Integrated genomic analyses of cutaneous T-cell lymphomas reveal the molecular bases for disease heterogeneity.

Cutaneous T-cell lymphomas (CTCLs) are a clinically heterogeneous collection of lymphomas of the skin-homing T cell. To identify molecular drivers of disease phenotypes, we assembled representative samples of CTCLs from patients with diverse disease subtypes and stages. Via DNA/RNA-sequencing, immunophenotyping, and ex vivo functional assays, we identified the landscape of putative driver genes, elucidated genetic relationships between CTCLs across disease stages, and inferred molecular subtypes in patients with stage-matched leukemic disease. Collectively, our analysis identified 86 putative driver genes, including 19 genes not previously implicated in this disease. Two mutations have never been described in any cancer. Functionally, multiple mutations augment T-cell receptor-dependent proliferation, highlighting the importance of this pathway in lymphomagenesis. To identify putative genetic causes of disease heterogeneity, we examined the distribution of driver genes across clinical cohorts. There are broad similarities across disease stages. Many driver genes are shared by mycosis fungoides (MF) and Sezary syndrome (SS). However, there are significantly more structural variants in leukemic disease, leading to highly recurrent deletions of putative tumor suppressors that are uncommon in early-stage skin-centered MF. For example, TP53 is deleted in 7% and 87% of MF and SS, respectively. In both human and mouse samples, PD1 mutations drive aggressive behavior. PD1 wild-type lymphomas show features of T-cell exhaustion. PD1 deletions are sufficient to reverse the exhaustion phenotype, promote a FOXM1-driven transcriptional signature, and predict significantly worse survival. Collectively, our findings clarify CTCL genetics and provide novel insights into pathways that drive diverse disease phenotypes.

Novel spa type and antimicrobial resistance gene profiles of Staphylococcus aureus strains isolated from bovine mastitis in Jordan.

Staphylococcu aureus is the most prevalent microorganism associated with mastitis in cattle. This study was designed to determine the spa types of Staph. aureus and to assess the resistance genes profile of isolated strains in dairy farms in Jordan. In total, 747 milk samples of cattle suffering from subclinical mastitis were collected from 37 dairy farms and tested for Staph. aureus. To detect antimicrobial resistance genes, all 219 strains of Staph. aureus were tested. Furthermore, 21 isolates of Staph. aureus were typed using spa typing. As a result, different proportions of resistance genes were found for Staph. aureus. High resistance genes were in tetK 100%, blaZ 99%, and tetM 97%. Moderate resistance genes were in aac(6')/aph(2'' 52%, ant(4')-Ia 48%, and ermC 41%. Low resistance genes were in ermA is 24%, aph(3')-III is 15%, and mecA is 15%. The spa typing of 21 isolates revealed six spa types, of which five were previously known. For the first time, a novel spa type (t17158) was identified as the main cause of mastitis in dairy cows in Jordan. The identification of resistance genes and spa types is helpful in determining the most effective treatments for cows and plays a significant role in reducing the transmission of pathogens.

Discovering a new part of the phenotypic spectrum of Coffin-Siris syndrome in a fetal cohort.

PURPOSE: Genome-wide sequencing is increasingly being performed during pregnancy to identify the genetic cause of congenital anomalies. The interpretation of prenatally identified variants can be challenging and is hampered by our often limited knowledge of prenatal phenotypes. To better delineate the prenatal phenotype of Coffin-Siris syndrome (CSS), we collected clinical data from patients with a prenatal phenotype and a pathogenic variant in one of the CSS-associated genes. METHODS: Clinical data was collected through an extensive web-based survey. RESULTS: We included 44 patients with a variant in a CSS-associated gene and a prenatal phenotype; 9 of these patients have been reported before. Prenatal anomalies that were frequently observed in our cohort include hydrocephalus, agenesis of the corpus callosum, hypoplastic left heart syndrome, persistent left vena cava, diaphragmatic hernia, renal agenesis, and intrauterine growth restriction. Anal anomalies were frequently identified after birth in patients with ARID1A variants (6/14, 43%). Interestingly, pathogenic ARID1A variants were much more frequently identified in the current prenatal cohort (16/44, 36%) than in postnatal CSS cohorts (5%-9%). CONCLUSION: Our data shed new light on the prenatal phenotype of patients with pathogenic variants in CSS genes.

