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.

DNA microarray analysis for the detection of mutations in hemophilia A.

BACKGROUND: Congenital deficiency of factor (F) VIII results in the inherited X-linked bleeding disorder hemophilia A. More than 900 different mutations are reported in the hemophilia A mutation database with the largest number of mutations being single nucleotide substitutions distributed throughout the gene. Complicating the molecular characterization of this disease is the complexity of the F8 gene, the mutational heterogeneity, and technical limitations of the current mutation detection techniques. OBJECTIVE: Development of a DNA oligonucleotide microarray-based technique for F8 gene analysis to detect hemophilia A mutations. METHODS: To construct the oligonucleotide DNA microarray system: a total of 720, one base pair overlapping, 25-mer perfect match probes were designed from six exons of the F8 gene. Twenty-two different F8 gene mutations previously identified by CSGE and DNA sequence analysis were tested by using a loss-of-signal analysis approach. Differentially labeled wild type and hemophilic samples were co-hybridized to the array. Sequence alterations were detected by quantifying relative losses of test sample hybridization signals to the perfectly matched probes. RESULTS: A total of 22 different F8 mutations were tested. To test the sensitivity of the system, a blinded study was performed on 16 of the samples. F8 gene mutations can be detected with 96% efficiency with this microarray system. CONCLUSION: This proof-of-principle study has demonstrated that a F8 DNA microarray platform is an alternative gene mutation analysis approach that has a high sensitivity, and reproducibility. The methodology is, however, expensive and time consuming, and with the reduction in sequencing costs, direct sequencing is now the most cost and time efficient strategy for hemophilia A mutation analysis.

Genetic linkage and association analysis in type 1 von Willebrand disease: results from the Canadian type 1 VWD study.

BACKGROUND: von Willebrand disease (VWD) is the most common bleeding disorder known in humans, with type 1 VWD representing the majority of cases. Unlike the other variant forms of VWD, type 1 disease represents a complex genetic trait, influenced by both genetic and environmental factors. AIM: To evaluate the contribution of the von Willebrand factor (VWF) and ABO blood group loci to the type 1 VWD phenotype, and to assess the potential for locus heterogeneity in this condition, we have performed genetic linkage and association studies on a large, unselected type 1 VWD population. METHOD: We initially collected samples from 194 Canadian type 1 VWD families for analysis. After the exclusion of families found to have either type 2 or type 3 VWD, and pedigrees with samples from single generations, linkage and association analysis was performed on 155 type 1 VWD families. RESULTS AND CONCLUSION: The linkage study has shown a low heterogeneity LOD score of 2.13 with the proportion of families linked to the VWF gene estimated to be 0.41. Linkage was not detected to the ABO locus in this type 1 VWD population. In the family-based association test, significant association was found between the type 1 VWD phenotype, the quantitative traits, VWF:Ag, VWF:RCo, and FVIII:C and the ABO 'O' and 'A' alleles and the VWF codon 1584 variant. There was also weak association with the -1185 promoter polymorphism and VWF:Ag, VWF:RCo, and FVIII:C plasma levels. These studies provide further evidence to support the role for genetic loci other than VWF and ABO in the pathogenesis of type 1 VWD.

Homozygotes and heterozygotes for ciliary neurotrophic factor null alleles do not show earlier onset of Huntington's disease.

The CAG repeat number on Huntington's disease (HD) chromosomes accounts for about 60% of the variance in the age at onset of HD. However, distinct familial factors may also influence the age at onset. HD is associated with loss of medium-sized GABA-ergic striatal output neurons. Intracerebral administration of human ciliary neurotrophic factor (CNTF) protects striatal output neurons in excitotoxic rodent and primate models of HD induced by intrastriatal quinolinic acid injection. We have examined the effect of a common null mutation in the human CNTF gene on the age of onset of HD using a multiple regression approach that takes into account the CAG repeat number on HD chromosomes. We failed to detect an earlier onset of HD in nine homozygotes and 71 heterozygotes with this CNTF mutation compared with 203 homozygotes with wild-type alleles.

Patients with features similar to Huntington's disease, without CAG expansion in huntingtin.

OBJECTIVE: To describe characteristics of gene-negative patients with clinical features of Huntington's disease (HD), exploring likely etiologies. BACKGROUND: When a direct gene test became definitive for diagnosis of HD, we discovered a number of patients in our clinics in Baltimore, MD, and Cambridge, UK, believed or suspected to have HD who did not have the triplet repeat expansion. METHODS: Patients were examined using standardized instruments, and given full neurologic and psychiatric evaluations. Those negative for HD were tested for dentatorubro-pallidoluysian atrophy, SCA-1, SCA-3, SCA-2, SCA-6, and other conditions as indicated. RESULTS: Of 15 patients, 7 received specific diagnoses or appear to be sporadic cases, 4 have a possible but uncertain relation to HD, and 4 have unknown familial progressive movement disorders. CONCLUSIONS: This last group of patients might be properly described as phenocopies of HD, some of which may be caused by unidentified triplet repeat expansions.

Intensive exposure to factor VIII is a risk factor for inhibitor development in mild hemophilia A.

