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).

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.

The role of AGG interruptions in the FMR1 gene stability: A survey in ethnic groups with low and high rate of consanguinity.

BACKGROUND: The prevalence and the role of AGG interruptions within the FMR1 gene in the normal population is unknown. In this study, we investigated the frequent of AGG loss, in one or two alleles within the normal population. The role of AGG in the FMR1 stability has been assessed by correlating AGG loss to the prevalence of premutation/full mutation in two ethnic groups differing in their consanguinity rate: high versus low consanguinity rate (HCR vs. LCR). METHODS: The CGG repeat allele size and AGG presence were measured in 6,865 and 6,204 females belonging to the LCR (5%) and HCR (>45%) groups, respectively, by Tripled-Primed-PCR technique. RESULTS: A lower prevalence of the premutation was observed in the HCR (1:158) as compared to the LCR group (1:128). No full mutation was found in the HCR females while in the LCR group the prevalence found was 1:1,149. Homozygosity rate was higher in the HCR population compared to the LCR group.The overall AGG loss was higher in the HCR population than in the LCR and increased with increased CGG repeat number in both ethnic groups. CONCLUSIONS: Although we observed a significantly higher rate of homozygosity and AGG loss in the HCR group, this did not affect the prevalence of the premutation and full mutation in this population. Their prevalence was significantly lower than in the LCR population. Finally, we discuss whether the loss of AGG could be also a polymorphic event but not only a stabilizing factor.

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.

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.

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.

Chromosomal Microarray Analysis Results From Pregnancies With Various Ultrasonographic Anomalies.

OBJECTIVE: To examine chromosomal microarray analysis results in pregnancies with various ultrasonographic anomalies and to characterize the copy number variants in diverse fetal phenotypes. METHODS: We retrospectively examined chromosomal microarray analyses of amniocenteses performed nationwide as a result of fetal ultrasonographic anomalies (structural defects, fetal growth restriction, and polyhydramnios) between January 2013 and September 2017. The rate of abnormal chromosomal microarray findings was compared between the different phenotypes and with a previously described control population of 15,225 pregnancies with normal ultrasonographic findings. RESULTS: Clinically significant chromosomal microarray aberrations were detected in 272 of 5,750 pregnancies (4.7%): 115 (2%) karyotype-detectable and 157 (2.7%) submicroscopic. Most commonly detected copy number variants were 22q11.21 deletions (0.4%) followed by 22q11.21 gain of copy number (0.2%). Specific copy number variants detected among pregnancies with abnormal ultrasonographic findings were up to 20-fold more prevalent compared with low-risk pregnancies. Some variants were associated with specific phenotypes (eg, 22q11.21 microdeletions with cardiovascular and 17q12 microdeletions with genitourinary defects). CONCLUSION: The rate of abnormal amniotic chromosomal microarray analysis results is twice that of karyotypic abnormalities in pregnancies with various abnormal ultrasonographic findings.

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.

Prenatal Diagnosis of Fragile X: Can a Full Mutation Allele in the FMR1 Gene Contract to a Normal Size?

Here we describe a case of a prenatal diagnosis of a male fetus that inherited the unstable allele from his full mutation mosaic mother (29, 160, >200 CGG repeats) reduced to a normal size range (19 CGG repeats). Haplotype analysis showed that the fetus 19 CGG repeats allele derived from the maternal unstable allele which was inherited from his maternal grandmother. No size mosaicism was detected by testing the DNA from in vitro cultured samples, including seventh passage culture as well as from two amniocentesis samples. Sequence analysis confirmed that the allele was 19 CGG repeats long. Methylation assay showed no methylation. Although none of the techniques used in this study can provide with absolute certainty the diagnosis, the results strongly indicate the presence in the fetus of an allele with a CGG repeat number in the normal range. Because this is a prenatal diagnosis case, the crucial question is whether the 19 CGG allele derived from the maternal unstable expanded allele, which contracted to the normal range, became a normal stable allele or a normal unstable allele which could expand in the next generation. It is also possible that allele size mosaicism of the FMR1 gene that went undetected exists. Because this is a prenatal diagnosis case, we cannot with certainty exclude the presence of an undetected expanded allele of the FMR1 gene, in addition to the 19 CGG allele derived from an unstable expanded allele, which contracted to the normal range.

Can cell proliferation of umbilical cord blood cells reflect environmental exposures?

