Clonal evolution through genetic bottlenecks and telomere attrition: Potential threats to in vitro data reproducibility.

Tissue cultures of immortalized human cells, also known as established cell lines, are broadly accessible and cost-efficient tools for biomedical research. We here review potential genetic sources of systematic error in cell line experiments due to clonal evolution in vitro. In particular, the authors highlight alterations in telomere function over prolonged culture and population bottlenecks, respectively, as two commonly overlooked phenomena that can result in significant alterations in cell line genotypes over just one or a few passages in vitro. These alterations may include changes in mutation status of oncogenes and large scale chromosomal imbalances. We introduce a simple list of factors to be avoided in order to reduce the risk of data misinterpretation due to clonal evolution, including unacknowledged in vitro selection pressures, prolonged culture per se, harsh population size reductions, experiments at early phases after establishment, and the employment of cell lines not sufficiently analyzed by high resolution genetic techniques.

TMEM70 deficiency: Novel mutation and hypercitrullinemia during metabolic decompensation.

Respiratory chain disorders comprise a heterogeneous group of diseases that are the result of mutations in nuclear or mitochondrial genes. TMEM70 encodes a nuclear protein involved in the assembly of respiratory chain complex V. Although mutations in various genes can result in isolated complex V deficiency; TMEM70 mutations represent the most common reported etiology. TMEM70 deficiency is known to cause a syndrome of neonatal mitochondrial encephalocardiomyopathy, accompanied by elevated lactate and hyperammonemia. Elevated citrulline has been reported previously in different inborn errors of metabolism, although uncommonly associated with TMEMT70 deficiency. We present a series of two siblings diagnosed with TMEM70 deficiency, and describe hypercitrullinemia during decompensation as a new finding in this condition. The cause of hyperammonemia in TMEM70 deficiency was previously assumed to be related to carbamoyl phosphate synthase 1 deficiency, but our finding of hypercitrullinemia rules out this possibility. We thus propose a different etiology for the hyperammonemia seen in these patients.

Combined CNV, haplotyping and whole exome sequencing enable identification of two distinct novel EYS mutations causing RP in a single inbred tribe.

Whole exome sequencing (WES) has become routine in clinical practice, especially in studies of recessive hereditary diseases in inbred consanguineous families, where homozygosity of a founder mutation is assumed. Multiple members of two consanguineous families of a single Bedouin tribe were diagnosed with apparently autosomal recessive/pseudo-dominant retinitis pigmentosa (RP). Affected individuals exhibited severe visual impairment with nyctalopia, marked constriction of visual fields, markedly reduced and delayed responses on electro-retinography (ERG) and eventual loss of central vision. Combined copy-number variant (CNV) analysis, haplotype reconstruction and WES of the kindred identified two distinct novel mutations in EYS (RP25): a p.(W1817*) nonsense mutation (identified through WES) and a large deletion encompassing 9 of the 43 exons, that was missed by WES and was identified through microarray CNV analysis. Segregation analysis of both mutations demonstrated that all affected individuals were either homozygous for one of the mutations, or compound heterozygous for both. The two mutations are predicted to cause loss of function of the encoded protein and were not present in screening of 200 ethnically-matched controls. Our findings of two distinct mutations in the same gene in a single inbred kindred, identified only through combined WES and microarray CNV analysis, highlight the limitations of either CNV or WES alone, as the heterozygous deletion had normal WES read-depth values. Moreover, they demonstrate pitfalls in homozygosity mapping for disease-causing variant identification in inbred communities.

Can bone marrow cellularity help in predicting prognosis in myelodysplastic syndromes?

OBJECTIVES: To ascertain the relevance of bone marrow cellularity (BMC) to the interpretation of blast percentage (blast%) in MDS prognostication. METHODS: We compared survival prediction based on blast% adjusted to different levels of cellularity, compared to the survival based on the original IPSS-R blast% grouping. RESULTS: We analyzed 355 consecutive MDS patients. Cellularity, in and of itself or its interaction with blast%, was not associated with overall survival (OS). In a small subset of patients with a hypercellular marrow (15%; n = 26), dismal prognosis was observed at lower levels of blast%. For these cases OS was similar to higher IPSS-R blast groups. For example, within the Intermediate group (blast% 5%-10%), those with a hypercellular marrow and >6% blasts had an OS of 10 m similar to 16 m in the High (blast% 10%-19%) blast group. These changes did not translate into a significant improvement in overall prognostic power of a cellularity-adjusted IPSS-R (C index 0.71 vs. 0.70). CONCLUSION: Adjusting blast% to cellularity did not improve prognostication. However, within IPSS-R-defined blast groups, a small subset of patients with relatively higher blast% and hypercellularity may have a worse prognosis than expected.

