Implementation of a new diagnostic service for congenital

adrenal hyperplasia

Charlene CrosbyWest Midlands Regional Genetics Laboratory

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

Dehydroeplandrosterone

(DHEA)

Cholesterol

Pregnenolone 17-hydroxypregnenolone

Progesterone 17-hydroxyprogesterone

(17-OHP)

Androstenedione

Deoxycorticosterone 11-deoxycortisol Testosterone

Corticosterone Cortisol

Aldosterone

21-hydroxylase 21-hydroxylase

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

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

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

C4A CYP21P C4B CYP21

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

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

Mini-Sequencing Protocol

Amplify Genomic DNA

SNaPshot Reaction

Remove Unincorporated ddNTPs

Electrophoresis

Data Analysed on GeneMapper Software v4.0

Remove dNTPs and Primers

NC

I2G G/G

I172N A/A

I2G G/A

Mini-Sequencing

*

*

*

I2G Q318XI2G Q318X

NC

Neg

I2G A/C/G

Q318X C/T* *

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

1 2 3 4 5 6 7 8 9 10

P30LI2G

I172NI236N V237EM239K

Q318XV281L

R356WF306+TP453S

∆8bp

I172N A/A P453S T/T

LTA C4A C4B CYP21CYP21P

TNXB CREBL1

1 2 3 4 5 6 7 8 9 10

1 probe 1 probe 3 probes 1 probe

TNXA

1 probe 1 probe1 probe5 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

MLPA

CAH 1 0.91 1.09 1.12 1.16 0.00 0.00 1.76 0.63 0.60 0.00 0.69 0.56

CAH 3 0.92 1.08 1.06 1.01 0.00 0.00 1.72 0.54 0.55 0.00 0.66 0.52

CAH 4 1.00 1.00 1.03 1.02 0.04 0.06 2.13 0.54 0.59 0.09 1.10 0.58

CAH 6 1.06 0.94 0.99 0.95 0.00 0.00 1.49 0.51 0.50 0.00 1.13 0.48

CAH 8 1.05 0.95 0.92 0.92 0.92 0.45 1.39 0.84 0.98 0.15 1.17 0.79

   Hom del Het del Dup 

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

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