Paloma Ropero

THE THALASSEMIA SYNDROMES: MOLECULAR CHARACTERIZATION IN THE SPANISH POPULATION

This work compiles the results of our research on α- and β-thalassemias, and includes a literature review of the molecular genetics of α- and β-thalassemias in Spain. We studied 1,564 subjects with thalassemia (294 with β-thalassemia and 1,264 with α-thalassemia) by molecular biology techniques. In relation to β-thalassemia, a total of 15 different mutations were characterized in a study of 308 chromosomes belonging to 294 unrelated subjects. Eleven were homozygotes (22 alleles), three compound heterozygotes (6 alleles), and the remaining 280 were heterozygotes (280 alleles). A total of 86.6% of the alleles identified can be grouped into five different mutations [IVS-I-1 (G → A), IVS-I-6 (T → C), IVS-I-110 (G → A), codon 39 (C → T), codons 8/9 (+G)]. In 14 subjects (4.5%), all heterozygotes, it was not possible to identify the alteration responsible for the β-thalassemia. For α-thalassemia, 911 subjects showed heterozygous α+-thalassemia (872 with −3.7 kb; 14 with −4.2 kb; two with the deletion of 3.5 kb of DNA, and 23 with nondeletional α-thalassemia). Two hundred and thirty-three subjects had homozygous α+-thalassemia (223 for –α−3.7/–α−3.7); one for –α−4.2/–α−4.2; six for –α−3.7/–α−4.2; one for –α−3.5/–α−3.7; one for ααNco/ααNco; one for αHphα/αHphα). One hundred patients presented with heterozygous α0-thalassemia (18 of whom were progenitors of patients with Hb H disease). The α0 determinant was found in 20 patients with Hb H disease associated with –α−3.7. From the DNA analysis were identified the ––MED, ––SEA, ––SPAN deletions and the ––CAL, ––CANT, and ––MA mutations; in three cases, a break that affects the distal portion of the short arm of chromosome 16; one of these was associated with the ATR-16 (α-thal with mental retardation) syndrome. Triplication of the α genes (ααα−3.7/αα) was found in 25 subjects, 16 of whom were associated with a heterozygous β-thalassemia. Only one patient was homozygous for the triplication of α genes (ααα−3.7/ααα−3.7) that was associated with a heterozygous β-thalassemia. In the Mediterranean region preventive programs for thalassemia, based on the detection of heterozygote carriers and genetic advice, are not sufficient to reduce the incidence of newborns with major thalassemia. Prenatal diagnosis of thalassemias has given a new dimension to the prevention of these, but in order to implement this, a knowledge of the mutations and the incidence of these, is essential. This study, therefore, aims to give a general picture of the molecular genetics of thalassemia and its geographical distribution in our area.

Cribado de hemoglobinopatías en una cohorte de recién nacidos en la Comunidad de Madrid

Fundamento y objetivo: Las hemoglobinopatias estructurales son un grupo de enfermedades cuya incidencia en la Comunidad de Madrid ha aumentado en los ultimos anos debido al creciente fenomeno de la inmigracion que esta viviendo. Dados los problemas de morbimortalidad que comportan y la posibilidad de instaurar tratamientos profilacticos tempranos, consideramos necesario realizar un estudio que demuestre la necesidad de la inclusion del cribado de hemoglobinopatias en dicha comunidad autonoma. Sujetos y metodo: Estudio de cohortes ambispectivo en 3.365 recien nacidos realizado en el Hospital Clinico San Carlos desde mayo de 2003 a abril de 2004. Todas las muestras se analizaron mediante cromatografia liquida de alta resolucion (HPLC) de intercambio ionico (Variant®, Bio-Rad) y en las patologicas se estudiaron las cadenas de globina mediante HPLC de fase reversa. Resultados: Se encontro una incidencia global de hemoglobinopatias de 7,7 de cada 1.000 recien nacidos, con un caso de hemoglobina S homocigota, 18 de hemoglobina S heterocigota, uno de hemoglobina E/A2, uno de hemoglobina C y 5 picos anomalos. Conclusiones: Consideramos que el cribado neonatal de hemoglobinopatias en la Comunidad de Madrid es necesario y que la HPLC de intercambio ionico Variant® es adecuada para el cribado de las hemoglobinopatias mas frecuentes, con ahorro de tiempo.

