Inherited disorders of biogenic amine metabolism are genetically determined conditions resulting in dysfunctions or lack of enzymes involved in the synthesis, degradation, or transport of dopamine, serotonin, adrenaline/noradrenaline, and their metabolites or defects of their cofactor or chaperone biosynthesis. They represent a group of treatable diseases presenting with complex patterns of movement disorders  associated with a delay in the emergence of postural reactions, global development delay, and autonomic dysregulation. The earlier the disease manifests, the more severe and widespread the impaired motor functions. Diagnosis mainly depends on measuring neurotransmitter metabolites in cerebrospinal fluid that may address the genetic confirmation. Correlations between the severity of phenotypes and genotypes may vary remarkably among the different diseases. Traditional pharmacological strategies are not disease-modifying in most cases. Gene therapy has provided promising results in patients with DYT-DDC and in vitro models of DYT/PARK-SLC6A3. The rarity of these diseases, combined with limited knowledge of their clinical, biochemical, and molecular genetic features, frequently leads to misdiagnosis or significant diagnostic delays. This review provides updates on these aspects with a final outlook on future perspectives. Inherited defects of biogenic amine neurotransmitter metabolism are ultrarare genetically determined conditions resulting in dysfunctions/lack of enzymes involved in the synthesis, degradation, or transport of dopamine, serotonin, adrenaline/noradrenaline, and their metabolites or defects of their cofactor or chaperone biosynthesis  1]. All. All1]. Several peculiarities justify the interest in these conditions: (a) as a group, they are among the most frequent genetic causes of movement disorders in children ,2,3,4; .A reliable estimate of the frequency of this set of conditions is available for hyperphenylalaninemia-associated disorders, which are intercepted by neonatal screening programs for phenylketonuria , all including 2–3% of the overall number of patients with neonatal hyperphenylalaninemia  ,7. A morhttp://intd-online.org, last access 7 October 2022), which collected 350 patients with disorders of biogenic amine metabolism: 161 patients with BH4 deficiency, 56 patients with DYT/PARK-PTS, 37 patients with DYT/PARK-QDPR, 36 patients with autosomal dominant DYT/PARK-GCH1, 18 patients with autosomal recessive DYT/PARK-GCH1, 14 patients with DYT/PARK-SR, 131 patients with DYT-DDC, 44 patients with DYT/PARK-TH, 5 with DYT/PARK-SLC6A3, 5 with DNAJC12 deficiency, and 4 patients with MAO A deficiency [No adequate epidemiological data are available for other primary biogenic amine disorders. An indirect estimate of their frequency can be drawn on the basis of cases entered in the International Working Group on Neurotransmitter Related Disorders (iNTD patient registry (DDC variants (19 homozygous and 39 compound heterozygous) suggested a global prevalence of 1800 living patients with DYT-DDC [The prevalence of DYT-DDC was also studied through other indirect evaluations ,11. The  DYT-DDC . An esti. Speech and language impairment are typical clinical traits of late or untreated conditions and, similar to other higher cortical functions, they respond less than movement disorders to pharmacological treatments [The pattern of neurological impairment, the age at the onset of symptoms, and their severity are usually correlated with the severity of biogenic amine depletion. The earlier the onset of the disease, the more severe and pervasive the neurological impairment is, resulting in a neurodevelopmental delay leading to intellectual disability, behavioral problems, and movement disorders ,12,13,14The onset of symptoms after three is usually associated with the selective impairment of motor functions, as usually happens for autosomal dominant DYT/PARK-GCH1 . An actuThe individual response to dopaminergic and serotoninergic treatment ,14 may rMovement disorders are a significant part of the clinical spectrum of inherited disorders of biogenic amine metabolism, both as an isolated disorder or in the context of infantile encephalopathy . A remarkable source of complication for their management includes the lack of consensus for the classification of movement disorders in children under 3 years of life and the relevant differences between their clinical presentations and those typical of adult-onset parkinsonism .Initial presentations may include rigidity of limbs and trunk hypotonia in patients with a global delay in developmental milestones . HypotonIn older children or adolescents, lead-pipe rigidity may often be combined with bradykinesia or focal arrhythmic jerks . AkinesiThis group of disorders includes diseases with hyperphenylalaninemia (detectable with newborn screening) and diseases without hyperphenylalaninemia .GCH1 (NM_000161.3) gene. GCH1  includes six exons and encodes for the GTPCH1 enzyme that catalyzes the first step in BH4 biosynthesis  [This autosomal recessive disease is due to pathogenic homozygous or compound heterozygous variants of the https://www.ncbi.nlm.nih.gov/clinvar/; last access 7 October 2022). Five of them are structural pathogenic/likely pathogenic variants involving other genes contiguous to GCH1, whereas another reported that the pathogenic complete deletion of the GCH1 gene leads to haploinsufficiency [n = 28), followed by frameshift (n = 18), splice site (n = 16), and nonsense ones (n = 11). The number of pathogenic variants reported in ClinVar drops to 56 after filtering, excluding those deposited without assertion criteria by submitters  are marked as pathogenic/likely pathogenic. Several variants associated with a suggestive clinical and biochemical phenotype in the literature or with a demonstrated disease-causing role after specific functional studies were not reviewed and reassessed in the two databases above. The recent review by Himmelreich et al. reported seven variants associated with clinical manifestations of GCH1 [The BioPKU database reports 329  of GCH1 . The intThe c.350T > G p.Leu117Arg) variant, which probably interferes with the access of GTP to its binding site, caused severe early onset motor dysfunctions in a recently developed homozygous mouse model mutant . This vaArg variaDystonia and tone abnormalities were reported as the initial signs in a proportion ranging between 25 and 40% of patients ,19,20. EDYT/PARK-PTS is an autosomal recessive disease due to pathogenic variants of the gene that is located on chromosome 11q22-3-q23.3 . The encPTS were missense, while splice sites, nonsense, and frameshift ones were less frequently described , which result in a pseudoexon [The ClinVar database (last access 7 October 22) reports 212 variants involving The BioPKU database reports 198 variants, including a multiexonic deletion whose breakpoint has not been characterized .A probable founder effect was suggested for variants c.155A > G (p.Asn52Ser), c.259C > T p.Pro87Ser), c.272A > G (p.Lys91Arg), c.286G > A (p.Asp96Asn) , c.84-29Ser, c.27A severe (85% of patients) and a mild phenotype (15% of patients) of DYT/PARK-PTS were defined according to the clinical presentation and biochemical alterations . VariantRecombinant expression and Western blot analyses in patients’ fibroblasts revealed the complete inactivation or the structural destabilization of the PTPS enzyme associated with variants c.200C > T/p.Thr67Met , c.385A Movement disorders were reported in about 25% of children with the severe phenotype . The mosDifferent patterns of clinical progression may be assessed after the neonatal-infantile period: (1) slowing of weight gain and developmental delays, diffuse dystonia with opisthotonus associated with lead-pipe rigidity or hypotonus, myoclonic jerks, and ataxia ,29,30,31Psychiatric disorders were recently reported in 39% of cases in an Italian cohort, including 28 patients . DYT/PARK-QDPR is an autosomal recessive disease due to pathogenic variants of the cited gene on chromosome 4p15.32  . A totalQDPR. No clear predominance of specific types of point mutations was highlighted, whereas 13 large deletions or multigenic duplications and an inversion of the entire gene were also deposited [ClinVar and BioPKU include, respectively, 257 and 141 variants involving eposited .Most reported variants are associated with inactive enzymes, with a few exceptions correlated with milder phenotypes. The variants c.451G > A(p.Gly151Ser) and c.635T > G(p.Phe212Cys) are associated with isolated abnormalities of serotonin metabolism, whereas c.199-1G > T is associated with an isolated mild hyperphenylalaninemia . DHPR loss or reduced function results in severe brain damage that might be caused by two main mechanisms: (1) the secondary accumulation of 7,8 dihydrobiopterin (BH2) and the subsequent inhibition of aromatic amino acid hydroxylases causing a low concentration of dopamine and serotonin in the brain; and (2) the reduced availability of 5-methyl-tetrahydrofolate because of a secondary defect of brain dihydrofolate reductase diverted to the synthesis of BH4 from BH2 to BH4 . The clinical presentation of DYT/PARK-QDPR is characterized by developmental delays, intellectual disability, and epilepsy, while movement disorders occur less frequently (less than 20% of infants and 30% of older children) ,38. In uPCBD1  gene on chromosome 10q22.1, which encodes an enzyme involved in the dehydration of pterin-4a-carbinolamine to quinoid dihydropteridine.  ,43. Diur-Neuromotor and cognitive regression with loss of head control, feeding disturbances, and diffuse athetoid movements ;-Delay in early motor milestones with pyramidal and extrapyramidal signs mimicking a cerebral palsy ,50,51;-Diurnal fluctuation of balance and tremors of limbs ;-Hypokinesia and drooling at the age of 9 months ;-Intermittent and fluctuating tiptoe walking evolving into gait difficulties and paroxysmal opisthotonus with akinesia lasting 2 to 3 h ;-Lower limb focal dystonia occurring between 2 and 3 years ,53.Early clinical onset is presumably underdiagnosed , and about 20–30% of patients are symptomatic within the age of 3 years ,45,46,47In our case, mild developmental delays during the first 2 years led to myoclonic jerks by the age of 3 years and myoclonic dystonia during adolescence in a boy carrying the variant c.671A > G (p.Lys224Arg) who had myoclonus-dystonia and resting or postural tremors of limbs in his paternal branch .DYT/PARK-SR is an autosomal recessive disease caused by pathogenic variants that may be distributed along the 3 exons of the gene (NM_003124.5) on chromosome 2p13.2. SR regulates the reduction of the pterin intermediate 6-pyruvoyl-tetrahydropterin included in de novo BH4 biosynthesis .Most of the 155 variants deposited on ClinVar are classified as VOUS, while very few are marked as pathogenic with provided assertion criteria and compatible associated clinical phenotypes .The compound heterozygous state involving variants c.512G > A(p.Cys171Tyr) and c.304 + 1_ + 12del (marked in A possible founder effect was suggested for c. 68G >A(p.Gly23Asp) in the Maltese population . The hetVariant c.