Integrated circuits utilize networked logic gates to compute Boolean logic operations that are the foundation of modern computation and electronics. With the emergence of flexible electronic materials and devices, an opportunity exists to formulate digital logic from compliant, conductive materials. Here, we introduce a general method of leveraging cellular, mechanical metamaterials composed of conductive polymers to realize all digital logic gates and gate assemblies. We establish a method for applying conductive polymer networks to metamaterial constituents and correlate mechanical buckling modes with network connectivity. With this foundation, each of the conventional logic gates is realized in an equivalent mechanical metamaterial, leading to soft, conductive matter that thinks about applied mechanical stress. These findings may advance the growing fields of soft robotics and smart mechanical matter, and may be leveraged across length scales and physics. A method to cultivate decision-making in soft materials would provide a key step to autonomous engineered matter. Here, the authors report a class of conductive polymer-based mechanical metamaterials that process information by digital logic and permit logic gate assembly. Liquid metal is often leveraged in compliant integrated circuits for the high conductivity and reversible self-healing behavior realized by liquid metal-based circuit interconnects8. Two-phase materials that combine conductive microparticles with a polymer substrate also support electrical function when subjected to large strains, while moreover being amenable to diverse fabrication practices12. To design the substrate for reconfiguration of a compliant integrated circuit, programmable mechanical deformation is often a candidate for reversible shape change4. For instance, kirigami13 and origami14 have inspired substrates that fold to tailor electrical behavior15. Elastic instabilities are also considered to transition electrical states in response to mechanical16 and thermal17 stresses.Structurally and materially compliant integrated circuits with reconfigurable electrical functions are an essential foundation for human-machine interfaces, soft robotics, and other future electronics that will serve medicine, science, engineering, and industry. These compliant conductors alleviate concerns of failure traditionally encountered when conventional metallic conductors are subjected to mechanical stress18. The search for logic-based information processing in artificial materials has led to concepts of discrete transmittance outputs resulting from elastic wave logic operations in mechanical metamaterials19 and from infrared wave logic operations in photonic metamaterials20. Logic functions in soft matter have also been emulated by high contrast colorimetric outputs from thermochromic elastomers subjected to input pressure states21.Reconfiguration of electrical networks is indeed the basis of digital logic, which is integral for information processing in modern computers as well as in the human brain23. Recently, discrete shape reconfigurations are cultivated by elastic beam buckling25. In formulations of buckling-based mechanologic, the two statically stable configurations represent the physical abstraction of discrete digital bits. Together, the digitized bits associated with mechanical buckling modes facilitate logic operations according to the design and assembly of the switch-able constituents26. This stratagem has been shown for mechanically27, chemically28, and humidity29 triggered elastic instabilities. Controller signaling for pressurized soft robots has also been demonstrated through mechanologic-based signal processing, suggesting one means for soft matter autonomy30. Yet, current embodiments of logic processing of mechanical stress inputs lack digital electrical outputs, which limits means to communicate with actuation and sensory mechanisms that may require electrical feedback to function27. Similar to anatomical realizations of intelligence and information processing to monitor and transmit sensory and motor impulses31, a means to cultivate decision-making capability in mechanically-robust compliant materials would provide a significant step towards autonomous soft matter able to assess and react to mechanical-electrical stimuli in a dynamic environment.Recent progress on embodiments of mechanologic provide another modality of information processing in soft matter. By the concept of mechanologic, a digital bit is abstracted as a reversible, mechanical, or material configurationThis report introduces a class of soft, conductive mechanical metamaterials with programmable elastic instabilities that function as electronic logic gates able to perform digital computations on mechanical stress input combinations. Here, we exploit reversible compact-to-deployed state transitions in conductive mechanical metamaterials through a strategic patterning of compliant substrate and soft, conductive material networks. We demonstrate physically reconfigurable metamaterials that respond to mechanical stress in discrete modes leading to changes in the compliant conductor network to realize all digital logic functions and logic assemblies.32 report that this geometry exhibits a negative Poisson’s ratio and a fully compacted state wherein the porosity of the compacted material is 0% in the ideal case of kinematic reconfiguration. In Fig. The metamaterials employ a constant cross-section consisting of a fundamental square tiling tessellation with square voids at the intersections. Shim et al.The elastomeric material substrates considered here are fabricated by casting liquid urethane rubber (Smooth-On VytaFlex 60) in 3D-printed molds (FlashForge Creator Pro) containing the negative of the sample architecture. The substrates also include surface channels in which the conductive networks are applied, Fig. 2 unit cells to be characterized more fully in the subsequent section of this report. An Ag-TPU trace pattern is applied to the elastomeric substrate. An electrical power supply and LED array are interfaced in series with the silver trace terminals at the sample leftmost and rightmost sides, respectively. By applying uniaxial displacement to the top of the metamaterial, the sample deforms into a fully compacted solid square Fig. The metamaterials studied in this report are referred to by the unit cell that constructs the constant cross-section architecture. In Fig. 2 unit cell geometry is shown in Fig. To characterize the elastic buckling deformation of the metamaterial unit cells, we develop a finite element model that simulates the material response observed in experiments (see Supplementary Information). The C2 unit cell illustrates three regimes during uniaxial compression, Fig. 2, the self-contact strain is The mechanical response for the C2 unit cells, Fig. 1 and R2 across the horizontal and vertical Ag-TPU terminal pairs, respectively.To demonstrate the relationship between the mechanical and electrical behavior by the compaction principle, we design a biaxially functional electrical switch Ag-TPU trace pattern on the surface of a metamaterial composed of C2 unit cell geometry and the Ag-TPU network on the sample, the metamaterial functions as a strain gated switch. For instance, Fig. 1 and R2 through the Ag-TPU trace pairs exhibit a nonconductive, infinite resistance that represents a 0 digital readout. Once the contact strain 1 and R2 to mean resistances near 3 Ω. The closed electrical connections therefore represent a 1 digital readout. A supporting kinematic model of the C2 unit cell confirms self-contact is induced near applied strain of 1 and R2 illustrates that the electrical connections may be achieved biaxially despite uniaxial mechanical input. These results introduce a technique to exploit transitions between uncompressed and compacted states of soft, conductive mechanical metamaterials to govern digital electrical signals.Using the C2 unit cells may exhibit two rotations in the lowest order buckling mode, Supplementary Fig. 1 that exploits a discontinuity with laterally adjacent unit cells in the left/right directions, Fig. We leverage this foundation to create logic gates controlled by mechanical deformation. Metamaterials composed from C1 unit cell is carried out to characterize the lowest order elastic buckling behavior. The unit cell top and bottom boundaries are relatively displaced with one side free to laterally displace in a given simulation. As shown in Fig. 1 unit cell exhibits a buckling mode at a critical strain of 0.0089. Two states of deformation may occur for the same critical strain. Mode 1(1) exhibits a clockwise rotation of the central bulk material block, while mode 1(2) exhibits a counterclockwise rotation. Realization of these modes is dependent upon the vector of shear and uniaxial compression applied respecting the horizontal mirror plane. Uniaxial compression simulations are conducted to determine the mechanical properties of the D1 unit cell. The results in Fig. 2 unit cell in Fig. 1 unit cell exhibits a lower critical strain 2 unit cell. A noticeable difference is also evident in the compacted states. Namely, the D1 unit cell compacts into a parallelogram cross-section Fig. 2 unit cell Fig. A modal analysis of the D1 unit cell as a digital input of 1 and clockwise rotation as a digital input of 0, according to electrical conduction through the D1 unit cell layer. The fundamental Buffer gate exemplifies this digitizing behavior as seen in Fig. 1 unit cell metamaterial outputs QBuffer of 1, and QBuffer of 0 for clockwise rotation. The Buffer metamaterial gate is realized by three D1 unit cells that are tiled together, keeping the discontinuity at the central bulk material block. The Ag-TPU trace applied to the surface realizes a switch that operates in agreement to Buffer logic in accordance with buckling modes of this metamaterial.Digital logic gates are provided with digital inputs and pass digital outputs. Our formulation of digital logic intrinsically couples discrete mechanical behavior with electrical signals passed through the metamaterials. Here, we consider a circuit powering node provided to an input terminal of the unit cell. The input node is highlighted in cyan color and labeled as NOT is 1 for the conductive Ag-TPU network in Fig. NOT of 0 is read out for the buckling mode with counterclockwise bulk material block rotation, Fig. 33, or critical strain, could be used for high sensitivity control34 of the combined mechanical and electrical functions of the soft, conductive logic gate.The NOT digital logic gate is an inverter of a digital signal, converting a 0 to a 1, and vice versa. The NOT gate is thus an inverted Buffer gate. In this embodiment of soft digital logic, output signal inversion is achieved by a horizontal or vertical mirroring of the conductive network. The metamaterial realization of the NOT logic gate that we explore here is shown in Fig. 1 unit cells each containing 5 unit cells to realize the metamaterial geometries shown in Fig. (1) requires shear to the right and then uniaxial compression, while mode 2(1) requires uniaxial compression then shear to the left near the critical strain, and vice versa for the mode 1(2) and mode 2(2). Thus, the logic inputs are represented by the layer-by-layer rotations, illustrated in Fig. 35.We build on this manifestation of soft, conductive matter-based digital logic to realize the remaining 6 logic gates: AND, NAND, OR, NOR, XOR, and XNOR. We use two rows of DAND = 1 when both layers rotate counterclockwise, or 1. Thus, a clear analogy exists between the circuitry schematic, the Ag-TPU network topology, and the mechanical buckling deformations. The Ag-TPU trace networks shown in Fig. The Ag-TPU trace networks required to realize the AND, NAND, OR, NOR, XOR, and XNOR gates in this formulation of soft digital logic are shown in Fig. Gates such as the NAND in Fig. To validate the functioning of the logic gates, samples are fabricated and compressed using the experimental setup in Supplementary Fig. AND = 1 for mode 1(2) when both mechanical inputs are counterclockwise rotations, and a digital output of QAND = 0 for all other combinations. The Supplementary Video The experimental digital outputs for each buckling mode behavior agree with the respective truth table entry for the ideal logic gates, Fig. 1 unit cell design explored here, metamaterial gates with n layers possess 2n self-contacting