Soluble biomarkers for Neuromyelitis Optica Spectrum Disorders: a mini review.

The Neuromyelitis Optica Spectrum Disorders (NMOSD) constitute a spectrum of rare autoimmune diseases of the central nervous system characterized by episodes of transverse myelitis, optic neuritis, and other demyelinating attacks. Previously thought to be a subtype of multiple sclerosis, NMOSD is now known to be a distinct disease with unique pathophysiology, clinical course, and treatment options. Although there have been significant recent advances in the diagnosis and treatment of NMOSD, the field still lacks clinically validated biomarkers that can be used to stratify disease severity, monitor disease activity, and inform treatment decisions. Here we review many emerging NMOSD biomarkers including markers of cellular damage, neutrophil-to-lymphocyte ratio, complement, and cytokines, with a focus on how each biomarker can potentially be used for initial diagnosis, relapse surveillance, disability prediction, and treatment monitoring.

A case of immunotherapy-responsive autoimmune hemichorea.

INTRODUCTION: Subacute adult-acquired hemichorea is a striking presentation with a broad differential, including ischemic, metabolic, and inflammatory causes. CASE: We encountered a 74-year-old woman with rapid onset of hemichorea and associated encephalopathy. Following a thorough workup without identification of clear imaging or laboratory abnormalities, we empirically treated with IVIg. Her hemichorea dramatically improved. Due to relapses of hemichorea, she required repeat immunotherapy with IVIg or high dose steroids followed by maintenance mycophenolate. DISCUSSION: This case of seronegative autoimmune hemichorea highlights the importance of a high index of suspicion for an inflammatory etiology of chorea when other causes are ruled out and performing an immunotherapy trial.

Comprehensive identification of somatic nucleotide variants in human brain tissue.

BACKGROUND: Post-zygotic mutations incurred during DNA replication, DNA repair, and other cellular processes lead to somatic mosaicism. Somatic mosaicism is an established cause of various diseases, including cancers. However, detecting mosaic variants in DNA from non-cancerous somatic tissues poses significant challenges, particularly if the variants only are present in a small fraction of cells. RESULTS: Here, the Brain Somatic Mosaicism Network conducts a coordinated, multi-institutional study to examine the ability of existing methods to detect simulated somatic single-nucleotide variants (SNVs) in DNA mixing experiments, generate multiple replicates of whole-genome sequencing data from the dorsolateral prefrontal cortex, other brain regions, dura mater, and dural fibroblasts of a single neurotypical individual, devise strategies to discover somatic SNVs, and apply various approaches to validate somatic SNVs. These efforts lead to the identification of 43 bona fide somatic SNVs that range in variant allele fractions from ~ 0.005 to ~ 0.28. Guided by these results, we devise best practices for calling mosaic SNVs from 250× whole-genome sequencing data in the accessible portion of the human genome that achieve 90% specificity and sensitivity. Finally, we demonstrate that analysis of multiple bulk DNA samples from a single individual allows the reconstruction of early developmental cell lineage trees. CONCLUSIONS: This study provides a unified set of best practices to detect somatic SNVs in non-cancerous tissues. The data and methods are freely available to the scientific community and should serve as a guide to assess the contributions of somatic SNVs to neuropsychiatric diseases.

MIPP-Seq: ultra-sensitive rapid detection and validation of low-frequency mosaic mutations.

BACKGROUND: Mosaic mutations contribute to numerous human disorders. As such, the identification and precise quantification of mosaic mutations is essential for a wide range of research applications, clinical diagnoses, and early detection of cancers. Currently, the low-throughput nature of single allele assays (e.g., allele-specific ddPCR) commonly used for genotyping known mutations at very low alternate allelic fractions (AAFs) have limited the integration of low-level mosaic analyses into clinical and research applications. The growing importance of mosaic mutations requires a more rapid, low-cost solution for mutation detection and validation. METHODS: To overcome these limitations, we developed Multiple Independent Primer PCR Sequencing (MIPP-Seq) which combines the power of ultra-deep sequencing and truly independent assays. The accuracy of MIPP-seq to quantifiable detect and measure extremely low allelic fractions was assessed using a combination of SNVs, insertions, and deletions at known allelic fractions in blood and brain derived DNA samples. RESULTS: The Independent amplicon analyses of MIPP-Seq markedly reduce the impact of allelic dropout, amplification bias, PCR-induced, and sequencing artifacts. Using low DNA inputs of either 25 ng or 50 ng of DNA, MIPP-Seq provides sensitive and quantitative assessments of AAFs as low as 0.025% for SNVs, insertion, and deletions. CONCLUSIONS: MIPP-Seq provides an ultra-sensitive, low-cost approach for detecting and validating known and novel mutations in a highly scalable system with broad utility spanning both research and clinical diagnostic testing applications. The scalability of MIPP-Seq allows for multiplexing mutations and samples, which dramatically reduce costs of variant validation when compared to methods like ddPCR. By leveraging the power of individual analyses of multiple unique and independent reactions, MIPP-Seq can validate and precisely quantitate extremely low AAFs across multiple tissues and mutational categories including both indels and SNVs. Furthermore, using Illumina sequencing technology, MIPP-seq provides a robust method for accurate detection of novel mutations at an extremely low AAF.

