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Esther TL Lau,1,2 Shirin H Tan,2,3 Yasmin J Antwertinger,1 Tony Hall,1 Lisa M Nissen1,21School of Clinical Sciences, Queensland University of Technology, Brisbane, QLD, Australia; 2School of Pharmacy, University of Queensland, Brisbane, QLD, Australia; 3Clinical Research Center, Sarawak General Hospital, Kuching, Sarawak, MalaysiaBackground: People living with persistent pain in Australia often cannot access adequate care to manage their pain. Therefore, as the most accessible healthcare professionals, community pharmacists have an important role to play in helping to improve patient outcomes. Hence, it is important to investigate patient needs and expectations in terms of counseling interactions with pharmacists, along with pharmacists’ approach to counseling interactions with these patients.Method: The nature of patient–pharmacist counseling interactions was explored with seven patients (one focus group), and 10 practicing pharmacists (two focus groups, three semi-structured interviews). The themes identified informed the development of an online survey that was advertised online to patients and pharmacists across Australia.Results: A total of 95 patients and 208 pharmacists completed the survey. Overall, more than half of patients (77/95) were satisfied with the care provided by their pharmacist, but only a third (71/205) of pharmacists were satisfied with the care they provided to patients. The majority of patients (67/94) reported that pharmacists provided good information about medications. This aligned with pharmacists’ responses, as most reported focusing on medication side effects (118/188) and instructions for taking pain medication (93/183) during patient interactions. However, when asked about empathy and rapport from pharmacists, only half to two-thirds (48–61/95) of patients expressed positive views. Overall, half of the patients (39/75) wanted a caring, empathetic, respectful, and private conversation with the pharmacist, and nearly half (40/89) perceived the pharmacist’s role as providing (new) information on alternative pharmacological and non-pharmacological therapies, including general advice on pain management.Conclusion: There was a disparity in the nature of the interaction and information that patients wanted from pharmacists, compared to what was provided by pharmacists. Training and education may help pharmacists to better engage in patient-centered care when interacting with people living with persistent pain, thereby improving health outcomes for these patients.Keywords: persistent, chronic, pain, pharmacist, counseling

Aida Sefidani Forough,1 Esther TL Lau,1 Kathryn J Steadman,1,2 Julie AY Cichero,1,2 Greg J Kyle,1 Jose Manuel Serrano Santos,1 Lisa M Nissen1 1School of Clinical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, QLD, Australia; 2School of Pharmacy, University of Queensland, Brisbane, QLD, Australia Abstract: Solid oral dosage forms such as tablets and capsules are generally the preferred method of drug delivery due to their convenience, cost, and acceptability. However, for many people, it can be a challenge to swallow solid oral medications, even those with healthy swallowing function. This review describes current strategies available to facilitate medication administration to otherwise healthy people with pill-swallowing difficulties. In general, restoring and maintaining the ability to swallow pills whole should ideally be the first choice in managing people with pill-swallowing difficulties. A number of strategies can potentially make it easier to swallow pills whole. These include postural adjustments, using pill-swallowing aids, and teaching pill-swallowing techniques. Where these are not successful or appropriate, then other approaches have to be considered using alternative formulations/routes of administration or deprescribing. If there is no other option, and it is not directly contraindicated for each specific medication dosage form, pills may be modified and mixed in foods and drinks to aid swallowing. In conclusion, people with pill-swallowing difficulties can benefit from a number of strategies designed to facilitate swallowing medications. However, these strategies should be further evaluated with regard to the evidence relating to both their efficacy and safety. Keywords: swallowing difficulties, pill swallowing, medication administration, oral dosage forms, tablets, capsules

Thi-My-Uyen Nguyen,1 Esther TL Lau,1,3 Kathryn J Steadman,1 Julie AY Cichero,1 Kaeleen Dingle,2 Lisa M Nissen1,3 1School of Pharmacy, University of Queensland, Brisbane, QLD, Australia; 2School of Public Health, Queensland University of Technology, Brisbane, QLD, Australia; 3School of Clinical Sciences, Queensland University of Technology, Brisbane, QLD, Australia Background: People often modify oral solid dosage forms when they experience difficulty swallowing them. Modifying dosage forms may cause adverse effects to the patient, and the person undertaking the modification. Pharmacists are often the first point of contact for people in the general community seeking advice regarding medications. Nurses are at the forefront of administering medications to patients and are likely to be most directly affected by a patient’s swallowing ability, while general practitioners (GPs) are expected to consider swallowing abilities when prescribing medications. Objective: To compare the perspectives and experiences of GPs, pharmacists, and nurses regarding medication dosage form modification and their knowledge of medication modification. Method: Questionnaires tailored to each profession were posted to 630 GPs, and links to an online version were distributed to 2,090 pharmacists and 505 nurses. Results: When compared to pharmacists and GPs, nurses perceived that a greater proportion of the general community modified solid dosage forms. Pharmacists and GPs were most likely to consider allergies and medical history when deciding whether to prescribe or dispense a medicine, while nurses’ priorities were allergies and swallowing problems when administering medications. While nurses were more likely to ask their patients about their ability to swallow medications, most health professionals reported that patients “rarely” or “never” volunteered information about swallowing difficulties. The majority of health professionals would advise a patient to crush or split noncoated non-sustained-release tablets, and would consult colleagues or reference sources for sustained-release or coated tablets. Health professionals appeared to rely heavily upon the suffix attached to medication names (which suggest modified release properties) to identify potential problems associated with modifying medications. Conclusion: The different professional roles and responsibilities of GPs, pharmacists, and nurses are associated with different perspectives of, and experiences with, people modifying medications in the general community and knowledge about consequences of medication modification. Keywords: crushing tablets, opening capsules, dosage form modification, health professional

Competing Interests: Intérêts concurrents: Rose Yeung a reçu une subvention de recherche du Fonds albertain Novo Nordisk pour le diabète (Fondation de l’Hôpital universitaire, société Novo Nordisk et ministère du Développement économique et du Commerce de l’Alberta). Rose Yeung est directrice médicale du Bureau de l’éducation permanente et du Programme de formation médicale de l’Université de l’Alberta; ce programme est subventionné par le gouvernement de l’Alberta. Darren Lau a reçu des honoraires du Collège des médecins de famille de l’Alberta. Aucun autre intérêt concurrent n’a été déclaré.

