Use of synthetic oligonucleotides for genomic DNA dot hybridization to split the DQw3 haplotype.

Comparison of two different HLA-DQ beta gene sequences from two DR4 individuals, probably corresponding to DQw3.2 (DQR4) and DQw3.1 (DQR5) specificities, has shown several nucleotide variations. Eight oligonucleotides (24 bases long), derived from these polymorphic areas, have been synthesized. Each oligonucleotide was hybridized to BamHI-digested DNA samples from eight families with HLA-DR4 individuals. Four polymorphic BamHI fragments were detected. Two of eight oligonucleotides gave a single signal (8.9 kilobases) on DQw3.2-positive haplotypes. We used one of these oligonucleotides in a genomic DNA dot hybridization and detected a hybridization signal only in DQw3.2-positive individuals. A very simple test like this allows the screening of a large population sample within a very short period.     

HLA-G gene polymorphism segregation within CEPH reference families

HLA-G, a nonclassical HLA class I antigen, presents tissue-restricted expression on human trophoblasts and may play an important role in immune tolerance of mother-versus-fetus. In this work we have demonstrated extensive HLA-G genomic polymorphism within three CEPH reference families, by PCR-SSCP analysis and direct sequencing. Among six unrelated parents we assigned eight HLA-G alleles, seven of which are new. We observed the segregation of HLA-G alleles of heterozygous parents among their offspring that matched the segregation of the HLA class I haplotypes. Only one of the mutations observed was found to be nonsynonymous indicating low polymorphism of the HLA-G molecule.     

Idiopathic Hemochromatosis: Linkage with HLA

Forty-eight unrelated patients with idiopathic hemochromatosis were found to have a significantly higher frequency of three HLA antigens (A3, B7 and B14) than 591 healthy controls. A significant association between HLA haplotypes and disease segregations was demonstrated in 14 family studies. A recessive inheritance of a strongly A3-linked disease gene responsible for abnormal iron stores in the heterozygote state is postulated. The lod score value (4.415 for θ= 0.025) is compatible with this hypothesis. However, the excess of HLA-identical pairs of affected sibs does not exclude the possibility of a pseudo-recessiveness due to two codominant genes both HLA-linked. For the first time, a means of screening for high risk subjects is available and therefore offers the possibility of a preventive approach.     

In vivo repopulation ability of genetically corrected bone marrow cells from Fanconi anemia patients

Fanconi anemia (FA) is a rare inherited genomic instability syndrome representing one of the best examples of hematopoietic stem cell deficiency. Although FA might be an excellent candidate for bone marrow (BM) genetic correction ex vivo, knockout animal models are not sufficient to guide preclinical steps, and gene therapy attempts have proven disappointing so far. Contributing to these poor results is a characteristic and dramatic early BM-cells die-off when placed in culture. We show here that human primary FA BM cell survival can be ameliorated by using specific culture conditions that limit oxidative stress. When coupled with retrovirus-mediated transfer of the main complementation group FANCA-cDNA, we could achieve long-term reconstitution of the stem cell compartment both in vitro and in vivo. Gene-corrected BM cultures grew for >120 days, and after cultured cell transplantation into NOD/SCID mice, clonogenic human cells carrying the FANCA transgene could be detected 6 months after transduction. By comparison, untransduced cells died in culture by 15 days. Of necessity for ethical reasons, experiments were conducted on a very limited number of primary BM cells. By using low cytokine regimen and conditions matching regulatory requirements, a contingent of gene-corrected cells slowly emerges with an unmet potential for in vivo engraftment. Future therapeutic applications of stem cells might be expanding from these data. In addition, we provide a model of gene-corrected human primary cell growth that carries the potential to better delineate the combined role of both DNA damage and oxidative stress in the pathogenesis of FA.     

Maternal traces of deep common ancestry and asymmetric gene flow between Pygmy hunter-gatherers and Bantu-speaking farmers

Two groups of populations with completely different lifestyles-the Pygmy hunter-gatherers and the Bantu-speaking farmers-coexist in Central Africa. We investigated the origins of these two groups and the interactions between them, by analyzing mtDNA variation in 1,404 individuals from 20 farming populations and 9 Pygmy populations from Central Africa, with the aim of shedding light on one of the most fascinating cultural transitions in human evolution (the transition from hunting and gathering to agriculture). Our data indicate that this region was colonized gradually, with an initial L1c-rich ancestral population ultimately giving rise to current-day farmers, who display various L1c clades, and to Pygmies, in whom L1c1a is the only surviving clade. Detailed phylogenetic analysis of complete mtDNA sequences for L1c1a showed this clade to be autochthonous to Central Africa, with its most recent branches shared between farmers and Pygmies. Coalescence analyses revealed that these two groups arose through a complex evolutionary process characterized by (i) initial divergence of the ancestors of contemporary Pygmies from an ancestral Central African population no more than approximately 70,000 years ago, (ii) a period of isolation between the two groups, accounting for their phenotypic differences, (iii) long-standing asymmetric maternal gene flow from Pygmies to the ancestors of the farming populations, beginning no more than approximately 40,000 years ago and persisting until a few thousand years ago, and (iv) enrichment of the maternal gene pool of the ancestors of the farming populations by the arrival and/or subsequent demographic expansion of L0a, L2, and L3 carriers.     

Subsets of malignant lymphomas in children related to the cell phenotype.

We studied the lymphomatous cells of 39 children presenting with the classical features of malignant lymphoma. Twenty-two had T lymphoblasts. We could classify these patients into three subsets: The T lymphoblasts from children group 1 displayed antigen(s) shared by a thymocyte subpopulation, had terminal deoxynucleotidyl transferase (TDT), but no affinity for peanut agglutinin (PNA). The T lymphoblasts from children group 2 lacked the thymocyte antigen(s), had no TDT, but showed affinity for PNA. The T lymphoblasts from children group 3 displayed mature T-cell antigens, had no TDT, and no affinity for PNA. Children from the three groups were similar in terms of clinical presentation, age and sex distribution, and cell morphology; however patients from the three groups might have a different prognosis. Fourteen children had B lymphoblasts that, in half of the cases, had affinity for Helix pomatia agglutinin. Three patients had lymphoblasts lacking specific marker. Two of them had cells displaying an antigen found on common acute lymphoblastic leukemia cells and had TDT.