Raffaele Sessa

Large Deletion Involving Exon 5 of the Arylsulfatase B Gene Caused Apparent Homozygosity in a Mucopolysaccharidosis Type VI Patient

Apparent homozygosity for the mutation p.R315X present on exon 5 of the arylsulfatase B (ARSB) gene in a mucopolysaccharidosis type VI patient was solved in this study by further testing for a second mutation. Patient cDNA analysis revealed that the entire exon 5 of the ARSB gene was lacking; this new mutation was identified as c.899-1142del. As the genomic DNA sequencing excluded the presence of splicing mutations, polymerase chain reaction analysis was performed for polymorphisms listed in the NCBI SNP database for the ARSB gene. This allowed the mutation at the genomic DNA level to be identified as g.99367-102002del; this gross deletion, involving the entire exon 5 of the gene and parts of introns 4 and 5 led to a frameshift starting at amino acid 300 and resulting in a protein with 39% amino acids different from the normal enzyme. We stress that extensive DNA analysis needs to be performed in case of apparent homozygosity to avoid potential errors in genetic counseling.

Identification and molecular characterization of the ‐‐CAMPANIA deletion, a novel α°‐thalassemic defect, in two unrelated Italian families

To the Editor: Alpha-thalassemia is characterized by decreased or complete lack of synthesis of a-globin chains. The a-globin gene cluster is located on the short arm of chromosome 16 and includes, in the 50-30 order, an embrionic globin gene (f2), three pseudogenes (pseudo-f, pseudo-a2, and pseudo-a1), two a-globin genes (a2 and a1), and a gene of undetermined function (y1). Most common defects result from large deletions involving one (-a) or both (--a) of the a-globin genes, although increasing number of point mutations have also been described [1–3]. Loss of function of three of four a-globin genes gives rise to HbH disease, an intermediate clinical form of athalassemia [4]. Mutations are population specific, and their distribution has been extensively determined, providing support for prevention programs [5,6]. During a study on the molecular basis for a-thalassemia in our region, we identified two patients with a novel type of a-thalassemia determinant resulting in the deletion of the entire f-a gene cluster. Clinical and hematological data (Supporting Information, Fig. S1A) were suggestive of HbH disease. Molecular analysis of the most common Mediterranean deletion forms of a-thalassemia indicated an apparent homozygosis for the -a deletion. As in both families only one parent was heterozygous for this mutation (Supporting information, Fig. S1B), the two patients were actually compound heterozygotes for the -a deletion and an uncommon a8 rearrangement. In both cases, hybridization with a f-globin probe on genomic DNA digested with Xba I failed to reveal any abnormal fragment (data not shown), whereas a L1 probe located 50 to the f-globin gene [7] showed an abnormal 6.3-kb fragment along with a normal 6.6-kb fragment suggestive of a deletion encompassing the entire f-a gene cluster (Supporting Information, Fig. S1C). This pattern was similar to that detected in heterozygotes for the --a deletion ( 6.0 kb/6.6 kb), first described in an Italian patient [8] and subsequently in a Spanish family [9]. An allele-specific PCR reaction previously described for the --a deletion [6] produced in both cases an amplicon of 2.1 kb instead of the 491bp size expected for this type of deletion (Supporting Information, Fig. S2A). Sequencing analysis of the 2.1-kb fragment allowed to identify the 31-kb region removed by this novel deletion and its resulting 50and 30-breakpoints located within the Alu Jb (coordinates 8635-8924) and Alu Sp (coordinates 39835-40133) repeats (Fig. 1A), respectively. As both patients were from Campania, a southern Italy region, the novel deletion was designated as CAMPANIA. Our results indicate that this rearrangement was produced by a mechanism of Alu-mediated recombination, which is rather common in this chromosomal region [10]. Definition of the specific Alu elements also demonstrates that the CAMPANIA and CAL deletions arose from independent mutational events, although their breakpoints lie very close to each other (Fig. 1B). Linkage analysis of polymorphic sites located upstream and downstream the deletion breakpoints (50HVR, XbaI RFLP, yPstI RFLP, and 30HVR) [11,12] showed for both patients the same junctional sequence and polymorphic genotypes in the regions surrounding the deletion: 50HVR (allele size of 830 bp), XbaI (2), yPstI (2), 30HVR (allele size of 2,100 bp), therefore supporting the hypothesis of a unique origin for this deletion (Fig. 1A and Supporting Information, Fig. S2D). Definition of the deletion breakpoints allowed to design an allele-specific amplification reaction for this novel deletion, which was found at the heterozygous condition in the obligate carriers (subjects I-2 from family a and I-2 from family b) and in another sibling from family b (II-2) (Supporting information, Fig. S2E). Our study contributes to define the wide spectrum of mutations that underlie the thalassemia syndromes in the Mediterranean area. Furthermore, involvement of Alu repeats remarks the high susceptibility of the a-globin gene cluster region to unequal crossing-over events. Therefore, given the high frequency with which these sequences occur within this region, the overall incidence of unusual deletional forms of a-thalassemia in our population is to be expected much higher than what was supposed so far [13]. Our study also indicates that exhaustive molecular studies are necessary to avoid potential pitfalls in genetic counseling. This requires specific, reliable, and easily accessible genetic tests to allow accurate large-scale screening programs and prenatal diagnosis services in regions where such uncommon a-thalassemic determinants could occur.

