In Silico, In Vitro, and In Vivo Evaluation of Caffeine-Coated Nanoparticles as a Promising Therapeutic Avenue for AML through NF-Kappa B and TRAIL Pathways Modulation.

BACKGROUND: Advancements in nanoscience have led to a profound paradigm shift in the therapeutic applications of medicinally important natural drugs. The goal of this research is to develop a nano-natural product for efficient cancer treatment. METHODS AND RESULTS: For this purpose, mesoporous silica nanoparticles (MSNPs) were formulated, characterized, and loaded with caffeine to develop a targeted drug delivery system, i.e., caffeine-coated nanoparticles (CcNPs). In silico docking studies were conducted to examine the binding efficiency of the CcNPs with different apoptotic targets followed by in vitro and in vivo bioassays in respective animal models. Caffeine, administered both as a free drug and in nanomedicine form, along with doxorubicin, was delivered intravenously to a benzene-induced AML model. The anti-leukemic potential was assessed through hematological profiling, enzymatic biomarker analysis, and RT-PCR examination of genetic alterations in leukemia markers. Docking studies show strong inter-molecular interactions between CcNPs and apoptotic markers. In vitro analysis exhibits statistically significant antioxidant activity, whereas in vivo analysis exhibits normalization of the genetic expression of leukemia biomarkers STMN1 and S1009A, accompanied by the restoration of the hematological and morphological traits of leukemic blood cells in nanomedicine-treated rats. Likewise, a substantial improvement in hepatic and renal biomarkers is also observed. In addition to these findings, the nanomedicine successfully normalizes the elevated expression of GAPDH and mTOR induced by exposure to benzene. Further, the nanomedicine downregulates pro-survival components of the NF-kappa B pathway and upregulated P53 expression. Additionally, in the TRAIL pathway, it enhances the expression of pro-apoptotic players TRAIL and DR5 and downregulates the anti-apoptotic protein cFLIP. CONCLUSIONS: Our data suggest that MSNPs loaded with caffeine, i.e., CcNP/nanomedicine, can potentially inhibit transformed cell proliferation and induce pro-apoptotic TRAIL machinery to counter benzene-induced leukemia. These results render our nanomedicine as a potentially excellent therapeutic agent against AML.

CYP2C19*17 association with higher plasma 4-hydroxy tamoxifen in Pakistani (estrogen-positive) breast cancer patients.

Breast cancer (BC) continues to be the most common cancer in the women worldwide. Since estrogen receptor (ER)-positive BC accounts for the majority of newly diagnosed cases, endocrine therapy is advised to utilize either tamoxifen (Tam) or aromatase inhibitors. The use of Tam as a monotherapy or in conjunction with an aromatase inhibitor following two or three years of endocrine therapy has long been recommended. When used adjuvantly, Tam medication reduces BC mortality and relapses, while it extends survival times in metastatic BC when used in conjunction with other treatments. Unfortunately, the efficiency of Tam varies considerably. This study was conducted to explore the influence of genetic polymorphisms in CYP2C19 gene on Tam's pharmacogenetics and pharmacokinetics in estrogen-positive BC patients. Data from healthy, unrelated individuals (n = 410; control group) and ER-positive BC patients (n = 430) receiving 20 mg of Tam per day were recruited. Steady-state plasma concentrations of Tam and its three metabolites were quantified using the high-performance liquid chromatography in the patients. The CYP2C19 polymorphisms were genotyped using an Amplification Refractory Mutation System-Polymerase Chain Reaction (ARMS-PCR) approach. More than 65% of healthy individuals were extensive metabolizers (*1/*1) for CYP2C19, whereas more than 70% of ER-positive BC patients were rapid and ultrarapid metabolizers (*1/17*, *17/17*). The polymorphism CYP2C19*17 is significantly associated with higher 4-hydroxytamoxifen (4-OH-Tam). Patients with the *17/*17 genotype exhibited 1- to 1.5-fold higher 4-OH-Tam, which was also high in patients with the *1/*2 and *2/*2 genotypes.

A cohort study investigating the role of Bisphenol A in the molecular pathogenesis of breast cancer.

