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Biallelic MFSD8 variants are an established cause of severe late-infantile subtype of neuronal ceroid lipofuscinosis (v-LINCL), a severe lysosomal storage disorder, but have also been associated with nonsyndromic adult-onset maculopathy. Here, we functionally characterized two novel MFSD8 variants found in a child with juvenile isolated maculopathy, in order to establish a refined prognosis. ABCA4 locus resequencing was followed by the analysis of other inherited retinal disease genes by whole exome sequencing (WES). Minigene assays and cDNA sequencing were used to assess the effect of a novel MFSD8 splice variant. MFSD8 expression was quantified with qPCR and overexpression studies were analyzed by immunoblotting. Transmission electron microscopy (TEM) was performed on a skin biopsy and ophthalmological and neurological re-examinations were conducted. WES revealed two novel MFSD8 variants: c.[590del];[439+3A>C] p.[Gly197Valfs*2];[Ile67Glufs*3]. Characterization of the c.439+3A>C variant via splice assays showed exon-skipping (p.Ile67Glufs*3), while overexpression studies of the corresponding protein indicated expression of a truncated polypeptide. In addition, a significantly reduced MFSD8 RNA expression was noted in patient's lymphocytes. TEM of a skin biopsy revealed typical v-LINCL lipopigment inclusions while neurological imaging of the proband displayed subtle cerebellar atrophy. Functional characterization demonstrated the pathogenicity of two novel MFSD8 variants, found in a child with an initial diagnosis of juvenile isolated maculopathy but likely evolving to v-LINCL with a protracted disease course. Our study allowed a refined neurological prognosis in the proband and expands the natural history of MFSD8-associated disease.

TANGO1 (transport and Golgi organization‐1 homolog) encodes a transmembrane protein, which is located at endoplasmic reticulum (ER) exit sites where it binds bulky cargo, such as collagens, in the lumen and recruits membranes from the ER‐Golgi intermediate compartment (ERGIC) to create an export route for cargo secretion. Mice lacking Mia3 (murine TANGO1 orthologue) show defective secretion of numerous procollagens and lead to neonatal lethality due to insufficient bone mineralization. Recently, aberrant expression of truncated TANGO1 in humans has been shown to cause a mild‐to‐moderate severe collagenopathy associated with dentinogenesis imperfecta, short stature, skeletal abnormalities, diabetes mellitus, and mild intellectual disability. We now show for the first time that complete loss of TANGO1 results in human embryonic lethality with near‐total bone loss and phenocopies the situation of Mia3 −/− mice. Whole‐exome sequencing on genomic DNA (gDNA) of an aborted fetus of Indian descent revealed a homozygous 4‐base pair (4‐bp) deletion in TANGO1 that is heterozygously present in both healthy parents. Parental fibroblast studies showed decreased TANGO1 mRNA expression and protein levels. Type I collagen secretion and extracellular matrix organization were normal, supporting a threshold model for clinical phenotype development. As such, our report broadens the phenotypic and mutational spectrum of TANGO1‐related collagenopathies, and underscores the crucial role of TANGO1 for normal bone development, of which deficiency results in a severe‐to‐lethal form of osteochondrodysplasia. © 2021 American Society for Bone and Mineral Research © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Type III collagen is a major fibrillar collagen consisting of three identical α1(III)-chains that is particularly present in tissues exhibiting elastic properties, such as the skin and the arterial wall. Heterozygous mutations in the COL3A1 gene result in vascular Ehlers-Danlos syndrome (vEDS), a severe, life-threatening disorder, characterized by thin, translucent skin and propensity to arterial, intestinal and uterine rupture. Most human vEDS cases result from a missense mutation substituting a crucial glycine residue in the triple helical domain of the α1(III)-chains. The mechanisms by which these mutant type III collagen molecules cause dermal and vascular fragility are not well understood. We generated a transgenic mouse line expressing mutant type III collagen, containing a typical helical glycine substitution (p.(Gly182Ser)). This Col3a1Tg-G182S mouse line displays a phenotype recapitulating characteristics of human vEDS patients with signs of dermal and vascular fragility. The Col3a1Tg-G182S mice develop severe transdermal skin wounds, resulting in early demise at 13-14weeks of age. We found that this phenotype was associated with a reduced total collagen content and an abnormal collagen III:I ratio, leading to the production of severely malformed collagen fibrils in the extracellular matrix of dermal and arterial tissues. These results indicate that expression of the glycine substitution in the α1(III)-chain disturbs formation of heterotypic type III:I collagen fibrils, and thereby demonstrate a key role for type III collagen in collagen fibrillogenesis in dermal and arterial tissues.

