Long-term effect of prospective detection of high genetic risk on couples' reproductive life: data for thalassaemia.

Prospective risk detection with availability of prenatal diagnosis is the best service currently available for couples at high genetic risk Here we describe the long term effect of this service on the reproductive life of 102 couples at risk of thalassaemia, whose risk was detected prospectively by carrier screening, who made use of prenatal diagnosis, and where the woman is now over 40. Overall outcome for couples is described in terms of number of favourable versus unfavourable pregnancy outcomes. (A favourable pregnancy outcome = unaffected livebirth, or affected livebirth resulting from informed parental choice.) The 102 couples had a total of 356 pregnancies, including 302 viable pregnancies, and 88% achieved a family unburdened by thalassaemia. 68% of viable pregnancies had a favourable outcome, but only 43% of couples had only favourable outcomes, and 26% lost two or more viable wanted pregnancies. When early losses are included 58% of pregnancies had a favourable outcome, but only 30% of couples had only favourable outcomes, and 41% lost two or more pregnancies. Even with the best available service, at risk couples remain victims of chance, and a significant minority experience great difficulty in obtaining even one healthy child. Research is needed on approaches that may allow couples better control of reproductive outcomes.

HTLV type I/II in British Columbia Amerindians: a seroprevalence study and sequence characterization of an HTLV type IIa isolate.

It has been established that the human T cell lymphotropic viruses type I and II (HTLV-I and HTLV-II) are both present in some indigenous peoples of the Americas. While HTLV-I has been identified in coastal British Columbia Indians (BCIs), HTLV-II has not been previously reported in the BCIs or other Canadian Amerindians. The prevalence of HTLV-I and HTLV-II in these populations has not been extensively studied. In this article, we examine a group of BCIs from Vancouver Island who belong to the Nuu-Chah-Nulth and are known to have an increased incidence of rheumatic disease. In 494 serum samples from this tribe, the levels of prevalence of HTLV-I and HTLV-II were 2.8 and 1.6%, respectively. No association could be made between arthropathy and HTLV-I infection. In addition, we characterized an HTLV-II isolate of a BCI from the coastal mainland of British Columbia and with a history of intravenous drug abuse. This case represents the first molecular characterization of a Canadian Amerindian HTLV-II isolate: a subtype IIa virus with phylogenetic affinity for intravenous drug user isolates and containing an extended form of the Tax protein. These results are consistent either with this strain having been sampled from a polymorphic ancestral pool of HTLV-II that gave rise to the current epidemic spread of this virus by intravenous drug use and sexual transmission, or with its being "back-transmitted" into the BC Amerindian population in association with intravenous drug use.

Mitochondrial genes and mammalian phylogenies: increasing the reliability of branch length estimation.

Since branch lengths provide important information about the timing and the extent of evolutionary divergence among taxa, accurate resolution of evolutionary history depends as much on branch length estimates as on recovery of the correct topology. However, the empirical relationship between the choice of genes to sequence and the quality of branch length estimation remains ill defined. To address this issue, we evaluated the accuracy of branch lengths estimated from subsets of the mitochondrial genome for a mammalian phylogeny with known subordinal relationships. Using maximum-likelihood methods, we estimated branch lengths from an 11-kb sequence of all 13 protein-coding genes and compared them with estimates from single genes (0.2-1.8 kb) and from 7 different combinations of genes (2-3.5 kb). For each sequence, we separated the component of the log-likelihood deviation due to branch length differences associated with alternative topologies from that due to those that are independent of the topology. Even among the sequences that recovered the same tree topology, some produced significantly better branch length estimates than others did. The combination of correct topology and significantly better branch length estimation suggests that these gene combinations may prove useful in estimating phylogenetic relationships for mammalian divergences below the ordinal level. Thus, the proper choice of genes to sequence is a critical factor for reliable estimation of evolutionary history from molecular data.

