Clinical and mutational investigations of tyrosinemia type II in Northern Tunisia: identification and structural characterization of two novel TAT mutations.

Abstract

Tyrosinemia type II or Richner-Hanhart Syndrome (RHS) is an autosomal recessive disorder characterized by keratitis, palmoplantar keratosis, mental retardation, and elevated blood tyrosine levels. The disease is due to a deficiency of hepatic cytosolic tyrosine aminotransferase (TATc), an enzyme involved in the tyrosine catabolic pathway. Because of the high rate of consanguinity this disorder seems to be relatively common among the Arab and Mediterranean populations. RHS is characterized by inter and intrafamilial phenotypic variability. A large spectrum of mutations within TATc gene has been shown to be responsible for RHS. In the present study, we report the clinical features and the molecular investigation of RHS in three unrelated consanguineous Tunisian families including 7 patients with confirmed biochemical diagnosis of tyrosinemia type II. Mutation analyses were performed and two novel missense mutations were identified (C151Y) and (L273P) within exon 5 and exon 8, respectively. The 3D-structural characterization of these mutations provides evidence of defective folding of the mutant proteins, and likely alteration of the enzymatic activity. Phenotype variability was observed even among individuals sharing the same pathogenic mutation.

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Molecular Genetics and Metabolism 88 (2006) 184–191
www.elsevier.com/locate/ymgme
1096-7192/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.ymgme.2006.02.006
Brief communication
Clinical and mutational investigations of tyrosinemia type II
in Northern Tunisia: IdentiWcation and structural characterization
of two novel TAT mutations
C. Charfeddinea, K. Monastirib, M. Moknic,d, A. Laadjimie, N. Kaabachif, O. Pering,
M. Nilgesg, S. Kassard,j, M. Keirallahe, M.N. Guedicheb, M.R. Kamounh, N. Tebibi,
M.F. Ben Dridii, S. Boubakerj, A. Ben Osmanc, S. Abdelhaka,¤
a “Molecular Investigation of Genetic Orphan Diseases” Research Unit, Institut Pasteur de Tunis, Tunis, Tunisia
b Department of Paediatrics, Hôpital de Monastir, Monastir, Tunisia
c Department of Dermatology, Hôpital de la Rabta de Tunis, Tunis, Tunisia
d “Study of Hereditary Keratinization Disorders” Research Unit, Hôpital de la Rabta de Tunis, Tunis, Tunisia
e Department of Ophtalmology, Hôpital Fatouma Bourghiba, Monastir, Tunisia
f Department of Biochemistry, Hôpital de la Rabta de Tunis, Tunis, Tunisia
g Structural Bioinformatics Group, Institut Pasteur and CNRS URA 2185, 25-28 rue du docteur Roux, F-75015 Paris, France
h Department of Dermatology, Hôpital Charles Nicole, Tunis, Tunisia
i Department of Paediatrics, Hôpital de la Rabta de Tunis, Tunis, Tunisia
j Department of Anatomo-pathology, Institut Pasteur de Tunis, Tunis, Tunisia
Received 22 November 2005; received in revised form 6 February 2006; accepted 6 February 2006
Available online 30 March 2006
Abstract
Tyrosinemia type II or Richner–Hanhart Syndrome (RHS) is an autosomal recessive disorder characterized by keratitis, palmoplantar
keratosis, mental retardation, and elevated blood tyrosine levels. The disease is due to a deWciency of hepatic cytosolic tyrosine amino-
transferase (TATc), an enzyme involved in the tyrosine catabolic pathway. Because of the high rate of consanguinity this disorder seems
to be relatively common among the Arab and Mediterranean populations. RHS is characterized by inter and intrafamilial phenotypic
variability. A large spectrum of mutations within TATc gene has been shown to be responsible for RHS. In the present study, we report
the clinical features and the molecular investigation of RHS in three unrelated consanguineous Tunisian families including 7 patients with
conWrmed biochemical diagnosis of tyrosinemia type II. Mutation analyses were performed and two novel missense mutations were iden-
tiWed (C151Y) and (L273P) within exon 5 and exon 8, respectively. The 3D-structural characterization of these mutations provides evi-
dence of defective folding of the mutant proteins, and likely alteration of the enzymatic activity. Phenotype variability was observed even
among individuals sharing the same pathogenic mutation.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Tyrosinemia type II; Amino acidemias; Structural models; Mutation screening; Haplotype analysis; Clinical heterogeneity
Introduction
Tyrosinemia type II, (MIM 276600), also referred to as
Richner–Hanhart Syndrome (RHS), is a recessive autosomal
genodermatosis described independently by Richner [1] and
Hanhart [2] as a distinct clinical syndrome. In 1973, Goldsmith
et al. [3] pointed out the similarity between the tyrosinemia
type II and the oculo-cutaneous syndrome. Because of the
high rate of consanguinity, this disorder seems to be relatively
common among the Arab and Mediterranean populations
[4,5,8]. However, no data is available on the disease prevalence.
The disorder shows a wide range of clinical presentations
aVecting skin (in 80% of reported cases) and eye (in 75% of
*Corresponding author. Fax: +216 71 791 833.
E-mail address: sonia.abdelhak@pasteur.rns.tn (S. Abdelhak).

