Rho-GTPase signaling drives melanoma cell plasticity

To investigate mechanisms that underlie different modes of tumor cell movement we have studied how regulation of the activity of the Rho family GTPases determines the mode of tumor cell movement. Guanine nucleotide exchange factors (GEFs) and GTPase accelerating proteins (GAPs) are key regulators of the activity of small GTPases with GEFs promoting activation to the GTP bound state and GAPs promoting inactivation by stimulating GTP hydrolysis. We identified two important signaling pathways regulating amoeboid and mesenchymal types of motility in melanoma. Here, we discuss our findings in the context of how specificity of Rho signaling is achieved by GEFs and GAPs.     

A simplified and efficient germline-specific CRISPR/Cas9 system for Drosophila genomic engineering

The type II CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated) has recently emerged as an efficient and simple tool for site-specific engineering of eukaryotic genomes. To improve its applications in Drosophila genome engineering, we simplified the standard two-component CRISPR/Cas9 system by generating a stable transgenic fly line expressing the Cas9 endonuclease in the germline (Vasa-Cas9 line). By injecting vectors expressing engineered target-specific guide RNAs into Vasa-Cas9 fly embryos, mutations were generated from site-specific DNA cleavages and efficiently transmitted into progenies. Because Cas9 endonuclease is the universal component of the type II CRISPR/Cas9 system, site-specific genomic engineering based on this improved platform can be achieved with lower complexity and toxicity, greater consistency, and excellent versatility.     

Quantitative DNA methylation analysis improves epigenotype-phenotype correlations in Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is a rare disorder characterized by overgrowth and predisposition to embryonal tumors. BWS is caused by various epigenetic and/or genetic alterations that dysregulate the imprinted genes on chromosome region 11p15.5. Molecular analysis is required to reinforce the clinical diagnosis of BWS and to identify BWS patients with cancer susceptibility. This is particularly crucial prenatally because most signs of BWS cannot be recognized in utero. We established a reliable molecular assay by pyrosequencing to quantitatively evaluate the methylation profiles of ICR1 and ICR2. We explored epigenotype-phenotype correlations in 19 patients that fulfilled the clinical diagnostic criteria for BWS, 22 patients with suspected BWS, and three fetuses with omphalocele. Abnormal methylation was observed in one prenatal case and 19 postnatal cases, including seven suspected BWS. Seven cases showed ICR1 hypermethylation, five cases showed ICR2 hypomethylation, and eight cases showed abnormal methylation of ICR1 and ICR2 indicating paternal uniparental disomy (UPD). More cases of ICR1 alterations and UPD were found than expected. This is likely due to the sensitivity of this approach, which can detect slight deviations in methylation from normal levels. There was a significant correlation (p < 0.001) between the percentage of ICR1 methylation and BWS features: severe hypermethylation (range: 75–86%) was associated with macroglossia, macrosomia, and visceromegaly, whereas mild hypermethylation (range: 55–59%) was associated with umbilical hernia and diastasis recti. Evaluation of ICR1 and ICR2 methylation by pyrosequencing in BWS can improve epigenotype-phenotype correlations, detection of methylation alterations in suspected cases, and identification of UPD.     

Characterization of Dinophysis spp. (Dinophyceae,Dinophysiales) from the mid-Atlantic region of the United States1

Due to the increasing prevalence of Dinophysis spp. and their toxins on every US coast in recent years, the need to identify and monitor for problematic Dinophysis populations has become apparent. Here, we present morphological analyses, using light and scanning electron microscopy, and rDNA sequence analysis, using a ~2-kb sequence of ribosomal ITS1, 5.8S, ITS2, and LSU DNA, of Dinophysis collected in mid-Atlantic estuarine and coastal waters from Virginia to New Jersey to better characterize local populations. In addition, we analyzed for diarrhetic shellfish poisoning (DSP) toxins in water and shellfish samples collected during blooms using liquid-chromatography tandem mass spectrometry and an in vitro protein phosphatase inhibition assay and compared this data to a toxin profile generated from a mid-Atlantic Dinophysis culture. Three distinct morphospecies were documented in mid-Atlantic surface waters: D. acuminata, D. norvegica, and a “small Dinophysis sp.” that was morphologically distinct based on multivariate analysis of morphometric data but was genetically consistent with D. acuminata. While mid-Atlantic D. acuminata could not be distinguished from the other species in the D. acuminata-complex (D. ovum from the Gulf of Mexico and D. sacculus from the western Mediterranean Sea) using the molecular markers chosen, it could be distinguished based on morphometrics. Okadaic acid, dinophysistoxin 1, and pectenotoxin 2 were found in filtered water and shellfish samples during Dinophysis blooms in the mid-Atlantic region, as well as in a locally isolated D. acuminata culture. However, DSP toxins exceeded regulatory guidance concentrations only a few times during the study period and only in noncommercial shellfish samples.     