MN1 C-terminal truncation syndrome is a novel neurodevelopmental and craniofacial disorder with partial rhombencephalosynapsis.

MN1 encodes a transcriptional co-regulator without homology to other proteins, previously implicated in acute myeloid leukaemia and development of the palate. Large deletions encompassing MN1 have been reported in individuals with variable neurodevelopmental anomalies and non-specific facial features. We identified a cluster of de novo truncating mutations in MN1 in a cohort of 23 individuals with strikingly similar dysmorphic facial features, especially midface hypoplasia, and intellectual disability with severe expressive language delay. Imaging revealed an atypical form of rhombencephalosynapsis, a distinctive brain malformation characterized by partial or complete loss of the cerebellar vermis with fusion of the cerebellar hemispheres, in 8/10 individuals. Rhombencephalosynapsis has no previously known definitive genetic or environmental causes. Other frequent features included perisylvian polymicrogyria, abnormal posterior clinoid processes and persistent trigeminal artery. MN1 is encoded by only two exons. All mutations, including the recurrent variant p.Arg1295* observed in 8/21 probands, fall in the terminal exon or the extreme 3' region of exon 1, and are therefore predicted to result in escape from nonsense-mediated mRNA decay. This was confirmed in fibroblasts from three individuals. We propose that the condition described here, MN1 C-terminal truncation (MCTT) syndrome, is not due to MN1 haploinsufficiency but rather is the result of dominantly acting C-terminally truncated MN1 protein. Our data show that MN1 plays a critical role in human craniofacial and brain development, and opens the door to understanding the biological mechanisms underlying rhombencephalosynapsis.

The impact of gender, age, race/ethnicity, and stage on quality of life in a spectrum of cutaneous lymphomas.

PURPOSE: Cutaneous lymphomas (CLs) are a group of rare, potentially disfiguring and disabling cancers that can have a significant impact on quality of life (QoL). While previous studies have shown that mycosis fungoides (MF) and Sézary syndrome (SS) impair QoL, the effect of other types of CL on QoL has not been evaluated. OBJECTIVE: To determine the impact of disease on QoL in all CL patients and to assess how QoL between the CL sub-types varies by demographic and clinical factors. METHODS: The Cutaneous Lymphoma Distress Questionnaire (CL-DQ) was used to assess QoL. All CL patients seen in a multidisciplinary CL clinic were screened for eligibility. Questionnaire responses were collected over a 22-month period between 2017 and 2019. A cross-sectional analysis of CL-DQ scores from an initial visit was performed to determine the effect of disease on QoL across CL sub-types and the potential impact of patient demographics, CL sub-type, and type of treatment. RESULTS: The study population consisted of 151 patients presenting with distinct types of cutaneous B- and T-cell lymphomas. Notable across the study population were the findings of frustration (44%), worry about progress/spread (43%), itching/pruritus (32%), and embarrassment/shame (28%). QoL was found to be most negatively affected in SS patients, females, younger patients, Black patients, and those with advanced stages of MF/SS. CONCLUSIONS: Impairment of QoL due to CL correlates with gender, age, race/ethnicity, and stage of MF/SS. While the negative impact on QoL is most pronounced in SS patients, other CL sub-types also affect QoL and impact psychosocial distress. Our findings highlight the need for QoL assessment in all CL patients and further examination of disparities noted across demographic groups.

Stroke symptoms impact on mental and physical health: A Lebanese population based study.

BACKGROUND/OBJECTIVE: Stroke symptoms in the absence of diagnosed stroke are common worldwide and associated with stroke risk factors and great impact on the physical and mental health functioning. The aim of this study was to assess, at the national level, the association of stroke symptoms with mental and physical health. METHODS: Quality of life was assessed using physical and mental component summary scores (PCS and MCS) of the Short Form 12v2 Health Survey in the Lebanese population. We assessed the differences in the mean PCS and MCS scores among asymptomatic individuals with no stroke/transient ischemic attack (TIA) history (n=1167), symptomatic individuals with no stroke/TIA history (n=125) and those with stroke/TIA history (n=46). Psychometric properties of the Lebanese version of the SF- 12v2 were evaluated using principal component analysis. RESULTS: Symptomatic individuals had an average PCS scores of 2.31 (95%CI: 0.75-3.88) points lower and those with stroke/TIA history had 3.26 (95%CI: 1.01-5.51) points lower when compared with asymptomatic individuals with no stroke/TIA history. Similarly, MCS scores for symptomatic individuals were 2.58 (95%CI: 1.02-4.13) points lower and those with stroke/TIA history had 3.28 (95%CI: 1.06-5.50) points lower than asymptomatic individuals. CONCLUSION: Physical and mental health functioning declined among symptomatic individuals and those with stroke/TIA history. Thus, frequent monitoring for the early detection of stroke symptoms may be recommended.

