Fanconi Anemia Patients from an Indigenous Community in Mexico Carry a New Founder Pathogenic Variant in FANCG.

Fanconi anemia (FA) is a rare genetic disorder caused by pathogenic variants (PV) in at least 22 genes, which cooperate in the Fanconi anemia/Breast Cancer (FA/BRCA) pathway to maintain genome stability. PV in FANCA, FANCC, and FANCG account for most cases (~90%). This study evaluated the chromosomal, molecular, and physical phenotypic findings of a novel founder FANCG PV, identified in three patients with FA from the Mixe community of Oaxaca, Mexico. All patients presented chromosomal instability and a homozygous PV, FANCG: c.511-3_511-2delCA, identified by next-generation sequencing analysis. Bioinformatic predictions suggest that this deletion disrupts a splice acceptor site promoting the exon 5 skipping. Analysis of Cytoscan 750 K arrays for haplotyping and global ancestry supported the Mexican origin and founder effect of the variant, reaffirming the high frequency of founder PV in FANCG. The degree of bone marrow failure and physical findings (described through the acronyms VACTERL-H and PHENOS) were used to depict the phenotype of the patients. Despite having a similar frequency of chromosomal aberrations and genetic constitution, the phenotype showed a wide spectrum of severity. The identification of a founder PV could help for a systematic and accurate genetic screening of patients with FA suspicion in this population.

Low bone mineral density and renal malformation in Mexican patients with Turner syndrome are associated with single nucleotide variants in vitamin D-metabolism genes.

Turner syndrome (TS) is a common genetic disorder. TS-phenotype includes short stature, gonadal dysgenesis, cardiac and kidney malformations, low bone mineral density (low-BMD) and thyroiditis. TS-phenotype varies from patient to patient and the cause is not clear, the genomic background may be an important contributor for this variability. Our aim was to identify the association of specific single nucleotide variants in the PTPN22, VDR, KL, and CYP27B1 genes and vitamin D-metabolism, heart malformation, renal malformation, thyroiditis, and low-BMD in 61 Mexican TS-patients. DNA samples were genotyped for SNVs: rs7975232 (VDR), rs9536282 (KL), rs4646536 (CYP27B1), and rs1599971 (PTPN22) using the KASP assay. Chi-square test under a recessive model and multifactorial dimensionality reduction method were used for analysis. We found a significant association between renal malformation and the rs9536282 (KL) variant and between rs4646536 (CYP27B1) and low-BMD, these variants may have modest effects on these characteristics but contribute to the variability of the TS phenotype. In addition, we identified gene-gene interactions between variants in genes KL, CYP27B1 and VDR related to vitamin D-metabolism and low-BMD in TS-patients. Our results support the idea that the genetic background of TS-patients contributes to the clinical variability seen in them.

Presence of 15p Marker D15Z1 on the Short Arm of Acrocentric Chromosomes is Associated with Aneuploid Offspring in Mexican Couples.

Variation in the location of the 15p region D15Z1 is recognized as a polymorphism in several human populations. We used high-stringency Fluorescence In Situ Hybridization (FISH) to detect D15Z1 in a Mexican cohort. Here, we report the presence of extra D15Z1 sequences on the p-arm of acrocentric chromosomes other than 15 in two groups of Mexican couples, one with healthy offspring (n = 75) and the other with aneuploid offspring (n = 87), mainly trisomy 21. The additional D15Z1 polymorphism was significantly increased in individuals with aneuploid offspring (26.4%), in comparison to individuals with healthy offspring (14%). The most frequent acceptor chromosome of D15Z1 was chromosome 13p, followed by 14p, and finally, 21p. Our results show an overall frequency of 21.6% of this polymorphism in the Mexican population and suggest that its presence might be associated with the mis-segregation of other acrocentric chromosomes and aneuploid offspring. The high frequency of the polymorphism of the D15Z1 sequence on acrocentric chromosomes other than 15 suggests a sequence homogenization of the acrocentric p arms, related to the important function of the centromere and the nucleolar organization region, which flank satellite III DNA.

Thiolated Au18 cluster: preferred Ag sites for doping, structures, and optical and chiroptical properties.

