Single-tube tetradecaplex panel of highly polymorphic microsatellite markers < 1 Mb from F8 for simplified preimplantation genetic diagnosis of hemophilia A.

Essentials Preimplantation genetic diagnosis (PGD) of severe hemophilia A relies on linkage analysis. Simultaneous multi-marker screening can simplify selection of informative markers in a couple. We developed a single-tube tetradecaplex panel of polymorphic markers for hemophilia A PGD use. Informative markers can be used for linkage analysis alone or combined with mutation detection. SUMMARY: Background It is currently not possible to perform single-cell preimplantation genetic diagnosis (PGD) to directly detect the common inversion mutations of the factor VIII (F8) gene responsible for severe hemophilia A (HEMA). As such, PGD for such inversion carriers relies on indirect analysis of linked polymorphic markers. Objectives To simplify linkage-based PGD of HEMA, we aimed to develop a panel of highly polymorphic microsatellite markers located near the F8 gene that could be simultaneously genotyped in a multiplex-PCR reaction. Methods We assessed the polymorphism of various microsatellite markers located ≤ 1 Mb from F8 in 177 female subjects. Highly polymorphic markers were selected for co-amplification with the AMELX/Y indel dimorphism in a single-tube reaction. Results Thirteen microsatellite markers located within 0.6 Mb of F8 were successfully co-amplified with AMELX/Y in a single-tube reaction. Observed heterozygosities of component markers ranged from 0.43 to 0.84, and ∼70-80% of individuals were heterozygous for ≥ 5 markers. The tetradecaplex panel successfully identified fully informative markers in a couple interested in PGD for HEMA because of an intragenic F8 point mutation, with haplotype phasing established through a carrier daughter. In-vitro fertilization (IVF)-PGD involved single-tube co-amplification of fully informative markers with AMELX/Y and the mutation-containing F8 amplicon, followed by microsatellite analysis and amplicon mutation-site minisequencing analysis. Conclusions The single-tube multiplex-PCR format of this highly polymorphic microsatellite marker panel simplifies identification and selection of informative markers for linkage-based PGD of HEMA. Informative markers can also be easily co-amplified with mutation-containing F8 amplicons for combined mutation detection and linkage analysis.

The effect of a forced-air warming blanket on patients' end-tidal and transcutaneous carbon dioxide partial pressures during eye surgery under local anaesthesia: a single-blind, randomised controlled trial.

Surgical drapes used during eye surgery are impermeable to air and hence risk trapping air underneath them. We investigated the effect of a forced-air warming blanket on carbon dioxide accumulation under the drapes in patients undergoing eye surgery under local anaesthesia without sedation. Forty patients of ASA physical status 1 and 2 were randomly assigned to either the forced-air warmer (n = 20) or a control heated overblanket (n = 20). All patients were given 1 l.min(-1) oxygen. We measured transcutaneous and end-tidal carbon dioxide partial pressures, heart rate, arterial pressure, respiratory rate, temperature and oxygen saturation before and after draping, then every 5 min thereafter for 30 min. The mean (SD) transcutaneous carbon dioxide partial pressure in the forced-air warming group stayed constant after draping at 5.7 (0.2) kPa but rose to a maximum of 6.4 (0.4) kPa in the heated overblanket group (p = 0.0001 for the difference at time points 15 min and later). We conclude that forced-air warming reduces carbon dioxide accumulation under the drapes in patients undergoing eye surgery under local anaesthesia.

Second and third year oral health and dental student perceptions of future professional work.

OBJECTIVES: To explore and compare the ways dental and oral health students characterise their future professional work (FPW) at the end of their second and third professional years. MATERIALS AND METHODS: Questionnaires were given to a cohort group of 48 dental students and 31 oral health students at the end of their second and third professional years at the University of Otago. Students' characterisations of their FPW were identified using an inductive approach, and the emphasis on each characterisation was confirmed using a 'weighted' table. RESULTS: Dental student response rates were 92% (in 2010) and 85% (in 2011); and oral health student response rates were 100% (in 2011) and 97% (in 2011). Students characterised their FPW in ten broad ways: in reference to treatment-related concerns, patient-related concerns, oral health promotion, oral health education, disease prevention and monitoring, communication, teamwork, maintaining an ideal clinical environment, maintaining a sense of self and improving quality of life. In both years, dental students emphasised treatment-related concerns as central to their FPW and dealing with patient-related concerns as a primary source of difficulty. Oral health students emphasised oral health promotion, oral health education, disease prevention and monitoring and restorative tasks as central to their FPW and dealing with patient-related concerns as a primary source of difficulty. CONCLUSION: Students' broad perceptions of their FPW changed little as they progressed through their programmes; however, their responses suggested the need for greater attention within their programmes to patient management and teamwork.

