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Functional defects of the mitochondrial translation machinery, as a result of mutations in nuclear-encoded genes, have been associated with combined oxidative phosphorylation (OXPHOS) deficiencies. We report siblings with congenital sensorineural deafness and lactic acidemia in association with combined respiratory chain (RC) deficiencies of complexes I, III and IV observed in fibroblasts and liver. One of the siblings had a more severe phenotype showing progressive hepatic and renal failure. Whole-exome sequencing revealed a homozygous mutation in the gene encoding mitochondrial ribosomal protein S7 (MRPS7), a c.550A>G transition that encodes a substitution of valine for a highly conserved methionine (p.Met184Val) in both affected siblings. MRPS7 is a 12S ribosomal RNA-binding subunit of the small mitochondrial ribosomal subunit, and is required for the assembly of the small ribosomal subunit. Pulse labeling of mitochondrial protein synthesis products revealed impaired mitochondrial protein synthesis in patient fibroblasts. Exogenous expression of wild-type MRPS7 in patient fibroblasts rescued complexes I and IV activities, demonstrating the deleterious effect of the mutation on RC function. Moreover, reduced 12S rRNA transcript levels observed in the patient's fibroblasts were also restored to normal levels by exogenous expression of wild-type MRPS7. Our data demonstrate the pathogenicity of the identified MRPS7 mutation as a novel cause of mitochondrial RC dysfunction, congenital sensorineural deafness and progressive hepatic and renal failure.

Inborn errors of metabolism (IEM) constitute inherited enzyme or transport protein defects, potentially leading to accumulation of toxic substrates or deficiency of essential products of any given process. There are often further consequences of the primary perturbation, leading to compensatory physiology or further interference in cellular processes. These biochemical effects can, in some instances, lead to fetal developmental abnormalities and in others to dramatic postnatal compromise with significant mortality and morbidity. This chapter discusses the following presentations: neonatal acute metabolic encephalopathy, neonatal epileptic epilepsy, liver disease, cardiomyopathy, nonimmune hydrops fetalis, and dysmorphic IEM (including some lysosomal and peroxisomal disorders and congenital disorders of glycosylation). Systematic investigation of these clinical presentations can often lead to a definitive diagnosis. Most of these disorders are rare, but recent medical advances have made many treatable. It is therefore imperative that these disorders are considered early in differential diagnoses, investigated rigorously and expeditiously, and managed appropriately. Newborn bloodspot screening protocols can also identify many of these conditions in the presymptomatic stage, and hence, knowledge of local screening strategies is imperative for those investigating neonates. Screening tests require definitive testing algorithms in order to correctly identify an affected infant.

Success rates for genomic analyses of highly heterogeneous disorders can be greatly improved if a large cohort of patient data is assembled to enhance collective capabilities for accurate sequence variant annotation, analysis, and interpretation. Indeed, molecular diagnostics requires the establishment of robust data resources to enable data sharing that informs accurate understanding of genes, variants, and phenotypes. The "Mitochondrial Disease Sequence Data Resource (MSeqDR) Consortium" is a grass-roots effort facilitated by the United Mitochondria] Disease Foundation to identify and prioritize specific genomic data analysis needs of the global mitochondrial disease clinical and research community. A central Web portal (https://mseqdr.org) facilitates the coherent compilation, organization, annotation, and analysis of sequence data from both nuclear and mitochondrial genomes of individuals and families with suspected mitochondria! disease. This Web portal provides users with a flexible and expandable suite of resources to enable variant-, gene-, and exome-level sequence analysis in a secure, Web-based, and user-friendly fashion. Users can also elect to share data with other MSeqDR Consortium members, or even the general public, either by custom annotation tracks or through the use of a convenient distributed annotation system (DAS) mechanism. A range of data visualization and analysis tools are provided to facilitate user interrogation and understanding of genomic, and ultimately phenotypic, data of relevance to mitochondrial biology and disease. Currently available tools for nuclear and mitochondrial gene analyses include an MSeqDR GBrowse instance that hosts optimized mitochondrial disease and mitochondrial DNA (mtDNA) specific annotation tracks, as well as an MSeqDR locus-specific database (LSDB) that curates variant data on more than 1300 genes that have been implicated in mitochondrial disease and/or encode mitochondria-localized proteins. MSeqDR is integrated with a diverse array of mtDNA data analysis tools that are both freestanding and incorporated into an online exome-level dataset curation and analysis resource (GEM.app) that is being optimized to support needs of the MSeqDR community. In addition, MSeqDR supports mitochondrial disease phenotyping and ontology tools, and provides variant pathogenicity assessment features that enable community review, feedback, and integration with the public ClinVar variant annotation resource. A centralized Web-based informed consent process is being developed, with implementation of a Global Unique Identifier (GUID) system to integrate data deposited on a given individual from different sources. Community-based data deposition into MSeqDR has already begun. Future efforts will enhance capabilities to incorporate phenotypic data that enhance genomic data analyses. MSeqDR will fill the existing void in bioinformatics tools and centralized knowledge that are necessary to enable efficient nuclear and mtDNA genomic data interpretation by a range of shareholders across both clinical diagnostic and research settings. Ultimately, MSeqDR is focused on empowering the global mitochondrial disease community to better define and explore mitochondrial diseases. (C) 2014 Elsevier Inc. All rights reserved.

Background: The basis of the pathogenicity of the H50Q variant α-synuclein is unknown. Results: The critical concentration of α-synuclein is decreased by 10-fold by the H50Q mutation, and its aggregation is modulated by the wild-type isoform. Conclusion: Key effects of the H50Q mutation on the aggregation of α-synuclein can be quantified. Significance: Our data provide insights into the mechanism of Lewy body formation in vivo., The conversion of α-synuclein from its intrinsically disordered monomeric state into the fibrillar cross-β aggregates characteristically present in Lewy bodies is largely unknown. The investigation of α-synuclein variants causative of familial forms of Parkinson disease can provide unique insights into the conditions that promote or inhibit aggregate formation. It has been shown recently that a newly identified pathogenic mutation of α-synuclein, H50Q, aggregates faster than the wild-type. We investigate here its aggregation propensity by using a sequence-based prediction algorithm, NMR chemical shift analysis of secondary structure populations in the monomeric state, and determination of thermodynamic stability of the fibrils. Our data show that the H50Q mutation induces only a small increment in polyproline II structure around the site of the mutation and a slight increase in the overall aggregation propensity. We also find, however, that the H50Q mutation strongly stabilizes α-synuclein fibrils by 5.0 ± 1.0 kJ mol−1, thus increasing the supersaturation of monomeric α-synuclein within the cell, and strongly favors its aggregation process. We further show that wild-type α-synuclein can decelerate the aggregation kinetics of the H50Q variant in a dose-dependent manner when coaggregating with it. These last findings suggest that the precise balance of α-synuclein synthesized from the wild-type and mutant alleles may influence the natural history and heterogeneous clinical phenotype of Parkinson disease.

