An integrated modelling methodology for estimating global incidence and prevalence of hereditary spastic paraplegia subtypes SPG4, SPG7, SPG11, and SPG15.

BACKGROUND: Hereditary spastic paraplegias (HSPs) are progressively debilitating neurodegenerative disorders that follow heterogenous patterns of Mendelian inheritance. Available epidemiological evidence provides limited incidence and prevalence data, especially at the genetic subtype level, preventing a realistic estimation of the true social burden of the disease. The objectives of this study were to (1) review the literature on epidemiology of HSPs; and (2) develop an epidemiological model of the prevalence of HSP, focusing on four common HSP genetic subtypes at the country and region-level. METHODS: A model was constructed estimating the incidence at birth, survival, and prevalence of four genetic subtypes of HSP based on the most appropriate published literature. The key model parameters were assessed by HSP clinical experts, who provided feedback on the validity of assumptions. A model was then finalized and validated through comparison of outputs against available evidence. The global, regional, and national prevalence and patient pool were calculated per geographic region and per genetic subtype. RESULTS: The HSP global prevalence was estimated to be 3.6 per 100,000 for all HSP forms, whilst the estimated global prevalence per genetic subtype was 0.90 (SPG4), 0.22 (SPG7), 0.34 (SPG11), and 0.13 (SPG15), respectively. This equates to an estimated 3365 (SPG4) and 872 (SPG11) symptomatic patients, respectively, in the USA. CONCLUSIONS: This is the first epidemiological model of HSP prevalence at the genetic subtype-level reported at multiple geographic levels. This study offers additional data to better capture the burden of illness due to mutations in common genes causing HSP, that can inform public health policy and healthcare service planning, especially in regions with higher estimated prevalence of HSP.

Natural history study and statistical modeling of disease progression in a preclinical model of myotubular myopathy.

Generating reliable preclinical data in animal models of disease is essential in therapy development. Here, we performed statistical analysis and joint longitudinal-survival modeling of the progressive phenotype observed in Mtm1-/y mice, a reliable model for myotubular myopathy. Analysis of historical data was used to generate a model for phenotype progression, which was then confirmed with phenotypic data from a new colony of mice derived via in vitro fertilization in an independent animal house, highlighting the reproducibility of disease phenotype in Mtm1-/y mice. These combined data were used to refine the phenotypic parameters analyzed in these mice and improve the model generated for expected disease progression. The disease progression model was then used to test the therapeutic efficacy of Dnm2 targeting. Dnm2 reduction by antisense oligonucleotides blocked or postponed disease development, and resulted in a significant dose-dependent improvement outside the expected disease progression in untreated Mtm1-/y mice. This provides an example of optimizing disease analysis and testing therapeutic efficacy in a preclinical model, which can be applied by scientists testing therapeutic approaches using neuromuscular disease models in different laboratories. This article has an associated First Person interview with the joint first authors of the paper.

Hierarchical Bayesian modelling of disease progression to inform clinical trial design in centronuclear myopathy.

BACKGROUND: Centronuclear myopathies are severe rare congenital diseases. The clinical variability and genetic heterogeneity of these myopathies result in major challenges in clinical trial design. Alternative strategies to large placebo-controlled trials that have been used in other rare diseases (e.g., the use of surrogate markers or of historical controls) have limitations that Bayesian statistics may address. Here we present a Bayesian model that uses each patient's own natural history study data to predict progression in the absence of treatment. This prospective multicentre natural history evaluated 4-year follow-up data from 59 patients carrying mutations in the MTM1 or DNM2 genes. METHODS: Our approach focused on evaluation of forced expiratory volume in 1 s (FEV1) in 6- to 18-year-old children. A patient was defined as a responder if an improvement was observed after treatment and the predictive probability of such improvement in absence of intervention was less than 0.01. An FEV1 response was considered clinically relevant if it corresponded to an increase of more than 8%. RESULTS: The key endpoint of a clinical trial using this model is the rate of response. The power of the study is based on the posterior probability that the rate of response observed is greater than the rate of response that would be observed in the absence of treatment predicted based on the individual patient's previous natural history. In order to appropriately control for Type 1 error, the threshold probability by which the difference in response rates exceeds zero was adapted to 91%, ensuring a 5% overall Type 1 error rate for the trial. CONCLUSIONS: Bayesian statistical analysis of natural history data allowed us to reliably simulate the evolution of symptoms for individual patients over time and to probabilistically compare these simulated trajectories to actual observed post-treatment outcomes. The proposed model adequately predicted the natural evolution of patients over the duration of the study and will facilitate a sufficiently powerful trial design that can cope with the disease's rarity. Further research and ongoing dialog with regulatory authorities are needed to allow for more applications of Bayesian statistics in orphan disease research.

