1471-2350-13-291471-2350Technical advance<p>Genotyping of a tri-allelic polymorphism by a novel melting curve assay in <it>MTHFD1L</it>: an association study of nonsyndromic Cleft in Ireland</p>MinguzziStefanomingu82@hotmail.itMolloyMAnneamolloy@tcd.iePeadarKirkepkirke@hrb.ieMillsJamesmillsj@exchange.nih.goveScottMJohnjscott@tcd.ieTroendleJamesjtroendlj@nhlbi.nih.govPangilinanFaithfaith@mail.nih.govBrodyLawrencelb68i@nih.govParle-McDermottAnneanne.parle-mcdermott@dcu.ie

Nutritional Genomics Group, School of Biotechnology, Dublin City University, Dublin, Ireland

School of Medicine, Trinity College Dublin, Dublin 2, Ireland

Child Health Epidemiology Unit, Health Research Board, Dublin, Ireland

Department of Health and Human Services, Eunice Kennedy Shriver National Institute of Health, Bethesda, MD, USA

School of Immunology & Biochemistry, Trinity College Dublin, Dublin 2, Ireland

Molecular Pathogenesis Section, Genome Technology Branch, National Human Genome Research Institute, Bethesda, MD, USA

BMC Medical Genetics1471-2350201213129http://www.biomedcentral.com/1471-2350/13/2910.1186/1471-2350-13-2922520921
19122011204201220420122012Minguzzi et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Polymorphisms within the MTHFD1L gene were previously associated with risk of neural tube defects in Ireland. We sought to test the most significant MTHFD1L polymorphisms for an association with risk of cleft in an Irish cohort. This required the development of a new melting curve assay to genotype the technically challenging MTHFD1L triallelic deletion/insertion polymorphism (rs3832406).

Methods

Melting curve analysis was used to genotype the MTHFD1L triallelic deletion/insertion polymorphism (rs3832406) and a Single Nucleotide Polymorphism rs17080476 in an Irish cohort consisting of 981 Irish case-parent trios and 1,008 controls. Tests for association with nonsyndromic cleft lip with or without cleft palate and cleft palate included case/control analysis, mother/control analysis and Transmission Disequilibrium Tests of case-parent trios.

Results

A successful melting curve genotyping assay was developed for the deletion/insertion polymorphism (rs3832406). The TDT analysis initially showed that the rs3832406 polymorphism was associated with isolated cleft lip with or without cleft palate. However, corrected p-values indicated that this association was not significant.

Conclusions

Melting Curve Analysis can be employed to successfully genotype challenging polymorphisms such as the MTHFD1L triallelic deletion/insertion polymorphism (DIP) reported here (rs3832406) and is a viable alternative to capillary electrophoresis. Corrected p-values indicate no association between MTHFD1L and risk of cleft in an Irish cohort.

Background

Cleft lip with or without cleft palate (CLP) and cleft palate only (CPO) are common birth defects of complex and heterogeneous aetiology. Previous studies suggest that folate deficiency before or during pregnancy can increase risk of clefting in the resulting offspring 1234. Folate supplementation in pregnancy has been shown to reduce the recurrence of CLP in families and to have a modest reduction in birth prevalence on a population basis 5. Nevertheless this association is still controversial 67. Numerous candidate gene association studies between clefts and folate related genes have shown mixed results and include methylenetetrahydrofolate reductase (MTHFR [Genbank: NP_005948.3]) 48910111213141516, methylenetetrahydrofolate dehydrogenase (NADP + dependent) (MTHFD1 [Genbank: NP_005947.3]) 11617, 5,10-methenyltetrahydrofolate synthetase (MTHFS [Genbank: NP_001186689.1]) and methionine synthase (MTR [Genbank: NP_000245.2]) 4171819. However, candidate gene studies to date have not considered MTHFD1L [Genbank: NP_001229696.1] in relation to nonsyndromic clefts. Environmental factors were reported for this cohort previously 16 and included data on the mother’s medication use, folic acid exposure, alcohol and smoking. No interaction between genotype and these environmental factors were found in that study.

