As compared to synthetic ones, polymers of natural origin generally present superior biocompatibility and bioactivity. Their assembly and further engineering give rise to a wide plethora of advanced supporting materials, accounting for systems based on hydrogels or scaffolds with either fibrous or porous architecture. The present review offers an overview of the various types of natural polymers currently adopted in bone tissue engineering, describing their manufacturing techniques and procedures of functionalization with active biomolecules, and listing the advantages and disadvantages in their respective use in order to critically compare their actual applicability potential. Their combination to other classes of materials  and other innovative strategies to reproduce physiological bone microenvironments in a more faithful way are also illustrated. The regeneration outcomes achieved in vitro and in vivo when the scaffolds are enriched with different cell types, as well as the preliminary clinical applications are presented, before the prospects in this research field are finally discussed. The collection of studies herein considered confirms that advances in natural polymer research will be determinant in designing translatable materials for efficient tissue regeneration with forthcoming impact expected in the treatment of bone defects.Despite considerable advances in microsurgical techniques over the past decades, bone tissue remains a challenging arena to obtain a satisfying functional and structural restoration after damage. Through the production of substituting materials mimicking the physical and biological properties of the healthy tissue, tissue engineering strategies address an urgent clinical need for therapeutic alternatives to bone autografts. By virtue of their structural versatility, polymers have a predominant role in generating the biodegradable matrices that hold the cells  Bone is an essential and multifunctional organ. While providing weight-bearing sustainment and assisting locomotion, the 206 bones in the adult human body have other defined biological roles, such as: generation of blood cells (haematopoiesis), physical protection of vital organs like brain or heart, and storage of minerals and growth factors Clarke, . Bone isin situ, re-create their biological microenvironments and sustain the growth of new tissue until integration into the transplantation area. SCs sense mechanical and biochemical signals from the niche they are located in Discher et al. (Novel strategies for the treatment and regeneration of bone tissue are offered by Tissue Engineering (TE), which aims at recovering, maintaining, and improving functions of damaged organs starting from a pool of regenerative cells, capable of both self-renewal, and differentiation in other cell types. Recent breakthroughs in the biology of stem cells (SCs), especially in relation to their trophic, regenerative and immunomodulatory functions, have raised tremendous enthusiasm in the development of innovative therapeutic solutions. Besides stem and progenitor cells, the backbone of TE relies on the investigation and optimization of cell stimulation strategies, and on the design of supporting materials  crystals are dispersed into a collagen fiber matrix  or polysaccharides  , describing the manufacturing techniques, procedures of functionalization with bioactive molecules and their To select the most appropriate biomaterial, the knowledge of the physicochemical architecture of native bone along with pertinent biomechanical features is critical. Some physico-mechanical properties of the natural bone and main BTE biomaterials are reported in 10(PO4)6(OH)2). Apart from free ions  and water, the remaining fraction is composed of organic components, type I collagen being the most abundant (nearly 90%) and serving as anchoring support for HA crystals  orchestration of osteoblast and osteoclast activity, (ii) endocrine regulation of phosphate homeostasis, (iii) sensing of local mechanical strains, and (iv) cell signaling. Residing inside lacunae within the mineralized matrix, osteocytes connect to the vasculature and bone surface by sending dendritic projections through tiny tunnels called canaliculi , which are made of sub-nanostructured crystals, collagens, and non-collagenous organic proteins. Sub-nano elements account for: HA plates in the size of 2 × 25 × 50 nm, 2–3 nm thick carbonate apatite and collagen molecules with diameter of 3–10 nm Jasiuk, . The mosScaling up from the nano to the micro-structural frame, larger structures are present. The collagen fibrils assemble into larger fibers. Lamellae, namely planar sheets resulting from the association of mineralized collagen fibers, are 3–7 μm wide. Concentric layers of few (3–8) lamellae wrapped around a central canal produce cylindrical tubes with an overall diameter of about 200–250 μm, termed as osteons or Haversian systems, running roughly parallel to the long axis of bone Jasiuk, . Size ofFinally, the arrangement of these microstructures determines the histological nature of the bone macro-structure: the arrangement of lamellae into sinuous and irregular convolutions creates cancellous bone, namely a spongy tissue with high porosity degree, where trabeculae with typical millimeter dimensions (0.1 mm diameter and 1 mm length) are found. The regular microstructure arising from ordered cylindrical lamellae constructs impart high density to the tissue, generating the compact, cortical bone , such as in 3D printing, where specific objects or components are produced by ink-jet printing a binder onto sequential powder layers  releases Si and Ca ions stimulating osteogenic differentiation and cell proliferation, and inducing the deposition of bone-like apatite on their surface after soaking in simulated body fluids (SBF)  are structurally based on SiOin vitro and in vivo  are reported to augment the tensile strength, the stress resistance and the apatite deposition ability in collagen composites, as well as to the enhance osteogenic differentiation of MSCs  as well as reinforcement elements in scaffolds constituted of other materials, various biodegradable synthetic polymers  have been extensively employed to fabricate collagenous BTE materials . This is due to its ability to swell without disintegrating and to incorporate hydrophobic drugs, as well as to a tuned degradation rate allowing for a controlled release. Release profiles of bFGF from collagen hydrogels were monitored and studied in relation to the proliferation ability and osteogenesis of seeded MSCs  showed improved migration with higher concentrations of released factor, cell-free SDF-1 containing hydrogels stimulated cell-homing in vivo and enhanced blood vessel formation.Current studies investigate: (i) integrated approaches combining more than one material with the collagen structures, (ii) loading with various cells, and (iii) enrichment with biofactors. The interaction of osteoblasts, pre-osteoblasts, and SCs with collagen inside BTE constructs was characterized Gelatin derives from an irreversible hydrolysis of collagen, reducing 300 kDa protein fibrils into smaller peptides, whose molecular weight range varies depending on the chosen denaturation method   is a natural polysaccharide deriving from chitin, the major component of crustacean exoskeleton  has been a promising strategy to overcome the mechanical weakness and the lack of osteoinductive properties  from the human jaw   comparable to that of dry bone (0.8–1.2 g/cm3)  Kumar, .in situ , the cell wall and intracellular space of seaweeds  remain the major sources. Alginates are linear unbranched polymers containing β-(1 → 4)-linked D-mannuronic acid (M) and α-(1 → 4)-linked L-guluronic acid (G) residues. The overall polysaccharide sequence contains blocks of consecutive G and M residues, or alternating MG residues. Sodium alginate is the main form of alginate used. The ability of alginates to form soft hydrogels in the presence of Cain situ co-precipitation elicits the integration of HA crystals  loaded with vascular stents for large bone defect regeneration in rabbit mandibles , encapsulated into oxidized alginate microbeads constituting an injectable and biodegradable scaffold for BTE  sequences from fibronectin were grafted on alginates in order to create functionalized gels that more closely recapitulate the chemistry of natural cell adhesive proteins and may be useful in developing optimized BTE scaffolds   is a fibrous protein owing unique mechanical properties, environmental stability, morphologic flexibility, tunable proteolytic biodegradability, along with the ability to support the osteogenic differentiation of MSCs.SF can be molded into diverse forms, chemically modified and combined synergistically with other minerals and polymers , also SF microparticles were employed to such scope, increasing the compressive modulus of SF hydrogels over6-folds in a 1:2 (matrix:particle) mixture, which in turn enhanced the osteogenic performance of MSCs as well as the calcium adsorption  is a glycosaminoglycan containing repeating disaccharide units of N-acetyl-D-glucosamine and D-glucuronic acid. Synthesized by hyaluronan synthases, it is a primary component of the ECM of the human connective tissue and the simplest one among the various GAGs  provided biphasic release kinetics of BMP-2 accounting for an initial burst followed by a sustained release and leading to ectopic bone formation  forms injectable and thermoreversible hydrogels with tunable mechanical properties useful for preparation of bilayered scaffolds for osteochondral regeneration  and proteins (like heparin or chondroitin sulfate) with relevance for BTE, are listed in Being very similar or identical to endogenous macromolecules, natural polymers are optimal constituents for implantation, not only because the biological environment is prepared to recognize and metabolically process them, but also because they enable to tailor the biomaterial functions at the molecular level Yannas, . The facBased on the collection of studies herein reviewed, one can distinguish the following promising research directions in natural polymeric BTE constructs: (i) strategies of controlled delivery of bioactive molecules to accelerate bone healing, (ii) modification of polymers to optimize the cell-matrix interactions and shape the scaffold topography, and (iii) engineering of GAMs to integrate advanced principles of gene therapy in the science of scaffold design.One of the most relevant perspectives in polymer science is represented by the creation of functional coatings improving the properties of biomaterials, such as bioceramics, which are ideal bone regeneration platforms by virtue of their drug-delivery ability and apatite-like formation  and bioreactor technologies to streamline and improve pre-implantation culture. Also, novel procedures for cell biophysical stimulation should be implemented to ease access to clinical use. In fact, despite several successful In conclusion, it is clear that a deeper knowledge about the dynamics of the bone tissue microenvironment should allow for a proper simulation, likely meeting the open challenges still limiting the natural polymers from entering into BTE clinically. However, these polymers offer a unique chance to create biomimetic biocompatible microenvironments for cells of the osteogenic niche. The strategies for engineering their properties are expected to further augment their clinical potential and become determinant in the design of efficient regenerative matrices to repair the skeletal lesions in the future.MF conceived the original idea of the manuscript, structured the outline, wrote the main text and prepared figures and tables. GB, MC, and AS edited the final manuscript. All authors approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Odobenus rosmarus), Phocidae , and Otariidae . Structural and functional chromosomal features were assessed with telomere repeat and ribosomal-DNA probes and by