Novel mutations in TTC37 associated with tricho-hepato-enteric syndrome.

Abstract

The Tricho-Hepato-Enteric (THE) syndrome is an autosomal recessive condition marked by early and intractable diarrhea, hair abnormalities, and immune defects. Mutations in TTC37, which encodes the putative protein Thespin, have recently been associated with THE syndrome. In this article, we extend the pattern of TTC37 mutations by the description of 11 novel mutations in 9 patients with a typical THE syndrome. The mutations were spread along the gene sequence, none of themrecurrent. Different types of mutation were observed: frameshift mutations, splice-site altering mutations, or missense mutations, most of them leading to the creation of a premature stop codon. Concurrently, we investigated the pattern of TTC37 expression in a panel of normal human tissues and showed that this gene is widely expressed, with high levels in vascular tissues, lymph node, pituitary, lung, and intestine. In contrast, TTC37 is not expressed in the liver, an organ that is not consistently affected in THE syndrome. Last, we suggested a model for the putative structure of the unknown Thespin protein.

Full text

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Référence pour les Maladies Digestives Rares de l'enfant, APHP,
Gastroentérologie et Nutrition Pédiatrique
Lemale, Julie; Hôpital Armand Trousseau, APHP, Gastroentérologie
et Nutrition Pédiatrique
Cezard, Jean-Pierre; Hôpital Robert Debré, Centre de Référence
pour les Maladies Digestives Rares de l'enfant, APHP,
Gastroentérologie et Nutrition Pédiatrique
Goulet, Olivier; Hôpital Necker-Enfants Malades, Centre de
Référence pour les Maladies Digestives Rares de l'enfant, APHP,
Gastroentérologie et Nutrition Pédiatrique
Sarles, Jacques; hôpital d'enfants de la Timone, Service de
pédiatrie multidisciplinaire
Levy, Nicolas; UMR 910, Faculté de médecine, université de la
Méditerranée
Badens, Catherine; UMR 910, Faculté de Médecine, Université de la
Méditerranée







Novel mutations in TTC37 associated with Tricho-Hepato-
Enteric syndrome


Journal: Human Mutation
Manuscript ID: humu-2010-0401.R1
Wiley - Manuscript type: Research Article
Date Submitted by the
Author:
04-Oct-2010
Complete List of Authors: Fabre, Alexandre; Hôpital d'enfants de la Timone, Service de
Pédiatrie Multidisciplinaire
Martinez-Vinson, Christine; Hôpital Robert Debré, Centre de
Référence pour les Maladies Digestives Rares de l'enfant, APHP,,
Gastroentérologie et Nutrition Pédiatrique
Roquelaure, Bertrand; hôpital d'enfants de la Timone, Service de
pédiatrie multidiciplinaire
Missirian, Chantal; Hôpital d'enfants de la Timone, Laboratoire de
Cytogénétique
Andre, Nicolas; Hôpital d'enfants de la Timone, Service d'oncologie
pédiatrique
Breton, Anne; Hôpital des Enfants, Gastro-entérologie, Hépatologie,
Nutrition, Diabétologie
Lachaux, Alain; hôpital Femme–Mère-Enfant du CHU de Lyon,
Hepatologie Gastroentérologie et Nutrition
Egritas, Odul; Gazi University School of Medicine, Department of
Pediatric Gastroenterology
Colomb, Virginie; Hôpital Necker-Enfants Malades, Centre de
John Wiley & Sons, Inc.
Human Mutation
peer-00614846, version 1 - 17 Aug 2011
Author manuscript, published in "Human Mutation 32, 3 (2011) 277"
DOI : 10.1002/humu.21420

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Key Words:
Syndromic diarrhea, Tricho-hepato-enteric syndrome, TTC37,
intractable diarrhea, Woolly hair, Thespin, Stankler syndrome




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Dr Catherine Badens
Laboratoire de Génétique moléculaire
Département de Génétique
Hôpital de la Timone
13385 Marseille cedex 5
France
1
Novel mutations in TTC37 associated with Tricho-Hepato-Enteric syndrome


Alexandre Fabre1,2, Christine Martinez-Vinson3 , Bertrand Roquelaure1, Chantal Missirian4,
Nicolas André5, Anne Breton6, Alain Lachaux7, Egritas Odul8, Virginie Colomb9, Julie
Lemale10, Jean-Pierre Cézard3, Olivier Goulet9, Jacques Sarles1, Nicolas Levy2,11, Catherine
Badens2,11.

