Clinical Trial-Ready Patient Cohorts for Multiple System Atrophy: Coupling Biospecimen and iPSC Banking to Longitudinal Deep-Phenotyping.

Multiple system atrophy (MSA) is a fatal neurodegenerative disease of unknown etiology characterized by widespread aggregation of the protein alpha-synuclein in neurons and glia. Its orphan status, biological relationship to Parkinson's disease (PD), and rapid progression have sparked interest in drug development. One significant obstacle to therapeutics is disease heterogeneity. Here, we share our process of developing a clinical trial-ready cohort of MSA patients (69 patients in 2 years) within an outpatient clinical setting, and recruiting 20 of these patients into a longitudinal "n-of-few" clinical trial paradigm. First, we deeply phenotype our patients with clinical scales (UMSARS, BARS, MoCA, NMSS, and UPSIT) and tests designed to establish early differential diagnosis (including volumetric MRI, FDG-PET, MIBG scan, polysomnography, genetic testing, autonomic function tests, skin biopsy) or disease activity (PBR06-TSPO). Second, we longitudinally collect biospecimens (blood, CSF, stool) and clinical, biometric, and imaging data to generate antecedent disease-progression scores. Third, in our Mass General Brigham SCiN study (stem cells in neurodegeneration), we generate induced pluripotent stem cell (iPSC) models from our patients, matched to biospecimens, including postmortem brain. We present 38 iPSC lines derived from MSA patients and relevant disease controls (spinocerebellar ataxia and PD, including alpha-synuclein triplication cases), 22 matched to whole-genome sequenced postmortem brain. iPSC models may facilitate matching patients to appropriate therapies, particularly in heterogeneous diseases for which patient-specific biology may elude animal models. We anticipate that deeply phenotyped and genotyped patient cohorts matched to cellular models will increase the likelihood of success in clinical trials for MSA.

Variants in STXBP3 are Associated with Very Early Onset Inflammatory Bowel Disease, Bilateral Sensorineural Hearing Loss and Immune Dysregulation.

BACKGROUND AND AIMS: Very early onset inflammatory bowel disease [VEOIBD] is characterized by intestinal inflammation affecting infants and children less than 6 years of age. To date, over 60 monogenic aetiologies of VEOIBD have been identified, many characterized by highly penetrant recessive or dominant variants in underlying immune and/or epithelial pathways. We sought to identify the genetic cause of VEOIBD in a subset of patients with a unique clinical presentation. METHODS: Whole exome sequencing was performed on five families with ten patients who presented with a similar constellation of symptoms including medically refractory infantile-onset IBD, bilateral sensorineural hearing loss and, in the majority, recurrent infections. Genetic aetiologies of VEOIBD were assessed and Sanger sequencing was performed to confirm novel genetic findings. Western analysis on peripheral blood mononuclear cells and functional studies with epithelial cell lines were employed. RESULTS: In each of the ten patients, we identified damaging heterozygous or biallelic variants in the Syntaxin-Binding Protein 3 gene [STXBP3], a protein known to regulate intracellular vesicular trafficking in the syntaxin-binding protein family of molecules, but not associated to date with either VEOIBD or sensorineural hearing loss. These mutations interfere with either intron splicing or protein stability and lead to reduced STXBP3 protein expression. Knock-down of STXBP3 in CaCo2 cells resulted in defects in cell polarity. CONCLUSION: Overall, we describe a novel genetic syndrome and identify a critical role for STXBP3 in VEOIBD, sensorineural hearing loss and immune dysregulation.

Moving Genomics to Routine Care: An Initial Pilot in Acute Cardiovascular Disease.

