Prenatal diagnosis of mucopolysaccharidosis type VII facilitating treatment in neonatal period.

Duo exome testing was performed on a fetus conceived via in vitro fertilization with an egg donor. The fetus presented with non-immune hydrops fetalis (NIHF) at 20 + 0 weeks gestation. Two variants were detected in the GUSB gene. Biallelic pathogenic variants cause mucopolysaccharidosis type VII (MPS-VII), which can present with NIHF prenatally. At the time of analysis and initial report, one variant was classified as likely pathogenic and the other as of uncertain clinical significance. Biochemical testing of the amniotic fluid supernatant showed elevated glycosaminoglycans and low ß-glucuronidase activity consistent with the diagnosis of MPS-VII. This evidence allowed the upgrade of the pathogenicity for both variants, confirming the diagnosis of MPS-VII. The infant was born at 36 + 5 weeks and enzyme replacement therapy (ERT) using vestronidase was initiated at 20 days with planning for hematopoietic stem cell transplant ongoing. The ERT therapy has been well tolerated, with decreasing quantitative urine glycosaminoglycans. Long-term follow up is required to determine whether treatment has been successful. This case demonstrates the utility of alternative testing methods to clarify the pathogenicity of variants and the clinical utility of obtaining a diagnosis antenatally in facilitating treatment in the neonatal period, and specifically highlights MPS-VII as a treatable cause of NIHF.

A novel SMOC1 pathogenic homozygous variant in a fetus with mesomelia of the lower limbs, micrognathia and hypertelorism and an incidental finding of CYP21A2-related congenital adrenal hyperplasia.

Trio exome sequencing was performed on a fetus with bilateral mesomelia of the lower limbs with significant angulation of the tibial bones, micrognathia and hypertelorism detected on ultrasound scan at 19 + 0 weeks gestation. The couple is consanguineous. A homozygous pathogenic frameshift variant in the SMOC1 gene (c.339_340del p.(Phe114Cysfs*40)) was detected and both parents were shown to be heterozygous. Pathogenic variants in the SMOC1 gene are associated with microphthalmia with limb anomalies which multidisciplinary team discussion determined to be causal of the scan anomalies detected. The fetus was also a compound heterozygote for CYP21A2 pathogenic variants, confirming a second diagnosis of non-classical congenital adrenal hyperplasia, which was felt incidental to the scan findings. The risk that this couple's next pregnancy would be affected by either of these disorders is 1 in 4 (25%) and demonstrates the importance of genetic diagnoses for the family and implications for future pregnancies.

Lessons learnt from prenatal exome sequencing.

BACKGROUND: Prenatal exome sequencing (ES) for monogenic disorders in fetuses with structural anomalies increases diagnostic yield. In England there is a national trio ES service delivered from two laboratories. To minimise incidental findings and reduce the number of variants investigated, analysis uses a panel of 1205 genes where pathogenic variants may cause abnormalities presenting prenatally. Here we review our laboratory's early experience developing and delivering ES to identify challenges in interpretation and reporting and inform service development. METHODS: A retrospective laboratory records review from 01.04.2020 to 31.05.2021. RESULTS: Twenty-four of 116 completed cases were identified as challenging including 13 resulting in difficulties in analysis and reporting, nine where trio inheritance filtering would have missed the diagnosis, and two with no prenatal diagnosis; one due to inadequate pipeline sensitivity, the other because the gene was not on the panel. Two cases with copy number variants identified were not detectable by microarray. CONCLUSIONS: Variant interpretation requires close communication between referring clinicians, with occasional additional examination of the fetus or parents and communication of evolving phenotypes. Inheritance filtering misses ∼5% of diagnoses. Panel analysis reduces but does not exclude incidental findings. Regular review of published literature is required to identify new reports that may aid classification.

ZMYND11-related syndromic intellectual disability: 16 patients delineating and expanding the phenotypic spectrum.

