Scalable Nanopore sequencing of human genomes provides a comprehensive view of haplotype-resolved variation and methylation.

Long-read sequencing technologies substantially overcome the limitations of short-reads but have not been considered as a feasible replacement for population-scale projects, being a combination of too expensive, not scalable enough or too error-prone. Here we develop an efficient and scalable wet lab and computational protocol, Napu, for Oxford Nanopore Technologies long-read sequencing that seeks to address those limitations. We applied our protocol to cell lines and brain tissue samples as part of a pilot project for the National Institutes of Health Center for Alzheimer's and Related Dementias. Using a single PromethION flow cell, we can detect single nucleotide polymorphisms with F1-score comparable to Illumina short-read sequencing. Small indel calling remains difficult within homopolymers and tandem repeats, but achieves good concordance to Illumina indel calls elsewhere. Further, we can discover structural variants with F1-score on par with state-of-the-art de novo assembly methods. Our protocol phases small and structural variants at megabase scales and produces highly accurate, haplotype-specific methylation calls.

Genome-wide structural variant analysis identifies risk loci for non-Alzheimer's dementias.

We characterized the role of structural variants, a largely unexplored type of genetic variation, in two non-Alzheimer's dementias, namely Lewy body dementia (LBD) and frontotemporal dementia (FTD)/amyotrophic lateral sclerosis (ALS). To do this, we applied an advanced structural variant calling pipeline (GATK-SV) to short-read whole-genome sequence data from 5,213 European-ancestry cases and 4,132 controls. We discovered, replicated, and validated a deletion in TPCN1 as a novel risk locus for LBD and detected the known structural variants at the C9orf72 and MAPT loci as associated with FTD/ALS. We also identified rare pathogenic structural variants in both LBD and FTD/ALS. Finally, we assembled a catalog of structural variants that can be mined for new insights into the pathogenesis of these understudied forms of dementia.

Scalable Nanopore sequencing of human genomes provides a comprehensive view of haplotype-resolved variation and methylation.

Long-read sequencing technologies substantially overcome the limitations of short-reads but to date have not been considered as feasible replacement at scale due to a combination of being too expensive, not scalable enough, or too error-prone. Here, we develop an efficient and scalable wet lab and computational protocol for Oxford Nanopore Technologies (ONT) long-read sequencing that seeks to provide a genuine alternative to short-reads for large-scale genomics projects. We applied our protocol to cell lines and brain tissue samples as part of a pilot project for the NIH Center for Alzheimer's and Related Dementias (CARD). Using a single PromethION flow cell, we can detect SNPs with F1-score better than Illumina short-read sequencing. Small indel calling remains to be difficult inside homopolymers and tandem repeats, but is comparable to Illumina calls elsewhere. Further, we can discover structural variants with F1-score comparable to state-of the-art methods involving Pacific Biosciences HiFi sequencing and trio information (but at a lower cost and greater throughput). Using ONT based phasing, we can then combine and phase small and structural variants at megabase scales. Our protocol also produces highly accurate, haplotype-specific methylation calls. Overall, this makes large-scale long-read sequencing projects feasible; the protocol is currently being used to sequence thousands of brain-based genomes as a part of the NIH CARD initiative. We provide the protocol and software as open-source integrated pipelines for generating phased variant calls and assemblies.

ATXN2 intermediate expansions in amyotrophic lateral sclerosis.

