Investigating diagnostic sequencing techniques for CADASIL diagnosis.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a cerebral small vessel disease caused by mutations in the NOTCH3 gene. Our laboratory has been undertaking genetic diagnostic testing for CADASIL since 1997. Work originally utilised Sanger sequencing methods targeting specific NOTCH3 exons. More recently, next-generation sequencing (NGS)-based technologies such as a targeted gene panel and whole exome sequencing (WES) have been used for improved genetic diagnostic testing. In this study, data from 680 patient samples was analysed for 764 tests utilising 3 different sequencing technologies. Sanger sequencing was performed for 407 tests, a targeted NGS gene panel which includes NOTCH3 exonic regions accounted for 354 tests, and WES with targeted analysis was performed for 3 tests. In total, 14.7% of patient samples (n = 100/680) were determined to have a mutation. Testing efficacy varied by method, with 10.8% (n = 44/407) of tests using Sanger sequencing able to identify mutations, with 15.8% (n = 56/354) of tests performed using the NGS custom panel successfully identifying mutations and a likely non-NOTCH3 pathogenic variant (n = 1/3) identified through WES. Further analysis was then performed through stratification of the number of mutations detected at our facility based on the number of exons, level of pathogenicity and the classification of mutations as known or novel. A systematic review of NOTCH3 mutation testing data from 1997 to 2017 determined the diagnostic rate of pathogenic findings and found the NGS-customised panel increases our ability to identify disease-causing mutations in NOTCH3.

Genetic variants associated with exercise performance in both moderately trained and highly trained individuals.

Adaptation to exercise training is a complex trait that may be influenced by genetic variants. We identified 36 single nucleotide polymorphisms (SNPs) that had been previously associated with endurance or strength performance, exercise-related phenotypes or exercise intolerant disorders. A MassARRAY multiplex genotyping assay was designed to identify associations with these SNPs against collected endurance fitness phenotype parameters obtained from two exercise cohorts (Gene SMART study; n = 58 and Hawaiian Ironman Triathlon 2008; n = 115). These parameters included peak power output (PP), a time trial (TT), lactate threshold (LT), maximal oxygen uptake (VO2 max) in recreationally active individuals and a triathlon time-to-completion (Hawaiian Ironman Triathlon cohort only). A nominal significance threshold of α < 0.05 was used to identify 17 variants (11 in the Gene SMART population and six in the Hawaiian Ironman Triathlon cohort) which were significantly associated with performance gains in highly trained individuals. The variant rs1474347 located in Interleukin 6 (IL6) was the only variant with a false discovery rate < 0.05 and was found to be associated with gains in VO2 max (additional 4.016 mL/(kg min) for each G allele inherited) after training in the Gene SMART cohort. In summary, this study found further evidence to suggest that genetic variance can influence training response in a moderately trained cohort and provides an example of the potential application of genomic research in the assessment of exercise trait response.

The effect of roasting on boron isotope ratio in coffee beans: Implications for provenance studies of roasted coffee.

We determined δ11B values of green and roasted coffee beans from 20 locations worldwide and conducted laboratory experiments with the aim to investigate boron isotope fractionation during roasting. Authentic single origin roasted coffees were found to be isotopically lighter than their green bean counterparts, with an average difference of 1.5‰. This isotope fractionation can be explained as arising from partial dissociation of boric acid in capillary water of green beans, where 11B isotopes are preferentially partitioned into molecules of undissociated boric acid and are then volatised during roasting. However, boron isotope fractionation induced by roasting was significantly smaller than between-origin variations in δ11B values of green coffee beans that had the range of ∼54‰. This implies that δ11B isotopic composition of roasted coffee retains the geographical origin information within δ11B values of green beans when regional differences in boron isotopic composition of coffee are considered.

Investigation of Mitochondrial Related Variants in a Cerebral Small Vessel Disease Cohort.