Molecular analysis of argininosuccinate lyase (ASAL) deficiency has led to the identification of a deletion hotspot in the ASL gene. Six individuals with ASAL deficiency had alleles that led to a complete absence of exon 13 from the ASL mRNA; each had a partial deletion of exon 13 in the genomic DNA. In all six patients, the deletions begin 18 bp upstream of the 3' end of exon 13. In four cases, the deletions were 13 bp in length, and ended within exon 13, whereas in two other patients the deletions were 25 bp and extended into intron 13. The sequence at which these deletions begin overlaps both a putative topoisomerase II recognition site and a DNA polymerase alpha mutation/frameshift site. Moreover, the topoisomerase II cut site is situated precisely at the beginning of the deletions, which are flanked by small (2- and 3-bp) direct repeats. We note that a similar concurrence of these two putative enzyme sites can be found in a number of other deletion sites in the human genome, most notably the DeltaF508 deletion in the CFTR gene. These findings suggest that the joint presence of these two enzyme sites represents a DNA sequence context that may favor the occurrence of small deletions.

Scoliosis is a common clinical manifestation of Rett syndrome, a neurodevelopmental disorder that almost exclusively affects girls. Following apparently normal development, these girls typically regress and lose previously attained cognitive, social, and motor skills. Severe intellectual and physical disabilities remain throughout life. Mutations in the methyl-CpG-binding protein 2 gene, MECP2, are detected in approximately 80% of cases and are associated with phenotypic variability. Population-based data on Australian cases were used to study the association between early developmental and genetic factors and the onset of scoliosis. The median age at scoliosis onset was 9.80 years, and three quarters of subjects had developed scoliosis by 13 years of age. Children with compromised early development before 6 months, those who were less mobile at 10 months, and those who never walked were more likely to have an earlier onset of scoliosis. When seven common point mutations and large genomic and C-terminal deletions were compared, the R294X mutation appeared to provide some protective effect against the development of scoliosis. (J Child Neurol 2006;21: 809—813; DOI 10.2310/7010.2006.00183).

In this review, we give a clinical overview of Rett syndrome (RTT), and provide a framework for clinical and molecular approaches to the diagnosis of this severe neurodevelopmental disorder. We also discuss issues that need to be considered in the management of RTT patients, and raise some of the challenges associated with genetic counselling.

A translocation that disrupted the Netrin G1 gene (NTNG1) was recently reported in a patient with the early seizure variant of Rett syndrome (RTT). The netrin G1 protein (NTNG1) has an important role in the developing central nervous system, particularly in axonal guidance, signalling and NMDA receptor function and was a good candidate gene for RTT. We recruited 115 patients with RTT (females: 25 classic and 84 atypical; 6 males) but no mutation in the MECP2 gene. For those 52 patients with epileptic seizure onset in the first six months of life, CDKL5 mutations were also excluded. We aimed to determine whether mutations in NTNG1 accounted for a significant subset of patients with RTT, particularly those with the early onset seizure variant and other atypical presentations. We sequenced the nine coding exons of NTNG1 and identified four sequence variants, none of which were likely to be pathogenic. Mutations in the NTNG1 gene appear to be a rare cause of RTT but NTNG1 function demands further investigation in relation to the central nervous system pathophysiology of the disorder.

Objective To examine the prevalence, cumulative incidence, and survival in an Australian cohort with Rett syndrome (RTT). Study design The Australian Rett Syndrome Database is a longitudinal data collection that included 276 verified female cases at the end of 2004. Survival was calculated using the Kaplan-Meier product limit method, and cumulative incidence was determined using the complement of the Kaplan-Meier method. Results Most cases (88.4%) have had MECP2 mutation testing, with positive results in 73%. The prevalence of RTT was .88 per 10,000 females in 5- to 18-year-olds, and the cumulative incidence was 1.09 per 10,000 females by 12 years of age. The cumulative incidence by the age of 5 years increased from .39 per 10,000 in the 1980 to 1984 cohort to .76 per 10,000 in birth cohorts beyond 1984. Survival was 77.8% at 25 years, compared with 99.96% survival in the Australian female population. Pneumonia (10/25) was the most common cause of death. Conclusions The availability of genetic testing has contributed to the changing pattern and timing of RTT diagnosis in Australia. Girls with RTT have worse survival compared with the general female population. When more data are available, it will be possible to evaluate the relationship between survival and specific MECP2 mutations.

Rett syndrome (RTT) is a debilitating neurological condition associated with mutations in the X-linked MECP2 gene, where apparently normal development is seen prior to the onset of cognitive and motor deterioration at 6-18 months of life. A targeted deletion of the methyl-CpG-binding domain (MBD) coding region and disruption of mRNA splicing was introduced in the mouse, resulting in a complete loss of Mecp2 transcripts and protein. Postnatal comparison of XO and XY mutant Mecp2 allele-containing null mice revealed similar effects on mouse growth and viability, suggesting that phenotypic manifestations are not modulated by the Y-chromosome. Further assessment of Mecp2-null XY mice highlighted cerebellar and hippocampal/amygdala-based learning deficits in addition to reduced motor dexterity and decreased anxiety levels. Brain tissues containing the hippocampal formation of XY Mecp2-null mice also displayed significant changes in genetic activity, which are related to the severity of the mutant phenotype.

Clinical phenotypes of congenital myasthenic syndromes and primary mitochondrial disorders share significant overlap in their clinical presentations, leading to challenges in making the correct diagnosis. Next generation sequencing is transforming molecular diagnosis of inherited neuromuscular disorders by identifying novel disease genes and by identifying previously known genes in undiagnosed patients. This is evident in two patients who were initially suspected to have a mitochondrial myopathy, but in whom a clear diagnosis of congenital myasthenic syndromes was made through whole exome sequencing. In patient 1, whole exome sequencing revealed compound heterozygous mutations c.1228C > T (p.Arg410Trp) and c.679C > T (p.Arg227*) in collagen-like tail subunit (single strand of homotrimer) of asymmetric acetylcholinesterase ( COLQ ). In patient 2, in whom a deletion of exon 52 in Dystrophin gene was previously detected by multiplex ligation-dependent probe amplification, Sanger sequencing revealed an additional homozygous mutation c.1511_1513delCTT (p.Pro504Argfs*183) in docking protein7 (DOK7). These case reports highlight the need for careful diagnosis of clinically heterogeneous syndromes like congenital myasthenic syndromes, which are treatable, and for which delayed diagnosis is likely to have implications for patient health. The report also demonstrates that whole exome sequencing is an effective diagnostic tool in providing molecular diagnosis in patients with complex phenotypes.