Objective: There is a lack of literature on indirect decompression in uniportal endoscopic posterolateral transforaminal lumbar interbody fusion (EPTLIF). Our aim is to evaluate the dimensions of the spinal canal and contralateral foramen before and after EPTLIF., Methods: This is a retrospective study of patients who underwent EPTLIF in a tertiary spine centre over a 2-year period. The cross-sectional area of the spinal canal and the contralateral foramen at the level of fusion were measured on magnetic resonance imaging scan at 1-day postoperation and at the final follow-up. Patients were grouped according to the decompression performed as per the clinician's judgement., Results: One hundred fifty-two levels of fusion were performed in 120 patients. There was a statistically significant clinical improvement in visual analogue scale and Oswestry Disability Index scores postoperation. The measurements of the spinal canal area were 106.0 mm2, 138.8 mm2, and 195.5 mm2; while contralateral foraminal area were 73.2 mm2, 104.4 mm2, and 120.7 mm2 at preoperation, 1-day postoperation, and at the final follow-up, respectively (p < 0.001). For the subgroup analyses, spinal canal area measurements for the bilateral decompression cohort (n = 35) were 57.0 mm2, 123.9 mm2, and 191.8 mm2; for the ipsilateral decompression cohort (n = 42) were 89.3 mm2, 128.9 mm2, 183.3 mm2; and for the cohort without any decompression and only cage inserted (n = 75) were 138.3 mm2, 151.2 mm2, and 204.1 mm2 (p < 0.001). Contralateral foraminal area measurements were 73.3 mm2, 106.4 mm2 and 120.4 mm2 in the bilateral decompression cohort; 69.5 mm2, 99.0 mm2, 116.9 mm2 in the ipsilateral decompression cohort; and 75.1 mm2, 106.5 mm2, 122.9 mm2 in the cohort without any decompression (p < 0.001)., Conclusion: Indirect decompression of both the spinal canal and the contralateral foramen can be achieved via EPTLIF. Decompression on an asymptomatic contralateral side is not necessary.

Pharmaceuticals and their packaging have a significant negative impact on the environment providing a very strong argument for action on the part of pharmacists and pharmacy technicians to engage with pro-environmental behaviours (PEBs) in their workplaces. The aims of this research were therefore to investigate in hospital pharmacists and pharmacy technicians, 1) factors affecting engagement with workplace PEBs, and 2) determine if legislated carbon reduction targets in the UK influenced workplace PEBs in the UK compared with Australia which does not have legislated carbon reduction targets. The environmentally responsible disposal of pharmaceutical waste was the PEB of interest in this study. A mixed methods research design was utilised and a conceptual model (key variables: environmental attitude, concern, and knowledge, and organisational factors) was developed to identify factors influencing workplace PEBs. Participants were from five hospitals in Queensland, Australia and five NHS hospitals in England, UK. There was no statistically significant difference in environmental attitude or concern between the two groups-most had a mid-environmental attitude score and low levels of environmental concern. Participants lacked knowledge of the issue and the link between the environment and public health. Both Australian and UK participants reported recycling packaging waste was not a priority in the hospital pharmacy workplace (even in hospitals with recycling capability) as hospitals focused on compliance with clinical (contaminated) and confidential waste streams. Environmental attitude, knowledge, and concern therefore appeared to be weak influences on intention to perform workplace PEBs with workplace social norms (compliance due to audits) appearing to be a significant mediator of action. The key difference between the cohorts was that UK pharmacists felt waste was not in the scope of their role, and therefore not their responsibility. This study identified that legislated carbon reduction targets did not influence hospital pharmacy workplace PEBs-neither cohort reported engaging significantly in workplace PEBs. UK Government and NHS sustainability policy did not appear to have disseminated to pharmacy department level of UK public hospitals to any great extent., Competing Interests: The authors have declared that no competing interests exist.

Background: In Australia, polypharmacy and medication-related problems are prevalent in the community. Therefore, medicines safety initiatives such as the Home Medicines Review (HMR) service are critical to health care provision. While the evidence continues to expand around HMR service, little is known of accredited pharmacists' experiences of HMR time investment., Objective: This study aimed to explore accredited pharmacists' experiences of HMR practice regarding time investment in the study's defined HMR Stages: 1 (initial paper-based assessment and review), 2 (in-home patient-accredited pharmacist consultation), and 3 (HMR report collation, generation, completion, and provision to the patient's General Practitioner, including any liaison time)., Methods: An electronic survey was developed and piloted by a panel of reviewers. Convenience sampling was used to distribute the final anonymous survey nationally via professional pharmacy organisations. Data were analyzed for frequency distributions and a chi-square test of independence was performed to evaluate any association between demographic variables relating to HMR time investment., Results: There was a total of 255 survey respondents, representing approximately 10% of national accredited pharmacist membership. The majority were experienced accredited pharmacists who had completed >100 HMRs (73%), were female (71%), and aged >40 years (60%). Regarding time investment for a typical instance of HMR, most spent: <30 minutes performing Stage 1 (46.7%), and 30-60 minutes performing Stage 2 (70.2%). In Stage 3, 40.0% invested 1-2 hours, and 27.1% invested 2-3 hours in HMR report collation and completion. Quantitative analysis revealed statistically significant (p=0.03) gender findings where females performed longer patient consultations than males (Stage 2). More HMR career experience resulted in statistically significant (p=0.01) less time performing Stage 1 (initial paper-based assessment and review); with a trend to less time performing Stage 3 (HMR report writing)., Conclusions: Accredited pharmacists invest significant time in performing comprehensive HMRs, especially during in-home patient consultations and during HMR report collation and completion. Their significant HMR time investment as medicines experts provides insight for program and workforce considerations and warrants further research to better understand their work processes for optimizing medicines use and improving health., Competing Interests: CONFLICT OF INTEREST None., (Copyright: © The Authors.)

