The Frequency of Smith-Lemli-Opitz Syndrome (SLOS) in the Population with Autism

Principal Investigator: Elaine Tierney

The investigators seek to examine specimens gathered by the Autism Genetic Resource Exchange (AGRE) from individuals with autism spectrum disorders (ASD) and their family members (when available), and American Type Culture Collection, other biomaterial repositories, and NIH studies and undergo the procedures listed above.
in order to:

1) determine, via gas chromatography/ mass spectrometry, the frequency of SLOS and hypocholesterolemia in samples from individuals with autism spectrum disorders (ASD) and their family members, non-ASD control individuals, and individuals who have fragile X syndrome or another genetic disorders associated with ASD;

2) perform mutation analyses to detect whether the samples reveal carrier status for mutations of the SLOS allele or polymorphisms of the HMG CoA reductase allele, ABCA1 allele, and ApoE allele;

3) correlate biochemical and molecular results with AGRE phenotypical and neurobehavioral data, including blood sterol levels, Autism Diagnostic Interview (ADI-R) scores, Autism Diagnostic Observation Schedule (ADOS) scores, Ravens IQ scores, and the presence of physical features;

4) for the hypocholesterolemic and non-hypocholesterolemic groups of AGRE ASD and their family members: we will analyze existing mapping data. Fine mapping will be performed in areas that are suggestive of linkage;

5) test for linkage in candidate regions using SNP array technology.

Study Related Procedures:

1. Biochemical Analyses of Blood Samples:
From the AGRE repository, we will perform sterol analyses on up to 2604 blood samples: 2504 from affected subjects, from unaffected and affected family members (siblings and parents), and up to 100 blood samples from subjects with fragile X syndrome or other chromosomal disorders.

Published data from other laboratories indicate that individuals with decreased dietary cholesterol intake will have increased levels of cholesterol precursors, indicated best by the level of lathosterol, as well as increased levels of phytosterols. Patients with decreased intrinsic rates of cholesterol synthesis will have low levels of lathosterol and increased levels of plant sterols. Lipoprotein analyses of the samples will provide evidence for any of several possible lipoprotein disturbances that might account for hypocholesterolemia, such as hypobetalipoproteinemia. It is expected that a small percentage of plasma samples may be abnormal due to medication use. If the results of any samples are found to be in the borderline range, lymphoblasts will be grown to test the cells’ ability to produce cholesterol.

2. DNA Mutation Analyses/Candidate Gene Approach
From the AGRE repository samples, we will perform analyses using up to 920 subjects’ DNA or cell lines: molecular approaches will be used to test for mutations/polymorphisms of genes known to be involved in cholesterol biosynthesis and homeostasis in the cohort of autistic patients with low cholesterol. These will include quantitative PCR, allelic discrimination using allele specific probes, and DNA sequencing. In addition, if sufficient power is present once the family structure and cholesterol phenotype of family members in the low cholesterol group have been defined, positional cloning will be considered;

a) For DHCR7 testing, DNA samples will be extracted and tested from cell lines from the 200 subjects with ASD who had blood testing. Control samples will be obtained from all of the approximately 400 unaffected siblings of the 200 subjects with ASD. Control samples from 100 subjects without ASD may also be obtained from the American Type Culture Collection (http://www.atcc.org), from other biomaterial repositories, and from NIH protocols. DHCR7 will be screened for the most common alleles that code for 7-dehydrocholesterol reductase: including IVS8-1G>C, T93M, W151X, V326L, R404C, R352W, and E448K using fluorogenic probe based allelic discrimination assays;

b) Other genes involved in cholesterol biosynthesis and homeostasis will be screened for polymorphisms in the DNA samples obtained from cell lines from the 40 subjects with ASD who were in the lowest quintile of the 200 blood samples. As controls, we will also test the 160 ASD in the upper 4 quintiles and approximately 80 unaffected siblings of the 40 in the lowest quintile of cholesterol levels. Several genes will be screened. ApoE genotype (E2, E3, E4) will be determined. Guided by the results of the sterol analysis, polymorphisms of HMG-CoA reductase, ABCA1 and other genes of cholesterol biosynthesis and regulation will be screened. Previously mapped ASD loci will be screened for additional genes that may affect cholesterol levels;

c) Quantitative PCR of a panel of candidate genes involved in cholesterol biosynthesis, absorption and homeostasis, or other cellular function related to genes in the candidate regions identified by our linkage analysis, will be performed with cell line samples from the 40 individuals with the lowest quintile of cholesterol levels, as well as with the cell lines from their approximately 200 family members (parents, unaffected and affected siblings), and 100 subjects without ASD. Evidence for reduced mRNA synthesis or stability would justify sequencing;