Intermediate CAG (polyQ) expansions in the gene ataxin-2 (ATXN2) are now recognized as a risk factor for amyotrophic lateral sclerosis. The threshold for increased risk is not yet firmly established, with reports ranging from 27 to 31 repeats. We investigated the presence of ATXN2 polyQ expansions in 9268 DNA samples collected from people with amyotrophic lateral sclerosis, amyotrophic lateral sclerosis with frontotemporal dementia, frontotemporal dementia alone, Lewy body dementia and age matched controls. This analysis confirmed ATXN2 intermediate polyQ expansions of ≥31 as a risk factor for amyotrophic lateral sclerosis with an odds ratio of 6.31. Expansions were an even greater risk for amyotrophic lateral sclerosis with frontotemporal dementia (odds ratio 27.59) and a somewhat lesser risk for frontotemporal dementia alone (odds ratio 3.14). There was no increased risk for Lewy body dementia. In a subset of 1362 patients with amyotrophic lateral sclerosis with complete clinical data, we could not confirm previous reports of earlier onset of amyotrophic lateral sclerosis or shorter survival in 25 patients with expansions. These new data confirm ≥31 polyQ repeats in ATXN2 increase the risk for amyotrophic lateral sclerosis, and also for the first time show an even greater risk for amyotrophic lateral sclerosis with frontotemporal dementia. The lack of a more aggressive phenotype in amyotrophic lateral sclerosis patients with expansions has implications for ongoing gene-silencing trials for amyotrophic lateral sclerosis.

Investigating RFC1 expansions in sporadic amyotrophic lateral sclerosis.

A homozygous AAGGG repeat expansion within the RFC1 gene was recently described as a common cause of CANVAS syndrome. We examined 1069 sporadic ALS patients for the presence of this repeat expansion. We did not discover any carriers of the homozygous AAGGG expansion in our ALS cohort, indicating that this form of RFC1 repeat expansions is not a common cause of sporadic ALS. However, our study did identify a novel repeat conformation and further expanded on the highly polymorphic nature of the RFC1 locus.

Genetic analysis of amyotrophic lateral sclerosis identifies contributing pathways and cell types.

Despite the considerable progress in unraveling the genetic causes of amyotrophic lateral sclerosis (ALS), we do not fully understand the molecular mechanisms underlying the disease. We analyzed genome-wide data involving 78,500 individuals using a polygenic risk score approach to identify the biological pathways and cell types involved in ALS. This data-driven approach identified multiple aspects of the biology underlying the disease that resolved into broader themes, namely, neuron projection morphogenesis, membrane trafficking, and signal transduction mediated by ribonucleotides. We also found that genomic risk in ALS maps consistently to GABAergic interneurons and oligodendrocytes, as confirmed in human single-nucleus RNA-seq data. Using two-sample Mendelian randomization, we nominated six differentially expressed genes (ATG16L2, ACSL5, MAP1LC3A, MAPKAPK3, PLXNB2, and SCFD1) within the significant pathways as relevant to ALS. We conclude that the disparate genetic etiologies of this fatal neurological disease converge on a smaller number of final common pathways and cell types.

Pathogenic Huntingtin Repeat Expansions in Patients with Frontotemporal Dementia and Amyotrophic Lateral Sclerosis.

We examined the role of repeat expansions in the pathogenesis of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) by analyzing whole-genome sequence data from 2,442 FTD/ALS patients, 2,599 Lewy body dementia (LBD) patients, and 3,158 neurologically healthy subjects. Pathogenic expansions (range, 40-64 CAG repeats) in the huntingtin (HTT) gene were found in three (0.12%) patients diagnosed with pure FTD/ALS syndromes but were not present in the LBD or healthy cohorts. We replicated our findings in an independent collection of 3,674 FTD/ALS patients. Postmortem evaluations of two patients revealed the classical TDP-43 pathology of FTD/ALS, as well as huntingtin-positive, ubiquitin-positive aggregates in the frontal cortex. The neostriatal atrophy that pathologically defines Huntington's disease was absent in both cases. Our findings reveal an etiological relationship between HTT repeat expansions and FTD/ALS syndromes and indicate that genetic screening of FTD/ALS patients for HTT repeat expansions should be considered.

Comparative clinical and genomic analysis of neurofibromatosis type 2-associated cranial and spinal meningiomas.

