L. Felicetti

Physical mapping evidence for a duplicated region on chromosome 10qter showing high homology with the facioscapulohumeral muscular dystrophy locus on chromosome 4qter.

Abstractp13E-11, a probe (D4F104S1 locus) derived from chromosome 4q35, detects -EcoRI-rearranged fragments less than 28 kb in both sporadic and familial cases of facioscapulohumeral muscular dystrophy (FSHD). These fragments are smaller than those observed in healthy individuals. The interpretation of Southern blots is complicated by the fact that pl3E-11 reveals two pairs of polymorphic alleles, one 4q35-specific and the other unlinked to 4q35, that sometimes overlap each other. We cloned a non-4q35 13-kb fragment not related to the disease from a sporadic FSHD patient of Italian origin. Haplotype analysis and in situ hybridization experiments showed that this fragment was located on the 10qter region. Restriction mapping of the 10qter clone, when compared with the 4q35 fragment, indicates a similar arrangement of KpnI tandemly repeated units and flanking sequences. However 4q35 and 10q26 EcoRI clones can be distinguished by restriction analysis with SfiI and SfyI. This observation could be exploited for future applications in the field of molecular diagnosis and genetic counseling. In addition the isolation of two 10q26 cosmid clones (D10S1484 and D10S1485) from a human genomic library and the construction of a detailed physical map, spanning about 40 kb, showed that the structural homology extended upstream of the EcoRI sites, suggesting that a duplicated FSHD locus resided in the subtelomeric region of the long arm of chromosome 10. We cannot exclude the involvement of the duplicated locus in the molecular mechanism of the disease and in the genetic heterogeneity of FSHD syndromes.

Sequence Homology between 4qter and 10qter Loci Facilitates the Instability of Subtelomeric KpnI Repeat Units Implicated in Facioscapulohumeral Muscular Dystrophy

Physical mapping and in situ hybridization experiments have shown that a duplicated locus with a structural organization similar to that of the 4q35 locus implicated in facioscapulohumeral muscular dystrophy is present in the subtelomeric portion of 10q. We performed sequence analysis of the p13E-11 probe and of the adjacent KpnI tandem-repeat unit derived from a 10qter cosmid clone and compared our results with those published, by other laboratories, for the 4q35 region. We found that the sequence homology range is 98%-100% and confirmed that the only difference that can be exploited for differentiation of the 10qter from the 4q35 alleles is the presence of an additional BlnI site within the 10qter KpnI repeat unit. In addition, we observed that the high degree of sequence homology does facilitate interchromosomal exchanges resulting in displacement of the whole set of BlnI-resistant or BlnI-sensitive KpnI repeats from one chromosome to the other. However, partial translocations escape detection if the latter simply relies on the hybridization pattern from double digestion with EcoRI/BlnI and with p13E-11 as a probe. We discovered that the restriction enzyme Tru9I cuts at both ends of the array of KpnI repeats of different chromosomal origins and allows the use of cloned KpnI sequences as a probe by eliminating other spurious fragments. This approach coupled with BlnI digestion permitted us to investigate the structural organization of BlnI-resistant and BlnI-sensitive units within translocated chromosomes of 4q35 and 10q26 origin. A priori, the possibility that partial translocations could play a role in the molecular mechanism of the disease cannot be excluded.

Molecular characterization of β-thalassemia mutations in Egypt

SummaryThe relative frequency of different β-thalassemia mutations and their association with β-globin haplotypes were studied in patients from the Nile delta region, Egypt, by means of the polymerase chain reaction, oligonucleotide hybridization and restriction analysis. We found that 8 mutations account for 77% of β-thalassemia chromosomes in this population, the commonest being IVS-1 nt 110, IVS-1 nt 6 and IVS-1 nt 1. Each mutation was associated with a specific haplotype, with the exception of IVS-1 nt 110, found on 3 different chromosomal backgrounds. Our data show that testing for the 8 detectable mutations makes feasible prenatal diagnosis in 65% of at risk couples and exclusion testing in an additional 25% of cases.