BACKGROUND: Inhibitors are rare in boys with mild hemophilia A (MHA; factor (F)VIII:C > 5%) but may arise following intense FVIII exposure, e.g. continuous infusion (CI). OBJECTIVES: To determine the impact of intense FVIII exposure in inhibitor formation in MHA at our institution and to compare this with previous reports. PATIENTS AND METHODS: We reviewed FVIII exposure and inhibitor development in boys (ages 0-18 years) with MHA followed at our institution from 1996 to 2001 and conducted a Medline search (1966-2002) on the experience of inhibitor development following intensive/CI exposure to FVIII. RESULTS: We identified 54 boys with MHA. Twenty-nine (54%) had been exposed to FVIII. Seven had received FVIII by CI. Four developed inhibitors; three high titer (at ages 10 years, 16 years and 17 years) and one low titer (at 1 month old). All four had received a CI of recombinant (r) FVIII of at least 6 days within 6 weeks of developing inhibitors. Baseline FVIII levels fell to < 1% in all cases and the three with high-titer inhibitors developed severe bleeding. Immune tolerance therapy (ITT) was attempted in two boys and was successful in one. Our literature search identified 35 cases (only four children) with MHA developing inhibitors following intense FVIII exposure often in the context of surgery. CONCLUSIONS: The incidence of inhibitors in our MHA population was 7.4%. If expressed according to exposure the incidence was significantly higher: 14% (4/29) for any exposure to FVIII and 57% (4/7) for exposure by CI. A prospective study to address whether CI is associated with an increased incidence of inhibitor development in MHA is warranted.

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 polyglutamine-coding repeats in the TATA-binding protein in different human populations and in patients with schizophrenia and bipolar affective disorder.

A new class of disease (including Huntington disease, Kennedy disease, and spinocerebellar ataxias types 1 and 3) results from abnormal expansions of CAG trinucleotides in the coding regions of genes. In all of these diseases the CAG repeats are thought to be translated into polyglutamine tracts. There is accumulating evidence arguing for CAG trinucleotide expansions as one of the causative disease mutations in schizophrenia and bipolar affective disorder. We and others believe that the TATA-binding protein (TBP) is an important candidate to investigate in these diseases as it contains a highly polymorphic stretch of glutamine codons, which are close to the threshold length where the polyglutamine tracts start to be associated with disease. Thus, we examined the lengths of this polyglutamine repeat in normal unrelated East Anglians, South African Blacks, sub-Saharan Africans mainly from Nigeria, and Asian Indians. We also examined 43 bipolar affective disorder patients and 65 schizophrenic patients. The range of polyglutamine tractlengths that we found in humans was from 26-42 codons. No patients with bipolar affective disorder and schizophrenia had abnormal expansions at this locus.

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.

The genetics of Canadian type 3 von Willebrand disease: further evidence for co-dominant inheritance of mutant alleles.

BACKGROUND: Type 3 von Willebrand disease (VWD) is the most severe form of the disease and is classically inherited in an autosomal recessive fashion. OBJECTIVES: The aim of the current study was to investigate the molecular pathogenesis of a Canadian cohort of type 3 VWD patients. PATIENTS AND METHODS: Thirty-four families comprised of 100 individuals were investigated. Phenotypic data, including bleeding scores (BS), von Willebrand factor (VWF) laboratory values and anti-VWF inhibitor status were included as well as sequence analysis. RESULTS: We identified 31 different mutations (20 novel): 8 frameshift, 5 splice site, 9 nonsense, 1 gene conversion, 6 missense and 2 partial gene deletion mutations. The majority of mutations identified were in the propeptide (42%); index cases (IC) with these mutations exhibited more severe bleeding (BS = 22) than those with mutations elsewhere in VWF (BS = 13). Sixty-two out of 68 (91%) mutant alleles were identified. Twenty-nine IC (85%) had a VWF null genotype identified; 17 homozygous, 12 compound heterozygous. In five IC (15%), two mutant VWF alleles were not identified to explain the type 3 VWD phenotype. In four ICs only one mutant VWF allele was identified and in one IC no mutant VWF alleles were identified. CONCLUSIONS: We have investigated the molecular pathogenesis of a Canadian cohort of type 3 VWD patients. Obligate carriers are not phenotypically silent in the Canadian population; 48% have been diagnosed with type 1 VWD. In approximately 50% of families in this study the inheritance pattern for type 3 VWD is co-dominant and not recessive.

Temporal and spatial variations in microclimate influence the larval foraging behaviors and performance of a conifer-feeding sawfly.

Herbivorous insects are often exposed to broad temporal and spatial variations in microclimate conditions within their host plants and have adapted a variety of behaviors, such as avoidance or basking, to either offset or benefit from such variation. Field experiments were carried out to investigate the influence of daily and intratree variations in microclimate on the behaviors (feeding, resting, dispersal, and hiding) and associated performance of late-instar larvae of the yellowheaded spruce sawfly, Pikonema alaskensis (Rohwer) (Hymenoptera: Tenthredinidae) within crowns of 1.25-1.5 m tall black spruce (Picea mariana [Miller] Britton Sterns Poggenburg); late instars feed on developing shoots of young spruce and are often exposed to microclimatic extremes with unknown effects on performance. Larvae fed diurnally from just after dawn (0800 h) until dusk (2000 h) and rested throughout the night, with brief periods of dispersal occurring in the morning and evening. Neither larval behavior nor abiotic conditions differed significantly between the upper and lower crowns of trees. Temperature, humidity, and solar insolation all explained >90% of variation in feeding; however, sunrise and sunset were the most likely cues influencing diurnal behavior. Most larvae (94%) fed on the bottom, shaded side of shoots, and field experiments indicated that this behavior is adaptive with respect to microclimate, probably reducing hygrothermal stress. Thus, behavioral adaptations by P. alaskensis to daily and within-shoot microclimatic variation may reduce the risk of hygrothermal stress during dispersal or feeding, while still allowing larvae to feed on the preferred and highly nutritious upper crown foliage of young spruce.