Novel variants in growth differentiation factor 9 in mothers of dizygotic twins

Context: Genes from the ovarian bone morphogenetic signaling pathway (GDF9 and BMP15) are critical for normal human fertility. We previously identified a deletion mutation in GDF9 in sisters with spontaneous dizygotic (DZ) twins, but the prevalence of rare GDF9 variants in twinning families is unknown. Objective: The objective was to evaluate the frequency of rare variants in GDF9 in families with a history of DZ twinning. Design and Subjects: We recruited 3450 individuals from 915 DZ twinning families (1693 mothers of twins) and 1512 controls of Caucasian origin. One mother of DZ twins was selected from 279 of the 915 families, and a DNA sample was screened for rare variants in GDF9 using denaturant HPLC. Variants were confirmed by DNA sequencing and genotyped in the entire sample by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. Results: We found two novel insertion/deletions (c.392-393insT, c.1268-1269delAA) and four missense alterations in the GDF9 sequence in mothers of twins. Two of the missense variants (c.307C > T, p.Pro103Ser and c.362C > T, p.Thr121Leu) were located in the proregion of GDF9 and two (c.1121C > T, p.Pro374Leu and c.1360C > T, p.Arg454Cys) in the mature protein region. For each variant, the frequencies were higher in cases compared with controls. The proportion of mothers of DZ twins carrying any variant (4.12%) was significantly higher (P < 0.0001) than the proportion of carriers in controls (2.29%). Conclusion: We describe new variants in the GDF9 gene that are significantly more common in mothers of DZ twins than controls, suggesting that rare GDF9 variants contribute to the likelihood of DZ twinning.

The refolding of different alpha-fetoprotein variants

The effect of glycosylation on AFP foldability was investigated by parallel quantitative and qualitative analyses of the refolding of glycosylated and nonglycosylated AFP variants. Both variants were successfully refolded by dialysis from the denatured-reduced state, attaining comparable ``refolded peak'' profiles and refolding yields as determined by reversed-phase HPLC analysis. Both refolded variants also showed comparable spectroscopic fingerprints to each other and to their native counterparts, as determined by circular dichroism spectroscopy. Inclusion body-derived AFP was also readily refolded via dilution under the same redox conditions as dialysis refolding, showing comparable circular dichroism fingerprints as native nonglycosylated AFP. Quantitative analyses of inclusion body-derived AFP showed sensitivity of AFP aggregation to proteinaceous and nonproteinaceous inclusion body contaminants, where refolding yields increased with increasing AFP purity. All of the refolded AFP variants showed positive responses in ELISA that corresponded with the attainment of a bioactive conformation. Contrary to previous reports that the denaturation of cord serum AFP is an irreversible process, these results clearly show the reversibility of AFP denaturation when refolded under a redox-controlled environment, which promotes correct oxidative disulfide shuffling. The successful refolding of inclusion body-derived AFP suggests that fatty acid binding may not be required for the attainment of a rigid AFP tertiary structure, contrary to earlier studies. The overall results from this work demonstrate that foldability of the AFP molecule from its denatured-reduced state is independent of its starting source, the presence or absence of glycosylation and fatty acids, and the refolding method used (dialysis or dilution).

EAAT2 splice variants in Alzheimer disease

Regional atrophy caused by neuronal loss is a characteristic of Alzheimer Disease (AD). Excitatory amino acid transporter-2 (EAAT2) is the major carrier responsible for clearing glutamate from the synaptic cleft in mammalian CNS. A localized attenuation of glutamate transport via reduced expression of functional forms of EAAT2 might contribute to regional excitotoxicity. The EAAT2 gene spans over 100 kb and encodes a 12-kb message. Several groups have identified alternative splice variants of EAAT2 in human brain tissue. These variants can affect transport by altering wild-type EAAT2 protein expression, localization, or transport efficiency. Alternative EAAT2 mRNA transcripts reportedly elicit a dominant-negative effect on glutamate uptake in cell culture. A 50% reduction in the expression in AD cortex of the truncated EAAT2 C-terminal isoform, EAAT2b, has been reported. We obtained cerebral cortex tissue, under informed written consent from the next of kin, from pathologically confirmed control, AD, and non-AD dementia cases. We aimed to determine the distribution and expression patterns of EAAT2 subtypes in susceptible and spared brain regions. We detected five alternate transcripts of EAAT2, two of which had not previously been reported. The relative contributions of novel variants, wild-type EAAT2, and previously discovered splice variants was investigated using Real-time PCR in AD, non-AD dementia, and age-matched control cortex. Our aim is to survey the relationship between these expression patterns and those of markers such as tau, GFAP, and b-amyloid, and to assess the correlation between variant-transporter expression and the level of cell loss.