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C. Charfeddine et al. / Molecular Genetics and Metabolism 88 (2006) 184–191
185
reported cases), associated to mental retardation (in over of
60% of reported cases) [6]. Skin manifestations of the disease
usually begin after the Wrst year of life, but may begin as early
as one month of age and consist of progressive painful, non-
pruritic, and hyperkeratotic plaques on soles and palms, often
associated with hyperhidrosis [7]. The eye symptoms typically
occur before skin lesions develop, include photophobia, red-
ness lacrimia, and pain. The mental retardation is variably
associated with RHS and ranges from severe retardation, asso-
ciated with microcephaly and other organ abnormalities in
some cases, to slight decrease in intelligence. There is no rela-
tionship between age of diagnosis and mental retardation.
However, the degree of mental retardation may be related to
the level of plasma tyrosine [4]. The presenting complaints and
the disease manifestations may be conWned exclusively to the
skin [9,10] or to the eyes [11,12]. RHS is an inborn error of
metabolism due to a deWciency in hepatic cytosolic amino-
transferase (TATc; L-tyrosine: 2-oxoglutarate aminotransfer-
ase, EC 2.6.1.5), an enzyme that catalyses the transamination
reaction converting tyrosine to p-hydroxyphenylpyruvate. The
absence or the signiWcant diminution of the activity of TAT
results in tyrosinemia, tyrosinuria, and increased level of
urinary tyrosine metabolites, namely p-hydroxyphenylacetate,
p-hydroxyphenylpyruvate, p-hydroxyphenyllactate, and N-
acetyl tyrosine [13]. An elevated plasma and/or urine tyrosine
level accompanying typical clinical signs and symptoms is gen-
erally suYcient for diagnosis [12]. Several studies showed that
tyrosine restricted and phenylalanine restricted diet provide
rapid resolution of oculocutaneous signs and symptoms in
RHS and may prevent mental retardation [12,14].
The TAT coding gene has been assigned to human chro-
mosome 16(q22.1–q22.3) [15–17]. A large spectrum of muta-
tions has been shown to be responsible for RHS [18,5]. The
R433Q and R433W substitutions identiWed in the TAT gene
were shown to cause, essentially, a complete loss of TAT
enzymatic activity when expressed as recombinant proteins
in Escherichia coli [5]. Two polymorphisms have also been
identiWed (P15S, S103S) [5]. Despite RHS cases being most
frequently reported among Arab and Mediterranean popu-
lations, little is currently known about the molecular genet-
ics of the disease in geographic areas outside the United
States and Europe. In the present study, we report the clini-
cal features and the molecular investigation of RHS in three
unrelated consanguineous Tunisian families including
patients with conWrmed biochemical diagnosis of tyrosine-
mia type II. Two novel homozygous missense mutations
C151Y and L273P in the TAT gene have been identiWed.
The 3D-structural characterization of these mutations pro-
vides evidence of defective folding of the mutant proteins,
and likely alteration of the enzymatic activity.
Materials and methods
Patients
Three unrelated consanguineous families (family 1, 2 and 3), with a
total of 7 patients and 21 unaVected family members, were investigated.
All patients were interviewed with a questionnaire including a pedigree
drawing to identify possible cases of RHS among their relatives. Available
members underwent ocular and dermatological examination. The clinical
and biochemical data of tyrosinemia type II patients were reviewed retro-
spectively. All patients were diagnosed on the basis of clinical symptoms of
RHS, the diagnosis of RHS was conWrmed biochemically with high tyro-
sine levels in plasma and/or urine.
Most patients are oVsprings of consanguineous intermarriage between
Wrst-degree cousins. No relationship between parents of patients VI-1 and
VI-2 for family RHS-2 could be established, although these parents had
the same surname and originated from the same city. All samples were col-
lected after informed consent from all the individuals participating in this
study or their parents for aVected children.
Histological examination
Skin biopsies were performed for at least one patient in each explored
family. Specimens were taken from the margins of the extending lesions of
soles. The samples were Wxed in 10% formaldehyde solution and paraYn
embedded. Four to Wve micrometer sections were processed and stained
with H–E (hematoxylin–eosin).
Genotyping and mutation analysis
Genomic DNA was isolated from whole blood leucocytes from healthy
subjects, patients and their parents when possible, according to standard
procedures. Haplotype and mutational analysis were performed as
described in Charfeddine et al. [19]. Three polymorphic microsatellites
markers encompassing the TAT locus: D16S515–D16S3067–D16S3026
were selected according to the Genethon mapping panel and the genetic
maps available on NCBI and
(www.ncbi.nlm.nih.gov) and (www.ensembl.org). Microsatellite markers
were selected based on their percentage of heterozygosity (>70%) and
closeness to the TAT gene. Intronic oligonucleotide primers Xanking the
TAT coding exons were designed and mutation screening was performed
by direct sequencing of corresponding PCR products. Primer sequences
are available upon request. Putative mutations were conWrmed by
sequencing of both strands. SpeciWc PCR was used to rule out their occur-
rence in at least 100 control chromosomes.
Ensembl genome browsers
Structural analysis
Since no three-dimensional structure of TAT is available, we predicted
a structure by comparative modelling based on homologous proteins and
proteins with similar sequence and related function. A BLAST [20] search
of the PDB database [21] to identify proteins with know three-dimensional
structure and signiWcant sequence similarity to TAT resulted in 14 possible
structural templates for modelling. Of these, we used the structures of the
11 proteins with known aminotransferase activity. We then prepared a
multiple sequence alignment between the TAT sequence, the template
sequences, and other similar sequences found in the non-redundant
sequence data base with the program T-COFFEE [22]. Structural models
of the wild-type protein and the mutants were then constructed with the
program MODELLER [23]. All sequence alignment and modelling was
performed in a semi-automated way using a home-written computer pro-
gram (BisKit, http://sourceforge.net/projects/biskit). The structures were
displayed and analysed using VMD [24].
Results
Clinical data
Family RHS-1
In 1991, patients V-1, V-2 and V-3 belonging to the large
consanguineous family RHS-1 from Northern Tunisia pre-
sented to the Dermatology Department for an evaluation

