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Waardenburg syndrome (WS) is a common genetic cause of syndromic hearing loss, accounting for 2-5% of congenital cases. It is characterized by hearing impairment and pigmentation abnormalities in the skin, hair, and eyes. Seven genes are associated with WS: PAX3, MITF, EDNRB, EDN3, SOX10, KITLG, and SNAI2. This study investigates the genetic causes of WS in three familial cases. Whole-exome sequencing (WES) was performed to identify single nucleotide variants (SNVs). Copy number variants (CNVs) were analyzed from the WES raw data and through multiplex ligation-dependent probe amplification (MLPA). The study identified one pathogenic SNV and two novel CNVs, corresponding to type I and type II WS patterns in the three families. The SNV, a nonsense variant (c.1198C>T p.Arg400*), was found in MITF and segregated in the affected father. The two CNVs were a deletion of exon 5 in PAX3 in a family with two affected members and a large novel deletion comprising seven genes, including SOX10, in a family with three affected members. These findings confirmed a WS diagnosis through genetic testing. The study emphasizes the importance of integrating multiple genetic testing approaches for accurate and reliable diagnosis, highlighting their role in improving patient management and providing tailored genetic counseling.
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During development, inner hair cells (IHCs) in the mammalian cochlea are unresponsive to acoustic stimuli but instead exhibit spontaneous activity. During this same period, neurons originating from the medial olivocochlear complex (MOC) transiently innervate IHCs, regulating their firing pattern which is crucial for the correct development of the auditory pathway. Although the MOC-IHC is a cholinergic synapse, previous evidence indicates the widespread presence of gamma-aminobutyric acid (GABA) signaling markers, including presynaptic GABAB receptors (GABABR). In this study, we explore the source of GABA by optogenetically activating either cholinergic or GABAergic fibers. The optogenetic stimulation of MOC terminals from GAD;ChR2-eYFP and ChAT;ChR2-eYFP mice evoked synaptic currents in IHCs that were blocked by α-bungarotoxin. This suggests that GABAergic fibers release ACh and activate α9α10 nicotinic acetylcholine receptors (nAChRs). Additionally, MOC cholinergic fibers release not only ACh but also GABA, as the effect of GABA on ACh response amplitude was prevented by applying the GABAB-R blocker (CGP 36216). Using optical neurotransmitter detection and calcium imaging techniques, we examined the extent of GABAergic modulation at the single synapse level. Our findings suggest heterogeneity in GABA modulation, as only 15 out of 31 recorded synaptic sites were modulated by applying the GABABR specific antagonist, CGP (100-200 µM). In conclusion, we provide compelling evidence that GABA and ACh are co-released from at least a subset of MOC terminals. In this circuit, GABA functions as a negative feedback mechanism, locally regulating the extent of cholinergic inhibition at certain efferent-IHC synapses during an immature stage.
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The α9α10 nicotinic cholinergic receptor (nAChR) is a ligand-gated pentameric cation-permeable ion channel that mediates synaptic transmission between descending efferent neurons and mechanosensory inner ear hair cells. When expressed in heterologous systems, α9 and α10 subunits can assemble into functional homomeric α9 and heteromeric α9α10 receptors. One of the differential properties between these nAChRs is the modulation of their ACh-evoked responses by extracellular calcium (Ca2+). While α9 nAChRs responses are blocked by Ca2+, ACh-evoked currents through α9α10 nAChRs are potentiated by Ca2+ in the micromolar range and blocked at millimolar concentrations. Using chimeric and mutant subunits, together with electrophysiological recordings under two-electrode voltage-clamp, we show that the TM2-TM3 loop of the rat α10 subunit contains key structural determinants responsible for the potentiation of the α9α10 nAChR by extracellular Ca2+. Moreover, molecular dynamics simulations reveal that the TM2-TM3 loop of α10 does not contribute to the Ca2+ potentiation phenotype through the formation of novel Ca2+ binding sites not present in the α9 receptor. These results suggest that the TM2-TM3 loop of α10 might act as a control element that facilitates the intramolecular rearrangements that follow ACh-evoked α9α10 nAChRs gating in response to local and transient changes of extracellular Ca2+ concentration. This finding might pave the way for the future rational design of drugs that target α9α10 nAChRs as otoprotectants.
