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.

Double Autosomal/Gonosomal Mosaic Trisomy 47,XXX/47,XX,+14 in a Newborn with Multiple Congenital Anomalies.

Chromosomal trisomies are the most frequent major chromosomal anomalies in humans and can be present in a mosaic or a non-mosaic constitution. We report the first case of a newborn girl presenting with multiple congenital anomalies and a double mosaic trisomy involving chromosome 14 and the X chromosome detected by array CGH. Karyotype analysis revealed a double mosaic with 2 independent abnormal cell lines and the absence of 46,XX and 48,XXX,+14 cell lineages. The patient showed most of the clinical characteristics of mosaic trisomy 14. Analysis of autosomal DNA markers in the proband's blood sample did not support the presence of chimerism. Further analysis of chromosome X DNA markers suggests that the extra X chromosome most probably arose as a consequence of nondisjunction in meiosis II in the maternal lineage.

Genetic Imbalances in Argentinean Patients with Congenital Conotruncal Heart Defects.

Congenital conotruncal heart defects (CCHD) are a subset of serious congenital heart defects (CHD) of the cardiac outflow tracts or great arteries. Its frequency is estimated in 1/1000 live births, accounting for approximately 10⁻30% of all CHD cases. Chromosomal abnormalities and copy number variants (CNVs) contribute to the disease risk in patients with syndromic and/or non-syndromic forms. Although largely studied in several populations, their frequencies are barely reported for Latin American countries. The aim of this study was to analyze chromosomal abnormalities, 22q11 deletions, and other genomic imbalances in a group of Argentinean patients with CCHD of unknown etiology. A cohort of 219 patients with isolated CCHD or associated with other major anomalies were referred from different provinces of Argentina. Cytogenetic studies, Multiplex-Ligation-Probe-Amplification (MLPA) and fluorescent in situ hybridization (FISH) analysis were performed. No cytogenetic abnormalities were found. 22q11 deletion was found in 23.5% of the patients from our cohort, 66% only had CHD with no other major anomalies. None of the patients with transposition of the great vessels (TGV) carried the 22q11 deletion. Other 4 clinically relevant CNVs were also observed: a distal low copy repeat (LCR)D-E 22q11 duplication, and 17p13.3, 4q35 and TBX1 deletions. In summary, 25.8% of CCHD patients presented imbalances associated with the disease.

CYP21A2 mutation update: Comprehensive analysis of databases and published genetic variants.

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders of adrenal steroidogenesis. Disorders in steroid 21-hydroxylation account for over 95% of patients with CAH. Clinically, the 21-hydroxylase deficiency has been classified in a broad spectrum of clinical forms, ranging from severe or classical, to mild late onset or non-classical. Known allelic variants in the disease causing CYP21A2 gene are spread among different sources. Until recently, most variants reported have been identified in the clinical setting, which presumably bias described variants to pathogenic ones, as those found in the CYPAlleles database. Nevertheless, a large number of variants are being described in massive genome projects, many of which are found in dbSNP, but lack functional implications and/or their phenotypic effect. In this work, we gathered a total of 1,340 GVs in the CYP21A2 gene, from which 899 variants were unique and 230 have an effect on human health, and compiled all this information in an integrated database. We also connected CYP21A2 sequence information to phenotypic effects for all available mutations, including double mutants in cis. Data compiled in the present work could help physicians in the genetic counseling of families affected with 21-hydroxylase deficiency.

Distribution of FMR1 and FMR2 Repeats in Argentinean Patients with Primary Ovarian Insufficiency.

The premutation state of FMR1 (Fragile X Mental Retardation 1) has been associated with primary ovarian insufficiency (POI), and is the most common known genetic cause for 46,XX patients. Nevertheless, very few studies have analyzed its frequency in Latin American populations. Additionally, a relationship between alleles carrying a cryptic microdeletion in the 5'UTR of FMR2 and the onset of POI has only been studied in one population. Our aim was to analyze the incidence of FMR1 premutations and putative microdeletions in exon 1 of FMR2 in a cohort of Argentinean women with POI. We studied 133 patients and 84 controls. Fluorescent PCR was performed, and the FMR2 exon 1 was further sequenced in samples presenting less than 11 repeats. We found the frequency of FMR1 premutations to be 6.7% and 2.9% for familial and sporadic patients, respectively. Among controls, 1/84 women presented a premutation. In addition, although we did not find microdeletions in FMR2, we observed a change (T >C) adjacent to the repeats in two sisters with POI. Given the repetitive nature of the sequence involved, we could not ascertain whether this represents a single nucleotide polymorphism (SNP) or a deletion. Therefore, a relationship between FMR2 and POI could not be established for 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.