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Identification of the cystic fibrosis gene: genetic analysis.

08 Sep 1989-Science (American Association for the Advancement of Science)-Vol. 245, Iss: 4922, pp 1073-1080
TL;DR: Extended haplotype data based on DNA markers closely linked to the putative disease gene locus suggest that the remainder of the cystic fibrosis mutant gene pool consists of multiple, different mutations.
Abstract: Approximately 70 percent of the mutations in cystic fibrosis patients correspond to a specific deletion of three base pairs, which results in the loss of a phenylalanine residue at amino acid position 508 of the putative product of the cystic fibrosis gene. Extended haplotype data based on DNA markers closely linked to the putative disease gene locus suggest that the remainder of the cystic fibrosis mutant gene pool consists of multiple, different mutations. A small set of these latter mutant alleles (about 8 percent) may confer residual pancreatic exocrine function in a subgroup of patients who are pancreatic sufficient. The ability to detect mutations in the cystic fibrosis gene at the DNA level has important implications for genetic diagnosis.

Summary (2 min read)

Group IV F

  • To obtain further information useful for understanding the nature of different CF mutations, AF508 was correlated with the extended DNA marker haplotypes (37) .
  • Most recombinations between haplotypes occurred between regions 1 and 2 and between 8 and 9, again in good agreement with the relatively long physical distance between these regions.
  • Other less frequent breakpoints occurred between short distance intervals, and they generally corresponded to the hot spots identified by pairwise allelic association studies (34) .
  • It is more likely that this group III chromosome represents a recurrent mutation event, a situation similar to the s and 1E mutations at the 3-globin locus (38) .
  • Together, the results of the oligonucleotide hybridization study and the haplotype analysis provide a strong indication that the gene locus described above is the CF locus and that the 3-bp deletion (AF508) is the most common mutation in CF.

Other CF mutations. Haplotype association with specific mutations has been demonstrated in studies of other genetic disorders (39). As indicated by these examples, knowledge of the haplotypes associated with the disease chromosomes facilitates studies on the molecular defects of each of the respective mutations.

  • The association of AF508 with one common and one rare CF haplotype provided further insight into the number of mutational events that could contribute to the present patient population.
  • On the basis of the extensive haplotype data, the two original chromosomes in which the AF508 mutation occurred are likely to carry the haplotype -AAAAAAA-(group Ia) and -CBAACBA-(group Ila), as defined (Table 3 ).
  • The other CF chromosomes in group I carrying the deletion are probably recombination products derived from the original chromosome.
  • As a result, a greater number of independent mutational events can be considered, and the data suggest that at least seven additional, putative mutations also contribute to the CF-PI phenotype.
  • The to be defined, none of these mutations severely affect the region corresponding to the oligonucleotide binding sites used in the PCRhybridization experiment (Fig. 2 and legend ).

Pancreatic sufficiency. CF-PS is defined clinically as sufficient pancreatic exocrine function for digestion of food; however, the level of residual pancreatic enzyme activity varies among patients (1, 40). Our previous haplotype data suggested that the CF-PI and CF-PS patients have different mutant alleles (19). Although the basic biochemical defect in

  • Thus, the residual exocrine function conferred by a mild (CF-PS) allele, although much lower than that of the normal gene product, would constitute a dominant phenotype over that of more severe (CF-PI) mutations with little or no function.
  • The observed frequencies for all five groups of patients were as expected from this hypothesis (Table 4 ).
  • The above analysis thus provides strong support for their hypothesis that CF-PI is due to the presence of two severe alleles and that a CF-PS patient carries either a single severe allele or two mild alleles.
  • All of them belong to the CF-PI subgroup, five of them homozygous and one heterozygous for AF508 (Table 5 ).
  • Previous DNA-based genetic testing for CF has only been available to families with affected children and to their close relatives (14, 43) .

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Identification of the Cystic Fibrosis Gene: Genetic Analysis
Author(s): Bat-sheva Kerem, Johanna M. Rommens, Janet A. Buchanan, Danuta Markiewicz,
Tara K. Cox, Aravinda Chakravarti, Manuel Buchwald, Lap-Chee Tsui
Reviewed work(s):
Source:
Science,
New Series, Vol. 245, No. 4922 (Sep. 8, 1989), pp. 1073-1080
Published by: American Association for the Advancement of Science
Stable URL: http://www.jstor.org/stable/1704306 .
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CFTR
122-136
Y
L G I G
L C
L L F I V R T L
GLNP 198-212
Y
L
I I
T
L
V
L
S
F I
L
R R
L
CFTR 307-319
S
S
A F F
F
S
G
F
F
V
V
F
COX 89-101
S E
V F
F F
A
G
F
F W
A F
CFTR 701-713
I L
N P
I N
S
I R K
F
S
I
NaCh 111-123
I
L T P
F N P
I R K
L A I
CFTR 1425-1442
D S I Q K L L N E R
S L F
R
Q
A
I S
raf
578-595
D S I K K L R D E R P L F
P Q I
L S
GLNP, glutamine
permease
of
E.
coli
[T.
Nohno,
T. Saito, J.
Hong, Mol.
Gen.
Genet.
205, 260
(1986)]; COX,
human
cytochrome
c oxidase polypeptide
III
[S.
Anderson
et al.,
Nature 290,
457
(1981)];
NaCh,
rat
brain sodium
channel III (32);
raf,
the serine-threonine
kinase
proto-oncogene
of Xenopus
laevis (31).
The first
two
sequences
are
within membrane
spanning
segments
and probably
reflect
only
coincidental
arrangements
of the hydrophobic
residues suited to
this
function.
