Amyotrophic lateral sclerosis onset is influenced by the burden of rare variants in known amyotrophic lateral sclerosis genes.

OBJECTIVE: To define the genetic landscape of amyotrophic lateral sclerosis (ALS) and assess the contribution of possible oligogenic inheritance, we aimed to comprehensively sequence 17 known ALS genes in 391 ALS patients from the United States. METHODS: Targeted pooled-sample sequencing was used to identify variants in 17 ALS genes. Fragment size analysis was used to define ATXN2 and C9ORF72 expansion sizes. Genotype-phenotype correlations were made with individual variants and total burden of variants. Rare variant associations for risk of ALS were investigated at both the single variant and gene level. RESULTS: A total of 64.3% of familial and 27.8% of sporadic subjects carried potentially pathogenic novel or rare coding variants identified by sequencing or an expanded repeat in C9ORF72 or ATXN2; 3.8% of subjects had variants in >1 ALS gene, and these individuals had disease onset 10 years earlier (p = 0.0046) than subjects with variants in a single gene. The number of potentially pathogenic coding variants did not influence disease duration or site of onset. INTERPRETATION: Rare and potentially pathogenic variants in known ALS genes are present in >25% of apparently sporadic and 64% of familial patients, significantly higher than previous reports using less comprehensive sequencing approaches. A significant number of subjects carried variants in >1 gene, which influenced the age of symptom onset and supports oligogenic inheritance as relevant to disease pathogenesis.

Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration.

Expanded hexanucleotide repeats in the chromosome 9 open reading frame 72 (C9orf72) gene are the most common genetic cause of ALS and frontotemporal degeneration (FTD). Here, we identify nuclear RNA foci containing the hexanucleotide expansion (GGGGCC) in patient cells, including white blood cells, fibroblasts, glia, and multiple neuronal cell types (spinal motor, cortical, hippocampal, and cerebellar neurons). RNA foci are not present in sporadic ALS, familial ALS/FTD caused by other mutations (SOD1, TDP-43, or tau), Parkinson disease, or nonneurological controls. Antisense oligonucleotides (ASOs) are identified that reduce GGGGCC-containing nuclear foci without altering overall C9orf72 RNA levels. By contrast, siRNAs fail to reduce nuclear RNA foci despite marked reduction in overall C9orf72 RNAs. Sustained ASO-mediated lowering of C9orf72 RNAs throughout the CNS of mice is demonstrated to be well tolerated, producing no behavioral or pathological features characteristic of ALS/FTD and only limited RNA expression alterations. Genome-wide RNA profiling identifies an RNA signature in fibroblasts from patients with C9orf72 expansion. ASOs targeting sense strand repeat-containing RNAs do not correct this signature, a failure that may be explained, at least in part, by discovery of abundant RNA foci with C9orf72 repeats transcribed in the antisense (GGCCCC) direction, which are not affected by sense strand-targeting ASOs. Taken together, these findings support a therapeutic approach by ASO administration to reduce hexanucleotide repeat-containing RNAs and raise the potential importance of targeting expanded RNAs transcribed in both directions.

Genetic effects at pleiotropic loci are context-dependent with consequences for the maintenance of genetic variation in populations.

Context-dependent genetic effects, including genotype-by-environment and genotype-by-sex interactions, are a potential mechanism by which genetic variation of complex traits is maintained in populations. Pleiotropic genetic effects are also thought to play an important role in evolution, reflecting functional and developmental relationships among traits. We examine context-dependent genetic effects at pleiotropic loci associated with normal variation in multiple metabolic syndrome (MetS) components (obesity, dyslipidemia, and diabetes-related traits). MetS prevalence is increasing in Western societies and, while environmental in origin, presents substantial variation in individual response. We identify 23 pleiotropic MetS quantitative trait loci (QTL) in an F(16) advanced intercross between the LG/J and SM/J inbred mouse strains (Wustl:LG,SM-G16; n = 1002). Half of each family was fed a high-fat diet and half fed a low-fat diet; and additive, dominance, and parent-of-origin imprinting genotypic effects were examined in animals partitioned into sex, diet, and sex-by-diet cohorts. We examine the context-dependency of the underlying additive, dominance, and imprinting genetic effects of the traits associated with these pleiotropic QTL. Further, we examine sequence polymorphisms (SNPs) between LG/J and SM/J as well as differential expression of positional candidate genes in these regions. We show that genetic associations are different in different sex, diet, and sex-by-diet settings. We also show that over- or underdominance and ecological cross-over interactions for single phenotypes may not be common, however multidimensional synthetic phenotypes at loci with pleiotropic effects can produce situations that favor the maintenance of genetic variation in populations. Our findings have important implications for evolution and the notion of personalized medicine.

