American Journal of Medical Genetics 105:20–22 (2001) Brief Research Communication Linkage Disequilibrium for Schizophrenia at the Chromosome 15q13-14 Locus of the ␣7-Nicotinic Acetylcholine Receptor Subunit Gene (CHRNA7) Robert Freedman,1* Sherry Leonard,1 Judith M. Gault,1 Jan Hopkins,1 C. Robert Cloninger,2 Charles A. Kaufmann,2 Ming T. Tsuang,2 Stephen V. Farone,2 Dolores Malaspina,2 Dragan M. Svrakic,2 Alan Sanders,3 and Pablo Gejman3 1Departments of Psychiatry and Pharmacology, University of Colorado Health Sciences Center and Denver VA Medical Center, Denver, Colorado 2NIMH Schizophrenia Genetics Initiative, Bethesda, Maryland 3Department of Psychiatry, University of Chicago, Chicago, Illinois The transmission/disequilibrium test was We reported an initial linkage for a neurophysiologi- used for fine mapping of the linkage of cal phenotype associated with schizophrenia to genetic schizophrenia to the chromosome 15q13-14 markers at chromosome 15q13-14 in nine multiply af- region, the site of a candidate gene, the ␣7 fected, European American families. The lod score for nicotinic acetylcholine receptor subunit the neurophysiological phenotype at D15S1360 was Z -P < 0.001; the lod score for schizophre .0.0 ס ␪ ,5.3 ס gene (CHRNA7), in parent–child triads from P < 0.007, in an ,0.07 ס ␪ ,1.3 ס the NIMH Schizophrenia Genetics Initiative nia was lower, Z families. This candidate gene was identified affecteds-only model [Freedman et al., 1997]. The lod from neurobiological studies of deficits in score for schizophrenia was less than the lod score for schizophrenics of the inhibitory gating of the neurophysiological phenotype, principally because the P50 auditory evoked potential. The neu- the neurophysiological phenotype diminished inhibi- robiological deficit was also used as a phe- tion of the P50 auditory evoked response to repeated notype for subsequent linkage analysis. In the present study, significant genotype-wise stimuli, has a highly penetrant dominant inheritance, disequilibrium (P < 0.007) was found at whereas schizophrenia is a less penetrant and more D15S165, a polymorphic simple sequence heterogeneous phenotype [Freedman et al., 1999]. Al- marker physically located within 1 mega- though biological phenotypes have been successfully base of both CHRNA7 and a partially dupli- used as surrogates for the genetic analyses of other cated, expressed sequence that includes ex- illnesses, including colon cancer (polyp formation), ons 5–10 of CHRNA7. Replication of this re- sickle-cell anemia (laboratory demonstration of red cell sult was found in an additional set of sickling), and hemochromatosis (serum iron levels), families. The results support this region as a this approach has not been widely employed for psy- chromosomal location involved in the ge- chiatric illnesses. The test of the value of this approach netic transmission of schizophrenia. Am. J. is whether it leads to replicable linkage findings and, Med. Genet. (Neuropsychiatr. Genet.) 105: ultimately, to the discovery of pathogenic molecular 20–22, 2001. © 2001 Wiley-Liss, Inc. changes. Because P50 inhibition is not generally mea- sured in genetic studies, replication of the finding in KEY WORDS: human chromosome pair 15; other samples has been performed using schizophrenia genetic linkage; nicotinic re- as the phenotype. A similar low positive score was re- ceptor; polymorphism; ported in the NIMH Genetics Initiative Schizophrenia P <0.002 .0.0 ס ␪ ,1.5 ס schizophrenia families by us at D15S1360, Z [Leonard et al., 1998]. The NIMH Genetics Initiative Contract grant sponsors: Veterans Affairs Medical Research investigators independently published a replication, Service, National Association for Research on Schizophrenia and with a multipoint linkage score derived from markers Affective Disorders, Colorado Associates, Inc., USPHS; Contract in the region; the Z score was higher for African Ameri- grant numbers: MH38321, DA09457. can than for European American families in the sample *Correspondence to: Robert Freedman, Department of Psychia- try C-268-71, University of Colorado Health Sciences Center, [Kaufmann et al., 1998]. A subsequent study of Bantu 4200 East Ninth Ave., Denver, CO 80262. families with D15S1360 revealed a positive nonpara- E-mail: [email protected] metric linkage score and significant transmission dis- Received 2 March 2000; Accepted 4 May 2000 equilibrium [Riley et al., 2000], and two other groups © 2001 Wiley-Liss, Inc. Chromosome 15 Disequilibrium in Schizophrenia 21 reported negative findings [Neves-Pereira et al., 1998; physical map confirms this location [Gault et al., 1998]. Curtis et al., 1999]. Interestingly, one of the groups Heterozygosity of the markers was also estimated from reporting negative findings has positive findings of CEPH families (D15S165 0.86; D15S1360 0.57; transmission disequilibrium with D15S1360 in their D15S144 0.69). The genetic distance between D15S165 bipolar families [Neves-Pereira et al., 1999]. The flank- and D15S144 is 6.4 cm; the physical distance is ap- ing marker ACTC has recently been reported to show a proximately 