Protein misfolding diseases: Prospects of pharmacological treatment.

Protein misfolding has been linked to numerous inherited diseases. Loss- and gain-of-function mutations (common features of genetic diseases) may cause the destabilization of proteins, leading to alterations in their properties and/or cellular location, resulting in their incorrect functioning. Misfolded proteins can, however, be rescued via the use of proteostasis regulators and/or pharmacological chaperones, suggesting that treatments with small molecules might be developed for a range of genetic diseases. This work describes the potential of these small molecules in this respect, including for the treatment of congenital disorder of glycosylation (CDG) due to phosphomannomutase 2 deficiency (PMM2-CDG).

Aging results in iron accumulations in the non-human primate choroid of the eye without an associated increase in zinc, copper or sulphur.

We present further analyses of a previous experiment published in 2016 where the distribution, concentration and correlation of iron, zinc, copper and sulphur in the choroid of the eye in young and aged old world primates (Macaca fascicularis) was studied with synchrotron X-ray fluorescence with a 2 µm resolution. The results indicate that iron accumulates in hotspots in the choroid with age with fluorescence intensity ranging from 2- to 7-fold (1002-3752 ppm) the mean level in the choroidal stroma (500 ppm) and maximum iron levels in blood vessel lumina. Iron hotspots with iron ppm > 1000 preferentially contained Fe3+ as demonstrated by Perls staining. There was a strong spatial co-localisation and correlation between copper and zinc (Pearson's correlation coefficient 0.97), and both elements with sulphur in the choroid of young animals. However, these are reduced in the choroid of aged animals and lost in the iron hotspots. The lack of proportional co-distribution suggests that iron accumulation does not induce a concomitant increase in zinc, copper or zinc-, copper-metalloproteins. It is possible that the iron hotspots are ferritin or hemosiderin molecules loaded with Fe3+ in stable, insoluble, non-toxic complexes without a significant oxidative environment.

Cognitive performance of people with traumatic spinal cord injury: a cross-sectional study comparing people with subacute and chronic injuries.

STUDY DESIGN: Cross-sectional study. OBJECTIVES: To assess the impact of spinal cord injury (SCI) on cognitive function in individuals with subacute and chronic SCI. SETTING: National Hospital for SCI patients (Spain). METHODS: The present investigation was designed to determine the nature, pattern, and extent of cognitive deficits in a group of participants with subacute (n = 32) and chronic (n = 34) SCI, using a comprehensive battery of reliable and validated neuropsychological assessments to study a broad range of cognitive functions. Twenty-seven able-bodied subjects matched to the groups with SCI for age and educational level formed the control group. RESULTS: The neuropsychological assessment showed alterations in the domain of attention, processing speed, memory and learning, executive functions, and in recognition in participants with SCI. The prevalence of cognitive dysfunction in the chronic stage was also confirmed at the individual level. The comparison of the neuropsychological assessment between the groups with subacute and chronic SCI showed a worsening of cognitive functions in those with chronic SCI compared to the group with subacute SCI. CONCLUSIONS: In participants with SCI, cognitive dysfunctions are present in the subacute stage and worsen over time. From a clinical point of view, we confirmed the presence of cognitive dysfunction that may interfere with the first stage of rehabilitation which is the most intense and important. Moreover, cognitive dysfunction may be important beyond the end of the first stage of rehabilitation as it can affect an individual's quality of life and possible integration to society.

Mitochondrial response to the BCKDK-deficiency: Some clues to understand the positive dietary response in this form of autism.

