Clinical implications of mosaicism: a 10-year retrospective review of 83 families in a university-affiliated genetics clinic.

Mosaicism refers to the coexistence of two or more genetically distinct cell populations in an individual from a single fertilized egg. We performed a retrospective analysis of all patients diagnosed with mosaic disorders between 2010 and 2021 in a university-affiliated genetics clinic, which attends to territory-wide genetic consultations. All patients with confirmed mosaic diagnoses through reproductive (n = 6), prenatal (n = 24), and postnatal (n = 53) testing were examined. We observed that mosaic 45, X (n = 31) and PIK3CA-related overgrowth spectrum (n = 16) disorders were among the most prevalent diagnoses in the clinic, and the total percentage of patients with mosaicism in our cohort was 2.0% (83/4157). A review of the diagnostic journey highlights the challenge in diagnosing mosaic disorders, whereby 38% of the subjects required more than one test sample, and 52% of the cases required more than one orthogonal method of detection to reach the correct diagnosis. While detection of mosaicism is passive through routine clinical testing, for example karyotyping in reproductive and prenatal care, in postnatal care, clinicians can more actively drive the detection of mosaicism. Therefore, we recommend a low threshold for additional genetic testing in suspected mosaicism for more accurate diagnosis and counselling.

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.

Silver-Russell syndrome in Hong Kong.

OBJECTIVES: To examine the molecular pathogenetic mechanisms, (epi)genotype-phenotype correlation, and the performance of the three clinical scoring systems-namely Netchine et al, Bartholdi et al, and Birmingham scores-for patients with Silver-Russell syndrome in Hong Kong. METHODS: This retrospective case series was conducted at two tertiary genetic clinics, the Clinical Genetic Service, Department of Health, and clinical genetic clinic in Queen Mary Hospital in Hong Kong. All records of patients with suspected Silver-Russell syndrome under the care of the two genetic clinics between January 2010 and September 2015 were retrieved from the computer database. RESULTS: Of the 28 live-birth patients with Silver-Russell syndrome, 35.7% had H19 loss of DNA methylation, 21.4% had maternal uniparental disomy of chromosome 7, 3.6% had mosaic maternal uniparental disomy of chromosome 11, and the remaining 39.3% were Silver-Russell syndrome of unexplained molecular origin. No significant correlation between (epi)genotype and phenotype could be identified between H19 loss of DNA methylation and maternal uniparental disomy of chromosome 7. Comparison of molecularly confirmed patients and patients with Silver-Russell syndrome of unexplained origin revealed that postnatal microcephaly and café-au-lait spots were more common in the latter group, and body and limb asymmetry was more common in the former group. Performance analysis showed the Netchine et al and Birmingham scoring systems had similar sensitivity in identifying Hong Kong Chinese subjects with Silver-Russell syndrome. CONCLUSION: This is the first territory-wide study of Silver-Russell syndrome in Hong Kong. The clinical features and the spectrum of underlying epigenetic defects were comparable to those reported in western populations.

Spread of X inactivation on chromosome 15 is associated with a more severe phenotype in a girl with an unbalanced t(X; 15) translocation.

We report on a baby girl with multiple congenital abnormalities, including cleft palate, intrauterine growth restriction, and double outlet right ventricle (DORV) with ventricular septal defect. She had an unbalanced chromosome translocation t (X;15) resulting in monosomy 15pter → p10 and trisomy Xq13.1 → q28. All three copies of Xq encompass the XIST gene. It is known that X chromosome inactivation could spread to the autosome part of an unbalanced translocation involving chromosome X and an autosome. To confirm the spread of X chromosome inactivation on chromosome 15, we evaluate the methylation change by the HumanMethylation450 BeadChip, a whole genome DNA methylation micorarray that includes 15,259 probes spanning 717 genes on chromosome 15. Results showed there was gain in DNA methylation of more than 20% in 586 CpG sites spanning the long arm of chromosome 15. We further examined the hypermethylated CpG sites located in CpG-island promoter, because genes subjected to X chromosome inactivation will have an increase in DNA methylation level in this region. A total of 75 sites representing 24 genes were hypermethylated. Nearly all of these probes are located in region proximal to the breakpoint, from 15q11.2 to 15q21.3 (35Mb) suggesting that X inactivation was spread to the proximal region of 15q. Gain of DNA methylation, especially in the CpG-island promoter, can result in functional inactivation of genes, and therefore could potentially worsen the phenotype of our patient.

Changes in rates and severity of compensation claims for asthma due to diisocyanates: a possible effect of medical surveillance measures.

