The effects of enoxaparin treatment in a xenograft mouse model of oral squamous cell carcinoma: A pilot study.

BACKGROUND: Recent studies suggest that enoxaparin may have therapeutic effects on oral squamous cell carcinoma. We aimed to assess this effect utilizing xenograft mouse model through evaluations of proliferation and angiogenesis markers at the RNA and protein levels. METHODS: Mice were divided into enoxaparin treatment (n = 4), positive control (n = 4) and negative control (n = 3) groups. Immunohistochemical analyses were performed utilizing Bcl-2, Bax and Ki-67 antibodies. Expression levels of proliferation and apoptosis related genes were calculated utilizing qRT-PCR. Time-dependent proliferation assays were performed in OSC-19 and HEK293 cell-lines. RESULTS: Bax antibody showed positive staining in the cytoplasm and nuclei of tumor cells, while Bcl-2 antibody displayed staining only in the cytoplasm. A proliferation index of 15%-20% was found in all groups with the Ki-67 marker indicating no metastasis. Enoxaparin treatment caused decrease in BCL2, BAX and CCNB1 genes' expressions. Compared to HEK293, proliferation assays demonstrated higher division rates in OSC-19 with a significant decrease in viability after 96 h. CONCLUSION: Reduced BCL-2 expression indicates a regression of tumor growth, but reduced BAX expression is not correlated with increased apoptosis. Despite the aggressive nature of OSC-19, our results showed a low cell viability with a high division rate when compared with the control HEK293. This paralleled our in vivo findings that showed absence of lymph node metastasis across all mice groups. This discrepancy with the literature suggests that further investigations of the underlying mechanisms and protein-level analyses are needed to draw definitive conclusions about the effect of enoxaparin on OSC-19 behavior.

Biallelic loss of EEF1D function links heat shock response pathway to autosomal recessive intellectual disability.

Intellectual disability (ID) is a genetically heterogeneous neurodevelopmental disorder characterised by significantly impaired intellectual and adaptive functioning. ID is commonly syndromic and associated with developmental, metabolic and/or neurological findings. Autosomal recessive ID (ARID) is a significant component of ID especially in the presence of parental consanguinity. Several ultra rare ARID associated variants in numerous genes specific almost to single families have been identified by unbiased next generation sequencing technologies. However, most of these new candidate ARID genes have not been replicated in new families due to the rarity of associated alleles in this highly heterogeneous condition. To determine the genetic component of ARID in a consanguineous family from Turkey, we have performed SNP-based linkage analysis in the family along with whole exome sequencing (WES) in an affected sibling. Eventually, we have identified a novel pathogenic variant in EEF1D, which has recently been recognised as a novel candidate gene for ARID in a single family. EEF1D encodes a ubiquitously expressed translational elongation factor functioning in the cytoplasm. Herein, we suggest that the loss of function variants exclusively targeting the long EEF1D isoform may explicate the ARID phenotype through the heat shock response pathway, rather than interfering with the canonical translational elongation.

Three Novel MEN1 Variants in AIP-Negative Familial Isolated Pituitary Adenoma Patients.

OBJECTIVES: Pituitary adenomas (PAs) may rarely occur in well-defined hereditary conditions, like multiple endocrine neoplasia type 1 (MEN1) syndrome and familial isolated pituitary adenoma (FIPA) associated with germline mutations in MEN1 and AIP, respectively. This study aimed to assess MEN1 genetic abnormalities in AIP mutation-negative FIPA patients, not associated with MEN1 components. METHODS: Among 20 patients evaluated in 13 FIPA families, 12 were previously reported as AIP mutation-negative. In this study, 6 new families with 8 patients were recruited. All patients were subjected to multiplex ligation-dependent probe amplification to detect copy number variations in AIP and MEN1, and AIP sequencing was performed in additional patients. AIP mutation-negative patients were subjected to MEN1 sequencing. RESULTS: Our cohort revealed only 3 novel heterozygous MEN1 variants including c.1846T>A p.(*616Argext*21), rs778272737:T>C, and rs972128957:C>T in 2 families, with patients diagnosed with Cushing disease, nonfunction al adenoma, and acromegaly, respectively. Among them, c.1846T>A p. (*616Argext*21) is a stop codon read-through, whereas the others are 3'UTR variations. MEN1 variation frequency was detected as 15%. CONCLUSIONS: MEN1 alterations can be of significance in FIPA patients and screening could be offered to AIP mutation-negative patients without MEN1 features. Further studies are needed to clarify the role of MEN1 in FIPA patients.

