Klinefelter syndrome is the most common sex chromosomal aneuploidy in males. It is well known that patients with this syndrome have greater mortality and morbidity compared to the general population due to cardiovascular diseases and endocrine metabolism disorders. This augmented risk is due both to hypogonadism and to the syndrome itself. Therefore, correct hormonal replacement therapy and early primary prevention are crucial to these patients. Even though different studies are available on this topic in adult patients, only a few authors have focused on the paediatric population. Thus, in this narrative review, we report the current knowledge of metabolic and nutritional aspects in children with Klinefelter syndrome. Klinefelter syndrome (KS) is the most common sex chromosomal aneuploidy in males. It is characterized by an additional X chromosome, which leads, in most cases, to a karyotype of 47 XXY ,2.The prevalence of KS is estimated between 1 in 448 to 1 in 917 male births ,4. The pThe supernumerary X-chromosome originates in 50% of the cases from the mother and in the other 50% of the cases from the father ,4. In boHistorically, KS was first described in 1942 by Dr Harry Klinefelter, who described a case series of men characterized by small testes, tall stature, narrow shoulders, broad hips, sparse body hair, gynecomastia and azoospermia . SeveralHypogonadism is a central element of KS. The testicular underdevelopment and dysfunction could be as early as infancy . AlreadyThe signs and symptoms of KS vary depending on age. In a few cases, newborns can show signs of hypogonadism, such as a micropenis, cryptorchidism and hypospadias . In the Patients with KS are diagnosed during adolescence due to small testicles, gynecomastia or delayed puberty. However, often, the diagnosis is made during adulthood during investigations for infertility or in patients who present signs and symptoms of hypogonadism .To date, we know there is an important discrepancy between the known prevalence of KS and the rate of clinical diagnosis . In factDuring adolescence, hormonal exams could be useful for diagnosis. In fact, testosterone may rise normally at the beginning of puberty; however, it usually decreases to lower-normal or subnormal levels. In response to this phenomenon, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) rise 1–2 years after the beginning of puberty. Moreover, inhibin B (INHB) and anti-Mullerian hormone (AMH) are low or undetectable [If KS is found early in infancy or even at the prenatal tests, the patient should undergo an adequate follow-up . In partCurrently, there are no universal guidelines that indicate when androgen replacement should be begun. Some authors suggest waiting until signs or symptoms of hypogonadism appear. Others prefer to start low testosterone doses at the first abnormal rise of LH. Testosterone is administered with depot injection or topical gel. In order to preserve the possibility of paternity, it is crucial to refer the patient to a centre specialized in advanced reproductive technology, in particular in testicular sperm extraction .It is extremely important to remind that KS is not only about hypogonadism and neurodevelopment.Different studies have focused on the long-term consequences of KS, showing that Klinefelter patients have greater morbidity and mortality ,11. The For these reasons, in this narrative review, we focus on the metabolic and nutritional aspects of KS, particularly in the paediatric population. These aspects are extremely important considering that early diagnosis will be more frequent due to prenatal tests. Therefore, a correct approach to nutrition and hormonal therapy in children and adolescents with KS will be necessary in order to reduce the cardiometabolic risk and, consequently, morbidity and mortality.A narrative review was performed starting on 29 January 2022 . The mosIt is well known that adult patients with hypogonadism present several metabolic alterations independently from KS. In fact, in the general adult male population, hypogonadism is associated with abnormal body composition, metabolic syndrome, insulin resistance, diabetes, elevated cardiovascular risk and osteoporosis . MoreoveDespite the clear connection between hypogonadism and metabolic alterations, in patients affected by KS, this interaction is extremely complex because hypogonadism is not the only factor that should be considered . In factFew studies have evaluated the metabolic aspects in paediatric patients affected by Klinefelter syndrome so far. There is a growing clinical and scientific interest in the metabolic alterations and the unfavourable changes in body composition present in KS patients since childhood. They typically have a tall stature, perhaps caused by SHOX duplication, and specific anthropometric findings, such as increased length, width and hip circumference .As mentioned beforehand, androgens play an important role in regulating adipogenesis and body fat distribution. Hypogonadism, common in adults with KS, has been found to be an independent risk factor for increasing truncal adiposity and diabetes in men with normal karyotypes ,28,29.Nevertheless, some authors reported an increased deposit of body fat, by the age of ten years in patients with KS, measuring subscapular and triceps skinfolds . In prepFurthermore, in a recent cross-sectional study, Aksglaede et al. showed that an unbalanced body composition among KS adults may already be present before puberty. In prepubertal subjects, with a median age of 11 years old, an increasing body fat mass (BFM), height and BF% were found upon a DEXA scan despite age-normal BMI, lean body mass (LBM) and weight for age, compared with normal boys, probably due to an unfavourable muscle-to-fat ratio. Moreover, increased BFM was demonstrated in both testosterone-treated and untreated KS children . HoweverThe findings of increased amounts of body fat in prepubertal boys suggest the weakness of the hypothesis that hypogonadism and low testosterone precede obesity. The multivariate analysis highlighted that abdominal obesity is primarily responsible for the decrease in insulin sensitivity, even when serum testosterone levels are in the normal range . In factPerhaps there is a self-perpetuating vicious circle between obesity and hypogonadism, in which increased abdominal fat reduces the insulin sensitivity of cells, in particular Leydig cells, thus causing a progressive decrease in testosterone production .Moreover, there is a possible genetic influence of body fat in KS in early childhood due to the overexpression of X-linked genes, skewed X-chromosome inactivation or CAG-repeat polymorphism in the androgen receptor as well as the influence of hypogonadism after puberty . FurtherIn conclusion, even though not all of the KS population develops obesity, unfavourable body composition is common and represents a key risk factor for morbidity ,21.Many studies have demonstrated an association between KS and diabetes, in both adulthood and childhood, although the risk factors have not been fully elucidated ,17. ThisInterestingly, type 2 diabetes (T2D) has some specific features in these patients. Diabetes seems to occur earlier in life compared to the healthy population, around the age of 30 years; it shows lower insulin sensitivity and higher insulin secretion ; blood gUsing the hyperinsulinemic euglycemic clamp test, Lee et al. reported that impaired peripheral insulin resistance was the underlying mechanism of impaired glucose tolerance in adult Korean patients with KS . AccordiWe do not know when the glucose metabolism impairment exactly starts; however, it seems reasonable to suppose that early adolescence is the most critical period.Davis et al. studied a group of adolescents with KS without a diagnosis of T2D and healthy controls between 10 and 17 years. They reported a higher HbA1c in KS patients than in healthy adolescents, which is indicative of chronically higher blood glucose . More stAs previously reported, hypogonadism is an independent risk factor for the development of diabetes in men with normal chromosomes ,38,39; hHowever, the chromosomal abnormality could act through other mechanisms in triggering IR, and these could be studied in KS boys at a prepubertal age. Indeed, they have similar circulating plasma levels of testosterone and gonadotropin at a prepubertal age to healthy age-matched controls of the same age despite presenting the genetic alteration typical of this disease since birth . AccordiFurthermore, autoimmunity is also frequent in KS. In support of this speculative hypothesis, a recent study showed that immunoreactivity directed against diabetes-specific autoantigens of type 1 diabetes was significantly higher in KS (8.2%) than in paired controls. However, the endocrine autoantibody profile of testosterone-treated and untreated KS patients was not different . MoreoveOther mechanisms, such as changes in body composition, inflammation status, high triglyceride levels, fatty liver and acute pancreatitis, might also play an important role in the development of diabetes mellitus in KS patients .However, the specific pathogenesis has yet to be elucidated, and further research is needed to clarify the complex interaction between genotype and metabolic phenotype in KS patients .Recent studies have evaluated MetS’ prevalence and occurrence since paediatric age in KS children.Bardsley et al. investigated the risk factors for MetS in 89 prepubertal KS boys age 4–12 years . Since dSimilarly, Davis et al. studied the relationship between gonadal and metabolic functions in a cohort of 93 prepubertal children (4–12 years) . DespiteSubsequently, the same authors hypothesized that exogenous androgen treatment would have a favourable effect on the cardio-metabolic health of KS children age 4 to 12 years from the same cohort. Thus, a randomized controlled trial with oxandrolone (Ox) versus placebo treatments was conducted. Even though a beneficial effect of Ox was appreciated for some features of MetS, after two years, the number of children who met the criteria for MetS did not differ between the groups .In line with previous studies, the increased prevalence of MetS (≥3 criteria) among KS adolescents age 10–18 years was confirmed in 30% of the subjects .Clarifying the risk for MetS among KS children and adolescents is crucial as features of MetS that present during childhood may track into adulthood . In factOverall, both the high prevalence of MetS and the risk of MetS independent from BMI in KS children need to be confirmed by broad cohort studies. Moreover, the impact of TRT on metabolic health during paediatric age calls for further study.Past epidemiological studies have found that men with Klinefelter syndrome have higher morbidity and mortality due to several conditions, including cardiovascular disease (CVD) ,11,43. Tp< 0.001) and triglycerides . Although underpowered, the authors performed a subgroup analysis regarding the presence or absence of TRT in KS adolescents. Those receiving TRT showed significantly lower systolic blood pressure percentile (p = 0.033) and approximately half of the prevalence in “three or more CM risk features” compared to those who were not on TRT (18% vs. 38%), even though it was not significant (p = 0.13). More interestingly, this study highlights how adolescents with normal-range BMI z scores (controls 0.36 ± 1.2 vs. KS 0.31 ± 1.3) might already have an increased CM risk compared to controls [Recently, a cohort study evaluated the cardio-metabolic risk in KS adolescents age 10 to 18 years compared to age- and BMI-matched healthy controls . To idencontrols .Overall, an increase in cardiometabolic risk markers was also confirmed in pre-pubertal children with KS ,18,19. MBesides cardio-metabolic outcomes, there is evidence that KS is associated with an increased risk of congenital heart malformations , althougConsidering the role of inflammation in increasing CV risk, future studies on paediatric populations should also evaluate inflammatory biomarkers as CRP concentrations. These were found to be significantly higher in KS adults compared to controls, yet testosterone therapy has a potential role in reducing them . MoreoveOverall, such CV abnormalities, together with an increased prevalence of metabolic disorders, may contribute to the increased CV morbidity and mortality observed in these patients. These data emphasize the importance of a dedicated analysis of CV in the management of patients with KS.Testosterone plays an important role, both in achieving peak bone mass by increasing serum levels during the critical period of puberty and in maintaining proper bone density, through the persistence of normal serum levels during adulthood . AksglaeAndrogens seem to be important in the regulation of bone mineral content, although the exact mechanism is not yet known and, moreover, they do not seem to be solely responsible. Hypogonadism, in fact, is more relative than absolute : a reducInterestingly, in a mouse model, it has been suggested that the extra X chromosome may itself cause abnormal bone structure in mice 12 to 34 months of age . AnotherAs previously shown, metabolic and cardiovascular