Sandra Ramos

DEB test for Fanconi anemia detection in patients with atypical phenotypes

Pancytopenia, hyperpigmentation, small stature, congenital abnormalities, and predisposition to neoplasia characterize Fanconi anemia (FA). The clinical phenotype is extremely variable, therefore the diagnosis is frequently delayed until the pancytopenia appears, making diagnosis difficult on the basis of clinical manifestations alone. Hypersensitivity of FA cells to the clastogenic effect of diepoxybutane (DEB) provides a unique marker for the diagnosis before the beginning of hematological manifestations. Our aim in this study was to detect FA in children with atypical manifestations to define which conditions should be routinely included in the DEB test screening. We performed the chromosomal breakage test in 34 patients with probable FA and 83 patients with clinical conditions that could suggest FA, but are not usually screened by the DEB test: 20 patients with aplastic anemia, 20 patients with VACTERL association, 20 with radial ray abnormalities, 7 with tracheo‐esophageal fistulae, 12 with anal atresia, and 4 with myelodysplastic syndrome. We found 18 DEB‐positive patients: 12 were in the group of probable FA and 6 in the other groups. Among the last ones: three were included because of aplastic anemia, without any other sign of FA, however when re‐examined, other anomalies were detected. The third patient had anal atresia, renal hypoplasia, pre‐axial polydactyly, and normal blood cell counts and was diagnosed as having VACTERL association. The other two patients lacking physical or hematological signs were identified among the group of radial ray abnormalities. Thus, our results highlight the need to increase the number of abnormalities indicating need for a DEB test. Delay in the diagnosis of FA may have serious consequences for the patients and their family members.

Detection of mosaicism in lymphocytes of parents of free trisomy 21 offspring

Down syndrome (DS) resulting from free trisomy 21 (FT21) has been largely associated with advanced maternal age. However, approximately 60% of FT21 cases are born to young couples. Thus, the etiological factors responsible for these FT21 children must differ from those proposed for maternal age-related FT21. These factors have not been defined. In this study, we analyzed the chromosomes of peripheral blood lymphocytes from three groups of couples aged < or =35 years, to identify chromosomal trisomies: Group I included 5 couples with normal offspring; Group II included 22 couples with one FT21 child; and Group III consisted of 3 couples with recurrent FT21. A total of 13,809 metaphases were analyzed with G-banding and 60,205 metaphases were analyzed with FISH using a 13/21 centromeric probe. Aneuploidy was significantly more frequent in Groups II and III. The frequencies of hyperdiploid cells were 0.19, 0.49 and 0.96% in Groups I-III, respectively. FISH analysis showed that trisomy 21 cell percentages were 0.08, 0.21 and 0.76 for Groups I-III, respectively, and were very similar to those obtained with G-banding. Trisomy 21 mosaicism was found in 2/22 couples with one FT21 offspring, and in 2/3 couples with recurrent FT21. Our data suggest that mosaicism is an important cause of FT21 offspring in young couples, and that aneuploidy is more frequent among couples with FT21 offspring. This may be related with age and other undetermined intrinsic and extrinsic factors.

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)(21pter → 21q10::22q10 → 22qter)(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.

Omphalocele, bladder exstrophy, imperforate anus, spine defects complex, and bilateral cleft lip and palate in one product of a triplet pregnancy obtained by in vitro fertilization: A case report†

Omphalocele, bladder exstrophy, imperforate anus, and spine defects (OEIS) complex was described by Carey et al. [1978] as an association of four severe congenital malformations (OEIS). The OEIS complex is rare, affecting 1 in 200,000 to 1 in 400,000 births [Hurwitz et al., 1987]. We report on an infant with major malformations as a part of OEIS complex with cleft lip and palate (CLP) obtained in a triplet pregnancy after in vitro fertilization (IVF). Our patient is the first case of OEIS with CLP. IVF may have been a contributing factor in this case. We present a female infant of 5 months, born in Panama, conceived after IVF with frozen sperm from the 43-year-old biological father who was in good health. The mother, also 43 years old, was diagnosed with endometriosis at age 27, and treated with danazol that resulted in early menopause. Four previous cycles of IVF were unsuccessful. IVF was successful utilizing ten fresh ova from a healthy 27-year-old donor. Four embryos were implanted, but only three survived. Sonograms were performed monthly, without abnormalities noted in the three fetuses. Amniocentesis and chorionic villi sampling (CVS) were not done. A normal maternal serum alpha-fetoprotein was recorded at the fifth month of pregnancy. The pregnancy was interrupted, by abdominal section, at 32 weeks of gestation because of abruption placentae. Our index case was the third child born. The normal triplets were female with normal weights for gestational age, and normal physical examinations. Our patient weighed 1,118 g and her length was 37 cm. with Apgar scores of 8/9. She hadbilateral CLP, and major malformations including in omphalocele, cloacal exstrophy, and imperforate anus, which fulfilled the clinical criteria of OEIS complex (Fig. 1). Correction of omphalocele, cloacal exstrophy, and imperforate anus were performed in the immediate newborn period. She was referred to our Institute, at 5 months because of mild developmental delay. Her weight, length, and head circumference were below the 3rd centile, she had a depressed nasal bridge, bilateral epicanthal folds, facial asymmetry, bilateral CLP. The labiamajorawere separated, the anuswaspatent and the right foot clubbed. The lip and palate were repaired at 9 months (Fig. 2). Symphysis pubis diastasis was present and cervical spine films revealed vertebral segmentation defects, including hemivertebras. A CT cerebral scan only showed mild cortical atrophy in frontal region. Kidney ultrasound and karyotype were normal. We propose that our patient has OEIS complex, a rare sporadic association between four severe congenital malformations OEIS that was first described by Carey et al. [1978]. The etiology is very heterogeneous, although most of the cases are sporadic, there has been observed recurrence in sibs [Smith et al., 1992] and monocygotic twin cases [Lee