Modelling Apoptotic cell clearance within the atherosclerotic plaque

The aged population have an increased susceptibility to infection, therefore function of the innate immune system may be impaired as we age. Macrophages, and their precursors monocytes, play an important role in host defence in the form of phagocytosis, and also link the innate and adaptive immune system via antigen presentation. Classically-activated 'M1' macrophages are pro-inflammatory, which can be induced by encountering pathogenic material or pro-inflammatory mediators. Alternatively activated 'M2' macrophages have a largely reparative role, including clearance of apoptotic bodies and debris from tissues. Despite some innate immune receptors being implicated in the clearance of apoptotic cells, the process has been observed to have a dominant anti-inflammatory phenotype with cytokines such as IL-10 and TGF-ß being implicated. The atherosclerotic plaque contains recruited monocytes and macrophages, and is a highly inflammatory environment despite high levels of apoptosis. At these sites, monocytes differentiate into macrophages and gorge on lipoproteins, resulting in formation of 'foam cells' which then undergo apoptosis, recruiting further monocytes. This project seeks to understand why, given high levels of apoptosis, the plaque is a pro-inflammatory environment. This phenomenon may be the result of the aged environment or an inability of foam cells to elicit an anti-inflammatory effect in response to dying cells. Here we demonstrate that lipoprotein treatment of macrophages in culture results in reduced capacity to clear apoptotic cells. The effect of lipoprotein treatment on apoptotic cell-mediated immune modulation of macrophage function is currently under study.

Reduced transferrin binding in Down syndrome:a route to senile plaque formation and dementia

Plasma transferrin binding in Down syndrome and Alzheimer's disease is significantly reduced compared with age matched controls and it was thought this may help elucidate a pathological time sequence for the onset of dementia in Down syndrome. In Down syndrome, there was a reduction in gallium and aluminium transferrin binding both with age and the onset of dementia. Non-transferrin bound gallium species were identified as non-transportable phosphate or silicate. Thus, the route of entry of metals into the brain must be via a transferrin mediated complex only. A clear sequence of pathological events has been demonstrated in Down syndrome which shows the pathway to development of plaques and dementia and this is believed to have an immunological origin.

Investigation of dyslexia and SLI risk variants in reading- and language-impaired subjects

Dyslexia (or reading disability) and specific language impairment (or SLI) are common childhood disorders that show considerable co-morbidity and diagnostic overlaps and have been suggested to share some genetic aetiology. Recently, genetic risk variants have been identified for SLI and dyslexia enabling the direct evaluation of possible shared genetic influences between these disorders. In this study we investigate the role of variants in these genes (namely MRPL19/C20RF3, ROBO1, DCDC2, KIAA0319, DYX1C1, CNTNAP2, ATP2C2 and CMIP) in the aetiology of SLI and dyslexia. We perform case–control and quantitative association analyses using measures of oral and written language skills in samples of SLI and dyslexic families and cases. We replicate association between KIAA0319 and DCDC2 and dyslexia and provide evidence to support a role for KIAA0319 in oral language ability. In addition, we find association between reading-related measures and variants in CNTNAP2 and CMIP in the SLI families.

Relationships between morphological types of plaque in Alzheimer’s disease as revealed in silver and immunostained preparations

The objective of this study was to determine the possible relationships between the morphological types of plaque revealed in silver and immunostained sections of Alzheimer’s disease (AD) tissue. The density of cored and uncored senile plaques in Glees and Marsland preparations, and of diffuse, primitive, classic and compact beta/A4 deposits in immunostained preparations were estimated. A principal components analysis (PCA) of the data suggested that three uncorrelated principal components accounted for 80% of the variation in lesion density in the tissues. This suggested that thee processes lead independently to the formation of: (1) the uncored Glees plaques; (2) the primitive beta/A4 deposits and most of the classic beta/A4 deposits and (3) the compact beta/A4 deposits and the remaining classic deposits. Hence, the uncored plaques revealed by the Glees stain and the primitive beta/A4 deposits represented distinct plaque populations. In addition, the classic beta/A4 deposits did not appear to represent a uniform plaque population but to originate from at least two pathological processes. The uncored Glees plaques appeared to the only plaque population closely related to the diffuse beta/A4 deposits.

The usefulness of spatial pattern analysis in understanding the pathogenesis of neurodegenerative disorders, with particular reference to plaque formation in Alzheimer's disease

Discrete, microscopic lesions are developed in the brain in a number of neurodegenerative diseases. These lesions may not be randomly distributed in the tissue but exhibit a spatial pattern, i.e., a departure from randomness towards regularlity or clustering. The spatial pattern of a lesion may reflect its development in relation to other brain lesions or to neuroanatomical structures. Hence, a study of spatial pattern may help to elucidate the pathogenesis of a lesion. A number of statistical methods can be used to study the spatial patterns of brain lesions. They range from simple tests of whether the distribution of a lesion departs from random to more complex methods which can detect clustering and the size, distribution and spacing of clusters. This paper reviews the uses and limitations of these methods as applied to neurodegenerative disorders, and in particular to senile plaque formation in Alzheimer's disease.