CBG  and CDAG (Chromomycin A3-DAPI after G-banding) methods. We demonstrated diversity of heterochromatin among pinniped karyotypes in terms of localization, size, and nucleotide composition. For the first time, an intrachromosomal rearrangement common for Otariidae and Odobenidae was revealed. We postulate that the order of evolutionarily conserved segments in the analyzed pinnipeds is the same as the order proposed for the ancestral Carnivora karyotype (2n = 38). The evolution of conserved genomes of pinnipeds has been accompanied by few fusion events (less than one rearrangement per 10 million years) and by novel intrachromosomal changes including the emergence of new centromeres and pericentric inversion/centromere repositioning. The observed interspecific diversity of pinniped karyotypes driven by constitutive heterochromatin variation likely has played an important role in karyotype evolution of pinnipeds, thereby contributing to the differences of pinnipeds’ chromosome sets.Pinnipedia karyotype evolution was studied here using human, domestic dog, and stone marten whole-chromosome painting probes to obtain comparative chromosome maps among species of Odobenidae ( Genome analysis for related species within taxonomic assemblages enriches our evolutionary perspective and provides new information about the speciation process. Comparative chromosome maps obtained by means of chromosome-painting probes reveal patterns of karyotype transformations during evolution and allow to formulate hypotheses about ancestral karyotype composition. Constitutive heterochromatin (CH), an important genome component with a cryptic role and poorly understood functions, is filled with diverse and often complex repetitive elements that are challenging for resolution and can confound bioinformatic assembly of genome sequences. Repeated sequences perform regulatory functions and ensure spatial organization of chromatin in the nucleus ,2. CH maIn recent decades, patterns of mammalian genome evolution were revealed by determining the rates of chromosome exchanges and by documenting the types of rearrangements within major orders that had shaped genomes of extant species on the basis of reconstructed ancestral karyotypes ,7,8,9,10Among Caniformia families, Pinnipedia  has a nearly unchanged ancestral karyotype. Pinnipeds represent a group of semiaquatic animals with advanced adaptations to life in water, thus raising a hypothesis that their conserved genome disposition is associated with acquired aquatic adaptations.Ulfur Arnason has revealed remarkable karyotype conservatism of this group by classic and banding cytogenetics ,12. ThisHere we investigated remarkable karyotype conservatism among pinnipeds by chromosome painting in a wider range of Otariidae and Phocidae species. With the help of high-resolution canine probes, we tested for the intrachromosomal rearrangements that could be present in otherwise conserved syntenic groups. We determined whether heterochromatin variation plays some part in the genomes featuring high levels of syntenic conservatism.Tissue samples from wild-caught animals were used here . The colStorage and transportation of the tissue samples, establishment of primary fibroblast cell lines, and chromosome preparation were performed as described before . We trieStandard GTG (G-bands by trypsin using Giemsa) banding staining was performed . CBG (C-Erignathus barbatus) and three eared seals . Some human and stone marten painting probes were used to clarify ambiguous mapping positions in the ringed seal (Phoca hispida), spotted seal (Phoca largha), and harbor seal (Phoca vitulina).Sets of human, domestic dog, and stone marten chromosome–specific painting probes have been described previously ,25,26. I5 and (CCCTAA)5 [A plasmid containing ribosomal DNA (rDNA)  was amplCCCTAA)5 .Digital images of hybridization signals were captured as described elsewhere ,26,29 usArctocephalus forsteri and Phocarctos hookeri karyotypes are described here for the first time. Each CFA probe stained one to five fragments in the karyotypes of the bearded seal, Australian fur seal, northern fur seal, and New Zealand sea lion. Sixty-eight homologous autosomal segments were detected in the genomes of all the studied species. X-chromosome synteny was conserved among these pinniped karyotypes. Chromosome maps of the New Zealand fur seal, New Zealand sea lion, northern fur seal, and bearded seal are presented in Dog (CFA) and some stone marten (MFO) chromosome-specific libraries were utilized to identify homologous segments in genomes of four pinniped species: one true seal and three eared seals. Phoca hispida (2n = 32) chromosome set has been published previously [Phoca largha (2n = 32) presented here are consistent with the literature data [P. hispida and P. largha are very similar to the pattern in the Baikal seal  and a new technique for the visualization of nucleotide composition of CH (CDAG method) were used as described in refs. ,23. RegiIn general, CH is scarce in genomes of pinnipeds. CBG staining yielded small pericentromeric blocks in walrus and true seal karyotypes except for the bearded seal a–d. CompUsing an array of staining assays, we determined the types, localization, and composition of heterochromatin areas in pinnipeds , Table 2Our analysis of the alternation of dog painting probes’ staining patterns and in heterochromatin regions uncovered patterns of variation in CH among pinnipeds. Species chromosomal sets proved to differ in size and composition of pericentromeric and intercalary heterochromatin and in the number of autosomes carrying additional heterochromatin segments on short arms. The dog painting probes helped to identify for the first time an intrachromosomal rearrangement common for eared seals and the walrus . Among tPhoca hispida.Male specimens were available for seven out of the 10 species included in this investigation. The Y chromosome is readily recognizable in the analyzed pinnipeds, being the smallest bi-armed chromosome of the complement . Only inPhoca hispida) and on autosome 6 of the harbor seal (Phoca vitulina). In both cases, these additional clusters of ribosomal genes were detectable in all of the analyzed cells and were substantially smaller than the main NOR site on PHIS 1p and PVIT 1p (s), respectively.In all of the species studied here, the NOR was situated on one homologous autosomal pair . RibosomThe first and subsequent cytogenetic studies have detected low variation in the diploid number 32–36) and similar G-banding patterns among pinniped karyotypes ,34,35,36 and simiConserved karyotypes of pinnipeds differ in the morphology of some autosomes shaped by varying heterochromatin regions. Heterochromatic regions are difficult to determine unless a special approach to the characterization of satellite DNA is used ,41. RegiWe revealed an interesting phenomenon of composition diversity of centromeric and pericentromeric CH on homologous chromosomes among pinnipeds . AlthougWe observed variation of p-arms’ heterochromatin  after deHeterochromatin segments may be conserved too, as in apomorphy. One such instance inferred from comparative chromosome maps is the chromosomes homologous to cat autosome Felis catus, CFA) A1q (HSA 5). This chromosomal segment carries an additional heterochromatic short arm in studied eared seal (except for AFOR 5p) and in other carnivores’ karyotypes analyzed by chromosome painting, indicating an ancestral origin of this heterochromatin segment ,32,42,43In summary, the majority of ancestral syntenies showed a wide variety of CH characteristics  despite small sizes of heterochromatin areas. Among the studied species, the numbers of autosomes affected by CH variation were different. Except for mustelids, the corresponding outgroup homologs in the stone marten, felids, and other carnivoran taxa do not feature such CH variation ,32. The Arctocephalus are known, but hybrid males have lower reproductive success [Karyotypes of all pinnipeds are similar, but at the same time, the species living together seem to retain reproductive isolation. For example, there are no cytogenetic reports about interspecific hybrids of three sympatric species: the ringed seal, spotted seal, and bearded seal. Interspecific hybrids of southern fur seals of the genus  success . It is pThe involvement of heterochromatin in genome functioning, adaptation, and speciation is still a fundamental problem of modern genetics. The notion that CH is a genetically inactive part of the genome has been refuted ,48,49,50We noticed differences in the distribution of CFA 12 on p- and q-arms in homologs of ancestral synteny ACK 6. In true seals, CFA 12 stained only the proximal part of the q-arm, but in the walrus and eared seals, this probe additionally highlighted a proximal small part of the p-arm . This fi12/1/12/1 in conserved synteny CFA 37/12/1 is common for at least three mustelid species  and centromere shifts have been detected in various vertebrate species  includinA potential rearrangement was detected here in the homologous segment on EBAR 10: painting probe CFA 11 highlighted a twofold larger portion of the chromosome than that in eared seals , likely Reconstruction of ancestral karyotypes for taxa at different levels is an important step for the compilation and classification of comparative cytogenetic data. The hypothesis about karyotype structure of the ancestor of all Carnivora has been corrected at each stage of cytogenetics development. At first, a “hypothetical primitive karyotype” with at least 2n = 34  was publBeing a basal canoid group, pinnipeds are an important lineage for ACK reconstruction if canoid families with substantially reorganized karyotypes  are ignored. Consequently, the new chromosome maps of pinnipeds confirm 2n = 38 for the ACK. Elements ACK3p/3q and ACK4p/4q are fused thereby reducing 2n from 42 to 38 . The ACKThe ancestral pinniped karyotype is identical to the ACK because no rearrangements have been identified on a branch leading from the ACK to Pinnipedia . The radOdobenus rosmarus (2n = 32), Phoca vitulina (2n = 32), Erignathus barbatus (2n = 34), Neomonachus schauinslandi , and Mirounga angustirostris  [Erignathus, Phoca, and Pusa here) inside Phocidae. There were no fusion/fission events during Monachinae formation as evidenced by pairwise comparative plots of chromosome level scaffolds between these species and E. barbatus.Recently, chromosome level genome assemblies for several pinniped species were released: for 2n = 34) . The monThe first cytogenetic studies on marine mammals in the 1970s–1980s revealed the conservatism of chromosome sets and the stability of diploid numbers: 2n = 44 for most Cetacea and 2n = 32 to 36 in pinnipeds . The obsIn addition, the evolution of the variable heterochromatic part of the genome in different groups of marine mammals has been researched actively in whales and less so in pinnipeds. Heterochromatin in whales reaches 25–30% of the genome and is composed of different families of repeated sequences ,80,81,82Here we expanded to eight the list of pinniped species with comparative chromosomal maps. The resulting data confirm the high conservatism of syntenic elements in this group of marine mammals relative to other carnivore groups. We postulate that the order of evolutionarily conserved segments is common among the analyzed pinnipeds and is the same as that in the proposed karyotype of the ancestor of the whole order Carnivora. Fusions of ancestral-karyotype elements are specific to each branch of pinnipeds. Evolutionary changes at the subchromosomal level were revealed in two events of evolutionarily new centromeres and one pericentric inversion/centromere repositioning. The interspecific diversity due to variations in the heterochromatin component of pinniped karyotypes are described for the first time. Thus, the karyotypic divergence of conserved genomes of pinnipeds has occurred largely at the level of heterochromatin or via intrachromosomal repositioning.