1 Service de Pédiatrie Multidisciplinaire, 2 Laboratoire de Génétique Moléculaire, Hôpital
d’enfants de la Timone, Marseille, 3 Service de Gastro-entérologie, Hôpital Robert Debré,
Paris, 4 Laboratoire de cytogénétique, Hôpital d’enfants de la Timone, Marseille, 5 Service de
Pédiatrie Oncologique, Hôpital d’enfants de la Timone, Marseille, 6 Service d’Hépatologie et
Maladies Métaboliques, Hôpital d’enfant de Toulouse, 7 Unité Fonctionnelle de Nutrition,
Hôpital Femme Mère Enfant de Lyon, 8 Gazi University School of Medicine, Department of
Pediatric Gastroenterology, Ankara, Turkey, 9 Service de Gastro-entérologie, Hôpital Necker-
Enfants Malades, Paris, 10 Gastroentérologie et Nutrition Pédiatrique, Hôpital Armand
Trousseau, Paris, 11 Unité Inserm U910, Faculté de Médecine, Université de la Méditerranée,
Marseille, France.

Key words: Syndromic Diarrhea, Intractable diarrhea, Tricho-Hepato-Enteric syndrome,
Stankler syndrome, TTC37, woolly hair,
Corresponding author :
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Abstract
The Tricho-Hepato-Enteric (THE) syndrome is an autosomal recessive condition marked by
early and intractable diarrhea, hair abnormalities and immune defects. Mutations in TTC37
which encodes the putative protein Thespin, have recently been associated with THE
syndrome. In this paper, we extend the pattern of TTC37 mutations by the description of 11
novel mutations in 9 patients with a typical THE syndrome. The mutations were spread along
the gene sequence, none of them being recurrent. Different types of mutation were observed:
frameshift mutations, splice site altering mutations or missense mutations, most of them
leading to the creation of a premature stop codon. Concurrently, we investigated the pattern of
TTC37 expression in a panel of normal human tissues and showed that this gene is widely
expressed, with high levels in vascular tissues, lymph node, pituitary, lung and intestine. In
contrast, TTC37 is not expressed in the liver, an organ which is not consistently affected in
THE syndrome. Lastly, we suggested a model for the putative structure of the unknown
Thespin protein.

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Very recently, Hartley et al described molecular defects causative for THE syndrome in
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Introduction
The Tricho-Hepato-Enteric syndrome (THE syndrome, OMIM n°222470) also known as
Syndromic Diarrhea, is a rare and severe autosomal recessive condition which associates
intractable diarrhea with facial dysmorphism, intrauterine growth retardation,
immunodeficiency with low serum concentrations of immunoglobulins, and hair
abnormalities characterized by woolly hair (Girault et al., 1994; Verloes et al., 1997).
Parenteral nutrition is usually started in the first weeks of life and maintained all life long in
most cases. The liver failure is inconstant and when present, it is observed initially or later on
in life. In the past, the presence or absence of liver failure led one to describe 2 different
syndromes but it is now admitted that the 2 entities represent 2 sides of the same disease
(Fabre et al, 2007, Goulet et al, 2008). Death can occur early in life even though some patients
can reach the third decade.
In the past few years, we collected samples for 12 patients from 11 families, all presenting a
typical phenotype of THE syndrome. We first excluded several functional candidate genes
(Fabre et al, 2009) and then performed a linkage analysis in order to unravel the genetic basis
of this syndrome. In 2 of the 3 consanguineous families, homozygosity mapping identified a
5Mb region in 5q as a potential locus for the disease.
TTC37 (NM_014639), a gene encoding for a putative protein named Thespin. Their study
reported 9 mutations transmitted in a recessive pattern in 12 patients presenting this
syndrome.
Since TTC37 maps in 5q, we assumed that this gene may be responsible for the disease in
some of our patients and so performed systematic sequencing of genomic DNA samples. In
this paper, we describe 11 novel TTC37 mutations in 9 patients out of 12 and provide clinical
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pattern was investigated by qPCR on a panel of normal transcripts from various tissues
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data for mutated and non mutated patients together with normal patterns of TTC 37 expression
in several tissues.

