A pseudoautosomal glycosylation disorder prompts the revision of dolichol biosynthesis.

Dolichol is a lipid critical for N-glycosylation as a carrier for activated sugars and nascent oligosaccharides. It is commonly thought to be directly produced from polyprenol by the enzyme SRD5A3. Instead, we found that dolichol synthesis requires a three-step detour involving additional metabolites, where SRD5A3 catalyzes only the second reaction. The first and third steps are performed by DHRSX, whose gene resides on the pseudoautosomal regions of the X and Y chromosomes. Accordingly, we report a pseudoautosomal-recessive disease presenting as a congenital disorder of glycosylation in patients with missense variants in DHRSX (DHRSX-CDG). Of note, DHRSX has a unique dual substrate and cofactor specificity, allowing it to act as a NAD+-dependent dehydrogenase and as a NADPH-dependent reductase in two non-consecutive steps. Thus, our work reveals unexpected complexity in the terminal steps of dolichol biosynthesis. Furthermore, we provide insights into the mechanism by which dolichol metabolism defects contribute to disease.

Loss of aryl hydrocarbon receptor potentiates FoxM1 signaling to enhance self-renewal of colonic stem and progenitor cells.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that senses xenobiotics, diet, and gut microbial-derived metabolites, is increasingly recognized as a key regulator of intestinal biology. However, its effects on the function of colonic stem and progenitor cells remain largely unexplored. Here, we observed that inducible deletion of AhR in Lgr5+ stem cells increases the percentage of colonic stem cells and enhances organoid initiating capacity and growth of sorted stem and progenitor cells, while AhR activation has the opposite effect. Moreover, intestinal-specific AhR knockout increases basal stem cell and crypt injury-induced cell proliferation and promotes colon tumorigenesis in a preclinical colitis-associated tumor model by upregulating FoxM1 signaling. Mechanistically, AhR transcriptionally suppresses FoxM1 expression. Activation of AhR in human organoids recapitulates phenotypes observed in mice, such as reduction in the percentage of colonic stem cells, promotion of stem cell differentiation, and attenuation of FoxM1 signaling. These findings indicate that the AhR-FoxM1 axis, at least in part, mediates colonic stem/progenitor cell behavior.

De novo variants in POLR3B cause ataxia, spasticity, and demyelinating neuropathy.

POLR3B encodes the second-largest catalytic subunit of RNA polymerase III, an enzyme involved in transcription. Bi-allelic pathogenic variants in POLR3B are a well-established cause of hypomyelinating leukodystrophy. We describe six unrelated individuals with de novo missense variants in POLR3B and a clinical presentation substantially different from POLR3-related leukodystrophy. These individuals had afferent ataxia, spasticity, variable intellectual disability and epilepsy, and predominantly demyelinating sensory motor peripheral neuropathy. Protein modeling and proteomic analysis revealed a distinct mechanism of pathogenicity; the de novo POLR3B variants caused aberrant association of individual enzyme subunits rather than affecting overall enzyme assembly or stability. We expand the spectrum of disorders associated with pathogenic variants in POLR3B to include a de novo heterozygous POLR3B-related disorder.

Evaluation of the diagnostic accuracy of exome sequencing and its impact on diagnostic thinking for patients with rare disease in a publicly funded health care system: A prospective cohort study.

PURPOSE: To evaluate the diagnostic utility of publicly funded clinical exome sequencing (ES) for patients with suspected rare genetic diseases. METHODS: We prospectively enrolled 297 probands who met eligibility criteria and received ES across 5 sites in Ontario, Canada, and extracted data from medical records and clinician surveys. Using the Fryback and Thornbury Efficacy Framework, we assessed diagnostic accuracy by examining laboratory interpretation of results and assessed diagnostic thinking by examining the clinical interpretation of results and whether clinical-molecular diagnoses would have been achieved via alternative hypothetical molecular tests. RESULTS: Laboratories reported 105 molecular diagnoses and 165 uncertain results in known and novel genes. Of these, clinicians interpreted 102 of 105 (97%) molecular diagnoses and 6 of 165 (4%) uncertain results as clinical-molecular diagnoses. The 108 clinical-molecular diagnoses were in 104 families (35% diagnostic yield). Each eligibility criteria resulted in diagnostic yields of 30% to 40%, and higher yields were achieved when >2 eligibility criteria were met (up to 45%). Hypothetical tests would have identified 61% of clinical-molecular diagnoses. CONCLUSION: We demonstrate robustness in eligibility criteria and high clinical validity of laboratory results from ES testing. The importance of ES was highlighted by the potential 40% of patients that would have gone undiagnosed without this test.

