Polycystic ovary syndrome (PCOS) is one of the common endocrine disorders in women, which causes numerous symptoms in women. The relationship of many micronutrients with this syndrome has been investigated. This study was conducted to examine the effects of magnesium supplementation on hyperandrogenism, hirsutism, and sleep quality in women with PCOS.n = 32) or placebo group (n = 32) for 10 weeks. Patients in the magnesium group received one 250 mg magnesium oxide tablet, per day. Hyperandrogenism, hirsutism, and sleep quality were measured at the beginning and end of the study. This randomized clinical trial was registered at https://www.IRCT.ir (IRCT20130903014551N8).In this parallel randomized clinical trial, 64 women with PCOS were randomly assigned to the magnesium group , hirsutism (p = 0.23), and sleep quality (p = 0.85) compared with placebo.Magnesium supplementation had no significant effect on hyperandrogenism (The present study showed that a single dose of magnesium supplementation elicited no beneficial effects on the mentioned symptoms in polycystic women. It is possible that the positive effects of magnesium observed in the former studies were due to the synergistic effects of other vitamins or minerals. More studies are needed in this area. The prevalence of PCOS is about 4%–21%, depending on the different diagnostic criteria.There is a lack of enough evidence regarding magnesium supplementation in women with PCOS. Indeed, previous studies have not focused on the effect of magnesium supplementation on sleep quality and hyperandrogenism in these patients, and although some studies examined the impact of magnesium supplementation on hirsutism, such studies used magnesium in combination with other nutrients and did not evaluate the effect of magnesium alone on hirsutism in PCOS. Therefore, the aim of the present clinical trial was to evaluate the effect of magnesium supplementation on hirsutism, hyperandrogenism, and sleep disorders in women with PCOS.22.1The present study was a parallel double‐blind randomized controlled trial, with a duration of 10 weeks, conducted between October 2020 to October 2021 in Isfahan, Iran. Women referred to Isfahan Shahid Beheshti's infertility clinic were assessed for inclusion and exclusion criteria. The inclusion criteria involved: (1) were between 18 and 45 years old; (2) had PCOS according to the Rotterdam criteriaThe following equation was used to estimate the required sample size:α = 0.05 and β = 0.20 (the power of the study was 80%). The serum concentration of dehydroepiandrosterone sulfate (DHEAS) was considered as the main variable. According to the previous studies, ∆ = 0.122 µmol/l and S2 = 0.19 µmol/l.n = 32 in each group). Included participants were randomly assigned in a ratio of 1:1 to either magnesium or placebo using a computer‐generated randomization sequence in permuted blocks of two, with individuals categorized according to the body mass index (≤24.9 and >24.9 kg/m2). Staff provided a randomization list and numbered supplement containers. All participants, care providers, and the analyzers of the outcomes were blinded to the study. All participants signed a written consent form, before study commencement. This study was ethically approved by The Research Council and Ethical Committee of Isfahan University of Medical Sciences, Isfahan, Iran (Code: IR.MUI.RESEARCH.REC.1399.404), and was registered at https://www.IRCT.ir (IRCT20130903014551N8).In this equation, 2.2Patients received sufficient information regarding the study. Women in the magnesium group received a 250 mg magnesium oxide tablet (Magni One® produced by DonyaDarou) per day for 10 weeks. women in the placebo group received a tablet containing 5 mg starch, and its color, appearance, smell, and taste were similar to the magnesium tablet. Participants in both groups were recommended to take the tablets after breakfast. Participants were monitored through a telephone call and virtual networks, and they were asked to give back empty containers of supplements to ensure that all supplements were used properly.A list of following dietary recommendations was provided for participants in both groups: (1) to avoid consuming refined or simple carbohydrates, such as white bread, white rice, sugar, and sweets; (2) consume higher amounts of fresh vegetables; (3) increase the number of meals and reduce the volume of each meal; (4) drink adequate fluid, especially water (at least eight glasses per day); (5) replace starchy vegetables, such as potatoes with nonstarchy vegetables, such as lettuce, cabbage, cucumber, and so on; one study has shown that consuming a low starch diet such as low starch carbohydrates resulted in weight loss, improved insulin sensitivity, and reduced testosterone in women with PCOS2.3The serum concentration of magnesium, DHEAs, and testosterone was measured at baseline, and DHEAs and testosterone were remeasured after 10 weeks. A 10 ml blood sample was drawn, and after serum isolation, magnesium was measured by the atomic absorption spectrophotometry method. Also, radioimmunoassay and high‐performance liquid chromatography were used to measure the level of testosterone and DHEAs, respectively.2.4Sleep quality was assessed using the Pittsburgh Sleep Quality Questionnaire (PSQI) completed by a trained staff. The validity and reliability of this questionnaire were evaluated and deemed to be acceptable.2.5A modified Ferriman–Gallwey questionnaire was used to assess the hirsutism status and was completed by a trained staff. The validity and reliability of this questionnaire were previously evaluated and shown to be acceptable.2.6To control the confounding effect of dietary intake and physical activity, five 1‐day food diaries and five 1‐day physical activity records were completed by each participant during the study. Records covered three usual days and two weekend days. Patients were visited at baseline, the 5th week, and at the end of the study. Food diaries and physical activity records were provided during these visits. Food records were analyzed using Nutritionist IV designed based on the USDA food composition database. Individuals were asked to report their activities, including walking, exercise, sleep, hours spent watching TV, housework, studying, bathing, and other activities, and subsequently, the total metabolic equivalent (MET) was calculated by multiplying the frequency, duration, and intensity of each physical activity in 24 h.2.7χ2‐test. Categorical variables were reported as percentages. Baseline and endpoint values were compared within each group by paired t‐test analysis. Intergroup comparisons were performed by independent Student's t‐test for numerical variables. Confounding variables  were adjusted by analysis of covariance. Numerical variables were reported as mean ± SD, except for log‐transformed variable , which was reported as geometric mean ± SD. All data analyses were conducted using Statistical Package for the Social Sciences (SPSS) version 21 statistical software from IBM company, with an a priori α‐level of 0.05. We used two‐sided tests for all variables.An intention‐to‐treat (ITT) analysis based on the linear regression method was applied in the present study.3n = 200; 0.2) patients declined to participate in the study (n = 62); (3) PCOS was treated (n = 34); (4) women were pregnant (n = 5); and (5) patients were on insemination or in vitro fertilization treatments (n = 15). Therefore, 64 patients were included in the study and were randomly assigned to the magnesium (n = 32) or placebo (n = 32) groups. Five patients were lost to follow‐up in the magnesium group because they: (1) declined to continue the study; (2) became pregnant; or (3) were not comfortable providing a blood sample. Similarly, five individuals were lost to follow‐up in the placebo group because (1) they declined to continue the study; (2) were not comfortable providing a blood sample; or (3) for personal reasons. Therefore, 54 patients completed the study. By using an ITT method, data from 64 participants (n = 32 in each group) were included in statistical analysis.Figure p = 0.62), weight (p = 0.92), height (p = 0.44), body mass index (p = 0.81), overweight or obesity (p = 0.99), educational status (p = 0.38), economic status (p = 0.17), marital status (p = 0.31), and the level of physical activity (p = 0.73) were not significantly different between two groups. However, baseline serum magnesium levels were significantly higher in the magnesium group than in the placebo group (p = 0.04).The general characteristics of the participants are shown in Table Table pb < 0.001 in the magnesium group and pb < 0.001 in the placebo group) increased significantly at the end of the trial compared with the baseline in both groups. Testosterone concentration was higher in the placebo group at the end of the study compared to the baseline (pb = 0.01). Contrary to our expectation, the hirsutism score decreased in the placebo group at the end of the trial (pb = 0.03). The quality of sleep did not show any significant change in any of the groups at the end of the study  for the magnesium group = 0.61, p value (pb) for the placebo group = 0.74). Comparison of measurements at the end of the study between the two groups did not show significant differences in terms of testosterone (pc = 0.20), DHEAs (pc = 0.16), hirsutism (pc = 0.42), and sleep quality (pc = 0.73). These findings did not change after adjusting the baseline values of each factor and serum magnesium .The effects of magnesium supplementation on hyperandrogenism, hirsutism, and sleep quality are shown in Table 4Results of this randomized clinical trial showed that supplementation with 250 mg/day of magnesium did not significantly improve hyperandrogenism, hirsutism, and sleep quality in women with PCOS. Consumption of dietary supplements has been increasing globallyIn the present study, we found that magnesium supplementation had no significant effect on hyperandrogenism and hirsutism. Although previous studies have reported that magnesium may have beneficial effects on hyperandrogenism and hirsutism, such studies have used magnesium in combination with other nutrients. Indeed, a clinical trial reported that cosupplementation of magnesium and vitamin E may improve hormonal status and hirsutism in patients with PCOS.The results of the present study revealed that magnesium supplementation could not change sleep quality. Although a clinical trial reported an improvement in insomnia following magnesium supplementation, magnesium was used in combination with melatonin and vitamin B complex.As mentioned, the present study did not observe a significant effect of magnesium supplementation on the improvement of hyperandrogenism, hirsutism, and sleep quality in women with PCOS. Some possible reasons for these nonsignificant findings are: (1) Most studies that reported a significant effect of magnesium supplementation used cosupplementation of magnesium and other nutrients. Therefore, it seems that synergistic interaction between nutrients may have contributed to beneficial effects. For example, vitamin E and magnesium are known to have a synergistic interaction and strengthen each other in biological pathways.Magnesium absorption is not fast with ≈80% of ingested magnesium being absorbed within 6–7 h.In the present study, some strengths and limitations should be acknowledged. Indeed, first and foremost, the Covid‐19 pandemic and subsequent lockdown had an adverse effect on the follow‐up of the patients. In addition, we utilized subjective data collection methods for some variables. Another limitation of the present study is the use of magnesium oxide despite its lower absorption in comparison with other types. Since we needed pure magnesium, a supplement without any other vitamins or minerals, for use in the study, the only available supplement, was magnesium oxide. Other magnesium compounds such as magnesium glycinate were mixed with other nutrients. Nevertheless, despite the limitations of the present work, a strength of this study is that we implemented an ITT method in statistical analysis and utilized well‐validated data collection methods. Furthermore, in instances of subjective data collection methods, these questionnaires/surveys were completed with the support of trained staff.In conclusion, 250 mg/day of magnesium supplement did not significantly improve hyperandrogenism, hirsutism, and sleep quality disorders in women with PCOS, and unnecessary supplementation should be avoided.Mahsa Gholizadeh‐Moghaddam: Data curation; investigation; software; writing – original draft; writing – review and editing. Hatav Ghasemi‐Tehrani: Conceptualization; data curation; methodology; resources; software; validation; writing – original draft. Gholamreza Askari: Conceptualization; funding acquisition; resources; validation; visualization; writing – original draft. Mahsima Jaripur: Data curation; investigation; methodology; software; writing – original draft. Cain C. T. Clark: Resources; validation; visualization; writing – original draft; writing – review and editing. Mohammad Hossein Rouhani: Conceptualization; data curation; formal analysis; methodology; project administration; writing – original draft. All authors have read and approved the final version of the manuscript. M. H. R. as the corresponding author had full access to all of the data in this study and takes complete responsibility for the integrity of the data and the accuracy of the data analysis.The authors declare no conflict of interest.This study was ethically approved by The Research Council and Ethical Committee of Isfahan University of Medical Sciences, Isfahan, Iran, (Code: IR.MUI.RESEARCH.REC.1399.404), and was registered at IRCT.ir on 2020‐10‐05 (Registration Code: IRCT20130903014551N8). Also, all participants completed an informed consent form.https://www.IRCT.ir on October 5, 2020 (Registration Code: IRCT20130903014551N8).Mohammed Hossein Rouhani as the lead author affirms that this manuscript is an honest, accurate, and transparent account of the study being reported. The reporting of this work is compliant with CONSORT guidelines. The lead author affirms that no important aspects of the study have been omitted and that any discrepancies from the study as planned. The Research Council and Ethical Committee of Isfahan University of Medical Sciences, Isfahan, Iran and Food Security Research Center, Isfahan University of Medical Sciences, Isfahan, Iran approved this study (Code: IR.MUI.RESEARCH.REC.1399.404). This randomized clinical trial was registered at


