Estimating human inbreeding coefficients: comparison of genealogical and marker heterozygosity approaches.

We have used genealogies and genomic polymorphisms to estimate individual inbreeding coefficients (F) in 50 subjects with an expected range (based on recent genealogies) of F from 0.0 to 0.0625. The estimates were based on two approaches, using genotypes respectively from 410 microsatellite markers (410-STR panel) and from 10,000 SNPs (10K-SNP panel). The latter was performed in a sub-sample of 15 individuals. We concluded that for both marker panels measures of inbreeding based on the excess of homozygosity over Hardy-Weinberg expectation were not closely correlated with 4-5 generation genealogical F-values. For the 10K-SNP panel we found two alternative measures which correlated more closely with F, based respectively on standard errors and on paired homozygosity of nearby SNPs over distances of 2-4 cM. We propose an empirical method for estimating standard errors and hence individual F-values, based on the variation between individual autosomes. This method could provide useful estimates of average F-values for groups of individuals in population-based studies of the effects of inbreeding/homozygosity on quantitative traits.

Extent of linkage disequilibrium in a Sardinian sub-isolate: sampling and methodological considerations.

The extent of linkage disequilibrium (LD) is an important factor when designing experiments for mapping disease or trait loci using LD mapping methods. It depends on the population history and hence is a characteristic of each population. Here, we have assessed the extent of LD in a sub-isolate of the general Sardinian population (775 members of one village) using 22 polymorphic markers on chromosome 19. We found high levels of disequilibrium that extended to 8 cM, when based on D', and 11 cM when based on the significance level of the allelic association. The fact that conclusions based on both methods are similar suggests that the estimates are quite robust. We have also shown, through a simple resampling technique, that small sample sizes can overestimate both the mean value of D' and its variance up to a factor of about 2 and 16, respectively, when the number of diplotypes (the pair of haplotypes that compose the genotype) decreased from 186 to 26. We evaluated the effect on D' of the depth of the pedigree available when using phased founders, and compared the estimates with those obtained when using unphased founders, and also the effect of grouping alleles on the value of D' and the significance level. Owing to the high sampling variance of LD, we recommend the use of at least 200 unrelated individuals when characterizing the extent of LD.

Power of linkage disequilibrium mapping to detect a quantitative trait locus (QTL) in selected samples of unrelated individuals.

We considered a strategy to map quantitative trait loci (QTLs) using linkage disequilibrium (LD) when the QTL and marker locus were multiallelic. The strategy involved phenotyping a large number of unrelated individuals and genotyping only selected individuals from the two tails of the trait distribution. Power to detect trait-marker association was assessed as a function of the number of QTL and marker alleles. Two patterns of LD were used to study their influence on power. When the frequency of the QTL allele with the largest effect and that of the marker allele linked in coupling were equal, power was maximum. In this case, increasing the number of QTL alleles reduced the power. The maximum difference in power between the two LD patterns studied was approximately 30%. For low QTL heritabilities (h2QTL<0.1) and single trait studies we recommend selecting around 5% of the upper and lower tails of the trait distribution.

Search for ethnic, geographic, and other factors in the epidemiology of Down syndrome in South America: analysis of data from the ECLAMC project, 1967-1997.

We have analyzed data on 3,157 cases of Down syndrome (DS) from nine South American countries in consecutive series of hospital live births over a 30-year period, with particular emphasis on possible ethnic or geographic variations in maternal age-adjusted incidence. The data constitute the largest series of DS cases assembled to date from an area lacking advanced health care systems. Absolute incidence rates were estimated from total hospital live births; relative rates were estimated from matched case-control data using conditional logistic regression. Maternal age-adjusted rates were closely similar to those reported elsewhere, and showed little or no dependency on other factors investigated, including paternal age, birth order, ancestral origin, country of birth, maternal educational level, maternal ABO and Rhesus blood groups, interval to and outcome of mother's previous pregnancy, and parental consanguinity. The absence of an effect of high birth order was particularly notable because of the relatively large number of grand multipara resulting from high fertility in this population. The study adds to a body of evidence suggesting that maternal age-adjusted DS rates vary little across human populations, and are therefore unlikely to be greatly influenced by genetic or environmental factors that differ between them. An unusual finding was of a markedly lower sex ratio (98 males per 100 females) than has been reported in other DS samples.

Population choice in mapping genes for complex diseases.

The difficulty of identifying susceptibility genes for common diseases has polarized geneticists' views on what disease models are appropriate and how best to proceed once high-density genome maps become available. Different disease models have different implications for using linkage or linkage-disequilibrium-based approaches for mapping complex disease genes. We argue that the choice of study population is a critical factor when designing a study, and that genetically simplified isolates are more useful than diverse continental populations under most assumptions.

