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  • Technical advance
  • Open Access
  • Open Peer Review

Discovery of rare ancestry-specific variants in the fetal genome that confer risk of preterm premature rupture of membranes (PPROM) and preterm birth

  • 1,
  • 2,
  • 3,
  • 1,
  • 3,
  • 4, 5,
  • 1, 3 and
  • 1, 3Email author
BMC Medical Genetics201819:181

https://doi.org/10.1186/s12881-018-0696-4

  • Received: 5 June 2018
  • Accepted: 25 September 2018
  • Published:
Open Peer Review reports

Abstract

Background

Preterm premature rupture of membranes (PPROM) is the leading identifiable cause of preterm birth, a complication that is more common in African Americans. Attempts to identify genetic loci associated with preterm birth using genome-wide association studies (GWAS) have only been successful with large numbers of cases and controls, and there has yet to be a convincing genetic association to explain racial/ethnic disparities. Indeed, the search for ancestry-specific variants associated with preterm birth has led to the conclusion that spontaneous preterm birth could be the consequence of multiple rare variants. The hypothesis that preterm birth is due to rare genetic variants that would go undetected in standard GWAS has been explored in the present study. The detection and validation of these rare variants present challenges because of the low allele frequency. However, some success in the identification of fetal loci/genes associated with preterm birth using whole genome sequencing and whole exome sequencing (WES) has recently been reported. While encouraging, this is currently an expensive technology, and methods to leverage the sequencing data to quickly identify and cost-effectively validate variants are needed.

Methods

We developed a WES data analysis strategy based on neonatal genomic DNA from PPROM cases and term controls that was unencumbered by preselection of candidate genes, and capable of identifying variants in African Americans worthy of focused evaluation to establish statistically significant associations.

Results

We describe this approach and the identification of damaging nonsense variants of African ancestry in the DEFB1 and MBL2 genes that encode anti-microbial proteins that presumably defend the fetal membranes from infectious agents. Our approach also enabled us to rule out a likely contribution of a predicted damaging nonsense variant in the METTL7B gene.

Conclusions

Our findings support the notion that multiple rare population-specific variants in the fetal genome contribute to preterm birth associated with PPROM.

Keywords

  • Preterm premature rupture of membranes
  • Defensin β1
  • Mannose binding lectin-2
  • Methyltransferase like 7B
  • Whole exome sequencing

Background

There are significant disparities in preterm birth rates in the United States, with African Americans experiencing an increased burden [1, 2]. Delivery after preterm premature rupture of the membranes (PPROM) is the leading identifiable cause of spontaneous preterm birth, and PPROM is more common in African-Americans. PPROM is believed to be caused, in part, by infection and inflammation, presumably incited by microbes ascending from the vagina, resulting in the release of pro-inflammatory cytokines and the activation of matrix-degrading proteases that breakdown the collagens that give the fetal membranes their tensile strength, resulting in unscheduled rupture [35].

Twin studies have revealed that both fetal and maternal genetic factors contribute to gestational age at delivery, but there is uncertainty about the roles played by specific fetal and maternal genes. Attempts to identify genetic loci associated with gestational age at delivery and preterm birth using genome-wide association studies (GWAS) have only been successful with large numbers of cases and controls (see reference [5] for a review). Moreover, these studies have not identified genes that could account for increased preterm births in African-Americans. Efforts to identify ancestry-specific variants using GWAS approaches have led to the conclusion that spontaneous preterm birth is likely to be the consequence of multiple common variants or rare variants not easily detected by GWAS [6]. This is not a surprising conclusion since GWAS is based the “common disease-common variant” hypothesis, positing that a significant proportion of the variance of common diseases are attributable to DNA variants that are present in > 1–5% of the population, and that there are many of these DNA variants, each contributing a small amount to the total risk to a particular disease [5].

As noted above, an alternative hypothesis is that diseases are associated with rare genetic variants that have relatively larger effect sizes that would go undetected in standard GWAS. The detection and validation of these rare variants presents challenges because of the low allele frequency. Some success in the identification of fetal loci/genes associated with preterm birth using whole genome sequencing and whole exome sequencing (WES) has recently been reported [79]. While encouraging, this is currently expensive technology and methods to leverage the sequencing data to quickly identify and cost-effectively validate variants are needed.

We recently pursued the approach of searching for rare variants in fetal genes that could contribute to risk of PPROM, employing WES to identify the burden of damaging mutations in African-American fetal (neonatal) samples [8, 9]. In one of our studies, our analysis of the WES data focused on genes that either negatively regulate the innate immune response or which encode proteins that protect the host against microbes and their noxious products. Rather than utilizing a prospective candidate gene filter, we decided to develop a WES analysis plan that was not encumbered by preselection and capable of identifying rare damaging variants in African-Americans worth focused evaluation to establish statistically significant association and the mechanism(s) underlying the mutation effect. We hoped to establish a cost effective simple process that could be applied to modest sample sizes.

Methods

Subjects

The subjects in the discovery WES (76 PPROM cases and 43 term controls) and initial confirmatory targeted genotyping for DEFB1 and MBL2 nonsense variants (188 PPROM cases and 175 term controls) have been previously described [8, 9]. The METTL7B SNPs were evaluated with the WES cohort. They were neonates born of self-reported African-American women. Term controls consisted of neonates born from uncomplicated singleton pregnancies (> 37 weeks gestation). PPROM cases were from singleton pregnancies prior to 37 weeks of completed gestation. The diagnosis of membrane rupture was based on pooling of amniotic fluid in the vagina, amniotic fluid ferning patterns and a positive nitrazine test. Women with multiple gestations, fetal anomalies, trauma, connective tissue diseases and medical complications of pregnancy requiring induction of labor were excluded as previously described [8, 9].

The previously published analysis of the DEFB1 and MBL2 nonsense variants included the WES cohort and initial confirmatory targeted genotyping cohort described above [9]. In the present study the METTL7B SNPs were evaluated with the original WES cohort. In addition, we performed targeted genotyping for the DEFB1 and MBL2 nonsense variants on 119 PPROM cases and 199 controls not previously reported. The subjects were recruited from the same populations as the previously reported cohorts using identical inclusion and exclusion criteria. Ninety-four of these PPROM cases and 94 term controls were used for genotyping METTL7B SNPs.

Whole exome sequencing and genotyping

The methods used for WES (50-100X coverage) and analysis of the sequencing data have been described in detail in previous publications [8, 9]. With the number of PPROM cases (76), we had 78% power to detect variants with a minor allele frequency of 0.005. Targeted genotyping was performed on the Agena (previously Sequenom) MassArray iPLEX platform [8, 9]. The primers used for METTL7B genotyping are presented in Additional file 1: Table S1. Only high confidence genotype calls were included in the analysis.

Estimation of African ancestry

To reduce the potential risk that population stratification biased the genetic association tests, the percent African ancestry of the PPROM neonates and term control neonates was determined using ancestry-informative markers as previously described [8, 9]. No significant differences in the percentage of African ancestry were found between PPROM cases and term controls (Means +/− S.D.; West African ancestry: PPROM cases: 0.695 +/− 0.073 (mean + S.D.); Term controls 0.698 +/− 0.087 (p > 0.10)) [9].

RT-PCR analysis

Detection of DEFB1 and METTL7B transcripts in fetal membrane RNA (1 μg) was accomplished by PCR as previously described [8]. The primers used for DEFB1 transcript amplification were forward 5’-CTGAAATCCTGGGTGTTGCC-3′ and reverse 5’-CTTCTGGTCACTCCCAGCTC-3′. The primers used for METTL7B transcript amplification were forward 5’-ACCTGCCTAGACCCAAATCC-3′ and reverse 5′- TTATTTGACAGCCTTTCCCATGA-3′. In both cases, PCR was run for 40 cycles and amplified bands were sequence verified to be the cognate transcripts.

Selection strategy

We developed the following simple screening method for analyzing the WES data: 1) Identify predicted damaging nonsense variants (gnomAD “high confidence”) present only in PPROM cases in the WES discovery panel; 2) Validate the nonsense variants by Sanger sequencing; 3) Verify it is a rare variant (minor allele frequency < 0.01) based on the genome aggregation database (gnomAD: http://gnomad.broadinstitute.org/); 4) Determine whether the variant/mutation is of African ancestry using a public database (gnomAD); 5) Determine whether the gene is under selective pressure, consistent with an essential role in a biological or pathophysiological process, by a literature review (PubMed: https://www.ncbi.nlm.nih.gov/pubmed/); 6) Assess whether heterozygous variants could potentially cause a biological effect by altering the expression level or activity of mature protein; 7) Determine whether the gene harboring the nonsense variant is expressed in fetal membranes; 8) Evaluate whether the gene could play a role in the existing pathophysiological concepts of PPROM from the literature; and 9) Conduct follow-up genotyping of the nonsense variant in independent cohorts to test the association of the identified variant with PPROM.