BACKGROUND: Whole-genome sequencing (WGS) costs are falling, yet, outside oncology, this information is seldom used in adult clinics. We piloted a rapid WGS (rWGS) workflow, focusing initially on estimating power for a feasibility study of introducing genome information into acute cardiovascular care. METHODS: A prospective implementation study was conducted to test the feasibility and clinical utility of rWGS in acute cardiovascular care. rWGS was performed on 50 adult patients with acute cardiovascular events and cardiac arrest survivors, testing for primary and secondary disease-causing variants, cardiovascular-related pharmacogenomics, and carrier status for recessive diseases. The impact of returning rWGS results on short-term clinical care of participants was investigated. The utility of polygenic risk scores to stratify coronary artery disease was also assessed. RESULTS: Pathogenic variants, typically secondary findings, were identified in 20% (95% CI, 11.7-34.3). About 60% (95% CI, 46.2-72.4) of participants were carriers for one or more recessive traits, most commonly in HFE and SERPINA1 genes. Although 64% (95% CI, 50.1-75.9) of participants carried at least one pharmacogenetic variant of cardiovascular relevance, these were actionable in only 14% (95% CI, 7-26.2). Coronary artery disease prevalence among participants at the 95th percentile of polygenic risk score was 88.2% (95% CI, 71.8-95.7). CONCLUSIONS: We demonstrated the feasibility of rWGS integration into the inpatient management of adults with acute cardiovascular events. Our pilot identified pathogenic variants in one out of 5 acute vascular patients. Integrating rWGS in clinical care will progressively increase actionability.

novoCaller: a Bayesian network approach for de novo variant calling from pedigree and population sequence data.

MOTIVATION: De novo mutations (i.e. newly occurring mutations) are a pre-dominant cause of sporadic dominant monogenic diseases and play a significant role in the genetics of complex disorders. De novo mutation studies also inform population genetics models and shed light on the biology of DNA replication and repair. Despite the broad interest, there is room for improvement with regard to the accuracy of de novo mutation calling. RESULTS: We designed novoCaller, a Bayesian variant calling algorithm that uses information from read-level data both in the pedigree and in unrelated samples. The method was extensively tested using large trio-sequencing studies, and it consistently achieved over 97% sensitivity. We applied the algorithm to 48 trio cases of suspected rare Mendelian disorders as part of the Brigham Genomic Medicine gene discovery initiative. Its application resulted in a significant reduction in the resources required for manual inspection and experimental validation of the calls. Three de novo variants were found in known genes associated with rare disorders, leading to rapid genetic diagnosis of the probands. Another 14 variants were found in genes that are likely to explain the phenotype, and could lead to novel disease-gene discovery. AVAILABILITY AND IMPLEMENTATION: Source code implemented in C++ and Python can be downloaded from https://github.com/bgm-cwg/novoCaller. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

An integrated clinical program and crowdsourcing strategy for genomic sequencing and Mendelian disease gene discovery.

Despite major progress in defining the genetic basis of Mendelian disorders, the molecular etiology of many cases remains unknown. Patients with these undiagnosed disorders often have complex presentations and require treatment by multiple health care specialists. Here, we describe an integrated clinical diagnostic and research program using whole-exome and whole-genome sequencing (WES/WGS) for Mendelian disease gene discovery. This program employs specific case ascertainment parameters, a WES/WGS computational analysis pipeline that is optimized for Mendelian disease gene discovery with variant callers tuned to specific inheritance modes, an interdisciplinary crowdsourcing strategy for genomic sequence analysis, matchmaking for additional cases, and integration of the findings regarding gene causality with the clinical management plan. The interdisciplinary gene discovery team includes clinical, computational, and experimental biomedical specialists who interact to identify the genetic etiology of the disease, and when so warranted, to devise improved or novel treatments for affected patients. This program effectively integrates the clinical and research missions of an academic medical center and affords both diagnostic and therapeutic options for patients suffering from genetic disease. It may therefore be germane to other academic medical institutions engaged in implementing genomic medicine programs.

Dual MAPK Inhibition Is an Effective Therapeutic Strategy for a Subset of Class II BRAF Mutant Melanomas.

