To obtain a crude chromosomal location of the gene or genes associated with a phenotype of interest. Genes or markers on different chromomes segregate independently. Any two genes or markers on the chromosome might be expected to segregate together especially if they are in close proximity.
New Developments in
W. Ted Brown, MD, PhD
Director, New York State
Institute for Basic Research
In Developmental Disabilities
NEW YORK STATE
A public-private partnership
5 initial goals:
Early identification and
Epidemiology and registry
Treatment research (lab
Epidemiology of Autism
Rates of autism are
The reasons are unclear. Better diagnosis?
How Common is Autism?
•The general observation is of a dramatic
•The standard figure up to about 1980
was ~ 1/2000.
• Since then, a consistent increase has been
• CDC (Feb 07) ⇒ 6.6/1000 or about 1/150
with 10.6/1000 in NJ.
What is Autism?
A poorly understood Developmental Disability
Hallmark: Severe impairment in social relations
DSM IV - 5 different Pervasive Dev Disorders
1. Autism - 6 or more of 12 features involving
repetitive & stereotypical behaviors
2. Onset prior to three yrs of age
3. Not Rett syndrome or CDD
(childhood disintegrative disorder)
Clinical and Research
Identification of Autism
ADI-R Autism Diagnostic Interview-
ADOS-G Autism Diagnostic
PDD-BI Pervasive Developmental
Disability – Behavior Inventory
- Marked impairment in social interactions &
eye contact, peer relations, social sharing.
- No delay in language, self help skills, or
general cognitive development.
- Apparently very common, but still is little
- Onset of regression >5 mo
- MECP2 gene mutation
PDD-NOS (Pervasive Developmental
Disorder - Not Otherwise Specified)
Reserved for cases not meeting the
full criteria for autism because of
later age of onset, atypical or
Concept of a broader spectrum:
Autistic Spectrum Disorders
Autism Spectrum Disorders
Time to give up on a single explanation for
autism. Happe et al. Nature Neurocience, Oct 2006
• 3,400 eight year old twin pairs were studied.
• Modest to low correlations between autistic-like traits in the
three core areas (social, language, sameness).
• Autism like traits are in a smooth continuum between
individuals with ASD and the general population-no humps
separating disease from not disease.
• Social/communication impairments correlated in the range
• Considerable numbers showed isolated difficulty in one
area - 59% with social difficulty had only social difficulty.
• Around 10% of all children showed only social impairment.
• Conclusion: largely nonoverlapping genes act on
each of these three traits.
Syndromal vs Pure Autism
• Pure Autism (non syndromal) -Limited to
moderate mental retardation to normal
intellectual functioning and no associated
signs or symptoms (except seizures).
• Syndromal autism- one or more
morphologic signs- estimated at ~20%
Cohen et al JADD 2005
Essential vs Complex Autism
Miles et al AJMG 2005
• Complex autism (260 total)
• 5% microcephalic
• 16% dysmorphic - total 21%
• Lower IQ p<.006
• More seizures p<.0008
• Less sib reoccurence 0 vs 4%
• Less relatives with autism 9 vs 20%
• Lower male to female ratio 3.2:1/6.5:1
Genetics of Autism
Identical twins with autism - 70-90%
Sibling recurrence risk - 5%
A high heritability relative to diabetes,
asthma, schizophrenia, hypertension, etc
A polygenetic (8-15 genes), multi-
factorial disorder with M:F ratio of 4:1
Autism: Genetic Etiologies?
• The high concordance in MZ twins
indicates a high degree of genetic
• The rapid fall off of concordance in DZ
twins suggested a multilocus, epistatic
• Caveats: Epigenetics & Mitochondria
Some Genetic Disorders
associated with Autism
• Fragile X ~25%
• Tuberous Sclerosis ~25%
• Down syndrome ~10%
• Angelman syndrome ~40%
• Prader-Willi syndrome ~25%
• San Filippo syndrome ~90%
• Smith-Magenis (del 17p11.2) ~90%
• VCF/ DiGeorge (del 22q11) ~25%
Folstein and Rosen-Sheidley, Nature Rev Genet, 2001
Genetics of Autism
Preliminary genome wide screenings
indicate loci on at least 21 chromosomes.
Chrs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 15, 16, 17, 18, 19, 20, 22, X
Cytogenetic Abnormalities have been
reported in 5-7% of cases
IMGSAC et al. Hum. Mol Genet. 7(3):571-578, 1998
Philippe et al. Hum Mol. Genet. 8(5);805-812, 1999
Liu et al. Am. J. Hum. Genet. 68;327-340, 2001
Yonan et al. Am J. Hum. Genet. 73(4):2003
Array-based comparative genomic hybridisation identifies
high frequency of cryptic chromosomal rearrangements
in patients with syndromic autism spectrum disorders.