Statistical analysis

The minor allele frequencies for the DEFB1 and MBL2 nonsense variants examined in this report would require a very large number of PPROM cases and controls for an association study to achieve a power of 0.8 and a p value = 0.05. Therefore, we combined all WES and genotyping data reported previously [9] with the results of the genotyping of the additional subjects for each nonsense variant in the analysis. Finding no genetic association with these samples sizes cannot rule out an association. However, the discovery of significant associations, albeit in a study of limited power, does not negate the findings, with the caveat that significant findings from low powered studies may not always replicate.

Associations were examined for statistical significance using Fisher’s Exact test (1-tailed) to determine whether the nonsense variant was overrepresented in PPROM cases. Nominal p values are reported. Correcting for multiple tests (Bonferroni adjustment) a, p value of < 0.017 would be considered the threshold for statistical significance, which was met for the DEFB1 and MBL2 nonsense variants studied.

Results

We detected more than 800 different nonsense variants (stop gain, stop loss, and start loss) in the discovery WES sample of PPROM cases and term controls, approximately 33% of which were unique to PPROM, the majority of which occurred in only one PPROM case, and 30% of the variant types were unique to term controls, with the majority occurring in one term control (Table 1) The remaining approximately one third of the nonsense variants occurred both in PPROM cases and controls, and not unexpectedly were nonsense variants with the highest allele frequency, suggesting that these variants might be tolerated and do not contribute to PPROM risk. Most of these nonsense variants have been previously detected in the human genome. More than 1400 coding sequence frameshift variants and splicing variants, predicted to be or possibly damaging were detected. Since a number of these variants were not previously known, it is uncertain whether they reflect sequencing errors in the WES. We suspect the latter since Sanger sequencing of a number of the DNA samples failed to confirm frameshift mutations. Consequently, we did not include the predicted damaging frameshift and splicing mutations in our screening paradigm.
Table 1

Nonsense Variants in PPROM Cases Identified by WES

Gene

Transcript

dbSNP141

Chr

Start

End

Ref

Obs

Casesa

Controlsa

One variant allele in PPROM Cohort

 ABCA7

NM_019112.3:p.Arg285a/c.853A > T

rs77403558

chr19

1043395

1043395

A

T

0,1,1

0,0,0

 ABCB5

NM_001163941.1:p.Lys626a/c.1876A > T

rs76179099

chr7

20725325

20725325

A

T

0,1,1

0,0,0

 ABCB5

NM_001163941.1:p.Gln1195a/c.3583C > T

rs146527949

chr7

20795056

20795056

C

T

0,1,1

0,0,0

 ABCC3

NM_003786.3:p.Gln132a/c.394C > T

rs201830141

chr17

48734452

48734452

C

T

0,1,1

0,0,0

 ACAD11

NM_032169.4:p.Glu13a/c.37G > T

rs151048899

chr3

132378559

132378559

C

A

0,1,1

0,0,0

 ACOXL

NM_001142807.1:p.Gly577a/c.1729G > T

rs189429375

chr2

111875379

111875379

G

T

0,1,1

0,0,0

 ACTN2

NM_001103.3:p.Glu120a/c.358G > T

 

chr1

236882310

236882310

G

T

0,1,1

0,0,0

 ADAMTS14

NM_139155.2:p.Arg317a/c.949C > T

rs199886417

chr10

72489128

72489128

C

T

0,1,1

0,0,0

 AHCTF1

NM_015446.4:p.Ser1120a/c.3359C > A

 

chr1

247030561

247030561

G

T

0,1,1

0,0,0

 ALDH3B1

NM_000694.3:p.Trp135a/c.405G > A

rs375063489

chr11

67786650

67786650

G

A

0,1,1

0,0,0

 ANGPTL7

NM_021146.3:p.Trp188a/c.563G > A

rs145750805

chr1

11253722

11253722

G

A

0,1,1

0,0,0

 ANKRD65

NM_001243535.1:p.Trp108a/c.323G > A

 

chr1

1354816

1354816

C

T

0,1,1

0,0,0

 AP3B1

NM_003664.4:p.Arg407a/c.1219C > T

 

chr5

77461445

77461445

G

A

0,1,1

0,0,0

 AP5Z1

NM_014855.2:p.Trp441a/c.1322G > A

rs373919408

chr7

4827275

4827275

G

A

0,1,1

0,0,0

 ATP2C2

NM_001286527.2:p.Tyr106a/c.318C > G

 

chr16

84438841

84438841

C

G

0,1,1

0,0,0

 AZU1

NM_001700.3:p.Arg236a/c.706C > T

rs112572343

chr19

831827

831827

C

T

0,1,1

0,0,0

 BCL2L12

NM_138639.1:p.Tyr44a/c.132 T > G

rs141156787

chr19

50169212

50169212

T

G

0,1,1

0,0,0

 BCLAF1

NM_014739.2:p.Arg298a/c.892C > T

rs138333275

chr6

136599127

136599127

G

A

0,1,1

0,0,0

 BPIFB6

NM_174897.2:p.Tyr434a/c.1302C > A

rs140595029

chr20

31631146

31631146

C

A

0,1,1

0,0,0

 C12orf40

NM_001031748.2:p.Gln568a/c.1702C > T

rs140530325

chr12

40114796

40114796

C

T

0,1,1

0,0,0

 C12orf42

NM_001099336.2:p.Glu13a/c.37G > T

rs202081871

chr12

103872168

103872168

C

A

0,1,1

0,0,0

 C12orf56

NM_001170633.1:p.Lys269a/c.805A > T

rs201295265

chr12

64712444

64712444

T

A

0,1,1

0,0,0

 C15orf32

NM_153040.2:p.Lys30a/c.88A > T

rs115999940

chr15

93015466

93015466

A

T

0,1,1

0,0,0

 C18orf54

NM_001288980.1:p.Ser530a/c.1589C > A

rs148065410

chr18

51904603

51904603

C

A

0,1,1

0,0,0

 C20orf173

NM_001145350.1:p.Arg36a/c.106C > T

rs141795719

chr20

34117097

34117097

G

A

0,1,1

0,0,0

 C20orf78

NM_001242671.1:p.Trp115a/c.345G > A

rs146528664

chr20

18790531

18790531

C

T

0,1,1

0,0,0

 C9orf50

NM_199350.3:p.Gln406a/c.1216C > T

rs374957154

chr9

132374706

132374706

G

A

0,1,1

0,0,0

 CACNA1A

NM_023035.2:p.Arg1918a/c.5752C > T

rs16044

chr19

13325415

13325415

G

A

0,1,1

0,0,0

 CAMK4

NM_001744.4:p.Glu439a/c.1315G > T

 

chr5

110820057

110820057

G

T

0,1,1

0,0,0

 CAPN11

NM_007058.3:p.Gln133a/c.397C > T

rs189429774

chr6

44137700

44137700

C

T

0,1,1

0,0,0

 CARD6

NM_032587.3:p.Leu560a/c.1679 T > G

rs150487186

chr5

40853113

40853113

T

G

0,1,1

0,0,0

 CCDC153

NM_001145018.1:p.Arg42a/c.124C > T

rs77842401

chr11

119065645

119065645

G

A

0,1,1

0,0,0

 CCDC168

NM_001146197.1:p.Tyr6396a/c.19188 T > G

rs73587211

chr13

103383859

103383859

A

C

0,1,1

0,0,0

 CCDC3

NM_031455.3:p.Ser188a/c.563C > A

rs150029612

chr10

12940666

12940666

G

T

0,1,1

0,0,0

 CCDC57

NM_198082.2:p.Arg676a/c.2026C > T

rs201336748

chr17

80121090

80121090

G

A

0,1,1

0,0,0

 CCDC60

NM_178499.3:p.Arg520a/c.1558C > T

rs78597191

chr12

119978425

119978425

C

T

0,1,1

0,0,0

 CCT8L2

NM_014406.4:p.Trp320a/c.959G > A

rs144853652

chr22

17072482

17072482

C

T

0,1,1

0,0,0

 CD1A

NM_001763.2:p.Arg249a/c.745C > T

rs149659983

chr1

158226716

158226716

C

T

0,1,1

0,0,0

 CEP135

NM_025009.4:p.Gln824a/c.2470C > T

 

chr4

56876034

56876034

C

T

0,1,1

0,0,0

 CETP

NM_000078.2:p.Glu133a/c.397G > T

 

