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Population variation in molecular polymorphisms of the short arm of the human X chromosome.

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AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 85:329-334 (19911
Population Variation in Molecular Polymorphisms of the Short
Arm of the Human X Chromosome
S.S.PAF'IHA, S.S. MASTANA, D.F. ROBERTS,
G.C. ONYEMELUKWE, AND S.S.BHATTACHARYA
Department of Human Genetics, University of Newcastle u on Tyne,
Newcastle upon Tyne NE2 4AA, England (S.S.P.,D.F.R., {S.B.);
Immunology Unit, Department of Medicine, Ahmadu Bella University
Hospital, Zaria, Nigeria (G.C.O.)
KEY WORDS
ABSTRACT
Five DNA probes (RC8, 754, X J 1-1,pert 87.8, and L1.28)
from the short arm of the human X chromosome were investigated in samples
from five populations (English, Nigerian, Chinese, Muslim, and Hindu from
India). The variation in the allele frequencies of several probes between
different groups was significant. The average heterozygosity in females of the
five populations ranged from 32% to 51%. The genetic distance between the
five groups was compatible with that using traditional polymorphic systems.
There is an interesting suggestion of longitudinal cline for allele "2 (9 kb)
detected with probe L1.28. The X-linked RFLPs are useful genetic markers for
anthropological studies.
Serological and biochemical polymorphisms have been extensively used to understand the phylogenetic relationships and the
genetic structure of various human opulations. In the last decade the search or new
enzyme/protein pol morphisms has made
little advance, main y because of the limitation of the technique of electrophoresis t o
differentiating only variation that accompanies a net change in the charge of the protein
molecule. However, the same techni ue has
com letely transformed the study of uman
DNL! polymorphisms, electro horesis being
ap lied to separate the smal fragments of
dif ferent sizes that result after treatment
with enzymes (restriction endonucleases) t o
cleave the total genomic DNA. The mutations that alter the nucleotide sequences,
both coding and noncodin sequences,
vide fragments of DNA of ifferent lengt in
polymorphic frequencies (Botstein et al.,
1980). Several hundreds of these restriction
fragment length polymorphisms (RFLPs)
are now known, widely distributed in the
human enome, and provide a powerful
means o carrier detection and prenatal diagnosis of various inherited diseases with
unidentified biochemical lesions (Hofker
et al., 1985).
Clinically the short arm of the human X
f!
T
P
cf
B
@ 1991 WILEY-LISS. INC
R
chromosome is of great interest. Several
highly polymorphic DNA probes localised
there have been isolated from genomic, Xchromosome-sorted, and hybrid cell line libraries (Willard et al., 1985; Wieacker et al.,
1984). These DNA olymorphisms have
helped to map man zlinked diseases such
as ichthyosis, Duc ennemecker muscular
7l
probes in healthy populations. There is no
conclusive heterogeneity in frequency of any
of these probes in European populations,
except for the 5.3 kb allele identified by RC8
probe (Schurmann et al., 1987).However, for
some of these probes, there are much wider
differences in fre uency in different races
(Summers, 1987; apiha et al., 1988; Wadhwa et al., 1989).Recent investigations with
DNA probes from the short arm of the X
chromosome linked t o the Duchenne muscular dystrophy gene have shown significant
variation among populations in different
continents, suggesting that it is important to
Y-
3
Received November 20,1989; accepted November 30, 1990.
S.S.
Mastana is now at Department of Human Sciences, Loughborough University of Technology, Loughborough, England.
