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.

Different clinical phenotype in triplets with haemophilia A.

Although many patients with haemophilia may have exactly the same residual clotting factor level, the clinical disease phenotype may vary greatly. This variation may be related to different genetic mutations responsible for haemophilia, environmental influences and co-inheritance of polymorphisms affecting the coagulation system. The study of siblings with haemophilia offers the opportunity to examine additional factors, other than genetic mutation and environment that may impact on the clinical phenotype of haemophilia. We present the unusual case of haemophilia occurring in fraternal triplets. Each of the triplets had a slightly different pattern of bleeding and response to treatment.

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.

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.

Distribution of FMR1 and FMR2 alleles in Javanese individuals with developmental disability and confirmation of a specific AGG-interruption pattern in Asian populations.

The number of trinucleotide repeats in the 5' untranslated regions of the FMR1 and FMR2 genes was determined by PCR in 254 Fragile XA-negative Javanese male children with developmental disabilities. The distribution of FMR1 and FMR2 trinucleotide repeat alleles was found to be significantly different in the Indonesian population with developmental disability compared to that in developmentally disabled populations in North America and Europe (p & 0.021). Sequence analysis was performed on the trinucleotide repeat arrays of the 27 individuals with FMR1 alleles in the 'grey zone' (35-54 repeats). A repeat array structure of 9A9A6A9 was found in 16 unrelated individuals with 36 repeats, confirming earlier observations in intellectually normal Japanese. We propose that this FMR1 array pattern is specific for Asian populations and that Javanese and Japanese populations arose from a single progenitor population.

Genetic diversity at the FMR1 locus in the Indonesian population.

We report an analysis of allelic diversity at short tandem repeat polymorphisms within the fragile XA locus in 1069 male volunteers from twelve Indonesian sub-populations. An odd numbered allele of DXS548 was found at high frequency in all Indonesian populations. Greater allelic diversity was identified at the loci under study than has been previously reported for an Asian population. These differences distinguish the Indonesian population from all previously reported Asian, European and African populations. A high frequency of small premutation alleles, 4/120 (3.3%, 95% CI 0.9-8.3%), was identified in the Moluccan population of Hiri Island.

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.

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.

Dentatorubral and pallidoluysian atrophy (DRPLA). Clinical and neuropathological findings in genetically confirmed North American and European pedigrees.

Dentatorubral and pallidoluysian atrophy (DRPLA) is an autosomal dominant disorder that clinically overlaps with Huntington's disease (HD) and manifests combinations of chorea, myoclonus, seizures, ataxia, and dementia. DRPLA is caused by a CAG triplet repeat (CTG-B37) expansion coding for polyglutamine on chromosome 12 and exhibits the genetic phenomenon of anticipation. This neurodegenerative disease has only rarely been reported in non-Japanese pedigrees, and there are only a few neuropathological studies in genetically confirmed patients. We report 10 cases of DRPLA from two North American and two British pedigrees in which CTG-B37 expansions have been demonstrated within each kindred (54-83 repeats), individually in 8 of the 10 cases, and describe the neuropathological findings in 4 cases. Members of DRPLA kindreds have a wide range of clinical phenotypes and markedly variable ages at onset. The neuropathological spectrum is centered around the cerebellifugal and pallidofugal systems, but neurodegenerative changes can be found in many nuclei, tracts, and systems. Evidence of CTG-B37 triplet repeat expansion should be sought in HD-like cases that are negative for expanded triplet repeats within the HD IT15 gene or in autopsy cases with degeneration of the dentatorubral or pallidoluysian systems.

Analysis of the 5' upstream sequence of the Huntington's disease (HD) gene shows six new rare alleles which are unrelated to the age at onset of HD.

The CAG repeat number in the Huntington's disease (HD) gene accounts for about 50% of the variation seen in age at onset of HD. In order to determine whether promoter sequence variation can contribute to the residual variation in age at onset, we studied the conserved 303 bp region upstream of the +1 translation start site in the HD gene in a population of 56 control East Anglians, 30 Africans, 34 Japanese, and 208 English Huntington's disease patients. A surprisingly high degree of variation was found. Seven alleles were identified, comprising four polymorphisms: two single base pair substitutions, a 6 bp VNTR present as one or two copies, and a 20 bp VNTR with one to three copies of the tandem repeat. No correlation between polymorphisms and age at onset of symptoms was found in HD patients. The 6 bp and 20 bp stretches are present only in single copies in the chimpanzees and gorilla, suggesting that these VNTRs have evolved by duplication of the core sequences in the human lineage.