Rapid radiation events in the family Ursidae indicated by likelihood phylogenetic estimation from multiple fragments of mtDNA.

The bear family (Ursidae) presents a number of phylogenetic ambiguities as the evolutionary relationships of the six youngest members (ursine bears) are largely unresolved. Recent mitochondrial DNA analyses have produced conflicting results with respect to the phylogeny of ursine bears. In an attempt to resolve these issues, we obtained 1916 nucleotides of mitochondrial DNA sequence data from six gene segments for all eight bear species and conducted maximum likelihood and maximum parsimony analyses on all fragments separately and combined. All six single-region gene trees gave different phylogenetic estimates; however, only for control region data was this significantly incongruent with the results from the combined data. The optimal phylogeny for the combined data set suggests that the giant panda is most basal followed by the spectacled bear. The sloth bear is the basal ursine bear, and there is weak support for a sister taxon relationship of the American and Asiatic black bears. The sun bear is sister taxon to the youngest clade containing brown bears and polar bears. Statistical analyses of alternate hypotheses revealed a lack of strong support for many of the relationships. We suggest that the difficulties surrounding the resolution of the evolutionary relationships of the Ursidae are linked to the existence of sequential rapid radiation events in bear evolution. Thus, unresolved branching orders during these time periods may represent an accurate representation of the evolutionary history of bear species.

Transcervical cells and the prenatal diagnosis of haemoglobin (Hb) mutations.

Prenatal diagnoses of haemoglobin (Hb) mutations were performed using transcervical cells, retrieved by aspiration from the endocervical canal of ten selected pregnant women at about 10 weeks of gestation, prior to chorionic villus sampling (CVS). Both parents were carriers of haemoglobinopathies (thalassaemia or HbS). Clumps of fetal cells were isolated by micromanipulation under an inverted microscope and aliquots of the extracted DNA tested separately for the presence of paternally derived chromosome markers and Hb mutations by quantitative fluorescent polymerase chain reaction (PCR). The correct prenatal diagnosis of Hb diseases, using selected single clumps of trophoblastic cellular elements free of maternal contaminating cells, was achieved in six out of ten cases.

Modelling the major histocompatibility complex susceptibility to RA using the MASC method.

To explain the association between HLA-DRB1 gene and rheumatoid arthritis (RA), two main hypotheses have been proposed. The first, the shared epitope hypothesis, assumes a direct role of DRB1 in RA susceptibility. The second hypothesis assumes a recessive disease susceptibility gene in linkage disequilibrium with DRB1. To investigate these two hypotheses, we analysed data on the HLA-DRB1 and TNF-LT loci in 49 affected sib-pairs. We used the Marker Association Segregation Chi-square (MASC) method in which the genotype distribution of markers among index cases and the haplotype sharing in affected sib-pairs are jointly taken into account. With DRB1 data alone, both hypotheses were shown to fit but with analysis of TNF data, both hypotheses were strongly rejected. Thus the TNF data provided additional information for a better understanding of genetic susceptibility to RA than was previously possible using only HLA-DR data. A theoretical standpoint is addressed here on the advisability of using different linked markers in a candidate region for modelling the contribution of this region in disease susceptibility.

Angiotensin-converting enzyme genotypes and risk for myocardial infarction in women.