buckling modes, in agreement with the number of binary input combinations for the n input gate assembly. This differentiating characteristic allows for a systematic metamaterial design process to generate arbitrary gate combinations via strategic conductive trace network topologies (see Supplementary Information), although the platforms are not as much assembled from modules as they are designed into a monolithic metamaterial circuit. Explorations of modularly assembled soft logic gates using our mechanical metamaterial modules may take inspiration from mechanical signal transmission concepts27 as well as techniques explored with pneumatic-mechanical realizations of soft logic30. Although the logic gates formulated here do not compete with the speed and component scaling of conventional digital microprocessors, the conductive metamaterial-based information processing may provide essential robustness of function and material compatibility for future autonomous soft matter that emulate numerous biological systems.This foundation is extensible to the formulation of logic gate combinations used in integrated circuits. For instance, Supplementary Fig. 36, the mechanical metamaterial building blocks exploited in this work may be considered as building blocks for future soft matter assemblies, such as to realize autonomous soft machines with integrated sensory, actuation, and decision-making functionalities37. The concepts embodied in this report are also free of length-scale dependence, by leveraging nominally kinematic rearrangement of cellular solids32, and suggest one means to process mechanical stress inputs without need for periodic sensor observation. Consequently, physical embodiments of these principles may be exploited for low-power information processing in fields as diverse as DNA origami38, electro-optical filters39, reconfigurable antenna40, and more.By establishing interfaces between soft mechanical metamaterials and digital logic gating, this work formulates a general method to synthesize discrete mechanical configurations with conductive networks to govern digital electrical outputs. As one manifestation of soft, smart matter2 and D1 unit cells are prepared utilizing this procedure.The elastomeric metamaterial substrates are fabricated by casting liquid urethane rubber (Smooth-On VytaFlex 60) in a two-part mold. The mold parts are designed in CAD software SOLIDWORKS and 3D-printed (FlashForge Creator Pro) with acrylonitrile butadiene styrene (ABS) filament. When assembled together, the two-part mold realizes the negative of the metamaterial substrate shape. The liquid urethane rubber utilized in this research is a two-part material, A and B parts that are initially mixed in a 1 A:1 B volume ratio and stirred by hand for 2 min. After the material is poured into the mold, it is cured for 24 h. The sample is then carefully demolded and prepared for testing. Metamaterials using CThe conductive ink utilized in the channels is a composite containing 35%  silver (Ag) microflakes  and 65% (v%) thermoplastic polyurethane (TPU) elastomer (BASF Elastollan Soft 35 A). To begin processing the conductive ink, Ag microflakes are first mixed in a glass vial with sufficient N-Methyl-2-pyrrolidone (NMP) solvent, and sonicated (Branson M2800 Ultrasonic Cleaner) for 60 min. TPU is then added to the Ag-NMP mixture, and planetary mixed (KK 300SS Mazerustar) at 225 Enamel-coated copper wire (22 gauge) leads are passed through small molded-in 1.50 mm diameter channels in the substrate to terminate at the front cross-sections of the metamaterials. Metamaterial photographs in Figs. 2 and D1 and metamaterials assembled from the unit cells are examined using distinct experimental methods by virtue of the unique mechanical behaviors that motivate distinct electrical circuit switching methods. The specimens are quasi-statically and vertically displaced −1, shown in Supplementary Fig. 2, the bottom specimen surface rests on a polished, fixed aluminum plate, Supplementary Fig. 2 the top and bottom surfaces of the specimen have limited motion in lateral displacements The unit cells C1, the bottom surface is presumed to slide in the simulations reported in the main text. To permit lateral sliding of metamaterials in the experimental characterization, we place a polished, rigid aluminum plate on smooth cylindrical shafts that act as a rolling surface for the bottom of the D1 samples, Supplementary Fig. 1 and R2 are evaluated across the horizontal and vertical Ag-TPU terminal pairs, respectively. Data is collected through an acquisition system (NI USB 6341 Multifunction DAQ) and analyzed in MATLAB.For metamaterial D−1 quasi-static experimental load frame loading rate is used. A hyperelastic Neo–Hookean material model is employed with Young’s modulus of 2.07 MPa and Poisson’s ratio of 0.499 for the samples considered here. Free quadrilateral elements are used for all geometry meshes with seed sizes proportional to the smallest geometric feature with at least 0.25 ratio of element characteristic dimension to substrate feature size.ABAQUS Linear Buckling Perturbation and Dynamic-Implicit simulations are conducted with 2D plane strain models based on the constant material cross section. For the Linear Buckling Perturbation simulations, a subspace eigensolver is used to determine the low-order buckling modes. For the nonlinear Dynamic-Implicit step, a time period that is proportional to the 0.5 mm.min2 unit cell, periodic boundary conditions are imposed on the outermost nodes while the bottom boundaries are fixed in 1 unit cell and metamaterial assemblies the boundaries are fixed in For the CBy applying compressive displacements All experiments and simulations are conducted a minimum of 6 times to confirm repeatability of trends and quantities.Supplementary InformationSupplementary Movie 1


Although several genes involved in the development of Tetralogy of Fallot have been identified, no genetic diagnosis is available for the majority of patients. Low statistical power may have prevented the identification of further causative genes in gene-by-gene survey analyses. Thus, bigger samples and/or novel analytic approaches may be necessary. We studied if a joint analysis of groups of functionally related genes might be a useful alternative approach. Our reanalysis of whole-exome sequencing data identified 12 groups of genes that exceedingly contribute to the burden of Tetralogy of Fallot. Further analysis of those groups showed that genes with high-impact variants tend to interact with each other. Thus, our results strongly suggest that additional candidate genes may be found by studying the protein interaction network of known causative genes. Moreover, our results show that the joint analysis of functionally related genes can be a useful complementary approach to classical single-gene analyses. Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart defect (CHD) . A full The joint analysis strategy attempts to overcome these difficulties: 1) the total number of tests is smaller, and (2) the effect size may be greater if the effects are in the same direction. Therefore, one essential consideration of a joint analysis is which genes need to be analysed together, and which ones should not be merged. Since most proteins interact with other proteins in order to carry out their function, we hypothesised that variants in either member of an interacting pair working together in a biological process might have similar effects. This hypothesis is supported by previous research that found that clusters of functionally related proteins were associated with particular diseases –11. Inde the totap value < 0.01; Fig. p value < 0.01; proportion test). A total of 165 patients (19.9 %) contain a high-impact variant in a single gene in those groupings, while 24 additional patients (2.9 %) contain variants in more than one gene. Thus, high-impact variants affecting those 12 functional groupings were found in 22.8% of the patients. In addition to biological processes assumed to be involved in TOF such as signalling pathways (19 patients) or regulation of transcription (130 patients), there were groupings involved in post-translational protein modification (41 patients), intracellular protein transport (10 patients), and cilium assembly (23 patients). This latter result in particular is in accord with the growing evidence showing that ciliopathies are linked to many cases of congenital heart disease, including TOF [p value < 0.05; Bonferroni-corrected permutation test), with some proteins bridging distinct biological processes.Our results show that 12 functional groupings exceed the number of expected patients with high-impact variants .Reassuringly, our approach recapitulated previous findings obtained in two gene-by-gene analyses of the same data , 5, i.e., CEP290 , KIAA058KIAA0586 , TCF12 [6 [TCF12 ), or oth2 [PSEN2  and CD36N2 [CD36 ). NonethOur analysis has not only been able to recapitulate the most prominent general biological processes known to be involved in the development of TOF such as signalling pathways and regulation of transcription but has also identified more specific processes known to be involved in CHD such as cilium assembly , 20. ImpSupplementary Table ISupplementary Table IISupplementary Table IIISupplementary Table IVSupplementary Table VSupplementary Figures S1–S3


Whole exome sequencing (WES) of matched tumor-normal pairs in rare tumors has the potential to identify genome-wide mutations and copy number alterations (CNAs). We evaluated 27 rare cancer patients with tumor-normal matching by WES and tumor-only next generation sequencing (NGS) as a comparator. Our goal was to: 1) identify known and novel variants and CNAs in rare cancers with comparison to common cancers; 2) examine differences between germline and somatic variants and how that functionally impacts rare tumors; 3) detect and characterize alleles in biologically relevant genes-pathways that may be of clinical importance but not represented in classical cancer genes. We identified 3343 germline single nucleotide variants (SNVs) and small indel variants—1670 in oncogenes and 1673 in tumor suppressor genes—generating an average of 124 germline variants/case. The number of somatic SNVs and small indels detected in all cases was 523:306 in oncogenes and 217 in tumor suppressor genes. Of the germline variants, six were identified to be pathogenic or likely pathogenic. In the 27 analyzed rare cancer cases, CNAs are variable depending on tumor type, germline pathogenic variants are more common. Cell fate pathway mutations  dominate pathogenesis and double hit (mutation + CNV) represent ~18% cases. Genomic analysis of tumors has dramatically reshaped cancer treatment through the identification of genetic variants that provide diagnostic and prognostic information and that aid in therapeutic selection . PatientResearch to identify causes or to develop strategies for prevention or early detection are also difficult. Despite these challenges, rare tumors offer the opportunity to identify novel targets and signaling pathways . A recenIn this study, we performed tumor and matched germline whole exome sequencing (WES) of rare tumors from 27 patients that were evaluated at our Early Phase Therapeutics Program. Each patient also had a biopsy sample tested using a clinically available tumor-only assay from a commercial vendor for comparison. Our aim was to further characterize and compare both differences and commonalities of rare and common cancers, to better inform prognosis, selection of potential therapeutic pathways and design trials for rare cancer patients.Twenty-seven rare cancer cases  were evaluated with whole exome sequencing (WES) for tumor and germline. There were 23 different rare cancer types represented in our survey, all of which met the criteria of a rare cancer .http://ongene.bioinfo-minzhao.org/) and the Tumor Suppressor Gene Database (https://bioinfo.uth.edu/TSGene/). All 694 oncogenes and 1016 tumor suppressor genes (129 genes were classified as both OGs and TSGs) are listed (https://www.cancergenomeinterpreter.org/home). Of the variants deemed to be driver mutations, there were a total of 72 germline variants  and 22 somatic variants  (BRCA2-Q2859Kfs (gray zone lymphoma), SDHA-R75* (spindle cell breast cancer), SDHC-A3Rfs , RUNX1-M151L (glioblastoma), FANCC-c.456+4A>T splice site/exon skipping (anaplastic astrocytoma), and MUTYH-G396D   and small indel variants. Of these, 