Implementation of a new diagnostic service for congenital adrenal hyperplasia

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. Cholesterol Pregnenolone 17-hydroxypregnenolone Progesterone 17-hydroxyprogesterone (17-OHP) Androstenedione 21-hydroxylase 21-hydroxylase Deoxycorticosterone 11-deoxycortisol Testosterone Corticosterone Cortisol Aldosterone Dehydroeplandrosterone (DHEA)

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. C4A CYP21P C4B CYP21 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

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

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

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

12. ∆8bp I236N V237E M239K V281L Q318X P30L I172N P453S R356W F306+T I2G 1 2 3 4 5 6 7 8 9 10 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

13. I172N A/A P453S T/T

14. 1 2 3 4 5 6 7 8 9 10 LTA C4A CYP21P C4B CYP21 TNXA TNXB CREBL1 1 probe 1 probe 3 probes 1 probe 1 probe 5 probes 1 probe 1 probe 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. Hom del Het del Dup MLPA

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