All LC/HP meals were delivered weekly to participants' homes, and participants in the control group consumed their habitual diet. Each participant underwent an oral glucose tolerance test (OGTT) to assess glucose tolerance, insulin, area under the curve (AUC) for glucose and insulin, Matsuda Index, glucose‐stimulated insulin secretion (GSIS), disposition index, and hepatic insulin extraction (HIE). Fasting blood lipid and inflammation were assessed, and body composition was estimated using dual‐energy x‐ray absorptiometry. A linear mixed model was used to evaluate the main effect of diet, time, and their interaction. Compared to the control group, participants in the LC/HP group had reduced total body fat mass , visceral fat mass , total‐ , and LDL‐cholesterol  . Regardless of group, AUCinsulin and peak insulin during the OGTT decreased, and HIE increased over time (ptime < 0.05). A trend for diet*time interaction was observed for glucoseOGTT120min  and peak C‐peptide . HDL‐cholesterol, lean body mass, Matsuda Index, fasting glucose, insulin, insulinOGTT120min, AUCglucose, pancreatic beta cell function , and inflammation  did not change over time. In conclusion, our results suggest that individuals with SCI and insulin resistance may adopt an LC/HP diet to improve body composition and lipid profiles. Its impact on glucose metabolism and inflammation remains inconclusive and warrants future investigations.We explored the impact of a low‐carbohydrate/high‐protein diet  on indices of metabolic function and body composition in individuals with chronic spinal cord injury (SCI). Adults with SCI  and insulin resistance or pre‐diabetes were randomly assigned to an 8‐week iso‐caloric LC/HP diet group ( Our results support a favorable change of the body composition and blood lipid profile after consuming a healthy LC/HP diet for 8 weeks in individuals with chronic SCI. This underscored the importance of dietary modifications for metabolic health in SCI. In the diet group, one participant was excluded due to weight loss before starting the study diet, and five participants discontinued the study diet . Among participants assigned to the control group, one developed a new condition unrelated to the study diet, and one was excluded due to starting new exercise regimens  were between the ages of 18 and 65 years; (2) had a traumatic SCI at the cervical, thoracic, or lumbar level (C5–L2) classified as American Spinal Injury Association impairment scale A, B, C, or D; (3) impaired glucose tolerance or insulin resistance; (4) no history of pre‐existing self‐reported type 2 diabetes, renal disease, or pressure injury; (5) were at least 3 years post‐injury ; (6) had not participated in a weight loss program for the last 6 months; (7) had not habitually consumed a HP diet (~30% energy from dietary protein); (8) had not been on antibiotics for at least 4 weeks before the study, and (9) had not taken medications for weight management, blood glucose, and lipid disorders. Interested participants underwent an oral glucose tolerance test (OGTT) to confirm eligibility  administered by the research coordinator. Initial calorie prescription was estimated using the Harris‐Benedict equation, and an activity factor of 1.2 was used. The study dietitian planned personalized menus consistent with the LC/HP diet parameters (for the LC/HP diet group) , three times  to confirm that there were no substantial changes in their diet during the study  ]*100% . This constant is generally consistent with the density of adipose tissue. Participants were scanned in light clothing while lying flat on their backs with their arms at their sides. Despite its wide use in assessing body composition in the general population, some limitations of DXA may introduce additional bias for individuals with SCI, such as interference of metal implants  using the enCORE software version 13.6   , and tumor necrosis factor‐α (TNF‐α) were analyzed in duplicate using the MesoScale Discovery Proinflammatory Kit. C‐reactive protein (CRP) was analyzed on a Sirrus Stanbio analyzer.2.9t‐tests and chi‐square tests were used to compare baseline group characteristics for continuous and categorical variables, respectively. A linear mixed‐effects model was built to compare dietary intake changes over time among participants in the control group. A linear mixed model analysis was constructed to test the effect of diet, time, and their interaction, with the participant as a random effect, and level of injury and sex as covariates. Pairwise post hoc comparisons were performed using the Tukey–Kramer multiple comparisons method to estimate within‐group changes. Statistical model assumptions were validated before data analysis. Statistical tests were two‐sided, and p < 0.05 was considered statistically significant. Statistical analyses were performed using the SAS, version 9.4 . Data presented are mean ± standard deviation unless otherwise stated. We intended to recruit 60 participants (30 for each group) to have 80% power to detect an effect size of 0.73 (Cohen's d) for between‐group differences in Matsuda index or the glucose concentration at minute 120 during the OGTT. However, due to COVID‐related interruptions, the final achieved sample size (n = 25) in this trial provides 80% power to detect an effect size of 1.18 at alpha level of 0.05. Thus, the results reported in this manuscript should be considered exploratory, rather than confirmatory.Descriptive statistics were calculated for all study variables of interest, with mean ± standard deviation reported for normally distributed continuous variables. Median and interquartile range (IQR) were reported for non‐normally distributed variables. Unpaired 33.1p > 0.05 for all) (Table ). On average, participants from both groups consumed diets with ~16% energy from dietary protein, comparable to the average protein intake among Americans  Table . On ave3.2p > 0.05 for both).Based on the checklist provided, participants in the diet group, on average, consumed 94% (IQR: 91%–97%) of the food items provided. Based on the dietary recalls, participants in the control group did not change their diet significantly over time Table . In addi3.3pdiet*time <0.05). Specifically, total body fat mass, percentage body fat, and visceral fat mass decreased by 5.9%, 2.9%, and 16.2% of their baseline values, respectively, in the LC/HP group . No significant changes in these outcomes were observed in the control group. Lean body mass did not change significantly over time in either group.Changes in body composition are shown in Figure 3.4pdiet*time = 0.09) and peak C‐peptide concentrations during the OGTT (pdiet*time = 0.07), where their concentrations decreased quantitatively among participants in the LC/HP group. Regardless of group assignment, insulin AUC (p = 0.04) and peak insulin concentrations (p = 0.03) decreased over time, and HIE (p = 0.03) increased over time. Though the diet‐by‐time interaction effect was not significant, the changes in insulin and HIE seem to be driven by those observed in the LC/HP group  and LDL‐c (p = 0.04). Among participants in the diet group, fasting total cholesterol decreased by −20.1 ± 28.0 mg/dl (p < 0.05), while the control group had minimal changes. LDL‐c quantitively decreased by 13.9 ± 26.8 mg/dl in the LC/HP group. No effects of diet, time, and their interaction were observed for triglycerides and HDL.Participants in the LC/HP diet group had favorable changes in lipid profiles compared to the control group Figure . A signi3.6Several markers of chronic inflammation, including CRP, IL‐6, IL‐8, IL‐10, and TNF‐alpha, did not change differently between groups over time Table .3.7No participant from either group reported any adverse events during the study.4Given the pathophysiological changes accompanied by SCI, such as muscle loss, increased adiposity, and impaired metabolism, unique nutritional considerations may be necessary to ameliorate and prevent the development of metabolic disorders  after the dietary intervention. This improvement may result from reduced total carbohydrate intake and changes in the type and quality of carbohydrates consumed, which collectively affect the meals' glycemic load (GL). The GL is estimated by multiplying the quality of carbohydrates in a given food by the amount of carbohydrates in a serving of that food. The study diet contained 40% energy from dietary carbohydrates. In comparison, participants consumed an average of 48% energy from dietary carbohydrate at baseline and had an excessive added sugar intake. The 2020 dietary guideline for Americans recommends limiting added sugar to less than 10% of calories per day , where glucose concentration at minute 120 during the OGTT decreased from 138.5 ± 11.2 to 117.7 ± 8.3 mg/d among participants in the diet group. The achieved effect size for the between‐group difference (Cohen's d = 0.70) is close to the expected effect size of 0.73 in our initial power calculation. Thus, the study diet may benefit glycemic control, but the current sample size is insufficient to reject the null hypothesis. Nevertheless, such a decrease is clinically meaningful. Upon closer examination of individual responses, at baseline, 5 participants in the LC/HP and 4 participants in the control group had pre‐diabetes. After the intervention, four out of five participants in the LC/HP group had remission of their pre‐diabetes and achieved normal glucose tolerance. In contrast, none of the four participants in the control group had remission of their pre‐diabetes. Furthermore, given that pancreatic beta bell function did not change over time based on the GSIS and DI data, the trend for reduced insulin responses  indicates improved insulin sensitivity. The increased HIE levels further support the potential benefit of the LC/HP diet on glucose metabolism. Previous studies showed that HIE is reduced in those with pre‐diabetes  as well as low diet quality . Thus, it is unclear whether the improvements in the study outcomes among participants in the LC/HP group were due to the differences in macronutrient composition or a combination of both macronutrient changes and a healthier dietary pattern. Second, due to the small sample size, it was not feasible to evaluate whether the impact of the diet differed for participants in different age groups, sexes, levels of injury, completeness of injury, or duration of the injury, as well as identify potential responders vs. non‐responders to the dietary intervention. As a result, the study result may not be generalizable to all individuals with SCI.5Our exploratory analysis supports a favorable change in the body composition and blood lipid profile after consuming a healthy LC/HP diet for 8 weeks in individuals with chronic SCI. The impact of the study diet on glucose metabolism and inflammation is inconclusive and warrants future evaluations. Nevertheless, our results underscored the importance of dietary modifications for metabolic health in SCI. Long‐term safety and efficacy diet trials for preventing and managing metabolic disorders in individuals with SCI are warranted.The authors confirm contribution to the paper as follows: study conception and design: Jia Li, Ceren Yarar‐Fisher, Amie McLain, and Barbara Gower, participant recruitment and data collection: Jia Li, analysis and interpretation of results: Jia Li and Ceren Yarar‐Fisher; draft manuscript preparation: Jia Li, Ceren Yarar‐Fisher, Amie McLain, and Barbara Gower. All authors reviewed the results and approved the final version of the manuscript.This work was supported by the National Institute on Disability, Independent Living, and Rehabilitation Research  and the National Center for Advancing Translational Sciences of the National Institutes of Health under award number UL1TR003096.The authors declare no competing interests during the course of this research.The University of Alabama at Birmingham institutional review board approved the study. We certify that all applicable institutional and governmental regulations concerning the ethical use of human volunteers/animals were followed.Table S1Click here for additional data file.


