A Dynamic Protein Interaction Landscape of the Human Centrosome-Cilium Interface.

The centrosome is the primary microtubule organizing center of the cells and templates the formation of cilia, thereby operating at a nexus of critical cellular functions. Here, we use proximity-dependent biotinylation (BioID) to map the centrosome-cilium interface; with 58 bait proteins we generate a protein topology network comprising >7,000 interactions. Analysis of interaction profiles coupled with high resolution phenotypic profiling implicates a number of protein modules in centriole duplication, ciliogenesis, and centriolar satellite biogenesis and highlights extensive interplay between these processes. By monitoring dynamic changes in the centrosome-cilium protein interaction landscape during ciliogenesis, we also identify satellite proteins that support cilia formation. Systematic profiling of proximity interactions combined with functional analysis thus provides a rich resource for better understanding human centrosome and cilia biology. Similar strategies may be applied to other complex biological structures or pathways.

A proximity-dependent biotinylation map of a human cell.

Compartmentalization is a defining characteristic of eukaryotic cells, and partitions distinct biochemical processes into discrete subcellular locations. Microscopy1 and biochemical fractionation coupled with mass spectrometry2-4 have defined the proteomes of a variety of different organelles, but many intracellular compartments have remained refractory to such approaches. Proximity-dependent biotinylation techniques such as BioID provide an alternative approach to define the composition of cellular compartments in living cells5-7. Here we present a BioID-based map of a human cell on the basis of 192 subcellular markers, and define the intracellular locations of 4,145 unique proteins in HEK293 cells. Our localization predictions exceed the specificity of previous approaches, and enabled the discovery of proteins at the interface between the mitochondrial outer membrane and the endoplasmic reticulum that are crucial for mitochondrial homeostasis. On the basis of this dataset, we created humancellmap.org as a community resource that provides online tools for localization analysis of user BioID data, and demonstrate how this resource can be used to understand BioID results better.

Properties of Stress Granule and P-Body Proteomes.

Stress granules and P-bodies are cytosolic biomolecular condensates that dynamically form by the phase separation of RNAs and proteins. They participate in translational control and buffer the proteome. Upon stress, global translation halts and mRNAs bound to the translational machinery and other proteins coalesce to form stress granules (SGs). Similarly, translationally stalled mRNAs devoid of translation initiation factors shuttle to P-bodies (PBs). Here, we review the cumulative progress made in defining the protein components that associate with mammalian SGs and PBs. We discuss the composition of SG and PB proteomes, supported by a new user-friendly database (http://rnagranuledb.lunenfeld.ca/) that curates current literature evidence for genes or proteins associated with SGs or PBs. As previously observed, the SG and PB proteomes are biased toward intrinsically disordered regions and have a high propensity to contain primary sequence features favoring phase separation. We also provide an outlook on how the various components of SGs and PBs may cooperate to organize and form membraneless organelles.

Interactome Rewiring Following Pharmacological Targeting of BET Bromodomains.

Targeting bromodomains (BRDs) of the bromo-and-extra-terminal (BET) family offers opportunities for therapeutic intervention in cancer and other diseases. Here, we profile the interactomes of BRD2, BRD3, BRD4, and BRDT following treatment with the pan-BET BRD inhibitor JQ1, revealing broad rewiring of the interaction landscape, with three distinct classes of behavior for the 603 unique interactors identified. A group of proteins associate in a JQ1-sensitive manner with BET BRDs through canonical and new binding modes, while two classes of extra-terminal (ET)-domain binding motifs mediate acetylation-independent interactions. Last, we identify an unexpected increase in several interactions following JQ1 treatment that define negative functions for BRD3 in the regulation of rRNA synthesis and potentially RNAPII-dependent gene expression that result in decreased cell proliferation. Together, our data highlight the contributions of BET protein modules to their interactomes allowing for a better understanding of pharmacological rewiring in response to JQ1.

Low-frequency inherited complement receptor variants are associated with purpura fulminans.

