ABSTRACT

Many eye diseases are affecting the people around the globe. Cataract is one of the

leading eye disease caused by the opacity of lens. It ultimately leads to the blindness.

Globally around 1.3 billion people are visually impaired out of that 20 million are blind

due to cataract. It is responsible for 51% of total blindness in the world. The present

study was designed for population based assessment of age related cataract among the

people of District Mansehra. This study was aimed for assessment of risk factors

responsible for cataract moreover to carry out the molecular study of CRYBB1 gene in

order to search if there is any mutation as it is reported widely. Data was collected

from the patients that visit the ophthalmologist for the treatment of cataract. Data from

volunteer patients were collected after the diagnosis of cataract, using a specially

designed questionnaire for this study. Later on collected data was analyzed on SPSS to

find out potential risk factors responsible for cataract. For DNA based analysis of ARC,

blood samples from 35 patients and 20 normal individuals were collected and DNA

extraction was carried out by phenol chloroform method. DNA of good quality was

obtained and quality and quantity of DNA was verified on 1% agrose gel

electrophoresis. Then it was used as template for PCR amplification. The blood

samples were collected from district Mansehra. The result of risk factors shows that

women were affected more due to Age Related Cataract (ARC) as compare to men.

While age group 3 i.e 66-75 years were at higher risk of ARC as compare to other age

groups. PCR amplification of CRYBB1 gene of exon 6 was performed by using specific

ix

primers. The results were verified through sequencing. The nucleotide analysis of

sequences was compared with references sequences taken from NCBI bank. MUSCLE

and Bioedit soft ware’s were used to analyze the results. In the analyzed samples no

mutations were detected in CRYBB1 gene for exon 6. It can be concluded that the risk

of cataract can be reduced by avoiding from the modifiable risk factors like exposure to

UV radiations, reducing the use of steroids and keeping away from smoke. While for

molecular study exon 6 of targeted gene CRYBB1 is not playing its role in the studied
population. Probably there is another exon of same gene or another gene is responsible

for causing cataract in the studied population. By selecting big sample size and

screening more genes, may help to know exact reason of cataract in the study area.

Chapter 1

INTRODUCTION

Eye diseases are one of the important health problems globally. When an

individual is unable to perform daily life activities due to visual disturbance, it is

called visual impairment. It is a major health problem because it affects all the

activities of daily life. The most common causes of vision loss are cataract,

refractive errors and glaucoma. Cataract is the major cause of blindness which

accounts for 51% of total blindness globally. Vision disorders affect people by

reducing their visual acuity and color vision. More than 161 million people in the

world are visually impaired, of which 37 million are blind. Vision disorders

occur due to variety of reasons such as inflammation, degeneration,

developmental problems (Murray et al., 2016).

The eye diseases are frequently varying from different regions and countries and

affecting the quality of life of all groups of ages (Moschos et al., 2014). Cataract

has different types which affect the people of all ages and geography. Congenital

cataract affects the children mostly while age related cataract effects the elderly.

Cataract is the opacity of the lens. The term cataract is derived from Greek word

which means” running water”. The cataract develops slowly. It may affect one

eye or often both eyes. The lens is made of mostly water and protein. The protein

is arranged in a particular way that maintains the lens clarity and let the light

pass through it. But some of the protein may clump, as the person ages and small

area of the lens starts to cloud. This is a cataract. Eventually the cataract becomes

more and more cloudy and grows larger making it difficult to see. Diabetes and
smoking contribute too. Mostly age-related cataracts develop due to protein

clumping. When a cataract is immature, the cloudiness affects only a small

portion of the lens. You may not early notice any changes in your vision.

Cataracts tend to grow slowly, so vision gets worse gradually. Over time, the

cloudy area in the lens may get prolonged, and the size of cataract may increases.

Seeing may become more difficult. Your vision may get duller or blurrier. At the

anterior surface on the lens is present single layer of epithelial cells. This layer

maintains transparency of the lens. Clinically cataract is a heterogeneous disease

(Moschos et al., 2014). The most important causes of age related cataract are age,

gender, diabetes, UV exposure, high B.P, steroid use, smoking eye injury or

inflammation. The risk factors also depend upon once daily life activities. The

common signs and symptoms of cataract include blurred vision or cloudy vision,

loss of peripheral vision, difficulty to see at night, high myopia (Zetterberg,

2015).

Figure 1.1 The picture shows the difference between normal and early stage

cataract eye.

As the person ages, experiences different changes in eye structure. The strength

of eye muscles decreases as with the age. The muscles of eyelid become weaker

with age and also reduce shape of the lens and the person is unable to see the

nearer objects. Aging not only affect the structure and function of the eye but also

destroy functioning of the structure of central nervous system that helps the

visual perceptions and daily activities of vision, perhaps the major age related

eye disease is cataract (Dagneile, 2013).

Figure 1.2 Different age related eye diseases in different parts of eyes.

In Pakistan the prevalence of mature cataract is 30%.An estimated 570, 000

people are bilaterally blind. About 79 million people will be blind by 2020,

according to an estimate, if no interruptions will made. The vision of WHO is to

eliminate the blindness of world by 2020 because it could be prevented (WHO

2014).

Globally as at 2010, it was estimated that cataract was responsible for fifty one %

of world blindness, about 19.9 million human with sixty % of people visually

impaired and eighty two % of all blind being fifty years old and older. The

concept of ARC is that cataract is the heritable cause of visual impairment after

uncorrected refractive errors. The percentage of which was 33% earlier and 43 %

later on (Gimble et al., 2011).

The prevalence of cataract is more in Punjab then other provinces. About 225,000

men and about 345,000 women are affected due to cataract blindness in Pakistan.

Women’s are at higher risk of cataract in the country. In KPK the total prevalence

of unilateral and bilateral cataract is 6.1% (Z Jadoon et al., 2007).

Types of Cataract

There are three major age related cataract types i.e. cortical cataract, nuclear

cataract and posterior sub capsular cataract (Wilson et al., 2003).

Nuclear Cataract

Nuclear cataract is associated with increases of the age and is the most common

form of age related cataract. Change in colour and progressive increase in

opacity leads to dense lens and the person may complain in gradual reduction of

vision acuity and changes in color perception. The lens becomes more and denser

with age. The increase colouration is due to protein aggregation in the nuclear

lens, decreasing its transparency and leading to various symptoms including

visual impairment and dull colour perception. When cataract matures with age

the nucleus opacity increases (Kawane et al., 2003).

Cortical Cataract

This type of cataract tends to occur more in person with diabetes. It is called

cortical cataract because it occur in the cortex of lens. These are whitish wedge

shaped opacities appear on the edge of lens cortex. Studies about cortical cataract
suggest that cortical opacities can induce hyperopic shifts and a significant

astigmatic shifts .With this type of cataract the most frequent symptom is

experiencing problem with glare. The changes can often have seen in the eye ball

as whitish patches. Histological cortical cataract is characterized by an

accumulation of pink staining esinophilic fluid between cortical fibers. This fluid

accumulation causes degeneration of broadening cells (Vasavada et al., 2004, and

Wilson et al., 2013).

Posterior Sub Capsular Cataract

They form on the back side of the lens. This type of cataract is more common in

people who have been taking oral steroid drugs. Near vision is more likely to be

affected than long vision. It is called sub capsular because it from beneath the

lens capsule. The early changes first appear as fine granules in the posterior sub

capsular area of the lens. Although posterior sub capsular cataract can occur

independently, as in this person it is frequently related to chronic intraocular

inflammation corticosteroid use, blunt ocular trauma radiation exposure and

electric shock’s tends to occur in younger patients and progress more quickly

than the other subtype cataract.

Risk Factors Responsible For Cataract

Cataract caused by both environmental and genetics factors. Cataract formation

is considered to be due to multi factorial diseases. Major risk factors for cataract

are:

Age

Aging is one of the important factors responsible for cataract .it affects the people

above 50 mostly. Cataract become more dense and severe with age and it is also

responsible for world blindness. With age lens protein starts to aggregate in

more amounts and lens becomes cloudier and person faces difficulty in vision or

blurred vision. More than90% of people aged 75 -85 have age related changes
(Gupta et at., 2014).

Gender

Studies show that women are at higher risk of lens opacities then men due to

deficiency of estrogen after menopause (Gamasky et al., 2011). The reduction of

estrogen in women results in many anti-aging difficulties. Studies shows that

estrogen has effect on lens therefore plays important role in cataract formation. A

higher risk of cortical cataract may explain the 10-20 % excess risk of cataract

among women (Celojevic D, 2015).

