Pilot study: Experimental plan and methods

P.I.: Zoran Brkanac, MD; University of Washington

Co–investigator: Wendy H. Raskind, MD, PhD; University of Washington

Characterization of translocation-induced genomic disruption of the

chromosome 22q11.2 region in an individual with autism and his family
 

The hypothesis for this proposal is that the gene(s) that is/are disrupted by a chromosomal 
translocation involving del22q11.2 in a proband with autism is an important candidate gene for 
autism and psychiatric disorders associated with del22q11.2 syndrome. This project aims to 
identify the exact location of the chromosome 22q11.2 break point at the DNA base pair level, 
and to fully characterize the proband and available family members using standardized 
psychiatric and behavioral assessment tools. These goals will be accomplished through the 
following specific aims (SA).

SA1: Complete the family ascertainment. Cytogenetic evaluation of the parents revealed that the 
father carries the same 46,XY,t(1;22)(p36.1;q11.23) balanced translocation as the proband. The 
paternal grandparents are deceased, but the father has four sisters and multiple nieces and 
nephews. Multiple family members have agreed to participate in the study, including cytogenetic, 
molecular genetic and phonotype assessments. Blood samples have been obtained and 
cytogenetic evaluations have been performed on one of the proband’s siblings, two of his paternal 
aunts and one cousin. One aunt and her daughter were found to carry the balanced translocation. 
Additional cytogenetic evaluations will be performed on at­risk relatives (SA 4), and at­risk 
nieces and nephews will be invited to participate in the study. 
SA2: Perform cognitive testing. We will complete the evaluation of the proband and evaluate all 
available family members. To date, only the proband has had formal cognitive testing. On 
previous testing, his Full Scale Intelligence Quotient was 76 at age 7 and 67 at age 9 measured 
with Wechsler Intelligence Scale for Children (WISC­III) . At age 16,  Wechsler Individual 
Achievement Test (WIAT­II) results were notable for a Mathematical Reasoning standard score 
(SS) of 40, Reading Comprehension SS 72 and Pseudoword Decoding SS of 111. There is an 
emerging consensus that del22q11.2 has a cognitive profile characterized by low FIQ and a 
complex pattern of strengths and weaknesses. The Verbal IQ (VIQ) is often higher than the 
Performance IQ (PIQ), with even greater differences when verbal comprehension factor scores 
are compared to perceptual organization scores. There is better verbal than visual memory and 
stronger achievement in reading and spelling compared to mathematical skills. Our proband’s 
cognitive profile with higher reading comprehension than mathematical reasoning and strong 
phonological skills is consistent with this pattern. We propose to administer a battery of tests to 
assess whether translocation carriers have a cognitive profile that is similar to that of del22q11.2 
and different from that of their karyotypically normal relatives. This battery will include tests of 
intelligence (WISC for children or WAIS for adults), achievement (WIAT) and memory 
(Wechsler memory scale). 
SA3: Perform psychiatric evaluation. The proband’s psychiatric history and examination were
consistent with past diagnoses of autistic disorder and schizophrenia. The father has not been
examined but has apparently normal intelligence and no history of psychiatric treatment or
hospitalization. To fully characterize the phenotype in the family, we will evaluate the at-risk
family members for the presence of psychiatric disorders and autism spectrum disorders (ASDs)
with standard assessment tools. Presence of psychiatric disorders will be evaluated with the
Structured Clinical Interview for DSM-IV Axis I Disorders (SCID) and Brief Psychiatric Rating
Scale (BPRS). We will assess ASDs with the Autism Diagnostic Interview-Revised (ADI-R), and
Autism Diagnostic Observation Scale (ADOS). In combination with SA2, comparison of profiles 
in translocation­carriers and those without the translocation will provide valuable information 
about the variable expressivity of the involved gene(s).
SA4: Cytogenetic evaluations. High band­resolution cytogenetic evaluation revealed the balanced 
translocation in the proband and several of his relatives. Similar analyses will be performed on all 
at­risk relatives using techniques that are standard in our laboratory (Sultana et al. 2002). 
SA5: Molecular characterization of the break point. 
        We will employ ultra­high resolution array 
painting technology (Gribble et al. 2007), to  The principle of array painting. The presence of
Chr. 22 material labeled with different color
characterize the breakpoint at the sequence level. This  allows for precise determination of the sequence
is a new application that combines commercially  location of the break.
available pre­designed low­resolution arrays and 
custom­designed ultra­high resolution arrays. First, 
Mayo Medical Laboratories Conversion Technology 
cell lines will be used to establish fused human­
hamster hybrid cell with EBV transformed cell line  QuickTimeª and a
TIFF (LZW) decompressor
are needed to see this picture.

from the proband that is already available in our 
laboratory. Conversion Technology is now 
commercially available from Mayo Medical 
Laboratories and its advantages that fused cell lines 
have lower preferential loss of human chromosomes. 
Separate somatic cell hybrid lines that have retained the derivative chromosome 22 (der22), and
the derivative chromosome 1 (der1), as the only chromosome 22 and chromosome 1 material,
will be isolated and expanded for DNA extraction. DNA from these cell line will be differentially
labeled with either Cy3-dCTP or Cy5-dCTP and hybridized to a commercially available whole
genome oligonucleotide array. These oligonucleotide array hybridizations are commercially
available from NimbleGen Systems, Madison, Wisconsin, USA. This hybridization will localize
the breakpoints at relatively low resolution and can also identify other genomic alterations, such
as submicroscopic deletions, that might be present on derivative chromosomes. Next, the
hybridization experiments will be repeated using a custom­designed oligonucleotide array that 
covers the chromosome 1 and 22 translocation regions with ultra­high resolution. Excluding 
repetitive sequenced, isothermally designed oligonucleotides will be spaced as close as every 
base pair of selected sequence. Depending on the content of the repetitive sequence in the region 
and the size of micro­deletion that sometimes accompanies translocations, such oligonucleotide 
array painting analysis successfully defined breakpoint regions with 55 to 7223 base­pair 
resolution (Gribble et al 2007). Finally, PCR primers spanning the breakpoint will be designed 
and long range PCR and sequencing on ABI 3100 DNA analyzer will be performed in our 
laboratory to establish the exact base­pair position of the chromosomal break.