CD33 rs2455069 SNP: Correlation with Alzheimer's Disease and Hypothesis of Functional Role.

The CD33 gene encodes for a member of the sialic-acid-binding immunoglobulin-type lectin (Siglec) family, and is one of the top-ranked Alzheimer's disease (AD) risk genes identified by genome-wide association studies (GWAS). Many CD33 polymorphisms are associated with an increased risk of AD, but the function and potential mechanism of many CD33 single-nucleotide polymorphisms (SNPs) in promoting AD have yet to be elucidated. We recently identified the CD33 SNP rs2455069-A>G (R69G) in a familial form of dementia. Here, we demonstrate an association between the G allele of the rs2455069 gene variant and the presence of AD in a cohort of 195 patients from southern Italy. We carried out in silico analysis of the 3D structures of CD33 carrying the identified SNP to provide insights into its functional effect. Structural models of the CD33 variant carrying the R69G amino acid change were compared to the CD33 wild type, and used for the docking analysis using sialic acid as the ligand. Our analysis demonstrated that the CD33-R69G variant may bind sialic acid at additional binding sites compared to the wild type, thus potentially increasing its affinity/specificity for this molecule. Our results led to a new hypothesis of rs2455069-A>G SNP as a risk factor for AD, suggesting that a long-term cumulative effect of the CD33-R69G variant results from the binding of sialic acid, acting as an enhancer of the CD33 inhibitory effects on amyloid plaque degradation.

LINC00473 as an Immediate Early Gene under the Control of the EGR1 Transcription Factor.

Immediate early genes play an essential role in cellular responses to different stimuli. Many of them are transcription factors that regulate the secondary response gene expression. Non-coding RNAs may also be involved in this regulatory cascade. In fact, they are emerging as key actors of gene expression regulation, and evidence suggests that their dysregulation may underly pathological states. We previously took a snapshot of both coding and long non-coding RNAs differentially expressed in neuronal cells after brain-derived neurotrophic factor stimulation. Among these, the transcription factor EGR1 (a well-known immediate early gene) and LINC00473 (a primate-specific long non-coding RNA) that has emerged as an interesting RNA candidate involved in neuronal function and in cancer. In this work, we demonstrated that LINC00473 gene expression kinetics resembled that of immediate early genes in SH-SY5Y and HEK293T cells under different cell stimulation conditions. Moreover, we showed that the expression of LINC00473 is under the control of the transcription factor EGR1, providing evidence for an interesting functional relationship in neuron function.

CD33 and SIGLECL1 Immunoglobulin Superfamily Involved in Dementia.

Sialic acid-binding immunoglobulin-type lectins, which are predominantly expressed in immune cells, represent a family of immunomodulatory receptors with inhibitory and activating signals, in both healthy and disease states. Genetic factors are important in all forms of dementia, especially in early onset dementia. CD33 was recently recognized as a genetic risk factor for Alzheimer disease (AD). Here, we present a 2-generation family with 4 members, the father and the 3 siblings, characterized by an early form of unusual dementia exhibiting a behavioral variant close to behavioral variant frontotemporal dementia phenotype and severe forms of memory loss suggestive of AD. We analyzed the CD33 gene in this family and identified 10 single nucleotide polymorphisms (SNPs) in a linkage disequilibrium block associated with the disease. We also identified a tag SNP, rs2455069-A>G, in CD33 exon 2 that could be involved with dementia risk. Additionally, we excluded the presence of C9orf72 expansion mutations and other mutations previously associated with sporadic FTD and AD. The tag SNP association was also analyzed in selected sporadic AD patients from the same Southern Italy region. We demonstrate that CD33 and SIGLECL1 have a significantly increased level of expression in these patients.

Clinical and Molecular Characterization of a Novel Progranulin Deletion Associated with Different Phenotypes.

BACKGROUND: Mutations in the GRN gene are causative for an autosomal dominant form of frontotemporal dementia. OBJECTIVE/METHODS: The objective of the present study is to describe clinical and molecular features of three siblings harboring the GRN deletion NM_002087.3:c.295_308delTGCCCACGGGGCTT, p.(Cys99Profs*15) identified with next generation sequencing. RESULTS: Our patients demonstrated heterogeneous clinical phenotypes, such as progressive supranuclear palsy-like in the proband and the behavioral variant of frontotemporal dementia in the two affected siblings. Progranulin haploinsufficiency was revealed by both gene expression and protein analyses. CONCLUSION: The pathogenicity of the novel GRN deletion c.295_308del TGCCCACGGGGCTT is confirmed by both functional analysis and segregation in three affected siblings.