Pathogenic variants in ZMYND11, which acts as a transcriptional repressor, have been associated with intellectual disability, behavioral abnormalities, and seizures. Only 11 affected individuals have been reported to date, and the phenotype associated with pathogenic variants in this gene have not been fully defined. Here, we present 16 additional patients with predicted pathogenic heterozygous variants in including four individuals from the same family, to further delineate and expand the genotypic and phenotypic spectrum of ZMYND11-related syndromic intellectual disability. The associated phenotype includes developmental delay, particularly affecting speech, mild-moderate intellectual disability, significant behavioral abnormalities, seizures, and hypotonia. There are subtle shared dysmorphic features, including prominent eyelashes and eyebrows, a depressed nasal bridge with bulbous nasal tip, anteverted nares, thin vermilion of the upper lip, and wide mouth. Novel features include brachydactyly and tooth enamel hypoplasia. Most identified variants are likely to result in premature truncation and/or nonsense-mediated decay. Two ZMYND11 variants located in the final exon-p.(Gln586*) (likely escaping nonsense-mediated decay) and p.(Cys574Arg)-are predicted to disrupt the MYND-type zinc-finger motif and likely interfere with binding to its interaction partners. Hence, the homogeneous phenotype likely results from a common mechanism of loss-of-function.

Case report: targeted whole exome sequencing enables the first prenatal diagnosis of the lethal skeletal dysplasia Osteocraniostenosis.

BACKGROUND: Osteocraniostenosis (OCS) is a rare genetic disorder characterised by premature closure of cranial sutures, gracile bones and perinatal lethality. Previously, diagnosis has only been possible postnatally on clinical and radiological features. This study describes the first prenatal diagnosis of OCS. CASE PRESENTATION: In this case prenatal ultrasound images were suggestive of a serious but non-lethal skeletal dysplasia. Due to the uncertain prognosis the parents were offered Whole Exome Sequencing (WES), which identified a specific gene mutation in the FAMIIIa gene. This mutation had previously been detected in two cases and was lethal in both perinatally. This established the diagnosis, a clear prognosis and allowed informed parental choice regarding ongoing pregnancy management. CONCLUSIONS: This case report supports the use of targeted WES prenatally to confirm the underlying cause and prognosis of sonographically suspected abnormalities.

Identification of PKD1 and PKD2 gene variants in a cohort of 125 Asian Indian patients of ADPKD.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) accounts for 2.6% of the patients with chronic kidney disease in India. ADPKD is caused by pathogenic variants in either PKD1 or PKD2 gene. There is no comprehensive genetic data from Indian subcontinent. We aimed to identify the pathogenic variants in the heterogeneous Indian population. PKD1 and PKD2 variants were identified by direct gene sequencing and/or multiplex ligation-dependent probe amplification (MLPA) in 125 unrelated patients of ADPKD. The pathogenic potential of the variants was evaluated computationally and were classified according to ACMG guidelines. Overall 300 variants were observed in PKD1 and PKD2 genes, of which 141 (47%) have been reported previously as benign. The remaining 159 variants were categorized into different classes based on their pathogenicity. Pathogenic variants were observed in 105 (84%) of 125 patients, of which 99 (94.3%) were linked to PKD1 gene and 6 (6.1%) to PKD2 gene. Of 159 variants, 97 were novel variants, of which 43 (44.33%) were pathogenic, and 10 (10.31%) were of uncertain significance. Our data demonstrate the diverse genotypic makeup of single gene disorders in India as compared to the West. These data would be valuable in counseling and further identification of probable donors among the relatives of patients with ADPKD.

TM4SF1 Regulates Pancreatic Cancer Migration and Invasion In Vitro and In Vivo.