330
S.S.PAPIHA ET AL
TABLE I . Allele frequencies of DNA polymorphisms for 754, X J l - I , 87.8, L.1.28, a n d RC8 in five population groups
European
(Northeast
England)
Probe and allele
754 (DXS84)
*1 (12 kb)
*2 (9 kb)
XJ1-l (DXS206)
*1 (3.8 kb)
*2 (3.1 kb)
Pert 87.8 (DXS164)
*I (3.8 kb)
*2 (1.1 and 2.7 kb)
L.1.28 (DXS7)
*I (12 kb)
"2 (9 kb)
RC8 (DXS9)
* I (3.2 kb)
*2 (5.3 kb)
*3 (3.0 kb)
(172)'
0.593 f 0.0372
0.407
(139)
0.252
0.748
+ 0.037
(150)
0.280
0.720
+ 0.037
(20)
0.318 f 0.104
0.682
(28)
0.857 0.066
0.143
(28)
0.214 f 0.078
0.786
(26)
0.538 0.098
0.462
(45)
0.560 f 0.074
0.440
(110)
0.773 f 0.040
0.227
*
5.1
9.9
16.9
16.5
3.6
25.1
(62)
0.806
0.194
*
(41)
0.875 f 0.052
0.115 0.050
0.010 f 0.016
754
x2
P<
European vs. African
European vs. Indians
European vs. Chinese
Indians vs. African
Indians vs. Chinese
Chinese vs. African
Asiatic Indians
Muslim
Hindu
(Hyderabad)
(Bombay)
African
(Nigeria)
33.1
5.7
2.1
15.5
0.0
13.5
0.0001
0.05
NS
NS
0.001
(54)
0.907 f 0.040
0.093
(53)
0.339 f 0.065
0.661
(51)
0.471 f 0.070
0.529
(57)
0.421 f 0.065
0.579
(59)
0.448 f 0.065
0.552
(59)
0.763 0.055
0.237
(53)
0.583 f 0.068
0.415
(51)
0.392 0.068
0.608
(56)
0.857 f 0.047
0.125 0.044
0.017 0.017
L1.28
x2
P<
(45)
0.378 f 0.072
0.622
(61)
0.721 f 0.057
0.279
(65)
0.661 0.059
0.338
Pert 87.8
x2
P<
P<
0.001
(62)
0.742 k 0.056
0.258
-
XJ1-1
0.05
0.001
0.001
0.001
NS
0.0001
+ 0.050
*
x2
Chinese
(Singapore)
0.2
12.2
33.2
6.3
7.0
18.0
NS
0.001
0.0001
0.05
0.05
0.0001
*
+
+
+
(65)
0.538
0.462
RC8
~
4.7
29.1
2.0
0.7
8.9
1.0
0.05
0.0001
NS
NS
0.01
NS
+ 0.062
-
x2
P<
7.7
0.1
9.8
10.1
14.3
0.0
0.05
NS
0.01
0.01
0.001
NS
:Numbers of chromosomes tested are in parentheses.
Standard errors.
establish the frequencies of these RFLPs in
various ethnic grou s in order to identify
which probe is more ikely to be informative
for clinical use in a particular population
(Papiha et al., 1989).In the present study we
have investi ated five DNA probes from the
short arm o the X chromosome in four racially different populations to establish the
extent of genetic diversity and affinity
among them.
P
f
MATERIALS AND METHODS
The blood sam les from English (European), Nigerian African), Singa ore Chinese, and Asiatic Indians (Hydera ad Muslim and Bombay Hindu) populations were
collected as part of ongoing genetic studies
(Wadhwa et al., 1989). Ten milliliter blood
sam les were collected in tubes containing
ED?A as anticoagulant. The samples were
frozen and transported in dry ice to our
laboratory in Newcastle upon Tyne. The Chinese samples were transported as DNA from
Singapore, but for the other samples the
extractions were carried out in Newcastle.
DNA was extracted from the lymphocytes
by the method of Kunkel et al. (1982). The
P
\
DNA sam les were digested to completion
using Taq and PstI enz mes following the
instructions given by t e manufacturers.
The digested samples were subjected to horizontal electrophoresis using 0.8% agarose
gel. The gels were denatured, neutralised,
and transferred to nitrocellulose filter by the
method of Southern (1975).The DNA probes
(754, XJ 1-1, 87.8, L1.28 and RC8) were'
labeled by random priming to a s ecific activity of 10' c d p g of DNA, an then the
bridised with radioactive
filters were
robes for 18 Kr at 65°C in ~ x S S C 5, x
benhart's solution, 0.1% SDS, and 100 pg
denatured salmon sperm DNA. The filters
were washed and autoradiographed on Fuji
film.
P
K
B
R
RESULTS
The number of chromosomes tested for
each opulation, the allele frequencies, and
the R LPs identified by each probe are given
in Table 1. For the female samples in each
population, there was a good agreement of
observed heterozy ote numbers with those
expected under ardy-Weinberg equilibrium.
P
w
331
HUMAN X CHROMOSOME MOLECULAR POLYMORPHISMS
z
m
z
H
a:
W
4 W
a:
z"
H
v1
z w
Fig. 1. Longtudinal cline of allele *2 (9 kb) detected with X-chromosome probe L 128.
Probe 754 (DXS 84)
The RFLPs identified by 754 were fragments of 12 kb (allele "1)and 9 kb (allele *2).
