Sex determination by PCR analysis of DNA extracted from incinerated, deciduous teeth.

Establishing the biological sex of human remains is a very important part of identifying victims of fire when severe soft tissue destruction has occurred. Deciduous (children's) teeth were exposed to a range of incineration temperatures 100-500 degrees C for 15 minutes. Polymerase Chain Reaction (PCR) amplification was used to identify specific human amelogenin regions. There was successful identification of human biological sex, from deciduous teeth exposed to incineration temperatures of 200 degrees C and below, using standard ethidium bromide gel staining. There was greater sensitivity using fragment analysis by laser induced fluorescence which achieved sex identification from some teeth heated to 400 degrees C.

A glycine to aspartic acid substitution of COL2A1 in a family with the Strudwick variant of spondyloepimetaphyseal dysplasia.

BACKGROUND: Spondyloepimetaphyseal dysplasia (SEMD) is one of a clinically heterogeneous group of skeletal disorders, characterized by defective growth and modelling of the spine and long bones. Common clinical features include disproportionate short stature, malformed vertebrae and abnormal epiphyses or metaphyses. Some cases have been associated with mutations in the COL2A1 gene. AIM: To determine whether the autosomal dominant Strudwick-type SEMD in a three-generation family, showing specific phenotypical features such as chest deformity, limb shortening, myopia and early-onset degenerative osteoarthrosis, might be caused by a novel COL2A1 mutation. DESIGN: Genetic testing and clinical examination of family members. METHODS: Direct sequencing of PCR-amplified genomic DNA from the COL2A1 gene. RESULTS: A point mutation within exon 20 of the COL2A1 gene was identified that substituted a glycine for an aspartic acid residue at codon 262. DISCUSSION: All previously reported autosomal dominant mutations causing SEMD have substituted an obligate glycine within the triple helix, in particular at codons 292, 304 and 709 in the three reported Strudwick-type patients. Additionally, a recurrent glycine substitution at codon 154 has been identified in two unrelated Finnish cases with radiological features consistent with the Strudwick subtype. Our sixth helical glycine substitution extends the mutational spectrum and genotype/phenotype correlations of Strudwick-type SEMD.

Vitreoretinopathy with phalangeal epiphyseal dysplasia, a type II collagenopathy resulting from a novel mutation in the C-propeptide region of the molecule.

A large family with dominantly inherited rhegmatogenous retinal detachment, premature arthropathy, and development of phalangeal epiphyseal dysplasia, resulting in brachydactyly was linked to COL2A1, the gene encoding proalpha1(II) collagen. Mutational analysis of the gene by exon sequencing identified a novel mutation in the C-propeptide region of the molecule. The glycine to aspartic acid change occurred in a region that is highly conserved in all fibrillar collagen molecules. The resulting phenotype does not fit easily into pre-existing subgroups of the type II collagenopathies, which includes spondyloepiphyseal dysplasia, and the Kniest, Strudwick, and Stickler dysplasias.

Pathology of early-onset Alzheimer's disease cases bearing the Thr113-114ins presenilin-1 mutation.

Most cases of familial presenile Alzheimer's disease are caused by mutations in the presenilin-1 (PSEN-1) gene, most of these mutations being missense mutations. A mutation in the splice donor site of intron 4 of PSEN-1 has been described recently which results in aberrant splicing of PSEN-1 mRNA, causing insertion of an additional amino acid, Thr113-114ins, into the protein. We studied the neuropathology of four cases bearing this mutation in an attempt to clarify the pathology of this hereditary form of Alzheimer's disease and to determine whether it differs from other familial forms of the disease. The disease presented as a progressive cognitive decline, myoclonus and seizures developing later in the disease, a feature common to PSEN-1-linked Alzheimer's disease. The course of the disease was relatively rapid, death occurring approximately 6 years after onset. Pathology in the intron 4 cases demonstrated a severe Alzheimer's disease pathology with abundant deposition of ss-amyloid (Ass) 1-42 senile plaques and the formation of neurofibrillary tangles. Amyloid angiopathy was present in these cases and was readily demonstrated by Ass 1-40 staining, particularly in the cerebellum. Cases with the intron 4 mutation appear clinically and pathologically similar to other cases of early-onset Alzheimer's disease bearing PSEN-1 mutations.

