Intersexual Twins due to Tetragametic Chimerism.

Disorders of or differences in sexual development (DSD) are defined by congenital conditions in which development of chromosomal, gonadal, or anatomic sex are atypical. Here, we report on monochorionic diamniotic twins delivered by caesarean section in the 36th week of pregnancy. Monochorionic twins are usually monozygous and thus should have the same sexual differentiation. In this case, one twin had female external genitalia, while the other showed ambiguous genitalia. At first, a diagnosis of mixed gonadal dysgenesis was proposed because of the obvious sexual discrepancy between the supposedly monozygous twins. Cytogenetic analyses were performed to assure the sex chromosome status for both children. Male and female cells were found subsequently in both children. While hematopoietic chimerism of monochorionic dizygous twins as a result of twin-to-twin blood transfusion is a rare but already well-documented phenomenon, to our knowledge this is the first case description of tetragametic chimerism that led to intersexuality.

Diagnostic applications of next generation sequencing in immunogenetics and molecular oncology.

SUMMARY: With the introduction of the next generation sequencing (NGS) technologies, remarkable new diagnostic applications have been established in daily routine. Implementation of NGS is challenging in clinical diagnostics, but definite advantages and new diagnostic possibilities make the switch to the technology inevitable. In addition to the higher sequencing capacity, clonal sequencing of single molecules, multiplexing of samples, higher diagnostic sensitivity, workflow miniaturization, and cost benefits are some of the valuable features of the technology. After the recent advances, NGS emerged as a proven alternative for classical Sanger sequencing in the typing of human leukocyte antigens (HLA). By virtue of the clonal amplification of single DNA molecules ambiguous typing results can be avoided. Simultaneously, a higher sample throughput can be achieved by tagging of DNA molecules with multiplex identifiers and pooling of PCR products before sequencing. In our experience, up to 380 samples can be typed for HLA-A, -B, and -DRB1 in high-resolution during every sequencing run. In molecular oncology, NGS shows a markedly increased sensitivity in comparison to the conventional Sanger sequencing and is developing to the standard diagnostic tool in detection of somatic mutations in cancer cells with great impact on personalized treatment of patients.

Disorders of implantation--are there diagnostic and therapeutic options?

Recent developments in reproductive medicine address oocyte morphology, sperm analysis and embryo selection. However, in a subgroup of infertile couples, it is the embryo implantation process that is disrupted. Diagnostic tools to identify patients at risk of implantation failure are limited and therapeutic options are far away from being established. In this review we focus on selected possible causes and treatments of failed implantation. Reproductive surgery allows a proper first step diagnosis and therapy of recurrent implantation failure (RIF). Possible anatomical malformations and associated diseases with treatment options are mentioned. Diagnostic procedures in patients often focus on defining gene polymorphisms (like hereditary thrombophilia and p53) and determinants of endometrial receptivity including endometrial gene expression profiles. Although significant differences in gene expression have been identified, the study populations are quite small, some of the data conflicting and clinical significance has yet to be proven. Implantation requires a close interaction between the fetal trophoblast and the maternal endometrium with natural killer cells (NK cells) playing a main part at the feto-maternal interface during early pregnancy. Therefore this review also focuses on NK cell receptor expression and new immunomodulatory treatment options like G-CSF in RIF patients.

Discrimination of HLA null and low expression alleles by cytokine-induced secretion of recombinant soluble HLA.

The disruption of disulfide bridges can decrease or abolish the cell surface expression of HLA class I molecules. Such disulfide bridges are formed by cysteine residues between amino acid (aa) positions 101/164 (alpha(2) domain) and 203/259 (alpha(3) domain). Sequence alterations in codons 101, 164, 203 and 259 have been observed in eleven HLA-A molecules. All of these variants except of A*3014L and A*3211Q have been reported to result in null expression alleles. In the case of HLA-A*3014L, a transversion at nucleotide position 563 replaces cysteine by serine at position 164 of the mature polypeptide. HLA-A*3014L is not detectable by standard microlymphocytotoxicity assay. To verify low or non-expression of this allele, we cloned soluble HLA-A*3014L and the reference allele HLA-A*3001 into a eukaryotic expression vector and transfected K562, C1R and HEK293 cells. Expression of soluble HLA-A*3014L and HLA-A*3001 was measured in the supernatants of transfected and untransfected cells incubated with or without IFN-gamma and/or TNF-alpha using a W6/32 and anti-beta(2)-microglobulin-based sandwich ELISA. Expression of mRNA transcripts of both alleles was determined by real-time RT-PCR. HLA-A*3014L was not detected in the supernatant of unstimulated transfectants. Stimulation with IFN-gamma and/or TNF-alpha led to an increase of HLA-A*3014L secretion to a detectable level and increased HLA-A*3001 expression up to 8-fold, but did not show any difference in the increase of mRNA levels between HLA-A*3014L and A*3001. Because of this lack of any difference in the mRNA transcription, the protein expression defect is most likely caused by the missing disulfide bond formation in the alpha2 domain. Thus, exposing the cells to cytokine stress allows to distinguish between low- and non-expressed alleles and to classify alleles with a questionable expression pattern (Q alleles). Classifying HLA alleles in expressed and non-expressed variants is essential for matching assessments. Additionally, this discrimination between cytokine inducible and non-inducible defect alleles may be important in allotransplant settings in which a cytokine storm usually occurs following pre-transplant myeloablative conditioning or post-transplant immunosuppressive therapy.

Disulfide bridge disruption in the alpha2 domain of the HLA class I molecule leads to low expression of the corresponding antigen.

Using sequence-based typing, we have identified a novel human leukocyte antigen (HLA)-A*30 allele, HLA-A*3014L, with a low expression pattern. The sequence of HLA-A*3014L is identical to that of HLA-A*3001 except for a G to C substitution in exon 3 at nucleotide position 563, resulting in an amino acid difference at position 164 (Cys to Ser). Due to the cysteine substitution, a disulfide bridge in the alpha2 domain of the HLA class I heavy chain cannot be formed. By using the standard microlymphocytotoxicity test, the HLA-A30 antigen cannot be detected. By flow cytometric analysis of the cell-surface expression at either 37 degrees C or 30 degrees C, a temperature-sensitive expression pattern of the HLA-A*3014L antigen was observed. Only by incubating the cells at 30 degrees C, which increases the stability of HLA class I heavy chains, was a weak but clearly detectable HLA-A*3014L expression found. The mRNA expression level of the HLA-A*3014L allele was not affected by the nucleotide substitution. The intrachain disulfide bond formation in the alpha2 domain is essential for the normal expression of the HLA molecules. Reduced protein expression is probably caused by incorrect HLA class I heavy chain folding and HLA class I complex assembly.

Simultaneous control of third-degree graft-versus-host disease and prevention of recurrence of juvenile myelomonocytic leukemia (JMML) with 6-mercaptopurine following fulminant JMML relapse early after KIR-mismatched bone marrow transplantation.

The authors describe a young boy with juvenile myelomonocytic leukemia (JMML) who relapsed 45 days after HLA and killer immunoglobulin-like receptor (KIR) mismatched unrelated donor bone marrow transplant (MMUD-BMT) and subsequently developed life-threatening graft-versus-host disease (GvHD). Treatment with 6-mercaptopurine (6-MP) appeared to control severe GvHD and possibly prevented recurrence of leukemic relapse.