Volume 116A, Issue 4 pp. 414-415
Correspondence
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Lack of association between ZIC2 and ZIC3 genes and the risk of neural tube defects (NTDs) in hispanic populations

Huiping Zhu

Huiping Zhu

Center for Environmental and Genetic Medicine, Institute of Bioscience and Technology, Texas A&M University System Health Science Center, Houston, Texas

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Wade M. Junker

Wade M. Junker

Center for Environmental and Genetic Medicine, Institute of Bioscience and Technology, Texas A&M University System Health Science Center, Houston, Texas

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Richard H. Finnell

Corresponding Author

Richard H. Finnell

Center for Environmental and Genetic Medicine, Institute of Bioscience and Technology, Texas A&M University System Health Science Center, Houston, Texas

Institute of Biosciences and Technology, Texas A&M University System Health Science Center, 2121 W. Holcombe Blvd., Houston, Texas 77030-3303.Search for more papers by this author
Stephen Brown

Stephen Brown

Department of Obstetrics and Gynecology, Columbia University, New York, New York

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Gary M. Shaw

Gary M. Shaw

March of Dimes Birth Defects Foundation, California Birth Defects Monitoring Program, Oakland, California

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Edward J. Lammer

Edward J. Lammer

Children's Hospital Research Institute, Oakland, California

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Mark Canfield

Mark Canfield

Texas Department of Health, Austin, Texas

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Kate Hendricks

Kate Hendricks

Texas Department of Health, Austin, Texas

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First published: 18 December 2002
Citations: 13

Contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

To the Editor:

The Zic genes are a family of transcription factors that are homologous to the Drosophila odd-paired (opa) genes. Mutations in the ZIC1, ZIC2 and ZIC3 genes have been previously described, and all result in neural malformations [Brown et al., 1998; Brown et al., 2001; 2002; Carrel et al., 2001]. Several studies indicated that mutations in the ZIC2 gene might cause holoprosencephaly (HPE) in humans [Brown et al., 1998; Brown et al., 2001]. The human ZIC2 gene is located at chromosome 13q32, which is a critical region in 13q-syndrome. Abnormalities of neural tube closure, including encephalocele and anencephaly, have been described in association with the 13q-syndrome [Brown et al., 1993, 1995]. In light of mouse studies showing that diminished expression of the Zic2 gene also resulted in lumbosacral neural tube defects (NTDs), ZIC2 may be an excellent candidate gene for human NTDs.

Studies of the mouse mutant, Bent tail (Bn), suggest a relationship between another Zic gene family member, Zic3, and the risk of NTDs [Klootwijk et al., 2000]. Bn is a mouse model for X-linked NTDs, as the fetuses present with exencephaly, an analogous defect of human anencephaly. Mice with Zic3 mutations were observed in more than 10% of all Bn embryos with NTDs. Mutations in human ZIC3 gene have been described in male patients with left-right axis malformations, and some of them had lumbosacral NTDs [Gebbia et al., 1997]. Carrel et al. [2001] sequenced the three exons of the ZIC3 gene in three families with X-linked spina bifida, yet failed to find mutations or any single nucleotide polymorphisms. The evidence from mouse studies and limited clinical observations suggests that the ZIC3 gene might also be worthy of study as a candidate gene for human NTDs.

Brown et al. [2002] recently described a possible association between a histidine tract polymorphism in ZIC2 and NTDs. However, their sample size was relatively small, which prevented definitive conclusions. We evaluated ZIC2 and ZIC3 genes in a group of NTD patients and controls, as well as their parents, in a Hispanic population from the Texas-Mexico border region. This population has a considerably higher prevalence of NTDs (16/10,000 live births) than is generally reported in the United States (8–10/10,000 live births). NTDs were defined as spina bifida or anencephaly. The study population has been described previously [Barber et al., 2000].

Subjects were selected from births and pregnancy terminations occurring between June 1, 1995 and September 30, 1998 in a largely Hispanic population from the 14 counties along the Texas-Mexico border. Cases were identified in hospitals, birthing centers, ultrasound centers, abortion centers, prenatal clinics, genetics clinics, and by birth attendants (midwives and nonhospital physicians). An NTD was defined as spina bifida or anencephaly. Controls were healthy livebirths that occurred in the same counties during that time period. Parents of NTD cases and control infants were also included.

Genomic DNA was obtained from Gutherie cards using the Puregene DNA Extraction Kit (Gentra, Minneapolis, MN). Primers designed to amplify the ZIC2 fragment encompassing the histidine tract were: upstream: 5′-aac tcc aca acc agt acg gc-3′; downstream: 5′-gca tat agc gga aaa agg ca-3′. The histidine tract polymorphism was determined by the fragment sizes (117bp or 120bp). The upstream primer was labeled at 5′ end by fluorescent dye 6-FAM (Integrated DNA Technology, Coralville, IA), so that the fragment size could be determined by Genescan analysis on an ABI3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). Exons 1, 2 and 3 of the ZIC3 gene were amplified using primers as designed by Carrel et al. [2001]. Samples were screened for sequence variants by automatic sequence analysis using the ABI3100 Genetic Analyzer.

Our results from the ZIC2 histidine track polymorphism analysis demonstrate that among 69 control infants, allele frequencies for 9H and 10H alleles were 89.9% and 10.1%, respectively. The frequency of the 10H allele among NTD case parents was 9.8%, while that of control parents was 11.4%. However, we did not observe a higher frequency of the 10H allele among infants with NTDs (7.1%, n1 = 28). In fact, considering homozygous wild-type 9H/9H as a reference, the odds ratio for homozygous mutant type (10H/10H) was 0.5 (95% confidence interval = 0.03–7.4), and for heterozygotes (9H/10H) was 2.7 (95% confidence interval = 0.6–12.8). Neither of these effect estimates, however, was precise. Compared to Brown et al. [2002], we found higher frequencies of 9H/10H heterozygotes, as well as 10H/10H homozygotes among our Hispanic control samples. Our data do not support the hypothesis of an association between the ZIC2 histidine track polymorphism and an increased risk of NTDs. However, it is clear that the frequency of the 10H variant varies more than 20-fold depending on the population studied.

For the ZIC3 gene, DNA samples of 35 NTD infants and 35 control infants were sequenced. We did not observe any mutations or single nucleotide polymorphisms (SNPs) in any of the three exons, suggesting that ZIC3 is unlikely to have substantial impact on the NTDs observed in this Hispanic population.

In conclusion, this study found no supporting evidence for an association between ZIC2 or ZIC3 and the risk of NTDs. Because most of our samples are from surviving spina bifida patients, it remains possible that these genes are related to failure of neural tube closure at a higher anatomic level, such as anencephaly. The biological function of the ZIC2 histidine track polymorphism remains unknown, but there is evidence that this polymorphism is differentially distributed in different populations. Further studies focusing on the function of the ZIC2 histidine track polymorphism and a possible association of this polymorphism with higher anatomic NTDs may be worth pursuing.

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