Complete Coding Region Research Articles

Characterization of a recombinant matrix metalloproteinase-2 from sea cucumber ( Stichopus japonicas ) and its application to prepare bioactive collagen hydrolysate

Abstract Matrix metalloproteinase(s) (MMPs) are involved in collagen metabolism in sea cucumber. In the present study, a complete coding region of MMP-2 gene was cloned from the body wall of sea cucumber (Stichopus japonicas) and the open reading frame contained 1887 bp encoding 629 amino acid residues. The deduced amino acid sequence of MMP-2 contained a signal peptide, a propeptide domain, a catalytic domain with three repeats of fibronectin-type II region and a C-terminal hemopexin-like domain. According to the domain prediction of MMP-2, its catalytic domain was successfully expressed in Escherichia coli. After refolding, the purified recombinant MMP-2 (rMMP-2) exhibited a single band both on SDS-PAGE and zymography gel. The molecular mass of rMMP-2 was approximately 40 kDa. Circular dichroism spectrum revealed that the denaturation temperature of rMMP-2 was 56.80 ± 0.25 °C. Enzymatic characterization of rMMP-2 showed that it hydrolyzed gelatin most effectively at pH 8.5 and 40 °C, suggesting it is a slight alkaline proteinase. In addition, Ca2+ was required for optimal gelatinolytic activity. The rMMP-2 degraded collagen effectively and the hydrolysate revealed scavenging activities on both 2, 2’-diphenyl-1-picrylhydrazyl free radical and hydrogen peroxide. These results indicated that rMMP-2 could potentially be applied for preparation of bioactive collagen hydrolysate.

Presence of two different bovine hepacivirus clusters in Germany.

During the last years, genetic information of hepaciviruses (family Flaviviridae), whose type species is the human hepatitis C virus, was detected in a wide range of primates and non-primate vertebrates. Here, samples collected from 263 German cattle kept in 22 different holdings were analysed for the presence of hepacivirus N (syn. bovine hepacivirus; BovHepV). One hundred eighty-six cattle that suffered from unspecific clinical signs such as fever and a reduced milk yield as well as 77 apparently healthy animals were included. A total of 39 cattle (14.8%) tested positive for BovHepV by real-time RT-PCR, but a correlation between clinical signs and virus infection could not be found. From 31 of the virus-positive samples, sequences of the NS3 coding region were generated and from two samples, viral sequences of the complete coding region were produced and compared to further European and African BovHepV sequences. Based on the NS3 genomic region, two distinct German BovHepV clusters were identified which differed between each other up to 20% at the nucleotide level, the diversity within the individual clusters reached up to 10%. Based on the full-length sequences, the newly detected virus variants group together with further German and African viruses in a sister relationship to other hepaciviruses from primates and further mammalians, but form distinct clusters within the BovHepV branch. In conclusion, highly diverse hepaciviruses were detected in German cattle further expanding the known phylogenetic diversity of the genus Hepacivirus.

Molecular and functional characterization of UDP-glucuronosyltransferase 1A in cynomolgus macaques

UDP-glucuronosyltransferases (UGTs) are drug-metabolizing enzymes essential for the metabolism of endogenous substrates and xenobiotics. Molecular characteristics of UGTs have been extensively investigated in humans, but in cynomolgus macaques, a non-human primate species widely used in drug metabolism studies, remain to be investigated. In this study, 12 UGT1A cDNAs (UGT1A1, 1A2, 1A4A, 1A4B, 1A5A, 1A5B, 1A5C, 1A6, 1A7, 1A8, 1A9, and 1A10) were isolated and characterized in cynomolgus macaques. UGT1A5C cDNA did not contain a complete coding region due to nonsense mutations, and was excluded from further analysis. Amino acid sequences of all 11 cynomolgus UGT1As had high sequence identities (92–95%) with human UGT1As and were phylogenetically close to human UGT1As. These cynomolgus UGT1A genes shared exons 2–5, and contained a variable exon 1 unique to each gene, similar to human UGT1A genes. Moreover, cynomolgus and human UGT1A gene clusters were located in corresponding regions in the genome. Among the 10 tissue types analyzed, cynomolgus UGT1A mRNAs were most abundantly expressed in the liver, jejunum, and/or kidney, the drug-metabolizing organs, similar to human UGT1As. Among these 11 cynomolgus UGT1A mRNAs, cynomolgus UGT1A2, UGT1A9, and UGT1A10 mRNAs were most abundantly expressed in the liver, kidney, and jejunum, respectively. Cynomolgus liver microsomes and UGT1A proteins catalyzed glucuronidation of the substrates human UGT1As catalyze, including 4-methylumbelliferone, 4-nitrophenol, estradiol, trifluoperazine, serotonin, and propofol, although trifluoperazine glucuronidation was not catalyzed by any cynomolgus UGT1A proteins. These results suggest that cynomolgus UGT1As are functional enzymes with molecular similarities to human UGT1As.

