Targeted next generation sequencing in SPAST-negative hereditary spastic paraplegia.

Molecular characterization is important for an accurate diagnosis in hereditary spastic paraplegia (HSP). Mutations in the gene SPAST (SPG4) are the most common cause of autosomal dominant forms. We performed targeted next generation sequencing (NGS) in a SPAST-negative HSP sample. Forty-four consecutive HSP patients were recruited from an adult neurogenetics clinic in Sydney, Australia. SPAST mutations were confirmed in 17 subjects, and therefore 27 SPAST-negative patients were entered into this study. Patients were screened according to mode of inheritance using a PCR-based library and NGS (Roche Junior 454 sequencing platform). The screening panel included ten autosomal dominant (AD) and nine autosomal recessive (AR) HSP-causing genes. A genetic cause for HSP was identified in 25.9 % (7/27) of patients, including 1/12 classified as AD and 6/15 as AR or sporadic inheritance. Several forms of HSP were identified, including one patient with SPG31, four with SPG7 (with one novel SPG7 mutation) and two with SPG5 (including two novel CYP7B1 frameshift mutations). Additional clinical features were noted, including optic atrophy and ataxia for patients with SPG5 and ataxia and a chronic progressive external ophthalmoplegia-like phenotype for SPG7. This protocol enabled the identification of a genetic cause in approximately 25 % of patients in whom one of the most common genetic forms of HSP (SPG4) was excluded. Targeted NGS may be a useful method to screen for mutations in multiple genes associated with HSP. More studies are warranted to determine the optimal approach to achieve a genetic diagnosis in this condition.

Mutant Parkin impairs mitochondrial function and morphology in human fibroblasts.

BACKGROUND: Mutations in Parkin are the most common cause of autosomal recessive Parkinson disease (PD). The mitochondrially localized E3 ubiquitin-protein ligase Parkin has been reported to be involved in respiratory chain function and mitochondrial dynamics. More recent publications also described a link between Parkin and mitophagy. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the impact of Parkin mutations on mitochondrial function and morphology in a human cellular model. Fibroblasts were obtained from three members of an Italian PD family with two mutations in Parkin (homozygous c.1072delT, homozygous delEx7, compound-heterozygous c.1072delT/delEx7), as well as from two relatives without mutations. Furthermore, three unrelated compound-heterozygous patients (delEx3-4/duplEx7-12, delEx4/c.924C>T and delEx1/c.924C>T) and three unrelated age-matched controls were included. Fibroblasts were cultured under basal or paraquat-induced oxidative stress conditions. ATP synthesis rates and cellular levels were detected luminometrically. Activities of complexes I-IV and citrate synthase were measured spectrophotometrically in mitochondrial preparations or cell lysates. The mitochondrial membrane potential was measured with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide. Oxidative stress levels were investigated with the OxyBlot technique. The mitochondrial network was investigated immunocytochemically and the degree of branching was determined with image processing methods. We observed a decrease in the production and overall concentration of ATP coinciding with increased mitochondrial mass in Parkin-mutant fibroblasts. After an oxidative insult, the membrane potential decreased in patient cells but not in controls. We further determined higher levels of oxidized proteins in the mutants both under basal and stress conditions. The degree of mitochondrial network branching was comparable in mutants and controls under basal conditions and decreased to a similar extent under paraquat-induced stress. CONCLUSIONS: Our results indicate that Parkin mutations cause abnormal mitochondrial function and morphology in non-neuronal human cells.

A novel CLCN1 mutation (G1652A) causing a mild phenotype of thomsen disease.

We investigated a 62-year-old man who had mild clinical features of myotonia congenita. He was found to have a novel heterozygous G-to-A nucleotide substitution at position 1652 in exon 15 of the CLCN1 gene. Clinicogenetic studies performed on his family revealed that his asymptomatic son also shared the mutation. We conclude that a novel chloride channel mutation (G1652A) has caused a mild form of autosomal-dominant myotonia congenita (Thomsen disease) in this family.

Mitochondrial DNA haplogroups J and K are not protective for Parkinson's disease in the Australian community.

MtDNA haplogroups J and K have been associated with a decreased risk of developing Parkinson's disease (PD). To confirm this finding, we compared the distribution of mtDNA haplogroups J and K in a large sample of Australian patients with PD (n = 890) to population-based controls (n = 3,491). We assigned subjects to haplogroups J or K using standard PCR/RFLP techniques. Of the 890 subjects with PD, 10.6% were haplogroup J (95% CI 8.6-12.8, n = 94) and 7.1% were haplogroup K (95% CI 5.5-8.9, n = 63). In our controls, 10.2% belonged to haplogroup J (95% CI 9.2-11.2, n = 356), and 7.8% were in haplogroup K (95% CI 6.9-8.7, n = 272). There was no significant difference in the prevalence of mtDNA haplogroup J or K in PD patients compared to population-based controls. Our findings indicate that mtDNA haplogroups J and K are not associated with a lower risk of PD.

Prevalence and clinical features of common LRRK2 mutations in Australians with Parkinson's disease.

We determined the prevalence of two common leucine-rich repeat kinase 2 (LRRK2) gene mutations in Australian patients with Parkinson's disease (PD). Of 830 affected patients, eight were heterozygous for the G2019S mutation, and two were heterozygous for the R1441H (4,322 G > A) mutation. In addition, one familial patient had a novel A1442P (4,324 G > C) mutation. Haplotype analysis showed that all LRRK2 G2019S-positive individuals carried the common founder haplotype 1 and a putative founder haplotype for the R1441H mutation carriers. Clinically, patients with LRRK2 mutations had typical levodopa responsive Parkinsonism with tremor being the commonest presenting feature. Patients with the G2019S mutation in our series had a similar age of onset of symptoms when compared with patients with other LRRK2 mutations or sporadic PD, although they were more likely to have a family history of PD (2.4% of Australian patients with familial PD and 0.3% of Australian patients with sporadic PD). Our results demonstrate that the G2019S mutation carriers share the same ancestors who migrated to Australia originally from Europe and that other LRRK2 mutations (R1441H and A1442P) can be found in this population.

Immunoreceptor transmembrane peptides and their effect on natural killer (NK) cell cytotoxicity.

Short peptides derived from the transmembrane sequence of NK activating receptors and associated molecules were tested in vitro for inhibition of NK cell cytotoxicity using a standard (51)Cr release assay in the absence or presence of peptides. NKL23 cell line was used as the NK effector and the target was the NKL23 sensitive 721.221 cell line. NKp46, NKp30, NKG2D and CD3-zeta peptides inhibited NK activity at higher concentration (100 microM) compared to controls by 6-13% (p<0.05). Modification of one non-effective peptide (NKP44) significantly enhanced inhibition by 30%, 17% and 11% at 100 microM, 50 microM and 10 microM respectively compared to controls. A T-cell antigen receptor-alpha chain transmembrane sequence derived peptide (CP) significantly inhibited NKL cell activation by 20-30% (p<0.05) at 50 microM and 100 microM concentrations compared to the control. The structural similarities between these immuno-receptors, and in particular the need for transmembrane electrostatic interactions for receptor function, provides the basis for current and future targeted therapeutic strategies.