Presented by : Priyanjali Ray Regn. No. : 11408031 Semester 7 Department of Genetic Engineering.

Introduction
What is Porphyria?
Also called Vampire Syndrome, is a group of rare disorders passed down through families, in which an important part of Hemoglobin, called heme, is not made properly.

What is Heme?
A prosthetic group that consists of an iron atom contained in the center

of a large heterocyclic organic ring called a porphyrin.

A part of Hemoglobin, which carries Oxygen to different parts of the

body.

Produced in the bone marrow and liver through a complex process

controlled by eight different enzymes.

Why does the disease occur?

I.

During production of Heme, different intermediate compounds or precursors are created and modified. If one of the eight essential enzymes in Heme production is deficient due to mutation in the gene coding for it, certain precursors may accumulate in bone marrow or liver, appear in excess in the blood, and get excreted in the urine or stool. This may lead to various physical symptoms or even damage of the nervous system. Mutation causing the reduction in enzyme activity also limits the amount of heme being produced. This may result in degenerative changes in the central nervous system. It also results in a decrease of the amount of Oxygen being sent to the heart for pumping and subsequently leads to reduction in the amount of oxygen being supplied to various parts of the body.

II.

Heme Biosynthesis Pathway

Glycine + Succinyl coA Gene : ALAS2 ALA Synthase Coproporphyrinogen III CPOX Coproporphyrinogen Oxidase

Delta-Aminolaevulinic acid (ALA) ALAD ALA Dehydratase

Protoporphyrinogen IX PPOX Protoporphyrinogen Oxidase

Porphobilinogen HMBS Porphobilinogen Deaminase Uroporphyrinogen III synthase

Protoporphyrin IX

Hydroxymethylbilane FECH Ferrochelatase UROS

Uroporphyrinogen III Uroporphyrinogen UROD Decarboxylase

Heme

Where does Heme biosynthesis occur in the cell?

Culture and History

Originally studied by Hippocrates but biochemically explained by Dr.

Felix Hoppe-Seyler in 1874.

detected in all races, multiple ethnic groups on every continent

including Africans, Asians, Australian aborigines, Caucasians, Peruvian, Mexican, Native Americans.

Links between porphyrias and mental illness have been noted for

decades. In the early 1950s patients with porphyrias and severe symptoms of depression were treated with electroshock.

suggested as an explanation for the origin of vampire and werewolf legends, based upon certain perceived similarities between the condition and the folklore.

about eight different types of porphyria have been observed up to date.

The most recent trend of treatment is using Heme Therapy wherein

Heme associated drugs are administered to the patient.

Eminent personalities who suffered from Porphyria

Vincent Willem Van Gogh (painter)

King George III (Britain)

How is Porphyria classified?

Classification systems are based on :
1.

Specific enzyme deficiency. For example : ALA Dehydratase deficiency

2.

Neurologic symptoms caused by the Porphyrias (acute porphyrias). For example : Hereditary coproporphyria, Acute intermittent porphyria

3.

Photosensitivity (cutaneous porphyrias). For example : Porphyria

cutanea tarda, variegate porphyria

4.

whether the excess precursors originate primarily in the liver (hepatic porphyrias) or primarily in the bone marrow (erythropoietic porphyrias).

Porphyria may lead to mental illness because :

Excess amounts of PBG or ALA may cause neurotoxicity.

Heme deficiency may result in degenerative changes in the central

nervous system.

Increased ALA concentrations in the brain may inhibit gammaaminobutyric acid release(GABA).

Clinical symptoms
1.

Abdominal pain or cramping (Acute Porphyria)

2.

Problems with the nervous system and muscles, seizures, mental disturbances, nerve damage (Acute Porphyria)

3.

Light sensitivity causing rashes, blistering, and scarring of the skin (Porphyria Cutanea tarda)

Other symptoms may include:

Muscle pain Muscle weakness or paralysis

Numbness or tingling
Pain in the arms or legs Pain in the back Personality changes Low blood pressure

Complications of the disease :

Coma or Paralysis Respiratory failure (due to weakness of chest muscles) Scarring of the skin

Blisters as observed in Porphyria cutanea tarda patients

Prevalence and Statistics

Around the world
The exact prevalence of porphyria is unknown, but it probably ranges

from 1 in 500 to 1 in 50,000 people worldwide. (until 2011)

For some forms of porphyria, the prevalence is unknown because many

people with a genetic mutation associated with the disease never experience signs or symptoms.

Acute intermittent porphyria (AIP) may occur more frequently in

northern European countries, such as Sweden, in the United Kingdom and in India.

 Hereditary coproporphyria, has been reported mostly in Europe and

North America.

Variegate porphyria is most common in the Afrikaner population of

South Africa; about 3 in 1,000 people in this population have the genetic change that causes this form of the disorder.

Data obtained courtesy of-http://ghr.nlm.nih.gov/condition=porphyria ; service of US National Library of Medicine.