In
contrast,
the latter
two sequences
are
both
in
polar
hydrophilic
regions
of the
proteins.
The large
extent of
amino acid
conservation
(11 of 13
residues) implies
some functional
relation between
these
short segments
of the primary
structure
of
the Na'
channel and CFTR. Similarities
between sequences
at the same
relative
locations
with
respect to the
COOH-termini
of the raf kinase and CFTR suggest
that
they may
also share
at least
a
small
facet
of their structures and
functions.
31. R.
LeGuellec,
K. LeGuellec,
J. Paris,
M. Philippe,
Nucleic Acids
Res. 16,
10357
(1988).
32.
M.
Noda
et
al.,
Nature
312,
121
(1984);
L. Salkoffet al.,
Science 237,
744 (1987).
33.
P.
R. Schofield
et al.,
Nature 328,
221 (1987).
34.
F. Sanger,
S. Nicklen,
A. R.
Coulsen,
Proc.
Natl. Acad. Sci.
U.S.A.
74,
5463
(1977).
35.
D. Eisenberg,
E. Schwarz,
M.
Komaramy,
R. Wall,
J. Mol.
Biol. 179,
125
(1984).
36. A. P. Feinberg
and B. Vogelstein,
Anal. Biochem.
132,
6
(1983).
37. J.
Rommens
et
al,,
Am.].
Hum.
Genet.
43, 645 (1988).
38. S. J.
Foote
et al.,
Cell 57,
921
(1989).
39.
J.
P.
McGrath
and
A.
Varshavsky,
Nature
340,
400
(1989).
40.
B.
P. Surin,
H.
Rosenberg,
G.
B. Cox,
J.
Bacteriol.
161,
189
(1985).
41.
C. F.
Higgins
et
al.,
Nature 298, 723 (1982).
42.
E. Gilson,
H. Nikaido,
M. Hofnung,
Nucleic
Acids Res. 10,
7449 (1982).
43.
I. D. Hiles,
M. P.
Gallagher,
D. H. Jamieson,
C.
F.
Higgins,
J. Mol.
Biol.
195,
125
(1987).
44.
A.
W. Bell
et al., J.
Biol.
Chem.
261,
7652 (1986).
45. R. F. Doolittle
et al.,
Nature
323,
451
(1986).
46.
I.
J.
Evans and
J.
A.
Downie,
Gene
43,
95
(1986).
47.
D. R. Gill,
G. H.
Hatfull, G.
P. C. Salmond,
Mol. Gen.
Genet.
205,
134
(1986).
48. J. E. Walker,
M.
Saraste,
M. J.
Runswick,
N.
J.
Gay,
EMBOJ.
1,
945 (1982).
49.
D. C. Fry,
S. A.
Kuby,
A. S. Mildvan,
Proc.
Natl.
Acad. Sci. U.S.A. 83,
907
(1986).
50.
We thank
0.
Augustinas
for
the
collection
of tissues;
T. Jensen
and R.
Baird for the
culturing
of
epithelial
cells;
L. Naismith
for
the
isolation of
RNA; D.
Kennedy and
D.
Markiewicz
for
technical
assistance
and M.
Buchwald
and
M. Dean
for
discussions.
Supported
by
NIH
grants
DK39690
(F.S.C.)
and
DK34944
(L.C.T.),
the Cystic
Fibrosis
Foundation
(U.S.A.),
the Canadian
Cystic
Fibrosis
Foundation,
and the
Sellers
Fund. J.M.R.
holds a
postdoctoral
fellowship
from the
Medical
Research Council
(MRC)
of Canada
and
F.S.C. is
an Associate
Investiga-
tor of the Howard
Hughes Medical
Institute.
7
August
1989;
accepted
18
August
1989
Identification
of
the
Cystic
Fibrosis
Gene:
Genetic
Analysis
BAT-SHEVA
KEREM, JOHANNA
M.
ROMMENS, JANET
A.
BucHANAN,
DANUTA
MARKIEWICZ,
TARA
K.
Cox,
ARAVINDA
CHAIRAVARTI,
MANUEL
BUCHWALD, LAP-CHEE
Tsui
Approximately 70
percent of
the mutations
in
cystic
fibrosis patients correspond
to a
specific
deletion of three
base pairs, which
results
in
the loss of
a
phenylalanine
residue at amino acid
position 508 of the
putative product
of the cystic fibrosis
gene. Extended haplotype
data based
on DNA markers
closely linked to
the putative disease
gene locus suggest
that the remainder of
the cystic fibrosis
mutant gene pool consists of
multiple,
different muta-
tions. A small set of these latter
mutant
alleles
(about
8
percent) may confer
residual pancreatic exocrine function
in
a
subgroup of patients who are
pancreatic sufficient.
The ability to detect mutations
in
the
cystic fibrosis gene
at
the DNA
level
has
important implications for genetic
diagnosis.
A
LTHOUGH THE FREQUENCY OF CYSTIC FIBROSIS (CF) IS
not uniformly high among all Caucasian populations, a
consensus estimate is that it occurs once in 2000 live births
(1). On
the
basis
of
the autosomal recessive mode of inheritance
for
this disease, a mutant allele frequency of 0.022 may be derived.
Several different mechanisms, including high mutation rate (2),
B. Kerem, J. M. Rommens, J. A. Buchanan, D. Markiewicz,
M. Buchwald and
L.-C.
Tsui
are in the
Department
of
Genetics,
Research
Institute,
The
Hospital
for Sick
Children, Toronto, Ontario M5G 1X8,
Canada.
T.
K. Cox and
A.