Mutations on the mitochondrial-expressed Branched Chain α-Keto acid Dehydrogenase Kinase (BCKDK) gene have been recently associated with a novel dietary-treatable form of autism. But, being a mitochondrial metabolism disease, little is known about the impact on mitochondrial performance. Here, we analyze the mitochondrial response to the BCKDK-deficiency in patient's primary fibroblasts by measuring bioenergetics, ultra-structural and dynamic parameters. A two-fold increase in superoxide anion production, together with a reduction in ATP-linked respiration and intracellular ATP levels (down to 60%) detected in mutants fibroblasts point to a general bioenergetics depletion that could affect the mitochondrial dynamics and cell fate. Ultrastructure analysis of BCKDK-deficient fibroblasts shows an increased number of elongated mitochondria, apparently associated with changes in the mediator of inner mitochondria membrane fusion, GTPase OPA1 forms, and in the outer mitochondrial membrane, mitofusin 2/MFN2. Our data support a possible hyperfusion response of BCKDK-deficient mitochondria to stress. Cellular fate also seems to be affected as these fibroblasts show an altered proportion of the cells on G0/G1 and G2/M phases. Knockdown of BCKDK gene in control fibroblasts recapitulates most of these features. Same BCKDK-knockdown in a MSUD patient fibroblasts unmasks the direct involvement of the accelerated BCAAs catabolism in the mitochondrial dysfunction. All these data give us a clue to understand the positive dietary response to an overload of branched-chain amino acids. We hypothesize that a combination of the current therapeutic option with a protocol that considers the oxidative damage and energy expenditure, addressing the patients' individuality, might be useful for the physicians.

Age-space-time CAR models in Bayesian disease mapping.

Mortality counts are usually aggregated over age groups assuming similar effects of both time and region, yet the spatio-temporal evolution of cancer mortality rates may depend on changing age structures. In this paper, mortality rates are analyzed by region, time period and age group, and models including space-time, space-age, and age-time interactions are considered. The integrated nested Laplace approximation method, known as INLA, is adopted for model fitting and inference in order to reduce computing time in comparison with Markov chain Monte Carlo (McMC) methods. The methodology provides full posterior distributions of the quantities of interest while avoiding complex simulation techniques. The proposed models are used to analyze prostate cancer mortality data in 50 Spanish provinces over the period 1986-2010. The results reveal a decline in mortality since the late 1990s, particularly in the age group [65,70), probably because of the inclusion of the PSA (prostate-specific antigen) test and better treatment of early-stage disease. The decline is not clearly observed in the oldest age groups. Copyright © 2016 John Wiley & Sons, Ltd.

Clostridium difficile infection after restorative proctocolectomy and ileal pouch anal anastomosis for ulcerative colitis.

AIM: Clostridium difficile infection (CDI) of the ileal pouch following restorative proctocolectomy (RPC) is becoming increasingly recognized. We aimed to understand better (i) the associated risk factors, (ii) treatment practices and (iii) the pouch diversion and failure rate in patients who developed CDI of the pouch after RPC for ulcerative colitis (UC). METHOD: Patients who tested positive for C. difficile of the pouch between 2007 and 2010 were included in the analysis. Data collected included patient demographics, time from RPC to documented CDI, the treatment of CDI and rate of excision of the pouch. RESULTS: Of 2785 patients recorded in the hospital CDI database, 15 had had an RPC with ileal pouch anal anastomosis. The median age was 44 years and the median interval from RPC to first documented episode of CDI was 3 years. Thirteen (81%) patients had had multiple episodes of pouchitis before and after CDI infection, and all were symptomatic at the time of testing for CDI. Within 30 days of the diagnosis of CDI, six (40%) patients were taking immunosuppressive medication, seven (47%) were taking a proton pump inhibitor and 12 (80%) had received antibiotics. Five patients required hospitalization for CDI and four had severe infections characterized by a serum creatinine more than 1.5 times baseline (n = 3) and a white cell count above 15 000 (n = 1). Six patients who underwent endoscopy had severe inflammation of the pouch including the presence of a pseudomembrane in one case. Ten patients were treated with metronidazole alone and five with vancomycin. Two patients had recurrent CDI of the pouch during a median follow-up period of 2.9 years and one had CDI refractory to medical management. This patient required diversion of the pouch with an ileostomy for refractory CDI but no patient required excision of the pouch. CONCLUSION: All 15 patients developing CDI of the pouch were successfully treated with antibiotics and only one required surgery in the form of an ileostomy.

