Novel and Recurrent PNPLA1 Mutations in Spanish Patients with Autosomal Recessive Congenital Ichthyosis; Evidence of a Founder Effect.

Autosomal recessive congenital ichthyosis (ARCI) is a group of rare non-syndrome diseases that affect cornification. PNPLA1 is one of the 12 related genes identified so far. Mutation screening of this gene has resulted in the identification of 13 individuals, from 10 families, who carried 7 different PNPLA1 mutations. These mutations included 2 missense, 2 frame-shift and 3 nonsense, 3 of them being novel. One of the identified variants, c.417_418delinsTC, was highly prevalent, as it was found in 6 out of 10 (60%) of our ARCI families with PNPLA1 mutations. Clinical manifestations varied significantly among patients, but altered sweating; erythema, palmar hyperlinearity and small whitish scales in flexor-extensor and facial areas were common symptoms. Haplotype analyses of c.417_418delinsTC carriers confirmed the existence of a common ancestor. This study expands the spectrum of the PNPLA1 disease, which causes variants and demonstrates that the c.417_418delinsTC mutation has founder effects in the Spanish population.

Clinical utility of next-generation sequencing in the aetiological diagnosis of sensorineural hearing loss in a Childhood Hearing Loss Unit. / Utilidad clínica de la secuenciación de nueva generación en el diagnóstico etiológico de la hipoacusia neurosensorial en una Unidad de Hipoacusia Infantil.

INTRODUCTION: Sensorineural hearing loss (SNL) is the most prevalent sensory deficit in our environment. Next generation genomic sequencing (NGS) enables an aetiological diagnosis in a high percentage of patients. Our pilot study shows the results of the systematic application of NGS in a Childhood Hearing Loss Unit, as well as its implications for the clinical management of patients and their families. MATERIAL AND METHOD: We included 27 patients diagnosed with SNL between 2014 and 2017, in which an environmental cause was ruled out. The genetic test consisted of a panel of genes analyzed by NGS (OTOgenicsTM panel). This panel has been designed to include genes associated with sensorineural or mixed hearing loss, early onset or late, syndromic and non-syndromic, regardless of their inheritance pattern. RESULTS: A genetic diagnosis was obtained in 56% (15/27) of the patients (62% in the case of bilateral SNL). Of the patients, 5/27 (19%) presented pathogenic variants in the GJB2 gene and the rest pathogenic and / or probably pathogenic variants in other genes associated with isolated SNL (PR2X2, TECTA and STRC), with syndromic SNL (CHD7, GATA3, COL4A5, MITF and SOX10) or with syndromic and non-syndromic SNL (BSND, ACTG1 and CDH23). DISCUSSION: The aetiological diagnosis of SNL is a challenge in clinical practice. Our series demonstrates that it is possible to implement genetic diagnosis in the care routine and that this information has prognostic and therapeutic implications.

Comprehensive genomic diagnosis of inherited retinal and optical nerve disorders reveals hidden syndromes and personalized therapeutic options.

PURPOSE: In the era of precision medicine, genomic characterization of blind patients is critical. Here, we evaluate the effects of comprehensive genetic analysis on the etiologic diagnosis of potentially hereditary vision loss and its impact on clinical management. METHODS: We studied 100 non-syndromic and syndromic Spanish patients with a clinical diagnosis of blindness caused by alterations on the retina, choroid, vitreous and/or optic nerve. We used a next-generation sequencing (NGS) panel (OFTALMOgenics™), developed and validated within this study, including up to 362 genes previously associated with these conditions. RESULTS: We identified the genetic cause of blindness in 45% of patients (45/100). A total of 28.9% of genetically diagnosed cases (13/45) were syndromic and, of those, in 30.8% (4/13) extraophthalmic features had been overlooked and/or not related to visual impairment before genetic testing, including cases with Mainzer-Saldino, Bardet-Biedl, mucolipidosis and MLCRD syndromes. In two additional cases-syndromic blindness had been proposed before, but not specifically diagnosed, and one patient with Heimler syndrome had been misdiagnosed as an Usher case before testing. 33.3% of the genetically diagnosed patients (15/45) had causative variants in genes targeted by clinical trials exploring the curative potential of gene therapy approaches. CONCLUSION: Comprehensive genomic testing provided clinically relevant insights in a large proportion of blind patients, identifying potential therapeutic opportunities or previously undiagnosed syndromes in 42.2% of the genetically diagnosed cases (19/45).

