American Journal of Medical Genetics 41:96-98 (1991)

Diagnosis of Twin Zygosity by Hypervariable RFLP Markers Atsushi Akane, Kazuo Matsubara, Hiroshi Shiono, Masao Yamada, and Yasuo Nakagome Department of Legal Medicine, Shimane Medical University, Izumo (A.A., K.M., H.S.) and Department of Congenital Abnormalities Research, National Children’s Medical Research Center, Tokyo (M.Y., Y.N.), Japan The application of variable number of tandem repeat (VNTR) markers to the determination of twin zygosity was investigated. In the first case, which was performed with the use of six VNTR markers, the probability of monozygosity, calculated from Essen-Moller’s formula 11, was 0.99972. In the other three cases in which four VNTR markers were analyzed, the probabilities were 0.98251-0.99557. These results suggest that VNTR markers are useful for determination of twin zygosity. KEY WORDS: DNA analysis, restriction fragment length polymorphism (RFLP), variable number of tandem repeat (VNTR)markers INTRODUCTION Zygosity diagnosis, determining whether the twins are mono- or di-zygotic, is performed by several methods such a s sex, blood group systems, dermatoglyphics, and restriction fragment length polymorphisms (RFLPs). Derom et al. [1985] first applied one triallelic and five diallelic RFLP markers to determine monozygosity of twins. Effective diagnosis by RFLPs generally depends upon their heterozygosities. If both genotypes of the parents are homozygous, the genetic markers are uninformative. When genotypes of parents are not examined, the probability of monozygosity depends on the frequencies of those alleles detected in the twins’ genotypes. Hypervariable RFLPs are thus expected to be more informative than conventional (diallelic) RFLPs because of higher heterozygosities and lower allele frequencies. Multiallelic RFLPs detected by variable number of tandem repeat (VNTR) markers [Nakamura et al., 1987al have been analyzed for such forensic purposes a s personal identification and parentage testing [Akane et Received for publication October 5,1990; revision received January 29, 1991. Address reprint requests to Dr. A. Akane, Department of Legal Medicine, Shimane Medical University, Izumo 693, Japan.

0 1991 Wiley-Liss, Inc.

al., 1990a, b; Yokoi et al., 19901. Analysis of VNTR markers can be similarly exploited for diagnosis of twin zygosity. The application of several VNTR markers to zygosity diagnoses of four sets of twins is described here.

SUBJECTS AND METHODS Case Reports DNA samples had been extracted from peripheral blood leukocytes of the examinees in four families by the method reported previously [Akane et al., 1990al. Since only DNA samples were supplied, other genetic markers such a s blood group systems were not examined. Analysis of RFLPs All VNTR probes were supplied by the Japanese Cancer Research Resources Bank [Nakamura et al., 1987a-c, 1988a, bl. RFLPs were analyzed by the Southern blotting method using 32P-labeled probes as described [Akane et al., 1990al. Probability of Monozygosity Probability of monozygosity, FWMZ) was calculated according to formula I1 of Essen-Moller [Inouye, 19621as Pr(MZ) =

1 n

1+ l/q*

3

IT (D,/MI)

i = l

where D or M is the probability that di- or mono-zygotic twins have the same genotypes at a genetic locus, and q is the rate of monozygotic and dizygotic twins in the population (q = 3.51, in Japanese). The ratio of D/M is determined by the combinations of genotypes of twins and their parents (Table I).

RESULTS AND DISCUSSION As shown in Table I, effective diagnosis of twin zygosity by RFLP analysis theoretically depends on the RFLP heterozygosities and allele frequencies. In case 1, genotypes were established for six VNTR markers in the twins, their brother, and parents (Table 111, resulting in a probability of monozygosity of 0.99972. No parental pair was observed such that both parents were homozygous for a given VNTR marker. In cases 2-4, genotypes of the twins and their parents were determined for four VNTR probes, and the probabilities of mono-

Diagnosis of Twin Zygosity by RFLPs

97

TABLE I. The Combinations of RFLP Genotypes and the Corresponding D/M Values Based on the Hardy-Weinberg Law Combination of Genotypes Parents Twins Hetero- and heterozygous“ Hetero- or homozygous Hetero- and heterozygousb Heterozygous Hetero- and heterozygousb Homozygous Hetero- or homozygous Hetero- and homozygous Hetero- or homozygous Homo- and homozygous (Unknown) and (unknown)

Heterozygous

(Unknown) and (unknown)

