Comparison Static and Dynamic Ultrasound Techniques of DDH: The Role of the Patient's Position.

Objectives: The ultrasound examination of the hip joint is performed in the static (Graf) technique in the lateral recumbent position and in the dynamic technique in the supine position. This study compares the two static and dynamic techniques and assesses the role of the patient's position in the examination of DDH. Methods: This cross-sectional study was conducted in 2020-2021 at Akbar Hospital, Mashhad University of Medical Sciences, Iran. 126 patients suspected of having DDH (199 hip) infants were enrolled in the study. All ultrasound examinations were performed with two static and dynamic techniques by a pediatric radiologist. Results: In the static and dynamic ultrasound examinations, the average alpha angle was 51.57 ± 6.41 degrees, and 53.41 ± 6.94 degrees, respectively. These changes were not statistically significant (P = 0.312). The relationship and agreement between instability with dynamic technique and instability with static technique (IIC unstable, D, III, and IV) were investigated. Significant agreement (Kappa=0.77 (95% CI: 0.66-0.87) with excellent clinical significance was obtained between the two ultrasound examination method. Also, in terms of DDH types in the static method with instability types in the dynamic method, a substantial agreement was found between the two examination methods (Kappa =0.67; (95% CI: 0.59-0.75) with good clinical significance. Conclusion: In the ultrasound examination of DDH with static and dynamic techniques, the change in the alpha angle was not statistically significant. Therefore, the hand of the radiologist is open in measuring alpha angles and there is no need to emphasize a specific position. The type of DDH in the static technique completely corresponded to the type of stability or instability in the dynamic technique.

GRAF1 Acts as a Downstream Mediator of Parkin to Regulate Mitophagy in Cardiomyocytes.

Cardiomyocytes rely on proper mitochondrial homeostasis to maintain contractility and achieve optimal cardiac performance. Mitochondrial homeostasis is controlled by mitochondrial fission, fusion, and mitochondrial autophagy (mitophagy). Mitophagy plays a particularly important role in promoting the degradation of dysfunctional mitochondria in terminally differentiated cells. However, the precise mechanisms by which this is achieved in cardiomyocytes remain opaque. Our study identifies GRAF1 as an important mediator in PINK1-Parkin pathway-dependent mitophagy. Depletion of GRAF1 (Arhgap26) in cardiomyocytes results in actin remodeling defects, suboptimal mitochondria clustering, and clearance. Mechanistically, GRAF1 promotes Parkin-LC3 complex formation and directs autophagosomes to damaged mitochondria. Herein, we found that these functions are regulated, at least in part, by the direct binding of GRAF1 to phosphoinositides (PI(3)P, PI(4)P, and PI(5)P) on autophagosomes. In addition, PINK1-dependent phosphorylation of Parkin promotes Parkin-GRAF1-LC3 complex formation, and PINK1-dependent phosphorylation of GRAF1 (on S668 and S671) facilitates the clustering and clearance of mitochondria. Herein, we developed new phosphor-specific antibodies to these sites and showed that these post-translational modifications are differentially modified in human hypertrophic cardiomyopathy and dilated cardiomyopathy. Furthermore, our metabolic studies using serum collected from isoproterenol-treated WT and GRAF1CKO mice revealed defects in mitophagy-dependent cardiomyocyte fuel flexibility that have widespread impacts on systemic metabolism. In summary, our study reveals that GRAF1 co-regulates actin and membrane dynamics to promote cardiomyocyte mitophagy and that dysregulation of GRAF1 post-translational modifications may underlie cardiac disease pathogenesis.

Length of treatment and ultrasound timing in infants with developmental dysplasia of the hip.