Methylmalonic aciduria cblB type: characterization of two novel mutations and mitochondrial dysfunction studies.

Methylmalonic aciduria (MMA) cblB type is caused by mutations in the MMAB gene, which codes for the enzyme adenosine triphosphate (ATP): cobalamin adenosyltransferase (ATR). This study reports differences in the metabolic and disease outcomes of two pairs of siblings with MMA cblB type, respectively harbouring the novel changes p.His183Leu/p.Arg190dup (P1 and P2) and the previously described mutations p.Ile96Thr/p.Ser174fs (P3 and P4). Expression analysis showed p.His183Leu and p.Arg190dup to be destabilizing mutations. Both were associated with reduced ATR stability and a shorter half-life than wild-type ATR. Analysis of several parameters related to oxidative stress and mitochondrial function showed an increase in reactive oxygen species (ROS) content, a decrease in mitochondrial respiration and changes in mitochondria morphology and structure in patient-derived fibroblasts compared to control cells. The impairment in energy production and the presence of oxidative stress and fission of the mitochondrial reticulum suggested mitochondrial dysfunction in cblB patients' fibroblasts. The recovery of mitochondrial function should be a goal in efforts to improve the clinical outcome of MMA cblB type.

Antisense-mediated therapeutic pseudoexon skipping in TMEM165-CDG.

Deficiencies in glycosyltransferases, glycosidases or nucleotide-sugar transporters involved in protein glycosylation lead to congenital disorders of glycosylation (CDG), a group of genetic diseases mostly showing multisystem phenotype. Despite recent advances in the biochemical and molecular knowledge of these diseases, no effective therapy exists for most. Efforts are now being directed toward therapies based on identifying new targets, which would allow to treat specific patients in a personalized way. This work presents proof-of concept for the antisense RNA rescue of the Golgi-resident protein TMEM165, a gene involved in a new type of CDG with a characteristic skeletal phenotype. Using a functional in vitro splicing assay based on minigenes, it was found that the deep intronic change c.792+182G>A is responsible for the insertion of an aberrant exon, corresponding to an intronic sequence. Antisense morpholino oligonucleotide therapy targeted toward TMEM165 mRNA recovered normal protein levels in the Golgi apparatus of patient-derived fibroblasts. This work expands the application of antisense oligonucleotide-mediated pseudoexon skipping to the treatment of a Golgi-resident protein, and opens up a promising treatment option for this specific TMEM165-CDG.

Functional analysis and in vitro correction of splicing FAH mutations causing tyrosinemia type I.

Hereditary tyrosinemia type I (HT1) is a rare disease caused by a deficiency of fumarylacetoacetate hydrolase (FAH) in the tyrosine catabolic pathway, resulting mainly in hepatic alterations due to accumulation of the toxic metabolites fumarylacetoacetate, maleylacetoacetate and succinylacetone. We have characterized using minigenes four splicing mutations affecting exonic or intronic nucleotides of the FAH gene identified in two HT1 patients. Two of the mutations are novel, c.82-1G>A and c.913G>C and the other two have been previously associated with a splicing defect (c.836A>G and c.1062+5G>A). All mutations were confirmed to affect splicing in minigenes, resulting in exon skipping or activation of a cryptic splice site. We have analyzed the effect of different compounds known to modulate splicing (valproic acid, phenyl butyrate, M344, EIPA, and resveratrol) and the overexpression of splice factors of the SR protein family on the transcriptional profile of the mutant minigenes. For the c.836A>G mutation, a partial recovery of the correctly spliced transcript was observed. These results confirm the relevance of performing functional studies for mutations potentially affecting the splicing process and open the possibility of supplementary therapeutic approaches to diseases caused by splicing defects.

Pregnancy in a methylmalonic acidemia patient with kidney transplantation: a case report.

