Identities and frequencies of variants in CYP1B1 causing primary congenital glaucoma in Pakistan.

Purpose: Primary congenital glaucoma (PCG) is a clinically and genetically heterogeneous disease. The present study was undertaken to find the genetic causes of PCG segregating in 36 large consanguineous Pakistani families. Methods: Ophthalmic examination including fundoscopy, or slit-lamp microscopy was performed to clinically characterize the PCG phenotype. Genomic nucleotide sequences of the CYP1B1 and LTBP2 genes were analyzed with either Sanger or whole exome sequencing. In silico prediction programs were used to assess the pathogenicity of identified alleles. ClustalW alignments were performed to determine evolutionary conservation, and three-dimensional (3D) modeling was performed using HOPE and Phyre2 software. Results: Among the known loci, mutations in CYP1B1 and LTBP2 are the common causes of PCG. Therefore, we analyzed the genomic nucleotide sequences of CYP1B1 and LTBP2, and detected probable pathogenic variants cosegregating with PCG in 14 families. These included the three novel (c.542T>A, c.1436A>G, and c.1325delC) and five known (c.868dupC, c.1168C>T, c.1169G>A, c.1209InsTCATGCCACC, and c.1310C>T) variants in CYP1B1. Two of the novel variants are missense substitutions [p.(Leu181Gln), p.(Gln479Arg)], which replaced evolutionary conserved amino acids, and are predicted to be pathogenic by various in silico programs, while the third variant (c.1325delC) is predicted to cause reading frameshift and premature truncation of the protein. A single mutation, p.(Arg390His), causes PCG in six (~43%) of the 14 CYP1B1 mutations harboring families, and thus, is the most common variant in this cohort. Surprisingly, we did not find any LTBP2 pathogenic variants in the families, which further supports the genetic heterogeneity of PCG in the Pakistani population. Conclusions: In conclusion, results of the present study enhance our understanding of the genetic basis of PCG, support the notion of a genetic modifier of CYP1B1, and contribute to the development of genetic testing protocols and genetic counseling for PCG in Pakistani families.

A new network representation for time series analysis from the perspective of combinatorial property of ordinal patterns.

Revealing system behavior from observed time series is a fundamental problem worthy of in-depth study and exploration, and has attracted extensive attention in a wide range of fields due to its wide application values. In this paper, we propose a novel network construction method for time series analysis, which is different from the existing ordinal network method concerning the transition probability of ordinal patterns in transition networks. The proposed network representation is based on the combinatorial property concerning the inversion number of ordinal patterns from the ordinal partitions of time series. For the proposed network construction method, the network nodes are represented by each ordinal partition of time series and the edge weight between network nodes is determined by a novel proximity relationship of ordinal patterns which is a newly defined metric based on the inversion number of ordinal patterns. Using random signals and chaotic signals as examples, we demonstrate the potential of the proposed network construction method for the network representation of time series. We also employ the proposed network construction method in quantitative EEG for the identification of three different physiological and pathological brain states. According to the results of AUC values, one can observe that the discriminating power of the AND of the proposed network construction method is slightly stronger than that of the available ordinal network. The experimental results illustrate that our proposed network construction method opens up a new pathway for network representation of time series, which is capable of quantifying time series for feature extraction and pattern learning for time series analysis.

Insights into nanoparticles-induced neurotoxicity and cope up strategies.

Nanoparticle applications are becoming increasingly popular in fields such as photonics, catalysis, magnetics, biotechnology, manufacturing of cosmetics, pharmaceuticals, and medicines. There is still a huge pile of undermining information about the potential toxicity of these products to humans, which can be encountered by neuroprotective antioxidants and anti-inflammatory compounds. Nanoparticles can be administered using a variety of methods, including oronasal, topical applications, and enteral and parenteral routes of administration. There are different properties of these nanomaterials that characterize different pathways. Crossing of the blood-brain barrier, a direct sensory nerve-to-brain pathway whose barriers are bypassed, these checks otherwise prevent the nanoparticles from entering the brain. This inflicts damage to sensory neurons and receptors by nanoparticles that lead to neurotoxicity of the central nervous system. A number of routes make nanoparticles able to penetrate through the skin. Exposure by various routes to these nanoparticles can result in oxidative stress, and immune suppression triggers inflammatory cascades and genome-level mutations after they are introduced into the body. To out-power, these complications, plant-based antioxidants, essential oils, and dietary supplements can be put into use. Direct nanoparticle transport pathways from sensory nerves to the brain via blood have been studied grossly. Recent findings regarding the direct pathways through which nanoparticles cross the blood-brain barriers, how nanoparticles elicit different responses on sensory receptors and nerves, how they cause central neurotoxicity and neurodegeneration through sensory nerve routes, and the possible mechanisms that outcast these effects are discussed.

