Daniela Loi

Elevation of serum creatine kinase as the only manifestation of an intragenic deletion of the dystrophin gene in three unrelated families

This study reports three children from three unrelated families, aged from 9 to 12 years, who were investigated because of the incidental finding of elevated serum creatine kinase (CK) levels and were found to have a dystrophinopathy. The molecular defect consisted of a deletion of variable extent within the central rod domain of the dystrophin gene, involving either exons 32-44 or 48-51 or 48-53. In each family we found the same deletion in at least one adult male relative aged from 40 to 77 years, who was either completely asymptomatic or had very mild muscle involvement (thin muscles and/or mild scoliosis), with normal or borderline CK levels. This study suggests once again that deletions of the central rod domain of dystrophin may be associated with elevation of serum CK as the only manifestation and that prediction of the clinical severity based solely on the molecular findings should be interpreted with caution.

Association of α globin gene quadruplication and heterozygous β thalassemia in patients with thalassemia intermedia

The degree of the globin chain imbalance is the pathogenetic clue to the clinical phenotype of thalassemia syndromes. This paper reports a duplication of the α globin gene locus in a group of hetereozygous β-thalassemia patients with the unexplained phenotype of thalassemia intermedia. Ten patients with thalassemia intermedia with variable severity and apparent simple heterozygosis for β0 39 C>T nonsense mutation were submitted to clinical, hematologic and molecular studies. The presence of an unknown molecular defect (silent β-thalassemia) unlinked to the β cluster interacting with the heterozygous β thalassemia, was previously postulated in these families. Analysis of the α globin gene cluster with PCR-based methods (MLPA, GAP-PCR, digestion with restriction enzymes) detected complex rearrangements in the α cluster. A duplication of the α globin gene locus, including the upstream regulatory region, was present in all the patients, associated in some of them with deletion or non-deletion α thalassemia. The variability of the clinical phenotype correlates with the degree of the globin chain imbalance. The presence of α globin cluster duplication should be considered in patients heterozygote for β-thalassemia with thalassemia intermedia phenotype and in the carriers of suspected silent β thalassemia.

Thalassaemia and Glucose-6-Phosphate Dehydrogenase Screening in 13- to 14-Year-Old Students of the Sardinian Population: Preliminary Findings

Objectives: In this paper we describe the outline and results of a 7-year screening programme for thalassaemias and glucose-6-phosphate dehydrogenase (G6PD) deficiency in 13- to 14-year-old students from the Sardinian population. Method: This programme had several steps: formal education on thalassaemia, request of informed consent by parents, blood testing and genetic counselling. Results:Out of 63,285 subjects tested, 6,521 (10.3%) were heterozygotes for β-thalassaemia, 16,175 (25.6%) for α-thalassaemia and 101 were carriers of a haemoglobin variant. One thousand four hundred and twenty (16.4%) males were hemizygotes for G6PD deficiency and 1,893 (20.6%) females were heterozygotes. Conclusion: The uptake of the programme was remarkably high and homogeneous across the island, indicating and confirming a great interest of the Sardinian population in any initiative directed at the prevention of homozygous β-thalassaemia.

Homozygous deletion of the major alpha-globin regulatory element (MCS-R2) responsible for a severe case of hemoglobin H disease

To the editor:nnAlpha-thalassemia commonly results from deletions or point mutations in one or both alpha-globin genes, located on chromosome 16p13.3. Rarely, alpha-thalassemia is caused by deletions in a region, located 30 to 70 kb upstream of the alpha-globin genes, containing 4 remote,

Complexity of the alpha-globin genotypes identified with thalassemia screening in Sardinia.

α-Thalassemia commonly results from deletions or point mutations in one or both α-globin genes located on chromosome 16p13.3 giving rise to complex and variable genotypes and phenotypes. Rarely, unusual non-deletion defects or atypical deletions down-regulate the expression of the α-globin gene. In the last decade of the program for β-thalassemia carrier screening and genetic counseling in Sardinia, the association of new techniques of molecular biology such as gene sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) to conventional methods has allowed to better define several thalassemic genotypes and the complex variability of the α-cluster with its flanking regions, with a high frequency of different genotypes and compound heterozygosity for two α mutations even in the same family. The exact molecular definition of the genotypes resulting from the interactions among the large number of α-thalassemia determinants and with β-thalassemia, is important for a correct correlation of genotype-phenotype and to prevent underdiagnosis of carrier status which could hamper the effectiveness of a screening program particularly in those regions where a high frequency of hemoglobinopathies is present.

