DIS3 depletion in multiple myeloma causes extensive perturbation in cell cycle progression and centrosome amplification.

DIS3 gene mutations occur in approximately 10% of patients with multiple myeloma (MM); furthermore, DIS3 expression can be affected by monosomy 13 and del(13q), found in roughly 40% of MM cases. Despite the high incidence of DIS3 mutations and deletions, the biological significance of DIS3 and its contribution to MM pathogenesis remain poorly understood. In this study we investigated the functional role of DIS3 in MM, by exploiting a loss-of-function approach in human MM cell lines. We found that DIS3 knockdown inhibits proliferation in MM cell lines and largely affects cell cycle progression of MM plasma cells, ultimately inducing a significant increase in the percentage of cells in the G0/G1 phase and a decrease in the S and G2/M phases. DIS3 plays an important role not only in the control of the MM plasma cell cycle, but also in the centrosome duplication cycle, which are strictly co-regulated in physiological conditions in the G1 phase. Indeed, DIS3 silencing leads to the formation of supernumerary centrosomes accompanied by the assembly of multipolar spindles during mitosis. In MM, centrosome amplification is present in about a third of patients and may represent a mechanism leading to genomic instability. These findings strongly prompt further studies investigating the relevance of DIS3 in the centrosome duplication process. Indeed, a combination of DIS3 defects and deficient spindle-assembly checkpoint can allow cells to progress through the cell cycle without proper chromosome segregation, generating aneuploid cells which ultimately lead to the development of MM.

Germline NUP98 Variants in Two Siblings with a Rothmund-Thomson-Like Spectrum: Protein Functional Changes Predicted by Molecular Modeling.

Two adult siblings born to first-cousin parents presented a clinical phenotype reminiscent of Rothmund-Thomson syndrome (RTS), implying fragile hair, absent eyelashes/eyebrows, bilateral cataracts, mottled pigmentation, dental decay, hypogonadism, and osteoporosis. As the clinical suspicion was not supported by the sequencing of RECQL4, the RTS2-causative gene, whole exome sequencing was applied and disclosed the homozygous variants c.83G>A (p.Gly28Asp) and c.2624A>C (p.Glu875Ala) in the nucleoporin 98 (NUP98) gene. Though both variants affect highly conserved amino acids, the c.83G>A looked more intriguing due to its higher pathogenicity score and location of the replaced amino acid between phenylalanine-glycine (FG) repeats within the first NUP98 intrinsically disordered region. Molecular modeling studies of the mutated NUP98 FG domain evidenced a dispersion of the intramolecular cohesion elements and a more elongated conformational state compared to the wild type. This different dynamic behavior may affect the NUP98 functions as the minor plasticity of the mutated FG domain undermines its role as a multi-docking station for RNA and proteins, and the impaired folding can lead to the weakening or the loss of specific interactions. The clinical overlap of NUP98-mutated and RTS2/RTS1 patients, accounted by converging dysregulated gene networks, supports this first-described constitutional NUP98 disorder, expanding the well-known role of NUP98 in cancer.

Expanding the Molecular Spectrum of ANKRD11 Gene Defects in 33 Patients with a Clinical Presentation of KBG Syndrome.

KBG syndrome (KBGS) is a neurodevelopmental disorder caused by the Ankyrin Repeat Domain 11 (ANKRD11) haploinsufficiency. Here, we report the molecular investigations performed on a cohort of 33 individuals with KBGS clinical suspicion. By using a multi-testing genomic approach, including gene sequencing, Chromosome Microarray Analysis (CMA), and RT-qPCR gene expression assay, we searched for pathogenic alterations in ANKRD11. A molecular diagnosis was obtained in 22 out of 33 patients (67%). ANKRD11 sequencing disclosed pathogenic or likely pathogenic variants in 18 out of 33 patients. CMA identified one full and one terminal ANKRD11 pathogenic deletions, and one partial duplication and one intronic microdeletion, with both possibly being pathogenic. The pathogenic effect was established by RT-qPCR, which confirmed ANKRD11 haploinsufficiency only for the three deletions. Moreover, RT-qPCR applied to six molecularly unsolved KBGS patients identified gene downregulation in a clinically typical patient with previous negative tests, and further molecular investigations revealed a cryptic deletion involving the gene promoter. In conclusion, ANKRD11 pathogenic variants could also involve the regulatory regions of the gene. Moreover, the application of a multi-test approach along with the innovative use of RT-qPCR improved the diagnostic yield in KBGS suspected patients.

