Using a combination of MLPA kits to detect chromosomal imbalances in patients with multiple congenital anomalies and mental retardation is a valuable choice for developing countries

Conventional karyotyping detects anomalies in 3-15% of patients with multiple congenital anomalies and mental retardation (MCA/MR). Whole-genome array screening (WGAS) has been consistently suggested as the first choice diagnostic test for this group of patients, but it is very costly for large-scale use in developing countries. We evaluated the use of a combination of Multiplex Ligation-dependent Probe Amplification (MLPA) kits to increase the detection rate of chromosomal abnormalities in MCA/MR patients. We screened 261 MCA/MR patients with two subtelomeric and one microdeletion kits. This would theoretically detect up to 70% of all submicroscopic abnormalities. Additionally we scored the de Vries score for 209 patients in an effort to find a suitable cut-off for MLPA screening. Our results reveal that chromosomal abnormalities were present in 87 (33.3%) patients, but only 57 (21.8%) were considered causative. Karyotyping detected 15 abnormalities (6.9%), while MLPA identified 54 (20.7%). Our combined MLPA screening raised the total detection number of pathogenic imbalances more than three times when compared to conventional karyotyping. We also show that using the de Vries score as a cutoff for this screening would only be suitable under financial restrictions. A decision analytic model was constructed with three possible strategies: karyotype, karyotype + MLPA and karyotype + WGAS. Karyotype + MLPA strategy detected anomalies in 19.8% of cases which account for 76.45% of the expected yield for karyotype + WGAS. Incremental Cost Effectiveness Ratio (ICER) of MLPA is three times lower than that of WGAS, which means that, for the same costs, we have three additional diagnoses with MLPA but only one with WGAS. We list all causative alterations found, including rare findings, such as reciprocal duplications of regions deleted in Sotos and Williams-Beuren syndromes. We also describe imbalances that were considered polymorphisms or rare variants, such as the new SNP that confounded the analysis of the 22q13.3 deletion syndrome. (C) 2011 Elsevier Masson SAS. All rights reserved.

Extending the Phenotype of Monosomy 1p36 Syndrome and Mapping of a Critical Region for Obesity and Hyperphagia

Rearrangements of 1p36 are the most frequently detected abnormalities in diagnostic testing for chromosomal cryptic imbalances and include variably sized simple terminal deletions, derivative chromosomes, interstitial deletions, and complex rearrangements. These rearrangements result in the specific pattern of malformation and neurodevelopmental disabilities that characterizes monosomy 1p36 syndrome. Thus far, no individual gene within this region has been conclusively determined to be causative of any component of the phenotype. Nor is it known if the rearrangements convey phenotypes via a haploinsufficiency mechanism or through a position effect. We have used multiplex ligation-dependent probe amplification to screen for deletions of 1p36 in a group of 154 hyperphagic and overweight/obese, PWS negative individuals, and in a separate group of 83 patients initially sent to investigate a variety of other conditions. The strategy allowed the identification and delineation of rearrangements in nine subjects with a wide spectrum of clinical presentations. Our work reinforces the association of monosomy 1p36 and obesity and hyperphagia, and further suggests that these features may be associated with non-classical manifestations of this disorder in addition to a submicroscopic deletion of similar to 2-3 Mb in size. Multiplex ligation probe amplification using the monosomy 1p36 syndrome-specific kit coupled to the subtelomeric kit is an effective approach to identify and delineate rearrangements at 1p36. (C) 2009 Wiley-Liss, Inc.

