Microsatellite evolution--evidence for directionality and variation in rate between species.

Microsatellite DNA sequences are rapidly becoming the dominant source of nuclear genetic markers for a wide range of applications, from genome mapping to forensic testing to population studies. If misinterpretation is to be avoided, it is vital that we understand fully the way in which microsatellite sequences evolve. We have therefore compared allele length distributions for 42 microsatellites in humans with their homologues in a range of related primates. We find a highly significant trend for the loci to be longer in humans, showing that microsatellites can evolve directionally and at different rates in closely related species.

Mutational bias provides a model for the evolution of Huntington's disease and predicts a general increase in disease prevalence.

Huntington's disease (HD) correlates with abnormal expansion in a block of CAG repeats in the Huntington's disease gene. We have investigated HD evolution by typing CAG alleles in several human populations and in a variety of primates. We find that human alleles have expanded from a shorter ancestral state and exhibit unusual asymmetric length distributions. Computer simulations are used to show that the human state can be derived readily from a primate ancestor, without the need to invoke natural selection. The key element is a simple length-dependent mutational bias towards longer alleles. Our model can explain a number of empirical observations, and predicts an ever-increasing incidence of HD.

Normal CAG and CCG repeats in the Huntington's disease genes of Parkinson's disease patients.

The clinical features of Parkinson's disease, particularly rigidity and bradykinesia and occasionally tremor, are seen in juvenile-onset Huntington's disease. Therefore, the CAG and CCG repeats in the Huntington's disease gene were investigated in 45 Parkinson's disease patients and compared to 40 control individuals. All of the Parkinson's disease chromosomes fell within the normal size ranges. In addition, the distributions of the two repeats in the Parkinson's disease patients did not differ significantly from those of the control population. Therefore, abnormalities of these trinucleotide repeats in the Huntington's disease gene are not likely to contribute to the pathogenesis of Parkinson's disease.

Analysis of thirteen trinucleotide repeat loci as candidate genes for schizophrenia and bipolar affective disorder.

A group of diseases are due to abnormal expansions of trinucleotide repeats. These diseases all affect the nervous system. In addition, they manifest the phenomenon of anticipation, in which the disease tends to present at an earlier age or with greater severity in successive generations. Many additional genes with trinucleotide repeats are believed to be expressed in the human brain. As anticipation has been reported in schizophrenia and bipolar affective disorder, we have examined allele distributions of 13 trinucleotide repeat-containing genes, many novel and all expressed in the brain, in genomic DNA from schizophrenic (n = 20-97) and bipolar affective disorder patients (23-30) and controls (n = 43-146). No evidence was obtained to implicate expanded alleles in these 13 genes as causal factors in these diseases.

Measurement of activity of urea resistant neutrophil alkaline phosphatase as an antenatal screening test for Down's syndrome.

OBJECTIVE: To investigate the value of measuring maternal urea resistant neutrophil alkaline phosphatase activity as an antenatal screening test for Down's syndrome. DESIGN: Case-control study of blood samples collected at nine to 27 weeks of pregnancy. SETTING: Antenatal clinics in London and Oxford. PATIENTS: 72 Women whose fetuses had been diagnosed by amniocentesis or chorionic villus sampling as having Down's syndrome and 156 women whose fetuses did not have the syndrome. Only singleton pregnancies were studied. MAIN OUTCOME MEASURE: Activity of urea resistant neutrophil alkaline phosphatase measured cytochemically. RESULTS: The median enzyme activity in the index patients was 1.65 times the expected median for the controls at the same duration of pregnancy (p less than 0.0001; 95% confidence interval 1.56 to 1.74). A cut off value that identified the 5% of control patients with the highest activities yielded a rate of detection of Down's syndrome of 79% (95% confidence interval 70 to 89%). CONCLUSION: Activity of urea resistant neutrophil alkaline phosphatase is an effective maternal blood marker for Down's syndrome. Its use in antenatal screening could lead to a substantial improvement in the detection of this disorder. Before introducing the test into routine medical practice it will have to be automated so that it can be used on a large scale and is less subjective.

The impact of national guidance for anomaly screening and invasive testing: unintended consequences.

