Reduced secretion and altered proteolytic processing caused by missense mutations in progranulin.

Progranulin (GRN) is a secreted growth factor involved in various cellular functions, and loss-of-function mutations are a major cause of frontotemporal lobar degeneration (FTLD) with TDP-43 positive pathology. Most FTLD-related GRN mutations are nonsense mutations resulting in reduced GRN expression. Nonsynonymous GRN missense mutations have been described as risk factor for neurodegenerative brain diseases, but their pathogenic nature remains largely elusive. We identified a double missense mutation in GRN leading to amino acid changes p.D33E and p.G35R in an FTLD patient from Turkish origin. Biochemical and cell biological analysis of the double-mutation together with 2 so-far uncharacterized GRN missense mutations (p.C105R and p.V514M) revealed a reduced secretion efficiency of the GRN p.D33E/p.G35R and p.C105R proteins. Furthermore, loss of the conserved cysteine residue affects protein folding and altered proteolytic processing by neutrophil elastase and proteinase 3. Our data indicate that the described variants may cause a loss-of-function, albeit to a lesser extent than GRN null mutations, and hence could be considered as low-penetrant risk factors for neurodegenerative diseases.

Reduced secreted clusterin as a mechanism for Alzheimer-associated CLU mutations.

BACKGROUND: The clusterin (CLU) gene has been identified as an important risk locus for Alzheimer's disease (AD). Although the actual risk-increasing polymorphisms at this locus remain to be identified, we previously observed an increased frequency of rare non-synonymous mutations and small insertion-deletions of CLU in AD patients, which specifically clustered in the ß-chain domain of CLU. Nonetheless the pathogenic nature of these variants remained unclear. Here we report a novel non-synonymous CLU mutation (p.I360N) in a Belgian Alzheimer patient and have explored the pathogenic nature of this and 10 additional CLU mutations on protein localization and secretion in vitro using immunocytochemistry, immunodetection and ELISAs. RESULTS: Three patient-specific CLU mutations in the ß-chain (p.I303NfsX13, p.R338W and p.I360N) caused an alteration of the subcellular CLU localization and diminished CLU transport through the secretory pathway, indicative of possible degradation mechanisms. For these mutations, significantly reduced CLU intensity was observed in the Golgi while almost all CLU protein was exclusively present in the endoplasmic reticulum. This was further confirmed by diminished CLU secretion in HEK293T and HEK293 FLp-In cell lines. CONCLUSIONS: Our data lend further support to the contribution of rare coding CLU mutations in the pathogenesis of neurodegenerative diseases. Functional analyses suggest reduced secretion of the CLU protein as the mode of action for three of the examined CLU mutations. One of those is a frameshift mutation leading to a loss of secreted protein, and the other two mutations are amino acid substitutions in the disulfide bridge region, possibly interfering with heterodimerization of the α- and ß-chain of CLU.

The genetics and neuropathology of frontotemporal lobar degeneration.

Frontotemporal lobar degeneration (FTLD) is a heterogeneous group of disorders characterized by disturbances of behavior and personality and different types of language impairment with or without concomitant features of motor neuron disease or parkinsonism. FTLD is characterized by atrophy of the frontal and anterior temporal brain lobes. Detailed neuropathological studies have elicited proteinopathies defined by inclusions of hyperphosphorylated microtubule-associated protein tau, TAR DNA-binding protein TDP-43, fused-in-sarcoma or yet unidentified proteins in affected brain regions. Rather than the type of proteinopathy, the site of neurodegeneration correlates relatively well with the clinical presentation of FTLD. Molecular genetic studies identified five disease genes, of which the gene encoding the tau protein (MAPT), the growth factor precursor gene granulin (GRN), and C9orf72 with unknown function are most frequently mutated. Rare mutations were also identified in the genes encoding valosin-containing protein (VCP) and charged multivesicular body protein 2B (CHMP2B). These genes are good markers to distinguish underlying neuropathological phenotypes. Due to the complex landscape of FTLD diseases, combined characterization of clinical, imaging, biological and genetic biomarkers is essential to establish a detailed diagnosis. Although major progress has been made in FTLD research in recent years, further studies are needed to completely map out and correlate the clinical, pathological and genetic entities, and to understand the underlying disease mechanisms. In this review, we summarize the current state of the rapidly progressing field of genetic, neuropathological and clinical research of this intriguing condition.

Genetic etiology of Parkinson disease associated with mutations in the SNCA, PARK2, PINK1, PARK7, and LRRK2 genes: a mutation update.

