264 Letters to the Editor

MAURIZIO MARGAGLIONE,6 GIUSEPPINA MAZZOLA 4GIOVANNI DI MINNO,3 AND GENEROSO ANDRIA1

Dipartimenti di 1Pediatria and 2Biochimica eBiotecnologie Mediche and 3Clinica Medica,Universita Federico II, Naples; 4Servizio diCoagulazione and 4Dipartimento di Medicina diLaboratorio, Istituto Scientifico IRCCS Ospedale SanRaffaele, Milan; and 6Laboratorio di Trombosi eAterosclerosi, IRCCS Casa Sollievo Della Sofferenza,San Giovanni Rotondo

Acknowledgments

This work was supported in part by Consiglio Nazionaledelle Ricerche, Italy.

References

Engbersen AMT, Franken DG, Boers GHJ, Stevens EMB, Trij-bels FJM, Blom HJ (1995) Thermolabile 5,10-methylenetet-rahydrofolate reductase as a cause of mild hyperhomocys-teinemia. Am J Hum Genet 56:142-150

Fermo I, Vigano D'Angelo S, Paroni R, Mazzola G, Calori G,D'Angelo A (1995) Prevalence of moderate hyperhomocys-t(e)inemia in patients with early-onset venous and arterialocclusive disease. Ann Intern Med 123:747-753

Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Mat-thews RG, Boers GJH, et al (1995) A candidate genetic riskfactor for vascular disease: a common mutation in methyl-enetetrahydrofolate reductase. Nat Genet 10:111-113

Kluijtmans L, van den Heuvel LPWJ, Boers GHJ, Frosst P,Stevens E, van Oost BA, den Heijer MJM, et al (1996)Molecular genetic analysis in mild hyperhomocysteinemia:a common mutation in the methylenetetrahydrofolate re-ductase gene is a genetic risk factor for cardiovascular dis-ease. Am J Hum Genet 58:35-41

Motulsky A (1996) Nutritional ecogenetics: homocysteine-re-lated arteriosclerotic vascular disease, neural tube defects,and folic acid. Am J Hum Genet 58:17-20

Address for correspondence and reprints: Dr. Generoso Andria, Departmentof Pediatrics, Federico II University, Via S. Pansini 5, 80131, Naples, Italy.C) 1996 by The American Society of Human Genetics. All rights reserved.0002-9297/96/5901-0037$02.00

Am. J. Hum. Genet. 59:264-268,1996

Evidence for Genetic Anticipation in Non-MendelianDiseases

To the Editor:Genetic anticipation (GA) is characterized by a reduc-tion in the age at disease onset (AAO) and also by anincrease in both the severity and proportion of affected

individuals in successive generations. Mott was the firstto coin the term "anticipation," in 1911 (Mott 1911).For almost 80 years the finding of GA was attributedto ascertainment biases. Although such biases may exist,it is difficult either to confirm their presence or to ex-clude them. This controversy was clarified, for a numberof rare Mendelian disorders, by the discovery of unstableDNA. Unstable DNA consists of multiple trinucleotiderepeats that tend to increase in subsequent generations(Mandel 1994). The ability of unstable triplets to in-crease in number across generations provided a molecu-lar basis for GA, and concern about ascertainment biasesdiminished.We noticed a reduction of AAO in younger genera-

tions, for familial breast cancer (BRCA), colon cancer(COCA), Alzheimer disease (AD), maturity-onset diabe-tes of the young (MODY), and insulin-dependent diabe-tes mellitus (IDDM). These disorders differ from unsta-ble DNA diseases because they belong to the categoryof common non-Mendelian disorders, with most casesoccurring sporadically. In addition, disease genes withtraditional DNA mutations (substitutions, deletions,and insertions) have been described for BRCA, COCA,and AD, with no evidence of trinucleotide expansion.

