Aicardi syndrome associated with autosomal genomic imbalance: coincidence or evidence for autosomal inheritance with sex-limited expression?

Aicardi syndrome (AIS), a rare neurodevelopmental disorder thought to be caused by an X-linked dominant mutation, is characterized by 3 main features: agenesis of corpus callosum, infantile spams and chorioretinal lacunae. A genome-wide study of a girl with AIS lead us to identify a 6q deletion;12q duplication, derived from a maternal 6q;12q translocation. The two intellectually impaired brothers of the proband showed the same genomic anomalies, but not the constellation of features characterizing the AIS. This could be either a coincidental observation of 2 rare conditions, but can also suggest an alternative hypothesis for the genetic etiology of AIS, indicating the existence of a subset of autosomal genes whose mutation could act in a sex-confined manner.

Familial pericentric inversion of chromosome 18: intrafamilial variability of the recombinant dup(18q).

A pericentric inversion of chromosome 18 [inv(18)(p11.32q22)] and its recombinants has been studied in a three-generation family. A mother/son couple, carrying the rec dup(18q), showed dysmorphisms and short stature but only the son had mild mental retardation and speech delay. Karyotype, FISH analysis with subtelomeric probes and a 0.8 Mb array-CGH investigations were used to analyze this recombinant, demonstrating no genomic differences between the two relatives. This is the first observation of familial transmission of a rec dup(18q), showing that this recombinant is associated with a mild phenotype with variable clinical picture.

A novel mutation in the SDHD gene responsible for familial paraganglioma. Medical and psychological implications.

Familial paragangliomas/pheochromocytomas are dominantly inherited disorders characterized by the development of highly vascularized tumors of the head and neck, derived from non-chromaffin cells of the extra-adrenal paraganglia, and tumors with endocrine activity, derived from chromaffin cells, usually located in the adrenal medulla and pre- and para-vertebral thoracoabdominal regions. Germline inactivating heterozygous mutations in one of the genes encoding for succinate dehydrogenase subunits B, C or D (SDHB, SDHC or SDHD) are responsible for hereditary paragangliomas (PGLs), accounting for nearly 70% of familial cases. Particularly in the SDHD gene, different types of mutations have been found, nevertheless, alterations other than point mutations and deletion leading to missense/nonsense/splicing mutations are extremely rare. Here we report a family with multiple cases of PGL which co-segregates with a novel SDHD gene mutation predictable to give rise to an abnormal gene product (CybS). The identification of the molecular event responsible for PGL in our family made genetic counseling particularly useful for younger first degree relatives at risk to develop this late-onset disease.

Formation of PML/RAR alpha high molecular weight nuclear complexes through the PML coiled-coil region is essential for the PML/RAR alpha-mediated retinoic acid response.

Retinoic Acid (RA) treatment induces disease remission of Acute Promyelocytic Leukaemia (APL) patients by triggering terminal differentiation of neoplastic cells. RA-sensitivity in APL is mediated by its oncogenic protein, which results from the recombination of the PML and the RA receptor alpha (RAR alpha) genes (PML/RAR alpha fusion protein). Ectopic expression of PML/RAR alpha into haemopoietic cell lines results in increased response to RA-induced differentiation. By structure-function analysis of PML/RAR alpha-mediated RA-differentiation, we demonstrated that fusion of PML and RAR alpha sequences and integrity of the PML dimerization domain and of the RAR alpha DNA binding region are required for the effect of PML/RAR alpha on RA-differentiation. Indeed, direct fusion of the PML dimerization domain to the N- or C-terminal extremities of RAR alpha retained full biological activity. All the biologically active PML/RAR alpha mutants formed high molecular weight complexes in vivo. Functional analysis of mutations within the PML dimerization domain revealed that the capacity to form PML/RAR alpha homodimers, but not PML/RAR alpha-PML heterodimers, correlated with the RA-response. These results suggest that targeting of RAR alpha sequences by the PML dimerization domain and formation of nuclear PML/RAR alpha homodimeric complexes are crucial for the ability of PML/RAR alpha to mediate RA-response.

Terminal megakaryocytic differentiation of TF-1 cells is induced by phorbol esters and thrombopoietin and is blocked by expression of PML/RARalpha fusion protein.

We have analyzed the differentiation program of growth factor-dependent TF-1 erythroleukemia cells as well as clones with inducible expression of the APL-specific PML/RARalpha protein. We have shown that TF-1 cells may be induced to megakaryocytic differentiation by phorbol ester (phorbol dibutyrate, PDB) addition, particularly when combined with thrombopoietin (Tpo). RT-PCR studies showed that Tpo induces Tpo receptor (TpoR or c-mpl), whose expression was further potentiated by PDB addition. When the cells are induced with both PDB and Tpo erythropoietin receptor (EpoR) expression was inhibited. In the absence of Zn2+-induced PML/RARalpha expression, PDB and Tpo induced megakaryocytic differentiation of TF-1 MTPR clones as observed in 'wild-type' TF-1 cells. Conversely, when PML/RARalpha expression was induced by Zn2+, PDB and Tpo treatment of these clones caused only a reduced level of megakaryocytic differentiation. These observations indicate that: (1) TF-1 cells as well as other erythroleukemic cells, possess the capacity to differentiate to megakaryocytic cells when grown in the presence of protein kinase (PKC) activators and more efficiently when combined with Tpo; (2) the PML/RARalpha gene has a wide capacity to interfere with the program of hematopoietic differentiation, including megakaryocytic differentiation. Finally, we also observed that PML/RARalpha expression in TF-1 cells induces an up-modulation of interleukin-3 receptor, c-kit and c-mpl, a phenomenon which may offer these cells a growth advantage.