Identification, Characterization, and Regulatory Mechanisms of a Novel EGR1 Splicing Isoform.

EGR1 is a transcription factor expressed in many cell types that regulates genes involved in different biological processes including growth, proliferation, and apoptosis. Dysregulation of EGR1 expression has been associated with many pathological conditions such as tumors and brain diseases. Known molecular mechanisms underlying the control of EGR1 function include regulation of transcription, mRNA and protein stability, and post-translational modifications. Here we describe the identification of a splicing isoform for the human EGR1 gene. The newly identified splicing transcript encodes a shorter protein compared to the canonical EGR1. This isoform lacks a region belonging to the N-terminal activation domain and although it is capable of entering the nucleus, it is unable to activate transcription fully relative to the canonical isoform.

Circulating levels of IL-1 family cytokines and receptors in Alzheimer's disease: new markers of disease progression?

BACKGROUND: Although the mechanisms underlying AD neurodegeneration are not fully understood, it is now recognised that inflammation could play a crucial role in the initiation and progression of AD neurodegeneration. A neuro-inflammatory network, based on the anomalous activation of microglial cells, includes the production of a number of inflammatory cytokines both locally and systemically. These may serve as diagnostic markers or therapeutic targets for AD neurodegeneration. METHODS: We have measured the levels of the inflammation-related cytokines and receptors of the IL-1 family in serum of subjects with AD, compared to mild cognitive impairment (MCI), subjective memory complaints (SMC), and normal healthy subjects (NHS). Using a custom-made multiplex ELISA array, we examined ten factors of the IL-1 family, the inflammation-related cytokines IL-1α, IL-1ß, IL-18, and IL-33, the natural inhibitors IL-1Ra and IL-18BP, and the soluble receptors sIL-1R1, sIL-1R2, sIL-1R3, and sIL-1R4. RESULTS: The inflammatory cytokines IL-1α and IL-1ß, their antagonist IL-1Ra, and their soluble receptor sIL-1R1 were increased in AD. The decoy IL-1 receptor sIL-1R2 was only increased in MCI. IL-33 and its soluble receptor sIL-1R4 were also significantly higher in AD. The soluble form of the accessory receptor for both IL-1 and IL-33 receptor complexes, sIL-1R3, was increased in SMC and even more in AD. Total IL-18 levels were unchanged, whereas the inhibitor IL-18BP was significantly reduced in MCI and SMC, and highly increased in AD. The levels of free IL-18 were significantly higher in MCI. CONCLUSIONS: AD is characterised by a significant alteration in the circulating levels of the cytokines and receptors of the IL-1 family. The elevation of sIL-1R4 in AD is in agreement with findings in other diseases and can be considered a marker of ongoing inflammation. Increased levels of IL-1Ra, sIL-1R1, sIL-1R4, and IL-18BP distinguished AD from MCI and SMC, and from other inflammatory diseases. Importantly, sIL-1R1, sIL-1R3, sIL-1R4, and IL-18BP negatively correlated with cognitive impairment. A significant elevation of circulating sIL-1R2 and free IL-18, not present in SMC, is characteristic of MCI and disappears in AD, making them additional interesting markers for evaluating progression from MCI to AD.

GRN deletion in familial frontotemporal dementia showing association with clinical variability in 3 familial cases.

Progranulin (GRN) gene mutations have been genetically associated with frontotemporal dementia (FTD) and are present in about 23% of patients with familial FTD. However, the neurobiology of this secreted glycoprotein remains unclear. Here, we report the identification of 3 pedigrees of Southern Italian extraction in whom FTD segregates with autosomal dominant inheritance patterns. We present evidence that all the available patients in these 3 familial cases are carrying the rare GRN gene exon 6 deletion g10325_10331delCTGCTGT (relative to nt 1 inNG_007886.1), alias Cys157LysfsX97. This mutation was previously described in 2 sporadic cases but was never associated with familial cases. Our patients demonstrate heterogeneous clinical phenotypes, such as the behavioral variant (bvFTD) in the affected men and the nonfluent/agrammatic variant of primary progressive aphasia (nfvPPA) in the affected woman. Haploinsufficiency was revealed by both quantitative real-time PCR of the gene and protein analyses. These findings provide further support for a previously proposed role for the GRN gene in the genetic etiology of FTD and its phenotypic variability.

Imidazole-stabilized gold nanoparticles induce neuronal apoptosis: an in vitro and in vivo study.

