chapter 2shodhganga.inflibnet.ac.in/bitstream/10603/31563/8/08_chapter 2.pdfmaterials and methods 68...

CHAPTER 2 MATERIALS AND METHODS

In this chapter, an attempt was made to describe the brief history and ethnographic

profile of Meitei community, Manipur. An attempt was also made to describe the

methodology for data & sample collection in the present study. Further, present

chapter also elaborates the standard techniques used for molecular analysis and its

statistical analysis.

2.1 Manipur

Manipur is a small state that lies in the slopes of the south flowing Sub-Himalayan

ranges in the north east region of India at the latitude 23.83ºN to 25.68ºN and

longitude 93.03ºE to 94.78º E. It is 790 m above the sea level and surrounded by hill

ranges in fold forming a valley in the middle of the state. It is one of the eight north

eastern states of India, occupying an area of 22,327 km2, surrounded by beautiful nine

hill ranges in fold. Its boundary is surrounded by the Indian states Nagaland in the

north, Assam in the west, Mizoram in the south-west and by the country Myanmar

(Burma) in the east and south. Through Manipur, Indian sub-continent is connected to

South East Asia. Manipur is separated from the Brahmaputra valley by its Western

hills and from the Chindwin valley of Myanmar by the eastern hills. The two main

physical features of Manipur are river valley and the western mountainous region. Its

boundary with rich cultural heritage, beautiful landscape with undulating hills,

emerald green valleys, blue lakes and dense forests described Manipur as the

“Switzerland of India” by Lord Irwin. Pandit Jawaharlal Nehru also called Manipur as

the “Jewel of India”.

MATERIALS AND METHODS

66 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India

Figure 2.1: Map showing location of Manipur 2.1.a Nomenclature

Manipur in early days was known by different names at various periods in its history,

such as, Tilli-Koktong, Poirei-Lam, Sanna-Leipak, Mitei-Leipak and Meitrabak.

Neighboring territories also called Manipur in different names such as Assamese

called it Moglai, the Burmese called it Kathe. The Shans knew Manipur as Kase and

to Ahoms it was Makeli (Kabui, 1991).

2.1.b Climate

The climate of Manipur can be broadly divided into Temperate (prevailing in higher

altitude), Sub-tropical (prevailing in lower altitudes and plain valley) and Tropical

(prevailing in foothills) which are influenced by the topography of the hilly region

that defines the geography of Manipur. In summer the average high temperature is

about 32-34°C, while in the winter temperatures can drop down to 1-2°C. Winter

season lasts from November to February and rainy season from May to September.


Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 67

January is the coldest month in Manipur and the warmest July. Spring and autumn

comes during these transition periods of March, April and October. Rainfall is

abundant, with about 65 inches (1650 mm) of precipitation occurring annually

although there are variations of rainfall across the state. November to February is the

state’s driest months. Having rich rainfall, rice cultivation is favourable and rice is the

staple food for all the communities of Manipur.

2.1.c Vegetation

The natural vegetation occupies an area of about 14,365 km² which is nearly 64% of

the total geographical area of the state. The vegetation consists of a large variety of

plants ranging from short and tall grasses, reeds and bamboos to trees of various

species. Broadly, there are four types of forests - Tropical Semi-evergreen, Dry

Temperate Forest, Sub-Tropical Pine and Tropical Moist Deciduous. Some of the

trees growing plenty in this region, which make a good forest resource, are teak, pine,

uningthou, leihao, bamboo etc. In addition to the above plants, rubber, tea, coffee,

orange, and cardamom are also grown in hilly areas. Major crops grown in Manipur

are maize, oil seeds, pulses, rice, sugarcane, wheat, rubber, coffee, cabbage, brinjal,

carrot, cauliflower, beans, potato, pea, radish and tomato.

2.1.d State Flower, Bird and Animal

Siroi lily (Lilium mackliniae) is found only in Manipur, in the upper reaches of the

Siroi hill ranges in the Ukhrul District of Manipur, at an elevation of 1730 m - 2590 m

above the sea level. This shade-loving lily has pale bluish-pink petals. It blooms in the

month of May-June and flowers in the monsoon months of June and July. Lily is the

state flower of Manipur.

The State bird of Manipur Nong-in is greatly endangered. The people of Manipur had

the belief that eternal soul of an orphan unable to bear hunger and thrust had

transformed itself into a bird to be known as Nong-in. In fact, this bird earned its

name Nong-in because of its ability in predicting changes in weather conditions (Nong

meaning rain, in meaning to follow i.e. one knowing about the rain). Nong-in is often

described as the most talented lover of song, dance and moonlights.



The Sangai is an endemic, rare and endangered Brow-antlered deer found only in

Manipur. Its common English name is Manipur Brow-antlered Deer and the scientific

name, Rucervus eldi eldi (IUNC red list of threatened Species). It lives in the marshy

wetland in Keibul Lamjao National Park about 45 km from Imphal, the capital of

Manipur. Sangai is the state animal of Manipur.

Figure 2.2: (a) State animal - Sangai (Source: james-raj.blogspot.in), (b) State -

bird Nong-in (Source: e-pao.net/education/science and technology/ (R.K. Birjit

Singh) and (c) State flower - Siroi Lily (Source: flowers-us.blogspot.in/2011/04/

siroi-lily.html)

2.1.e Transportation

Manipur remains somewhat isolated from the rest of India, even the development

works for transportation have been taken up at very fast speed and communications

within the state are poor. One of the national highways passing through Manipur, the

highway from Tamu on the Myanmar border in the south via Imphal to Dimapur (in

Nagaland) in the north is the important route linked to the important Asian countries;

this highway also connects Imphal with the Northeast Frontier Railway near Dimapur.

Air transportations are available from Calcutta, New Delhi, Guwahati and Silchar but

much beyond the reach of commoners.

2.1.f Loktak Lake

It is the largest fresh water lake in North-East India, also called the only Floating Lake

in the world due to the floating phumdis (heterogeneous mass of vegetation, soil, and

organic matters at various stages of decomposition). It is located towards the south

(a) (b) (c)



western end of the valley extending over an area of more than 75 km2. It plays an

important role in the economy of Manipur and also serves as a source of water for

hydropower generation, irrigation and drinking water supply. The lake is also a source

of livelihood for the rural fishermen who live in the surrounding areas and on

phumdis.

Figure 2.3: Aerial view of Loktak Lake

(Source: manipuronline.com/gallery/loktak-lake-4/)

2.2 History of Manipur

The history of Manipur can be divided briefly into four main periods- the ancient

(Before Christ), the early period (1st - 13th century AD), the medieval period (15th -

18th century AD) and the modern period (19th - 21st century AD).

2.2.a The Ancient Period (Before Christ)

The first Meitei kingdom was established by Tang-Ja-Leela Pakhangba during 1445-

1405 BC. Tang-Ja Leela can be translated as ‘Tang-Ja’ short name of ‘Tang-Shang’

dynasty, ‘Leela’ who followed the Ireel trail, ‘Pa’ means forefathers and ‘Kangba’

means to know his forefathers. The Chief of Tang-Shang group who came to Manipur

from China and settled down at the origin of Ireel river (Kabui, 1991). He married the

daughter of Lei-Hou Chief, Sinbee Leima and they settled together at Koubru hill

ranges. Kangba, the son of Tang-Ja-Leela Pakhangba introduced ‘Sagol Kang-Jei’,

Polo. Hence, the name of king Kangba was used in naming the stick ‘Kang-jei’ and



‘Kang-droom’ for the ball. Meitei Calendar (Cheiroba) known as Mari-Fam (MF) was

introduced during Koi-Koi time, the son of King Kangba. Around 934 BC Meidingu

Korou Nongdren Pakhangba administered the kingdom very well and all people lived

peacefully. During Chingkhou Poireiton (34-18BC) reign, the kingdom was known as

Poirei-lam (the land of Poireiton) and the people were called Poirei-Meitei (Kabui,

1991).

Figure 2.4: Photograph showing Sagol Kangjei (Polo)

(Source: www.firstpost.com/topic/place/manipur-polo-players-of-manipur-india)

2.2.b The Early Period (1st - 13th century AD)

Nongda Lairen Pakhangba, an able descendent of Ningthou Kangba and Tanja Leela

Pakhangba reigned at the low lying areas named Kangla (dry land). From him, the

Mangang clan originated. He was a great patronage of Sanna-Mahi and attained the

status of a Lai-Ningthou (God King). Cheitharol Knmbaba Chronicle started from

him. Khamlang Atonba, the son of Meidingu Chenglei Lanthaba, ascended the throne

after his father in 965 AD. His wife Lairenjam Chanu Mubisu, the daughter of

Louthog-pak chief was a master of weaving and embroidery. She invented Khoi-

Mayek style of Meitei-Phanek Mapan Naiba, one of the best and favorite designs for

Manipur ladies.

2.2.c The Medieval Period (15th - 18th century AD)

Meidingu Senbi Kiyamba, the son of Ningthou Khomba and Leima Linthoingambi

became the king at the age of 24 in 1467 AD. He had good relations with king of

Pong (Shan Kingdom) which is evidence from the existence of Pheiya (Almighty) at

Lamangdong (presently known as Bishnupur) presented by the king of Pong.



Meidingu Pamheiba, the son of Meidingu Charairongba and Sapam Chanu Ningthil

Chaibi became the king on 23rd August, 1708 AD. He was influenced by Hindu

religion which had reached to Manipur around 15th - 16th century AD. He burnt all the

Holy books, ‘Puya’ related to Sanna-Mahi religion (called Puya-Meithaba) and

converted himself to the Ramanandi Sect of Vaisnavism. With the Sanskritization

process of Meiteis, after conversion of the kings to Hinduism towards the beginning

of 18th century, Meitei Gods were transformed to the gods of Hindu mythology

(Ibugohal and Khelchandra, 1967).

The name ‘Manipur’ for ‘Meitrabak’ or ‘Sana-Leipak’ came to existence in 1774 AD

when Warren Hastings was the Governor General of India. Mr. Rendel assigned the

name and the kingdom extended from Ningthi in the east to Chittagong in the south

and up to Bhramaputra area in the North and Cachar in the East.

