Genome-wide linkage in Utah autism pedigrees.

Genetic studies of autism over the past decade suggest a complex landscape of multiple genes. In the face of this heterogeneity, studies that include large extended pedigrees may offer valuable insights, as the relatively few susceptibility genes within single large families may be more easily discerned. This genome-wide screen of 70 families includes 20 large extended pedigrees of 6-9 generations, 6 moderate-sized families of 4-5 generations and 44 smaller families of 2-3 generations. The Center for Inherited Disease Research (CIDR) provided genotyping using the Illumina Linkage Panel 12, a 6K single-nucleotide polymorphism (SNP) platform. Results from 192 subjects with an autism spectrum disorder (ASD) and 461 of their relatives revealed genome-wide significance on chromosome 15q, with three possibly distinct peaks: 15q13.1-q14 (heterogeneity LOD (HLOD)=4.09 at 29 459 872 bp); 15q14-q21.1 (HLOD=3.59 at 36 837 208 bp); and 15q21.1-q22.2 (HLOD=5.31 at 55 629 733 bp). Two of these peaks replicate earlier findings. There were additional suggestive results on chromosomes 2p25.3-p24.1 (HLOD=1.87), 7q31.31-q32.3 (HLOD=1.97) and 13q12.11-q12.3 (HLOD=1.93). Affected subjects in families supporting the linkage peaks found in this study did not reveal strong evidence for distinct phenotypic subgroups.

A high-density SNP genome-wide linkage scan in a large autism extended pedigree.

We performed a high-density, single nucleotide polymorphism (SNP), genome-wide scan on a six-generation pedigree from Utah with seven affected males, diagnosed with autism spectrum disorder. Using a two-stage linkage design, we first performed a nonparametric analysis on the entire genome using a 10K SNP chip to identify potential regions of interest. To confirm potentially interesting regions, we eliminated SNPs in high linkage disequilibrium (LD) using a principal components analysis (PCA) method and repeated the linkage results. Three regions met genome-wide significance criteria after controlling for LD: 3q13.2-q13.31 (nonparametric linkage (NPL), 5.58), 3q26.31-q27.3 (NPL, 4.85) and 20q11.21-q13.12 (NPL, 5.56). Two regions met suggestive criteria for significance 7p14.1-p11.22 (NPL, 3.18) and 9p24.3 (NPL, 3.44). All five chromosomal regions are consistent with other published findings. Haplotype sharing results showed that five of the affected subjects shared more than a single chromosomal region of interest with other affected subjects. Although no common autism susceptibility genes were found for all seven autism cases, these results suggest that multiple genetic loci within these regions may contribute to the autism phenotype in this family, and further follow-up of these chromosomal regions is warranted.

Absence of linkage and linkage disequilibrium to chromosome 15q11-q13 markers in 139 multiplex families with autism.

Chromosomal region 15q11-q13 has been implicated to harbor a susceptibility gene or genes underlying autism. Evidence has been derived from the existence of cytogenetic anomalies in this region associated with autism, and the report of linkage in a modest collection of multiplex families. Most recently, linkage disequilibrium with the marker GABRB3-155CA2 in the candidate locus GABRB3, located in this region, has been reported. We searched for linkage using eight microsatellite markers located in this region of chromosome 15 in 147 affected sib-pairs from 139 multiplex autism families. We also tested for linkage disequilibrium in the same set of families with the same markers. We found no evidence for excess allele sharing (linkage) for the markers in this region. Also, we found no evidence of linkage disequilibrium, including for the locus GABRB3-155CA2. Thus, it appears that the role of this region of chromosome 15 is minor, at best, in the majority of individuals with autism.

A genomic screen of autism: evidence for a multilocus etiology.

We have conducted a genome screen of autism, by linkage analysis in an initial set of 90 multiplex sibships, with parents, containing 97 independent affected sib pairs (ASPs), with follow-up in 49 additional multiplex sibships, containing 50 ASPs. In total, 519 markers were genotyped, including 362 for the initial screen, and an additional 157 were genotyped in the follow-up. As a control, we also included in the analysis unaffected sibs, which provided 51 discordant sib pairs (DSPs) for the initial screen and 29 for the follow-up. In the initial phase of the work, we observed increased identity by descent (IBD) in the ASPs (sharing of 51.6%) compared with the DSPs (sharing of 50.8%). The excess sharing in the ASPs could not be attributed to the effect of a small number of loci but, rather, was due to the modest increase in the entire distribution of IBD. These results are most compatible with a model specifying a large number of loci (perhaps >/=15) and are less compatible with models specifying

Exclusion of linkage to the HLA region in ninety multiplex sibships with autism.

