Article

IFRD1 is a candidate gene for SMNA on chromosome 7q22–q23

Department of Psychiatry and Behavioral Sciences, University of Washington School of Medicine, Seattle, WA 98104, USA.
The American Journal of Human Genetics (Impact Factor: 10.93). 06/2009; 84(5):692-7. DOI: 10.1016/j.ajhg.2009.04.008
Source: PubMed
ABSTRACT
We have established strong linkage evidence that supports mapping autosomal-dominant sensory/motor neuropathy with ataxia (SMNA) to chromosome 7q22-q32. SMNA is a rare neurological disorder whose phenotype encompasses both the central and the peripheral nervous system. In order to identify a gene responsible for SMNA, we have undertaken a comprehensive genomic evaluation of the region of linkage, including evaluation for repeat expansion and small deletions or duplications, capillary sequencing of candidate genes, and massively parallel sequencing of all coding exons. We excluded repeat expansion and small deletions or duplications as causative, and through microarray-based hybrid capture and massively parallel short-read sequencing, we identified a nonsynonymous variant in the human interferon-related developmental regulator gene 1 (IFRD1) as a disease-causing candidate. Sequence conservation, animal models, and protein structure evaluation support the involvement of IFRD1 in SMNA. Mutation analysis of IFRD1 in additional patients with similar phenotypes is needed for demonstration of causality and further evaluation of its importance in neurological diseases.

Full-text

Available from: Wendy H Raskind, Apr 26, 2014
REPORT
IFRD1 Is a Candidate Gene for SMNA
on Chromosome 7q22-q23
Zoran Brkanac,
1,
*
David Spencer,
2
Jay Shendure,
2
Peggy D. Robertson,
2
Mark Matsushita,
3
Tiffany Vu,
3
Thomas D. Bird,
3,4,5
Maynard V. Olson,
2
and Wendy H. Raskind
1,2,3,5
We have established strong linkage evidence that supports mapping autosomal-dominant sensory/motor neuropathy with ataxia
(SMNA) to chromosome 7q22-q32. SMNA is a rare neurological disorder whose phenotype encompasses both the central and the periph-
eral nervous system. In order to identify a gene responsible for SMNA, we have undertaken a comprehensive genomic evaluation of the
region of linkage, including evaluation for repeat expansion and small deletions or duplications, capillary sequencing of candidate
genes, and massively parallel sequencing of all coding exons. We excluded repeat expansion and small deletions or duplications as caus-
ative, and through microarray-based hybrid capture and massively parallel short-read sequencing, we identified a nonsynonymous
variant in the human interferon-related developmental regulator gene 1 (IFRD1) as a disease-causing candidate. Sequence conservation,
animal models, and protein structure evaluation support the involvement of IFRD1 in SMNA. Mutation analysis of IFRD1 in additional
patients with similar phenotypes is needed for demonstration of causality and further evaluation of its importance in neurological
diseases.
We have described a five-generation American family of
Northern European ancestry with an autosomal-dominant
sensory/motor neuropathy with ataxia (SMNA, MIM
607458). There is no evidence of genetic anticipation,
mental retardation, or dementia in this family. The unique
and complex phenotype of SMNA includes axonal sensory
neuropathy present in all affected family members as its
earliest sign. Later in the disease, cerebellar and motor-tract
dysfunction develops. In persons with cerebellar dysfunc-
tion for whom MRI was performed, mild cerebellar atrophy
was seen. Both upper and lower motor neurons are impli-
cated in motor-tract dysfunction, as evidenced by Babinski
sign, muscle atrophy, and EMG findings. Full phenotypic
description and MRI findings were reported previously.
1
The phenotype in this family overlaps with those of hered-
itary ataxias and neuropathies.
Hereditary ataxias are best categorized by the mode of
inheritance and the causative gene or linked locus, if the
gene has not been identified. The identification of at least
28 genes or loci for autosomal-dominant spinocerebellar
ataxias (SCAs)
2
attests to the wide heterogeneity of this
group of disorders. Hereditary neuropathies have shown
even greater clinical and genetic heterogeneity. Charcot-
Marie-Tooth (CMT) neuropathy encompassing at least 40
genes or loci is clinically characterized primarily by
progressive motor neuropathy but also includes sensory
findings. Hereditary sensor y neuropathies have prominent
autonomic features, and so far five genes or loci have been
identified.