Resistance Modulation of Individual and Polymicrobial Culture of S. aureus and E. coli through Nanoparticle-Coupled Antibiotics.

Polymicrobial mastitis is now becoming very common in dairy animals, resulting in exaggerated resistance to multiple antibiotics. The current study was executed to find drug responses in individual and mixed Culture of Staphylococcus aureus and Escherichia coli isolated from milk samples, as well as to evaluate the antibacterial potential of tungsten oxide nanoparticles. These isolates (alone and in mixed culture) were further processed for their responses to antibiotics using the disc diffusion method. On the other hand, tungsten oxide WO3 (W) nanoparticles coupled with antibiotics (ampicillin, A, and oxytetracycline, O) were prepared through the chemical method and characterized by X-ray diffraction, scanning electron microscopy (SEM), and UV-visible techniques. The preparations consisting of nanoparticles alone (W) and coupled with ampicillin (WA) and oxytetracycline (WO) were tested against individual and mixed Culture through the well diffusion and broth microdilution methods. The findings of the current study showed the highest resistance in E. coli was against penicillin (60%) and ampicillin (50%), while amikacin, erythromycin, ciprofloxacin, and oxytetracycline were the most effective antibiotics. S. aureus showed the highest resistance against penicillin (50%), oxytetracycline (40%), and ciprofloxacin (40%), while, except for ampicillin, the sensitive strains of S. aureus were in the range of 40-60% against the rest of antibiotics. The highest zones of inhibition (ZOI) against mixed Culture were shown by imipenem and ampicillin, whereas the highest percentage decrease in ZOI was noted in cases of ciprofloxacin (-240%) and gentamicin (-119.4%) in comparison to individual Culture of S. aureus and E. coli. It was noteworthy that the increase in ZOI was not more than 38% against mixed Culture as compared to the individual Culture. On the other hand, there was a significant reduction in the minimum inhibitory concentration (MIC) of nanoparticle-coupled antibiotics compared to nanoparticles alone for individual and mixed-culture bacteria, while MICs in the case of mixed Culture remained consistently high throughout the trial. This study therefore concluded that diverse drug resistance was present in both individual and mixed-culture bacteria, whereas the application of tungsten oxide nanoparticle-coupled antibiotics proved to be an effective candidate in reversing the drug resistance in bacterial strains.

Design and Analysis of an O+E-Band Hybrid Optical Amplifier for CWDM Systems.

Broadband amplification in the O+E-band is very desirable nowadays as a way of coping with increasing bandwidth demands. The main issue with doped fiber amplifiers working in this band such as the bismuth-doped fiber amplifier is that they are costly and not widely available. Therefore, a wideband and flat-gain hybrid optical amplifier (HOA) covering the O+E-band based on a parallel combination of a praseodymium-doped fiber amplifier (PDFA) and a semiconductor optical amplifier (SOA) is proposed and demonstrated through numerical simulations. The praseodymium-doped fiber (PDF) core is pumped using a laser diode with a power of 500 mW that is centered at a wavelength of 1030 nm. The SOA is driven by an injection current of 60 mA. The performance of the HOA is analyzed by the optimization of various parameters such as the PDF length, Pr3+ concentration, pump wavelength, and injection current. A flat average gain of 24 dB with a flatness of 1 dB and an output power of 9.6 dBm is observed over a wavelength range of 1270-1450 nm. The noise figure (NF) varies from a minimum of 4 dB to a maximum of 5.9 dB for a signal power of 0 dBm. A gain reduction of around 4 dB is observed for an O-band signal at a wavelength of 1290 nm by considering the up-conversion effect. The transmission performance of the designed HOA as a pre-amplifier is evaluated based on the bit-error rate (BER) analysis for a coarse wavelength-division multiplexing (CWDM) system of eight on-off keying (OOK)-modulated channels, each having a data rate of 10 Gbps. An error-free transmission over 60 km of standard single-mode fiber (SMF) is achieved for different data rates of 5 Gbps, 7.5 Gbps, and 10 Gbps.

Delineating the Molecular and Phenotypic Spectrum of the CNGA3-Related Cone Photoreceptor Disorder in Pakistani Families.

