Whole genome sequencing reveals a frameshift mutation and a large deletion in YY1AP1 in a girl with a panvascular artery disease.

BACKGROUND: Rare diseases are pathologies that affect less than 1 in 2000 people. They are difficult to diagnose due to their low frequency and their often highly heterogeneous symptoms. Rare diseases have in general a high impact on the quality of life and life expectancy of patients, which are in general children or young people. The advent of high-throughput sequencing techniques has improved diagnosis in several different areas, from pediatrics, achieving a diagnostic rate of 41% with whole genome sequencing (WGS) and 36% with whole exome sequencing, to neurology, achieving a diagnostic rate between 47 and 48.5% with WGS. This evidence has encouraged our group to pursue a molecular diagnosis using WGS for this and several other patients with rare diseases. RESULTS: We used whole genome sequencing to achieve a molecular diagnosis of a 7-year-old girl with a severe panvascular artery disease that remained for several years undiagnosed. We found a frameshift variant in one copy and a large deletion involving two exons in the other copy of a gene called YY1AP1. This gene is related to Grange syndrome, a recessive rare disease, whose symptoms include stenosis or occlusion of multiple arteries, congenital heart defects, brachydactyly, syndactyly, bone fragility, and learning disabilities. Bioinformatic analyses propose these mutations as the most likely cause of the disease, according to its frequency, in silico predictors, conservation analyses, and effect on the protein product. Additionally, we confirmed one mutation in each parent, supporting a compound heterozygous status in the child. CONCLUSIONS: In general, we think that this finding can contribute to the use of whole genome sequencing as a diagnosis tool of rare diseases, and in particular, it can enhance the set of known mutations associated with different diseases.

Novel frameshift mutation in LIS1 gene is a probable cause of lissencephaly: a case report.

BACKGROUND: Lissencephaly (LIS) is a cortical malformation, characterized by smooth or nearly smooth cerebral surface and a shortage of gyral and sulcal development, which is caused by deficient neuronal migration during embryogenesis. Neuronal migration involves many gene products, among which is the product of the PAFAH1B1 gene, associated with this disease. LIS is a rare disease, characterized by low population frequency, and with non-specific clinical symptoms such as early epilepsy, developmental delay or cerebral palsy-like motor problems. Given that high-throughput sequencing techniques have been improving diagnosis, we have chosen this technique for addressing this patient. CASE PRESENTATION: We present the case of a seven years old male patient with an undiagnosed rare disease, with non-specific clinical symptoms possibly compatible with lissencephaly. The patient was enrolled in a study that included the sequencing of his whole genome. Sequence data was analyzed following a bioinformatic pipeline. The variants obtained were annotated and then subjected to different filters for prioritization. Also mitochondrial genome was analyzed. A novel candidate frameshift insertion in known PAFAH1B1 gene was found, explaining the index case phenotype. The assessment through in silico tools reported that it causes nonsense mediated mechanisms and that it is damaging with high confidence scores. The insertion causes a change in the reading frame, and produces a premature stop codon, severely affecting the protein function and probably the silencing of one allele. The healthy mother did not carry the mutation, and the unaffected father was not available for analysis. CONCLUSIONS: Through this work we found a novel de novo mutation in LIS1/PAFAH1B1 gene, as a likely cause of a rare disease in a young boy with non-specific clinical symptoms. The mutation found correlates with the phenotype studied since the loss of function in the gene product has already been described in this condition. Since there are no other variants in the PAFAH1B1 gene with low population frequency and due to family history, a de novo disease mechanism is proposed.

Phenotypical, Clinical, and Molecular Aspects of Adults and Children With Homozygous Familial Hypercholesterolemia in Iberoamerica.