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of complaint of painful palmar and plantar keratosis at the
age of 9, 8, and 6 years, respectively. Cutaneous symptoms
were the Wrst manifestations and appeared in early infancy
as bullae and erosions that progress to discrete hyperkera-
totic papules and plaques with irregular border. Probands
were screened for tyrosinemia type II on the basis of
plasma and urinary tyrosine levels. Untreated plasma tyro-
sine concentrations were 1255, 1183, and 975?mol/L
(normal, 23–69?mol/L) for patients V-1, V-2, and V-3 of
RHS-1, respectively. A low-tyrosine diet was initiated at
age of diagnosis. After 3 months of good compliance,
serum tyrosine levels were 1129, 915, and 893?mol/L for
patients RHS 1, 2, 3, respectively. This drop was accompa-
nied by a parallel improvement in the clinical status of the
patients. Unfortunately, the patients stopped the low phen-
ylalanine and tyrosine diet. They were lost out of sight until
1997 (4 years after diagnosis) and re-evaluated at age of 15,
14, and 12 years, respectively, for V-1, V-2, and V-3 patients
of the RHS-1 family. On physical examination, common
clinical Wndings were observed in the three sisters. Painful
focal palmoplantar keratosis plaques localized to the palms,
mainly on peripheral pressure bearing areas (tips of Wngers,
thenar and hypothenar eminences) were observed (Fig. 1).
Sharply demarcated yellowish white hyperkeratotic papules
and plaques surrounded by erythema were located on the
plantar surfaces of feet. The lesions were tending combined
with Wssuring and sometimes painful enough to impair
standing and walking. All patients have palmoplantar hyper-
hidrosis between the toes with fetid odor.
However, no abnormalities were detected on ophthal-
mologic examination for both V-1 and V-3 RHS-1 patients,
a decreased visual acuity and a history of recurrent “red
eyes” without lacrimination since infancy was noted for
their sister V-2.
Skin symptoms for the 3 patients and eye redness partic-
ularly for patient V-2 improved spontaneously in summer,
but relapsed in autumn and winter.
Patients had mild mental impairment and a mild lan-
guage disorder that had been observed at age of 11, 10, and
8 years, respectively, for V-1, V-2, and V-3. Intelligence test-
ing revealed low average intelligence with a scale IQ of 60,
67, and 70 for patients V-1, V-2, and V-3 of RHS-1 family,
respectively. They had been constrained to stop their study
early during their infancy. Liver and renal function test
were normal.
Family RHS-2
Patient VII-2 was a 2-year-old second child of Wrst-
cousin parents from Central Tunisia. He was issued from
normal dizygote twin pregnancy. His twin and older
brother were both healthy. At 4 months of age, he was seen
in the pediatric department for evaluation of bilateral kera-
titis and four months later for a mild Wngertip keratoderma,
leading to the putative diagnosis of tyrosinemia type II. The
diagnosis of RHS was established by the age of 9 months,
on the basis of an abnormal urinary tyrosine metabolites
accumulation. Untreated plasma tyrosine concentration
was at 760?mol/L for patients VII-2.
During interviewing family members, two other aVected
individuals VI-1 and VI-2, 20 and 23-year-old daughters,
respectively, (Wrst cousins of patient VII-1 and VII-2) were
identiWed. Early in infancy, they developed repeated epi-
sodes of photophobia followed up with cutaneous symp-
toms that worsened with age. These patients underwent
dermatological examinations and exhibited a painful focal
palmoplantar hyperkeratosis predominant on Wngertips
and fetid odor hyperhidrosis. Keratotic plaques were local-
ized to pressure points on the plantar surface for both feet
in VI-1 and VI-2 patients in family RHS-2. Plantar was
more involved than palm in both patients. Ophthalmologic
examination did not reveal any abnormalities in the two
patients. Skin symptoms for the two patients and eye red-
ness particularly for patient VI-2 improved spontaneously
in summer.
Family RHS-3
Patient V-1 of family RHS-3 was 3-year-old Wrst
oVspring of Wrst-cousin parents from a city of Central
Tunisia. The patient was born at term by spontaneous
vaginal delivery complicated by neonatal distress. At 4
months of age, he developed bilateral keratitis. The diag-
nosis of RHS was conWrmed at the age of 15 months.
Untreated plasma tyrosine level was at 1939?mol/L, and
the gas chromatography-mass spectrometry showed a
markedly abnormal accumulation of urinary tyrosine
metabolites corresponding to 4-hydroxyphenyllactic,
4-hydroxy-phenylpyruvic, 4-hydroxy-phenylacetic and
N-acetyl tyrosine. At the age of two and a half years, his
keratitis improved; however, he showed a mild psychomo-
tor developmental delay with hemiparesis and seizures.
No cutaneous involvement was noticed. The patient was
started on a low-protein diet. When the patient’s mother
started a new pregnancy, she asked for a postnatal diag-
nosis for RHS.
Fig. 1. Sharply demarcated yellowish white hyperkeratotic plaques located
on the hypothenar eminence and on Wngertips observed in patient V-1 in
family RHS-1. (For interpretation of the references to colour in this Wgure
legend, the reader is referred to the web version of this paper.)