Myostatin: a Circulating Biomarker Correlating with Disease in Myotubular Myopathy Mice and Patients.

Myotubular myopathy, also called X-linked centronuclear myopathy (XL-CNM), is a severe congenital disease targeted for therapeutic trials. To date, biomarkers to monitor disease progression and therapy efficacy are lacking. The Mtm1 -/y mouse is a faithful model for XL-CNM, due to myotubularin 1 (MTM1) loss-of-function mutations. Using both an unbiased approach (RNA sequencing [RNA-seq]) and a directed approach (qRT-PCR and protein level), we identified decreased Mstn levels in Mtm1 -/y muscle, leading to low levels of myostatin in muscle and plasma. Myostatin (Mstn or growth differentiation factor 8 [Gdf8]) is a protein released by myocytes and inhibiting muscle growth and differentiation. Decreasing Dnm2 by genetic cross with Dnm2 +/- mice or by antisense oligonucleotides blocked or postponed disease progression and resulted in an increase in circulating myostatin. In addition, plasma myostatin levels inversely correlated with disease severity and with Dnm2 mRNA levels in muscles. Altered Mstn levels were associated with a generalized disruption of the myostatin pathway. Importantly, in two different forms of CNMs we identified reduced circulating myostatin levels in plasma from patients. This provides evidence of a blood-based biomarker that may be used to monitor disease state in XL-CNM mice and patients and supports monitoring circulating myostatin during clinical trials for myotubular myopathy.

Translational medicine in neuromuscular disorders: from academia to industry.

Although around half of US Food and Drug Administration (FDA)-approved drugs originate from discoveries made in academic research laboratories, the US National Institutes of Health (NIH) estimates that nearly 90% of therapies developed in preclinical stages never reach clinical trials. From those in clinical trials, only 10% obtain marketing approval. Despite the recent advances in our understanding and diagnosis of neuromuscular disease, and the development of rational therapies in clinical trials, these numbers have not changed dramatically over the past two decades. This article discusses the advantages and challenges for translational research initiated from academia, and the trend towards bridging the gap between discovery and translation to the clinic. A focus is made on recent advances in therapeutic development for neuromuscular disorders.

Alterations in mucosal immunity identified in the colon of patients with irritable bowel syndrome.

BACKGROUND & AIMS: Irritable bowel syndrome (IBS) has been associated with mucosal dysfunction, mild inflammation, and altered colonic bacteria. We used microarray expression profiling of sigmoid colon mucosa to assess whether there are stably expressed sets of genes that suggest there are objective molecular biomarkers associated with IBS. METHODS: Gene expression profiling was performed using Human Genome U133 Plus 2.0 (Affymetrix) GeneChips with RNA from sigmoid colon mucosal biopsy specimens from 36 IBS patients and 25 healthy control subjects. Real-time quantitative polymerase chain reaction was used to confirm the data in 12 genes of interest. Statistical methods for microarray data were applied to search for differentially expressed genes, and to assess the stability of molecular signatures in IBS patients. RESULTS: Mucosal gene expression profiles were consistent across different sites within the sigmoid colon and were stable on repeat biopsy over approximately 3 months. Differentially expressed genes suggest functional alterations of several components of the host mucosal immune response to microbial pathogens. The most strikingly increased expression involved a yet uncharacterized gene, DKFZP564O0823. Identified specific genes suggest the hypothesis that molecular signatures may enable distinction of a subset of IBS patients from healthy controls. By using 75% of the biopsy specimens as a validation set to develop a gene profile, the test set (25%) was predicted correctly with approximately 70% accuracy. CONCLUSIONS: Mucosal gene expression analysis shows there are relatively stable alterations in colonic mucosal immunity in IBS. These molecular alterations provide the basis to test the hypothesis that objective biomarkers may be identified in IBS and enhance understanding of the disease.

