Using RNA sequencing for identifying gene imprinting and random monoallelic expression in human placenta

Figures & data

Figure 1. Schematic view to identify imprinted and monoallelically expressed genes. The pipeline shows the steps involved in analyzing and detecting imprinted and monoallelically expressed genes using RNA-Seq data and R scripts. Arrows show the data flow. Data sources (including RNA-Seq, SNP array data and external sources) are labeled by light gray boxes, while dark gray box shows the final result about imprinted and monoallelically expressed genes. UCSC: University of California Santa Cruz database.¹⁷

Table 1. Candidate imprinted genes after RNA-Seq and exome genotyping

No.	Gene symbol	Gene name	Location	Maternal reads (%)	Paternal reads	p-value	q-value
1	RHOBTB3	Rho-related BTB domain containing 3	5q15	251 (16.2)	1302	4.8e-171	1.2e-167
2	PEG10	paternally expressed 10	7q21.3	20 (3.5)	550	2.1e-135	2.5e-132
3	ZFAT-AS1	ZFAT antisense RNA 1	8q24.22	45 (9.6)	422	7.2e-78	5.8e-75
4	AIM1	absent in melanoma 1	6q21	29 (8.9)	298	2.1e-57	1.3e-54
5	HLA-DRB1	major histocompatibility complex, class II, DR β 1	6p21.32	0 (0)	47	1.4e-14	5.7e-12
6	TLR3	toll-like receptor 3	4q35.1	86 (28.8)	213	1.4e-13	4.7e-11
7	DLG5	discs, large homolog 5 (Drosophila)	10q22.3	316 (66.1)	162	1.7e-12	5.0e-10
8	GRHL1	grainyhead-like 1 (Drosophila)	2p25.1	185 (35.2)	340	1.3e-11	3.4e-09
9	LGALS14	lectin, galactoside-binding, soluble, 14	19q13.2	164 (34.7)	309	2.5e-11	6.0e-09
10	SPTLC3	serine palmitoyltransferase, long chain base subunit 3	20p12.1	149 (34.3)	286	4.9e-11	1.1e-08
11	CEP170	centrosomal protein 170kDa	1q43	0 (0)	33	2.3e-10	4.7e-08
12	ABP1	amiloride binding protein 1	7q36.1	524 (60.0)	349	3.5e-09	6.4e-07
13	AKAP7	A kinase (PRKA) anchor protein 7	6q23.2	45 (90.0)	5	4.2e-09	7.0e-07
14	ZFAT	zinc finger and AT hook domain containing	8q24.22	40 (26.3)	112	4.4e-09	7.0e-07
15	PAPPA2	pappalysin 2	1q25.2	144 (69.6)	63	1.8e-08	2.7e-06
16	CHD1	chromodomain helicase DNA binding protein 1	5q21.1	13 (20.0)	52	1.2e-06	1.6e-04
17	PTGFRN	prostaglandin F2 receptor inhibitor	1p13.1	35 (28.7)	87	2.8e-06	3.7e-04
18	BCLAF1	BCL2-associated transcription factor 1	6q23.3	130 (66.3)	66	5.7e-06	7.2e-04
19	SEC16A	SEC16 homolog A (S. cerevisiae)	9q34.3	14 (21.9)	50	7.1e-06	8.5e-04
20	KIAA1191	KIAA1191	5q35.2	18 (100.0)	0	7.6e-06	8.7e-04
21	CLASP1	cytoplasmic linker associated protein 1	2q14.3	1 (5.0)	19	4.0e-05	4.4e-03
22	LGALS8	lectin, galactoside-binding, soluble, 8	1q43	95 (66.9)	47	6.9e-05	7.2e-03
23	HEG1	heart development protein with EGF-like domains 1	3q21.2	130 (64.0)	73	7.7e-05	7.7e-03
24	KIAA1919	KIAA1919	6q21	16 (25.4)	47	1.2e-04	1.1e-02
25	CEP63	centrosomal protein 63kDa	3q22.2	20 (27.8)	52	2.1e-04	1.9e-02
26	IFI30	interferon, gamma-inducible protein 30	19p13.11	350 (57.6)	258	2.2e-04	1.9e-02
27	SYNE2	spectrin repeat containing, nuclear envelope 2	14q23.2	697 (45.3)	842	2.4e-04	2.0e-02
28	ELMO2	engulfment and cell motility 2	20q13.12	13 (100.0)	0	2.4e-04	2.0e-02
29	OR51B5	olfactory receptor, family 51, subfamily B, member 5	11p15.4	16 (94.1)	1	2.7e-04	2.1e-02
30	CASC5	cancer susceptibility candidate 5	15q15.1	3 (12.5)	21	2.8e-04	2.1e-02
31	OVCH2	ovochymase 2 (gene/pseudogene)	11p15.4	35 (76.1)	11	5.4e-04	4.0e-02
32	ANLN	anillin, actin binding protein	7p14.2	9 (22.5)	31	6.8e-04	4.9e-02
33	KIAA1551	KIAA1551	12p11.21	270 (57.9)	196	7.0e-04	5.0e-02

