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Genome‐scale high‐throughput sequencing enables the detection of unprecedented numbers of sequence variants. Variant filtering and interpretation are facilitated by mutation databases, in silico tools, and population‐based reference datasets such as ExAC/gnomAD, while variants are classified using the ACMG/AMP guidelines. These methods, however, pose clinically relevant challenges. We queried the gnomAD dataset for (likely) pathogenic variants in genes causing autosomal‐dominant disorders. Furthermore, focusing on the fibrillinopathies Marfan syndrome (MFS) and congenital contractural arachnodactyly (CCA), we screened 500 genomes of our patients for co‐occurring variants in FBN1 and FBN2. In gnomAD, we detected 2653 (likely) pathogenic variants in 253 genes associated with autosomal‐dominant disorders, enabling the estimation of variant‐filtering thresholds and disease predisposition/prevalence rates. In our database, we discovered two families with hitherto unreported co‐occurrence of FBN1/FBN2 variants causing phenotypes with mixed or modified MFS/CCA clinical features. We show that (likely) pathogenic gnomAD variants may be more frequent than expected and are challenging to classify according to the ACMG/AMP guidelines as well as that fibrillinopathies are likely underdiagnosed and may co‐occur. Consequently, selection of appropriate frequency cutoffs, recognition of digenic variants, and variant classification represent considerable challenges in variant interpretation. Neglecting these challenges may lead to incomplete or missed diagnoses.

Pharmacogenetics represents a major driver of precision medicine, promising individualized drug selection and dosing. Traditionally, pharmacogenetic profiling has been performed using targeted genotyping that focuses on common/known variants. Recently, whole-genome sequencing (WGS) is emerging as a more comprehensive short-read next-generation sequencing approach, enabling both gene diagnostics and pharmacogenetic profiling, including rare/novel variants, in a single assay. Using the example of the pharmacogene CYP2D6, we demonstrate the potential of WGS-based pharmacogenetic profiling as well as emphasize the limitations of short-read next-generation sequencing. In the near future, we envision a shift toward long-read sequencing as the predominant method for gene diagnostics and pharmacogenetic profiling, providing unprecedented data quality and improving patient care.

Although several pharmacogenetic (PGx) predispositions affecting drug efficacy and safety are well established, drug selection and dosing as well as clinical trials are often performed in a non-pharmacogenetically-stratified manner, ultimately burdening healthcare systems. Pre-emptive PGx testing offers a solution which is often performed using microarrays or targeted gene panels, testing for common/known PGx variants. However, as an added value, whole-genome sequencing (WGS) could detect not only disease-causing but also pharmacogenetically-relevant variants in a single assay. Here, we present our WGS-based pipeline that extends the genetic testing of Mendelian diseases with PGx profiling, enabling the detection of rare/novel PGx variants as well. From our in-house WGS (PCR-free 60× PE150) data of 547 individuals we extracted PGx variants with drug-dosing recommendations of the Dutch Pharmacogenetics Working Group (DPWG). Furthermore, we explored the landscape of DPWG pharmacogenes in gnomAD and our in-house cohort as well as compared bioinformatic tools for WGS-based structural variant detection in CYP2D6. We show that although common/known PGx variants comprise the vast majority of detected DPWG pharmacogene alleles, for better precision medicine, PGx testing should move towards WGS-based approaches. Indeed, WGS-based PGx profiling is not only feasible and future-oriented but also the most comprehensive all-in-one approach without generating significant additional costs., International Journal of Molecular Sciences, 21 (7), ISSN:1422-0067

Aims Antihypertensive drugs are included in the medical therapy of vascular Ehlers–Danlos syndrome (vEDS). The β-blocker celiprolol has been suggested to prevent arterial damage in vEDS, but the underlying mechanism remains unclear. It is also unknown whether the widely used angiotensin II receptor type 1 antagonist losartan has a therapeutic effect in vEDS. Here, we evaluated the impact of celiprolol and losartan on the biomechanical integrity of the vEDS thoracic aorta. Methods and results We established a new approach to measure the maximum tensile force at rupture of uniaxially stretched murine thoracic aortic rings. In a vEDS model, which we (re-)characterized here at molecular level, heterozygous mice showed a significant reduction in the rupture force compared to wild-type mice, reflecting the increased mortality due to aortic rupture. For the assessment of treatment effects, heterozygous mice at 4 weeks of age underwent a 4-week treatment with celiprolol, losartan, and, as a proof-of-concept drug, the matrix metalloproteinase inhibitor doxycycline. Compared to age- and sex-matched untreated heterozygous mice, treatment with doxycycline or celiprolol resulted in a significant increase of rupture force, whereas no significant change was detected upon losartan treatment. Conclusions In a vEDS model, celiprolol or doxycycline, but not losartan, can improve the biomechanical integrity of the aortic wall, thereby potentially reducing the risk of dissection and rupture. As doxycycline is a broad-spectrum antibiotic with considerable side effects, celiprolol may be more suitable for a long-term therapy and thus rather indicated for the medication of patients with vEDS., Cardiovascular Research, 116 (2), ISSN:1755-3245, ISSN:0008-6363