Since our initial publication of SMNA, no other families
with neurological phenotypes have been reported to map
within the same region. We were confident in our mapping
study, because the five-generation pedigree provided
a LOD score of 6.36 (p ¼ 1/10
Lod
¼ 4.36 3 10
7
) and the
linked haplotype showed complete segregation with the
disease. We were not able to narrow the region by identi-
fying additional family members, and the region of
interest remained large, from 109 to 131 Mbp on chromo-
some 7 (NCBI build 36.2). Consequently, our approach has
been to comprehensively evaluate the region of interest for
the presence of genetic changes that could be causative.
The institutional review board of the University of Wash-
ington approved research protocols.
To evaluate for nucleotide repeat expansions, we used
the University of California Santa Cruz Genome Browser
(UCSC GB) ‘simple repeats’ track to query the critical
region for the presence of sequences with potential for
expansion. We defined those sequences as CTG/CAG,
CGG/CCG, GAA/TTC, GAC/GTC, CCTG/CAGG, and
ATTCT/AGAAT repeats with more than ten uninterrupted
repeat units present in the reference sequence. For eight
sequences in the region with potential for expansion,
PCR amplification and visualization on agarose gel of
DNA from two affected and two unaffected family
members did not reveal evidence for repeat expansion.
In order to identify microdeletions and microduplica-
tions, we performed comparative genomic hybridization
(CGH) on a custom 385 K high-definition microarray. For
our region of 22 Mb, this represents an average tiling
density of one probe every 57 base pairs. The hybridization
experiments were performed at the NimbleGen facility,
and data were analyzed with SigMAP software. Initial
CGH analysis of one of the affected individuals indicated
two putative microdeletions of 2650 and 924 bp, respec-
tively. However, sequencing of these two locations in the
same individual revealed heterozygosity for alleles present
1
Department of Psychiatry and Behavioral Sciences,
2
Department of Genome Sciences,
3
Department of Medicine,
4
Department of Neurology, University
of Washington School of Medicine, Seattle, WA 98104, USA;
5
Veterans Administration Puget Sound Health Care Center, GRECC and MIRECC, Seattle,
WA 98108, USA
*Correspondence: zbrkanac@u.washington.edu
DOI 10.1016/j.ajhg.2009.04.008. ª2009 by The American Society of Human Genetics. All rights reserved.
692 The American Journal of Human Genetics 84, 692–697, May 15, 2009
Page 1
in dbSNPs, establishing that putative deletions were false
positives and excluding microdeletions as causative.
To evaluate genes in the region, we first initiated capil-
lary sequencing of selected candidate genes. Depending
on annotation, the region encompasses more than 150
genes. On the basis of the known or presumed gene func-
tion, tissue-expression data, similarities to the model-
organism genes, existence of animal models, involvement
in human diseases, and other cues from the literature, we
ranked candidate genes to prioritize sequencing. Primer 3
software was used in designing primers for the exons of
selected genes. Exons were PCR amplified and cycle
sequenced with ABI Prism BigDye terminator kits. Samples
were electrophoresed on an ABI 310 or 3130XL DNA
Analyzer and analyzed with Sequencher software by two
independent persons as previously described.
3
The candi-
date-gene analysis encompassed exons of 70 genes.
As massively parallel sequencing became available, we
sought to expand the analysis to cover all the genes in
the region. Based on NCBI build 36.1, UCSC GB gene-
annotation algorithms indicated 297 genes with 1300
exons in the region. This total contains overlaps due to
different splice variants. We also included intronic and
exon flanking sequences and regions that were identified
by sno and miRNA track, because they might be relevant
for neurological disorders. In total, we identified 3.7 Mb
of target bases. With the identified sequence, we designed
an oligonucleotide capture array with 385,000 probes
(NimbleGen).