Aberrant splicing in the presenilin-1 intron 4 mutation causes presenile Alzheimer's disease by increased Abeta42 secretion.

We previously described a splice donor site mutation in intron 4 of presenilin-1 (PSEN1) in two patients with autopsy-confirmed early-onset Alzheimer's disease (AD). Here we provide evidence that the intron 4 mutation is present in four additional unrelated early-onset AD cases, that the mutation segregates in an autosomal dominant manner and that all cases have one common ancestor. We demonstrate that the intron 4 mutation produces three different transcripts, two deletion transcripts (Delta4 and Delta4cryptic) and one insertion transcript (insTAC), by aberrant splicing. The deletion transcripts result in the formation of C-truncated (approximately 7 kDa) PSEN1 proteins while the insertion transcript produces a full-length PSEN1 with one extra amino acid (Thr) inserted between codons 113 and 114 (PSEN1 T113-114ins). The truncated proteins were not detectable in vivo in brain homogenates or lymphoblast lysates of mutation carriers. In vitro HEK-293 cells overexpressing Delta4, Delta4cryptic or insTACPSEN1 cDNAs showed increased Abeta42 secretion (approximately 3.4 times) only for the insertion cDNA construct. Increased Abeta42 production was also observed in brain homogenates. Our data indicate that in the case of intron 4 mutation, the AD pathophysiology results from the presence of the PSEN1 T113-114ins protein comparable with cases carrying dominant PSEN1 missense mutations.

Apo E genotypes and risk of dementia in Down syndrome.

The relationship between apo E genotypes and risk of dementia in Down syndrome is not clear. Accordingly, we have analysed this locus in 20 demented and 25 nondemented individuals with Down syndrome and combined these data with other studies in a meta-analysis. The meta-analysis revealed an estimated odds ratio for dementia of 2.74 (95% CI 1.34-5.58) (P =.0004) for apo epsilon4 carriers compared with apo epsilon3/epsilon3, similar to that observed in late-onset Alzheimer's disease. An additional parallel with late-onset Alzheimer's disease was shown by the apo epsilon2 allele, which was associated with decreased dementia risk in Down syndrome (odds ratio for apo epsilon2/epsilon2 + epsilon2/epsilon3 = 0.37 (95% CI 0.14-0. 96)). Thus, apo E genotypes are associated with similar risk effects in Down syndrome dementia and late-onset Alzheimer's disease.

Apo E and Apo CI loci are associated with dementia in younger but not older late-onset cases.

Numerous groups have confirmed that apolipoprotein E allelic variation accounts for a proportion of the genetic risk for late-onset Alzheimer's disease (AD). However, there is a paucity of data on the impact of this locus on the overall risk of dementia (as opposed to AD) in the elderly. Most studies have ascertained specifically AD cases from hospital clinics or brain banks and many demented cases have vascular dementia or mixed AD and vascular pathology. We have examined the closely linked apo E and apo CI loci in demented cases and non-demented controls from two community-based aged Cambridgeshire populations: the rural Ely population (cohort 1) comprised 60 pairs of demented and non-demented elderly individuals, with a mean age of 84.2 years (SD = 6.11); the Cambridge city population (cohort 2) comprised 81 pairs all aged over 84 with a mean age of 87.7 years (SD = 2.9). The younger Ely cohort showed significant allelic associations with dementia at the apo E and apo CI loci, which were not replicated in the older Cambridge cohort. These data suggest the possibility of age-dependent penetrance for different candidate genes in late-onset dementia. We propose a number of explanations to account for the stronger associations we observed between dementia and apo CI, compared to the neighbouring apo E locus. Our data are compatible with the possibility that specific alleles or genotypes may confer different risks for overall dementia, compared to AD.

A presenilin-1 truncating mutation is present in two cases with autopsy-confirmed early-onset Alzheimer disease.

