American Journal of Medical Genetics 37551-557 (1990)

Sexual Discordance in Monozygotic Twins Elizabeth J. Perlman, Gail Stetten, Cathy M. Tuck-Miiller, Rosann A. Farber, Wilma L. Neuman, Karin J. Blakemore, and Grover M. Hutchins Departments of Pathology (E.J.P.,G.M.H.), Gynecology and Obstetrics (G.S.,C.M.T.-M.,K. J.B.), T h e J o h n s Hopkins Medical Institutions, Baltimore Maryland, T h e Department of Medicine, T h e University of Chicago, Chicago Illinois (W.L.N.), and T h e Department of Pathology, T h e University of North Carolina, Chapel Hill, North Carolina (R.A.F.). We report on monozygotic (MZ) twins who were discordant for phenotypic sex and U11rich-Turner syndrome (UTS). The nonviable female was hydropic with cystic hygromas, ventricular septa1 defect, bicuspid aortic valve, polysplenia, intestinal malrotation, and small ovaries. The male was phenotypically normal. The monochorionic, diamniotic placenta had hydropic changes limited to the UTS infant's side. Skin samples from the infants and blood from their parents were obtained for cytogenetic and molecular analysis. Karyotypes of the twins were 45,X and 46,XY. Quinacrine polymorphisms on 7 chromosomes and RFLP analysis at 8 loci showed complete identity. MZ twins discordant for phenotypic sex have been described previously. Most of these show evidence of mosaicism in a 45,X patient with a normal 46,XY cell line, and a normal 46,XY male. While the issue of mosaicism in our case cannot be fully resolved, no mosaicism was found in 50 cells analyzed cytogenetically from each culture or by PCR analysis of a Y-specific sequence. The twins probably originated from either postzygotic nondisjunction or anaphase lag, followed or accompanied by twinning. The discordant placental morphology suggests an embryonic origin of at least part of the placental mesenchymal core. KEY WORDS: monozygotic twins, placenta disease, polymorphism (genetics), Turner syndrome INTRODUCTION Monozygotic (MZ) twins who are cytogenetically different are rare, and are thought to be the result of a n Received for publication December 15, 1989; revision received April 13, 1990. Address reprint requests to Elizabeth J. Perlman, M.D., Department of Pathology, The Johns Hopkins Hospital, 600 N. Wolfe Street, Baltimore, MD 21205.

0 1990 Wiley-Liss, Inc.

early postzygotic mitotic error. The timing of this mitotic error with respect to the twinning event determines the presence and degree of mosaicism in one or both twins. Heterokaryotic twinning involves the X chromosome in the majority of cases; consequently the twins are concordant or discordant for the U1lrich-Turner syndrome (UTS) [Benirschke and Kim, 19731. There have been 7 reported cases of twins discordant for both phenotypic sex and UTS [Edwards et al., 1966; Gonsoulin et al., 1990; Karp et al., 1975; Reindollar et al., 1987; Russell et al., 1966; Schmidt et al., 1976; Turpin et al., 19611. The current study reports inviable premature twins discordant for phenotypic sex and UTS, with no detected mosaicism. Quinacrine polymorphic variants and restriction fragment length polymorphism (RFLP) analysis are combined with the placental pathology to establish monozygosity and to shed some light on the possible timing and sequence of the twinning event and the mitotic error. CLINICAL REPORT The mother of the twins, a 25-year-old white primigravida, presented with uterine contractions. Sonography demonstrated a twin gestation of approximately 26 weeks. The preterm labor was further complicated by mild maternal hypertension and vaginal bleeding, and a cesarean section was performed for suspected placental abruption. Twin A was a markedly hydropic female infant weighing 920 g who died within minutes of delivery. Twin B was a normally developed 420 g male infant with Apgar scores of 4 and 6 a t 1and 5 minutes, respectively. He required intubation and mechanical ventilation, but deteriorated with hyaline membrane disease and severe prematurity. Despite maximal support, the infant died 18 hours after birth. AUTOPSY RESULTS At autopsy, twin A was a grossly hydropic female infant with a n approximate anatomic age of 22 weeks (weight 920 g, crown-heel length 28 cm, crown-rump length 18 cm, right foot length 34 mm). Three large cystic hygromas were present in the posterior nuchal region (Fig. 1A). Radiopaque material injected into the right cystic hygroma demonstrated no communication with other hygromas or with the local lymphatic channels. The lungs were markedly hypoplastic, with a com-