Recently, the X-ray determined structure of the thiolated Au18 cluster has been reported. In this communication, we addressed a study of structures and chiroptical properties of thiolated Au18 cluster doped with up to ten Ag atoms, which have been calculated by Time Dependent Density Functional Theory (TD-DFT). The number of Ag atoms was steadily varied and more stable isomers showed optical and Circular Dichroism (CD) spectra distinct from that found for the parent Au18 cluster. Doping with more than four Ag atoms results in enhancement of the oscillator strength of the HOMO-LUMO peak and it is expected that this feature can be exploited for photoluminescence applications.

NFE2L2 Gene Variants and Arsenic Susceptibility: A Lymphoblastoid Model.

Arsenic (As) exposure is a major risk for several types of cancer and metabolic diseases such as diabetes. The transcription factor nuclear factor erythroid 2-related factor (Nrf2) is a key mediator in the cellular defense against As-induced adverse effects. The -653G/A and -617C/A gene variants modulate expression levels of the Nrf2 coding gene (NFE2L2) and are postulated to be associated with several illnesses. In this study the functional effect of these polymorphisms was investigated in the cellular sensitivity to As-mediated effects. Using quantitative real-time polymerase chain reaction (qRT-PCR) the basal levels of NFE2L2 mRNA and the induced levels of NFE2L2 and its target gene NQO1 were measured in lymphoblastoid cells carrying different genotypes for -653G/A and -617C/A polymorphisms following As exposure. The effects of different NFE2L2 gene genotypes on cell proliferation were also explored after chronic metal exposure. A tendency toward reduction in basal levels of NFE2L2 mRNA was noted in the heterozygous (GA/CA) and risk homozygous (AA/AA) genotypes of both polymorphisms in immortalized lymphoblastoid cells. Although the expression of NFE2L2 and NQO1 after acute acute iAs exposure was not markedly influenced by -653G/A and -617C/A genotype, it was found that these single-nucleotide polymorphisms (SNPs) were correlated with a differential sensitivity to chronic exposure to the metalloid. Further studies are needed to completely understand the role of -653G/A and -617C/A SNPs in regulation of the NFE2L2 gene.

Reversion from basal histone H4 hypoacetylation at the replication fork increases DNA damage in FANCA deficient cells.

The FA/BRCA pathway safeguards DNA replication by repairing interstrand crosslinks (ICL) and maintaining replication fork stability. Chromatin structure, which is in part regulated by histones posttranslational modifications (PTMs), has a role in maintaining genomic integrity through stabilization of the DNA replication fork and promotion of DNA repair. An appropriate balance of PTMs, especially acetylation of histones H4 in nascent chromatin, is required to preserve a stable DNA replication fork. To evaluate the acetylation status of histone H4 at the replication fork of FANCA deficient cells, we compared histone acetylation status at the DNA replication fork of isogenic FANCA deficient and FANCA proficient cell lines by using accelerated native immunoprecipitation of nascent DNA (aniPOND) and in situ protein interactions in the replication fork (SIRF) assays. We found basal hypoacetylation of multiple residues of histone H4 in FA replication forks, together with increased levels of Histone Deacetylase 1 (HDAC1). Interestingly, high-dose short-term treatment with mitomycin C (MMC) had no effect over H4 acetylation abundance at the replication fork. However, chemical inhibition of histone deacetylases (HDAC) with Suberoylanilide hydroxamic acid (SAHA) induced acetylation of the FANCA deficient DNA replication forks to levels comparable to their isogenic control counterparts. This forced permanence of acetylation impacted FA cells homeostasis by inducing DNA damage and promoting G2 cell cycle arrest. Altogether, this caused reduced RAD51 foci formation and increased markers of replication stress, including phospho-RPA-S33. Hypoacetylation of the FANCA deficient replication fork, is part of the cellular phenotype, the perturbation of this feature by agents that prevent deacetylation, such as SAHA, have a deleterious effect over the delicate equilibrium they have reached to perdure despite a defective FA/BRCA pathway.

A Boolean network model of the FA/BRCA pathway.