Chakravarti are in the
Department of Human Genetics, University of Pittsburgh, Pittsburgh,
PA
15261.
M.
Buchwald and L.-C. Tsui are
also
members
of
the
Departments
of Medical Genetics and
Medical Biophysics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
8
SEPTEMBER
i989
heterozygote advantage (3), genetic drift (4), multiple
loci
(5),
and
reproductive compensation (6), have been proposed
in
attempts to
explain the high incidence and, indirectly, the nature
of
the CF
mutations. Although some of these hypotheses could not be further
addressed because
of
the lack of knowledge about the basic defect
in
CF, several important observations have
been made
during
the
past
few years through genetic analysis
of
the families of affected
individuals
(7-20).
Extensive linkage analysis provides evidence for the existence of
a
single CF locus
on
human chromosome 7 (region q31) (7-10, 21).
The detection
of
allelic and haplotype association between the CF
RESEARCH ARTICLES 1073

locus (CF) and closely linked DNA markers further suggests
a small
number of mutations in CF (12-16, 19). A single mutational
event
may account for most CF mutations in Northern European
popula-
tions (12); another mutation appears to be prevalent
in some
Southern European populations (15). Additional haplotype
data
indicate that there could be several other mutations (15,
16, 19, 20).
The two clinical subgroups
of
CF patients, those with
pancreatic
insufficiency (CF-PI) and those with pancreatic sufficiency
(CF-PS),
can
be explained by different mutations on the basis of family
studies
(18) and haplotype data (19, 20). Patients with CF-PI,
which
constitute the larger proportion (about 85 percent)
of the CF
population, appear
to be
genetically
more
homogeneous
than those
with CF-PS (about 15 percent); most individuals
with CF-PS
appear to be compound heterozygotes (19). We must
emphasize,
however, that these studies were based on linked DNA
markers
whose exact relation to CF was not entirely certain.
Further
understanding
of
the
pathophysiology, haplotype
association, and
population distribution of
CF
would require detailed
molecular
knowledge about the mutations.
The extensive
genetic
and
physical mapping
data made
it
possible
for molecular
cloning
studies
to
be focused on
a small
segment
of
DNA on chromosome 7 (12, 13, 17, 21, 22).
A
putative
CF gene has
now been identified through chromosome walking and
jumping
(23)
and
cDNA
clones have been
isolated
and characterized
(24).
Because of the lack
of
chromosome deletions and
rearrangements
in
CF
and
the lack of
a
well-developed
functional
assay
for
the CF
gene
product,
the
identification of
the
CF locus required
a
detailed
characterization
of the locus
itself
and
comparison
between
the CF
and normal (N)
alleles.
Random, phenotypically normal,
individuals
could
not be included as
controls
in
the
comparison
because
of the
high frequency
of
symptomless
carriers
in
the
population.
As
a
result, only parents of CF patients,
each
of
whom by definition
carries
an N
and a CF chromosome, were suitable
for the
analysis.
Moreover, because
of the
strong
allelic association
observed be-
tween CF and
some of
the
closely
linked
DNA markers
(12-15, 21),
it was necessary
to
exclude
the
possibility
that
sequence
differences
detected between N and CF
were
polymorphisms
associated
with
the disease
locus.
Identification
of RFLP's and
family
studies.
To determine the
relation
of each of the
DNA
segments
isolated
from the
chromo-
some
walking
and
jumping experiments
to
CF,
restriction
fragment
length polymorphisms (RFLP's)
were identified and used
to
study
families
where
crossover
events between CF and other
flanking
DNA markers had
previously
been detected
(25, 26).
In
all,
18
RFLP's
were detected
in
the
500-kb
region
of
chromosome
7;
17
of
them
(from
E6
to CE
1.0)
are listed
in Table
1;
some
of them
corresponded
to markers
previously reported (12, 13).
Five of
the
RFLP's
were identified with cDNA and
genomic
DNA
probes
derived from the
putative
CF
locus, namely 10-1X.6,
T6/20, H1.3,
and CE1.0
(23, 24).
The
RFLP
data
(Table 1)
are
presented
with
markers
in
the
regions
of MET and D7S8 included
for
comparison.
The
physical
distances between
these
markers
as
well as their
relation
to
MET
and
D7S8
are
shown
in
Fig.
1.
Because
families
with
informative crossovers
only gradually
be-
came
available
[through
the
courtesy
of
investigators (27)]
over
the
course of the
study,
not
all
the DNA markers
were
examined
in
these
families
(28).
The recombination
breakpoints
were localized
for the
two families
[out
of
four
that
showed
a crossover between D7S23
and
CF
(26)]
that were informative
for
the DNA markers
tested.
One of them showed
a
crossover between
the sites defined
by
JG2E1 (also known
as KM19) and E2.6 (29), and
the other
between JG2E1 and E4.1 (Mp6d.9) (30) (Fig. 1). These results
indicated that CF mapped to the telomeric side of
JG2E1.
The two
other families were not informative in the analysis. Only a single
1074
family
(SLC1380)
was documented to show a recombination
event
between CF and D7S8 (25), and more recently the crossover
point
was
narrowed to between CF and D7S424, a jump clone
derived
from D7S8 (31); no crossover was detected with the above-
described DNA markers in this family. CF was therefore
delimited
to
the region between JG2E1 and D7S424, spanning less than
900
kb
(Fig. 1). Because
of
the small number of recombinant
families
available for the analysis, which was expected from the close
distance
between the markers and CF, and because of the possibility
of
misdiagnosis, alternative approaches were necessary in further
fine
mapping of the CF locus.