Investigative genetic genealogy for human remains identification.

Investigative genetic genealogy (IGG) has emerged as a highly effective tool for tying a forensic DNA sample to an identity. While much of the attention paid to IGG has focused on cases where the DNA is from an unknown suspect, IGG has also been used to help close hundreds of unidentified human remains (UHR) cases. Genome-wide single-nucleotide polymorphism (SNP) genotype data can be obtained from forensic samples using microarray genotyping or whole-genome sequencing (WGS) with protocols optimized for degraded DNA. After bioinformatic processing, the SNP data can be uploaded to public GG databases that allow law enforcement usage, where it can be compared with other users' data to find distant relatives. A genetic genealogist can then build the family trees of the relatives to narrow down the identity of the source of the forensic DNA sample. To date, 367 UHR identifications using IGG have been publicly announced. The same IGG techniques developed and refined for UHR cases have significant potential for disaster victim identification, where DNA is often extremely compromised, and close family references may not be available. This paper reviews the laboratory, bioinformatic, and genealogical techniques used in IGG for UHR cases and presents three case studies that demonstrate how IGG is assisting with remains identification.

Epistatic Features and Machine Learning Improve Alzheimer's Disease Risk Prediction Over Polygenic Risk Scores.

Background: Polygenic risk scores (PRS) are linear combinations of genetic markers weighted by effect size that are commonly used to predict disease risk. For complex heritable diseases such as late-onset Alzheimer's disease (LOAD), PRS models fail to capture much of the heritability. Additionally, PRS models are highly dependent on the population structure of the data on which effect sizes are assessed and have poor generalizability to new data. Objective: The goal of this study is to construct a paragenic risk score that, in addition to single genetic marker data used in PRS, incorporates epistatic interaction features and machine learning methods to predict risk for LOAD. Methods: We construct a new state-of-the-art genetic model for risk of Alzheimer's disease. Our approach innovates over PRS models in two ways: First, by directly incorporating epistatic interactions between SNP loci using an evolutionary algorithm guided by shared pathway information; and second, by estimating risk via an ensemble of non-linear machine learning models rather than a single linear model. We compare the paragenic model to several PRS models from the literature trained on the same dataset. Results: The paragenic model is significantly more accurate than the PRS models under 10-fold cross-validation, obtaining an AUC of 83% and near-clinically significant matched sensitivity/specificity of 75%. It remains significantly more accurate when evaluated on an independent holdout dataset and maintains accuracy within APOE genotype strata. Conclusions: Paragenic models show potential for improving disease risk prediction for complex heritable diseases such as LOAD over PRS models.

Familial isolated clubfoot is associated with recurrent chromosome 17q23.1q23.2 microduplications containing TBX4.

Clubfoot is a common musculoskeletal birth defect for which few causative genes have been identified. To identify the genes responsible for isolated clubfoot, we screened for genomic copy-number variants with the Affymetrix Genome-wide Human SNP Array 6.0. A recurrent chromosome 17q23.1q23.2 microduplication was identified in 3 of 66 probands with familial isolated clubfoot. The chromosome 17q23.1q23.2 microduplication segregated with autosomal-dominant clubfoot in all three families but with reduced penetrance. Mild short stature was common and one female had developmental hip dysplasia. Subtle skeletal abnormalities consisted of broad and shortened metatarsals and calcanei, small distal tibial epiphyses, and thickened ischia. Several skeletal features were opposite to those described in the reciprocal chromosome 17q23.1q23.2 microdeletion syndrome associated with developmental delay and cardiac and limb abnormalities. Of note, during our study, we also identified a microdeletion at the locus in a sibling pair with isolated clubfoot. The chromosome 17q23.1q23.2 region contains the T-box transcription factor TBX4, a likely target of the bicoid-related transcription factor PITX1 previously implicated in clubfoot etiology. Our result suggests that this chromosome 17q23.1q23.2 microduplication is a relatively common cause of familial isolated clubfoot and provides strong evidence linking clubfoot etiology to abnormal early limb development.

Epistatic Features and Machine Learning Improve Alzheimer's Risk Prediction Over Polygenic Risk Scores.

