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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”.
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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.
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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.
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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)
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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
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70 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
‘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.
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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.
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72 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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.
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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
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74 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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
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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.
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76 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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.
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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
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78 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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)
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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)
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80 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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)
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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.
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82 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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
MATERIALS AND METHODS
Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 83
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).
MATERIALS AND METHODS
84 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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.
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Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 85
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.
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86 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
Figure 2.17: Diagrammatic representation of Alcohol Distillation, a traditional
method of alcohol distillation in Manipur
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Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 87
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)
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88 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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.
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Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 89
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,
MATERIALS AND METHODS
90 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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
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Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 91
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
MATERIALS AND METHODS
92 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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
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Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 93
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
MATERIALS AND METHODS
94 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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
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Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 95
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
MATERIALS AND METHODS
96 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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
MATERIALS AND METHODS
Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 97
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.
MATERIALS AND METHODS
98 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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
MATERIALS AND METHODS
Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 99
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.
MATERIALS AND METHODS
100 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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)
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Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 101
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
MATERIALS AND METHODS
102 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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)
Autoclaved and stored at 40C
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
MATERIALS AND METHODS
Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 103
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)
MATERIALS AND METHODS
104 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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)
MATERIALS AND METHODS
Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 105
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)
MATERIALS AND METHODS
106 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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)
MATERIALS AND METHODS
Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 107
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.
MATERIALS AND METHODS
108 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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
Autoclaved and stored at 40C
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
Autoclaved and stored at 40C
1 Stock TBE 50ml
2 Autoclaved Triple Distilled Water 450ml
Dissolved in a Final volume of 500 ml Distilled water and
stored at 40C
MATERIALS AND METHODS
Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 109
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 –
MATERIALS AND METHODS
110 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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.
MATERIALS AND METHODS
Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 111
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
MATERIALS AND METHODS
112 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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
MATERIALS AND METHODS
Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 113
2.10.b ADH1B Arg47His Polymorphism
This polymorphism (rs1229984) is detected by PCR amplification of exon 3 region of
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:
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 Condition:
Condition Temperature Time
Initial Denaturation 95oC 5 min
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
MATERIALS AND METHODS
114 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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
This polymorphism (rs671) is detected by PCR amplification of exon 12 region of
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`
MATERIALS AND METHODS
Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 115
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:
The desired PCR product size is 430bp. It was checked in 2% agarose gel. The
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
les Denaturation 95oC 30 sec
Annealing 64oC 30 sec Extension 72oC 30 sec
Final extension 72oC 5 min Storage 4oC For ever
MATERIALS AND METHODS
116 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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:
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 7µl of DNA sample (10ng/µL)
PCR Conditions:
Condition Temperature Time
Initial Denaturation 94oC 4 min
35 C
ycle
s Denaturation 94oC 30 sec
Annealing 63.2oC 30 sec
Extension 72oC 1 min
Final extension 72oC 2 min
Storage 40C For ever
MATERIALS AND METHODS
Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 117
The desired PCR product size is 207bp. It was checked in 2% agarose gel. The
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’
MATERIALS AND METHODS
118 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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:
The desired PCR product size is 156bp. It was checked in 2% agarose gel. The
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
les Denaturation 94oC 30 sec
Annealing 62oC 30 sec Extension 72oC 1 min
Final extension 72oC 3 min Storage 4oC For ever
MATERIALS AND METHODS
Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 119
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:
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:
Condition Temperature Time
Initial Denaturation 94oC 3 min
30 C
ycle
s Denaturation 94oC 30 sec
Annealing 61oC 30 sec
Extension 72oC 1 min
Final extension 72oC 2 min
Storage 4oC For ever
MATERIALS AND METHODS
120 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
The desired PCR product size is 236bp. It was checked in 2% agarose gel. The
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
enzyme TaqI (Grandy et al., 1993). The details are as follows -
Primer Sequence -
Forward primer: 5′ - GAT GTG TAG GAA TTA GCC AGG- 3`
Reverse primer: 5′ - GAT ACC CAC TTC AGG AAG TC– 3`
MATERIALS AND METHODS
Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 121
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:
The desired PCR product size is 459bp. It was checked in 2% agarose gel. The
successful PCR product was digested with Taq1 restriction enzyme at 65oC for 2
hour. The digested product was genotyped by electrophoresis in 2% agarose gel. The
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
les Denaturation 94oC 30 sec
Annealing 59oC 30 sec Extension 72oC 1 min
Final extension 72oC 2 min Storage 4oC For ever
MATERIALS AND METHODS
122 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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
enzyme TaqI (Grandy et al., 1993). The details are as follows -
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:
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:
Condition Temperature Time
Initial Denaturation 94oC 3 min
30 C
ycle
s Denaturation 94oC 30 sec
Annealing 58oC 30 sec
Extension 72oC 1 sec
Final extension 72oC 2 min
Storage 4oC For ever
MATERIALS AND METHODS
Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 123
The desired PCR product size is 300bp. It was checked in 2% agarose gel. The
successful PCR product was digested with Taq1 restriction enzyme at 65oC for 2
hour. The digested product was genotyped by electrophoresis in 2% agarose gel. The
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.
MATERIALS AND METHODS
124 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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.
MATERIALS AND METHODS
Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 125
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
MATERIALS AND METHODS
126 Alcoholism: Its Genetics and Impact on Health – A study among Meiteis of Manipur, India
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).
MATERIALS AND METHODS
Alcoholism: Its Genetics and Impact on Health - A study among Meiteis of Manipur, India 127
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