The utility for liquid biopsy of tumor‐associated circulating single‐nucleotide variants, as opposed to mutations, of the mitochondrial (mt) and nuclear genomes in neuroblastoma (NB) is unknown.Variants of the mt and nuclear genomes from tumor, blood cells, and consecutive plasma samples of five patients with metastatic NB that relapsed or progressed were analyzed. Targeted parallel sequencing results of the mt genome, and of the coding region of 139 nuclear genes and 22 miRNAs implicated in NB, were correlated with clinical imaging and laboratory data.All tumors harbored multiple somatic mt and nuclear single nucleotide variants with low allelic frequency, most of them not detected in the circulation. In one patient a tumor‐associated mt somatic variant was detected in the plasma before and during progressive disease. In a second patient a circulating nuclear tumor‐associated DNA variant heralded clinical relapse. In all patients somatic mt and nuclear variants not evident in the tumor biopsy at time of diagnosis were found circulating at varying timepoints. This suggests either tumor heterogeneity, evolution of tumor variants or a confounding contribution of normal tissues to somatic variants in patient plasma. The number and allelic frequency of the circulating variants did not reflect the clinical course of the tumors. Mutational signatures of mt and nuclear somatic variants differed. They varied between patients and were detected in the circulation without mirroring the patients' course.In this limited cohort of NB patients clinically informative tumor‐associated mt and nuclear circulating variants were detected by targeted parallel sequencing in a minority of patients. A motif provides additional, contextual information about the variant. The six possible classes of base mutations can form 96 possible motifs. By calculating the frequencies of such mutational motifs a mutational signature is obtained.MYCN and segmental chromosome alterations, which can be detected by liquid biopsy in patients with NB.Molecularly, NB is characterized mainly by changes in copy number alterations, many of which carry a poor prognosis and are used to stratify patients into high‐risk groups. Prominent among these are amplification of ALK, ATRX, LMO1, genes of the RAS‐RAF‐MAPK pathway, as well as genomic rearrangements of TERT.In contrast to copy number alterations, recurrent mutations are rare in NB. These include mutations of Many NB harbor tumor‐associated mt variantsIn addition to tumor DNA, microRNAs have been detected in the blood of mice and NB patients, thus constituting potential circulating tumor‐associated biomarkers for NB.In a small cohort of patients with progressing or relapsing NB we detected informative circulating tumor‐associated mt and nuclear variants by targeted sequencing in a minority of patients.22.1Figure 2.2All patients with NB treated at the Department of Pediatrics and Adolescent Medicine of the University Medical Center Ulm between October 2012 and March 2018, and from whom sufficient tumor DNA, germline DNA and consecutive plasma samples were available in adequate quality, were included in the investigation. The study was approved by the local ethics committee and informed consent was obtained. A total of five patients were chosen based on having stage 4 disease, reasoning that initial metastatic disease facilitates detection of circulating variants. One patient went into complete remission (CR) during monitoring and then relapsed, while four showed partial remission (PR) and then progressed. A detailed description of the patients is given in Table 2.3Vanillylmandelic acid and homovanillic acid in spot urine, and bone marrow cytology and histology were analyzed by standard methods.Tumors were imaged by magnetic resonance imaging using a 3T device . MIBG scanning was performed by a dual‐head gamma camera  4 and 24 h after intravenous application of Iodine‐123‐MIBG (Metaiodobenzyl‐guanidin). In sum, 77 MRIs and 2 CTs of the whole body or abdomen as well as 33 MIBG scans were evaluated.The mintLesion program  was used to calculate tumor burden at each time point. Briefly, tumor volume of primary tumor and metastases in lymphnodes, liver, lung, brain, and peritoneal cavity was determined. The volume of bone marrow metastases was added. The volume of bone marrow metastases was calculated incorporating related approaches form other imaging methods.2.4Tumor samples were obtained at the time of surgery. DNA samples from the NB were either provided by the Tumorbank Embryonale Tumoren, Unikinderklinik Köln, Cologne, Germany or isolated from formalin‐fixed paraffin‐embedded tumor tissue slides provided by the Department of Pathology, University Medical Center Ulm, Germany. DNA was isolated using the GeneJet FFPE DNA Purification Kit  from three or four slides per patient measuring 8 mm in depth.Germline DNA of each patient was extracted from mononuclear blood cells after diagnosis with a time lag to surgery, using the QIAamp DNA Blood Mini Kit .g for 10 min at 4°C. The plasma was subjected to a second round of centrifugation at 14.000 rpm for 10 min at 4°C. The supernatant was aliquoted and stored at −80°C. Ct DNA was isolated from one aliquot of plasma with the QIAamp Circulating Nucleic Acid Kit  following the manufacturer's instructions. Following elution with 30 μl a second elution step was performed with 20 μl, resulting in a total volume of 50 μl per sample.From each patient, consecutive peripheral blood samples were collected into EDTA tubes and processed within 1.5 h. Depending on the size of the child, 1.6 to 4.9 ml blood was collected. Samples were centrifuged at 8002.5http://target.nci.nih.gov/dataMatrix/), and finally genes not implicated in NB so far but deemed to be potentially relevant based on their function. The 139 nuclear genes and 22 miRNAs are listed in Table ATRX and TERT loci, were included in the panel  were prepared utilizing ThruPLEX Plasma‐Seq or ThruPLEX Tag‐Seq kits , respectively. Libraries were prepared according to the manufacturer's protocol. 6–18 ng DNA from cell‐free plasma samples, and 5–50 ng DNA from tumor and blood cells were used. Each library was amplified with 10 cycles and assigned an individual index. A unique molecular tag was added to identify PCR duplicates.Target regions were enriched using the SureSelectXT kit with the custom capture panel. Hybridization and capture were performed according to the manufacturer's protocol, with modifications. IDT xGen universal blocking oligonucleotides  were added to the SureSelect Block Mix before hybridization. The postcapture target amplification step was modified by omitting the indices and switching to P5 and P7 primers  instead of the indexing postcapture primer. The optimal cycle number for target amplification was determined to be 12, as assessed by the KAPA library quantification kit . DNA concentration and average fragment size were measured by Tape Station , and libraries were pooled equimolarily to 4 nM. Sequencing was performed on a NextSeq 500 device (Ilumina) with 75 PE and high‐output kit mode.2.7GATK guidelines.http://www.ncbi.nlm.nih.gov/projects/SNP/index.html) and 1000 genomes (http://www.1000genomes.org/). Duplicate removal was performed via Picard (http://broadinstitute.github.io/picard/). Somatic nuclear variants or mutations were filtered using Mutect2 (https://gatk.broadinstitute.org/hc/en-us/articles/360037593851-Mutect2) by comparing to the germline control sample. MitoSeek version 1.3http://www.biobase-international.com/product/hgmd). Individual signatures were generated using R and subjected to principal component analysis as a statistical approach to capture the main sources of variation in each patient, which are henceforth called principal signatures. Principal component analysis was done using the Bioconductor package SomaticSignatures.Bioconductor package gplots.An in‐house bioinformatics pipeline was used for sequence analysis and annotation following 33.1Mean depth coverage of the targeted nuclear regions was 296× in blood cells (germ line), 117× in tumor samples and 169× in plasma samples. Mean depth of coverage of the mt genome was 7774× in blood cells, 7827× in tumor samples, and 990× in plasma samples.3.2Tumor‐associated mt variants were found in all tumors, with their number and allelic frequency ranging from 21 to 40 (median 29), and 1.1% to 49.3%, respectively. Tumor‐associated somatic nuclear variants were also present in all tumors. Their number and allelic frequency ranged from 32 to 70 median 58), and 1.1% to 90%, respectively ; investigation (lead); writing – original draft ; writing – review and editing . Medhanie Mulaw: Conceptualization ; formal analysis ; investigation ; methodology ; writing – original draft ; writing – review and editing . Katharina Kneer: Investigation ; methodology ; writing – original draft . Meinhard Beer: Supervision (supporting). Ambros Beer: Supervision (supporting). Thomas F. Barth: Investigation (supporting); writing – original draft (supporting). Vladimir Benes: Investigation (supporting). Johannes Schulte: Resources (supporting); validation (supporting); writing – original draft (supporting). Matthias Fischer: Validation (supporting); writing – original draft (supporting); writing – review and editing (supporting). Klaus‐Michael Debatin: Supervision (supporting); validation (supporting). Christian Beltinger: Conceptualization (lead); funding acquisition (lead); supervision (lead); writing – original draft (lead); writing – review and editing (lead).The authors declare that they have no competing interests.The study was approved by the ethics committee of the University of Ulm (protocol code 25/14) and written informed consent was obtained.FIGURE S1 No tumor‐associated circulating variants in a patient with lethal cerebral metastasis.Click here for additional data file.(A) Clinical course. Chemo, chemotherapy; ASCT, autologous stem cell transplantation; cis‐RA, cis‐retinoic acid differentiation therapy; PR, partial remission; PROG; progression; D, death; MIBG trend, response assessment by MIBG; BM, bone marrow samples (filled circle: positive for NB cells); Plasma samples, time of plasma sampling; SUR, surgery and biopsy; VMA, vanillylmandelic acid; HVA, homovanillic acid.(B) High number of mt and nuclear somatic variants with low allelic frequency in tumor and consecutive plasma samples. T, tumor; P1–5, consecutive plasma samples.(C) Mt and nuclear principal signatures differ. Mt and nuclear principal signatures S1, S2, and S3 are shown.(D) Circulating mt and nuclear principal signatures do not reflect the clinical course. Unsupervised hierarchical clustering of the principal signatures S1, S2, and S3 by the consecutive samples T (tumor) and P1–5 (plasma samples) are depictedClick here for additional data file.TABLE S1 Patient characteristicsClick here for additional data file.TABLE S2 Nuclear genes sequencedClick here for additional data file.TABLE S3 Chromosomal location of regions for structural analyses included in the custom NB panelClick here for additional data file.TABLE S4 Mitochondrial and nuclear somatic variants are found in tumors (T) and consecutive plasma samples (P1–P5). Allelic frequencies are shown as %Click here for additional data file.


