Phylogenetic analysis and identification of Aeromonas species based on sequencing of the cpn60 universal target

An analysis of the universal target (UT) sequence from the cpn60 gene was performed in order to evaluate its usefulness in phylogenetic and taxonomic studies and as an identification marker for the genus Aeromonas. Sequences of 555 bp, corresponding to the UT region, were obtained from a collection of 35 strains representing all of the species and subspecies of Aeromonas. From the analysis of these sequences, a range of divergence of 0-23.3% was obtained, with a mean of 11.2±0.9 %. Comparative analyses between cpn60 and gyrB, rpoD and 16S rRNA gene sequences were carried out from the same Aeromonas strain collection. Sequences of the cpn60 UT region showed similar discriminatory power to gyrB and rpoD sequences. The phylogenetic relationships inferred from cpn60 sequence distances indicated an excellent correlation with the present affiliation of Aeromonas species with the exception of Aeromonas hydrophila subsp. dhakensis, which appeared in a separate phylogenetic line, and Aeromonas sharmana, which exhibited a very loose phylogenetic relationship to the genus Aeromonas. Sequencing of cpn60 from 33 additional Aeromonas strains also allowed us to establish intra- and interspecific threshold values. Intraspecific divergence rates were ≤3.5 %, while interspecific divergence rates fell between 3.7 and 16.9 %, excluding A. sharmana. In this study, cpn60 UT sequencing was shown to be a universal, useful, simple and rapid method for the identification and phylogenetic affiliation of Aeromonas strains

Chiari malformation type I: a case-control association study of 58 developmental genes

Chiari malformation type I (CMI) is a disorder characterized by hindbrain overcrowding into an underdeveloped posterior cranial fossa (PCF), often causing progressive neurological symptoms. The etiology of CMI remains unclear and is most likely multifactorial. A putative genetic contribution to CMI is suggested by familial aggregation and twin studies. Experimental models and human morphometric studies have suggested an underlying paraxial mesoderm insufficiency. We performed a case-control association study of 303 tag single nucleotide polymorphisms (SNP) across 58 candidate genes involved in early paraxial mesoderm development in a sample of 415 CMI patients and 524 sex-matched controls. A subgroup of patients diagnosed with classical, small-PCF CMI by means of MRI-based PCF morphometry (n = 186), underwent additional analysis. The genes selected are involved in signalling gradients occurring during segmental patterning of the occipital somites (FGF8, Wnt, and retinoic acid pathways and from bone morphogenetic proteins or BMP, Notch, Cdx and Hox pathways) or in placental angiogenesis, sclerotome development or CMI-associated syndromes. Single-marker analysis identified nominal associations with 18 SNPs in 14 genes (CDX1, FLT1, RARG, NKD2, MSGN1, RBPJ1, FGFR1, RDH10, NOG, RARA, LFNG, KDR, ALDH1A2, BMPR1A) considering the whole CMI sample. None of these overcame corrections for multiple comparisons, in contrast with four SNPs in CDX1, FLT1 and ALDH1A2 in the classical CMI group. Multiple marker analysis identified a risk haplotype for classical CMI in ALDH1A2 and CDX1. Furthermore, we analyzed the possible contributions of the most significantly associated SNPs to different PCF morphometric traits. These findings suggest that common variants in genes involved in somitogenesis and fetal vascular development may confer susceptibility to CMI

Rare functional genetic variants in COL7A1, COL6A5, COL1A2 and COL5A2 frequently occur in Chiari Malformation Type 1

