STR mutations on chromosome 15q cause thyrotropin resistance by activating a primate-specific enhancer of MIR7-2/MIR1179.

Thyrotropin (TSH) is the master regulator of thyroid gland growth and function. Resistance to TSH (RTSH) describes conditions with reduced sensitivity to TSH. Dominantly inherited RTSH has been linked to a locus on chromosome 15q, but its genetic basis has remained elusive. Here we show that non-coding mutations in a (TTTG)4 short tandem repeat (STR) underlie dominantly inherited RTSH in all 82 affected participants from 12 unrelated families. The STR is contained in a primate-specific Alu retrotransposon with thyroid-specific cis-regulatory chromatin features. Fiber-seq and RNA-seq studies revealed that the mutant STR activates a thyroid-specific enhancer cluster, leading to haplotype-specific upregulation of the bicistronic MIR7-2/MIR1179 locus 35 kb downstream and overexpression of its microRNA products in the participants' thyrocytes. An imbalance in signaling pathways targeted by these micro-RNAs provides a working model for this cause of RTSH. This finding broadens our current knowledge of genetic defects altering pituitary-thyroid feedback regulation.

Implementing Evidence-Based Assertions of Clinical Actionability in the Context of Secondary Findings: Updates from the ClinGen Actionability Working Group.

PURPOSE: The ClinGen Actionability Working Group (AWG) developed an evidence-based framework to generate actionability reports and scores of gene-condition pairs in the context of secondary findings from genome sequencing. Here we describe the expansion of the framework to include actionability assertions. METHODS: Initial development of the actionability rubric was based on previously scored adult gene-condition pairs and individual expert evaluation. Rubric refinement was iterative and based on evaluation, feedback, and discussion. The final rubric was pragmatically evaluated via integration into actionability assessments for 27 gene-condition pairs. RESULTS: The resulting rubric has a four-point scale (limited, moderate, strong, definitive) and uses the highest-scoring outcome-intervention pair of each gene-condition pair to generate a preliminary assertion. During AWG discussions, pre-defined criteria and factors guide discussion to produce a consensus assertion for a gene-condition pair, which may differ from the preliminary assertion. The AWG has retrospectively generated assertions for all previously scored gene-condition pairs and are prospectively asserting on gene-condition pairs under assessment, having completed over 170 adult and 188 pediatric gene-condition pairs. CONCLUSION: The AWG expanded its framework to provide actionability assertions to enhance the clinical value of their resources and increase their utility as decision aids regarding return of secondary findings.

Functional categorization of gene regulatory variants that cause Mendelian conditions.

Much of our current understanding of rare human diseases is driven by coding genetic variants. However, non-coding genetic variants play a pivotal role in numerous rare human diseases, resulting in diverse functional impacts ranging from altered gene regulation, splicing, and/or transcript stability. With the increasing use of genome sequencing in clinical practice, it is paramount to have a clear framework for understanding how non-coding genetic variants cause disease. To this end, we have synthesized the literature on hundreds of non-coding genetic variants that cause rare Mendelian conditions via the disruption of gene regulatory patterns and propose a functional classification system. Specifically, we have adapted the functional classification framework used for coding variants (i.e., loss-of-function, gain-of-function, and dominant-negative) to account for features unique to non-coding gene regulatory variants. We identify that non-coding gene regulatory variants can be split into three distinct categories by functional impact: (1) non-modular loss-of-expression (LOE) variants; (2) modular loss-of-expression (mLOE) variants; and (3) gain-of-ectopic-expression (GOE) variants. Whereas LOE variants have a direct corollary with coding loss-of-function variants, mLOE and GOE variants represent disease mechanisms that are largely unique to non-coding variants. These functional classifications aim to provide a unified terminology for categorizing the functional impact of non-coding variants that disrupt gene regulatory patterns in Mendelian conditions.

Single-nucleoid architecture reveals heterogeneous packaging of mitochondrial DNA.