Vitamin D supplementation is proposed as a potentially effective nutritional intervention to mitigate the risk of sarcopenia. The aim of this systematic review and meta‐analysis was to investigate the impact of vitamin D supplementation monotherapy on indices of sarcopenia in community‐dwelling older adults.A comprehensive search of the literature was conducted in PubMed, Web of Science, Scopus, and Cochrane Library. Eligible randomized controlled trials (RCTs) compared the effect of vitamin D supplementation (as monotherapy) with placebo on indices of sarcopenia in older (>50 years) adults. Using the random effects inverse‐variance model, we calculated the mean difference (MD) in handgrip strength (HGS), short physical performance battery (SPPB), timed up and go (TUG), and appendicular lean mass (ALM) between groups. We also calculated the standardized mean difference (SMD) in general muscle strength and general physical performance  between groups.I2 = 0%; P = 0.007). Vitamin D supplementation conferred no effect on HGS , TUG , ALM , general muscle strength , or general physical performance .Ten RCTs were included in the meta‐analysis. A significant decrease in SPPB scores was observed with vitamin D supplementation compared with placebo (MD: −0.23; 95% CI −0.40 to −0.06; Vitamin D supplementation did not improve any sarcopenia indices in community‐dwelling older adults and may compromise some aspects of physical performance. Future studies are warranted to investigate the impact of vitamin D supplementation on individual indices of SPPB, including mobility and balance, in older adults. Optimal body composition and skeletal muscle function are key contributors to healthy ageing. Ageing is associated with the gradual loss of muscle mass and strength called sarcopenia,in vivo studies have linked VDR overexpression with skeletal muscle hypertrophy.Vitamin D is a fat‐soluble secosteroid that is primarily synthesized following solar ultraviolet light exposure and is most commonly implicated in regulating bone health by increasing intestinal absorption of calcium and phosphate.The independent effect of vitamin D supplementation on indices of sarcopenia has not been comprehensively established in community‐dwelling older adults, and so the aim of this systematic review and meta‐analysis was to investigate the effect of vitamin D supplementation as a monotherapy on muscle mass, strength, and physical performance in this population.This systematic review and meta‐analysis was conducted in accordance with Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) guidelines.TableTwo independent reviewers (K. P. and K. K. T.) searched PubMed, Embase, Web of Science, and Cochrane library from inception until March 2021. The full search strategy and the search terms used are described in the Supporting Information, Studies were included based on the following criteria: (i) RCTs; (ii) healthy and/or community‐dwelling adults; (iii) intervention group received vitamin D supplementation as monotherapy; (iv) control group received placebo; (v) participants aged ≥50 years  extracted data independently which included name of first author, date of publication, country of origin, number of participants, outcome measurements, and treatment type, dose, and duration. Disagreements between authors were resolved by two independent reviewers (P. G. and K. S. K.). The quality of included studies was evaluated using the risk‐of‐bias 2 (RoB2) toolOur meta‐analysis compared changes in HGS, SPPB, TUG, ALM, and general muscle strength and physical performance in participants randomized to vitamin D supplementation or placebo.t and P values or by calculating a correlation coefficient from a known change from baseline standard deviation derived from a similar study.Quantitative data were treated as continuous measurements, and changes in outcomes from baseline to follow‐up were compared between groups to calculate mean differences. When units of measurements were inconsistent and could not be converted to units required for be included in the analysis and/or outcomes measured the same aspects of muscle health, standardized mean differences were used. When numerical data were not reported, graphical values were estimated using DigitizIt 2.5 Software. Statistical significance was assessed using the random effects model and inverse‐variance method. Any missing standard deviations for changes between baseline and follow‐up among outcome measurements were estimated depending on availability of either confidence intervals, standard errors, Q (χ2 test) and I2. The classification of data as moderately heterogeneous was based on I2 from 30 to 49% and highly heterogeneous from 50% and above.Statistical heterogeneity of outcome measurements between different studies was assessed using the overlap of their confidence interval (95% CI) and expressed as measurements of Cochran's Q χ2 test anFigureTableet al. where calcitriol was administered.et al.2D] was measured. According to the cut off values established by the Endocrine Society,The initial literature search yielded 6255 publications. After duplicates and abstracts were excluded, 25 full‐texts were identified, and 10 studies were deemed eligible for the systematic review and meta‐analysis2) and assessed with the use of hydraulic or pneumatic hand dynamometers.2).2D was expressed in pmol/litre (pmol/L). When expressed in nmol/L, serum 25(OH)D concentration was converted to ng/mL using a factor of 2.5.Handgrip strength was expressed in kilograms (kg) or pounds/square inch        D and 1,25(OH)) Figure. Our ana) Figure. Vitamin) Figure. Changes) Figure, TUG (MD) Figure, and gen) Figure.FigureI2 = 65%, P = 0.94) or women alone , or in studies performed in the USA  or the rest of the world . Studies administering moderate doses of vitamin D supplementation also had no effect on HGS . No effect of vitamin D supplementation on HGS was observed for studies with treatment duration of more than 6 months , but treatment duration of <6 months had a significant negative effect on HGS ,A series of subgroup analyses based on sex , treatment duration , and dose of vitamin D supplementation (moderate: 853–1667 IU/day), and geographic origin of study  was performed for HGS, and the effect remained unchanged Figure. No effeFigureI2 = 59%, P = 0.68) and serum vitamin D  concentrations  were similar to those observed in primary analyses. Type of vitamin D supplementation and study risk of bias also had no effect on our findings , global muscle strength , and serum vitamin D  concentrations .Lifestyle factors also had no effect on our findings Figure,B; afters Figure–C; resulThis systematic review and meta‐analysis examined the effects of vitamin D supplementation as a monotherapy, compared to placebo, on muscle mass, strength and physical performance in community‐dwelling older adults. Vitamin D supplementation decreased SPPB scores  but had no effect on muscle mass, strength, or any other physical performance parameters. These observations were based on findings from 10 studies with a low overall risk of bias.et al. have proposed meaningful decline estimates of 0.27–0.55 for SPPB score, which suggests that the between‐group difference in change in SPPB score of −0.23 from baseline to follow‐up we observed for vitamin D compared with placebo could be clinically relevant.Vitamin D supplementation led to a significant reduction in SPPB scores among community‐dwelling older adults compared to placebo. Perera The age‐related decline in muscle mass and strength may place older adults at an increased risk of falls and fractures.et al. revealed a small but significant effect of vitamin D supplementation on general muscle strength, when combining multiple measures of muscle strength.et al. previously reported that the risk of falls and fracture in older women receiving intermittent annual high‐dose (500 000 IU) vitamin D monotherapy increased in the first 3 months following vitamin D supplementation.Vitamin D supplementation had no effect on HGS compared to placebo, although negative effects of treatment durations <6 months were observed. HGS has been proposed as a valuable and reliable assessment of muscle strength due to its low cost and practicability in clinical and community healthcare settings.According to the EWGSOP and AWGS definitions, the evaluation of ALM as a surrogate measurement of muscle mass constitutes a valuable parameter for the diagnosis of sarcopenia.This is the first study to examine the effect of vitamin D supplementation as a monotherapy on indices of sarcopenia  in community‐dwelling older adults, compared with placebo. Previous studies have only investigated vitamin D effects without controlling for confounders such as calcium co‐supplementation and co‐morbidity status. Increased calcium intake has been associated with lower odds of sarcopenia,This systematic review and meta‐analysis revealed no effect of vitamin D supplementation on muscle mass or strength, but resulted in a significant decrease in SPPB score, indicating potential deleterious effects on lower‐limb physical function, when administered as a monotherapy in community‐dwelling older adults. A sub‐group analysis also revealed decreases in HGS following vitamin D supplementation regimens of <6 months. Future studies that supplement vitamin D in populations with low vitamin D status and/or low baseline physical performance are warranted to provide insight on its effects on sarcopenia indices in these groups, particularly for balance and mobility measures which comprise the SPPB. Further research is also necessary to confirm effects of short‐term vitamin D supplementation on muscle function in older adults. The lack of homogeneity in sarcopenia indices highlights the need for a global consensus regarding clinically relevant measurement tools for assessing muscle mass, strength, and physical performance in older populations. However, based on current evidence, the impact of vitamin D supplementation in mitigating risk of sarcopenia may be considered negligible, at least in community‐dwelling older adults.There are no conflict of interest.Table S1. Search terms employed to screen different electronic databases for the literature search.Click here for additional data file.Figure S1. Quality assessment of the included studies according to the Cochrane risk‐of‐bias 2 tool.Figure S2. Effect of vitamin D supplementation on changes in serum vitamin D (25‐hydroxyvitamin D [25(OH)D] and 1,25‐dihydroxyvitamin D ) levels compared to placebo.Figure S3. Subgroup analysis of handgrip strength changes in response to vitamin D supplementation based on (A) sex, vitamin D treatment (B) duration and (C) dose and (D) geographic origin of study, compared to placebo.Figure S4. Sensitivity analysis based on the effect of lifestyle factors on changes in (A) handgrip strength (B) serum vitamin D (25‐hydroxyvitamin D [25(OH)D] and 1,25‐dihydroxyvitamin D ), in response to vitamin D supplementation compared to placebo.Figure S5. Sensitivity analysis based on the effect of type of vitamin D supplementation or risk of bias on changes in (A) handgrip strength (HGS), (B) general muscle strength (HGS and knee extension test at 180 degrees (KET)) and (C) serum vitamin D (25‐hydroxyvitamin D [25(OH)D] and 1,25‐dihydroxyvitamin D ) levels, in response to vitamin D supplementation compared to placebo.Click here for additional data file.