d) Once both the family structure and the cholesterol phenotype of family members of the 40 subjects with the lowest quintile of cholesterol levels is defined, positional mapping of the cholesterol phenotype is planned if sufficient power is present. DNA samples would be obtained from the cell lines from all 40 individuals with the lowest quintile of cholesterol levels and the estimated 160 unaffected family members of these individuals;

e) The technology to simultaneously genotype hundreds of thousands of single nucleotide polymorphisms (SNPs) in a single assay has been developed, and in familial linkage studies, high-density SNP genotyping has already been proven to speed up mutation identification of Mendelian traits several fold. We will test for linkage in candidate regions using SNP array technology. Patterns of linkage disequilibrium will be investigated, and the transmission of haplotypes across candidate genes will be tested for association. This may be performed with the 720 cell lines from families with ASD detailed directly above in section number 2 and the 200 detailed directly below and may occur at KKI or at NIH by JHUSOM IRB approved co-investigators;

f) In order to compare the cellular function of the subjects and their families with other populations, up to 100 cell lines from controls without ASD and up to 100 blood samples from subjects with fragile X syndrome or other chromosomal disorders will be examined as noted above.

Study Subjects.
Previously banked plasma and cell line samples will be obtained from AGRE. These samples were banked from individuals with ASD and their family members (when available). A catalog from AGRE that describes the program was submitted with the original IRB application in 2001. Cell lines will be obtained from the American Type Culture Collection, from other biomaterial repositories, and NIH protocols. These samples will be provided with only codes as identifiers.

Inclusion criteria.

1. Biochemical Analyses of AGRE Blood Samples:

a) up to 604 blood samples from individuals who have ASD who had the ADI-R performed.

b) up to 600 blood samples will be from unaffected and affected family members (siblings and parents) of the up to 120 subjects from multiplex families who were in the lowest quintile of the 600 samples tested from the individuals with ASD who had the full ADI-R performed.

c) up to 600 blood samples will be from unaffected and affected family members (siblings and parents) of 140 subjects who were randomly selected from the middle 3 quintiles of cholesterol levels of the 704 samples already tested from individuals who had the ADI-R performed.

d) up to 600 blood samples from family members of the 120 subjects who were in the highest quintile of cholesterol levels.

e) up to 100 samples will be from subjects who are non-ASD controls.

f) up to 100 blood samples will be from subjects with fragile X syndrome or other chromosomal disorders.

Approved with 2008 CRA:
We request permission to increase the number of biological samples and family data to be analyzed as part of this protocol from 2804 subjects and 920 cell lines to a total of 14,804 of each subjects’ blood, cell line, genotypic and phenotypic data (an additional 4,000 affected subjects and their 8,000 family members). AGRE has added and continues to add large numbers of subjects and we also seek to add subjects from National Institute of Health’s Genetic Repository.

2. DNA Mutation Analyses/Candidate Gene Approach/ SNP genotyping.

a) For DHCR7 testing, DNA samples will be extracted and tested from cell lines from the 200 subjects with ASD who had blood testing. Control samples will be obtained from approximately 400 unaffected siblings of the 200 subjects with ASD;

b) Screening for polymorphisms of genes involved in cholesterol biosynthesis and homeostasis will occur in DNA samples obtained from cell lines from the 40 subjects with ASD who were in the lowest quintile of the 200 blood samples. As controls, we will also test the 160 ASD in the upper 4 quintiles and approximately 80 unaffected siblings of the 40 in the lowest quintile of cholesterol levels;

c) Quantitative PCR of a panel of candidate genes will be performed with cell line samples from the 40 individuals with the lowest quintile of cholesterol levels, as well as with the cell lines from their approximately 200 family members (parents, unaffected and affected siblings);

d) Once both the family structure and the cholesterol phenotype of family members of the 40 subjects with the lowest quintile of cholesterol levels is defined, positional mapping of the cholesterol phenotype is planned if sufficient power is present. DNA samples would be obtained from the cell lines from all 40 probands and the estimated 160 unaffected family members;

e) SNP genotyping may be performed using cell lines from the subjects detailed above and directly below;

f) Up to 100 cell line samples will be obtained from the American Type Culture Collection, other biomaterial repositories, and NIH studies and undergo the procedures listed above.

Exclusion criteria.
Subjects with fragile X syndrome or other chromosomal disorder will be excluded from the analyses of ASD of unknown etiology, but will be analyzed separately.

The study does not screen for eligibility.

An application will be made to AGRE asking permission to obtain the samples. When the parents/guardians of subjects with ASD banked these samples, they gave consent to AGRE that samples may be used for a wide variety of studies and that they would not be contacted to give additional consent for these individual studies. The AGRE board consists of distinguished researchers in the field of autism. AGRE uses the Rutgers University Cell and DNA Repository. There is no data safety monitoring committee.