In its late stages, prostate cancer (PCa) is characterized by a high propensity to form osteoblastic bone metastases, mainly treated by palliative approaches. In a previous work, we demonstrated that a gap junctional protein, connexin43 (Cx43) is implicated both in the increase of aggressiveness of PCa cells and in their impact on bone. To analyze the reciprocal part of the dialogue, the current study addresses the role of Cx43 in the impact of bone microenvironment on PCa cells abilities. Using Cx43-overexpressing PCa cell lines, we determined that Cx43 is necessary for promigratory effect induced by osteoblastic conditioned media (ObCM) on individual cells. Next, we demonstrated the requirement of Cx43 membrane localization at the leading edge and the involvement of the cytoplasmic part in this ObCM-induced migration. Overall, our findings precise the role of Cx43 during PCa progression and its putative use as aggressiveness marker and as potential therapeutic targets.Among the different interacting molecules implicated in bone metastases, connexin43 (Cx43) may increase sensitivity of prostate cancer (PCa) cells to bone microenvironment, as suggested by our in silico and human tissue samples analyses that revealed increased level of Cx43 expression with PCa progression and a Cx43 specific expression in bone secondary sites. The goal of the present study was to understand how Cx43 influences PCa cells sensitivity and aggressiveness to bone microenvironment. By means of Cx43-overexpressing PCa cell lines, we revealed a Cx43-dependent promigratory effect of osteoblastic conditioned media (ObCM). This effect on directional migration relied on the presence of Cx43 at the plasma membrane and not on gap junctional intercellular communication and hemichannel functions. ObCM stimulation induced Rac1 activation and Cx43 interaction with cortactin in protrusions of migrating PCa cells. Finally, by transfecting two different truncated forms of Cx43 in LNCaP cells, we determined that the carboxy terminal (CT) part of Cx43 is crucial for the responsiveness of PCa cells to ObCM. Our study demonstrates that Cx43 level and its membrane localization modulate the phenotypic response of PCa cells to osteoblastic microenvironment and that its CT domain plays a pivotal role. Prostate cancer (PCa) is the most common male cancer in term of worldwide incidence and represents one of the major causes from cancer death in men . This androgen-sensitive cancer is characterized by a significant rise in case number with age and a distinct feature of human PCa is the net propensity to disseminate towards bone and to develop metastasis. Bone metastases, present in more than 70% of prostate cancer patients in the advanced stages, are associated with dramatic complications mainly skeletal-related events (SRE) linked to both osteogenesis and osteolysis  was obtained as pGEX-2 T fusion genes  and produced as described. Recombinant proteins were prepared as glutathione S-transferase fusion proteins in Escherichia coli (BL21 strain), purified using glutathione-sepharose beads , and used as GST-fusion proteins. To measure Rac1 activity, LNCaP cells were cultured and stimulated under the same conditions as for migration, except that they were seeded in 10 cm-diameter culture dishes. After 17 h of stimulation with or without ObCM, cells were lysed using a cell lysis buffer containing 50 mM Tris-HCl pH 7.4, 100 mM NaCl, 2 mM MgCleference .t-test was used to compare two experimental groups. Data were taken statistically significant for a value of p < 0.05 .Results were expressed as the mean of n experiments ± the standard error of the mean (SEM). Statistical analyzes were carried out using GraphPad Prism software  using data from at least 3 independent experiments. Analysis of variance  followed by a Bonferroni post-test was used for the analysis of at least three experimental groups. Unpaired In the bone metastatic context, our results clearly demonstrate that Cx43 level and its membrane localization can modulate the phenotypic response of PCa cells to an osteoblastic microenvironment and that the CT domain of Cx43 plays a pivotal role in this phenomenon. Our study also reveals the presence of a N-terminally truncated Cx43 isoform (Gja1-20k) in LNCaP cells whose expression level depends on ObCM stimulation and may regulate the traffic of full-length Cx43 to plasma membrane and, subsequently, the promigratory effect.


Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Increasing studies showed that long non-coding RNAs (lncRNAs), a novel class of RNAs that are greater than 200 nucleotides in length but lack the ability to encode proteins, exert crucial roles in the occurrence and progression of HCC. LncRNAs promote the proliferation, migration, invasion, autophagy, and apoptosis of tumor cells by regulating downstream target gene expression and cancer-related signaling pathways. Meanwhile, lncRNA can be used as biomarkers to predict the efficacy of HCC treatment strategies, such as surgery, radiotherapy, chemotherapy, and immunotherapy, and as a potential individualized tool for HCC diagnosis and treatment. In this review, we overview up-to-date findings on lncRNAs as potential biomarkers for HCC surgery, radiotherapy, chemotherapy resistance, target therapy, and immunotherapy, and discuss the potential clinical application of lncRNA as tools for HCC diagnosis and treatment. Hepatocellular carcinoma (HCC), one of the primary liver cancers, ranks sixth in the incidence of global malignant tumors and ranks third in the mortality rate Figure Table 1) Table 2) Long non-coding RNAs (lncRNAs) are a class of RNAs with length longer than 200 nucleotides and lacking the ability to encode proteins In this review, we overview and discuss the up-to-date findings of the role of lncRNAs as potential diagnostic biomarkers and therapeutic targets for HCC diagnosis and treatment.Serum alpha fetal protein (AFP) is the common and golden standard tumor biomarker for screening and diagnosing of HCC, however, recent studies showed that the sensitivity and specificity of using AFP as biomarker for early diagnosis of HCC are not satisfactory Serum levels of several lncRNAs were upregulated in HCC patients. Xu et al. detected serum exosomes levels of LINC00635 and ENSG00000258332.1 in HCC patients, hepatic cirrhosis patients, chronic hepatitis B patients, and healthy subjects, and found that these lncRNAs were up-regulated in HCC group, and high serum levels of these lncRNAs are associated with poor prognosis The upregulation of some lncRNAs also can be as potential biomarkers for diagnosing of HCC tumor stages and overall survival. Wang et al. found that the serum levels of LRB1, accompanied by the levels of protein markers AFP, were obviously increased in HCC patients, and using LRB1 combined with AFP as biomarkers can significantly improve the diagnostic accuracy of HCC Surgical treatments, including liver transplantation (LT) and partial liver resection (LR), are efficient methods for HCC treatment; however, the post-treatment prognosis remains poor due to metastasis and recurrence. Recent studies showed that lncRNAs have a great effect on the prediction of recurrence and survival time in HCC patients after surgery.in vitro studies indicated that down-regulateded HOTTIP expression impairs the invasiveness and increases the chemosensitivity of tumor cells. In addition, two single nucleotide polymorphisms (SNPs) (rs2071265 and rs142077238) in the HOTTIP gene were genotyped in 102 peripheral blood samples of HCC patients, COX regression analysis found SNP rs2071265 is an independent predictor for HCC recurrence and is associated with early recurrence of HCC patients. These results indicate that HOTTIP expression levels and SNP type are predictive indicators for tumor recurrence after liver transplantation in HCC patients Wu et al. collected blood and tumor tissue samples from 155 HCC patients who underwent LT treatment, the authors found that HOTTIP have a high expression level in HCC tissues Yuan et al. found that the MVIH expression levels were markedly upregulated in cancer tissues of HCC patients undergoing hepatectomy NEAT1 is oncogenic lncRNA in diverse kinds of tumorigenesis, including gastric cancer, breast cancer, lung cancer and colorectal cancer MALAT1 is the most widely researched lncRNAs in the treatment of cancer HOTAIR is the antisense strand of HOX gene cluster, and is an oncogenic lncRNA in cancer Figure 2).Radiotherapy is one of the main traditional methods for HCC treatment In HCC tissues and cells, Chen et al. found that the levels of lncRNA NEAT1_2 and oncogenic protein WEE1 was upregulated Chen et al. investigated the roles of the linc-ROR in HCC, and found that linc-ROR promotes HCC metastasis by mediating epithelial-mesenchymal transition. Further study found that radiation-tolerant HCC cells showed higher levels of linc-ROR and lower levels of γ-H2AX, a DNA damage marker In vitro, knockdown of SUMO1P3 expression of HCC cells, and explosion of these cells to radiation could significantly reduce the survival rates of HCC cells, indicating that knockdown of SUMO1P3 promotes the radiosensitivity of HCC cells. Meanwhile, knockdown of SUMO1P3 could also inhibit the growth of tumor cells, reduce invasiveness and promote apoptosis SUMO1P3 is a novel identified lncRNA that was up-regulated in several types of cancer, including gastric cancer, bladder cancer, and non-small cell lung cancer, and is a prognostic and therapeutic target these cancers LncRNA H19 was firstly identified as mammalian development regulator Zhang and colleagues detected the expression level of LINC00473 in HCC cell lines and normal hepatocytes, and the results indicated that the expression level of LINC00473 in HCC cell lines was significantly higher than that of normal hepatocytes In vitro studies showed that lncRNA TP73-AS1 down-regulated the expression of PTEN, enhanced the phosphorylation level of AKT, and reduced the radiosensitivity of HCC cells. The same results have also appeared in vivo experiments. After silencing lncRNA TP73-AS1, the tumor growth rate of mice was significantly slowed down, and the tumor volume was also significantly smaller than the control wildtype group LncRNA TP73-AS1 is located on human chromosome 1p36 Figure 3).Chemotherapy is a traditional and efficient method for cancer therapy and the most common drugs for treating HCC include 5-fluorouracil (FU), cisplatin (CDDP), oxaliplatin, doxorubicin, gemcitabine, capecitabine, and mitoxantrone By performing lncRNA microarray analyses, Wu et al. found that the mRNA levels of KRAL were significantly reduced in 5-FU-resistant HCC cells et al. first compared the expression of MALAT1 in 5-FU-sensitive and resistant HCC cell lines, and the results revealed that MALAT1 mRNA levels and the levels of hypoxia-inducible factor (HIF)-2α were significantly increased in 5-FU resistant cells LncRNA MALAT1 was initially identified as an indicator index of poor clinical outcome in early-stage non-small cell lung cancer (NSCLC) patients Wu et al. established the drug-resistant HCC cell lines and performed lncRNA microarray analysis to explore the expression patterns of lncRNAs in CDDP-resistant cancer cells GAS5 was initially identified in a subtractive complementary DNA (cDNA) library, which was used to sequence RNA expressed in growth-arrested cells LINC01234 has been proven to be an indicator of prognosis in ovarian cancer in vivo experiments, where TPTEP1 inhibited tumor growth in mice, reduced the number of lung and liver metastases, and was down-regulated in mouse tumor tissues TPTEP1 is located on chromosome 22 and is regulated by DNA methylation in cancer Huang et al. performed microarray analysis on four pairs of oxaliplatin-resistant and sensitive HCC cells, and discovered that NR2F1-AS1 was highly expressed in oxaliplatin-resistant HCC cells HULC was first identified as the most up-regulated lncRNA in HCC Xiao et al. found a novel lncRNA named as HCC associated long non-coding RNA (HANR), and found that the expression levels of HANR in HCC cell