POLR3B is associated with a developmental and epileptic encephalopathy with myoclonic-atonic seizures and ataxia.

OBJECTIVE: POLR3B encodes the second largest subunit of RNA polymerase III, which is essential for transcription of small non-coding RNAs. Biallelic pathogenic variants in POLR3B are associated with an inherited hypomyelinating leukodystrophy. Recently, de novo heterozygous variants in POLR3B were reported in six individuals with ataxia, spasticity, and demyelinating peripheral neuropathy. Three of these individuals had epileptic seizures. The aim of this article is to precisely define the epilepsy phenotype associated with de novo heterozygous POLR3B variants. METHODS: We used online gene-matching tools to identify 13 patients with de novo POLR3B variants. We systematically collected genotype and phenotype data from clinicians using two standardized proformas. RESULTS: All 13 patients had novel POLR3B variants. Twelve of 13 variants were classified as pathogenic or likely pathogenic as per American College of Medical Genetics (ACMG) criteria. Patients presented with generalized myoclonic, myoclonic-atonic, atypical absence, or tonic-clonic seizures between the ages of six months and 4 years. Epilepsy was classified as epilepsy with myoclonic-atonic seizures (EMAtS) in seven patients and "probable EMAtS" in two more. Seizures were treatment resistant in all cases. Three patients became seizure-free. All patients had some degree of developmental delay or intellectual disability. In most cases developmental delay was apparent before the onset of seizures. Three of 13 cases were reported to have developmental stagnation or regression in association with seizure onset. Treatments for epilepsy that were reported by clinicians to be effective were: sodium valproate, which was effective in five of nine patients (5/9) who tried it; rufinamide (2/3); and ketogenic diet (2/3). Additional features were ataxia/incoordination (8/13); microcephaly (7/13); peripheral neuropathy (4/13), and spasticity/hypertonia (6/13). SIGNIFICANCE: POLR3B is a novel genetic developmental and epileptic encephalopathy (DEE) in which EMAtS is the predominant epilepsy phenotype. Ataxia, neuropathy, and hypertonia may be variously observed in these patients.

SMPD4 regulates mitotic nuclear envelope dynamics and its loss causes microcephaly and diabetes.

Biallelic loss-of-function variants in SMPD4 cause a rare and severe neurodevelopmental disorder with progressive congenital microcephaly and early death. SMPD4 encodes a sphingomyelinase that hydrolyses sphingomyelin into ceramide at neutral pH and can thereby affect membrane lipid homeostasis. SMPD4 localizes to the membranes of the endoplasmic reticulum and nuclear envelope and interacts with nuclear pore complexes (NPC). We refine the clinical phenotype of loss-of-function SMPD4 variants by describing five individuals from three unrelated families with longitudinal data due to prolonged survival. All individuals surviving beyond infancy developed insulin-dependent diabetes, besides presenting with a severe neurodevelopmental disorder and microcephaly, making diabetes one of the most frequent age-dependent non-cerebral abnormalities. We studied the function of SMPD4 at the cellular and organ levels. Knock-down of SMPD4 in human neural stem cells causes reduced proliferation rates and prolonged mitosis. Moreover, SMPD4 depletion results in abnormal nuclear envelope breakdown and reassembly during mitosis and decreased post-mitotic NPC insertion. Fibroblasts from affected individuals show deficient SMPD4-specific neutral sphingomyelinase activity, without changing (sub)cellular lipidome fractions, which suggests a local function of SMPD4 on the nuclear envelope. In embryonic mouse brain, knockdown of Smpd4 impairs cortical progenitor proliferation and induces premature differentiation by altering the balance between neurogenic and proliferative progenitor cell divisions. We hypothesize that, in individuals with SMPD4-related disease, nuclear envelope bending, which is needed to insert NPCs in the nuclear envelope, is impaired in the absence of SMPD4 and interferes with cerebral corticogenesis and survival of pancreatic beta cells.

The clinical and molecular spectrum of ZFYVE26-associated hereditary spastic paraplegia: SPG15.

