SORL1 mutations in early- and late-onset Alzheimer disease.

OBJECTIVE: To characterize the clinical and molecular effect of mutations in the sortilin-related receptor (SORL1) gene. METHODS: We performed whole-exome sequencing in early-onset Alzheimer disease (EOAD) and late-onset Alzheimer disease (LOAD) families followed by functional studies of select variants. The phenotypic consequences associated with SORL1 mutations were characterized based on clinical reviews of medical records. Functional studies were completed to evaluate β-amyloid (Aβ) production and amyloid precursor protein (APP) trafficking associated with SORL1 mutations. RESULTS: SORL1 alterations were present in 2 EOAD families. In one, a SORL1 T588I change was identified in 4 individuals with AD, 2 of whom had parkinsonian features. In the second, an SORL1 T2134 alteration was found in 3 of 4 AD cases, one of whom had postmortem Lewy bodies. Among LOAD cases, 4 individuals with either SORL1 A528T or T947M alterations had parkinsonian features. Functionally, the variants weaken the interaction of the SORL1 protein with full-length APP, altering levels of Aβ and interfering with APP trafficking. CONCLUSIONS: The findings from this study support an important role for SORL1 mutations in AD pathogenesis by way of altering Aβ levels and interfering with APP trafficking. In addition, the presence of parkinsonian features among select individuals with AD and SORL1 mutations merits further investigation

Copy Number Variants in Extended Autism Spectrum Disorder Families Reveal Candidates Potentially Involved in Autism Risk

Copy number variations (CNVs) are a major cause of genetic disruption in the human genome with far more nucleotides being altered by duplications and deletions than by single nucleotide polymorphisms (SNPs). In the multifaceted etiology of autism spectrum disorders (ASDs), CNVs appear to contribute significantly to our understanding of the pathogenesis of this complex disease. A unique resource of 42 extended ASD families was genotyped for over 1 million SNPs to detect CNVs that may contribute to ASD susceptibility. Each family has at least one avuncular or cousin pair with ASD. Families were then evaluated for co-segregation of CNVs in ASD patients. We identified a total of five deletions and seven duplications in eleven families that co-segregated with ASD. Two of the CNVs overlap with regions on 7p21.3 and 15q24.1 that have been previously reported in ASD individuals and two additional CNVs on 3p26.3 and 12q24.32 occur near regions associated with schizophrenia. These findings provide further evidence for the involvement of ICA1 and NXPH1 on 7p21.3 in ASD susceptibility and highlight novel ASD candidates, including CHL1, FGFBP3 and POUF41. These studies highlight the power of using extended families for gene discovery in traits with a complex etiology

Evidence of novel finescale structural variation at autism spectrum disorder candidate loci

Background: Autism spectrum disorders (ASD) represent a group of neurodevelopmental disorders characterized by a core set of social-communicative and behavioral impairments. Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain, acting primarily via the GABA receptors (GABR). Multiple lines of evidence, including altered GABA and GABA receptor expression in autistic patients, indicate that the GABAergic system may be involved in the etiology of autism. Methods: As copy number variations (CNVs), particularly rare and de novo CNVs, have now been implicated in ASD risk, we examined the GABA receptors and genes in related pathways for structural variation that may be associated with autism. We further extended our candidate gene set to include 19 genes and regions that had either been directly implicated in the autism literature or were directly related (via function or ancestry) to these primary candidates. For the high resolution CNV screen we employed custom-designed 244 k comparative genomic hybridization (CGH) arrays. Collectively, our probes spanned a total of 11 Mb of GABA-related and additional candidate regions with a density of approximately one probe every 200 nucleotides, allowing a theoretical resolution for detection of CNVs of approximately 1 kb or greater on average. One hundred and sixty-eight autism cases and 149 control individuals were screened for structural variants. Prioritized CNV events were confirmed using quantitative PCR, and confirmed loci were evaluated on an additional set of 170 cases and 170 control individuals that were not included in the original discovery set. Loci that remained interesting were subsequently screened via quantitative PCR on an additional set of 755 cases and 1,809 unaffected family members. Results: Results include rare deletions in autistic individuals at JAKMIP1, NRXN1, Neuroligin4Y, OXTR, and ABAT. Common insertion/deletion polymorphisms were detected at several loci, including GABBR2 and NRXN3. Overall, statistically significant enrichment in affected vs. unaffected individuals was observed for NRXN1 deletions. Conclusions: These results provide additional support for the role of rare structural variation in ASD

