The Genetic Basis of AutoimmuneThe Genetic Basis of Autoimmune Thyroid DiseaseThyroid Disease : : A Bioinformatics A Bioinformatics

ApproachApproach

Project Done By:

POUSALI MUKHERJEEM.Sc. In Genetic Engineering

2nd Year 4th SemesterRoll No.:072101887019

Registration no.: 072101887201019Institute of Genetic Engineering

West Bengal University of Technology

Thyroid GlandThyroid GlandThe The thyroidthyroid is one of the largest is one of the largest endocrine glands in the body.endocrine glands in the body.

This gland is found in the neck inferior This gland is found in the neck inferior to (below) the thyroid cartilage (also to (below) the thyroid cartilage (also known as the Adam’s Apple in men) and known as the Adam’s Apple in men) and at approximately the same level as the at approximately the same level as the cricoid cartilagecricoid cartilage

The thyroid controls how quickly the The thyroid controls how quickly the body burns energy, makes proteins, and body burns energy, makes proteins, and how sensitive the body should be to how sensitive the body should be to other hormones.other hormones.

The thyroid is controlled by the The thyroid is controlled by the hypothalamus and pitutary. hypothalamus and pitutary.

The thyroid participates in processes by The thyroid participates in processes by producing thyroid hormones, principally producing thyroid hormones, principally thyroxine (T4) and triiodothyronine (T3). thyroxine (T4) and triiodothyronine (T3).

These hormones regulate the rate of These hormones regulate the rate of metabolism and affect the growth and rate of metabolism and affect the growth and rate of function of many other systems in the bodyfunction of many other systems in the body. .

Iodine is an essential component of both T3 Iodine is an essential component of both T3 and T4. The thyroid also produces the and T4. The thyroid also produces the hormone calcitonin, which plays a role in hormone calcitonin, which plays a role in calcium homeostasis.calcium homeostasis.

The thyroid cells thus have three functions:The thyroid cells thus have three functions: They collect and transport iodineThey collect and transport iodine They synthesize thyroglobulin and secrets into They synthesize thyroglobulin and secrets into

the colloid andthe colloid and They remove the thyroid hormones from They remove the thyroid hormones from

thyroglobulin and secrete them into the thyroglobulin and secrete them into the circulation.circulation.

Thyroxine

Triiodothyronine

Thyroid Hormone SynthesisThyroid Hormone Synthesis

T3 and T4 RegulationT3 and T4 RegulationThe production of thyroxine and triiodothyronin is regulated by The production of thyroxine and triiodothyronin is regulated by

thyroid stimulating hormone (TSH), released by the anterior thyroid stimulating hormone (TSH), released by the anterior pitutary (that is in turn released as a result of TRH release by the pitutary (that is in turn released as a result of TRH release by the hypothalamus). TSH production is suppressed when the T4 hypothalamus). TSH production is suppressed when the T4 levels are high, and vice versa.levels are high, and vice versa.

The TSH production itself is modulated by thyrotropin releasing The TSH production itself is modulated by thyrotropin releasing hormone (TRH), which is produced by the hypothalamus and hormone (TRH), which is produced by the hypothalamus and secreted at an increased rate in situations such as cold (in which secreted at an increased rate in situations such as cold (in which an accelerated metabolism would generate more heat). an accelerated metabolism would generate more heat).

TSH production is blunted by Somatostatin, rising levels of glucocorticoids and TSH production is blunted by Somatostatin, rising levels of glucocorticoids and sex hormones (estrogen and testesterone), and excessively high blood iodide sex hormones (estrogen and testesterone), and excessively high blood iodide concentration.concentration.

Thyroid DysfunctionThyroid DysfunctionThyroid disease occurs when the thyroid gland doesn't supply Thyroid disease occurs when the thyroid gland doesn't supply

the proper amount of hormones needed by the body. the proper amount of hormones needed by the body.

HyperthyroidismHyperthyroidism Hypothyroidism

•"Hyper" is from the Greek, meaning "over or "above."

