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. ( cell biology ) degrees from Albert Einstein College of Medicine, did 
his internship and residency in pediatrics at the University of Pittsburgh, and held a fellowship in medical 
genetics at the University of Washington. After seven years as a faculty member at the Children 's Hospital 
of Harvard Medical School in Boston, he joined the University of Michigan Medical Center. 
WE are interested in the two major aspects of 
Down syndrome, namely the etiology and 
pathogenesis of this disorder. 
Etiology 
Why does a woman's risk of having an offspring 
with Down syndrome increase dramatically with 
age? There are two competing theories. The 
"older egg" model states that as women age, they 
produce more abnormal (aneusomic) eggs. The 
"relaxed selection" model states that as women 
age, they lose the ability to abort abnormal con- 
ceptuses. To determine which of these hypothe- 
ses, or whether a combination of the two, is 
correct, it is necessary to study many cases of 
Down syndrome. 
Our laboratory examines the molecular aspects 
of such a study. We utilize polymerase chain reac- 
tion (PGR) techniques that analyze polymorphic 
variability due to differences in the quantity of 
interspersed d(A,C) :d(G,T) sequences and to dif- 
ferences in the length and/or sequence of the 3' 
end of human interspersed highly repetitive Alu 
sequences. Two major advantages of these tech- 
niques are the extensive variability of these se- 
quences and the requirement of only small 
amounts of subject DNA for the PCR-based analy- 
sis. The latter point is particularly important, as 
blood from even small children becomes almost 
an inexhaustible resource with this technology. 
To detect useful variability on chromosome 
21, we hybridized cosmids containing flow- 
sorted DNA enriched for this chromosome (cour- 
tesy of Lawrence Livermore Laboratories and Im- 
perial Cancer Research Fund) with a poly 
d(A,C):d(G,T) probe. In addition, we isolated 
several cosmids and yeast anificial chromosomes 
(YACs) that contain variable oligo d(A,C) :d(G,T) 
and Alu sequences near the centromere of 21q. 
Variability detected by oligomers that abut these 
sequences on 2 Iq along with variability detected 
by more standard restriction fragment length 
polymorphisms has resulted in two important 
(and unexpected) findings: 1) 94 percent of 
nondisjunction errors are maternal in origin, a sig- 
nificantly higher percentage than the 75 percent 
figure based on subjective cytogenetic observa- 
tions that must have been in error; 2) a significant 
plurality of Down syndrome cases show no detect- 
able crossing over on chromosome 2 1 in the par- 
ent responsible for nondisjunction. 
It will be especially important to distinguish 
between meiosis I and II nondisjunction errors. 
To accomplish this, we have performed locus ex- 
pansion on D21S16 and D21S120 and isolated 
various YACs that in aggregate comprise the most 
proximal known markers on 21q. We are 
currently extracting variable markers from these 
clones. The establishment of molecular variabil- 
ity near the centromere of chromosome 2 1 will 
enable us to determine whether an error is mater- 
nal meiosis I, maternal meiosis II, paternal meio- 
sis I, or paternal meiosis II. By comparing these 
rates among women of different age groups, it 
will be possible to determine whether the older 
egg or relaxed selection model (or a combination 
of the two) is correct. Furthermore, it is too early 
to determine whether the lack of crossing over 
observed in a plurality of cases reflects no chias- 
mata or terminalization of chiasmata and whether 
this depression of crossing over is associated with 
advanced maternal age. To answer these ques- 
tions, it will be necessary to examine variable mo- 
lecular probes that span the long arm of chromo- 
some 21, including markers at the distal tip of 
2 Iq. Whatever the results, the biology of nondis- 
junction is of even greater interest and complex- 
ity than imagined originally. 
Pathogenesis 
To isolate genes encoded by chromosome 2 1 , 
we use a novel recombination-based methodol- 
ogy. Genomic fragments isolated from chromo- 
some 2 1 are cloned into a plasmid vector with 
the genetic marker supY. RNA isolated from a vari- 
ety of human tissues was used to construct bacte- 
riophage X cDNA libraries, which are then in- 
fected into cells harboring supY plasmids 
carrying individual nonrepeated genomic frag- 
ments on chromosome 2 1 . If any member of the 
genie cDNA library shares homology with the ge- 
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