Neurofibromatosis type 2 (NF2) is an autosomal dominant Mendelian tumor predisposition disorder caused by germline pathogenic variants in the tumor suppressor NF2. Meningiomas are the second most common neoplasm in NF2, often occurring in multiple intracranial and spinal locations within the same patient. In this prospective longitudinal study, we assessed volumes and growth rates of ten spinal and ten cranial benign meningiomas in seven NF2 patients that concluded with surgical resection and performed whole-exome sequencing and copy-number variant (CNV) analysis of the tumors. Our comparison of the volume and the growth rate of NF2-associated spinal and cranial meningiomas point to the differences in timing of tumor initiation and/or to the differences in tumor progression (e.g., non-linear, saltatory growth) at these two anatomical locations. Genomic investigation of these tumors revealed that somatic inactivation of NF2 is the principal and perhaps the only driver of tumor initiation; and that tumor progression likely occurs via accumulation of CNVs, rather than point mutations. Results of this study contribute to a better understanding of NF2-associated meningiomas clinical behavior and their genetic underpinnings.

First insight into the somatic mutation burden of neurofibromatosis type 2-associated grade I and grade II meningiomas: a case report comprehensive genomic study of two cranial meningiomas with vastly different clinical presentation.

BACKGROUND: Neurofibromatosis type 2 (NF2) is a rare autosomal dominant nervous system tumor predisposition disorder caused by constitutive inactivation of one of the two copies of NF2. Meningiomas affect about one half of NF2 patients, and are associated with a higher disease burden. Currently, the somatic mutation landscape in NF2-associated meningiomas remains largely unexamined. CASE PRESENTATION: Here, we present an in-depth genomic study of benign and atypical meningiomas, both from a single NF2 patient. While the grade I tumor was asymptomatic, the grade II tumor exhibited an unusually high growth rate: expanding to 335 times its initial volume within one year. The genomes of both tumors were examined by whole-exome sequencing (WES) complemented with spectral karyotyping (SKY) and SNP-array copy-number analyses. To better understand the clonal composition of the atypical meningioma, the tumor was divided in four sections and each section was investigated independently. Both tumors had second copy inactivation of NF2, confirming the central role of the gene in meningioma formation. The genome of the benign tumor closely resembled that of a normal diploid cell and had only one other deleterious mutation (EPHB3). In contrast, the chromosomal architecture of the grade II tumor was highly re-arranged, yet uniform among all analyzed fragments, implying that this large and fast growing tumor was composed of relatively few clones. Besides multiple gains and losses, the grade II meningioma harbored numerous chromosomal translocations. WES analysis of the atypical tumor identified deleterious mutations in two genes: ADAMTSL3 and CAPN5 in all fragments, indicating that the mutations were present in the cell undergoing fast clonal expansion CONCLUSIONS: This is the first WES study of NF2-associated meningiomas. Besides second NF2 copy inactivation, we found low somatic burden in both tumors and high level of genomic instability in the atypical meningioma. Genomic instability resulting in altered gene dosage and compromised structural integrity of multiple genes may be the primary reason of the high growth rate for the grade II tumor. Further study of ADAMTSL3 and CAPN5 may lead to elucidation of their molecular implications in meningioma pathogenesis.

Mice Lacking M1 and M3 Muscarinic Acetylcholine Receptors Have Impaired Odor Discrimination and Learning.

The cholinergic system has extensive projections to the olfactory bulb (OB) where it produces a state-dependent regulation of sensory gating. Previous work has shown a prominent role of muscarinic acetylcholine (ACh) receptors (mAChRs) in regulating the excitability of OB neurons, in particular the M1 receptor. Here, we examined the contribution of M1 and M3 mAChR subtypes to olfactory processing using mice with a genetic deletion of these receptors, the M1-/- and the M1/M3-/- knockout (KO) mice. Genetic ablation of the M1 and M3 mAChRs resulted in a significant deficit in odor discrimination of closely related molecules, including stereoisomers. However, the discrimination of dissimilar molecules, social odors (e.g., urine) and novel object recognition was not affected. In addition the KO mice showed impaired learning in an associative odor-learning task, learning to discriminate odors at a slower rate, indicating that both short and long-term memory is disrupted by mAChR dysfunction. Interestingly, the KO mice exhibited decreased olfactory neurogenesis at younger ages, a deficit that was not maintained in older animals. In older animals, the olfactory deficit could be restored by increasing the number of new born neurons integrated into the OB after exposing them to an olfactory enriched environment, suggesting that muscarinic modulation and adult neurogenesis could be two different mechanism used by the olfactory system to improve olfactory processing.