lines and HCC tissues were up-regulated. In addition, Kaplan-Meier analysis showed that high HANR expression was connected with poor prognosis LncARSR was firstly found in renal cell carcinoma (RCC) cells, and was related with resistance to sunitinib in RCC patients Figure 3).Molecular targeted therapy is a new method for treating cancer that has been accepted by people in recent years. Many targeted drugs, including sorafenib (SOR), lenvatinib, regorafenib, cabozantinib, and ramucirumab, currently were used to treat HCC SNHG1 is highly expressed in HepG2 and Huh7 cells in vitro cell model and in vivo xenograft tumor model data showed that knockdown of SNHG16 enhanced SOR chemosensitivity in HCC LncRNA SNHG16 is a novel cancer-related lncRNA and promotes tumor cell growth and metastasis in multiple cancers, including HCC cells and tissues Zhang et al. found that the IC50 Value of SOR was significantly increased in SOR-treated SNHG3-overexpressing HCC cell, indicating that SNHG3 enhance SOR resistance in vitro and in HepG2-SOR resistant xenografts in vivo. Further in vitro study showed that knockdown of TUC338 upregulated the expression level of RAS GTPase activating protein (RasGAP) gene RASAL1 LncRNA TUC338 was characterized as an oncogene in several types of cancer, such as prostate carcinoma and lung cancer in vitro experiment showed that overexpression of NEAT1 suppress the miR-335 expression in HCC cell lines Chen et al. found that knockdown of NEAT1 promoted SOR-induced cancer cell death Figure 4). In addition to the regulatory effect of lncRNA in T cells, it also affects macrophages and other immune cells. Tumor-associated macrophages (TAM) are important participants in tumor immunity, and the polarization of macrophages is one of the research hotspots in tumor therapy. M1 type macrophages have anti-tumor effects through direct killing mechanism and antibody-dependent cell-mediated cytotoxicity (ADCC) kills tumor cells Immunotherapy has become one of the promising options for HCC treatment following surgery, radiotherapy, chemotherapy, and targeted therapy + T cell suppressed cell apoptosis and enhanced the lytic activity to tumor cells by inhibiting Tim-3 expression in vitro studies showed that miR-155 is a target of NEAT1, and miR155 inhibition alleviated knocking down of NEAT1-induced CD8+ T cell apoptosis, and rescued Tim-3 levels + T cell function and promotes HCC immune escape, which may act as an immunotherapy target for HCC patients Yan et al. found that expression levels NEAT1 were up-regulated in PBMC of HCC patients. Subsequent studies showed that knocking down of NEAT1 expression of CD8+ T cells in TILs + TILs, and binding of lnc-Tim3 with Tim3 leads to release of Bat3 from Tim3, thereby promoting cell cycle arrest of CD8+ T cells, and contributing to the survival of CD8+ T cells + T cells.Using high-throughput screening, Ji et al. explored the associations between mRNAs and lncRNAs in the tumor infiltrated lymphocytes (TIL) of HCC patients, and found that Lnc-Tim3 was highly up-regulated and negatively correlated with the IFN-γ-secreting activity of CD8+ T cells were highly up-regulated + T cells correlated with increased proportion of T regulatory (Treg) cells within HCC tissues In order to prove the effect of lncRNAs in connecting T cells and HCC, Jiang et al. investigated the associations between mRNAs and lncRNAs in the tumor infiltrated lymphocytes (TILs) of patients with hepatocellular carcinoma and found that lnc-EGFR levels in CD4Previous studies have shown that FENDRR down-regulates the expression of GPC3 and inhibits cell proliferation and migration LncRNA Cox-2 is a lncRNA with a size of about 50 kb Previous studies have shown that lncRNA TUC339 is enriched in exosomes derived from HCC cells in vivo experiments. The M2 macrophages polarization ability was found to be significantly weakened in mice transplanted with heterotopic liver tumors of silenced MALAT1 gene. These results suggest that MALAT1 promotes the polarization of M2 macrophages through 466 miR-140 to produce anti-cancer effect Hou and colleagues found that after knocking out MALAT1 in HCC cell lines, the ability of macrophages to polarize toward the M1 subgroup was increased, while the ability to polarize toward the M2 subgroup was weakened The morbidity and mortality of HCC have been dramatically increased in recent decades. Despite advances in the diagnosis and treatment of HCC, effective treatment strategies were still not well, as metastasis and recurrence rate are still very high. More and more preclinical studies showed that lncRNAs are good diagnostic and prognostic biomarker candidates for HCC therapy. Although preclinical data showed strong relationship between lncRNAs and HCC, by now, no clinical studies were reported about using lncRNAs as biomarkers for the diagnosis and prognosis of HCC. Future clinical studies may investigate the serum lncRNA profiles to explore more specific and sensitive lncRNA as biomarkers for HCC diagnosis and prognosis. To identify new lncRNAs as biomarkers for HCC, and to elucidate the molecular mechanisms of how lncRNAs work in HCC, future studies may need to focused on the development of experimental techniques, such as new sequencing techniques, and the development of lncRNA bioinformatics databases.While lncRNAs have great potential in the diagnosis and prognosis of HCC, we also face a series of challenges when lncRNAs are used as a potential drug candidate or therapeutic target for HCC treatment. First, the molecular weight and negative charge of nucleic acids make it difficult to make lncRNAs pass through biofilms. Second, RNA is easily degraded by RNase enzymes in the human body, recognized by the immune system, and quickly eliminated by the liver and kidneys. Finally, it is easy to lose function in the endosomes. At present, there are two main methods to solve the delivery problem: one is to modify nucleic acid molecules to stabilize and avoid recognition by the immune system, the other is to use drug delivery systems, such as protamine vector technology, nanoparticle vector technology, adeno-associated viral vector (AAV) technology, etc., among which almost all natural AAV capsid proteins can trigger effective transgene expression in the liver, so recombinant AAV targeted to the liver is a treatment HCC provides an excellent gene delivery platform. However, these delivery systems still have defects such as immune system rejection and reduced protein expression efficiency, and future research should focus on developing novel methods to overcome these disadvantages.


Although abnormal cortical gyrification has been consistently reported in patients with schizophrenia, whether gyrification abnormalities reflect a genetic risk for the disorder remains unknown. This study investigated differences in cortical gyrification between unaffected relatives (URs) with high genetic loading for schizophrenia and healthy controls (HCs) to identify potential genetic vulnerability markers. A total of 50 URs of schizophrenia patients and 50 matched HCs underwent T1-weighted magnetic resonance imaging to compare whole-brain gyrification using the local gyrification index (lGI). Then, the lGI clusters showing significant differences were compared between the UR subgroups based on the number of first-degree relatives with schizophrenia to identify the effect of genetic loading on cortical gyrification changes. The URs exhibited significantly lower cortical gyrification than the HCs in clusters including medial parieto-occipital and cingulate regions comprising the bilateral precuneus, cuneus, pericalcarine, lingual, isthmus cingulate, and posterior cingulate gyri. Moreover, URs who had two or more first-degree relatives with schizophrenia showed greater gyrification reductions in these clusters than those who had at least one first-degree relative with schizophrenia. Our findings of reduced gyrification in URs, which are consistent with accumulated evidence of hypogyria observed in regions showing patient-control differences in previous studies, highlight that such hypogyria in posteromedial regions may serve as a genetic vulnerability marker and reflect early neurodevelopmental abnormalities resulting from a genetic risk for schizophrenia. Endophenotype strategies have been adopted in an effort to identify genetic underpinnings in the etiology of schizophrenia to allow early detection and prevention of the disease2. An endophenotype is a state-independent and measurable biological trait found in unaffected relatives (URs) of probands at a higher rate than in the general population. Abnormalities in gyrification, the developmental process of forming cortical sulcal and gyral patterns, have been proposed as a candidate structural endophenotype for schizophrenia3, and this hypothesis has been supported by its characteristics of high heritability4 and consistent observations in schizophrenia patients9. In particular, schizophrenia is widely accepted as a disease attributable to genetic factors that initiate abnormal neurodevelopmental processes before the onset of illness11. Given that the formation of cortical folding is largely determined during early neurodevelopment and strongly influenced by genetic factors14, investigating gyrification changes in URs may help identify a genetic vulnerability marker reflecting the early neurodevelopmental etiopathology of schizophrenia.Schizophrenia is highly heritable in that biological relatives of patients have an increased risk of developing the disease, and genetic factors constitute significant risk factors in schizophrenia15. While several inconsistent results have been reported17, reduced gyrification was the main finding in most lGI studies18, and both patients with schizophrenia and patients with first-episode psychosis (FEP) showed common widespread hypogyria in the medial parieto-occipital and cingulate regions19. However, with respect to gyrification abnormalities reported in URs of schizophrenia patients24, the results have been conflicting and inconclusive, and only one existing lGI study reported a negative finding , which might have reduced the probability of finding crucial cortical folding differences caused by genetic risk. Moreover, relatives of schizophrenia patients with different symptomatic and functional statuses have been included as ‘unaffected relatives of patients with schizophrenia’, complicating identification of the pure genetic effects of schizophrenia on gyrification differences that exclude the influence of other psychiatric symptoms. For instance, the Edinburgh High Risk Study reported that more than 40% of URs of schizophrenia patients have psychiatric symptoms26, suggesting the possibility of the inclusion of URs with prodromal symptoms in previous studies, which might have also influenced their general functioning. In particular, negative results may have been observed in an lGI study of first-degree relatives of psychotic disorder probands with schizophrenia, psychotic bipolar, and schizoaffective disorders because these first-degree relatives had relatively low genetic loading for schizophrenia and were recruited based on relationships with probands who had different psychotic illnesses19.The local gyrification index (lGI) method, which considers the intrinsic three-dimensional morphology of the cortical surface, has enabled accurate examination of regionally specific gyral abnormalities in the whole-brain19. As they were characterized by traits such as schizotypy, which is considered a genetic risk factor for schizophrenia, this result suggests that hypogyria in the cingulate may serve as a genetic vulnerability marker for schizophrenia. Furthermore, patients with 22q11 deletion syndrome (22q11DS), which reflects another major genetic liability for schizophrenia, also showed significantly decreased gyrification in a large medial cluster extending from the posterior cingulate to the precuneus regions, supporting the notion that hypogyria in the medial parietal lobe is possibly associated with a genetic risk for schizophrenia15. However, it remains