Genetic and clinical characteristics including occurrence of testicular adrenal rest tumors in Slovak and Slovenian patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

**Background:** Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder primarily caused by 21-hydroxylase deficiency (21-OH). The CYP21A2 gene is located near its pseudogene (CYP21A2P), with up to 75% of pathogenic variants resulting from gene recombination and conversion events. This study aimed to analyze the mutational spectrum, clinical characteristics, genotype-phenotype correlations, and testicular adrenal rest tumor (TART) prevalence in CAH patients from Slovakia and Slovenia, where one country (Slovakia) has had newborn screening (NBS) since 2003 and the other (Slovenia) had not introduced it at the time of study.

**Methods:** Data were obtained from 104 patients with CAH registered in Slovak and Slovenian databases (40 from Slovakia, 60 from Slovenia). Low-resolution genotyping (qPCR, MLPA, SnaPshot) detected common point mutations; high-resolution genotyping (Southern blotting, allele-specific long-range PCR, SNPCheck, sequencing) detected deletions, conversions, and other sequence changes. Genotypes were classified according to residual 21-hydroxylase activity into groups 0 (null), A, B, and C. TARTs were diagnosed by scrotal ultrasound performed by pediatric radiologists approximately every 2 years.

**Key Results:** Complete genotype and phenotype data were available for 100 patients. The cohort comprised 63% salt-wasting (SW-CAH), 16% simple virilizing (SV-CAH), and 21% non-classic (NC-CAH) forms. CYP21A2 gene deletion/conversion and c.293-13A/C>G together accounted for 55.5% of affected alleles. In SW-CAH, the most common variants were c.293-13A/C>G (44.53%) and deletion/conversion (32.03%). In SV-CAH, p.Ile172Asn was most common (28.13%). In NC-CAH, p.Val282Leu was most frequent (33.33%). The frequency of alleles with multiple pathogenic variants was higher in Slovenian patients (15.83% of all alleles). Severe genotypes (0 and A) correlated well with SW-CAH (94.74% and 97.3%, respectively), while less severe genotypes showed weaker correlation (50% for group B with SV-CAH, 70.83% for group C with NC-CAH). The median age at diagnosis for SW-CAH was 6 days in Slovakia versus 28.5 days in Slovenia (p=0.01). Most Slovak patients (24/29) were detected by NBS. TARTs were identified in 7 of 24 male patients (29.2%), all of whom had SW-CAH and poor hormonal control. The median age at TART diagnosis was 13 years (range 8-24). The average 17-OHP level at TART diagnosis was 217.88 nmol/l.

**Clinical Implications:** This study confirms the critical importance of newborn screening for CAH, particularly in accelerating diagnosis of severe forms during the critical neonatal period—the median age at diagnosis was 22.5 days lower in screened versus unscreened populations. The genotype-phenotype correlation was strong for severe variants but less reliable for milder variants, consistent with other European populations. Given the 29.2% prevalence of TARTs in male SW-CAH patients and the potential for remission with early detection and improved compliance, the authors strongly recommend annual scrotal ultrasound screening for all male CAH patients. The findings support the introduction of newborn screening in Slovenia and highlight the need for ongoing genetic surveillance to map less frequent pathogenic variants across regions.