-13G > A, which is in the 5’UTR, was described in a patient with autosomal dominant responsive dopa dystonia. Functional studies on patients’ fibroblasts showed a decrease in SR enzyme activity and the amount of synthesized protein . The onset of symptoms ranged between 7 months and 6 years in a 43-patient multicentric retrospective study . Early cTH gene spans about 8 Kb and includes 14 exons [TH is predominantly expressed in dopaminergic neurons of areas regulating motor functions  but also motivation, addiction, and reward  [DYT/PARK-TH is an autosomal recessive disease caused by pathogenic variants of the gene  on chromosome 11p15.5 . The TH 14 exons . The enc14 exons . TH is p cortex) . A pathogenic role was reported for 66 variants, including 14 nonsense mutations and 19 mutations in the promoter ,64. TH gene with a demonstrated pathogenic role  and 237 VOUS. No clear genotype–phenotype correlations are demonstrated [ClinVar reports 64 variants of the nstrated ,64. SeveTwo partially overlapping phenotypes of tyrosine hydroxylase deficiency were suggested in a 2010 retrospective study: in the former (type A), motor functions are more selectively involved, while the latter (type B) identifies an “encephalopathic” form, including a minority of children with a more generalized impairment of neurological functions and development . The recPatients with mild phenotypes usually present with developmental delays, generalized hypokinetic-rigid syndrome, and fluctuating dystonia during the first weeks or months of life . DystoniOne of our patients presented with normal psychomotor development until the second year of life when toe-walking gait, frequent falls, and delayed language suggested the diagnosis of spastic paraplegia . Later, Patients with more severe phenotypes display an early derangement of neurological development, a severe hypotonic-hypokinetic syndrome with dystonic movements and posture, oculogyric crises, ptosis, focal or generalized dystonic features, and jerky movements such as tremors and myoclonus . The ons65. Fluctuations in neurological symptoms include a periodical worsening of dystonic crises or lethargy-irritability crises [Sometimes, severe hypokinesia and hypotonia may remind the patterns of muscle diseases or spinal muscle atrophyy crises . Several reported patients presented with complex phenotypes ,65,70. ADYT-DDC is an autosomal recessive inherited disease due to pathogenic gene variants  on chromosome 7p12.2-p12.1 . AADC isThe BioPKU database includes 422 variants: 7% of them are identified as pathogenic, 32% as likely pathogenic, 58% as VOUS, 1% as likely benign, and 1% as benign . A totalVariants c.304G > A (p.Gly102Ser), c.1040G > A(p. Arg347Gln) , and c.4The variant c.304G > A(p.Gly102Ser) was found with an allele frequency of 8% of patients in a 78-patient cohort and is associated with an AADC enzyme activity of 16% when compared to the wild-type protein . In the The clinical presentation of DYT-DDC includes combined signs of dopaminergic , serotoninergic , and noradrenergic  dysfunctions . SymptomEarly movement disorders, including delay in postural reactions with severe hypotonia and various dystonic presentations with a prominence of oculogyric crises, occur in early infancy in 95–97% of cases aged 2–12 years .The index patients, i.e., two male monozygotic twins born to related parents at 2 months, presented with severe hypotonia, crying-induced dystonic postures of the arms and the legs, oculogyric crises, and occasional choreoathetosis movements of the extremities . The folClinical observations in two of our female patients with mild symptoms evidenced a spontaneous improvement of motor symptoms occurring during the transition from the early stages of life into adolescence and young adulthood . One of A recent retrospective study in a 9-patient Italian cohort evidenced a susceptibility to psychopathological and psychiatric disturbances, including generalized anxiety disorder, depressive disorder, obsessive–compulsive disorder, oppositional defiant disorder, and attention deficit hyperactivity disorder .The two isoenzymes monoamine oxidases A and B, located in secretory neurotransmitter vesicles, are key enzymes responsible for the oxidative deamination of biogenic amines . Genes eMAOA deficiency presents with intellectual disabilities, autonomic dysregulation, and behavioral disturbances . MAO-B dMost pathogenic variants deposited in ClinVar include large duplications involving many genes and extending more than 1 kb, with only a few mutations smaller than 50 bp . The de DBH gene  on chromosome 9q34.2, is a membrane-bound enzyme that converts dopamine to norepinephrine in presynaptic vesicles. To date, 25 patients from 20 families are described in the literature, mainly in the USA and the Netherlands [Dopamine-β-hydroxylase deficiency (DBH-D), encoded by the herlands . The molecular genetic diagnosis was achieved in 19 patients from 16 families, and 10 different pathogenic variants were identified . A compoSevere orthostatic hypotension, eyelid ptosis, sporadic dysmorphic features, rare reproductive dysfunctions, and normal cognitive development represented the most common clinical hallmarks . SLC6A3 gene  on chromosome 5p15.33, modulates the active reuptake of dopamine from the synapse and is a principal regulator of dopaminergic neurotransmission [The dopamine transporter (DAT), encoded by the smission ,90,91. Lsmission ,90,91. DYT/PARK-SLC6A3 is an autosomal recessive disease that was diagnosed in 14 published patients ,92,93. NSLC6A3 gene, and only three of them were deposited with assertion criteria  variants of the criteria . Most ofOther recently reported likely pathogenic variants, which were not deposited in ClinVar, include c.934A > T (p.Ile312Phe), causing a structural perturbation with an indirect impact on catalytic activity, c.1261G > A (p.Asp421Asn), compromising the sodium binding site, and c.1639dupC (p.His547Profs*56), which influences the structure of the transporter.DYT/PARK-SLC6A3 manifests during the first months of life with two of the following alternative patterns of neuromotor derangement: (1) a severe akinetic-rigid syndrome and (2) a severe dyskinetic syndrome ,91,92.Among all the different monoamine neurotransmitters related to movement disorders, this is probably the only disorder manifesting with such extreme and precocious hypokinesia associated with remarkable impairment of gross motor and intentional skill development ,92,93. FHypomimia is particularly remarkable in DYT/PARK-SLC6A3, sometimes contrasting with an incongruous and persistent motionless smile and with oculogyric crises or other ocular dyskinesia, such as ocular fluttering and opsoclonus coupled with an increase in saccade latency . In the SLC18A2  on chromosome 10q25.3. DYT/PARK-SLC18A2 is an autosomal recessive disease affecting a protein involved in the transport of biogenic amines inside the presynaptic vesicles . The encThe index family included eight affected members . All theMore recent studies extended the total number of patients to 58 affected individuals (16 of them died before the age of 13) carrying 19 homozygous variants and presenting with global developmental delays, hypotonia, dystonia, oculogyric crises, and autonomic symptoms ,101,102.DNAJC12 gene  on chromosome 10q21.3. DNAJC12 is a heat shock cochaperone protein interacting with PAH, TH, and TPH [DNAJC12 and PAH in some Spanish patients suggested the possible role of DNAJC12 as a modifier of PAH deficiency [The recently reported DNAJC12 deficiency is caused by autosomal recessively transmitted pathogenic variants of the  and TPH . An impa and TPH . The coeficiency . In thisficiency . Forty-one patients were reported in the literature, aged between 2 and 40 years ,106. The clinical phenotype is highly variable, including different degrees of hyperphenylalaninemia, juvenile parkinsonism, dystonia, autism, intellectual disabilities, attention deficit hyperactivity disorder, psychiatric symptoms, and no symptoms in 18 patients ,108. Twelve pathogenic nonstructural variants with direct and demonstrated effects on phenylalaninaemia were deposited in ClinVar. Most of them were inserted without assertion criteria . Other vSeveral biochemical markers  are essential for a correct address to genetic confirmation . The meaThe measurement of urine levels of neopterin, biopterin, and sepiapterin may detect pterin defects, while high urine levels of vanillylactic acid may reveal DYT-DDC .In some cases, biochemical markers may correlate with the clinical severity with possible prognostic implications. CSF HVA and HVA/5HIAA ratios significantly correlated with more severe phenotypes in a cohort of 36 patients with DYT/PARK-TH . PatientA recent proteomic study on the CSF of 90 patients revealed novel possible severity biomarkers, including decreased levels of apolipoprotein D for DYT-DDC, a decrease in apolipoprotein H, an increase in oligodendrocyte myelin glycoprotein for tyrosine hydroxylase deficiency, and a decrease in collagen6A3 for BH4 deficiency .Brain neuroimaging is often expected, even if a recent systematic re-evaluation of 87 MRIs of 70 patients evidenced different abnormalities: diffuse cortical/subcortical atrophy in 24 patients, T2-hyperintensity of central tegmental tracts in 9 patients, changes in arterial watershed zones involving parieto-occipital and cerebellar areas in 8 patients (DYT/PARK-QDPR), myelination delay in 4 patients (DYT-DDC and DYT/PARK-TH), signs of profound hypoxic-ischemic injuries, and a combination of deep gray matter and watershed injuries in 2 patients (DYT-DDC) . VolumetThe loss of DAT activity in the basal ganglia, as measured by single-photon emission tomography of DAT, may be a useful diagnostic marker in patients with DYT/PARK-SLC6A3 [Inherited monoamine neurotransmitter disorders include a group of treatable diseases . Almost 115. Robust evidence for these treatments’ efficacy is lacking; consequently, doses and the whole therapeutic strategies tend to rely more on expert recommendations [ndations ,74,115. PTS gene (164-672C > T and c.164-716A > T) [DDC copies [Antisense oligonucleotide treatment resulted in the complete rescue of PTPS proteins in the fibroblasts of patients carrying two deep intronic variants activating pseudoexons on the 16A > T) . More ad16A > T) ,118,119.C copies ,118,119.C copies ,118,119.C copies ,118,119.Similar strategies based on lentiviral and adenoviral vectors provided the same promising results in patient-derived induced pluripotent stem cells and mouse models of DYT/PARK-SLC6A3 . Enzyme Primary disorders of biogenic amine metabolism represent an interesting model of how clinical practice should be integrated with biochemical and molecular genetic techniques to improve patient outcomes in severe neurological disorders. Therapeutic progress, up to the enormous potentialities showed by gene therapy for DYT-DDC, will probably contribute to the achievement of this aim.


