Post on 16-Mar-2016
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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