The Population-Based Hungarian Twin Registry: An Update.

Between 2006 and 2021, the Hungarian Twin Registry (HTR) operated a volunteer twin registry of all age groups (50% monozygotic [MZ], 50% dizygotic [DZ], 70% female, average age 34 ± 22 years), including 1044 twin pairs, 24 triplets and one quadruplet set. In 2021, the HTR transformed from a volunteer registry into a population-based one, and it was established in the Medical Imaging Centre of Semmelweis University in Budapest. Semmelweis University's innovation fund supported the development of information technology, a phone bank and voicemail infrastructure, administrative materials, and a new website was established where twins and their relatives (parent, foster parent or caregiver) can register. The HTR's biobank was also established: 157,751 individuals with a likely twin-sibling living in Hungary (77,042 twins, 1194 triplets, 20 quadruplets, and one quintuplet) were contacted between February and March of 2021 via sealed letters. Until November 20, 2022, 12,001 twin individuals and their parents or guardians (6724 adult twins, 3009 parents/guardians and 5277 minor twins) registered, mostly online. Based on simple self-reports, 37.6% of the registered adults were MZ twins and 56.8% were DZ; 1.12% were triplets and 4.5% were unidentified. Of the registered children, 22.3% were MZ, 72.7% were DZ, 1.93% were triplets, and 3.05% were unidentified. Of the registered twins, 59.9% were female (including both the adult and minor twins). The registration questionnaire consists of eight parts, including socio-demographic and anthropometric data, smoking habits and medical questions (diseases, operations, therapies). Hungary's twin registry has become the sole and largest population-based twin registry in Central Eastern Europe. This new resource will facilitate performing world-class modern genetic research.

The Hungarian Twin Registry Update: Turning From a Voluntary to a Population-Based Registry.

Since our last report on the voluntary Hungarian Twin Registry (HTR) in 2012, the number of pairs or multiplets included increased from 310 to 1044. Efforts to turn the registry into a population-based one are on the way. Nearly 128,000 twins living in Hungary (98,500 adults) will be mailed information on how to register on the new HTR website. Twins will be asked to invite their spouses and immediate family members. Meanwhile, strong cooperation through exchange programs has been developed with other foreign twin registries. Current research focuses on radiogenomics, musculoskeletal, cardiovascular and respiratory diseases, gut microbiome as well as basic molecular research and yielded new awards and further publications.

Arsenic in drinking water and congenital heart anomalies in Hungary.

Inorganic arsenic can get easily through the placenta however there are very few human data on congenital anomalies related to arsenic exposure. Objective of our study was to explore the associations between arsenic content of drinking water and prevalence of some congenital anomalies. Four anomalies reported to the Hungarian Congenital Anomalies Registry between 1987 and 2003 were chosen to be analysed in relation to arsenic exposure: congenital anomalies of the circulatory system (n=9734) were considered as cases, while Down syndrome, club foot and multiple congenital malformations were used as controls (n=5880). Arsenic exposure of the mothers during pregnancy was estimated by using archive measurement data for each year and for each settlement where the mothers lived. Analysis of the associations between the prevalence of congenital heart anomalies and arsenic exposure during pregnancy was performed by logistic regression. The child's gender and age of the mother were adjusted for. The associations were evaluated by using the present EU health limit value of 10.0 µg/L arsenic concentration as a cut-off point. Regular consumption of drinking water with arsenic concentration above 10 µg/L during pregnancy was associated with an increased risk of congenital heart anomalies in general (adjusted OR=1.41; 95% C.I.: 1.28-1.56), and especially that of ductus Botalli persistens (adjusted OR=1.81, 95%C.I.: 1.54-2.11) and atrial septal defect (adjusted OR=1.79; 95%C.I.: 1.59-2.01). The presented results showed an increased risk of congenital heart anomalies among infants whose mothers were exposed to drinking water with arsenic content above 10 µg/L during pregnancy. Further studies of possible similar effects of concentrations below 10 µg/L are warranted.

Bioimpedance analysis of body composition in an international twin cohort.