Encapsulation enhances protoplast fusant stability

A barrier to cost-efficient biomanufacturing is the instability of engineered genetic elements, such as plasmids. Instability can also manifest at the whole-genome level, when fungal dikaryons revert to parental species due to nuclear segregation during cell division. Here, we show that by encapsulating Saccharomyces cerevisiae-Pichia stipitis dikaryons in an alginate matrix, we can limit cell division and preserve their expanded metabolic capabilities. As a proxy to cellulosic ethanol production, we tested the capacity of such cells to carry out ethanologenic fermentation of glucose and xylose, examining substrate use, ploidy, and cell viability in relation to planktonic fusants, as well as in relation to planktonic and encapsulated cell cultures consisting of mixtures of these species. Glucose and xylose consumption and ethanol production by encapsulated dikaryons were greater than planktonic controls. Simultaneous co-fermentation did not occur; rather the order and kinetics of glucose and xylose catabolism by encapsulated dikaryons were similar to cultures where the two species were encapsulated together. Over repeated cycles of fed-batch culture, encapsulated S. cerevisiae-P. stipitis fusants exhibited a dramatic increase in genomic stability, relative to planktonic fusants. Encapsulation also increased the stability of antibiotic-resistance plasmids used to mark each species and preserved a fixed ratio of S. cerevisiae to P. stipitis cells in mixed cultures. Our data demonstrate how encapsulating cells in an extracellular matrix restricts cell division and, thereby, preserves the stability and biological activity of entities ranging from genomes to plasmids to mixed populations, each of which can be essential to cost-efficient biomanufacturing.     

Angelman syndrome and severe infections in a patient with de novo 15q11.2–q13.1 deletion and maternally inherited 2q21.3 microdeletion

Angelman syndrome is a neurodevelopmental disorder characterized by mental retardation, severe speech disorder, facial dysmorphism, secondary microcephaly, ataxia, seizures, and abnormal behaviors such as easily provoked laughter. It is most frequently caused by a de novo maternal deletion of chromosome 15q11–q13 (about 70–90%), but can also be caused by paternal uniparental disomy of chromosome 15q11–q13 (3–7%), an imprinting defect (2–4%) or in mutations in the ubiquitin protein ligase E3A gene UBE3A mostly leading to frame shift mutation. In addition, for patients with overlapping clinical features (Angelman-like syndrome), mutations in methyl-CpG binding protein 2 gene MECP2 and cyclin-dependent kinase-like 5 gene CDKL5 as well as a microdeletion of 2q23.1 including the methyl-CpG binding domain protein 5 gene MBD5 have been described. Here, we describe a patient who carries a de novo 5 Mb-deletion of chromosome 15q11.2–q13.1 known to be associated with Angelman syndrome and a further, maternally inherited deletion 2q21.3 (~ 364 kb) of unknown significance. In addition to classic features of Angelman syndrome, she presented with severe infections in the first year of life, a symptom that has not been described in patients with Angelman syndrome. The 15q11.2–q13.1 deletion contains genes critical for Prader–Willi syndrome, the Angelman syndrome causing genes UBE3A and ATP10A/C, and several non-imprinted genes: GABRB3 and GABRA5 (both encoding subunits of GABA A receptor), GOLGA6L2, HERC2 and OCA2 (associated with oculocutaneous albinism II). The deletion 2q21.3 includes exons of the genes RAB3GAP1 (associated with Warburg Micro syndrome) and ZRANB3 (not disease-associated). Despite the normal phenotype of the mother, the relevance of the 2q21.3 microdeletion for the phenotype of the patient cannot be excluded, and further case reports will need to address this point.     