Genetic diversity of Paramecium species on the basis of multiple loci analysis and ITS secondary structure models.

Among ciliates, Paramecium has become a privileged model for the study of "species problem" particularly in the case of the "Paramecium aurelia complex" that has been intensely investigated. Despite extensive studies, the taxonomy of Paramecium is still challenging. The major problem is an uneven sampling of Paramecium with relatively few representatives of each species. To investigate species from the less discovered region (Pakistan), 10 isolates of Paramecium species including a standing-alone FT8 strain previously isolated by some of us were subjected to molecular characterization. Fragments of 18S recombinant DNA (rDNA), ITS1-5.8S-ITS2-5'LSU rDNA, cytochrome c oxidase subunit II, and hsp70 genes were used as molecular markers for phylogenetic analysis of particular isolates. The nucleotide sequences of polymerase chain reaction products of all markers were compared with the available sequences of relevant markers of other Paramecium species from GenBank. Phylogenetic trees based on all molecular markers showed that all the nine strains had a very close relationship with Paramecium primaurelia except for the FT8 strain. FT8 consistently showed its unique position in comparison to all other species in the phylogenetic trees. Available sequences of internal transcribed spacer 1 (ITS1) and ITS2 and some other ciliate sequences from GenBank were used for the construction of secondary models. Two highly conserved helices supported by compensatory base changes among all ciliates of ITS2 secondary structures were found similar to other eukaryotes. Therefore, the most conserved 120 to 180 base pairs regions were identified for their comparative studies. We found that out of the three helices in ITS1 structure, helix B was more conserved in Paramecium species. Despite various substitutions in the primary sequence, it was observed that secondary structures of ITS1 and ITS2 could be helpful in interpreting the phylogenetic relationships both at species as well as at generic level.

ACTB Loss-of-Function Mutations Result in a Pleiotropic Developmental Disorder.

ACTB encodes ß-actin, an abundant cytoskeletal housekeeping protein. In humans, postulated gain-of-function missense mutations cause Baraitser-Winter syndrome (BRWS), characterized by intellectual disability, cortical malformations, coloboma, sensorineural deafness, and typical facial features. To date, the consequences of loss-of-function ACTB mutations have not been proven conclusively. We describe heterozygous ACTB deletions and nonsense and frameshift mutations in 33 individuals with developmental delay, apparent intellectual disability, increased frequency of internal organ malformations (including those of the heart and the renal tract), growth retardation, and a recognizable facial gestalt (interrupted wavy eyebrows, dense eyelashes, wide nose, wide mouth, and a prominent chin) that is distinct from characteristics of individuals with BRWS. Strikingly, this spectrum overlaps with that of several chromatin-remodeling developmental disorders. In wild-type mouse embryos, ß-actin expression was prominent in the kidney, heart, and brain. ACTB mRNA expression levels in lymphoblastic lines and fibroblasts derived from affected individuals were decreased in comparison to those in control cells. Fibroblasts derived from an affected individual and ACTB siRNA knockdown in wild-type fibroblasts showed altered cell shape and migration, consistent with known roles of cytoplasmic ß-actin. We also demonstrate that ACTB haploinsufficiency leads to reduced cell proliferation, altered expression of cell-cycle genes, and decreased amounts of nuclear, but not cytoplasmic, ß-actin. In conclusion, we show that heterozygous loss-of-function ACTB mutations cause a distinct pleiotropic malformation syndrome with intellectual disability. Our biological studies suggest that a critically reduced amount of this protein alters cell shape, migration, proliferation, and gene expression to the detriment of brain, heart, and kidney development.

Lysyl oxidase-like 2 is a regulator of angiogenesis through modulation of endothelial-to-mesenchymal transition.

