Clinical genomics expands the morbid genome of intellectual disability and offers a high diagnostic yield.

Intellectual disability (ID) is a measurable phenotypic consequence of genetic and environmental factors. In this study, we prospectively assessed the diagnostic yield of genomic tools (molecular karyotyping, multi-gene panel and exome sequencing) in a cohort of 337 ID subjects as a first-tier test and compared it with a standard clinical evaluation performed in parallel. Standard clinical evaluation suggested a diagnosis in 16% of cases (54/337) but only 70% of these (38/54) were subsequently confirmed. On the other hand, the genomic approach revealed a likely diagnosis in 58% (n=196). These included copy number variants in 14% (n=54, 15% are novel), and point mutations revealed by multi-gene panel and exome sequencing in the remaining 43% (1% were found to have Fragile-X). The identified point mutations were mostly recessive (n=117, 81%), consistent with the high consanguinity of the study cohort, but also X-linked (n=8, 6%) and de novo dominant (n=19, 13%). When applied directly on all cases with negative molecular karyotyping, the diagnostic yield of exome sequencing was 60% (77/129). Exome sequencing also identified likely pathogenic variants in three novel candidate genes (DENND5A, NEMF and DNHD1) each of which harbored independent homozygous mutations in patients with overlapping phenotypes. In addition, exome sequencing revealed de novo and recessive variants in 32 genes (MAMDC2, TUBAL3, CPNE6, KLHL24, USP2, PIP5K1A, UBE4A, TP53TG5, ATOH1, C16ORF90, SLC39A14, TRERF1, RGL1, CDH11, SYDE2, HIRA, FEZF2, PROCA1, PIANP, PLK2, QRFPR, AP3B2, NUDT2, UFC1, BTN3A2, TADA1, ARFGEF3, FAM160B1, ZMYM5, SLC45A1, ARHGAP33 and CAPS2), which we highlight as potential candidates on the basis of several lines of evidence, and one of these genes (SLC39A14) was biallelically inactivated in a potentially treatable form of hypermanganesemia and neurodegeneration. Finally, likely causal variants in previously published candidate genes were identified (ASTN1, HELZ, THOC6, WDR45B, ADRA2B and CLIP1), thus supporting their involvement in ID pathogenesis. Our results expand the morbid genome of ID and support the adoption of genomics as a first-tier test for individuals with ID.

The early wear susceptibility of cementum, enamel and dentine in vitro.

OBJECTIVES: To assess the differential early wear susceptibility of cementum, enamel and dentine at a micron level. METHODS: Whole human molar buccal surfaces incorporating natural enamel and cementum (n = 20) confirmed by imaging (digital microscopy: Keyence, VHX-7000 Milton Keynes, UK), were mounted, scanned (profilometry: XYRIS 4000, Taicaan, Southampton, UK), and allocated to receive erosion (citric acid, pH 2.7, 30 min (n = 10)) or erosion/abrasion challenges (3 cycles of (citric acid, pH 2.7, 10 min, 60 300 g linear abrasion strokes), n=10). Samples were polished and the experiment repeated on polished enamel, and polished coronal and radicular dentine within the same tooth. Profilometric wear data were obtained using superimposition: GeoMagic (3Dsystems, Darmstadt, Germany) and subtraction: MountainsMap (DigitalSurf, Besancon, France). Data were normal. A general linear model was used to assess differences between groups and substrates. RESULTS: The mean step height (SD) for natural enamel was 8.82 µm (2.53) for erosion and 11.48 µm (2.95) for erosion/abrasion. For natural cementum, the mean step height was 6.00 µm (2.29) for erosion and 4.67 µm (1.58) for erosion/abrasion. Dentine step heights ranged from 7.20 µm (1.53) for erosion and 9.79 µm (1.01) for erosion/abrasion with no statistical differences in dentine wear. Natural cementum surfaces had the lowest wear (p<0.001). Dentine had significantly less wear than natural enamel (p<0.02). CONCLUSIONS: Cementum surfaces demonstrated the most wear resistance, followed by dentine under erosion dominant conditions in this in vitro study. Further in-vivo investigations are needed to confirm the intraoral stability of cementum. CLINICAL SIGNIFICANCE: Cementum may be the least susceptible of dental substrates to wear and dentine does not wear at a faster rate than enamel under erosive conditions. This adds to our knowledge on the development of non-carious cervical lesions and questions whether wear rates will accelerate once dentine is exposed.