Introduction of Viral Hemorrhagic Septicemia Virus into Freshwater Cultured Rainbow Trout Is Followed by Bursts of Adaptive Evolution.

Viral hemorrhagic septicemia virus (VHSV), a rhabdovirus infecting teleost fish, has repeatedly crossed the boundary from marine fish species to freshwater cultured rainbow trout. These naturally replicated cross-species transmission events permit the study of general and repeatable evolutionary events occurring in connection with viral emergence in a novel host species. The purpose of the present study was to investigate the adaptive molecular evolution of the VHSV glycoprotein, one of the key virus proteins involved in viral emergence, following emergence from marine species into freshwater cultured rainbow trout. A comprehensive phylogenetic reconstruction of the complete coding region of the VHSV glycoprotein was conducted, and adaptive molecular evolution was investigated using a maximum likelihood approach to compare different codon substitution models allowing for heterogeneous substitution rate ratios among amino acid sites. Evidence of positive selection was detected at six amino acid sites of the VHSV glycoprotein, within the signal peptide, the confirmation-dependent major neutralizing epitope, and the intracellular tail. Evidence of positive selection was found exclusively in rainbow trout-adapted virus isolates, and amino acid combinations found at the six sites under positive selection pressure differentiated rainbow trout- from non-rainbow trout-adapted isolates. Furthermore, four adaptive sites revealed signs of recurring identical changes across phylogenetic groups of rainbow trout-adapted isolates, suggesting that repeated VHSV emergence in freshwater cultured rainbow trout was established through convergent routes of evolution that are associated with immune escape.IMPORTANCE This study is the first to demonstrate that VHSV emergence from marine species into freshwater cultured rainbow trout has been accompanied by bursts of adaptive evolution in the VHSV glycoprotein. Furthermore, repeated detection of the same adaptive amino acid sites across phylogenetic groups of rainbow trout-adapted isolates indicates that adaptation to rainbow trout was established through parallel evolution. In addition, signals of convergent evolution toward the maintenance of genetic variation were detected in the conformation-dependent neutralizing epitope or in close proximity to disulfide bonds involved in the structural conformation of the neutralizing epitope, indicating adaptation to immune response-related genetic variation across freshwater cultured rainbow trout.

Analysis of carboxylesterase 2 transcript variants in cynomolgus macaque liver

Carboxylesterase (CES) is important for the detoxification of a wide range of drugs and xenobiotics. In this study, the hepatic level of CES2 mRNA was examined in cynomolgus macaques used widely in preclinical studies for drug metabolism.Three CES2 mRNAs were present in cynomolgus macaque liver. The mRNA level was highest for cynomolgus CES2A (formerly CES2v3), much lower for cynomolgus CES2B (formerly CES2v1) and extremely low for cynomolgus CES2C (formerly CES2v2). Most various transcript variants produced from cynomolgus CES2B gene did not contain a complete coding region. Thus, CES2A is the major CES2 enzyme in cynomolgus liver.A new transcript variant of CES2A, CES2Av2, was identified. CES2Av2 contained exon 3 region different from wild-type (CES2Av1). In cynomolgus macaques expressing only CES2Av2 transcript, CES2A contained the sequence of CES2B in exon 3 and vicinity, probably due to gene conversion.On genotyping, this CES2Av2 allele was prevalent in Indochinese cynomolgus macaques, but not in Indonesian cynomolgus or rhesus macaques. CES2Av2 recombinant protein showed similar activity to CES2Av1 protein for several substrates.It is concluded that CES2A is the major cynomolgus hepatic CES2, and new transcript variant, CES2Av2, has similar functions to CES2Av1.