Human lactoferrin induces apoptosis-like cell death in Candida albicans: critical role of K+-channel-mediated K+ efflux.

Human lactoferrin (hLf) induced an apoptosis-like phenotype in Candida albicans cells, which includes phosphatidylserine externalization, nuclear chromatin condensation, DNA degradation, and increased reactive oxygen species (ROS) production. Intracellular ROS accumulation was seen to correlate with candidacidal activity in hLf-treated cells. Mitochondrial activity was involved as indicated by mitochondrial depolarization and increased hLf resistance of cells preincubated with sordarin or erythromycin, the latter of which inhibits protein synthesis in mitoribosomes. Interestingly, Cl(-)- and K(+)-channel blockers prevented the hLf antimicrobial activity, but only when cells were pretreated with the blocking agent (tetraethylammonium) prior to the hLf-induced K(+)-release period. These results indicate for the first time that K(+)-channel-mediated K(+) efflux is required for the progression of apoptosis-like process in yeast, suggesting that this essential apoptotic event of higher eukaryotes has been evolutionary conserved among species ranging from yeasts to humans.

Comprehensive genomic diagnosis of non-syndromic and syndromic hereditary hearing loss in Spanish patients.

BACKGROUND: Sensorineural hearing loss (SNHL) is the most common sensory impairment. Comprehensive next-generation sequencing (NGS) has become the standard for the etiological diagnosis of early-onset SNHL. However, accurate selection of target genomic regions (gene panel/exome/genome), analytical performance and variant interpretation remain relevant difficulties for its clinical implementation. METHODS: We developed a novel NGS panel with 199 genes associated with non-syndromic and/or syndromic SNHL. We evaluated the analytical sensitivity and specificity of the panel on 1624 known single nucleotide variants (SNVs) and indels on a mixture of genomic DNA from 10 previously characterized lymphoblastoid cell lines, and analyzed 50 Spanish patients with presumed hereditary SNHL not caused by GJB2/GJB6, OTOF nor MT-RNR1 mutations. RESULTS: The analytical sensitivity of the test to detect SNVs and indels on the DNA mixture from the cell lines was > 99.5%, with a specificity > 99.9%. The diagnostic yield on the SNHL patients was 42% (21/50): 47.6% (10/21) with autosomal recessive inheritance pattern (BSND, CDH23, MYO15A, STRC [n = 2], USH2A [n = 3], RDX, SLC26A4); 38.1% (8/21) autosomal dominant (ACTG1 [n = 3; 2 de novo], CHD7, GATA3 [de novo], MITF, P2RX2, SOX10), and 14.3% (3/21) X-linked (COL4A5 [de novo], POU3F4, PRPS1). 46.9% of causative variants (15/32) were not in the databases. 28.6% of genetically diagnosed cases (6/21) had previously undetected syndromes (Barakat, Usher type 2A [n = 3] and Waardenburg [n = 2]). 19% of genetic diagnoses (4/21) were attributable to large deletions/duplications (STRC deletion [n = 2]; partial CDH23 duplication; RDX exon 2 deletion). CONCLUSIONS: In the era of precision medicine, obtaining an etiologic diagnosis of SNHL is imperative. Here, we contribute to show that, with the right methodology, NGS can be transferred to the clinical practice, boosting the yield of SNHL genetic diagnosis to 50-60% (including GJB2/GJB6 alterations), improving diagnostic/prognostic accuracy, refining genetic and reproductive counseling and revealing clinically relevant undiagnosed syndromes.

