UNIVERSITY OF CINCINNATI Date: 28-May-2010 I, Emily Sites , hereby submit this original work as part of the requirements for the degree of: Master of Science in Genetic Counseling It is entitled: Copy Number Variation in Monozygotic Twins with NF1 Student Signature: Emily Sites This work and its defense approved by: Committee Chair: Lisa Martin, PhD Lisa Martin, PhD Teresa Smolarek, PhD Teresa Smolarek, PhD Elizabeth Schorry, MD Elizabeth Schorry, MD 5/27/2010 808 Copy Number Variation in Monozygotic Twins with NF1 Emily Sites M.S. University of Cincinnati December, 2008 University of Cincinnati Genetic Counseling Graduate Program College of Medicine Master’s Degree Thesis May 28th, 2010 Thesis Committee: Elizabeth Schorry, M.D. Teresa Smolarek, Ph.D. Lisa Martin, Ph.D. (Chair) Emily Sites Genetic Counseling Graduate Program Abstract for master’s thesis: Copy Number Variation in Monozygotic Twins with NF1 May 28th, 2010 General Abstract A major challenge of managing patients with Neurofibromatosis Type 1 (NF1) is the extreme variability of its phenotype, with no way to predict which patients are at high risk for serious complications. Family members and even monozygotic (MZ) twins with NF1 demonstrate inconsistent expression of the disease. The underlying mechanism for this discordance has never been elucidated. We propose that DNA copy number variants (CNVs), small deletions and duplications of genomic material, may contribute to the variability of disease manifestation. CNVs are known to differ between MZ twins and have recently been implicated in the etiology of several disorders including autism and schizophrenia Hypothesis: MZ twins with NF1 will have within-pair differences in CNVs that may explain their discordant NF1 complications, and CNVs will be present in larger numbers in NF1 patients compared to the general population. Methods: MZ twins with NF1, ages 5 to 18 years, were recruited from the CCHMC NF clinic population. Extensive data was collected for each twin’s NF1 features and complications. The Illumina 610k SNP microarray chip was used to identify and compare CNVs in peripheral blood from the twins and their parents. Age-matched controls were selected from a pre-existing CNV study population. Results: Of the five twin pairs reported here, three were discordant for optic pathway glioma, three for number of plexiform neurofibromas, one for pectus deformity, one for scoliosis, one for malignancy, and one pair was concordant for all parameters. We identified 43 CNVs meeting our conservative criteria, 18 of which overlap known or predicted genes. The average number of CNVs per twin pair was 8.6 with a range from 3 to 12. Of interest were five previously unreported areas of copy number change, two of which contain genes. We have yet to identify a de novo (non-familial) CNV. A larger study population would be needed to identify a correlation between familial CNVs and specific complications. Conclusions: Additional data is needed to determine if there is a correlative relationship between CNVs and NF1 phenotype. iii iv TABLE OF CONTENTS TITLE PAGE Introduction 1 Methods 6 Results 8 Discussion 12 Tables 18 Figures 22 References 24 v INTRODUCTION Neurofibromatosis Type 1 (NF1) is a tumor predisposition syndrome that affects one in thirty-five hundred live births. Individuals with NF1 are at an increased risk of developing both benign and malignant tumors. Other characteristics of the disease include café au lait spots, Lisch nodules of the iris, optic pathway gliomas, scoliosis, pectus deformities, other bone and vascular abnormalities and learning disabilities (Friedman and Birch 1997). Neurofibromatosis Type 1 was first described in 1882, and the gene responsible was cloned in 1990 (Ober 1978; Wallace, Marchuk et al. 1990). The 350kb NF1 gene is located at 17q11.2 (Daston, Scrable et al. 1992). One of the most challenging aspects of managing patients with NF1 is the extreme variability of phenotype. Currently, there is no way to predict which patients are at high risk for serious complications like malignancy, and to appropriately manage those at higher risk. Family members and even monozygotic (MZ) twins with NF1 demonstrate inconsistent expression of the disease. The underlying mechanism for this discordance has never been elucidated, although several have been proposed. These include: modifying genes, stochastic “2nd hit” events, environmental agents, epigenetic alterations, and post-zygotic (somatic) mutations. Despite extensive research, little evidence has been found to identify these factors or events. The search for other modifying genes has yielded little success to date. Second- hit, loss-of-heterozygosity events have been well-documented in several tumor types of NF1 and in at least one type of skeletal complication (Stevenson, Zhou et al. 2006), but are unlikely to explain the entire spectrum of NF1 features. Indeed, in NF1 1 neurofibromas, less than 50% of tumors have been found to have 2nd hit events in the NF1 gene, implying an additional mechanism at play (Wiest, Eisenbarth et al. 2003). Full gene deletions