ABCA7 frameshift deletion associated with Alzheimer disease in African Americans

Objective: To identify a causative variant(s) that may contribute to Alzheimer disease (AD) in African Americans (AA) in the ATP-binding cassette, subfamily A (ABC1), member 7 (ABCA7) gene, a known risk factor for late-onset AD. Methods: Custom capture sequencing was performed on ∼150 kb encompassing ABCA7 in 40 AA cases and 37 AA controls carrying the AA risk allele (rs115550680). Association testing was performed for an ABCA7 deletion identified in large AA data sets (discovery n = 1,068; replication n = 1,749) and whole exome sequencing of Caribbean Hispanic (CH) AD families. Results: A 44-base pair deletion (rs142076058) was identified in all 77 risk genotype carriers, which shows that the deletion is in high linkage disequilibrium with the risk allele. The deletion was assessed in a large data set (531 cases and 527 controls) and, after adjustments for age, sex, and APOE status, was significantly associated with disease (p = 0.0002, odds ratio [OR] = 2.13 [95% confidence interval (CI): 1.42–3.20]). An independent data set replicated the association (447 cases and 880 controls, p = 0.0117, OR = 1.65 [95% CI: 1.12–2.44]), and joint analysis increased the significance (p = 1.414 × 10−5, OR = 1.81 [95% CI: 1.38–2.37]). The deletion is common in AA cases (15.2%) and AA controls (9.74%), but in only 0.12% of our non-Hispanic white cohort. Whole exome sequencing of multiplex, CH families identified the deletion cosegregating with disease in a large sibship. The deleted allele produces a stable, detectable RNA strand and is predicted to result in a frameshift mutation (p.Arg578Alafs) that could interfere with protein function. Conclusions: This common ABCA7 deletion could represent an ethnic-specific pathogenic alteration in AD

R-Ras Regulates Migration through an Interaction with Filamin A in Melanoma Cells

Changes in cell adhesion and migration in the tumor microenvironment are key in the initiation and progression of metastasis. R-Ras is one of several small GTPases that regulate cell adhesion and migration on the extracellular matrix, however the mechanism has not been completely elucidated. Using a yeast two-hybrid approach we sought to identify novel R-Ras binding proteins that might mediate its effects on integrins.We identified Filamin A (FLNa) as a candidate interacting protein. FLNa is an actin-binding scaffold protein that also binds to integrin β1, β2 and β7 tails and is associated with diverse cell processes including cell migration. Indeed, M2 melanoma cells require FLNa for motility. We further show that R-Ras and FLNa interact in co-immunoprecipitations and pull-down assays. Deletion of FLNa repeat 3 (FLNaΔ3) abrogated this interaction. In M2 melanoma cells active R-Ras co-localized with FLNa but did not co-localize with FLNa lacking repeat 3. Thus, activated R-Ras binds repeat 3 of FLNa. The functional consequence of this interaction was that active R-Ras and FLNa coordinately increased cell migration. In contrast, co-expression of R-Ras and FLNaΔ3 had a significantly reduced effect on migration. While there was enhancement of integrin activation and fibronectin matrix assembly, cell adhesion was not altered. Finally, siRNA knockdown of endogenous R-Ras impaired FLNa-dependent fibronectin matrix assembly.These data support a model in which R-Ras functionally associates with FLNa and thereby regulates integrin-dependent migration. Thus in melanoma cells R-Ras and FLNa may cooperatively promote metastasis by enhancing cell migration

Blood Derived Induced Pluripotent Stem Cells (iPSCs): Benefits, Challenges and the Road Ahead

Since the creation of induced Pluripotent Stem Cells (iPSCs) ten years ago, hundreds of publications have demonstrated their considerable impact on disease modeling and therapy. In this commentary, we will summarize key milestones, benefits and challenges in the iPSC field. Furthermore, we will highlight blood as an effective and easily accessible source for patient-specific iPSCs derivation in the context of work done in our laboratory and others

Genetic modifiers of MeCP2 function in Drosophila.