Rett syndrome is one of many severe neurodevelopmental disorders with feeding difficulties. In this study, associations between feeding difficulties, age, MECP2 genotype, and utilization of gastrostomy were investigated. Weight change and family satisfaction following gastrostomy were explored. Data from the longitudinal Australian Rett Syndrome Database whose parents provided data in the 2011 family questionnaire (n = 229) were interrogated. We used logistic regression to model relationships between feeding difficulties, age group, and genotype. Content analysis was used to analyze data on satisfaction following gastrostomy. In those who had never had gastrostomy and who fed orally (n = 166/229), parents of girls

Background: Rett syndrome is a neurodevelopmental disorder mostly affecting females and caused by mutations in the MECP2 gene. Originally the syndrome was characterised as having a normal prenatal and perinatal period with later regression. Previous work has speculated that the girl with Rett syndrome may not be normal at birth. Aims: to examine whether early development between birth and ten months varies by genotype in Rett syndrome. Methods: cases were sourced from two databases, the Australian Rett Syndrome Database (est. 1993) and the newly formed InterRett - IRSA Rett Phenotype Database. Data available on 320 cases included information provided by parents on perinatal problems, early developmental behaviour and mobility. Problem scores, mobility scores and a total composite score for each mutation were generated and compared. Results: overall, 58% of respondents noted unusual behaviour during the first six months and 70.6% from the period between 6 and 10 months of life. Statistically significant differences were detected between some of the common mutations. Infants with R294X (P=0.05) and R133C (P=0.03) were less likely than those with R255X to have problems in the perinatal period. The most severe profile overall for early development was associated with mutations R255X and R270X. Conclusion: This is the largest study to date examining the effects of individual mutations in Rett syndrome. With the ongoing case ascertainment and expansion of InterRett, sample size will increase rapidly and provide improved statistical power for future analyses. Results from this study will contribute to understanding the mechanism of early development in Rett syndrome and determining if and at which time(s) early intervention might be feasible.

T70N human lysozyme is the only known naturally occurring destabilised lysozyme variant that has not been detected in amyloid deposits in human patients. Its study and a comparison of its properties with those of the amyloidogenic variants of lysozyme is therefore important for understanding the determinants of amyloid disease. We report here the X-ray crystal structure and the solution dynamics of T70N lysozyme, as monitored by hydrogen/deuterium exchange and NMR relaxation experiments. The X-ray crystal structure shows that a substantial structural rearrangement results from the amino acid substitution, involving residues 45-51 and 68-75 in particular, and gives rise to a concomitant separation of these two loops of up to 6.5A. A marked decrease in the magnitudes of the generalised order parameter (S2) values of the amide nitrogen atom, for residues 70-74, shows that the T70N substitution increases the flexibility of the peptide backbone around the site of mutation. Hydrogen/deuterium exchange protection factors measured by NMR spectroscopy were calculated for the T70N variant and the wild-type protein. The protection factors for many of backbone amide groups in the beta-domain of the T70N variant are decreased relative to those in the wild-type protein, whereas those in the alpha-domain display wild-type-like values. In pulse-labelled hydrogen/deuterium exchange experiments monitored by mass spectrometry, transient but locally cooperative unfolding of the beta-domain of the T70N variant and the wild-type protein was observed, but at higher temperatures than for the amyloidogenic variants I56T and D67H. These findings reveal that such partial unfolding is an intrinsic property of the human lysozyme structure, and suggest that the readiness with which it occurs is a critical feature determining whether or not amyloid deposition occurs in vivo.

Among cases in the Australian Rett Syndrome Database, the nonsense mutation p.R270X is one of the most commonly occurring single pathogenic MECP2 mutations. In two recent published reports of the MECP2 mutational spectrum the p.R270X appeared to be under represented. We hypothesised that increased mortality arising from this mutation may underlie this apparent discrepancy. We investigated our hypothesis in two independent study groups from Australia and the UK with prospective data collections (total n=524). Only females with Rett syndrome and an identified MECP2 mutation were included. Significant differences in survival were detected among Rett syndrome cases grouped for the eight most frequent mutations (log-rank chi(2) (7)=15.71, P=0.03). Moreover, survival among cases with p.R270X, when compared with survival among cases with all the other mutations was reduced (log-rank chi(2) (2)=6.94, P=0.01). Our observation of a reduced survival associated with the p.R270X mutation offers an explanation for the under representation of p.R270X in older subjects with Rett syndrome.

Skewed X chromosome inactivation (XCI) has been implicated in modulating the severity of Rett syndrome (RTT), although studies by different groups have yielded conflicting results. In this study we have characterised the XCI pattern in various neuroanatomical regions of nine RTT brains and non-neural tissue in two of these patients to determine whether or not variable XCI patterns occur in different brain regions or somatic tissues of the same patient. The mean XCI patterns for frontal and occipital cortex were compared between RTT and control subjects, and showed no significant differences when comparing RTT frontal to control frontal cortex or RTT occipital to control occipital cortex. However, one RTT subject displayed variability across the different neuroanatomical regions of the brain and skewing in some non-neural tissues. This observation adds another dimension to the epigenetic factors that may contribute to the phenotype in RTT. It also mandates that caution should be exercised in factoring XCI, including assumptions based on the blood XCI pattern, into the development of phenotype-genotype correlations.

Rett syndrome (RTT) is an X-linked disorder caused by mutations in the methyl CpG binding protein 2 (MECP2) gene. The pattern of X-chromosome inactivation (XCI) is thought to play a role in phenotypic severity. In the present study, patterns of XCI were assessed by lacZ staining of embryos and adult brains of mice heterozygous for a X-linked Hmgcr-nls-lacZ transgene on a mutant mouse model of RTT. We found that there was no difference between the lacZ staining patterns in the brain of wild-type and heterozygous mutant embryos at embryonic day 9.5 (E9.5) suggesting that Mecp2 has no effect on the primary pattern of XCI. At 20 weeks of age, there was no significant difference between XCI patterns in the Purkinje cells in the cerebellum of heterozygous mutant and wild-type mice when the mutant allele was inherited from the mother. However, when the mutant allele was paternally inherited, a significant difference was detected. Thus, parental origin of the mutation may have a bearing on phenotype through XCI patterns. An estimation of the Purkinje cell precursor number based on XCI mosaicism revealed that, when the mutation was paternally inherited, the precursor number was less than that in the wild-type mice. Therefore, it is likely that the number of precursor cells allocated to the Purkinje cell lineage is affected by a paternally inherited mutation in Mecp2. We also observed that the pattern of XCI in cultured fibroblasts was significantly correlated with patterns in the Purkinje cells in mutant animals but not in wild-type mice.

