SnapShot: Genetics of ALS and FTD.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are considered to be part of a spectrum. Clinically, FTD patients present with dementia frequently characterized by behavioral and speech problems. ALS patients exhibit alterations of voluntary movements caused by degeneration of motor neurons. Both syndromes can be present within the same family or even in the same person. The genetic findings for both diseases also support the existence of a continuum, with mutations in the same genes being found in patients with FTD, ALS, or FTD/ALS.

In situ proximity labeling identifies Lewy pathology molecular interactions in the human brain.

The intracellular misfolding and accumulation of alpha-synuclein into structures collectively called Lewy pathology (LP) is a central phenomenon for the pathogenesis of synucleinopathies, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Understanding the molecular architecture of LP is crucial for understanding synucleinopathy disease origins and progression. Here we used a technique called biotinylation by antibody recognition (BAR) to label total (BAR-SYN1) and pathological alpha-synuclein (BAR-PSER129) in situ for subsequent mass spectrometry analysis. Results showed superior immunohistochemical detection of LP following the BAR-PSER129 protocol, particularly for fibers and punctate pathology within the striatum and cortex. Mass spectrometry analysis of BAR-PSER129-labeled LP identified 261 significantly enriched proteins in the synucleinopathy brain when compared to nonsynucleinopathy brains. In contrast, BAR-SYN1 did not differentiate between disease and nonsynucleinopathy brains. Pathway analysis of BAR-PSER129-enriched proteins revealed enrichment for 718 pathways; notably, the most significant KEGG pathway was PD, and Gene Ontology (GO) cellular compartments were the vesicle, extracellular vesicle, extracellular exosome, and extracellular organelle. Pathway clustering revealed several superpathways, including metabolism, mitochondria, lysosome, and intracellular vesicle transport. Validation of the BAR-PSER129-identified protein hemoglobin beta (HBB) by immunohistochemistry confirmed the interaction of HBB with PSER129 Lewy neurites and Lewy bodies. In summary, BAR can be used to enrich for LP from formalin-fixed human primary tissues, which allowed the determination of molecular signatures of LP. This technique has broad potential to help understand the phenomenon of LP in primary human tissue and animal models.

Lysosomal polygenic risk is associated with the severity of neuropathology in Lewy body disease.

Intraneuronal accumulation of misfolded α-synuclein is the pathological hallmark of Parkinson's disease and dementia with Lewy bodies, often co-occurring with variable degrees of Alzheimer's disease related neuropathology. Genetic association studies have successfully identified common variants associated with disease risk and phenotypic traits in Lewy body disease, yet little is known about the genetic contribution to neuropathological heterogeneity. Using summary statistics from Parkinson's disease and Alzheimer's disease genome-wide association studies, we calculated polygenic risk scores and investigated the relationship with Lewy, amyloid-ß and tau pathology. Associations were nominated in neuropathologically defined samples with Lewy body disease from the Netherlands Brain Bank (n = 217) and followed up in an independent sample series from the Mayo Clinic Brain Bank (n = 394). We also generated stratified polygenic risk scores based on single-nucleotide polymorphisms annotated to eight functional pathways or cell types previously implicated in Parkinson's disease and assessed for association with Lewy pathology in subgroups with and without significant Alzheimer's disease co-pathology. In an ordinal logistic regression model, the Alzheimer's disease polygenic risk score was associated with concomitant amyloid-ß and tau pathology in both cohorts. Moreover, both cohorts showed a significant association between lysosomal pathway polygenic risk and Lewy pathology, which was more consistent than the association with a general Parkinson's disease risk score and specific to the subset of samples without significant concomitant Alzheimer's disease related neuropathology. Our findings provide proof of principle that the specific risk alleles a patient carries for Parkinson's and Alzheimer's disease also influence key aspects of the underlying neuropathology in Lewy body disease. The interrelations between genetic architecture and neuropathology are complex, as our results implicate lysosomal risk loci specifically in the subset of samples without Alzheimer's disease co-pathology. Our findings hold promise that genetic profiling may help predict the vulnerability to specific neuropathologies in Lewy body disease, with potential relevance for the further development of precision medicine in these disorders.

