Molecular Analysis of Down Syndrome 
David M. Kumit, M.D., Ph.D. — Investigator 
Dr. Kumit is also Professor of Pediatrics and Human Genetics at the University of Michigan Medical 
School. He received his M.D. and Ph.D. degrees in cell biology from Albert Einstein College of Medicine. 
He did his internship and residency in pediatrics at the University of Pittsburgh and his fellowship in 
medical genetics at the University of Washington. After seven years as a Harvard Medical School faculty 
member at Children's Hospital, he joined the University of Michigan Medical Center. 
WE are primarily interested in the etiology 
and pathogenesis of Down syndrome. 
Why does the risk of having an offspring with 
Down syndrome increase dramatically as women 
age? There are two competing theories. The 
"older egg" model states that the older a woman 
becomes, the more abnormal (aneusomic) eggs 
she produces. The "relaxed selection" model 
states that as women age, they lose the ability to 
abort abnormal conceptuses. To determine the 
correct hypothesis, or whether a combination of 
the two applies, it is necessary to study a large 
number of Down syndrome cases. 
Given our important (and unexpected) finding 
that 94 percent of nondisjunction errors are ma- 
ternal in origin, resolution of the above contro- 
versy requires an ability to distinguish the stage 
of maternal cell division (meiosis I or II) in 
which the error occurs. To accomplish this, we 
will analyze four distinct probes (H6-5-6, 1 26-4- 
1, D21S120, and GT14) that detect polymorphic 
oligo d(A,C) sequences in the pericentromeric 
region of chromosome 2 1 . Conditions and sizes 
have been achieved that allow each of these 
probes to be multiplexed in a single lane without 
co-interference. Using four different fluorescent 
markers, it is feasible to analyze electrophoreti- 
cally in a single gel lane a molecular weight stan- 
dard and a family consisting of father, mother, 
and their child with Down syndrome. Pouring the 
gels is the labor-intensive procedure in this pro- 
tocol, and the ability to multiplex three family 
members and four polymorphisms in a single 
lane will yield a 12-fold savings in gel-pouring 
effort. This savings is made worthwhile by 
the fact that we must study at least 500 fami- 
lies to comprehend the biological basis for non- 
disjunction. This work was also supported by a 
grant from the National Institutes of Health. 
To study pathogenesis, we first isolated genes 
encoded by chromosome 21, using a novel re- 
combination-based methodology. Genomic frag- 
ments isolated from yeast artificial chromosomes 
(YACs) that map to chromosome 21 are cloned 
into a plasmid vector with the genetic marker 
sup¥. RNA isolated from a variety of 20-week fetal 
human tissues was used to construct bacterio- 
phage X cDNA libraries, which are then infected 
into cells harboring a supY plasmid carrying a 
nonrepeated genomic fragment on chromosome 
21 . (Using tissues recently obtained from earlier 
abortus specimens, we will now be able to make 
complex cDNA libraries from small amounts of 
these tissues by polymerase chain reaction tech- 
niques.) If any member of the genie cDNA library 
in X shares homology with the genomic DNA frag- 
ment in the plasmid, then recombination me- 
diated by that homology will ensue. 
Following recombination between the bacte- 
riophage and the plasmid, selection for bacterio- 
phages carrying a given plasmid with sup¥ will 
result in selection for bacteriophages carrying a 
cDNA that is homologous to a genomic DNA 
cloned in the plasmid. In other words the system 
is designed to select for genomic sequences that 
are transcribed. The system is designed to stand 
alone or to interdigitate with the genomic initia- 
tive as it proceeds. In the latter case, as sequenc- 
ing detects open reading frames that represent 
candidates for transcription, the recombination- 
based assay is designed to delineate analytically 
the tissue and timing of transcription and to re- 
sult preparatively in isolation of the transcribed 
sequence. 
The bulk of cDNA (gene) libraries are contami- 
nated with small amounts of ubiquitous DNA se- 
quences from plasmid pBR322. To circumvent 
this problem, we have cloned the sup¥ gene into 
an R6K-derived plasmid that lacks homology 
with pBR322 and have made other improvements 
that should increase the applicability of this pro- 
cedure. We should now be able to screen a wide 
variety of extant cDNA libraries for transcription 
by chromosome-specific elements. 
This systematology applied in model experi- 
ments has demonstrated that we can perform 
both selection and counterselection appropri- 
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