High-throughput sequencing (HTS) has revolutionized genetics by enabling the detection of sequence variants at hitherto unprecedented large scale. Despite these advances, however, there are still remaining challenges in the complete coverage of targeted regions (genes, exome or genome) as well as in HTS data analysis and interpretation. Moreover, it is easy to get overwhelmed by the plethora of available methods and tools for HTS. Here, we review the step-by-step process from the generation of sequence data to molecular diagnosis of Mendelian diseases. Highlighting advantages and limitations, this review addresses the current state of (1) HTS technologies, considering targeted, whole-exome, and whole-genome sequencing on short- and long-read platforms; (2) read alignment, variant calling and interpretation; as well as (3) regulatory issues related to genetic counseling, reimbursement, and data storage.

In the current genomics era, technological advances have led to the identification of an increasing number of gene–disease associations. In a recent issue of the Journal , Renard et al. [(1)][1] assessed and clinically validated genes that may predispose to syndromic or nonsyndromic forms of

In the current high-throughput genomics era, efficient and accurate analysis of large-scale whole-genome sequencing (WGS) data constitutes a computational bottleneck. Johnston et al. (1) introduce the PEMapper/PECaller software package for short-read WGS alignment and variant calling, promising faster analyses with reduced output file sizes and “nearly identical (or better)” variant calling accuracy compared with the de facto standard Burrows–Wheeler aligner/Genome Analysis Toolkit (BWA/GATK) best-practices pipeline (2). However, we cannot confirm this promised BWA/GATK-like accuracy of PEMapper/PECaller, and there are other pipelines offering ultrafast WGS data analyses with small disk footprints, as we show in this correspondence. To assess sensitivity/recall, precision, computation time, and disk footprint of four corresponding pipelines, we performed alignment and variant calling for the reference short-read WGS data of NA12878 and the Ashkenazim trio (3, 4). The four pipelines included the downloadable PEMapper/PECaller (1) and BWA/GATK (2) as well as the commercially available Isaac (5) and GENALICE MAP (genalice.com) software packages (versions and settings specified in Fig. 1). To largely reduce systematic errors and alignment artifacts, we limited our benchmarking of whole-genome variant … [↵][1]2To whom correspondence should be addressed. Email: matyas{at}genetikzentrum.ch. [1]: #xref-corresp-1-1

Eruptions fed from subsurface reservoirs commonly construct volcanic edifices at the surface, and the growth of an edifice will in turn modify the subsurface stress state that dictates the conditions under which subsequent rupture of the inflating reservoir can occur. We re-examine this problem using axisymmetric finite element models of ellipsoidal reservoirs beneath conical edifices, explicitly incorporating factors (e.g., full gravitational loading conditions, an elastic edifice instead of a surface load, reservoir pressures sufficient to induce tensile rupture) that compromise previous solutions to illustrate why variations in rupture behavior can occur. Relative to half-space model results, the presence of an edifice generally rotates rupture toward the crest of a spherical reservoir, with increasing flank slope (for an edifice of constant volume) and larger edifices (or greater reservoir scaled depths) normally serving to enhance this trend. When non-spherical reservoirs are considered, the presence of an edifice amplifies previously identified half-space failure characteristics, shifting rupture to the crest more rapidly for prolate reservoirs while forcing rupture closer to the midpoint of oblate reservoirs. Rupture is always observed to occur in the σ t orientation, and depending on where initial failure occurs rupture favors the initial emplacement of either lateral sills, circumferential intrusions or vertically ascending dikes. Ultimately, integration of our numerical model results with other information, for instance the sequence of intrusion/eruption events observed at a given volcano, can provide useful new insight into how a volcano's subsurface magma plumbing system evolved. We demonstrate this process through application of our model to Summer Coon, a well-studied stratocone on Earth, and Ilithyia Mons, a large conical shield volcano on Venus.

Elastic, gravitationally loaded finite element models of an ellipsoidal magma reservoir in mechanically layered host rock yield new insight into the effect of layer stiffness, thickness, and spatial configuration on the resulting surface displacement curve and tensile rupture of the reservoir wall. Decreasing layer stiffness enhances surface and subsurface deformation, focusing pressure across a narrow area, and generally decreases the likelihood of vertical magma intrusion and surface eruption. In contrast, increasing layer stiffness impedes uplift and distributes strain across a broad area, enhancing the likelihood of vertical magma intrusion. Application of the model to deformation that occurred between 1985 and 1999 at the Long Valley caldera, California shows that the predicted reservoir radius and depth change by 16% and 12% respectively when mechanical layers inferred to be present are included in the model, highlighting the importance of explicitly incorporating mechanical layering. Analysis of rupture conditions for the reservoirs modeled in the present paper indicates that vertical intrusions are likely. In addition, analysis of rupture conditions for Long Valley magma reservoirs recently inferred from surface displacement inversions indicates that they will fail before achieving sufficient pressure to reproduce measured surface displacement.

