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

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

4. Testing for Autosomal Recessive Disorders Atypical, rare or private mutation Atypical, rare or private mutation Usual screening strategy may have lower sensitivity Usual screening strategy may have lower sensitivity Negative result may not significantly reduce likelihood of diagnosis Negative result may not significantly reduce likelihood of diagnosis Need for additional screening (availability/cost) Need for additional screening (availability/cost) Affecteds expected to be homozygous Affecteds expected to be homozygous Confirmation of homozygosity Confirmation of homozygosity Autozygosity analysis may be of use 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. Complex multi-system disorder that may affect the respiratory, pancreatic, gastro-intestinal and reproductive organ systems. Incidence 1/2,500 (Caucasians) Incidence 1/2,500 (Caucasians) Less frequent in other populations Less frequent in other populations Carrier Frequency Carrier Frequency 1/20 - 1/25 (Caucasians) 1/20 - 1/25 (Caucasians) Mutations in CFTR gene Mutations in CFTR gene >1600 different mutations reported >1600 different mutations reported

6. Cystic Fibrosis Initial screening for 29-32 mutations Initial screening for 29-32 mutations 80-90% mutations (Caucasian) 80-90% mutations (Caucasian) No mutation is detected No mutation is detected Reduces likelihood (~2% affected CF patients would give this result) Reduces likelihood (~2% affected CF patients would give this result) Single mutation detected Single mutation detected Increases likelihood (but doesnt confirm) Increases likelihood (but doesnt confirm) However, if consanguineous: However, if consanguineous: % mutations detected <80% % mutations detected <80% Standard interpretation of negative result inaccurate Standard interpretation of negative result inaccurate Potential homozygosity for rarer mutation in kit Potential homozygosity for rarer mutation in kit Confirmation of atypical result Confirmation of atypical result Testing of parental samples Testing of parental samples Interpretation of heterozygosity Interpretation of heterozygosity

7. Cystic Fibrosis Additional studies in consanguineous pedigrees Additional studies in consanguineous pedigrees Linked markers Linked markers Autozygosity analysis Autozygosity analysis Support or exclude autozygous inheritance Support or exclude autozygous inheritance Full sequencing Full sequencing Implications for neonatal screening Implications for neonatal screening 4 most common Caucasian mutations screened only 4 most common Caucasian mutations screened only 2nd raised IRT result for high likelihood/clinical referral 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 Degeneration and loss of the proximal anterior horn cells in the spinal cord Muscle wasting and atrophy Muscle wasting and atrophy Incidence: 1/10,000 Incidence: 1/10,000 Carrier frequency: 1/50 Carrier frequency: 1/50 SMN1 gene SMN1 gene >95% homozygous for deletion exon 7 (most exon 8 also deleted) >95% homozygous for deletion exon 7 (most exon 8 also deleted) Compound hets deletion/point mutation Compound hets deletion/point mutation

9. Spinal Muscular Atrophy (SMA) First level test: screen for deletion of SMN1 exon 7 and exon 8 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 If no deletion detected report as diagnosis highly unlikely or extremely unlikely However, if consanguineous However, if consanguineous Possible homozygosity for point mutation Possible homozygosity for point mutation More cautious interpretation More cautious interpretation reduces likelihood but cannot exclude a diagnosis reduces likelihood but cannot exclude a diagnosis Linkage/autozygosity analysis Linkage/autozygosity analysis Screen for point mutations - no UK lab offering? 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 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 Coefficient of Relationship (R) - proportion of genes shared by related individuals F = R x 1/2 F = R x 1/2 If one parent transmits a particular allele what is probability the other parent will transmit same allele If one parent transmits a particular allele what is probability the other parent will transmit same allele

11. Risk Calculations First Cousins: R=1/8 First Cousins: R=1/8 First Cousins: R=1/8 First Cousins: R=1/8

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

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

16. Risk Calculations Increased impact for rarer disorders Increased impact for rarer disorders

17. Risk Calculations 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) R=1/8

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

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

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

21. Heteroduplex Analysis Denature & Re-anneal

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

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

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

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

26. Spiking of PCRs with Wildtype DNA 1/3 vol. test and wildtype (all 3 columns equal vol.) Denature & re-anneal, then analyse (1 v 2 and 3 v 2)

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

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

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