Lysyl oxidase-like 2 (LOXL2) belongs to the family of lysyl oxidases, and as such promotes crosslinking of collagens and elastin by oxidative deamination of lysine residues. In endothelial cells (ECs), LOXL2 is involved in crosslinking and scaffolding of collagen IV. Additionally, several reports have shown a role for LOXL2 in other processes, including regulation of gene expression, tumor metastasis, and epithelial-to-mesenchymal transition (EMT). Here, we demonstrate an additional role for LOXL2 in the regulation of angiogenesis by modulation of endothelial-to-mesenchymal transition (EndMT). LOXL2 knockdown in ECs results in decreased migration and sprouting, and concordantly, LOXL2 overexpression leads to an increase in migration and sprouting, independent of its catalytic activity. Furthermore, LOXL2 knockdown resulted in a reduced expression of EndMT markers, and inhibition of transforming growth factor-ß (TGF-ß)-mediated induction of EndMT. Interestingly, unlike in EMT, overexpression of LOXL2 alone is insufficient to induce EndMT. Further investigation revealed that LOXL2 expression regulates protein kinase B (PKB)/Akt and focal adhesion kinase (FAK) signaling, both pathways that have been implicated in the regulation of EMT. Altogether, our studies reveal a role for LOXL2 in angiogenesis through the modulation of EndMT in ECs, independent of its enzymatic crosslinking activity.

A distinct neurodevelopmental syndrome with intellectual disability, autism spectrum disorder, characteristic facies, and macrocephaly is caused by defects in CHD8.

A decade ago, we described novel de novo submicroscopic deletions of chromosome 14q11.2 in three children with developmental delay, cognitive impairment, and similar dysmorphic features, including widely-spaced eyes, short nose with flat nasal bridge, long philtrum, prominent Cupid's bow of the upper lip, full lower lip, and auricular anomalies. We suggested that this constituted a new multiple congenital anomaly-intellectual disability syndrome due to defects in CHD8 and/or SUPT16H. The three patients in our original cohort were between 2 years and 3 years of age at the time. Here we present a fourth patient and clinical updates on our previous patients. To document the longitudinal course more fully, we integrate published reports of other patients and describe genotype-phenotype correlations among them. Children with the disorder present with developmental delay, intellectual disability, and/or autism spectrum disorder in addition to characteristic facies. Gastrointestinal and sleep problems are notable. The identification of multiple patients with the same genetic defect and characteristic clinical phenotype, confirms our suggestion that this is a syndromic disorder caused by haploinsufficiency or heterozygous loss of function of CHD8.

Homozygous mutation in ELMO2 may cause Ramon syndrome.

We report on a girl, born to first cousin Lebanese parents, with intellectual disability, seizures, repeated gingivorrhagia, enlarged lower and upper jaws, overgrowth of the gums, high arched and narrow palate, crowded teeth, hirsutism of the back, large abdomen and a small umbilical hernia. Cysts of the mandible, fibrous dysplasia of bones, and enlarged adenoids causing around 60% narrowing of the nasopharyngeal airways were noted at radiographic examination. Her brother presented with the same features in addition to a short stature, an ostium secundum, and more pronounced intellectual disability. He died at the age of 8 years from a severe pulmonary infection and repeated bleeding episodes. A clinical diagnosis of Ramon syndrome was made. Whole exome sequencing studies performed on the family revealed the presence of a novel homozygous missense mutation in ELMO2 gene, p.I606S in the affected individuals. Loss of function mutations in ELMO2 have been recently described in another clinically distinct condition: primary intraosseous vascular malformation or intraosseous hemangioma, called VMOS. Review of the literature and differential diagnoses are discussed.

Comprehensive whole genome sequence analyses yields novel genetic and structural insights for Intellectual Disability.