Monogenic forms of cerebral small vessel disease (CSVD) can be caused by both variants in nuclear DNA and mitochondrial DNA (mtDNA). Mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is known to have a phenotype similar to Cerebral Autosomal Dominant Arteriopathy with Sub-cortical Infarcts and Leukoencephalopathy (CADASIL), and can be caused by variants in the mitochondrial genome and in several nuclear-encoded mitochondrial protein (NEMP) genes. The aim of this study was to screen for variants in the mitochondrial genome and NEMP genes in a NOTCH3-negative CADASIL cohort, to identify a potential link between mitochondrial dysfunction and CSVD pathology. Whole exome sequencing was performed for 50 patients with CADASIL-like symptomology on the Ion Torrent system. Mitochondrial sequencing was performed using an in-house designed protocol with sequencing run on the Ion GeneStudio S5 Plus (S5 +). NEMP genes and mitochondrial sequencing data were examined for rare (MAF < 0.001), non-synonymous variants that were predicted to have a deleterious effect on the protein. We identified 29 candidate NEMP variants that had links to either MELAS-, encephalopathy-, or Alzheimer's disease-related phenotypes. Based on these changes, variants affecting POLG, MTO1, LONP1, NDUFAF6, NDUFB3, and TCIRG1 were thought to play a potential role in CSVD pathology in this cohort. Overall, the exploration of the mitochondrial genome identified a potential role for mitochondrial related proteins and mtDNA variants contributing to CSVD pathologies.

Polar motion from laser tracking of artificial satellites.

Measurements of the range to the Beacon Explorer C spacecraft from a single laser tracking system at Goddard Space Flight Center have been used to determine the change in latitude of the station arising from polar motion. A precision of 0.03 arc second was obtained for the latitude during a 5-month period in 1970.

A whole-genome assembly of Drosophila.

We report on the quality of a whole-genome assembly of Drosophila melanogaster and the nature of the computer algorithms that accomplished it. Three independent external data sources essentially agree with and support the assembly's sequence and ordering of contigs across the euchromatic portion of the genome. In addition, there are isolated contigs that we believe represent nonrepetitive pockets within the heterochromatin of the centromeres. Comparison with a previously sequenced 2.9- megabase region indicates that sequencing accuracy within nonrepetitive segments is greater than 99. 99% without manual curation. As such, this initial reconstruction of the Drosophila sequence should be of substantial value to the scientific community.

Genomic strategies for defining and dissecting developmental and physiological pathways.

A major challenge in genetics research is defining and dissecting the diversity of developmental and physiological pathways that lie between genes and traits. New functional genomics methods are transforming these studies by providing comprehensive and systematic approaches that complement traditional methods of formal genetics, biochemistry, and cell biology. Together, these complementary approaches will test whether reductionism can account for the complex web of interactions that lead from genetic variation to morphological, physiological, and behavioral traits in health and disease.

The association between diabetic retinopathy and skeletal muscle capillary basal lamina thickening corrected for the influence of age and duration of diabetes.

The association between an objective measure of diabetic retinopathy and skeletal muscle capillary basal lamina thickness was examined in a group of 30 male insulin-treated diabetic subjects, mean age (+/- SD) 44.6 +/- 13.2 yr, duration of diabetes 21.2 +/- 11.2 yr, % ideal body weight (% IBW) 106 +/- 11%. In addition, muscle capillary basal lamina width was measured in a group of 18 nondiabeitc men, mean age 40.7 +/- 16.3 yr and % IBW 118 +/- 23%. The muscle capillary width of the diabetic subjects was significantly greater than that of the nondiabetic group (P less than 0.01), but the values of the two overlapped considerably. In the diabetic group, there was a significant association of basal lamina width with age (P less than 0.01) but not with duration of diabetes. The association between extent of retinopathy and muscle capillary basal lamina width was not strong. The findings of the study do not therefore support the use of an estimate of muscle capillary basal lamina thickness as a single representative measure of diabetic microangiopathy.

Hemoglobin A1c compared with three conventional measures of diabetes control.