4
For isolation and sequencing of targeted
DNA, shotgun Illumina sequencing libraries were con-
structed from genomic DNA of an affected male, his
affected father, and his unaffected mother (Figure 1A)
and amplified in accordance with the manufacturer’s
protocol. The libraries were hybridized to the capture array,
and after washing, hybridized material was eluted, in
accordance with a modified version of the manufacturer’s
protocol. After elution, the enriched sequencing libraries
were sequenced on an Illumina Genome Analyzer I.
The resulting 36 bp single-end sequencing reads were
mapped to the human reference genome (build 36.2)
with the MAQ software package.
5
Consensus calls for
variant identification were also carried out with MAQ. A
single lane produced 167 Mb, 213 Mb, and 249 Mb of
sequence that could be uniquely mapped to the reference
genome (MAQ mapping score > 0) for the proband,
affected father, and unaffected mother, respectively. Of
these, 42% (71 Mb), 37% (79 Mb), and 52% (128 Mb) map-
ped within the 22 Mb interval of interest. Mean coverage
of the 3.7 Mb of targeted bases within the interval was
18-fold, 20-fold, and 32-fold. Applying a MAQ consensus
quality threshold of 40 resulted in consensus calling at
82%, 88%, and 92% of the targeted bases. However, the
consensus-call rate at coding subsequences was higher.
For 141 kb of targeted sequence within the interval defined
as coding by the NCBI’s CCDS database (Sept 2008
version), 95% and 97% were sufficiently covered for
consensus calling with the same thresholds in the proband
and affected father, respectively. Across the 3.7 Mb of tar-
geted sequence, 2295, 2429, and 2543 variants were iden-
tified. In all three samples, 92% of identified variants were
previously documented in dbSNP (version 129). Across the
interval, a total of five, four, and three nonsynonymous,
heterozygous variants that were not in dbSNP were discov-
ered in the proband, affected father, and unaffected
mother, respectively. Three of the variants are present in
all affected family members and were confirmed by PCR
and capillary sequencing.
A variant, NM_002851.2:g.140329G/A (C7bp121,
440,723), in the PTPRZ1 (MIM 176891) gene does not
affect a conserved nucleotide (conservation score 0.002).
A second variant, NM_152556.2:g.44215T/A (C7bp112,
322,954), is in an uncharacterized transcript, C7orf60.
This variant has a conservation score of 0.974. Lastly,
a NM_001007245.1:g35795A/G (C7bp111,886,256) vari-
ant with a conservation score 1.00 in exon 5 of the IFRD1
(MIM 603502) gene was identified. The same IFRD1
variant was also observed during capillary sequencing of
candidate genes (Figure 1B). None of the three variants
above were observed in 1255 unrelated American healthy
control individuals of European descent during testing
with TaqMan assays as previously described.
6
The 12 exons
of the IFRD1 gene were sequenced in 83 case individuals
with ataxia without neuropathy, and no new nonsynony-
mous variants were found.
On the basis of the exclusion of other genomic mecha-
nisms for disease causation and the consideration of
IFRD1 biological function, its involvement in pathways
where mutations result in phenotypes with neuropathy,
and animal knockout models, we propose IFRD1 as
a candidate gene for SMNA. The IFRD1 gene has 1791
bp organized in 12 exons and encodes a protein of 451
amino acids. The rat IFRD1 ortholog Pc4 was initially iso-
lated in PC12 cells because it was upregulated during
nerve growth factor (NGF)-induced neuronal differentia-
tion.
7
In mouse, Tis7/Ifrd1 was identified as an immediate
early gene that is induced by tetradecynol phorbol
acetate, epidermal growth factor, and fibroblast growth
factor stimulation,
8,9
and it is 89% and 93% identical to
the human ortholog on the nucleotide and protein levels.
In human tissues IFRD1 is ubiquitously expressed with
high levels of expression in the brain,
10
including expres-
sion in the cerebellum and spinal cord.
11
In the mouse, in
early gestation, Ifrd1 is expressed in restricted structures,
including the brain, spinal cord and spinal ganglia. At
late gestation, the gene is ubiquitously expressed.
10
Trophic factor stimulation upregulates Ifrd1 expression
and results in translocation of the protein from cytoplasm
to the nucleus.
12
In the nucleus, Ifrd1 acts as a transcrip-
tional corepressor and interacts with proteins of the
SIN3 histone deacetylase complex.