Recent guidance from the UK National Screening Committee (NSC) and the Fetal Anomaly Screening Programme (FASP) has led to important changes in prenatal ultrasound diagnosis and invasive testing. These relate to prenatal ultrasound investigation of what were previously known as 'soft markers' for Down's syndrome at the time of the detailed anomaly scan and as to whether full karyotype or FISH (fluorescent in situ hybridisation)/QFPCR (quantitative fluorescence PCR) testing for trisomies should be carried out when an invasive test is performed. Neither recommendation is directly related to the other but both in combination could have profound implications for the detection of chromosomal abnormalities other than trisomy 21 (Down's syndrome). In the light of two cases recently managed in one regional fetal medicine unit, we retrospectively reviewed cases where, with correct application of the NSC and FASP recommendations, non-lethal and clinically important chromosomal abnormalities would most likely not have been detected.

Potter's syndrome in the second trimester--prenatal screening and pathological findings in 60 cases of oligohydramnios sequence.

Fifty-two second-trimester and eight third-trimester (> 28/40) autopsies with clinical or pathological evidence of oligohydramnios sequence ('Potter's syndrome') were reviewed. Twenty-eight cases had renal anomalies (71 per cent in terminations following prenatal ultrasound), 27 had no renal malformation (35 per cent with chorioamnionitis), and five had external assessments only. In 15 cases, the renal lesion was part of a multiple malformation syndrome. Seven cases had a lesion which either recurred in a sibling in the same family or was a recognized autosomal recessive syndrome. Three cases had an abnormal karyotype, two of which had renal anomalies. Maternal serum alpha-fetoprotein (AFP) did not discriminate between cases with renal malformations and those without. Pulmonary hypoplasia was commoner in third-trimester than in second-trimester cases. External appearance and absent umbilical artery were not reliable predictors of underlying internal anomalies. These findings reflect the shift from postnatal to prenatal diagnosis in modern practice. In this series, mainly second-trimester cases, 50 per cent of cases had no malformations, in a condition which is traditionally associated with renal disease. The high incidence of chorioamnionitis suggests that the mechanism of oligohydramnios is occult amniotic fluid leakage. Prenatal diagnosis of oligohydramnios in the second trimester is dependent on ultrasound scanning and a full post-mortem examination is necessary to identify any underlying fetal cause.

Congenital nephropathy and ventriculomegaly: a report of four cases.

Congenital nephrotic syndrome with ventriculomegaly and a normal karyotype is a rare association. We report four cases, three of which were conceived consecutively by one couple. All the cases were associated with elevated maternal serum alpha-fetoprotein. Renal histology in one fetus demonstrated colloid filled cysts distributed in the corticomedullary area. Transmission electron microscopy of the glomeruli showed normally developed foot processes and confirmatory genetic studies excluded Finnish congenital nephrotic syndrome. It is probable that congenital nephropathy in conjunction with ventriculomegaly is the result of an autosomal recessive syndrome.

Haplotype analysis of the delta 2642 and (CAG)n polymorphisms in the Huntington's disease (HD) gene provides an explanation for an apparent 'founder' HD haplotype.

The discovery of the intragenic delta 2642 deletion/insertion polymorphism in the Huntington's disease (HD) gene provides a tool to explore HD evolution, as the deletion is rare in normal chromosomes but overrepresented in HD chromosomes. Thus, delta 2642 deletion alleles were thought to mark normal chromosomes that are particularly prone to becoming HD chromosomes. We have examined this polymorphism in a range of human and non-human primate populations. Our results suggest that the deletion event probably occurred in the human lineage on a chromosome with a CAG allele length at the upper end of the normal size range, as the deletion seemed to be only associated with human chromosomes with 20 or more repeats. These deletion chromosomes are prone to expansion into the HD range because they are at the upper end of the normal range. These data explain the apparent 'founder' HD haplotype defined by the deletion and suggest that chromosomes carrying the deletion are no more mutable than non-deletion chromosomes with similar sized CAG repeats.

Genetic association between monoamine oxidase A microsatellite and RFLP alleles and bipolar affective disorder: analysis and meta-analysis.

