Clinical consequences of age-related skin barrier dysfunction. Part II. Approach to barrier dysfunction in the geriatric population.

Aging is associated with significant changes to skin structure and function which lead to distinct clinical needs in geriatric dermatology patients. By understanding the relationship between altered skin function with aging and the clinical implications for geriatric dermatology patients, dermatologists can guide patients in developing a preventive regimen and managing age-related skin pathology. The second article of this 2-part continuing medical education series reviews the most common conditions affecting geriatric dermatology patients and specific treatment considerations with a focus on the impact of age-related skin barrier dysfunction.

Clinical consequences of age-related skin barrier dysfunction. Part I. Structural, molecular and physiologic changes with cutaneous aging.

Aging is associated with significant changes to skin structure and function. As the United States population ages, dermatologists are increasingly presented with the clinical consequences of these changes. Understanding the biology of aging skin allows dermatologists to best guide patients towards proactive treatment of age-related skin disease. The first article of this 2-part continuing medical education series reviews the structural, molecular and functional changes associated with skin aging.

Detection of viral gene expression in risk-stratified biopsies reveals no active HPV in cutaneous squamous cell carcinoma.

BACKGROUND: Human papillomavirus (HPV) infection is known to promote the development of mucosal squamous cell carcinoma (mSCC), including pathologically high-grade lesions, but its role in cutaneous squamous cell carcinoma (cuSCC) remains unclear, particularly in lesions that are considered high risk. OBJECTIVE: We aimed to determine whether enhanced HPV transcriptional activity can be detected in high-risk cuSCC samples compared with low-grade SCC samples or normal skin. METHODS: We performed RNA sequencing of cuSCC across 23 risk-stratified skin lesions. A subset of samples was tested for the presence of HPV DNA. High-quality, non-human reads from each sample group were used for viral analysis using Microbiome Coverage Profiler. RESULTS: None of the samples analysed had detectable expression of HPV RNA, while 64% of samples tested positive for HPV DNA. All samples were found to have expression of human endogenous retrovirus, and multiple samples showed expression of other viruses. CONCLUSIONS: Viral and prophage gene expression can be monitored in cuSCC or normal skin biopsies, yet no sample in our study showed evidence of active HPV gene expression despite evidence of HPV genome presence. This suggests HPV transcription does not play a role in differentiating high-risk cuSCCs from low-risk cuSCCs or normal skin.

Mitochondrial DNA deletion mutations increase exponentially with age in human skeletal muscle.

BACKGROUND: Mitochondrial DNA (mtDNA) deletion mutations lead to electron transport chain-deficient cells and age-induced cell loss in multiple tissues and mammalian species. Accurate quantitation of somatic mtDNA deletion mutations could serve as an index of age-induced cell loss. Quantitation of mtDNA deletion molecules is confounded by their low abundance in tissue homogenates, the diversity of deletion breakpoints, stochastic accumulation in single cells, and mosaic distribution between cells. AIMS: Translate a pre-clinical assay to quantitate mtDNA deletions for use in human DNA samples, with technical and biological validation, and test this assay on human subjects of different ages. METHODS: We developed and validated a high-throughput droplet digital PCR assay that quantitates human mtDNA deletion frequency. RESULTS: Analysis of human quadriceps muscle samples from 14 male subjects demonstrated that mtDNA deletion frequency increases exponentially with age-on average, a 98-fold increase from age 20-80. Sequence analysis of amplification products confirmed the specificity of the assay for human mtDNA deletion breakpoints. Titration of synthetic mutation mixtures found a lower limit of detection of at least 0.6 parts per million. Using muscle DNA from 6-month-old mtDNA mutator mice, we measured a 6.4-fold increase in mtDNA deletion frequency (i.e., compared to wild-type mice), biologically validating the approach. DISCUSSION/CONCLUSIONS: The exponential increase in mtDNA deletion frequency is concomitant with the known muscle fiber loss and accelerating mortality that occurs with age. The improved assay permits the accurate and sensitive quantification of deletion mutations from DNA samples and is sufficient to measure changes in mtDNA deletion mutation frequency in healthy individuals across the lifespan and, therefore, patients with suspected mitochondrial diseases.

Alopecia areata in a patient with WNT10A heterozygous ectodermal dysplasia.

