Ilaria Guella

Effects of PCSK9 genetic variants on plasma LDL cholesterol levels and risk of premature myocardial infarction in the Italian population

The R46L variant in the proprotein-convertase subtilisin-kexin type 9 (PCSK9) gene was associated with reduced levels of LDL and total cholesterol and with a lower risk of coronary artery disease. We investigated the association of R46L with myocardial infarction (MI) in 1,880 Italian patients with premature MI and 1,880 controls. A trend toward a protective effect of the L46 allele was observed [odds ratio (OR) = 0.75, 95% confidence interval (CI) = 0.49–1.13; P = 0.17], although the association with MI was not significant. This is probably due to the combined effect of the low frequency of R46L among Italians and of the young age of the analyzed cohort for whom the impact of coronary atherosclerosis is less important. This hypothesis was indirectly confirmed by the significant association found after including 1,056 additional older controls (OR = 0.67, 95% CI = 0.46-0.97; P = 0.036). LDL cholesterol was significantly lower in L46 carriers (116.2 ± 34.7 mg/dl) than in noncarriers (137.4 ± 47.3 mg/dl; P = 0.00022); a similar reduction was observed for total cholesterol (191.7 ± 37.7 vs. 211.7 ± 49 mg/dl; P = 0.00019). Analysis of 23 additional polymorphisms in the PCSK9 region identified another single nucleotide polymorphism (SNP) (rs11206510) associated with cholesterol levels. We confirmed that the L46 allele not only decreases LDL cholesterol but also protects against MI. Moreover, we replicated the association of total and LDL cholesterol with the SNP rs11206510.

The association of factor V Leiden with myocardial infarction is replicated in 1880 patients with premature disease

See also Tosetto A. Thrombophilic mutations and cardiovascular disease: the case is still open. This issue, pp 2113–5.

α-synuclein genetic variability: A biomarker for dementia in Parkinson disease

The relationship between Parkinson disease (PD), PD with dementia (PDD), and dementia with Lewy bodies (DLB) has long been debated. Although PD is primarily considered a motor disorder, cognitive impairment is often present at diagnosis, and only ∼20% of patients remain cognitively intact in the long term. Alpha‐synuclein (SNCA) was first implicated in the pathogenesis of the disease when point mutations and locus multiplications were identified in familial parkinsonism with dementia. In worldwide populations, SNCA genetic variability remains the most reproducible risk factor for idiopathic PD. However, few investigators have looked at SNCA variability in terms of cognitive outcomes.

Disease Penetrance of Late-Onset Parkinsonism: A Meta-analysis

IMPORTANCE Mutations in SNCA, LRRK2, VPS35, EIF4G1, and DNAJC13 have been implicated in late-onset familial parkinsonism. However, the estimated disease penetrance of these mutations varies widely. OBJECTIVE To compare penetrance of various mutations reported in published genetic studies to improve the understanding of late-onset parkinsonism. DATA SOURCES Forty-nine previously published studies, including 709 participants, were included for all original and subsequent articles in ISI Web of Science, PubMed electronic databases, and extracted information about number of mutation carriers within families and sporadic cases worldwide for pathogenic mutations in SNCA, LRRK2, VPS35, EIF4G1, and DNAJC13. The end-of-search date was January 31, 2014. STUDY SELECTION Published studies were included if there was information on the ethnicity of the patient or unaffected individual, confirmation of mutation, age of patient or unaffected individual, age at onset, and first motor symptom of patient. Autosomal recessive parkinsonism and genes implicated without significant genetic linkage were excluded from this study. DATA EXTRACTION AND SYNTHESIS The age-associated cumulative incidence was estimated using the Kaplan-Meier method with age at onset as the time variable; asymptomatic carriers were right censored at the age at last contact or age at death. MAIN OUTCOMES AND MEASURES Comparative measures were obtained with log-rank tests, and each penetrance estimate was given separately with 95% confidence intervals. RESULTS All the assessed autosomal dominant Parkinson disease mutations have significantly different age-dependent cumulative incidences (P < .001). In particular, penetrance of SNCA duplications was comparable to point mutations (log-rank P = .97) and driven by inclusion of SNCA p.A53T (mean age at onset, 45.9 years; 95% CI, 43-49 years). In addition, Israeli Ashkenazi Jewish LRRK2 p.G2019S carriers (mean age at onset, 57.9 years; 95% CI, 54-63 years) were comparable to Tunisian Arab Berbers (mean age at onset, 57.1 years; 95% CI, 45.5-68.7 years) (P = .58), whereas Norwegian carriers (mean age at onset, 63 years; 95% CI, 51.4-74.6 years) were significantly different from the other groups (P < .001). CONCLUSIONS AND RELEVANCE Parkinson disease pathogenic mutations have an age-dependent penetrance that could be ameliorated or exacerbated by modifier genes or environmental factors in different populations.