risks in KS patients are an important issue that may occur even during paediatric age. Obesity, diabetes, dyslipidaemia and metabolic syndrome are usually referred to as non-communicable diseases (NCDs) since they are non-infectious diseases that progress slowly but become chronic ,58,59. TAccording to the Guidelines on Klinefelter Syndrome from the European Academy of Andrology (EAA), it is highly recommended to provide the patients correct information on lifestyle intervention to minimize cardiovascular risk factors, to monitor cardiovascular risk factors regularly and to treat obesity, diabetes and dyslipidaemia . CounselOnce obesity or MetS are present, a referral to weight management programs is recommended . In thisRegarding bone mineralization, the EAA suggests, after determination of vitamin D blood levels, an adequate vitamin D and calcium supplementation in pre-pubertal children with KS . Howeverp < 0.05) higher risk of developing hypogonadism compared to those with the lowest (Q1) [Considering testosterone deficiency in KS children and adolescents, deepening the knowledge of the link between diet and testosterone might be interesting. Recently, few studies have evaluated the association between dietary patterns and testosterone levels among the adult population ,69. Hu eest (Q1) .The relationship between the male sex hormone, testosterone, and obesity is complex, and dietary-related factors may serve as important intermediates . HoweverEven though testosterone treatment is recommended by most endocrinologists starting from the peripubertal period, no randomized placebo-controlled trials have ever been conducted ,4,7.Most of the studies on the outcome of TRT are observational studies that compared Klinefelter patients to healthy controls . Almost There are only a few studies regarding testosterone treatment in children. Samango-Sprouse et al. evaluated the long-term neurological outcome of three months of treatment with 25 mg of testosterone in infants between 4 and 15 months of age. They found positive effects on cognitive function, in particular motor function at 6 years of age . AnotherAs partially reported above, only two studies have investigated the effect of TRT on BMI in children. One randomized controlled trial by Davis et al. showed a reduction of 15% in adiposity in 10 infants with KS treated with 25 mg of testosterone monthly for 3 months between 2 and 5 months old . A seconFrom the review of the literature, it appears clear that Klinefelter patients are at an increased risk of mortality, especially for metabolic abnormalities. As described above, these alterations are present since infancy, and they might be only partially explained by hypogonadism. Probably, the syndrome itself is a causative factor for genetic elements that are not yet fully understood. Considering the relevance of metabolic alterations that emerged in this review, we suggest to consider performing regular anthropometric assessments and health education to promote a healthy lifestyle since the paediatric age in addition to the evaluations recommended by the current guidelines on the management of Klinefelter syndrome ,60 Figu.Finally, further studies are needed to better understand the etiopathogenesis of the metabolic alterations in this genetic disease. Moreover, more studies should be focused on the paediatric population in order to arrange early interventions that could improve the natural course of the disease.


N‑heterocyclic carbene complexes form a basis for molecular gyroscopes in which solid-state emission is quenched by axle rotation.Organizing molecular rotors in highly ordered crystals enables the use of molecular motion to control physical properties. Now,  Designing systems with this functionality, however, remains a challenge. Now, Mingoo Jin, Hajime Ito, and colleagues from Hokkaido University, Japan and University of California Los Angeles, USA, construct molecular gyroscopes composed of binuclear N‑heterocyclic carbene complexes, in which rotary behavior and optical properties are fine-tuned by the nature of the transition metal ions that link the rotator unit to the static units (10.1021/jacs.0c11981)4.Incorporating rotational elements into crystals provides the opportunity to use molecular motion to tune material properties. Solid-state emission can be controlled in amphidynamic crystals—those that accommodate 2-fold rotational motion—through rotation-induced luminescence quenchingN‑heterocyclic carbene (NHC) ligands could shield a coaxially coordinated central pyrazine rotator  complex, is shown to stabilize the transition state.The team envisioned that bulky, concave tor Fig. , therebyThe emission properties are also tuned by switching the metal ion. While the Au(I) rotor exhibits blue luminescence, greater electronic delocalization in the Cu(I) complex causes a distinct red-shift in the solid-state emission. In both cases, molecular rotation is found to induce emission quenching.Thanks to the ease of synthesis of these gyroscopic rotors, and considering the vast library of NHC ligands that organometallic chemists have designed to date, it isn’t difficult to imagine that this system could serve as a platform for the construction of a range of tunable amphidynamic crystals. “Chiral NHC ligands possessing asymmetric structural moieties may allow for the design and formation of chiral space in the crystalline phase, which could contribute to exploring non-linear photophysical properties in the solid-state.”, comments Mingoo Jin.


Molecular Cytogenetics Editorial Board, passed away. It is to note that she was among the founders of Molecular Cytogenetics and was intrinsically involved in the developing journal’s aims, scope and original policy [Molecular Cytogenetics is hard to estimate.On 31 August, 2021, Professor Svetlana Grigorievna Vorsanova  centromeric one-color/two-color/multicolor fluorescence in situ hybridization (FISH), which was used to uncover aneuploidies, to decipher marker chromosomes and to analysis of structural chromosome abnormalities involving centromeric DNAs –21 and  , 81–85.Another local story of Svetlana’s contribution to diagnostic research that resulted in important contribution to biomedicine was related to introducing cytogenomic microarray analysis of copy number variations (CNVs) in Russia. Svetlana was among the leaders of the research group, which was the first one that introduced microarray analysis of CNVs in Russia , 87. TheSvetlana’s research merging bioinformatics and molecular cytogenetics (in silico molecular cytogenetics) was dedicated to unraveling mechanisms of diseases associated with CNVs or chromosome abnormalities, to determine causes and consequences of chromosomal and/or genomic instability, to simulate consequences of genomic variations and to interpret CNVs –97. ThesThe latest interests of Svetlana were related to ‘syndrome-specific’ chromosomal mosaicism in neurodevelopmental diseases. Thus, Turner’s syndrome mosaicism  and KlinSvetlana made serious efforts in elevating the educational level in cytogenetics. She received numerous thanks and acknowledgements of Russian-spoken specialists from all over the world because of her co-authored books (textbooks) , 108–114It is not a big secret that Svetlana’s family is the core of research groups performed such a body of biomedical research. Her son, Dr. Ilia V. Soloviev, a pioneer of molecular cytogenetic and cytogenomic research (the results of his brilliant work and his ideas formed our research directions), tragically passed away in the 1990s. Her husband Professor Yuri B. Yurov passed away five years ago . Unaccep


Star-shaped 2,4,6-tris-1,3,5-triazine molecules self-assemble at the solid–liquid interface into a compact hexagonal nanoarchitecture on graphite. High resolution scanning tunneling microscopy (STM) images of the molecules reveal intramolecular features. Comparison of the experimental data with calculated molecular charge density contours shows that the molecular features in the STM images correspond to molecular LUMO+2. Intramolecular contrast in the STM images of 2,4,6-tris-1,3,5-triazine molecules recorded at room-temperature and at the liquid–solid interface. Star-shaped molecules are promising building blocks to realize self-assembled structures. Star-shaped molecules usually constitute a central ring surrounded with three organic straight arms.20–43 High resolution imaging techniques are required not only to elucidate molecular assembly but also assess the electronic properties of individual molecules in the organic structure.Engineering novel two-dimensional (2D) organic nanoarchitectures is the focus of intense research interest2 and D2 functionalized tips have been used to improve the resolution of non-contact atomic force microscopy (nc-AFM) in vacuum at low temperature.44–62 NaCl functionalised STM tip has been used to enhanced intramolecular features in organic and hybrid nanoarchitectures on a metal surface at room temperature in vacuum.63 It was thus possible to reveal that the intramolecular features correspond to the lowest unoccupied molecular orbitals (LUMO). SPM tips can hardly be functionalized at the solid–liquid interface at room temperature, but intramolecular features in organic layers have been sometimes reported.64,65 However these features are usually not assigned to the electronic or topographic properties of the molecule.Recent technical developments have been perfected to improve the resolution of scanning probe microscopy (SPM) to separate surface topographic from electronic contribution in the SPM images of 2D nanostructures. Xe-functionalized and CO, HIn this paper we investigate the self-assembly of 2,4,6-tris-1,3,5-triazine molecules at the 1-phenyloctane/graphite interface. This star-shaped molecule self-assembles into a compact nanoarchitecture on graphite at the solid–liquid interface. STM at room temperature reveals intramolecular details, corresponding to one of the molecular LUMOs states.266Nearly saturated solutions of 2,4,6-tris-1,3,5-triazine (Aldrich) in 1-phenyloctane (Aldrich) were prepared. A droplet of the solution was then deposited on a highly oriented pyrolytic graphite substrate (HOPG). STM imaging of the samples was performed at the liquid–solid interface using a Pico-SPM  scanning tunneling microscope. The surfaces were imaged using STM 1 h after molecular deposition. Cut Pt/Ir tips were used to obtain constant current images at room temperature with a bias voltage applied to the sample. STM images were processed and analyzed using the application FabViewer.3The 2,4,6-tris-1,3,5-triazine molecule is 3-fold symmetry molecule. Its chemical structure is presented in 67,68The calculated charge density contours of molecular four first lowest unoccupied molecular orbitals (LUMOs) and highest occupied molecular orbitals (HOMOs) for a free standing molecule are presented in the 69 This structure has a 1.4 nm hexagonal unit cell constant. The model of the molecular arrangement is presented in Vt ∼0.3 V, are presented in Vs = +0.30 V, whereas the STM in Vs = +0.27 V. The two STM images are revealing intramolecular details. As a guide for the eyes, molecular scheme has been superimposed to the two STM images in Vs = +0.30 V, the intensity of the bright features in the peripheral benzene rings and the central triazine ring is similar in the STM images, Vs = +0.27 V, Vs = +0.30 V, Vs = +0.30 V, the carbon atoms (A) of the molecular methyl-groups appear as bright as the labelled 1,3,5,6 carbon atoms of the neighboring benzene ring, Vs = +0.30 V, the nitrogen atoms appear as bright as the side 1,3,5,6 carbon atoms of the benzene ring, whereas at Vs = +0.27 V they appear brighter.High resolution STM images of the 2,4,6-tris-1,3,5-triazine molecule recorded for high resolution STM images were recorded at 470 but these data are rarely concretely compared to the molecular structure and molecular density of states. The experimental STM images in Vs = +0.30 V, their intensity is low and these features are darker than the other ones in the STM image, Vs = +0.27 V, 2,4,6-Tris-1,3,5-triazine molecules self-assemble into a compact arrangement on graphite. Scanning tunneling microscopy surprisingly reveals intramolecular details, despite the measurements have been performed at room temperature and at the solid–liquid interface. STM images recorded in the constant-current mode result from the convolution between the surface topography and the variation of surface integrated density of states. Very low temperature and vacuum conditions are usually required to acquire STM images with consistent submolecular resolution. Atomic details in molecular skeleton have been observed at the solid/liquid interface at room temperature5To summarize, scanning tunneling microscopy showed that star-shaped 2,4,6-tris-1,3,5-triazine molecules self-assemble into a close-packed nanoarchitecture on graphite at the liquid–solid interface. Intramolecular features are observed in the STM images at room temperature. Comparison of experimental molecular images with calculated molecular charge density contours reveals that molecular LUMO+2 is observed in the STM images.There are no conflicts to declare.


