Roberta Lelis Dutra

FGFR4 profile as a prognostic marker in squamous cell carcinoma of the mouth and oropharynx.

Background Fibroblast growth factor receptor 4 (FGFR4) is a member of a receptor tyrosine kinase family of enzymes involved in cell cycle control and proliferation. A common single nucleotide polymorphism (SNP) Gly388Arg variant has been associated with increased tumor cell motility and progression of breast cancer, head and neck cancer and soft tissue sarcomas. The present study evaluated the prognostic significance of FGFR4 in oral and oropharynx carcinomas, finding an association of FGFR4 expression and Gly388Arg genotype with tumor onset and prognosis. Patients and Methods DNA from peripheral blood of 122 patients with oral and oropharyngeal squamous cell carcinomas was used to determine FGFR4 genotype by PCR-RFLP. Protein expression was assessed by immunohistochemistry (IHC) on paraffin-embedded tissue microarrays. Results Presence of allele Arg388 was associated with lymphatic embolization and with disease related premature death. In addition, FGFR4 low expression was related with lymph node positivity and premature relapse of disease, as well as disease related death. Conclusion Our results propose FGFR4 profile, measured by the Gly388Arg genotype and expression, as a novel marker of prognosis in squamous cell carcinoma of the mouth and oropharynx.

Detection of deletions at 7q11.23 in Williams-Beuren syndrome by polymorphic markers

INTRODUCTION: Williams-Beuren syndrome (WBS; OMIM 194050) is caused by a hemizygous contiguous gene microdeletion at 7q11.23. Supravalvular aortic stenosis, mental retardation, overfriendliness, and ocular and renal abnormalities comprise typical symptoms in WBS. Although fluorescence in situ hybridization is widely used for diagnostic confirmation, microsatellite DNA markers are considered highly informative and easily manageable. OBJECTIVES: This study aimed to test the microsatellite markers for the diagnosis of Williams-Beuren syndrome, to determine the size and parental origin of microdeletion, compare the clinical characteristics between patients with different sizes of the deletion and parental origin. METHODS: We studied 97 patients with clinical diagnosis of Williams-Beuren syndrome using five microsatellite markers: D7S1870, D7S489, D7S613, D7S2476 and D7S489_A. RESULTS AND DISCUSSION: Using five markers together, the result was informative in all patients. The most informative marker was D7S1870 (78.4%), followed by D7S613 (75.3%), D7S489 (70.1%) and D7S2476 (62.9%). The microdeletion was present in 84 (86.6%) patients and absent in 13 (13.4%) patients. Maternal deletions were found in 52.4% of patients and paternal deletions in 47.6% of patients. The observed size of deletions was 1.55 Mb in 76/84 patients (90.5%) and 1.84 Mb in 8/84 patients (9.5%). SVAS as well as ocular and urinary abnormalities were more frequent in the patients with a deletion. There were no clinical differences in relation to either the size or parental origin of the deletion. CONCLUSION: Using these five selected microsatellite markers was informative in all patients, thus can be considered an alternative method for molecular diagnosis in Williams-Beuren syndrome.

Investigation of Copy Number Variation in Children with Conotruncal Heart Defects

Background Congenital heart defects (CHD) are the most prevalent group of structural abnormalities at birth and one of the main causes of infant morbidity and mortality. Studies have shown a contribution of the copy number variation in the genesis of cardiac malformations. Objectives Investigate gene copy number variation (CNV) in children with conotruncal heart defect. Methods Multiplex ligation-dependent probe amplification (MLPA) was performed in 39 patients with conotruncal heart defect. Clinical and laboratory assessments were conducted in all patients. The parents of the probands who presented abnormal findings were also investigated. Results Gene copy number variation was detected in 7/39 patients: 22q11.2 deletion, 22q11.2 duplication, 15q11.2 duplication, 20p12.2 duplication, 19p deletion, 15q and 8p23.2 duplication with 10p12.31 duplication. The clinical characteristics were consistent with those reported in the literature associated with the encountered microdeletion/microduplication. None of these changes was inherited from the parents. Conclusions Our results demonstrate that the technique of MLPA is useful in the investigation of microdeletions and microduplications in conotruncal congenital heart defects. Early diagnosis of the copy number variation in patients with congenital heart defect assists in the prevention of morbidity and decreased mortality in these patients.

Congenital Heart Disease as a Warning Sign for the Diagnosis of the 22q11.2 Deletion