1670 were found in oncogenes and 1673 were in tumor suppressor genes, generating an average of 124 germline variants per case . This lie listed . Variant in OGs) . From gesarcoma) .SPEN-Q3621* nonsense variant that was identified in this tumor likely truncates the protein product , resulting in loss of a portion of a domain that is necessary for interactions with other nuclear co-repressors. While SPEN mutations are reported in 8% (8/97) of MCC samples in COSMIC, this variant is not functionally characterized and its effect on protein function is unknown.Among the 27 rare tumor patient cases included in our study, there were a total of 27 actionable somatic variants. Eight of the 27 patients in which we identified a variant of clinical significance had multiple actionable variants. One of the three tumors without an actionable variant—Merkel Cell Carcinoma (MCC), stained positive for Merkel cell polyoma virus and therefore was expected to have a very low tumor mutation burden . InteresActionable variants found in the patient cohort were classified in the four categories: FDA-approved for rare cancer type, FDA-approved for different cancer type, clinical trial for rare cancer type, clinical trial for different cancer type. All reported variants were deemed to be somatic.Unsurprisingly, for the majority of rare tumor patients in our study, available FDA approved medications and/or clinical trials were not available for use in the patient’s tumor type. In fact, only one patient had a variant with an FDA approved therapy, and three had a variant with a clinical trial for their tumor type . This isAdditionally, our tumor/germline approach identified a single pathogenic variant that turned out to be germline in origin. This frameshift variant (A3fs) in the C subunit of the succinate dehydrogenase (SDH) gene was found in a patient with gastrointestinal stromal tumor (GIST). This variant occurs early in the gene and is predicted to lead to nonsense mediated decay (loss of function), which generates oncometabolites that dysregulate epigenetic repression . The patp = 0.01800). Given the small sample size of our rare tumor cohort, we analyzed other rare tumors in these same databases to confirm the higher frequency of germline variants. There is a consistent yet slight increase in the overall percentage of germline variants detected in rare tumors in our cohort when compared to the other rare tumor cohorts  cancers . Among o cohorts . Of noteCopy number analysis (CNA) performed using Sequenza  on all WWhen mapping the locations of all CNVs on to each chromosome, we find that there appears to be several protected regions as well as hot spots incurring gains and losses of copies at a much greater frequency. While all chromosomes showed some level of amplification, there is a greater frequency of amplification events clustering near the tips of both the p and q arms A. There We used the TCGA containing 3149 common and 2120 rare cancers showed amplifications are more prevalent than deletions: rare (2.25-fold) and common cancers (3-fold) . Common To determine the alteration frequencies of pathways known to be involved in cancer, we mapped all variants to genes in 11 canonical signaling pathways . Figure TP53, which saw an increase of 17% to 28% from germline to driver. The number total of cases showing a predicted germline driver mutation was limited to 23, with (n = 5) cases each showing variants in WNT, Notch (n = 4) cases and each in RTK-RAS and TP53 (n = 3) in Hippo. No other pathway showed a germline driver mutation in more than 1 case. Of the somatic variants, the predicted drivers clustered around the Notch and p53 pathways with four and five respectively, with one case showing a double hit in TP53. We also saw three cases with a somatic driver in the RTK-RAS pathway, one of which also had a double hit.When the analysis was limited to only variants predicted to be driver mutations, we saw 22% of germline variants in the WNT signaling pathway and 13% in the Hippo pathway, with no somatic driver variants detected . All othTP53.Finally, we investigated if any variants classified as drivers had also undergone any additional copy number variation. This analysis identified 5 total cases (18.5%) experiencing a double hit . Of the Deep genomic profiling of rare tumors is critical to understand the molecular architecture of each specific cancer type to attain clear and actionable therapies due to difficulties in early diagnosis and limited or no standard of care therapies . Tumor-oRUNX1-M151L deemed likely pathogenic  but nothenotype . Over-expathways . Given trategies ,38.IDH1 and PIK3CA gene variants which occurred at the same site. The IDH1 variant occurred at amino acid 132 in the IDH1 gene in two sarcoma cases; chondrosarcoma R132C and pleiomorphic sarcoma R132G substitution respectively. The pleomorphic sarcoma with a concurrent IDH1 R132G mutation and a SUFU splice site mutation may indicate that the Hedgehog (Hh) pathway is operant in these cells independent of SHH ligand expression [SUFU was found in our metastatic mucoepidermoid sarcoma patient indicative of active Hh signaling amenable for therapeutic intervention. The mutant IDH1 makes it a highly promising candidate for the IDH1 small molecule inhibitor ivosidenib [PIK3CA variant occurred at amino acid E542K in two rare salivary gland tumors, myoepithelioma and mucoepidermoid tumor. A concurrent HRAS mutation identified in salivary mucoepidermoid tumor patient will most likely not respond to a PI3KCA inhibitor, however, the myoepithelioma patient could benefit from off-label therapy. There were nine genes in our cases that occurred in more than a single rare cancer case of which TP53 is the most prevalent occurring in seven cases. TP53 is the most common gene to carry pathogenic variants in common cancers with an average of 44.4%. In addition, we found two relatively common cancer genes in more than one rare cancer case: ARID1A (occurred in two cases and twice in one case) and CDKN2A (occurred in two cases). ARID1A and CDKN2A are also on the top 20 cancer gene list in breast, colon, and lung cancers, and are found in an average of 7.4% and 4.2% cases, respectively, across all four common cancer types. Both ARID1A and CDKN2A mutations are context dependent tumor suppressor genes that may be targetable in a synthetic lethal pair such as with a EGLN (prolyl hydroxylase) inhibitor (targeting HIF1α) or MTAP deletions with a PRMT5 (arginine N-methyltransferase 5) inhibitor dependent tumors respectively [Our cohort of rare tumors affected 18 different tissues of the body , with onpression  since SUpression . Furtherosidenib  approvedFOXL2, TNFAIP3, CHD1L, HRAS, KIT, CTCF—that occurred in 12 cases that are not on the top 20 list of any of the four common cancers used for comparison. These genes are found in <5% of breast, colon, prostate, or lung cancer cases. FOXL2 mutations was found in Granulosa cell ovarian cancer case, ~5% of ovarian cancer, which functions as a DNA binding forkhead transcription factor required for granulosa cell differentiation. The Cys134Trp mutation in FOXL2 is associated increased cell cycling and downregulation of genes associated with apoptosis [TNFAIP3 [CHD1L [TNFAIP3 is a ubiquitin-editing enzyme that binds and inhibits E3 ubiquitin ligase RNF168 responsible for regulating histone H2A turnover key to proper DNA repair, while CHD1L is a DNA helicase possessing chromatin remodeling functions important for early embryonic development and promotes cell proliferation and anti-apoptosis. The GIST patient also has a tumor suppressor-oncogene pair, CTCF [KIT [KIT mutations  are well established in GIST. We are the first to document a concurrent loss of CTCF and a KIT activating mutation in GIST, however, in SDH-deficient GIST, CTCF may be epigenetically silenced [Further, we found six genes—poptosis . The tum[TNFAIP3  and CHD13 [CHD1L  both havir, CTCF  and KIT TCF [KIT  both witTCF [KIT . Oncogensilenced .Copy number alterations (CNAs) play a role in cancer type  , tumor pAnalysis restricted to amplifications of oncogenes and loss of tumor suppressor genes showed the same patterns in both rare and common cancers . SarcomaThis study is limited by the relatively small sample size and by methodology that only targets the protein-coding regions of the genome. This limitation did not allow us to assess many important questions concerning the overall stability of some genomic regions compared to others in rare cancer, any impacts of epigenetic regulation, etc. In addition, there are difficulties in comparing rare and common cancers from different data sets. Despite these limitations our study suggests that tumor-germline matched analysis may have particular advantages in the case of rare tumors.n = 27, see Graphical Abstract) were evaluated at the University of Arizona Cancer Center Clinic and consented to an IRB approved protocol. All procedures performed, including buccal samples for germline DNA, involving human participants were done in accordance with the ethical standards of the University of Arizona Institutional Review Board . Tumor biopsies for all patients were evaluated by University of Arizona Genetics Core , where WES was performed. Paraffin blocks from the same tumor samples  were sent to Caris Life Sciences for NGS on a 592-gene panel (Caris MI/X platform).This study involved a consecutive group of rare tumor patients referred to the Early Phase Therapeutics Program regarding molecular profiling-based basket and umbrella clinical trials and/or off-label therapies. All patients . Matching germline DNA was isolated from buccal swabs. DNA was quantified using the Qubit quantitation system with standard curve as per the supplier protocol  and all samples were further tested for quality using the Fragment Analyzer  following the manufacturer-recommended protocols. The estimated fraction of tumor cells in the selected tissue ranged from 10% to 90%. Whole exome sequencing (WES) was performed by array capture of 60 Mb of exome target sequence using the SureSelectXT Human All Exon V6 enrichment  or a Nextera kit . All exome library builds were followed by paired-end sequencing (2 × 100 bp reads) on an Illumina HiSeq 2500 or NextSeq 550.https://github.com/vcflib/vcflib#vcflib). For MuTect2 results, variants were filtered based on the MuTect2-assigned filter flag, quality score, and read depth . For freebayes results, tumor and germline variants were separated into individual vcf files using “vcfsamplediff” function in vcflib. For freebayes somatic results, variants were filtered based on the quality score, read depth, and somatic score fields . For freebayes germline results, variants were filtered based on quality score and read depth (QUAL > 20 and DP > 20). The filtered variants were annotated using ANNOVAR release version “2018Apr16” [The mean coverage over the exon target regions was 154-fold for the tumor sample and 99-fold for the normal sample. Sequences were aligned to the human genome using standard methods , followeUsing vcftools, the remaining variants were restricted to either known oncosignaling genes for pathway analysis  or knownSomatic copy number alterations (CNAs) were identified using Sequenza v2.1.2  for R (vWES of tumor-normal allows for better CNV analysis in addition to identifying germline pathogenic mutations. In addition, WES of rare tumors provides information that is outside the current search space of gene panels but may ultimately provide clinically useful data to be used now or in the future. Although there is currently a relative shortage of tumor-specific FDA approved therapies and clinical trials for rare tumor patients, as novel targeted therapies become more readily available, specification of somatic vs. germline mutations and CNA in newly linked cancer genes will be critical in determining the best possible treatment options. This is especially important given the possibility of positive treatment outcomes for rare tumors with targeted and immune checkpoint therapies. Taken together, our study highlights the urgent need for exploration of tumorigenesis pathways in rare tumors and the subsequent need for development of specific clinical trials to determine optimal treatment strategies. As shown in our study, rare tumor patients may disproportionately benefit from both tumor-germline and WES approaches to potentially allow for better treatment options via future tumor genome-guided discoveries and therapies.


