Lower limit of amplification of the in-house HIV-1 genotyping assay.

<p>Lower limit of amplification of the in-house HIV-1 genotyping assay.</p

Primers for the amplification and sequencing of the HIV-1 protease and reverse transcriptase.

<p>Primers for the amplification and sequencing of the HIV-1 protease and reverse transcriptase.</p

General mechanisms for <i>ATXN2</i> gene <i>de novo</i> mutagenesis in the population.

<p>Two models can be proposed for explanation of <i>de novo</i> CAG expansions in <i>ATXN2</i>. Both involve loss of the CAA interruption in large alleles resulting in a minimal length of pure repeat within the CAG expansion. CAA interruptions break the CAG tract in discrete repeat arrays protecting it from instability <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070560#pone.0070560-Ross1" target="_blank">[17]</a>. According to this study, the minimal length of the internal pure repeat leading to <i>de novo</i> mutations is 8 CAG.</p

Genetic markers for <i>ATXN2</i> haplotyping and gene sequencing.

<p>A) <i>ATXN2</i> gene schematic maps and microsatellite, D12S1333 (telomeric at 200 kb from <i>ATXN2</i>), D12S1672 (intragenic at exon 1) and D12S1332 (centromeric at 350 kb from <i>ATXN2</i>) and SNIPs markers, rs695871 (at 177 bp upstream CAG expansion), rs695872 (at 106 bp upstream CAG expansion) and rs390624 (within the expanded CAG) used for haplotyping cases involved in <i>de novo</i> mutations, the polymorphic (CCG)nCCC/poly-proline adjacent to the CAG expansion is also indicated. B) Sequencing for case II-10, mother of the proband III-16 (25 CAG with only one CAA interruption). C) Relative position for other SNPs situated either within or near the expanded CAG.</p

Phenotype and <i>ATXN2</i> genotype for ALS case series.

<p>Affected by history and genotype inferred from daugther.</p>*<p>De novo mutation causing disease. <b>EERC</b>: El Escorial Revised Classification.</p

<i>De Novo</i> Mutations in Ataxin-2 Gene and ALS Risk

<div><p>Pathogenic CAG repeat expansion in the ataxin-2 gene (<i>ATXN2</i>) is the genetic cause of spinocerebellar ataxia type 2 (SCA2). Recently, it has been associated with Parkinsonism and increased genetic risk for amyotrophic lateral sclerosis (ALS). Here we report the association of <i>de novo</i> mutations in <i>ATXN2</i> with autosomal dominant ALS. These findings support our previous conjectures based on population studies on the role of large normal <i>ATXN2</i> alleles as the source for new mutations being involved in neurodegenerative pathologies associated with CAG expansions. The <i>de novo</i> mutations expanded from ALS/SCA2 non-risk alleles as proven by meta-analysis method. The ALS risk was associated with SCA2 alleles as well as with intermediate CAG lengths in the <i>ATXN2</i>. Higher risk for ALS was associated with pathogenic CAG repeat as revealed by meta-analysis.</p></div

Genetics, EMG and MRI analysis.

<p>A) Electrophoresis of fluorescent fragment analysis of <i>ATXN2</i> CAG repeat. In each lane, the content is specified. B) Fasciculation patterns in proband’s tongue and in biceps brachii recorded by EMG. C) Midsagittal MRI image of proband, no cerebellar atrophy is evident. D) Representative 3% agarose electrophoresis of <i>C9ORF72</i> analysis in index case and parents (lanes 2, 3, 4). Lanes 1and 8: MW markers (Ready Load™ 1–12.216 Kb ladder and 250–3500 bp ladder in multiples of 250 bp (Invitrogen), respectively; Lane 7: mock; lanes 2, 3, 4: case III-16, and both parents II-9 and II-10, respectively. Note that each DNA showed two defined bands despite PCR products with 7-deaza-2-deoxy GTP stain poorly with ethidium bromide. These 3 samples were heterozygous with bands higher than 250 bp but bellow 350 bp (hex repeat ∼11units) using Renton et al. primers anchoring 280 bp from hex-repeat. Lanes 5, 6 are unrelated ALS cases from the Cuban population.</p

Meta-analyses for large and intermediate CAG lengths in <i>ATXN2</i>.

<p>Galbraith (A–C) and forest plots (D–F) for different <i>ATXN2</i> CAG length across populations. A) and D) ≥24/27 CAG. B) and E) ≥30 CAG. C) and F) ≥32 CAG.</p

Table. 2. Microsatellite haplotypes.

<p>NA: Not available, Inferred haplotype are bracketed,</p>*<p>sick by history.</p