ABSTRACT: Extreme disease phenotypes can provide key insights into the pathophysiology of common conditions, but studying such cases is challenging due to their rarity and the limited statistical power of existing methods. Herein, we used a novel approach to pathway-based mutational burden testing, the rare variant trend test (RVTT), to investigate genetic risk factors for an extreme form of sepsis-induced coagulopathy, infectious purpura fulminans (PF). In addition to prospective patient sample collection, we electronically screened over 10.4 million medical records from 4 large hospital systems and identified historical cases of PF for which archived specimens were available to perform germline whole-exome sequencing. We found a significantly increased burden of low-frequency, putatively function-altering variants in the complement system in patients with PF compared with unselected patients with sepsis (P = .01). A multivariable logistic regression analysis found that the number of complement system variants per patient was independently associated with PF after controlling for age, sex, and disease acuity (P = .01). Functional characterization of PF-associated variants in the immunomodulatory complement receptors CR3 and CR4 revealed that they result in partial or complete loss of anti-inflammatory CR3 function and/or gain of proinflammatory CR4 function. Taken together, these findings suggest that inherited defects in CR3 and CR4 predispose to the maladaptive hyperinflammation that characterizes severe sepsis with coagulopathy.

High-Density Proximity Mapping Reveals the Subcellular Organization of mRNA-Associated Granules and Bodies.

mRNA processing, transport, translation, and ultimately degradation involve a series of dedicated protein complexes that often assemble into large membraneless structures such as stress granules (SGs) and processing bodies (PBs). Here, systematic in vivo proximity-dependent biotinylation (BioID) analysis of 119 human proteins associated with different aspects of mRNA biology uncovers 7424 unique proximity interactions with 1,792 proteins. Classical bait-prey analysis reveals connections of hundreds of proteins to distinct mRNA-associated processes or complexes, including the splicing and transcriptional elongation machineries (protein phosphatase 4) and the CCR4-NOT deadenylase complex (CEP85, RNF219, and KIAA0355). Analysis of correlated patterns between endogenous preys uncovers the spatial organization of RNA regulatory structures and enables the definition of 144 core components of SGs and PBs. We report preexisting contacts between most core SG proteins under normal growth conditions and demonstrate that several core SG proteins (UBAP2L, CSDE1, and PRRC2C) are critical for the formation of microscopically visible SGs.

HERV1-env Induces Unfolded Protein Response Activation in Autoimmune Liver Disease: A Potential Mechanism for Regulatory T Cell Dysfunction.

Regulatory T cells (Tregs) are not terminally differentiated but can acquire effector properties. Here we report an increased expression of human endogenous retrovirus 1 (HERV1-env) proteins in Tregs of patients with de novo autoimmune hepatitis and autoimmune hepatitis, which induces endoplasmic reticulum (ER) stress. HERV1-env-triggered ER stress activates all three branches (IRE1, ATF6, and PERK) of the unfolded protein response (UPR). Our coimmunoprecipitation studies show an interaction between HERV1-env proteins and the ATF6 branch of the UPR. The activated form of ATF6α activates the expression of RORC and STAT3 by binding to promoter sequences and induces IL-17A production. Silencing of HERV1-env results in recovery of Treg suppressive function. These findings identify ER stress and UPR activation as key factors driving Treg plasticity (species: human).

Activated sputum eosinophils associated with exacerbations in children on mepolizumab.

BACKGROUND: MUPPITS-2 was a randomized, placebo-controlled clinical trial that demonstrated mepolizumab (anti-IL-5) reduced exacerbations and blood and airway eosinophils in urban children with severe eosinophilic asthma. Despite this reduction in eosinophilia, exacerbation risk persisted in certain patients treated with mepolizumab. This raises the possibility that subpopulations of airway eosinophils exist that contribute to breakthrough exacerbations. OBJECTIVE: We aimed to determine the effect of mepolizumab on airway eosinophils in childhood asthma. METHODS: Sputum samples were obtained from 53 MUPPITS-2 participants. Airway eosinophils were characterized using mass cytometry and grouped into subpopulations using unsupervised clustering analyses of 38 surface and intracellular markers. Differences in frequency and immunophenotype of sputum eosinophil subpopulations were assessed based on treatment arm and frequency of exacerbations. RESULTS: Median sputum eosinophils were significantly lower among participants treated with mepolizumab compared with placebo (58% lower, 0.35% difference [95% CI 0.01, 0.74], P = .04). Clustering analysis identified 3 subpopulations of sputum eosinophils with varied expression of CD62L. CD62Lint and CD62Lhi eosinophils exhibited significantly elevated activation marker and eosinophil peroxidase expression, respectively. In mepolizumab-treated participants, CD62Lint and CD62Lhi eosinophils were more abundant in participants who experienced exacerbations than in those who did not (100% higher for CD62Lint, 0.04% difference [95% CI 0.0, 0.13], P = .04; 93% higher for CD62Lhi, 0.21% difference [95% CI 0.0, 0.77], P = .04). CONCLUSIONS: Children with eosinophilic asthma treated with mepolizumab had significantly lower sputum eosinophils. However, CD62Lint and CD62Lhi eosinophils were significantly elevated in children on mepolizumab who had exacerbations, suggesting that eosinophil subpopulations exist that contribute to exacerbations despite anti-IL-5 treatment.