International variation in reported livebirth prevalence rates of Down syndrome, adjusted for maternal age.

Reported livebirth prevalence of Down syndrome (DS) may be affected by the maternal age distribution of the population, completeness of ascertainment, accuracy of diagnosis, extent of selective prenatal termination of affected pregnancies, and as yet unidentified genetic and environmental factors. To search for evidence of the latter, we reviewed all published reports in which it was possible to adjust both for effects of maternal age and for selective termination (where relevant). We constructed indices that allowed direct comparisons of prevalence rates after standardising for maternal age. Reference rates were derived from studies previously identified as having near complete ascertainment. An index value significantly different from 1 may result from random fluctuations, as well as from variations in the factors listed above. We found 49 population groups for which an index could be calculated. Methodological descriptions suggested that low values could often be attributed to under-ascertainment. A possible exception concerned African-American groups, though even among these most acceptable studies were compatible with an index value of 1. As we have reported elsewhere, there was also a suggestive increase in rates among US residents of Mexican or Central American origin. Nevertheless, our results suggest that "real" variation between population groups reported to date probably amounts to no more than +/-25%. However, reliable data in many human populations are lacking including, surprisingly, some jurisdictions with relatively advanced health care systems. We suggest that future reports of DS livebirth prevalence should routinely present data that allow calculation of an index standardised for maternal age and adjusted for elective prenatal terminations.

Elevated maternal age-specific rates of Down syndrome liveborn offspring of women of Mexican and Central American origin in California.

BACKGROUND: Maternal age-specific rates of Down syndrome livebirths are widely utilized in personal and policy decisions concerning provision of and election of prenatal cytogenetic diagnostic services. The only extensive reference data available are on those of primarily European ancestral origin. In the absence of definitive evidence of any ethnic, racial or environmental influence upon rates (other than those associated with age) these rate schedules have been widely applied to those of all national origins. METHODS: Available material age-specific data on livebirths from intensive studies on those of Hispanic (primarily of Mexican and Central American background) and of other origin in populations in the U.S.A. with likely complete ascertainment were analysed. The numbers observed were compared with (i) those predicted from established published rate schedules in those of primarily European origin, and (ii) with the observations on livebirths of non-Hispanic European origin in the same population as the Hispanic live births. RESULTS: In comparisons with the numbers predicted from published rates, observed numbers of case among Hispanic live births were increased by 19 per cent (SE 0.06) in younger mothers, 23 per cent in older mothers (SE 0.07) and 20 per cent (SE 0.04) in those of all ages. Comparisons with observed rates in those of Hispanic origin with those observed in non-Hispanic births in the same time intervals and populations indicated that the excess rates in Hispanics were not attributable to some local factor increasing rates in all ethnic groups at least among those under 35. CONCLUSIONS: Data on mothers of Mexican and Central American origin residing in the U.S.A. indicate maternal age-specific rates of Down syndrome in live births about 20 per cent greater than those in published rate schedules on Down syndrome, widely used in decisions concerning election or provison of prenatal diagnostic services. The reason for this difference remains unknown.

Trends in prenatal diagnosis of Down syndrome and other autosomal trisomies in Scotland 1990 to 1994, with associated cytogenetic and epidemiological findings.

The present report summarizes findings on 670 cases of autosomal trisomy diagnosed in Scotland, with actual or expected dates of delivery in 1990 to 1994 inclusive. Cases were notified by cytogenetic service laboratories. There were 277 prenatal and 369 postnatal diagnoses and 24 spontaneous losses. Excluding the latter, numbers diagnosed with trisomy 21, trisomy 18, trisomy 13, and other trisomies were, respectively, 470 (72.8%), 108 (16.7%), 36 (5.6%), and 32 (5.0%). Estimated maternal age-specific birth rates for trisomy 21 were close to published values from other jurisdictions. However, comparisons with a clinically based national register of congenital anomalies suggested that 3-4% of Down syndrome births were never karyotyped, most being early neonatal deaths. There was a striking increase over the period in the proportion of cases detected prenatally, associated with increased maternal serum screening in mothers <35 years old. Over the 3 final years (1992-1994), prenatal screening followed by elective termination was estimated to reduce the birth rate in trisomy 21 by 24% in mothers aged <35 years, by 57% in older mothers, and by 35% in all mothers. The crude incidence per 1,000 births fell from 1.08 in 1990-1991 to 0.77 in 1992-1994, in spite of an upward shift in the overall maternal age distribution. For trisomies 18 and 13, the estimated overall reductions in the birth rate over the whole 5-year period were respectively, 26 and 17%. In free trisomy 18, there was a significant reduction in the sex ratio (male/female) to 0.65, in line with earlier studies.

