Orsetta Zuffardi

Localization of factors controlling spermatogenesis in the nonfluorescent portion of the human y chromosome long arm

SummaryA deletion of the Y chromosome at the distal portion of band q11 was found in 6 men with normal male habitus but with azoospermia. Five of them were found during a survey of 1170 subfertile males while the sixth was karyotyped because of slight bone abnormalities. These findings, together with a review of the literature, suggest that on the distal portion of the nonfluorescent segment of the long arm of the Y, factors are located controlling spermatogenesis.

Human Bone Marrow–Derived Mesenchymal Stem Cells Do Not Undergo Transformation after Long-term In vitro Culture and Do Not Exhibit Telomere Maintenance Mechanisms

Significant improvement in the understanding of mesenchymal stem cell (MSC) biology has opened the way to their clinical use. However, concerns regarding the possibility that MSCs undergo malignant transformation have been raised. We investigated the susceptibility to transformation of human bone marrow (BM)-derived MSCs at different in vitro culture time points. MSCs were isolated from BM of 10 healthy donors and propagated in vitro until reaching either senescence or passage (P) 25. MSCs in the senescence phase were closely monitored for 8 to 12 weeks before interrupting the cultures. The genetic characterization of MSCs was investigated through array-comparative genomic hybridization (array-CGH), conventional karyotyping, and subtelomeric fluorescent in situ hybridization analysis both before and after prolonged culture. MSCs were tested for the expression of telomerase activity, human telomerase reverse transcriptase (hTERT) transcripts, and alternative lengthening of telomere (ALT) mechanism at different passages. A huge variability in terms of proliferative capacity and MSCs life span was noted between donors. In eight of 10 donors, MSCs displayed a progressive decrease in proliferative capacity until reaching senescence. In the remaining two MSC samples, the cultures were interrupted at P25 to pursue data analysis. Array-CGH and cytogenetic analyses showed that MSCs expanded in vitro did not show chromosomal abnormalities. Telomerase activity and hTERT transcripts were not expressed in any of the examined cultures and telomeres shortened during the culture period. ALT was not evidenced in the MSCs tested. BM-derived MSCs can be safely expanded in vitro and are not susceptible to malignant transformation, thus rendering these cells suitable for cell therapy approaches.

A recurrent 15q13.3 microdeletion syndrome associated with mental retardation and seizures

We report a recurrent microdeletion syndrome causing mental retardation, epilepsy and variable facial and digital dysmorphisms. We describe nine affected individuals, including six probands: two with de novo deletions, two who inherited the deletion from an affected parent and two with unknown inheritance. The proximal breakpoint of the largest deletion is contiguous with breakpoint 3 (BP3) of the Prader-Willi and Angelman syndrome region, extending 3.95 Mb distally to BP5. A smaller 1.5-Mb deletion has a proximal breakpoint within the larger deletion (BP4) and shares the same distal BP5. This recurrent 1.5-Mb deletion contains six genes, including a candidate gene for epilepsy (CHRNA7) that is probably responsible for the observed seizure phenotype. The BP4–BP5 region undergoes frequent inversion, suggesting a possible link between this inversion polymorphism and recurrent deletion. The frequency of these microdeletions in mental retardation cases is ∼0.3% (6/2,082 tested), a prevalence comparable to that of Williams, Angelman and Prader-Willi syndromes.

Olfactory Receptor–Gene Clusters, Genomic-Inversion Polymorphisms, and Common Chromosome Rearrangements

The olfactory receptor (OR)-gene superfamily is the largest in the mammalian genome. Several of the human OR genes appear in clusters with > or = 10 members located on almost all human chromosomes, and some chromosomes contain more than one cluster. We demonstrate, by experimental and in silico data, that unequal crossovers between two OR gene clusters in 8p are responsible for the formation of three recurrent chromosome macrorearrangements and a submicroscopic inversion polymorphism. The first two macrorearrangements are the inverted duplication of 8p, inv dup(8p), which is associated with a distinct phenotype, and a supernumerary marker chromosome, +der(8)(8p23.1pter), which is also a recurrent rearrangement and is associated with minor anomalies. We demonstrate that it is the reciprocal of the inv dup(8p). The third macrorearrangment is a recurrent 8p23 interstitial deletion associated with heart defect. Since inv dup(8p)s originate consistently in maternal meiosis, we investigated the maternal chromosomes 8 in eight mothers of subjects with inv dup(8p) and in the mother of one subject with +der(8), by means of probes included between the two 8p-OR gene clusters. All the mothers were heterozygous for an 8p submicroscopic inversion that was delimited by the 8p-OR gene clusters and was present, in heterozygous state, in 26% of a population of European descent. Thus, inversion heterozygosity may cause susceptibility to unequal recombination, leading to the formation of the inv dup(8p) or to its reciprocal product, the +der(8p). After the Yp inversion polymorphism, which is the preferential background for the PRKX/PRKY translocation in XX males and XY females, the OR-8p inversion is the second genomic polymorphism that confers susceptibility to the formation of common chromosome rearrangements. Accordingly, it may be possible to develop a profile of the individual risk of having progeny with chromosome rearrangements.

Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute.

There is great interest in mesenchymal stromal cells (MSCs) for cell‐therapy and tissue engineering approaches. MSCs are currently expanded in vitro in the presence of fetal calf serum (FCS); however, FCS raises concerns when used in clinical grade preparations. The aim of this study was to evaluate whether MSCs expanded in medium supplemented with platelet‐lysate (PL), already shown to promote MSC growth, are endowed with biological properties appropriate for cell‐therapy approaches. We confirm previously published data showing that MSCs expanded in either FCS or PL display comparable morphology, phenotype, and differentiation capacity, while PL‐MSCs were superior in terms of clonogenic efficiency and proliferative capacity. We further extended these data by investigating the immune‐regulatory effect of MSCs on the alloantigen‐specific immune response in mixed lymphocyte culture (MLC). We found that MSCs‐PL are comparable to MSCs‐FCS in their capacity to: (i) decrease alloantigen‐induced cytotoxic activity; (ii) favor differentiation of CD4+ T‐cell subsets expressing a Treg phenotype; (iii) increase early secretion of IL‐10 in MLC supernatant, as well as induce a striking augmentation of IL‐6 production. As compared with MSCs‐PL, MSCs‐FCS were more efficient in suppressing alloantigen‐induced lymphocyte subset proliferation and reducing early IFNγ‐secretion. Resistance to spontaneous transformation into tumor cells of expanded MSCs was demonstrated by molecular karyotyping and maintenance of normal morphology/phenotype after prolonged in vitro culture. Our data support the immunological functional plasticity of MSCs and suggest that MSCs‐PL can be used as an alternative to MSCs‐FCS, although these latter cells might be more suitable for preventing/treating alloreactivity‐related immune complications. J. Cell. Physiol. 211: 121–130, 2007.

Cryptic deletions are a common finding in “balanced” reciprocal and complex chromosome rearrangements: a study of 59 patients

Using array comparative genome hybridisation (CGH) 41 de novo reciprocal translocations and 18 de novo complex chromosome rearrangements (CCRs) were screened. All cases had been interpreted as “balanced” by conventional cytogenetics. In all, 27 cases of reciprocal translocations were detected in patients with an abnormal phenotype, and after array CGH analysis, 11 were found to be unbalanced. Thus 40% (11 of 27) of patients with a “chromosomal phenotype” and an apparently balanced translocation were in fact unbalanced, and 18% (5 of 27) of the reciprocal translocations were instead complex rearrangements with >3 breakpoints. Fourteen fetuses with de novo, apparently balanced translocations, all but two with normal ultrasound findings, were also analysed and all were found to be normal using array CGH. Thirteen CCRs were detected in patients with abnormal phenotypes, two in women who had experienced repeated spontaneous abortions and three in fetuses. Sixteen patients were found to have unbalanced mutations, with up to 4 deletions. These results suggest that genome-wide array CGH may be advisable in all carriers of “balanced” CCRs. The parental origin of the deletions was investigated in 5 reciprocal translocations and 11 CCRs; all were found to be paternal. Using customised platforms in seven cases of CCRs, the deletion breakpoints were narrowed down to regions of a few hundred base pairs in length. No susceptibility motifs were associated with the imbalances. These results show that the phenotypic abnormalities of apparently balanced de novo CCRs are mainly due to cryptic deletions and that spermatogenesis is more prone to generate multiple chaotic chromosome imbalances and reciprocal translocations than oogenesis.

Disruption of the ProSAP2 Gene in a t(12;22)(q24.1;q13.3) Is Associated with the 22q13.3 Deletion Syndrome

The terminal 22q13.3 deletion syndrome is characterized by severe expressive-language delay, mild mental retardation, hypotonia, joint laxity, dolichocephaly, and minor facial dysmorphisms. We identified a child with all the features of 22q13.3 deletion syndrome. The patients karyotype showed a de novo balanced translocation between chromosomes 12 and 22, with the breakpoint in the 22q13.3 critical region of the 22q distal deletion syndrome [46, XY, t(12;22)(q24.1;q13.3)]. FISH investigations revealed that the translocation was reciprocal, with the chromosome 22 breakpoint within the 22q subtelomeric cosmid 106G1220 and the chromosome 12q breakpoint near STS D12S317. Using Southern blot analysis and inverse PCR, we located the chromosome 12 breakpoint in an intron of the FLJ10659 gene and located the chromosome 22 breakpoint within exon 21 of the human homologue of the ProSAP2 gene. Short homologous sequences (5-bp, CTG[C/A]C) were found at the breakpoint on both derivative chromosomes. The translocation does not lead to the loss of any portion of DNA. Northern blot analysis of human tissues, using the rat ProSAP2 cDNA, showed that full-length transcripts were found only in the cerebral cortex and the cerebellum. The FLJ10659 gene is expressed in various tissues and does not show tissue-specific isoforms. The finding that ProSAP2 is included in the critical region of the 22q deletion syndrome and that our proband displays all signs and symptoms of the syndrome suggests that ProSAP2 haploinsufficiency is the cause of the 22q13.3 deletion syndrome. ProSAP2 is a good candidate for this syndrome, because it is preferentially expressed in the cerebral cortex and the cerebellum and encodes a scaffold protein involved in the postsynaptic density of excitatory synapses.