Black pepper production in Malaysia was restricted by various diseases. Hazardous chemical products appear to be the best solution to control diseases in black pepper cultivation. However, persistence of chemical residues in peppercorns could affect the quality of exports and consumptions. Application of fertilizers is crucial to sustain pepper growth and high yield. But, continuous use of chemical fertilizers could affect the soil ecosystem and eventually restrict nutrient uptake by pepper roots. Therefore, we propose biological approaches as an alternative solution instead of chemical products to sustain pepper cultivation in Malaysia. In this study, we have isolated a total of seven indigenous rhizobacteria antagonistic to soil-borne Fusarium solani, the causal fungus of slow decline, the most serious debilitating disease of black pepper in Malaysia. The isolated bacteria were identified as Bacillus subtilis, Bacillus siamensis, Brevibacillus gelatini, Pseudomonas geniculata, Pseudomonas beteli, Burkholderia ubonensis and Burkholderia territorii. These bacteria were effective in production of antifungal siderophore with the amount of 53.4 %-73.5 % per 0.5 mL of cell-free supernatants. The bacteria also produced appreciable amount of chitinase with chitinolytic index was ranged from 1.19 to 1.76. The bacteria have shown phosphate solubilizing index within 1.61 to 2.01. They were also efficient in ACC deaminase (0.52 mM-0.62 mM) and ammonia (60.3 mM-75.3 mM) production. The isolated antagonists were efficacious in stimulation of black pepper plant growth and root development through IAA (10.5 μg/mL-42.6 μg/mL) secretion. In conclusion, the isolated rhizobacteria are potent to be developed not only as biocontrol agents to minimize the utilization of hazardous chemicals in black pepper disease management, but also developed as bio-fertilizers to improve black pepper plant growth due to their capabilities in plant growth-promotion., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Black pepper is an important commodity crop in Malaysia that generates millions of annual revenue for the country. However, black pepper yield is affected by slow decline disease caused by a soil-borne fungus Fusarium solani. RNA sequencing transcriptomics approach has been employed in this study to explore the differential gene expression in susceptible Piper nigrum L. and resistant Piper colubrinum Link. Gene expression comparative analysis of the two pepper species has yielded 2,361 differentially expressed genes (DEGs). Among them, higher expression of 1,426 DEGs was detected in resistant plant. These DEGs practically demonstrated the major branches of plant-pathogen interaction pathway (Path: ko04626). We selected five groups of defence-related DEGs for downstream qRT-PCR analysis. Cf-9, the gene responsible for recognizing fungal avirulence protein activity was found inexpressible in susceptible plant. However, this gene exhibited promising expression in resistant plant. Inactivation of Cf-9 could be the factor that causes susceptible plant fail in recognition of F. solani and subsequently delay activation of adaptive response to fungal invasion. This vital study advance the understanding of pepper plant defence in response to F. solani and aid in identifying potential solution to manage slow decline disease in black pepper cultivation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Black pepper ( Piper nigrum ) is a vital spice crop with uses ranging from culinary to pharmacological applications. However, limited genetic information has constrained the understanding of the molecular regulation of flower and fruit development in black pepper. In this study, a comparison among three different black pepper varieties, Semengok Aman (SA), Kuching (KC), and Semengok 1 (S1), with varying fruit characteristics was used to provide insight on the genetic regulation of flower and fruit development. Next-generation sequencing (NGS) technology was used to determine the flower and fruit transcriptomes by sequencing on an Illumina HiSeq 2500 platform followed by de novo assembly using SOAPdenovo-Trans. The high-quality assembly of 66,906 of unigenes included 64.4% of gene sequences (43,115) with similarity to one or more protein sequences from the GenBank database. Annotation with Blast2Go assigned 37,377 genes to one or more Gene Ontology terms. Of these genes, 5,874 genes were further associated with the biological pathways recorded in the KEGG database. Comparison of flower and fruit transcriptome data from the three different black pepper varieties revealed a large number of DEGs between flower and fruit of the SA variety. Gene Ontology (GO) enrichment analysis further supports functions of DEGs between flower and fruit in the categories of carbohydrate metabolic processes, embryo development, and DNA metabolic processes while the DEGs in fruit relate to biosynthetic process, secondary metabolic process, and catabolic process. The enrichment of DEGs in KEGG pathways was also investigated, and a large number of genes were found to belong to the nucleotide metabolism and carbohydrate metabolism categories. Gene expression profiling of flower formation-related genes reveals that other than regulating the flowering in black pepper, the flowering genes might also be implicated in the fruit development process. Transcriptional analysis of sugar transporter and carbohydrate metabolism genes in different fruit varieties suggested that the carbohydrate metabolism in black pepper fruit is developmentally regulated, and some genes might serve as potential genes for future crop quality improvement. Study on the piperine-related gene expression analysis suggested that lysine-derived products might present in all stages of fruit development, but the transportation was only active at the early stage of fruit development. These results indicate several candidate genes related to the development of flower and fruit in black pepper and provide a resource for future functional analysis and potentially for future crop improvement., Competing Interests: The authors confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome., (Copyright © 2020 Choy Yuen Khew et al.)

Background: A comparative survey from patients and radiographers of the new slump sitting flexion posture and the conventional standing forward bending posture. This study was performed to compare the technical and logistical aspects of the slump sitting versus the forward bending posture. Slump sitting flexes the lumbar spine more than the forward bending and increases the diagnosis rate of sagittal spinal instability up to 40% depending on the diagnostic criteria used. This should not come at the expense of patient safety and comfort nor burden the radiographers., Methods: Sixty patients were recruited from a single tertiary spine centre. Patients were block randomised into two groups with either the forward bending or the slump sitting being performed first. Feedback was obtained through self-administered questionnaires from patients regarding perceived safety, convenience and comfort, plus from radiographers regarding the imaging process, proxy measures of radiographer ability and scan difficulty., Results: There was no significant difference between the baseline characteristics in both groups. Majority (63%) of patients preferred slump sitting and felt that forward bending caused pain (P=0.025). Overall, slump sitting was equivalent in comfort, perceived safety and ease to forward bending. Despite requiring more logistics (P=0.031), more effort to set up (P=0.002) and explain (P=0.012), the majority of radiographers (83%) preferred slump sitting. This method was felt to be less dangerous (P=0.015) and easier to maintain (P<0.001)., Conclusions: This study showed that the superiority of slump sitting in allowing more lumbar flexion compared to the forward bending comes with patient safety or comfort. The technical demands of the learning curve can be offset with training. As such, slump sitting flexion views should be adopted as the standardized method for assessing spinal instability., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare.