Background To alert for the diagnosis of the 22q11.2 deletion syndrome (22q11.2DS) in patients with congenital heart disease (CHD). Objective To describe the main CHDs, as well as phenotypic, metabolic and immunological findings in a series of 60 patients diagnosed with 22q11.2DS. Methods The study included 60 patients with 22q11.2DS evaluated between 2007 and 2013 (M:F=1.3, age range 14 days to 20 years and 3 months) at a pediatric reference center for primary immunodeficiencies. The diagnosis was established by detection of the 22q11.2 microdeletion using FISH (n = 18) and/or MLPA (n = 42), in association with clinical and laboratory information. Associated CHDs, progression of phenotypic facial features, hypocalcemia and immunological changes were analyzed. Results CHDs were detected in 77% of the patients and the most frequent type was tetralogy of Fallot (38.3%). Surgical correction of CHD was performed in 34 patients. Craniofacial dysmorphisms were detected in 41 patients: elongated face (60%) and/or elongated nose (53.3%), narrow palpebral fissure (50%), dysplastic, overfolded ears (48.3%), thin lips (41.6%), elongated fingers (38.3%) and short stature (36.6%). Hypocalcemia was detected in 64.2% and decreased parathyroid hormone (PTH) level in 25.9%. Decrease in total lymphocytes, CD4 and CD8 counts were present in 40%, 53.3% and 33.3%, respectively. Hypogammaglobulinemia was detected in one patient and decreased concentrations of immunoglobulin M (IgM) in two other patients. Conclusion Suspicion for 22q11.2DS should be raised in all patients with CHD associated with hypocalcemia and/or facial dysmorphisms, considering that many of these changes may evolve with age. The 22q11.2 microdeletion should be confirmed by molecular testing in all patients.

Complex structural rearrangement features suggesting chromoanagenesis mechanism in a case of 1p36 deletion syndrome

AbstractGenome rearrangements are caused by the erroneous repair of DNA double-strand breaks, leading to several alterations that result in loss or gain of the structural genomic of a dosage-sensitive genes. However, the mechanisms that promote the complexity of rearrangements of congenital or developmental defects in human disease are unclear. The investigation of complex genomic abnormalities could help to elucidate the mechanisms and causes for the formation and facilitate the understanding of congenital or developmental defects in human disease. We here report one case of a patient with atypical clinical features of the 1p36 syndrome and the use of cytogenomic techniques to characterize the genomic alterations. Analysis by multiplex ligation-dependent probe amplification and array revealed a complex rearrangement in the 1p36.3 region with deletions and duplication interspaced by normal sequences. We also suggest that chromoanagenesis could be a possible mechanism involved in the repair and stabilization of this rearrangement.

Williams-Beuren Syndrome: A Clinical Study of 55 Brazilian Patients and the Diagnostic Use of MLPA

Williams-Beuren syndrome (WBS) is a genetic disease caused by a microdeletion in the 7q11.23 region. It is characterized by congenital heart disease, mainly supravalvular aortic stenosis, mental retardation, mild short stature, facial dysmorphisms, and variable abnormalities in different systems. Objectives. To report the clinical findings of 55 Brazilian patients confirmed by multiplex ligation-dependent probe amplification (MLPA). Methods. Patients were followed up for 4 years at the Genetics Unit of the Instituto da Criança of the Hospital das Clínicas, FMUSP, Brazil. A kit specific for WBS was used to detect the 7q11.23 microdeletion. Results. Two patients with negative FISH results had positive MLPA results for WBS. The characteristics of the patients with the deletion were as follows: typical WBS facies (98.2%), neuropsychomotor delay (98.2%), hypersocial behavior (94.5%), hyperacusis (94.5%), and congenital heart disease (81.8%). Conclusions. MLPA was effective in detecting the microdeletion in the 7q11.23 region to confirm the diagnosis of WBS. MLPA was also able to confirm the diagnosis of WBS in two patients with typical clinical characteristics but negative FISH results. Thus, MLPA is a promising method in the diagnostic investigation of WBS. WBS is a multisystemic disorder and therefore requires multidisciplinary care and specific follow-up to prevent complications.

Post-mortem cytogenomic investigations in patients with congenital malformations.

Congenital anomalies are the second highest cause of infant deaths, and, in most cases, diagnosis is a challenge. In this study, we characterize patterns of DNA copy number aberrations in different samples of post-mortem tissues from patients with congenital malformations. Twenty-eight patients undergoing autopsy were cytogenomically evaluated using several methods, specifically, Multiplex Ligation-dependent Probe Amplification (MLPA), microsatellite marker analysis with a MiniFiler kit, FISH, a cytogenomic array technique and bidirectional Sanger sequencing, which were performed on samples of different tissues (brain, heart, liver, skin and diaphragm) preserved in RNAlater, in formaldehyde or by paraffin-embedding. The results identified 13 patients with pathogenic copy number variations (CNVs). Of these, eight presented aneuploidies involving chromosomes 13, 18, 21, X and Y (two presented inter- and intra-tissue mosaicism). In addition, other abnormalities were found, including duplication of the TYMS gene (18p11.32); deletion of the CHL1 gene (3p26.3); deletion of the HIC1 gene (17p13.3); and deletion of the TOM1L2 gene (17p11.2). One patient had a pathogenic missense mutation of g.8535C>G (c.746C>G) in exon 7 of the FGFR3 gene consistent with Thanatophoric Dysplasia type I. Cytogenomic techniques were reliable for the analysis of autopsy material and allowed the identification of inter- and intra-tissue mosaicism and a better understanding of the pathogenesis of congenital malformations.

A possible role of different PTPN genes in immune regulation.

