Disease‐causing mutations in the promoter and enhancer of the ornithine transcarbamylase gene Received: 15 September 2017 Revised: 19 December 2017 Accepted: 21 December 2017 DOI:...

Disease‐causing mutations in the promoter and enhancer of the ornithine transca
amylase gene
Received: 15 September 2017 Revised: 19 December 2017 Accepted: 21 December 2017
DOI: XXXXXXXXXX/humu.23394
R E S E A RCH ART I C L E
Disease-causingmutations in the promoter and enhance
of the ornithine transca
amylase gene
Yoon J. Jang1 Abigail L. LaBella2 Timothy P. Feeney3 Nancy Braverman4
Mendel Tuchman1 HirokiMorizono1 Nicholas AhMew5 Ljubica Caldovic1
1Center forGeneticMedicine Research, Chil-
dren'sNational Health System,Washington,
District of Columbia
2Department of Biological Sciences, Vande
ilt
University, Nashville, Tennessee
3Harvard T.H. Chan School of PublicHealth,
HarvardUniversity, Cam
idge,Massachusetts
4McGill UniversityHealthCentre,McGill Uni-
versity,Montreal, Quebec, Canada
5Center for Translational Sciences, Children's
National Health System,Washington, District of
Columbia
Co
espondence
Dr. LjubicaCaldovic,Center forGenetic
MedicineResearch,Children'sNationalMed-
icalCenter, 111MichiganAveNW,Washington
DC,20010.
Email: XXXXXXXXXX
Funding information
MarylandHHMIUndergraduateResearch
Fellowship;RashidFamilyFund
Contract grant sponsor:National Institute
ofDiabetesDigestive andKidneyDiseases
(R01DK047870,R01DK064913).
Communicatedby JohannesZschocke
Abstract
The ornithine transca
amylase (OTC) gene is on the X chromosome and its product catalyzes
the formation of citrulline from ornithine and ca
amylphosphate in the urea cycle. About 10%–
15% of patients, clinically diagnosed with OTC deficiency (OTCD), lack identifiable mutations in
the coding region or splice junctions of the OTC gene on routine molecular testing. We collected
DNA from such patients via retrospective review and by prospective enrollment. In nine of 38
subjects (24%), we identified a sequence variant in theOTC regulatory regions. Eight subjects had
unique sequence variants in theOTCpromoter andone subject had a novel sequence variant in the
OTC enhancer. All sequence variants affect positions that are highly conserved inmammalianOTC
genes. Functional studies revealed reduced reporter gene expression with all sequence variants.
Two sequence variants caused decreased binding of the HNF4 transcription factor to its mutated
inding site. Bioinformatic analyses combined with functional assays can be used to identify and
authenticate pathogenic sequence variants in regulatory regions of the OTC gene, in other urea
cycle disorders or other inborn e
ors of metabolism.
K EYWORDS
enhancer mutation, gene expression gene regulation, hyperammonemia, ornithine transca
amy-
lase, ornithine transca
amylase deficiency, promoter mutation, urea cycle
1 INTRODUCTION
The urea cycle functions in the liver where it converts ammonia, a neu-
otoxic product of protein catabolism, into urea, which is excreted in
urine (Brusilow & Horwich, XXXXXXXXXXOrnithine transca
amylase (OTC;
EC XXXXXXXXXX, MIM# XXXXXXXXXXis a mitochondrial enzyme that catalyzes
the second reaction of the urea cycle: the formation of L-citrulline from
L-ornithine and ca
amylphosphate. The human OTC gene, located on
the short arm of the X chromosome (Xp11.4), is 70 kb long and has
10 exons that contain a 1,062 bp coding sequence (Hata et al., 1986).
This gene codes for a protein comprised of 354 amino acids, includ-
ing a 32 amino acid mitochondrial targeting peptide at its N-terminus
(Horwich et al., 1984; Horwich, Kalousek, Fenton, Pollock, & Rosen-
erg, XXXXXXXXXXPrimary OTC deficiency (OTCD;MIM# XXXXXXXXXXis caused
y mutations in the OTC gene that lead to either reduced or absent
functional OTC enzyme, thus limiting ammonia flux through the urea
cycle. This results in the accumulation of blood ammonia, which may
manifest as lethargy, vomiting, behavioral and neurological abnormal-
ities, and, in severe cases, coma and death (Breningstall, XXXXXXXXXXIn
addition to high plasma ammonia, biochemical abnormalities associ-
ated with OTCD include elevated plasma glutamine, low or absent cit-
ulline, and increased excretion of orotic acid and orotidine in the urine
(Brusilow&Horwich, 2001).