In order to verify this observation statistically, wecollected the AAO for these non-Mendelian diseases(age at diagnosis for cancers) from available publishedpedigrees. On the basis of previous anticipation studies(McInnis et al. 1993), we chose two sampling strategies.First, direct transmission of disease in only parent-childpairs was analyzed, and the AAO in the parental genera-tion (Gi) was compared with that in the child's genera-tion (G2); (this corresponds to "scheme 4," describedelsewhere [McInnis et al. 1993]). In addition, for oneBRCA study (Hall et al. 1990), we analyzed the datawith all possible transmitting pairs-that is, each af-fected member of G1 was matched to each affectedmember of G2 ("scheme 3" [McInnis et al. 1993]).Scheme 4 was adopted for all the other analyses becausethe same highly statistically significant result was ob-tained for both sampling strategies. For some diseasesthe distribution of AAO was not normal; thus a Wil-coxon matched-pair analysis was used in addition to apaired t-test.

For BRCA, only females were included, and in casesof bilateral pathology the AAO of the earliest tumorwas used. Initial analysis of directly transmittingmother-daughter pairs from a large BRCA linkage study("BRCA Hall" [Hall et al. 1990]) was followed by anal-ysis of all other recent available BRCA pedigrees to-gether ("BRCA others" [Zuppan et al. 1991; Arason etal. 1993; Bowcock et al. 1993; Chamberlain et al. 1993;Cohen et al. 1993; Deville et al. 1993; Feunteun et al.1993; Kelsell et al. 1993; Lindblom et al. 1993a; Ma-zoyer et al. 1993; Smith et al. 1993; Spurr et al. 1993;

Letters to the Editor

Table 1

Evaluation of Intergenerational Differences in AAO in Non-Mendelian Diseases

MEAN ± SD AAO(years) % (No. OF PAIRS)

No. OF G1 - G2aDISEASE PAIRS G1 G2 (years) WILCOXON RANK PAIRED t-TEST G1 > G2b G1 < G2C G1 = G2d

BRCA Hall 59 54.3 ± 12.7 47.8 ± 10.9 6.5 Z = 3.14, P = .0017 t = 3.46, P = .001 66 (39) 33 (20) 0BRCA Hall EAOO 18 48.7 ± 10.2 40.4 ± 6.8 8.3 Z = 3.44, P = .0006 t = 5.42, P = .000 83 (15) 17 (3) 0BRCA others 159 50.5 ± 12.2 41.6 ± 11.7 8.9 Z = 6.87, P = .0000 t = 7.96, P = .0000 72 (115) 25 (39) 3 (5)COCA 67 52.5 ± 13.5 45.2 ± 11.8 7.3 Z = 3.99, P = .0001 t = 4.21, P = .000 72 (48) 28 (19) 0AD 129 55.4 ± 13.1 52.9 ± 12.1 2.5 Z = 2.63, P = .0085 t = 2.88, P = .005 53 (68) 37 (48) 10 (13)MODY-non-IDDM 72 33.3 ± 13.6 22.3 ± 14.4 11.0 Z = 5.63, P = .0000 t = 7.04, P = .000 85 (61) 15 (11) 0IDDM 51 21.6 ± 10.3 7.2 ± 4.4 14.4 Z = 5.99, P = .0000 t = 11.0, P = .000 92 (47) 6 (3) 2 (1)

'Mean age difference, in AAO, between G1 and G2.b % (no. of pairs) where AAO in G1 is greater than that in G2.c % (no. of pairs) where AAO in G1 is less than that in G2.d % (no. of pairs) where AAO in G1 equals that in G2.

