Objective
Excessive oxidative stress has been demonstrated as a mechanism for neural tube defects (NTDs). The current exploratory study sought to examine sequence variations in the superoxide dismutase 1 ( SOD1 ) and 2 ( SOD2 ) genes in patients with myelomeningocele and to identify variants altering risk for myelomeningocele.
Study Design
We sequenced deoxyribonucleic acid from 96 patients with myelomeningocele. The 11 exons were amplified by polymerase chain reaction, and the products were sequenced with the Sanger method. Results were compared with reference sequences ( NM_000454 , NM_000636 , and NM_001024466 ) obtained from University of California Santa Cruz Genome Browser. Observed alleles that differed from the reference sequences were considered novel variants.
Results
We found 1 novel variant and 1 variant only recently described in phase 1 of the 1000 Genomes Project but not yet validated. The novel variant is located in the 3′-untranslated region (UTR) of SOD2 and is present in 2 of 96 patients (1.0% allele frequency). The other variant is located in the 3′-UTR of SOD1 and is present in 2 of 96 patients (1.0% allele frequency). Minor allele frequencies of known single nucleotide polymorphisms were compared with unaffected population controls.
Conclusion
We identified 1 novel variant and made the second report of an additional variant in the SOD genes studied. The variant located in the 3′-UTR of SOD1 is predicted to alter microribonucleic acid (miRNA) binding. The variant located in the 3′-UTR of SOD2 is predicted to alter 2 miRNA binding sites and potentially affects messenger ribonucleic acid production. We also identified 2 known single-nucleotide polymorphisms that occur in significantly different frequency compared with the unaffected population controls.
Neural tube defects (NTDs) are heterogeneous congenital malformations of the brain and spinal cord resulting from failure of normal neural tube to closure by 28 days after conception. NTDs (both anencephaly and spina bifida) have a worldwide incidence at birth of approximately 0.5-1 in 1000 and are the most common structural malformations of the central nervous system in humans. A common and severe NTD phenotype, myelomeningocele, represents more than 90% of live-born infants with spina bifida. Most individuals living with myelomeningocele have a multiple disabilities and a shortened life expectancy. However, in spite of a high prevalence and major morbidity, the etiology of NTDs is not well understood.
The exact cause of an individual NTD is typically not known, but clustering within families as well as associations with specific genetic syndromes suggests that these phenotypes are related and are caused by an underlying genetic susceptibility. NTDs exhibit a multifactorial inheritance pattern, with genetic and environmental influences. Identifying the hereditary basis of NTDs and the contributing genetic factors is critical for characterizing the interactions between genes and the environment.
The role of folate metabolism in the development of NTDs prompted the US Food and Drug Administration to mandate fortification of all cereal grains with folate in 1998. The result was a significant decrease in the rate of NTDs by 20-30%. However, in spite of folate supplementation, NTDs still occur with a prevalence of 3.5 per 10,000, likely involving other genetic and environmental influences.
It has been demonstrated that increased oxidative stress (such as seen with maternal diabetes) influences a change in embryonic gene expression and enzymatic pathways that contribute to key developmental processes, resulting in an increased risk of congenital defects. The immature antioxidant defense mechanism characteristic of early embryonic development compounds the impact of reactive oxygen species on the developing embryo.
The superoxide dismutase (SOD) enzymes, which produce hydrogen peroxide from superoxide radicals, are key protective mechanisms in the body’s defense against reactive oxygen species. Studies have found that that addition of SOD or antioxidants to diabetic culture medium reduces the frequency of embryonic defects. Dietary copper deficiency has also been associated with malformed embryos and considerably reduced SOD activity, supporting a contributing environmental influence on NTD formation. Transgenic murine studies have also demonstrated that overexpression of SOD enzymes results in reduced neural malformation rates in embryos exposed to high levels of oxidative stress. Aberrations of SOD function are therefore a possible mechanism by which oxidative stress may contribute to the risk of congenital anomalies, including NTDs.