Hypertension

High blood pressure increases with age so it affects the structure of eye and

function. Optic nerves face many structural changes as a result of high blood

pressure (Soji et at., 2014). Studies have shown that high B.P is associated with

age related vision impairment. Because structure and function of eye undergo

through various changes due to high BP (Sato et al., 2018).

Diabetes

Diabetes have close relation with cataract formation .diabetic patient are at

higher risk of cataract formation i.e 2-5 time more frequent in person with

diabetes. Diabetic patient tend to develop cataract at an early age and may be

more prone to develop posterior sub capsular cataract (Gupta et al., 2014).Control

of blood glucose level can lower the risk of diabetic retinopathy. The physiology

and morphology of eye changes in person with diabetes. The diabetic people are

at higher risk of cortical cataract (Javaadi et al., 2008). The percentage of diabetes

is 46.6 % for patients of nuclear cataract and 15.2 % for cortical cataract and 14.2

% for sub capsular cataract .The eye lens take its nutrients from aqueous humour

, fluid filling front portion off eye .Aqueous humors provide oxygen and glucose

when have no good control over glucose level. Sugar level rise in aqueous

humour and in lens t causes it to swell, affecting clarity of vision (Fahim et al .,

2016 ).
Figure 1.3 Relation between diabetes and cataract with age.

UV exposure

Studies showed that cortical cataract is associated with UV radiations. It has a

variable effect on cataract severity. UV radiations are absorbed in the lens .UV

radiations cause the protein to clump that is found in the lens (Fahim et al., 2016).

Smoking

Smoking’s have double risk of formation of cataract. It harms vital structure of

the eyes. Smoking leads to accumulation of heavy metals like cadmium in the

lens. Smoking is associated with 17% increased risk of cataract depends upon the

dose of smoking (Gakamsky et al., 2011).

Steroid use

Systematic steroid use has been associated with posterior sub capsular cataract.

Higher doses and prolonged uses increased the risk of cataract (Fahimet al.,

2016). Other risk factors of cataract include family history, diagnosis of cataract

visual acuity test used to performed doing eye chart exam the doctor will ask to

read letter from distance to find how sharp the vision is. The eye being examined

using slit lamp to detect measurement of lens opacity (Gakamskyetal.,2011).

Smoking, sunlight exposure and systemic disease management are modifiable

risk factors. Taking measures to changes these factors can delay the onset and

progression of cataract formation. Cataract may also be result of direct physical

injury to the lens or its capsule such as blunt ocular trauma or a penetrating

injury. These forms of cataract usually occur within months of injury and can

result in a rapid and significant reduction in visual function (Kim et al., 2017).

Genetic basis of cataract

Although the cataract is considered as an eye disease caused by environmental

effects, but it has the genetics basis as well. There are around 38 genes

10

responsible for different types of cataract. Eye lens consists of different types of
proteins. They are arranged in such a manner to maintain the transparency of

lens. Normal function of lens is controlled by genetic components in the genome.

When there are changes in these genetic components this can result in lens

opacification, these changes are thus related with age related phenomena.

Crystallin is the major lens protein. It is essential for lens maintance. Usually lens

contains 3 types of crystalline, alpha beta and gamma crystallins.

Alpha crystalline is the largest beta and gammas have similar amino acid

sequences and similar structures. The beta crystalline has 5 protein chains, (3

CRYBB and 4 CRYAB),

CRYBB1, BB2, BB3 and BB4. The lens opacity involves biochemical mechanism in

the aging process including unfolding and protein aggregation. The crystalline

are responsible for protein aggregation (Gupta PD et al., 2006). There are

different types of genes involve in cataract formation.CRYBB1.It is the major

crystalline consist of 9% of the soluble protein. Mutations for CRYBB1 are

associated with variety of ocular defects and opacities including recessive

nuclear cataract and pulverulent dominant cataract (Wiatow 2012). The

CRYBB1/βB1 gene located on chromosome no 22q12.1. The gene for βB1

crystallin is an 18.6 kb gene with 6 exons, a structure common to all three of the

genes for β B-crystalline and CRYBA4 (Yang et al., 2008). Studies have shown

that, genetic risk factors are responsible for heritable age related nuclear cataract

11

the percentage for heredity is 14 % -48% and 24%- 75% for age related cortical

cataract (Shiels et al., 2008).

Diagnosis of cataract

Although cataract can be observed commonly but only ophthalmologist can

confirm it after the examination of eye. Different tests are performed to diagnose

cataract including brief eye examination (Henderson et al., 2014). Following are

the diagnosis method to verify the cataract.

Visual acuity
The test is performed to measure vision quality at certain distance. Doctor will

ask to read letters of different size from the chart. The visual acuity test is easy,

pain less and fast way for cataract diagnosis (Henderson et al., 2014).

12

Contrast sensitivity

It is similar to visual acuity test. It is performed to diagnose that how much

cataract decreases, the image contrast due to light scattering and glare caused by

cataract (Pelli 2014).

Slit lamp

This test is by using special microscope .the microscope magnifies the eye to

severity of cataract on the lens. The patient will be asked to place the chin on chin

rest of split lens and light will be directed to the lens of patient’s eye. The doctor

will examine the cloudiness of lens by looking through the slit lamp (Gargallo

2014).

Pupil dilation

It is the common test for diagnosis of cataract. It is a thorough examination of the

lens. The test will measure that weather the cataract is affecting quality of vision

or not.

Treatment of cataract

Cataract surgery is the major option to treat it. The surgery includes lens

implantation (Aao, 2016).The new lens is placed in the eye so the person is able

to see clearly and cloudiness of the vision is treated. Modern cataract operation

provides restoration of vision by the removal of the opaque lens nucleus and

cortex, followed by implantation of an intraocular lens in the lens capsule.

13

During surgery the lens nucleus and cortex are extracted through an opening in

the anterior part of the lens capsule but the main part of the capsule remains in

the eye in order to secure the implanted IOP. Intraocular lenses have been made

improved throughout the years since the first IOP was implanted by Sir Harold.
Modern IOPS are foldable and therefore can be implanted by small incision,

which minimizes tissue trauma to the eye (Spalton et al., 2016).The modern

cataract surgeries are divided into following types.

Phacoemulsification

It is common method of extraction of cataract in the developed world. The

process involves minute incisions with the use of narrow probe to carefully

breakup and slupt crystalline lens using, ultrasonic vibrations. This method can

be considered as extra capsular cataract extraction (ECEC) because capsule is left

largely intact however ECEC refers to the procedure that doesn’t involve an

ultrasound device .This method is expensive .The machine used are of high cost

(Sundaraj et al., 2013).

Intracapsular cataract extraction

It is the oldest method of cataract extraction. It involves pushing of whole lens

within its capsule into posterior segment of the eye this allows the removal of

lens opacity and improved vision function the method involves whole lens

removal from eye, it is called intra capsular cataract extraction (Abdusalam

2015).

14

Extra Capsular Cataract Extraction

In this type of surgery eye lens is removed while the capsule that contain lens is

left intact partially to allow intraocular lens implantation (Abdusalam 2015).

Objectives of Study

1. To assess the risk factor responsible for age related cataract in the study area.

2. To detect the mutations at exon 6 of CRYBB1 gene responsible for age related cataract.

15

Chapter 2

MATERIAL AND METHODS

Ethical Approval

Ethical approval for this study was taken from the bio ethical committee of
Hazara University Mansehra before starting the study.

Informed Consent

Before getting the data and blood samples, patients were briefed about this study

and the use of data and blood sample. After being agreed to share the data and

blood sample, informed consent was taken on the prescribed form.

Data Collection

The data from the age related cataract patients was collected through a specially

designed questionnaire prepared with the consultation of ophthalmologist. The

questionnaire contains two sections, the first section contains information about

the patient and section portion contain signs and symptoms and causes of

cataract. Data was collected from 1000 patients that belong to Mansehra District.

Inclusion Criteria

For data and sample collection, only age related cataract patients were recruited

that reside in Mansehra District and were diagnosed with age related cataract.

Exclusion Criteria

For data collection and sample collection, only age related cataract patients were

included. Patients for other eye diseases were excluded.

16

Sample collection

Blood samples were collected from age related cataract patients. Around 3-5 mL

of blood sample was collected from each patient.