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Fig. 1. Twin A (45,X), 920 g, with hydrops and cervical cystic hygromas (A). Twin B (46,XY),490 g, morphologically normal (B).

bined weight of2.8 g, and showed bilateral trilobation. A ventricular septa1 defect and a bicuspid aortic valve were present. The liver had a midline position and 2 spleens of equal size were present on either side of the mesogastrium. Intestinal malrotation was present, with the cecum located in the left upper quadrant. The genitourinary tract was without gross abnormalities. The ovaries were small and histologically consistent with UTS a t this gestational age. Twin B a t autopsy was a normally formed male fetus with an approximate anatomic age of 23 weeks (weight 490 g, crown-heel length 28 cm, crown-rump length 18 cm, right foot length 41 mm) (Fig. 1B). The lungs were of normal size and histologically demonstrated diffuse severe hyaline membrane disease and focal intraparenchymal hemorrhage. The abdominal organs were without abnormalities. The testes were grossly and histologically normal. Cerebral hemorrhage was present in the lateral, third, and fourth ventricles of the brain. The single placenta was divided by a translucent membrane. Histologic sections of the dividing membrane demonstrated two apposed amnions without a n intervening chorion. The maternal aspect of the placenta corresponding to twin A contained multiple foci of hydropic villi (Fig. 2). The umbilical cord of twin A contained a single umbilical artery. The placenta and umbilical cord of twin B were grossly and microscopically normal. Given the discrepant weights of the twins, and the monochorionic, diamniotic placenta, the possibility of twin-twin transfusion was considered. The placenta showed several small-caliber vessels crossing from the placenta of twin A to twin B. Nevertheless, the hemoglobin level of twin B was 17.2 m d d l at birth. therefore, twin-twin transfusion syndrome is unlikely.

Cytogenetic Analysis Cytogenetic studies were carried out on cultured skin fibroblasts established from biopsies obtained at autopsy on the twins, and on peripheral lymphocytes from their parents. Chromosome banding was produced with quinacrine fluorescence [Caspersson et al., 19711 or a heat Wright’s stain method [Yunis et al., 19791. Chromosomal polymorphisms a t the centromere and/or satel, 22 were lite of chromosomes 1 , 3 , 4 , 9 , 1 3 , 1 4 , 1 5 , 2 1and scored for size and intensity. Comparisons were made by assigning a letter to each different variant type seen (Fig. 3). Seven sets of informative homologs were examined (Table I), and for each the twins showed no genetic differences.

Fig. 2. Placenta of twin A showing multiple hydropic villi.

Discordant Monozygotic Twins

Fig. 3. Quinacrine polymorphic variants seen on chromosome 14 of the parents and twins. The “a” variant shows a brightly fluorescent satellite, the “b” variant shows a nonfluorescent satellite, and variant “c” shows no satellite. Each twin inherited the “a” and “b” variants.