A familial t(4;8) translocation segregates with epilepsy and migraine with aura.

Three relatives carrying a t(4;8)(p15.2;p23.2) translocation had juvenile myoclonic epilepsy, self-limited photosensitive occipital epilepsy and migraine with aura. The t(4;8) translocation interrupted the coding sequence of CSMD1 gene and occurred immediately to the 3'UTR of STIM2 gene. STIM2 was overexpressed in the patient carrying the unbalanced translocation, and all three individuals had a single functional copy of CSMD1. Array CGH study disclosed that these three individuals also carried a deletion at 5q12.3 that involves the RGS7BP gene. The overall results favor the view that CSMD1, STIM2, and RGS7BP genes could contribute to epilepsy and migraine phenotypes in our family.

13q mosaic deletion including RB1 associated to mild phenotype and no cancer outcome - case report and review of the literature.

BACKGROUND: The 13q deletion syndrome is a rare chromosome disorder associated with wide phenotypic spectrum, which is related to size and location of the deleted region and includes intellectual disability, growth retardation, craniofacial dysmorphisms, congenital malformations, and increased risk of retinoblastoma. CASE PRESENTATION: Here, we report on a teenage boy with a mild phenotype characterized by obesity, hyperactivity, dysphagia, dysgraphia, sleep disturbance, and minor dysmorphic features (round face, bushy eyebrows, and stubby hands). Array Comparative Genomic Hybridization on blood identified a mosaic 13q14.13-13q31.1 deletion, with a mosaicism rate around 40%, which was confirmed by quantitative PCR and interphase Fluorescent In Situ Hybridization (iFISH) on both blood genomic DNA and cultured/uncultured blood lymphocytes, respectively. Conversely, karyotype analysis on blood estimated a mosaicism rate of 24% and iFISH on buccal smears revealed a borderline value of 0.4%, suggesting the absence of 13q deletion in this cell line. CONCLUSIONS: The comparison with previous patients carrying similar deletions informed that the proband clinical presentation is the mildest reported to date, thus supporting the burden of mosaicism in modulating the phenotype also in case of large chromosomal rearrangements. Characterization of further cases by in-depth mosaicism rate in tissues with different embryonic origins might contribute in the future to a better definition of genotype-phenotype correlation, including tumor risk.

Rothmund-Thomson Syndrome: Insights from New Patients on the Genetic Variability Underpinning Clinical Presentation and Cancer Outcome.

Biallelic mutations in RECQL4 gene, a caretaker of the genome, cause Rothmund-Thomson type-II syndrome (RTS-II) and confer increased cancer risk if they damage the helicase domain. We describe five families exemplifying clinical and allelic heterogeneity of RTS-II, and report the effect of pathogenic RECQL4 variants by in silico predictions and transcripts analyses. Complete phenotype of patients #39 and #42 whose affected siblings developed osteosarcoma correlates with their c.[1048_1049del], c.[1878+32_1878+55del] and c.[1568G>C;1573delT], c.[3021_3022del] variants which damage the helicase domain. Literature survey highlights enrichment of these variants affecting the helicase domain in patients with cancer outcome raising the issue of strict oncological surveillance. Conversely, patients #29 and #19 have a mild phenotype and carry, respectively, the unreported homozygous c.3265G>T and c.3054A>G variants, both sparing the helicase domain. Finally, despite matching several criteria for RTS clinical diagnosis, patient #38 is heterozygous for c.2412_2414del; no pathogenic CNVs out of those evidenced by high-resolution CGH-array, emerged as contributors to her phenotype.

Constitutional de novo deletion of the FBXW7 gene in a patient with focal segmental glomerulosclerosis and multiple primitive tumors.

Multiple primary malignant neoplasms are rare entities in the clinical setting, but represent an important issue in the clinical management of patients since they could be expression of a genetic predisposition to malignancy. A high resolution genome wide array CGH led us to identify the first case of a de novo constitutional deletion confined to the FBXW7 gene, a well known tumor suppressor, in a patient with a syndromic phenotype characterized by focal segmental glomerulosclerosis and multiple primary early/atypical onset tumors, including Hodgkin's lymphoma, Wilms tumor and breast cancer. Other genetic defects may be associated with patient's phenotype. In this light, constitutional mutations at BRCA1, BRCA2, TP53, PALB2 and WT1 genes were excluded by performing sequencing and MLPA analysis; similarly, we ruled out constitutional abnormalities at the imprinted 11p15 region by methylation specific -MLPA assay. Our observations sustain the role of FBXW7 as cancer predisposition gene and expand the spectrum of its possible associated diseases.

