Enhanced Optic Nerve Expansion and Altered Ultrastructure of Elastic Fibers Induced by Lysyl Oxidase Inhibition in a Mouse Model of Marfan Syndrome.

Two major constituents of exfoliation material, fibrillin-1 and lysyl oxidase-like 1 (encoded by FBN1 and LOXL1), are implicated in exfoliation glaucoma, yet their individual contributions to ocular phenotype are minor. To test the hypothesis that a combination of FBN1 mutation and LOXL1 deficiency exacerbates ocular phenotypes, the pan-lysyl oxidase inhibitor ß-aminopropionitrile (BAPN) was used to treat adult wild-type (WT) mice and mice heterozygous for a missense mutation in Fbn1 (Fbn1C1041G/+) for 8 weeks and their eyes were examined. Although intraocular pressure did not change and exfoliation material was not detected in the eyes, BAPN treatment worsened optic nerve and axon expansion in Fbn1C1041G/+ mice, an early sign of axonal damage in rodent models of glaucoma. Disruption of elastic fibers was detected only in Fbn1C1041G/+ mice, which increased with BAPN treatment, as shown by histologic and immunohistochemical staining of the optic nerve pia mater. Transmission electron microscopy showed that Fbn1C1041G/+ mice had fewer microfibrils, smaller elastin cores, and a lower density of elastic fibers compared with WT mice in control groups. BAPN treatment led to elastin core expansion in both WT and Fbn1C1041G/+ mice, but an increase in the density of elastic fiber was confined to Fbn1C1041G/+ mice. LOX inhibition had a stronger effect on optic nerve and elastic fiber parameters in the context of Fbn1 mutation, indicating the Marfan mouse model with LOX inhibition warrants further investigation for exfoliation glaucoma pathogenesis.

Wnt Signaling Inhibition Prevents Postnatal Inflammation and Disease Progression in Mouse Congenital Myxomatous Valve Disease.

BACKGROUND: Myxomatous valve disease (MVD) is the most common cause of mitral regurgitation, leading to impaired cardiac function and heart failure. MVD in a mouse model of Marfan syndrome includes valve leaflet thickening and progressive valve degeneration. However, the underlying mechanisms by which the disease progresses remain undefined. METHODS: Mice with Fibrillin 1 gene variant Fbn1C1039G/+ recapitulate histopathologic features of Marfan syndrome, and Wnt (Wingless-related integration site) signaling activity was detected in TCF/Lef-lacZ (T-cell factor/lymphoid enhancer factor-ß-galactosidase) reporter mice. Single-cell RNA sequencing was performed from mitral valves of wild-type and Fbn1C1039G/+ mice at 1 month of age. Inhibition of Wnt signaling was achieved by conditional induction of the secreted Wnt inhibitor Dkk1 (Dickkopf-1) expression in periostin-expressing valve interstitial cells of Periostin-Cre; tetO-Dkk1; R26rtTA; TCF/Lef-lacZ; Fbn1C1039G/+ mice. Dietary doxycycline was administered for 1 month beginning with MVD initiation (1-month-old) or MVD progression (2-month-old). Histological evaluation and immunofluorescence for ECM (extracellular matrix) and immune cells were performed. RESULTS: Wnt signaling is activated early in mitral valve disease progression, before immune cell infiltration in Fbn1C1039G/+ mice. Single-cell transcriptomics revealed similar mitral valve cell heterogeneity between wild-type and Fbn1C1039G/+ mice at 1 month of age. Wnt pathway genes were predominantly expressed in valve interstitial cells and valve endothelial cells of Fbn1C1039G/+ mice. Inhibition of Wnt signaling in Fbn1C1039G/+ mice at 1 month of age prevented the initiation of MVD as indicated by improved ECM remodeling and reduced valve leaflet thickness with decreased infiltrating macrophages. However, later, Wnt inhibition starting at 2 months did not prevent the progression of MVD. CONCLUSIONS: Wnt signaling is involved in the initiation of mitral valve abnormalities and inflammation but is not responsible for later-stage valve disease progression once it has been initiated. Thus, Wnt signaling contributes to MVD progression in a time-dependent manner and provides a promising therapeutic target for the early treatment of congenital MVD in Marfan syndrome.

