Fragile X Fragile X Laboratory Testing:Laboratory Testing:Background and Quality Background and Quality

Improvement OpportunitiesImprovement Opportunities(part 1 of 2)(part 1 of 2)

Elaine Lyon, Ph.D.University of Utah/ARUP Laboratories

Association for Molecular Pathology, Chair, Clinical Practice Committee

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OutlineOutline

Clinical Features of Fragile X Molecular Basis of Disease Molecular Testing/Interpretation Opportunities for Improvement

Quality control material Interlaboratory study (AMP, CDC, NIST)

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Fragile X SyndromeFragile 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.

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FeaturesFeatures 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

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

Chromosome LevelChromosome Level

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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.

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Molecular SchematicMolecular Schematic

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Protein LevelProtein 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

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TransmissionTransmission

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

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Risk of Expansion byRisk of Expansion byPre-mutation Size Pre-mutation Size

Number of Maternal Pre-Mutation CGG Repeats

Approximate % Risk that a Son Will be Affected with Fragile X Syndrome

56-59 7%

60-69 10%

70-79 29%

80-89 36%

90-99 47%

> 100 50%

Adapted originally from Warren & Nelson 1994; modified according to Nolin et al. 1996.

GENEReviews at www.genetests.org, FMR1-Related Disorders.

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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

Jacquemont, JAMA 2004

Penetrance is age related

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Distribution of Subjects

020406080

100120140160180

18 - 29 30 - 39 40 - 49 50+

Age at Interview

Nu

mb

er

of

Su

bje

cts

Full Mutation

Premutation

Control

Proportion of Subjects Experiencing POF

0 0 0 0

0.05

0.11

0.22

0.26

0.020 0 0

0

0.05

0.1

0.15

0.2

0.25

0.3

18 - 29 30 - 39 40 - 49 50+

Age at Interview

Pro

po

rtio

n o

f S

ub

ject

s

Allingham-Hawkins, AJMG, 199912

Premature Ovarian Failure (POF)

Cessation of menses before age 40

21% of females with pre-mutations