HELLP babies link a novel lincRNA to the trophoblast cell cycle

Article (PDF Available)inThe Journal of clinical investigation 122(11) · October 2012with43 Reads
DOI: 10.1172/JCI65171 · Source: PubMed
Abstract
The HELLP syndrome is a pregnancy-associated disease inducing hemolysis, elevated liver enzymes, and low platelets in the mother. Although the HELLP symptoms occur in the third trimester in the mother, the origin of the disease can be found in the first trimester fetal placenta. A locus for the HELLP syndrome is present on chromosome 12q23 near PAH. Here, by multipoint nonparametric linkage, pedigree structure allele sharing, and haplotype association analysis of affected sisters and cousins, we demonstrate that the HELLP locus is in an intergenic region on 12q23.2 between PMCH and IGF1. We identified a novel long intergenic noncoding RNA (lincRNA) transcript of 205,012 bases with (peri)nuclear expression in the extravillous trophoblast using strand-specific RT-PCR complemented with RACE and FISH. siRNA-mediated knockdown followed by RNA-sequencing, revealed that the HELLP lincRNA activated a large set of genes that are involved in the cell cycle. Furthermore, blocking potential mutation sites identified in HELLP families decreased the invasion capacity of extravillous trophoblasts. This is the first large noncoding gene to be linked to a Mendelian disorder with autosomal-recessive inheritance.
Research article
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 11 November 2012 4003
HELLP babies link a novel lincRNA
to the trophoblast cell cycle
Marie van Dijk,
1,2
Hari K. Thulluru,
1,2
Joyce Mulders,
1
Omar J. Michel,
1
Ankie Poutsma,
1
Sandra Windhorst,
1
Gunilla Kleiverda,
3
Daoud Sie,
4
Augusta M.A. Lachmeijer,
5
and Cees B.M. Oudejans
1,2
1
Department of Clinical Chemistry and
2
Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands.
3
Department of Gynecology, Flevoziekenhuis, Almere, The Netherlands.
4
Department of Pathology and
5
Department of Clinical Genetics,
VU University Medical Center, Amsterdam, The Netherlands.
The HELLP syndrome is a pregnancy-associated disease inducing hemolysis, elevated liver enzymes, and low
platelets in the mother. Although the HELLP symptoms occur in the third trimester in the mother, the origin
of the disease can be found in the first trimester fetal placenta. A locus for the HELLP syndrome is present on
chromosome 12q23 near PAH. Here, by multipoint nonparametric linkage, pedigree structure allele sharing,
and haplotype association analysis of affected sisters and cousins, we demonstrate that the HELLP locus is in
an intergenic region on 12q23.2 between PMCH and IGF1. We identified a novel long intergenic noncoding
RNA (lincRNA) transcript of 205,012 bases with (peri)nuclear expression in the extravillous trophoblast using
strand-specific RT-PCR complemented with RACE and FISH. siRNA-mediated knockdown followed by RNA-
sequencing, revealed that the HELLP lincRNA activated a large set of genes that are involved in the cell cycle.
Furthermore, blocking potential mutation sites identified in HELLP families decreased the invasion capacity
of extravillous trophoblasts. This is the first large noncoding gene to be linked to a Mendelian disorder with
autosomal-recessive inheritance.
Introduction
Preeclampsia (new-onset hypertension in pregnancy with pro-
teinuria) and hemolysis, elevated liver enzymes, low platelets
(HELLP) syndrome share a similar onset in the early placenta
(1–3). Local placental dysfunction is followed by activation of
compensatory pathways in the placenta (PlGF-sFLT1-endoglin
axis) and mother (TGF-β–mediated NOS-dependent vasodila-
tion) that ultimately trigger the appearance of systemic mater-
nal symptoms (4–6). Genetically, the HELLP syndrome and
preeclampsia are distinct entities (7). The familiar forms of
preeclampsia involve the STOX1 gene (8). The HELLP gene is
unknown, but 3 putative loci have been identified by genome-
wide linkage analysis (7). Given the consistent observation in
monozygotic parous twins that lack of concordancy for pro-
teinuric hypertension is the rule rather than the exception, we
searched these loci for the presence and identity of the HELLP
gene, with the assumption that fetal (i.e., placental) contribu-
tions are essential for any genetic basis of HELLP (9).
Here we showed, by multipoint nonparametric linkage, pedi-
gree structure allele sharing, and haplotype association analysis
of affected sisters and cousins, that the HELLP locus resides in
an intergenic region on chromosome 12q23.2 between PMCH
and IGF1. By strand-specific RT-PCR complemented with rapid
amplification of cDNA ends (RACE) and FISH, a novel long
intergenic noncoding RNA (lincRNA) transcript with expres-
sion in the placental extravillous trophoblast was identified. By
siRNA-mediated knockdown followed by RNA sequencing, the
HELLP lincRNA was found to be involved in the cell cycle. By
using morpholinos to block potential mutation sites, as identi-
fied in HELLP families, we observed a decreased invasion capac-
ity of extravillous trophoblasts.
Results
Analysis of HELLP families reveals linkage to an intergenic region on
12q23. Using the original cohort of families with the HELLP syn-
drome (n = 34; ref. 7), the 3 loci previously found to have nominal
linkage were reanalyzed using additional microsatellite markers.
The nonparametric lod scores for 12p12 and 20p12 decreased
(from 1.55 to 1.08) and disappeared (from 1.70 to 0.37), respec-
tively. The lod score for the 12q23 region increased from nominal
to suggestive (i.e., from 2.1 to 2.37).