Maternal/paternal reads (%) – number of RNA-Seq reads that overlapped with SNPs in the respective gene and were previously determined to correspond to either maternal or paternal allele, and the proportion (%) of maternal reads from the total number of reads; p-value – binomial test p-value; q-value – FDR-corrected p-value; q-value < 0.05 was considered significant to detect allele-specific expression of a gene.

Table 2. Imprinted and monoallelically expressed genes found in the current study

No.	Gene	Heterozygous SNPs	Significant ASE-SNPs	Significant ASE%	Imprinting or monoallelic expression	Previously reported imprinting ^Reference
1	RHOBTB3	25	25	100.0%	IP	Paternal expression ^11
2	ZFAT-AS1	10	10	100.0%	IP	Paternal expression ^9
3	BCLAF1	4	4	100.0%	RM
4	ZFAT	20	19	95.0%	RM	Paternal expression ^9
5	IFI30	26	23	88.5%	RM
6	PEG10	17	15	88.2%	IP	Paternal expression^50,51
7	PAPPA2	51	42	82.4%	IM
8	LGALS14	16	13	81.2%	IP
9	AIM1	35	28	80.0%	IP	Paternal expression ^19
10	LGALS8	114	91	79.8%	IM
11	SPTLC3	47	37	78.7%	IP
12	ABP1	39	30	76.9%	RM

Heterozygous SNPs – number of placental transcriptome heterozygous SNPs where child has at least 1% minor allele frequency (MAF) from dbSNP database and 3% MAF from RNA-Seq, also detecting the same alleles as in dbSNP database (see Materials and Methods for more details about this filtering step); Significant ASE-SNPs – number of heterozygous SNPs in children showing statistically significant allele-specific expression (ASE) in placental transcriptome; Significant ASE% – proportion of SNPs with statistically significant ASE; and Previously reported imprinting – previously reported imprinted and monoallelically expressed genes, showing the parental specificity in gene expression.

IP – Imprinted and paternal expression, IM – Imprinted and maternal expression and RM – Random monoallelic expression.