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C. Charfeddine et al. / Molecular Genetics and Metabolism 88 (2006) 184–191
187
Histological Wndings
Histological examinations showed marked hyperkerato-
sis with foci of parakeratosis in all samples. Moderate to
important acanthosis accompanied by thickening of the
granular cell layer was observed. Papillomatosis with a mild
hyperplasia of the suprabal cells was present in some cases.
A mild inWltrate of mononuclear cells surrounding the
blood vessels of the dermis was seen. No hyphes or yeast-
like were observed in the superWcial layers of the epidermis.
Haplotype analysis
RHS patients and family members when available were
subtyped with the three microsatellites markers
(D16S515, D16S3067, and D16S3026) overlapping the
TAT gene region. For each family, the most likely haplo-
type was constructed by visual inspection. AVected
oVsprings in each family showed homozygosity by descent
at the TAT locus, whereas none of the unaVected was
homozygous for the two markers Xanking TAT gene
(Fig. 2). All aVected members in each family presented the
same homozygous disease haplotype for the two closest
markers to TAT, D16S515 and D16S3067. AVected
oVsprings of unrelated families RHS-2 and RHS-3 shared
the same disease haplotype (144–226) for the two closest
markers to TAT. This suggests that families RHS-2 and
RHS-3 likely share a common mutation inherited from a
common ancestor as they originate from the same town in
Central Tunisia.
Fig. 2. Pedigrees and haplotype analysis for 3 investigated RHS Tunisian families with microsatellites markers Xanking TAT gene. The disease haplotype
is indicated by shading. The allele size of each marker is given in base pair. nd, not determined.
4
2
1
158 172
144 144
226 226
III
IV
V
VI
VII
II
I
158 172
144 138
226 240
158 160
144 138
226 228
2
158 172
144 144
226 226
D16S3050
D16S3067
D16S515
3
158 172
144 138
226 240
3
158 158
144 146
226 226