•The body use up energy morequickly than it should, and chemical activity

(like metabolism) in the cells speeds up.

•"Hypo" means "under" or "below." •The amount of hormone

released into the bloodstream is below normal, the body uses up energy more slowly,and chemical activity (metabolism)

in the cells slows down.

Types Thyroid DysfunctionTypes Thyroid Dysfunction

Graves’ DiseaseGraves’ Disease-- This is the most common cause of This is the most common cause of hyperthyroidism.hyperthyroidism.

It is a chronic disorder in which the affected person’s It is a chronic disorder in which the affected person’s immune system produces antibodies that attack the immune system produces antibodies that attack the thyroid, causing inflammation, damage, and the thyroid, causing inflammation, damage, and the production of excessive amounts of thyroid hormone.production of excessive amounts of thyroid hormone.

Hashimoto’s ThyroiditisHashimoto’s Thyroiditis – – This is the most common This is the most common cause of hypothyroidism in the United States. cause of hypothyroidism in the United States.

Like Graves’ disease, it is a chronic autoimmune Like Graves’ disease, it is a chronic autoimmune condition related to the production of antibodies that condition related to the production of antibodies that target the thyroid and cause inflammation and damage.target the thyroid and cause inflammation and damage.

With Hashimoto’s thyroiditis, however, the body makes With Hashimoto’s thyroiditis, however, the body makes decreased amounts of thyroid hormone. decreased amounts of thyroid hormone.

Contd..Contd..

ThyroiditisThyroiditis——Thyroiditis is an inflammation of the thyroid gland. It Thyroiditis is an inflammation of the thyroid gland. It may be associated with either hypo- or hyperthyroidism. It may be may be associated with either hypo- or hyperthyroidism. It may be painful, feeling like a sore throat, or painless. painful, feeling like a sore throat, or painless.

Thyroiditis may be due to autoimmune activity, an infection, Thyroiditis may be due to autoimmune activity, an infection, exposure to a chemical that is toxic to the thyroid, or an unkown exposure to a chemical that is toxic to the thyroid, or an unkown cause.cause.

Depending on the cause, it can be acute but transient or chronic. Depending on the cause, it can be acute but transient or chronic.

Thyroid Thyroid NodulesNodules——A thyroid nodule is a small lump on the thyroid A thyroid nodule is a small lump on the thyroid gland that may be solid or a fluid-filled cyst. As many as 4% of gland that may be solid or a fluid-filled cyst. As many as 4% of women and 1% of men will have one or more thyroid nodules; women and 1% of men will have one or more thyroid nodules; however, the overwhelming majority of these nodules are however, the overwhelming majority of these nodules are harmless. harmless.

Occasionally, thyroid nodules can be cancerous and need to be Occasionally, thyroid nodules can be cancerous and need to be treated.treated.

Contd..Contd..

GoitersGoiters——A thyroid goiter is a visible enlargement of the thyroid gland. In the A thyroid goiter is a visible enlargement of the thyroid gland. In the past, this condition was relatively common and was due to a lack of iodine in past, this condition was relatively common and was due to a lack of iodine in the diet. the diet.

Iodine is a necessary component of thyroid hormone production. Iodine is a necessary component of thyroid hormone production. Iodine-related goiters are common and represent the most common cause of Iodine-related goiters are common and represent the most common cause of

hypothyroidism in some countries. hypothyroidism in some countries.

Any of the diseases listed above can also cause goiters. Goiters may compress vital Any of the diseases listed above can also cause goiters. Goiters may compress vital structures of the neck, including the trachea and esophagus. This compression structures of the neck, including the trachea and esophagus. This compression can make it difficult to breathe and swallow. can make it difficult to breathe and swallow.