Click Chemistry: Reaction Rates and Their Suitability for Biomedical Applications.

Click chemistry has become a commonly used synthetic method due to the simplicity, efficiency, and high selectivity of this class of chemical reactions. Since their initial discovery, further click chemistry methods have been identified and added to the toolbox of click chemistry reactions for biomedical applications. However, selecting the most suitable reaction for a specific application is often challenging, as multiple factors must be considered, including selectivity, reactivity, biocompatibility, and stability. Thus, this review provides an overview of the benefits and limitations of well-established click chemistry reactions with a particular focus on the importance of considering reaction rates, an often overlooked criterion with little available guidance. The importance of understanding each click chemistry reaction beyond simply the reaction speed is discussed comprehensively with reference to recent biomedical research which utilized click chemistry. This review aims to provide a practical resource for researchers to guide the selection of click chemistry classes for different biomedical applications.

In Vivo PET Detection of Lung Micrometastasis in Mice by Targeting Endothelial VCAM-1 Using a Dual-Contrast PET/MRI Probe.

Current clinical diagnostic imaging methods for lung metastases are sensitive only to large tumours (1-2 mm cross-sectional diameter), and early detection can dramatically improve treatment. We have previously demonstrated that an antibody-targeted MRI contrast agent based on microparticles of iron oxide (MPIO; 1 µm diameter) enables the imaging of endothelial vascular cell adhesion molecule-1 (VCAM-1). Using a mouse model of lung metastasis, upregulation of endothelial VCAM-1 expression was demonstrated in micrometastasis-associated vessels but not in normal lung tissue, and binding of VCAM-MPIO to these vessels was evident histologically. Owing to the lack of proton MRI signals in the lungs, we modified the VCAM-MPIO to include zirconium-89 (89Zr, t1/2 = 78.4 h) in order to allow the in vivo detection of lung metastases by positron emission tomography (PET). Using this new agent (89Zr-DFO-VCAM-MPIO), it was possible to detect the presence of micrometastases within the lung in vivo from ca. 140 µm in diameter. Histological analysis combined with autoradiography confirmed the specific binding of the agent to the VCAM-1 expressing vasculature at the sites of pulmonary micrometastases. By retaining the original VCAM-MPIO as the basis for this new molecular contrast agent, we have created a dual-modality (PET/MRI) agent for the concurrent detection of lung and brain micrometastases.

Synthesis and In Vivo Evaluation of a Site-specifically Labeled Radioimmunoconjugate for Dual-Modal (PET/NIRF) Imaging of MT1-MMP in Sarcomas.

Bone sarcomas are devastating primary bone cancers that mostly affect children, young adults, and the elderly. These aggressive tumors are associated with poor survival, and surgery remains the mainstay of treatment. Surgical planning is increasingly informed by positron emission tomography (PET), and tumor margin identification during surgery is aided by near-infrared fluorescence (NIRF) imaging, yet these investigations are confounded by probes that lack specificity for sarcoma biomarkers. We report the development of a dual-modal (PET/NIRF) immunoconjugate ([89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW) that targets MT1-MMP, a matrix metalloproteinase overexpressed in high-grade sarcomas. [89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW was synthesized via site-specific chemoenzymatic glycan modification, characterized, and isolated in high specific activity and radiochemical purity. Saturation binding and immunoreactivity assays indicated only minor perturbation of binding properties. A novel mouse model of dedifferentiated chondrosarcoma based on intrafemoral inoculation of HT1080 WT or KO cells (high and low MT1-MMP expression, respectively) was used to evaluate target binding and biodistribution. Fluorescence and Cerenkov luminescence images of [89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW showed preferential uptake in HT1080 WT tumors. Ex vivo gamma counting revealed that uptake in MT1-MMP-positive tumors was significantly higher than that in control groups. Taken together, [89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW is a promising dual-modal sarcoma imaging agent for pre-operative surgical planning and intraoperative surgical guidance.