OBJECTIVE: Multiple twin studies have demonstrated the heritability of anthropometric and metabolic traits. However, assessment of body composition parameters by bioimpedance analysis (BIA) has not been routinely performed in this setting. DESIGN: A cross-sectional study. SETTING: Study subjects were recruited and assessed at twin festivals or at major university hospitals in Italy, Hungary, and the United States to estimate the influence of genetic and environmental components on body composition parameters in a large, wide age range, international twin cohort by using bioelectrical impedance analysis. SUBJECTS: 380 adult twin pairs (230 monozygotic and 150 dizygotic pairs; male:female ratio, 68:32; age years 49.1 ± 15.4; mean ± standard deviation; age range 18-82) were included in the analysis. RESULTS: Heritability was calculated for weight (82%; 95% confidence interval [CI]: 78-85), waist and hip circumferences (74%; 95%CI: 68-79), body fat percentage (74%; 95%CI: 69-79), fat-free mass (74%; 95%CI: 69-79) and body mass index (79%; 95%CI: 74-83). The completely environmental model showed no impact of shared environmental effects on the variance, while unshared environmental effects were estimated as between 18% and 26%. CONCLUSIONS: BIA findings provide additional evidence to the heritability of anthropometric attributes related to obesity and indicate the practical value of this simple method in supporting efforts to prevent obesity-related adverse health events.

50 years of the Hungarian Congenital Abnormality Registry.

The mandatory notification of patients ("cases") with different congenital abnormalities (CAs) diagnosed from birth until the end of the first postnatal year by medical doctors was ordered by the Ministry of Health in Hungary in 1962 and this CA-registry was continued as the Hungarian Congenital Abnormality Registry (HCAR) based on the international recommendation from 1970. The primary objective of the HCAR has been to determine the baseline birth prevalence rate of different CAs as reliably as possible, with three secondary objectives: (i) to detect temporal and/or spatial clusters of CAs; (ii) to evaluate increasing or decreasing time trends of CAs; and (iii) to assist in the planning of medical and social services for children and families affected by CA so that appropriate resources are allocated efficiently and effectively. This paper summarizes the activities and the evolution of the HCAR over the past 50 years (1962-2011) including the Hungarian Case-Control Surveillance of Congenital Abnormalities for postmarketing surveillance of drug teratogenicity and prevention of CAs; the special evaluation of unidentified multiple CAs; the Hungarian Surveillance of Germinal Mutations and several international collaborations. In conclusion, Hungary enjoyed optimal conditions for the HCAR due to a centralized state health system; all deliveries took place in inpatient clinics; the quality of pediatric care was high and pediatricians notified most CAs. Autopsy was mandatory in infant death, the staff of the HCAR did not consider this CA-registry only as a statistical system but the Hungarian Center for Congenital Anomaly Control and the Hungarian Case-Control Surveillance of Congenital Abnormalities based on the HCAR worked with close collaboration with the parents in order to promote the possible good quality of life of their affected children and to prevent their risk of recurrence.

Genetic impact dominates over environmental effects in development of carotid artery stiffness: a twin study.

Arterial stiffness is an independent predictor of cardiovascular, cerebrovascular and all-cause mortality. Quantifying the genetic influence on the stiff arterial phenotype allows us to better predict the development of arterial stiffness. In this study, we aimed to determine the heritability of carotid artery stiffness in healthy twins. We studied 98 twin pairs of both sexes. We determined carotid artery stiffness locally using echo tracking and applanation tonometry. We estimated the heritability of stiffness parameters using structural equation modeling. The carotid distensibility coefficient showed the highest heritability (64%, 95% confidence interval 45-77%). The incremental elastic modulus, compliance and stiffness index ß also showed substantial heritability (62%, 61% and 58%, respectively). The remaining 36-42% phenotypic variance was attributed to unshared environmental effects. Genetic influence appears to dominate over environmental factors in the development of carotid artery stiffness. Environmental factors may have an important role in favorably influencing the genetic predisposition for accelerated arterial stiffening.

[Hungarian twin studies: results of four decades]. / Magyarországi ikerkutatások: négy évtized eredményei.

