Deterioration of muscle force and contractile characteristics are early pathological events in spinal and bulbar muscular atrophy mice

Spinal and bulbar muscular atrophy (SBMA), also known as Kennedy's Disease, is a late-onset, X-linked, progressive neuromuscular disease, which predominantly affects males. The pathological hallmarks of the disease are defined by selective loss of spinal and bulbar motor neurons, accompanied by weakness, atrophy and fasciculations of bulbar and limb muscles. SBMA is caused by a CAG repeat expansion in the gene that encodes the androgen receptor (AR) protein. Disease manifestation is androgen dependent and results principally from a toxic gain of AR function. There are currently no effective treatments for this debilitating disease. It is important to understand the course of the disease in order to target therapeutics to key pathological stages. This is especially relevant in disorders such as SBMA, where disease can be identified prior to symptom onset, through family history and genetic testing. To fully characterise the role of muscle in SBMA, we undertook a longitudinal physiological and histological characterisation of disease progression in the AR100 mouse model of SBMA. Our results show that the disease first manifests in skeletal muscle, prior to any motor neuron degeneration, which only occurs in late stage disease. These findings reveal alterations in muscle function, including reduced muscle force and changes in contractile characteristics, are early pathological events in SBMA mice and suggest that muscle-targeted therapeutics may be effective in SBMA

Gene expression analysis reveals early dysregulation of disease pathways and links Chmp7 to pathogenesis of spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) results from a CAG repeat expansion within the androgen receptor gene (AR). It is unclear why motor neurons selectively degenerate and there are currently no treatments for this debilitating disease. To uncover the causative genes and pathways involved in motor neuron dysfunction, we undertook transcriptomic profiling of primary embryonic motor neurons from SBMA mice. We show that transcriptional dysregulation occurs early during development in SBMA motor neurons. One gene found to be dysregulated, Chmp7, was also altered in vivo in spinal cord before symptom onset in SBMA mice, and crucially in motor neuron precursor cells derived from SBMA patient stem cells, suggesting that Chmp7 may play a causal role in disease pathogenesis by disrupting the endosome-lysosome system. Furthermore, genes were enriched in SBMA motor neurons in several key pathways including p53, DNA repair, WNT and mitochondrial function. SBMA embryonic motor neurons also displayed dysfunctional mitochondria along with DNA damage, possibly resulting from DNA repair gene dysregulation and/or mitochondrial dysfunction. This indicates that a coordinated dysregulation of multiple pathways leads to development of SBMA. Importantly, our findings suggest that the identified pathways and genes, in particular Chmp7, may serve as potential therapeutic targets in SBMA

FUS is not dysregulated by the spinal bulbar muscular atrophy androgen receptor polyglutamine repeat expansion.

Spinal bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis are two distinct forms of motor neuron disease with different genetic causes, pathology, and clinical course. However, both disorders are characterized by the progressive loss of lower motor neurons and by a similar protective response to growth factors in animal models, therefore raising the possibility of an overlap in the final pathogenic cascade. Mutations in the FUS gene and fused in sarcoma (FUS) protein pathology have now been identified in some amyotrophic lateral sclerosis cases, while a CAG expansion in the androgen receptor gene is known to cause SBMA. Recently, multiple lines of evidence have identified FUS as a major target of the androgen receptor, suggesting that FUS could be dysregulated in SBMA motor neurons. We have investigated this possibility by using a well-established mouse model of SBMA and our analysis of primary motor neuron cultures, spinal cords, and microdissected motor neurons show no evidence for FUS dysregulation

CTCF cis-Regulates Trinucleotide Repeat Instability in an Epigenetic Manner: A Novel Basis for Mutational Hot Spot Determination