DNA Extraction

DNA extraction of blood samples was carried out using Phenol-Chloroform

method that was optimized earlier in the same lab. The genomic DNA was

extraction by means of phenol chloroform method. Labeled eppendroff tubes

were taken and blood samples were defreezed before extraction and about 200

dilutions were added. After dilution 300µllysis buffer was added. For 2-3

minutes at room temperature tubes were left by adding beta marcepto ethanol

and PK. By using the pipette tip supernant was shifted to the labeled eppendorff
tubes and amount of isopropanol was equally added. For 60 minutes the

incubation of samples were done at 20ºC for overnight. For 20 minutes

centrifugation was done at 8000rpm. After centrifugation pellet was air dried by

carefully removing the ethanol. About 60 ddH20 was added to pellet. To check

the presence of DNA Gel electrophoresis was performed.

Selection of primers

Amplification of CRYBB1 gene was performed with reversed and forward

primers. The double distilled water was used to dilute the primers. So the final

concentration became 100µI.

17

Table 2.1 Primers for exon 6 of CRYBB1 gene.

REVERSE PRIMERS (RP) 5’AGCGAGGAAGTCACATCCCAGT 3’

FORWARD PRIMER (FP) 5’ CCTAGAAAAGGAAACCGAGGC 3’

The Targeted Gene PCR Amplification

The PCR amplification of acquired DNA was carried out using it as a template.

The PCR reagents were prepared by using following components.

TABLE 2.2 Reagents for PCR

Sr

no

List of products added Volume of

reagents

1 10 x Taq buffer 2.0 μl

2 Dntps 2.5 μl

3 Forward primer 2.5µ1

4 TaqDNA polymerase 2.0µ1

5 Template 2.0µ1

6 ddh20 0.5µ1

7 Mgcl2 1.5µ1

8 Reverse primer 12μl

Final volume 25μl

18

The circumstances required for PCR amplification were

35cycle

94°C 94°C 72 °C 72°C

5mint 30sec 53ºC 1mint 5mint HOLD

1mint 10ºC

PCR Product Electrophoresis by Agrose Gel

The 15 % agrose gel was used to check PCR amplified product. With 4µl of

loading dye the product was mixed and loaded on the gel. For 30 minutes gel

electrophoresis was done at 400volt. In UV light apparatus, gel was loaded to

find the presence of desired PCR bands. The photographs of bands were

captured.

Record of DNA Amplification

PCR product was analyzed through agarose gel.1.5% agarose gel was prepared by

adding 0.75g of agarose powder, 2ml of 50X TAE and 48ml of distilled water.

Solution was heated for 2 minutes so that solute dissolved completely in solvent.

About 25µL of Ethidium Bromide was added after cooling the solution. Gel tank

was use to transfer the gel. The combs were inserted and the solution was left for

solidification. Combs were removed after wells formation. With PCR product

about 2µl dye was mixed. The product was loaded on wells after that. The gel

19

electrophoresis was carried out for 30-40 minutes. The obtained product was

observed under UV tech and pictures were captured.

Sequence analysis of PCR product

The amplified PCR product was sent to Beijing Genomic Institute China. The

product was sent along with primers for further sequencing of fragments.

Analysis of nucleotide sequence

The sequencing of blood samples was carried out at Beijing Genomic Institute
China (BGI). The sequence of cataract patients and normal individuals were

compared with control sequence at NCBI.

Mutational analysis

The data of sequencing was analyzed for finding more about mutations if any by

using bioinformatics software bioedit.

Statistical Analysis

All the collected data was statistically analyzed by using SPSS software for the

risk factor assessment. While MUSCLE and Bio edit were used for the analysis of

sequences.

20

Chapter 3

RESULTS

Data collection and Risk Factors Study

The risk factors responsible for cataract were recorded on the basis of

information from the patients. The structure survey was carried out through

gathering the information from ARC patients that were regularly visiting the

hospital. In this study following risk factors are responsible for age related

cataract.

AGE

In this study, age has been found the most important factors associated with

cataract. Patients were divided into 3 age groups, i.egroup1 (51-65)years old

patients, group 2 (66-75), group 3 (76<).Therefore the highest number of patients

with age related cataract wasranged from 66-75 years, followed by 51-65 years to

76< as shown in figure 1.3.

21

Figure 3.1 Cataracts present in different age groups

Gender of Patient

The collected data shows that female are affected more 51.8 % as compare to the
males. It can be seen in fig 3.2

Figure3.2 Gender wise distribution of cataract patients

22

Frequency Ration of Cataract Types

All the sub types of cataract were found in the individuals of study area. Along

with these, the highest ratio among types of cataract was of cortical i.e (55.0%)

followed by nuclear cataract (36.8%) and sub- capsular cataract (4.4%), while

congenital cataract was lowest 3.9%.

Figure 3.3 Ratio of different type of cataract in study area.

Risk Factors Assessment

Various risk factors are responsible for cataract. Steroid use was found as highest

risk factor accounts 52.4 % of all the cases. It depends upon the amount of

23

dosage, so prolong use of steroids play an important role in development of

cataract. Hyper tension is second most potential risk factor of cataract .around

485 individuals i-e 48.5% has chances of cataract due to hypertension. Changes in

the lens protein occur as a result of hypertension, leading to cataract formation.

Next important risk factor is diabetes mellitus. Out of 1000 patients 393 were

diabetic. Exposure to ultraviolet rays (UV) is another potential risk factor of

cataract. People of district Mansehra have more UV exposure due to daily

activities during summer therefore cataract formation is more common among

them. Almost 30.9% individuals are affected with cataract due to UV exposure.

Next factor of cataract is smoking. A total of 8.7 % patients of cataract were

involved in smoking.

Table 3.1 Potential risk factors responsible for age related cataract

Parameter Frequency Valid Percentage

Steroid Use

Hypertension

Diabetes
UV Exposure

Smoking

Family History

524 / 1000

485 / 1000

393 / 1000

309 / 1000

87 / 1000

153/ 1000

52.4%

48.5%

39.3%

30.9%

8.7 %

1.53 %

24

Symptoms of Cataract

The frequency of symptoms of cataract is mentioned in following table. The table

contain common symptoms for cataract.

Table 3.2 Represents the frequency and percentage of symptoms of cataract.

DNA Based Analysis of Cataract

Data was recorded on the designed questionnaire from cataract patients,

clarifying the type of research work. Blood sample were collected from 35

patients of age related cataract, with the help of ophthalmologist from Mehboob

charity Vision Hospital, Gandiyan Mansehra.

Parameters

Loss Of Peripheral Vision

Sensitive To Light

High Myopia
Double Vision In One Eye

Pupils appear grey

Blurred vision

Frequent onset of glasses

Family History

Frequencies

984/ 1000

974

968

245

882

877

51

153

Valid Percentage

98.4%

97.4%

96.8%

24.5%

8.82%

8.87%

5.1%

1.53%

25

Genomic DNA Extraction

The DNA was isolated from blood by using standard protocol method. This

work was perfomed in the Human Genetics Lab Hazara University Mansehra.

The figures shows result of extracted DNA. The quality and quantity of DNA

was checked on 1% agarose gel.

Figure 3.4 The extracted genomic DNA results of age related cataract patients.

Amplification of CRYBB1 Gene

The extracted genomic DNA was used as template for amplification of desired

gene i.eCRYBB1 gene.

Gel electrophoresis of PCR products.

An agarose gel represented of amplified PCR product using CRYBB1 gene

primers (CRB1) beside the genomic DNA of cataract patients.

26

Figure 3.5 Agarose gel representation of PCR amplified product using CRYBB1

primers.

Analysis of Nucleotide Sequence

The sequencing of samples carried out at Beijing Genomic Institute China. The

sequence of cataract patients and normal individuals were compared with

control sequence at NCBI, database.

The sequences were obtained from BGI, China. The sequences were BLAST

against NCBI to check the similarity. The results were aligned with reference

sequence of exon 6. The alignment results of disease and control were shown

from exon 6 of CRYBB1 gene from NCBI.

Reference sequence of exon 6 of CRYBB1gene for primer CRB1

GCTGTGGCCAGGGTTTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTGCAGTGATC

CAGGCGAGGTCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCCAGCTTCCCCTTTGTG

CCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGGGG

AGTCCCCAGGTAAGAGGGAACCCACAGCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGAAAAGG

GAGACAGAGAGAGGGAGGCAGAGATAGCAGTGCCATGAGAGTCAGAGAGCAGGGCCATGGAGAT

AAAGACGGAAGTGGAGAGAGGAAGAAAGGGAGGGAGAGGGAGAGGGGAGAGAGTGAGAAAAG

ACAGAAATAGAGAGAGAAGTAAATATAATTAAAAAGGACAGATAGAGAGATAAGCAAAGATACA

ATGAGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGGGAGAGAAA

27

Figure 3.6 Nucleotide sequence peaks representingCRYBB1 gene exon 6region.