BACKGROUND: Breast cancer is an abnormal division of breast cells. Bisphenol A (BPA), an environmental toxicant, is identified as an emerging risk factor for breast cancer development. However, to the best of our knowledge, no previous study has investigated the BPA levels in breast cancer patients in Pakistan. The present study sought to explore the role of BPA in tumor growth among the Pakistani population. METHODS: The levels of BPA were analyzed in the serum samples of breast cancer patients and controls by using HPLC. To elucidate the role of BPA to initiate tumorigenic events in breast tissue different biochemical assays along with expression analysis of tumor markers were performed. RESULTS: The level of BPA in the serum samples of breast cancer patients was significantly higher than control. Histological analysis of breast cancer tissue samples revealed distinct subtypes of tumor, such as ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). There was a significant increase in ROS level while a significant decrease in the levels of superoxide dismutase (SOD) and catalase (CAT) enzymes in malignant breast tissue samples as compared to control tissue samples. We found upregulated expression of p53, ZEB1 and WNT1 genes at mRNA level in malignant breast tissue samples by 17 folds, 328 folds and 35 folds, respectively. p53 protein expression in malignant breast tissue samples was also enhanced at the translational level. CONCLUSION: Current findings suggest a relationship between BPA and the progression of breast cancer among the Pakistani population.

Effect of Iron-fortified Wheat Flour on Hemoglobin Levels among Women of Reproductive Age Group in Mansehra, KPK, Pakistan.

Micronutrient deficiencies continue to affect approximately 25% of the World's population. Fortification of staple foods is recognized as one of the most effective interventions to combat micronutrient deficiencies such as iron deficiency. The objective of the current research was to elucidate the effect of iron-fortified wheat flour on the mean hemoglobin levels of women of the reproductive age group (15-49 years) in the Mansehra district, KPK, Pakistan. The study sample consisted of 280 women whose baseline hemoglobin levels were determined at the start of the study. They were fed with iron-fortified wheat flour for a period of 120 days after which their hemoglobin levels were measured again. A 24-hour dietary recall was also taken from the study participants to determine the amounts and frequencies of major foods consumed during the last 24 hours. The study results showed that the consumption of iron-fortified wheat flour had significantly increased the mean hemoglobin levels of women. The study concluded that the consumption of iron-fortified wheat flour could be an effective strategy to combat the problem of iron deficiency in Pakistan.

Co-administration of Inulin and Iron Fortificants improves Iron Deficiency Biomarkers in Female Sprague Dawley Rats.

Micronutrient deficiencies affect approximately 2 billion people worldwide and iron deficiency anemia is one of them. The instant research was an attempt to determine the efficacy of co-administration of two iron fortificants (NaFeEDTA and FeSO4) and inulin (a prebiotic) on serum iron, ferritin, transferrin, and total iron-binding capacity in iron-deficient female Sprague Dawley rats. For this research, rats were divided into ten groups, (two control and eight treatment groups). Treatment groups were made iron deficient by feeding them with triapine, an iron binder for two weeks. All treatment groups were fed with inulin at two different dosage levels along with iron fortificants. The study results showed that serum ferritin and serum iron levels significantly improved from initiation to termination of study. Also, mean values of total iron-binding capacity and serum transferrin showed a steady decline over a period of three months indicating that iron stores were being improved. It was concluded that co-administration of inulin and iron fortificants helped improve iron deficiency biomarkers in female Sprague Dawley rats.

Combined efficacy of Cinnamomum zeylanicum and doxorubicin against leukemia through regulation of TRAIL and NF-kappa B pathways in rat model.