We have examined genomic DNA from 40 cases of autopsy-confirmed early-onset Alzheimer disease (EOAD) (age at onset <=65 years) that were all unselected for family history. We have sequenced the 10 exons and flanking intronic sequences of the presenilin-1 (PS-1) gene for all 40 individuals. A single mutation, a deletion of a G from the intron 4 splice-donor consensus sequence, was detected in two individuals in this study. The mutation was associated with two shortened transcripts, both with shifted reading frames resulting in premature-termination codons. All the PS-1 mutations described elsewhere have been missense or in-frame splice mutations, and recent data suggest that these result in disease by gain-of-function or dominant-negative mechanisms. The mutation that we have identified is likely to result in haploinsufficiency and would be most consistent with other mutations acting in a dominant-negative manner. However, we cannot exclude the possibility that the small amounts of truncated transcripts exert a gain of function. Since no other mutations or polymorphisms were detected in our patients, mutations in the coding regions and splice consensus sequences of PS-1 are likely to be rare in EOAD cases unselected for family history.

Analysis of alpha-1 antichymotrypsin, presenilin-1, angiotensin-converting enzyme, and methylenetetrahydrofolate reductase loci as candidates for dementia.

The genetic factors which predispose individuals to dementia in old age have not been fully defined. Although the apolipoprotein E4 allele accounts for a proportion of the genetic risk for late-onset Alzheimer disease (AD), it is neither necessary nor sufficient to cause this disease. Recent suggestions that other loci are involved in dementia risk have been supported by findings of associations of genotypes at the alpha-1 antichymotrypsin (ACT) and presenilin-1 (PS-1) loci with AD. We investigated these loci in two community-based aged Cambridgeshire populations: the rural Ely population (cohort 1) comprised 60 pairs of demented and nondemented elderly individuals, with a mean age of 84.2 years; and the Cambridge city population (cohort 2) comprised 81 pairs all over age 84, with a mean age of 87.3 years. Since vascular risk factors are likely to impact on dementia risk, we also examined the angiotensin-converting enzyme (ACE) and methylenetetrahydrofolate reductase (MTHFR) genes as candidates. ACE, ACT, PS-1, and MTHFR genotype and allele frequencies were not significantly different in cases and matched controls. These data support the doubts which have been raised about the involvement of the PS-1 and ACT polymorphisms in late-onset dementia.

Analysis of the apo E/apo C-I, angiotensin converting enzyme and methylenetetrahydrofolate reductase genes as candidates affecting human longevity.

Genetic factors are likely to affect human survival, since twin studies have shown greater concordance for age of death in monozygotic compared to dizygotic twins. Coronary artery disease is an important contributor to premature mortality in the UK. Accordingly, we have chosen genes associated with cardiovascular risk, apo E/apo C-I, angiotensin converting enzyme (ACE) and methylenetetrahydrofolate reductase (MTHFR), as candidates which may affect longevity/survival into old age. An association study was performed by comparing allele and genotype frequencies at polymorphic loci associated with these genes in 182 women and 100 men aged 84 years and older with 100 boys and 100 girls younger than 17 years. MTHFR allele and genotype frequencies were similar in the elderly and young populations. Apo C-I allele and genotype frequencies were significantly different in the elderly women compared to the younger sample (P < 0.05). No difference was observed in the elderly men. At the neighbouring apo E gene, we only observed a difference between genotypes in the elderly women and the young sample; however, this did not retain significance when the genotype frequencies of the young sample were adjusted to values expected from the allele frequencies on the basis of Hardy-Weinberg equilibrium and compared to observed genotypes in elderly men and women. In contrast to previous studies, apo E2 was not overrepresented in the elderly men or women. Thus, the proposition that apo E2, E3 and E4 protein isoforms are themselves functionally associated with increasing risks for early death, may be too simplistic. The I/I ACE was depleted in the elderly males but not the elderly females. Furthermore, significant differences were observed between ACE genotypes in elderly men and elderly women. These data suggest that the penetrance of loci which influence survival may vary according to sex. The depletion of the ACE I/I genotype in elderly men is generally consistent with a previous study which found decreased frequencies of the I allele in French centenarians compared to younger controls. However, these results are apparently paradoxical, since others have suggested that the I allele is associated with increased cardiovascular risk. Clarification of the overall effect of a genotype on survival will be vital if therapies are to be considered which target specific genetic variants.

