Diabetologia (1986) 29: 667- 669
Diabetoiogia
Rapid communications
Restriction fragment length polymorphism (RFLP) of the human insulin
receptor gene in Japanese: its possible usefulness as a genetic marker
J. Takeda 1, Y. Seino 2, Y. Y o s h i m a s a 1, H. F u k u m o t o 1, G. K o h 1, H. K u z u y a ~, H. I m u r a 1 and S. Seino 3
Second Division, Department of Internal Medicine and
2Division of Metabolism and Clinical Nutrition, Kyoto University School of Medicine, Kyoto, Japan;
3 Department of Biochemistry/Molecular Biology, University of Chicago, Chicago, Illinois, USA
Summary. Restriction fragment length polymorphism of the
human insulin receptor gene was analyzed with a 4.2 Kb
cDNA probe in Japanese normal subjects and Type 2 (non-in-
sulin-dependent) diabetic patients. Restriction endonuclease
Rsa I digestion showed polymorphism of the human insulin
receptor gene, with a band at 6.7 Kb, 6.2 Kb or 3.6 Kb. The
frequency of the 6.7 Kb band was less than that in Caucasians.
Furthermore, 15% of all the Japanese subjects examined
lacked a 3.6 Kb band, which is commonly found in Cauca-
sians. We have also detected restriction fragment length poly-
morphism in the human insulin receptor gene by Pvu II or Stu
I digestion. Although no significant association of restriction
Genetic factors contribute to susceptibility o f diabetes
mellitus. One o f the characteristic features o f Type 2
(non-insulin-dependent) diabetes mellitus is the "insu-
lin resistant state" due to a possible target tissue defect
in insulin action. It is, therefore, reasonable to assume
that the structural a b n o r m a l i t y o f the h u m a n insulin
receptor ( I N S R ) gene a n d its flanking region m a y pro-
duce insulin receptor dysfunction through either abnor-
mal moiety of the I N S R or a b n o r m a l gene expression,
either o f which in turn might cause an "insulin resistant
state". Recently, the p r i m a r y structure o f the I N S R gene
has b e e n determined f r o m the nucleotide sequence of
the h u m a n insulin receptor c D N A [1]. Furthermore, the
I N S R gene has been s h o w n to have restriction fragment
length p o l y m o r p h i s m (RFLP) in Caucasians [2]. Since
the entire sequence o f the I N S R gene is not k n o w n at
present, it would be interesting to see whether there is
any difference in the R F L P pattern o f the I N S R gene
between different ethnic groups, a n d also whether there
is any correlation b e t w e e n R F L P a n d Type 2 diabetes.
In the present study, we have e x a m i n e d the pattern and
frequency of R F L P in J a p a n e s e n o r m a l subjects and
Type 2 diabetic patients with Southern blotting analysis
[3] using h u m a n insulin receptor c D N A .
© Springer-Verlag 1986
fragment length polymorphism with Type2 diabetes was
found in the present study, our results suggest that the re-
striction fragment length polymorphism in the human insulin
receptor gene varies among ethnic groups, and that the re-
striction fragment length polymorphism linked tO the human
insulin receptor gene might be a useful marker for the linkage
study of the genes located close to the human insulin receptor
gene on chromosome 19.
Key words: Restriction fragment length polymorphism
(RFLP), insulin receptor gene, Type2 diabetes mellitus,
Southern blotting, racial difference.
Subjects and methods
Nineteen Japanese normal subjects, 10 males and 9 females, aged 32
to 80, and 26 Type 2 diabetic patients, 15 males and 11 females, aged
40 to 61, were studied. Genomic DNA was isolated from nucleated
white blood cells Obtained from 10 ml of heparinized peripheral ve-
nous blood. Three to five p~gof the DNA samples were digested with
an excessive dose of restriction endonuclease Rsa I, Pvu II, or Stu I
(Toyobo, Osaka, Japan), and subjected to 0.7°/(0or 1% agarose gel elec-
trophoresis. Separated DNA fragments were transferred to nitrocellu-
lose filters (Schleicher and Schell) by the method of Southern [3]. Fil-
ters were prehybridized for 24h at 42°C in 50% Formamide,
5 x NaCl/Cit, 5 x Denhardt solution, 50 mmol/I sodium phosphate
(pH 6.5), 0.25% NaDodSO4, and 250 l.tg/ml sonicated salmon sperm
DNA. Hybridization was successivelyperformed for 24 h at 42 °C in
50% Formamide, 5 x NaC1/Cit, 2 x Denhardt solution, 20 mmol/1 so-
dium phosphate (pH 6.5), 0.25% NaDodSO4, 100 ixg/ml sonicated
salmon sperm DNA, 10% Dextran sulfate and nick-translated cDNA
by [32p]-a-dCTP(Amersham) [4]. The probe used, which was kindly
supplied by Dr. G.I.Bell (Chiron Corporation, Emeryville, Calif,
USA) is 4.2 kb and covers from amino acid 295 to the poly A tract of
the INSR gene. Filters were washed with 0.1 x NaC1/Cit and 0.1%
NaDodSO4 at room temperature for 1 h and then at 60 °C for %-1 h,
and exposed to X-ray films with an intensifying screen at - 70 °C for
3 to 7 days.
Results and discussion
As shown in Figure I a, the Rsa I digestion showed allel-
ic p o l y m o r p h i s m with a b a n d at 6.7 Kb, 6.2 K b or
668 J. Takeda et al.: Restriction fragment length polymorphism in Japanese