We recently reported that 5 single-nucleotide polymorphisms (SNPs) in the SOD1 and SOD2 genes were implicated in myelomeningocele risk. Four of the 5 SNPs were located in the intronic regions remote from the exons, and one (rs2070424) is approximately 260 bp upstream from exon 4 of SOD1 . As a follow-up study, we sequenced the 11 exons of the SOD1 and SOD2 genes of deoxyribonucleic acid (DNA) obtained from a selected population of children born after 1998 with myelomeningocele to develop a more comprehensive understanding of variants in the SOD1 and SOD2 genes potentially altering myelomeningocele risk.
Materials and Methods
The children with a myelomeningocele in our study are a subset from a larger cohort of 864 myelomeningocele patients enrolled in ongoing genetic studies in our laboratory. The study was approved by the institutional review board of the University of Texas Health Science Center at Houston. The study population characteristics are further described in detail elsewhere.
The 96 children with nonsyndromic myelomeningocele tested in this study included 48 whites of European descent and 48 Hispanics of Mexican descent living in the United States or Canada. Hispanics of Mexican descent are of particular importance because they are underrepresented in genetic databases and have the highest prevalence of NTDs in the United States (approximately 4 per 10,000).
A sample size of 96 was utilized because of the configuration of the standard polymerase chain reaction (PCR) plate (8 rows × 12 columns = 96). Additionally, each child has 2 alleles (96 + 96 = 192 total alleles) so that rare variants with an approximately 0.5% population frequency (1 of 192) may be detected. Patients were selected with the goal of having an equal representation of white and Hispanic individuals and to maximize those patients in whom DNA was also available from both parents. We included only myelomeningocele affected patients born after the 1998 mandatory folic acid fortification of the US diet to select those patients whose mothers were more likely to have adequate dietary folate.
Blood and saliva samples were obtained from the patients and their parents. Genomic DNA was extracted from whole blood using the Puregene DNA purification kit (Gentra Systems, Minneapolis, MN). DNA from saliva was extracted using the Oragene DNA preparation kit (DNA Genotek; Kanata, Ontario, Canada). Stock DNA was then stored at –80°C. Working DNA aliquots of 10 ng/μL were prepared for PCR at the start of the study following whole genome amplification (GenomiPhi; GE Healthcare, Piscataway, NJ).
PCR and nested-sequencing primers were designed based on the reference genomic sequences of SOD1 ( NM_000454 ) and SOD2 ( NM_000636 , NM_001024466 ) extracted from the University of Santa Cruz Genome Browser (University of California Santa Cruz Genome Browser; Available at: http://genome.ucsc.edu/cgibin/hgGateway . Accessed Feb. 5, 2013). We included approximately 50-100 bases flanking the boundaries of exons to also examine the splice donors and acceptors. A total of 6 PCR primer pairs were designed for SOD1 and 9 primer pairs for SOD2 (available upon request) synthesized by Integrated DNA Technologies USA (Commercial Park, Coralville, IA).
PCR amplification of exons was performed using the MJ Research PTC-100 Programmable Thermal Cycler (MJ Research, Waltham, MA). We confirmed the PCR product sizes by 1.8% agarose gel electrophoresis. The amplified exon DNA was then treated with T7 exonuclease and shrimp alkaline phosphatase (United States Biochemicals, Affymetrix, Cleveland, OH) to remove excess primers and nucleotides. The digested product was subjected to Sanger sequencing using the BigDyeTerminator (Applied Biosystems, Foster City, CA) reagents with nested sequencing primers and resolved on the ABI3100 Genetic Analyzer (Life Technologies Inc, Grand Island, NY).
