Between Light and Dark, the Chimera Comes Out

Background  Chimerism, especially in the absence of sexual ambiguity, is extremely rare in humans. We report the case of a 6-year-old boy whose skin pigmentary abnormalities revealed chimerism.

Observations  The boy had no remarkable previous medical history, and he had normal intelligence and development. On examination, we found a disorder of the skin pigmentation that was difficult to categorize; there was a lighter-appearing skin patch in the median frontal area and also on one-half of the abdominal area, with a sharp midline demarcation. He also had 2 lighter Blaschko-linear bands on the lower extremities and an indefinable mixture of lighter and darker skin on the back and the lateral part of the trunk. It was not possible to ascertain by means of clinical examination of the patient, his parents, and his brother which of the 2 shades was his normal skin color. Because this pattern of pigmentation might be related to mosaicism, we determined his karyotype. We found that his lymphocytes had a normal number of chromosomes, half of them being either 46,XX or 46,XY. In contrast, his fibroblasts were exclusively XY. The chimerism was confirmed by the analysis of the red blood cell antigens, which revealed the presence of 2 different populations. The characterization of the HLA haplotypes of the lymphocytes showed that the boy inherited 2 HLA haplotypes from his mother but only 1 from his father. Interestingly, the ratio of XX to XY cells was expanded in the T-cell subset compared with other peripheral blood mononuclear cell populations.

Conclusions  This is an exceptional case of human chimerism revealed by abnormal skin pigmentation. This boy displayed 2 normal shades of skin color, which we suggest be termed cutis bicolor, as a result of 2 different genetic backgrounds. He also had immune chimerism, which challenges our current comprehension of antigen presentation and tolerance.

In Greek mythology, the chimera was a fire-breathing female monster resembling a lion in the forepart, a goat in the middle, and a dragon behind. She devastated Caria and Lycia until she was slain by Bellerophon.

In medicine, the term chimera designates a person composed of 2 genetically distinct types of cells. During pregnancy, fetal cells pass into the mother, where they persist for decades after the pregnancy, leading to fetal microchimerism. Maternal cells also pass into the fetus, where they can persist long after the birth of the child into adulthood, leading to maternal microchimerism.¹ Microchimerism occurs in healthy individuals, but it can also be involved in the pathogenesis of some skin disorders, such as pityriasis lichenoides or polymorphic eruptions of pregnancy, and in autoimmune disorders, such as scleroderma.^2-4

However, microchimerism is defined by the presence within an individual of a low level of cells (about 1:500 000 cells) derived from a different individual, and it is thus different from chimerism.¹ Chimerism occurs, for example, after bone marrow or hematopoietic stem cell transplantation. Very exceptionally, chimerism can occur spontaneously, and it is then the result from the amalgamation of 2 different zygotes in a single embryo, which leads to tetragametic chimerism.⁵

Most reported cases of tetragametic chimerism occurring in individuals without sexual ambiguity were detected when blood group screening revealed an unusual phenotype.^6,7 We report the case of a young patient in whom skin pigmentary abnormalities revealed chimerism.

A 6-year-old-boy was referred for evaluation of white spots on his skin. He was born after a healthy pregnancy, his development was normal, and his parents related no significant medical event. He had never received a blood transfusion. On examination, his skin had 2 shades of color. He had lighter skin on the front; parts of the trunk, with a sharp midline delimitation on the abdomen; and on bands distributed along the lines of Blaschko on the legs (Figure 1 and Figure 2). He had an indefinable mixture of lighter and darker skin on the back and the lateral part of the trunk (Figure 3), and his skin was darker on the rest of the body. Even after examination of his brother and his parents, it was not possible to ascertain which of the 2 shades was his normal skin color. Otherwise, examination findings were unremarkable, and there was no clinical sexual ambiguity.

This very unusual type of pigment distribution strongly suggested a mosaicism or chimerism, so we performed cytogenetic investigations. Lymphocyte karyotyping showed that 50% of lymphocytes were 46,XY and 50% were 46,XX. In contrast, skin fibroblasts from a biopsy specimen of lighter-appearing skin displayed a normal 46,XY karyotype. Blood group determination revealed a double population (Table 1).

Serological HLA typing also revealed the presence of 2 lymphocyte populations, each expressing 1 different HLA haplotype, whereas the second HLA haplotype was common to all cells, making a total of 3 expressed HLA haplotypes. The HLA typing of the parents demonstrated that the common HLA haplotype was inherited from the father, whereas the 2 other haplotypes had a maternal origin. Genotyping confirmed the presence of the 3 HLA haplotypes in his blood cells (Table 2 and Table 3). Interestingly, 1 maternal haplotype expressed HLA-B*49 molecules, which are recognized by anti–HLA-Bw4 antibodies, whereas the other 2 expressed HLA-B molecules recognized by anti–HLA-Bw6 antibodies. The HLA typing of the skin fibroblast cell line (XY) demonstrated that the male cells were Bw4+Bw6+, and thus, by exclusion, that the XX cells were Bw6+/Bw6+. This property was used to characterize more precisely the ratios of XX and XY cells among peripheral blood mononuclear cell populations (see the next paragraph). These cells were stained with an anti-Bw4 monoclonal antibody and monoclonal antibodies specific to different blood cell populations. Flow cytometry analysis showed that 90% of the monocytes, B cells, and NK cells, but only 40% of the T lymphocytes, were HLA-Bw4+ and consequently XY. Control experiments using an anti-Bw6 monoclonal antibody confirmed that 100% of the cells were Bw6+.

