HIBCH mutations can cause Leigh-like disease with combined deficiency of multiple mitochondrial respiratory chain enzymes and pyruvate dehydrogenase

Background: Deficiency of 3-hydroxy-isobutyryl-CoA hydrolase (HIBCH) caused by HIBCH mutations is a rare cerebral organic aciduria caused by disturbance of valine catabolism. Multiple mitochondrial respiratory chain (RC) enzyme deficiencies can arise from a number of mechanisms, including defective maintenance or expression of mitochondrial DNA. Impaired biosynthesis of iron-sulphur clusters and lipoic acid can lead to pyruvate dehydrogenase complex (PDHc) deficiency in addition to multiple RC deficiencies, known as the multiple mitochondrial dysfunctions syndrome. Methods: Two brothers born to distantly related Pakistani parents presenting in early infancy with a progressive neurodegenerative disorder, associated with basal ganglia changes on brain magnetic resonance imaging, were investigated for suspected Leigh-like mitochondrial disease. The index case had deficiencies of multiple RC enzymes and PDHc in skeletal muscle and fibroblasts respectively, but these were normal in his younger brother. The observation of persistently elevated hydroxy-C4-carnitine levels in the younger brother led to suspicion of HIBCH deficiency, which was investigated by biochemical assay in cultured skin fibroblasts and molecular genetic analysis. Results: Specific spectrophotometric enzyme assay revealed HIBCH activity to be below detectable limits in cultured skin fibroblasts from both brothers. Direct Sanger sequence analysis demonstrated a novel homozygous pathogenic missense mutation c.950G <A; p.Gly317Glu in the HIBCH gene, which segregated with infantile-onset neurodegeneration within the family. Conclusions: HIBCH deficiency, a disorder of valine catabolism, is a novel cause of the multiple mitochondrial dysfunctions syndrome, and should be considered in the differential diagnosis of patients presenting with multiple RC deficiencies and/or pyruvate dehydrogenase deficiency

Differential phenotypic expression of a novel PDHA1 mutation in a female monozygotic twin pair

Pyruvate dehydrogenase complex (PDC) deficiency caused by mutations in the X-linked PDHA1 gene has a broad clinical presentation, and the pattern of X-chromosome inactivation has been proposed as a major factor contributing to its variable expressivity in heterozygous females. Here, we report the first set of monozygotic twin females with PDC deficiency, caused by a novel, de novo heterozygous missense mutation in exon 11 of PDHA1 (NM_000284.3: c.1100A>T). Both twins presented in infancy with a similar clinical phenotype including developmental delay, episodes of hypotonia or encephalopathy, epilepsy, and slowly progressive motor impairment due to pyramidal, extrapyramidal, and cerebellar involvement. However, they exhibited clear differences in disease severity that correlated well with residual PDC activities (approximately 60% and 20% of mean control values, respectively) and levels of immunoreactive E1α subunit in cultured skin fibroblasts. To address whether the observed clinical and biochemical differences could be explained by the pattern of X-chromosome inactivation, we undertook an androgen receptor assay in peripheral blood. In the less severely affected twin, a significant bias in the relative activity of the two X chromosomes with a ratio of approximately 75:25 was detected, while the ratio was close to 50:50 in the other twin. Although it may be difficult to extrapolate these results to other tissues, our observation provides further support to the hypothesis that the pattern of X-chromosome inactivation may influence the phenotypic expression of the same mutation in heterozygous females and broadens the clinical and genetic spectrum of PDC deficiency

Differential phenotypic expression of a novel PDHA1 mutation in a female monozygotic twin pair.

Pyruvate dehydrogenase complex (PDC) deficiency caused by mutations in the X-linked PDHA1 gene has a broad clinical presentation, and the pattern of X-chromosome inactivation has been proposed as a major factor contributing to its variable expressivity in heterozygous females. Here, we report the first set of monozygotic twin females with PDC deficiency, caused by a novel, de novo heterozygous missense mutation in exon 11 of PDHA1 (NM_000284.3: c.1100A>T). Both twins presented in infancy with a similar clinical phenotype including developmental delay, episodes of hypotonia or encephalopathy, epilepsy, and slowly progressive motor impairment due to pyramidal, extrapyramidal, and cerebellar involvement. However, they exhibited clear differences in disease severity that correlated well with residual PDC activities (approximately 60% and 20% of mean control values, respectively) and levels of immunoreactive E1α subunit in cultured skin fibroblasts. To address whether the observed clinical and biochemical differences could be explained by the pattern of X-chromosome inactivation, we undertook an androgen receptor assay in peripheral blood. In the less severely affected twin, a significant bias in the relative activity of the two X chromosomes with a ratio of approximately 75:25 was detected, while the ratio was close to 50:50 in the other twin. Although it may be difficult to extrapolate these results to other tissues, our observation provides further support to the hypothesis that the pattern of X-chromosome inactivation may influence the phenotypic expression of the same mutation in heterozygous females and broadens the clinical and genetic spectrum of PDC deficiency