Neonatal imaging studies report corpus callosum abnormalities after neonatal hypoxic–ischaemic encephalopathy (HIE), but corpus callosum development and relation to cognition in childhood are unknown. Using magnetic resonance imaging (MRI), we examined the relationship between corpus callosum size, microstructure and cognitive and motor outcomes at early school‐age children cooled for HIE (cases) without cerebral palsy compared to healthy, matched controls. A secondary aim was to examine the impact of HIE‐related neonatal brain injury on corpus callosum size, microstructure and growth.Participants aged 6–8 years underwent MRI, the Movement Assessment Battery for Children Second Edition and Wechsler Intelligence Scale for Children Fourth Edition. Cross‐sectional area, volume, fractional anisotropy and radial diffusivity of the corpus callosum and five subdivisions were measured. Multivariable regression was used to assess associations between total motor score, full‐scale IQ (FSIQ) and imaging metrics.N = 40) compared to controls (N = 39) demonstrated reduced whole corpus callosum area , volume , fractional anisotropy and increased radial diffusivity (P < 0.05) within segments II–V. In cases, segment V area , volume , whole corpus callosum fractional anisotropy  and radial diffusivity  were associated with FSIQ. Growth of the corpus callosum was restricted in cases with a FSIQ ≤85, and volume was reduced in cases with mild neonatal multifocal injury compared to white matter injury alone.Adjusting for age, sex and intracranial volume, cases (Following neonatal HIE, morphological and microstructural changes in the corpus callosum are associated with reduced cognitive function at early school age. Therapeutic hypothermia (TH), reducing the body temperature to 33.5°C for 72 h, has reduced the incidenceTo address these issues, we compared CC morphometry and microstructure derived from structural and diffusion‐weighted magnetic resonance imaging (MRI) at early school age (6–8 years) between children without CP cooled for HIE (cases) and matched controls. We then examined the impact of neonatal hypoxic–ischaemic brain injury on CC morphology at early school age, and the association between cognitive and motor impairment at early school age and CC growth. Finally, we investigated the association between CC morphology, microstructure and cognitive and motor function at early school age in cases and controls.The South West‐Frenchay NRES Committee and Health Research Authority, UK (15/SW/0148) approved the study. We recruited a prospective cohort after obtaining informed parental consent. The study was conducted at the University of Bristol, UK.Sixty‐nine eligible case children were identified from a patient database at St Michael's Hospital, Bristol, UK. Of those contactable, 50 agreed to participate. We included children aged 6–8 years, born >35 weeks' gestation at St Michael's Hospital who underwent TH within 6 h of birth for encephalopathy, confirmed by neurological examination and amplitude‐integrated electroencephalogram (aEEG),Children aged 6–8 years, born >35 weeks' gestation without neonatal HIE were recruited via local schools in Bristol. Of the 65 parents who registered interest in the study, we recruited 43 eligible children most comparable to cases. Cases and controls were matched at the group level by age, sex and socio‐economic status as determined by the index of multiple deprivations, based on a weighted combination of seven domains, as defined by the UK Government.b = 0, 500, 1000 s/mm2). T1‐W, DWI and T2‐W scans were used for qualitative inspection of the CC and sagittal T1‐W images were used for manual morphometric CC analysis.Neonatal MRI scans were performed using a 1.5T Siemens Symphony  between 4 and 15 days after birth, during natural sleep following a feed or with chloral hydrate if required. All infants underwent T1‐weighted (T1‐W) and T2‐W imaging using turbo spin echo sequences (slice thickness 4 mm), echo time (TE)/repetition time (TR) = 7.7/400 ms, flip angle 90° and TE/TR = 99/3520 msec. Axial diffusion‐weighted imaging (DWI) was obtained using TE/TR: 112/3700 msec, flip angle 90° and three diffusion weightings .Participants underwent MRI using a 3T Siemens Skyra. The protocol included a T1‐W sequence (magnetisation prepared rapid gradient echo): 176 slices, 1.0 × 1.0 × 1.0 mm voxels, TE/TR = 2.19/1500 msec, inversion time = 800 msec, flip angle 9°, field of view (FoV) 234 × 250 mm, GeneRalized Autocalibrating Partially Parallel Acquisitions (GRAPPA) acceleration factor 4Before scanning, children were shown a video demonstrating the MRI procedure and chose a DVD to watch during scanning. Scans were individually inspected by two raters (J. C. Brooks and A. P. Spencer) blinded to case–control status for suitability for analysis, excluding scans with significant motion artefacts.The presence and extent of brain injury on neonatal and childhood MRI was assessed by a perinatal neurologist experienced in MRI interpretation (F. M. Cowan) blinded to case–control status and independently of the neonatal scans. Neonatal brain injury was quantified using the Rutherford classification system,Childhood MRI scans were assessed for the presence of abnormalities, particularly focal central grey matter (GM) lesions and WM hyperintensities on T2‐W images.As CC size and thickness depend on axonal fibre density,Thirty‐six (85.7%) cases with childhood MRI had neonatal imaging suitable for morphometric analysis. Manual calculations were undertaken by a single assessor (G. Geary). Longitudinal total and anterior‐, mid‐ and posterior‐third cross‐sectional CC area and supratentorial brain area (STBA) were calculated from mid‐sagittal T1‐W images by manually demarcating the boundaries of the CC and brain using Osirix.The process was repeated three times and averaged. As the Hofer and Frahm classification has not been validated in neonatal cohorts, we used anterior, middle and posterior CC subdivisions commonly reported with manual segmentation. We further subdivided the middle‐third into the anterior‐third and posterior two‐thirds as this latter section carries most motor fibres. We computed the ratio of whole and CC subdivisions area to the STBA to adjust for differences in brain size.Manual calculations were performed by the same assessor (G. Geary) blinded to case–control status, using the method described above.http://jerlab.psych.sc.edu/NeurodevelopmentalMRIDatabase/) using rigid body transformation with FSL's FLIRT.x = 0) and four adjacent parasagittal slices  to provide a median value for the area for each subject. The area of each of the five segments of the CC according to Hofer and Frahm was calculated.Morphometric analysis was conducted using ‘C8’, a fully automated MATLAB program for quantifying the area of the human CC.Callosal volume estimates were generated by summating the areas calculated for the five slices (each 1 mm thick) spanning the midline. Corresponding volumes for each of the five Hofer and Frahm segments were estimated by multiplying the whole CC volume by the relative proportions for each of the segment areas.3), ignoring all voxels with a value of zero. The total volume of each tissue type was summed to produce ICV. Due to movement and poor alignment between scans, ICV could not be calculated for the neonatal data set.Intracranial volumes  were calculated for childhood scans using partial volume estimate (PVE) segmentations obtained from FAST. Using the brain‐extracted images, the mean voxel PVE for GM, WM and cerebrospinal fluid across the whole image for each subject were multiplied by the total image volume  using the Wechsler Intelligence Scale for Children 4th Edition (WISC‐IV).t test was used to compare the normally distributed variables and Mann–Whitney U to compare variables of skewed distribution. Proportions were compared using chi‐squared tests. To reduce bias and improve reliability, the relationship and agreement between childhood manual and automated whole CC area measurements was assessed using a scatter plot, Pearson correlation coefficient and Bland–Altman plot.Normality distribution for demographics in the neonatal and childhood period was assessed using the Shapiro–Wilk's test. Independent samples P< 0.05.The relationship between case status and whole/individual CC segment areas and volumes derived from C8 was analysed using univariable linear regression and adjusted for age at scan, sex and ICV using multivariable linear regression. Using stepwise multivariable linear regression, the effect of sex on whole or individual CC segment areas and volumes/ICV ratio was explored with case–control status, age at scan and the interaction variable ‘case status*sex’ as covariates. For microstructural analysis with TBSS, a voxelwise comparison of diffusion metrics and association with cognitive and motor scores was performed using FSL's non‐parametric permutation testing software, RANDOMISE.Association