Oral Mucoceles in Children--Analysis of 56 New Cases.

BACKGROUND: Mucoceles are common cystic lesions of the oral mucosa. Extravasation mucoceles (EMs) are mainly found in the lower lip of young patients, whereas retention mucoceles (RMs) are usually located in the cheek or palate of older patients. This study was undertaken to more fully characterize the clinicopathologic features of mucoceles in pediatric patients. METHODS: The records of 56 pediatric patients with mucoceles were included in the study. Age, sex, history of trauma, intraoral site, size, and method of treatment were evaluated. Histopathologically the lesions were classified as being EMs or RMs. RESULTS: The age range was 1.5 to 16 years (mean age 11.2 yrs). Of the 56 patients, 24 (43%) were males and 32 (57%) were females, with a male:female ratio of 1:1.33. A history of trauma was recorded in 32 (57%) patients of the lower lip. The intraoral sites were the lower lip (38 [68%]), tongue (10 [18%]), and floor of the mouth (8 [14%]). Of the 56 patients, 44 (79%) were EM and 12 (21%) were ranulas. No RMs were found. Mucoceles ranged from 0.3 to 3.8 cm in diameter (mean 0.9 cm). The treatment of EMs was surgical excision. Cryosurgey, electrosurgery, and carbon dioxide laser were also used. CONCLUSION: In contrast to adults, where EM and RM types can be found, among children all cases are of the EM type. The disparate site and age incidences of EMs and RMs of the oral mucosa suggest that these two types are not related and possibly have a different etiopathogenesis.

Primary ciliary dyskinesia caused by homozygous mutation in DNAL1, encoding dynein light chain 1.

In primary ciliary dyskinesia (PCD), genetic defects affecting motility of cilia and flagella cause chronic destructive airway disease, randomization of left-right body asymmetry, and, frequently, male infertility. The most frequent defects involve outer and inner dynein arms (ODAs and IDAs) that are large multiprotein complexes responsible for cilia-beat generation and regulation, respectively. Although it has long been suspected that mutations in DNAL1 encoding the ODA light chain1 might cause PCD such mutations were not found. We demonstrate here that a homozygous point mutation in this gene is associated with PCD with absent or markedly shortened ODA. The mutation (NM_031427.3: c.449A>G; p.Asn150Ser) changes the Asn at position150, which is critical for the proper tight turn between the ß strand and the α helix of the leucine-rich repeat in the hydrophobic face that connects to the dynein heavy chain. The mutation reduces the stability of the axonemal dynein light chain 1 and damages its interactions with dynein heavy chain and with tubulin. This study adds another important component to understanding the types of mutations that cause PCD and provides clinical information regarding a specific mutation in a gene not yet known to be associated with PCD.

Association between prenatal exposure to metals and neonatal morbidity.

An association between prenatal exposure to (semi-)metals and of neonatal morbidity was assessed by introducing an oxidative stress as a possible intermediate step. An oxidative stress was measured by cell proliferation (CP) ratio in umbilical cord blood cells. Urine samples of 18 out of 58 enrolled women (31%) were positive for (semi-)metals; 25.9% of women were positive for aluminum (Al). The CP ratio was higher (1) in subjects with Al, (2) in mothers to newborns diagnosed as small-for-gestational age (p value = .052), (3) neonates that weighed less (p value = .079), and (4) in women who experienced repeated abortions (p value = .049). Our findings suggest the possibility of metal-induced oxidative stress.

O-linked ß-N-acetylglucosaminylation (O-GlcNAcylation) in primary and metastatic colorectal cancer clones and effect of N-acetyl-ß-D-glucosaminidase silencing on cell phenotype and transcriptome.

