Cytogenetics investigation in 151 Brazilian infertile male patients and genomic analysis in selected cases: experience of 14 years in a public genetic service.

OBJECTIVES: Male infertility accounts for approximately 30% of cases of reproductive failure. The characterization of genetic variants using cytogenomic techniques is essential for the adequate clinical management of these patients. We aimed to conduct a cytogenetic investigation of numerical and structural rearrangements and a genomic study of Y chromosome microdeletions/microduplications in infertile men derived from a single centre with over 14 years of experience. RESULTS: We evaluated 151 infertile men in a transversal study using peripheral blood karyotypes and 15 patients with normal karyotypes through genomic investigation by multiplex ligation-dependent probe amplification (MLPA) or polymerase chain reaction of sequence-tagged sites (PCR-STS) techniques. Out of the 151 patients evaluated by karyotype, 13 presented chromosomal abnormalities: two had numerical alterations, and 11 had structural chromosomal rearrangements. PCR-STS detected a BPY2 gene region and RBMY2DP pseudogene region microdeletion in one patient. MLPA analysis allowed the identification of one patient with CDY2B_1 and CDY2B_2 probe duplications (CDY2B and NLGN4Y genes) and one patient with BPY2_1, BPY2_2, and BPY2_4 probe duplications (PRY and RBMY1J genes).

Interaction between PTSD-PRS and trauma affects PTSD likelihood in women victims of sexual assault.

OBJECTIVE: Post-traumatic stress disorder (PTSD) is triggered by traumatic events, but genetic vulnerability and a history of childhood trauma are additional factors that may increase the risk of PTSD. Thus, our study focused on exploring the interaction between genetic susceptibility, as assessed by polygenic risk score (PRS), and traumatic events. METHODS: We evaluated 68 women with PTSD who had been sexually assaulted and 63 healthy controls without a history of sexual assault. DNA was genotyped using the Infinium Global Screening Array (Illumina), and PRS analysis was performed using PRSice. Furthermore, logistic regression models were employed to examine the interaction between childhood trauma, traumatic life events, and PTSD-PRS and how they contribute to the risk of developing PTSD. RESULTS: We found a significant association between PRS, childhood trauma (p = 0.03; OR = 1.241), and PTSD. Additionally, an interaction was observed between PRS, traumatic life events, and childhood trauma, particularly relating to physical and emotional neglect (p = 0.028; OR = 1.010). When examining neglect separately, we found a modest association between emotional neglect and PTSD (p = 0.014; OR = 1.086). CONCLUSIONS: Our findings highlight the importance of considering genetic vulnerability and traumatic experiences in understanding the etiology of PTSD.

Cri-du-Chat Syndrome: Revealing a Familial Atypical Deletion in 5p.

Introduction: Cri-du-chat syndrome is generally diagnosed when patients present a high-pitched cry at birth, microcephaly, ocular hypertelorism, and prominent nasal bridge. The karyotype is useful to confirm deletions in the short arm of chromosome 5 (5p-) greater than 10 Mb. In cases of smaller deletions, it is necessary to resort to other molecular techniques such as fluorescence in situ hybridization, multiplex ligation-dependent probe amplification (MLPA) or genomic array. Case Presentation: We report a family with an atypical deletion in 5p (mother and 2 children) and variable phenotypes compared with the literature. We applied a P064 MLPA kit to evaluate 5p- in the mother and the 2 children, and we used the Infinium CytoSNP-850K BeadChip genomic array to evaluate the siblings, an 11-year-old boy and a 13-year-old girl, to better define the 5p breakpoints. Both children presented a high-pitched cry at birth, but they did not present any of the typical physical features of 5p- syndrome. The MLPA technique with 5 probes for the 5p region revealed that the patients and their mother presented an atypical deletion with only 4 probes deleted (TERT_ex2, TERT_ex13, CLPTM1L, and IRX4). The genomic array performed in the siblings' samples revealed a 6.2-Mb terminal deletion in 5p15.33p15.32, which was likely inherited from their mother, who presented similar molecular features, seen in MLPA. Discussion: The sparing of the CTNND2 gene, which is associated with cerebral development, in both siblings may explain why these 2 patients had features such as better communication skills which most patients with larger 5p deletions usually do not present. In addition, both patients had smaller deletions than those found in patients with a typical 5p- phenotype. This report demonstrates the utility of genomic arrays as a diagnostic tool to better characterize atypical deletions in known syndromes such as 5p- syndrome, which will allow a better understanding of the genotype-phenotype correlations.