Prevalence of metallo-ß-lactamase-producing Pseudomonas aeruginosa isolated from diabetic foot infections in Iraq.

Metallo-ß-lactamase (MBL)-producing Pseudomonas aeruginosa is a major cause of nosocomial infections. However, there is little information in Iraq regarding its prevalence in patients with diabetic foot ulcer. Carbapenems are efficient antibiotics against extended-spectrum ß-lactamase-producing P. aeruginosa. However, there are many potential health risks associated with carbapenem-resistant P. aeruginosa. We aimed to determine MBL-producing P. aeruginosa isolated from diabetic foot ulcer infections. A total of 97 P. aeruginosa isolates were isolated from pus and deep tissue swabs of 282 patients admitted to Al-Sader hospital, Najaf City, Iraq, with diabetic foot infections from October 2017 to January 2018. All P. aeruginosa isolates were tested by the Kirby-Bauer disc diffusion method for evaluating 13 antibiotics. Phenotypic carbapenem resistance was confirmed by the combined disc test, double-disc synergy test, modified Hodge test and CHROMagar KPC agar. All phenotypic MBL-producing P. aeruginosa isolates were screened for bla IMP, bla NDM, bla SIM, bla SPM and bla VIM genes by multiplex PCR. Of the 97 P. aeruginosa isolates, combined disc test and modified Hodge test revealed 12 isolates (12.4%) to be MBL producers, and ten (10.3%) displayed MBL production as accessed by CHROMagar KPC agar test. Nine isolates (9.3%) were carbapenemase producers by the imipenem and ceftizoxime double-disc synergy test. Of 12 phenotypic MBL-producing P. aeruginosa, PCR amplification confirmed 4 (33.3%) and 3 (25%) isolates harbouring bla VIM and bla IMP gene respectively, but none carried the bla NDM, bla SIM or bla SPM genes. The steady and rapid increase of MBL production is worrisome and needs to be controlled through extensive studies and more judicious selection of antibiotics, especially carbapenems.

Effect of methotrexate administration on status of some essential trace elements and antioxidant enzymes in pregnant rats in late gestation.

OBJECTIVE: Folate antagonists are widely used in the treatment of various cancerous states. Paucity of data on effect of administration of one such widely used drug, methotrexate (MTX), on the status of essential trace elements and antioxidant enzymes in pregnant women or in pregnant animals prompted us to undertake this study. METHODS: MTX at a concentration of 5 mg/kg body weight was administered intraperitoneally as single dose to pregnant Sprague-Dawley rats for three consequitive days from day 17 of pregnancy. Control group of pregnant rats received single dose of saline instead of the anti-cancer drug on all the 3 days. After receiving the third dose of drug, the treated rats and control group rats were sacrificed, 1 h after intraperitoneal injection of a cocktail of essential trace elements namely, Cu, Se and Zn administered as a single bolus dose. Blood samples were collected 30 min of trace element cocktail injection, after decapitation and concentrations of trace elements in serum samples were determined by atomic absorption spectrophotometry. Concentrations of antioxidant enzymes, such as superoxide dismutase, glutathione peroxidase and total antioxidant status were determined by specific analytical kits, using spectrophotometry. RESULTS: In control group(n = 6), serum concentrations of Cu, Se and Zn averaged 2330.5, 614.8 and 2773.2 microg/l, while in study group (n = 6) the concentrations of trace elements averaged 2294, 596 and 2713 microg/l, respectively. Student's t-test did not show any statistical significance (p > 0.05) between various trace element concentrations in control and treated groups. Cu:Zn ratios of control and treated group of rats did not vary significantly as well. Concentrations of superoxide dismutase, glutathione peroxidase in whole blood samples in control rats averaged 165 and 43,260 U/ml, respectively, while in MTX-treated group of animals the corresponding antioxidant enzymes averaged 330.6 and 67,101 U/ml respectively. SOD and GPX values were significantly higher in drug-treated animals compared to controls (Student's t-test, p < 0.05) However, total antioxidant activity was shown to be significantly lower (Student's t-test; p < 0.05) in the drug-treated group compared to control. CONCLUSIONS: We report for the first time that effect of MTX administration in pregnancy is not associated with significant alteration in disposition of essential trace elements. However, the effect of drug administration on antioxidant enzyme status in pregnant women cannot be excluded while using the drug in clinical states.

