Genetic Analysis Algorithm for the Study of Patients with Multiple Congenital Anomalies and Isolated Congenital Heart Disease.

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.

Análisis de anomalías cromosómicas, desbalances genómicos y variantes de secuencia como causa de cardiopatías congénitas / Analysis of chromosomal abnormalities, genomic imbalances, and sequence variants as cause of congenital heart defects

INTRODUCCIÓN: Las cardiopatías congénitas (CC) son causadas por el desarrollo anómalo del corazón durante el período embriofetal. Abarcan un amplio espectro de anomalías estructurales de las cavidades cardíacas o de los grandes vasos, con una prevalencia mundial de 6 a 9 por 1000 nacimientos. En Argentina constituyen un tercio de las anomalías congénitas (AC) al nacimiento. Si bien su etiología es heterogénea, se ha observado recurrencia familiar acorde con la influencia de factores genéticos. El objetivo del estudio fue evaluar la presencia de anomalías cromosómicas, desbalances genómicos o variantes de secuencias en una muestra de niños afectados con CC en Argentina. MÉTODOS: Se incluyó a 289 pacientes con CC de hasta 16 años. Se realizó un cariotipo para pacientes con otras AC y análisis por amplificación múltiple de sondas dependiente de ligación (MLPA) de regiones genómicas asociadas a CC para aquellos con CC conotroncales. En muestras seleccionadas, se analizaron desbalances genómicos por microarreglos de ADN (array-CGH) o variantes de secuencia en el gen NKX2-5. RESULTADOS: Hubo 9 pacientes que presentaron anomalías cromosómicas, 21 desbalances por MLPA y 8 por array-CGH. No se hallaron variantes patogénicas en NKX2-5 en los casos estudiados. DISCUSIÓN: Se halló la causa de la afección en el 13% de los casos analizados. El estudio de pacientes con CC aisladas o asociadas a otras AC no había sido abordado previamente en Argentina mediante este algoritmo

An update on genetic variants of the NKX2-5.

NKX2-5 is a homeodomain transcription factor that plays a crucial role in heart development. It is the first gene where a single genetic variant (GV) was found to be associated with congenital heart diseases in humans. In this study, we carried out a comprehensive survey of NKX2-5 GVs to build a unified, curated, and updated compilation of all available GVs. We retrieved a total of 1,380 unique GVs. From these, 970 had information on their frequency in the general population and 143 have been linked to pathogenic phenotypes in humans. In vitro effect was ascertained for 38 GVs. The homeodomain had the biggest cluster of pathogenic variants in the protein: 49 GVs in 60 residues, 23 in its third α-helix, where 11 missense variants may affect protein-DNA interaction or the hydrophobic core. We also pinpointed the likely location of pathogenic GVs in four linear motifs. These analyses allowed us to assign a putative explanation for the effect of 90 GVs. This study pointed to reliable pathogenicity for GVs in helix 3 of the homeodomain and may broaden the scope of functional and structural studies that can be done to better understand the effect of GVs in NKX2-5 function.

Genetic characterization of a large cohort of Argentine 21-hydroxylase Deficiency.

CONTEXT: 21-hydroxylase deficiency is the most common cause of Congenital Adrenal Hyperplasia. It presents as severe or classical forms-salt wasting and simple virilizing-and a mild or nonclassical (NC). Several studies have reported the frequency of pathogenic variants in different populations, although few of them included a large number of NC patients. OBJECTIVE: To analyse the CYP21A2 gene defects in a large cohort of Argentine patients. DESIGN: Molecular characterization of 628 patients (168 classical, 460 nonclassical, representing 1203 nonrelated alleles), 398 relatives, 126 partners. METHODS: Genetic variants were assessed by allele-specific PCR, PCR-RFLP or direct sequencing. Deletions, duplications and large gene conversions (LGC) were studied by Southern blot/MLPA or long-range PCR. Biological implications of novel variants were analysed by structure-based in silico studies. RESULTS: The most frequent pathogenic variants were p.V282L (58%) in NC alleles and c.293-13C>G (31.8%) and p.I173N (21.1%) in classical. Deletions and LGC were found at low frequency (6.2%), 57 alleles had rare pathogenic variants, and 3 had novel variants: p.(S166F); p.(P189R), p.(R436L). Genotype-phenotype correlation was observed in 98.6% of the cases, 11 asymptomatic first-degree relatives had pathogenic variants in both alleles, and 21/126 partners were carriers. CONCLUSIONS: We conducted a comprehensive genetic characterization of the largest cohort of 21-hydroxylase patients from the region. In particular, we add to the molecular characterization of a large number of NC patients and to the estimation of the disease carrier's frequency in our population.