PURPOSE: Dual MAPK pathway inhibition (dMAPKi) with BRAF and MEK inhibitors improves survival in BRAF V600E/K mutant melanoma, but the efficacy of dMAPKi in non-V600 BRAF mutant tumors is poorly understood. We sought to characterize the responsiveness of class II (enhanced kinase activity, dimerization dependent) BRAF mutant melanoma to dMAPKi. EXPERIMENTAL DESIGN: Tumors from patients with BRAF wild-type (WT), V600E (class I), and L597S (class II) metastatic melanoma were used to generate patient-derived xenografts (PDX). We assembled a panel of melanoma cell lines with class IIa (activation segment) or IIb (p-loop) mutations and compared these with WT or V600E/K BRAF mutant cells. Cell lines and PDXs were treated with BRAFi (vemurafenib, dabrafenib, encorafenib, and LY3009120), MEKi (cobimetinib, trametinib, and binimetinib), or the combination. We identified 2 patients with BRAF L597S metastatic melanoma who were treated with dMAPKi. RESULTS: BRAFi impaired MAPK signaling and cell growth in class I and II BRAF mutant cells. dMAPKi was more effective than either single MAPKi at inhibiting cell growth in all class II BRAF mutant cells tested. dMAPKi caused tumor regression in two melanoma PDXs with class II BRAF mutations and prolonged survival of mice with class II BRAF mutant melanoma brain metastases. Two patients with BRAF L597S mutant melanoma clinically responded to dMAPKi. CONCLUSIONS: Class II BRAF mutant melanoma is growth inhibited by dMAPKi. Responses to dMAPKi have been observed in 2 patients with class II BRAF mutant melanoma. These data provide rationale for clinical investigation of dMAPKi in patients with class II BRAF mutant metastatic melanoma.See related commentary by Johnson and Dahlman, p. 6107.

Actionable gene-based classification toward precision medicine in gastric cancer.

BACKGROUND: Intertumoral heterogeneity represents a significant hurdle to identifying optimized targeted therapies in gastric cancer (GC). To realize precision medicine for GC patients, an actionable gene alteration-based molecular classification that directly associates GCs with targeted therapies is needed. METHODS: A total of 207 Japanese patients with GC were included in this study. Formalin-fixed, paraffin-embedded (FFPE) tumor tissues were obtained from surgical or biopsy specimens and were subjected to DNA extraction. We generated comprehensive genomic profiling data using a 435-gene panel including 69 actionable genes paired with US Food and Drug Administration-approved targeted therapies, and the evaluation of Epstein-Barr virus (EBV) infection and microsatellite instability (MSI) status. RESULTS: Comprehensive genomic sequencing detected at least one alteration of 435 cancer-related genes in 194 GCs (93.7%) and of 69 actionable genes in 141 GCs (68.1%). We classified the 207 GCs into four The Cancer Genome Atlas (TCGA) subtypes using the genomic profiling data; EBV (N = 9), MSI (N = 17), chromosomal instability (N = 119), and genomically stable subtype (N = 62). Actionable gene alterations were not specific and were widely observed throughout all TCGA subtypes. To discover a novel classification which more precisely selects candidates for targeted therapies, 207 GCs were classified using hypermutated phenotype and the mutation profile of 69 actionable genes. We identified a hypermutated group (N = 32), while the others (N = 175) were sub-divided into six clusters including five with actionable gene alterations: ERBB2 (N = 25), CDKN2A, and CDKN2B (N = 10), KRAS (N = 10), BRCA2 (N = 9), and ATM cluster (N = 12). The clinical utility of this classification was demonstrated by a case of unresectable GC with a remarkable response to anti-HER2 therapy in the ERBB2 cluster. CONCLUSIONS: This actionable gene-based classification creates a framework for further studies for realizing precision medicine in GC.

Loss of function mutation in LOX causes thoracic aortic aneurysm and dissection in humans.

Thoracic aortic aneurysms and dissections (TAAD) represent a substantial cause of morbidity and mortality worldwide. Many individuals presenting with an inherited form of TAAD do not have causal mutations in the set of genes known to underlie disease. Using whole-genome sequencing in two first cousins with TAAD, we identified a missense mutation in the lysyl oxidase (LOX) gene (c.893T > G encoding p.Met298Arg) that cosegregated with disease in the family. Using clustered regularly interspaced short palindromic repeats (CRISPR)/clustered regularly interspaced short palindromic repeats-associated protein-9 nuclease (Cas9) genome engineering tools, we introduced the human mutation into the homologous position in the mouse genome, creating mice that were heterozygous and homozygous for the human allele. Mutant mice that were heterozygous for the human allele displayed disorganized ultrastructural properties of the aortic wall characterized by fragmented elastic lamellae, whereas mice homozygous for the human allele died shortly after parturition from ascending aortic aneurysm and spontaneous hemorrhage. These data suggest that a missense mutation in LOX is associated with aortic disease in humans, likely through insufficient cross-linking of elastin and collagen in the aortic wall. Mutation carriers may be predisposed to vascular diseases because of weakened vessel walls under stress conditions. LOX sequencing for clinical TAAD may identify additional mutation carriers in the future. Additional studies using our mouse model of LOX-associated TAAD have the potential to clarify the mechanism of disease and identify novel therapeutics specific to this genetic cause.