Based on its association with neural tube defects (NTDs) 20, and the previously detected association of its cytoplasmic homologue MTHFD1 in our cleft cohort, we considered the mitochondrial enzyme MTHFD1L to be a prime candidate for consideration for association with cleft. The relevance of this gene is increasing given its identification in genome wide association screens as being associated with coronary artery disease 2122 and Alzheimer’s disease 23. Moreover a previous study has shown that MTHFD1L is upregulated in human colon adenocarcinoma 24. The MTHFD1L gene encodes the mitochondrial C1-Tetrahydrofolate(THF) Synthase protein which has a monofunctional 10-formyl-THF synthetase activity while lacking the 5,10-methylene-THF dehydrogenase and 5,10-methenyl-THF cyclohydrolase activities typically found in the trifunctional cytoplasmic protein encoded by MTHFD1 25. It has been shown that the MTHFD1L gene produces 2 alternatively spliced mRNAs with the shorter transcript lacking synthetase activity 26. Previously, we reported that the MTHFD1L rs3832406 DIP and numerous SNPs in linkage disequilibrium (LD) are associated with the risk of NTDs in the Irish population 20. We proposed that the DIP polymorphism is the direct disease causing variant within the associated LD block by affecting alternative splicing of the gene 20.

In this study, we genotyped the MTHFD1L DIP rs3832406 and the most statistically significant NTD-associated SNP in the adjacent LD block i.e., rs17080476, in 981 Irish case-parent trios affected by CPL or CPO. We developed a melting curve method capable of genotyping deletion/insertion polymorphisms without the need for capillary electrophoresis.

Methods

Subjects

Buccal swab or blood samples were obtained at the Dublin Cleft Centre in Ireland as previously described 16 from subjects with cleft palate only (CPO) or cleft lip with or without cleft palate (CLP) along with their mothers and fathers. A total of 2,688 samples including 758 complete triads and 223 incomplete triads were collected for this study. Out of the total number of cleft cases this included 347 (33.8%) isolated CPO cases plus an additional 108 (10.5%) with multiple defects and 531 (51.7%) isolated CLP cases plus an additional 42 (4%) with multiple defects. All the cases of this study were non-syndromic. Multiple cases included children with one or multiple defects along with cleft. Chromosomal anomalies and other conditions (i.e. mother had diabetes or epilepsy or was exposed to potentially teratogenic drugs) were excluded. Control samples (n = 1,008) were collected from a population of 56,049 pregnant women attending the three main maternity hospitals in the Dublin area between 1986 and 1990 as previously described 1627. Written informed consent was obtained from all participants. Ethical approval was granted by the Research Ethics Committees of the Health Research Board of Ireland, the participating hospitals, and the Institutional Review Board at NIH.

Genotyping

Genomic DNA was extracted from blood or buccal swab collected samples using a QIAamp DNA Blood Mini Kit (Qiagen, UK). HybProbe melting curve assays were designed to genotype DIP rs3832406 and SNP rs17080476 on a LightCycler 480 Real Time PCR machine (Roche) and are described in more detail below. Genotyping quality was verified by repeat genotyping of at least 10% of samples with agreement rate of ≫99% and overall success rate of ≫99%. In addition, 10% of the controls were genotyped by the HybProbe melting curve assays described here and compared to the assays used previously 20. Comparison of control genotype calls gave a 95.7% agreement for DIP rs3832406 and 99% agreement for SNP rs17080476. All discrepant genotype calls for any sample were resolved by re-genotyping or were left out of the final analysis.

SNP rs17080476 assay

SNP rs17080476 reagents and analysis conditions are: Forward Primer 5′-GCAACTTTGTTTAGTATGAAAATTTGAT-3′ (4 μM), reverse primer5′-TCTGTCTTCACCCAGCC (2 μM), anchor probe 5′-Bodipy630/650-AAGAGGGGAAAAAAAACCTTTCTCCATTATTCCTA-PHO-3′(0.4 μM), sensor probe 5′-ATTCATTTCTTTACAGCAGTGGGATTATGAAA-Fluorescein 3′ (0.2 μM), pre-incubation 10 minutes at 95°C, amplification 45 cycles of 15 seconds at 95°C, 15 seconds at 56°C, 15 seconds at 72°C, melting curve 1 minute at 95°C, 2 minutes at 50°C, acquisition ramp up to 80°C (0.11°C/s, 5 acquisitions per°C).

DIP rs3832406 assay

DIP rs3832406 reagents and analysis conditions are: forward primer 5′-AAGCTTCCTGTTACCAC-3′ (4 μM), reverse primer 5′-AGGAGAATCACTTCAACC-3′ (2 μM), anchor probe: 5′-AGCCCCACGTTTGAATTTTATGTTTTTCCTAAAGT-Fluorescein-3′ (0.2 μM). Sensor probe: 5′BODIPY630/650-AGGGAAGATTATTATTATTATTATTATTATTATTTTCTTTTTCAGACGGA-Phosphate-3′ (0.2 μM),pre-incubation 10 minutes at 95°C, amplification 45 Cycles of 10 seconds at 95°C, 10 seconds at 56°C, 10 seconds at 72°C, melting curve 10 seconds at 95°C, 1 minute at 50°C, acquisition ramp up to 70°C (0.02°C/s, 30 acquisitions per°C).