At least 25 inherited disorders in humans result from microsatellite repeat expansion. Dramatic variation in repeat instability occurs at different disease loci and between different tissues; however, cis-elements and trans-factors regulating the instability process remain undefined. Genomic fragments from the human spinocerebellar ataxia type 7 (SCA7) locus, containing a highly unstable CAG tract, were previously introduced into mice to localize cis-acting “instability elements,” and revealed that genomic context is required for repeat instability. The critical instability-inducing region contained binding sites for CTCF—a regulatory factor implicated in genomic imprinting, chromatin remodeling, and DNA conformation change. To evaluate the role of CTCF in repeat instability, we derived transgenic mice carrying SCA7 genomic fragments with CTCF binding-site mutations. We found that CTCF binding-site mutation promotes triplet repeat instability both in the germ line and in somatic tissues, and that CpG methylation of CTCF binding sites can further destabilize triplet repeat expansions. As CTCF binding sites are associated with a number of highly unstable repeat loci, our findings suggest a novel basis for demarcation and regulation of mutational hot spots and implicate CTCF in the modulation of genetic repeat instability

AIB1 gene amplification and the instability of polyQ encoding sequence in breast cancer cell lines

BACKGROUND: The poly Q polymorphism in AIB1 (amplified in breast cancer) gene is usually assessed by fragment length analysis which does not reveal the actual sequence variation. The purpose of this study is to investigate the sequence variation of poly Q encoding region in breast cancer cell lines at single molecule level, and to determine if the sequence variation is related to AIB1 gene amplification. METHODS: The polymorphic poly Q encoding region of AIB1 gene was investigated at the single molecule level by PCR cloning/sequencing. The amplification of AIB1 gene in various breast cancer cell lines were studied by real-time quantitative PCR. RESULTS: Significant amplifications (5–23 folds) of AIB1 gene were found in 2 out of 9 (22%) ER positive cell lines (in BT-474 and MCF-7 but not in BT-20, ZR-75-1, T47D, BT483, MDA-MB-361, MDA-MB-468 and MDA-MB-330). The AIB1 gene was not amplified in any of the ER negative cell lines. Different passages of MCF-7 cell lines and their derivatives maintained the feature of AIB1 amplification. When the cells were selected for hormone independence (LCC1) and resistance to 4-hydroxy tamoxifen (4-OH TAM) (LCC2 and R27), ICI 182,780 (LCC9) or 4-OH TAM, KEO and LY 117018 (LY-2), AIB1 copy number decreased but still remained highly amplified. Sequencing analysis of poly Q encoding region of AIB1 gene did not reveal specific patterns that could be correlated with AIB1 gene amplification. However, about 72% of the breast cancer cell lines had at least one under represented (<20%) extra poly Q encoding sequence patterns that were derived from the original allele, presumably due to somatic instability. Although all MCF-7 cells and their variants had the same predominant poly Q encoding sequence pattern of (CAG)(3)CAA(CAG)(9)(CAACAG)(3)(CAACAGCAG)(2)CAA of the original cell line, a number of altered poly Q encoding sequences were found in the derivatives of MCF-7 cell lines. CONCLUSION: These data suggest that poly Q encoding region of AIB1 gene is somatic unstable in breast cancer cell lines. The instability and the sequence characteristics, however, do not appear to be associated with the level of the gene amplification

Mechanistic insight into the pathology of polyalanine expansion disorders revealed by a mouse model for x linked hypopituitarism

Extent: 9 p.Polyalanine expansions in transcription factors have been associated with eight distinct congenital human diseases. It is thought that in each case the polyalanine expansion causes misfolding of the protein that abrogates protein function. Misfolded proteins form aggregates when expressed in vitro; however, it is less clear whether aggregation is of relevance to these diseases in vivo. To investigate this issue, we used targeted mutagenesis of embryonic stem (ES) cells to generate mice with a polyalanine expansion mutation in Sox3 (Sox3-26ala) that is associated with X-linked Hypopituitarism (XH) in humans. By investigating both ES cells and chimeric mice, we show that endogenous polyalanine expanded SOX3 does not form protein aggregates in vivo but rather is present at dramatically reduced levels within the nucleus of mutant cells. Importantly, the residual mutant protein of chimeric embryos is able to rescue a block in gastrulation but is not sufficient for normal development of the hypothalamus, a region that is functionally compromised in Sox3 null embryos and individuals with XH. Together, these data provide the first definitive example of a disease-relevant PA mutant protein that is both nuclear and functional, thereby manifesting as a partial loss-of-function allele.James Hughes Sandra Piltz, Nicholas Rogers, Dale McAninch, Lynn Rowley and Paul Thoma