Gold nanoparticles are increasingly being employed in innovative biological applications thanks to their advantages of material- and size-dependent physics and chemical interactions with the cellular systems. On the other hand, growing concern has emerged on the toxicity which would render gold-based nanoparticles harmful to cell cultures, animals, and humans. Emerging attention is focused on the interaction of gold nanoparticles with nervous system, especially regarding the ability to overcome the blood-brain barrier (BBB) which represents the major impediment to the delivery of therapeutics into the brain. We synthesized highly stable 2-mercapto-1-methylimidazole-stabilized gold-nanoparticles (AuNPs)-mmi to investigate their entry, accumulation, and toxicity in vitro (SH-SY5Y human neuroblastoma cells) and in vivo (brain of C57BL/6 mice) through optical and electron microscopy. After incubation in the cell culture medium at the lowest dose of 0.1 mg/mL the (AuNPs)-mmi nanoparticles were found compacted and recruited into endosome/lysosomes (1 h) before their fusion (2 h) and the onset of neuronal death by apoptosis (4 h) as proved by terminal-transferase-mediated dUTP nick end labeling assay and caspase-3 immunoreactivity. The ability of (AuNPs)-mmi to cross the BBB was assessed by injection in the caudal vein of C57BL/6 mice. Among different brain regions, the nanoparticles were found in the CaudatoPutamen area, mainly in the striatal neurons 4 h after injection. These neurons showed the typical hallmarks of apoptosis. Our findings provide, for the first time, the dynamic of 2-mercapto-1-methylimidazole nanogold uptake. The molecular mechanism which underlies the nanogold-driven apoptotic event is analyzed and discussed in order to take into account when designing nanomaterials to interface with biological structures.

Donor splice-site mutation in CUL4B is likely cause of X-linked intellectual disability.

X-linked intellectual disability is the most common form of cognitive disability in males. Syndromic intellectual disability encompasses cognitive deficits with other medical and behavioral manifestations. Recently, a large family with a novel form of syndromic X-linked intellectual disability was characterized. Eight of 24 members of the family are male and had cognitive dysfunction, short stature, aphasia, skeletal abnormalities, and minor anomalies. To identify the causative gene(s), we performed exome sequencing in three affected boys, both parents, and an unaffected sister. We identified a haplotype consisting of eight variants located in cis within the linkage region that segregated with affected members in the family. Of these variants, two were novel. The first was at the splice-donor site of intron 7 (c.974+1G>T) in the cullin-RING ubiquitin ligase (E3) gene, CUL4B. This variant is predicted to result in failure to splice and remove intron 7 from the primary transcript. The second variant mapped to the 3'-UTR region of the KAISO gene (c.1127T>G). Sanger sequencing validated the variants in these relatives as well as in three affected males and five carriers. The KAISO gene variant was predicted to create a binding site for the microRNAs miR-4999 and miR-4774; however, luciferase expression assays failed to validate increased targeting of these miRNAs to the variant 3'-UTR. This SNP may affect 3'-UTR structure leading to decreased mRNA stability. Our results suggest that the intellectual disability phenotype in this family is caused by aberrant splicing and removal of intron 7 from CUL4B gene primary transcript.

Cortical metabolic deficits in a rat model of cholinergic basal forebrain degeneration.

Evidence indicates that the degeneration of basal forebrain cholinergic neurons may represent an important factor underlying the progressive cognitive decline characterizing Alzheimer's disease (AD). However, the nature of the relationship between cholinergic depletion and AD is not fully elucidated. This study aimed at clarifying some aspects of the relation existing between deficits in cerebral energy metabolism and degeneration of cholinergic system in AD, by investigating the neuronal metabolic activity of several cortical areas after depletion of basal forebrain cholinergic neurons. In cholinergically depleted rats, we evaluated the neuronal metabolic activity by assaying cytochrome oxidase (CO) activity in frontal, parietal and posterior parietal cortices at four different time-points after unilateral injection of 192 IgG-saporin in the nucleus basalis magnocellularis. Unilateral depletion of cholinergic cells in the basal forebrain induced a bilateral decrease of metabolic activity in all the analyzed areas. Frontal and parietal cortices showed decreased metabolic activity even 3 days after the lesion, when the cholinergic degeneration was still incomplete. In posterior parietal cortex metabolic activity decreased only 7 days after the lesion. The possible molecular mechanisms underlying these findings were also investigated. Real-time PCR showed an increase of CO mRNA levels at 3, 7 and 15 days after the lesion both in frontal and parietal cortices, followed by normalization at 30 days. Western Blot analysis did not show any change in CO protein levels at any time-point after the lesion. Our findings support a link between metabolic deficit and cholinergic hypofunctionality characterizing AD pathology. The present model of cholinergic hypofunctionality provides a useful means to study the complex mechanisms linking two fundamental and interrelated phenomena characterizing AD from the early stages.

