Identifying the genetic causes for prenatally diagnosed structural congenital anomalies (SCAs) by whole-exome sequencing (WES).

BACKGROUND: Whole-exome sequencing (WES) has become an invaluable tool for genetic diagnosis in paediatrics. However, it has not been widely adopted in the prenatal setting. This study evaluated the use of WES in prenatal genetic diagnosis in fetuses with structural congenital anomalies (SCAs) detected on prenatal ultrasound. METHOD: Thirty-three families with fetal SCAs on prenatal ultrasonography and normal chromosomal microarray results were recruited. Genomic DNA was extracted from various fetal samples including amniotic fluid, chorionic villi, and placental tissue. Parental DNA was extracted from peripheral blood when available. We used WES to sequence the coding regions of parental-fetal trios and to identify the causal variants based on the ultrasonographic features of the fetus. RESULTS: Pathogenic mutations were identified in three families (n = 3/33, 9.1%), including mutations in DNAH11, RAF1 and CHD7, which were associated with primary ciliary dyskinesia, Noonan syndrome, and CHARGE syndrome, respectively. In addition, variants of unknown significance (VUSs) were detected in six families (18.2%), in which genetic changes only partly explained prenatal features. CONCLUSION: WES identified pathogenic mutations in 9.1% of fetuses with SCAs and normal chromosomal microarray results. Databases for fetal genotype-phenotype correlations and standardized guidelines for variant interpretation in prenatal diagnosis need to be established to facilitate the use of WES for routine testing in prenatal diagnosis.

Causal somatic mutations in urine DNA from persons with the CLOVES subgroup of the PIK3CA-related overgrowth spectrum.

Congenital lipomatous overgrowth with vascular, epidermal, and skeletal (CLOVES) anomalies and Klippel-Trenaunay (KTS) syndromes are caused by somatic gain-of-function mutations in PIK3CA, encoding a catalytic subunit of phosphoinositide 3-kinase. Affected tissue is needed to find mutations, as mutant alleles are not detectable in blood. Because some patients with CLOVES develop Wilms tumor, we tested urine as a source of DNA for mutation detection. We extracted DNA from the urine of 17 and 24 individuals with CLOVES and KTS, respectively, and screened 5 common PIK3CA mutation hotspots using droplet digital polymerase chain reaction. Six of 17 CLOVES participants (35%) had mutant PIK3CA alleles in urine. Among 8 individuals in whom a mutation had been previously identified in affected tissue, 4 had the same mutant allele in the urine. One study participant with CLOVES had been treated for Wilms tumor. We detected the same PIK3CA mutation in her affected tissue, urine, and tumor, indicating Wilms tumors probably arise from PIK3CA mutant cells in patients with CLOVES. No urine sample from a participant with KTS had detectable PIK3CA mutations. We suggest that urine, which has the advantage of being collected non-invasively, is useful when searching for mutations in individuals with CLOVES syndrome.

Hospitalization among workers compensated for occupational asthma.

Occupational asthma (OA) can cause persistent symptoms, but populations with OA have not been followed for the development of serious outcomes such as hospitalization. Subjects receiving compensation for OA during 1980-1993, and a comparison sample of workers with musculoskeletal injuries (INJ) were identified from the Ontario Workers' Compensation Board. We also identified for comparison a group of asthmatic patients (AP) seen at a tertiary care hospital clinic during the same period. The file was matched with the Ontario Ministry of Health data base of hospitalizations through 1996. We compared the frequency of hospitalization of the subgroups with that expected in the general population using standardized morbidity ratios (SMRs), and directly by proportional hazards regression. The study group included 844 OA claimants, 1,556 INJ claimants, and 402 AP. Although admissions for all causes combined and respiratory disease among INJ were less than expected in the general population, admissions for all causes combined exceeded that expected among OA and AP. Admissions for respiratory disease were markedly greater than expected among OA (SMR 9.2) and AP (SMR 17) because of even greater excess admissions for asthma (SMRs 45 and 81, respectively). Compared with those with INJ, those with OA were more likely to be hospitalized for all causes combined (adjusted relative risk [RR] 1.4, 95% confidence interval [CI] 1.2 to 16); cardiovascular disease (RR 1.4, 95% CI 0.9 to 2.0); respiratory disease (RR 5.4, 95% CI 3.8 to 7.7); and asthma (RR 28.1, 95% CI 10.2 to 77.2) but not for malignancies (RR 1.0) or injuries (RR 0.9). Those with OA were admitted to hospital about half as frequently as AP for respiratory disease and asthma (although this was modified by smoking status and sex), but were 30% more likely to be admitted for ischemic heart disease (IHD). Among the OA claimants, factors that were significantly associated with hospitalization for asthma included older age and exposure to agents other than isocyanates. Those with OA became less likely to be hospitalized for asthma with increasing time after onset, particularly after 5 or more years. We conclude that subjects with OA suffer higher rates of hospitalizations for all causes combined, respiratory disease, and asthma than other workers, although less than among AP seen at a tertiary care center.

