Jan Traeger-Synodinos

Systematic documentation and analysis of human genetic variation in hemoglobinopathies using the microattribution approach

We developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to hemoglobinopathies and thalassemia and implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories. A total of 1,941 unique genetic variants in 37 genes, encoding globins and other erythroid proteins, are currently documented in these databases, with reciprocal attribution of microcitations to data contributors. Our project provides the first example of implementing microattribution to incentivise submission of all known genetic variation in a defined system. It has demonstrably increased the reporting of human variants, leading to a comprehensive online resource for systematically describing human genetic variation in the globin genes and other genes contributing to hemoglobinopathies and thalassemias. The principles established here will serve as a model for other systems and for the analysis of other common and/or complex human genetic diseases.

Unstable and Thalassemic α Chain Hemoglobin Variants: A Cause of Hb H Disease and Thalassemia Intermedia

We report an update of the α-globin gene point mutations resulting in structural modification associated with an α-thalassemia (α-thal) phenotype. These variants, barely symptomatic in the heterozygous state, are either unstable due to folding defects and/or defects in binding to α-hemoglobin stabilizing protein (AHSP). This is predicted to result in precipitation of the unstable α chains or Hb variant, a concomitant decrease in the overall quantity of normal α-globin in the red cells and a potential degree of anemia and possibly, hemolysis. Genotype/phenotype correlation and potential genetic risk in combination with common or less common α-thal defects are discussed.

Advances in technologies for screening and diagnosis of hemoglobinopathies

Hemoglobinopathies constitute the most common monogenic disorders worldwide, caused by mutations in the globin genes that synthesize the globin chains of hemoglobin. Synthesis may be reduced (thalassemia) or underlie abnormal hemoglobins. Mutation interactions produce a wide range of disorders. For neonatal and antenatal screening, identification of affected newborns or carriers is achieved by hematological tests. DNA analysis supports definitive diagnosis, and additionally facilitates prenatal diagnosis procedures. Most methods used today have been developed over several decades, with few recent advances in hematology methods. However, DNA methods evolve continuously. With global migration and multiethnic societies the trend is from targeted, population-specific methods towards generic methods, such as Sanger sequencing (point mutations) and multiplex ligation probe amplification (deletions). DNA microarrays constitute an advanced DNA method for some mutation categories. The newest DNA technology is next-generation sequencing. Although not completely ready for routine use currently, next-generation sequencing may soon become a reality for some hemoglobin diagnostic laboratories.

A Minimal Set of SNPs for the Noninvasive Prenatal Diagnosis of β-Thalassaemia

β‐thalassaemia is one of the commonest autosomal recessive single‐gene disorders worldwide. Prenatal tests use invasive methods, posing a risk for the pregnancy itself. Development of a noninvasive prenatal diagnostic method is, therefore, of paramount importance. The aim of the present study is to identify high‐heterozygote informative single‐nucleotide polymorphisms (SNPs), suitable for the development of noninvasive prenatal diagnosis (NIPD) of β‐thalassaemia. SNP genotyping analysis was performed on 75 random samples from the Cypriot population for 140 SNPs across the β‐globin cluster. Shortlisted, highly heterozygous SNPs were then examined in 101 carrier families for their applicability in the noninvasive detection of paternally inherited alleles. Forty‐nine SNPs displayed more than 6% heterozygosity and were selected for NIPD analysis, revealing 72.28% of the carrier families eligible for qualitative SNP‐based NIPD, and 92% for quantitative detection. Moreover, inference of haplotypes showed predominant haplotypes and many subhaplotypes with sufficient prevalence for diagnostic exploitation. SNP‐based analyses are sensitive and specific for the detection of the paternally inherited allele in maternal plasma. This study provides proof of concept for this approach, highlighting its superiority to NIPD based on single markers and thus providing a blueprint for the general development of noninvasive prenatal diagnostic assays for β‐thalassaemia.

Two new β-thalassemia deletions compromising prenatal diagnosis in an Italian and a Turkish couple seeking prevention

Two novel deletions in the beta gene cluster were identified by Multiplex Ligation-dependent Probe Amplification in two at-risk couples seeking prevention. This study exemplifies a successful diagnostic approach in case one member of the couple is an atypical thalassemia carrier. When the molecular background of couples requesting prevention is unclear, family analysis and tools to define rare mutations are essential. We report two novel deletion defects observed in an Italian and in a Turkish couple. The first proband presented with microcytic hypochromic parameters without iron deficiency, a normal HbA2 and an elevated HbF (10.6%). His father presented with a similar phenotype and his wife was heterozygous for the common Mediterranean codon 39 (HBB:c.118C>T) mutation. Having excluded point mutations and common deletions, Multiplex Ligation-dependent Probe Amplification was performed revealing an unknown Gγ(Aγδβ)0-thalassemia defect spanning from the Aγ gene to downstream of the β-globin gene provisionally named Leiden 69.5 kb deletion. In the second case, the wife presented with a mild thalassemic picture, normal HbA2, elevated HbF (18.5%) and a β/α globin chain synthesis ratio of 0.62, without iron deficiency or any known β-thalassemia defect, while the husband was a simple carrier of the common Mediterranean IVS-I-110 (HBB:c.93-21 G>A) mutation. A new large deletion involving the β-gene and part of the δ-gene was identified by Multiplex Ligation-dependent Probe Amplification provisionally named “Leiden 7.4 kb”.

