Tyrosine kinase inhibitors and interferon-α increase tunneling nanotube (TNT) formation and cell adhesion in chronic myeloid leukemia (CML) cell lines.

Chronic myeloid leukemia (CML) is a stem cell disease of the bone marrow where mechanisms of inter-leukemic communication and cell-to-cell interactions are proposed to be important for optimal therapy response. Tunneling nanotubes (TNTs) are novel intercellular communication structures transporting different cargos with potential implications in therapy resistance. Here, we have investigated TNTs in CML cells and following treatment with the highly effective CML therapeutics tyrosine kinase inhibitors (TKIs) and interferon-α (IFNα). CML cells from chronic phase CML patients as well as the blast crisis phase cell lines, Kcl-22 and K562, formed few or no TNTs. Treatment with imatinib increased TNT formation in both Kcl-22 and K562 cells, while nilotinib or IFNα increased TNTs in Kcl-22 cells only where the TNT increase was associated with adherence to fibronectin-coated surfaces, altered morphology, and reduced movement involving ß1integrin. Ex vivo treated cells from chronic phase CML patients showed limited changes in TNT formation similarly to bone marrow cells from healthy individuals. Interestingly, in vivo nilotinib treatment in a Kcl-22 subcutaneous mouse model resulted in morphological changes and TNT-like structures in the tumor-derived Kcl-22 cells. Our results demonstrate that CML cells express low levels of TNTs, but CML therapeutics increase TNT formation in designated cell models indicating TNT functionality in bone marrow derived malignancies and their microenvironment.

Partial Response to Sorafenib in a Child With a Myeloid/Lymphoid Neoplasm, Eosinophilia, and a ZMYM2-FLT3 Fusion.

Dysregulated tyrosine kinases in myeloid/lymphoid neoplasms with eosinophilia are rare, but do occur in children. To increase awareness of this diagnosis, we present a child who was diagnosed after a 3-year disease history. The patient was initially treated according to a T-cell lymphoblastic lymphoma protocol, but genetic analyses at recurrence revealed microdeletions resulting in an in-frame fusion of ZMYM2 and FLT3. Treatment with sorafenib, an FLT3 tyrosine kinase inhibitor, rapidly resulted in significant reduction of lymphadenopathy and normalization of white blood cell and eosinophil counts. At 17 months of treatment, he remains in complete hematologic, but not molecular remission.

Haploinsufficiency of MEIS2 is associated with orofacial clefting and learning disability.

MEIS2 is a homeodomain-containing transcription factor of the TALE superfamily that has been proven important for development. We confirm and extend a recent single clinical report stating that deletions in MEIS2 can cause cleft palate [Crowley et al. (2010); Am J Med Genet 152A:1326-1327]. Here we report on five additional patients with 15q14 deletions of sizes 0.6, 0.6, 1.0, 1.9, and 4.8 Mb, respectively, all involving MEIS2. In addition, we present a family with four affected individuals and an intragenic 58 kb direct duplication disrupting MEIS2. In total, 7/9 cases had clefting, from mild (submucous cleft palate) to severe (cleft lip and palate), and 3/9 cases had ventricular septal defects. All cases had delayed motor development and most had learning disability, at worst in the mild intellectual disability range. The cases had overlapping facial features (broad forehead, finely arched eyebrows, mildly shortened philtrum, and tented upper lip) but individually they were not considered to be dysmorphic. Our results show that MEIS2 is a gene needed for palate closure. In syndromic cases of cleft palate, MEIS2 should be considered among the candidate genes, for example, in cases without 22q11.2 deletions.

Disruption of a long distance regulatory region upstream of SOX9 in isolated disorders of sex development.

BACKGROUND: The early gonad is bipotential and can differentiate into either a testis or an ovary. In XY embryos, the SRY gene triggers testicular differentiation and subsequent male development via its action on a single gene, SOX9. The supporting cell lineage of the bipotential gonad will differentiate as testicular Sertoli cells if SOX9 is expressed and conversely will differentiate as ovarian granulosa cells when SOX9 expression is switched off. RESULTS: Through copy number variation mapping this study identified duplications upstream of the SOX9 gene in three families with an isolated 46,XX disorder of sex development (DSD) and an overlapping deletion in one family with two probands with an isolated 46,XY DSD. The region of overlap between these genomic alterations, and previously reported deletions and duplications at the SOX9 locus associated with syndromic and isolated cases of 46,XX and 46,XY DSD, reveal a minimal non-coding 78 kb sex determining region located in a gene desert 517-595 kb upstream of the SOX9 promoter. CONCLUSIONS: These data indicate that a non-coding regulatory region critical for gonadal SOX9 expression and subsequent normal sex development is located far upstream of the SOX9 promoter. Its copy number variations are the genetic basis of isolated 46,XX and 46,XY DSDs of variable severity (ranging from mild to complete sex reversal). It is proposed that this region contains a gonad specific SOX9 transcriptional enhancer(s), the gain or loss of which results in genomic imbalance sufficient to activate or inactivate SOX9 gonadal expression in a tissue specific manner, switch sex determination, and result in isolated DSD.

