Mutation of HOXA13 in hand-foot-genital syndrome.

There are several human syndromes which involve defects of the limbs and the Müllerian ducts or its derivatives. The hand-foot-genital (HFG) syndrome is an autosomal dominant, fully penetrant disorder that was originally described by Stern et al. Additional reports describing other affected families have also been published. Limb anomalies include short first metacarpals of normal thickness, small distal phalanges of the thumbs, short middle phalanges of the fifth fingers, and fusion or delayed ossification of wrist bones. In the feet, the great toe is shorter due to a short first metatarsal and a small, pointed distal phalanx. Uterine anomalies are common in females with HFG, and typically involve a partially divided (bicornuate) or completely divided (didelphic) uterus, representing defects of Müllerian duct fusion. Urinary tract malformations in affected HFG females include a displaced urethral opening and malposition of ureteral orifices in the bladder wall; affected males may have hypospadias (ventrally misplaced urethral opening) of variable severity. We report the identification of a HOXA13 nonsense mutation in a family with hand-foot-genital syndrome. The mutation converts a highly conserved tryptophan residue in the homeodomain to a stop codon, which truncates 20 amino acids from the protein and likely eliminates or greatly reduces the ability of the protein to bind to DNA.

The molecular basis of hypodactyly (Hd): a deletion in Hoxa 13 leads to arrest of digital arch formation.

Hypodactyly (Hd) is a semidominant mutation in mice that maps in a genetic interval overlapping the Hoxa cluster. The profound deficiency of digital arch structures in Hd/Hd mice is consistent with a defect in a gene activated late in limb morphogenesis. We have determined the structure of the Hoxa13 gene and describe a 50-base pair deletion in the first exon of the Hd allele that probably arose from unequal recombination or misalignment between triplet repeats. It is predicted that no Hoxa13 protein is made from Hd mRNA. The hypodactyly limb phenotype is similar to that of Hoxd13-deficient mice in sharing defects along multiple axes and alterations in cartilage maturation; however, the overall effects on digital arch formation are more severe in Hd/Hd mice. Our results confirm the critical role of AbdB-like Hox genes in the development of the autopod, and add to the spectrum of mutations involving triplet repeats.

Follow-up examination of linkage and association to chromosome 1q43 in multiple sclerosis.

Multiple sclerosis (MS) is a debilitating neuroimmunological and neurodegenerative disease affecting >4,00,000 individuals in the United States. Population and family-based studies have suggested that there is a strong genetic component. Numerous genomic linkage screens have identified regions of interest for MS loci. Our own second-generation genome-wide linkage study identified a handful of non-major histocompatibility complex regions with suggestive linkage. Several of these regions were further examined using single-nucleotide polymorphisms (SNPs) with average spacing between SNPs of approximately 1.0 Mb in a dataset of 173 multiplex families. The results of that study provided further evidence for the involvement of the chromosome 1q43 region. This region is of particular interest given linkage evidence in studies of other autoimmune and inflammatory diseases including rheumatoid arthritis and systemic lupus erythematosus. In this follow-up study, we saturated the region with approximately 700 SNPs (average spacing of 10 kb per SNP) in search of disease-associated variation within this region. We found preliminary evidence to suggest that common variation within the RGS7 locus may be involved in disease susceptibility.

Limb development: molecular dysmorphology is at hand!

We present a review of limb development integrating current molecular information and selected genetic disorders to illustrate the advances made in this field over the last few years. With this knowledge, clinical geneticists can now begin to consider molecular mechanisms and pathways when investigating patients with limb malformation syndromes.

Evolution of N-terminal sequences of the vertebrate HOXA13 protein.