Fetal cell microchimerism: a protective role in autoimmune thyroid diseases.

OBJECTIVE: The physiological persistence of fetal cells in the circulation and tissue of a previously pregnant woman is called fetal cell microchimerism (FCM). It has been hypothesized to play a role in systemic autoimmune disease; however, only limited data are available regarding its role in autoimmune thyroid disease (AITD). DESIGN: Circulating FCM was analyzed in a large series of previously pregnant women with Graves' disease (GD), Hashimoto's thyroiditis (HT), or no disease (healthy controls (HCs)). To exclude the possible bias related to placental factors, the polymorphic pattern of human leukocyte antigen-G (HLA-G) gene, which is known to be involved in the tolerance of fetal cells by the maternal immune system, was investigated. METHODS: FCM was evaluated by PCR in the peripheral blood, and the Y chromosome was identified by fluorescence in situ hybridization in some GD tissues. HLA-G polymorphism typing was assessed by real-time PCR. RESULTS: FCM was significantly more frequent in HC (63.6%) than in GD (33.3%) or HT (27.8%) women (P=0.0004 and P=0.001 respectively). A quantitative analysis confirmed that circulating male DNA was more abundant in HC than it was in GD or HT. Microchimeric cells were documented in vessels and in thyroid follicles. In neither GD/HT patients nor HC women was the HLA-G typing different between FCM-positive and FCM-negative cases. CONCLUSION: The higher prevalence of FCM in HC as compared to GD and HT patients suggests that it plays a possible protective role in autoimmune thyroid disorders. Placental factors have been excluded as determinants of the differences found. The vascular and tissue localization of microchimeric cells further highlights the ability of those cells to migrate to damaged tissues.

Complex de novo chromosomal rearrangement at 15q11-q13 involving an intrachromosomal triplication in a patient with a severe neuropsychological phenotype: clinical report and review of the literature.

Interstitial triplications of 15q11-q13, leading to tetrasomy of the involved region, are very rare, with only 11 cases reported to date. Their pathogenicity is independent of the parental origin of the rearranged chromosome. The associated phenotype resembles, but is less severe, than that of patients bearing inv dup(15) marker chromosomes. Here, we describe a boy of 3 years and 9 months of age who exhibited very mild craniofacial dysmorphism (arched eyebrows, hypertelorism, and a wide mouth), developmental delay, generalized hypotonia, ataxic gait, severe intellectual disability, and autism. Array comparative genomic hybridization (CGH) analysis identified a heterozygous duplication of 1.1 Mb at 15q11.2 (between low-copy repeats BP1 and BP2), and a heterozygous triplication of 6.8 Mb at 15q11.2-q13.1 (BP2-BP4). Both acquisitions were de novo and contiguous. Microsatellite polymorphism analysis revealed the maternal origin of the triplication and the involvement of both maternal chromosomes 15. Furthermore, fluorescence in situ hybridization (FISH) analysis using BAC clones revealed that the rearrangement was complex, containing three differently sized tandem repeats of which the middle one was inverted. Our study confirms and extends the model proposed to explain the formation of intrachromosomal triplications through recombination events between non-allelic duplicons. The comparison of the proband's clinical presentation with those of previously described cases attests the existence of endophenotypes due to the parental origin of the 15q11-q13 triplicated segment and suggests a timetable for achievement of developmental milestones, thereby contributing to improved genotype-phenotype correlations.

A new structural rearrangement associated to Wolfram syndrome in a child with a partial phenotype.