Pathogenic variants affecting the TB5 domain of the fibrillin-1 protein: not only in geleophysic/acromicric dysplasias but also in Marfan syndrome.

BACKGROUND: Marfan syndrome (MFS) is a multisystem disease with a unique combination of skeletal, cardiovascular and ocular features. Geleophysic/acromicric dysplasias (GPHYSD/ACMICD), characterised by short stature and extremities, are described as 'the mirror image' of MFS. The numerous FBN1 pathogenic variants identified in MFS are located all along the gene and lead to the same final pathogenic sequence. Conversely, in GPHYSD/ACMICD, the 28 known heterozygous FBN1 pathogenic variants all affect exons 41-42 encoding TGFß-binding protein-like domain 5 (TB5). METHODS: Since 1996, more than 5000 consecutive probands have been referred nationwide to our laboratory for molecular diagnosis of suspected MFS. RESULTS: We identified five MFS probands carrying distinct heterozygous pathogenic in-frame variants affecting the TB5 domain of FBN1. The clinical data showed that the probands displayed a classical form of MFS. Strikingly, one missense variant affects an amino acid that was previously involved in GPHYSD. CONCLUSION: Surprisingly, pathogenic variants in the TB5 domain of FBN1 can lead to two opposite phenotypes: GPHYSD/ACMICD and MFS, suggesting the existence of different pathogenic sequences with the involvement of tissue specificity. Further functional studies are ongoing to determine the precise role of this domain in the physiopathology of each disease.

A novel de novo intragenic duplication in FBN1 associated with early-onset Marfan syndrome in a 16-month-old: A case report and review of the literature.

Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder due to pathogenic variants in Fibrillin-1 (FBN1) affecting nearly one in every 10,000 individuals. We report a 16-month-old female with early-onset MFS heterozygous for an 11.2 kb de novo duplication within the FBN1 gene. Tandem location of the duplication was further confirmed by optical genome mapping in addition to genetic sequencing and chromosomal microarray. This is the third reported case of a large multi-exon duplication in FBN1, and the only one confirmed to be in tandem. As the vast majority of pathogenic variants associated with MFS are point mutations, this expands the landscape of known FBN1 pathogenic variants and supports consistent use of genetic testing strategies that can detect large, indel-type variants.

EFEMP1 haploinsufficiency causes a Marfan-like hereditary connective tissue disorder.

Phenotypic features of a hereditary connective tissue disorder, including craniofacial characteristics, hyperextensible skin, joint laxity, kyphoscoliosis, arachnodactyly, inguinal hernia, and diverticulosis associated with biallelic pathogenic variants in EFEMP1 have been previously described in four patients. Genome sequencing on a proband and her mother with comparable phenotypic features revealed that both patients were heterozygous for a stop-gain variant c.1084C>T (p.Arg362*). Complementary RNA-seq on fibroblasts revealed significantly reduced levels of mutant EFEMP1 transcript. Considering the absence of other molecular explanations, we extrapolated that EFEMP1 could be the cause of the patient's phenotypes. Furthermore, nonsense-mediated decay was demonstrated for the mutant allele as the principal mechanism for decreased levels of EFEMP1 mRNA. We provide strong clinical and genetic evidence for the haploinsufficiency of EFEMP1 due to nonsense-medicated decay to cause severe kyphoscoliosis, generalized hypermobility of joints, high and narrow arched palate, and potentially severe diverticulosis. To the best of our knowledge, this is the first report of an autosomal dominant EFEMP1-associated hereditary connective tissue disorder and therefore expands the phenotypic spectrum of EFEMP1 related disorders.

Effects of Age, Sex, and Extracellular Matrix Integrity on Aortic Dilatation and Rupture in a Mouse Model of Marfan Syndrome.