2.2.d The Modern Period (19th - 21st century AD)

When Marjeet was the king of Manipur, Manipuris faced the invasion of Burmese

fiercely for seven days in 1819 AD. This period is known as Chahi-Taret Khuntakpa,

1819-1825 AD (seven years of Manipur anarchy). In 1825, Manipuris attacked

Burmese led by Gambir Sing (the brother of Marjeet) and drove them beyond Ningthi

(Chindwin) river. On 26th June 1825, he declared himself as the king of Manipur.

Maharaja Chandrakirti, the only son of Maharaja Gambir Singh and Maisnam Chanu

Kumudini Ponglen-Khombi, ascended the throne at the age of 2 years with his uncle

Narasing as a caretaker. During his reign, capital was shifted from Langthaban to

Kangla at Imphal on 9th May, 1844 AD. Chandrakirti introduced “Sagol Kangjei”,

Manipuri Polo, to the British. Re-demarcation of Manipur’s boundary (present day

map) was done again on 13th December, 1873 AD with Dr. Brown (Political Agent).

Dr. Brown published the Meitei script for the first time in 1877 AD for the Asiatic

Society of Bengal. The then Bengal Government started teaching Bengali script and

English.



Figure 2.5: Ancient capital of Manipur, Kangla

(Source: bharatiyaculture.blogspot.in/ 2010/04/kangla-fort-)

Maharaja Surchand Singh, the eldest son of Chandrakirti ascended the throne after his

father. In 1890, his younger brothers, Zillangamba and Angousana revolted against

him along with Jubaraj Tikendrajit. Kullachandra, the elder brother of Tikendrajit,

became the king. Surchand and his brothers requested the British Government to

restore their throne. Lord Landsdowne, the viceroy of India ordered Mr. J. W.

Quinton, Governor of Assam, to recognize Kullachandra as the King but to arrest

Jubaraj Tikendrajit. Accordingly Mr. Quinton and his army raided the residence of

Jubaraj without prior notice. However, they could not capture Tikendrajit. In a further

attempt, Mr. Quinton and Mr. Grimwood, the political agent along with five other

British officers were killed.

The British Government waged open war against Manipur and it is known as Anglo-

Manipuri war. Major Paona Brajabashi lost his life on the Khongjom war and British

conquered Manipur and lost its independence on 27th April, 1891 AD. Jubaraj

Tikendrajit and Thangal General were hanged by neck on 13th August, 1891 AD at

Mapan Kangjei-bung (Polo ground). Later on, the place became historical site and the

statue with three heads of kanglasha, known as Saheed Minar, was laid down to

remember the great heroes of Manipur.



Figure 2.6: Saheed Minar - Statue of three heads of Kanglasha

(Source: en.wikipedia.org/wiki/File:Kanglasha.jpg)

Several movements led by Neta Irabot sprang up in the demand for self rule of

Manipur against the British Government. On 28th August 1947 AD, the British

handed over Manipur to Maharaja Budhachandra Singh and Maharani Iroshi

Devi. Maharaja entered Kangla at Imphal and hoisted the National Flag of Manipur

bearing the Dragon God Pakhangba. However, the newly formed independent India

and its Government in New Delhi pressured the King to sign a merger agreement with

India under very unusual circumstances. Maharaja signed the documents on 21st

September 1949 AD at Shillong without prior consideration and approval from

elected members of the Manipur Assembly.

2.2.e Present Manipur

Since 1956 Manipur had been a union territory of India and became a full-fledged

state on 21st January 1972 with a legislative assembly of 60 seats of which 20 are

reserved for Scheduled tribe and 1 reserved for scheduled caste. The state is

represented in the Lok Sabha by two members and by one member in the Rajya

Sabha. Administratively, the state has 9 districts namely Imphal East, Imphal West,

Thoubal, Bishenpur (Valley Districts), Ukhrul, Senapati, Tamenglong, Chandel and

Churchandpur (Hill Districts). According to the Census 2011, Manipur has been

divided into 37 sub divisions and there are 36 blocks, 31 towns and 2391 villages

comprising 166 gram panchayats. Its major towns are Moreh, Churachandpur, Andro,

Jiribam, Thoubal, Kakching, Imphal, Ukhrul, Mao, Tamenglong, Kongpokpi, Chandal



and Moirang. Manipur’s population is largely rural, Imphal capital of the state being

the only city of any size.

2.3 Peoples of Manipur

Manipur has been the home of more than thirty ethnic groups in historical times, with

varying responses to the changing socio-cultural environment, forming a

heterogeneous population including both tribal and non tribal population with a total

population of 27,21,756 (Census of India, 2011). Peopling of Manipur could be

divided broadly into two based on its geographical location such as hill dwellers as

the tribes and valley dwellers non-tribal at large. Valley is mainly inhabited by

Meiteis including Meitei Brahmins (Bamons), Manipuri Muslims (Pangal) and some

other tribal populations and non-Manipuris commonly call as Mayang (For examples

Nepalis, Bengalis, Marwaris, Punjabis and other Indian communities).

Table 2.1: General profile of Manipur showing the distribution pattern of

population and literacy rate in different districts of Manipur

District Headquarters Literacy rate

Area (sq.km)

Population 2011

Population Percentage

Imphal West Imphal 86.70 519 5,14,683 18.91

Imphal East Imphal 82.81 709 4,52,661 16.63

Thoubal Thoubal 76.66 514 4,20,517 15.45

Senapati Senapati 75.00 3271 3,54,972 13.04

Churachandpur Churachandpur 84.29 4570 2,71,274 9.97

Bishnupur Bishnupur 76.35 496 2,40,363 8.83

Ukhrul Ukhrul 81.87 4544 1,83,115 6.73

Chandel Chandel 70.85 3313 1,44,028 5.29

Tamenglong Tamenglong 70.40 4391 1,40,143 5.20

TOTAL 22,327 27,21,756 100



On the other hand hilly regions are mainly inhabited by tribal populations which are

dividing into two main ethno-denominations namely Nagas and Kuki. According to

Gazette of India (No.10 of 2003) there are as many as 33 scheduled tribes in Manipur.

Some of the important tribal population groups are Tangkhul, Thadou, Zeliangrong

(composed of three tribal populations Zemei, Laingmei, Roungmei or Kabuis), Mao,

Maram, Tarao, Poumai, Paite, Hmar, Maring, Anal, Aimol, Angami, Chiru, Chothe,

Gangte, Monsang, Moyon, Kom, Purum, Ralte, Sema, Simte, Salte, Vaiphei,

Lamgang, Zhou, Koirao, Koireng, Thangal and Lamgang.

2.3.a Meitei

The word ‘Meitei’ is believed to be derived from the Kachin word Moi and Siamese

Tai (Hodgson, 1853) showing a possible link between the Meitei with people like the

Moi / Man, Tai, Shans, Myanmaries and Chinese who are classified as Mongoloid

stocks. Meiteis were known by various names, such as Mi-tei, Poirei-Mitei, Meetei or

Maitei. Meiteis exhibit mongoloid morphology (Brown, 1974 and Hodson, 1908)

having medium stature and broad (brachycephalic) type of head. Skin colour was

found to be ranging from brownish to yellow brown with epicanthic eye fold. They

have prominent flattened nose and dark black hair (Bhasin and Bhasin, 2002).

2.3.b Origin of Meitei

Meitei community was a hard nucleus group who ruled the land meticulously and had

solely contributed in State formation. It was belief that Meiteis were originated

somewhere in the north from a cave. Although there is still no definitive indication of

the ethno-historical account of the original settlers of Manipur valley, it is certain that

the Meitei, a major community, inhabits the parts of the valley since very early days.

In early remote days, some diverse communities called Khuman, Luwang, Mangang,

Moirang, Meitei, Chenglei, Chakpa, Khaba-Nganba and Angom took their settlements

at different parts of the valley in different ages and later on these smaller principalities

were amalgamated under the Meitei supremacy (Hodson, 1908). These subjugated

groups have become components of the Meitei as 7 exogamous clans. These

subsequently came together under the dominance of the Meitei ningthouja yek. The

yeks had their own divinities (Lai) some of which may have been defined ancestors.



The reverence of all these Lai continues to play an important role in Meitei society

(Parratt and Parratt, 1997). The Meiteis are distributed throughout the valley and make

up about 60% of the total population of Manipur.

2.3.c Language

Since Meiteis are the major community, culturally and economically, Meiteilon

(Meitei language) has become to be known as Manipuri after the name Manipur was

introduced in the eight schedule as one of the 18 official languages of India (71st

amendment of the Indian Constitution, 1992) and become state’s official language.

More than three‐fifths of the people speak Manipuri, which, along with English, is the

official language of the state. Meiteilon is one of the branches of Tibeto Burman sub-

family of Sino-Tibetan family (Grierson, 1903).

2.3.d Religion

Since the 16th century, a large section of the Meiteis who had their traditional belief in

Sanamahi religion got converted into Hinduism and highly Sanskritised. However,

most of the Meiteis, Meitei Brahmins and other scheduled Meiteis follow the

traditional worship of Sanamahi inside their houses as a part of their religion after the

revival of the old Sanamahi faith and Pakhangba cult. The Meiteis’ conversion into

Hindu Vaisnavism, under the patronage of the Maharaja Meidingu Pamheiba (1708-

1747AD), has also brought in an impact on their socio-cultural life.

2.3.e Occupation

The Meiteis are primarily agriculturalists. Agriculture and forestry are the main

sources of income. Rice is the major crop, and the rich soil also supports corn (maize),

sugarcane, mustard, tobacco, orchard fruits, and pulses (legumes). Fruits such as

pineapple, mango, orange, lemon, guava, and jackfruit are also cultivated. Any kind

of large scale industry is not available in the state. Traditional and house hold type

industries based on locally available raw materials such as bamboos, canes, jutes, etc

are advancing in the state through the help of Self Help Groups supported/financed by

non-governmental organizations (NGOs) and other micro financial groups.