Several studies have suggested a role for the histocompatibility complex of loci (HLA) in the genetic susceptibility to autism. We have tested this hypothesis by linkage analysis using genetic marker loci in the HLA region on chromosome 6p in multiplex families with autism. We have examined sharing of alleles identical by descent in 97 affected sib pairs from 90 families. Results demonstrate no deviation from the null expectation of 50% sharing of alleles in this region; in fact, for most marker loci, the observed sharing was less than 50%. Thus, it is unlikely that loci in this region contribute to the genetic etiology of autism to any significant extent in our families.

Autism and the X chromosome. Multipoint sib-pair analysis.

BACKGROUND: Genetic factors undoubtedly play a major etiologic role in autism, but how it is inherited remains unanswered. The increased incidence in males suggests possible involvement of the X chromosome. METHODS: Using data from 38 multiplex families with autism (2 or more autistic siblings), we performed a multipoint sib-pair linkage analysis between autism and 35 microsatellite markers located on the X chromosome. The model included a single parameter, the risk ratio lambda xs (i.e., ratio of risk to siblings compared with the population prevalence), owing to an X-linked gene. Different lambda xs values were assumed and regions of exclusion were established. RESULTS: The entire X chromosome could be excluded for a lambda xs value of 4. The ability to exclude an X-linked gene decreased with smaller lambda xs values, and some positive evidence was obtained with smaller values. A maximum lod score of 1.24 was obtained at locus DXS424 with a lambda xs value of 1.5. CONCLUSIONS: We were able to exclude any moderate to strong gene effect causing autism on the X chromosome. Smaller gene effects (lambda xs < 4) could not be excluded, in particular, a gene of small effect located between DXS453 and DXS1001.

Male-to-male transmission in extended pedigrees with multiple cases of autism.

Despite strong genetic influences in autism, the true mode of inheritance remains unknown. Sex differences in autism have been described in both singleton and multiplex families [Lord et al., 1982; Volkmar et al., 1993; McLennan et al., 1993; Lord, 1992]: Boys outnumber girls by 3 or 4 to 1, and so a sex-linked mode of transmission must also be considered. The key characteristic of X-linkage is that all sons of affected men are unaffected (no male-to-male transmission). In the present study, which is part of an ongoing linkage project in autism, we describe 77 multiplex autism families, 11 of who are affected cousin or half-sibling families. By using these families, it is possible to trace the path of genetic transmission and observe whether the hypothesis of X-linkage is tenable. Of 11 extended pedigrees from 77 multiplex families, six show male-to-male transmission; in these families, X-linkage can be excluded as the genetic basis for their autism. The data from the other five families are compatible with either an autosomal or an X-linked mode of transmission. The key point to emerge, then, is that autism cannot be exclusively an X-linked disorder; there must be an autosomal mode of transmission at least in some families. Thus we must consider the alternative hypotheses that autism is either entirely autosomal, or it is genetically heterogeneous, involving at least one autosomal locus with genderspecific expression, as well as a possible locus on the X-chromosome.

Molecular analysis and test of linkage between the FMR-1 gene and infantile autism in multiplex families.

Approximately 2%-5% of autistic children show cytogenetic evidence of the fragile X syndrome. This report tests whether infantile autism in multiplex autism families arises from an unusual manifestion of the fragile X syndrome. This could arise either by expansion of the (CGG)n trinucleotide repeat in FMR-1 or from a mutation elsewhere in the gene. We studied 35 families that met stringent criteria for multiplex autism. Amplification of the trinucleotide repeat and analysis of methylation status were performed in 79 autistic children and in 31 of their unaffected siblings, by Southern blot analysis. No examples of amplified repeats were seen in the autistic or control children or in their parents or grandparents. We next examined the hypothesis that there was a mutation elsewhere in the FMR-1 gene, by linkage analysis in 32 of these families. We tested four different dominant models and a recessive model. Linkage to FMR-1 could be excluded (lod score between -24 and -62) in all models by using probes DXS548, FRAXAC1, and FRAXAC2 and the CGG repeat itself. Tests for heterogeneity in this sample were negative, and the occurrence of positive lod scores in this data set could be attributed to chance. Analysis of the data by the affected-sib method also did not show evidence for linkage of any marker to autism. These results enable us to reject the hypothesis that multiplex autism arises from expansion of the (CGG)n trinucleotide repeat in FMR-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetics of autism: characteristics of affected and unaffected children from 37 multiplex families.