Expanding the Phenotype of 8p23.1 Deletion Syndrome: Eight New Cases Resembling the Clinical Spectrum of 22q11.2 Microdeletion.

OBJECTIVE: To report the effectiveness of early molecular diagnosis in the clinical management of rare diseases, presenting 8 patients with 8p23.1DS who have clinical features that overlap the phenotypic spectrum of 22q11.2DS. STUDY DESIGN: This report is part of a previous study that aims to provide a precocious molecular diagnosis of the 22q11.2 deletion syndrome in 118 infants with congenital heart disease. To confirm the clinical diagnosis, patients underwent comparative genomic screening by the multiplex ligation-dependent probe amplification (MLPA) assay with the SALSA MLPA probemix kits P064-B2, P036-E1, P070-B2, P356-A1, and P250- B1. Subsequently, the patients performed the genomic microarray using the Infinium CytoSNP-850K BeadChip to confirm the deletion, determine the breakpoints of the deletion, and search for genomic copy number variations. RESULTS: MLPA performed with 3 different kits revealed the 8p23.1 typical deletion involving the PPP1R3B, MSRA, and GATA4 genes in the 5 patients. The array analysis was performed on these 5 patients and 3 other patients (8 patients) who also had clinical suspicion of 22q11 deletion (8 patients) allowed a precise definition of the breakpoints and excluded other genomic abnormalities. CONCLUSIONS: Cytogenomic screening was efficient in establishing a differential diagnosis and ruling out the presence of other concomitant syndromes. The clinical picture of the 8p23.1 deletion syndrome is challenging; however, cytogenomic tools can provide an exact diagnosis and help to clarify the genotype-phenotype complexity of these patients. Our reports underline the importance of early diagnosis and clinical follow-up of microdeletion syndromes.

Novel rearrangements between different chromosomes with direct impact on the diagnosis of 5p- syndrome.

OBJECTIVES: Copy Number Variations (CNVs) in the human genome account for common populational variations but can also be responsible for genetic syndromes depending on the affected region. Although a deletion in 5p is responsible for a syndrome with highly recognizable phenotypical features, other chromosomal abnormalities might overlap phenotypes, especially considering that most studies in 5p use traditional cytogenetic techniques and not molecular techniques. METHODS: The authors have investigated 29 patients with clinical suspicion of 5p- syndrome using Chromosomal Microarray (CMA), and have gathered information on previous tests, clinical signs, symptoms, and development of the patients. RESULTS: The results showed 23 pure terminal deletions, one interstitial deletion, one deletion followed by a 3 Mb duplication in 5p, three cases of 5p deletion concomitant to duplications larger than 20 Mb in chromosomes 2, 9, and 18, and one 5p deletion with a chromosome Y deletion. CMA showed relevant CNVs not typically associated with 5p- that may have contributed to the final phenotype in these patients. CONCLUSIONS: The authors have identified three novel rearrangements between chromosomes 5 and 2 (Patient 27), 5 and 18 (Patient 11), and 5 and Y (Patient 22), with breakpoints and overlapped phenotypes that were not previously described. The authors also highlight the need for further molecular investigation using CMA, in different chromosomes beyond chromosome 5 (since those cases did not show only the typical deletion expected for the 5p- syndrome) to explain discordant chromosomal features and overlapped phenotypes to unravel the cause of the syndrome in atypical cases.