Preliminary report of mortality among workers compensated for work-related asthma.

BACKGROUND: Although fatalities due to asthma have been reported among subjects with occupational asthma (OA) associated with re-exposure, groups of subjects with work-related asthma have not been systematically followed up for mortality. During a review of compensation claims for asthma in Ontario, we identified 3 respiratory deaths among subjects previously compensated for OA for whom their surviving spouses received death benefits. This suspected "cluster" prompted us to undertake an investigation to examine mortality pattern among workers compensated for work-related asthma. METHODS: Subjects receiving compensation for OA or aggravation of asthma (AA) between 1980 and 1993, and a comparison sample of workers with claims for musculoskeletal injuries during the same period were identified from the Ontario Workers' Compensation Board. We also identified another comparison group of non-compensated asthmatic patients seen at a hospital clinic during the same period. The files of those with work-related asthma were reviewed to determine if OA or AA was adequately documented. Mortality was ascertained by linkage with the Mortality Database at the Ontario Cancer Registry through 1996. We compared the mortality of the three groups with that expected in the general population of Ontario using SMRs, and directly by proportional-hazards regression. RESULTS: The study included 3,070 subjects: 1,112 with work-related OA/AA with adequate documentation, 1,556 with work-related injuries, and 402 patients with non-work-related asthma. Of the 66 deaths identified, only 2 deaths were due to asthma, both in the work-related asthma group: one from the index cluster and one not previously identified. A second index death was coded as dying from COPD not elsewhere classified (ICD9 496), while the third index death also died of asthma but there was not sufficient information documenting OA to include the subject in the analyses. As compared with the general population, there were fewer deaths than expected from most causes, except for deaths among the work-related asthma claimants and the nonwork-related asthma patients from respiratory diseases (SMRs 1.3 and 5.9, respectively; 0.5 among injury claimants), all chronic obstructive lung disease (ICD9 490-496; SMRs 2.3 and 7.7, respectively), and asthma (SMRs 18.2 and 0, respectively). In direct comparison of the work-related asthma claimants with the injury claimants, the risk of death appeared elevated from respiratory disease (RR 2.6) and ischemic heart disease (IHD) (RR 2.8) but the confidence intervals included unity. CONCLUSIONS: This preliminary report raises the possibility that serious outcomes, including excess deaths from respiratory disease, in particular asthma, may occur among those with work-related asthma even in the absence of re-exposure. However, the findings are inconclusive given that the number of deaths was small and we identified only one new asthma death in addition to the index cluster. We also observed for the first time that deaths due to circulatory disease, particularly IHD, may also be increased among such workers; this needs to be confirmed elsewhere.

Policy development for compensating workers exposed to crystalline silica in Ontario, Canada.

The Ontario Workers' Compensation Board develops policy for diseases by considering scientific information within legal, political, and social contexts. The purpose of this paper is to describe the process used to develop a policy for lung cancer among gold miners and to examine the extent to which this process assists the development of similar guidelines for workers with silica dust exposure. The scientific and policy questions are similar, both requiring consultation with stakeholders. To improve the development process for the gold miner policy, consultation for silica and lung cancer needs to be more inclusive. The resulting procedures would also need to be precise enough to assist adjudicators to make decisions without limiting their ability to decide each claim on the merits of the case. The major challenge is to ensure that the final policy is scientifically and legally supportable and acceptable to both workers and employers.

The effect of lowering faecal pH on the rate of proliferation of the normal colonic mucosa.

Epidemiological and animal studies suggest that faecal pH may be a risk factor for colorectal cancer with low faecal pH associated with a lower incidence of the disease. The aim of this study was to determine whether faecal pH (or dietary fibre) affects the short-term risk factors for colon cancer. Sixty-nine normal volunteers were randomized into three equal groups (A-C). They provided food records, faecal specimens and submitted to rectal biopsy for thymidine labelling studies before and after a 2-week intervention. Group A received a placebo of fruit juice. Group B, approximately 3.0 g d-1 sodium sulphate in juice. Group C, 30 g d-1 supplementary dietary fibre as wheat bran in bread. Age, sex, weight, height and intake of macronutrients and minerals were similar in the groups prior to intervention. Faecal pH was similar for the three groups before and was reduced in Group B after intervention (P = 0.001) with a relative reduction of 0.5 pH units. The labelling index for the three groups was similar prior to intervention; after, it was lowest in Group B with a relative reduction of 0.5% points, although this difference was not statistically significant. The results thus do not support the hypothesis that an acidification of faecal pH leads to a reduction in risk markers for colon cancer.