India
In India, AIP has been reported from various parts of the country and

some specific communities have been found to be especially susceptible.

Fifteen patients of AIP were detected out of 2,500 persons of the

Maheshwari community surveyed in Sri-Dungargarh municipal area of Bikaner. The fifteen patients belonged to 11 families and the female to male ratio was 9:6. Most of them were encountered in the age group 11 -20 years.

(Rajashekar Reddi, Nitin K Sethi, Ish Anand, PK Sethi., 2002, Acute Intermittent Porphyria : Management Aspects., JIACM, J Assoc Physicians India)

Genetic Basis
Each form of porphyria results from mutations in one of these

genes: ALAD, ALAS2, CPOX, FECH, HMBS, PPOX, UROD, or UROS.

1.

These genes are necessary for coding for the enzymes needed to produce heme.

2.

The mutations reduce enzyme activity, which limits the amount of heme produced.

3.

porphyrins and porphyrin precursors, formed during the process of heme production, accumulate in liver and other organs. On accumulation in the skin, the precursors interact with sunlight and cause the cutaneous forms of porphyria.

4.

The acute forms of the disease occur when porphyrins and porphyrin precursors build up in and damage the nervous system.

Type
ALA-Dehydratase Porphyria (ADP) Acute Intermittent Porphyria (AIP) Congenital Erythropoietic Porphyria (CEP)

Inheritance

Gene

Gene Locus
9q33.1

Enzyme
ALA dehydratase

Autosomal recessive ALAD

Autosomal dominant HMBS Autosomal recessive UROS

11q23.3 10q25.2-q26.3

PBG deaminase Uroporphyrinogen III synthase Uroporphyrinogen III decarboxylase

Porphyria Cutanea Autosomal dominant UROD Tarda (PCT), familial form

1p34

Hepatoerythropoietic Autosomal recessive UROD Porphyria (HEP) Hereditary Autosomal dominant CPOX Coproporphyria (HCP) Variegate Porphyria Autosomal dominant PPOX (VP) Erythropoietic Autosomal recessive FECH Protoporphyria (EPP)
X-linked Protoporphyria (XLP) X-linked ALAS2

1p34 3q12

Uroporphyrinogen III decarboxylase Coproporphyrinogen III oxidase

Protoporphyrinogen oxidase Ferrochelatase ALA synthase

1q22 18q21.3 Xp11.21

Note : One type of porphyria, porphyria cutanea tarda, results from both
genetic and nongenetic factors. About 20 percent of cases are related to mutations in the UROD gene. The remaining cases are not associated with UROD gene mutations and are classified as sporadic. Factors contributing to the development of porphyria cutanea tarda are :
1.

An increased amount of Iron in the liver. Mutations in the HFE gene (which cause an iron overload disorder) are also associated with porphyria cutanea tarda.

2.

Hepatitis C or HIV infection.

3.

Other, as-yet-unidentified genetic factors may also play a role in this form of porphyria.

Representation of loci of genes involved in some common forms of Porphyria

short arm of chromosome 1; position 34 PCT (UroD gene)

long arm of chromosome 11; position 23.3 AIP (HMBS gene)

Molecular Basis
Acute Intermittent Porphyria
It is a hepatic porphyria.

Characterized by a deficiency of the enzyme porphobilinogen

deaminase (PBGD) also known as hydroxymethylbilane synthase (HMBS).

Over 200 PBGD mutations have been reported worldwide, including

small insertions and deletions, missense mutations, as well as mutations in the PBGD promoter region

HMBS/PBGD gene
comprises 8,673 bases. 15 exons. There are 2 types of transcripts : Housekeeping and Erythroid-specific :

formed by alternate splicing.

Type I mutations

Patients exhibit reduced enzymatic activity as well as reduced PBGD protein content, both at approximately 50 percent of normal. Single base substitutions or deletions leading to a single amino acid change, resulting in the loss of expression of the enzyme protein.

Type II mutations

They are characterized by decreased PBGD activity in nonerythroid cells (eg liver) at approximately 50 percent of normal, but with normal erythroid PBGD activity. single base substitutions which occur in the exon/intron boundary of exon 1

Type III mutations

Occurs in exons 10 and 12, which are regions thought to be essential for catalytic activity of the enzyme

Diagnosis
Biochemical Diagnosis

Blood tests can be done to measure Porphyrin levels

Urinalysis

Urinalysis
Urinalysis is the physical, chemical, and microscopic examination of

urine. It involves a number of tests to detect and measure various compounds that pass through the urine.
A special stick ("dipstick") tests for various substances in the urine. The

stick contains little pads of chemicals that change color when they come in contact with the substances of interest. It is used to detect presence of porphyrin precursors.

Upon further chemcical analysis and quantification, it has been found that : Aminolevulinic acid Normal urinary excretion of ALA is <7 mg. It increases to 25 to 100 mg. Porphobilinogen Normal urinary excretion of PBG has been variously given as <2 mg. It increases to 50 to 200 mg.

