Holt-Oram syndrome is a genetically heterogeneous disease with one locus mapping to human chromosome 12q.

Holt-Oram syndrome (HOS) is an autosomal dominant condition affecting the heart and upper limbs. We have sought to identify the location of this gene using microsatellite DNA markers in a linkage study. Of seven families analysed, five show linkage between HOS and markers on chromosome 12q. But the two remaining families, phenotypically indistinguishable from the others, do not show this linkage. Analysis with the computer program HOMOG indicates that HOS is a heterogeneous disease. Our analysis places one HOS locus in a 21 cM interval in the distal region of chromosome 12q. The localization of a gene for HOS, reported here, represents an important step towards a better understanding of limb and cardiac development.

Holt-Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family.

Holt-Oram syndrome is a developmental disorder affecting the heart and upper limb, the gene for which was mapped to chromosome 12 two years ago. We have now identified a gene for this disorder (HOS1). The gene (TBX5) is a member of the Brachyury (T) family corresponding to the mouse Tbx5 gene. We have identified six mutations, three in HOS families and three in sporadic HOS cases. Each of the mutations introduces a premature stop codon in the TBX5 gene product. Tissue in situ hybridization studies on human embryos from days 26 to 52 of gestation reveal expression of TBX5 in heart and limb, consistent with a role in human embryonic development.

T-Box Genes in Human Development and Disease.

T-box genes are important development regulators in vertebrates with specific patterns of expression and precise roles during embryogenesis. They encode transcription factors that regulate gene transcription, often in the early stages of development. The hallmark of this family of proteins is the presence of a conserved DNA binding motif, the "T-domain." Mutations in T-box genes can cause developmental disorders in humans, mostly due to functional deficiency of the relevant proteins. Recent studies have also highlighted the role of some T-box genes in cancer and in cardiomyopathy, extending their role in human disease. In this review, we focus on ten T-box genes with a special emphasis on their roles in human disease.

In vivo co-localisation of MBNL protein with DMPK expanded-repeat transcripts.

Myotonic dystrophy (DM1) is the most common form of adult muscular dystrophy and is inherited as an autosomal dominant trait. The genetic basis of DM1 is the expansion of a CTG repeat in the 3' untranslated region of a protein kinase gene (DMPK). The molecular mechanism by which this expanded repeat produces the pathophysiology of DM1 remains unknown. Transcripts from the expanded allele accumulate as foci in the nucleus of DM1 cells and it has been suggested that these transcript foci sequester cellular proteins that are required for normal nuclear function. We have investigated the role of three RNA-binding proteins, CUG-BP, hnRNP C and MBNL, as possible sequestered factors. Using a combination of indirect immunofluorescence to detect endogenous proteins and overexpression of proteins with green fluorescent protein (GFP) tags we have shown that CUG-BP and hnRNP C do not co-localise with expanded repeat foci in DM1 cell lines. However, GFP-tagged MBNL does itself form foci in DM1 cell lines and co-localises with the foci of expanded repeat transcripts. GFP-tagged MBNL does not appear as foci in non-DM1 cell lines. This work provides further support for the involvement of MBNL in DM1.

High levels of allele loss at the FHIT and ATM genes in non-comedo ductal carcinoma in situ and grade I tubular invasive breast cancers.

Fifty-four grade 1 tubular breast cancers and nine non-comedo ductal carcinoma in situ samples have been analyzed for loss of heterozygosity using a series of microsatellite markers. Markers mapping to regions of the genome for which loss of heterozygosity has been documented previously in higher-grade breast cancers were selected for this analysis. Even within this group of good prognostic early breast cancers, genetic events are very common. The highest levels of loss were observed for D3S1300, which maps within an intron of the recently identified FHIT gene. High levels of loss were also observed within the ATM gene. These findings indicate that allele loss at FHIT and ATM may be an important early event in the development of sporadic breast cancer.

The effect of 4CMB on germ cells of the mouse.