Prion-like α-synuclein pathology in the brain of infants with Krabbe disease.

Krabbe disease is an infantile neurodegenerative disorder resulting from pathogenic variants in the GALC gene that causes accumulation of the toxic sphingolipid psychosine. GALC variants are also associated with Lewy body diseases, an umbrella term for age-associated neurodegenerative diseases in which the protein α-synuclein aggregates into Lewy bodies. To explore whether α-synuclein in Krabbe disease has pathological similarities to that in Lewy body disease, we performed an observational post-mortem study of Krabbe disease brain tissue (n = 4) compared to infant controls (n = 4) and identified widespread accumulations of α-synuclein. To determine whether α-synuclein in Krabbe disease brain displayed disease-associated pathogenic properties we evaluated its seeding capacity using the real-time quaking-induced conversion assay in two cases for which frozen tissue was available and strikingly identified aggregation into fibrils similar to those observed in Lewy body disease, confirming the prion-like capacity of Krabbe disease-derived α-synuclein. These observations constitute the first report of prion-like α-synuclein in the brain tissue of infants and challenge the putative view that α-synuclein pathology is merely an age-associated phenomenon, instead suggesting it results from alterations to biological pathways, such as sphingolipid metabolism. Our findings have important implications for understanding the mechanisms underlying Lewy body formation in Lewy body disease.

Whole-exome sequencing reveals PSEN1 and ATP7B combined variants as a possible cause of early-onset Lewy body dementia: a case study of genotype-phenotype correlation.

Dementia with Lewy bodies is a neurodegenerative disease, sharing features with Parkinson's and Alzheimer's diseases. We report a case of a patient dementia with Lewy bodies carrying combined PSEN1 and ATP7B mutations. A man developed dementia with Lewy bodies starting at the age of 60 years. CSF biomarkers were of Alzheimer's disease and DaTSCAN was abnormal. Whole-exome sequencing revealed a heterozygous p.Ile408Thr PSEN1 variant and a homozygous p.Arg616Trp ATP7B variant. This case reinstates the need of considering ATP7B mutations when evaluating a patient with parkinsonism and supports p.Ile408Thr as a pathogenic PSEN1 variant.

MED27, SLC6A7, and MPPE1 Variants in a Complex Neurodevelopmental Disorder with Severe Dystonia.

BACKGROUND: Despite advances in next generation sequencing technologies, the identification of variants of uncertain significance (VUS) can often hinder definitive diagnosis in patients with complex neurodevelopmental disorders. OBJECTIVE: The objective of this study was to identify and characterize the underlying cause of disease in a family with two children with severe developmental delay associated with generalized dystonia and episodic status dystonicus, chorea, epilepsy, and cataracts. METHODS: Candidate genes identified by autozygosity mapping and whole-exome sequencing were characterized using cellular and vertebrate model systems. RESULTS: Homozygous variants were found in three candidate genes: MED27, SLC6A7, and MPPE1. Although the patients had features of MED27-related disorder, the SLC6A7 and MPPE1 variants were functionally investigated. SLC6A7 variant in vitro overexpression caused decreased proline transport as a result of reduced cell-surface expression, and zebrafish knockdown of slc6a7 exhibited developmental delay and fragile motor neuron morphology that could not be rescued by L-proline transporter-G396S RNA. Lastly, patient fibroblasts displayed reduced cell-surface expression of glycophosphatidylinositol-anchored proteins linked to MPPE1 dysfunction. CONCLUSIONS: We report a family harboring a homozygous MED27 variant with additional loss-of-function SLC6A7 and MPPE1 gene variants, which potentially contribute to a blended phenotype caused by multilocus pathogenic variants. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

A comprehensive analysis of copy number variation in a Turkish dementia cohort.