The monoamine oxidase A locus (MAOA) at Xp11 was considered a good candidate to investigate in bipolar affective disorder since this enzyme plays an important role in the degradation of various neurotransmitters and a mutation in this gene has been associated with borderline mental retardation and a behavioural phenotype that has some resemblance to the manic syndrome. Previous association studies comparing allele frequencies of a microsatellite and RFLP at the monoamine oxidase A locus in bipolar affective disorder cases and controls in the UK have yielded conflicting results: Lim and colleagues reported a positive association, while no evidence for allelic association was obtained by Cradock and co-workers. A significant allelic association was observed between Japanese bipolar cases and controls at the MAOA microsatellite but different alleles seemed to be overrepresented in the bipolar cases in this population compared to the UK. In order to resolve these differences, we have examined this locus in our series of unrelated bipolar cases and age- and sex-matched controls and found significantly different MAOA microsatellite allele frequencies. In addition, we have pooled the data from the two previous UK studies with ours to create a total data set including 67 males and 113 females with bipolar affective disorder and a similar number of matched controls. No evidence for heterogeneity was observed for the control MAOA microsatellite or RFLP allele frequencies in these three studies. However, we found a significant difference between the pooled normal and bipolar allele frequencies both for the microsatellite and the RFLP at MAOA.

Numbers or words? A randomized controlled trial of presenting screen negative results to pregnant women.

The Objective of this study was to test the hypothesis that presenting risk information using numbers rather than words is a more effective way of communicating the residual risk inherent in a screen negative test result. We used a randomised controlled trial in a large UK teaching hospital. Two hundred and twenty pregnant women who received negative results on serum screening for Down syndrome participated. Presentation of screen negative test results were given either as a numerical probability (e.g. you have a 1:650 chance of having a baby with Down syndrome) or as a verbal probability (your chance of having a baby with Down syndrome is low). In both interventions the verbal anchor 'it is unlikely that your baby has Down syndrome' was used. Our aims were to measure the understanding of the residual risk in a screen negative result, and anxiety. Immediately after receipt of the results, 97% of those receiving their results in the form of a numerical probability and 91% of those receiving their results in the form of a verbal probability correctly understood that their baby probably did not have Down syndrome (95% CI for difference: 0% to 12%; p=0.04). All those who were incorrect believed that their baby definitely did not have Down syndrome. Subgroup analysis showed that this effect was confined to those with lower levels of education (i.e. those without a university degree), amongst whom understanding was poorer. There was no difference between intervention groups in understanding the results at four months. There were no differences between intervention groups in the levels of anxiety at one week or four months after receiving their results. In conclusion, communicating residual risks using numbers rather than words has a small beneficial effect of increasing awareness of residual risks without raising anxiety. Further work is needed to estimate the size of this effect in less well-informed and less highly educated populations.

Apathy and hypersomnia are common features of myotonic dystrophy.

OBJECTIVES: Myotonic dystrophy is a disease characterised by myotonia and muscle weakness. Psychiatric disorder and sleep problems have also been considered important features of the illness. This study investigated the extent to which apathy, major depression, and hypersomnolence were present. The objective was to clarify if the apathy reported anecdotally was a feature of CNS involvement or if this was attributable to major depression, hypersomnolence, or a consequence of chronic muscle weakness. METHODS: These features were studied in 36 adults with non-congenital myotonic dystrophy and 13 patients with Charcot-Marie-Tooth disease. By using patients with Charcot-Marie-Tooth disease as a comparison group the aim was to control for the disabling effects of having an inherited chronic neurological disease causing muscle weakness. Standardised assessment instruments were used wherever possible to facilitate comparison with other groups reported in the medical literature. RESULTS: There was no excess of major depression on cross sectional analysis in these patients with mild myotonic dystrophy. However, apathy was a prominent feature of myotonic dystrophy in comparison with a similarly disabled group of patients with Charcot-Marie-Tooth disease (clinician rated score; Mann Whitney U test, p=0.0005). Rates of hypersomnolence were greater in the myotonic dystrophy group, occurring in 39% of myotonic dystrophy patients, but there was no correlation with apathy. CONCLUSION: These data suggest that apathy and hypersomnia are independent and common features of myotonic dystrophy. Apathy cannot be accounted for by clinical depression or peripheral muscle weakness and is therefore likely to reflect CNS involvement. These features of the disease impair quality of life and may be treatable.

Mild myotonic dystrophy is associated with memory impairment in the context of normal general intelligence.

Previous investigations of cognitive function in myotonic dystrophy (DM) suggested that DM subjects have impairment of general intelligence and severe defects in executive function. These studies had to neglect patients with mild clinical disease, since they were generally performed before the availability of DNA testing for DM. We have investigated a group of milder DM cases (defined both by their degree of disability and CTG expansion size) and have not found any defect in general intelligence using the National Adult Reading Test, Standard Progressive Matrices, and Mini-Mental State examination. Three executive function tests were used. The cognitive estimates test and the Wisconsin Card Sorting Test showed tendencies towards impairment, which were of borderline significance, but the verbal fluency task was performed normally. However, in the face of normal general intelligence, mild DM subjects as a group show impairment of memory function. This suggests that memory function may be more sensitive to small CTG repeat expansions than general intelligence.