Presenilin-1 intron 8 polymorphism is not associated with autopsy-confirmed late-onset Alzheimer's disease.

Mutations in the presenilin-1 (PS-1) gene may account for the majority of familial early-onset Alzheimer's disease (EOAD) cases. However, there is controversy as to whether the bi-allelic intron 8 PS-1 polymorphism plays a role in late-onset AD (LOAD). As previous association studies with this polymorphism have all investigated clinically diagnosed LOAD cases, we have analysed the frequency of the PS-1 intronic polymorphism in a series of autopsy-confirmed early- (n = 54) and late-onset (n = 199) cases and a large control population of non-demented, aged individuals (n = 215). Our sample size should have had the power to reveal effects of the size previously reported for the PS-1 polymorphism, but we detected no significant increase in the 1/1 risk genotype distribution in EOAD or LOAD cases. Thus, we have been unable to find an association between the PS-1 intronic polymorphism and early- or late-onset AD within this autopsy-confirmed population.

The tRNA(Gln) 4336 mitochondrial DNA variant is not a high penetrance mutation which predisposes to dementia before the age of 75 years.

The genetic factors that predispose to Alzheimer's disease (AD) are heterogeneous. Two recent reports have suggested that a mitochondrial DNA mutation within the tRNAGln gene, located at position 4336, may be a risk factor for AD, as it was found in 10/256 (3.9%) cases with AD confirmed by necropsy. Although low prevalences of this mutation were detected in non-demented subjects in both of these studies, the controls were not carefully matched with the AD cases. We have investigated the frequency of this mutation in two community based elderly cohorts in Cambridgeshire, who have participated in longitudinal studies of cognitive function. The 4336 mitochondrial mutation was detected in 8/ 443 people examined. These people were found to be non-demented at ages 74, 81, 84, 86, 89, 90, 91, and 102 years, in contrast to the previously described cases whose onset of dementia occurred between 60 and 76 years (mean 68). Accordingly, we believe that this mitochondrial variant is not a high penetrance mutation which predisposes to dementia before the age of 76 years.

Bias in mean frequency estimation of Doppler signals due to wall clutter filters.

Wall clutter filters used in Doppler ultrasound systems give rise to a bias in the mean frequency estimation. This effect has been explored for two second order filters: the initialised infinite impulse response (IIR)-type filter, and the finite impulse response (FIR) echo-canceller filter. These are used in conjunction with two mean frequency estimation algorithms: the FFT-based intensity-weighted-mean algorithm, and the autocorrelation algorithm. The bias is shown to be caused by the finite settling times of the filters. The IIR filter, although having a much better frequency response than the FIR filter, has a greater bias associated with it. A compromise between bias and frequency response is suggested and a simple method of bias correction is described.

Human cytomegalovirus infection of the monocyte/macrophage lineage in bone marrow.

Peripheral blood monocytes (PBM) are one site of persistence of human cytomegalovirus (HCMV) in healthy carriers. However, because PBM circulate only briefly before entering the tissues and are difficult to infect with HCMV, it has been suggested that they may acquire HCMV during development in the bone marrow. Consistent with this, we show evidence that bone marrow progenitors from healthy HCMV carriers contain endogenous HCMV DNA as detected by PCR. We also show that bone marrow precursors are readily infected by clinical isolates of HCMV in vitro but that no viral gene expression occurs until these cells become differentiated. In contrast, incubation of these cells at any developmental stage with the laboratory strain AD169 resulted in few cells expressing viral immediate-early genes, and this correlated with a lack of entry of AD169 virus. These observations are consistent with bone marrow progenitors acting as a reservoir for HCMV and transmitting the viral genome to PBM, in the absence of lytic-gene expression, until they leave the circulation and undergo tissue-specific differentiation to macrophages.