Microshear bond strength of different restorative materials to teeth with molar-incisor-hypomineralisation (MIH): a pilot study.

AIM: To compare the microshear bond strength of resin based composite (RBC) and resin modified glass ionomer cement (RMGIC) restorations when bonded to teeth with molar incisor hypomineralisation (MIH). METHODS: Eleven first permanent molars with MIH were included in this study. Teeth were sectioned mesio-distally producing a total of 22 surfaces for testing. Each specimen was placed inside a plastic ring with the flattened surface in contact with a glass slab. The plastic ring was filled with autopolymerising acrylic resin to imbed the specimen leaving the enamel surface exposed. Each surface was then bonded to 0.96 mm diameter RBC and light cured RMGIC following the manufacturers' instructions. Microshear bond testing was performed after 24 h storage in distilled water at 37 °C. A Bisco shear tester was used to apply shear stress of 1 mm/min until failure. Wilcoxon signed-rank paired-test was used for comparison of bond strength values. RESULTS: Microshear bond strength of RBC (30.80 ± 8.19 MPa) was significantly higher than that of RMGIC (11.13 ± 6.91 MPa) when bonded to hypomineralised permanent first molars (p < 0.001). CONCLUSION: The microshear bond strength of RBC is significantly higher than that of RMGIC when bonded to MIH affected teeth. Therefore, RBC can be recommended as the restoration of choice for teeth with MIH whenever the clinical scenario allows.

Transport kinetics of chromium in perfused human placental lobule in late gestation: in vitro study.

Objective: Reports relating to maternal-fetal transport kinetics of chromium, an essential trace element in the human pregnancies are scanty. Hence, we thought it interesting to investigate the transport kinetics of this trace element in the human placenta in late gestation in vitro. Methods: Human placentae were collected immediately after delivery from normal uncomplicated pregnancies. Chromium chloride solution (GFS Chem Inc, Ohio, USA) at 10 times the physiological concentrations and antipyrine (Sigma Chem Co., St. Louis, USA) as internal reference marker was injected as a single bolus (100 µl) into the maternal arterial circulation of perfused placental lobules and perfusate samples were collected from maternal and fetal circulations over a study period of 5 minutes. National culture and Tissue collection medium, diluted with Earle's buffered salt solution was used as the perfusate. Serial perfusate samples were collected from fetal venous perfusate for a period of 30 minutes. Chromium concentration in perfusate samples was determined using atomic absorption spectrophotometry and the concentration of reference marker, antipyrine was measured by spectrophotometry. Transport kinetics and transport parameters of study and reference markers were assessed using well-established parameters. Results: Differential transport rates of chromium and antipyrine in 10 perfusions differed significantly for 10 and 50% efflux fractions (ANOVA test, p < .05) while those of 25, 75, and 90% efflux fractions were not significantly different between the study and reference substances. Chromium transport fraction (TF) averaged 54.9% of bolus dose in 10 perfusions while that of antipyrine averaged 89% of bolus dose, representing 61.80% of reference marker TF. The difference observed in TF values of chromium and antipyrine was statistically significant (Student's t-test, p < .05). Pharmacokinetic parameters such as area under the curve, clearance, absorption rate, elimination rate of chromium compared to reference marker was significantly different (ANOVA test, p < .05) between the study and reference substances. Conclusions: Our studies report for the first time maternal-fetal transport kinetics of chromium in human placenta in vitro. Considering the restricted transfer of this essential trace element from maternal to fetal circulation despite its small molecular weight, we hypothesize an active transport of chromium across the human placental membrane. Further studies relating to placental transport kinetics of this trace element in various pregnancy-related disease states are in progress.