BACKGROUND: Recent discoveries in cancer therapeutics have proven combination therapies more effective than individual drugs. This study describes the efficacy of the combination of Cinnamomum zeylanicum and doxorubicin against benzene-induced leukemia. METHODS AND RESULTS: Brine shrimp assay was used to assess the cytotoxicity of C. zeylanicum, doxorubicin and their combination. After AML induction in Sprague Dawley rats, the same drugs were given to rat groups. Changes in organ weight, haematological profile, and hepatic enzymes were determined. Real-time PCR was used to elucidate the effect on the expression of STMN1, GAPDH, P53 and various TRAIL and NF-kappaB components. C. zeylanicum reduced the cytotoxicity of doxorubicin. The combination treatment showed better anti-leukemic results than any of the individual drugs as evident from STMN1 expression (p < 0.001). It was particularly effective in reducing total white blood cell counts and recovering lymphocytes, monocytes and eosinophils along with hepatic enzymes ALT and AST (p < 0.001). All doses recovered relative organ weights and improved blood parameters. The combination therapy was particularly effective in inducing apoptosis, inhibition of proliferation marker GAPDH (p < 0.001) and NF-kappaB pathway components Rel-A (p < 0.001) and Rel-B (p < 0.01). Expressions of TRAIL components c-FLIP (p < 0.001), TRAIL ligand (p < 0.001) and caspase 8 (p < 0.01) were also altered. CONCLUSION: Cinnamomum zeylanicum in combination with doxorubicin helps to counter benzene-induced cellular and hepatic toxicity and improves haematological profile. The anti-leukemic effects are potentially due to inhibition of GAPDH and NF-kappa B pathway, and through regulation of TRAIL pathway. Our data suggests the use of C. zeylanicum with doxorubicin to improve anti-leukemic therapeutic regimes.

Comparative analysis of the serum microbiome of HIV infected individuals.

HIV infects the CD4 cells which marks the suppression of our immune system. DNA from serum of healthy, treated and untreated HIV infected individuals was extracted. The DNA was subjected to 16S metagenomic sequencing and analyzed using QIIME2 pipeline. 16S sequencing analysis showed serum microbiome was dominated by Firmicutes, Proteobacteria, Bacteroidota and Actinobacteria. Treated HIV infection showed highest abundance of Firmicutes (66.40%) significantly higher than untreated HIV infection (35.88%) and control (41.89%). Bacilli was most abundant class in treated (63.59%) and second most abundant in untreated (34.53%) while control group showed highest abundance of class Gamma-proteobacteria (45.86%). Untreated HIV infection group showed Enterococcus (10.72%) and Streptococcus (6.599%) as the most abundant species. Untreated HIV infection showed significantly higher (p = 0.0039) species richness than treated and control groups. An altered serum microbiome of treated HIV infection and higher microbial abundance in serum of untreated HIV infection was observed.

Association of cell free mitochondrial DNA and caspase-1 expression with disease severity and ARTs efficacy in HIV infection.

HIV infection is a global health concern. Current HIV-diagnostics provide information about the disease progression and efficacy of anti-retroviral therapies (ARVs), but this information is very limited and sometimes imprecise. Present study assessed the potential role of mononuclear cell (MNC) death, expression of caspases (1&3) and cell free mitochondrial DNA (CF mt-DNA) in HIV infected individuals. Apoptosis, cell-count, expression of caspases and CF mt-DNA were measured through flow cytometry and qPCR, respectively, in HIV infected individuals (n = 120) divided in two groups i.e. ARVs-receiving (treated, n = 87), ART-naïve (untreated, n = 37) and healthy individuals (n = 47). Data showed significant (p < 0.0001) cell death in untreated individuals than treated and healthy individuals. CD4-positive T-cell percentage declined (p < 0.0001) in untreated as compared to treated individuals. Caspase-1, an indicator of pyroptosis, and CF mt-DNA were also elevated in untreated HIV infected individuals. Untreated individuals when administered with ARVs showed improved CD4-positive T-cell percentage, lower caspase-1, CF mt-DNA and cell death. Data elucidated positive co-relation between cell death and CF mt-DNA in treated and untreated HIV infected individuals. While CD4-positive T-cell percentage was negatively correlated with caspase-1 expression and CF mt-DNA. Elevated levels of CF mt-DNA and caspase-1 in HIV infected individuals, positive correlation between cell death and CF mt-DNA, negative correlation of CD4-positive T-cell percentage with CF mt-DNA and caspase-1 expression clearly indicated the potential of CF mt-DNA and caspase-1 as a novel disease progression and ARTs effectiveness biomarkers in HIV.

