RPGR protein has different interacting partners to function in ciliary protein trafficking. In this review, we specifically focus on RPGR and its two interacting proteins: RPGRIP1 and RPGRIP1L. We summarize the function of the three proteins and highlight recent studies that provide insight into the cellular function of those proteins.Ciliopathies encompass a group of genetic disorders characterized by defects in the formation, maintenance, or function of cilia. Retinitis pigmentosa (RP) is frequently one of the clinical features presented in diverse ciliopathies. RP is a heterogeneous group of inherited retinal disorders, characterized by the death of photoreceptors and affecting more than one million individuals worldwide. The  Cilia are tiny, hair-like structures protruding from the cell surface. They are highly conserved organelles and serve a variety of sensory functions in both unicellular and multicellular organisms. Historically, cilia have been classified into two categories: motile and nonmotile cilia (primary cilia), depending on the arrangement of the microtubule triplets, termed the axoneme. The axoneme of motile cilia is built in the classical 9+2 arrangement, where nine outer microtubule doublets surround a central pair of singlet microtubules. The axoneme of primary cilia lacks the central pairs of microtubules and is arrayed in a 9+0 configuration . Primary Chlamydomonas reinhardtii, in which anterograde and retrograde movements of particles were observed through differential interference contrast microscopy [ C. elegans to primates [All protein synthesizing machineries are restricted to the cytoplasm, so continued elongation of the cilium requires the selective import and transport of ciliary proteins by intraflagellar transport (IFT), which mediates bidirectional movement of multiprotein loaded particles along the axoneme. IFT was initially discovered in flagella ofcroscopy . IFT polprimates , 5. The primates , 6 and tprimates , 8. Baseprimates . IFT comprimates . Most ofPhotoreceptors are the photosensitive cells of the retina responsible for converting incident light into electric signals that are transmitted to the brain via the optic nerves and are interpreted by the nervous system. Rods and cones are the two types of photoreceptors, classified according to their shape and function. The human retina contains over 120 million rods and 6 million cones. Rods and cones are unevenly distributed across the retina. Some organisms, such as mouse, also have a rod dominant retina; others, such as zebrafish and squirrel, have a cone dominant retina. In humans, the central region of the retina is the macula. The central part of the macula is referred to as the “fovea,” which only contains cones and is the region showing highest visual acuity . Rods arPhotoreceptors consist of four distinct components: a synaptic terminal, an outer segment (OS), an inner segment (IS), and a connecting cilium (CC) that connects the IS and OS . The OS The cilium proteome contains hundreds of different proteins involved in cilia protein trafficking, structure, and signal transductions , 16. DefPhenotypic manifestations of ciliopathies can range from isolated blindness or renal disease to multiorgan system disorders such as retinitis pigmentosa, Meckel-Gruber syndrome (MKS), Joubert syndrome, or Bardet-Biedl syndrome (BBS). Defects of ciliary proteins can give rise to broad range of defects, including retinitis pigmentosa, hepatic, pancreatic, and kidney cyst formation, polydactyly, situs inversus, brain malformations, encephalocele, hydrocephalus, sensory defects, and skeletal abnormalities. However, mutations in the same ciliary gene can give rise to heterogeneous clinical phenotypes with different levels of severity . Convershttp://www.sph.uth.tmc.edu/Retnet/). Symptoms of RP include accumulation of intraretinal pigment-like deposits, retinal vessel attenuation, and characteristic changes in electroretinogram (ERG) patterns [Retinitis pigmentosa (RP) encompasses a group of inherited retinal degenerations that show progressive loss of photoreceptors with highly variable clinical and genetic heterogeneity. It is one of the most heterogeneous genetic disorders known in man , resultipatterns . The typpatterns . RPGR gene are the major cause of RP, accounting for more than 70% of XLRP and over 20% of nonsyndromic RP in North American families [ RPGR mutations are responsible for 8.8% of Japanese RP patients [ RPGR gene is located in chromosomal region Xp21.1, spanning 172 kilobases. It was first cloned in 1996 and was initially reported to consist of 19 exons [ RPGRex1–19 transcript contains 19 exons, coding a protein with 815 amino acids with an isoprenylation motif at the C-terminus; the RPGRORF15 has 15 exons, encoding a protein with 1152 amino acids. It shares exons 1–14 with RPGRex1–19 plus the exon ORF15, encoding 567 amino acids with a repetitive glycine and glutamic acid-rich domain and a conserved basic C-terminal domain , was identified by yeast two-hybrid screening mouse embryonic cDNA library using the RCC1-like domain [ORF15 interacts with whirlin, whose mutations cause Usher syndrome, and with nucleophosmin, which functions in cell division [ε, γ tubulin, kinesin II subunit KIF3A and KAP3, dynein subunit intermediate chain, dynactin subunits: P150Glued and P50-dynamitin, IQCB1 (NPHP5), and CEP290 (NPHP6) ( IQCB1 gene cause Senior-Loken syndrome (SLS), an inherited disorder characterized by RP and renal diseases [Localization of RPGR in the retina is species dependent. RPGR is localized predominantly in the connecting cilia of mouse photoreceptors, but, in human and bovine photoreceptors, localization has been reported in the outer segments of both rod and cone cells , 35. Oute domain . Using ae domain –41. Coime domain . The C-tdivision , 43. Coi (NPHP6)  [42, 44,diseases –48. IQCBdiseases . RPGRIP1 lead to LCA, a severe retinal dystrophy causing blindness or severe visual defects at birth or in early childhood [ RPGRIP1L cause either the lethal Meckel syndrome (MKSS) or Joubert syndrome type B (JBTS), which show much broader cilia defects [ RPGRIP1 gene is located on chromosome 14q11 and encodes a protein with 1286 residues. RPGRIP1L gene is located on chromosome 16q12.2 and encodes a protein of 1315 residues [+-binding site in RPGRIP1 C-terminal C2 (C2-C) domain, but the recent crystal structure of RPGR-interacting domain of RPGRIP1 did not support the prediction [Mutations inhildhood . Defects defects , 52. Humresidues . RPGRIP1residues , 54. Theresidues , 51, 52.residues , 51, 52.ediction . Both RPediction . Mutatioediction . More reediction . Misloca RPGR morphants include shorter Kupffer's vesicle cilia, a shortened body axis, kinked tail, and hydrocephaly [ RPGR knockout (KO) mice showed a slow retinal degeneration with photoreceptor death noted by 6 months of age [Large amounts of data from studies in cell lines and animal models suggest RPGR and interacting proteins play a critical role in cilia genesis, maintenance, and function. Knockdown of RPGR in hTERT-RPE1 cells resulted in defects in ciliogenesis with a reduced number of cilia or shortened cilia , 59. Morocephaly , 61. RPGs of age . Althoug RPGRIP1 in mouse (referred to as RPGRIP1tm1Tili) resulted in early retinal degeneration with most of photoreceptors degenerating by 3 months of age, resembling the phenotypes in LCA patients. The abnormal photoreceptors were apparent at the age of postnatal day 15 (P15), showing disorganized outer segments and pyknotic nuclei, a sign of ongoing cell death [ RPGRIP1 KO mice presented a normal structure of connecting cilia, suggesting RPGRIP1 is not needed for the connecting cilia development and maintenance. RPGR, however, lost localization to connecting cilia in RPGRIP1 KO mice, suggesting RPGR correct localization is dependent on RPGRIP1. A recent N-ethyl-N-nitrosourea-induced RPGRIP1 null mouse model (referred to as RPGRIP1nmf247) showed a more severe retinal degeneration when compared to that of RPGRIP1tm1Tili mice. RPGRIP1tm1Tili mice showed a rapid and progressive photoreceptor cell death. Outer segments were not seen in RPGRIP1nmf247 mice at P7 and P14. Only 3-4 photoreceptor nuclei layers remained by P21. Rhodopsin mislocalization was detected at P12, the earliest time point examined. Other outer segment proteins, ROM1, transducin, and arrestin, were also mislocalized, but neither transducin nor arrestin mislocalized in RPGRIP1tm1Tili mice [ RPGRIP1tm1Tili mice suggested that RPGRIP1 is essential for rod outer segment morphogenesis, whereas phenotypes in the RPGRIP1nmf247 mice suggested RPGRIP1 is essential not only for rod outer segment disc morphogenesis but also for outer segment formation. The phenotypic difference between the two mouse mutant strains was caused by a short alternative splice variant in RPGRIP1tm1Tili mice, which may have a role in outer segment formation.Knockout ofll death . RPGRIP1ili mice , 64. The RPGRIP1L (Ftm) gene results in the death of mouse at midgestation and recapitulating similar malformation observed in human MKS fetues, including brain, liver, kidney, limb, and eye developmental defects [ RPGRIP1L in zebrafish showed a broad range of ciliary defects, including shortened body axis, curved body, malformed somites, kinked notochord, abnormal tail extension, and hydrocephaly [RPGRIP1L is implicated in different types of ciliopathies. Inactivation of the defects , 65. Morocephaly , 66. In  RPGR or RPGRIP1 KO mice and in a patient carrying an RPGR mutation, suggesting RPGR and its interacting proteins are involved in protein trafficking in cilia [δ, which is loaded with farnesylated cargo [ RPGRIP1 KO mice (RPGRIP1nmf247) [Mislocalization of outer segment proteins has been observed inin cilia –64, 67. ed cargo . Since Red cargo , was obsnmf247) . RPGRIP1nmf247) .α (PDGFR α), mTOR, and Hippo signaling pathways [The primary cilium is a coordinating hub for different signaling pathways, such as Wnt , sonic hedgehog (SHH), fibroblast growth factor (FGF), Notch, platelet derived growth factor receptor pathways . RPGR anpathways . The nonpathways . RPGRIP1pathways . NPHP4, pathways . In factpathways . RPGR gene are the most common cause of RP, frequently associated with ciliopathies. RPGR directly or indirectly interacts with ciliary proteins to form one or more protein complexes. RPGR and its interacting proteins may regulate cilia genesis, maintenance, and function mainly through recruiting ciliary protein to cilia. RPGR protein complex is involved in cilia regulatory signaling pathways, but understanding the complicated RPGR-involved molecular mechanisms remains a challenge.Mutations in the