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dextran sulfate, 1x modified Denhardt’s solution [Denhardt, 19661, 0.02 M sodium phosphate, pH 6.7, 50% formamide, and 250 pg/ml sonicated salmon sperm DNA. Membranes were then washed twice at room temperature in 2 x SSC, 0.1% SDS, once at room temperature in 0.1 x SSC, 0.1% SDS, and then twice a t 63°C in the same solution. They were exposed to Kodak XAR-5 X-ray film backed with Dupont Lightning-Plus intensifying screens for 1-5 days. The probes used were pYNH24, pYNZ132, and pTHH39 [Nakamura et al., 1987a1,pTP5E LFarber et al., 19881,pmetD [White et al., 19861, pYNB3.1R [O’Connell et al., 19871, a-globin 3’HVR [Reeders et al., 19851,andpRMU3 [Nakamura et al., 1987133. Polymerase chain reactions (PCR) were done in a 100 ~1 reaction mixture containing 1pg genomic DNA, 100 pmol of each oligonucleotide primer, and each of the four dNTPs a t 20 pM, in 10 mM Tris, pH 8.3,50 mM KC1,2.5 mM MgC12, 0.01% gelatin. After this mixture was heated for 7 minutes a t 95”C, 2 units Taq polymerase (Perkin-Elmer Cetus) was added. Thirty cycles of annealing and extension (63°C for 5 minutes) and denaturation (94°C for 1minute) were carried out. Amplification products were visualized on ethidium bromide-stained 1.5% agarose gels. The hybridization patternso f 8 polymorphic probes to DNA extracted from each Of the twins and their parents were analyzed (Fig. 4). The restriction bands were desig-

Molecular Analysis DNA was extracted from cultured skin fibroblasts from each twin, and from blood samples from their parents. Briefly, cell pellets were lysed, followed by phenol, chloroform and ether extraction. The extracted DNA was then ethanol precipitated and resuspended in 10 mM “his, pH 7.5, 1 mM EDTA. The extracted DNA was analyzed for RFLP by Southern blotting [Southern, 19751. Restriction endonucleases were obtained from New England Biolabs (Beverly, MA). DNA was digested according to the manufacturer’s instructions using a 7.5-fold excess of enzyme. Restricted DNA was subjected to electrophoresis in 0.8% agarose gels and transferred to MSI nylon hybridization membrane (Fisher) in l o x SSC (1.5 M NaC1, 0.15 M sodium citrate). DNA probes were 32P-labeledusing the Multiprime DNA labeling system (Amersham International, UK). The labeled probes were allowed to hybridize to membrane-bound DNA for 24 hours a t 42°C in 5 x SSC, 10% TABLE I Quinacrine Variant Analysis Chromosome 1 3 9 13 14 15 22

Mother _ ___ aa ab ab aa ab aa ab

Twin A Twin B Father _ _ _45,X _ _ _ -46,XY ab aa aa aa ab ab aa ab ab ab ab ab bc ab ab ab ab ab aa aa aa ~

Fig 4 Autoradiograph of Southern blot of DNA from twins and parents, probed with u-globln 3’HVR Lane (a) 45,X twin, (b) 46,XY twin, (c) father, (d) mother

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Perlman et al. TABLE 11. Restriction Fragment Length Polymorphism Analysis Probe YNH24 TP5E metD YNB3.1R YNZ132 THH39 3’HVR RMU3

Chromosomal location

Enzyme

2 5q 7q 7q 8 14 16P 17q

MspI TaqI TaqI RsaI TaqI MspI RsuI TaqI

nated according to size by numbers, one being the l a r g est DNA fragment (Table 11). None of the 8 probes showed genetic differences between the twins. To test for the presence of a small fraction of XY cells, amplification of a Y chromosome-specific repeated sequence [Nakahori et al., 19861 was performed on DNA from the UTS twin by the PCR technique [Saiki et al., 19881,utilizing reported primer sequences [Kogan et al., 19871. An Alu sequence was amplified on parallel samples as a control. No evidence for the presence of Y-specific sequences in the UTS twin’s DNA was found.