Structural chromosomal variations in neurological diseases.

BACKGROUND: Significant advancement in the identification of genetic mutations and molecular pathways underlying Mendelian neurologic disorders was accomplished by using the methods of linkage, gene cloning, sequencing, mutation, and functional analyses, in the 1990s. Subsequently, the Human Genome Project defined the entire sequence of the genome providing reference for any pathologic condition, and identified single nucleotide polymorphisms as a means for whole genome association studies and linkage disequilibrium mapping in common, complex trait diseases. Simultaneously, data also emerged describing the structural chromosomal variations, and it became increasingly recognized that in addition to the more traditional mutation types, gene copy number variations (CNVs) contribute to normal variability in human phenotypes and may underlie the development of diseases with Mendelian inheritance, complex trait, or sporadic presentation. REVIEW SUMMARY: Here we describe the occurrence of CNVs in the human genome, and discuss their importance in health and disease. Targeting the practicing neurologist, we review the presently known CNVs with pathogenic significance in common neurologic disorders, and highlight new research directions in complex trait diseases. CONCLUSIONS: The role of chromosomal structural variations in the pathogenesis of neurologic disorders is increasingly recognized. Available data may only capture a small subgroup of conditions related to these recently discovered genetic variations. The ongoing genome studies are expected to reveal structural chromosomal alterations as an underlying cause of many as yet poorly understood common diseases. New challenges include defining chromosomal break points, evaluating biologic consequences of gene dosage effect, and using molecular genetics for personalized therapeutic intervention. This survey of the CNV literature was closed in September 2008.

Variants of ST8SIA1 are associated with risk of developing multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system of unknown etiology with both genetic and environmental factors playing a role in susceptibility. To date, the HLA DR15/DQ6 haplotype within the major histocompatibility complex on chromosome 6p, is the strongest genetic risk factor associated with MS susceptibility. Additional alleles of IL7 and IL2 have been identified as risk factors for MS with small effect. Here we present two independent studies supporting an allelic association of MS with polymorphisms in the ST8SIA1 gene, located on chromosome 12p12 and encoding ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1. The initial association was made in a single three-generation family where a single-nucleotide polymorphism (SNP) rs4762896, was segregating together with HLA DR15/DQ6 in MS patients. A study of 274 family trios (affected child and both unaffected parents) from Australia validated the association of ST8SIA1 in individuals with MS, showing transmission disequilibrium of the paternal alleles for three additional SNPs, namely rs704219, rs2041906, and rs1558793, with p = 0.001, p = 0.01 and p = 0.01 respectively. These findings implicate ST8SIA1 as a possible novel susceptibility gene for MS.

Highly variable penetrance in subjects affected with cavernous cerebral angiomas (CCM) carrying novel CCM1 and CCM2 mutations.

Cavernous vascular malformations may affect brain and out-of-brain tissues. In most cases, cerebral cavernous malformations (CCMs) involve the brain alone, and are rarely associated with skin hemangiomas, spinal cord, retinal, hepatic or vertebral lesions. CCMs can cause seizures, intracranial and spinal haemorrhages, focal neurological deficits, and migraine-like headaches. After collecting CCM families of Italian origin and investigating the genetic basis of the disorder we disclosed two novel molecular variations in the KRIT1 and MGC4607 genes. We found a novel CCM1 gene mutation (Q66X) in a family with apparently asymptomatic old-aged mutation carriers and patients who either had skin angiomas alone or the full association of cerebral, spinal, and skin lesions. In this family we report the highest variability in mutation penetrance so far described, including the presence of CCM in one subject since birth (surgery at 19 months of age), a condition to our knowledge so far unreported. In a CCM2 affected family, we also report a novel causative mutation, (54_55delAC) in exon 2 of the MGC4607 gene, that produces a truncated protein containing only 22 amino acids. These data describe novel CCM mutations associated with a particularly high variability of the penetrance causing, in some cases, reduced expression of clinical symptoms and sporadic cases with apparent negative family history. Hence they emphasize the importance of DNA-based diagnostics and genetic counseling to identify unaffected mutation carriers subjects, even at advanced age.

Arvanil and anandamide up-regulate CD36 expression in human peripheral blood mononuclear cells.