Control of diabetes was studied during an 8-wk camp program in 18 insulin-dependent counselors with a mean age of 19.3 yr and a mean duration of diabetes of 11.4 yr. A composite score was obtained for each subject derived from three factors: percent sugar-free urine tests, 24-h glucose excretion as percent of carbohydrate intake, and mean preprandial blood glucose (MPBG). The mean hemoglobin A1c (HbA1c) at the end of the period was 8.3 +/- 1.6% (+/ SD) (normal range, 4-6%). Scores ranging from 24 (fair control) to 45 (excellent control) showed a significant inverse correlation with HbA1c (r = 0.807, P less than 0.001) and MPBG (r = -0.674, P less than 0.01). HbA1c showed a significant correlation with the MPBG (r = 0.693, P less than 0.01). The HbA1c level was predicted better by percent sugar-free urine tests than by the 24-h glucose excretion. Thus it appears that accurate quantification of control may be obtained by using a scoring system. Critical comparison of HbA1c levels to various glycemic indices may provide useful alternatives for estimating diabetes control.

Stability of hemoglobin AIc levels on repetitive determination in diabetic out-patients.

Hemoglobin (Hb) AIc levels were measured biweekly for 14 weeks in 49 diabetic out-patients and 20 nondiabetic subjects. As conventional indices of diabetes control, urine tests were performed four times per day, and plasma glucose concentrations were measured 2 h post breakfast. The diabetic group ranged in age from 15-73 yr and in duration of diabetes from 6 months to 45 yr. Eleven subjects were on diet therapy alone, three were on oral hypoglycemic agents, and 35 were on insulin therapy. The nondiabetic group ranged in age from 18-65 yr. On entry to the study, the mean (+/-SD) Hb AIc and plasma glucose levels of the diabetic group (8.60 +/- 2.11% and 142.5 +/- 99.9 mg/dl) were significantly higher than in the control group (4.68 +/- 0.60% and 102.4 +/- 21.7 mg/dl; P less than 0.001) and remained so throughout the study. The mean coefficient of variation for Hb AIc did not differ significantly between the control group and either the diet therapy or insulin therapy diabetic groups. Urine test values averaged over 2-week periods for each diabetic subject showed a high degree of stability. The mean Hb AIc levels for individual diabetic subjects correlated with the mean plasma glucose levels (r = 0.544; P less than 0.001), proportion of 2% urine tests (r = -0.798; P less than 0.001). These data provide further support for Hb AIc as a measure of diabetes control and, in addition, provide the first direct evidence that a single Hb AIc determination in a 3-month period is adequate for this purpose when the subjects are on a stable therapeutic regimen.

Pancreatic endocrine tissue in benign mediastinal teratoma.

Four cases of benign cystic mediastinal teratoma containing pancreatic tissue have been studied using immunohistochemical and morphometric techniques. The different pancreatic endocrine cell types were stained using antibodies to insulin, glucagon, somatostatin, and pancreatic polypeptide, and the volume density of each cell type was estimated by point counting. Sections from different regions of the normal adult pancreas were also examined and the results compared. There was an increased total volume density of endocrine cells in the teratomatous pancreas, with a pronounced increase in the proportion of somatostatin containing D cells. The results are similar to those described for pancreatic tissue in neonates, and it is suggested that this is further evidence of altered functional differentiation of the pancreatic tissue found in teratomas.

Recurrent insulinoma syndrome with metastatic glucagonoma.

A case is reported of a patient who presented with symptomatic hypoglycaemia and who had three pancreatic tumours resected over the ensuing eight years. Immunocytochemistry demonstrated two of these to be insulinomas and the third to be a glucagonoma. In addition metastatic spread of cells positive for glucagon had occurred to a lymph node and multiple nodules staining positively for glucagon were present in the remainder of the pancreas.

Pituitary adenomas producing growth hormone in acromegalic patients.