13
Genes in the NGF-IFRD1 pathway have roles in other
neuropathies. A homozygous mutation in NGF subunit
beta NGFB (MIM 162030) has been identified in a Swedish
family whose affected members exhibit peripheral
The American Journal of Human Genetics 84, 692–697, May 15, 2009 693
Page 2
neuropathy with a loss of deep pain and temperature
perception,
14
best fitting into the category of hereditary
sensory and autonomic neuropathy type V (HSAN5,
MIM 608654). The phenotype of heterozygous carriers
does not include loss of pain perception and ranges from
asymptomatic cases to disabling polyneuropathy.
15
Consistent with this finding, homozygous NGF receptor
knockout mice have a phenotype that includes decreased
peripheral sensory innervation.
16
Mutations in neuro-
tropic tyrosine kinase receptor 1 (NTRK1 , MIM 256800)
a protein that associates with NGF receptor NGFR (MIM
162010), have been found in multiple families with
HSAN4 (MIM 256800), an autosomal-recessive neurop-
athy that is characterized by mental retardation, anhydro-
sis, and congenital insensitivity to pain.
17
Altogether,
these data indicate involvement of NGF signaling in
A
B
C
Figure 1. SMNA Family Pedigree and Mutational Analysis
(A) Five-generation SMNA pedigree. Circled individuals were resequenced. Individual VI-1 was recently diagnosed with SMNA.
(B) Sequence analysis of IFRD1 indicating heterozygous nucleotide substitution 743A/G, which causes amino acid Ile172Val change,
marked with an arrowhead.
(C) Multiple protein-sequence alignment of IFRD1 with its orthologs from UCSD 17-way conservation panel. Ile172 position is marked with
an arrowhead.
694 The American Journal of Human Genetics 84, 692–697, May 15, 2009
Page 3
neuronal differentiation and survival and in pathological
mechanisms of neuropathies.
Homozygous Ifrd1 knockout mice exhibit muscle
atrophy and an increase in the number of central nuclei,
a small fiber diameter, and a lower total number of fibers,
a phenotype that resembles neurogenic muscle atrophy
that was found in one muscle biopsy performed in the
SMNA family. The mice also show muscle-regeneration
abnormalities, with delays in the reinnervation process
and deficits in nerve-evoked stimulations, further impli-
cating neurogenic abnormalities.
18
The function of Ifrd1
has been studied in dorsal root ganglion (DRG) neuronal
cultures, where it is expressed.
19
In Ifrd1
/
DRG cultures,
axon initiation, outgrowth, and elongation are dimin-
ished. Stimulation of DRGs with NGF results in increased
axon initiation, outgrowth, and branching. These experi-
ments further implicate Ifrd1 as a downstream effector of
NGF and highlight its role in axonal growth and function
and in the DRG sensory system. It is notable that periph-
eral sensory impairment is a most prominent neurological
deficit in our family.
In our family, we observed a missense mutation,
NP_001541:p.I172V, which substitutes valine for isoleu-
cine. The identified change, NM_001550:c743A/G,
affects a highly conserved nucleotide (conservation score
1.00), as determined by the UCSC browser and the phast-
Cons program that identifies cross-species conserved
nucleotides in multiply aligned sequences.
20
However,
valine is the amino acid at residue 172 in several species,
including elephant, chicken, Xenopus tropicalis, and zebra-
fish (Figure 1C). This change might not severely affect the
biochemical properties of the protein, given that both
amino acids are aliphatic and hydrophobic. However,
isoleucine-to-valine substitutions are pathogenic in other
diseases. In Noonan syndrome (MIM 163950), the I282V
substitution in PTPN11 (MIM 176876) has been found in
multiple cases
21
and was shown to perturb the stability
of the SH2 tyrosine phosphatase catalytic domain.
22
An
early onset form of Alzheimer disease (MIM 104300) is
caused by mutations in the APP gene (MIM 104760). The
most common causal mutation is a reverse V717I substitu-
tion, but I716V that affects beta-amyloid plaque formation
has also been described.
23
Additionally valine-to-isoleu-
cine mutations in the PRNP gene (MIM 176640) have
been found in Creutzfeldt-Jakob disease (CJD, MIM
123400). The V180I mutation found in sporadic cases is
associated with a distinct phenotype,
24
and the V210I
mutation was characterized with incomplete pene-
trance.