In this study we analysed the regulation of gene expression by arvanil and anandamide in human peripheral blood mononuclear cells (PBMCs) to clarify their immunosuppressive properties. PBMCs were activated, leading to CD36 down regulation, that was normalized by arvanil and anandamide. We used microarray technology to identify a regulatory pattern associated with cell proliferation in the presence of both substances. CD3-CD28 stimulated PBMCs showed a pattern of up-regulated and down-regulated genes after treatment with these substances. We selected and analysed several genes chosen by their function in the regulation of cell proliferation. We showed a transcriptional control of the CD36 gene by arvanil and anandamide associated with an increased protein expression, thus suggesting a possible role of CD36 in anandamide and arvanil anti-inflammatory pattern.

A novel frameshift mutation of FOXC2 gene in a family with hereditary lymphedema-distichiasis syndrome associated with renal disease and diabetes mellitus.

Lymphedema-distichiasis (LD) syndrome is a clinically variable autosomal dominant disorder. The disorder is caused by mutations in the forkhead transcription factor FOXC2 gene on chromosome band 16q24.3. Here, we report the sequence of the FOXC2 gene in a German-Irish family with LD in six affected relatives over three generations and identify a single adenine base pair insertion at nt 1006--1007. This insertion creates a frameshift mutation that predicts a premature stop at codon 462. In addition to LD, four of the affected family members have renal disease and three have diabetes mellitus (DM), not usually seen in the LD syndrome. Polymorphisms of FOXC2 in diabetics have been studied in different populations. Our sequence analysis of the 5' untranslated region (UTR) C-512T shows the homozygous T allele in all family members tested. The sequencing data in this family suggests the possibility of a novel phenotype-haplotype. This novel phenotype, LD/renal disease/type 2 diabetes, might be the result of a combination of the nt 1006--1007 insA and the upstream UTR homozygous T polymorphism.

Second family with hearing impairment linked to 19q13 and refined DFNA4 localisation.

Until now, over 30 loci have been identified by linkage analysis of affected families that segregate non-syndromic and dominantly inherited forms of hearing impairment (DFNA). A German family with a non-syndromic progressive hearing impairment transmitted in autosomal dominant mode was linked to 19q13.3-q13.4 by a genome-wide scan. Due to the low lod-score (1.85 at theta=0.05) for APOC2-locus we extended the fine mapping attempt with further markers in the same chromosomal region. This resulted in significant evidence for linkage to the markers D19S246 and D19S553 (two-point lod-score of 4.05 and 3.55 at theta=0.0) and a candidate critical region of 14 cM between markers D19S412 and D19S571. This region shows partial overlap with the previously reported DFNA4 critical region. The human gene BAX is orthologous to the rodent Bcl2-related apoptosis gene that is temporally expressed during the postnatal period in the developing inner ear of the mouse. BAX, mapping at a distance of no more than 0.73 cM distally to marker D19S553 appeared a likely candidate in our pedigree but genomic sequencing of coding regions and exon/intron boundaries excluded disease-related mutations. However, additional ESTs in the same region remain to be tested.

Linkage analysis conditional on HLA status in a large North American pedigree supports the presence of a multiple sclerosis susceptibility locus on chromosome 12p12.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system with a probable immune-mediated pathogenesis. Strong evidence supports the hypothesis that MS is determined by genetic and environmental factors, but these factors remain largely undefined. The genetic component is suggested by a higher concordance rate in monozygotic (28%) versus dizygotic (5%) twins as well as familial recurrence risk. Several studies have shown association of MS with the histocompatibility leukocyte antigen (HLA) class II region, specifically DR15, DQ6. However, there is no convincing evidence of a common susceptibility locus. We have identified a pedigree of Pennsylvania Dutch extraction, in which MS segregates with an autosomal dominant inheritance pattern. We have collected blood samples from 18 family members, seven of whom show typical signs of MS lesions by magnetic resonance imaging. The 18 individuals were serotyped for HLA class I and II and analyzed by a genome-wide screen for linkage analysis. We have found evidence for suggestive linkage to markers on 12p12 with a maximum multipoint LOD score of 2.71, conditional on the presence of DR15, DQ6. Contingency table analysis showed that all MS affected individuals have both the DR15, DQ6 allele and the 12p12 haplotype whereas the unaffected individuals have either one or neither of these markers (P = 0.00011). Our data suggests that both HLA DR15, DQ6 and a novel locus on chromosome 12p12 may be necessary for development of MS in this family.