Francesca Blasi

A genomewide screen for autism: Strong evidence for linkage to chromosomes 2q, 7q, and 16p

Autism is characterized by impairments in reciprocal communication and social interaction and by repetitive and stereotyped patterns of activities and interests. Evidence for a strong underlying genetic predisposition comes from twin and family studies, although susceptibility genes have not yet been identified. A whole-genome screen for linkage, using 83 sib pairs with autism, has been completed, and 119 markers have been genotyped in 13 candidate regions in a further 69 sib pairs. The addition of new families and markers provides further support for previous reports of linkages on chromosomes 7q and 16p. Two new regions of linkage have also been identified on chromosomes 2q and 17q. The most significant finding was a multipoint maximum LOD score (MLS) of 3.74 at marker D2S2188 on chromosome 2; this MLS increased to 4.80 when only sib pairs fulfilling strict diagnostic criteria were included. The susceptibility region on chromosome 7 was the next most significant, generating a multipoint MLS of 3.20 at marker D7S477. Chromosome 16 generated a multipoint MLS of 2.93 at D16S3102, whereas chromosome 17 generated a multipoint MLS of 2.34 at HTTINT2. With the addition of new families, there was no increased allele sharing at a number of other loci originally showing some evidence of linkage. These results support the continuing collection of multiplex sib-pair families to identify autism-susceptibility genes.

Absence of coding mutations in the X-linked genes Neuroligin 3 and Neuroligin 4 in individuals with autism from the IMGSAC collection.

Neuroligin abnormalities have been recently implicated in the aetiology of autism spectrum disorders (ASD), given the finding of point mutations in the two X‐linked genes NLGN3 and NLGN4X and the important role of neuroligins in synaptogenesis. To enquire on the relevance and frequency of neuroligin mutations in ASD, we performed a mutation screening of NLGN3 and NLGN4X in a sample of 124 autism probands from the International Molecular Genetic Study of Autism Consortium (IMGSAC). We identified a new non‐synonymous variant in NLGN3 (Thr632Ala), which is likely to be a rare polymorphism. Our data indicate that coding mutations in these genes are very rarely associated to ASD.

Mutation screening and association analysis of six candidate genes for autism on chromosome 7q.

Genetic studies have provided evidence for an autism susceptibility locus (AUTS1) on chromosome 7q. Screening for mutations in six genes mapping to 7q, CUTL1, SRPK2, SYPL, LAMB1, NRCAM and PTPRZ1 in 48 unrelated individuals with autism led to the identification of several new coding variants in the genes CUTL1, LAMB1 and PTPRZ1. Analysis of genetic variants provided evidence for association with autism for one of the new missense changes identified in LAMB1; this effect was stronger in a subgroup of affected male sibling pair families, implying a possible specific sex-related effect for this variant. Association was also detected for several polymorphisms in the promoter and untranslated region of NRCAM, suggesting that alterations in expression of this gene may be linked to autism susceptibility.

SLC25A12 and CMYA3 gene variants are not associated with autism in the IMGSAC multiplex family sample

Autism is a severe neurodevelopmental disorder with a complex genetic predisposition. Linkage findings from several genome scans suggest the presence of an autism susceptibility locus on chromosome 2q24–q33, making this region the focus of candidate gene and association studies. Recently, significant association with autism has been reported for single-nucleotide polymorphisms (SNPs) in the SLC25A12 and CMYA3 genes on chromosome 2q. We attempted to replicate these findings in the collection of families from the International Molecular Genetic Study of Autism Consortium (IMGSAC), using the transmission disequilibrium test and case–control comparison. Our study failed to reveal any significant association for the SNPs tested at either locus, suggesting that these variants are unlikely to play a major role in genetic susceptibility to autism in our sample.