2.7 mb, contained within overlapping lod score of 3.46 in a selected population of lithium- YAC’s 940c5 and 810f11. The families of the NIMH responsive bipolar families [Turecki et al., 2000]. Genetics Initiative, whose ascertainment has previ- D15S1360 is a simple sequence repeat that is within ously been described, were studied [Kaufmann et al., 120 kb of the coding region of the ␣7 nicotinic acetyl- 1998]. Sixty-two parent–child triads in 50 families choline receptor subunit gene (CHRNA 7) [Gault et al., were examined. Genotyping with these markers has 1998]. This marker was chosen because preliminary been previously described [Leonard et al., 1998]. analysis had shown evidence for linkage of the P50 Table I demonstrates significant genotype-wise inhibitory abnormality at chromosome 15q14, which transmission disequilibrium for D15S165 (genotype- df, P < 0.007). The empirical P-value 24 ,44.5 ס was subsequently shown to be the locus of CHRNA7 wise ␹2 [Coon et al., 1993; Chini et al., 1994]. CHRNA7 is a from 1,000 Monte Carlo simulations was P < 0.0310 ± viable candidate gene because of neurobiological evi- 0.0055 (SE). Because there is preexisting information dence indicating the involvement in P50 inhibition of about linkage in this region, the analysis was repeated the nicotinic receptor that is coded for by this gene using only the oldest affected individual in each sibling [Freedman et al., 1994]. CHRNA7 itself has a compli- pair. The allele-wise significance was P < 0.044 and the cated genomic structure. The gene has 10 exons that genotype-wise significance was P < 0.040, demonstrat- are conserved across chick and other mammalian spe- ing a positive test of association with D15S165. Repli- cies. However, the human gene is part of a duplicated cation of the D15S165 transmission disequilibrium was cassette of expressed sequences; the duplicons are lo- assessed in an independent sample of 47 triads from 47 cated nearly adjacent to each other within approxi- families, 45 of which were European American [Cao et mately 1 mb of DNA at chromosome 15q14. The dupli- al., 1997]; a genotype-wise significance of P < 0.019 was con that contains CHRNA7, and is adjacent to found. D15S1360, is telomeric of a duplicon that contains an Stratification of the NIMH sample into African expressed sequence which has four novel exons fol- Americans and European Americans revealed similar lowed by six exons which duplicate exons 5–10 of the genotype-wise disequilibrium in both groups at this full-length CHRNA7. The sequence homology between marker. Transmission disequilibrium of D15S144, these last six exons and CHRNA7 is over 99% [Gault et which was not significant in the entire sample, was al., 1998]. The functional significance of this duplica- significantly responsible for transmission disequilib- tion is unknown. Mutation screening of the coding re- rium in the European American sample. Transmission gion of the full-length CHRNA7 reveals no polymor- disequilibrium of D15S1360 was not significant in ei- phism that is uniquely found in a high proportion of ther the entire sample or in the two subsets. Examina- people with schizophrenia [Gault et al., 1999]. There- tion of the transmission of specific alleles of each fore, additional mapping and sequencing outside the marker showed that a rare allele of D15S165 (206 bp, coding region of CHRNA7 is required to find the mo- CEPH frequency 0.01) was transmitted to 14 of 15 lecular alterations that give rise to the linkage signal schizophrenics in three African American and four Eu- and to the dysfunction of the ␣7 nicotinic receptor in ropean American pedigrees (P < 0.000006). In addition, schizophrenia. the 190 bp allele of D15S165 (CEPH frequency 0.03) The fine mapping of complex traits to genetic loci is was significantly transmitted in African Americans frequently necessary in the discovery of the molecular DNA polymorphisms that are responsible for patho- genic alterations in gene function. For rare genetic ill- TABLE I. Linkage Disequilibrium Mapping of Schizophrenia at nesses, single recombinations are often informative be- Chromosome 15q14 cause the rareness of the illness makes it unlikely that Significance (P) of Transmission Disequilibrium: an allelic or genetic variant or even a phenocopy is Marker Allele-wise Genotype-wise responsible for the apparent recombination. However, in a common, complex trait like schizophrenia, these Total sample D15S165 0.071 0.0069* alternative explanations are as likely, if not more D15S1360 0.18 0.26 likely, than a recombination, particularly in families D15S144 0.16 0.17 ascertained by an affected sibpair. Therefore, linkage African Americans disequilibrium was selected as a strategy for fine map- D15S165 0.25 0.066 ping. All probability inferences are from ␹2 tests, un- D15S1360 0.32 0.23 less otherwise noted. D15S144 0.94 0.87 European Americans The extended transmission disequilibrium test D15S165 0.058 0.059 (ETDT) [Sham and Curtis, 1995] was used for fine D15S1360 0.24 0.33 mapping of the region indicated to be involved in link- D15S144 0.036 0.012* age.