Phylogenetic relationship and molecular dating of Indian pangolin (Manis crassicaudata) with other extant pangolin species based on complete cytochrome b mitochondrial gene

Pangolins are a group of eight endangered mammalian species included in the family Manidae (Order Pholidota) and divided into four each African (Arboreal species; Phataginus tetradactyla, Phataginus tricuspis, Ground dwelling species; Smutsia gigantea and Smutsia temminckii) and Asian (Ground dwelling; Manis crassicaudata, Manis pentadactyla, Manis javanica and Manis culionensis) species. The taxonomy of all the eight extant pangolin species based on molecular genetics studies, remains unresolved and poorly examined. The present study is to address this lacuna by studying the phylogenetic, taxonomic status and molecular dating of Indian pangolin with other six out of eight extant pangolins (Sunda pangolin possibly extinct) based on complete coding region of mitochondrial cytochrome b gene. Overall sequences divergence among pangolins ranged between 0.01 ± 0.01 and 0.26 ± 0.03, where within ‘Manis’ it ranged between 0.01 ± 0.01 and 0.14 ± 0.03. Cytochrome b sequences based phylogenetic tree revealed, the division of seven pangolin species into two paraphyletic clades of African and Asian species, further these two paraphyletic clades were divided into three well-supported monophyletic clades, first for the genus ‘Smutsia’ with two African ground pangolins, second for the genus ‘Phataginus’ with two African arboreal pangolins and third for ‘Manis’ with three Asian species. Within clade of ‘Manis’, Chinese pangolin and Malayan pangolin are basal where Indian pangolin is present as a sister clade and furthermore, molecular dating analysis suggested that pangolins diverged from Carnivora at ∼87.2 MYA, followed by the split of Asian pangolins and African pangolins at ∼36.1 MYA and Indian pangolin split from Chinese pangolin and Malayan pangolin at ∼16.7 MYA.

Detection and Characterization of Homologues of Human Hepatitis Viruses and Pegiviruses in Rodents and Bats in Vietnam.

Rodents and bats are now widely recognised as important sources of zoonotic virus infections in other mammals, including humans. Numerous surveys have expanded our knowledge of diverse viruses in a range of rodent and bat species, including their origins, evolution, and range of hosts. In this study of pegivirus and human hepatitis-related viruses, liver and serum samples from Vietnamese rodents and bats were examined by PCR and sequencing. Nucleic acids homologous to human hepatitis B, C, E viruses were detected in liver samples of 2 (1.3%) of 157 bats, 38 (8.1%), and 14 (3%) of 470 rodents, respectively. Hepacivirus-like viruses were frequently detected (42.7%) in the bamboo rat, Rhizomys pruinosus, while pegivirus RNA was only evident in 2 (0.3%) of 638 rodent serum samples. Complete or near-complete genome sequences of HBV, HEV and pegivirus homologues closely resembled those previously reported from rodents and bats. However, complete coding region sequences of the rodent hepacivirus-like viruses substantially diverged from all of the currently classified variants and potentially represent a new species in the Hepacivirus genus. Of the viruses identified, their routes of transmission and potential to establish zoonoses remain to be determined.