Modified Linggui Zhugan Decoction (MLZD) is a Traditional Chinese Medicine prescription developed from Linggui Zhugan Decoction (LZD) that has been used for the clinical treatment of ischemic cardiovascular diseases. However, the cardioprotective mechanism of MLZD against post-myocardial infarction (MI) ventricular remodeling remains unclear.We explored the effects of MLZD on ventricular remodeling and their underlying mechanisms, respectively, in SD rats with MI models and in H9c2 cardiomyocytes with oxygen-glucose deprivation (OGD) models. The cardiac structure and function of rats were measured by echocardiography, HE staining, and Masson staining. Apoptosis, inflammation, mitochondrial structure and function, and sirtuin 3 (SIRT3) expression were additionally examined.MLZD treatment significantly ameliorated cardiac structure and function, and thus reversed ventricular remodeling, compared with the control. Further research showed that MLZD ameliorated mitochondrial structural disruption, protected against mitochondrial dynamics disorder, restored impaired mitochondrial function, inhibited inflammation, and thus inhibited apoptosis. Moreover, the decreased expression level of SIRT3 was enhanced after MLZD treatment. The protective effects of MLZD on SIRT3 and mitochondria, nevertheless, were blocked by 3-TYP, a selective inhibitor of SIRT3.These findings together revealed that MLZD could improve the ventricular remodeling of MI rats by ameliorating mitochondrial damage and its associated apoptosis, which might exert protective effects by targeting SIRT3. Coronary artery disease (CAD) remains the leading cause of death in developed as well as developing countries , among w2+ bursts, non-selective mitochondrial permeability transition pore (MPTP) opening, and mitochondrial membrane potential (MMP) collapse  Bge.], Panax ginseng [Panax ginseng C. A. Mey.], Ramulus cinnamomi [Cinnamomum cassia Presl], Poria [Poria cocos (Schw.) Wolf], Rhizoma atractylodis macrocephalae [Atractylodes macrocephala Koidz.], Rhizoma alismatis [Alisma orientalis (Sam.) Juzep.], Radix salviae miltiorrhizae [Salvia miltiorrhiza Bunge.], Pericarpium areca [Areca catechu L.], Semen lepidii [Lepidium apetalum Willd.], and Radix angelicae sinensis [Angelica sinensis (Oliv.) Diels], which exerts crucial protections on the heart. For example, an extract of Radix astragali, calycosin, was reported to inhibit neutrophil infiltration and protect heart integrity in isoproterenol-induced MI by synergizing with gallic acid  is regarded as complementary and alternative medicine for the primary and secondary prevention of cardiovascular diseases . Lingguilic acid . The carlic acid . The ethlic acid , 24. Cinlic acid , 26, waslic acid , and to lic acid . Atractylic acid .Nevertheless, what remains to be further explored is whether MLZD can protect against ventricular remodeling after MI, and the mechanisms through which it acts. Investigating these important questions may be helpful in providing a promising therapy for these heart diseases. In this work, we investigated the underlying mechanisms of MLZD in ventricular remodeling, respectively using SD rats with MI models and H9c2 cells with OGD models, seeking to verify our hypothesis that MLZD exerts pharmacological effects targeting mitochondrial damage and apoptosis, thereby alleviating post-MI ventricular remodeling.1 the chemical constituents of 10 traditional Chinese medicines in MLZD were sequentially retrieved. Referring to the screening conditions of TCMSP, that is, oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18, eligible potential active ingredients were obtained. LC-MS analysis was subsequently performed. A hundred milligrams of MLZD powder was dissolved in water (5 ml) with the assistance of ultrasound. Then the solution was filtered. The 50 μl of the filtrate was diluted with MeCN to 1 ml, then it was analyzed by LC-Mass (Waters Acquity ultra-performance LC). The data was collected from the spectrum of positive charges.Modified Linggui Zhugan Decoction ingredients were screened via network pharmacology and qualitatively analyzed via liquid chromatography-mass spectrometry (LC-MS) analysis. From the Traditional Chinese Medicine System Pharmacology (TCMSP) database,n = 10), the surviving and successfully modeled rats were randomly divided into the model group  and the Modified Linggui Zhugan Decoction group . All of the animals were housed under the same conditions in a temperature-controlled room (24 ± 1°C) with a natural day/night cycle light and were given ad libitum access to standard chow and water for 4 weeks. All the experimental procedures were approved by the Institutional Animal Care and Use Committee of Guang’anmen Hospital, China Academy of Chinese Medical Sciences, in accordance with the regulations on the management and use of experimental animals.Male SD rats (200–220 g) were obtained from Beijing Huafukang Biotechnology Co., Ltd  2020-0004) and fed adaptively for 3 days. A MI model was induced through proximal left anterior descending coronary artery (LAD) ligation as has been described , 31, whiThe 10 drugs of the MLZD formula, including Radix astragali (21081961), Panax ginseng (21040161), Ramulus cinnamomic (21092511), Poria (21102131), Rhizoma atractylodis macrocephalae (21100541), Rhizoma alismatis (21090051), Radix salviae miltiorrhizae (21080651), Pericarpium areca (21081271), Semen lepidii (21071351) and Radix angelicae sinensis (21082271), were purchased from Jiangyin Tianjiang Pharmaceutical Co. Ltd.  or Sichuan New Green Pharmaceutical Science and Technology Development Co. Ltd. , which met the grade standards of the Chinese Pharmacopoeia. These drugs were provided by the Chinese Pharmacy of Guang’anmen Hospital, China Academy of Chinese Medical Sciences , and the voucher specimens of all drugs were deposited at the Cardiovascular Laboratory, Guang’anmen Hospital, China Academy of Chinese Medical Sciences . The clinical dose of MLZD was 190 g, with 30, 10, 10, 30, 15, 30, 20, 15, 15, and 15 g for Radix astragali, Panax ginseng, Ramulus cinnamomi, Poria, Rhizoma atractylodis macrocephalae, Rhizoma alismatis, Radix salviae miltiorrhizae, Pericarpium areca, Semen lepidii, and Radix angelicae sinensis, respectively. In reference to previous studies , the MLZAfter 28 days of continuous intragastric administration, the cardiac structure, and function of rats were determined by non-invasive transthoracic echocardiography in M-mode, implementing a Vevo-2100 high-resolution echocardiography system . Following being anesthetized with sodium pentobarbital (40 mg/kg), two-dimensional echocardiograms of the left ventricular (LV) long-axis were recorded at the level of the papillary muscle tips for detecting LV ejection fraction (LVEF), LV fractional shortening (LVFS), LV end-diastolic anterior wall thicknesses , LV end-systolic anterior wall thicknesses , LV end-diastolic internal diameters , LV end-systolic internal diameters , LV end-diastolic volume  and LV end-systolic volume . These echocardiographic parameters were obtained by averaging the corresponding parameters of three cardiac cycles.After echocardiography, the rats were immediately euthanized and their hearts were excised, cut off attachments, irrigated clean with cold saline buffer, measured for weight and size, and transected into two parts at the maximum transverse diameter. The upper part of cardiac tissue was fixed with 4% paraformaldehyde  at 4°C for examinations like histopathology and immunohistochemistry, and the apex part was stored at −80°C or fixed with 2.5% glutaraldehyde  for western blot analyses, transmission electron microscopy, and the like. The tetramethylrhodamine methyl ester (TMRM) staining should be performed immediately with fresh heart tissue.After being fixed in 4% paraformaldehyde overnight, myocardium, liver, and kidney specimens were dehydrated, rendered transparent, embedded with paraffin, and cut into 4-μm-thick transverse sections for hematoxylin and eosin (HE) and Masson staining. The specimens were eventually observed under an optical microscope to assess histopathological changes, and Image J software  was used to quantify the ratio of the blue-positive stained region to the entire surface, which reflected the severity of cardiac fibrosis.For detecting several crucial proteins through immunohistochemistry, the paraffin slices were boiled in an autoclave for 3 min to repair the antigen. The slices were afterward placed at 60°C for 2 h, deparaffinized, and hydrated with xylene and ethanol, followed by phosphate-buffered saline (PBS) and double-distilled water to wash the retrieved nuclear antigen. Following that, the samples were incubated with primary antibodies overnight at 4°C: SIRT3 antibody , anti-mitofusin 2 antibody , phospho-Drp1 (p-Drp1) antibody , anti-Bax antibody , and anti-Bcl-2 antibody . The samples were then interacted with HRP-conjugated goat anti-rabbit immunoglobulin G (IgG) at 37°C for 30 min, followed by staining with diaminobenzidine (DAB) detection kit .Apoptosis in cardiac tissue was eventually analyzed via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining using a TUNEL kit  according to kit protocols. Images were observed under a microscope , and the ratio of apoptosis in randomly selected visual fields was quantified using Image J software .3, fixed in 2.5% glutaraldehyde  for 24 h, and then washed in PBS. After that, the tissues were secondary fixed with 1% osmium tetroxide, dehydrated with graded ethanol, embedded in the ultra-thin epoxy part, sliced, uranyl acetate stained, and eventually photographed using a transmission electron microscope . The mitochondrial numbers and average mitochondrial areas were analyzed and quantified with Image J software.The ultrastructure of cardiomyocytes was observed utilizing a transmission electron microscope (TEM) as has been described –36. Brie2 atmosphere. Oxygen-glucose deprivation (OGD) was performed to emulate the MI model in vitro , at 37°C for 4 h. After that, the culture solution was replaced with standard culture media or treated with MLZD, in normoxic conditions for another 24 h. The cells were divided into seven groups: CON, OGD, 3-TYP , OGD+3-TYP, MLZD, OGD+MLZD (0.5 mg/ml), and OGD+MLZD+3-TYP. 3- pyridine (3-TYP) is a selective inhibitor of SIRT3  were cultured in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum  and 1% streptomycin-penicillin  at 37°C in a 5% COin vitro . Brieflyof SIRT3 , 40, and450 values in the control group, which was set at 100%.Cell viability was evaluated with the Cell Counting Kit-8 (CCK-8)  according to the manufacturer’s instructions. Briefly, H9c2 were inoculated in 96-well plates at a density of 3,000 cells/well and cultured for 48 h. Then, the cardiomyocytes were treated as described above. Hundred microliter mixture (90 μl DMEM+10 μl CCK8 solution) was then added to each well and incubated at 37°C for 1–4 h. The absorbance was eventually measured with a microplate reader  at 450 nm. Cell viability was expressed as the percentage of OD450 values were detected