We report a case of a patient with ectodermal dysplasia attributed to a heterozygous 321C>A mutation in WNT10A who developed overlying autoimmune mediated hair loss. To the best of our knowledge this is the first reported case of alopecia areata in a patient with WNT10A heterozygous ectodermal dysplasia. This case highlights the importance of considering multiple pathways of hair loss in patients with underlying genetic defects and raises the possibility of a shared genetic predisposition.

Blood Leukocyte DNA Methylation Predicts Risk of Future Myocardial Infarction and Coronary Heart Disease.

BACKGROUND: DNA methylation is implicated in coronary heart disease (CHD), but current evidence is based on small, cross-sectional studies. We examined blood DNA methylation in relation to incident CHD across multiple prospective cohorts. METHODS: Nine population-based cohorts from the United States and Europe profiled epigenome-wide blood leukocyte DNA methylation using the Illumina Infinium 450k microarray, and prospectively ascertained CHD events including coronary insufficiency/unstable angina, recognized myocardial infarction, coronary revascularization, and coronary death. Cohorts conducted race-specific analyses adjusted for age, sex, smoking, education, body mass index, blood cell type proportions, and technical variables. We conducted fixed-effect meta-analyses across cohorts. RESULTS: Among 11 461 individuals (mean age 64 years, 67% women, 35% African American) free of CHD at baseline, 1895 developed CHD during a mean follow-up of 11.2 years. Methylation levels at 52 CpG (cytosine-phosphate-guanine) sites were associated with incident CHD or myocardial infarction (false discovery rate<0.05). These CpGs map to genes with key roles in calcium regulation (ATP2B2, CASR, GUCA1B, HPCAL1), and genes identified in genome- and epigenome-wide studies of serum calcium (CASR), serum calcium-related risk of CHD (CASR), coronary artery calcified plaque (PTPRN2), and kidney function (CDH23, HPCAL1), among others. Mendelian randomization analyses supported a causal effect of DNA methylation on incident CHD; these CpGs map to active regulatory regions proximal to long non-coding RNA transcripts. CONCLUSION: Methylation of blood-derived DNA is associated with risk of future CHD across diverse populations and may serve as an informative tool for gaining further insight on the development of CHD.

GeMes, clusters of DNA methylation under genetic control, can inform genetic and epigenetic analysis of disease.

Epigenetic marks such as DNA methylation have generated great interest in the study of human disease. However, studies of DNA methylation have not established population-epigenetics principles to guide design, efficient statistics, or interpretation. Here, we show that the clustering of correlated DNA methylation at CpGs was similar to that of linkage-disequilibrium (LD) correlation in genetic SNP variation but for much shorter distances. Some clustering of methylated CpGs appeared to be genetically driven. Further, a set of correlated methylated CpGs related to a single SNP-based LD block was not always physically contiguous-segments of uncorrelated methylation as long as 300 kb could be interspersed in the cluster. Thus, we denoted these sets of correlated CpGs as GeMes, defined as potentially noncontiguous methylation clusters under the control of one or more methylation quantitative trait loci. This type of correlated methylation structure has implications for both biological functions of DNA methylation and for the design, analysis, and interpretation of epigenome-wide association studies.

Nanopore sequencing identifies a higher frequency and expanded spectrum of mitochondrial DNA deletion mutations in human aging.

Mitochondrial DNA (mtDNA) deletion mutations cause many human diseases and are linked to age-induced mitochondrial dysfunction. Mapping the mutation spectrum and quantifying mtDNA deletion mutation frequency is challenging with next-generation sequencing methods. We hypothesized that long-read sequencing of human mtDNA across the lifespan would detect a broader spectrum of mtDNA rearrangements and provide a more accurate measurement of their frequency. We employed nanopore Cas9-targeted sequencing (nCATS) to map and quantitate mtDNA deletion mutations and develop analyses that are fit-for-purpose. We analyzed total DNA from vastus lateralis muscle in 15 males ranging from 20 to 81 years of age and substantia nigra from three 20-year-old and three 79-year-old men. We found that mtDNA deletion mutations detected by nCATS increased exponentially with age and mapped to a wider region of the mitochondrial genome than previously reported. Using simulated data, we observed that large deletions are often reported as chimeric alignments. To address this, we developed two algorithms for deletion identification which yield consistent deletion mapping and identify both previously reported and novel mtDNA deletion breakpoints. The identified mtDNA deletion frequency measured by nCATS correlates strongly with chronological age and predicts the deletion frequency as measured by digital PCR approaches. In substantia nigra, we observed a similar frequency of age-related mtDNA deletions to those observed in muscle samples, but noted a distinct spectrum of deletion breakpoints. NCATS-mtDNA sequencing allows the identification of mtDNA deletions on a single-molecule level, characterizing the strong relationship between mtDNA deletion frequency and chronological aging.