OBJECTIVE: Characterize homozygous familial hypercholesterolemia (HoFH) individuals from Iberoamerica. Approach and Results: In a cross-sectional retrospective evaluation 134 individuals with a HoFH phenotype, 71 adults (age 39.3±15.8 years, 38.0% males), and 63 children (age 8.8±4.0 years, 50.8% males) were studied. Genetic characterization was available in 129 (96%). The majority (91%) were true homozygotes (true HoFH, n=79, 43.0% children, 46.8% males) or compound heterozygotes (compound heterozygous familial hypercholesterolemia, n=39, 51.3% children, 46.2% males) with putative pathogenic variants in the LDLR. True HoFH due to LDLR variants had higher total (P=0.015) and LDL (low-density lipoprotein)-cholesterol (P=0.008) compared with compound heterozygous familial hypercholesterolemia. Children with true HoFH (n=34) tended to be diagnosed earlier (P=0.051) and had a greater frequency of xanthomas (P=0.016) than those with compound heterozygous familial hypercholesterolemia (n=20). Previous major cardiovascular events were present in 25 (48%) of 52 children (missing information in 2 cases), and in 43 (67%) of 64 adults with LDLR variants. Children who are true HoFH had higher frequency of major cardiovascular events (P=0.02), coronary heart (P=0.013), and aortic/supra-aortic valve diseases (P=0.022) than compound heterozygous familial hypercholesterolemia. In adults, no differences were observed in major cardiovascular events according to type of LDLR variant. From 118 subjects with LDLR variants, 76 (64%) had 2 likely pathogenic or pathogenic variants. In 89 subjects with 2 LDLR variants, those with at least one null allele were younger (P=0.003) and had a greater frequency of major cardiovascular events (P=0.038) occurring at an earlier age (P=0.001). CONCLUSIONS: There was a high frequency of cardiovascular disease even in children. Phenotype and cardiovascular complications were heterogeneous and associated with the type of molecular defect.

Genomes of Fasciola hepatica from the Americas Reveal Colonization with Neorickettsia Endobacteria Related to the Agents of Potomac Horse and Human Sennetsu Fevers.

Food borne trematodes (FBTs) are an assemblage of platyhelminth parasites transmitted through the food chain, four of which are recognized as neglected tropical diseases (NTDs). Fascioliasis stands out among the other NTDs due to its broad and significant impact on both human and animal health, as Fasciola sp., are also considered major pathogens of domesticated ruminants. Here we present a reference genome sequence of the common liver fluke, Fasciola hepatica isolated from sheep, complementing previously reported isolate from cattle. A total of 14,642 genes were predicted from the 1.14 GB genome of the liver fluke. Comparative genomics indicated that F. hepatica Oregon and related food-borne trematodes are metabolically less constrained than schistosomes and cestodes, taking advantage of the richer millieux offered by the hepatobiliary organs. Protease families differentially expanded between diverse trematodes may facilitate migration and survival within the heterogeneous environments and niches within the mammalian host. Surprisingly, the sequencing of Oregon and Uruguay F. hepatica isolates led to the first discovery of an endobacteria in this species. Two contigs from the F. hepatica Oregon assembly were joined to complete the 859,205 bp genome of a novel Neorickettsia endobacterium (nFh) closely related to the etiological agents of human Sennetsu and Potomac horse fevers. Immunohistochemical studies targeting a Neorickettsia surface protein found nFh in specific organs and tissues of the adult trematode including the female reproductive tract, eggs, the Mehlis' gland, seminal vesicle, and oral suckers, suggesting putative routes for fluke-to-fluke and fluke-to-host transmission. The genomes of F. hepatica and nFh will serve as a resource for further exploration of the biology of F. hepatica, and specifically its newly discovered trans-kingdom interaction with nFh and the impact of both species on disease in ruminants and humans.

Two mutations in the SBDS gene reveal a diagnosis of Shwachman-Diamond syndrome in a patient with atypical symptoms.