that correspond to the complex IV concentration.Concentrations of cytochrome c oxidase (Complex IV) in cardiomyocytes were determined using a Rat Cytochrome C Oxidase ELISA kit . The standard substance at known concentrations, cell supernatant and enzyme-conjugate reagents were pipetted into the wells of microplate strips, and incubated for 60 min at 37°C. Then bound enzyme and chromogenic substrate was added successively. Finally, the OD340 values in 10 s and 2 min were recorded, respectively, as A1 and A2. Complex I activity (U/mg prot) = 2680 × (A1 − A2)/Cpr. Where Cpr was the protein concentration of samples.According to the manufacturer’s instructions of Mitochondrial Complex I Activity Detection Kit , cells were collected to the extracting solution for homogenization, centrifugation, and ultrasonic crushing. After adding samples and detection reagents into the 96-well plate, the ODBased on the instructions of Adenosine Triphosphate (ATP) Chemiluminescence Assay Kit , the cells were collected and mixed in the extracting solution, then bathed in boiling water for 10 minutes, centrifuged, and diluted of the supernatant. After adding enzyme reagents, standard solutions, and samples into the 96-well enzyme label plate, the fluorescence values of each well were measured on the chemiluminescence detector.Mitochondrial membrane potential was detected by tetramethylrhodamine methyl ester(TMRM) staining as described in a previous study . Fresh mA total of 20 μg of protein extract from myocardial tissues and cardiomyocytes was separated using a 10% Omni-easy™ one-step PAGE gel rapid preparation kit  and then transferred to the 0.22 or 0.45 μm PVDF membranes . The PVDF membranes were subsequently blocked with 5% nonfat dry milk  at room temperature for 2 h and incubated with primary antibodies at 4°C overnight: anti-SIRT3 antibody , anti-SOD2/MnSOD antibody ,anti-mitofusin 2 antibody  or anti-mitofusin 2 antibody , phospho-Drp1 (p-Drp1) antibody , Drp1 antibody , COX IV antibody , PGC-1α antibody , PPAR-γ , cytochrome c antibody, cleaved caspase-3 antibody , anti-Bax antibody, anti-Bcl-2-antibody , NLRP3 , ASC , caspase-1 , NFκB p50 , NFκB p65  and IL-1β . We next incubated the PVDF membranes with the secondary antibody HRP-conjugated goat anti-rabbit IgG (H&L)  or goat anti-mouse IgG (H&L)  at room temperature for 1 h. The gels were finally visualized utilizing Gel Doc™ XR+ System  and Image Lab Software  with clarity max western ECL substrate , followed by quantitative analysis with Image J software, and the results expressed as density values were normalized to GAPDH or tubulin.P values <0.05 considered to be statistically significant.Graphpad Prism software (version 8.0) was used for statistical analysis, and the detection was repeated at least three times independently. The data were presented as the mean ± standard deviation revealed slightly enlarged hearts in MI rats and improved conditions after MLZD intervention , B. ThroW revealeTo further verify MI-induced myocardial damage and the role of MLZD in this process, we examined whether apoptosis occurred and its related pathways. As shown by results for TUNEL staining in the heart tissue , B, the To verify the regulating effect of MLZD on myocardial inflammation, inflammation-related proteins including NLRP3, caspase-1, ASC, IL-1β, NFκB p65, and NFκB p50 in the myocardial tissue of rats in three groups were detected. As shown in To examine the effects of MLZD on mitochondrial morphology and dynamics changes, mitochondrial ultrastructure was observed through an electron microscope, and Mfn2, p-Drp1, PGC-1α, and PPAR-γ expression were detected by western blot or immunohistochemistry. Intact outer membrane and dense cristae of mitochondria were mainly shown in the normal group, while more abnormal mitochondrial morphologies were provoked in the MI rats along with decreased mitochondria volume, disarrayed cristae, and swollen matrix. The MLZD treatment gradually recovered the mitochondrial structure . FurtherTo clarify the possible pathways by which MLZD ameliorates mitochondrial abnormalities, the relative protein expression of SIRT3 in the cardiac tissue was detected considering its regulation of mitochondria. The results revealed the downregulated generation of SIRT3 in the MI rats compared with the healthy ones, while treatment of MLZD blocked the effect . These rTo determine the role of SIRT3 in MLZD-mediated cardioprotection, H9c2 was treated with 3-TYP, a selective inhibitor of SIRT3. The expression level of SIRT3 and cell viability of treated cardiomyocytes were detected. As shown in The effects of MLZD on mitochondria in OGD cells and the role of 3-TYP in this process were evaluated by detecting Drp1, p-Drp1, Mfn2, PGC-1α, cytochrome c oxidase subunit 4 (COX IV), mitochondrial electron transport chain complex I and IV, ATP content, and MMP. The results showed that in OGD model cells MLZD alleviated increased Drp1 and p-Drp1 expression, while it promoted Mfn2, PGC-1α, and COX IV expression. However, this effect was reversed by the SIRT3 inhibitor 3-TYP ,B. And 3Due to a lack of effective interventions, MI and MI-caused ventricular remodeling have become serious health threats worldwide. As a complementary and alternative medicine, TCM has great potential to alleviate this health burden. In this pharmacological study through in vivo experiments in rats, we found that MLZD protected against post-MI ventricular remodeling, in part by alleviating mitochondrial injury and mitochondrial-associated apoptosis. Further experimental verification revealed that the cardioprotective mechanism of MLZD may be exerted partly by promoting SIRT3 expression.In vitro experiments, additionally, we first examined the viability of H9c2 cardiomyocytes treated with MLZD alone without OGD, and subsequently tested the effects of MLZD on OGD, finding that MLZD had a protective effect on cell viability  is a prominent deacetylase mainly found in mitochondria and influences almost all the main aspects of mitochondrial function . It is iEvaluating the collective evidence, it indicated that MLZD treatment could ameliorate post-myocardial infarction ventricular remodeling by inhibiting apoptosis induced by mitochondrial abnormalities. This research described a promising therapy for MLZD, indicating that it prevented apoptosis by protecting mitochondria, including ameliorating mitochondrial structural disruption, protecting against mitochondrial dynamics disorder, restoring impaired mitochondrial function and inhibiting inflammation, which may be exerted by promoting SIRT3 expression . MLZD miThe original contributions presented in this study are included in the article/This animal study was reviewed and approved by Institutional Animal Care and Use Committee of Guang’anmen Hospital, China Academy of Chinese Medical Sciences.MX wrote the main text. XZ, YL, YZ, FD, LLv, YW, and ZS retrieved and organized the documents. XC and LLi made great contributions to revisions, polishing this manuscript, and with revising figures. All authors contributed to the article and approved the submitted version.


To identify the potential key genes and molecular pathways associated with keratoconus and allergic disease.  The pubmed2ensembl database was used to identify the text mining genes (TMGs) collectively involved in keratoconus and allergic disease. The GeneCodis program was used to perform the Gene Ontology (GO) biological process and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of TMGs. The protein-protein interaction (PPI) network of the TMGs was established by STRING; the significant gene modules and hub genes of PPI were further performed using the Cytoscape software. The DAVID database was used to perform the GO and KEGG analyses of the significant module.  In total, 98 TMGs collectively involved in keratoconus and allergic disease were identified. 19 enriched biological processes including 71 genes and 25 enriched KEGG pathways including 59 genes were obtained. A TMG PPI network was constructed, and 51 genes/nodes were identified with 110 edges; 3 most significant modules and 12 hub genes were chosen from the PPIs. GO enrichment analysis showed that the TMGs were primarily associated with collagen catabolic process, extracellular matrix organization and disassembly, cell adhesion and migration, collagen-containing extracellular matrix, extracellular matrix, and structure organization. KEGG pathway analysis showed that these DEGs were mainly involved in the IL-17 signaling pathway, inflammatory bowel disease, rheumatoid arthritis, allograft rejection, T cell receptor signaling pathway, cytokine-cytokine receptor interaction, and TNF signaling pathway. IL10, IL6, MMP9, MMP1, HGF, VEGFA, MMP3, MMP2, TGFB1, IL4, IL2, and IFNG were potential key genes involved in keratoconus. IL-17 signaling pathway was the potential pathways accounting for pathogenesis and development of keratoconus. The results revealed that  Keratoconus (KC) is a corneal ectasia disease characterized by thinning and steepening, which would cause irregular astigmatism and progressive myopia, leading to further loss of vision . KC is cAt present, various treatments for KC exist, including spectacles, contact lenses, corneal collagen cross-linking (CXL), and corneal surgery. However, these treatments present several limitations. In its early stages, spectacles or rigid contact lenses only improve the visual impairment but cannot delay or prevent the degeneration of KC . CXL canKC is a multifactorial disease that involves several genes and environmental factors . AllergiCorneal stroma, which consists of keratocytes and extracellular matrix (ECM), is the main structural fraction of the cornea accounting for 90% of corneal thickness . And theText mining, an effective method to quickly extract critical information from a large amount of the biomedical literature, has been widely used to explore novel associations between genes and pathologies . In recehttp://pubmed2ensembl.ls.manchester.ac.uk/) [The pubmed2ensembl website (.ac.uk/)  is an onP value cut-off (P = 0.00001) was set.The GeneCodis  was usedhttps://string-db.org/), a database includes 3 billion interactions associated with 24.6 million proteins referred to 5090 organs [In order to assess functional associations among the products of the selected TMGs, the STRING  was used to visualize the PPI network . Then, tMoreover, to select hub genes from the PPI network, the cytoHubba plugin of Cytoscape was used, which was computed by four ranking methods: EPC (edge percolated component), MCC , MNC , and DMNC (density of maximum neighborhood component) . The tophttps://david.ncifcrf.gov/) was used to perform GO enrichment analysis and KEGG pathway enrichment analysis. The GO enrichment analysis includes biological process (BP), cellular component (CC), and molecular function (MF) [P < 0.05 was considered statistically significant.To clarify the functions of the significant genes module, the DAVID database (ion (MF) . The funion (MF) . P < 0.0Based on the data mining strategy that is described