Large mosaic copy number variations confer autism risk.

Although germline de novo copy number variants (CNVs) are known causes of autism spectrum disorder (ASD), the contribution of mosaic (early-developmental) copy number variants (mCNVs) has not been explored. In this study, we assessed the contribution of mCNVs to ASD by ascertaining mCNVs in genotype array intensity data from 12,077 probands with ASD and 5,500 unaffected siblings. We detected 46 mCNVs in probands and 19 mCNVs in siblings, affecting 2.8-73.8% of cells. Probands carried a significant burden of large (>4-Mb) mCNVs, which were detected in 25 probands but only one sibling (odds ratio = 11.4, 95% confidence interval = 1.5-84.2, P = 7.4 × 10-4). Event size positively correlated with severity of ASD symptoms (P = 0.016). Surprisingly, we did not observe mosaic analogues of the short de novo CNVs recurrently observed in ASD (eg, 16p11.2). We further experimentally validated two mCNVs in postmortem brain tissue from 59 additional probands. These results indicate that mCNVs contribute a previously unexplained component of ASD risk.

The landscape of somatic mutation in cerebral cortex of autistic and neurotypical individuals revealed by ultra-deep whole-genome sequencing.

We characterize the landscape of somatic mutations-mutations occurring after fertilization-in the human brain using ultra-deep (~250×) whole-genome sequencing of prefrontal cortex from 59 donors with autism spectrum disorder (ASD) and 15 control donors. We observe a mean of 26 somatic single-nucleotide variants per brain present in ≥4% of cells, with enrichment of mutations in coding and putative regulatory regions. Our analysis reveals that the first cell division after fertilization produces ~3.4 mutations, followed by 2-3 mutations in subsequent generations. This suggests that a typical individual possesses ~80 somatic single-nucleotide variants present in ≥2% of cells-comparable to the number of de novo germline mutations per generation-with about half of individuals having at least one potentially function-altering somatic mutation somewhere in the cortex. ASD brains show an excess of somatic mutations in neural enhancer sequences compared with controls, suggesting that mosaic enhancer mutations may contribute to ASD risk.

Parallel RNA and DNA analysis after deep sequencing (PRDD-seq) reveals cell type-specific lineage patterns in human brain.

Elucidating the lineage relationships among different cell types is key to understanding human brain development. Here we developed parallel RNA and DNA analysis after deep sequencing (PRDD-seq), which combines RNA analysis of neuronal cell types with analysis of nested spontaneous DNA somatic mutations as cell lineage markers, identified from joint analysis of single-cell and bulk DNA sequencing by single-cell MosaicHunter (scMH). PRDD-seq enables simultaneous reconstruction of neuronal cell type, cell lineage, and sequential neuronal formation ("birthdate") in postmortem human cerebral cortex. Analysis of two human brains showed remarkable quantitative details that relate mutation mosaic frequency to clonal patterns, confirming an early divergence of precursors for excitatory and inhibitory neurons, and an "inside-out" layer formation of excitatory neurons as seen in other species. In addition our analysis allows an estimate of excitatory neuron-restricted precursors (about 10) that generate the excitatory neurons within a cortical column. Inhibitory neurons showed complex, subtype-specific patterns of neurogenesis, including some patterns of development conserved relative to mouse, but also some aspects of primate cortical interneuron development not seen in mouse. PRDD-seq can be broadly applied to characterize cell identity and lineage from diverse archival samples with single-cell resolution and in potentially any developmental or disease condition.

Accurate detection of mosaic variants in sequencing data without matched controls.

Detection of mosaic mutations that arise in normal development is challenging, as such mutations are typically present in only a minute fraction of cells and there is no clear matched control for removing germline variants and systematic artifacts. We present MosaicForecast, a machine-learning method that leverages read-based phasing and read-level features to accurately detect mosaic single-nucleotide variants and indels, achieving a multifold increase in specificity compared with existing algorithms. Using single-cell sequencing and targeted sequencing, we validated 80-90% of the mosaic single-nucleotide variants and 60-80% of indels detected in human brain whole-genome sequencing data. Our method should help elucidate the contribution of mosaic somatic mutations to the origin and development of disease.

Linked-read analysis identifies mutations in single-cell DNA-sequencing data.

Whole-genome sequencing of DNA from single cells has the potential to reshape our understanding of mutational heterogeneity in normal and diseased tissues. However, a major difficulty is distinguishing amplification artifacts from biologically derived somatic mutations. Here, we describe linked-read analysis (LiRA), a method that accurately identifies somatic single-nucleotide variants (sSNVs) by using read-level phasing with nearby germline heterozygous polymorphisms, thereby enabling the characterization of mutational signatures and estimation of somatic mutation rates in single cells.

Aging and neurodegeneration are associated with increased mutations in single human neurons.