Homozygous

DIM 0.25 0.50 0.25 0.50 1.00

+ 3b + 1‘ + b +1) 9a2 + 6a + 1 4(1 + a)’

lOab + 3a 4(ab + a

heterozygous genotypes (only found in multiallelic RFLPs). The same heterozygous genotypes. ‘a and b are frequencies of alleles detected in twins’ genotypes. a Different

zygosity were 0.99557, 0..98251, and 0.99118, respectively (Table 111). Although the 4-allelic RFLP probe pTHH59 was uninformative as the genotypes of both parents were homozygous in cases 3 and 4, the probabilities were sufficiently high to establish zygosity. If the genotypes of parents had been unknown, the D/M values, which were calculated from the allele frequencies, would be higher, reducing the probabilities of monozygosity (Tables11,111).Thus, zygosity of twins can be determined with greater certainty when the genetic markers of the twins and their parents are analyzed simultaneously. VNTR markers were found to be highly informative for diagnosis of twin zygosity. However, since a marker may be uninformative when the genotypes of parents are homozygous, more than four markers should be analyzed. Zygosity diagnosis has also been performed by DNA fingerprinting, which can determine monozygosity with high probability in a single test [Asaka and Honma, 1988; Hill and Jeffreys, 1985; Jones et al., 1987; Motomura et al., 19871. However, incomplete digestion of DNA samples by restriction enzymes is a considerable problem in DNA fingerprinting. For example, Jones et al. [19871described a photograph of DNA “fingerprints” of monozygotic twins, in which a fragment was detected in one twin but not in the other. This difference, which had been explained by the authors as incomplete digestion caused by a DNA methylation change, might be

explained if the twins were dizygotic. Since DNA fingerprinting detects many fragments, it is difficult to distinguish the false bands caused by incomplete digestion. On the contrary, when a partially digested DNA sample is hybridized with a VNTR probe, false bands are distinguishable: Each VNTR probe only detects two alleles, and any incompletely digested fragments will be proportionally longer than the true alleles. This characteristic of VNTR markers renders them more reliable than the minisatellite probes used in DNA fingerprinting for personal identification and parentage testing, as well as for determination of zygosity in twins. Another problem is the possibility of somatic mutation a t the earliest embryonic stage. In analyses using hypervariable minisatellite probes, the possibility of a mutation event should be always taken into consideration [Jeffreys et al., 1988;Wolff et al., 19881.The de novo generation of new alleles is thought to occur predominantly during meiosis. Such a mutation may also arise in mitosis although the incidence is very low per allele per mitosis) [Armour et al., 19891. If a somatic mutation occurred in an embryo immediately after the polyembryony, the resulting monozygotic twins might have different alleles. The probability of such an event is small but should not be ignored. In contrast to DNA fingerprinting, analysis using VNTR markers allows any mutant VNTR allele to be distinguished from wild type [Akane et al. 1990al.

TABLE 11. Case 1: RFLP Genotypes in VNTR Markers and DIM* Probe“ pYNH24 pTHH59 pEFD75.0 pEFD126.3 pYNZ22 pYNZ2

(MspI) (PvuII) (TaqI) (TaqI) (MspI) (RsaI)

Father

Twin A

3.35,2.00 1.78J.50 3.20,2.41 2.70,2.70 0.98,0.90 2.05J.60

3.80,3.35 1.78,1.78 3.20,2.41 2.70,2.70 0.90,0.78 2.05,2.05

* Each genotype is represented a

Twin B

Brother

3.80,3.35 3.80,3.35 1.78J.78 1.50,1.50 3.20,2.41 3.20,2.41 2.70,2.70 2.70,2.70 0.90,0.78 0.90,0.78 2.05.2.05 2.05.2.05

Mother 3.80,2.40 1.78,1.50 3.20,2.41 2.80,2.70 0.96,0.78 2.35.2.05

D/Mb 0.25 (0.29129) 0.25 (0.42581) 0.50 (0.45359) 0.50 (0.77919) 0.25 (0.32319) 0.25 (0.54615)

as a pair of restriction fragment lengths (kb) of alleles. Restriction enzymes used in parentheses. Each number in parenthesis is DIM when the genotypes of both parents are assumed to be unknown.

98

Akane et al. TABLE 111. Cases 2-4: RFLP Genotypes in VNTR Markers and D/M* Probe Case 2 pYNH24 pTHH59 pEFD75.1 pYNZ2 Pr(MZ)‘ Case 3 pYNH24 pTHH59 pEFD75.1 pYNZ2 Pr(MZ) Case 4 pYNH24 pTHH59 pEFD75.1 pYNZ2 Pr(MZ)

Father

Thin A

Twin B

Mother

DIM“

2.40,2.50 1.78,0.72 2.38,2.17 2.05,1.60

2.30,2.40 1.78,0.72 2.38,2.38 2.05,1.60

2.30,2.40 1.78,0.72 2.38,2.38 2.05.1.60

2.30,3.45 1.78J.78 2.38,2.17 1.60,1.60

0.25 (0.31791) 0.50 (0.38330) 0.25 (0.54264) 0.50 (0.65834) 0.99557 (0.98775)

2.35,2.35 1.78,1.78 2.38,2.38 2.05.1.60

2.35,2.45 1.78,1.78 2.38,2.38 2.05,2.05

2.35,2.45 1.78J.78 2.38,2.38 2.05.2.05

2.45,2.85 1.78,1.78 2.38,2.17 2.05,1.60

0.50 (0.31791) 1.00 (0.42581) 0.50 (0.54264) 0.25 (0.54615) 0.98251 (0.98870)

2.90,3.30 1.50,1.50 3.20,2.38 2.05,1.60

2.90,3.65 1.50,1.50 2.38,2.17 2.05.1.60

2.90,3.65 1.50,1.50 2.38,2.17 2.05.1.60

2.25,3.65 1.50,1.50 3.20,2.17 2.05.1.60

0.25 (0.27154) 1.00 (0.66434) 0.25 (0.54240) 0.50 (0.65834) 0.99118 (0.98198)

* Each genotype is represented as a pair of restriction fragment lengths (kb)of alleles. a

Each number in parenthesis is DIM value when the genotypes ofboth parentsare assumed to be unknown. Probability of monozygosity.

