THE SCREENING OF HSD17B5 GENE AND DERMATOGLYPHIC STUDY IN PCOS

WOMEN

Arman Firoz, Prathap Srirangan, Nishu Sekar, V.G.Abilash*

Division of Biomolecules and Genetics,

School of Biosciences and Technology,

VIT University, Vellore

abilash.vg@vit.ac.in

ABSTRACT: One in every four couples had been found to be

suffering from infertility in developing countries. Polycystic

ovary disease is an endocrine disorder in female that can be one

of the prominent causes of female infertility. The objective of

our study was to determine the single nucleotide polymorphisms of

the 17b-hydroxysteroid dehydrogenase type 5 genes in PCOS

patients of South Indian population and to analyze the

dermatoglyphics pattern variation of patients with PCOS compare

to normal female to find any relation between PCOS cases and

dermatoglyphics. 10 PCOS patients and 10 controls were studied in

this study. Blood samples were taken to perform Molecular study

and dermatoglyphic prints was taken to analyses patterns.

Genotyping of HSD17B5 in PCOS patients and controls was carried

out by the polymerase chain reaction method. Our studies suggest

that there is no relation of HSD17B5 variants with the

development of PCOS in the South Indian population, whereas the

dermatoglyphics study showed significant variation in PCOS

patients and normal control. This can be of great advantage for

clinicians as a non-invasive technique for screening, while to

the patients a relief from financial and psychosocial stress at

the time of treatment.

1. INTRODUCTION

Polycystic ovary syndrome

(PCOS) is one of the

genetically complex and the

very most common endocrine

disorders (hormonal disorders)

in females of reproductive age

of 12–45 years and affects

about 1 in 15 women worldwide.

Although the clinical features

of polycystic ovary syndrome

(PCOS) are diverse, the

classical characteristic

features of this syndrome

include anovulation, hyper

production of androgen and

obesity. Some studies have

shown that PCOS is related

with insulin resistance and

diabetes. PCOS may not be

diagnosed until well into

adulthood, even though it may

start showing in adolescence.

The factors which contribute

to the aetiology of PCOS are

believed to be both genetic

predisposition as well as life

style.

Major enzymes of the

androgen metabolism pathway

encodes number of genes, such

as 17b-hydroxysteroid

dehydrogenase type 5,

cytochrome P19 (CYP450,

CYP11a), have been analyzed,

and relation have been

reported. The activity of

HSD17b5 is inevitable for the

testosterone biosynthesis and

it is located at chromosome

10, location 10p15-p14. In

females, it is mainly found in

the uterine endometrium

epithelial cells, corpora

lutea and also in the mammary

gland. Whereas in males, major

expression was found in the

adrenal gland and in Leydig

cells of testis as well. The

HSD17B5 gene is also involved

in metabolism of estrogen.

Dermatoglyphics (DGs) is

the study of epidermal ridge

patterns of palms,

fingerprints and soles. The

term dermatoglyphics is

derived from Greek derma

(skin) and glyphic (carvings).

Dermatoglyphics had wide

spread medical interest during

last few decades. Screening of

genetically transmitted

diseases by using

dermatoglyphics as a

diagnostic tool is becoming

popular since, Ridge formation

is genetically determined.

This is an attempt made

probably for the first time to

study dermatoglyphics in

infertile female patients with

PCOS.

In this present study we

examined the functional impact

of HSD17B5 in developing PCOS

and attempt to study

dermatoglyphics in patients

with PCOS.

2. MATERIAL AND METHODS

A. Study subjects

10 PCOS patient samples and 10

age and sex matched controls

samples were recruited from

Sandhiya infertility clinic,

Vellore for the present study.

The age groups of patients were

18-26. Blood samples were

collected in EDTA vacutainers

from all the patients. Blood

samples from each patient were

collected by the Physicians with

the written consent.

B. Genotyping

Genomic DNA was extracted from

the blood taken from

10patients and 10 controls.