unknown whether reduced gyrification represents an endophenotype specifically for schizophrenia. To our knowledge, there has been no direct investigation of whole-brain gyrification in asymptomatic URs with high genetic loading for schizophrenia, which could increase the probability of detecting pure effects of genetic risk for schizophrenia on cortical gyrification changes.However, URs with axis II cluster A personality disorders exhibited reduced gyrification with a trend approaching statistical significance in regions of hypogyria evidenced in their probands19, we hypothesized that our URs of schizophrenia patients would exhibit hypogyrification patterns in the medial parieto-occipital and cingulate regions compared to HCs as a genetic vulnerability marker. Furthermore, to strengthen the influence of genetic liability on reduced cortical folding, URs were divided into two subgroups according to the number of first-degree relatives with schizophrenia; in this exploratory analysis, we assessed whether URs with higher genetic loading for schizophrenia showed greater hypogyria in the lGI clusters with significant differences between URs and HCs.In this study, we compared cortical gyrification in asymptomatic, high-functioning URs with high genetic loading for schizophrenia and healthy controls (HCs) using the lGI to identify potential endophenotypes for schizophrenia. Based on previous lGI studies reporting reduced gyrification in regions of case-control differences in schizophrenia patients and their relatives28: (1) the “multiplex” group included URs who had at least two first-degree relatives or monozygotic twins with schizophrenia (n = 9) and (2) the “simplex” group included URs who had only one first-degree relative with schizophrenia and at least one other affected second- to third-degree relative (n = 41). No significant group differences were found between the multiplex and simplex UR subgroups except for the sex ratio .The demographic and clinical backgrounds of the groups are presented in Table p < 0.001) had a peak vertex located within the lingual gyrus covering portions of the precuneus, cuneus, pericalcarine, lingual, isthmus cingulate, parahippocampal, and posterior cingulate gyri. The right cluster showing a significantly reduced lGI (p < 0.001) had a peak vertex within the precuneus gyrus comprising portions of the precuneus, cuneus, pericalcarine, lingual, isthmus cingulate, and posterior cingulate gyri. No region had a significantly increased lGI in the URs compared to the HCs  and right precuneus cluster  were demonstrated in the URs compared to the HCs. The results are shown as box plots in Fig. Additionally, significantly reduced mean lGI values in the left lingual cluster . The right precuneus cluster also showed a tendency toward decreased gyrification in the multiplex group , although the difference was not statistically significant. The results are shown as box plots in Fig. To demonstrate the effect of genetic loading on reduced gyrification in the URs as an exploratory analysis, the comparison between the two subgroups of URs in terms of the extracted mean lGI values from the clusters showing significant differences between the URs and HCs was conducted. Compared to the simplex group, the multiplex group demonstrated a significantly reduced mean lGI in the left lingual cluster  connectivity in the precuneus and anterior cingulate cortex (ACC) than the simplex group, suggesting that dysfunction in these regions is related to genetic risk for schizophrenia27. Consistently, our findings demonstrated the effects of genetic loading for schizophrenia on the amount of reduced cortical folding in the posteromedial cortex, which supports the notion that the precuneus/posterior cingulate regions reflect one of the most genetically vulnerable regions for schizophrenia.Notably, our URs with two or more first-degree relatives with schizophrenia  showed greater hypogyrification patterns in the medial parieto-occipital and cingulate regions than those with one first-degree relative and at least one second- to third-degree relative with schizophrenia . It has been hypothesized that the genetic risk for schizophrenia increases proportionally with familial closeness and the number of affected probands19. However, previous studies of URs of schizophrenia patients have reported conflicting results regarding gyrification patterns, mainly due to the heterogeneous characteristics of URs and different methods used to measure gyrification. In particular, the previous work by Nanda et al. (2014), one of the most prominent lGI studies in this line, examined gyrification in first-degree relatives of patients with different psychotic disorders. However, they treated the relatives of patients with schizophrenia, schizoaffective disorder, and psychotic bipolar disorder as an overall group in the same sample to examine whether gyrification qualifies as an endophenotype broadly marking psychosis liability. Furthermore, this group may have included relatives with relatively low genetic loading for schizophrenia , which might also have reduced the probability of finding significant gyrification differences caused by genetic risk for schizophrenia, and thereby resulted in a negative finding. To investigate whether reduced gyrification represents an endophenotype specifically for schizophrenia, URs with high genetic loading for schizophrenia were recruited in our study by including URs who had at least one first-degree relative with schizophrenia and one or more other affected first- to third-degree relatives. In addition, some of the URs in previous studies may have had certain psychiatric symptoms or prodromal syndromes that might also have influenced their general functioning, making it difficult to identify a pure effect of genetic risk for schizophrenia on changes in cortical folding. Compared to these previous studies, our findings were further bolstered by applying strict inclusion criteria for subjects in that URs having prodromal symptoms were screened out by measuring Structured Interview for Prodromal Syndromes (SIPS) scores, and subjects who had any general psychopathology were excluded by assessing Brief Psychiatric Rating Scale (BPRS), Hamilton Rating Scale for Anxiety (HAMA), and Hamilton Rating Scale for Depression (HAMD) scores to ensure they were all asymptomatic; Global Assessment of Functioning (GAF) scores were also evaluated to confirm that all URs were well functioning. Taken together, these analyses allowed us to investigate the pure effect of shared genetic factors of schizophrenia on cortical gyrification changes as a biological trait that was free from the aforementioned confounding factors, which is consistently evidenced in other genetic models for schizophrenia19.Abnormal gyrification has been repeatedly suggested as a candidate endophenotype for schizophrenia11. Longitudinal studies of subjects at genetic high risk have indicated that progressive brain structural alterations predate the onset of psychosis33, suggesting that genetic vulnerability to schizophrenia might affect cortical changes during neurodevelopment. In fact, prenatal genetic insults are known to influence embryonic and early neurodevelopmental processes, such as neuronal proliferation and differentiation, synaptic formation, and myelination, leading to prolonged alterations in cortical structure and connectivity in schizophrenia34. As the formation of gyral and sulcal patterns occurs mainly in early neurodevelopment and is strongly influenced by genetic factors during this period13, investigating gyrification differences in asymptomatic URs with high genetic loading may help reveal the effects of genetic risk on the neurodevelopmental etiopathology of schizophrenia. Because cortical gyrification mainly increases before and shortly after birth and undergoes minor decreases until becoming relatively stable in early childhood14, reduced gyrification in URs relative to HCs may result from deficits in an earlier phase of increasing cortical maturation or an excessive decrease in later neurodevelopmental processes. Furthermore, the alterations in gyrification may reflect underlying deficits in white matter integrity during cortical maturation37, which are known to be disrupted in both schizophrenia patients and their URs39.Schizophrenia is widely accepted as a neurodevelopmental disease in that deficits of the brain are determined by genetic factors early in life but are not fully established until adolescenceThere are several limitations of the present study that should be considered. The cross-sectional nature of the study makes it difficult to investigate whether abnormal cortical folding increases the possibility of developing schizophrenia. Thus, longitudinal MRI analyses in URs of patients with schizophrenia would be required to identify alterations in neurodevelopmental trajectories and confirm the role of gyrification deficits as a vulnerability marker for schizophrenia. Moreover, the sample size for the multiplex UR subgroup was relatively small. Thus, our findings of the effect of the level of genetic loading for schizophrenia on the degree of hypogyria should be considered exploratory and further tested in a larger cohort of both simplex and multiplex UR subgroups.In conclusion, our findings of asymptomatic, high-functioning URs with high genetic loading for schizophrenia demonstrate that genetic vulnerability for schizophrenia is characterized by reduced gyrification, and, in particular, the posteromedial regions may serve as the most vulnerable regions to genetic liability in schizophrenia. Taken together with previous findings in patients with schizophrenia, our findings confirm that such hypogyria may represent a trait characteristic of schizophrenia and reflect early neurodevelopmental disturbances resulting from a genetic risk for schizophrenia. Our findings of the relationship between higher genetic loading for schizophrenia and greater cortical gyrification reduction further support the possibility of hypogyria as a candidate structural endophenotype that may contribute to the understanding of the genetic underpinnings of a neurobiological etiology in schizophrenia. Future investigations of genes involved in the neurodevelopmental mechanisms of cortical gyrification associated with increased risk for developing schizophrenia will identify the genetic influence on the neurodevelopmental etiopathology of schizophrenia.40, a center for prospective, longitudinal investigation of people at high risk for schizophrenia, and from the inpatient and outpatient clinics of the Department of Psychiatry at Seoul National University Hospital. The URs were defined as asymptomatic subjects with high genetic risk who had at least one first-degree relative with schizophrenia and one or more other affected first- to third-degree relatives. This standard has the benefit of including URs with high genetic loading and excluding relatives of patients with sporadic schizophrenia. The probands of URs were assessed for schizophrenia according to the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) by trained psychiatrists. The family history of psychiatric disorders and the degree of genetic loading for schizophrenia were evaluated in URs using the Family Interview for Genetic Studies41. To assess clinical status upon admission into the study, the Structured Interview for Prodromal Syndromes (SIPS)42 was used to screen for prodromal psychotic symptoms. URs with SIPS scores exceeding certain criteria for clinical high risk (CHR) for psychosis (any P1–P5 ≥ 3) were excluded from this study. The URs were also administered the Brief Psychiatric Rating Scale (BPRS) to measure general psychopathologic symptom severity and the Hamilton Rating Scale for Depression (HAMD)43 and the Hamilton Rating Scale for Anxiety (HAMA)44 to assess the severity of depression and anxiety, respectively. In addition, the Global Assessment of Functioning (GAF) scale was also assessed to evaluate the overall functioning of subjects.Fifty URs of schizophrenia patients were recruited from