D-bifunctional protein deficiency caused by splicing variants in a neonate with severe peroxisomal dysfunction and persistent hypoglycemia.

D-bifunctional protein (DBP) deficiency is a rare, autosomal recessive peroxisomal enzyme deficiency resulting in a high burden of morbidity and early mortality. Patients with DBP deficiency resemble those with a severe Zellweger phenotype, with neonatal hypotonia, seizures, craniofacial dysmorphisms, psychomotor delay, deafness, blindness, and death typically within the first 2 years of life, although patients with residual enzyme function can survive longer. The clinical severity of the disease depends on the degree of enzyme deficiency. Loss-of-function variants typically result in no residual enzyme activity; however, splice variants may result in protein with residual function. We describe a full-term newborn presenting with hypotonia, seizures, and unexplained hypoglycemia, who was later found to have rickets at follow up. Rapid whole genome sequencing identified two HSD17B4 variants in trans; one likely pathogenic variant and one variant of uncertain significance (VUS) located in the polypyrimidine tract of intron 13. To determine the functional consequence of the VUS, we analyzed RNA from the patient's father with RNA-seq which showed skipping of Exon 14, resulting in a frameshift mutation three amino acids from the new reading frame. This RNA-seq analysis was correlated with virtually absent enzyme activity, elevated very-long-chain fatty acids in fibroblasts, and a clinically severe phenotype. Both variants are reclassified as pathogenic. Due to the clinical spectrum of DBP deficiency, this provides important prognostic information, including early mortality. Furthermore, we add persistent hypoglycemia to the clinical spectrum of the disease, and advocate for the early management of fat-soluble vitamin deficiencies to reduce complications.

The public economic burden of suboptimal type 2 diabetes control upon taxpayers in Sweden: Looking beyond health costs.

AIM: To estimate the fiscal burden for taxpayers in Sweden associated with type 2 diabetes (T2D) attributed to diabetes-related complications in patients failing to meet HbA1c targets. MATERIAL AND METHODS: We developed a public economic framework to assess how changes in diabetes-related complications influenced projected tax contributions and government disability payments for people with T2D. The analysis applied accepted disease-modelling practices to estimate different rates of diabetes-related complications based on an HbA1c of 6.9% (52 mmol/mol) and of 6.0% (42 mmol/mol). We adjusted the employment activity rates for those experiencing T2D-related events, applying age-specific earnings to estimate lifetime tax losses. Furthermore, the likelihood of receiving payments for health-related employment inactivity was estimated. Direct healthcare costs are excluded from this analysis. RESULTS: The estimated per person earnings loss for immediate and delayed HbA1c control was Swedish krona (SEK) 42 299 and SEK 44 157, respectively, over 10 years. The lost employment activity of people with T2D translates to lost tax revenues of SEK 23 265 and SEK 24 287 for immediate and delayed control, respectively. The estimated difference in disability payments was SEK 538. Combining the tax revenue loss and excess disability payments defines the broader fiscal costs, where we observe combined fiscal losses that favour immediate and sustained control by SEK 1560 over 10 years. CONCLUSIONS: We show that conducting fiscal analysis of diabetes interventions offers an enriched perspective capturing a range of costs that fall on government in relation to lost tax revenue and disability payments. Tax-financed health systems may benefit from broadening the consideration of costs and benefits when evaluating new interventions and treatment practices.

Genomic sites hypersensitive to ultraviolet radiation.

If the genome contains outlier sequences extraordinarily sensitive to environmental agents, these would be sentinels for monitoring personal carcinogen exposure and might drive direct changes in cell physiology rather than acting through rare mutations. New methods, adductSeq and freqSeq, provided statistical resolution to quantify rare lesions at single-base resolution across the genome. Primary human melanocytes, but not fibroblasts, carried spontaneous apurinic sites and TG sequence lesions more frequent than ultraviolet (UV)-induced cyclobutane pyrimidine dimers (CPDs). UV exposure revealed hyperhotspots acquiring CPDs up to 170-fold more frequently than the genomic average; these sites were more prevalent in melanocytes. Hyperhotspots were disproportionately located near genes, particularly for RNA-binding proteins, with the most-recurrent hyperhotspots at a fixed position within 2 motifs. One motif occurs at ETS family transcription factor binding sites, known to be UV targets and now shown to be among the most sensitive in the genome, and at sites of mTOR/5' terminal oligopyrimidine-tract translation regulation. The second occurs at A2-15TTCTY, which developed "dark CPDs" long after UV exposure, repaired CPDs slowly, and had accumulated CPDs prior to the experiment. Motif locations active as hyperhotspots differed between cell types. Melanocyte CPD hyperhotspots aligned precisely with recurrent UV signature mutations in individual gene promoters of melanomas and with known cancer drivers. At sunburn levels of UV exposure, every cell would have a hyperhotspot CPD in each of the ∼20 targeted cell pathways, letting hyperhotspots act as epigenetic marks that create phenome instability; high prevalence favors cooccurring mutations, which would allow tumor evolution to use weak drivers.

