Marion Phylipsen

Nine unknown rearrangements in 16p13.3 and 11p15.4 causing α- and β-thalassaemia characterised by high resolution multiplex ligation-dependent probe amplification

Background: Approximately 80% of the α- and 10% of the β-thalassaemias are caused by genomic deletions involving the α- and β-globin gene clusters on chromosomes 16p13.3 and 11p15.5, respectively. Gap-PCR, Southern blot analysis, and fluorescent in situ hybridisation are commonly used to identify these deletions; however, many deletions go undetected using conventional techniques. Methods: Patient samples for which no abnormalities had been found using conventional DNA techniques were analysed by a three colour multiplex ligation-dependent probe amplification assay. Two sets of 35 and 50 probes, covering a region of 700 kb of the α- and 500 kb of the β-globin gene cluster, respectively, were designed to detect rearrangements in the α- and β-globin gene clusters. Results: In 19 out of 38 patient samples, we found 11 different α-thalassaemia deletions, six of which were not previously described. Two novel deletions leaving the α-globin gene cluster intact were found to cause a complete downregulation of the downstream α-genes. Similarly, 31 out of 51 patient samples were found to carry 10 different deletions involving the β-globin gene cluster, three of which were not previously described. One involves the deletion of the locus control region leaving the β-globin gene cluster intact. Conclusions: These deletions, which are not easily detected by conventional techniques, may have clinical implications during pregnancy ranging from mild to life threatening microcytic haemolytic anaemia in neonates. The approach as described here is a rapid and sensitive method for high resolution analysis of the globin gene clusters and for any region of the genome.

Combined TLR2 and TLR4 ligation in the context of bacterial or helminth extracts in human monocyte derived dendritic cells: Molecular correlates for Th1/Th2 polarization

BackgroundRecognition of pathogens by dendritic cells (DCs) through interaction with pattern recognition receptors, including Toll like receptors (TLR), is crucial for the initiation of appropriate polarized T helper (Th) cell responses. Yet, the characteristics and differences in molecular profiles of DCs with different T cell polarizing capacities are still poorly defined. To address this issue, the molecular profile of human monocyte derived DCs was characterized after exposure to TLR4 ligand LPS in combination with the Th1 promoting bacterial extracts from Listeria monocytogenes and Escherichia coli or the Th2 promoting helminth derived phospholipids from Schistosoma mansoni and Ascaris lumbricoides, all with TLR2 activating capacity.ResultsWith regard to the signalling pathways activated upon exposure to LPS and the TLR2 activating compounds, we find that the ratio of activated Mitogen Activated Protein Kinases (MAPK) p-ERK/p-p38 is lower in DCs stimulated with the bacterial products compared to DCs stimulated with the helminth products, which correlates with the Th1 and Th2 polarizing capacity of these compounds. Furthermore, analysis of the mRNA expression levels of a set of 25 carefully selected genes potentially involved in modulation of T cell polarization revealed that the mRNA expression of notch ligand delta-4 and transcription factor c-fos are differentially regulated and show a strong correlation with Th1 and Th2 polarization, respectively.ConclusionThis study shows that combined TLR2 and TLR4 activation in the context of different antigen sources can induce very distinct molecular profiles in DCs and suggests that the Th1/Th2 polarizing capacity of compounds can be predicted with the molecular signature they induce in DCs.

Non‐invasive prenatal diagnosis of beta‐thalassemia and sickle‐cell disease using pyrophosphorolysis‐activated polymerization and melting curve analysis

The aim of this study was to develop a pyrophosphorolysis‐activated polymerization (PAP) assay for non‐invasive prenatal diagnosis (NIPD) of β‐thalassemia major and sickle‐cell disease (SCD). PAP is able to detect mutations in free fetal DNA in a highly contaminating environment of maternal plasma DNA.

Fine‐tiling array CGH to improve diagnostics for α‐ and β‐thalassemia rearrangements

Implementation of multiplex ligation‐dependent probe amplification (MLPA) for thalassemia causing deletions has lead to the detection of new rearrangements. Knowledge of the exact breakpoint sequences should give more insight into the molecular mechanisms underlying these rearrangements, and would facilitate the design of gap‐PCRs. We have designed a custom fine‐tiling array with oligonucleotides covering the complete globin gene clusters. We hybridized 27 DNA samples containing newly identified deletions and nine positive controls. We designed specific primers to amplify relatively short fragments containing the breakpoint sequence and analyzed these by direct sequencing. Results from nine positive controls showed that array comparative genomic hybridization (aCGH) is suitable to detect small and large rearrangements. We were able to locate all breakpoints to a region of approximately 2 kb. We designed breakpoint primers for 22 cases and amplification was successful in 19 cases. For 12 of these, the exact locations of the breakpoints were determined. Seven of these deletions have not been reported before. aCGH is a valuable tool for high‐resolution breakpoint characterization. The combination of MLPA and aCGH has lead to relatively cheap and easy to perform PCR assays, which might be of use for laboratories as an alternative for MLPA in populations where only a limited number of specific deletions occur with high frequency. Hum Mutat 33:272–280, 2012.