Because the human OTC gene is located on the X-chromosome,
severe OTCD primarily affects hemizygous males and accounts fo
approximately one-half of all urea cycle disorders (Lindgren, de Mar-
tinville, Horwich, Rosenberg, & Francke, XXXXXXXXXXMale hemizygotes
with mutations that a
ogate or severely impair OTC function invari-
ably exhibit hyperammonemia within the first week of life (Ah Mew
et al., 2013; McCullough et al., XXXXXXXXXXMales with hypomorphic OTC
alleles that retain residual enzyme activity, or female heterozygotes
with skewed lyonization (Caldovic, Abdikarim, Narain, Tuchman, &
Morizono, 2015; McCullough et al., 2000) typically present symp-
tomatically after the first week of life (Caldovic et al., 2015; Numata
HumanMutation. 2018;39:527–536. c© 2017Wiley Periodicals, Inc. 527wileyonlineli
ary.com/journal/humu
http:
orcid.org/ XXXXXXXXXX
528 JANG ET AL.
et al., XXXXXXXXXXThe true prevalence of OTCD is unknown, but it has
een estimated to be between one in 14,000 and one in 76,000
(Balasu
amaniam et al., 2010; Brusilow & Maestri, 1996; Dionisi-
Vici et al., 2002; Nettesheim et al., 2017; Summar et al., XXXXXXXXXXIn
85%–90% of patients with the biochemical phenotype of OTCD, a
mutation can be identified through commercially available sequencing
or deletion/duplication testing. In the remaining 10%–15% of affected
individuals, a molecular cause of OTCD cannot be ascertained through
clinical testing. In a few such instances, mutations were ultimately
identified in intronic or regulatory regions (Caldovic, Abdikarim,
Narain, Tuchman, & Morizono, 2015; Engel et al., 2008; Luksan et al.,
2010). Mutations in these regions may, in fact, be responsible fo
a large proportion of OTCD, in patients with no identified variants
via cu
ent clinical methods. However, sequencing of “deep” intronic
or regulatory regions is cu
ently neither routine nor clinically
available.
OTC is expressed in the liver and intestine of humans and othe
mammals (Brusilow&Horwich, XXXXXXXXXXTranscription of the humanOTC
gene appears to initiate at multiple transcription start sites (Luksan
et al., 2010),whereas transcriptionof themouseand ratOtcgenes initi-
ate 136 and 98 bp upstreamof the translation initiation codon, respec-
tively (Takiguchi, Murakami, Miura, & Mori, 1987; Veres, Craigen, &
Caskey, XXXXXXXXXXIn the rat Otc promoter, four regions, A–D, bind tran-
scription factors that regulate expression of the Otc gene (Figure 1;
(Murakami, Nishiyori, Takiguchi, & Mori, XXXXXXXXXXThe rat Otc promote
was sufficient to direct expression of transgenes in the liver and intes-
tine of transgenic mice, but expression was higher in the intestine
than in the liver (Jones et al., 1990; Murakami, Takiguchi, Inomoto,
Yamamura, & Mori, XXXXXXXXXXExpression studies in cultured cells and
transgenic animals revealed that an enhancer, located approximately
11 kb upstream of the first exon of the rat Otc gene, is essential for a
high level of expression of the Otc gene in the liver (Murakami et al.,
1990). In vitro binding studies revealed four sites, designated I–IV, that
are important for the function of the rat −11 kb enhancer (Figure 2
(Murakami et al., 1990)).
In this study, we screened conserved upstream regulatory regions
of the OTC gene in 38 subjects with clinically diagnosed OTCD, but in
whom no deleteriousOTCmutation was identified by clinical sequenc-
ing of the coding regionor splice junctions. Eight of these subjectswere
found to ha
or one of six unique sequence variants in the OTC pro-
moter. One subject had a novel sequence variant in the OTC enhancer.
Five sequence variants were within either known or predicted tran-
scription factor binding sites and the remaining two affected base
pairs that are highly conserved in verte
ates. Six sequence variants
were not found in any of the databases of single nucleotide polymor-
phisms. Functional testing confirmed that all seven sequence variants
described herein cause either reduced expression of reporter gene
in cultured cells or reduced binding of the hepatic nuclear factor 4
(HNF4) transcription factor. These results highlight the importance of
seeking sequence variants in regulatory regions of genes of patients
with OTCD without identifiable mutations as well as the need for bet-
ter and simpler functional testing of regulatory mutations in disease-
causing genes.
