The Fbn1 gene variant governs passive ascending aortic mechanics in the mgΔlpn mouse model of Marfan syndrome when superimposed to perlecan haploinsufficiency.

Introduction: Ascending thoracic aortic aneurysms arise from pathological tissue remodeling that leads to abnormal wall dilation and increases the risk of fatal dissection/rupture. Large variability in disease manifestations across family members who carry a causative genetic variant for thoracic aortic aneurysms suggests that genetic modifiers may exacerbate clinical outcomes. Decreased perlecan expression in the aorta of mgΔlpn mice with severe Marfan syndrome phenotype advocates for exploring perlecan-encoding Hspg2 as a candidate modifier gene. Methods: To determine the effect of concurrent Hspg2 and Fbn1 mutations on the progression of thoracic aortopathy, we characterized the microstructure and passive mechanical response of the ascending thoracic aorta in female mice of four genetic backgrounds: wild-type, heterozygous with a mutation in the Fbn1 gene (mgΔlpn), heterozygous with a mutation in the Hspg2 gene (Hspg2+/-), and double mutants carrying both the Fbn1 and Hspg2 variants (dMut). Results: Elastic fiber fragmentation and medial disarray progress from the internal elastic lamina outward as the ascending thoracic aorta dilates in mgΔlpn and dMut mice. Concurrent increase in total collagen content relative to elastin reduces energy storage capacity and cyclic distensibility of aortic tissues from mice that carry the Fbn1 variant. Inherent circumferential tissue stiffening strongly correlates with the severity of aortic dilatation in mgΔlpn and dMut mice. Perlecan haploinsufficiency superimposed to the mgΔlpn mutation curbs the viability of dMut mice, increases the occurrence of aortic enlargement, and reduces the axial stretch in aortic tissues. Discussion: Overall, our findings show that dMut mice are more vulnerable than mgΔlpn mice without an Hspg2 mutation, yet later endpoints and additional structural and functional readouts are needed to identify causative mechanisms.

Creating an HLA-homozygous iPS cell bank for the Brazilian population: Challenges and opportunities.

Identifying human leukocyte antigen (HLA) haplotype-homozygous donors for the generation of induced pluripotent stem (iPS) cell lines permits the construction of biobanks immunologically compatible with significant numbers of individuals for use in therapy. However, two questions must be addressed to create such a bank: how many cell lines are necessary to match most of the recipient population and how many people should be tested to find these donors? In Japan and the UK, 50 and 100 distinct HLA-A, -B, and -DRB1 triple-homozygous haplotypes would cover 90% of those populations, respectively. Using data from the Brazilian National Registry of Bone Marrow Donors (REDOME), encompassing 4,017,239 individuals, we identified 1,906 distinct triple-homozygous HLA haplotypes. In Brazil, 559 triple-homozygous cell lines cover 95% of the population, and 3.8 million people would have to be screened. Finally, we show the contribution of the 30 most frequent triple-homozygous HLA haplotypes in Brazil to populations of different countries.

Extracellular matrix and vascular dynamics in the kidney of a murine model for Marfan syndrome.

Fibrillin-1 is a pivotal structural component of the kidney's glomerulus and peritubular tissue. Mutations in the fibrillin-1 gene result in Marfan syndrome (MFS), an autosomal dominant disease of the connective tissue. Although the kidney is not considered a classically affected organ in MFS, several case reports describe glomerular disease in patients. Therefore, this study aimed to characterize the kidney in the mgΔlpn-mouse model of MFS. Affected animals presented a significant reduction of glomerulus, glomerulus-capillary, and urinary space, and a significant reduction of fibrillin-1 and fibronectin in the glomerulus. Transmission electron microscopy and 3D-ultrastructure analysis revealed decreased amounts of microfibrils which also appeared fragmented in the MFS mice. Increased collagen fibers types I and III, MMP-9, and α-actin were also observed in affected animals, suggesting a tissue-remodeling process in the kidney. Video microscopy analysis showed an increase of microvessel distribution coupled with reduction of blood-flow velocity, while ultrasound flow analysis revealed significantly lower blood flow in the kidney artery and vein of the MFS mice. The structural and hemodynamic changes of the kidney indicate the presence of kidney remodeling and vascular resistance in this MFS model. Both processes are associated with hypertension which is expected to worsen the cardiovascular phenotype in MFS.