25,26
Functional studies of V210I have shown that
the mechanism of disease is the increase in helical and
aggregation propensities of the helix-3 sequence of
PRNP
27
.
To further investigate the I172V protein change and
IFRD1 protein structure, we used Phyre.
28
Phyre combines
different protein homology and analogy detection
methods, including known tridimensional structural
profiles, in a meta-server in order to identify query-
template homologies. Phyre analysis of wild-type (WT)
IFRD1 protein identified homologies with structures
whose folds are characterized by alpha-alpha superhelix
patterns that belong to Armadillo and HEAT repeat fami-
lies. Homology analysis indicated that amino acids 162–
177 form an alpha helix and that amino acid positions
P171 and L173 may form a cavity or a cleft (Figure 2). In
addition, analysis of WT and I172L mutant proteins pre-
dicted differences with respect to their pairing with
complex protein structures. Homology of WT IFRD1 to
S. ceviseae exportin, complexed with importin and ranGtp,
was observed. This homology was not observed for the
I172V mutant that, in turn, was found to have homologies
to H. sapiens transportin complexed with basic nuclear
localization signal proteins. This kind of discrepancy in
homologies was not observed when WT protein was
compared to an A144V mutant, the only nonsynonymous
IFRD1 mutation present in dbSNP. This observation
suggests that the mutation might affect the folding proper-
ties of IFRD1, as well as its affinity for binding and trans-
port through the nuclear pore complex.
In conclusion, by genomic analysis of the region linked
to SMNA, we identified IFRD1 as a candidate gene.
Evidence that supports IFRD1 as the candidate gene
includes the strong statistical evidence for linkage in the
Figure 2. Phyre Analysis of IFRD1 Mutation Region
Phyre analysis of IFRD1 showing that amino acids 162–177 form an alpha helix. Homology with S. cerevisiae Karyopherin alpha protein
(d1wa5b_Sequence) indicates that amino acid positions 171 and 173, flanking the mutated residue, may form a cavity or cleft.
The American Journal of Human Genetics 84, 692–697, May 15, 2009 695
Page 4
region (p ¼ 4.36 3 10
7
); absence of a genomic mechanism
that might cause the disease, such as repeat expansions,
deletions, and/or duplications; and extensive coding-
sequence analysis of the region. Additionally, on the basis
of its role in the NGF pathway and animal studies, IFRD1 is
a strong a priori biological candidate for SMNA. Mutation in
genes that are part of NGF pathway, NTRK1 and NGFB,
cause HSAN IV and HSAN V. Existing knockout-animal
models have neurological phenotypes such as muscle
atrophy and axonal-growth dysregulation, consistent
with its possible role in a neurodegenerative disorder like
SMNA. Our bioinformatic analysis of mutant IFRD1 indi-
cates that the observed mutation might affect binding
and transport through the nuclear pore complexes. Altered
membrane trafficking of signaling molecules has been
proposed as a mechanism that when dysregulated can
lead to neuropathies because of extreme polarity and size
of peripheral neurons. The weakness of the study is that
we have not unequivocally demonstrated that mutation
in IFRD1 causes SMNA. It remains possible that IFRD1
I172V is a rare variant and that some other change in the
original interval undetected by our approach is causative.
Also, we have not excluded changes in PTPRZ1 and
C7orf60 as causative. However, PTPRZ1 is unlikely to be
causal, given that the gene and the identified variant are
poorly conserved. The C7orf60 gene and the variant identi-
fied are not as well conserved as IFRD1. Functional studies of
the IFRD1 I172V mutant gene could provide support for its
pathogenicity and provide additional insights into mecha-
nisms by which the mutant protein causes disease.
However, ultimately, proof that mutations in IFRD1 cause
SMNA will be provided by identification of other familial
and sporadic cases with similar phenotypes. For 83 patients
with ataxia available in our laboratory we have screened all
the exons of IFRD1 and have not identified any additional
mutations. However, neuropathy is not a component of
ataxias in our sample. Further screening of IFRD1 for muta-
tion in patients with related phenotypes that include
neuropathy is warranted.
