Mutation of a nuclear succinate dehydrogenase gene results in mitochondrial respiratory chain deficiency.

We now report a mutation in the nuclear-encoded flavoprotein (Fp) subunit gene of the succinate dehydrogenase (SDH) in two siblings with complex II deficiency presenting as Leigh syndrome. Both patients were homozygous for an Arg554Trp substitution in the Fp subunit. Their parents (first cousins) were heterozygous for the mutation that occurred in a conserved domain of the protein and was absent from 120 controls. The deleterious effect of the Arg to Trp substitution on the catalytic activity of SDH was observed in a SDH- yeast strain transformed with mutant Fp cDNA. The Fp subunit gene is duplicated in the human genome (3q29; 5p15), with only the gene on chromosome 5 expressed in human-hamster somatic cell hybrids. This is the first report of a nuclear gene mutation causing a mitochondrial respiratory chain deficiency in humans.

LAG-3, a novel lymphocyte activation gene closely related to CD4.

We have identified a novel human gene of the Ig superfamily, designated LAG-3. Expression of this gene is undetectable in resting PBL, while it is found (a 2-kb message) in activated T and NK cells. The LAG-3 gene includes eight exons; the corresponding cDNA encodes a 498-amino acid membrane protein with four extracellular IgSF domains. The first one belongs to the V-SET; it is particular since it includes an extra loop in the middle of the domain and an unusual intrachain disulphide bridge. The three other domains belong to the C2-SET. Strong internal homologies are found in the LAG-3 molecule between domains 1 and 3, as well as between domains 2 and 4. It is therefore likely that LAG-3 has evolved by duplication of a pre-existing gene encoding a two IgSF-domain structure. The compared analysis of LAG-3 and CD4, with respect to both their peptidic sequence as well as their exon/intron organization, indicated that the two molecules are closely related. This point is strengthened by the finding that both genes are located on the distal part of the short arm of chromosome 12.

Delayed and incomplete reprogramming of chromosome methylation patterns in bovine cloned embryos.

Full-term development has now been achieved in several mammalian species by transfer of somatic nuclei into enucleated oocytes [1, 2]. Although a high proportion of such reconstructed embryos can evolve until the blastocyst stage, only a few percent develop into live offspring, which often exhibit developmental abnormalities [3, 4]. Regulatory epigenetic markers such as DNA methylation are imposed on embryonic cells as normal development proceeds, creating differentiated cell states. Cloned embryos require the erasure of their somatic epigenetic markers so as to regain a totipotent state [5]. Here we report on differences in the dynamics of chromosome methylation between cloned and normal bovine embryos before implantation. We show that cloned embryos fail to reproduce distinguishable parental-chromosome methylation patterns after fusion and maintain their somatic pattern during subsequent stages, mainly by a highly reduced efficiency of the passive demethylation process. Surprisingly, chromosomes appear constantly undermethylated on euchromatin in morulae and blastocysts, while centromeric heterochromatin remains more methylated than that of normal embryos. We propose that the abnormal time-dependent methylation events spanning the preimplantation development of clones may significantly interfere with the epigenetic reprogramming, contributing to the high incidence of physiological anomalies occurring later during pregnancy or after clone birth.

The epigenetic imprinting defect of patients with Beckwith-Wiedemann syndrome born after assisted reproductive technology is not restricted to the 11p15 region.

BACKGROUND: Genomic imprinting refers to an epigenetic marking resulting in monoallelic gene expression and has a critical role in fetal development. Various imprinting diseases have recently been reported in humans and animals born after the use of assisted reproductive technology (ART). All the epimutations implicated involve a loss of methylation of the maternal allele (demethylation of KvDMR1/KCNQ1OT1 in Beckwith-Wiedemann syndrome (BWS), demethylation of SNRPN in Angelman syndrome and demethylation of DMR2/IGF2R in large offspring syndrome), suggesting that ART impairs the acquisition or maintenance of methylation marks on maternal imprinted genes. However, it is unknown whether this epigenetic imprinting error is random or restricted to a specific imprinted domain. AIM: To analyse the methylation status of various imprinted genes (IGF2R gene at 6q26, PEG1/MEST at 7q32, KCNQ1OT1 and H19 at 11p15.5, and SNRPN at 15q11-13) in 40 patients with BWS showing a loss of methylation at KCNQ1OT1 (11 patients with BWS born after the use of ART and 29 patients with BWS conceived naturally). RESULTS: 3 of the 11 (27%) patients conceived using ART and 7 of the 29 (24%) patients conceived normally displayed an abnormal methylation at a locus other than KCNQ1OT1. CONCLUSIONS: Some patients with BWS show abnormal methylation at loci other than the 11p15 region, and the involvement of other loci is not restricted to patients with BWS born after ART was used. Moreover, the mosaic distribution of epimutations suggests that imprinting is lost after fertilisation owing to a failure to maintain methylation marks during pre-implantation development.