BACKGROUND: Intellectual Disability (ID) is among the most common global disorders, yet etiology is unknown in ~30% of patients despite clinical assessment. Whole genome sequencing (WGS) is able to interrogate the entire genome, providing potential to diagnose idiopathic patients. METHODS: We conducted WGS on eight children with idiopathic ID and brain structural defects, and their normal parents; carrying out an extensive data analyses, using standard and discovery approaches. RESULTS: We verified de novo pathogenic single nucleotide variants (SNV) in ARID1B c.1595delG and PHF6 c.820C > T, potentially causative de novo two base indels in SQSTM1 c.115_116delinsTA and UPF1 c.1576_1577delinsA, and de novo SNVs in CACNB3 c.1289G > A, and SPRY4 c.508 T > A, of uncertain significance. We report results from a large secondary control study of 2081 exomes probing the pathogenicity of the above genes. We analyzed structural variation by four different algorithms including de novo genome assembly. We confirmed a likely contributory 165 kb de novo heterozygous 1q43 microdeletion missed by clinical microarray. The de novo assembly resulted in unmasking hidden genome instability that was missed by standard re-alignment based algorithms. We also interrogated regulatory sequence variation for known and hypothesized ID genes and present useful strategies for WGS data analyses for non-coding variation. CONCLUSION: This study provides an extensive analysis of WGS in the context of ID, providing genetic and structural insights into ID and yielding diagnoses.

CXCR4 blockade with AMD3100 enhances Taxol chemotherapy to limit ovarian cancer cell growth.

The standard of care for ovarian cancer includes initial treatment with chemotherapy. Despite initial efficacy, over 70% of patients develop recurrence; thus, there is a need to identify novel approaches that can improve therapeutic outcomes. We evaluated AMD3100 (Plerixafor), an FDA-approved CXCR4 inhibitor, as a potential adjunctive therapy for low-dose Taxol (Paclitaxel) by assessing the impact on in-vitro ovarian cancer cell proliferation. Proliferation was a measure for both human TOV-112D and murine ID8 ovarian cancer cells incubated with AMD3100 and Taxol, either individually or in combination. Impact of treatment was first determined for the simultaneous administration of AMD3100 and Taxol. We next assessed a sequential application of AMD3100 pretreatment, followed by AMD3100, Taxol, or a combination to test for sensitization to Taxol. In addition, we measured the impact of AMD3100 and Taxol, individually and in combination, on colony formation, an in-vitro model assay of tumor growth. Expression data, as measured by flow cytometry, show that both ID8 and TOV-112D cells are positive for CXCR4, CXCR7, and CXCL12. Combination treatment with AMD3100 (≤10 µmol/l) sensitized both ID8 and TOV-112D cells to low concentrations of Taxol (≤5 nmol/l), limiting cell proliferation and colony formation in vitro. Pretreatment with AMD3100 significantly increased the sensitivity of human ovarian cancer to low-dose Taxol or the combination of AMD3100 and Taxol, although this effect was not evident in murine cells. Importantly, for both human and murine cells, incubation with a combination of AMD3100 and Taxol had the largest impact on limiting cell proliferation. AMD3100 in combination with low-dose Taxol offers improved efficacy and the potential of reduced toxicity for the treatment of ovarian cancer.

Squamous neoplasms arising within tattoos: clinical presentation, histopathology and management.

Tattooing, which involves the placement of ink into the skin, is an ancient decorative technique that has remained popular in modern society. Tattoos have long been known to cause cutaneous reactions, which include the emergence of neoplasms such as keratoacanthoma (KA) and squamous cell carcinoma (SCC) in tattooed areas of the skin. We review the clinical presentations, histology and treatment options for squamous neoplasms, primarily KA and SCC, arising in tattoos.

Effect of simulated pulpal blood flow rate on the rise in pulp chamber temperature during direct fabrication of exothermic provisional restorations.

AIM: To evaluate ex vivo the effect of several simulated pulpal blood flow rates on the change in pulp chamber temperature during direct fabrication of a provisional restoration using a polymethylmethacrylate (PMMA) resin. METHODOLOGY: Fifteen noncarious human premolars were prepared for complete coverage restorations. A curved needle connected to a peristaltic pump simulated the pulp blood flow. Two K-type thermocouples connected to a digital thermometer were placed in the pulp chamber, and the assembly was placed in an incubator at 37 °C. Three provisional crowns were made for each specimen using no water flow (group 1), a 1-mL min-1 flow rate (group 2) and a 0.5-mL/min-1 flow rate (group 3). The pulp chamber temperature was recorded continuously during polymerization until the temperature increase peaked and started to decrease and reached the baseline temperature (37 °C). The temperature increase was measured for the three water flow conditions. Data were analysed statistically using descriptive statistics, repeated measures one-way analysis of variance (anova) with Greenhouse-Geisser correction and Bonferroni tests. The level of significance was set at P < 0.05. RESULTS: All of the groups were associated with an increased pulp chamber temperature. Groups with flow rates at 1 and 0.5 mL min-1 had a significantly lower temperature rise when compared to the group without water flow (P < 0.001). CONCLUSIONS: Direct fabrication of provisional restorations can cause a critical increase in pulp chamber temperature. However, in the presence of simulated pulpal blood flow rates of 1 or 0.5 mL min-1 , the increase in pulp chamber temperature did not exceed the critical threshold (5.6 °C).