In cases in which new SNPs were identified, we utilized custom TaqMan SNP genotyping assays with fluorescently labeled probes (Life Technologies) following the manufacturer’s protocol to interrogate our total cohort of 864 patients. We added as controls 92 Mexican-American volunteers recruited from the Houston area and 93 white individuals from the Human Variation Panel-Caucasian Panel of 100 (HD100CAU; Coriell Institute for Medical Research, Camden, NJ) with no personal or family history of NTDs. The genotyping results were read using the 7900HT Fast real-time PCR system (Life Technologies) and analyzed using the 7900HT sequence detection system software.
Sequence results were analyzed using the DNA Sequencing Analysis Software version 5.1 from Applied Biosystems. To identify variants in sequences, we manually compared results with the reference sequences ( NM_000454 , NM_000636 , NM_001024466 ) obtained from the University of California Santa Cruz (UCSC) Genome Browser. The white reference population (CEU) includes unaffected whites from Utah included in the Centre d’Étude du Polymorphisme Humain Collection for mapping genetic markers. The Mexican American (MEX) reference population includes unaffected Mexican Americans from Los Angeles, CA, used in the International HapMap project, the single-nucleotide polymorphism database (dbSNP). These reference populations have been shown to be genetically very similar to the 2 patients group in our study.
Allele frequencies of variants were tallied, and any variant not reported in the dbSNP was considered a novel variant. Potential variants were subjected to a repeat PCR, and a reverse sequencing reaction to confirm the validity of the variant. The allele frequencies of known SNPs among myelomeningocele patients were compared with the SNP frequencies among ethnically matched unaffected population controls reported in the dbSNP and compared with a Fisher test (when possible). A 2-tailed P < .05 was considered significant.
Results
In sequencing the exons of the SOD1 and SOD2 genes among 96 children with myelomeningocele, we found a novel variant at c.*68G>A within the 3′-untranslated region (UTR) of SOD2 not previously reported in public SNP databases ( Figure 1 , A). Aligning sequences of the SOD2 3′-UTR variant ( NM_001024466 ; c.*68G>A) in humans to 23 different available vertebrates using the UCSC Genome Browser (Available at: http://genome.ucsc.edu/index.html ; Accessed March 14, 2013) shows the reference allele c.*68G to be highly conserved, particularly among primates ( Figure 1 , B).
We additionally noted a variant only recently described in the phase 1 of the 1000 Genomes Project, and not yet validate at c.*153T>G (rs188029963), located in the 3′-UTR of SOD1 ( Figure 2 , A). Aligning sequences of the SOD1 3′-UTR of 30 different vertebrate species using the UCSC Genome Browser shows the T allele (reference allele) at c.*153T>G to be highly conserved and only different in the lizard and pika ( Figure 2 , B). In both cases, DNA from each affected individual’s parents was available for sequencing, and this variant was present in 1 parent of each affected child.
We evaluated 25 SNPs within or flanking the exons of SOD1 and 35 SNPs within or flanking the exons of SOD2 . We report those SNPs in which a rare allele was identified among our cohort ( Tables 1 and 2 ). Sixteen of the 25 SNPs evaluated in SOD1 do not have population frequencies reported for the Caucasian reference population (CEU), and 24 of 25 do not have frequencies reported for the reference Mexican American (MEX) populations. Only 3 SNPs and 1 insertion/deletion demonstrated heterozygosity among white American children, and only 1 SNP demonstrated heterozygosity among Mexican American children for SOD1 ( Table 1 ). Thirteen of the 35 SNPs studied in SOD2 do not have population frequencies reported for the CEU reference population, and 26 of the 35 SNPs studied do not have frequencies reported for the reference MEX populations.