BACKGROUND: Transmural failure of the aorta is responsible for substantial morbidity and mortality; it occurs when mechanical stress exceeds strength. The aortic root and ascending aorta are susceptible to dissection and rupture in Marfan syndrome, a connective tissue disorder characterized by a progressive reduction in elastic fiber integrity. Whereas competent elastic fibers endow the aorta with compliance and resilience, cross-linked collagen fibers confer stiffness and strength. We hypothesized that postnatal reductions in matrix cross-linking increase aortopathy when turnover rates are high. METHODS: We combined ex vivo biaxial mechanical testing with multimodality histological examinations to quantify expected age- and sex-dependent structural vulnerability of the ascending aorta in Fbn1C1041G/+ Marfan versus wild-type mice without and with 4-week exposures to ß-aminopropionitrile, an inhibitor of lysyl oxidase-mediated cross-linking of newly synthesized elastic and collagen fibers. RESULTS: We found a strong ß-aminopropionitrile-associated sexual dimorphism in aortic dilatation in Marfan mice and aortic rupture in wild-type mice, with dilatation correlating with compromised elastic fiber integrity and rupture correlating with compromised collagen fibril organization. A lower incidence of rupture of ß-aminopropionitrile-exposed Marfan aortas associated with increased lysyl oxidase, suggesting a compensatory remodeling of collagen that slows disease progression in the otherwise compromised Fbn1C1041G/+ aorta. CONCLUSIONS: Collagen fiber structure and function in the Marfan aorta are augmented, in part, by increased lysyl oxidase in female and especially male mice, which improves structural integrity, particularly via fibrils in the adventitia. Preserving or promoting collagen cross-linking may represent a therapeutic target for an otherwise vulnerable aorta.

IL11 (Interleukin-11) Causes Emphysematous Lung Disease in a Mouse Model of Marfan Syndrome.

BACKGROUND: Marfan Syndrome (MFS) is an inherited connective tissue disorder caused by mutations in the FBN1 (fibrillin-1) gene. Lung abnormalities are common in MFS, but their pathogenesis is poorly understood. IL11 (interleukin-11) causes aortic disease in a mouse model of MFS and was studied here in the lung. METHODS: We examined histological and molecular phenotypes in the lungs of Fbn1C1041G/+ mice (mouse model of Marfan Syndrome [mMFS]), an established mouse model of MFS. To identify IL11-expressing cells, we used immunohistochemistry on lungs of 4- and 16-week-old Fbn1C1041G/+:Il11EGFP/+ reporter mice. We studied the effects of IL11 inhibition by RT-qPCR, immunoblots and histopathology in lungs from genetic or pharmacologic models: (1) 16-week-old IL11 receptor (IL11RA) knockout mMFS mice (Fbn1C1041G/+:Il11ra1-/- mice) and (2) in mMFS mice administered IgG control or interleukin-11 receptor antibodies twice weekly from 4 to 24 weeks of age. RESULTS: mMFS lungs showed progressive loss and enlargement of distal airspaces associated with increased proinflammatory and profibrotic gene expression as well as matrix metalloproteinases 2, 9, and 12. IL11 was increased in mMFS lungs and localized to smooth muscle and endothelial cells in young mMFS mice in the Fbn1C1041G/+:Il11EGFP/+ reporter strain and in fibroblasts, in older mice. In mMFS mice, genetic (Fbn1C1041G/+:Il11ra1-/-) or pharmacologic (anti-interleukin-11 receptor) inhibition of IL11 signaling reduced lung emphysema, fibrosis, and inflammation. This protective effect was associated with reduced pathogenic ERK1/2 signaling and lower metalloproteinase 2, 9, and 12 expression. CONCLUSIONS: IL11 causes lung disease in mMFS. This reveals a shared IL11-driven disease mechanism in lung and aorta in MFS and suggests inhibition of IL11 signaling as a holistic approach for treating multiorgan morbidity in MFS.

Impact of pathogenic FBN1 variant types on the development of severe scoliosis in patients with Marfan syndrome.

BACKGROUND: Among the several musculoskeletal manifestations in patients with Marfan syndrome, spinal deformity causes pain and respiratory impairment and is a great hindrance to patients' daily activities. The present study elucidates the genetic risk factors for the development of severe scoliosis in patients with Marfan syndrome. METHODS: We retrospectively evaluated 278 patients with pathogenic or likely pathogenic FBN1 variants. The patients were divided into those with (n=57) or without (n=221) severe scoliosis. Severe scoliosis was defined as (1) patients undergoing surgery before 50 years of age or (2) patients with a Cobb angle exceeding 50° before 50 years of age. The variants were classified as protein-truncating variants (PTVs), which included variants creating premature termination codons and inframe exon-skipping, or non-PTVs, based on their location and predicted amino acid alterations, and the effect of the FBN1 genotype on the development of severe scoliosis was examined. The impact of location of FBN1 variants on the development of severe scoliosis was also investigated. RESULTS: Univariate and multivariate analyses revealed that female sex, PTVs of FBN1 and variants in the neonatal region (exons 25-33) were all independent significant predictive factors for the development of severe scoliosis. Furthermore, these factors were identified as predictors of progression of existing scoliosis into severe state. CONCLUSIONS: We elucidated the genetic risk factors for the development of severe scoliosis in patients with Marfan syndrome. Patients harbouring pathogenic FBN1 variants with these genetic risk factors should be monitored carefully for scoliosis progression.