Establishment of iPSC lines and zebrafish with loss-of-function AHDC1 variants: Models for Xia-Gibbs syndrome.

Xia-Gibbs syndrome (XGS) is a syndromic form of intellectual disability caused by heterozygous AHDC1 variants, but the pathophysiological mechanisms underlying this syndrome are still unclear. In this manuscript, we describe the development of two different functional models: three induced pluripotent stem cell (iPSC) lines with different loss-of-function (LoF) AHDC1 variants, derived by reprogramming peripheral blood mononuclear cells from XGS patients, and a zebrafish strain with a LoF variant in the ortholog gene (ahdc1) obtained through CRISPR/Cas9-mediated editing. The three iPSC lines showed expression of pluripotency factors (SOX2, SSEA-4, OCT3/4, and NANOG). To verify the capacity of iPSC to differentiate into the three germ layers, we obtained embryoid bodies (EBs), induced their differentiation, and confirmed the mRNA expression of ectodermal, mesodermal, and endodermal markers using the TaqMan hPSC Scorecard. The iPSC lines were also approved for the following quality tests: chromosomal microarray analysis (CMA), mycoplasma testing, and short tandem repeat (STR) DNA profiling. The zebrafish model has an insertion of four base pairs in the ahdc1 gene, is fertile, and breeding between heterozygous and wild-type (WT) animals generated offspring in a genotypic proportion in agreement with Mendelian law. The established iPSC and zebrafish lines were deposited on the hpscreg.eu and zfin.org platforms, respectively. These biological models are the first for XGS and will be used in future studies that investigate the pathophysiology of this syndrome, unraveling its underlying molecular mechanisms.

Indigenous people from Amazon show genetic signatures of pathogen-driven selection.

Ecological conditions in the Amazon rainforests are historically favorable for the transmission of numerous tropical diseases, especially vector-borne diseases. The high diversity of pathogens likely contributes to the strong selective pressures for human survival and reproduction in this region. However, the genetic basis of human adaptation to this complex ecosystem remains unclear. This study investigates the possible footprints of genetic adaptation to the Amazon rainforest environment by analyzing the genomic data of 19 native populations. The results based on genomic and functional analysis showed an intense signal of natural selection in a set of genes related to Trypanosoma cruzi infection, which is the pathogen responsible for Chagas disease, a neglected tropical parasitic disease native to the Americas that is currently spreading worldwide.

The fibrotic niche impairs satellite cell function and muscle regeneration in mouse models of Marfan syndrome.

AIM: It has been suggested that the proliferation and early differentiation of myoblasts are impaired in Marfan syndrome (MFS) mice during muscle regeneration. However, the underlying cellular and molecular mechanisms remain poorly understood. Here, we investigated muscle regeneration in MFS mouse models by analyzing the influence of the fibrotic niche on satellite cell function. METHODS: In vivo, ex vivo, and in vitro experiments were performed. In addition, we evaluated the effect of the pharmacological inhibition of fibrosis using Ang-(1-7) on regenerating skeletal muscles of MFS mice. RESULTS: The skeletal muscle of MFS mice shows an increased accumulation of collagen fibers (81.2%), number of fibroblasts (157.1%), and Smad2/3 signaling (110.5%), as well as an aberrant number of fibro-adipogenic progenitor cells in response to injury compared with wild-type mice. There was an increased number of proinflammatory and anti-inflammatory macrophages (3.6- and 3.1-fold, respectively) in regenerating muscles of wild-type mice, but not in the regenerating muscles of MFS mice. Our data show that proliferation and differentiation of satellite cells are altered (p ≤ 0.05) in MFS mice. Myoblast transplantation assay revealed that the regenerating muscles from MFS mice have reduced satellite cell self-renewal capacity (74.7%). In addition, we found that treatment with Ang-(1-7) reduces fibrosis (71.6%) and ameliorates satellite cell dysfunction (p ≤ 0.05) and muscle contractile function (p ≤ 0.05) in MFS mice. CONCLUSION: The fibrotic niche, caused by Fbn1 mutations, reduces the myogenic potential of satellite cells, affecting structural and functional muscle regeneration. In addition, the fibrosis inhibitor Ang-(1-7) partially counteracts satellite cell abnormalities and restores myofiber size and contractile force in regenerating muscles.