Whole exome sequencing reveals a C-terminal germline variant in CEBPA-associated acute myeloid leukemia: 45-year follow up of a large family.

Familial acute myeloid leukemia is rare and linked to germline mutations in RUNX1, GATA2 or CCAAT/enhancer binding protein-α (CEBPA). We re-evaluated a large family with acute myeloid leukemia originally seen at NIH in 1969. We used whole exome sequencing to study this family, and conducted in silico bioinformatics analysis, protein structural modeling and laboratory experiments to assess the impact of the identified CEBPA Q311P mutation. Unlike most previously identified germline mutations in CEBPA, which were N-terminal frameshift mutations, we identified a novel Q311P variant that was located in the C-terminal bZip domain of C/EBPα. Protein structural modeling suggested that the Q311P mutation alters the ability of the CEBPA dimer to bind DNA. Electrophoretic mobility shift assays showed that the Q311P mu-tant had attenuated binding to DNA, as predicted by the protein modeling. Consistent with these findings, we found that the Q311P mutation has reduced transactivation, consistent with a loss-of-function mutation. From 45 years of follow up, we observed incomplete penetrance (46%) of CEBPA Q311P. This study of a large multi-generational pedigree reveals that a germline mutation in the C-terminal bZip domain can alter the ability of C/EBP-α to bind DNA and reduces transactivation, leading to acute myeloid leukemia.

Tumors displaying hybrid schwannoma and neurofibroma features in patients with neurofibromatosis type 2.

Although schwannoma and neurofibroma tumors are generally reported as distinct pathologic diagnoses, sporadic schwannoma/neurofibroma hybrid nerve sheath tumors have been reported in the general population with components of both entities. We report the clinicopathological features of these hybrid nerve sheath tumors in patients with neurofibromatosis type 2 (NF2). A retrospective review of nerve sheath tumor surgical specimens from patients with NF2 enrolled at the National Institutes of Health was performed. Those specimens reported to have schwannoma-like and neurofibromalike features were selected for further characterization by morphology, immunohistochemical panel (CD34, S100, neurofilament triplet protein (immunostain) (NFTP), epithelial membrane antigen (EMA)), and confirmation as hybrid tumors. Of 43 total NF2 patients undergoing resection of nerve sheath tumors, 11 specimens from 11 (26%) patients were found to be benign nerve sheath tumors exhibiting hybrid features of both neurofibroma and schwannoma. Immunohistochemical studies showed the schwannoma component to be S100+, CD 34- while the neurofibroma component was CD34+, variable S100+. Our experience emphasizes the importance of including this distinct tumor subtype, the schwannoma/neurofibroma hybrid tumor, in the differential diagnosis of nerve sheath tumors in NF2 patients and suggests that the relationship between neurofibroma and schwannoma tumors is closer than previously suspected..

Juvenile myelomonocytic leukemia due to a germline CBL Y371C mutation: 35-year follow-up of a large family.

Juvenile myelomonocytic leukemia (JMML) is a pediatric myeloproliferative neoplasm that arises from malignant transformation of the stem cell compartment and results in increased production of myeloid cells. Somatic and germline variants in CBL (Casitas B-lineage lymphoma proto-oncogene) have been associated with JMML. We report an incompletely penetrant CBL Y371C mutation discovered by whole-exome sequencing in three individuals with JMML in a large pedigree with 35 years of follow-up. The Y371 residue is highly evolutionarily conserved among CBL orthologs and paralogs. In silico bioinformatics prediction programs suggested that the Y371C mutation is highly deleterious. Protein structural modeling revealed that the Y371C mutation abrogated the ability of the CBL protein to adopt a conformation that is required for ubiquitination. Clinically, the three mutation-positive JMML individuals exhibited variable clinical courses; in two out of three, primary hematologic abnormalities persisted into adulthood with minimal clinical symptoms. The penetrance of the CBL Y371C mutation was 30% for JMML and 40% for all leukemia. Of the 8 mutation carriers in the family with available photographs, only one had significant dysmorphic features; we found no evidence of a clinical phenotype consistent with a "CBL syndrome". Although CBL Y371C has been previously reported in familial JMML, we are the first group to follow a complete pedigree harboring this mutation for an extended period, revealing additional information about this variant's penetrance, function and natural history.