Statistical methods

Power calculations to detect an odds ratio of 1.5 assuming a dominant model for the case–control analyses were as follows: rs3832406 Allele 1 60%, Allele 2 95%, Allele 3 93%; rs17080476 G 95%. Assuming a recessive model: rs3832406 Allele 1 96%, Allele 2 36%, Allele 3 23%; rs17080476 G 34%. Our primary analysis was carried out with isolated nonsyndromic cases of CLP and CPO and their parents. A secondary analysis was then carried out including nonsyndromic cleft cases with other defects. Hardy-Weinberg equilibrium (HWE) was tested within each subject class (case, mother, father and controls) for each polymorphism by chi-squared test. Associations with CLP and CPO were tested for each polymorphism in cases/controls and separately in mothers/controls by logistic regression and odds ratios using either a dominant or recessive genetic disease model. Triads (case, mother, and father) were used to perform the Transmission Disequilibrium Test (TDT) of Spielman et al. 28. The TDT P-values were adjusted using permutational correction 29.

Results and discussion

Development of a novel assay to genotype DIP rs3832406 by Melting Curve Analysis

The MTHFD1L gene has received particular attention in recent years owing to its association with coronary artery disease, Alzheimer’s disease and NTDs. Our previous study, demonstrated that the MTHFD1L rs3832406 DIP is functional by impacting on alternative splicing efficiency 20. We report a new modified melting curve assay to genotype this functionally relevant triallelic MTHFD1L polymorphism without the need for traditional capillary electrophoresis methods. A single assay which is able to distinguish 3 alleles contemporaneously was developed taking advantage of the GC-rich regions flanking the DIP (Figure 1). As described previously 20, this polymorphism is a repeated “ATT” sequence that has three common alleles, Allele 1 (ATT7) Allele2 (ATT8) and Allele 3 (ATT9). A wide 50-base sensor probe was designed to perfectly match Allele 3 with 9 ATT repeats and its flanking regions, producing a melting temperature (Tm) of 63°C (Figure 1c). The same probe produces a 3-base mismatched bubble on Allele 2 and a 6-base mismatched bubble on Allele 1 causing a Tm of 60.3°C and 58.8°C respectively (Figure 1a-b). The probe pairing starts from the GC-rich external regions allowing the formation of an internal mismatched bubble for Alleles 1 and 2. A slow acquisition ramp allowed melting peaks for each homozygote and heterozygote genotype to be distinguished (Figure 1d-e).

<p>Figure 1</p>

Modified Melting Curve Analysis for DIP rs3832406

Modified Melting Curve Analysis for DIP rs3832406. The Sensor probe design for detection of all three alleles of DIP rs3832406 is shown. The Sensor probe is designed to perfectly match the complement of Allele 3. A. Sensor probe bound to Allele 1 has a Tm of 58.8°C. B. Sensor probe bound to Allele 2 has a Tm of 60.3°C. C. Sensor probe bound to Allele 3 has a Tm of 63°C. D. Examples of homozygote melting peaks for each of the three alleles. E. Examples of heterozygote melting peaks for all three allele combinations.

DIP rs3832406, SNP rs17080476 and risk of CLP

We genotyped rs3832406 DIP and SNP rs17080476 in an Irish cleft cohort in a bid to test for association. The genotype frequencies of SNP rs17080476 and DIP rs3832406 in our CLP, CPO and control samples are shown in Table 1. Genotype distributions in all groups were in HWE. DIP rs3832406 showed an association with CLP case status based on TDT analysis (Table 2). The TDT analysis showed that Allele 1 is transmitted to the offspring 55.2% of times (p = 0.037) in isolated CLP cases, indicating that this allele is associated with increased disease risk. The addition of multiple case families to this analysis enhances the statistical significance (56.1% transmission, p = 0.011). Allele 3 has the lowest frequency and was passed to the offspring only 42.8% of times (p = 0.035) in multiple CLP cases, appearing to have a protective role against the disease. However, correction of these significant p-values using permutational adjustment resulted in loss of statistical significance. We did not observe statistical significance with SNP rs17080476 which shares a D’ value of 0.61 with DIP rs3832406 and represented the most statistically compelling variant from this genomic region in our NTD study 20 (Table 2). The majority of other analyses performed showed no significant association with the risk of cleft (Table 3).

