Mutations in known disease genes account for the majority of autosomal recessive retinal dystrophies.

Retinal dystrophies (RDs) are hereditary blinding eye conditions that are highly variable in their clinical presentation. The remarkable genetic heterogeneity that characterizes RD was a major challenge in establishing the molecular diagnosis in these patients until the recent advent of next-generation sequencing. It remains unclear, however, what percentage of autosomal recessive RD remain undiagnosed when all established RD genes are sequenced. We enrolled 75 families in which RD segregates in an apparently autosomal recessive manner. We show that the yield of a multigene panel that contains known RD genes is 67.5%. The higher yield (82.3%) when whole exome sequencing was implemented instead was often due to hits in genes that were not included in the original design of the panel. We also show the value of homozygosity mapping even during the era of exome sequencing in uncovering cryptic mutations. In total, we describe 45 unique likely deleterious variants (of which 18 are novel including one deep intronic and one genomic deletion mutation). Our study suggests that the genetic heterogeneity of autosomal recessive RD is approaching saturation and that any new RD genes will probably account for only a minor role in the mutation burden.

Sociodemographic characteristics and sexual behavior as risk factors for human papillomavirus infection in Saudi Arabia.

OBJECTIVES: To determine the prevalence and the sociodemographic characteristics and sexual behavior risk factors for human papillomavirus (HPV) infection in a hospital-based cohort of women in Saudi Arabia. METHODS: Cervical specimens and questionnaire data were collected from women attending clinics in Riyadh, Saudi Arabia. Cervical specimens were examined for abnormal cytology using a standard Pap test and for the presence of HPV-DNA using PCR and reverse line blot hybridization tests. RESULTS: Approximately 73% of the 400 women tested were Saudi nationals. Nearly 50% were under 40 years old (range 22-80 years, mean±standard deviation 41.20±10.43 years). Approximately 17% of the women were HPV-positive. The most commonly detected HPV types were HPV-18 (34%) and HPV-16 (19%), with multiple infections detected in 10% of positive specimens. Multivariate analyses revealed that smoking and multiple partners were significant risk factors for HPV infection (p<0.01). CONCLUSIONS: Because of societal challenges and an unsubstantiated assumption of low HPV prevalence, few studies have examined sociodemographic characteristics or sexual behaviors associated with HPV in Saudi women. However, a high prevalence of HPV infection was found, with smoking and multiple partners as significant risk factors, in this hospital-based cohort of predominantly Saudi women.

Randomised trial of cervical cerclage, with and without occlusion, for the prevention of preterm birth in women suspected for cervical insufficiency.

OBJECTIVE: To evaluate the effect of cerclage, with and without cervical occlusion. DESIGN: Multicentre, stratified, randomised controlled trial. SETTING: Hospital-based multicentre study with 18 tertiary centres from nine countries. POPULATION: Women with a history of cervical insufficiency (prophylactic trial) and women with a short cervix (therapeutic trial) were recruited from August 2006 to August 2011. METHODS: A centralised telephone randomisation service with a computer system was used to randomise women to cervical cerclage with or without cervical occlusion. Only the analyst performing the interim analyses was blinded. MAIN OUTCOME MEASURES: The take-home baby rate (number of infants discharged alive from the hospital), gestational age at delivery, and the number of days in the neonatal intensive care unit (NICU). RESULTS: Women (n = 309) were stratified into the prophylactic trial (n = 213) or the therapeutic trial (n = 96). The trial stopped early due to slow recruitment and an interim analysis showing no benefit of occlusion. Final analysis comprised 197 women in the prophylactic trial and 87 women in the therapeutic trial. No added effect of cervical occlusion was found in terms of the take-home baby rate in the prophylactic trial (92 versus 90%, RR 1.03, 95% CI 0.94-1.12) or in the therapeutic trial (81 versus 85%, RR 0.96, 95% CI 0.79-1.16). No effect of cervical occlusion was found in terms of gestational age at delivery and number of days the neonate spent in the NICU. Cervical occlusion was associated with no harm. CONCLUSIONS: Cervical occlusion with cerclage had no significant additional effect.

Clinical, biochemical and molecular characterization of peroxisomal diseases in Arabs.

Peroxisomes are single membrane-bound cellular organelles that carry out critical metabolic reactions perturbation of which leads to an array of clinical phenotypes known as peroxisomal disorders (PD). In this study, the largest of its kind in the Middle East, we sought to comprehensively characterize these rare disorders at the clinical, biochemical and molecular levels. Over a 2-year period, we have enrolled 17 patients representing 16 Arab families. Zellweger-spectrum phenotype was observed in 12 patients and the remaining 5 had the rhizomelic chondrodysplasia punctata phenotype. We show that homozygosity mapping is a cost-effective strategy that enabled the identification of the underlying genetic defect in 100% of the cases. The pathogenic nature of the mutations identified was confirmed by immunofluorescence and complementation assays. We confirm the genetic heterogeneity of PD in our population, expand the pool of pathogenic alleles and draw some phenotype/genotype correlations.

