SummaryReproducibility in molecular and cellular studies is fundamental to scientific discovery. To establish the reproducibility of a well-defined long-term neuronal differentiation protocol, we repeated the cellular and molecular comparison of the same two iPSC lines across five distinct laboratories. Despite uncovering acceptable variability within individual laboratories, we detect poor cross-site reproducibility of the differential gene expression signature between these two lines. Factor analysis identifies the laboratory as the largest source of variation along with several variation-inflating confounders such as passaging effects and progenitor storage. Single-cell transcriptomics shows substantial cellular heterogeneity underlying inter-laboratory variability and being responsible for biases in differential gene expression inference. Factor analysis-based normalization of the combined dataset can remove the nuisance technical effects, enabling the execution of robust hypothesis-generating studies. Our study shows that multi-center collaborations can expose systematic biases and identify critical factors to be standardized when publishing novel protocols, contributing to increased cross-site reproducibility.

Mutations have been identified in four patients, independently classified as congenital Rett variants from France, Spain and Latvia. Clinical data have been compared with the two previously reported patients with mutations in FOXG1. In all cases hypotonia, irresponsiveness and irritability were present in the neonatal period. At birth, head circumference was normal while a deceleration of growth was recognised soon afterwards, leading to severe microcephaly. Motor development was severely impaired and voluntary hand use was absent. In contrast with classic Rett, patients showed poor eye contact. Typical stereotypic hand movements with hand washing and hand mouthing activities were present continuously. Some patients showed abnormal movements of the tongue and jerky movements of the limbs. Brain magnetic resonance imaging showed corpus callosum hypoplasia in most cases, while epilepsy was a variable sign. Scoliosis was present and severe in the older patients. Neurovegetative symptoms typical of Rett were frequently present.

Rett syndrome (RTT) is a progressive neurologic disorder representing one of the most common causes of mental retardation in females. To date mutations in three genes have been associated with this condition. Classic RTT is caused by mutations in the MECP2 gene, whereas variants can be due to mutations in either MECP2 or FOXG1 or CDKL5. Mutations in CDKL5 have been identified both in females with the early onset seizure variant of RTT and in males with X-linked epileptic encephalopathy. CDKL5 is a kinase protein highly expressed in neurons, but its exact function inside the cell is unknown. To address this issue we established a human cellular model for CDKL5-related disease using the recently developed technology of induced pluripotent stem cells (iPSCs). iPSCs can be expanded indefinitely and differentiated in vitro into many different cell types, including neurons. These features make them the ideal tool to study disease mechanisms directly on the primarily affected neuronal cells. We derived iPSCs from fibroblasts of one female with p.Q347X and one male with p.T288I mutation, affected by early onset seizure variant and X-linked epileptic encephalopathy, respectively. We demonstrated that female CDKL5-mutated iPSCs maintain X-chromosome inactivation and clones express either the mutant CDKL5 allele or the wild-type allele that serve as an ideal experimental control. Array CGH indicates normal isogenic molecular karyotypes without detection of de novo CNVs in the CDKL5-mutated iPSCs. Furthermore, the iPS cells can be differentiated into neurons and are thus suitable to model disease pathogenesis in vitro. INTRODUCTIONRett syndrome (RTT) is a progressive neurological disorder that affects 1 in 10 000 girls worldwide and represents one of the most common causes of mental retardation in females. RTT is characterized by an apparently normal development for the first 6-18 months of life, followed by regression with the onset of clinical signs including mental retardation, loss of speech, acquired microcephaly, growth retardation, autistic features, seizures, ataxia and hand stereotypies. 1 Beside the classic form, several RTT variants have been described including the Zappella variant, the congenital form, the 'forme fruste' and the early onset seizures variant. [2][3][4][5] In past years, mutations in three genes have been associated with classic and/or variant RTT: MECP2 and CDKL5, located on the X chromosome, and FOXG1, on chromosome 14. 1,6,7 About 80% of classic RTT cases are caused by mutations in MECP2 that encodes for a methyl-CpG-binding protein involved in the regulation of gene expression. 8,9 To investigate the molecular mechanisms leading from MECP2 mutations to RTT onset, different mouse models have been generated and extensively characterized. 10-13 These models allowed identification of specific alterations in glutamatergic neurons: 13 cells lacking Mecp2 have reduced synapse numbers and, accordingly, they show a reduced synaptic response. The opposite effects are elicited by Mecp2 over-expression....