Rett syndrome (RS) is a severe neurodevelopmental disorder that contributes significantly to severe intellectual disability in females worldwide. It is caused by mutations in MECP2 in the majority of cases, but a proportion of atypical cases may result from mutations in CDKL5, particularly the early onset seizure variant. The relationship between MECP2 and CDKL5, and whether they cause RS through the same or different mechanisms is unknown, but is worthy of investigation. Mutations in MECP2 appear to give a growth disadvantage to both neuronal and lymphoblast cells, often resulting in skewing of X inactivation that may contribute to the large degree of phenotypic variation. MeCP2 was originally thought to be a global transcriptional repressor, but recent evidence suggests that it may have a role in regulating neuronal activity dependent expression of specific genes such as Hairy2a in Xenopus and Bdnf in mouse and rat.

Over 170 known mutations of the mitochondrial genome are responsible for disease. Due to the unique features of mitochondrial genetics, such patients are clinically diverse and difficult to diagnose. As pathogenic mitochondrial DNA (mtDNA) mutations are mostly heteroplasmic, denaturing high-performance liquid chromatography (DHPLC) could be used to detect these heteroplasmic species and therefore act as a rapid screening test for mtDNA mutations. The entire mitochondrial genome was amplified by PCR in 40 overlapping regions. In addition, known mtDNA mutants were constructed for each of these regions using a PCR-based site-directed mutagenesis approach. These mutants were used as positive controls and showed a detection limit of 3-10% heteroplasmy by DHPLC (depending on the specific mutation) compared to 40% for conventional sequencing. To further validate the screening test, mtDNA from 17 patients with seven different pathogenic mutations was used to compare mutation detection by DHPLC and conventional sequencing. DHPLC had a sensitivity of 88% compared to 82% for sequencing. This increased to 100% sensitivity for DHPLC when excluding the m.8993T>G mutation. DHPLC analysis is therefore a sensitive, rapid and cost-effective method to screen for mutations in the mitochondrial genome. The role of pyrosequencing in the quantitation of mutant load for known mtDNA mutations was highlighted using the m.3243A>G mutation as an illustrative example. Pyrosequencing analysis was able to discriminate samples containing as little as 5% heteroplasmy and proved to be an accurate and reproducible method for estimation of mutant load.

Rett syndrome (RTT) is a severe neurodevelopmental disorder caused, in most classic cases, by mutations in the X-linked methyl-CpG-binding protein 2 gene (MECP2). A large degree of phenotypic variation has been observed in patients with RTT, both those with and without MECP2 mutations. We describe a family consisting of a proband with a phenotype that showed considerable overlap with that of RTT, her identical twin sister with autistic disorder and mild-to-moderate intellectual disability, and a brother with profound intellectual disability and seizures. No pathogenic MECP2 mutations were found in this family, and the Xq28 region that contains the MECP2 gene was not shared by the affected siblings. Three other candidate regions were identified by microsatellite mapping, including 10.3 Mb at Xp22.31-pter between Xpter and DXS1135, 19.7 Mb at Xp22.12-p22.11 between DXS1135 and DXS1214, and 16.4 Mb at Xq21.33 between DXS1196 and DXS1191. The ARX and CDKL5 genes, both of which are located within the Xp22 region, were sequenced in the affected family members, and a deletion of nucleotide 183 of the coding sequence (c.183delT) was identified in CDKL5 in the affected family members. In a screen of 44 RTT cases, a single splice-site mutation, IVS13-1G--A, was identified in a girl with a severe phenotype overlapping RTT. In the mouse brain, Cdkl5 expression overlaps--but is not identical to--that of Mecp2, and its expression is unaffected by the loss of Mecp2. These findings confirm CDKL5 as another locus associated with epilepsy and X-linked mental retardation. These results also suggest that mutations in CDKL5 can lead to a clinical phenotype that overlaps RTT. However, it remains to be determined whether CDKL5 mutations are more prevalent in specific clinical subgroups of RTT or in other clinical presentations.

We have screened 13 patients with neurological abnormalities and 3-methylglutaconic aciduria (3MGA) for mutations in the OPA3 gene, which are known to be the cause of Costeff syndrome (optic atrophy, chorea and spasticity; type III 3MGA). We aimed to explore whether mutations in the OPA3 gene are present in patients with 3MGA but without classic Costeff syndrome. OPA3 mutations (IVS1-1G>C) were identified in 2 patients with the classic phenotype of type III 3MGA, but not in the other 11 patients with differing non-Costeff phenotypes associated with developmental delay and neurological signs and symptoms as described. We identified a previously described sequence variation in the OPA3 gene (c.231T>C) in 12/13 patients. The alteration was homozygous in 8/12 and heterozygous in 4/12. This alteration was also found in 60 of 98 normal control alleles. In a single patient, a novel sequence variation in the 5' UTR was identified, (c.-38A>G). Our studies suggest that the c.231T>C sequence variation is of no clinical significance, whereas the significance of the 5' UTR sequence variation remains open to speculation. Our study of the OPA3 gene in patients with 3MGA without Costeff syndrome suggests that mutations in OPA3 are not a common cause of 3MGA in the absence of signs of Costeff syndrome.

Rett syndrome is a severe neurodevelopmental disorder generally affecting girls. Affected individuals are apparently normal at birth but later pass through a period of regression with loss of hand and communication skills and the development of hand stereotypies and dyspraxia. Mutations in the methyl-CpG binding protein 2 (MECP2) gene, have now been found to cause Rett syndrome in up to 80% of classical cases. We report a girl with Down syndrome, one of three children with birth defects in a family of five. From the age of 18 months she developed symptomatology considered by her primary physician to be very characteristic of Rett syndrome. However, this remained a clinical diagnosis till the age of 12 years. Laboratory confirmation of the dual diagnosis, which includes a R168X mutation in the MECP2 gene in addition to trisomy 21, has now been possible. The presence of one neurological or developmental disorder does not necessarily preclude a diagnosis of Rett syndrome.

Immunoglobulin (Ig)-like proteins have been shown to fold following formation of a nucleus comprising interactions between residues that are distant in the primary sequence. What role do the loops connecting these nucleus residues play? Here, the importance of loops connecting beta-strands in different sheets of the Ig fold is investigated, by insertion of five glycine residues into the B-C loop of an Ig domain from human titin, TI I27. The folding pathway of this elongated 'pseudo wild-type' TI I27 is probed using protein engineering and Phi-value analysis. The Phi-values calculated for mutants within the pseudo wild-type protein indicate that the folding nucleus in wild-type TI I27 is conserved, supporting the hypothesis that the inter-sheet loop is not critical to the formation of a long-range folding nucleus.

