Analysis of HELLP families reveals linkage to an intergenic region on 12q23.
Using the original cohort of families with the HELLP syndrome (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), respectively. 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 linkage analysis using Smnallele
confirmed the 4-marker region between PAH and D12S1647 (lod and NPL scores >3; Figure A) and indicated recessive inheritance (in descending order: Smnallele
; ref. 10
). Pedigree analysis narrowed this region to 2 minimal 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 (cousins and their mothers) was restricted to the first region near D12S1030 (Supplemental File 1; supplemental material available online with this article; doi:
Identification of the HELLP locus on chromosome 12q23.2.
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, 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 polymorphisms (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 B 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 characteristic 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. Phenotypically affected females born from phenotypically affected mothers were genetically considered cases, as the disease genotypes of the former are homozygous or compound heterozygous; phenotypically affected females born from nonaffected mothers were considered controls, as the former are unaffected, heterozygous carriers. 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 intergenic region between C12orf48 and IGF1 on 12q23.2 (Figure C), a finding that was confirmed to be significant by permutation testing (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 nonaffected 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 phenotype (Figure ).
Parent-child segregation of 12q23.2 region in monozygotic twin sisters discordant for HELLP.
The HELLP transcript is a lincRNA expressed in extravillous trophoblasts.
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 cellular 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 ). By FISH, expression was found to be nuclear, perinuclear, and cytoplasmic, both in vitro — in the extravillous trophoblast cell line (SGHPL-5) used for its identification (Figure , A–C) — and in vivo (i.e., first trimester placenta tissue), whereas no signal was detected using a sense probe as negative control (Figure D). In the anchoring villi of human first-trimester placenta tissue, (peri)nuclear expression was restricted to extravillous trophoblast cells and their precursors (i.e., column extravillous cytotrophoblasts; Figure E). In first-trimester placentabed 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 trophoblast surrounding maternal spiral arteries (Figure , F and G). Comparison with the transcriptome assemblies of 17 adult tissues (in addition to placenta) in the human lincRNA catalog (
) indicated that the HELLP lincRNA was preferentially, if not exclusively, expressed in the placenta (Supplemental File 3).
Location of the HELLP lincRNA on chromosome 12q23, identified by overlapping strand-specific PCR amplifications supplemented by RACE experiments to identify the 5′ and 3′ ends.
Localization of the HELLP lincRNA.
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 RNA-sequence (RNA-Seq) analysis after siRNA-mediated downregulation of the HELLP transcript in SGHPL-5 cells. The differential 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 validation analysis, the following selection criteria were used: q value threshold, <0.05; log2 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 quantitative 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 control. One of the upregulated genes, AZIN1, consists of 2 transcripts of which only 1 is upregulated, while the other is downregulated but with a log2 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 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 ). 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).
Top 10 networks from ingenuity pathway analysis
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 (C→T at position 101,120,459), HAPLO378 (A→G at position 101,319,702), and HAPLO215Rev (G→C at position 101,241,930), were not found in 200 control chromosomes of nonaffected individuals of similar race and therefore qualified as mutations (Figure A). 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 B). Using the combination of HAPLO378 with HAPLO215Rev morpholinos (being compound 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 followed 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 C). 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 combination at 2.5 μM each. Furthermore, HAPLO215Rev showed a clear trend toward a decrease in invaded cells (P = 0.0645; Figure D).
Blocking potential mutation sites decreases extravillous trophoblast invasion.