Capra ibex) population of the Bargy massif, reservoir of a virulent outbreak of brucellosis. We analysed the polymorphism and associations with disease resistance of the MHC Class II Drb gene and several non-MHC genes  involved in the innate immune response to Brucella in domestic ungulates. We found a very low neutral genetic diversity and a unique MHC Drb haplotype in this population founded few decades ago from a small number of individuals. By contrast, other immunity-related genes have maintained polymorphism and some showed significant associations with the brucellosis infection status hence suggesting a predominant role of pathogen-mediated selection in their recent evolutionary trajectory. Our results highlight the need to monitor immunogenetic variation in wildlife epidemiological studies and to look beyond the MHC.While it is now broadly accepted that inter-individual variation in the outcomes of host–pathogen interactions is at least partially genetically controlled, host immunogenetic characteristics are rarely investigated in wildlife epidemiological studies. Furthermore, most immunogenetic studies in the wild focused solely on the major histocompatibility complex (MHC) diversity despite it accounts for only a fraction of the genetic variation in pathogen resistance. Here, we investigated immunogenetic diversity of the Alpine ibex ( Often neglected, inter-individual variation in host susceptibility plays a key role in this dynamics since, depending on their biology , their spatial or social behaviours and/or their genetic characteristics, some individuals contribute far more than others to disease spread2. Therefore, identifying individuals and their characteristics that are most responsible for disease transmission is an important step for improving epidemiological surveillance and management of wildlife diseases3.The epidemiological dynamics of infectious diseases results from a complex interplay between the pathogen, the host and the environment4. Pathogen-mediated selective pressures shape the genetic components of host immunity and give rise to inter-individual variation in resistance to infectious diseases5. This immunogenetic variation is particularly well documented in humans and domestic animals but much less in wild animals. Furthermore, most studies focused solely on the adaptive branch of the immune system, particularly on the major histocompatibility complex (MHC)7. Although there is no doubt that MHC plays a key role in individual susceptibility to many diseases8, it accounts for only a fraction of the genetic variation in pathogen resistance9. Before the adaptive immunity intervenes in the host response, other genes involved in the innate immunity such as the Toll-like receptors (TLRs) genes are major players in the host frontline defense against a wide range of microparasites including vectors of zoonotic diseases such as Coxiella brunetii (Q fever) or Borrelia sp. (Lyme disease)12.Host genetic and immunogenetic characteristics are rarely investigated in wildlife epidemiological studies despite evidence that inter-individual variation in the outcomes of host–pathogen interactions is at least partially genetically controlled13. Populations that went through severe bottlenecks may show low levels of genetic diversity at the MHC  or TLR loci . However pathogen-mediated balancing selection may in some cases buffer the effect of drift to maintain polymorphism17. Several not-mutually exclusive mechanisms may act in synergy to maintain immunity-related diversity, the three more widespread being: the “negative frequency-dependence”, the “heterozygous advantage” and the “fluctuating selection”. The “negative frequency-dependent selection”  hypothesis suggests that the fitness-advantage of alleles is inversely proportional to their frequency in the population as a result of the host-pathogen18 or prey-predator co-evolutionary dynamics19. The “heterozygote advantage” hypothesis predicts a correlation between individual heterozygosity and fitness or disease susceptibility21. The “fluctuating selection” hypothesis23 suggest that population genetic diversity is maintained when spatially and/or temporally varying directional selection occurs24.Genetic variation at the MHC and other immunity-related genes encoding pathogen recognition receptors is shaped by both neutral  and selective processes. The relative importance of these evolutionary forces depends on both the population demographic history that influences the strength of the genetic drift, and the (multi)pathogen challenge that determines the type and intensity of selection processesCapra ibex) population from the Bargy area in France. This ibex population from the northern French Alps has undergone a major outbreak of brucellosis (Brucella melitensis). Until very recently, European wild ungulates were seen as dead-end hosts for this pathogen25 and all previous outbreaks of brucellosis in wild ruminants spontaneously faded out28. However, the situation in Bargy is alarming since the disease has affected an unprecedented high proportion of the population . Brucellosis persisted at high levels in the following years despite intense culling of animals which led to the removal of 40% of the population . Beyond the conservation and ethical issues, this outbreak has raised public health and economic concerns since, for the first time, cattle and humans were infected by the wildlife reservoir30. In this context, it was critical to identify the drivers of pathogen persistence in this population and individual characteristics favoring its spread. Previous works showed that host-infectiousness vary significantly across age and sex and among socio-spatial units within the Bargy massif29. However, genetic factors of susceptibility to brucellosis in Alpine ibex have never been investigated while several associations with innate gene polymorphism have been discovered in domestic ungulates32.Here, we investigated the neutral and immunity-related genetic diversity of the Alpine ibex  involved in the innate immune response to Brucella sp. in domestic ungulates36. The second objective was to investigate associations between individual brucellosis infection status and genetic/immunogenetic profile. We first assessed the role of multi-locus heterozygosity at neutral microsatellites as a proxy of inbreeding (heterozygosity-fitness correlation). We then looked at potential associations with the heterozygosity status (heterozygous advantage hypothesis) and/or specific haplotype of immunity-related genes. In both domestic and wild ungulates, brucellosis is well-known to cause abortion and reproductive failures38 with known impacts on individual fitness. Therefore, these associations may reflect pathogen-mediated balancing or directional selective pressure on immunity-related gene polymorphism.The first aim of this study was to assess the neutral and immunity-related genetic variation of the Bargy population. We screened the neutral microsatellite diversity to test the hypothesis that the intrinsic susceptibility to brucellosis of ibex at Bargy results from a particular low genetic diversity in comparison with other reintroduced ibex populations. We also evaluated the polymorphism of the commonly used E was 0.43 ± 0.17 (mean ± standard error) and mean NA was 3.12 over all microsatellite loci. Neutral variation did not vary significantly before  and after  the culling operations that occurred in 2013–2015 .The tests for Hardy-Weinberg equilibrium, after correction for multiple testing, revealed that SR-CRSP07 STR locus showed a significant excess of homozygotes. This marker was thus removed from further analysis. HMhc-Drb gene on 144 individuals revealed a unique haplotype in Bargy corresponding to CaIb-DRB*1 haplotype, previously isolated by Schalschl et al.39 and Grossen et al.40. The Sanger sequencing of the three Tlr genes generated an average of 2,169 bp of sequence covering almost the entire coding region of the genes (92% on average). Tlr genes exhibited seven SNPS among which five were non-synonymous. We isolated 1 SNPs for Tlr1 and revealed two functional haplotypes (i.e. encoding different amino acid sequences) including a rare haplotype (5%) observed in only 14 individuals. The Tlr2 gene exhibited three SNPs (two non-synonymous) and showed three functional haplotypes with a common haplotype (Tlr2a = 72%) and two haplotypes with low frequencies (Tlr2b = 15% and Tlr2c = 13%). Lastly, we found three SNPs (two non-synonymous) in Tlr4 gene and identified four functional haplotypes among which three haplotypes showed similar moderate frequencies ranging between 25% and 35% while the fourth one was rare (9%). Expected heterozygosity (HE) (given differences in haplotype frequencies) varied greatly among Tlrs ranging from 0.09 for Tlr1 to 0.71 for Tlr4  with frequencies of 75% and 25% respectively (HE = 0.37).The Illumina sequencing of the exon 2 of the SLC11A1 genotype and brucellosis status  had a lower probability to be seropositive than heterozygous individuals . Tlr1 heterozygous status was also associated with ibex brucellosis status  or of the heterozygous/homozygous status (heterozygote advantage) . The probability to be seropositive was lower in males than in females and reached a maximum in the core area of the massif   Table .Figure 140. By contrast, other immunity-related genes involved in the innate immunity such as Tlr genes  and Slc11A1 have maintained polymorphism despite serial founder events occurring throughout the Alpine ibex reintroduction history. We revealed significant associations between the polymorphism of some of these genes (Tlr1 and Slc11A1) and Brucella infection status, which may suggest a role of pathogen-mediated selection in their recent evolution.The aim of this study was to assess the neutral and immunity-related genetic diversity of the Alpine ibex population of the Bargy massif, wildlife reservoir of a persistent and virulent outbreak of brucellosis. As expected, this population established a few decades ago from a very limited number of founders (<15 ibex) shows very low genetic variation at neutral microsatellite loci and exhibits a single MHC Class II haplotype similarly to most ibex populations of the Western AlpsE) of ~40% and an average number of alleles (MNA) per microsatellite loci <4, the ibex population of Bargy shows particularly low levels of microsatellite genetic variation, compared to values reported in goat breeds  and in other wild European ungulate populations . However, these differences may arise because the microsatellite loci used in the different species underwent different ascertainment bias. Our results are consistent with the levels previously observed in other Alpine ibex populations reintroduced in Switzerland during the XXth century42 and in particular, for the Mont Pleureur population (HE = 0.42) from which the Bargy founders came from. Low genetic diversity might reduce individual fitness and population demographic performance due to reduced evolutionary potential44. For example, in the Gran Paradiso ibex population, there is a direct relationship between multi-locus microsatellite heterozygosity, male body mass and gastrointestinal nematode infestation  (i.e. Heterozygosity-fitness correlation)35. In our case, we did not observe any relationship between the genome-wide heterozygosity (MLH) of ibex and their Brucella infection status. This is in agreement with the recent study of Brambilla et al.45 that did not find any effect of neutral heterozygosity on infectious keratoconjunctivitis symptoms, another bacterial disease recurrently affecting Alpine ibex populations. Similar low levels of neutral genetic diversity were observed in other