First Detection of Hb Taybe [α38(C3) or α39(C4) Thr→0 (α1)] in An Italian Child

Hb Taybe [α38(C3) or α39(C4) Thr→0 (α1)] is an unstable hemoglobin (Hb) variant caused by a deletion of a threonine residue at codon 39 of the α1-globin chain. Usually asymptomatic or with minimal hematological abnormalities in the heterozygous state, Hb Taybe becomes clinically evident in compound heterozygosity with α-thalassemia (α-thal) or in homozygous patients. To date, Hb Taybe has been described in Israeli-Arab and Greek individuals. We report, for the first time, a patient with chronic hemolytic anemia due to the presence of Hb Taybe in trans to the α2 initiation codon mutation ATG>ACG in an Italian child. Hb Taybe was not evident at Hb analysis with cellulose acetate electrophoresis and high performance liquid chromatography (HPLC). Globin biosynthetic studies revealed an α/β-globin ratio in the range of β-thal trait. Consequently, an investigation of the α- and β-globin genes was requested in order to avoid missing any rare globin chain variant and to offer accurate genetic counseling.

Two atypical forms of HbH disease in Sardinia

Hemoglobin H disease is usually caused by deletion or inactivation of three α-globin genes, leaving only one α-globin gene intact and active.1 The most frequent defects responsible for HbH disease in Sardinia are the coinheritance of the --Med deletion in one chromosome and the -α3.7 Kb deletion or, less frequently, the α2 initiation codon mutation ATG>ACG (α2NcoI) in the other chromosome.2,3 HbH disease due to deletions including the major upstream regulatory element (MCS-R2) and leaving intact both α-globin genes have also been described.4,5 We report here two new α0 deletions, both located on the short arm of chromosome 16, responsible for HbH disease in two different Sardinian families. These unusual deletions were respectively associated with the common α2NcoI mutation and -α3.7 deletion in trans. n nTable 1 shows the hematologic and molecular data of the probands and their family members. All patients had severe microcytic anemia (Hb 2.6–9.5 g/dL, MCV 52.0–75.7 fl). Jaundice, spleen enlargement, sporadic hemolytic and aplastic crisis due to B19 parvovirus infection requiring red blood cell transfusions were detected in patients II-1 and II-2 of Family A. A mild thalassemia-like facies was present only in II-1 of the same family. Molecular screening for the most common α-globin gene deletion and non-deletion defects revealed the apparent homozygosity for the α2NcoI mutation in the proband of Family A and in her sister, and the apparent homozygosity for the -α3.7 deletion in the proband of Family B. In spite of that, the α2NcoI mutation was present only in the father of the Family A proband and the -α3.7 deletion was present only in the mother of the Family B proband. n n n nTable 1. n nHematologic data and genotype of the patients and their parents. n n n nIn Family A, MLPA analysis, made using MLPA kit (HBA140-B3 MCR-Holland), revealed a deletion of at least 7535 bp beginning in the region between α1-pseudo-globin gene and α2-globin gene and extending to 2.4 kb downstream of α1-globin gene in the proband, in her sister and in their mother. Sequencing analysis of an ~500 bp breakpoint fragment, obtained using specific primers around MLPA deleted probes, allowed us to define the exact deletion breakpoint at position 161276/7 (5′) and 170485/6 (3′). This deletion removed a region of 9209 nt involving both α-globin genes and part of the first exon of the θ gene. In addition, an insertion of six nucleotides (ATTAGT) at position 161216 before the 5′ breakpoint was detected. No orphan sequence was found. The 5′ breakpoint is shifted 2 nt up and the 3′ breakpoint is shifted 1032 nt down, as compared to the breakpoints of the α0 thalassemia deletion found in a recently reported Dutch family.6 n nIn Family B, MLPA analysis revealed a larger deletion which removes all the MLPA probes specific for the sub-telomeric region, including an α-globin gene cluster with all regulatory elements, in the proband and in her father. CGH-array analysis with oligonucleotides (8x60K Agilent Technologies) and SNP genotyping allowed us to define the 3′ breakpoint between the 4th exon of the NME4 gene and the IVSII of the DECR2 gene (389660 and 395647 coordinates) (Figure 1). n n n nFigure 1. n nSchematic representation of the short arm of chromosome 16 (16p13.3) and of the α0 deletions in the two families. The α-globin regulatory region (MCS-R 1 to 4) is indicated as black dots. Black bars represent deleted DNA regions. White ... n n n nThe greater severity of the α2NcoI non-deletion defect as compared to the -α3.7 deletion in trans to α0 deletions, could be the reason for the different phenotypes in the HbH patients of the two families.2 The different size of alpha0 deletions and the loss of genes located in the deleted region in Family B do not seem to interfere in the determination of specific phenotype. n nSeveral large deletions involving the α-globin gene cluster have been recently described.7–10 Although these deletions also remove other genes, affected heterozygotes appear phenotypically normal, apart from α-thalassemia carrier phenotype; however, an HbH patient with a telomeric deletion of ~285 kb associated with the common -α3.7 deletion in trans presented scoliosis, the severity of which remains unexplained.8 A region on chromosome 16p for which haploinsufficiency leads to mental retardation typical of ATR16 has been narrowed down to a region ~0.9 and 1.5–1.7 Mb from telomere. Alu-family repeats, frequent in the genome and particularly common in and around the α-globin gene cluster, facilitate DNA strand exchanges during replication and non-homologous recombinations which are a frequent cause of α0 deletions.9–11 In addition to the common conventional molecular techniques, recent alternative methods, such as MLPA and CGH, become essential for a correct α-globin genotype definition. The exact identification of uncommon and unknown alpha deletion defects, although rare, allows appropriate genetic counselling to be offered to couples at risk for HbH disease or hemoglobin Bart’s hydrops fetalis syndrome, especially in Sardinia were small isolated communities at risk can still be found.