Maternal-foetal status of copper, iron, molybdenum, selenium and zinc in obese gestational diabetic pregnancies.

Obesity is well known to be a contributory risk factor for several disease states, including diabetes mellitus. Paucity of data on maternal-foetal status of essential trace elements in obese diabetic pregnancies prompted us to undertake this study. Maternal venous and umbilical arterial and venous blood samples were collected from obese gestational diabetic patients (Body Mass Index (BMI) >30) and control obese pregnant women (BMI>30) at time of spontaneous delivery or caesarean sections and concentrations of essential trace elements such as Cu, Fe, Mo, Se and Zn were determined in various samples by atomic absorption spectrophotometry. Activities of antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPX) and total antioxidant (TAO) in maternal and umbilical blood were assessed using appropriate reagent kits. Maternal-foetal disposition and exchange parameters of elements studied were assessed using established criteria. Concentrations of Cu, Fe, Mo, Se and Zn in serum of control obese pregnant women (n=10) averaged 2404, 2663, 11.0, 89.0 and 666 microg/l respectively, while in the obese diabetic group (n=11), the corresponding values averaged 2441, 2580, 13.3, 85.1 and 610 microg/l respectively. Activities of antioxidant enzymes such as SOD, GPX and TAO were not significantly different in maternal veins of control and diabetic groups. Varying differences were noted in the case of antioxidant enzyme activities in umbilical blood samples of control and study groups. We conclude that obesity is not associated with significant alterations in antioxidant enzyme status in gestational diabetes and only with relatively minor alterations in status of some essential trace elements.

Effect of hyperglycaemic load on maternal-foetal transport of L-leucine in perfused human placental lobule: in vitro study.

The effect of hyperglycaemic loads on L-leucine transport in the maternal-foetal direction has been investigated in human placenta in vitro, using perfusion of isolated placental lobules. National Culture and Tissue Collection medium diluted with Earle's buffered salt solution was used as the perfusate. (14)C-labelled L-leucine along with tritiated water as reference were injected as a 100-microl bolus into the maternal circulation and serial perfusate samples were collected over a 5-min study period. In 6 successful perfusions, the differential transport rate of leucine for 10, 25, 50, 75 and 90% of efflux in the foetal vein averaged 1.26, 1.10, 1.19, 1.10 and 1.04 times respectively that of reference in the control euglycaemic phase. In the experimental hyperglycaemic phases of 27.8 and 55.6 mM/l, leucine transport for the corresponding efflux periods averaged 1.37, 1.33, 1.28, 1.22 and 1.26 times and 1.16, 0.97, 1.08, 1.08 and 1.08 times respectively that of the reference marker. Analysis of variance (ANOVA) test showed that the difference between the three groups was not statistically significant. In the control euglycaemic phase, leucine transport fraction (TF) averaged 40.90+/-3.51% of corresponding water TF while in experimental hyperglycaemic phases, TF of the amino acid averaged 37.80+/-4.82% and 35.61+/-3.21% of water TF respectively. The difference between the control and hyperglycaemic perfusion phases was not statistically significant. Further, no statistical difference could be shown between the two hyperglycaemic perfusion series themselves. Similarly, kinetic parameters such as absorption rate, elimination rate and absorption rate:elimination rate ratio were not significantly different in the hyperglycaemic perfusion phases compared to control phase. Our studies seem to indicate that leucine transport kinetics in in vitro conditions are not altered significantly in placentas exposed to hyperglycaemic loads.