Material and methods:
Patients
Patients and family members were assessed under approved human subject protocols and all
participants provided informed consent.
Molecular studies
DNA was isolated from blood via a standard manufacturer’s protocol (QIAamp DNA blood
minikit, QIAgen). To analyse the TTC37 gene, direct sequencing was performed, after PCR
amplification of the 43 exons and intronic flanking sequences, on an ABI 3130XL (Applied
Biosystems).
The TTC37 genomic sequence from GenBank accession numbers NM_014639 was used as
reference sequence. Detailed protocols and primer sequences are available on request. Two
non-mutated patients and one heterozygous were tested by CGH array using the commercial
Agilent 2x400K SurePrint G3 Human CGH Microarray (Agilent Technologies) array with the
overall median probe spacing of 5,3 kb. Total RNA from 3 patient samples was prepared from
lymphoblastoid cells and reverse transcribed using M-MLV (Sygma). Normal expression
(Rapid-Scan cDNA panel, OriGene) using a probe located on exon-intron junction 35-36 of
TTC37 and compared to GAPDH expression in duplex reactions. qPCR were done in
triplicates and 2 sets of experiments were performed.
Bioinformatics analysis
The topology of normal and mutated Thespin protein was analysed using the algorithms
proposed at TMpred (http://www.ch.embnet.org/software/TMPRED_form.html), TOPPRED
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mutations. All the parents of mutated patients were investigated and carried a mutation in
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(http://www.mobyle.pasteur.fr/cgi-bin/portal.py?form=Toppred), PSORT II
(http://psort.ims.u-tokyo.ac.jp) and SMART (http://smart.embl.de/smart/show_motifs.pl)
Predicted effects of missense mutations were obtained on Polyphen website
(http://genetics.bwh.harvard.edu/pph).
Results
Patients phenotype
The clinical features of the 12 patients are summarised in table 1. Detailed clinical data were
previously published for 3 of them (Fabre et al, 2007, Egritas et al 2010). All the 12 patients
presented the 3 major signs of THE syndrome i.e. dysmorphism, hair abnormalities and
intractable diarrhea. Among the 12 patients, one died of septicemia at age 10, 2 have stopped
parenteral nutrition and 1 never required it.
Identification and characterization of novel TTC37 mutations in THE patients
Screening for mutations of TTC37 coding sequence and intron-exon junctions of DNA
samples from THE patients identified 10 novel non ambiguous mutations in homozygous or
compound heterozygous condition, in 8 (out of 12) patients. In addition, one patient presented
only a heterozygous missense mutation. The mutations were spread along the gene: 3 were
nonsense or frameshift mutations in the coding sequence leading to premature stop codon, 5
were mutations affecting splice sites either by substitution or deletion and 3 were missense
heterozygous condition. The location and predicted consequences of the mutations on protein
expression or function are reported in table 2.
Two of the 3 patients without mutation and the one with a single heterozygous mutation were
investigated by whole genome CGH array with 17 probes mapping in TTC37. No CNV of the
TTC37 region or of other regions could be identified.
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peptide repeats (TPR) domains are predicted and their number varies from 5 to 22.
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The effects of splice sites mutations on sequence transcripts were analysed by direct
sequencing of RNA transcripts from lymphoblastoid cells, for 3 patients. The tested samples
exhibited abnormal sequence due to the modification of splicing: skipping exon 23 in the case
of the c.2515+1 C>G mutation, leading to a frameshift and creation of a premature stop
codon (figure 1a); skipping exon 25 in the case of the c.2577-3_-7delTTTT, leading to the
deletion of 19 amino-acids in frame (figure 1b); cryptic splice activation in exon 42 in case of
the c.4620+1 G>C mutation promoting an alternative splicing and the replacement of the 41
terminal amino-acids by 61 others.
Pattern of expression
Quantitative expression in normal tissues was assessed in a panel of 48 different tissues and
revealed that TTC37 is widely expressed with the highest levels observed in vascular tissues,
lymph node, pituitary, lung and intestine. Noticeably, we did not find any expression in the
liver (figure 2).
Bioinformatics
In silico protein predictions were performed to propose a putative structure for Thespin and to
evaluate the potential effect of TTC37 mutations identified in this study. The algorithm results
are in accordance to predict that Thespin is mostly cytosolic and may contain 4
transmembrane domains (Figure 3). With all algorithms used in this study, several tetratrico-
Discussion
Mutations previously identified in THE syndrome are heterogeneous and include frameshift,
nonsense and splice site mutations. Here, we describe further 11 different mutations that add
