Clinical heterogeneity in retinitis pigmentosa caused by variants in RP1 and RLBP1 in five extended consanguineous pedigrees.

Purpose: The aim of this study is to identify disease-causing variants in five consanguineous Jordanian families with a history of autosomal recessive retinitis pigmentosa (RP), and to investigate the clinical variability across the affected individuals. Methods: Exome sequencing (ES) and ophthalmic examinations were performed to classify the underlying RP-causative variants and their pathogenic consequences. The candidate variants in the affected and unaffected family members underwent segregation analyses with Sanger sequencing. Results: We described four variants in the RP1 and RLBP1 genes as disease-causing across the five families, including novel (c.398delC; p.Pro133GlnfsTer126) and recurrent (c.79delA; p.Thr27ProfsTer26) variants in RLBP1 and two previously reported variants in RP1 ((c.1126C>T; p.Arg376Ter) and (c.607G>A; p.Gly203Arg)). The consequent clinical manifestations were thoroughly investigated using a battery of ophthalmic tests, including electroretinography (ERG), optical coherence tomography (OCT), visual acuity (VA), and fundus examination. The phenotypes indicated clinical heterogeneity, typical RP for variants in RP1, and retinitis punctata albescens (RPA) for variants in RLBP1. Conclusions: This study extends the pathogenic variant spectrum for the RP1 and RLBP1 genes. The study also revealed the consequent clinical progression, severity, and presentation of RP. Furthermore, we confirm that ES is an efficient molecular diagnostic approach for RP.

Three-dimensional cultures of gingival fibroblasts on fibrin-based scaffolds for gingival augmentation: A proof-of-concept study.

OBJECTIVE: Gingival tissue regeneration is associated with several challenges. Tissue engineering regenerates the different components of the tissues, providing three major elements: living cells, appropriate scaffolds, and tissue-inducing substances. This study aimed to regenerate the gingival connective tissue in vitro, using human gingival fibroblasts cultured in three-dimensional fibrin gel scaffolds. DESIGN: Human gingival fibroblasts were seeded in a novel three-dimensional fibrin gel scaffold and maintained in two media types: platelet lysate media (control) and collagen-stimulating media (test). Cellular viability and proliferation were assessed, and the production of collagen and other extracellular matrix components in these constructs was investigated and compared. RESULTS: Human gingival fibroblasts cultured in three-dimensional cultures were metabolically active and proliferated in both media. Furthermore, histologic sections, scanning electron microscopy, and quantitative polymerase chain reaction confirmed the production of higher levels of collagen and other extracellular matrix fibers in three-dimensional constructs cultured in collagen-stimulating media. CONCLUSIONS: Culturing human gingival fibroblasts in a novel three-dimensional fibrin gel scaffold containing collagen-stimulating media resulted in a tissue-equivalent construct that mimics human gingival connective tissue. The impact of these results should be considered for further investigations, which may help to develop a compatible scaffold for gingival soft tissue regeneration and treatment of mucogingival deformities.

Generation of a human induced pluripotent stem cell (iPSC) line (JUCTCi019-A) from a patient with Charcot-Marie-Tooth disease type 2A2 (CMT2A2) due to a heterozygous missense substitution c.2119C > T (p.Arg707Trp) in MFN2 gene.

Charcot-Marie-Tooth disease (CMT) is an inherited neurological disorder characterized by the progressive damage of the peripheral nerves. We generated a human induced pluripotent stem cell (iPSC) line JUCTCi019-A using dermal fibroblasts-derived from a 50-year-old CMT2A2 patient carrying a heterozygous missense substitution c.2119C > T (p.Arg707Trp) in the MFN2 gene. Fibroblasts were reprogrammed by Sendai viruses encoding for the reprogramming factors: OCT4, SOX2, KLF4 and c-MYC. Characterization showed normal iPSC morphology and karyotype, expression of pluripotency markers and differentiation into three-germ layers. This iPSC line represents an ideal source for disease modelling and drug development of CMT2A2 disease.

The utility of whole-exome sequencing in accurate diagnosis of neuromuscular disorders in consanguineous families in Jordan.