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Calcio , Receptores Nicotínicos , Animales , Ratas , Acetilcolina/metabolismo , Acetilcolina/farmacología , Secuencia de Aminoácidos , Sitios de Unión , Calcio/metabolismo , Simulación de Dinámica Molecular , Técnicas de Placa-Clamp , Subunidades de Proteína/metabolismo , Subunidades de Proteína/genética , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/química , Xenopus laevisRESUMEN
Non-syndromic hearing impairment (NSHI) is a very heterogeneous genetic condition, involving over 130 genes. Mutations in GJB2, encoding connexin-26, are a major cause of NSHI (the DFNB1 type), but few other genes have significant epidemiological contributions. Mutations in the STRC gene result in the DFNB16 type of autosomal recessive NSHI, a common cause of moderate hearing loss. STRC is located in a tandem duplicated region that includes the STRCP1 pseudogene, and so it is prone to rearrangements causing structural variations. Firstly, we screened a cohort of 122 Spanish familial cases of non-DFNB1 NSHI with at least two affected siblings and unaffected parents, and with different degrees of hearing loss (mild to profound). Secondly, we screened a cohort of 64 Spanish sporadic non-DFNB1 cases, and a cohort of 35 Argentinean non-DFNB1 cases, all of them with moderate hearing loss. Amplification of marker D15S784, massively parallel DNA sequencing, multiplex ligation-dependent probe amplification and long-range gene-specific PCR followed by Sanger sequencing were used to search and confirm single-nucleotide variants (SNVs) and deletions involving STRC. Causative variants were found in 13 Spanish familial cases (10.7%), 5 Spanish simplex cases (7.8%) and 2 Argentinean cases (5.7%). In all, 34 deleted alleles and 6 SNVs, 5 of which are novel. All affected subjects had moderate hearing impairment. Our results further support this strong genotype-phenotype correlation and highlight the significant contribution of STRC mutations to moderate NSHI in the Spanish population.
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Listeria monocytogenes is an opportunistic foodborne pathogen. It can resist stress conditions by adapting through the production of biofilms, which represents a serious problem for the food industry. It is classified into 14 serotypes, although only four (1/2a, 1/2b, 1/2c, and 4b) account for 89.0-98.0% of listeriosis cases worldwide. The objective of this study was to detect and serotype L.monocytogenes isolated from different food matrices from processing plants in Argentina. In the period 2016-2021, 1832 samples (meat, ready-to-eat foods, ice cream, dairy foods, and frozen vegetables) were analyzed, of which 226 (12.34%) isolates compatible with L.monocytogenes were detected. At the same time, environmental and surface samplings were performed in processing plants for ready-to-eat foods, sausages and dairy products, where environmental contamination with L.monocytogenes was detected in numerous critical points of the process, yielding a positivity rate of 22.7%. The molecular analysis of serogroups was performed, where it was observed that serogroup IIb was the most frequent with 66.5% (n=107), and in descending order IIc with 22.3% (n=36), and IIa (n=9) and IVb (n=9) with 5.6%. The serogroup mostly isolated in environmental monitoring was IIb. This work highlights the importance of the detection and serotyping of L.monocytogenes for taking actionable measures and identifying outbreaks, and is the first study in Argentina to describe an extensive study in food matrices.
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Listeria monocytogenes , Listeria monocytogenes/genética , Serotipificación , Contaminación de Alimentos , Microbiología de Alimentos , Argentina/epidemiología , Reacción en Cadena de la PolimerasaRESUMEN
The present study investigates the spectrum and analysis of mitochondrial DNA (mtDNA) variants associated with Leber hereditary optic neuropathy (LHON) in an Argentinean cohort, analyzing 3 LHON-associated mitochondrial genes. In 32% of the cases, molecular confirmation of the diagnosis could be established, due to the identification of disease-causing variants. A total of 54 variants were observed in a cohort of 100 patients tested with direct sequencing analysis. The frequent causative mutations m.11778G>A in MT-ND4, m.3460G>A in MT-ND1, and m.14484T>C in MT-ND6 were identified in 28% of the cases of our cohort. Secondary mutations in this Argentinean LHON cohort were m.11253T>C p.Ile165Thr in MT-ND4, identified in three patients (3/100, 3%) and m.3395A>G p.Tyr30Cys in MT-ND1, in one of the patients studied (1%). This study shows, for the first time, the analysis of mtDNA variants in patients with a probable diagnosis of LHON in Argentina. Standard molecular methods are an effective first approach in order to achieve genetic diagnosis of the disease, leaving NGS tests for those patients with negative results.