Allelic
association. Allelic association (linkage
disequilibrium)
Table
1.
RIFLP's associated with the CF locus.
Probe
Frag-
name
Enzyme
ment
N*
CF-PI*
Alt Alt
Source
name
~~~(kb)
metD BanI 7.6 28 48 0.60 0.10 (11); TS?
6.8 59
25
metD Taq
I
6.2 74 75 0.66 0.06 (9); TS
4.8 19
4
metH
Taq
I
7.5 45 49
0.35 0.05 (9); TS
4.0 38 20
E6
Taq
I
4.4
58 62 0.45 0.06 (19); TS
3.6
42
17
E7
Taq
I
3.9
40 16
0.47 0.07 TS
3+0.9 51 57
pHl31
Hinf
I
0.4 81 33
0.73 0.15 (13); TS
0.3 18 47
W3D1.4 Hind III 20 82
33
0.68 0.13
(19);
TS
10
22
47
H2.3A
Taq
I
2.1 39
53
0.64
0.09 (12); TS
(XV2C)
1.4 37
11
EG1.4 Hinc II 3.8
31 69
0.89 0.17 TS
2.8 56 7
EG1.4
Bgl
II
20
27 69
0.89 0.18
TS
15
62 9
JG2E1
Pst
I
7.8
69
10
0.88
0.18
(12, 19);
TS
(KM19)
6.6 30
70
E2.6 Msp
I
13
34
6 0.85 0.14
TS
(E.9)
8.5
26
55
H2.8A Nco
I
25
22
55
0.87 0.18
TS
8
52
9
E4.1
Msp
I
12 37
8
0.77 0.11
(29); TS
(Mp6d.9)
8.5+3.5
38
64
J44
Xba
I
15.3
40
70 0.86 0.13 TS
15+0.3
44
6
10-lX.6
Acc
I 6.5
67
15 0.90 0.24 TS
3.5+3
14
60
10-lX.6
Hae III
1.2
14 61 0.91 0.25 TS
0.6 72 15
T6/20
Msp
I
8
56
66
0.51 0.54
TS
4.3 21 8
H1.3
Nco
I
2.4
53
7 0.87 0.15 TS
1+1.4
35
69
CEl.0
Nde
I
5.5 81 73 0.41 0.03 TS
4.7+0.8 8
3
J32
Sac
d
15 21 24
0.17
0.02
(31);
TS
6
47 38
J3.11
Msp
I
4.2
36 38
0.29 0.04
(10);
TS
1.8
62 36
J29
Pvu
II
9
26 36
0.36
0.06
(31);
TS
6 55
36
*The number
of N
and
CF-PI
(CF
with
pancreatic insufficiency)
chromosomes
was
derived from
the
parents
in
the families
used
in
our
linkage analysis (36).
tStandard-
ized association
(A),
which is less
influenced
by
the
fluctuation
of DNA
marker
allele
distribution
among
the
N
chromosomes,
is
used here
for
the
comparison.
Yule's
association
coefficient
(A)
=
(ad
-
bc)/(ad
+
bc),
where
a, b, c,
and
d
are the
numbers of
N chromosomes with DNA marker allele
1, CF with 1, N with 2, and CF with
2,
respectively. Relative risk can be calculated from the relation RR
-
(1 + A)/( 1 - A)
or
its reverse.
tLAllelic
association (A), calculated according to (33) on the basis of
a
frequency of 0.02 for CF chromosomes in the population, is included for
compari-
son. ?TS, this
study.
SCIENCE,
VOL. 245

Locus
MET
D7S 122 D7S23
D7S399 CF
Probe
metD metH
E6 E7
pH131
W3D1.4
H2.3A EG1.4 JG2E1 E2.6
H2.8 E4.1
J44
10-1X.6
T6/20
H1.3
CE1.0
(XV2C)
(CS.7) (KM19) (E.9)
(Mp6d.9)
RFLP
Ban
T Ian
spI
Taq
I
Taq IHint
I
Hind
III
Taqi
BgI
(Hha)
PstI
MspI
Nco
I
Msp
I Xba I
AccIHae
Msp
I
Nco I
Nde
I
RF P
Taq
I
Taq
I
\
~
H
in
j/c
__ _ __ _ __ _ __ _ _
kb g
12.51
3I300101
20
~5
125
20
515
20 30
25
35
35 80
>50
I650
-10 >10
Fig. 1. Map of the
RFLP's closely
linked to the CF locus.
Details
of the RFLP's are
shown
in Table
1.
The
inverted
triannle
indicat.es
-the
location
of the
AFR-
mutation.
has been detected for many closely
linked
DNA
markers
(32, 33).
Although the utility of using
allelic association for
measuring
genetic distance is uncertain,
an overall correlation
has
been ob-
served between CF and the flanking
DNA
markers.
A
strong
association with CF was noted
for
the
closer
DNA
markers D7S23
(12, 14) and D7S122 (13),
whereas little or
no association was
detected
for the more
distant
markers
MET, D7S8,
or D7S424
(21,
31; Fig. 1).
The degree
of
association between
DNA
markers and CF
(as
measured by the Yule's
association
coefficient)
increased
from
0.35
for metH and
0.17
for
J32
to
0.91
for 10- IX.6
[only
CF-PI families
were used
in
the analysis
as
they appeared
to be
genetically
more
homogeneous
than CF-PS
(19)].
The association
coefficients
ap-
peared to be rather
constant
over the 300-kb
interval between
EGI.4 and H1.3;
the
fluctuations
detected at several
locations,
most
notably at H2.3A, E4.1, and
T6/20,
were
probably due
to
the
variation
in
the allelic distribution among
the N chromosomes
(Table 1). These data are therefore
consistent
with
the result from
the study of recombinant
families
(Fig. 1).
