Whole Exome and Transcriptome Sequencing in Sporadic ALS

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a fatal and devastating neurodegenerative disorder that causes the progressive death of upper and lower motor neurons. Although many efforts have been done to elucidate molecular factors involved in the onset and progression of the disorder, the causes of ALS are yet unknown and undefined. Transcriptome studies, based mostly on microarrays, have revealed multiple perturbations of the motor neuron function, supporting the current idea that several cellular events contribute to the pathobiology of the disease, including mitochondrial dysfunction, enhanced apoptosis, glutamate-mediated excitotoxicity, free radical injury, protein misfolding, abnormal calcium metabolism and altered axonal transport. In the present study, we have deeply sequenced the whole transcriptome of ventral horns of the human lumbar spinal cord from matched control and ALS post-mortem donors. Whole exome sequencing from the same donors has also been performed to exclude known genetic variants associated to the familiar form of ALS. In addition, to characterize the ALS transcriptome we have sequenced the RNA fraction at low molecular weight in the same tissues and individuals. Genomic and transcriptomic reads have been generated using the Illumina HiSeq2000 sequencer.]]>
We collected age- and sex- matched post-autopsy samples of ventral horns from lumbar spinal cord from 3 ALS and 3 controls. Samples were provided from NICHD Brain and Tissue Bank for Developmental Disorders (http://medschool.umaryland.edu/btbank/). Age was in the range 54-61 years old. Gender was male in all cases. Post-mortem delay was less than 10 hours for ALS samples. Race was Caucasian. ALS donor IDs (according to NICHD bank) were: 5388, 925 and 4762. Control donor IDs (according to NICHD bank) were: 5393, 5356 and 5362. Sample 5362 was identified as outlier based on our RNA-Seq experiments. We have extended the initial study collecting additional age- and sex- matched post-autopsy samples of ventral horns from lumbar spinal cord. Samples were obtained from the NICHD Brain and Tissue Bank for Developmental Disorders (University of Maryland - http://medschool.umaryland.edu/btbank/) and the Human Brain and Spinal Fluid Resource Center (Los Angeles, CA). Age was in the range 42-69 years old. Gender was male in all cases. Post-mortem delay was less than 19 hours and race was Caucasian. ALS donor IDs from NICHD bank were: 5595 and 1292. ALS donor IDs from HBSFRC bank were: 5215, 5187, 5165 and 5223. Control donor IDs from NICHD bank were: 5456, 5663, 5458, 5361 and 5617. ]]>
We started a pilot study to characterize the transcriptome of ventral horn tissue from post-mortem ALS patients in 2011, analyzing 3 controls and 3 diseased samples. In this first attempt, we sequenced only the polyA+ fraction of RNAs. Whole exome sequencing of same samples was also performed to confirm their sporadic nature. In 2015 we extended the previous study including 5 controls and 6 ALS samples that have been sequenced according to the TruSeq Stranded Total RNA Sample Prep Kit on an Illumina NextSeq500 platform.]]>