PURPOSE: Developmental dysplasia of the hip (DDH) joint is a complex condition that may lead to severe problems. Ultrasound scan (USS) in the first four-to-six weeks of life is considered the gold-standard for diagnosis while the Pavlik harness (PH) is a widely utilized method as first-line treatment. The purpose of this study is to compare clinical outcomes of infants with DDH in relation to the timing and frequency of USS following application of the PH. METHODS: Retrospective data were collected over a 5-year period from February 2017 to February 2022. We included patients who underwent the first USS post-diagnosis and PH application in two, three, four and six weeks. Two-hundred-twenty-five patients were included and divided in four groups according to timing of the first follow-up: week-2, n = 13; week-3, n = 66; week-4, n = 95; and week-6, n = 51. For every patient Graf classification, treatment length and number of follow-ups were documented. RESULTS: Week-3 and week-4 groups displayed a statistically significant shorter treatment length compared to week-6 group (p value < 0.001), while also demonstrating a lower number of sonographic follow-ups per patient compared to both week-6 (p value < 0.001) and week-2 (p value = 0.002 vs week-3; p value < 0.001 vs week 4). Week-4 group presented the highest treatment completion (56%) on first visit post-diagnosis. Conservative treatment with PH failed in 1.8% (4/225) and displayed no significant difference among all subgroups. CONCLUSIONS: Differences in timing of first USS post-DDH diagnosis and initiation of treatment can lead to discrete outcomes with implications to the clinical outcome and cost effectiveness.

Ultrasonographic screening for developmental dysplasia of the hip: the Graf method revisited.

Until the 1980s, the diagnosis of developmental dysplasia of the hip (DDH) was based on clinical examination and radiographic imaging. In 1980, Reinhard Graf developed his own ultrasonographic method for the examination of the infant hip joint. Graf's method evaluates the osseous and cartilaginous coverage of the femoral head by the acetabulum in the infantile hip joint by measuring the angles α and ß. The validity of Graf method is that with these measurements the hip joint is further classified by Graf classification into types I to IV that guide treatment. Currently, Graf method is considered the gold standard examination for the diagnosis of DDH in many European countries. This review article aims to discuss the incidence, risk factors and pathophysiology of DDH, and to emphasize on the Graf method for the evaluation, classification, prevention and further management of this entity.

The risk of DDH between breech and cephalic-delivered neonates using Graf ultrasonography.

PURPOSE: Developmental dysplasia of the hip (DDH) is one of the most common musculoskeletal disorder in infants. The most significant risk factors include female gender, breech presentation, left hip and family history. In this study, we utilized the Graf method at different time intervals to evaluate both breech-delivered and cephalic-born newborns. The objectives were to compare the incidence of DDH in cephalic and breech-delivered neonates and investigate whether the hip joints of neonates delivered in the breech position exhibit a distinct maturation pattern. MATERIAL AND METHODS: We studied prospectively 618 hip joints (309 newborns). Each hip joint was examined with the Graf method in four time periods as follows: Phase #1 (0-1 weeks), Phase #2 (1-4 weeks), Phase #3 (4-7 weeks), and Phase #4 (7-10 weeks). The α and ß angles for each hip joint were measured, and the hips were classified according to Graf classification. With our statistical analysis within the different phases, we were able to investigate potential variations in the maturation patterns between newborns delivered in the breech and cephalic delivery positions. RESULTS: A significant difference (at the 5% level) was observed in Phase 1 between breech and cephalic-delivered neonates (35.6-8.6%). This difference tended to decrease in next phases (13.6-1% in Phase 2, 2.5-0% in Phase 3 and 1.7-0% in Phase 4). A significant difference (at the 5% level) for cephalic-delivered neonates was also observed between Phase 1 and Phase 4 (8.5-0%), but the percentages were low. Additionally, the breech-delivered had extreme difference in incidence of DDH from Phase 1 to Phase 4 (35.6-11.9%, 2.5%, and 1.7%, respectively). CONCLUSION: It appears that there is an actual difference in the incidence of DDH between breech-delivered and cephalic-delivered neonates, although the difference may be less significant than previously considered. The majority of the breech-delivered neonates that were initially considered as pathological (Phase 1) are, in fact, healthy. This is ascertained in subsequent ultrasound examinations conducted in later phases (Phases 2-4), when the incidence of pathological cases decreases. This could be attributed to potential different maturation pattern between these groups.