A
similar conclusion
could also be made by inspection
of the
extended
DNA marker
haplotypes associated with the CF chromosomes (as discussed
below). However,
the
strong
allelic association detected over the
large physical distance between EG1.4 and H1.3 did not allow
further refined
mapping
of
the
CF locus.
Since
J44
was
the
last
genomic
DNA
clone isolated by
chromosome
walking
and
jumping
before
a
cDNA clone was identified
(24),
the
strong
allelic associa-
tion detected for the
JG2E1-J44
interval
prompted
us to search for
candidate gene sequences over this entire interval. The highest
degree of allelic association was in fact detected between CF and the
two RFLP's detected by 10-1X.6, a region near the major CF
mutation.
Strong
allelic association was
also detected
among subgroups
of
RFLP's on both
the
CF
and
N
chromosomes, namely,
between
adjacent markers
E6 and
E7,
between
pH131
and
W3D1.4,
be-
tween the
Acc
I
and Hae
III
polymorphic
sites
detected
by
10-1X.6
and
among EG1.4, JG2E1,
E2.6
(E.9), H2.8,
and E4.1
(34).
The
two
groups
of
distal
markers in the
MET
and
D7S8
regions
also
showed
some
degree
of
linkage disequilibrium among themselves,
but
they
showed little association with markers
from E6 to
CELO,
consistent
with the distant locations for
MET and
D7S8.
The lack of
association between
DNA markers that are
physically
close
may
indicate the
presence
of
recombination
hot
spots (33, 34). Examples
of
these
potential
hot
spots
are the
region
between E7 and
pH131,
around H2.3A, and between
J44
and
the
regions
covered
by
the
probes
10-1X.6
and
T6/20 (Fig. 1).
These
regions, containing
frequent recombination breakpoints, were useful in the subsequent
analysis of extended haplotype data
for
the CF region.
The
major CF mutation.
Molecular
cloning experiments have
allowed us to
identify a gene
in
the J44-D7S424 interval (23, 24).
Sequence analysis
of
overlapping
cDNA
clones
of
this gene predict-
ed a
protein with properties consistent with membrane association
8
SEPTEMBER
I989
D7S424
D7S8
D7S426
J32
p3H-3
pJ3.11
J29
Sac I
Pvu
11
Msp
I Pvu
11
-60
10
-100
Table 2.
Distribution
of CF and
non-CF (N) chromosomes
with
and
without the
3-bp
deletion (AF508).
The data for
the CF-PI and
CF-PS
chromosomes
were de-_-_--
rived from
the CF fam-
ilies included
in
our link-
age analysis
(36).
These
CF
N
families
were
originally
selected
without our
Without the
deletion
knowledge
about
PI
or
CF-PI
24
PS;
the
14
CF-PS
fam- CF-PS
9
ilies subsequently
identi-
Unclassified
36
fied
were not included
as
Subtotal
69 (32%)
198
part
of
this calculation.
The unclassified
CF
With the
deletion
chromosomes
were de-
CF-PI
62
rived from
CF
families
CF-PS
5
for
which
pancreatic
Unclassified
78
fimction
data
were not
Subtotal 145 (68%)
0
available.
Total 214
198
and
nucleotide
binding
(24).
Comparison
of
the nucleotide
se-
quences
of
the
cDNA clones derived from
N
and CF
individuals
revealed
a 3-bp deletion
in
the
CF gene, resulting
in
the
loss of a
single phenylalanine
residue (position
508)
in
the predicted
amino
acid
sequence
(24).
To
investigate
the proportion
of CF
patients
carrying
this
dele-
tion
(AFso8),
we used genomic
DNA
samples
from patients
and
their
parents.
Each
sample
was
amplified
with oligonucleotide
primers flanking
the
mutation in
a polymerase
chain reaction
(PCR)
(35)
and hybridized
to 32P-labeled
oligonucleotides
specific
for the
normal
and
the putative
mutant
sequences (Fig.
2). The
result of
this
analysis
showed
that
68 percent
(145/214)
of CF chromosomes
in the
general
patient
population
had
the
AF508
mutation
(Table 2).
In
contrast,
none
(0/198)
of the N chromosomes
had the
deletion
(Table
2)
(X2
=
207,
P
<
10-57.5!),
suggesting
that this
sequence
alteration
is
specific
to CF and that it is the major
mutation
causing
the
disease. No
recombination
has been detected
between
the
AF508
mutation site and CF.
However,
the possibility
that this
sequence
difference corresponded
to a tightly
linked DNA
polymorphism
had
yet
to
be excluded.
Haplotype
analysis.
Extended
haplotypes
based on 23
DNA
markers
were generated
for
the
CF and
N
chromosomes
in
the
collection of families
previously
used
for
linkage
analysis (36).
Assuming recombination
between
chromosomes
of different
haplo-
types,
it was
possible
to construct several
lineages of the
observed
RESEARCH ARTICLES
I075

Table 3.
DNA marker
haplotypes spanning
the CF
locus.