We present the case of a 53-yr-old woman with an inherited bone marrow failure coexisting with uncommon extrahematological symptoms, such as cirrhosis and skin abnormalities. Whole-exome sequencing revealed a diagnosis of Shwachman-Diamond syndrome (SDS) with an atypical presentation. Unexpected was the age of disease expression, normally around the pediatric age, with a predominantly median survival age of 36 yr. To our knowledge, she was the first adult patient with a molecular diagnosis of Shwachman-Diamond in Uruguay. The patient was referred to our service when she was 43-yr-old with a history of bone marrow failure with anemia and thrombocytopenia. All secondary causes of pancytopenia were excluded. Bone marrow aspirate and biopsy specimens were hypocellular for the patient's age. Numerous dysplastic features were observed in the three lineages. She had a normal karyotype and normal chromosomal fragility. A diagnosis of low-risk hypoplastic MDS was made. Dermatological examination revealed reticulate skin pigmentation with hypopigmented macules involving the face, neck, and extremities; nail dystrophy; premature graying; and thin hair. Extrahematological manifestations were present (e.g., learning difficulties, short stature). Last, she was diagnosed with cryptogenic liver cirrhosis CHILD C. This rules out all other possible causes of chronic liver disease. This clinical presentation initially oriented the diagnosis toward telomeropathy, so we did a telomeropathy NGS panel that came up negative. Finally, we did an exome sequencing that confirmed the diagnosis of SDS. Using whole-exome sequencing, we were able to find two compound heterozygous mutations in the SBDS gene that were responsible for the phenotype of a patient that was undiagnosed for 10 years. An earlier genetic diagnosis could have influenced our patient's outcome.

Survey of transcripts expressed by the invasive juvenile stage of the liver fluke Fasciola hepatica.

BACKGROUND: The common liver fluke Fasciola hepatica is the agent of a zoonosis with significant economic consequences in livestock production worldwide, and increasing relevance to human health in developing countries. Although flukicidal drugs are available, re-infection and emerging resistance are demanding new efficient and inexpensive control strategies. Understanding the molecular mechanisms underlying the host-parasite interaction provide relevant clues in this search, while enlightening the physiological adaptations to parasitism. Genomics and transcriptomics are still in their infancy in F. hepatica, with very scarce information available from the invasive newly excysted juveniles (NEJ). Here we provide an initial glimpse to the transcriptomics of the NEJ, the first stage to interact with the mammalian host. RESULTS: We catalogued more than 500 clusters generated from the analysis of F. hepatica juvenile expressed sequence tags (EST), several of them not detected in the adult stage. A set of putative F. hepatica specific transcripts, and a group of sequences conserved exclusively in flatworms were identified. These novel sequences along with a set of parasite transcripts absent in the host genomes are putative new targets for future anti-parasitic drugs or vaccine development. Comparisons of the F. hepatica sequences with other metazoans genomes or EST databases were consistent with the basal positioning of flatworms in the bilaterian phylogeny. Notably, GC content, codon usage and amino acid frequencies are remarkably different in Schistosomes to F. hepatica and other trematodes. Functional annotation of predicted proteins showed a general representation of diverse biological functions. Besides proteases and antioxidant enzymes expected to participate in the early interaction with the host, various proteins involved in gene expression, protein synthesis, cell signaling and mitochondrial enzymes were identified. Differential expression of secreted protease gene family members between juvenile and adult stages may respond to different needs during host colonization. CONCLUSION: The knowledge of the genes expressed by the invasive stage of Fasciola hepatica is a starting point to unravel key aspects of this parasite's biology. The integration of the emerging transcriptomics, and proteomics data and the advent of functional genomics tools in this organism are positioning F. hepatica as an interesting model for trematode biology.

Gene Silencing in the Liver Fluke Fasciola hepatica: RNA Interference.

The chronic infection with the liver fluke of the genus Fasciola spp. is the most prevalent foodborne trematodiasis, affecting at least one-fourth of the world livestock grazing in areas where the parasite is present. Moreover, fascioliasis is considered a major zoonosis mainly in rural areas of central South America, Northern Africa, and Central Asia. Increasing evidences of resistance against triclabendazole may compromise its use as drug of choice; thus, novel control strategies are desperately needed. Functional genomic approaches play a key role in the validation and characterization of new targets for drug and vaccine development. So far, RNA interference has been the only gene silencing approach successfully employed in liver flukes of the genus Fasciola spp. Herein, we describe a detailed step-by-step protocol to perform gene silencing mediated by RNAi in Fasciola hepatica.