Quantification of N-acetylaspartic acid in urine by LC-MS/MS for the diagnosis of Canavan disease.

Canavan disease is an autosomal recessive leukodystrophy characterized by excessive excretion of N-acetylaspartic acid (NAA) in urine. The disease is caused by deficiency of aspartoacylase, the enzyme responsible for the hydrolysis of NAA into acetate and l-aspartate. Patients, who are often asymptomatic in their early months, show a wide spectrum of clinical presentation thereafter that includes macrocephaly, poor head control, seizures, abnormal muscle tone, optic atrophy, significant developmental delay and death. In this work, we describe a simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of NAA in urine. The internal standard d3-NAA was added to untreated urine and the mixture was injected into the LC-MS/MS system operated in the negative ion mode. Detection was achieved in multiple reaction monitoring (MRM) mode by monitoring m/z 174 --> 88, 174 --> 130 and 174 --> 58 for NAA and 177 --> 89 for the internal standard. Separation was carried out on a C8 column (2.1 x 150 mm) using a mixture of acetonitrile and water (1:1 v/v) containing 0.05% formic acid at a flow rate of 0.25 ml/min. NAA was eluted at 1.6 min and the run time was approximately 2 min. Using spiked urine, the assay was linear up to 2 mmol/L with limit of quantification at 1 micromol/L (S/N = 12). NAA in patients' urine (n = 17) ranged between 366 and 21,235 mmol/mol creatinine compared to controls of <39 mmol/mol creatinine (n = 159). This LC-MS/MS method for NAA as described involved no extraction and no derivatization, showed no interference, and gave excellent recovery with low variability and short analytical time.

Non-immune fetal hydrops: Are we doing the appropriate tests each time?

Non-immune fetal hydrops is diagnosed when there is fluid accumulation in more than one extravascular space. A long list of etiologies has been found in association with non-immune hydrops. Thorough investigations are needed to be able to identify an underlying cause. There are many recent reports indicating that non-immune hydrops can be an extreme presentation of a number of metabolic disorders, mostly lysosomal storage diseases. The fetal hydrops associated with metabolic disorders is usually severe with very thick skin, massive ascitis, other feature could be seen such as contracture deformities, skeletal abnormalities, hepatosplenomegaly, renal abnormalities, and enlarged nuchal translucency. The diagnosis of a metabolic disorder can be done by a variety of different tests: measuring the level of the specific enzyme or metabolite, histological examination of different organs, or mutation identification. An index case is usually needed to confirm the diagnosis. In-utero diagnosis of a metabolic disorder in the absence of an index case is difficult and only available in selected laboratories around the world. In populations with high consanguinity, these diseases are much more commonly present than what we might think. Routine screening for metabolic diseases especially lysosomal storage diseases should be considered in these populations, and definitely in cases of recurrent hydrops in the same family. More efforts should be spent on identifying causative mutations in different ethnic groups. Every effort should be made to identify the etiology in an index case in the family, as this might be the best opportunity for improving future care.

A prospective management study of slow-release aspirin in the palliation of uteroplacental insufficiency predicted by uterine artery Doppler at 20 weeks.

OBJECTIVE: To investigate the effect of low-dose, slow-release aspirin in reducing the incidence and/or severity of pregnancy complications in women identified as high risk of developing problems associated with uteroplacental insufficiency, namely pre-eclampsia or delivering a small-for-gestational age (SGA) baby. DESIGN: A prospective, randomized management study. One thousand and twenty-two women of mixed parity underwent color flow/pulsed Doppler (CFPD) imaging of the uterine arteries at the time of the 17-23 week (mean 19.9) anomaly scan. Women who were screen positive were randomized to a control or treatment group. The treatment group was given 100-mg slow-release aspirin (Disprin CV) daily and followed up at regular intervals. Women in the routine group received routine antenatal care. Main outcome measures were pre-eclampsia and SGA < 3rd centile. Secondary outcome measures were: SGA < 10th centile, pre-eclampsia requiring delivery before 34 weeks, placental abruption, an Apgar score < 7 at 5 min, admission to neonatal intensive care unit or a pregnancy that resulted in a stillbirth or neonatal death. Odds ratios (OR) with 95% confidence intervals (CI) were calculated for severe and any complications. RESULTS: Two hundred and sixteen women were screen positive according to the defined criteria. One hundred and three women were assigned to the treatment group and 113 to the control group. The difference in the incidence of pre-eclampsia and SGA < 3rd centile between the control and treatment groups did not reach statistical significance. There was a statistically significant reduction in any (OR 0.41 (CI 0.35-0.45), P < 0.01) and severe pregnancy complications (OR 0.43 (CI 0.21-0.84), P < 0.05) in the treatment group compared with the controls. CONCLUSIONS: The administration of slow-release aspirin to women identified as high risk, using color Doppler imaging of the uterine arteries at 20 weeks' gestation, did not significantly alter the incidence of pre-eclampsia or delivery of a SGA baby. It did, however, improve the outcome by reducing the overall incidence of complications associated with uteroplacental insufficiency.

