Author(s): Domenico Dell’Edera*, Francesca Mangia, Rosalba Ardea Dell’Edera, Maria Teresa Dell’Edera, Francesco Larocca, Annunziata Anna Epifania and Arianna Allegretti
Hemoglobinopathies are a major health problem worldwide. These disorders are characterized by a variable clinical and hematological situation related to a phenotypic heterogeneity. Moreover, in order to have an exact correlation between the biochemical picture and the genetic defect associated with it, it is useful and often indispensable the molecular study of alpha and beta globin genes. The present case report concerns a pregnant woman of Moroccan ethnicity who came to our observation to undergo combined first-trimester testing useful for screening of major chromosomal aneuploidies. Study of hemoglobins A, A2, and F by High performance liquid chromatography (HPLC) revealed the presence of a proportion of abnormal hemoglobin associated with hemoglobin S. Molecular investigation of globin genes excluded that the biochemical variant in question was related to hemoglobin S. In fact, molecular investigation of beta globin genes, found that the observed variant was due to the presence of a genetic defect leading to hemoglobin O Arab synthesis. Evidence suggests that molecular analysis of globin genes provides the most effective and correct way to correlate the detected biochemical picture with its associated genetic defect. The only biochemical study in the presented case determines an incorrect clinical evaluation with consequent inaccurate prognosis. The mutation detected in this work can be identified using a simple and inexpensive kit. This would generate, in economic terms, significant savings associated with a correct diagnosis.
Hemoglobinopathies are a heterogeneous group of diseases with autosomal recessive transmission. Mutations in beta globin genes can lead to changes in amino acid sequence with heterogeneous clinical effects.
This paper aimed to highlight how the study based on biochemical data alone, in some cases, is not reliable to correctly highlight the related genetic defect. This could lead to an incorrect clinical assessment resulting in an inaccurate prognosis [1,2].
The molecular study of alpha and beta globin genes is essential to have a correct relationship between the detected biochemical picture and the associated genetic defect [3].
The present case report concerns a pregnant woman of a thirtyone-years-old patient of Moroccan ethnicity who came to our observation to undergo combined first-trimester combined test was performed at 12 weeks of gestation. The final risk was 1:1000 for The present case report concerns a pregnant woman of a thirtyone-years-old patient of Moroccan ethnicity who came to our observation to undergo combined first-trimester combined test was performed at 12 weeks of gestation. The final risk was 1:1000 for
The history taken of the husband did not reveal that he had a biochemical picture associated with malfunctioning beta globin genes. To be certain of what we observed, we repeated hematologic screening and molecular study of globin genes to the couple.
The examinations repeated to the couple are as follows:
The molecular analysis of the globin genes was performed following the DNA isolation, starting from 25 ?l of blood, using the extraction kit of Promega Italy S.r.l. (DNA IQTM System, cod.C6701).
The polymerase chain reaction (PCR) and reverse-hybridization was performed. The procedure includes two steps:
PCR amplification using biotinylated primers and hybridization of amplification products to a test strip containing allele-specific oligonucleotide probes immobilized as an array of parallel lines. Bound biotinylated sequences are detected using streptavidinalkaline phosphatase and color substrates. The amplification and the reverse hybridization on a strip were obtained with the use of commercial kits produced by Nuclear Laser Medicine, Milan Italy. The genetic tests were aimed at checking 39 mutations in the β-globin genes (code n. AC104 and AC91 - figure 1A and 1B): -101[C>T]; -92[C>T];-86 [C>A]; -87[C>G]; -30[T>A]; -29 [A>G]; cap+1[A>C]; cap+33 [C>G]; HbG San José: 23 [A>G]; HbD Ouled Rabah: 60 [C>A]; HbE: 79 [G>A]; HbG Copenaghen: 142 [G>A]; Hb Camperdown: 315 [G>C]; HbD-Punjab: 364 [G>C]; HbO Arab: 364[G>A]; Hb Neapolis: 380 [T>G]; Hb Lepore-BW: [NG_000007.3: g63632_7104del]; Hb Siciliana ()-O [NG_000007.3: g64336_77738del13403]; codon 5[-CT]; HbC: codon 6[G>A]; HbS: codon 6[A>T]; codon 6 [-A]; codon 8[-AA]; codon 8/9[+G]; codon 30 [G>C]; IVS 1.1 [G>A]; IVS 1.2 [T>A]; IVS 1.5[G>C]; IVS 1.6[T>C]; IVS 1.110[G>A]; IVS 1.116[T>G]; IVS 1-25; codon 39[C>T]; codon 44[-C]; codon 76 -C; IVS 2.1[G>A]; IVS 2.654 C>T; IVS 2.745 [C>G]; IVS 2.844[C>G].