2
158 172
144 144
226 226
1
158 172
144 138
226 240
nd nd
144 144
236 240
nd nd
138 144
236 228
I
III
IV
V
II
1
158 158
144 144
232 232
2
158 158
144 144
232 232
3
158 158
144 144
232 232
D16S3050
D16S3067
D16S515
Family RHS1
Family RHS2

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C. Charfeddine et al. / Molecular Genetics and Metabolism 88 (2006) 184–191
Mutation analysis
The search for mutation was started by screening one
patient within a given RHS family or as a representative
of a common haplotype. The segregation of the identiWed
mutation was conWrmed throughout other aVected family
members or other patients sharing the same haplotype.
Mutation analyses were performed by direct sequencing
of genomic DNA for all the 12 coding exons of TAT gene.
Two novel homozygous missense mutations were identi-
Wed. The mutation detected in patient V-1 in family RHS-
1 was a G to A transition at nucleotide 548 of exon 5. This
mutation results in the substitution of a cysteine (TGT)
for tyrosine residue (TAT) at codon 151 (C151Y) (Fig. 3).
The C151Y missense mutation has been conWrmed in the
two aVected sisters (V-2 and V-3) in family RHS-1. The
second mutation Wrst detected in patients VII-2 of family
RHS-2 was a T to C substitution at nucleotide 914 of
exon 8 in TAT gene. This mutation leads to a conversion
of a leucine residue (CTG) with a proline residue (CCG)
at amino acid 278 (L273P) (Fig. 3). The L273P was also
identiWed in the aVected cousins VI-1 and VI-2 of patient
VII-2 within family RHS-2 and in patient V-1 of family
RHS-3, thus conWrming previous haplotype investiga-
tions. Fifty unrelated healthy individuals (a panel of 100
chromosomes) derived from the general population did
not carry C151Y or L273P, conWrming that these muta-
tions do not correspond to polymorphisms. Furthermore,
based on the dbSNP (www.ncbi.nlm.nih.gov/SNP), none
of these missense mutations was previously reported as a
polymorphism.
Structural analysis of TAT protein
The search of the PDB data base with the human TAT
protein sequence resulted in 11 possible structural tem-
plates with aminotransferase
(ATTY_TRYCR), 1W7L
(Q95VY4_AEDAE), 1D2F (MALY_ECOLI),
(BDL_ECOLI), 1XOM
(Q9P9M8_PYRFU), 1GDE (O59096_PYRHO (AAT)),
1J32 (Q8RR70_PHOLP (AAT)), 1B5P (AAT_THET8),
1O4S (AAT_THEMA). Only 1BW0 is the structure of a
tyrosine aminotransferase. The sequence identities ranged
from 16 to 30% in a multiple sequence alignment between
human TAT and the templates, highlighting some residues
in the active site and the mutations described in this paper
(Fig. 4A). Fig. 4B shows a view of three-dimensional mod-
els of the wild-type protein and the two mutations. A view
onto the location of the two mutations is presented, with
the main chain of the protein in a cartoon representation.
Of the side-chains, only the active site residues and the two
mutated amino acids are shown in orange and green,
respectively. Interestingly, although the two mutations are
separated by more than 100 residues, they aVect exactly the
same region of the protein in the 3D structure. They are in
fact packing against each other.
Both mutations are located in the interior of the protein
but not very far from the surface, in regions with moderate
sequence conservation. They are neither directly involved in
the chemical reaction catalysed by the protein, nor are they
particularly close to the active site. We suggest therefore
that the mutations prevent the correct fold of the protein to
activity: 1BW0
1YIZ
1UO8
1XI9
(KAT1_HUMAN),
(O57946_PYRHO),
2
160 170
144 144
230 240
160 170
144 144
230 236