BSCL2/seipin gene, exhibits the most severe lipodystrophic phenotype with loss of both metabolic and mechanical adipose depots. The majority of Seipin mutations cause C-terminal truncations, along with a handful of point mutations. Seipin localizes to the ER and is composed of a conserved region including a luminal loop and two transmembrane domains, plus cytosolic N- and C-termini. Animal models deficient in seipin recapitulate the human lipodystrophic phenotype. Cells isolated from seipin knockout mouse models also exhibit impaired adipogenesis. Mechanistically, seipin appears to function as a scaffolding protein to bring together interacting partners essential for lipid metabolism and LD (lipid droplet) formation during adipocyte development. Moreover, cell line and genetic studies indicate that seipin functions in a cell-autonomous manner. Here we will provide a brief overview of the genetic association of the CGLs, and focus on the current understanding of differential contributions of distinct seipin domains to lipid storage and adipogenesis. We will also discuss the roles of seipin-interacting partners, including lipin 1 and 14-3-3β, in mediating seipin-dependent regulation of cellular pathways such as actin cytoskeletal remodelling.CGL  is a genetic disorder characterized by near complete loss of adipose tissue along with increased ectopic fat storage in other organs including liver and muscle. Of the four CGL types, BSCL2 , resulting from mutations in the  Lipodystrophy is characterized by the selective loss of adipose tissue, with manifestation of clinically heterogeneous phenotypes. Since a key function of adipose tissue is the storage of excess energy in the form of lipids, extreme loss of fat in lipodystrophy results in hypertriglyceridemia and ectopic accumulation of TAG (triacylglycerol) in other organs, particularly liver, leading to a severe metabolic condition. Not surprisingly, lipodystrophy is often accompanied by insulin resistance and its complications of diabetes mellitus, hepatic steatosis, acanthosis nigricans and dyslipidaemia . Other mAGPAT2 (BSCL1), Seipin (BSCL2), CAV1 (BSCL3) and PTRF (polymerase I and transcript release factor)/Cavin (BSCL4), with majority of patients (~95%) studied thus far possessing mutations in BSCL1 or BSCL2. Here we will summarize the current knowledge on each of the causative genes, and focus on the mechanistic understanding of BSCL2 in this review.BSCL is a rare autosomal recessive disorder, with an estimated prevalence of 1 in 10 million worldwide, discovered by Berardinelli and Seip in 1954 and 1959, respectively . PromineLoss of ‘metabolically active’ adipose tissue is observed in BSCL1 patients, specifically at subcutaneous, intermuscular, intra-abdominal, intrathoracic and bone marrow areas ,8. BSCL1AGPAT2 (1-acylglycerol-3-phosphate-O-acyl-transferase 2) gene, on chromosome 9q34, was identified as the cause of BSCL1 [−/− mouse model revealed reduced amount of WAT (white adipose tissue), mimicking observations in human counterparts [Aberrant expression of the of BSCL1 ,11. Exprof BSCL1 ,12. AGPAof BSCL1 ,14. Agarof BSCL1 . Nine otof BSCL1 . Haque eterparts . Taken tseipin mutations is not linked to a specific ethnic type, and BSCL2 patients studied originate from various races, including white ethnicities of Europeans, Mediterranean and Middle Eastern Arabs [BSCL2 patients exhibit the most severe phenotype of lipodystrophy among the four BSCLs. In addition to the loss of metabolically active adipose tissue, BSCL2 patients also exhibit loss of mechanical adipose tissue, which serves protective or supportive functions of body parts. Loss of these mechanical adipose tissue was observed in the palms, soles, scalp, orbits  and periarticular regions (around the joints) ,8. So farn Arabs ,15,17, Arn Arabs  and Japarn Arabs .seipin to be the responsible gene of BSCL2 in 2001 [BSCL2 causing the Silver syndrome or distal hereditary motor neuropathy type V (collectively named Seipinopathy) [seipin are involved in two seemingly distinct disorders: lipodystrophy and motor neuropathy. It is worth noting that there is higher prevalence of cardiomyopathy and intellectual impairment in BSCL2 versus BSCL1 patients [Through a genome-wide analysis of linkage and homozygosity mapping, Magre et al. first identified  in 2001 . Coincidnopathy) ,22. In spatients ,23. In fpatients , suggestpatients ,25.seipin is ubiquitously expressed, lipodystrophic mutations in seipin are considered loss-of-function, whereas mutations associated with motor neuropathies are deemed gain-of-function. It was originally thought that specific mutations in seipin are explicit to either lipodystrophy or seipinopathy. However, a recent finding of a novel c.985C>T nonsense mutation that results in Y289LfsX64 (tyrosine 289 mutated to leucine with 64 amino acid frameshift including stop codon) revealed a lipodystrophic phenotype, severe neuronal damage and early death at 7–8 years of age in both compound heterozygous (n=4) and homozygous children (n=2) [Although en (n=2) . PhysiolThe majority of identified mutations in seipin are nonsense, producing truncated protein products, with only a handful being missense. As such, it is reasonable that literatures to date have generally associated aberrant seipin expression to be loss-of-function mutations.Cav1 (7q31), caused by the 112G◇T nucleotide change, was found in a homozygous Brazilian female patient [CAV1 exhibit a typical BSCL phenotype, with the exception of one heterozygous patient carrying the -88delC frameshift mutation, who exhibits partial lipodystrophy of upper body subcutaneous fat [AGPAT2 or seipin was found in the subject, CAV1 was suggested as the causative gene of BSCL3. The affected patient exhibits near total loss of subcutaneous and visceral adipose tissue depots, muscular hypertrophy, hypertriglyceridemia, hepatosplenomegaly, hepatic steatosis and severe insulin resistance, typical of BSCL phenotype. CAV1 is a major structural component of caveolae, flask-shaped invaginations in the plasma membrane involved in endocytosis, lipid regulation and signal transduction [A recent study by Kim et al. linked BSCL mutation of the scaffolding protein Cav1 (Caveolin-1) . The non patient . Intrigueous fat . As no msduction . The abisduction –33. Remisduction –36. Thessduction ,38PTRF/cavin-1 gene essential for caveolae formation [BSCL4 –41 was iormation . Four paormation . What isormation . Causatiormation ,44.In the following sub-sections, we will discuss the current understanding of the molecular functions of seipin and potential pathogenic mechanisms of seipin in lipodystrophy.Seipin is located on chromosome 11q13 and exists in three isoforms. Isoform 1 has 11 exons and translates to 462 amino acids. Isoform 2 lacks exon 1 and translates a shorter product of 398 amino acids without the 64 amino acids at the N-terminus, whereas isoform 3 splices out exon 7 and generates an even smaller 287 amino acid product with a shortened C-terminus [seipin in mammals, fruit fly and yeast contain a conserved central domain, including the TMDs and the central loop, of about 230 amino acids [seipin, and lacks both N- and C- termini  and adipocytes (preadipocytes treated with adipogenic cocktail and mature adipocytes).In 2008, Fei et al. showed that in yeast, depletion of a putative seipin orthologue FLD1 (FLD1Δ) exhibited LD-associated defects . These din vitro, and potentially increase the risk for liver cirrhosis and hepatocellular carcinoma in BSCL2 patients.A recent interesting study examined the relations of seipin and HCV (hepatitis C virus). By overexpressing wild-type seipin in a hepatocellular carcinoma cell line Huh7, the authors showed increased size and reduced number of LDs per cell. This led to reduced total outer surface area of LDs, which was correlated with reduced production of HCV particles . This fi−/− MEF cells during adipogenesis, phosphorylation of hormone-sensitive lipase and perilipin 1A, target substrates of PKA (protein kinase A), is increased [−/− MEF cells, as evidenced by elevated levels of C/EBPα, PPARγ and aP2 [In BSCL2ncreased . It was  and aP2 . Another and aP2 . Levels  and aP2 . Conside and aP2 ,54.in vivo models thus far have highlighted the differential roles of seipin in LD formation in non-adipocytes and adipocytes. Knockdown of seipin leads to (1) enhanced TAG synthesis and LD formation in non-adipocytes, and (2) dysfunction of adipocyte development and LD formation through enhanced lipolysis or inhibition of TAG production. While the absence of seipin expression evidently inhibits LD development during adipogenesis, excess expression of wild-type seipin appears to have a similar detrimental effect. In a mouse model overexpressing 150% more seipin in adipose tissue (aP2-seipin), MRI and histological analysis showed significant reduction in subcutaneous and intra-abdominal fat and up to 50% increase in TAG storage in the liver [Three he liver . Insteadhe liver ,68. The 2+-ATPase) that transport calcium from cytosol to ER lumen [Taken into consideration of our recent studies that seipin regulates lipid homoeostasis by preventing lipid overloading in non-adipocytes while promoting lipid storage in adipocytes , and thaER lumen . AlthougAmong potential pathological features of CGL/BSCL is the inflammatory activation. So far no report has yet to tie BSCL disorders to inflammation, although there was a report of elevated circulating TNFα (tumour necrosis factor α) levels in patients of HIV-associated lipodystrophy . AdditioAdipose tissue macrophage infiltration and inflammatory response are often associated with an obesity phenotype, where enlarged adipocytes release free fatty acids through lipolysis and pro-inflammatory cytokines TNFα, IL-6 and MCP-1, resulting in development of insulin resistance. For instance, IL-6 inhibits insulin receptor substrate 1, impairs insulin stimulated glucose transport, and causes insulin resistance ,74. ResuThe role of BSCL2/seipin seems to be complex and studies to date suggested that this enigmatic protein may differentially regulate lipid homoeostasis by inhibiting LD formation in non-adipocytes while promoting adipogenesis to store excess lipid. Depletion of seipin leads to increased TAG accumulation in non-adipocytes but inhibition of adipogenesis. However, expression of the seipin mutant, specifically A212P, has no effect on TAG accumulation in non-adipocytes yet inhibits adipogenesis. Mechanistically, the C-terminus of seipin appears to play an essential role in both LD biogenesis and adipogenic programming, shedding light on the pathogenesis of non-functional truncated protein due to nonsense mutations, such as R275X, I262fs and Q391X, which lack the C-terminus. However, other point mutations, T78A, L91P and A212P, seem to retain the C-terminal region. In our model , the con