brucellosis-infected ibex populations (Grand Paradiso) in which the prevalence level has remained low and infection vanished spontaneously26. Therefore, contrary to our hypothesis, the low level of neutral diversity at Bargy is in itself not sufficient to explain its potential higher sensitivity to brucellosis compared to other ibex populations. However, a growing number of studies illustrated the poor predictive power of microsatellite loci to evaluate functional diversity and showed the importance of accounting for immunogenetic variation to get a better understanding of the population’s adaptive response potential to pathogen-mediated selection47.With an expected heterozygosity  play a fundamental role in vertebrate innate immune defense against intracellular and extracellular micropathogens including viruses, bacteria, protozoa or fungi50. In particular, these genes are involved in the recognition and immune response to pathogenic bacteria that recurrently cause outbreaks such as Mycoplasma sp. (causing pneumonia or infectious kerotoconjunctivitis infections)52 or Brucella abortus54. Contrary to our expectation and despite serial founder events throughout the species reintroduction history, the three studied Tlr genes and the SlC11A1 have all maintained several haplotypes while the MHC Class II DRB (second exon) is monomorphic in Bargy. There are very few studies that investigate Tlr polymorphism in ungulates populations: levels of Tlr haplotype diversity in Bargy ibex  appears similar to those reported for the same genes in roe deer populations (NH = 3–5)17, domestic goat (NH = 1–4)55 or cattle (NH = 2–5)56. Tlr4 showed four functional haplotypes (i.e. encoding different amino-acid sequences) including three alleles with moderate to high frequencies (>25%). This balanced polymorphism which contrasts with the very low neutral and MHC diversity in Bargy, suggests that balancing pathogen-mediated selection may have favoured the maintenance of genetic variation as showed in other free-ranging animal species58. Furthermore, it is worth noting that the presence of balanced polymorphism does not mean that the gene is involved in brucellosis resistance, which can be demonstrated only by studying associations between immunogenetic patterns and pathogen prevalence59.Our study is the first to our knowledge to reveal immunity-related polymorphism in non-MHC genes in Alpine ibex. Previous work on ibex focused exclusively on MHC Class II geneset al.35, we did not find evidence of correlation between multi-locus heterozygosity at neutral microsatellites and brucellosis infection status . This means that a higher individual multi-locus heterozygosity (a proxy of the level of inbreeding) does not confer a resistance advantage regarding Brucella infection. In contrast, we found associations between single immunity-related genes  and ibex infection status hence supporting the “local effect” hypothesis in agreement with Brambilla et al.45. These authors observed a lower susceptibility of MHC Drb heterozygous to infectious keratoconjunctivitis (heterozygote advantage hypothesis) while we revealed here an effect of specific alleles. Ibex carrying two copies of the less frequent allele of the Slc11A1 gene were less likely to develop the Brucella infection, which suggests that this “resistance allele” is recessive. Associations between a specific Slc11A1 allele and Brucella infection has been already reported in domestic goat36 and water buffalo31. Moreover, in an in-vitro experiment on water buffalo’s monocytes infected by several Brucella species including B. melitensis, Borriello et al.60 demonstrated that some Slc11A1 genetic variants confer a higher Slc11A1 mRNA expression and a higher ability in controlling the intracellular replication of the Brucella. The “resistant allele” (A330) showed similar frequency in the Bargy and Aravis populations (25% and 21% respectively) but is much more frequent in other Alpine populations (e.g. 54% in Belledonne massif (northern French Alps), 40% in Grand Paradiso National Park – results not shown). Further comparison with other recently reintroduced ibex populations should be performed to investigate whether the particularly low frequency of this resistant variant in Bargy may have favored the emergence of this brucellosis outbreak together with other, non-genetic factors such as spatial ibex distribution and abundance.In contrast to Brambilla Tlr1 (96%) conferred a resistance advantage against Brucella infection. However, this resistance advantage is relative: although 92% of ibex in Bargy are homozygous for this allele, 37% are still seropositive. Therefore, individuals that carry out this allele have a lower probability to get infected after a contact with the bacteria, but do not show a complete resistance. Such relationship between Tlr1 genotype and Brucella infection has been already evidence in Cattle by Prakash et al.32. Tlr1 interact with Brucella sp. by recognizing specific pathogen-associated molecular patterns. Individuals carrying the rare allele (Tlr1b) may have a decreased ability to recognize Brucella and thus to trigger an adequate immune response. Further work is needed to clarify the functional role of these immunity-related genes against brucellosis in Alpine ibex and in particular how these polymorphisms affects the detection and the replication of the bacteria . Particularly, in vitro studies of ibex white blood cells and their response to Brucella36 would help to confirm a variation linked to immune genes and to identity cellular pathways to resistance.Our results also suggest that the most frequent haplotype of Slc11A1 and Tlr1) and brucellosis infection status in Alpine ibex. However, this does not in itself provide evidence for directional pathogen-mediated selection because we did not actually demonstrate that these alleles confer a selective advantage. Seropositive ibex were euthanized, so there is no data available on the impact of B. melitensis infection on survival and reproduction rates in this population. However, brucellosis is well-known to cause abortion and reproductive failures in other domestic and wild ungulates38. So we can reasonably hypothesize that B. melitensis infection actually affects the fitness of ibex in Bargy and may ultimately lead to selection on these genes.In agreement with our hypothesis and the literature on domestic ungulates, we showed significant associations between immunity-related genes  involve micropathogens  and MHC Class II often plays a minor role in the immune response against these pathogens49. Our study highlights the need to look beyond MHC class II and to explore the diversity of other candidate immunity-related genes5, in particular MHC class I, cytokine and Toll-like receptors genes that are centrally involved in the innate immune response against micro pathogens in humans and domestic species.Genetic monitoring is widely recognized as a valuable tool for the management and conservation of populations65 is recommended to prevent inbreeding depression and increase both genome-wide45 and immunity-related diversity47. Further, these results raise the question of the interplay between sanitary management and population genetic structure. Here, the test-and-cull management procedure was expected to affect the genetic structure of the population, by selectively eliminating infected individuals carrying specific alleles. In the same line, some authors suggested that artificial selection of specific Slc11A1 or Tlr genotypes in breeding programs may help to increase population natural resistance and limit spread of brucellosis in domestic ungulates32. However, it is important to keep in mind that natural populations are exposed to multi-pathogen pressures (e.g. brucellosis and Q fever in the Bargy population) and that most immunity-related genes are involved in the immune response against several pathogens. Therefore, artificially selecting a specific genotype to increase population resistance to a pathogen may lead to the loss of key haplotypes in the host-control of other diseases, particularly in species with very low genetic diversity. Incorporating data on immunogenetic variation in epidemiological models should help to predict how variation in allele frequency may affect the spread dynamics of the disease, and how disease management may affect genetic structure of the population.In a conservation framework, our results call for immediate management actions to increase the neutral and immunity-related diversity of the Bargy ibex population, whose census size had been halved  over the monitoring period. Translocations of unrelated individuals from the Gran Paradiso source population or from other genetic units of Alpine ibex66. Since the discovery of the outbreak in 2012, the population has been continuously monitored by Capture-Mark-Recapture (CMR)29. Using GPS data on marked individuals, Marchand et al.29 demonstrated that females are structured in five distinct socio-spatial units . Animals were captured by dart-gun xylazine-ketamine anesthesia  following protocols described in Lambert et al.67. During captures, test-and-cull was implemented on the basis of serological tests  as part of management measures decided by the French Authorities . Seronegative individuals were marked and released while seropositive ones were euthanized. All captured animals were handled by professionals from the French Game and Wildlife management agency  according to the ethical conditions detailed in the specific accreditations delivered by the Préfecture de Paris , by the French Minister of Ecology, Sustainable Development and Energy  and by the Préfecture de la Haute-Savoie  in accordance with the French environmental code (Art. R421-15 to 421-31 and R422-92 to 422-94-1). Euthanasia was performed by veterinarians in accordance with the requirements of these accrediting authorities, and slaughtering operations were performed in accordance with accreditations delivered by the Préfecture de la Haute-Savoie . We hence confirm that we treated animals in accordance with relevant guidelines and regulations and that our protocols were approved by the National Committee for Nature Protection (CNPN in French), a group of independent experts elicited by the ministry of the environment and in charge of evaluating capture/destruction of the protected species.Alpine ibex were reintroduced in the Bargy Massif (France) during three release events in 1974 and 1976 comprising a total of 14 released individuals  translocated from the Mont Pleureur population in Switzerland29.In addition to CMR monitoring, French Authorities implemented selective culling of animals with observed clinical signs and mass indiscriminate slaughter of individuals of five years and more: culling operations occurred between autumn 2013 and spring 2014 (N = 256 killed) and then in autumn 2015 (N = 70). The population size dropped from 567 individuals (95% CI: [487–660]) in 2013 to 310 [275–352] in 2014 and 277 [220–351] in 201568. In particular, since 2014, the rapid Laminar Flow Immune-chromatographic Assay (LFIA)  was performed in the field. This test had been validated on ibex samples by the EU/OIE/FAO and National reference laboratory . Further details on the validation method and the congruence of the FLIA with reference tests  were provided in Anses (2014). Seropositive ibex were considered as infected by B. melitensis, but not necessarily infectious at the time of capture67.Brucellosis serologic status of individuals was evaluated using a range of serological tests42 . The same individuals were also typed at a microsatellite located at the 3′UTR of the Solute Carrier Family 11 member A1 (SLC11A1) gene using the following