in The 98 TMGs were analyzed for GO biological processes (BP) using GeneCodis to identify the most enriched terms closely related to the pathology of keratoconus. As shown in Next, the 71 TMGs were further analyzed for KEGG pathways using GeneCodis. The KEGG pathway enrichment analysis identified 25 significant pathways that involved 59 TMGs. The top 10 KEGG pathway contained malaria, allograft rejection, rheumatoid arthritis, cytokine-cytokine receptor interaction, pathways in cancer, AGE-RAGE signaling pathway in diabetic complications, graft-versus-host disease, type I diabetes mellitus, and inflammatory bowel disease .The PPI network of the 59 selected TMGs was constructed using the STRING database, which had a total of 51 nodes with 110 edges . FurtherIL10, IL6, MMP9, MMP1, HGF, VEGFA, MMP3, MMP2, TGFB1, IL4, IL2, and IFNG, were considered to be hub genes  family, which is composed of IL-17A–F, plays a crucial role in acute and chronic inflammatory reactions . IL-17 hd C/EBPs . Gomes ed C/EBPs . Karolakd C/EBPs . The prod C/EBPs . Therefoβ includes three isoforms in mammals, TGF-β1, TGF-β2, and TGF-β3, and is a key mediator of fibrogenesis [β markers in severe keratoconus patients [β contributes to upregulate the expression of MMP2 by modulating Smad2 [β. These genes are the potential key genes that may be involved in KC.Interleukin-10 (IL-10) is an anti-inflammatory cytokine with important roles in preventing T helper type 1 cells from producing cytokine. Several studies have shown that there was no obvious change in IL-10 in tears of keratoconus and control subjects –53, whilogenesis . A recenpatients . It was ng Smad2 . Interfeng Smad2 . It was Strengths of our study include the fact that it is one of the first to use text mining and bioinformatics analysis to identify the potential crucial genes and key pathways of KC based on the common genes involved in KC and allergic disease. Limitations of this study must also be acknowledged. Firstly, this study explored the molecular mechanism of keratoconus in the gene level using bioinformatics analysis; further experimental studies are required to verify the results. Secondly, adolescents' population is a high-risk group for KC, but we did not include the age factor in this research; future investigations taking the age into consideration may provide more accurate and comprehensive conclusions.IL10, IL6, MMP9, MMP1, HGF, VEGFA, MMP3, MMP2, TGFB1, IL4, IL2, and IFNG, that may be involved in the keratoconus as well as in allergic diseases. These genes were enriched in the HIF-1 signaling pathway, T cell receptor signaling pathway, and TNF signaling pathway. Extracellular matrix remodeling and immune inflammatory response may be the key alterations in KC. The absence of experimental validation is a limitation of this study, and further studies are needed.In conclusion, we identified 12 hub genes,


TNFRSF13C, a gene encoding B-cell activating factor receptor (BAFFR). Significant reduced blood B-cell numbers and BAFFR levels were observed in line with a dysregulation in BAFF–BAFFR signaling. The elevated frequency of PD-1+ dysfunctional Tfh cells composed predominantly by Th1 phenotype was observed at disease onset and during follow-up. This case report describes a patient progressing to T1D on a BAFFR-mediated immunodysregulatory background, suggesting a role of BAFF–BAFFR signaling in islet-specific tolerance and T1D progression.The immunological events leading to type 1 diabetes (T1D) are complex and heterogeneous, underscoring the necessity to study rare cases to improve our understanding. Here, we report the case of a 16-year-old patient who showed glycosuria during a regular checkup. Upon further evaluation, stage 2 T1D, autoimmune thrombocytopenic purpura (AITP), and common variable immunodeficiency (CVID) were diagnosed. The patient underwent low carb diet, losing > 8 kg, and was placed on Ig replacement therapy. Anti-CD20 monoclonal antibody  was administered 2 years after diagnosis to treat peripheral polyneuropathy, whereas an atypical mycobacteriosis manifested 4 years after diagnosis and was managed with prolonged antibiotic treatment. In the fifth year of monitoring, the patient progressed to insulin dependency despite ZnT8A autoantibody resolution and IA-2A and GADA autoantibody decline. The patient had low T1D genetic risk score (GRS = 0.22817) and absence of human leukocyte antigen (HLA) DR3/DR4-DQ8. Genetic analysis identified the monoallelic mutation H159Y in  Type 1 diabetes (T1D) is a disease of multifactorial origin caused by the autoimmune destruction of insulin-producing pancreatic β cells. Several immune players have been identified as contributors to the disease immunopathogenesis, involving both the innate and adaptive arms of the immune system . T cellsINS, PTPN22, IL2RA, IFIH1, and CTLA4, are considered additional contributing genetic factors  in non-HLA genes, such as  factors , 9. Rece factors , 11.Common variable immunodeficiency (CVID) is a heterogenous disease classified as predominantly antibody deficiency , with a A healthy 16-year-old man with a Caucasian ethnic background underwent a medical visit for a pre-participation sport evaluation. As part of the checkup, urinalysis was performed, resulting positive for glycosuria (99 mg/dl) but negative for ketones. Biochemical analysis revealed the presence of prediabetes  associated with mild thrombocytopenia  and microcytemia (MCV 78 fl) that was treated with iron supplementation for 1.5 months. Of note, glycosuria (252 mg/dl), not further addressed, and a platelet count at the lower limit of normal  were present at the age of 12 years, according to his medical records. Stage 2 T1D was diagnosed by the presence of three islet AAbs , dysglycemia , glucose intolerance ), and a partially impaired insulin secretion . Family history included autoimmune Hashimoto’s thyroiditis (treated with levothyroxine) (father), anti-thyroid peroxidase antibodies (younger brother), and T2DM . No signs of celiac disease, atrophic gastritis, or autoimmune thyroid disease were found in the patient. A low-carb diet was recommended with a consequent decline in weight (> 8 kg in a 3-month period) and blood glucose normalization. Concomitant to stage 2 T1D, immune thrombocytopenia (ITP) , and hypogammaglobulinemia  were diagnosed . Bone maDuring a 5-year follow-up, he did not experience any ITP relapses and his platelet count remained stable between 100,000 and 150,000/µl.Two years after CVID and T1D stage 2 diagnosis, the patient was admitted to the hospital for asymmetric axonal sensitive polyneuropathy, probably triggered by CMV infection, which was managed with high-dose IVIg, RTX, Pregabalin, and Duloxetin. Steroids were not considered due to his comorbidities . Neurological improvement occurred with a mild persistence of sensitive alterations.Two years later, an atypical mycobacterial pulmonary infection associated with generalized lymphadenopathy and worsening splenomegaly was discovered and treated with long-time pluri-antibiotic therapy.The patient remained insulin free for 4 years after the initial prediabetes diagnosis when the dysglycemia evolved into stage 3 T1D (at 21 years of age) marking the start of insulin therapy.Despite receiving three doses of the anti–SARS-CoV-2 vaccine (the last dose in December 2021) and showing a good humoral and cellular response , the patThe complete timeline from the time of diagnosis (07/2016) to now is shown in BAFFR (H159Y) was identified and confirmed by Sanger sequencing. The mother carried the wild-type allele, whereas the father carried the same mutation. Additionally, T1D GRS was calculated by typing 30 common HLA and non-HLA genetic variants associated with T1D, as previously described (reg) surface as compared with HC. BAFFR decrease was more pronounced in B cells (MFI reduction 82.1%) than in T cells  (The index patient underwent genetic screening by whole exome sequencing (WES). A monoallelic mutation in escribed . The indThe diagnosis of CVID was confirmed by the patient’s immunological profile. The patient showed mild lymphopenia with a global decrease and altered distribution of the B- and T-cell compartment already at disease onset and during follow-up as compared with age- and gender-matched healthy donors (HC) Table 1.+CD4+ cells, the frequency of Circulating T follicular helper cell (cTfh) (CXCR5+FoxP3-), Circulating T follicular regulatory cell (cTfr) (CXCR5+FoxP3+), and cTreg (CXCR5-FoxP3+) cells was determined. While cTfr cell frequencies in the patient were within the normal range, cTreg cell frequencies were within the lower range at first but returned to average normal values in subsequent FUs   (reg cells (CD25+CD127-/loFoxP3+) and FoxP3 levels (γMFI) in the proband were reduced at disease (By assessing the expression of CXCR5 and FoxP3 among CD312–5.68) Table 1.+CCR6-) cells at the expense of the Tfh2 and Tfh17 subsets  , whereas ICOS+ cTfh cell frequency remained higher since disease onset   . The frequency of B memory cells (CD19+CD27+) was lower than HC . Potentially autoreactive B cells defined as CD19+CD21lowCD38low B cells  and heterologous settings (CVID B cells with HC Tfh cells or vice versa) and the percentage of CD20-CD38+ plasmablasts together with IgM and IgG levels were evaluated after a week . On the other hand, IgM production by B naïve cells was similar to HC . Tfh cells co-cultured either with autologous or heterologous B naïve cells were unable to induce class switching and IgG production in vitro  and non-Tfh (CD4+CXCR5-) cell functional status was also evaluated in vitro by intracellular cytokine profile. Total PBMCs were activated with PMA/Ionomycin and the expression of interferon-γ (IFN-γ), interleukin-17 (IL-17), and IL-21 was evaluated by flow cytometry (FC) . On the contrary, higher frequencies of IFN-γ and IL-21 producing cells were observed within the CXCR5- compartment (Tfh (CD4 with HC Table 1.BAFFR (H159Y) inherited from the father. Two years after CVID and T1D stage 2 diagnosis, RTX was administered to treat peripheral polyneuropathy with a potentially positive impact on diabetes progression. Additional diet adjustment (hypoglycemic/ketogenic) led to an 8-kg weight loss that possibly impacted the disease course. Eventually, the patient progressed to insulin dependency, despite a decline in islet AAbs levels. The patient displayed the typical immunological signs of CVID, that is, reduction in circulating B cells, switched memory B cells, and an increase in autoreactive CD21lowCD38low B cells. B cell counts remained low during a 5-year FU. The patient was positive for SARS-CoV-2 antibodies prior to infection and vaccination, probably secondary to the presence of these antibodies in IVIg. After receiving three vaccine doses and natural SARS-CoV-2 infection, his anti–SARS-CoV-2 antibodies remained detectable. Generally, CVID patients, especially those with autoimmunity, have variable alterations in humoral responses against vaccines, including against SARS-CoV-2, that could account for a low specific response to some infections and vaccination . Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.BL and LPac contributed equally. Conception and design: BL, LPac, CCa, and GF. Development of methodology: BL, LPac, and GM. Acquisition of data: BL, LP, GM, TJ, SC, JG, IMa, IMe, MB, EZ, BR, CCi, FB, CG, PZ, NR, AD, GA, LPas, PC, PP, AF, VL, PC, and RS. Analysis and interpretation of data: BL, LPac, SG, MPC, GDM, AA, LPi, CCa, and GF. Writing, review, and revision of the manuscript: BL, LPas, SG, GA, LPa, MPC, GDM, AA, LPi, CCa, and GF. Study supervision: GF. All authors contributed to the article and approved the submitted version.This work was supported from 5x1000 OSR pilot & seed grant and GR-2016-02365089 to GF and MPC.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