| Variable | Minor allele (A2) frequency | ||||||
|---|---|---|---|---|---|---|---|
| CEU | MEX | CEU/MEX | |||||
| SNP | A1/A2 | All MM | MM | Referent | MM | Referent a | P value |
| SOD1 | |||||||
| 21:33036248 | A/ATAAACAG | 0.50% (1/190) | 1.00% (1/96) | 0.44% (36/8218) b | 0.00% | ND | .34/– |
| rs2234694 | A/C | 2.10% (4/190) | 3.20% (3/94) | 4.20% (5/120) c | 1.00% (1/96) | ND | .79/– |
| rs188029963 | T/G | 1.00% (2/192) | 2.10% (2/96) | ND | 0.00% | ND | –/– |
| rs17880487 | C/T | 1.60% (3/190) | 1.00% (1/96) | 0.40% (1/226) b | 2.10% (2/94) | 4.20% (5/116) | .44/.46 |
a The MEX population consists of Mexican ancestry from Los Angeles, CA (International HapMap Project).
c The CEU population consists of Utah residents with northern and western European ancestry from the Centre d’Etude du Polymorphisme Humain collection (International HapMap Project).
| Variable | Minor allele (A2) frequency | ||||||
|---|---|---|---|---|---|---|---|
| CEU | MEX | CEU/MEX | |||||
| SOD2 | |||||||
| SNP | A1/A2 | All MM | MM | Referent | MM | Referent a | P value |
| rs5746092 | G/C | 20.1% (37/184) | 22.90% (22/96) | 25.80% (31/120) b | 17.00% (15/88) | ND | .63/– |
| rs5746093 | C/G | 0.54% (1/188) | 1.00% (1/96) | ND | 0.00% | ND | –/– |
| rs5746094 | T/C | 20.21% (38/188) | 22. 9% (22/96) | 25.00% (30/120) b | 17.40% (16/92) | ND | .75/– |
| rs4880 | A/G | 52.63% (100/190) | 42.71% (41/96) | 43.94% (145/330) c | 62.77% (59/94) | 63.53% (108/170) | .14/1.00 |
| rs4987023 | C/T | 0.00% | 0.00% | 0.00% | 0.00% | 1.70% (2/116) | –/– |
| rs2758332 | G/T | 53.13% (102/192) | 47.92% (46/96) | 41.70% (50/120) b | 58.33% (56/96) | ND | .33/– |
| rs5746133 | G/C | 0.00% | 0.00% | 2.30% (4/174) c | 0.00% | ND | –/– |
| rs5746134* | C/T | 4.17% (8/192) | 1.04% (1/96) | 0.30% (1/330) c | 7.29% (7/96) | 0.60% (1/166) | .24/.004 |
| rs5746135 | G/A | 4.17% (8/192) | 1.04% (1/96) | 0.30% (1/328) c | 7.29% (7/96) | 13.53% (23/170) | .40/.15 |
| rs5746136 | G/A | 25.52% (49/192) | 29.17% (28/96) | 31.82% (105/330) c | 21.88% (21/96) | 25.58% (44/172) | .71/.55 |
| rs5746137 | A/T | 1.04% (2/192) | 2.08% (2/96) | 1.70% (2/120) b | 0.00% | ND | 1.00/– |
| rs5746138 | A/G | 4.17% (8/192) | 3.13% (3/96) | 7.50% (9/120) b | 5.21% (5/96) | ND | .23/– |
| rs5746140 | C/A | 7.29% (14/192) | 1.04% (1/96) | ND | 13.54% (13/96) | 9.87% (15/152) | –/.41 |
| rs5746141* | G/A | 2.63% (5/190) | 2.08% (2/96) | 9.39% (31/330) c | 3.19% (3/94) | 2.91% (5/172) | .028/1.00 |
| rs7855 | T/C | 5.21% (10/192) | 6.25% (6/96) | 6.06% (20/330) c | 4.17% (4/96) | 1.74% (3/172) | 1.00/.43 |
| 6:160100282 | G/A | 1.04% (2/192) | 2.08% (2/96) | ND | 0.00% | ND | –/– |
| rs2842980 | A/T | 16.15% (31/192) | 20.80% (20/96) | 24.70% (81/328) c | 11.46% (11/96) | 11.31% (19/169) | .50/1.00 |
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