Clinical and molecular aspects of familial hypercholesterolemia in Ibero-American countries.

BACKGROUND: There is little information about familial hypercholesterolemia (FH) epidemiology and care in Ibero-American countries. The Ibero-American FH network aims at reducing the gap on diagnosis and treatment of this disease in the region. OBJECTIVE: To describe clinical, molecular, and organizational characteristics of FH diagnosis in Argentina, Brazil, Chile, Colombia, Mexico, Portugal, Spain, and Uruguay. METHODS: Descriptive analysis of country data related to FH cascade screening, molecular diagnosis, clinical practice guidelines, and patient organization presence in Ibero-America. RESULTS: From a conservative estimation of an FH prevalence of 1 of 500 individuals, there should be 1.2 million heterozygous FH individuals in Ibero-America and roughly 27,400 were diagnosed so far. Only Spain, Brazil, Portugal, and Uruguay have active cascade screening programs. The prevalence of cardiovascular disease ranged from 10% to 42% in member countries, and the highest molecular identification rates are seen in Spain, 8.3%, followed by Portugal, 3.8%, and Uruguay with 2.5%. In the 3 countries with more FH patients identified (Spain, Portugal, and Brazil) between 10 and 15 mutations are responsible for 30% to 47% of all FH cases. Spain and Portugal share 5 of the 10 most common mutations (4 in low density lipoprotein receptor [LDLR] and the APOB3527). Spain and Spanish-speaking Latin American countries share 6 of the most common LDLR mutations and the APOB3527. LDL apheresis is available only in Spain and Portugal and not all countries have specific FH diagnostic and treatment guidelines as well as patient organizations. CONCLUSIONS: Ibero-American countries share similar mutations and gaps in FH care.

Identification of Chalcones as Fasciola hepatica Cathepsin L Inhibitors Using a Comprehensive Experimental and Computational Approach.

BACKGROUND: Increased reports of human infections have led fasciolosis, a widespread disease of cattle and sheep caused by the liver flukes Fasciola hepatica and Fasciola gigantica, to be considered an emerging zoonotic disease. Chemotherapy is the main control measure available, and triclabendazole is the preferred drug since is effective against both juvenile and mature parasites. However, resistance to triclabendazole has been reported in several countries urging the search of new chemical entities and target molecules to control fluke infections. METHODOLOGY/PRINCIPLE FINDINGS: We searched a library of forty flavonoid derivatives for inhibitors of key stage specific Fasciola hepatica cysteine proteases (FhCL3 and FhCL1). Chalcones substituted with phenyl and naphtyl groups emerged as good cathepsin L inhibitors, interacting more frequently with two putative binding sites within the active site cleft of the enzymes. One of the compounds, C34, tightly bounds to juvenile specific FhCL3 with an IC50 of 5.6 µM. We demonstrated that C34 is a slow-reversible inhibitor that interacts with the Cys-His catalytic dyad and key S2 and S3 pocket residues, determinants of the substrate specificity of this family of cysteine proteases. Interestingly, C34 induces a reduction in NEJ ability to migrate through the gut wall and a loss of motility phenotype that leads to NEJ death within a week in vitro, while it is not cytotoxic to bovine cells. CONCLUSIONS/SIGNIFICANCE: Up to date there are no reports of in vitro screening for non-peptidic inhibitors of Fasciola hepatica cathepsins, while in general these are considered as the best strategy for in vivo inhibition. We have identified chalcones as novel inhibitors of the two main Cathepsins secreted by juvenile and adult liver flukes. Interestingly, one compound (C34) is highly active towards the juvenile enzyme reducing larval ability to penetrate the gut wall and decreasing NEJ´s viability in vitro. These findings open new avenues for the development of novel agents to control fluke infection and possibly other helminthic diseases.