OBJECTIVES: We tested for an association between the angiotensin-converting enzyme (ACE) DD polymorphic genotype and myocardial infarction (MI) in a sample group composed exclusively of women. BACKGROUND: The human ACE gene occurs with either an insertion (I allele) or a deletion (D allele) of a 287-base pair (bp) Alu element. Part of the variance in serum ACE levels may be accounted for by this polymorphism. Also, the DD genotype has been associated with an increased risk of MI in predominantly male populations. However, the risk in women is poorly defined. METHODS: Genomic DNA was extracted from buffy coat blood using a phenol/chloroform method. Angiotensin-converting enzyme alleles were identified using primers to bracket the insertion region in intron 16. Amplification using polymerase chain reaction allowed identification of a 490-bp (I allele) or a 190-bp (D allele) product, or both. RESULTS: Allelic and genotypic frequencies in control subjects were similar to those reported in mostly male populations, and frequencies of genotypes were in the Hardy-Weinberg equilibrium. In contrast, the distribution of genotypes in patients with MI diverged from the equilibrium. Specifically, DD genotypic frequency was increased in women with (n = 141) versus without (n = 338) a previous MI (39% vs. 29%, odds ratio [OR] 1.54, 95% confidence interval 1.02 to 2.32, p < 0.04). Risk was particularly increased in women <60 years old (OR 2.04, p < 0.05). In contrast, the DD genotype did not predict angiographic coronary artery disease. CONCLUSIONS: Consistent with findings in male-dominated populations, a modest association of the ACE DD genotype with MI was found in women. The basis for this association requires further study.

Audit of prenatal diagnosis for haemoglobin disorders in the United Kingdom: the first 20 years.

OBJECTIVES: To audit services for prenatal diagnosis for haemoglobin disorders in the United Kingdom. DESIGN: Comparison of the annual number of cases recorded in a United Kingdom register of prenatal diagnoses for haemoglobin disorders, with the annual number of pregnancies at risk of these disorders, by ethnic group and regional health authority. The number of pregnancies at risk was estimated using data on ethnic group from the 1991 census and data from the United Kingdom thalassaemia register, which records the number of babies born with thalassaemia. SETTING: The three national prenatal diagnosis centres for haemoglobin disorders. SUBJECTS: 2068 cases of prenatal diagnosis for haemoglobin disorders in the United Kingdom from 1974 to 1994. MAIN OUTCOME MEASURES: Utilisation of prenatal diagnosis by risk, ethnic group, and regional health authority. Proportion of referrals in the first trimester and before the birth of any affected child. RESULTS: National utilisation of prenatal diagnosis for haemoglobin disorders was around 20%. During the past 10 years it has remained steady at about 50% for thalassaemias and risen from 7% to 13% for sickle cell disorders. Utilisation for sickle cell disorders varies regionally from 2% to 20%. Utilisation for thalassaemias varies by ethnic group. It is almost 90% for Cypriots and ranges regionally for British Pakistanis from 0% to over 60%. About 60% of first prenatal diagnoses are done for couples without an affected child. Less than 50% of first referrals are in the first trimester. CONCLUSIONS: National utilisation of prenatal diagnosis for haemoglobin disorders is far lower than expected, and there are wide regional variations. A high proportion of referrals are still in the second trimester and after the birth of an affected child. The findings point to serious shortcomings in present antenatal screening practice and in local screening policies and to inadequate counselling resources, especially for British Pakistanis.

The risk of preterm birth across generations.

OBJECTIVE: To examine the risk of preterm birth for mothers who themselves were born before term. METHODS: Data were taken from a linked data base of birth certificates composed of two cohorts: 1) a parental cohort of women born between 1947 and 1957 and 2) their offspring born between 1970 and 1992. "Preterm mothers" were women in the parental cohort who were born at less than 37 weeks' gestation. "Term mothers" were women in the parental cohort born at or after 38 weeks' gestation. Preterm mothers and term mothers were matched for birth year, county of birth, marital status, parity, and age. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated for the risk of preterm delivery in preterm mothers. Multiple logistic regression was used to assess the interaction of concomitant variables with the risk of premature delivery. RESULTS: The risk of preterm birth was significantly higher in preterm mothers than in term mothers (OR 1.18; 95% CI 1.02, 1.37). The risk increased as the gestational age at the mothers' birth decreased (less than 30 weeks'; OR 2.38; 95% CI 1.37, 4.16). The interaction between maternal age and parity increased the risk of preterm delivery at less than 34 weeks in some age and parity strata. CONCLUSION: An increased risk of preterm delivery exists for women who themselves were born before 37 weeks' gestation. The risk is inversely correlated with the maternal gestational age at birth and is influenced by maternal age and parity.