primers: Fw- TCTGGACCTGTCTCATCACC and Rv- ACTCCCTCTCCATCTTGCTG70. The SLC11A1 gene, formerly known as natural resistance-associated macrophage protein 1 (NRAMP1) gene, is involved in the innate resistance to intra-cellular pathogens71. Polymorphism in microsatellites of the 3′ UTR of the gene were associated with resistance to Brucella infection in water buffalo Bubalus bubalis36 and in the domestic goat31, a species closely related to the Alpine ibex. In particular, some SLC11A1 genotypes have a higher ability of controlling the intracellular replication of Brucella in vitro60.DNA was extracted from blood using the DNeasy Blood and Tissue kit (QIAGEN). All individuals were genotyped at 25 polymorphic putatively neutral microsatellitesMhc-Drb and Tlr genes for a subset of the individuals (N = 146) sampled between 2012 and 2014. The second exon of the Mhc-Drb class II gene encoding the ligand-binding domain of the protein was amplified and sequenced using Illumina MiSeq system following the procedure detailed in Quéméré et al.17. Haplotypes and individual genotypes were identified using the SESAME barcode software72. Tlr genes  were genotyped using the two-step procedure described in Quéméré et al.17. A pilot study on 30 individuals was first performed to identify polymorphic sites (SNPs). We screened almost the entire coding region of the three Tlr genes (92% in average) including the leucine-rich extracellular region of receptors involved in antigen-binding. Details on primer sequences and accession numbers are provided in Table 73.We also genotyped E) and the number of alleles/haplotypes (NA) as implemented in GENETIX software v4.05.274. For each locus, a test for Hardy–Weinberg proportions was performed based on 1000 permutations. To allow comparison with other ibex populations42 and between the two monitoring periods (before and after culling operations), we used FSTAT version 2.9.375 to calculate the allelic richness (sNA), a standardized measure of number of alleles corrected for differences in samples size. Lastly, standardized multi-locus heterozygosity at neutral microsatellites (MLH) was calculated for each individual as the ratio of its heterozygosity to the mean heterozygosity of the population76. Our set of microsatellite loci showed significant identity disequilibrium , (e.g. a measure of covariance in heterozygosity), a condition for detecting heterozygosity-fitness correlations. Both MLH and g2 were calculated using the R package inbreedR77. To assess whether management operations  had led to a decrease of the genetic diversity, we performed a comparison (using Student’s t-tests) of the allelic richness (sNA) and expected heterozygosity (HE) of ibex sampled before (between autumn 2012 and autumn 2013) and after (years 2016–2017) the slaughtering operations that occurred between autumn 2013 and autumn 2015.The genetic diversity of both microsatellites and immunity-related genes was evaluated by calculating the expected heterozygosity  of the binomial family (using a logit link function). The serological status of each individual was coded as 1  or 0 .vs homozygote) and the “haplotype advantage” hypothesis by considering associations between the infection status and the number of copies  of Tlr or Slc11A1 haplotypes with frequency >10%. Concerning non-genetic factors, following Marchand et al.29, we included the effects of ibex sex as a categorical variable and age as a continuous variable in years (linear and quadratic terms). We also fitted a “year” effect as a categorical variable to test for varying epidemiological situations across years and a “location” effect because seroprevalence has been shown to vary among socio-spatial units29.We fitted models testing for the effect of genetic effects, while taking into account other biological and environmental factors that possibly affect pathogen exposure. We tested the “heterozygosity fitness-correlation” hypothesis  by fitting individual standardized multi-locus microsatellite heterozygosity (MLH) as a fixed effect. For each immunity-related gene, we tested the “heterozygote advantage hypothesis” by fitting a binary fixed effect . We conducted an initial exploration of our data to ascertain their distribution and spread, to identify outliers and examine relationships between variables81. We used variance inflation factors (VIF) to assess which explanatory variables were collinear, and should be retained in the analyses 81. To minimize multicollinearity, the effects of the most frequent haplotype of a gene and its heterozygosity status were evaluated in separate models.We used a multimodel inference approaches to establish which explanatory variables had an effect, averaged over plausible modelset al.79 and using the “zero method” (i.e. a zero is substituted into models where the parameter is absent). Analyses were performed using the package lme482, MUMIN v1.7.783 and AICcmodavg v1.2584 in R version 3.3.3 .Model averaged parameter estimates are provided with their unconditional standard errors (SE) and 95% confidence intervals, after averaging model with ΔAIC < 7 (relative to the best model) as recommended by Burnham Table S1— S5Table S6.Table S7.


The natural history of adrenal function in autoimmune Addison disease once diagnosed and treated has not been systematically studied, but several case reports of recovery from established adrenal failure suggest it may not be uniform.To ascertain steroidogenic function in autoimmune Addison disease immediately following diagnosis and during prolonged treatment.1-24 in 20 newly diagnosed autoimmune Addison disease patients at first presentation and then again within a month. We also studied 37 patients with established Addison disease (for between 7 months and 44 years) in a medication-free state, measuring peak serum cortisol responses to ACTH1-24 and the urine LC-MS steroid metabolome.We studied peak serum cortisol in response to ACTHP < 0.003).Adrenal steroidogenesis declined rapidly after steroid replacement treatment for newly diagnosed Addison disease was started, with a peak serum cortisol falling from 138 ± 19 nmol/L (SEM) at presentation to 63 ± 13 nmol/L over 4 weeks (Six of 37 participants (16%) with established Addison disease had detectable serum cortisol and urine glucocorticoid and mineralocorticoid metabolites during repeat testing, indicating variable degrees of residual adrenal function.Autoimmune Addison disease is a heterogeneous condition, showing a rapid decline in adrenal steroidogenesis during the first few weeks following diagnosis, but low-level residual function in a minority of patients, which appears to persist for many years. Autoimmune Addison disease (AAD) is a rare endocrinopathy characterized by immune-mediated destruction of the adrenal cortex . Without1-24-stimulated serum cortisol in 10 patients, but concluded that there was no useful recovery of adrenal function in any subject  test within 4 weeks of the commencement of oral glucocorticoid replacement. These participants were recruited for enrollment into 2 different studies  that attempted to improve adrenal function early on in the natural history of the condition . Patients with primary adrenal failure resulting from adrenal gland infiltration or infection, with secondary adrenal failure, or with autoimmune polyglandular syndrome type 1 were excluded (Thirty-seven patients with established AAD (diagnosis established more than 6 months before study participation) were recruited from the endocrine clinic at our center . A diagnosis of AAD was confirmed by the subjects having either a low basal cortisol level with a high ACTH level or a subnormal response to the short Synacthen test , lower limit of detection 20 nmol/L. A healthy peak cortisol response to ACTHeviously  and analeviously . To asset-test. Categorical data were analyzed by Fisher exact test. Nonparametric continuous data  were analyzed by Mann-Whitney U test. Correlation between serum cortisol and urinary steroid metabolites was performed using a Spearman rank test. All analysis was performed using Prism v0.8.2.0 software.Comparison of parametric continuous data  was performed using unpaired Student 1-24–stimulated serum cortisol measurements. Plasma ACTH was increased in all patients at presentation . They were subsequently treated with hydrocortisone, recruited for the therapeutic clinical trials and ACTH1-24–stimulated serum cortisol measurements were repeated 14 to 28 days later (median 26 days), before any other intervention. In 19 of the 20 cases, stimulated serum cortisol dropped substantially over the first few weeks of hydrocortisone treatment, from a median at presentation of 125 nmol/L (range < 23-257) to 39.5 nmol/L (range < 23-265),   were studied on the day of first presentation with adrenal insufficiency, either with random or ACTH U test) .Thirty-seven patients  with established Addison disease from between 7 months and 44 years duration were studied . Thirty-Interestingly, patient B , Fig. 2 1-24 stimulation (peak 22 nmol/L) had comparable levels of urine glucocorticoid precursors to several individuals with easily detectable serum cortisol, suggesting that urine steroid analysis may represent a more sensitive indicator of residual steroidogenesis  AAD patients had some evidence of residual adrenal function with detectable serum cortisol and urine glucocorticoid and mineralocorticoid metabolites. In addition, at least 1 other participant had clearly detectable urine steroid metabolites in the absence of a significant serum cortisol response. In common with the study of Vulto et al. , we founOur results indicate that AAD is not a homogeneous condition and that a sizeable minority of patients with AAD may have low-level residual adrenal function. Whether these patients represent a distinct cohort either in terms of their etiopathogenesis, requirement for steroid replacement or resilience to adrenal crisis and other complications remains unknown. However, it seems likely that there is no relationship between time since diagnosis and the presence of residual adrenal function, with several individuals identified with residual function by this study having had AAD treated for more than 10 years. Furthermore, we have been able to study one individual over 4 years and demonstrate that the residual adrenal function was indeed persistent. This would fit with the idea that the adrenocortical stem or progenitor cells remain intact and are spared the autoimmune attack in these individuals . In contThe strength of this study lies in the design of testing participants in a medication-free state, which has not been done previously, and in measuring both stimulated serum cortisol concentrations and the urine steroid metabolome. The weaknesses are that all but one patient were studied at just a single time-point, and that although the largest cohort studied in this way to date, it still remains a relatively small cohort of AAD patients.In conclusion, our study illuminates several novel aspects of the natural history of AAD. First, that patients with newly diagnosed AAD have a rapid decline in steroidogenic capacity once replacement glucocorticoid medication is commenced, presumably owing to reduced adrenal ACTH exposure. We also show that a sizeable minority of AAD patients have durable low-level residual adrenal function even many years from their original diagnosis and institution of replacement therapy. This may be sensitively detected by serum 11-deoxycortisol or its major urinary metabolite tetrahydro-11-deoxycortisol. These observations should help plan future intervention studies that could target specifically patients with residual adrenal function to ameliorate this chronic condition.