While the the role of the homeodomain in HOX function has been evaluated extensively, little attention has been given to the non-homeodomain portions of the HOX proteins. To investigate the evolution of the HOXA13 protein and to identify conserved residues in the N-terminal region of the protein with potential functional significance, N-terminal Hoxa13 coding sequences were PCR-amplified from fish, amphibian, reptile, chicken, and marsupial and eutherian mammal genomic DNA. Compared with fish HOXA13, the mammalian protein has increased in size by 35% primarily owing to the accumulation of alanine repeats and flanking segments rich in proline, glycine, or serine within the first 215 amino acids. Certain residues and amino acid motifs were strongly conserved, and several HOXA13 N-terminal domains were also shared in the paralogous HOXB 13 and HOXD13 genes; however, other conserved regions appear to be unique to HOXA13. Two domains highly conserved in HOXA13 orthologs are shared with Drosophila AbdB and other vertebrate AbdB-like proteins. Marsupial and eutherian mammalian HOXA13 proteins have three large homopolymeric alanine repeats of 14, 12, and 17-18 residues that are absent in reptiles, birds, and fish. Thus, the repeats arose after the divergence of reptiles from the lineage that would give rise to the mammals. In contrast, other short homopolymeric alanine repeats in mammalian HOXA13 have remained virtually the same length, suggesting that forces driving or limiting repeat expansion are context dependent. Consecutive stretches of identical third-base usage in alanine codons within the large repeats were found, supporting replication slippage as a mechanism for their generation. However, numerous species-specific base substitutions affecting third-base alanine repeat codon positions were observed, particularly in the largest repeat. Therefore, if the large alanine repeats were present prior to eutherian mammal development as is suggested by the opossum data, then a dynamic process of recurring replication slippage and point mutation within alanine repeat codons must be considered to reconcile these observations. This model might also explain why the alanine repeats are flanked by proline, serine, and glycine-rich sequences, and it reveals a biological mechanism that promotes increases in protein size and, potentially, acquisition of new functions.

An efficient method for isolating putative promoters and 5'-transcribed sequences from large genomic clones.

Efficient strategies to isolate promoters and flanking exons from large genomic clones would facilitate the assembly of transcription units, complement existing techniques to isolate expressed sequences, and provide 5' regulatory elements. We have developed a rapid and simple method to isolate promoters from large mammalian genomic DNA clones by exploiting the abundance of binding sites for the ubiquitous transcription factor Sp1 in gene promoters. Using this method, putative promoter sequences with Sp1-binding sites are enriched approximately 100-fold from fragmented P1 clone DNA. Based on the abundance of Sp1-binding motifs in promoters, we predict that a significant subset of vertebrate promoters could be isolated by this method.

Molecular cloning and transformation of cyclodiene resistance in Drosophila: an invertebrate gamma-aminobutyric acid subtype A receptor locus.

Cyclodiene resistance represents 60% of the reported cases of insecticide resistance and is also present in vertebrates. Resistance is due to insensitivity of the cyclodiene/picrotoxinin binding site on the gamma-aminobutyric acid subtype A (GABAA) receptor-chloride ionophore complex. Following isolation of cyclodiene-resistant Drosophila mutants, we report the cloning of the locus conferring resistance via a "chromosomal walk" and rescue of the susceptible phenotype by P-element-mediated germ-line transformation. Amino acid sequence analysis of a cDNA from the locus reveals homology with vertebrate GABAA subunits. To our knowledge, this represents the first cloning of an invertebrate GABA receptor and also allows us to manipulate the resistance status of an insect via germ-line transformation. This gene may be useful as a selectable marker in other insect systems.

Novel HOXA13 mutations and the phenotypic spectrum of hand-foot-genital syndrome.

Hand-foot-genital syndrome (HFGS) is a rare, dominantly inherited condition affecting the distal limbs and genitourinary tract. A nonsense mutation in the homeobox of HOXA13 has been identified in one affected family, making HFGS the second human syndrome shown to be caused by a HOX gene mutation. We have therefore examined HOXA13 in two new and four previously reported families with features of HFGS. In families 1, 2, and 3, nonsense mutations truncating the encoded protein N-terminal to or within the homeodomain produce typical limb and genitourinary abnormalities; in family 4, an expansion of an N-terminal polyalanine tract produces a similar phenotype; in family 5, a missense mutation, which alters an invariant domain, produces an exceptionally severe limb phenotype; and in family 6, in which limb abnormalities were atypical, no HOXA13 mutation could be detected. Mutations in HOXA13 can therefore cause more-severe limb abnormalities than previously suspected and may act by more than one mechanism.