Teare et al. 1993; Friedman et al. 1994; Inoue et al.1994; Simard et al. 1994; Tonin et al. 1994; Narod etal. 1995]). Pedigrees with familial COCA (Lynch et al.1991a, 1991b, 1992; Aaltonen et al. 1993; Peltomakiet al. 1993; Lindblom et al. 1993b; Green et al. 1994)and families with AD (Cook et al. 1979; Bird et al. 1988;Fitch et al. 1988; Kamino et al. 1992; Schellenberg etal. 1992; Sorbi et al. 1993; Haltia et al. 1994; van Duijnet al. 1994) were collected from the literature. Two stud-ies of MODY were found (Bell et al. 1991; Vaxillaireet al. 1995), and only patients with non-IDDM orMODY were included-that is, those with impairedglucose tolerance or IDDM were excluded. Only onestudy provided data on IDDM (Soltesz and The Hungar-ian Childhood Diabetes Epidemiology Study Group1994). Disease severity, as well as the affected propor-tion of successive generations, have not been examinedhere, because of limited published data regarding thesemeasures. Care was taken to prevent duplicate analysisof pedigrees published more than once.Although there is no cause to suspect GA in these

non-Mendelian diseases, the analysis of these publishedpedigrees has demonstrated highly statistically signifi-cant AAO differences across generations (table 1). Thisfinding may either result from common ascertainmentbiases or represent a universal feature of these diseases.

Evidence for GA may be weakened by unaffected indi-viduals from younger generations becoming affectedafter ascertainment (Ridley et al. 1988). To test for thisbias, we analyzed BRCA families 1-7 who had early-AAO BRCA (mean family AAO <45 years) from onestudy (Hall et al. 1990); these families also demonstratedevidence of anticipation (see "BRCA Hall EAAO" intable 1). The mean age at diagnosis for all women withBRCA in these EAAO families was 40.9 years, comparedwith the mean age of unaffected women in these fami-lies, 58.8 years. Only 3 of the 20 unaffected women

were <40 years of age, 11 of them were >60 years ofage, while only 1 case of BRCA occurred at >60 yearsof age in these families. This provides evidence that themajority of unaffected women in these families havepassed the age of risk in these families, and ascertain-ment bias, which would exclude women with late-onsetdisease, is reduced.

Preferential ascertainment of early-onset cases, whothen fail to have children (Ridley et al. 1988), is unlikelyto significantly bias the results for BRCA, COCA, andAD, for which the mean AAO is >40 years. Also, thesediseases should not influence the number of offspringsignificantly. Prophylactic screening may be responsiblefor earlier detection of BRCA and COCA in youngergenerations, but this is unlikely to account for the aver-age difference between generations, 6.5-8.9 and 7.3years, respectively. To control for this, it would be neces-sary to know the stage of disease at diagnosis; however,this information has not been published. Neither a longprodromal period before clinical diagnosis (which canlead to ambiguities with regard to AAO, especially inolder generations) nor bilineal transmission is applicableto cancer families.

Diabetes is likely to be subject to a larger number ofascertainment biases: the relatively early AAO in IDDMcan influence reproductive fitness. There is strong evi-dence for secular trends in non-IDDM (Hanson et al.1995). More family studies examining reproductive fit-ness and cohort effects are required in order to test forthese biases.

In AD, the proportion of "Gi<G2" is the highest,compared with that in the other diseases (table 1), al-though it is still less than the proportion "G1>G2" forAD. Therefore, the evidence for GA cannot be explainedas regression to the mean (McInnis et al. 1994). In addi-tion, we performed a comparative analysis of the degreeof direct and reverse anticipation (A) in the direct

26S

266 Letters to the Editor

(Gi>G2) and reverse (Gl<G2) anticipating pairs. Inregression to the mean, the mean A AAO would be ofequal magnitude but in the opposite direction. For AD,the G1>G2 pairs demonstrated a larger A in compari-son with G1<G2 pairs (mean ± SD = 9.0 ± 7.49 yearsin Gl>G2, vs. 6.25 ± 4.97 years in G1<G2 [t-test t= 2.21, P = .029; Mann-Whitney Z = 2.02, P = .03 8]).All the other diseases presented in table 1 showed statis-tically significant differences between A of G1>G2 andG1<G2 pairs (Mann-Whitney test; P < .05), arguingagainst the possibility that regression to the mean ac-counts for our findings.The criterion of genomic imprinting (GI), a differen-

tial effect of the sex of the disease-transmitting parent,has been suggested as a means of distinguishing betweentrue GA and statistical artifact (Ridley et al. 1988). Innon-Mendelian diseases, evidence for GI could bemasked because of multiple genes being involved in thedisease (Petronis et al. 1995b). Depending on the pro-portion of maternally and paternally imprinted genes,GI may not be observed. The criterion of GI is applicableonly when genetic loci are identified by linkage analysis,and then evidence for GI can be sought in geneticallyhomogeneous samples.