Background: Chromosomal microarray offers superior sensitivity for identification of submicroscopic copy number variants (CNV) and it is advocated to be the first tier genetic testing for patients with autism spectrum disorder (ASD). In this regard, diagnostic yield of array comparative genomic hybridization (CGH) for ASD patients is determined in a cohort of Chinese patients in Hong Kong., Methods: A combined adult and paediatric cohort of 68 Chinese ASD patients (41 patients in adult group and 27 patients in paediatric group). The genomic DNA extracted from blood samples were analysed by array CGH using NimbleGen CGX-135K oligonucleotide array., Results: We identified 15 CNV and eight of them were clinically significant. The overall diagnostic yield was 11.8 %. Five clinically significant CNV were detected in the adult group and three were in the paediatric group, providing diagnostic yields of 12.2 and 11.1 % respectively. The most frequently detected CNV was 16p13.11 duplications which were present in 4 patients (5.9 % of the cohort)., Conclusions: In this study, a satisfactory diagnostic yield of array CGH was demonstrated in a Chinese ASD patient cohort which supported the clinical usefulness of array CGH as the first line testing of ASD in Hong Kong.

Objective: To evaluate the clinical impact of chromosomal microarray (CMA) on the management of paediatric patients in Hong Kong., Methods: We performed NimbleGen 135k oligonucleotide array on 327 children with intellectual disability (ID)/developmental delay (DD), autism spectrum disorders (ASD), and/or multiple congenital anomalies (MCAs) in a university-affiliated paediatric unit from January 2011 to May 2013. The medical records of patients were reviewed in September 2013, focusing on the pathogenic/likely pathogenic CMA findings and their "clinical actionability" based on established criteria., Results: Thirty-seven patients were reported to have pathogenic/likely pathogenic results, while 40 had findings of unknown significance. This gives a detection rate of 11% for clinically significant (pathogenic/likely pathogenic) findings. The significant findings have prompted clinical actions in 28 out of 37 patients (75.7%), while the findings with unknown significance have led to further management recommendation in only 1 patient (p < 0.001). Nineteen out of the 28 management recommendations are "evidence-based" on either practice guidelines endorsed by a professional society (n = 9, Level 1) or peer-reviewed publications making medical management recommendation (n = 10, Level 2). CMA results impact medical management by precipitating referral to a specialist (n = 24); diagnostic testing (n = 25), surveillance of complications (n = 19), interventional procedure (n = 7), medication (n = 15) or lifestyle modification (n = 12)., Conclusion: The application of CMA in children with ID/DD, ASD, and/or MCAs in Hong Kong results in a diagnostic yield of ∼ 11% for pathogenic/likely pathogenic results. Importantly the yield for clinically actionable results is 8.6%. We advocate using diagnostic yield of clinically actionable results to evaluate CMA as it provides information of both clinical validity and clinical utility. Furthermore, it incorporates evidence-based medicine into the practice of genomic medicine. The same framework can be applied to other genomic testing strategies enabled by next-generation sequencing.

22q11.2 deletion syndrome (22q11.2DS) is a multi-systemic disorder with high phenotypic variability. Under-diagnosis in adults is common and recognition of facial dysmorphic features can be affected by age and ethnicity. This study aims to determine the prevalence of undiagnosed 22q11.2DS in adult Chinese patients with conotruncal anomalies and to delineate their facial dysmorphisms and extra-cardiac manifestations. We recruited consecutively 156 patients with conotruncal anomalies in an adult congenital heart disease (CHD) clinic in Hong Kong and screened for 22q11.2DS using fluorescence-PCR and fluorescence in-situ hybridization. Assessment for dysmorphic features was performed by a cardiologist at initial screening and then by a clinical geneticist upon result disclosure. Clinical photographs were taken and childhood photographs collected. Eighteen patients (11.5%) were diagnosed with 22q11.2DS, translating into 1 previously unrecognized diagnosis of 22q11.2DS in every 10 adult patients with conotruncal anomalies. While dysmorphic features were detected by our clinical geneticist in all patients, only two-thirds were considered dysmorphic by our cardiologist upon first assessment. Evolution of facial dysmorphic features was noted with age. Extra-cardiac manifestations included velopharyngeal incompetence or cleft palate (44%), hypocalcemia (39%), neurodevelopmental anomalies (33%), thrombocytopenia (28%), psychiatric disorders (17%), epilepsy (17%) and hearing loss (17%). We conclude that under-diagnosis of 22q11.2DS in Chinese adults with conotruncal defects is common and facial dysmorphic features may not be reliably recognized in the setting of adult CHD clinic, referral for genetic evaluation and molecular testing for 22q11.2DS should be offered to patients with conotruncal defects., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Objective: To evaluate the effectiveness of whole-genome array comparative genomic hybridization (aCGH) in prenatal diagnosis in Hong Kong., Methods: Array CGH was performed on 220 samples recruited prospectively as the first-tier test study. In addition 150 prenatal samples with abnormal fetal ultrasound findings found to have normal karyotypes were analyzed as a 'further-test' study using NimbleGen CGX-135K oligonucleotide arrays., Results: Array CGH findings were concordant with conventional cytogenetic results with the exception of one case of triploidy. It was found in the first-tier test study that aCGH detected 20% (44/220) clinically significant copy number variants (CNV), of which 21 were common aneuploidies and 23 had other chromosomal imbalances. There were 3.2% (7/220) samples with CNVs detected by aCGH but not by conventional cytogenetics. In the 'further-test' study, the additional diagnostic yield of detecting chromosome imbalance was 6% (9/150). The overall detection for CNVs of unclear clinical significance was 2.7% (10/370) with 0.9% found to be de novo. Eleven loci of common CNVs were found in the local population., Conclusion: Whole-genome aCGH offered a higher resolution diagnostic capacity than conventional karyotyping for prenatal diagnosis either as a first-tier test or as a 'further-test' for pregnancies with fetal ultrasound anomalies. We propose replacing conventional cytogenetics with aCGH for all pregnancies undergoing invasive diagnostic procedures after excluding common aneuploidies and triploidies by quantitative fluorescent PCR. Conventional cytogenetics can be reserved for visualization of clinically significant CNVs.