Jacquemont et al. J Med Genet, 43: 843-9, 2006
Abnormalities found in 8/29 (27.5%) patients: 6 deletions and 2
duplications. Altered segments ranged in size from 1.4 to 16 Mb.
Strong Association of De Novo Copy Number Mutations
with Autism Sebat, et al. Science 316, 445-9, (2007)
• Sampled 264 families, 118 simplex, 47 multiplex, and 99
control families. Majority came from AGRE. Genome scans
were performed by a form of comparative genomic
hybridization (CGH) providing a mean resolution of 35 kb.
• CNVs were identified in 12 of 118 (10%) of simplex, 2 of
77 (3%) multiplex, and in 2 of 196 (1%) of controls.
• CNVs were in the range of 100kb-12mb.
• Adding the known rate of cytogenetically visible
abnormalities, the total frequency of de novo variation
detectable in sporadic cases is ~15% at this resolution.
• Affected genomic regions were highly heterogeneous and
included mutations of single genes.
• These findings establish de novo germline mutation as a
more significant risk factor for ASD than previously
Structural Variation of Chromosomes in Autism
Spectrum Disorder Marshall et al. AJHG 82, 477-88, (2008)
• Sampled 427 families. Genome scans were performed by
• 277 unbalance CNVs were found in 44% of ASD families,
not present in controls,.
• de novo CNVs were identified in ~7% of simplex, ~2% of
• CNVs were in the range of 100kb-20mb.
• Affected genomic regions were highly heterogeneous and
included mutations of single genes (NLGN4, SHANK3,
NRXN1,, etc) and regions previously associated with
syndromes (22q11.2, 16p11.2, etc).
CNVs may also be involved with
Although complex disorders such as schizophrenia have
a heritable component, identifying the genetic components
associated has been very difficult. Walsh et al (Sci
April 08) found that multiple, individually rare, structural
mutations (genomic microdeletions and microduplications)
occurred more frequently in 150 individuals with
schizophrenia than in controls. The enrichment was more
than threefold among schizophrenia cases generally and
more than fourfold among schizophrenia cases with onset
by age 18. The genes disrupted by the genomic
breakpoints of mutations in the schizophrenia patients
were not random, but were disproportionately members of
pathways controlling neuronal signaling and brain
Autistic children linked to same sperm
Gwenyth Jackaway is a professor at New York's Fordham
University. She's single but had always wanted to have a child.
So she contacted California Cryobank, one of the largest sperm
donor banks in the country. Cryobank doesn't reveal the identities
of donors but allows people to choose based on the traits they'd
like their child to have. Jackaway decided on "Donor X". What she
couldn't know then is that her son would have autism. So she
started to wonder whether Donor X might carry a gene that could
have contributed. In six families Jackaway contacted that had
used Donor X, three of the children are autistic, and one is
showing signs of autism.
The cause or causes of autism are not known and are hotly
debated. Most experts believe that genetics are a component,
making a child predisposed to autism or responsive to an
Researchers have found some genetic areas associated with
autism, but it could take years before the gene or genes that
cause autism or contribute to it will be determined. Until then,
Geri Dawson, chief science officer for Autism Speaks, says
there's no way to screen for those genes and prevent them fromCNN April 04
Offspring of men 40 years or older were 5.75 times (95% confidence interval, 2.65-12.46;
P<.001) more likely to have ASD compared with offspring of men younger than 30 years,
after controlling for year of birth, socioeconomic status, and maternal age. Advancing
maternal age showed no association with ASD after adjusting for paternal age.
Advancing Paternal Age and Autism. Reichenberg et al. Arch Gen Psych 63:1026,2006.
Paternal age US 'controls' a
AGRE autistic first child
n=2 492 308 n=312
30–39 41.9 54.7
40–49 3.0 3.7
Paternal age and autism are associated in a family-based sample.
Cantor et al. Mol Psych 12:419-21,2007.
PATERNAL AGE AND AUTISM
Advanced paternal age has been associated with several congenital
disorders, including Apert syndrome, craniosynostosis,
neurofibromatosis, progeria, situs inversus, syndactyly, cleft lip
and/or palate, hydrocephalus, and neural tube defects . In addition,
advanced paternal age has been associated with schizophrenia and
decreased intellectual capacities in the offspring.
The most widely proposed mechanism underlying these congenital
anomalies is known as the “copy error” hypothesis, first proposed by
Penrose. After puberty, spermatocytes divide every 16 days, and by
the age of 35 years, approximately 540 cell divisions have occurred.
