The HLXB9 homeobox gene was recently identified as a locus for autosomal dominant Currarino syndrome, also known as hereditary sacral agenesis (HSA). This gene specifies a 403-amino acid protein containing a homeodomain preceded by a very highly conserved 82-amino acid domain of unknown function; the remainder of the protein is not well conserved. Here we report an extensive mutation survey that has identified mutations in the HLXB9 gene in 20 of 21 patients tested with familial Currarino syndrome. Mutations were also detected in two of seven sporadic Currarino syndrome patients; the remainder could be explained by undetected mosaicism for an HLXB9 mutation or by genetic heterogeneity in the sporadic patients. Of the mutations identified in the 22 index patients, 19 were intragenic and included 11 mutations that could lead to the introduction of a premature termination codon. The other eight mutations were missense mutations that were significantly clustered in the homeodomain, resulting, in each patient, in nonconservative substitution of a highly conserved amino acid. All of the intragenic mutations were associated with comparable phenotypes. The only genotype-phenotype correlation appeared to be the occurrence of developmental delay in the case of three patients with microdeletions. HLXB9 expression was analyzed during early human development in a period spanning Carnegie stages 12-21. Signal was detected in the basal plate of the spinal cord and hindbrain and in the pharynx, esophagus, stomach, and pancreas. Significant spatial and temporal expression differences were evident when compared with expression of the mouse Hlxb9 gene, which may partly explain the significant human-mouse differences in mutant phenotype.

Clues regarding candidate genes which influence susceptibility to spina bifida and anencephaly come from the identification of folate-associated risk factors and from studies of mouse mutants showing neural tube anomalies. On this basis we selected five candidate genes ; CBS, MS, MTHFR, T (Brachyury) and BRCA1 for genetic analysis in 31 Dutch and 48 British NTD families. Ten polymorphisms, two for each gene, were used in transmission tests for disequilibrium (TDT). In six instances more than 50 transmissions from heterozygous parents could be examined. Using TDT we find evidence for an association between an allele at the T gene and liability to NTD in the embryo. Data from British and Dutch populations showed the same trend and in combination gave a χ# TDT l 4.89, P l 0.03 (OR 2.39, CI 95 % 1.02-5.61). No association, in either population group, was found for CBS, MS and MTHFR, the enzymes most directly associated with the known risk factors in folate metabolism. The possibility of complex genetic interactions was explored ; the data show that a Gly919 MS variant occurs more frequently in combination with the MTHFR thermolabile variant in mothers of NTD offspring (OR 3.94, CI 95 % 1.0-16.3).

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We describe a genetic analysis of the human homologue (T) of the mouse T (Brachyury) gene; human T was recently cloned in our laboratory. The protein product of the T gene is a transcription factor crucial in vertebrates for the formation of normal mesoderm. T mutant Brachyury mice die in midgestation with severe defects in posterior mesodermal tissues; heterozygous mice are viable but have posterior axial malformations. In addition to its importance in development, T has intrigued geneticists because of its association with the mouse t-haplotype; this haplotype is a variant form of the t-complex and is characterized by transmission ratio distortion, male sterility and recombination suppression. We have identified a common polymorphism of human T by single strand conformation polymorphism (SSCP) and used this in mapping studies and to re-investigate the idea that human T is involved in susceptibility to the multifactorial, neural tube defect, spina bifida. Our mapping data show that human T maps to 6q27 and lies between two other genes of the t-complex, TCP1 and TCP10. These data add to the evidence that in man the genes of the t-complex are split into two main locations on the short and long arms of chromosome 6. We have used an allele association test which is independent of mode of inheritance and penetrance to analyse data from the spina bifida families. Using this test we find evidence for a significant (p = 0.02) association between transmission of the TIVS7-2 allele of the human T gene and spina bifida.

T is a transcription factor which activates transcription by binding to repeated arrangements of the dodecamer 5'-AGGTGTGAAATT-3'. Using in vitro synthesised T protein, we have demonstrated that T binds to its target DNA as a homodimer and that truncated protein containing only the Nterminal 233 amino-acid residues, which comprise the DNAbinding domain, can form a dimer. We also report a common human polymorphism, Gly-177-Asp, within the DNA-binding domain at a position which is a conserved glycine residue in T homologues from other vertebrates. The proposition that T forms heterodimers with other members of the T-box transcription factor family and the implications for disorders of axial development are discussed.© 1997 Federation of European Biochemical Societies.