RIRI alters the balance of both axes so that RAS can affect RIRI-induced AKI. In overall, the alteration of Ang II/AT1R and AKI by RIRI is important to consider. This review has looked for the effects and interactions of RAS activities during RIRI conditions.Renal ischemia-reperfusion injury (RIRI) is a sequence of complicated events that is defined as a reduction of the blood supply followed by reperfusion. RIRI is the leading cause of acute kidney injury (AKI). Among the diverse mediators that take part in RIRI-induced AKI, the renin-angiotensin system (RAS) plays an important role via conventional , angiotensin (Ang) II, and Ang II type 1 receptor (AT Renal ischemia-reperfusion injury (RIRI) is a sequence of complicated events that is defined as a reduction of the blood supply followed by the recovery of perfusion and reoxygenation to the kidney . The mos1R), and the nonconventional RAS include angiotensin 1-7 (Ang 1-7), angiotensin 1-9 (Ang 1-9), ACE2, AT2R, and MasR [1R and ACE2/Ang 1–7/MasR axes [RAS is a hormonal system with coordinated effects on the heart, vessels, and kidneys, and it is involved in regulating blood pressure and homeostasis of fluid and electrolytes , 20. Theand MasR . Many stasR axes , 24. HenAGT is a glycosylated protein that is mainly produced by the liver, and it is secreted into plasma. However, intrarenal AGT mRNA and protein are located primarily in proximal tubular cells, and AGT can be secreted to the tubular lumen , 26. AGTβ1 adrenergic receptors in the sympathetic nervous system [Renin, a protease synthesized by the juxtaglomerular apparatus of the afferent arteriole which is secreted in response to hypotension detected by baroreceptors, decreased the sodium delivered to distal nephrons and activation of the s system . Renin cs system . RIRI was system . Increass system . The rens system . Renin ps system , 40, 41.1R and AT2R. There are two subtypes of AT1R (AT1a and AT1b) in rodents. AT1R is expressed in the proximal tubule brush border, basolateral membranes and the cortical CD of the kidney, vascular smooth muscle cells of all renal vascular segments, including the afferent and efferent arterioles [Ang I, made from AGT, is converted to Ang II by an Ang-converting enzyme (ACE) in lung endothelial cells . In additerioles , 47. Locterioles , 27.1R antagonist) at various dosages did not protect against RIRI lesions. These results indicate that the effects of captopril or enalapril on RIRI are not mediated by the AT1R, and it may be mediated by AT2R or mechanisms independent of Ang II. The findings of this investigation support earlier results on using ACEIs, captopril, or enalapril to prevent RIRI in rats. The ACEIs reduce bradykinin breakdown, which leads to B2 kinin receptor activation and vasodilation. This impact is thought to be caused by the generation of NO [Ischemia, or a reduction in blood flow, is the main cause of many serious disorders, including organ transplantation. According to animal studies, reperfusion of ischemic regions, particularly reoxygenation, adds to further tissue injury (reperfusion injury) , 49. RIR1R contribute to the effects of alcohol on the myocardium through oxidative stress damage, the mechanism of which may be achieved by regulating nicotinamide adenine dinucleotide oxidase (NOX) [1R expression reduction was indicated in the ischemic kidney at 24 h reperfusion and recovery to sham levels at 72 h reperfusion [It is interesting that in other organs, such as the heart, other effects were seen in this regard. A study in alcoholic cardiomyopathy (ACM) shows that Ang II and ATse (NOX) . Ang II se (NOX) , renal mse (NOX) . Numerouse (NOX) , 53, 54.se (NOX) . AT1R ex1R in the damaged kidney upregulates several proinflammatory genes and also develops tubulointerstitial fibrosis [1R inhibition by losartan before RIRI reduces inflammatory factors such as interleukin 1 beta (IL-1β), interleukin-17 (IL-17), tumor necrosis factor-α (TNF-α) and fibrosis induced by RIRI, and as a result, tubular injury and vascular obstruction improve within 24 h of reperfusion [α) [α (oxygen-sensitive subunit of HIF), which is primarily expressed in tubular epithelial cells, by losartan as a renoprotective mechanism can reduce renal damage in RIRI [1R promotes autophagy in renal proximal tubular cells and increases renal tolerance to RIRI, thereby protecting the kidneys against RIR [1R antagonist, has shown a renoprotective effect in RIRI; telmisartan pretreatment significantly decreased blood urea nitrogen (BUN) and serum creatinine (Cr) levels, inhibited the depletion of the antioxidant factors, and decreased lipid peroxidation induced by RIRI in the kidney [After RIRI, the vasoconstrictor effect of Ang II decreases, possibly due to receptor reduction in vascular and tubular regions of the ischemic kidney . In RIRIfibrosis , but AT1erfusion –58. Moresion [α) , 58. Uprsion [α) . Activat in RIRI . Indeed, in RIRI . This ef in RIRI . Autopha in RIRI , 62. Zha in RIRI . In the e kidney .1R signalling, plays a role in vascular homeostasis and injury, it is unclear whether clinically relevant endogenous-biased antagonism of AT1R signalling exists in physiological and pathophysiological circumstances. Explorations of the endogenous-biased antagonism of AT1R or other GPCRs may provide innovative treatment options for cardiovascular disorders [Although compelling evidence indicates that biased activation of G protein-coupled receptor (GPCR) signalling, like ATisorders .ACE is a central component of the RAS, which converts the inactive Ang I to the active peptide Ang II and metabolizes other vasoactive peptides, such as bradykinin . ACE als2R is another functional Ang II receptor in RAS, and it is similar in molecular structure to the G protein-coupled receptor superfamily, which consists of 7 transmembrane regions [2R encodes a protein of 363 amino acids with molecular weight of 41,220 Da; 34% of the amino acid sequence of AT2R is homologous with AT1R [2R mRNA is not expressed in adult animals, the AT2R protein has been detected in fetal and newborn rat kidneys by using immunohistochemistry and Western blot analysis [2R was observed mainly in the vascular and tubular parts, especially the proximal tubule, collecting duct, afferent arterioles, arcuate arteries, and outer medullary descending vasa recta [2R mRNA was detected in the tubular organization, vessels, and glomeruli [2R neutralizes most of the AT1R actions by suppressing cell proliferation and differentiation [1R via increasing the formation of kinins , NO, and guanosine cyclic 3′5′monophosphate (cGMP) and eventually dilating blood vessels and lowering blood pressure [1R and AT2R activation can play an important role in regulating the physiological functions of the renal and cardiovascular systems [2R antagonists and agonists have shown a protective role for this receptor in RIRI [2R increased significantly [2R blockade by PD123319 increased the RBF response to Ang II infusion in a dose-related manner in female rats [2R due to the increased NO production [α [2R activation exerts anti-inflammatory effects and protects organs from injuries, including AKI [2R, inverted these changes. Also, C21 increased CD4+FoxP3+ (regulatory T cells) and CD4+IL-10+ cells and decreased Cr, BUN, NGAL, and cell-associated kidney injury molecule-1 (KIM-1) as the markers of renal epithelial injury. These results indicate the role of AT2R in protecting kidneys against RIRI [AT regions . The cDNith AT1R . Althouganalysis . The rensa recta , 80. In lomeruli . The stintiation , 83. It pressure , 82–85.  systems , 82, 86. in RIRI , 88. In ficantly . After Rale rats , which moduction . Infiltroduction . RIRI cading AKI . Treatme2R in RIRI increased and its stimulation had beneficial renal effects. C21 pretreatment reduced renal dysfunction and preservation of tubular architecture, while PD123319 pretreatment decreased renal function in rats subjected to RIRI [2R induced renoprotective effect against AKI induced by RIRI [2R activation with agonist exerts a protective effect in RIRI-related cardiohepatic dysfunction as showed by inhibited oxidative stress, downregulated inflammatory cytokines such as IL-6, IL-1, IL-17A, monocyte chemoattractant protein-1 (MCP-1), and TNF-α, and improved cardiohepatic depressor arm of RAS under diabetes mellitus (DM) and nondiabetes mellitus conditions [Lack of cytoskeletal integrity and interruption of intercellular junctions associated with ATP depletion take place in AKI . The exp to RIRI , 95. Dow by RIRI . AT2R acnditions .ACE2, as the main component of the ACE2/Ang1-7/MasR axis, is a multifunctional enzyme that exerts its physiological effect through interaction with Ang I and Ang II peptides . ACE2 caα, chemokines, monocyte chemoattractant protein-1 (MPC-1), apoptosis, and oxidative stress increased following RIRI [1R activation [Available studies have proven a link between reducing renal mRNA expression or activity of ACE2 in RIRI , 106–108ing RIRI , 111. Thing RIRI . The bening RIRI –114. In tivation .Ang 1–7 is a biologically active heptapeptide found in the circulation and many tissues, such as the heart and kidney . The pro−/− animals had reduced RBF, increased renal vascular tone, and increased GFR and urinary albumin excretion [+ accumulation, and hypertension and generates structural and molecular modifications that promote renal fibrosis [MasR, a functional receptor axis of the RAS presser, is found in multiple tissues, including the brain, heart, kidney, lung, liver, spleen, tongue, testis, and skeletal muscle . In the xcretion . The genfibrosis .−/−) did not alter the degree of renal dysfunction and inflammatory response, such as the number of systemic and renal neutrophils [RIRI in mice injected with Ang1-7 caused more significant tissue damage, which was revealed by infiltration of inflammatory cells, more severe diffuse matrix deposition, intraglomerular protein deposition, and increased cellularity . The gentrophils . These rtrophils .1R, the renoprotective role of ACEIs and the AT1R blockers in the deleterious effects of activation of this axis against ischemic AKI has been demonstrated. In the RIRI situation, Ang II via stimulation of the AT1R in the damaged kidney upregulates several proinflammatory genes and also develops tubulointerstitial fibrosis; so the use of losartan (AT1R antagonist) before RIRI improves ischemic damage by reducing inflammatory factors and apoptosis and improving renal hemodynamic response (increased RBF). In addition, ACEIs through antioxidant, antiapoptosis, and anti-inflammatory effects and also by decreasing protein oxidation, lipid peroxidation, and acute tubular injury play a protective role against RIRI-induced renal damage. Although the studies on the components of the nonconventional axis (ACE2/Ang 1-7/Ang 1-9/AT2R and MasR) of the RAS are less than those of the conventional axis, using activators and inhibitors has shown a protective role for the ACE2, AT2R, and MasR against AKI induced by RIRI. The expression of AT2R and MasR in RIRI is increased, which may be a compensatory mechanism for the kidney to protect itself against ischemic injury. Thus, further understanding of the role of RAS components by focusing on identifying the signalling pathways of inflammation, apoptosis, oxidant, functional impairment, and hemodynamic responses due to activation of the RAS in RIRI can play an important role in the use of agonists/antagonists or activators/inhibitors of these components against AKI caused by RIRI.Recent studies have identified RAS activation as an influential factor in RIRI-induced AKI. Investigation of RAS components, especially its receptors using its agonists and antagonists, has played an important role in understanding the function of this system in RIRI-induced AKI. In the conventional RAS containing Ang II/ACE/AT


