Molecular Resolution of a TNXA / TNXB CH-1 Chimera and the Non-Chimeric TNXB Allele in CAH-X

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency can co-occur with CAH-X syndrome, a connective-tissue disorder caused by TNXB haploinsufficiency, resulting from TNXA/TNXB chimeric genes within the RCCX module. Here we performed a family-based molecular investigation to define a complex RCCX rearrangement, confirm the TNXA/TNXB chimera architecture, and independently assess the non-chimeric TNXB allele in a phenotypically discordant father-daughter pair. The proband underwent targeted CYP21A2 variant screening, trio gene-specific CYP21A2 sequencing, and copy-number analysis by multiplex ligation-dependent probe amplification. TNXA/TNXB short-read data were examined at pseudogene-informative positions, and the rearranged allele was characterized by RCCX-spanning long-range PCR using CYP779F and Tena32F followed by sequencing. A separate 2,536-bp allele-specific PCR was used to interrogate the 3' region of the non-chimeric TNXB allele. Gene-specific sequencing identified a maternally inherited c.137_138delinsGA p.(Pro46Arg) variant, classified as a variant of uncertain significance, while P050-C1 MLPA demonstrated a paternally inherited deletion involving all tested CYP21A2 exons and TNXB exon 35. Broader TNXB analysis with P155 showed no additional exon-level copy-number abnormality. Relative read balance at selected TNXA/TNXB-informative positions supported the suspected rearrangement. Sequencing of the approximately 8.5-kb RCCX-spanning amplicon established a CH-1-compatible architecture and restricted the recombination breakpoint to a 371-bp interval. No additional potentially clinically relevant variant was identified within the sequenced region of the non-chimeric TNXB allele. This study provides detailed molecular resolution of a TNXA/TNXB CH-1 rearrangement and illustrates the value and limitations of complementary allele-specific analyses in phenotypically discordant CAH-X families.