2.3.f Dance and Culture

Manipur has its rich art and culture. The most obliging aspect of Manipuri culture is

that, it has retained the ancient ritual based dances and folk dances along with the later

developed classical Manipuri dance style. Among the classical categories, 'Ras Leela',

the epitome of Manipur classical dance is based on the spiritual and eternal love of

Radha and Krishna, the full incarnation of God. This dance enacts their sublime and

transcendental love and the selfless devotion of the milkmaids of Vrindavan to the

Lord. Another important characteristic of Manipuri dance is the Khamba-Thoibi

dance which is a duet performance of male and female dancers.

Figure 2.7: Ras Leela performed by Radha and Krishna

(Source: http://www.nartanalay.com/manipuri_dance.html)

2.3.g Festivals

Manipur is a land of festivities, fun and frolic all the year round. A year in Manipur

represents a cycle of festivals. Some of the common festivals celebrated by Meiteis in

Manipur are as follows -

2.3.g.i Ningol Chakouba

It is a remarkable social festival of the Meiteis celebrated to strengthen relationship

between brothers and sisters. It is celebrated on the second day of the new moon in

the month of Hiyangei (November). On this particular day married women of the

family come to the parental house along with their children and enjoy sumptuous

feast. Brothers present gifts to their sisters after the feast and get blessing from the



sisters. In recent times, this festival has become quite popular and is being practiced

by most of the communities in Manipur.

Figure 2.8: Photograph showing Ningol Chak-kouba

(Source: kockyn.com/best-wishes-on-ningol-chakkouba)

2.3.g.ii Yaoshang (Holi)

It is premier festival of Manipur Hindus and also called as Dol Jatra celebrated for

five days commencing from the full moon day of Phalgun, Langban

(February/March). The main function is collection of donations by singing religious

songs from house to house by women folk and blocking roads with long rob by girls.

Another event during this festival is “Thabal Chongba”, a kind of Manipuri folk

dance in which boys and girls hold hands and dance in circle. However, at present a

slight change in this festival is observed. During these five days, games and sports

meet are organized by local bodies or clubs in almost all localities encouraging youths

in the field of sports.

Figure 2.9: Yaoshang Pala March

(Source: www.flickr.com/photos/ Photo Courtesy Poknapham)



2.3.g.iii Cheiraoba

It is observed for celebrating New Year in Manipur, the first day of the month Sajibu

(April). During the festival, people clean and decorate their houses and prepare

special festive dishes which are first offered to various deities. After meal at afternoon

peoples come out and climb nearest hill tops as a part of the ritual entails and worship

deities. It is also believed that climbing hill tops will enable them to rise to greater

heights in their worldly life.

Figure 2.10: Variety of special dishes offered to the deities

(Source: Manipur.gov.in/festivals.html)

2.3.g.iv Kang Chingba

One of the greatest festivals of the Hindus of Manipur, the festival is celebrated for

ten days in the month of Ingen (June/July). It is also commonly known as “Ratha Jatra

of Manipur” in which Lord Jagannath leaves his temple in a Rath locally known as

'Kang' pulled by pilgrims who vie with one another for this honour.

Figure 2.11: Kang Chingba (Rath Jatra) of Lord Jagannath

(Source: www.e-pao.net/epGallery.asp)



2.3.g.v Heikru Hidongba

It is a festival of joy, with religious significance along a 16 meter wide boat. Long

narrow boats are used to accommodate a large number of rowers. Idol of Lord Shri

Bishnu is installed in the boat and begins the boat race. It is celebrated in the month of

Langban (September).

Figure 2.12: Racing of boat at the event of Heigru Hidongba

(Source: www.e-pao.net/epGallery.asp-Hidongba)

2.3.g.vi Lai-Haraoba

Celebrated in hour of the sylvan deities known as Umang Lai, the festival represents

the worship of traditional deities and ancestors for prosperity and good fortune and

protection of the whole locality it reigns. A number of dances by both men and

women are performed before the ancient divinities. The Lai Haraoba of God-

Thangjing, the ruling deity of Moirang, is the most famous one and attracts huge

gatherings. It is held in the month of Kalen (May).

Figure 2.13: Performance of traditional rituals during the Lai Haraoba

(Source: www.Sumansarawgi.com/ManipurCulture.html)



2.3.vii Pung Cholom

Nupa Pala (Kartal cholom or Cymbal dance) is ramification of the Manipuri style of

dance and music. It is normally performed by male artists using cymbals and wearing

white pheijom (dhoti) and spherical turbans. They sing and dance to the rhythm of

Pung (Mridanga).

Figure 2.14: Photograph showing Pung Cholom

(Source: www.Sumansarawgi.com/ManipurCulture.html)

2.3.h Dietary Habits

Rice is the staple food and there is certain restriction in the consumption of meat..

Fish is the common article of diet. Rice beer (Yu) is the most common alcoholic

beverage consumed by people. Meitei diet has been influenced by many other cultures

due to various socio political reasons. Sanskritisation is one of the biggest factors that

influences the change of dietary habit. From the meat eater they became fish eater and

those who accepted the Hinduism to its extreme even gave up fish and became pure

vegetarian. Meitei loves vegetable chutney Iromba/boiled delicacy, champhut and

kangshoi. This is a mixture of various boiled vegetables mashed with fermented fish

and chillis.



2.4 Meitei Women

The Meitei women control the markets and trades for vegetables and traditional

clothings. Ima Market or Kwairamban Keithel (Women Market) is an exclusively

Women's market of its own kind. Meitei women conduct most of the trade in the

valley and enjoy high social status. The women also take important roles in

safeguarding the communities and their children. They were effective in blocking

unwanted drug and many other substance abusers like pan, tobacco, tablet etc.

through Meira Paibi, a women organisation named after their activities for protecting

their own communities from the malice of drugs, atrocities from antisocial element

etc. by holding torches at the time of night. But, with the society becoming more

complex, the traditional roles of the women are waning. They often revolted against

alcohol addiction and other substance abuse from time to time. At present, different

organizations and NGOs are taking a serious action on alcohol problem in the state.

Figure 2.15: Photograph showing Meira Paibi

(Source: e-pao.net/epSubPageExtractor.asp – Spot light on women)

2.5 Preparation of Alcohol in Manipur

Alcohol is a special form of embodied material culture and the most widely used

psychoactive agent in the world. It has been a fundamentally important social,

economic, political, and religious artifact for millennia. Alcoholic beverages of

different tribal communities have received attention of several ethno botanists and

anthropologists. Yu the traditional alcoholic beverage of Manipur also called local

liquor is a distilled product of the fermented local rice, a locally available cheap raw



material. Traditionally, the local liquor is prepared in different communities of

Manipur and mainly restricted to the scheduled tribes (ST) and scheduled castes (SC).

At present, the so called local liquor production is also carried out by communities

other than SCs and STs (Singh and Singh, 2006). Earlier, preparation of local alcohol

was mainly for domestic purposes with limited usage. Now, the trend of alcohol

distillation has been changed and the concept turned out to commercial purpose with

the rise of economic advancement as well as socio-cultural transformation.

Interestingly, as on today local liquor is still produced in some parts of the valley

especially Andro, Pheiyeng and Sekmai for self-commercial profits. Manipur is a dry

state but the paradox is that you can get the best brand names in the world.

Preparation of distilled alcohol follows certain steps. A brief account of alcohol

distillation from rice has been described in the following paragraphs.

2.5.a Preparation of Hamei

Hamei is a dry, round-to-flattened solid ball-like used to prepare rice-based beverage,

locally called atingba - fermented beverage and distilled clear liquor called yu in

Manipur (Singh and Singh, 2006). Hamei is prepared by homogenously mixing

powder form of white rice and filtrate of dried bark plant call Yanglee (Albizia

myriophylla). Finely grinded rice powder called yam, where the rice was previously

soaked in water for 2-3 hrs is thoroughly mixed with the brownish filtrate of bark

powders of Yanglee plant. For example, for 1kg rice, around 8-10 gm Yanglee is

added. From this paste mass, a cake like structure in the form of rounded flattened

mass is prepared known as hamei.

The prepared hamei is sun dried or stored for 4-5 days over a hearth till the material is

completely dried. Hamei can be stored in cool, dry place for over a year. After 4-5

days of storage (fermentation), after fine water drop/droplets appear over its surface, it

is taken out and air dried. The whole process is done under the diffused or dim light.

The hamei is ready for use only when the alcoholic smell comes from it. The

flavoring agents that developed during storage are diacetyl, volatile phenols and esters

(Prescott and Dunn, 1959; Koizumi, 1974 and Lehtamen, 1982).



Figure 2.16: Diagrammatic flowchart showing preparation of Hamei

Hamei is a popular domestic business for the people of scheduled caste and tribes, as

it is also used as an ingredient of cattle foods. The traditional practitioners believe

that, the quality of hamei fermentation will be responsible for the quality and quantity

of ethyl alcohol. Therefore, it is generally said that even a drop of sweat that falls over

hamei during its preparation will spoil the whole mass.



2.5.b Fermented Beverages

There are two common types of fermented beverages such as yu angouba and

atingba. Yu angouba were prepared by soaking rice in water for around 2-3hrs along

with some germinated paddy. After this, the water is drained out and the soaked rice

is crushed till powder form. In another vessel water is boiled and in this boiled water

the crushed rice is added with continuous stirring till it gets cooled then it is covered

by cloth and kept for 2-3 days without any disturbance. Within these days foam

started coming out and a typical flavor and odor is released indicating that yu angouba

is now ready to consume. It cannot be stored for a longer period of time.

Atingba, in this type of alcoholic beverage rice is cooked and spread in a tray made of

bamboo. Hamei is mixed properly along with the cooked rice. The whole content is

transferred in a vessel then a little amount of water is poured just to dip it and covered

with a cloth. Heat is released for 2-3 days (Irabot, 2005). After which water is again

poured to ease the heat. Atingba is ready to drink after 6-8 hrs of pouring the water. A

proper atingba is formed after 4-5 days of fermentation during summer and after 7-8

days in winter. This kind of alcoholic beverage can be consumed for only 1-2 days

after fermentation but can be kept for around 1-2 months which is used for preparing

yu.

