Thomas B. Friedman

Correlation between Waardenburg syndrome phenotype and genotype in a population of individuals with identified PAX3 mutations

Waardenburg syndrome (WS) type 1 is an autosomal dominant disorder characterized by sensorineural hearing loss, pigmentary abnormalities of the eye, hair, and skin, and dystopia canthorum. The phenotype is variable and affected individuals may exhibit only one or a combination of several of the associated features. To assess the relationship between phenotype and gene defect, clinical and genotype data on 48 families (271 WS individuals) collected by members of the Waardenburg Consortium were pooled. Forty-two unique mutations in the PAX3 gene, previously identified in these families, were grouped in five mutation categories: amino acid (AA) substitution in the paired domain, AA substitution in the homeodomain, deletion of the Ser-Thr-Pro-rich region, deletion of the homeodomain and the Ser-Thr-Pro-rich region, and deletion of the entire gene. These mutation classes are based on the structure of the PAX3 gene and were chosen to group mutations predicted to have similar defects in the gene product. Association between mutation class and the presence of hearing loss, eye pigment abnormality, skin hypopigmentation, or white forelock was evaluated using generalized estimating equations, which allowed for incorporation of a correlation structure that accounts for potential similarity among members of the same family. Odds for the presence of eye pigment abnormality, white forelock, and skin hypopigmentation were 2, 8, and 5 times greater, respectively, for individuals with deletions of the homeodomain and the Pro-Ser-Thr-rich region compared to individuals with an AA substitution in the homeodomain. Odds ratios that differ significantly from 1.0 for these traits may indicate that the gene products resulting from different classes of mutations act differently in the expression of WS. Although a suggestive association was detected for hearing loss with an odds ratio of 2.6 for AA substitution in the paired domain compared with AA substitution in the homeodomain, this odds ratio did not differ significantly from 1.0.

On the loss of uricolytic activity during primate evolution—I. Silencing of urate oxidase in a hominoid ancestor

Urate oxidase activity is not detectable in liver homogenates from the gibbon, orangutan, chimpanzee, gorilla and human. Liver homogenates from five genera of Old World and two genera of New World monkeys have easily detectable levels of urate oxidase activity. There is no evidence for extant detectable intermediate steps in the loss of urate oxidase activity in the hominoids. Urate oxidase activity from Old World and New World monkeys is stable, a simple observation which debunks a long-standing myth. Urate oxidase activity was silenced in an ancestor to the five living genera of hominoids after divergence from the Old World monkeys.

Missense mutation in the paired domain of PAX3 causes craniofacial-deafness-hand syndrome.

Craniofacial‐deafness‐hand syndrome (MIM 122880) is inherited as an autosomal dominant mutation characterized by the absence or hypoplasia of the nasal bones, profound sensorineural deafness, a small and short nose with slitlike nares, hypertelorism, short palpebral fissures, and limited movement at the wrist and ulnar deviations of the fingers. In a family of three affected individuals with this syndrome, a mother and two children, a missense mutation (Asn47Lys) in the paired domain of PAX3 was initially detected by SSCP analysis. PCR amplification using an oligonucleotide with a terminal 3′‐residue match for the C‐to‐G transversion in codon 47 showed the presence of this mutation in the DNA from all affected members. The DNA from unaffected members were refractory to PCR amplification with the mutation‐specific oligonucleotide but did amplify a control primer pair in the same PCR reaction tube. A previously described missense mutation in this same codon (Asn47His) is associated with Waardenburg syndrome type 3 (Hoth et al., 1993). Substitution of a basic amino acid for asparagine at residue 47, conserved in all known murine Pax and human PAX genes, appears to have a more drastic effect on the phenotype than missense, frameshift and deletion mutations of PAX3 that cause Waardenburg syndrome type 1.

Donor age influences the growth of rabbit lens epithelial cells in vitro

Abstract Experiments were designed to optimize the conditions required for the establishment of cell lines from individual rabbit and human lenses. The influence of donor age and the response of the epithelia to various media, sera, substrata and growth factors were determined. Epithelial cells were obtained from 4–6-day-, 3-, 5-, 8-, 10-week- and 4-months- to 4-yr-old rabbits. Cell lines from animals aged 4 months or older were maintained in culture for over 2 yr, had a population doubling time of ca. 2.5 days, required serum for growth, and contained polypeptides that comigrated with and had an apparent molecular weight of known lens crystallins. Explants or enzymatically dissociated cells from 4–6-day-, 3-or 5-week-old donors exhibited little mitosis. This limited growth occurred in cells cultured in Eagles MEM, BME. Dulbeccos modified MEM, medium 199, RPMI-1640, Hams F-14, Leibovitzs L-15, KEI-4 or in a modified Ca2+-free MEM, supplemented with heat-inactivated rabbit or bovine sera or in these sera-containing-media supplemented with growth factors. Cells from the 4–6-day-olds migrated from the explant, synthesized DNA, enlarged, but did not show a marked increase in cell number. Analysis of video tapes indicated that the outgrowth from the explants of 4–6-day-olds was due solely to cell migration. Under similar culture conditions fibroblasts from 4–6-day-old rabbits or lens epithelia from rabbits 4 months or older were capable of generating continuous cell lines. Under like culture conditions, lens epithelia from humans aged 30–89 yr underwent a maximum of only three subcultures, enlarged and did not divide. Human lens cells have been maintained in a viable non-dividing condition for 1 yr. Cell lines possessing lens-specific proteins could be obtained from 4–6-day-old or 3-week-old rabbits if the cells were cultured in MEM containing non-heat inactivated rabbit serum. Clearly, the growth requirements of lens epithelial cells from younger rabbits can be markedly different from those of their older counterparts.