Fig. I a-e. Restriction fragment

length polymorphisms (RFLP)
linked to the human insulin receptor
(INSR) gene are shown, a Rsa I
polyrnorphism: the 3,6 kb, the 6.2 kb
and the 6.7 kb bands are polymor-
phic and noted in lanes 1, 3; 2, 3, 4;
and 2 respectively. Other smaller
bands were constantly ob-
served, b Stu I polymorphism: the
polymorphic 7.2 kb band is observed
in lane 1, and the 6.6 kb band in lane
2. e Pvu II polymorphism: lane 2
lacks a 3.8 kb band and has an addi-
tional 4,4 kb band

Table 1. RFLP of Japanese normal subjects and Type 2 diabetic pat- stricted DNA. To avoid possible cross-hybridizations
ients using endonuclease Rsa I, Stu I or Pvu II with INSR-like sequences, we have made RFLP analy-
Subjects (n) ses under as stringent conditions as possible. A strong
signal, therefore, might be generated due to the overlap-
a RsaI 6.7kb 6.2kb 3.6kb
ping of fragments of the same molecular size or much
Normal subjects (19) 21.1 (4) 73.7 (14) 84.2 (16) continuous homology to the cDNA. Likewise, a frag-
Type 2 diabetic patients (21) 9.5 (2) 71.4 (15) 85.7 (18) ment with less homology would show a weak signal due
Total (40) 15 (6) 72.5 (29) 85 (34)
to stringent conditions. Moreover, we have analyzed a
b Stu I 7.2 kb 6.6 kb few pedigrees to ascertain RFLP and obtained an infor-
Normal subjects (18) 11.1 (2) 88.8 (16)
mation of co-dominant inheritance. On the other hand,
Type 2 diabetic patients (17) 11.7 (2) 88.2 (15) when we compared the RFLP pattern frequencies of
Total (35) 11.4 (4) 88.5 (31) normal subjects and Type 2 patients, no significant as-
sociation of RFLP with Type 2 diabetic patients was
c Pvu II 4.4 kb 3.8 kb
found (Table 1).
Normal subjects (17) 17.6 (3) 82.3 (14) As a genetic marker of diabetes mellitus, RFLP of
Type 2 diabetic patients (26) 11.5 (3) 92.3 (24) the insulin gene has been extensively studied [5]. Some
Total (43) 13.9 (6) 88.3 (38)
reported the association of RFLP with susceptibility to
Percentages of individuals with each polymorphic band are indicated diabetes mellitus [6, 7], whereas others have not [8, 9],
and therefore, the significance of RFLP is still elusive. It
has become clear from these studies, however, that there
3.6 Kb. The frequency of these bands in Japanese nor- are racial differences in RFLP of the insulin gene [6-9].
mal subjects (Table 1 a) is different from that reported
previously in Caucasians [2]. We found a low frequency The present study using Southern blotting technique
of the 6.7 Kb band (21.1%) and a high frequency of the [3] has demonstrated that the INSR gene is markedly
6.2 Kb band (73.7%); Caucasians showed almost the polymorphic both in normal and diabetic Japanese sub-
same frequency of the 6.7 Kb and the 6.2 Kb band [2]. jects. When compared with RFLP of the INSR gene in
Furthermore, 15% of all the subjects examined lacked a Caucasians [2], racial differences are evident. It is still
3.6 Kb band, which is commonly observed in Cauca- unknown, however, where polymorphic regions exist in
sians [2]. These results suggest that the RFLP in the the INSR gene and what is its possible significance in
INSR gene obtained by Rsa I digestion varies substan- the expression of the gene, because organization of the
tially among ethnic groups. The Stu I digestion revealed INSR gene has not been elucidated yet.
RFLP with allelic fragments at 7.2 Kb and 6.6 Kb. The The present study has also suggested that there is no
frequency of the 7.2Kb band is approximately 11% obvious difference between Type 2 diabetic patients and
(Fig. I b, Table 1 b), compatible with that found in Cau- normal subjects studied. It is yet possible, however, that
casians (15%) (personal communication). Pvu II poly- the genotypic polymol~hism in the INSR gene has a re-
morphism has been also detected as a band of 3.8 Kb lation with the phenotypic polymorphism such as that
(88.3%) or 4.4Kb (13.9%), as shown in Figure l c and involved in the production of abnormal insulin receptor
Table 1 c. In addition, various signal intensities have moiety or abnormal gene expression. We might indi-
been observed in both polymorphic and non-polymor- cate, therefore, that RFLP of the INSR gene is a useful
phic bands, as is the case with Rsa I, Stu I and Pvu II re- marker for the linkage analysis of the relationship of the
J. Takeda et al.: Restriction fragment length polymorphism in Japanese
INSR gene with other genes located close to the INSR
gene on chromosome 19 [10] in the study of the genetic
background of diabetes mellitus.
Acknowledgement. We wish to express our appreciation to Dr.
G. I. Bell for having supplied us with the cDNA clones, to T. Kayano
for helpful discussion, to S. Ogaya and N. Sawai for technical assis-
tance, and to H. Tachikawa for secretarial service.
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Received: 10 June 1986
and in revised form: 14 July 1986
Dr. Jun Takeda
Second Division
Department of Medicine
Kyoto University, School of Medicine
54 Shogoin Kawahara-cho
Sakyo-ku, Kyoto 606
Japan