Effect of high-fat and low-fiber meals on the cell proliferation activity of colorectal mucosa.

Normal healthy volunteers (n = 43) were divided into four groups that received diets providing low or high levels of dietary fat (33 or 96 g/day) and low or high levels of dietary fiber (6 and 41 g/day) for a period of five days. Proliferation was assessed with tritiated thymidine labeling of three rectal biopsies. After five days on the prescribed diets, the average thymidine labeling index (LI) of the group on the high fat-low fiber diet was only 25% higher than the average LI of the group on the low fat-high fiber diet, a difference that is not statistically significant. We conclude that a short-term increase in dietary fat and decrease in dietary fiber does not result in a large increase in cell proliferation rate.

Comparisons of diet and biochemical characteristics of stool and urine between Chinese populations with low and high colorectal cancer rates.

In an investigation of the roles of diet and stool biochemistry in human colorectal carcinogenesis, 24-hour food, urine, and stool samples were collected from randomly selected participants from two populations with a fourfold difference in colorectal cancer risk: Chinese in Sha Giao, People's Republic of China (low risk), and Chinese-Americans of similar ages in San Francisco County, Calif, in the United States (high risk). The findings supported the hypotheses that colorectal cancer risk is increased by the consumption of high-fat, high-protein, and low-carbohydrate diets and is associated with high levels of cholesterol in stool as well as increased daily outputs of 3-methyl-histidine and malonaldehyde in urine. However, risk does not increase with low stool bulk and low total stool fibers.

Effect of calcium supplementation on mucosal cell proliferation in high risk patients for colon cancer.

Recent findings suggest that supplemental calcium could lower the abnormally high proliferation rate found in the colonic mucosa of subjects at high risk for colon cancer. In this double blind controlled study, this effect in volunteers previously operated upon for a colorectal adenocarcinoma was tested. Thirty subjects were randomised to receive either elemental calcium 1200 mg/day or a placebo. Mucosal proliferation was measured with tritiated thymidine labelling before and after the 30 day intervention period. Diets, faecal pH and the concentration of calcium and bile acids in the aqueous phase of feaces were also measured. Labelling index did not differ significantly in the two groups before intervention (placebo 4.0(2.4) v calcium 4.9(2.9), but the difference approached significance afterwards (4.4(2.4) v 6.5(3.4), p = 0.06). Individual changes occurring with intervention were tabulated and comparison of the means for the groups was not significant (delta = 0.3 vs delta = 1.8, p = 0.11). Calcium concentration, faecal pH and deoxycholic acid concentration increased in the calcium group (p = 0.02, 0.005 and 0.004 respectively). Calcium does not show any effect in decreasing colonic mucosal proliferation in this high risk group for colon cancer; it may increase faecal pH and the production of deoxycholic acid in the colon.

Proliferative activity of rectal mucosa and soluble fecal bile acids in patients with normal colons and in patients with colonic polyps or cancer.

Rectal biopsies and fecal collections were obtained from a consecutive series of 34 outpatients prior to colonoscopy at a gastroenterology clinic. Subsequently, 14 were found to have no colonic pathology, 13 had adenomatous polyps, (3 of those had a previous history of colon cancer) and 7 were diagnosed with colon cancer. In confirmation of earlier studies the tritiated thymidine labelling index was higher in patients with tumors than in those without pathology (7.9% vs. 5.8% with P = 0.06). The patients with colonic tumors also had significantly higher levels of deoxycholic acid (P = 0.01) and lithocholic acid (P = 0.005) in the aqueous extract of their feces. This study shows that these biochemical measures may indicate colon cancer risk.

Assessment of past diet in epidemiologic studies.

The reproducibility of recall of diet was examined for 44 men in Toronto, Ontario, Canada, by comparing estimates of consumption obtained from a dietary questionnaire in 1982 with estimates of consumption made by recalling the original diet at an interview conducted one year later in 1983. Estimates of average consumption obtained by recall were significantly lower than those originally reported for most foods and nutrients, but the magnitude of the differences was never greater than 20% of the original estimate. Correlations between individuals' levels of consumption were greater than 0.7 for nine of the 13 foods and nutrients studied. Current diet, assessed from two-day food records, was also associated with consumption originally reported for some nutrients. Fecal levels of hemicellulose were associated with fiber consumption originally reported and with current fiber consumption, and urine levels of 3-methylhistidine were associated with past meat consumption. The best prediction of past consumption of fiber and fat, however, was obtained from the recalled diet. No significant additional contribution to the prediction was made from estimates of current consumption or from biochemical measures.