Association of genetic variations in apolipoprotein B with hypercholesterolemia, coronary artery disease, and receptor binding of low density lipoproteins.

To search for unique mutations in the apolipoprotein B (apoB) gene that disrupt the binding of LDL to its receptor and cause hypercholesterolemia, we examined more than 800 patients with high LDL cholesterol levels and/or coronary artery disease (CAD). Analysis of patient DNA by single-strand conformation polymorphism and allele-specific oligonucleotide hybridization of the sequence surrounding the putative receptor- binding domain of apoB (amino acid positions 2965 to 3534) revealed seven variations. LDL from heterozygotes with either Arg 3500 Gln or Arg 3531 Cys bound defectively with the LDL receptor in competitive binding assays. The Arg 3500 Gln substitution was statistically more prevalent in patients with hypercholesterolemia (P = 0.0003). Total cholesterol and LDL-cholesterol were significantly higher (P< 0.0004) in 34 apoB 3500 Gln carriers than in the controls. The allele encoding the Arg 3531 Cys substitution was more prevalent (0.8%) in the CAD group (P = 0.05) than in the controls. A Ser 3252 Gly variant was statistically more prevalent in the hypercholesterolemic group (P = 0.03), but LDL with this mutation had normal LDL receptor-binding activity. The other four variants identified (Leu 3350 Leu, Gln 3405 Glu, Val 3396 Met, and Ser 3455 Arg) were not associated with defective LDL-receptor binding, hypercholesterolemia, or CAD, nor were the apoB mutations associated with elevated lipid levels in family members. The surprising result that only two mutations of apoB in the receptor-binding domain (Arg 3500 Gln and Arg 3531 Cys) were associated with defective LDL binding, hypercholesterolemia, or CAD is in stark contrast with familial hypercholesterolemia, where nearly 150 mutations of the LDL receptor have been described that disrupt its function. This study strongly suggests that a limited number of mutations of apoB markedly influence LDL binding to its receptor.

Molecular systematics of short-horned lizards: biogeography and taxonomy of a widespread species complex.

We surveyed mitochondrial DNA (mtDNA) sequence variation in short-horned lizards (Phrynosoma douglasi) from throughout western North America and used these data to estimate an intraspecific phylogeny and to assess biogeographic scenarios underlying the geographic structure of lineages in this species. We sequenced 783 base pairs from 38 populations of P. douglasi and three putative outgroups (P. ditmarsi, P. orbiculare, P. platyrhinos). We detected high levels of nucleotide variation among populations and a spatial distribution of mtDNA lineages compatible with major geographic regions. The phylogenetic hypotheses best supported by the data suggest that P. douglasi, as currently described, is paraphyletic with respect to P. ditmarsi. Populations of P. douglasi from the Pacific Northwest (ID, CA, OR, WA) form a monophyletic group that is sister to the subsequent radiation of P. ditmarsi and other P. douglasi clades. These results suggest that divergences within this widespread species are fairly old. We focused on the genetic structure of populations of P. douglasi from a geographic perspective and interpreted the intraspecific phylogeny in light of geologic and climatic changes in western North America during the last 20 million years. The generally high levels of genetic variation found in these population comparisons are in accord with high levels of morphological variation in this species group; however, only in the Pacific Northwest region is there spatial congruence between these phylogenetic results and subspecific ranges based on previous morphological studies. We compared the evolutionary units delineated in this study with previously described subspecies of P. douglasi and evaluated the support (from morphology and mtDNA) for each population lineage in the phylogeny and the implications for the taxonomy of this group.

Prenatal detection of Hb mutations using transcervical cells.