Thyroid Dysfunction and AutoimmunityThyroid Dysfunction and AutoimmunityAutoimmunityAutoimmunity is the failure of an is the failure of an

organism to organism to recognize its own recognize its own constituent parts as constituent parts as selfself, which , which results in an immune response results in an immune response against its own cells and tissues. against its own cells and tissues. Any disease that results from Any disease that results from such an aberrant immune such an aberrant immune response is termed an response is termed an autoimmune diseaseautoimmune disease..

Autoimmune Thyroid disease is Autoimmune Thyroid disease is associated with antibodies that associated with antibodies that block or stimulate the natural block or stimulate the natural occurring thyroid stimulator, occurring thyroid stimulator, thyroid stimulating hormone, or thyroid stimulating hormone, or TSH. TSH.

The Genetics of Autoimmune Thyroid The Genetics of Autoimmune Thyroid Disease (AITD)Disease (AITD)

Autoimmune thyroid disease (AITD) comprises a series of interrelated Autoimmune thyroid disease (AITD) comprises a series of interrelated conditions including conditions including hyperthyroid Graves’ diseasehyperthyroid Graves’ disease, , Hashimoto’s Hashimoto’s (goitrous) thyroiditis(goitrous) thyroiditis, , atrophic autoimmune hypothyroidismatrophic autoimmune hypothyroidism, , postpartum thyroiditispostpartum thyroiditis and and thyroid-associated orbitopathythyroid-associated orbitopathy..

These different manifestations of AITD may occur synchronously.These different manifestations of AITD may occur synchronously.

Autoimmune thyroid diseases are the commonest autoimmune Autoimmune thyroid diseases are the commonest autoimmune disorders in the population, affecting between disorders in the population, affecting between 2% and 4%2% and 4% of of women and up to women and up to 1%1% of men. of men.

Autoimmune thyroid diseases prevalence increases with advancing Autoimmune thyroid diseases prevalence increases with advancing age.age.

Is There Any Relationship Between Is There Any Relationship Between AITD and Gender?AITD and Gender?

It is an observed fact that females are more It is an observed fact that females are more affected than males. affected than males.

From the early age to the later years (50-59 years) From the early age to the later years (50-59 years) the thyroid dysfunction is the thyroid dysfunction is predominant in femalespredominant in females. .

The age group that is mostly affected by the The age group that is mostly affected by the thyroid dysfunction is 40-49 years both in case of thyroid dysfunction is 40-49 years both in case of males and females males and females

0

5

10

15

20

25

10 to19

20-29 30-39 40-49 50-59 60-69 >70

TSH high Male

TSH high Female

TSH normal Male

TSH normal Female

TSH low Male

TSH low Female

Relation of Thyroid Dysfunction With Relation of Thyroid Dysfunction With Age And SexAge And Sex

Why Females are More Affected With Why Females are More Affected With AITD?AITD?

X-chromosome inactivation is a X-chromosome inactivation is a possible explanation for the possible explanation for the female predominance in the female predominance in the prevalence of prevalence of autoimmune autoimmune thyroid diseasesthyroid diseases..

Women are more complex in that Women are more complex in that they have two similar X they have two similar X chromosomes.chromosomes.

This results in a double dose of often This results in a double dose of often similar genes among the 1098 similar genes among the 1098 genes coded for on the X genes coded for on the X chromosomechromosome

XY XX

X X

XX

Daughter

X-chromosome InactivationX-chromosome Inactivation

X-inactivation (also called lyonization) is a process by X-inactivation (also called lyonization) is a process by which one of the two copies of the X chromosome which one of the two copies of the X chromosome present in female mammals is inactivated. present in female mammals is inactivated.

The inactive X chromosome is silenced by packaging into The inactive X chromosome is silenced by packaging into transcriptionally inactive heterochromatin. transcriptionally inactive heterochromatin.

X-inactivation occurs so that the female, with two X X-inactivation occurs so that the female, with two X chromosomes, does not have twice as many X chromosomes, does not have twice as many X chromosome gene products as the male, which only chromosome gene products as the male, which only possess a single copy of the X chromosome. possess a single copy of the X chromosome.