the Seoul Youth ClinicFifty HCs matched for age, sex, handedness, intelligence quotient (IQ), and years of education were recruited via internet advertisements and screened for the presence of psychiatric disorders or symptoms using the Structured Clinical Interview for DSM-IV–Non-Patient Version (SCID-NP). Potential HCs were excluded if they reported past or current axis-I diagnoses and had any first- to third-degree biological relatives with a lifetime history of any major psychiatric disorders.The IQ of all participants was measured with the Korean version of the Wechsler Adult Intelligence Scale (K-WAIS) to estimate intelligence. The exclusion criteria for all participants included a lifetime diagnosis of a psychotic disorder, substance abuse or dependence, neurological disease or head injury, evidence of any other mental illness, or intellectual disability (IQ < 70). The present study was also approved by the Institutional Review Board at Seoul National University Hospital, and written informed consent was obtained from all subjects and from the parents of subjects under 18 years of age after the procedures had been fully explained.3; field of view, 250 mm; flip angle, 9°; and 208 slices. To ensure quality control, the acquired MRI images were visually inspected for any artifacts or malformation of brain structures.The subjects were scanned, and T1-weighted (T1) images were acquired using a Siemens 3T MAGNETOM Trio MR scanner . For the T1 images, a three-dimensional (3D) magnetization-prepared rapid acquisition gradient-echo (MPRAGE) sequence and the following imaging parameters were used: repetition time, 1670 ms; echo time (TE), 1.89 ms; voxel size, 1.0 × 0.98 × 0.98 mmhttp://surfer.nmr.mgh.harvard.edu/) according to the standard and automatic reconstruction algorithm45. This processing stream consists of automated transformation to Talairach space, normalization of intensity, removal of nonbrain tissue, and segmentation of gray/white matter tissue resulting in a white mesh and a pial mesh composed of approximately 150,000 vertices for each hemisphere.The preprocessing of T1 images was performed using FreeSurfer version 5.3.0  on the smoothed outer surface and of their corresponding paired circular ROIs on the pial surface. The lGI value at each vertex of the cortical surface was computed as the ratio between the smoothed outer surface and buried cortex resulting in the measurement of individual maps.p < 0.05, and clusters were corrected for multiple comparisons by Monte Carlo simulation. For clusters showing significant group differences, mean lGI values were extracted for each participant, and independent t-tests were performed to compare gyrification patterns between the groups.Data were analyzed using Statistical Package for Social Sciences (IBM SPSS Version 22). The demographic and clinical characteristics between URs and HCs were examined with independent t-tests or χ2 tests. A whole-brain lGI comparison between groups was performed with FreeSurfer. The lGI values were mapped on the average template subject (fsaverage) for each subject to perform the contrasts of the vertexwise analysis in Query Design Estimate Contrast (QDEC). A general linear model (GLM) for surface-based group analyses was conducted to assess regional group differences in lGI at each vertex for the right and left hemispheres separately while controlling for age and sex. A smoothing Gaussian kernel of 5-mm full-width at half-maximum (FWHM) was applied to the maps. The significant cluster-forming threshold was set to To further investigate the effect of genetic loading on clusters showing significant differences between the URs and HCs, the URs were divided into two subgroups based on the number of their first-degree relatives with schizophrenia. The demographic and clinical variables and the mean lGI values were compared between the two subgroups of URs using nonparametric Mann-Whitney U tests or χ2 tests due to the small sample size.Further information on research design is available in the Reporting Summary


Autism spectrum disorder (ASD) is a neurological and developmental disorder characterized by social impairment and restricted interactive and communicative behaviors. It may occur as an isolated disorder or in the context of other neurological, psychiatric, developmental, and genetic disorders. Due to rapid developments in genomics and imaging technologies, imaging genetics studies of ASD have evolved in the last few years. Increased risk for ASD diagnosis is found to be related to many specific single-nucleotide polymorphisms, and the study of genetic mechanisms and noninvasive imaging has opened various approaches that can help diagnose ASD at the nascent level. Identifying risk genes related to structural and functional changes in the brain of ASD patients provide a better understanding of the disease’s neuropsychiatry and can help identify targets for therapeutic intervention that could be useful for the clinical management of ASD patients. A recent study estimated prevalence of ASD in the United States in 2014–2016 was 2.47% among adolescents and children2. While in the United Kingdom, the annual prevalence rate for children aged 8 years between 2004 and 2010 was 3.8/1000 for boys and 0.8/1000 for girls3. Recent studies have shown that the pooled ASD prevalence estimate in Asia is 0.36%, including data from nine countries 4. The prevalence of ASD in the Middle East region was documented to be 1.4 per 10,000 children in Oman5, 4.3 per 10,000 children in Bahrain6, 1/167 in Saudi Arabia7, and a recent study reported ASD prevalence to be 1.14 % in children aged 6–11 years in Qatar8.Autism spectrum disorder (ASD) is a neurological and developmental disorder consisting of a wide range of symptoms and disability that develop in early childhood and persists throughout life. The common symptoms of ASD include limited activities, lower engagement and communication, talking and learning problems, and repetitive behavior. According to the World Health Organization, the global burden of ASD is continuously growing, with a current prevalence rate of 1 in 160 children. Reported prevalence rates vary widely from country to country though. Recent data from the Centers for Disease Control and Prevention showed that about 1 in 68 children in the United States had been identified with some form of ASD, with more than 3 million people affected9. The exact cause of ASD remains unclear; however, it is thought that both genetic and environmental factors play essential roles. The effect of ASD on society is enormous and multifaceted as it affects not only the child but also the siblings and parents and significantly disturbs the functioning of family routine life. Individuals with ASD are very likely to encounter the criminal justice system, mostly due to a lack of knowledge of their social and communication difficulties. There are also financial pressures associated with the recovery and decreased opportunities for jobs. Various studies have focused on the problems faced by families of autistic children10. It may affect the performance of the child at school, based on the severity of the disorder, and if untreated or undiagnosed, it may continue into adulthood, affecting both personal and professional life. Although there is no standard treatment for ASD, early detection and use of successful therapies can make a difference in the management and improvement of symptoms, and thus the overall quality of life of children with ASD. Before the clinical symptoms occur, therefore, a consensus is reached in treating ASD based on early detection.ASD incidence is 4–5 times greater in males than in females11. Historically, the genetic essence of ASD has been identified in family and twin studies13. The study of family exome sequencing has found some rare de novo mutations in people with ASD as opposed to healthy controls. It is estimated that ~1000 genes are involved in autism15. On the other hand, the main variants associated with autism16 are yet to be identified in genome-wide association studies. By understanding the evidence at gene expression, we will obtain a complete understanding of autism which can aid in the clinical treatment of patients with ASD.Recent cohort studies have reported that hereditary factors can be traced to more than 50% of autism cases17 and can help develop therapeutic approaches to prevent or reverse ASD brain changes. In this review article, as demonstrated in the imaging, we illustrate how genetic variations are correlated with the structural and functional changes in the brain.On the other hand, noninvasive imaging provides information on the structural and functional changes of autism in the brain. Studies have shown that connecting genomics to imaging can provide a better understanding of ASD’s pathophysiology18. Young patients with autism showed abnormally enlarged neurons in the cerebellar nuclei, inferior olive, and vertical limb of the diagonal band of broca18. Another postmortem study found an increased number of neurons in the prefrontal cortex in the brain of ASD children19. Purkinje cell count was found to be decreased in the archicerebellar and neocerebellar cortices of the cerebellar hemisphere in autistic patients20. Another study observed a reduced number of small pale neurons in the brains of adult autistic patients21. A postmortem study of the brain of patients with ASD also reported altered axonal density and the presence of impaired myelin in white matter (WM)22. Overall, the pathological findings suggest a restricted brain development pattern in ASD patients.Postmortem studies conducted on autistic brains helped to identify the neuroanatomical changes associated with ASD. Smaller cell size and increased density of cells in the hippocampus, limbic system, entorhinal cortex, and amygdala have been observed in all ages of ASD individuals23. A comparison of the brain morphometric features in 3–4 years old ASD children with typically developing (TD) children and developmentally delayed children24 was conducted using high-resolution 3-D MRI. Increased cerebellar volume was found in children with ASD compared to TD, and developmentally delayed children and amygdala enlargement with increased cerebral volume was seen in ASD children24. Another study observed cerebral enlargement in both boys and girls with ASD compared to controls, and boys with regressive autism showed more structural alterations as compared to girls25. Volumetric MR studies demonstrated a 5–10% increase in brain volume of ASD children who were scanned between 18 months and 4 years26.Brain changes in ASD patients have been primarily studied using magnetic resonance imaging (MRI). MRI is the most widely used noninvasive modality to examine the structural and functional brain changes in different neurological and neurodegenerative disorders. Various MR-based methods are applied to study the morphological, functional, and metabolic changes in the brain of ASD. MRI findings in children with ASD ranging from 2 to 5 years of age revealed that there is an abnormal development of frontal and temporal lobes, lower gray matter (GM), WM, and amygdala volume compared to same-age healthy controls27. Another study showed increased cortical thickness in the parietal and temporal lobes of autistic children compared to healthy controls28. An MRI study of adults with ASD, who had delayed language development, also called high‐functioning autism (HFA), observed increased cortical thickness in frontal, occipital, temporal, parietal, cingulate, and fusiform gyri while decreased cortical thickness was observed in the post- and paracentral gyrus29. Surface-based morphometry (SBM) analysis is also used to measure cortical thickness in ASD patients. An SBM based study found decreased cortical thickness in the left orbitofrontal and parahippocampal gyrus, left frontal pole, and pars triangularis, while increased cortical thickness was observed in the left precuneus and anterior cingulate cortex30. Another SBM based study reported reduced cortical volume in the left middle temporal gyrus, reduced