O-linked ß-N-acetylglucosamine (O-GlcNAc) glycosylation is a regulatory post-translational modification occurring on the serine or threonine residues of nucleocytoplasmic proteins. O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase and O-GlcNAcase (OGA), which are responsible for O-GlcNAc addition and removal, respectively. Although O-GlcNAcylation was found to play a significant role in several pathologies such as type II diabetes and neurodegenerative diseases, the role of O-GlcNAcylation in the etiology and progression of cancer remains vague. Here, we followed O-GlcNAcylation and its catalytic machinery in metastatic clones of human colorectal cancer and the effect of OGA knockdown on cellular phenotype and on the transcriptome. The colorectal cancer SW620 metastatic clone exhibited increased O-GlcNAcylation and decreased OGA expression compared with its primary clone, SW480. O-GlcNAcylation elevation in SW620 cells, through RNA interference of OGA, resulted in phenotypic alterations that included acquisition of a fibroblast-like morphology, which coincides with epithelial metastatic progression and growth retardation. Microarray analysis revealed that OGA silencing altered the expression of about 1300 genes, mostly involved in cell movement and growth, and specifically affected metabolic pathways of lipids and carbohydrates. These findings support the involvement of O-GlcNAcylation in various aspects of tumor cell physiology and suggest that this modification may serve as a link between metabolic changes and cancer.

Autosomal-recessive hypophosphatemic rickets is associated with an inactivation mutation in the ENPP1 gene.

Human disorders of phosphate (Pi) handling and hypophosphatemic rickets have been shown to result from mutations in PHEX, FGF23, and DMP1, presenting as X-linked recessive, autosomal-dominant, and autosomal-recessive patterns, respectively. We present the identification of an inactivating mutation in the ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene causing autosomal-recessive hypophosphatemic rickets (ARHR) with phosphaturia by positional cloning. ENPP1 generates inorganic pyrophosphate (PPi), an essential physiologic inhibitor of calcification, and previously described inactivating mutations in this gene were shown to cause aberrant ectopic calcification disorders, whereas no aberrant calcifications were present in our patients. Our surprising result suggests a different pathway involved in the generation of ARHR and possible additional functions for ENPP1.

Cytogenetics of central giant cell granuloma of the mandible.

PURPOSE: Central giant cell granuloma is a benign entity that commonly occurs in the mandible and maxilla. It is usually treated by surgical excision, varying from curettage to en bloc resection. Because the entity is more common in diseases such as neurofibromatosis, a genetic element may be involved in its pathogenesis. Cytogenetic studies of central giant cell granuloma affecting bone are rare, and to the authors' knowledge, there are none reported in the literature for central giant cell granuloma of the mandible. MATERIALS AND METHODS: The authors investigated the cytogenetic profile of a case occurring in the mandible. Fresh biopsy tissue was minced and cultured in RPMI-1640 medium. Cells were fixed and stained, and cytogenetic analysis was performed according to standard procedures. RESULTS: A clone with t(1;17;18) and other random numerical chromosomal changes was found. CONCLUSIONS: The significance of these findings in diagnosis and prognosis is currently unclear and further karyotyping studies are needed to more fully understand this tumor.

Pleomorphic adenoma occurring outside the submandibular gland: a case report of an accessory submandibular gland.

The accessory submandibular gland is a rare anatomic variant and the incidence of pathology reported within an accessory submandibular gland is even rarer. This report describes the case of a 22-year-old woman who presented with a slowly enlarging mass in the submandibular triangle, which on ultrasound examination suggested that it was close to, but not arising from, the submandibular gland. Fine-needle aspiration cytology was consistent with a pleomorphic adenoma. At surgery, the tumor was found to be entirely separate from the submandibular gland. This case presents an extremely rare occurrence of an accessory submandibular gland and, to the authors' knowledge, is the first report of a pleomorphic adenoma occurring within an accessory submandibular gland.

Metachronous pleomorphic adenomas occurring in the parotid and a minor salivary gland with genetic changes detected by comparative genomic hybridization.

A 32-year-old female underwent an extracapsular dissection for a pleomorphic adenoma (PA) of the parotid gland. Six months later, she presented with an increasing lump on the upper lip that, following excision, was confirmed to be a second PA. Formalin-fixed paraffin embedded tissues were analyzed for chromosomal aberrations. Comparative genomic hybridization analysis showed multiple chromosomal aberrations in the parotid PA. In comparison, no chromosomal aberrations were found in the lip PA. To our knowledge, metachronous benign pleomorphic adenomas occurring in both a major and minor salivary gland is unreported, and furthermore, there are no comparative genomic hybridization reports of this rare occurrence. We discuss the clinicopathological implications.

Trisomy 7 as sole aberration in peripheral ameloblastoma of the mandible.