Melatonin abated Bisphenol A-induced neurotoxicity via p53/PUMA/Drp-1 signaling.

Bisphenol A (BPA), an endocrine disruptor, is widely used in the manufacture of different daily life products. Accumulating evidence supports the association between the increasing incidence of neurodegenerative diseases and the BPA level in the environment. In the present study, we aimed to evaluate the neuroprotective role of melatonin against BPA-induced mitochondrial dysfunction-mediated apoptosis in the brain. Herein, adult Sprague Dawley rats were administrated (subcutaneously) with BPA (100 µg/kg BW, 1 mg/kg BW, and 10 mg/kg BW) and melatonin (4 mg/kg BW) for 16 days. Our results showed BPA exposure significantly increased the oxidative stress as demonstrated by increased free radicals (ROS), TBARs level, disrupted cellular architecture, and decreased antioxidant enzymes including SOD, CAT, APX, POD, and GSH levels. Additionally, BPA treatment increased the expression of PUMA, p53, and Drp-1 resulting in apoptosis in the brain tissue of rats. However, melatonin treatment significantly attenuated BPA-induced toxic effects by scavenging ROS, boosting antioxidant enzyme activities, and interestingly enervated brain apoptosis by normalizing p53, PUMA, and Drp-1 expressions at both transcriptional and translational level. Moreover, the brain tissue histology also revealed the therapeutic potential of melatonin by normalizing the cellular architecture. Conclusively, our finding suggests that melatonin could alleviate oxidative stress and mitochondrial dysfunction-linked apoptosis, rendering its neuroprotective potential against BPA-induced toxicity.

Naphthoquinones from Handroanthus impetiginosus promote skin wound healing through Sirt3 regulation.

OBJECTIVES: Lapachone is a natural naphthoquinone-derived compound found in Tabebuia avellanedae. It is well-known for its analgesic, anti-inflammatory, anti-microbial, diuretic, and anti-cancerous effects. However, the wound-healing effects of this compound are not known yet. The aim of this study was to investigate the wound healing activity of naphthoquinones (α-lapachone and ß-lapachone) from Handroanthus impetiginosus. MATERIALS AND METHODS: Expression of Sirt3, migration-related proteins (Rac1, Cdc42, α-Pak) and angiogenesis-related protein of vascular endothelial growth factor (VEGF) was monitored using western blot analysis. Blood vessel formation and tissue development were monitored by angiogenesis assay and hematoxylin & eosin (H & E) staining, respectively on mouse skin tissue samples. Both α-lapachone and ß-lapachone increased Sirt3 expression in vivo, but only ß-lapachone increased Sirt3 expression in vitro. RESULTS: Both the compounds accelerated wound healing in cultured skin cells as well as mouse skin; however, ß-lapachone was more effective at lower concentrations. Both of the compounds increased the expression of migration-related proteins both in vitro and in vivo. Similarly, α-lapachone and ß-lapachone increased VEGF expression, tissue development and blood vessel formation in mouse skin. CONCLUSION: These findings indicated that α-lapachone and ß-lapachone are novel Sirt3 activators, and Sirt3 has a role in wound healing. Thus, Sirt3 and its regulators come out as a novel target and potential drug candidates, respectively in the important field of cutaneous wound healing.

Pistacia integerrima alleviated Bisphenol A induced toxicity through Ubc13/p53 signalling.