MOTIVATION: Fanconi anemia (FA) is a chromosomal instability syndrome originated by inherited mutations that impair the Fanconi Anemia/Breast Cancer (FA/BRCA) pathway, which is committed to the repair of DNA interstrand cross-links (ICLs). The disease displays increased spontaneous chromosomal aberrations and hypersensitivity to agents that create DNA interstrand cross-links. In spite of DNA damage, FA/BRCA-deficient cells are able to progress throughout the cell cycle, probably due to the activity of alternative DNA repair pathways, or due to defects in the checkpoints that monitor DNA integrity. RESULTS: We propose a Boolean network model of the FA/BRCA pathway, Checkpoint proteins and some alternative DNA repair pathways. To our knowledge, this is the largest network model incorporating a DNA repair pathway. Our model is able to simulate the ICL repair process mediated by the FA/BRCA pathway, the activation of Checkpoint proteins observed by recurrent DNA damage, as well as the repair of DNA double-strand breaks and DNA adducts. We generated a series of simulations for mutants, some of which have never been reported and thus constitute predictions about the function of the FA/BRCA pathway. Finally, our model suggests alternative DNA repair pathways that become active whenever the FA/BRCA pathway is defective.

Frequent copy number variants in a cohort of Mexican-Mestizo individuals.

BACKGROUND: The human genome presents variation at distinct levels, copy number variants (CNVs) are DNA segments of variable lengths that range from several base pairs to megabases and are present at a variable number of copies in human genomes. Common CNVs have no apparent influence on the phenotype; however, some rare CNVs have been associated with phenotypic traits, depending on their size and gene content. CNVs are detected by microarrays of different densities and are generally visualized, and their frequencies analysed using the HapMap as default reference population. Nevertheless, this default reference is inadequate when the samples analysed are from people from Mexico, since population with a Hispanic genetic background are minimally represented. In this work, we describe the variation in the frequencies of four common CNVs in Mexican-Mestizo individuals. RESULTS: In a cohort of 147 unrelated Mexican-Mestizo individuals, we found that the common CNVs 2p11.2 (99.6%), 8p11.22 (54.5%), 14q32.33 (100%), and 15q11.2 (71.1%) appeared with unexpectedly high frequencies when contrasted with the HapMap reference (ChAS). Yet, while when comparing to an ethnically related reference population, these differences were significantly reduced or even disappeared. CONCLUSION: The findings in this work contribute to (1) a better description of the CNVs characteristics of the Mexican Mestizo population and enhance the knowledge of genome variation in different ethnic groups. (2) emphasize the importance of contrasting CNVs identified in studied individuals against a reference group that-as best as possible-share the same ethnicity while keeping this relevant information in mind when conducting CNV studies at the population or clinical level.

On the π coordination of organometallic fullerene complexes.

Novel organometallic complexes of fullerene C80 and aryl ligands were simulated. The nature and characteristics of this family of complexes involving π coordination between the fullerene and a metal centre have been studied from a theoretical point of view. We are particularly interested in complexes where η6 coordination is present, this being the strangest manifestation of known coordinations, and thus we have studied several known and simulated compounds of this kind in order to understand the lack of examples. The presence of other η6 or η5 ligands on the opposite side seems to be an important element aiding the stabilization of these complexes, also inducing the conductive and semiconductive behaviour of the studied species.

Hybridization vs. bond stretching isomerism in Ru(II) cyclometalated complexes of 2-phenylpyridine.

The phenomenon of formation of two isomers, yellow and orange, of the cyclometalated Ru(II) complex, [Ru(o-C6H4-py)(MeCN)4]⁺, was investigated by EELS spectroscopy and theoretical calculations. Both forms show very similar structures and spectroscopic properties, but slight differences in X-ray data and absorption between them were noted. No double minimum on the potential energy surface was found and thus these two forms cannot be considered as bond stretching isomers. However, the DFT study revealed the change in the hybridization of the carbon in trans-position of one of acetonitrile ligands. This effect can be responsible for the difference in colour. The results of the theoretical modelling coincide well with the experimental EELS data.

Capture of Fullerenes in Cages and Rings by Forming Metal-π Bond Arene Interactions.