Spinal cord stimulation for treatment of chronic neuropathic pain in adolescent patients: a single-institution series, systematic review, and individual participant data meta-analysis.

OBJECTIVE: Neuropathic pain is undertreated in children. Neurosurgical treatments of pediatric chronic pain are limited by the absence of both US Food and Drug Administration approval and pediatric-specific hardware, as well as weak referral patterns due to a lack of physician education. This study presents a single-institution retrospective case series of spinal cord stimulation (SCS) in children ≤ 19 years of age and a systematic review of SCS in children. The authors' findings may further validate the role of SCS as an effective treatment modality for varied neuropathic pain syndromes found in pediatric patients. METHODS: The study was a single-center, single-surgeon, retrospective case series of individuals treated between July 2017 and May 2022. The outcomes for pediatric patients with chronic neuropathic pain syndromes indicated by the multidisciplinary pain clinic for evaluation for SCS were cataloged. A systematic review and individual participant data (IPD) meta-analysis was performed for cases treated until May 2022, using PubMed, EMBASE, and Scopus to characterize outcomes of children with neuropathic pain treated with SCS. RESULTS: Twelve patients were evaluated and 9 were indicated for percutaneous or buried lead trials. Seven female and 2 male patients between the ages of 13 and 19 years were implanted with trial leads. Eight of 9 (89%) patients went on to receive permanent systems. The average trial length was 6 days, and the length of stay for both trial and implant was less than 1 day. Complication rates due to CSF leaks were 22% and 0% for trial and implant, respectively. Visual analog scale pain scores decreased from 9.2 to 2.9 (p = 0.0002) and the number of medications decreased from 4.9 to 2.1 (p = 0.0005). Functional status also improved for each patient. A systematic review identified 13 studies describing pediatric patients with SCS, including 12 providing IPD on 30 patients. In the IPD meta-analysis, pain was reduced in 16/16 (100%) of patients following surgery and in 25/26 (96.2%) at last follow-up. Medication use was decreased in 16/21 (76.2%), and functional outcomes were improved in 29/29 (100%). The complication rate was 5/30 (16.7%). CONCLUSIONS: SCS effectively decreases pain and medication use for pediatric neuropathic pain syndromes. Patients also report improved functional status, including improved matriculation, gainful employment, and physical activity. There is minimal high-quality literature describing neuromodulation for pain in children. Neuromodulation should be considered earlier as a viable alternative to escalating use of multiple drugs and as a potential mechanism to address tolerance, dependence, and addiction in pediatric patients.

Prognostic factors for surgically managed intramedullary spinal cord tumours: a single-centre case series.

PURPOSE: Intramedullary spinal cord tumours (IMSCTs) are comparatively rare neoplasms. We present a single-centre clinical case series of adult patients with surgically managed IMSCTs. METHODS: We performed a retrospective analysis of electronic patient records in the time period spanning July 2010 to July 2021. All adult patients that had undergone surgical management for IMSCTs were eligible for inclusion. Baseline and post-operative clinical and radiological characteristics, along with follow-up data, were assessed. We also performed a literature review with a focus on surgical outcomes for IMSCTs. RESULTS: Sixty-six patients matched our selection criteria, with a median age of 42 years (range 23-85). Thirty-four ependymomas, 17 haemangioblastomas, 12 astrocytomas, 2 lymphomas and 1 teratoma were included. Statistical analysis yielded several significant findings: IMSCTs spanning a greater number of vertebral levels are significantly associated with poor McCormick outcomes (p = 0.03), presence of gait disturbance before surgery is significantly associated with poor outcome for both post-operative McCormick and Nurick scores (p = 0.007), and radicular pain present pre-operatively is significantly associated with a good post-operative McCormick score (p = 0.045). Haemangioblastomas are significantly more likely to have a clear intra-operative dissection plane compared to ependymomas and astrocytomas (p = 0.009). However, astrocytomas have a significantly higher prevalence of good McCormick outcomes compared to ependymomas and haemangioblastomas (p = 0.03). CONCLUSION: Histological diagnosis, cranio-caudal extent of the tumour and the presence or absence of baseline deficits-such as gait impairment and radicular pain-are significant in determining neurological outcomes after surgery for IMSCTs.