Cytogenomics Investigation of Infants with Congenital Heart Disease: Experience of a Brazilian Center. / Investigação Citogenômica de Crianças com Doença Cardíaca Congênita: Experiência de um Centro no Brasil.

BACKGROUND: Some syndromes have specific and easily recognizable features, while others may be more complex to identify and may present different phenotypic manifestations, for example. An etiological diagnosis is important to understand the nature of the disease, to establish the prognosis and to start the treatment, allowing the inclusion of patients in society and reducing the financial cost of such diseases. OBJECTIVE: The initial proposal of this study was cytogenetic screening for the detection of the 22q11.2 deletion syndrome in consecutive newborns and infants with congenital heart disease using the multiplex ligation-dependent probe amplification (MLPA) technique. Therefore, throughout our research, other genomic alterations were identified in these cardiac patients. Thus, our objective was extended to investigate these other cytogenetic alterations. METHODS: We investigated 118 neonates with congenital heart diseases born consecutively during one year using the MLPA technique. RESULTS: The MLPA technique allowed the detection of 22q11.2DS in 10/118 patients (8.5%). Other genomic alterations were also identified in 6/118 patients (5%): 1p36 del, 8p23 del (2 cases), 7q dup, 12 dup and 8q24 dup. CONCLUSION: This study highlights the relevance of detecting genomic alterations that are present in newborns and infants with congenital cardiac diseases using cytogenomic tools.

FUNDAMENTO: Algumas síndromes têm características específicas e facilmente reconhecíveis, enquanto outras podem ser mais complexas de se identificar e podem apresentar diferentes manifestações fenotípicas, por exemplo. Um diagnóstico etiológico é importante para entender a natureza da doença, para estabelecer o prognóstico e para começar o tratamento, permitindo a inclusão de pacientes na sociedade e reduzindo o custo financeiro dessas doenças. OBJETIVO: A proposta inicial deste estudo foi a triagem citogenética para detectar a síndrome de deleção 22q11.2 (SD22q11.2) em recém-nascidos e crianças com doença cardíaca congênita utilizando a técnica da amplificação multiplex de sondas dependente de ligação (MLPA). Assim, por meio da pesquisa, outras mudanças genômicas foram identificadas nesses pacientes cardíacos. Nosso objetivo se estendeu a investigar essas outras mudanças citogenéticas. MÉTODOS: Investigamos 118 recém-nascidos com doenças cardíacas congênitas nascidos consecutivamente durante um ano, utilizando a técnica da MLPA. RESULTADOS: A técnica da MLPA permitiu a detecção da SD22q11.2 em 10/118 pacientes (8,5%). Outras alterações genômicas foram identificadas em 6/118 pacientes (5%): 1p36 del, 8p23 del (2 casos), 7q dup, 12 dup e 8q24 dup. CONCLUSÃO: Este estudo ressalta a relevância da detecção de alterações genômicas que estão presentes em recém-nascidos e crianças com doenças cardíacas congênitas por meio de ferramentas citogenômicas.

Investigação Citogenômica de Crianças com Doença Cardíaca Congênita: Experiência de um Centro no Brasil / Cytogenomics Investigation of Infants with Congenital Heart Disease: Experience of a Brazilian Center