The effect of 4CMB on meiosis in the mouse was studied using both male and female test systems. Females were superovulated and treated with 4CMB at metaphase I and oocytes sampled at metaphase II. Similarly with males, chromosome analysis was made at metaphase II for spermatocytes treated at metaphase I. No increase in the frequency of structural of numerical chromosome abnormalities was noted for treated mice as compared to controls.

Testing for the chemical induction of aneuploidy in the male mouse.

Dyad scores of metaphase II spermatocytes in the mouse have been used as an end point to assess the aneuploidy-inducing potential of three different chemicals; p-fluorophyalanine, phenylalanine and 6-mercaptopurine. The sensitivities of three different spermatogenic stages have been tested; pre-leptotene, zygotene and metaphase I. No effect was found at any treated stage for 6-mercaptopurine and phenylalanine. p-Fluorophenylalanine, when compared to control treatments, did, however, induce non-disjunction when applied at metaphase I. It also caused a delay to spermatogenesis when applied at this stage. The potential of mammalian test systems for the routine screening of chemicals as non-disjunction inducers, is discussed.

Testing of 3 chemical compounds for aneuploidy induction in the female mouse.

The 3 chemicals, 6 mercaptopurine (6-MCP), phenylalanine and para-fluorophenylalanine (pFPA) have been tested on mouse oocytes of the Swiss strain for possible aneuploidy-inducing effects. Tests were made at the dictyate stage in young and aged females and at the preovulatory (diakinesis/MI) stage in aged females only. Metaphase II chromosome complements were analysed for aneuploidy resulting from segregational errors arising at the first meiotic division. No evidence of non-disjunction was found either in treated or control groups up to the age of 40 weeks tested. The need to select for gametogenic stage and strain when using a mouse model system for aneuploidy testing, is considered.

Virtual cloning and physical mapping of a human T-box gene, TBX4.

The Tbx2/3/4/5 subfamily is one of the largest subgroupings within the T-box gene family, the members of which encode developmentally critical transcription factors. TBX4, a human member of the Tbx2/3/4/5 subfamily, has been identified and characterized from a high-throughput genomic sequence. The genomic organization of TBX4 was elucidated by computational sequence analysis, and the putative cDNA sequence was assembled. The genomic organization of TBX4 is very similar to that of TBX5, as is the situation for TBX2 and TBX3. The physical configuration of the TBX4-TBX2 cluster on human chromosome 17q21-q22 is similar to that of the TBX5-TBX3 cluster on chromosome 12q23-q24. The assembled TBX4 cDNA sequence was searched against the EST databases, and a TBX4 EST was identified.

Assignment of two of the translation initiation factor-4E (EIF4EL1 and EIF4EL2) genes to human chromosomes 4 and 20.

The eukaryotic translation initiation factor (eIF-4E) has recently been cloned from human, mouse, and yeast. This polypeptide is the rate-limiting component of the eukaryotic translation apparatus and is involved in the mRNA-ribosome binding step of eukaryotic protein synthesis. We have designed oligonucleotide primers to the 3' untranslated region of the gene encoding eIF-4E and specifically amplified the human gene in human/rodent somatic cell hybrids using the polymerase chain reaction. By this method, one of the human eIF-4E genes (EIF4EL1, eukaryotic translation initiation factor 4E-like 1) has been mapped to human chromosome 4qter-4p15. In addition, we have localized a second eIF-4E gene (EIF4EL2, eukaryotic translation initiation factor 4E-like 2) to human chromosome 20 by Southern blot analysis of mapping panels established from human/rodent somatic cell hybrids.

The identification of exons from the MED/PSACH region of human chromosome 19.