OBJECTIVES: A medical surveillance programme was introduced into Ontario for workers exposed to diisocyanates in 1983, but no mandated surveillance programme is in effect in this province for other occupational respiratory sensitisers. This study assesses changes in incidence and severity of compensated claims for occupational asthma (OA) due to diisocyanates compared with other causes, which have occurred since the introduction of this surveillance programme. METHODS: New claims for OA compensated by the Ontario Workers' Compensation Board (WCB) between 1980 and 1993 were retrospectively reviewed. Linkage was made between these data and an Ontario Ministry of Health database to assess hospital admissions for asthma from the date of onset of OA until the end of 1996. RESULTS: Numbers of claims for OA induced by diisocyanates ranged from 9-15/year in 1980-83, increased up to 55-58 claims/year in 1988-90, then fell to 19-20 claims/year by 1992-93. By contrast yearly numbers of claims for OA due to other causes increased up to 1985-87 then remained relatively stable. Duration of symptoms for OA induced by diisocyanates was shorter than for other claims and there were fewer hospital admissions among those with OA induced by diisocyanates than among those with OA induced by other causes. Occupational asthma from all causes was diagnosed earlier in claims for 1987-93 compared with 1980-86, and indicators of severity of asthma were also milder in accepted claims during 1987-93 than in earlier claims. CONCLUSIONS: Although engineering and industrial hygiene measures may have contributed to these changes, our findings are also consistent with a beneficial contribution from the medical surveillance programme for workers exposed to diisocyanates.

Structure-activity relationship studies of a bisbenzimidazole-based, Zn(2+)-dependent inhibitor of HCV NS3 serine protease.

A survey of isosteric replacements of the phosphonoalanine side chain coupled with a process of conformational constraint of a bisbenzimidazole-based, Zn(2+)-dependent inhibitor of hepatitis C virus (HCV) NS3 serine protease resulted in the identification of novel series of active compounds with extended side chains. However, Zn(2+)-dependent HCV NS3 inhibition was relatively insensitive to the structural variations examined but dependent on the presence of negatively charged functionality. This result was interpreted in the context of an initial electrostatic interaction between protease and inhibitor that is subsequently consolidated by Zn(2+), with binding facilitated by the featureless active site and proximal regions of the HCV NS3 protein.

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.

Near-infrared spectroscopy for bioprocess monitoring and control.

This article describes the calibration of a spectroscopic scanning instrument for the measurement of selected contaminants in a complex biological process stream. Its use is for the monitoring of a process in which contaminants are to be removed selectively by flocculation from yeast cell homogenate. The main contaminants are cell debris, protein, and RNA. A low-cost instrument has been developed for sensitivity in the region of the NIR spectrum (from 1900 to 2500 nm) where preliminary work found NIR signatures from cell debris, protein, and RNA. Calibration models have been derived using a multivariate method for concentrations of these contaminants, such as would be found after the flocculation process. Two strategies were compared for calibrating the NIR instrument. In one case, samples were prepared by adding materials representative of the contaminants to clarified yeast homogenate so the contaminant levels were well known but outside the range of interest. In the other case, where samples were like those from the process stream after flocculation and floc removal, there was uncertainty of analysis of contaminant level, but the calibration was in the range of interest. Calibration using process stream samples gave results close to those derived from traditional assays. When the calibration models were used to predict the contaminant concentrations in previously unseen samples, the correlation coefficients between measurements and predictions were above 90% in all cases but one. The prediction errors were similar to the errors in the traditional assays.

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.

Effect of high fat consumption on cell proliferation activity of colorectal mucosa and on soluble faecal bile acids.

To assess the effect of fat consumption on the proliferation of the rectal mucosa, 30 normal volunteers (22 to 71 years) were randomly allocated to three groups: (a) basal low fat diet containing 30 g of fat per day; (b) the basal diet with doses of 30 g corn oil taken with each of the three meals: 120 g fat/day; (c) the basal diet with one dose of 90 g corn oil after the last meal: 120 g fat/day. Rectal biopsies were taken 15 cm from the anal verge after five days on the diets and mucosal cell proliferation was measured by labelling index (LI). The LI was significantly (p less than 0.01) higher in group (c) (9.2) than in group (a) (5.9), with group (b) intermediate (6.8). In multiple stepwise regression analysis, the data were best fitted with age and the variable indicating fat consumed as a bolus as predictors of LI (r2 = 0.39, p less than 0.001). In separate analyses the regression coefficient with age in the fat bolus group was 0.23, p less than 0.001. There was some tendency towards lower bile acids in the faecal water in group (a) than in groups (b) and (c) following the diets and between the bile acids and LI (for lithocholic acid r = 0.48, p = 0.01). These data show that dietary fat given as a bolus can lead to an increase in the proliferation of human colonic cells, possibly as a consequence of raised levels of cytotoxic acidic lipids in the faecal stream.

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.