Mutational Spectrum in Egyptian CAH Patients

1. Mutational Spectrum in Egyptian CAH Patients AMANY IBRAHIM LECTURER OF PEDIATRICS DEMPU Cairo University, Cairo, Egypt

2. Introduction • Congenital adrenal hyperplasia (CAH), is a family of autosomal recessive disorders involving impaired synthesis of cortisol from cholesterol by the adrenal cortex. • 21-hydroxylase deficiency (21-OHD) is the most common cause of CAH in which excessive adrenal androgen biosynthesis results in virilization in all individuals and salt wasting in some individuals. • A classic form with severe enzyme deficiency and prenatal onset of virilization is distinguished from a non-classic form with mild enzyme deficiency and postnatal onset. The classic form is further divided into the simple virilizing form (~25% of affected individuals) and the salt-wasting form, in which aldosterone production is inadequate (≥75% of individuals).

3. Molecular Genetic Testing • CYP21A2 is the only gene in which mutations are known to cause 21-OHD CAH. • Common mutations often included: c.293-13A>G, c.293-13C>G, p.Pro31Leu, p.Ile173Asn, exon6 mutation cluster p.[Ile237Asn;Val238Glu;Met240Lys], p.Val282Leu, p.Leu308PhefsTer6, p.Gln319Ter, p.Arg357Trp, p.Pro454Ser, p.Gly111ValfsTer21, and the 30-kb chimeric pseudogene. Many of these common mutations arise as a result of gene conversion • The majority of individuals from heterogeneous populations with 21-OHD CAH are compound heterozygotes

4. Gene structure • The functional gene for adrenal 21-hydroxylase, CYP21A2, is located approximately 30 kb from a nonfunctional pseudogene, CYP21A2P, on chromosome 6p in the human leukocyte antigen (HLA) gene cluster. • CYP21A2 and CYP21A2P, the latter of which is inactive because of the presence of multiple deleterious mutations, share a high level of nucleotide sequence identity (98% between exons and 96% between introns). Both the functional gene and the pseudogene comprise ten exons.

5. Normal and Abnormal Gene Products • Normal gene product: The encoded protein is predicted to contain 494 amino acids with a molecular weight of 55 kd. The enzyme is at most 28% homologous to other cytochrome P450 enzymes. • Abnormal gene product: Aberration of the gene product depends on the specific mutation. Approximately 20% of the mutations are meiotic recombinations deleting a 30-kb gene segment that encompasses the 3' end of the CYP21A1Ppseudogene, all of the adjacent C4B complement gene, and the 5' end of CYP21A2, producing a nonfunctional chimeric pseudogene.

6. Allelic Variants • Five benign variants of the functional geneCYP21A2 are known. • Pathogenic allelic variants:CYP21A2 and CYP21A2P occur in a region of other repeated (duplicated) genes arranged in tandem. This arrangement facilitates recombination events between repeated sequences. Such recombination events are a major cause of CYP21A2 mutations that result in 21-OHD CAH. Recombination resulting from unequal crossing over during meiosis between the functional CYP21A2 homologs can result in gross CYP21A2deletion or duplication. The high degree of sequence similarity between CYP21A2 and CYP21A2P facilitates gene conversion, a phenomenon whereby a segment of functional CYP21A2 is replaced by a segment copied from the CYP21A2Ppseudogene. Therefore, the segment of the converted CYP21A2 has sequence variants typical of the pseudogene. These variants are pathogenic and inactivate normal CYP21A2 expression and/or translation of normal protein.