In European opulations the allele "2 ranges
from 34% in 8ermans to 46% in French, and
the frequency of 41% found in our Sam le
from Northeast England fits well in t is
ran e. In both the Indian populations this
alle e is relatively low in frequency, ran n
from 19%to 26%, but is more infrequent 9%
in the Chinese. In the Nigerians the frequency of this allele is the highest so far
re orted (68%). The distributions of 754
RPLPs are not sigificantly different between
the two Indian samples or between the Indian and Chinese samples, but the paired
comparisons of all other populations show
significant heterogeneity.
Probe XJ 1-1 (DXS 206)
The probe XJ 1-1identifies a diallelic pol morphism (3.8 kb allele "1 and 3.1 kb allefe
"2). The allele *2 is most common in the
English sample (75%).In the Indian population its frequency ranges from 53% to 66%, a
frequency similar to that found in the Chinese sample. However, in the Nigerian population this allele is infrequent (14%),a fre-
K
P
r5
quency difference from the other three racial
grou s that is highly significant (European
vs. dgerian, x2 = 33.1,P < 0.0001;Indianvs.
Nigerian, x2 = 15.5, P < 0.001; Chinese vs.
Nigerian, x2 = 13.2,P < 0.001).
Probe pert 87.8 (DXS 164)
Like the previous two probes, 87.8 also
identifies a diallelic polymorphism. The allele "1 is characterized by fragment size 3.8
kb and the allele *2 by two fragments 2.7 and
1.1 kb. The difference between the fre uencies of the allele *2 in the English and igerian populations is slight and not significant
(72% and 79%, respectively) and so is that
between the two Indian samples (Muslim,
58% and Hindu, 42%).However, in the Chinese the frequency of allele *2 is low (28%),
and a similar low frequency has also been
reported for J a anese (Akita et al., 1987).
The frequency iifferences between the Enlish and Indian, English and Chinese, and
Ehinese and Nigerian are all highly significant.
Probe L 1.28 (DXS 7)
The fragment size of allele "1identified by
L 1.28 is 12 kb and for allele "2 is 9 kb. The
xr
332
S.S.PAPIHA ET AL.
TABLE 2. Heterozygosity of D N A polymorphisms for Pert 87.8, 754, XJl-1, and L.1.28 in four differentpopulations
Restriction
enzyme
Probe
Pst
Obs
EXP
TaqI Obs
EXP
Pert 87.8
TaqI Obs
EXP
L 1.28
TaqI Obs
EXP
Average heterozygosity
754
XJ1.l
European
Asiatic
Indians
African
Chinese
0.479
(0.483)
0.406
(0.377)
0.297
(0.308)
0.340
(0.350)
0.381
0.370
(0.313)
0.500
(0.448)
0.500
(0.488)
0.579
(0.495)
0.487
0.400
(0.434)
0.143
(0.245)
0.286
(0.336)
0.462
(0.497)
0.323
0.071
(0.168)
0.769
(0.470)
0.563
(0.402)
0.632
(0.447)
0.509
allele "2 reaches its hi hest frequency in the
Indian samples (Musfm, 55% and Hindu,
61%),followed by the Nigerian and Chinese
samples (46% and 34%, respectivel 1. The
lowest frequency (23%)occurs in the nglish
population. The difference between the Indian and English samples is highly significant (x2 = 29.1, P < 0.0001). The gene frequencies in the Chinese of Singa ore is
similar to those in Thais and Han hinese
(Summers, 1987). Figure 1 suggests that
there may be an east-west cline.
Probe RC8 (0x5' 9)
The RFLPs identified by RC8 show two
common alleles (allele *1, 3.2 kb; and allele
*2,5.3 kb) and a third rare allele (3.9 kb). The
lowest frequency of allele "2 occurs in the
English sample (12%) and the highest frequency in the Nigerian and Chinese sam les
(44% and 46%, respectivel 1. In the Injian
samples the frequency of a1 ele *2 is intermediate, but, though the Muslim frequency of
allele "2 is twice (24%) that in the Hindu
sample (12%),this difference is not significant. The frequency differences between the
English and Chinese, between Indian and
Chinese, and between Indian and Nigerian
are all significant. The Thai, Papua New
Guineans, and Australian aborigines show
frequencies ranging from 17% to 24%, but
the Han Chinese have a very low frequency
of this allele (4%) and this is significantly
different from other PO ulations (Summers,
1987).The allele "3 (3 k ) of this system was
first described in the French population
(Rahuel et al., 19841, but the same allele
outside Euro e was first seen in the Hindu
population o Bombay, India.