Seqüenciació de gens; Genòmica; Imatges per ressonància magnèticaSecuenciación de genes; Genómica; Imágenes por resonancia magnéticaGene sequencing; Genomics; Magnetic resonance imagingChiari Malformation Type 1 (CM-1) is characterized by herniation of the cerebellar tonsils below the foramen magnum and the presence of headaches and other neurologic symptoms. Cranial bone constriction is suspected to be the most common biologic mechanism leading to CM-1. However, other mechanisms may also contribute, particularly in the presence of connective tissue disorders (CTDs), such as Ehlers Danlos Syndrome (EDS). Accumulating data suggest CM-1 with connective tissue disorders (CTD+) may have a different patho-mechanism and different genetic risk factors than CM-1 without CTDs (CTD-). To identify CM-1 genetic risk variants, we performed whole exome sequencing on a single large, multiplex family from Spain and targeted sequencing on a cohort of 186 unrelated adult, Caucasian females with CM-1. Targeted sequencing captured the coding regions of 21 CM-1 and EDS candidate genes, including two genes identified in the Spanish family. Using gene burden analysis, we compared the frequency of rare, functional variants detected in CM-1 cases versus publically available ethnically-matched controls from gnomAD. A secondary analysis compared the presence of rare variants in these genes between CTD+ and CTD- CM-1 cases. In the Spanish family, rare variants co-segregated with CM-1 in COL6A5, ADGRB3 and DST. A variant in COL7A1 was present in affected and unaffected family members. In the targeted sequencing analysis, rare variants in six genes (COL7A1, COL5A2, COL6A5, COL1A2, VEGFB, FLT1) were significantly more frequent in CM-1 cases compared to public controls. In total, 47% of CM-1 cases presented with rare variants in at least one of the four significant collagen genes and 10% of cases harbored variants in multiple significant collagen genes. Moreover, 26% of CM-1 cases presented with rare variants in the COL6A5 gene. We also identified two genes (COL7A1, COL3A1) for which the burden of rare variants differed significantly between CTD+ and CTD- CM-1 cases. A higher percentage of CTD+ patients had variants in COL7A1 compared to CTD+ patients, while CTD+ patients had fewer rare variants in COL3A1 than did CTD- patients. In summary, rare variants in several collagen genes are particularly frequent in CM-1 cases and those in COL6A5 co-segregated with CM-1 in a Spanish multiplex family. COL6A5 has been previously associated with musculoskeletal phenotypes, but this is the first association with CM-1. Our findings underscore the contribution of rare genetic variants in collagen genes to CM-1, and suggest that CM-1 in the presence and absence of CTD symptoms is driven by different genes.This work was supported by a grant from Conquer Chiari to AAK. Collection of the Chiari1000 study participants utilized in this study was supported by a grant from Conquer Chiari to FL at University of Akron. Collection of the Duke study participants utilized in this study was supported by a grant from the National Institutes of Health (NS063273). A.U. was the recipient of a Postdoctoral Fellowship from Fundación Ramón Areces (Spain). RL is the Executive Director of Conquer Chiari which provided some of the funding for this work. For the manuscript, he assisted with revising and editing the manuscript. The funders did have a role in study design, but had no role in data collection and analysis, decision to publish, or preparation of the manuscript

Expression Levels of an Alpha-Synuclein Transcript in Blood May Distinguish between Early Dementia with Lewy Bodies and Parkinson's Disease

Lewy body diseases (LBD) including dementia with Lewy bodies (DLB) and Parkinson disease (PD) are characterized by alpha-synuclein pathology. DLB is difficult to diagnose and peripheral biomarkers are urgently needed. Therefore, we analyzed the expression of five alpha-synuclein gene (SNCA) transcripts, SNCAtv1, SNCAtv2, SNCAtv3, SNCA126, and SNCA112, in 45 LBD and control temporal cortex samples and in the blood of 72 DLB, 59 PD, and 54 control subjects. The results revealed overexpression of SNCAtv1 and SNCA112 in DLB, and SNCAtv2 in PD temporal cortices. In DLB blood, diminution of all SNCA transcripts was observed. SNCAtv1 and SNCAtv2 were diminished in PD with disease onset before 70 years. SNCAtv3, driven by its own promoter, showed opposite expression in early DLB and PD, suggesting that its amount may be an early, DLB specific biomarker. Correlation between blood transcript levels and disease duration was positive in DLB and negative in PD, possibly reflecting differences in brain alpha-synuclein aggregation rates associated with differences in disease courses. In conclusion, SNCA transcripts showed a disease-specific increase in the brain and were diminished in blood of LBD patients. SNCAtv3 expression was decreased in early DLB and increased in early PD and could be a biomarker for early DLB diagnosis

Is oligoprogression a potentially curable disease in epidermal growth factor receptor mutant lung adenocarcinoma?