Antibody-mediated rejection (AMR) is caused by antibodies that recognize donor human leukocyte antigen (HLA) or other targets. As knowledge of AMR pathophysiology has increased, a combination of factors is necessary to confirm the diagnosis and phenotype. However, frequent modifications to the AMR definition have made it difficult to compare data and evaluate associations between AMR and graft outcome. The present paper was developed following a Broad Scientific Advice request from the European Society for Organ Transplantation (ESOT) to the European Medicines Agency (EMA), which explored whether updating guidelines on clinical trial endpoints would encourage innovations in kidney transplantation research. ESOT considers that an AMR diagnosis must be based on a combination of histopathological factors and presence of donor-specific HLA antibodies in the recipient. Evidence for associations between individual features of AMR and impaired graft outcome is noted for microvascular inflammation scores ≥2 and glomerular basement membrane splitting of >10% of the entire tuft in the most severely affected glomerulus. Together, these should form the basis for AMR-related endpoints in clinical trials of kidney transplantation, although modifications and restrictions to the Banff diagnostic definition of AMR are proposed for this purpose. The EMA provided recommendations based on this Broad Scientific Advice request in December 2020; further discussion, and consensus on the restricted definition of the AMR endpoint, is required. AMR is recognized as a spectrum of discrete injury patterns, as outlined below.Advances in the development of sensitive assays for DSA identification have improved our understanding of AMR histopathology , 12. AMRThe detrimental impact of AMR on kidney transplantation outcome has been known for decades, as illustrated by the early routine implementation of crossmatching to avoid this rejection phenotype . The theThe next advancement followed the introduction of C4d staining, which documented histopathogenetic links between circulating DSA and organ damage, by detecting complement activation by DSA fixed to surface antigens on the endothelial cell . The 200Chronic (c)AMR subtypes were first recognized in the Banff 2005 update, as chronic active (ca)AMR, with transplant glomerulopathy (TG) and/or severe peritubular capillary basement membrane multilayering (PTCML) and/or simple interstitial fibrosis and tubular atrophy and/or arterial fibrous intimal thickening, with C4d positivity . EvidencCurrently, AMR diagnosis within Banff Classification category 2 is based on four partially overlapping components: histological features of AMR activity; evidence of antibody interaction with graft vascular endothelium; histological features of AMR chronicity; and DSA or equivalents  19, 21), 2119, 2Updates to the Banff Classification of AMR over time make it difficult to maintain long-term follow-up registries or compare literature. Moreover, the interobserver agreement (κ-statistic) of the most important lesion scores for AMR was quoted as 0.39 for Banff Lesion Score g, 0.38 for ptc, and 0.48 for cg—at best a fair-to-moderate agreement, even among very experienced transplant nephropathologists .Several problems arise when reviewing evidence of an association between AMR and allograft outcome. Firstly, AMR definitions have changed very frequently since 2001, as outlined above, making it difficult to compare data from studies conducted over the last 2 decades. Secondly, Banff diagnostic criteria and categories are adjusted based on antecedent literature, and as they arise as a synthesis of several different studies, rarely fully align with literature on which they are based. Conversely, outcomes of AMR diagnoses according to their strict definitions in the Banff Classification have rarely been investigated for either their association with outcome, or their success in delineating which patients are eligible for a specific therapy; where this has been done, results show improved prediction of outcome with the 2013 version compared to the 2003/2007 Classification, and with the 2013 version compared to the 2017 Classification , 26. InsdnDSA, preformed DSA, or no HLA-DSA must also be known.To our knowledge, no method of transcriptome analysis has been formally recognized as thoroughly validated by Banff. No transplant centers have obtained adequate clinical validation to use transcript analysis for defining AMR, as required by Banff consensus. In addition, although the Banff Classification makes no distinction between AMR in patients with preformed DSA (high-risk) and non-sensitized (low-risk) transplant recipients, the diagnosis and treatment pathways for both groups might be quite different, as are the underlying biology and clinical phenotypes. Consequently, AMR classification may need to include more than histology, because identical histological diagnoses in the kidney (such as TG or TMA) can be the consequence of different disease entities . FinallyThus, although the basic principles of diagnosing AMR have generally remained constant, given the considerable changes to Banff definitions of AMR, longitudinal comparison of literature findings is more challenging for AMR than for aTCMR. Interpretation of the AMR literature must be undertaken cautiously, taking account of these limitations. In reviewing evidence that could serve as background for the definition of AMR, first it is important to evaluate studies that have assessed outcomes associated with various combinations of biopsy features. In the following sections, we divide this information into subcategories broadly based on the Banff classification. After evaluating the literature on allograft outcome, we consider data relating to associations between outcome and individual biopsy features that are components of AMR . MoreoveThis subcategory is discussed in conjunction with C4d positivity with acute tubular injury (ATI) in the absence of other apparent cause.Much of the evidence for an association between aAMR and outcome  comes from publications that only include components of aAMR   29–34).–34.29–34Given the heterogeneity of definitions, overall, the quality of evidence that strictly defines the association between aAMR and increased graft loss is low; in recipients of HLA-incompatible grafts, quality of evidence is higher. However, if one evaluates the literature with less stringency about the exact AMR definition, there is consensus that aAMR is an important risk factor for graft failure , 28. MorIn the absence of dysfunction , the outcome used for features of aAMR is usually TG rather than graft loss, although a retrospective study indicated that subclinical AMR in 1-year protocol biopsies had a detrimental impact on graft survival . There iOur proposal is that aAMR, exactly as defined by the current Banff classification, cannot be adopted as a surrogate endpoint for future cAMR and graft loss in low-risk situations, i.e., non-sensitized graft recipients, without DSA against the graft. Conversely, in high-risk patients, i.e., sensitized patients with DSA against the graft, evidence supports features of aAMR as a surrogate for graft loss, especially if associated with graft dysfunction. Future research should aim to establish outcome  in patients with aAMR, strictly defined according to Banff criteria and specifically excluding cases with features of chronicity. Such research should involve retrospective and prospective studies, and high- and conventional-risk transplantations. Data from randomized controlled trials investigating aAMR treatment regimens would also be particularly valuable. Although further data are awaited, there is broad consensus on the clinical relevance and impact of aAMR after kidney transplantation. Since aAMR leads to therapeutic interventions, treatment burden, associated morbidity, and increased cost, features of aAMR represent a key endpoint for interventional studies.According to the 2017 Banff Classification, a diagnosis of cAMR or caAMR can only be established based on presence of TG (Banff Lesion Score cg > 0) and/or severe PTCML. For caAMR, this must be accompanied by “evidence of antibody interaction with tissue” and “DSA or equivalent”; for cAMR, this must be in conjunction with “a prior documented diagnosis of aAMR or caAMR or documented prior evidence of DSA.” The ill-defined transplant vasculopathy is no longer considered a chronicity parameter for these diagnoses . Data foA retrospective study of 44 patients with TG examined the outcome of graft loss, with TG defined as Banff cg > 0 (glomerular basement membrane splitting of >10% of the entire tuft in the most severely affected glomerulus) ; this deTo investigate associations between TG and other parameters, as well as with outcomes, using archetypal analysis, a retrospective study of 385 patients with TG identified five distinct immunological, histological, and functional profiles of TG that were associated with allograft failure . AnotherBecause of repeated revisions to Banff criteria , the incidence of caAMR is under-reported. No studies fulfill all criteria for this diagnosis according to Banff 2017 or have a sufficient follow-up to use strictly defined Banff caAMR as an endpoint .We therefore recommend that clinical trials in kidney transplantation using caAMR as an endpoint or an inclusion criterion strictly adhere to Banff consensus criteria and report granular histological features of Banff Lesion Scores, to allow between-trial comparisons. Additional research is needed, in high- and conventional-risk transplantation scenarios that consider the effect of treating aAMR earlier, equally defined according to the strict Banff Classification.A noteworthy change to the Banff Classification in 2017 was its