28

Nucleotide Sequence Analysis For 4G1

GGGTTTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTGCAGTGATCCAGGCGAGGT

CAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCCAGCTTCCCCTTTGTGCCACAGACTA

TAGGGCCTGATGTCTGCCAGACTATAAAATGGGGGGTCGGACTCTAGTCACCCATAGCACCTGCCC

GCCTGCCTGTCCTCCAGCAGGAAGTAGCAAGTCCCCAGGTAAGAGGGAACCCACAGCCCAGCTGA

GAGCTGAAGGGGAGGGGAAGAGAAAAGGGAGACAGAGAGAGGGAGGCAGAGATAGCAGTGCCA

TGAGAGTCAGAGAGCAGGGCCATGGAGATAAAGACGGAAGTGGAGAGAGGAAGAAAGGGAGGG

AGAGGGAGAGGGGAGAGAGTGAGAAAAGACAGAAATAGAGAGAGAAGTAAATATAATTAAAAA

GGACAGATAGAGAGATAAGCAAAGATACAATGAGGGAGAGATAGTGGGGGAGAGAGGAACAGA

GAGGGAGAGAA

Reference Sequence Comparison And 4G1

Ex6 GGGTTTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTGCAGTGATCCAG

4-G1 GGGTTTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTGCAGTGATCCAG

************************************************************

Ex6 GCGAGGTCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCCAGCTTCCCCTT

4-G1 GCGAGGTCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCCAGCTTCCCCTT

************************************************************

Ex6 TGTGCCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGGGGGTCGGACTCTA

4-G1 TGTGCCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGGGGGTCGGACTCTA

************************************************************

Ex6 GTCACCCATAGCACCTGCCCGCCTGCCTGTCCTCCAGCAGGAAGTAGCAAGTCCCCAGGT

4-G1 GTCACCCATAGCACCTGCCCGCCTGCCTGTCCTCCAGCAGGAAGTAGCAAGTCCCCAGGT

************************************************************

Ex6 AAGAGGGAACCCACAGCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGAAAAGGGAGACAG

4-G1 AAGAGGGAACCCACAGCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGAAAAGGGAGACAG

************************************************************

Ex6 AGAGAGGGAGGCAGAGATAGCAGTGCCATGAGAGTCAGAGAGCAGGGCCATGGAGATAAA

4-G1 AGAGAGGGAGGCAGAGATAGCAGTGCCATGAGAGTCAGAGAGCAGGGCCATGGAGATAAA
29

************************************************************

Ex6 GACGGAAGTGGAGAGAGGAAGAAAGGGAGGGAGAGGGAGAGGGGAGAGAGTGAGAAAAGA

4-G1 GACGGAAGTGGAGAGAGGAAGAAAGGGAGGGAGAGGGAGAGGGGAGAGAGTGAGAAAAGA

************************************************************

Ex6 CAGAAATAGAGAGAGAAGTAAATATAATTAAAAAGGACAGATAGAGAGATAAGCAAAGAT

4-G1 CAGAAATAGAGAGAGAAGTAAATATAATTAAAAAGGACAGATAGAGAGATAAGCAAAGAT

************************************************************

Ex6 ACAATGAGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGGGAGAGAAA

4-G1 ACAATGAGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGGGAGAGAAA

*************************************************

Figure 3.7 Nucleotide sequence peaks representingCRYBB1 gene at exon 6 region

Nucleotide Sequence Analysis of 4G2

GCTGTGGCCAGGGTTTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTGCAGTGATCCAGGCG

AGGTCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCCAGCTTCCCCTTTGTGCCACAGACTAT

AGGGCCTGATGTCTGCCAGACTATAAAATGGGGGGTCGGACTCTAGTCACCCATAGCACCTGCCCGCCTGCC

TGTCCTCCAGCAGGAAGTAGCAAGTCCCCAGGTAAGAGGGAACCCACAGCCCAGCTGAGAGCTGAAGGGG

AGGGGAAGAGAAAAGGGAGACAGAGAGAGGGAGGCAGAGATAGCAGTGCCATGAGAGTCAGAGAGCAG

GGCCATGGAGATAAAGACGGAAGTGGAGAGAGGAAGAAAGGGAGGGAGAGGGAGAGGGGAGAGAGTGA

GAAAAGACAGAAATAGAGAGAGAAGTAAATATAATTAAAAAGGACAGATAGAGAGATAAGCAAAGATAC

AATGAGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGGGAGAGAAACAGACAGGCAGATAGG

30

Reference Sequence Comparison and 4G2

Ex6 GCTGTGGCCAGGGTTTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTGC

4-G2 GCTGTGGCCAGGGTTTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTGC

************************************************************

Ex6 AGTGATCCAGGCGAGGTCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCCA

4-G2 AGTGATCCAGGCGAGGTCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCCA

************************************************************
Ex6 GCTTCCCCTTTGTGCCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGGGGG

4-G2 GCTTCCCCTTTGTGCCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGGGGG

************************************************************

Ex6 TCGGACTCTAGTCACCCATAGCACCTGCCCGCCTGCCTGTCCTCCAGCAGGAAGTAGCAA

4-G2 TCGGACTCTAGTCACCCATAGCACCTGCCCGCCTGCCTGTCCTCCAGCAGGAAGTAGCAA

************************************************************

Ex6 GTCCCCAGGTAAGAGGGAACCCACAGCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGAAA

4-G2 GTCCCCAGGTAAGAGGGAACCCACAGCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGAAA

************************************************************

Ex6 AGGGAGACAGAGAGAGGGAGGCAGAGATAGCAGTGCCATGAGAGTCAGAGAGCAGGGCCA

4-G2 AGGGAGACAGAGAGAGGGAGGCAGAGATAGCAGTGCCATGAGAGTCAGAGAGCAGGGCCA

************************************************************

Ex6 TGGAGATAAAGACGGAAGTGGAGAGAGGAAGAAAGGGAGGGAGAGGGAGAGGGGAGAGAG

4-G2 TGGAGATAAAGACGGAAGTGGAGAGAGGAAGAAAGGGAGGGAGAGGGAGAGGGGAGAGAG

************************************************************

Ex6 TGAGAAAAGACAGAAATAGAGAGAGAAGTAAATATAATTAAAAAGGACAGATAGAGAGAT

4-G2 TGAGAAAAGACAGAAATAGAGAGAGAAGTAAATATAATTAAAAAGGACAGATAGAGAGAT

************************************************************

Ex6 AAGCAAAGATACAATGAGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGGGA

4-G2 AAGCAAAGATACAATGAGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGGGA

*****************************************************

31

Figure 3.8Nucleotide sequence peaks ofCRYBB1 gene representing exon 6region.

Nucleotide Sequence analysis of 4G3

ATACAATGAGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGGGAGAGAAACAGACAGGCAGA

TAGGCATGCTGTGGCCAGGGTTTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTGC

AGTGATCCAGGCGAGGTCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCCAGCTTCC

CCTTTGTGCCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGGGGGTCGGACTCTAGTC

ACCCATAGCACCTGCCCGCCTGCCTGTCCTCCAGCAGGAAGTAGCAAGTCCCCAGGTAAGAGGGA
ACCCACAGCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGAAAAGGGAGACAGAGAGAGGGAGG

CAGAGATAGCAGTGCCATGAGAGTCAGAGAGCAGGGCCATGGAGATAAAGACGGAAGTGGAGAG

AGGAAGAAAGGGAGGGAGAGGGAGAGGGGAGAGAGTGAGAAAAGACAGAAATAGAGAGAGAA
GTAAATATAATTAAAAAGGACAGATAGAGAGATAA

Comparison of Reference Sequence and 4G3

Ex6 -GCTGTGGCCAGGGTTTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTG

4G3 TGCTGTGGCCAGGGTTTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTG

***********************************************************

Ex6 CAGTGATCCAGGCGAGGTCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCC

4G3 CAGTGATCCAGGCGAGGTCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCC

************************************************************

Ex6 AGCTTCCCCTTTGTGCCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGGGG

4G3 AGCTTCCCCTTTGTGCCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGGGG

************************************************************

Ex6 GTCGGACTCTAGTCACCCATAGCACCTGCCCGCCTGCCTGTCCTCCAGCAGGAAGTAGCA

32

4G3 GTCGGACTCTAGTCACCCATAGCACCTGCCCGCCTGCCTGTCCTCCAGCAGGAAGTAGCA

************************************************************

Ex6 AGTCCCCAGGTAAGAGGGAACCCACAGCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGAA

4G3 AGTCCCCAGGTAAGAGGGAACCCACAGCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGAA

************************************************************

Ex6 AAGGGAGACAGAGAGAGGGAGGCAGAGATAGCAGTGCCATGAGAGTCAGAGAGCAGGGCC

4G3 AAGGGAGACAGAGAGAGGGAGGCAGAGATAGCAGTGCCATGAGAGTCAGAGAGCAGGGCC

************************************************************

Ex6 ATGGAGATAAAGACGGAAGTGGAGAGAGGAAGAAAGGGAGGGAGAGGGAGAGGGGAGAGA

4G3 ATGGAGATAAAGACGGAAGTGGAGAGAGGAAGAAAGGGAGGGAGAGGGAGAGGGGAGAGA

************************************************************

Ex6 GTGAGAAAAGACAGAAATAGAGAGAGAAGTAAATATAATTAAAAAGGACAGATAGAGAGA

4G3 GTGAGAAAAGACAGAAATAGAGAGAGAAGTAAATATAATTAAAAAGGACAGATAGAGAGA
 ************************************************************

REF TAAGCAAAGATACAATGAGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGGGAGAGAAA

4G3 TAAGCAAAGATACAATGAGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGGGAGAGAAA

************************************************************

Figure 3.9Nucleotide sequence peaks ofCRYBB1 gene representing exon 6region.