Cellular metabolism relies on the regulation and maintenance of mitochondrial DNA (mtDNA). Hundreds to thousands of copies of mtDNA exist in each cell, yet because mitochondria lack histones or other machinery important for nuclear genome compaction, it remains unresolved how mtDNA is packaged into individual nucleoids. In this study, we used long-read single-molecule accessibility mapping to measure the compaction of individual full-length mtDNA molecules at near single-nucleotide resolution. We found that, unlike the nuclear genome, human mtDNA largely undergoes all-or-none global compaction, with most nucleoids existing in an inaccessible, inactive state. Highly accessible mitochondrial nucleoids are co-occupied by transcription and replication components and selectively form a triple-stranded displacement loop structure. In addition, we showed that the primary nucleoid-associated protein TFAM directly modulates the fraction of inaccessible nucleoids both in vivo and in vitro, acting consistently with a nucleation-and-spreading mechanism to coat and compact mitochondrial nucleoids. Together, these findings reveal the primary architecture of mtDNA packaging and regulation in human cells.

Synchronized long-read genome, methylome, epigenome, and transcriptome for resolving a Mendelian condition.

Resolving the molecular basis of a Mendelian condition (MC) remains challenging owing to the diverse mechanisms by which genetic variants cause disease. To address this, we developed a synchronized long-read genome, methylome, epigenome, and transcriptome sequencing approach, which enables accurate single-nucleotide, insertion-deletion, and structural variant calling and diploid de novo genome assembly, and permits the simultaneous elucidation of haplotype-resolved CpG methylation, chromatin accessibility, and full-length transcript information in a single long-read sequencing run. Application of this approach to an Undiagnosed Diseases Network (UDN) participant with a chromosome X;13 balanced translocation of uncertain significance revealed that this translocation disrupted the functioning of four separate genes (NBEA, PDK3, MAB21L1, and RB1) previously associated with single-gene MCs. Notably, the function of each gene was disrupted via a distinct mechanism that required integration of the four 'omes' to resolve. These included nonsense-mediated decay, fusion transcript formation, enhancer adoption, transcriptional readthrough silencing, and inappropriate X chromosome inactivation of autosomal genes. Overall, this highlights the utility of synchronized long-read multi-omic profiling for mechanistically resolving complex phenotypes.

Full-length Isoform Sequencing for Resolving the Molecular Basis of Charcot-Marie-Tooth 2A.

Objectives: Transcript sequencing of patient-derived samples has been shown to improve the diagnostic yield for solving cases of suspected Mendelian conditions, yet the added benefit of full-length long-read transcript sequencing is largely unexplored. Methods: We applied short-read and full-length transcript sequencing and mitochondrial functional studies to a patient-derived fibroblast cell line from an individual with neuropathy that previously lacked a molecular diagnosis. Results: We identified an intronic homozygous MFN2 c.600-31T>G variant that disrupts the branch point critical for intron 6 splicing. Full-length long-read isoform complementary DNA (cDNA) sequencing after treatment with a nonsense-mediated mRNA decay (NMD) inhibitor revealed that this variant creates 5 distinct altered splicing transcripts. All 5 altered splicing transcripts have disrupted open reading frames and are subject to NMD. Furthermore, a patient-derived fibroblast line demonstrated abnormal lipid droplet formation, consistent with MFN2 dysfunction. Although correctly spliced full-length MFN2 transcripts are still produced, this branch point variant results in deficient MFN2 levels and autosomal recessive Charcot-Marie-Tooth disease, axonal, type 2A (CMT2A). Discussion: This case highlights the utility of full-length isoform sequencing for characterizing the molecular mechanism of undiagnosed rare diseases and expands our understanding of the genetic basis for CMT2A.

DNA-m6A calling and integrated long-read epigenetic and genetic analysis with fibertools.

Long-read DNA sequencing has recently emerged as a powerful tool for studying both genetic and epigenetic architectures at single-molecule and single-nucleotide resolution. Long-read epigenetic studies encompass both the direct identification of native cytosine methylation as well as the identification of exogenously placed DNA N6-methyladenine (DNA-m6A). However, detecting DNA-m6A modifications using single-molecule sequencing, as well as co-processing single-molecule genetic and epigenetic architectures, is limited by computational demands and a lack of supporting tools. Here, we introduce fibertools, a state-of-the-art toolkit that features a semi-supervised convolutional neural network for fast and accurate identification of m6A-marked bases using PacBio single-molecule long-read sequencing, as well as the co-processing of long-read genetic and epigenetic data produced using either PacBio or Oxford Nanopore sequencing platforms. We demonstrate accurate DNA-m6A identification (>90% precision and recall) along >20 kilobase long DNA molecules with a ~1,000-fold improvement in speed. In addition, we demonstrate that fibertools can readily integrate genetic and epigenetic data at single-molecule resolution, including the seamless conversion between molecular and reference coordinate systems, allowing for accurate genetic and epigenetic analyses of long-read data within structurally and somatically variable genomic regions.