Haplotypes*
CFt
N
Region
AF508
Others
1 2
3 4
5 6
7 8
9
PI
PS
PI
PS
Group
Ia
A A A A
A A
A A
A 10
1
A A A A
A A
-
A
A 3
A
A A A
-
A A
-
A
1
A A A A
-
-
A
- A
1
A
A A A
A A
A A
B
10
1
A
A - A
A A
A A
B 4
A A A A
- A
A A
B
1
A
A
- A
A A
A A
C
1
B
A
A A
A A
A A
A 4
B
A
- A
A A
A A
A
1
B
A
A A
- A
A A
A
1
B A
A A
A A
A A
- 1
B
A
A A - A
A
-
A
1
A
B A
A
A A
A A
A
1
A
D A
A
A A
A A
A
1
A G A A A A
A A A
1
B
B
A A
A A
A A
A
1
B
C
A A
A A
A A
A 2
E B A A
-
-
A
-
A
1
D
B
A A
-
A
-
A
A
1
D
B
B A
A A
A A
A
1
B A
-
A A
A A A B
1
C
A
-
A A A
A A B
1
A
D A A A A A A
B
1
D C
A
A A A
A A
B
1
A
D A A
- A
A A
B
1
D D
-
A A A
A
A
B
1
B B
-
A A A
-
A
B
1
A B
A
A A A
A A E 2
A B
- A
A
A
A A
E
1 1
A
E B
A A A
A A
E
1
A C A A A A
A A B
1
A C - C
-
A A A
B
1
A B A
B
A A
A
-
A
1
B
C
B
A - A A
A/D B
1
Group
lb
A C
-
A
A
A A A A
1
A C
A A
A
A A A
-
1
D C - A A A
A A
B
1
D C
A
A A A
A A
D
1
F C - A A A
A A
B
1
B
C
A A A
A
A
A
B 3
Group
Ic
B
C
A B C A
A D A
1
B C
A
B
C A
A
D B
1
F
C
A B C A
A D B
1
F A A
B
C A A
D
B
1
A
B A B
C A
A
D B
1
B B A
B
C A A
D B
1
B D A B
C A
-
D
C
1
A
B
A B A A
-
D A
1
Group
Id
D
B A A A A
A
C
A
1
B
C
B
C
A A
A C
B
1
Subtotals
57
5 7
1
14
Group
Ha
B A
-
B
B B
A C
B
1
-
B/C B
B B
B A
C B
1
B A
- B
-
B A A/C
B
1
A
B B
B B B
A C B
1
B
B
B B B
B A C
A
3
A C
B
B B B
A C
A
1
A C
B B
B B
-
C A
1
F
C
B
B B
B A C A
1
A C
B
B B B A C
B
1
A C
- B B B
-
C
C
1
B
C
B
B
-
B
A C
C
1
B
C
B B B B
A
C
B
1
B
C
B B B B A C
A
1
B
C
B B B B A
C
D
1
B
C
- B B
B A
C
B
1
B C
B B B B
- C B
1
D C
B B B B
A C B
2
D - B
B - B
A C B
1
F C
B B B
B A C
B
1
C C - B
B B
A C B
1
I076
Table 3
continued.
Haplotypes*
CFt
N
Region
t\F508
Others
1 2 3
4
5 6
7 8
9
PI
PS
PI
PS
A
A A
B
B B
A
C
B
1
B G
A
B
B B
A C
B
1
F
A
-
B
B B
A
C
B
1
B H
-
B
B B
A
C
B
1
B B
-
B
B B
A C
B
1
A B A B
B B
A C
B
1
A B A B
B
B A C
A
1
F D A B
B
B
A C
B
1
C
D
A B B
B
A C
A
1
B D A
B B
B
A C
A
1
B
C
A B
B B
A C
A
2
A C A B
B B
A C
B
1
A C
A B B B
-
C
B
1
A
C
A B
B B
A C
C
1
B
C
A B
B B
A C
B
1
D
B/C
- B B B
A
C
A
2
C C
A
B B B A
C
A
1
D
B
- B
B B
A A/C
B
1
D B A B
- B
A C
B
1
A
G
A
B B B A C
A
1
B
C
- B B B A A/C A
1
A C
B
D B
B A C
B
1
A C
- D -
B A C
B
1
B B B
E
B B
A C
C
1
F
D
A
B
B B
A
C
C
1
A A A
A
A
B A C
D
1
-
B/C A
B
C
B A C
B
1
Group
IIb
A C A
B B B A
B E
1
A C
- B
B B A B B
1
Group
HIc
B D
-
B
-
B
A A
A
1
Subtotals
0
0 6
4
45
Group
IIIa
B
C
B A A
C
B
A
B
1
Group
IIb
B A B A A
C
B A
B
1
B
C
B
A
A
C
B A
A
1
B
C
B A A
C
B A
B
1
B C
-
A A
C
B A
B
2
A
B --A A
C
B A
B 1
A B
-
A
A C
B A C
1
B
B B A
A
C
B
A B
2
D C
B
A
A
C B
A
A
1
A
B B C A
C
B A
B
1
B B A A
A
C
B
A B
2
1
B
B - A A
C
B
A
B
1
1
B
B
A A
A
C B A
A
1
D
A
A A A
C
B A
B
1
D C A A
A
C B
A
B
2
A C
- A A C
B
A
B
1
1
D B A
A A
C
-
A C
1
Group
IIIc
A A
A B
B
C
B A
-
1
F
B B B
B
C
B A
B
1
D B
B B
B
C
B A
A
1
Subtotals
1
0
7
2
17
Group
IV
F C
B A
A
C B C
A
1
B C
A A A
C B
C -
1
A
B
A
A A
C
-
C
B 1
A
H B A -
C -
C
B 1
D
B B B
B
C
B C
B
1
Subtotals
0
0 0
1
4
Group
V
B C B B B
C
A C
A
1
A C
B
B -
-
A
-
A 1
B B B B B C
A C
B
1
B
C
B B
B
C
A
C
B
1
B
C
- B
B
C
A
C
B
1
D - A B
B C
- C B
1
B C A B
C C
A C A
1
B
C - B
C C -
_C
D
1
Subtotals
0
0 2 1
5
SCIENCE,
VOL. 245

Citations
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Journal ArticleDOI
08 Sep 1989-Science
TL;DR: A deletion of three base pairs that results in the omission of a phenylalanine residue at the center of the first predicted nucleotide-binding domain was detected in CF patients.
