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Implementation of a new diagnostic service for congenital adrenal hyperplasia. Charlene Crosby West 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 - PowerPoint PPT Presentation
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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