Effects of human lactoferrin on the cytoplasmic membrane of Candida albicans cells related with its candidacidal activity.

Human lactoferrin is an innate host defence protein with antimicrobial activity that exerts a candidacidal effect in a cation concentration-dependent manner. We investigated the ability of this cationic protein (with an isoelectric point of 8.7) to permeabilize the cytoplasmic membrane of Candida albicans cells. Despite minor K(+)-release in lactoferrin-treated C. albicans cells, the killing effect was not related to an extensive membrane permeabilization, as indicated by: (a) the non-release of macromolecular cytosolic constituents; (b) the non-permeabilization for extracellular propidium iodide nor for intracellular accumulated calcein; and (c) the inability to disrupt the phospholipid bilayer of 8-aminonaphthalene-1,3,6, trisulfonic acid/p-xylene-bis-pyridiniumbromide-loaded liposomes. These results suggest that lactoferrin exerts its candidacidal effect through a mechanism different from membrane permeabilization described for other cationic peptides.

Different anti-Candida activities of two human lactoferrin-derived peptides, Lfpep and kaliocin-1.

The synthetic peptides Lfpep and kaliocin-1 include the sequences from positions 18 to 40 and 153 to 183 of human lactoferrin, respectively. Lfpep is a cationic peptide with bactericidal and giardicidal effects, whereas kaliocin-1 is a novel bactericidal peptide that corresponds to a highly homologous sequence present in the transferrin family of proteins. Both peptides presented fungicidal activity against Candida spp., including fluconazole- and amphotericin B-resistant clinical isolates. Lfpep exhibited higher antifungal activity (8- to 30-fold) and salt resistance than kaliocin-1. The killing activity of Lfpep was mediated by its permeabilizing activity on Candida albicans cells, whereas kaliocin-1 was unable to disrupt the cytoplasmic membrane, as indicated by its inability to allow permeation of propidium iodide and the small amount of K+ released. The amino acid sequence of kaliocin-1 includes the "multidimensional antimicrobial signature" conserved in disulfide-containing antimicrobial peptides and a striking similarity to brevinin-1Sa, an antimicrobial peptide from frog skin secretions, exhibiting a "Rana box"-like sequence. These features may be of interest in the design of new antifungals.

Antibiotic tolerance induced by lactoferrin in clinical Pseudomonas aeruginosa isolates from cystic fibrosis patients.

Lactoferrin-induced cell depolarization and a delayed tobramycin-killing effect on Pseudomonas aeruginosa cells were correlated. This antibiotic tolerance effect (ATE) reflects the ability of a defense protein to modify the activity of an antibiotic as a result of its modulatory effect on bacterial physiology. P. aeruginosa isolates from cystic fibrosis patients showed higher ATE values (< or = 6-fold) than other clinical strains.

Modulation of in vitro fungicidal activity of human lactoferrin against Candida albicans by extracellular cation concentration and target cell metabolic activity.

The anti-Candida activity of the innate defense protein human lactoferrin was investigated. Lactoferrin displayed a clear fungicidal effect against Candida albicans only under low-strength conditions. This candidacidal activity was inversely correlated with the extracellular concentration of the monovalent cations and was prevented by Na(+) and K(+) (> or 30 mM) and by divalent cations (Ca(2+) and Mg(2+) at > or 4 mM). A slight cellular release of K(+), cytosolic acidification, and a change in the membrane potential were observed in C. albicans cells treated with lactoferrin, suggesting that this protein directly or indirectly interacts with the cytoplasmic membrane. Mitochondrial inhibitors (carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, azide, and antimycin) as well as anaerobic conditions significantly reduced the killing effect of lactoferrin. These results suggest that low-strength conditions and the cellular metabolic state may modulate the candidacidal activity of human lactoferrin.