Abstract: Overlapping complementary DNA clones were isolated from epithelial cell libraries with a genomic DNA segment containing a portion of the putative cystic fibrosis (CF) locus, which is on chromosome 7 Transcripts, approximately 6500 nucleotides in size, were detectable in the tissues affected in patients with CF The predicted protein consists of two similar motifs, each with (i) a domain having properties consistent with membrane association and (ii) a domain believed to be involved in ATP (adenosine triphosphate) binding A deletion of three base pairs that results in the omission of a phenylalanine residue at the center of the first predicted nucleotide-binding domain was detected in CF patients

6,731 citations

Journal ArticleDOI
John W. Belmont1, Paul Hardenbol, Thomas D. Willis, Fuli Yu1, Huanming Yang2, Lan Yang Ch'Ang, Wei Huang3, Bin Liu2, Yan Shen3, Paul K.H. Tam4, Lap-Chee Tsui4, Mary M.Y. Waye5, Jeffrey Tze Fei Wong6, Changqing Zeng2, Qingrun Zhang2, Mark S. Chee7, Luana Galver7, Semyon Kruglyak7, Sarah S. Murray7, Arnold Oliphant7, Alexandre Montpetit8, Fanny Chagnon8, Vincent Ferretti8, Martin Leboeuf8, Michael S. Phillips8, Andrei Verner8, Shenghui Duan9, Denise L. Lind10, Raymond D. Miller9, John P. Rice9, Nancy L. Saccone9, Patricia Taillon-Miller9, Ming Xiao10, Akihiro Sekine, Koki Sorimachi, Yoichi Tanaka, Tatsuhiko Tsunoda, Eiji Yoshino, David R. Bentley11, Sarah E. Hunt11, Don Powell11, Houcan Zhang12, Ichiro Matsuda13, Yoshimitsu Fukushima14, Darryl Macer15, Eiko Suda15, Charles N. Rotimi16, Clement Adebamowo17, Toyin Aniagwu17, Patricia A. Marshall18, Olayemi Matthew17, Chibuzor Nkwodimmah17, Charmaine D.M. Royal16, Mark Leppert19, Missy Dixon19, Fiona Cunningham20, Ardavan Kanani20, Gudmundur A. Thorisson20, Peter E. Chen21, David J. Cutler21, Carl S. Kashuk21, Peter Donnelly22, Jonathan Marchini22, Gilean McVean22, Simon Myers22, Lon R. Cardon22, Andrew P. Morris22, Bruce S. Weir23, James C. Mullikin24, Michael Feolo24, Mark J. Daly25, Renzong Qiu26, Alastair Kent, Georgia M. Dunston16, Kazuto Kato27, Norio Niikawa28, Jessica Watkin29, Richard A. Gibbs1, Erica Sodergren1, George M. Weinstock1, Richard K. Wilson9, Lucinda Fulton9, Jane Rogers11, Bruce W. Birren25, Hua Han2, Hongguang Wang, Martin Godbout30, John C. Wallenburg8, Paul L'Archevêque, Guy Bellemare, Kazuo Todani, Takashi Fujita, Satoshi Tanaka, Arthur L. Holden, Francis S. Collins24, Lisa D. Brooks24, Jean E. McEwen24, Mark S. Guyer24, Elke Jordan31, Jane Peterson24, Jack Spiegel24, Lawrence M. Sung32, Lynn F. Zacharia24, Karen Kennedy29, Michael Dunn29, Richard Seabrook29, Mark Shillito, Barbara Skene29, John Stewart29, David Valle21, Ellen Wright Clayton33, Lynn B. Jorde19, Aravinda Chakravarti21, Mildred K. Cho34, Troy Duster35, Troy Duster36, Morris W. Foster37, Maria Jasperse38, Bartha Maria Knoppers39, Pui-Yan Kwok10, Julio Licinio40, Jeffrey C. Long41, Pilar N. Ossorio42, Vivian Ota Wang33, Charles N. Rotimi16, Patricia Spallone43, Patricia Spallone29, Sharon F. Terry44, Eric S. Lander25, Eric H. Lai45, Deborah A. Nickerson46, Gonçalo R. Abecasis41, David Altshuler47, Michael Boehnke41, Panos Deloukas11, Julie A. Douglas41, Stacey Gabriel25, Richard R. Hudson48, Thomas J. Hudson8, Leonid Kruglyak49, Yusuke Nakamura50, Robert L. Nussbaum24, Stephen F. Schaffner25, Stephen T. Sherry24, Lincoln Stein20, Toshihiro Tanaka 
18 Dec 2003-Nature
TL;DR: The HapMap will allow the discovery of sequence variants that affect common disease, will facilitate development of diagnostic tools, and will enhance the ability to choose targets for therapeutic intervention.
Abstract: The goal of the International HapMap Project is to determine the common patterns of DNA sequence variation in the human genome and to make this information freely available in the public domain. An international consortium is developing a map of these patterns across the genome by determining the genotypes of one million or more sequence variants, their frequencies and the degree of association between them, in DNA samples from populations with ancestry from parts of Africa, Asia and Europe. The HapMap will allow the discovery of sequence variants that affect common disease, will facilitate development of diagnostic tools, and will enhance our ability to choose targets for therapeutic intervention.