<p>Table 1</p>

DIPrs3832406

Isolated defects

Multiple defects

CLP

Fathers

Mothers

Cases

Fathers

Mothers

Cases

Controls

n

%

n

%

n

%

n

%

n

%

n

%

n

%

11

162

41.8

187

40.1

209

41.9

174

42.0

203

40.8

231

43.5

419

42.1

12

102

26.3

128

27.5

149

29.9

116

28.0

140

28.1

163

30.7

267

26.8

13

76

19.6

89

19.1

84

16.8

82

19.8

97

19.5

91

17.1

196

19.7

22

18

4.6

24

5.2

18

3.6

18

4.3

24

4.8

18

3.4

40

4.0

23

24

6.2

31

6.7

28

5.6

24

5.8

34

6.8

28

5.3

52

5.2

33

6

1.5

7

1.5

11

2.2

6

1.4

7

1.4

11

2.1

21

2.1

Total

388

466

499

420

505

542

995

CPO

Fathers

Mothers

Cases

Fathers

Mothers

Cases

n

%

n

%

n

%

n

%

n

%

n

%

11

98

37.3

118

38.1

134

41.7

145

40.8

154

36.9

175

41.0

12

78

29.7

82

26.5

88

27.4

97

27.3

110

26.4

118

27.6

13

60

22.8

69

22.3

62

19.3

77

21.7

99

23.7

86

20.1

22

8

3.0

12

3.9

7

2.2

8

2.3

18

4.3

9

2.1

23

18

6.8

20

6.5

23

7.2

25

7.0

26

6.2

29

6.8

33

1

0.4

9

2.9

7

2.2

3

0.8

10

2.4

10

2.3

Total

263

310

321

355

417

427

SNPrs17080476

Isolated defects

Multiple defects

CLP

Fathers

Mothers

Cases

Fathers

Mothers

Cases

Controls

n

%

n

%

n

%

n

%

n

%

n

%

n

%

AA

268

68.2

296

63.1

320

63.1

285

67.1

320

62.9

344

62.5

660

65.9

AG

110

28.0

160

34.1

170

33.5

124

29.2

176

34.6

188

34.2

308

30.8

GG

15

3.8

13

2.8

17

3.4

16

3.8

13

2.6

18

3.3

33

3.3

Total

393

469

507

425

509

550

1001

Isolated defects

Multiple defects

CPO

Fathers

Mothers

Cases

Fathers

Mothers

Cases

n

%

n

%

n

%

n

%

n

%

n

%

AA

173

65.5

205

65.7

214

65.6

234

65.9

274

65.6

288

66.8

AG

86

32.6

93

29.8

100

30.7

115

32.4

127

30.4

125

29.0

GG

5

1.9

14

4.5

12

3.7

6

1.7

17

4.1

18

4.2

Total

264

312

326

355

418

431

MTHFD1LGenotyping Results in Triads (Cases, Mother and Fathers) and Controls for CLP and CPO (Isolated) or with Other Defects (Multiple)

<p>Table 2</p>

Allele

Passed

Not Passed

GRR1 (95% CI)

P-value

1 GRR = genotype relative risk

2 CI = confidence interval

3 Significant values are marked in bold.

DIP rs3832406

n

%

n

%

Isolated CLP

1

194

55.6

155

44.4

1.3 (1.0, 1.5)

0.0372

2

119

47.2

133

52.8

0.9 (0.7, 1.1)

0.3781

3

88

43.8

113

56.2

0.8 (0.6, 1.0)

0.0786

Total

401

401

Multiple CLP

1

216

56.5

166

43.5

1.3 (1.1, 1.6)

0.0107

2

128

46.5

147

53.5

0.9 (0.7, 1.1)

0.2523

3

92

42.8

123

57.2

0.7 (0.6,1.0)

0.0351

Total

436

436

Isolated CPO

1

145

52.5

131

47.5

1.1 (0.9, 1.4)

0.3996

2

86

47.8

94

52.2

0.9 (0.7, 1.2)

0.5511

3

77

48.1

83

51.9

0.9 (0.7, 1.3)

0.6353

Total

308

308

Multiple CPO

1

188

52.2

172

47.8

1.1 (0.9, 1.3)

0.3992

2

115

48.9

120

51.1

1.0 (0.7, 1.2)

0.7443

3

101

47.4

112

52.6

0.9 (0.7, 1.2)

