Autozygosity mapping in Leeds – examples

1. Autozygosity mapping in Leeds – examples

2. Consanguinity in Bradford Babies parents relatedness Mum’s parents relatedness

3. Mean a value for babies = 0.033 38.9% 1o cousin 7.1% 1o-2o cousin 5.2% 2o cousins Mean a value for mothers = 0.021 28.7% 1o cousins Mean a value in Pakistan = 0.33 (1991) 50.4% 1o cousins 10.9% 2o cousins Attock 1923 41.6% 1o cousins 18.7% 1o-4o cousin (Punjab census 1923) Consanguinity

4. Patient 1 • Aged two years: • Not walking, generalised delay • Generalised seizures • Tremor, Ataxia, • OFC < 3ocentile Height and weight << 0.4th • Not dysmorphic • Hearing loss • Cone rod dystrophy • Develops renal tubulopathy: • Persistently low serum potassium and magnesium • Evidence of increased urinary excretion of potassium and magnesium • Requires salt supplementation • Magnesium losing tubulopathy

6. Analysis • Disorder consistent with AR inheritance • Genotyped family using Xba142 10k affymetrix platform • Affecteds only approach • Cheap! • Data analysed using AutoSNPa and IBD finder • Most valuable analysis in this case with IBD finder

7. IBD finder output for chromosome 1

8. Note overlapping haplotypes in affecteds Minimal region defined by six SNPs

13. Check for presence in large number of ethnically matched controls (192 in this case) Preservation ? Functional data Abnormal BAERs Abnormal ERGs Confirm significance of variant

14. Heterologous expression of KNCJ10 wildtype (WT) and mutants (R65P, G77R) in Xenopus oocytes measured by two-electrode voltage clamp. Currents were measured at -80 mV. Note significant decrease of specific currents in mutant KCNJ10 indicated by asterisks. The number of experiments is indicated by n. Experimental model

15. Genes

16. KCNJ11 Hyperinsulinemic hypoglycemia Permanent neonatal diabetes KCNJ13 Snowflake vitreoretinal degeneration KCNJ2 Andersen syndrome KCNJ1 Antenatal Barrter syndrome KCNJ10 Ataxia Mental retardation Visual disturbance Seizures Magnesium losing tubulopathy Inwardly rectifying postassium channels

17. Patient 2 • Seen aged 9 months • Profound delay • Seizures • Optic nerve hyoplasia • Now aged 5 • Unable to sit up • No speech • seizures

20. Analysis • Family structure compatible with AR inheritance • Affecteds only genotyped • 10k Affymetrix platform as before • Analysed with AutoSNPa and IBDfinder • AutoSNPa most useful in this case

23. Candidates? Tuba8 lies within the homozygous region Mutations in Tuba3 found in another form of brain dysplasia- lissencephaly

24. -14bp deletion in intron 2 • -11 bp upstream of the exon 2 splice junction • eliminates the acceptor site polypyrimidine tract • In-silico prediction- loss of exon 2

25. 4r(i) 4r(ii) 1 2 3 4 5 1↔4ri 1↔5 1↔2 1↔3 Amplicon exons: N H P N H P N H P N H P LCL RNA: 1↔4r(ii) − N P 1000 1000 700 Fibroblast RNA: 500 500 RT-PCR of cDNA from lymphoblastoid cell lines (upper panel) and fibroblasts from control (N) obligate heterozygote (H) and affected (P) with 11bpTUBA8 deletion

26. Further confirmatory work

27. Autozygosity mapping-success • Highly dependant on family structure • Absolute requirement is clear phenotype • IBD finder and AutoSNPa allow visual inspection of data • Possible to use to identify shared ancestral haplotypes between families • You’ve still got to find and confirm mutations!