Inherited CHST11/MIR3922 deletion is associated with a novel recessive syndrome presenting with skeletal malformation and malignant lymphoproliferative disease.

Glycosaminoglycans (GAGs) such as chondroitin are ubiquitous disaccharide carbohydrate chains that contribute to the formation and function of proteoglycans at the cell membrane and in the extracellular matrix. Although GAG-modifying enzymes are required for diverse cellular functions, the role of these proteins in human development and disease is less well understood. Here, we describe two sisters out of seven siblings affected by congenital limb malformation and malignant lymphoproliferative disease. Using Whole-Genome Sequencing (WGS), we identified in the proband deletion of a 55 kb region within chromosome 12q23 that encompasses part of CHST11 (encoding chondroitin-4-sulfotransferase 1) and an embedded microRNA (MIR3922). The deletion was homozygous in the proband but not in each of three unaffected siblings. Genotyping data from the 1000 Genomes Project suggest that deletions inclusive of both CHST11 and MIR3922 are rare events. Given that CHST11 deficiency causes severe chondrodysplasia in mice that is similar to human limb malformation, these results underscore the importance of chondroitin modification in normal skeletal development. Our findings also potentially reveal an unexpected role for CHST11 and/or MIR3922 as tumor suppressors whose disruption may contribute to malignant lymphoproliferative disease.

Balancing between affinity and speed in target DNA search by zinc-finger proteins via modulation of dynamic conformational ensemble.

Although engineering of transcription factors and DNA-modifying enzymes has drawn substantial attention for artificial gene regulation and genome editing, most efforts focus on affinity and specificity of the DNA-binding proteins, typically overlooking the kinetic properties of these proteins. However, a simplistic pursuit of high affinity can lead to kinetically deficient proteins that spend too much time at nonspecific sites before reaching their targets on DNA. We demonstrate that structural dynamic knowledge of the DNA-scanning process allows for kinetically and thermodynamically balanced engineering of DNA-binding proteins. Our current study of the zinc-finger protein Egr-1 (also known as Zif268) and its nuclease derivatives reveals kinetic and thermodynamic roles of the dynamic conformational equilibrium between two modes during the DNA-scanning process: one mode suitable for search and the other for recognition. By mutagenesis, we were able to shift this equilibrium, as confirmed by NMR spectroscopy. Using fluorescence and biochemical assays as well as computational simulations, we analyzed how the shifts of the conformational equilibrium influence binding affinity, target search kinetics, and efficiency in displacing other proteins from the target sites. A shift toward the recognition mode caused an increase in affinity for DNA and a decrease in search efficiency. In contrast, a shift toward the search mode caused a decrease in affinity and an increase in search efficiency. This accelerated site-specific DNA cleavage by the zinc-finger nuclease, without enhancing off-target cleavage. Our study shows that appropriate modulation of the dynamic conformational ensemble can greatly improve zinc-finger technology, which has used Egr-1 (Zif268) as a major scaffold for engineering.

Asymmetrical roles of zinc fingers in dynamic DNA-scanning process by the inducible transcription factor Egr-1.

Egr-1 is an inducible transcription factor that recognizes 9-bp target DNA sites via three zinc finger domains and activates genes in response to cellular stimuli such as synaptic signals and vascular stresses. Using spectroscopic and computational approaches, we have studied structural, dynamic, and kinetic aspects of the DNA-scanning process in which Egr-1 is nonspecifically bound to DNA and perpetually changes its location on DNA. Our NMR data indicate that Egr-1 undergoes highly dynamic domain motions when scanning DNA. In particular, the zinc finger 1 (ZF1) of Egr-1 in the nonspecific complex is mainly dissociated from DNA and undergoes collective motions on a nanosecond timescale, whereas zinc fingers 2 and 3 (ZF2 and ZF3, respectively) are bound to DNA. This was totally unexpected because the previous crystallographic studies of the specific complex indicated that all of Egr-1's three zinc fingers are equally involved in binding to a target DNA site. Mutations that are expected to enhance ZF1's interactions with DNA and with ZF2 were found to reduce ZF1's domain motions in the nonspecific complex suggesting that these interactions dictate the dynamic behavior of ZF1. By experiment and computation, we have also investigated kinetics of Egr-1's translocation between two nonspecific DNA duplexes. Our data on the wild type and mutant proteins suggest that the domain dynamics facilitate Egr-1's intersegment transfer that involves transient bridging of two DNA sites. These results shed light on asymmetrical roles of the zinc finger domains for Egr-1 to scan DNA efficiently in the nucleus.