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In retinoblastoma, two RB1 mutations are necessary for tumor development. Recurrent genomic rearrangements may represent subsequent events required for retinoblastoma progression. Array-comparative genomic hybridization was carried out in 18 eye samples, 10 from bilateral and eight from unilateral retinoblastoma patients. Two unilateral cases also showed areas of retinoma. The most frequent imbalance in retinoblastomas was 6p gain (40%), followed by gains at 1q12-q25.3, 2p24.3-p24.2, 9q22.2, and 9q33.1 and losses at 11q24.3, 13q13.2-q22.3, and 16q12.1-q21. Bilateral cases showed a lower number of imbalances than unilateral cases (P = 0.002). Unilateral cases were divided into low-level (£4) and high-level (³7) chromosomal instability groups. The first group presented with younger age at diagnosis (mean 511 days) compared with the second group (mean 1606 days). In one retinoma case ophthalmoscopically diagnosed as a benign lesion no rearrangements were detected, whereas the adjacent retinoblastoma displayed seven aberrations. The other retinoma case identified by retrospective histopathological examination shared three rearrangements with the adjacent retinoblastoma. Two other gene-free rearrangements were retinoma specific. One rearrangement, dup5p, was retinoblastoma specific and included the SKP2 gene. Genomic profiling indicated that the first retinoma was a pretumoral lesion, whereas the other represents a subclone of cells bearing 'benign' rearrangements overwhelmed by another subclone presenting aberrations with higher 'oncogenic' potential. In summary, the present study shows that bilateral and unilateral retinoblastoma have different chromosomal instability that correlates with the age of tumor onset in unilateral cases. This is the first report of genomic profiling in retinoma tissue, shedding light on the different nature of lesions named 'retinoma'. (Cancer Sci 2009; 100: 465-471) R etinoblastoma (RB, OMIM#180200) is the most common primary intraocular malignancy in children, initiated by the inactivation of both alleles of the RB1 tumor-suppressor gene. (1,2) Approximately 40% of RB patients carry a predisposing germline mutation transmitted as an autosomal-dominant trait. In these patients, inactivation of the second RB1 allele occurs in the retinal cells and generally results in multiple and often bilateral tumors. In the remaining 60% of children, both mutational events occur in the same retinal cell leading to unilateral sporadic RB. Retinoma (RN), a benign retinal lesion, is considered to be the precursor of RB.(4,5) Unlike RB, which is typically opaque white, RN appears as a translucent gray retinal mass, frequently associated with calcification and retinal pigment epithelial hyperplasia. (6) The histopathology of RN includes foci of photoreceptor differentiation (fleurettes), momomorphic round nuclei, abundant fibrillar eosinophilic stroma, and absence of mitotic activity.Recently, it has been demonstrated that the two mutational events inactivating the RB1 gene are already present in RN.(4) U...

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Mutations in the Forkhead box G1 (FOXG1) gene, a brain specific transcriptional factor, are responsible for the congenital variant of Rett syndrome. Until now FOXG1 point mutations have been reported in 12 Rett patients. Recently seven additional patients have been reported with a quite homogeneous severe phenotype designated as the FOXG1 syndrome. Here we describe two unrelated patients with a de novo FOXG1 point mutation, p.Gln46X and p.Tyr400X, respectively, having a milder phenotype and sharing a distinctive facial appearance. Although FoxG1 action depends critically on its binding to chromatin, very little is known about the dynamics of this process. Using fluorescence recovery after photobleaching, we showed that most of the GFP-FoxG1 fusion protein associates reversibly to chromatin whereas the remaining fraction is bound irreversibly. Furthermore, we showed that the two pathologic derivatives of FoxG1 described in this paper present a dramatic alteration in chromatin affinity and irreversibly bound fraction in comparison with Ser323fsX325 mutant (associated with a severe phenotype) and wild type Foxg1 protein. Our observations suggest that alterations in the kinetics of FoxG1 binding to chromatin might contribute to the pathological effects of FOXG1 mutations.

We present a social learning experiment in which subjects predict the value of a good in sequence. We elicit each subject's belief twice: …rst ("…rst belief"), after he observes his predecessors' prediction; second, after he also observes a private signal. Our main result is that subjects update on their signal asymmetrically. They weigh the private signal as a Bayesian agent when it con…rms their …rst belief and overweight it when it contradicts their …rst belief. This way of updating, incompatible with Bayesianism, can be explained by ambiguous beliefs (multiple priors on the predecessor's rationality) and a generalization of the Maximum Likelihood Updating rule. Our experiment allows for a better understanding of the overweighting of private information documented in previous studies.