DISCUSSION MZ heterokaryotic twins are exceptional, with 19 cases reported [Benirshke and Kim, 1973; Gonsoulin et al., 1990; Karp et al., 1975; Reindollar et al., 1987; Schmidt et al., 19761. Most ofthese cases (15twin pairs) involve discordance for gonadal dysgenesis, and the remaining involve discordance for the Down syndrome. Unlike MZ twins with Down or Klinefelter syndromes, in which phenotypic and karyotypic concordance is the rule, most MZ twins with UTS show phenotypic and karyotypic discordance and/or mosaicism. The theoretical explanation for this is that Down or Klinefelter syndrome is usually the result of a prezygotic nondisjunction, whereas UTS is considered to be a postzygotic nondisjunction. Nance and Uchida 119641noted that MZ twinning is more frequent in families of patients with UTS than it is in the population a t large. These observations have led to the suggestion that MZ twinning and mitotic nondisjunction in these cases may be related, although the pathogenesis of this relationship is not clearly understood. MZ twinning is considered in some cases to result from a n abnormality of a n early cleavage [Verp et al., 19881. Similarly the 45,X chromosome constitution may also arise during a n abnormal postzygotic cleavage, and a single factor may play a role in both. These observations are supported by the high incidence of mosaicism in UTS [Ferrier et al., 19701 and by the extreme phenotypic variability seen in this syndrome. Previously described MZ twins with concordance or discordance for UTS syndrome are listed in Table 111. Other twin pairs may belong in this table, but they have not been investigated thoroughly enough to either establish monozygosity or to verify the chromosomal status in both twins. Of these 20 twin pairs, 7 show concordance, with both twins having signs of UTS; 12 show discordance, with one UTS female and one phenotypically normal individual; and one case involves a

Mother

Father

Twin A 45,x

111

213 112 1/1 112 112 3I4 314 1I3

113 112 1/2 112 111 114 114 213

111 1/2

2 12 111 112 112 212

Twin B -~ 46,X -

113 112 112 112 lil 114 114 213

UTS female and a sexually ambiguous infant. The frequency of mosaicism in this group is striking. As shown in Table 111,of the 13pairs of twins discordant for UTS, 7 show clear evidence of mosaicism. Of the remaining, in 4 pairs only lymphocyte cultures were performed, or only one fibroblast culture from one or both infants was examined. When addressing MZ twins, lymphocyte cultures may be misleading due to placental vascular anastomoses, which enable blood and stem cells to be transferred from one infant to the other, resulting in pseudomosaicism or chimerism. In addition, because of the high incidence of mosaicism in sex chromosomal abnormalities, multiple tissue cultures are required to increase the likelihood of detecting mosaicism. The MZ twins described here appear to be nonmosaic, however multiple tissue samples were not examined. The full expression of UTS in the patient we report suggests that this infant was indeed nonmosaic. PCR studies searching for Y chromosome sequences in the skin from the UTS infant were negative. Other tissues would need to be studied to more fully rule out a Y-bearing cell line. Seven other cases of MZ twins with discordance of phenotypic sex have been described and are listed in Table 111, as they also involve gonadal dysgenesis. Turpin et al. [1961] described 17-year-old twins in whom both skin fibroblast cultures and fascia lata cultures showed nonmosaic 45,X and 46,XY in a UTS female and normal male respectively. However, lymphocyte cultures were 46,XY in the UTS twin and 46,XY (18 cells) and 45,X (2 cells) in the normal male. This may reflect low level mosaicism detected only in lymphocytes, or it may reflect blood chimerism with selection for the chromosomally complete cell lines. Verp et al. [19881 reported a growth disadvantage of 45,X skin fibroblasts when cocultured with 46,XX cells. Perhaps in vivo there is a similar progressive decrease in the percentage of abnormal cell lines in mosaic individuals, particularly in lymphocytes with their relatively rapid turnover. Edwards et al. [1966] reported MZ heterokaryotic twins with a UTS female and a normal male phenotype. The male, despite normal sexual development to age 21, had two skin fibroblast cultures and two lymphocyte cultures showing a nonmosaic 45,X karyotype. The authors concluded that this patient would probably show mosaicism, and the presence of Y chromosomal material, if gonadal tissue was sampled. Four other reports also described MZ twins with a UTS female and a normal male (Table 111).Only Reindollar et al. [1987] and Gonsoulin et al. 119903 examined

