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The gene loci for adrenal hypoplasia congenita (AHC) and glycerol kinase deficiency (GK) map in Xp21 distal to Duchenne muscular dystrophy (DMD), and proximal to DXS28 (C7), by analysis of patient deletions. We have constructed a yeast artificial chromosome (YAC) contig encompassing a 1.2 Mb region extending distally from DMD, and containing DXS708 (JC-1), the distal junction clone of a patient with GK and DMD. A pulsed-field gel electrophoresis map of the YAC contig identified 3 potential CpG islands. Whole YAC hybridization identified cosmids both for construction of cosmid contigs, and isolation of single copy probes. Thirteen new single copy probes and DXS28 and DXS708 were hybridized on a panel of patients; the deletion mapping indicates that the YAC contig contains both GK and at least part of AHC, and together with the physical map defines a GK critical region of 50-250 kb. In one AHC patient with a cytogenetically detectable deletion we used the new probes to characterize a complex double deletion. Non-overlapping deletions observed in other unrelated AHC patients indicate that the AHC gene is large, extending over at least 200-500 kb. This mapping provides the basis for the identification of the AHC and GK genes

Kelly J Perkins,1,2 Kay E Davies21Sir William Dunn School of Pathology, 2MRC Functional Genomics Unit, University of Oxford, Oxford, UKAbstract: Duchenne muscular dystrophy (DMD), an allelic X-linked progressive muscle-wasting disease, is one of the most common single-gene disorders in the developed world. Despite knowledge of the underlying genetic causation and resultant pathophysiology from lack of dystrophin protein at the muscle sarcolemma, clinical intervention is currently restricted to symptom management. In recent years, however, unprecedented advances in strategies devised to correct the primary defect through gene- and cell-based therapeutics hold particular promise for treating dystrophic muscle. Conventional gene replacement and endogenous modification strategies have greatly benefited from continued improvements in encapsidation capacity, transduction efficiency, and systemic delivery. In particular, RNA-based modifying approaches such as exon skipping enable expression of a shorter but functional dystrophin protein and rapid progress toward clinical application. Emerging combined gene- and cell-therapy strategies also illustrate particular promise in enabling ex vivo genetic correction and autologous transplantation to circumvent a number of immune challenges. These approaches are complemented by a vast array of pharmacological approaches, in particular the successful identification of molecules that enable functional replacement or ameliorate secondary DMD pathology. Animal models have been instrumental in providing proof of principle for many of these strategies, leading to several recent trials that have investigated their efficacy in DMD patients. Although none has reached the point of clinical use, rapid improvements in experimental technology and design draw this goal ever closer. Here, we review therapeutic approaches to DMD, with particular emphasis on recent progress in strategic development, preclinical evaluation and establishment of clinic

Forkhead-box protein P2 is a transcription factor that has been associated with intriguing aspects of cognitive function in humans, non-human mammals, and song-learning birds. Heterozygous mutations of the human FOXP2 gene cause a monogenic speech and language disorder. Reduced functional dosage of the mouse version (Foxp2) causes deficient cortico-striatal synaptic plasticity and impairs motor-skill learning. Moreover, the songbird orthologue appears critically important for vocal learning. Across diverse vertebrate species, this well-conserved transcription factor is highly expressed in the developing and adult central nervous system. Very little is known about the mechanisms regulated by Foxp2 during brain development. We used an integrated functional genomics strategy to robustly define Foxp2-dependent pathways, both direct and indirect targets, in the embryonic brain. Specifically, we performed genome-wide in vivo ChIP-chip screens for Foxp2-binding and thereby identified a set of 264 high-confidence neural targets under strict, empirically derived significance thresholds. The findings, coupled to expression profiling and in situ hybridization of brain tissue from wild-type and mutant mouse embryos, strongly highlighted gene networks linked to neurite development. We followed up our genomics data with functional experiments, showing that Foxp2 impacts on neurite outgrowth in primary neurons and in neuronal cell models. Our data indicate that Foxp2 modulates neuronal network formation, by directly and indirectly regulating mRNAs involved in the development and plasticity of neuronal connections.