Novel PTEN germline mutation in a family with mild phenotype: Difficulties in genetic counseling

PTEN gene (phosphatase and tensin homolog deleted on chromosome ten, MIM 601628) is a tumor suppressor gene implicated in PTEN hamartoma tumor syndromes (PHTS) including Cowden syndrome, Bannayan–Riley–Ruvalcaba syndrome and Proteus-like syndrome. PTEN mutations have been more recently reported in children with macrocephaly and autism spectrum disorders or mental retardation, without other symptoms of PHTS. Although tumor risk has not been evaluated in these patients and their relatives, the same surveillance as for Cowden syndrome is usually proposed. We report a family including patients carrying a novel PTEN mutation and presenting with a mild phenotype consisting of macrocephaly, hypotonia during the first year of life and mild learning disabilities, without autistic features. None of these patients exhibited PTHS-related symptoms such as tumors, lipomas, vascular malformations or pigmented macules of the glans penis. This report raises the question of extending the indications of PTEN mutation screening to familial macrocephaly with learning disabilities. Detection of a mutation in this family led to difficult questions about surveillance, genetic counseling and familial information since the mother declined tumor screening and disclosure of genetic risk information to at-risk relatives.     

Exceptional complex chromosomal rearrangement and microdeletions at the 4q22.3q23 and 14q31.1q31.3 regions in a patient with azoospermia

In this report we describe the first patient ever found to have azoospermia in association with both exceptional complex chromosomal rearrangements and microdeletions at two translocation breakpoints. A 36-year-old male who had been suffering from male factor infertility was admitted to our clinic. The patient also displayed mild dysmorphia. An analysis of the patient's semen revealed azoospermia. GTG banding revealed the presence of an exceptional complex chromosomal rearrangement involving chromosomes 1, 4, 10 and 14. Using subtelomeric FISH analysis, the patient's karyotype was designated as 46,XY,t(1;10)(q43q44;q21q26.1)(CEB108/T7+,D1S3738-;10PTEL006+,D10S2290+, D1S3738+), ins(14;4) (q31.3;q23q33)(D14S1420+; D4S3359+, D4S2930+). Array-CGH analysis revealed two microdeletions at the 4q22.3q23 and 14q31.1q31.3 chromosomal regions. We suggest that microdeletions at the 4q22.3q23 and 14q31.1q31.3 chromosomal regions associated with both an exceptional complex chromosomal rearrangement and the Homo sapiens chromosome 4 open reading frame 37 (C4orf37) gene located at the 4q22.3q23 region might be associated with male factor infertility.     

Phenotype and Micro-array characterization of duplication 11q22.1-q25 and review of the literature

Partial duplication of 11q is related to several malformations like growth retardation, intellectual disability, hypoplasia of corpus callosum, short nose, palate defects, cardiac, urinary tract abnormalities and neural tube defects. We have studied the clinical and molecular characteristics of a patient with severe intellectual disabilities, dysmorphic features, congenital inguinal hernia and congenital cerebral malformation which is referred to as cytogenetic exploration. We have used FISH and array CGH analysis for a better understanding of the double chromosomic aberration involving a 7p microdeletion along with a partial duplication of 11q due to adjacent segregation of a paternal reciprocal translocation t(7;11)(p22;q21) revealed after banding analysis. The patient's karyotype formula was: 46,XY,der(7)t(7;11)(p22;q21)pat. FISH study confirmed these rearrangement and array CGH technique showed precisely the loss of at least 140 Kb on chromosome7p22.3pter and 33.4 Mb on chromosome11q22.1q25. Dysmorphic features, severe intellectual disability and brain malformations could result from the 11q22.1q25 trisomy. Our study provides an additional case for better understanding and delineating the partial duplication 11q.     

Prenatal diagnosis and molecular cytogenetic characterization of a de novo proximal interstitial deletion of chromosome 4p (4p15.2→p14)

We present prenatal diagnosis of de novo proximal interstitial deletion of chromosome 4p (4p15.2→p14) and molecular cytogenetic characterization of the deletion using uncultured amniocytes. We review the phenotypic abnormalities of previously reported patients with similar proximal interstitial 4p deletions, and we discuss the functions of the genes of RBPJ, CCKAR, STIM2, PCDH7 and ARAP2 that are deleted within this region.