Christina Vrettou

Phenotypic and molecular diversity of haemoglobin H disease: a Greek experience

Haemoglobin H (Hb H) disease is the severest form of α‐thalassaemia compatible with post‐natal life and occurs when α‐thalassaemia mutations interact to reduce α‐globin synthesis to levels approximately equivalent to the output of a single α‐globin gene. Hb H disease has variable clinical expression, mainly related to underlying genotypes. The spectrum of α‐thalassaemia determinants in Greece appears greater than in any other population studied and, in 75 Greek Hb H disease patients, we found 12 α‐thalassaemia mutations interacting to produce 15 Hb H disease genotypes. Evaluation of haematological, biochemical and clinical findings, and correlation with genotypes, defined genetic predictors of disease severity and factors involved in disease progression. In accordance with previous reports, patients with non‐deletion α‐thalassaemia mutations had more severe clinical expression. Additionally, we found that all patients with the most severe phenotypes had α‐thalassaemic globin variants. Phenotypic severity was not simply related to the degree of α‐globin deficiency: high Hb H levels were found to exacerbate anaemia by negatively influencing tissue oxygenation, and both Hb H and α‐thalassaemic haemoglobin variants appear to reduce red cell survival within the bone marrow and circulation. Together with the long‐term follow‐up in many patients, this report provides comprehensive information for management of Hb H disease and appropriate family counselling.

De Novo Interstitial Duplication of the 15q11.2-q14 PWS/AS Region of Maternal Origin: Clinical Description, Array CGH Analysis, and Review of the Literature

The 15q11‐q13 PWS/AS critical region involves genes that are characterized by genomic imprinting. Multiple repeat elements within the region mediate rearrangements, including interstitial duplications, interstitial triplications, and supernumerary isodicentric marker chromosomes, as well as the deletions that cause Prader–Willi syndrome (PWS) and Angelman syndrome (AS). Recently, duplications of maternal origin concerning the same critical region have been implicated in autism spectrum disorders (ASD). We present a 6‐month‐old girl carrying a de novo duplication of maternal origin of the 15q11.2‐q14 PWS/AS region (17.73u2009Mb in size) [46,XX,dup(15)(q11.2‐q14)] detected with a high‐resolution microarray‐based comparative genomic hybridization (array‐CGH). The patient is characterized by severe hypotonia, obesity, microstomia, long eyelashes, hirsutism, microretrognathia, short nose, severe psychomotor retardation, and multiple episodes of drug‐resistant epileptic seizures, while her brain magnetic resonance imaging (MRI) documented partial corpus callosum dysplasia. In our patient the duplicated region is quite large extending beyond the Prader–Willi–Angelman critical region (PWACR), containing a number of genes that have been shown to be involved in ASD, exhibiting a severe phenotype, beyond the typical PWS/AS clinical manifestations. Reporting of similar well‐characterized clinical cases with clearly delineated breakpoints of the duplicated region will clarify the contribution of specific genes to the phenotype.

Interaction of an α+-Thalassemia Deletion with Either a Highly Unstable α-Globin Variant (α2, Codon 59, GGC→GAC) or a Nondeletional α-Thalassemia Mutation (AATAA→AATAAG): Comparison of Phenotypes Illustrating “Dominant” α-Thalassemia

Thalassemia syndromes and unstable hemoglobins traditionally represent two phenotypically separate disorders of hemoglobin synthesis. Highly unstable hemoglobin variants, however, often have phenotypic characteristics associated with both ineffective erythropoiesis (thalassemias) and peripheral hemolysis (unstable hemoglobins). Many highly unstable β chain variants cause a dominant thalassemia-like phenotype, in which simple heterozygotes for such mutations have a clinical expression similar to thalassemia intermedia. The phenotypic expression of highly unstable α-globin variants is usually less severe, due mainly to a gene dosage effect, and they are often only characterized on interaction with other α-thalassemia mutations, whence they are classified as nondeletional α-thalassemia determinants. This study reports the clinical and hematological findings in five cases with rare α-thalassemia genotypes: a single patient with the thalassemic α2-globin gene codon 59 Gly→Asp hemoglobin variant in trans to an ...