The Brain-Lung-Thyroid syndrome (BLTS): A novel deletion in chromosome 14q13.2-q21.1 expands the phenotype to humoral immunodeficiency

Genetic defects of NKX2-1 are classically associated with hypothyroidism, benign chorea and neonatal respiratory distress. The purpose of this study was to identify the genetic pathogenesis of the “NKX2-1 triad” in a 10 year-old female presenting additional features barely described in the disorder. In the neonatal period, she presented with generalized hypotonia and respiratory distress, with later episodes of frequent wheezing. At 3 month-age developmental dysplasia of the hip was diagnosed and at 10 months, primary hypothyroidism was detected and treated. Subsequently, delayed achievement of developmental milestones and then subtle choreic movements of extremities were identified at 2 years of age. Furthermore, delayed teeth eruption and agenesis of some dental pieces, short stature and joint hyperlaxity were also noticed. At 10 years, a poor immune response to polysaccharide antigens and hypogammaglobulinemia, including all IgG subclasses were detected. Surprisingly, no mutations were identified in the complete coding region of NKX2-1 by PCR and Sanger sequencing. MLPA showed a de novo loss of gene dosage in all 3 probes located in NKX2-1 exons. A CGH-array identified a deletion of 3.32 Mb in chromosome 14q13.2-q21.1 containing 20 genes, including NKX2-1, PAX9 and two candidate genes (NFKB1A and PPP2R3C) involved in immune response. The Brain-Lung-Thyroid syndrome (OMIM#610978; ORPHA:209905) associated with other clinical phenotypes should suggest monoallelic deletions of chromosome 14 causing haploinsufficiency of NKX2-1, and other contiguous genes like PAX9 (hypodontia) or other dosage-sensitive genes in the chromosomal vicinity that emerge as candidates for hypogammaglobulinemia, mainly NFKBIA.

Identification and whole-genome characterization of a recombinant Enterovirus B69 isolated from a patient with Acute Flaccid Paralysis in Niger, 2015

Enterovirus B69 (EV-B69) is a rarely reported type and till date, only the full-length genome sequence of the prototype strain is available. Besides the prototype strain, only limited VP1 sequences of this virus from Africa and India are available in GenBank. In this study, we analyzed the full-length genome sequence of an EV-B69 strain recovered from a patient with acute flaccid paralysis in Niger. Compared with the EV-B69 prototype strain, it had 79.6% and 76.3% nucleotide identity in the complete genome and VP1 coding region, respectively. VP1 sequence analyses revealed also high variation in nucleotide similarity (68.9%–82.8%) with previously isolated EV-B69 strains in India and Africa. The great genetic divergence among EV-B69 strains indicates that this type is not a newly emergent virus, but has circulated for many years at low epidemic strength. Phylogenetic incongruity between structural and non-structural regions and similarity plot analyses revealed that multiple recombination events occurred during its evolution. This study expands the number of EV-B69 whole genome sequences which would help genomic comparison for future studies to understand the biological and pathogenic properties of this virus, assess its potential public health impact and comprehend the role of recombination in the evolution of enteroviruses.

Diagnosis of Fanconi Anaemia by ionising radiation- or mitomycin C-induced micronuclei

Fanconi Anaemia (FA) is an autosomal recessive disorder characterised by defects in DNA repair, associated with chromosomal instability and cellular hypersensitivity to DNA cross-linking agents such as mitomycin C (MMC). The FA repair pathway involves complex DNA repair mechanisms crucial for genomic stability. Deficiencies in DNA repair genes give rise to chromosomal radiosensitivity. FA patients have shown increased clinical radiosensitivity by exhibiting adverse normal tissue side-effects. The study aimed to investigate chromosomal radiosensitivity of homozygous and heterozygous carriers of FA mutations using three micronucleus (MN) assays. The G0 and S/G2MN assays are cytogenetic assays to evaluate DNA damage induced by ionising radiation in different phases of the cell cycle. The MMC MN assay detects DNA damage induced by a crosslinking agent in the G0 phase. Patients with a clinical diagnosis of FA and their parents were screened for the complete coding region of 20 FA genes. Blood samples of all FA patients and parents were exposed to ionising radiation of 2 and 4Gy. Chromosomal radiosensitivity was evaluated in the G0 and S/G2 phase. Most of our patients were homozygous for the founder mutation FANCG c.637_643delTACCGCC; p.(Tyr213Lysfs*6) while one patient was compound heterozygous for FANCG c.637_643delTACCGCC and FANCG c.1379G > A, p.(Gly460Asp), a novel missense mutation. Another patient was compound heterozygous for two deleterious FANCA mutations. In FA patients, the G0- and S/G2-MN assays show significantly increased chromosomal radiosensitivity and genomic instability. Moreover, chromosomal damage was significantly elevated in MMC treated FA cells. We also observed an increase in chromosomal radiosensitivity and genomic instability in the parents using 3 assays. The effect was significant using the MMC MN assay. The MMC MN assay is advantageous as it is less labour intense, time effective and has potential as a reliable alternative method for detecting FA patients from parents and controls.