We subsequently tested 57 individuals (7 families with affected
sib-pairs, 4 families with affected cousin-pairs, and 2 discordant
monozygous twin sisters with their partners, of which 36 females
were affected) with 26 microsatellite markers (D12S309–D12S395)
in the 23.6-Mb region on 12q23. Nonparametric multipoint link-
age analysis using S
mnallele
confirmed the 4-marker region between
PAH and D12S1647 (lod and NPL scores >3; Figure 1A) and indi-
cated recessive inheritance (in descending order: S
mnallele
, S
pairs
, S
all
,
S
robdom
; ref. 10). Pedigree analysis narrowed this region to 2 mini-
mal critical regions: D12S1607–PAH and D12S338–D123S317,
each about 1 Mb. In the highly informative family 93113, in which
2 sisters married 2 brothers from an unrelated family, maximal
allele sharing in all affected females from 2 generations (cous-
ins and their mothers) was restricted to the first region near
D12S1030 (Supplemental File 1; supplemental material available
online with this article; doi:10.1172/JCI65171DS1).
We found no mutations in the coding sequences of the 38
known and predicted genes within or near these 2 regions:
DRAM, CCDC53, NUP37, C12orf48 (also referred to as PARPBP),
PMCH, IGF1, PAH, ASCL1, C12orf42, BC041342, STAB2, NT5DC3,
Authorship note: Marie van Dijk and Hari K. Thulluru contributed equally to this
work.
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2012;122(11):4003–4011. doi:10.1172/JCI65171.
Related Commentary, page 3837
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4004 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 11 November 2012
Figure 1
Identication of the HELLP locus on chromosome 12q23.2. Multipoint nonparametric linkage analysis (A; see also Supplemental File 1), pedigree
structure analysis with SNP/INDEL markers (B; see also Supplemental File 2), and HaploView analysis (C) dened the HELLP locus region as
residing in a intergenic region of about 170 kb between C12orf48 and IGF1 on 12q23.2.
research article
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 11 November 2012 4005
LOC253724 (also referred to as GNN), HSP90B1, TDG, GLT8D2,
HCFC2, NFYB, TXNRD1, EID3, CHST11, BC030271, SLC41A2,
C12orf45, APPL2, OCC-1, RFX4, ACACB, FOXN4, UBE3B, ANAPC7,
CCDC63, RPH3A, OAS1, P/OKCL.4, OAS2, DDX54, and LHX5.
Instead, using the 181 SNPs and insertion/deletion polymor-
phisms (INDEL) identified, we found the HELLP locus to reside
in an intergenic region of 154 kb (chromosome 12: 101,114,674–
101,268,434 bp; UCSC assembly NCBI36/hg18) between C12orf48
and IGF1 (Figure 1B and Supplemental File 2).
We confirmed this region by haplotype association analysis
(HaploView) following deep sequencing. We tested 26 singletons
(sisters and cousins) with 405 SNP markers. We took advantage of
the placental genotype-maternal phenotype discrepancy character-
istic for preeclampsia and HELLP (the maternal endophenotype is
caused by the placental effector genotype, i.e., the genotype of the
child born from the affected pregnancy), as this permitted case-
control association analysis within the family cohort. Phenotypi-
cally affected females born from phenotypically affected mothers
were genetically considered cases, as the disease genotypes of the
former are homozygous or compound heterozygous; phenotypi-
cally affected females born from nonaffected mothers were con-
sidered controls, as the former are unaffected, heterozygous carri-
ers. Using the “Solid Spine of LD” algorithm to evaluate blocks of
linkage disequilibrium (LD) with a minimum D value of 0.8, the
single block identified (chromosome 12: 101,150,849–101,320,921
bp) confirmed that the HELLP gene was present within the inter-
genic region between C12orf48 and IGF1 on 12q23.2 (Figure 1C), a
finding that was confirmed to be significant by permutation test-
ing (P = 0.0334; n = 5,000).
Discordant monozygotic twins confirm placental origin of the HELLP
syndrome. In the discordant monozygotic twin sister family, the
HELLP linkage region presented as a cluster of minor alleles
with heterozygous sharing between the affected twin sister and
her partner, while completely absent in the partner of the non-
affected twin sister. Using 3 informative SNPs in this cluster,
we observed complete agreement with the placental model: the
presence of a fetal susceptibility gene, expressed in placenta cells
in contact with maternal vessels, determined the maternal phe-
notype (Figure 2).