C-myb proto-oncogene: evidence for intermolecular recombination of coding sequences.

We have characterized a novel chicken c-myb exon whose sequences are specifically expressed in thymic cells. In situ hybridization experiments indicate that this thymus-specific coding exon is localized on a small chromosome, distinct from the large acrocentric chromosome 3 on which we recently mapped the bulk of 15 exons, common to the c-myb mRNA species expressed in hematopoietic cells of both B and T lineages. These observations indicate that intermolecular recombination is required for the tissue-specific expression of the c-myb proto-oncogene. We also show that these thymus-specific sequences are conserved in human DNA and lie on chromosome 17q25, whereas the human c-myb locus is localized on chromosome 6q22-23. Sequencing data obtained from genomic DNA and PCR analyses performed with c-myb mRNA species expressed in chicken thymic cells strongly suggest that a repeated decameric sequence plays a key role in the recombination process.

Co-amplification of transcriptionally active epidermal growth factor receptor and ribosomal genes in the human hepatoma cell line Li7A.

A high level of expression of the functional product of the epidermal growth factor receptor (EGFR) gene was detected in the human hepatocarcinoma cell line Li7A and it was found to correlate with gene amplification. The karyotype was paratriploid, with 15 rearranged chromosomes, several of which contained abnormally banded regions (ABRs). The search for DNA sequences co-amplified with the EGFR gene, using the in-gel renaturation technique, allowed us to detect an amplified DNA band (La1) of about 30 kb. This DNA was used as a probe for in situ hybridization on chromosomes, to locate the amplified segment. In normal lymphocytes, the DNA of band La1 hybridized to chromosome regions in which repetitive DNAs are located, i.e. on juxtacentromeric regions, the site of alphoid and CCATT satellite DNA, and on the short arms of acrocentrics, the site of ribosomal RNA (RNR) genes. In Li7A cells, it hybridized to the same regions and, in addition, to several chromosome arms corresponding to ABRs. The same ABRs hybridized to EGFR and RNR probes, but neither Alu sequences nor various probes for other repetitive sequences were recognized. They also exhibited nucleolus organizer region staining characterizing functionally active (RNR) genes. It was concluded that transcriptionally active genes were co-amplified in the same ABRs, although they originated from different chromosomes, i.e. chromosome 7 for EGFR and acrocentrics for RNR genes.

Physical mapping of human loci homologous to the chicken nov proto-oncogene.

The human locus (novH) corresponding to the nov protooncogene overexpressed in avian nephroblastoma has been identified and mapped on chromosome 8q24.1. Another locus sharing homology with novH and corresponding to the connective tissue growth factor (CTGF) gene has also been mapped on chromosome 6q23.1. The chromosomal assignment of nov and CTGF proximal to c-myc and c-myb respectively is of interest because chromosomal abnormalities involving these regions have been associated with different human tumors including Wilms'.

DNMT3B mutations and DNA methylation defect define two types of ICF syndrome.

ICF syndrome is a rare autosomal recessive disease characterized by variable immunodeficiency, centromeric instability, and facial abnormalities. Mutations in the catalytic domain of DNMT3B, a gene encoding a de novo DNA methyltransferase, have been recognized in a subset of patients. ICF syndrome is a genetic disease directly related to a genomic methylation defect that mainly affects classical satellites 2 and 3, both components of constitutive heterochromatin. The variable incidence of DNMT3B mutations and the differential methylation defect of alpha satellites allow the identification of two types of patients, both showing an undermethylation of classical satellite DNA. This classification illustrates the specificity of the methylation process and raises questions about the genetic heterogeneity of the ICF syndrome.

Cloning and expression of a lymphocyte activation gene (LAG-1).

Using subtractive hybridization of a cDNA library we have identified a human gene, termed LAG-1 (for "Lymphocyte Activation Gene-1"). This cDNA codes for a 69 amino-acid polypeptide which belongs to a new class of recently described proteins secreted by activated lymphocytes and/or monocytes. The LAG-1 gene was cloned, sequenced and its chromosomal location assigned to chromosome 17 (q21 band). The promoter region of the LAG-1 gene was shown to include a GM-CSF-like decanucleotide sequence. Using a baculovirus vector expression system, we found that a 10 kDa recombinant LAG-1 protein is secreted by AcNPV infected SF9 cells, as determined in Western blot experiments by the reactivity of specific anti-peptidic heteroantibodies. Finally the natural LAG-1 protein was precipitated from the supernatant of internally labeled activated Nk cells and shown to migrate as a single entity of 14 kDa in SDS-PAGE analysis.

cDNA cloning, tissue distribution and chromosomal localization of the human ID4 gene.

