Scientific collaborations

HLA DNA sequencing versus HLA SSO/SSP genotyping for improving donor-recipient immunogenetics (2004)

To improve the donor-recipient HLA match, 3 molecular biology methods were evaluated in terms of HLA genotyping for kidney donors and peripheral hematopoietic stem cell donors. We were interested in the impact of HLA matching at the allele level for clinical diagnosis and for the prognosis of kidney and bone marrow allograft survival. The comparison of HLA DNA direct sequencing – SSP/SSO HLA genotyping revealed a matching performance of HLA alleles with different degrees of expression for direct sequencing between donors and recipients.

Result

• Direct HLA sequencing resolved all SSO/SSP allelic ambiguities in 48% of cases (donor-recipients)
• In 4% of cases, direct HLA sequencing deciphered new alleles.

Conclusions

Preliminary data demonstrate that optimal HLA matching still has a lot to offer in terms of reducing the frequency of acute rejection and has an indisputable impact on the long-term survival of renal and bone marrow allografts. Through direct sequencing, there is a chance of discovering new HLA alleles in both the donor and the recipient. A more accurate HLA match allows avoiding increased doses of immunosuppressive agents and their adverse effects in the post-transplant period, thus improving the patient’s quality of life. Direct HLA sequencing: identifies null alleles, identifies insertions and deletions, resolves heterozygous ambiguities, identifies weakly expressed alleles, detects the soluble form of alleles.

The preliminary conclusions of HBV genotyping in Romania are: HBV genotype A is associated with inactive, asymptomatic carrier status and is present in double HBV-VHD infection.

HBV genotype D is associated with active viral infection and evolution towards hepatocellular carcinoma; genotype D is associated with the development of hepatocellular carcinoma.

Currently, several mutations are known that occur at the level of nucleotide sequences in the HBV genome, during the infection, and which have important clinical and virological consequences.
Thus, the mutation in the S gene that includes the substitution of glycine with arginine at codon position 145 (G145R) was frequently encountered in newborns from HBsAg positive mothers, in whom HBV infection developed despite vaccination at birth. Also, this mutation was detected in liver transplant patients who had recurrent HBV infection even though HIBg immunoglobulin was administered to them. The “core promoter” and “precore” variants produce HBV virions that do not secrete HBeAg.
The most frequent “precore promoter” mutation has a dual change A1762T and G1764A that decreases “precore” mRNA and thus HBeAg secretion.

These variants are found in particular in patients who do not have serum HBeAg present, having HBV genotypes B, C, and D. The G1896A mutation, premature stop codon, should also be mentioned.

In severe cases of acute and chronic hepatitis, “precore” HBV mutants have been described.
Mutations in the polymerase gene have been identified in patients who are on antiviral therapy and who develop antiviral resistance. The best characterized mutants in the polymerase gene were identified during lamivudine therapy.

The mutations M552V, M552I, L528M, (YMDD) occur in the catalytic domain of the polymerase that confers resistance to lamivudine and related nucleoside analogues.

Preliminary sequencing data of circulating strains of HBV in Romania were relevant by our working group and colleagues from Great Britain (I. Constantinescu, TJ Harrison, R. Ling – unpublished data, 2000).

The patients with HBV infection selected for sequencing have different forms of the disease, either aggressive or mild biochemically and histologically, our goal being to find a possible correlation between possible HBV mutants in the “core promoter” region and clinical and paraclinical aspects.

The preliminary data showed in these cases stop codon type mutations: 28 Tryptophan -> stop codon; 131 Arginine -> Lysine (aac->aaa); 145 Glycine-> Arginine (gga->aga).

This study is ongoing and the final results will provide new aspects related to mutations in the “core promoter” region and the clinical and histopathological evolution of chronic infection induced by HBV.

Molecular virology studies of HBV provide a unique understanding of the virus-host relationship with an important impact on the evolution of hepatitis infection and prognosis, but especially on the selection of patients for antiviral therapy.

In conclusion, without a molecular virology diagnosis we cannot successfully understand and treat a chronic HBV infection.

HBV carries out its DNA genome replication process via a reverse transcription process that has gaps in the reading activity, thus reaching the quasi-species form of HBV.
HBV genotypes A and D have different replication efficiencies in cell cultures and in vivo these characteristics are preserved.

Frequently, the mutant forms (especially those in the “precore”, “core promoter” region) come to predominate over time in the wild virus strain.

For viral dominance, both viral intrapopulation and intra-host factors must be taken into consideration. Mutants can develop a selection advantage within the infected host during chronic infection. The negative HBeAg mutants have a biological advantage over the wild strain of the virus, so these strains are selected throughout evolution.

In the general population, the wild type of HBV still predominates over the mutant forms.
Also, the mutated form of the stop codon in the “precore” region is associated with fulminant hepatitis and is much more frequently found in HBV genotype D.

In such cases, the therapy of choice is lamivudine, which acts on the dominant variant mutated into “precore”.

It is known that IFN has a potent antiviral action only on the wild virus strain. Therefore, the therapeutic decision must be made individually, only after a molecular virology diagnosis, in order to have a sustained virological response, in fact, a therapeutic success.