Expanding the clinical phenotype of IARS2-related mitochondrial disease.

BACKGROUND: IARS2 encodes a mitochondrial isoleucyl-tRNA synthetase, a highly conserved nuclear-encoded enzyme required for the charging of tRNAs with their cognate amino acid for translation. Recently, pathogenic IARS2 variants have been identified in a number of patients presenting broad clinical phenotypes with autosomal recessive inheritance. These phenotypes range from Leigh and West syndrome to a new syndrome abbreviated CAGSSS that is characterised by cataracts, growth hormone deficiency, sensory neuropathy, sensorineural hearing loss, and skeletal dysplasia, as well as cataract with no additional anomalies. METHODS: Genomic DNA from Iranian probands from two families with consanguineous parental background and overlapping CAGSSS features were subjected to exome sequencing and bioinformatics analysis. RESULTS: Exome sequencing and data analysis revealed a novel homozygous missense variant (c.2625C > T, p.Pro909Ser, NM_018060.3) within a 14.3 Mb run of homozygosity in proband 1 and a novel homozygous missense variant (c.2282A > G, p.His761Arg) residing in an ~ 8 Mb region of homozygosity in a proband of the second family. Patient-derived fibroblasts from proband 1 showed normal respiratory chain enzyme activity, as well as unchanged oxidative phosphorylation protein subunits and IARS2 levels. Homology modelling of the known and novel amino acid residue substitutions in IARS2 provided insight into the possible consequence of these variants on function and structure of the protein. CONCLUSIONS: This study further expands the phenotypic spectrum of IARS2 pathogenic variants to include two patients (patients 2 and 3) with cataract and skeletal dysplasia and no other features of CAGSSS to the possible presentation of the defects in IARS2. Additionally, this study suggests that adult patients with CAGSSS may manifest central adrenal insufficiency and type II esophageal achalasia and proposes that a variable sensorineural hearing loss onset, proportionate short stature, polyneuropathy, and mild dysmorphic features are possible, as seen in patient 1. Our findings support that even though biallelic IARS2 pathogenic variants can result in a distinctive, clinically recognisable phenotype in humans, it can also show a wide range of clinical presentation from severe pediatric neurological disorders of Leigh and West syndrome to both non-syndromic cataract and cataract accompanied by skeletal dysplasia

Isolation and Cultivation of Adult Human Keratinocyte Stem Cells for Regeneration of Epidermal Sheets

Background: Keratinocyte stem cell is one of the adult stem cells that inhabits the skin and contributes to skin function and renewal. Adult stem cells are best defined by their capacity to self-renew, and to maintain tissue function for a long period of time. These findings indicate the importance of these cells for clinical applications including regenerative medicine, tissue engineering and gene therapy. In full-thickness damage or injury including burns, the  cultured epidermal autografts (CEAs) may be placed directly onto muscle or fascia. Methods: A small split thickness skin biopsy (1 12أ—'> 2 cm) was obtained aseptically to isolate stem cells. The biopsy was cut into thin pieces and treated with trypsin at 4° C overnight (cold trypsin method) to obtain a single-cell suspension. The cells were seeded at a density of 3×104 cells/cm2 onto a preformed mitomycine-C treated 3T3 cell as feeder  layer  in DMEM medium supplemented with 10% fetal  bovine serum (FBS) and other special supplements. Clonogenic keratinocytes divided and colonies quickly expanded and pushed away the 3T3 feeder layer cells, which then detached from the culture vessel and eliminated with medium changes. Primary cultures were usually subcultured when the cells were in exponential growth phase .Colonies of keratinocytes were expanded and after 7-10 days fused and formed a coherent stratified epithelium. Confluent  cultured  epithelia were detached enzymatically as coherent sheets from the surface of  the culture flasks and transferred onto petrolatum- impregnated gauze.Histological studies of cultured epithelium were also carried out. Results: In our experience from 1 cm2 of skin sample, 2,5- 4×106 cells were obtained. It resulted in keratinocytes suspensions which consisted at least 90% single cells. Cultured keratinocytes proliferated and after 8-10 days became confluent. The area of cultured epithelium detached from T-25 and T-75 culture flasks was approximately 12-15 cm2 and 35-40 cm2 respectively. Histological studies showed that 10-day old cultured epithelium had 3-4 cell layers consisting of small basal cells and big scquamous cells with large nucleus. Also in the basal layer few melanocytes with melanin pigments in the cells cytoplasm were found.The 20-day old cultured epithelium had 8-10 layers consisting of small and round basal cells, scquamous cells and 2-3 layers of keratinized cells. Conclusion: Culture of keratinocyte stem cells could result in multilayer epithelium that creates a good cosmetic appearance upon transplantation. This could re-generate an epidermis that is resistant to trauma and infections. It can be considered as an appropriate substitution in skin loss conditions.   Keywords: Epidermal Sheets, Skin Adult Stem cells, Keratinocyte