The genetic tests were aimed at checking 21 mutations in the α-globin genes (code n. AC099): 3.7 single gene deletion, 4.2 single gene deletion, MED double gene deletion, SEA double gene deletion, CAL double gene deletion, FIL double gene deletion, 20.5kb double gene deletion, anti-3.7 gene triplication, α1 cd 14 G>A, α1 cd 59 (Hb Adana), α1 CD 142 Hb Hasharon, α2 init cd, α2 cd 19 [-G], α2 IVS1 [-5nt], α2 cd 59 (Hb Adana), α2 cd 125 (Hb Quong Sze), α2 cd 142 (Hb Constant Spring), α2 cd 142 (Hb Icaria), α2 cd 142 (Hb Pakse), α2 cd 142 (Hb Koya Dora), α2 polyA1[AATAAA-AATAAG], α2 poly-A2[AATAAA-AATGAA].
The outcome of biochemical and molecular tests excluded that the husband had a globin gene alteration.
Abnormal hemoglobin was detected in the wife and was identified as “variant HbS (β6 Glu→Val)” by HPLC (Figure 1). This last datum suggested that the pregnant woman was a healthy carrier of sickle-cell anemia. Molecular analysis of the globin genes revealed that the variant highlighted by HPLC was not due to the presence of hemoglobin S, but to the presence, in heterozygosity, of hemoglobin HbO Arab (β121 Glu→Lys) (figure 2A and figure 2B). In the condition of heterozygosity, HbO Arab is completely silent.
Figure 1: Abnormal hemoglobin was detected in the wife and was identified as “variant HbS by HPLC
Figure 2: List of mutations researched in the beta globin genes and mutations detected. A) Molecular analysis of the globin genes revealed that the variant highlighted by HPLC was not due to the presence of hemoglobin S, B) Presence, in heterozygosity, of hemoglobin HbO Arab.
The homozygosity condition is rare and has only occasionally been reported to cause very mild hemolytic anemia with some splenomegaly. Significant hemolysis and/or anemia or microcytosis with hemoglobin O-Arab should prompt further investigation for co-inheritance of thalassemia or sickle cell hemoglobin. Interestingly, the position of the substituted amino acid in HbO Arab (β121 Glu → Lys) is the same as in HbD Los Angeles (β121 Glu → Gln). In HbO Arab the amino acid present at position 121 is lysine (Lys), whereas in HbD Los Angeles the amino acid present at position 121 is glutamine (Gln). This represents a critical point for tetramer assembly, and the resulting hemoglobin is slightly unstable, resulting in mild hemolysis in both variants.
Arab HbO was reported for the first time in an Arab family in Israel4. Regarding the origin of Arab HbO, it has been shown that it appeared about 2000 years ago on a rare haplotype, characteristic of Greek Pomaks. Its frequency increased as a consequence of high genetic drift within this population, and it subsequently spread throughout the Mediterranean basin and the Middle East 5.
The case we describe demonstrates that biochemical screening alone is in many cases insufficient to accurately determine the genetic defect causing a hemoglobinopathy. In these cases, a precise diagnosis will allow to offer exact and detailed information on the implications related to the genetic defect in question
Thus, correct identification of the healthy carrier and extension of the analysis to the spouse and family members is necessary. In our case, the molecular study of globin genes, led not only to a correct molecular diagnosis, but also to define a genetic condition that from the biochemical point of view had been confused for HbS [4,5].
This paper has shown that biochemical diagnosis alone, in some cases, is not reliable enough to detect carriers of hemoglobinopathies/thalassemias.
Molecular analysis of globin genes provides the most effective way to detect carriers of hemoglobinopathy with certainty. He mutations detected in this work can be identified with a simple and inexpensive kit. This means, in economic terms, significant savings to healthcare spending
We can say that in the field of prevention, knowing also means recognizing a specific condition, which means faster diagnosis, prevention of complications, a great impact on the quality of life of patients and reduction of social costs of a potentially disabling disease.
This research was not supported
The authors declare that they have no competing interests.
The data of the present paper are available
As they are anonymous data it was not necessary to ask for approval.
As they are anonymous data it was not necessary to ask for approval.
As they are anonymous data it was not necessary to ask for approval.
The authors thank “Association Gian Franco Lupo - Un Sorriso Alla Vita” (ONLUS: no-profit organization of social utility)