Structure-based activity prediction of CYP21A2 stability variants: A survey of available gene variations.

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency accounts for 90-95% of CAH cases. In this work we performed an extensive survey of mutations and SNPs modifying the coding sequence of the CYP21A2 gene. Using bioinformatic tools and two plausible CYP21A2 structures as templates, we initially classified all known mutants (n = 343) according to their putative functional impacts, which were either reported in the literature or inferred from structural models. We then performed a detailed analysis on the subset of mutations believed to exclusively impact protein stability. For those mutants, the predicted stability was calculated and correlated with the variant's expected activity. A high concordance was obtained when comparing our predictions with available in vitro residual activities and/or the patient's phenotype. The predicted stability and derived activity of all reported mutations and SNPs lacking functional assays (n = 108) were assessed. As expected, most of the SNPs (52/76) showed no biological implications. Moreover, this approach was applied to evaluate the putative synergy that could emerge when two mutations occurred in cis. In addition, we propose a putative pathogenic effect of five novel mutations, p.L107Q, p.L122R, p.R132H, p.P335L and p.H466fs, found in 21-hydroxylase deficient patients of our cohort.

Misregulation effect of a novel allelic variant in the Z promoter region found in cis with the CYP21A2 p.P482S mutation: implications for 21-hydroxylase deficiency.

The aim of the current study was to search for the presence of genetic variants in the CYP21A2 Z promoter regulatory region in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Screening of the 10 most frequent pseudogene-derived mutations was followed by direct sequencing of the entire coding sequence, the proximal promoter, and a distal regulatory region in DNA samples from patients with at least one non-determined allele. We report three non-classical patients that presented a novel genetic variant-g.15626A>G-within the Z promoter regulatory region. In all the patients, the novel variant was found in cis with the mild, less frequent, p.P482S mutation located in the exon 10 of the CYP21A2 gene. The putative pathogenic implication of the novel variant was assessed by in silico analyses and in vitro assays. Topological analyses showed differences in the curvature and bendability of the DNA region bearing the novel variant. By performing functional studies, a significantly decreased activity of a reporter gene placed downstream from the regulatory region was found by the G transition. Our results may suggest that the activity of an allele bearing the p.P482S mutation may be influenced by the misregulated CYP21A2 transcriptional activity exerted by the Z promoter A>G variation.

Functional studies of p.R132C, p.R149C, p.M283V, p.E431K, and a novel c.652-2A>G mutations of the CYP21A2 gene.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most frequent inborn error of metabolism and accounts for 90-95% of CAH cases. In the present work, we analyzed the functional consequence of four novel previously reported point CYP21A2 mutations -p.R132C, p.R149C, p.M283V, p.E431K- found in Argentinean 21-hydroxylase deficient patients. In addition, we report an acceptor splice site novel point mutation, c.652-2A>G, found in a classical patient in compound heterozygosity with the rare p.R483Q mutation. We performed bioinformatic and functional assays to evaluate the biological implication of the novel mutation. Our analyses revealed that the residual enzymatic activity of the isolated mutants coding for CYP21A2 aminoacidic substitutions was reduced to a lesser than 50% of the wild type with both progesterone and 17-OH progesterone as substrates. Accordingly, all the variants would predict mild non-classical alleles. In one non-classical patient, the p.E431K mutation was found in cis with the p.D322G one. The highest decrease in enzyme activity was obtained when both mutations were assayed in the same construction, with a residual activity most likely related to the simple virilizing form of the disease. For the c.652-2A>G mutation, bioinformatic tools predicted the putative use of two different cryptic splicing sites. Nevertheless, functional analyses revealed the use of only one cryptic splice acceptor site located within exon 6, leading to the appearance of an mRNA with a 16 nt deletion. A severe allele is strongly suggested due to the presence of a premature stop codon in the protein only 12 nt downstream.