BACKGROUND/AIMS: The cell surface protein transmembrane 4 L6 family member 1 (TM4SF1) has been detected in various tumors and plays a major role in the development of cancer. We aimed to investigate the effects of TM4SF1 on the migration and invasion of pancreatic cancer in vitro and in vivo and explore its related molecular mechanisms. METHODS: qRT-PCR and immunohistochemical analyses were used to measure the expression of TM4SF1 in pancreatic cancer tissues and adjacent tissues. TM4SF1 was silenced using siRNA and shRNA to investigate the role of this protein in the proliferation and metastasis of pancreatic cancer cells. MTS and Transwell assays were used to examine the effect of TM4SF1 on pancreatic cancer cell lines. The expression and activity of MMP-2 and MMP-9 were determined by qRT-PCR, western blots and gelatin zymography. In vivo, orthotopic pancreatic tumor models were used to examine the formation of metastasis. RESULTS: qRT-PCR and immunohistochemical analyses showed that TM4SF1 was highly expressed in pancreatic cancer tissues compared with the adjacent tissues. In in vitro experiments the silencing of TM4SF1 reduced cell migration and invasion and down-regulated the expression and activity of MMP-2 and MMP-9. However, no significant difference in cell proliferation was detected after silencing TM4SF1. Additionally, knocking down TM4SF1 decreased the formation of lung and liver metastases in orthotopic pancreatic tumor models. CONCLUSION: Our results demonstrate that the expression of TM4SF1 is higher in pancreatic cancer tissues and pancreatic cancer cell lines than controls. Knockdown of TM4SF1 inhibited the migration and invasion of pancreatic cancer cells by regulating the expression and activity of MMP-2 and MMP-9, which suggests that TM4SF1 may play a significant role in metastasis in pancreatic cancer.

Animal Models of Gastrointestinal and Liver Diseases. The difficulty of animal modeling of pancreatic cancer for preclinical evaluation of therapeutics.

Pancreatic ductal adenocarcinoma (PDAC) is relatively rare but extremely lethal. Standard cytotoxic therapeutics provide little benefit. To date, newer targeted therapeutics have also not been highly successful. Often novel therapeutics that have appeared to perform well in preclinical models have failed in the clinic. Many factors contribute to these failures, but the one most often attributed is the shortcomings of the preclinical models. A plethora of animal models now exist for PDAC, including cell line xenografts, patient-derived xenografts, a wide variety of genetic mouse models, and syngeneic xenografts. These models have generated a tremendous amount of information useful for the understanding of PDAC. Yet none seems to well predict clinical outcomes of new treatments. This review will discuss how genetic instability and cellular heterogeneity make this disease so difficult to model accurately. We will also discuss the strengths and weaknesses of many of the popular models. Ultimately we will argue that there is no perfect model and that the best approach to understanding clinical performance is the use of multiple preclinical models with an understanding of their salient features.

An enhanced proportional resonance controller design for the PMSM based electric vehicle drive system.

Permanent magnet synchronous machine (PMSM) has proven to be a more economical traction drive system for electric vehicle (EV) applications owing to increased efficiency and high-power density. However, the drive system requires more efficient control schemes to deliver better dynamic performance irrespective of dynamic changes in the motor speed, machine parameters and disturbances. Hence, to tackle the dynamic changes, to enhance the wider operating speed, to achieve precise speed tracking capability, and improved efficiency, a novel control algorithm for the PMSM based EV is proposed in this paper. The control algorithm is implemented by adopting the merits of conventional proportional resonance (PR) and proportional integral (PI) controller. The proposed control strategy is designed with an outer PI speed regulator and the inner enhanced PR (EPR) current regulator. The uniqueness of the proposed EPR controller is that the controller is designed to damp the torsional mode oscillation owing to dynamic changes such as speed and torque regulation evading the additional control loop. The effectiveness of the control scheme is tested in MATLAB Simulink and hardware-in-loop (HIL) real time simulator RT5700. To validate the effectiveness of the proposed control scheme the results are compared with the conventional control schemes. The results presented show that the proposed control technique successfully enhances the static and dynamic performance, and resilience of the EV system. Also, the proposed scheme significantly reduces the flux ripples, torque ripples, current jitter, peak overshoot, undershoot compared to the conventional current controllers.

Adrenomedullin is up-regulated in patients with pancreatic cancer and causes insulin resistance in ß cells and mice.