The miRnome of Fasciola hepatica juveniles endorses the existence of a reduced set of highly divergent micro RNAs in parasitic flatworms.

The liver fluke Fasciola hepatica is a foodborne zoonotic parasite affecting livestock worldwide, with increasing relevance in human health. The first developmental stage that the host meets after ingestion of the parasite is the newly excysted juvenile, that actively transverses the gut wall and migrates to its final location in the liver. The regulation of the early developmental events in newly excysted juveniles is still poorly understood and a relevant target for control strategies. Here we investigated the putative involvement of small regulatory RNAs in the invasion process. The small RNA population of the newly excysted juvenile fall into two classes, one represented by micro (mi)RNAs and a secondary group of larger (32-33 nucleotides) tRNA-derived sequences. We identified 40 different miRNAs, most of those belonging to ancient miRNAs conserved in protostomes and metazoans, notably with a highly predominant miR-125b variant. Remarkably, several protostomian and metazoan conserved families were not detected in consonance with previous reports of drastic miRnome reduction in parasitic flatworms. Additionally, a set of five novel miRNAs was identified, probably associated with specific gene regulation expression needs in F. hepatica. While sequence conservation in mature miRNA is high across the metazoan tree, we observed that flatworm miRNAs are more divergent, suggesting that mutation rates in parasitic flatworms could be high. Finally, the distinctive presence of tRNA-derived sequences, mostly 5' tRNA halves of selected tRNAs in the small RNA population of newly excysted juveniles, raises the possibility that both miRNA and tRNA fragments participate in the regulation of gene expression in this parasite.

Genética de la hipercolesterolemia familiar / Genetics of familial hypercholesterolemia / Genética da hipercolesterolemia familiar

Resumen: La hipercolesterolemia familiar (HF) es un trastorno hereditario caracterizado por niveles séricos anormalmente altos de colesterol desde el nacimiento. Por este motivo, los pacientes con HF presentan un riesgo muy alto de sufrir eventos cardiovasculares prematuros. Con una prevalencia de 1 en 250, la HF es considerada hoy día una de las enfermedades genéticas más frecuentes en la población mundial. Sin embargo, a pesar de su elevada frecuencia y de que existen tratamientos efectivos para controlarla, la HF continúa siendo subtratada y subdiagnosticada. La HF es un trastorno oligogénico del metabolismo lipídico determinado principalmente por mutaciones en el gen LDLR (90% de los casos) y de herencia autosómica dominante. Con menos frecuencia se han encontrado mutaciones en genes APOB, PCSK9, STAP1 y APOE (también de herencia autosómica dominante) y en el gen LDLRAP1 (de herencia autosómica recesiva). En los pacientes sin mutaciones en estos genes, el aumento del colesterol puede ser explicado por la suma de alelos de riesgo en distintos loci, configurando una HF poligénica. Esta diversidad de mutaciones mayores y variantes comunes polimórficas conduce a un amplio espectro de presentaciones clínicas. La presencia de mutaciones en el gen LDLR aumenta más de veinte veces el riesgo de sufrir eventos coronarios. La identificación de una variante patogénica permite iniciar el diagnóstico familiar en cascada, que es la estrategia más costo-efectiva para la identificación de afectados. Es por esto que en los últimos años se ha destacado la importancia del diagnóstico molecular como conductor de la intervención médica y del seguimiento familiar.

Summary: Familial hypercholesterolemia is an inherited disorder characterized by abnormally high serum cholesterol levels from birth. For this reason, patients have a very high risk of suffering premature cardiovascular events. With a prevalence of 1 in 250, it is now considered one of the most frequent genetic diseases in the world. However, despite its high frequency and effective treatments, it continues to be under-treated and under-diagnosed worldwide. Familial hypercholesterolemia is an oligogenic disorder of lipid metabolism mainly determined by mutations in LDLR gene (90% of cases) and of autosomal dominant inheritance. Less frequently, mutations have been found in APOB, PCSK9, STAP1 and APOE genes (also of autosomal dominant inheritance) and in the LDLRAP1 gene (of autosomal recessive inheritance). In patients without mutations in these genes, the increase in their cholesterol can be explained by the sum of risk alleles in different loci, configuring a polygenic disease. This diversity of major mutations and common polymorphic variants lead to a wide spectrum of clinical presentations. The presence of mutations in the LDLR gene increases the risk of coronary events over twenty times. The identification of a pathogenic variant allows family cascade screening, which is the most cost-effective strategy for the identification of affected persons. That is why in recent years the importance of molecular diagnosis as a driver of medical intervention and family monitoring has been highlighted.