Growth hormone was shown in histological sections of 25 pituitary adenomas from acromegalic patients by means of the unlabelled peroxidase-antiperoxidase (PAP) technique. On the basis of the numbers of cytoplasmic granules, the cells of the adenomas were of two types: densely granulated and sparsely granulated. The densely granulated cells had abundant cytoplasm containing numerous granules, whereas the sparsely granulated cells had little cytoplasm with scanty granules. Depending on the predominant cell type the adenomas were also classified as densely granulated or sparsely granulated: 21 of the 25 adenomas (84%) were densely granulated and four (16%) sparsely granulated. There was some variation, however, in the relative numbers of the two types of cell from one part of an adenoma to another, a feature consistent with one type of cell in different phases of activity. There was no significant difference in mean serum growth hormone concentrations between the two groups, and granularity of the adenomas in histological sections did not therefore correlate with secretory activity. Nine adenomas showed extrasellar extension. The mean serum growth hormone concentration in these cases was lower than the mean of the adenomas confined to the sella turcica. Thus the size of the tumour did not correlate with the serum growth hormone concentration. Three of the four adenomas in the sparsely granulated group showed extrasellar extension, compared with 6 of 21 classified as densely granulated. This suggests that sparsely granulated adenomas have a more aggressive pattern of behaviour, but histological evidence for this was lacking.

Further development and automation of a high pressure liquid chromatography method for the determination of glycosylated hemoglobins.

A previously described high pressure liquid chromatography system for the determination of glycosylated hemoglobin concentrations has been automated and simplified. With this methodology it is possible to perform up to 60 analyses per day for hemoglobin A1a+b% and hemoglobin A1c%. Where an estimate of the total fast hemoglobin alone is required, then a considerably greater number of analyses can be performed. The individual values are calculated directly with an electronic integrator. The mean coefficient of variation of the duplicate determinations of 48 samples was 0.63 +/- 0.83% (mean +/- SD). Aliquots of pooled hemolysates have been maintained in liquid nitrogen at -90 degrees C and run at the beginning and end of each daily analytical run over an 18-mo period. Both the inter- and intra-run coefficients of variation of these values have remained consistently less than 3%. Therefore, the methodology offers a reliable and accurate method of containing glycosylated hemoglobin values for clinical use.

A rapid method for the determination of glycosylated hemoglobins using high pressure liquid chromatography.

Hemoglobin (Hb) Alc is a minor component of Hb found in normal individuals but elevated two or threefold in patients with diabetes mellitus. Limited studies have suggested that the level of Hb Alc is proportional to the integrated concentration of glucose over time. Thus it could serve as an index of hyperglycemia. Its measurement may enable a more objective approach to assessing whether or not the control of hyperglycemia can be correlated with the severity of complications of diabetes. Large scale clinicab studies of Hb Alc have not been undertaken for lack of a rapid assay system. This article describes a method of high pressure liquid chromatography (HPLC) which enables the isolation of Hb Alc in 27 min using only 12 microgram of Hb (100 microliter of blood) and a second method for the isolation of total fast Hb components (also elevated in diabetes) in 11 min. Using the first method, a total of 36 assays were performed on the blood of a single normal volunteer over a one month period. the mean level of Hb Alc was 4.95 +/- 0.12% (SD) +/- 0.02% (SEM), while the coefficient of variation (C.V.) was 2.4%. The mean Hb Alc & b level was 1.65 +/- 0.06% +/- 0.01% (C.V. = 3.6%). Values for Hb Alc in 10 normal individuals were 5.06 (mean) +/- 0.32% (SD) +/- 0.01% (SEM). Hb Alc values in 15 patients with diabetes mellitus ranged from 6.8 to 20.0%. The second method was designed to assay Hb Ala, Hb Alb, and Hb Alc as a single peak and yielded results identical to the sum of these components as determined by the first method ( r = 0.98; p less than 0.001).

The determination of glycosylated hemoglobins in rats using high pressure liquid chromatography.

Glycosylated hemoglobins (GHb) or fast hemoglobins (FH) are minor components of hemoglobin that so far have been quantified in men, monkeys, and mice, and they are elevated in diabetic subjects of all these species. Since the rat is a useful model for experimental diabetic studies, hemolysates from streptozotocin-induced diabetic rats were analyzed for FH fractions using a high pressure liquid chromatography method. In a long-term study (3 mo), the maximal increment of the FH fractions was achieved after 5 wk of diabetes (from 5.67% +/- 0.41% SD to 10.80% +/- 0.74%) supporting the notion that the biosynthesis of these compounds occurs continuously during the lifespan of the red cell. In a short-term study, however, an elevation of the FH by 11% after 2 days and by 26% after 6 days of diabetes was noticed suggesting that a rapid increase of the FH may occur in relation to rapid changes of the glucose level.