Comparison of outcomes of different Graf grades of developmental dysplasia of the hip in infants treated with Tubingen splint versus Pavlik harness - A systematic review.

This systematic review was designed to compare the outcomes of the two braces against each other classified by the Graf method. The databases sources included PubMed, Embase, and Google Scholar. The keywords included "DDH Tubingen versus Pavlik" and Tubingen and Pavlik separately. Included papers provided specific data regarding success and failure rate, avascular necrosis (AVN), duration, and age of intervention. The excluded studies discussed surgeries, diagnosis and mechanism, and ones that weren't in English. Total of 20 papers were included, resulting in 1243 Tubingen and 420 Pavlik samples. It was seen that the Tubingen splint had a statistically significant greater success rate and lower failure rate for Graf 2, D, and 3 hips, while both braces were not very successful for Graf 4 at success rates less than 60 %. Tubingen also had a lower incidence of AVN. Both braces shared similar ages of intervention, duration, and time per day. Both braces are very comparable to each other, each having better success rates for lower Graf grades, which points to the importance of bracing earlier to improve the success rates. The Tubingen splint had a higher success rate, lower failure rate, and lower AVN rate compared to the Pavlik harness. This points to the Tubingen splint potentially being the preferred option for bracing in infants.

Universal in-house neonatal hips ultrasonography screening in the United Arab Emirates.

OBJECTIVES: To investigate if the incidence of developmental dysplasia of the hip (DDH) differs considering the geographical origin, clinical picture, or presence of risk factors in homogenous cohort of neonates born in Mediclinic Al Jowhara hospital, Al Ain, United Arab of Emirates (UAE). METHODS: Universal ultrasonography hips screening of the neonates in the maternity ward of Mediclinic Al Jowhara hospital, Al Ain, UAE, was carried out using the Graf method. The average age of the neonates was 3 days. Two groups were formed for comparison: I) the Gulf Cooperation Council (GCC) group (n=169, 47.7%), and II) the non-GCC group (n=185, 52.3%). RESULTS: The incidence of DDH was 1.7%. It was higher among neonates from the GCC region (2.9%) and significantly higher among girls from this region (6.3%). The incidence of immature hips (type IIa) was 9% and was similar regardless of origin or gender. CONCLUSION: Neonates from the GCC region, girls in particular, have a higher incidence of DDH. These results highlight the emergency to establish a national ultrasonography DDH screening program.

Chondro-osseous border in baby hip ultrasonography for developmental dysplasia of the hip: an indispensable litmus paper for the accuracy of scientific publications.

PURPOSE: The aim of our study was to use the chondro-osseous border (COB) as an indispensable assessment criterion to evaluate the quality of baby hip ultrasonography (US) images in the literature pertaining to the application of Graf's technique. MATERIALS AND METHODS: Our literature review search yielded 144 articles. Of these, 41 contained images that were stated to be based on the application of Graf's technique. Two reviewers, a radiologist and an orthopaedic surgeon, both course instructors for the use of baby hip US for developmental dysplasia of the hip (DDH), independently evaluated the articles to assess the diagnostic validity of the published images on the basis of a single criterion: the identification of the COB. RESULTS: Of the 41 articles which were analyzed, 15 contained images without a COB, which corresponds to 36% (roughly, one out of three). Articles from countries where universal screening is performed (Germany, Italy, Switzerland, Mongolia) were all correct. All the articles from Turkey, a country which has made significant contribution with published material about Graf's technique over many years, and most of the articles from China (80%), where Graf's technique has become popular in recent years, contained correct images. CONCLUSION: Published literature lacks strict criteria for the publication of correct images corresponding to Graf's technique in baby hip US. This fact raises concerns about the quality of published material (and, as a corollary, the quality of clinical application of the technique) and should have ramifications on scientific journal policies regarding the publication of such manuscripts.