The hereditary cerebellar ataxias are a diverse group of neurodegenerative disorders primarily characterised by loss of balance and coordination due to dysfunction of the cerebellum and its associated pathways. Although many genetic mutations causing inherited cerebellar ataxia have been identified, a significant percentage of patients remain whose cause is unknown. The transient receptor potential (TRP) family member TRPC3 is a non-selective cation channel linked to key signalling pathways that are affected in cerebellar ataxia. Furthermore, genetic mouse models of TRPC3 dysfunction display cerebellar ataxia, making the TRPC3 gene an excellent candidate for screening ataxic patients with unknown genetic aetiology. Here, we report a genetic screen for TRPC3 mutations in a cohort of 98 patients with genetically undefined late-onset cerebellar ataxia and further ten patients with undefined episodic ataxia. We identified a number of variants but no causative mutations in TRPC3. Our findings suggest that mutations in TRPC3 do not significantly contribute to the cause of late-onset and episodic human cerebellar ataxias.

The subplate lays the foundation of the developing cerebral cortex, and abnormalities have been suggested to contribute to various brain developmental disorders. The causal relationship between cellular pathologies and cognitive disorders remains unclear, and therefore, a better understanding of the role of subplate cells in cortical development is essential. Only by determining the molecular taxonomy of this diverse class of neurons can we identify the subpopulations that may contribute differentially to cortical development. We identified novel markers for murine subplate cells by comparing gene expression of subplate and layer 6 of primary visual and somatosensory cortical areas of postnatal day (P)8 old mice using a microarray-based approach. We examined the utility of these markers in well-characterized mutants (reeler, scrambler, and p35-KO) where the subplate is displaced in relation to the cortical plate. In situ hybridization or immunohistochemistry confirmed subplate-selective expression of complexin 3, connective tissue growth factor, nuclear receptor-related 1/Nr4a2, and monooxygenase Dbh-like 1 while transmembrane protein 163 also had additional expression in layer 5, and DOPA decarboxylase was also present in the white matter. Localization of marker-positive cells in the reeler and p35-KO cortices suggests different subpopulations of subplate cells. These new markers open up possibilities for further identification of subplate subpopulations in research and in neuropathological diagnosis.

Mouse models for genetic diseases are among the most powerful tools available for developing and testing new treatment strategies. ADAM12 is a disintegrin and metalloprotease, previously demonstrated to significantly alleviate the pathology of mdx mice, a model for Duchenne muscular dystrophy in humans. More specifically ADAM12 appeared to prevent muscle cell necrosis in the mdx mice as evidenced by morphological analysis and by the reduced levels of serum creatine kinase. In the present study we demonstrated that ADAM12 may compensate for the dystrophin deficiency in mdx mice by increasing the expression and redistribution of several components of the muscle cell-adhesion complexes. First, we analyzed transgenic mice that overexpress ADAM12 and found mild myopathic changes and accelerated regeneration following acute injury. We then analyzed changes in gene-expression profiles in mdx/ADAM12 transgenic mice compared with their littermate controls and found only a few genes with an expression change greater than 2-fold between mdx/ADAM12 and mdx. The small changes in gene expression were unexpected, considering the marked improvement of the mdx pathology when ADAM12 is overexpressed, and suggested that significant changes in mdx/ADAM12 muscle might occur post-transcriptionally. Indeed, by immunostaining and immunoblotting we found an approximately 2-fold increase in expression, and distinct extrasynaptic localization, of alpha 7B integrin and utrophin, the functional homolog of dystrophin. The expression of the dystrophin-associated glycoproteins was also increased. In conclusion, these results demonstrate a novel way to alleviate dystrophin deficiency in mice, and may stimulate the development of new approaches to compensate for dystrophin deficiency in animals and humans.

