A Patient with Sjogren's Syndrome and Subsequent Diagnosis of Inclusion Body Myositis and Light-Chain Amyloidosis.

We discuss a challenging case of a 58-year-old Vietnamese-American woman who presented to her new primary care provider with an 8-year history of slowly progressive dysphagia, hoarseness, muscle weakness with associated frequent falls, and weight loss. She eventually reported dry eyes and dry mouth, and she was diagnosed with Sjogren's syndrome. Subsequently, she was additionally diagnosed with inclusion body myositis and gastric light-chain (AL) amyloidosis. Although inclusion body myositis has been previously associated with Sjogren's syndrome, inclusion body myositis is rare in non-Caucasians, and the trio of Sjogren's syndrome, inclusion body myositis, and AL amyloidosis has not been previously reported. Sjogren's syndrome is a systemic autoimmune condition characterized by ocular and oral dryness. It is one of the most common rheumatologic disorders in the USA and worldwide. Early diagnosis of Sjogren's is particularly important given the frequency and variety of associated autoimmune diseases and extraglandular manifestations. Furthermore, although inclusion body myositis has a low prevalence, it is the most common inflammatory myopathy in older adults and is unfortunately associated with long delays in diagnosis, so knowledge of this disorder is also crucial for practicing internists.

Manipulating Cx43 expression triggers gene reprogramming events in dermal fibroblasts from oculodentodigital dysplasia patients.

Oculodentodigital dysplasia (ODDD) is primarily an autosomal dominant disorder linked to over 70 GJA1 gene [connexin43 (Cx43)] mutations. For nearly a decade, our laboratory has been investigating the relationship between Cx43 and ODDD by expressing disease-linked mutants in reference cells, tissue-relevant cell lines, 3D organ cultures and by using genetically modified mouse models of human disease. Although salient features of Cx43 mutants have been revealed, these models do not necessarily reflect the complexity of the human context. To further overcome these limitations, we have acquired dermal fibroblasts from two ODDD-affected individuals harbouring D3N and V216L mutations in Cx43, along with familial controls. Using these ODDD patient dermal fibroblasts, which naturally produce less GJA1 gene product, along with RNAi and RNA activation (RNAa) approaches, we show that manipulating Cx43 expression triggers cellular gene reprogramming. Quantitative RT-PCR, Western blot and immunofluorescent analysis of ODDD patient fibroblasts show unusually high levels of extracellular matrix (ECM)-interacting proteins, including integrin α5ß1, matrix metalloproteinases as well as secreted ECM proteins collagen-I and laminin. Cx43 knockdown in familial control cells produces similar effects on ECM expression, whereas Cx43 transcriptional up-regulation using RNAa decreases production of collagen-I. Interestingly, the enhanced levels of ECM-associated proteins in ODDD V216L fibroblasts is not only a consequence of increased ECM gene expression, but also due to an apparent deficit in collagen-I secretion which may further contribute to impaired collagen gel contraction in ODDD fibroblasts. These findings further illuminate the altered function of Cx43 in ODDD-affected individuals and highlight the impact of manipulating Cx43 expression in human cells.

The G60S Cx43 mutant enhances keratinocyte proliferation and differentiation.

Transient knock-down of the gap junction protein Cx43 by antisense and siRNA, or gap junction block with mimetic peptides, have been shown to enhance epidermal wound healing. However, patients with oculodentodigital dysplasia (ODDD) express mutant Cx43 that leads to a chronic reduction in gap junctional intercellular communication. To determine whether mutant Cx43 in keratinocytes would impact upon the wound healing process, we localized Cx43 in human and mouse skin tissue expressing mutant Cx43 and assessed the ability of primary keratinocytes derived from a mouse model of ODDD to proliferate, migrate and differentiate. In the epidermis from an ODDD patient and in the epidermis of mice expressing the G60S mutant or in keratinocytes obtained from mutant mice, Cx43 was frequently found within intracellular compartments and rarely localized to punctate sites of cell-cell apposition. Primary keratinocytes derived from G60S mutant mice proliferated faster but migrated similarly to keratinocytes derived from wild-type control mice. Keratinocytes derived from mutant mice expressed abundant Cx43 and higher levels of involucrin and loricrin under low calcium conditions. However, after calcium-induced differentiation, similar levels of Cx43, involucrin and loricrin were observed. Thus, we conclude that during wound healing, mutant Cx43 may enhance keratinocyte proliferation and promote early differentiation of keratinocytes.