Summary The bone disorder osteogenesis imperfecta (OI) is genetically heterogeneous. Most affected individuals have an autosomal dominant disorder caused by heterozygous variants in either of the type I collagen genes (COL1A1 or COL1A2). To date, two reports have linked Mesoderm Development LRP Chaperone (MESD) to autosomal recessive OI type XX. Four different biallelic pathogenic variants in MESD were shown to cause a progressively deforming phenotype, associated with recurrent fractures and oligodontia in five individuals in five families. Recently, compound heterozygosity for a frameshift predicted to lead to a premature termination codon in exon 2 of the 3-exon gene and a second frameshift in the terminal exon in MESD were detected in three stillbirths in one family with severe OI consistent with the neonatal lethal phenotype. We have identified four additional individuals from four independent families with biallelic variants in MESD: the earlier reported c.632dupA (p.Lys212Glufs∗19) and c.676C>T (p.Arg226∗)—which are associated with a severe form of OI—and one new pathogenic variant, c.603-606delTAAA (p.Asn201Lysfs∗15), which causes a neonatal lethal form of OI. MESD acts in the WNT signaling pathway, where it is thought to play a role in the folding of the WNT co-receptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/LRP6) and in chaperoning their transit to the cell surface. Our report broadens the phenotypic and genetic spectrum of MESD-related OI, provides additional insight into the pathogenic pathways, and underscores the necessity of MESD for normal WNT signaling in bone formation., Mesoderm Development LRP Chaperone (MESD) has been linked to autosomal recessive osteogenesis imperfecta type XX. Our report describes four new individuals and broadens the phenotypic and genetic spectrum of MESD-related OI, provides additional insight into the pathogenic pathways, and underscores the necessity of MESD for normal WNT signaling in bone formation.

Inactivating variants in the centrosomal CEP78 gene have been found in cone-rod dystrophy with hearing loss (CRDHL), a particular phenotype distinct from Usher syndrome. Here, we identified and functionally characterized the first CEP78 missense variant c.449T>C, p.(Leu150Ser) in three CRDHL families. The variant was found in a biallelic state in two Belgian families and in a compound heterozygous state-in trans with c.1462-1G>T-in a third German family. Haplotype reconstruction showed a founder effect. Homology modeling revealed a detrimental effect of p.(Leu150Ser) on protein stability, which was corroborated in patients' fibroblasts. Elongated primary cilia without clear ultrastructural abnormalities in sperm or nasal brushes suggest impaired cilia assembly. Two affected males from different families displayed sperm abnormalities causing infertility. One of these is a heterozygous carrier of a complex allele in SPAG17, a ciliary gene previously associated with autosomal recessive male infertility. Taken together, our data indicate that a missense founder allele in CEP78 underlies the same sensorineural CRDHL phenotype previously associated with inactivating variants. Interestingly, the CEP78 phenotype has been possibly expanded with male infertility. Finally, CEP78 loss-of-function variants may have an underestimated role in misdiagnosed Usher syndrome, with or without sperm abnormalities. ispartof: Human Mutation vol:41 issue:5 pages:998-1011 ispartof: location:United States status: published

Background Proteoglycans are large and structurally complex macromolecules which can be found in abundancy in the extracellular matrix and on the surface of all animal cells. Mutations in the genes encoding the enzymes responsible for the formation of the tetrasaccharide linker region between the proteoglycan core protein and the glycosaminoglycan side chains lead to a spectrum of severe and overlapping autosomal recessive connective tissue disorders, collectively coined the ‘glycosaminoglycan linkeropathies’. Results We report the clinical findings of two novel patients with a complex linkeropathy due to biallelic mutations in B3GAT3, the gene that encodes glucuronosyltransferase I, which catalyzes the addition of the ultimate saccharide to the linker region. We identified a previously reported c.667G > A missense mutation and an unreported homozygous c.416C > T missense mutation. We also performed a genotype and phenotype-oriented literature overview of all hitherto reported patients harbouring B3GAT3 mutations. A total of 23 patients from 10 families harbouring bi-allelic mutations and one patient with a heterozygeous splice-site mutation in B3GAT3 have been reported. They all display a complex phenotype characterized by consistent presence of skeletal dysplasia (including short stature, kyphosis, scoliosis and deformity of the long bones), facial dysmorphology, and spatulate distal phalanges. More variably present are cardiac defects, joint hypermobility, joint dislocations/contractures and fractures. Seven different B3GAT3 mutations have been reported, and although the number of patients is still limited, some phenotype-genotype correlations start to emerge. The more severe phenotypes seem to have mutations located in the substrate acceptor subdomain of the catalytic domain of the glucuronosyltransferase I protein while more mildly affected phenotypes seem to have mutations in the NTP-sugar donor substrate binding subdomain. Conclusions Loss-of-function mutations in B3GAT3 are associated with a complex connective tissue phenotype characterized by disproportionate short stature, skeletal dysplasia, facial dysmorphism, spatulate distal phalanges and -to a lesser extent- joint contractures, joint hypermobility with dislocations, cardiac defects and bone fragility. Based on the limited number of reported patients, some genotype-phenotype correlations start to emerge.