A cDNA e encoding the human Id4 protein has been isolated from an astrocytoma library. The predicted protein product shares 98% identity with the mouse Id4 protein and is markedly different from that already reported. By FISH analysis, the human ID4 gene was more precisely mapped to chromosome 6p22.3-p23. Northern blot analysis showed that ID4 is mainly expressed in thyroid, brain and fetal tissue and in some nervous system tumor cell lines.

Chromosomal reallocation of the chicken c-myb locus and organization of 3'-proximal coding exons.

In the course of our studies concerning the tissue-specific expression of the c-myb proto-oncogene, we have established the nucleotide sequence of the chicken c-myb 3'-proximal coding exons. In situ hybridization performed with different genomic DNA probes corresponding to nearly all the c-myb gene allowed us to localize the corresponding locus on the large acrocentric chromosome 3 in chicken. Our sequencing data also indicate that the 3'-proximal noncoding sequences represented in c-myb mRNA species are derived from non-contiguous exons.

Construction and characterization of a partial library of yeast artificial chromosomes from human chromosome 21.

We report a protocol for cloning large DNA fragments in yeast artificial chromosomes (YAC). A partial library has been constructed from a somatic hybrid containing chromosome 21 as the single source of human DNA. About 4.0 Mb of human DNA was recovered in 17 YAC clones. Three clones were analyzed by in situ hybridization and mapped on chromosome 21. One clone hybridized with the chromosome 21 centromeric region and may provide new insight both on the molecular structure of centromere and on the localization of Alzheimer disease gene.

Cytogenetic study of five cases of lung adenosquamous carcinomas.

The cytogenetic study of five cases of untreated adenosquamous carcinoma of the lung allows us to propose a number of characteristic anomalies. All tumor cells were hyperdiploid, with a mean chromosome number ranging from 59 to 83, and had many clonal chromosome rearrangements. The chromosomes the most frequently affected by these rearrangements were, by decreasing order, 1, 3, and 15; 7 and 8; and 17. No recurrent breakpoints were observed in euchromatic regions, most breaks (45/66) involving juxtacentromeric heterochromatin or immediately adjacent regions. Although chromosome 3 was frequently rearranged, no recurrent deletions of its short arm were observed.

Recurrent chromosome aberrations in human lung squamous cell carcinomas.

Cytogenetic study of seven cases of previously untreated lung squamous cell carcinomas (SQC) is reported. Chromosome numbers vary from 38 to 538, with a majority of hypotriploid karyotypes with complex rearrangements. The numbers of recurrent imbalances were evaluated in considering the average number of chromosomes or chromosome segments in each analyzed metaphase and for each case. In decreasing order of frequency, deficiencies for 3p, 5q, 8p, Y, 5p, 10p, 13, and, to a lesser degree, for 8q, 9, 10q, 11pter, 14, 15, and 21 were observed; the excesses principally involve 1q, 3q, and 7q. In three tumors, homogeneously staining regions were observed at various chromosome sites. Most chromosome rearrangements occurred after breakage in constitutive heterochromatin, and no recurrent breakpoints were found in euchromatin except 11p15. The major consequences of these anomalies may be chromosomal imbalances, leading to hemizygosity and perhaps related to gene dosage, rather than to alterations of genes.

Recurrent homogeneously staining regions in 8p1 in breast cancer and lack of amplification of POLB, LHRH, and PLAT genes.

In a cytogenetic study of 125 primary and untreated breast cancers, 107 were selected for the quality of their metaphases permitting detection of amplifications:homogeneously staining regions (HSRs), abnormally banded region (ABRs), and double minutes (dmins). HSRs and ABRs were detected in 62 cases (58%), but no cases of dmins were observed. The localizations of HSRs and ABRs were not random because they were observed in the 8p1 position in 14 cases. The possible amplifications of five sequences, MOS (8q1), LHRH (8p21.1), POLB (8p11.2), PLAT (8p12), and D8Z2 (8c) were investigated in three tumors with HSR on the short arm of chromosome 8. Because these sequences were not amplified, two interpretations can be proposed: 1) there is a frequent amplification of a sequence from the 8p1 region, located between the investigated sequences; and 2) the amplifications do not occur in 8p1, but HSRs or ABRs of undetermined origin have a strong tendency to be translocated onto 8p. Because cases with HSR(8p) have less complex karyotypes than with HSRs in other locations, the first interpretation is the most likely: HSRs may be formed in 8p and further translocated on other chromosomes in the course of tumor progression.

