Publications

Hexanucleotide expansions in the C9ORF72 gene are frequently found in patients with amyotrophic lateral sclerosis, frontotemporal dementia or both, some of whom exhibit concurrent extrapyramidal symptoms. To determine if repeat expansions are a cause of Parkinson's disease (PD), we used repeat-primed polymerase chain reaction to investigate the frequency of C9ORF72 repeat expansions in a cohort of 478 patients with PD and 662 control subjects. Three control subjects were found to be expansion carriers, and no expansions were found among patients, suggesting that C9ORF72 expansions are not a common cause of PD.

Cerebrospinal fluid (CSF) tau, tau phosphorylated at threonine 181 (ptau), and Aβ₄₂ are established biomarkers for Alzheimer's disease (AD) and have been used as quantitative traits for genetic analyses. We performed the largest genome-wide association study for cerebrospinal fluid (CSF) tau/ptau levels published to date (n = 1,269), identifying three genome-wide significant loci for CSF tau and ptau: rs9877502 (p = 4.89 × 10⁻⁹ for tau) located at 3q28 between GEMC1 and OSTN, rs514716 (p = 1.07 × 10⁻⁸ and p = 3.22 × 10⁻⁹ for tau and ptau, respectively), located at 9p24.2 within GLIS3 and rs6922617 (p = 3.58 × 10⁻⁸ for CSF ptau) at 6p21.1 within the TREM gene cluster, a region recently reported to harbor rare variants that increase AD risk. In independent data sets, rs9877502 showed a strong association with risk for AD, tangle pathology, and global cognitive decline (p = 2.67 × 10⁻⁴, 0.039, 4.86 × 10⁻⁵, respectively) illustrating how this endophenotype-based approach can be used to identify new AD risk loci.

IMPORTANCE: Hexanucleotide repeat expansions in the chromosome 9 open reading frame 72 (C9orf72) gene underlie a significant fraction of frontotemporal dementia and amyotrophic lateral sclerosis.

OBJECTIVE: To investigate the frequency of C9orf72 repeat expansions in clinically diagnosed late-onset Alzheimer disease (AD).

DESIGN, SETTING, AND PATIENTS: This case-control study genotyped the C9orf72 repeat expansion in 872 unrelated familial AD cases and 888 control subjects recruited as part of the National Institute on Aging Late-Onset Alzheimer Disease Family Study cohort, a multisite collaboration studying 1000 families with 2 or more individuals clinically diagnosed as having late-onset AD.

MAIN OUTCOMES AND MEASURES: We determined the presence or absence of the C9orf72 repeat expansion by repeat-primed polymerase chain reaction, the length of the longest nonexpanded allele, segregation of the genotype with disease, and clinical features of repeat expansion carriers. RESULTS Three families showed large C9orf72 hexanucleotide repeat expansions. Two additional families carried more than 30 repeats. Segregation with disease could be demonstrated in 3 families. One affected expansion carrier had neuropathology compatible with AD. In the National Institute on Aging Late-Onset Alzheimer Disease Family Study series, the C9orf72 repeat expansions constituted the second most common pathogenic mutation, just behind the PSEN1 A79V mutation, highlighting the heterogeneity of clinical presentations associated with repeat expansions.

CONCLUSIONS AND RELEVANCE: C9orf72 repeat expansions explain a small proportion of patients with a clinical presentation indistinguishable from AD, and they highlight the necessity of screening frontotemporal dementia genes in clinical AD cases with strong family history.

Retinitis Pigmentosa (RP) involves a group of genetically determined retinal diseases caused by a large number of mutations that result in rod photoreceptor cell death followed by gradual death of cone cells. Most cases of RP are monogenic, with more than 80 associated genes identified so far. The high number of genes and variants involved in RP, among other factors, is making the molecular characterization of RP a real challenge for many patients. Although HRM has been used for the analysis of isolated variants or single RP genes, as far as we are concerned, this is the first study that uses HRM analysis for a high-throughput screening of several RP genes. Our main goal was to test the suitability of HRM analysis as a genetic screening technique in RP, and to compare its performance with two of the most widely used NGS platforms, Illumina and PGM-Ion Torrent technologies. RP patients (n = 96) were clinically diagnosed at the Ophthalmology Department of Donostia University Hospital, Spain. We analyzed a total of 16 RP genes that meet the following inclusion criteria: 1) size: genes with transcripts of less than 4 kb; 2) number of exons: genes with up to 22 exons; and 3) prevalence: genes reported to account for, at least, 0.4% of total RP cases worldwide. For comparison purposes, RHO gene was also sequenced with Illumina (GAII; Illumina), Ion semiconductor technologies (PGM; Life Technologies) and Sanger sequencing (ABI 3130xl platform; Applied Biosystems). Detected variants were confirmed in all cases by Sanger sequencing and tested for co-segregation in the family of affected probands. We identified a total of 65 genetic variants, 15 of which (23%) were novel, in 49 out of 96 patients. Among them, 14 (4 novel) are probable disease-causing genetic variants in 7 RP genes, affecting 15 patients. Our HRM analysis-based study, proved to be a cost-effective and rapid method that provides an accurate identification of genetic RP variants. This approach is effective for medium sized (<4 kb transcript) RP genes, which constitute over 80% of the total of known RP genes.

INTRODUCTION: Some familial Alzheimer's disease (AD) cases are caused by rare and highly-penetrant mutations in APP, PSEN1, and PSEN2. Mutations in GRN and MAPT, two genes associated with frontotemporal dementia (FTD), have been found in clinically diagnosed AD cases. Due to the dramatic developments in next-generation sequencing (NGS), high-throughput sequencing of targeted genomic regions of the human genome in many individuals in a single run is now cheap and feasible. Recent findings favor the rare variant-common disease hypothesis by which the combination effects of rare variants could explain a large proportion of the heritability. We utilized NGS to identify rare and pathogenic variants in APP, PSEN1, PSEN2, GRN, and MAPT in an Ibero-American cohort.

METHODS: We performed pooled-DNA sequencing of each exon and flanking sequences in APP, PSEN1, PSEN2, MAPT and GRN in 167 clinical and 5 autopsy-confirmed AD cases (15 familial early-onset, 136 sporadic early-onset and 16 familial late-onset) from Spain and Uruguay using NGS. Follow-up genotyping was used to validate variants. After genotyping additional controls, we performed segregation and functional analyses to determine the pathogenicity of validated variants.

RESULTS: We identified a novel G to T transition (g.38816G>T) in exon 6 of PSEN1 in a sporadic early-onset AD case, resulting in a previously described pathogenic p.L173F mutation. A pathogenic p.L392V mutation in exon 11 was found in one familial early-onset AD case. We also identified a novel CC insertion (g.10974_10975insCC) in exon 8 of GRN, which introduced a premature stop codon, resulting in nonsense-mediated mRNA decay. This GRN mutation was associated with lower GRN plasma levels, as previously reported for other GRN pathogenic mutations. We found two variants in MAPT (p.A152T, p.S318L) present only in three AD cases but not controls, suggesting that these variants could be risk factors for the disease.

CONCLUSIONS: We found pathogenic mutations in PSEN1, GRN and MAPT in 2.33% of the screened cases. This study suggests that pathogenic mutations or risk variants in MAPT and in GRN are as frequent in clinical AD cases as mutations in APP, PSEN1 and PSEN2, highlighting that pleiotropy of MAPT or GRN mutations can influence both FTD and AD phenotypic traits.