Evidence of polyclonality in neurofibromatosis type 2-associated multilobulated vestibular schwannomas.

BACKGROUND: Neurofibromatosis type 2 (NF2) is a tumor syndrome that results from mutation of the NF2 tumor suppressor gene. The hallmark of NF2 is the presence of bilateral vestibular schwannoma (VS). Though NF2-associated and sporadic VS share identical histopathologic findings and cytogenetic alterations, NF2-associated VS often appears multilobulated, is less responsive to radiosurgery, and has worse surgical outcomes. Temporal bone autopsy specimens and MRI of the inner ear performed on NF2 patients suggest that multiple discrete tumors may be present within the labyrinth and cerebellopontine angle. METHODS: Treatment-naïve ears in patients enrolled in a prospective NF2 natural history study (NIH#08-N-0044) were included for MRI analysis. T2-weighted and postcontrast T1-weighted MRIs were evaluated for the presence of multiple discrete tumors or a multilobulated mass. Peripheral blood (germline) and regional samples of tumor tissue were procured from consecutive patients enrolled in this study undergoing resection of a multilobulated VS (MVS). Histopathologic evaluation and genetic analysis (single nucleotide polymorphism array analysis, NF2 sequencing) were performed on each specimen. RESULTS: Over half of NF2 ears harbored either an MVS (60/139 ears) or multiple discrete masses (19/139 ears). For 4 successive MVSs, genetic analysis revealed an admixture of cell populations, each with its own somatic NF2 mutation or deletion. CONCLUSIONS: These findings suggest that the majority of NF2-associated VSs are polyclonal, such that the tumor mass represents a collision of multiple, distinct tumor clones. This explains the characteristic lobulated gross appearance of NF2-associated VS, and may also explain the substantially different treatment outcomes compared with sporadic VS.

Audiovestibular Characteristics of Small Cochleovestibular Schwannomas in Neurofibromatosis Type 2.

OBJECTIVE: Describe the relationship between cochleovestibular schwannoma (CVS) volume, audiovestibular characteristics, and magnetic resonance imaging (MRI) findings in patients with neurofibromatosis type 2 (NF2). STUDY DESIGN: Subgroup analysis of NF2 prospective natural history study from 2008 to 2011. SETTING: Quaternary medical research institute. SUBJECTS AND METHODS: NF2 patients with small treatment-naive CVSs (volume <1000 mm(3)) by ear; N = 49 ears (32 patients). Cross-sectional analysis of the following parameters was performed: tumor size, auditory brainstem response (ABR), 4-frequency pure-tone average (4f-PTA; 0.5, 1, 2, and 4KHz), cervical vestibular evoked myogenic potential (cVEMP), caloric testing, 240° velocity step test (VST), and MRI findings. RESULTS: For all physiologic measures but the 4f-PTA, larger tumors correlated with abnormal responses (P < .05). For abnormal ABR, mean tumor volume was 405 vs 151 mm(3) (P = .0007) for normal ABR. Similarly, larger tumors correlated with weak caloric responses (mean 521 vs 165 mm(3); P = .0007) and weak cVEMP (mean 357 vs 192 mm(3); P = .0262). Tumor volume was not significantly correlated with 4f-PTA. Elevated intralabyrinthine protein on MRI fluid-attenuated inversion recovery sequences was correlated with larger tumor volume (mean 333 vs 55 mm(3); P = .001) and abnormal ABR and 4f-PTA (P < .05) but did not correlate with cVEMP, VST, or caloric responses. CONCLUSION: In our cohort, ABR, caloric response, cVEMP, and elevated intralabyrinthine protein correlated with tumor volume, but 4f-PTA did not. Abnormal ABR and 4f-PTA correlated with elevated intralabyrinthine protein. These findings may provide insight on the effect of small CVS on the inner ear and cochleovestibular nerves, which may aid in their optimal management.