The HELLP transcript is a lincRNA expressed in extravillous tro-
phoblasts. We screened the intergenic region for transcription
in SGHPL-5, a diploid cell line representative of first-trimester
extravillous trophoblast, the fetal cell central in the etiology of
preeclampsia and HELLP (11). We performed a complete analysis
of the number, size, 5- and 3-ends, splicing pattern, and cellu-
lar location of the transcript(s) involved. This identified a single,
unspliced large transcript with 5-cap structure and 3-polyA-tail
of 205,012 bases (chromosome 12: 101,115,493–101,320,504 bp;
UCSC assembly hg18) with no coding potential (CPC, –0.874346;
ref. 12 and Figure 3). By FISH, expression was found to be nucle-
ar, perinuclear, and cytoplasmic, both in vitro — in the extravil-
lous trophoblast cell line (SGHPL-5) used for its identification
(Figure 4, A–C) — and in vivo (i.e., first trimester placenta tissue),
whereas no signal was detected using a sense probe as negative
control (Figure 4D). In the anchoring villi of human first-tri-
mester placenta tissue, (peri)nuclear expression was restricted to
extravillous trophoblast cells and their precursors (i.e., column
extravillous cytotrophoblasts; Figure 4E). In first-trimester pla-
centabed showing a front of extravillous trophoblast invasion
into the myometrium, invasive extravillous trophoblast cells
actively involved in maternal spiral artery modification showed
a distinct difference in subcellular localization: predominantly
nuclear in the endovascular trophoblasts within maternal spiral
arteries, while perinuclear and cytoplasmic in the interstitial tro-
phoblast surrounding maternal spiral arteries (Figure 4, F and
G). Comparison with the transcriptome assemblies of 17 adult
Figure 2
Parent-child segregation of 12q23.2 region in monozygotic twin sisters discordant for HELLP. 3 informative SNPs located within the HELLP region
with linkage were analyzed for parent-child segregation in discordant, monozygotic twin sisters. Only 1 sister (patient 1) developed the HELLP
syndrome during pregnancy. The other sister (patient 3) had 2 normal pregnancies. The minor alleles (red) were homozygous in the child born of
affected patient 1 (patient A). In contrast, homozygosity for the major alleles (green) was seen in the children born of normal patient 3 (patients
B and C). This pattern supports the presence of a placental susceptibility gene for the HELLP syndrome, in which the fetal genotype expressed
in the placenta determines the maternal phenotype.
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4006 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 11 November 2012
tissues (in addition to placenta) in the human lincRNA catalog
(http://www.broadinstitute.org/genome_bio/human_lincrnas/)
indicated that the HELLP lincRNA was preferentially, if not
exclusively, expressed in the placenta (Supplemental File 3).
Genome-wide RNA sequencing indicates involvement in the cell cycle.
Given its unknown function, its large size, and the absence of
additional landmarks preventing prioritization of regions for
functional and mutational analyses, we performed genome-wide
Figure 3
Location of the HELLP lincRNA on chromosome 12q23, identied by overlapping strand-specic PCR amplications supplemented by RACE
experiments to identify the 5 and 3 ends.
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The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 11 November 2012 4007
RNA-sequence (RNA-Seq) analysis after siRNA-mediated down-
regulation of the HELLP transcript in SGHPL-5 cells. The differ-
ential expression induced after HELLP transcript knockdown was
analyzed using TopHat and Cufflinks software, calculating the
significant differential expressions at the gene level. In this list, 3
significant gene_id hits corresponded with downregulation of the
HELLP transcript on chromosome 12q23. These hits had q values
(P value corrected for false discovery rate [FDR]) of 0.019, 0.030,
and 0.031 and showed expression values in the control sample of
0.21, 0.12, and 0.15, respectively, whereas the siRNA-mediated
knockdown sample value was 0 for all 3 hits. For subsequent vali-
dation analysis, the following selection criteria were used: q value
threshold, <0.05; log
2
fold change, ≥2; and a value of at least 1 of
either the control sample or the siRNA-mediated knockdown
sample. Furthermore, unannotated transcripts were excluded.
By cross-checking the list of genes with the results obtained in
an untransfected sample, the hits originating from transfection
effects were omitted. This yielded 4 upregulated genes and 1,364
downregulated genes upon knockdown of the HELLP transcript.
Validation of the RNA-Seq results was performed by quantita-
tive RT-PCR assays on 14 transcripts, including all 4 upregulated
genes, 5 downregulated genes with a q value of 0, a selection of 3
genes with q values between 0 and 0.05, and a gene that did not get
through the additional selection criteria (GRB10) as a negative con-
trol. One of the upregulated genes, AZIN1, consists of 2 transcripts
of which only 1 is upregulated, while the other is downregulated
but with a log
2
fold change of –1.47; therefore, this gene also did
not get through the additional selection criteria. Quantitative RT-
PCR (Supplemental File 4) showed that differential expression
could be validated for genes with a q value below 0.01, with one
exception; the upregulated gene MEG8 could not be validated with
the opposite expression pattern observed likely to be caused and
complicated by the 35 SNORD genes downstream of MEG8. The
remaining transcripts — 1 with upregulated expression and 8 with
Figure 4
Localization of the HELLP lincRNA. FISH showed nuclear (A), perinuclear (B), and cytoplasmic (C) localization in SGHPL-5 cells; (D) no signals
were seen using a sense probe as negative control. (E) In rst-trimester placental tissue, nuclear and perinuclear expression was found in the
anchoring villous, consisting of column extravillous trophoblasts and villous cytotrophoblasts. (F) In rst-trimester placentabed, localization was
found to be nuclear in endovascular extravillous trophoblasts (arrows), whereas interstitial trophoblasts showed predominant perinuclear and cyto-
plasmic expression (arrowheads). (G) First-trimester placentabed showing the front (dotted line) of the extravillous trophoblast invasion into the
myometrium. Inset: nuclear endovascular expression of the HELLP lincRNA (enlarged ×4.5). Scale bars: 5 μm (AD); 30 μm (E and F); 100 μm (G).
