Genotyping in patients with congenital adrenal hyperplasia by sequencing of newborn bloodspot samples

Objectives: Genotype-phenotype correlation in congenital adrenal hyperplasia (CAH) caused by 21-hydroxylase deficiency ranges from 45 to 97 %. We performed massively parallel sequencing ofCYP21A2 on stored newborn blood-spot samples to catalogue the genotypes present in our patients with CAH and enable genotype-phenotype comparison. Methods: Participants <= 15 years old with clinically diagnosed CAH were recruited from The Sydney Children's Hospitals Network. Phenotype was classified from clinical and biochemical details in the medical record as salt wasting (SW), simple virilising (SV), non-classic (NC) or an intermediate phenotype (SW/SV; SV/NC). Amplicon-based sequencing for CYP21A2 was performed on stored newborn bloodspot samples by the New South Wales Newborn Bloodspot Screening Laboratory on MiSeq (TM) Dx (Illumina, California). Available genetic test results were also obtained from the medical records. Results: Samples from 67 participants (43 % female, age 0.3-15 years) were sequenced, including 9 sibships. SW phenotype was present in 33/67 participants (49 %), SV in 9(13 %) and NC in 16 (24 %). Intermediate phenotypes included SW/SV in seven participants (10 %) and SV/NC in two (3 %).Variants were identified in 90/116 alleles (78 %). A complete genotype was available in 47/67 participants (70 %). The most common genotype was homozygous c.293-13A/C>G (I2G) in 7/47 participants (15 %). Genotype correlated with the most commonly reported phenotype in 36/44 cases (82 %).Correlation was higher in SW and NC phenotypes. Conclusions:This study uses genetic testing of newborn bloodspots to identify and characterise the genotypes present in an ethnically diverse Australian population with CAH. It further strengthens our knowledge of genotype-phenotype correlations in CAH.