Resumo: A hipercolesterolemia familiar é um distúrbio hereditário caracterizado por níveis séricos de colesterol anormalmente altos desde o nascimento. Por esse motivo, os pacientes apresentam um risco muito alto de sofrer eventos cardiovasculares prematuros. Com prevalência de 1 em 250, é hoje considerada uma das doenças genéticas mais frequentes na população mundial. No entanto, apesar de sua alta frequência e da existência de tratamentos eficazes para controlá-la, ela continua sendo subtratada e subdiagnosticada em todo o mundo. É um distúrbio oligogênico do metabolismo lipídico, determinado principalmente por mutações no gene LDLR (90% dos casos) e herança autossômica dominante. Menos frequentemente, foram encontradas mutações nos genes APOB, PCSK9, STAP1 e APOE (também de herança autossômica dominante) e no gene LDLRAP1 (de herança autossômica recessiva). Em pacientes sem mutações nesses genes, o aumento do colesterol pode ser explicado pela soma dos alelos de risco em diferentes loci, configurando uma hipercolesterolemia familiar poligênica. Essa diversidade de grandes mutações e variantes polimórficas comuns leva a um amplo espectro de apresentações clínicas. A presença de mutações no gene LDLR aumenta o risco de eventos coronarianos mais de vinte vezes. A identificação de uma variante patogênica permite iniciar o diagnóstico da família em cascata, que é a estratégia mais econômica para a identificação das pessoas afetadas. É por isso que, nos últimos anos, foi destacada a importância do diagnóstico molecular como fator de intervenção médica e monitoramento familiar.

Síndrome branquio-oto-renal. Presentación de un caso / Branchio-oto-renal syndrome. Case study / Síndrome brânquio-otorrenal. Relato de caso

Resumen: El síndrome branquio-oto-renal (BOR) es un trastorno hereditario autosómico dominante, poco frecuente, que se presenta con expresividad variable y penetrancia reducida, con gran heterogeneidad clínica inter e intrafamiliar. Es causado por alteraciones del desarrollo del oído, riñones y de los segundos arcos branquiales, estructura que en el embrión en desarrollo da origen a tejidos del cuello. Los hallazgos clínicos más comunes son las fístulas o quistes en el cuello, déficit auditivo, malformaciones del oído externo con apéndices u hoyuelos preauriculares y afectación renal uni o bilateral que va de moderada a severa. El síndrome es causado principalmente por mutaciones en el gen EYA1 que actúa como regulador transcripcional durante la embriogénesis, las cuales se detectan en el 40% de los pacientes. Presentamos un caso ilustrativo de síndrome de BOR que se mostró con hallazgos clínicos sugestivos como fositas preauriculares, fístulas branquiales, hipoacusia severa con anomalías anatómicas del oído, compromiso renal y antecedentes familiares. En este paciente se encontró una variante patogénica en el gen EYA1: c.1081C>T (p.Arg361TER). Esta es considerada una variante nula, ya que provoca un codón de terminación prematuro que conduce a una pérdida de la función de la proteína. Dada la expresividad variable del síndrome de BOR, el diagnóstico molecular cobra importancia para evitar errores diagnósticos, iniciar el seguimiento familiar en cascada con el fin de identificar familiares afectados y para implementar medidas preventivas tendientes a disminuir la morbilidad y mortalidad causadas por este síndrome.

