This case report describes a novel intronic mutation, CYP21A2:c.738+75C>T (rs1463196531), identified in a 4-year-old male with congenital adrenal insufficiency, and expands the known mutation spectrum associated with this condition. The patient, born full-term to unrelated parents, presented with adrenal failure within the first month of life, characterized by acute adrenal crisis symptoms such as vomiting, dehydration, weight loss, hypotension, and electrolyte imbalances. Hormonal evaluations confirmed primary adrenocortical insufficiency, necessitating ongoing hydrocortisone and fludrocortisone therapy. Using family trio-based amplicon sequencing of the CYP21A2 gene, we identified compound heterozygosity consisting of a full gene deletion and a novel pathogenic intronic mutation. Additionally, analysis of WGS data was performed to rule out pathogenic variants in genes that might lead to a similar phenotype, thereby eliminating the possibility of other genes contributing to the proband’s disease. This case demonstrates the potential of using amplicon sequencing in molecular genetic diagnostic testing to detect rare intronic variants in the CYP21A2 gene in cases of early-onset adrenal failure. It also contributes to a better understanding of the genetic basis of congenital adrenal hyperplasia (CAH), which remains a significant autosomal recessive disorder affecting cortisol and aldosterone production, with an incidence of 1 in 10,000 to 1 in 15,000 globally.

Cone dystrophy with supernormal rod responses (CDSRR) is a rare autosomal recessive hereditary retinal dystrophy caused by biallelic KCNV2 variants. Accurate prevalence data remain limited because current estimates rely on clinically ascertained cases. This study aimed to estimate its population prevalence by integrating curated variant evidence and large-scale population genomics resources. The key methodological feature of this study is a multi-tiered pathogenicity framework combined with estimation of a biologically plausible prevalence range: Known pathogenic variants define conservative estimates, whereas probably pathogenic variants and strong variants of uncertain significance define broader estimates. Allele frequencies were analyzed in 807,162 individuals from the gnomAD database and 144,127 Russian genomes from GDB and EvogenDB. A Bayesian framework was applied to calculate disease prevalence from aggregated carrier frequencies across predefined variant tiers. Conservative estimates based on known pathogenic variants, were 1 in 343,926 globally and 1 in 1,911,347 in Russia. Including broader tiers increased estimates to 1 in 14,620 and 1 in 32,764, respectively. Compared to previous clinically ascertained prevalence of 1 in 865,000, these estimates define a wider biologically plausible prevalence range and are consistent with possible clinical underascertainment. This tier-based approach accounts for uncertainty in variant classification when estimating rare disease prevalence.

Malignant hyperthermia susceptibility (MHS; OMIM 145600, 601887) is a pharmacogenetic condition in which exposure to volatile anesthetics and/or succinylcholine can trigger a potentially life-threatening hypermetabolic crisis of skeletal muscle. MHS is predominantly associated with pathogenic variants in RYR1, whereas CACNA1S contributes a substantially smaller proportion of genetically confirmed cases. Estimating the population prevalence of MHS is challenging because manifestation requires exposure to a triggering agent, penetrance is incomplete, and the evidentiary strength supporting individual variants varies considerably. In this study, MHS-associated RYR1 and CACNA1S variants were compiled from four complementary resources—EMHG, ClinGen MHS VCEP, HGMD, and ClinVar—and subjected to source-specific pathogenicity and phenotype filtering. A total of 13,918 unique reported variants were initially identified. Progressive evidence-based curation generated three nested variant tiers: a high-confidence Expert tier comprising 102 variants; an expanded HGMD-inclusive tier comprising 446 variants; and a full expanded ClinVar-inclusive tier comprising 593 variants. Population allele frequencies were evaluated using gnomAD v4.1.0 and the Russian GDB v1.3.4 database. The high-confidence Expert tier yielded genetically inferred MHS frequencies of approximately 1 in 807 in gnomAD and 1 in 1078 in GDB. Inclusion of HGMD-derived variants increased the corresponding estimates to approximately 1 in 50 and 1 in 104, respectively.

The standard-of-car and genetic testing for 21-hydroxylase deficiency congenital adrenal hyperplasia (CAH) is Sanger sequencing of a long-range CYP21A2 amplicon combined with multiplex ligation-dependent probe amplification (MLPA). Many Sanger sequencing protocols capture only selected regions of CYP21A2 and require several sequencing reactions. This retrospective study explores the potential of short-read next-generation sequencing (SRS) to replace Sanger sequencing in CAH genetic testing. A total of 216 participants, including CAH patients, their parents and individuals with no family history of CAH, were included. We assessed the performance of standard SRS analysis and improved upon it with developed computational software AmpliconPipe, which includes copy number calling and caller harmonization for precise variant detection. With these procedures, SRS of the CYP21A2 long-range amplicon achieved 100% sensitivity (CI 95% [94.9, 100]) and 100% specificity (CI 95% [97.4, 100]) for CAH diagnosis, and 100% sensitivity (CI 95% [94.9, 100]) with 98.6% specificity (CI 95% [95.0, 99.6]) for CAH-carrier diagnosis, with better gene coverage outperforming the standard-of-care method, discovering novel variants, and detecting CYP21A2 copy number alterations with high sensitivity. These findings establish SRS as a practical, accurate, and immediately implementable alternative to Sanger sequencing for CAH genetic testing.

NHLRC2-associated FINCA disease is an ultra-rare autosomal recessive multisystem disorder caused by biallelic pathogenic variants in NHLRC2. Its mutational spectrum and genotype–phenotype correlations remain incompletely defined, and the contribution of non-coding variants is poorly understood. Here, we report a male infant with a severe FINCA-like phenotype, including early-onset hemolytic anemia, pulmonary involvement, neurodevelopmental impairment, growth failure, recurrent infections, and fatal progression at 8.5 months. Whole-genome sequencing identified a compound heterozygous NHLRC2 genotype comprising the previously reported pathogenic missense variant c.442G>T (p.Asp148Tyr) and a novel deep intronic variant, c.331+6863A>G. Segregation analysis confirmed inheritance from different parents. Integrated genomic and splicing analysis predicted that c.331+6863A>G creates a strong cryptic donor splice site and supports pseudoexon inclusion. Reconstruction of the predicted aberrant transcript indicated premature termination and potential susceptibility to nonsense-mediated mRNA decay. To our knowledge, this is the first reported deep intronic NHLRC2 variant predicted to activate pseudoexon inclusion. Although experimental validation was unavailable, convergent clinical, segregation, population, and computational evidence supports c.331+6863A>G as the most plausible second disease-associated allele. This case expands the genomic spectrum of NHLRC2-associated FINCA disease and highlights the diagnostic value of phenotype-driven whole-genome sequencing.