The centrosomal/basal body protein OFD1 is required for microtubule organization and cell cycle progression.

Oral-Facial-Digital type I (OFD1) is a rare inherited form of renal cystic disease associated with ciliary dysfunction. This disorder is due to mutations in the OFD1 gene that encodes a protein localized to centrosomes and basal bodies in different cell types. Immunofluorescence analysis demonstrated that OFD1 displays a dynamic distribution during cell cycle. High-content microscopy analysis of Ofd1-depleted fibroblasts revealed impaired cell cycle progression. Immunofluorescence analysis and cell proliferation assays also indicated the presence of a variety of defects such as centrosome accumulation, nuclear abnormalities and aneuploidy. In addition, Ofd1-depleted cells displayed an abnormal microtubule network that may underlie these defects. All together our results suggest that OFD1 contributes to the function of the microtubule organizing center (MTOC) in the cell, controlling cell cycle progression both in vitro and in vivo.

Characterization of the most frequent ATP7B mutation causing Wilson disease in hepatocytes from patient induced pluripotent stem cells.

H1069Q substitution represents the most frequent mutation of the copper transporter ATP7B causing Wilson disease in Caucasian population. ATP7B localizes to the Golgi complex in hepatocytes but moves in response to copper overload to the endo-lysosomal compartment to support copper excretion via bile canaliculi. In heterologous or hepatoma-derived cell lines, overexpressed ATP7B-H1069Q is strongly retained in the ER and fails to move to the post-Golgi sites, resulting in toxic copper accumulation. However, this pathogenic mechanism has never been tested in patients' hepatocytes, while animal models recapitulating this form of WD are still lacking. To reach this goal, we have reprogrammed skin fibroblasts of homozygous ATP7B-H1069Q patients into induced pluripotent stem cells and differentiated them into hepatocyte-like cells. Surprisingly, in HLCs we found one third of ATP7B-H1069Q localized in the Golgi complex and able to move to the endo-lysosomal compartment upon copper stimulation. However, despite normal mRNA levels, the expression of the mutant protein was only 20% compared to the control because of endoplasmic reticulum-associated degradation. These results pinpoint rapid degradation as the major cause for loss of ATP7B function in H1069Q patients, and thus as the primary target for designing therapeutic strategies to rescue ATP7B-H1069Q function.

MePR: a novel human mesenchymal progenitor model with characteristics of pluripotency.

Human embryo stem cells or adult tissues are excellent models for discovery and characterization of differentiation processes. The aims of regenerative medicine are to define the molecular and physiological mechanisms that govern stem cells and differentiation. Human mesenchymal stem cells (hMSCs) are multipotent adult stem cells that are able to differentiate into a variety of cell types under controlled conditions both in vivo and in vitro, and they have the remarkable ability of self-renewal. hMSCs derived from amniotic fluid and characterized by the expression of Oct-4 and Nanog, typical markers of pluripotent cells, represent an excellent model for studies on stemness. Unfortunately, the limited amount of cells available from each donation and, above all, the limited number of replications do not allow for detailed studies. Here, we report on the immortalization and characterization of novel mesenchymal progenitor (MePR) cell lines from amniotic fluid-derived hMSCs, whose biological properties are similar to primary amniocytes. Our data indicate that MePR cells display the multipotency potential and differentiation rates of hMSCs, thus representing a useful model to study both mechanisms of differentiation and pharmacological approaches to induce selective differentiation. In particular, MePR-2B cells, which carry a bona fide normal karyotype, might be used in basic stem cell research, leading to the development of new approaches for stem cell therapy and tissue engineering.

Contiguous gene syndrome due to an interstitial deletion in Xp22.3 in a boy with ichthyosis, chondrodysplasia punctata, mental retardation and ADHD.

Microdeletions of Xp22.3 can result in contiguous gene syndromes, showing the variable association of apparently unrelated clinical manifestations such as ichthyosis, chondrodysplasia punctata, hypogonadotropic hypogonadism, anosmia, ocular albinism, short stature and mental retardation. We report on a boy with ichthyosis, dysmorphic features and mental retardation with ADHD. The patient was born at term after a pregnancy complicated by threatened abortion; decreased fetal movements and low estriol serum levels were reported during the last trimester. The boy was referred to us at the age of 13 years. He presented with aggressive and hyperactive behavior. He had dry hair, a flat face, bilateral lens opacities, a small nose with hypoplastic tip, alae nasi and nares, a high-arched palate with a very small cleft, mixed dentition with 7 unerupted permanent teeth, left sensorineural and right mixed hearing loss with a calcified plaque of the tympanic membrane, marked shortness of terminal phalanges of hands and feet, ichthyosis of trunk and limbs. The genomic interval between AFM248th5 and KAL1 was investigated. PCR analysis showed a deletion in Xp22.3, with the distal breakpoint between the marker AFM248th5 and PABX and the proximal one between DXS278 and KAL1. Array-CGH and FISH analysis confirmed the interstitial deletion (of about 5.5 Mb) and refined the breakpoints. We discuss the phenotype of our patient in relationship to the deleted segment and the possibility of mental retardation and ADHD genes in the region.

Mosaic 13q13.2-ter deletion restricted to tissues of ectodermal and mesodermal origins.

The '13q-' syndrome shows widely variable manifestations. Investigation of the involvement of different tissues has never been reported in patients with 13q- syndrome previously. We describe a patient with mosaicism for del(13q) and clinical features of 13q- syndrome. The mother of the patient was professionally exposed to aniline colorants and glue components during the whole pregnancy. The patient had dysmorphic features, skeletal anomalies and brain malformations with agenesis of the corpus callosum, vermian hypoplasia and IVth ventricular system abnormalities. Eye examination revealed chorioretinal coloboma and irregular dispersion of retinal pigment in the right eye. The karyotype analyses and the molecular studies performed on peripheral lymphocytes, oral swab and cells of urinary tract were normal whereas a deletion of the long arm of chromosome 13 (13q13.2) was found in skin fibroblasts and in hair cells. We hypothesized that the 13q deletion arose during the third week after conception possibly due to a teratogenic effect and that tissue of mesodermal and ectodermal origin are involved. We suggest analysing a fibroblast karyotype when a diagnosis of 13q- syndrome is suspected on clinical ground. The role of teratogens in causing this type of mosaic chromosome abnormality also warrants further investigation.