between TIS assessed from neonatal MRI and childhood CC area and volume normalised to ICV was assessed with simple linear regression. The Kruskal–Wallis test was used to compare childhood whole CC area and volume normalised to ICV, RD and FA between controls and (1) cases with TIS score 0–3 or TIS score 4–6, and (2) cases with no neonatal BGT/WM injury, BGT injury only, WM injury only and both BGT and WM injury. Post‐hoc tests were corrected for multiple hypothesis testing using Benjamini‐Hochberg. A false discovery rate (FDR) ≤5% was considered significant.FSIQ, MABC‐2 total score, WISC‐IV and MABC‐2 domain scores were compared between cases and controls using independent samples t tests and multiple comparisons correction using FDR (as above). Manually parcellated areas of the whole and three CC subdivisions on neonatal and childhood scans were used to assess CC growth. Multivariate regression analyses for repeated measures  using generalised estimating equations were applied using Gaussian family distribution, linked by participant identification number, and independent correlation structure with robust standard error adjustment to analyse whether the growth of the whole CC and CC subdivision areas were related to FSIQ≤ or >85 or MABC‐2 ≤ or >15th centile controlled for age at scan as a covariate. Stepwise multivariable linear regression was used to determine associations between childhood whole CC and segments area, volume, FA, RD and outcome variables FSIQ and MABC‐2 total score adjusted for covariates sex, age at scan, case status and ICV. This was computed for cases and controls together and separately.https://www.graphpad.com/scientific‐software/prism/). All P‐values reported are two‐tailed and <0.05 is considered significant.All analyses were conducted using IBM SPSS Statistics for Windows, Version 27.0  and graphs were produced in GraphPad Prism 8.0 for Windows  did not want to participate in the MRI scan. Of the 82 scans obtained, 3 cases (0 controls) failed T1‐W quality control, leaving 79 participants (40 cases) with suitable quality T1‐W data for morphometric analysis. Of the 82 DWI scans acquired, 4 cases had incomplete data, and 7 cases and 3 controls failed DWI quality control, leaving 68 participants (32 cases) for analysis  were obtained at a mean (standard deviation [SD]) age of 7.9 (1.97) days; 12/42 (28.6%) showed no evidence of brain injury (TIS = 0). BGT scores of 2 and 1 were ascribed to 2 and 6 scans respectively (8/42 = 19%) and WM scores of 3, 2 and 1 were ascribed to 3, 11 and 16 scans respectively (30/42 = 71.4%). No CC signal abnormalities were seen on any image. All eight scans with evidence of BGT injury also showed WM injury . The remaining 22 scans with WM injury  had normal BGT.Scans  and one control had a small focal lesion with an increased T2 signal in the right trigone.r2 = 0.87, P = 0.0001) with manual measurements smaller on average: mean difference (SD) 32.64 mm2 (39.62). The limits of agreement were −1110.3 to 45.01 with three data points (3.6%) outside. To reduce human error, we used automated CC area and volume measurements for all analyses except CC growth, as data from automated analysis of neonatal CC area were unavailable.In 79 participants (40 cases), manual and automated whole CC area measurements were correlated , whole CC area  and volume , area of CC segments I , II , III  and V  and volume of CC segments I , II , III  and V . When adjusting for ICV, sex and age at scan, cases demonstrated reduced CC segment II area  and volume , and whole CC area  and volume  compared to controls.Cases had lower ICV ; there was no sex difference in controls . In the multivariable regression, all males compared to all females had lower CC segment III area/ICV ratio , segment III volume/ICV ratio  and whole CC area/ICV ratio  independent of the age of scan and case status without interaction with case status. However, male cases compared to female cases and controls had lower whole CC volume/ICV ratio  showing an interaction between case status and sex.In cases, males had higher ICV than females (mean difference [95% CI] 138.8 cmP < 0.05) in CC segments II, III, IV and V in cases compared to controls  in the same segments  compared to controls , volume , FA  and higher RD . Compared to children with TIS 0–3, children with TIS 4–6 had lower whole CC area  and volume . Controls, cases with no BGT or WM injury and cases with isolated WM injury differed to cases with BGT and WM injury in the whole CC area, volume and RD  or controls .Corpus callosum area and volume were expressed as a proportion of ICV to account for case–control differences. Of the 42 children with neonatal scans, 40 had morphometric data and 32 had diffusion measurements at early school age. There was no significant linear association between neonatal MRI TIS and whole CC area, volume, RD and FA on early school‐age scans . Cases with an FSIQ ≤85 compared to those >85 had a lower slope of anterior, mid, posterior third and whole CC area growth  compared to healthy matched controls. Reduced CC growth trajectory from birth to childhood was observed in cases at risk of cognitive impairment (FSIQ ≤85). As reduced CC area, volume and altered diffusion metrics in childhood were associated with combined although mild BGT/WM injury on neonatal MRI in the absence of overt CC injury, these differences observed in the CC are likely linked to Wallerian degenerationIn childhood, the CC plays a fundamental role in facilitating cognitive and motor functioning by integrating information across the hemispheres.Consistent with our findings in a smaller sample of the current cohort, cases obtained lower scores on tests of motor and cognitive function at school age.The microstructural analyses echoed the findings of morphological differences in the CC between cases and controls. Comparison of diffusion metrics demonstrated reduced FA and increased RD spanning segments II–V in cases, implicating a role of de‐ or dysmyelination for which RD is sensitive.In the current study, regression analyses revealed that FA and RD across the entire CC were associated with reduced cognitive performance in cases but not controls, suggesting fewer interhemispheric fibres or reduced axonal myelination contribute to impaired cognition. This is consistent with previous findings in this cohort demonstrating case–control differences in the association between structural connectivity and FSIQ in a network comprising exclusively interhemispheric WM connections.In this cohort, mild neonatal brain injury linked to combined BGT/WM damage was associated with reduced CC volume and increased RD in childhood. While CC thinning following global hypoxic–ischaemic brain injury has been described,Finally, we also found evidence of sexual dimorphism. In male cases, there was an interaction between case status and sex on the whole CC volume. In a longitudinal study examining volumetric CC trajectories from infancy to early adulthood, ratios of total CC and genu, posterior midbody and splenium volumes to the whole brain were lower in males than females during childhood.Our relatively small sample size may have precluded detecting an association between motor scores and CC metrics. Further, due to the neonatal acquisition sequences used, we were unable to compute the trajectory of CC volume and microstructure from neonate to childhood, which would have provided a more holistic picture of CC development in this population. Finally, we excluded children with CP from our cohort, limiting the range of data. However, we aimed to investigate changes related to functional outcomes in children without overt physical disabilities in mainstream schooling who may not be identified by current follow‐up protocols following TH for HIE. Given 85% of survivors cooled for HIE do not have CP,Smaller posterior CC area, volume and altered diffusion metrics observed in cases were associated with combined BGT/WM injury on neonatal MRI and lower cognitive scores in childhood. These results help provide a link between lesions on neonatal MRI and cognitive outcome at childhood following neonatal HIE through impaired corpus callosal development, which may support further research into predicting outcome and enabling earlier intervention in this population. Although TH has been successful in reducing deep GM injury following HIE, there are still WM developmental alterations. Therefore, therapeutic interventions should focus on WM protection and development. Ongoing assessments of children's abilities are needed in those without CP attending mainstream school and greater additional support may be required before school age for children without CP following HIE at birth.MT, FMC, JCWB and EC were responsible for the conception and design of the study. HB, APCS, GG, SJ, JCWB and EC were responsible for the acquisition and analysis of data. All authors contributed to writing and editing the manuscript and figures.None declared.