A novel route to substituted 4-methylene-4,5-dihydroisoxazoles mediated by hafnium(IV) chloride.

Heating alkyl vinyl ketones and N-tert-butylarylmethylideneamine N-oxides in the presence of HfCl4 results in the formation of 4-methylene-4,5-dihydroisoxazoles in good yield.

The prognostic significance of urinary albumin in Polynesians with non-insulin-dependent diabetes.

Polynesian (59 Maori and 30 Pacific Island) patients were identified from two diabetes clinic registers and followed for a mean of 4.8 years, in order to determine the prognostic significance of urinary albumin excretion. Events were defined as death or entry onto a renal replacement programme. Fourteen events occurred during the period of follow-up. Urinary albumin/creatinine ratio was treated as a continuous variable in a proportional hazards analysis. A 10-fold increase in albumin/creatinine ratio was associated with a 5-fold increase in the risk of an event (95% C.I. = 2.05-12.09). In conclusion, elevated urinary albumin/creatinine predicted mortality and renal morbidity in Maori and Pacific Island patients with non-insulin-dependent diabetes.

Assessment of Micral-Test microalbuminuria test strip in the laboratory and in diabetic outpatients.

We have evaluated a semi-quantitative dry immunochemical screening method (Micral-Test) for the detection of low concentrations of albumin in urine. The stability of Micral-Test strips on storage was good, especially with regard to temperature, light and humidity. Changes in urine osmolality (urea and creatinine concentration), pH and sodium and potassium concentration did not have a significant analytical effect on the Micral-Test measurement; extremes of temperature altered the rate of colour development. The depth of dipping the strip into the sample and the timing of reading colour development were critical. We measured the albumin concentration in 184 urine samples from diabetic outpatients by the Micral-Test and by our in-house immunoturbidimetric method; a Micral-Test result of 20 mg/L had a sensitivity of 91% and specificity of 97% to predict a discriminating urine albumin concentration greater than 30 mg/L by the in house method. The Micral-Test is suitable for use by non-laboratory personnel and is capable of producing analytically acceptable results for use in diabetes clinics and by general practitioners.

Current state of metabolic control achieved in a New Zealand diabetes clinic.

AIMS: To report on current degree of metabolic control achieved in diabetic patients in a New Zealand diabetes clinic. METHODS: All metabolic data were reviewed for patients attending the Waikato diabetes clinic from 1 January 1992 to 1 January 1995. Values were obtained from 409 insulin treated patients (IDDM), with a mean age of 39 (SD 15 yr); 200 patients who transferred from diet or pills to insulin (NIDDM-I) with a mean age of 59 (SD 12 yr); and 290 noninsulin dependent patients (NIDDM), mean age 54 (SD 13 yr). Results. Percentage of fructosamine values in the normal range: IDDM 10%, NIDDM-I 12.5% and NIDDM 37.9%. Mean values (SD). IDDM group: fructosamine 376 (78) mmol/L, cholesterol 5.24 (1.1) mmol/L, HDL cholesterol 1.47 (0.45) mmol/L, triglycerides 1.71 (1.35) mmol/L. NIDDM-I group: fructosamine 359 (67) mmol/L, cholesterol 5.82 (1.1) mmol/L, HDL cholesterol 1.21 (0.45) mmol/L, triglycerides 2.92 (3.8) mmol/L. NIDDM group: fructosamine, 312 (73) mmol/L, cholesterol 5.83 (1.24) mmol/L, HDL cholesterol 1.1 (0.33) mmol/L, triglycerides 3.11 (3.59) mmol/L. Percentage of smokers in each group: IDDM 24%, NIDDM-I 8.5%, NIDDM 17.2%. CONCLUSIONS: the majority of patients fail to achieve normal fructosamine values. Lipid control is poor, other than in the IDDM group. A significant number of patients continue to smoke. Considerable scope for improvement is noted.