Intrinsically disordered regions as affinity tuners in protein-DNA interactions.

Intrinsically disordered regions, terminal tails, and flexible linkers are abundant in DNA-binding proteins and play a crucial role by increasing the affinity and specificity of DNA binding. Disordered tails often undergo a disorder-to-order transition during interactions with DNA and improve both the kinetics and thermodynamics of specific DNA binding. The DNA search by proteins that interact nonspecifically with DNA can be supported by disordered tails as well. The disordered tail may increase the overall protein-DNA interface and thus increase the affinity of the protein to the DNA and its sliding propensity while slowing linear diffusion. The exact effect of the disordered tails on the sliding rate depends on the degree of positive charge clustering, as has been shown for homeodomains and p53 transcription factors. The disordered tails, which may be viewed as DNA recognizing subdomains, can facilitate intersegment transfer events that occur via a "monkey bar" mechanism in which the domains bridge two different DNA fragments simultaneously. The "monkey bar" mechanism can be facilitated by internal disordered linkers in multidomain proteins that mediate the cross-talks between the constituent domains and especially their brachiation dynamics and thus their overall capability to search DNA efficiently. The residue sequence of the disordered tails has unique characteristics that were evolutionarily selected to achieve the optimized function that is unique to each protein. Perturbation of the electrostatic characteristics of the disordered tails by post-translational modifications, such as acetylation and phosphorylation, may affect protein affinity to DNA and therefore can serve to regulate DNA recognition. Modifying the disordered protein tails or the flexibility of the inter-domain linkers of multidomain proteins may affect the cross-talk between the constituent domains so as to facilitate the search kinetics of non-specific DNA sequences and increase affinity to the specific sequences.

Modulating protein-DNA interactions by post-translational modifications at disordered regions.

Intrinsically disordered regions, particularly disordered tails, are very common in DNA-binding proteins (DBPs). The ability of disordered tails to modulate specific and nonspecific interactions with DNA is tightly linked to their being rich in positively charged residues that are often non-randomly distributed along the tail. Perturbing the composition and distribution of charged residues in the disordered regions by post-translational modifications, such as phosphorylation and acetylation, may impair the ability of the tail to interact nonspecifically with DNA by reducing its DNA affinity. In this study, we analyzed datasets of 3398 and 8943 human proteins that undergo acetylation or phosphorylation, respectively. Both modifications are common on the disordered tails of DBPs (3.1 ± 0.2 (0.07 ± 0.007) and 2.0 ± 0.2 (0.02 ± 0.003) acetylation and phosphorylation sites per tail (per tail residue), respectively). Phosphorylation sites are abundant in disordered regions and particularly in flexible tails for both DBPs and non-DBPs. While acetylation sites are also frequently occurred in the disordered tails of DBPs, in non-DBPs they are often found in ordered regions. This difference may indicate that acetylation has different function in DBPs and non-DBPs. Post-translational modifications, which often take place at disordered sites of DBPs, can modulate the interactions of proteins with DNA by changing the local and global properties of the tails. The effect of the modulation can be tuned by adjusting the number of modifications and the cross-talks between them.

DNA search efficiency is modulated by charge composition and distribution in the intrinsically disordered tail.