Presently pregnancy is no more exceptional in women with metabolic diseases. However, it still poses significant medical problems both before and after childbirth. The challenge is even greater if the mother has undergone organ transplantation, because of her metabolic disease. We report on a case of pregnancy in a patient 29-year-old with methylmalonic acidemia cblA type (OMIM 251100) who received a renal transplantation at the age of 17 for end-stage renal disease (ESRD) caused by her primary disease. During pregnancy neither metabolic crises nor renal function changes were observed in the mother, with the only exception of a mild increase of her systemic blood pressure. To the fetus pregnancy was uneventful and during the first 30 months after birth the baby's neuropsychomotor development was normal and there were no episodes of metabolic derangement. This is evidence that methylmalonicacidemia cblA, even when treated with renal transplantation for inherent ESRD, is no contraindication to pregnancy. It is even possible that a functioning transplanted kidney contributes to improve metabolic parameters.

Wernicke-like encephalopathy during classic maple syrup urine disease decompensation.

We describe a new neuroradiologic picture observed during metabolic decompensation in two maple syrup urine disease (MSUD) patients that resembles Wernicke encephalopathy (WE). Clinical observations and the review of the literature regarding WE and MSUD pathophysiology prompted us to hypothesize a pathogenic link between these two disorders. Based on these findings, clinicians and neuroradiologists should be aware of MSUD as a possible predisposing factor of WE in children.

Mutation analysis in 54 propionic acidemia patients.

Deficiency of propionyl CoA carboxylase (PCC), a dodecamer of alpha and beta subunits, causes inherited propionic acidemia. We have studied, at the molecular level, PCC in 54 patients from 48 families comprised of 96 independent alleles. These patients of various ethnic backgrounds came from research centers and hospitals in Germany, Austria and Switzerland. The thorough clinical characterization of these patients was described in the accompanying paper (Grünert et al. 2012). In all 54 patients, many of whom originated from consanguineous families, the entire PCCB gene was examined by genomic DNA sequencing and in 39 individuals the PCCA gene was also studied. In three patients we found mutations in both PCC genes. In addition, in many patients RT-PCR analysis of lymphoblast RNA, lymphoblast enzyme assays, and expression of new mutations in E.coli were carried out. Eight new and eight previously detected mutations were identified in the PCCA gene while 15 new and 13 previously detected mutations were found in the PCCB gene. One missense mutation, p.V288I in the PCCB gene, when expressed in E.coli, yielded 134% of control activity and was consequently classified as a polymorphism in the coding region. Numerous new intronic polymorphisms in both PCC genes were identified. This study adds a considerable amount of new molecular data to the studies of this disease.

Identifying extreme COVID-19 mortality risks in English small areas: a disease cluster approach.

The COVID-19 pandemic is having a huge impact worldwide and has highlighted the extent of health inequalities between countries but also in small areas within a country. Identifying areas with high mortality is important both of public health mitigation in COVID-19 outbreaks, and of longer term efforts to tackle social inequalities in health. In this paper we consider different statistical models and an extension of a recent method to analyze COVID-19 related mortality in English small areas during the first wave of the epidemic in the first half of 2020. We seek to identify hotspots, and where they are most geographically concentrated, taking account of observed area factors as well as spatial correlation and clustering in regression residuals, while also allowing for spatial discontinuities. Results show an excess of COVID-19 mortality cases in small areas surrounding London and in other small areas in North-East and and North-West of England. Models alleviating spatial confounding show ethnic isolation, air quality and area morbidity covariates having a significant and broadly similar impact on COVID-19 mortality, whereas nursing home location seems to be slightly less important.

Hepatocyte-like cells differentiated from methylmalonic aciduria cblB type induced pluripotent stem cells: A platform for the evaluation of pharmacochaperoning.