Dominant-negative variant in SLC1A4 causes an autosomal dominant epilepsy syndrome.

SLC1A4 is a trimeric neutral amino acid transporter essential for shuttling L-serine from astrocytes into neurons. Individuals with biallelic variants in SLC1A4 are known to have spastic tetraplegia, thin corpus callosum, and progressive microcephaly (SPATCCM) syndrome, but individuals with heterozygous variants are not thought to have disease. We identify an 8-year-old patient with global developmental delay, spasticity, epilepsy, and microcephaly who has a de novo heterozygous three amino acid duplication in SLC1A4 (L86_M88dup). We demonstrate that L86_M88dup causes a dominant-negative N-glycosylation defect of SLC1A4, which in turn reduces the plasma membrane localization of SLC1A4 and the transport rate of SLC1A4 for L-serine.

Evaluation of N 6-methyldeoxyadenosine antibody-based genomic profiling in eukaryotes.

Low-level DNA N 6-methyldeoxyadenosine (DNA-m6A) has recently been reported across various eukaryotes. Although anti-m6A antibody-based approaches are commonly used to measure DNA-m6A levels, this approach is known to be confounded by DNA secondary structures, RNA contamination, and bacterial contamination. To evaluate for these confounding features, we introduce an approach for systematically validating the selectivity of antibody-based DNA-m6A methods and use a highly selective anti-DNA-m6A antibody to reexamine patterns of DNA-m6A in C. reinhardtii, A. thaliana, and D. melanogaster Our findings raise caution about the use of antibody-based methods for endogenous m6A quantification and mapping in eukaryotes.

Full-length isoform sequencing for resolving the molecular basis of Charcot-Marie-Tooth 2A.

Objectives: Transcript sequencing of patient derived samples has been shown to improve the diagnostic yield for solving cases of likely Mendelian disorders, yet the added benefit of full-length long-read transcript sequencing is largely unexplored. Methods: We applied short-read and full-length isoform cDNA sequencing and mitochondrial functional studies to a patient-derived fibroblast cell line from an individual with neuropathy that previously lacked a molecular diagnosis. Results: We identified an intronic homozygous MFN2 c.600-31T>G variant that disrupts a branch point critical for intron 6 spicing. Full-length long-read isoform cDNA sequencing after treatment with a nonsense-mediated mRNA decay (NMD) inhibitor revealed that this variant creates five distinct altered splicing transcripts. All five altered splicing transcripts have disrupted open reading frames and are subject to NMD. Furthermore, a patient-derived fibroblast line demonstrated abnormal lipid droplet formation, consistent with MFN2 dysfunction. Although correctly spliced full-length MFN2 transcripts are still produced, this branch point variant results in deficient MFN2 protein levels and autosomal recessive Charcot-Marie-Tooth disease, axonal, type 2A (CMT2A). Discussion: This case highlights the utility of full-length isoform sequencing for characterizing the molecular mechanism of undiagnosed rare diseases and expands our understanding of the genetic basis for CMT2A.

RNA polymerases reshape chromatin and coordinate transcription on individual fibers.

During eukaryotic transcription, RNA polymerases must initiate and pause within a crowded, complex environment, surrounded by nucleosomes and other transcriptional activity. This environment creates a spatial arrangement along individual chromatin fibers ripe for both competition and coordination, yet these relationships remain largely unknown owing to the inherent limitations of traditional structural and sequencing methodologies. To address these limitations, we employed long-read chromatin fiber sequencing (Fiber-seq) to visualize RNA polymerases within their native chromatin context at single-molecule and near single-nucleotide resolution along up to 30 kb fibers. We demonstrate that Fiber-seq enables the identification of single-molecule RNA Polymerase (Pol) II and III transcription associated footprints, which, in aggregate, mirror bulk short-read sequencing-based measurements of transcription. We show that Pol II pausing destabilizes downstream nucleosomes, with frequently paused genes maintaining a short-term memory of these destabilized nucleosomes. Furthermore, we demonstrate pervasive direct coordination and anti-coordination between nearby Pol II genes, Pol III genes, transcribed enhancers, and insulator elements. This coordination is largely limited to spatially organized elements within 5 kb of each other, implicating short-range chromatin environments as a predominant determinant of coordinated polymerase initiation. Overall, transcription initiation reshapes surrounding nucleosome architecture and coordinates nearby transcriptional machinery along individual chromatin fibers.