3
158
144
226
II
I
170 158
144 144
236 240
170 158
144 144
236 226
160 158
144 144
230 226
158 170
144 144
226 240

III
IV
V
D16S3050
D16S3067
D16S515
2
170
144
236
170 158
142 144
238 230

160 170
144 142
226 238

160 160
144 138
226 230
1
158 160
144 144
226 226
2
170 160
144 138
236 230
170 158
144 144
226 230
170 158
142 144
238 230
158 162
144 142
230 232
Family RHS3
Fig. 2. (continued).

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C. Charfeddine et al. / Molecular Genetics and Metabolism 88 (2006) 184–191
189
be formed and that they thus indirectly aVect the eYciency
of the enzyme.
Discussion
While the RHS is a relatively rare metabolic disease,
patients diagnosed as having RHS were described in
diVerent ethnic groups, mostly in the Arab world, which is
characterized by a high rate of consanguineous marriages
(range between 20 and 60%, mostly between Wrst cousins),
which contribute to an increase in the prevalence of reces-
sive genetic disorders [25,26]. We identiWed two novel
homozygous missense mutations, C151Y and L273P,
within the TAT gene. The C151Y and L273P mutations
appear to be speciWc to the RHS Tunisian families as none
of these mutations have been reported so far in other pop-
ulations. These mutations occurred in exon 5 and 8 of the
TAT gene, where no mutations have been previously
reported.
While it is diYcult to predict a molecular scenario with
certainty, in particular for the C151Y mutation, the eVects
of the mutations can be rationalized based on the primary
sequence alignment (Fig. 4A) and the 3D models
(Fig. 4B). Mutation C151Y introduces a bulky side-chain
into a conWned space. Position 151 is usually occupied by
a smaller and less rigid hydrophobic residue than tyrosine.
Mutation L273P is likely to aVect the stability of the pro-
tein in two ways: Wrst, a proline residue has less interac-
tion surface than a leucine, hence, the mutation reduces
the stability of the tertiary fold (in the multiple alignment,
this position in the sequence is always occupied by a
larger hydrophobic residue). Second, the residue is located
in a beta sheet. A proline residue can only form one
hydrogen bond, and the secondary structure is locally
destabilized.
A variable clinical expression of the disease has been
observed among aVected individuals who carried the
same pathogenic mutation. For family RHS-1 in which
all aVected individuals carried the C151Y mutation,
patient V-2 presented the full range of the RHS clinical
features including ocular lesions, painful palmoplantar
hyperkeratosis and mental retardation, whereas her
aVected sisters (V-1 and V-3) showed no ocular involve-
ment. Patients VII-2 in family RHS-2 and V-1 in RHS-3
exhibited only keratitis, patients VI-2 and VI-3 in family
RHS-2 presented the association of cutaneous and
ocular involvement, although all those patients carried
the L273P mutation. Discrepancy in early clinical mani-
festation of RHS is noticeable among the diVerent
reports in the literature. Macsai et al. [12] reported RHS
cases for which the ophthalmologic features of tyrosine-
mia type II occur before skin lesions development with
the early signs. Other reports described RHS cases
having Wrst ophthalmologic manifestation in their forties
[27].
Patients having dermatologic manifestations showed
progressivity of the hyperkeratotic skin lesions [7]. This
has been reported previously in RHS patients [28]. An
improvement in skin symptoms during the summer
months was observed in all patients, which may be due to
a seasonal change in dietary behaviour, as suggested by
others [28].
Fig. 3. Mutations identiWed within TAT gene in Tunisian patients with RHS. Sequence electropherograms corresponding to each mutation from patients
(upper panel) are compared with the wild-type sequence of unrelated control (lower panel), respectively. (A) Genomic DNA sequence of exon 5 for 3
patients of family RHS1 that show the G! A homozygous missense substitution at nucleotide 548 leading to amino acid change from cysteine (TGT) to
tyrosine (TAT) at codon 151 (C151Y). (B) Genomic DNA sequence of exon 8 for 4 patients of family RHS2 and RHS3 showing the T ! C homozygous
missense transversion at nucleotide 914 resulting in amino acid change from leucine (CTG) to proline (CCG) at codon 278 (L278P). Their respective par-
ents are heterozygous for the L278P mutation (middle panel).