The diagnosis of twin zygosity by RFLP was described here, demonstrating the utility of hypervariable VNTR markers for the purpose.

ACKNOWLEDGMENTS The authors are grateful to Dr. S. B. England for reviewing the manuscript. This work was supported in part by grants from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare of Japan. All DNA probes were kindly supplied from the Japanese Cancer Research Resources Bank. REFERENCES Akane A, Matsubara K, Shiono H, Yuasa I, Yokota S, Yamada M, Nakagome Y (1990a):Paternity testing: Blood group systems and DNA analysis by VNTR markers. J Forensic Sci 35:1217-1225. Akane A, Nakajima H, Shiono H, Matsubara K, Yamada M, Nakagome Y (1990b): A case of maternity testing: exclusion by polymorphic VNTR markers of DNA. Jpn J Human Genet 35319-323. Armour JAL, Pate1 I, Thein SL, Fey MF, Jeffreys AJ (1989):Analysis of somatic mutations at human minisatellite loci in tumors and cell lines. Genomics 4:328-334. Asaka A, Honma M (1988):Zygosity diagnosis and DNA fingerprints. Igaku no Ayumi 144:707-709. Derom C, Bakker E, Vlietinck R, Derom R, Van Den Berghe H, Thiery M, Pearson P (1985): Zygosity determination in new-born twins using DNA variants. J Med Genet 22:279-82. Hill AVS, Jeffreys AJ (1985): Use of minisatellite DNA probes for determination of twin zygosity a t birth. Lancet 21394-1395. Inouye E (1962):Zygosity diagnosis of Japanese twins by Essen-Moller’s formula 11. In Fujita K (ed): “Studies on Twins, 111.” Tokyo: Nihon-Gakujutsu-Shinkokai, pp 1-13. Jeffreys AJ, Royle NJ, Wilson V, Wong Z (1988):Spontaneous mutation

rates to new length alleles a t tandem-repetitive hyper-variable loci in human DNA. Nature 332278-281. Jones L, Thein SL, Jeffreys AJ, Apperley JF, Catovsky D, Goldman JM (1987):Identical twin marrow transplantation for 5 patients with chronic myeloid leukaemia: Role of DNA fingerprinting to confirm monozygosity in 3 cases. Eur J Haematol 39:144-147. Motomura K, Tateishi H, Nishisho I, Okazaki M, Miki T, Tonomura A, Takai S, Mori T, Jeffreys AJ (1987):The zygosity determination of Japanese twins using a minisatellite core probe. J p n J Human Genet 32:9-14. Nakamura Y, Leppert M, OConnell P, Wolff R, Holm T, Culver M, Martin C, Fujimoto E, Hoff M, Kumkin E, White R (1987a):Variable number of tandem repeat (VNTR) markers for human gene mapping. Science 2351616-1622. Nakamura Y, Gillilan S, O’Connel P, Leppert M, Lathrop GM, Lalouel J-M, White R (1987b):Isolation and mapping of a polymorphic DNA sequence pYNH24 on chromosome 2 (D2S44). Nucleic Acids Res 15:10073. Nakamura Y, Fujimoto E, O’Connel P, Leppert M, Lathrop GM, Lalouel J-M, White R (1987~): Isolation and mapping of a polymorphic DNA sequence (pEFD126.3) on chromosome 9q (D9S7). Nucleic Acids Res 15:10607. Nakamura Y, Holm T, Gillilan S, Leppert M, OConnel P, Lathrop GM, Lalouel J-M, White R (1988a): Isolation and mapping of a polymorphic DNA sequence (pTHH59) on chromosome 17q (D17S4). Nucleic Acids Res 16:3598. Nakamura Y, Culver M, Sergeant L, Leppert M, OConnel P, Lathrop GM, Lalouel J-M, White R (1988b): Isolation and mapping of a polymorphic DNA sequence (pYNZ2)on chromosome l p (DlS57). Nucleic Acids Res 16:4747. Wolff RK, Nakamura Y,White R (1988):Molecular characterization of a spontaneously generated new allele at a VNTR locus: No exchange of flanking DNA sequences. Genornics 3:347-351. Yokoi T, Nata M, Odaira T, Sagisaka K (1990): Hypervariable polymorphic VNTR loci for parentage testing and individual identification. Jpn J Human Genet 35179-188.