SNP bearing region of HSD17B5

gene is amplified using

polymerase chain reaction

(PCR) technique. The PCR

amplification was carried out

in a volume of 20µl (each). It

contains 5µl genomic DNA, 1µl

of each primer (forward and

reverse), 6µl of 2x master-

Red, 7µl of MilliQ water. The

PCR done to get a product of

468bp fragment. The

amplification was carried out

for 30 cycles with a condition

of initial denaturation at

94°C for 5 minutes,

denaturation at 94°C for 30

seconds, annealing at 40°C for

30 seconds, initial extension

at 72°C for 60 seconds and

final extension at 72°C for 7

minutes. The products obtained

by PCR were verified and DNA

fragments were separated by

using 0.8% Agarose gel

electrophoresis stained with

ETBR (ethidium bromide) and

the picture was taken using

gel docking machine. The PCR

product was kept for further

process of restriction

fragment length polymorphism

(RFLP) to screen the sample

for the suspected SNP of

HSD17B5 gene. Table 1 shows

the master mix preparation and

table 2 shows the conditions

for PCR.

Table 1: Preparation of PCR

Master Mix

Steps of PCS Conditions

Initiation 94⁰C for 5 minutes

Denaturation 94⁰C for 30 seconds

Annealing(standardized at)

40⁰C for 1 minute

Elongation (Initial Extension)

72⁰C for 1 minute

Final Extension 72⁰C for 7 minutes

Hold 4⁰C

Table 2: PCR conditions

C. Dermatoglyphics

Dermatoglyphic prints

were made by ‘INK METHOD’

which is described by Cummins

in 1936. The materials

required for dermatoglyphics

prints are Stamp pad, A3 size

paper, cotton, Spirit, Soap &

water. Instruments which were

used for this study are

Scanner, laptop, compound

magnifying lens, pencil &

Scale. Hands of the subjects

were asked to clean with soap

& water before taking print.

Stamp pad was used to print of

entire palm, digit and the

hypothenar border. Prints of

hands of subject from proximal

to distal end were taken on

the A3 sheet, which is kept on

a flat surface and back side

of the palm was pressed softly

in such a way that the hollow

of palm is well printed. The

palm was slowly lifted from

the paper to get the desired

print. The Dermatoglyphic

Content Quantity

2X Master Mix-

Red

6µl

Forward Primer 1µl

Reverse Primer 1µl

MilliQ Water 7µl

Master mix + DNA 15µl + 5µl

Total 20µl/sample

prints of 10 patients and 10

controls were scanned and

detailed Dermatoglyphic

analysis were done. It is

again analyzed using

magnifying hand lens for the

confirmation. Qualitative and

quantitative parameters of

fingerprint were taken for

more concise information. The

Non-invasive technique of

dermatoglyphics is shown in

figure 1.

Figure 1: Dermatoglyphic technique

3. RESULTS

From the patients, we obtained

the results after checking the

obesity according to its

severity which is depicted as

figure 2. Hormonal variations

was seen in patients with PCOS

which is represented in figure

3

and we found some of the PCOS

patients got family background

of disorders, which is plotted

in figure 4. One of the

research paper suggested the

association of HSD17b5 gene

and obesity (Hai-xiang Sun,

M.D et,al.)

Figure 2: Severity of obesity and number of people suffering from

obesity.

Number of patients

Number of patie

FSH LH PRL1 T3 T4 TSH0

2

4

6

8

10

12

Hormone profile chart Less than nomal rangeHormone profile chart Normal rangeHormone profile chart More than normal range

Figure-3: Hormone profile of patients

Hormonal profile showed slight

variations in the hormone

level and family history

showed parents of some of the

patients have some kind of

disorders such as

reproductive, insulin

resistant, cardio and obesity,

which is represented in figure

4.

Number of pati

Cons

angu

init

y(pa

rent

s)

Repr

oduc

tive

Insu

lin

Obes

ity

Card

io

family history of following

0

1

2

3

Series1

Figure-4: Family history of various disorders

The DNA was isolated from

the 10 PCOS case and 10

controls and the qualitative

analysis were checked in 0.8%

Agarose gel electrophoresis

and the picture shown in

figure-5.

Figure-5: Qualitative analysis

of extracted DNA of PCOS

patients on 0.8% Agarose Gel

Electrophoresis.