We have used exon amplification to identify putative transcribed sequences from an 823-kb contig consisting of 28 cosmids that form a minimum tiling path from the interval 19p12-p13.1. This region contains the genes responsible for multiple epiphyseal dysplasia (MED) and pseudoachondroplasia (PSACH). We have trapped 66 exons (an average of 2.4 exons per cosmid) from pools of 2 or 3 cosmids. The majority of exons (51.5%) show only weak similarity or no similarity (36.3%) to sequences in current databases. Six of 8 exons examined from these groups, however, show cross-species sequence conservation, indicating that many of them probably represent authentic exons. Eight exons show identity or significant similarity to ESTs or known genes, including the human TNF receptor 3 '-flanking region gene, human epoxide hydrolase (EPHX), human growth/differentiation factor (GOF-1), human myocyte-specific enhancer factor 2, the rat neurocan gene, and the human cartilage oligomeric matrix protein gene (COMP). Mutations in this latter gene have recently been shown to be responsible for MED and PSACH.

Maternal ageing and aneuploid embryos--evidence from the mouse that biological and not chronological age is the important influence.

Maternal ageing remains the overwhelming factor in the aetiology of human aneuploidy. Whether aberrant meiotic chromosome segregation in the oocyte relates causally to ovarian physiological ageing or to some factor dependent on the passage of chronological time, remains to be determined. The present experimental studies in the mouse indicate the former. An earlier cessation of reproductive life, brought on by unilateral ovariectomy in CBA females, resulted in the earlier onset of irregular cyclicity and an earlier rise in aneuploidy. The results could not be explained on the basis of the "production line" hypothesis. The clinical implications are that the probability of conceiving a Down foetus will be determined by distance in time from the approaching menopause, rather than by the chronological age of the woman per se.

Chromosomal assignment of c-MEL, a human transforming oncogene, to chromosome 19 (p13.2-q13.2).

The human malignant melanoma cell line NK14 contains a novel transforming gene which was identified using DNA transfection into NIH3T3 cells (1). This gene has been assigned to chromosome 19p13.2-q13.2 using human-mouse and human-hamster somatic cell hybrids.

Mapping human chromosomes in somatic cell hybrids using a low-copy-number repetitive sequence.

We have isolated a human-specific repetitive sequence with a copy number of several hundred (p11L26). Using Southern blots of EcoR1-digested DNA from somatic cell hybrids containing one or a few human chromosomes, band patterns specific for those chromosomes can be generated by hybridization with p11L26. Genomic copies of the sequence have also been mapped to subchromosomal regions using translocations. The probe offers a useful addition to the standard techniques for mapping human chromosomes in hybrid cell lines.

Long-range restriction map of a region of human chromosome 19 containing the apolipoprotein genes, a CLL-associated translocation breakpoint, and two polymorphic MluI sites.

The apolipoprotein gene cluster on human chromosome 19 (APOC1, APOC2, APOE) has been localised by pulsed-field gel electrophoresis to within 200 kb of a chronic lymphocytic leukemia-associated translocation breakpoint. A restriction map covering 1300 kb around these loci has been constructed and contains two polymorphic MluI sites, which appear to show Mendelian inheritance. The orientation of the map on the chromosome has been established as 19cen - CLL breakpoint - APOC2 - 19qter. Pedigree analysis using APOC2, a probe derived from the CLL breakpoint, and other localised markers on 19q suggests that the myotonic dystrophy locus is distal to APOC2 on 19q.

Transcriptional abnormality in myotonic dystrophy affects DMPK but not neighboring genes.

Myotonic dystrophy (DM) is caused by the expansion of a trinucleotide repeat, CTG, in the 3' untranslated region of a protein kinase gene, DMPK. We set out to determine what effect this expanded repeat has on RNA processing. The subcellular fractionation of RNA and the separate analysis of DMPK transcripts from each allele reveals that transcripts from expanded DMPK alleles are retained within the nucleus and are absent from the cytoplasm of DM cell lines. The nuclear retention of DMPK transcripts occurs above a critical threshold between 80 and 400 CTGs. Further analysis of the nuclear RNA reveals an apparent reduction in the proportion of expansion-derived DMPK transcripts after poly(A)+ selection. Quantitative analysis of RNA also indicates that although the level of cytoplasmic DMPK transcript is altered in DM patients, the levels of transcripts from 59 and DMAHP, two genes that immediately flank DMPK, are unaffected in DM cell lines.