Intrinsically disordered tails are common in DNA-binding proteins and can affect their search efficiency on nonspecific DNA by promoting the brachiation dynamics of intersegment transfer. During brachiation, the protein jumps between distant DNA regions via an intermediate state in which the tail and globular moieties are bound to different DNA segments. While the disordered tail must be long and positively charged to facilitate DNA search, the effect of its residue sequence on brachiation is unknown. We explored this issue using the NK-2 and Antp homeodomain transcription factors. We designed 566 NK-2 tail-variants and 55 Antp tail-variants having different net charges and positive charge distributions and studied their dynamics and DNA search efficiencies using coarse-grained molecular dynamics simulations. More intersegment transfers occur when the tail is moderately positively charged and the positive charges are clustered together in the middle of the tail or towards its N terminus. The presence of a negatively charged residue does not significantly affect protein brachiation, although it is likely that the presence of many negatively charged residues will complicate the DNA search mechanism. A bioinformatic analysis of 1,384 wild-type homeodomains illustrates that the charge composition and distribution in their N-tail sequences are consistent with an optimal charge pattern to promote intersegment transfer. Our study thus indicates that the residue sequence of the disordered tails of DNA-binding proteins has unique characteristics that were evolutionarily selected to achieve optimized function and suggests that the sequence-structure-function paradigm known for structured proteins is valid for intrinsically disordered proteins as well.

Facilitated DNA search by multidomain transcription factors: cross talk via a flexible linker.

More than 70% of eukaryotic proteins are composed of multiple domains. However, most studies of the search for DNA focus on individual protein domains and do not consider potential cross talk within a multidomain transcription factor. In this study, the molecular features of the DNA search mechanism were explored for two multidomain transcription factors: human Pax6 and Oct-1. Using a simple computational model, we compared a DNA search of multidomain proteins with a search of isolated domains. Furthermore, we studied how manipulating the binding affinity of a single domain to DNA can affect the overall DNA search of the multidomain protein. Tethering the two domains via a flexible linker increases their affinity to the DNA, resulting in a higher propensity for sliding along the DNA, which is more significant for the domain with the weaker DNA-binding affinity. In this case, the domain that binds DNA more tightly anchors the multidomain protein to the DNA and, via the linker, increases the local concentration of the weak DNA-binding domain (DBD). The tethered domains directly exchange between two parallel DNA molecules via a bridged intermediate, where intersegmental transfer is promoted by the weaker DBD. We found that, in general, the relative affinity of the two domains can significantly affect the cross talk between them and thus their overall capability to search DNA efficiently. The results we obtained by examining various multidomain DNA-binding proteins support the necessity of discrepancies between the DNA-binding affinities of the constituent domains.

Searching DNA via a "Monkey Bar" mechanism: the significance of disordered tails.

The search through nonspecific DNA for a specific site by proteins is known to be facilitated by sliding, hopping, and intersegment transfer between separate DNA strands, yet the driving forces of these protein dynamics from the molecular perspective are unclear. In this study, molecular features of the DNA search mechanism were explored for three homologous proteins (the HoxD9, Antp, and NK-2 homeodomains) using a simple computational model in which protein-DNA interactions are represented solely by electrostatic forces. In particular, we studied the impact that disordered N-terminal tails (N-tails), which are more common in DNA-binding proteins than in other proteins, have on the efficiency of DNA search. While the three homeodomain proteins were found to use similar binding interfaces in specific and nonspecific interactions with DNAs, their different electrostatic potentials affect the nature of their sliding dynamics. The different lengths and net charges of the N-tails of the homeodomains affect their motion along the DNA. The presence of an N-tail increases sliding propensity but slows linear diffusion along the DNA. When the search is performed in the presence of two parallel DNA molecules, a direct transfer, which is facilitated by the protein tail, from one nonspecific DNA to another occurs. The tailed proteins jump between two DNA molecules through an intermediate in which the recognition helix of the protein is adsorbed to one DNA fragment and the N-tail is adsorbed to the second, suggesting a "monkey bar" mechanism. Our study illustrates how the molecular architecture of proteins controls the efficiency of DNA scanning.

Double-hybrid functionals for thermochemical kinetics.

We propose two new double-hybrid functionals, denoted B2K-PLYP and mPW2K-PLYP, which yield thermochemical performance comparable to existing double-hybrid functionals but offer superior performance for barrier heights of various kinds. We show that the new functionals yield excellent performance for all of the following: (a) main-group thermochemistry; (b) main-group thermochemical kinetics; (c) late transition metal reactions. In addition, B2K-PLYP performs well for weak interactions.