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Teresa Lamb Clinical Scientist Leeds DNA Laboratory

Implications of Consanguinity for Routine Diagnostic Testing and Development of Specialist Services. Teresa Lamb Clinical Scientist Leeds DNA Laboratory. Outline. Relevance of consanguinity to diagnostic molecular genetic laboratories Routine testing Autosomal recessive disorders

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Teresa Lamb Clinical Scientist Leeds DNA Laboratory

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  1. Implications of Consanguinity for Routine Diagnostic Testing and Development of Specialist Services Teresa Lamb Clinical Scientist Leeds DNA Laboratory

  2. Outline • Relevance of consanguinity to diagnostic molecular genetic laboratories • Routine testing • Autosomal recessive disorders • Risk calculations • Specialist service design and provision • Choice of screening strategy • Problems and pitfalls

  3. Relevance of Consanguinity • Diverse populations served by each lab • Range of ethnic groups practice consanguineous unions • Consanguinity may alter testing strategy and/or interpretation of results

  4. Testing for Autosomal Recessive Disorders • Atypical, rare or private mutation • Usual screening strategy may have lower sensitivity • Negative result may not significantly reduce likelihood of diagnosis • Need for additional screening (availability/cost) • Affecteds expected to be homozygous • Confirmation of homozygosity • Autozygosity analysis may be of use

  5. Cystic Fibrosis • Complex multi-system disorder that may affect the respiratory, pancreatic, gastro-intestinal and reproductive organ systems. • Incidence 1/2,500 (Caucasians) • Less frequent in other populations • Carrier Frequency • 1/20 - 1/25 (Caucasians) • Mutations in CFTR gene • >1600 different mutations reported

  6. Cystic Fibrosis • Initial screening for 29-32 mutations • 80-90% mutations (Caucasian) • No mutation is detected • Reduces likelihood (~2% affected CF patients would give this result) • Single mutation detected • Increases likelihood (but doesn’t confirm) • However, if consanguineous: • % mutations detected <80% • Standard interpretation of negative result inaccurate • Potential homozygosity for rarer mutation in kit • Confirmation of atypical result • Testing of parental samples • Interpretation of heterozygosity

  7. Cystic Fibrosis • Additional studies in consanguineous pedigrees • Linked markers • Autozygosity analysis • Support or exclude autozygous inheritance • Full sequencing • Implications for neonatal screening • 4 most common Caucasian mutations screened only • 2nd raised IRT result for “high likelihood”/clinical referral

  8. Spinal Muscular Atrophy (SMA) • Degeneration and loss of the proximal anterior horn cells in the spinal cord • Muscle wasting and atrophy • Incidence: 1/10,000 • Carrier frequency: 1/50 • SMN1 gene • >95% homozygous for deletion exon 7 (most exon 8 also deleted) • Compound hets deletion/point mutation

  9. Spinal Muscular Atrophy (SMA) • First level test: screen for deletion of SMN1 exon 7 and exon 8 • If no deletion detected report as diagnosis “highly unlikely” or “extremely unlikely” • However, if consanguineous • Possible homozygosity for point mutation • More cautious interpretation • “reduces likelihood but cannot exclude a diagnosis” • Linkage/autozygosity analysis • Screen for point mutations - no UK lab offering?

  10. Risk Calculations • Coefficient Inbreeding (F) - probability that child of consanguineous union will be homozygous for allele derived from common ancestor • Coefficient of Relationship (R) - proportion of genes shared by related individuals • F = R x 1/2 • If one parent transmits a particular allele what is probability the other parent will transmit same allele

  11. FirstCousins: FirstCousins: FirstCousins: FirstCousins: R=1/8 R=1/8 R=1/8 R=1/8 Risk Calculations

  12. Risk Calculations FirstCousins Once Removed: R=1/16

  13. Risk Calculations SecondCousins: R=1/32

  14. Risk Calculations CF: No family history Non-consanguineous = 1/22 x 1/22 x 1/4 = 1/1936

  15. Risk Calculations R=1/8 CF: No family history Consanguineous = 1/22 x 1/8 x 1/4 = 1/704

  16. Risk Calculations • Increased impact for rarer disorders

  17. Risk Calculations R=1/8 Known carrier of CF mutation: Consanguineous = 1 x 1/8 x 1/4 = 1/32 (Non-consanguineous = 1x 1/22 x 1/4 = 1/88)

  18. Developing and Running a Diagnostic Service for Rare Recessive Disorders

  19. Assay Design • Gene structure • Mutation spectrum • Trinucleotide repeat expansions • Large re-arrangements • Point mutations • Mutation distribution • Recurrent mutations (founder effects) • Mutation hot-spots

  20. Mutation Scanning • Private mutations • Whole gene screening • Scanning technique • Different behaviour of heteroduplexes • CSCE • dHPLC • HRM

  21. Heteroduplex Analysis Denature & Re-anneal Denature & Re-anneal

  22. Denature & Re-anneal ? Denature & Re-anneal Heteroduplex Analysis for Rare Recessive Disorders • Problem • Reduced sensitivity • Sequence/mutation specific – ALMS1 example

  23. Heteroduplex Analysis - Reduced Sensitivity for Homozygous Changes • HRM analysis • Blind trial of 14 previously tested patients • 10 different amplicons • Each patient had mutation or variant in at least one amplicon but not variant for each amplicon • 3 false negatives • All 3 were homozygous changes • Other homozygous changes were detected • Dependent on nature of variant and sequence context

  24. Heteroduplex Analysis for Rare Recessive Disorders • Problem • Reduced sensitivity • Sequence/mutation specific – ALMS1 example • Solutions • Screen parents • Spiking – ALMS1 example

  25. Spiking of PCRs with Wildtype DNA • To enable detection of homozygote variants by facilitating heteroduplex formation • Pre-PCR spiking • Post-PCR spiking • Visualise amplification of all samples • Analyse samples before and after spiking (prevent missing heterozygous changes masked by WT alleles)

  26. Wildtype controls 1.5xreaction volume 1/3 vol. test and wildtype (all 3 columns equal vol.) Denature & re-anneal, then analyse (1 v 2 and 3 v 2) Test samples (incl. blank & controls) 1.5xreaction volume Spiking of PCRs with Wildtype DNA

  27. Heteroduplex Analysis for Rare Recessive Disorders • Problem • Reduced sensitivity • Sequence/mutation specific – ALMS1 example • Solutions • Screen parents • Spiking – ALMS1 example • Large number of WT controls per run (cost and DNA availability) • Lack of batching (need group of WT samples to give normal pattern to compare against)

  28. Interpretation of Results • Pathogenicity of private mutation • Limited mutations published • Less functional data • Fewer orthologues • Heterozyote • Possibility of two different mutations within family • Genetic heterogeneity • Possibility of mutation in two different genes giving similar phenotype in family

  29. Conclusions • Consanguinity is likely to be encountered by all diagnostic laboratories. • Awareness of consanguinity important to enable provision of suitable tests and accurate interpretation of results. • Consanguinity has effect on risk calculations. • Services for rare recessive disorders must be designed to detect homozygous variants with high sensitivity.

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