Chemical and biomechanical characterization of hyperhomocysteinemic bone disease in an animal model

BACKGROUND: Classical homocystinuria is an autosomal recessive disorder caused by cystathionine β-synthase (CBS) deficiency and characterized by distinctive alterations of bone growth and skeletal development. Skeletal changes include a reduction in bone density, making it a potentially attractive model for the study of idiopathic osteoporosis. METHODS: To investigate this aspect of hyperhomocysteinemia, we supplemented developing chicks (n = 8) with 0.6% dl-homocysteine (hCySH) for the first 8 weeks of life in comparison to controls (n = 10), and studied biochemical, biomechanical and morphologic effects of this nutritional intervention. RESULTS: hCySH-fed animals grew faster and had longer tibiae at the end of the study. Plasma levels of hCySH, methionine, cystathionine, and inorganic sulfate were higher, but calcium, phosphate, and other indices of osteoblast metabolism were not different. Radiographs of the lower limbs showed generalized osteopenia and accelerated epiphyseal ossification with distinct metaphyseal and suprametaphyseal lucencies similar to those found in human homocystinurics. Although biomechanical testing of the tibiae, including maximal load to failure and bone stiffness, indicated stronger bone, strength was proportional to the increased length and cortical thickness in the hCySH-supplemented group. Bone ash weights and IR-spectroscopy of cortical bone showed no difference in mineral content, but there were higher Ca(2+)/PO(4)(3- )and lower Ca(2+)/CO(3)(2- )molar ratios than in controls. Mineral crystallization was unchanged. CONCLUSION: In this chick model, hyperhomocysteinemia causes greater radial and longitudinal bone growth, despite normal indices of bone formation. Although there is also evidence for an abnormal matrix and altered bone composition, our finding of normal biomechanical bone strength, once corrected for altered morphometry, suggests that any increase in the risk of long bone fracture in human hyperhomocysteinemic disease is small. We also conclude that the hCySH-supplemented chick is a promising model for study of the connective tissue abnormalities associated with homocystinuria and an important alternative model to the CBS knock-out mouse

Epigenetic Analysis of KSHV Latent and Lytic Genomes

Epigenetic modifications of the herpesviral genome play a key role in the transcriptional control of latent and lytic genes during a productive viral lifecycle. In this study, we describe for the first time a comprehensive genome-wide ChIP-on-Chip analysis of the chromatin associated with the Kaposi's sarcoma-associated herpesvirus (KSHV) genome during latency and lytic reactivation. Depending on the gene expression class, different combinations of activating [acetylated H3 (AcH3) and H3K4me3] and repressive [H3K9me3 and H3K27me3] histone modifications are associated with the viral latent genome, which changes upon reactivation in a manner that is correlated with their expression. Specifically, both the activating marks co-localize on the KSHV latent genome, as do the repressive marks. However, the activating and repressive histone modifications are mutually exclusive of each other on the bulk of the latent KSHV genome. The genomic region encoding the IE genes ORF50 and ORF48 possesses the features of a bivalent chromatin structure characterized by the concomitant presence of the activating H3K4me3 and the repressive H3K27me3 marks during latency, which rapidly changes upon reactivation with increasing AcH3 and H3K4me3 marks and decreasing H3K27me3. Furthermore, EZH2, the H3K27me3 histone methyltransferase of the Polycomb group proteins (PcG), colocalizes with the H3K27me3 mark on the entire KSHV genome during latency, whereas RTA-mediated reactivation induces EZH2 dissociation from the genomic regions encoding IE and E genes concurrent with decreasing H3K27me3 level and increasing IE/E lytic gene expression. Moreover, either the inhibition of EZH2 expression by a small molecule inhibitor DZNep and RNAi knockdown, or the expression of H3K27me3-specific histone demethylases apparently induced the KSHV lytic gene expression cascade. These data indicate that histone modifications associated with the KSHV latent genome are involved in the regulation of latency and ultimately in the control of the temporal and sequential expression of the lytic gene cascade. In addition, the PcG proteins play a critical role in the control of KSHV latency by maintaining a reversible heterochromatin on the KSHV lytic genes. Thus, the regulation of the spatial and temporal association of the PcG proteins with the KSHV genome may be crucial for propagating the KSHV lifecycle

Dentifrices, mouthwashes, and remineralization/caries arrestment strategies

While our knowledge of the dental caries process and its prevention has greatly advanced over the past fifty years, it is fair to state that the management of this disease at the level of the individual patient remains largely empirical. Recommendations for fluoride use by patients at different levels of caries risk are mainly based on the adage that more is better. There is a general understanding that the fluoride compound, concentration, frequency of use, duration of exposure, and method of delivery can influence fluoride efficacy. Two important factors are (1) the initial interaction of relatively high concentrations of fluoride with the tooth surface and plaque during application and (2) the retention of fluoride in oral fluids after application