Apo E genotypes in multiple sclerosis, Parkinson's disease, schwannomas and late-onset Alzheimer's disease.

Apolipoprotein E (apo E) exists in three allelic, functionally distinct isoforms (apo E2, E3 and E4). Recent work has suggested that apo-E-dependent uptake of lipoproteins may play important roles in the development and maintenance of the nervous system and in the responses to both peripheral and central nervous system injury. If apo-E-mediated transport of lipids were a rate-limiting step in these processes, one might expect that the functional differences between the alleles would be associated with varying predispositions to neurodegenerative and demyelinating diseases. Thus, we looked for an association between particular apo E genotypes and susceptibility to multiple sclerosis and Parkinson's disease. If apo-E-mediated cholesterol uptake were limiting in neuronal growth, one might also expect that apo E2 alleles would slow CNS tumour growth. Accordingly, apo E genotypes were investigated in individuals with sporadic vestibular schwannomas and neurofibromatosis type 2 (NF-2). No significant alteration in the apo E allele distributions was observed in any of these conditions, nor did the apo E genotypes correlate with disease severity. However, we confirmed the previous findings of an over-representation of the apo E4 allele in autopsy-diagnosed late-onset Alzheimer's disease patients. In addition, our data supported the recent observations that apo E2 may be associated with a protective effect for late-onset Alzheimer's disease. These contrasting risks associated with the apo E2 and E4 alleles strengthen the suggestions that this gene is directly involved in the pathogenesis of Alzheimer's disease.

Study of the Huntington's disease (HD) gene CAG repeats in schizophrenic patients shows overlap of the normal and HD affected ranges but absence of correlation with schizophrenia.

The CAG repeats in the Huntington's disease gene were investigated in chromosomes from 71 unrelated schizophrenic persons and 18 patients with schizoaffective disorder in order to determine if any of these patients had abnormal expansions. All of the probands had repeat sizes in the normal range (< 35 repeats) and there was no significant difference between the allele distributions of these patients and the normal controls. The families of two patients with 32 repeats and one patient with 34 repeats were investigated further and showed no uniform segregation of the disease with the large repeat alleles. The proband with 34 repeats inherited a chromosome that originally had 36 repeats in her father. The presence of 36 CAG repeats in members of her family and in HD patients suggests that there is an overlap between the normal and Huntington's disease CAG repeat size ranges. The more recently described CCG polymorphism in this gene was also examined in the schizophrenic and schizoaffective persons. All patients had alleles in the normal range.

Second-trimester maternal serum screening using alpha-fetoprotein, human chorionic gonadotrophin, and unconjugated oestriol: experience of a regional programme.

Over a 2-year period from January 1991 to December 1992, second-trimester maternal serum screening for Down's syndrome using alpha-fetoprotein (alpha FP), human chorionic gonadotrophin (hCG), and unconjugated oestriol (uE3) was made available to five health districts in East Anglia, with a total population of 1.2 million. Amniocentesis was offered when the risk of Down's syndrome at term was 1:200 or greater. 25,359 singleton pregnancies were screened, representing an uptake of 77 per cent. The recall rate for the 24 per cent of women who had not had a dating scan prior to the test was 9.4 per cent compared with 3.9 per cent for those who had been scanned (P < 0.0005). Seventy-five per cent (36/48) of Down's syndrome pregnancies were detected for a false-positive rate of 4.0 per cent. Twenty-five out of 36 of detected Down's syndrome pregnancies were dated by scan prior to sampling, and in the 11 remaining cases, the dates were confirmed by scan after a high-risk result was obtained. The exclusion of uE3 from the screening protocol would have reduced the detection rate to 52 per cent (25/48) for the same false-positive rate. Eighty-five per cent of women identified at high risk accepted the offer of an amniocentesis. Other fetal abnormalities detected were trisomy 18 (3), trisomy 13 (2), 45,X (6), 69,XXX (5), other chromosome abnormalities (9), open neural tube defects (26), hydrocephalus (7), abdominal wall defects (4), and steroid sulphatase deficiency (6).