Mutations in the prostate specific antigen (PSA/KLK3) correlate with male infertility

Prostate specific antigen (PSA/KLK3) is known to be the chief executor of the fragmentation of semenogelins, dissolution of semen coagulum, thereby releasing sperm for active motility. Recent research has found that semenogelins also play significant roles in sperm fertility by affecting hyaluronidase activity, capacitation and motility, thereby making PSA important for sperm fertility beyond simple semen liquefaction. PSA level in semen has been shown to correlate with sperm motility, suggesting that PSA level/activity can affect fertility. However, no study investigating the genetic variations in the KLK3/PSA gene in male fertility has been undertaken. We analyzed the complete coding region of the KLK3 gene in ethnically matched 875 infertile and 290 fertile men to find if genetic variations in KLK3 correlate with infertility. Interestingly, this study identified 28 substitutions, of which 8 were novel (not available in public databases). Statistical comparison of the genotype frequencies showed that five SNPs, rs266881 (OR = 2.92, P < 0.0001), rs174776 (OR = 1.91, P < 0.0001), rs266875 (OR = 1.44, P = 0.016), rs35192866 (OR = 4.48, P = 0.025) and rs1810020 (OR = 2.08, P = 0.034) correlated with an increased risk of infertility. On the other hand, c.206 + 235 T > C, was more freuqent in the control group, showing protective association. Our findings suggest that polymorphisms in the KLK3 gene correlate with infertility risk.

Additional value of screening for minor genes and copy number variants in hypertrophic cardiomyopathy.

IntroductionHypertrophic cardiomyopathy (HCM) is the most prevalent inherited heart disease. Next-generation sequencing (NGS) is the preferred genetic test, but the diagnostic value of screening for minor and candidate genes, and the role of copy number variants (CNVs) deserves further evaluation.MethodsThree hundred and eighty-seven consecutive unrelated patients with HCM were screened for genetic variants in the 5 most frequent genes (MYBPC3, MYH7, TNNT2, TNNI3 and TPM1) using Sanger sequencing (N = 84) or NGS (N = 303). In the NGS cohort we analyzed 20 additional minor or candidate genes, and applied a proprietary bioinformatics algorithm for detecting CNVs. Additionally, the rate and classification of TTN variants in HCM were compared with 427 patients without structural heart disease.ResultsThe percentage of patients with pathogenic/likely pathogenic (P/LP) variants in the main genes was 33.3%, without significant differences between the Sanger sequencing and NGS cohorts. The screening for 20 additional genes revealed LP variants in ACTC1, MYL2, MYL3, TNNC1, GLA and PRKAG2 in 12 patients. This approach resulted in more inconclusive tests (36.0% vs. 9.6%, p<0.001), mostly due to variants of unknown significance (VUS) in TTN. The detection rate of rare variants in TTN was not significantly different to that found in the group of patients without structural heart disease. In the NGS cohort, 4 patients (1.3%) had pathogenic CNVs: 2 deletions in MYBPC3 and 2 deletions involving the complete coding region of PLN.ConclusionsA small percentage of HCM cases without point mutations in the 5 main genes are explained by P/LP variants in minor or candidate genes and CNVs. Screening for variants in TTN in HCM patients drastically increases the number of inconclusive tests, and shows a rate of VUS that is similar to patients without structural heart disease, suggesting that this gene should not be analyzed for clinical purposes in HCM.

Characterization of Eptesipoxvirus, a novel poxvirus from a microchiropteran bat.