33

Nucleotide Sequence Analysis of 4G4

CTGCTGTGGCCAGGGTTTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTGCAGTGA

TCCAGGCGAGGTCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCCAGCTTCCCCTTTG

TGCCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGGGGGTCGGACTCTAGTCACCCA

TAGCACCTGCCCGCCTGCCTGTCCTCCAGCAGGAAGTAGCAAGTCCCCAGGTAAGAGGGAACCCAC

AGCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGAAAAGGGAGACAGAGAGAGGGAGGCAGAGA

TAGCAGTGCCATGAGAGTCAGAGAGCAGGGCCATGGAGATAAAGACGGAAGTGGAGAGAGGAAG

AAAGGGAGGGAGAAGGAGAGGGGAGAGAGTGTTTATAGACAGAAATAGAGAGAGAAGTAAATAT

AATTAAAAAGGACAGATAGAGAGATAAGCAAAGATACAATGAGGGAGAGATAGTGGGGGAGAGA

GGAACAGAGAGGGAGAGAAACAGACAGGCAGATAGGCA

Comparison of Reference Sequence and 4G4

EX6 --GCTGTGGCCAGGGTTTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCT

4-G4 CTGCTGTGGCCAGGGTTTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCT

**********************************************************

EX6 GCAGTGATCCAGGCGAGGTCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGC

4-G4 GCAGTGATCCAGGCGAGGTCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGC

************************************************************

Ex6 CAGCTTCCCCTTTGTGCCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGGG

4-G4 CAGCTTCCCCTTTGTGCCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGGG

************************************************************

Ex6 GGTCGGACTCTAGTCACCCATAGCACCTGCCCGCCTGCCTGTCCTCCAGCAGGAAGTAGC

4-G4 GGTCGGACTCTAGTCACCCATAGCACCTGCCCGCCTGCCTGTCCTCCAGCAGGAAGTAGC

************************************************************
Ex6 AAGTCCCCAGGTAAGAGGGAACCCACAGCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGA

4-G4 AAGTCCCCAGGTAAGAGGGAACCCACAGCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGA

************************************************************

Ex6 AAAGGGAGACAGAGAGAGGGAGGCAGAGATAGCAGTGCCATGAGAGTCAGAGAGCAGGGC

34

4-G4 AAAGGGAGACAGAGAGAGGGAGGCAGAGATAGCAGTGCCATGAGAGTCAGAGAGCAGGGC

************************************************************

Ex6 CATGGAGATAAAGACGGAAGTGGAGAGAGGAAGAAAGGGAGGGAGAGGG-----------

4-G4 CATGGAGATAAAGACGGAAGTGGAGAGAGGAAGAAAGGGAGGGAGAAAG-----------

*************************************************

Ex6 AGTGAGAAAAGACAGAAATAGAGAGAGAAGTAAATATAATTAAAAAGGACAGATAGAGAG

4-G4 ---------AGACAGAAATAGAGAGAGAAGTAAATATAATTAAAAAGGACAGATAGAGAG

***************************************************

Ex6 ATAAGCAAAGATACAATGAGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGGGAGAGAA

4-G4 ATAAGCAAAGATACAATGAGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGGGAGAGAA

************************************************************

Figure 3.10 Nucleotide sequence peaks ofCRYBB1 gene representing exon 6

region.

Nucleotide sequence analysis of 4G5

AAGTGCTGCTGTCTGCAGTGATCCAGGCGAGGTCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCC

TGCTGAGCCAGCTTCCCCTTTGTGCCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGG

GGGTCGGACTCTAGTCACCCATAGCACCTGCCCGCCTGCCTGTCCTCCAGCAGGAAGTAGCAAGTC

CCCAGGTAAGAGGGAACCCACAGCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGAAAAGGGAG

ACAGAGAGAGGGAGGCAGAGATAGCAGTGCCATGAGAGTCAGAGAGCAGGGCCATGGAGATAAA

GACGGAAGTGGAGAGAGGAAGAAAGGGAGGGAGAGGGAGAGGGGAGAGAGTGAGAAAAGACA

GAAATAGAGAGAGAAGTAAATATAATTAAAAAGGACAGATAGAGAGATAAGCAAAGATACAATG

AGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGGGAGAGAAACAGACAGGCCAGAATAGGCA

35
Comparison of Reference Sequence and 4G5

Ex6 AAGGAAAGTGCTGCTGTCTGCAGTGATCCAGGCGAGGTCAGGGCCCCAGTCCCCCACTCC

4-G5 -----AAGTGCTGCTGTCTGCAGTGATCCAGGCGAGGTCAGGGCCCCAGTCCCCCACTCC

*******************************************************

Ex6 CTGCCCAGGCCTGCTGAGCCAGCTTCCCCTTTGTGCCACAGACTATAGGGCCTGATGTCT

4-G5 CTGCCCAGGCCTGCTGAGCCAGCTTCCCCTTTGTGCCACAGACTATAGGGCCTGATGTCT

************************************************************

Ex6 GCCAGACTATAAAATGGGGGGTCGGACTCTAGTCACCCATAGCACCTGCCCGCCTGCCTG

4-G5 GCCAGACTATAAAATGGGGGGTCGGACTCTAGTCACCCATAGCACCTGCCCGCCTGCCTG

************************************************************

Ex6 TCCTCCAGCAGGAAGTAGCAAGTCCCCAGGTAAGAGGGAACCCACAGCCCAGCTGAGAGC

4-G5 TCCTCCAGCAGGAAGTAGCAAGTCCCCAGGTAAGAGGGAACCCACAGCCCAGCTGAGAGC

************************************************************

Ex6 TGAAGGGGAGGGGAAGAGAAAAGGGAGACAGAGAGAGGGAGGCAGAGATAGCAGTGCCAT

4-G5 TGAAGGGGAGGGGAAGAGAAAAGGGAGACAGAGAGAGGGAGGCAGAGATAGCAGTGCCAT

************************************************************

Ex6 GAGAGTCAGAGAGCAGGGCCATGGAGATAAAGACGGAAGTGGAGAGAGGAAGAAAGGGAG

4-G5 GAGAGTCAGAGAGCAGGGCCATGGAGATAAAGACGGAAGTGGAGAGAGGAAGAAAGGGAG

************************************************************

Ex6 GGAGAGGGAGAGGGGAGAGAGTGAGAAAAGACAGAAATAGAGAGAGAAGTAAATATAATT

4-G5 GGAGAGGGAGAGGGGAGAGAGTGAGAAAAGACAGAAATAGAGAGAGAAGTAAATATAATT

************************************************************

Ex6 AAAAAGGACAGATAGAGAGATAAGCAAAGATACAATGAGGGAGAGATAGTGGGGGAGAGA

4-G5 AAAAAGGACAGATAGAGAGATAAGCAAAGATACAATGAGGGAGAGATAGTGGGGGAGAGA

************************************************************

Ex6 GGAACAGAGAGGGAGAGAAACAGACAGGC

4-G5 GGAACAGAGAGGGAGAGAAACAGACAGGC

*****************************

36
Figure 3.11 Nucleotide sequence peaks of CRYBB1 gene representing exon 6

region.