chr16

57003551

57003551

G

T

0,1,1

0,0,0

 CFHR4

NM_001201550.2:p.Arg41a/c.121A > T

rs199547603

chr1

196871610

196871610

A

T

0,1,1

0,0,0

 CHD1L

NM_004284.4:p.Arg611a/c.1831C > T

 

chr1

146756149

146756149

C

T

0,1,1

0,0,0

 CLEC2A

NM_001130711.1:p.Trp137a/c.411G > A

rs142033208

chr12

10066279

10066279

C

T

0,1,1

0,0,0

 CLEC4A

NM_016184.3:p.Trp176a/c.528G > A

rs115176426

chr12

8289461

8289461

G

A

0,1,1

0,0,0

 COL6A5

NM_001278298.1:p.Gln1184a/c.3550C > T

rs115380050

chr3

130114290

130114290

C

T

0,1,1

0,0,0

 CPA3

NM_001870.2:p.Arg178a/c.532C > T

rs145845146

chr3

148597632

148597632

C

T

0,1,1

0,0,0

 CPA6

NM_020361.4:p.Arg311a/c.931C > T

rs139145929

chr8

68346383

68346383

G

A

0,1,1

0,0,0

 CPO

NM_173077.2:p.Trp102a/c.305G > A

rs138166151

chr2

207823062

207823062

G

A

0,1,1

0,0,0

 CTSV

NM_001201575.1:p.Arg96a/c.286C > T

 

chr9

99799644

99799644

G

A

0,1,1

0,0,0

 CYP1A2

NM_000761.4:p.Tyr495a/c.1485C > A

rs143193369

chr15

75047363

75047363

C

A

0,1,1

0,0,0

 DACT2

NM_001286351.1:p.Ser264a/c.791C > G

 

chr6

168694817

168694817

G

C

0,1,1

0,0,0

 DCDC5

NM_020869.3:p.Arg864a/c.2590C > T

rs200380430

chr11

30900214

30900214

G

A

0,1,1

0,0,0

 DCHS2

NM_017639.3:p.Arg1343a/c.4027C > T

rs150179829

chr4

155226252

155226252

G

A

0,1,1

0,0,0

 DEFB1

NM_005218.3:c.111C>A

rs5743490

chr8

6728299

6728299

G

T

0,1,1

0,0,0

 DLEC1

NM_007337.2:p.Cys1696a/c.5088C > A

rs201487599

chr3

38163847

38163847

C

A

0,1,1

0,0,0

 DNAH10

NM_207437.3:p.Arg3898a/c.11692C > T

 

chr12

124411308

124411308

C

T

0,1,1

0,0,0

 DNAH14

NM_001373.1:p.Glu19a/c.55G > T

rs61745064

chr1

225140459

225140459

G

T

0,1,1

0,0,0

 DRD4

NM_000797.3:p.Glu62a/c.184G > T

 

chr11

637488

637488

G

T

0,1,1

0,0,0

 DTHD1

NM_001170700.2:p.Trp678a/c.2034G > A

rs149895631

chr4

36345134

36345134

G

A

0,1,1

0,0,0

 EBLN2

NM_018029.3:p.Tyr164a/c.492 T > G

rs2231925

chr3

73111724

73111724

T

G

0,1,1

0,0,0

 ECHDC2

NM_001198961.1:p.Trp11a/c.33G > A

rs368731634

chr1

53387313

53387313

C

T

0,1,1

0,0,0

 EDDM3A

NM_006683.4:p.Arg43a/c.127C > T

rs138978934

chr14

21215866

21215866

C

T

0,1,1

0,0,0

 EFCAB13

NM_152347.4:p.Arg236a/c.706C > T

rs78865644

chr17

45438788

45438788

C

T

0,1,1

0,0,0

 EFCAB13

NM_152347.4:p.Lys433a/c.1297A > T

rs74969489

chr17

45452257

45452257

A

T

0,1,1

0,0,0

 EGF

NM_001963.4:p.Gln1095a/c.3283C > T

rs138244768

chr4

110925770

110925770

C

T

0,1,1

0,0,0

 EIF3J

NM_003758.3:p.Glu192a/c.574G > T

 

chr15

44852449

44852449

G

T

0,1,1

0,0,0

 ELOVL5

NM_001242830.1:p.Gly246a/c.736G > T

rs41273878

chr6

53133964

53133964

C

A

0,1,1

0,0,0

 ELOVL5

NM_001242828.1:p.Gln102a/c.304C > T

rs150583340

chr6

53152683

53152683

G

A

0,1,1

0,0,0

 ELP4

NM_001288725.1:p.Gln385a/c.1153C > T

 

chr11

31784965

31784965

C

T

0,1,1

0,0,0

 ENGASE

NM_001042573.2:p.Arg352a/c.1054C > T

rs149186913

chr17

77079117

77079117

C

T

0,1,1

0,0,0

 EOGT

NM_001278689.1:p.Lys188a/c.562A > T

rs116711473

chr3

69053587

69053587

T

A

0,1,1

0,0,0

 EPB41L4A

NM_022140.3:p.Arg348a/c.1042C > T

rs368151776

chr5

111570376

111570376

G

A

0,1,1

0,0,0

 EVC2

NM_147127.4:p.Lys342a/c.1024A > T

 

chr4

5664955

5664955

T

A

0,1,1

0,0,0

 EVPL

NM_001988.2:p.Gln938a/c.2812C > T

rs151046085

chr17

74006474

74006474

G

A

0,1,1

0,0,0

 EVPLL

NM_001145127.1:p.Trp209a/c.627G > A

rs182498101

chr17

18286454

18286454

G

A

0,1,1

0,0,0

 EYS

NM_001292009.1:p.Trp2090a/c.6270G > A

 

chr6

64940639

64940639

C

T

0,1,1

0,0,0

 FAM179A

NM_199280.2:p.Arg162a/c.484C > T

rs183676260

chr2

29225458

29225458

C

T

0,1,1

0,0,0

 FAM187B

NM_152481.1:p.Trp231a/c.693G > A

rs35001809

chr19

35718891

35718891

C

T

0,1,1

0,0,0

 FAM200A

NM_145111.3:p.Leu61a/c.182 T > G

 

chr7

99145849

99145849

A

C

0,1,1

0,0,0

 FAM227B

NM_152647.2:p.Arg5a/c.13C > T

rs140471517

chr15

49907356

49907356

G

A

0,1,1

0,0,0

 FAM60A

NM_001135811.1:p.Ser70a/c.209C > A

 

chr12

31448187

31448187

G

T

0,1,1

0,0,0

 FASTKD1

NM_024622.4:p.Ser768a/c.2303C > G

rs34291832

chr2

170387886

170387886

G

C

0,1,1

0,0,0

 FBXL21

NM_012159.4:p.Trp72a/c.215G > A

rs148275750

chr5

135272498

135272498

G

A

0,1,1

0,0,0

 FBXO48

NM_001024680.1:p.Glu134a/c.400G > T

rs148116960

chr2

68691409

68691409

C

A

0,1,1

0,0,0

 FCRL6

NM_001004310.2:p.Gln406a/c.1216C > T

 

chr1

159785362

159785362

C

T

0,1,1

0,0,0

 FDXR

NM_001258013.2:p.Gln66a/c.196C > T

rs187001043

chr17

72868325

72868325

G

A

0,1,1

0,0,0

 FMO1

NM_001282692.1:p.Arg506a/c.1516C > T

rs60639054

chr1

171254588

171254588

C

T

0,1,1

0,0,0

 FREM3

NM_001168235.1:p.Gln166a/c.496C > T

 

chr4

144621333

144621333

G

A

0,1,1

0,0,0

 FTCD

NM_006657.2:p.Arg71a/c.211C > T

rs8133955

chr21

47574090

47574090

G

A

0,1,1

0,0,0

 FXYD3

NM_001136007.1:p.Arg45a/c.133C > T

 

chr19

35610131

35610131

C

T

0,1,1

0,0,0

 GAL3ST4

NM_024637.4:p.Trp289a/c.867G > A

rs147809354

chr7

99758145

99758145

C

T

0,1,1

0,0,0

 GCAT

NM_001171690.1:p.Trp2a/c.5G > A

rs202183602

chr22

38203979

38203979

G

A

0,1,1

0,0,0

 GNB3

NM_002075.3:p.Lys89a/c.265A > T

 