It is not possible to exclude all potential ascertainmentbiases for these diseases. Nevertheless, the evidence forGA in non-Mendelian diseases cannot be rejected solelybecause of the absence of DNA expansion or becauseof phenomenological dissimilarities with rare Mendeliandiseases. The universality of evidence for GA across non-Mendelian diseases is surprising. AAO dynamics consis-tent with GA have been shown in major psychosis(McInnis et al. 1993; Bassett and Honer 1994; Petronisand Kennedy 1995a), Parkinson disease (Payami et al.1995), hypertrophic cardiomyopathy (Gregor andCerny 1992), olivopontocerebellar atrophy (Dai 1991),paraganglioma (van der Mey et al. 1989), and two dis-eases with an autoimmune component: rheumatoid ar-thritis (Deighton et al. 1994) and Blau syndrome (Ra-phael et al. 1993).The absence of trinucleotide repeats in non-Mende-

lian diseases does not exclude GA-the molecular mech-anism that controls AAO may vary in different diseases.For cancer or diabetes, another genetic factor related tothe disease gene may control the timing of the patho-genic process. Finally, the causal relationship betweenunstable DNA expansion and AAO remains to beproved. Experimental data show that the degree ofexpansion correlates inversely with AAO, but there isno direct evidence that larger expansions directly causeearlier AAO. Unfolding the temporal genetic programis one of the most challenging issues of developmentalbiology. We believe that both basic and clinical sciencemay benefit from a more comprehensive study of GA inhuman genetic diseases. Mott's "law of anticipation,"

which received almost no attention until recently, mayprove to be one of the most universal laws of morbidhuman genetics.

ANDREW D. PATERSON, JAMES L. KENNEDY, ANDARTURAS PETRONIS

Neurogenetics Section, Clarke Institute of Psychiatry,Toronto

Acknowledgments

We thank Kathryn Tzimika, Joshua Garfinkel, Vadim Kour-ktchi, and the library staff at the Clarke Institute of Psychiatryfor their assistance. This work was supported by grants fromthe Scottish Rite Schizophrenia Program, the Ontario Friendsof Schizophrenics, and the Ontario Mental Health Foundation(all to J.L.K. and A.P). A.P. is an MRC/SSC Fellow. A.D.P.was supported by the McIntosh private foundation.

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Chamberlain JS, Boehnke M, Frank TS, Kiousis S, Xu J, GuoS-W, Hauser ER, et al (1993) BRCA1 maps proximal toD17S579 on chromosome 17q21 by genetic analysis. Am JHum Genet 52:792-798

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Feunteun J, Narod SA, Lynch HT, Watson P, Conway T,Lynch J, Parboosingh J, et al (1993) A breast-ovarian cancersusceptibility gene maps to chromosome 17q21. Am J HumGenet 52:736-742

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Friedman LS, Ostermeyer EA, Szabo CI, Dowd P, Lynch ED,Rowell SE, King M-C (1994) Confirmation of BRCA1 byanalysis of germline mutations linked to breast and ovariancancer in ten families. Nat Genet 8:399-404

Green RC, Narod SA, Morasse J, Young T-L, Cox J, FitzgeraldGWN, Tonin P. et al (1994) Hereditary nonpolyposis coloncancer: analysis of linkage to 2pi5-16 places the COCA1locus telomeric to D2S123 and reveals genetic heterogeneityin seven Canadian families. Am J Hum Genet 54:1067-1077