Acknowledgments
We are grateful to the members of the family for their participation
in these studies and to Elizabeth Thompson for her intellectual
contributions during the early stages of the research. Skillful tech-
nical assistance was provided by Ruolan Qiu, Choli Lee, Emily
Turner, Sarah Summer, Zarshid Arbibi, Ruben Burbank, Catherine
Morgan, Jane Ranchalis, and John Wolf. We thank Chang-En Yu
for generous provision of control samples. The research was sup-
ported in part by funds and resources from the National Organiza-
tion for Rare Disorders, the Department of Veterans Affairs, the
Mary Gates and the Herschel and Caryl Roman Endowments for
Students, and NIH/NHGRI grant R21HG004749 to J.S.
Received: February 6, 2009
Revised: April 4, 2009
Accepted: April 13, 2009
Published online: April 30, 2009
Web Resources
The URLs for data presented herein are as follows:
GeneReviews at GeneTests, http://www.genetests.org
Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.
nlm.nih.gov/Omim/
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    • "In contrast, since the FC group includes affected brothers of autistic girls, FC signals are best interpreted not as female-specific, but as female-affecting risk loci, in accord with a hypothesis that only a subset of ASD risk loci are penetrant in females [7,91011121365]. The significant MO and FC signals identified here implicate regions containing promising candidate genes that warrant further exploration by targeted re-sequencing (for example,66676869). The MO peak at 1p31.3 is located directly over NFIA [GenBank:NG_011787], whose gene product has transcription factor activity and has been implicated in central nervous system development [55,56]. "
    [Show abstract] [Hide abstract] ABSTRACT: Autism spectrum disorders (ASDs) are male-biased and genetically heterogeneous. While sequencing of sporadic cases has identified de novo risk variants, the heritable genetic contribution and mechanisms driving the male bias are less understood. Here, we aimed to identify familial and sex-differential risk loci in the largest available, uniformly ascertained, densely genotyped sample of multiplex ASD families from the Autism Genetics Resource Exchange (AGRE), and to compare results with earlier findings from AGRE. From a total sample of 1,008 multiplex families, we performed genome-wide, non-parametric linkage analysis in a discovery sample of 847 families, and separately on subsets of families with only male, affected children (male-only, MO) or with at least one female, affected child (female-containing, FC). Loci showing evidence for suggestive linkage (logarithm of odds >=2.2) in this discovery sample, or in previous AGRE samples, were re-evaluated in an extension study utilizing all 1,008 available families. For regions with genome-wide significant linkage signal in the discovery stage, those families not included in the corresponding discovery sample were then evaluated for independent replication of linkage. Association testing of common single nucleotide polymorphisms (SNPs) was also performed within suggestive linkage regions. We observed an independent replication of previously observed linkage at chromosome 20p13 (P < 0.01), while loci at 6q27 and 8q13.2 showed suggestive linkage in our extended sample. Suggestive sex-differential linkage was observed at 1p31.3 (MO), 8p21.2 (FC), and 8p12 (FC) in our discovery sample, and the MO signal at 1p31.3 was supported in our expanded sample. No sex-differential signals met replication criteria, and no common SNPs were significantly associated with ASD within any identified linkage regions. With few exceptions, analyses of subsets of families from the AGRE cohort identify different risk loci, consistent with extreme locus heterogeneity in ASD. Large samples appear to yield more consistent results, and sex-stratified analyses facilitate the identification of sex-differential risk loci, suggesting that linkage analyses in large cohorts are useful for identifying heritable risk loci. Additional work, such as targeted re-sequencing, is needed to identify the specific variants within these loci that are responsible for increasing ASD risk.