Analysis of X chromosome inactivation in autism spectrum disorders

Autism spectrum disorders (ASD) are complex genetic disorders more frequently observed in males. Skewed X chromosome inactivation (XCI) is observed in heterozygous females carrying gene mutations involved in several X‐linked syndromes. In this study, we aimed to estimate the role of X‐linked genes in ASD susceptibility by ascertaining the XCI pattern in a sample of 543 informative mothers of children with ASD and in a sample of 163 affected girls. The XCI pattern was also determined in two control groups (144 adult females and 40 young females) with a similar age distribution to the mothers sample and affected girls sample, respectively. We observed no significant excess of skewed XCI in families with ASD. Interestingly, two mothers and one girl carrying known mutations in X‐linked genes (NLGN3, ATRX, MECP2) showed highly skewed XCI, suggesting that ascertainment of XCI could reveal families with X‐linked mutations. Linkage analysis was carried out in the subgroup of multiplex families with skewed XCI (≥80:20) and a modest increased allele sharing was obtained in the Xq27‐Xq28 region, with a peak Z‐score of 1.75 close to rs719489. In summary, our results suggest that there is no major X‐linked gene subject to XCI and expressed in blood cells conferring susceptibility to ASD. However, the possibility that rare mutations in X‐linked genes could contribute to ASD cannot be excluded. We propose that the XCI profile could be a useful criteria to prioritize families for mutation screening of X‐linked candidate genes.

CONSTITUTIVE EXPRESSION OF INTERLEUKIN-1BETA (IL-1BETA ) IN RAT OLIGODENDROCYTES

Abstract The RT-PCR analysis of RNA from progenitor and differentiated primary rat oligodendrocytes, and from the oligodendrocyte CG-4 cell line, shows the presence of the IL-1β mRNA, the type I IL-1 receptor and the IL-1 receptor accessory protein in these cells. In situ hybridization of a rat IL-1β probe to primary progenitor and differentiated rat oligodendrocytes results in a positive signal. The double hybridization of the IL-1β probe, together with an oligodendrocyte-specific differentiation marker, to sections of postnatal rat brain at different stages of differentiation is also positive. The double immuno-labelling technique utilized indicates coincidence of the signals on the brain slices. The results show that IL-1β mRNA is constitutively expressed in rat brain oligodendrocytes from 1 day after birth onward. In agreement with this observation, CG-4 cells, primary progenitor and differentiated rat oligodendrocytes are positively stained by antibodies against IL-1β. Postnatal brain slices from 1 and 4 day old and adult rats, labelled with a double immunofluorescence technique, are also stained by antibodies against IL-1β. This signal coincides with that of antibodies against oligodendrocyte-specific surface markers. We conclude that IL-1β is constitutively expressed in rat brain progenitor and differentiated oligodendrocytes.

Stage-specific gene expression in early differentiating oligodendrocytes

The screening of a differential library from precursor and differentiated oligodendrocytes, obtained through the representational difference analysis (RDA) technique, has generated a number of cDNA recombinants corresponding to mRNA coding for known and unknown proteins: (1) mRNA coding for proteins involved in protein synthesis, (2) mRNA coding for proteins involved in the organization of the cytoskeleton, and (3) mRNA coding for proteins of unknown function. The expression profile of the mRNA was studied by Northern blot hybridization to the poly‐A+ mRNA from primary rat progenitor and differentiated oligodendrocytes. In most cases, hybridization to the precursor was higher than hybridization to the differentiated mRNA, supporting the validity of the differential screening. Hybridization of the cDNA to rat cerebral hemisphere and brain stem poly‐A+ mRNA, isolated from 1‐ to 90‐day‐old rats, confirms the results obtained with the mRNA from differentiating oligodendrocytes. The intensity of the hybridization bands decreases as differentiation proceeds. The pattern of expression observed in oligodendrocytes is different from that found in the brain only in the case of the nexin‐1 mRNA, the level of which remains essentially constant throughout differentiation both in the brain stem and in the cerebral hemispheres, in agreement with the published data. In contrast, the intensity of hybridization to the oligodendrocyte mRNA is dramatically lower in the differentiated cells compared with the progenitor oligodendrocyte cells. Some of the recombinant cDNA represent mRNA sequences present at high frequency distribution in the cells, while others belong to the rare sequences group. Six recombinants code for proteins of the ribosomal family, suggesting that of approximately 70 known ribosomal proteins, only a few are upregulated during oligodendrocyte differentiation. The third category of open reading frame (ORF) is represented by rare messengers coding for proteins of unknown functions and includes six clones: RDA 279, 11, 95, 96, 254, and 288. GLIA 39:114–123, 2002.