Health promotion board-ministry of health clinical practice guidelines: treating tobacco use and dependence.

The Health Promotion Board (HPB) has updated the clinical practice guidelines on Treating Tobacco Use and Dependence to provide health professionals in Singapore with evidence-based interventions for smoking cessation. This article reproduces the introduction and executive summary of key guideline recommendations (with recommendations from the guidelines) from the HPB-MOH Clinical Practice Guidelines on Treating Tobacco Use and Dependence, for the information of SMJ readers. Chapters and page numbers mentioned in the reproduced extract refer to the full text of the guidelines, which are available from the Health Promotion Board website: http://www.hpb.gov.sg/cpg-smoking-cessation. The recommendations should be used with reference to the full text of the guidelines. Following this article are multiple choice questions based on the full text of the guidelines.

Efficacy of iontophoresis with glycopyrronium bromide for treatment of primary palmar hyperhidrosis.

BACKGROUND: There is limited data on the efficacy of iontophoretic treatment of primary palmar hyperhidrosis using glycopyrronium bromide. The first line treatment for primary palmar hyperhidrosis is usually topical aluminium chloride, but clinical experience indicates that it is not effective for more severe disease. OBJECTIVE: To evaluate the efficacy of using glycopyrronium bromide iontophoresis in the treatment of primary palmar hyperhidrosis, and to evaluate if the benefit of treatment varies with the severity of disease. METHODS: This is an open-label study involving patients undergoing weekly treatment of iontophoresis with glycopyrronium bromide for 4 weeks. Gravimetric measurements of sweat production and subjective scores of palmar sweatiness were recorded prior to starting treatment and 1 week after the last treatment. Side-effects were monitored weekly. RESULTS: Twenty two of the 25 patients recruited completed the 4-week treatment. There was a significant mean improvement of 23.4 mg/min (P = 0.001) between baseline and post-treatment gravimetric measurements. Patients with a higher baseline sweat output demonstrated a trend towards a greater reduction in sweat production (Pearson's correlation correlation coefficient, r = 0.41). The patients experienced dryness of the palms for a mean duration of 5 days after iontophoresis. All patients reported an improvement in satisfaction scores and 81.8% reported an improvement in subjective severity scores. No serious side-effects were encountered during the study. CONCLUSIONS: Iontophoresis using glycopyrronium bromide is an effective and well-tolerated treatment for primary palmar hyperhidrosis. The possibility of its greater benefit in patients with more severe baseline disease requires verification.

Rapid aneuploidy screening with fluorescence in-situ hybridisation: is it a sufficiently robust stand-alone test for prenatal diagnosis?

OBJECTIVES: To assess the clinical utility of fluorescence in-situ hybridisation with chromosomes 13, 18, 21, X and Y as a stand-alone test in detecting chromosomal abnormalities, and the types of chromosomal abnormalities missed. DESIGN: Retrospective analysis. SETTING: A restructured Government hospital in Singapore and an academic hospital in the United States. PARTICIPANTS: Cytogenetic data of prenatal specimens and results of fluorescence in-situ hybridisation of 5883 patients performed between January 2000 and August 2007 were reviewed. RESULTS: Fluorescence in-situ hybridisation detected 558 (9.5%) patients with chromosomal abnormalities. Abnormal ultrasounds (70%) and maternal serum screens (21%) were the most indicative of chromosomal abnormalities. When comparing fluorescence in-situ hybridisation data with karyotype results for the five chromosomes of interest, the sensitivity and specificity were 99.3% and 99.9%, respectively. When comparing fluorescence in-situ hybridisation data with karyotype results for all chromosomes, the sensitivity decreased to 86.8%, whereas the specificity remained at 99.9%. Of 643 cases with karyotype abnormalities, 85 were fluorescence in-situ hybridisation-negative (false negative rate, 13.2%), which included structural rearrangements, chromosome mosaicism, and other trisomies. Despite abnormal ultrasound indications, fluorescence in-situ hybridisation missed 32 cases which included structural rearrangements, mosaicisms, and other trisomies. CONCLUSION: This study does not support fluorescence in-situ hybridisation as a stand-alone test. Institutions supporting fluorescence in-situ hybridisation as a stand-alone test must seriously consider the risks of a missed diagnosis.