Multi-site validation of a functional assay to adjudicate SCN5A Brugada Syndrome-associated variants.

Brugada Syndrome (BrS) is an inheritable arrhythmia condition that is associated with rare, loss-of-function variants in the cardiac sodium channel gene, SCN5A. Interpreting the pathogenicity of SCN5A missense variants is challenging and ~79% of SCN5A missense variants in ClinVar are currently classified as Variants of Uncertain Significance (VUS). An in vitro SCN5A-BrS automated patch clamp assay was generated for high-throughput functional studies of NaV1.5. The assay was independently studied at two separate research sites - Vanderbilt University Medical Center and Victor Chang Cardiac Research Institute - revealing strong correlations, including peak INa density (R2=0.86). The assay was calibrated according to ClinGen Sequence Variant Interpretation recommendations using high-confidence variant controls (n=49). Normal and abnormal ranges of function were established based on the distribution of benign variant assay results. The assay accurately distinguished benign controls (24/25) from pathogenic controls (23/24). Odds of Pathogenicity values derived from the experimental results yielded 0.042 for normal function (BS3 criterion) and 24.0 for abnormal function (PS3 criterion), resulting in up to strong evidence for both ACMG criteria. The calibrated assay was then used to study SCN5A VUS observed in four families with BrS and other arrhythmia phenotypes associated with SCN5A loss-of-function. The assay revealed loss-of-function for three of four variants, enabling reclassification to likely pathogenic. This validated APC assay provides clinical-grade functional evidence for the reclassification of current VUS and will aid future SCN5A-BrS variant classification.

Single cell biology-a Keystone Symposia report.

Single cell biology has the potential to elucidate many critical biological processes and diseases, from development and regeneration to cancer. Single cell analyses are uncovering the molecular diversity of cells, revealing a clearer picture of the variation among and between different cell types. New techniques are beginning to unravel how differences in cell state-transcriptional, epigenetic, and other characteristics-can lead to different cell fates among genetically identical cells, which underlies complex processes such as embryonic development, drug resistance, response to injury, and cellular reprogramming. Single cell technologies also pose significant challenges relating to processing and analyzing vast amounts of data collected. To realize the potential of single cell technologies, new computational approaches are needed. On March 17-19, 2021, experts in single cell biology met virtually for the Keystone eSymposium "Single Cell Biology" to discuss advances both in single cell applications and technologies.

Rewiring of human neurodevelopmental gene regulatory programs by human accelerated regions.

Human accelerated regions (HARs) are the fastest-evolving regions of the human genome, and many are hypothesized to function as regulatory elements that drive human-specific gene regulatory programs. We interrogate the in vitro enhancer activity and in vivo epigenetic landscape of more than 3,100 HARs during human neurodevelopment, demonstrating that many HARs appear to act as neurodevelopmental enhancers and that sequence divergence at HARs has largely augmented their neuronal enhancer activity. Furthermore, we demonstrate PPP1R17 to be a putative HAR-regulated gene that has undergone remarkable rewiring of its cell type and developmental expression patterns between non-primates and primates and between non-human primates and humans. Finally, we show that PPP1R17 slows neural progenitor cell cycle progression, paralleling the cell cycle length increase seen predominantly in primate and especially human neurodevelopment. Our findings establish HARs as key components in rewiring human-specific neurodevelopmental gene regulatory programs and provide an integrated resource to study enhancer activity of specific HARs.

Clinical utility of brain MRS imaging of patients with adult-onset non-cirrhotic hyperammonemia.

Adult-onset non-cirrhotic hyperammonemia (NCH) is a rare, but often fatal condition that can result in both reversible and irreversible neurological defects. Here we present five cases of adult-onset non-cirrhotic hyperammonemia wherein brain magnetic resonance spectroscopy (MRS) scans for cerebral glutamine (Gln) and myo-inositol (mI) levels helped guide clinical management. Specifically, we demonstrate that when combined with traditional brain magnetic resonance imaging (MRI) scans, cerebral Gln and mI MRS can help disentangle the reversible from irreversible neurological defects associated with hyperammonemic crisis. Specifically, we demonstrate that whereas an elevated brain MRS Gln level is associated with reversible neurological defects, markedly low mI levels are associated with a risk for irreversible neurological defects such as central pontine myelinolysis. Overall, our findings indicate the utility of brain MRS in guiding clinical care and prognosis in patients with adult-onset non-cirrhotic hyperammonemia.