heterogeneity to the molecular genetics of THE syndrome (figure 3). These 11 novel
mutations, in addition to the 9 mutations previously described, indicate strongly that TTC37 is
the main gene responsible for THE syndrome. There is no clear hot spot region for mutations
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conservation in canis (92%), bos (92%), rattus (83%), mus (82%), gallus (66%), danio (55%)
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even though, in 70% of the mutated alleles (Hartley et al 2010 and our study), the mutation is
localised in the 3’ half of the gene. Most of the mutations are nonsense, frameshift or splice
mutations that are predicted to produce premature stop codon. At least, 2 of the 3 missense
mutations might affect splicing as well, as they are located in the vicinity of splice sites. For
one of the patients, a single missense mutation in heterozygous condition has been identified
to date. As we exclude the presence of an intragenic deletion or duplication on the other
allele, we assume that a non-identified intronic substitution producing an aberrant splicing is
probably associated with the missense mutation.
The phenotypes of the 3 patients who did not carry any mutation in TTC37 have been
carefully re-evaluated and were confirmed as being typical THE syndrome, suggesting
strongly that at least one other gene may be implicated in this disease.
Multi-tissue transcript expression analysis showed wide expression of TTC37 mRNA with a
high expression level in intestinal tissue but not in the liver, indicating that liver dysfunction
observed in THE syndrome is probably secondary to another genetic cause or to long term
parenteral nutrition. More surprising is the high level of expression observed in vein, arteria
and lung as no abnormalities have been noticed in these tissues.
Up to date, the function of Thespin is unknown. We identified orthologs of human TTC37
using HomoloGene (www.ncbi.nlm.nih.gov/homologene) and found a nucleotide sequence
and drosophila (27%) in favour of an essential role for Thespin.
Protein motifs and domains are predicted in Thespin by several algorithms such as TMpred,
PSORT and TOPPRED. Four transmembrane domains located in the C-terminal half of the
protein are predicted, the last one in the C-terminal part being predicted with a stronger score.
The putative protein also contains several tetratrico-peptide repeats (TPR) domains which are
structural motifs consisting of 34 Amino acid residues and found in over 300 human proteins
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specific disease as done for THE syndrome, may enable one to describe new protein functions
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(D’andrea et al, 2003). Despite the fact that the amino-acids are poorly conserved, these
motifs assemble into a characteristic Helix-Turn-Helix structure. The basic function of these
TPR motifs is to mediate protein-protein interactions and therefore, can be involved in a
variety of biological process such as cell-cycle regulation, transcriptional control, protein
transport or folding. Consequently, no hint is given by the presence of TPR domains
regarding a specific function. Interestingly, one of the splice site mutations of this series leads
to a deletion of 19 residues in frame, following the skipping of exon 25. This deletion does
not modify the predicted transmembrane domains but deleted several putative TPR domains
of the C-terminal part, suggesting a major functional role for these structures.
Beside the TPR domains, neither the gene nor its derived protein show any significant
sequence similarity to other known human DNA or protein sequences. In the literature,
mutations in other TPRs containing proteins have been described as causative in several
neurological human diseases such as Leber congenital amaurosis (Sohocki et al, 2000) or
CMT type 4C (Senderek et al, 2003) but no clear link can be made between these proteins and
their putative function.
Collectively, our results confirmed the role of TTC37 in THE syndrome, extended the pattern
of mutations associated with this syndrome and provided new data on the normal expression
pattern of TTC37 and the putative structure of Thespin. Assigning an unknown protein to a
and will lead surely to characterize the localisation and functional properties of Thespin.
Acknowledgements
We are grateful to the patients and family members for their participation in the study and
warmly acknowledge Patrice Bourgeois, Pierre Cau and Claudine Schiff for their helpful
discussion, Benedicte Arnaud-Pigneur and Noël Peretti for providing patient samples. This
work was supported by the Assistance Publique-Hôpitaux de Marseille (AORC 2009).
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Hartley JL, Zachos NC, Dawood B, Donowitz M, Forman J, Pollitt RJ, Morgan NV, Tee L,
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a: Scheme representation of TTC37 with mutations described previously (underneath) and in
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Legends