omission of ‘suspicious for aAMR’ and ‘suspicious for caAMR’ categories . The mosUntil 2019, no publication presented outcomes for patients with “suspicious for aAMR.” Then, the evidence appeared, with a caveat, because the 123 DSA− patients with AMR included six patients with TG (Banff Lesion Score cg ≥ 1); irrespective of C4d status, outcomes for patients with histological features of AMR but without DSA were no different than for controls without AMR . AlthougThe literature offers even less information about the diagnostic subcategory of “suspicious for caAMR,” eliminated from Banff in 2017. One study involving 21 DSA− patients showed an average transplant survival after diagnosis of 3.7 years .Some DSA− cases “suspicious for AMR” could be explained by non-HLA antibodies. Without any hard evidence, standardized tests, or validated assay and cut-off value to screen for non-HLA-DSA, we do not recommend that non-HLA-DSA be considered in the diagnosis of AMR. Further research is needed before non-HLA antibodies can be included in the definition of endpoints for registration studies.Overall, we do not recommend using cases in the “suspicious for” categories as endpoints.This section reports on two category 2 diagnoses that are separated in the Banff 2017/2019 Classification . FirstlyTechnically, according to the Banff classification, a biopsy that is C4d+ with DSA but with a reasonable other cause of ATI  is not AMR, yet publications have not assessed for (or reported on) other causes of ATI. Some early reports on C4d staining date from before widespread recognition of the full spectrum of histological features of aAMR, therefore descriptions of poor outcomes for C4d+ cases “without features of rejection” must be handled cautiously.The 2001 Banff meeting recognized a form of AMR with no or little inflammation, included in the list of category 2 diagnoses as “acute tubular necrosis-like minimal inflammation, C4d+.” It was stated that “acute humoral rejection may be manifested only by ATI without other evidence of rejection (seen in 10% of cases).” The evidence cited  describeThe 2007 Banff meeting  describeAdditional publications investigating the link between ATI-AMR and C4d+ without evidence of rejection, with outcome data, are presented in It is impossible to give a guideline recommendation because of inconsistent findings in the argument that C4d+ ATI without evidence of rejection is associated with increased risk of graft loss. In addition, there is no recent consensus definition of ATI, or of degrees of severity of ATI, or of what reasonably constitutes exclusion of other causes of ATI. Therefore, we recommend that the C4d+ ATI-only form of AMR and C4d+ without evidence of rejection subcategory of AMR should not be used as an efficacy measure in clinical trials. We also recommend that future research incorporates definitions of ATI and assessments of its severity, based on definitions agreed in the context of international collaborations . Such research should include both patients with preformed antibodies (sensitized) and non-sensitized patients, with representation of early and late post-transplant periods.Endarteritis is also a feature of aTCMR that initially was not included in AMR definitions; this makes findings from early studies difficult to interpret for the given purpose . Although endarteritis was described as a risk factor for graft loss , there abona fide diagnosis of AMR-associated TMA. In some patients with dnTMA, an underlying genetic defect in complement regulation might be relevant, although only one case series suggested this . During the 2015 Banff meeting, a working group was formed  to help ted this .We are unaware of sufficient published data about the outcomes of adequately investigated cases of AMR-associated TMA. The largest case series describes 33 patients with TMA and C4d positivity, 40% of whom experienced transplant failure within 2 years of diagnosis . Since CMVI is the main histological feature indicating activity in aAMR and caAMR. The Banff criteria for AMR use cut-off values of MVI >0 and >1, respectively, to establish C4d+ and C4d− aAMR; these values were established by consensus, based on published evidence . MVI aboBased on the low-quality evidence that MVI is an independent predictor of graft loss, we cautiously recommend that the MVI score is used as an efficacy marker for clinical trials in kidney transplantation. We also recommend that further research is undertaken to confirm the effect of MVI on outcome, in prospective randomized controlled trials, with granular histological data for Banff Lesion Scores and DSA.There are caveats to the prognostic value of C4d status: thresholds for positivity scoring differ, depending on antibody and study. For example, the monoclonal antibody used on frozen tissue is particularly sensitive; therefore >10% of PTC must be positive, whereas for the polyclonal anti-C4d antibody used on formalin-fixed paraffin-embedded tissue samples, any percentage and intensity of PTC positivity sufficiently describes a biopsy as C4d+ .Literature findings related to the potential prognostic value of C4d status are varied, likely because of the dynamic process  3, 63, , 79. A lAs TG is the main feature indicating chronicity in the diagnosis of caAMR and cAMR, much of the evidence indicating that this feature is an indicator of outcome has been covered above . As withp < 0.0001) (p < 0.01); this study used a lower threshold (Banff Lesion Score cg > 0) (In 55 patients with TG (Banff Lesion Score cg ≥ 1b)  there wa 0.0001) . Similar cg > 0) , equival cg > 0) , 23, but cg > 0) .et al. identified ∼50% graft loss within 3 years after the index biopsy , it is impossible to give a guideline recommendation or consensus-based statement. Different gene sets/classifiers are used across the studies, with no unifying set of genes agreed on for future validation in prospective research. Also, to our knowledge, no transplant centers have clinical validation for use of transcript analysis for AMR, especially for improving the prediction of graft outcome. Consequently, we recommend that gene transcripts/classifiers strongly associated with AMR are not used as efficacy measures for clinical trials. Future research in the context of international collaborations on agreed gene sets/classifiers  should assess non-selective populations of transplant biopsies and determine clinically meaningful molecular scoring systems that predict cAMR development and graft failure. These studies should include multivariate analyses in combination with traditional clinical, histopathological, or immunogenetic parameters.dnDSA. Given that subclinical AMR in protocol biopsies appears to be associated with impaired graft survival, but protocol biopsies are not universally performed, and the management of subclinical AMR is heterogeneous, it is unsurprising that consensus documents do not provide guidance , 2219, 2• Evidence relating to the relationship between AMR and outcomes is largely based on retrospective analyses that do not utilize the strict, most recent Banff categories of AMR, but instead investigate individual features of AMR, combinations of individual features of AMR, or combined Banff categories (such as combining aAMR and caAMR).○ Strongest evidence for associations between individual features and impaired graft outcome is noted for MVI score ≥2  and cg>10% (>10% of the most severely affected glomerulus).○ Together with presence of HLA-DSA, these parameters should be the basis for AMR endpoints, acknowledging their limitations .• Based on evidence for association between individual features of AMR and outcome, AMR diagnosed in indication or protocol biopsies should be considered as a primary endpoint in clinical trials for kidney transplantation.• However, based on available evidence, we suggest refinement of the Banff 2017 definition for AMR diagnosis to the following three AMR-related endpoints:○ Restricted aAMR, defined by the conservative threshold of at least moderate MVI  and DSA positivity (anti-HLA antibodies) with or without C4d positivity (C4d ≥ 1 on paraffin tissue or ≥2 on frozen tissue).○ Restricted caAMR, defined by the conservative threshold of cg ≥ 1 according to Banff 2011  plus at least moderate MVI  and DSA positivity (anti-HLA antibodies) with or without C4d positivity .○ Restricted cAMR, defined by the conservative threshold of cg ≥ 1 according to Banff 2011  and current or past DSA positivity (anti-HLA antibodies) with or without C4d positivity .• Other features of AMR used in Banff AMR definitions  show less-robust evidence than MVI score ≥2 and cg>10%.○ In isolation, without the other features of AMR described above, these features should not be considered as efficacy endpoints for clinical trials.• The use of histology as endpoint for studies after kidney transplantation needs to consider that histological scoring reproducibility is at best moderate.• There is a clear need for additional investigations of outcomes for all features and all categories of AMR.○ Any such studies should follow the Banff 2017 recommendations on best practice for pathology endpoints in clinical trials , in part• The CHMP recognized the issues in defining AMR.• The CHMP welcomed and endorsed the suggestion to initiate a discussion on the use of the Banff classification as a tool to define AMR as an endpoint in clinical trials, in addition to its diagnostic and research value.• The rationale behind the restricted definitions of aAMR and caAMR for use as primary endpoints was well received by the CHMP.○ For this to happen, evidence-based classification and state-of-the-art, transparent, and standardized review processes of scientific data are required to demonstrate the usefulness of the restricted Banff definitions for AMR.