Magma flow paths in sill-fed dikes of the Ferrar large igneous province (LIP), contrast with those predicted by classic models of dike transport in LIPs and magmatic rift settings. We examine anisotropy of magnetic susceptibility (AMS) flow paths in dike networks at Terra Cotta Mountain and Mt. Gran, which intruded at paleodepths of ~ 2.5 and ~ 1.5 km. These intrusions (up to 30 m thick) exhibit irregular, interconnected dike-sill geometries and adjoin larger sills (~ 200–300 m thick) at different stratigraphic levels. Both shallowly dipping and sub-vertical magma flow components are interpreted from AMS measurements across individual intrusions, and often match macroscopic flow indicators and variations in dike attitudes. Flow paths suggest that intrusive patterns and magma flow directions depended on varying stress concentrations and rotations during dike and sill propagation, whereas a regional extensional tectonic control was negligible or absent. Unlike giant dike swarms in LIPs elsewhere (e.g., 1270 Ma MacKenzie LIP), dikes of the Ferrar LIP show no regionally consistent vertical or lateral flow patterns, suggesting these intrusion were not responsible for long-distance transport in the province. In the absence of regionally significant, colinear dike swarms, or observed intrusions at crustal depths ≥ 4 km, we suggest that long distance magma transport occurred in sills within Beacon Supergroup sedimentary rocks. This interpretation is consistent with existing geochemical data and thermal constraints, which support lateral magma flow for ~ 3,500 km across the Gondwana supercontinent before freezing.

There are many congenital malformations of the mitral valve which produce valvar insufficiency. From a surgical point of view, systems based exclusively on anatomic analysis are not always entirely appropriate for assessment of these lesions. With this in mind, Carpentier proposed a functional approach for analysis based upon the motion of the valvar leaflets. From 1969 to 1994, 135 children under the age of 12 (mean age: 5.8 + 3.15 Y, 0.6–12Y) underwent surgery in our department, basing treatment on such analysis. Since motion of the leaflets during the operation is compromised by cardioplegia, and sometimes exposure can be however difficult, preoperative echocardiography was a mandatory part of the diagnostic cascade.Normal motion of the leaflets was present in 41 patients, with deformation of the annulus in 14, a cleft in 21, and partial agenesis in 6. Prolapse of leaflets was present in 42 patients. Leaflet motion was restricted in 28 patients. These were divided in two groups, one with normal papillary muscles and commissural fusion or short cords. The other with abnormal papillary muscles producing a parachute arrangement in 6 and a hammock valve in 9. Associated lesions were present in 47% of the patients. Conservative surgical procedures following the precepts developed by Carpentier were used in 127 patients. Valvar replacement was necessary in 8 patients. Operative mortality was 4%. Mean follow up was 8.4 ± 5.3 years (1–23Y). Actuarial survival at 5 years was 90 ± 6% and, at this time, was stable. No thromboembolic events occurred after conservative surgery. The reoperation rate was 5% for those undergoing repair (6 patients). We conclude that the functional classification developed by Carpentier is a reliable and robust approach to these complex lesions. Conservative surgery is feasible in most of the cases presenting with congenital mitral valvar insufficiency. Results are stable and reliable. Surgery should be undertaken before the onset of left ventricular deterioration.

Human breast cancers are known to preferentially metastasize to skeletal sites, however, the mechanisms that mediate the skeletal preference (orthotropism) of specific types of cancers remains poorly understood. There is a significant clinical correlation between the expression of bone sialoprotein (BSP) and skeletal metastasis of breast cancers. Our laboratory, as well as others, have proposed the concept that skeletal selective metastasis and associated disease may be attributable to a mimicry of skeletal cellular phenotypes by metastasizing cancer cells. We hypothesize that breast cancer cell expression of phenotypic properties of skeletal cell types, including BSP as one component of that phenotype, is the result of ectopic expression or activity of one or more central transcriptional regulators of bone cell gene expression. To test this hypothesis, we examined the molecular mechanisms that regulate bsp expression in human breast cancer cell lines with previously characterized metastatic potentials. Our results demonstrate that the majority of the distal bsp promoter sequences act to repress BSP expression in cancer cells and that most of the promoter activity resides in the proximal -110 bp of the bsp promoter. In this region, we identified a putative Runx binding element providing a basis for a mechanism for skeletal gene activation. Our results demonstrate that Runx2 is ectopically expressed in breast cancer cells and that one isoform of Runx2 can activate bsp expression in these cells. In addition, we observe that bsp expression is additionally regulated by the homeodomain factor Msx2, another regulator of osteoblast-associated genes. Thus, this is the first report of osteoblast-related transcription factors being expressed in human breast cancer cells and provides a component of a mechanism that may explain the osteoblastic phenotype of human breast cancer cells that preferentially metastasize to bone.