Treatment With Methotrexate Associated With Lipid Core Nanoparticles Prevents Aortic Dilation in a Murine Model of Marfan Syndrome.

In Marfan syndrome (MFS), dilation, dissection, and rupture of the aorta occur. Inflammation can be involved in the pathogenicity of aortic defects and can thus be a therapeutic target for MFS. Previously, we showed that the formulation of methotrexate (MTX) associated with lipid nanoparticles (LDE) has potent anti-inflammatory effects without toxicity. To investigate whether LDEMTX treatment can prevent the development of aortic lesions in the MFS murine model. MgΔloxPneo MFS (n = 40) and wild-type (WT, n = 60) mice were allocated to 6 groups weekly injected with IP solutions of: (1) only LDE; (2) commercial MTX; (3) LDEMTX (dose = 1mg/kg) between 3rd and 6th months of life. After 12 weeks of treatments, animals were examined by echocardiography and euthanatized for morphometric and molecular studies. MFS mice treated with LDEMTX showed narrower lumens in the aortic arch, as well as in the ascending and descending aorta. LDEMTX reduced fibrosis and the number of dissections in MFS but not the number of elastic fiber disruptions. In MFS mice, LDEMTX treatment lowered protein expression of pro-inflammatory factors macrophages (CD68), T-lymphocytes (CD3), tumor necrosis factor-α (TNF-α), apoptotic factor cleaved-caspase 3, and type 1 collagen and lowered the protein expression of the transforming growth factor-ß (TGF-ß), extracellular signal-regulated kinases ½ (ERK1/2), and SMAD3. Protein expression of CD68 and CD3 had a positive correlation with an area of aortic lumen (r 2 = 0.36; p < 0.001), suggesting the importance of inflammation in the causative mechanisms of aortic dilation. Enhanced adenosine availability by LDEMTX was suggested by higher aortic expression of an anti-adenosine A2a receptor (A2a) and lower adenosine deaminase expression. Commercial MTX had negligible effects. LDEMTX prevented the development of MFS-associated aortic defects and can thus be a candidate for testing in clinical studies.

Defective hematopoietic differentiation of immune aplastic anemia patient-derived iPSCs.

In acquired immune aplastic anemia (AA), pathogenic cytotoxic Th1 cells are activated and expanded, driving an immune response against the hematopoietic stem and progenitor cells (HSPCs) that provokes cell depletion and causes bone marrow failure. However, additional HSPC defects may contribute to hematopoietic failure, reflecting on disease outcomes and response to immunosuppression. Here we derived induced pluripotent stem cells (iPSCs) from peripheral blood (PB) erythroblasts obtained from patients diagnosed with immune AA using non-integrating plasmids to model the disease. Erythroblasts were harvested after hematologic response to immunosuppression was achieved. Patients were screened for germline pathogenic variants in bone marrow failure-related genes and no variant was identified. Reprogramming was equally successful for erythroblasts collected from the three immune AA patients and the three healthy subjects. However, the hematopoietic differentiation potential of AA-iPSCs was significantly reduced both quantitatively and qualitatively as compared to healthy-iPSCs, reliably recapitulating disease: differentiation appeared to be more severely affected in cells from the two patients with partial response as compared to the one patient with complete response. Telomere elongation and the telomerase machinery were preserved during reprogramming and differentiation in all AA-iPSCs. Our results indicate that iPSCs are a reliable platform to model immune AA and recapitulate clinical phenotypes. We propose that the immune attack may cause specific epigenetic changes in the HSPCs that limit adequate proliferation and differentiation.

Effects of Magnetite Nanoparticles and Static Magnetic Field on Neural Differentiation of Pluripotent Stem Cells.