Gender differences in brain morphology have previously been reported in the temporal lobe and an 'X-chromosome dosage effect' has been described in Turner syndrome (45,X). To examine this further, we investigated temporal lobe morphology, metabolism and function in nine children with non-mosaic Turner syndrome using magnetic resonance imaging, (1)H magnetic resonance spectroscopy and neuropsychological testing and compared outcomes with results from nine age-matched control girls (46,XX). Turner subjects were found to have significantly larger superior temporal lobes (P = 0.004) and middle temporal lobes (P = 0.047) than controls. The size of the temporal lobe was found to correlate negatively with temporal lobe choline-containing compounds suggesting that increased temporal lobe size is associated with larger cells and/or decreased dendrites. This suggests a developmental failure to prune neurons. The degree of enlargement correlates negatively with functional performance on temporal-lobe associated tasks, suggesting that the enlargement may be a compensatory mechanism, or possibly causative in the case of semantic fluency performance. These temporal lobe abnormalities are discussed with reference to genes which are absent in Turner syndrome and to hormonal differences between Turner syndrome subjects and 46,XX controls.

In 2001, the International Rett Syndrome Association funded the establishment of a World Wide Web—based database to collect and display the genetic data of children and adults with Rett syndrome from around the world. RettBASE () encompasses both published and unpublished data; includes pathogenic mutations, benign polymorphisms, and sequence variations of uncertain significance; and has a range of query capabilities, allowing for simple or complex interrogation of the database. To undertake genotype—phenotype correlations and to identify the likely subtle differences in phenotype, detailed phenotype data on large samples will be provided by the International Rett Syndrome Association International Phenotype database InterRett. InterRett is under development by the Australian Rett syndrome study group at the Telethon Institute for Child Health Research in Perth, Western Australia. It will collect data from clinicians and families and provide deidentified, collated data on the Internet (). Data records will be linked with RettBASE through a common unique identifier. An international reference panel is advising on the development of the database. Data collection procedures from families and clinicians are currently being piloted. Full data collection from both groups began in the second half of 2003. Concurrently, the output database will be developed to provide deidentified individual records and collated data for clinicians and researchers and collated data for families and the general public. This Web-based database will be an invaluable resource for understanding the nature of the disorder and managing children and adults with Rett syndrome. ( J Child Neurol 2003;18:709—713).

Rett syndrome (RTT) is a neurodevelopmental disorder affecting primarily females, with an incidence of around 1 in 15,000 females. In 1999, mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2) were first reported in RTT subjects, and since that time there have been a number of publications describing cohorts of patients and their mutations. In addition, MECP2 mutations have been reported in patients who do not fit the diagnostic criteria for Rett syndrome. We have developed a new locus-specific database, RettBASE (http://mecp2.chw.edu.au/), loosely based on the PAHdb website. The aim is to obtain data relating to all known instances of MECP2 variations, including published ta and data directly submitted by one of various means (either by using an online submission form, or by sending the same form in Adobe portable document format (pdf) or Microsoft Word format by email or fax to the database curators). The database has a range of query capabilities, allowing for simple or complex interrogation of the database. To address the issue of patient confidentiality, we have incorporated an Excel spreadsheet algorithm that allows the generation of a unique number based on the subject's name and date of birth. We believe this database will prove to be a useful resource, allowing the development of accurate prevalence data for disease-causing mutations, providing a catalog of polymorphisms, and potentially allowing more accurate phenotype-genotype correlations to be drawn.

Background Coronary calcium detected noninvasively is an attractive way to diagnose atherosclerosis before the development of symptoms. This study examines the prognostic value of coronary calcium in asymptomatic subjects with usual cardiovascular risk. Methods and Results In 425 asymptomatic subjects, 229 men (aged 45.1 ± 14 years) and 196 women (aged 42.7 ± 13 years), coronary calcium presence was studied by digital cinefluoroscopy. The majority (76%) had no or at most one risk factor. Subjects were followed up for 58.4 ± 12.7 months for cardiac events. Coronary calcium was present in 76 of 425 (17.9%) subjects. Cardiac events were observed in 21 subjects: 2 cardiac deaths, 7 acute myocardial infarctions, 3 coronary artery bypass grafts, 3 coronary angioplasty procedures, 3 events of unstable angina, and 10 events of stable angina pectoris. Survival curve analysis showed significant differences in all the studied end points between subjects with and those without calcium. Coronary calcium was an independent predictor of all events (3.6-fold increase, P < .008), cardiac death/myocardial infarction/revascularization (13.9-fold increase, P < .02), and stable angina (7.4-fold increase, P < .007). However, calcium did not independently predict cardiac death/myocardial infarction or acute coronary syndromes. Conclusions Coronary calcium in asymptomatic subjects with usual cardiovascular risk adds significant incremental information to risk factors information for the development of symptomatic coronary artery disease. (Am Heart J 2003;145:542-8.)

We report a 7½-year-old girl with an ∼9.5 Mb duplication of proximal 17p. Her clinical features include moderately severe developmental delay, absence of speech, talipes, congenital dislocation of the hips, premature adrenarche, dysmorphic facial features, deep palmar creases, and signs and symptoms of peripheral neuropathy consistent with Charcot–Marie–Tooth disease type 1A (CMT1A). Chromosome analysis revealed a partially duplicated 17p with two centromeres on the derivative chromosome. Fluorescence in situ hybridization (FISH) analysis demonstrated the tandemly duplicated segment spans 17p10-p12, including the entire Smith–Magenis syndrome (SMS) critical region and a portion of the CMT1A critical region. One breakpoint mapped within the centromere and the second breakpoint mapped within the CMT1A critical region, distal to the PMP22 gene. Microsatellite polymorphism studies showed that the duplicated chromosome is of maternal origin. We compare the clinical features of our patient to those of individuals with partial trisomy of proximal 17p to further delineate the genotype–phenotype correlation associated with segmental duplication of this chromosomal region. © 2003 Wiley-Liss, Inc.

Inborn errors of metabolism are individually rare, but collectively are responsible for significant levels of paediatric morbidity and mortality. More than 400 biochemically diverse inborn errors of metabolism have been identified. Recent advances in the diagnosis and treatment of these disorders have substantially improved the prognosis for many of them. Paediatricians and neonatologists play a vital role in identifying which patients need to be investigated. The diagnosis of an inborn error of metabolism often needs to be established quickly in order to prevent death or permanent neurological sequelae, and this should be carried out in collaboration with a specialized unit. The present review provides a practical approach to the recognition and investigation of neonates in whom an inborn error may be present. We also provide guidelines for the stabilization and initial management of infants at high risk of a metabolic disorder.

Mitochondrial fatty acid beta-oxidation (FAO) disorders are clinically and biochemically heterogeneous diseases mainly associated with intolerance to catabolic stress. These disorders can now be detected pre-symptomatically by newborn screening, and thus the clinical phenotype in an individual patient may be unclear. Correlation of clinical severity with concentrations of acylcarnitine species was investigated in fibroblasts from FAO-deficient patients presenting with various phenotypes and asymptomatic neonates detected by newborn screening. Intact cells were incubated in medium containing deuterium-labelled hexadecanoic acid and L-carnitine for 72h, and the accumulated acylcarnitines in the culture medium analysed using electrospray tandem mass spectrometery. Fibroblasts from patients with long-chain FAO disorders presenting at an early age and with poor clinical outcomes accumulated higher concentrations of long-chain acylcarnitine species compared with those from patients with milder phenotypes. This suggests that the in vitro quantitative acylcarnitine profiling could perhaps predict the prognosis of some FAO defects. This would be particularly useful information for the asymptomatic/pre-symptomatic FAO-deficient infant detected by the expanded newborn screening program, in whom the risk of developing symptoms later in life is not known.