To date, molecular genetic analyses have identified over 500 distinct DNA variants in five disease genes associated with familial Parkinson disease; alpha-synuclein (SNCA), parkin (PARK2), PTEN-induced putative kinase 1 (PINK1), DJ-1 (PARK7), and Leucine-rich repeat kinase 2 (LRRK2). These genetic variants include approximately 82% simple mutations and approximately 18% copy number variations. Some mutation subtypes are likely underestimated because only few studies reported extensive mutation analyses of all five genes, by both exonic sequencing and dosage analyses. Here we present an update of all mutations published to date in the literature, systematically organized in a novel mutation database (http://www.molgen.ua.ac.be/PDmutDB). In addition, we address the biological relevance of putative pathogenic mutations. This review emphasizes the need for comprehensive genetic screening of Parkinson patients followed by an insightful study of the functional relevance of observed genetic variants. Moreover, while capturing existing data from the literature it became apparent that several of the five Parkinson genes were also contributing to the genetic etiology of other Lewy Body Diseases and Parkinson-plus syndromes, indicating that mutation screening is recommendable in these patient groups.

Serum biomarker for progranulin-associated frontotemporal lobar degeneration.

OBJECTIVE: Mutations that lead to a loss of progranulin (PGRN) explain a considerable portion of the occurrence of frontotemporal lobar degeneration. We tested a biomarker allowing rapid detection of a loss of PGRN. METHODS: We used an enzyme-linked immunosorbent assay to measure in serum the PGRN protein levels of six affected and eight unaffected carriers from within an extended Belgian founder family segregating the null mutation IVS1+5G>C. Further, we measured serum PGRN levels in 2 patients with another null mutation (a Met1 and a frameshift mutation), in 4 patients carrying a predicted pathogenic missense mutation and in 5 patients carrying a benign missense polymorphism, in 9 unaffected noncarrier relatives, and in 22 community controls. RESULTS: Serum PGRN levels were reduced in both affected and unaffected null mutation carriers compared with noncarrier relatives (p(exact) < 0.0001), and allowed perfect discrimination between carriers and noncarriers (sensitivity: 1.0; 1 - specificity: 0.0). Serum PGRN levels in Cys139Arg and Arg564Cys mutation carriers were significantly lower than in controls, but greater than in null mutation carriers, fitting the hypothesis of partial loss of function caused by these missense mutations. As expected, levels for carriers of benign missense polymorphisms were not significantly different from controls. INTERPRETATION: Our results indicate that the serum PGRN level is a reliable biomarker for diagnosing and early detection of frontotemporal lobar degeneration caused by PGRN null mutations, and provided the first in vivo evidence that at least some missense mutations in PGRN may lead to a (partial) loss of PGRN.

Frontotemporal lobar degeneration with ubiquitin-positive inclusions: a molecular genetic update.

Frontotemporal lobar degeneration (FTLD) is a clinically, pathologically and genetically highly complex disorder. In the last few years enormous progress has been made in dissecting the genetic etiology of FTLD. Mutations have been identified in the progranulin gene (PGRN), the charged multivesicular body protein 2B gene (CHMP2B) and the valosin-containing protein gene (VCP). Mutations in these genes all lead to FTLD pathology characterized by ubiquitin-immunoreactive neuronal cytoplasmic and intranuclear lentiform inclusions (FTLD-U). The similar pathology suggests that these genes may be connected trough a common disease pathway leading to neurodegeneration and the formation of these pathognomic inclusions. This review focuses on the molecular genetic processes underlying FTLD-U pathology.

Progranulin mutations in ubiquitin-positive frontotemporal dementia linked to chromosome 17q21.

Two genetically distinct types of frontotemporal dementia (FTD) are linked to chromosome 17q21. FTD with parkinsonism (FTDP-17) results from mutations in the gene encoding microtubule associated protein tau (MAPT) and is associated with tau deposition in the patient's brain. An increasing number of FTD families are linked to 17q21 in the absence of a demonstrable MAPT mutation. Brains of these patients do not show tau deposits, but tau-negative intra- and perinuclear inclusions of unknown composition that are immunoreactive to ubiquitin (FTDU-17). These ubiquitin inclusions are located in the cytoplasm or nucleus of predominantly neuronal cells of affected brain regions. By extensive segregation analyses in conclusively linked FTDU-17 families, the candidate region was reduced to a 6.2 Mb segment containing MAPT; however, genomic sequencing of MAPT in FTDU-17 patients excluded disease-causing mutations. Further, the linked region was characterized by the presence of multiple low-copy repeat regions associated with genomic instability. However, we excluded genomic rearrangements as the cause of FTDU-17. Subsequent sequencing of positional candidate genes identified loss-of-function mutations in the gene encoding progranulin (PGRN), a growth factor involved in multiple physiological processes such as cellular proliferation and survival and tissue repair, and pathological processes including tumorigenesis. In a Belgian FTD patient series, the prevalence of PGRN mutations was 3.5 times higher than that of MAPT mutations underscoring a major role for PGRN in FTD pathogenesis. Together, mutation data provided convincing evidence that PGRN haploinsufficiency leads to neurodegeneration because of reduced PGRN-mediated neuronal survival. The PGRN protein is not deposited in the ubiquitin-positive inclusions, the nature of which remains unknown. Due to the functions of PGRN in neuronal survival and the clinicopathological overlaps between FTD and other dementias it is likely that reduced PGRN expression is associated with the progression of other neurodegenerative brain diseases including Alzheimer's disease. These findings open promising novel targets for therapeutic intervention against neurodegeneration.

Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21.

Frontotemporal dementia (FTD) with ubiquitin-immunoreactive neuronal inclusions (both cytoplasmic and nuclear) of unknown nature has been linked to a chromosome 17q21 region (FTDU-17) containing MAPT (microtubule-associated protein tau). FTDU-17 patients have consistently been shown to lack a tau-immunoreactive pathology, a feature characteristic of FTD with parkinsonism linked to mutations in MAPT (FTDP-17). Furthermore, in FTDU-17 patients, mutations in MAPT and genomic rearrangements in the MAPT region have been excluded by both genomic sequencing and fluorescence in situ hybridization on mechanically stretched chromosomes. Here we demonstrate that FTDU-17 is caused by mutations in the gene coding for progranulin (PGRN), a growth factor involved in multiple physiological and pathological processes including tumorigenesis. Besides the production of truncated PGRN proteins due to premature stop codons, we identified a mutation within the splice donor site of intron 0 (IVS0 + 5G > C), indicating loss of the mutant transcript by nuclear degradation. The finding was made within an extensively documented Belgian FTDU-17 founder family. Transcript and protein analyses confirmed the absence of the mutant allele and a reduction in the expression of PGRN. We also identified a mutation (c.3G > A) in the Met1 translation initiation codon, indicating loss of PGRN due to lack of translation of the mutant allele. Our data provide evidence that PGRN haploinsufficiency leads to neurodegeneration because of reduced PGRN-mediated neuronal survival. Furthermore, in a Belgian series of familial FTD patients, PGRN mutations were 3.5 times more frequent than mutations in MAPT, underscoring a principal involvement of PGRN in FTD pathogenesis.

novoSNP, a novel computational tool for sequence variation discovery.

Technological improvements shifted sequencing from low-throughput, work-intensive, gel-based systems to high-throughput capillary systems. This resulted in a broad use of genomic resequencing to identify sequence variations in genes and regulatory, as well as extended genomic regions. We describe a software package, novoSNP, that conscientiously discovers single nucleotide polymorphisms (SNPs) and insertion-deletion polymorphisms (INDELs) in sequence trace files in a fast, reliable, and user-friendly way. We compared the performance of novoSNP with that of PolyPhred and PolyBayes on two data sets. The first data set comprised 1028 sequence trace files obtained from diagnostic mutation analyses of SCN1A (neuronal voltage-gated sodium channel alpha-subunit type I gene). The second data set comprised 9062 sequence trace files from a genomic resequencing project aiming at the construction of a high-density SNP map of MAPT (microtubule-associated protein tau gene). Visual inspection of these data sets had identified 38 sequence variations for SCN1A and 488 for MAPT. novoSNP automatically identified all 38 SCN1A variations including five INDELs, while for MAPT only 15 of the 488 variations were not correctly marked. PolyPhred detected far fewer SNPs as compared to novoSNP and missed nearly all INDELs. PolyBayes, designed for the sequence analysis of cloned templates, detected only a limited number of the variations present in the data set. Besides the significant improvement in the automated detection of sequence variations both in diagnostic mutation analyses and in SNP discovery projects, novoSNP also offers a user-friendly interface for inspecting possible genetic variations.

APOE epsilon4 and Alzheimer's disease: positive association in a Colombian clinical series and review of the Latin-American studies

OBJECTIVE: As the strength of the association between the APOE epsilon4 allele and Alzheimer's disease (AD) varies across ethnic groups, we studied if there was such an association in Colombian patients. METHOD: We performed apolipoprotein E (APOE) genotyping in a clinical sample of 83 unrelated AD patients, predominantly late-onset (>65 yrs) including familial ( n =30) and sporadic AD cases (n= 53) diagnosed according to NINCDS-ADRDA criteria and assessed by a multi-disciplinary team. Control subjects (n = 44) had no significant cognitive impairment by medical interview and neuro-psychological testing. RESULTS: We found a high association (OR= 5.1 95 percentCI 1.9 -13.6) between APOE epsilon4 and AD, in this series with predominantly late-onset cases with familial aggregation in 24 cases (28.9 percent). A significant negative association was found between epsilon2 and AD (OR= 0.2 95 percent CI 0.05-0.75). CONCLUSION: Further population-based surveys in Colombia are warranted to precise a possible dose effect of APOE epsilon4