Wolfram syndrome (WS) is a rare autosomal recessive disorder characterized by diabetes insipidus (DI), insulin-dependent diabetes mellitus (DM), optic atrophy (OA) and deafness caused by mutations in WFS1 gene (4p16.1), which encodes an endoplasmic reticulum protein, called Wolframin. We describe the case of an infant who presented hypernatremia and severe hypoplasia of the left eyeball with alteration of visual evoked potentials. Persistent hypernatremia, iposmolar polyuria and high plasma osmolality suggested DI, confirmed by a normal urine concentration after vasopressin test. Treatment with vasopressin allowed a normalization of sodium levels and urine output. Brain magnetic resonance imaging showed absence of the neurohypophysis hyperintense signal, normal adenohypophysis and optic tracts hypoplasia. The concomitant presence of DI and OA, even in the absence of DM and deafness, prompted the suspicion of WS and complete genetic analysis was performed. Genomic DNA sequencing of WFS1 showed no inactivating mutations described to date, but suggested a structural mutation as markers genotyping revealed a segmental paternal heterodisomy involving the upstream regulatory region (promoter and 5'UTR). cDNA sequencing revealed the coexistence of the wild-type transcript and two splice variants; one variant, probably benign, is known in literature and the other one causes the loss of exon 2, containing the translation initiation site. Western blot confirmed a marked protein reduction. During the clinical follow-up child's condition remained stable and glucose metabolism is still in the standard. In conclusion, the phenotype associated with this structural rearrangement, which substantially reduces the synthesis of Wolframin, confirms a tissue-specific pattern of expression of WFS1, suggests the presence of a different protein dosage sensitivity in different tissues and could be causative of DI and OA in our patient. The "incomplete" phenotype here described, usually absent in typical WS cases, is explained by the residual Wolframin expression that would preserve other organs, i.e. pancreatic islets. A careful longitudinal clinical follow-up will assess any changes in the phenotypic penetrance in our patient.

Increased loss of the Y chromosome in peripheral blood cells in male patients with autoimmune thyroiditis.

Multiple mechanisms have been proposed to explain the peculiar distribution of autoimmune thyroiditis (AIT) among women and men. Most attention has been focused on the detection of the role of estrogens and the X chromosome. Specifically, a potential role for X haploinsufficiency has been proposed in the female patient population and an association with the disease has been confirmed. Our knowledge of the etiopathogenesis of autoimmunity in male patients remains, however, limited. Next to the possible role of androgens and their imbalances, the Y chromosome appears as a potential candidate for influence of the immune function in men. Herein we analyzed a population of male patients with AIT (n=31) and healthy controls (n=88) to define a potential association of disease and the loss of the Y chromosome. Y chromosome loss increases in AIT compared to unaffected subjects; these phenomenon increases with aging as expected, however, the degree of loss is significantly increased in the patient population compared to the healthy controls. We were, thus, able to confirm the existence of an analogous mechanism in the male population to previously identified X haploinsufficiency in female patients with AIT. We propose that this commonality might represent a relevant feature in the etiopathogenesis of AIT that should be further investigated.

Combined characterization of a pituitary adenoma and a subcutaneous lipoma in a MEN1 patient with a whole gene deletion.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant hereditary disorder associated with mutations of the MEN1 gene, which is characterized by combined tumors of the parathyroid glands, pancreatic islet cells, and the anterior pituitary. A significant number of patients with the clinical features of MEN1, however, do not show MEN1 mutations upon direct sequencing. We describe a young woman who fulfilled the clinical and biochemical criteria for MEN1 syndrome, but DNA sequencing did not indicate any MEN1 mutations. She developed a prolactin-secreting pituitary macroadenoma, primary hyperparathyroidism with parathyroid hyperplasia, pancreatic lesions, and two subcutaneous lipomas. Array comparative genomic hybridization (aCGH) analysis of peripheral blood DNA revealed a heterozygous germline deletion at 11q13.1 that spanned at least 22.23 kilobases and contained the entire MEN1 gene. Integrated aCGH and cytogenetic analyses of the adenoma and lipoma tissues revealed somatic inactivation of the wild-type MEN1 allele by different routes: the second hit of MEN1 recessive oncogenesis leading to adenoma implied a loss of heterozygosity, whereas a balanced translocation deleting the wild-type MEN1 allele primed the lipoma development. These findings show that aCGH is a valuable means of optimizing genetic testing in MEN1 patients which complements other technologic approaches to elucidating the pathologic mechanisms of MEN1 tumors.

Misbehaviour of XIST RNA in breast cancer cells.