GLA. The phenotype varies depending on the GLA variant, alpha‐galactosidase residual activity, patient's age and gender and, for females, X chromosome inactivation. Over 1000 variants have been identified, many through screening protocols more susceptible to disclose non‐pathogenic variants or variants of unknown significance (VUS). This, together with the non‐specificity of some FD symptoms, challenges physicians attempting to interpret GLA variants. The traditional way to interpreting pathogenicity is based on a combined approach using allele frequencies, genomic databases, global and disease‐specific clinical databases, and in silico tools proposed by the American College of Medical Genetics and Genomics. Here, a panel of FD specialists convened to study how expertise may compare with the traditional approach. Several GLA VUS, highly controversial in the literature , were re‐analyzed through reviews of patients' charts. The same was done for pathogenic GLA variants with some specificities. Our data suggest that input of geneticists and physicians with wide expertise in disease phenotypes, prevalence, inheritance, biomarkers, alleles frequencies, disease‐specific databases, and literature greatly contribute to a more accurate interpretation of the pathogenicity of variants, bringing a significant additional value over the traditional approach.Fabry disease (FD) is an X‐linked genetic disease due to pathogenic variants in   Quantitative/functional α‐Gal A deficiency leads to progressive accumulation of its undegraded substrates globotriaosylceramide (Gb3) and its deacylated derivative globotriaosylsphingosine (lyso‐Gb3), in tissues and body fluids.Fabry disease  is a rare X‐linked inborn error of glycosphingolipid metabolism, caused by pathogenic variants in the GLA variant, age, gender and residual α‐Gal A activity.FD has two main forms, “classic” and “later‐onset.”GLA variants. Understanding variant pathogenicity is the key to accurate prevalence estimation, diagnosis, and management of FD. Revised interpretation of GLA variants, previously inaptly classified “pathogenic,” has decreased the estimates of FD prevalence (Table High‐throughput next generation sequencing (NGS)‐based screening programs in high‐risk populations and newborns have identified several novel ce Table .5, 6GLA variants have been reported,Over 1000 GLA VUS. This paper illustrates the challenges in interpretating pathogenicity of GLA VUS through real‐life cases, highlights the input of highly specialized experts and provide practical recommendations for accurate diagnosis of FD.A panel of French physicians involved in the diagnosis and management of FD , convened to discuss the diagnostic challenges posed by 2GLA alleles and using “form” or “phenotype” for clinical description of the disease . The terms “mutation” and “polymorphism” should be avoided, due to implied pathogenicity and benignity, respectively. Variants are categorized as benign (class 1), likely benign (class 2), of uncertain significance , likely pathogenic (class 4), or pathogenic (class 5).In FD, the term “variant” had been historically used to describe the two main forms/phenotypes, creating confusion between genetic and clinical levels when the Human Genome Variation Society (HGVS) advised to replace “mutation” by the term “variant.”33.1The “classic” phenotype typically manifests during childhood,Absent/very low residual α‐Gal A activity is associated with the more severe classic phenotype, earlier onset of symptoms, and multi‐organ involvement.3.2Alpha‐Gal A activity can be measured in leukocytes, plasma, or dried blood spot (DBS) cards. Reference values vary widely depending on pre‐analytical and technical conditions. The activity of another lysosomal enzyme, e.g., β‐galactosidase or hexosaminidase, should be simultaneously determined for quality control. Alpha‐Gal A deficiency is expressed in micromole/L/h.Globotriaosylsphingosine or lyso‐Gb3 ‐β‐d‐galactopyranosyl‐‐β‐d‐glucopyranosyl‐‐‐2‐amino‐octadec‐4‐ene‐1,3‐diol) is the deacylated form of Gb3. Increased plasma lyso‐Gb3 levels in male FD patients were first reported in 2008.3. Lyso‐Gb3 has progressively superseded Gb3 as the main biomarker for diagnosing and monitoring FD.3.2.13 in the pathogenesis of FD, possibly through inflammation.3 to cultured smooth muscle cells induces cell proliferation, suggesting its role in the vascular remodeling characteristic of FD.3 on endothelial nitric oxide synthase may also contribute to vascular dysfunction in FD patients.3 may induce glomerular damage through activation of Notch1 signaling and TGF‐β1‐mediated production of extracellular matrix by podocytes.3 in target tissues including myocardium and kidney. In a recently proposed model of cardiac FD staging, myocardial inflammation can also precede the effects of Gb3 storage on cardiac wall thickness and function.Increasing evidence implicates lyso‐Gb3.2.23 levels are dramatically increased in male patients with classic FD .3 levels are higher in those with frameshift and nonsense variants than in those with missense variants.3 can reliably distinguish classically affected male and female FD patients from individuals without FD.3 levels are insufficient to exclude FD in women, but are highly unlikely in male FD patients. Interestingly, several studies have demonstrated normal plasma lyso‐Gb3 levels in individuals carrying benign GLA variants.3 levels was reported to indicate FD in patients with an uncertain diagnosis of FD carrying a VUS, while normal plasma lyso‐Gb3 levels were observed in individuals with negative tissue biopsies.Plasma lyso‐Gb3 levels and clinical FD manifestations. In both male and female FD patients, a correlation between plasma lyso‐Gb3 exposure and disease severity was reported.3 concentration was identified as an independent risk factor for cerebrovascular white matter lesions in male patients and left ventricular hypertrophy in female patients.3 concentrations and left ventricular mass index, but not kidney function.3 levels correlated with left ventricular mass, glomerular filtration rate, and overall disease severity.3 levels and severe clinical events has been recently reported.3 levels are also increased in FD patients, and correlate with Gb3 levels.3 concentration, total urinary concentration of lyso‐Gb3 and its structural analogs represent a specific diagnostic biomarker, and is elevated in both classical and non‐classical FD patients.Multiple studies have investigated the association between plasma lyso‐Gb3.2.33 levels within 3 months, thereafter, remaining relatively stable.3 levels was reported for female FD patients.3 clearance. In male patients with classic FD, lyso‐Gb3 levels a year after ERT initiation were lower in patients who began ERT before age 25, versus those who started later in life.3.3 levels.3 and α‐Gal A activity) is crucial to adjust treatment approach, and patient clinical control at least every 6–12 months.3 is a useful biomarker both for diagnosing and monitoring FD patients.In hemizygous FD patients, enzyme replacement therapy (ERT) markedly reduces  plasma lyso‐Gb3.3GLA variant.GLA or another disease‐causing gene influencing FD phenotype.Multiple factors may contribute to the phenotypic heterogeneity associated with a given GLA variant in female FD patients.The clinical presentation is more variable in heterozygous females than hemizygous males, likely due to epigenetics.4GLA high allelic heterogeneity , c.679C>T; p.(Arg227*), c.901C>T; p.(Arg301*)] and missense variants impacting the active site or key residues of the enzyme .GLA variants may, in contrast, be associated with later‐onset FD and a residual enzyme activity between 3% and 25% of normal in male patients, including c.337T>C; p.(Phe113Leu)/p.(F113L)Classic FD with absent or negligible α‐Gal A activity is associated with several nonsense GLA variants have been classified as VUS, partly because many of them are private to one or few families and because ACMG‐AMP classification is difficult to implement in routine practice. Interpreting variants is particularly complex in the later‐onset phenotype, which may mimic isolated HCM and lack other FD features.GLA variants has been extensively discussed in the literature. However, for some of them it remains unclear, requiring insights from the experts for an accurate interpretation.Some GLA variants' pathogenicity, and the insights from field experts, are presented. Written informed consent was obtained for all cases.Hereafter, eight cases illustrating the challenges in interpretation of 4.14.1.1cornea verticillata), renal or ear involvement were all absent. Holter‐ECG (24‐h) was normal. Cardiac MRI revealed a normal left ventricular mass (81 g/m2) and gadolinium late‐enhancement showed subendocardial  fibrosis of inferolateral, anterolateral, and septal walls, indicative of ischemia rather than FD. Significant obstructive coronary disease was absent. Plasma α‐Gal A levels were slightly reduced (6.7 μkat/kg) with normal plasma lyso‐Gb3  and urinary Gb3. Molecular genetic analyses identified a hemizygous missense GLA variant c.427G>A; p.(Ala143Thr).A 41‐year‐old man was referred for transient ischemic attack (TIA) with dysarthria and central paralysis of the left VII cranial nerve. Brain magnetic resonance imaging (MRI) revealed sequelae of a recent minor stroke and multifocal punctate white matter lesions, mainly in the periventricular area. Medical history included hypertension and tobacco use. Classic FD signs  carriers, suggesting the non‐pathogenicity of this Considering the low specificity of a single disease symptom, high residual enzyme activity and recent literature strongly questioning the pathogenicity of the p.(Ala143Thr) variant, diagnosis of FD in this patient was reconsidered and finally ruled out/excluded. Other etiologies for cardiac fibrosis are being investigated.GLA analysis identified the p.(Ala143Thr) variant. Investigation revealed arthritis of the second and third metacarpophalangeal joints, but no signs of FD. Alpha‐Gal A activity and plasma lyso‐Gb3  were normal. Family screening identified p.(Ala143Thr) in the patient's 15‐ and 21‐year‐old sons and 35‐year‐old sister. Neither of her sons had FD signs/symptoms and therefore refused further investigations including enzymatic assay, which would have been diagnostic. Extensive investigations in the patient's sister, who had multiple sclerosis, did not reveal any sign of FD. The index case was subsequently diagnosed with spondylarthritis, while FD was excluded.A 44‐year‐old female patient with a history of pain due to polyarthralgia and rachialgia, and ischemic stroke of unknown etiology consulted a rheumatology department. Molecular 3 . Extensive analysis of a panel of sarcomeric genes involved in hereditary cardiomyopathies revealed c.6327_6332del; p.(2109_2111del), a pathogenic variant in the FLNC gene . This FLNC variant was inherited from his 57‐year‐old mother, who will be further investigated.A 25‐year‐old male patient with moderate HCM was referred for further analyses. Septal wall thickness was 17 mm. Ventricular obstruction, dilation and rhythm anomalies were absent. Left ventricular ejection fraction was 60%. There were no features of FD. Family history revealed that patient's maternal uncle, with the p.(Ala143Thr) variant, had been considered 12 years earlier with a possible diagnosis of FD, which was subsequently reconsidered and excluded on the basis of α‐Gal A activity only reduced to 35%  and normal urinary GbGLA missense variants. Those patients had normal lyso‐Gb3 levels, no typical FD symptoms, Gb3 negative biopsies and generally an absence of stroke/TIA in other family members carrying p.