Stephen A. Wilcox

Disruption of a novel member of a sodium/hydrogen exchanger family and DOCK3 is associated with an attention deficit hyperactivity disorder-like phenotype

Background: Attention deficit hyperactivity disorder (ADHD) is a complex condition with high heritability. However, both biochemical investigations and association and linkage studies have failed to define fully the underlying genetic factors associated with ADHD. We have identified a family co-segregating an early onset behavioural/developmental condition, with features of ADHD and intellectual disability, with a pericentric inversion of chromosome 3, 46N inv(3)(p14:q21). Methods: We hypothesised that the inversion breakpoints affect a gene or genes that cause the observed phenotype. Large genomic clones (P1 derived/yeast/bacterial artificial chromosomes) were assembled into contigs across the two inversion breakpoints using molecular and bioinformatic technologies. Restriction fragments crossing the junctions were identified by Southern analysis and these fragments were amplified using inverse PCR. Results: The amplification products were subsequently sequenced to reveal that the breakpoints lay within an intron of the dedicator of cytokinesis 3 (DOCK3) gene at the p arm breakpoint, and an intron of a novel member of the solute carrier family 9 (sodium/hydrogen exchanger) isoform 9 (SLC9A9) at the q arm. Both genes are expressed in the brain, but neither of the genes has previously been implicated in developmental or behavioural disorders. Conclusion: These two disrupted genes are candidates for involvement in the pathway leading to the neuropsychological condition in this family.

The human/mouse imprinted genesIGF2, H19, SNRPN andZNF127 map to two conserved autosomal clusters in a marsupial

The four genesIGF2, H19, SNRPN andZNF127 are imprinted in mouse and human.IGF2 andH19 form one conserved cluster on the distal part of mouse chromosome 7 and human chromosome 11p15.5, whereasSNRPN andZNF127 form another on the middle of mouse chromosome 7 and on human chromosome 15q11-13. We have explored the evolution of these imprinted regions by cloning and mappingIGF2, H19, SNRPN andZNF127 homeologues in marsupials. Specifically, we wished to determine whether the arrangements were shared in eutherian and marsupial mammals, and to determine whether they lay on autosomes, or on the X, as might be predicted by the hypothesis that imprinting evolved from X inactivation. Using fluorescencein situ hybridization, we localized the marsupial homeologues ofIGF2 andH19 to the distal part of tammar wallaby chromosome 2p and the marsupial homeologues ofSNRPN andZNF127 to the middle of chromosome 1q. Thus, these genes were originally organized in two separate autosomal clusters in the therian ancestor 180 million years ago, the conservation of which may suggest a functional relationship. The autosomal location of these clusters does not suggest a recent evolutionary relationship between imprinting and X chromosome inactivation.

Genes Located In and Near the Human Pseudoautosomal Region are Located in the X-Y Pairing Region in Dog and Sheep

We cloned and mapped the dog and/or sheep homologues of two human pseudoautosomal genes CSF2RA and ANT3. We also cloned and mapped dog and/or sheep homologues of STS and PRKX, which are located nearby on the differential region of the human X and have related genes or pseudogenes on the Y. STS, as well as CSF2RA, mapped to the tips of the short arm of the sheep X and Y (Xp and Yp), and STS and PRKX, as well as ANT3, mapped to the tips of the dog Xp and Y long arm (Yq). These locations within the X-Y pairing regions suggest that the regions containing all these human Xp22.3-Xpter genes are pseudoautosomal in dog and sheep. This supports the hypothesis that a larger pseudoautosomal region (PAR) shared by eutherian groups was disrupted by chromosomal rearrangements during primate evolution. The absence of STS and ANT3 from the sex chromosomes in two prosimian lemur species must therefore represent a recent translocation from their ancestral PAR, rather than retention of a smaller ancestral PAR shared by mouse.

Statistical analysis of in situ hybridization data: Derivation and use of the Zmax test

There are many situations in which grain distributions resulting from in situ hybridization of radioactively labeled probes to unique genes should be subjected to a statistical analysis. However, the problems posed by analysis of in situ hybridization data are not straightforward, and no completely satisfying method is currently available. We have developed a procedure in which the major and any number of minor site(s) of hybridization may be specifically located and the significance of each tested. This zmax procedure first tests the overall distribution for departure from randomness and then identifies significantly overlabeled whole chromosomes (or chromosome arms or other large segments), a process that may be repeated to pinpoint significantly overlabeled regions within these chromosomes. We describe in detail the derivation of the zmax statistic, present tables of significant zmax levels, and show with examples how zmax is used in tests of significance of in situ hybridization data.

A eutherian X-linked gene, PDHA1, is autosomal in marsupials: A model for the evolution of a second, testis-specific variant in eutherian mammals

We report the cloning and mapping of a gene (PDHA) for the pyruvate dehydrogenase E1 alpha subunit in marsupials. In situ hybridization and Southern blot analysis show that PDHA is autosomal in marsupials, mapping to chromosome 3q in Sminthopsis macroura and 5p in Macropus eugenii. Since these locations represent a region that was translocated to the p arm of the human X chromosome following marsupial/eutherian divergence, we suggest that the marsupial PDHA gene is homologous to PDHA1, the somatic eutherian isoform located on human Xp and mouse X. Only one copy of PDHA is found in marsupials, whereas a second, testis-specific, intronless form is observed in eutherian mammals. We also suggest that translocation of PDHA to the eutherian X chromosome, which is inactivated during spermatogenesis, led to the evolution of a second testis-specific locus by retroposition to an autosome.

Connexin26 deafness in several interconnected families

Mutations in the connexin26 gene are the basis of much autosomal recessive sensorineural deafness. There is a high frequency of mutant alleles, largely accounted for by one common mutation, 35delG. We have studied a group of families, who had been brought together through marriages between Deaf persons, in which there are more than 30 Deaf people in four generations. We show that many of the several cases of deafness are the result of 35delG homozygosity or 35delG/Q57X compound heterozygosity at the connexin26 locus. A considerable range of audiographic phenotypes was observed. The combined effects of a high population frequency of mutant alleles, and of positive assortative marriage among the Deaf, led to an infrequently observed recessive pedigree pattern.

Shared synteny between human chromosome 10 and chromosome 1 of the marsupial tammar wallaby, Macropus eugenii

Marsupial homologs of the human chromosome 10 loci IL2RA, HK1, and PLAU have been cloned and mapped by fluorescence in situ hybridization to chromosome 1q of the tammar wallaby, Macropus eugenii. Relative distance measurements of the hybridization signals on M. eugenii chromosome 1 show that marsupial homologs of human (HSA) 10p IL2RA and 10q HK1/PLAU flank the marsupial homologs of the human 5q gene IL5 and the human 15q imprinted genes SNRPN and ZNF127. The shared synteny, therefore, does not necessarily mean that HSA 10 represents an ancestral grouping; rather, it suggests that HSA 10p and HSA 10q represent two different ancestral mammalian units which fused directly in primates and were incorporated independently into two different regions of the same chromosome in marsupials.