Molecular Studies of β-Thalassemia Heterozygotes with Raised Hb F Levels

Hb F levels in β-thalassemia heterozygotes are usually less than 2%, but amongst 1,059 patients studied, 73 (7%) had Hb F levels above 2.5% (2.6–14.0%). To investigate factors that may influence the increase of Hb F levels in these heterozygotes, we characterized the β-thalassemia mutations and their chromosomal background, γ-globin gene promoter variations, and α-globin genotypes. All 73 β-thalassemia heterozygotes carried β-thalassemia point mutations previously observed in the Greek population; gene mapping excluded b gene cluster deletions; only two cases had an additional γ-globin gene (γγγ/γγ). Five α-globin genes (ααα/αα) were detected in 17/73 cases (23%) as compared to a carrier rate of 1.76% in the general population. Molecular, hematological, and biosynthetic findings in these compound heterozygotes indicated that the raised Hb F levels were caused by cell selection due to ineffective erythropoiesis. In the remaining 56 simple β-thalassemia heterozygotes, 11 β-thalassemia mutations were observed, each on the expected haplotype(s), and analysis of the γ gene promoters revealed three known polymorphisms (in linkage disequilibrium), with minimal influence on γ-globin levels. However, the overall distribution of β-thalassemia mutations in the 56 simple β-thalassemia heterozygotes was significantly different (P<0.0002) compared to that in 986 simple β-thalassemia heterozygotes with <2.5% Hb F., implicating an association between β-thalassemia mutations and moderately increased Hb F levels, most notably codon 39 (C→T), IVS-II-1 (G→A), codon 6 (-A), and codon 8 (-AA), which accounted for 41/56 (73%) cases with >2.5% Hb F. In the remaining 15/56 (27%) cases, no common underlying globin genotypes could explain the raised Hb F levels. Overall, this study indicates that the control of Hb F levels in β-thalassemia heterozygotes is heterogeneous and multi-factorial.

The interaction of α° thalassaemia with Hb Icaria: three unusual cases of haemoglobinopathy H

The clinical, haematological, biosynthetic and molecular data of three Greek haemoglobin H (HbH) disease patients with a distinctive clinical phenotype are described. During infancy all three patients had unusually severe clinical manifestations for HbH disease, with anaemia necessitating blood transfusions, signs of bone changes, growth impairment, and splenomegaly. Molecular analysis identified a rare α‐thalassaemia genotype (−u2003−Med/αu2003Icα) . Splenectomy resulted in marked amelioration of the clinical signs; post splenectomy all three patients preserve adequate haemoglobin levels (9–10u2003g/dl) with growth restored to normal. Despite the initial severe clinical phenotype in these patients, our experience indicates that splenectomy modifies the clinical course to that of mild thalassaemia intermedia. This observation should be considered carefully when giving genetic counselling to families carrying the rare Hb Icaria mutation and an α° thalassaemia mutation.

Prenatal, noninvasive and preimplantation genetic diagnosis of inherited disorders: hemoglobinopathies.

Disorders of hemoglobin synthesis have been used as a prototype for the development of most approaches for prenatal diagnosis (PND). PND for hemoglobinopathies based on molecular analysis of trophoblast or amniocyte DNA has accumulated approximately 30 years of experience. Disadvantages with conventional PND include ‘invasive’ fetal sampling and the need to terminate affected ongoing pregnancies. New developments are directed towards improving both the timing and/or safety of procedures. Preimplantation genetic diagnosis, an established procedure with 20 years of clinical application, avoids the need to terminate affected pregnancies through the identification and selective transfer of unaffected in vitro fertilization embryos. Approaches towards ‘noninvasive’ PND, through analyzing fetal cells or free fetal DNA present in the circulation of pregnant women, are a focus of ongoing research. Overall, PND, preimplantation genetic diagnosis (and potentially ‘noninvasive’ PND) represent valuable reproductive options for couples at risk of having a child affected with a severe inherited disease.

An alpha-thalassemic hemoglobinopathy: homozygosity for the HB Agrinio alpha 2-globin chain variant.

