The initial HLA-specific PCR assay was developed to investigate maternal microchimerism in a 49-year-old woman with scleroderma, and targeted the HLA class I antigen HLA-B44. The patient’s class II haplotypes were DRB1*1104-DRB3*02-DQA1*0501-DQB1*0301 and DRB1*1501-DRB5*0101-DQA1*0102-DQB1*0602, and the mother was homozygous for the former haplotype; thus, no class II allele could be targeted to test for maternal DNA. However, at the HLA-B locus the mother had B44 and B8, whereas the patient had B35 and B8. The mother’s and patient’s HLA-B alleles were determined by sequencing to be B*4403,*0801, and B*3508,*0801, respectively. HLA-B44–specific primers were developed, and the difference in B alleles were exploited to test for maternal-specific DNA in the patient. As seen in Figure , the 190-bp HLA-B44–specific PCR product was readily detected in DNA extracted from PBMCs of the patient. Blood samples from 3 different draw dates spanning 2.5 years (2 of which are shown in Figure ) were positive for the noninherited maternal DNA. The HLA-specific PCR product was eluted, purified, and subjected to sequencing. The sequence obtained (Table ) was identical to that from the mother (both from DNA extracted from PBMCs and from sequencing the product from the mother after the HLA-B44–specific assay). The sequence corresponds to amino acids 110–172 encoded by the third exon of HLA-B*4403. No HLA-B44–specific DNA was detected in another scleroderma patient for whom the mother had a nonshared HLA-B44 antigen. Two children represent alternative sources of microchimerism in this patient.
Figure 1 HLA-B44–specific PCR of DNA extracted from PBMCs of a 49-year-old scleroderma patient who was B44-negative and whose mother was B44-positive. Lanes 3 and 5 are patient samples from 2 different draw dates. Lane 7 is a positive control from the (more ...)
Sequencing result for the HLA-specific product shown above and deduced amino acid sequence
Additional HLA-specific assays were developed targeting HLA class II sequences of DRB5
, and DRB1*11
. The DRB5
locus is absent on all HLA haplotypes, except for those that have DRB1
alleles that encode for DR2
molecules. Primers were developed that were specific for DRB5*01
, which is present on haplotypes that encode DRB1*15
, and were employed to test all scleroderma patients and healthy subjects for whom the mother had DRB5*01
that was not shared by the subject. DR2
molecules are encoded by DRB1*15
alleles; none of our families had DRB1*16
alleles, so no other DRB5-
encoded molecules were present in the families studied. DRB5*01
-specific DNA was detected in 2 of 3 SSC patients. DRB5*01
-specific DNA was also detected in 2 of 3 healthy normal subjects. The scleroderma patient shown in Figure had positive results in all aliquots tested and in an additional assay (each assay is done in duplicate); the control subject had positive results in 1 aliquot but not in another aliquot from a different draw date. Sequencing of the 210-bp product revealed identity of the subjects and the mothers’ alleles (Table ). The sequence corresponds to amino acids 7–76 of DRB5*01
. In the scleroderma patient shown in Figure , DRB5*01
-specific DNA could also have originated from a child, because this patient had 3 sons, all of whom inherited DRB5*01
from their father. However, the patient had no other pregnancies, and multiple tests for male DNA using both a quantitative assay (6
) and a very sensitive nested PCR to test for male DNA (17
) consistently revealed no male DNA in peripheral blood samples from this patient. In this scleroderma family, it is interesting that the haplotype the patient did not inherit from her mother was identical to the paternally inherited haplotype of her sons for DRB1
, and HLA-B.
Figure 2 HLA-DRB5*01-specific PCR of DNA extracted from PBMCs of a scleroderma patient and a normal subject. The DRB5 gene is present only on some HLA haplotypes. Both mothers had DRB5*01 and both subjects did not. Lanes 3 and 4 are patient samples (more ...)
Sequencing result for the products of HLA-specific PCR shown above and deduced amino acid sequence
DRB1*04-specific and DRB1*11-specific assays identified maternal microchimerism in 10 of 18 and 2 of 5 subjects, respectively. Two of 3 scleroderma patients had maternal-specific DRB1*04 detected, as did 8 of 15 controls. In the normal subjects, the only potential source of DRB1*04-specific DNA was from the mother. In 1 scleroderma patient, an alternative potential source was the patient’s daughter, who inherited DRB1*04 from her father. However, the child’s DRB1*04 allele was DRB1*0404, whereas that of the patient’s mother was DRB1*0401. Additional testing was conducted in which a DRB1*04-specific primer was used in conjunction with 1 of 2 different primers that target a difference between DRB1*0404 and DRB1*0401, based on a dimorphism at position 258. The results clearly indicated that the chimeric DNA was DRB1*0401 (maternal); no DRB1*0404 DNA was detected (data not shown). One scleroderma patient and 1 of 4 controls tested for DRB1*11 had maternal microchimerism.
Overall maternal-specific DNA was detected in 17 of 31 (55%) study subjects, without a significant difference in scleroderma patients and controls. The age range of scleroderma patients with persistent maternal microchimerism was 11–49 years (mean age: 34 years). The age range of all normal subjects with persistent maternal microchimerism was 9–46 years (mean age: 25 years).
PBMC cytospin preparations from 5 male subjects were subjected to in situ hybridization with double labeling for X and Y chromosome–specific sequences. Male subjects included 3 scleroderma patients (1 with scleroderma/myositis overlap) and 2 normal subjects. Two female cells were seen in 1 of the scleroderma patients among 69,745 cells counted, and 1 female cell was seen among 88,567 cells counted in the patient with scleroderma/myositis overlap (Figure ). No female cells were seen in a normal male among 32,622 cells counted, in a second normal male among 32,622 cells counted, or in the other male scleroderma patient among 40,687 cells counted.
Figure 3 In situ hybridization with double labeling for X and Y chromosome–specific sequences of PBMCs from male patients with scleroderma (above) and scleroderma/myositis overlap (below). A cell with 2 X chromosomes (green) is seen among male cells that (more ...)