A role of X chromosome inactivation process in the development of breast cancer have been suggested. In particular, the relationship between the breast cancer predisposing gene BRCA1 and XIST, the main mediator of X chromosome inactivation, has been intensely investigated, but still remains controversial. We investigated this topic by assessing XIST behaviour in different groups of breast carcinomas and in a panel of breast cancer cell lines both BRCA1 mutant and wild type. In addition, we evaluated the occurrence of broader defects of heterochromatin in relation to BRCA1 status in breast cancer cells. We provide evidence that in breast cancer cells BRCA1 is involved in XIST regulation on the active X chromosome, but not in its localization as previously suggested, and that XIST can be unusually expressed by an active X and can decorate it. This indicates that the detection of XIST cloud in cancer cell is not synonymous of the presence of an inactive X chromosome. Moreover, we show that global heterochromatin defects observed in breast tumor cells are independent of BRCA1 status. Our observations sheds light on a possible previously uncharacterized mechanism of breast carcinogenesis mediated by XIST misbehaviour, particularly in BRCA1-related cancers. Moreover, the significant higher levels of XIST-RNA detected in BRCA1-associated respect to sporadic basal-like cancers, opens the possibility to use XIST expression as a marker to discriminate between the two groups of tumors.

Fluorescence in situ hybridization dissection of a chronic myeloid leukemia case bearing the apparently balanced translocations (9;22)(q34;q11.2) and (11;11)(p15;q13).

We report on a patient with chronic myeloid leukemia (CML), which was detected by conventional cytogenetic analysis, to carry two different acquired and apparently balanced translocations, (9;22)(q34;q11.2) and (11;11)(p15;q13). By fluorescence in situ hybridization characterization, we were able to finely map the genomic regions involved in the translocation breakpoints and to disclose concomitant deletions adjacent to the breakpoints on the two derivative chromosomes 11 and the derivative chromosome 22, and the insertion of a segment from chromosome band 11q12.2 into the derivative chromosome 9. We discuss the putative mechanism that could have led to the formation of this complex rearrangement and speculate on the role in leukemogenesis played by the genes mapping at the breakpoints and within the deleted regions.

Fetal cell microchimerism in papillary thyroid cancer: a possible role in tumor damage and tissue repair.

Fetal cells enter the maternal circulation during pregnancy and can persist in the maternal blood or tissues for decades, creating a physiologic microchimerism. Because papillary thyroid cancer (PTC) is more frequent in women, the role of persisting fetal male cells in this tumor has been investigated. Tumor tissue specimens were obtained from 63 women with PTC who had a male pregnancy before the diagnosis. Male cells, identified by PCR amplification of a male-specific gene, the sex-determining region Y, was detected in 47.5% of women. By fluorescence in situ hybridization (FISH) analyses, the total number of microchimeric cells was significantly higher in neoplastic tissue than in controlateral normal sections. By combined FISH and immunohistochemistry (immuno-FISH), male cells expressing thyroglobulin were found in tumor and normal tissues, whereas male microchimeric cells stained with the CD45 antigen were detected only in tumor sections. Microchimeric cells negative for either marker were detected both in tumor and normal tissues. Moreover, both CD45(+) and Tg(+) fetal cells did not express MHC II antigens. In conclusion, fetal microchimerism has been documented in a high proportion of women with PTC. The immuno-FISH studies indicate that CD45(+)/MHC II(-) male cells found in neoplastic tissues might be committed to destroy tumor cells, whereas Tg(+)/MHC II(-) cells could have a repair function. Finally, microchimeric cells negative for either CD45 or Tg could have "progenitor-like" properties able to transdifferentiate in different cellular types. Although a pathogenetic mechanism cannot be excluded, the whole of the present results indicates a protective role of microchimerism in thyroid cancer.

13q Deletion and central nervous system anomalies: further insights from karyotype-phenotype analyses of 14 patients.

BACKGROUND: Chromosome 13q deletion is associated with varying phenotypes, which seem to depend on the location of the deleted segment. Although various attempts have been made to link the 13q deletion intervals to distinct phenotypes, there is still no acknowledged consensus correlation between the monosomy of distinct 13q regions and specific clinical features. METHODS: 14 Italian patients carrying partial de novo 13q deletions were studied. Molecular-cytogenetic characterisation was carried out by means of array-comparative genomic hybridisation (array-CGH) or fluorescent in situ hybridisation (FISH). RESULTS: Our 14 patients showed mental retardation ranging from profound-severe to moderate-mild: eight had central nervous system (CNS) anomalies, including neural tube defects (NTDs), six had eye abnormalities, nine had facial dysmorphisms and 10 had hand or feet anomalies. The size of the deleted regions varied from 4.2 to 75.7 Mb. CONCLUSION: This study is the first systematic molecular characterisation of de novo 13q deletions, and offers a karyotype-phenotype correlation based on detailed clinical studies and molecular determinations of the deleted regions. Analyses confirm that patients lacking the 13q32 band are the most seriously affected, and critical intervals have been preliminarily assigned for CNS malformations. Dose-sensitive genes proximal to q33.2 may be involved in NTDs. The minimal deletion interval associated with the Dandy-Walker malformation (DWM) was narrowed to the 13q32.2-33.2 region, in which the ZIC2 and ZIC5 genes proposed as underlying various CNS malformations are mapped.