chr12

6952399

6952399

A

T

0,1,1

0,0,0

 GP6

NM_001083899.2:p.Trp429a/c.1287G > A

rs74697203

chr19

55526026

55526026

C

T

0,1,1

0,0,0

 GPR148

NM_207364.2:p.Arg81a/c.241C > T

rs140681574

chr2

131486965

131486965

C

T

0,1,1

0,0,0

 GSG1

NM_001080555.2:p.Arg160a/c.478A > T

 

chr12

13241799

13241799

T

A

0,1,1

0,0,0

 GTDC1

NM_001284238.1:p.Trp9a/c.26G > A

rs145066970

chr2

144934759

144934759

C

T

0,1,1

0,0,0

 GUCA1C

NM_005459.3:p.Glu18a/c.52G > T

rs143174402

chr3

108672558

108672558

C

A

0,1,1

0,0,0

 HEPHL1

NM_001098672.1:p.Trp96a/c.288G > A

 

chr11

93778956

93778956

G

A

0,1,1

0,0,0

 HERC6

NM_017912.3:p.Gln1021a/c.3061C > T

rs4413373

chr4

89363604

89363604

C

T

0,1,1

0,0,0

 HIST1H1T

NM_005323.3:p.Arg168a/c.502A > T

rs35191055

chr6

26107820

26107820

T

A

0,1,1

0,0,0

 HKDC1

NM_025130.3:p.Trp721a/c.2163G > A

rs147565138

chr10

71018662

71018662

G

A

0,1,1

0,0,0

 HMCN2

NM_001291815.1:p.Arg3622a/c.10864C > T

 

chr9

133281539

133281539

C

T

0,1,1

0,0,0

 HRG

NM_000412.3:p.Glu294a/c.880G > T

rs140336956

chr3

186394974

186394974

G

T

0,1,1

0,0,0

 HSD17B14

NM_016246.2:p.Arg79a/c.235C > T

rs139341223

chr19

49335965

49335965

G

A

0,1,1

0,0,0

 IQCE

NM_001287499.1:p.Ser366a/c.1097C > A

rs367705543

chr7

2629593

2629593

C

A

0,1,1

0,0,0

 ITGA10

NM_003637.3:p.Arg313a/c.937C > T

 

chr1

145532484

145532484

C

T

0,1,1

0,0,0

 JMJD7-PLA2G4B

NM_005090.3:p.Arg486a/c.1456C > T

rs199962342

chr15

42135893

42135893

C

T

0,1,1

0,0,0

 KCNJ16

NM_001291622.1:p.Gly168a/c.502G > T

 

chr17

68128625

68128625

G

T

0,1,1

0,0,0

 KCNJ16

NM_001291622.1:p.Arg337a/c.1009C > T

rs142625269

chr17

68129132

68129132

C

T

0,1,1

0,0,0

 KCNJ18

NM_001194958.2:p.Arg399a/c.1195C > T

rs144702327

chr17

21319849

21319849

C

T

0,1,1

0,0,0

 KCNU1

NM_001031836.2:p.Trp768a/c.2303G > A

 

chr8

36767025

36767025

G

A

0,1,1

0,0,0

 KIAA0319L

NM_024874.4:p.Arg1019a/c.3055C > T

 

chr1

35900590

35900590

G

A

0,1,1

0,0,0

 KIAA0753

NM_014804.2:p.Gln896a/c.2686C > T

rs149782904

chr17

6493199

6493199

G

A

0,1,1

0,0,0

 KIF27

NM_017576.2:p.Arg1336a/c.4006C > T

rs371473677

chr9

86452116

86452116

G

A

0,1,1

0,0,0

 KLHDC7A

NM_152375.2:p.Gln252a/c.754C > T

rs115859684

chr1

18808229

18808229

C

T

0,1,1

0,0,0

 KLHDC9

NM_152366.4:p.Trp223a/c.669G > A

rs150493322

chr1

161069277

161069277

G

A

0,1,1

0,0,0

 KLHL33

NM_001109997.2:p.Arg230a/c.688C > T

 

chr14

20898147

20898147

G

A

0,1,1

0,0,0

 KLK4

NM_004917.3:p.Trp153a/c.458G > A

rs104894704

chr19

51411852

51411852

C

T

0,1,1

0,0,0

 KLRF1

NM_016523.2:p.Trp128a/c.383G > A

 

chr12

9994456

9994456

G

A

0,1,1

0,0,0

 LFNG

NM_001166355.1:p.Ser27a/c.80C > A

rs372947239

chr7

2552823

2552823

C

A

0,1,1

0,0,0

 LHX4

NM_033343.3:p.Gln29a/c.85C > T

 

chr1

180217428

180217428

C

T

0,1,1

0,0,0

 LILRB2

NM_005874.4:p.Glu161a/c.481G > T

rs370409653

chr19

54783377

54783377

C

A

0,1,1

0,0,0

 LTBP4

NM_001042544.1:p.Trp1176a/c.3527G > A

rs35079932

chr19

41128415

41128415

G

A

0,1,1

0,0,0

 LY9

NM_002348.3:p.Arg478a/c.1432C > T

rs145664274

chr1

160788097

160788097

C

T

0,1,1

0,0,0

 MAFA

NM_201589.3:p.Lys346a/c.1036A > T

 

chr8

144511541

144511541

T

A

0,1,1

0,0,0

 MALRD1

NM_001142308.2:p.Gln888a/c.2662C > T

 

chr10

19498280

19498280

C

T

0,1,1

0,0,0

 MARK1

NM_001286124.1:p.Arg548a/c.1642C > T

 

chr1

220825398

220825398

C

T

0,1,1

0,0,0

 MBL2

NM_000242.2:p.Glu210ac.628G > T

rs74754826

chr10

54528016

54528016

C

A

0,1,1

0,0,0

 MCEMP1

NM_174918.2:p.Gln183a/c.547C > T

rs113286748

chr19

7743869

7743869

C

T

0,1,1

0,0,0

 MDM1

NM_017440.4:p.Arg643a/c.1927C > T

rs147627177

chr12

68696445

68696445

G

A

0,1,1

0,0,0

 MDN1

NM_014611.2:p.Glu4974a/c.14920G > T

 

chr6

90368430

90368430

C

A

0,1,1

0,0,0

 METTL2A

NM_181725.3:p.Arg291a/c.871C > T

rs147656413

chr17

60522259

60522259

C

T

0,1,1

0,0,0

 MGAT4D

NM_001277353.1:p.Lys372a/c.1114A > T

 

chr4

141372566

141372566

T

A

0,1,1

0,0,0

 MIB1

NM_020774.3:p.Gln219a/c.655C > T

 

chr18

19358082

19358082

C

T

0,1,1

0,0,0

 MIB2

NM_001170689.1:p.Gln739a/c.2215C > T

rs146481628

chr1

1565047

1565047

C

T

0,1,1

0,0,0

 MLANA

NM_005511.1:p.Arg51a/c.151C > T

 

chr9

5897630

5897630

C

T

0,1,1

0,0,0

 MLF1

NM_001195432.1:p.Arg194a/c.580C > T

 

chr3

158317881

158317881

C

T

0,1,1

0,0,0

 MROH2A

NM_001287395.1:p.Gln944a/c.2830C > T

 

chr2

234723288

234723288

C

T

0,1,1

0,0,0

 MST1R

NM_002447.2:p.Gln690a/c.2068C > T

rs61734381

chr3

49934828

49934828

G

A

0,1,1

0,0,0

 MST1R

NM_002447.2:p.Lys621a/c.1861A > T

rs9819888

chr3

49935503

49935503

T

A

0,1,1

0,0,0

 MUC12

NM_001164462.1:p.Arg171a/c.511C > T

 

chr7

100634355

100634355

C

T

0,1,1

0,0,0

 MUC19

NM_173600.2:p.Arg7595a/c.22783C > T

rs183548726

chr12

40938465

40938465

C

T

0,1,1

0,0,0

 MYOM3

NM_152372.3:p.Arg513a/c.1537C > T

 

chr1

24416105

24416105

G

A

0,1,1

0,0,0

 NARR

NM_001256281.1:p.Gln177a/c.529C > T

rs140500150

chr17

27043980

27043980

G

A

0,1,1

0,0,0

 NCOA1

NM_003743.4:p.Arg1122a/c.3364C > T

 

chr2

24964713

24964713

C

T

0,1,1

0,0,0

 NGB

NM_021257.3:p.Gln11a/c.31C > T

 

chr14

77737250

77737250

G

A

0,1,1

0,0,0

 NIPSNAP3A

NM_015469.1:p.Arg96a/c.286C > T

rs34856872

chr9

107515201

107515201

C

T

0,1,1

0,0,0

 NKX1–2

NM_001146340.1:p.Trp4a/c.12G > A

 