We describe data on a 7-year-old girl with congenital dyserythropoietic anemia (CDA), who also had familial Mediterranean fever (FMF). Repeated transfusions required since the age of 6 months to treat her CDA led to iron overload and a persistently high ferritin level. Her relapsing FMF made effective iron chelation therapy very difficult. Consequently, at the age of 4 years, she underwent allogeneic, sibling bone marrow transplantation (BMT). During conditioning for her BMT, symptoms of FMF, including splenomegaly, arthritis, and recurrent abdominal pain, began to resolve and she was gradually weaned off colchicine. Now, 2 years after the transplantation, she remains free from FMF symptomatology and is off all immunosuppressants. This case demonstrates that symptoms of FMF can be alleviated by the therapy used during allogeneic BMT. In this patient it is likely that the missing factor in FMF is now being provided by granulocytes derived from the stem cells within transplanted bone marrow.

We have developed a flow cytometric assay to measure the oxidative capacity of cultured lymphoblasts as a possible screening test for patients suspected of having a defect of the mitochondrial respiratory chain. Cells were incubated overnight in serum free media, followed by incubation with dihydroethidium with and without rotenone, and then analysed using flow cytometry to measure fluorescence. Inhibition with rotenone gave an increase in fluorescence compared to uninhibited cells. The change in fluorescence was significantly lower in four of the six patient cell lines, with a correlation between the activity of complex I and change in fluorescence. This method may be applicable to cell lines with defects in other complexes of the respiratory chain.

Mitochondrial fatty acid [beta ]-oxidation (FAO) is coupled to the respiratory chain (RC). Functional defects of one pathway may lead to secondary alteration in flux through the other. We investigated the acylcarnitine profiles in cultured fibroblasts obtained from 14 healthy subjects, 31 patients with 8 different primary enzyme deficiencies of FAO, and 16 patients with primary RC defects including both isolated and multiple enzyme complex defects. Intact cells were incubated in media containing deuterium-labeled hexadecanoic acid and L-carnitine, and the acylcarnitines analysed using an electrospray tandem mass spectrometer. All FAO-deficient cell lines revealed disease-specific acylcarnitine profiles related to the sites of defects. Some cell lines from patients with RC defects showed profiles similar to those of controls, whereas others had abnormal profiles mimicking those found in FAO disorders. The acylcarnitine profiles of patients with RC enzyme defects were not predictable, and in some patients defects caused by mutations in either nuclear-encoded or mitochondrial DNA were associated with acylcarnitine abnormalities. While in vitro acylcarnitine profiling is useful for the diagnosis of FAO deficiencies, abnormal profiles do not exclusively indicate these disorders, and primary defects of the RC remain a possibility. Awareness of this diagnostic pitfall will aid in the selection of subsequent confirmatory tests and therapeutic options.

Objective To describe the experiences of Australian paediatricians while caring for children with rare diseases, and their educational and resource needs. Design A brief online survey was developed and deployed to a representative sample of 679 paediatricians from the Australian Paediatric Surveillance Unit database. Results Of the 679 paediatricians, 242 (36%) completed the survey. The respondents were representative of all states and territories of Australia, urban and rural regions, and hospital and private practice. Almost all respondents (93%) had seen children with one or more of >350 different rare diseases during their career; 74% had seen a new patient with rare disease in the last 6 months. The most common problems encountered while caring for patients were: diagnostic delays (65%), lack of available treatments (40%), clinical guidelines (36%) and uncertainty where to refer for peer support (35%). Few paediatricians said that rare diseases were adequately covered during university (40%) or the Fellowship of the Royal Australasian College of Physicians (50%) training, and 28% felt unprepared to care for patients with rare diseases. Paediatricians wanted lists of specialist referral services (82%) and online educational modules about rare diseases (78%) that could be accessed via one online portal that consolidated multiple resources. Smartphone applications on rare diseases were favoured by paediatricians aged

Successful protein folding is central to all biological cellular processes with a large portion of the proteome able to begin to acquire its three-dimensional structure in a co-translational manner during its biosynthesis on the ribosome. The vectorial emergence of the nascent polypeptide from the exit tunnel and its attachment to its parent ribosome results in differences between the details of the folding process of isolated polypeptides and that on the ribosome.We have developed a strategy to enable the study of co-translational folding using solution-state NMR spectroscopy, the only technique able to characterize this dynamic process at atomic resolution. Using isotopically-labelled ribosome-nascent chain complexes (RNCs), we have determined a high-resolution, structural description of protein folding on the ribosome via snapshots that mimic the emergence of an immunoglobulin-like domain within a multidomain protein. Our NMR results reveal the structure and dynamic features of how conformational space is sampled by a fledgling nascent polypeptide as it converts into its folded state and demonstrate that the entire immunoglobulin domain has to be emerged from the tunnel before native folding is possible. These findings contrast with analogous studies of C-terminal truncations of this domain, indicating significant differences between folding on the ribosome and that in bulk solution. The ribosome itself is shown to influence the process of folding; we use a combination of protein engineering and NMR dynamics to describe residue-specific sites of nascent-chain-ribosome interactions of different magnitude of strength, and chemical shift-restrained MD simulations of RNCs to underpin our spectral observations. The studies presented are providing the first high-resolution insights of these fundamental processes and begin to shape our understanding of the energy landscape sampled by a nascent chain on the cusp of initiation of folding on the ribosome.

Primary flavin mono-oxygenase 3 deficiency, an inborn error of choline metabolism, leads to an accumulation of trimethylamine, which because of its associated pungent odour of rotting fish, is a socially crippling disorder. Although it often has its onset in early childhood, it may take years or even decades before the diagnosis is established. In this review the clinical biochemical and genetic features of the disorder are reported. The principles of therapy will also be covered, including dietary, pharmacological approaches, as well as techniques used to manipulate the gastrointestinal environment as a strategy to reduce the gastrointestinal load of trimethylamine.