Copy number gain of 17p13.3 has been shown to be associated with developmental delay/autism and Split-Hand-Foot malformation. We report a case of fetus with bilateral split-hand malformation detected on prenatal ultrasound. Array comparative genomic hybridization detected 2 maternally inherited copy number gains in the 17p13.3 region with one of them involving the BHLHA9 gene and part of the YWHAE gene. The mother is normal in intelligence with mild right foot anomaly only. Although the BHLHA9 copy gain is known to be associated with split-hand-foot malformation, the penetrance and expressivity is highly variable. More challenging is the effect of partial YWHAE copy number gain on neurodevelopment is inconclusive based on current literature. This case highlights the difficulties of prenatal genetic counseling in array comparative genomic hybridization findings in clinical situation with incomplete understanding of genotype-phenotype correlation., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Zein was investigated for use as an oral-drug delivery system by loading prednisolone into zein microparticles using coacervation. To investigate the adaptability of this method to other drugs, zein microparticles were loaded with hydrocortisone, which is structurally related to prednisolone; or mesalazine, which is structurally different having a smaller LogP and ionizable functional groups. Investigations into the in vitro digestibility, and the electrophoretic profile of zein, and zein microparticles were conducted to shed further insight on using this protein as a drug delivery system. Hydrocortisone loading into zein microparticles was comparable with that reported for prednisolone, but mesalazine loading was highly variable. Depending on the starting quantities of hydrocortisone and zein, the average amount of microparticles equivalent to 4 mg hydrocortisone, (a clinically used dose), ranged from 60-115 mg, which is realistic and practical for oral dosing. Comparatively, an average of 2.5 g of microparticles was required to deliver 250 mg of mesalazine (a clinically used dose), so alternate encapsulation methods that can produce higher and more precise mesalazine loading are required. In vitro protein digestibility revealed that zein microparticles were more resistant to digestion compared to the zein raw material, and that individual zein peptides are not preferentially coacervated into the microparticles. In combination, these results suggest that there is potential to formulate a delivery system based on zein microparticles made using specific subunits of zein that is more resistant to digestion as starting material, to deliver drugs to the lower gastrointestinal tract.

Fetal DNA is present in the plasma of pregnant women. Massively parallel sequencing of maternal plasma DNA has been used to detect fetal trisomies 21, 18, 13 and selected sex chromosomal aneuploidies noninvasively. Case reports describing the detection of fetal microdeletions from maternal plasma using massively parallel sequencing have been reported. However, these previous reports were either polymorphism-dependent or used statistical analyses which were confined to one or a small number of selected parts of the genome. In this report, we reported a procedure for performing noninvasive prenatal karyotyping at 3 Mb resolution across the whole genome through the massively parallel sequencing of maternal plasma DNA. This method has been used to analyze the plasma obtained from 6 cases. In three cases, fetal microdeletions have been detected successfully from maternal plasma. In two cases, fetal microduplications have been detected successfully from maternal plasma. In the remaining case, the plasma DNA sequencing result was consistent with the pregnant mother being a carrier of a microduplication. Simulation analyses were performed for determining the number of plasma DNA molecules that would need to be sequenced and aligned for enhancing the diagnostic resolution of noninvasive prenatal karyotyping to 2 Mb and 1 Mb. In conclusion, noninvasive prenatal molecular karyotyping from maternal plasma by massively parallel sequencing is feasible and would enhance the diagnostic spectrum of noninvasive prenatal testing.

Objective: To evaluate the performance of the locally developed universal Down syndrome screening programme., Design: Population-based cohort study in the period July 2010 to June 2011 inclusive., Setting: Four Hong Kong Hospital Authority Departments of Obstetrics and Gynaecology and a central university-based laboratory for maternal serum processing and risk determination., Participants: Women were offered either a first-trimester combined test (nuchal translucency, free beta human chorionic gonadotropin, and pregnancy-associated plasma protein-A) or nuchal-translucency-only test, or a second-trimester double test (alpha-fetoprotein and total human chorionic gonadotropin) for detection of Down syndrome according to their gestational age. Those with a trisomy 21 term risk of 1:250 or higher were offered a diagnostic test., Results: A total of 16 205 pregnancies were screened of which 13 331 (82.3%) had a first-trimester combined test, 125 (0.8%) had a nuchal-translucency test only, and 2749 (17.0%) had a second-trimester double test. There were 38 pregnancies affected by Down syndrome. The first-trimester screening tests had a 91.2% (31/34) detection rate with a screen-positive rate of 5.1% (690/13 456). The second-trimester test had a 100% (4/4) detection rate with a screen-positive rate of 6.3% (172/2749). There were seven (0.9%) pregnancies that miscarried following an invasive diagnostic test. There were two Down syndrome-affected live births, both with an estimated first-trimester trisomy 21 term risk lower than 1:250., Conclusion: The universal screening programme offered at the four units was effective and achieved the expected detection rates and low false-positive rates, and to maintain these, the current emphasis on training, quality control, and regular auditing must continue.