Establishment of epithelial cell lines from individual rabbit lenses

We describe a method of initiating primary cell cultures from lenses of rabbits by an explant-outgrowth technique. These primary cultures routinely yield epithelial cell lines when subcultured with collagenase-trypsin chicken serum. Most lines examined retain the diploid chromosome number through at least 30 passages.

Autosomal and X-Linked Auditory Disorders

Our conceptual thinking about hearing is confined, in part, by the “deafness” genes that have and have not identified. The proper development of the auditory system and its associated electromechanical processes requires the orchestrated temporal and spatial expression of numerous different genes. Further characterization of the genes for hearing loss will provide a clearer vision of the structure and function of the auditory system in health and disease. It is hoped that these discoveries will establish a conceptual basis for the rational therapy of hearing loss and deafness.

The urate oxidase gene of Drosophila pseudoobscura and Drosophila melanogaster: Evolutionary changes of sequence and regulation

SummaryThe urate oxidase (UO) transcription unit of Drosophila pseudoobscura was cloned, sequenced, and compared to the UO transcription unit from Drosophila melanogaster. In both species the UO coding region is divided into two exons of approximately equal size. The deduced D. pseudoobscura and D. melanogaster UO peptides have 346 and 352 amino acid residues, respectively. The nucleotide sequences of the D. pseudoobscura and D. melanogaster UO protein-coding regions are 82.2% identical whereas the deduced amino acid sequences are 87.6% identical with 42 amino acid changes, 33 of which occur in the first exon. Although the UO gene is expressed exclusively within the cells of the Malpighian tubules in both of these species, the temporal patterns of UO gene activity during development are markedly different. UO enzyme activity, UO protein, and UO mRNA are found in the third instar larva and adult of D. melanogaster but only in the adult stage of D. pseudoobscura. The intronic sequences and the extragenic 5′ and 3′ flanking regions of the D. pseudoobscura and D. melanogaster UO genes are highly divergent with the exception of eight small islands of conserved sequence along 772 by 5′ of the UO protein-coding region. These islands of conserved sequence are possible UO cis-acting regulatory elements as they reside along the 5′ flanking DNA of the D. melanogaster UO gene that is capable of conferring a wild-type D. melanogaster pattern of UO regulation on a UO-lacZ fusion gene.

Three Mutations in the Paired Homeodomain of PAX3 That Cause Waardenburg Syndrome Type 1

Genomic DNA from probands of various Waardenburg syndrome (WS) families were PCR-amplified using primers flanking the 8 exons of PAX3. The PCR fragments were screened for sequence variants, and subsequently cycle sequenced. Mutations were detected in exon 6 for 3 probands of WS type 1 families. These mutations all occur in the paired homeodomain DNA-binding motif.

THE FAINT BAND/INTERBAND REGION 28C2 TO 28C4-5/(-) OF THE DROSOPHILA MELANOGASTER SALIVARY GLAND POLYTENE CHROMOSOMES IS RICH IN TRANSCRIPTS

SummaryUrate oxidase mRNA and five other transcripts map along 38 kb of DNA in the region 28C on the Drosophila melanogaster second chromosome. Three biotinylated restriction fragments from this 38 kb of DNA, one from each end and one from the middle, were individually hybridized in situ to slightly stretched salivary gland polytene chromosomes. The data from these in situ hybridizations in combination with the transcription map of the 38 kb of DNA indicate that: (i) there are six discrete RNA species encoded along the 38 kb of DNA and (ii) these six transcripts map to the faint band/interband region which includes the proximal edge of 280, the three faint bands, 28C2, 280 and 28C4-5(−), and the adjacent interband chromatin. Our data are consistent with the few published studies directly demonstrating that faint band/interband regions of the Drosophila melanogaster salivary gland polytene chromosomes code for a high density of transcripts.

Cloning a cDNA for Drosophila melanogaster urate oxidase

A cDNA library from third-instar larval Malpighian tubules of Drosophila melanogaster was constructed and screened for urate oxidase (UO) clones by hybridization selection. The coding sequence for UO was mapped by in situ hybridization to position 28C on the left arm of chromosome 2. The UO activity in Drosophila shows a complex developmental profile. A UO cDNA was used as a probe of Northern blots of poly(A) + RNA from various stages of development. The data show that there is a direct correlation between the transcriptional activity of the UO locus as evidenced by the quantitative changes of UO mRNA and the levels of UO activity and protein during development.