BACKGROUND & AIMS: New-onset diabetes in patients with pancreatic cancer is likely to be a paraneoplastic phenomenon caused by tumor-secreted products. We aimed to identify the diabetogenic secretory product(s) of pancreatic cancer. METHODS: Using microarray analysis, we identified adrenomedullin as a potential mediator of diabetes in patients with pancreatic cancer. Adrenomedullin was up-regulated in pancreatic cancer cell lines, in which supernatants reduced insulin signaling in beta cell lines. We performed quantitative reverse-transcriptase polymerase chain reaction and immunohistochemistry on human pancreatic cancer and healthy pancreatic tissues (controls) to determine expression of adrenomedullin messenger RNA and protein, respectively. We studied the effects of adrenomedullin on insulin secretion by beta cell lines and whole islets from mice and on glucose tolerance in pancreatic xenografts in mice. We measured plasma levels of adrenomedullin in patients with pancreatic cancer, patients with type 2 diabetes mellitus, and individuals with normal fasting glucose levels (controls). RESULTS: Levels of adrenomedullin messenger RNA and protein were increased in human pancreatic cancer samples compared with controls. Adrenomedullin and conditioned media from pancreatic cell lines inhibited glucose-stimulated insulin secretion from beta cell lines and islets isolated from mice; the effects of conditioned media from pancreatic cancer cells were reduced by small hairpin RNA-mediated knockdown of adrenomedullin. Conversely, overexpression of adrenomedullin in mice with pancreatic cancer led to glucose intolerance. Mean plasma levels of adrenomedullin (femtomoles per liter) were higher in patients with pancreatic cancer compared with patients with diabetes or controls. Levels of adrenomedullin were higher in patients with pancreatic cancer who developed diabetes compared those who did not. CONCLUSIONS: Adrenomedullin is up-regulated in patients with pancreatic cancer and causes insulin resistance in ß cells and mice.

Analysis of Ellis van Creveld syndrome gene products: implications for cardiovascular development and disease.

Mutations identified in a cohort of patients with atrioventricular septal defects as a part of Ellis van Creveld syndrome (EvC syndrome) led us to study the role of two non-homologous genes, EVC and LBN, in heart development and disease pathogenesis. To address the cause of locus heterogeneity resulting in an indistinguishable heart-hand phenotype, we carried out in situ hybridization and immunofluorescence and identified co-localization of Evc and Lbn mRNA and protein. In the heart, expression was identified to be strongest in the secondary heart field, including both the outflow tract and the dorsal mesenchymal protrusion, but was also found in mesenchymal structures of the atrial septum and the atrioventricular cushions. Finally, we studied the transcriptional hierarchy of EVC and LBN but did not find any evidence of direct transcriptional interregulation between the two. Due to the locus heterogeneity of human mutations predicted to result in a loss of protein function, a bidirectional genomic organization and overlapping expression patterns, we speculate that these proteins function coordinately in cardiac development and that loss of this coordinate function results in the characteristics of EvC syndrome.

Characterization of novel neutralizing mouse monoclonal antibody JM1-24-3 developed against MUC18 in metastatic melanoma.

BACKGROUND: MUC18 is a glycoprotein highly expressed on the surface of melanoma and other cancers which promotes tumor progression and metastasis. However, its mechanism of action and suitability as a therapeutic target are unknown. METHODS: A monoclonal antibody (mAb) (JM1-24-3) was generated from metastatic melanoma tumor live cell immunization, and high-throughput screening identified MUC18 as the target. RESULTS: Analysis of molecular interactions between MUC18 and JM1-24-3 revealed that the downstream signaling events depended on binding of the mAb to a conformational epitope on the extracellular domain of MUC18. JM1-24-3 inhibited melanoma cell proliferation, migration and invasion in vitro and reduced tumor growth and metastasis in vivo. CONCLUSION: These results confirm that MUC18 is mechanistically important in melanoma growth and metastasis, suggest that the MUC18 epitope identified is a promising therapeutic target, and that the JM1-24-3 mAb may serve as the basis for a potential therapeutic agent.

Neuropilin-2-mediated tumor growth and angiogenesis in pancreatic adenocarcinoma.