SNCA and MAPT genes: Independent and joint effects in Parkinson disease in the Italian population

Background Significant efforts have been focused on investigating the contribution of common variants to Parkinson disease (PD) risk. Several independent GWAS and metanalysis studies have shown a genome-wide significant association of single nucleotide polymorphisms (SNPs) in the α-synuclein (SNCA) and microtubule-associated protein tau (MAPT) regions. Here we investigated the role of SNCA and MAPT as PD susceptibility genes in a large Italian population of 904 patients and 891 controls. An evaluation of gene–gene and gene-environment interactions in association with PD was also attempted. Methods The SNCA Rep1 microsatellite was genotyped by a fluorescent PCR assay, whereas the SNPlex genotyping system was used to genotype 12 additional markers across the SNCA gene, and 2 SNPs tagging the risk MAPT H1 haplotype. Results Single-marker analysis demonstrated nominal evidence of association for: i) the 261-bp-long allele of Rep1; ii) 7 SNPs in the SNCA region (top SNP: rs356186, P = 3.08 × 10−04, intron 4); iii) both SNPs identifying the MAPT H1 haplotype (P = 4.63 × 10−04 and P = 4.23 × 10−04 for rs1800547 and rs9468, respectively). Moreover, we found a highly significant protective haplotype spanning ∼83 kb from intron 4 to the 3′ end of SNCA (P = 1.29 × 10−05). Conclusions Our findings strongly confirm SNCA and MAPT as major PD susceptibility genes for idiopathic PD in the Italian population. Interaction analyses did not evidence either epistatic effects between the two loci or gene-environment interactions.

Glucocerebrosidase mutations in primary parkinsonism

Introduction Mutations in the lysosomal glucocerebrosidase (GBA) gene increase the risk of Parkinsons Disease (PD). We determined the frequency and relative risk of major GBA mutations in a large series of Italian patients with primary parkinsonism. Methods We studied 2766 unrelated consecutive patients with clinical diagnosis of primary degenerative parkinsonism (including 2350 PD), and 1111 controls. The entire cohort was screened for mutations in GBA exons 9 and 10, covering approximately 70% of mutations, including the two most frequent defects, p.N370S and p.L444P. Results Four known mutations were identified in heterozygous state: 3 missense mutations (p.N370S, p.L444P, and p.D443N), and the splicing mutation IVS10+1G>T, which results in the in-frame exon-10 skipping. Molecular characterization of 2 additional rare variants, potentially interfering with splicing, suggested a neutral effect. GBA mutations were more frequent in PD (4.5%, RR = 7.2, CI = 3.3–15.3) and in Dementia with Lewy Bodies (DLB) (13.8%, RR = 21.9, CI = 6.8–70.7) than in controls (0.63%). but not in the other forms of parkinsonism such as Progressive Supranuclear Palsy (PSP, 2%), and Corticobasal Degeneration (CBD, 0%). Considering only the PD group, GBA-carriers were younger at onset (52 ± 10 vs. 57 ± 10 years, P < 0.0001) and were more likely to have a positive family history of PD (34% vs. 20%, P < 0.001). Conclusion GBA dysfunction is relevant for synucleinopathies, such as PD and DLB, except for MSA, in which pathology involves oligodendrocytes, and the tauopathies PSP and CBD. The risk of developing DLB is three-fold higher than PD, suggesting a more aggressive phenotype.

Defining neurodegeneration on Guam by targeted genomic sequencing.

Amyotrophic lateral sclerosis/parkinsonism–dementia complex has been described in Guam, Western Papua, and the Kii Peninsula of Japan. The etiology and pathogenesis of this complex neurodegenerative disease remains enigmatic.