A case of hereditary persistence of fetal hemoglobin caused by a gene not linked to the β-globin cluster

The pattern of inheritance of several polymorphic restriction sites associated with the β-gene cluster, and spanning a region of 52 kb, demonstrates that a determinant for hereditary persistence of fetal hemoglobin (HPFH) segregates independently from the non-α globin gene cluster, as we postulated several years ago on purely genetical grounds. This finding provides additional evidence for the existence of diffusible factors affecting γ-chain expression. Moreover, we have identified a “private” HinccII polymorphism, in the vicinity of the ɛ gene in the family studied.SummaryThe pattern of inheritance of several polymorphic restriction sites associated with the β-gene cluster, and spanning a region of 52 kb, demonstrates that a determinant for hereditary persistence of fetal hemoglobin (HPFH) segregates independently from the non-α globin gene cluster, as we postulated several years ago on purely genetical grounds. This finding provides additional evidence for the existence of diffusible factors affecting γ-chain expression. Moreover, we have identified a “private” HinccII polymorphism, in the vicinity of the ɛ gene in the family studied.

A New β-Thalassemia Mutation Produced by a Single Nucleotide Substitution in the Conserved Dinucleotide Sequence of the IVS-I Consensus Acceptor Site (Ag→AA)

An Egyptian child with thalassemia major was found to carry two different haplotypes (I and VI) associated with two beta-thalassemic chromosomes. Analysis with several oligonucleotides and restriction enzymes, which identify the mutations most common in the Mediterranean area, allowed the identification of only one mutation, namely T----C at position 6 of the first intervening sequence (IVS-I). In order to characterize the other mutation the beta gene was amplified with polymerase chain reaction and sequenced. A G----A substitution was found at position 130 of the IVS-I which alters the conserved dinucleotide AG present in the consensus acceptor sequence, thus producing a beta (0)-thalassemia. This mutation was further confirmed by restriction analysis since it creates a new restriction site for the enzyme Afl II. It is concluded that this subject carries the IVS-I-6 mutation associated with haplotype VI, frequently observed in Mediterranean areas, and a new mutation at the acceptor site of the IVS-I, which has not been described before, associated with haplotype I. This thalassemic gene can be added to the list of mutations that can be identified by Southern analysis using Afl II.

Frequency and molecular types of deletional α-thalassemia in Egypt

SummaryThe frequency of deletional α-thalassemia in the Egyptian population was estimated at 0.08 by DNA analysis of a newborn random sample. No α0 determinants were found. The most frequent α+ determinant was the −α3.7 type I in association with the medium allele at inter-zeta HVR. The −α4.2 and αααanti 3.7 arrangements were found at very low frequencies.

Frequency and types of deletional α+ in Northern Sardinia

SummaryWe determined by restriction mapping the frequency of the −α3.7 determinant in a random sample of 48 adults in Northern Sardinia. We found a frequency of 0.18±0.04 and demonstrated that only type I crossover as determined by Apa I digestion (Higgs et al. 1984) is present. Moreover, we showed that this haplotype is not associated with an Rsa I polymorphism 5′ to the α2-globin gene. These data support the hypothesis of a unique origin of this deletion in Sardinia.

Molecular characterization of HbH disease in the Cuban population

SummaryMolecular characterization of the α-thalassemia mutations present in nine HbH subjects from Cuba was achieved by digestion with Bam HI, Bgl II, and Apa I and hybridization with α- and ζ-specific probes. The results show that the molecular basis of the genetic defect is quite homogeneous, all the subjects carrying the −α3.7 type I/--SEA genotype. Variations are observed in the size of the ζ polymorphic fragments.

Differential Segregation of β+ IVS‐I‐110 (G→A), Aγ − 117 (G→A), and Gγ − 158 (C→T) Mutations in Members of an Albanian Family

In the past few decades, many varieties ( 20) of point mutations in the promoters of the gor g-globin genes that produce an overexpression of fetal hemoglobin (Hb) in adults, have been identified at the molecular level (1,2). The co-inheritance in cis of the g 196 (C!T) with the b codon 39 (C!T) mutations, the most frequent b-thalassemia (thal) in Sardinia, was shown to be the genetic basis for the Sardinian db-thal (3,4). Another point mutation, the g 158 (C!T), if linked to particular b-globin cluster haplotypes, attenuates the clinical phenotype of sickle cell disease by increasing production of Hb F (5–7). The same mutation ameliorates the clinical picture of b-thal homozygote patients resulting in b-thal intermedia (8,9). Several cases have been described of association in trans of g-globin promoter mutations and b-thal. Here we report on a family of Albanian descent, in which two mutations of the g-globin gene promoters [g 158 (C!T) and g 117 (G!A)] are associated in trans with a bþ-thal defect [IVS-I-110 (G!A)], that result in increased levels of Hb F (Fig. 1).