Nowadays, the task of the selectively capture of fullerene molecules from soot is the subject of several studies. The low solubility of fullerenes represents a drawback when the goal is to purify them and to carry out chemical procedures where they participate. There are different molecules that can act as a kind of cocoon, giving shelter to the fullerene cages in such a way that they can be included in a solution or can be extracted from a mix. In this work, a theoretical study of some known and new proposed organic molecules of this kind is presented. In all cases, the interaction occurs with the help of a metallic atom or ion which plays the role of a bridge, providing a place for a metallocene like interaction to occur. The thermodynamic arguments favoring the formation of this adduct species are addressed as well as the nature of the bond by means QTAIM parameters and frontier molecular orbitals analysis.

MYC Promotes Bone Marrow Stem Cell Dysfunction in Fanconi Anemia.

Bone marrow failure (BMF) in Fanconi anemia (FA) patients results from dysfunctional hematopoietic stem and progenitor cells (HSPCs). To identify determinants of BMF, we performed single-cell transcriptome profiling of primary HSPCs from FA patients. In addition to overexpression of p53 and TGF-ß pathway genes, we identified high levels of MYC expression. We correspondingly observed coexistence of distinct HSPC subpopulations expressing high levels of TP53 or MYC in FA bone marrow (BM). Inhibiting MYC expression with the BET bromodomain inhibitor (+)-JQ1 reduced the clonogenic potential of FA patient HSPCs but rescued physiological and genotoxic stress in HSPCs from FA mice, showing that MYC promotes proliferation while increasing DNA damage. MYC-high HSPCs showed significant downregulation of cell adhesion genes, consistent with enhanced egress of FA HSPCs from bone marrow to peripheral blood. We speculate that MYC overexpression impairs HSPC function in FA patients and contributes to exhaustion in FA bone marrow.

Detection of short-term chromosomal damage due to therapeutic 131I exposure in patients with thyroid cancer.

We evaluated the chromosomal aberration (CA) frequencies in the peripheral blood lymphocytes of ten female patients, age average 43.7 +/- 12.9, with thyroid cancer (TC) who had been exposed to 100-200 mCi therapeutic doses of 131I. The blood samples were obtained before-treatment and at 2 and 24 h after-treatment. Radiation was measured in the samples by means of dysprosium-activated calcium sulfate thermoluminescent dosimetry. The maximum radiation levels were detected in the samples taken 2 h after treatment. A positive correlation was found between the sample-emitted radiation values and the frequencies of CAs (r = 0.495; p < 0.01). The average baseline frequency of aberrations found in the ten studied patients was 0.009 per cell. Upon application of the 131I therapeutic dose, this frequency increased to 0.04 and 0.02 CAs/cell at 2 and 24 h after-treatment, respectively (p < 0.05). Break-type aberrations experienced a peak at 2 h after-treatment, whereas rejoined aberrations, such as dicentrics, rings, and radial figures, increased with sampling time. Seven patients with metastases had high amounts of CAs at 2 and 24 h after-treatment, in comparison to three patients without metastases who had a lower frequency of CAs at 24h aftertreatment. This difference could be due to the fact that circulating lymphocytes were exposed to a greater cancerous tissue mass, which retains 131I during the diagnostic and therapeutic processes. These results demonstrate the importance of detecting and surgically removing the largest possible amount of thyroid tissue in order to diminish the exposure of normal cells to radiation.

Non-classical 1p36 deletion in a patient with Duane retraction syndrome: case report and literature review.

BACKGROUND: Monosomy of 1p36 is considered the most common terminal microdeletion syndrome. It is characterized by intellectual disability, growth retardation, seizures, congenital anomalies, and distinctive facial features that are absent when the deletion is proximal, beyond the 1p36.32 region. In patients with proximal deletions, little is known about the associated phenotype, since only a few cases have been reported in the literature. Ocular manifestations in patients with classical 1p36 monosomy are frequent and include strabismus, myopia, hypermetropia, and nystagmus. However, as of today only one patient with 1p36 deletion and Duane retraction syndrome (DRS) has been reported. CASE PRESENTATION: We describe a patient with intellectual disability, facial dysmorphism, and bilateral Duane retraction syndrome (DRS) type 1. Array CGH showed a 7.2 Mb de novo deletion from 1p36.31 to 1p36.21. DISCUSSION: Our patient displayed DRS, which is not part of the classical phenotype and is not a common clinical feature in 1p36 deletion syndrome; we hypothesized that this could be associated with the overlapping deletion between the distal and proximal 1p36 regions. DRS is one of the Congenital Cranial Dysinnervation Disorders, and a genetic basis for the syndrome has been extensively reported. The HES3 gene is located at 1p36.31 and could be associated with oculomotor alterations, including DRS, since this gene is involved in the development of the 3rd cranial nerve and the 6th cranial nerve's nucleus. We propose that oculomotor anomalies, including DRS, could be related to proximal 1p36 deletion, warranting a detailed ophthalmologic evaluation of these patients.