SPON2, TGM2, MMP16, GPC4, SULF1), cell-cell adhesion , immune/inflammatory/pain responses , and essential for redox balance . Our findings provide a picture of the gene expression profile and dysregulated pathways in JHS/EDS-HT skin fibroblasts that correlate well with the systemic phenotype of the patients.Joint hypermobility syndrome/Ehlers–Danlos syndrome hypermobility type (JHS/EDS-HT), is likely the most common systemic heritable connective tissue disorder, and is mostly recognized by generalized joint hypermobility, joint instability complications, minor skin changes and a wide range of satellite features. JHS/EDS-HT is considered an autosomal dominant trait but is still without a defined molecular basis. The absence of (a) causative gene(s) for JHS/EDS-HT is likely attributable to marked genetic heterogeneity and/or interaction of multiple loci. In order to help in deciphering such a complex molecular background, we carried out a comprehensive immunofluorescence analysis and gene expression profiling in cultured skin fibroblasts from five women affected with JHS/EDS-HT. Protein study revealed disarray of several matrix structural components such as fibrillins, tenascins, elastin, collagens, fibronectin, and their integrin receptors. Transcriptome analysis indicated perturbation of different signaling cascades that are required for homeostatic regulation either during development or in adult tissues as well as altered expression of several genes involved in maintenance of extracellular matrix architecture and homeostasis (e.g.,  Ehlers-Danlos syndromes (EDS) are a heterogeneous group of heritable connective tissue disorders (HCTDs) sharing a variable combination of skin hyperextensibility, internal organ and vessel fragility and dysfunctions, and generalized joint hypermobility (gJHM) , 2. Six The actual difficulties in recognizing JHS/EDS-HT are due to the low specificity of available diagnostic criteria and the lack of any confirmatory test. Clinical variability is wide and now includes functional gastrointestinal disorders, cardiovascular dysautonomia, and gynecological manifestations, all not comprised in the available diagnostic criteria –10. WhilTo gain insights into the pathogenesis of JHS/EDS-HT, we performed a transcriptome-wide expression profiling in five skin fibroblast strains, derived from adult patients with full-blown characteristics and which showed a common disarray of several extracellular matrix (ECM) structural proteins. Our work adds insights into etiopathogenesis of JHS/EDS-HT for future studies aimed at deciphering the molecular basis of such a protean disorder.This study was approved by the local Ethical Committee “Comitato Etico Provincia di Brescia, ASST Spedali Civili, Brescia, Italia”, registration number NP2378 and performed in accordance with the Declaration of Helsinki Principles. Written informed consent was obtained from all patients and controls to the study and for skin biopsy. Patients selected for this study were clinically evaluated in the Centre of Heritable Connective tissue disorders and Ehlers-Danlos syndromes of the Spedali Civili of Brescia. Criteria included in the Villefranche nosology for EDS-HT and Brighton criteria for JHS were used for assessing JHS/EDS-HT patients. gJHM was evaluated using the Beighton score (BS); patients with negative BS (≤ 5/9) were investigated for historical JHM using the 5-point questionnaire . Intensiin vitro at 37° C in a 5% CO2 atmosphere in Earle’s Modified Eagle Medium (MEM) supplemented with 2 mM L-glutamine, 10% FBS, 100 μg/ml penicillin and streptomycin . Fibroblasts were expanded until full confluency and then harvested by 0.25% trypsin/0.02% EDTA treatment at the same passage number (from 3rd to 4th).Skin fibroblast cultures from five JHS/EDS-HT female patients, four positive both for the Villefranche and Brighton criteria and one for the Brighton criteria, and six unrelated sex-matched healthy donors were established in our lab from skin biopsies by standard protocols. Controls had a negative BS and did match neither the Villefranche nor the Brighton criteria, as well as those for the other HCTDs. Dermal fibroblasts were grown Goat anti-type I collagen (COLLI) polyclonal antibody (Ab), rabbit anti-type III collagen (COLLIII) Ab, mouse anti-elastin (ELN) (clone 10B8), anti-α5β1 (clone JBS5), anti-αvβ3 (clone LM609), and anti-α2β1 (clone BHA.2) integrin monoclonal antibodies (mAbs) were from Millipore-Chemicon Int. . Goat anti-type V collagen (COLLV) Ab was purchased from LifeSpan BioSciences, Inc. . Anti-fibrillins (FBNs) (clone 11C1.3) mAb was from NeoMarkers . The rabbit Ab against human fibronectin (FN) and mAb against all of the human isoforms of tenascin (TNs) (clone BC-24) were from Sigma Chemicals . Rhodamine-conjugated anti-goat secondary Ab was obtained from Calbiochem-Novabiochem INTL, Alexa Fluor 488 anti-rabbit and Alexa Fluor 594 anti-mouse were from Life Technologies.To analyze the FN, COLLI, COLLIII, COLLV, and TNs ECM organization, JHS/EDS-HT fibroblasts were immunoreacted as described previously , 14. In Cells were then incubated for 1 h with anti-mouse or anti-rabbit secondary Abs conjugated to Alexa Fluor 594 and 488, or with anti-goat IgG. IF signals were acquired by a CCD black-and-white TV camera  mounted on a Zeiss fluorescence Axiovert microscope and digitalized by Image Pro Plus software . All experiments were repeated three times.in vitro transcription, cRNA were reverse transcribed and the corresponding cDNA was fragmented, biotin labeled, and hybridized over night at 45° C onto the arrays. The chips were then washed in the Fluidics station FS 450, scanned using the scanner 3000 7G system, and analyzed with the Affymetrix GeneChip Operating Software. Analysis of miRNA expression profile was performed on patients’ and controls’ fibroblasts in accordance with manufacturer’s instructions, starting from 250 ng of total RNA labeled with the Affymetrix Flash Tag Biotin Labeling Kit, followed by the hybridization on the GeneChip miRNA 3.0 array. The resulting CEL files were analyzed using Partek Genomics Suite software, version 6.6 Copyright; 2014 . One-way ANOVA analysis was conducted to identify the differentially expressed genes (DEGs) between patients and controls by using a combination of fold change value greater than 1.5 and a false discovery rate (FDR) ≤0.3, according to the Benjamini-Hochberg procedure [Total RNA was extracted from skin fibroblasts of patients and controls using the Qiagen RNeasy kit according to manufacturer’s instructions . RNA quality control was assessed on an Agilent 2100 BioAnalyzer . Transcriptome-wide expression profiling was performed using the Affymetrix Gene 1.0 ST platform. Microarray analysis was performed starting from 250 ng of total RNA per sample; labeled targets were prepared using Ambion Whole Transcript Expression Kit (Life Technologies) and GeneChip WT Terminal Labeling and Controls Kit  in accordance with manufacturers’ instructions. In brief, total RNA was primed with synthetic primers containing a T7 promoter sequence, reverse transcribed into first-strand cDNA and converted into double-stranded cDNA. Following the rocedure . One-wayThe miRNA target prediction databases miRWalk, TargetScan, and miRDB were queried to correlate the differentially expressed miRNAs with the DEGs. All microarray data are MIAME compliant, and the raw data have been deposited in the MIAME compliant GEO database with the accession numbers GSE77753 and GSE77756.HPRT, GAPDH, ATP5B, CYC1, and RPLP0 reference genes were also amplified for normalization of cDNA loading. Relative mRNA expression levels were normalized to the geometric mean of these reference genes and analyzed using the 2-(ΔΔCt) method. Expression of miRNA was assayed using stem-loop RT–PCR starting from 50 ng of total RNA in a final volume of 15 μl followed by TaqMan based qPCR profiling in accordance with manufacturer’s instructions (Life Technologies). The qPCR reaction contained 1.3 μl of reverse transcriptase product, 10 μl of TaqMan 2x Universal PCR Master Mix, and 1 μl of the appropriate TaqMan MicroRNA assay containing primers and probes for the target miRNA. Expression of selected miRNAs was based on the 2-(ΔΔCt) method by using RNU66 as endogenous control, and qPCR reactions were run in triplicate. Statistical analyses were performed with GraphPad Prism software . Results were expressed as the mean value of relative quantification ± SEM. Statistical significance between groups was determined using one sample t test .Relative expression levels of a series of selected genes/miRNAs identified by array analysis were confirmed by quantitative real-time PCR (qPCR) by using different RNA extractions. In particular, 3 μg of total RNA were reverse-transcribed with random primers by standard procedure. qPCR were performed with SYBR Green qPCR Master Mix (Life Technologies), 10 ng of cDNA, and with 10 μM of each primers set. qPCR were performed using the ABI PRISM 7500 Real-Time PCR System by standard thermal cycling conditions. Clinical findings of the five women are summarized in To investigate the organization of different structural proteins into the ECM of JHS/EDS-HT and control skin fibroblasts, expression of COLLI, COLLIII, COLLV, FN, TNs, FBNs and ELN, and distribution of α2β1, α5β1, and αvβ3 integrin receptors were analyzed by IF. As shown in In order to identify genes potentially involved in the molecular mechanisms underlying the complex pathogenetic basis of JHS/EDS-HT, transcriptome-wide expression analysis was carried out comparing gene expression pattern between patients’ and controls’ skin fibroblasts and using the Benjamini-Hochberg procedure to control the false discovery rate .A total of 208 DEGs were identified by applying a fold change threshold ≥1.5 with a FDR ≤0.3. In particular, 46 genes were significantly up-regulated and 162 down-regulated . Panel AAPOL1, AQP9, PTGER4, ENPP2, PPARG, APOBEC3G, COLEC12, and CFD. A range of genes, i.e., AKR1C3, AKR1C2, PHYHD1, MAOB, ADH1C, ND6, and GSTM5 were related to “oxidation reduction process” and involved in different aspects of cellular metabolism, such as “metabolism of xenobiotics by cytochrome P450”. Biological processes related to chemical homeostasis, including AQP9, ADM, PPARG, CLDN11, and SLC40A1 were also over-represented in JHS/EDS-HT fibroblasts.To identify biological processes that are over- or under-represented in patients’ fibroblasts, we classified all up- and down-regulated genes according to the GO categories. This analysis, performed on up-regulated genes, generated 4 distinct GO clusters . The mosIL6, SMAD7, KLF10, NR4A2, NFKBIA, NR4A1, NR4A3, FOSB, JUNB, LIF, HES1, INHBA, GATA2, FOS, and involved in regulation of kinase activity, i.e., SPRY2, ERCC6, PRLR, MET, SPHK1, TGFA, PDGFC, DGKI, GADD45B, TRIB1, VLDLR, and CBS. Processes related to regulation of inflammatory and defense responses with different involved transcripts, i.e., PVR, IL6, TGM2, NFKBIA, ITGA2, and IDO1, were also under-represented in patients’ cells. Different down-regulated transcripts, such as PCDHB8, PCDHB16, TEK, PCDH9, ROR2, ITGA4, CDH2, CDH10, SHROOM3, CHRM2, SMAD7, FHL2, DSP, OXTR, ITGA2, SSX2IP, and HOMER1, were involved in cell adhesion. This GO term was particularly enriched in genes that encode either protocadherins, i.e., PCDHB8, PCDH9, and PCDHB16, or cadherins, i.e., CDH2, and CDH10, a superfamily of Ca2+-dependent transmembrane proteins that act as cell adhesion molecules involved in homophilic cell adhesion and cell-cell junction organization , prolactin receptor , integrin receptors alpha 2 , and alpha 4 , G protein-coupled receptor CHRM2 (-3.36), transcription factor NR4A1 (-4.19), v-myc avian