High-mobility group A2 gene expression is frequently induced in non-functioning pituitary adenomas (NFPAs), even in the absence of chromosome 12 polysomy.

The high-mobility group A2 (HMGA2) gene has a critical role in benign tumors where it is frequently rearranged, and in malignant tumors, where it is overexpressed in the absence of structural modification of the HMGA2 locus. By previous fluorescence in situ hybridization (FISH) and reverse transcriptase PCR analyses on human prolactin-secreting pituitary adenomas we detected rearrangement of the HMGA2 gene and amplification of its native region associated with activated expression. These data indicated a role for the HMGA2 gene in the development of human pituitary prolactinomas, since they are consistent with the appearance of prolactin/growth hormone adenomas in transgenic mice overexpressing the HMGA2 gene. To assess a more general role for HMGA2 in pituitary oncogenesis, we investigated HMGA2 amplification and expression in a panel of non-functioning pituitary adenomas (NFPAs) which account for 25% of all pituitary adenomas. We provide evidence that out of 18 NFPA tumors tested, 12 expressed HMGA2, but, different from prolactinomas, only in two cases the upregulation of the gene could be associated with amplification and/or rearrangement of the HMGA2 locus. Increased dosage of chromosome 12 was found in the expressing and non-expressing NFPAs, confirming that this sole event is insufficient to drive up activation of the HMGA2 gene. A role for chromosome 12 polysomy to promote structural instability of HMGA2 is confirmed, but the mechanism via trisomy is less prevalent in the frequently diploid NFPAs than in the usually hyperdiploid prolactinomas. Micro-rearrangements of HMGA2 gene not detectable by FISH analysis and/or sequence alterations could contribute to upregulation of HMGA2 gene in pituitary adenomas of the NFPA subtype. However, it cannot be excluded that the HMGA2 overexpression may be due, in some NFPA patients, to the same, still mainly unknown, mechanisms responsible for HMGA2 overexpression in malignant neoplasias.

Narrowing the candidate region of Albright hereditary osteodystrophy-like syndrome by deletion mapping in a patient with an unbalanced cryptic translocation t(2;6)(q37.3;q26).

We here describe a submicroscopic translocation affecting the subtelomeric regions of chromosomes 2q and 6q identified in a patient referred to us because of mental retardation, obesity, brachydactyly, and short stature. FISH analysis using subtelomeric probes showed a 46,XY,der(2)t(2;6)(q37.3;q26) in the propositus, and a balanced t(2;6) in his father and sister. FISH with region-specific genomic clones made it possible to map the 2q37.3 breakpoint precisely to the region covered by BAC 585E12, and the 6q26 breakpoint to between the regions encompassed by 414A5 and 480A20. The 2q subtelomeric deletion has often been found in patients with Albright hereditary osteodystrophy (AHO)-like syndrome but, to the best of our knowledge, the 2q37.3-qter monosomy ascertained in our patient is the smallest so far described within the syndrome's critical interval, and may thus enhance the search for the responsible genes.

The High Mobility Group A2 gene is amplified and overexpressed in human prolactinomas.

Trisomy of chromosome 12 is a nonrandom chromosomal change in pituitary adenomas, particularly prolactinomas. This and the finding that prolactin-secreting pituitary adenomas develop in transgenic mice overexpressing the wild-type HMGA2 gene (which maps to 12q14-15) prompted us to investigate HMGA2 rearrangements and expression in human prolactinomas. By dual-color interphase fluorescence in situ hybridization analysis using HMGA2-specific PACs and BACs, we found that the HMGA2 locus was amplified in seven of the eight prolactinoma samples examined. The cytogenetic manifestations of elevated HMGA2 concentrations ranged from simple trisomy to tetrasomy 12 and der(12) chromosomes to marker chromosomes bearing 12q14-15-derived regions. Reverse transcription-PCR, Western blot and immunohistochemical analysis showed HMGA2 overexpression in a number of prolactinomas bearing rearrangement of regions 12q14-15. These data suggest a critical role of the HMGA2 overexpression in the generation of prolactin-secreting pituitary adenomas in humans.