Pathogenic TNNI1 variants disrupt sarcomere contractility resulting in hypo- and hypercontractile muscle disease.

Troponin I (TnI) regulates thin filament activation and muscle contraction. Two isoforms, TnI-fast (TNNI2) and TnI-slow (TNNI1), are predominantly expressed in fast- and slow-twitch myofibers, respectively. TNNI2 variants are a rare cause of arthrogryposis, whereas TNNI1 variants have not been conclusively established to cause skeletal myopathy. We identified recessive loss-of-function TNNI1 variants as well as dominant gain-of-function TNNI1 variants as a cause of muscle disease, each with distinct physiological consequences and disease mechanisms. We identified three families with biallelic TNNI1 variants (F1: p.R14H/c.190-9G>A, F2 and F3: homozygous p.R14C), resulting in loss of function, manifesting with early-onset progressive muscle weakness and rod formation on histology. We also identified two families with a dominantly acting heterozygous TNNI1 variant (F4: p.R174Q and F5: p.K176del), resulting in gain of function, manifesting with muscle cramping, myalgias, and rod formation in F5. In zebrafish, TnI proteins with either of the missense variants (p.R14H; p.R174Q) incorporated into thin filaments. Molecular dynamics simulations suggested that the loss-of-function p.R14H variant decouples TnI from TnC, which was supported by functional studies showing a reduced force response of sarcomeres to submaximal [Ca2+] in patient myofibers. This contractile deficit could be reversed by a slow skeletal muscle troponin activator. In contrast, patient myofibers with the gain-of-function p.R174Q variant showed an increased force to submaximal [Ca2+], which was reversed by the small-molecule drug mavacamten. Our findings demonstrated that TNNI1 variants can cause muscle disease with variant-specific pathomechanisms, manifesting as either a hypo- or a hypercontractile phenotype, suggesting rational therapeutic strategies for each mechanism.

A Novel Truncating Mutation in PAX1 Gene Causes Otofaciocervical Syndrome Without Immunodeficiency.

Otofaciocervical syndrome (OTFCS) is a rare genetic disorder of both autosomal recessive and autosomal dominant patterns of inheritance. It is caused by biallelic or monoallelic mutations in PAX1 or EYA1 genes, respectively. Here, we report an OTFCS2 female patient of 1st consanguineous healthy parents. She manifested facial dysmorphism, hearing loss, intellectual disability (ID), and delayed language development (DLD) as the main clinical phenotype. The novel homozygous variant c.1212dup (p.Gly405Argfs*51) in the PAX1 gene was identified by whole exome sequencing (WES), and family segregation confirmed the heterozygous status of the mutation in the parents using the Sanger sequencing. The study recorded a novel PAX1 variant representing the sixth report of OTFCS2 worldwide and the first Egyptian study expanding the geographic area where the disorder was confined.

Clinical and molecular study of Egyptian patients with Treacher Collins syndrome.

Treacher Collins syndrome (TCS) is a rare disorder of craniofacial development following different patterns of inheritance. To date, mutations in four genes ( TCOF1, POLR1D, POLR1C , and POLR1B ) have been found to cause the condition. The molecular defect remains unidentified in a significant proportion of patients. In the current study, whole exome sequencing including analysis of copy number variants was applied for genetic testing of eight Egyptian patients with typical TCS phenotype, representing the first molecular analysis of TCS patients in Egypt as well as in Arab countries. Five heterozygous frameshift mutations were reported, including four variants in the TCOF1 gene (c.3676_3694delinsCTCTGG, c.3984_3985delGA, c.4366_4369delGAAA, and c.3388delC) and one variant in the POLR1D gene (c.60dupA). Four variants were novel extending the disease mutation spectrum. In three affected individuals, no variants of interest were identified in genes associated with TCS or clinically overlapping conditions. Additionally, no relevant variant was detected in genes encoding other subunits of RNA polymerase (pol) I. Molecular analysis is important to provide accurate genetic counseling. It would also contribute to reduced disease incidence. Further studies should be designed to investigate other possible etiologies when no pathogenic variants were revealed in either of the known genes.

Clinical and molecular spectrum of a large Egyptian cohort with ALS2-related disorders of infantile-onset of clinical continuum IAHSP/JPLS.

