Longitudinal change in mitochondrial heteroplasmy exhibits positive selection for deleterious variants.

A common feature of human aging is the acquisition of somatic mutations, and mitochondria are particularly prone to mutation due to their inefficient DNA repair and close proximity to reactive oxygen species, leading to a state of mitochondrial DNA heteroplasmy1,2. Cross-sectional studies have demonstrated that detection of heteroplasmy increases with participant age3, a phenomenon that has been attributed to genetic drift4-7. In this first large-scale longitudinal study, we measured heteroplasmy in two prospective cohorts (combined n=1405) at two timepoints (mean time between visits, 8.6 years), demonstrating that deleterious heteroplasmies were more likely to increase in variant allele fraction (VAF). We further demonstrated that increase in VAF was associated with increased risk of overall mortality. These results challenge the claim that somatic mtDNA mutations arise mainly due to genetic drift, instead demonstrating positive selection for predicted deleterious mutations at the cellular level, despite an negative impact on overall mortality.

Disruption of TUFT1, a Desmosome-Associated Protein, Causes Skin Fragility, Woolly Hair, and Palmoplantar Keratoderma.

Desmosomes are dynamic complex protein structures involved in cellular adhesion. Disruption of these structures by loss-of-function variants in desmosomal genes leads to a variety of skin- and heart-related phenotypes. In this study, we report TUFT1 as a desmosome-associated protein, implicated in epidermal integrity. In two siblings with mild skin fragility, woolly hair, and mild palmoplantar keratoderma but without a cardiac phenotype, we identified a homozygous splice-site variant in the TUFT1 gene, leading to aberrant mRNA splicing and loss of TUFT1 protein. Patients' skin and keratinocytes showed acantholysis, perinuclear retraction of intermediate filaments, and reduced mechanical stress resistance. Immunolabeling and transfection studies showed that TUFT1 is positioned within the desmosome and that its location is dependent on the presence of the desmoplakin carboxy-terminal tail. A Tuft1-knockout mouse model mimicked the patients' phenotypes. Altogether, this study reveals TUFT1 as a desmosome-associated protein, whose absence causes skin fragility, woolly hair, and palmoplantar keratoderma.

Whole Exome Sequencing in Two Southeast Asian Families With Atypical Femur Fractures.

Atypical femur fractures (AFFs) are rare complications of anti-resorptive therapy. Devastating to the affected individual, they pose a public health concern because of reduced uptake of an effective treatment for osteoporosis due to patient concern. The risk of AFF is increased sixfold to sevenfold in patients of Asian ethnicity compared with Europeans. Genetic factors may underlie the AFF phenotype. Given the rarity of AFFs, studying familial AFF cases is valuable in providing insights into any genetic predisposition. We present two Singaporean families, one comprising a mother (1-a) and a daughter (1-b), and the other comprising two sisters (2-a and 2-b). All four cases presented with bisphosphonate-associated AFF. Whole-exome sequencing (WES) was performed on 1-b, 2-a, and 2-b. DNA for 1-a was not available. Variants were examined using a candidate gene approach comprising a list of genes previously associated with AFF in the literature, as well as using unbiased filtering based on dominant and/or recessive inheritance patterns. Using a candidate gene approach, rare variants shared between all three cases were not identified. A rare variant in TMEM25, shared by the two sisters (2-a and 2-b), was identified. A rare heterozygous PLOD2 variant was present in the daughter case with AFF (1-b), but not in the sisters. A list of potential genetic variants for AFF was identified after variant filtering and annotation analysis of the two sisters (2-a and 2-b), including a Gly35Arg variant in TRAF4, a gene required for normal skeletal development. Although the findings from this genetic analysis are inconclusive, a familial aggregation of AFFs is suggestive of a genetic component in AFF pathogenesis. We provide a comprehensive list of rare variants identified in these AFF familial cases to aid future genetic studies. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

A bioinformatics pipeline for estimating mitochondrial DNA copy number and heteroplasmy levels from whole genome sequencing data.

Mitochondrial diseases are a heterogeneous group of disorders that can be caused by mutations in the nuclear or mitochondrial genome. Mitochondrial DNA (mtDNA) variants may exist in a state of heteroplasmy, where a percentage of DNA molecules harbor a variant, or homoplasmy, where all DNA molecules have the same variant. The relative quantity of mtDNA in a cell, or copy number (mtDNA-CN), is associated with mitochondrial function, human disease, and mortality. To facilitate accurate identification of heteroplasmy and quantify mtDNA-CN, we built a bioinformatics pipeline that takes whole genome sequencing data and outputs mitochondrial variants, and mtDNA-CN. We incorporate variant annotations to facilitate determination of variant significance. Our pipeline yields uniform coverage by remapping to a circularized chrM and by recovering reads falsely mapped to nuclear-encoded mitochondrial sequences. Notably, we construct a consensus chrM sequence for each sample and recall heteroplasmy against the sample's unique mitochondrial genome. We observe an approximately 3-fold increased association with age for heteroplasmic variants in non-homopolymer regions and, are better able to capture genetic variation in the D-loop of chrM compared to existing software. Our bioinformatics pipeline more accurately captures features of mitochondrial genetics than existing pipelines that are important in understanding how mitochondrial dysfunction contributes to disease.