Cone photoreceptor dysfunction represents a clinically heterogenous group of disorders characterized by nystagmus, photophobia, reduced central or color vision, and macular dystrophy. Here, we described the molecular findings and clinical manifestations of achromatopsia, a partial or total absence of color vision, co-segregating with three known missense variants of CNGA3 in three large consanguineous Pakistani families. Fundus examination and optical coherence tomography (OCT) imaging revealed myopia, thin retina, retinal pigment epithelial cells loss at fovea/perifovea, and macular atrophy. Combination of Sanger and whole exome sequencing revealed three known homozygous missense variants (c.827A>G, p.(Asn276Ser); c.847C>T, p.(Arg283Trp); c.1279C>T, p.(Arg427Cys)) in CNGA3, the α-subunit of the cyclic nucleotide-gated cation channel in cone photoreceptor cells. All three variants are predicted to replace evolutionary conserved amino acids, and to be pathogenic by specific in silico programs, consistent with the observed altered membrane targeting of CNGA3 in heterologous cells. Insights from our study will facilitate counseling regarding the molecular and phenotypic landscape of CNGA3-related cone dystrophies.

Genetic Causes of Oculocutaneous Albinism in Pakistani Population.

Melanin pigment helps protect our body from broad wavelength solar radiation and skin cancer. Among other pigmentation disorders in humans, albinism is reported to manifest in both syndromic and nonsyndromic forms as well as with varying inheritance patterns. Oculocutaneous albinism (OCA), an autosomal recessive nonsyndromic form of albinism, presents as partial to complete loss of melanin in the skin, hair, and iris. OCA has been known to be caused by pathogenic variants in seven different genes, so far, according to all the currently published population studies. However, the detection rate of alleles causing OCA varies from 50% to 90%. One of the significant challenges of uncovering the pathological variant underlying disease etiology is inter- and intra-familial locus heterogeneity. This problem is especially pertinent in highly inbred populations. As examples of such familial locus heterogeneity, we present nine consanguineous Pakistani families with segregating OCA due to variants in one or two different known albinism-associated genes. All of the identified variants are predicted to be pathogenic, which was corroborated by several in silico algorithms and association with diverse clinical phenotypes. We report an individual affected with OCA carries heterozygous, likely pathogenic variants in TYR and OCA2, raising the question of a possible digenic inheritance. Altogether, our study highlights the significance of exome sequencing for the complete genetic diagnosis of inbred families and provides the ramifications of potential genetic interaction and digenic inheritance of variants in the TYR and OCA2 genes.

Molecular outcomes, clinical consequences, and genetic diagnosis of Oculocutaneous Albinism in Pakistani population.

Nonsyndromic oculocutaneous Albinism (nsOCA) is clinically characterized by the loss of pigmentation in the skin, hair, and iris. OCA is amongst the most common causes of vision impairment in children. To date, pathogenic variants in six genes have been identified in individuals with nsOCA. Here, we determined the identities, frequencies, and clinical consequences of OCA alleles in 94 previously unreported Pakistani families. Combination of Sanger and Exome sequencing revealed 38 alleles, including 22 novel variants, segregating with nsOCA phenotype in 80 families. Variants of TYR and OCA2 genes were the most common cause of nsOCA, occurring in 43 and 30 families, respectively. Twenty-two novel variants include nine missense, four splice site, two non-sense, one insertion and six gross deletions. In vitro studies revealed retention of OCA proteins harboring novel missense alleles in the endoplasmic reticulum (ER) of transfected cells. Exon-trapping assays with constructs containing splice site alleles revealed errors in splicing. As eight alleles account for approximately 56% (95% CI: 46.52-65.24%) of nsOCA cases, primarily enrolled from Punjab province of Pakistan, hierarchical strategies for variant detection would be feasible and cost-efficient genetic tests for OCA in families with similar origin. Thus, we developed Tetra-primer ARMS assays for rapid, reliable, reproducible and economical screening of most of these common alleles.

Homozygous missense variant in the human CNGA3 channel causes cone-rod dystrophy.