Twin studies play a role in examining the contribution of genetic variations and environmental factors responsible for the determination of phenotypic variables and of genetic linkage between genotypes. Hungarian twin studies, supported by three twin registries (among them two twin-database), date back to 1970s. Studies mainly focused on various congenital abnormalities, the effect of contraceptive pills and folic acid on the frequency of twin pregnancies, as well as psychosexual and alcohol consumptional behaviors. Monogenic Mendelian inheritance of lactose (mal)absorption was demonstrated for the first time. Hungarian Twin Registry was founded in 2007, which contributed to the current understanding on the background of several disorders, e.g. metabolic syndrome and atherosclerosis. As part of an international twin study, among others, arterial stiffness, central blood pressure, carotid intima/media thickness, venous biomechanics, body composition, lung function and smoking characteristics were also assessed. Absence of genetic background in non-alcoholic fatty liver disease and high inheritance of carotid plaque characteristics were demonstrated for the first time. The review also aims to summarize future plans of the Hungarian Twin Registry.

The Hungarian Twin Registry.

The first Hungarian Twin Registry was established in Budapest in 1970 through the mandatory reporting of multiple-births. In the 1980s a second, volunteer adult registry was also founded. Unfortunately, both registries ceased to exist in the 1990s. Efforts started in 2006 to revive a Hungarian twin registry. The team spearheading this effort reports here on this progress. Currently, the voluntary Hungarian Twin Registry consists of 310 adult twin pairs and multiplets. Current research focuses on cardiovascular and respiratory health and yielded multiple awards and publications. Efforts are on the way to expand into social, psychological, and obesity studies.

Evidence for a strong genetic influence on carotid plaque characteristics: an international twin study.

BACKGROUND AND PURPOSE: Few family studies reported moderate genetic impact on the presence and scores of carotid plaques. However, the heritability of carotid plaque characteristics remains still unclear. Twin studies more reliably estimate the relative contribution of genes to these traits in contrast to family study design. METHODS: One hundred ninety-two monozygotic and 83 dizygotic adult twin pairs (age 49±15 years) from Italy, Hungary, and the United States underwent B-mode and color Doppler ultrasound of bilateral common, internal, and external carotid arteries. RESULTS: Age-, sex-, and country-adjusted heritability was 78% for the presence of carotid plaque (95% CI, 55%-90%), 74% for plaque echogenicity (hypoechoic, hyperechoic, or mixed; 95% CI, 38%-87%), 69% for plaque size (area in mm2 in longitudinal plane; 50 percentile; 95% CI, 16%-86%), 74% for plaque sidedness (unilateral or bilateral; 95% CI, 25%-90%), 74% for plaque numerosity (95% CI, 26%-86%), 68% (95% CI, 40%-84%), and 66% (95% CI, 32%-90%) for the presence of plaque in carotid bulbs and proximal internal carotid arteries. No role of shared environmental factors was found. Unique environmental factors were responsible for the remaining variance (22%-34%). Controlling for relevant covariates did not change the results significantly. CONCLUSIONS: The heritability of ultrasound characteristics of carotid plaque is high. Unshared environmental effects account for a modest portion of the variance. Our findings should stimulate the search for genes responsible for these traits.

Prevalence of esophageal atresia among 18 international birth defects surveillance programs.

BACKGROUND: The prevalence of esophageal atresia (EA) has been shown to vary across different geographical settings. Investigation of geographical differences may provide an insight into the underlying etiology of EA. METHODS: The study population comprised infants diagnosed with EA during 1998 to 2007 from 18 of the 46 birth defects surveillance programs, members of the International Clearinghouse for Birth Defects Surveillance and Research. Total prevalence per 10,000 births for EA was defined as the total number of cases in live births, stillbirths, and elective termination of pregnancy for fetal anomaly (ETOPFA) divided by the total number of all births in the population. RESULTS: Among the participating programs, a total of 2943 cases of EA were diagnosed with an average prevalence of 2.44 (95% confidence interval [CI], 2.35-2.53) per 10,000 births, ranging between 1.77 and 3.68 per 10,000 births. Of all infants diagnosed with EA, 2761 (93.8%) were live births, 82 (2.8%) stillbirths, 89 (3.0%) ETOPFA, and 11 (0.4%) had unknown outcomes. The majority of cases (2020, 68.6%), had a reported EA with fistula, 749 (25.5%) were without fistula, and 174 (5.9%) were registered with an unspecified code. CONCLUSIONS: On average, EA affected 1 in 4099 births (95% CI, 1 in 3954-4251 births) with prevalence varying across different geographical settings, but relatively consistent over time and comparable between surveillance programs. Findings suggest that differences in the prevalence observed among programs are likely to be attributable to variability in population ethnic compositions or issues in reporting or registration procedures of EA, rather than a real risk occurrence difference. Birth Defects Research (Part A), 2012.