Prenatal diagnoses were performed on six selected pairs of parents known to be carriers of Hb mutations by testing transcervical cells (TCCs) retrieved, prior to chorionic villus sampling (CVS), by aspiration of the cervical mucus from the pregnant mothers at 10-12 weeks of gestation. A concordance between the results of testing chorionic villus cells and isolated clumps of trophoblastic cellular elements was observed in four of the six cases.

Data linkage methods used in maternally-linked birth and infant death surveillance data sets from the United States (Georgia, Missouri, Utah and Washington State), Israel, Norway, Scotland and Western Australia.

In this paper we describe the methods used to link birth and infant mortality and morbidity surveillance data sets into sibships using deterministic or multistage probabilistic linkage methods. We describe nine linked data sets: four in the United States (Georgia, Missouri, Utah and Washington State), and four elsewhere (Scotland, Norway, Israel and Western Australia). Norway and Israel use deterministic methods to link births and deaths into sibships. The deterministic linkage is usually dependent on the availability of national identification numbers. In both countries they assign these numbers at birth. Deterministic linkage is usually highly successful, and the major problem is the validation of linkages. In the United States, Western Australia and UK linkage is multistage and probabilistic. This approach is usually dependent on the calculation linkage weights from sociodemographic variables. The success rates of probabilistic methods are above 80%. Maternally-linked perinatal data open new vistas for epidemiological research. Recurrence of poor perinatal outcomes is more appropriately studied using longitudinally-linked data sets. In addition, the emergence of risk factors and the recurrence of risk factors can be studied.

Comparison of sulfasalazine and placebo in the treatment of ankylosing spondylitis. A Department of Veterans Affairs Cooperative Study.

OBJECTIVE: To determine whether sulfasalazine (SSZ) at a dosage of 2,000 mg/day is effective for the treatment of active ankylosing spondylitis (AS) that is not controlled with nonsteroidal antiinflammatory drug therapy. METHODS: Two hundred sixty-four patients with AS were recruited from 15 clinics, randomized (double-blind) to SSZ or placebo treatment, and followed up for 36 weeks. Treatment response was based on morning stiffness, back pain, and physician and patient global assessments. RESULTS: While longitudinal analysis revealed a trend favoring SSZ in the middle of treatment, no difference was seen at the end of treatment. Response rates were 38.2% for SSZ and 36.1% for placebo (P = 0.73). The Westergren erythrocyte sedimentation rate declined more with SSZ treatment than with placebo (P < 0.0001). AS patients with associated peripheral arthritis showed improvement that favored SSZ (P = 0.02). Adverse reactions were fewer than expected and were mainly due to nonspecific gastrointestinal complaints. CONCLUSION: SSZ at a dosage of 2,000 mg/day does not seem to be more effective than placebo in the treatment of AS patients with chronic, longstanding disease. SSZ is well tolerated and may be more effective than placebo in the treatment of AS patients with peripheral joint involvement. This effect is more pronounced in treatment of the peripheral arthritis in this subgroup of AS patients.

Comparison of sulfasalazine and placebo in the treatment of psoriatic arthritis. A Department of Veterans Affairs Cooperative Study.

OBJECTIVE: To determine whether sulfasalazine (SSZ) at a dosage of 2,000 mg/day is effective for the treatment of active psoriatic arthritis (PsA) resistant to nonsteroidal antiinflammatory drug therapy. METHODS: Two hundred twenty-one patients with PsA were recruited from 15 clinics, randomized (double-blind) to SSZ or placebo treatment, and followed up for 36 weeks. Treatment response was based on joint pain/ tenderness and swelling scores and physician and patient global assessments. RESULTS: Longitudinal analysis revealed a trend favoring SSZ treatment (P = 0.13). At the end of treatment, response rates were 57.8% for SSZ compared with 44.6% for placebo (P = 0.05). The Westergren erythrocyte sedimentation rate declined more in the PsA patients taking SSZ than in those taking placebo (P < 0.0001). Adverse reactions were fewer than expected and were mainly due to nonspecific gastrointestinal complaints, including dyspepsia, nausea, vomiting, and diarrhea. CONCLUSION: SSZ at a dosage of 2,000 mg/day is well tolerated and may be more effective than placebo in the treatment of patients with PsA.