Table 1
Top 10 networks from ingenuity pathway analysis
Network Score
A
Focus genes
B
Cellular function and maintenance, cell morphology, nervous system development and function 34 32
Cell cycle, DNA replication, recombination, and repair, cellular assembly and organization 32 31
Gene expression, DNA replication, recombination, and repair, cell cycle 30 30
Cancer, cellular development, cellular growth and proliferation 28 29
Cell cycle, DNA replication, recombination, and repair, cellular assembly and organization 27 28
Cell death, liver necrosis/cell death, molecular transport 25 27
Cellular movement, cardiovascular system development and function, cellular assembly and organization 22 25
Antigen presentation, cell-to-cell signaling and interaction, hematological system development and function 20 24
Cell morphology, embryonic development, organ development 15 20
RNA damage and repair, molecular transport, RNA trafficking 15 20
A
Signicance score of the network, shown as the negative log of the P value. The score indicates the signicance of the assembly of the set of focus genes
in the network.
B
Number of genes from the submitted gene list that are present in the network.
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4008 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 11 November 2012
downregulated expression after siRNA-mediated knockdown —
could be validated. The gene list selected with the criteria used for
validation analysis was accordingly adjusted to a q value threshold
of <0.01. This validated set, excluding MEG8, consisted of 1,198
upregulated genes and 1 downregulated gene (Supplemental File
5) and was submitted to Ingenuity Pathway Analysis (IPA; see
Methods). The top 10 networks from network analysis showed
they were predominantly associated with the cell cycle (Table 1).
Furthermore, function annotation analysis revealed significant
increases in functions related to G1/S phase and cell death, whereas
significant decreases were found for functions related to G2/M
phase, cell survival, and migration (Supplemental File 6).
Blocking potential mutation sites decreases extravillous trophoblast
invasion. Although we have not yet identified all disease-causing
mutations in the complete set of HELLP families, we identified
a couple that segregate correctly with the disease phenotype in
all generations with compound heterozygosity in the patients
involved. These sequence variants, RW18-5 (CT at position
101,120,459), HAPLO378 (AG at position 101,319,702),
and HAPLO215Rev (GC at position 101,241,930), were not
found in 200 control chromosomes of nonaffected individu-
als of similar race and therefore qualified as mutations (Figure
5A). Functional studies were performed using morpholinos
to block the regions containing these sites. Validation of their
experimental effect showed that blocking the different mutation
sites using optimal morpholino concentrations (5 μM) seem to
prevent HELLP lincRNA degradation, leading to a net increase
in HELLP lincRNA levels (Figure 5B). Using the combination
of HAPLO378 with HAPLO215Rev morpholinos (being com-
pound heterozygotes in family 9265) with a concentration of 2.5
μM each did not show this effect as clearly. Single morpholinos
at a concentration of 2.5 μM could also not show this increase
in HELLP lincRNA levels (data not shown). If these sites are of
functional importance and critical in the pathophysiology of the
HELLP syndrome, the morpholino blocking effects should (a)
be seen in trophoblast cells; (b) affect the expression of the same
genes as identified by RNA-Seq, but in the opposite direction, as
blocking shows an upregulation of the HELLP lincRNA instead
of siRNA-mediated knockdown; (c) exhibit a greater effect when
the mutations that segregate in the same patient are tested in
combination. Finally, the transcriptional effects should be fol-
lowed by a functional cellular effect, i.e., defective trophoblast
invasion. This was exactly the situation observed when using
the different morpholinos separately or in combination. The
increase in HELLP lincRNA was followed by increased transcript
levels of downstream effector genes when using the RW18-5 or
HAPLO215Rev morpholino, while this was not observed for
HAPLO378 (Figure 5C). In addition, while the individual effects
of suboptimal doses (2.5 μM) of the morpholinos when tested
separately were incomplete (data not shown), the effect was
complete when HAPLO378 and HAPLO215Rev were tested in
combination. Finally, we performed Matrigel invasion assays in
the SGHPL-5 cells to test whether these transcriptional changes
are accompanied by a phenotypic effect mimicking the primary
clinical defect (i.e., decrease in trophoblast invasion). The results
confirmed our findings: HAPLO378 did not have an effect on
invasion, whereas RW18-5 significantly decreased the number of
invaded cells, as did HAPLO378 and HAPLO215Rev in combina-
tion at 2.5 μM each. Furthermore, HAPLO215Rev showed a clear
trend toward a decrease in invaded cells (P = 0.0645; Figure 5D).
Discussion
Here we show by multipoint nonparametric linkage, pedigree struc-
ture allele sharing, and haplotype association analysis of HELLP-
affected sisters and cousins that the locus of the HELLP syndrome
resides in an intergenic region on chromosome 12q23.2 between
PMCH and IGF1. By strand-specific RT-PCR complemented with
RACE experiments, we identified an unspliced noncoding RNA
transcript of 205 kb in length located within this intergenic region.
The transcript was furthermore found to be expressed in extravil-
lous trophoblasts. In first-trimester placenta, the expression was
predominantly nuclear in the endovascular extravillous tropho-
blasts, while interstitial extravillous trophoblasts mainly showed
perinuclear and cytoplasmic expression. Results of whole-genome
RNA-Seq indicated that the HELLP lincRNA is involved in 1 or
more processes in the cell cycle, in which siRNA-mediated knock-
down of the transcript leads to reduced activity of the G2/M phase
of the cell cycle, while the G1/S phase shows an increase in activity.
By using morpholinos to block potential mutation sites as iden-
tified in HELLP families, we found that they were able to upregu-
late the expression of the HELLP lincRNA. This suggests that the
morpholinos are blocking the breakdown of the transcript itself.