Contd..Contd.. A multifunctional domain on the X chromosome to be A multifunctional domain on the X chromosome to be

inactivated, called the inactivated, called the X inactivation centerX inactivation center (XIC), is (XIC), is present in the Xq13 region. present in the Xq13 region.

Using transgenic and knockout mice, this domain has Using transgenic and knockout mice, this domain has been further refined to reveal the XIST gene.been further refined to reveal the XIST gene.

Which encodes an untranslated RNA that coats the X Which encodes an untranslated RNA that coats the X chromosome and, together with some associated factors, chromosome and, together with some associated factors, silences itsilences it

Females frequently exhibit a random 50:50 ratio of the Females frequently exhibit a random 50:50 ratio of the two cell lines two cell lines

However deciding which chromosome becomes However deciding which chromosome becomes

inactive is complex and appears to be mostly random inactive is complex and appears to be mostly random

X-Chromosome Inactivation and AITDX-Chromosome Inactivation and AITD

Skewed X-Chromosome Inactivation (XCI) in the etiology of AITD Skewed X-Chromosome Inactivation (XCI) in the etiology of AITD explains the female preponderance of AITD.explains the female preponderance of AITD.

A skewed XCI is a deviation from the original ratio and is A skewed XCI is a deviation from the original ratio and is arbitrarily defined, as a pattern where 80% or more of the cells arbitrarily defined, as a pattern where 80% or more of the cells inactivate the same X chromosome.inactivate the same X chromosome.

This can yield a situation in which self-antigens on one X This can yield a situation in which self-antigens on one X chromosome may fail to be expressed at sufficiently high levels in chromosome may fail to be expressed at sufficiently high levels in the thymus. the thymus.

FOXP3FOXP3 is a key gene in the development of regulatory T cells is a key gene in the development of regulatory T cells

The The FOXP3FOXP3 gene is associated with thyroid autoimmunity gene is associated with thyroid autoimmunity which is among the typical autoimmune diseases that develop which is among the typical autoimmune diseases that develop

in individuals with in individuals with FOXP3FOXP3 mutations mutations ((Yoshiyuki Yoshiyuki et alet al 20072007). ).

Association of FOXP3 and AITDAssociation of FOXP3 and AITD

FOXP3 gene can be a cause of FOXP3 gene can be a cause of autoimmune thyroid disease.autoimmune thyroid disease.

The The FOXP3FOXP3 gene is located within an X- gene is located within an X-chromosome locus (Xp11.23).chromosome locus (Xp11.23).

This region has been previously reported This region has been previously reported to be linked with autoimmune thyroid to be linked with autoimmune thyroid diseases (AITD). diseases (AITD).

The official name of this gene is The official name of this gene is “forkhead box P3.” “forkhead box P3.”

Location and Related SNPsLocation and Related SNPs

Ideogram

Genes/500kb

mRNA

Genotyped SNPs

What is the normal function of What is the normal function of FOXP3 gene?FOXP3 gene?

The FOXP3 gene provides instructions for producing the forkhead The FOXP3 gene provides instructions for producing the forkhead box P3 (FOXP3) protein. box P3 (FOXP3) protein.

The FOXP3 protein attaches (binds) to specific regions of DNA The FOXP3 protein attaches (binds) to specific regions of DNA and helps control the activity of genes that are involved in and helps control the activity of genes that are involved in regulating the immune system.regulating the immune system.

On the basis of this role, the On the basis of this role, the FOXP3 protein is called a FOXP3 protein is called a transcription factor.transcription factor.

This protein is essential for the production and normal function of This protein is essential for the production and normal function of certain immune cells called regulatory T cells, which play an certain immune cells called regulatory T cells, which play an important role in preventing autoimmunity. important role in preventing autoimmunity.

The FOXP3 protein is found primarily in an immune system gland The FOXP3 protein is found primarily in an immune system gland called the thymus, where regulatory T cells are produced. called the thymus, where regulatory T cells are produced.

