Breakpoint mapping of 13 large parkin deletions/duplications reveals an exon 4 deletion and an exon 7 duplication as founder mutations

Early-onset Parkinson’s disease (EOPD) has been associated with recessive mutations in parkin (PARK2). About half of the mutations found in parkin are genomic rearrangements, i.e., large deletions or duplications. Although many different rearrangements have been found in parkin before, the exact breakpoints involving these rearrangements are rarely mapped. In the present study, the exact breakpoints of 13 different parkin deletions/duplications, detected in 13 patients out of a total screened sample of 116 EOPD patients using Multiple Ligation Probe Amplification (MLPA) analysis, were mapped using real time quantitative polymerase chain reaction (PCR), long-range PCR and sequence analysis. Deletion/duplication-specific PCR tests were developed as a rapid and low cost tool to confirm MLPA results and to test family members or patients with similar parkin deletions/duplications. Besides several different deletions, an exon 3 deletion, an exon 4 deletion and an exon 7 duplication were found in multiple families. Haplotype analysis in four families showed that a common haplotype of 1.2 Mb could be distinguished for the exon 7 duplication and a common haplotype of 6.3 Mb for the deletion of exon 4. These findings suggest common founder effects for distinct large rearrangements in parkin

GLYCOPROTEIN HORMONE ALPHA‐SUBUNIT AND PROLACTIN RELEASE BY CULTURED PITUITARY ADENOMA CELLS FROM ACROMEGALIC PATIENTS:CORRELATION WITH GH RELEASE

In‐vitro data of pituitary adenoma cells from 28 acromegalic patients were evaluated. In addition to GH, PRL was produced by 16 adenomas (57%) and alpha‐subunit by 15 adenomas (54%) while there was a significantly higher incidence of tumours producing PRL and alpha‐subunit simultaneously. From 26 pituitary adenomas enough cells were obtained in order to perform secretion studies. Percentage basal hormone release (medium: (medium + intra‐cellular hormone)) ± 100% of GH and alpha‐subunit by 11 adenomas showed a close correlation while such a correlation for GH and PRL was present only in a subgroup of 10 of 13 adenomas. The responses of GH and alpha‐subunit release to 10nM SMS201–995, 10nM bromocriptine, 100nM TRH and 10nM GHRH were closely related in that a response or an absent response of GH release to the four secretagogues was virtually always attended with a response or an absent response respectively of alpha‐subunit release. Such a relationship was less evident with respect to the effects of SMS201–995, bromocriptine, TRH and GHRH on GH and PRL release. We conclude that basal and secretagogue‐induced alpha‐subunit release by cultured pituitary adenoma cells from acromegalic patients closely follows the pattern of GH release while such a relationship for GH and PRL is present only in a subgroup of the adenomas secreting GH and PRL simultaneously.</p

Heterogeneity of pituitary adenoma cell subpopulations from acromegalic patients obtained by Percoll density gradient centrifugation

Pituitary adenoma cells from 6 acromegalic patients were separated on continuous Percoll density gradients according to differences in their density. Two adenomas produced GH only in culture, the other 4 adenomas produced either GH and PRL (one adenoma) or GH and α-subunit (one adenoma) or GH, PRL and α-subunit (2 adenomas). The cell subpopulations obtained by this technique differed in the amount of hormone production per 105 cells: GH release decreased from the low density fractions to the higher density fractions in 5 of 6 adenomas. Intracellular GH levels completely followed this profile. In the mixed GH/α-subunit adenomas the α-subunit profile completely paralleled the GH profile, whereas in the mixed GH/PRL adenomas the PRL profile showed a pattern different from that of GH (and α-subunit). In neither of the adenomas did we find any differences between the subpopulations with respect to the responsiveness of GH, PRL or α-subunit release to GHRH, TRH and the somatostatin analogue SMS 201-995. Conclusions: 1. Within pituitary adenomas from acromegalic patients heterogeneity exists with respect to hormone production per cell. 2. The cell subpopulations obtained by density gradient centrifugation are not different in their responsiveness to SMS 201-995, GHRH or TRH. 3. Because GH and α-subunit release by the fractions from the mixed GH/α-subunit secreting adenomas were completely parallel, further evidence for co-release of GH and α-subunit by the same tumoural cells is provided.</p

Long-term culture of rat mammotrope and somatotrope subpopulations separated on continuous Percoll density gradients:Effects of dopamine, TRH, GHRH and somatostatin

Normal adult female rat mammotrope and somatotrope subpopulations were separated on continuous Percoll density gradients according to differences in their density. Viable cells were recovered in 16 fractions. The cells from each fraction were cultured during 7 days after which period 4-h incubations were performed. rPRL secretion per cell increased towards the higher density fractions. No major difference in TRH, dopamine and somatostatin responsiveness was observed between mammotropes that were recovered in the different gradient fractions. In addition, no differences in somatostatin responsiveness between the somatotrope cells in the different gradient fractions were observed. However, somatotropes that were recovered in the highest density region of the gradient appeared to be more responsive to GHRH than the lower density somatotropes. In the various gradient fractions there were no paradoxical effects of TRH and dopamine on rGH release and of GHRH on rPRL release. Conclusions: 1. In long-term cultures there is no evidence for functionally different subpopulations of mammotropes and somatotropes, separated according to differences in their density, with regard to dopamine and TRH responsiveness and with regard to somatostatin responsiveness, respectively. 2. There is no evidence for a (mammosomatotrope?) subpopulation of cells showing paradoxical responses of PRL or GH release to GHRH and dopamine or TRH, respectively.</p

Differential splicing of human androgen receptor pre-mRNA in X-linked Reifenstein syndrome, because of a deletion involving a putative branch site.

The analysis of the androgen receptor (AR) gene, mRNA, and protein in a subject with X-linked Reifenstein syndrome (partial androgen insensitivity) is reported. The presence of two mature AR transcripts in genital skin fibroblasts of the patient is established, and, by reverse transcriptase-PCR and RNase transcription analysis, the wild-type transcript and a transcript in which exon 3 sequences are absent without disruption of the translational reading frame are identified. Sequencing and hybridization analysis show a deletion of > 6 kb in intron 2 of the human AR gene, starting 18 bp upstream of exon 3. The deletion includes the putative branch-point sequence (BPS) but not the acceptor splice site on the intron 2/exon 3 boundary. The deletion of the putative intron 2 BPS results in 90% inhibition of wild-type splicing. The mutant transcript encodes an AR protein lacking the second zinc finger of the DNA-binding domain. Western/immunoblotting analysis is used to show that the mutant AR protein is expressed in genital skin fibroblasts of the patient. The residual 10% wild-type transcript can be the result of the use of a cryptic BPS located 63 bp upstream of the intron 2/exon 3 boundary of the mutant AR gene. The mutated AR protein has no transcription-activating potential and does not influence the transactivating properties of the wild-type AR, as tested in cotransfection studies. It is concluded that the partial androgen-insensitivity syndrome of this patient is the consequence of the limited amount of wild-type AR protein expressed in androgen target cells, resulting from the deletion of the intron 2 putative BPS