gyrification index in the left supramarginal gyrus and increased cortical thickness in pars opercularis of inferior frontal gyrus in HFA compared to TD individuals31. The SBM approach was also used to compare the cortical abnormalities in individuals with low‐functioning autism (LFA) and HFA and Asperger’s syndrome with TD controls. In HFA individuals, shape abnormality was observed bilaterally in parietal operculum and ventral postcentral gyrus. LFA individuals showed shape abnormality in the pars opercularis located in the inferior frontal gyrus, and individuals with Asperger’s syndrome showed bilateral abnormalities in the intraparietal sulcus region32. Voxel-based morphometry analysis observed increased GM volume in frontal, parietal, temporal lobes and limbic system and reduced WM volume in limbic system, frontal and temporal lobes of autistic subjects33. Another voxel-based morphometry study showed reduced GM volumes in frontal, temporal, and parietal lobes in HFA individuals compared to healthy controls34.Imaging studies have observed both decreased and increased cortical thickness in patients with ASD. One study, performed on eight autistic subjects, showed decreased cortical thickness in the regions of inferior frontal, occipital, supramarginal, and post- and precentral gyrus, anterior cingulate, prefrontal, parietal, and temporal cortex35. DTI is widely used to track the changes in the microstructural properties of WM such as axonal density, axonal injury, and myelination in various human brain’ disorders. Provided that ASD is a developmental disorder, DTI can be used to identify variations in WM to better define developmental trajectories in individuals with ASD36. Increased FA was observed in the frontal lobes and corpus callosum regions of children aged 2–3 years with ASD compared to healthy controls37. While, another study showed reduced FA in frontal–posterior tracts in the brain of children with ASD, aged 10–18 years38. DTI studies showed a difference in trajectories of FA in infants aged 6–24 months who were at higher family risk of developing ASD. FA was higher in infants who had ASD diagnosis at the age of 6 months, while FA was found to be lower at the age of 24 months39. One study reported decreased FA in the parietal lobes, temporal lobes, lateral occipital cortex, left anterior cingulate, middle frontal cortex, and in the corticospinal tract of individuals with HFA as compared to the controls34. DTI tractography confirmed abnormality of the WM structures in the regions of the corpus callosum, inferior longitudinal and fronto-occipital fasciculus, and superior longitudinal fasciculus in ASD patients40.Diffusion tensor imaging (DTI), a noninvasive MRI technique, exploits the diffusion properties of a water molecule to map the microstructural, architectural, and compositional characteristics of the tissue. Fractional anisotropy (FA) and mean diffusivity are two commonly used DTI metrices in mapping the restricted movement of water molecules in the tissue, where higher FA associates with the greater directionality and tissue integrity42  is a major cell adhesion protein that plays a vital role in the development of the synapse, and it has also been implicated in ASD. An ex vivo MRI study showed a reduction in various WM and GM regions of NL3 knockin mice, with deficits in social and anxiety-related behaviors43. A similar study used MRI to image mouse models with mutations implicated in autism, such as NL3 knockin, MECP2, and integrin β3 (ITGB3) homozygous knockout mouse models to investigate the effects of these mutations on brain morphology44. The study found enlarged WM, and GM volumes of crus II lobule in NL3 mutants increased cerebellar volumes in MECP2 mutants and reduced cerebellar structures in ITGB3 mutants44. A neuroimaging study reported reduced total brain volume and hippocampal size with enlarged basal ganglia structures in SHANK3 knockout mice compared to a prenatal zinc-deficient mouse model of ASD45.Neuroligin-3  is a rare neuronal migration disorder, and patients with CDFE exhibit autistic characteristics. A study reported a homozygous mutation in the CNTNAP2 gene in all CDFE patients, which is responsible for an early developmental insult, mainly in the frontal and temporal neocortex74. Another study showed that the impact of CNTNAP2 missense variants on axonal growth as the loss of CNTNAP2 allele contributes to the disruption in axonal growth75. An MRI study investigated the homozygous c.3709DelG mutation in CNTNAP2 in the forebrain organoids generated from human induced pluripotent stem cells that were derived from patients with syndromic ASD. The study found that individuals with c.3709DelG mutation in CNTNAP2 displayed increased head circumference and increased GM volume76. An imaging genetic study found an association of CNTNAP2 rs779475 single-nucleotide polymorphism with WM and GM morphology in healthy controls and observed reduced volume of GM in the regions of frontal and occipital lobes and cerebellum. However, there was a reduction in the WM volume in the regions of right rostral cingulum, right caudal inferior fronto-occipital fasciculus, and the posterior thalamic radiations in homozygotes for the risk allele (rs779475 T/T) as compared to the nonrisk homozygotes and a group of rs779475T heterozygotes77.Mutations in CHD8) gene, which plays a vital role in the chromatin remodeling process, are amongst the most commonly reported mutations in ASD. CHD8 mutations have also been linked with axonal and dendritic growth, and a study showed that CHD8 is highly expressed in a microtubule-associated-protein 2 positive neurons and parvalbumin neurons which plays an essential role in the development of neocortex in the human brain. The findings of the study suggested that CHD8 mutations in humans can result in neuronal deficits that can contribute to ASD pathophysiology51.Mutations in the chromodomain helicase DNA binding protein 8 (MET), as a candidate risk gene for ASD. In order to find the association of MET with cortical development, a structural MRI study investigated the relationship between a single-nucleotide polymorphism within the MET promoter  and the development of cortical thickness in ASD individuals. The study observed cortical thickness reduction in the regions of ventral precentral and postcentral gyri, superior and middle temporal gyri, anterior cingulate cortex, and in the right fronto-polar cortex with increasing C allele dose78. Another study found that MET risk allele carrier (rs1858830) individuals with ASD and TD showed higher activation of amygdala, reduced WM integrity, and reduced connectivity between the posterior cingulate cortex and medial prefrontal cortex (mPFC)79.Studies have proposed the association of MET proto-oncogene, receptor tyrosine kinase (SLC6A4) involving a serotonin-transporter related polymorphic region (5HTTLPR) results in the generation of short or long alleles. A study by Wassink et al. found that the 5HTTLPR short allele significantly influences the cortical GM volumes in male autistic children80. Several neuroimaging studies have investigated the effects of different oxytocin receptor (OXTR) risk alleles  on brain morphological changes in healthy adults89. OXTR risk allele carriers (rs2254298) showed increased amygdala volume89, decreased GM volume in the hypothalamus83, and right insula84. A study showed that dysregulation in the β-catenin/BRN2 transcriptional cascade was associated with an increased number of neurons and proliferation of neural progenitor cells derived from pluripotent stem cells of ASD individuals and also contributed to increased brain volumes90.A common polymorphism in the serotonin-transporter gene 97, facial processing 98, response inhibition (simple inhibition/1-back inhibition task)99 and visuomotor coordination 100 in ASD individuals. Task-based study based on social processing found hypoactivation in social brain nodes in the regions of the mPFC, frontal gyrus, insula, temporal sulcus, amygdala, and the fusiform gyrus101.Resting-state fMRI (rsfMRI) is a powerful tool that is being used for mapping the neuronal activity of various neurological disorders. rsfMRI studies have revealed abnormalities in the neuronal network in ASD patients that help in understanding the underlying neurobiology of ASD. An rsfMRI study observed altered functional connectivity between the cerebral cortex and dentate nucleus in individuals with ASD, which is suggestive of impaired interaction between the cerebellum and social cortical regions of the brain102 and examining the neural activity during the detection of senseless sentences103 have also shown communication impairments such as decreased brain synchrony in language processing regions in ASD103. Task-based studies focusing on reward processing showed impairments in mesolimbic and meso cortical brain regions in ASD. Both over connectivity and underconnectivity between frontal and posterior regions was observed in task-based functional connectivity study104. An fMRI study revealed that ASD individuals had reduced activation of higher-order social  and executive processing  of the brain and higher activation of lower-order structures that are involved in the mediation of primary motor and sensory processing during emotional task performance105. While some task-based studies such as source recognition task106 and emotional face processing task107 have reported increased connectivity between the brain regions of ASD individuals107.Task-based fMRI studies focused on cognitive control reported fronto-striatal dysfunction in ASD, specifically in frontal gyrus, basal ganglia, and anterior cingulate cortex regions. Task-based studies that involved examining brain responses associated with figures of speech like punsNeuroimaging can help bridge the gap between genes, environment, and different ASD behavioral phenotypes. Although functional imaging techniques such as fMRI have revolutionized the understanding of various mental disorders, the fMRI technique also contains several limitations. The big challenge in current fMRI-based methods is the impact of age/gender and the development of scanning techniques for the diagnosis of ASD across heterogeneous populations. One of the limitations of fMRI is that it has no role in measuring the direct neuronal activity instead it provides changes in the signal based on the blood oxygen level, which may lead to the false-positive or -negative signal specially in patients with abrupt behavioral changes such as ASD. Secondly, an individual’s performance on a specific task must be considered in a task-based fMRI and must be well-thought-out when analyzing the fMRI data. The fMRI data are also analyzed as group averages; therefore, it is not straightforward to diagnose individual autistic patients. This could be par by introducing an individualized diagnostic model based on artificial intelligence.Furthermore, the data obtained from fMRI may be compromised to some extent due to head motion artifacts that alter voxel and stable state magnetization leading to distortion of anatomical locations and may show false regions associated with brain activity. In addition, fMRI data can only demonstrate that a particular region of the brain is involved in a specific cognitive function but cannot determine whether it is a cause or a consequence of that specific cognitive function. The lack of longitudinal data also makes it challenging to identify the abnormal developmental course in ASD during infancy. Nevertheless, the integration of multiple sites and data sources and the increase in the number of subjects and intra-variability among subjects can improve the feasibility of the fMRI technique in ASD.