Maternal-fetal transport kinetics of L-Leucine in vitro in gestational diabetic pregnancies.

OBJECTIVE: Paucity of data relating to transport of amino acids in gestational diabetic pregnancies prompted us to undertake this study. Transport kinetics of a model amino acid, L-leucine was investigated in gestational diabetic pregnancies in vitro, using perfusion of isolated placental lobules. METHODS: Placentae from diabetic and control pregnant women were collected post-partum. Suitable placental lobules were then perfused, using National Culture and Tissue Collection (NCTC) medium, diluted with Earle's buffered salt solution as perfusate. 14C-labelled L-leucine along with tritiated water as reference were injected as a 100 ul bolus into the maternal circulation and serial perfusate samples collected over a 5-minute study period. RESULTS: In 6 successful perfusions, differential transport rate of L-leucine for 10, 25, 50, 75 and 90% of efflux in the fetal vein averaged 1.17, 1.12, 1.22, 1.20 and 1.17 times respectively that of reference in the diabetic group. In the control group (n=6), leucine transport indices for the corresponding efflux periods averaged 1.13, 1.15, 1.18, 1.17 and 1.16 times respectively that of the reference marker. Student's 't' test showed that the difference between the two groups was not statistically significant (p > 0.05) for all the efflux fractions studied. In the diabetic series, leucine transport fraction (TF) averaged 41.2 +/- 4.5% of corresponding water TF while in control group, the amino acid TF averaged 46.5 +/- 6.5% of water TF. The difference between the two series, however was not statistically significant (p > 0.05). Similarly, kinetic parameters as area under the curve, clearance, elimination constant, time for maximum response, absorption rate, and elimination rate in the diabetic and control groups, were not significantly different (p > 0.05) as well. CONCLUSION: Our study seems to indicate that transport kinetics of leucine under in vitro conditions, do not differ significantly in placentae of gestational diabetic women compared to controls.

Maternal-fetal status of copper, iron, molybdenum, selenium and zinc in patients with gestational diabetes.

OBJECTIVE: The status of essential trace elements such as copper, iron, zinc, selenium and molybdenum was investigated in gestational diabetic pregnancies at term, and data were compared to control pregnancies. Fetal/maternal ratios of the elements and copper/zinc ratio were also computed in control and study populations. METHODS: Samples from maternal venous, umbilical artery and umbilical vein were collected from gestational diabetic and control pregnant women, at the time of spontaneous delivery or Cesarean section, and concentrations of various trace elements were determined by atomic absorption spectrophotometry. RESULTS: The concentrations of copper, iron, molybdenum, selenium and zinc averaged 2156.2 microg/l, 2020.1 microg/l, 13.1 microg/l, 102.3 microg/l and 656.2 microg/l, respectively, in maternal venous blood in control pregnant women at term (n=15) while in the corresponding gestational diabetic group (n=15), concentrations of the trace elements averaged 2345.8 microg/l, 2061.6 microg/l, 15.0 microg/l, 75.2 microg/l and 610.3 microg/l, respectively. Student's t test showed that the selenium concentration was significantly lower (p<0.05)in the diabetic group compared to controls. Values of other elements were not significantly different. Umbilical blood/maternal blood ratios of the trace elements showed varying differences. The Cu/Zn ratio was found to be significantly different between umbilical and maternal samples of control and study groups, indicating a possibility of compromised antioxidant function in diabetic pregnancies. CONCLUSIONS: We speculate that altered maternal-fetal status of some essential trace elements in gestational diabetes patients could have deleterious influences on the health of the mother as well as the fetus and newborn.

Assessment of maternal-fetal status of some essential trace elements in pregnant women in late gestation: relationship with birth weight and placental weight.