2.5.c Yu - Distilled Beverage

It is prepared from atingba. This atingba is poured in an aluminum pot and is cooked

in low flame. Above to this pot an aluminum funnel is placed and from this a pipe is

connected to the other part of the pot. This pipe is used for collecting yu. The pot is

covered tightly with an aluminum plate. On top of this another aluminum pot is

placed containing cold water. All the connecting points are sealed properly with cow

dung paste. Distillation continues until all the alcohol present in the content is out.

This can be checked by dipping a small stick into the boiling atingba and lit it, if

produced green flame than it indicates that the alcohol content is more (Chatradhari

and Irabot, 1996). So, based on this technique the distillation process is continued.

The remaining content after the extraction of yu is used as pig feed. This type of

alcoholic beverage is very hard as compared to the others stated above.



Figure 2.17: Diagrammatic representation of Alcohol Distillation, a traditional

method of alcohol distillation in Manipur



2.6 Selection of Field Area

The present study was conducted among the Meitei community, an endogamous

group, from four districts namely Imphal East, Imphal West, Bishnupur and Thoubal

of Manipur where Meiteis are predominantly settled.

2.6.a Bishnupur District

History of Bishnupur recorded the facts of its origin since king Kyamba ascended

throne of Manipur in 1467 AD. The original name of Bishnupur was known as

Lumlangdong, now called as Lamangdong.

The Bishnupur with it’s headquarter at Bishnupur (27 km from Imphal) was opened

on 25th May 1983. It lies between 93.43ºE and 93.53ºE Longitudes and 24.18ºN and

24.44ºN Latitudes bounded on the North by Imphal West District, on the South by

Churachandpur District, on

the East by Imphal and

Thoubal Districts. There are

64 Revenue villages in the

District. For a better and

convenient administration

the District is divided into

three Sub-Divisions, viz (1)

Bishnupur Sub-Division with

its head quarter (HQ) at

Bishnupur and (2) Moirang

Sub-Division (opened on 12-

12-83) with its HQ at

Moirang and (3) Nambol

Sub-Division with its HQ at

Nambol.

Figure 2.18: District map of Bishnupur, Manipur

(Source: www.mapofindia.com)



2.6.b Thoubal District

The Thoubal district lies between 2345'N and 2445'N latitude and 9345'E and

9415'E longitude bounded on the north by Imphal district, on the east by Ukhrul and

Chandel districts, on the south by Chandel and Churachandpur districts and on the

west by the districts of Imphal and Bishnupur. The district came into existence in

May, 1983 through a notification of the Government of Manipur, under the Manipur

Land Revenue and Land Reforms Act 1960. In November 1983, Thoubal was

bifurcated into Thoubal and Kakching sub-divisions comprising of Kakching and

Waikhong Tahsils with all their existing villages.

The district has two community development blocks one within each sub-division. It

has 9 main towns namely Lilong, Thoubal, Yairipok, Shikhong Sekmai, Wangjing,

Heirok, Kakching, Kakching Khunou and Sugnu and a part of Samurou whose major

portion is in the Imphal District. Thoubal and Kakching are Municipalities. The

national highway 39 (Indo-Burma Road), passes through the heart of the district.

Through the district runs an international road that leads to Myanmar (Burma) via

Moreh and Tammu.

Although little is known about

its ancient history, the district

has in recent past. It is in this

district, at Khongjom, that the

last battle of the independence

of Manipur was fought in April,

1891 by a few and ill-equipped

soldiers of Manipur against the

might of the British Empire

where the sun does not set, as

the saying goes.



2.6.c Imphal East

Imphal East District came into existence on 18th June 1997 with its head quarters at

Porompat occupying the eastern part of Imphal District. The District is situated at an

altitude 790 m above the mean sea level and it lies between 24.480N latitude and

93.570E longitude. The District is situated in two separate valleys of the state namely

Central Valley and Jiribam Valley. The District is connected with three national

highways (NH) such as NH 39, NH 53 and NH 150.

There are four Revenue Sub-Divisions

in the district namely (1) Porompat Sub-

Division; (2) Sawombung Sub-Division;

(3) Keirao Bitra Sub-Division and (4)

Jiribam Sub-Division. There are 237

Revenue villages in the district. The

total number of urban local bodies is

four comprising of two Municipalities

(Imphal and Jiribam) and two Nagar

Panchayats (Andro and Lamlai).

2.6.d Imphal West

The Imphal West District falls in

the Category of Manipur valley

region. Imphal City, the State

Capital is the nodal functional

centre of this District. It lies

between 24.300N to 25.000N

latitude and 93.450E to 94.150E

longitude. It is surrounded by

Senapati District on the north, on

the east by Imphal East and

Thoubal districts, on the south by

Thoubal and Bishnupur Districts,



and on the west by Senapati and Bishnupur Districts. The district is connected with

other districts and states through two national highways such as NH 39 (Indo-

Burma/Myanmar Road) and NH 53 (New Cachar Road).

The field areas were randomly selected for field work: 11 localities from Thoubal

district, 20 localities from Imphal East district, 19 localities from Imphal West district

and 3 localities from Bishnupur district. The details of the selected are listed below

(Table 2.2).

Table 2.2: Area covered within the four districts of Manipur

Sl. No. Name of the Locality Rural/Urban Name of the District

1 Thoubal Bazar Urban Thoubal

2 Thoubal Ningombam Urban Thoubal

3 Thoubal Leishangthem Rural Thoubal

4 Okram Urban Thoubal

5 Yairipok Vishnunaha Urban Thoubal

6 Athokpam Urban Thoubal

7 Wangjing Urban Thoubal

8 Kakching Urban Thoubal

9 Langmeithet Rural Thoubal

10 Wabagai Rural Thoubal

11 Thoubal Khunou Urban Thoubal

12 Wangkhei Urban Imphal East

13 Palace Compound Urban Imphal East

14 Khongman Urban Imphal East

15 Brahmapur Nahabam Leikai Urban Imphal East

16 Khurai Urban Imphal East

17 Kongpal Porompat Urban Imphal East

18 Khabam Rural Imphal East

19 Moirang Kampu Urban Imphal East

20 Naharup Rural Imphal East

21 Thambalkhong Rural Imphal East

22 New Checkon Urban Imphal East



23 Top Rural Imphal East

24 Bamon Kampu Rural Imphal East

25 Sambei Rural Imphal East

26 Chingkhu Rural Imphal East

27 Yairipok Poiroukhongjin Rural Imphal East

28 Langdum Rural Imphal East

29 Andro Urban Imphal East

30 Lamlai Yorabung Urban Imphal East

31 Nongmeibung Urban Imphal East

32 Haobam Marak Urban Imphal West

33 Kwakeithel Urban Imphal West

34 Heirangkhoithong Urban Imphal West

35 Sagoldband Urban Imphal West

36 Tera Rural Imphal West

37 Shamusang Rural Imphal West

38 Kakwa Lamdaibung Urban Imphal West

39 Uripok Urban Imphal West

40 Lalambung Urban Imphal West

41 Naoremthong Urban Imphal West

42 Lilong Urban Imphal West

43 Moirangkhom Urban Imphal West

44 Singjamei Urban Imphal West

45 Takyel Khongban Rural Imphal West

46 Lamboi Khongnangkhong Urban Imphal West

47 Chingmeirong Urban Imphal West

48 Thangmeiband Urban Imphal West

49 Nagamapal Urban Imphal West

50 Iroisemba Rural Imphal West

51 Nambol Bazar Urban Bishnupur

52 Moirang Rural Bishnupur

53 Thanga Rural Bishnupur



2.7 Work Plan

The present study involves two phases namely pilot survey and field work. Prior to

the pilot survey, necessary interview schedules are formulated and ethical clearance

(Annexure I) is also obtained from the ethical committee, Department of

Anthropology, University of Delhi, Delhi. A brief description of methodology used in

the present study is shown in the figure given below (Figure 2.22).

2.7.a Phase I: Pilot Survey

A pilot survey was conducted in the month of September, 2009 to establish a good

rapport with the local authorities and also people to explore the feasibility for to

carrying out the research work in the selected population. Major works covered

during the pilot survey are as follows -

1. Geographical areas basically inhabiting by Meitei community were identified.

2. Required permissions were taken from the concerned authorities of village,

rehabilitation centers and hospitals to carry out the field work.

3. A good rapport was established with villagers, patients and medical personnel

of the area under study to facilitate the data collection.

4. Tentative interview schedules were pre-tested among the subjects of the

present study. Modifications were made wherever necessary.

Figure 2.22: Depiction of brief Methodology



2.7.b Phase II: Field Work

The second phase of field work is broadly categorized under two heads - Primary

Data Collection and Laboratory Analysis.

2.7.c Primary Data Collection

The pretested interview schedules were administered to collect information related

with complete profile of an individual. Separate interview schedules based on DMS-

IV criteria for classifying alcohol dependence were also administered (Annexure II).

It encompasses the following sectors:

1. Personal information – name, age, sex, surname, education and occupation

2. Life style variable – smoking status, tobacco and history of alcohol

consumption

3. DSM-IV criteria for alcohol dependence

4. Disease profile – name of illness, age of onset, mode of treatment, available

hospital reports of the undergoing treatment of the individuals related with

alcohol dependence

5. Anthropometric and physiological variables – weight, height, waist and hip

circumference and blood pressure

Figure 2.23: Diagrammatic representation of study design used in the present

study



To understand the complex etiology of alcohol dependence, a case-control study was

conducted in the present study following the brief study design shown above (Figure

2.23). Primary data pertaining to complete profile of an individual were collected

during different phases of field work at different periods of time intervals (Table 2.3).