−/− mice study showed abnormal development of the cortex and impaired synaptic plasticity in the prefrontal cortex region with impaired social and emotional responses108 (Table AVPR1A) were linked to autism. Impaired social recognition and reduced anxiety behaviors were observed in knockout mice of AVPR1A, while overexpression of AVPR1A in mice led to increased social memory110. There is also an inversion of cortical layering due to neuronal migration defects in the Reelin (RELN) knockout mice111, resulting in various behavioral abnormalities, such as aggressive behavior and memory and learning impairments112. CNVs of the cytoplasmic FMR1-interacting protein 1 (CYFIP1) was found to be associated with ASD and other neuropsychiatric disorders such as schizophrenia. CYFIP1 plays an essential part in regional brain connectivity and corpus callosum function. A study found that the CYFIP1-heterozygous mice displayed reduced bilateral functional connectivity across the entire brain and defects in the WM architecture. In these mice, reduced myelination is also observed in the callosal axons, an altered presynaptic feature that contributes to the motor and sensorimotor abnormalities113.Reduced levels of oxytocin have been found in ASD patients, and CD38 is a vital enzyme that controls oxytocin production. A CD38CNTNAP2 and may contribute to the development of autistic-like features and other neurodevelopment disorders114.Another rsfMRI study showed decreased local and long-range functional connectivity in prefrontal and midbrain of mice with the homozygous loss of NRXN1) is one of the ASD risk genes that are found to influence the brain structure and functions, and its polymorphisms are found to be associated with structural alterations in the prefrontal–thalamic circuitry in healthy individuals, conferring a potential risk of developing ASD or schizophrenia115. Single-cell analysis of neural stem cells from an autistic patient with bi-allelic NRXN1-α deletion observed a phenotypic shift toward radial glia-like cell with impaired maturation action potential and reduced calcium signaling116. On the other hand, CNTNAP2 polymorphisms are found to be associated with altered brain connectivity in regions that are involved in reward and language development117  showed increased activation in the right inferior frontal gyrus and right lateral temporal cortex118. Mutations in the genes involved in the oxytocin/vasopressin system may also contribute to ASD risk as they influence the function of brain regions such as the amygdala and hypothalamus that are associated with emotional and social processing120 , autism, and the impact of oxytocin levels on brain response to social stimuli121. An fMRI study observed increased activation of the left fusiform gyrus in homozygotes for CD38 genetic variant rs3796863119. The MET gene variants are also found to be involved in altering the connectivity and integrity of WM in the temporo-parieto-occipital regions that are involved in high neurological functions such as working memory, language processing, and face and object recognition, all that may predispose an individual to ASD79. Another study reported that the loss of CNTNAP2 contributed to the reduction in long-range connectivity in the brain regions of mice, mainly affecting the prefrontal regions that act as connectivity hubs in the brain122 and this hypoconnectivity in the fronto-posterior region lead to impaired social behaviors, predisposing to autism risk114. The OXTR polymorphism rs1042778 is found to be associated with two common clinical phenotypes of ASD, such as aggression and panic in male ASD patients. As compared to GG homozygotes, the T-allele carriers were at higher risk of possessing aggressive and panic behaviors123. Some of the task-based fMRI studies in healthy individuals carrying the OXTR risk alleles showed increased activation of inferior occipital gyrus during a fear processing task86, decreased activation in the mesolimbic reward circuitry in a reward anticipation task85 and increased amygdala activity during a direct gaze processing task87. One study found elevated levels of AVP with reduced levels of apelin in ASD patients that point to the vasopressinergic dysfunction in autism124. Another study proposed that the shorter alleles of RS1 polymorphism lead to reduced transcription of AVPR1A that may increase the susceptibility to ASD125. An fMRI study found an association between ASD and AVPR1A genetic variants and reported differential activation of amygdala during a face-matching task in RS1 and RS3 risk allele carriers120.Imaging genetics studies have shown the involvement of ASD risk genes in altering the brain circuits that control reward and language processing, and social behavior Table . Neurexi120 Fig. . StudiesMTX2), neurofilament light polypeptide (NEFL), and solute carrier family 25, member 27 (SLC25A27) was found in the anterior cingulate gyrus, the motor cortex, and thalamus regions of autistic patients. NEFL plays a vital role in the maintenance and assembly of the axonal cytoskeleton126, while SLC25A27 is expressed in the central nervous system and has been found to play an essential role in neuronal cell differentiation127, apoptosis inhibition128, and reduction of reactive oxygen species129.Reduced expression of three genes namely metaxin 2  with diagnostic models based on volumetric morphometry involving four different classification methods and classification based on thickness was found to be more efficient and predictive of ASD compared to classification based on volume30. rsfMRI pipelines have been used to extract predictive biomarkers in autism by constructing participant-specific connectomes and then comparing these connectomes across participants to learn connectivity patterns that may identify ASD individuals138. The results suggested that rsfMRI data collected from different sites could reveal robust functional connectivity biomarkers of ASD.MRI-based diagnostic models are used for the behavioral assessment of autistic patients. These diagnostic model studies involve three steps, including extraction of properties from MR images, construction of diagnostic model using statistical models followed by evaluation and validation by researchers. Several studies have focussed on MRI-based diagnostic models for the detection and classification of ASDNRXN superfamily genes115. Neuropeptide signaling and emotional functioning was found to be influenced by the oxytocin and arginine vasopressin receptor genes via structural and functional modification in the amygdala–hypothalamus circuitry77. One study showed a relationship between frontal lobe connectivity and common genetic variants in CNTNAP2 using a functional neuroimaging study117 and the study found that ASD and TD individuals who were nonrisk allele carriers showed more reduction in the activation of mPFC during an fMRI task as compared to risk allele carriers117. Another study showed decreased functional connectivity in the prefrontal cortex, cortical spinal tract, corpus callosum, and decreased integrity of WM in children and adolescents carrying MET rs1858830, C risk allele. Such studies suggest that the genes affect the brain regions that are involved in social and emotional processing139.Imaging genetics in ASD has proven useful, and pathways that include common genetic variation in TD individuals at risk of developing ASD have been characterized. Prenatal transcription regulation and synapse formation in the developing brain is impacted by the genes associated with ASD Table . Alterat139 can be achieved. Ultimately, imaging genetics in ASD might be promising for the clinical management of ASD-affected patients.More detailed brain analysis combined with visualization, will promote the elucidation of the relationship between genes and altered neuronal circuits. Also, researching the time-dependent impacts of ASD-involved genetic variants that are the cause of neurodevelopmental modification can aid in the progress of ASD imaging genetics. In addition, the plasticity that is altered in different neuropsychiatric disorders such as ASD can be measured with high resolution by combining electroencephalography or fMRI with transcranial magnetic stimulation. Advances in noninvasive brain imaging techniques and scans during rest or task may help in infant neuroimaging that are at a higher risk to develop ASD. With studies investigating genetic effects on brain development using high-resolution imaging, studying brain-behavior connections and the impact of new therapies on brain development, further translational progress will happen in the future. By neuroimaging endophenotypes, a greater understanding of the impact of ASD risk genes on the brain circuitryThe field of imaging genetics has exponentially grown in recent decades from its candidate gene studies to large-scale longitudinal studies, cross-modal investigations, and translational animal models of various psychiatric disorders. In addition, imaging genetics has begun integrating transcriptomic data and analytical methods for assessing pathway enrichment, such as the score system for pathway regulation. Of addition to translational animal research and pharmacological intervention in vitro and in vivo, imaging genetics can also provide an insight into various behavioral and genetic factors that contribute to the risk of ASDs.140. It is important to properly replicate the studies, particularly those with false-positive results, to address the impact of a genetic variation in a disease, and this problem can be solved by correcting genome-wide associations with large sample size imaging phenotypes.One of the challenges facing imaging genetics is the conceptualization of endophenotypes, which states that endophenotypes are heritable and associated with psychiatric disorders and may impede research on brain-based associations by limiting imaging genetic research to genes previously associated with a psychiatric disorder141. Also, the differences in age and severity of the ASD cohort may lead to disparity in findings, and so large-scale studies are required that concentrate on exploring the developmental trajectories that are crucial to understanding the neurobiology underlying ASD. Such large-scale study databases can be obtained from various consortia, such as autism brain imaging data exchange and National Autism Research Database (NDAR). The heterogeneity of the ASD phenotypes, therefore, leads to the difficulties faced in ASD neuroimaging genetics as there is a high degree of variability in frequency, the number of behavioral symptoms reported, and the associated characteristics in ASD. In addition, the brain responses produced as a result of a particular task stimulus are inadequate to determine if these responses result from a neural processing disorder or are due to the introduction of an extrinsic factor.Another challenge in ASD neuroimaging research is the discrepancy in the brain connectivity findings due to motion artifacts. The motion artifacts in the rsfMRI data can misrepresent group differences in connectivity metrics which can lead to incorrect interpretation of the altered brain connectivity data in ASD. Other methodological variables such as pipeline type, field of view, and dataset can significantly impact (in terms of over-or underconnectivity) the functional connectivity MRI studies in ASD142 that can be used for the clinical management of ASD patients.In addition, more development in data analytical methods such as machine learning algorithms or software can allow data from multiple modalities to be incorporated into a heterogeneous population, and can also recognize and detect digital biomarkersTo explain the origin of ASD’s underlying pathophysiology, the study of genetic alterations and the relationship between genes and environmental factors is crucial. Imaging genetics studies have revealed important information about the pathophysiology of ASD. One of the key aims of imaging genetics studies in ASD is to elucidate neural pathways that give rise to phenotypical heterogeneity. Enhanced risk of ASD is shown to be related to several different single-nucleotide polymorphisms, and the stratification of neuroimaging data by specific genetic risk factors occurring in ASD may help to understand the neurobiology underlying this disorder. Such studies will help to elucidate ASD from various perspectives and subsequently will pave the way for personalized treatment for patients with ASD.