Automatic and Human Level Graf's Type Identification for Detecting Developmental Dysplasia of the Hip.

BACKGROUND: Developmental dysplasia of the hip (DDH) is a common congenital disorder that may lead to hip dislocation and requires surgical intervention if left untreated. Ultrasonography is the preferred method for DDH screening; however, the lack of experienced operators impedes its application in universal neonatal screening. METHODS: We developed a deep neural network tool to automatically register the five keypoints that mark important anatomical structures of the hip and provide a reference for measuring alpha and beta angles following Graf's guidelines, which is an ultrasound classification system for DDH in infants. Two-dimensional (2D) ultrasonography images were obtained from 986 neonates aged 0-6 months. A total of 2,406 images from 921 patients were labeled with ground truth keypoints by senior orthopedists. RESULTS: Our model demonstrated precise keypoint localization. The mean absolute error was approximately 1 mm, and the derived alpha angle measurement had a correlation coefficient of R = 0.89 between the model and ground truth. The model achieved an area under the receiver operating characteristic curve of 0.937 and 0.974 for classifying alpha < 60° (abnormal hip) and < 50° (dysplastic hip), respectively. On average, the experts agreed with 96% of the inferenced images, and the model could generalize its prediction on newly collected images with a correlation coefficient higher than 0.85. CONCLUSIONS: Precise localization and highly correlated performance metrics suggest that the model can be an efficient tool for assisting DDH diagnosis in clinical settings.

Erratum: Comparison of treatment outcomes of stable and unstable developmental dysplasia of the hip with the Tübingen splint.

[This corrects the article DOI: 10.3389/fped.2022.976367.].

Assessment for late developmental hip dysplasia in a cohort of infants with risk factors and normal hip ultrasound.

Background: Developmental dysplasia of the hip (DDH) is a known orthopedic pathology of newborns that, if not diagnosed and treated, can lead to debilitating long-term consequences. Ultrasound has proven to be an effective method for the early diagnosis of this condition. Recently, reports of late DDH in populations at risk (breech presentation) and after negative ultrasound examination have emerged in the literature. Aim: The objective of the study was to assess the possible appearance of late DDH in Italian children with risk factors but negative ultrasound screening. Materials and methods: We selected patients with risk factors for DDH and a negative hip ultrasound from the medical records of children referred to the Hip Ultrasound Clinic (Rome, Italy) from January 2018 to November 2021. To identify possible cases of late DDH, from February 2022 to July 2022, all patients who met the inclusion criteria were submitted to orthopedic follow-up clinical evaluation. In the case of a pathological objective examination, radiography was performed. Results: Fifty-five patients (52.7% female, 52.7% with breech presentation, and 41.8% with a positive family history) met the inclusion criteria. The median age of gait onset was 13 months. The median age of orthopedic follow-up examination was 45 months. Only three patients (5.5%) had a pathological examination, but no x-ray were pathological. Conclusion: Our study has not documented cases of late DDH. Considering the small study population and the only clinical follow-up, further studies are needed to clarify the possible late development of this condition.

GRAF1 Regulates Brown and Beige Adipose Differentiation and Function.

Adipose tissue, which is crucial for the regulation of energy within the body, contains both white and brown adipocytes. White adipose tissue (WAT) primarily stores energy, while brown adipose tissue (BAT) plays a critical role in energy dissipation as heat, offering potential for therapies aimed at enhancing metabolic health. Regulation of the RhoA/ROCK pathway is crucial for appropriate specification, differentiation and maturation of both white and brown adipocytes. However, our knowledge of how this pathway is controlled within specific adipose depots remains unclear, and to date a RhoA regulator that selectively controls adipocyte browning has not been identified. Our study shows that expression of GRAF1, a RhoGAP highly expressed in metabolically active tissues, closely correlates with brown adipocyte differentiation in culture and in vivo. Mice with either global or adipocyte-specific GRAF1 deficiency exhibit impaired BAT maturation, reduced capacity for WAT browning, and compromised cold-induced thermogenesis. Moreover, defects in differentiation of mouse or human GRAF1-deficient brown preadipocytes can be rescued by treatment with a Rho kinase inhibitor. Collectively, these studies indicate that GRAF1 can selectively induce brown and beige adipocyte differentiation and suggest that manipulating GRAF1 activity may hold promise for the future treatment of diseases related to metabolic dysfunction.