Fentanyl dose for the insertion of Classic Laryngeal Mask Airways in non-paralysed patients induced with propofol 2.5 mg/kg.

The aim of this randomised, controlled trial was to determine the optimum dose of fentanyl in combination with propofol 2.5 mg x kg(-1) when inserting the Classic Laryngeal Mask Airway. Seventy-five ASA I or II patients were randomly assigned to five groups of fentanyl dosage: 0 microg x kg(-1) (placebo), 0.5 microg x kg(-1), 1.0 microg x kg(-1), 1.5 microg x kg(-1) and 2.0 microg x kg(-1). Anaesthesia was induced by first injecting the study drug over 10 seconds. Three minutes after the study drug was injected, propofol (2.5 mg x kg(-1)) was injected over 10 seconds. The Classic Laryngeal Mask Airway was inserted four minutes and 30 seconds after injection of the study drug. Insertion conditions were evaluated using a four-category score. Thirty-nine males and 36 females aged 19 to 59 years were studied. The incidence of prolonged apnoea increased as fentanyl dose increased. We found that there was a high rate of successful first attempt at insertion with 1 microg x kg(-1) and 1.5 microg x kg(-1), 93% and 87% respectively, compared to 87% in the 2.0 microg x kg(-1) group. The 1.0 microg x kg(-1) group also achieved an 80% optimal insertion conditions score of 4, compared to 73% in the 1.5 microg x kg(-1) group and 80% in the 2 microg x kg(-1) group. Therefore we recommend 1.0 microg x kg(-1) as the optimal dose of fentanyl when used in addition to propofol 2.5 mg/kg for the insertion of the Classic Laryngeal Mask Airway.

Difficulties with prenatal diagnosis of the Walker-Warburg syndrome.

We describe a postnatally diagnosed case of Walker-Warburg syndrome--a form of congenital muscular dystrophy with lissencephaly and eye abnormalities. We reviewed the literature to highlight its clinico-radiological diagnostic features and discuss the difficulties encountered with prenatal diagnosis, especially in cases with no positive family history. An increased awareness of this rare but lethal condition, and a high index of suspicion during routine antenatal ultrasound, could prompt further advanced fetal ultrasonography and magnetic resonance imaging, and aid in timely prenatal diagnosis, management, and counseling.

Array comparative genomic hybridisation analysis of boys with X linked hypopituitarism identifies a 3.9 Mb duplicated critical region at Xq27 containing SOX3.

INTRODUCTION: Array comparative genomic hybridisation (array CGH) is a powerful method that detects alteration of gene copy number with greater resolution and efficiency than traditional methods. However, its ability to detect disease causing duplications in constitutional genomic DNA has not been shown. We developed an array CGH assay for X linked hypopituitarism, which is associated with duplication of Xq26-q27. METHODS: We generated custom BAC/PAC arrays that spanned the 7.3 Mb critical region at Xq26.1-q27.3, and used them to search for duplications in three previously uncharacterised families with X linked hypopituitarism. RESULTS: Validation experiments clearly identified Xq26-q27 duplications that we had previously mapped by fluorescence in situ hybridisation. Array CGH analysis of novel XH families identified three different Xq26-q27 duplications, which together refine the critical region to a 3.9 Mb interval at Xq27.2-q27.3. Expression analysis of six orthologous mouse genes from this region revealed that the transcription factor Sox3 is expressed at 11.5 and 12.5 days after conception in the infundibulum of the developing pituitary and the presumptive hypothalamus. DISCUSSION: Array CGH is a robust and sensitive method for identifying X chromosome duplications. The existence of different, overlapping Xq duplications in five kindreds indicates that X linked hypopituitarism is caused by increased gene dosage. Interestingly, all X linked hypopituitarism duplications contain SOX3. As mutation of this gene in human beings and mice results in hypopituitarism, we hypothesise that increased dosage of Sox3 causes perturbation of pituitary and hypothalamic development and may be the causative mechanism for X linked hypopituitarism.