PURPOSE: Neuropilin-2 (NRP-2) is a coreceptor for vascular endothelial growth factor (VEGF) on endothelial cells. NRP-2 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) cells relative to nonmalignant ductal epithelium. This study determined the role of NRP-2 in PDAC cells. EXPERIMENTAL DESIGN: NRP-2 expression was reduced in PDAC cells with stable short-hairpin RNA (shRNA) transfection. Western blotting was done to evaluate signaling intermediates. Migration and invasion studies were carried out in Boyden chambers. Anchorage-independent growth was assessed by soft-agar colony formation. In vivo growth was evaluated using murine subcutaneous and orthotopic xenograft models. Immunohistochemical analysis evaluated in vivo proliferation and angiogenesis. RESULTS: shRNA-NRP-2 decreased NRP-2 levels without affecting neuropilin-1 levels. Akt activation was decreased in clones with reduced NRP-2 (shRNA-NRP-2). shRNA-NRP-2 cells showed decreased migration, invasion, and anchorage-independent growth compared with control cells. In vitro proliferation rates were similar in control- and shRNA-transfected cells. Subcutaneous and orthotopic xenografts from shRNA-transfected cells were significantly smaller than those resulting from control-transfected cells (P < 0.05). Furthermore, shRNA-NRP-2 tumors exhibited less cellular proliferation and decreased microvascular area relative to control tumors (P < 0.05). Constitutive expression of the angiogenic mediator Jagged-1 was reduced in shRNA-NRP-2 cells, whereas vascular endothelial growth factor levels were unchanged. CONCLUSION: Reduction of NRP-2 expression in PDAC cells decreased survival signaling, migration, invasion, and ability to grow under anchorage-independent conditions. In vivo, reduction of NRP-2 led to decreased growth of xenograft tumors and decreased vascular area, which was associated with decreased Jagged-1 levels. NRP-2 is a potential therapeutic target on PDAC cells.

Nuclear factor-kappaB p65/relA silencing induces apoptosis and increases gemcitabine effectiveness in a subset of pancreatic cancer cells.

PURPOSE: Nuclear factor kappaB (NFkappaB) activity may increase survival and protect cancer cells from chemotherapy. Therefore, NFkappaB activity may be prognostic, and inhibition of NFkappaB may be useful for pancreatic cancer therapy. To test these hypotheses, we examined NFkappaB activity and the effects of inhibiting NFkappaB in several pancreatic cancer cell lines with differing sensitivities to gemcitabine. EXPERIMENTAL DESIGN: The gemcitabine sensitivity of pancreatic cancer cell lines BxPC-3, L3.6pl, CFPAC-1, MPanc-96, PANC-1, and MIA PaCa-2 were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and fluorescence-activated cell sorting assays. NFkappaB levels were determined by electrophoretic mobility shift assay and reporter assays. The effects of gemcitabine on NFkappaB activity were determined in vitro and in vivo. NFkappaB was inhibited by silencing of the p65/relA subunit using small interfering RNA in vitro and by neutral liposomal delivery of small interfering RNA in vivo, and the effects were evaluated on gemcitabine sensitivity. RESULTS: The cell lines L3.6pl, BxPC-3, and CFPAC-1 were sensitive, whereas MPanc-96, PANC-1, and MIA PaCa-2 were resistant to gemcitabine. No significant correlation was observed between basal NFkappaB activity and gemcitabine sensitivity. Gemcitabine treatment did not activate NFkappaB either in vitro or in vivo. Silencing of p65/relA induced apoptosis and increased gemcitabine killing of all gemcitabine-sensitive pancreatic cancer cells. No significant effects, however, were observed on gemcitabine-resistant pancreatic cancer cell lines either in vitro or in vivo. CONCLUSIONS: NFkappaB activity did not correlate with sensitivity to gemcitabine. Silencing of p65/relA was effective alone and in combination with gemcitabine in gemcitabine-sensitive but not gemcitabine-resistant pancreatic cancer cells. Thus, NFkappaB may be a useful therapeutic target for a subset of pancreatic cancers.

