HUMAN GENETIC DISEASE 
YuET Wai Kan, M.D., Investigator 
The work in Dr. Kan's laboratory is focused pri- 
marily on the study of the pathophysiology and mo- 
lecular diagnosis of genetic diseases of the red 
blood cell. 
I. Abnormal Hemoglobins and Thalassemia. 
A. Prenatal diagnosis. The method of prenatal diag- 
nosis for sickle cell anemia and thalassemia contin- 
ues to be refined, with the aim of simplifying it for 
routine use in areas where these diseases are com- 
mon and automating the diagnostic procedure so 
that large numbers of screenings can be performed. 
Nonradioactive probes have been used to sim- 
plify the method and detect specific mutations in 
P-thalassemia. In a collaboration with researchers in 
China, horseradish peroxidase-labeled oligonucleo- 
tide probes were used to detect the common 
P-thalassemia mutations in southern China. Another 
nonradioactive method, denaturing gradient gel 
electrophoresis, identified all 12 ^-thalassemia 
mutations described in the Chinese population. Be- 
cause each mutation produces a distinctive band on 
the denaturing gradient gel, individuals hetero2y- 
gous or homozygous for one mutation or doubly 
hetero2ygous for two mutations can be distinguished 
on the basis of their different patterns. This method 
is currently being tested in collaboration with investi- 
gators in Greece. 
In a collaboration with Dr. Farid Chehab, a fluo- 
rescent label for the polymerase chain reaction 
(PCR) has been developed in which the primers 
used for PCR are labeled with one or more fluores- 
cent dyes. Several different colored dyes have been 
used to detect gene deletions, chromosome rear- 
rangements, and point mutations. The advantages 
of this method are that the result can be observed 
by eye immediately after amplification and the pro- 
cedure lends itself to automation by real-time laser 
scanner on electrophoresis. The availability of an 
automated test would facilitate the implementation 
of the test in newborn screening programs. In addi- 
tion to its application to genetic and neoplastic dis- 
eases, color PCR could also be applied to detect in- 
fectious agents. 
B. Spontaneous mutations in ^thalassemia. The 
first case of spontaneous mutation in (3-thalassemia 
was described 15 years ago. The P-globin gene from 
this patient has now been cloned and directly se- 
quenced. Results indicate that the mutation occurs 
in the father's germline and is due to a single base 
deletion, resulting in a frameshift mutation at the 
codon. 
C. Globin gene expression. The globin genes are 
expressed in a tissue-specific manner in erythroid 
cells. Four major hypersensitive sites (HS) upstream 
from the (3-globin gene cluster enhance P-globin 
gene expression in erythroid cells, although their 
mode of action is not understood and is currently 
under investigation. One of the four sites, HSII, dis- 
plays enhancer activity in transfection assays, as 
well as in transgenic mice. The activity was found to 
reside in a 700 bp fragment. Footprinting and gel 
mobility shift assays demonstrated protein binding 
to a direct repeat that contains the consensus se- 
quence of an API protein-binding site. Deletion of 
this site from the 700 bp fragment abolishes en- 
hancer activity. Hence enhancement appears to be 
mediated through interaction with the API pro- 
tein-binding site. Further studies are in progress to 
characterize these protein interactions. 
II. Other Red Cell Proteins. 
A. Protein 4. 1. In a collaboration with Drs. John Con- 
boy and Mohandas Narla, the complex mechanism of 
alternate splicing in the protein 4. 1 gene in erythroid 
cells continues to be studied. Protein 4.1 is a compo- 
nent of the cytoskeletal protein found in erythroid 
and nonerythroid cells. Many different isoforms of 
this protein are produced from a single gene by alter- 
nate splicing mechanisms that involve at least six dif- 
ferent exons, two of which appear to be important 
functionally. One splicing involves a 21-amino acid 
coding block at the actin/spectrin-binding domain. 
The erythroid form of protein 4.1 includes these 21 
amino acids, while the nonerythroid form does not. 
Another splicing occurs at the amino terminal, where 
the removal of 80 nucleotides upstream from the nor- 
mal AUG and the addition of 17 nucleotides further 
upstream introduces a new AUG codon that adds 209 
amino acids to the amino end of the protein. Antibod- 
ies prepared against the amino extension indicate 
that the protein is located in the cell nucleus. Further 
studies are in progress to determine its function. 
B. Glucose-6-phosphate dehydrogenase (G6PD). 
The enzyme G6PD may be an example of a novel 
post-transcriptional modification of a gene product. 
It has been known for some time that G6PD is en- 
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