We assessed a large consanguineous Pakistani family (PKAB157) segregating early onset low vision problems. Funduscopic and electroretinographic evaluation of affected individuals revealed juvenile cone-rod dystrophy (CRD) with maculopathy. Other clinical symptoms included loss of color discrimination, photophobia and nystagmus. Whole-exome sequencing, segregation and haplotype analyses demonstrated that a transition variant (c.955T>C; p.(Cys319Arg)) in CNGA3 co-segregated with the CRD phenotype in family PKAB157. The ability of CNGA3 channel to influx calcium in response to agonist, when expressed either alone or together with the wild-type CNGB3 subunit in HEK293 cells, was completely abolished due to p.Cys319Arg variant. Western blotting and immunolocalization studies suggest that a decreased channel density in the HEK293 cell membrane due to impaired folding and/or trafficking of the CNGA3 protein is the main pathogenic effect of the p.Cys319Arg variant. Mutant alleles of the human cone photoreceptor cyclic nucleotide-gated channel (CNGA3) are frequently associated with achromatopsia. In rare cases, variants in CNGA3 are also associated with cone dystrophy, Leber's congenital amaurosis and oligo cone trichromacy. The identification of predicted p.(Cys319Arg) missense variant in CNGA3 expands the repertoire of the known genetic causes of CRD and phenotypic spectrum of CNGA3 alleles.

Identification and functional characterization of natural human melanocortin 1 receptor mutant alleles in Pakistani population.

Melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor of the melanocyte's plasma membrane, is a major determinant of skin pigmentation and phototype. Upon activation by α-melanocyte stimulating hormone, MC1R triggers the cAMP cascade to stimulate eumelanogenesis. We used whole-exome sequencing to identify causative alleles in Pakistani families with skin and hair hypopigmentation. Six MC1R mutations segregated with the phenotype in seven families, including a p.Val174del in-frame deletion and a p.Tyr298* nonsense mutation, that were analyzed for function in heterologous HEK293 cells. p.Tyr298* MC1R showed no agonist-induced signaling to the cAMP or ERK pathways, nor detectable agonist binding. Conversely, signaling was comparable for p.Val174del and wild-type in HEK cells overexpressing the proteins, but binding analysis suggested impaired cell surface expression. Flow cytometry and confocal imaging studies revealed reduced plasma membrane expression of p.Val174del and p.Tyr298*. Therefore, p.Tyr298* was a total loss-of-function (LOF) allele, while p.Val174del displayed a partial LOF attribute.

Molecular genetic studies and delineation of the oculocutaneous albinism phenotype in the Pakistani population.

BACKGROUND: Oculocutaneous albinism (OCA) is caused by a group of genetically heterogeneous inherited defects that result in the loss of pigmentation in the eyes, skin and hair. Mutations in the TYR, OCA2, TYRP1 and SLC45A2 genes have been shown to cause isolated OCA. No comprehensive analysis has been conducted to study the spectrum of OCA alleles prevailing in Pakistani albino populations. METHODS: We enrolled 40 large Pakistani families and screened them for OCA genes and a candidate gene, SLC24A5. Protein function effects were evaluated using in silico prediction algorithms and ex vivo studies in human melanocytes. The effects of splice-site mutations were determined using an exon-trapping assay. RESULTS: Screening of the TYR gene revealed four known (p.Arg299His, p.Pro406Leu, p.Gly419Arg, p.Arg278*) and three novel mutations (p.Pro21Leu, p.Cys35Arg, p.Tyr411His) in ten families. Ex vivo studies revealed the retention of an EGFP-tagged mutant (p.Pro21Leu, p.Cys35Arg or p.Tyr411His) tyrosinase in the endoplasmic reticulum (ER) at 37°C, but a significant fraction of p.Cys35Arg and p.Tyr411His left the ER in cells grown at a permissive temperature (31°C). Three novel (p.Asp486Tyr, p.Leu527Arg, c.1045-15 T > G) and two known mutations (p.Pro743Leu, p.Ala787Thr) of OCA2 were found in fourteen families. Exon-trapping assays with a construct containing a novel c.1045-15 T > G mutation revealed an error in splicing. No mutation in TYRP1, SLC45A2, and SLC24A5 was found in the remaining 16 families. Clinical evaluation of the families segregating either TYR or OCA2 mutations showed nystagmus, photophobia, and loss of pigmentation in the skin or hair follicles. Most of the affected individuals had grayish-blue colored eyes. CONCLUSIONS: Our results show that ten and fourteen families harbored mutations in the TYR and OCA2 genes, respectively. Our findings, along with the results of previous studies, indicate that the p.Cys35Arg, p.Arg278* and p.Gly419Arg alleles of TYR and the p.Asp486Tyr and c.1045-15 T > G alleles of OCA2 are the most common causes of OCA in Pakistani families. To the best of our knowledge, this study represents the first documentation of OCA2 alleles in the Pakistani population. A significant proportion of our cohort did not have mutations in known OCA genes. Overall, our study contributes to the development of genetic testing protocols and genetic counseling for OCA in Pakistani families.