myelocytomatosis viral oncogene homolog , and by different members of receptor tyrosine kinases including MET proto-oncogene-receptor tyrosine kinase , and TEK tyrosine kinase .To identify differentially expressed pathways, enrichment analysis was carried out on all DEGs by using both Partek pathways algorithm and DAVID database with a significance threshold of p-value  tissues . In partINHBA, -3.40), and intracellular effectors, such as SMAD family member 7 , and inhibitor of DNA binding 1 , and 3 .Concerning TGFβ signaling, patients’ cells showed decreased expression of genes encoding cytokines, such as inhibin beta A , suppressor of cytokine signaling 3 , sprouty RTK signaling antagonist 2 , PRLR, and MYC mentioned above, were down-regulated.Pathway analysis also revealed perturbation of JAK-STAT signaling. Different related genes encoding cytokines and receptors, such as FOS, -2.25; and FOSB, -2.07) and jun B proto-oncogene . Other dysregulated genes that participate in this pathway were four and a half LIM domains 2 , inhibitor of NFkB signaling , and peroxisome proliferator activated receptor γ .Pathways analysis also identified perturbation of osteoclast differentiation, as shown by the down-regulation of Fos/activator protein-1 osteoclastogenic transcription factors including Fos (+/Ca2+exchanger), member 1 , oxytocin receptor , and different second messengers i.e., cholinergic receptor muscarinic 2 , sphingosine kinase 1 , and calcium-dependent enzymes such as phosphodiesterase 1C, calmodulin-dependent 70 kDa , and myosin light chain kinase .Pathways enrichment analysis also highlighted that the signaling calcium pathways seems to be altered in patients’ fibroblasts. Indeed, different members of this signaling cascade showed a decreased expression including calcium exchanger solute carrier family 8 , glutathione S-transferase , flavin monoamine oxidase , and aldo/keto reductase .Enrichment analyses further revealed perturbation of pathways involved in regulation of redox homeostasis and xenobiotics metabolism mediated by cytochrome P450 contained only up-regulated genes, such as members of the alcohol dehydrogenase family . Differential expression of CLDN11, DSP, FLG, ITGA2, and ITGA4 all playing a pivotal role in ECM-cell interaction and cell adhesion, were also confirmed  that encodes an ECM protein that functions as a regulator of cellular growth, differentiation, and apoptosis, and plays a critical role in innate immune response by acting as an opsonin to direct macrophage phagocytosis [Patients’ cells showed a decreased expression of  complex . On the ocytosis , 32. Accocytosis –36. SPONocytosis . In thisocytosis . Moreoveocytosis .The disarray of different ECM structural proteins and the altered expression of genes involved in ECM remodeling should play a role not only in the generation of inflammation, but also in the neuronal plasticity and hyperexcitability of nociceptive neurons and in the chronification of pain observed in JHS/EDS-HT patients . It is wCLDN11, which encodes a member of the claudin family that are molecules related to tight junctions, which are fundamental for maintenance of tissues architecture and morphogenesis [DSP that encodes desmoplakin, a member of cytolinker proteins family, showed decreased expression in patients’ cells. Desmoplakin is essential in epidermal sheet formation and is required for assembly of functional desmosomes, maintaining cytoskeletal architecture and reinforcing membrane attachments allowing for stable intercellular adhesion [CDH2, CHD10, PCDH9, PCDHB16, and PCDHB18. During embryonic development, cadherins control separation of distinct tissue layers, formation of tissue boundaries, and synapses between neurons. In adult tissues, they are involved in orderly turnover of rapidly growing tissues, such as lining of the gut and the epidermis, regulation of epithelial and endothelial cell junctions, and maintenance of the stable tissue organization [Cell adhesion, a fundamental process for formation and maintenance of tissues morphogenesis, seems to be altered in JHS/EDS-HT fibroblasts. In particular, we observed an increased expression of ogenesis . On the adhesion . As a funization . Cadherinization . NotablyFLG, AQP9, and AKR1C3. JHS/EDS-HT cells showed decreased expression of FLG that encodes filaggrin, a key protein that plays a central role in the formation of cornified cell envelope, which is critical for an effective skin barrier [FLG confer risk for associated allergic diseases including food allergy, and atopic asthma and are the most significant known genetic risk factor for the atopic dermatitis (AD) development, a common chronic inflammatory skin disease characterized by epidermal barrier dysfunction and immunological alterations [FLG, suggesting that dysfunction of pathways involved in skin barrier integrity, including keratinocyte differentiation may contribute to the AD pathogenesis [FLG, together with other unrecognized genetic and environmental factors, may contribute to a defective epidermal barrier increasing risk of atopic asthma and AD in JHS/EDS-HT patients. In this regard, earlier studies suggested increased prevalence of atopy and asthmatic symptoms in patients with different EDS types also comprising JHS/EDS-HT, in association with various pulmonary physiological abnormalities [AKR1C3 and AQP9: AKR1C3 encodes an enzyme of aldo-keto reductase family that promotes inflammation in skin lesions of AD patients [AQP9 codes for a member of the aquaporins that enhances skin barrier function and antimicrobial defenses [AQP9 as a novel marker of chronic inflammation underlying these diseases [Microarray revealed differential expression of several genes involved in epidermal development and keratinocyte differentiation, such as  barrier . Filaggr barrier . Mutatioerations , 48. Mulogenesis . Consistmalities . On the patients , while Adefenses . Furtherdiseases .CFD, COLEC12, NR4A1, NR4A2, NR4A3, and HES1. JHS/EDS-HT cells displayed an increased expression of complement factor D (CFD), a component of the alternative complement pathway that has a role in the inflammatory response and tissue injury [COLEC12 codes for a cell surface glycoprotein that acts as a scavenger receptor that is involved in the clearance of glycoproteins released by degranulation of neutrophils at sites of inflammation [NR4A1, NR4A2, and NR4A3 are ligand-dependent transcription factors that modulate NF-kB activity in a dynamic fashion, either repressing or enhancing target gene expression leading to altered inflammatory outcome [HES1 is an early marker of differentiation of multiple endocrine cell types in the developing stomach and gut, which transcriptional activity is regulated by SFRP2, a member of the secreted frizzled-related proteins that can modulate Wnt signaling [SFRP2, and PRICKLE1 were down-regulated, whereas receptor frizzled 3 (FZD3), required for the Wnt signaling cascade, showed an increased expression. These findings suggest that a perturbation of this transduction pathway, which is known to be involved in the development and renewal of the intestinal epithelium [Transcriptome data also highlighted significant expression changes of several genes related to inflammatory and immune responses that include e injury . This see injury . In agree injury . CFD ande injury  demonstrammation . NR4A1,  outcome . HES1 isignaling . In JHS/ithelium , might aithelium .PRLR encodes prolactin receptor, a member of the cytokine receptor superfamily that is expressed in a variety of immune cells, in which this hormone can be pro-inflammatory or anti-inflammatory by regulating proliferation, survival, and release of inflammatory mediators [KCNQ5 is a member of the K+ channels family involved in attenuation of the thermal hyperalgesia-induced inflammatory pain [ADM codes for adrenomedullin that is an important mediator for pathological pain, as its expression is enhanced both in acute and chronic inflammation, which triggers up-regulation of pronociceptive mediators and down-regulation of pain-inhibiting molecule in a cascade contributing to the development of morphine tolerance [Concerning pain, ediators . Serum eediators . PRL canediators . Thus, iediators . About tediators  showed tediators . In addiediators , 67, in ediators , 69. KCNory pain . ADM codolerance . Of noteGSTM5, ADH1B, ADH1C, SELENBP1, and MAOB. GSTM5, encoding glutathione S-transferase mu 5, plays a role in detoxification of drugs and products of oxidative stress [ADH1B, and ADH1C, encode enzymes of alcohol dehydrogenase family that metabolize a wide variety of substrates, including lipid peroxidation products [SELENBP1) is involved in selenium transport, an essential nutrient which displays neuroprotective and antioxidant activities in preventing certain neurologic diseases, such as schizophrenia and bipolar disorder. In this regard, up-regulated expression of SELENBP1 has been reported in both blood and brain of schizophrenic patients resulting a strong candidate biomarker for schizophrenia [MAOB encodes a monoamine oxidase responsible for the oxidative deamination of different neurotransmitters, such as serotonin, melatonin and dopamine. Increased expression of this enzyme was reported in age-related neurodegenerative diseases wherein it is associated with oxidative stress and vulnerability of the brain dopamine system [Our results showed up-regulation of several metabolic genes related to oxidant/antioxidant balance, i.e., e stress . Likewisproducts . Seleniuophrenia . Of noteophrenia , 75. MAOe system .In conclusion, although our data were obtained in a connective tissue cell model, this study pointed out significant gene expression changes that should perturb numerous biological processes, finally leading to the systemic clinical manifestations of JHS/EDS-HT patients. Future investigations on a larger cohort of patients are needed to corroborate the present results and also to identify potential biomarkers that may be supportive to the clinical diagnosis of this neglected disorder.S1 Figvs the statistical significance  on the y-axis is shown; up-regulated genes are reported in red, and down-regulated genes are in blue. (B) Hierarchical clustering of 208 DEGs identified in patients’ skin fibroblasts. Red color represents high gene expression, and blue indicates low gene expression. P: patients; C: controls.(A) Volcano plot depicts all statistically significant DEGs identified in JHS/EDS-HT cells. The fold-change of DEGs on the x-axis (TIF)Click here for additional data file.S2 FigRNU66 as an internal normalization transcript. The bars represent the mean ratio of the target miRNA expression in patients’ fibroblasts compared with five unrelated healthy individuals. qPCR was performed in triplicate, and the results are expressed as the mean ± SEM. Statistical significance was calculated with one sample t test .The expression levels of a selection of up-regulated and down-regulated miRNAs were evaluated using (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 Table(DOC)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.