As a result, de novo genetic mutations that result from replication
errors and defective DNA repair mechanisms are believed to
propagate in successive clones of spermatocytes. These mutations
accumulate with advancing paternal age and thus help explain how
this disorder, which has a large genetic component, can be
maintained in the population despite reduced reproduction in
Prenatal and perinatal risk factors for autism: a review and integration of findings.
Kolevzon A, Gross R, Reichenberg A. Arch Pediatr Adolesc Med. 161:326-33. 2007
Advancing paternal age has been consistently
associated with increased risk of autism and ASDs.
25% of very premature babies show
A study of toddlers found that about one-quarter (26 of 91) of
babies born very prematurely had signs of autism on an early
The research is preliminary since formal autism testing wasn't
done. But the results are provocative, suggesting that tiny
preemies may face greater risks of developing autism than
That suggests autism may be an under-appreciated
consequence of medical advances enabling the tiniest of
premature babies to survive, said lead author Dr
Limperopoulos, a researcher at McGill University in Montreal
and Children's Hospital in Boston. Pediatrics April 08
Fever Improves Autism Symptoms
Children with autism appear to improve when they have a fever,
according to intriguing new research that could lead to a better
understanding of the disorder.
Fever was associated with less hyperactivity, improved
communication, and less irritability in the study involving children with
autism and related disorders.
Anecdotal reports of improvements in autism symptoms related to
fever have circulated for years, but the research represents the first
scientific investigation into the observed association.
While kids with autism might be expected to be calmer and less
hyperactive when they have fevers, the improvement in
communication and socialization seen in the study suggests that fever
directly affects brain function says pediatric neurologist Andrew
Zimmerman, MD, of Baltimore's Kennedy Krieger Institute
Curran et al. Pediatrics Dec 07
What Is Autism Caused by?
Dr Tom Insel, NIMH Director
and chair of the NIH Interagency Autism
Coordinating Committee, suggested:
In Autism, the phenotype is diverse:
Language development is diverse.
Social cognition is variable.
Regressive form seen in ~30%.
CNV differences multiplex vs. simplex.
Male only vs. female in family.
Male ratio of 4:1
– higher in high functioning.
The recent discovery of a new class of mammalian small regulatory
RNAs termed PIWI-interacting RNA (piRNA) has extended the
diverse family of small regulatory RNAs.
piRNAs are a novel class of small RNAs isolated from mammalian
germline cells. piRNAs interact with the Piwi subfamily of proteins
and form a ribonucleoprotein complex called Piwi-interacting RNA
complex (piRC). piRNAs consist of more than 50,000
(perhaps >200,000 @ Betel PLoS 07) different species, in
contrast to several hundred species of miRNAs.
piRNABank is a web analysis system and resource
Sai Lakshmi S, Agrawal S.
Nucleic Acids Research 2007 Sep 18
Small non-coding RNAs may play a key role
PIWI-interacting RNAs (piRNAs) are a novel class of small (30 nt) noncoding
RNAs identified in mammalian germline cells and constitute the most abundant
known class of genes with over 32,000 elements in humans. We have examined
piRNAs' organization in the human genome and have found that currently known
human piRNAs map to 70,736 sites and are structured in about 400 clusters,
containing each at least 10 piRNAs. A large proportion of the piRNA loci (about
65%) are located within repeat sequences, mainly LTRs and LINE sequences, and
over 50% contain repeated units of a single piRNA, 71 being composed of tandem
copies of a unique piRNAs. Surprisingly, over 25% of total piRNAs are located in
regions that contain segmental duplications (SDs) and about 37% are within copy
number variant (CNVs) regions. In addition, 233 (58%) and 220 (55%) piRNA
clusters are within SDs and CNVs, respectively. Similarly, a significant subset
of SDs (43%), especially those with the highest level of nucleotide identity,
contains piRNAs. Finally, we have confirmed experimentally that the genomic
sequences in which piRNAs are embedded in vary in copy number in humans.
Since SDs and CNVs account for 5% and 12% of the human genome sequence,
respectively, the significant enrichment of piRNAs is suggestive of a
functional role of these elements. This association provides the first link
between SDs and CNVs with elements that could have a putative functional role in
the integrity of the genome.
Enrichment and variability of PIWI-interacting RNAs (piRNAs) in segmental
duplications and copy number variants (CNVs) suggest a functional role in the
integrity of the genome. Armengol, Caceres, Brunet, X. Estivill (#153 ASHG)
The Fragile X Syndrome
The leading cause of inherited mental
retardation. It is associated with autism.
This common mental retardation
syndrome is due to the silencing of the
FMR1 gene on the X chromosome.
The FMR1 protein (FMRP) is an RNA
binding protein and modifies the
expression of a set of other genes.
