Our current topics of research are: Large unstable repeat expansions Pathogenic mutations involving DNA repeat expansions are responsible for over 20 different neurodegenerative and neuromuscular diseases. All result from expanded tracts of repeated DNA sequences (microsatellites) that become unstable beyond a critical length, and lead to different pathologies in human. Our research is focused on the study of a subclass of such disorders; those that result from particularly large repeat expansions. Large unstable repeat expansions have several features in common: • reside within non-coding regions of genes • reveal extensive germline and somatic instability • embedded within CG-rich regions in the DNA (CpG islands) • are accompanied by abnormal DNA methylation and chromatin condensation • tend to increase with maternal transmission Our long term goal is to better understand how large repeat expansions lead to DNA methylation and heterochromatin induction, and how such alterations contribute to mis-expression of genes and to disease pathogenesis. • Fragile X Syndrome Fragile X syndrome (FXS) is the most common heritable form of cognitive impairment. It is caused by a deficiency in the fragile X mental retardation protein (FMRP). Most patients lack FMRP due to an unstable expansion of a CGG tri-nucleotide repeat number (200 to 4,000 copies), and in the 5'-untranslated region of the FMR1 gene. When the CGG expands, it results in local gain of aberrant DNA methylation and histone modifications in a developmentally regulated process. These changes lead to FMR1 gene inactivation and consequently to FMRP deficiency. Early in development, while the gene is still active, the CGG number tends to change, leading to a high degree of mosaicism for expansion size between and within tissues of affected individuals. This phenomenon, termed somatic instability, which contributes to disease severity and age of onset in other unstable repeat pathologies, is tightly correlated with FMR1 transcription and is lost with the aberrant acquisition of CpG methylation. The mechanism by which CGG expansions elicit hypermethylation and consequently result in gene inactivation is unclear. Nor are the mechanisms/factors that promote/regulate CGG repeat instability in FXS, due to the lack of appropriate animal and cellular models. The long term goal of our research is to resolve the mechanistic link between FMR1 transcription, epigenetic modifications and repeat instability. For this we are using a large set of human embryonic stem cell lines that we established from FXS affected embryos. The great advantage of these cell lines is that they offer the unique ability to investigate the mechanistic link between these factors by uncoupling these features from each other in ways that are not feasible in patient somatic cells. • Myotonic Dystrophy Type 1 Myotonic dystrophy type 1 (DM1) is the most common autosomal dominant myopathy in adults. It is characterized by myotonia, muscle wasting, cataracts, endocrine changes, and frequently by heart conduction defects (80% of patients), arrhythmias and cardiac myopathy. DM1 results from a tri-nucleotide CTG repeat expansion (50 to 4,000 copies) at the 3'-untranslated region of the dystrophia myotonica-protein kinase gene (DMPK). Several mechanisms have been proposed to underlie DM1 pathogenesis, yet no single model fully recapitulates the complexity of DM1 pathobiology. Previous studies have shown that CTG expansions lead to local changes in repressive epigenetic marks, including CpG methylation, in the DMPK gene region. However, the contribution of these alterations to gene transcription, and consequently to disease manifestation, remains unclear. The aim of our research is to determine when and how CTG expansions lead to aberrant DNA methylation at the 3'-end of DMPK during early embryo development. In addition, it aims to explore how such alterations contribute to DM1 pathogenesis, specifically in cardiac muscle cells. We are taking advantage of our large cohort of DM1-affecetd HESC lines, to address these unresolved questions. • C9/ALS-FTD Amyotrophic lateral sclerosis (ALS) and/or Frontotemporal dementia (FTD) is an autosomal dominant neurodegenerative disorder characterized by adult onset of one or both of these features in an affected individual. The leading cause for ALS-FTD is a G4C2 repeat expansion in the first intron of the C9orf72 gene, between noncoding exons 1a and 1b (24%–37% of familial and 6%–7% of sporadic cases in whites). The G4C2 expansion, when large enough (>30 repeat copies), coincides with hypermethylation of a CpG island near the repeats. Nevertheless, the contribution of this epigenetic change to disease pathogenesis is yet unclear. The long term goal of our research is to uncover how G4C2 repeat expansions trigger local CpG hypermethylation in C9orf72 gene during development, and explore how such epigenetic alterations contribute to ALS-FTD pathogenesis. Thus far we established two mutant HESC lines from preimplantation embryos with a G4C2 expansion at the C9orf72 gene. We intend to use these exceptional cell lines, in addition to iPS cells derived from patient's fibroblasts, to explore: when CpG hypermethylation is aberrantly acquired at the C9orf72 gene and, what is the consequence of aberrant hypermethylation on local gene transcription. • Dyskeratosis Congenita Dyskeratosis Congenita (DC) is a heritable condition characterized by premature ageing, bone marrow failure, and predisposition for cancer. It is caused by defects in the structures that protect the ends of the chromosomes, called telomeres, which are composed of long streches of DNA repeats (TTAGGG), and are normally maintained by telomerase, a cellular reverse transcriptase that adds telomere repeats to the ends of chromosomes. Some of the defects in DC may be introduced in the young embryo, and cause cancer only much later in life. The long-term goal of our research is to understand how telomeres are normally maintained during early human embryonic development, and how this process is compromised in DC embryonic cells. For this we are taking advantage of a unique cell resource; three human embryonic stem cell (HESC) lines that were established from in vitro fertilized human embryos that were found to carry a dominant mutation in the Telomerase gene (hTERT) by preimplantation genetic diagnosis. Our DC HESC lines provide an invaluable opportunity to explore how telomere maintenance and function are executed during early human development in health and disease, and understand how genomic defects occurring very early in life increase the risk for cancer as we grow up and age. The study of these processes will contribute to our understanding of premature ageing disorders and cancer development in general, and to our ability to develop approaches for early diagnosis and prevention.