Background: Polygenic risk scores (PRS) are linear combinations of genetic markers weighted by effect size that are commonly used to predict disease risk. For complex heritable diseases such as late onset Alzheimer's disease (LOAD), PRS models fail to capture much of the heritability. Additionally, PRS models are highly dependent on the population structure of data on which effect sizes are assessed, and have poor generalizability to new data. Objective: The goal of this study is to construct a paragenic risk score that, in addition to single genetic marker data used in PRS, incorporates epistatic interaction features and machine learning methods to predict lifetime risk for LOAD. Methods: We construct a new state-of-the-art genetic model for lifetime risk of Alzheimer's disease. Our approach innovates over PRS models in two ways: First, by directly incorporating epistatic interactions between SNP loci using an evolutionary algorithm guided by shared pathway information; and second, by estimating risk via an ensemble of machine learning models (gradient boosting machines and deep learning) instead of simple logistic regression. We compare the paragenic model to a PRS model from the literature trained on the same dataset. Results: The paragenic model is significantly more accurate than the PRS model under 10-fold cross-validation, obtaining an AUC of 83% and near-clinically significant matched sensitivity/specificity of 75%, and remains significantly more accurate when evaluated on an independent holdout dataset. Additionally, the paragenic model maintains accuracy within APOE genotypes. Conclusion: Paragenic models show potential for improving lifetime disease risk prediction for complex heritable diseases such as LOAD over PRS models.

Extended kinship analysis of historical remains using SNP capture.

DNA-assisted identification of historical remains requires the genetic analysis of highly degraded DNA, along with a comparison to DNA from known relatives. This can be achieved by targeting single nucleotide polymorphisms (SNPs) using a hybridization capture and next-generation sequencing approach suitable for degraded skeletal samples. In the present study, two SNP capture panels were designed to target ~ 25,000 (25 K) and ~ 95,000 (95 K) nuclear SNPs, respectively, to enable distant kinship estimation (up to 4th degree relatives). Low-coverage SNP data were successfully recovered from 14 skeletal elements 75 years postmortem using an Illumina MiSeq benchtop sequencer. All samples contained degraded DNA but were of varying quality with mean fragment lengths ranging from 32 bp to 170 bp across the 14 samples. SNP comparison with DNA from known family references was performed in the Parabon Fx Forensic Analysis Platform, which utilizes a likelihood approach for kinship prediction that was optimized for low-coverage sequencing data with cytosine deamination. The 25 K panel produced 15,000 SNPs on average, which allowed for accurate kinship prediction with strong statistical support in 16 of the 21 pairwise comparisons. The 95 K panel increased the average SNPs to 42,000 and resulted in an additional accurate kinship prediction with strong statistical support (17 of 21 pairwise comparisons). This study demonstrates that SNP capture combined with massively parallel sequencing on a benchtop platform can yield sufficient SNP recovery from compromised samples, enabling accurate, extended kinship predictions.

A Qualitative Study of Factors That Influence Contraceptive Choice among Adolescent School-Based Health Center Patients.

STUDY OBJECTIVE: Long-acting reversible contraceptive (LARC) methods can prevent teen pregnancy yet remain underutilized by adolescents in the United States. Pediatric providers are well positioned to discuss LARCs with adolescents, but little is known about how counseling should occur in pediatric primary care settings. We explored adolescent womens' attitudes and experiences with LARCs to inform the development of adolescent-centered LARC counseling strategies. DESIGN: Qualitative analysis of one-on-one interviews. SETTING: Participants were recruited from 2 urban school-based, primary care centers. PARTICIPANTS: Thirty adolescent women aged 14-18 years, diverse in race/ethnicity, and sexual experience. INTERVENTIONS: Interviews were audio-recorded, transcribed, and coded using inductive and deductive coding. MAIN OUTCOME MEASURE: Major themes were identified to integrate LARC-specific adolescent preferences into existing counseling approaches. RESULTS: Participants (mean age, 16.2 years; range, 14-18 years) represented a diverse range of racial and/or ethnic identities. Half (15/30) were sexually active and 17% (5/30) reported current or past LARC use. Five themes emerged regarding key factors that influence LARC choice, including: (1) strong preferences about device-specific characteristics; (2) previous exposure to information about LARCs from peers, family members, or health counseling sessions; (3) knowledge gaps about LARC methods that affect informed decision-making; (4) personal circumstances or experiences that motivate a desire for effective and/or long-acting contraception; and (5) environmental constraints and supports that might influence adolescent access to LARCs. CONCLUSION: We identified 5 factors that influence LARC choice among adolescent women and propose a framework for incorporating these factors into contraceptive counseling services in pediatric primary care settings.

Knowledge and Acceptability of Long-Acting Reversible Contraception Among Adolescent Women Receiving School-Based Primary Care Services.