Four decades of the Graf method in screening for developmental dysplasia of the hip (part I): Rightly the gold standard or of dubious benefit?

Introduction: The method of infant hip sonography according to Graf is used for general hip screening in Serbia, Austria, and Germany and is considered the gold standard. In other countries, such as the USA, however, it is not well accepted and is claimed to lead to high costs and overtreatment. The aim of this study was to investigate how many of the mentioned sources in a recent review article contained sonograms that met the quality criteria as taught in Graf's ultrasound courses. Methods: A systematic review published by Sakkers et al. was analyzed in terms of addressing the quality criteria of Graf's method. Studies that were suitable by title, abstract, manuscript, that contained an image of sonographic hip examination, and that were accessible were included into analysis. Results: Within the described review, there were 22 papers on the Graf method. Of these, 10 contained hip sonograms and were applicable for final analysis. The quality criteria according to Graf were not fulfilled within 5 of these 10 papers. Within these papers, there are examples of schematic sonograms that do not correspond with the quality criteria either. Conclusion: Skepticism regarding the Graf method may be based on user errors and insufficient application of the Graf quality assessment algorithm, which results in high intra- and inter-observer variations. Based on these findings, a working group was initiated to evaluate further work according to the same procedure (currently approximately 130 papers).

Impact of the coronavirus disease 2019 pandemic on ultrasound screening of developmental dysplasia of the hip and follow-up imaging: a report from southeastern Turkey.

OBJECTIVE: To investigate the effect of coronavirus disease 2019 restrictions on ultrasound (US) screening of developmental dysplasia of the hip (DDH) in a children's hospital. METHODS: The records of US screening of DDH were retrospectively evaluated in the pandemic period (April 2020 to July 2021) and the pre-pandemic period (January 2019 to February 2020). The monthly US number, sex, radiologist number, and age at the initial examination were recorded. RESULTS: A total of 6107 US scans were performed during the pre-pandemic period, which significantly decreased to 3340 during the pandemic. The number of monthly US scans performed did not change between the pre-pandemic (142.7/month) and pandemic (128.2/month) periods. The number of delayed examinations in the total population did not significantly change between the two periods. However, the number of delayed examinations in patients with abnormal hips was significantly increased during the pandemic compared with that in the pre-pandemic period. CONCLUSIONS: Coronavirus disease 2019 restrictions decreased the US screening rate of DDH by almost half, but the number of US scans performed by each radiologist was unchanged. The compliance with follow-up recommendations was reduced by half, which may lead to an increase in the incidence of delayed and untreated DDH cases.