Blood Test (Porphyrin level)

A blood sample is needed.
Three porphyrins can normally be measured in small amounts in

human blood. They are:

1. 2. 3.

Coproporphyrin Protoporphyrin Uroporphyrin

Protoporphyrin is normally found in the highest amount. More tests are needed to show the levels of specific porphyrins.

 This test specifically measures total porphyrin levels, but reference

values (a range of values seen in a group of healthy people) for the individual components are also included:
Total porphyrin levels: 16 to 60 mcg/dL

Coproporphyrin levels: < 2 mcg/dL

Protoporphyrin levels: 16 to 60 mcg/dL

Uroporphyrin levels: < 2 mcg/dL

Note: mcg/dL = micrograms per deciliter

What Abnormal Results Mean

Increased levels of coproporphyrins may be a sign of: Hepatic coproporphyria Increased protoporphyrin levels may be a sign of: Congenital erythropoietic protoporphyria Variegate porphyria Increased uroporphyrin levels may be a sign of: Porphyria cutanea tarda

Molecular Diagnosis
Acute intermittent porphyria (AIP) is an autosomal dominant disorder

caused by a porphobilinogen (PBG) deaminase deficiency. A denaturing gradient gel electrophoresis (PCR-DGGE) analysis followed by direct sequencing of the DNA fragments can be performed to investigate molecular defect.

According to this, sensitivity of the DGGE screening method was close

to 100%.

What is Denaturing Gel Electrophoresis (DGGE)?

Denaturing gradient gels are used to detect mutations. The genomic fragments are run on a low to high denaturant gradient

acrylamide gel; initially the fragments move according to molecular weight, but as they progress into higher denaturing conditions, each (depending on its sequence composition) reaches a point where the DNA begins to melt and forces DNA molecules to unwind
The melting severely retards the progress of the molecule in the gel,

and a mobility shift is observed.

It is the mobility shift which can differ for slightly different sequences

(depending on the sequence, as little as a single bp change can cause a mobility shift). Alleles are detected by differences in mobility. This can also be used to differentiate diseased condition from normal using known genes responsible for a particular disease.

Treatment
Note : Porphyria does not kill usually. It affects the quality of life.
Some of the medicines used to treat porphyria may include:
Heme therapy Panhematin (US), Heme-arginate.

{Hematin administered intravenously while Panhematin is consumed; form of alkaline heme; corrects heme deficiency in liver, represses production of precursors e.g., decreases level of PBG in urine (important in AIP)}.
Propranolol to control the heartbeat as the heartbeat may get affected

along with blood pressure in some cases of Porhyria.

 Other treatments may include: Chloroquine - famous for use in malaria treatment, has also been

observed to regulate ALA Synthase activity, causes reduction in precursor formation and accumulation.
Fluids and glucose to boost carbohydrate levels, which helps limit the

production of porphyrins.

 Depending on the type of porphyria, the doctor may suggest to take the

following preventive measures to control the discomfort and symptoms:

Avoid alcohol
Avoid injuring the skin

Avoid sunlight as much as possible and use sunscreen when outside

Eat a high-carbohydrate diet

An interesting fact to note :

Research studies have been done on mice models of Congenital

Erythropoietic Porphyria for Gene Therapy.

The UROS gene is located on chromosome 10. codes for uroporphyrinogen synthase (the 4th enzyme in the pathway). Lentivirus-mediated transfer of the human UROS cDNA into

hematopoietic stem cells (HSCs) from Uros(mut248) mice was done. complete and long-term enzymatic, metabolic, and phenotypic correction of the disease, favoured by a survival advantage of corrected red blood cells.

Prevention of the disease :

Genetic counselling may benefit people who want to have children and who have a family history of any type of Porphyria as most of them are genetically inherited.

Current Research

It was observed that inhibition of delta-aminolevulinic acid synthase

led to hepatic heme depletion via induction or suicidal inactivation of cytochrome P450. The process is now sufficiently understood such that prediction of porphyria from structural and functional information of the enzymes in an individual may be possible in the near future.

Foundations and Associations

Around the World
American Porphyria Foundation: Laboratory Testing for

Porphyria [porphyriafoundation.com]
The British Porphyria Association [porphyria.org.uk]

European Porphyria Initiative [irondisorders.org]

Iron Overload Diseases Association [ironoverload.org] American Liver Foundation [liverfoundation.org]

Case Reports and Institutes in INDIA doing research on Porphyria

J.L.N. Medical College & Hospital, Ajmer. C. Kothari Medical & Research Institute, Bikaner, Rajasthan. S.P. Medical College, Bikaner, Rajasthan. Silchar Medical College, Silchar, Assam. Bankura Sammilani Medical College, West Bengal. R.G. Kar Medical College, Kolkata, West Bengal.

Thank You