KMT2D is found. As somatic KMT2D variants can be found in 5–10% of tumors a tumor predisposition in Kabuki syndrome is discussed. So far less than 20 patients with Kabuki syndrome and a concomitant malignancy have been published. Here we report on a female patient with Kabuki syndrome and a c.2558_2559delCT germline variant in KMT2D who developed an embryonal rhabdomyosarcoma (ERMS) at 10 years. On tumor tissue we performed DNA-methylation profiling and exome sequencing (ES). Copy number analyses revealed aneuploidies typical for ERMS including  gains of chromosomes 2, 3, 7, 8, 12, 15, and 20 and 3 focal deletions of chromosome 11p. DNA methylation profiling mapped the case to ERMS by a DNA methylation-based sarcoma classifier. Sequencing suggested gain of the wild-type KMT2D allele in the trisomy 12. Including our patient literature review identified 18 patients with Kabuki syndrome and a malignancy. Overall, the landscape of malignancies in patients with Kabuki syndrome was reminiscent of that of the pediatric population in general. Histopathological and molecular data were only infrequently reported and no report included next generation sequencing and/or DNA-methylation profiling. Although we found no strong arguments pointing towards KS as a tumor predisposition syndrome, based on the small numbers any relation cannot be fully excluded. Further planned studies including profiling of additional tumors and long term follow-up of KS-patients into adulthood could provide further insights.Kabuki syndrome is a well-recognized syndrome characterized by facial dysmorphism and developmental delay/intellectual disability and in the majority of patients a germline variant in The online version contains supplementary material available at 10.1007/s10689-022-00306-z. KMT2D  or KDM6A  can be identified [KMT2D variants are observed in approximately 5–10% of all cancers [KMT2D are supposed to be driver events in various tumor types [Kabuki syndrome (KS), also known as Niikawa-Kuroki syndrome or Kabuki-make-up syndrome, is a syndrome where affected persons present with a characteristic face, including arched eyebrows with a sparse lateral one-third and long palpebral fissures with eversion of the lower eyelids. Other features are hypotonia and developmental delay/intellectual disability . In the entified . This maentified , 7. Such cancers . This fr cancers  and mutaor types . To dateor types . KMT2D for types  and is ior types , 38. Alsor types . NeverthThe clinical history of the patient is described in the results section. The parents of the patient provided written informed consent for the use of archival tissue for further analyses and consent for publication.FOXO1 and EWSR1 break-apart probes were conducted as part of routine clinical diagnostics and the former re-evaluated by two bone- and soft-tissue pathologists (M.v.d.H and Ra.S.).Histopathological, immunohistochemical and FISH analyses with For the present study, after consent from the parents of the patient, additional molecular analyses were performed on fresh-frozen tumor material from the primary biopsy. For this DNA was extracted from fresh-frozen tumor tissue with a Promega Maxwell RSC DNA FFPE kit  according to manufacturer’s instructions.DNA-methylation profiling: For DNA methylation analysis of the tumor DNA the Illumina Infinium® array technology  using the Infinium Methylation EPIC BeadChip (850K array) was used following the manufacturer’s instructions. Raw methylation data was processed as analogous to Wagener et al. and further described in the Supporting methods [https://www.molecularneuropathology.org/msp/ [ methods , 41. Fororg/msp/ . DNA metorg/msp/ . For coporg/msp/ . Subsequorg/msp/ .Exome sequencing was performed as described in the Supporting methods. In brief, tumor-only exome sequencing was performed on a NextSeq  with Illumina Nextera™ Exome Kit. For data-analysis evaluation was restricted to a virtual gene panel of 95 cancer predisposition related genes  with addition of KMT2D and KDM6A, as well as 10 genes recurrently mutated in embryonal and/or fusion negative rhabdomyosarcoma (Supporting Table S1) [able S1) –48. The The English literature published till September 1st 2021 was searched for publications with patients with a diagnosis of KS and a concomitant malignancy. The search strategy is outlined in detail in the Supporting Methods.KMT2D (g.49444907_49444908) predicted to result in a p.(Pro853Argfs*3) change at the protein level . She had the classical facial features of the syndrome as assessed by an expert dysmorphologist (C.T.R.M.S.). Her height growth followed -2 SD and she had a nasal speech. She was included in a clinical trial investigating the metabolic effect of growth hormone in children with KS [A 10-year old girl had a clinical and molecular diagnosis of KS with a c.2558_2559delCT pathogenic variant in FOXO1 and EWSR1 break negative.The pre-treatment core needle biopsy and post-chemotherapy excisional specimen were histopathologically analysed Fig. a–d and nDNA-methylation analyses: the methylation profile obtained with genome-wide epigenomic profiling of the current case was analysed using the “DKFZ-sarcoma classifier” and showed a methylation class of rhabdomyosarcoma  confirming the histopathological diagnosis. DNA-methylation based CNV-analysis showed amongst others,  gains of chromosomes 2, 3, 7, 8, 12, 15, and 20 and 3 focal losses in chromosome 11p /differentially methylated region 1 (DMR1) and a hypomethylation of KCNQ1/KCNQ1OT1 imprinting center 2 (IC2)/differentially methylated region 2 (DMR2) was seen .Exome sequencing revealed, amongst others, sequence variants in ERCC5 and TP53. In KDM6A one synonymous and in KMT2D one synonymous and one intronic variant were observed (Supporting Table S3). The germline variant in KMT2D was not identified in the tumor-DNA as a result of low-coverage at this position  but confirmed with Sanger sequencing . The Sanger sequence, moreover, suggests a gain of the wild-type and not the mutated allele in the trisomy 12  or KDM6A (n = 1) variant was identified. One patient, patient no.8, had a clinical diagnosis of KS but was negative for KMT2D and KDM6A variants by exome sequencing and array-CGH [MLL2 (KMT2D) and KDM6A as cause of KS in 2010 and 2012/2013 [With literature review we identified, including the patient from the present study, 18 patients with a clinical and/or molecular diagnosis of KS who developed a malignancy (Table rray-CGH . In othe012/2013 –56. In e012/2013  and not 012/2013 ) and neuThe reported malignancies in patients with a clinical and/or molecular diagnosis of KS are bone- and soft-tissue tumors (n = 5), hematologic malignancies (n = 5), embryonal tumors (n = 4) and tumors not belonging to any of the aforementioned malignancy groups (n = 4). Among the bone- and soft-tissue tumors, 3 of the 5 cases were reported as sarcoma . The most frequent hematologic malignancy was Burkitt lymphoma which was seen in 3 patients. Precursor B-cell acute lymphoblastic leukemia (pre-B-ALL) and Hodgkin’s lymphoma were seen in one patient each. Two patients presented with a neuroblastoma, and Wilms tumor, fetal-type hepatoblastoma, ependymoma, hepatocellular carcinoma, carcinoma of unknown primary origin and endometrial cancer were seen in one patient each.could point to a familial predisposition for leukemia. Patient 16 [In addition, the reports were screened for potential tumor predisposing and/or contributing factors other than KS. In case 10 , with prtient 16 , hepatoctient 16  had a EBtient 16  developetient 16  an EBV-aKDM6A variant) most reports lacked a detailed