Third-generation epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) have shown impressive results in EGFR mutant lung cancer (LC) patients in terms of disease control rate with a positive impact on overall survival. Nevertheless, after months of treatment with targeted therapy, progression inevitably occurs. Some patients develop oligoprogression and local treatment is required for optimal disease control while maintaining EGFR-TKIs. This work features a clinical case of a patient harboring an EGFR mutant LC undergoing oligoprogression to EGFR-TKIs, first into the brain and afterward to the primary tumor, requiring local ablative strategies, including primary tumor resection three years after the start of osimertinib. Currently, the patient is still alive and continues with a complete response upon EGFR-TKIs maintenance. Hence, oligoprogression, even in driven oncogenic tumors, represents a distinct biological entity and potential curative disease that deserves particular consideration in multidisciplinary tumor boards. In this case, tumor primary resection after three years of the initial diagnosis represents a paradigm shift in the treatment of EGFR mutant patients

Bases genètiques de la malformació de Chiari

La Malformació de Chiari tipus I (MCI) ha estat definida tradicionalment com la herniació de les amígdales cerebel•loses d’almenys 5mm, a través del forat mange. En general, els símptomes es posen de manifest durant la segona o tercera dècada de vida, tot i que s’han descrit casos pediàtrics. Donada la complexitat del quadre clínic, per realitzar un diagnòstic adient es requereix avaluació clínica i estudi de neuroimatge. La tècnica de preferència és la ressonància magnètica d’imatge, considerant-se actualment com a pacients de MCI aquells que presenten un descens de les amígdales superior a 3mm per sota del forat magne. L'existència de casos asimptomàtics dificulta establir una prevalença concreta, però s’ha estimat que podria estar entre 1/1000 a 1/5000 sent major en dones que en homes (2:1 aproximadament). Fins el moment, es desconeix l’etiologia de la malaltia però la hipòtesi més acceptada és que MCI és deguda al desenvolupament insuficient del mesoderm paraxial. Diferents estudis realitzats fins el moment evidencien que almenys, un subgrup de pacients amb MCI són deguts a contribució genètica: 1) casos d’agregació familiar amb afectes en tres generacions; 2) estudis de bessons 3) associació amb síndromes genètics coneguts amb herència mendeliana produïts per anomalies óssies que donen suport a la hipòtesi de la insuficiència del mesoderm com a causa de MCI. Davant l’evidència clara d’un component genètic com a principal causant de l’etiologia de MCI, l’objectiu del projecte va ser la identificació de les bases genètiques de la MCI, tant en gens responsables de les formes mendelianes com en gens responsables de les formes complexes de MCI mitjançant dues estratègies: 1-Identificació de variants genètiques de susceptibilitat en pacients amb MCI mitjançant estudis d’associació de tipus cas-control. 2-Anàlisi genètic de formes monogèniques mitjançant l’anàlisi de lligament a marcardors polimòrfics i la seqüenciació del DNA a gran escala.Chiari malformation type I (CMI) has been traditionally defined as the chronic tonsillar herniation of at least 5 mm below the foramen magnum (FM). In general, the symptoms manifest during the second or third decade of life, however, several pediatric cases have been described. Given the complexity of this symptoms, to perform a proper diagnostic of CMI, a clinical evaluation and a neuroimage analysis is required. The preferred diagnostic method is the Magnetic Resonance image (MRI), considering as CMI those patients showing a tonsilar descent upper 3mm through the FM. The existence of asymptomatic cases of CMI difficult to establish its prevalence, however, it has been estimated to be in the range of 1/1000 to 1/5000 individuals, being higher in women than in men (2:1, approximately). The etiology of this disease remains unknown, but the most accepted hypothesis is that CMI is the result of an insufficient development of the paraxial mesoderm. Several evidences suggest that, at least in a subset of CMI cases, genetic factors may play an important role: 1) cases of family aggregation with individuals affected in at least three different generations; 2) twin studies were monozygotic twins reveal a higher degree of concordance; 3) cases were CMI associated as part of known Mendelian syndromes, some of which feature prominent bony abnormalities, supporting the mesodermal origin of the disorder. Considering the clear evidences for a genetic contribution in the origin of CMI, this project was focused on identifying the genetic basis underlying the CMI, detecting the genes responsible for either the mendelian and the complex forms of CMI. To achive this main goal, two different strategies were followed: 1- Identification of susceptibility genetic variants in CMI patients using a case-control association approach. 2- Genetic analyses of monogenic forms by linkage analysis with polymorphic markers and high-throughput DNA sequencing