Schistosoma japonicum insulin receptors (SjIRs) have been identified as encouraging vaccine candidates. Interrupting or blocking the binding between host insulin and the schistosome insulin receptors (IRs) may result in reduced glucose uptake leading to starvation and stunting of worms with a reduction in egg output. To further understand how schistosomes are able to exploit host insulin for development and growth, and whether these parasites and their mammalian hosts compete for the same insulin source, we identified insulin binding sites on the SjIRs. Based on sequence analysis and the predicted antigenic structure of the primary sequences of the SjIRs, we designed nine and eleven peptide analogues from SjIR-1 and SjIR-2, respectively. Using the Octet RED system, we identified analogues derived from SjIR-1 (10) and SjIR-2  with insulin-binding sequences specific for S. japonicum. Nevertheless, the human insulin receptor (HIR) may compete with the SjIRs in binding human insulin in other positions which are important for HIR binding to insulin. However, no binding occurred between insulin and parasite analogues derived from SjIR-1  and SjIR-2  at the same locations as HIR sequences which have been shown to have strong insulin binding affinities. Importantly, we found two analogues (1 and 3), derived from SjIR-1, and two analogues (13 and 15) derived from SjIR-2, were responsible for the major insulin binding affinity in S. japonicum. These peptide analogues were shown to have more than 10 times  stronger binding capacity for human insulin compared with peptides derived from the HIR in the same sequence positions. Paradoxically, analogues 1, 3, 13 and 15 do not appear to contain major antigenic determinants which resulted in poor antibody responses to native S. japonicum protein. This argues against their future development as peptide-vaccine candidates. Schistosoma mansoni and S. haematobium, only three molecules, S. mansoni fatty acid binding protein (Sm14), S. mansoni tetraspanin-2 (Sm-TSP-2) and S. haematobium glutathione S-transferase (Sh28GST) have entered human clinical trials   was added, the wells were incubated at room temperature for 20 mins and read on a plate reader using Microplate manager software . Data are presented as antibody endpoint titres, defined as the highest dilution of test serum that yielded an average O.D. two standard deviations (SDs) greater than that obtained in the absence of primary antibody.Antibodies were raised commercially against peptide analogues 1, 3, 13, and 15 in mice by GensCript Biotech Corporation . Briefly, mice received a primary immunization with 50 μg peptide conjugated with ovalbumin. Three boosts at three weekly intervals were commenced two weeks after the primary immunization. The injections were delivered subcutaneously at multiple sites along the neck and spine. Blood was collected two weeks after the final boost. The titre of the antibody was determined using an enzyme-linked immunosorbent assay (ELISA). Briefly, Maxisorb immunoplates  were coated overnight at 4°C in coating buffer (100 μl /well) with peptide (100 μl of 4 μg/ml) or adult worm antigen preparation (SWAP) from S. japonicum antigen extract. The crude antigen was prepared from adult worms of S. japonicum freshly perfused from the intestinal veins of mice percutaneously infected with 60 cercariae six weeks previously. After three washes in perfusion buffer (8.5 g NaCl and 15 g NaCitrate in 1 l of water), to minimise contamination of the schistosome protein extract with host components, an adult worm antigen preparation (SWAP) was made as described [The mouse anti-peptide serum was used in Western blotting to probe the native protein in a separated crude escribed . The SWAescribed .S. japonicum [S. mansoni [E. multilcularis [Drosophila melanogaster IR [S. japonicum, S. mansoni, D. melanogaster and Homo sapiens [To date, IRs have been characterized from only three flatworm species: aponicum , S. mans mansoni  and E. mlcularis . As a reaster IR  has been sapiens  and used sapiens . Peptide+2 1576.9 peak was identified in the mass spectrum . RP-HPLC and mass spectrum results for the 24 peptides  were acquired with an average purity of 98%, as determined by RP-HPLC and MS.Peptide analogue 13 was used as a representative example to illustrate peptide purity and quality. As shown in in vitro between human insulin and some of the peptide analogues, including 1, 13, 3, 15, 21, 20, 10, and 22  from SjIR-1 and 15 (KD = 6.2E-07) from SjIR-2, which are located at the same sequence position as analogue 29 in the HIR (KD = 4.48E-05) , also boThe similar insulin binding positions in the HIR and the SjIRs, and the relatively high sequence identity (39-41%) for analogues 1, 13, 15  between Overall, the results of the binding assays showed that the SjIRs have a stronger competitive capacity to bind host insulin  than HIR by sharing the same major insulin binding sites I  and II  in SjIR-1 and 2, respectively. It has been well established that a competitive relationship amongst growth factors occurs ubiquitously in mammalian cells allowing them to interact with different growth factor receptors containing a similar structure . HoweverS. japonicum-specific insulin binding peptides  in ELISA or western-blot.Titres of mouse anti analogue 1, 3, 13, 15 antibodies were 1:256,000, 1:64,000, 1:8,000, 1:64,000, respectively. We found that none of the antisera bound consistently a S. japonicum and then stimulated the cells with these peptides. T cell-mediated immune responses  were determined using flow-cytometric staining, but no significant responses were detected, further reflecting the lack of antigenicity of these peptides (data not shown).Although our structural modelling predictions indicated these four analogues are located on the surface of the SjIRs  and theyS. japonicum cercarial challenge model [It is important to emphasize, however, that immunization with the ligand domain of the SjIR-1 (rSjLD-1) and SjIR-2 (rSjLD-2) fusion proteins, which include analogues 1, 3 and analogues 13, 15, respectively, induced a significant retardation in worm growth, depressed fecundity and the release of 75% immature intestinal eggs in a murine vaccine/ge model ,13. FurtS. japonicum. Further, these peptides have more than 10 times stronger binding capacity for human insulin when compared with peptide analogues derived from the HIR in the same sequence positions. However, these analogues do not appear to contain major antigenic determinants as they generated poor antibody responses to native S. japonicum protein. Whereas this argues against the future development of these synthetic analogues as vaccine candidates, our study provides additional information about the IRs from these parasites, further supporting the development of a recombinantly-derived SjIR vaccine.Our study has shown that analogues 1 and 3 derived from SjIR-1 and analogues 13 and 15 from SjIR-2 are responsible for the major insulin binding affinity in S1 FigS. mansoni (SmIR-1 and 2), E.multilocularis (EmIR), D. melanogaster (DmIR) and Homo sapiens (HIR). Black boxes indicate identical amino acids and grey boxes denote sequence similarity. Peptides P13, P1, P28, P15, P3, P29, P21, P22, P10, P9 and P20 are shown boxed in different colours. These peptides all bound human insulin using the Octet RED system.The extracellular regions of SjIR-1 and 2 were aligned with those from insulin receptors in (PDF)Click here for additional data file.S2 Fig13; Right panel: MS chromatogram of analogue 13.The peptide (ADVRHSSSLTKLSRCTVIEGDLFIVFTR) was analyzed by RP-HPLC and MS. Left panel: RP-HPLC chromatogram of analogue (TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Despite modest improvements in survival over the last several decades, the treatment of AML continues to present a formidable challenge. Most patients are elderly, and these individuals, as well as those with secondary, therapy-related, or relapsed/refractory AML, are particularly difficult to treat, owing to both aggressive disease biology and the high toxicity of current chemotherapeutic regimens. It has become increasingly apparent in recent years that coordinated interruption of cooperative survival signaling pathways in malignant cells is necessary for optimal therapeutic results. The modest efficacy of monotherapy with both cytotoxic and targeted agents in AML testifies to this. As the complex biology of AML continues to be elucidated, many “synthetic lethal” strategies involving rational combinations of targeted agents have been developed. Unfortunately, relatively few of these have been tested clinically, although there is growing interest in this area. In this article, the preclinical and, where available, clinical data on some of the most promising rational combinations of targeted agents in AML are summarized. While new molecules should continue to be combined with conventional genotoxic drugs of proven efficacy, there is perhaps a need to rethink traditional philosophies of clinical trial development and regulatory approval with a focus on mechanism-based, synergistic strategies. Blood 2013; 122:360), etc.Despite significant progress in recent years in unraveling the genetic basis of AML , resultiMalignant cells are particularly vulnerable to the simultaneous disruption of multiple, cooperative survival signaling pathways ,16. In rA particularly popular combination has been that of DNA methyltransferase inhibitors (DNMTIs) with histone deacetylase inhibitors (HDACIs). That epigenetic processes play a fundamental role in cancer causation and progression has been recognized for over a decade now . ChromatFLT3-mutated AML and 0–7 (median 2) prior therapies, the ORR was 46%, including 27% CR/CRi [FLT3 mutations based on in vitro evidence of synergy against FLT3-internal tandem duplication (FLT3-ITD+) cells [DNMTIs have also been combined with many other classes of targeted agents in AML. Based on the ability of the proteasome inhibitor bortezomib to up-regulate miR-29b, resulting in loss of transcriptional activation of several genes relevant to myeloid leukemogenesis, including DNA methyltransferases and receptor tyrosine kinases, a phase I trial of bortezomib and decitabine was conducted . Seven o% CR/CRi . FLT3 li% CR/CRi . Decitab+) cells . Sequent+) cells . 