Constitutive Activation of PKA Catalytic Subunit in Adrenal Cushing's Syndrome

BACKGROUND Corticotropin-independent Cushings syndrome is caused by tumors or hyperplasia of the adrenal cortex. The molecular pathogenesis of cortisol-producing adrenal adenomas is not well understood. METHODS We performed exome sequencing of tumor-tissue specimens from 10 patients with cortisol-producing adrenal adenomas and evaluated recurrent mutations in candidate genes in an additional 171 patients with adrenocortical tumors. We also performed genomewide copy-number analysis in 35 patients with cortisol-secreting bilateral adrenal hyperplasias. We studied the effects of these genetic defects both clinically and in vitro. RESULTS Exome sequencing revealed somatic mutations in PRKACA, which encodes the catalytic subunit of cyclic AMP-dependent protein kinase (protein kinase A [PKA]), in 8 of 10 adenomas (c.617A→C in 7 and c.595_596insCAC in 1). Overall, PRKACA somatic mutations were identified in 22 of 59 unilateral adenomas (37%) from patients with overt Cushings syndrome; these mutations were not detectable in 40 patients with subclinical hypercortisolism or in 82 patients with other adrenal tumors. Among 35 patients with cortisol-producing hyperplasias, 5 (including 2 first-degree relatives) carried a germline copy-number gain (duplication) of the genomic region on chromosome 19 that includes PRKACA. In vitro studies showed impaired inhibition of both PKA catalytic subunit mutants by the PKA regulatory subunit, whereas cells from patients with germline chromosomal gains showed increased protein levels of the PKA catalytic subunit; in both instances, basal PKA activity was increased. CONCLUSIONS Genetic alterations of the catalytic subunit of PKA were found to be associated with human disease. Germline duplications of this gene resulted in bilateral adrenal hyperplasias, whereas somatic PRKACA mutations resulted in unilateral cortisol-producing adrenal adenomas. (Funded by the European Commission Seventh Framework Program and others.).

Heterozygous submicroscopic inversions involving olfactory receptor-gene clusters mediate the recurrent t(4;8)(p16;p23) translocation.

The t(4;8)(p16;p23) translocation, in either the balanced form or the unbalanced form, has been reported several times. Taking into consideration the fact that this translocation may be undetected in routine cytogenetics, we find that it may be the most frequent translocation after t(11q;22q), which is the most common reciprocal translocation in humans. Case subjects with der(4) have the Wolf-Hirschhorn syndrome, whereas case subjects with der(8) show a milder spectrum of dysmorphic features. Two pairs of the many olfactory receptor (OR)-gene clusters are located close to each other, on both 4p16 and 8p23. Previously, we demonstrated that an inversion polymorphism of the OR region at 8p23 plays a crucial role in the generation of chromosomal imbalances through unusual meiotic exchanges. These findings prompted us to investigate whether OR-related inversion polymorphisms at 4p16 and 8p23 might also be involved in the origin of the t(4;8)(p16;p23) translocation. In seven case subjects (five of whom both represented de novo cases and were of maternal origin), including individuals with unbalanced and balanced translocations, we demonstrated that the breakpoints fell within the 4p and 8p OR-gene clusters. FISH experiments with appropriate bacterial-artificial-chromosome probes detected heterozygous submicroscopic inversions of both 4p and 8p regions in all the five mothers of the de novo case subjects. Heterozygous inversions on 4p16 and 8p23 were detected in 12.5% and 26% of control subjects, respectively, whereas 2.5% of them were scored as doubly heterozygous. These novel data emphasize the importance of segmental duplications and large-scale genomic polymorphisms in the evolution and pathology of the human genome.

Identification and mapping of human cDNAs homologous to Drosophila mutant genes through EST database searching

Cross–species comparison is an effective tool used to identify genes and study their function in both normal and pathological conditions. We have applied the power of Drosophila genetics to the vast resource of human cDNAs represented in the expressed sequence tag (EST) database (dbEST) to identify novel human genes of high biological interest. Sixty–six human cDNAs showing significant homology to genes causing Drosophila mutant phenotypes were identified by screening dbEST using the ‘text string’ option, and their map position was determined using both fluorescence in situ hybridization (FISH) and radiation hybrid mapping. Comparison between these genes and their putative partners in Drosophila may provide important insights into their function in mammals. Furthermore, integration of these genes into the transcription map of the human genome contributes to the positional candidate approach for disease gene identification.