Association of Fragile X and Autism
• 17 screening studies of 1,243 autistic
subjects for FX had a range of 0 -16%,
and a mean of 4%.
• 14 studies of 479 FX males had a
range of 7- 60%, and a mean of 24%
• A recent study by Don Bailey of 57
boys with FX found 25% with autism.
Association of Fragile X and Autism
• Behavioral characteristics found in
many FX males are similar to that in
autism (perseveration, stereotypies,
regulating affect, attention, hyperactivity).
• Anxiety-based symptoms more
common in FX (gaze aversion).
• Social interests, perception of others
emotions, attachment to parents are
higher in FX.
A maternally inherited chromosomal
duplication of the region 15q11-q13 has
been found in 1-3% of several large
This region is paternally deleted in
Angelman syndrome, and maternally
deleted in Prader-Willi syndrome.
Contains several potentially relevant
genes: Gaba receptor, Ubiquitin related
• Angelman Syndrome –Develpmental
delay, retardation speech impairment,
gait ataxia and happy behavior. Often
microcephaly and seizures. 1/12000
• Silence or disruption of UBE3A
maternally derived gene
• Autism in Angelman ~40%
• Prader-Willi is the lack of paternal
contribution within the same region
• Hypotonia, dev delay, and MR.
• Prevalence 1/29,000
• Autism in PW =25%
• Phenotype includes: MR, seizures,
language disorders, autism, hypotonia
• One series- 8/9 exhibited autism
• Brain size tends towards small 20%
• Autism shown to have larger brain size
on neuropathologic exam,
neuroimaging and is present in first
Brain Abnormalities in
Rare postmortem studies in autism (<30).
Typically larger brain size 10-20%.
Brain size enlarged by 10-20% using MRI
during 2-4 yrs, then falls off after 8-10 yrs.
Smaller nerve cell size and more dense
packing seen in internal regions: limbic system
and brain stem nuclei.
Minicolumns are smaller and less compact.
More white matter in frontal lobes, the
cerebellum, various other areas, and
associated with signs of inflamation.
Brain Abnormalities in
Dr. George Wegiel at IBR has studied
14 autism cases and age matched
Finds smaller and decreased numbers
of neurons in many areas.
Finds elevated levels of intracellular
Beta-Amyloid in 40% of autistic
specimens, unrelated to age.
14 brains of autistic subjects and
14 age matched control brains
Age range: 4 to 64 years.
Autism Tissue Program (Princeton) coordinated selection of material for
morphometric study of cortical and subcortical subdivisions in several
European and US research centers engaged in Autism Brain Atlas Project.
Inclusion/exclusion criteria. Selection of brains was based on clinical,
neuropathological and technical inclusion and exclusion criteria.
This study of historically the largest collection of brains of autistic
individuals and age matched control subjects is the product of coordinated
efforts of several institutions and dozens of unnamed contributors.
Amygdala: Lateral, basal, accessory and central nucleus
Cerebellum: Purkinje cells
Brain stem: Nucleus of facial nerve and
Nucleus olivaris inferior
Entorhinal cortex: Layers II (stellate neurons), III, V, and VI.
Structures to be examined: Examination of 11 other structures
(in progress) will complete comprehensive mapping of
developmental and age-associated changes in brains of autistic
GRADING OF DEVELOPMENTAL DEFICITS
IN THE SIZE OF NEURONAL BODY IN
4-7 YEAR OLD AUTISTIC CHILDREN
Nucleus accumbens: -46%
Amygdala (lateral basal nucleus): -29%
Dentate nucleus: -25%
Nucleus of facial nerve: +5% (not significant)
PERCENTAGE OF SMALL NEURONAL NUCLEI
IN AUTISTIC AND CONTROL PEOPLE
Structure Neuronal Autism Control Ratio
autism/ volume μm3
Amygdala. Lateral n <300 45% 15% 2.5x
Amygdala. Lat.Bas.n. <300 46% 15% 3.0x
N. accumbens <200 82% 12% 6.8x
Putamen <200 66% 31% 2.1x
Caudate <200 72% 27% 2.7x
Dentate n <300 55% 18% 3.0x
N. olivaris <400 47% 16% 2.9x
Average 59% 20% 3.0x
N. Facial n. <1,500 50% 58% 0.9x
Triple Hit Hypothesis
• UNDERLYING VULNERABILITY
• EXOGENOUS STRESSOR
• CRITICAL PERIOD OF BRAIN
Some fundamental questions
• How many autisms are there?
• Is there is a “real” autism and one or
• Should autism be studied as an entity
or as part of a continuum?
• Where do we draw the lines? Who are
• What do words like co-morbid and dual
diagnosis really mean?