Molecular Diagnoses of Tyrosinemia Type II;

Identification and Characterization of Tyrosine

Aminotransferase (TAT) Gene Mutations Among
Suspected Patients
Semi-essential amino acid.
 Sources:
-protein hydrolysis.
-hydroxylation of phenylalanine.
 Precursor for the synthesis of
catecholamines, thyroid hormones, and melanogenesis.
Enzymatic conversion of
phenylalanine to tyrosine

COO- Phenylalanine COO-

+H N hydroxylase +H N
3 CH + O2 3
CH
+ H2 O
CH2 CH2
H+ + NADPH
NADP+

OH
tyrosine
phenylalanine
 Tyrosinemia

Causes:
 Vitamin C deficiency or immature
liver enzymes in premature infants.
 Hyperthyroidism.
 Liver failure.
 Hereditary inborn errors of the
tyrosine degradation pathway.
Tyrosine Metabolism
Tyrosinemia type II

 First described by Richner (1938) and Hanhart

(1947).
 More than 50 cases have been reported
worldwide, half of them from Italy.
 No data is available on disease prevalence among
Arabs.
 Several cases were reported from Saudi Arabia,
Kuwait, Tunisia, Palestine, and Iran.
Patients’ Diagnosis
 Lab tests
1. Plasma tyrosine level >500uM (Normal: 30-
130uM). The level of other amino acids remains
normal.
2. High conc. of tyrosine in urine (Normal: 26-83
umol/l).
3. Increased excretion of tyrosine metabolites in
urine.
4. Reduction or complete absence of TAT activity
in liver biopsies.
Clinical Symptoms
Dermatologic findings:
 Hyperkeratotic papules & plaques of the palms and soles.
 Age at onset of skin lesions range from the first few weeks
of life to the second decade.
Opthalmologic features:
 Occur typically before skin lesions, or simultaneously.
 Signs: corneal clouding, neovascularization, dendritic
ulcers & scarring.
 May be misdiagnosed as herpes simplex virus infection.
Mental retardation:
 May range from severe retardation to slight decreases in
intelligence.
 Pathology
 Local deposition of tyrosine crystals
causes eye & skin lesions.
 Accumulation of tyrosine leads to
increased synthesis of HVA which may
cause neurological dysfunction.
 High accumulation of tyrosine & its
metabolites (phenolic acids) may cause
tissue damage.
Control & Management
1. Dietary management:
A tyrosine and phenylalanine-restricted diet.

2. Drug management:
XPhen,Tyr and Tyrosidon.

3. Prevention programs & Prenatal diagnosis:

Genetic counseling and carrier detection.
Prenatal Diagnosis
 Tyrosine and its degradation products do
not accumulate in amniotic fluid.

 TAT is not expressed in chorionic villi

or in amniocytes.

 Molecular analysis is the only possible

approach to identify prenatal diagnosis.
The Molecular
Basis of Tyrosine
Aminotransferase
(TAT) gene.
 hTAT gene was assigned to
chromosome 16.

Organization of the human TAT gene

84 247 105
63 159 139 53 153 129 84 99 1434

A B C D E F G H I J K L
A
II III
I

B A B C D E F G H I J K L ALU
I. Patients
 Seven patients from six families were
diagnosed at Al-Yamamah Hospital to
have RHS based on elevated blood
tyrosine levels & clinical features.
 An eighth patient with normal blood
tyrosine level was included in the
study.
 Demographic, clinical features and plasma tyrosine levels of Richner _

Hanhart Syndrome patients before and after dietary treatment

Related information about the patients at the time

Related information about the patients at the age of of performing this study
Patients
diagnosis

Plasma
Plasma tyrosin
Cuta Cutan Develo
Occular tyrosine e after
neous Developme- Occular eous pme-
Symbol Gender Age involvemen before Age treatme
involv ntal delay involvement involve ntal
t treatment nt
ement ment delay
(µmol/L) (µmol/
L)

TYR1 F 3m - - - 757 9m - - - 761

TYR2A M 5y + + + 1261 14 y ? + + ND

TYR2B M 2y + + + ND 7y ? + + ND

TYR3 M 2y + + + 2015 7y - + + 1150

TYR4 M 14 m + + - 1109 3y - - - 1117

TYR5 M 8m + - - 1825 4y - - - 1044

TYR6 M 7m + - - 1060 5y - - - 952

 Mother

Father Family 1

TYR1

1 2 3 4 5 6 78 9

Sister
TYR2A
Cont.
Brother
TYR5 Brother Family 2
II. Sample Processing

Family Sister
5 Brother
Brother
TYR2B
Genomic DNA purification,

Family TYR6 TYR3

6 Sister Family 3
Sister
Quantitation, and Qualification.

Sister
TYR4
1 2 3 4 5 6 7 8 9
III. Identification of Mutations
Polymerase Chain reaction (PCR).

Marker
THIJ6

TKL6
TEF6

THI6
TA6

TC6

TG6
TB6

TD6
1 2 3 4 5 6 7 8 9 10
PCR product purification
Before purification After purification

Marker

TA1

TA5
TA5
TA1
DNA sequence analysis.
 BLAST alignment (gi37541544)
Alignment for the query sequence PCR product containing exon D
BLAST Results
Identification of G → T splicing mutation in exon K
(11)

Identification of a C → T nonsense mutation in

exon L (12)

Identification of 6 polymorphisms.
BLAST alignment for the query sequence of PCR product
containing exon K with TAT gene sequence for patients TYR1,
TYR3, TYR4, TYR5 and TYR6

T408T

IVS11+143a>g
BLAST alignment for the query sequence of PCR product
containing exons L with TAT gene sequence for patients TYR2A
and TYR2B

R417X
.
Restriction Analysis
A method Used for the identification of a particular
mutation in a DNA fragment by use of specific
restriction endonuclease that recognize specific 4
to 8 bp sequences called restriction sites and then
cleave both DNA strands at this site.
Restriction analysis of the exon K+L PCR
product with AlwNI enzyme for the presence
of T408T splicing mutation.

Marker

Mother
TYR1

Father
499 600
279
220
100
Mutations reported in the TAT Gene {Natt et al.,
1987. Natt et al., 1992. Huhn et al., 1998. Charfeddine et al.,
2006. Maydan et al., 2006 & our study 2007}

T 408 T TYR1/3/
IVS 2 – 4 a>g IVS 8+2 t>g
Palestine 4/5/6

A BC D E F G H I J K L
TAT gene

R 417 X TYR2A/
R 57 X R119W S 223 X G 362 V 2B
France

-7,-8 CT deletion F411X

L 201 R insertion

C151Y L 273 P
R433Q R433W
polymorphisms reported in TAT Gene {Huhn
et al., 1998. Maydan et al., 2006 & our study 2007}

(S 103S) G>A IVS 11 +143 a>g

French patient Palestinian patients
)TYR2A/2B) TYR1/3/4/5/6

A BC D E F G H I J K L
TAT gene

IVS 8 +113 t>c

(G>A) P 15 S Palestinian patients
g>t @-17 TYR1/3/4/6
TYR2A/2B
IVS 9 -73 g>t
IVS 7 +84 c>g All patients &control
All patients &control
Results highlights
Early diagnosis of tyrosinemia type II is important
to avoid the risk of complications and mental
retardation; most patients have improved on low
protein diet.

The final clinical expression of the disease results

from potential interaction between genomic and
non-genomic factors; patients with the same splicing
mutation show variations in signs and symptoms.
 Results highlights

This investigation represents an expended molecular

analysis of tyrosinemia type II among Palestinian
patients; the molecular basis has been identified only
in Tunisia & Palestine.
Thank
You