Discordant Monozygotic Twins

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TABLE 111. Monozygotic Twins with Turner Syndrome*

Reference Turner twin Second ____~_ .-___ ~ _twin _ _. _~_ ~ .~ ~

Turner and Zanartu [19621 Lemli and Smith [1963] Decourt et al. [19641 Pescia et al. [19751 Riekhof et al. [1972] Ferrier et al. [19701 Bourdy [19691 Potter and Taitz [19721 Ross et al. [19691

Mikkelsen et al. [1963] Aurias [1973] Pedersen et al. [1980] Weiss et al. [19821 Turpin et al. [19611 Edwards et al. [1966] Karp et al. [19751 Schmidt et al. [19761 Reindollar et al. [1987] Gonsoulin et al. [19901 Russell e t al. [1966] *

45,X (B + S ) 45,X (B) 4 5 3 (B) 45,X (B) 45,X (B + S ) 45,Xl46,XXqil46,XX (B + S ) 45,Xl46,XX (B) 45,x ( S ) 45,Xl46,XX (B) 45,x ( S ) 45,Xl47,XXX (B) 45,X/46,XX (B + S ) 45,Xl46,XXl46,XiXq (B) 46,XiXql47148149 ( S ) 45,Xl46,XX (B) 45,x ( S ) 45,X/46,XX (B) 46,XY (B) 45,X ( S and fascia) 45,X/46,XY (B) 45,x ( S ) 45,X/46,XY (B) 45,Xl46,XY (GI 46,XY (B) 45,X (S+ G ) 45,X/46,XY (B + S + G) (sexually ambiguous) 45,X @ + A ) 45,X/46,XY (B) 45,X (B + S )

45,X (B + S ) (UTS) 45,X (B) (UTS) 45,X (B) iUTS) 45,X (B) (UTS) 45,X (B + S) (UTS) 45,Xl46,XX/47XXX (B + S ) (UTS) 45,X/46,XX (B) (UTS) 46,XX (S) 45,X/46,XX (B) (nl female) 46,XX (S) 45,X/47,XXX (B) in1 female) 4 7 , x x x (S) 45,X/46,XX (B + S ) (nl female) 46,XX (nl female) 46,XX (B) (nl female) 46,XX (B) (nl fernale) 45,X/46,XY (B) (nl male) 46,X (S and fascia lata) 45,X (Bx2 and Sx2) (nl male) 45,X/46,XY (B) (nl male) 46,XY (B) (nl male) 45,X/46,XY (B + S ) (nl male) 46,XY (G) 46,XY ( S + A) (nl male) 45,Xl46,XY (B) 45,Xl46,XY (B + S + G) (intersex)

A, amniotic fluid; B, lymphocytes; S, skin fibroblasts; G, gonadal tissue.

tissue fibroblasts of the normal male. Reindollar et al. [1987] reported 2 separate cultures of penile skin showing a single 45,X cell, with 49 cells 46,XY, and cultures of gonadal tissue showing 100% 46,XY. This suggests that low level mosaicism may exist in the phenotypically normal twin in twin pairs discordant for UTS. Gonsoulin et al. 119901 reported discordant twins in utero. Cultures of the kidney, skin, lung, and placenta of the normal male were examined, all of which were 100% 46,XY. The same tissues in the twin with UTS were 100% 45,X. Russell et al. [1966] described MZ twins in whom one had UTS manifestations, and the other twin was sexually ambiguous, with a 45,X/46,XY karyotype from skin and gonad. Female sex was assigned to both twins. Also of interest are MZ triplets described by Dallapiccola et al. 119851, in whom there were two phenotypically and karyotypically normal boys and a n UTS female with a single fibroblast culture showing nonmosaic 45.X. I n the currently reported case of MZ twins discordant for sex, it appears that 2 events are occurring, the twinning event and a mitotic error resulting in loss of a Y chromosome. Correlating the placental pathology with what is known of early embryology may shed some light on the timing and sequence of these events [Benirschke and Kim, 1973; Bulmer, 19701. The inner cell mass first segregates from the trophoblast on days 3-4 after fertilization, followed by growth of the inner cell mass and development of the small amniotic cavity a t about day 7.