Thalassemia in Western Australia: 11 novel deletions characterized by Multiplex Ligation-dependent Probe Amplification

The number of immigrants in Western Australia from many different areas where hemoglobinopathies are endemic has increased dramatically since the 1970s. Therefore, many different thalassemia mutations have been introduced in the country, which add a technological diagnostic problem to the serious burden of hemoglobinopathy management and to public health care. Recently, we have developed a rapid and simple technique based on Multiplex Ligation-dependent Probe Amplification to detect deletions causing alpha-and beta-thalassemia, deltabeta-thalassemia and Hereditary Persistence of Fetal Hemoglobin. A screening for (unknown) deletions was performed in a cohort of patients of different ethnic backgrounds preselected for their thalassemia phenotype, in which common deletions and point mutations were excluded. Out of 37 cases suspected to carry a deletion, 27 were found to carry 17 different deletion types of which 6 causing alpha-thalassemia and 5 causing beta-thalassemia were novel. For 3 of the deletions, we have been able to characterize the exact breakpoint sequences by long-range PCR and direct sequencing. These results show that MLPA is a suitable technology to detect unknown and uncommon deletions. These could represent a diagnostic problem when offering prevention to couples at risk presenting with unclear phenotypes and might result in a serious fetal problem when the deletion involves embryonic genes.

Occurrence of common and rare δ-globin gene defects in two multiethnic populations: thirteen new mutations and the significance of δ-globin gene defects in β-thalassemia diagnostics

Introduction:  The aim of this review is to study the frequency of common and the occurrence of rare and novel mutations of the delta‐globin gene and of Hb Lepore defects that might interfere with thalassemia diagnostics and to report the rationale of HbA2 estimation in the presence of delta‐ or alpha‐gene mutations.

A new alpha(0)-thalassemia deletion found in a Dutch family (--(AW)).

Alpha-thalassemia is an inherited hemoglobin disorder characterized by a microcytic hypochromic anemia caused by a quantitative reduction of the alpha-globin chain. The majority of the alpha-thalassemias is caused by deletions in the alpha-globin gene cluster. A deletion in the alpha-globin gene cluster, which was found in a Dutch family, was characterized by MLPA, long-range PCR and direct sequencing. We describe the molecular characterization of a novel 8.2kb deletion (--(AW)), involving both alpha-globin genes in cis. The deletion is caused by a non-homologous recombination event between an Alu and an L1-repeat sequence. This deletion is the third example of a non-homologous recombination event involving an Alu and an L1 repeat, and the first described in the human alpha-globin gene cluster. Because of a 25% risk of Hb Barts with hydrops foetalis in the offspring when in combination with another alpha(0)-thalassemia allele, it is important to diagnose this deletion.

A Single-Tube Multiplex Gap-Polymerase Chain Reaction for the Detection of Eight β-Globin Gene Cluster Deletions Common in Southeast Asia

Up to now, more than 200 different β-thalassemia (β-thal) mutations have been characterized. The majority are point mutations causing expression defects. Only approximately 10.0% of the defects are caused by large deletions involving the β-globin gene cluster causing β0-thal, (δβ)0-thal, Gγ(Aγδβ)0-thal and other conditions with or without persistence of fetal hemoglobin (Hb). For the prevention of severe forms of β-thal intermedia and β-thal major, it is important to identify carriers of point mutations as well as carriers of deletions. β-Thalassemia and related disorders are most commonly present among populations from all Mediterranean countries as well as Southeast Asia, India, Africa, Central America and the Middle East. Twelve relatively frequently occurring deletion types have been described involving the β-globin gene cluster. These include the 105 bp β0-thal deletion, the 619 bp deletion, the 3.5 kb deletion, the Southeast Asian (SEA) deletion, the Filipino deletion, Hb Lepore, the Thai (δβ)0-thal, the Siriraj J Gγ(Aγδβ)0-thal, the Chinese Gγ(Aγδβ)0-thal, the Asian Indian deletion-inversion Gγ(Aγδβ)0-thal as well as the (hereditary persistence of fetal hemoglobin) HPFH-6 and HPFH-7 deletions. To improve the rapid detection of the eight common β-globin cluster deletions in Southeast Asian countries, a simple molecular technique based on a single-tube multiplex gap-polymerase chain reaction (PCR) has been developed in this study. This technique provides a fast, simple and cost effective diagnostic test for deletion types of β-thal that can be applied in every molecular diagnostic laboratory having standard PCR equipment.

Two New α1-Globin Gene Point Mutations: Hb Nedlands (HBA1:c.86C>T) [α28(B9)Ala→Val] and Hb Queens Park (HBA1:c.98T>A) [α32(B13)Met→Lys]

We report two new point mutations of the α1-globin gene found in a Greek and a Burmese patient, both living in Western Australia. The patients were initially selected for their microcytic hypochromic parameters as belonging to a group suspected for uncommon (deletion) defects. Gap-polymerase chain reaction (gap-PCR) and multiplex ligation-dependent probe amplification (MLPA) technologies were applied, and in those cases not showing deletions, direct sequencing was performed. We have found 1) HBA1:c.86C>T, Hb Nedlands [α28(B9)Ala→Val] which, based on the red cell indices and phenotype prediction scores, is presumed to be clinically silent, and 2) HBA1:c.98T>A, Hb Queens Park [α32(B13)Met→Lys] which seems to be associated with a mild α-thalassemia (α-thal) phenotype. The phenotype/genotype correlation is briefly described.

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”.