Clinical validation of novel lightning dose optimizer for gamma knife radiosurgery of irregular-shaped arteriovenous malformations and pituitary adenomas.

PURPOSE: To demonstrate the clinical feasibility of a novel treatment planning algorithm via lightning dose optimizer (LDO) on Leksell Gamma Knife (LGK) GammaPlan with significantly faster planning times for stereotactic radiosurgery (SRS) of the complex and difficult arteriovenous malformations (AVMs) and pituitary adenomas. METHODS AND MATERIALS: After completing the in-house end-to-end phantom testing and independent dose verification of the recently upgraded LDO algorithm on GammaPlan using the MD Anderson's IROC anthropomorphic SRS head phantom irradiation credentialing, 20 previously treated GK-SRS patients (10 AVM, average volume 3.61 cm3 and 10 pituitary adenomas, average volume 0.86 cm3 ) who underwent manual forward planning on GammaPlan were retrospectively replanned via LDO. These pathologies were included because of the need for adequate dose delivery with organs at risk in very close proximity. LDO finds the target curvature boundary by well-formulated linear programing objectives and inversely optimizes the GK-SRS plan by isocenter placement, optimization, and sequencing. For identical target coverage, the LDO and original manual plans were compared for target conformity, gradient index, dose to critical organs, and surrounding normal brain. Additionally, various treatment delivery parameters, including beam-on time were recorded. RESULTS: For both patient cohorts, LDO provided similar target coverage with better dose conformity, tighter radiosurgical dose distribution with a lower value of gradient indices (all p < 0.001), and lower dose to critical organs. For AVMs, there was a significant reduction of normal brain V10Gy , V12Gy , and V14Gy by 4.74, 3.67, and 2.67 cm3 (all p < 0.001). LDO had twice the number of shots (p < 0.001), and longer beam-on time (p = 0.012) by a factor of 1.44. For pituitary adenomas, LDO provided systematically lower values of V10Gy , V12Gy , and V14Gy by 1.08, 0.86, and 0.68 cm3 (all p < 0.001), and lower maximum dose to optic pathway by 0.7 Gy (p = 0.005), but had almost twice the numbers of shots (p < 0.001) and increased beam-on time (p = 0.005) by a factor of 1.2. However, for both patient groups, the average planning time for the LDO was <5 min, compared to the estimated 30-90 min of manual planning times. CONCLUSION: GK-SRS treatment on Leksell Perfexion GammaPlan using the LDO provided highly conformal target coverage with a steep dose gradient, spared critical organs, and significantly reduced normal brain dose for complex targets at the cost of slightly higher treatment times. LDO generated high-quality treatment plans and could significantly reduce planning time. If available, the LDO algorithm is suggested for validation and clinical use for complex and difficult GK cases.

Genetics of agenesis/hypoplasia of the uterus and vagina: narrowing down the number of candidate genes for Mayer-Rokitansky-Küster-Hauser Syndrome.

PURPOSE: Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome consists of congenital absence of the uterus and vagina and is often associated with renal, skeletal, cardiac, and auditory defects. The genetic basis is largely unknown except for rare variants in several genes. Many candidate genes have been suggested by mouse models and human studies. The purpose of this study was to narrow down the number of candidate genes. METHODS: Whole exome sequencing was performed on 111 unrelated individuals with MRKH; variant analysis focused on 72 genes suggested by mouse models, human studies of physiological candidates, or located near translocation breakpoints in t(3;16). Candidate variants (CV) predicted to be deleterious were confirmed by Sanger sequencing. RESULTS: Sanger sequencing verified 54 heterozygous CV from genes identified through mouse (13 CV in 6 genes), human (22 CV in seven genes), and translocation breakpoint (19 CV in 11 genes) studies. Twelve patients had ≥ 2 CVs, including four patients with two variants in the same gene. One likely digenic combination of LAMC1 and MMP14 was identified. CONCLUSION: We narrowed 72 candidate genes to 10 genes that appear more likely implicated. These candidate genes will require further investigation to elucidate their role in the development of MRKH.