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التحليل الجزيئي للجين CYP21A2 في المرضى العراقيين المصابين بتضخم الغدة الكظرية الولادي == Molecular Analysis of CYP21A2 Gene in Iraqi Patients with Congenital Adrenal Hyperplasia
Author name:
رقية غياث ياسين العبيدي
Supervisor name:
بسام موسى صادق الموسوي
General topic:
Medicine
Specific topic:
Diseases - Genetics
Degree:
Master
University:
University of Baghdad - Faculty Of Medicine
Language:
English
University location:
Baghdad
First pages:
19T1613 - p.pdf
Abstract:
Background : Congenital adrenal hyperplasia (CAH) is a group of autosomal - recessive disorders that result from a genetic defect in the pathway of steroidogenesis. The most common type is 21 - hydroxylase enzyme deficiency (21 - OHD), which accounts for 90 - 95% of cases and is categorized into two clinical forms : severe classic early onset (salt wasting and simple virilizing) and milder non - classic late onset forms. The molecular analysis of mutations affecting CYP21A2 gene coding for 21 - OH enzyme was not tried earlier in Iraqi patients.Aim of Study : To analyze the commonly expected mutations affecting CYP21A2 gene causing 21 - hydroxylase and thus congenital adrenal hyperplasia in Iraqi children and to determine the frequency of each type and their clinical impact.Patients, Materials & Methods : Fifty - four children with a clinical diagnosis of CAH were recruited from the Pediatric Endocrine Consultation Clinic / Children Welfare Hospital - Baghdad and the Genetic Consultation Clinic - Teaching Laboratories / Medical City - Baghdad during the period between September 14th, 2014 and March 14th, 2015. The clinical diagnosis of 21 - hydroxylase deficiency was made when the patients presented with ambiguous genitalia in females with or without dehydration, or vomiting and dehydration with normal male phenotype early in life, or pseudo - precocious puberty later in life.Peripheral blood samples were collected from all patients. Two samples were taken from those with ambiguous genitalia; one for chromosomal analysis and sex determination and the other for DNA analysis. For the other cases, a single sample was taken for DNA analysis.The other tests, namely electrolytes and hormonal assessment were sent for to be performed by another laboratory (governmental or private).Two cases with ambiguous genitalia showing male karyotypes were excluded from this study as they do not fit in the criteria of 21OHD.Molecular analysis of CYP21A2 gene was performed by PCR amplification using biotinylated primers and hybridization of amplification products to a test strip containing allele - specific oligonucleotide probes immobilized as an array of parallel lines. The bound biotinylated sequences are detected using streptavidin - alkaline phosphatase and color substrates according to the manufacturers’ instructions.Results : CYP21A2 gene was analyzed by CAH StripAssay® for detection of 11 common point mutations, and 50% of deletions and conversions in all 52 cases. Mutations (point mutations and deletions/conversions) were detected in 36 (69.2%) cases while 16 (30.8%) cases had undetected mutations. The most frequent mutations were : large deletions / conversions in 11 (21.15%), Q318X in 7 (13.46%), I2 Splice in 6 (11.54%), I172N in 5 (9.6%), Del 8bp in 1 (1.9%), V281L in 2 (3.85%), P453S in 1 (1.9%), R483P in 1 (1.9%) and multiple mutations in 2 cases (3.85%). Among those with large deletions/conversions, deletion/conversion (P30L, I2Splice, Del8bp) found in 3 (5.8%) cases, Deletion/conversion (P30L, I2 Splice, Del8bp, I172N) in 2 (3.85%) cases, Deletion/conversion (Cluster E6, V281L, L307 frameshift, Q318X, R356W) in 4 (7.7%) cases, complete gene deletion in 2 (3.85%) cases.Four mutations (namely P30L, R356W, Cluster E6, L307 frameshift) were not detected in any of our cases.Most cases demonstrated good correlations between genotypes and phenotypes. In the salt wasting group, the correlation was found in all cases with homozygous genotype, while among the simple virilizing group, the concordance of genotype/phenotype was found in all cases of group B (5 cases with homozygous I172N) and the single case in group A (homozygous I2 Splice), while it was absent in group null (one case withhomozygous deletion/conversion) and in group C (one case with homozygous V281L).Conclusions : 1. The majority of 21 - hydroxylase deficiency cases were females presented early in life with ambiguous genitalia with/without salt losing crisis, while a smaller number of patients were males with normal genitalia and salt wasting with/without precocious puberty; the patients have a high rate of parental consanguinity.2. There is a diversity of mutations affecting CYP21A2 gene in Iraqi 21 - hydroxylase deficiency patients, with a good percentage of undetected mutations.3. The diagnosis of 21 - OHD in CAH patients will be made more accurate when based on results of molecular testing.4. The data of molecular studies are useful in genetic counseling, in prenatal diagnosis and offering accurate treatment plan during pregnancy and in detection of carriers in families having previously affected child, diminishing the complications for these families
