X-linked Cornelia de Lange syndrome owing to SMC1L1 mutations.

Cornelia de Lange syndrome is a multisystem developmental disorder characterized by facial dysmorphisms, upper limb abnormalities, growth delay and cognitive retardation. Mutations in the NIPBL gene, a component of the cohesin complex, account for approximately half of the affected individuals. We report here that mutations in SMC1L1 (also known as SMC1), which encodes a different subunit of the cohesin complex, are responsible for CdLS in three male members of an affected family and in one sporadic case.

Cornelia de Lange syndrome mutations in SMC1A or SMC3 affect binding to DNA.

Cornelia de Lange syndrome (CdLS) is a clinically heterogeneous developmental disorder characterized by facial dysmorphia, upper limb malformations, growth and cognitive retardation. Mutations in the sister chromatid cohesion factor genes NIPBL, SMC1A and SMC3 are present in approximately 65% of CdLS patients. In addition to their canonical roles in chromosome segregation, the cohesin proteins are involved in other biological processes such as regulation of gene expression, DNA repair and maintenance of genome stability. To gain insights into the molecular basis of CdLS, we analyzed the affinity of mutated SMC1A and SMC3 hinge domains for DNA. Mutated hinge dimers bind DNA with higher affinity than wild-type proteins. SMC1A- and SMC3-mutated CdLS cell lines display genomic instability and sensitivity to ionizing radiation and interstrand crosslinking agents. We propose that SMC1A and SMC3 CdLS mutations affect the dynamic association between SMC proteins and DNA, providing new clues to the underlying molecular cause of CdLS.

Claspin inhibition leads to fragile site expression.

Fragile sites are hot spots for sister chromatid exchanges, translocations, deletions, complex rearrangements, and gene amplification. It has been hypothesized that rearrangements at fragile sites derive from unreplicated regions resulting from stalled forks that escape the ATR replication checkpoint. In the present study, we investigated the role of the Claspin (CLSPN) gene, which codes for an adaptor protein in the ATR pathway, during DNA replication stress in human cells. We show that the inhibition of the CLSPN gene leads to both genome instability and fragile site expression. Following aphidicolin treatment, we found a transient increase of Claspin synthesis due to its requirement to checkpoint activation. However, Claspin synthesis decreased after a prolonged aphidicolin treatment. We propose that CLSPN modulation, following an extreme replication block, allows rare cells to escape checkpoint mechanisms and enter mitosis with a defect in genome assembly. Our observations provide the basis for a better understanding of cell cycle checkpoints deregulation in cancer.

SMC1 inhibition results in FRA3B expression but has no effect on its delayed replication.

Cellular processes involved in fragile site expression have been investigated by studying the effect on the replication pattern of the commonest fragile site FRA3B of RNA interference (RNAi)-mediated sister maintenance chromosome 1 (SMC1) inhibition in normal human fibroblasts. Replication timing of FRA3B in G2 was studied by bromodeoxyuridine (BrdU) labeling for the final 2h of cell culture whereas in the S phase was investigated by a fluorescence in situ hybridization (FISH)-based approach through the analysis of clones spanning the FRA3B region. Results showed that FRA3B is normally late replicated even though it is not expressed in untreated cells. On the other hand, SMC1 inhibition leads to FRA3B expression even if the percent of late replicated cells is comparable to control cells. These results obtained by analysing the commonest fragile site suggest that SMC1 plays a role in protecting late replicating regions from stresses occurring in the final steps of genome replication and that delayed replication is necessary but not sufficient for inducing fragile site expression.

Chromosome transplantation as a novel approach for correcting complex genomic disorders.

Genomic disorders resulting from large rearrangements of the genome remain an important unsolved issue in gene therapy. Chromosome transplantation, defined as the perfect replacement of an endogenous chromosome with a homologous one, has the potential of curing this kind of disorders. Here we report the first successful case of chromosome transplantation by replacement of an endogenous X chromosome carrying a mutation in the Hprt genewith a normal one in mouse embryonic stem cells (ESCs), correcting the genetic defect. The defect was also corrected by replacing the Y chromosome with an X chromosome. Chromosome transplanted clones maintained in vitro and in vivo features of stemness and contributed to chimera formation. Genome integrity was confirmed by cytogenetic and molecular genome analysis. The approach here proposed, with some modifications, might be used to cure various disorders due to other X chromosome aberrations in induced pluripotent stem (iPS) cells derived from affected patients.

Targeted Gene Correction in Osteopetrotic-Induced Pluripotent Stem Cells for the Generation of Functional Osteoclasts.

Autosomal recessive osteopetrosis is a human bone disease mainly caused by TCIRG1 gene mutations that prevent osteoclasts resorbing activity, recapitulated by the oc/oc mouse model. Bone marrow transplantation is the only available treatment, limited by the need for a matched donor. The use of induced pluripotent stem cells (iPSCs) as an unlimited source of autologous cells to generate gene corrected osteoclasts might represent a powerful alternative. We generated iPSCs from oc/oc mice, corrected the mutation using a BAC carrying the entire Tcirg1 gene locus as a template for homologous recombination, and induced hematopoietic differentiation. Similarly to physiologic fetal hematopoiesis, iPSC-derived CD41(+) cells gradually gave rise to CD45(+) cells, which comprised both mature myeloid cells and high proliferative potential colony-forming cells. Finally, we differentiated the gene corrected iPSC-derived myeloid cells into osteoclasts with rescued bone resorbing activity. These results are promising for a future translation into the human clinical setting.

SMC1 involvement in fragile site expression.

Common fragile sites have been involved in neoplastic transformation, although their molecular basis is still poorly understood. Here, we demonstrate that inhibition of the SMC1 by RNAi is sufficient to induce fragile site expression. By investigating normal, ATM- and ATR-deficient cell lines, we provide evidence that the contribution of SMC1 in preventing the collapse of stalled replication fork is an Atr-dependent pathway. Using a fluorescent antibody specific for gamma-H2AX, we show that very rare discrete nuclear foci appear 1 and 2 h after exposure to aphidicolin and/or RNAi-SMC1, but became more numerous and distinct after longer treatment times. In this context, fragile sites might be viewed as an in vitro phenomenon originating from double-strand breaks formed because of a stalled DNA replication that lasted too long to be managed by physiological rescue acting through the Atr/Smc1 axis. We propose that in vivo, following an extreme replication block, rare cells could escape checkpoint mechanisms and enter mitosis with a defect in genome assembly, eventually leading to neoplastic transformation.