BACKGROUND: Copy number variants (CNVs) include deletions or multiplications spanning genomic regions. These regions vary in size and may span genes known to play a role in human diseases. As examples, duplications and triplications of SNCA have been shown to cause forms of Parkinson's disease, while duplications of APP cause early onset Alzheimer's disease (AD). RESULTS: Here, we performed a systematic analysis of CNVs in a Turkish dementia cohort in order to further characterize the genetic causes of dementia in this population. One hundred twenty-four Turkish individuals, either at risk of dementia due to family history, diagnosed with mild cognitive impairment, AD, or frontotemporal dementia, were whole-genome genotyped and CNVs were detected. We integrated family analysis with a comprehensive assessment of potentially disease-associated CNVs in this Turkish dementia cohort. We also utilized both dementia and non-dementia individuals from the UK Biobank in order to further elucidate the potential role of the identified CNVs in neurodegenerative diseases. We report CNVs overlapping the previously implicated genes ZNF804A, SNORA70B, USP34, XPO1, and a locus on chromosome 9 which includes a cluster of olfactory receptors and ABCA1. Additionally, we also describe novel CNVs potentially associated with dementia, overlapping the genes AFG1L, SNX3, VWDE, and BC039545. CONCLUSIONS: Genotyping data from understudied populations can be utilized to identify copy number variation which may contribute to dementia.

Rare variants in TP73 in a frontotemporal dementia cohort link this gene with primary progressive aphasia phenotypes.

BACKGROUND AND PURPOSE: TP73 was recently reported to cause amyotrophic lateral sclerosis (ALS). ALS and frontotemporal dementia (FTD) are considered to form part of a continuum. We aimed to investigate whether TP73 variants may be associated with FTD. METHODS: We studied a thoroughly investigated cohort of 65 Portuguese patients with frontotemporal dementia using whole-exome sequencing. The patients had no other known genetic cause for their disease (C9orf72 expansion was also excluded). RESULTS: Of the 65 patients studied, two had rare variants in TP73 (p.Gly605Ser and p.Arg347Trp). Both variants had minor allele frequency <0.001 and were predicted to be pathogenic in silico. The two patients displayed a phenotype that included predominant language impairment, suggestive of non-fluent progressive aphasia. CONCLUSION: We show that two thoroughly studied patients without other known genetic changes harbored TP73 rare variants, which are pathogenic in silico. This adds evidence to support the role of TP73 in the ALS-FTD spectrum, especially in primary progressive aphasia cases.

Genetic analysis reveals novel variants for vascular cognitive impairment.

OBJECTIVES: The genetic background of vascular cognitive impairment (VCI) is poorly understood compared to other dementia disorders. The aim of the study was to investigate the genetic background of VCI in a well-characterized Finnish cohort. MATERIALS & METHODS: Whole-exome sequencing (WES) was applied in 45 Finnish VCI patients. Copy-number variant (CNV) analysis using a SNP array was performed in 80 VCI patients. This study also examined the prevalence of variants at the miR-29 binding site of COL4A1 in 73 Finnish VCI patients. RESULTS: In 40% (18/45) of the cases, WES detected possibly causative variants in genes associated with cerebral small vessel disease (CSVD) or other neurological or stroke-related disorders. These variants included HTRA1:c.847G>A p.(Gly283Arg), TREX1:c.1079A>G, p.(Tyr360Cys), COLGALT1:c.1411C>T, p.(Arg471Trp), PRNP: c.713C>T, p.(Pro238Leu), and MTHFR:c.1061G>C, p.(Gly354Ala). Additionally, screening of variants in the 3'UTR of COL4A1 gene in a sub-cohort of 73 VCI patients identified a novel variant c.*36T>A. CNV analysis showed that pathogenic CNVs are uncommon in VCI. CONCLUSIONS: These data support pathogenic roles of variants in HTRA1, TREX1 and in the 3'UTR of COL4A1 in CSVD and VCI, and suggest that vascular pathogenic mechanisms are linked to neurodegeneration, expanding the understanding of the genetic background of VCI.