Heritability of central blood pressure and arterial stiffness: a twin study.

OBJECTIVE: Central blood pressure and aortic stiffness have been consistently reported as strong cardiovascular risk factors. Twin studies by comparing identical with nonidentical twins produce information on the relative contribution of genes and environment. METHODS: One hundred and fifty-four monozygotic (MZ) and 42 dizygotic (DZ) twin pairs (age 43 ± 17 years) from Hungary and the United States underwent brachial and central augmentation index (AIx), brachial and central pressure, and aortic pulse wave velocity (PWV) measurements with the invasively validated Arteriograph device. Bivariate Cholesky decomposition models were applied. RESULTS: Age-adjusted, sex-adjusted and country-adjusted heritability was 60.0% for central SBP [95% confidence interval (CI), 44.8-69.6%], 50.1% for aortic PWV (95%CI, 26.0-66.8%), 48.7% for aortic AIx (95%CI, 1.7-74.0%), 46.8% for brachial AIx (95%CI, 1.1-73.8%), 46.7% for central pulse pressure (PP) (95%CI, 12.4-61.4%), and 30.0% for brachial PP (95%CI, 0.0-53.4%). Central SBP and PP had strong bivariate correlations with brachial (r = 0.461 and 0.425) and central AIx (r = 0.457 and 0.419), as well as with aortic PWV (r = 0.341 and 0.292, all P < 0.001). Brachial PP had a weak correlation with brachial AIx (r = -0.118, P < 0.05), central AIx (r = -0.122, P < 0.05), and none with aortic PWV (r = 0.08, P = n.s.). Genetic factors explained a moderate phenotypic correlation between central PP, SBP, brachial SBP and aortic PWV. CONCLUSIONS: Central systolic and PPs, brachial PP, AIx, aortic PWV are moderately heritable. A moderate genetic covariance among aortic PWV and central PP, central SBP and brachial SBP was found.

Effect of genetic and environmental influences on cardiometabolic risk factors: a twin study.

BACKGROUND: Both genetic and environmental factors play a role in the pathogenesis of type 2 diabetes and cardiovascular diseases. The magnitude of genetic and environmental influences may vary in different populations and can be investigated by twin studies. METHODS: In this cross-sectional study, 101 (63 monozygotic and 38 dizygotic) adult twin pairs (n = 202; mean age: 44.3 ± 15.8 years) were investigated. Past medical history was recorded and physical examination was performed. Fasting venous blood samples were taken for measuring laboratory parameters. For assessing heritability of 14 cardiovascular risk factors, the structural equation (A-C-E) model was used. RESULTS: The following risk factors were highly (> 70.0%) or moderately (50.0 - 69.0%) heritable: weight (88.1%), waist circumference (71.0%), systolic blood pressure (57.1%), diastolic blood pressure (57.7%), serum creatinine (64.1%), fibrinogen (59.9%), and serum C-reactive protein (51.9%). On the other hand, shared and unique environmental influences had the highest proportion of total phenotypic variance in serum total cholesterol (46.8% and 53.2%), serum HDL-cholesterol (58.1% and 14.9%), triglycerides (0.0% and 55.9%), fasting blood glucose (57.1% and 42.9%), fasting insulin (45.4% and 54.5%), serum uric acid (46.0% and 31.3%), and serum homocysteine (71.8% and 28.2%, respectively). CONCLUSION: Some cardiometabolic risk factors have strong heritability while others are substantially influenced by environmental factors. Understanding the special heritability characteristics of a particular risk factor can substantiate further investigations, especially in molecular genetics. Moreover, identifying genetic and environmental contribution to certain cardiometabolic risk factors can help in designing prevention and treatment strategies in the population investigated.

Conjoined twins: a worldwide collaborative epidemiological study of the International Clearinghouse for Birth Defects Surveillance and Research.