Although obesity is considered as the main cause of Type II diabetes (T2DM), non-obese individuals may still develop T2DM and obese individuals may not.PI3K/AKT axis from 100 non-obese and obese participants with insulin sensitivity and insulin resistance states were compared in this study toward the understanding of obesity heterogeneity molecular mechanism.The mRNA expression of IRS1 and PTEN between groups, whereas PI3K, AKT2 and GLUT4 genes were expressed at a lower level in obese diabetic group compared to other groups and were statistically significant. PDK1 gene was expressed at a higher level in non-obese diabetic group compared to obese diabetic and non-obese non-diabetics groups. No statistically significant difference was identified in gene expression pattern of PI3K/AKT pathway between obese non-diabetics and non-obese non-diabetics.In present study, there was no statistically significant difference in gene expression levels of PI3K/AKT pathway which is related to the fasting state, showed reduced expression in obese diabetic group due to the chronic over-nutrition which may induced insensitivity and reduced gene expression. The pathogenesis of insulin resistance in the absence of obesity in non-obese diabetic group could be due to disturbance in another pathway related to the non-fasting state like gluconeogenesis. Therefore, the molecular mechanism of insulin signalling in non-obese diabetic individuals is different from obese diabetics which more investigations are required to study insulin signalling pathways in greater depth, in order to assess nutritional factors, contribute to insulin resistance in obese diabetic and non-obese diabetic individuals.The components of  The relationship between insulin resistance and obesity is well recognised while the responsible mechanisms of obesity-related insulin resistance remain poorly understood –3. Even PI3K/AKT-dependent pathway (Phosphatidylinositol 3-kinase/Protein Kinase B) which results in GLUT4 (Glucose Transporter 4) translocation to the plasma membrane to mediate glucose uptake and activation of glycogen synthase [PI3K phosphorylates membrane phospholipids and converts PIP2  to PIP3 . This complex phosphorylates/activates the PDK1 and PDK2 (Phosphoinositide-Dependent Kinase) leading to activation of AKT/PKB and PKC (Protein Kinase C) phosphorylation to translocate GLUT4 to the plasma membrane from intracellular vesicular compartment [PI3K/AKT pathway can be reversely dephosphorylated by PTEN phosphatase (Phosphatase and TENsin homolog deleted on chromosome 10) through converting PIP3 back to PIP2 [Recent progress in research methodology on signaling pathways has identified the insulin signal transduction and documented its alterations in insulin-resistance state –7. Insulsynthase . By stimpartment . PI3K/AK to PIP2 .IR, IRS, PI3K, AKT and GLUT4 have been reported to be reduced in Type II diabetes [Insulin resistance in Type II diabetes has been characterized by several defects in the insulin signaling cascade , 12–14. diabetes –19. UndeType II diabetes is a multifaceted disease resulting from the interaction of genetics, epigenetics, lifestyle such as diet and environmental as the contributing factors. These risk factors induce or suppress expression of genes involved in insulin signaling . NutritiPI3K/AKT pathway in obese and non-obese metabolically healthy individuals and compare this pattern with obese and non-obese diabetics to propose molecular mechanistic insights into how differential regulation of PI3K/AKT pathway is responsible for obesity heterogeneity in Type II diabetes.The aim of this study was to investigate the gene expression pattern of PI3K/AKT pathway. In diabetic and non-diabetic group, 50% were normal/overweight (BMI < 30 kg/m2) and 50% were obese (BMI of > 30 kg/m2). This weight distribution was not significantly different between four groups. The age ranged from 35 to 60 years and there was no statistically significant difference between groups (P>0.05). Participants who had cancers, nephropathy complications, thyroid & parathyroid diseases and pregnant women were excluded from this study. Also, participants were excluded if they reported using any form of tobacco, cigarettes and alcoholic beverages. The study was approved by the ethics committee of Universiti Putra Malaysia and National Medical Research Register (NMRR & MREC). Also, the study was conducted in accordance to the Declaration of Helsinki in its currently applicable version and the guidelines of the International Conference on Harmonization of Good Clinical Practice (ICH-GCP). All participants gave written informed consent before taking part in the study.A total of 50 Type II diabetic and 50 non-diabetic individuals recruited in the context of a cross-sectional study on insulin signal transduction at the Universiti Putra Malaysia and Serdang Hospital. Diabetic and non-diabetic participants were divided to two groups based on category of body mass index (BMI < 30 and BMI > 30) to find out differences in molecular mechanism underlying regulation of The mRNA expression of insulin signalling components from i) obese Type II diabetic participants (OD) and obese non-diabetics (OND), ii) non-obese Type II diabetic participants (NOD) and non-obese non-diabetics (NOND), iii) obese Type II diabetic participants (OD) and non-obese diabetics (NOD), iv) obese non-diabetic participants (OND) and non-obese non-diabetics (NOND) were compared in this study toward the understanding of obesity heterogeneity molecular mechanism.Assessment of clinical and metabolic characteristics of participants. FBS (Cat.No:04657527) was tested by colorimetric enzymatic method using Roche Diagnostics GmbH Cobas-c 311 Germany machine. HbA1c analysis was performed by the colorimetric method  and C-peptide level was determined by ELISA Kit .Blood samples were collected after an overnight fast. Total RNA was isolated by using PAXgene Blood RNA Tubes  according to the manufacturer protocol and it was converted to cDNA by using the Qiagen cDNA Synthesis Kit . Real time PCR (RT-PCR) reactions were performed by using SYBR green technology and QuantiTect Rev. Transcription Kit . The final volume of the RT-PCR reaction mixture was 20 μl and contained 2 μl cDNA, 1 μM of each primer, 10 μl Master Mix and RNase-free water up to 20 μl. RNA concentration and purity were measured by using a Nano Drop ND1000 spectrophotometer at A260:A280 ratio . RNA quality and integrity were determined via agarose gel electrophoresis. A positive control containing all the components of the reaction in except of template and a negative control containing template without reverse transcriptase were used to ensure quality of run and confirm the absence of contamination. RT-PCR was performed on a Bio-Rad cycler  and cycling conditions were as follows: 95 °C for 5 min ; 95 °C for 10 s (denaturation); 60 °C for 30 s ; followed by 40 cycles.GAPDH and β-ACTIN as housekeeping genes.Real-time PCR results were calculated according to the ∆∆CT method by using P < 0.05.Each experiment was performed triplicate and all data were expressed as means ± SE. Statistical analysis was performed by using SPSS 21.0 statistical software package . The univariate normality assumptions were verified with the Shapiro-Wilk test. Independent-samples t-test was applied to compare the means between two groups and threshold of significance was defined as a Clinical and metabolic characteristic data of participants involved in this study is summarised in Table PI3K/AKT pathway is responsible for glucose uptake from blood stream to the target cells, the underlying mechanism has not been clarified yet. In current study, gene expression pattern of the components of this pathway has been identified and compared among the participants based on obesity and insulin sensitivity state, as demonstrated in Tables Although the PI3K, AKT2 and GLUT4 expression levels were significantly lower in obese diabetic participants compared to obese non-diabetics. PDK1 expression level was significantly higher in non-obese diabetic participants compared to non-obese non-diabetics. The obese diabetic and non-obese diabetic groups were significantly different based on PI3K, AKT2, GLUT4 and PDK1 gene expression levels. The gene expression levels of PI3K, AKT2 and GLUT4 were significantly lower in obese diabetic participants compared to non-obese diabetics whereas the gene expression level of PDK1 was significantly higher in non-obese diabetics compared to obese diabetics. No statistically significant gene expression difference was identified between non-obese non-diabetics and obese non-diabetics.These results provide evidence that non-obese diabetics might be different from obese diabetics, also obese diabetics might be different from obese non-diabetics in mechanism of insulin signal transduction which causes obesity heterogeneity.PI3K, AKT2 and GLUT4 were significantly lower in obese Type II diabetic participants in comparison to the obese non-diabetics.Expression levels of PI3K/AKT pathway between non-obese diabetic and non-obese non-diabetics in except of higher expression level of PDK1 in non-obese diabetics.There was no significant difference in gene expression pattern of PI3K, AKT2 and GLUT4 were significantly lower in obese diabetic participants compared to non-obese diabetics whereas the gene expression level of PDK1 was significantly higher in non-obese diabetics.The gene expression levels of PI3K/AKT pathway genes.The non-obese non-diabetic and obese non-diabetic groups were not significantly different based on Likewise, there was no difference in the trends of the data when men and women were analysed separately.PI3K/AKT pathway toward the identification of genetic determinants of obesity heterogeneity in Type II diabetes. Consideration of only PI3K/AKT axis of insulin signalling pathways limited interpretations to