Molecular Psychiatry (1999) 4, 58–63 1999 Stockton Press All rights reserved 1359–4184/99 $12.00 dentatorubral-pallidoluysian atrophy, and Hunt- ORIGINAL RESEARCH ARTICLE ington’s disease (HD).1,2 CAG expansion has also been reported in schizophrenia,3–5 suggesting a role for PGE. CAG expansion in schizophrenia was difficult to repli- Detection of polyglutamine cate,6 suggesting that locus and allelic heterogeneity in expansion in a new acidic schizophrenia may lead to complex inheritance for which genetic associations are difficult to detect.7 In protein: a candidate for this respect, the use of nuclear families comprising a patient affected by earlier onset and/or a severe form childhood onset of a psychiatric disorder is considered an increasingly attractive method for identifying susceptibility genes.7 schizophrenia? We searched for PGE in childhood onset schizophrenia 8–11 1 1 1 (COS), a rare form of the disease. COS can be S Morinie`re , C Saada , S Holbert , detected using unmodified DSM-IIIR8,9 and shows the 2 3 4 E Sidransky , A Galat , El Ginns , following main features: (i) greater disease severity 2 1 JL Rapoport and C Ne´ri than adult onset schizophrenia (AOS); (ii) disruption of multiple developmental domains before the appear- 1Fondation Jean Dausset-CEPH, Paris, France; 2Child ance of psychotic symptoms, and, occasionally, pro- Neurogenetic Branch, National Institute of Mental Health gression of brain abnormalities after onset of psy- (NIMH), Bethesda, MD, USA; 3Commissariat a` l’Energie chosis;9–11 and (iii) clinical9 and neurobiological11 Atomique, De´partement D’Inge´nierie des Prote´ines continuity with AOS. Therefore, the study of COS may (CEA/DIEP), Saclay, France; 4Child Psychiatry Branch, provide new insights into the genetic susceptibility to NIMH, Bethesda, MD, USA schizophrenia in general. Lymphoblastoid cell lines (LCLs) for 32 unrelated Keywords: polyglutamine; schizophrenia; candidate COS patients recruited all over the United States (ongoing NIMH study, see Ref. 8) were tested by West- Polyglutamine expansion (PGE) encoded by a CAG ern blot analysis with the TATA-binding protein (TBP) repeat underlies eight inherited neurodegenerative dis- monoclonal antibody mAb1C2. MAb1C2 specifically eases, among which is Huntington’s disease. CAG 12 expansion has also been reported in schizophrenia, recognizes long polyglutamines (polyglns). Above the suggesting a role for PGE. To investigate the potential detection threshold (33–35 Glns), the intensity of the 12 role of PGE as a candidate for schizophrenia, we signal increases with the number of glutamines. searched for PGE in nuclear families comprising a Sixty-kDa PGE signals were observed in eight of 11 patient affected by childhood onset schizophrenia unrelated black American COS patients (Table 1). The (COS, a rare and severe form of the disease) as a vari- intensity of the 60-kDa band was strong in two ation of the candidate gene approach for identifying (patients 14 and 18) of the eight positive COS patients. susceptibility genes. We tested lymphoblastoid cell In the nuclear families (NFI and NFII) of patients 14 lines from COS patients (n = 32) by Western blot analy- and 18, a weak 60-kDa band (indicative of a small PGE) sis with 1C2, a monoclonal antibody that specifically was detected in some unaffected parents and sibs, sug- recognizes long polyglutamines. Eight of 11 unrelated gesting that the weaker 60-kDa band is not indicative black American COS patients showed a 60-kDa (approximately) band indicative of PGE. A strong 60-kDa of COS (Figure 1a and b). Using high resolution, long band (suggestive of a large PGE) was detected in two SDS-PAGE (10%) gels, the weak band clearly migrated of the eight positive patients. A weaker 60-kDa band at a slightly higher position than the strong band in (suggestive of a smaller and non pathogenic PGE) was NFI (Figure 1a, bottom panel), suggesting that the weak detected in some unaffected parents or sibs of these signal does not correspond to the same protein with a two COS patients, and in six other black American COS shorter polygln tract and that the strong and weak PGE patients. The strong and weak PGE signals were found signals arise from two different proteins. The detection to correspond to two different proteins. Unrelated black profiles for NFI and NFII were reproduced using three Americans unaffected by COS (n = 38) were negative for different batches of LCL extracts. the strong 60-kDa PGE signal. Healthy white Americans = Long polygln stretches in human proteins induce a (n 53) were negative for both the strong and weak 60- migration schift towards high molecular weights.12 For kDa PGE signals. Two-dimensional gel analysis