Isolated p.H62L Mutation in the CYP21A2 Gene in a Simple Virilizing 21-Hydroxylase Deficient Patient.

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency accounts for 90%-95% of cases. This autosomal recessive disorder has a broad spectrum of clinical forms, ranging from severe or classical, which includes the salt-wasting and simple virilizing forms, to the mild late onset or nonclassical form. Most of the disease-causing mutations described are likely to be the consequence of nonhomologous recombination or gene conversion events between the active CYP21A2 gene and its homologous CYP21A1P pseudogene. Nevertheless, an increasing number of naturally occurring mutations have been found. The change p.H62L is one of the most frequent rare mutations of the CYP21A2 gene. It was suggested that the p.H62L represents a mild mutation that may be responsible for a more severe enzymatic impairment when presented with another mild mutation on the same allele. In this report, a 20-year-old woman carrying an isolated p.H62L mutation in compound heterozygosity with c.283-13A/C>G mutation is described. Although a mildly nonclassical phenotype was expected, clinical signs and hormonal profile of the patient are consistent with a more severe simple virilizing form of 21-hydroxylase deficiency. The study of genotype-phenotype correlation in additional patients would help in defining the role of p.H62L in disease manifestation.

Structure-based analysis of five novel disease-causing mutations in 21-hydroxylase-deficient patients.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most frequent inborn error of metabolism, and accounts for 90-95% of CAH cases. The affected enzyme, P450C21, is encoded by the CYP21A2 gene, located together with a 98% nucleotide sequence identity CYP21A1P pseudogene, on chromosome 6p21.3. Even though most patients carry CYP21A1P-derived mutations, an increasing number of novel and rare mutations in disease causing alleles were found in the last years. In the present work, we describe five CYP21A2 novel mutations, p.R132C, p.149C, p.M283V, p.E431K and a frameshift g.2511_2512delGG, in four non-classical and one salt wasting patients from Argentina. All novel point mutations are located in CYP21 protein residues that are conserved throughout mammalian species, and none of them were found in control individuals. The putative pathogenic mechanisms of the novel variants were analyzed in silico. A three-dimensional CYP21 structure was generated by homology modeling and the protein design algorithm FoldX was used to calculate changes in stability of CYP21A2 protein. Our analysis revealed changes in protein stability or in the surface charge of the mutant enzymes, which could be related to the clinical manifestation found in patients.

Mechanism of action of novel naphthofuranquinones on rat liver microsomal peroxidation.

In order to elucidate the effect on mammal systems of new derivatives from 2-hydroxy-3-allyl-naphthoquinone, alpha-iodinated naphthofuranquinone (NPPN-3223), beta-iodinated naphthofuranquinone (NPPN-3222) and beta-methyl naphthofuranquinone (NPPN-3226) synthesized as possible trypanocidal agents, their effect on rat liver microsomal lipid peroxidation was investigated. They (a) inhibited NADPH-dependent, iron-catalyzed microsomal rat liver lipid peroxidation; (b) did not inhibit the tert-butyl hydroperoxide-dependent lipid peroxidation; (c) did not inhibit ascorbate-lipid peroxidation with the exception of NPPN-3226 which did inhibit it; (d) stimulated NADPH oxidation and microsomal oxygen uptake; (e) increased superoxide anion formation by NADPH-supplemented microsomes and (f) stimulated ascorbate oxidation. The three drugs were reduced to their seminaphthofuranquinone radical by the liver NADPH-P450 reductase system, as detected by ESR measurements. These results support the hypothesis that naphthofuranquinones reduction by microsomal NADPH-P450 reductase and semiquinone oxidation by molecular oxygen diverts electrons, preventing microsomal lipid peroxidation. In addition, hydroquinones and/or semiquinones formed by naphthofuranquinones reduction would be capable of lipid peroxidation inhibition and on interacting with the lipid peroxide radicals can lead to an antioxidant effect as we suggested for NPPN-3226 in close agreement to the inhibition of ascorbate-lipid peroxidation. Due to the properties of these molecules and their incoming structure developments, naphthofuranquinones would be considered as potentially promising therapeutic agents, mainly against Chagas disease.