A retrospective study of adult patients with noncirrhotic hyperammonemia.

Adult-onset noncirrhotic hyperammonemia (NCH) is poorly understood and has a high morbidity and mortality. To elucidate the etiology and management of NCH, we performed a retrospective analysis of 23 adults (median age 51) with NCH treated between 2014 and 2020 at two academic medical centers. Hyperammonemia was diagnosed in all cases during the evaluation of altered mental status, with 22% presenting with seizures. Peak ammonia levels were >200 µmol/L in 70% of cases. Defects in ammonia metabolism were assessed using urea cycle biochemical testing, germline genetic testing, and testing for urease-producing infectious agents. Ammonia metabolism defects in these cases appear attributable to four major sources: (a) infection with urease-producing organism (n = 5); (b) previously undiagnosed inborn errors of metabolism (IEMs) (n = 4); (c) clinical exposures causing acquired urea cycle dysfunction (n = 6); and (d) unexplained acquired urea cycle dysfunction (uaUCD) (n = 8), as evidenced by biochemical signatures of urea cycle dysfunction without a genetic or clinical exposure. Severe protein malnutrition appeared to be a reversible risk factor for uaUCD. Overall, 13% of our cohort died prior to resolution of hyperammonemia, 26% died after hyperammonemia resolution, 57% survived after having reversible neurological changes, and 4% survived with irreversible neurological changes. Renal replacement therapy for ammonia clearance was often utilized for patients with an ammonia level above 250 µmol/L and patients were frequently empirically treated with antibiotics targeting urea-splitting organisms. Our study demonstrates that acquired urea cycle dysfunction, IEMs and urease-producing infections are major sources of adult-onset NCH and highlights successful management strategies for adult-onset NCH.

Single-molecule regulatory architectures captured by chromatin fiber sequencing.

Gene regulation is chiefly determined at the level of individual linear chromatin molecules, yet our current understanding of cis-regulatory architectures derives from fragmented sampling of large numbers of disparate molecules. We developed an approach for precisely stenciling the structure of individual chromatin fibers onto their composite DNA templates using nonspecific DNA N6-adenine methyltransferases. Single-molecule long-read sequencing of chromatin stencils enabled nucleotide-resolution readout of the primary architecture of multikilobase chromatin fibers (Fiber-seq). Fiber-seq exposed widespread plasticity in the linear organization of individual chromatin fibers and illuminated principles guiding regulatory DNA actuation, the coordinated actuation of neighboring regulatory elements, single-molecule nucleosome positioning, and single-molecule transcription factor occupancy. Our approach and results open new vistas on the primary architecture of gene regulation.

Noncirrhotic hyperammonemia after deceased donor kidney transplantation: A case report.

A 72-year-old woman with end-stage kidney disease due to recurrent urinary tract infections and obstructive uropathy of a solitary kidney presented to our hospital for renal transplantation. She underwent successful transplantation of a deceased donor allograft, but developed acute mental status deterioration on the fifth postoperative day. Her serum ammonia was found to be markedly elevated to 447 µmol/L in the setting of normal hepatic function. She was treated with emergent dialysis and empiric antibiotics targeting urea-splitting organisms, and ultimately made a full neurologic recovery with stable renal allograft function. Noncirrhotic hyperammonemia (NCH) is an exceedingly rare clinical entity but seems to have a predilection for patients who have undergone solid organ transplantation. This report emphasizes the importance of rapid diagnosis and initiation of treatment for NCH, which is associated with a high rate of mortality and irreversible neurological morbidity. We outline the successful workup and management approach for this patient.

Recurrent SLC1A2 variants cause epilepsy via a dominant negative mechanism.

SLC1A2 is a trimeric transporter essential for clearing glutamate from neuronal synapses. Recurrent de novo SLC1A2 missense variants cause a severe, early onset developmental and epileptic encephalopathy via an unclear mechanism. We demonstrate that all 3 variants implicated in this condition localize to the trimerization domain of SLC1A2, and that the Leu85Pro variant acts via a dominant negative mechanism to reduce, but not eliminate, wild-type SLC1A2 protein localization and function. Finally, we demonstrate that treatment of a 20-month-old SLC1A2-related epilepsy patient with the SLC1A2-modulating agent ceftriaxone did not result in a significant change in daily spasm count. ANN NEUROL 2019;85:921-926.