Therefore, a zygote that twins before days 3-4 will develop 2 chorions and amnions, resulting in a dichorionic, diamniotic placenta. When the twinning event occurs between days 3 and 7, after the chorion is differentiated but before the amnion develops, a monochorionic, diamniotic placenta results. Twinning after day 7 results in a monochorionic, monoamniotic placenta. Because the placenta of the current case was monochorionic diamniotic, the twinning event necessarily occurred between days 3 and 7. The absence of detectable mosaicism provides a clue to the timing of the mitotic error with respect to the twinning event. Russell et al. [1966] suggested 4 karyotypic possibilities in twins derived from a n XY zygote. Type 1 (X and XY), Type 2 (X/XY and XY), Type 3 (X and WXY), and Type 4 (XIXY and XIXY). I n Type 1, the twinning event and mitotic error in theory occur simultaneously shortly after the formation of the inner cell mass. In Type 2, the mitotic error occurs after the twinning event, resulting in a mosaic UTS infant and a normal male. It is also possible for the mitotic error to occur shortly before twinning in an older zygote, with segregation of the abnormal cellis into one inner cell mass and not the other. Type 3 most likely involves a mitotic error followed by twinning with unequal segregation of cells. Type 4 is the result of mitotic error occurring before the twinning event, resulting in 2 mosaic progeny. This is a convenient, albeit simplistic, approach, and selective cell death may be involved in

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any karyotypic combination. With the current case, in which no mosaicism was detected, the twinning event occurred between days 3 and 7, when the inner cell mass grows from approximately 3 to 30 cells. It is apparent that in order to have no, or low level mosaicism, without evoking selective cell death, the twinning event would have to occur very close to the time of the mitotic error. In addition, both events would have to occur when the inner cell mass was composed of very few cells. So it is most likely that in this case both twinning and mitotic nondisjunction occurred a t around 3-4 days following fertilization. Another interesting facet of this case is the discrepancy in the placental morphology. Hydropic villi are commonly seen in infants with UTS. However, these have been attributed to the presence of 45,X cell lines in the placenta, as well as the infant, resulting in hydropic changes in both. As discussed previously, the mitotic error in this case by necessity occurred in the inner cell mass, and did not involve the placental cell lines. Therefore, it seems theoretically possible that a t least part of the mesenchymal core is derived from cells originating in the embryo. The exact origin of the mesenchymal core is unclear [Blakemore, 19881. Some consider it to be a delamination of the inner wall of the trophoblast itself [Hertig, 19351, and others regard the extraembryonic mesoderm to originate from the mesoderm of the embryo itself [Luckett, 19711. Kalousek et al. [1987] suggest that chorionic villi are derived from 3 cell lineages: the trophoectoderm, the extraembryonic mesoderm, and the primitive embryonic streak. They described mosaicism confined to the chorion, presumably due to chromosomal abnormalities that arise in cells which are not progenitors of the embryo proper. It is possible that in the twins we describe the opposite has occurred with the cytogenetically abnormal cells present in the fetus and in the primitive embryonic streak which migrated into the chorionic villi, resulting in the observed hydropic villi. These issues obviously have great importance as chorionic villus sampling becomes more prevalent. Unfortunately, placental tissue in this case was not submitted for cytogenetic evaluation. In summary, MZ twins were discordant for phenotypic sex and UTS. Chromosomal polymorphism analysis and DNA fingerprinting were utilized to establish monozygosity. No mosaicism was detected; however, only one skin fibroblast culture was examined from each infant, and therefore mosaicism cannot be excluded.