The localization of the HRX/ALL1 protein to specific nuclear subdomains is altered by fusion with its eps15 translocation partner.

Translocations involving the HRX/ALL1 locus at chromosomal region 11q23 are among the most frequent cytogenetic abnormalities in acute leukemias. 11q23 translocations involve different chromosome partners and lead to the formation of HRX/ALL1 fusion proteins. The HRX/ALL1 protein is a putative transcription factor that has been implicated in developmental regulation in mammals. We report here the cellular localization of the HRX/ALL1 protein as well as that of the HRX/ALL1-eps15 fusion protein, the result of the t(1;11) (p32-q23) translocation of acute myeloid leukemias. The HRX/ALL1 protein was localized to both the cytoplasm and the nucleus. The nuclear pattern was characterized by diffuse staining, perinuclear accumulation, and localization within nuclear bodies of variable size, morphology, and number. The HRX/ALL1-eps15 localized exclusively to the nucleus within bodies that were smaller and more numerous than the HRX/ALL1 nuclear bodies. HRX/ALL1 fusion with an unknown partner in leukemia blasts with 11q23 abnormalities had similar morphological features. Thus, the fusion with eps15 alters the cellular compartmentalization of HRX/ALL1, providing a putative mechanism for activation of HRX/ALL1 by 11q23 abnormalities.

Effects on differentiation by the promyelocytic leukemia PML/RARalpha protein depend on the fusion of the PML protein dimerization and RARalpha DNA binding domains.

The block of terminal differentiation is a prominent feature of acute promyelocytic leukemia (APL) and its release by retinoic acid correlates with disease remission. Expression of the APL-specific PML/RARalpha fusion protein in hematopoietic precursor cell lines blocks terminal differentiation, suggesting that PML/ RARalpha may have the same activity in APL blasts. We expressed different PML/RARalpha mutants in U937 and TF-1 cells and demonstrated that the integrity of the PML protein dimerization and RARalpha DNA binding domains is crucial for the differentiation block induced by PML/RARalpha, and that these domains exert their functions only within the context of the fusion protein. Analysis of the in vivo dimerization and cell localization properties of the PML/RARalpha mutants revealed that PML/RARalpha--PML and PML/RARalpha--RXR heterodimers are not necessary for PML/RARalpha activity on differentiation. We propose that a crucial mechanism underlying PML/RARalpha oncogenic activity is the deregulation of a transcription factor, RARalpha, through its fusion with the dimerization interface of another nuclear protein, PML.

The acute promyelocytic leukemia-specific PML/RAR alpha fusion protein reduces the frequency of commitment to apoptosis upon growth factor deprivation of GM-CSF-dependent myeloid cells.

PML/RAR alpha is the putative transforming sequence of acute promyelocytic leukemias. We investigated the effects of PML/RAR alpha on cell survival by expressing the fusion protein in the growth factor-dependent TF-1 cell line and analyzing the kinetics of cell death after GM-CSF deprivation. Results showed that PML/RAR alpha expression markedly delayed apoptotic cell death (3 weeks vs 1 week) without inducing growth factor independence. Growth factor deprivation caused rapid and massive apoptosis of control TF-1 cells (>95% apoptotic cells after 4-5 days). Factor-deprived control cells were synchronously and irreversibly committed to apoptosis as shown by their inability to re-enter the cell cycle after GM-CSF re-addition. The percentage of apoptotic cells in the PML/RAR alpha expressing cells was low (approximately 30-35%) and constant over the 4 weeks of factor deprivation. GM-CSF re-addition produced rapid increase in cell number at all time points during the 4 weeks of factor deprivation, suggesting that commitment to apoptosis was asynchronous and delayed in PML/RAR alpha-expressing TF-1 cells. We conclude that PML/RAR alpha interferes with the genetic pathways which regulate survival by reducing the frequency of commitment to apoptosis. This biological effect of PML/RAR alpha may contribute to its leukemogenetic potential.

Acute promyelocytic leukaemia cells resistant to retinoic acid show further perturbation of the RAR alpha signal transduction system.

Acute promyelocytic leukaemia (APL) cell lines resistant to all-trans retinoic acid (ATRA) have been previously derived from the NB4 cell line, and characterized as having lost the expression of the intact pml/RAR alpha fusion protein. To confirm the association between ATRA-resistance and alteration in the fusion protein at the clonal level, 16 clones were generated from ATRA-resistant APL cell lines. All clones show immunological (HLA class I and II, CD11b and c, CD13 and 33), molecular and growth features similar to the parental cell lines. To investigate whether the irradiation protocol used to generate the previously reported retinoic acid-resistant NB4.306 cell line induced additional alterations that could render these cells able to escape the anti-proliferative effect of retinoic acid (ATRA), an additional ATRA-resistant APL cell line, [NB4.007/6], was generated, under the selective pressure of ATRA, from the NB4 cell line without previous radiation. This cell line shows resistance to the anti-proliferative and differentiating action of ATRA. The NB4.007/6 cell line contains the t(15;17) chromosome translocation, shows the usual pml/RAR alpha hybrid DNA but expresses no detectable amount of the usual pml/RAR alpha protein in Western blot analysis, similarly to the NB4.306 cell line. Finally, the relative resistance to ATRA of NB4.306 and NB4.007/6 was evaluated by comparing the phenotypic (CD11b) changes induced by ATRA in these two lines with those induced in the parental, ATRA-sensitive, NB4 cell line. It is estimated that NB4.306 and NB4.007/6 are about 300 and 70 times less sensitive to ATRA than the original NB4 cell line.