The gene loci for adrenal hypoplasia congenita (AHC) and glycerol kinase deficiency (GK) map in Xp21 distal to Duchenne muscular dystrophy (DMD), and proximal to DXS28 (C7), by analysis of patient deletions. We have constructed a yeast artificial chromosome (YAC) contig encompassing a 1.2 Mb region extending distally from DMD, and containing DXS708 (JC-1), the distal junction clone of a patient with GK and DMD. A pulsed-field gel electrophoresis map of the YAC contig identified 3 potential CpG islands. Whole YAC hybridization identified cosmids both for construction of cosmid contigs, and isolation of single copy probes. Thirteen new single copy probes and DXS28 and DXS708 were hybridized on a panel of patients; the deletion mapping indicates that the YAC contig contains both GK and at least part of AHC, and together with the physical map defines a GK critical region of 50-250 kb. In one AHC patient with a cytogenetically detectable deletion we used the new probes to characterize a complex double deletion. Non-overlapping deletions observed in other unrelated AHC patients indicate that the AHC gene is large, extending over at least 200-500 kb. This mapping provides the basis for the identification of the AHC and GK genes

We have studied a cohort of nemaline myopathy (NM) patients with mutations in the muscle α-skeletal actin gene (ACTA1). Immunoblot analysis of patient muscle demonstrates increased γ-filamin, myotilin, desmin and α-actinin in many NM patients, consistent with accumulation of Z line-derived nemaline bodies. We demonstrate that nebulin can appear abnormal secondary to a primary defect in actin, and show by isoelectric focusing that mutant actin isoforms are present within insoluble actin filaments isolated from muscle from two ACTA1 NM patients. Transfection of C2C12 myoblasts with mutant actinEGFP constructs resulted in abnormal cytoplasmic and intranuclear actin aggregates. Intranuclear aggregates were observed with V163L-, V163M- and R183G-actinEGFP constructs, and modeling shows these residues to be adjacent to the nuclear export signal of actin. V163L and V163M actin mutants are known to cause intranuclear rod myopathy, however, intranuclear bodies were not reported in patient R183G. Transfection studies in C2C12 myoblasts showed significant alterations in the ability of V136L and R183G actin mutants to polymerize and contribute to insoluble actin filaments. Thus, we provide direct evidence for a dominant-negative effect of mutant actin in NM. In vitro studies suggest that abnormal folding, altered polymerization and aggregation of mutant actin isoforms are common properties of NM ACTA1 mutants. Some of these effects are mutation-specific, and likely result in variations in the severity of muscle weakness seen in individual patients. A combination of these effects contributes to the common pathological hallmarks of NM, namely intranuclear and cytoplasmic rod formation, accumulation of thin filaments and myofibrillar disorganization

The gene loci for adrenal hypoplasia congenita (AHC) and glycerol kinase deficiency (GK) map in Xp21 distal to Duchenne muscular dystrophy (DMD), and proximal to DXS28 (C7), by analysis of patient deletions. We have constructed a yeast artificial chromosome (YAC) contig encompassing a 1.2 Mb region extending distally from DMD, and containing DXS708 (JC-1), the distal junction clone of a patient with GK and DMD. A pulsed-field gel electrophoresis map of the YAC contig identified 3 potential CpG islands. Whole YAC hybridization identified cosmids both for construction of cosmid contigs, and isolation of single copy probes. Thirteen new single copy probes and DXS28 and DXS708 were hybridized on a panel of patients; the deletion mapping indicates that the YAC contig contains both GK and at least part of AHC, and together with the physical map defines a GK critical region of 50-250 kb. In one AHC patient with a cytogenetically detectable deletion we used the new probes to characterize a complex double deletion. Non-overlapping deletions observed in other unrelated AHC patients indicate that the AHC gene is large, extending over at least 200-500 kb. This mapping provides the basis for the identification of the AHC and GK genes