chr10

126138501

126138501

C

T

0,1,1

0,0,0

 NLRP12

NM_001277126.1:p.Arg1017a/c.3049C > T

rs35064500

chr19

54299165

54299165

G

A

0,1,1

0,0,0

 NME3

NM_002513.2:p.Trp159a/c.477G > A

rs140703991

chr16

1820683

1820683

C

T

0,1,1

0,0,0

 NUDT7

NM_001105663.2:p.Glu8a/c.22G > T

rs182579196

chr16

77756501

77756501

G

T

0,1,1

0,0,0

 OAS1

NM_001032409.1:p.Arg73a/c.217C > T

rs147431531

chr12

113346377

113346377

C

T

0,1,1

0,0,0

 OR10K1

NM_001004473.1:p.Tyr259a/c.777C > A

rs143219550

chr1

158436128

158436128

C

A

0,1,1

0,0,0

 OR10R2

NM_001004472.1:p.Lys73a/c.217A > T

 

chr1

158449884

158449884

A

T

0,1,1

0,0,0

 OR1G1

NM_003555.1:p.Ser95a/c.284C > G

 

chr17

3030562

3030562

G

C

0,1,1

0,0,0

 OR2M4

NM_017504.1:p.Arg223a/c.667C > T

rs143728385

chr1

248402897

248402897

C

T

0,1,1

0,0,0

 OR4M2

NM_001004719.2:p.Tyr177a/c.531C > G

rs148183880

chr15

22369106

22369106

C

G

0,1,1

0,0,0

 OR5A1

NM_001004728.1:p.Arg125a/c.373C > T

rs150073749

chr11

59211014

59211014

C

T

0,1,1

0,0,0

 OR5M11

NM_001005245.1:p.Tyr126a/c.378 T > A

rs17547284

chr11

56310356

56310356

A

T

0,1,1

0,0,0

 OR8I2

NM_001003750.1:p.Tyr289a/c.867C > G

rs61887097

chr11

55861650

55861650

C

G

0,1,1

0,0,0

 OR8U8

NM_001013356.2:p.Trp270a/c.810G > A

rs140673261

chr11

56143919

56143919

G

A

0,1,1

0,0,0

 P2RY4

NM_002565.3:p.Trp348a/c.1043G > A

rs41310667

chrX

69478432

69478432

C

T

0,1,1

0,0,0

 PADI2

NM_007365.2:p.Gln340a/c.1018C > T

rs142403504

chr1

17410253

17410253

G

A

0,1,1

0,0,0

 PCDHGA10

NM_018913.2:p.Tyr331a/c.993 T > G

 

chr5

140793735

140793735

T

G

0,1,1

0,0,0

 PCOLCE2

NM_013363.3:p.Arg204a/c.610C > T

rs143280691

chr3

142557712

142557712

G

A

0,1,1

0,0,0

 PCSK6

NM_002570.4:p.Arg413a/c.1237C > T

rs77239269

chr15

101929740

101929740

G

A

0,1,1

0,0,0

 PCSK9

NM_174936.3:p.Cys679a/c.2037C > A

rs28362286

chr1

55529215

55529215

C

A

0,1,1

0,0,0

 PDE5A

NM_001083.3:p.Gln860a/c.2578C > T

rs140289122

chr4

120419806

120419806

G

A

0,1,1

0,0,0

 PDIA6

NM_001282705.1:p.Arg13a/c.37C > T

 

chr2

10977695

10977695

G

A

0,1,1

0,0,0

 PEG3

NM_001146184.1:p.Trp16a/c.48G > A

 

chr19

57335976

57335976

C

T

0,1,1

0,0,0

 PEG3

NM_001146184.1:p.Lys14a/c.40A > T

 

chr19

57335984

57335984

T

A

0,1,1

0,0,0

 PEX7

NM_000288.3:p.Leu292a/c.875 T > A

rs1805137

chr6

137219351

137219351

T

A

0,1,1

0,0,0

 PIF1

NM_001286497.1:p.Glu49a/c.145G > T

rs75683534

chr15

65116390

65116390

C

A

0,1,1

0,0,0

 PLA2R1

NM_007366.4:p.Cys1377a/c.4131 T > A

rs145354671

chr2

160801430

160801430

A

T

0,1,1

0,0,0

 PLCD4

NM_032726.3:p.Arg117a/c.349C > T

rs146112514

chr2

219483469

219483469

C

T

0,1,1

0,0,0

 PLCL2

NM_001144382.1:p.Arg1115a/c.3343C > T

rs149144281

chr3

17131374

17131374

C

T

0,1,1

0,0,0

 POLR3B

NM_018082.5:p.Glu374a/c.1120G > T

 

chr12

106820993

106820993

G

T

0,1,1

0,0,0

 PROSER3

NM_001039887.2:p.Arg439a/c.1315C > T

rs375920305

chr19

36259319

36259319

C

T

0,1,1

0,0,0

 PRSS41

NM_001135086.1:p.Gln69a/c.205C > T

 

chr16

2849034

2849034

C

T

0,1,1

0,0,0

 PSME1

NM_176783.2:p.Gln229a/c.685C > T

rs370880151

chr14

24607785

24607785

C

T

0,1,1

0,0,0

 PSRC1

NM_001005290.3:p.Lys249a/c.745A > T

rs116389032

chr1

109823457

109823457

T

A

0,1,1

0,0,0

 PTBP3

NM_001244898.1:p.Arg56a/c.166C > T

rs143872137

chr9

115038264

115038264

G

A

0,1,1

0,0,0

 PTGDR

NM_000953.2:p.Trp48a/c.143G > A

rs41533946

chr14

52734675

52734675

G

A

0,1,1

0,0,0

 PXDNL

NM_144651.4:p.Cys1258a/c.3774 T > A

rs117752382

chr8

52284560

52284560

A

T

0,1,1

0,0,0

 PZP

NM_002864.2:p.Gln1168a/c.3502C > T

rs143616823

chr12

9309819

9309819

G

A

0,1,1

0,0,0

 RABEP1

NM_004703.5:p.Arg270a/c.808C > T

 

chr17

5253769

5253769

C

T

0,1,1

0,0,0

 RABEPK

NM_001174152.1:p.Arg113a/c.337C > T

rs199553121

chr9

127975774

127975774

C

T

0,1,1

0,0,0

 RGS11

NM_183337.2:p.Arg133a/c.397C > T

rs149201684

chr16

324075

324075

G

A

0,1,1

0,0,0

 RNF133

NM_139175.1:p.Arg240a/c.718C > T

rs141697772

chr7

122338255

122338255

G

A

0,1,1

0,0,0

 RNPC3

NM_017619.3:p.Gln185a/c.553C > T

 

chr1

104078061

104078061

C

T

0,1,1

0,0,0

 RPGRIP1

NM_020366.3:p.Arg52a/c.154C > T

rs192003551

chr14

21762904

21762904

C

T

0,1,1

0,0,0

 RPTN

NM_001122965.1:p.Arg771a/c.2311C > T

rs192865821

chr1

152127264

152127264

G

A

0,1,1

0,0,0

 SCAND1

NM_033630.2:p.Trp43a/c.128G > A

 

chr20

34542376

34542376

C

T

0,1,1

0,0,0

 SCD5

NM_024906.2:p.Gln192a/c.574C > T

 

chr4

83582226

83582226

G

A

0,1,1

0,0,0

 SCUBE2

NM_001170690.1:p.Cys548a/c.1644C > A

 

chr11

9055237

9055237

G

T

0,1,1

0,0,0

 SDSL

NM_138432.2:p.Leu280a/c.839 T > G

 

chr12

113875733

113875733

T

G

0,1,1

0,0,0

 SFTPD

NM_003019.4:p.Gln80a/c.238C > T

rs79085361

chr10

81702597

81702597

G

A

0,1,1

0,0,0

 SLC16A4

NM_004696.2:p.Trp482a/c.1446G > A

rs114581294

chr1

110906406

110906406

C

T

0,1,1

0,0,0

 SLC22A11

NM_018484.2:p.Arg48a/c.142C > T

rs35008345

chr11

64323613

64323613

C

T

0,1,1

0,0,0

 SLC22A24

NM_001136506.2:p.Arg347a/c.1039C > T

rs374095536

chr11

62863494

62863494

G

A

0,1,1

0,0,0

 SLC2A5

NM_003039.2:p.Ser291a/c.872C > A

 

chr1

9099872

9099872

G

T

0,1,1

0,0,0

 SLC5A8

NM_145913.3:p.Lys183a/c.547A > T

 