Nucleotide Sequence Analysis of 4G6

TTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTGCAGTGATCCAGGCGAGGTCAGG

GCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCCAGCTTCCCCTTTGTGCCACAGACTATAGG

GCCTGATGTCTGCCAGACTATAAAATGGGGGGTCGGACTCTAGTCACCCATAGCACCTGCCCGCCT

GCCTGTCCTCCAGCAGGAAGTAGCAAGTCCCCAGGTAAGAGGGAACCCACAGCCCAGCTGAGAGC

TGAAGGGGAGGGGAAGAGAAAAGGGAGACAGAGAGAGGGAGGCAGAGATAGCAGTGCCATGAG

AGTCAGAGAGCAGGGCCATGGAGATAAAGACGGAAGTGGAGAGAGGAAGAAAGGGAGGGAGAG

GGAGAGGGGAGAGAGTGAGAAAAGACAGAAATAGAGAGAGAAGTAAATATAATTAAAAAGGAC

AGATAGAGAGATAAGCAAAGATACAATGAGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGG

GAGAGAAACAGACAGGCAGATAGG

Comparison of Reference Sequence and 4G6

Ex6 AAGGAAAGTGCTGCTGTCTGCAGTGATCCAGGCGAGGTCAGGGCCCCAGTCCCCCACTCC

4-G6 AAGGAAAGTGCTGCTGTCTGCAGTGATCCAGGCGAGGTCAGGGCCCCAGTCCCCCACTCC

************************************************************

Ex6 CTGCCCAGGCCTGCTGAGCCAGCTTCCCCTTTGTGCCACAGACTATAGGGCCTGATGTCT

4-G6 CTGCCCAGGCCTGCTGAGCCAGCTTCCCCTTTGTGCCACAGACTATAGGGCCTGATGTCT

************************************************************

37

Ex6 GCCAGACTATAAAATGGGGGGTCGGACTCTAGTCACCCATAGCACCTGCCCGCCTGCCTG

4-G6 GCCAGACTATAAAATGGGGGGTCGGACTCTAGTCACCCATAGCACCTGCCCGCCTGCCTG

************************************************************

Ex6 TCCTCCAGCAGGAAGTAGCAAGTCCCCAGGTAAGAGGGAACCCACAGCCCAGCTGAGAGC

4-G6 TCCTCCAGCAGGAAGTAGCAAGTCCCCAGGTAAGAGGGAACCCACAGCCCAGCTGAGAGC

************************************************************

Ex6 TGAAGGGGAGGGGAAGAGAAAAGGGAGACAGAGAGAGGGAGGCAGAGATAGCAGTGCCAT

4-G6 TGAAGGGGAGGGGAAGAGAAAAGGGAGACAGAGAGAGGGAGGCAGAGATAGCAGTGCCAT

************************************************************
Ex6 GAGAGTCAGAGAGCAGGGCCATGGAGATAAAGACGGAAGTGGAGAGAGGAAGAAAGGGAG

4-G6 GAGAGTCAGAGAGCAGGGCCATGGAGATAAAGACGGAAGTGGAGAGAGGAAGAAAGGGAG

************************************************************

Ex6 GGAGAGGGAGAGGGGAGAGAGTGAGAAAAGACAGAAATAGAGAGAGAAGTAAATATAATT

4-G6 GGAGAGGGAGAGGGGAGAGAGTGAGAAAAGACAGAAATAGAGAGAGAAGTAAATATAATT

************************************************************

Ex6 AAAAAGGACAGATAGAGAGATAAGCAAAGATACAATGAGGGAGAGATAGTGGGGGAGAGA

4-G6 AAAAAGGACAGATAGAGAGATAAGCAAAGATACAATGAGGGAGAGATAGTGGGGGAGAGA

************************************************************

Ex6 GGAACAGAGAGGGAGAGAAACAGACAGGCAGATAGG

4-G6 GGAACAGAGAGGGAGAGAAACAGACAGGCAGATAGG

************************************

38

Figure 3.12 Nucleotide sequence peaks of CRYBB1 gene representing exon 6

region.

Nucleotide ssequence aanalysis of 4G7

CCTTTGTGCCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGGGGGTCGGACTCTAGTC

ACCCATAGCACCTGCCCGCCTGCCTGTCCTCCAGCAGGAAGTAGCAAGTCCCCAGGTAAGAGGGA

ACCCACAGCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGAAAAGGGAGACAGAGAGAGGGAGG

CAGAGATAGCAGTGCCATGAGAGTCAGAGAGCAGGGCCATGGAGATAAAGACGGAAGTGGAGAG

AGGAAGAAAGGGAGGGAGAGGGAGAGGGGAGAGAGTGAGAAAAGACAGAAATAGAGAGAGAA

GTAAATATAATTAAAAAGGACAGATAGAGAGATAAGCAAAGATACAATGAGGGAGAGATAGTGG
GGGAGAGAGGAACAGAGAGGGAGAGAAAC

Comparison of Reference Sequence and 4G7

Ex7 CCTTTGTGCCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGGGGGTCGGAC

4-G7 CCTTTGTGCCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGGGGGTCGGAC

************************************************************

Ex7 TCTAGTCACCCATAGCACCTGCCCGCCTGCCTGTCCTCCAGCAGGAAGTAGCAAGTCCCC

4-G7 TCTAGTCACCCATAGCACCTGCCCGCCTGCCTGTCCTCCAGCAGGAAGTAGCAAGTCCCC
 ************************************************************

Ex7 AGGTAAGAGGGAACCCACAGCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGAAAAGGGAG

4-G7 AGGTAAGAGGGAACCCACAGCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGAAAAGGGAG

************************************************************

Ex7 ACAGAGAGAGGGAGGCAGAGATAGCAGTGCCATGAGAGTCAGAGAGCAGGGCCATGGAGA

4-G7 ACAGAGAGAGGGAGGCAGAGATAGCAGTGCCATGAGAGTCAGAGAGCAGGGCCATGGAGA

************************************************************

Ex7 TAAAGACGGAAGTGGAGAGAGGAAGAAAGGGAGGGAGAGGGAGAGGGGAGAGAGTGAGAA

4-G7 TAAAGACGGAAGTGGAGAGAGGAAGAAAGGGAGGGAGAGGGAGAGGGGAGAGAGTGAGAA

************************************************************

Ex7 AAGACAGAAATAGAGAGAGAAGTAAATATAATTAAAAAGGACAGATAGAGAGATAAGCAA

4-G7 AAGACAGAAATAGAGAGAGAAGTAAATATAATTAAAAAGGACAGATAGAGAGATAAGCAA

************************************************************

39

Ex7 AGATACAATGAGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGGGAGAGAAAC

4-G7 AGATACAATGAGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGGGAGAGAAAC

******************************************************

40

Figure 3.13 Nucleotide sequence peaks of CRYBB1 gene representing exon 6

region

Nucleotide sequence analysis of 4G8

TTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTGCAGTGATCCAGGCGAGGTCAGG

GCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCCAGCTTCCCCTTTGTGCCACAGACTATAGG

GCCTGATGTCTGCCAGACTATAAAATGGGGGGTCGGACTCTAGTCACCCATAGCACCTGCCCGCCT

GCCTGTCCTCCAGCAGGAAGTAGCAAGTCCCCAGGTAAGAGGGAACCCACAGCCCAGCTGAGAGC

TGAAGGGGAGGGGAAGAGAAAAGGGAGACAGAGAGAGGGAGGCAGAGATAGCAGTGCCATGAG

AGTCAGAGAGCAGGGCCATGGAGATAAAGACGGAAGTGGAGAGAGGAAGAAAGGGAGGGAGAG

GGAGAGGGGAGAGAGTGAGAAAAGACAGAAATAGAGAGAGAAGTAAATATAATTAAAAAGGAC

AGATAGAGAGATAAGCAAAGATACAATGAGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGG
GAGAGAAACAGACAGGCAGATAGG

Comparison of reference sequence and 4G8

REF TTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTGCAGTGATCCAGGCGA

4-G8 TTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTGCAGTGATCCAGGCGA

************************************************************

REF GGTCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCCAGCTTCCCCTTTGTG

4-G8 GGTCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCCAGCTTCCCCTTTGTG

************************************************************

Ex6 CCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGGGGGTCGGACTCTAGTCA

4-G8 CCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGGGGGTCGGACTCTAGTCA

41

************************************************************

Ex6 CCCATAGCACCTGCCCGCCTGCCTGTCCTCCAGCAGGAAGTAGCAAGTCCCCAGGTAAGA

4-G8 CCCATAGCACCTGCCCGCCTGCCTGTCCTCCAGCAGGAAGTAGCAAGTCCCCAGGTAAGA

************************************************************

Ex6 GGGAACCCACAGCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGAAAAGGGAGACAGAGAG

4-G8 GGGAACCCACAGCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGAAAAGGGAGACAGAGAG

************************************************************

Ex6 AGGGAGGCAGAGATAGCAGTGCCATGAGAGTCAGAGAGCAGGGCCATGGAGATAAAGACG

4-G8 AGGGAGGCAGAGATAGCAGTGCCATGAGAGTCAGAGAGCAGGGCCATGGAGATAAAGACG

************************************************************

Ex6 GAAGTGGAGAGAGGAAGAAAGGGAGGGAGAGGGAGAGGGGAGAGAGTGAGAAAAGACAGA

4-G8 GAAGTGGAGAGAGGAAGAAAGGGAGGGAGAGGGAGAGGGGAGAGAGTGAGAAAAGACAGA

************************************************************

Ex6 AATAGAGAGAGAAGTAAATATAATTAAAAAGGACAGATAGAGAGATAAGCAAAGATACAA

4-G8 AATAGAGAGAGAAGTAAATATAATTAAAAAGGACAGATAGAGAGATAAGCAAAGATACAA

************************************************************

Ex6 TGAGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGGGAGAGAAACAGACAGGCAGATAG

4-G8 TGAGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGGGAGAGAAACAGACAGGCAGATAG
 ************************************************************

Ex6 G

4-G8 G

Figure 3.14 Nucleotide sequence peaks of CRYBB1 gene representing exon 6

region

42

Nucleotide sequence analysis of 4G9

AGGGTTTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTGCAGTGATCCAGGCGAGG

TCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCCAGCTTCCCCTTTGTGCCACAGACT

ATAGGGCCTGATGTCTGCCAGACTATAAAATGGGGGGTCGGACTCTAGTCACCCATAGCACCTGCC

CGCCTGCCTGTCCTCCAGCAGGAAGTAGCAAGTCCCCAGGTAAGAGGGAACCCACAGCCCAGCTG

AGAGCTGAAGGGGAGGGGAAGAGAAAAGGGAGACAGAGAGAGGGAGGCAGAGATAGCAGTGCC

ATGAGAGTCAGAGAGCAGGGCCATGGAGATAAAGACGGAAGTGGAGAGAGGAAGAAAGGGAGG

GAGAGGGAGAGGGGAGAGAGTGAGAAAAGACAGAAATAGAGAGAGAAGTAAATATAATTAAAA

AGGACAGATAGAGAGATAAGCAAAGATACAATGAGGGAGAGATAGTGGGGGAGAGAGGAACAG

AGAGGGAGAGAAACAGACAGGCAGATAGG

Comparison of reference sequence and 4G9

Ex6 AGGGTTTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTGCAGTGATCCA

4-G9 AGGGTTTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTGCAGTGATCCA

************************************************************

Ex6 GGCGAGGTCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCCAGCTTCCCCT

4-G9 GGCGAGGTCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCCAGCTTCCCCT

************************************************************

Ex6 TTGTGCCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGGGGGTCGGACTCT

4-G9 TTGTGCCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGGGGGTCGGACTCT

************************************************************

Ex6 AGTCACCCATAGCACCTGCCCGCCTGCCTGTCCTCCAGCAGGAAGTAGCAAGTCCCCAGG

4-G9 AGTCACCCATAGCACCTGCCCGCCTGCCTGTCCTCCAGCAGGAAGTAGCAAGTCCCCAGG

************************************************************
Ex6 TAAGAGGGAACCCACAGCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGAAAAGGGAGACA

4-G9 TAAGAGGGAACCCACAGCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGAAAAGGGAGACA

************************************************************

43

Ex6 GAGAGAGGGAGGCAGAGATAGCAGTGCCATGAGAGTCAGAGAGCAGGGCCATGGAGATAA

4-G9 GAGAGAGGGAGGCAGAGATAGCAGTGCCATGAGAGTCAGAGAGCAGGGCCATGGAGATAA

************************************************************

Ex6 AGACGGAAGTGGAGAGAGGAAGAAAGGGAGGGAGAGGGAGAGGGGAGAGAGTGAGAAAAG

4-G9 AGACGGAAGTGGAGAGAGGAAGAAAGGGAGGGAGAGGGAGAGGGGAGAGAGTGAGAAAAG

************************************************************

Ex6 ACAGAAATAGAGAGAGAAGTAAATATAATTAAAAAGGACAGATAGAGAGATAAGCAAAGA

4-G9 ACAGAAATAGAGAGAGAAGTAAATATAATTAAAAAGGACAGATAGAGAGATAAGCAAAGA

************************************************************

Ex6 TACAATGAGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGGGAGAGAAACAGACAGGCA

4-G9 TACAATGAGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGGGAGAGAAACAGACAGGCA

************************************************************

Ex6 GATAGG

4-G9 GATAGG

******

Figure 3.15 Nucleotide sequence peaks of CRYBB1 gene representing exon

6region.

44

Nucleotide sequence analysis of 4G10

GCTGTGGCCAGGGTTTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTGCAGTGATC

CAGGCGAGGTCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCCAGCTTCCCCTTTGTG

CCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGGGGGTCGGACTCTAGTCACCCATA

GCACCTGCCCGCCTGCCTGTCCTCCAGCAGGAAGTAGCAAGTCCCCAGGTAAGAGGGAACCCACA

GCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGAAAAGGGAGACAGAGAGAGGGAGGCAGAGAT

AGCAGTGCCATGAGAGTCAGAGAGCAGGGCCATGGAGATAAAGACGGAAGTGGAGAGAGGAAGA
AAGGGAGGGAGAGGGAGAGGGGAGAGAGTGAGAAAAGACAGAAATAGAGAGAGAAGTAAATAT

AATTAAAAAGGACAGATAGAGAGATAAGCAAAGATACAATGAGGGAGAGATAGTGGGGGAGAGA

GGAACAGAGAGGGAGAGAAACAGACA

Comparison of reference sequence and 4G10

Ex6 GCTGTGGCCAGGGTTTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTGC

4-G10 GCTGTGGCCAGGGTTTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTGC

************************************************************

Ex6 AGTGATCCAGGCGAGGTCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCCA

4-G10 AGTGATCCAGGCGAGGTCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCCA

************************************************************

Ex6 GCTTCCCCTTTGTGCCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGGGGG

4-G10 GCTTCCCCTTTGTGCCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGGGGG

************************************************************

Ex6 TCGGACTCTAGTCACCCATAGCACCTGCCCGCCTGCCTGTCCTCCAGCAGGAAGTAGCAA

4-G10 TCGGACTCTAGTCACCCATAGCACCTGCCCGCCTGCCTGTCCTCCAGCAGGAAGTAGCAA

************************************************************

Ex6 GTCCCCAGGTAAGAGGGAACCCACAGCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGAAA

4-G10 GTCCCCAGGTAAGAGGGAACCCACAGCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGAAA

************************************************************

Ex6 AGGGAGACAGAGAGAGGGAGGCAGAGATAGCAGTGCCATGAGAGTCAGAGAGCAGGGCCA

4-G10 AGGGAGACAGAGAGAGGGAGGCAGAGATAGCAGTGCCATGAGAGTCAGAGAGCAGGGCCA

45

************************************************************

Ex6 TGGAGATAAAGACGGAAGTGGAGAGAGGAAGAAAGGGAGGGAGAGGGAGAGGGGAGAGAG

4-G10 TGGAGATAAAGACGGAAGTGGAGAGAGGAAGAAAGGGAGGGAGAGGGAGAGGGGAGAGAG

************************************************************

Ex6 TGAGAAAAGACAGAAATAGAGAGAGAAGTAAATATAATTAAAAAGGACAGATAGAGAGAT

4-G10 TGAGAAAAGACAGAAATAGAGAGAGAAGTAAATATAATTAAAAAGGACAGATAGAGAGAT

************************************************************
Ex6 AAGCAAAGATACAATGAGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGGGAGAGAAAC

4-G10 AAGCAAAGATACAATGAGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGGGAGAGAAAC

************************************************************

Ex6 AGACA

4-G10 AGACA

*****

Figure 3.16 Nucleotide sequence peaks of CRYBB1 gene representing exon 6

region

46

Nucleotide Sequence Analysis of H1

CAGGGTTTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTGCAGTGATCCAGGCGAG

GTCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCCAGCTTCCCCTTTGTGCCACAGAC

