A number of studies have indicated that 8p22–p12 is likely to harbor schizophrenia susceptibility loci. In this region, the candidate gene of interest, neuregulin 1 (NRG1), may play a role in the pathogenesis of schizophrenia. Then in the present study, we performed the linkage disequilibrium to determine the association between three genetic variants (SNPs: rs3924999, rs2954041, SNP8NRG221533) on NRG1 gene and schizophrenia in 246 Chinese Han schizophrenic family trios using PCR-based restriction fragment length polymorphism method and denaturing high-performance liquid chromatography. The transmission disequilibrium test analysis for each variant showed a significant difference between two transmitted alleles even after Bonferroni correction (rs3924999, P=0.007752; rs2954041, P=0.0009309; SNP8NRG221533, P=0.012606). The global χ² test for haplotype transmission also revealed a strong association (χ²=46.068, df=7, P<0.000001). Our results suggest that the NRG1 gene may play a role in conferring susceptibility to the disease.

Schizophrenia is a complex disease, a common mental disorder with a prevalence of 0.5–1% in the general population. It is clinically characterized by disturbed thought processes, delusions, hallucinations, and/or reduced social skills.¹ A number of studies indicate a genetic component contributing to schizophrenia, but the mode of inheritance does not follow a simple Mendelian pattern. In spite of the failure to identify a schizophrenia gene of major import in multiply affected families, more than one data set from genome-wide scans provides convincing evidence that 8p22–p21 may harbor candidate genes which may confer susceptibility to schizophrenia to an individual.^2,3,4,5 Interestingly, two studies carried out by Stefansson et al^6,7 reported that the gene coding for neuregulin 1 (NRG1) was located in 8p21; its position as well as function strongly supported NRG1 gene as a susceptibility gene for schizophrenia. In their study, however, they had not found a clear pathogenic mutation. Several dozens of single nucleotide polymorphisms (SNPs) have been identified within the locus (http://www.ncbi.nlm.nih.gov/SNP/), of which we randomly selected two that can be detected by the restriction fragment length polymorphism (RFLP) analysis. Of the two SNPs, one SNP (rs3924999, G38A), a G to A base change, occurs in position 12 within the second exon of the NRG1 gene, and the database (http://www.ncbi.nlm.nih.gov/SNP/) shows that the SNP changes the amino acid from arginine (Arg) to glutamine (Gln) (Arg 38 Gln). Another SNP (rs2954041), located in fifth intron, may still be informative, for example, by being in linkage disequilibrium with a causal locus for schizophrenia. Therefore, it is valuable to investigate the possibility that these polymorphism sites on the NRG1 locus may be associated with schizophrenia. We also genotyped the single most significant SNP (SNP8NRG221533) described in Stefansson's findings^6,7 by denaturing high-performance liquid chromatography (dHPLC) in order to clarify the similarities or differences of SNP8NRG221533 polymorphism in the different populations.

In the present study, we conducted association analysis to determine the relationship between NRG1 gene and schizophrenia. As the transmission disequilibrium test (TDT), which evaluates allele transmissions from heterozygous parents to affected individuals, provides an index of association which is unbiased by population structure. Therefore, we performed a family-based test in the present study.

We analyzed the three polymorphisms of NRG1 gene in 246 Chinese Han schizophrenic family trios by the PCR-based RFLP and dHPLC. The genotype distributions and allelic frequencies are presented in Table 1. The TDT analysis for each locus showed a significant difference between two transmitted alleles (Table 2) even after Bonferroni correction (rs3924999, P= 0.007752; rs2954041, P=0.0009309; SNP8NRG221533, P=0.012606). As haplotype has more accuracy and statistical power than individual SNPs in linkage-disequilibrium-based association study, we estimated haplotype frequencies of the three SNPs with TRANSMIT software.⁸ The global χ² test for haplotype transmission also revealed a strong association between the NRG1 locus and schizophrenia (χ²=46.068, df=7, P<0.000001). As shown in Table 3, the 1-df test for individual haplotypes demonstrated that an excess of the TAG haplotype was transmitted by the parent to the affected offspring (χ²=17.134, df=1, P<0.000035).

NRG1 is one family member of structurally related glycoproteins that include NRG2, NRG3, and NRG4.⁹ NRG1 plays a role as a multifunctional factor in the neurodevelopment and many aspects such as cell differentiation. In mouse embryos of 14.5 days, Orr-Urtreger et al¹⁰ found that NRG1 expression was confined predominantly to the central and peripheral nervous systems, including the neuroepithelium lining the lateral ventricles of the brain, the ventral horn of the spinal cord, and the intestinal as well as dorsal root ganglia. Through a ribozyme-based strategy for the perturbation of NRG1 function in developing chick embryos, Zhao and Lemke¹¹ discovered that NRG1 could promote both muscle cell differentiation in the heart and neuronal differentiation in the central nervous system. Recently, Huang et al¹² reported that neuregulins and their receptors, the ERBB protein tyrosine kinases, are essential for neuronal development and involved in long-term potentiation in the hippocampal CA1 region without affecting basal synaptic transmission, the brain mechanisms considered important for memory formation, and they also considered that the signaling role of NRG in the central nervous system of adults may be involved in the modulation of synaptic plasticity.