A novel homozygous GALC variant has been associated with Krabbe disease in a consanguineous family.

Krabbe disease (KD) or globoid cell leukodystrophy is an autosomal recessive lysosomal storage disorder involving the white matter of the peripheral and the central nervous systems. It is caused by a deficiency of galactocerebrosidase enzyme activity. The most common manifestation is the classical early onset KD that leads to patient's loss before the age of 2. Herein, we report the evaluation of a consanguineous family with three affected children manifesting severe neurological findings that ended with death before the age of 2, in an attempt to provide genetic diagnosis to the family. One of the children underwent detailed physical and neurological examinations, including brain magnetic resonance imaging (MRI) and scalp electroencephalography (EEG) evaluations. GALC genetic testing on this child enabled identification of a novel homozygous variant (NM_000153.3: c.1394C>T; p.(Thr465Ile)), which confirmed diagnosis as KD. Familial segregation of this variant was performed by PCR amplification and Sanger sequencing that revealed the parents as heterozygous carriers. We believe this novel GALC variant will not only help in genetic counseling to this family but will also aid in identification of future KD cases.

Whole Exome Sequencing Revealed Paternal Inheritance of Obesity-related Genetic Variants in a Family with an Exclusively Breastfed Infant.

Objectives: Obesity is a serious health problem, that progressively affects individuals' lives with comorbidities involving heart disease, stroke, and diabetes mellitus. Since its prevalence increases particularly in children under age-of-five years, its genetic and environmental causes should be determined for prevention and control of the disease. This study aimed to detect underlying genetic risk factors in a family with an exclusively breastfed obese infant. Methods: A three-generation family was recruited to be evaluated for obesity. Detailed examinations along with body mass indexcalculations were performed on available family members. Whole exome sequencing was performed on 7-month-oldobese infant utilizing Illumina-NextSeq550. Bioinformatic analyses were performed on the Genomize SEQ platform with variant filtering at minor allele frequencies (MAF)<1% for all normal populations. Sanger sequencing was applied in variant confirmation and family segregation. Results: Neuro-motor developmental features were normal and genetic syndromes were excluded from the index. Early-onset severe obesity (4.25SDS weight-for-height) was obvious in index case, where his father and grandmother were also obese (BMIs: 38.1kg/m2 and 31.3kg/m2, respectively). WES analysis revealed deleterious variants in SH2B1, PDE11A, ADCY3, and CAPN10 genes previously associated with obesity. All variants were evaluated as novel candidates for obesity except PDE11A and family segregation confirmed paternal inheritance. Conclusion: This study confirmed the paternal inheritance of all potentially deleterious obesity-related variants. The cumulative effect of individual variants might explain the obesity phenotype in this family. The infant is recommended to be under periodic follow-up due to increased risk for later childhood obesity.

Whole exome sequencing reveals novel candidate variants for endometriosis utilizing multiple affected members in a single family.

BACKGROUND: Endometriosis is an estrogen-dependent, chronic inflammatory disease that affects 10% of women during the reproductive ages. Despite the estimated 50% heritability for the condition, only 26% was associated with common genetic variants. Thus, necessity of identifying rare variants for the missing heritability is implicated in the literature. Therefore, our study aimed to identify novel rare genetic variants involved in the pathogenesis of endometriosis utilizing a family of multiple affected members. METHODS: A family composed of four affected women along with their two unaffected mothers were recruited at a single gynecology and infertility clinic specialized in endometriosis. All patients presented with endometriomas, which was visualized by transvaginal ultrasonography. Two affected individuals had received laparoscopic endometrioma excision and therefore were diagnosed with recurrent disease. One mother had a history of endometrial serous adenocarcinoma (ESC) for which she underwent hysterectomy with bilateral oophorectomy. Three endometriosis cases were whole exome sequenced on Illumina NextSeq 550 platform with an average of 90% coverage. Candidate genes were confirmed by Sanger sequencing and followed-up with family segregation. RESULTS: Novel rare variants were identified in TNFRSF1B (NM_001066.3: c.1072G>A, p.(Ala358Thr)) and GEN1 (NM_001130009.3: c.1574C>T, p.(Ser525Leu)) as possible genetic causes of endometriosis. A third novel rare variant was identified in CRABP1 (NM_004378.3:c.54G>C, p.(Glu18Asp)) only on the mother with ESC history and her daughters. CONCLUSION: Novel candidate genetic variants that might contribute to endometriosis were suggested that need replication through independent cohorts or validation by functional studies. The family has also received genetic counseling and that the affected daughters are on clinical follow-up, accordingly.