Exposure to environmental toxicants such as Bisphenol A (BPA) has raised serious health issues globally particularly in developing countries. It is ubiquitously used in the manufacturing of canned food and feeding bottles. BPA generated reactive oxygen species can lead to several diseases including cardiotoxicity. However, the endpoints stimulated in BPA cardiotoxicity yet need to be investigated. The current study was aimed to investigate the underlying molecular pathways which may contribute in revealing the protective effects of Pistacia integerrima against BPA induced oxidative stress. The dose of 100 µg/kg BW of BPA, 200 mg/kg BW P. integerrima, and 4 mg/kg BW melatonin was administered to Sprague Dawley rats. Present results of western blotting and qRT-PCR showed the increased expression of p53, PUMA and Drp1, while downregulation of Ubc13 in heart tissues of BPA treated group whereas the levels were reversed upon treatment with P. integerrima. The role of BPA in heart tissue apoptosis was further confirmed by the increased level of P-p53, cytochrome C and disrupted cellular architecture whereas the P. integerrima has shown its ameliorative potential by mitigating the adverse effects of BPA. Moreover, the oxidant, antioxidant, lipid, and liver markers profile has also revealed the therapeutic potential of P. integerrima by maintaining the levels in the normal range. However, melatonin has also manifested the normalized expression of apoptotic markers, biochemical markers, and tissue architecture. Conclusively, the data suggest that P. integerrima may be a potential candidate for the treatment of BPA induced toxicity by neutralizing the oxidative stress through Ubc13/p53 pathway.

Assessment of cell free mitochondrial DNA as a biomarker of disease severity in different viral infections.

OBJECTIVE: Cell Free mitochondrial DNA (CF mt-DNA) has emerged as a novel biomarker to investigate disease pathophysiology of different infections. The present study was designed to elucidate the association between CF mt-DNA, IL-6 and viral load in HIV, HBV and HCV infections and predict its role as a potential biomarker to assess the disease severity in viral infections. METHODS: Total 120 blood samples were collected from January 2018 to December 2018 of HIV, HBV and HCV patients and healthy controls (30 samples in each group). DNA and RNA were extracted from the serum to determine the levels of CF mt-DNA and viral load, respectively. IL-6 from the serum of infected individuals was quantified with ELISA. RESULTS: HCV patients showed the highest levels of CF mt-DNA, IL-6 and viral load, followed by HBV and HIV. Significant correlation was found between CF mt-DNA and IL-6 among the HBV patients (p=0.017). However, no significant correlation of CF mt-DNA was observed with IL-6 in HIV and HCV or with the viral load in any of the three infections. CONCLUSION: Elevated CF mt-DNA indicates its role in severity of viral infections. Independence of CF mt-DNA expression from viral load and IL-6 in case of HIV and HCV suggests involvement of other inflammatory pathways regulating CF mt-DNA elevation.

Biallelic variants in four genes underlying recessive osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is an inherited heterogeneous rare skeletal disorder characterized by increased bone fragility and low bone mass. The disorder mostly segregates in an autosomal dominant manner. However, several rare autosomal recessive and X-linked forms, caused by mutations in 18 different genes, have also been described in the literature. Here, we present five consanguineous families segregating OI in an autosomal recessive pattern. Affected individuals in the five families presented severe forms of skeletal deformities. It included frequent bone fractures with abnormal healing, short stature, facial dysmorphism, osteopenia, joint laxity, and severe scoliosis. In order to search for the causative variants, DNA of at least one affected individual in three families (A-C) were subjected to whole exome sequencing (WES). In two other families (D-E), linkage analysis using highly polymorphic microsatellite markers was followed by Sanger sequencing. Sequence analysis revealed two novels and three previously reported disease-causing variants. The two novel homozygous variants including [c.824G > A; p.(Cys275Tyr)] in the SP7 gene and [c.397C > T, p.(Gln133*)] in the SERPINF1 gene were identified in families A and B, respectively. The three previously reported homozygous variants including [c.497G > A; p.(Arg166His)] in the SPARC gene, (c.359-3C > G; intron 2) and [c.677C > T; p.(Ser226Leu)] in the WNT1 gene were identified in family C, D, and E. In conclusion, our findings provided additional evidence of involvement of homozygous sequence variants in the SP7, SERPINF1, SPARC and WNT1 genes causing severe OI. It also highlights the importance of extensive genetic investigations to search for the culprit gene in each case of skeletal deformity.

Apoptosis of Leukemia Cells by Ocimum basilicum Fractions Following TNF alpha Induced Activation of JNK and Caspase 3.