How understudied populations have contributed to our understanding of Alzheimer's disease genetics.

The majority of genome-wide association studies have been conducted using samples with a broadly European genetic background. As a field, we acknowledge this limitation and the need to increase the diversity of populations studied. A major challenge when designing and conducting such studies is to assimilate large samples sizes so that we attain enough statistical power to detect variants associated with disease, particularly when trying to identify variants with low and rare minor allele frequencies. In this review, we aimed to illustrate the benefits to genetic characterization of Alzheimer's disease, in researching currently understudied populations. This is important for both fair representation of world populations and the translatability of findings. To that end, we conducted a literature search to understand the contributions of studies, on different populations, to Alzheimer's disease genetics. Using both PubMed and Alzforum Mutation Database, we systematically quantified the number of studies reporting variants in known disease-causing genes, in a worldwide manner, and discuss the contributions of research in understudied populations to the identification of novel genetic factors in this disease. Additionally, we compared the effects of genome-wide significant single nucleotide polymorphisms across populations by focusing on loci that show different association profiles between populations (a key example being APOE). Reports of variants in APP, PSEN1 and PSEN2 can initially determine whether patients from a country have been studied for Alzheimer's disease genetics. Most genome-wide significant associations in non-Hispanic white genome-wide association studies do not reach genome-wide significance in such studies of other populations, with some suggesting an opposite effect direction; this is likely due to much smaller sample sizes attained. There are, however, genome-wide significant associations first identified in understudied populations which have yet to be replicated. Familial studies in understudied populations have identified rare, high effect variants, which have been replicated in other populations. This work functions to both highlight how understudied populations have furthered our understanding of Alzheimer's disease genetics, and to help us gauge our progress in understanding the genetic architecture of this disease in all populations.

Congenital ataxia due to novel variant in ATP8A2.

Congenital ataxias are a heterogeneous group of disorders characterized by congenital or early-onset ataxia. Here, we describe two siblings with congenital ataxia, who acquired independent gait by age 4 years. After 16 years of follow-up they presented near normal cognition, cerebellar ataxia, mild pyramidal signs, and dystonia. On exome sequencing, a novel homozygous variant (c.1580-18C > G - intron 17) in ATP8A2 was identified. A new acceptor splice site was predicted by bioinformatics tools, and functionally characterized through a minigene assay. Minigene constructs were generated by PCR-amplification of genomic sequences surrounding the variant of interest and cloning into the pCMVdi vector. Altered splicing was evaluated by expressing these constructs in HEK293T cells. The construct with the c.1580-18C > G homozygous variant produced an aberrant transcript, leading to retention of 17 bp of intron 17, by the use of an alternative acceptor splice site, resulting in a premature stop codon by insertion of four amino acids. These results allowed us to establish this as a disease-causing variant and expand ATP8A2-related disorders to include less severe forms of congenital ataxia.

Genetics of synucleins in neurodegenerative diseases.

The SNCA locus currently has an indisputable role in Parkinson's disease and other synucleinopathies. The role of genetic variability in the other members of the synuclein family (SNCB and SNCG) in disease is far less clear. In this review, we critically assess the pathogenicity, main characteristics, and roles of genetic variants in these genes reported to be causative of synucleinopathies. We also summarize the different association signals identified in the SNCA locus that have been associated with risk for disease. We take a bird's eye view of the variability currently reported in the general population for the three genes and use these data to infer on the potential relationship between each of the genes and human disease.

A rare loss-of-function variant of ADAM17 is associated with late-onset familial Alzheimer disease.