Although multiple actionable genes have been identified in melanoma, 38–42% of patients are still not druggable based on current research. In our study, DNA-NGS and RNA-NGS were utilized to construct molecular profiles of a Chinese cohort of 469 melanoma patients. Up to 11.7% (7/60) of patients in the undruggable group could be recognized as actionable by DNA and RNA sequential sequencing. Additionally, the use of RNA-NGS enhanced the proportion of druggable fusions from 2.56% to 17.27%. In total, the use of RNA-NGS increased the druggable proportion from 75% to 78%. Our study systemically analyzed the genetic landscape of Asian melanoma and demonstrated how DNA and RNA sequential sequencing is essential in bringing clinical benefits to more melanoma patients.n = 469) were sequentially sequenced by DNA-NGS and RNA-NGS. The genomic alterations were determined, and potentially actionable targets were investigated. Results: Patients with potential druggable targets were identified in 75% of Chinese melanoma patients by DNA-NGS based on OncoKB, which was much higher than in a previous Asian study. NRG1 fusions were first identified in melanoma. In addition, up to 11.7% (7/60) of patients in the undruggable group could be recognized as actionable by including RNA-NGS analysis. By comparing the fusion detection rate between DNA-NGS and RNA-NGS, all available samples after DNA-NGS detection were further verified by RNA-NGS. The use of RNA-NGS enhanced the proportion of druggable fusions from 2.56% to 17.27%. In total, the use of RNA-NGS increased the druggable proportion from 75% to 78%. Conclusions: In this study, we systemically analyzed the actionable landscape of melanoma in the largest Asian cohort. In addition, we first demonstrated how DNA and RNA sequential sequencing is essential in bringing clinical benefits to more patients with melanoma.Background: In contrast to Caucasian melanoma, which has been extensively studied, there are few studies on melanoma in Asian populations. Sporadic studies reported that only 40% of Asian melanoma patients could be druggable, which was much lower than that in Caucasians. More studies are required to refine this conclusion. Methods: Chinese melanoma patients ( BRAF V600E could be targeted with dabrafenib and trametinib, which have been approved by the FDA for pan-cancer treatment [NRTK fusions could be inhibited by TRK inhibitors [The morbidity rate of melanoma has rapidly increased in recent years, causing more than 57,000 deaths worldwide in 2020 . DNA-basreatment ,9. For ehibitors ,11,12,13Although multiple actionable genes have been identified in melanoma, 38–42% of patients are still not druggable based on current research ,15. In nTo identify underlying genomic targets with clinical significance, DNA-NGS and RNA-NGS were utilized to construct molecular profiles of a Chinese cohort of 469 melanoma patients. We systematically delineated the molecular genetic landscape of Chinese melanoma patients and stratified patients into druggable or undruggable groups based on actionable genes, TMB status, and MSI status. Furthermore, potential actionable genes were investigated in the undruggable group, and we demonstrated how RNA sequencing is essential in bringing benefits to more melanoma patients.Tumor histological specimens  obtained from multi-centers ) between March 2018 and May 2020 were used for NGS detection. This study has been approved by the Ethics Committee of the Zhejiang Cancer Hospital (IRB-2021-3). All patients were diagnosed via pathology, imaging, and clinical findings.Genomic DNA was extracted from tumor samples using a QIAamp DNA FFPE Tissue Kit  and fragmented to construct a library using KAPA Hyper Prep kits . DNA fragments of 200~400 bp were purified by an Agencourt AMPure XP Kit . Fragmented genomic DNA libraries were constructed with the KAPA HTP Library Preparation Kit  according to the instruction manual. After determining the concentration by a Qubit dsDNA HS Assay kit, DNA libraries were analyzed using Onco PanScan™ , which is an 825-gene panel that includes major tumor-related genes.Total RNA was isolated from FFPE samples using the AllPrep DNA/RNA Mini Kit . The RNA quality and quantity were determined by the Qubit RNA HS Assay  and the 4200 TapeStation System , respectively. Qualified RNA was used to synthesize double-stranded cDNA using a SuperScript VILO cDNA Synthesis kit  and an Abclonal Second Strand Synthesis Module  according to the recommended protocol. To prepare genomic libraries, the double-stranded cDNA was fragmented and constructed by a KAPA HTP Library Preparation Kit . The prepared libraries were captured with FusionCapture™ , which contains 395 cancer-related genes, and then subjected to Illumina NovaSeq6000  for paired-end sequencing. The sequencing reads were mapped to UCSC hg19 through HISAT2-2.0.5, and FusionMap was used to identify gene fusions.All statistical analyses were conducted using GraphPad Prism (version 8.02) software. The results are expressed as the mean ± SEM. The data were analyzed using a t test or one-way ANOVA, followed by the appropriate post-hoc tests .BRAF mutation, RAS mutation , and NF1 mutation, which are included in the KRAS/RAF pathway, were found in 120 (26%), 115 (25%), and 46 (10%) samples, respectively. Mutations in PTEN and PIK3CA from the PIK3CA pathway were identified in 28 (6%) and 14 (3%) patients, respectively. The genes from the CDKN2A pathway were also analyzed, and TP53 , CDK4 , MDM2 , and CDKN2A  were the most frequently mutated genes. Furthermore, two receptor tyrosine kinase fusion genes not related to the above pathways, ALK and NRG1, were found in our cohort, accounting for 1.5% (n = 7) and 0.6% (n = 3), respectively. In addition, other top six mutated genes in the cohort were KIT , CCND1 , FAT3 , LRP1B , MYC (5%), and TERT (4%) (Melanoma samples were obtained from 256 men (55%) and 213 women (45%) with ages ranging from 5 to 89 years (average age = 57 years). Collectively, 469 resected melanoma samples were screened by DNA sequencing. Numerous pathways were involved in the landscape. The ERT (4%) A. Apart http://oncokb.org/, accessed on 1 January 2022). Seventy-five percent (352/469) of patients had actionable genes after matching those genes with actionable mutations in OncoKB. The druggable patients were then grouped based on the highest level of drug response evidence in OncoKB. Approximately 47% (164/352) of patients could be classified as OncoKB level 1. This was followed by level 2 , level 3 , and level 4   B.BRAF, NRAS, and NF1 mutations were the top three in the SNV/Indel group, accounting for more than half of the total. In the CNV group, the proportions of CDK4 and MDM2 amplification were obviously predominant. Only three druggable tyrosine kinase fusion genes  were identified at the DNA level, and the BRAF fusions were dominant  in the fusion group. Furthermore, the top genes in the three groups were analyzed, and the number of gene mutations was counted based on the highest actionable level from OncoKB. The top actionable mutation genes in the study were BRAF, NRAS, NF1, and CDK4. Most of the genes only had one highest level due to a single variation. However, BRAF, FGFR1, and KIT presented two highest levels because of various mutations /Indel, CNV (copy number variant), and fusion, which were found in 85% (299/352), 17% (59/352), and 3% (10/352) of patients, respectively C. The pautations D.BRAF , RAF1 , ALK , FGFR1 , ETV6 , AKT3 , TCF12 , ATF , ZCCHC7 , ARHGEF2 , KDM6A , and WHSC1L1  . All fusions with druggable potential could be targeted by drugs from clinical trials  A. These , n = 1) B. Based l trials C. In totALK (n = 4), BRAF (n = 10), NRG1 (n = 2), RAF1 (n = 2), BCR (n = 1), NAB2 (n = 2), RELA (n = 2), and TERT (n = 2) (n = 110), regardless of whether they were druggable. At the RNA level, positive fusions were validated in 38 patients: ALK (n = 3), BRAF (n = 10), FGFR1 (n = 1), NTRK2 (n = 1), NTRK3 (n = 1), RAF1 (n = 3), AKT3 (n = 1), ATF1 (n = 1), CBFB (n = 1), ETS1 (n = 2), ETV6 (n = 2), JAZF1 (n = 2), KDM6A (n = 1), MAG (n = 1), METFA (n = 1), NF1 (n = 1), PAX5 (n = 1), RB1 (n = 1), SMARCB1 (n = 1), TCF12 (n = 1), WHSC1L1 (n = 1), and ZCCHC7 (n = 1) :  (n = 2) A. We exp (n = 1) A. RNA-NG (n = 1) A. If onl (n = 1) A. Overal (n = 1) B.The significant enhancement of fusion detection by RNA-NGS was attributed to the different designs of the DNA-NGS and RNA-NGS panels, which could be categorized into four groups: breakpoints that were covered by only the RNA-NGS panel (RNA cover only), breakpoints that were covered by DNA-NGS but failed to identify fusions (DNA cover but negative), the fusions that were verified by both DNA-NGS and RNA-NGS (Consistency), and the different partners that were validated by DNA-NGS and RNA-NGS (RNA correction) A. In theBRAF-WDFY1 to CUX1-BRAF and LMCD1-RAF1 to ERC1-RAF1 (BRAF-WDFY1 fusion detected by DNA NGS with “antisense rearrangements” juxtaposing the 5′ portion of the kinase genes and the 3′ portion of intergenic partners (BRAF lacked the tyrosine kinase domain (TKD), and the patient could not be referred to RAF tyrosine kinase domain (TKIs). However, the RNA-NGS results revealed that CUX1 exon 11 was fused to BRAF exon 9 with an intact TKD. The inconsistency between the DNA and RNA results was perhaps due to the reciprocal rearrangement in which DNA-NGS detected only the opposite breakpoint downstream of BRAF rather than the primary junction constituting CUX1 and BRAF. In Patient 2, DNA-NGS revealed that the partner LMCD1 was fused