PURPOSE: Leukemia, one of the major cancers, affects a large proportion of people around the world. Better treatment options for leukemia are required due to a large number of side effects associated with current therapeutic regimens. In the present study, we sought to determine the pathway of triggering apoptosis of leukemic cells by Ocimum basilicum (O. basilicum) plant extract. MATERIALS/METHODS: Methanolic extract of the O. basilicum plant material was prepared. The crude extract was fractionated into several fractions through column chromatography using ethyl acetate and n-hexane as eluting solvents. Cell viability of leukemic cells was assessed via Cell titer GLO assay and apoptosis was measured through Annexin V/PI staining. Two apoptotic molecules JNK and caspases were analyzed through western blotting while pro-inflammatory cytokines TNFα, CCL2 and CXCL8 using qPCR. Fractions were characterized through LC-MS. RESULTS: The most potent with lowest IC50 values among the fractions were BF2 (2:8 n-hexane:ethyl acetate) and BF3 (3:7 n-hexane:ethyl acetate). Cytotoxicity was associated with apoptosis. Apoptosis was found caspasedependent and P-JNK activation was detected sustained. A significant increase in the level of TNF α and a decrease in the level of CXCL8 were observed in BF2 and BF3 treated cells. CONCLUSION: The fractions of O. basilicum extract were found to kill cells following JNK pathway activation. Excellent results were obtained with BF2 and BF3 probably due to predominant Epicatechin and Cinnamic acid derivatives in these fractions.

Correction to: Novel mutation G324C in WNT1 mapped in a large Pakistani family with severe recessively inherited Osteogenesis Imperfecta.

In the original publication of this article [1], there are two errors in the article which the cDNA position of the pathogenic variant WNT1 p.Gly324Cys should be c.970G>T instead of c.1168G>T.

A Novel Homozygous Frameshift Variant in XYLT2 Causes Spondyloocular Syndrome in a Consanguineous Pakistani Family.

We report on three new patients with spondyloocular syndrome (SOS) in a consanguineous Pakistani family. All three patients present progressive generalized osteoporosis, short stature, recurrent fractures, hearing loss and visual impairments. WES revealed a novel homozygous frameshift variant in exon 11 of XYLT2 (NG 012175.1, NP_071450.2) resulting in loss of evolutionary conserved amino acid sequences (840 - 865/865) at C-terminus p.R840fs∗115. Sanger Sequencing confirmed the presence of the novel homozygous mutation in all three patients while the parents were heterozygous carriers of the mutation, in accordance with an autosomal recessive inheritance pattern. Only nine variants worldwide have previously been reported in XYLT2 in patients with SOS phenotype. These three patients with novel homozygous variant extend the genotypic and phenotypic spectrum of SOS.

Recessive multiple epiphyseal dysplasia - Clinical characteristics caused by rare compound heterozygous SLC26A2 genotypes.

Pathogenic sequence variants in the solute carrier family 26 member 2 (SLC26A2) gene result in lethal (achondrogenesis Ib and atelosteogenesis II) and non-lethal (diastrophic dysplasia and recessive multiple epiphyseal dysplasia, rMED) chondrodysplasias. We report on two new patients with rMED and very rare compound heterozygous mutation combinations in non-consanguineous families. Patient I presented in childhood with waddling gait and joint stiffness. Radiographs showed epiphyseal changes, bilateral coxa plana-deformity and knee valgus deformity, for which he underwent surgeries. At present 33 years his height is 165 cm. Patient II presented with cleft palate, small jaw, short limbs, underdeveloped thumbs and on radiographs, cervical kyphosis with an underdeveloped C4. He also developed severe scoliosis but has grown at -2.9 SD curve. Molecular analysis revealed that patient I is heterozygous for two known pathogenic variants in SLC26A2, a splice site variant c.-26+2T > C and a missense variant c.1957T > A (p.Cys653Ser), while patient II is compound heterozygous for missense variants c.835C > T (p.Arg279Trp) and c.1535C > A (p.Thr512Lys). These patients further elucidate the variability of the phenotypic and genetic presentations of rMED.

Interplay of N acetyl cysteine and melatonin in regulating oxidative stress-induced cardiac hypertrophic factors and microRNAs.