chr12

101587548

101587548

T

A

0,1,1

0,0,0

 SLC6A18

NM_182632.2:p.Gln249a/c.745C > T

rs200802505

chr5

1239577

1239577

C

T

0,1,1

0,0,0

 SPATA24

NM_194296.1:p.Gln185a/c.553C > T

rs183526939

chr5

138732554

138732554

G

A

0,1,1

0,0,0

 SPERT

NM_152719.2:p.Trp135a/c.405G > A

 

chr13

46287565

46287565

G

A

0,1,1

0,0,0

 SSPO

NM_198455.2:p.Gln2048a/c.6142C > T

rs200402989

chr7

149491941

149491941

C

T

0,1,1

0,0,0

 STMND1

NM_001190766.1:p.Gln60a/c.178C > T

rs146229126

chr6

17115289

17115289

C

T

0,1,1

0,0,0

 TAS2R19

NM_176888.1:p.Arg39a/c.115C > T

rs146593308

chr12

11175056

11175056

G

A

0,1,1

0,0,0

 TAS2R20

NM_176889.2:p.Trp35a/c.104G > A

rs116400924

chr12

11150371

11150371

C

T

0,1,1

0,0,0

 TMEM150B

NM_001085488.2:p.Cys45a/c.135C > A

 

chr19

55831820

55831820

G

T

0,1,1

0,0,0

 TMEM70

NM_017866.5:p.Arg80a/c.238C > T

rs387907070

chr8

74891018

74891018

C

T

0,1,1

0,0,0

 TMX4

NM_021156.2:p.Gln69a/c.205C > T

rs373356438

chr20

7990934

7990934

G

A

0,1,1

0,0,0

 TOR1AIP1

NM_001267578.1:p.Ser50a/c.149C > A

 

chr1

179851786

179851786

C

A

0,1,1

0,0,0

 TREX2

NM_080701.3:p.Arg87a/c.259C > T

rs141078733

chrX

152710630

152710630

G

A

0,1,1

0,0,0

 TRIOBP

NM_001039141.2:p.Arg1025a/c.3073C > T

 

chr22

38121636

38121636

C

T

0,1,1

0,0,0

 TSPAN19

NM_001100917.1:p.Gly19a/c.55G > T

rs188656791

chr12

85423670

85423670

C

A

0,1,1

0,0,0

 TTC22

NM_017904.3:p.Arg342a/c.1024C > T

rs2270002

chr1

55251314

55251314

G

A

0,1,1

0,0,0

 TTC25

NM_031421.3:p.Glu602a/c.1804G > T

rs375330943

chr17

40117478

40117478

G

T

0,1,1

0,0,0

 TTLL3

NM_001025930.3:p.Arg704a/c.2110C > T

rs115917139

chr3

9874914

9874914

C

T

0,1,1

0,0,0

 UBAP1L

NM_001163692.1:p.Glu34a/c.100G > T

 

chr15

65398454

65398454

C

A

0,1,1

0,0,0

 UGT1A7

NM_019077.2:p.Tyr81a/c.243C > A

rs149618508

chr2

234590826

234590826

C

A

0,1,1

0,0,0

 UGT2A1

NM_001252274.2:p.Tyr192a/c.576 T > A

rs111696697

chr4

70512787

70512787

A

T

0,1,1

0,0,0

 UMODL1

NM_173568.3:p.Trp1379a/c.4136G > A

rs376098587

chr21

43549884

43549884

G

A

0,1,1

0,0,0

 UPK3A

NM_006953.3:p.Ser87a/c.260C > A

rs138918236

chr22

45683104

45683104

C

A

0,1,1

0,0,0

 UTS2B

NM_198152.3:p.Arg111a/c.331C > T

rs16866426

chr3

190993044

190993044

G

A

0,1,1

0,0,0

 VSIG10L

NM_001163922.1:p.Gly340a/c.1018G > T

 

chr19

51843858

51843858

C

A

0,1,1

0,0,0

 VWCE

NM_152718.2:p.Gln866a/c.2596C > T

rs61729958

chr11

61026419

61026419

G

A

0,1,1

0,0,0

 WDR33

NM_001006622.2:p.Trp265a/c.794G > A

 

chr2

128522234

128522234

C

T

0,1,1

0,0,0

 WDR49

NM_178824.3:p.Ser330a/c.989C > A

 