(Ala143Thr).https://evs.gs.washington.edu/), p.(Ala143Thr) is the paradigm of a coincidental finding while exploring a variety of genetic diseases, such as MYH‐associated polyposis, achromatopsia, hereditary cancer‐predisposing syndrome, and renal dysplasia, where it should not be considered causal (similarly to the aforementioned strokes/TIA).The missense p.Ala143Thr) variant was first described in 1997Thr variaThe high residual enzyme activity associated with p.Ala143Thr) is in favor of a pseudo‐deficiency rather than a disease‐causing allele per se.Thr is in4.1.23 . The GLA variant c.937G>T; p.(Asp313Tyr) was identified, but an expert geneticist (D.P.G.) advised to exclude FD, since there was no other clinical sign of FD or relevant family history, apart from slightly decreased α‐Gal A activity . Renal biopsy was not suggestive of FD.A 67‐year‐old female had undergone chronic hemodialysis since age 26, due to biopsy‐confirmed membranoproliferative glomerulonephritis with unclear etiology. DBS analysis demonstrated reduced α‐Gal A activity  and normal lyso‐GbGLA variant p.(Asp313Tyr)/Tyr313, also identified in his two sisters affected with familial cardiomyopathy and his asymptomatic mother. Notably, the patient's father had died of a cardiac arrest, which raises suspicion of an autosomal dominant transmission. Extensive investigations did not reveal any FD feature. In contrast, additional molecular studies using a panel of five sarcomeric genes identified a pathogenic variant in the TNNT2 troponin T gene. This variant was likely responsible for the familial cardiomyopathy, inherited from the patient's father whereas the p.(Asp313Tyr) GLA variant inherited from patient's mother was coincidental.A 50‐year‐old male consulted due to a family history of HCM and GLA gene, though clinical description was not provided. However, another group subsequently published a case with the additional presence of another GLA variant , creating an uncertainty regarding pathogenicity of p.(Asp313Tyr).GLA variants, that is, c.352C>T; p.(Arg118Cys)/p.(R118C) (24.5%),The missense p.Asp313Tyr) variant was historically considered pathogenic for a while.13Tyr var4.1.3GLA variant. His sister, an asymptomatic p.(Ser126Gly) GLA carrier, was already considered for FD‐specific treatment in Italy. Extensive investigations did not reveal any sign or symptom of FD; α‐Gal A levels were 57% of normal in two independent assays. Brain MRI, glomerular filtration rate and lyso‐Gb3 were normal and FD was consequently excluded.A 45‐year‐old Italian male was referred to the French Referral Center for Fabry disease for a suspicion of familial FD in association to the p.(Ser126Gly) n = 60; genomes frequency of 0.063%, n = 14) in GnomAD, above the global frequency of FD (Table p.(Ser126Gly) has been found in 74 cases , which was categorized as pathogenic. Patient had elevated lyso‐Gb3 . Investigations found no renal/neurological involvement, angiokeratoma, or cornea verticillata. Her two sisters, (both with HCM) and rest of family were unavailable for further evaluation. FD was confirmed and disease‐specific treatment initiated. The frameshift associated with this variant was predicted to result in classic FD even though patient's symptoms were mostly limited to the heart, illustrating the phenotypic variability of many genetic diseases.A 48‐year‐old Algerian female was referred for evaluation of isolated HCM. The patient reported acroparesthesia during childhood. NGS sequencing of genes involved in hereditary cardiomyopathies identified a novel, previously undescribed frameshift 4.2.23 levels . Fabry disease diagnosis was confirmed, and disease‐specific treatment initiated, together with close clinical and biochemical follow‐up. Additional tests showed microalbuminuria, presence of T2/FLAIR signal alterations in the periventricular white matter on brain MRI, normal audiogram, and absence of cornea verticillata. Family screening is ongoing for his two sisters (aged 57 and 59), his brother (aged 42), and his two daughters (aged 26 and 27).A 54‐year‐old male was hospitalized for syncope, due to complete atrioventricular block; a dual chamber pacemaker was implanted. He had no FD relevant medical history (except for clustered angiokeratomas). His mother had died from sudden cardiac arrest at age 69. Investigations revealed markedly reduced leukocyte α‐Gal A activity  and elevated plasma lyso‐GbGLA genotyping revealed a novel hemizygous 4‐base insertion (c.1281_1282insCTTA) predicted to preserve the entire coding sequence of α‐galactosidase A, with the exception of a conservative change of the last amino‐acid (leucine to isoleucine) at codon 429, together with the addition of a 22‐amino‐acid tail. A frameshift variant is theoretically predicted to cause a severe full‐blown classic phenotype in a 54‐year‐old male patient, which was not the case in this hemizygote with predominantly cardiac symptoms. This may be due to the unique nature of the genetic variant preserving the coding sequence until the last codon of the protein.4.3https://varsome.com) with the addition of “BS1” . It changed again from “VUS” to “pathogenic” on August 3rd, 2020, with the addition of the “PS3” (Pathogenic Strong criterion number 3), as a Varsome user reported this variant as “likely pathogenic” in a paper supported by a functional expression study, with additional removal of both criteria BS1 and BS2 criteria. Furthermore, p.(Ala143Thr) is classified as benign in dbFGP, with conflicting results in other databases   can help in determining the pathogenicity of missense variants, their partial reliability is well‐known as confirmed with/for the five most common (likely) benign ts Table .GLA variant pathogenicity  from 22 individuals with the p.(Arg118Cys) on Table .59 Caron Table .GLA variant was first suggested to cause FD over 20 years ago,3 and normal tissue biopsies. Frequency of this variant in Asian populations may reach 0.83% in Japan and 1% in South Korea, which is much higher than FD global prevalence.GLA variant.While the p.(Glu66Gln) 5Distinguishing disease‐causing variants from benign bystanders is probably one of the biggest challenges in contemporary clinical genetics. Below, the authors provide practical recommendations for accurate diagnosis of FD Table , which m5.13 levels may help in the interpretation, although the possibility of normal lyso‐Gb3 levels in heterozygotes affected with the later‐onset phenotype should be considered.In FD and other genetic diseases of metabolism, surrogate biomarkers are available, which, when properly used, can optimize interpretation of the clinical relevance of a genetic variant. In male patients, α‐Gal A levels are key to confirm or exclude pathogenicity of a variant. In both genders, plasma lyso‐GbGLA variant does not necessarily imply pathogenicity, since the available amenability table includes proven non‐pathogenic variants such as p.(Arg118Cys), p.(Ala143Thr) and p.(Asp313Tyr) .In the case of FD, where a “pharmacogenetic” specific treatment exists, it should be noted that “amenability” of a given 5.2GLA sequencing is conducted in two contexts: clinical suspicion of FD, or screening at‐risk populations . Screening of large at‐risk populations will therefore disclose GLA VUS with relatively high prevalence. In patients with GLA VUS and a non‐specific phenotype, other common genetic causes should be systematically searched . Erroneous classification of benign variants as pathogenic delays correct diagnosis and can prompt expensive unnecessary treatments. In contrast, detection of a likely benign GLA variant in a patient with very strong clinical suspicion of FD, should prompt search for a second GLA variant.3 inclusions in some cells does not necessarily imply pathogenicity. Electron microscopy can better assess the amount of stored material, although the sampled tissue and the cellular type  may yield different results and interpretation should consider phenocopies of FD .5.3GLA variants (p.(Asn215Ser) in Caucasians and IVS4 + 919G>A in Asians) occur indeed at a frequency <1%. However, this rule does not always apply, as exemplified by FD, where many GLA variants considered or proven as benign (p.(Glu66Gln), p.(Arg118Cys), p.(Ser126Gly), p.(Ala143Thr), p.(Asp313Tyr)) do not meet the >1% frequency threshold.Traditionally, an AF < 1% supports pathogenicity and >1% benignity. The most frequent pathogenic GLA variant, if the variant's AF is higher than 0.0125%  and higher than the frequency of the most common pathogenic allele (indicated as the “Popmax Filtering AF” in GnomAD), the variant should be considered as “likely benign.”http://cardiodb.org/allelefrequencyapp/) can help compute the maximum expected AF and provide better estimates of disease‐causing variants in the general population (maximum credible population AF).When considering the pathogenicity of a GLA VUS can be performed for further information. Absence of co‐segregation strongly suggests benignity while presence of co‐segregation is in favor pathogenicity.Once pathogenicity of a missense variant has been verified, the next step is to determine its association with a specific phenotype. Whether the “classic” or “later‐onset” phenotype of FD is more prevalent is still unclear; several arguments exist for both hypotheses. The total number of genetic variants responsible for classic FD overwhelms the number of genetic variants associated with later‐onset FD Figure .69 How5.4It is important to periodically review the literature and perform a specific database search for additional evidence on pathogenicity/benignity of variants, since with growing evidence over time, some variants originally designated VUS may be reclassified as either likely pathogenic/pathogenic or in contrast likely benign/benign and in the latter case, no longer considered to be FD‐causing variants.6GLA variants integrates information from in vitro prediction softwares, AF and gene databases.GLA variants. Misdiagnosis prolongs patient diagnostic odyssey, resulting in higher morbidity and adding unnecessary financial burden to the healthcare system.Since the publication of ACMG‐AMP,D.P.G.: Consultant for Sanofi‐Genzyme, Idorsia, Takeda; speaker's honoraria from Takeda, Amicus, Sanofi‐Genzyme. T.L.: Hotel/travel grants from Sanofi‐Genzyme, Takeda, BioMarin, Enzyvant, Orphan. E.H.: Speaker's honoraria and/or travel grants from Sanofi‐ Genzyme, GSK, Actelion, Sobi; B.K.: Speaker's honoraria from Travere, Sanofi, Alnylam, Reata; hotel/travel grants from Sanofi, Travere, Reata; D.L.: Speaker's honoraria and hotel/travel expenses from Amicus, Sanofi‐Genzyme. V.L.S.: Speaker's honoraria from Sanofi‐Genzyme; hotel/travel grants from Amicus, Orphan, Takeda, Sanofi‐Genzyme; K.N.: Speaker's honoraria, travel grants from Sanofi Genzyme; grants from Amicus therapeutics; E.N.: Speaker's honoraria and/or travel grants from Amicus Therapeutics, Sanofi‐Genzyme. J.P.R.: Consultant for Sanofi‐Genzyme; hotel/travel expenses from Amicus Therapeutics; speaker's honoraria from Amgen.Each author participated in the meetings, analyzing literature, revising, and interpreting the presented cases with current data, establishing recommendations and approved the manuscript. Dominique P. Germain designed and supervised the study, substantially contributed to developing the manuscript and wrote the revised version.