Neurodevelopmental processes of pluripotent cells, such as proliferation and differentiation, are influenced by external natural forces. Despite the presence of biogenic magnetite nanoparticles in the central nervous system and constant exposure to the Earth's magnetic fields and other sources, there is scant knowledge regarding the role of electromagnetic stimuli in neurogenesis. Moreover, emerging applications of electrical and magnetic stimulation to treat neurological disorders emphasize the relevance of understanding the impact and mechanisms behind these stimuli. Here, the effects of magnetic nanoparticles (MNPs) in polymeric coatings and the static external magnetic field (EMF) were investigated on neural induction of murine embryonic stem cells (mESCs) and human induced pluripotent stem cells (hiPSCs). The results show that the presence of 0.5% MNPs in collagen-based coatings facilitates the migration and neuronal maturation of mESCs and hiPSCs in vitro. Furthermore, the application of 0.4 Tesla EMF perpendicularly to the cell culture plane, discernibly stimulates proliferation and guide fate decisions of the pluripotent stem cells, depending on the origin of stem cells and their developmental stage. Mechanistic analysis reveals that modulation of ionic homeostasis and the expression of proteins involved in cytostructural, liposomal and cell cycle checkpoint functions provide a principal underpinning for the impact of electromagnetic stimuli on neural lineage specification and proliferation. These findings not only explore the potential of the magnetic stimuli as neural differentiation and function modulator but also highlight the risks that immoderate magnetic stimulation may affect more susceptible neurons, such as dopaminergic neurons.

Precision medicine implementation challenges for APOL1 testing in chronic kidney disease in admixed populations.

Chronic Kidney Disease (CKD) is a public health problem that presents genetic and environmental risk factors. Two alleles in the Apolipoprotein L1 (APOL1) gene were associated with chronic kidney disease; these alleles are common in individuals of African ancestry but rare in European descendants. Genomic studies on Afro-Americans have indicated a higher prevalence and severity of chronic kidney disease in people of African ancestry when compared to other ethnic groups. However, estimates in low- and middle-income countries are still limited. Precision medicine approaches could improve clinical outcomes in carriers of risk alleles in the Apolipoprotein L1 gene through early diagnosis and specific therapies. Nevertheless, to enhance the definition of studies on these variants, it would be necessary to include individuals with different ancestry profiles in the sample, such as Latinos, African Americans, and Indigenous peoples. There is evidence that measuring genetic ancestry improves clinical care for admixed people. For chronic kidney disease, this knowledge could help establish public health strategies for monitoring patients and understanding the impact of the Apolipoprotein L1 genetic variants in admixed populations. Therefore, researchers need to develop resources, methodologies, and incentives for vulnerable and disadvantaged communities, to develop and implement precision medicine strategies and contribute to consolidating diversity in science and precision medicine in clinical practice.

An in vitro stem cell model of human epiblast and yolk sac interaction.

Human embryogenesis entails complex signalling interactions between embryonic and extra-embryonic cells. However, how extra-embryonic cells direct morphogenesis within the human embryo remains largely unknown due to a lack of relevant stem cell models. Here, we have established conditions to differentiate human pluripotent stem cells (hPSCs) into yolk sac-like cells (YSLCs) that resemble the post-implantation human hypoblast molecularly and functionally. YSLCs induce the expression of pluripotency and anterior ectoderm markers in human embryonic stem cells (hESCs) at the expense of mesoderm and endoderm markers. This activity is mediated by the release of BMP and WNT signalling pathway inhibitors, and, therefore, resembles the functioning of the anterior visceral endoderm signalling centre of the mouse embryo, which establishes the anterior-posterior axis. Our results implicate the yolk sac in epiblast cell fate specification in the human embryo and propose YSLCs as a tool for studying post-implantation human embryo development in vitro.

Generation of two human induced pluripotent stem cell (hiPSC) lines derived from unrelated Marfan Syndrome patients.

Marfan Syndrome (MFS) is a pleiotropic and autosomal dominant condition caused by pathogenic variants in FBN1. Although fully penetrant, clinical variability is frequently observed among patients and there are only few genotype-phenotype correlations described so far. Here, we describe the generation and characterization of hiPSC lines derived from two unrelated MFS patients harboring heterozygous variants in FBN1. Human iPSCs were obtained from erythroblasts reprogrammed with episomal vectors carrying the reprogramming factors OCT4, SOX2, KLF4, c-MYC and LIN-28, and characterized according to established criteria. Differentiated cells demonstrated different patterns of fibrillin-1 expression suggesting different molecular mechanisms between the two patients.

Hyperkyphosis is not dependent on bone mass and quality in the mouse model of Marfan syndrome.