This study presents 46 patients from 23 unrelated Egyptian families with ALS2-related disorders without evidence of lower motor neuron involvement. Age at onset ranged from 10 months to 2.5 years, featuring progressive upper motor neuron signs. Detailed clinical phenotypes demonstrated inter- and intrafamilial variability. We identified 16 homozygous disease-causing ALS2 variants; sorted as splice-site, missense, frameshift, nonsense and in-frame in eight, seven, four, three, and one families, respectively. Seven of these variants were novel, expanding the mutational spectrum of the ALS2 gene. As expected, clinical severity was positively correlated with disease onset (p = 0.004). This work provides clinical and molecular profiles of a large single ethnic cohort of patients with ALS2 mutations, and suggests that infantile ascending hereditary spastic paralysis (IAHSP) and juvenile primary lateral sclerosis (JPLS) are belonged to one entity with no phenotype-genotype correlation.

Variable predicted pathogenic mechanisms for novel MECP2 variants in RTT patients.

BACKGROUND: Methyl CpG binding protein 2 (MeCP2) is essential for the normal function of mature neurons. Mutations in the MECP2 gene are the main cause of Rett syndrome (RTT). Gene mutations have been identified throughout the gene and the mutation effect is mainly correlated with its type and location. METHODS: In this study, a series of in silico algorithms were applied for analyzing the functional consequences of 3 novel gene missense mutations (D121A, S359Y, and P403S) and a rarely reported one with suspicious effect (R133H) on RettBASE. Besides, a ROC curve analysis was performed to investigate the critical factors affecting variant pathogenicity. RESULTS: (1) The ROC curve analysis for a retrieved set of MeCP2 variants showed that physicochemical characters do not significantly affect variant pathogenicity; (2) PREM PDI tool revealed that both D121A and R133H mainly contribute to disease progression via reducing MeCP2 affinity to DNA; (3) GPS v5.0 software indicated that P403S may correlate with altered protein phosphorylation; however, no defective protein interaction has been already documented. (4) The applied computational algorithms failed to explore any informative pathogenic mechanism for the S359Y variant. CONCLUSION: The conducted approach might provide an efficient prediction model for the effect of MECP2 variants that are located in MBD and CTD.

Potential Value of miR-23a for Discriminating Neuromyelitis Optica Spectrum Disorder from Multiple Sclerosis.

BACKGROUND: Until now, no laboratory test or test set can guarantee the diagnosis of multiple sclerosis (MS) at early disease stages, and the disease symptoms may interfere with many other disease conditions. Analyzing the expression of circulating miRNAs may provide a useful approach for early and differential MS diagnosis. The main objective is assessment of the potential of serum miR-23a, miR-155, and miR-572 to differentiate between MS and other neuroinflammatory diseases. METHODS: Serum miRNAs were obtained from 37 MS patients and 25 healthy age-matched controls, along with patients with neuromyelitis optica spectrum disorder (NMOSD) [n = 13] and neuropsychiatric systemic lupus erythematosus (NPSLE) [n = 10]. miRNA expression levels were analyzed using real-time polymerase chain reaction (PCR) and pairwise comparisons were made to reveal the diagnostic/distinguishing potential of the analyzed miRNAs. RESULTS: In the study cohort, the three investigated miRNAs failed to display significant dysregulation in MS patients. However, they could significantly discriminate patients with NMOSD and NPSLE [median (IQR): 8.1 (6.1-9.2) and 8.8 (7.9-9.7) for miR-23a, 7.5 (5.3-8.3) and 8.0 (7.5-9.5) for miR-155 and 6.9 (5.0-8.8) and 6.4 (5.3-8.8) for miR-572 in NMOSD and NPSLE, respectively] from healthy subjects [median (IQR): 3.4 (1.5-4.3), 3.1 (1.1-5.6) and 3.5 (1.7-5.6) for miR-23a, miR-155 and miR-572, respectively], with area under the curve (AUC) ≤0.8. Remarkably, miR-23a has been emerging as a prospective biomarker for differentiation of MS from NMOSD as well as NPSLE (AUC<0.9). The miRNA combined use contributed to enhanced diagnostic and discriminatory performance in the study groups. CONCLUSION: Certain miRNA expression levels would contribute to discriminating MS from other neuroinflammatory diseases.

Osteogenic Differentiation Potential of Human Bone Marrow and Amniotic Fluid-Derived Mesenchymal Stem Cells in Vitro & in Vivo.