Molecular Basis of Thalassemia

Hemoglobinopathies are a heterogeneous group of monogenic disorders widespread overall. They are commonly subdivided into three partially overlapping subgroups: structural variants which comprise the sickle cell anemia syndrome; thalassemias, characterized by a reduced rate of synthesis of one or more globin chains of hemoglobin; conditions of high persistence of fetal hemoglobin in adulthood (HPFH) (Weatherall & Clegg, 2001). As a group, they are the commonest monogenic disorders in the world population. It is thought that the high prevalence of these defects could be due to selective advantage of the carrier state to malaria infection. However, in spite of epidemiological evidences supporting this hypothesis as well as of extensive hematological studies, the mechanisms underlying this protection still remain unknown. It is, however, evident that as a consequence of this positive selection, these diseases are mostly common in geographic areas extending from the Mediterranean region through tropical countries including Sub-Saharian Africa, the Middle East, India, Southeast Asia and Indonesia, where malaria was or still is endemic (Weatherall & Clegg, 2001). In many of these areas the estimated frequencies of these disorders range from 3 to 10 percent, even though in some specific areas the carrier frequencies may be higher, reaching 80-90% in some tribal populations in India (Harteveld & Higgs, 2010). Because of their high frequencies, different hemoglobin defects may be co-inherited, giving rise to an extremely complex series of genotypes and clinical phenotypes. In fact, in many regions thalassemic defects coexist with structural Hb variants; it is also quite common for individuals from areas at high frequency of thalassemic defects to inherit genes for more than one type of thalassemia. Furthermore, some Hb variants are synthesized at reduced rate or are highly instable, leading both to functional and structural deficiency of the affected globin chain, thus resulting in a thalassemic condition, generally showing dominant inheritance. These complex interactions contribute to generate a wide range of clinical disorders that, taken together, constitute the thalassemic syndromes (Weatherall, 2001). The complex and heterogeneous spectrum of molecular defects underlying these inherited conditions is regionally specific and in most cases the geographic and ethnic distributions have been determined, providing support for prevention programs based on screening, genetic counselling and prenatal diagnosis in couples at risk.

Role of the cold shock domain protein A in the transcriptional regulation of HBG expression

Impaired switching from fetal haemoglobin (HbF) to adult globin gene expression leads to hereditary persistence of fetal haemoglobin (HPFH) in adult life. This is of prime interest because elevated HbF levels ameliorate β‐thalassaemia and sickle cell anaemia. Fetal haemoglobin levels are regulated by complex mechanisms involving factors linked or not to the β‐globin gene (HBB) locus. To search for factors putatively involved in the expression of the γ‐globin genes (HBG1, HBG2), we examined the reticulocyte transcriptome of three siblings who had different HbF levels and different degrees of β‐thalassaemia severity although they had the same ΗBA‐ and ΗΒB cluster genotypes. By mRNA differential display we isolated the cDNA coding for the cold shock domain protein A (CSDA), also known as dbpA, previously reported to interact in vitro with the HBG2 promoter. Expression studies performed in K562 and in primary erythroid cells showed an inverse relationship between HBG and CSDA expression levels. Functional studies performed by Chromatin Immunoprecipitation and reporter gene assays in K562 cells demonstrated that CSDA is able to bind the HBG2 promoter and suppress its expression. Therefore, our study demonstrated that CSDA is a trans‐acting repressor factor of HBG expression and modulates the HPFH phenotype.

Defective mRNA levels are responsible for a β-thalassemia phenotype associated with Hb Federico II, a novel hemoglobin variant [β-106 (G8) Leu→Val]

Hemoglobinopathies are widespread monogenic disorders that encompass complex and partially overlapping hemoglobin disorders and thalassemia syndromes. About 960 hemoglobin variants have been identified, some of which are reported to be unstable.[1][1] Various mechanisms for the decreased stability

research paper: Role of the cold shock domain protein A in the transcriptional regulation of HBG expression

Impaired switching from fetal haemoglobin (HbF) to adult globin gene expression leads to hereditary persistence of fetal haemoglobin (HPFH) in adult life. This is of prime interest because elevated HbF levels ameliorate β‐thalassaemia and sickle cell anaemia. Fetal haemoglobin levels are regulated by complex mechanisms involving factors linked or not to the β‐globin gene (HBB) locus. To search for factors putatively involved in the expression of the γ‐globin genes (HBG1, HBG2), we examined the reticulocyte transcriptome of three siblings who had different HbF levels and different degrees of β‐thalassaemia severity although they had the same ΗBA‐ and ΗΒB cluster genotypes. By mRNA differential display we isolated the cDNA coding for the cold shock domain protein A (CSDA), also known as dbpA, previously reported to interact in vitro with the HBG2 promoter. Expression studies performed in K562 and in primary erythroid cells showed an inverse relationship between HBG and CSDA expression levels. Functional studies performed by Chromatin Immunoprecipitation and reporter gene assays in K562 cells demonstrated that CSDA is able to bind the HBG2 promoter and suppress its expression. Therefore, our study demonstrated that CSDA is a trans‐acting repressor factor of HBG expression and modulates the HPFH phenotype.