Large-scale cis- and trans-eQTL analyses identify thousands of genetic loci and polygenic scores that regulate blood gene expression.

Trait-associated genetic variants affect complex phenotypes primarily via regulatory mechanisms on the transcriptome. To investigate the genetics of gene expression, we performed cis- and trans-expression quantitative trait locus (eQTL) analyses using blood-derived expression from 31,684 individuals through the eQTLGen Consortium. We detected cis-eQTL for 88% of genes, and these were replicable in numerous tissues. Distal trans-eQTL (detected for 37% of 10,317 trait-associated variants tested) showed lower replication rates, partially due to low replication power and confounding by cell type composition. However, replication analyses in single-cell RNA-seq data prioritized intracellular trans-eQTL. Trans-eQTL exerted their effects via several mechanisms, primarily through regulation by transcription factors. Expression of 13% of the genes correlated with polygenic scores for 1,263 phenotypes, pinpointing potential drivers for those traits. In summary, this work represents a large eQTL resource, and its results serve as a starting point for in-depth interpretation of complex phenotypes.

A comparison of genotyping arrays.

Array technology to genotype single-nucleotide variants (SNVs) is widely used in genome-wide association studies (GWAS), clinical diagnostics, and linkage studies. Arrays have undergone a tremendous growth in both number and content over recent years making a comprehensive comparison all the more important. We have compared 28 genotyping arrays on their overall content, genome-wide coverage, imputation quality, presence of known GWAS loci, mtDNA variants and clinically relevant genes (i.e., American College of Medical Genetics (ACMG) actionable genes, pharmacogenetic genes, human leukocyte antigen (HLA) genes and SNV density). Our comparison shows that genome-wide coverage is highly correlated with the number of SNVs on the array but does not correlate with imputation quality, which is the main determinant of GWAS usability. Average imputation quality for all tested arrays was similar for European and African populations, indicating that this is not a good criterion for choosing a genotyping array. Rather, the additional content on the array, such as pharmacogenetics or HLA variants, should be the deciding factor. As the research question of a study will in large part determine which class of genes are of interest, there is not just one perfect array for all different research questions. This study can thus help as a guideline to determine which array best suits a study's requirements.

Composition of cutaneous bacterial microbiome in seborrheic dermatitis patients: A cross-sectional study.

BACKGROUND: Seborrheic dermatitis (SD) is a chronic inflammatory skin disease with a multifactorial aetiology. Malassezia yeasts have been associated with the disease but the role of bacterial composition in SD has not been thoroughly investigated. OBJECTIVES: To profile the bacterial microbiome of SD patients and compare this with the microbiome of individuals with no inflammatory skin disease (controls). METHODS: This was a cross sectional study embedded in a population-based study. Skin swabs were taken from naso-labial fold from patients with seborrheic dermatitis (lesional skin: n = 22; non-lesional skin SD: n = 75) and controls (n = 465). Sample collection began in 2016 at the research facility and is still ongoing. Shannon and Chao1 α- diversity metrics were calculated per group. Associations between the microbiome composition of cases and controls was calculated using multivariate statistics (permANOVA) and univariate statistics. RESULTS: We found an increased α-diversity between SD lesional cases versus controls (Shannon diversity: Kruskal-Wallis rank sum: Chi-squared: 19.06; global p-value = 7.7x10-5). Multivariate statistical analysis showed significant associations in microbiome composition when comparing lesional SD skin to controls (p-value = 0.03;R2 = 0.1%). Seven out of 13 amplicon sequence variants (ASVs) that were significantly different between controls and lesional cases were members of the genus Staphylococcus, most of which showed increased composition in lesional cases, and were closely related to S. capitis S. caprae and S. epidermidis. CONCLUSION: Microbiome composition differs in patients with seborrheic dermatitis and individuals without diseases. Differences were mainly found in the genus Staphylococcus.

Congenital hypopituitarism in two brothers with a duplication of the 'acrogigantism gene' GPR101: clinical findings and review of the literature.