TATAGGGCCTGATGTCTGCCAGACTATAAAATGGGGGGTCGGACTCTAGTCACCCATAGCACCTGC

CCGCCTGCCTGTCCTCCAGCAGGAAGTAGCAAGTCCCCAGGTAAGAGGGAACCCACAGCCCAGCT

GAGAGCTGAAGGGGAGGGGAAGAGAAAAGGGAGACAGAGAGAGGGAGGCAGAGATAGCAGTGC

CATGAGAGTCAGAGAGCAGGGCCATGGAGATAAAGACGGAAGTGGAGAGAGGAAGAAAGGGAG

GGAGAGGGAGAGGGGAGAGAGTGAGAAAAGACAGAAATAGAGAGAGAAGTAAATATAATTAAA

AAGGACAGATAGAGAGATAAGCAAAGATACAATGAGGGAGAGATAGTGGGGGAGAGAGGAACA

GAGAGGGAGAGAAACAGACAGGCAGATAGG

Comparison of Reference Sequence and 4H1

EX6 CAGGGTTTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTGCAGTGATCC

4-H1 CAGGGTTTCTGCTGTGTGTGCTGGTCTCACAAAGGAAAGTGCTGCTGTCTGCAGTGATCC

************************************************************

EX6 AGGCGAGGTCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCCAGCTTCCCC

4-H1 AGGCGAGGTCAGGGCCCCAGTCCCCCACTCCCTGCCCAGGCCTGCTGAGCCAGCTTCCCC

************************************************************

EX6 TTTGTGCCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGGGGGTCGGACTC

4-H1 TTTGTGCCACAGACTATAGGGCCTGATGTCTGCCAGACTATAAAATGGGGGGTCGGACTC

************************************************************
EX6 TAGTCACCCATAGCACCTGCCCGCCTGCCTGTCCTCCAGCAGGAAGTAGCAAGTCCCCAG

4-H1 TAGTCACCCATAGCACCTGCCCGCCTGCCTGTCCTCCAGCAGGAAGTAGCAAGTCCCCAG

************************************************************

EX6 GTAAGAGGGAACCCACAGCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGAAAAGGGAGAC

4-H1 GTAAGAGGGAACCCACAGCCCAGCTGAGAGCTGAAGGGGAGGGGAAGAGAAAAGGGAGAC

************************************************************

REF AGAGAGAGGGAGGCAGAGATAGCAGTGCCATGAGAGTCAGAGAGCAGGGCCATGGAGATA

4-H1 AGAGAGAGGGAGGCAGAGATAGCAGTGCCATGAGAGTCAGAGAGCAGGGCCATGGAGATA

47

************************************************************

EX6 AAGACGGAAGTGGAGAGAGGAAGAAAGGGAGGGAGAGGGAGAGGGGAGAGAGTGAGAAAA

4-H1 AAGACGGAAGTGGAGAGAGGAAGAAAGGGAGGGAGAGGGAGAGGGGAGAGAGTGAGAAAA

************************************************************

Ex6 GACAGAAATAGAGAGAGAAGTAAATATAATTAAAAAGGACAGATAGAGAGATAAGCAAAG

4-H1 GACAGAAATAGAGAGAGAAGTAAATATAATTAAAAAGGACAGATAGAGAGATAAGCAAAG

************************************************************

Ex6 ATACAATGAGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGGGAGAGAAACAGACAGGC

4-H1 ATACAATGAGGGAGAGATAGTGGGGGAGAGAGGAACAGAGAGGGAGAGAAACAGACAGGC

************************************************************

Ex6 AGATAGG

4-H1 AGATAGG

*******

Figure 3.17 Nucleotide sequence peaks of CRYBB1 gene representing exon 6

region

48

Chapter 4

DISCUSSIONS

Cataract is characterized by the changes in the lens architecture and aggregation

of protein. Age related cataract is distributed into different types due to its
morphology. In Pakistan the prevalence of cataract is 30%.The rate of prevalence

of cataract is increasing 1 million per year. This study shows that people of old

age are at higher risk of having cataract as compare to younger’s. According to

WHO cataract is the leading cause of reversible blindness and the rate of

blindness due to cataract will be more than 40 million in 2020.According to

estimate 38.8% men and 45.9 % women over the age of 75 years are at higher risk

of having cataract (WHO, 2014).

Several risk factors found to be related with age related cataract. The result

shows that individual from age group II (66-75) year are at higher risk of age

related cataract, as compared to others. Epidemiological studies demonstrated

that people over the age of 50 years get blindness mainly due to cataract and

from other eye diseases (Taylor et al .,2013). Gender also plays important role for

cataract formation. This study shows that females are at higher risk 51%of having

cataract as compared to males 482%. It is probably due to decrease in the

estrogen level after menopause which usually occurs after 50 years. These results

are supported by the study carried out by Zetterberg et al., (2014).

49

In this study, age related cataract is classified into four types. Cortical cataract,

nuclear cataract, subs capsular cataract and congenital cataract. The percentage

of cortical cataract is higher i-e 55.0% in this study area as compared to other sub

types. Other studies showed that the genetic elements increase the incidence of

age related cortical cataract accounting for 53-58 % (Hammad et al., 2001).

According to Kouzis et al (2018) cortical and nuclear cataract is higher risk for age

related cataract formation as compared to other types of cataract.

The results show that steroid use by cataract patients plays an important role in

cataract genesis. About 52.4% are getting cataract due to prolong steroid use.

Other epidemiological studies show that corticosteroids and cataract have strong

association, while prolong steroid use causes posterior sub capsular cataract

(Condon et al., 2006). Oral corticosteroids are used for treatment of inflammatory
and immune diseases like arthritis asthma (Alison et al., 2006).

Hypertension is the second most important risk factor causing cataract in the

study area. About 48.5% cataract patients reported to have hypertension, which

probably led them to cataract. Another study on cataract demonstrated that

hypertension is greatly associated with nuclear cataract (Mamatha et al., 2018).

Dieu (2017) studied the incident of cataract by hypertension and the results

revealed that individuals from 50-70 years specifically gain the cataract due to

increase in hypertension.

Diabetes mellitus is followed by the hypertension in term of cataract causing risk

factor. The results of this study reveals that 39.3% cataract patients were

50

suffering from diabetes before getting the cataract, which shows that it is one of

the potential risk factor. Studies carried out on age related cataract shows the

similar results as that of current study. Diabetes was found to be common cause

of cataract in old age and diabetic patients are 2-3 times more likely to have

cataract as compare to non-diabetics. Diabetes affects the whole morphology of

the eye lens (Jaavadi et al., 2008).

Ultraviolet light appears to be one of the important risk factor which accounts for

30.9% of all the persons. Other study shows that as the person ages the eyes are

more affected due to UV radiations. UV exposure causes higher chances of

cortical cataract according to Truscott (2005).

Smoking has mild association with age related cataract. Total of 8.7% causes of

cataract due to smoking in this study. The result of the study carried out by

Nindhi are in agreement with this study that smoking increase the prevalence of

cortical and nuclear cataract and leads to damage to lens (Nindhi et al., 2018).

Family history is also an important risk factor of cataract. Present study shows

that only 1.53% of heritability causes are responsible cataract. Other study shows

that mutations in same or even different families lead to cataract formation

(Hejtmancik et al., 2013).

Present study was design to determine the association of CRYBB1 gene for ARC

patients. Samples of nucleotide sequences were compared with reference

sequence of exon 6 taken from NCBI. Further molecular analysis shows no

mutation for CRYBB1 gene. The results of this study are supported by Jiao (2008)

51

who carried out similar study and reported no mutation at exon 6 of CRYBB1

gene. Hence this study verified it. So there is a possibility that either any other

exon of CRYBB1 or other gene is responsible for the age related cataract in the

study area.

52

CONCLUSIONS

The present study shows the major risk factors associated with age related

cataract and mutations among CRYBB1 gene in district Mansehra .The age

related cataract is a major ocular disorder affecting the daily activities of the

people. Women are more affected by ARC as compare to men. Aging, steroid

use, diabetes, UV exposure, hypertension and smoking found to be the potential

risk factor for the ARC in the study area.

The molecular study performed shows that there is no mutation at exon 6 of

CRYBB1 gene. It is possible that either any other exon of CRYBB1 or any other

gene is responsible for ARC in the population of study area.

53

RECOMMENDATIONS

The risk factors responsible for cataract like UV exposure, steroid use and

smoking are modifiable and can be decreased by the awareness to the public. It

may reduce the formation of age related cataract.

To find out such mutations further research should be done. Such mutations

could find out by increasing the blood sample size taken from patients. The

further mutations can also be detected by studying the other exons of CRYBB1

gene. By increasing the amount of data collection and more interaction with
patients will be able to find out major or accurate risk factors for ARC. The

preventable risk factors like smoking, UV exposure and hypertension could be

minimized and also non modifiable causes of ARC could be minimized by giving

awareness.