Environmental hazards were shown to have an impact on cell proliferation (CP). We investigated CP of lymphocytes in umbilical cord blood in relation to prenatal environmental exposures in a sample of 346 Arab-Bedouin women giving birth in a local hospital. Information on subjects' addresses at pregnancy, potential household exposures and demographical status was collected in an interview during hospitalization. This population is usually featured by high rates of neonatal morbidity and multiple environmental exposures, originating from the local industrial park (IP), household hazards and frequent male smoking. A geometric mean CP ratio 2.17 (2.06; 2.29), and was high in women residing in a direction of prevailing winds from the local IP (p value = 0.094) and who gave birth during fall-winter season (p value = 0.024). Women complaining on disturbing exposure to noise had lower CP (p value = 0.015), compared to other women. CP was not indicative of neonatal morbidity. However, our findings suggest that CP of umbilical cord might be modified by environmental exposures. A long-term follow-up of the children is required to assess their developmental outcomes.

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.

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.

Oral squamous cell carcinoma in patients twenty years of age or younger--review and analysis of 186 reported cases.

To review the literature on reported cases of squamous cell carcinoma (SCC) of the oral cavity in patients twenty-years-of-age or younger. All well-documented cases of oral SCC in patients twenty-years-of-age or less, published between 1936 and 2012, were collected and the clinicopathologic features were evaluated. Primary cases of oral SCC were selected. Age, gender, intra-oral subsite were recorded. A total of 88 articles describing 186 cases were included. The group of otherwise healthy patients had a mean age of 14.08years (range newborn-20years), the m:f ratio was 1.36; the oral subsites were the tongue, gingiva, and lower lip. A second group of patients who have disorders that predispose to cancer development, such as xeroderma pigmentosum, Fanconi's anemia, and a history of bone marrow transplant, had a mean age of 13.17years (range 5-20years); the m:f ratio was 1.23; the oral subsite was mainly the tongue. There was a slight difference between otherwise healthy patients and patients with predisposing systemic factors, but this difference was not statistically significant. It contrast to adults, there is only a weak predilection for males (m:f ratio of 1.23-1.36). In the young population SCC occurs most frequently in the tongue, followed by gingiva and lips. Unlike in adults, SCC is very uncommon in the floor of mouth. Oral SCC may, indeed, occur in patients younger than 20years and clinicians should take cognizance of this. Periodical examination of the oral cavity of young patients is recommended in cases of systemic diseases that predispose to cancer development such as xeroderma pigmentosum, Fanconi's anemia, and a history of bone marrow transplant.

Study of plasma-induced peripheral blood mononuclear cells survival using Fourier transform infrared microspectroscopy.

Components present in the acellular fraction of blood influence the blood cell survival and function and the response to biotic and abiotic factors. Human plasma and sera have been used as therapeutic agents and are known to increase cell survival. White blood cells in normal blood are exposed to plasma components in vivo, but the effect of such plasma components in vitro on adherent peripheral blood mononuclear cells (PBMCs) that includes monocytes has not been fully investigated. We cultured human PBMCs with autologous plasma and observed structural variation due to plasma addition in PBMCs along with increased cell survival. Light microscopy of the cells showed increased granularity in plasma-treated cells. Fourier transform infrared (FTIR) spectroscopy was used to elucidate the possible mechanism by studying the changes in the biochemical composition of the cells that explained the observations. FTIR spectroscopy of plasma-treated cells show altered spectral pattern in the mid-IR region, indicating increased phospholipid levels. Heat-stable components in the plasma possibly increase the differentiation of PBMCs, as evident by increased phospholipid metabolism. The data suggest that plasma-stimulated membrane biogenesis may contribute to PBMC survival by inducing them to differentiate into antigen presenting cells (APCs) like macrophages and dendritic cells.

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.

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.

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.

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.

Pathological fracture of the mandible in a paediatric patient with congenital insensitivity to pain with anhidrosis (CIPA).

Congenital insensitivity to pain with anhidrosis (CIPA) is a rare hereditary sensory and autonomic neuropathy (HSAN) characterized by pain, self-mutilating behaviour, anhidrosis and recurrent hyperthermia. CIPA has a multisystem involvement, including fractures of the extremities with slow healing, immunologic abnormalities, and a chronic inflammatory state. The mandible is reported to have a higher incidence of osteomyelitis, though mandibular fracture among CIPA patients, is very rare, with to our knowledge no reports in children. A case of pathological fracture of the mandible in a 6-year-old child with CIPA treated by ORIF is reported. In contrast to the slow healing reported in long bones, the mandible healed very quickly, possibly indicating that the osteoporotic mandible in this group of patients is different from that seen in the elderly. Furthermore, the standard ORIF technique can be safely used in this rare group.

Chromosomal aberrations in adenomatoid hyperplasia of palatal minor salivary gland.

Adenomatoid hyperplasia of minor salivary glands is rare, idiopathic, and benign, and typically presents as a tumour-like mass in the hard or soft palate. Its exact nature is not clear and histological examination usually shows an excess of normal-appearing minor salivary glands. To our knowledge, cytogenetic analysis of it in a minor salivary gland of the palate has not previously been reported. We present the cytogenetic analysis of adenomatoid hyperplasia in the hard palate of a 52-year-old woman.