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INTRODUCTION

  1. Top of page
  2. INTRODUCTION
  3. CASE
  4. DISCUSSION
  5. REFERENCES

This letter reports an unexpected cell-free fetal DNA testing result.

Prenatal determination of fetal sex using cell-free fetal DNA is now commonly used as a noninvasive way of early detection of fetal sex. The technique is routinely used for fetuses at risk of X-linked disorders. The detection of Y-chromosome sequences in maternal blood plasma denotes the presence of a male fetus, whereas its absence suggests that the fetus is female. For sexing using maternal plasma, two targets on the Y chromosome are commonly used;[1] The genomic probe (DYS14), which binds to a multi-copy sequence located within the testis-specific protein Y-encoded (TSPY) gene and sex-determining region Y (SRY), a single copy gene.

CASE

  1. Top of page
  2. INTRODUCTION
  3. CASE
  4. DISCUSSION
  5. REFERENCES

The 29 year old patient was undergoing prenatal testing as she was known to have an interstitial deletion affecting the short arm of one of her X chromosomes (karyotype result 46,X,del (X)(p22.1p22.3)). The initial proband in this family was her sister, who had been found to have this deletion after being investigated for premature ovarian failure. It was in the process of cascade screening that our patient was diagnosed. Women with this deletion appear to be phenotypically normal but with an increased risk of premature ovarian failure. It was predicted that men with this deletion are likely to have significant abnormalities as they would be affected by numerous X-linked disorders. For this reason, our patient undertook prenatal testing.

A blood sample was taken from the patient at 9 weeks gestation to determine the sex of the fetus. Quantitative polymerase chain reaction (PCR) analysis of DNA extracted from a maternal serum contained both the DYS14 and SRY gene sequence at an unexpectedly high level. Using real-time quantitative PCR, the typical number of cycles required for the florescent signal to cross the threshold level for the DYS14 probe, using DNA extracted from maternal plasma, is 34 to 37. The number of cycles required in our case before threshold was reached ranged from 27.65 to 28.29. As this was a lot less than is usually required, it suggested that the maternal serum contained higher levels of the DYS14 gene sequence than that typically seen from carrying a male fetus. A similar pattern was also seen for the SRY gene sequence. DNA was then extracted from the buffy coat of the blood sample and the test repeated. This indicated that the mother possesses the DYS14 and SRY gene sequence in her own DNA. Fluorescence in situ hybridization, using the Vysis SRY probe, confirmed the presence of the SRY gene on the abnormal chromosome X carried by the mother.

The presence of the SRY gene in the maternal DNA would mask the presence of any fetal SRY making the result impossible to interpret. The patient was therefore offered the option of a chorionic villous sample in order to clarify the situation. This found the fetus to be female with two normal copies of the X chromosome.

As a consequence of this cell-free fetal DNA testing result, further studies were performed on the initial proband in this family. Microarray analysis [Nimblegen CGX microarray (HG18, Build 36) Roche NimbleGen, Madison, Wisconsin, USA] detected a 28 Mb deletion (1443 probes) that mapped to the short arm of chromosome X at the position p22.32p21.1 (2,75,239-31,166,969). Chromosome Y material encompassing a 7 Mb region (344 probes) was also mapped to a 7 Mb region at position p11.31p11.2 (2,806,349-10,071,857). The Xp telomere was still present indicating a translocation of the SRY region into the short arm of one of the X chromosomes. This confirmed the presence of an XY translocation 46,X,der(X)t(X;Y)(p22.33p21.1;p11.2) (Figure 1).

image

Figure 1. Partial karyotype of G-band chromosome X with the abnormal chromosome on the right hand side. Also, fluorescence in situ hybridization (FISH) of chromosome X with blue 4',6-diamidino-2-phenylindole (DAPI) counterstain, green X centromere signals and red sex-determining region Y (SRY) signal on the abnormal X

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DISCUSSION

  1. Top of page
  2. INTRODUCTION
  3. CASE
  4. DISCUSSION
  5. REFERENCES

The interstitial deletion (X)(p22.3p22.1) was also accompanied by insertion of material from the Y chromosome including the SRY gene, which is usually located at Yp11.3. This forms an unbalanced XY translocation which probably occurred during recombination as part of the process of meiosis in an intervening male relative. Recombination between the X and Y chromosome is usually limited to the pseudoautosomal region 1 and 2.[2] Occasionally, recombination outside of this region can occur because of homologous sequences between the X and Y chromosome.[3]

The presence of SRY on the X chromosome can be associated with variable sexual phenotype including both fertile men and women, 46,XX maleness and in rare cases 46,XX true hermaphrodites.[2, 5, 6] It has also been reported to cause variable sexual phenotype within the same family.[5] In people with 46,XX testicular disorders of sexual development, the SRY gene is present in approximately 80%, usually on one of the X chromosomes.[4] This phenotypic variation may be due to reduced or variable SRY expression resulting from either spreading of X inactivation into the translocated Yp segment, a mutation within the SRY gene or a position effect due to the close proximity of a translocation breakpoint.[5] This positional effect may reduce the SRY expression below a critical threshold such that variation in the expression level of interacting genes, such as SRY box 9 (SOX9) and dosage sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX1), could result in variation in testis-determination and sexual phenotype.[5] The additional SRY gene in this family appears to be nonfunctional and the cause of this could be for any of these factors. There are very few reported cases of XX SRY-positive women in the literature suggesting that this family is either extremely rare, or that because the phenotype is relatively benign, formal testing is very rarely requested. More cases of XX SRY positive women may come to light with improved genetic testing methods such as microarray, which will give us a better understanding of the phenotype.

In conclusion, this case details an unexpected cell-free fetal DNA testing result, which was only detected through employment of the quantitative PCR technique. If this was not the case, the correct karyotype would not have been reported. This is likely to be a very rare occurrence, but if a similar result is found again, we would recommend further testing of the maternal chromosomes to look for a translocation encompassing the SRY gene.

WHAT'S ALREADY KNOWN ABOUT THIS TOPIC?

  • Cell-free fetal DNA testing is commonly used as a noninvasive method of determing fetal sex.
  • Presence of sex-determining region Y (SRY) on the X chromosome is associated with a variable sexual phenotype.

WHAT DOES THIS STUDY ADD?

  • Details an unexpected cell-free fetal DNA sexing result.
  • Highlights the importance of using the technique of quantitative polymerase chain reaction in free fetal DNA testing.
  • If a similar result is found again, we would recommend further testing of the maternal chromosomes to look for a translocation encompassing the sex-determining region Y (SYR) gene.

REFERENCES

  1. Top of page
  2. INTRODUCTION
  3. CASE
  4. DISCUSSION
  5. REFERENCES