In the field of hereditary spastic paraplegia (HSP), progress in molecular diagnostics needs to be translated into robust phenotyping studies to understand genetic and phenotypic heterogeneity and to support interventional trials. ZFYVE26-associated hereditary spastic paraplegia (HSP-ZFYVE26, SPG15) is a rare, early-onset complex HSP, characterized by progressive spasticity and a variety of other neurological symptoms. While prior reports, often in populations with high rates of consanguinity, have established a general phenotype, there is a lack of systematic investigations and a limited understanding of age-dependent manifestation of symptoms. Here we delineate the clinical, neuroimaging and molecular features of 44 individuals from 36 families, the largest cohort assembled to date. Median age at last follow-up was 23.8 years covering a wide age range (11-61 years). While symptom onset often occurred in early childhood [median: 24 months, interquartile range (IQR) = 24], a molecular diagnosis was reached at a median age of 18.8 years (IQR = 8), indicating significant diagnostic delay. We demonstrate that most patients present with motor and/or speech delay or learning disabilities. Importantly, these developmental symptoms preceded the onset of motor symptoms by several years. Progressive spasticity in the lower extremities, the hallmark feature of HSP-ZFYVE26, typically presents in adolescence and involves the distal lower limbs before progressing proximally. Spasticity in the upper extremities was seen in 64%. We found a high prevalence of extrapyramidal movement disorders including cerebellar ataxia (64%) and dystonia (11%). Parkinsonism (16%) was present in a subset and showed no sustained response to levodopa. Cognitive decline and neurogenic bladder dysfunction progressed over time in most patients. A systematic analysis of brain MRI features revealed a common diagnostic signature consisting of thinning of the anterior corpus callosum, signal changes of the anterior forceps and non-specific cortical and cerebellar atrophy. The molecular spectrum included 45 distinct variants, distributed across the protein structure without mutational hotspots. Spastic Paraplegia Rating Scale scores, SPATAX Disability Scores and the Four Stage Functional Mobility Score showed moderate strength in representing the proportion of variation between disease duration and motor dysfunction. Plasma neurofilament light chain levels were significantly elevated in all patients (Mann-Whitney U-test, P < 0.0001) and were correlated inversely with age (Spearman's rank correlation coefficient r = -0.65, P = 0.01). In summary, our systematic cross-sectional analysis of HSP-ZFYVE26 patients across a wide age-range, delineates core clinical, neuroimaging and molecular features and identifies markers of disease severity. These results raise awareness to this rare disease, facilitate an early diagnosis and create clinical trial readiness.

Characterization of Cardiac-Onset Initial Presentation in Friedreich Ataxia.

We examined the clinical features of Friedreich ataxia (FRDA) patients who present first with cardiac disease in order to understand the earliest features of the diagnostic journey in FRDA. We identified a group of subjects in the FACOMS natural history study whose first identified clinical feature was cardiac. Only 0.5% of the total cohort belonged to this group, which was younger on average at the time of presentation. Their cardiac symptoms ranged from asymptomatic features to heart failure with severe systolic dysfunction. Two of those individuals with severe dysfunction proceeded to heart transplantation, but others spontaneously recovered. In most cases, diagnosis of FRDA was not made until well after cardiac presentation. The present study shows that some FRDA patients present based on cardiac features, suggesting that earlier identification of FRDA might occur through enhancing awareness of FRDA among pediatric cardiologists who see such patients. This is important in the context of newly identified therapies for FRDA.

De Novo variants in EEF2 cause a neurodevelopmental disorder with benign external hydrocephalus.

Eukaryotic translation elongation factor 2 (eEF2) is a key regulatory factor in gene expression that catalyzes the elongation stage of translation. A functionally impaired eEF2, due to a heterozygous missense variant in the EEF2 gene, was previously reported in one family with spinocerebellar ataxia-26 (SCA26), an autosomal dominant adult-onset pure cerebellar ataxia. Clinical exome sequencing identified de novo EEF2 variants in three unrelated children presenting with a neurodevelopmental disorder (NDD). Individuals shared a mild phenotype comprising motor delay and relative macrocephaly associated with ventriculomegaly. Populational data and bioinformatic analysis underscored the pathogenicity of all de novo missense variants. The eEF2 yeast model strains demonstrated that patient-derived variants affect cellular growth, sensitivity to translation inhibitors and translational fidelity. Consequently, we propose that pathogenic variants in the EEF2 gene, so far exclusively associated with late-onset SCA26, can cause a broader spectrum of neurologic disorders, including childhood-onset NDDs and benign external hydrocephalus.

Heterozygous Variants in KDM4B Lead to Global Developmental Delay and Neuroanatomical Defects.