description. (Molecular)cytogenetic data were provided only in three published cases. For patient 6 [KMT2D variant was present in both germ-line and tumor DNA in a heterozygous state. Apart from the case included in the present study, in none of the cases next-generation sequencing and/or methylation profiling on the tumor were performed. Although most of the tumors do not meet (current) World Health Organization (WHO) Classification of Tumours diagnostic criteria for the reported tumor entities and therefore caution has to be made with drawing conclusions based on (some of the) provided diagnoses, it appears that overall the clinical presentation of the tumors in the patients does not seem to be very unusual with regard to age and sex distribution, site of presentation and histopathological characteristics . Although patient 18 (KDM6A variant) developed endometrial cancer (subtype not provided) at a young age (≤ 31 years), approximately ≤5% of endometrial cancers are reported to occur in women younger than 40 years [Although histopathological features were reported in 10 of the 16 patients  oncogenic potential of KMT2D and its role in cancer we refer to recent articles [In our study we present the history of a patient with Kabuki syndrome (KS) with a germline articles –68 and rarticles , 69, 70.articles , a diseaarticles , 73. AltKMT2D would, if being a genuine tumor predisposition gene (TPG), predispose to a rather wide range of malignancies including bone- and soft-tissue tumors, hematological malignancies, embryonal tumors and carcinoma’s. Although many TPGs predispose to a single or limited types of tumors , based on the observed tumor spectrum, KMT2D would belong to a group of TPGs predisposing to a broader spectrum of tumours like seen for TP53, PTEN, STK11 and DICER1.First, from a pediatric-oncology perspective it seems that the landscape of reported malignancies occurring in patients with KS broadly resembles that of the pediatric population in general. In the literature review we identified 18 patients with KS presenting with in total 19 malignancies. This included solid tumors in 13 patients  and hematologic malignancies (n = 5). The one additional patient with KS but without accompanying clinical information had a diagnosis of Burkitt lymphoma  (SupportKMT2D variants are recurrent but not highly frequent in Burkitt lymphoma occurring in ≤ 15% patients [Kmt2d-loss mouse models which showed (after immunization) an enhanced germinal-center response with an increase in the number of germinal-center B-cells with increased proliferation [KMT2D variants may not have a direct classic tumor predisposition effect but may instead increase the chance of developing Burkitt lymphoma by an increased germinal-center response and proliferation.Of interest, although hematologic malignancies are common in both patients with KS and the general pediatric population the distribution of reported malignancies is different: for KS (including patient S1 from Table S4) 4 patients with Burkitt lymphoma –27 and 1patients –81. Intrpatients , 82—and patients –85 and tpatients . In lineferation , 87 it mAs three EBV + malignancies  were reported it might be speculated that the combination of immune deficiency in patients with KS and EBV infection could—in analogy to other inborn errors errors of immunity—contribute to an increased susceptibility to develop EBV + lymphoproliferations and tumors , 89. HowKMT2D variants are mostly only infrequently reported. In contrast, malignancies in which somatic KMT2D variants are highly recurrent typically do not or only infrequently occur in patients with KS. E.g., somatic variants involving KMT2D are only infrequently reported in  rhabdomyosarcoma [KMT2D variants can be identified in approximately 5% [KMT2D variants varies strongly between individual genetic subgroups and is high(er) in e.g. the ERG/DUX4 and ZNF384 [KMT2D variants only (very) infrequently occur. In contrast, in other cancers including, amongst others, pediatric- and adult diffuse large B-cell lymphoma (DLBCL) (20–35%) [KMT2D variants are (highly) recurrent but these cancers have not been reported in patients with KS (yet). However, with a lack of longitudinal studies it remains unclear whether KS patients reach the ages at which many of these tumor types are most prevalent. In cancer truncating KMT2D variants have been reported with varying frequencies [KMT2D variants found 80% of the germline variants causing KS to be protein truncating. On the other hand, somatic KMT2D variants in cancer where predominantly missense with only 35% were predicted to be protein truncating [KMT2D missense variants [KMT2D variants no variant classification is provided [KMT2D variants has been reported which might be relevant considering the (relatively) frequent germline origin of KMT2D missense variants initially detected with sequencing of tumor material [KMT2D variants non-mutually exclusive parallels can be drawn with the situation for ARID1A/B- and SMARCB1. E.g. for ARID1A/B truncating germline variants cause Coffin-Siris-Syndrome but may not predispose to cancer although truncating somatic variants are frequently present in cancer [SMARCB1 both the type (truncating versus non-truncating missense) and location in the gene determine the phenotype  [Second,  for other malignancies reported in patients with KS, besides Burkitt lymphoma, somatic osarcoma –48, 90, osarcoma –95 and oosarcoma –81. Hepaately 5% –99. In pd ZNF384 , 101 read ZNF384 , 100–103d ZNF384 –106, neud ZNF384 , 108 andd ZNF384 , 109–111(20–35%) , 113, ad(20–35%) , 15, nod(20–35%) –116, (no(20–35%) , 117, up(20–35%) –120, eso(20–35%) , 122 and(20–35%) –125 somaquencies , 70, 126uncating  it can nvariants  a dominavariants . Moreoveprovided  and depeprovided , 130–134material . Regardin cancer , 137. InKMT2D as overall in cancer bi-allelic variants are rare and most variants are present in heterozygous state [KMT2D we cannot exclude such variant because of incomplete coverage (95%) of the gene. The genome wide epigenetic profiling  and CNV-analysis  revealed a for ERMS typical aberrations and profile [KMT2D variant due to insufficient coverage at this position and in this region. However, we confirmed the variant with Sanger sequencing. Although taking the intrinsic limitations of quantification and Sanger sequencing into account the pattern of the peak-heights of the wild-type and mutated-sequence is suggestive for a gain of the wild-type and not the mutated allele.Third, when interpreting the molecular data it should be taken into account that, in contrast to some other tumor predisposition genes, a functional read-out like bi-allelic involvement (e.g. Lynch syndrome) is not useful for us state . In addi profile , 47, 138In conclusion we present the first exome wide genomic and genome wide epigenomic analyses of a malignancy occurring in a patient with KS. Our molecular findings and observations from the literature neither prove nor rule out a potential tumor predisposition for KS. Regarding the tumor spectrum and age of onset of the tumor in patients with Kabuki syndrome it was observed that this broadly resembled that of the pediatric population in general. However, even an in vitro or in vivo oncogenic effect of KMT2D perturbation might not directly translate to a clinical relevant tumor predisposition. Regarding the latter, one should consider that if in KS the cancer-frequency would exceed a reasonable threshold for tumor surveillance (e.g. ≥ 1 ~ 5% for other pediatric cancer predisposition syndromes ) and if Supplementary file1 (PDF 502 kb)Below is the link to the electronic supplementary material.


