1. Fragile X Laboratory Testing: Background and Quality Improvement Opportunities (part 1 of 2) Elaine Lyon, Ph.D. University of Utah/ARUP Laboratories Association for Molecular Pathology, Chair, Clinical Practice Committee 1

2. Outline Clinical Features of Fragile X Molecular Basis of Disease Molecular Testing/Interpretation Opportunities for Improvement Quality control material Interlaboratory study (AMP, CDC, NIST) 2

3. Fragile X Syndrome Most common inherited form of mental retardation. Incidence 1:4000 males and 1:8000 females. Affected males have mental retardation, characteristic physical features and behavior. Affected females exhibit a less severe phenotype. Found in all populations. 3

4. Features Mental impairment Attention deficit/autistic-like Long, thin face - prominent forehead Large ears Flexible joints Low muscle tone Enlarged testicles Jones KL. Smith’s Recognizable Patterns of Human Malformation, 4 th Ed. 4

5. Folate-sensitive fragile site at Xq27.3 (FRAXA). Other sites: FRAXD, FRAXE, & FRAXF. Chromosome Level 5

6. Molecular Level Tri Nucleotide Repeat (CGG) at the 5' Untranslated Region (UTR). A small expansion (pre-mutation) associated with increased mRNA A large expansion associated with methylation, inactivating gene expression. 6

7. Molecular Schematic 7

8. Protein Level Normal – protein widely expressed (nerve, brain, etc.) RNA binding protein Pre-mutation – normal protein, increased mRNA Full mutation – no protein produced Protein expression by immunohistochemistry (IHC) suspected deletions/point mutations in males 8

9. Transmission Female pre-mutation carriers 50/50 chance of transmitting unstable allele May stay within pre-mutation range May expand to full mutation (higher pre-mutations more likely to fully expand in one generation) Male pre-mutation carriers Will transmit pre-mutation to all daughters Unlikely to expand Intermediate May expand to pre-mutation, but not full mutation, in one generation 9

10. Risk of Expansion by Pre-mutation Size Number of Maternal Pre-Mutation CGG Repeats Approximate % Risk that a Son Will be Affected with Fragile X Syndrome 56-597% 60-6910% 70-7929% 80-8936% 90-9947% > 10050% Adapted originally from Warren & Nelson 1994; modified according to Nolin et al. 1996. GENEReviews at www.genetests.org, FMR1-Related Disorders.www.genetests.org 10

11. Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) Symptoms Late-onset, progressive cerebellar ataxia/intention tremor Short-term memory loss, executive function deficits, cognitive decline Lower-limb proximal muscle weakness, and autonomic dysfunction Genetics FMR1 pre-mutation mRNA accumulation 11

12. Allingham-Hawkins, AJMG, 1999 12 Premature Ovarian Failure (POF) Cessation of menses before age 40 21% of females with pre- mutations