A systematic study and literature review of parental somatic mosaicism of FBN1 pathogenic variants in Marfan syndrome.

BACKGROUND: A proportion of de novo variants in patients affected by genetic disorders, particularly those with autosomal dominant (AD) inheritance, could be the consequence of somatic mosaicism in one of the progenitors. There is growing evidence that germline and somatic mosaicism are more common and play a greater role in genetic disorders than previously acknowledged. In Marfan syndrome (MFS), caused by pathogenic variants in the fibrillin-1 gene (FBN1) gene, approximately 25% of the disease-causing variants are reported as de novo. Only a few cases of parental mosaicism have been reported in MFS. METHODS: Employing an amplicon-based deep sequencing (ADS) method, we carried out a systematic analysis of 60 parents of 30 FBN1 positive, consecutive patients with MFS with an apparently de novo pathogenic variant. RESULTS: Out of the 60 parents studied (30 families), the majority (n=51, 85%) had a systemic score of 0, seven had a score of 1 and two a score of 2, all due to minor criteria common in the normal population. We detected two families with somatic mosaicism in one of the progenitors, with a rate of 6.6% (2/30) of apparently de novo cases. CONCLUSIONS: The search for parental somatic mosaicism should be routinely implemented in de novo cases of MFS, to offer appropriate genetic and reproductive counselling as well as to reveal masked, isolated clinical signs of MFS in progenitors that may require specific follow-up.

Hyperuricaemia Does Not Interfere with Aortopathy in a Murine Model of Marfan Syndrome.

Redox stress is involved in the aortic aneurysm pathogenesis in Marfan syndrome (MFS). We recently reported that allopurinol, a xanthine oxidoreductase inhibitor, blocked aortopathy in a MFS mouse model acting as an antioxidant without altering uric acid (UA) plasma levels. Hyperuricaemia is ambiguously associated with cardiovascular injuries as UA, having antioxidant or pro-oxidant properties depending on the concentration and accumulation site. We aimed to evaluate whether hyperuricaemia causes harm or relief in MFS aortopathy pathogenesis. Two-month-old male wild-type (WT) and MFS mice (Fbn1C1041G/+) were injected intraperitoneally for several weeks with potassium oxonate (PO), an inhibitor of uricase (an enzyme that catabolises UA to allantoin). Plasma UA and allantoin levels were measured via several techniques, aortic root diameter and cardiac parameters by ultrasonography, aortic wall structure by histopathology, and pNRF2 and 3-NT levels by immunofluorescence. PO induced a significant increase in UA in blood plasma both in WT and MFS mice, reaching a peak at three and four months of age but decaying at six months. Hyperuricaemic MFS mice showed no change in the characteristic aortic aneurysm progression or aortic wall disarray evidenced by large elastic laminae ruptures. There were no changes in cardiac parameters or the redox stress-induced nuclear translocation of pNRF2 in the aortic tunica media. Altogether, the results suggest that hyperuricaemia interferes neither with aortopathy nor cardiopathy in MFS mice.

Age and sex dependency of thoracic aortopathy in a mouse model of Marfan syndrome.