Osteogenesis imperfecta (OI) is a heritable connective tissue disorder, mainly characterized by bone fragility and low bone mass. Defects in the type I procollagen-encoding genes account for the majority of OI, but increasingly more rare autosomal recessive (AR) forms are being identified, which are caused by defects in genes involved in collagen metabolism, bone mineralization, or osteoblast differentiation. Bi-allelic mutations in WNT1 have been associated with a rare form of AR OI, characterized by severe osteoporosis, vertebral compression, scoliosis, fractures, short stature, and variable neurological problems. Heterozygous WNT1 mutations have been linked to autosomal dominant early-onset osteoporosis. In this study, we describe the clinical and molecular findings in 10 new patients with AR WNT1-related OI. Thorough revision of the clinical symptoms of these 10 novel patients and previously published AR WNT1 OI cases highlight ptosis as a unique hallmark in the diagnosis of this OI subtype.

International audience; Proteoglycans are among the most abundant and structurally complex biomacromolecules and play critical roles in connective tissues. They are composed of a core protein onto which glycosaminoglycan (GAG) side chains are attached via a linker region. Biallelic mutations in B3GALT6, encoding one of the linker region glycosyltransferases, are known to cause either spondyloepimetaphyseal dysplasia (SEMD) or a severe pleiotropic form of Ehlers-Danlos syndromes (EDS). This study provides clinical, molecular and biochemical data on 12 patients with biallelic B3GALT6 mutations. Notably, all patients have features of both EDS and SEMD. In addition, some patients have severe and potential life-threatening complications such as aortic dilatation and aneurysm, cervical spine instability and respiratory insufficiency. Whole-exome sequencing, next generation panel sequencing and direct sequencing identified biallelic B3GALT6 mutations in all patients. We show that these mutations reduce the amount of β3GalT6 protein and lead to a complete loss of galactosyltransferase activity. In turn, this leads to deficient GAG synthesis, and ultrastructural abnormalities in collagen fibril organization. In conclusion, this study redefines the phenotype associated with B3GALT6 mutations on the basis of clinical, molecular and biochemical data in 12 patients, and provides an in-depth assessment of β3GalT6 activity and GAG synthesis to better understand this rare condition.

Significance Type I collagenopathies are a heterogenous group of connective tissue disorders, caused by genetic defects in type I collagen. Inherent to these disorders is a large clinical variability, of which the underlying molecular basis remains undefined. By systematically analyzing skeletal phenotypes in a large set of type I collagen zebrafish mutants, we show that zebrafish models are able to both genocopy and phenocopy different forms of human type I collagenopathies, arguing for a similar pathogenetic basis. This study illustrates the future potential of zebrafish as a tool to further dissect the molecular basis of phenotypic variability in human type I collagenopathies, to improve diagnostic strategies as well as promote the discovery of new targetable pathways for pharmacological intervention of these disorders., The type I collagenopathies are a group of heterogeneous connective tissue disorders, that are caused by mutations in the genes encoding type I collagen and include specific forms of osteogenesis imperfecta (OI) and the Ehlers–Danlos syndrome (EDS). These disorders present with a broad disease spectrum and large clinical variability of which the underlying genetic basis is still poorly understood. In this study, we systematically analyzed skeletal phenotypes in a large set of zebrafish, with diverse mutations in the genes encoding type I collagen, representing different genetic forms of human OI, and a zebrafish model resembling human EDS, which harbors a number of soft connective tissues defects, typical of EDS. Furthermore, we provide insight into how zebrafish and human type I collagen are compositionally and functionally related, which is relevant in the interpretation of human type I collagen-related disease models. Our studies reveal a high degree of intergenotype variability in phenotypic expressivity that closely correlates with associated OI severity. Furthermore, we demonstrate the potential for select mutations to give rise to phenotypic variability, mirroring the clinical variability associated with human disease pathology. Therefore, our work suggests the future potential for zebrafish to aid in identifying unknown genetic modifiers and mechanisms underlying the phenotypic variability in OI and related disorders. This will improve diagnostic strategies and enable the discovery of new targetable pathways for pharmacological intervention.