2 MATERIALS AND METHODS
2.1 Study subjects
This project, which included retrospective and prospective compo-
nents, was conducted with the approval of the Institutional Review
Board of the Children's National Health System. Inclusion criteria
were: suspected urea cycle disorder with reduced or absent OTC
enzymatic activity in the liver biopsy and/or urinary orotate above
30 ?mol/mmol of urinary creatinine, but absence of a disease-causing
mutation in the exons and intron/exon boundaries of the OTC gene.
When available, gender, age of disease onset, enzymatic activity of
CPS1 in the liver biopsy, and concentrations of ammonia and citrulline
in the plasma were collected for eligible patients. All participants
were probands. Participants and their families were also prospectively
enrolled into the study after refe
al by their metabolic physicians. In
the retrospective study component, participants were selected from a
database of de-identified patients who were screened for mutations
in the coding region and intron/exon junctions of the OTC gene at the
Children's National Health SystemMolecular Genetics Laboratory. Of
1,035 patients in the database, 106 were eligible for this study, 43
were diagnosed with OTCD based on reduced or absent OTC activity
in the liver, and the remaining 63 had a documented history of hyper-
ammonemia and elevated urinary orotate. DNAwas available from 24
subjects. Subjects 1–4 were ascertained retrospectively and subjects
5–9were participants in the prospective study.
2.2 Subjects with sequence changes in the
egulatory regions of theOTC gene
Subject 1 was a male diagnosed with late-onset OTCD based on OTC
activity of 6.5 ?mol min−1 g−1, which was approximately one-tenth of
the control value (56.7 ?molmin−1 g−1 of liver tissue). TheCPS1 activ-
ity in the same liver sample was normal (2.9 ?mol min−1 g−1). Values
of urinary orotate, plasma ammonia, glutamine, and citrullinewere not
available.
Subject 2was amale diagnosedwith late onsetOTCDbased onOTC
activity of 10.3 ?mol min−1 g−1, which was approximately one-tenth
of the control value (110.6 ?mol min−1 g−1 of liver tissue). The CPS1
activity in the same liver biopsy was normal (6.1 ?mol min−1 g−1). His
urinary orotate concentration was 77 ?mol/mmol creatinine (normal
1.2), and plasma ammonia, glutamine, and citrulline concentrations
were 92 ?M (normal <32), 1,064 ?M (normal 205–756), and 17 ?M
(normal 12–55), respectively.
Subject 3, a male, was included in the study based on the high con-
centration of urinary orotate (261 ?mol/mmol creatinine), and plasma
ammonia, glutamine, and citrulline concentrations of 1,564, 2,715, and
37 ?M, respectively.
Subject 4 was a male with neonatal onset hyperammonemia and
no measurable OTC activity in the liver. The CPS1 activity in the
liver biopsy was also absent. His urinary orotate concentration was
1,624 ?mol/mmol creatinine, whereas plasma ammonia and glutamine
concentrations were 683 and 4,442 ?M, respectively.
JANG ET AL. 529
A
B
TTTACTATAC CTTCTCTATC ATCTTGCACC CCCAAAATAG CTTCCAGGGC ACTTCTTTCT ATTTGTTTTT GTGGAAAGAC TGGCAATTAG AGGTAGAAAA
XXXXXXXXXX
GTGAAATAAA TGGAAATAGT ACTACTCAGG ACTGTCACAT CTACATCTGT GTTTTTGCAG TGCCAATTTG CATTTTCTGA GTGAGTTACT TCTACTCACC
150 200
TTCACAGCAG CCGGTACCGC AGTGCCTTGC ATATATTATA TCCTCAATGA GTACTTGTCA ATTGATTTTG TACATGCGTG TGACAGTATA AATATATTAT
250 Region A 300
HNF-4, COUP-TF
GAAAAATGAG GAGGCCAGGC AATAAAAGAG TCAGGATTTC TTCCAAAAAA AATACACAGC GGTGGAGCTT GGCATAAAGT TCAAATGCTC CTACACCCTG
350 Region B 400
GATA HNF-4, COUP-TF
CCCTGCAGTA TCTCTAACCA GGGGACTTTG ATAAGGAAGC TGAAGGGTGA TATTACCTTT GCTCCCTCAC TGCAACTGAA CACATTTCTT