HLA haplotying

 
Technically, PGD for HLA typing is a difficult procedure due to the extreme polymorphism of the HLA region. Taking into account also the complexity of the region (presence of a large number of loci and alleles) the use of a direct HLA typing approach would require standardization of a PCR protocol specific for each family, presenting different HLA allele combinations, making it time consuming and unfeasible. The use of a preimplantation HLA matching protocol irrespective of the specific genotypes involved facilitates notably the procedure.

Our Centre has developed a new strategy based on the use of a flexible indirect HLA typing protocol applicable to a wide spectrum of possible HLA genotypes. The approach involves testing of single blastomeres by fluorescent multiplex polymerase chain reaction (PCR) analysis of polymorphic short tandem repeat (STR) markers, scattered throughout the HLA complex, obtaining a “fingerprint” of the entire HLA region.

By selecting a consistent number of STR markers evenly spaced throughout the HLA complex, an accurate mapping of the whole region can be achieved. Because genes in the HLA complex are tightly linked and usually inherited in block, profiles obtained from such markers in father, mother and affected child allow the determination of specific haplotypes. Thus, the HLA region can be indirectly typed by segregation analysis of the STR alleles and the HLA identity of the embryos with the affected sibling can be ascertained evaluating the inheritance of the matching haplotypes.

The use of microsatellite markers for this purpose is very useful, since they may provide information on identity over a greater distance within the HLA region compared to matching strictly for the classic HLA genes, making haplotyping more accurate in predicting compatibility.

The strategy presented here enables the selection of HLA-matched embryos can be performed for any genotype combination, without the need to develop a specific diagnostic experimental design for each couple, because the selected panel of STR markers have already been worked out and can be used for other patients. As a consequence, a substantial shortening of the preliminary phase can be achieved.
It is important to keep the time to develop a family-specific PCR protocol as short as possible; using STR markers enables to work quickly and safely. Obviously there is a positive impact on overall cost-effectiveness but there are also clinical arguments. Patients with congenital anemias can survive for many years. However, they will develop a number of time-dependent complications causing direct morbidity and mortality and reducing the success-rate of an eventual transplant procedure. For patients with congenital immune deficiencies, the continuous risk of potentially dangerous infections will decrease if the transplant is performed earlier. In urgent cases such as leukemia, the children are in remission but they can relapse suddenly and therefore there is no time to lose. Finally it is important to speed up the preclinical work to reduce the psychological stress the family is confronted with while waiting.

An example of preimplantation HLA matching procedure using STR haplotyping, in combination with PGD for b-thalassaemia, is shown in Figure 2. A total of 15 cumulus-oocyte complexes (COCs) were retrieved for this PGD cycle, 12 mature oocytes were inseminated by ICSI and 12 became fertilised. Three days later 12 embryos were selected for embryo biopsy on the basis of regular development and morphology. Two blastomeres were removed and analysed from each embryo. Eleven embryos yielded conclusive results, 8 of which resulted HLA non-identical (2 affected, 5 carrier, 1 normal). Only three embryos appeared to be both healthy and HLA identical and were therefore transferred, resulting in the birth of two twins HLA matched with the affected sibling. Stem cells collected from the umbilical cord blood of both twins were transplanted to the affected child, who is no longer blood transfusion dependent.

Figure 3 presents the results of preimplantation HLA matching performed in combination with PGD for WAS. A total of 15 regularly fertilized oocytes resulted after inseminating 19 oocytes. On the third day, 13 embryo were suitable for biopsy, in 6 of which only 1 cell was removed. From 7 embryos 2 blastomeres were collected. Eleven embryos produced conclusive results, 9 of which resulted HLA non-identical (2 affected, 1 carrier, 6 normal). Only 2 HLA-matched embryos were obtained, but only one (embryo 14) appeared also unaffected and was therefore transferred, resulting in birth of a carrier female, HLA matched with the affected sibling. Stem cell transplantation was performed in the affected child, resulting in a successful hematopoietic reconstruction.

Another important advantage of using STR markers in preimplantation HLA matching is that the whole HLA complex can be covered and this allows the detection of recombination events between HLA genes. Recombination occurrences, if not detected, could strongly affect the accuracy of the HLA matching procedure.

The importance of detecting recombination within the HLA region is demonstrated in Figure 4, where are described the results of a cycle involving preimplantation HLA matching, without PGD of a causative gene, performed for a couple having a child affected by a sporadic form of DBA. In this case, embryos were genotyped using a panel of 14 different STR markers, evenly distributed along the whole HLA region. Recombination between flanking markers of the paternal or maternal haplotype was detected in 2 (embryos 1 and 6) of the 15 embryos tested. In one of them (embryo 1), a single recombination occurred in the maternal haplotype, between the alleles of the markers D6S105 and MIB. In the other embryo (embryo 6), initially appearing to be HLA matched with the affected sibling, a double recombination event was observed between markers D6S1683 and D6S265. This occurrence, detected by using a consistent number of STR markers able to determine a fine mapping of the whole HLA region, could lead to a HLA-genotyping misdiagnosis if not detected, and the embryo would be erroneously diagnosed as HLA identical. Hence, the reliability of the procedure is strongly correlated with the number of STR markers used for HLA haplotyping.

The combined use of a multiplex HLA STR marker system also allows detection of aneuploidies of chromosome 6. The relevance of aneuploidy testing for chromosome 6 is seen in Figure 4. One of the 15 embryos tested in this case appeared to have only one maternal chromosome 6 (embryos 5), and one (embryo 13) had an extra maternal chromosome, consistent with a diagnosis of monosomy 6 and trisomy 6, respectively, making them unacceptable for transfer.

 

Next : Example of PGD for HLA matching