Nonclonal Chromosome Aberrations and Genome Chaos in Somatic and Germ Cells from Patients and Survivors of Hodgkin Lymphoma.

Anticancer regimens for Hodgkin lymphoma (HL) patients include highly genotoxic drugs that have been very successful in killing tumor cells and providing a 90% disease-free survival at five years. However, some of these treatments do not have a specific cell target, damaging both cancerous and normal cells. Thus, HL survivors have a high risk of developing new primary cancers, both hematologic and solid tumors, which have been related to treatment. Several studies have shown that after treatment, HL patients and survivors present persistent chromosomal instability, including nonclonal chromosomal aberrations. The frequency and type of chromosomal abnormalities appear to depend on the type of therapy and the cell type examined. For example, MOPP chemotherapy affects hematopoietic and germ stem cells leading to long-term genotoxic effects and azoospermia, while ABVD chemotherapy affects transiently sperm cells, with most of the patients showing recovery of spermatogenesis. Both regimens have long-term effects in somatic cells, presenting nonclonal chromosomal aberrations and genomic chaos in a fraction of noncancerous cells. This is a source of karyotypic heterogeneity that could eventually generate a more stable population acquiring clonal chromosomal aberrations and leading towards the development of a new cancer.

FANCC Dutch founder mutation in a Mennonite family from Tamaulipas, México.

BACKGROUND: Fanconi anemia (FA) (OMIM #227650) is a rare hereditary disease characterized by genomic instability. The clinical phenotype involves malformations, bone marrow failure, and cancer predisposition. Genetic heterogeneity is a remarkable feature of FA; at least 22 FANC genes are known to cooperate in a unique FA/BRCA repair pathway. A common rule on the mutations found in these genes is allelic heterogeneity, except for mutations known to have arisen from a founder effect like the FANCC c.67delG in the Dutch Mennonite Community. Here, we present an 11-year-old male patient, member of the Mennonite Community of Tamaulipas México, with a clinical and cytogenetic diagnosis of FA. METHOD: Chromosome fragility test was performed in all siblings. Genomic DNA was obtained from peripheral blood samples. Sanger sequencing was used to identify the FANCC c.67delG mutation (NC_000009.11(NM_000136.2):c.67delG p.(Asp23IlefsTer23)) and its accompanying haplotype. RESULTS: The FANCC c.67delG mutation in 13 members of his family confirmed a FA diagnosis in two of his siblings and identified heterozygous carriers. Haplotype analysis supports that in this family, FA is caused by the founder mutation that initially appeared in Mennonite Dutch and followed this population's migrations through Canada and further to Mexico. CONCLUSION: The identification of the FANCC c.67delG mutation in this family not only allows proper genetic counseling, but it also grants the possibility to raise awareness of FA risk among the Mennonite community living in Mexico.

Acrocentric cryptic translocation associated with nondisjunction of chromosome 21.

Down syndrome is the most frequent autosome aneuploidy in live newborns. It was recently proposed that pericentromeric cryptic translocations might be a cause of chromosome nondisjunction. We describe here a phenotypically normal subject with a cryptic translocation involving the short arms of chromosomes 13 or 21 and 22, who had a son with Down syndrome. Fluorescent in situ hybridization (FISH) on paternal metaphase chromosomes showed a chromosome 22 centromere positive for both 13/21 and 14/22 centromeric probes. The same probes hybridized on different and contiguous sites of chromatin fibers, eliminating cross-hybridization artifacts. This confirmed the presence of a cryptic translocation generating a dicentric chromosome 22: fib ish dic(21;22)(21 pter --> 21q10::22q10 --> 22 qter)(D13/21Z1+;D14/22Z1+). Microsatellite STR segregation analysis confirmed the paternal origin of the additional chromosome 21 in the Down syndrome patient. To determine whether the father showed a higher-than-normal frequency of chromosome 21 nondisjunction, FISH analysis of spermatozoa was performed using a sequence specific probe (21q22.13-q22.2). The frequency of disomy 21 spermatozoa was twofold higher in the cryptic translocation carrier as compared to normal subjects (P < 0.014), suggesting that the rearrangement favored the nondisjunction of chromosome 21. This is the first report associating a pericentromeric cryptic translocation of acrocentric chromosomes with the generation of aneuploidy, supporting the hypothesis that this type of rearrangement may contribute to abnormal chromosomal segregation.