0.4512

Total

404

404

SNP rs17080476

Isolated CLP

G

132

54.3

111

45.7

1.2 (0.9, 1.5)

0.1785

A

111

45.7

132

54.3

Total

243

243

Multiple CLP

G

144

54.3

121

45.7

1.2 (0.9, 1.5)

0.1582

A

121

45.7

144

54.3

Total

265

265

Isolated CPO

G

87

50.6

85

49.4

1.0 (0.8, 1.4)

0.8788

A

85

49.4

87

50.6

Total

172

172

Multiple CPO

G

119

50.9

115

49.1

1.0 (0.8, 1.3)

0.7937

A

115

49.1

119

50.9

Total

234

234

TDT analysis for DIP rs3832406 and SNP rs17080476 in all cleft sample

<p>Table 3</p>

Polymorphism/Allele

Name

Dominant

Recessive

Multiplicative

1 OR = odds ratio

2 CI = confidence interval

3 Significant values are marked in bold.

OR1(95% CI2)

p- value

OR(95% CI)

p-value

OR (95% CI)

p-value

Isolated CLP

DIP Allele 1

Case-CTRL

1.1 (0.8, 1.5)

0.6159

1 (0.8, 1.3)

0.8468

1 (0.9, 1.2)

0.7022

Mother-CTRL

0.9 (0.6, 1.2)

0.3917

0.9 (0.7, 1.1)

0.4774

0.9 (0.8, 1.1)

0.3476

DIP Allele 2

Case-CTRL

1.1 (0.9, 1.4)

0.3358

0.8 (0.5, 1.4)

0.4926

1.1 (0.9, 1.3)

0.5534

Mother-CTRL

1.1 (0.9, 1.4)

0.2363

1.2 (0.7, 2)

0.5077

1.1 (0.9, 1.4)

0.2176

DIP Allele 3

Case-CTRL

0.9 (0.7, 1.1)

0.1928

1 (0.5, 2)

0.9158

0.9 (0.7, 1.1)

0.2349

Mother-CTRL

1 (0.8, 1.3)

0.9044

0.7 (0.3, 1.5)

0.3311

1 (0.8, 1.2)

0.8726

SNP

Case-CTRL

1 (0.6, 1.8)

0.9798

0.9 (0.7, 1.1)

0.1816

0.9 (0.7, 1.1)

0.2494

Mother-CTRL

1.3 (0.7, 2.5)

0.4286

0.9 (0.7, 1.1)

0.2382

0.9 (0.8, 1.1)

0.4336

Multiple CLP

DIP Allele 1

Case-CTRL

1 (0.7, 1.4)

0.9697

1 (0.8, 1.2)

0.9333

1 (0.8, 1.2)

0.9358

Mother-CTRL

0.8 (0.6, 1.2)

0.2857

0.9 (0.7, 1.2)

0.4737

0.9 (0.8, 1.1)

0.2955

DIP Allele 2

Case-CTRL

1.1 (0.9, 1.4)

0.258

0.9 (0.5, 1.6)

0.6969

1.1 (0.9, 1.3)

0.4046

Mother-CTRL

1.1 (0.9, 1.4)

0.2396

1.3 (0.8, 2.2)

0.3264

1.1 (0.9, 1.4)

0.1809

DIP Allele 3

Case-CTRL

0.9 (0.7, 1.1)

0.323

1 (0.5, 2.2)

0.9053

0.9 (0.7, 1.1)

0.3998

Mother-CTRL

1 (0.8, 1.3)

0.9303

0.7 (0.3, 1.7)

0.4316

1 (0.8, 1.2)

0.8883

SNP

Case-CTRL

1 (0.5, 1.8)

0.9538

0.9 (0.7, 1.1)

0.2786

0.9 (0.8, 1.1)

0.3381

Mother-CTRL

1.2 (0.6, 2.3)

0.5906

0.9 (0.7, 1.1)

0.2905

0.9 (0.8, 1.1)

0.4526

Isolated CPO

DIP Allele 1

Case-CTRL

1 (0.7, 1.4)

0.9498

1 (0.8, 1.2)

0.6933

1 (0.8, 1.2)

0.7915

Mother-CTRL

0.9 (0.6, 1.2)

0.3981

0.8 (0.6, 1)

0.0708

0.9 (0.7, 1)

0.0808

DIP Allele 2

Case-CTRL

1 (0.8, 1.3)

0.8703

0.5 (0.2, 1.1)

0.0749

1 (0.8, 1.2)