BACKGROUND: Neuromuscular disorders (NMDs) encompass a large group of genetic and acquired diseases affecting muscles, leading to progressive muscular weakness. These disorders are frequently inherited in an autosomal-recessive (AR) pattern with extreme heterogeneity and various clinical presentations. Consanguinity increases the likelihood of AR disorders, with high rates of cousin inbreeding in Jordan and other Arab countries. In Jordan, the implementation of genetic diagnosis is limited, with delayed or misdiagnosis of genetic disorders. Thus, the lack of genetic counselling and specialized treatment options is frequently encountered in the country. METHODS: Whole-exome sequencing (WES) was conducted for eleven probands from ten Jordanian families who have been formerly diagnosed with limb-girdle dystrophy (LGMD) and Charcot-Marie-Tooth disease (CMT). The previous diagnoses were established principally on clinical examination in the absence of genetic testing. Additionally, Sanger sequencing and segregation analysis were used to validate the resulted pathogenic variants. RESULTS: Multiple variants were identified using WES: For DYSF gene, a missense variant (c. 4076 T > C, p.Leu1359Pro) in exon 38; a nonsense variant (c. 4321C > T, p.Gln1441Ter) in exon 39; a single-nucleotide deletion (c. 5711delG, p.Gly1904AlafsTer101) in exon 51. Other variants included a missense variant (c. 122G > A, p.Arg41Gln) in exon 3 of MPV17 gene, a single-nucleotide deletion (c. 859 delC, p.Lue287Ser fs14*) in exon 6 of SGCB gene, a missense variant (c. 311G > A, p.Gly104Asp) in exon 2 of SLC25A46 gene, a nonsense variant (c. 496C > T, p.Arg166Ter) in exon 5 of SGCG gene, and a nonsense variant (c.3202C > T, p.Gln1068Ter) in exon 13 of SH3TC2 gene. CONCLUSION: Utilization of WES is helpful to facilitate rapid and accurate NMDs diagnosis, complementing a thorough clinical evaluation. This approach can be invaluable to aid in the identification of genetic risks among consanguineous couples. Subsequently, well-informed genetic counselling and potential individualized treatment can be provided.

Unique Variant Spectrum in a Jordanian Cohort with Inherited Retinal Dystrophies.

Whole Exome Sequencing (WES) is a powerful approach for detecting sequence variations in the human genome. The aim of this study was to investigate the genetic defects in Jordanian patients with inherited retinal dystrophies (IRDs) using WES. WES was performed on proband patients' DNA samples from 55 Jordanian families. Sanger sequencing was used for validation and segregation analysis of the detected, potential disease-causing variants (DCVs). Thirty-five putatively causative variants (6 novel and 29 known) in 21 IRD-associated genes were identified in 71% of probands (39 of the 55 families). Three families showed phenotypes different from the typically reported clinical findings associated with the causative genes. To our knowledge, this is the largest genetic analysis of IRDs in the Jordanian population to date. Our study also confirms that WES is a powerful tool for the molecular diagnosis of IRDs in large patient cohorts.

Generation of an induced pluripotent stem cell (iPSC) line (JUCTCi017-A) from a patient with limb-girdle muscular dystrophy (LGMD) due to a homozygous p.Lue287Ser fs14* mutation in the SGCB gene.

Limb-girdle muscular dystrophies (LGMDs) are a large group of heterogenous genetic diseases characterized by muscle weakness. In this study, an induced pluripotent stem cell (iPSC) line was generated from LGMD patient's skin dermal fibroblasts, carrying a homozygous mutation in the Sarcoglycan Beta (SGCB) gene; chr4:52890221, c. 859 delC, p.Lue 287Ser fs14*. The reprogramming process was carried out using Sendai viruses encoding for Yamanaka factors. The resulting iPSCs showed normal morphology and karyotype, expressed pluripotency markers, demonstrated the potential to differentiate in vitro into three germ layers and retained the disease-causing SGCB mutation. This iPSC line represents an ideal source of cells for the investigation of LGMD disease mechanisms.

Generation of a human induced pluripotent stem cell (iPSC) line (JUCTCi011-A) from skin fibroblasts of a healthy Jordanian male subject.

Human induced pluripotent stem cell line (JUCTCi011-A) was generated from skin fibroblasts obtained from a 34-year-old healthy male subject from Jordan. The generated iPSCs showed typical embryonic-like characteristics. They retained their normal karyotype similar to their parental dermal fibroblast cells, expressed pluripotency markers and showed a differentiation potential into three germ layers as demonstrated by immunostaining and flow cytometry. This generated cell line can be used in disease modeling studies, to serve as a healthy control line and to help in developing novel therapeutic strategies for patients with hereditary neuromuscular diseases.