Spinocerebellar ataxia type 2 with focal epilepsy--an unusual association.

INTRODUCTION: The spinocerebellar ataxias are a rare group of inherited neurodegenerative disorders. Epilepsy has not previously been associated with spinocerebellar ataxia type 2 (SCA2). CLINICAL PICTURE: We describe a family with 3 affected members who had typical phenotypic and MRI features of SCA2. Two had focal epilepsy with complex partial seizures and epileptiform discharges on electroencephalography. Trinucleotide expansions in the pathological range were found in the SCA2 gene, confirming SCA2. Sequencing of the expanded SCA2 gene did not reveal any new mutations that could account for epilepsy. TREATMENT AND OUTCOME: The focal epilepsy was well-controlled with carbamazepine. CONCLUSION: We hypothesise that the new feature of focal epilepsy is due to co-existence of a separate unlinked epilepsy susceptibility gene with the expanded SCA2 gene. Under this oligogenic model, both genes must be present, and co-inheritance of this susceptibility gene with the expanded SCA2 gene causes a complex interaction which triggers epilepsy.

Audit of 'crash' emergency caesarean sections due to cord prolapse in terms of response time and perinatal outcome.

OBJECTIVE: The objective was to audit 'crash' emergency caesarean sections (CS) with respect to response time (the diagnosis to delivery interval [DDI]) and perinatal outcome. MATERIALS AND METHODS: The computerised database at the Singapore General Hospital (SGH) delivery suite was used to identify all cases of 'crash' emergency CS activated for the diagnosis of cord prolapse from 1992 to 2002. Patients' case notes and neonatal charts were reviewed and the following variables were evaluated: parity, gestational age at the time of delivery and the DDI. Neonatal outcome was measured by Apgar scores at 1 and 5 minutes, cord pH and admission to the neonatal intensive care unit (NICU). RESULTS: A total of 34 cases of umbilical cord prolapse were identified from 29,867 deliveries, giving an incidence of 0.11% (1 in 900). The median gestational age was 38.5 weeks (range, 25 to 41 weeks). The median time from diagnosis to delivery was 20 minutes (range, 10 to 40 minutes). Seventy-six percent (19/30) were delivered within 30 minutes. The time of diagnosis was not recorded for 5 cases. Sixty-three percent of neonates had an Apgar score < or = 7 at 1 minute of life, increasing to 97% at 5 minutes. There were 3 NICU admissions for reasons of prematurity. There was no perinatal mortality. Cord pH was not performed for 47% of (14/30) neonates. Among the remaining 16 neonates, an umbilical cord pH of < or = 7.20 was found in 62% (10/16). There was poor correlation between the DDI and umbilical cord pH. CONCLUSION: Three-quarters of our 'crash' emergency CS for cord prolapse were performed within 30 minutes with a good perinatal outcome. However, we have identified areas for improvement to optimise further the operational efficiency of 'crash' emergency CS.

Changing trends in indications for caesarean sections in a tertiary hospital.

OBJECTIVES: To study the caesarean section rate and the trends in indications for caesarean delivery at the Singapore General Hospital (SGH) during two study periods of 6 months each. MATERIALS AND METHODS: The percentages of caesarean sections attributable to specific indications were computed for the first 6 months of 1998 and the last 6 months of 2001. Subgroup analysis of "less common indications" was performed. RESULTS: In the first half of 1998, 170 caesarean sections were performed giving a rate of 16.77%. 54.12% of women were multiparous. The main indication for caesarean section was dystocia (4.24% of deliveries). Two hundred and sixty caesarean sections were performed in the later half of 2001 giving a caesarean section rate of 25.10%. 53.46% of women were multiparous. The main indication for caesarean section was dystocia (5.41% of deliveries). Increase in caesarean section rate in 2001 was attributed to statistically significant increase in caesarean section for previous caesarean section and placenta previa major. Other changes in practice included shorter operating time which may be related to decision not to perform peritoneal closure. CONCLUSION: Studying indications for caesarean section are useful for hospitals, clinicians and researchers in determining strategies to lower primary and repeat caesarean section rate.