MLPA analysis in 30 Sotos syndrome patients revealed one total NSD1 deletion and two partial deletions not previously reported

Sotos syndrome (MIM #117550) is an autosomal dominant condition characterized by pre and postnatal overgrowth, macrocephaly and typical facial gestalt with frontal bossing, hypertelorism, antimongoloid slant of the palpebral fissures, prominent jaw and high and narrow palate. This syndrome is also frequently associated with brain, cardiovascular, and urinary anomalies and is occasionally accompanied by malignant lesions such as Wilms turnout and hepatocarcinoma. The syndrome is known to be caused by mutations or deletions of the NSD1 gene. To detect both 5q35 microdeletions and partial NSD1 gene deletions we screened 30 Brazilian patients with clinical diagnosis of Sotos syndrome by multiplex ligation dependent probe amplification. We identified one patient with a total deletion of NSD1 and neighbouring FGFR4, other with missing NSD1 exons 13-14 and another with a deletion involving FGFR4 and spanning up to NSD1 exon 17. All deletions were de novo. The two NSD1 partial deletions have not been previously reported. The clinical features of the three patients included a typical facial gestalt with frontal bossing, prominent jaw and high anterior hairline; macrocephaly, dolichocephaly, large hands; neonatal hypotonia and jaundice. All presented normal growth at birth but postnatal overgrowth. Two patients with NSD1 and FGFR4 gene deletions presented congenital heart anomalies. (C) 2009 Elsevier Masson SAS. All rights reserved.

De novo copy number variants identify new genes and loci in isolated sporadic tetralogy of Fallot

Tetralogy of Fallot (TOF), the most common severe congenital heart malformation, occurs sporadically, without other anomaly, and from unknown cause in 70% of cases. Through a genome-wide survey of 114 subjects with TOF and their unaffected parents, we identified 11 de novo copy number variants (CNVs) that were absent or extremely rare (<0.1%) in 2,265 controls. We then examined a second, independent TOF cohort (n = 398) for additional CNVs at these loci. We identified CNVs at chromosome 1q21.1 in 1% (5/512, P = 0.0002, OR = 22.3) of nonsyndromic sporadic TOF cases. We also identified recurrent CNVs at 3p25.1, 7p21.3 and 22q11.2. CNVs in a single subject with TOF occurred at six loci, two that encode known (NOTCH1, JAG1) disease-associated genes. Our findings predict that at least 10% (4.5-15.5%, 95% confidence interval) of sporadic nonsyndromic TOF cases result from de novo CNVs and suggest that mutations within these loci might be etiologic in other cases of TOF.

Epstein-Barr virus detection in invasive and pre-invasive lesions of the uterine cervix

In the present study, our aim was to investigate whether EBV DNA could be found in association with invasive and pre-invasive cervical cancer lesions. We hypothesize that EBV is not merely a commensal agent when present in malignant cervical lesions. DNA was extracted from cervical scrapings followed by nested PCR-based amplification. The patients were 66 women with high grade cervical intraepithelial neoplasia and 14 women with invasive cervical cancer. The control group consisted of 89 women with a normal Pap smear and colposcopy as well as a negative HPV DNA test. Analysis of our results, in conjunction with the work of other authors, leads us to propose that EBV is not merely a commensal agent when present in malignant cervical lesions. The presence of DNA from EBV is significantly associated with cervical cancer.

COL1A1 Sp1-binding site polymorphism as a risk factor for genital prolapse

The objective of this study was to verify the possible association between the Sp1-binding site polymorphism and genital prolapse. A case-control study was conducted in 107 patients with stages III and IV genital prolapse. The control group included 209 women with stages 0 and I. The polymorphism of type I collagen Sp1-binding site was identified by amplification of the first intron of the COL1A1 gene. We did not find differences in the prevalence of the GT and TT genotypes between the groups (p=0.34), even when we grouped patients with at least one polymorphic allele (GT and TT) and compared them with patients without the polymorphic allele (GG; p=0.17) The presence of at least one vaginal delivery, family history for prolapse, and macrosomatic fetus were independent risk factors for prolapse. In conclusion, the COL1A1 Sp1-binding site was not significantly associated with genital prolapse among our study subjects.