Genotypes at the GluR6 kainate receptor locus are associated with variation in the age of onset of Huntington disease.

Huntington disease (HD) is associated with abnormal expansions of a CAG repeat close to the 5' end of the IT15 gene. We have assembled a set of 293 HD subjects whose ages of onset were known and sized their HD CAG repeats. These repeats accounted for 69% of the variance of age of onset when we used the most parsimonious model, which relates the logarithm of age of onset to a function of CAG repeat number. Since other familial factors have been proposed to influence the age of onset of HD, we have examined a number of candidate loci. The CAG repeat number on normal chromosomes, the delta2642 polymorphism in the HD gene, and apolipoprotein E genotypes did not affect the age of onset of HD. Although mitochondrial energy production defects in HD have led to suggestions that variants in the mitochondrial genome may be associated with clinical variability in HD, this suggestion was not supported by our preliminary experiments that examined the DdeI mitochondrial restriction fragment length polymorphism at position 10,394. Excitotoxicity has been a favored mechanism to explain the cell death in HD, particularly since intrastriatal injection of excitatory amino acids in animals creates HD-like pathology. Accordingly, we investigated the GluR6 kainate receptor. Of the variance in the age of onset of HD that was not accounted for by the CAG repeats, 13% could be attributed to GluR6 genotype variation. These data implicate GluR6-mediated excitotoxicity in the pathogenesis of HD and highlight the potential importance of this process in other polyglutamine repeat expansion diseases.

Non-Mendelian transmission at the Machado-Joseph disease locus in normal females: preferential transmission of alleles with smaller CAG repeats.

Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3, is a neurodegenerative disorder which is associated with a CAG repeat expansion in the MJD1 gene on chromosome 14q32.1. A recent study reported an excess of transmission of disease chromosomes relative to normal chromosomes from affected fathers, while this phenomenon was not observed in female meioses. These data were compatible with meiotic drive. We investigated the transmission of alleles with larger versus smaller CAG repeat numbers in the MJD1 gene in normal heterozygotes from the 40 CEPH families. Our data suggest that there was no segregation distortion in male meioses, while the smaller CAG allele was inherited in 57% of female meioses (p < 0.016). The pattern of inheritance of smaller versus larger CAG alleles at this locus was significantly different when male and female meioses were compared (p = 0.0139). While previous data suggest that meiotic drive may be a feature of certain human diseases, including the trinucleotide diseases MJD, myotonic dystrophy, and dentatorubral-pallidoluysian atrophy, these data are compatible with meiotic drive also occurring among non-disease associated CAG sizes.

Analysis of spinocerebellar ataxia types 1, 2, 3, and 6, dentatorubral-pallidoluysian atrophy, and Friedreich's ataxia genes in spinocerebellar ataxia patients in the UK.

Accurate clinical diagnosis of the spinocerebellar ataxias (SCAs) can be difficult because of overlap in phenotype with other disorders and variation in clinical manifestations. Six SCA loci have been mapped and four disease causing genes identified, in addition to the causative gene for Friedreich's ataxia (FA). All of the identified mutations are expansions of trinucleotide repeat tracts. The SCA2 and SCA6 genes were published recently. The extent of the normal CAG size ranges at these loci and the relative frequencies of the known causes of SCA in the UK are not known. This study first investigated the normal size ranges of the SCA2 and SCA6 loci by genotyping control populations of West African and South African subjects, since African populations generally show the greatest allelic diversity. We found one allele larger than the previously determined normal range for SCA2, and our results at the SCA6 locus agreed with the previously reported normal range. The second component of the study assessed the relative frequencies of the SCA1, 2, 3, and 6, DRPLA, and FA trinucleotide repeat mutations in 146 patients presenting with SCA-like symptoms referred to genetic diagnostic laboratories in the UK. We detected mutations in 14% of patients referred with a diagnosis of autosomal dominant SCA, and in 15% of patients referred with spinocerebellar ataxia where we did not have sufficient family history data available to allow categorisation as familial or sporadic cases. Friedreich's ataxia accounted for 3% of the latter category of cases in our sample, but the most common causes of SCA were SCA2 and SCA6.

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.