Summary: The branchio-oto-renal syndrome (BOR) is an uncommon autosomal dominant genetic disorder with variable expressivity, reduced penetrance and significant clinical and intra-familial heterogeneity. It is caused by alterations in the development of the ears, kidneys and second branchial arches, a structure that in developing embryos produces neck tissue. The most common clinical findings are neck fistulas or cysts, hearing loss, malformations of the outer ear with appendices and/or pre-auricular dimples and unilateral or bilateral moderate to severe renal involvement. The syndrome is mainly caused by mutations in the EYA1 gene that acts as a transcriptional regulator during embryogenesis, and which are detected in 40% of patients. We hereby introduce a representative case of BOR syndrome that showed suggestive clinical findings such as preauricular holes, branchial fistulas, severe hearing loss with anatomical anomalies of the ear, and renal impairment. In this case, we found a pathogenic variant in the EYA1 gene: c.1081C>T (p.Arg361TER). This is considered a null variant, since it causes a premature stop codon that leads to protein function loss. Given the BOR Syndrome variable expressivity, molecular diagnosis is relevant to prevent diagnostic errors, initiate familial cascade screening in order to identify affected relatives and to implement preventive measures aimed at reducing the morbidity and mortality caused by this syndrome.

Resumo: A síndrome branquio-oto-renal (BOR) é uma desordem genética autossômica dominante incomum com expressividade variável, penetrância reduzida e significativa heterogeneidade clínica e intrafamiliar. É causada por alterações no desenvolvimento das orelhas, rins e segundos arcos branquiais, as quais durante o desenvolvimento do embrião produzem tecido no pescoço. Os achados clínicos mais comuns são fístulas ou cistos no pescoço, déficit auditivo, malformações da orelha externa com apêndices e / ou seios pré-auriculares e comprometimento renal moderado a grave unilateral ou bilateral. A síndrome é causada principalmente por mutações no gene EYA1, que atua como regulador da transcrição durante a embriogênese e é detectada em 40% dos pacientes. Introduzimos aqui um caso representativo da síndrome BOR que apresentou achados clínicos sugestivos, como fossas pré-auriculares, fístulas branquiais, perda auditiva grave com anomalias anatômicas da orelha e comprometimento renal. Nesse caso, encontramos uma variante patogênica no gene EYA1: c.1081C> T (p.Arg361TER). Isso é considerado uma variante nula, pois causa um códon de parada prematuro que leva a uma perda da função da proteína. Dada a expressividade variável da síndrome do ROR, o diagnóstico molecular é relevante para evitar erros de diagnóstico, iniciar o rastreamento em cascata familiar, a fim de identificar os familiares afetados e implementar medidas preventivas destinadas a reduzir a morbimortalidade causada pela síndrome.

RNA interference in Fasciola hepatica newly excysted juveniles: long dsRNA induces more persistent silencing than siRNA.

In trematodes RNA interference is the current tool of choice for functional analysis of genes since classical reverse genetic approaches remain unavailable. Whereas this approach has been optimized in schistosomes, few reports are available for other trematodes, likely reflecting the difficulties in the establishment of the technology. Here we standardized conditions for RNAi in the liver fluke Fasciola hepatica, the causative agent of fasciolosis, one of the most problematic infections affecting livestock worldwide. Targeting a single copy gene, encoding leucine aminopeptidase (LAP) as a model, we refined delivery conditions which identified electro-soaking, i.e. electroporation and subsequent incubation as efficient for introduction of small RNAs into the fluke. Knock down of LAP was achieved with as little as 2.5 µg/ml dsRNA concentrations, which may reduce or obviate off-target effects. However, at these concentrations, tracking incorporation by fluorescent labeling was difficult. While both long dsRNA and short interfering RNA (siRNA) are equally effective at inducing a short-term knock down, dsRNA induced persistent silencing up to 21 days after treatment, suggesting that mechanisms of amplification of the interfering signal can be present in this pathogen. Persistent silencing of the invasive stage for up to 3 weeks (close to what it takes for the fluke to reach the liver) opens the possibility of using RNAi for the validation of putative therapeutic targets.