chr3

167250675

167250675

G

T

0,1,1

0,0,0

 WRN

NM_000553.4:p.Arg369a/c.1105C > T

rs17847577

chr8

30938648

30938648

C

T

0,1,1

0,0,0

 ZBED6CL

NM_138434.2:p.Gln29a/c.85C > T

rs73474332

chr7

150027578

150027578

C

T

0,1,1

0,0,0

 ZIM3

NM_052882.1:p.Lys438a/c.1312A > T

rs111350153

chr19

57646393

57646393

T

A

0,1,1

0,0,0

 ZNF107

NM_001282359.1:p.Ser181a/c.542C > G

rs200723270

chr7

64167017

64167017

C

G

0,1,1

0,0,0

 ZNF135

NM_007134.1:p.Glu221a/c.661G > T

rs148932599

chr19

58578441

58578441

G

T

0,1,1

0,0,0

 ZNF154

NM_001085384.2:p.Arg192a/c.574C > T

rs74939505

chr19

58213743

58213743

G

A

0,1,1

0,0,0

 ZNF200

NM_003454.3:p.Arg392a/c.1174C > T

rs138531369

chr16

3273906

3273906

G

A

0,1,1

0,0,0

 ZNF211

NM_001265597.1:p.Tyr602a/c.1806 T > A

rs146505315

chr19

58153465

58153465

T

A

0,1,1

0,0,0

 ZNF665

NM_024733.3:p.Gln154a/c.460C > T

rs74974920

chr19

53669283

53669283

G

A

0,1,1

0,0,0

 ZNF718

NM_001289930.1:p.Gln278a/c.832C > T

rs116083456

chr4

155530

155530

C

T

0,1,1

0,0,0

 ZNF781

NM_152605.3:p.Arg53a/c.157C > T

rs140682866

chr19

38160893

38160893

G

A

0,1,1

0,0,0

 ZSCAN9

NM_001199479.1:p.Arg193a/c.577C > T

rs76542212

chr6

28198122

28198122

C

T

0,1,1

0,0,0

More than one allele in PPROM Cohort

 ABCB5

NM_001163941.1:p.Arg353a/c.1057C > T

rs150279505

chr7

20687233

20687233

C

T

0,2,2

0,0,0

 ACSM3

NM_005622.3:p.Trp292a/c.875G > A

rs52817836

chr16

20792388

20792388

G

A

0,2,2

0,0,0

 ALPK1

NM_001102406.1:p.Trp595a/c.1785G > A

rs116802171

chr4

113352488

113352488

G

A

0,2,2

0,0,0

 AMZ1

NM_001284355.1:p.Arg292a/c.874C > T

rs55919423

chr7

2752059

2752059

C

T

0,4,4

0,0,0

 AOAH

NM_001177506.1:p.Gln556a/c.1666C > T

rs145455591

chr7

36554130

36554130

G

A

0,2,2

0,0,0

 C9orf129

NM_001098808.1:p.Gln170a/c.508C > T

rs115115786

chr9

96080763

96080763

G

A

0,3,3

0,0,0

 CLEC6A

NM_001007033.1:p.Ser200a/c.599C > A

rs114953954

chr12

8630029

8630029

C

A

0,2,2

0,0,0

 COL6A6

NM_001102608.1:p.Gly1434a/c.4300G > T

rs140872639

chr3

130311412

130311412

G

T

0,2,2

0,0,0

 COQ6

NM_182480.2:p.Trp14a/c.41G > A

rs17094161

chr14

74416836

74416836

G

A

0,6,6

0,0,0

 EFCAB5

NM_198529.3:p.Gln952a/c.2854C > T

rs73274829

chr17

28405349

28405349

C

T

0,2,2

0,0,0

 ERVMER34–1

NM_001242690.1:p.Trp68a/c.204G > A

rs61731313

chr4

53611484

53611484

C

T

0,2,2

0,0,0

 HSD17B13

NM_178135.4:p.Trp150a/c.450G > A

rs61748262

chr4

88238244

88238244

C

T

0,2,2

0,0,0

 HTR4

NM_001286410.1:p.Lys389a/c.1165A > T

rs58336229

chr5

147861104

147861104

T

A

0,2,2

0,0,0

 KCNMB3

NM_014407.3:p.Trp106a/c.317G > A

rs145138176

chr3

178962425

178962425

C

T

0,2,2

0,0,0

 KRT74

NM_175053.3:p.Gln285a/c.853C > T

rs147781415

chr12

52964608

52964608

G

A

0,2,2

0,0,0

 MAGEE2

NM_138703.4:p.Glu120a/c.358G > T

rs1343879

chrX

75004529

75004529

C

A

1,0,2

0,0,0

 MATK

NM_002378.3:p.Arg9a/c.25C > T

rs74830030

chr19

3789321

3789321

G

A

0,2,2

0,0,0

 METTL7B

NM_152637.2:p.Arg224a/c.670C > T

rs115687886

chr12

56077768

56077768

C

T

0,7,7

0,0,0

 MROH2B

NM_173489.4:p.Trp191a/c.572G > A

rs1023840

chr5

41061715

41061715

C

T

0,3,3

0,0,0

 MUC19

NM_173600.2:p.Gln8113a/c.24337C > T

rs75211948

chr12

40961512

40961512

C

T

0,3,3

0,0,0

 OPRM1

NM_001008503.2:p.Arg401a/c.1201C > T

rs34427887

chr6

154567863

154567863

C

T

0,2,2

0,0,0

 OR10Z1

NM_001004478.1:p.Tyr153a/c.459C > A

rs148998855

chr1

158576687

158576687

C

A

0,3,3

0,0,0

 OR6C6

NM_001005493.1:p.Leu200a/c.599 T > A

rs76796682

chr12

55688418

55688418

A

T

0,2,2

0,0,0

 PHF19

NM_001009936.2:p.Gln180a/c.538C > T

rs112858270

chr9

123632050

123632050

G

A

0,3,3

0,0,0

 PKD1L2

NM_052892.3:p.Trp1184a/c.3551G > A

rs147079883

chr16

81194437

81194437

C

T

1,1,3

0,0,0

 PLCXD2

NM_001185106.1:p.Trp292a/c.876G > A

rs77085054

chr3

111451475

111451475

G

A

0,2,2

0,0,0

 RNF212

NM_001193318.2:p.Gln188a/c.562C > T

rs60035268

chr4

1087487

1087487

G

A

0,4,4

0,0,0

 SLC10A5

NM_001010893.2:p.Leu201a/c.602 T > A

rs112999969

chr8

82606606

82606606

A

T

0,2,2

0,0,0

 TAS2R46

NM_176887.2:p.Gln288a/c.862C > T

rs150894148

chr12

11214032

11214032

G

A

0,2,2

0,0,0

 TCHHL1

NM_001008536.1:p.Gln294a/c.880C > T

rs61749316

chr1

152059278

152059278

G

A

0,4,4

0,0,0

 ULBP3

NM_024518.1:p.Glu110a/c.328G > T

rs34672740

chr6

150387059

150387059

C

A

0,2,2

0,0,0

 ZAN

NM_003386.2:p.Trp1883a/c.5649G > A

rs2293766

chr7

100371358

100371358

G

A

1,4,6

0,0,0

 ZNF486

NM_052852.3:p.Tyr210a/c.630C > G

rs184976796

chr19

20308149

20308149

C

G

0,2,2

0,0,0

 ZNF594

NM_032530.1:p.Glu684a/c.2050G > T

rs114754534

chr17

5085502

5085502

C

A

0,2,2

0,0,0

 ZP4

NM_021186.3:p.Arg252a/c.754C > T

 

chr1

238050156

238050156

G

T

0,2,2

0,0,0

WES (50–100 X coverage) data were analyzed as described in (Modi et al., 2017a,b) to extract nonsense variants

All nonsense variants were identified by WES and only selected variants were confirmed by Sanger sequencing

Chr chromosome

aAllele count: Homozygous, Heterozygous, Total Alleles

A heterozygous nonsense variant (rs5743490) of African ancestry in the Defensin Beta 1 (DEFB1) gene, which encodes a small cysteine-rich cationic peptide that damages the cellular membranes of bacteria and some viruses, was found in PPROM cases in our initial WES and targeted genotyping [9], but not in neonates born at term (Tables 1, 2, 3 and 4). No other loss of function variants, including splicing variants and frameshift variants, were identified in DEFB1 in our WES. DEFB1 is expressed by the fetal membranes [9] (Additional file 2: Figure S1).
Table 2

SNPs Evaluated

 

rs Number

Nucleotide Change

Protein Sequence Change

Ancestry

MAF

DEFB1

rs5743490

G/T

p.Cys37Ter

African

0.0008586

MBL2

rs74754826

C/A

p.Glu210Ter

African

0.000594

METTL7B

rs115687886

C/T

p.Arg224Ter

African

0.004004

rs138407179

G/T

p.Gly80Ter

African

7.941e-05

rs146636131

G/T

p.Arg224Leu

African

0.0005122

MAF Minor allele frequency from gnomAD

Table 3

Putative Ancestry-Specific Variants Conferring Risk of PPROM

 

DEFB1 rs5743490

MBL2 rs74754826

METTL7B rs115687886

Present in cases only in WES?

Yesa

Yesa

Yes

Validated by Sanger sequencing?

Yesa

Yesa

Yes

Rare?

Yes

Yes

Yes

African ancestry?

Yes

Yes

Yes

Under selective pressure?

Yes

Yes

?

Heterozygous impact?

Plausible

Plausible

?

Expressed in fetal membranes?

Yes

Yesa

Yes

Plausible pathophysiology?

Yes

Yes

?

Replication?

Yes

Yes

No

aData derived from the present report and Modi et al. [9]

Table 4

Allele Counts and Minor Allele Frequencies

rs Number

Term

PPROM

MAC/TA

Homo

Het

MAF

MAC/TA

Homo

Het

MAF

p Value (nominal)

rs5743490 Combineda

1/751

0

1

0.0013

10/694

1

8

0 .0144

(p < 0.004)

rs74754826 Combineda

1/751

0

1

0.0013

8/694

1

8

0.0115

(p < 0.015)

rs115687886 Combined

10(9)/254

1

8(7)

0.035

20(19)/318

0

20(19)

0.060

(p > 0.05)

MAC/TA Minor allele count/total alleles, MAF Minor allele frequency, Homo Homozygous, Het Heterozygous

(9) = rs115687886 nonsense mutations subtracting out those with an adjacent rs1466636131 minor allele

a Data derived from the present study and Modi et al. [9]

The DEFB1 rs5743490 SNP has two alternative minor alleles, C/T (African ancestry), which creates a stop codon; and G/A (Latino ancestry), which produces a synonymous amino acid change. We verified by Sanger sequencing that the minor allele of rs5743490 that we detected encoded a stop codon [9]. This DEFB1 nonsense variant truncates the DEFB1 protein 4 amino acids into the mature peptide amino acid sequence so that no functional DEFB1 would be made [10]. However, the mutant protein, if expressed, could have dominant negative activity by preventing proteolytic processing of the un-mutated pro-peptide encoded by the normal allele. Thus, heterozygous mutations could possibly be functionally significant.

An additional 115 PPROM cases and 191 controls were subsequently genotyped for the DEFB1 nonsense mutation, yielding more nonsense mutations in PPROM cases, including a neonate with a homozygous DEFB1 nonsense variant, and only one mutant allele in a term control (Table 4). A statistically significant association of the rs5743490 nonsense mutation and PPROM was present in the combined cohorts (Table 4) (p < 0.004 by Fisher’s Exact test, 1-tailed).

We discovered another rare nonsense variant of African ancestry (rs74754826) in the MBL2 gene, which encodes mannose binding lectin-2, a protein involved in anti-microbial host defense [9]. It was only detected in PPROM cases (WES and initial targeted genotyping), and it met the screening criteria for being a PPROM candidate gene (Tables 1, 2 and 3). One hundred and nineteen PPROM cases and 199 term controls were genotyped in the present study for this nonsense variant, and a statistically significant association of the minor allele with PPROM was found (P < 0.015 by Fisher’s Exact test, 1-tailed) (Table 4).

We then applied our WES screening approach to look for other PPROM candidate genes, including genes where the nonsense mutation was of relatively high allele frequency in PPROM cases. We detected 7 heterozygous nonsense variants in the Methyltransferase Like 7B (METTL7B) gene in the WES discovery panel in PPROM cases, and none in term controls. This was the largest number of unique “PPROM mutation alleles”. METTL7B transcripts were detected in human placenta and amnion by PCR with sequence verification of the amplicon (Additional file 2: Figure S1).

A METTL7B SNP (rs146636131) adjacent to rs115687886 that is in phase modifies the codon to create a benign missense variant (p.Arg224Leu). Subjects with both rs115687886 and rs146636131 minor alleles were considered to have the missense variant rather than the nonsense mutation (Tables 1, 2, 3 and 4). Another rarer nonsense variant (rs138407179) was also detected. Both METTL7B nonsense variants are identified as causing loss of function with “high confidence” in the gnomAD database. rs138407179 has two alternate minor alleles, one encoding the stop codon of African ancestry (G/T) and another (G/A), which encodes a predicted damaging variant of South Asian ancestry.