Table 3. Summary of detected imprinted and monoallelically expressed genes

Gene symbol	Gene name	Parental preference	Main function	(Patho)physiological associations ^Reference
I. Cellular apoptosis and tissue development
PEG10	Paternally expressed 10	Paternal	Antiapoptotic factor	· Underexpressed in low birth weight babies^52,53 · Overexpressed in hepatocellular carcinomas^54
AIM1	Antigen or absent in melanoma 1	Paternal	Factor regulating the function of endoplasmic reticulum and cellular differentiation	· Human pigmentation variation^55 · Mutation in oculocutaneous albinism^56 · Metabolic activity of melanomas^57 and prostate cancer^58 · Biological behavior of smooth muscle cells of ductus arteriosus (For a review see ref. 59)
BCLAF1	Bcl^-2 associated transcription factor 1	Random monoallelic	Apoptotic factor and transcriptional repressor	· Suppressed function in carcinomas^21,22 and myelomas^60 · Genetic polymorphisms in increased risk of B-cell lymphomas^61,62 · Developmental processes of tissues and T-cell homeostasis independently from apoptosis^63 · Apoptosis associated with differentiation of spermatozoa^64
PAPPA2	Pappalysin 2; pregnancy associated plasma protein A2	Maternal	Cleaves IGFBP5 and thereby regulates bioavailability of IGFs	· Elevated expression in placenta in preeclampsia (For a review see ref. 31)
II. Inflammation and modulation of immune system
IFI30	INF-γ-inducible protein 30	Random monoallelic	Lysosomal thiol reductase involved in MHC-II restricted antigen processing and presenting	· Involved in central and peripheral CD4+ T-cell tolerance^33,35 · Regulation of inflammation, hyperglycemia and obesity^65,66
LGALS14	Galactoside-binding soluble lectin 14	Paternal	Differentiation, adhesion, growth and apoptosis	· Function not studied for member 14, but predominantly expressed in placenta^36
LGALS8	Galactoside-binding soluble lectin 8	Maternal	Cytosolic lectin detecting bacterial invasion	· Expressed in villous and extra-villous trophoblasts^39
ZFAT; ZFAT-AS1	Zinc finger and AT hook domain containing and ZFAT antisense RNA 1	Paternal for ZFAT-AS1 and random monoallelic for ZFAT	Zn-finger transcription factor involved in apoptosis and cell survival of immune cells and trophoblasts	· Necessary in processes of embryonic cell survival, including placental angiogenesis^9
III. Proteins mediating metabolic processes
ABP1	Amiloride binding protein 1 (amine oxidase copper-containing)	Random monoallelic	Metal-binding membrane glycoprotein with oxidative deaminase properties	· In the complex of Na^+ cannels in seminal vesicles, colon and lung pneumocytes^67 · Target for diuretic amiloride · Higher expression in inflammatory bowel disease^68
SPTLC3	Serine palmitoyltransferase, long chain base subunit 3	Paternal	Synthesis of sphingolipids that mediate a range of cellular signaling functions	· Highly expressed in placenta^69 · Sphingolipid-associated disorders in neurological, psychiatric and metabolic diseases^71
IV. Regulating cell cycle
RHOBTB3	Rho-related BTB domain containing 3	Paternal	Rho GTPase, a regulator of cytoskeleton and involved in cell cycle regulation and vesicle transport	· Involved in carcinogenic processes and cellular homeostasis^71 · By inhibiting the serotonin receptor degradation, contributes to variety of central nervous functions^72 · Involved in embryonal development and function of innate immune system^73

IGF – insulin-like growth factor, IGFBP5 – IGF binding protein 5, INF-γ - interferon gamma and IUGR – intrauterine growth restriction.

Figure 2. Allele-specific expression in different placental samples. Each grid shows the allele-specific expression for each of the 12 imprinted and monoallelically expressed genes in different placental samples. The X-axis is the genomic position of the SNP and the Y-axis shows the percentage of maternal reads, with 100% refers to complete maternal expression, 0% corresponds to complete paternal expression and 50% refers to perfect biallelic expression. Statistically significant results are shown in red (maternal expression) and blue (paternal expression). From all non-significant results (depicted in black), only those are shown where the total number of RNA-Seq reads was at least 10. P001 - P014 are family trios analyzed.

Figure 3. Density curve for gene expression. Red lines show the distribution of expression levels for all 12 imprinted and monoallelically expressed genes detected in the current study (see Table 2 for details). Median expression of all genes is normalized to 1 (blue line).

Figure 4. Genome visualization of the imprinted and monoallelically expressed genes. Previously reported placental imprinted genes are in black (Table S1), candidate imprinted genes are marked in blue (Table 1) and imprinted and monoallelically expressed genes found in our analysis are marked in red (Table 2). This image was generated using Idiographica web service.⁴⁹

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