Conjoined twins (CT) are a very rare developmental accident of uncertain etiology. Prevalence has been previously estimated to be 1 in 50,000 to 1 in 100,000 births. The process by which monozygotic twins do not fully separate but form CT is not well understood. The purpose of the present study was to analyze diverse epidemiological aspects of CT, including the different variables listed in the Introduction Section of this issue of the Journal. The study was made possible using the International Clearinghouse for Birth Defects Surveillance and Research (ICBDSR) structure. This multicenter worldwide research includes the largest sample of CT ever studied. A total of 383 carefully reviewed sets of CT obtained from 26,138,837 births reported by 21 Clearinghouse Surveillance Programs (SP) were included in the analysis. Total prevalence was 1.47 per 100,000 births (95% CI: 1.32-1.62). Salient findings including an evident variation in prevalence among SPs: a marked variation in the type of pregnancy outcome, a similarity in the proportion of CT types among programs: a significant female predominance in CT: particularly of the thoracopagus type and a significant male predominance in parapagus and parasitic types: significant differences in prevalence by ethnicity and an apparent increasing prevalence trend in South American countries. No genetic, environmental or demographic significant associated factors were identified. Further work in epidemiology and molecular research is necessary to understand the etiology and pathogenesis involved in the development of this fascinating phenomenon of nature.

[Heritability of the risk factors characteristic for the metabolic syndrome: a twin study]. / A metabolikus szindróma összetevoinek genetikai meghatározottsága: ikervizsgálatok eredményei.

UNLABELLED: Both genetic and environmental factors play role in the pathogenesis of the metabolic syndrome. The magnitude of genetic and environmental influences on the components of metabolic syndrome may vary in different populations. AIMS: The present study was aimed to determine the effects of genetic and environmental factors on risk factors characteristic for the metabolic syndrome. METHODS: A total of 101 (63 monozygotic and 38 dizygotic) adult twin pairs (n=202; mean age: 43.3±15.8 years) were investigated. Medical history was recorded and physical examination was carried out for each subject. Fasting venous blood samples were used for measuring laboratory parameters. The presented estimates include the heritability structural equation (A-C-E) model results. In Model-1, all presented parameters are age- and gender- corrected. In Model-2, parameters were corrected for age, gender, body mass index and waist circumference. RESULTS: Heritability in waist circumference (as well as in other anthropometric parameters such as weight and height) was high (Model-1: 71.0-88.1%). Similarly, genetic factors had the highest proportion of total phenotypic variance in systolic and diastolic blood pressure (Model-2: 57.1% and 57.7%, respectively). Based on the results of Model-2, unique environmental factors dominate alterations in serum triglycerides values (55.9%) while shared environmental factors proved to be substantial in alterations of HDL-cholesterol and fasting blood glucose values (58.1% and 57.1%, respectively). Comparing the results of Model-1 and Model-2, the difference in A-C-E model varied from 0.0% to 17.1%, indicating that only a minor proportion of genetic and environmental influences can be explained by the effects of anthropometric parameters. CONCLUSIONS: Among adult Hungarian healthy people, genetic factors have substantial influence on waist circumference and blood pressure values while environmental factors dominate alterations in serum triglycerides, HDL-cholesterol and fasting blood glucose values. The different heritability of individual risk factors challenges the original unifying concept of the metabolic syndrome. The results may be useful for establishing and implementing primary cardiovascular prevention both at individual and population levels.

Psychosexual study of communist era Hungarian twins.

Our aim in this study is to describe the characteristics of sexual development in twins and estimate the role of heritability and environmental factors as causes of certain sexual disorders. Two hundred and ten adult same-sex twin pairs (92 monozygotic [MZ] female, 41 MZ male, 55 dizygotic [DZ] female and 22 DZ male pairs) were involved in the study. Data were collected in 1982 by self-administered questionnaires that included items on sexual maturation, sexual life, contraception, mutual sexual activity within twin pairs and alcohol use. The ratio of married to unmarried twins was nearly the same in MZs and DZs, with the exception that the divorce rate was higher in MZ female twins (14%), and DZ and male twins were slightly more likely to be single. Menarche was later in twins compared to non-twin Hungarian women. 57% of MZs experienced menarche within 3 months of each other, 77% within 6 months while it occurred for 30% and 43% respectively in DZs. The first seminal emission indicated some delay in male twins compared with the Hungarian general population sample. MZ first kisses occurred later than DZ's first kisses. The same was true for the first petting, masturbation and first sexual intercourse. Anorgasmy is 27% heritable but the estimate is not statistically significant. Concordance rate for premature ejaculation in MZs was greater than in DZs but the structural equation model showed significant misfit. Age at menarche appeared to be strongly heritable.