Primary retroperitoneal lipoma: A soft tissue pathology heresy? Report of a case with classic histologic, cytogenetics, and molecular genetic features

Adipose tissue tumors of the retroperitoneum showing no identifiable cytologic atypia are usually classified as lipoma-like well-differentiated liposarcoma. Whether a subset of these tumors represents true examples of retroperitoneal lipoma remains a controversial subject, because the diagnostic liposarcoma cells may be of difficult identification, even after extensive sampling. Herein, we describe a large retroperitoneal lipoma with classic histopathologic, cytogenetic, molecular cytogenetic, and molecular genetic features. Extensive morphologic inspection showed no evidence of cytologic atypia. Cytogenetic analysis performed on fresh tissue material revealed the classic lipoma chromosome t(3;12)(q27;q14-15). Fluorescence in situ hybridization on multiple sections excluded the presence of MDM2 and CDK4 amplification, but showed HMGA2 balanced rearrangement in most cells. Reverse-transcriptase polymerase chain reaction followed by sequencing analysis confirmed the presence of the HMGA2-LPP fusion gene, a characteristic and the most common fusion product found in lipoma. The patient has been followed for 2.5 years without evidence of recurrence or metastasis. These results indicate that retroperitoneal lipomata do exist, but their diagnosis must rely on stringent histologic, cytogenetic, and molecular genetic analysis.

The Hus1 homologue of Leishmania major encodes a nuclear protein that participates in DNA damage response

The protozoan parasite Leishmania presents a dynamic and plastic genome in which gene amplification and chromosome translocations are common phenomena. Such plasticity hints at the necessity of dependable genome maintenance pathways. Eukaryotic cells have evolved checkpoint control systems that recognize altered DNA structures and halt cell cycle progression allowing DNA repair to take place. In these cells, the PCNA-related heterotrimeric complex formed by the proteins Hus1, Rad9, and Rad1 is known to participate in the early steps of replicative stress sensing and signaling. Here we show that the Hus1 homolog of Leishmania major is a nuclear protein that improves the cell capability to cope with replicative stress. Overexpression of LmHus1 confers resistance to the genotoxic drugs hydroxyurea (HU) and methyl methanesulfonate (MMS) and resistance to HU correlates to reduced net DNA damage upon LmHus1 expression. (C) 2011 Elsevier B.V. All rights reserved.

Indirect immune detection of ecdysone receptor (EcR) during the formation of DNA puffs in Bradysia hygida (Diptera, Sciaridae)

Gene amplification occurs in Bradysia hygida salivary glands, at the end of the fourth larval instar. The hormone 20-hydroxyecdysone (20E) triggers this process, which results in DNA puff formation. Amplified genes are activated in two distinct groups. The activity of the first group is dependent on high levels of 20E, while the second group needs low hormone levels. Consequently, the salivary glands of B. hygida constitute an interesting biological model to study how 20E, and its receptors, affect gene amplification and activity. We produced polyclonal antibodies against B. hygida EcR (BhEcR). In western blots a polypeptide of about 66 kDa was detected in salivary gland extracts. The antibodies were also used for indirect immune-localization of BhEcR in polytene chromosomes. RNA-polymerase II was also immune-detected. We did not detect the receptor in chromosome C where the first and second groups of DNA puffs form during DNA puff anlage formation, but it was present during puff expansion. During the active phase of both groups of DNA puffs, RNA polymerase II co-localized with BhEcR. After puff regression, these antigens were not detected. Apparently, EcR plays a direct role in the transcription of amplified genes, but its role in gene amplification remains enigmatic.

Maternal embryonic leucine zipper kinase transcript abundance correlates with malignancy grade in human astrocytomas