A likelihood-based approach to the estimation of relative DNA copy number by comparative genomic hybridization.

A general approach is described for efficiently and objectively analyzing comparative genomic hybridization (CGH) profile data based on the use of realistic statistical models and the application of standard likelihood-based inference. In contrast to other methods in current use, the approach provides a most parsimonious explanation by identifying the smallest number of relative DNA copy number changes consistent with the data, together with estimates of their levels and positions and of their standard errors. By making efficient use of available data, it has the potential to enhance the resolution of CGH technology. The computational feasibility of the method is illustrated by application to real CGH profile data from human chromosome 4.

Evidence for safety and efficacy of DOTAP cationic liposome mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis.

In cystic fibrosis (CF), mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene results in defective transepithelial ion transport, leading to life shortening inflammatory lung disease. Before lung studies, we tested the safety and efficacy of gene delivery to the nasal epithelium of CF patients using pCMV-CFTR-DOTAP cationic liposome complex. A single dose of 400 micrograms pCMV-CFTR:2.4 mg DOTAP was administered in a randomised, double-blinded fashion to the nasal epithelium of eight CF patients, with a further eight receiving buffer only. Patients were monitored for signs and symptoms for 2 weeks before treatment and 4 weeks after treatment. Inflammatory cells were quantified in a nasal biopsy taken 3 days after treatment. There was no evidence for excess nasal inflammation, circulating inflammatory markers or other adverse events ascribable to active treatment. Gene transfer and expression were assayed by the polymerase chain reaction. Transgene DNA was detected in seven of the eight treated patients up to 28 days after treatment and vector derived CFTR mRNA in two of the seven patients at +3 and +7 days. Transepithelial ion transport was assayed before and after treatment by nasal potential difference during drug perfusion and by SPQ fluorescence halide ion conductance. Partial, sustained correction of CFTR-related functional changes toward normal values were detected in two treated patients. The level of gene transfer and functional correction were comparable to those reported previously using adenoviral vectors or another DNA-liposome complex, but here were sustained and uncompromised by false positives. These results justify further studies with pCMV-CFTR-DOTAP aimed at treating CF lung disease.

Use of computer simulation to evaluate a putative cluster of genetic or teratologic outcomes: adjustment for "multiple hypotheses" and application to a reported excess of Down's syndrome.

The identification of an apparent excess of a genetic outcome in a particular area and/or a particular time often provokes considerable public alarm about the presence of an environmental mutagen. It is often difficult to determine in any particular case whether the observation, whatever its nominal statistical significance, is due to chance concatenation of events or to an environmental factor. Statistical evaluation is made more difficult by the profuse number of possible hypotheses that could have triggered concern about an excess. This renders it difficult to calculate the actual probability of the observation (or one more extreme). By attempting to identify similar types of outcomes that could have provoked an apparent excess and then undertaking computer simulations assuming random deviations from a constant rate, one may attempt to adjust for the problem of multiple hypotheses. We apply this approach to a reported excess of Down's syndrome in Norway in 1985-1986 in younger mothers, and conclude that there is a high probability that it arose by chance.

Cancer risk associated with germline DNA mismatch repair gene mutations.

The autosomal dominant syndrome of Hereditary Nonpolyposis Colorectal Cancer (HNPCC) is due to germline DNA mismatch repair gene mutations in most cases. However, the penetrance of such mutations outwith classical HNPCC kindreds is unknown because families studied to date have been specifically selected for research purposes. Using a population-based strategy, we have calculated the lifetime cancer risk associated with germline DNA mismatch repair gene mutations, irrespective of their family history. We identified 67 gene carriers whose risk to age 70 for all cancers was 91% for males and 69% for females. The risk of developing colorectal cancer was significantly greater for males than for females (74% versus 30%, P= 0.006). The risk of uterine cancer (42%) exceeded that for colorectal cancer in females, emphasising the need for uterine screening. Our findings give further insight into the biological effect of defective DNA mismatch repair. We have demonstrated a systematic approach to identifying individuals at high risk of cancer but who may not be part of classical HNPCC families. The risk estimates derived from these analyses provide a rational basis on which to guide genetic counselling and to tailor clinical surveillance.

Laboratory and clinical studies in support of cystic fibrosis gene therapy using pCMV-CFTR-DOTAP.