α-Synuclein is an intrinsically disordered protein whose aggregation is implicated in Parkinson’s disease. A second member of the synuclein family, β-synuclein, shares significant sequence similarity with α-synuclein but is much more resistant to aggregation. β-Synuclein is missing an 11-residue stretch in the central non-β-amyloid component region that forms the core of α-synuclein amyloid fibrils, yet insertion of these residues into β-synuclein to produce the βSHC construct does not markedly increase the aggregation propensity. To investigate the structural basis of these different behaviors, quantitative nuclear magnetic resonance data, in the form of paramagnetic relaxation enhancement-derived interatomic distances, are combined with molecular dynamics simulations to generate ensembles of structures representative of the solution states of α-synuclein, β-synuclein, and βSHC. Comparison of these ensembles reveals that the differing aggregation propensities of α-synuclein and β-synuclein are associated with differences in the degree of residual structure in the C-terminus coupled to the shorter separation between the N- and C-termini in β-synuclein and βSHC, making protective intramolecular contacts more likely.

Rett syndrome (RTT) is a neurodevelopmental disorder associated with mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2) and consequent dysregulation of brain maturation. Patients suffer from a range of debilitating physical symptoms, however, behavioral and emotional symptoms also severely affect their quality of life. Here, we present previously unreported and clinically relevant affective dysfunction in the female heterozygous Mecp2(tm1Tam) mouse model of RTT (129sv and C57BL6 mixed background). The affective dysfunction and aberrant anxiety-related behavior of the Mecp2(+/-) mice were found to be reversible with environmental enrichment (EE) from 4 weeks of age. The effect of exercise alone (via wheel running) was also explored, providing the first evidence that increased voluntary physical activity in an animal model of RTT is beneficial for some phenotypes. Mecp2(+/-) mutants displayed elevated corticosterone despite decreased Crh expression, demonstrating hypothalamic-pituitary-adrenal axis dysregulation. EE of Mecp2(+/-) mice normalized basal serum corticosterone and hippocampal BDNF protein levels. The enrichment-induced rescue appears independent of the transcriptional regulation of the MeCP2 targets Bdnf exon 4 and Crh. These findings provide new insight into the neurodevelopmental role of MeCP2 and pathogenesis of RTT, in particular the affective dysfunction. The positive outcomes of environmental stimulation and physical exercise have implications for the development of therapies targeting the affective symptoms, as well as behavioral and cognitive dimensions, of this devastating neurodevelopmental disorder.

SummaryObjective TSH suppression therapy in patients with differentiated thyroid cancer (DTC) has been associated with adverse effects on areal bone mineral density (aBMD) only in postmenopausal women. The purpose of study was to examine the effect of TSH suppression therapy on skeletal integrity using peripheral quantitative computed tomography (pQCT) at the radius and tibia in pre- and postmenopausal women with DTC and controls. Study design and patients Subjects included 80 women with DTC (40 pre- and 40 postmenopausal) and 89 (29 and 60, respectively) controls. pQCT was performed at the radius and tibia, Dual-energy X-ray absorptiometry (DXA) at the hip and lumbar spine, while samples were taken for calciotropic hormones and bone markers. Results No differences were observed concerning aBMD by DXA. In premenopausal women, there were no significant differences concerning vBMD, while cortical thickness was higher at the radius in patients with DTC (P

We evaluated family satisfaction following spinal fusion in girls with Rett syndrome.Families participating in the population-based and longitudinal Australian Rett Syndrome Database whose daughter had undergone spinal fusion provided data on satisfaction overall, care processes and expected changes in health and function. Content analysis of responses to open-ended questions was conducted.Families reported high levels of overall satisfaction and consistently high ratings in relation to surgical and ICU care. Outstanding clinical care and the development of strong partnerships with clinical staff were much appreciated by families, whereas poor information exchange and inconsistent care caused concerns.Family satisfaction is an important outcome within a patient-centred quality of care framework. Our findings suggest strategies to inform the delivery of care in relation to spinal fusion for Rett syndrome and could also inform the hospital care of other children with disability and a high risk of hospitalization.

Correct protein folding is key to a functional proteome, as globular proteins must acquire their correct three-dimensional native fold in order to fulfil their biological role. A significant amount of folding occurs co-translationally on the ribosome and this process is likely to differ from folding of isolated proteins not least through the attachment to the ribosome and the vectorial emergence of the nascent polypeptide from the ribosomal exit tunnel. We are using solution-state Nuclear Magnetic Resonance (NMR) spectroscopy to study structural aspects of the co-translational protein folding of stalled ribosome-bound nascent chain complexes (RNCs) which mimic the progressive emergence of the nascent polypeptide and its acquisition of structure. The current state of our study on the co-translational folding of a model immunoglobulin domain will be presented including some of the structural models built from chemical shift data of RNCs. These findings contrast with analogous studies of C-terminal truncatio...

MBF1 (multi-protein bridging factor 1) is a protein containing a conserved HTH (helix–turn–helix) domain in both eukaryotes and archaea. Eukaryotic MBF1 has been reported to function as a transcriptional co-activator that physically bridges transcription regulators with the core transcription initiation machinery of RNA polymerase II. In addition, MBF1 has been found to be associated with polyadenylated mRNA in yeast as well as in mammalian cells. aMBF1 (archaeal MBF1) is very well conserved among most archaeal lineages; however, its function has so far remained elusive. To address this, we have conducted a molecular characterization of this aMBF1. Affinity purification of interacting proteins indicates that aMBF1 binds to ribosomal subunits. On sucrose density gradients, aMBF1 co-fractionates with free 30S ribosomal subunits as well as with 70S ribosomes engaged in translation. Binding of aMBF1 to ribosomes does not inhibit translation. Using NMR spectroscopy, we show that aMBF1 contains a long intrinsically disordered linker connecting the predicted N-terminal zinc-ribbon domain with the C-terminal HTH domain. The HTH domain, which is conserved in all archaeal and eukaryotic MBF1 homologues, is directly involved in the association of aMBF1 with ribosomes. The disordered linker of the ribosome-bound aMBF1 provides the N-terminal domain with high flexibility in the aMBF1–ribosome complex. Overall, our findings suggest a role for aMBF1 in the archaeal translation process.