of NF1 can be associated with a more severe phenotype and higher tumor burden (Upadhyaya, Ruggieri et al. 1998), but no other significant genotype-phenotype correlations have been found. This full gene deletion, also referred to as NF1 microdeletion, occurs in only 5-10% of patients and predisposes individuals to more severe learning disabilities and facial dysmorphology, earlier or greater burden of cutaneous neurofibromas and possibly malignancy (Lopez Correa, Brems et al. 2000). It has been suggested that the severity of NF1 microdeletion is due to the concomitant loss of a group of genes adjacent to the NF1 gene, including CENTA2, RAB11FIP4, C17orf79, and UTP6 (Bartelt-Kirbach, Wuepping et al. 2009), but this has not been demonstrated conclusively. Twin studies have historically been a valuable tool for studying genetic disorders. Early genetic studies compared monozygotic twins raised separately to determine heritability of specific traits. The literature reports at least 24 pairs of proven MZ twins with NF1 [see References, Case Studies]. Easton et al. (Easton, Ponder et al. 1993) studied a large NF1 population, and found that NF1 features varied to a greater degree with increasing distance from a proband, with 6 pairs of MZ twins having the closest agreement in traits such as presence of neurofibromas, head circumference, and learning disabilities, compared to more distant relatives with NF1. They concluded that there was significant evidence for modifying loci affecting NF1 expression. Most other twin reports have been case reports. Some have shown remarkable concordance for NF1 features, although others have shown equally intriguing 2 discordancies. Most MZ twin pairs have been generally concordant for overall numbers of café-au-lait spots and cutaneous neurofibromas, implying control by genetic factors, although not solely by the NF1 gene. Presence and location of plexiform neurofibromas has generally been discordant in twin pairs, as expected if these tumors required a random, second-hit event. There have been surprising reports of concordance in other NF1 tumors such as optic nerve glioma (ONG; 6 pairs concordant, 5 pairs discordant) (Cartwright 1982; Crawford and Buckler 1983; Pascual-Castroviejo, Verdu et al. 1988; Kelly, Sproul et al. 1998), more than expected from the prevalence of ONG in 15% of children with NF1. Although MZ twins were historically assumed to be genetically identical, recent studies have shown remarkable differences in MZ twins in areas such as copy number variants (CNV)(Bruder, Piotrowski et al. 2008). Copy number variants are microduplications or deletions of DNA that are typically defined as being larger than 1 kilobase (kb), and now known to be widespread throughout the human genome (Scherer, Lee et al. 2007). There is mounting evidence that CNVs have played a significant role in normal population variation, evolution, and disease predisposition (Wong, deLeeuw et al. 2007; McCarroll, Kuruvilla et al. 2008). Genes involved in immunity and defense, sensory perception, and cell adhesion have been found to be associated with CNVs (Conrad, Andrews et al. 2006). Significant CNV associations have been found in schizophrenia and autism (Sebat, Lakshmi et al. 2007; Xu, Roos et al. 2008). Some CNVs represent recurrent polymorphisms occurring at a low rate in the population and have no known clinical significance. However, more than 50% of CNVs occur in areas of known genes and could potentially cause significant phenotypic effect 3 (Iafrate, Feuk et al. 2004). The average individual in the general population will be found to have at least 10-50 CNVs using current SNP-based microarrays, with the majority of those (>90%) being familial (Iafrate, Feuk et al. 2004; Conrad, Andrews et al. 2006; McCarroll, Kuruvilla et al. 2008). The incidence of CNVs depends greatly on methodology used, and it is likely that the numbers demonstrated to date represent only the tip of the iceberg. De novo CNVs are those found in a proband, but neither parent, and in our case perhaps only one twin. These are thought to arise either in a germ cell or in the early embryo. If a CNV were present in only one of a pair of MZ twins, we would expect that the change occurred sometime after division of the 2 embryos. De novo CNVs which occur during post-fertilization may have a mosaic distribution thoughout the body. This could potentially be detected by sampling tissues representative of multiple germ layers. We sampled both blood (mesoderm) and buccal (ectoderm) cells to investigate this possibility. De novo CNVs are generally thought to be a likely cause of phenotypic abnormality. Bruder et al. studied whole-genome CNV in MZ twin pairs discordant for Parkinsonian disease and compared them to CNVs in unselected and concordant MZ twin pairs. They found multiple CNVs in all twin pairs, most in mosaic form, and concluded that these differences represented an example of somatic mosaicism. This group utilized both the Illumina 300k SNP array and 32K BAC array platforms and were reportedly able to detect CNVs with as low as 10% mosaicism.