The genome of Eptesipoxvirus (EPTV) is the first poxvirus genome isolated from a microbat. The 176,688 nt sequence, which is believed to encompass the complete coding region of the virus, is 67% A+T and is predicted to encode 191 genes. 11 of these genes have no counterpart in GenBank and are therefore unique to EPTV. The presence of a distantly related ortholog of Vaccinia virus F5L in EPTV uncovered a link with fragmented F5L orthologs in Molluscum contagiosum virus/squirrelpox and clade II viruses. Consistent with the unique position of EPTV approximately mid-point between the orthopoxviruses and the clade II viruses, EPTV has 11 genes that are specific to the orthopoxviruses and 13 genes that are typical, if not exclusive, to the clade II poxviruses. This mosaic nature of EPTV blurs the distinction between the old description of the orthopoxvirus and clade II groups. Genome annotation and characterization failed to find any common virulence genes shared with the other poxvirus isolated from bat (pteropoxvirus); however, EPTV encodes 3 genes that may have been transferred to or from deerpox and squirrelpox viruses; 2 of these, a putative endothelin-like protein and a MHC class I-like protein are likely to have immunomodulatory roles.Electronic supplementary materialThe online version of this article (doi:10.1007/s11262-017-1485-4) contains supplementary material, which is available to authorized users.

Testing Convergent Evolution in Auditory Processing Genes between Echolocating Mammals and the Aye-Aye, a Percussive-Foraging Primate.

Several taxonomically distinct mammalian groups—certain microbats and cetaceans (e.g., dolphins)—share both morphological adaptations related to echolocation behavior and strong signatures of convergent evolution at the amino acid level across seven genes related to auditory processing. Aye-ayes (Daubentonia madagascariensis) are nocturnal lemurs with a specialized auditory processing system. Aye-ayes tap rapidly along the surfaces of trees, listening to reverberations to identify the mines of wood-boring insect larvae; this behavior has been hypothesized to functionally mimic echolocation. Here we investigated whether there are signals of convergence in auditory processing genes between aye-ayes and known mammalian echolocators. We developed a computational pipeline (Basic Exon Assembly Tool) that produces consensus sequences for regions of interest from shotgun genomic sequencing data for nonmodel organisms without requiring de novo genome assembly. We reconstructed complete coding region sequences for the seven convergent echolocating bat–dolphin genes for aye-ayes and another lemur. We compared sequences from these two lemurs in a phylogenetic framework with those of bat and dolphin echolocators and appropriate nonecholocating outgroups. Our analysis reaffirms the existence of amino acid convergence at these loci among echolocating bats and dolphins; some methods also detected signals of convergence between echolocating bats and both mice and elephants. However, we observed no significant signal of amino acid convergence between aye-ayes and echolocating bats and dolphins, suggesting that aye-aye tap-foraging auditory adaptations represent distinct evolutionary innovations. These results are also consistent with a developing consensus that convergent behavioral ecology does not reliably predict convergent molecular evolution.

Genetic variations of the bovine MX1 and their association with mastitis

The primary agent of mastitis is a wide spectrum of bacterial strains; however, viral-related mastitis has also been reported. The MX dynamin-like GTPase 1 (MX1) gene has been demonstrated to confer positive antiviral responses to many viruses, and may be a suitable candidate gene for the study of disease resistance in dairy cattle. The present study was conducted to investigate the genetic diversity of the MX1 gene in Chinese cattle breeds and its effects on mastitis in Holstein cows. First, polymorphisms were identified in the complete coding region of the bovine MX1 gene in 14 Chinese cattle breeds. An association study was then carried out, utilizing polymorphisms detected in Holstein cows to determine the associations of these single nucleotide polymorphisms (SNPs) with mastitis. We identified 13 previously reported SNPs in Chinese domestic cattle and four of them in Holstein cattle. A novel 12 bp indel was also discovered in Holstein cattle. In addition, haplotype frequencies and linkage disequilibrium of four SNPs detected in Holstein cows were investigated. Analysis of these four SNPs in Chinese Holstein cows revealed two SNPs (g.143181370 T&amp;gt;C and g.143182088 C&amp;gt;T)&lt;br /&gt; significantly (P &amp;lt; 0.05) associated with somatic cell score (SCS). The results indicated that SNPs in the MX1 gene might contribute to the variations observed in the SCS of dairy cattle. Therefore, implementation of these two mutations in selection indexes of the dairy industry might be beneficial by favouring the selection individuals with lower SCS.