A total of four hundred and eighty four (484) individuals were recruited from four

districts of Manipur comprising both rural and urban populations. Only males above

25 years were included in the present study since alcohol consumption by women is

socially unacceptable in this society (Singh et al., 2005). Of the 484 individuals

recruited in the study, 155 were alcohol dependent (AD) cases, recruited either

directly from de-addiction centers or through population based house hold survey. In

both ways, diagnosis of alcohol dependence was determined using the DSM-IV

criteria (American Psychiatric Association, 1994, 2000). Age matched 329 controls

without any histories of substance dependence were randomly recruited from the

same geographical region and same community through house hold survey. The

selected control group included individuals consuming alcohol who were not

categorized as alcohol dependence as per DSM-IV criteria, rather they were alcohol

abusers. Among alcohol abusers different patterns of alcohol consumption were

found. This could be broadly classified into two different phenotypes of alcohol

drinking on the basis of quantity of alcohol consumed such as occasional drinkers

(ODs=116) and moderate drinkers (MDs=84). However, absolute non drinkers

(NDs=129) were also included in the present study. They were interviewed for the

collection of above primary data. Anthropometric and physiological measurements

were also collected from the same individual from whom primary data was collected.

Table 2.3: Different periods of field work conducted during the year 2009-2011

Sl. No Month Period

1 September – November, 2009 3 months

3 August – October, 2010 3 months

4 February – April, 2011 3 months

TOTAL 9 months



2.7.d Anthropometric Measurements

Anthropometric measurements undertaken during present study are collected using

the following methods.

Height vertex or Stature: It measures the vertical distance from vertex to

floor.

Vertex (v): It is the highest point on the head when the head is in eye-ear

plane.

Instrument used: Anthropometric Rod

Weight: Weight is taken by means of standard weighing machine with fine

accuracy. The subject is advised to wear minimum number of clothes at the

time of weighing and is not weighed right after meals.

Instrument used: Weighing Machine

Waist Girth: It measures the circumference of the abdomen at the most lateral

contour of the body between ribs and intestine.

Instrument used: Measuring Tape

Hip Girth: it measures the circumference of the hip at their widest portion.

Instrument used: Measuring Tape

2.7.e Physiological Variables (Blood Pressure)

It is the pressure exerted by blood on the arterial wall. When left ventricle contracts,

blood enters the aorta which is already filled with blood. As more and more blood

enters the aorta, the blood flow exerts pressure on the elastic arterial wall. This

pressure is called the blood pressure.

Diastolic Blood Pressure (DBP): As blood fills up the aorta, the pressure

which remains in the arteries during the relaxation of the heart is called as

diastolic blood pressure.

Systolic Blood Pressure (SBP): It is the measure of pressure exerted by the

blood on the wall of the vessel during each contraction of the ventricular

muscles.

Instrument used: Sphygmomanometer and Stethoscope



Table 2.4: Inclusion and Exclusion Criteria for the selection of AD cases and

controls of the present study

Inclusion Criteria

Exclusion Criteria

2.8 Blood Sample Collection

In the present study, 5ml of overnight fasting intravenous blood samples were

collected from 484 male individuals who are unrelated upto 1st cousin and from whom

data related to anthropometric, physiological and clinical profiles have already been

collected. Of 484, 155 individuals were AD cases and 329 were controls according to

the inclusion and exclusion criteria of the present study (Table 2.4). Trained clinical

personnel were hired for collecting the blood samples and informed written consent

(Annexure III) was obtained prior to blood collection from each individual. Of 5ml

intravenous blood sample, 2ml of blood was used for biochemical (lipid profile -

cholesterol, triglyceride, high density lipoprotein, low density lipoprotein, very low

Sl.

No Cases Controls

1 Who follow DSM-IV criteria for alcohol dependence

Normal individuals who do not follow DSM-IV criteria for alcohol dependence

2 Age ≥ 25 yrs Age ≥ 25 yrs

3 Belonging to Meitei Community Belonging to Meitei Community

Sl.

No Cases Controls

1 Drinkers who does not follow DSM-IV criteria for alcohol dependence

Individual with neuropsychological disorders/symptoms

2 Age < 25 yrs Age < 25 yrs

3 Multi drug users Multi drug users

4 Meitei Women Meitei Women

5 Not Belonging to Meitei Community

Not Belonging to Meitei Community



density lipoprotein and fasting glucose level) liver enzyme (aspartate

aminotransferase and alanine aminotransferase) analysis. Other 3ml of intravenous

blood samples was collected in ethylenediaminetetraacetic acid (EDTA) coated

vacutainers (Figure 2.24).

Figure 2.24: Description of blood sample collection used for different

biochemical and molecular analysis

All the biochemical analysis were done outsourcing on the same day of blood

collection at CECIL Medical Laboratory, Regional Institute of Medical Sciences

(RIMS) Gate, Imphal, Manipur using various techniques such as Glucose-Oxidase

method (GOD/POD), Wybenga & Pileggi and enzymatic GOP method. Collected

blood samples for molecular analysis were stored in ice box and transported to the

Molecular Anthropology Laboratory, Department of Anthropology, University of

Delhi by air with the proper permission from airport authorities. Samples were

subjected to Deoxyribonucleic acid (DNA) extraction within one week of collection

to get proper yield of DNA. Extraction of DNA and further molecular analysis were

done in the Molecular Anthropology Laboratory, Department of Anthropology,

University of Delhi.



Table 2.5: Different biochemical parameters taken in present study and their

normal cut of values according to the method employed

Biochemical Parameters Method Unit Normal Range

Cholesterol Wybenga & Pileggi mg/dl 150-250

Triglyceride GOP mg/dl 36-200

High Density Lipoprotein Wybenga & Pileggi mg/dl 35-65

Low Density Lipoprotein Indirect mg/dl Upto 150

Very Low Density

Lipoprotein

Indirect mg/dl 5-40

Fasting Blood Sugar GOD/POD mg/dl 60-105

Of 484, 472 individuals were included in the present study for analyzing molecular

genetics since extraction of DNA from twelve individuals sample was not successful.

Of 472, 126 individuals were NDs, 116 were ODs, 84 were MDs and 146 were AD

cases.

2.9 Laboratory Analysis

The wet laboratory techniques carried out in the present study can be broadly divided

into the following steps:

1. DNA extraction from the whole blood samples

2. Amplification of required fragment of DNA for selected genes using the

appropriate oligonucleotide primers by polymerase chain reaction (PCR).

3. Restriction digestion of the PCR products using restriction enzymes.

4. Genotyping of the digested product using agarose and polyacrylamide gel

electrophoresis (PAGE)

5. Gel documentation using UV light



2.9.a DNA Extraction

Extraction of DNA of the collected blood samples was done according to Miller et al.

(1988) protocol. Isolated DNA from white blood cells (WBCs) were re-suspended in

Tris EDTA (TE) buffer and made dissolved by keeping at 370C overnight. Dissolved

DNA samples were quantified using spectrophotometer Nanodrop and then preserved

in -800C for further molecular analysis.

PROTOCOL FOR DNA EXTRACTION (Miller et al. 1988)

1. Collected blood samples were centrifuged at 2000 rpm for 10 min and plasma

was separated in a micro-centrifuge tube with the help of pipette, without

disturbing the beneath layers, and stored in -800C.

2. Transferred the above remaining sample into 50 ml falcon tube and red blood

cell (RBC) lysis working buffer was added to make the final volume upto 40

ml in order to haemolyse the RBCs.

3. Suspension was mixed by inverting the tube several times with the help of

rocker until the solution becomes translucent.

4. After keeping the sample at room temperature for about 10 min, the solution

was centrifuged at 1000 rpm for 10 min and then the supernatant was

discarded.

5. Again 15 ml of RBC lysis working buffer was added and vortexed it to ensure

complete lysis of RBCs present in the solution and centrifuged at 1000 rpm for

10 min.

6. After discarding the supernatant 7.5 ml of nuclei lysis buffer was added to lyse

the nucleus of the WBCs.

7. 400 µl of 10% SDS (cleansing agent) and 25 µl of Proteinase K (to remove

extra proteins) was added and vortexed carefully for proper mixing of reagents

with the above solution.

8. The above solution was incubated at 650C for 2 hours in a water bath (to

increase the activity of the above reagents) and then 2 ml of saturated NaCl

(6M) solution was added to remove impurities.

9. After shaking vigorously for 15 sec the solution was immediately centrifuged

at 3100 rpm for 25 min.



10. Absolute ethanol was added to make the final volume upto 30 ml for

precipitating the DNA.

11. DNA was precipitated by inverting the tube 10-20 times very gently and

collected in a microcentrifuge tube with the help of a pipette.

12. 70% ethanol was added to the transferred DNA for washing and centrifuged at

13000 rpm for 10 min.

13. After supernatant was carefully removed 1 ml of 70% ethanol was added and

carefully vortexed to ensure thorough washing of DNA.

14. Centrifuged at 13000 rpm for 10 min and supernatant was carefully removed

and kept air dried for evaporating the ethanol completely from the sample.

15. The DNA sample was re-suspended in 0.5 ml Tris EDTA (TE) buffer and

made to dissolve by keeping at 370C for overnight.

16. Dissolved DNA samples were ultimately stored at -800C for further molecular

analysis.

Figure 2.25: Simplified diagrammatic representation of DNA extraction

using salting out method (Miller et al., 1988)



Chemicals Used in DNA Extraction

I. RBC Lysis Stock Buffer (10X)

NH4Cl 8.20 g

NaHCO3 0.84 g

EDTA 0.37 g

Dissolve in final volume of 100 ml of triple distilled water

(TDW). Autoclaved and stored at 40C

1X RBC lysis working buffer was prepared by 1:9 ratio and stored at 40C.

II. Nucleic Lysis Buffer (10X)

III. 1M Tris HCl

6.05 g of Tris salt was dissolved in 30 ml of triple distilled water.

Adjusted pH value at 8 with the help of HCl

Final volume was made upto 50 ml and stored at 40C after autoclave.

IV. 5M Saturated Sodium Chloride (NaCl) Solution:

29.22 g of NaCl was dissolved in triple distilled water.

Final volume was made upto 100 ml and stored at 40C after autoclave.

V. 0.5M EDTA

23.26 gm of EDTA was dissolved in 100 ml of triple distilled water

Adjusted pH value at 8 with the help of NaOH pellets and stored at 40C.