Association between gene polymorphisms in the cyclophosphamide metabolism pathway with complications after haploidentical hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for patients with hematologic malignances. Haploidentical HSCT (Haplo-HSCT) is an alternative option for patients who do not have an HLA-matched donor. The use of post-transplantation high dose cyclophosphamide (PT-Cy) is commonly employed for graft-versus-host disease (GVHD) prophylaxis in haplo-HSCT. Cyclophosphamide (Cy) is an alkylating agent with antineoplastic and immunosuppressive activity, whose bioactivation requires the activity of polymorphic enzymes in the liver to produce phosphoramide mustard, which is a DNA alkylating agent. To identify polymorphisms in the genes of Cy metabolism and correlate them with post-HSCT complications [GVHD, sinusoidal obstruction syndrome (SOS), hemorrhagic cystitis (HC) and transplant-related mortality (TRM)], we designed a custom next-generation sequencing panel with Cy metabolism enzymes. We analyzed 182 patients treated with haplo-HSCT with PT-Cy from 2007 to 2019, detecting 40 variants in 11 Cy metabolism genes. Polymorphisms in CYP2B6, a major enzyme involved in Cy activation, were associated with decreased activity of this enzyme and a higher risk of Graf-versus-host disease (GVHD). Variants in other activation enzymes (CYP2A6, CYP2C8, CYP2C9, CYP2C19) lead to decreased enzyme activity and were associated with GVHD. Polymorphisms in detoxification genes such as glutathione S-transferases decreased the ability to detoxify cyclophosphamide metabolites due to lower enzyme activity, which leads to increased amounts of toxic metabolites and the development of III-IV acute GVHD. GSMT1*0 a single nucleotide polymorphism previously recognized as a risk factor for SOS was associated with a higher risk of SOS. We conclude that polymorphisms of genes involved in the metabolism of cyclophosphamide in our series are associated with severe grades of GVHD and toxicities (SOS and TRM) after haplo-HSCT and could be used to improve the clinical management of transplanted patients.

Four decades of developmental dysplastic hip screening according to Graf: What have we learned?

Purpose: Sonographic hip examination according to Graf is widely accepted standard for diagnosing developmental dysplastic hips (DDH) but it is criticized for alleged intra- and interobserver variations. This review was conducted to evaluate whether objective quality criteria according to the Graf method are fulfilled within scientific literature. Methods: A systematic literature search on Pubmed was performed using the search string: [(DDH) OR (Hip dysplasia)] AND (Graf). Studies suitable by title, abstract, manuscript, containing an image of sonographic hip examination and online accessibility were included into analysis. Results: 131 studies were included into final analysis. Only 68 (51.9%) presented correct sonographic images according Graf's criteria. 98 (74.8%) studies plotted alpha-angles (angle between bony roof line and base line) but only 85 (64.8%) studies beta-angles (angle between cartilage roof line and base line). Studies were contributed from 25 countries. Conclusion: Assumingly, skepticism regarding the Graf method is based on user errors and insufficient application of the Graf quality assessment algorithm resulting in high intra- and interobserver variations. When performed correctly, the Graf method is of high diagnostic value.

Comparison of treatment outcomes of stable and unstable developmental dysplasia of the hip with the Tübingen splint.

Background: The Tübingen splint was initially developed for the treatment of stable developmental hip dysplasia (DDH). Later on, some authors expanded its include for the treatment of unstable DDH, but there remain some controversies in the literature. This study aims to compare the outcome between stable and unstable DDH treated with a Tübingen splint. Methods: Epidemiological data and ultrasonographic data of all infants diagnosed with DDH and initially treated with a Tübingen splint at our institution between May 2017 and February 2020 were assessed retrospectively. We divided the population into stable and unstable hips using the Graf classification. Age at treatment initiation, duration of treatment, complications, and radiological outcome between 12 and 24 months were investigated. Results: We included a total of 45 patients (57 hips) affected by DDH treated with the Tübingen splint. Treatment has been successful in 93% of stable hips and only 40% of unstable hips. Radiological outcome at 1-year follow-up significantly correlated with initial Graf classification (p < 0.001). Conclusion: The Tübingen splint is a safe and effective treatment for stable hips, nevertheless, for unstable hips, closed reduction, and spica cast remains the gold standard.

The Anatomical Position of Graf's Standard Plane and Its Relationship With Pelvic Morphology: A Computed Tomography-Based Study.