The first phase I study of cystic fibrosis gene therapy using cationic liposomes to deliver the cystic fibrosis conductance regulator gene to the nose reported partial and transient correction of the nasal transepithelial ion transport defect, While encouraging, further improvements will be required if this form of treatment is to be of therapeutic value. We tested a new formulation, pCMV-CFTR-DOTAP. The complex is stable for 10 days and effective at correcting the electrophysiological deficit in the trachea of CF mutant mice at 8 or 9 days after intratracheal instillation. Reliable protocols for consistent detection of as few as 10 molecules of CFTR mRNA and DNA in nasal brushing samples are described, Both vector and DNA have been produced to Good Manufacturing Practice standard, Nasal potential difference measurements developed at the National Heart and Lung Institute to assess the CFTR ion channel activity in CF patients replicated well at the Scottish Adult Cystic Fibrosis Service. The SPO fluorescence assay for halide ion conductance in nasal brushings has also been tested. These establish baseline conditions in the Scottish CF cohort from which evidence for correction can be judged under clinical trial conditions. These studies formed the basis for regulatory approval of a randomised, placebo controlled double-blind phase I research study.

Microsatellite instability and the role of hMSH2 in sporadic colorectalcancer.

Microsatellite instability (MSI) occurs in most tumours from patients with hereditary non-polyposis colorectal cancer (HNPCC) and in around 17% of sporadic colorectal cancers. Germline defects in mismatch repair (MMR) genes are responsible for the majority of large HNPCC families, with hMSH2 accounting for at least 50%. MMR gene defects also occur in a small proportion of sporadic colorectal tumours with MSI. Here we report a systematic analysis of mismatch repair deficiency in 215 Scottish patients with sporadic colorectal tumours. We found that 16.4% of tumours exhibited MSI; survival analysis by Cox proportional hazards method showed a substantial survival advantage for patients with tumours showing MSI, independent of other prognostic factors. Tumours with MSI were screened for hMSH2 mutations and although 61% were found to have alterations, of these only 1/24 was exonic. The majority of these changes were reductions in length at intronic mononucleotide tracts and we postulate that these alterations are the result of a genetic defect elsewhere, although they may compromise hMSH2 function as a second step in tumourigenesis. Our findings indicate that instability confers an improved prognosis in colorectal cancer and, despite the fact that these two groups of tumours share similar biological characteristics, the genetic basis of HNPCC and sporadic colorectal cancer with MSI is different.

Methods for maternal age-standardization of the incidence of congenital abnormalities.

In order to compare the birth incidences of particular congenital abnormalities in different populations, it is often necessary to allow for the effects of maternal age. Three age-adjusted indices are defined, based on analogous indices from the literature on mortality studies, namely, the Standardized Mortality Ratio, the Comparative Mortality Figure and Kerridge's Inverse Method. In most practical situations the differences between them are likely to be trival. However, the first index is maximally efficient under multiplicative risk models and is easily adjusted for incomplete data. The second is the only one to provide valid comparisons under additive, as well as multiplicative, models. The third has the advantage that it does not require a knowledge of the maternal age distribution of all births in the population. The use of the three indices is illustrated with published data on Down's syndrome.

Factors affecting the timing and imprinting of replication on a mammalian chromosome.

Fluorescence in situ hybridisation has been used to follow replication of the short arm of human chromosome 11 using chromosome anomalies to distinguish the maternally-and paternally-derived homologues. The temporal difference in replication timing within and between chromosomes has been estimated by combining S phase detection with dual colour fluorescence in situ hybridisation. Proximal regions of 11p, including the WT1 gene, tend to replicate earlier on the maternally-derived chromosome than on the paternally-derived homologue. More distal parts of 11p (including the IGF2 gene) have the opposite imprint. The average difference in replication timing between homologous loci in the population of cells is small compared to the differences between loci along a single chromosome. The imprint is not strictly adhered to since many nuclei have hybridisation patterns opposite to the trend within the population. The nature of the imprinting signal has been investigated. Absolute replication time, but not the imprint, was affected by azacytidine, an inhibitor of DNA methylation. The replication imprint was modified by treatments that inhibit histone deacetylation. We suggest that replication imprinting reflects differences in chromatin structure between homologues.

Models of DNA replication timing in interphase nuclei: an exercise in inferring process from state.

Patterns of spots, produced by fluorescence in situ hybridization, can be used to infer the order and timing of replication of DNA sequences in S-phase nuclei, sampled randomly from asynchronous cell populations. We describe statistical models that provide estimates of replication timings and completion rates and of their standard errors. By applying the technique to experimental data from diploid cell lines, we show how it can be used to compare the replication timings either of different sequences on the same chromosome, or of the same sequence on a pair of homologous chromosomes.