KDM4B is a lysine-specific demethylase with a preferential activity on H3K9 tri/di-methylation (H3K9me3/2)-modified histones. H3K9 tri/di-demethylation is an important epigenetic mechanism responsible for silencing of gene expression in animal development and cancer. However, the role of KDM4B on human development is still poorly characterized. Through international data sharing, we gathered a cohort of nine individuals with mono-allelic de novo or inherited variants in KDM4B. All individuals presented with dysmorphic features and global developmental delay (GDD) with language and motor skills most affected. Three individuals had a history of seizures, and four had anomalies on brain imaging ranging from agenesis of the corpus callosum with hydrocephalus to cystic formations, abnormal hippocampi, and polymicrogyria. In mice, lysine demethylase 4B is expressed during brain development with high levels in the hippocampus, a region important for learning and memory. To understand how KDM4B variants can lead to GDD in humans, we assessed the effect of KDM4B disruption on brain anatomy and behavior through an in vivo heterozygous mouse model (Kdm4b+/-), focusing on neuroanatomical changes. In mutant mice, the total brain volume was significantly reduced with decreased size of the hippocampal dentate gyrus, partial agenesis of the corpus callosum, and ventriculomegaly. This report demonstrates that variants in KDM4B are associated with GDD/ intellectual disability and neuroanatomical defects. Our findings suggest that KDM4B variation leads to a chromatinopathy, broadening the spectrum of this group of Mendelian disorders caused by alterations in epigenetic machinery.

De Novo Variants in the ATPase Module of MORC2 Cause a Neurodevelopmental Disorder with Growth Retardation and Variable Craniofacial Dysmorphism.

MORC2 encodes an ATPase that plays a role in chromatin remodeling, DNA repair, and transcriptional regulation. Heterozygous variants in MORC2 have been reported in individuals with autosomal-dominant Charcot-Marie-Tooth disease type 2Z and spinal muscular atrophy, and the onset of symptoms ranges from infancy to the second decade of life. Here, we present a cohort of 20 individuals referred for exome sequencing who harbor pathogenic variants in the ATPase module of MORC2. Individuals presented with a similar phenotype consisting of developmental delay, intellectual disability, growth retardation, microcephaly, and variable craniofacial dysmorphism. Weakness, hyporeflexia, and electrophysiologic abnormalities suggestive of neuropathy were frequently observed but were not the predominant feature. Five of 18 individuals for whom brain imaging was available had lesions reminiscent of those observed in Leigh syndrome, and five of six individuals who had dilated eye exams had retinal pigmentary abnormalities. Functional assays revealed that these MORC2 variants result in hyperactivation of epigenetic silencing by the HUSH complex, supporting their pathogenicity. The described set of morphological, growth, developmental, and neurological findings and medical concerns expands the spectrum of genetic disorders resulting from pathogenic variants in MORC2.

Plasma Neurofilament Light Chain Is Elevated in Adaptor Protein Complex 4-Related Hereditary Spastic Paraplegia.

BACKGROUND: Adaptor protein complex 4-associated hereditary spastic paraplegia (AP-4-HSP) is caused by pathogenic biallelic variants in AP4B1, AP4M1, AP4E1, and AP4S1. OBJECTIVE: The aim was to explore blood markers of neuroaxonal damage in AP-4-HSP. METHODS: Plasma neurofilament light chain (pNfL) and glial fibrillary acidic protein (GFAP) levels were measured in samples from patients and age- and sex-matched controls (NfL: n = 46 vs. n = 46; GFAP: n = 14 vs. n = 21) using single-molecule array assays. Patients' phenotypes were systematically assessed using the AP-4-HSP natural history study questionnaires, the Spastic Paraplegia Rating Scale, and the SPATAX disability score. RESULTS: pNfL levels increased in AP-4-HSP patients, allowing differentiation from controls (Mann-Whitney U test: P = 3.0e-10; area under the curve = 0.87 with a 95% confidence interval of 0.80-0.94). Phenotypic cluster analyses revealed a subgroup of individuals with severe generalized-onset seizures and developmental stagnation, who showed differentially higher pNfL levels (Mann-Whitney U test between two identified clusters: P = 2.5e-6). Plasma GFAP levels were unchanged in patients with AP-4-HSP. CONCLUSIONS: pNfL is a potential disease marker in AP-4-HSP and can help differentiate between phenotypic subgroups. © 2023 International Parkinson and Movement Disorder Society.

XRCC1 mutation is associated with PARP1 hyperactivation and cerebellar ataxia.