BACKGROUND: A key strategy to reduce unintended adolescent pregnancies is to expand access to long-acting reversible contraceptive (LARC) methods, including intrauterine devices and subdermal contraceptive implants. LARC services can be provided to adolescents in school-based health and other primary care settings, yet limited knowledge and negative attitudes about LARC methods may influence adolescents' utilization of these methods. This study aimed to evaluate correlates of knowledge and acceptability of LARC methods among adolescent women at a school-based health center (SBHC). METHODS: In this cross-sectional study, female patients receiving care at 2 SBHCs in Seattle, Washington completed an electronic survey about sexual and reproductive health. Primary outcomes were (1) LARC knowledge as measured by percentage correct of 10 true-false questions and (2) LARC acceptability as measured by participants reporting either liking the idea of having an intrauterine device (IUD)/subdermal implant or currently using one. RESULTS: A total of 102 students diverse in race/ethnicity and socioeconomic backgrounds completed the survey (mean age 16.2 years, range 14.4-19.1 years). Approximately half reported a lifetime history of vaginal sex. Greater LARC knowledge was associated with white race (regression coefficient [coef] = 26.8; 95% CI 13.3-40.4; P < .001), history of vaginal intercourse (coef = 29.9; 95% CI 17.1-42.7; P < .001), and current/prior LARC use (coef = 22.8; 95% CI 6.5-40.0; P = .007). Older age was associated with lower IUD acceptability (odds ratio = 0.53, 95% CI 0.30-0.94; P = .029) while history of intercourse was associated with greater implant acceptability (odds ratio 5.66, 95% CI 1.46-22.0; P = .012). DISCUSSION: Adolescent women in this SBHC setting had variable knowledge and acceptability of LARC. A history of vaginal intercourse was the strongest predictor of LARC acceptability. Our findings suggest a need for LARC counseling and education strategies, particularly for young women from diverse cultural backgrounds and those with less sexual experience.

Providing long-acting reversible contraception services in Seattle school-based health centers: key themes for facilitating implementation.

PURPOSE: The purpose of this study was to describe the implementation of a program that provides long-acting reversible contraception (LARC) services within school-based health centers (SBHCs) and to identify barriers and facilitators to implementation as reported by SBHC clinicians and administrators, public health officials, and community partners. METHODS: We conducted 14 semistructured interviews with key informants involved in the implementation of LARC services. Key informants included SBHC clinicians and administrators, public health officials, and community partners. We used a content analysis approach to analyze interview transcripts for themes. We explored barriers to and facilitators of LARC service delivery across and within key informant groups. RESULTS: The most cited barriers across key informant groups were as follows: perceived lack of provider procedural skills and bias and negative attitudes about LARC methods. The most common facilitators identified across groups were as follows: clear communication strategies, contraceptive counseling practice changes, provider trainings, and stakeholder engagement. Two additional barriers emerged in specific key informant groups. Technical and logistical barriers to LARC service delivery were cited heavily by SBHC administrative staff, community partners, and public health officials. Expense and billing was a major barrier to SBHC administrative staff. CONCLUSIONS: LARC counseling and procedural services can be implemented in an SBHC setting to promote access to effective contraceptive options for adolescent women.

C9orf72 BAC Transgenic Mice Display Typical Pathologic Features of ALS/FTD.

Noncoding expansions of a hexanucleotide repeat (GGGGCC) in the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia. Here we report transgenic mice carrying a bacterial artificial chromosome (BAC) containing the full human C9orf72 gene with either a normal allele (15 repeats) or disease-associated expansion (∼100-1,000 repeats; C9-BACexp). C9-BACexp mice displayed pathologic features seen in C9orf72 expansion patients, including widespread RNA foci and repeat-associated non-ATG (RAN) translated dipeptides, which were suppressed by antisense oligonucleotides targeting human C9orf72. Nucleolin distribution was altered, supporting that either C9orf72 transcripts or RAN dipeptides promote nucleolar dysfunction. Despite early and widespread production of RNA foci and RAN dipeptides in C9-BACexp mice, behavioral abnormalities and neurodegeneration were not observed even at advanced ages, supporting the hypothesis that RNA foci and RAN dipeptides occur presymptomatically and are not sufficient to drive neurodegeneration in mice at levels seen in patients.

Exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways.

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment. We report the results of a moderate-scale sequencing study aimed at increasing the number of genes known to contribute to predisposition for ALS. We performed whole-exome sequencing of 2869 ALS patients and 6405 controls. Several known ALS genes were found to be associated, and TBK1 (the gene encoding TANK-binding kinase 1) was identified as an ALS gene. TBK1 is known to bind to and phosphorylate a number of proteins involved in innate immunity and autophagy, including optineurin (OPTN) and p62 (SQSTM1/sequestosome), both of which have also been implicated in ALS. These observations reveal a key role of the autophagic pathway in ALS and suggest specific targets for therapeutic intervention.