E
EUROPEAN
INDIAN
1
8
9
I
CHINE
AFRICAN
Fig. 2. Genetic relationship of European, African,
Mon oloid, and Asiatic Indian populations (Harpending
and fenkins' Method, 1973).
somes analysed, so the sampling errors are
relatively high and the interpretation of the
results requires caution. However, all five
RFLPs studied here show conclusive differences in frequency among the different racial
groups. Similar variation has also been demonstrated in other studies of other RFLPs
(Bowcocketal., 1987; Summers, 1987; Shimmot0 et al., 1988). Although the differences
between the two Indian samples are not
significant] the observed slight variation in
frequency in all the RFLP systems suggests
the possibility of a considerable local variation in India.
To assess the genetic affinity among the
four racial roups, measures that analyse
DISCUSSION
several alle e frequencies simultaneously
The present estimates of allele frequencies were adopted. Genetic affinity and differenare based on limited numbers of chromo- tiation may be measured by relative het-
g
P
K
333
HUMAN X CHROMOSOME MOLECULAR POLYMORPHISMS
TABLE 3. Huruendine's distance matrix
English
European (English)
West Africa
India (Muslim)
India (Hindu)
Singapore (Chinese)
West Africa
-
0.456
0.444
0.661
0.229
0.355
i
CPP
P
R
a
P
Hindu
Chinese
0.053
0.156
0.200
-
-
0.448
0.150
erozygosity and the allele frequency. The
observed and expected heterozygosity for
four RFLP systems in the females of the four
racial groups are given in Table 2. The exected heterozygosity ranges in English
From 30% to 48%; Nigerians, 24% to 50%;
Indians, 21% to 50%; and Chinese, 16% to
47%. The average heterozygosity (H) also
varies from 32% to 51%. There are as et no
estimates of heterozygosit available fyor the
DNA polymor hisms o f t e X chromosome
with which t e present estimates can be
compared.
Genetic affinity of the four samples of the
different racial groups may be further assessed by another measure of differentiation. The euclidian distance between each
pair of o ulations was computed using the
metho o Harpending and Jenkins (1973).
The genetic relationship of these four populations is shown as a dendrogram (Fig. 2)
derived from the distance matrix (Table 3) b
using the complete linkage method (Sneat
and Sokal, 1973). The two Indian PO ulations form a single cluster and show af inity
to the Europeans, while the African PO ulation is the most remote from all the ot ers.
The genetic relationship of the four racial
groups shown by these five DNA polymorphisms appears similar to that by analysis
with several traditional polymorphic systems (Nei and Roychoudhury, 1974).
For probe L 1.28 several representative
populations of the world have been investiated (Figure 1).The frequency of allele *2 (9
b fragment) in European populations
ranges from 23% to 30% from which level
there appears to be a west-east cline of
increasing frequency to the highest, 64%
(Summers, 1987).
To conclude, the probes of the short arm of
human X chromosome show a wide ran e of
variation in frequencies of various RF Ps.
Explanations for this can only be s eculative. These DNA polymor hisms are t ought
to be functionally neutra so their variation
may derive from historical and prehistoric
movements and settlement or some early
K
Muslim
K
f
h"
random events, e.g., founder effect or drift.
Or it may be that they are linked with disease susceptibility genes and so are subject
to different selective pressure in different
populations and environments. Whatever
their ex lanation, this analysis of X-linked
DNA PO ymorphisms confirms that RFLPs
are a useful anthropological tool to help elucidate genetic affinity among human populations.
P
ACKNOWLEDGMENTS
ateful to our colleagues from
N. Saha from Singapore for
helping us with the blood samples and DNA
respectively.
LITERATURE CITED
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Friedlander JS, and Cavalli-Sforza LL (1987)Study of
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Harpending HC, and Jenkins T (1973)Genetic distance
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and PL Workman (eds):&ethods and theories of Anthropological Genetics. Alburquerque: University of
New Mexico Press, pp. 177-179.
Hofker MH, Wa enaar MC, Goor N, Bakker B, Vanommen GJB, andPPearson PL (1985) Isolation of probes
detectin restriction fragment length polymorphisms
from X ckromosome specific libraries: potential use for
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Genet. 70:148-156.
Kunkel LM, Tantravhi U, Eisenhard M, and Latt S
(1982)Regional localization on the human X of DNA
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S.S. PAPIHA ET AL
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Southern E (1975) Detection of s ecific sequences as
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