Genotyping of a tri-allelic polymorphism by a novel melting curve assay in MTHFD1L: an association study of nonsyndromic Cleft in Ireland

  • Stefano Minguzzi1,

    Affiliated with

    • Anne M Molloy2,

      Affiliated with

      • Kirke Peadar3,

        Affiliated with

        • James Mills4,

          Affiliated with

          • John M Scott5,

            Affiliated with

            • James Troendle4,

              Affiliated with

              • Faith Pangilinan6,

                Affiliated with

                • Lawrence Brody6 and

                  Affiliated with

                  • Anne Parle-McDermott1Email author

                    Affiliated with

                    BMC Medical Genetics201213:29

                    DOI: 10.1186/1471-2350-13-29

                    Received: 19 December 2011

                    Accepted: 20 April 2012

                    Published: 20 April 2012

                    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 [14]. 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 [6, 7]. Numerous candidate gene association studies between clefts and folate related genes have shown mixed results and include methylenetetrahydrofolate reductase (MTHFR [Genbank: NP_005948.3]) [4, 816], methylenetetrahydrofolate dehydrogenase (NADP + dependent) (MTHFD1 [Genbank: NP_005947.3]) [1, 16, 17], 5,10-methenyltetrahydrofolate synthetase (MTHFS [Genbank: NP_001186689.1]) and methionine synthase (MTR [Genbank: NP_000245.2]) [4, 1719]. 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 [21, 22] 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 [16, 27]. 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).
                    http://static-content.springer.com/image/art%3A10.1186%2F1471-2350-13-29/MediaObjects/12881_2011_944_Fig1_HTML.jpg
                    Figure 1

                    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).
                    Table 1

                    MTHFD1L Genotyping Results in Triads (Cases, Mother and Fathers) and Controls for CLP and CPO (Isolated) or with Other Defects (Multiple)

                    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

                       
                    Table 2

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

                      

                    Allele

                    Passed

                    Not Passed

                    GRR1 (95% CI)

                    P-value

                    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

                       

                    1 GRR = genotype relative risk

                    2 CI = confidence interval

                    3 Significant values are marked in bold.

                    Table 3

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

                     

                    Polymorphism/Allele

                    Name

                    Dominant

                    Recessive

                    Multiplicative

                       

                    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)

                    0.0431 3

                    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

                    1 OR = odds ratio

                    2 CI = confidence interval

                    3 Significant values are marked in bold.

                    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.

                    Declarations

                    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.

                    Authors’ Affiliations

                    (1)
                    Nutritional Genomics Group, School of Biotechnology, Dublin City University
                    (2)
                    School of Medicine, Trinity College Dublin
                    (3)
                    Child Health Epidemiology Unit, Health Research Board
                    (4)
                    Department of Health and Human Services, Eunice Kennedy Shriver National Institute of Health
                    (5)
                    School of Immunology & Biochemistry, Trinity College Dublin
                    (6)
                    Molecular Pathogenesis Section, Genome Technology Branch, National Human Genome Research Institute

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                    30. Pre-publication history

                      1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1471-2350/​13/​29/​prepub

                    Copyright

                    © Minguzzi et al.; licensee BioMed Central Ltd. 2012