Figure 1:
Sequencing analysis of transcripts in 2 patients with splice mutations:
a: scheme representation of the splice site mutation c.2515+1 C>G, cDNA PCR amplification
with primers located in exon 22 and 24 for the homozygous patient, his heterozygous mother
and a normal control and sequence obtained for the patient showing exon 23 skipping.
b: scheme representation of the splice site mutation c.2577-3_-7delTTTTT, cDNA PCR
amplification with primers located in exon 23 and 26 for the homozygous patient and a
normal control and sequence (forward and reverse) obtained for the patient showing exon 25
skipping.

Figure 2:
qPCR analysis on normal human tissues: 48 tissues have been tested, 34 are shown on the
graph. Experiments have been done twice with probe located on exons 35-36 junction. RQ:
Relative Quantification to GAPDH expression.

Figure 3:
our study (above); red: non sense or frameshift, blue missense, green: splicing mutations.
b: Model of human Thespin as predicted by TMPred algorithm.



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trichorrhexis nodosa. NR: not reported
Patients with
mutations in
TTC37
Patients without
mutations in
TTC37
Hartley et al. 2010
Number of patients (families)
Consanguinity
Median age
Male/female
Hair abnormalities
Dysmorphism
Intractable diarrhea
Onset in the first month
Parenteral nutrition
Immunodeficiency
IUGR
Weight<3percentile
Liver involvement
Mental retardation

9 (8)
4/8
10 y
4/5
9/9
9/9
9/9
7/9
9/9
9/9
7/9
6/9
4/9 (initial 1/4)
5/8

3 (3)
1/3
11 y
0/3
3/3
3/3
3/3
2/3
2/3
2/3
2/3
2/3
2/3 (initial 2/3)
2/3

12 (11)
7/11
3 y
6/6
12/12
12/12
12/12
NR
12/12
12/12
10/11
NR
5/10
7/9


Table 1:
Clinical features of the 12 patients of this study and the 12 previously published by Hartley et
al, 2010. Facial dysmorphism is characterized by hypertelorism, broad flat nasal bridge,
prominent forehead; Observed hair abnormalities are sparse, fragile and uncombable hair with

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Table 2. Mutations in TTC37, geographical origin and consanguinity in the families of the
patients with THE syndrome (NM_014639; NP_055454)
Family
identifier
(number of
affected
individuals)
1 (2)

2 (1)

3 (1)

4 (1)


Mutation 1


Mutation 2


Consanguinity


Geographical
origin
c.326_330delTGCCT c.326_330delTGCCT
p.Leu96TrpfsX10
c.1168delA
p.Val390PheFsX419
c.2515+1G>C
p.Cys813ValfsX56
c.2577-3_-
7DelTTTTT
p.Asn860_878GluDel
Yes

No

Yes

No
Middle-East

France

North Africa

France
p.Leu96TrpfsX10
c.3564-2A>G
(nd)
c.2515+1G>C
p.Cys813ValfsX56
c.4620+1G>C
p.Trp1524_1564
DelIns61
c.4454T>G
p.Leu1485Arg,
possibly damaging
c.3808C>G
p.Pro1270Ala,
probably damaging
c.3960C>A
p.Tyr1320X



5 (1)

c.3015-1C>T
nd
No

France

6 (1)

c.3808C>G
p.Pro1270Ala,
probably damaging
c.3960C>A
p.Tyr1320X
c.3230C>A
p.Ala1077Asp,
probably damaging
Yes

North Africa

7 (1)

8 (1)

Yes

No

North Africa

France


Regarding missense mutations, predictions at the protein level, were done using Polyphen
algorithm. For frameshift mutations, predictions are theoretical and for splice site mutations,
experimental at the transcript level. Nucleotide numbering reflects cDNA numbering with +1
corresponding to the A of the ATG translation initiation codon in the reference sequence. The
initiation codon is codon 1.

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