Objectives: To validate the clinical efficacy and practical feasibility of massively parallel maternal plasma DNA sequencing to screen for fetal trisomy 21 among high risk pregnancies clinically indicated for amniocentesis or chorionic villus sampling., Design: Diagnostic accuracy validated against full karyotyping, using prospectively collected or archived maternal plasma samples., Setting: Prenatal diagnostic units in Hong Kong, United Kingdom, and the Netherlands., Participants: 753 pregnant women at high risk for fetal trisomy 21 who underwent definitive diagnosis by full karyotyping, of whom 86 had a fetus with trisomy 21. Intervention Multiplexed massively parallel sequencing of DNA molecules in maternal plasma according to two protocols with different levels of sample throughput: 2-plex and 8-plex sequencing., Main Outcome Measures: Proportion of DNA molecules that originated from chromosome 21. A trisomy 21 fetus was diagnosed when the z score for the proportion of chromosome 21 DNA molecules was >3. Diagnostic sensitivity, specificity, positive predictive value, and negative predictive value were calculated for trisomy 21 detection., Results: Results were available from 753 pregnancies with the 8-plex sequencing protocol and from 314 pregnancies with the 2-plex protocol. The performance of the 2-plex protocol was superior to that of the 8-plex protocol. With the 2-plex protocol, trisomy 21 fetuses were detected at 100% sensitivity and 97.9% specificity, which resulted in a positive predictive value of 96.6% and negative predictive value of 100%. The 8-plex protocol detected 79.1% of the trisomy 21 fetuses and 98.9% specificity, giving a positive predictive value of 91.9% and negative predictive value of 96.9%., Conclusion: Multiplexed maternal plasma DNA sequencing analysis could be used to rule out fetal trisomy 21 among high risk pregnancies. If referrals for amniocentesis or chorionic villus sampling were based on the sequencing test results, about 98% of the invasive diagnostic procedures could be avoided.

Massively parallel sequencing of DNA molecules in the plasma of pregnant women has been shown to allow accurate and noninvasive prenatal detection of fetal trisomy 21. However, whether the sequencing approach is as accurate for the noninvasive prenatal diagnosis of trisomy 13 and 18 is unclear due to the lack of data from a large sample set. We studied 392 pregnancies, among which 25 involved a trisomy 13 fetus and 37 involved a trisomy 18 fetus, by massively parallel sequencing. By using our previously reported standard z-score approach, we demonstrated that this approach could identify 36.0% and 73.0% of trisomy 13 and 18 at specificities of 92.4% and 97.2%, respectively. We aimed to improve the detection of trisomy 13 and 18 by using a non-repeat-masked reference human genome instead of a repeat-masked one to increase the number of aligned sequence reads for each sample. We then applied a bioinformatics approach to correct GC content bias in the sequencing data. With these measures, we detected all (25 out of 25) trisomy 13 fetuses at a specificity of 98.9% (261 out of 264 non-trisomy 13 cases), and 91.9% (34 out of 37) of the trisomy 18 fetuses at 98.0% specificity (247 out of 252 non-trisomy 18 cases). These data indicate that with appropriate bioinformatics analysis, noninvasive prenatal diagnosis of trisomy 13 and trisomy 18 by maternal plasma DNA sequencing is achievable.

Background: Noninvasive prenatal diagnosis of fetal aneuploidy by maternal plasma analysis is challenging owing to the low fractional and absolute concentrations of fetal DNA in maternal plasma. Previously, we demonstrated for the first time that fetal DNA in maternal plasma could be specifically targeted by epigenetic (DNA methylation) signatures in the placenta. By comparing one such methylated fetal epigenetic marker located on chromosome 21 with another fetal genetic marker located on a reference chromosome in maternal plasma, we could infer the relative dosage of fetal chromosome 21 and noninvasively detect fetal trisomy 21. Here we apply this epigenetic-genetic (EGG) chromosome dosage approach to detect Edwards syndrome (trisomy 18) in the fetus noninvasively., Principal Findings: We have systematically identified methylated fetal epigenetic markers on chromosome 18 by methylated DNA immunoprecipitation (MeDIP) and tiling array analysis with confirmation using quantitative DNA methylation assays. Methylated DNA sequences from an intergenic region between the VAPA and APCDD1 genes (the VAPA-APCDD1 DNA) were detected in pre-delivery, but not post-delivery, maternal plasma samples. The concentrations correlated positively with those of an established fetal genetic marker, ZFY, in pre-delivery maternal plasma. The ratios of methylated VAPA-APCDD1(chr18) to ZFY(chrY) were higher in maternal plasma samples of 9 male trisomy 18 fetuses than those of 27 male euploid fetuses (Mann-Whitney test, P=0.029). We defined the cutoff value for detecting trisomy 18 fetuses as mean+1.96 SD of the EGG ratios of the euploid cases. Eight of 9 trisomy 18 and 1 of 27 euploid cases showed EGG ratios higher than the cutoff value, giving a sensitivity of 88.9% and a specificity of 96.3%., Conclusions: Our data have shown that the methylated VAPA-APCDD1 DNA in maternal plasma is predominantly derived from the fetus. We have demonstrated that this novel fetal epigenetic marker in maternal plasma is useful for the noninvasive detection of fetal trisomy 18.

Objective: To report the type and frequency of chromosomal anomalies and Y-microdeletions among Hong Kong Chinese subfertile men with sperm concentrations lower than 5 million/mL. DESIGN. Retrospective study., Setting: A reproductive centre in Hong Kong., Participants: A total of 295 Chinese subfertile men who underwent both karyotyping and Y-microdeletion studies from 2000 to 2007 were categorised as having non-obstructive azoospermia (n=71), very severe oligospermia (sperm concentration>0 and 2 and <5 million/mL, n=66)., Main Outcome Measures: Karyotyping and Y-microdeletion studies., Results: The prevalence of chromosomal anomalies and Y-microdeletions in the study population were 8.5% (25/295; 95% confidence interval, 5.6-12.3%) and 6.4% (19/295; 3.9-9.9%), respectively. The total prevalence of chromosomal anomalies and Y-microdeletions was 13.2% (39/295; 95% confidence interval, 9.6-17.6%) as five cases of non-obstructive azoospermia showed both Y structural alterations and AZFbc deletion. The corresponding figures for chromosomal anomalies in the groups with non-obstructive azoospermia, very severe oligospermia, and severe oligospermia were 21.1% (15/71; 95% confidence interval, 12.3-32.4%), 5.7% (9/158; 2.6-10.5%), and 1.5% (1/66; 0.0-8.2%). While for Y-microdeletions they were 8.5% (6/71; 3.2-17.5%), 8.2% (13/158; 4.5-13.7%) and 0% (0/66; 0.0-4.4%), respectively. The respective overall prevalence rates for chromosomal anomalies and Y-microdeletions in these groups were: 22.5% (16/71; 13.5-34.0%), 13.9% (22/158; 8.9-20.3%), and 1.5% (1/66; 0.0-8.2%)., Conclusions: Our findings strongly support the recommendation for both karyotyping and Y-microdeletion analyses in subfertile men with sperm concentrations of 2 million/mL or lower before they undergo assisted reproduction treatment.