A Genetically Heterogeneous Rat Model with Divergent Mitochondrial Genomes.

We generated a genetically heterogenous rat model by a 4-way cross strategy using 4 inbred strains (Brown Norway [BN], Fischer 344 [F344], Lewis [LEW], and Wistar Kyoto [KY]) to provide investigators with a highly genetically diverse rat model from commercially available inbred rats. We made reciprocal crosses between males and females from the 2 F1 hybrids to generate genetically heterogeneous rats with mitochondrial genomes from either the BN (OKC-HETB, a.k.a "B" genotype) or WKY (OKC-HETW a.k.a "W" genotype) parental strains. These two mitochondrial genomes differ at 94 nucleotides, more akin to human mitochondrial genome diversity than that available in classical laboratory mouse strains. Body weights of the B and W genotypes were similar. However, mitochondrial genotype antagonistically affected grip strength and treadmill endurance in females only. In addition, mitochondrial genotype significantly affected multiple responses to a high-fat diet (HFD) and treatment with 17α-estradiol. Contrary to findings in mice in which males only are affected by 17α-estradiol supplementation, female rats fed a HFD beneficially responded to 17α-estradiol treatment as evidenced by declines in body mass, adiposity, and liver mass. Male rats, by contrast, differed in a mitochondrial genotype-specific manner, with only B males responding to 17α-estradiol treatment. Mitochondrial genotype and sex differences were also observed in features of brain-specific antioxidant response to a HFD and 17α-estradiol as shown by hippocampal levels of Sod2 acetylation, JNK, and FoxO3a. These results emphasize the importance of mitochondrial genotype in assessing responses to putative interventions in aging processes.

Long read mitochondrial genome sequencing using Cas9-guided adaptor ligation.

The mitochondrial genome (mtDNA) is an important source of disease-causing genetic variability, but existing sequencing methods limit understanding, precluding phased measurement of mutations and clear detection of large sporadic deletions. We adapted a method for amplification-free sequence enrichment using Cas9 cleavage to obtain full length nanopore reads of mtDNA. We then utilized the long reads to phase mutations in a patient with an mtDNA-linked syndrome and demonstrated that this method can map age-induced mtDNA deletions. We believe this method will offer deeper insight into our understanding of mtDNA variation.

Pressure-Driven Mitochondrial Transfer Pipeline Generates Mammalian Cells of Desired Genetic Combinations and Fates.

Generating mammalian cells with desired mitochondrial DNA (mtDNA) sequences is enabling for studies of mitochondria, disease modeling, and potential regenerative therapies. MitoPunch, a high-throughput mitochondrial transfer device, produces cells with specific mtDNA-nuclear DNA (nDNA) combinations by transferring isolated mitochondria from mouse or human cells into primary or immortal mtDNA-deficient (ρ0) cells. Stable isolated mitochondrial recipient (SIMR) cells isolated in restrictive media permanently retain donor mtDNA and reacquire respiration. However, SIMR fibroblasts maintain a ρ0-like cell metabolome and transcriptome despite growth in restrictive media. We reprogrammed non-immortal SIMR fibroblasts into induced pluripotent stem cells (iPSCs) with subsequent differentiation into diverse functional cell types, including mesenchymal stem cells (MSCs), adipocytes, osteoblasts, and chondrocytes. Remarkably, after reprogramming and differentiation, SIMR fibroblasts molecularly and phenotypically resemble unmanipulated control fibroblasts carried through the same protocol. Thus, our MitoPunch "pipeline" enables the production of SIMR cells with unique mtDNA-nDNA combinations for additional studies and applications in multiple cell types.

Two cases of alopecia areata treated with ruxolitinib: a discussion of ideal dosing and laboratory monitoring.

BACKGROUND: Alopecia areata is a relatively common condition affecting patients seen in community dermatology clinics. A 2014 study implicated the JAK1/JAK2 inhibitor, ruxolitinib in short-term treatment of alopecia, however little information exists about the long-term use in otherwise healthy individuals in the community setting. METHODS: A patient with chronic alopecia areata and a patient with acute onset alopecia universalis were treated with oral ruxolitinib for over a year. RESULTS: Both patients experienced sustained, near-complete regrowth without hematologic or other complications after one year of treatment. Oral ruxolitinib effectively and safely treated alopecia in two women. CONCLUSIONS: Ruxolitinib should be considered for cases of unresponsive alopecia in the community.