Marfan syndrome (MFS) is an autosomal dominant disease affecting cardiovascular, ocular and skeletal systems. It is caused by mutations in the fibrillin-1 (FBN1) gene, leading to structural defects of connective tissue and increased activation of TGF-ß. Angiotensin II (ang-II) is involved in TGF-ß activity and in bone mass regulation. Inhibition of TGF-ß signaling by blockage of the ang-II receptor 1 (AT1R) via losartan administration leads to improvement of cardiovascular and pulmonary phenotypes, but has no effect on skeletal phenotype in the haploinsufficient mouse model of MFS mgR, suggesting a distinct mechanism of pathogenesis in the skeletal system. Here we characterized the skeletal phenotypes of the dominant-negative model for MFS mgΔlpn and tested the effect of inhibition of ang-II signaling in improving those phenotypes. As previously shown, heterozygous mice present hyperkyphosis, however we now show that only males also present osteopenia. Inhibition of ang-II production by ramipril minimized the kyphotic deformity, but had no effect on bone microstructure in male mutant animals. Histological analysis revealed increased thickness of the anterior longitudinal ligament (ALL) of the spine in mutant animals (25.8 ± 6.3 vs. 29.7 ± 7.7 µm), coupled with a reduction in type I (164.1 ± 8.7 vs. 139.0 ± 4.4) and increase in type III (86.5 ± 10.2 vs. 140.4 ± 5.6) collagen in the extracellular matrix of this ligament. In addition, we identified in the MFS mice alterations in the erector spinae muscles which presented thinner muscle fibers (1035.0 ± 420.6 vs. 655.6 ± 239.5 µm2) surrounded by increased area of connective tissue (58.17 ± 6.52 vs. 105.0 ± 44.54 µm2). Interestingly, these phenotypes were ameliorated by ramipril treatment. Our results reveal a sex-dependency of bone phenotype in MFS, where females do not present alterations in bone microstructure. More importantly, they indicate that hyperkyphosis is not a result of osteopenia in the MFS mouse model, and suggest that incompetent spine ligaments and muscles are responsible for the development of that phenotype.

Generation of 6 lines of human pluripotent stem cells from hypertensive patients and 3 lines of human pluripotent stem cell from normotensive patients.

Hypertension is a complex multifactorial disease characterized by a chronic increase of arterial pressure. Ninety percent of the cases are idiopathic and thus classified as essential hypertension. Uncontrolled arterial pressure has devasting consequences including cardiac insufficiency, stroke, dementia, chronic renal disease, ischemic heart disease and death. The hiPSC lines described here from six hypertensive patients and three controls were characterized according to established criteria and were shown to maintain pluripotency, differentiation into the three germ layers and genomic integrity. These cell lines can contribute to the understanding of the molecular mechanisms involved in hypertension in different cell types.

Generation of genetically modified human induced pluripotent stem cell lines harboring haploin sufficient or dominant negative variants in the FBN1 gene.

Marfan Syndrome (MFS) is an autosomal dominant connective tissue disorder caused by mutations in the FBN1 gene. To investigate the molecular mechanisms of pathogenesis for the syndrome, we genetically modified the FBN1 gene in a line of induced pluripotent stem cells (hiPSCs) derived from a healthy donor using the CRISPR/Cas9 gene editing technology. The sublines described here were characterized according to established criteria and were shown to maintain pluripotency, three germ layer differentiation potential and genomic integrity. These clones can now be used to better understand the pathogenesis of MFS in different cell types.

Concurrent X chromosome inactivation and upregulation during non-human primate preimplantation development revealed by single-cell RNA-sequencing.

In mammals, dosage compensation of X-linked gene expression between males and females is achieved by inactivation of a single X chromosome in females, while upregulation of the single active X in males and females leads to X:autosome dosage balance. Studies in human embryos revealed that random X chromosome inactivation starts at the preimplantation stage and is not complete by day 12 of development. Alternatively, others proposed that dosage compensation in human preimplantation embryos is achieved by dampening expression from the two X chromosomes in females. Here, we characterize X-linked dosage compensation in another primate, the marmoset (Callithrix jacchus). Analyzing scRNA-seq data from preimplantation embryos, we detected upregulation of XIST at the morula stage, where female embryos presented a significantly higher expression of XIST than males. Moreover, we show an increase of X-linked monoallelically expressed genes in female embryos between the morula and late blastocyst stages, indicative of XCI. Nevertheless, dosage compensation was not achieved by the late blastocyst stage. Finally, we show that X:autosome dosage compensation is achieved at the 8-cell stage, and demonstrate that X chromosome dampening in females does not take place in the marmoset. Our work contributes to the elucidation of primate X-linked dosage compensation.