We have performed cDNA microarray analyses to identify gene expression differences between highly invasive glioblastoma multiforme (GBM) and typically benign pilocytic astrocytomas (PA). Despite the significant clinical and pathological differences between the 2 tumor types, only 63 genes were found to exhibit 2-fold or greater overexpression in GBM as compared to PA. Forty percent of these genes are related to the regulation of the cell cycle and mitosis. QT-PCR validation of 6 overexpressed genes: MELK, AUKB, ASPM, PRC1, IL13RA2 and KIAA0101 confirmed at least a 5-fold increase in the average expression levels in GBM. Maternal embryonic leucine zipper kinase (MELK) exhibited the most statistically significant difference. A more detailed investigation of MELK expression was undertaken to study its oncogenic relevance. In the examination of more than 100 tumors of the central nervous system, we found progressively higher expression of MELK with astrocytoma grade and a noteworthy uniformity of high level expression in GBM. Similar level of overexpression was also observed in medulloblastoma. We found neither gene promoter hypomethylation nor amplification to be a factor in MELK expression, but were able to demonstrate that MELK knockdown in malignant astrocytoma cell lines caused a reduction in proliferation and anchorage-independent growth in in vitro assays. Our results indicate that GBM and PA differ by the expression of surprisingly few genes. Among them, MELK correlated with malignancy grade in astrocytomas and represents a therapeutic target for the management of the most frequent brain tumors in adult and children. (C) 2007 Wiley-Liss, Inc.

Evaluation of Adult Chronic Chagas` Heart Disease Diagnosis by Molecular and Serological Methods

Chagas` disease caused by Trypanosoma cruzi is endemic in Latin America. T. cruzi presents heterogeneous populations and comprises two main genetic lineages, named T. cruzi I and T. cruzi II. Diagnosis in the chronic phase is based on conventional serological tests, including indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA), and diagnosis in the acute phase based on parasitological methods, including hemoculture. The objective of this study was to evaluate the diagnostic procedures of Chagas` disease in adult patients in the chronic phase by using a PCR assay and conventional serological tests, including TESA-blot as the gold standard. Samples were obtained from 240 clinical chronic chagasic patients. The sensitivities, compared to that of TESA-blot, were 70% for PCR using the kinetoplast region, 75% for PCR using the nuclear repetitive region, 99% for IIF, and 95% for ELISA. According to the serological tests results, we recommend that researchers assess the reliability and sensitivity of the commercial kit Chagatest ELISA recombinant, version 3.0 (Chagatest Rec v3.0; Wiener Lab, Rosario, Argentina), due to the lack of sensitivity. Based on our analysis, we concluded that PCR cannot be validated as a conventional diagnostic technique for Chagas` disease. These data have been corroborated by low levels of concordance with serology test results. It is recommended that PCR be used only for alternative diagnostic support. Using the nuclear repetitive region of T. cruzi, PCR could also be applicable for monitoring patients receiving etiologic treatment.

Prospective analysis between the therapy of immunosuppressive medication and allogeneic microchimerism after liver transplantation

After liver transplantation, migration of donor-derived hematopoietic cells to recipient can be detected in pheripheral blood. This state is termed microchimerism. The aim of this study was to investigate prospectively the presence of allogeneic microchimerism, the occurrence of acute cellular rejection and the level of immunosuppression in transplanted patients. Microchimerism occurrence between 10 days and 12 months after liver transplantation was analyzed in 47 patients aged between 15 and 65 by a two-stage nested PCR/SSP technique to detect donor MHC HLA-DR gene specifically. A pre-transplant blood sample was colleted from each patient to serve as individual negative control. Microchimerism was demonstrated in 32 (68%) of the 47 patients; of these, only 10 patients (31.2%) presented rejection. Early microchimerism was observed in 25 patients (78.12%) and late microchimerism in 7 patients (21.8%). Among the patients with microchimerism, 14 were given CyA and 18 were given FK506. In the group without microchimerism, 12 patients were given CyA and 03 were given FK506. There was a significant association between the presence of microchimerism and the absence of rejection (p=0.02) and also between microchimerism and the type of immunosuppression used. Our data indicate that microchimerism and probably differentiation of donor-derived leukocytes can have relevant immunologic effects both in terms of sensitization of recipient and in terms of immunomodulation toward tolerance induction. (C) 2008 Elsevier B.V. All rights reserved.