sug- example, TBP is a 37.2-kDa protein which migrates at gested that the strong PGE signal corresponds to an acidic (pI 4 approximately) protein and resulted in a 50 kDa approximately. This suggests that the strong 60- more precise estimation (52–57 kDa) of its relative mass. kDa PGE signal corresponds to a protein with a true This protein appeared to be not represented in Gen- mass of about 47 kDa. When probed with mAb1C2, it bank, as suggested by the exclusion of several candi- was previously reported that the normal HD protein is date CAG repeats. Our data suggest that this acidic pro- barely detectable after gross overexposure if the tein might be a candidate for COS. polygln contains more than 27 units.12 Using SDS- Polyglutamine expansion (PGE) encoded by a CAG PAGE (7%, 10%) gels with prolonged migration and repeat underlies eight inherited neurodegenerative dis- long exposure, the mAb1C2-reactive 60-kDa protein in eases including spinobulbar muscular atrophy, spino- COS patient 14 was not detected as a doublet (data not cerebellar ataxia 1 (SCA1), SCA2, SCA3, SCA6, SCA7, shown), suggesting that the unexpanded allelic protein Detection of polyglutamine expansion S Morinie`re et al 59 Table 1 PGE in black American COS patients Patient Sexa Ethnic background Age of onset Age of blood draw 60-kDab PGE signal 1 F white American 9 14 − 2 F white American 11 15 − 3 M white American 10 12 − 4 M white American 8 13 − 5 M black American 10 14 + 6 M white American 10 14 − 7 M white American 7 12 − 8 M white Iranian 11 13 − 9 M white American 11 17 − 10 F black American 10 16 − 11 M white American 11 14 − 12 F white American 9 10 − 13 M black American 11 16 + 14 F black American 12 14 ++ 15 M white American 11 15 − 16 F black American 12 16 + 17 M white American 11 16 − 18 F black American 11 13 ++ 19 F black American 12 17 − 20 M white American 7 14 − 21 F black American 5 9 + 22 M white American 6 21 − 23 F Philipino 11 12 − 24 F white Hispanic 11 16 − 25 F white American 10 13 − 26 F white Hispanic 12 14 − 27 F white Hispanic 9 17 − 28 M white American 11 12 − 29 M black American 12 19 + 30 M black American 12 16 + 31 M indian American 12 15 − 32 M black American 12 15 − Thirty-two COS patients were tested for PGE by Western blot analysis with mAb1C2 (see Figure 1). Eight of the 11 black American COS patients are positive for PGE. aF, female; M, male. b − + ++ Approximate Mr , negative; , weak intensity (also observed in black American individuals unaffected by COS); , strong intensity (observed in COS patients only). contains less than 28 glutamines.12 In control experi- protein. The PGE signal was not detected in the PGE- ments, a 60-kDa band was not observed in three heal- negative COS patient 10 (data not shown). The PGE sig- thy white families (CEPH Utah pedigrees 1334, 1344, nal was not detected in COS patient 16 either, suggest- and 1420; 53 individuals in total; data not shown) pre- ing that the weak 60-kDa band corresponds to a protein viously reported to show CAG expansion.13 We also (pI not in the range tested) different from the acidic tested 38 unrelated black American controls with no protein detected in COS patients 14 and 18 (data not history of psychiatric diseases (see Methods). Except shown). This observation was consistent with the for 11 of them (showing a weak 60-kDa band), these observations previously made in NFI using high resol- individuals were negative for 60-kDa PGE signals as ution 1D gel electrophoresis (see paragraph above). illustrated in Figure 1c. To identify the gene for the polygln expanded in To further characterize the protein which carries the COS patients 14 and 18, we selected 27 proteins carry- strong PGE signal, we performed two-dimensional (2D) ing 8–34 consecutive Glns by using Genbank scanning, gel electrophoresis (pI 10–3 gradient). Using two differ- and one acidic protein, DAN2614 (52.5 kDa, pI 4.8), was ent batches of LCL extract of COS patient 14, the com- retained for PCR screening in COS family NFI (see parison of silver-stained IEF/SDS PAGE (Figure 2a) Methods). Twenty-seven CAG repeats found in with 1C2 immunoblots (Figure 2b) suggested that PGE cDNAs15–17 and CAG/CTG repeats isolated from gen- is carried by an acidic protein (isoelectrofocusing point omic DNA18 consisting of more than 10–15 consecutive [pI] of 4 approximately). Two-dimensional gel electro- units were also tested for expansion in NFI (see phoresis experiments also resulted in a more precise Methods). All the candidates tested showed unen- estimation (52–57 kDa) of the relative mass (Mr) of this larged CAG repeat alleles, and no difference in the size Detection of polyglutamine expansion S Morinie`re et al 60 Figure 1 Detection of PGE in COS.