Bases genètiques en la malformació de Chiari tipus i

[cat] La malformació de Chiari de tipus I (MCI) s'ha definit tradicionalment com una alteració congènita caracteritzada pel descens de les amígdales cerebel.loses a travès del forat magne en 3 o més mm. Es creu que és conseqüència d'una fosa cranial posterior (FCP) hipoplàstica, amb un os occipital anormalment curt, que resulta d'un mesoderma paraxial insuficient. La simptomatologia descrita pels pacients és molt variada: mals de cap occipitals, cervicàlgies, marejos, vertígens, pèrdua de sensibilitat i de força a les extremitats, alteracions de la son i problemes en la deglució entre d'altres, atribuïts a la compressió de les estructures neurològiques contingudes en la FCP i a l'alteració de la circulació del líquid cefaloraquidi. S'ha estimat la seva prevalença en 1/1280 i, tot i que es desconeix la seva etiologia, diversos estudis apunten a una component genètica com la causa més probable. En la MCI molts dels casos són esporàdics, però s'han identificat famílies amb diversos individus afectats en diferents generacions. Per aquest motiu, aquest treball va tenir com a objectiu principal estudiar les bases genètiques de la patologia. Es van reclutar més de 530 pacients, pertanyents a 450 famílies, i es van desenvolupar dues estratègies: 1) estudi de les formes aparentment complexes de la patologia mitjançant un estudi d'associació genètica, en què es van avaluar variants de susceptibilitat en gens implicats en el desenvolupament del mesoderma paraxial, somites, endoteli vascular i os occipital; i 2) estudi de les formes aparentment monogèniques, mitjançant un estudi mutacional del gen SUZ12 que, en haploinsuficiència en un model murí, produeix fenotips similars als de les malformacions de Chiari tipus I i II. Es van seqüenciar també els exornes de dos pacients pertanyents a una mateixa família. Donat que el descens amigdalar cerebel.lós pot ser produït per diferents causes, es va realitzar també un estudi morfomètric de la FCP en imatges de ressonància magnètica amb la finalitat de definir un mètode de diagnòstic més específic que l'existent, i que permetés diferenciar els pacients amb MCI produïda per una FCP hipoplàstica de la produïda de forma secundària, per altres mecanismes. Es va caracteritzar també la cavitat orofaríngia dels pacients. Els resultats que es van obtenir demostren que és necessari establir un nou criteri diagnòstic que permeti diferenciar els pacients amb MCI en funció de la seva causa, la qual cosa facilitaria l'estudi de la base genètica d'aquesta malformació. S'ha trobat correlació entre certes alteracions a la cavitat orofaríngia i símptomes típics de MCI com les apnees de la son o problemes en la deglució. També s'han identificat variants de susceptibilitat en diferents gens implicats en la via de síntesi de l'àcid retinoic. Però els estudis de cribatge mutacional del gen candidat i de seqüenciació d'exornes en casos familiars no han permès identificar gens responsables de la MCI.[eng] Chiari malformation type I (CMI) is a congenital disease that has been traditionally defined as the chronic tonsillar herniation of at least 3 mm below the foramen magnum. It is characterized by an overcrowding of a normally developed hindbrain within a hypoplastic PCF, although both age at presentation and symptoms show high variability. The etiology of the disease remains unknown, but the most accepted hypothesis is that CMI is the result of an insufficient development of the paraxial mesoderm. Several evidences suggest that, at least in a subset of CMI patients, genetic factors may play an important role. This project focused on the identification of genetic risk factors for CMI, including genes responsible for the mendelian and also the complex forms of the disease. To achieve this goal, two different strategies were followed: 1) Identification of susceptibility genetic variants in CMI patients using a casecontrol association approach. 2) Genetic analyses of monogenic forms by a mutational screen of the candidate gene SUZ12 and by exome sequencing in two CMI patients ofthe same family. We recruited 530 CMI patients from 450 unrelated families. Given that tonsilar herniation can be caused by five distinct mechanisms, we developed a probability model to establish a diagnosis with 93% confidence in those patients with a hypoplastic PCF. This diagnostic tool allowed to define a clinical homogeneous group for further genetic studies. A cephalometric oropharynx morphometric study was also performed to identify the possible underlying anatomical anomalies leading to less known symptoms. The results of the genetics association study paint to a putative role for defective retinoic acid signalling and fetal vasculogenesis in causing hypoplasia of the basichondrocranium. However, when we studied the monogenic forms, we did not find any causal variant