A serosurvey for hantavirus infection in wild rodents from the states of Rio de Janeiro and Pernambuco, Brazil

Sera from 269 rodents obtained during the routine surveillance operations in plague areas of Rio de Janeiro and Pernambuco states, Brazil were tested by ELISA for specific IgG antibodies against a recombinant nucleocapsid (N) protein of Araraquara hantavirus. ELISA-positive sera were submitted to reverse transcriptase-polymerase chain reaction (RT-PCR) for amplification of the virus genome and later sequencing for identification of the viral variant. The samples from the state of Pernambuco were antibody negative, and although four from Rio de Janeiro were ELISA-positive, they failed to yield viral cDNA by RT-PCR. This is the first report of the presence of antibodies to a hantavirus among rodents from Rio de Janeiro and suggests the possibility of human cases of hantavirus pulmonary syndrome (HPS) in that state, although no case has yet been reported. (C) 2008 Elsevier B.V. All rights reserved.

A novel homozygous splice acceptor site mutation of KISS1R in two siblings with normosmic isolated hypogonadotropic hypogonadism

Context: Loss-of-function mutations of the kisspeptin-1 receptor gene, KISS1R, have been identified in patients with normosmic isolated hypogonadotropic hypogonadism (nIHH). Objective: To investigate KISS1R defects in patients with absent or delayed puberty. Patients: We investigated KISS1R gene defects in a cohort of 99 Brazilian patients with nIHH or constitutional delay of puberty (CDP). Methods: The entire coding region of KISS1R was amplified by PCR followed by automatic sequencing. In addition, screening for KISS1R exonic deletions was performed by multiplex ligation-dependent probe amplification. Results: One novel homozygous KISS1R mutation was identified in two siblings with nIHH. This variant was an insertion/deletion (indel) mutation characterized by the deletion of three nucleotides (GCA) at position -2 to -4, and by the insertion of seven nucleotides (ACCGGCT) at the same position, within the 30 splice acceptor site of intron 2 of KISS1R. The brothers who carried this KISS1R mutation had no clinical evidence of pubertal development at the ages of 14 and 20 years. Computational analysis of this indel mutation predicted the generation of an abnormal protein. In addition, a new heterozygous KISS1R variant (p.E252Q) was identified in a male patient with sporadic nIHH. However, in vitro studies of this variant did not demonstrate functional impairment. Only known polymorphisms were identified in patients with CDP. Conclusion: Loss-of-function mutations of KISS1R represents a rare cause of nIHH, and was absent in patients with CDP. We have described a novel KISS1R homozygous splice acceptor site mutation in the familial form of nIHH.

Global poly(A) mRNA expression profile measured in individual bovine oocytes and cleavage embryos

The objective of this article was to estimate quantitative differences for GAPDH transcripts and poly(A) mRNA: (i) between oocytes collected from cumulus-oocyte complexes (COCs) qualified morphologically as grades A and B; (ii) between grade A oocytes before and after in vitro maturation (IVM); and (iii) among in vitro-produced embryos at different developmental stages. To achieve this objective a new approach was developed to estimate differences between poly(A) mRNA when using small samples. The approach consisted of full-length cDNA amplification (acDNA) monitored by real-time PCR, in which the cDNA from half of an oocyte or embryo was used as a template. The GAPDH gene was amplified as a reverse transcription control and samples that were not positive for GAPDH transcripts were discarded. The fold differences between two samples were estimated using delta Ct and statistical analysis and were obtained using the pairwise fixed reallocation randomization test. It was found that the oocytes recovered from grade B COCs had quantitatively less poly(A) mRNA (p < 0.01) transcripts compared with grade A COCs (1 arbitrary unit expression rate). In the comparison with immature oocytes (I arbitrary unit expression rate), the quantity of poly(A) mRNA did not change during IVM, but declined following IVF and varied with embryo culture (p < 0.05). Amplification of cDNA by real-time PCR was an efficient method to estimate differences in the amount of poly(A) mRNA between oocytes and embryos. The results obtained from individual oocytes suggested an association between poly(A) mRNA abundance and different morphological qualities of oocytes from COCs. In addition, a poly(A) mRNA profile was characterized from oocytes undergoing IVM, fertilization and blastocyst heating.