Establishment of (JUCTCi007-A) iPSC line from a patient with congenital myasthenic syndrome (CMS) carrying a homozygous mutation p.Arg331Trp (c.991C > T) in the CHRNE gene.

Induced pluripotent stem cells (iPSCs) were generated from skin fibroblasts obtained from a 24-year-old female diagnosed with hereditary congenital myasthenic syndrome (CMS), caused by p.Arg331Trp (c.991C > T) homozygous mutation in the gene coding for the epsilon subunit of the acetylcholine receptor (CHRNE). The generated iPSCs shared similar karyotype with the parental dermal fibroblast cells, expressed pluripotency stem cell markers, and demonstrated differentiation potential into the three germ layers. This cell line can be used as an ideal model to facilitate the understanding of the pathogenic disease mechanisms underlying the congenital myasthenic syndrome and for developing novel therapeutic strategies.

Derivation of three human induced pluripotent stem cell lines (JUCTCi014-A, JUCTCi015-A, JUCTCi016-A) from mesenchymal stem cells (MSCs) derived from bone marrow, adipose tissue and Wharton's jelly samples.

Mesenchymal stem cells (MSCs) are recognized as a valuable source of cells in clinical treatment and tissue engineering applications. In this study, we created human induced pluripotent stem cells (hiPSCs) from different MSC sources to evaluate the capacity of MSC-derived iPSCs to differentiate into any cell type of the human body and to serve as an alternative source for iPSC generation. Here in, the generated hiPSC lines retained their normal karyotype and showed similar STR-based identities to the parental cells. Reprogrammed cells also showed positive expression of the pluripotency markers and the ability to differentiate into the three germ layers.

Establishment of a human induced pluripotent stem cell (iPSC) line (JUCTCi010-A) from a healthy Jordanian female skin dermal fibroblasts.

Human integration-free induced pluripotent pluripotent stem cells (hiPSCs) were generated from skin fibroblasts obtained from a 27-year-old healthy Jordanian female. The resulting iPSCs expressed the most common pluripotency stem cell markers, they retained the normal karyotype similar to the original fibroblasts and showed the potential to differentiate into three germ layers in vitro. This iPSC line could serve as a wild-type control that can be used in hereditary disease modeling studies and in optimization of different differentiation protocols.

Establishment of a human induced pluripotent stem cell line, JUCTCi012-A, from multiple symmetric lipomatosis (MSL) patient carrying a homozygous Arg707Trp (c.2119C > T) mutation in the MFN2 gene.

Induced pluripotent stem cells (iPSCs) were generated from skin fibroblasts collected from a 39-year-old multiple symmetric lipomatosis (MLS) female patient carrying a point mutation in MFN2 gene (c.2119C > T). The resulting iPSCs showed typical embryonic-like morphology, expressed pluripotency stem cell markers, retained the normal karyotype after reprogramming and showed the potential to differentiate into three germ layers. This iPSC line can be used for studying MSL disease mechanisms.

Generation and characterization of induced pluripotent stem cell (iPSC) line (JUCTCi002-A) from a patient with ataxia with oculomotor apraxia type 1 (AOA1) harboring a homozygous mutation in the APTX gene.

Ataxia with Oculomotor Apraxia Type 1 (AOA1) is an autosomal-recessive cerebellar ataxia characterized by early-onset cerebellar atrophy and axonal sensorimotor polyneuropathy. AOA1 is related to mutations in the aprataxin (APTX) gene encoding for the aprataxin protein. The aprataxin protein has been reported to be involved in DNA single-strand break repair (SSBR) machinery and it localizes to the mitochondria to preserve the mitochondrial function. Here, we demonstrate the generation of induced pluripotent stem cell (iPSC) line (JUCTCi002-A) from AOA1 patient's skin dermal fibroblasts. The selected line showed normal karyotype, expression of pluripotency markers and the ability to differentiatie in vitro into the three germ layers.

Extending the spectrum of CLRN1- and ABCA4-associated inherited retinal dystrophies caused by novel and recurrent variants using exome sequencing.