PURPOSE: Congenital hypopituitarism (CH) can cause significant morbidity or even mortality. In the majority of patients, the etiology of CH is unknown. Understanding the etiology of CH is important for anticipation of clinical problems and for genetic counselling. Our previous studies showed that only a small proportion of cases have mutations in the known 'CH genes'. In the current project, we present the results of SNP array based copy number variant analysis in a family with unexplained congenital hypopituitarism. METHODS: DNA samples of two affected brothers with idiopathic CH and their mother were simultaneously analyzed by SNP arrays for copy number variant analysis and Whole Exome Sequencing (WES) for mutation screening. DNA of the father was not available. RESULTS: We found a 6 Mb duplication including GPR101 and SOX3 on the X-chromosome (Xq26.2-q27.1) in the two siblings and their mother, leading to 2 copies of this region in the affected boys and 3 copies in the mother. Duplications of GPR101 are associated with X-linked acrogigantism (the phenotypic 'opposite' of the affected brothers), whereas alterations in SOX3 are associated with X-linked hypopituitarism. CONCLUSION: In our patients with hypopituitarism we found a 6 Mb duplication which includes GPR101, a gene associated with X- linked gigantism, and SOX3, a gene involved in early pituitary organogenesis that is associated with variable degrees of hypopituitarism. Our findings show that in duplications containing both GPR101 and SOX3, the growth hormone deficiency phenotype is dominant. This suggests that, if GPR101 is duplicated, it might not be expressed phenotypically when early patterning of the embryonic pituitary is affected due to SOX3 duplication. These results, together with the review of the literature, shed a new light on the role of GPR101 and SOX3 in pituitary function.

Reduced penetrance of pathogenic ACMG variants in a deeply phenotyped cohort study and evaluation of ClinVar classification over time.

PURPOSE: We studied the penetrance of pathogenically classified variants in an elderly Dutch population from the Rotterdam Study, for which deep phenotyping is available. We screened the 59 actionable genes for which reporting of known pathogenic variants was recommended by the American College of Medical Genetics and Genomics (ACMG), and demonstrate that determining what constitutes a known pathogenic variant can be quite challenging. METHODS: We defined "known pathogenic" as classified pathogenic by both ClinVar and the Human Gene Mutation Database (HGMD). In 2628 individuals, we performed exome sequencing and identified known pathogenic variants. We investigated the clinical records of carriers and evaluated clinical events during 25 years of follow-up for evidence of variant pathogenicity. RESULTS: Of 3815 variants detected in the 59 ACMG genes, 17 variants were considered known pathogenic. For 14/17 variants the ClinVar classification had changed over time. Of 24 confirmed carriers of these variants, we observed at least one clinical event possibly caused by the variant in only three participants (13%). CONCLUSION: We show that the definition of "known pathogenic" is often unclear and should be approached carefully. Additionally variants marked as known pathogenic do not always have clinical impact on their carriers. Definition and classification of true (individual) expected pathogenic impact should be defined carefully.

Unique near-complete deletion of GLI2 in a patient with combined pituitary hormone deficiency and post-axial polydactyly.

INTRODUCTION: Combined pituitary hormone deficiency (CPHD) can cause a broad spectrum of health problems, ranging from short stature only, to convulsions or even death. In the majority of patients, the cause is unknown. METHODS: The idex case had unexplained CPHD, pituitary anomalies on MRI and polydactyly. In the patients and her unaffected parents, we performed SNP array analysis and Whole Exome Sequencing, after candidate gene analysis turned out negative. RESULTS: We found a unique de novo heterozygous 229.9 kb deletion in the index case on chr. 2q14.2. This deletion covered 12 out of the 13 coding exons of the GLI2 gene, a transcription factor involved in midline formation and previously associated with CPHD. As reported GLI2 deletions and mutations show a large phenotypic variability, we performed a genotype-phenotype analysis. This revealed that GLI2 missense mutations usually present with a 'ppp-only' phenotype (pituitary anomalies ± postaxial polydactyly without brain phenotype), whereas the 'ppp-plus' phenotype (with major brain malformations and/or intellectual disabilities) is more frequent in patients with larger deletions, and those with frameshift mutations/point mutations or splice variants resulting in a stop codon (p < .001). CONCLUSION: The present case shows that a deletion of the GLI2 gene only (not affecting any of the adjacent genes) causes pituitary anomalies without brain phenotype. This suggests that brain phenotype only occurs when additional genes adjacent to GLI2 are deleted, or when mutations result in truncated GLI2 mRNA/protein. However, due to the lack of functional data for many GLI2 mutations and based on the available information regarding variable penetrance, phenotype-genotype correlations need to be made with caution.

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.