Common variants of about 20 genes contributing to AD risk have so far been identified through genome-wide association studies (GWAS). However, there is still a large proportion of heritability that might be explained by rare but functionally important variants. One of the so far identified genes with rare AD causing variants is ADAM10. Using whole-genome sequencing we now identified a single rare nonsynonymous variant (SNV) rs142946965 [p.R215I] in ADAM17 co-segregating with an autosomal-dominant pattern of late-onset AD in one family. Subsequent genotyping and analysis of available whole-exome sequencing data of additional case/control samples from Germany, UK, and USA identified five variant carriers among AD patients only. The mutation inhibits pro-protein cleavage and the formation of the active enzyme, thus leading to loss-of-function of ADAM17 alpha-secretase. Further, we identified a strong negative correlation between ADAM17 and APP gene expression in human brain and present in vitro evidence that ADAM17 negatively controls the expression of APP. As a consequence, p.R215I mutation of ADAM17 leads to elevated Aß formation in vitro. Together our data supports a causative association of the identified ADAM17 variant in the pathogenesis of AD.

Genetic architecture of common non-Alzheimer's disease dementias.

Frontotemporal dementia (FTD), dementia with Lewy bodies (DLB) and vascular dementia (VaD) are the most common forms of dementia after Alzheimer's disease (AD). The heterogeneity of these disorders and/or the clinical overlap with other diseases hinder the study of their genetic components. Even though Mendelian dementias are rare, the study of these forms of disease can have a significant impact in the lives of patients and families and have successfully brought to the fore many of the genes currently known to be involved in FTD and VaD, starting to give us a glimpse of the molecular mechanisms underlying these phenotypes. More recently, genome-wide association studies have also pointed to disease risk-associated loci. This has been particularly important for DLB where familial forms of disease are very rarely described. In this review we systematically describe the Mendelian and risk genes involved in these non-AD dementias in an effort to contribute to a better understanding of their genetic architecture, find differences and commonalities between different dementia phenotypes, and uncover areas that would benefit from more intense research endeavors.

Rare coding variants in the phospholipase D3 gene confer risk for Alzheimer's disease.

Genome-wide association studies (GWAS) have identified several risk variants for late-onset Alzheimer's disease (LOAD). These common variants have replicable but small effects on LOAD risk and generally do not have obvious functional effects. Low-frequency coding variants, not detected by GWAS, are predicted to include functional variants with larger effects on risk. To identify low-frequency coding variants with large effects on LOAD risk, we carried out whole-exome sequencing (WES) in 14 large LOAD families and follow-up analyses of the candidate variants in several large LOAD case-control data sets. A rare variant in PLD3 (phospholipase D3; Val232Met) segregated with disease status in two independent families and doubled risk for Alzheimer's disease in seven independent case-control series with a total of more than 11,000 cases and controls of European descent. Gene-based burden analyses in 4,387 cases and controls of European descent and 302 African American cases and controls, with complete sequence data for PLD3, reveal that several variants in this gene increase risk for Alzheimer's disease in both populations. PLD3 is highly expressed in brain regions that are vulnerable to Alzheimer's disease pathology, including hippocampus and cortex, and is expressed at significantly lower levels in neurons from Alzheimer's disease brains compared to control brains. Overexpression of PLD3 leads to a significant decrease in intracellular amyloid-ß precursor protein (APP) and extracellular Aß42 and Aß40 (the 42- and 40-residue isoforms of the amyloid-ß peptide), and knockdown of PLD3 leads to a significant increase in extracellular Aß42 and Aß40. Together, our genetic and functional data indicate that carriers of PLD3 coding variants have a twofold increased risk for LOAD and that PLD3 influences APP processing. This study provides an example of how densely affected families may help to identify rare variants with large effects on risk for disease or other complex traits.

A novel human pain insensitivity disorder caused by a point mutation in ZFHX2.