BACKGROUND: Cell therapies offer a promising potential in promoting bone regeneration. Stem cell therapy presents attractive care modality in treating degenerative conditions or tissue injuries. The rationale behind this is both the expansion potential of stem cells into a large cell population size and its differentiation abilities into a wide variety of tissue types, when given the proper stimuli. A progenitor stem cell is a promising source of cell therapy in regenerative medicine and bone tissue engineering. AIM: This study aimed to compare the osteogenic differentiation and regenerative potentials of human mesenchymal stem cells derived from human bone marrow (hBM-MSCs) or amniotic fluid (hAF-MSCs), both in vitro and in vivo studies. SUBJECTS AND METHODS: Human MSCs, used in this study, were successfully isolated from two human sources; the bone marrow (BM) and amniotic fluid (AF) collected at the gestational ages of second or third trimesters. RESULTS: The stem cells derived from amniotic fluid seemed to be the most promising type of progenitor cells for clinical applications. In a pre-clinical experiment, attempting to explore the therapeutic application of MSCs in bone regeneration, Rat lumbar spines defects were surgically created and treated with undifferentiated and osteogenically differentiated MSCs, derived from BM and second trimester AF. Cells were loaded on gel-foam scaffolds, inserted and fixed in the area of the surgical defect. X-Ray radiography follows up, and histopathological analysis was done three-four months post- operation. The transplantation of AF-MSCs or BM-MSCs into induced bony defects showed promising results. The AF-MSCs are offering a better healing effect increasing the likelihood of achieving successful spinal fusion. Some bone changes were observed in rats transplanted with osteoblasts differentiated cells but not in rats transplanted with undifferentiated MSCs. Longer observational periods are required to evaluate a true bone formation. The findings of this study suggested that the different sources; hBM-MSCs or hAF-MSCs exhibited remarkably different signature regarding the cell morphology, proliferation capacity and osteogenic differentiation potential. CONCLUSIONS: AF-MSCs have a better performance in vivo bone healing than that of BM-MSCs. Hence, AF derived MSCs is highly recommended as an alternative source to BM-MSCs in bone regeneration and spine fusion surgeries. Moreover, the usage of gel-foam as a scaffold proved as an efficient cell carrier that showed bio-compatibility with cells, bio-degradability and osteoinductivity in vivo.

Extracellular miR-145, miR-223 and miR-326 expression signature allow for differential diagnosis of immune-mediated neuroinflammatory diseases.

BACKGROUND: Although misdiagnosis of neuromyelitis optica spectrum disorder (NMOSD) with neuropsychiatric systemic lupus erythematosus (NPSLE) or multiple sclerosis (MS) is not infrequent, reliable biomarkers remains an unmet need. Extracellular microRNAs (miRNAs) represent a worthy avenue to identify biomarkers for differential diagnosis. We aimed to explore the potential role of some selected circulating miRNAs as biomarkers for the differential diagnosis in immune-mediated neuroinflammatory diseases. METHODS: A total of 80 subjects were enrolled in the present study, including 37 patients with MS (relapsing-remitting MS [RRMS; n=18] and secondary progressive MS [SPMS; n=19]), 10 patients with NMOSD and 10 patients with NPSLE as well as 23 healthy subjects. Serum expression levels of three selected miRNAs (miR-145, miR-223 and miR-326) were measured using quantitative real-time polymerase chain reaction (qRT-PCR). Whole blood expression levels of cellular immune response-relevant target genes, including signaling mother against decapentaplegic peptide 3 (SMAD3) and specificity protein 1 (SP1), were also measured using qRT-PCR. RESULTS: In comparison to healthy subjects, only miR-145 and miR-223 were significantly up-regulated in MS patients, whereas, all the analyzed miRNAs revealed insignificant upregulation in NMOSD patients. All the examined miRNAs were significantly down-regulated in NPSLE patients compared to healthy subjects. miR-145, miR-223 and miR-326 expression profile is a promising diagnostic biomarker for MS and NPSLE, but not for NMOSD. This expression profile is capable of differentiating not only among MS, NMOSD and NPSLE, but also between RRMS and SPMS. CONCLUSION: Specific circulating miRNAs expression signature may have the potential to differentially diagnose immune-mediated neuroinflammatory diseases.

The Modulatory Effects of Mesenchymal Stem Cells on Osteoclastogenesis.

The effect of mesenchymal stem cells (MSCs) on bone formation has been extensively demonstrated through several in vitro and in vivo studies. However, few studies addressed the effect of MSCs on osteoclastogenesis and bone resorption. Under physiological conditions, MSCs support osteoclastogenesis through producing the main osteoclastogenic cytokines, RANKL and M-CSF. However, during inflammation, MSCs suppress osteoclast formation and activity, partly via secretion of the key anti-osteoclastogenic factor, osteoprotegerin (OPG). In vitro, co-culture of MSCs with osteoclasts in the presence of high concentrations of osteoclast-inducing factors might reflect the in vivo inflammatory pathology and prompt MSCs to exert an osteoclastogenic suppressive effect. MSCs thus seem to have a dual effect, by stimulating or inhibiting osteoclastogenesis, depending on the inflammatory milieu. This effect of MSCs on osteoclast formation seems to mirror the effect of MSCs on other immune cells, and may be exploited for the therapeutic potential of MSCs in bone loss associated inflammatory diseases.