Early and specific diagnosis of oxidative stress linked diseases as cardiac heart diseases remains a major dilemma for researchers and clinicians. MicroRNAs may serve as a better tool for specific early diagnostics and propose their utilization in future molecular medicines. We aimed to measure the microRNAs expressions in oxidative stress linked cardiac hypertrophic condition induced through stimulants as Endothelin and Isoproterenol. Cardiac hypertrophic animal models were confirmed by BNP, GATA4 expression, histological assays, and increased cell surface area. High oxidative stress (ROS level) and decreased antioxidant activities were assessed in hypertrophied groups. Enhanced expression of miR-152, miR-212/132 while decreased miR-142-3p expression was observed in hypertrophic condition. Similar pattern of these microRNAs was detected in HL-1 cells treated with H2O2. Upon administration of antioxidants, the miRNAs expression pattern altered from that of the cardiac hypertrophied model. Present investigation suggests that oxidative stress generated during the cardiac pathology may directly or indirectly regulate anti-hypertrophy pathway elements through microRNAs including antioxidant enzymes, which need further investigation. The down-regulation of free radical scavengers make it easier for the oxidative stress to play a key role in disease progression.

Novel mutation G324C in WNT1 mapped in a large Pakistani family with severe recessively inherited Osteogenesis Imperfecta.

INTRODUCTION: Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous disease with skeletal fragility and variable extra-skeletal manifestations. To date several point mutations in 18 different genes causing different types of OI have been identified. Mutations in WNT1 compromise activity of the osteoblasts leading to disturbed bone mass accrual, fragility fractures and progressive skeletal abnormalities. The present study was conducted to determine the underlying genetic cause of an autosomal recessive skeletal dysplasia in a large consanguineous family from Chinute, Pakistan. MATERIALS AND METHODS: Blood was collected from 24 individuals of affected family along with clinical data. Homozygosity mapping was performed to confirm consanguinity. SNPs were identified, followed by whole exome and Sanger sequencing. In silico characterization of WNT1 mutation was performed using multiple platforms. RESULTS: Nine affected family members exhibited severe bone deformities, recurrent fractures, short stature and low bone mineral density. SNP array data revealed homozygous segments > 1 Mb in length accounting for 2.1-12.7% of the genome in affected individuals and their siblings and a single 6,344,821 bp homozygous region in all affected individuals on chromosome 12q12-q13. This region includes two potential OI candidate genes WNT1 and VDR. We did whole-exome sequencing for both genes in two patients and identified a novel damaging missense mutation in exon 4 of WNT1: c.1168G > T (NM_005430) resulting in p.G324C. Sanger sequencing confirmed segregation of mutation with the disease in family. CONCLUSION: We report a novel mutation responsible for OI and our investigation expands the spectrum of disease-causing WNT1 mutations and the resulting OI phenotypes.

Interplay of mitochondria apoptosis regulatory factors and microRNAs in valvular heart disease.

Valvular heart disease (VHD) is an active process involving a wide range of pathological changes. The major complications of VHD are stenosis and regurgitation, which are macroscopic phenomena, induced in part through cellular changes. Altered expression of mitochondria associated genes causes membrane potential depolarization, leading to the increased levels of apoptosis observed in cardiac dysfunction. Objective of this study is to find molecular medicine candidates that can control expression of the key mitochondria apoptosis regulatory genes. Present study aims to assess the way microRNA are involved in regulating mitochondrial apoptosis regulatory genes and observation of their expression in the heart valve dysfunction. Apoptotic genes PUMA and DRP1 were found to be highly expressed, whereas anti-apoptotic gene ARC was down regulated. The expression level of GATA-4 transcription factor was also reduced in cardiac valve tissues. MicroRNAs miR-15a and miR-29a were repressed, while miR-214 was up regulated. Furthermore, study showed that PUMA, DRP1 and ARC expression might be attenuated by their respective miRNAs. Our results indicate that mitochondria regulatory genes might be controlled by miR-15a, miR-29a and miR-214, in VHD patients. Present study may provide platform for future research regarding potential therapeutic role of miRNAs in CVDs.