Differential expression of TP53 associated genes in Fanconi anemia cells after mitomycin C and hydroxyurea treatment.

Fanconi anemia (FA) is a rare, heritable chromosomal instability disease characterized by several congenital defects and cancer predisposition. Functional interactions between specific FA proteins and DNA damage response and repair activities have been reported, but the interplay between these mechanisms for maintaining genomic stability are not well understood. Many DNA damage response proteins are transcriptionally regulated by the tumor suppressor protein p53 (TP53), suggesting an important regulatory role for the DNA damage and stress response pathway. To better understand the association between FA and the DNA damage stress response we analyzed the levels of chromosomal damage and damage mediated gene transcription responses in lymphoblastoid cells derived from normal individuals and patients carrying the most common FA complementation group (FA-A). Chromosomal aberrations were first measured after exposure to mitomicyn C (MMC) or hydroxyurea (HU). Aliquots of the same cell were than assayed for the transcriptional response of 21 DNA damage and stress response genes using quantitative real-time PCR. The FA-A lymphoblastoid cells showed significant increases in the frequency of chromosome aberrations relative to non-FA-A lymphoblastoid lines after MMC treatment. The MMC induced damage was correlated with a general increase in expression of TP53-modulated DNA damage stress response genes involved in processes such as DNA repair, cell cycle progression, and apoptosis. Following HU treatment FA cells showed a decreased induction of CAs with much less transcriptional differences between targeted genes. Overall, the differences between the normal and FA-A cells after genotoxic treatments imply an increased activation and reliance of FA cells on the down-stream activities of TP53 for prevention of cell killing and chromosome damage from interstrand crosslinks but not for general replication arrest and double strand breaks. Furthermore, these results imply a regulatory connection between the FA pathway and activation of TP53 for responding to DNA damage. Alterations in the regulation of the DNA damage response may be related to the complex phenotypes seen in FA patients.

Genomic chaos in peripheral blood lymphocytes of Hodgkin's lymphoma patients one year after ABVD chemotherapy/radiotherapy.

Hodgkin's lymphoma (HL) is a lymphoid malignancy representing 5% of all cancers in children, 16% in adolescents, and 30-40% of all malignant lymphomas and has a survival rate of ~95% at 10 years. One of the most common treatment schemes uses a cocktail of genotoxic agents including adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD) with or without radiotherapy. We investigated the occurrence of chromosomal damage in peripheral blood lymphocytes from five patients diagnosed with HL who provided samples before (BT), during chemotherapy (DT) and ~1 year after ABVD chemotherapy/radiotherapy (AT). Five healthy subjects served as controls. Chromosomal abnormalities were evaluated by multicolor fluorescence in situ hybridization. The average frequencies of structural chromosomal aberrations in HL samples were 0.11, 0.22, and 0.96 per cell in BT, DT, and AT samples, respectively. These frequencies were significantly different (P < 0.0001) with respect to control subjects (0.02 per cell). Interestingly, the highest frequency of structural damage, including genomic chaos and nonclonal abnormalities, was observed in the AT samples indicating that new aberrations were continuously produced. Rejoined structural chromosomal aberrations were the most common type of aberrations, although aneuploidies were also significantly increased. Finally, we found several chromosomal abnormalities linked to cancer secondary to treatment in all five HL patients. Our results show that ABVD chemotherapy plus radiotherapy is inducing genomic chaos in vivo; moreover, the persistence of genomic instability in the hematopoietic stem cells from HL patients may play a role in the occurrence of secondary cancer that is observed in 5-20% of HL patients. Environ. Mol. Mutagen. 59:755-768, 2018. © 2018 Wiley Periodicals, Inc.