We defined the genetic landscape of balanced chromosomal rearrangements at nucleotide resolution by sequencing 141 breakpoints from cytogenetically-interpreted translocations and inversions. We confirm that the recently described phenomenon of “chromothripsis” (massive chromosomal shattering and reorganization) is not unique to cancer cells but also occurs in the germline where it can resolve to a karyotypically balanced state with frequent inversions. We detected a high incidence of complex rearrangements (19.2%) and substantially less reliance on microhomology (31%) than previously observed in benign CNVs. We compared these results to experimentally-generated DNA breakage-repair by sequencing seven transgenic animals, and revealed extensive rearrangement of the transgene and host genome with similar complexity to human germline alterations. Inversion is the most common rearrangement, suggesting that a combined mechanism involving template switching and non-homologous repair mediates the formation of balanced complex rearrangements that are viable, stably replicated and transmitted unaltered to subsequent generations.

Huntington's disease (HD) is an inherited autosomal dominant neurodegenerative disorder caused by an expansion of a CAG trinucleotide repeat in the huntingtin (HTT) gene [Huntington's Disease Collaborative Research Group (1993) A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. The Huntington's Disease Collaborative Research Group. Cell, 72, 971-983]. Despite identification of the gene in 1993, the underlying life-long disease process and effective treatments to prevent or delay it remain elusive. In an effort to fast-track treatment strategies for HD into clinical trials, we have developed a new large-animal HD transgenic ovine model. Sheep, Ovis aries L., were selected because the developmental pattern of the ovine basal ganglia and cortex (the regions primarily affected in HD) is similar to the analogous regions of the human brain. Microinjection of a full-length human HTT cDNA containing 73 polyglutamine repeats under the control of the human promotor resulted in six transgenic founders varying in copy number of the transgene. Analysis of offspring (at 1 and 7 months of age) from one of the founders showed robust expression of the full-length human HTT protein in both CNS and non-CNS tissue. Further, preliminary immunohistochemical analysis demonstrated the organization of the caudate nucleus and putamen and revealed decreased expression of medium size spiny neuron marker DARPP-32 at 7 months of age. It is anticipated that this novel transgenic animal will represent a practical model for drug/clinical trials and surgical interventions especially aimed at delaying or preventing HD initiation. New sequence accession number for ovine HTT mRNA: FJ457100.

The neurodegenerative disorder Huntington's disease (HD) is typically characterized by extensive loss of striatal neurons and the midlife onset of debilitating and progressive chorea, dementia, and psychological disturbance. HD is caused by a CAG repeat expansion in the Huntingtin (HTT) gene, translating to an elongated glutamine tract in the huntingtin protein. The pathogenic mechanism resulting in cell dysfunction and death beyond the causative mutation is not well defined. To further delineate the early molecular events in HD, we performed RNA-sequencing (RNA-seq) on striatal tissue from a cohort of 5-y-old OVT73-line sheep expressing a human CAG-expansion HTT cDNA transgene. Our HD OVT73 sheep are a prodromal model and exhibit minimal pathology and no detectable neuronal loss. We identified significantly increased levels of the urea transporter SLC14A1 in the OVT73 striatum, along with other important osmotic regulators. Further investigation revealed elevated levels of the metabolite urea in the OVT73 striatum and cerebellum, consistent with our recently published observation of increased urea in postmortem human brain from HD cases. Extending that finding, we demonstrate that postmortem human brain urea levels are elevated in a larger cohort of HD cases, including those with low-level neuropathology (Vonsattel grade 0/1). This elevation indicates increased protein catabolism, possibly as an alternate energy source given the generalized metabolic defect in HD. Increased urea and ammonia levels due to dysregulation of the urea cycle are known to cause neurologic impairment. Taken together, our findings indicate that aberrant urea metabolism could be the primary biochemical disruption initiating neuropathogenesis in HD.is a dominantly inherited neurological disorder typified by chorea, psychological disturbance, and dementia. The symptoms progress and result in premature death, typically 10-15 y after onset. Currently no available treatment can delay or prevent the onset of HD. The gene responsible, Huntingtin (HTT), is ubiquitously expressed and encodes the large and multifunctional huntingtin protein.The disease-causing mutation is an expanded CAG repeat in exon 1 of HTT, coding for a glutamine tract within the protein (1). The disease-causing repeat lower length threshold is 36 units and is fully penetrant at 40 units and above (2, 3). There is an inverse correlation between expanded CAG repeat size and age at onset of symptoms (4-6). Although the mutation is well defined, the pathogenic process is not sufficiently understood to enable effective treatment. The majority of HD research focuses on the brain where there is characteristic neuropathology, primarily atrophy of the striatum (7).Alongside the striking neurological phenotype of HD, there is a generalized metabolic disruption. HD mutation carriers weigh less on average than non-HD individuals (8). Weight loss begins presymptomatically (9, 10), and in symptomatic individuals, energy expenditure far exceeds that utilized in movement, despite high calor...

The enzyme peptidylarginine deiminase (PAD; EC 3.5.3.15) is responsible for the formation of protein-bound citrulline, a major amino acid in the inner root sheath (IRS) and in the medulla of the hair follicle. From mainly biochemical evidence, it is known that the substrate for the enzyme is trichohyalin and that trichohyalin granules gradually disappear to form a matrix with intermediate-like filaments in the IRS cells. In the medulla, the granules aggregate into large masses without filaments. The proteins in both the IRS and medulla are finally cross-linked by transglutaminase. A corollary of the apparent central role of PAD acting on the trichohyalin protein in these processes is that it should be present in the IRS and medulla cells, coincident with trichohyalin. Hair-follicle PAD has not previously been isolated. In the current study, the enzyme was isolated from wool follicles of adult sheep and peptide sequences were used to design DNA primers for the synthesis of PCR products from follicle mRNA. Subsequently, a PAD-specific complementary RNA probe and a trichohyalin complementary RNA probe were prepared for localization studies by in situ hybridization in wool follicles and the epithelia of the rumen, embryonic hoof, and tongue papillae. The experiments have revealed a striking co-expression of PAD and trichohyalin in all of these tissues. The amino acid sequence of the wool-follicle PAD molecule has been deduced from sequencing of the cloned PCR products.

Insidious changes in behaviour herald the onset of progressive neurodegenerative disorders such as Huntington's disease (HD), sometimes years before overt symptoms are seen. Sleep and circadian disturbances are particularly disruptive symptoms in patients with neurological disorders, but they are difficult to measure in humans. Here we studied circadian behaviour in transgenic HD sheep expressing the full-length human huntingtin protein with an expanded CAG repeat mutation in the juvenile range. Young HD sheep with no other symptoms exhibited circadian behavioural abnormalities that worsened with age. The most obvious change was a disturbed evening behaviour reminiscent of 'sundowning' that is seen in some patients with dementia. There were no structural abnormalities seen with magnetic resonance imaging, even in 5-year-old HD sheep. Interestingly, detection of the circadian abnormalities depended upon their social grouping. Abnormalities emerged in sheep kept in an 'HD-only' flock, whereas the behaviour of HD sheep kept mixed with normal sheep was relatively normal. Sleep-wake abnormalities in HD patients are also likely to be hidden, and may precede overt symptoms by many years. Sleep disruption has deleterious effects, even in normal people. The knock-on effects of sleep-wake disturbance may exacerbate, or even cause symptoms such as irritability and depression that are common in early stage HD patients. HD sheep will be useful models for probing the mechanisms underlying circadian behavioural disorder in HD.