Adrenomedullin is expressed in pancreatic cancer and stimulates cell proliferation and invasion in an autocrine manner via the adrenomedullin receptor, ADMR.

The current study investigated adrenomedullin as a potential autocrine regulator of pancreatic cancer cell function. Adrenomedullin was localized in the neoplastic epithelium of 90% (43 of 48) of human pancreatic adenocarcinomas analyzed by immunohistochemistry and was expressed by 100% (8 of 8) of pancreatic cancer cell lines analyzed by reverse transcription-PCR. Pancreatic cancer cell lines also secreted adrenomedullin into the culture medium as determined by ELISA (5 of 5). Exogenous adrenomedullin treatment of Panc-1, BxPC3, and MPanc96 cells in vitro stimulated cell proliferation, invasion, and nuclear factor kappaB activity, indicating the ability of the cells to respond to adrenomedullin. Treatment of the cell cultures with an adrenomedullin antagonist inhibited basal levels of proliferation and nuclear factor kappaB activity, supporting the autocrine function of this molecule. Furthermore, increasing adrenomedullin levels by gene transfer to Panc-1 cells increased, whereas adrenomedullin small hairpin RNA silencing in MPanc96 cells inhibited tumor growth and metastasis in vivo. Adrenomedullin is able to act through at least two different receptors, adrenomedullin receptor (ADMR) and calcitonin receptor-like receptor (CRLR). Reverse transcription-PCR and Western blotting indicated that pancreatic cancer cells expressed only ADMR but not CRLR. In contrast, cells found in the tumor microenvironment, primary human pancreatic stellate and endothelial (HUVEC) cells, expressed both ADMR and CRLR. Small hairpin RNA silencing of ADMR in pancreatic cancer cells blocked adrenomedullin-induced growth and invasion, indicating that this receptor is involved in the autocrine actions of adrenomedullin. These data indicate that adrenomedullin acting via ADMR increases the aggressiveness of pancreatic cancer cells and suggests that these molecules may be useful therapeutic targets.

Suppression of stromal-derived Dickkopf-3 (DKK3) inhibits tumor progression and prolongs survival in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, and it is unclear whether its stromal infiltrate contributes to its aggressiveness. Here, we demonstrate that Dickkopf-3 (DKK3) is produced by pancreatic stellate cells and is present in most human PDAC. DKK3 stimulates PDAC growth, metastasis, and resistance to chemotherapy with both paracrine and autocrine mechanisms through NF-κB activation. Genetic ablation of DKK3 in an autochthonous model of PDAC inhibited tumor growth, induced a peritumoral infiltration of CD8+ T cells, and more than doubled survival. Treatment with a DKK3-blocking monoclonal antibody inhibited PDAC progression and chemoresistance and prolonged survival. The combination of DKK3 inhibition with immune checkpoint inhibition was more effective in reducing tumor growth than either treatment alone and resulted in a durable improvement in survival, suggesting that DKK3 neutralization may be effective as a single targeted agent or in combination with chemotherapy or immunotherapy for PDAC.

Does the private sector care about AIDS? Evidence from firm surveys in East Africa.

OBJECTIVE: Our objective was to identify the determinants of HIV/AIDS prevention activity and pre-employment health checks by private firms in Kenya, Uganda and Tanzania. DESIGN: We used data from the World Bank Enterprise Surveys for Uganda, Kenya and Tanzania, encompassing 860 formally registered firms in the manufacturing sector. METHODS: Econometric analysis of firm survey data was used to identify the determinants of HIV/AIDS prevention including condom distribution and voluntary counselling and testing (VCT). Multivariate regression analysis was the main tool used to determine statistical significance. RESULTS: Approximately a third of enterprises invest in HIV/AIDS prevention. Prevention activity increases with size, most likely because larger firms and firms with higher skilled workers have greater replacement costs. Even in the category of larger firms, less than 50% provide VCT. We found that the propensity of firms to carry out pre-employment health checks of workers also varies by the size of firm and skill level of the workforce. Finally, data from worker surveys showed a high degree of willingness on the part of workers to be tested for HIV in the three East African countries.