Thoracic aortic aneurysm is one of the manifestations of Marfan syndrome (MFS) that is known to affect men more severely than women. However, the incidence of MFS is similar between men and women. The aim of this study is to show that during pathological aortic dilation, sex-dependent severity of thoracic aortopathy in a mouse model of MFS translates into sex-dependent alterations in cells and matrix of the ascending aorta, consequently affecting aortic biomechanics. Fibrillin-1 C1041G/+ (Het) mice were used as a mouse model of MFS. Ultrasound measurements from 3 to 12 mo showed increased aortic diameter in Het aorta, with larger percentage increase in diameter for males compared with females. Immunohistochemistry showed decreased contractile smooth muscle cells in Het aortic wall compared with healthy aorta, which was accompanied by decreased contractility measured by wire myography. Elastin autofluorescence, second-harmonic generation microscopy of collagen fibers, and passive biomechanical assessments using myography showed more severe damage to elastin fibers, increased medial fibrosis, and increased stiffness of the aortic wall in MFS males but not females. Male and female Het mice showed increased expression of Sca-1-positive adventitial progenitor cells versus controls at young ages. In agreement with clinical data, Het mice demonstrate sex-dependent severity of thoracic aortopathy. It was also shown that aging exacerbates the disease state especially for males. Our findings suggest that female mice are protected from progression of aortic dilation at early ages, leading to a lag in aneurysm growth.NEW & NOTEWORTHY Male Fbn1C1041G/+ mice show more severe thoracic aortic changes compared with females, especially at 12 mo of age. Up to 6 mo of age, Sca-1+ smooth muscle progenitor cells are more abundant in the adventitia of both male and female Fbn1 Het mice compared with wild types (WTs). Male and female Het mice show similar patterns of expression of Sca-1+ cells at early ages.

Macromolecular crowding enhances fibrillin-1 deposition in the extracellular matrix.

Biochemical and biophysical factors need consideration when modelling in vivo cellular behaviour using in vitro cell culture systems. One underappreciated factor is the high concentration of macromolecules present in vivo, which is typically not simulated under standard cell culture conditions. This disparity is especially relevant when studying biochemical processes that govern extracellular matrix (ECM) deposition, which may be altered due to dilution of secreted macromolecules by the relatively large volumes of culture medium required for cell maintenance in vitro. Macromolecular crowding (MMC) utilises the addition of inert macromolecules to cell culture medium to mimic such high concentration environments found in vivo. The present study induced MMC using the sucrose polymer Ficoll and examined whether fibrillin-1 deposition by human lung fibroblasts could be augmented. Fibrillin-1 forms extracellular microfibrils, which are versatile scaffolds required for elastic fibre formation, deposition of other ECM proteins and growth factor regulation. Pathogenic variants in the fibrillin-1 gene (FBN1) cause Marfan syndrome, where ECM deposition of fibrillin-1 can be compromised. Using immunocytochemistry, significantly enhanced fibrillin-1 deposition was observed when lung fibroblasts were cultured under MMC conditions. MMC also augmented fibrillin-1 deposition in Marfan syndrome patient-derived skin fibroblasts in a cell line- and likely FBN1 variant-specific manner. The ability of MMC to increase fibrillin-1 deposition suggested potential applications for tissue-engineering approaches, e.g. to generate tendon or vascular tissues, where fibrillin-1 microfibrils and elastic fibres are key determinants of their biomechanical properties. Moreover, it suggested the potency of MMC to better mimic in vivo ECM environments in cell culture studies.

Deletion of AT1a (Angiotensin II Type 1a) Receptor or Inhibition of Angiotensinogen Synthesis Attenuates Thoracic Aortopathies in Fibrillin1C1041G/+ Mice.

Objective: A cardinal feature of Marfan syndrome is thoracic aortic aneurysm. The contribution of the renin-angiotensin system via AT1aR (Ang II [angiotensin II] receptor type 1a) to thoracic aortic aneurysm progression remains controversial because the beneficial effects of angiotensin receptor blockers have been ascribed to off-target effects. This study used genetic and pharmacological modes of attenuating angiotensin receptor and ligand, respectively, to determine their roles on thoracic aortic aneurysm in mice with fibrillin-1 haploinsufficiency (Fbn1C1041G/+). Approach and Results: Thoracic aortic aneurysm in Fbn1C1041G/+ mice was found to be strikingly sexual dimorphic. Males displayed aortic dilation over 12 months while aortic dilation in Fbn1C1041G/+ females did not differ significantly from wild-type mice. To determine the role of AT1aR, Fbn1C1041G/+ mice that were either +/+ or -/- for AT1aR were generated. AT1aR deletion reduced expansion of ascending aorta and aortic root diameter from 1 to 12 months of age in males. Medial thickening and elastin fragmentation were attenuated. An antisense oligonucleotide against angiotensinogen was administered to male Fbn1C1041G/+ mice to determine the effects of Ang II depletion. Antisense oligonucleotide against angiotensinogen administration attenuated dilation of the ascending aorta and aortic root and reduced extracellular remodeling. Aortic transcriptome analyses identified potential targets by which inhibition of the renin-angiotensin system reduced aortic dilation in Fbn1C1041G/+ mice. Conclusions: Deletion of AT1aR or inhibition of Ang II production exerted similar effects in attenuating pathologies in the proximal thoracic aorta of male Fbn1C1041G/+ mice. Inhibition of the renin-angiotensin system attenuated dysregulation of genes within the aorta related to pathology of Fbn1C1041G/+ mice.