ACKNOWLEDGMENTS DNA probes were generously provided by Dr. Y. Nakamura, Cancer Center, Tokyo, Dr. G. Vande Woude, Frederick Cancer Research Facility, and Dr. D. R. Higgs, University of Oxford. This work was supported in part by Grant CA 40046. W. N. was supported by USPHS postdoctoral training grant CA 09273. REFERENCES Aurias A (1974):Contribution a l’etude du mecanisme et de la signification due monozygotisme heterocaryote. These pour le Doctorat en Medecine, Paris.

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Discordant Monozygotic Twins Pedersen IK, Philip J, Sele V, Starup J (1980):Monozygotic twins with dissimilar phenotypes and chromosome complements. Acta Obstet Gynecol Scand 59:459-462. Pescia G, Ferrier PE, Wyss-Hutin D, Klein D (1975):45,X Turner’s syndrome in monozygotic twin sisters. J Med Genet 12:390-396. Potter AM, Taitz LS (1972):Turner’s syndrome in one of monozygotic twins with mosaicism. Acta Paediatr Scand 61:473-476. k e d e r s ST, Breuning MH, Davies KE, Nicholls RD, J a r m a n AP, Higgs DR, Pearson PL, Weatherall DJ (19851:A highly polymorphic DNA market linked to adult polycystic kidney disease on chromosome 16. Nature 317:542-544. Reindollar RH, Byrd J R , Hahn DH, Haseltine FP, McDonough PG (1987):A cytogenetic and endocrinologic study of a set of monozygotic isokaryotic 45,X/46,XY twins discordant for phenotypic sex: Mosaicism versus chimerism. Fertil Steril 47:626--633. Riekhof PL, Horton WA, Harris DJ, Schimke RN (1972):Monozygotic twins with the l r n e r syndrome. Am J Obstet Gynecol 112:59-61. Ross GT, Tjio J H , Lipsett MB (1969):Cytogenetic studies of presumptively monozygotic twin girls discordant for gonadal dysgenesis. J Clin Endocrinol 29:440-445. Russell A, Moschos A, Butler U,Abraham J M (19661:Gonadal dysgenesis and its unilateral variant with testis in monozygous twins: Related to discordance in sex chromosomal status. J Clin Endocrinol 26:1282-1292. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis

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KB, Erlich HA i19881: Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491. Schmidt R, Sobel EH, Nitowsky HM, Dar H, Allen FH J r (1976): Monozygotic twins discordant for sex. J Med Genet 13:64-79. Southern EM (1975):Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503-517. Turner HH, Zanartu J (1962):Ovarian dysgenesis in identical twins: Discrepancy between nuclear chromatin pattern i n somatic cells and in blood cells. J Clin Endocrinol 22660-665. Turpin R, Lejeune J, Lafourcade J, Chigot PL, Salmon C (1961):Presomption de monozygotism en depit d u n dimorphisme sexuel; sujet masculin XY e t sujet neutre Haplo X. C R Acad Sci iD) (Paris) 252:2945-2946. Verp MS, Rosinsky B, LeBeau MM, Martin AO, Kaplan R, Wallemark C-B, Otano L, Simpson J L (1988):Growth disadvantage of45,X and 46,X,del(X)(pll)fibroblasts. Clin Genet 33:277-285. Weiss E, Loevy H, Saunders A, Pruzansky S, Rosenthal IM (1982): Monozygotic twins discordant for Ullrich-Turner syndrome. Am J Med Genet 13:389-399. White R, Leppert M, O’Connell P, Nakamura Y, Woodward S, Hoff M, Herbst J, Dean M, Vande Woude G, Lathrop GM, Lalouel J-M (1986):Further linkage data on cystic fibrosis: The Utah study. Am J Hum Genet 39:694-698. Yunis JJ, Ball DW, Sawyer J R (1979): G-banding patterns of high resolution human chromosomes 6-22,X and Y. Hum Genet 49:291-306.