Using PCR technique, the

annealing temperature of

amplification was standardized

and the standardized

temperature is 46°C. The

product size were found to be

468 bp and shown in figure-6.

Figure 6: 2% Agarose gel

showing the PCR amplification

of SNP bearing region of

HSD17B5 gene in PCOS patients.

The significant findings were

found in the PCOS patients and

controls by dermatoglyphics

study. Normally whorl patterns

are seen in thumb finger of

normal people whereas if there

is any genetic mutation it may

vary. In this study we found

high variations of patterns of

thumb finger in patients as

compared to thumb finger of

control. The data of patients

and controls dermatoglyphic

pattern is shown in figure-7.

Figure-7: Dermatoglyphic analysis of patients and control

4. DISCUSSION

Many previous studies

reported that Dermatoglyphic

and chromosomal findings have

definite correlation and also

reported that the

dermatoglyphics patterns are

under genetic influence. The

dermatoglyphics patterns in

diagnosis are bright

especially in chromosomal

abnormalities. In the present

study, we can’t justify the

correlation between the

dermatoglyphics and the

molecular study, due to very

small number of population (10

PCOS cases and 10 controls).

But we found high variation in

Number of patient

thumb finger pattern of

patients compared to control.

The PCR product standardized

and the polymorphism of

HSD17B5 gene will detect in

the present population by

using RFLP. The

dermatoglyphics study will

carry out in the large number

of population.

5. CONCLUSION

. Our studies suggest that

there is no relation of

HSD17B5 variants with the

development of PCOS in the

South Indian population,

whereas the dermatoglyphics

study showed significant

variation in PCOS patients and

normal control. Hormone

variation, obesity severity

variation reveals that the

obesity and hormone levels

strongly relative with PCOS.

Further study is needed with

larger number of population to

come up with the contribution

of HSD17B5 gene variants to

PCOS.

6. ACKNOWLEDGEMENT

We would like to thank

the VIT UNIVERSITY, Vellore

for providing us the

facilities needed for this

project.

7. REFERENCES

1. Zucchero TM et,al. 2004.

Interferon regulatory factor 6

(IRF6) gene variants and the

risk of isolated cleft lip or

palate. N Engl J Med 351:769-

780.

2. Meenakshi S,

Balasubramanyam V, Sayee

Rajangam. Dermatoglyphics in

Amenorrhea – qualitative

analysis. J.Obstet Gynecol

India 2006 vol.56, no.3,

P.250-254.

3. Dr. Aaditi Shah*, Dr.

Vasanti Arole. Chromosomes,

Dermatoglyphics and Polycystic

Ovary Syndrome (pcos) Int J

Biol Med Res. 2014; 5(1):

3850-3854

4. Lu Ke et,al. Polymorphisms

of the HSD17B6 and HSD17B5

Genes in Chinese Women with

Polycystic Ovary Syndrome.

Journal of women’s health.

Volume 19, Number 12, 2010 ª

Mary Ann Liebert, Inc. DOI:

10.1089=jwh.2009.1902.

5. Tsuyoshi et,al. Polycystic

ovary syndrome is associated

with genetic polymorphism in

the insulin signalling gene

IRS-1 but not ENPP1 in a

Japanese population. Life

Sciences 81 (2007) 850–854.

6. Rennaisance SJ, Disaia PJ,

Hammond CB et al. Danforth’s

Obstetrics and Gynecology.

Philadelphia. Lippincott.

William and Wilkins. 1999:

601.

7. Schaumann B, Alter M.

Dermatoglyphics in medical

disorders. New York. Springer-

Verlag, 1976.

doi:10.1016/j.fertnstert .2013

.04.001.

8. Mutalik GS, Lokhandwala VA,

Anjeneyulu R. Dermatoglyphical

findings in primary

amenorrhea. J Obstet Gynecol

India 1968; 18:738-43.

9. Kenan Qin et,al.

Identification of a Functional

Polymorphism of the Human Type

5 17β-Hydroxysteroid

Dehydrogenase Gene Associated

with Polycystic Ovary

Syndrome.