XRCC1 is a molecular scaffold protein that assembles multi-protein complexes involved in DNA single-strand break repair. Here we show that biallelic mutations in the human XRCC1 gene are associated with ocular motor apraxia, axonal neuropathy, and progressive cerebellar ataxia. Cells from a patient with mutations in XRCC1 exhibited not only reduced rates of single-strand break repair but also elevated levels of protein ADP-ribosylation. This latter phenotype is recapitulated in a related syndrome caused by mutations in the XRCC1 partner protein PNKP and implicates hyperactivation of poly(ADP-ribose) polymerase/s as a cause of cerebellar ataxia. Indeed, remarkably, genetic deletion of Parp1 rescued normal cerebellar ADP-ribose levels and reduced the loss of cerebellar neurons and ataxia in Xrcc1-defective mice, identifying a molecular mechanism by which endogenous single-strand breaks trigger neuropathology. Collectively, these data establish the importance of XRCC1 protein complexes for normal neurological function and identify PARP1 as a therapeutic target in DNA strand break repair-defective disease.

FXR1-related congenital myopathy: expansion of the clinical and genetic spectrum.

BACKGROUND: Biallelic pathogenic variants in FXR1 have recently been associated with two congenital myopathy phenotypes: a severe form associated with hypotonia, long bone fractures, respiratory insufficiency and infantile death, and a milder form characterised by proximal muscle weakness with survival into adulthood. OBJECTIVE: We report eight patients from four unrelated families with biallelic pathogenic variants in exon 15 of FXR1. METHODS: Whole exome sequencing was used to detect variants in FXR1. RESULTS: Common clinical features were noted for all patients, which included proximal myopathy, normal serum creatine kinase levels and diffuse muscle atrophy with relative preservation of the quadriceps femoris muscle on muscle imaging. Additionally, some patients with FXR1-related myopathy had respiratory involvement and required bilevel positive airway pressure support. Muscle biopsy showed multi-minicores and type I fibre predominance with internalised nuclei. CONCLUSION: FXR1-related congenital myopathy is an emerging entity that is clinically recognisable. Phenotypic variability associated with variants in FXR1 can result from differences in variant location and type and is also observed between patients homozygous for the same variant, rendering specific genotype-phenotype correlations difficult. Our work broadens the phenotypic spectrum of FXR1-related congenital myopathy.

The Clinician-reported Genetic testing Utility InDEx (C-GUIDE): Preliminary evidence of validity and reliability.

PURPOSE: Demonstrating the clinical utility of genetic testing is fundamental to clinical adoption and reimbursement, but standardized definitions and measurement strategies for this construct do not exist. The Clinician-reported Genetic testing Utility InDEx (C-GUIDE) offers a novel measure to fill this gap. This study assessed its validity and inter-rater reliability. METHODS: Genetics professionals completed C-GUIDE after disclosure of test results to patients. Construct validity was assessed using regression analysis to measure associations between C-GUIDE and global item scores as well as potentially explanatory variables. Inter-rater reliability was assessed by administering a vignette-based survey to genetics professionals and calculating Krippendorff's α. RESULTS: On average, a 1-point increase in the global item score was associated with an increase of 3.0 in the C-GUIDE score (P < .001). Compared with diagnostic results, partially/potentially diagnostic and nondiagnostic results were associated with a reduction in C-GUIDE score of 9.5 (P < .001) and 10.2 (P < .001), respectively. Across 19 vignettes, Krippendorff's α was 0.68 (95% CI: 0.63-0.72). CONCLUSION: C-GUIDE showed acceptable validity and inter-rater reliability. Although further evaluation is required, C-GUIDE version 1.2 can be useful as a standardized approach to assess the clinical utility of genetic testing.

Heterozygous variants in MYH10 associated with neurodevelopmental disorders and congenital anomalies with evidence for primary cilia-dependent defects in Hedgehog signaling.

PURPOSE: Nonmuscle myosin II complexes are master regulators of actin dynamics that play essential roles during embryogenesis with vertebrates possessing 3 nonmuscle myosin II heavy chain genes, MYH9, MYH10, and MYH14. As opposed to MYH9 and MYH14, no recognizable disorder has been associated with MYH10. We sought to define the clinical characteristics and molecular mechanism of a novel autosomal dominant disorder related to MYH10. METHODS: An international collaboration identified the patient cohort. CAS9-mediated knockout cell models were used to explore the mechanism of disease pathogenesis. RESULTS: We identified a cohort of 16 individuals with heterozygous MYH10 variants presenting with a broad spectrum of neurodevelopmental disorders and variable congenital anomalies that affect most organ systems and were recapitulated in animal models of altered MYH10 activity. Variants were typically de novo missense changes with clustering observed in the motor domain. MYH10 knockout cells showed defects in primary ciliogenesis and reduced ciliary length with impaired Hedgehog signaling. MYH10 variant overexpression produced a dominant-negative effect on ciliary length. CONCLUSION: These data presented a novel genetic cause of isolated and syndromic neurodevelopmental disorders related to heterozygous variants in the MYH10 gene with implications for disrupted primary cilia length control and altered Hedgehog signaling in disease pathogenesis.