A new glucose 6 phosphate dehydrogenase variant, G6PD Sinnai (34 G→T)

In this paper we report a male infant heterozygous for thalassemia with a mild glucose 6 phosphate dehydrogenase deficiency. The molecular basis of this new Class III G6PD variant is a G→T mutation at nucleotide 34 in the exon 2, which predicts a Val→Leu amino acid substitution at codon 12. We designated this variant as G6PD Sinnai from the place of birth of the propositus. Hum Mutat 12:72–73, 1998.

Two novel mutations (10410 T→G; 10296 del C) at carboxy‐terminus of the dystrophin gene associated with mental retardation

As part of a search for causative genes of familial pancreatic carcinoma, the p16 genes were sequenced in members of 21 families with a phenotype of familial pancreatic carcinoma (2 or more first degree relatives affected). One family was found in which members carried a novel p16 allele with a G to T transversion at position 451, creating a missense amino acid change at codon 145 (Asp to Cys) and possibly disrupting the donor splice site of the exon 2/3 boundary. This coding change is not a known polymorphism, and occurs at a codon position in which another missese/splicing change has been shown to be linked to familial melanoma/pancreas cancer.In this study we have carried out a mutational screening of exons 62‐79 of the dystrophin gene by SSCP in 38 italian patients with DMD/BMD and found two novel mutations at exon 70, in 2 mentally retarded DMD patients. Hum Mutat 12:70, 1998.

A new glucose 6 phosphate dehydrogenase variant G6PD Sinnai (34 G-->T). Mutations in brief no. 156. Online.

In this paper we report a male infant heterozygous for thalassemia with a mild glucose 6 phosphate dehydrogenase deficiency. The molecular basis of this new Class III G6PD variant is a G→T mutation at nucleotide 34 in the exon 2, which predicts a Val→Leu amino acid substitution at codon 12. We designated this variant as G6PD Sinnai from the place of birth of the propositus. Hum Mutat 12:72–73, 1998.