Maternal demographic and socioeconomic characteristics of live-born infants with isolated ocular congenital abnormalities.

PURPOSE: To evaluate maternal age and birth order, in addition socioeconomic status and finally occupational background of mothers who delivered babies with different isolated ocular congenital abnormalities. METHODS: The data set of the Hungarian Case-Control Surveillance of Congenital Abnormalities, 1980-2002 was used and the evaluation of maternal variables was based on both medical records and maternal information. RESULTS: Mean maternal age and birth order was lower in the mothers of cases with isolated an/microphthalmia. The mean birth order was also lower in the mothers with isolated congenital cataract compared with the control groups. The mothers of cases with isolated coloboma had the usual mean maternal age with a very high proportion of second birth order. The proportion of unmarried women, low maternal socio-economic status and unemployment was larger in the groups of isolated an/microphthalmia and isolated primary congenital glaucoma and these mothers frequently worked in the agriculture. CONCLUSIONS: Cases with different isolated ocular congenital abnormalities showed different maternal characteristics as the reference controls, therefore it is necessary to evaluate each isolated ocular congenital abnormality separately and maternal characteristics can be considered as potential confounders in the analyses of ocular congenital abnormalities.

Drug treatment during pregnancy and isolated orofacial clefts in hungary.

OBJECTIVE: To evaluate the possible association between all kinds of drug treatments during pregnancy and isolated cleft lip with or without cleft palate (CL/P) and posterior cleft palate (PCP) in the offspring. SETTING: The dataset of the large population-based Hungarian Case-Control Surveillance of Congenital Abnormalities, 1980-1996, was evaluated. PARTICIPANTS: One thousand three hundred seventy-four cases with isolated CL/P and 601 with PCP, plus 38,151 population controls (without birth defects) and 20,868 malformed controls with other defects. INTERVENTION: In this observation case-control study the data collection was based on prospective medical records particularly prenatal logbook, retrospective maternal data via a self-reported questionnaire, and home visits of nonresponding mothers. MAIN OUTCOME MEASURES: Isolated CL/P and PCP associated with drug treatments during pregnancy. RESULTS: An increased risk for isolated CL/P was found in cases born to mothers treated with amoxicillin, phenytoin, oxprenolol, and thiethylperazine during the second and third month of pregnancy, i.e., the critical period of isolated CL/P. Risk of isolated PCP was increased in mothers with oxytetracycline and carbamazepine treatment during the third and fourth month of pregnancy, i.e., the critical period of PCP. CONCLUSIONS: This study confirmed the orofacial cleft (OFC) inducing effect of phenytoin, carbamazepine, oxytetracycline, and thiethylperazine and suggested a possible association between OFCs and oxprenolol and amoxicillin. However, drugs may have only a limited role in the origin of isolated OFCs.

Gastroschisis and associated defects: an international study.

Our objective was to evaluate the frequency and type of malformations associated with gastroschisis in a large pool of international data, to identify malformation patterns, and to evaluate the role of maternal age in non-isolated cases. Case-by-case information from 24 registries, all members of the International Clearinghouse for Birth Defects Surveillance and Research (ICBDSR), were evaluated. After the exclusion of other abdominal wall defects cases were classified as: (a) isolated; (b) recognizable syndrome, chromosomal or not; (c) multiple congenital anomalies (MCA). Our results showed that out of 3,322 total cases 469 non-isolated cases were registered (14.1%): 41 chromosomal syndromes, 24 other syndromes, and 404 MCA. Among MCA four groups of anomalies were most frequent: CNS (4.5%), cardio-vascular (2.5%), limb (2.2%), and kidney anomalies (1.9%). No similar patterns emerged except two patterns resembling limb-body wall complex and OEIS. In both of them the gastroschisis could be however misclassified. Chromosomal trisomies and possibly non-syndromic MCA are associated with an older maternal age more than isolated cases. On consideration of our data and the most valid studies published in the literature, the best estimate of the proportion of gastroschisis associated with major unrelated defects is about 10%, with a few cases associated to recognizable syndromes. Recognized syndromes with gastroschisis seem to be so exceptional that the well documented and validated cases are worth being published as interesting case report. An appropriate case definition in etiological studies should include only isolated gastroschisis after an appropriate definition of isolated and non-isolated cases and a thorough case-by-case review.