Chronic pain is a major global public health issue causing a severe impact on both the quality of life for sufferers and the wider economy. Despite the significant clinical burden, little progress has been made in terms of therapeutic development. A unique approach to identifying new human-validated analgesic drug targets is to study rare families with inherited pain insensitivity. Here we have analysed an otherwise normal family where six affected individuals display a pain insensitive phenotype that is characterized by hyposensitivity to noxious heat and painless bone fractures. This autosomal dominant disorder is found in three generations and is not associated with a peripheral neuropathy. A novel point mutation in ZFHX2, encoding a putative transcription factor expressed in small diameter sensory neurons, was identified by whole exome sequencing that segregates with the pain insensitivity. The mutation is predicted to change an evolutionarily highly conserved arginine residue 1913 to a lysine within a homeodomain. Bacterial artificial chromosome (BAC) transgenic mice bearing the orthologous murine p.R1907K mutation, as well as Zfhx2 null mutant mice, have significant deficits in pain sensitivity. Gene expression analyses in dorsal root ganglia from mutant and wild-type mice show altered expression of genes implicated in peripheral pain mechanisms. The ZFHX2 variant and downstream regulated genes associated with a human pain-insensitive phenotype are therefore potential novel targets for the development of new analgesic drugs.awx326media15680039660001.

Microdeletion in a FAAH pseudogene identified in a patient with high anandamide concentrations and pain insensitivity.

The study of rare families with inherited pain insensitivity can identify new human-validated analgesic drug targets. Here, a 66-yr-old female presented with nil requirement for postoperative analgesia after a normally painful orthopaedic hand surgery (trapeziectomy). Further investigations revealed a lifelong history of painless injuries, such as frequent cuts and burns, which were observed to heal quickly. We report the causative mutations for this new pain insensitivity disorder: the co-inheritance of (i) a microdeletion in dorsal root ganglia and brain-expressed pseudogene, FAAH-OUT, which we cloned from the fatty-acid amide hydrolase (FAAH) chromosomal region; and (ii) a common functional single-nucleotide polymorphism in FAAH conferring reduced expression and activity. Circulating concentrations of anandamide and related fatty-acid amides (palmitoylethanolamide and oleoylethanolamine) that are all normally degraded by FAAH were significantly elevated in peripheral blood compared with normal control carriers of the hypomorphic single-nucleotide polymorphism. The genetic findings and elevated circulating fatty-acid amides are consistent with a phenotype resulting from enhanced endocannabinoid signalling and a loss of function of FAAH. Our results highlight previously unknown complexity at the FAAH genomic locus involving the expression of FAAH-OUT, a novel pseudogene and long non-coding RNA. These data suggest new routes to develop FAAH-based analgesia by targeting of FAAH-OUT, which could significantly improve the treatment of postoperative pain and potentially chronic pain and anxiety disorders.

Additional rare variant analysis in Parkinson's disease cases with and without known pathogenic mutations: evidence for oligogenic inheritance.

Oligogenic inheritance implies a role for several genetic factors in disease etiology. We studied oligogenic inheritance in Parkinson's (PD) by assessing the potential burden of additional rare variants in established Mendelian genes and/or GBA, in individuals with and without a primary pathogenic genetic cause in two large independent cohorts totaling 7,900 PD cases and 6,166 controls. An excess (≥30%) of cases with a recognised primary genetic cause had ≥1 additional rare variants in Mendelian PD genes, as compared with no known mutation PD cases (17%) and unaffected controls (16%), supporting our hypothesis. Carriers of additional Mendelian gene variants have younger ages at onset (AAO). The effect of additional Mendelian variants in LRRK2 G2019S mutation carriers, of which ATP13A2 variation is particularly common, may account for some of the variation in penetrance. About 10% of No Known Mutation-PD cases harbour a rare GBA variant compared to known pathogenic mutation PD cases (8%) and controls (5%), with carriers having earlier AAOs. Together, the data suggest that the oligogenic inheritance of rare Mendelian variants may be important in patient with a primary pathogenic cause, whereas GBA increases risk across all forms of PD. This study highlights the potential genetic complexity of Mendelian PD. The identification of potential modifying variants provides new insights into disease mechanisms by potentially separating relevant from benign variants and by the interaction between genes in specific pathways. In the future this may be relevant to genetic testing and counselling of patients with PD and their families.