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Haltia M, Viitanen M, Sulkava R, Ala-Hurula, V, PoyhonenM, Goldfarb L, Brown P (1994) Chromosome 14-encodedAlzheimer's disease: genetic and clinicopathological descrip-tion. Ann Neurol 36:362-367

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Address for correspondence and reprints: Dr. Arturas Petronis, NeurogeneticsSection, Clarke Institute of Psychiatry, 250 College Street, Toronto, OntarioMST 1R8, Canada. E-mail: petronisa@cs.clarke-inst.on.caO 1996 by The American Society of Human Genetics. All rights reserved.0002-9297/96/5901-0038$02.00

Am. J. Hum. Genet. 59:268-269, 1996

When Does Maternal Age-Dependent Trisomy 21Arise Relative to Meiosis?

To the Editor:Polymorphic DNA markers have recently been used toestimate the fraction of trisomy 21 (Down syndrome)cases that may be attributable to postzygotic nondis-junction-indicative of a loss in the fidelity of the firstfew cell divisions after fertilization (Antonarakis et al.1993; Sherman et al. 1994). In these studies, a postzy-gotic nondisjunction is defined as a case in which twochromosomes of the trisomic set are homozygous for allinformative markers (i.e., for those markers that wereheterozygous in their parent of origin). These studiesestimate that the postzygotic mutation mechanism ac-counts for 4.5% (11/238) and 3.5% (9/255) of their

cases, respectively, but their estimates may actually beconservative, since all noninformative haplotypes (fre-quency not reported) are arbitrarily attributed to meiosisII-type nondisjunction. Nevertheless, even the conser-vative estimates would, if confirmed, constitute a newand nonnegligible source of chromosomal segregationerrors leading to trisomy. These studies' conclusions aresupported by the observation that the 20 reported "post-zygotic" cases (5 paternal and 15 maternal) appear tobe less dependent on maternal age (mean maternal age28.4 years) than maternal meiosis I-type failures (meanmaternal age 31.2 years). However, given the limitedsample size involved, one should be cautious in positingthe absence of a maternal age effect.

In the absence of evidence for somatic mosaicism (An-tonarakis et al. 1993; Sherman et al. 1994), it seemsprudent to use linkage analysis to test for the proposedpostzygotic origin of the extra chromosomes, becausea truly postzygotic origin for homozygous segregantsshould result in their being recombined in approxi-mately half of the cases. For the sake of simplicity, weassume (fig. 1) that only a single crossover has occurredbetween the relevant pair of chromosomes in each germcell, yielding two recombined chromatids and two non-recombined ones. If the nondisjunction were postzygoticin origin, the probability that a recombined chromatidwould be transmitted to the zygote should be the same(50%) as the probability that a nonrecombined onewould be transmitted from the same meiosis. Subse-quently, both recombined and nonrecombined chromo-somes should be subject to postzygotic nondisjunctionto the same extent. If the observed proportion of recom-bined chromosomes were close to 50%, the postzygotic-nondisjunction hypothesis would be upheld. If, on theother hand, the observed proportion were to differ sig-nificantly from the expectation (i.e., if the majority ofcases showed duplication of a nonrecombinant chromo-some), then alternative mechanisms should be consid-ered more plausible.

Specifically, it may be the case that nondisjunctionoccurred before meiosis in the parental germ lines, ratherthan postmeiotically (Vig 1984; Zheng and Byers 1992;Sensi and Ricci 1993). Chromosomes that had sufferednondisjunction prior to meiosis would proceed into mei-osis in the manner typical of trisomies, forming either atrivalent or a bivalent-plus-univalent configuration dur-ing meiotic prophase. In either case, two identical chro-mosomes that had arisen within the premeiotic cellularlineage and had then undergone recombination with oneanother in meiosis would remain identical in theirmarker configurations. They would then falsely appearto represent the products of meiosis II-type nondisjunc-tion with no recombination ("postzygotic-type nondis-junction"; see fig. 1). On the other hand, if either dupli-cated chromosome had exchanged marker alleles with