0.6497

Mother-CTRL

1 (0.8, 1.3)

0.7614

1.1 (0.6, 1.9)

0.798

1 (0.8, 1.3)

0.7284

DIP Allele 3

Case-CTRL

1.1 (0.9, 1.4)

0.3873

1.1 (0.5, 2.4)

0.7843

1.1 (0.9, 1.4)

0.3963

Mother-CTRL

1.3 (1, 1.7)

0.04313

1.1 (0.5, 2.4)

0.7368

1.2 (1, 1.5)

0.0576

SNP

Case-CTRL

0.8 (0.4, 1.4)

0.4111

1 (0.8, 1.3)

0.7454

1 (0.8, 1.2)

1

Mother-CTRL

0.8 (0.4, 1.5)

0.4739

1 (0.8, 1.3)

0.8894

1 (0.8, 1.2)

0.7186

Multiple CPO

DIP Allele 1

Case-CTRL

1 (0.7, 1.5)

0.9335

1 (0.8, 1.3)

0.9081

1 (0.8, 1.2)

0.8998

Mother-CTRL

0.8 (0.6, 1.2)

0.3735

0.8 (0.7, 1.1)

0.2065

0.9 (0.7, 1.1)

0.1723

DIP Allele 2

Case-CTRL

1 (0.8, 1.3)

0.8253

0.5 (0.2, 1.2)

0.1285

1 (0.8, 1.2)

0.7474

Mother-CTRL

1 (0.8, 1.3)

0.824

1 (0.5, 1.9)

0.9076

1 (0.8, 1.3)

0.882

DIP Allele 3

Case-CTRL

1.1 (0.8, 1.4)

0.5704

1 (0.4, 2.5)

0.9392

1.1 (0.8, 1.4)

0.598

Mother-CTRL

1.2 (0.9, 1.6)

0.1179

1.4 (0.6, 3.1)

0.4179

1.2 (1, 1.6)

0.1044

SNP

Case-CTRL

0.9 (0.5, 1.7)

0.7393

1 (0.8, 1.3)

0.9235

1 (0.8, 1.2)

0.847

Mother-CTRL

0.7 (0.4, 1.4)

0.3247

1 (0.8, 1.3)

0.9406

1 (0.8, 1.2)

0.6925

Logistic regression analysis of case/controls and mother/controls for DIP rs3832406 and SNP rs17080476 in all cleft samples

Conclusion

Our analysis shows no strong association between specific polymorphisms within the MTHFD1L gene and risk of cleft in an Irish cohort. The main limitation of our study would be sample size and the uncorrected p-values do indicate a possible association between the rs3832406 DIP and risk of CLP. However, we suggest further screening of rs3832406 DIP in a larger cohort and describe a new assay that will facilitate this. We have demonstrated that the modified Melting Curve Analysis developed for DIP rs3832406 could be a valid alternative to capillary electrophoresis for the genotyping of multiple allele deletion/insertion polymorphisms and can be employed by any laboratory with a Real-Time PCR instrument with melting curve capacity.

Abbreviations

CLP, Cleft lip with or without cleft palate; CPO, Cleft palate only; DIP, Deletion/insertion polymorphism; HWE, Hardy-Weinberg equilibrium; LD, Linkage disequilibrium; MTHFD1, Methylenetetrahydrofolate dehydrogenase (NADP + dependent); MTHFD1L, Methylenetetrahydrofolate dehydrogenase (NADP + dependent) 1-like; MTHFR, Methylenetetrahydrofolate reductase; MTR, Methionine synthase; MTHFS, Methenyltetrahydrofolate synthetase; NTD, Neural tube defect; SNP, Single nucleotide polymorphism; TDT, Transmission disequilibrium test.

Competing interest

The authors declare that they have no competing interests.

Authors’ contributions

SM designed and carried out the genotyping assays, performed data analysis and drafted the paper. AM participated in the study design and execution, and commented on the manuscript. PK participated in the study design. JM participated in the study design and commented on the manuscript. JS participated in the study design. JT performed the statistical analyses. FP participated in the study design and commented on the manuscript. LB participated in the study design. APM participated in genotyping assay and study design, data interpretation and drafting of the paper. All authors read and approved the final manuscript.

Acknowledgements

This work was supported by the IRCSET Embark initiative. The authors sincerely thank all the patients and families who participated in the study, the Cleft Lip and Palate Association of Ireland, and the Dublin Cleft Centre team. Recruitment of the Irish cleft cohort was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development and the Health Research Board of Ireland.

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