Haemoglobinopathies with high oxygen affinity. Experience of Erythropathology Cooperative Spanish Group

Haemoglobinopathies are the world’s most frequently found monogenic disorders. In the cases with high oxygen affinity, the decrease in the liberation of the oxygen determines a secondary erythrocytosis. In this work, we present 17 unrelated families of Caucasian race and of Spanish origin, with ten variants of haemoglobin or haemoglobinopathies with high oxygen affinity which were diagnosed in our laboratory. Of the ten haemoglobinopathies, in four (the Hb San Diego, the Hb Johnstown, the Hb Malmö and the Hb Columbia-Missouri), the change of amino acid affects zones of the contact α1β2; in two variants (the Hb Strasbourg and the Hb Syracuse), it affects the unions with 2,3-DPG in the central cavity; in the other two (the Hb Badalona and the Hb La Coruña), the cavity of contact with the group haem is affected; in one (Hb Bethesda), it affects the zone of contact α1β1; and in one (Hb Olympia), the position 20 of the chain in the helix B in the surface of the protein is affected. In all cases, the change of amino acid, though of different form, facilitates that the quaternary structure of the haemoglobin becomes stable in its relaxed configuration so the transfer of oxygen and the P50 value are decreased. All cases were sent to our laboratory because of shown erythrocytosis. In the majority of them, the diagnosis was done during an analysis of routine or for being relatives of the first ones.

Frequency of the C282Y and H63D mutations of the hemochromatosis gene (HFE) in a cohort of 1,000 neonates in Madrid (Spain).

For centuries in Europe, population movements have contributed to ethnic groups, cultures, and consequently, inheritance mixing. There are certain genetic diseases such as hereditary hemochromatosis whose distribution is directly related to the population movements. The objective of the present investigation was to determine the C282Y and H63D mutation frequency of the HFE gene in a cohort study of 1,000 neonates in the Community of Madrid (Spain), thus contributing to the HFE gene mutations distribution research in Europe and establishing the origin of the mutations in Spain. The allelic frequency of C282Y mutation was 1.7% (CI 95% 1.1–2.3) and the H63D allele was present in 16.4% of chromosomes (CI 95% 14.8–18). In Spain, the presence of C282Y mutation and its distribution could be due more to Celtic than to Viking legacy, whereas it is assumed that the one in relation to the H63D variant occurred in the Basque Country during the Paleolithic Period.

La mutacion H63D del gen HFE se asocia con un riesgo aumentado de carcinoma hepatocelular

AIM To disclose whether mutations in the HFE gene inducing liver iron overload are related to the risk of hepatocellular carcinoma (HCC) in otherwise predisposed patients. PATIENTS AND METHODS One hundred and ninety-six patients (161 males) diagnosed with HCC and 181 healthy controls were included in the study. All subjects were white Spaniards.C282Y and H63D mutations in the HFE gene were identified in leucocyte genomic DNA using a polymerase chain reaction (PCR) and specific restriction enzymes. RESULTS (CASES/CONTROLS): 1. Genotype distribution: a) C282Y mutation: homozygotes 1/0, heterozygotes 12/23, wild type 183/158 (p = 0.07, non significant); b) H63D mutation: homozygotes 9/5, heterozygotes 85/52, wild type 102/124 (0dds ratio 2.00, 95% C.I. 1.29-3.12, p = 0.002. Four cases and 6 controls were carriers of heterozygous mixed genotypes. 2. Allele frequencies: a) C282Y mutation: wild type allele 378/339, mutated allele 14/23 (p = 0.11, non significant); b) H63D mutation: wild type allele 289/300, mutated allele 103/62 (0dds ratio 1.72, 95% C.I. 1.19-2.50, p = 0.004). Age at diagnosis, gender and etiology of the underlying liver disease do not influence these findings. CONCLUSION The C282Y mutation in the HFE gene is not related to the risk of HCC in non-hemochromatosis patients. The H63D mutation is associated with a higher risk of HCC in cirrhotic patients irrespective of their underlying liver disease.

THE FIRST CASE OF Hb E-SASKATOON ASSOCIATED WITH Hb LEPORE-BALTIMORE FOUND IN SPAIN

Hb E-Saskatoon [β22(B4)Glu→Lys] does not cause any clinical symptoms in the heterozygous state. The homozygous state shows moderate phenotype expression. It has also been detected in association with β-thalassemia. We present the first case of Hb E-Saskatoon associated with Hb Lepore-Baltimore. This unusual combination of mutations does not aggravate the clinical picture, as only microcytosis and hypochromia have been observed. Hb E-Saskatoon can only be correctly characterized by ion exchange high performance liquid chromatography (HPLC) or by DNA sequencing.

Effects of hemoglobin variants on hemoglobin a1c values measured using a high-performance liquid chromatography method.