Objective The Graf method is the most widely used ultrasonographic method for evaluating developmental dysplasia of the hip (DDH), and it relies on a set standard plane. However, no previous reports have discussed the detailed anatomical location of the plane. The aim of this study was to evaluate the exact anatomical position of Graf's standard plane in the pelvis and to ascertain the correlation between this position and pelvic morphology in children without abnormal pelvic morphology. Methods We retrospectively assessed the pelvic CT data of 32 children (64 hips) aged three to five years without abnormal pelvic morphology and measured the pelvic winging and acetabular anteversion and coverage. We defined the coronal plane that passed through the center of the bilateral femoral head as plane A. We determined that Graf's standard plane could be approximated by rotating plane A until the outer wall of the ilium was parallel to the sagittal plane, and we defined this as plane A'. AA' was defined as the angle from plane A to plane A' on the sagittal plane. The anterior rotational angle (clockwise, viewing from the right side) was measured as the positive AA'. Moreover, we measured the pelvic rotation, acetabular anteversion, and acetabular coverage and evaluated the correlation between AA' and these morphological parameters. Results The average AA' was -8.27° and AA' had a significant correlation with acetabular anteversion (Spearman's ρ=0.40**, p<0.01). Conclusions We found that Graf's standard plane, as determined by the CT scan, tilts slightly posteriorly. This information may be useful in improving the ease of ultrasonographic examination of DDH.

Rho GTPase-activating protein 10 (ARHGAP10/GRAF2) is a novel autoantibody target in patients with autoimmune encephalitis.

BACKGROUND: In 2010, we described a novel immunoglobulin G (IgG) autoantibody (termed anti-Ca after the index case) targeting Rho GTPase-activating protein 26 (ARHGAP26, also termed GTPase regulator associated with focal adhesion kinase [GRAF], or oligophrenin-like protein 1 [OPHN1L]) in autoimmune cerebellar ataxia (ACA). Later, ARHGAP26-IgG/anti-Ca was reported in patients with limbic encephalitis/cognitive decline or peripheral neuropathy. In several of the reported cases, the syndrome was associated with cancer. ARHGAP10/GRAF2, which is expressed throughout the central nervous system, shares significant sequence homology with ARHGAP26/GRAF. Mutations in the ARHGAP10 gene have been linked to cognitive and psychiatric symptoms and schizophrenia. OBJECTIVE: To assess whether ARHGAP26-IgG/anti-Ca co-reacts with ARHGAP10. METHODS: Serological testing for ARHGAP10/GRAF2 autoantibodies by recombinant cell-based assays and isotype and IgG subclass analyses. RESULTS: 26/31 serum samples (84%) from 9/12 (75%) ARHGAP26-IgG/anti-Ca-positive patients and 4/6 ARHGAP26-IgG/anti-Ca-positive CSF samples from four patients were positive also for ARHGAP10-IgG. ARHGAP10-IgG (termed anti-Ca2) remained detectable in the long-term (up to 109 months) and belonged mainly to the complement-activating IgG1 subclass. Median ARHGAP26-IgG/anti-Ca and median ARHGAP10-IgG/anti-Ca2 serum titres were 1:3200 and 1:1000, respectively, with extraordinarily high titres in some samples (ARHGAP26-IgG/anti-Ca: up to 1:1000,000; ARHGAP10-IgG: up to 1:32,000). ARHGAP26/anti-Ca serum titres exceeded those of ARHGAP10-IgG in all samples but one. A subset of patients was positive also for ARHGAP10-IgM and ARHGAP10-IgA. CSF/serum ratios and antibody index calculation suggested intrathecal production of ARHGAP26-IgG/anti-Ca and anti-ARHGAP10. Of 101 control samples, 100 were completely negative for ARHGAP10-IgG; a single control sample bound weakly (1:10) to the ARHGAP10-transfected cells. CONCLUSIONS: We demonstrate that a substantial proportion of patients with ARHGAP26-IgG/anti-Ca-positive autoimmune encephalitis co-react with ARHGAP10. Further studies on the clinical and diagnostic implications of ARHGAP10-IgG/anti-Ca2 seropositivity in patients with autoimmune encephalitis are warranted.