Combined evaluation of proliferation and apoptosis to calculate IC50 of VPA-induced PANC-1 cells and assessing its effect on the Wnt signaling pathway.

Pancreatic ductal adenocarcinoma (PDAC) is among the most deadly cancers. Since most patients develop resistance to conventional treatments, new approaches are in urgency. Valproic acid (VPA) was shown to induce apoptosis and reduce proliferation in PANC-1 cells. Wnt signaling pathway is known to be involved in apoptosis and PDAC onset. However, VPA-induced apoptosis and its impact on Wnt signaling in PDACs are not linked, yet. We aimed to calculate IC50 of VPA-induced PANC-1 cells by combined analyses of proliferation and apoptosis, while assessing its effect on Wnt signaling pathway. PANC-1 was induced with increased VPA doses and time points. Three independent proliferation and apoptosis assays were performed utilizing carboxyfluorescein succinimidyl ester and Annexin V/PI staining, respectively. Flow cytometry measurements were analyzed by CellQuest and NovoExpress. Taqman hydrolysis probes and SYBR Green PCR Mastermix were assessed in expression analyses of Wnt components utilizing 2-ΔΔCt method. Cell proliferation was inhibited by 50% at 2.5 mM VPA that evoked a significant apoptotic response. Among the screened Wnt components and target genes, only LEF1 exhibited significant four-fold upregulation at this concentration. In conclusion, cancer studies mostly utilize MTT or BrdU assays in estimating cell proliferation and calculating IC50 of drugs, which provided conflicting VPA dosages utilizing PANC-1 cells. Our novel combined approach enabled specific, accurate and reproducible IC50 calculation at single cell basis with no apparent effect on Wnt signaling components. Future studies are needed to clarify the role of LEF1 in this model.

Screening of AIP Gene Variations in a Cohort of Turkish Patients with Young-Onset Sporadic Hormone-Secreting Pituitary Adenomas.

Aims: Aryl hydrocarbon receptor-interacting protein (AIP) gene mutations have long been associated with apparently sporadic pituitary adenomas (PAs) with a prevalence range of 0-12%. The aim of this study was to evaluate the frequency of germline AIP variations in a large cohort of apparently sporadic PAs diagnosed before the age of 40 years, who did not exhibit hypercalcemia and/or MEN1 syndrome components during long-term follow-up. Materials and Methods: A total of 97 patients, diagnosed with functional PAs ≤40 years old, composed of somatotropinoma (n = 55), prolactinoma (n = 25), and corticotrophinoma (n = 17), were recruited for this study. Fifty-one of these patients [somatotropinoma (n = 30), prolactinoma (n = 15), and corticotrophinoma (n = 11)] were previously reported as AIP mutation-negative by Sanger sequencing. The entire coding sequence of the AIP gene, along with exon/intron boundaries and the untranslated regions of 41 newly recruited patients, were sequenced for germline variations. In addition, all patients were subjected to multiplex ligation-dependent probe amplification to detect copy number variations in the AIP gene. Results: The AIP c.911G>A: p.Arg304Gln (rs104894190) variant was detected in only two patients with functional PA: one with somatotropinoma [in 1/55 (1.8%)] and one with prolactinoma [in 1/25 (4%)]. None of the corticotrophinomas revealed AIP gene alterations. Thus, the overall prevalence of AIP variation was 2.1% in our cohort. Conclusions: Germline AIP gene variations among Turkish patients with apparently sporadic PAs are relatively rare among patients ≤40 years old. None of the patients in our cohort revealed any obviously pathogenic AIP variants.