Consistent with upregulated expression of the lincRNA, both the
RW18-5 and especially the HAPLO215Rev morpholino were able to
upregulate downstream effector genes found to be downregulated
upon siRNA-mediated knockdown of the transcript. The combina-
tion of HAPLO378 and HAPLO215Rev morpholinos, as found in
one of the HELLP families, showed similar effects. Finally, by using
invasion assays, we found that morpholino RW18-5 and the combi-
nation of HAPLO378 with HAPLO215Rev significantly decreased
the amount of invaded extravillous trophoblast cells, whereas HAP-
LO215Rev showed a trend toward a reduction in extravillous troph-
oblast invasion. This would fit the placental origin of the HELLP
syndrome, in which extravillous trophoblasts show reduced inva-
sion into the maternal decidua, caused by reduced differentiation
of the extravillous trophoblasts from a proliferative toward an inva-
sive phenotype. This concept was already indicated by the RNA-Seq
results showing effects on the cell cycle, in which siRNA-mediated
knockdown of the HELLP lincRNA led to decreased activity of the
G2/M phase, whereas the G1/S phase showed increased activity.
In summary, we showed a region on chromosome 12q23 to be
associated with the HELLP syndrome by genome-wide linkage anal-
ysis of families with the disease. The region contains a noncoding
RNA transcript more than 205 kb in length. This lincRNA was local-
ized in early placenta extravillous trophoblasts and, by genome-wide
RNA analysis followed by pathway analysis, found to classify as an
activator affecting a large set of genes involved in the cell cycle, in
particular in activating the G2/M phase while deactivating the G1/S
phase. Blocking potential mutation sites as identified in HELLP fam-
ilies decreased the invasion capacity of extravillous trophoblasts, con-
sistent with the placental origin of the HELLP syndrome. lincRNAs
have so far been implicated in chromatin remodeling, transcriptional
control, and post-transcriptional processing (13–15). The next step is
to identify the precise function of this lincRNA as a regulating non-
coding RNA and its dysfunction in the HELLP syndrome.
Methods
Patient recruitment. In our genetic studies, we included all HELLP families
available to us that included at least 2 affected females (sisters or cousins).
The original cohort of families (n = 67) with preeclampsia, eclampsia, the
HELLP syndrome, or pregnancy-induced hypertension (PIH), recruited
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The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 11 November 2012 4009
Figure 5
Blocking potential mutation sites
decreases extravillous trophoblast
invasion. (A) 3 potential mutations were
investigated in detail. RW18-5 (CT at
position 101,120,459) occurred in family
9401; the mutation on the other allele
has not yet been identied. HAPLO378
(AG at position 101,319,702) and
HAPLO215Rev (GC at position
101,241,930) occurred as compound
heterozygous mutations in family 9265.
HAPLO378 was also present in fam-
ily 93113. Women affected by HELLP
are indicated by black symbols; chil-
dren born of HELLP pregnancies are
indicated by gray symbols. Potential
mutagenic alleles are also shaded gray.
(B) Morpholinos at a concentration of
5 μM blocking the potential mutagenic
sites upregulated expression of the
HELLP lincRNA in SGHPL-5 cells. (C)
Upregulation of the HELLP lincRNA led
to upregulation of downstream effec-
tor genes that showed downregulation
after siRNA-mediated knockdown of the
transcript when the RW18-5 or HAP-
LO215Rev morpholino was used at a
concentration of 5 μM. The combination
of HAPLO378 and HAPLO215Rev (2.5
μM each) also upregulated the down-
stream effector genes. (D) Invasion of
SGHPL-5 cells signicantly decreased
when RW18-5 morpholinos were intro-
duced, whereas HAPLO215Rev trend-
ed toward a signicant decrease in inva-
sion. HAPLO378 did not affect invasion.
The combination of HAPLO215Rev and
HAPLO378 (2.5 μM each) signicantly
decreased the amount of invaded cells.
*P < 0.05; **P < 0.01.
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4010 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 11 November 2012
don, London, United Kingdom), RNA was isolated using the RNeasy mini
kit (Qiagen) including on-column DNase treatment, and reverse tran-
scription was performed using the SuperScript III First-Strand Synthesis
System for RT-PCR (Invitrogen) using random hexamers as well as gene-
specific reverse primers to confirm strand specificity. Overlapping PCRs
covering the complete area between the genes PMCH and IGF1 was per-
formed on cDNA using Platinum Taq DNA Polymerase High Fidelity (Inv-
itrogen). To cover the complete transcript, 42 PCRs were needed yielding
2- to 7-kb PCR products. Confirmation of the correct products amplified
was done by sequencing using BigDye Terminators and analyzed on an
ABI3130XL genetic analyzer (Applied Biosystems). On the 5 and 3 ends
of the transcript, where PCR products could no longer be amplified, 5and
3 RACE experiments were performed using the Generacer kit according to
the manufacturer’s instructions (Invitrogen). The exact transcript bound-
aries were confirmed by sequencing. See Supplemental File 7 for primer
sequences. The HELLP transcript sequence described herein was deposited
in GenBank (accession no. JX088243).
Placental tissue collection. First-trimester placentas were obtained at the
time of elective termination of pregnancy. Informed consent was obtained
from each patient, and collections were approved by the Ethical Commit-
tee of the VU University Medical Center and the Flevoziekenhuis Almere.
Tissue was collected into ice-cold PBS. After extensive washing the placen-
tal tissues were submerged in Tissue-Tek and plated at –80°C.