The levels of methyl-CpG-binding protein 2 (MeCP2) are critical for normal post-natal development and function of the nervous system. Loss of function of MeCP2, a transcriptional regulator involved in chromatin remodeling, causes classic Rett syndrome (RTT) as well as other related conditions characterized by autism, learning disabilities, or mental retardation. Increased dosage of MeCP2 also leads to clinically similar neurological disorders and mental retardation. To identify molecular mechanisms capable of compensating for altered MeCP2 levels, we generated transgenic Drosophila overexpressing human MeCP2. We find that MeCP2 associates with chromatin and is phosphorylated at serine 423 in Drosophila, as is found in mammals. MeCP2 overexpression leads to anatomical (i.e., disorganized eyes, ectopic wing veins) and behavioral (i.e., motor dysfunction) abnormalities. We used a candidate gene approach to identify genes that are able to compensate for abnormal phenotypes caused by MeCP2 increased activity. These genetic modifiers include other chromatin remodeling genes (Additional sex combs, corto, osa, Sex combs on midleg, and trithorax), the kinase tricornered, the UBE3A target pebble, and Drosophila homologues of the MeCP2 physical interactors Sin3a, REST, and N-CoR. These findings demonstrate that anatomical and behavioral phenotypes caused by MeCP2 activity can be ameliorated by altering other factors that might be more amenable to manipulation than MeCP2 itself

Derivation of stem cell line UMi028-A-2 containing a CRISPR/Cas9 induced Alzheimer’s disease risk variant p.S1038C in the TTC3 gene

The UMi028-A-2 human induced pluripotent stem cell line carries a homozygous mutation (rs377155188, C>G, p.S1038C) in the tetratricopeptide repeat domain 3 (TTC3) gene that was introduced via CRISPR/Cas9 genome editing. The line was originally derived from a neurologically normal male and has been thoroughly characterized following editing. The p.S1038C variant has been shown to potentially contribute to the risk of late onset Alzheimer’s disease and is a resource to further investigate the consequences of TTC3 and this alteration in disease pathology

Examining the impact of a rare protein‐truncating SORL1 variant on AD pathology

Background Recent analyses of rare variants using whole exome sequencing have found an enrichment of SORL1 loss‐of‐function (LoF) variants in early onset Alzheimer’s disease (EOAD). This makes SORL1 one of the highest risk factors for AD, along with other candidate EOAD genes such as APP, PS1, and PS2. SORL1 encodes an endocytic receptor involved in the trafficking of amyloid‐β precursor protein (APP) and the secretion of amyloid‐β. We identified a family with multiple individuals affected with EOAD carrying a single base pair deletion (c.4293del) in SORL1, resulting in a frameshift and premature termination of the protein (p.Cys1431fs; Kunkle et al. JAMA Neurol. 2017). Several recent studies have demonstrated that haploinsufficiency of SORL1 can induce AD‐related phenotypes in cultured neurons. However, the functional consequences of specific variants remain largely undefined. In this study, we used patient‐specific induced pluripotent stem cell (iPSC)‐derived neurons to evaluate the effect of a rare LoF SORL1 variant on AD‐related pathology. Method Patient‐specific iPSC lines were derived from two related heterozygous p.Cys1431fs carriers with EOAD. Each line was validated for pluripotency through immunocytochemical staining and RT‐PCR. Karyotypic stability was assessed by G‐banding. To evaluate the functional consequences of the SORL1 deletion, patient and control iPSC lines were differentiated into cortical neurons and assessed for AD‐related phenotypes. Result The iPSC lines were successfully differentiated into cortical neurons, characterized by immunostaining for various neuronal markers. Our data show that neurons bearing the p.Cys1431fs SORL1 mutation have increased levels of APP accumulated in EEA1+ endosomes compared to neurons from cognitively intact individuals. Additionally, analysis of synaptic density revealed that neurons carrying the p.Cys1431fs variant show a significant reduction of SYN1+ puncta compared to cognitively intact individuals. Conclusion Our results indicate that patient‐derived neurons carrying the p.Cys1431fs variant have cellular defects associated with AD pathology while replicating, at least in part, previous in vitro findings on SORL1 haploinsufficiency