Prenatal diagnosis of haemoglobinopathies: our experience of 523 cases.

Abstract Background: We performed counselling for prenatal diagnosis (PD) of haemoglobinopathies in 372 couples. Thirty-four out of 372 (9.1%) did not undergo PD: six due to spontaneous abortion; nine because it was too difficult to make a decision if PD was positive; 18 because counselling excluded the carrier status of one or both parents; and one because parental mutations were mild. Methods: Eleven out of 338 (3.3%) couples underwent PD because they had a thalassaemic child; 106 (31.4%) were found to be at high risk during pre-conceptional screening; 221 (65.4%) because of familiarity. Of 523 PDs in 486 (92.9%), including six dichorionic twin pregnancies, PD was performed on DNA from chorionic villi (CV), and in 37 from amniocytes (7.1%). In 1/523 cases, PD was not completed because DNA from CV was not sufficient; in two cases single tandem repeat analysis revealed maternal contamination of foetal DNA; in 7/522 (1.3%) cases PD revealed non-paternity. In 435/522 (83.3%) cases, PD was performed using reverse dot-blot and ARMS; 34/522 (6.5%) required sequencing. In 53/522 (10.2%) cases it was necessary to test globin loci for large rearrangements. Results: One hundred and twenty out of 522 (23.0%) PDs revealed an affected foetus. In all but two cases the couple interrupted pregnancy. In the six twin pregnancies PD revealed a normal and a carrier foetus (two cases), carrier status in both foetuses (two cases) and a carrier and an affected foetus (two cases). In these latter cases the couple planned selective interruption. Conclusions: Our PD procedure is successful and reliable, and is useful in high-risk areas characterised by molecular heterogeneity.

Role of the Cold Shock Domain Protein A in the transcriptional regulation of gamma-globin gene expression.

Impaired switching from fetal haemoglobin (HbF) to adult globin gene expression leads to hereditary persistence of fetal haemoglobin (HPFH) in adult life. This is of prime interest because elevated HbF levels ameliorate β‐thalassaemia and sickle cell anaemia. Fetal haemoglobin levels are regulated by complex mechanisms involving factors linked or not to the β‐globin gene (HBB) locus. To search for factors putatively involved in the expression of the γ‐globin genes (HBG1, HBG2), we examined the reticulocyte transcriptome of three siblings who had different HbF levels and different degrees of β‐thalassaemia severity although they had the same ΗBA‐ and ΗΒB cluster genotypes. By mRNA differential display we isolated the cDNA coding for the cold shock domain protein A (CSDA), also known as dbpA, previously reported to interact in vitro with the HBG2 promoter. Expression studies performed in K562 and in primary erythroid cells showed an inverse relationship between HBG and CSDA expression levels. Functional studies performed by Chromatin Immunoprecipitation and reporter gene assays in K562 cells demonstrated that CSDA is able to bind the HBG2 promoter and suppress its expression. Therefore, our study demonstrated that CSDA is a trans‐acting repressor factor of HBG expression and modulates the HPFH phenotype.

research paper: Role of the cold shock domain protein A in the transcriptional regulation of HBG expression: Role of CSDA in Modulation of Globin Gene Expression

Impaired switching from fetal haemoglobin (HbF) to adult globin gene expression leads to hereditary persistence of fetal haemoglobin (HPFH) in adult life. This is of prime interest because elevated HbF levels ameliorate β‐thalassaemia and sickle cell anaemia. Fetal haemoglobin levels are regulated by complex mechanisms involving factors linked or not to the β‐globin gene (HBB) locus. To search for factors putatively involved in the expression of the γ‐globin genes (HBG1, HBG2), we examined the reticulocyte transcriptome of three siblings who had different HbF levels and different degrees of β‐thalassaemia severity although they had the same ΗBA‐ and ΗΒB cluster genotypes. By mRNA differential display we isolated the cDNA coding for the cold shock domain protein A (CSDA), also known as dbpA, previously reported to interact in vitro with the HBG2 promoter. Expression studies performed in K562 and in primary erythroid cells showed an inverse relationship between HBG and CSDA expression levels. Functional studies performed by Chromatin Immunoprecipitation and reporter gene assays in K562 cells demonstrated that CSDA is able to bind the HBG2 promoter and suppress its expression. Therefore, our study demonstrated that CSDA is a trans‐acting repressor factor of HBG expression and modulates the HPFH phenotype.