5,926 citations

Journal ArticleDOI
21 Jun 2002-Science
TL;DR: It is shown that the human genome can be parsed objectively into haplotype blocks: sizable regions over which there is little evidence for historical recombination and within which only a few common haplotypes are observed.
Abstract: Haplotype-based methods offer a powerful approach to disease gene mapping, based on the association between causal mutations and the ancestral haplotypes on which they arose. As part of The SNP Consortium Allele Frequency Projects, we characterized haplotype patterns across 51 autosomal regions (spanning 13 megabases of the human genome) in samples from Africa, Europe, and Asia. We show that the human genome can be parsed objectively into haplotype blocks: sizable regions over which there is little evidence for historical recombination and within which only a few common haplotypes are observed. The boundaries of blocks and specific haplotypes they contain are highly correlated across populations. We demonstrate that such haplotype frameworks provide substantial statistical power in association studies of common genetic variation across each region. Our results provide a foundation for the construction of a haplotype map of the human genome, facilitating comprehensive genetic association studies of human disease.

5,634 citations

Journal ArticleDOI
John W. Belmont1, Andrew Boudreau, Suzanne M. Leal1, Paul Hardenbol  +229 moreInstitutions (40)
27 Oct 2005
TL;DR: A public database of common variation in the human genome: more than one million single nucleotide polymorphisms for which accurate and complete genotypes have been obtained in 269 DNA samples from four populations, including ten 500-kilobase regions in which essentially all information about common DNA variation has been extracted.
Abstract: Inherited genetic variation has a critical but as yet largely uncharacterized role in human disease. Here we report a public database of common variation in the human genome: more than one million single nucleotide polymorphisms (SNPs) for which accurate and complete genotypes have been obtained in 269 DNA samples from four populations, including ten 500-kilobase regions in which essentially all information about common DNA variation has been extracted. These data document the generality of recombination hotspots, a block-like structure of linkage disequilibrium and low haplotype diversity, leading to substantial correlations of SNPs with many of their neighbours. We show how the HapMap resource can guide the design and analysis of genetic association studies, shed light on structural variation and recombination, and identify loci that may have been subject to natural selection during human evolution.

5,479 citations


Additional excerpts

  • ..., Hirschsprung’s disease, cystic fibrosis) (13, 24)....

    [...]

Journal ArticleDOI
29 Jun 1995-Nature
TL;DR: A minimal cosegregating region containing the AD3 gene is defined, and at least 19 different transcripts encoded within this region corresponds to a novel gene whose product is predicted to contain multiple transmembrane domains and resembles an integral membrane protein.
Abstract: Some cases of Alzheimer's disease are inherited as an autosomal dominant trait. Genetic linkage studies have mapped a locus (AD3) associated with susceptibility to a very aggressive form of Alzheimer's disease to chromosome 14q24.3. We have defined a minimal cosegregating region containing the AD3 gene, and isolated at least 19 different transcripts encoded within this region. One of these transcripts (S182) corresponds to a novel gene whose product is predicted to contain multiple transmembrane domains and resembles an integral membrane protein. Five different missense mutations have been found that cosegregate with early-onset familial Alzheimer's disease. Because these changes occurred in conserved domains of this gene, and are not present in normal controls, they are likely to be causative of AD3.

4,110 citations

References
More filters
Journal ArticleDOI
29 Jan 1988-Science
TL;DR: A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction, which significantly improves the specificity, yield, sensitivity, and length of products that can be amplified.
Abstract: A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.

17,663 citations

Journal ArticleDOI
20 Dec 1985-Science
TL;DR: Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia, using primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase of target DNA copies.
Abstract: Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia. The first involves the primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase (220,000 times) of target DNA copies. In the second technique, the presence of the beta A and beta S alleles is determined by restriction endonuclease digestion of an end-labeled oligonucleotide probe hybridized in solution to the amplified beta-globin sequences. The beta-globin genotype can be determined in less than 1 day on samples containing significantly less than 1 microgram of genomic DNA.

9,107 citations

Book
01 Jan 1972
TL;DR: The metabolic basis of inherited disease, the metabolic basis for inherited disease as mentioned in this paper, The metabolic basis in inherited disease and inherited diseases, and inherited disease diagnosis and management, in the context of inherited diseases
Abstract: The metabolic basis of inherited disease , The metabolic basis of inherited disease , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی

7,724 citations

Journal ArticleDOI
08 Sep 1989-Science
TL;DR: A deletion of three base pairs that results in the omission of a phenylalanine residue at the center of the first predicted nucleotide-binding domain was detected in CF patients.
Abstract: Overlapping complementary DNA clones were isolated from epithelial cell libraries with a genomic DNA segment containing a portion of the putative cystic fibrosis (CF) locus, which is on chromosome 7 Transcripts, approximately 6500 nucleotides in size, were detectable in the tissues affected in patients with CF The predicted protein consists of two similar motifs, each with (i) a domain having properties consistent with membrane association and (ii) a domain believed to be involved in ATP (adenosine triphosphate) binding A deletion of three base pairs that results in the omission of a phenylalanine residue at the center of the first predicted nucleotide-binding domain was detected in CF patients

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01 Jan 1989

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Trending Questions (1)
Can cystic fibrosis be traced to a single gene?

Extended haplotype data based on DNA markers closely linked to the putative disease gene locus suggest that the remainder of the cystic fibrosis mutant gene pool consists of multiple, different mutations.