    Full-text · Article · Feb 2014 · Molecular Autism
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    • "c o m / l o c a t e / b b a d i s to numbers of growth factors such as epidermal growth factor and nerve growth factor in vitro [9] . Although IFRD1 modulates the pathophysiology of human cystic fibrosis lung disease through regulation of the neutrophil effector function [10], IFRD1 is a candidate gene for autosomal-dominant sensory/motor neuropathy with ataxia, which is a rare neurological disorder whose phenotype involves both the central and peripheral nervous systems in humans [11]. Accordingly, TIS7 has been implicated in the regulation of cell growth and differentiation, in addition to the pathogenesis of various diseases, through modulating patterns of gene expression. "
    [Show abstract] [Hide abstract] ABSTRACT: Although tetradecanoyl phorbol acetate induced sequence-7 (TIS7) has been identified as a co-activator/repressor of gene transcription in different eukaryotic cells, little attention has been paid to the functionality of TIS7 in adipocytes. Here, we evaluated the possible role of TIS7 in mechanisms underlying the regulation of adipogenesis. TIS7 expression was preferentially up-regulated in white adipose tissues (WAT) of obesity model mice as well as in pre-adipocytic 3T3-L1 cells cultured under hypoxic conditions. TIS7 promoter activity was selectively enhanced by activating transcription factor-6 (ATF6) among different transcription factors tested, while induction of TIS7 by hypoxic stress was markedly prevented by knockdown of ATF6 by shRNA in 3T3-L1 cells. Overexpression of TIS7 markedly inhibited Oil Red O staining and expression of particular adipogenic genes in 3T3-L1 cells. TIS7 synergistically promoted gene transactivation mediated by Wingless-type mouse mammary tumor virus integration site family (Wnt)/β-catenin, while blockade of the Wnt/β-catenin pathway by a dominant negative form of T-cell factor-4 (DN-TCF4) markedly prevented the inhibition of adipogenesis in 3T3-L1 cells with TIS7 overexpression. TIS7 predominantly interacted with β-catenin in the nucleus of WAT in the genetically obese ob/ob mice as well as in 3T3-L1 cells cultured under hypoxic conditions. Both knockdown of TIS7 by shRNA and introduction of DN-TCF4 similarly reversed the hypoxia-induced inhibition of adipogenic gene expression in 3T3-L1 cells. These findings suggest that TIS7 could play a pivotal role in adipogenesis through interacting with β-catenin to promote the canonical Wnt signaling in pre-adipocytes under hypoxic stress such as obesity.
    Full-text · Article · Mar 2013 · Biochimica et Biophysica Acta
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    • "High-throughput sequencing (HTS) has emerged as a powerful tool to study undiagnosed diseases. Many recent publications describe new genes discovered by whole exome sequencing [Bilguvar et al., 2010; Bonnefond et al., 2010; Choi et al., 2009; Erlich et al., 2011; Hoischen et al., 2010; Kalay et al., 2011; Klein et al., 2011; Krawitz et al., 2010; Lalonde et al., 2010; Ng et al., 2010a; Ng et al., 2010b; Puente et al., 2011; Simpson et al., 2011; Sobreira et al., 2010; Walsh et al., 2010; Wei et al., 2011; Worthey et al., 2011] , and additional publications report genes identified by related techniques [Brkanac et al., 2009; Johnston et al., 2010; Kahrizi et al., 2011; Lupski et al., 2010; Nikopoulos et al., 2010; Rehman et al., 2010; Rios et al., 2010; Summerer et al., 2010; Volpi et al., 2010]. HTS methods produce a list of genotype calls numbering on the order of 10 4 per exome, 10 5 for the combined exomes of a small family, and 10 6 per genome. "
    [Show abstract] [Hide abstract] ABSTRACT: The Undiagnosed Diseases Program at the National Institutes of Health uses high-throughput sequencing (HTS) to diagnose rare and novel diseases. HTS techniques generate large numbers of DNA sequence variants, which must be analyzed and filtered to find candidates for disease causation. Despite the publication of an increasing number of successful exome-based projects, there has been little formal discussion of the analytic steps applied to HTS variant lists. We present the results of our experience with over 30 families for whom HTS sequencing was used in an attempt to find clinical diagnoses. For each family, exome sequence was augmented with high-density SNP-array data. We present a discussion of the theory and practical application of each analytic step and provide example data to illustrate our approach. The article is designed to provide an analytic roadmap for variant analysis, thereby enabling a wide range of researchers and clinical genetics practitioners to perform direct analysis of HTS data for their patients and projects. Hum Mutat 33:599–608, 2012. © 2012 Wiley Periodicals, Inc. †
    Full-text · Article · Apr 2012 · Human Mutation
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