It has long been hypothesized that aging and neurodegeneration are associated with somatic mutation in neurons; however, methodological hurdles have prevented testing this hypothesis directly. We used single-cell whole-genome sequencing to perform genome-wide somatic single-nucleotide variant (sSNV) identification on DNA from 161 single neurons from the prefrontal cortex and hippocampus of 15 normal individuals (aged 4 months to 82 years), as well as 9 individuals affected by early-onset neurodegeneration due to genetic disorders of DNA repair (Cockayne syndrome and xeroderma pigmentosum). sSNVs increased approximately linearly with age in both areas (with a higher rate in hippocampus) and were more abundant in neurodegenerative disease. The accumulation of somatic mutations with age-which we term genosenium-shows age-related, region-related, and disease-related molecular signatures and may be important in other human age-associated conditions.

Integrated genome and transcriptome sequencing identifies a noncoding mutation in the genome replication factor DONSON as the cause of microcephaly-micromelia syndrome.

While next-generation sequencing has accelerated the discovery of human disease genes, progress has been largely limited to the "low hanging fruit" of mutations with obvious exonic coding or canonical splice site impact. In contrast, the lack of high-throughput, unbiased approaches for functional assessment of most noncoding variants has bottlenecked gene discovery. We report the integration of transcriptome sequencing (RNA-seq), which surveys all mRNAs to reveal functional impacts of variants at the transcription level, into the gene discovery framework for a unique human disease, microcephaly-micromelia syndrome (MMS). MMS is an autosomal recessive condition described thus far in only a single First Nations population and causes intrauterine growth restriction, severe microcephaly, craniofacial anomalies, skeletal dysplasia, and neonatal lethality. Linkage analysis of affected families, including a very large pedigree, identified a single locus on Chromosome 21 linked to the disease (LOD > 9). Comprehensive genome sequencing did not reveal any pathogenic coding or canonical splicing mutations within the linkage region but identified several nonconserved noncoding variants. RNA-seq analysis detected aberrant splicing in DONSON due to one of these noncoding variants, showing a causative role for DONSON disruption in MMS. We show that DONSON is expressed in progenitor cells of embryonic human brain and other proliferating tissues, is co-expressed with components of the DNA replication machinery, and that Donson is essential for early embryonic development in mice as well, suggesting an essential conserved role for DONSON in the cell cycle. Our results demonstrate the utility of integrating transcriptomics into the study of human genetic disease when DNA sequencing alone is not sufficient to reveal the underlying pathogenic mutation.

Group II metabotropic glutamate receptor interactions with NHERF scaffold proteins: Implications for receptor localization in brain.

The group II metabotropic glutamate receptors mGluR2 and mGluR3 are key modulators of glutamatergic neurotransmission. In order to identify novel Group II metabotropic glutamate receptor (mGluR)-interacting partners, we screened the C-termini of mGluR2 and mGluR3 for interactions with an array of PDZ domains. These screens identified the Na+/H+ exchanger regulatory factors 1 and 2 (NHERF-1 & -2) as candidate interacting partners. Follow-up co-immunoprecipitation studies demonstrated that both mGluR2 and mGluR3 can associate with NHERF-1 and NHERF-2 in a cellular context. Functional studies revealed that disruption of PDZ interactions with mGluR2 enhanced receptor signaling to Akt. However, further studies of mGluR2 and mGluR3 signaling in astrocytes in which NHERF expression was reduced by gene knockout (KO) and/or siRNA knockdown techniques revealed that the observed differences in signaling between WT and mutant mGluR2 were likely not due to disruption of interactions with the NHERF proteins. Electron microscopic analyses revealed that Group II mGluRs were primarily expressed in glia and unmyelinated axons in WT, NHERF-1 and NHERF-2 KO mice, but the relative proportion of labeled axons over glial processes was higher in NHERF-2 KO mice than in controls and NHERF-1 KO mice. Interestingly, our anatomical studies also revealed that loss of either NHERF protein results in ventriculomegaly, which may be related to the high incidence of hydrocephaly that has previously been observed in NHERF-1 KO mice. Together, these studies support a role for NHERF-1 and NHERF-2 in regulating the distribution of Group II mGluRs in the murine brain, while conversely the effects of the mGluR2/3 PDZ-binding motifs on receptor signaling are likely mediated by interactions with other PDZ scaffold proteins beyond the NHERF proteins.

Intersection of diverse neuronal genomes and neuropsychiatric disease: The Brain Somatic Mosaicism Network.

Neuropsychiatric disorders have a complex genetic architecture. Human genetic population-based studies have identified numerous heritable sequence and structural genomic variants associated with susceptibility to neuropsychiatric disease. However, these germline variants do not fully account for disease risk. During brain development, progenitor cells undergo billions of cell divisions to generate the ~80 billion neurons in the brain. The failure to accurately repair DNA damage arising during replication, transcription, and cellular metabolism amid this dramatic cellular expansion can lead to somatic mutations. Somatic mutations that alter subsets of neuronal transcriptomes and proteomes can, in turn, affect cell proliferation and survival and lead to neurodevelopmental disorders. The long life span of individual neurons and the direct relationship between neural circuits and behavior suggest that somatic mutations in small populations of neurons can significantly affect individual neurodevelopment. The Brain Somatic Mosaicism Network has been founded to study somatic mosaicism both in neurotypical human brains and in the context of complex neuropsychiatric disorders.