To the Editor: We have recently evaluated the presence of autoimmunity in 42 molecularly confirmed patients diagnosed with RASopathies [1], a class of common autosomaldominant syndromes with neurofaciocutaneous and developmental involvement that include Noonan syndrome and Noonan-related disorders [2]. We were surprised with the high frequency (14%) of the patients presenting an association with autoimmune diseases, including systemic lupus erythematosus, thyroiditis, autoimmune hepatitis and others, in a such young population (average age of 19 years). This represents a twoto threefold increase in frequency when compared with the normal population (5–8%) [3]. All of the patients with such association presented a pathogenic mutation in the PTPN11 gene, which encodes a tyrosine phosphorylation enzyme that belongs to the same family of PTPN22, widely known for its participation in autoimmunity [4]. Additionally, we also found alterations in the levels of immunoglobulins (Igs) in 14 (52%) of the 27 patients with mutations in the PTPN11 gene in whom the Igs were measured (Table 1). These patients had at least one immunoglobulin out of the normal range: IgA and IgM levels were below normal limits for age in, respectively, one patient each (respectively, 38.1 and 35 mg ⁄ dl) patients. IgG, IgM and IgE levels were elevated, respectively, in three (1607–1740 mg ⁄ dl), one (237.6 mg ⁄ dl) and 11 (188–160 mg ⁄ dl) patients. None of them presented clinical criteria for the diagnosis of an immunodeficiency, although this inquiry was not the primary focus of our study. We were not able to establish a genotype–phenotype correlation considering the level of the Igs nor a statistically relevant (P < 0.05) difference between the groups of patients because the number of patients, though considerable for a genetic disorder, was small to reach a statistical significance. More than 60% of the 107 known PTPNs are expressed in the leucocytes and play a key role in regulating many signalling pathways and processes, including signalling, activation and tolerance [5, 6]. The high frequency of autoimmunity and alterations in the levels of Igs in patients harbouring mutations in PTPN11 may suggest the involvement of other PTPNs, besides PTPN22, in the regulation of the immunity. While we wait for more studies to clarify the participations of other PTPNs in the regulation of immunity and to determine the real extent in the clinical outcomes, it is important for the clinicians to be aware of the possible association of immune dysregulation when dealing with RASopathic patients.

Cytogenomic assessment of the diagnosis of 93 patients with developmental delay and multiple congenital abnormalities: The Brazilian experience

OBJECTIVE: The human genome contains several types of variations, such as copy number variations, that can generate specific clinical abnormalities. Different techniques are used to detect these changes, and obtaining an unequivocal diagnosis is important to understand the physiopathology of the diseases. The objective of this study was to assess the diagnostic capacity of multiplex ligation-dependent probe amplification and array techniques for etiologic diagnosis of syndromic patients. METHODS: We analyzed 93 patients with developmental delay and multiple congenital abnormalities using multiplex ligation-dependent probe amplifications and arrays. RESULTS: Multiplex ligation-dependent probe amplification using different kits revealed several changes in approximately 33.3% of patients. The use of arrays with different platforms showed an approximately 53.75% detection rate for at least one pathogenic change and a 46.25% detection rate for patients with benign changes. A concomitant assessment of the two techniques showed an approximately 97.8% rate of concordance, although the results were not the same in all cases. In contrast with the array results, the MLPA technique detected ∼70.6% of pathogenic changes. CONCLUSION: The obtained results corroborated data reported in the literature, but the overall detection rate was higher than the rates previously reported, due in part to the criteria used to select patients. Although arrays are the most efficient tool for diagnosis, they are not always suitable as a first-line diagnostic approach because of their high cost for large-scale use in developing countries. Thus, clinical and laboratory interactions with skilled technicians are required to target patients for the most effective and beneficial molecular diagnosis.

Subtelomeric Copy Number Variations: The Importance of 4p/4q Deletions in Patients with Congenital Anomalies and Developmental Disability.

The most prevalent structural variations in the human genome are copy number variations (CNVs), which appear predominantly in the subtelomeric regions. Variable sizes of 4p/4q CNVs have been associated with several different psychiatric findings and developmental disability (DD). We analyzed 105 patients with congenital anomalies (CA) and developmental and/or intellectual disabilities (DD/ID) using MLPA subtelomeric specific kits (P036 /P070) and 4 of them using microarrays. We found abnormal subtelomeric CNVs in 15 patients (14.3%), including 8 patients with subtelomeric deletions at 4p/4q (53.3%). Additional genomic changes were observed at 1p36, 2q37.3, 5p15.3, 5q35.3, 8p23.3, 13q11, 14q32.3, 15q11.2, and Xq28/Yq12. This indicates the prevalence of independent deletions at 4p/4q, involving PIGG, TRIML2, and FRG1. Furthermore, we identified 15 genes with changes in copy number that contribute to neurological development and/or function, among them CRMP1, SORCS2, SLC25A4, and HELT. Our results highlight the association of genes with changes in copy number at 4p and 4q subtelomeric regions and the DD phenotype. Cytogenomic characterization of additional cases with distal deletions should help clarifying the role of subtelomeric CNVs in neurological diseases.