Human dermal fibroblasts derived from oculodentodigital dysplasia patients suggest that patients may have wound-healing defects.

Oculodentodigital dysplasia (ODDD) is primarily an autosomal dominant human disease caused by any one of over 60 mutations in the GJA1 gene encoding the gap junction protein Cx43. In the present study, wound healing was investigated in a G60S ODDD mutant mouse model and by using dermal fibroblasts isolated from two ODDD patients harboring the p.D3N and p.V216L mutants along with dermal fibroblasts isolated from their respective unaffected relatives. Punch biopsies revealed a delay in wound closure in the G60S mutant mice in comparison to wild-type littermates, and this delay appeared to be due to defects in the dermal fibroblasts. Although both the p.D3N and p.V216L mutants reduced gap junctional intercellular communication in human dermal fibroblasts, immunolocalization studies revealed that Cx43 gap junctions were prevalent at the cell surface of p.D3N expressing fibroblasts but greatly reduced in p.V216L expressing fibroblasts. Mutant expressing fibroblasts were further found to have reduced proliferation and migration capabilities. Finally, in response to TGFß1, mutant expressing fibroblasts expressed significantly less alpha smooth muscle actin suggesting they were inefficient in their ability to differentiate into myofibroblasts. Collectively, our results suggest that ODDD patients may have subclinical defects in wound healing due to impaired function of dermal fibroblasts.

Connexin43 Mutant Patient-Derived Induced Pluripotent Stem Cells Exhibit Altered Differentiation Potential.

We present for the first time the generation of induced pluripotent stem cells (iPSCs) from a patient with a connexin-linked disease. The importance of gap junctional intercellular communication in bone homeostasis is exemplified by the autosomal dominant developmental disorder oculodentodigital dysplasia (ODDD), which is linked to mutations in the GJA1 (Cx43) gene. ODDD is characterized by craniofacial malformations, ophthalmic deficits, enamel hypoplasia, and syndactyly. In addition to harboring a Cx43 p.V216L mutation, ODDD iPSCs exhibit reduced Cx43 mRNA and protein abundance when compared to control iPSCs and display impaired channel function. Osteogenic differentiation involved an early, and dramatic downregulation of Cx43 followed by a slight upregulation during the final stages of differentiation. Interestingly, osteoblast differentiation was delayed in ODDD iPSCs. Moreover, Cx43 subcellular localization was altered during chondrogenic differentiation of ODDD iPSCs compared to controls and this may have contributed to the more compact cartilage pellet morphology found in differentiated ODDD iPSCs. These studies highlight the importance of Cx43 expression and function during osteoblast and chondrocyte differentiation, and establish a potential mechanism for how ODDD-associated Cx43 mutations may have altered cell lineages involved in bone and cartilage development. © 2017 American Society for Bone and Mineral Research.

Specific functional pathologies of Cx43 mutations associated with oculodentodigital dysplasia.

Oculodentodigital dysplasia (ODDD) is a rare genetic disease that affects the development of multiple organs in the human body. More than 70 mutations in the gap junction connexin43 (Cx43) gene, GJA1, are associated with ODDD, most of which are inherited in an autosomal dominant manner. Many patients exhibit similar clinical presentations. However, there is high intrafamilial and interfamilial phenotypic variability. To better understand this variability, we established primary human dermal fibroblast cultures from several ODDD patients and unaffected controls. In the present study, we characterized three fibroblast lines expressing heterozygous p.L7V, p.G138R, and p.G143S Cx43 variants. All ODDD fibroblasts exhibited slower growth, reduced migration, and defective cell polarization, traits common to all ODDD fibroblasts studied so far. However, we found striking differences in overall expression levels, with p.L7V down-regulated at the mRNA and protein level. Although all of the Cx43 variants could traffic to the cell surface, there were stark differences in gap junction plaque formation, gap junctional intercellular communication, Cx43 phosphorylation, and hemichannel activity among Cx43 variants, as well as subtle differences in myofibroblast differentiation. Together these findings enabled us to discover mutation-specific pathologies that may help to predict future clinical outcomes.