Sensitive assay for the determination of cefazolin or ceftriaxone in plasma utilizing LC.

A rapid, specific and very sensitive liquid chromatographic assay using standard ultraviolet detection has been developed to measure cefazolin (CFZ) or ceftriaxone (CFX) in small samples (200 microl) of plasma using either drug as the internal standard for measurement of the other. A rapid extraction was performed using C18 bonded Sep Pak cartridges with high extraction efficiency for both drugs. The chromatographic system employed the use of a Nova-Pak C18 4-microm cartridge with a radial compression system preceded by a Guard-Pak with a C18 insert. The mobile phase consisted of an aqueous solution containing 10 mM of dibasic potassium phosphate and 10 mM cetyltrimethylammonium bromide (pH 6.5) with acetonitrile (73:27 v/v). The drug and internal standard (CFZ/CFX) were detected using a UV detector set at a wavelength of 274 nm. Assay results were linearly related to the concentration (r > 0.997) for the wide range which was examined (0.005-120 microg/ml) for either drug. We report the precision, accuracy, recovery, linearity, sensitivity and specificity of this assay. The intra-run and inter-run CV was less than 9.02%. This method is currently being used for clinical therapeutic monitoring and pharmacokinetic studies of CFZ and CFX in patients undergoing cesarean section.

The role of color Doppler imaging of the uterine arteries at 20 weeks' gestation in stratifying antenatal care.

OBJECTIVE: To determine the value of one-stop color Doppler imaging of the uterine arteries at the time of the 20-week anomaly scan, to select women at risk of developing pre-eclampsia and intrauterine growth restriction (IUGR). PATIENTS AND METHODS: A total of 1022 unselected women had color Doppler imaging of both uterine arteries at the time of their dating/anomaly scan (19-21 weeks' gestation). The presence or absence of notching of the flow velocity waveform (FVW) was noted, and the resistance index (RI) was measured. The main outcome measures were pre-eclampsia, birth weight, placental abruption and stillbirth. RESULTS: The outcome in 946 women (92.6%) was available for analysis. Of these, 216 (23%) had abnormal uterine artery Doppler studies, 117 (12.4%) with bilateral (right and left FVW) notches; 21 (2.2%) women developed pre-eclampsia, and 57 (6.0%) neonates were small for gestational age (SGA; < 5th centile), at birth. For women with bilateral notches the odds ratio (OR) for developing pre-eclampsia was 12.8 (95% confidence interval (CI) 5.3-30.8), and 52.6 (95% CI 6.4-430.1) for pre-eclampsia requiring delivery before 37 weeks' gestation. If the uterine artery Doppler studies were normal, the odds ratio for developing pre-eclampsia was 0.11 (95% CI 0.04-0.28), and 0.3 (95% CI 0.17-0.51) for the delivery of an SGA baby less than the 5th centile. In women with bilateral notches with mean RI greater than 0.55, the positive predictive value for the main outcome measures was 46%. CONCLUSION: Women with normal uterine artery color/pulsed Doppler studies at 20 weeks' gestation constitute a group that have a low risk of developing obstetric complications related to uteroplacental insufficiency. Women with high resistance in both uterine arteries (bilateral notches) have an increased risk of the subsequent development of such complications, in particular those requiring delivery before term. The addition of color Doppler imaging of the uterine arteries at the time of the routine 20-week dating/anomaly scan may be of use in determining the type and level of antenatal care that is offered to women.

Fetal heart rate and umbilico-placental Doppler flow velocity waveforms in early pregnancies with a chromosomal abnormality and/or an increased nuchal translucency thickness.

Fetal heart rate, umbilical artery pulsatility index, end-diastolic flow, nuchal translucency thickness and placental thickness were recorded in 250 women with a viable singleton pregnancy undergoing chorionic villous sampling for fetal karyotyping at 11-14 weeks of gestation. The fetal karyotype was normal in 210 cases and abnormal in 40, including 21 with trisomy 21, 13 with trisomy 18, three with triploidy, two with monosomy X and one with trisomy 13. A total of 52 fetuses with a normal karyotype had a nuchal translucency > or = 3 mm and were considered separately. There was a stable and significant increase in the mean fetal heart rate in trisomy 21 pregnancies compared to controls. No significant difference was found for the other variables between the groups. In chromosomally normal fetuses with an increased nuchal thickness, the development of fetal heart rate and compliance of the umbilico-placental circulation were within the normal ranges. Some fetuses with trisomy 18 or triploidy had an increased resistance to blood flow in the umbilical artery, which was probably due to abnormal placental development.