Congenital anomalies (CA) affect 3–5% of newborns, representing the second-leading cause of infant mortality in Argentina. Multiple congenital anomalies (MCA) have a prevalence of 2.26/1000 births in newborns, while congenital heart diseases (CHD) are the most frequent CA with a prevalence of 4.06/1000 births. The aim of this study was to identify the genetic causes in Argentinian patients with MCA and isolated CHD. We recruited 366 patients (172 with MCA and 194 with isolated CHD) born between June 2015 and August 2019 at public hospitals. DNA from peripheral blood was obtained from all patients, while karyotyping was performed in patients with MCA. Samples from patients presenting conotruncal CHD or DiGeorge phenotype (n = 137) were studied using MLPA. Ninety-three samples were studied by array-CGH and 18 by targeted or exome next-generation sequencing (NGS). A total of 240 patients were successfully studied using at least one technique. Cytogenetic abnormalities were observed in 13 patients, while 18 had clinically relevant imbalances detected by array-CGH. After MLPA, 26 patients presented 22q11 deletions or duplications and one presented a TBX1 gene deletion. Following NGS analysis, 12 patients presented pathogenic or likely pathogenic genetic variants, five of them, found in KAT6B, SHH, MYH11, MYH7 and EP300 genes, are novel. Using an algorithm that combines molecular techniques with clinical and genetic assessment, we determined the genetic contribution in 27.5% of the analyzed patients.

The gene CYP21A2, mapped to the RCCX module in 6p21.3, is responsible for the 21-hydroxylase deficiency (21OHD). In this work, we used Oxford Nanopore Technology (ONT) Long Reads (LR) sequencing to analyze samples from a large Argentinian cohort of 21OHD. Our goal was to gain additional information about the GVs involved in the rearrangements, to obtain higher resolution for the breakpoints of converted alleles and to retrieve new data of TNX and CYP21A1P genes. A total of 34 samples were sequenced in 2 amplicons of 8.5 Kb covering the centromeric and telomeric RCCX modules. The number of GVs found varied between 17-106 and all expected pathogenic GVs and new ones were obtained with the LR sequencing workflow developed. From the 21 alleles with macroconverted or chimeric genes containing the promoter of the CYP21A1P, we uncovered 5 of them lacking GVs commonly expected to be involved in 21OHD rearrangements. Importantly, we reclassified one patient with a deletion that had been overlooked with the current diagnostic technologies applied. These new findings change the genetic counseling for the patient and his family related to the presence of a carrier allele for Congenital Adrenal Hyperplasia and Ehlers-Danlos Syndrome (CAH-X). Furthermore, by addressing the study of the centromeric and the telomeric RCCX modules, we found 19 GVs for CYP21A1P and 29 GVs for TNXA not previously described in Latin American populations or not reported in any of the consulted databases. This study may represent the first one applying ONT LR in clinical studies in Latin America, highlighting the importance of LR sequencing as a high-resolution method of diagnosis and better cost-effective balance. It allows us to better understand the diversity of the RCCX modules and to gain knowledge of the molecular pathogenesis of the disease.

In this work, we aim to identify the genetic causes of pathogenesis in Argentinean patients with multiple congenital anomalies (MCA) and isolated Congenital Heart Disease (iCHD). We recruited 174 MCA and 194 iCHD patients from 15 public hospitals. Karyotyping was performed for MCA patients, and MLPA for conotruncal CHD or suspected 2q11 Deletion Syndrome (22q11DS). Selected samples were analyzed by array-CGH (Comparative genomic hybridization) (n = 89) and/or Next-Generation Sequencing (NGS) (n = 18). We successfully analyzed 252/368 patients: 14 had cytogenetic abnormalities, 27 had imbalances in 22q11, and 16 had other clinically relevant copy number variations (CNVs). NGS revealed 12 relevant nucleotide variants (five novels). Combining molecular, clinical and genetic evaluations, the diagnostic yield was 26.2%.