FISH. RNA FISH was carried out on SGHPL-5 cells and 5-μm-thin pla-
cental tissue cryostat sections, using oligonucleotide probes (Supplemen-
tal File 7) against the HELLP lincRNA labeled with digoxigenin (DIG) at
the 5 and 3 end (Eurogentec), as described previously (20). Briefly, after
fixation, slides were prehybridized for 30 minutes at 41°C (25°C below
the T
m
of the probe) in a humidified chamber with prehybridization buf-
fer, then incubated with hybridization buffer containing an antisense or a
sense probe with final concentrations of 100 ng/ml for 1 hour. Posthybrid-
ization washes with SSC buffer were done at 5°C higher than the hybrid-
ization temperature. Slides were incubated in blocking buffer (Roche) for
30 minutes, followed by anti-DIG/HRP antibody (Roche) preabsorbed at
1:500 dilution for another 30 minutes in a humidified chamber at room
temperature. The signals were amplified using tyramide signal amplifi-
cation (TSA, 1:50 dilution; PerkinElmer) for 5 minutes in a humidified
chamber. Slides were dehydrated in ethanol and mounted in Vectashield
containing DAPI nuclear stain (Vector Laboratories). Visualization was
performed on a Leica DM5000B microscope.
siRNA-mediated knockdown. The SGHPL-5 cell line was transfected with
either scrambled siRNA or a combination of 4 siRNAs against the lincRNA
evenly spread across the transcript (Supplemental File 7), using Lipofectamine
RNAiMAX according to the manufacturer’s instructions (Invitrogen). siRNA
knockdown was assessed by quantitative RT-PCR on RNA from transfected
cells using primers and a probe (Supplemental File 7) specific for the tran-
script using the Taqman EZ RT-PCR kit (Applied Biosystems). The log
2
fold
change knockdown value was at least –5 in all transfections used.
RNA-Seq and pathway analysis after siRNA-mediated downregulation. RNA-Seq
was performed on 1 untransfected sample and 2 independent transfection
couples consisting of a scrambled control sample and an siRNA-mediated
knockdown sample as described above. Total RNA (1 μg/sample, DNase-treat-
ed, RIN ≥9.8) was subjected to a double round of poly-A mRNA purification,
fragmented, and primed for cDNA library synthesis using the TruSeq RNA
sample preparation kit (FC-122-1001). All procedures were done according to
the manufacturer’s instructions (Illumina). Following validation (Agilent 2100
Bioanalyzer, DNA High Sensitivity) and normalization (AUC 200- to 500-bp
fragments), samples were clustered (TruSeq paired-end cluster kit v3-cBot-
HS, PE-401-3001) followed by paired-end sequencing (100 bp; TruSeq SBS
kit v3-HS 200 cycles, FC-401-3001) on a HiSeq2000. To maximize coverage
from 22 hospitals in the Netherlands and used for genome-wide linkage
analysis, has been described elsewhere (7). We collected blood samples with
informed consent of all affected females and their parents, when available.
We collected blood samples from a monozygotic twin sister pair and their
partners. The twin sister pair was discordant for the HELLP syndrome:
only one developed the disease during pregnancy. The nonaffected twin
sister had two normal pregnancies. We collected buccal swabs from all chil-
dren available to us born from affected pregnancies and, if available, from
nonaffected pregnancies from the same families.
In order for a family to be included, the clinical profiles of the affected
females had to meet the strict criteria: (a) LDH ≥600 IU/l, ASAT ≥70 IU/l, ALAT
≥70 IU/l, and ≤100 platelets × 10
9
/l; (b) at least 2 affected females in each family
(single exception: monozygotic twin sisters); and (c) DNA available from at
least 2 generations. For most families, DNA was available from 3 generations.
All families were of mixed European descent, with no apparent relationship
among families except for family 93113, in which 2 brothers married 2 sisters
from an unrelated family. The daughters born from these couples both devel-
oped HELLP. These double–first cousins were informative for the fact that
the IBD alleles shared were maximal within the minimal critical region. Ethics
committee approval was secured from all participating hospitals. All of the
participants or their legal representatives gave informed consent.
Multipoint linkage analysis with microsatellite markers. We performed nonpara-
metric allele-sharing linkage analysis with 26 microsatellite markers located
on 12q23 between D12S309 and D12S395 on 53 individuals (35 affected
females). The marker alleles in individual samples were analyzed by PCR with
oligonucleotide primers labeled with 6-FAM or HEX. PCR product lengths
were determined by capillary electrophoresis using ABI3100 or ABI3130XL
genetic analyzer (Applied Biosystems) and analyzed using GeneScan or Gen-
emapper (version 4.0; Applied Biosystems). DNA from Centre d’Etude du
Polymorphisme Humain (CEPH) individual 1347/02 was considered the
standard for DNA fragment lengths. Nonparametric lod scores were calcu-
lated using Allegro version 2 (16). Allele frequencies were obtained from the
CEPH genotype database (version 10.0; www.cephb.fr). The order and posi-
tion of the markers were derived from the Decode genetic map (17). Marker,
linkage, and family data are shown in Supplemental File 1.
Identity-by-descent allele-sharing analysis with SNPs and INDEL polymorphism
markers. We designed primers using Primer 3 (http://frodo.wi.mit.edu) or
ExonPrimer (http://ihg.gsf.de/ihg/ExonPrimer.html) to amplify exons of
the 38 known and predicted genes in the regions with linkage. We analyzed
exon fragments by cycle sequencing on an ABI3130XL Genetic Analyzer
(Applied Biosystems). We analyzed the data by Sequencing Analysis (ver-
sion 3.7; Applied Biosystems) or 4Peaks software (http://www.mekentosj.
com/science/4peaks). The SNPs and INDEL polymorphisms we identified
were used for pedigree structure analysis of the regions with nonparamet-
ric lod scores >3 (Supplemental File 2).