Sequence, distribution and quantification of the motilin precursor in the cat.

Due to motilin's relation to the migrating motor complex (MMC), the physiology of motilin has been mostly studied in man and dog. The cat does not have an MMC pattern, and little is known about cat motilin. Therefore we identified the cat motilin precursor (GenBank accession no. AF127917) and developed a quantitative polymerase chain reaction (PCR) to explore its distribution in the gastrointestinal tract and in the central nervous system (CNS). The precursor is closely related to the dog precursor and consists of an open reading frame of 348bp encoding the signal peptide (25 amino acids), the motilin sequence (22 amino acids) and the motilin associated peptide (69 amino acids). One amino acid of the signal peptide was subject to gene polymorphism. Quantification of motilin messenger RNA (mRNA) was for the first time achieved. It is most abundant in the gastrointestinal tract, with the highest concentration in the duodenum, the lowest in the colon and is not detectable in the corpus. However an important expression was also observed in several regions of the CNS, except the striatum and cerebral cortex. The highest level was in the hypothalamus (although 23-fold lower than in the duodenum), the lowest level in the pons. Moderate levels were found in the thyroid. These data suggest that the physiological role of motilin may extend beyond its effect on gastrointestinal motility.

Mechanisms involved in the loss of excitatory post-stimulus responses by inflammation.

AIM: Electrical stimulation of colonic muscles elicits a response during the stimulation period, and a transient excitation after the stimulus. Post-stimulus or "rebound" excitation has been linked to pathways involving inhibitory neurotransmitters, prostaglandins and substance P but the mechanism is incompletely understood. Because rabbit colitis is characterized by a loss of inhibitory neurotransmission we hypothesized it might affect the rebound response. Therefore we characterized rebound responses in non-inflamed and inflamed tissue by comparing the effect of antagonists/blockers of putative (nitric oxide [NO], ATP, substance P, prostaglandins) and new (serotonin) neurotransmitters. METHODS: Strips from rabbits with colitis induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) were subjected to electrical field stimulation. Because rebound responses are more prominent under nonadrenergic noncholinergic (NANC) conditions, the effect of specific antagonists (N(omega)-nitro-L-arginine methyl ester (L-NAME), indomethacin, SR140333, methiothepin) on the rebound response was compared under normal and NANC conditions. RESULTS: NANC-conditions increased rebound responses in non-inflamed strips, but this effect was reduced or abolished in inflamed strips. Rebound responses were reduced by pretreatment with the NO-synthase inhibitor, L-NAME, under NANC conditions in non-inflamed strips but not affected in inflamed tissue. In contrast, the P(2) purine receptor antagonist, suramin, did not affect rebound responses in inflamed and non-inflamed strips. The effect of the cyclo-oxygenase inhibitor (COX), indomethacin, on rebound responses was reversed from excitatory to inhibitory by inflammation. Under NANC conditions rebound contractions were also reduced by the neurokinin-1 (NK(1)) antagonist, SR140333, both in normal and inflamed strips. The most pronounced reduction in rebound responses in inflamed and non-inflamed strips under normal conditions was observed with the 5-hydroxytryptamin (1,2) (5-HT(1,2)) antagonist, methiothepin. CONCLUSION: Rebound responses are mainly non-cholinergic and involve NO, substance P, serotonin and inhibitory prostaglandins. In inflamed tissue the nitrergic pathway is absent, excitatory prostaglandins prevail and the cholinergic and tachykinergic components are relatively more important. However there remains an important serotonergic contribution. Our data suggest that inflammation damages different neural pathways to a different extent and is most selective for nitrergic pathways.

Inactivation of the constitutively active ghrelin receptor attenuates limbic seizure activity in rodents.