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ADN Mitocondrial , Atrofia Óptica Hereditaria de Leber , Humanos , ADN Mitocondrial/genética , Atrofia Óptica Hereditaria de Leber/diagnóstico , Atrofia Óptica Hereditaria de Leber/genética , Argentina , Mitocondrias/genética , MutaciónRESUMEN
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.
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Pérdida Auditiva/genética , Audición/genética , Mutación , Adolescente , Adulto , Niño , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Técnicas de Genotipaje , Pérdida Auditiva/diagnóstico , Pérdida Auditiva/metabolismo , Pérdida Auditiva/fisiopatología , Herencia , Humanos , Lactante , Masculino , Modelos Moleculares , Linaje , Fenotipo , Conformación Proteica , Relación Estructura-Actividad , Secuenciación del Exoma , Adulto JovenRESUMEN
Dystrophinopathies cover a spectrum of rare progressive X-linked muscle diseases, arising from DMD mutations. They are among the most common pediatric muscular dystrophies, being Duchenne muscular dystrophy (DMD) the most severe form. Despite the fact that there is still no cure for these serious diseases, unprecedented advances are being made for the development of therapies for DMD. Some of which are already conditionally approved: exon skipping and premature stop codon read-through. The present work aimed to characterize the mutational spectrum of DMD in an Argentinian cohort, to identify candidates for available pharmacogenetic treatments and finally, to conduct a comparative analysis of the Latin American (LA) frequencies of mutations amenable for available DMD therapies. We studied 400 patients with clinical diagnosis of dystrophinopathy, implementing a diagnostic molecular algorithm including: MLPA/PCR/Sanger/Exome and bioinformatics. We also performed a meta-analysis of LA's metrics for DMD available therapies. The employed algorithm resulted effective for the achievement of differential diagnosis, reaching a detection rate of 97%. Because of this, corticosteroid treatment was correctly indicated and validated in 371 patients with genetic confirmation of dystrophinopathy. Also, 20 were eligible for exon skipping of exon 51, 21 for exon 53, 12 for exon 45 and another 70 for premature stop codon read-through therapy. We determined that 87.5% of DMD patients will restore the reading frame with the skipping of only one exon. Regarding nonsense variants, UGA turned out to be the most frequent premature stop codon observed (47%). According to the meta-analysis, only four LA countries (Argentina, Brazil, Colombia and Mexico) provide the complete molecular algorithm for dystrophinopathies. We observed different relations among the available targets for exon skipping in the analyzed populations, but a more even proportion of nonsense variants (â¼40%). In conclusion, this manuscript describes the theragnosis carried out in Argentinian dystrophinopathy patients. The implemented molecular algorithm proved to be efficient for the achievement of differential diagnosis, which plays a crucial role in patient management, determination of the standard of care and genetic counseling. Finally, this work contributes with the international efforts to characterize the frequencies and variants in LA, pillars of drug development and theragnosis.
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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.
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Conexina 26/genética , Conexina 30/genética , Variación Genética , Genoma Humano , Genómica/métodos , Pérdida Auditiva/genética , Argentina/epidemiología , Estudios de Cohortes , Femenino , Pruebas Genéticas , Pérdida Auditiva/epidemiología , Pérdida Auditiva/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , MasculinoRESUMEN
BACKGROUND: Keratitis-Ichthyosis-Deafness (KID) syndrome is a rare condition characterized by pre-lingual sensorineural deafness with skin hyperkeratinization. The primary cause of the disease is a loss-of-function mutation in the GJB2 gene. Mutations in Argentinean patients have not been described. CASE PRESENTATION: We studied a 2 year-old boy with bilateral congenital sensorineural deafness with dry skin over the entire body, hypotrichosis of the scalp, thin and light-blond hair. Analysis of the GJB2 gene nucleotide sequence revealed the substitution of guanine-148 by adenine predicted to result in an Asp50Asn amino acid substitution. CONCLUSION: This is the first KID report in a patient from Argentina. This de novo mutation proved to be the cause of keratitis-ichthyosis-deafness syndrome (KID-syndrome) in the patient, and has implications in medical genetic practice.