Background: Hemoglobin A1c (HbA1c) is routinely used to monitor long-term glycemic control and for diagnosing diabetes mellitus. However, hemoglobin (Hb) gene variants/modifications can affect the accuracy of some methods. The potential effect of Hb variants on HbA1c measurements was investigated using a high-performance liquid chromatography (HPLC) method compared with an immunoturbimetric assay. Methods: Fasting plasma glucose (FPG) and HbA1c levels were measured in 42 371 blood samples. Samples producing abnormal chromatograms were further analyzed to characterize any Hb variants. Fructosamine levels were determined in place of HbA1c levels when unstable Hb variants were identified. Results: Abnormal HPLC chromatograms were obtained for 160 of 42 371 samples. In 26 samples HbS was identified and HbA1c results correlated with FPG. In the remaining 134 samples HbD, Hb Louisville, Hb Las Palmas, Hb N-Baltimore, or Hb Porto Alegre were identified and HbA1c did not correlate with FPG. These samples were retested using an immunoturbidimetric assay and the majority of results were accurate; only 3 (with the unstable Hb Louisville trait) gave aberrant HbA1c results. Conclusions: Hb variants can affect determination of HbA1c levels with some methods. Laboratories should be aware of Hb variants occurring locally and choose an appropriate HbA1c testing method.

Study of Three Families with Hb Agrinio [α29(B10)Leu→Pro, CTG>CCG (α2)] in the Spanish Population: Three Homozygous Cases

Most α-thalassemia (α-thal) mechanisms are deletions of one or both α-globin genes and less than 5.0-10.0% are point mutations. Hb Agrinio [α29(B10)Leu→Pro, CTG>CCG (α2)] is a hyperunstable α chain structural variant in which the thalassemic phenotype is determined by a post translational precipitation of the structurally anomalous chain in erythroid precursors. This study involved 14 cases with Hb Agrinio from three families. Selective sequencing of the α2 gene showed a CTG(Leu)>CCG(Pro) mutation at codon 29. The mutation was found in a heterozygous state in 11 cases and in a homozygous state in three cases. These are the first cases with Hb Agrinio described in Spain. In all cases where a leucine is exchanged for a proline, an unstable hemoglobin (Hb) will occur both in the α and the β chain. Some of these are as unstable as Hb Agrinio and their presence is difficult to detect except by DNA sequencing.

Hemoglobina Stanleyville II [ α78(EF7)Asn → Lys]. Primer caso descrito en España

Resumen Fundamento y objetivo Las hemoglobinopatias estructurales son el resultado de mutaciones en los genes de globina que determinan una alteracion cualitativa en la expresion de dichos genes. En la mayoria de ellas la alteracion estructural no condiciona ningun cambio significativo, por lo que cursan de forma silente o asintomatica. En este trabajo presentamos un nuevo caso de hemoglobina (Hb) Stanleyville II. Pacientes y metodo El probando es una mujer de 72 anos, raza blanca y origen canario. En la analitica presentaba Hb de 14,3 g/dl, hematocrito del 44,4%, volumen corpuscular medio de 85,8 fl, Hb corpuscular media de 27,7 pg y concentracion de Hb corpuscular media de 32,2 g/l; el indice de anisocitosis era del 15,1%, reticulocitos del 1,2%, HbA2 del 3,1% y HbF del 1,6%. En la electroforesis en acetato de celulosa a pH alcalino y en el isoelectroenfoque se separo una Hb anormal a la altura de la HbS. En agar citrato a pH acido la Hb anormal no se separaba de la HbA. Por cromatografia liquida de alta resolucion de fase reversa se eluyo una cadena anormal mas precoz que la normal. Resultados En el analisis molecular, que se completo con la secuenciacion de los productos de amplificacion por reaccion en cadena de la polimerasa de los genes α 1 y α 2 , se demostro la mutacion AAC → AAA en el codon 78 del segundo exon del gen 2 en estado heterocigoto, que determina un cambio de asparagina por lisina. Conclusiones La sustitucion de un aminoacido con carga neutra, como la asparagina, por otro con carga muy positiva, como la lisina, en el segmento EF, que corresponde a la superficie externa de la estructura terciaria de la cadena de globina, determina un cambio neto en la carga de la cadena. Esto permite su facil diferenciacion por metodos electroforeticos y cromatograficos. Sin embargo, como la localizacion no es fundamental para la estabilidad, solubilidad y afinidad por el oxigeno del tetramero, cursa de forma silente o asintomatica. La Hb Stanleyville II se habia descrito hasta ahora en familias de raza negra del Congo, Uganda, Zaire, EE.UU., Alsacia y Brasil. Este caso representa el primero descrito en Espana.

Hb Johnstown [β109 (G11) Val→Leu]: Second case described and associated for the first time with β0‐thalassemia in two Spanish families

Hb Johnstown, a high oxygen affinity hemoglobin, was identified in four members from two unrelated Spanish families with erythrocytosis and left‐shifted hemoglobin–oxygen dissociation curve. This hemoglobin variant, electrophoretically silent, was analyzed by reverse‐phase high‐performance liquid chromatography, and the mutation was characterized at the DNA level by β gene sequencing. In one of these families, two members are affected with Hb Johnstown in association with β0‐thalassemia. In these cases the erythrocytosis and low values for P50 due to Hb Johnstown remain in spite of the β‐thalassemia. Am. J. Hematol. 65:298–301, 2000.