Methylmalonic aciduria cblB type (MMA cblB type, MMAB OMIM #251110), caused by a deficiency in the enzyme ATP:cob(I)alamin adenosyltransferase (ATR, E.C_2. 5.1.17), is a severe metabolic disorder with a poor prognosis despite treatment. We recently described the potential therapeutic use of pharmacological chaperones (PCs) after increasing the residual activity of ATR in patient-derived fibroblasts. The present work reports the successful generation of hepatocyte-like cells (HLCs) differentiated from two healthy and two MMAB induced pluripotent stem cell (iPSC) lines, and the use of this platform for testing the effects of PCs. The MMAB cells produced little ATR, showed reduced residual ATR activity, and had higher concentrations of methylmalonic acid compared to healthy HLCs. Differential proteome analysis revealed the two MMAB HCLs to show reproducible differentiation, but this was not so for the healthy HLCs. Interestingly, PC treatment in combination with vitamin B12 increased the amount of ATR available, and subsequently ATR activity, in both MMAB HLCs. More importantly, the treatment significantly reduced the methylmalonic acid content of both. In summary, the HLC model would appear to be an excellent candidate for the pharmacological testing of the described PCs, for analyzing the effects of new drugs, and investigating the repurposing of older drugs, before testing in animal models.

Overexpression of adapted U1snRNA in patients' cells to correct a 5' splice site mutation in propionic acidemia.

Splicing defects account for 16% of the mutant alleles in the PCCA and PCCB genes, encoding both subunits of the propionyl-CoA carboxylase (PCC) enzyme, defective in propionic acidemia, one of the most frequent organic acidemias causing variable neurological impairment. Most of the splicing mutations identified affect the conserved 3' splice (3' ss) or 5' splice (5' ss) sites, the latter predictably through lowering the strength of base pairing with U1snRNA. Among the 5' ss mutations we have focused on the c.1209+3A>G (IVS13+3A>G) mutation in the PCCA gene, identified in four patients (three homozygous and one heterozygous) of common geographical origin and causing exon 13 skipping. To study the potential of splicing modulation to restore PCC function, we analyzed the effect of transient transfections in patients' cells with modified U1snRNA adapted to compensate the mutant change and other mismatches at different positions of the 5' ss. Using this strategy normal transcript could be efficiently recovered with the concomitant disappearance of the aberrant exon skipping transcript, as observed after standard RT-PCR and sequence analysis or using fluorescent primers and semiquantitative RT-PCR. Different efficiencies with up to 100% exon inclusion were observed depending on the transfection conditions and specifically on the adapted U1snRNA used, confirming previously reported dependencies between nucleotides at the 5' ss for its correct recognition by the spliceosome. The reversal of the splicing defect did not result in a significant increase in enzyme activity, suggesting other factors must be taken into account for the application of overexpression of splice factors such as U1 as therapeutic strategy for splice defects.

Propionic and methylmalonic acidemia: antisense therapeutics for intronic variations causing aberrantly spliced messenger RNA.

We describe the use of antisense morpholino oligonucleotides (AMOs) to restore normal splicing caused by intronic molecular defects identified in methylmalonic acidemia (MMA) and propionic acidemia (PA). The three new point mutations described in deep intronic regions increase the splicing scores of pseudoexons or generate consensus binding motifs for splicing factors, such as SRp40, which favor the intronic inclusions in MUT (r.1957ins76), PCCA (r.1284ins84), or PCCB (r.654ins72) messenger RNAs (mRNAs). Experimental confirmation that these changes are pathogenic and cause the activation of the pseudoexons was obtained by use of minigenes. AMOs were targeted to the 5? or 3? cryptic splice sites to block access of the splicing machinery to the pseudoexonic regions in the pre-mRNA. Using this antisense therapeutics, we have obtained correctly spliced mRNA that was effectively translated, and propionyl coenzyme A (CoA) carboxylase (PCC) or methylmalonylCoA mutase (MCM) activities were rescued in patients' fibroblasts. The effect of AMOs was sequence and dose dependent. In the affected patient with MUT mutation, close to 100% of MCM activity, measured by incorporation of (14)C-propionate, was obtained after 48 h, and correctly spliced MUT mRNA was still detected 15 d after treatment. In the PCCA-mutated and PCCB-mutated cell lines, 100% of PCC activity was measured after 72 h of AMO delivery, and the presence of biotinylated PCCA protein was detected by western blot in treated PCCA-deficient cells. Our results demonstrate that the aberrant inclusions of the intronic sequences are disease-causing mutations in these patients. These findings provide a new therapeutic strategy in these genetic disorders, potentially applicable to a large number of cases with deep intronic changes that, at the moment, remain undetected by standard mutation-detection techniques.