TREM2 variant p.R47H as a risk factor for sporadic amyotrophic lateral sclerosis.

IMPORTANCE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which microglia play a significant and active role. Recently, a rare missense variant (p.R47H) in the microglial activating gene TREM2 was found to increase the risk of several neurodegenerative diseases, including Alzheimer disease. Whether the p.R47H variant is a risk factor for ALS is not known. OBJECTIVES: To determine whether p.R47H (rs75932628) in TREM2 is a risk factor for ALS and assess whether TREM2 expression is dysregulated in disease. DESIGN, SETTING, AND PARTICIPANTS: Samples of DNA from 923 individuals with sporadic ALS and 1854 healthy control individuals self-reported as non-Hispanic white were collected from ALS clinics in the United States and genotyped for the p.R47H variant in TREM2. Clinical data were obtained on ALS participants for genotype/phenotype correlations. Expression of TREM2 was measured by quantitative polymerase chain reaction and compared in spinal cord samples from 18 autopsied patients with ALS and 12 neurologically healthy controls, as well as from wild-type and transgenic SOD1G93A mice. MAIN OUTCOMES AND MEASURES: Minor allele frequency of rs75932628 and relative expression of TREM2. RESULTS: The TREM2 variant p.R47H was more common in patients with ALS than in the controls and is therefore a significant risk factor for ALS (odds ratio, 2.40; 95% CI, 1.29-4.15; P = 4.1×10-3). Furthermore, TREM2 expression was increased in spinal cord samples from ALS patients and SOD1G93A mice (P = 2.8×10-4 and P = 2.8×10-9, respectively), confirming dysregulated TREM2 in disease. Expression of TREM2 in the human spinal cord was negatively correlated with survival (P = .04) but not with other phenotypic aspects of disease. CONCLUSIONS AND RELEVANCE: This study demonstrates that the TREM2 p.R47H variant is a potent risk factor for sporadic ALS. To our knowledge, these findings identify the first genetic influence on neuroinflammation in ALS and highlight the TREM2 signaling pathway as a therapeutic target in ALS and other neurodegenerative diseases.

Lack of C9ORF72 coding mutations supports a gain of function for repeat expansions in amyotrophic lateral sclerosis.

Hexanucleotide repeat expansions in C9ORF72 are a common cause of familial and apparently sporadic amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). The mechanism by which expansions cause neurodegeneration is unknown, but current evidence supports both loss-of-function and gain-of-function mechanisms. We used pooled next-generation sequencing of the C9ORF72 gene in 389 ALS patients to look for traditional loss-of-function mutations. Although rare variants were identified, none were likely to be pathogenic, suggesting that mutations other than the repeat expansion are not a common cause of ALS, and providing supportive evidence for a gain-of-function mechanism. We also show by repeat-primed PCR genotyping that the C9ORF72 expansion frequency varies by geographical region within the United States, with an unexpectedly high frequency in the Mid-West. Finally we also show evidence of somatic instability of the expansion size by Southern blot, with the largest expansions occurring in brain tissue.

Expression of novel Alzheimer's disease risk genes in control and Alzheimer's disease brains.

Late onset Alzheimer's disease (LOAD) etiology is influenced by complex interactions between genetic and environmental risk factors. Large-scale genome wide association studies (GWAS) for LOAD have identified 10 novel risk genes: ABCA7, BIN1, CD2AP, CD33, CLU, CR1, EPHA1, MS4A6A, MS4A6E, and PICALM. We sought to measure the influence of GWAS single nucleotide polymorphisms (SNPs) and gene expression levels on clinical and pathological measures of AD in brain tissue from the parietal lobe of AD cases and age-matched, cognitively normal controls. We found that ABCA7, CD33, and CR1 expression levels were associated with clinical dementia rating (CDR), with higher expression being associated with more advanced cognitive decline. BIN1 expression levels were associated with disease progression, where higher expression was associated with a delayed age at onset. CD33, CLU, and CR1 expression levels were associated with disease status, where elevated expression levels were associated with AD. Additionally, MS4A6A expression levels were associated with Braak tangle and Braak plaque scores, with elevated expression levels being associated with more advanced brain pathology. We failed to detect an association between GWAS SNPs and gene expression levels in our brain series. The minor allele of rs3764650 in ABCA7 is associated with age at onset and disease duration, and the minor allele of rs670139 in MS4A6E was associated with Braak tangle and Braak plaque score. These findings suggest that expression of some GWAS genes, namely ABCA7, BIN1, CD33, CLU, CR1 and the MS4A family, are altered in AD brains.