1. Implementation of a new diagnostic service for congenital adrenal hyperplasia Charlene Crosby West Midlands Regional Genetics Laboratory

2. Congenital Adrenal Hyperplasia Classic congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder with an incidence of 1 in 7,000-15,000 Non-classic CAH is less severe and effects 1 in 500- 1000 individuals 90-95% of cases are caused by deficiency of 21- hydroxylase, which catalyses the synthesis of cortisol and aldosterone from cholesterol

3. Dehydroeplandrosterone (DHEA) Cholesterol Pregnenolone17-hydroxypregnenolone Progesterone17- hydroxyprogesterone (17-OHP) Androstenedione Deoxycorticosterone11-deoxycortisolTestosterone CorticosteroneCortisol Aldosterone 21-hydroxylase

4. Clinical Presentation Clinical severity depends on degree of 21- hydroxylase deficiency –Good genotype phenotype correlations Classical CAH –Simple Virilsing: Ambiguous genitalia in females –Salt Wasting: Dehydration, vomiting and diarrhoea. If untreated can prove fatal Non-classical CAH –Milder than classical CAH –Androgen excess can cause precocious puberty in either sex –Males are often undiagnosed/asymptomatic

5. Treatment Glucocorticoids which suppress ACTH, are used to reduce the levels of adrenal sex steroids in the blood Individuals with salt wasting CAH also require mineralcorticoids and sodium chloride supplements Surgery on virilised females Growth monitoring to detect over and under treatment Dexamethosone can be used to prevent/reduce prenatal virilisation. Side effects for the mother include weight gain, irritability and oedema

6. The 21-Hydroxylase Gene The 21-hydroxylase (CYP21) gene and its pseudogene (CYP21P) are located at 6p21.3 Analysis of CYP21 is complicated due to the high sequence homology between CYP21 and CYP21P 95% of mutations are generated by recombination –20% deletions –75% point mutations C4ACYP21PC4BCYP21

7. Strategy Common strategies used to test for CAH diagnostically –ARMS PCR or sequencing –MLPA or Southern blotting A mini-sequencing method using the ABI Prism SNaPshot multiplex kit was validated to detect the common CYP21 point mutations (27 positive controls) MLPA used to detect deletions/gene conversions (30 positive controls) Together, these two techniques will detect 90-95% of mutations in CYP21 which lead to CAH

8. Mini-Sequencing Mutation specific primer anneals directly adjacent to the mutation being investigated Single base extension occurs with the addition of the complementary dye-labelled ddNTP Primers are synthetically elongated with polyT tracts of different lengths –Products range from 18 to 91 nucleotides in size Wild type and mutant alleles slightly differ in size due to the different molecular weights of the dyes

9. Mini-Sequencing Protocol Amplify Genomic DNA SNaPshot Reaction Remove Unincorporated ddNTPs Electrophoresis Data Analysed on GeneMapper Software v4.0 Remove dNTPs and Primers

10. NC I2G G/G I172N A/A I2G G/A Mini-Sequencing * * *

11. NC Neg I2G A/C/G Q318X C/T **

12. Sequencing The common point mutations are amplified in four nested PCRs from the primary 3 Kb PCR fragment Alternatively, the CYP21 gene can be amplified in two fragments followed by five nested PCRs 1234 5 678910 P30L I2G I172N I236N V237E M239K Q318XV281L R356W F306+T P453S ∆8bp

13. I172N A/AP453S T/T

14. LTAC4AC4BCYP21CYP21P TNXBCREBL1 12345678910 1 probe 3 probes1 probe TNXA 1 probe 5 probes Promoter (pseudogenic promoter reduces transcription) Exon 3 (8bp deletion in pseudogene) Exon 4 (I172N missense mutation in pseudogene) Exon 6 (I236N ex6 cluster mutation in pseudogene) Exon 8 (Q318X nonsense mutation in pseudogene) MLPA

15. CAH 10.911.091.121.160.00 1.760.630.600.000.690.56 CAH 30.921.081.061.010.00 1.720.540.550.000.660.52 CAH 41.00 1.031.020.040.062.130.540.590.091.100.58 CAH 61.060.940.990.950.00 1.490.510.500.001.130.48 CAH 81.050.950.92 0.451.390.840.980.151.170.79 Hom delHet delDup

16. Results & Discussion Using mini-sequencing and MLPA, all mutations in the controls were correctly identified –one additional mutation was detected Identification of pathogenic CYP21 mutations in cis –highlights the importance of determining phase when two heterozygous point mutations are detected Currently conventional sequencing and MLPA are being used for mutation detection Testing for the ten common point mutations and deletions will detect 90-95% of mutations which cause 21-hydroxylase deficiency

17. Acknowledgements University of Birmingham –Dr Nils Krone Manchester Regional Genetics Service –Helene Schlect –Simon Tobi Yorkshire Regional Genetics Service –Ian Berry West Midlands Regional Genetics Laboratory –Yvonne Wallis –Fiona Macdonald –Jennie Bell –Richard Barber