Peripheral ameloblastoma (PA) is a rare, extraosseous odontogenic tumor with histologic features similar to those of the more common intraosseous ameloblastoma. The exact nature and tumorigenesis of PA are unclear. Although there are some reports on the cytogenetics of intraosseous ameloblastoma, to the authors' knowledge, there are no studies on the cytogenetic analysis of PA. The cytogenetic analysis of a PA occurring in the gingiva of a 56-year-old man is presented. Trisomy 7 was the only cytogenetic aberration.

[Introsseous schwannoma of the mandible]. / Intraosser schwannoma a mandibulában.

Schwannoma is a benign neoplasm originating from the neural sheath and occuring most often in the soft tissues of the head and neck. Intraosseous schwannoma (IS) is extremely rare, most commonly occurring in the mandible. This paper documents a case of IS and the histopathology, karyotyping, CT, and MRI in the diagnostic work-up. Histologically it was a classic schwannoma. The karyotype was normal. CT demonstrated destruction of the mandibular canal while MRI detected encasement of the inferior alveolar nerve by the tumor, and consequently the need to resect the nerve together with the tumor. Using CT and MRI, the morbidity associated with the anticipated surgery can precisely be determined preoperatively.

Cystic lesions of the jaws - a clinicopathological study of 322 cases and review of the literature.

Three hundred and twenty-two patients (192 male and 130 female) with cystic lesions of the jaw were successfully diagnosed and treated. One hundred and fifty-five (48%) were radicular cysts, 80 (25%) were dentigerous cysts, 23 (7%) were odontogenic keratocyst (=keratocystic odontogenic tumor), 19 (6%) were eruption cysts, 16 (5%) were traumatic bone cysts, and 29 (9%) were non-odontogenic cysts. There were 95 in the pediatric age group (1 month to 16 years) and 227 in the adult age group (17 years and older). Male to female ratio was 1 in the pediatric age group and 1.7 in the adult age group. The treatment modalities were: marsupialization, enucleation, enucleation with bone grafting, or resection. The distribution and characteristics of jaw cysts in children are different from those in adults. In children there is a relatively high rate of developmental cysts, whereas in adults the inflammatory cysts are more common. Following enucleation of a cystic jaw lesion, the entire surgical specimen and not only a biopsy specimen, should be examined histopathologically to prevent any possibility of an intramural squamous cell carcinoma that may be overlooked. The differences in prevalence of each type of jaw cyst during a lifetime may point toward a multifactorial polygenic pattern rather than a monogenic pattern.

Chromosomal aberrations in minor salivary gland pleomorphic adenoma.

PURPOSE: Cytogenetic analysis of a pleomorphic adenoma (PA) arising in the major salivary glands, in particular the parotid, is well documented, with chromosome 8 being the most commonly involved aberration, mainly in t(3;8). However, cytogenetic studies of PA in the minor salivary glands (MSGs) are rare and, to the authors' knowledge, only 3 reports have been published. The authors investigated the cytogenetic abnormalities of a series of 6 PAs arising from MSGs and compared these with published findings from the parotid gland to determine whether the karyotype was the same in the 2 sites. MATERIALS AND METHODS: Six fresh samples of MSG PA were examined by classic cytogenetic analysis. The tissue was minced and cultured in RPMI-1640 medium. The cells were fixed after 2 to 8 days of culture and analyzed according to standard procedures. More than 25 metaphases were analyzed on G-banded slides, and the karyotype was described according to International System for Human Cytogenetic Numenclature guidelines. RESULTS: The spectrum of chromosomal aberrations found in the MSG PAs was similar to those reported in the major salivary glands in all 6 cases. CONCLUSIONS: Cytogenetically, there would seem to be no clear differences in PAs arising from the major salivary glands versus the MSGs. It is unknown whether the underlying tumorigenesis and chromosomal aberrations of PAs from major salivary glands and MSGs are similar, although the proportion of malignant tumors arising from the MSGs is much larger compared with the parotid. Further studies are needed in this area.

Cutaneous sinus tract of dental origin in children-a report of 28 new cases.