Intragenic CNVs for epigenetic regulatory genes in intellectual disability: Survey identifies pathogenic and benign single exon changes.

The disruption of genes involved in epigenetic regulation is well known to cause Intellectual Disability (ID). We reported a custom microarray study that interrogated among others, the epigenetic regulatory gene-class, at single exon resolution. Here we elaborate on identified intragenic CNVs involving epigenetic regulatory genes; specifically discussing those in three genes previously unreported in ID etiology-ARID2, KDM3A, and ARID4B. The changes in ARID2 and KDM3A are likely pathogenic while the ARID4B variant is uncertain. Previously, we found a CNV involving only exon 6 of the JARID2 gene occurred apparently de novo in seven patients. JARID2 is known to cause ID and other neurodevelopmental conditions. However, exon 6 of this gene encodes one of a series of repeated motifs. We therefore, investigated the impact of this variant in two cohorts and present a genotype-phenotype assessment. We find the JARID2 exon 6 CNV is benign, with a high population frequency (>14%), but nevertheless could have a contributory effect. We also present results from an interrogation of the exomes of 2,044 patients with neurocognitive phenotypes for the incidence of potentially damaging mutation in the epigenetic regulatory gene-class. This paper provides a survey of the fine-scale CNV landscape for epigenetic regulatory genes in the context of ID, describing likely pathogenic as well as benign single exon imbalances. © 2016 Wiley Periodicals, Inc.

Arsenic processing of yeast isolates IIB-As1 & IIB-As2 and production of glutathione under stress conditions.

Four arsenic resistant yeast were isolated from the industrial wastewater. Two strains IIB-As1 and IIB-As2 identified as Candida tropicalis and Saccharomyces cerevisiae, respectively. IIB-As1 and IIB-As2 showed maximum arsenic resistance. IIB-As1 showed maximum growth at 35 °C whereas it was 30 °C for IIB-As2. The yeast isolate showed typical growth curves, but arsenic extended the lag phase. Glutathione plays an important role in metal tolerance. In the present study, As increased the level glutathione and non-protein thiols in yeast isolates. Removal of As from supernatant was analyzed using the atomic absorption spectrophotometer. They removed arsenic from the medium after 72 h of incubation. Both yeast strains efficiently removed arsenic from the industrial effluent when used individually or in consortia.

Variability in secondary structure of 18S ribosomal RNA as topological marker for identification of Paramecium species.

Besides cytological and molecular applications, Paramecium is being used in water quality assessment and for determination of saprobic levels. An unambiguous identification of these unicellular eukaryotes is not only essential, but its ecological diversity must also be explored in the local environment. 18SrRNA genes of all the strains of Paramecium species isolated from waste water were amplified, cloned and sequenced. Phylogenetic comparison of the nucleotide sequences of these strains with 23 closely related Paramecium species from GenBank Database enabled identification of Paramecium multimicronucleatum and Paramecium jenningsi. Some isolates did not show significant close association with other Paramecium species, and because of their unique position in the phylogenetic tree, they were considered new to the field. In the present report, these isolates are being designated as Paramecium caudatum pakistanicus. In this article, secondary structure of 18SrRNA has also been analyzed as an additional and perhaps more reliable topological marker for species discrimination and for determining possible phylogenetic relationship between the ciliate species. On the basis of comparison of secondary structure of 18SrRNA of various isolated Paramacium strains, and among Paramecium caudatum pakistanicus, Tetrahymena thermophila, Drosophila melanogaster, and Homo sapiens, it can be deduced that variable regions are more helpful in differentiating the species at interspecific level rather than at intraspecific level. It was concluded that V3 was the least variable region in all the organisms, V2 and V7 were the longest expansion segments of D. melanogaster and there was continuous mutational bias towards G.C base pairing in H. sapiens.