Detection of translocations of 10p by non-radioactive in situ hybridization of VIM gene in SV40-transformed human cell lines.

SV40-transformed human fibroblasts exhibit characteristic chromosome imbalances, fairly well correlated with the activity of enzymes encoded by genes located on chromosome segments either in deficiency or in excess. However, a major discrepancy existed for the expression of vimentin gene (VIM), which was high, even though the map location of the gene (10p) was missing in many cell lines. An in situ hybridization technique using a biotinylated probe for the human VIM was applied to detect eventual cryptic translocations, as chromosome 10p is difficult to identify. In two cell lines (WI 98 and HEL1 HBLT) in which a loss of copy number of 10p was assumed after karyotyping, a signal for VIM was detected in unidentified short arms of derivative chromosomes. This exemplifies that in situ hybridization is a powerful complement to classical cytogenetics to detect rearrangements in highly rearranged karyotypes from transformed or cancerous cells. These results also strengthen the interpretation of the correlation between karyotypic and metabolic imbalances in transformed cells.

Distribution of the various radiation-induced chromosomal rearrangements in relation to the dose and sampling time.

The quantitative analysis of the chromosome rearrangements detected in 2128 R-banded metaphases, obtained from gamma-irradiated human lymphocytes after 48 to 96 h in culture is reported. Depending on the culture time, and possibly on the dose of radiation (from 1 to 3 Gy), the most frequent type of rearrangement was either dicentrics or reciprocal translocations. In first generation mitoses, the frequency of cells without rearrangement ranged from 0.66 to 0.18, and the mean number of rearranged chromosomes per cell from 0.79 to 3.28. The dose-response curve follows a quadratic function for dicentric aberration yields, but not for other rearrangements.

Identification and chromosomal localization of a DNA fragment implicated in the partial correction of the Fanconi anemia group D cellular defect.

Fanconi anemia (FA) cells, complementation group D, which had been transfected with mouse genomic DNA were partially corrected for their mitomycin C (MMC) hypersensitivity. A genomic DNA fragment which complements the resistance of FA(D) cells to MMC close to normal level has been cloned; it has no correcting activity in FA group A cells. It contains two highly conserved regions between the mouse and human genome, which flank mouse repeated DNA. This DNA fragment detects a 3.6-4-kb mRNA transcript in human cells. Moreover this fragment maps to chromosome 11q23, a region of particular interest since several genes involved in the control of major cellular functions are located in this area. This DNA fragment may belong to a gene directly or indirectly involved in FA(D) function.

Risk of chromosomal disease due to radiation. Tentative estimate from the study of radiation-induced translocations in human fibroblasts.

A sample of 214 reciprocal 2-break translocations observed in fibroblasts, both after accidental 'in vivo', and experimental 'in vitro' gamma-irradiation, was studied. The distribution of the breaks along the chromosomes does not seem at random. The minimal possible imbalance that these translocations could induce by malsegregation, if they existed in germ cells, was estimated. These imbalances were compared with the chromosomal trisomies and monosomies known to be compatible with life after birth in man. It is concluded that about 2/5 of the radiation-induced translocations might induce a viable trisomy and/or monosomy. This result, similar to that previously obtained in human lymphocytes, indicates the validity of the extrapolation from one tissue to another, and hopefully to germ cells.

Tentative estimate of the risk of chromosomal disease due to radiation-induced translocations in man.

An attempt to estimate one of the parameters establishing the risk of occurrence of abnormal live-born progeny by malsegregation of radiation-induced translocation is reported. A sample of 247 2-break translocations induced by gamma-rays in human lymphocytes was studied in relation to the minimal possible imbalance they could induce in gametogenesis. These imbalances were compared with chromosomal trisomies and monosomies known to be compatible with life after birth in man. It is concluded that at least 106 out of 247 translocations should not give viable products in cases of malsegregation. A second comparison, with translocations ascertained in human subjects for various reasons, led to the conclusion that about 2/5 of the radiation-induced translocations might involve a risk of partial trisomies or monosomies. Cell survival and frequency of meiotic malsegregations are other parameters needed to make a correct estimate. A short discussion shows the difficulty of such estimates from inter-specific comparisons.