Comparison of sulfasalazine and placebo in the treatment of reactive arthritis (Reiter's syndrome). A Department of Veterans Affairs Cooperative Study.

OBJECTIVE: To determine whether sulfasalazine (SSZ) at a dosage of 2,000 mg/day is effective in the treatment of reactive arthritis (ReA) that has been unresponsive to nonsteroidal antiinflammatory drug (NSAID) therapy. METHODS: One hundred thirty-four patients with ReA who had failed to respond to NSAIDs were recruited from 19 clinics, randomized (double-blind) to receive either SSZ or placebo, and followed up for 36 weeks. The definition of treatment response was based on joint pain/tenderness and swelling scores and physician and patient global assessments. RESULTS: Longitudinal analysis revealed improvement in the patients taking SSZ compared with those taking placebo, which appeared at 4 weeks and continued through the trial (P = 0.02). At the end of treatment, response rates were 62.3% for SSZ treatment compared with 47.7% for placebo treatment. The Westergren erythrocyte sedimentation rate declined more with SSZ treatment than with placebo (P < 0.0001). Adverse reactions were fewer than expected and were mainly due to nonspecific gastrointestinal complaints. CONCLUSION: SSZ at a dosage of 2,000 mg/day is well tolerated and effective in patients with chronically active ReA.

Genetic variability in the tumor necrosis factor-lymphotoxin region influences susceptibility to rheumatoid arthritis.

The major histocompatibility complex class III tumor necrosis factor-lymphotoxin (TNF-LT) region (6p21.3) was investigated as a possible susceptibility locus for rheumatoid arthritis (RA). Inheritance of five TNF microsatellite markers was determined in 50 multiplex families. Overall, 47 different haplotypes were observed. One of these, the TNF a6, b5, c1, d3, e3 (H1) haplotype, was present in 35.3% of affected, but in only 20.5% of unaffected, individuals (P < .005). This haplotype accounted for 21.5% of the parental haplotypes transmitted to affected offspring and only 7.3% not transmitted to affected offspring (P = .0003). The TNF a6 and TNF c1 alleles were individually associated with RA (P = .0005 and .0008, respectively), as were the HLA-DRB1 "shared epitope" (SE) (P = .0001) and HLA-DRB1*0401 (P = .0018). Both univariate and bivariate conditional logistic regression analysis showed significant effects of TNF c1 and SE in increasing risk to RA (P < .001). Stratification by the presence of SE indicated an independent effect of the TNFc1 allele (P = .0003) and the HLA A1, B8, DR3 extended haplotype (always TNFa2, b3, c1, d1, e3) (P = .0027) in SE heterozygotes, while the H1 haplotype was associated with RA in SE homozygotes (P = .0018). The TNF-LT region appears to influence susceptibility to RA, distinct from HLA-DR.

The retrieval of ancient human DNA sequences.

DNA was extracted from approximately 600-year-old human remains found at an archaeological site in the southwestern United States, and mtDNA fragments were amplified by PCR. When these fragments were sequenced directly, multiple sequences seemed to be present. From three representative individuals, DNA fragments of different lengths were quantified and short overlapping amplification products cloned. When amplifications started from <40 molecules, clones contained several different sequences. In contrast, when they were initiated by a few thousand molecules, unambiguous and reproducible results were achieved. These results show that more experimental work than is often applied is necessary to ensure that DNA sequences amplified from ancient human remains are authentic. In particular, quantitation of the numbers of amplifiable molecules is a useful tool to determine the role of contaminating contemporary molecules and PCR errors in amplifications from ancient DNA.