7. Pathogenic allelic variants • Small-scale gene conversions account for some of the common mutations, such as a combination of p.Pro31Leu, c.293-13A or C>G, and p.Gly111ValfsTer21 on the same allele, detected by allele specificpolymerase chain reaction method. • Large-scale gene conversions also occur, some of which may require additional testing (see Molecular Genetic Testing, Interpretation of test results). • Approximately 20%-30% of mutant alleles are the result of meiotic recombination between repeated sequences that result in a 30-kb deletion that encompasses the 3' end of the CYP21A1Ppseudogene, all of the adjacentC4B complement gene, and the 5' end of CYP21A2, thereby producing a nonfunctional chimeric pseudogene. • Another common mutation is c.293-13A>G or c.293-13C>G, occurring with a frequency of 20%-30%, leading to aberrant splicing and truncated small or unusual protein. • Nine disease-causing mutations in the nonfunctional pseudogene inactivate the functional gene when transferred fromCYP21A2P to CYP21A2 by gene conversion. These nine mutations, together with CYP21A2deletionand apparent large gene conversions, account for approximately 95% of all disease-causing CYP21 alleles

8. Selected CYP21A2 Allelic Variants

9. Grouping of Common CYP21A2 Mutations by Residual Enzyme Activity

10. Summary of Molecular Genetic Testing Used in 21-OHD CAH

11. Objectives • To determine the mutational spectrum in Egyptian CAH patients attending DEMPU. • To determine the genetic profile for family members of CAH patients. • To test the carrier rate of 11 common mutations causing CAH in a group of apparently healthy Egyptians. • Genotype-phenotype correlation.

12. Statement of proposed research • The use of strip hybridization assay method newly commercially available will help easy, rapid and cost effective testing for CAH patients, their families and detection of the carrier rate for the 11 common mutations tested for. • Comparison between the results obtained in the current study and previous results for some mutations detected by either RT-PCR or sequencing will be carried out. • Establishment of this screening method for molecular diagnosis of CAH will facilitate both using this method as routine molecular diagnostic method for this disorder and providing genetic counseling for families of CAH patients.

14. Methods and Procedures • The study will include 200 CAH patients diagnosed on clinical and biochemical basis as 21-OH deficiency CAH. Family members of 40 patients will be included in the study. • Informed consent was obtained from the parents of all patients. • A group of 200 apparently healthy age matched children will be included in the study. They will be tested for the common CAH mutations and will serve to give indication about the carrier rate after having an informed consent from them also. • Blood samples will be obtained from all groups in the study for DNA separation and molecular analysis.

15. Clinical and Lab teams • The clinical team of the project will be responsible for: • Case selection and recruitment. • Full clinical sheet preparation including phenotypic diagnosis. • The lab team will be responsible for: • Sample collection • DNA separation • Purchasing strip assay kits and other needed consumable and minor equipments. • Performing the molecular analysis in timed order. • Interpretation of lab results.

16. Collaboration between clinical and lab teams • Statistical analysis of data obtained. • genotype-phenotype correlation. • Comparison between the results obtained in the current study and previous results for some mutations detected by either RT-PCR or sequencing will be carried out. • Writing the annual report. • Scientific publications.

17. Clinical Team includes • Professor Dr. Mona Hafez • Dr. Amany Ibrahim • Dr. Noha Musa • Dr. Hend Mehawed

18. Lab Team includes • Professor Dr. Fatma El-Mogy

19. Method of Assay • The strip hybridization assay method will be used in this study. • The 11 common mutations that can be screened for are: c.293-13A>G, c.293-13C>G, p.Pro31Leu, p.Ile173Asn, exon6 mutation cluster p.[Ile237Asn;Val238Glu;Met240Lys], p.Val282Leu, p.Leu308PhefsTer6, p.Gln319Ter, p.Arg357Trp, p.Pro454Ser, p.Gly111ValfsTer21

20. The method to used include • Isolation of the genomic DNA from the blood samples. • Multiple PCR amplification of specific DNA sequences and simultaneous biotin labeling. • Precise selective hybridization of specific sequences onto strip assay. • Easy and clear identification of hybridized sequences of interest. • Biotinylated sequences are detected by streptavidin-alkaline phosphatase. • Automated result interpretation.