Proteostasis regulators as potential rescuers of PMM2 activity.

Phosphomannomutase 2 deficiency (PMM2-CDG) is the most common N-glycosylation disorder. To date there is no treatment. Following the identification of a number of destabilizing pathogenic variants, our group suggested PMM2-CDG to be a conformational disease. The aim of the present study was to evaluate the possible use of proteostasis network regulators to increase the stability, and subsequently the enzymatic activity, of misfolded PMM2 mutant proteins. Patient-derived fibroblasts transduced with their own PMM2 folding or oligomerization variants were treated with different concentrations of the proteostasis regulators celastrol or MG132. Celastrol treatment led to a significant increase in mutant PMM2 protein concentration and activity, while MG132 had a small effect on protein concentration only. The increase in enzymatic activity with celastrol correlated with an increase in the transcriptional and proteome levels of the heat shock proteins Hsp90 and Hsp70. The use of specific Hsp70 or Hsp90 inhibitors showed the positive effect of celastrol on PMM2 stability and activity to occur through Hsp90-driven modulation of the proteostasis network. The synergistic effect of celastrol and a previously described pharmacological chaperone was also examined, and a mutation-dependent synergistic effect on PMM2 activity was noted. These results provide proof-of-concept regarding the potential treatment of PMM2-CDG by proteostasis regulators, either alone or in combination with pharmacological chaperones.

Pseudoexon exclusion by antisense therapy in methylmalonic aciduria (MMAuria).

Development of pseudoexon exclusion therapies by antisense modification of pre-mRNA splicing represents a type of personalized genetic medicine. Here we present the cellular antisense therapy and the cell-based splicing assays to investigate the effect of two novel deep intronic changes c.1957-898A>G and c.1957-920C>A identified in the methylmalonyl-coenzyme A (CoA) mutase (MUT) gene. The results show that the nucleotide change c.1957-898A>G is a pathological mutation activating pseudoexon insertion and that antisense morpholino oligonucleotide (AMO) treatment in patient fibroblasts leads to recovery of MUT activity to levels 25 to 100% of control range. On the contrary, the change c.1957-920C>A, identified in two fibroblasts cell lines in cis with c.1885A>G (p.R629G) or c.458T>A (p.D153V), appears to be a rare variant of uncertain clinical significance. The functional analysis of c.1885A>G and c.458T>A indicate that they are the disease-causing mutations in these two patients. The results presented here highlight the necessity of scanning the described intronic region for mutations in MUT-affected patients, followed by functional analyses to demonstrate the pathogenicity of the identified changes, and extend previous work of the applicability of the antisense approach in methylmalonic aciduria (MMAuria) for a novel intronic mutation.

Flexible Bayesian P-splines for smoothing age-specific spatio-temporal mortality patterns.

In this paper age-space-time models based on one and two-dimensional P-splines with B-spline bases are proposed for smoothing mortality rates, where both fixed relative scale and scale invariant two-dimensional penalties are examined. Model fitting and inference are carried out using integrated nested Laplace approximations, a recent Bayesian technique that speeds up computations compared to McMC methods. The models will be illustrated with Spanish breast cancer mortality data during the period 1985-2010, where a general decline in breast cancer mortality has been observed in Spanish provinces in the last decades. The results reveal that mortality rates for the oldest age groups do not decrease in all provinces.