Identification of somatic and germ-line DICER1 mutations in pleuropulmonary blastoma, cystic nephroma and rhabdomyosarcoma tumors within a DICER1 syndrome pedigree

BackgroundDICER1 syndrome is a pediatric cancer predisposition condition causing a variety of tumor types in children and young adults. In this report we studied a family with two relatives presenting a variety of neoplastic conditions at childhood.MethodsGerm-line mutation screening of the complete coding region of the DICER1 gene in genomic DNA from the proband was performed. The presence of somatic DICER1 mutation and further alterations in driver genes was investigated in genomic DNA obtained from available tumor samples.ResultsA nonsense germ-line mutation in DICER1 causing a truncated protein at the IIIb domain level was identified segregating within a family including two affected relatives who developed in one case cystic nephroma and pleuropulmonary blastoma, and rhabdomyosarcoma and multinodular goiter in the other. Additional in trans DICER1 missense somatic mutations in the IIIb DICER1 domain were found both in the cystic nephroma and in the rhabdomyosarcoma, suggesting that neoplasms in this family might arise from the unusual two-hit mechanism for DICER-derived tumorigenesis in which after the presence of a truncated constitutive protein, a neomorphic DICER1 activity is somatically adquired. Additional genetic alterations, such as TP53 mutations, were identified in the rhabdomyosarcoma.ConclusionsBesides DICER1 loss of standard activity, oncogenic cooperation of other genes, as mutated TP53, may involve developing higher grade tumors within this syndrome. Given the broad clinical spectrum that may arise, genetic counseling and close surveillance must be offered to all family members at risk of DICER1 syndrome.

Molecular characterization and tissue expression profiles of prepro-vasoactive intestinal peptide in the Chinese alligator (Alligator sinensis) during the active and hibernating periods.

The Chinese alligator (Alligator sinensis), a freshwater crocodilian endemic to China, is one of the most endangered crocodilian species; up to this date, very little is known about the endocrine regulation of its metabolic activities during different physiological states. In this study, we characterized the structure of the prepro-vasoactive intestinal peptide in Chinese alligator (prepro-caVIP) for the first time and examined its expression profiles in various tissues during the active and hibernating periods. The prepro-caVIP cDNA consists of a 221-bp 5'-untranslated region (UTR), a 606-bp complete coding region (CDS), and a 312-bp 3'-UTR, which encodes the 201-amino acid prepro-caVIP containing a 28-amino acid vasoactive intestinal peptide (VIP) and a 27-amino acid PHI (peptide histidine isoleucine). Multiple alignment analysis showed that VIP shares 100% identity with the given birds, reptiles, and African clawed frog, and 89% identity with mammals, 96% with fishes. Real-time quantitative PCR showed that the prepro-caVIP is widely expressed in all the examined tissues, and the expression level is significantly higher in small intestine, stomach, pancreas, lung, and skeletal muscle, whereas lower in heart, liver, spleen, kidney, ovary, and oviduct. During hibernation, the expression level of caVIP was significantly decreased in small intestine (P < 0.01), pancreas, and skeletal muscle (P < 0.05), whereas significantly increased in liver, spleen, and lung (P < 0.01). The wide distribution of caVIP and its differential expression changes in various tissues during hibernation implicated that it might play multiple effects in Chinese alligator and participate in the physiological adaptation of various organs in a paracrine and/or neurocrine manner.

Targeted Capture of Complete Coding Regions across Divergent Species.