Objectives: The application of rapid aneuploidy testing as a stand-alone approach in prenatal diagnosis is much debated. The major criticism of this targeted approach is that it will not detect other chromosomal abnormalities that will be picked up by traditional karyotyping. This study aimed to study the nature of such chromosomal abnormalities and whether parents would choose to terminate affected pregnancies., Design: Retrospective study on a cytogenetic database., Setting: Eight public hospitals in Hong Kong., Participants: The karyotype results of 19 517 amniotic fluid cultures performed for advanced maternal age (>or=35 years) from 1997 to 2002 were classified according to whether they were detectable by rapid aneuploidy testing. The outcomes of pregnancies with abnormal karyotypes were reviewed from patient records., Results: In all, 333 (1.7%) amniotic fluid cultures yielded abnormal karyotypes; 175 (52.6%) of these were detected by rapid aneuploidy testing, and included trisomy 21 (n=94, 28.2%), trisomy 18 or 13 (n=21, 6.3%), and sex chromosome abnormalities (n=60, 18.0%). The other 158 (47.4%) chromosomal abnormalities were not detectable by rapid aneuploidy testing, of which 63 (18.9%) were regarded to be of potential clinical significance and 95 (28.5%) of no clinical significance. Pregnancy outcomes in 327/333 (98.2%) of these patients were retrieved. In total, 143 (42.9%) of these pregnancies were terminated: 93/94 (98.9%) for trisomy 21, 20/21 (95.2%) for trisomy 18 or 13, 19/60 (31.7%) for sex chromosome abnormalities, and 11/63 (17.5%) for other chromosomal abnormalities with potential clinical significance. There were no terminations in the 95 pregnancies in which karyotyping results were regarded to be of no clinical significance., Conclusions: 'Knowing less' by the rapid aneuploidy stand-alone testing could miss about half of all chromosomal abnormalities detectable by amniocentesis performed for advanced maternal age. Findings from two fifths of the latter were of potential clinical significance, and the parents chose to terminate one out of six of the corresponding pregnancies. If both techniques are available, parents could have enhanced autonomy to choose.

A retrospective study was performed to compare the detection rate of chromosomal abnormalities by different approaches of full karyotyping, rapid aneuploidy diagnosis (RAD) or both when invasive prenatal testing is performed for diagnosis of thalassaemia. The karyotype results of 1120 prenatal samples obtained from thalassaemia couples from January 1985 to December 2002 in a referral centre for prenatal diagnosis were studied. The detection rate of chromosomal abnormalities by four different approaches were compared: (i) karyotyping for all samples; (ii) RAD (21,18,13,X,Y) for all samples; (iii) RAD for all samples + karyotyping for cases with ultrasound abnormalities; and (iv) RAD (21,18,13) for all + RAD (X,Y) for cases with ultrasound abnormalities consistent with Turner syndrome + karyotyping for cases with ultrasound abnormalities. Normal karyotypes were found in 1103 samples (98.5%). There were 17 cases (1.5%) of chromosomal abnormalities: four cases (0.36%) were clinically significant, eight cases (0.7%) were of borderline clinical significance and five cases (0.44%) were not confirmed by subsequent prenatal or postnatal tests. The incidences of autosomal (7/1120 = 0.63%) and sex chromosomal (5/1120 = 0.45%) abnormalities were not higher than those (0.41 and 0.22%, respectively) from newborn surveys (Hook and Hamerton, 1977) (P = 0.398 and 0.216, respectively). Approach 1 would detect all 17 chromosomal abnormalities. Approach 2 would detect three of four clinically significant chromosomal abnormalities but not detect six of eight chromosomal abnormalities of borderline clinical significance and three of five chromosomal abnormalities not confirmed by subsequent prenatal or postnatal tests. Approach 3, in addition, would be able to detect all four clinically significant chromosomal abnormalities. Approach 4 would detect all four clinically significant chromosomal abnormalities but would not detect seven of eight chromosomal abnormalities of borderline clinical significance and four of five chromosomal abnormalities not confirmed by subsequent prenatal or postnatal tests. RAD (21,18,13) for all + RAD (X,Y) for cases with ultrasound abnormalities consistent with Turner syndrome + karyotyping for cases with ultrasound abnormalities seemed to be the best approach for the detection of chromosomal abnormalities when invasive prenatal testing is performed for diagnosis of thalassaemia.

The accuracy of new molecular diagnostics, fluoresence in situ hybridization or quantitative fluorescence-PCR (collectively known as rapid aneuploidy screening), in prenatal diagnosis has already been demonstrated in a number of large studies. The challenge now is how to apply them clinically in the most cost-effective manner. It is now time to appraise whether rapid aneuploidy screening can replace traditional karyotyping when amniocenteses are performed for increased risk of Down's syndrome by maternal serum screening or advanced maternal age in the absence of ultrasound abnormality. The ten most recent studies from the literature within this research theme are reviewed and the pros and cons of this new approach in prenatal diagnosis are discussed, including the suggestion of future studies.

This paper presents the first two successful cases of pre-implantation genetic diagnosis in Hong Kong and discusses the indications and the advantages over prenatal diagnosis. Patients should be informed about the procedure and extensively counselled about the possibility of misdiagnosis and the need for conventional prenatal diagnosis during pregnancy.