Ghrelin is a pleiotropic neuropeptide that has been recently implicated in epilepsy. Animal studies performed to date indicate that ghrelin has anticonvulsant properties; however, its mechanism of anticonvulsant action is unknown. Here we show that the anticonvulsant effects of ghrelin are mediated via the growth hormone secretagogue receptor (GHSR). To our surprise, however, we found that the GHSR knockout mice had a higher seizure threshold than their wild-type littermates when treated with pilocarpine. Using both in vivo and in vitro models, we further discovered that inverse agonism and desensitization/internalization of the GHSR attenuate limbic seizures in rats and epileptiform activity in hippocampal slices. This constitutes a novel mechanism of anticonvulsant action, whereby an endogenous agonist reduces the activity of a constitutively active receptor.

The hepatocarcinoma cell line HepG2 does not express a GHS-R1a-type ghrelin receptor.

The interaction of ghrelin, a 28-residue acylated peptide, with the growth hormone secretagogue receptor 1a (GHS-R1a) has been studied mostly in cells expressing a recombinant GHS-R1a. As awareness is growing on the importance to study G protein-coupled receptors in a natural environment, we studied the effect of ghrelin in the human hepatocellular HepG2 cell line because it has been described in literature to respond to ghrelin. Despite extensive efforts, we were not able to confirm mRNA expression of GHS-R1a by reverse transcription PCR, radioligand binding, or ghrelin-induced GHS-R1a receptor activation; therefore, we conclude that HepG2 cells do not express GHS-R1a. On the other hand, we confirmed a modest effect of ghrelin on the up-regulation of IRS-1 phosphorylation, which might suggest the existence of an alternative ghrelin receptor in HepG2 cells.

Alterations in expression of p11 and SERT in mucosal biopsy specimens of patients with irritable bowel syndrome.

BACKGROUND & AIMS: The pathophysiology of irritable bowel syndrome (IBS) remains enigmatic; abnormalities in serotonin metabolism have been implicated. Two proteins that influence the function of serotonin and serotonergic receptors are serotonin transporter protein (SERT or soluble carrier protein, SLC6A4) and p11 (S-100A10, or calpactin I light chain). Both proteins are reported to be associated with depression-like states, a frequent comorbid condition in IBS. We explored the hypothesis that expression of these 2 proteins in colonic and rectal mucosa is abnormal in patients with IBS as compared with healthy controls. METHODS: Messenger RNA (mRNA) expression of SLC6A4 and p11 was measured in sigmoid and rectal mucosal biopsy specimens. Genotype of the promoter for SLC6A4 was also assessed in all participants. Validation studies explored reproducibility of 2 biopsy specimens taken from the same region and biopsy specimens taken an average of approximately 3 months apart. RESULTS: We found normal colonic mucosal expression of SLC6A4 in diarrhea (IBS-D)- or constipation-predominant IBS (IBS-C). On the other hand, p11 expression was increased in IBS. No significant effect on p11 mRNA expression in sigmoid colon or rectum was noted from antidepressant treatment in any of the analyzed subgroups. CONCLUSIONS: Colonic mucosal expression of SLC6A4 in IBS is normal. Given that overexpression of p11 can increase serotonergic receptor functions (eg, 5-HT(1B) receptors), these data support the need for further study of the interaction between p11 expression in health and disease and its role in the therapeutic response to serotonergic agents, including antidepressants.

Desensitization of the human motilin receptor by motilides.