Despite continued advances in sequencing technologies, there is a need for methods that can efficiently sequence large numbers of genes from diverse species. One approach to accomplish this is targeted capture (hybrid enrichment). While these methods are well established for genome resequencing projects, cross-species capture strategies are still being developed and generally focus on the capture of conserved regions, rather than complete coding regions from specific genes of interest. The resulting data is thus useful for phylogenetic studies, but the wealth of comparative data that could be used for evolutionary and functional studies is lost. Here, we design and implement a targeted capture method that enables recovery of complete coding regions across broad taxonomic scales. Capture probes were designed from multiple reference species and extensively tiled in order to facilitate cross-species capture. Using novel bioinformatics pipelines we were able to recover nearly all of the targeted genes with high completeness from species that were up to 200 myr divergent. Increased probe diversity and tiling for a subset of genes had a large positive effect on both recovery and completeness. The resulting data produced an accurate species tree, but importantly this same data can also be applied to studies of molecular evolution and function that will allow researchers to ask larger questions in broader phylogenetic contexts. Our method demonstrates the utility of cross-species approaches for the capture of full length coding sequences, and will substantially improve the ability for researchers to conduct large-scale comparative studies of molecular evolution and function.

Using a Next Generation Sequencing Panel to Discover the Obscure Causes of Hereditary Hemolytic Anemias

Hereditary hemolytic anemias are a heterogeneous group of disorders with consequences ranging from non-anemic hemolysis to severe life-threatening anemia. However, the late morbidity in patients without transfusions is often underappreciated because of erythropoietic compensatory stimulation inducing hematopoiesis by erythroferrone/hepcidin axis. Principal causes of hereditary hemolytic anemias are germline mutations of red cell cytoskeleton (e.g. hereditary spherocytosis and elliptocytosis/pyropoikilocytosis) or enzyme deficiencies (e.g. Glucose 6 phosphate dehydrogenase deficiency and pyruvate kinase deficiency). Routine morphological and biochemical analysis may be inconclusive and misleading particularly in transfusion-dependent infants and children. Molecular studies have not been extensively used to diagnose these disorders due to the complex genetic nature of these disorders, and multi-gene disorders. In these cases, patients may undergo multiple rounds of single gene testing, which can be very costly and time consuming. The advent of next generation sequencing (NGS) methods in the clinical laboratory has made diagnosing complex genetic disorders feasible. Our diagnostic panel includes 28 genes encoding cytoskeletal proteins and enzymes, and covers the complete coding region, splice site junctions, and, where appropriate, deep intronic or regulatory regions. Targeted gene capture and library construction for next-generation sequencing (NGS) was performed using Sure Select kit (Agilent Technologies, Santa Clara, USA). Prior to sequencing on the Illumina Next Seq, (Illumina Inc) instrument, indexed samples are quantified using qPCR and then pooled. Samples were sequenced using 2x150 paired end sequencing. We now report the first 68 patients evaluated using our NGS panel. The age of the patients ranged from newborn to 62 years. These patients presented with symptoms ranging from mild lifelong anemia to severe hemolytic anemia with extreme hyperbilirubinemia. Genetic variants were classified using the American College of Medical Genetics (ACMG) guidelines. We identified pathogenic variants in 11 patients and likely pathogenic variants in 12 others, the majority of these were novel. Many variants with unknown significance were also identified that could potentially contribute to disease. The most commonly mutated genes were SPTB and SPTA1, encoding spectrin subunits. Some complex interactions were uncovered i.e. SPTA1 mutations along with alpha LELY leading to hereditary pyropoikilocytosis; Spectrin variants along with Gilbert syndrome causing severe hyperbilirubinemia in neonates; and Spectrin variants in combination with PKLR and G6PD variants. Our results demonstrate that many patients with hemolytic anemia harbor complex combinations of known and novel mutations in RBC cytoskeleton/enzyme genes, but their clinical significance is further augmented by polymorphisms of UGT1A1 gene contributing to severe neonatal hyperbilirubinemia and its consequences. To conclude, next-generation sequencing provides a cost-effective and relatively rapid approach to molecular diagnosis, especially in instances where traditional testing failed. We have used this technology successfully to determine the molecular causes of hemolytic anemia in many cases with no prior family history. DisclosuresYaish:Octapharma: Other: Study investigator.

Role of FIT2 in porcine intramuscular preadipocyte differentiation

AbstractFat-inducing transcript 2 (FIT2) plays an important role in the formation of intramuscular lipid droplets in skeletal muscle. However, its role in porcine intramuscular preadipocyte differentiation remains unclear. In the present study, a 789-bp fragment covering the complete coding region of porcine FIT2 (