Peripheral blood mononuclear cells were prepared by centrifugation on a Ficoll cushion (Flobio SA, Courbevoie, France), stained with an anti-Bw4 or an anti-Bw6 monoclonal antibody, and counterstained with phycoerythrin-conjugated F(ab′)₂ polyclonal goat anti-mouse antibodies. After a blocking step with mouse serum, cells were labeled with fluorescein isothiocyanate (FITC) conjugated monoclonal antibodies specific to CD19 (B cells), CD3 (T cells), or CD14 (monocytes). To analyze NK lymphocytes (CD3⁻/CD16⁺ or CD56⁺), cells were labeled with 3 monoclonal antibodies (anti-CD16 and anti-CD56, both FITC-conjugated, and anti-CD3, phycoerythrin-cyanine5–conjugated). Cells were analyzed using a flow cytometer (Facscalibur; Becton-Dickinson, Mountain View, California). The Bw4+ positivity allowed analysis of XY cells of parental origin.

Tetragametic chimerism is exceptional in individuals without sexual ambiguity, and previously reported cases were detected during an investigation of unusual blood phenotypes or of a disputed maternity.^6-8 A MEDLINE search did not identify reports of cases revealed by abnormal skin pigmentation, although it has been stipulated for years that chimerism could result in abnormal skin pigmentation.⁹

Eleven years ago, Happle et al¹⁰ coined the term cutis tricolor to designate hyperpigmented and hypopigmented patches in a boy with multiple other congenital defects. We suggest naming the anomaly reported herein cutis bicolor, because our patient actually had 2 normal shades of color associated with 2 normal genetic backgrounds. Yet, it is true that every form of cutaneous chimerism or mosaicism can result in 2 shades of color, and thus the term cutis bicolor is not specific. Our patient had 2 Blaschko-linear lighter bands on a limb and lighter skin sharply demarcated on the midline in the abdominal area, probably resulting from the migration of skin stem cells from a single origin during embryogenesis. Those 2 patterns and the lighter patch on the median front fit with the classification developed by Happle et al¹⁰ of skin patterns observed in mosaicism and correspond respectively to skin types Ia, II, and IV.¹¹ However, on the rest of his body, there was an indefinable mixture of lighter and darker skin. Thus, the pattern of pigmentation in this boy reflects the complex regulation of skin pigmentation during embryogenesis in humans. Given the neural crest origin of melanocytes, the absence of sexual ambiguity is remarkable.

Karyotype analysis revealed the presence of equal numbers of XX and XY peripheral lymphocytes, whereas HLA typing of blood lymphocytes demonstrated the inheritance of 2 HLA haplotypes from his mother and 1 from his father. Surprisingly, 60% of the T cells but only 10% of other mononuclear cells had the HLA haplotype of XX cells. Thus, the XX T cells seemed to be expanded, which points to puzzling mechanisms of the regulation of homeostasis of these T lymphocytes. Another interesting issue is the lack of NK-mediated autoimmunity directed against cells of different haplotypes, although it is known that in chimeric individuals the 2 different cell populations are tolerated immunologically. Indeed, usually NK cells target and kill cells of different haplotypes. We have not investigated the repertoire of the inhibitory receptor of NK cells, but it was demonstrated¹² that, in normal situations, a subpopulation of NK cells, not equipped with inhibitory receptors for self-HLA class I molecules, can be present and not autoreactive. This observation can possibly explain the absence of reactivity of XY NK cells toward XX cells in this young boy.

In summary, this boy did not have 1 normal and 1 abnormal skin color but rather 2 shades of colors related to 2 different genetic backgrounds as a consequence of chimerism. We suggest calling this anomaly cutis bicolor. The prevalence of this phenomenon in humans is unknown, and published reports in apparently healthy individuals without sexual ambiguity are exceptional.⁵ The immune chimerism in this boy challenges our conception of antigen presentation and tolerance, and his long-term prognosis seems unpredictable and will need cautious follow-up at puberty.

Correspondence: Dan Lipsker, MD, PhD, Clinique Dermatologique, 1 Place de l’hôpital, 67091 Strasbourg CEDEX, France (dan.lipsker@chru-strasbourg.fr).

Accepted for Publication: October 16, 2007.

Author Contributions:Study concept and design: Lipsker and de la Salle. Acquisition of data: Lipsker and de la Salle. Analysis and interpretation of data: Lipsker, Flory, Wiesel, Hanau, and de la Salle. Drafting of the manuscript: Lipsker and de la Salle. Critical revision of the manuscript for important intellectual content: Flory, Wiesel, and Hanau. Administrative, technical, and material support: Lipsker and de la Salle.

Financial Disclosure: None reported.