Little is known about the characteristics of cutaneous sinus tract (CST) of dental origin in children. 28 cases of CST of dental origin in children were evaluated. Age, sex, site of skin lesion, duration until diagnosis, teeth involved, etiology, treatment, and outcome were recorded. The last dental care visit was also recorded. The mean age was 10.25 years (range 4-16). The male to female ratio was 1:1.74. The mandibular-submandibular area was the most common site of skin lesions. The mandibular first molar was the most involved tooth, followed by the mandibular incisor. Caries was the most common etiology. The mean duration of lesions until correct diagnosis was 6.5 months (range 0.3-12 mos). The treatment was root canal therapy or extraction. After appropriate dental treatment, CSTs resolve rapidly. Surgical revision of the scars were indicated in eight (29%) patients, to provide better cosmetic results. In these patients, the duration of lesions were longer. Preventive dental care, as indicated by last dental care visit, was poor. CST in children is different from that in adults in terms of sites of skin lesions, duration of lesions, and involved tooth and similar to that in adults in terms of etiology and treatment modality. Early treatment of the dental infection may cause healing of the cutaneous lesion spontaneously, without a scar.

Metastatic neuroblastoma of the mandible: a cytogenetic and molecular genetic study.

Neuroblastoma (NB) jaw metastases are rare. Here, we report on cytogenetic and genetic studies on metastatic NB to the mandible. A 7-year-old boy, with an abdominal neuroblastoma, presented with a mass of the left body of the mandible. Cytogenetic analysis of the original tumor and the mandibular lesion biopsies revealed similar heterogenous subclones with 42 ~ 47,XY,+der(1)(q11 → qter),-2,del(7)(q21.1 → qter),-8,-9,-10,-11,del(11)(q13.3 → qter),-13,-14,-15,-17, + 18-18,der(18)(?),+21,+m1,+m2,+m3,+m4,+m5,+m6,+m7[cp25]. The different markers were identified by SKY analysis. Most of the cells carried 3-6 of these translocations: der(1;21), der(2;9;17), der(2;15;18), der(2;15;Y), der(8;10), der(10;17). Molecular examination using Neuroblastoma MLPA kit (MRC-Holland) revealed gain of 1q25, 1q42, 2q33, 2p23, 2p24 (N-myc), and 21q22, and loss of 11q22, 11q23, 17p13, and 17q11. FISH analysis using N-myc probe showed high amplification levels of N-myc. The cytogenetic and molecular genetic work-ups revealed that the mandibular lesion is a metastasis of the original abdominal tumor and not a second primary caused by the aggressive treatment. Clinical parameters such as : patient's age, site of primary tumor and the mandibular metastasis, together with poor prognosis genetic markers explain the patient's short-term survival.

A DAPI-Based Modified C-banding Technique for a Rapid Achieving High Photographic Contrast of Centromeres on Chromosomes.

Many chromosome assays rely on the quantification of chromosome abnormalities in cells, and one important abnormality is the existence of more than one centromere for each chromosome. The quantification of such abnormalities has been studied before. However, this process is labor-intensive and time consuming. Thus, this assay is challenging for ex-laboratory applications, where speed is required. We present a visualization method that uses a cheap stain-DAPI, long (e.g., high-resolution) chromosomes and our modified C-banding method for labeling chromosomes. The labeled chromosomes can then be easily seen with a conventional and readily available fluorescence microscopy system. This method achieves an acceleration of the detection of the presence of constitutive heterochromatin in chromosomal centromeres by more than 10 times, to ~2 h, in Human lymphocyte cells and in cells of the human Jurkat line. This new procedure will ultimately provide an easier and cheaper alternative to FISH/PNA probes, or the classic Giemsa staining method. Simplification and reduction in time of the overall procedure will enable the utilization of centromere-counting assays in laboratory and ex-laboratory applications, including in emergency response.

De Novo Large Deletion Leading to Fragile X Syndrome.

Fragile X syndrome (FXS) is the most frequent cause of X-linked inherited intellectual disabilities (ID) and the most frequent monogenic form of autism spectrum disorders. It is caused by an expansion of a CGG trinucleotide repeat located in the 5'UTR of the FMR1 gene, resulting in the absence of the fragile X mental retardation protein, FMRP. Other mechanisms such as deletions or point mutations of the FMR1 gene have been described and account for approximately 1% of individuals with FXS. Here, we report a 7-year-old boy with FXS with a de novo deletion of approximately 1.1 Mb encompassing several genes, including the FMR1 and the ASFMR1 genes, and several miRNAs, whose lack of function could result in the observed proband phenotypes. In addition, we also demonstrate that FMR4 completely overlaps with ASFMR1, and there are no sequencing differences between both transcripts (i.e., ASFMR1/FMR4 throughout the article).