this study.The main strength of this study is the inclusion of non-obese and obese participants with insulin sensitivity and insulin resistance states. This study design allowed for a simultaneous investigation on the influence of obesity and diabetes on PI3K/AKT pathway in NOD group is more closely resemble to the gene expression pattern of OND and NOND groups and differs from OD group. Indeed, significant higher gene expression level of PDK1 in NOD group compared to other groups emphasizes disturbance in another signaling pathway in NOD individuals.Even though other biochemical and physiological differences may exist between OD and NOD group beyond their BMI, the results of this study suggest that the gene expression pattern of PI3K/AKT pathway gene expression and insulin resistance and Type II diabetes is not necessarily associated with obesity and alterations in PI3K/AKT pathway gene expression. However, this study has revealed that disturbed PI3K/AKT pathway genes expression was associated with obesity and insulin resistance. Classification of obesity into metabolically healthy obesity and metabolically unhealthy obesity can also be explained by this hypothesis. Insulin resistance occurs when obesity is accompanied by reduced gene expression of PI3K/AKT2/GLUT4 pathway. An obese individual with a normal PI3K/AKT2/GLUT4 gene expression level would be still metabolically healthy until the time body is not able to keep this ability. Another assumption is that their adipocytes are able to switch from glucose to fatty acid metabolism which may be beneficial in combating metabolic syndrome [PI3K/AKT pathway in NOD individuals, disturbances in other insulin signalling pathways are proposed.Therefore, it can be hypothesized that insulin resistance and obesity might be distinct risk factors for metabolic disturbances . Furthersyndrome  or the csyndrome , hence, PI3K/AKT (phosphatidylinositol 3-kinase/protein kinase B) and MAPK (mitogen-activated protein kinases) pathways [PI3K/AKT pathway has been considered which is related to the fasting state, hence the components of this pathway showed alterations in OD group as a result of over nutrition. Over-stimulated PI3K/AKT pathway due to the chronic over-nutrition may induced insensitivity. This “overstimulation-induced insensitivity” phenomenon is commonly present in almost all of the metabolic disorders [PI3K/AKT pathway in OD group. Therefore, pathogenesis of insulin resistance in the absence of obesity in NOD individuals could be related to other pathways of insulin signaling [Two major downstream pathways of insulin action are the pathways , 27. In isorders  which maignaling , 30.PI3K, AKT2 and GLUT4 in OD group compared to other groups. Nevertheless, there was no significant difference in gene expression level of PTEN and IRS1 in OD, NOD, OND and NOND groups.The findings of present study showed reduced expression level of IRS1 expression level might be due to the collaboration of various mechanisms including signal amplification as a compensatory mechanism and convergence of other signalling pathways. However, the role of negative feedback loops cannot be neglected, as control of insulin signalling can be achieved by autoregulation whereby downstream elements inhibit upstream components [No significant difference in mponents , 32.PTEN decreases insulin-stimulated PI3K/AKT pathway, GLUT4 translocation and glucose uptake into the cells [PTEN antibody increases insulin-stimulated GLUT4 translocation to the cell membrane and glucose uptake [PTEN expression level might be related to the collaboration or interaction of PTEN with other phosphatases in antagonizing PI3K/AKT pathway and induction of diabetes. Therefore, further studies are required to differentiate the roles of different phosphatases, their interactions and collaborations. Since the main role of PTEN is performed by dephosphorylating the stimulated form of insulin receptor and modulating post-receptor signalling through converting PIP3 back into PIP2 and reversing the effects of PI3K/AKT pathway [It has been documented that overexpression of he cells , 33. Mic pathway –36, its  pathway –39.MAPK insulin signalling pathway was not investigated in this study, previous data from researchers [MAPK signal transduction contributes to development of Type II diabetes as well. The coordinated regulation of glucose uptake from circulation and endogenous glucose production is indispensable to maintain constant blood glucose level. The liver contributes to this process through controlling the gluconeogenesis and glycogenolysis. Suppression of endogenous glucose production during the postprandial state is attributed to insulin. Moreover, insulin activates glycogen synthase while simultaneously inactivates phosphorylase a and phosphorylase kinase to control glycogen metabolism through the MAPK pathway [MAPK phosphatase-1 exhibited hepatic insulin resistance and increased gluconeogenesis [PI3K/AKT pathway components). Transcription factor FOX01, has been identified as a main regulator of gluconeogenesis [PI3K/AKT axis and subsequently phosphorylates/inactivates FOX01 to inhibit gluconeogenesis [AKT could be corrected by concomitant hepatic deletion of FOX01 [FOX01 in the liver of mice caused reduced gluconeogenic genes expression level and fasting glucose. Also, glucose clamp studies demonstrated that FOX01 ablation impairs fasting gluconeogenesis and glycogenolysis.Although earchers  support  pathway . Mice laogenesis . Thus, togenesis . In the ogenesis . Hyperglof FOX01 , 44. SpePKC is an activator of gluconeogenesis through activation of MAPK pathway [MAPK pathway stimulates FOX01 axis to increase Glucose-6-phosphatase and PEP carboxykinase. PI3K via PDK1 activates PKC, which stimulates gluconeogenesis and contributes to insulin resistance by converting free fatty acids to Acetyl-CoA, which activates pyruvate carboxylase [It has been known that  pathway , 46 besi pathway . Activatboxylase  Fig. 1)PKC is anPI3K/AKT and MAPK pathways [PKC axis instead of AKT (PKB) axis. Though, this study did not directly address the effect of PKC activation on selective insulin resistance, contribution of endogenous glucose production is proposed in NOD individuals as a result of increased gene expression level of PDK1 in this group which is the main regulator of PKC axis. This selective model of insulin resistance might be induced by different nutritional stimulators and a combination of nutritional and hormonal model that includes multiple modes of insulin signalling pathways regulations is proposed [Consequently, the insulin regulation of endogenous and exogenous glucose metabolism and maintenance of glucose homeostasis are mediated though pathways . In thisproposed . This moIt has been suggested that ketogenic diets and high glycemic index or glycemic load diets develop Type II diabetes through endogenous glucose production and emphasizes the role of nutritional induction for obesity paradox in diabetes.PKC [PKC were protected from diet-induced insulin resistance, even with increased hepatic lipid content [FOX01 is increased in non-alcoholic steatohepatitis individuals that emphasizes the role of disturbed lipid metabolism in dysregulation of gluconeogenesis [ketogenic diets induce severe hepatic insulin resistance in mice through impairments in insulin suppression of endogenous glucose production during a hyper-insulinemic euglycemic clamp. These researchers claimed that hepatic insulin resistance could be attributed to the increase of hepatic triglycerides and diacylglycerol content which may cause reduced insulin signalling through activation of PKC , 49. Mic content . Also, gogenesis . ContradPKC axis [It has been suggested that chronic exposure to high glycemic index and high glycemic load diets stimulate more production of insulin, resulting in hyper-insulinemia and insulin resistance due to the pancreas exhaustion and failure . MoreovePKC axis .Another proof of induced insulin resistance through endogenous glucose production is hyperglycemia induced by psychiatric stress that play role in pathogenesis of Type II diabetes . The levMAPK pathway that controls metabolism and contributes in regulation of gluconeogenesis. Therefore, the pathogenesis of insulin resistance in the absence of obesity in NOD individuals could be due to the other pathways related to the non-fasting states like gluconeogenesis. Indeed, the contribution of endogenous glucose production may have been underestimated in NOD individuals.Another molecular mechanism associated with insulin receptor regulation which is not considered in this study is Evidently, diabetes and obesity are a complex disease with a large pool of genes that are involved in its development. It can be postulated that obesity is an independent or distinct risk factor of Type II diabetes while the impaired gene expression of different insulin signalling pathways would be the main risk factors which some of these disorders may accompany by obesity and some may not. This hypothesis offers an effective explanation for the noted paradoxes of diabetes and obesity.PI3K/AKT pathway has been considered which is related to the fasting state, and its components showed reduced expression in obese diabetic group due to the chronic over-nutrition which may induce insensitivity and reduced gene expression level.In this study the Although evidence of differential regulation of insulin signalling pathway based on nutritional stimulations is available, molecular mechanisms responsible for insulin resistance and obesity heterogeneity are largely speculative at this time. Future investigations are undertaken to address the question that whether nutritional mediators stimulate different molecular mechanisms of insulin signalling or endogenous glucose production is responsible for insulin resistance and these paradoxical effects for devising treatments.