57% of +) cells . Phase I+) cells  and with+) cells  in patie+) cells  and conc+) cells  administ+) cells ,37.In vitro, however, short interfering RNA (siRNA) silencing of the anti-apoptotic proteins Mcl-1 and Bcl-xL, but not Bcl-2, exhibited variable synergy with azacytidine [Given their efficacy and safety, and consequent widespread use as single agents for the treatment of patients with AML who are elderly and/or unfit for conventional chemotherapy, azacytidine and decitabine are increasingly being combined in clinical trials with a plethora of emerging, investigational targeted agents. Examples include volasertib (NCT02003573), a polo-like kinase 1 (PLK-1) inhibitor recently designated an “orphan drug” for AML , the fircytidine .FLT3-ITD+ AML cells and overcame FLT3 TKI resistance-conferring mutations such as F691L and D835V [A relatively recent development in the field of epigenetic targeting of chromatin networks in cancer has been the discovery of potent and specific small-molecule BET  inhibitors . The brond D835V . Furthernd D835V .Distinct from their role as epigenetic modifiers, HDACIs exert a plethora of other actions  in neoplAs noted above, the single-agent activity of HDACIs in AML is modest , and it 2 IV of bortezomib on days 1, 4, 8 and 11 and 1000 mg/m2 IV of belinostat on days 1–5 and 8–12 of a 3-week cycle. Of 35 response-evaluable subjects, one patient with mixed lineage leukemia (MLL)-rearranged biphenotypic acute leukemia refractory to “7 + 3” and FLAG-Ida  achieved a CR and went on to an allogeneic HSCT; 4 had a partial remission (PR), 14 achieved stable disease (SD), and 16 had disease progression. Four subjects discontinued study treatment due to adverse events (AEs). Overall, the regimen was very well-tolerated. One patient with Janus kinase 2 (JAK2)-mutated myelofibrosis transformed to AML remains on treatment beyond 31 cycles (manuscript in preparation). Efforts are currently underway to better characterize (at a genomic level) the leukemias of the two patients who did exceptionally well on this study.In pre-clinical studies, co-administration of sub-micromolar concentrations of the pan-HDACI belinostat with low nanomolar concentrations of bortezomib sharply increased apoptosis in AML and cell lines and primary blasts . Synergii.e., those that inhibit cyclin T/CDK9, additionally inhibit global cellular transcription [Cyclin-dependent kinase inhibitors (CDKIs) represent an interesting class of agents capable of inducing cell cycle arrest and apoptosis in malignant cells, particularly hematologic tumor types, which may be more susceptible to inhibition of cell cycling and apoptosis induction . Some ofcription , thus docription .WAF1/CIP1. These observations led to a phase I trial of the combination of alvocidib and vorinostat in patients with relapsed/refractory or poor prognosis newly diagnosed acute leukemia or higher risk MDS [2 IV load over 30 min followed by 20 mg/m2 infused over 4 h  on daysstration . No objestration .+ cells, including those bearing the gatekeeper mutation T315I, as well as in AML cells, both FLT3-mutated and -wild type . Considering that mutations that confer resistance to one of the most promising FLT3 TKIs, quizartinib, have been described [As discussed above, HDACIs, especially those that inhibit HDAC6, acetylate and thereby interfere with the function of chaperone proteins, in particular Hsp90, consequently down-regulating several pro-growth and pro-survival Hsp90 “client” proteins of critical importance in myeloid leukemias, e.g., breakpoint cluster region-Abelson (Bcr-Abl), mutant FLT3, c-Raf and Akt . In the escribed , this stCell cycle checkpoints, part of the DNA damage response (DDR) network, are in-built safety mechanisms the activation of which helps preserve genomic integrity by halting cell division upon the occurrence of DNA damage and allowing time for DNA repair . If repap53-wild type and -deficient, as well as in those bearing FLT3-ITD [+CD38−CD123+) population [Given that HDACIs induce DNA damage  and inhiFLT3-ITD . Furtherpulation . However15 to inactivate it, and Wee1 inhibition causes forced activation of CDK1, premature mitotic entry and impairment of homologous recombination [i.e., Tyr15 and Thr14, and dephosphorylation of both sites is necessary for full cyclin B/CDK1 (cdc2) activation. Upon G2/M checkpoint activation, ATR/Chk1 phosphorylates (and thereby inhibits) CDC25A, -B and -C, thus preventing premature mitotic entry [The Wee1 kinase has recently emerged as a novel therapeutic target in AML ,117,118.bination . Activatbination . Notablyic entry .14 [p53 and FLT3 mutational status, including in “LIC”s, primary AML blasts and in a xenograft mouse (flank) model [15 dephosphorylation, it also induced Chk1 activation and Thr14 phosphorylation [15 and Thr14 dephosphorylation, and full cyclin B/CDK1 (cdc2) activation, accompanied by premature mitotic entry and DNA damage [While Chk1 is a positive regulator of Wee1 (through stimulatory phosphorylation), Wee1 inhibition results in compensatory activation of Chk1, leading to phosphorylation of cyclin B/CDK1 (cdc2) at Thr14 , a thera14 . While t14 ,126,127,k) model . Importarylation . HoweverA damage . These d+ cells, both in vitro and in vivo [Aside from the strategies discussed above, HDACIs may potentially be successfully combined with a number of other investigational agents in AML. The first-in-class polo-like kinase 1 (PLK1) inhibitor volasertib was recently granted first “breakthrough” , and the in vivo . Enhance in vivo . Given t in vivo , the comThe first-in-class inhibitor of protein “neddylation”, MLN4924 , is anot3°8/Ser473) variably occurs in 50–80% of patients [CIP1/WAF1 [The phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is a cellular growth, proliferation, motility and survival signaling axis  that reppatients ,156; henpatients . Althougpatients ,159, haspatients ,161. ComIP1/WAF1 . Inactivi.e., they displace the apoptosis effectors from their association with the anti-apoptotic proteins [The Bcl-2 family of proteins stands at the crossroads of cellular survival and death, and the pro- and anti-apoptotic members of this family regulate the intrinsic, or mitochondrial, pathway of apoptosis . The semproteins , a functproteins .The discovery of ABT-737, a specific “BH3-mimetic” antagonist of Bcl-2 and Bcl-xL, demonstrated for the first time that specific protein-protein interactions could be targeted by small molecules, and ushered in a new era in cell death research . Subsequi.e., Mcl-1 and Bcl-2/-xL [in vitro (including in “LICs”) and in vivo, through the same mechanism, i.e., down-regulation of Mcl-1, which is induced by ABT-737 via ERK activation [Neither ABT-199 (GDC-0199) nor navitoclax inhibits Mcl-1, which is fundamental to the pathogenesis and maintenance of AML  and the cl-2/-xL ,184. Sincl-2/-xL  or transcl-2/-xL , respectcl-2/-xL . In the cl-2/-xL  and obatcl-2/-xL  have beecl-2/-xL . In thescl-2/-xL ,188. HDAcl-2/-xL  and obatcl-2/-xL . HDACI-icl-2/-xL . Both mocl-2/-xL . Finallytivation .FasL and Bim), Mdm2, glycogen synthase kinase 3 (GSK3) isoforms (which down-regulate cyclin D1 and Myc), procaspase 9, IκB kinase (the negative regulator of NF-κB), the endogenous CDKI p27KIP1 and Chk1 [A particularly strong rationale exists  for comband Chk1 ,194. Impand Chk1 , and conand Chk1 . Some ofand Chk1 ,198. Finand Chk1 ,161 and and Chk1 , in AML and Chk1 , throughand Chk1  and mTORand Chk1  demonstrand Chk1 . Indeed,and Chk1 . PI3K/mTand Chk1 . Respondand Chk1 . StudiesWAF1/CIP1 [The PI-CDKI combination of bortezomib and alvocidib is synergistic in myeloid leukemia cells ,205 but,AF1/CIP1 . In CML Dramatic potentiation of CDKI-induced apoptosis by inhibitors of PI3K has been demonstrated in AML cell lines and primary patient-derived blasts, accompanied by diminished Bad phosphorylation, induction of Bcl-2 cleavage, and down-regulation of XIAP and Mcl-1 . In contin vitro and in vivo, along with a requirement for ERK1/2 activation in progression across the G2/M boundary and through mitosis [p53 mutational status [The observation that inhibition of Chk1 triggers marked ERK1/2 activation, which can be blocked by MEK inhibitors  or Ras-t mitosis , as well mitosis ,215 prov mitosis . Furtherl status . Howeverl status ,211,212 l status , combinel status , could aThe number of rational combinations of targeted agents that are possible in AML and may be effective, at least in theory, is virtually limitless. The biggest challenge, therefore, is how to most judiciously choose the most promising combinations and bring them forward into clinical trials, which are costly and time-consuming. For these reasons, consideration should also be given to identifying the biologic subtypes of AML most likely to benefit from a given combination, as illustrated by the suggestion that basal Akt activation might predict for efficacy of a strategy simultaneously targeting Bcl-2/-xL nd PI3K/mTOR. Additionally, attention needs to be paid to better trial designs, e.g., adaptive designs, to get us answers to the biggest challenges confronting our patients in the most expeditious manner possible. Finally, the current paradigm for regulatory approval of new drugs in the United States discourages manufacturers from venturing into combinations of unapproved agents, and slows the pace of therapeutic progress. As a result, very few of the combinations discussed in this article have been tested in patients. While a complex problem, this is one that will require a concerted effort by lawmakers, researchers, industry and the concerned public to effect real change in the fight against cancer in general, and AML in particular.