Evidence from twin and family studies strongly suggests that genetic factors play a prominent role in the etiology of some cases of infantile autism. Genetic factors would be expected to be especially strong in families with multiple autistic members (multiplex families). This report describes the identification and evaluation of 44 families with two or more autistic children collected as part of a genetic linkage study in autism. Families were referred with a presumptive classification of multiplex autism. Children referred as autistic, as well as their presumptively normal siblings, were assessed using the Autism Diagnostic Interview (ADI) and the Autism Diagnostic Observation Scale (ADOS). Thirty-seven of the 44 families (87%) had at least two children who met diagnostic criteria for autism on the ADI. Of the total group of 117 children evaluated in those families, 83 (71%) met all ADI criteria and could be unambiguously classified as autistic (affected), 26 (22%) met none of the ADI criteria and were classified as not autistic (unaffected), and 8 (7%) were classified as uncertain because they met one or more but not all of the ADI cutpoints. Autistic siblings were not significantly concordant for most autism characteristics, for IQ, or for verbal ability. Significant concordances were found, however, for behaviors related to rituals and repetitive play, and for social impairments in the expression and understanding of facial expressions of emotion.(ABSTRACT TRUNCATED AT 250 WORDS)

Possible association of the extended MHC haplotype B44-SC30-DR4 with autism.

We previously reported that the complement C4B null allele appears to be associated with infantile autism. Since the C4B null allele is known to be part of the extended or ancestral haplotype [B44-SC30-DR4], we investigated the incidence of [B44-SC30-DR4] in 21 autistic children and their parents. This extended haplotype was increased by almost six-fold in the autistic subjects as compared with healthy controls. Moreover, the total number of extended haplotypes expressed on chromosomes of autistic subjects was significantly increased as compared with those expressed on chromosomes of healthy subjects. We conclude that a gene related to, or included in, the extended major histocompatibility complex may be associated with autism.

Complex segregation analysis of autism.

A complex segregation analysis of autism in 185 Utah families was carried out using the mixed model. The 209 affected individuals in these families represent nearly complete ascertainment of the autistic cases born in Utah between 1965 and 1984. The sibling recurrence risk for autism was 4.5% (95% confidence limits 2.8%-6.2%). Likelihoods were maximized for major-gene models, a polygenic model, a sibling-effect model, and a mixed model consisting of major-gene and shared-sibling effects. The analysis provided no evidence for major-locus inheritance of autism. Subdivision of the sample according to the probands' IQ levels showed that sibling recurrence risk did not vary consistently with IQ level. A segregation analysis of families in which the proband had an IQ less than 50 also failed to provide evidence for a major locus. However, because of the etiologic heterogeneity of this disorder, genetic analysis of other meaningful subsets of families could prove informative.

Increased frequency of the null allele at the complement C4b locus in autism.

Associations between C4 deficiency and autoimmune disorders have been found over the past several years. Since autism has several autoimmune features, the frequencies of null (no protein produced) alleles at the C4A and C4B loci were studied in 19 subjects with autism and their family members. The autistic subjects and their mothers had significantly increased phenotypic frequencies of the C4B null allele (58% in both the autistic subjects and mothers, compared with 27% in control subjects). The siblings of the autistic subjects also had an increased frequency of the C4B null allele, but this increase was not significant. The fathers had normal frequencies of this null allele. All family members had normal frequencies of the C4A null allele, all normal C4A and C4B alleles and all BF and C2 alleles.

The UCLA-University of Utah epidemiologic survey of autism: the etiologic role of rare diseases.

Twelve rare diseases known to cause CNS pathology were found in 26 (11%) of 233 autistic probands identified during a recent epidemiologic survey of Utah. These 26 probands had significantly lower mean IQs than the remaining patients (43 versus 60) but similar sex distribution and prevalence of abnormal EEGs and seizures. The rarity and diversity of these 12 diseases make it highly unlikely that they randomly occurred with autism. Their presence in this epidemiologic survey is the most compelling evidence to date to support the hypothesis that different diseases producing different types of CNS pathology can play an etiologic role in autism.

Detection of maternal antibodies in infantile autism.