The Multiple Functions of Fibrillin-1 Microfibrils in Organismal Physiology.

Fibrillin-1 is the major structural component of the 10 nm-diameter microfibrils that confer key physical and mechanical properties to virtually every tissue, alone and together with elastin in the elastic fibers. Mutations in fibrillin-1 cause pleiotropic manifestations in Marfan syndrome (MFS), including dissecting thoracic aortic aneurysms, myocardial dysfunction, progressive bone loss, disproportionate skeletal growth, and the dislocation of the crystalline lens. The characterization of these MFS manifestations in mice, that replicate the human phenotype, have revealed that the underlying mechanisms are distinct and organ-specific. This brief review summarizes relevant findings supporting this conclusion.

[Analysis of FBN1 genemutations in a pedigree with Marfan syndrome].

Mutations in fibrillin-1 (FBN1) were detected in an autosomal dominant Marfan syndrome (MFS) pedigree. The related phenotypes and the significance of mutation screening were discussed. Complete medical and cardiovascular examinations for all pedigree members were performed. Whole exons sequencing (WES) was used to sequence the DNA of the patients and their relatives. The potential pathogenic mutation sites were screened by bioinformatics method. Sanger sequencing was used to verify the mutation sites in the pedigree. The results showed that FBN1 missense mutation was c.6806 T>C in exon 56, resulting in isoleucine being replaced by threonine (p. Ile2269Thr). This mutation has not been reported in Chinese Han population. The occurrence of the mutations strongly correlated with the phenotypes of the patients. The results expand the mutation spectrum of FBN1, and it is helpful to further explore the molecular pathogenesis of MFS and MFS related diseases.

Deficiency of Circulating Monocytes Ameliorates the Progression of Myxomatous Valve Degeneration in Marfan Syndrome.

BACKGROUND: Myxomatous valve degeneration (MVD) involves the progressive thickening and degeneration of the heart valves, leading to valve prolapse, regurgitant blood flow, and impaired cardiac function. Leukocytes composed primarily of macrophages have recently been detected in myxomatous valves, but the timing of the presence and the contributions of these cells in MVD progression are not known. METHODS: We examined MVD progression, macrophages, and the valve microenvironment in the context of Marfan syndrome (MFS) using mitral valves from MFS mice (Fbn1C1039G/+), gene-edited MFS pigs (FBN1Glu433AsnfsX98/+), and patients with MFS. Additional histological and transcriptomic evaluation was performed by using nonsyndromic human and canine myxomatous valves, respectively. Macrophage ontogeny was determined using MFS mice transplanted with mTomato+ bone marrow or MFS mice harboring RFP (red fluorescent protein)-tagged C-C chemokine receptor type 2 (CCR2) monocytes. Mice deficient in recruited macrophages (Fbn1C1039G/+;Ccr2RFP/RFP) were generated to determine the requirements of recruited macrophages to MVD progression. RESULTS: MFS mice recapitulated histopathological features of myxomatous valve disease by 2 months of age, including mitral valve thickening, increased leaflet cellularity, and extracellular matrix abnormalities characterized by proteoglycan accumulation and collagen fragmentation. Diseased mitral valves of MFS mice concurrently exhibited a marked increase of infiltrating (MHCII+, CCR2+) and resident macrophages (CD206+, CCR2-), along with increased chemokine activity and inflammatory extracellular matrix modification. Likewise, mitral valve specimens obtained from gene-edited MFS pigs and human patients with MFS exhibited increased monocytes and macrophages (CD14+, CD64+, CD68+, CD163+) detected by immunofluorescence. In addition, comparative transcriptomic evaluation of both genetic (MFS mice) and acquired forms of MVD (humans and dogs) unveiled a shared upregulated inflammatory response in diseased valves. Remarkably, the deficiency of monocytes was protective against MVD progression, resulting in a significant reduction of MHCII macrophages, minimal leaflet thickening, and preserved mitral valve integrity. CONCLUSIONS: All together, our results suggest sterile inflammation as a novel paradigm to disease progression, and we identify, for the first time, monocytes as a viable candidate for targeted therapy in MVD.