OBJECTIVE: The status of the essential trace elements copper (Cu), iron (Fe), zinc (Zn), selenium (Se) and molybdenum (Mo) has been investigated in maternal and umbilical cord blood in control, uncomplicated pregnancies at term, and the possibility assessed of a relationship between blood levels of these trace elements and newborn weight and placental weight. Fetal-maternal ratios of the elements were also computed to establish baseline values for the Kuwaiti obstetric population. METHODS: Blood samples were collected from a maternal vein, the umbilical artery and umbilical vein of normal pregnant women at the time of spontaneous delivery or Cesarean section, and the concentrations of various trace elements determined by atomic absorption spectrophotometry. RESULTS: The concentration of Cu, Fe, Mo, Se and Zn averaged 2406.1, 3252.1, 11.6, 107.3 and 696.2 microg/l, respectively, in maternal venous blood in the pregnant women (n=39) at term. Umbilical venous/maternal venous ratios of Cu, Fe, Mo, Se and Zn averaged 0.32, 1.96, 1.03, 0.83 and 1.55, respectively. Neonatal birth weight did not correlate with maternal blood levels of the trace elements (p>0.05) in the mother-child pairs studied. However, neonatal weight correlated negatively (p<0.05) with umbilical venous Cu level. Placental weight correlated positively (p<0.05) with Fe and Mo levels and negatively with Zn level in umbilical venous blood. CONCLUSIONS: Our results indicate an active placental transport for Fe and Zn, while Cu, Mo and Se appear to be exchanged passively between mother and fetus. Evaluation of Fe, Mo, Se and Zn levels in maternal and umbilical cord blood does not appear to be useful in the assessment of fetal growth.

Use of through-and-through guidewire for delivering large stent-grafts into the distal aortic arch.

The availability of large diameter stent-grafts is now allowing the endovascular treatment of thoracic aortic aneurysms. Most aneurysms are closely related to the distal arch and it is thus necessary to pass the delivery systems into the arch to effectively cover the proximal neck. Even with extra-stiff guidewires in position, it may still be difficult to achieve this, as a result of tortuosity at the iliac arteries and the aorta. We detail a technique where a stiff guidewire is passed from a brachial entry point through the aorta and out at the femoral arteriotomy site. This allows extra-support and may enable the delivery system to be passed further into the aortic arch than it could with just the regular guidewire position.

Transcatheter regional urokinase therapy in the management of inferior vena cava thrombosis.

PURPOSE: To study the efficacy of local infusion of urokinase (UK) in the treatment of symptomatic inferior vena cava (IVC) thrombosis. MATERIALS AND METHODS: Eight patients (five men and three women) who ranged in age from 19 years to 75 years (mean, 56 years) with symptomatic IVC thrombosis underwent local catheter-directed infusion of UK with use of up to three access sites. Infrarenal IVC thrombus and iliac vein thrombus was identified in all patients. Four patients had extension of thrombus proximal to the renal veins. Seven of eight patients had at least one risk factor for IVC thrombosis: hypercoagulable state (n = 3), IVC filter (n = 3), malignancy (n = 2), recent surgery (n = 2), and oral contraceptive use (n = 1). No serious procedure-related complications were encountered, although one patient died 5 days after UK therapy of pulmonary failure due to advanced lung cancer. UK was infused for an average of 79 hours (range, 24-140 hours) and a mean total dose of 7.4 million U of UK (range, 2.9-14.4 million U). Adjunctive balloon angioplasty was performed in three patients. No vascular stents were placed. Clinical and/or radiographic follow-up was obtained in all eight patients. RESULTS: Thrombolysis was successful in seven of eight (88%) IVCs with no or minimal residual thrombus. The remaining seven patients had no lower extremity swelling 2-24 months (mean, 11 months) after the procedure. Three of seven patients had computed tomographic or venographic follow-up (mean, 9 months; range, 1.5-15 months), demonstrating unchanged or improved IVC patency. CONCLUSIONS: Transcatheter regional infusion of UK for re-establishing venous patency in acute IVC thrombosis appears to be effective with good short-term and mid-term clinical benefit.