Haplotype block analysis. Tag SNPs were designed with Tagger (18) and
sequenced as described above. See Supplemental File 7 for primer sequenc-
es. Case-control association analysis was performed for the familial cohort
studied. Affected females born from affected mothers were considered cases
(homozygous or compound heterozygous patients); affected females born
from nonaffected mothers were considered controls (heterozygous carriers).
We tested 26 singletons (sisters and cousins) with 405 SNP markers. We
used Haploview version 4.2 (19) to evaluate blocks of LD using the “Solid
Spine of LD” algorithm with a minimum D value of 0.8. The Solid Spine
of LD method internal to Haploview defines a block when the first and last
markers are in strong LD with all intermediate markers. Marker settings
were as follows: HW P value, cutoff 1.0; minimum percentage genotype,
100; maximum number Mendelian errors, 1; minor allele frequency, 0.05.
Detailed transcript analysis. From the extravillous trophoblast cell line
SGHPL-5 (11), provided by J. Cartwright (St. George’s University of Lon-
research article
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 11 November 2012 4011
facturer’s instructions. Efficient morpholino delivery was monitored using a
fluorescein-labeled control. Cytotoxicity was observed at morpholino concen-
trations greater than 5 μM. RNA was isolated after 48 hours, followed by quan-
titative RT-PCR experiments for the HELLP lincRNA, HMMR, USP1, NUF2,
ERBB2IP, C12orf48, SMC2, RECQL, and MRE11A and normalized for GAPDH,
all as described above. Data were obtained from 3 independent experiments.
In the invasion assays, morpholinos were delivered in serum-starved SGHPL-5
cells using EndoPorter. 50,000 cells were plated on 100 μl diluted Matrigel-
coated (BD) 8.0-μm cell culture inserts, and the cells invaded for 48 hours.
The insert membranes were fixed, covered in Vectashield with DAPI (Vector
Laboratories), and coverslipped, after which the whole underside membrane
was counted. Data were obtained from 6 independent experiments.
Statistics. Unless otherwise indicated, statistical analyses were performed
using Graphpad Prism software and a 1-sample t test with theoretical mean
of 0. P values less than 0.05 were considered significant. Results are pre-
sented as mean ± SEM.
Study approval. The studies performed on human samples were approved
by the Ethical Committee of the VU University Medical Center and, in the
case of placental tissues, also by the Ethical Committee of the Flevozieken-
huis Almere. Informed consent was obtained from all patients.
Acknowledgments
We are grateful for the collaboration of many colleagues and, most
of all, for the contributions of the HELLP patients and their fami-
lies. We further thank the donors and all participating staff of the
Flevoziekenhuis Almere for the human placental tissue specimens
used in this study. This work was supported by the Netherlands
Organization for Scientific Research (NWO grants no. 950-10-612
and 91611177), European Union (SAFE LSHB-CT-2004-503243),
and Foundation for Translational Research (STR).
Received for publication June 4, 2012, and accepted in revised
form September 13, 2012.
Address correspondence to: Cees B.M. Oudejans, Department of
Clinical Chemistry, VU University Medical Center, De Boelelaan 1117,
1081 HV Amsterdam, The Netherlands. Phone: 31.20.444.3867; Fax:
31.20.444.3895; E-mail: cbm.oudejans@vumc.nl.
with inclusion of low-abundance transcripts (21, 22), each lane contained a
single DNA library. Cluster densities were 623–970 K/mm
2
, q scores (≥Q30)
43.2%–80.5%, and FastQ output 44.44–69.17 gigabase for both forward and
reverse reads. RNA-Seq reads were aligned to the preassembled reference
genome (Illumina iGenome, data source UCSC assembly hg18; June 20, 2011)
using Tophat (version 1.4.0) in combination with Bowtie (version 0.12.5) and
SAMtools (version 0.1.18) using the default settings (23). Transcript assembly,
abundance estimation (defined as fragments per kilobase of exon per mil-
lion fragments mapped; FPKM), and differential expression was performed
by sequential analysis of Tophat output (accepted_hits.bam). For this, tran-
scripts were assembled using Cufflinks (version 1.3.0) under conditions (RABT
assembly; ref. 24) permitting the identification of novel unannotated tran-
scripts (transcripts.gtf) and with correction for fragment bias to account for
biases in library preparation (25). The assemblies to be compared were merged
(Cuffmerge), generating a transcript index (merged.gtf). Subsequently, dif-
ferential analysis of significant changes in transcript expression, splicing and
promoter use was performed (Cuffdiff) in the different transfection couples
(scrambled vs. siRNA-mediated knockdown). All transfected samples were
also compared with the untransfected sample. These data were checked for the
occurrence of the HELLP lincRNA and downregulation of this transcript in
the siRNA-mediated knockdown samples. The transfection couple of scram-
bled and siRNA-mediated knockdown samples with the highest difference
was used in subsequent validation and pathway analysis in combination with
the analyses with the untransfected sample to remove transfection-induced
effects. Genes with significant differential expression were sorted by q value (P
value corrected for FDR) and log
2
(fold change). IPA (build 140500; Ingenu-
ity Systems Inc.) was performed on the gene list with differentially expressed
genes, including the additional selection criteria described above.