Treatment of urinary tract infection with gentamicin once or three times daily.

AIM: To examine the safety and efficacy of once-daily (OD) gentamicin treatment compared with conventional 8-hourly dosing (TDS) for urinary tract infection (UTI). METHODS: This was a prospective, randomized, controlled trial of children 1 mo to 13 y of age with presumed UTI. Children were randomly assigned to OD gentamicin 5 mg kg(-1) d(-1) or TDS gentamicin 6 mg kg(-1) d(-1) divided 8 hourly. Microbiological efficacy, nephrotoxicity, ototoxicity and renal scarring were assessed at the end of treatment. RESULTS: 210 patients with presumed UTI were recruited, of whom 172 were analysable (OD 84, TDS 88). The median age was 7 mo, 50% were male and 74% (n = 127) of patients had pyelonephritis. The majority of infections were due to Escherichia coli (n = 153, 89%), of which 9 (5.2%) were bacteraemic. Comparing the two groups, there was no significant difference in age, gender, duration of fever before admission, pyuria, nitrite positivity or initial total white blood cell count. All patients had negative urine cultures after 2-3 d of treatment, demonstrating 100% microbiological efficacy. There was no difference between the two groups in terms of ototoxicity, nephrotoxicity, duration of gentamicin treatment or time to fever defervescence. CONCLUSION: OD gentamicin is as efficacious as TDS gentamicin in the treatment of UTI in children, with no difference in ototoxicity and nephrotoxicity.

Gestational diabetes mellitus: a call for systematic tracing.

INTRODUCTION: Women with history of gestational diabetes mellitus (GDM) have up to 50% lifetime risk of developing frank diabetes mellitus (DM). They are an ideal group of patients to implement early interventional measures to halt the progression to diabetes. The success of any early intervention programme would depend largely on postpartum follow-up. We set out to study the response rate to postpartum oral glucose tolerance test (OGTT) and to profile the non-responders on 105 women who attended our Gestational Diabetes Joint Clinic (GDJC). MATERIALS AND METHODS: We divided these women into 3 groups according to their response to postpartum OGTT and compared their weights, glycaemic parameters and other clinical characteristics during gestation. Group A comprised non-responders or those who did not turn up for postpartum OGTT; group B comprised responders with a normal postpartum OGTT; and group C comprised responders with an abnormal postpartum OGTT defined as 2-hour plasma glucose equal or more than 7.8 mmol/L. RESULTS: The non-respondent rate to postpartum diabetes screening was 37.1%. The non-responders were found to be significantly heavier, with more severe hyperglycaemia during their pregnancy (in terms of glycosylated haemoglobin and results of antepartum OGTT) and had bigger babies compared to the responders with normal postpartum OGTT. Their features instead resembled those who had failed their postpartum OGTT. CONCLUSION: The group of non-responders was probably at similar risk of developing glucose intolerance postpartum as those who were tested abnormal. A more effective call and recall system and education programme is, therefore, needed to ensure postpartum attendance of all patients with GDM.

Pregnancy after renal transplantation: experience in Singapore General Hospital.

INTRODUCTION: Renal transplantation offers the best hope for those women with end-stage renal disease who wish to have children. However, pregnancy after renal transplantation is associated with increased maternal and fetal morbidity. The aim of this retrospective study was to review the outcome of pregnancy in renal transplant patients in Singapore General Hospital. MATERIALS AND METHODS: Forty-two pregnancies, occurring between December 1986 and December 2000, in 25 out of 141 renal transplant women in their reproductive age group (18 to 45 years old) were identified from our high-risk pregnancy record and retrospectively analysed. RESULTS: Thirteen (31%) pregnancies were unsuccessful; 10 abortions, 2 ectopic pregnancies and 1 stillbirth. The remaining 29(69%) successful pregnancies were complicated by maternal anaemia (65.5%), superimposed hypertension (44.8%), premature rupture of membranes (27.6%), urinary (17.2%) and lower genital tract (13.8%) infections, abnormal glucose tolerance test (13.8%), premature delivery (44.8%), low-birth-weight babies (44.8%), small-for-gestational-age babies (20.7%) and intrauterine growth restriction (20.7%). There were no documented cases of multiple pregnancies, congenital anomalies or deterioration of renal function. The outcome of pregnancy was not statistically influenced by preconception renal function and transplant-conception interval. CONCLUSIONS: Successful pregnancy is possible in women after renal transplantation. Such pregnancy is often associated with increased maternal and fetal complications and should be managed by a multidisciplinary approach in a tertiary centre. The function and survival of renal allograft was not adversely affected by pregnancy.