A Generic, Flexible Protocol for Preimplantation Human Leukocyte Antigen Typing Alone or in Combination with a Monogenic Disease, for Rapid Case Work-up and Application

Abstract Human leukocyte antigen (HLA) typing of in vitro fertilization (IVF) embryos, aims to establish a pregnancy that is HLA compatible with an affected sibling who requires hematopoietic stem cell transplantation (HSCT). It can be performed with or without preimplantation genetic diagnosis (PGD) for exclusion of a single-gene disorder (SGD) and it is a multistep, technically challenging procedure at every stage. Our purpose was to address the difficulties of genetic analysis by developing a fast, reliable and accurate PGD-HLA protocol, to simplify patient work-up and PGD application, while providing high flexibility for combination with any SGD. Requests included PGD-HLA for β-thalassemia (β-thal)/sickle cell disease (most common request), Diamond-Blackfan anemia (DBA), chronic granulomatous disease (CGD) and preimplantation-HLA typing only. For HLA haplotyping, we selected a panel of 26 short tandem repeats (STRs) distributed across the entire HLA locus, following PGD guidelines. When required, mutation detection was performed by both a direct and indirect approach. To support concurrent SGD exclusion and HLA typing, a one-step, single-tube, multiplex fluorescent touchdown-polymerase chain reaction (PCR) was optimized. The described touchdown-PCR was successfully applied for all PGD-HLA protocols. Eight clinical cycles were performed with a diagnosis achieved for 94.7% of amplified biopsied blastomeres. Embryo transfer took place in six cycles, with two pregnancies achieved and two healthy female infants (from a twin pregnancy) born so far. Our protocol enables HLA typing in a single PCR, reducing the risk of contamination and the cost, and providing faster results. It requires minimum optimization before clinical application, irrespective of the SGD involved, decreasing the waiting time from referral to treatment for all PGD-HLA cases.

Association of TLR4 single-nucleotide polymorphisms and sarcoidosis in Greek patients.

The present study investigates the potential role of Toll-like receptor 4 (TLR4) Asp299Gly and Thr399Ile single-nucleotide polymorphisms (SNPs) as risk factors in the development of sarcoidosis using a novel high-throughput microtiter well-based bioluminometric genotyping assay. One hundred and nineteen Greek patients with sarcoidosis and 209 control subjects were genotyped for the two SNPs of the TLR4 gene. The genotypes observed were in Hardy-Weinberg equilibrium. The heterozygote frequency for both SNPs in sarcoidosis group and control population was 13.4% (16/119) and 10.5% (22/209), respectively. The minor genotype was found to be the same for both sarcoidosis and control groups and similar to that found in other Caucasian populations. No significant association of Asp299Gly and Thr399Ile polymorphisms with increased susceptibility to sarcoidosis was found (p = 0.61 and odds ratio = 1.183). In conclusion, genotype data for the TLR4 Asp299Gly and Thr399Ile polymorphisms in the Greek population were found to be in linkage disequilibrium, and no contribution in the pathogenesis of sarcoidosis was established. Further, in course of the present study, we demonstrated a very simple and sensitive high-throughput bioluminometric assay for genotyping Asp299Gly and Thr399Ile polymorphisms in the TLR4 gene.

A novel α0-thalassemia deletion in a Greek patient with HbH disease and β-thalassemia trait: A novel α0-thalassemia deletion

Objectives:  To determine the molecular basis in a Greek child suspected of having HbH disease and β‐thalassemia trait.

Bioluminometric assay for relative quantification of mutant allele burden: application to the oncogenic somatic point mutation JAK2 V617F.

Unlike the inherited mutations, which are present in all cells, somatic (acquired) mutations occur only in certain cells of the body and, quite often, are oncogenic. Quantification of mutant allele burden (percentage of the mutant allele) is critical for diagnosis, monitoring of therapy, and detection of minimal residual disease. With point mutations, the challenge is to quantify the mutant allele while discriminating from a large excess of the normal allele that differs in a single base-pair. To this end, we report the first bioluminometric assay for quantification of the allele burden and its application to JAK2 V617F somatic point mutation, which is a recently (2005) discovered molecular marker for myeloproliferative neoplasms. The method is performed in microtiter wells and involves a single PCR, for amplification of both alleles, followed by primer extension reactions with allele-specific primers. The products are captured in microtiter wells and detected by oligo(dT)-conjugated photoprotein aequorin. The photoprotein is measured within seconds by simply adding Ca(2+). We have demonstrated that the percent (%) luminescence signal due to the mutant allele is linearly related to the allele burden. As low as 0.85% of mutant allele can be detected and the linearity extends to 100%. The assay is complete within 50 min after the amplification step.

Absolute quantification of the alleles in somatic point mutations by bioluminometric methods based on competitive polymerase chain reaction in the presence of a locked nucleic acid blocker or an allele-specific primer.

In somatic (acquired) point mutations, the challenge is to quantify minute amounts of the mutant allele in the presence of a large excess of the normal allele that differs only in a single base pair. We report two bioluminometric methods that enable absolute quantification of the alleles. The first method exploits the ability of a locked nucleic acid (LNA) oligonucleotide to bind to and inhibit effectively the polymerase chain reaction (PCR) amplification of the normal allele while the amplification of the mutant allele remains unaffected. The second method employs allele-specific PCR primers, thereby allowing the amplification of the corresponding allele only. DNA internal standards (competitors) are added to the PCR mixture to compensate for any sample-to-sample variation in the amplification efficiency. The amplification products from the two alleles and the internal standards are quantified by a microtiter well-based bioluminometric hybridization assay using the photoprotein aequorin as a reporter. The methods allow absolute quantification of less than 300 copies of the mutant allele even in samples containing less than 1% of the mutant allele.