An integrated modelling methodology for estimating global incidence and prevalence of hereditary spastic paraplegia subtypes SPG4, SPG7, SPG11, and SPG15.

BACKGROUND: Hereditary spastic paraplegias (HSPs) are progressively debilitating neurodegenerative disorders that follow heterogenous patterns of Mendelian inheritance. Available epidemiological evidence provides limited incidence and prevalence data, especially at the genetic subtype level, preventing a realistic estimation of the true social burden of the disease. The objectives of this study were to (1) review the literature on epidemiology of HSPs; and (2) develop an epidemiological model of the prevalence of HSP, focusing on four common HSP genetic subtypes at the country and region-level. METHODS: A model was constructed estimating the incidence at birth, survival, and prevalence of four genetic subtypes of HSP based on the most appropriate published literature. The key model parameters were assessed by HSP clinical experts, who provided feedback on the validity of assumptions. A model was then finalized and validated through comparison of outputs against available evidence. The global, regional, and national prevalence and patient pool were calculated per geographic region and per genetic subtype. RESULTS: The HSP global prevalence was estimated to be 3.6 per 100,000 for all HSP forms, whilst the estimated global prevalence per genetic subtype was 0.90 (SPG4), 0.22 (SPG7), 0.34 (SPG11), and 0.13 (SPG15), respectively. This equates to an estimated 3365 (SPG4) and 872 (SPG11) symptomatic patients, respectively, in the USA. CONCLUSIONS: This is the first epidemiological model of HSP prevalence at the genetic subtype-level reported at multiple geographic levels. This study offers additional data to better capture the burden of illness due to mutations in common genes causing HSP, that can inform public health policy and healthcare service planning, especially in regions with higher estimated prevalence of HSP.

A rare case of diffuse crescents in fibrillary glomerulonephritis
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Diffuse crescentic involvement in fibrillary glomerulonephritis (FGN) is very rare. We describe a case of FGN with diffuse crescents in a patient who presented with clinical findings concerning for rapidly progressive kidney failure and pathologic findings suggestive of anti-glomerular basement membrane (GBM) disease. Serologies for anti-neutrophil cytoplasmic antibody (ANCA) and anti-GBM were negative. IgG subtyping showed IgG1 dominance, which has not been described in FGN. We present this unique case to emphasize the importance of considering FGN in biopsies showing diffuse crescentic glomerulonephritis with linear IgG staining of glomerular capillary walls, especially in the absence of other significant proliferative changes.

Myeloid bodies is not an uncommon ultrastructural finding.

The presence of myeloid bodies (MBs) is classically associated with Fabry disease (FD). However, MBs are also identified in patients without clinical evidence of FD. We attempt to further understand the clinicopathologic significance of incidental MBs in those without FD. Among the 4400 renal biopsies accessioned at the University of Rochester Medical Center from 2010 to 2021, we identified 32 cases showing MBs, 6 of which had FD. Medications were compared between a non-FG and a control-group of randomly selected cases without MBs (non-MBs). Both Fabry-group (FG) and non-Fabry-group (non-FG) were predominantly middle-aged (mean 48 years vs 56, respectively). Non-FG had slight female predominance (1:4), while all in FG were female. The majority of both non-FG and non-MBs cohort were on the same medications reported to cause phospholipidosis except sertraline and hydralazine (p = .04), which were more frequent in non-FG. Ultrastructurally, non-FG tended to show focal MBs in predominantly podocytes, while FG showed more extensive MBs in not only podocytes but also parietal, tubular, endothelial, and myocyte cells (p = .03). In addition, half of FG had another superimposed renal disease including kappa-light chain deposition disease, thin-basement membrane nephropathy, and lithium-related changes. MBs are encountered not only in FD but in other settings including CADs, toxins, and other inheritable diseases. Although secondary causes of MBs typically show less extensive involvement compared to FD, these features overlap. Given the challenges in diagnosing female carriers, the finding of MBs, though not specific to FD, may be the only clue that leads to further work-up and timely diagnosis, underscoring the importance of considering FD among other etiologies in differential diagnosis.