to the 3′ portion of RAF1 and that the breakpoint was in intron 9 of RAF1. At the RNA level, CUX1-BRAF was found, in which exon 8 of RAF1 was involved  from the Park et al. study [BRAF fusion and one ALK fusion were identified in the largest Chinese cohort (n = 81) from the Huang et al. study [n = 469). The leading number of fusions with clinical significance in our cohort was that of BRAF , which was slightly higher than that in the Caucasian population (1.1%) [KIAA1549-BRAF is the most common BRAF fusion [BRAF fusions in our present study were discrete. This result demonstrated that there was no enriched BRAF fusion type in melanoma. Furthermore, the RAF1 fusions  in the same pathway as BRAF were first reported in the Asian cohort. Among these fusions, four (three BRAF fusions and one RAF1 fusion) were novel. Additionally, our study identified NRG1 fusions in melanoma, which have never been found before. Surprisingly, we found high numbers of ALK rearrangements in melanoma (n = 4) for the first time. However, compared to the results in lung cancer [ALK fusions in our study were totally different, and none of them were the canonical type (EML4-ALK). Couts et al. demonstrated that melanoma expressing EML4-ALK was sensitive to ALK inhibitors in vitro and in vivo [ALK fusions from our study.Melanoma has been studied extensively in the Caucasian population ,18,19,20l. study . Only onl. study . In contn (1.1%) . It is iF fusion . The parg cancer , the par in vivo . This reBRAF fusions. In our study, RNA-NGS sequencing was systemically performed on patients without potential clinical utility at the DNA level for the first time, and comprehensive fusion types were investigated. In addition to the BRAF fusions, ALK, RAF1, and FGFR1 fusions were detected in the cohort. Briefly, up to 11.7% (7/60) of patients in the undruggable group could benefit from RNA-NGS based on the results. Overall, the use of RNA-NGS increased the proportion of treatable patients from 75% to 78%.Although more than half of the population could be targetable based on DNA-NGS results from our findings and the abovementioned studies ,15, apprNTRK3 is close to 100 kb in length (UCSC Genome Browser), which is close to 10% of the total size of the panel. Tiling such introns is not only technically challenging but also not practical in terms of the overall sequencing throughput. Additionally, introns with low frequency will not be tiled due to the cost factor. For example, ALK fusions have been frequently found in introns 18, 19, and 20 [ALK breakpoint in intron 17 was first identified in our study, so it is unrealistic to be covered as a frequent intron in gene rearrangement.The increased proportion of fusion detection by RNA-seq could be attributed to several causes. Large introns could not be covered by the panel assay due to their length. For example, intron 13 of , and 20 . To our CD74, the most frequent partner in the ROS1 fusion, is usually covered by the DNA-seq panel. However, the partners in the BRAF fusions were too diverse to be covered.On the other hand, eight fusions only detected by RNA-NGS were expected to be identified by DNA-NGS. Due to the complexity of the introns, the rearrangement that takes place in an intron may not be successfully detected by DNA-NGS. Five of those fusions involved BRAF on introns 7, 8, and 9. Similar to ROS1, the repeat sequence in those introns can be present at many other sites in the genome, and the inclusion of baits for these regions would simply result in unmappable reads ,30. TherSeveral limitations should be discussed. The clinicopathological parameters, such as clinical subtypes and disease stages were missed in the study. Those parameters could bring more a comprehensive understanding of Asian melanoma. Due to limitations in the amount of specimens, only samples with enough specimens were further sequenced by RNA-NGS. The resulting increased proportion of potential druggable patients in the undruggable group may be biased. However, more than half of the samples in the undruggable group had been analyzed by RNA-NGS, so the results did not induce significant bias. In addition, this study was a retrospective study, and patients with positive fusion detected by RNA-NGS were not able to receive targeted therapy, especially those with novel fusions with potential clinical significance. Therefore, further prospective trials of applying DNA and RNA sequential sequencing in targeted therapy for melanoma patients are needed in the future.This study systemically analyzed the genetic landscape of Asian melanoma and demonstrated how DNA and RNA sequential sequencing is essential in bringing clinical benefits to more melanoma patients.


The relationship between advanced nonalcoholic steatohepatitis (NASH) and plasma fatty acid composition remains unknown. We aimed to examine the plasma fatty acid composition in biopsy-confirmed nonalcoholic fatty liver disease (NAFLD) and evaluate the relationship between histological findings and fatty acid composition. Overall, 235 patients (134 women) with NAFLD were enrolled. Comprehensive blood chemistry tests and histological examinations of liver samples were conducted. Multivariate analyses adjusted for age, sex, body mass index, alanine aminotransferase, hemoglobin A1c, creatinine, total cholesterol, triglyceride, and NAFLD Activity Score values showed that lower levels of arachidic, behenic, α-linolenic, eicosatetraenoic, docosapentaenoic, and docosahexaenoic acids and higher levels of mead acid were associated with fibrosis stage 3–4. Furthermore, higher lauric acid, myristic acid, and palmitic acid levels and monounsaturated fatty acids such as palmitoleic acid and oleic acid were significantly associated with high NAS in analyses adjusted for the same factors and fibrosis stage. The plasma fatty acid composition was associated with the histological evidence of NASH. Increased synthesis of fatty acids is associated with NASH; insufficient intake of n-3 essential fatty acids and reduced elongation of fatty acids are associated with fibrosis in NASH. These features may help clinicians to understand and treat advanced NASH cases. Unhealthy eating habits and lack of exercise are associated with increasing rates of nonalcoholic fatty liver disease (NAFLD). Many patients with NAFLD retain a normal quality of life . HoweverFatty acids are the substrates of triglycerides and are important for fatty liver formation. Moreover, the fatty acid composition is associated with various diseases. The higher palmitic acid (16:0), palmitoleic acid 16:1n–7), and stearic acid 18:0) levels in plasma and plasma phospholipids (PL) have been associated with insulin resistance and diabetes .,19.18,19t-test and the Tukey–Kramer test. Conversely, other fatty acids, which were non-normally distributed, were analyzed using the Wilcoxon test and Steel–Dwass test. Spearman’s correlation coefficients were used to assess the correlation between the fatty acids and the clinical variables. Crude odds ratios (ORs) and their 95% confidence intervals for the association between histological findings and fatty acid plasma levels were derived in logistic regression analysis. Multiple logistic regression analysis was adjusted for age, sex, BMI, ALT, HbA1c, Cre, TC, TG, and NAS values or fibrosis stage, which were selected a priori as potential confounding factors. Data are shown as medians (IQR) or as counts (percentages). p-values of <0.05 were considered statistically significant.Statistical analyses were performed using the JMP software, version 14.2 . Since the continuous variables, such as behenic and lignoceric acids, proved to be normally distributed, they were analyzed using an unpaired The patients’ characteristics are presented in Patients with advanced fibrosis were significantly older, more obese, and had higher GGT and HbA1c levels and lower TC levels compared to patients with non-advanced fibrosis . ConversThe comparisons of fatty acid levels between non-advanced fibrosis stage and advanced fibrosis stage or between low and high NAS groups are shown in Lower levels of saturated fatty acids, such as arachidic acid, behenic acid, and lignoceric acid, and polyunsaturated fatty acids, such as linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid, and higher levels of mead acid were significantly associated with fibrosis stage 3–4 . In addiConcurrently, saturated fatty acids, such as lauric acid, myristic acid, palmitic acid, stearic acid, and arachidic acid, monounsaturated fatty acids, such as myristoleic acid, palmitoleic acid, oleic acid, and eicosenoic acid, and polyunsaturated fatty acids, such as linoleic acid, α-linolenic acid, eicosadienoic acid, mead acid, arachidonic acid, adrenic acid, and docosapentaenoic acid were associated with high NAS values . MultivaThis study showed that very-long-chain fatty acids and n-3 polyunsaturated fatty acid levels were decreased and that mead acid levels associated with essential fatty acid shortages  were incPrevious studies have reported on the association between NAFLD and fatty acid fraction. Araya et al. enrolled 11 control participants, 10 patients with NAFL, and nine patients with NASH to examine the levels of polyunsaturated fatty acid in total liver lipids, TG, and phospholipids, and revealed that the n-6:n-3 ratio (weight/weight) in liver total lipids in the NASH group was higher than those in the control and NAFL groups . Puri etThe changes in fatty acid composition may be involved in the pathogenesis of NASH. Our study showed that the levels of plasma very-long-chain saturated fatty acids, such as arachidic acid (20:0) and behenic acid (22:0), and polyunsaturated fatty acids, such as α-linolenic acid 18:3n3), eicosapentaenoic acid (20:5n3), docosapentaenoic acid (22:5n3), and docosahexaenoic acid (22:6n3), were decreased in advanced fibrosis, while the levels of plasma lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid 16:1n7), and oleic acid 18:1n9) were increased in patients with high NAS. Increased levels of plasma saturated fatty acids, such as palmitic acid (16:0) and stearic acid (18:0), and monounsaturated fatty acids, such as palmitoleic acid (16:1n–7), are associated with increased insulin resistance and diabetes risk [, and ole:3n3, eic were incThe strength of this study is the relatively large sample size and inclusion of patients with advanced fibrosis that is clinically problematic. However, this study has some limitations. First, the fatty acid composition of lipids in the liver remains unclear. However, plasma fatty acid composition in CE, TG, and PL reflects the composition of TG in the liver . The plaIn conclusion, this study has shown that the plasma fatty acid composition was associated with histological findings of NASH. These features may be useful for identifying advanced and highly active NASH. Furthermore, affecting the enzymes associated with the increased synthesis of fatty acids or the intake of deficient fatty acids could improve NAFLD pathogenesis and potentially lead to the development of treatment options for NAFLD.