Despite the adenoids are regularly removed in patients with mucopolysaccharidoses (MPS), the underlying tissue and cellular pathologies remain understudied. We characterized an (immuno)histopathologic and ultrastructural phenotype dominated by lysosomal storage changes in a specific subset of adenotonsillar paracortical cells in 8 MPS patients (3 MPS I, 3 MPS II, and 2 MPS IIIA). These abnormal cells were effectively detected by an antibody targeting the lysosomal membrane tetraspanin CD63. Important, CD63+ storage vacuoles in these cells lacked the monocytes/macrophages lysosomal marker CD68. Such a distinct patterning of CD63 and CD68 was not present in a patient with infantile neurovisceral variant of acid sphingomyelinase deficiency. The CD63+ storage pathology was absent in two MPS I patients who either received enzyme-replacement therapy or underwent hematopoietic stem cells transplantation prior the adenoidectomy. Our study demonstrates novel features of lysosomal storage patterning and suggests diagnostic utility of CD63 detection in adenotonsillar lymphoid tissue of MPS patients.

• Keywords
• Adenoidectomy, CD63, CD68, Enzyme-replacement therapy, Hematopoietic stem cells transplantation, Mucopolysaccharidoses,
• MeSH
• Tetraspanin 30 MeSH
• Enzyme Replacement Therapy MeSH
• Humans MeSH
• Lymphoid Tissue pathology   MeSH
• Lysosomes MeSH
• Mucopolysaccharidoses * diagnosis   drug therapy   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Tetraspanin 30 MeSH
• CD63 protein, human MeSH Browser

PURPOSE: To report molecular genetic findings in six probands with congenital hereditary endothelial dystrophy (CHED) variably associated with hearing loss (also known as Harboyan syndrome). Furthermore, we developed a cellular model to determine if disease-associated variants induce aberrant SLC4A11 pre-mRNA splicing. METHODS: Direct sequencing of the entire SLC4A11 coding region was performed in five probands. In one individual, whole genome sequencing was undertaken. The effect of c.2240+5G>A on pre-mRNA splicing was evaluated in a corneal endothelial-like (CE-like) cell model expressing SLC4A11. CE-like cells were derived from autologous induced pluripotent stem cells (iPSCs) via neural crest cells exposed to B27, PDGF-BB, and DKK-2. Total RNA was extracted, and RT-PCR was performed followed by Sanger and a targeted next generation sequencing (NGS) approach to identify and quantify the relative abundance of alternatively spliced transcripts. RESULTS: In total, 11 different mutations in SLC4A11 evaluated as pathogenic were identified; of these, c.1237G>A, c.2003T>C, c.1216+1G>A, and c.2240+5G>A were novel. The c.2240+5G>A variant was demonstrated to result in aberrant pre-mRNA splicing. A targeted NGS approach confirmed that the variant introduces a leaky cryptic splice donor site leading to the production of a transcript containing an insertion of six base pairs with the subsequent introduction of a premature stop codon (p.Thr747*). Furthermore, a subset of transcripts comprising full retention of intron 16 also were observed, leading to the same functionally null allele. CONCLUSIONS: This proof-of-concept study highlights the potential of using CE-like cells to investigate the pathogenic consequences of SLC4A11 disease-associated variants.

• MeSH
• Antiporters biosynthesis   genetics   MeSH
• Cell Differentiation MeSH
• Corneal Dystrophies, Hereditary genetics   metabolism   pathology   MeSH
• Child MeSH
• Adult MeSH
• Induced Pluripotent Stem Cells cytology   metabolism   MeSH
• Cells, Cultured MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Hearing Loss, Sensorineural genetics   metabolism   pathology   MeSH
• Child, Preschool MeSH
• RNA Precursors MeSH
• Anion Transport Proteins biosynthesis   genetics   MeSH
• Gene Expression Regulation * MeSH
• RNA genetics   MeSH
• Pedigree MeSH
• Endothelium, Corneal metabolism   pathology   MeSH
• Aged MeSH
• RNA Splicing MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Child, Preschool MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antiporters MeSH
• RNA Precursors MeSH
• Anion Transport Proteins MeSH
• RNA MeSH
• SLC4A11 protein, human MeSH Browser

The human amniotic membrane (HAM) is widely used for its wound healing effect in clinical practice, as a feeder for the cell cultivation, or a source of cells to be used in cell therapy. The aim of this study was to find effective and safe enzymatic HAM de-epithelialization method leading to harvesting of both denuded undamaged HAM and viable human amniotic epithelial cells (hAECs). The efficiency of de-epithelialization using TrypLE Express, trypsin/ ethylenediaminetetraacetic (EDTA), and thermolysin was monitored by hematoxylin and eosin staining and by the measurement of DNA concentration. The cell viability was determined by trypan blue staining. Scanning electron microscopy and immunodetection of collagen type IV and laminin α5 chain were used to check the basement membrane integrity. De-epithelialized hAECs were cultured and their stemness properties and proliferation potential was assessed after each passage. The HAM was successfully de-epithelialized using all three types of reagents, but morphological changes in basement membrane and stroma were observed after the thermolysin application. About 60% of cells remained viable using trypsin/EDTA, approximately 6% using TrypLE Express, and all cells were lethally damaged after thermolysin application. The hAECs isolated using trypsin/EDTA were successfully cultured up to the 5th passage with increasing proliferation potential and decreased stem cell markers expression (NANOG, SOX2) in prolonged cell culture. Trypsin/EDTA technique was the most efficient for obtaining both undamaged denuded HAM and viable hAECs for consequent culture.

• MeSH
• Amnion cytology   metabolism   pathology   MeSH
• DNA analysis   isolation & purification   MeSH
• Edetic Acid chemistry   MeSH
• Epithelial Cells cytology   metabolism   pathology   MeSH
• Collagen Type IV metabolism   MeSH
• Cells, Cultured MeSH
• Laminin metabolism   MeSH
• Humans MeSH
• Microscopy, Electron, Scanning MeSH
• Nanog Homeobox Protein metabolism   MeSH
• Cell Proliferation MeSH
• Re-Epithelialization MeSH
• SOXB1 Transcription Factors metabolism   MeSH
• Trypsin metabolism   MeSH
• Cell Survival MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• Edetic Acid MeSH
• Collagen Type IV MeSH
• laminin alpha5 MeSH Browser
• Laminin MeSH
• Nanog Homeobox Protein MeSH
• SOXB1 Transcription Factors MeSH
• Trypsin MeSH