Aldose reductase (AR), the first enzyme in the polyol pathway, has been implicated in the pathogenesis of diabetic complications, although its physiological role is unclear. In mice, besides AR, two AR-like proteins, mouse vas deferens protein (MVDP) and fibroblast growth factor-regulated protein (FR-1), have been reported recently. Tissue-specific expression of these two genes was examined using the RNase protection assay method. Contrary to previous reports, MVDP was detected in a variety of tissues besides the vas deferens. High levels of MVDP mRNA were found in the adrenal glands, and low levels of expression were detected in eye, intestine, seminal vesicle, kidney, liver, testis and lung. The major gene expression pattern for FR-1 was slightly different from that of MVDP, with the highest levels of mRNA detected in testis, heart, adrenal gland, and ovary; less was found in the lung and it was barely detectable in eye, intestine, liver and seminal vesicle tissue. Mouse embryos, as early as 10.5 days post coitum, expressed both genes, although the levels of expression were different. Human AR mRNA was found in human vas deferens, although not at the high level found in mice. The localization of both MVDP and FR-1 transcripts in the adrenal cortex by in situ hybridization led to the speculation that these two AR-like proteins could be related to hormone production.

The entire mouse collagen X gene (Col10a-1) has been isolated. The gene is composed of three exons and two introns spanning 7.0 kb of the DNA sequence. Exons 2 and 3 together encode 15-bp of 5' untranslated sequence, a 2040-bp open reading frame and an 895-nucleotide 3' non-coding region. In the 5' flanking region of the gene, two consensus TATA-box sequences were found. Identification of the first exon by ribonuclease-protection assays and the determination of the 5' end of Col10a-1 mRNA transcripts by primer-extension analyses show that the more 3' TATA box is probably predominantly used and that there are at least three transcription start sites in the exon 1 sequence 3' to this, resulting in 5' untranslated regions of 78, 77 and 55 nucleotides. By means of rapid amplification of cDNA ends by polymerase chain reaction, an additional mRNA species was detected which overlapped the other Col10a-1 transcripts, including the 3' TATA box sequence, giving a 5' untranslated sequence of approximately 235 bases. This latter transcript starts approximately 20 bp 3' to the more 5' TATA box. The data suggest alternative use of promoters and transcription starts for the Col10a-1 gene. Comparison of the combined nucleotide and deduced amino acid sequences of exons 2 and 3 with chicken, bovine and human collagen X genes, showed a high degree of similarity indicating conservation of this gene throughout evolution. Mouse Col10a-1 mRNA was shown to be approximately 3.0 kb and the pepsinized protein, as detected by SDS/PAGE, was approximately 45 kDa. The mRNA and protein sizes correlate with that predicted by the open reading frame. Reverse-transcription polymerase chain reaction assays indicate that the mouse collagen X gene is first expressed at 13.5 days post coitum, temporally preceding the onset of endochondral ossification. In agreement with the generally accepted association of type-X collagen with endochondral ossification, in situ hybridization analyses indicate that Col10a-1 mRNA are restricted to the hypertrophic regions of growth cartilage.

The mouse alpha 1(II) collagen gene has been isolated and a 5' portion of the gene which has low homology to other collagen genes was used to study the pattern of expression during mouse embryogenesis. In situ hybridization studies show that in the mouse, like the chick, alpha 1(II) collagen is expressed in chondrogenic tissues in advance of chondrocyte differentiation. The gene is expressed early in embryogenesis at 9.5 days both in the cranial mesenchyme destined for the chondrocranium, and the sclerotome of the somites, and at 12.5 days in the primordia of the hyoid and the laryngeal cartilage. Type II collagen gene transcripts were found in all the chondrogenic tissues of the axial and appendicular skeleton until the onset of endochondral ossification. Expression of alpha 1(II) collagen mRNA was also observed in non-chondrogenic tissues such as the notochord which may be responsible for inducing chondrogenesis in somitic mesoderm, neural retina, the corneal and conjunctival epithelia and sclera of the developing eye. Expression in the tail tendon was late, at 16.5-18.5 days. Transient expression was also found in the heart at 9.5-12.5 days, the epidermis at 10.5-14.5 days, the calvarial mesenchyme at 12.5-16.5 days, the inner ear at 14.5 days and the fetal brain from 9.5-14.5 days. Within the neural tube, alpha 1(II) collagen mRNA was localized in the proliferative ventricular cells of the forebrain and midbrain of 9.5- to 10.5-day embryos. Subsequently, transcription of the alpha 1(II) collagen gene was confined to restricted areas of the rhombencephalic basal plate, the ventricular layer of the hindbrain and the cervical spinal cord. These examples of expression of the type II collagen gene in the developing nervous system seem to suggest that active transcription of this gene might be associated with early stages of neuroblast differentiation. Type II collagen may therefore have additional roles in development unrelated to chondrogenesis.

The high-temperature production (HTP) regulon of Escherichia coli consists of a set of operons that are induced coordinately by a shift to a high temperature under the control of a single chromosomal gene called htpR or hin. To identify more components of this regulon, the rates of synthesis of many polypeptides resolved on two-dimensional polyacrylamide gels were measured in various strains by pulse-labeling after a temperature shift-up. A total of 13 polypeptides were found to be heat inducible only in cells bearing a normal htpR gene on the chromosome or on a plasmid; on this basis these polypeptides were designated products of the HTP regulon. Several hybrid plasmids that contain segments of the E. coli chromosome in the 75-min region were found to carry the htpR gene. A restriction map of this region was constructed, and selected fragments were subcloned and tested for the ability to complement an htpR mutant. The polypeptides encoded by these fragments were detected by permitting expression in maxicells, minicells, and chloramphenicol-treated cells. Complementation was accompanied by production of a polypeptide having a molecular weight of approximately 33,000. This polypeptide, designated F33.4, was markedly reduced in amount in an htpR mutant expected to contain very little htpR gene product. Polypeptide F33.4 is postulated to be the product of htpR and to be an effector that controls heat induction of the HTP regulon.