Tris HCl (1M) pH=8.0 4.0 ml

NaCl (5M) 32.0 ml

EDTA (0.5M) pH=8.0 1.6 ml

Final volume made up to 400 ml with triple distilled water

Autoclaved and stored at 40C



VI. Proteinase K Solution:

20 mg proteinase K was dissolved in 1 ml of triple distilled water.

200 µL was aliquoted in microcentrifuge tubes and stored at -200C.

VII. 10% Sodium Dodecyl Sulphate (SDS):

10 mg of SDS was dissolved in triple distilled water.

Final volume was made upto 100 ml and stored at room temperature.

VIII. 6M Saturated Sodium Chloride (NaCl) Solution:

35.064 g of NaCl was dissolved in triple distilled water.

Final volume was made upto 100 ml and stored at room temperature.

IX. Absolute ethanol stored at room temperature.

X. 70% absolute ethanol was made by 7:3 ratio of absolute ethanol and triple

distilled water and stored at -20˚C.

XI. Tris EDTA (TE) Buffer (200 ml)

1 Tris HCl (1M) 2.0 ml

2 EDTA (0.5M) 200 µl

Dissolve in final volume of 200 ml triple distilled water (pH = 8)


2.9.b DNA Quantification

In every molecular analysis, quantification of DNA/RNA is required to have known

concentration for further downstream applications. Quantification also increases the

rate of success in every reaction through checking its purity level. It calculates the

concentration of the DNA in a particular volume. Therefore, extracted DNA samples

have been quantified using spectophotometre (NanoDrop) before proceeding to

molecular analysis. Readings are taken in different wavelengths and NanoDrop has

the capability to measure highly concentrated samples without dilution. Two micro

liters (2µL) of stock DNA sample is used for the process. Along with DNA quantity,

the amount of protein contamination and salt impurity can also be obtained



simultaneously. To assess the purity of DNA, absorbance at 260/280 nm is used and a

ratio of ~1.8 is generally accepted as “pure” for DNA. If the ratio is lower, it may

indicate the presence of protein, phenol or other contaminants that absorb strongly at

or near 280 nm. Similarly the ratio of absorbance at 260 and 230 nm is used to assess

the purity of nucleic acid and value lies commonly in the range of 1.8-2.2 which are

often higher than the respective 260/280 value. If the 260/230 value is lower than 1.8,

it indicates the presence of co-purified contaminants.

PROTOCOL FOR DNA QUANTIFICATION USING Spectrophotometer

1. The sample surfaces of the spectrophotometer were cleaned before the start of

software module with triple distilled water (TDW) to remove any dried sample

or dust particles that might be present.

2. The NanoDrop program and the appropriate module for quantifying DNA

were opened.

3. The top and bottom sensors of the instrument were wiped off with tissue paper

and 2 µL of TDW was pipetted onto the sensor. The lever arm was brought

down.

4. The onscreen prompts were followed to calibrate.

5. Wiped the sensors and pipette 2 µL of TE buffer to

make blank since DNA samples were dissolved in TE

buffer.

6. Followed the onscreen prompts to blank.

7. After wiping the sensors, pipette 2 µL of the DNA

sample.

8. Following the onscreen prompts, the concentration was measured.

9. Concentration of the sample is given in ng/µL unit based on the absorbance at

260nm.

10. For DNA, the peak should be at 260 nm, and as a general rule, the 260/280

ratio should be between 1.8 and 2.0.

11. The equation for calculating DNA in the presence of contaminates is –

A [260] /A [280] = pure dsDNA

Figure 2.26: Nanodrop (Spectophotometer)



12. The higher the ratio, the more pure the DNA sample. It is acceptable to have a

ratio between 1.8 and 2.0.

For making 10ng/uL aliquots of 100 µL volume

After calculating the volume of DNA (from stock) from the above formula, this

amount of DNA (x uL) from stock was pippeted into fresh 200uL PCR strips and the

final volume was made upto 100 uL after adding triple distilled water (TDW).

Aliquots of 10ng/µL were prepared for all the samples and stored at -200C for further

molecular analysis.

2.9.c DNA Amplification - Polymerase Chain Reaction (PCR)

Revolutionary change emerged in the field of genetics with the invention of

polymerase chain reaction (PCR) technique during 1980’s. PCR was first invented by

Kary B. Mullis for which he was awarded Nobel Prize in chemistry in 1993 (Mullis,

1990). The method relies on thermal cycling, consisting of cycles of repeated heating

and cooling of the reaction for DNA melting and enzymatic replication of the DNA.

PCR allows amplification of a specific target DNA sequence which is then used for

further analysis. It requires specific oligonucleotide primers for amplifying the region

of interest, PCR buffer for providing a suitable chemical environment for optimum

activity and stability of the DNA polymerase, magnesium chloride (MgCl2) for

providing Mg2+ ion which acts as a catalyst in the reaction, deoxynucleotide

triphosphates (dNTPs), the building blocks for new DNA strand and enzyme called

Thermus aquaticus (Taq) polymerase which catalyzes the reaction of complementary

sequence synthesis. During a PCR, changes in temperature are used to control the

activity of the polymerase and the binding of primers.

DNA concentration at OD260 X Volume of DNA (stock DNA)

=

Desired concentration (10ng/uL) X 100ul (Final volume)



A DNA sample can be copied many times (amplified) in vitro and exponential growth

of the region of interest is achieved by 3-step cycle such as –

1. Denaturation - Heating the DNA to separate the double-stranded molecule

into single strands.

2. Annealing - Cool the mixture to allow complementary primers to hydrogen-

bond to the single strands.

3. Extension - Add DNA polymerase and nucleotides to replicate each strand.

Denaturation occurs at around 950C, where the double stranded DNA will be

separated to form single strands. This allows the primer to act on it in order to form

complimentary strand. Annealing takes place generally at around 50-650C. In this

phase primer forms the hydrogen bond with ends of target sequence. Extension occurs

at about 72oC. DNA polymerase adds nucleotides to the 3’ end of each primer.

Figure 2.27: Diagrammatic depiction of PCR

(Source: http://bio1151.nicerweb.com/Locked/media/ch20/PCR.html)



There are many ways of detecting a variant/SNP/mutation at particular site on the

DNA strand. In present study, restriction fragment length polymorphism (RFLP) that

is PCR technique followed by restriction digestion was used for amplifying and

detecting present or absence of the variation/SNP/mutation in all the selected genetic

markers.

2.9.d Restriction Enzyme Digestion

Restriction enzymes are naturally found in bacteria that defend against the

incorporation of foreign DNA. Werner Arber was the first scientist to postulate the

existence of restriction enzymes in the early 1960s (Arber and Dussoix, 1962). He

noticed that viral-DNA of a bacteriophage was cut into smaller pieces when entered

into a host bacterium. For this, he theorized the presence of “restriction enzyme”.

However, the restriction enzyme of E. coli was the first bacterial enzyme to be

isolated and studied (Meselson and Yuan, 1968). Subsequently after two years, a new

restriction enzyme having high-quality than E. coli called HindII was isolated from

the bacterium Haemophilus influenza called as "endonuclease R (Kelly and Smith,

1970 & Smith and Wilcox, 1970).

The mode of action of enzyme is cleavage of DNA at a particular sequence

recognition site, providing an identifying landmark that may differentiate it from other

DNA samples. When used at a polymorphic site, differentiation in the DNA samples

can be obtained due to success or failure of cleavage by the enzyme, resulting in

fragment lengths of different sizes. The different fragment sizes may then be detected

using gel electrophoresis. These particular sites are termed restriction fragment length

polymorphism (RFLPs).

Figure 2.28: Diagrammatic depiction of Restriction digestion (Source: http://academic.wsc.edu/mathsci/christensen_d/bigsceam2.html)



Specific restriction enzyme for each polymorphic site was used for detecting the

presence or absence of the mutant allele in the sample. Such restriction enzymes are

activated and start working at a particular temperature. After polymerase chain

reaction i.e. PCR products were treated with restriction enzymes and incubated at a

particular temperature for 1-2 hours/overnight to undergo proper digestion of the

enzyme. After digestion, genotyping was done.

2.9.e Genotyping

The digested products obtained after PCR and restriction digestion were genotyped

using agarose or polyacrylamide gel electrophoresis technique based on the desired

band size. Gel electrophoresis allows the separation and identification of DNA

fragments of various sizes. Polyacrylamide gels have a small range of separation, but

very high resolving power. So, it can separate the smaller band sizes. While agarose

gels have a large range of separation, but relatively low resolving power and are good

for separating larger band sizes. So, the gel was chosen based on the expected band

size. The digested product was mixed with loading dye and loaded into the wells in a

supporting matrix of agarose or polyacrylamide and then exposed to an electrical

field.

Loading dye acts as both coloring substance for naked eye visualization and also adds

weight to the product into the well in order to prevent from floating out as submarine

gels are used. The negatively charged DNA molecules migrate through the pores in

the agarose or polyacrylamide matrix to the positive anode, their velocity decreases as

their fragment size increases. The location of the DNA within the gel was determined

by staining with a fluorescent intercalating dye such as ethidium bromide (EtBr) and

viewed under a UV light source. In case of agarose gel, EtBr was added in making the

gel but in case of polyacrylamide, the staining process was done after the

electrophoresis run was completed. The gel was dipped in the solution of EtBr for 10

minutes.



Chemicals Used in Genotyping

I.a Tris-acetate-EDTA (TAE) Stock Buffer (50X)

I.b Tris-acetate-EDTA (TAE) Working Buffer

1 TAE Stock Buffer 10 ml

2 Autoclaved Triple Distilled Water 490 ml

Final volume of 500 ml (1:50) and stored at 40C

II. 40% Acrylamide solution

III. Tris-Boric acid-EDTA (TBE) Buffer (Stock)

IV. Tris-Boric acid-EDTA (TBE) Buffer working (1:9)

1 Tris 12.1 g

2 Glacial Acetic Acid 2.85 ml

3 EDTA 5 ml

Dissolved in a final volume of 50 ml Triple Distilled Water after adjusting the PH of 7-8 with absolute hydrochloric acid (HCl) Autoclaved and stored at 40C

1 Acrylamide 7.7 g

2 NN Methylenebisacrylamide 0.4 g

Dissolved I a final volume of 20 ml Triple Distilled Water


1 Tris 10.8 g

2 Boric acid 5.5 g

3 EDTA 0.58 g

Dissolved in final volume of 100 ml Triple Distilled


1 Stock TBE 50ml

2 Autoclaved Triple Distilled Water 450ml

Dissolved in a Final volume of 500 ml Distilled water and

stored at 40C



V. 10% Ammonium persulphate

100 mg of Ammonium persulphate dissolved in 1 ml of triple distilled water.