An integrated clinical and molecular study of a cohort of Turkish patients with Marfan syndrome harboring known and novel FBN1 variants.

Marfan syndrome (MFS) is an autosomal dominant genetic condition that mainly affects connective tissue in many parts of the body. Cardinal manifestations involve the ocular, skeletal, and cardiovascular systems. The diagnosis of MFS relies on the revised Ghent criteria, outlined by international expert opinion to facilitate accurate recognition of this syndrome as well as to improve patient management and counseling. However, it may not always be possible to make a definitive diagnosis according to these criteria in each patient and thus molecular confirmation is necessary in subjects with suspected MFS. This debilitating, if not fatal, disorder is caused by mutations in FBN1, which encodes a major constitutive element of extracellular microfibrils. Here, we present a detailed clinical and molecular analysis of 76 Turkish patients with definitive or suspected MFS diagnosed at our center between 2014 and 2019. We were able to identify a total of 51 different FBN1 variants in our cohort, 31 of which have previously been reported in the relevant scientific literature. The remaining 20 variants have not been documented to date. In one patient, we detected a large deletion including the entire FBN1 gene using the array CGH approach. Currently, there are very few studies on the genotype-phenotype correlation of patients with MFS, and no clear genotype-phenotype maps for MFS have been constructed so far, except for some cases. We believe that our findings will make a rich and peculiar contribution to the elusive genotype-phenotype relationship in MFS, especially in this large and populous ethnic group.

Positional cloning and comprehensive mutation analysis identified a novel KDM2B mutation in a Japanese family with minor malformations, intellectual disability, and schizophrenia.

The importance of epigenetic control in the development of the central nervous system has recently been attracting attention. Methylation patterns of lysine 4 and lysine 36 in histone H3 (H3K4 and H3K36) in the central nervous system are highly conserved among species. Numerous complications of body malformations and neuropsychiatric disorders are due to abnormal histone H3 methylation modifiers. In this study, we analyzed a Japanese family with a dominant inheritance of symptoms including Marfan syndrome-like minor physical anomalies (MPAs), intellectual disability, and schizophrenia (SCZ). We performed positional cloning for this family using a single nucleotide polymorphism (SNP) array and whole-exome sequencing, which revealed a missense coding strand mutation (rs1555289644, NM_032590.4: c.2173G>A, p.A725T) in exon 15 on the plant homeodomain of the KDM2B gene as a possible cause of the disease in the family. The exome sequencing revealed that within the coding region, only a point mutation in KDM2B was present in the region with the highest logarithm of odds score of 2.41 resulting from whole genome linkage analysis. Haplotype analysis revealed co-segregation with four affected family members (IV-9, III-4, IV-5, and IV-8). Lymphoblastoid cell lines from the proband with this mutation showed approximately halved KDM2B expression in comparison with healthy controls. KDM2B acts as an H3K4 and H3K36 histone demethylase. Our findings suggest that haploinsufficiency of KDM2B in the process of development, like other H3K4 and H3K36 methylation modifiers, may have caused MPAs, intellectual disability, and SCZ in this Japanese family.

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.

Toward precision medicine in vascular connective tissue disorders.

Tremendous progress has been made in understanding the etiology, pathogenesis, and treatment of inherited vascular connective tissue disorders. While new insights regarding disease etiology and pathogenesis have informed patient counseling and care, there are numerous obstacles that need to be overcome in order to achieve the full promise of precision medicine. In this review, these issues will be discussed in the context of Marfan syndrome and Loeys-Dietz syndrome, with additional emphasis on the pioneering contributions made by Victor McKusick.