorphic PALB2 allele

Absence of Rad51 foci in fibroblasts from patients II-4 & II-5.

<p>Following exposure to either 3Gy IR or 50ngml^-1 mitomycin C no Rad51 foci were detected in fibroblasts from II-4 and II-5. γH2AX foci indicate the presence of DNA DSB. The residual foci at 24h following IR are consistent with a DNA repair deficiency.</p

Co-immunoprecipitation of BRCA2 with mutant PALB2 proteins.

<p>A) Cultures of U2OS cell lines that inducibly express FLAG-tagged WT or mutant PALB2 proteins were transfected with PALB2 siRNA and induced to express the tagged PALB2 proteins by treatment with doxycycline. Samples were incubated with anti-FLAG antibody and immunoprecipitates electrophoresed and blotted serially with mouse anti-BRCA2 (top panel), rabbit anti-PALB2 246A antibody (middle panel) and then rabbit anti-PALB2 247A antibody (bottom panel). 246A antibody was not stripped before blotting with 247A. A band approximating to 460kDa and detected with anti-BRCA2 antibody was immunoprecipitated with FLAG-tagged WT, T839_K862del (exon 6 deleted) and PALB2 revertant (Exon4delRev) proteins, in those samples that were incubated with anti-FLAG antibody. Anti-PALB2 antibodies identified FLAG-tagged WT protein at ~170kDa as expected (lane 1), with the exon 6 deleted protein appearing to be slightly smaller (lane 2). The FLAG-tagged Q559RfsTer2 and Y551Ter proteins were found at their expected sizes (middle panel). The FLAG-tagged PALB2 revertant protein (Exon4delRev) was evident with Ab 247A in bottom panel. B) Samples of the NETN lysates were taken as ‘input’ samples before immunoprecipitation, and 5% of total lysate immunoprecipitated was subjected to PAGE and Western blotting. The filters were blotted serially with different antibodies as in A. These blots show BRCA2 in all samples before immunoprecipitation. Anti-PALB2 antibodies identify FLAG-tagged PALB2 protein in all WT and mutant expressing cell lines. The aprataxin protein indicates the similarity of loading.</p

Recruitment of Rad51 foci to sites of damage in U2OS cells expressing FLAG-tagged exon 6 deleted p.T839_K862del mutant PALB2.

<p>Recruitment of Rad51 foci to sites of damage in U2OS cells that expressed FLAG-tagged WT, revertant PALB2 lacking exon 4 (Exon4delRev) or exon 6 deleted T839_K862del mutant PALB2, but not in cells expressing Y551Ter (present in the EUFA1341 cell line) or the Q559RfsTer2 PALB2, following depletion of endogenous PALB2 and MMC or IR exposure.</p

Analysis of expression of PALB2 protein in a family with previously unrecognised FA-N patients.

<p>A) Western blot showing loss of full length PALB2 in the affected patient II-5 (Ab 301-246A)(lane 3). However, there is the presence of a faint signal for a slightly smaller protein, probably corresponding to the exon six deletion mutation. Also present in the patient lane is a much smaller band at 90kDa. Lane 4 is lysate of cells from a FA-D1 (biallelic mutation of BRCA2) patient. B) All relatives of the patient II-5 show bands for WT PALB2. Also found in the lanes for the mother (I-2) and sibling II-3, (lanes 3 and 7) is the smaller dense 90kD protein band consistent with I-2 and II-3 carrying the c.1676_1677delAAinsG mutation. In addition, a less dense band of the same size, 90kD, appears as a consequence of the antibody cross reacting with another protein. This cross reacting band is visible in the normal control (both left and right), I-1, II-7 and II-6. The father carries the c.2586+1G>A, although the exon 6 deleted protein cannot be seen against the background of the normal full length protein from his WT allele.</p

Clinical and cellular features of affected siblings.

<p>A) Pedigree of family showing affected siblings. It is not known whether sibling II-2 also had the same syndrome as II-4 and II-5. B) Colony forming assay following exposure of fibroblasts to ¹³⁷Cs γ-rays, C) Colony forming assay following exposure of fibroblasts to Mitomycin C. The proportion (%) of surviving colonies was plotted against dose. The assay was repeated at least three times for each cell strain. Error bars show the s.e.m of survival at each dose.</p

<i>PALB2</i> mutations in the affected family.

<p>Electropherograms of <i>PALB2</i> mutations in DNA in the affected siblings. A) Top panel, c.1676_1677delAAinsG in genomic DNA; lower panel, <i>wt</i> sequence at that position. B) Top panel mutant c.2586+1G>A in genomic DNA; lower panel, <i>wt</i> sequence at that position. C) cDNA sequencing showing that the c.2586+1G>A mutation causes exon 6 to be omitted during RNA splicing. D) Diagramatic representation of the PALB2 protein and predicted effects of mutations. Diagram of PALB2 showing the epitopes to which the two anti-PALB2 antibodies bind and also the locations of the mutations in the patients. Note that because the epitope for the PALB2 antibody 246A is in exon 4, the exon revertant protein (Exon4delRev) deletion mutant cannot be detected with this antibody. Similarly, because the Y551Ter and Q559RfsTer2 mutants truncate at residues 551 and 559 respectively, they do not possess the epitopes for the 247A antibody. Only wild type and T839_K862del exon six deletion PALB2 are detectable with both antibodies.</p