Serological profile of foot-and-mouth disease in wildlife populations of West and Central Africa with special reference to Syncerus caffer subspecies

The role which West and Central African wildlife populations might play in the transmission dynamics of FMD is not known nor have studies been performed in order to assess the distribution and prevalence of FMD in wild animal species inhabiting those specific regions of Africa. This study reports the FMD serological profile extracted from samples (n = 696) collected from wildlife of West and Central Africa between 1999 and 2003. An overall prevalence of FMDV NSP reactive sera of 31.0% (216/696) was estimated, where a significant difference in seropositivity (p = 0.000) was reported for buffalo (64.8%) as opposed to other wild animal species tested (17.8%). Different levels of exposure to the FMDV resulted for each of the buffalo subspecies sampled (p = 0.031): 68.4%, 50.0% and 0% for Nile Buffalo, West African Buffalo and African Forest Buffalo, respectively. The characterisation of the FMDV serotypes tested for buffalo found presence of antibodies against all the six FMDV serotypes tested, although high estimates for type O and SAT 3 were reported for Central Africa. Different patterns of reaction to the six FMDV serotypes tested were recorded, from sera only positive for a single serotype to multiple reactivities. The results confirmed that FMDV circulates in wild ruminants populating both West and Central Africa rangelands and in particular in buffalo, also suggesting that multiple FMDV serotypes might be involved with type O, SAT 2 and SAT 1 being dominant. Differences in serotype and spill-over risk between wildlife and livestock likely reflect regional geography, historical circulation and differing trade and livestock systems

Microdissection of Shoot Meristem Functional Domains

The shoot apical meristem (SAM) maintains a pool of indeterminate cells within the SAM proper, while lateral organs are initiated from the SAM periphery. Laser microdissection–microarray technology was used to compare transcriptional profiles within these SAM domains to identify novel maize genes that function during leaf development. Nine hundred and sixty-two differentially expressed maize genes were detected; control genes known to be upregulated in the initiating leaf (P0/P1) or in the SAM proper verified the precision of the microdissections. Genes involved in cell division/growth, cell wall biosynthesis, chromatin remodeling, RNA binding, and translation are especially upregulated in initiating leaves, whereas genes functioning during protein fate and DNA repair are more abundant in the SAM proper. In situ hybridization analyses confirmed the expression patterns of six previously uncharacterized maize genes upregulated in the P0/P1. P0/P1-upregulated genes that were also shown to be downregulated in leaf-arrested shoots treated with an auxin transport inhibitor are especially implicated to function during early events in maize leaf initiation. Reverse genetic analyses of asceapen1 (asc1), a maize D4-cyclin gene upregulated in the P0/P1, revealed novel leaf phenotypes, less genetic redundancy, and expanded D4-CYCLIN function during maize shoot development as compared to Arabidopsis. These analyses generated a unique SAM domain-specific database that provides new insight into SAM function and a useful platform for reverse genetic analyses of shoot development in maize

Heterochromatin Protein 1β (HP1β) has distinct functions and distinct nuclear distribution in pluripotent versus differentiated cells

Background: Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into every cell type and to self-renew. These characteristics correlate with a distinct nuclear architecture, epigenetic signatures enriched for active chromatin marks and hyperdynamic binding of structural chromatin proteins. Recently, several chromatin-related proteins have been shown to regulate ESC pluripotency and/or differentiation, yet the role of the major heterochromatin proteins in pluripotency is unknown. Results: Here we identify Heterochromatin Protein 1β (HP1β) as an essential protein for proper differentiation, and, unexpectedly, for the maintenance of pluripotency in ESCs. In pluripotent and differentiated cells HP1β is differentially localized and differentially associated with chromatin. Deletion of HP1β, but not HP1aα, in ESCs provokes a loss of the morphological and proliferative characteristics of embryonic pluripotent cells, reduces expression of pluripotency factors and causes aberrant differentiation. However, in differentiated cells, loss of HP1β has the opposite effect, perturbing maintenance of the differentiation state and facilitating reprogramming to an induced pluripotent state. Microscopy, biochemical fractionation and chromatin immunoprecipitation reveal a diffuse nucleoplasmic distribution, weak association with chromatin and high expression levels for HP1β in ESCs. The minor fraction of HP1β that is chromatin-bound in ESCs is enriched within exons, unlike the situation in differentiated cells, where it binds heterochromatic satellite repeats and chromocenters. Conclusions: We demonstrate an unexpected duality in the role of HP1β: it is essential in ESCs for maintaining pluripotency, while it is required for proper differentiation in differentiated cells. Thus, HP1β function both depends on, and regulates, the pluripotent state