Antimicrobial resistance (AMR) represents one of the most crucial threats to public health and modern health care. Previous studies have identified challenges with estimating the magnitude of the problem and its downstream effect on human health and mortality. To our knowledge, this study presents the most comprehensive set of regional and country-level estimates of AMR burden in the WHO European region to date.We estimated deaths and disability-adjusted life-years attributable to and associated with AMR for 23 bacterial pathogens and 88 pathogen–drug combinations for the WHO European region and its countries in 2019. Our methodological approach consisted of five broad components: the number of deaths in which infection had a role, the proportion of infectious deaths attributable to a given infectious syndrome, the proportion of infectious syndrome deaths attributable to a given pathogen, the percentage of a given pathogen resistant to an antimicrobial drug of interest, and the excess risk of mortality (or duration of an infection) associated with this resistance. These components were then used to estimate the disease burden by using two counterfactual scenarios: deaths attributable to AMR  and deaths associated with AMR . Data were solicited from a wide array of international stakeholders; these included research hospitals, surveillance networks, and infection databases maintained by private laboratories and medical technology companies. We generated 95% uncertainty intervals (UIs) for final estimates as the 25th and 975th ordered values across 1000 posterior draws, and models were cross-validated for out-of-sample predictive validity.Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecium, Streptococcus pneumoniae, and Acinetobacter baumannii. Methicillin-resistant S aureus was shown to be the leading pathogen–drug combination in 27 countries for deaths attributable to AMR, while aminopenicillin-resistant E coli predominated in 47 countries for deaths associated with AMR.We estimated 541 000 deaths (95% UI 370 000–763 000) associated with bacterial AMR and 133 000 deaths (90 100–188 000) attributable to bacterial AMR in the whole WHO European region in 2019. The largest fatal burden of AMR in the region came from bloodstream infections, with 195 000 deaths (104 000–333 000) associated with resistance, followed by intra-abdominal infections (127 000 deaths [81 900–185 000]) and respiratory infections (120 000 deaths [94 500–154 000]). Seven leading pathogens were responsible for about 457 000 deaths associated with resistance in 53 countries of this region; these pathogens were, in descending order of mortality, The high levels of resistance for several important bacterial pathogens and pathogen–drug combinations, together with the high mortality rates associated with these pathogens, show that AMR is a serious threat to public health in the WHO European region. Our regional and cross-country analyses open the door for strategies that can be tailored to leading pathogen–drug combinations and the available resources in a specific location. These results underscore that the most effective way to tackle AMR in this region will require targeted efforts and investments in conjunction with continuous outcome-based research endeavours.Bill & Melinda Gates Foundation, Wellcome Trust, and Department of Health and Social Care using UK aid funding managed by the Fleming Fund. Review on Antimicrobial Resistance estimated that AMR could result in the global loss of 10 million lives per year by 2050, with substantial economic ramifications.Antimicrobial resistance (AMR) represents a salient challenge of our time, as the loss of effective antimicrobials might result in common infections becoming life threatening and hinder the ability to perform common surgical procedures and other medical treatments. The Sustainable Development Goals (SDGs) consider AMR to be both a global public health problem and a societal issue.Evidence before this studyReview on Antimicrobial Resistance from 2016 concluded that as many as 10 million people could die annually from AMR by 2050. This concerning trend was recently confirmed by the 2019 global burden of AMR study, which showed that AMR is a leading cause of death around the world, particularly in low-resource settings. Through an in-depth search of the articles available in PubMed covering exposure to resistant bacterial agents  represents an emerging and alarming threat to human health worldwide. The highly cited Added value of this studyTo our knowledge, this study provides the most comprehensive analysis of the burden of AMR in the WHO European region to date, providing estimates for 53 countries, 23 bacterial pathogens, and 88 pathogen–drug combinations in 2019. These estimates advance previous related work in the WHO European region in several ways. First, this study offers an expanded scope, including additional countries in Europe, and an increased number of pathogen–drug combinations. Second, we use the major methodological innovations put forth in the 2019 global burden of bacterial AMR study. Third, we draw on two different counterfactual scenarios, acknowledged previously in other studies, to describe the magnitude of the problem within the WHO European region. Fourth, this study allows for comparability with other causes of death since it builds on estimates of disease incidence, prevalence, and mortality from the Global Burden of Diseases, Injuries, and Risk Factors Study 2019.Implications of all the available evidenceOur estimates indicate that bacterial AMR is a substantial problem in the WHO European region, with stark differences between subregions and specific countries. Such estimates of the impact of AMR on morbidity and mortality are indispensable for informing public health investment decisions for each country in this region. Additionally, highlighting specific pathogens and pathogen–drug combinations with the highest estimated burden might specifically inform policy targets and policy design—concentrating on expanding infection prevention and control programmes, patient awareness, and informing research priorities in the field of antibiotic and vaccine development.Coordinated global-level, regional-level, and country-level strategies are necessary to attenuate the emergence and spread of AMR; however, considering different trends in various parts of the world,Most AMR studies have focused on the EU and European Economic Area (EEA); hence, more comparable information and a comprehensive, pan-European overview is needed, especially if we aim to compare data before and during the COVID-19 pandemic in the future. Previous ECDC surveillance reports have also concentrated on invasive isolates  of eight bacterial species deemed of public health importance in EuropeCassini and colleaguesThis study extends the results of the 2019 global burden of AMR studyStreptococcus pneumoniae. We used clinical breakpoints and methods from the Clinical and Laboratory Standards Institute (CLSI) as guidance for classification of isolates into categories of susceptible or resistant.Our input data consisted of 471 million individual records or isolates covering 7585 study-location-years obtained from surveillance systems, hospital systems, systematic literature reviews, and other sources . From thOur overall approach can be divided into five broad components: the number of deaths in which infection was implicated, the proportion of infectious deaths attributable to a given infectious syndrome, the proportion of infectious syndrome deaths attributable to a given pathogen, the percentage of a given pathogen resistant to an antimicrobial drug of interest, and the excess risk of death or duration of an infection associated with this resistance.We followed GATHERIn our approach, ten estimation steps took place within the aforementioned five broad modelling components . In estiTo generate burden estimates of multiple pathogen–drug combinations that were mutually exclusive within a pathogen (and could thus be added), we introduced a population-attributable fraction (PAF) for each resistance profile with resistance to at least one drug. This metric considers prevalence of resistance, excess risk, and a redistribution of burden to each antibiotic on the basis of the respective excess risk.In estimation step ten, we computed two counterfactual scenarios to quantify the benefit of eliminating drug-resistant infections, estimating the drug-resistant burden as deaths and DALYs directly attributable to bacterial AMR on the basis of the counterfactual of drug-sensitive infection, and deaths and DALYs associated with bacterial AMR on the basis of the counterfactual of no infection .In a scenario in which drug-resistant infections are replaced with drug-susceptible ones, we considered the excess risk of resistance, known as the attributable to AMR counterfactual scenario. Deaths attributable to AMR were calculated by multiplying the number of deaths for each underlying cause by the fraction of these deaths in which infection was implicated, followed by multiplying the fraction of infectious deaths attributable to each infectious syndrome. This was then multiplied by the fraction of infectious syndrome deaths attributable to each pathogen and by the PAF for each location-year and pathogen–drug combination.Under the no-infection counterfactual scenario, infections that are resistant would not occur; this is also termed the associated with AMR scenario. Calculations here closely follow the process described for the attributable to AMR counterfactual, except the PAF is replaced with the prevalence of resistance for each location-year and pathogen–drug combination. We used a similar approach to calculate DALYs for both counterfactual scenarios .Details on our modelling approach can be found in the 2019 global burden of AMR studyGiven the relationship between antibiotic consumption levels and AMR rates,Consistent with GBD methods,The funders of the study had no role in study design, data collection, data analysis, data interpretation, or the writing of the report.We estimated 1·2 million  deaths in 2019 involving one of 11 infectious syndromes  as an underlying or an intermediate cause of death in the WHO European region. Of these, we focused on 0·9 million (0·6–1·3) deaths that were caused by both susceptible and resistant bacterial agents to estimate AMR burden. The largest fatal burden in the region came from bloodstream infections (319 000 deaths [169 000–548 000] or 34%), followed by respiratory infections (231 000 deaths [186 000–293 000] or 25%) and peritoneal and intra-abdominal infections  associated with AMR and 133 000 deaths (90 100–188 000) attributable to AMR . Among 3Escherichia coli (153 982 deaths), Staphylococcus aureus (83 325 deaths), Klebsiella pneumoniae (68 994 deaths), Pseudomonas aeruginosa (43 801 deaths), Enterococcus faecium (40 800 deaths), S pneumoniae (39 383 deaths), and Acinetobacter baumannii  attributable to and 31 977 deaths (20 183–47 980) associated with AMR. This was followed by K pneumoniae (5865 deaths [3836–8647] attributable to and 16 496 deaths [10 845–23 828] associated with AMR) and S aureus  had the highest mortality counts for strains of E coli resistant to any antibiotic were seen in Bulgaria, with mortality rates per 100 000 of 7·29 (95% UI 4·01–12·13) attributable to and 29·29 (16·51–46·72) associated with AMR. Bulgaria also had the highest crude mortality rates for resistant K pneumoniae, with 4·59 deaths (2·63–7·46) attributable to and 14·70 deaths (8·85–22·95) associated with AMR, per 100 000 population. The highest crude mortality rates for resistant S aureus were observed in Portugal, with 5·65 deaths (3·33–8·52) attributable to and 24·39 deaths (19·26–30·91) associated with AMR, per 100 000 population  and in Türkiye for deaths associated with AMR (7·04 per 100 000 [4·56–10·66]). For S aureus, the lowest crude mortality rates were observed in Sweden for deaths both attributable to (0·45 per 100 000 [0·28–0·70]) and associated with AMR (2·10 per 100 000 ) and associated with AMR , 13·6 deaths (for K pneumoniae), and 12·8 deaths (for S aureus) per 100 000. Uzbekistan also had the highest age-standardised mortality rates per 100 000 attributable to AMR for E coli (4·1 deaths) and K pneumoniae (3·7 deaths), whereas Türkiye had the highest AMR-associated S aureus mortality, with 2·9 deaths per 100 000. The lowest age-standardised mortality rates per 100 000 population for E coli, K pneumoniae, and S aureus were observed in Finland (4·2 deaths associated with and 0·9 deaths attributable to AMR for E coli), Switzerland (0·9 deaths associated with and 0·2 deaths attributable to AMR for K pneumoniae), and Sweden (0·9 deaths associated with and 0·2 deaths attributable to AMR for S aureus).The highest age-standardised mortality rates associated with AMR for S aureus was the leading combination in 27 countries (51% of the whole region) for deaths attributable to AMR, followed by E faecium resistant to fluoroquinolones in eight countries, and E coli resistant to third-generation cephalosporins in six countries (E coli predominated as the leading pathogen–drug combination in 47 countries (89% of the region) for deaths associated with AMR , which was proposed in 2019 and will be further informed by studies akin to ours. A notable difference with previous European results is that our results indicate a larger burden of aminopenicillin-resistant E coli than that which is third-generation cephalosporin resistant, whereas Cassini and colleagues found the reverse.K pneumoniae in the region should be noted as well, reaching almost 20 000 attributable and almost 70 000 associated deaths. This is in line with other studies highlighting this pathogen as the fastest growing AMR threat in Europe in terms of human morbidity and mortality,Where specific pathogens are concerned, we have shown that E coli), and that resistance rates to this antimicrobial can remain high despite decades of reduced consumption—primarily due to resistance co-selection in bacteria driven by genomically colocalised and yet laterally transferable resistance determinants, low fitness cost, and other mechanisms.Patient exposure to antimicrobial agents is one of the most important factors in the selection of drug-resistant bacterial mutants. Generally, a positive correlation between use and resistance was evident for the WHO European region as a whole for most antimicrobials, and the relationship was usually the strongest for countries in western and central Europe. One of the exceptions was trimethoprim–sulfamethoxazole, but studies have already shown that variations in its use are not a major factor in determining the prevalence of resistance among several species . Hence, further efforts should be made to swiftly develop and implement national action plans in countries where this process is lagging. Prioritising dedication to and sustainability of these efforts and setting transparent review schedules .These sources represent the backbone of ECDC reports, as well as a 2022 joint ECDC-WHO–Europe report,P aeruginosa or A baumannii due to our approach to multidrug-resistant infections .RA reports consulting fees from AbbVie; and payment or honoraria for lectures, presentations, speaker's bureaus, manuscript writing, or educational events from AbbVie, B Braun, Sandoz, and Laropharm, all outside the submitted work. CH and AP report grants from the Romanian National Authority for Scientific Research and Innovation, CNDS-UEFISCDI, for project number PN-III-P4-ID-PCCF-2016-0084 and project number PN-III-P2-2.1-SOL-2020-2-0351, outside the submitted work. CH also reports grants from the Romanian Ministry of Research Innovation and Digitalization MCID for project number ID-585-CTR-42-PFE-2021, outside the submitted work. A-FAM reports grants or contracts from MilkSafe, research co-financed by the EU European Regional Development Fund and Greek national funds through the operational programme Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH–CREATE–INNOVATE (T2EDK-02222), as well as from ELIDEK ; stocks in a family winery; and other financial or non-financial interests in the BGI Group as a scientific officer, all outside of the submitted work. BS reports grants or contracts from the Fleming Fund; and leadership or fiduciary role in other board, society, committee or advocacy group, paid or unpaid, with the GBD Scientific Council and WHO RGHS, all outside the submitted work. AS reports support for the present manuscript from the Bill & Melinda Gates Foundation as payment to their institution, and paid participation on a Scientific Advisory Board for Vivli, outside the submitted work. All other authors declare no competing interests.