Follow-up targeted genotyping of the METTL7B SNPs of interest on 94 PPROM cases and 94 term controls detected the nonsense variant in term controls, including a homozygote mutant. Statistical analysis of the combined analysis WES data and follow-up genotyping revealed no statistically significant association of the METTL7B rs115687886 nonsense mutation with PPROM (Tables 3 and 4), a finding that was not unexpected based on the fact that the minor allele of rs115687886 did not robustly meet all screening criteria as noted above.

Discussion

The simple screening approach outlined above may be useful to others seeking rare variants with moderate to high effect size associated with preterm birth in specific populations. The approach can also be used to identify rare mutations that are protective for PPROM by starting the screening with selection of variants found only in term controls, not in PPROM, and applying the subsequent filters. The fact that the majority of WES nonsense variants were detected in a single PPROM case, but each individual case harbored multiple nonsense variants allows for a test of genetic burden to be conducted as we have done in our previous studies [8, 9] in addition to the more focused examination of the contributions of individual variants. Importantly, the patterns of nonsense mutations among PPROM cases and term controls could also point to pathways and gene networks that when disrupted promote PPROM or protect against it.

Our findings suggest that a rare damaging DEFB1 variant of African ancestry may have a role in the pathophysiology of PPROM, presumably because it facilitates a dysbiotic reproductive tract flora that invades and or inflames the fetal membranes leading to premature rupture. The DEFB1 gene has been under selective pressure [11, 12], and has rare loss of function variants with four different ancestries (African, Latino, East Asian, European) reported in the genomAD database. It will be of interest to determine in the future if the other DEFB1 loss of function variants play a role in preterm birth after PPROM in the respective populations.

The discovery that the DEFB1 nonsense variant is associated PPROM prompted us to examine damaging variants in other beta defensin genes in our WES study. A stop-loss variant of African ancestry was identified in DEFB119 (rs12329612) in 19 PPROM cases, including 1 homozygote (20 alleles/152 total alleles), and 5 term controls, including 1 homozygote. A start-loss variant detected in DEFB128 (rs145944118) was found in one PPROM case, and another start-loss variant (rs18818350) was detected in DEFB132 in one term control. The functional significance of these variants and their relationship to preterm birth are currently unknown.

We discovered a significant association between a nonsense variant in an anti-microbial gene, MBL2, and PPROM. This association is consistent with the work of others who examined common MBL2 polymorphisms in fetal DNA from European populations and found increased risk of preterm birth [13, 14]. MBL-2 presumably functions as part of the host defense system, including DEFB1, which prevents or limits infections that cause chorioamnionitis and PPROM.

In contrast to our findings with the DEFB1 and MBL2 nonsense variants, the nonsense variant (rs115687886) in the METTL7B gene did not stand up to further scrutiny as a PPROM candidate. METTL7B encodes a putative methyltransferase whose transcript is elevated in blood leukocytes in the context of infection in pregnancy, providing a potential link to PPROM mechanisms [15]. However, the METTL7B was initially reported to be a lipid droplet-associated protein whose function with respect to lipid metabolism remains obscure [16].

The METTL7B rs115687886 minor allele (in the absence of the adjacent in phase SNP) truncates the protein at amino acid position 224 of the 244-amino acid protein. This truncation is outside of the methyltransferase domain (amino acids 75–172). The functional significance of this protein truncation has not been established to the best of our knowledge, which could make the mutation “ineligible” in our screening criteria. Moreover, the rs115687886 minor allele frequencies in our African-American term controls and PPROM cases are relatively high (Table 4) and outside of our definition of “rare” (Allele frequency < 0.01). No splicing or frameshift variants predicted to disrupt the protein coding sequence were detected in METTL7B the WES sample.

The other nonsense minor allele we examined (rs138407179), found in both a PPROM case and a term control, truncates the protein at amino acid residue 80, which likely damages the protein. Genotyping of additional PPROM cases and controls is required to determine if this nonsense variant is associated with PPROM. We could find no information in the literature regarding selective pressures impinging on the METTL7B gene.

Although our proposed strategy is consistent with guidelines for investigating causality of sequence variants in human disease, our approach has limitations including the current cost of WES and the use of modest sample sizes which may not have the power to detect pathophysiologic important rare variants/mutations [17]. The focus on nonsense variants found only in cases might exclude important PPROM-associated mutations from consideration if there was by chance a nonsense variant in the control group but not the cases. Importantly, the analysis strategy used in this report did not encompass other potentially damaging variants including frameshift, splicing and damaging missense variants, including variants that cause gain of function. These variants/mutations could, of course, be incorporated into the screening algorithm. In addition, WES would not identify intragenic regulatory elements that have an impact on gene expression levels. Another limitation of our study is the absence of direct evidence for disrupted function of the DEFB1 and MBL-2 proteins derived from the respective mutant transcripts. That said, the DEFB1 nonsense mutation would not lead to production of a mature peptide, so it is most certainly damaging. However, its potential to be a dominant negative inhibiting processing of full length DEFB1 pro-peptide from the major allele in heterozygous mutants remains to be explored. Likewise, the impact of the MBL2 nonsense mutation on protein function is only predicted, and studies need to be conducted with recombinant proteins to prove loss of function.

Our findings on the DEFB1 and MBL2 nonsense mutations are consistent with the notion that rare fetal mutations contribute to the disparities in preterm birth among African-Americans, and support the mining of rare mutations identified in WES as a portal to discovery of genes playing a role in preterm birth. A similar approach could be applied to other populations focusing on ancestry-enriched damaging variants. For example, there are rare damaging Latino (rs759177517; p. Tyr5Ter) and East Asian (rs140403947, p. Tyr60Ter) nonsense variants in the DEFB1 gene that could be evaluated for association with PPROM in the respective populations.

Conclusions

Our findings based on a simple and cost-effective data analysis strategy support the notion that multiple rare population-specific variants in the fetal genome contribute to preterm birth associated with PPROM.

Abbreviations

DEFB1: 

Defensin beta 1

MBL2: 

Mannose-binding lectin-2

METTL7B: 

Methyltransferase like 7B

PPROM: 

Preterm premature rupture of membranes

WES: 

Whole exome sequencing

Declarations

Acknowledgements

The authors wish to thank Sonya Washington for her support in subject recruitment.

Funding

This research was funded by National Institutes of Health Grants R01 HD073555 and P60 MD002256. This research was also supported, in part, by the Perinatology Research Branch, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services (NICHD/ NIH); and, in part, with Federal funds from NICHD, NIH under Contract No. HSN275201300006C. Funding from the Burroughs Wellcome Fund Preterm Birth Initiative (Grant No. 1015040) to Timothy York supported this research in part.

Availability of data and materials

The entire WES data set represents protected health information which cannot be shared publicly. The authors will gladly provide information on selected genes or genetic variants.

Authors’ contributions

All authors have read and approved the manuscript. JS, TY, HP BM and RR conceived the study and wrote the manuscript. BM and HP carried out bioinformatics analysis of the whole exome sequencing. BM, MT, RK, LJ performed genotyping and RNA expression studies and participated in writing the manuscript.

Ethics approval and consent to participate

Subjects were self-reported African-American women and their neonates receiving obstetrical care at MCV Hospitals, Richmond, VA (all samples in the WES) and Hutzel Hospital in Detroit, MI. The study was approved by the Institutional Review Boards of MCV Hospitals, Richmond, VA (IRB Number: HM15009); Wayne State University (IRB Numbers: 103897MP2F (5R), 082403MP2F (5R), 110605MP4F, 103108MP2F, 052308MP2F) as well as NICHD (National Institute of Child Health and Human Development) (IRB Numbers: 0H97-CH-N065, OH98-CH-N001, OH97-CH-N067, OH99-CH-N056, OH09-CH-N014). Subjects from Hutzel Hospital, Detroit, MI were enrolled under both Wayne State University as well as NICHD protocols and thus respective IRB numbers for both institutes are provided. Written informed consent was obtained from mothers before sample collection.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Authors’ Affiliations

(1)
Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA, USA
(2)
Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA, USA
(3)
Department of Obstetrics and Gynecology, Virginia Commonwealth University School of Medicine, Sanger Hall 11-029, 1101 East Marshall Street, Richmond, VA 23298, USA
(4)
Perinatology Research Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, MD, USA
(5)
Perinatology Research Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Detroit, MI, USA

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Copyright

© The Author(s). 2018

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