How are changes in the FOXP3 How are changes in the FOXP3 gene related to AITD?gene related to AITD?

At least At least 21 mutations21 mutations in the FOXP3 gene have been found to in the FOXP3 gene have been found to cause auto immune diseases and X-linked (IPEX) syndrome. cause auto immune diseases and X-linked (IPEX) syndrome.

Most mutations Most mutations change one protein building blockchange one protein building block (amino (amino acid) in the region of the FOXP3 protein that binds to DNA acid) in the region of the FOXP3 protein that binds to DNA or lead to the production of an abnormally short, or lead to the production of an abnormally short, nonfunctional protein. nonfunctional protein.

Mutations in the FOXP3 gene result in Mutations in the FOXP3 gene result in reduced numbers or reduced numbers or a complete absence of regulatory T cellsa complete absence of regulatory T cells. .

Without the proper number of regulatory T cells, the body Without the proper number of regulatory T cells, the body cannot control immune responses. Normal body tissues and cannot control immune responses. Normal body tissues and organs are attacked, causing the multiple autoimmune organs are attacked, causing the multiple autoimmune disorders.disorders.

Genomic regions, transcripts, and products of FOXP3 gene

FOXP3 gene and its relation with X-FOXP3 gene and its relation with X-linked autoimmunitylinked autoimmunity

The protein encoded by this gene is The protein encoded by this gene is a member of the forkhead/winged-a member of the forkhead/winged-helix family of transcriptional helix family of transcriptional regulators.regulators.

Defects in this gene are the cause Defects in this gene are the cause X-linked autoimmunityX-linked autoimmunity

Genomic Size:Genomic Size: 14392    14392   

Exon Count:Exon Count: 12    12   

Coding Exon Count:Coding Exon Count: 11 11

Front

Side

Top

Conserved domains on forkhead box Conserved domains on forkhead box P3 (FOXP3)isoform a [P3 (FOXP3)isoform a [Homo sapiensHomo sapiens]]

Forkhead (FH), also known as a "winged Forkhead (FH), also known as a "winged helix".helix".

FH is named for the Drosophila fork head protein, a FH is named for the Drosophila fork head protein, a transcription factor which promotes terminal rather transcription factor which promotes terminal rather than segmental development. than segmental development.

This family of transcription factor domains, which This family of transcription factor domains, which bind to B-DNA as monomers, are also found in the bind to B-DNA as monomers, are also found in the Hepatocyte nuclear factor (HNF) proteins, which Hepatocyte nuclear factor (HNF) proteins, which provide tissue-specific gene regulation. provide tissue-specific gene regulation.

The structure contains 2 flexible loops or "wings" in The structure contains 2 flexible loops or "wings" in the C-terminal region, hence the term winged helix. the C-terminal region, hence the term winged helix.

Gene Model InformationGene Model Information

2397RNA size NoSelenocysteine

NoStop codon in genome YesHas stop codon

NoFrame shift in genomeYesHas start codon

NoCDS single in intron: 1296ORF size

NoCDS single in 3' UTR 12Exon count

NoNonsense-mediated-decay

CodingCategory

Orthologous Genes in Other Orthologous Genes in Other SpeciesSpecies

Orthologies between human, mouse, and rat are Orthologies between human, mouse, and rat are computed by taking the best BLASTP hit, and filtering computed by taking the best BLASTP hit, and filtering out non-syntenic hits. out non-syntenic hits.

But there is absence of an ortholog.But there is absence of an ortholog. This absence may reflect incomplete annotations in the This absence may reflect incomplete annotations in the

other species rather than a true absence of the other species rather than a true absence of the orthologous gene.orthologous gene.

FOXP3 Gene and EthnicityFOXP3 Gene and Ethnicity

Previous reports suggest that polymorphisms of the FOXP3 Previous reports suggest that polymorphisms of the FOXP3 gene may play a role in the genetic susceptibility to AITD in gene may play a role in the genetic susceptibility to AITD in Caucasians, perhaps by altering FOXP3 function and/or Caucasians, perhaps by altering FOXP3 function and/or expression (expression (Ban Y Ban Y et alet al, 2007, 2007).).