Validation experiments by quantitative RT-PCR. Quantitative RT-PCR
using gene expression assays (Applied Biosystems) for MEG8, GRB10,
HMMR, USP1, NUF2, ERBB2IP, C12orf48, SMC2, RECQL, MRE11A, CRYAB,
LOC388564, AZIN1 (transcript 1), and AZIN1 (transcript 2) were per-
formed on an ABI7300. Normalization was done with gene expression
assays for GAPDH.
Morpholino-mediated transcript block and Matrigel invasion assays. Morpholinos
blocking 3 different potential mutation sites (Supplemental File 7) and the
standard negative control morpholino (Gene Tools) were delivered into the
SGHPL-5 cell line using EndoPorter (Gene Tools) according to the manu-
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    • "In the heart, Fendrr (Fetal-lethal noncoding developmental regulatory RNA) is an excellent example for the role of lncRNAs in cardiac development as intraventricular septal heart defects were observed embryonically in Fendrr-deficient mice [55]. Role of other lncRNAs in CVDs is demonstrated by lncRNA MIAT, which is associated with increased risk of myocardial infarction [56]; lncRNA ANRIL is associated with increased risk to coronary heart disease [57] ; lncRNA DBE- T localizes to the facioscapulohumeral muscular dystrophy (FSHD) locus [58]; and a novel lncRNA is identified in association with HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets) [59]. Furthermore, vascular lincRNA-p21 represses proliferation and induces apoptosis in vitro and in vivo in vascular smooth muscle cells [60]. "
    [Show abstract] [Hide abstract] ABSTRACT: Objective . To evaluate the relationship between TGF β signaling and endothelial lncRNA expression. Methods. Human umbilical vein endothelial cell (HUVECs) lncRNAs and mRNAs were profiled with the Arraystar Human lncRNA Expression Microarray V3.0 after 24 hours of exposure to TGF β 1 (10 ng/mL). Results . Of the 30,584 lncRNAs screened, 2,051 were significantly upregulated and 2,393 were appreciably downregulated ( P < 0.05 ) in response to TGF β . In the same HUVEC samples, 2,148 of the 26,106 mRNAs screened were upregulated and 1,290 were downregulated. Of these 2,051 differentially expressed upregulated lncRNAs, MALAT1, which is known to be induced by TGF β in endothelial cells, was the most (~220-fold) upregulated lncRNA. Bioinformatics analyses indicated that the differentially expressed upregulated mRNAs are primarily enriched in hippo signaling, Wnt signaling, focal adhesion, neuroactive ligand-receptor interaction, and pathways in cancer. The most downregulated are notably involved in olfactory transduction, PI3-Akt signaling, Ras signaling, neuroactive ligand-receptor interaction, and apoptosis. Conclusions. This is the first lncRNA and mRNA transcriptome profile of TGF β -mediated changes in human endothelial cells. These observations may reveal potential new targets of TGF β in endothelial cells and novel therapeutic avenues for cardiovascular disease-associated endothelial dysfunction.
    Full-text · Article · Apr 2016
    • "However, the origin of the disease can be traced back to the first trimester fetal placenta. Genome-wide linkage analysis of families with HELLP syndrome identified a region on chromosome 12q23 between PMCH and IGF1 to be associated with the disease [15]. The region contains a lncRNA more than 205 kb in length which is expressed in early placenta extravillous trophoblasts. "
    [Show abstract] [Hide abstract] ABSTRACT: Since decades it has been known that non-protein-coding RNAs have important cellular functions. Deep sequencing recently facilitated the discovery of thousands of novel transcripts, now classified as long noncoding RNAs (lncRNAs), in many vertebrate and invertebrate species. LncRNAs are involved in a wide range of cellular mechanisms, from almost all aspects of gene expression to protein translation and stability. Recent findings implicate lncRNAs as key players of cellular differentiation, cell lineage choice, organogenesis and tissue homeostasis. Moreover, lncRNAs are involved in pathological conditions such as cancer and cardiovascular disease, and therefore provide novel biomarkers and pharmaceutical targets. Here we discuss examples illustrating the versatility of lncRNAs in gene control, development and differentiation, as well as in human disease.
    Full-text · Article · Mar 2016
    • "However, the origin of the disease can be traced back to the first trimester fetal placenta. Genome-wide linkage analysis of families with HELLP syndrome identified a region on chromosome 12q23 between PMCH and IGF1 to be associated with the disease [15]. The region contains a lncRNA more than 205 kb in length which is expressed in early placenta extravillous trophoblasts. "
    [Show abstract] [Hide abstract] ABSTRACT: While the vast majority of the genome is transcribed into RNA, only a small fraction of these transcripts have protein-coding potential. A large fraction of the transcribed RNA belongs to the class known as long non-coding RNAs (lncRNAs). Several recent studies have shown that at least some of these lncRNA transcripts represent functional RNA molecules. LncRNAs can utilize a wide range of mechanisms to regulate the RNA and/or the protein content of a cell on the transcriptional and the post-transcriptional levels. So far, many studies have identified differentially expressed lncRNAs in various physiological contexts, genetic disorders and human diseases. A steadily increasing number of studies could establish functional roles for some of these lncRNAs in developmental processes, cancer and tissue homeostasis. Taken together, these functions provide an additional layer of gene regulation and contribute to the high complexity of physiological and disease-related phenotypes.
    Article · Mar 2016
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