Maternal antibodies reactive with antigenic proteins expressed on the cell surface of paternal lymphocytes can be detected in couples with histories of more than one miscarriage or stillbirth. It is possible, but not proven, that these antibodies also react with tissues of the fetus and result in fetal death. Since many mothers of autistic children have a history of pregnancy disorder, antibodies were studied in 11 mothers of autistic children who were 6 years of age or younger. Six of the mothers had antibodies that reacted with lymphocytes of the autistic child. Five of these six mothers had a history of pregnancy disorder. Since antigens expressed on lymphocytes are found on cells of the central nervous system and, perhaps, other tissues of the developing embryo, it is suggested that aberrant maternal immunity may be associated with the development of some cases of infantile autism.

The UCLA-University of Utah epidemiologic survey of autism: prenatal, perinatal, and postnatal factors.

In a recent epidemiologic survey conducted in Utah, 241 autistic patients (DSM-III criteria) were found. Medical records of 233 autistics were surveyed for the presence of 36 potentially pathologic prenatal, perinatal, and postnatal factors. These results were compared with those of an identical survey of 62 of their nonautistic siblings, with the results of four previously published surveys, and with normative data. No potentially pathologic factor or group of factors occurred significantly more frequently among the autistic patients. Also, previous observations of significant differences in the occurrence of certain factors in the histories single vs multiple siblings with autism were not confirmed, with the exception of increased viral-type illness during gestation in single-incidence cases. Thus, the etiology of the brain pathology that characteristically disrupts normal development and produces the syndrome of autism remains obscure. Other data from the epidemiologic survey, however, suggest that the role of genetic factors needs to be explored further.

The UCLA-University of Utah epidemiologic survey of autism: genealogical analysis of familial aggregation.

To assess familial aggregation of autism, 86 autistic subjects were linked to the Utah Genealogical Database. Kinship coefficients were estimated for all possible pairs of autistic subjects and then averaged. Fifty replicate sets of matched control subjects (86 members in each set) were drawn randomly from the database, and the average kinship coefficient was computed for all possible pairs of individuals in each set. The average kinship coefficient for the autistic subjects was approximately 1/1,000, while the average kinship coefficients for the 50 control groups ranged from 4/100,000 to 1.6./10,000. These results indicate a strong tendency for autism to cluster in families. When kinship was analyzed by specific degrees of relationship, it was shown that the familial aggregation of autism is confined exclusively to sib pairs and does not extend to more remote degrees of relationship. This finding indicates that a single-gene model is unlikely to account for most cases of autism.

The UCLA-University of Utah epidemiologic survey of autism: recurrence risk estimates and genetic counseling.

The authors recently reported, in this journal, an epidemiologic survey of autism in Utah. Twenty (9.7%) of the 207 families ascertained had more than one autistic child. Analyses of these data revealed that autism is 215 times more frequent among the siblings of autistic patients than in the general population. The overall recurrence risk estimate (the chance that each sibling born after an autistic child will develop autism) is 8.6%. If the first autistic child is a male the recurrence risk estimate is 7%, and if a female 14.5%. These new recurrence risk estimates should be made available to all individuals who have autistic children and are interested in family planning.

Psychometric assessment of first-degree relatives of 62 autistic probands in Utah.

The Wechsler Intelligence Scales, Wide Range Achievement Test, and the Shipley-Hartford Test were administered to 122 parents and 153 siblings of 62 autistic probands in Utah. Scores were distributed as expected within the published normative ranges for each scale. Parents' scores correlated with those of their nonautistic children, but neither parents' nor siblings' scores correlated with the IQ level of the autistic probands. These results do not confirm prior reports from England and the United States of a high rate of cognitive and learning problems in the siblings of autistic individuals, nor the aggregation of such problems in the siblings of probands with high or low levels of cognitive function.

The UCLA-University of Utah epidemiologic survey of autism: prevalence.

The authors conducted an epidemiologic survey in Utah using a four-level ascertainment system, blind current diagnostic assessments, and DSM-III criteria. Of 483 individuals ascertained, 241 were diagnosed as having autism. The best estimate for the prevalence rate was 4 per 10,000 population. Autism was not associated with parental education, occupation, racial origin, or religion. Sixty-six percent of the autistic subjects scored below 70 on standardized IQ tests, and females scored proportionately lower than males. Twenty (9.7%) of 207 families had more than one autistic sibling, which supports the authors' previous finding that there may be a familial subtype of autism.