H19/Igf2 methylation and transcription in porcine cloned embryos treated with trichostatin A (TSA), and examined H19/Igf2 imprinting patterns in cloned fetuses and piglets. Our results showed that compared with the maintenance of H19/Igf2 methylation in fertilized embryos, cloned embryos displayed aberrant H19/Igf2 methylation and lower H19/Igf2 transcripts. When TSA enhanced the development of cloned embryos, the disrupted H19/Igf2 imprinting was largely rescued in these treated embryos, more similar to those detected in fertilized counterparts. Further studies displayed that TSA effectively rescued the disrupted imprinting of H19/Igf2 in cloned fetuses and piglets, prevented the occurrence of cloned fetus and piglet abnormalities, and enhanced the full-term development of cloned embryos. In conclusion, our results demonstrated that aberrant imprinting induced by SCNT led to the abnormalities of cloned fetuses and piglets and low cloning efficiency, and TSA rescued the disrupted imprinting in cloned embryos, fetuses and piglets, and prevented the occurrence of cloned fetus and piglet abnormalities, thereby improving the development of cloned embryos. This study would have important implications in improving cloning efficiency and the health of cloned animals.Imprinting disorders induced by somatic cell nuclear transfer (SCNT) usually lead to the abnormalities of cloned animals and low cloning efficiency. Histone deacetylase inhibitors have been shown to improve gene expression, genomic methylation reprogramming and the development of cloned embryos, however, the imprinting statuses in these treated embryos and during their subsequent development remain poorly studied. In this study, we investigated the dynamics of  Though somatic cell nuclear transfer (SCNT) has been achieved in many species, overall cloning efficiency is still low, and the developmental abnormalities, including low birth rates, placental defect and large offspring syndrome, etc., frequently occur –3, limitin vitro produced embryos [It is generally believed that the developmental abnormalities of cloned animals and low cloning efficiency are largely due to incomplete epigenetic reprogramming, among which, genomic imprinting is a valuable genetic marker for understanding epigenetic reprogramming and evaluating the developmental competence and normality of  embryos –6.Genomic imprinting is an epigenetic regulatory mechanism, showing a monoallelic, parental-specific expression, and crucial for embryo development . In normTo facilitate epigenetic reprogramming and cloning efficiency, epigenetic modification agents are usually applied, among which, histone deacetylase inhibitors could improve DNA damage repair, gene expression and genomic imprinting, etc., in cloned embryos, leading to the enhanced development of cloned embryos –13. Our H19/Igf2, representing genomic imprinting, is most studied in animals, and also well-known in pigs [H19 and Igf2, controlled by the differentially methylated region (DMR) of H19, are maternal and paternal specific, respectively [Igf2. Otherwise, H19 expression has a silencing effect on Igf2 expression. This mechanism allows for the precise control of H19 and Igf2 expression. In this study, the DMR3 of H19/Igf2 (widely accepted) was selected to investigate the effects of TSA on the imprinting statuses of cloned embryos, fetuses and piglets [At present,  in pigs , 17. Preectively , 19. TheChemicals were purchased from Sigma Aldrich Corporation , and disposable and sterile plasticware was obtained from Nunclon , unless otherwise stated.All the treatments of piglets were approved by the Animal Care and Use Commission of Northeast Agriculture University, according to animal welfare laws, guidelines and policies. All pigs involved in this research were raised and bred according to the guideline of Animal Husbandry Department of Heilongjiang, China.PAFs were isolated from the ear of an American Large White boar. After removal of skin tissues and gristle, the remaining tissues were finely minced into pieces, digested with 0.25% trypsin-0.04% ethylenediaminetetraacetic acid solution (GIBCO), and dispersed in high glucose enriched Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum  and 1% penicillin-streptomycin (GIBCO). Then, the dispersed cells were centrifuged, resuspended and cultured in DMEM. Until confluence, PAFs were digested, centrifuged, resuspended in FBS containing 10% dimethyl sulfoxide and stored in liquid nitrogen until use. Prior to SCNT, PAFs were thawed, cultured and subsequently used in 3–5 passages.Oocyte maturation has been described previously . BrieflyThe procedures for porcine IVF and SCNT have been described in one of our previous reports . BrieflyEmbryo transfer has been described . About 2-1 Proteinase K at 56°C for 1 h. For PAFs, fetus and piglet tongue tissues, a Universal Genomic DNA Extraction Kit (TaKaRa) was used to extract genomic DNA. For samples of 200 MII oocytes and 200 1-cell, 100 2-cell, 50 4-cell, 30 8-cell and 10 blastocyst stage pooled zona pellucida-removed embryos, digestion was performed in M-Digestion Buffer plus with Proteinase K at 50°C for 20 min. After treatment, CT (cytosine to thymine) conversion reagents were added to all the samples at 98°C for 10 min and 64°C for 2.5 h. Then, the samples were desalted, purified and diluted with M-Elution Buffer. Subsequently, nested PCR was carried out to amplify DMR3 of H19/Igf2 using previously reported primers as described in Bisulfite sequencing has been reported . Briefly−ΔΔCT method.Measurement of gene expression with quantitative real time PCR has been applied in our previous studies , 21. BriDifferences in data (mean ± SEM) were analyzed with SPSS statistical software. Statistical analysis of data regarding embryo development was performed using t-text. The data for gene expression and DNA methylation were analyzed with one-way ANOVA. For all analyses, differences were considered to be statistically significant when P<0.05.H19/Igf2 in sperm and MII oocytes were examined, showing hypermethylation in sperm and hypomethylation in MII oocytes (H19/Igf2 methylation did not obviously change at the 1-cell stage in comparison with the mean methylation level of sperm and oocytes (53.57% vs 49.11%), and was maintained from the 1-cell to blastocyst stage  and DNA methylation (Dnmt1 and Dnmt3a) related genes were significantly improved , while the normal cloned fetuses showed significantly (P<0.05) higher H19/Igf2 methylation levels than the IVF fetuses and no significant differences from IV fetuses. And, Igf2 transcripts in the abnormal cloned fetuses were significantly lower than the normal cloned fetuses, IVF and IV fetuses , NT-TSA-N and IVF groups , IVF and normal cloned piglets  can represent genomic imprinting to be a valuable genetic marker for evaluating the developmental competence and normality of in vitro produced embryos [H19/Igf2 was chosen to evaluate the reprogramming of genomic imprinting during pig cloning.Genomic imprinting is an epigenetic mechanism that ensures parental monoallelic expression, and crucial for embryo development, fetus growth and postnatal behaviors , and the embryos , 19. HerH19/Igf2, coinciding with previous reports [Naturally, genomic imprinting is established during gametogenesis and maintained through the subsequent development, however, SCNT bypasses this step and destroys genomic imprinting , 8, 23.  reports , whereas reports . Moreove reports , 23, 24.H19/Igf2 transcripts were obviously upregulated in cloned blastocysts after TSA treatment, possibly benefit for the post-implantation development [Our previous studies have demonstrated that histone deacetylase inhibitors can improve genomic methylation reprogramming and the development of cloned embryos , 15, andelopment . Thus, Telopment , furtherH19/Igf2 were usually observed in abnormal cloned fetuses. This may be due to that the downregulated methylation levels of H19/Igf2 led to the reduced Igf2 expression, further resulting in the small and abnormal fetuses [H19/Igf2 compared with those in the NT-TSA group. Thus, to some extent, the imprinting improvement by TSA treatment promotes the development of cloned fetuses.The improvement of genomic imprinting in early cloned embryos would enhance their subsequent development or viability , 11. The fetuses , 27. IntH19/Igf2, similar to Beckwith Wiedemann syndrome in human assisted reproduction [H19/Igf2 DMR3 is upregulated, further leading to high Igf2 expression and piglet abnormality [H19/Igf2 were normal in the NT-TSA group, suggesting that TSA has a correcting effect on genomic imprinting. And more, TSA has been reported to normalize gene expression in cloned animals [When birth, most cloned piglets died of macroglossia in the NT-CON group, and the weight was higher than those of the live cloned or fertilized piglets. Notably, these cloned piglets with macroglossia were accompanied with the high methylation and expression levels of oduction , 29. Preoduction , 31. Thuormality , 27, it  animals . Thus, T animals , thus, mIn conclusion, our results demonstrated that aberrant imprinting induced by SCNT led to the abnormalities of cloned fetuses and piglets, and TSA rescued the disrupted imprinting in cloned embryos, fetuses and piglets, and prevented the occurrence of cloned fetus and piglet abnormalities, thereby enhancing the development of cloned embryos.S1 FigA, hypermethylation in sperm, B, hypomethylation in MII stage oocytes, and C, moderate methylation in PAFs. Black or white circles represent methylated or unmethylated CpG sites.(TIF)Click here for additional data file.S2 Figa-bValues with different superscripts differ significantly (P<0.05).The expression patterns of Hat1 (A), Hdac1 (B), Dnmt1 (C) and Dnmt3a (D) at the 1-cell, 2-cell, 4-cell, 8-cell and blastocyst stages of IVF, NT-CON and NT-TSA embryos. In comparison with IVF embryos, cloned embryos displayed the disrupted expression patterns of Hat1, Hdac1, Dnmt1 and Dnmt3a, while these gene expression profiles in NT-TSA embryos were almost normal. The transcript abundance in MII stage oocytes was considered to be the control. The data were expressed as mean ± SEM. (TIF)Click here for additional data file.S3 FigA, blastocysts (×40) derived from cloned embryos, and B, blastocysts (×40) derived from cloned embryos treated with 40 nM TSA.(TIF)Click here for additional data file.S4 FigIV-D, the dead piglet derived from the IV group, and NT-TSA-D, the dead piglet derived from the NT-TSA group. The IV, IV-D and NT-TSA-D piglets displayed nearly normal H19/Igf2 methylation. Black and white circles represent methylated and unmethylated CpG sites.(TIF)Click here for additional data file.S1 TableThe primer sequence, amplified length and gene accession number for bisulfite sequencing and quantitative real time PCR.(PDF)Click here for additional data file.