D4R has also been investigated as a target for antipsychotics due to its high affinity for clozapine. As opposed to the closely related dopamine D2 receptor (D2R), dopamine-induced arrestin recruitment and desensitization at the D4R have not been studied in detail. Indeed, some earlier investigations could not detect arrestin recruitment and desensitization of this receptor upon its activation by agonist. Here, we used a novel nanoluciferase complementation assay to study dopamine-induced recruitment of β-arrestin2  and G protein-coupled receptor kinase-2 (GRK2) to the D4R in HEK293T cells. We also studied desensitization of D4R-evoked G protein-coupled inward rectifier potassium  current responses in Xenopus oocytes. Furthermore, the effect of coexpression of GRK2 on βarr2 recruitment and GIRK response desensitization was examined. The results suggest that coexpression of GRK2 enhanced the potency of dopamine to induce βarr2 recruitment to the D4R and accelerated the rate of desensitization of D4R-evoked GIRK responses. The present study reveals new details about the regulation of arrestin recruitment to the D4R and thus increases our understanding of the signaling and desensitization of this receptor.The dopamine D The D4R is encoded by the DRD4 gene, which is expressed most abundantly in the retina, with moderate expression in brain tissue  channels have been repeatedly demonstrated  and D2R-NP  were in pcDNA3.1+ . D4R-NP was designed to be analogous to D2R-NP, which was previously described by Laschet et al.  to promote surface expression, followed by a FLAG tag (DYKDDDDK). For oocyte experiments, D4R, RGS4, GIRK1 (Kir3.1), GIRK4 (Kir3.4), and GRK2 were in pXOOM . Both D4R constructs were based on the four-repeat variant of human D4R (D4.4R) and were, along with GRK2-LgBiT  and untagged human βarr2 and GRK2, synthesized by Genscript, Inc.  and cloned into pXOOM. LgBiT-βarr2 , followed by in vitro transcription using the T7 mMessage mMachine kit . The concentration and purity of cRNA were determined by spectrophotometry.in pNBe3, Promega, Madison, WI) was also a gift from Drs. Julien Hanson and Céline Laschet. For oocyte experiments, the plasmids were linearized using the appropriate restriction enzymes  were incubated at 37 °C with 5% CO2 in 10 cm culture plates  containing Dulbecco’s modified eagle medium  supplemented with 0.01% penicillin/streptomycin (Thermo Fisher Scientific) and 10% FBS (Thermo Fisher Scientific). Cells were transfected with 1 µg/dish D4R-NP or D2R-NP and 1 µg/dish LgBiT-βarr2/GRK2-LgBiT along with 10 µg untagged GRK2, when indicated, using linear polyethylenimine , dissolved in DMSO and diluted in HBSS, were injected to yield a final concentration of 10 µM (clozapine) or 1 µM (raclopride). HBSS injection was used as control.Xenopus laevis, were surgically isolated as previously described . When applicable, 0.6–19 ng/oocyte of βarr2 and 0.3 ng/oocyte of GRK2 cRNA were also injected.The oocytes of the African clawed toad, escribed . The sur2, 0.4 mM CaCl2, 15 mM HEPES, 1 mM ascorbic acid, adjusted to pH 7.4 with NaOH), yielding a K+ reversal potential of ∼−40 mV. Ascorbic acid was included to prevent spontaneous oxidation of DA.Following RNA injection and 6 days of incubation at 12 °C, electrophysiological experiments were performed on the oocytes using the parallel eight-channel, two-electrode voltage-clamp OpusXpress 6000A  . Continu4R and D2R constructs was evaluated essentially as described by .Y is the response, Top is the maximal response as a fraction of the mean control (HBSS) luminescence and X is the logarithm of dopamine concentration. Data points are presented as mean ± SEM throughout. pEC50 values from individual experiments were compared using Student’s paired t-test, testing differences in pEC50s obtained from experiments performed in the presence or absence of exogenous GRK2. Experiments with and without GRK2 were carried out on the same day with cells of the same passage number.The following equation was used for fitting:Electrophysiology recordings were initially screened for inclusion in Clampfit 10.6 (Molecular Devices). Recordings in which holding currents at −40 mV were stable before and after the −80 mV step and in which the current (at −80 mV) after dopamine washout was equal to or smaller than before dopamine application were included in the analysis. Recordings were peak normalized and time-averaged using Matrix Laboratory 2018b .Statistical analysis was performed in GraphPad Prism 8 using the two-tailed, paried or unpaired Student’s t-test, one-way ANOVA with Dunnett’s test for multiple comparisons, or repeated measures ANOVA with Sidak’s test for multiple comparisons, as appropriate, for normally distributed data. Mann-Whitney test, Wilcoxon matched-pairs signed rank test, or Kruskal–Wallis test with Dunn’s correction for multiple comparisons were used when normal distribution of data could not be assumed. Normality was assessed using the Shapiro-Wilk test. The significance threshold was set to 0.05.4R. To this end, we adapted a modified nanoBiT assay, which was recently demonstrated to work well with D2R (4R. As the higher-affinity nanoluciferase fragment 'native peptide’ (NP) was reported to result in a higher signal-to-noise ratio compared to the lower-affinity SmBiT . The larger complementary fragment (LgBiT) was fused to the N-terminus of βarr2 (LgBiT-βarr2). In HEK293T cells transfected with D4R-NP and LgBiT-βarr2, a concentration-dependent increase in luminescence was observed when cells were stimulated with increasing dopamine in the presence of a constant concentration of the substrate furimazine  revealed a dopamine concentration-dependent increase of luminescence (4R-NP. The luminescence response in cells coexpressing D4R-NP and GRK2-LgBiT returned towards baseline (in the continued presence of dopamine) much faster than what was observed in cells co-expressing D4R-NP and LgBiT-βarr2 . For comparison, we also assessed the recruitment of GRK2 to the D2R. Similar to the findings with D4R-NP, dopamine concentration-dependently increased luminescence in cells transfected with GRK2-LgBiT and D2R-NP, although the fold-over-baseline increase in luminescence was greater than with the D4R-based construct (2R-NP (2R antagonist raclopride (1 µM) rapidly reversed the dopamine-induced luminescence increase  are expressed in the central nervous system and capable of interacting with D2-like receptors, βarr2 has been found to play the major role in D2R signaling and desensitization in vivo  and less likelihood of steric interference of the enzyme fragments with the interaction between the two tagged proteins.We note that some previous investigations that did not detect Dentation  or fluorentation . On the entation  or bioluractions . Assay sractions  in the practions  may haveXenopus oocyte GIRK assay is another highly sensitive assay that allows for kinetic, live-cell experiments. The dosage-dependent increase in GIRK response decay rate observed with increasing amounts of coinjected βarr2 cRNA suggests that βarr2 mediates desensitization of GIRK responses evoked by D4R, similar to what has been described for D2R in the corresponding assay , a higher affinity of GRK2 for unphosphorylated vs. phosphorylated D4R (with GRK2- D4R interaction decreasing once the receptor is phosphorylated), or both. Interestingly, the luminescence response in corresponding experiments with D2R-NP decayed considerably slower  remained similar in the presence or absence of exogenous GRK2. Rather, we would speculate that the decrease in maximal dopamine-induced luminescence signal is due to competition between GRK2 and βarr2 for binding to the receptor, as was also suggested by (2R. The stronger effect of GRK2 coexpression on D2R signalling would seem consistent with the higher fold-over-baseline increase in luminescence and the slower response decay in experiments with GRK2-LgBiT and D2R-NP as compared to D4R-NP, as noted above.The effect of GRK2 coexpression on LgBiT-βarr2 recruitment was more pronounced at the D the D4R . This si the D4R , suggestested by , who rep4R. We also demonstrated that βarr2 recruitment is enhanced by GRK2 coexpression, although this effect is less pronounced than at the D2R. In addition, the potentiating action of GRK2 was observed at the level of βarr2-mediated desensitization of GIRK current responses evoked by the D4R. Finally, evidence was obtained for transient dopamine-induced recruitment of GRK2 to the D4R. The nanoluciferase complementation assay employed here may be useful for drug discovery efforts targeting the D4R. In addition, the new information regarding GRK2 regulation of D4R may prove relevant for understanding the biological functions of this relatively little-explored dopamine receptor.In this study, we presented a nanoluciferase complementation assay able to report on dopamine-mediated βarr2 recruitment to the D


The Körner's septum (KS), persistent petrosquamosal lamina, is a bony lamina  that extends from the articular fossa to the mastoid apex, above the middle ear, and runs inferiorly and laterally to the facial nerve canal. The petrous and squamous bones meet at this septum. The anatomical structure of KS, which most frequently occurs at the level of the head of the malleus and/or the anterior semicircular canal, is described in depth in this work. The embryological elements of the temporal bone development that result in the formation of KS are taken into consideration. Clinically KS is considered an important anatomical variation, in the development of chronic diseases of the ear like chronic otitis media, especially attic retraction pockets, and cholesteatoma as it can contribute to attic blockage. Also, studies have found a significant association between tympanosclerosis and KS. High-resolution computed tomography (HRCT) and cone-beam computed tomography are the two imaging methods most commonly used to identify KS. It is observed that KS was associated with an increased risk for chronic otitis media, and residual cholesteatoma. The purpose of this review article is to provide a general overview of the KS and its clinical implication, as well as to summarize and discuss the latest clinical data regarding this entity. The Körner's septum (KS) is an osseous lamina that divides the mastoid's air cells within the temporal bone. In 1887, Hartmann described KS as a thin bony wall that lies in between the antrum petrosum and the antrum mastoideum. Later scholars noted its presence, but it was not until Körner that the clinical importance of the septum was brought up . The potThe KS is regarded as a component of the petrosquamosal suture since it is situated at the point where the petrous and squamous portions of the temporal bone meet. The petrosquamosal suture is divided into three segments, with different names in different publications, according to the literature -8. The mThe KS extends from the petrosquamosal fissure in the posterior part of the mandibular fossa, travels over the middle ear cavity , and then directs downward, laterally to the facial nerve canal , and into the direction of the mastoid apex. Mastoid cells are divided into deep and superficial cells by the posterior portion of KS. According to Proctor B, KS continuity may be broken at multiple points . The sepAnatomy of KS Mastoid cells are divided into deep and superficial cells by KS, which corresponds to the persistent petrosquamosal suture . AnalysiWojciechowski et al. state that the KS is divided into three sections: the anterior, which is the most constant and is located at the level of the head of the malleus, the superior, which is located at the level of the superior semicircular canal, and the posterior  . Some wrSquamous chronic otitis media and KSAccording to the Browning classification, there are two forms of squamous COM: inactive (squamous) COM-pars tensa/flaccida retraction and retained debris. Additionally, there is an active (squamous) COM with pars flaccida/tensa retraction, squamous-type epithelial debris, and inflammatory, pus-filled middle ear mucosa . KS can Diagnostic imaging of KSHRCT and cone-beam computed tomography (CBCT) of the temporal bone provides the clearest images of KS ,15. WithClinical relevance of KSKS could be a serious clinical issue. The antrum and epitympano-mastoid canal appear to be a false medial wall during antromastoidectomy ,5. The pIt also has a big impact on middle ear surgery since there is a higher probability of leaving behind residual cholesteatoma in the cog-covered posterior portion of the epitympanum. Therefore, it is recommended to conduct a mastoidectomy with anterosuperior tympanostomy in cases of broad KS . In pattThe prevalence of KS in healthy ears was reported to be 6.58%, compared to 30.4% in ears with retraction pockets and/or tympanic membrane adhesion, and 17.4% in cases of chronic otitis media without retraction pockets by Göksu et al., who did not validate these findings ,19. In 2Additionally, the bone plate known as the KS, which separates the mastoid cells, is called the petrosquamous lamina (at antrum). KS is more persistent in adults than in children in the temporal bone. In a few investigations, KS was observed in chronic otitis media with squamosal disease . Middle This study provides a better understanding of one of the important etiopathogenesis associated with COM especially the squamosal type, the KS. The causes of the displacement of the squamous portion of the growing temporal bone that results in KS during the second and third trimesters of pregnancy are yet unknown. However, credible research findings and a sizable number of clinical case reports support the hypothesis that the presence of KS increases the risk of developing inflammatory middle ear conditions like cholesteatoma, retraction pocket, and tympanosclerosis, likely as a result of decreased ventilation of the middle ear cavities. Clinically, it is crucial to perform thorough pre-surgical imaging diagnostics of the temporal bones using HRCT and/or CBCT. These tests enable otorhinolaryngologists to determine whether KS is present and, if it is, to categorize it as a complete or partial septum. However, more investigations are necessary to determine the impact of KS, a developmental variant and a serious clinical issue, on the course of middle ear inflammatory processes naturally.