The prevalence of autoimmune thyroid disease is lowest in The prevalence of autoimmune thyroid disease is lowest in blacks and highest in whites. blacks and highest in whites.

DiscussionDiscussion

This study focuses on theThis study focuses on the predominance of autoimmune thyroid predominance of autoimmune thyroid disease in females.disease in females.

This predominance can be mostly explained by the X chromosome This predominance can be mostly explained by the X chromosome inactivation.inactivation.

However, it has been previously reported that Xp11.23 is linked However, it has been previously reported that Xp11.23 is linked with autoimmune diseases.with autoimmune diseases.

Hence, a probable relation can be drawn between AITD and Hence, a probable relation can be drawn between AITD and FOXP3 gene. The mutation of this gene results in the X-linked FOXP3 gene. The mutation of this gene results in the X-linked autoimmune disorders. autoimmune disorders.

ConclusionConclusion

From this study this conclusion can be drawn that:From this study this conclusion can be drawn that:o AITD is predominant in female of age group 40-49 AITD is predominant in female of age group 40-49

years.years.o The most probable reason behind this is skewed X-The most probable reason behind this is skewed X-

chromosome inactivation.chromosome inactivation.o This can be hypothesized that skewing of X-This can be hypothesized that skewing of X-

chromosome is due to the mutation in the FOXP3 chromosome is due to the mutation in the FOXP3 gene.gene.

o The prevalence of autoimmune thyroid disease is The prevalence of autoimmune thyroid disease is however not estimated in Indian population till now.however not estimated in Indian population till now.

Anderson’s Pathology, Volume-I, 8th-Edition.Anderson’s Pathology, Volume-I, 8th-Edition. Williams- A Text Book of Endocrinology, 6th editionWilliams- A Text Book of Endocrinology, 6th edition Ganong- Review of Medical Physiology, 22nd editionGanong- Review of Medical Physiology, 22nd edition Harrison's Principles of Internal Medicine, 17e Part Fifteen: Endocrinology and Harrison's Principles of Internal Medicine, 17e Part Fifteen: Endocrinology and

Metabolism > Section 1: Endocrinology >Metabolism > Section 1: Endocrinology > Pathophysiology of Disease: An Introduction to Clinical Medicine, 5th EditionPathophysiology of Disease: An Introduction to Clinical Medicine, 5th Edition J Clin June 2003, Polymorphism in Thyroid Hormone Pathway Genes, Endocrinol J Clin June 2003, Polymorphism in Thyroid Hormone Pathway Genes, Endocrinol

Metab 88(6):2880-2888Metab 88(6):2880-2888 J Clin july 2004, Heritability of the Thyroid Hormones, Endocrinol Metab J Clin july 2004, Heritability of the Thyroid Hormones, Endocrinol Metab

89(7):3276–328489(7):3276–3284 W. Jean Dodds, Thyroid Disease and Autoimmune Thyroiditis, DVM 938 Stanford W. Jean Dodds, Thyroid Disease and Autoimmune Thyroiditis, DVM 938 Stanford

StreetSanta Monica, CA 90403StreetSanta Monica, CA 90403 The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 12 5385-5397The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 12 5385-5397 Hot Thyroidology - Journal owned by the European Thyroid AssociationHot Thyroidology - Journal owned by the European Thyroid Association Arch Dis Child 1998;Arch Dis Child 1998;79:79:242-245 ( September )242-245 ( September )

ReferenceReference

AcknowledgementAcknowledgement

I am very grateful to Prof. Amit Chakraborty

our principal Sir and Dr. Sudipa Chakraborty our vice-principal Madam

for their valuable guidance to complete my project work.

I am also grateful to all the And above all I am thankful to my family for their

support