BACKGROUND: Inherited retinal dystrophies (IRDs) are characterized by extreme genetic and clinical heterogeneity. There are many genes that are known to cause IRD which makes the identification of the underlying genetic causes quite challenging. And in view of the emergence of therapeutic options, it is essential to combine molecular and clinical data to correctly diagnose IRD patients. In this study, we aimed to identify the disease-causing variants (DCVs) in four consanguineous Jordanian families with IRDs and describe genotype-phenotype correlations. METHODS: Exome sequencing (ES) was employed on the proband patients of each family, followed by segregation analysis of candidate variants in affected and unaffected family members by Sanger sequencing. Simulation analysis was done on one novel CLRN1 variant to characterize its effect on mRNA processing. Clinical evaluation included history, slit-lamp biomicroscopy, and indirect ophthalmoscopy. RESULTS: We identified two novel variants in CLRN1 [(c.433+1G>A) and (c.323T>C, p.Leu108Pro)], and two recurrent variants in ABCA4 [(c.1648G>A, p.Gly550Arg) and (c.5460+1G>A)]. Two families with the same DCV were found to have different phenotypes and another family was shown to have sector RP. Moreover, simulation analysis for the CLRN1 splice donor variant (c.433+1G>A) showed that the variant might affect mRNA processing resulting in the formation of an abnormal receptor. Also, a family that was previously diagnosed with nonsyndromic RP was found to have Usher syndrome based on their genetic assessment and audiometry. CONCLUSION: Our findings extend the spectrum of CLRN1- and ABCA4-associated IRDs and describe new phenotypes for these genes. We also highlighted the importance of combining molecular and clinical data to correctly diagnose IRDs and the utility of simulation analysis to predict the effect of splice donor variants on protein formation and function.

The effect of platelet lysate in culture of PDLSCs: an in vitro comparative study.

BACKGROUND: Cellular therapy clinical applications require large-scale production of stem cells. Therefore, abundance, ease of isolation, and proliferative potential are the most important factors in choosing the appropriate source of cells for transplantation studies. Multipotent stem cells obtained from periodontal ligament (PDL) can be used in periodontal tissue regeneration. In this study, we aimed to evaluate and compare the characteristics of periodontal ligament stem cells (PDLSCs), extracted by either enzymatic digestion or explant methods, and expanded using two different serum types: fetal bovine serum (FBS) and xeno-free platelet lysate (PL). METHODS: Expanded PDLSCs were assessed for their proliferation capacity, surface markers expression, colony formation, differentiation potential and ability to self-renewal. Most importantly, PDLSCs were evaluated for their ability to produce osteoblasts in vitro. RESULTS: PDLSCs isolated by explant method and expanded in PL serve as a promising source of stem cells for osteoblasts regeneration. These cells showed higher proliferation capacity, they retained their stemness characteristics throughout the passages and they revealed an increase in the expression level of osteogenic markers, without showing any karyotypic abnormalities after cell expansion. CONCLUSIONS: PDLSCs produced using explant extraction method and expanded in cell culture media supplemented with PL provide an excellent source of xeno-free cells for the generation of functional osteoblasts.

Novel CERKL variant in consanguineous Jordanian pedigrees with inherited retinal dystrophies.

OBJECTIVE: To identify the disease-causing variants in 2 families with autosomal recessive inherited retinal dystrophies (IRDs) and to characterize phenotypic variability across the affected family members. DESIGN: Exome sequencing and ophthalmic clinical examination study. PARTICIPANTS: Six members from 2 consanguineous Jordanian families with IRD. METHODS: Ophthalmic examinations and whole-exome sequencing (WES) were performed to identify IRD-causing variants in affected individuals from each family, followed by segregation analysis of candidate variants in affected and unaffected family members by Sanger sequencing. RESULTS: We identified 2 different homozygous deletion variants in CERKL in each family: a novel pathogenic variant, c.450_451delAT, and a known variant, c.1187_1188delTG. Both variants co-segregated with the disease in all affected family members. The resulting phenotypes further supported that CERKL is associated with cone-rod dystrophy (CRD) rather than retinitis pigmentosa (RP), as originally established. CONCLUSION: Our study expands the genotypic spectra of CERKL variants, providing insights into the relevant pathogenesis of RP/CRD. We also confirm that the WES approach is a valuable tool for the molecular diagnosis of retinopathies.