The misfolding of intrinsically disordered proteins such asa-synuclein, tau and the Abpeptide has been associated withmany highly debilitating neurodegenerative syndromes including Parkinson’s and Alzheimer’s diseases. Therapeutictargeting of the monomeric state of such intrinsically disordered proteins by small molecules has, however, been a majorchallenge because of their heterogeneous conformational properties. We show here that a combination of computationaland experimental techniques has led to the identification of a drug-like phenyl-sulfonamide compound (ELN484228), thattargetsa-synuclein, a key protein in Parkinson’s disease. We found that this compound has substantial biological activity incellular models ofa-synuclein-mediated dysfunction, including rescue ofa-synuclein-induced disruption of vesicletrafficking and dopaminergic neuronal loss and neurite retraction most likely by reducing the amount ofa-synucleintargeted to sites of vesicle mobilization such as the synapse in neurons or the site of bead engulfment in microglial cells.These results indicate that targetinga-synuclein by small molecules represents a promising approach to the developmentof therapeutic treatments of Parkinson’s disease and related conditions. Fil: Toth, Gergely. University of Cambridge; Reino Unido. Elan Pharmaceuticals. San Francisco; Estados Unidos Fil: Gardai, Shyra J.. Elan Pharmaceuticals. San Francisco; Estados Unidos Fil: Zago, Wagner. Elan Pharmaceuticals. San Francisco; Estados Unidos Fil: Bertoncini, Carlos Walter. University of Cambridge; Reino Unido. Universidad Nacional de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Cremades, Nunilo. University of Cambridge; Reino Unido Fil: Roy, Susan L.. Purdue University; Estados Unidos Fil: Tambe, Mitali A.. Purdue University; Estados Unidos Fil: Roche, Jean Christophe. Purdue University; Estados Unidos Fil: Galvagnion, Celine. University of Cambridge; Reino Unido Fil: Skibinski, Gaia. Gladstone Institute of Neurological Disease. San Francisco; Estados Unidos. Taube-Koret Center for Neurodegenerative Disease Researc. San Francisco; Estados Unidos Fil: Finkbeiner, Steven. Taube-Koret Center for Neurodegenerative Disease Researc. San Francisco; Estados Unidos. Gladstone Institute of Neurological Disease. San Francisco; Estados Unidos. Departments of Neurology and Physiology. San Francisco; Estados Unidos Fil: Bova, Michael. Elan Pharmaceuticals. San Francisco; Estados Unidos Fil: Regnstrom, Karin. Elan Pharmaceuticals. San Francisco; Estados Unidos Fil: Chiou, San-San. Elan Pharmaceuticals. San Francisco; Estados Unidos Fil: Johnston, Jennifer. Elan Pharmaceuticals. San Francisco; Estados Unidos Fil: Callaway, Kari. Elan Pharmaceuticals. San Francisco; Estados Unidos Fil: Anderson, John P.. Elan Pharmaceuticals. San Francisco; Estados Unidos Fil: Jobling, Michael F.. Elan Pharmaceuticals. San Francisco; Estados Unidos Fil: Buel, Alexander K.. University of Cambridge; Reino Unido Fil: Yednock, Ted A.. Elan Pharmaceuticals. San Francisco; Estados Unidos Fil: Knowles, Tuomas P. J.. University of Cambridge; Reino Unido Fil: Vendruscolo, Michele. University of Cambridge; Reino Unido Fil: Christodoulou, John. University College London; Estados Unidos Fil: Dobson, Christopher M.. University of Cambridge; Reino Unido Fil: Schenk, Dale. Elan Pharmaceuticals. San Francisco; Estados Unidos Fil: McConlogue, LIsa. Elan Pharmaceuticals. San Francisco; Estados Unidos

Recent developments in the field of reprogramming somatic cells to induced pluripotent stem cells (iPSC) has brought the field closer to the exciting possibility of their future clinical application. A number of challenges remain to be addressed before clinical application of reprogrammed stem cells becomes a reality. The main issues are the threat of cancer, control of differentiation to tissue specific stem cells or body organs and the immune response of iPSC-derived cells. The future use of iPSC will require that they are cancer free, do not proliferate and can be contained to the specific injection site for tissue regeneration. This review discusses the recent advances that address these main challenges in the field of cellular reprogramming

The relation of coronary artery calcification with the presence of symptoms of coronary artery disease and its angiographic severity is not clear. We studied 37 apparently healthy, asymptomatic subjects that were found by digital cinefluoroscopy to have coronary calcium and compared to age- and sex-matched group of patients with coronary calcium and symptomatic coronary artery disease. Normal coronary arteries and non-obstructive lesions only were found in 12/37 (32.4%) and 11/37 (29.7%) asymptomatic subjects vs. 1/37 (2.7%) and 2/37 (5.4%) patients; P

Purpose : Rett syndrome is a neurodevelopmental disorder that occurs almost exclusively in females. In recent years there has been increased knowledge concerning the multidisciplinary management of individuals with Rett syndrome. The aim of this paper is to provide an update of the clinical phenotype, natural history and current genetic understanding of the disorder. Results / Conclusion : Rett syndrome is thought to be the second most common cause of severe mental retardation in females after Down syndrome. it now appears that females with RS present with a much broader phenotype than originally described. Recently, mutations in the MECP2 gene encoding X-linked methyl-CpG-binding-protein 2 have been identified in some females with Rett syndrome.

Background Mutations in the mitochondrial tyrosyl-tRNA synthetase (YARS2) gene have previously been identified as a cause of the tissue specific mitochondrial respiratory chain (RC) disorder, Myopathy, Lactic Acidosis, Sideroblastic Anaemia (MLASA). In this study, a cohort of patients with a mitochondrial RC disorder for who anaemia was a feature, were screened for mutations in YARS2. Methods Twelve patients were screened for YARS2 mutations by Sanger sequencing. Clinical data were compared. Functional assays were performed to confirm the pathogenicity of the novel mutations and to investigate tissue specific effects. Results PathogenicYARS2 mutations were identified in three of twelve patients screened. Two patients were found to be homozygous for the previously reported p.Phe52Leu mutation, one severely and one mildly affected. These patients had different mtDNA haplogroups which may contribute to the observed phenotypic variability. A mildly affected patient was a compound heterozygote for two novel YARS2 mutations, p.Gly191Asp and p.Arg360X. The p.Gly191Asp mutation resulted in a 38-fold loss in YARS2 catalytic efficiency and the p.Arg360X mutation did not produce a stable protein. The p.Phe52Leu and p.Gly191Asp/p.Arg360X mutations resulted in more severe RC deficiency of complexes I, III and IV in muscle cells compared to fibroblasts, but had relatively normal YARS2 protein levels. The muscle-specific RC deficiency can be related to the increased requirement for RC complexes in muscle. There was also a failure of mtDNA proliferation upon myogenesis in patient cells which may compound the RC defect. Patient muscle had increased levels of PGC1-α and TFAM suggesting mitochondrial biogenesis was activated as a potential compensatory mechanism. Conclusion In this study we have identified novel YARS2 mutations and noted marked phenotypic variability among YARS2 MLASA patients, with phenotypes ranging from mild to lethal, and we suggest that the background mtDNA haplotype may be contributing to the phenotypic variability. These findings have implications for diagnosis and prognostication of the MLASA and related phenotypes.

Genetic mitochondrial defects of the respiratory chain show marked phenotypic variability. Laboratory diagnosis is complicated and includes biochemical screening tests, tissue histopathology, functional enzyme studies, and molecular tests where available. Normal respiratory chain function necessitates the co-ordinated expression of over 100 different gene loci, and the interaction of two genetic systems, the nuclear and mitochondrial genomes. Thus genetic counselling for the mitochondrial disorders is extremely challenging. In this review, the classes of mitochondrial and nuclear defects that give rise to functional abnormalities of the mitochondrial respiratory chain are discussed, with specific instructive examples described in some detail.