Obtaining miRNA from Saliva-Comparison of Sampling and Purification Methods

The use of saliva as a biomarker source has advantages over other biofluids and imaging techniques, and miRNAs are ideal biomarker candidates. They are involved in numerous cellular processes, and their altered expression suggests that miRNAs play a crucial regulatory role in disease development. We wanted to find an easily reproducible and executable miRNA-obtaining methodology suitable for quantification. Three commercial miRNA extraction kits (mirVana, Nucleospin and miRNeasy) and three saliva collectors (50 mL tubes, Salimetrics and Oragene) were tested. Several features, including RNA quality and technical parameters, were evaluated. The expression of five synthetic spike-in controls and seven saliva-miRNAs was analyzed independently and grouped by the collectors and the extraction kits. The combination of Oragene and miRNeasy assured the most sensitive detection of all seven saliva miRNAs. Testing different combinations of saliva collectors and RNA purification kits permitted the establishment of combinations for different uses. The results of our study highlight that optimization of resources for biomarker studies is possible after careful planning of each study

Chiari Malformation Type I: A Case-Control Association Study of 58 Developmental Genes

Chiari malformation type I (CMI) is a disorder characterized by hindbrain overcrowding into an underdeveloped posterior cranial fossa (PCF), often causing progressive neurological symptoms. The etiology of CMI remains unclear and is most likely multifactorial. A putative genetic contribution to CMI is suggested by familial aggregation and twin studies. Experimental models and human morphometric studies have suggested an underlying paraxial mesoderm insufficiency. We performed a case-control association study of 303 tag single nucleotide polymorphisms (SNP) across 58 candidate genes involved in early paraxial mesoderm development in a sample of 415 CMI patients and 524 sex-matched controls. A subgroup of patients diagnosed with classical, small-PCF CMI by means of MRI-based PCF morphometry (n = 186), underwent additional analysis. The genes selected are involved in signalling gradients occurring during segmental patterning of the occipital somites (FGF8, Wnt, and retinoic acid pathways and from bone morphogenetic proteins or BMP, Notch, Cdx and Hox pathways) or in placental angiogenesis, sclerotome development or CMI-associated syndromes. Single-marker analysi

A Retrospective 2D Morphometric Analysis of Adult Female Chiari Type I Patients with Commonly Reported and Related Conditions

Purpose: Researchers have sought to better understand Chiari type I malformation (CMI) through morphometric measurements beyond tonsillar position (TP). Soft tissue and bone structures within the brain and craniocervical junction have been shown to be different for CMI patients compared to healthy controls. Yet, several morphological characteristics have not been consistently associated with CMI. CMI is also associated with different prevalent conditions (PCs) such as syringomyelia, pseudotumor, Ehlers-Danlos syndrome (EDS), scoliosis, and craniocervical instability. The goal of this study was two-fold: (1) to identify unique morphological characteristics of PCs, and (2) to better explain inconsistent results from case-control comparisons of CMI.Methods: Image, demographic, and PC information was obtained through the Chiari1000, a self-report web-accessed database. Twenty-eight morphometric measurements (MMs) were performed on the cranial MR images of 236 pre-surgery adult female CMI participants and 140 female healthy control participants. Custom software was used to measure 28 structures within the posterior cranial fossa (PCF) compartment, craniocervical junction, oral cavity, and intracranial area on midsagittal MR images for each participant.Results: Morphometric analysis of adult females indicated a smaller McRae line length in CMI participants with syringomyelia compared to those without syringomyelia. TP was reduced in CMI participants with EDS than those without EDS. Basion to posterior axial line was significantly longer in CMI participants with scoliosis compared to those without scoliosis. No additional MMs were found to differ between CMI participants with and without a specific PC. Four morphometric differences were found to be consistently different between CMI participants and healthy controls regardless of PC: larger TP and a smaller clivus length, fastigium, and corpus callosum height in CMI participants.Conclusion: Syringomyelia, EDS, and scoliosis were the only PCs that showed significant morphometric differences between CMI participants. Additionally, four midsagittal MR-based MMs were found to be significantly different between healthy controls and CMI participants regardless of the presence of one or more PCs. This study suggests that the prevalence of comorbid conditions are not strongly related to CMI morphology, and that inconsistent findings in the radiographic literature cannot be explained by varying prevalence of comorbid conditions in CMI study samples