Hydroxyurea induces chromosomal damage in G2 and enhances the clastogenic effect of mitomycin C in Fanconi anemia cells

Fanconis anemia (FA) is a recessive disease; 16 genes are currently recognized in FA. FA proteins participate in the FA/BRCA pathway that plays a crucial role in the repair of DNA damage induced by crosslinking compounds. Hydroxyurea (HU) is an agent that induces replicative stress by inhibiting ribonucleotide reductase (RNR), which synthesizes deoxyribonucleotide triphosphates (dNTPs) necessary for DNA replication and repair. HU is known to activate the FA pathway; however, its clastogenic effects are not well characterized. We have investigated the effects of HU treatment alone or in sequential combination with mitomycin‐C (MMC) on FA patient‐derived lymphoblastoid cell lines from groups FA‐A, B, C, D1/BRCA2, and E and on lymphocytes from two unclassified FA patients. All FA cells showed a significant increase (P < 0.05) in chromosomal aberrations following treatment with HU during the last 3 h before mitosis. Furthermore, when FA cells previously exposed to MMC were treated with HU, we observed an increase of MMC‐induced DNA damage that was characterized by high occurrence of DNA breaks and a reduction in rejoined chromosomal aberrations. These findings show that exposure to HU during G2 induces chromosomal aberrations by a mechanism that is independent of its well‐known role in replication fork stalling during S‐phase and that HU interfered mainly with the rejoining process of DNA damage. We suggest that impaired oxidative stress response, lack of an adequate amount of dNTPs for DNA repair due to RNR inhibition, and interference with cell cycle control checkpoints underlie the clastogenic activity of HU in FA cells. Environ. Mol. Mutagen. 56:457–467, 2015.

Genomic chaos in peripheral blood lymphocytes of Hodgkin's lymphoma patients one year after ABVD chemotherapy/radiotherapy: Genomic Chaos in Peripheral Blood Lymphocytes

Hodgkins 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.

Unique association of hypochondroplasia with craniosynostosis and cleft palate in a Mexican family

Hypochondroplasia (HCH) is a skeletal dysplasia caused by an abnormal function of the fibroblast growth factor receptor 3. Although believed to be relatively common, its prevalence and phenotype are not well established owing to its clinical, radiological, and genetic heterogeneity. Here we report on a molecularly proven HCH family with an affected father and two children. The siblings (male and female) with HCH also had craniosynostosis and cleft palate, respectively. The present report supports the conclusion that the full clinical spectrum of HCH is not completely delineated. It also suggests that secondary, as yet unknown, modifying factors can influence the final phenotype.

A possible centromeric 21/22 translocation as an alternative cause of nondisjunction in trisomy 21

Two cases with normal karyotype and an additional signal in chromosome 22 with the 13/21 alpha satellite DNA probe have been reported in the literature. In both, one of the parents had the same marker, so an inherited polymorphism was considered. Here we describe another family case which is the first related to aneuploidy.The patient is a 16 month old boy with Down syndrome, he is the second product of young non consanguineous parents, there is family history of a paternal cousin who was a malformed stillbirth. The G banding karyotype was 47, XY+21 and FISH with centromeric 13/21 probe showed 6 signals instead of 5. Cytogenetics analysis with G and C bands and dual FISH with 13/21 and 14/22 probes evidenced that the extra signal was in a chromosome 22, which was inherited from the father because he revealed 5 marks with the 13/21 probe and 4 with the 14/22.This rearrangement could be explained by an inherited polymorphism, cross-hybridization or a translocation between the centromeric region of chromosomes 21 and 22. In this case, the father could be a new mutation or the product of an adyacent 1 segregation inherited from an ancestor, without an abnormal phenotype but with the risk of nondisjunction during the pairing at meiosis. In order to prove this hypothesis for genetic counseling, it is necessary to establish the parental origin of the patients extra chromosome 21, determine the frequency of nondisjunction in the fathers semen by FISH analysis and complete cytogenetic studies in other paternal relatives.

Chronic Burr-Hole ECoG and SEEG in the Assessment of Surgical Treatment for Epilepsy

Present day surgical treatment of epilepsy is based on the failure of medical treatment and the localization of epileptic focus. The most accurate technic is stereo-electro-encephalography (SEEG) consisting of chronic implantation of deep electrodes by a stereotactical technique. However the inconvenience of the high cost and risk of cerebral damage due to the introduction of necessarily numerous electrodes must be borne in mind.