FLT3-ITD mutations in acute myeloid leukaemia - molecular characteristics, distribution and numerical variation.

Recurrent somatic internal tandem duplications (ITD) in the FMS-like tyrosine kinase 3 (FLT3) gene characterise approximately one third of patients with acute myeloid leukaemia (AML), and FLT3-ITD mutation status guides risk-adapted treatment strategies. The aim of this work was to characterise FLT3-ITD variant distribution in relation to molecular and clinical features, and overall survival in adult AML patients. We performed two parallel retrospective cohort studies investigating FLT3-ITD length and expression by cDNA fragment analysis, followed by Sanger sequencing in a subset of samples. In the two cohorts, a total of 139 and 172 mutant alleles were identified in 111 and 123 patients, respectively, with 22% and 28% of patients presenting with more than one mutated allele. Further, 15% and 32% of samples had a FLT3-ITD total variant allele frequency (VAF) < 0.3, while 24% and 16% had a total VAF ≥ 0.7. Most of the assessed clinical features did not significantly correlate to FLT3-ITD numerical variation nor VAF. Low VAF was, however, associated with lower white blood cell count, while increasing VAF correlated with inferior overall survival in one of the cohorts. In the other cohort, ITD length above 50 bp was identified to correlate with inferior overall survival. Our report corroborates the poor prognostic association with high FLT3-ITD disease burden, as well as extensive inter- and intrapatient heterogeneity in the molecular features of FLT3-ITD. We suggest that future use of FLT3-targeted therapy could be accompanied with thorough molecular diagnostics and follow-up to better predict optimal therapy responders.

Mosaic upd(14)pat in a patient with mild features of Kagami-Ogata syndrome.

We report a Norwegian girl with mild clinical features of Kagami-Ogata syndrome (KOS) and mosaic upd(14)pat. To our knowledge, this is the first report describing a mosaic patient with KOS. These results imply that mosaic uniparental disomy should be examined in patients with mild features of imprinted disorders.

Angioma serpiginosum with oesophageal papillomatosis is an X-linked dominant condition that maps to Xp11.3-Xq12.

We report on a four-generation family with localized subepidermal telangiectasias following Blaschko's lines (angioma serpiginosum). The vascular streaks are present at birth and progress slowly thereafter. In several family members papillomatosis of the entire oesophagus was found to be part of the condition. Mild nail and hair dystrophy added to the resemblance of Goltz-Gorlin syndrome (focal dermal hypoplasia), suggesting that the present condition could be a mild variant. All affected family members are females, there is no increased miscarriage rate, and X-inactivation in affected females is highly skewed, compatible with X-linked dominant inheritance with very early in utero lethality in males. In the family, 11 informative meioses were available to study the segregation of X-chromosome markers. Significant linkage (LOD score 3.31) was found to a region flanked by markers DXS8026 and DXS106 (44-67 Mb from Xpter) that includes the centromere.

RevSex duplication-induced and sex-related differences in the SOX9 regulatory region chromatin landscape in human fibroblasts.

It was recently shown that duplications of the RevSex element, located 0.5 Mb upstream of SOX9, cause XX-disorder of sex development (DSD), and that deletions cause XY-DSD. To explore how a 148 kb RevSex duplication could have turned on gonadal SOX9 expression in the absence of SRY in an XX-male, we examined the chromatin landscape in primary skin fibroblast cultures from the index, his RevSex duplication-carrier father and six controls. The ENCODE project supports the notion that chromatin state maps show overlap between different cell types, i.e., that our study of fibroblasts could be of biological relevance. We examined the SOX9 regulatory region by high-resolution ChIP-on-chip experiments (a kind of "chromatin-CGH") and DNA methylation investigations. The RevSex duplication was associated with chromatin changes predicting better accessibility of the SRY-responsive TESCO enhancer region 14-15 kb upstream of SOX9. Four kb downstream of the TESCO evolutionary conserved region, a peak of the enhancer/promoter-associated H3K4me3 mark was found together with a major dip of the repressive H3K9me3 chromatin mark. Similar differences were also found when three control males were compared with three control females. A marked male/female difference was a more open chromatin signature in males starting ~400 kb upstream of SOX9 and increasing toward the SOX9 promoter. In the RevSex duplication-carrier father, two positions of DNA hypomethylation were also found, one corresponding to the H3K4me3 peak mentioned above. Our results suggest that the RevSex duplication could operate by inducing long-range epigenetic changes. Furthermore, the differences in chromatin state maps between males and females suggest that the Y chromosome or X chromosome dosage may affect chromatin conformation, i.e., that sex-dependent gene regulation may take place by chromatin modification.