The evolutionarily conserved transmembrane anterior posterior transformation 1 protein, encoded by TAPT1, is involved in murine axial skeletal patterning, but its cellular function remains unknown. Our study demonstrates that TAPT1 mutations underlie a complex congenital syndrome, showing clinical overlap between lethal skeletal dysplasias and ciliopathies. This syndrome is characterized by fetal lethality, severe hypomineralization of the entire skeleton and intra-uterine fractures, and multiple congenital developmental anomalies affecting the brain, lungs, and kidneys. We establish that wild-type TAPT1 localizes to the centrosome and/or ciliary basal body, whereas defective TAPT1 mislocalizes to the cytoplasm and disrupts Golgi morphology and trafficking and normal primary cilium formation. Knockdown of tapt1b in zebrafish induces severe craniofacial cartilage malformations and delayed ossification, which is shown to be associated with aberrant differentiation of cranial neural crest cells.

Whereas the vast majority of osteogenesis imperfecta (OI) is caused by autosomal dominant defects in the genes encoding type I procollagen, mutations in a myriad of genes affecting type I procollagen biosynthesis or bone formation and homeostasis have now been associated with rare autosomal recessive OI forms. Recently, homozygous or compound heterozygous mutations in BMP1, encoding the metalloproteases bone morphogenetic protein-1 (BMP1) and its longer isoform mammalian Tolloid (mTLD), were identified in 5 children with a severe autosomal recessive form of OI and in 4 individuals with mild to moderate bone fragility. BMP1/mTLD functions as the procollagen carboxy-(C)-proteinase for types I to III procollagen but was also suggested to participate in amino-(N)-propeptide cleavage of types V and XI procollagens and in proteolytic trimming of other extracellular matrix (ECM) substrates. We report the phenotypic characteristics and natural history of 4 adults with severe, progressive OI characterized by numerous fractures, short stature with rhizomelic shortening, and deformity of the limbs and variable kyphoscoliosis, in whom we identified novel biallelic missense and frameshift mutations in BMP1. We show that BMP1/mTLD-deficiency in humans not only results in delayed cleavage of the type I procollagen C-propeptide but also hampers the processing of the small leucine-rich proteoglycan prodecorin, a regulator of collagen fibrillogenesis. Immunofluorescent staining of types I and V collagen and transmission electron microscopy of the dermis show impaired assembly of heterotypic type I/V collagen fibrils in the ECM. Our study thus highlights the severe and progressive nature of BMP1-associated OI in adults and broadens insights into the functional consequences of BMP1/mTLD-deficiency on ECM organization.

Contains fulltext : 169752.pdf (Publisher’s version ) (Closed access) Defects of the V-type proton (H+) ATPase (V-ATPase) impair acidification and intracellular trafficking of membrane-enclosed compartments, including secretory granules, endosomes, and lysosomes. Whole-exome sequencing in five families affected by mild to severe cutis laxa, dysmorphic facial features, and cardiopulmonary involvement identified biallelic missense mutations in ATP6V1E1 and ATP6V1A, which encode the E1 and A subunits, respectively, of the V1 domain of the heteromultimeric V-ATPase complex. Structural modeling indicated that all substitutions affect critical residues and inter- or intrasubunit interactions. Furthermore, complexome profiling, a method combining blue-native gel electrophoresis and liquid chromatography tandem mass spectrometry, showed that they disturb either the assembly or the stability of the V-ATPase complex. Protein glycosylation was variably affected. Abnormal vesicular trafficking was evidenced by delayed retrograde transport after brefeldin A treatment and abnormal swelling and fragmentation of the Golgi apparatus. In addition to showing reduced and fragmented elastic fibers, the histopathological hallmark of cutis laxa, transmission electron microscopy of the dermis also showed pronounced changes in the structure and organization of the collagen fibers. Our findings expand the clinical and molecular spectrum of metabolic cutis laxa syndromes and further link defective extracellular matrix assembly to faulty protein processing and cellular trafficking caused by genetic defects in the V-ATPase complex.