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. 2001 Mar;60(3):248–253. doi: 10.1136/ard.60.3.248

Lack of evidence for an increased microchimerism in the circulation of patients with Sjögren's syndrome

I Toda 1, M Kuwana 1, K Tsubota 1, Y Kawakami 1
PMCID: PMC1753575  PMID: 11171687

Abstract

OBJECTIVE—To examine the hypothesis that fetal microchimerism plays a part in the pathogenic process of Sjögren's syndrome (SS).
METHODS—Genomic DNA samples were extracted from peripheral blood whole nucleated cells and the CD34+ cell enriched fraction of patients with SS and healthy women who had male offspring as well as nulliparous women. A Y chromosome-specific sequence was detected as a marker for fetal cells by a nested polymerase chain reaction (PCR) and by DNA hybridisation combined with PCR using specific primers and probes. All procedures were performed with great care to avoid the contamination of male DNA.
RESULTS—A nested PCR and DNA hybridisation combined with PCR was established that can detect a single male cell out of 1.67×105 female cells. It was not possible to increase the sensitivity further because the amount of template DNA held in the PCR was limited. When these methods were used, no fetal cells were detected in any samples from patients with SS, though they were detected in whole nucleated cells from two healthy women who had delivered sons previously.
CONCLUSIONS—The findings indicate that circulating fetal cells in patients with SS are uncommon (<1 in 1.67×105), if they exist. With the conventional PCR based methods that were used, it is difficult to evaluate the quantitative difference in circulating fetal cells between patients with SS and healthy women.



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Figure 1  .

Figure 1  

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Amplification of the TSPY gene using male DNA as a template in the presence of serial amounts of background female DNA. Serial amounts of female genomic DNA (100 ng—10 µg) were mixed with 10 ng male genomic DNA and used as templates for the first polymerase chain reaction (PCR). The PCR products were fractionated on a 1.5% agarose gel and stained with ethidium bromide. Lane 1: 100 ng, lane 2: 300 ng, lane 3: 600 ng, lane 4: 1 µg, lane 5: 2 µg, lane 6: 5 µg, and lane 7: 10 µg of background female DNA. Lane 8: molecular weight markers (100 bp ladder).

Figure 2  .

Figure 2  

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Detection of the TSPY gene by nested polymerase chain reaction (PCR) using serial amounts of male DNA as templates. Serial amounts of male DNA (10−3-10−10 µg) in 1 µg of female DNA were amplified by the nested PCR. The PCR products were fractionated on 1.5% agarose gel and stained with ethidium bromide. Lane 1: 10−3 µg, lane 2: 10−4 µg, lane 3: 10−5 µg, lane 4: 10−6 µg, lane 5: 10−7 µg, lane 6: 10−8 µg, lane 7: 10−9 µg, and lane 8: 10−10 µg of male DNA. Lane 9: no template for the first PCR and lane 10: no template for the second PCR. Lane 11: molecular weight markers (100 bp ladder).

Figure 3  .

Figure 3  

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Detection of male cells in peripheral blood whole nucleated cells from women who had delivered sons within the past 24 hours by a nested polymerase chain reaction (PCR). Genomic DNA from peripheral blood whole nucleated cells obtained from three women one day after delivery (lanes 1-3: 1 µg), and control male DNA (lane 4: 10−5 µg and lane 5: 10−2 µg) were applied for the nested PCR. The PCR products were fractionated on 1.5% agarose gel and stained with ethidium bromide. Lane 6: no template for the first PCR and lane 7: no template for the second PCR. Lane 8: molecular weight markers (100 bp ladder).

Figure 4  .

Figure 4  

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Detection of male cells in peripheral blood whole nucleated cells from healthy women with no sons, healthy women with sons, and patients with SS with sons by a nested polymerase chain reaction (PCR). Genomic DNA (1 µg) from these subjects was amplified by a nested PCR, and the PCR products were fractionated on 1.5% agarose gel and stained with ethidium bromide. Lanes 1-6: healthy women with no sons, lanes 7-17: healthy women with sons, and lanes 18-36: patients with SS with sons. Lane 37: male DNA (10−5 µg), lane 38: DNA from a female stem cell transplantation (SCT) recipient from a male donor (1 µg), lane 39: no template for the first PCR, and lane 40: no template for the second PCR. Lane 41: molecular weight markers (100 bp ladder).

Figure 5  .

Figure 5  

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Detection of male cells in peripheral blood whole nucleated cells from patients with Sjögren's syndrome with sons, healthy women with sons, and healthy women with no sons by DNA hybridisation combined with a polymerase chain reaction (PCR). The PCR products of the first PCR were fractionated by electrophoresis and hybridised with probes specific for the TSPY gene. Reactivities were visualised with a chemiluminescence detection system. Lanes 1-18: patients with SS with sons, lanes 19-31: healthy women with sons, and lanes 32-36: healthy women with no sons. Lane 37: male DNA (10−2 µg) in the first PCR and lane 38: no template for the first PCR. Lane 39: molecular weight markers (100 bp ladder).

Selected References

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