Expression and characterisation of Fmr1 splice variants during folliculogenesis in the rat.

CONTEXT: The FMR1 gene consists of 17 exons and codes for the FMRP protein. FMR1 is involved in four genetic disorders depending on the CGG repeats length in its 5'UTR: the full mutation is responsible for the Fragile X syndrome while the premutation is associated with the Fragile X-associated Tremor/Ataxia Syndrome, Fragile X-associated Primary Ovarian Insufficiency (FXPOI) and Fragile X-associated neuropsychiatric disorders. FMR1 presents multiple isoforms resulting from skipping of exons 12 and 14 and the use of alternative splice sites in exons 15 and 17. AIMS: To investigate the expression of Fmr1 splicing variants during folliculogenesis in the rat. METHODS: We used preantral, early antral and preovulatory follicles to isolate RNA and characterise, by fluorescent PCR followed by sequencing, all the isoforms present in the different follicular stages. KEY RESULTS: We identified two isoforms resulting from splicing of exon 12, six isoforms resulting from splicing of exon 14 and 15 and one isoform for exon 17. CONCLUSIONS: The expression levels of the isoforms vary within each follicular stage but not between different stages of folliculogenesis. Importantly, we identify for the first time in rat, an isoform that contains exon 12 and two isoforms, one that includes and one that excludes exon 14 and use the third acceptor site in exon 15. IMPLICATIONS: Characterisation of the different FMR1 variants expressed during folliculogenesis will help to understand the potential distinct cellular roles of each of them and the possible implication in the development of FXPOI.

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.

Predicting pathogenicity for novel hearing loss mutations based on genetic and protein structure approaches.

Hearing loss is a heterogeneous disorder. Identification of causative mutations is demanding due to genetic heterogeneity. In this study, we investigated the genetic cause of sensorineural hearing loss in patients with severe/profound deafness. After the exclusion of GJB2-GJB6 mutations, we performed whole exome sequencing in 32 unrelated Argentinean families. Mutations were detected in 16 known deafness genes in 20 patients: ACTG1, ADGRV1 (GPR98), CDH23, COL4A3, COL4A5, DFNA5 (GSDDE), EYA4, LARS2, LOXHD1, MITF, MYO6, MYO7A, TECTA, TMPRSS3, USH2A and WSF1. Notably, 11 variants affecting 9 different non-GJB2 genes resulted novel: c.12829C > T, p.(Arg4277*) in ADGRV1; c.337del, p.(Asp109*) and c.3352del, p.(Gly1118Alafs*7) in CDH23; c.3500G > A, p.(Gly1167Glu) in COL4A3; c.1183C > T, p.(Pro395Ser) and c.1759C > T, p.(Pro587Ser) in COL4A5; c.580 + 2 T > C in EYA4; c.1481dup, p.(Leu495Profs*31) in LARS2; c.1939 T > C, p.(Phe647Leu), in MYO6; c.733C > T, p.(Gln245*) in MYO7A and c.242C > G, p.(Ser81*) in TMPRSS3 genes. To predict the effect of these variants, novel protein modeling and protein stability analysis were employed. These results highlight the value of whole exome sequencing to identify candidate variants, as well as bioinformatic strategies to infer their pathogenicity.

GJB2 and GJB6 Genetic Variant Curation in an Argentinean Non-Syndromic Hearing-Impaired Cohort.

Genetic variants in GJB2 and GJB6 genes are the most frequent causes of hereditary hearing loss among several deaf populations worldwide. Molecular diagnosis enables proper genetic counseling and medical prognosis to patients. In this study, we present an update of testing results in a cohort of Argentinean non-syndromic hearing-impaired individuals. A total of 48 different sequence variants were detected in genomic DNA from patients referred to our laboratory. They were manually curated and classified based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology ACMG/AMP standards and hearing-loss-gene-specific criteria of the ClinGen Hearing Loss Expert Panel. More than 50% of sequence variants were reclassified from their previous categorization in ClinVar. These results provide an accurately interpreted set of variants to be taken into account by clinicians and the scientific community, and hence, aid the precise genetic counseling to patients.

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.

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.