First-trimester ultrasonographic scans of a 28-year-old primigravida with a naturally fertilized twin pregnancy showed unequivocal monochorionic diamniotic placentation at 6 weeks of gestation, indicative of monozygosity. From 14 weeks, serial scans showed phenotypic sex discordance in structurally normal twins, with Fetus 1 appearing to be male and Fetus 2 appearing to be female. Retrospective review of the results obtained on first ultrasonographic imaging and pathological review of the placenta confirmed the pregnancy to be monochorionic. Mid-trimester amniocentesis was performed on each amniotic sac to investigate for mosaicism and to validate zygosity. Each parent provided written informed consent for the genomic analyses of each twin and for the analysis of her or his own genome.

G-banded karyotyping on cultured cells showed 46,XX/46,XY chimerism in each twin. Quantitation by means of single-nucleotide polymorphism (SNP) array (Illumina HumanCytoSNP-12, version 2.1; effective resolution, 0.20 Mb) and fluorescence in situ hybridization (FISH) (Vysis probes DXZ1, DYZ3, and SRY [Abbott Molecular]) on cultured and uncultured amniocytes confirmed 46,XX/46,XY chimerism in each twin. FISH showed an XX/XY chimerism ratio of 47:53 in Twin 1 and 90:10 in Twin 2, and SNP karyotyping resulted in similar ratios (50:50 and 93:7, respectively). Near-similar ratios were later confirmed in cord tissue from each twin, whereas ratios in postnatal blood lymphocytes were similar in the two twins (XX/XY chimerism ratio, 81:19 in Twin 1 and 78:22 in Twin 2), which is explained by the shared circulation during gestation (Table S1 in the Supplementary Appendix, available with the full text of this article at NEJM.org).

Noninvasive zygosity testing was performed by means of targeted sequencing (Roche NimbleGen hybridization capture–based target enrichment followed by paired-end Illumina sequencing) of circulating DNA in maternal plasma. Sequencing data were interpreted with the use of the FetalQuant algorithm5,6 to identify and quantify the proportion of minor alleles detected across sequenced SNP sites per chromosome, termed the “apparent fractional fetal DNA concentration.” The concentrations of fetal DNA at different loci varied to a greater extent than is normally the case, with 46% of tested SNPs showing either greater representation or lesser representation than the predicted stochastic value for monozygotic twins, which indicated that the twins were not monozygous (Fig. S1 in the Supplementary Appendix). However, the genotyping tracks in the SNP array of mosaic amniotic DNA from each twin showed allelic contributions from genetically different persons only approximately half the time, in contrast to the 75% nonsharing expected in dizygotic siblings.

Figure 1. Figure 1. Results of Pairwise SNP Analysis. As shown in Panel A, an analysis that used the SNPduo Web tool (Pevsner Laboratory, Baltimore) compared genotypes between Twin 1 and Twin 2 and showed regions of nonshared genotypes (identity by state [IBS] 1 [IBS1]) and shared genotypes (IBS2) across chromosome 11 (data on other chromosomes are available in Fig. S4 in the Supplementary Appendix). Panels B and C show the results of a modified pairwise SNP analysis that compared only the A and B alleles from each parent with those of Twin 1 and Twin 2 across chromosome 11. The paternal haplotypes were shared between twins at some loci (IBS1) and differed at others (IBS0). Maternal haplotypes were identical across all chromosomes between twins and therefore shared a single allele (IBS1).

To investigate this unusual genetic concordance in nonidentical twins, peripheral-blood DNA from each parent was analyzed by means of SNP array, and parental alleles were compared with those of each twin to determine identity by state with the use of the SNPduo Web tool (Pevsner Laboratory, Baltimore). By this method, we concluded that the twins shared either one or two identical alleles at each locus. There were no loci at which the twins had no identical alleles, as should be observed for approximately 50% of loci in dizygotic twins. The same pairwise analysis was used to evaluate SNP data between both the maternal and paternal alleles and between one cell line derived from Twin 1 and one derived from Twin 2. This analysis revealed identical maternal genotypes across all autosomes of each twin (i.e., both twins had the same maternal copy) but a mix of identical and nonidentical paternal genotypes (i.e., different paternal copies) in each twin (Figure 1A through 1C, and Figs. S2 and S3 in the Supplementary Appendix). Analysis of SNPs from twin amniotic fluid and parental blood showed that 265,489 maternal and 179,205 paternal SNPs were informative for parent of origin. Of these, the twins shared 265,400 maternal and 139,155 paternal SNPs, which makes them 100% maternally identical and 77.7% paternally identical.

To show that each twin had three distinct haplotypes, as inferred from the pairwise SNP analysis, we used linked-read sequencing to directly determine phased haplotypes from amniotic-fluid DNA of each twin. Long DNA molecules were partitioned into different droplets and tagged by a unique sequence barcode for all DNA subfragments with the use of linked-read technology (10x Genomics). The 10x Genomics–based library was further captured by probes that covered common SNPs present in chromosomes 1, 2, 3, 5, 8, 15, and 22. Sequencing results that enabled the assembly of haplotype blocks as fragments sharing a common “barcode” were derived from the same haplotype block. Bioinformatic strategies were then used to identify regions that harbored three haplotypes. Sequenced short reads that were each tagged by a unique barcode were mapped to the human reference genome and assembled to synthetic long reads on the basis of the barcode information. Next, long reads were clustered if they mapped to the same genome region. For long molecules in the same cluster, we determined haplotype information and counted the number of haplotypes detected.

In Twin 1, we identified 715 regions (median block size, 25.5 kb; interquartile range, 3.9 to 171.1), or 14.5% of the directly phased haplotypes, that were composed of three distinct haplotypes (which suggests the existence of chimerism, in which some cells of Twin 1 had one paternal haplotype and other cells of this twin had the other paternal haplotype). In Twin 2, we identified 357 regions (median block size, 8.6 kb; interquartile range, 1.6 to 195.9), or 10.7% of the haplotypes, that were composed of three haplotypes (Fig. S5 and Table S2 in the Supplementary Appendix). The locations of these “polyploid” loci were consistent with those shown by the pairwise SNP analysis and provide support for the findings that each twin had the same maternal haplotype and that each was chimeric for two distinct paternal haplotypes.

The parents were counseled about the risk of genital ambiguity and mixed gonadal dysgenesis, and the pregnancy progressed uneventfully until 33 weeks of gestation, when, in the presence of fetal growth discordance, the smaller Twin 1 had a low amniotic-fluid volume and reduced fetal movements. Umbilical arterial Doppler studies suggested fetal compromise. The twins were delivered by cesarean section and had normal Apgar scores of 7 to 9. Histologic analysis confirmed monochorionic diamniotic placentation.

Postnatal examination and ultrasonography of the genital tract confirmed Twin 1 as phenotypically male and Twin 2 as female, with no evidence of sexual ambiguity. Soon after birth, a purpuric rash was noted in Twin 2, which extended from the right mid-humerus to the right hand. She received a diagnosis of a right brachial artery thromboembolism caused by a paradoxical embolus from an inferior vena cava thrombus. On review of the obstetric ultrasonographic images on the day of delivery, the thrombus was believed to have arisen just before birth. Despite enoxaparin therapy, the limb could not be saved, and she underwent a below-shoulder amputation at 4 weeks of age. A comprehensive thrombophilia screening in Twin 2 was negative. During follow-up at 3 years of age, routine ovarian surveillance showed Twin 2 to have gonadal dysgenesis, and prophylactic oophorectomy was performed. Otherwise, both twins were developmentally normal and without physical evidence of mosaicism.