Despite the limited number of cases described in the literature with 17q12 rearrangements, our proband's phenotypic features both overlap and expand on previously reported cases. Since syndrome-specific DNA sequencing studies failed to provide an explanation for this patient's unusual habitus, we postulate that this case represents an expansion of the 17q12 microdeletion phenotype. Further analysis of the deleted interval is recommended for new genotype-phenotype correlations.Copy number variations involving the 17q12 region have been associated with developmental and speech delay, autism, aggression, self-injury, biting and hitting, oppositional defiance, inappropriate language, and auditory hallucinations. We present a tall-appearing 17-year-old boy with marfanoid habitus, hypermobile joints, mild scoliosis, pectus deformity, widely spaced nipples, pes cavus, autism spectrum disorder, intellectual disability, and psychiatric manifestations including physical and verbal aggression, obsessive-compulsive behaviors, and oppositional defiance. An echocardiogram showed borderline increased aortic root size. An abdominal ultrasound revealed a small pancreas, mild splenomegaly with a 1.3 cm accessory splenule, and normal kidneys and liver. A testing panel for Marfan, aneurysm, and related disorders was negative. Subsequently, a 400 K array-based comparative genomic hybridization (aCGH) + SNP analysis was performed which identified a  The 17q12 region contains copy number variations previously reported in association with a variety of clinical findings, most frequently renal cystic disease, maturity onset diabetes of the young type 5, pancreatic atrophy, Mullerian aplasia in females, and variable cognitive involvement . Renal cMoreno-De-Luca et al.  propose Herein, we report a patient with the 17q12 deletion using chromosomal microarray analysis presenting with autism spectrum disorder, behavioral difficulties, cognitive impairment, and joint laxity with a remarkable marfanoid body habitus, not previously described in reported cases of pathogenic copy number variations in this chromosomal region.The proband was a 17-year-old Caucasian male. His mother and father were 40 years old, and his mother was gravida 3, para 0 at the time of conception. The 32-week gestation was complicated by preeclampsia and maternal uterine fibroids with delivery by Cesarean section. At birth, the proband weighed 2 pounds 8 ounces (1.134 kg) (3rd centile) and measured 17.5 inches (44.45 cm) (70th centile) in length  detected on chromosomal microarray analysis who had speech delay and dyspraxia, mild learning difficulties, and autism spectrum disorder, a 4-year-old male with a 2.07 Mb deletion  who had speech delay, mild hypotonia, coronal hypospadias, and dysmorphic features including telecanthus, blepharophimosis, ptosis, epicanthus inversus, anteverted nares, depressed nasal bridge, long philtrum, and small ears with overfolding of the helices, and a 7-month-old male with a 1.6 Mb maternally inherited deletion  with a head lag and inability to sit independently. All patients had renal cysts and hypercalcemia during the neonatal period [Several reports support the association of cognitive impairment with 17q12 deletion. In a study of 4 patients with 17q12 deletion, Nagamani et al.  found 3  de novo deletions ranging from 1.49 to 1.85 Mb in size, including HNF1B and 19 other genes. They had early onset developmental delay, social interaction difficulties, restricted and repetitive behaviors, and communication delays. All were diagnosed with renal cysts [Loirat et al.  studied  HNF1B and LHX1 genes. The proband had attention deficit hyperactivity, disruptive behavior, and learning difficulty with no renal abnormalities, while her brother was found to have the same 17q12 deletion but only mild developmental delay and bilateral renal cysts. Their mother had the same deletion with learning difficulty but no renal cysts [Variable features have been reported among family members with the same 17q12 deletion. George et al. [While some reports have found an association between growth retardation and 17q12 deletion, no previous reports included tall stature or marfanoid body habitus. Hinkes et al. , however HNF1B gene has been frequently reported in association with maturity onset diabetes of the young type 5 (MODY5), cystic renal disease, renal dilations, pancreatic atrophy, and liver abnormalities also seen in this deletion syndrome. Other case reports of individuals with deletions and duplications at the 17q12 critical region have included the involvement of a critical region including the AATF, ACACA, CCL3L, C17orf78, DDX52, DUSP14, DHRS11, GGNBP2, HNF1B, LHX1, LOC284100, TBC1D3G, MRM1, MYO19, PIGW, SYNRG, TADA2A, and ZNHIT3 genes [TheT3 genes   may be responsible for these manifestations. Nevertheless, we also cannot rule out additional genetic phenomena such as variable expressivity, penetrance, and digenic inheritance due to the mutation of a second gene. Although clinical suspicion for the patient's microdeletion is responsible for his clinical presentation, further gene sequencing in this case, such as with whole clinical exome sequencing, is still warranted to rule out any additional contributing genetic factors. The authors encourage the reporting of other patients with this chromosome abnormality using microarray analysis to further delineate the genetic and clinical characteristics of pathogenic copy number alterations in this region.In summary, deletions of 17q12 have been reported in fewer than 100 individuals, and to our knowledge none have manifested a distinctly marfanoid habitus. We propose that our proband's unique phenotype is an expansion of the described 17q12 clinical spectrum. In support of this conclusion, it is very likely that poorly understood genes involved in our proband's deletion (such as


Here, we report 40 new whole-genome sequences of influenza A(H1N1)pdm09 viruses isolated from Finnish patients during 2009 to 2014. A preliminary analysis of these and 186 other whole genomes of influenza A(H1N1)pdm09 viruses isolated from hospitalized and nonhospitalized patients during 2009 to 2014 in Finland revealed several viral mutations that might be associated with patient hospitalizations. The most recent influenza pandemic and the epidemics following it were associated with influenza A(H1N1)pdm09 viruses. Infections with these viruses in most cases were self-limiting and led to the recovery of infected individuals within 1 week. However, in some cases, infections with A(H1N1)pdm09 viruses resulted in hospitalizations. Many different factors might be associated with the hospitalization of patients infected with A(H1N1)pdm09 viruses, including viral and host genetics, underlying health conditions, and lifestyle and environmental factors.Human influenza A viruses cause epidemics and pandemics . Ten mutations were found in viral hemagglutinin (HA)  and neuraminidase (NA)  glycoproteins. Thirteen signatures were found in polymerase subunits polymerase acidic (PA) (N321K), polymerase basic 1 (PB1) , and PB2 . Two signatures were found in matrix 1 (M1) (V80I and K230I), one in M2 (D21G), one in nonstructural 1 (NS1) (L90I), one in nucleoprotein (NP) (S498N), and one in PA-X (R221Q). No signatures were found in NS2/NEP.In this study, we searched for viral genetic factors that might be associated with patient hospitalizations. In particular, we sequenced the whole genomes of 40 influenza AH1N1)pdm09 strains isolated from nonhospitalized and hospitalized patients  who were diagnosed with influenza A virus infection. The sequencing procedure is described elsewhere , 106, 241, and 369 of NA, 321 of PA, 344 and 354 of PB2, 80 of M1, and their combinations were shown to enhance virus replication in human cells and mice and contribute to an increased severity of A(H1N1)pdm09 virus infections in patients admitted to hospitals in England during 2009 to 2011 68. We werWe found that the viruses that circulated in 2013/2014 in Finland drifted from the earliest Finnish A(H1N1)pdm09 isolate (A/Helsinki/P15/2009), for which the whole-genome sequence is available, by ca. 1.3% at the amino acid level. This indicates that novel mutations associated with patient hospitalizations may emerge and should therefore be monitored.KM437818 to KM437825, KM437826 to KM437833, KM437770 to KM437777, KM437778 to KM437785, KM437706 to KM437713, KM437842 to KM437849, KM437786 to KM437793, KM437794 to KM437801, KM437682 to KM437689, KM437658 to KM437665, KM437666 to KM437673, KM437674 to KM437681, KM437690 to KM437697, KM437698 to KM437705, KM437722 to KM437729, KM366559 to KM366566, KM366351 to KM366358, KM366359 to KM366366, KM366367 to KM366374, KM366391 to KM366398, KM366399 to KM366406, KM366407 to KM366414, KM366415 to KM366422, KM366423 to KM366430, KM366431 to KM366438, KM366439 to KM366446, KM366447 to KM366454, KM366463 to KM366470, KM366479 to KM366486, KM366487 to KM366494, KM366495 to KM366502, KM366503 to KM366510, KM366511 to KM366518, KM366519 to KM366526, KM366527 to KM366534, KM366543 to KM366550, KM366551 to KM366558, KM366567 to KM366574, KM366575 to KM366582, and KM366383 to KM366390.The full-genome sequences of influenza A viruses have been deposited in GenBank under the accession numbers