Outcome of pregnancy in Asian women with systemic lupus erythematosus: experience of a single perinatal centre in Singapore.

OBJECTIVE: To study maternal and fetal outcomes in women with systemic lupus erythematosus (SLE). MATERIALS AND METHODS: Retrospective study of 27 pregnancies in 18 women with SLE in a single centre. RESULTS: The mean age was 30 years and most patients were nulliparous. Twenty-six of 27 pregnancies were in disease remission at the time of booking. Renal impairment was present in 7 pregnancies (6 women), of whom 2 were in end-stage renal disease on dialysis. Gestational diabetes developed in 4 pregnancies. There were 6 cases of pre-existing hypertension and 5 with superimposed pre-eclampsia. One woman developed intrapartum eclampsia. Two women had secondary antiphospholipid syndrome (APS) and suffered late fetal losses; in addition, they also developed SLE flares in the form of autoimmune haemolytic anaemia in the postpartum period. There was no maternal mortality. There was one termination of pregnancy for severe renal disease. The median gestational age at delivery was 38 weeks (range, 24 to 40 weeks) and the mean birth weight was 3047 g; the median Apgar scores were 8 and 9 at 1 and 5 minutes of life, respectively. There were 5 cases of intrauterine growth restriction (IUGR), 4 of which occurred in women with renal impairment. There were no cases of congenital heart block or neonatal lupus. There was a late fetal loss at 24 weeks in a woman with secondary APS. There were 2 preterm deliveries (7.4%) due to intervention for IUGR. CONCLUSION: Good pregnancy outcomes can be expected in women with SLE in remission. Pre-pregnancy counselling is crucial to achieve this. All pregnancies should still be considered high risk and be managed jointly between the obstetricians, the perinatologists and the physicians. In particular, those with renal impairment are at increased risk of IUGR, superimposed pre-eclampsia and preterm births. Co-existing APS augurs a poorer prognosis for pregnancy outcome, and may present atypically as autoimmune haemolytic anaemia in the postpartum period.

The course of pregnancy in a patient with nail-patella syndrome.

INTRODUCTION: The nail-patella syndrome is a rare autosomal dominant condition with high penetrance. Pregnancy in such a patient is rare and we believe this to be the first report of a live birth occurring in a patient with nail-patella syndrome. CLINICAL PICTURE: A 25-year-old patient presented in her first pregnancy with nephrotic syndrome associated with characteristic bone abnormalities and nail dysplasia and was later diagnosed to have nail-patella syndrome. In her second pregnancy, the course of her pregnancy was complicated by further deterioration of renal function with superimposed pre-eclampsia resulting in early delivery at 28 weeks. CONCLUSION: Such pregnancies should be regarded as high risk and managed jointly with the renal physician in a tertiary care centre to ensure an optimal outcome to the mother and baby.

Clinical management of PCOS.

The polycystic ovary syndrome (PCOS) is a heterogeneous condition with genetic predisposition. It is characterized by a myriad of symptoms including oligomenorrhea or amenorrhea, anovulation or infertility, hirsutism or acne. Insulin resistance appears to be an important factor in PCOS though the lack of an etiology has led to symptom oriented therapy which includes lifestyle modification, the use of cyclical progestagens and antiandrogens. Ovulation induction by drug therapy and ovarian drilling aims to treat subfertility in women with PCOS. Therapeutic approaches to PCOS remain an ongoing source of debate. Insulin sensitizing agents may bring new hope in therapy. Future research is aimed at shedding light on the pathophysiology so as to optimize treatment of women with PCOS.