Association and Functional Analyses of MEF2A as a Susceptibility Gene for Premature Myocardial Infarction and Coronary Artery Disease

Background—Mutations in the MEF2A gene, coding for a member of the myocyte enhancer factor 2 family of transcription factors, have been reported in patients with coronary artery disease and myocardial infarction (MI). In particular, a 21-bp deletion and 3 missense mutations were demonstrated either to reduce MEF2A transcriptional activity or to impair its nuclear translocation. However, the association of MEF2A with coronary artery disease/MI was not confirmed in other studies. We analyzed the role of MEF2A in the pathogenesis of MI in 2008 Italian patients with premature MI and in 2008 controls. Methods and Results—Mutational screening of exon 8 (containing all so-far reported point mutations) disclosed 5 novel and 2 previously described missense mutations. Microsatellite genotyping and sequencing revealed the presence of the 21-bp deletion (located in exon 12) in 5 cases and in none of the controls. Functional studies on mutant proteins showed no alteration, neither in the transactivating properties (all mutants) nor in the nuclear localization (21-bp deletion). Furthermore, an association analysis performed using 3 microsatellites at the MEF2A locus showed no significant association with MI. These results were confirmed in a replication study performed on an independent Italian population with coronary artery disease. Conclusions—All together, our data do not support MEF2A as a susceptibility gene for coronary artery disease/MI in the Italian population.

Molecular characterization of in-frame and out-of-frame alternative splicings in coagulation factor XI pre-mRNA

Alternative splicing of pre-mRNAs is a central process to the generation of proteome complexity. However, many alternative mRNA isoforms carry premature termination codons (PTCs) rendering them possible targets for the nonsense-mediated mRNA decay (NMD) pathway. The F11 gene consists of 15 exons spanning approximately 23 kb on chromosome 4q35 and codes for coagulation factor XI (FXI), a 160-kDa dimeric zymogen composed of 4 apple domains and a serine protease domain. Here, we characterized the F11 splicing pattern in human liver and platelets identifying multiple in-frame and out-of-frame splicing events. Inhibition of NMD resulted in the up-regulation of all unproductively spliced F11 transcripts, thus providing evidence that these PTC-containing mRNAs are under the control of NMD. Among in-frame alternatively spliced transcripts, the one skipping exons 6 and 7 would lead to the synthesis of a FXI protein lacking 1 apple domain (FXI-Delta6/7). Ex vivo expression in mammalian cells demonstrated that FXI-Delta6/7 is mostly retained intracellularly, and secreted only in low amounts. Traces of this FXI isoform were detectable in human plasma. Our results suggest that the coupling of alternative splicing and NMD may play a role in regulating F11 expression, and point to the existence of a novel FXI isoform.

Molecular characterization of two novel mutations causing factor XI deficiency: A splicing defect and a missense mutation responsible for a CRM+ defect

Severe factor XI (FXI) deficiency is a bleeding disorder generally inherited as an autosomal recessive trait and characterized by haemorrhagic symptoms mainly associated with injury or surgery. So far, more than 150 causative molecular defects have been identified throughout the F11 gene. In the present study, we investigated the molecular basis of FXI deficiency in two Italian patients. Mutational screening of the F11 gene disclosed a novel missense substitution (Arg184Gly) in exon 7 and two splicing mutations: a novel G>A transition affecting the last nucleotide of exon 4 (325G>A), and the already known IVS6+3A>G. RT-PCR assays were performed on total RNA extracted from platelets and lymphocytes of each patient. Sequencing of RT-PCR products demonstrated that both 325G>A and IVS6+3A>G mutations abolish the corresponding donor splice site, causing the skipping of the affected exon; this in turn results in a frameshift introducing a premature termination codon. Expression of recombinant FXI-Arg184Gly revealed a 70% reduction in FXI activity, suggesting that the Arg184Gly mutation might cause a cross-reactive material positive (CRM+) deficiency. In conclusion, the functional consequences of two splicing mutations leading to FXI deficiency have been elucidated. Moreover, we report a novel missense mutation in the FIX-binding region of the FXI A3 domain leading to a CRM+ deficiency.