The mgΔlpn mouse model for Marfan syndrome recapitulates the ocular phenotypes of the disease.

PURPOSE: Fibrillin-1 and -2 are major components of tissue microfibrils that compose the ciliary zonule and cornea. While mutations in human fibrillin-1 lead to ectopia lentis, a major manifestation of Marfan syndrome (MFS), in mice fibrillin-2 can compensate for reduced/lack of fibrillin-1 and maintain the integrity of ocular structures. Here we examine the consequences of a heterozygous dominant-negative mutation in the Fbn1 gene in the ocular system of the mgΔlpn mouse model for MFS. METHODS: Eyes from mgΔlpn and wild-type mice at 3 and 6 months of age were analyzed by histology. The ciliary zonule was analyzed by scanning electron microscopy (SEM) and immunofluorescence. RESULTS: Mutant mice presented a significantly larger distance of the ciliary body to the lens at 3 and 6 months of age when compared to wild-type, and ectopia lentis. Immunofluorescence and SEM corroborated those findings in MFS mice, revealing a disorganized mesh of microfibrils on the floor of the ciliary body. Moreover, mutant mice also had a larger volume of the anterior chamber, possibly due to excess aqueous humor. Finally, losartan treatment had limited efficacy in improving ocular phenotypes. CONCLUSIONS: In contrast with null or hypomorphic mutations, expression of a dominant-negative form of fibrillin-1 leads to disruption of microfibrils in the zonule of mice. This in turn causes lens dislocation and enlargement of the anterior chamber. Therefore, heterozygous mgΔlpn mice recapitulate the major ocular phenotypes of MFS and can be instrumental in understanding the development of the disease.

Human induced pluripotent stem cells as a tool for disease modeling and drug screening for COVID-19.

The emergence of the new corona virus (SARS-CoV-2) and the resulting COVID-19 pandemic requires fast development of novel prevention and therapeutic strategies. These rely on understanding the biology of the virus and its interaction with the host, and on agnostic phenotypic screening for compounds that prevent viral infection. In vitro screenings of compounds are usually performed in human or animal-derived tumor or immortalized cell lines due to their ease of culturing. However, these platforms may not represent the tissues affected by the disease in vivo, and therefore better models are needed to validate and expedite drug development, especially in face of the COVID-19 pandemic. In this scenario, human induced pluripotent stem cells (hiPSCs) are a powerful research tool due to their ability to generate normal differentiated cell types relevant for the disease. Here we discuss the different ways hiPSCs can contribute to COVID-19 related research, including modeling the disease in vitro and serving as a platform for drug screening.

Roadmap for Establishing Large-Scale Genomic Medicine Initiatives in Low- and Middle-Income Countries.

In the post-genomic era, genomic medicine interventions as a key component of personalized medicine and tailored-made health care are greatly anticipated following recent scientific and technological advances. Indeed, large-scale sequencing efforts that explore human genomic variation have been initiated in several, mostly developed, countries across the globe, such as the United States, the United Kingdom, and a few others. Here, we highlight the successful implementation of large-scale national genomic initiatives, namely the Genome of Greece (GoGreece) and the DNA do Brasil (DNABr), aiming to emphasize the importance of implementing such initiatives in developing countries. Based on this experience, we also provide a roadmap for replicating these projects in other low-resource settings, thereby bringing genomic medicine in these countries closer to clinical fruition.

Is HSPG2 a modifier gene for Marfan syndrome?

Marfan syndrome (MFS) is a connective tissue disease caused by variants in the FBN1 gene. Nevertheless, other genes influence the manifestations of the disease, characterized by high clinical variability even within families. We mapped modifier loci for cardiovascular and skeletal manifestations in the mg∆loxPneo mouse model for MFS and the synthenic loci in the human genome. Corroborating our findings, one of those loci was identified also as a modifier locus in MFS patients. Here, we investigate the HSPG2 gene, located in this region, as a candidate modifier gene for MFS. We show a correlation between Fbn1 and Hspg2 expression in spinal column and aorta in non-isogenic mg∆loxPneo mice. Moreover, we show that mice with severe phenotypes present lower expression of Hspg2 than those mildly affected. Thus, we propose that HSPG2 is a strong candidate modifier gene for MFS and its role in modulating disease severity should be investigated in patients.