DNA methylation-based measures of biological age: meta-analysis predicting time to death.

Estimates of biological age based on DNA methylation patterns, often referred to as "epigenetic age", "DNAm age", have been shown to be robust biomarkers of age in humans. We previously demonstrated that independent of chronological age, epigenetic age assessed in blood predicted all-cause mortality in four human cohorts. Here, we expanded our original observation to 13 different cohorts for a total sample size of 13,089 individuals, including three racial/ethnic groups. In addition, we examined whether incorporating information on blood cell composition into the epigenetic age metrics improves their predictive power for mortality. All considered measures of epigenetic age acceleration were predictive of mortality (p≤8.2x10-9), independent of chronological age, even after adjusting for additional risk factors (p<5.4x10-4), and within the racial/ethnic groups that we examined (non-Hispanic whites, Hispanics, African Americans). Epigenetic age estimates that incorporated information on blood cell composition led to the smallest p-values for time to death (p=7.5x10-43). Overall, this study a) strengthens the evidence that epigenetic age predicts all-cause mortality above and beyond chronological age and traditional risk factors, and b) demonstrates that epigenetic age estimates that incorporate information on blood cell counts lead to highly significant associations with all-cause mortality.

DNA methylation is stable during replication and cell cycle arrest.

DNA methylation is an epigenetic modification with important functions in development. Large-scale loss of DNA methylation is a hallmark of cancer. Recent work has identified large genomic blocks of hypomethylation associated with cancer, EBV transformation and replicative senescence, all of which change the proportion of actively proliferating cells within the population measured. We asked if replication or cell-cycle arrest affects the global levels of methylation or leads to hypomethylated blocks as observed in other settings. We used fluorescence activated cell sorting to isolate primary dermal fibroblasts in G0, G1 and G2 based on DNA content and Ki67 staining. We additionally examined G0 cells arrested by contact inhibition for one week to determine the effects of extended arrest. We analyzed genome wide DNA methylation from sorted cells using whole genome bisulfite sequencing. This analysis demonstrated no global changes or large-scale hypomethylated blocks in any of the examined cell cycle phases, indicating that global levels of methylation are stable with replication and arrest.

Age and sun exposure-related widespread genomic blocks of hypomethylation in nonmalignant skin.

BACKGROUND: Aging and sun exposure are the leading causes of skin cancer. It has been shown that epigenetic changes, such as DNA methylation, are well established mechanisms for cancer, and also have emerging roles in aging and common disease. Here, we directly ask whether DNA methylation is altered following skin aging and/or chronic sun exposure in humans. RESULTS: We compare epidermis and dermis of both sun-protected and sun-exposed skin derived from younger subjects (under 35 years old) and older subjects (over 60 years old), using the Infinium HumanMethylation450 array and whole genome bisulfite sequencing. We observe large blocks of the genome that are hypomethylated in older, sun-exposed epidermal samples, with the degree of hypomethylation associated with clinical measures of photo-aging. We replicate these findings using whole genome bisulfite sequencing, comparing epidermis from an additional set of younger and older subjects. These blocks largely overlap known hypomethylated blocks in colon cancer and we observe that these same regions are similarly hypomethylated in squamous cell carcinoma samples. CONCLUSIONS: These data implicate large scale epigenomic change in mediating the effects of environmental damage with photo-aging.

Model-oriented ocean tomography using higher frequency, bottom-mounted hydrophones.

A tomographic scheme is presented that ingests ocean acoustic measurements into an ocean model using data from bottom-mounted hydrophones. The short distances between source-receiver pairs (1-10 km) means arrival times at frequencies of 8-11 kHz are readily detectable and often distinguishable. The influence of ocean surface motion causes considerable variability in acoustic travel times. Techniques are presented for measuring travel times and removing the variability due to surface waves. An assimilation technique is investigated that uses differences in measured and modeled acoustic travel times to impose corrections on the oceanographic model. Equations relating travel time differences to oceanographic variables are derived, and techniques are presented for estimating the acoustic and ocean model error covariance matrices. One test case using a single source-receiver pair shows that the tomographic information can have an impact on constraining the solution of the ocean circulation model but can also introduce biases in the predictions. A second test case utilizes knowledge of a bias in a model-predicted variable to limit grid cells that are impacted by the tomographic data. In this case, using the tomographic data results in significant improvements in the model predictions without introducing any biases.