Molecular characterization of the new clinical entity associated with congenital adrenal hyperplasia: the CAH-X syndrome in the Spanish population

Abstract

Objectives: The chimeras causing the CAH-X syndrome (SCAH-X) result from recombination between CYP21A2-TNXB and their respective pseudogenes (CYP21A1P-TNXA). The clinical manifestations of this syndrome include congenital adrenal hyperplasia (CAH) and Ehlers-Danlos syndrome (EDS). Since SCAH-X has been recently described, the number of publications available is limited. The objective of this study was to set up a molecular approach and a screening algorithm for detecting CAH-X chimeras, determine their frequency and distribution in the Spanish population, and assess their clinical pattern of occurrence in a group of patients.

Methods: A total of 186 patients were eligible for CAH-X molecular genetic testing. Testing included MLPA, heterodimer detection by capillary gel electrophoresis, and sequencing of exons 40, 41, and 43 of TNXB. A review was performed of the medical history of 20 patients from three hospitals of reference and the signs and symptoms of EDS they exhibited.

Results: In total, 78 CAH patients were carriers of CAH-X chimeras (41.9 %). Forty-six patients were carriers of CH1 (24.7 %), 24 of CH2 (12.9 %), and 8 of CH3 (4.3 %), with a heterogeneous geographical distribution. Seven (35 %) of the 20 carriers of a CAH-X chimera who underwent clinical examination experienced clinical manifestations of EDS.

Conclusions: The impact of SCAH-X in the Spanish population was assessed by genetic testing. In the light of the clinical pattern of occurrence and significant prevalence of SCAH-X in the Spanish population, early diagnosis of this entity is essential for an appropriate follow-up of clinical manifestations.

Keywords: CAH-X syndrome; CYP21A2; TNXB; congenital adrenal hyperplasia; hypermobility-type Ehlers–Danlos syndrome; tenascin.

Figure 1:

Genomic organization of locus 6p21.3, CAH-X chimeras and their molecular characterization. (A) Locus 6p21.3 and RCCX module showing the genes and respective pseudogenes (Adaptation from Marino, 2021). (B) Representation of the three types of CAH-X chimeras (CH1, CH2, and CH3) identified to date. The squares in clear gray represent TNXB exons, whereas TNXA exons are represented in dark gray. The molecular variants that characterize each chimera are indicated (Adaptation from Marino, 2021). (C) Scheme displaying the different techniques used for TNXB exon amplification for SCAH-X molecular characterization. The amplification primers used in PCRs are indicated next to the publication where they were suggested (Adaptation from Marino, 2021). (D) Scheme of the amplicons obtained by PCR and primers used for the sequencing of exons 35, 40, 41, and 43 of TNXB.

Figure 2:

Molecular strategy for CAH-X quimeras. (A–B) Capillary gel electrophoresis. (A) Example of a run with several positive samples (tracks 2, 6, and 7), which exhibit the heteroduplex band (indicated in mustard color). The remainder of samples was negative for Del120pb. (B) Profile of the chromatogram obtained when screening was positive for heteroduplex. The peaks corresponding to alignment markers (in green), the amplicon (in maroon), and the resulting heteroduplex (in mustard) are indicated. (C) MLPA results: visualization of the results obtained for a Del120pb carrier (exon 35 TNXB). In green: probes corresponding to the CYP21A2 gene. In yellow: probes corresponding to the TNXB gene. Red arrows indicate the probes that hybridize against exon 35 of the TNXB gene. (D–F) Sequences corresponding to the regions containing SCAH-X characteristic variants. (D) Exon 40 of TNXB, where position c.12174C>G (p.Cys4058Trp) is indicated with an arrow, corresponding to a patient who was heterozygous for the variant analyzed. (E) Exon 41 of the TNXB gene. The arrow indicates position c.12218G>A (p.Arg4073His), which is characteristic of the CAH-X CH3 chimera. (F) Region corresponding to exon 43 of the TNXB gene. The arrows indicate positions c.12514G>A (p.Asp4172Asn) and c.12524G>A (p.Ser4175Asn), respectively (the two were heterozygous for the analyzed variants).

Figure 3:

Haplotypes with the different variants of the CAH-X chimeras found in Spanish patients. (A) Variants of the CAH-X CH1 chimera. The most frequent allele was the one that harbored Del120pb, along with another four variants. An allele only included Del120pb. Another allele harbored Del120pb and the c.12174C>G variant (p.Cys4058Trp). In the case of the CAH-X chimera, complete molecular analysis was performed of only six alleles. The reason is that, in the case of these chimeras, once the presence of Del120pb was detected by capillary gel electrophoresis/MLPA, the molecular study of exons 40, 41, and 43 was not performed. (B) The most frequent variant of the CAH-X CH2 chimera was the one that harbored the c.12174C>G mutation (p.Cys4058Trp), along with the molecular abnormalities present in exons 41 and 43. The second most frequent variant was the one that only included the abnormality that is characteristic of this chimera. (C) As it occurs with the other two chimeras, the most frequent variant of the CAH-X CH3 chimera was the one that included the cluster that characterizes it. Another two types of variants were identified: a variant that only contained an abnormality in exon 41 c.12218G>A (p.Arg4073His) and another variant that had an abnormality in exon 41 and one of the occasional abnormalities of exon 43 c.12524G>A (p.Ser4175Asn).