VI. Ethidium Bromide (EtBr) Solution

2 mg EtBr dissolved in 1 ml Autoclaved triple Distilled Water.

Vortex the micro centrifuge tube and store at room temperature after covering

it with aluminum foil.

2.9.f Preparation of Gel

Agarose gel: Agarose was weighted according to the desired concentration and the

size of the gel to be used. It is cooked in the beaker with corresponding volume of

TAE working buffer. 3% of EtBr was added in the cooked agarose for staining the

DNA fragments. It was then poured in the gel casting tray. Precautions were taken to

avoid bubble formation. Wells were made using comb for loading samples and then it

was left to solidify. The electrophoresis was carried out in horizontals electrophoretic

apparatus where the gel was submerged in TAE buffer.

Polyacrylamide Gel: Polyacrylamide gels are chemically cross-linked gels formed by

the polymerization of acrylamide with a cross-linking agent, N,N'-

methylenebisacrylamide (Bis). The reaction is a free radical polymerization, carried

out with ammonium persulfate as the initiator and N,N,N',N'-tetramethylenediamine

(TEMED) as the catalyst.

Polyacrylamide gel electrophoresis was carried out in vertical electrophoretic

apparatus (MICROKIN). Two glass slides were fixed on the apparatus with the spacer

in between the slides on both sides. A layer of agarose was used to prevent the

leakage. According to the desired concentration, 40% acrylamide solution, TBE

buffer (stock) and water were taken in a beaker. 10% APS and TEMED were added to

initiate the gelling process and the solution was poured in-between the two glass

slides. Comb was inserted from top and thereby wells were made for loading the

samples. In the present study, 8 % polyacrylamide gel solution was prepared using

the following protocol –



Reagent Working Volume

Acrylamide (40%) 3ml

TBE Stock (10X) 1.5ml

APS (10%) 90µl

Temed 13µl

Final volume was made upto 15 ml with distill water

Samples were mixed with the loading dye and loaded into the wells and

electrophoresis was carried out in TBE buffer medium. Loading dye acts both as

colored substance for naked eye visualization and also weight to the product into the

well in order to prevent them from floating out. The negatively charged DNA

molecules migrate through the pores in the polyacrylamide matrix to the positive

anode, their velocity decreases as their fragment size increases. The DNA

visualization within the gel was determined by staining for 10 minutes in fluorescent

intercalating dye such as ethidium bromide (EtBr) and viewed under a UV light

source.

2.9.g Gel Documentation System

Both the gels agarose and polyacrylamide were checked in UV light after proper

running with constant voltage (100V). The DNA molecules formed distinct bands,

after digesting with specific restriction enzymes, in the gel according to their fragment

sizes. Genotyping was done with respect to the band i.e. fragment of base pair (bp).

Instrument used for checking the gel is G-BOX with genesnape software.

2.10 Detailed Protocols Used for Selected Genetic Markers

In present study, four genes were selected on the basis of enzymes responsible for

alcohol metabolism and neurotransmitter receptors associated with neurobehavioural

mechanisms. The details of the polymorphic sites of studied genes (ADH1B

Arg47His, ADH1C Ile349Val, ALDH2 Glu487Lys, DRD2 (TaqI B, TaqI D and -

141C Ins/Del), ANKK1 TaqI A and DRD1 -48A/G) are listed in table 2.6.



Table 2.6: Characteristics of the selected eight SNPs in the present study

The detail PCR protocol used in the present study for each genetic marker is listed

below -

2.10.a ADH1C Ile349Val Polymorphism

This polymorphism (rs698) is detected by PCR amplification of exon 8 region of

chromosome 4q (21-24) followed by restriction digestion using restriction enzyme

SspI (Osier et al., 2002). The details are as follows -

Primer sequences

Forward primer – 5’ TTG TTT ATC TGT GAT TTT TTT TGT 3’

Reverse primer – 5’ CGT TAC TGT AGA ATA CAA AGC 3’

PCR mix

Gene Chrom Location SNP SNP

Location Polymer Site

Ancestral Allele Reference

ADH1B Arg47His 4q21-24 rs1229984 Exon 3 G/A G (Arg) Hayashida et

al., 2010 ADH1C Ile349Val 4q21-24 rs698 Exon 8 G/A G (Val) Osier et al.,

2002 ALDH2 Glu487Lys 12q24.2 rs671 Exon 12 G/A G (Glu) Hayashida et

al., 2010

ANKK1 TaqI A 11q23 rs1800497 exon 8 T/C T Grandy et al.,

1993

DRD2 TaqI B 11q23 rs17294542 Intron1 G/A G Grandy et al.,

1993

DRD2 TaqI D 11q23 rs1800498 Intron2 C/T C Grandy et al.,

1993

DRD2 -141C Ins/Del

11q23 rs1799732 Promoter Ins/Del Ins Gelernter et al., 1998

DRD1.1 -48A/G 5q35 rs4532 5’UTR A/G A Cichon et al.,

1994



Reagent Working Concentration

Taq Buffer A 1X MgCl2 1.5mM dNTPs 200µM Forward primer 0.5µM Reverse primer 0.5µM Taq DNA Polymerase 1U All the ingredients were subjected to PCR in 10µl reaction volume containing 5µl of DNA sample (10ng/µL).

PCR Conditions

The desired PCR product size is 378bp. It was checked in 2% agarose gel. Amplified

PCR product was digested with SspI restriction enzyme for 2 hour in 370C. The

product was again genotyped in 2% agarose gel. The required digested product sizes

are: ADH1C*2/*2=378bp; ADH1C*1/*2=378bp, 274bp and 104bp;

ADH1C*1/*1=274bp and 104bp

Figure 2.29: Agarose gel electrophoresis showing ADH1C Ile349Val (rs698)

polymorphism

Conditions Temperature Time

Initial Denaturation 95oC 5 min

40

Cyc

les Denaturation 95oC 15 sec

Annealing 55oC 15 sec Extension 72oC 75 sec

Final extension 72oC 10 min Storage 4oC For ever



2.10.b ADH1B Arg47His Polymorphism


chromosome 4q (21-24) followed by restriction digestion using restriction enzyme

RseI (Hayashida et al., 2010). The details are as follows -

Primer sequence:

Forward primer: – 5′ - CCT TGG GGA TAA ACT GAA TCT T – 3’

Reverse primer: 5′- GAA ATC CTG GAT GGT GAA CC – 3`

PCR mix:


Taq Buffer A 1X

MgCl2 0.5mM

DNTPs 200µM

Forward primer 0.2µM

Reverse primer 0.2µM

Taq DNA Polymerase 1U

All the ingredients were subjected to PCR in 15µl reaction volume

containing 5µl of DNA sample (10ng/µL).

PCR Condition:

Condition Temperature Time


35 C

ycle

s Denaturation 95oC 30 sec

Annealing 64oC 30 sec

Extension 72oC 30 sec

Final extension 72oC 5 min

Storage 4oC For ever



The desired PCR product size is 348bp. It was checked in 2% agarose gel. The

successful PCR product was digested with RseI restriction enzyme at 37oC for 2 hour.

The digested product was genotyped by electrophoresis in 2% agarose gel. The

desired band sizes were: ADH1B*1/*1=348bp; ADH1B*1/*2=348bp, 185bp and

163bp; ADH1B*2/*2=185bp and 163bp

Figure 2.30: Agarose gel electrophoresis showing ADH1B Arg47His (rs1229984)

polymorphism

2.10.c ALDH2 Glu487Lys Polymorphism


chromosome 12q (24.2) followed by restriction digestion using restriction enzyme

Eco571 (Hayashida et al., 2010). The details are as follows -

Primer Sequence:

Forward primer: 5′ - TCA AAT TAC AGG GTC AAC TGC T - 3`

Reverse primer: 5′ - GGC TGG GTC TTT ACC CTC TC – 3`



PCR mix: Reagent Working Concentration Taq Buffer A 1X MgCl2 0.5mM DNTPs 200µM Forward primer 0.2µM Reverse primer 0.2µM Taq DNA Polymerase 1U All the ingredients were subjected to PCR in 15µl reaction volume containing 5µl of DNA sample (10ng/µL).

PCR Conditions:


successful PCR product was digested with Eco571 restriction enzyme at 37oC for 2

hour. The digested product was genotyped by electrophoresis in 2% agarose gel. The

desired band sizes were: ALDH2*2/*2=430bp; ALDH2*1/*2=430bp, 296bp and

134bp; ALDH2*1/*1=296bp and 134bp

Figure 2.31: Agarose gel electrophoresis showing ALDH2 Glu487Lys (rs671) polymorphism

Condition Temperature Time Initial Denaturation 95oC 5 min

35

Cyc


Annealing 64oC 30 sec Extension 72oC 30 sec




2.10.d DRD1 -48A/G gene polymorphism

This polymorphism (rs4532) is detected by PCR amplification of 5’ untranslated

(UTR) region of chromosome 5q (35) followed by restriction digestion using

restriction enzyme Ddel (Cichon et al., 1994). The details are as follows -

Primer Sequence

Forward primer: 5′ - ACT GAC CCC TAT TCC CTG CT - 3`

Reverse primer: 5′ - AGC ACA GAC CAG CGT GTT C – 3`

PCR mix:


Taq Buffer A 1X

MgCl2 0.5mM

DNTPs 200µM




All the ingredients were subjected to PCR in 15µl reaction

volume containing 7µl of DNA sample (10ng/µL)

PCR Conditions:



35 C

ycle


Annealing 63.2oC 30 sec

Extension 72oC 1 min


Storage 40C For ever




successful PCR product was digested with Ddel restriction enzyme at 37oC for 2 hour.