In Stefansson et al's study,^6,7 they found that a core at-risk haplotype represented a block of linkage disequilibrium containing the 5′exon of NRG1 and upstream; however, the only SNP (SNP8NRG433E1006) in the exon of NRG1 was not found in significant excess in patients. In the present study, we conducted an association analysis to examine the relationship between the NRG1 locus and schizophrenia in a Chinese population. Both the TDT and the haplotype analysis showed a strong association between the NRG1 locus and schizophrenia. In our sample, the SNP8NRG221533, which gave the best single marker association in the study of Stefansson et al showed significant evidence, although its significance was less statistically than the other two polymorphisms. These results not only confirmed the finding reported by Stefansson et al, but also extended the linked haplotype block from the 5′exon of NRG1 to the fifth intron in Chinese Han people. In the haplotype block, the SNP rs3924999 is located in the second exon of the NRG1 gene (nucleotide sequence site 1200844), which is conservative in all isoforms of NRG1, and the database (http://www.ncbi.nlm.nih.gov/SNP/) shows that this SNP is a functional polymorphism (Arg 38 Gln). However, to examine whether the SNP is a clear pathogenic mutation in the protein level would be required. The other SNP rs2954041 also showed the linkage disequilibrium. Located in the fifth intron, the result suggested that this site might be linked with functional loci, either SNP rs3924999 or other loci. Thus, we could not exclude the other linkage polymorphic sites in NRG1 gene such as enhancers or other controlling sites.

In summary, our results, along with Stefansson's findings, suggested that the NRG1 gene might either play a role in predisposing an individual to schizophrenia or be in linkage disequilibrium with a causal locus for schizophrenia. Therefore, it is necessary to carry out further investigation to confirm these findings by replicating studies as well as genotyping haplotypes within the NRG1 and adjacent loci.

Subjects

Schizophrenic patients and their biological parents were recruited for the present study at the Institute of Mental Health, Peking University, China. All the subjects were Chinese of Han descent. All patients met the ICD-10 criteria. Of the patients, 138 (56%) were male and 108 (44%) female, mean age 29 years. The mean duration of illness was 5 years. All subjects gave informed consent for genetic analysis. The study was approved by the Ethics Committee of Peking University.

Peripheral blood samples were obtained from subjects and genomic DNA was extracted using the phenol–chloroform method.

Genotyping

PCR-RFLP

Two SNPs (rs3924999, rs2954041) were genotyped by the PCR-based RFLP analysis for all subjects. Primers and restriction enzymes used are described in Table 4. PCR amplification was performed in 25-μl reaction volume containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.001% (w/v) gelatin, 200 μM of each dNTP, 0.4 μM of each primer, 1.0 U of Taq DNA polymerase, and 40 ng genomic DNA. The conditions used for PCR amplification included an initial denaturation at 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, 55–62°C for 40 s, 72°C for 1 min, and a final elongation at 72°C for 10 min. A 15-μl aliquot of the PCR product mixtures was completely digested with 5 U of restriction enzyme and then separated on 4% (SNP1) and 5% (SNP2) agarose gels.

dHPLC

The DNA sequence containing the SNP8 NRG221533 was amplified with a pair of primers: 5′ GCA TTA GAA CTA GAA CTT GCG TGA 3′ and 5′ TGG GAA CTC TCC ATC TCT TTC A 3′. Before dHPLC analysis, PCR products were denatured by heating to 94°C for 1 min, and were then allowed to reanneal by cooling to 50°C over 25 min.

dHPLC. was performed using a WAVE DNA fragment analyzer (Transgenomic). Column temperature for dHPLC analysis was determined using software that is available free of charge over the Internet (http://insertion.standford.edu/melt.html).¹³ To ensure maximum sensitivity, in addition to the temperature suggested by the software (n°C), each fragment was also run at n±2°C. Where dHPLC analysis suggested that a subject was heterozygous, the PCR products from that individual were sequenced to determine the nature of the polymorphism using the Big Dye Terminator Cycle Sequencing Kit (Perkin-Elmer Applied Biosystems) as described by the manufacturer.

Statistical analysis

The TDT¹⁴ was applied to analyze allelic association in family trios consisting of father, mother, and affected offspring with schizophrenia, where preferential allelic transmission from heterozygous parents to affected offspring is tested by applying the (b−c)²/(b+c) statistics and the χ² (McNemar's test). The null hypothesis of no association indicates that each allele carried by a heterozygous parent has a 50% chance for transmission to an affected child. If the allele plays a causal role in the development of the disorder, then its transmission should exceed 50%. Haplotype frequency was estimated by TRANSMIT program, version 2.5.2.⁸ We applied Bonferroni corrections for all multiple statistical tests.

We express our gratitude to all patients and their family members, without whose cooperation, this work would not have been possible. We also thank Professor Lian Zhang, Professor Kaifeng Pang in Beijing Institute for Cancer Research in helping us to carry out dHPLC. This work has been supported by Grant H010210180112 from Beijing Biomedical R & D Innovation Program, Grant 30000059 from the National Natural Scientific Foundation of China, Grant 2000-A-A32 from Peking University Center for Human Disease Genomics, and Grant 2002BA711A07-06 from National Key Project.

Author notes

1. • J Z Yang
   •  & T M Si

   Contributed equally to this work.

Affiliations

1. Institute of Mental Health, Peking University, Beijing 100083, China

   • J Z Yang
   • , T M Si
   • , Y Ruan
   • , Y S Ling
   • , Y H Han
   • , X L Wang
   • , M Zhou
   • , H Y Zhang
   • , Q M Kong
   • , C Liu
   • , D R Zhang
   • , L Shu
   •  & D Zhang

2. The Center for Genomic Medicine, University of Jilin, Changchun 130021, China

   • Y Q Yu
   • , S Z Liu
   •  & G Z Ju

3. Peking University Center for Human Disease Genomics, Beijing 100083, China

   • D L Ma

Corresponding author

Correspondence to D Zhang.

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Publication history

DOI

https://doi.org/10.1038/sj.mp.4001377