Temporal Retinal Nerve Fiber Loss in Patients with Spinocerebellar Ataxia Type 1

BACKGROUND: Autosomal dominant spinocerebellar ataxia type 1 is an adult onset progressive disorder with well characterized neurodegeneration in the cerebellum and brainstem. Beyond brain atrophy, few data exist concerning retinal and optic nerve involvement. OBJECTIVE: To evaluate retinal changes in SCA1 patients compared to age and gender matched healthy controls. METHODOLOGY/PRINCIPAL FINDINGS: Nine patients with SCA1 were prospectively recruited from the ataxia clinic and were compared to nine age and gender matched healthy controls. Both cohorts received assessment of visually evoked potentials and eye examination by optical coherence tomography to determine retinal nerve fiber layer thickness and total macular volume. While no differences were found in visually evoked potentials, SCA1 patients showed a significant reduction of mean retinal nerve fiber layer thickness (RNFLT) compared to healthy controls (84±13 µm vs. 97±8 µm, p = 0.004). Temporal areas showed the most prominent RNFLT reduction with high statistical significances (temporal-inferior: p<0.001, temporal: p<0.001, temporal-superior: p = 0.005) whereas RNFLT in nasal areas was in the range of the control group. From six SCA1 patients an additional macular scan was obtained. The comparison to the corresponding healthy control showed a slight but not significant reduction in TMV (8.22±0.68 mm(3) vs. 8.61±0.41 mm(3), p = 0.15). CONCLUSION: In SCA1 patients, we found evidence for degeneration of retinal nerve fibers. The temporal focus of the observed retinal nerve fiber layer reduction suggests an involvement of the papillo-macular bundle which resembles pathology found in toxic or mitochondrial optic nerve disease such as Leber's hereditary optic neuropathy (LHON) or dominant optic atrophy (DOA)

CAG and GGC repeat polymorphisms in the androgen receptor gene and breast cancer susceptibility in BRCA1/2 carriers and non-carriers

Variation in the penetrance estimates for BRCA1 and BRCA2 mutations carriers suggests that other genetic polymorphisms may modify the cancer risk in carriers. A previous study has suggested that BRCA1 carriers with longer lengths of the CAG repeat in the androgen receptor (AR) gene are at increased risk of breast cancer (BC). We genotyped 188 BRCA1/2 carriers (122 affected and 66 unaffected with breast cancer), 158 of them of Ashkenazi origin, 166 BC cases without BRCA1/2 mutations and 156 Ashkenazi control individuals aged over 56 for the AR CAG and GGC repeats. In carriers, risk analyses were conducted using a variant of the log-rank test, assuming two sets of risk estimates in carriers: penetrance estimates based on the Breast Cancer Linkage Consortium (BCLC) studies of multiple case families, and lower estimates as suggested by population-based studies. We found no association of the CAG and GGC repeats with BC risk in either BRCA1/2 carriers or in the general population. Assuming BRCA1/2 penetrance estimates appropriate to the Ashkenazi population, the estimated RR per repeat adjusted for ethnic group (Ashkenazi and non-Ashkenazi) was 1.05 (95%CI 0.97–1.17) for BC and 1.00 (95%CI 0.83–1.20) for ovarian cancer (OC) for CAG repeats and 0.96 (95%CI 0.80–1.15) and 0.90 (95%CI 0.60–1.22) respectively for GGC repeats. The corresponding RR estimates for the unselected case–control series were 1.00 (95%CI 0.91–1.10) for the CAG and 1.05 (95%CI 0.90–1.22) for the GGC repeats. The estimated relative risk of BC in carriers associated with ≥28 CAG repeats was 1.08 (95%CI 0.45–2.61). Furthermore, no significant association was found if attention was restricted to the Ashkenazi carriers, or only to BRCA1 or BRCA2 carriers. We conclude that, in contrast to previous observations, if there is any effect of the AR repeat length on BRCA1 penetrance, it is likely to be weak. © 2001 Cancer Research Campaign http://www.bjcancer.co