The digested product was genotyped by electrophoresis in 8% polyacrylamide gel

electrophoresis. The desired band sizes were: GG=146bp and 61bp; AG=146bp, 61bp,

42bp and 19bp; AA=146bp, 42bp and 19bp

Figure 2.32: Polyacrylamide gel electrophoresis showing DRD1 -48A/G (rs4532)

polymorphism

2.10.e DRD2 -141C Ins/Del Polymorphism

This polymorphism (rs1799732) is detected by PCR amplification of promoter region

of chromosome 11q (23) followed by restriction digestion using restriction enzyme

MvaI (Gelernter et al., 1998). The details are as follows -

Primer sequences:

Forward primer – 5’ GAC CCA GCC TGC AAT CAC 3’

Reverse primer – 5’ AGG AGC TGT ACC TCC TCG G 3’



PCR mix:

Reagent Working Concentration Taq Buffer A 1X MgCl2 1.5mM DNTPs 200µM Forward primer 0.2µM Reverse primer 0.2µM Taq DNA Polymerase 1U All the ingredients were subjected to PCR in 15µl reaction volume containing 5µl of DNA sample (10ng/µL)

PCR Conditions:


successful PCR product was digested with MvaI restriction enzyme at 37oC for 1

hour. The digested product was genotyped by electrophoresis in 2.5% agarose gel.

The desired band sizes were: Del=156bp; Ins-Del=156bp, 124bp and 32bp; Ins=

124bp and 32bp

Figure 2.33: Agarose gel electrophoresis showing DRD2 -141C Ins/Del

(rs1799732) polymorphism

Conditions Temperature Time Initial Denaturation 94oC 3 min

35

Cyc


Annealing 62oC 30 sec Extension 72oC 1 min




2.10.f ANKK1 TaqI A Gene Polymorphism

This polymorphism (rs1800497) is detected by PCR amplification of 3’ UTR of

chromosome number 11q (23) followed by restriction digestion using restriction

enzyme TaqI (Grandy et al., 1993). The details are as follows -

Primer Sequence -

Forward primer: 5′ - CCT TCC TGA GTG TCA TCA AC - 3`

Reverse primer: 5′ - ACG GCT CCT TGC CCT CTA G – 3`

PCR mix:


Taq Buffer A 1X

MgCl2 0.5mM

DNTPs 200µM






PCR Conditions:



30 C

ycle



Extension 72oC 1 min






successful PCR product was digested with TaqI restriction enzyme at 65oC for 2 hour.

The digested product was genotyped by electrophoresis in 2% agarose gel. The

desired band sizes were: A2A2=124bp and 112bp; A2A1=236bp, 124bp and 112bp;

A1A1 =236bp

Figure 2.34: Agarose gel electrophoresis showing ANKK1 TaqI A (rs1800497)

polymorphism

2.10.g DRD2 TaqI B Gene Polymorphism

This polymorphism (rs17294542) is detected by PCR amplification of intron 1 region

of chromosome number 11q (23) followed by restriction digestion using restriction


Primer Sequence -

Forward primer: 5′ - GAT GTG TAG GAA TTA GCC AGG- 3`

Reverse primer: 5′ - GAT ACC CAC TTC AGG AAG TC– 3`



PCR mix: Reagent Working Concentration Taq Buffer A 1X MgCl2 0.5mM DNTPs 200µM Forward primer 0.2µM Reverse primer 0.2µM Taq DNA Polymerase 1U All the ingredients were subjected to PCR in 10µl reaction volume containing 5µl of DNA sample (10ng/µL)

PCR Conditions:


successful PCR product was digested with Taq1 restriction enzyme at 65oC for 2


desired band sizes were: B1B1=459bp; B1B2=459bp, 267bp and 192bp B2B2=267bp

and 192bp

Figure 2.35: Agarose gel electrophoresis showing DRD2 TaqI B (rs1079597) polymorphism

Condition Temperature Time Initial Denaturation 94oC 3 min

30

Cyc


Annealing 59oC 30 sec Extension 72oC 1 min




2.10.h DRD2 TaqI D Gene Polymorphism

This polymorphism (rs1800498) is detected by PCR amplification of intron 2 region

of chromosome number 11q (23) followed by restriction digestion using restriction


Primer Sequence -

Forward primer: 5′ - CCT CTG AGG CTT ACT GTC TG - 3`

Reverse primer: 5′ - AAA ACT AGG GAG GGT CAG AG – 3`

PCR Mix:


Taq Buffer A 1X

MgCl2 0.5mM

DNTPs 200µM






PCR Conditions:



30 C

ycle



Extension 72oC 1 sec






successful PCR product was digested with Taq1 restriction enzyme at 65oC for 2


desired band sizes were: D1D1=300bp; D1D2=300bp, 212bp and 84bp; D2D2=212bp

and 84bp

Figure 2.36: Agarose gel photograph showing DRD2 TaqI D (rs 800498)

polymorphism

2.11 Statistic Analysis

Statistical analysis for data obtained during present study was performed using

various appropriate statistical tools and softwares. Data related with anthropometric,

physiological, demographic and biochemical parameters were analyzed using MS

Excel 2007 and SPSS 16.0 version (Software Package for Social Sciences). For

genetic and molecular data analysis genotypes were counted through gene counting

method and their respective allele frequencies were also calculated using the

following mathematical formulae.

22AA AB

freq A pN

2

2BB AB

freq B qN

Where, A and B are the different alleles and N is the total number of samples studied.



2.11.a Hardy-Weinberg Equilibrium Test

Mathematical formula used to test Hardy-Weinberg Equilibrium was calculated as-

2expexp

observed ectedHWE

ected

Expected genotype frequencies were also calculated using following mathematical

formula to test for in accordance with Hardy-Weinberg equilibrium (HWE). Whether

a population or a group of study (i.e. case or control) is following HWE or not is

based on the Chi-square (χ2) test (a test of significance).

Expected frequency (A/A) = p2 Expected frequency (B/B) = q2

Expected frequency (A/B) = 2pq

The chi square statistic is a nonparametric statistical technique used to determine if a

distribution of observed frequencies differs from the theoretical expected frequencies.

Chi-square statistics use nominal (categorical) or ordinal level data.

Two types of chi-square test –

1. Chi-square test for goodness of fit which compares the expected and observed

values to determine how well an experimenters predictor fit the data.

2. Chi-square test for independence which compares two sets of categories to

determine whether the two groups are distributed differently among the

categories (McGibbon, 2006).

χ2 test enables to determine the degree of deviation between observed frequencies and

theoretical frequencies. Its p value concludes whether the deviation between observed

and expected frequencies is due to error of sampling or due to chance. The p value

below 0.05 is considered to be significant which means that the observed differences

or greater differences would occur by chance in only 1/20 similar cases. Statistically

significant means findings of a study are unlikely to have arisen because of chance.



2.11.b ODD Ratio (OR)

Odd ratio is one of a range of statistics used to assess the risk of a particular outcome

(or disease) if a certain factor (or exposure) is present. It is a way of comparing

whether the probability of a certain event is the same for two groups. It is a ratio

between members within a population expressing a trait or not, relative to their

exposure to a related risk [i.e. has trait (exposed) that trait (not exposed) x lacks trait

(exposed)/lacks trait (not exposed)]. It is just the cross-product ratio of the entries in

the 2-by-2 table as follows

The odds ratio is a relative measure of risk and its OR value signifies the degree of

risk. Value may vary from zero (0) to infinity, if OR is 1 then there is no relationship

between exposure and response variable or the event is equally likely in both groups.

Degree of relative risk increases with the increase in OR and it is calculated with a

chi-square statistic for a two by two table to test the null hypothesis at 95%

confidential interval (CI) using online program available at

http://www.hutchon.net/ConfidOR.html. An OR less than one implies that the event is

less likely in the first group and an OR greater than one implies that the event is more

likely in the first group. Mathematical formula used to calculate odd ratio was as

follows -

Exposure Variable Response Variable

+ -

+ A1 B1

- A2 B2



The estimation of OR gives us a notion about the measure of the association. As pre

dominant model, a single copy of mutant allele is enough to modify the risk, whereas

co-dominant model allows every genotype to give a different and non-additive risk. In

the recessive model, the risk of mutant homozygote is compared whereas the over-

dominant model shows the risk for heterozygotes.

2.11.e Logistic Regression

Logistic Regression (logistic model) is used for prediction of the probability of

occurrence of an event by fitting data to a logistic curve. Through logistic regression,

effects of a number of risk factors on the development or otherwise of a disease are

estimated. It is a variation of ordinary regression useful when the observed outcome is

restricted to two values, which usually represent the occurrence or non-occurrence of

some outcome events, (usually coded as 1 or 0 respectively). It produces a formula

that predicts the probability of the occurrence as a function of the independent

variables. It also produces odd ratios associated with each predictor value from

logistic regression coefficients.

Logistic regression makes use of several predictor variables that may be either

numerical or categorical. It is useful for situations in which one wants to predict the

presence or absence of a characteristic or outcome based on values of a set of

predictor variables. It is a useful way of describing the relationship between one or

more risk factors and an outcome such as disease (which only takes two possible

values: having disease or not having disease).



Software used for analysis of different variables taken in the present study is listed in

table 2.7.

Table 2.7: Software used for the analysis of demographic, anthropometric,

physiological, biochemical and genetics data

Software Measures Reference

POPGENE 1.32 Allele frequency, Hardy

Weinberg equilibrium

and heterozygosity

Yeh and Yang,1999

SNP Stats Haplotype Frequencies Freely available online

MS Excel 2007 Calculation of mean,

percentage and filtration

of data, drawing of table,

graphs, figure etc.

Microsoft Cooperation

SPSS 16 Multivariate analysis

Logistic Regression

SPSS Inc. 2008

Odd Ratio Relative Risk Freely available online

Linkage Disequilibrium Linkage Analysis Majumdar & Majumder, 1999

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