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C. Charfeddine et al. / Molecular Genetics and Metabolism 88 (2006) 184–191
To our knowledge, this investigation is the Wrst molec-
ular analysis of RHS in the Northern African and Mid-
dle Eastern populations. The two new identiWed missense
mutations (C151Y and L273P) should be investigated in
other Southern Mediterranean and Middle Eastern
patients as they might be carried by other populations
sharing the same ethnical background. Taking into
account the cultural tendency for inbreeding in Tunisian
population, early neonatal diagnosis should be proposed
for families at risk. In the family RHS-3, the healthy
sib V-2, brother of the patient V-1 was screened negative
for the L273P mutation, thus conWrming the status of
the individual. Early screening of siblings in families
with RHS history is indicated even in the absence of
clinical Wndings. Careful dietary control of maternal
plasma tyrosine level should be considered during future
pregnancy for female patients [8,29]. When possible, an
early institution of a phenylalanine-tyrosine restricted
diet in infancy is currently the most eVective therapy
available to promptly reverse ocular and cutaneous
abnormalities and prevent from the risk of mental retar-
dation.
Acknowledgments
We thank the patients and family members. This work
was supported by the Tunisian Ministry of ScientiWc and
Technological Research and, Development of Competen-
cies (Research Unit on “Molecular Investigation of
Genetic Orphan Diseases” UR 26/04 and Research Unit
on “Study of Hereditary Keratinization Disorders” UR
24/04).
References
[1] H. Richner, HorhautaVektion bei Keratoma palmare et plantare
hereditarium, Klin. Mbl. Augenheilk. 100 (1938) 580–588.
[2] E. Hanhart, Neue Sonderformen von Keratosis palmo-plantaris u.a. eine
regelmaessig dominante mit systematisierten Lipomen, ferner zwei einf-
Fig. 4. (A) Alignment of the sequence of the human TAT protein against a structural alignment of other aminotransferases. The mutated residues (151,
273) and active site residues (145, 172, 278–280, 288) are indicated. Conserved hydrophobic residues are marked in blue, positively charged residues in red,
negatively charged residues in magenta, polar residues in green, glycines in orange, prolines in yellow, tyrosines and histidines in cyan. The Wgure was pro-
duced with Jalview [30]. (B) 3D models of human TAT, with a view onto the mutated residues. The protein backbone is shown in ribbon representation,
with beta sheets in yellow, helices in magenta, and other residues involved in hydrogen bonds in blue. The active site residues are shown as orange spheres,
the residues 151 and 273 as green spheres. The Wgure was produced with VMD [24]. (For interpretation of the references to colour in this Wgure legend, the
reader is referred to the web version of this paper.)

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