Tachyphylaxis may have contributed to the failure of the motilide ABT-229 [N-ethyl, N-methyl 4'' deoxy erythromycin (EM)-B enolether] in clinical trials. We compared the desensitizing potency of structurally related motilides [EM-A, EM-A enolether (ME4), N-ethyl, N-methyl EM-A (ME36), EM-B enolether (ME67), N-ethyl, N-methyl EM-A enolether (EM523), ABT-229 and 4'' deoxy EM-A enolether (KOS1326)] in a Chinese hamster ovary (CHO)-K1 cell line expressing the human motilin receptor (MTLR) and in rabbit duodenal segments. CHO-MTLR cells were preincubated with motilides prior to stimulation with motilin. The negative logarithm of the preincubation concentration reducing the maximal motilin-induced Ca(2+) flux to 50% was calculated (pDC(50)). Internalization was visualized in CHO-K1 cells containing an enhanced green fluorescent protein (EGFP)-tagged MTLR and quantified in binding experiments. The contractile response of repeated stimulations was measured in duodenal segments. In CHO-MTLR cells, the pDC(50) was ABT-229 (8.78) > motilin (7.77) > EM-A (4.78), different from their order of potency to induce Ca(2+) release (pEC(50)): motilin (9.39) > ABT-229 (8.46) > EM-A (7.11). In cells with the EGFP-tagged MTLR, ABT-229 decreased membrane fluorescence by 25 +/- 2% compared with 16 +/- 2% for motilin and 8 +/- 2% for EM-A. Binding studies confirmed that EM-A did not induce MTLR internalization (residual binding 96 +/- 4% compared with motilin, 31 +/- 3% and ABT-229, 21 +/- 1%). Comparison of the pDC(50) and pEC(50) values of the other motilides ME4 (5.90; 8.08), ME67 (6.03; 8.12), ME36 (3.32; 6.62), EM-523 (6.02; 8.22), and KOS1326 (7.32; 8.14) suggested that the strong desensitizing properties of ABT-229 are mostly related to the removal of the 4''-OH of the cladinose sugar. The decline of the contractile response in duodenal segments correlated with the pDC(50). The ability to desensitize and internalize the MTLR is not only determined by potency. This may be an important criterion for the development of a clinically useful compound.

Interaction of the growth hormone-releasing peptides ghrelin and growth hormone-releasing peptide-6 with the motilin receptor in the rabbit gastric antrum.

The structural relationship between the motilin and the growth hormone secretagogue receptor (GHS-R), and between their respective ligands, motilin and ghrelin, prompted us to investigate whether ghrelin and the GHS-R agonist growth hormone-releasing peptide-6 (GHRP-6), could interact with the motilin receptor. The interaction was evaluated in the rabbit gastric antrum with binding studies on membrane preparations and with contraction studies on muscle strips in the presence of selective antagonists under conditions of electrical field stimulation (EFS) or not. Binding studies indicated that the affinity (pK(d)) for the motilin receptor was in the order of ghrelin (4.23 +/- 0.07) < GHRP-6 (5.54 +/- 0.08) < motilin (9.13 +/- 0.03). The interaction of ghrelin with the motilin receptor requires the octanoyl group. Motilin induced smooth muscle contractile responses but ghrelin and GHRP-6 were ineffective. EFS elicited on- and off-responses that were increased by motilin already at 10(-9) M, but not by 10(-5) M ghrelin. In contrast, GHRP-6 also enhanced the on- and off-responses. The motilin antagonist Phe-cyclo[Lys-Tyr(3-tBu)-betaAla-] trifluoroacetate (GM-109) blocked the effect of GHRP-6 on the off-responses but not on the on-responses. Under nonadrenergic noncholinergic conditions, the effects of motilin and GHRP-6 on the on-responses were abolished; those on the off-responses were preserved. All responses were blocked by neurokinin (NK)(1) and NK(2) antagonists. In conclusion, ghrelin is unable to induce contractions via the motilin receptor. However, GHRP-6 enhances neural contractile responses, partially via interaction with the motilin receptor on noncholinergic nerves with tachykinins as mediator, and partially via another receptor that may be a GHS-R subtype on cholinergic nerves that corelease tachykinins.

The motilin pharmacophore in CHO cells expressing the human motilin receptor.

We performed a structure-activity study with the human motilin receptor, which was recently cloned from thyroid tissue. N-terminal fragments, Ala-analogs of motilin, and motilides were tested in a cell line that expresses the cloned human motilin receptor and apoaequorin. Full potency to induce calcium fluxes was obtained with N-terminal fragments of 14 amino acids. Motilin fragments 1-14 in which residues 1 (Phe), 4 (Ile), and 7 (Tyr) were replaced by Ala showed the largest reduction in potency. Only motilides with an enol configuration had markedly higher potencies compared to erythromycin A. The potencies to induce Ca(2+) fluxes correlated strongly with rabbit binding and contractility data, suggesting that the cloned receptor is indeed the motilin receptor, responsible for contractile effects. Conservation of the motilin pharmacophore in evolution indicates an important physiological role of motilin.