This report describes the first case of homozygosity for the Hb Agrinio [alpha 29(B10)Leu-->Pro] alpha 2-globin gene variant (codon 29, CTG-->CCG) in a Greek patient. At 12 months of age, the proband presented with a marked hypochromic, microcytic anemia, a very low level of Hb H (< 2.5%), rare Hb H inclusions, and a balanced alpha/non-alpha biosynthesis ratio. The mother had hematological findings and globin biosynthesis consistent with heterozygous beta-thalassemia, but paradoxically, red cell morphology demonstrated very rare Hb H inclusions. The father had mild microcytosis and hypochromia. Analysis of alpha- and beta-globin genotypes demonstrated that the patient was homozygous for the highly unstable Hb Agrinio variant, caused by a T-->C mutation in codon 29 of the alpha 2-globin gene. At the age of 13 years, the proband had a clinical phenotype compatible with mild thalassemia intermedia with moderate anemia (Hb 7-8 g/dL), normal growth and development, slight splenomegaly, and minimal bone changes, while Hb H and inclusion bodies were not detected.

Sensitive Monogenic Noninvasive Prenatal Diagnosis by Targeted Haplotyping

During pregnancy, cell-free DNA (cfDNA) in maternal blood encompasses a small percentage of cell-free fetal DNA (cffDNA), an easily accessible source for determination of fetal disease status in risk families through non-invasive procedures. In case of monogenic heritable disease, background maternal cfDNA prohibits direct observation of the maternally inherited allele. Non-invasive prenatal diagnostics (NIPD) of monogenic diseases therefore relies on parental haplotyping and statistical assessment of inherited alleles from cffDNA, techniques currently unavailable for routine clinical practice. Here, we present monogenic NIPD (MG-NIPD), which requires a blood sample from both parents, for targeted locus amplification (TLA)-based phasing of heterozygous variants selectively at a gene of interest. Capture probes-based targeted sequencing of cfDNA from the pregnant mother and a tailored statistical analysis enables predicting fetal gene inheritance. MG-NIPD was validated for 18 pregnancies, focusing on CFTR, CYP21A2, and HBB. In all cases we could predict the inherited alleles with >98% confidence, even at relatively early stages (8 weeks) of pregnancy. This prediction and the accuracy of parental haplotyping was confirmed by sequencing of fetal material obtained by parallel invasive procedures. MG-NIPD is a robust method that requires standard instrumentation and can be implemented in any clinic to provide families carrying a severe monogenic disease with a prenatal diagnostic test based on a simple blood draw.

Complex preimplantation genetic diagnosis for beta-thalassaemia, sideroblastic anaemia, and human leukocyte antigen (HLA)-typing

Abstract Preimplantation genetic diagnosis (PGD) to select histocompatible siblings to facilitate curative haematopoeitic stem-cell transplantation (HSCT) is now an acceptable option in the absence of an available human leukocyte antigen (HLA) compatible donor. We describe a case where the couple who requested HLA-PGD, were both carriers of two serious haematological diseases, beta-thalassaemia and sideroblastic anaemia. Their daughter, affected with sideroblastic anaemia, was programmed to have HSCT. A multiplex-fluorescent-touchdown-PCR protocol was optimized for the simultaneous amplification of: the two HBB-gene mutated regions (c.118Cu2009>u2009T, c.25-26delAA), four short tandem repeats (STRs) in chr11p15.5 linked to the HBB gene, the SLC25A38 gene mutation (c.726Cu2009>u2009T), two STRs in chr3p22.1 linked to the SLC25A38 gene, plus eleven informative STRs for HLA-haplotyping (chr6p22.1-21.3). This was followed by real-time nested PCR and high-resolution melting analysis (HRMA) for the detection of HBB and SLC25A38 gene mutations, as well as the analysis of all STRs on an automatic genetic analyzer (sequencer). The couple completed four clinical in vitro fertilization (IVF)/PGD cycles. At least one matched unaffected embryo was identified and transferred in each cycle. A twin pregnancy was established in the fourth PGD cycle and genotyping results at all loci were confirmed by prenatal diagnosis. Two healthy baby girls were delivered at week 38 of pregnancy. The need to exclude two familial disorders for HLA-PGD is rarely encountered. The methodological approach described here is fast, accurate, clinically-validated, and of relatively low cost.

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.