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© 2019 by the authors. Leishmaniasis is caused by the flagellated protozoan Leishmania, and is a neglected tropical disease (NTD), as defined by theWorld Health Organisation (WHO). Bisulphite conversion technology converts all genomic material to a simplified form during the lysis step of the nucleic acid extraction process, and increases the efficiency of multiplex quantitative polymerase chain reaction (qPCR) reactions. Through utilization of qPCR real-time probes, in conjunction with bisulphite conversion, a new duplex assay targeting the 18S rDNA gene region was designed to detect all Leishmania species. The assay was validated against previously extracted DNA, from seven quantitated DNA and cell standards for pan-Leishmania analytical sensitivity data, and 67 cutaneous clinical samples for cutaneous clinical sensitivity data. Specificity was evaluated by testing 76 negative clinical samples and 43 bacterial, viral, protozoan and fungal species. The assay was also trialed in a side-by-side experiment against a conventional PCR (cPCR), based on the Internal transcribed spacer region 1 (ITS1 region). Ninety-seven percent of specimens from patients that previously tested positive for Leishmania were positive for Leishmania spp. with the bisulphite conversion assay, and a limit of detection (LOD) of 10 copies per PCR was achieved, while the LOD of the ITS1 methodology was 10 cells/1000 genomic copies per PCR. This method of rapid, accurate and simple detection of Leishmania can lead to improved diagnosis, treatment and public health outcomes.

Nemaline myopathy (NM) is a disorder of the skeletal muscle thin filament characterised by muscle dysfunction and electron-dense protein accumulations (nemaline bodies). Pathogenic mutations have been described in nine genes to date, but the genetic basis remains unknown in many cases. We used whole exome sequencing (WES) in two families with NM and subsequent gene sequencing in over 540 additional genetically unresolved NM patients to identify and characterise a new genetic cause of NM. We developed a knock-down zebrafish model of this condition and used immunohistochemistry, western blotting, single-fibre contractility studies and recombinant protein studies to characterise the expression, localisation and biochemical functions of the new disease-related protein. We identified homozygous or compound heterozygous variants in LMOD3, which encodes leiomodin-3 (Lmod3) in 21 patients from 14 families. Affected individuals had severe generalised weakness and hypotonia, and most affected individuals died in the neonatal period. We demonstrated that Lmod3 is expressed from early muscle differentiation, localises to thin filaments with enrichment at the pointed ends, and has strong actin nucleating activity. Loss of Lmod3 in patient muscle results in shortening and disorganisation of thin filaments. Knockdown of lmod3 in the zebrafish replicates this phenotype. These findings define a new genetic subtype of congenital myopathy and demonstrate an essential, previously unrecognised role for Lmod3 in the regulation of sarcomeric thin filaments in skeletal muscle.

Genome-wide association studies have been successful in identifying common variants that influence the susceptibility to complex diseases. From these studies, it has emerged that there is substantial overlap in susceptibility loci between diseases. In line with those findings, we hypothesized that shared genetic pathways may exist between multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). While both diseases may have inflammatory and neurodegenerative features, epidemiological studies have indicated an increased co-occurrence within individuals and families. To this purpose, we combined genome-wide data from 4088 MS patients, 3762 ALS patients and 12 030 healthy control individuals in whom 5 440 446 single-nucleotide polymorphisms (SNPs) were successfully genotyped or imputed. We tested these SNPs for the excess association shared between MS and ALS and also explored whether polygenic models of SNPs below genome-wide significance could explain some of the observed trait variance between diseases. Genome-wide association meta-analysis of SNPs as well as polygenic analyses fails to provide evidence in favor of an overlap in genetic susceptibility between MS and ALS. Hence, our findings do not support a shared genetic background of common risk variants in MS and ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative disease characterized by loss of upper and lower motor neurons. ALS is considered to be a complex trait and genome-wide association studies (GWAS) have implicated a few susceptibility loci. However, many more causal loci remain to be discovered. Since it has been shown that genetic variants associated with complex traits are more likely to be eQTLs than frequency-matched variants from GWAS platforms, we conducted a two-stage genome-wide screening for eQTLs associated with ALS. In addition, we applied an eQTL analysis to finemap association loci. Expression profiles using peripheral blood of 323 sporadic ALS patients and 413 controls were mapped to genome-wide genotyping data. Subsequently, data from a two-stage GWAS (3,568 patients and 10,163 controls) were used to prioritize eQTLs identified in the first stage (162 ALS, 207 controls). These prioritized eQTLs were carried

Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative disease characterized by loss of upper and lower motor neurons. ALS is considered to be a complex trait and genome-wide association studies (GWAS) have implicated a few susceptibility loci. However, many more causal loci remain to be discovered. Since it has been shown that genetic variants associated with complex traits are more likely to be eQTLs than frequency-matched variants from GWAS platforms, we conducted a two-stage genome-wide screening for eQTLs associated with ALS. In addition, we applied an eQTL analysis to finemap association loci. Expression profiles using peripheral blood of 323 sporadic ALS patients and 413 controls were mapped to genome-wide genotyping data. Subsequently, data from a two-stage GWAS (3,568 patients and 10,163 controls) were used to prioritize eQTLs identified in the first stage (162 ALS, 207 controls). These prioritized eQTLs were carried forward to the second sample with both gene-expression and genotyping data (161 ALS, 206 controls). Replicated eQTL SNPs were then tested for association in the second-stage GWAS data to find SNPs associated with disease, that survived correction for multiple testing. We thus identified twelve cis eQTLs with nominally significant associations in the second-stage GWAS data. Eight SNP-transcript pairs of highest significance (lowest p = 1.27 x 10(-51)) withstood multiple-testing correction in the second stage and modulated CYP27A1 gene expression. Additionally, we show that C9orf72 appears to be the only gene in the 9p21.2 locus that is regulated in cis, showing the potential of this approach in identifying causative genes in association loci in ALS. This study has identified candidate genes for sporadic ALS, most notably CYP27A1. Mutations in CYP27A1 are causal to cerebrotendinous xanthomatosis which can present as a clinical mimic of ALS with progressive upper motor neuron loss, making it a plausible susceptibility gene for ALS

Spinal muscular atrophy (SMA) is a clinically and genetically heterogeneous disease characterized by the degeneration of lower motor neurons. The most frequent form is linked to mutations in SMN1. Childhood SMA associated with progressive myoclonic epilepsy (SMA-PME) has been reported as a rare autosomal-recessive condition unlinked to mutations in SMN1. Through linkage analysis, homozygosity mapping, and exome sequencing in three unrelated SMA-PME-affected families, we identified a homozygous missense mutation (c.125C>T [p.Thr42Met]) in exon 2 of ASAH1 in the affected children of two families and the same mutation associated with a deletion of the whole gene in the third family. Expression studies of the c.125C>T mutant cDNA in Farber fibroblasts showed that acid-ceramidase activity was only 32% of that generated by normal cDNA. This reduced activity was able to normalize the ceramide level in Farber cells, raising the question of the pathogenic mechanism underlying the CNS involvement in deficient cells. Morpholino knockdown of the ASAH1 ortholog in zebrafish led to a marked loss of motor-neuron axonal branching, a loss that is associated with increased apoptosis in the spinal cord. Our results reveal a wide phenotypic spectrum associated with ASAH1 mutations. An acid-ceramidase activity below 10% results in Farber disease, an early-onset disease starting with subcutaneous lipogranulomata, joint pain, and hoarseness of the voice, whereas a higher residual activity might be responsible for SMA-PME, a later-onset phenotype restricted to the CNS and starting with lower-motor-neuron disease.