Resumo Fundamento Algumas síndromes têm características específicas e facilmente reconhecíveis, enquanto outras podem ser mais complexas de se identificar e podem apresentar diferentes manifestações fenotípicas, por exemplo. Um diagnóstico etiológico é importante para entender a natureza da doença, para estabelecer o prognóstico e para começar o tratamento, permitindo a inclusão de pacientes na sociedade e reduzindo o custo financeiro dessas doenças. Objetivo A proposta inicial deste estudo foi a triagem citogenética para detectar a síndrome de deleção 22q11.2 (SD22q11.2) em recém-nascidos e crianças com doença cardíaca congênita utilizando a técnica da amplificação multiplex de sondas dependente de ligação (MLPA). Assim, por meio da pesquisa, outras mudanças genômicas foram identificadas nesses pacientes cardíacos. Nosso objetivo se estendeu a investigar essas outras mudanças citogenéticas. Métodos Investigamos 118 recém-nascidos com doenças cardíacas congênitas nascidos consecutivamente durante um ano, utilizando a técnica da MLPA. Resultados A técnica da MLPA permitiu a detecção da SD22q11.2 em 10/118 pacientes (8,5%). Outras alterações genômicas foram identificadas em 6/118 pacientes (5%): 1p36 del, 8p23 del (2 casos), 7q dup, 12 dup e 8q24 dup. Conclusão Este estudo ressalta a relevância da detecção de alterações genômicas que estão presentes em recém-nascidos e crianças com doenças cardíacas congênitas por meio de ferramentas citogenômicas.

Abstract Background Some syndromes have specific and easily recognizable features, while others may be more complex to identify and may present different phenotypic manifestations, for example. An etiological diagnosis is important to understand the nature of the disease, to establish the prognosis and to start the treatment, allowing the inclusion of patients in society and reducing the financial cost of such diseases. Objective The initial proposal of this study was cytogenetic screening for the detection of the 22q11.2 deletion syndrome in consecutive newborns and infants with congenital heart disease using the multiplex ligation-dependent probe amplification (MLPA) technique. Therefore, throughout our research, other genomic alterations were identified in these cardiac patients. Thus, our objective was extended to investigate these other cytogenetic alterations. Methods We investigated 118 neonates with congenital heart diseases born consecutively during one year using the MLPA technique. Results The MLPA technique allowed the detection of 22q11.2DS in 10/118 patients (8.5%). Other genomic alterations were also identified in 6/118 patients (5%): 1p36 del, 8p23 del (2 cases), 7q dup, 12 dup and 8q24 dup. Conclusion This study highlights the relevance of detecting genomic alterations that are present in newborns and infants with congenital cardiac diseases using cytogenomic tools.

Cardiovascular findings in Williams-Beuren Syndrome: Experience of a single center with 127 cases.

Williams-Beuren syndrome (WBS) is a rare, microdeletion syndrome characterized by facial dysmorphisms, intellectual disability, a friendly personality, cardiovascular and other abnormalities. Cardiovascular defects (CVD) are among the most prevalent characteristics in WBS, being supravalvular aortic stenosis (SVAS) the most frequent, followed by peripheral pulmonary stenosis (PPS). A comprehensive retrospective review of medical records of 127 patients with molecular diagnosis of WBS, in a period of 20 years, was done to evaluate the incidence, the natural history of cardiovascular disease, and the need for surgical intervention, including heart transplantation (HT). A total of 94/127 patients presented with CVD. Of these 94 patients, 50% presented with SVAS and 22.3% needed heart surgery and/or cardiac catheterization including one that required HT due to severe SVAS-related heart failure at 19 years of age. The patient died in the postoperative period due to infectious complications. Cardiovascular problems are the major cause of sudden death in patients with WBS, who have a significantly higher mortality risk associated with surgical interventions. There is a higher risk for anesthesia-related adverse events and for major adverse cardiac events following surgery. End-stage heart failure due to myocardial ischemia has been described in WBS patients and it is important to consider that HT can become their only viable option. To our knowledge, the case mentioned here is the first HT reported in an adolescent with WBS. HT can be a viable therapeutic option in WBS patients with adequate evaluation, planning, and a multidisciplinary team to provide the required perioperative care and follow-up.

Novel rearrangements between different chromosomes with direct impact on the diagnosis of 5p- syndrome

Abstract Objectives Copy Number Variations (CNVs) in the human genome account for common populational variations but can also be responsible for genetic syndromes depending on the affected region. Although a deletion in 5p is responsible for a syndrome with highly recognizable phenotypical features, other chromosomal abnormalities might overlap phenotypes, especially considering that most studies in 5p use traditional cytogenetic techniques and not molecular techniques. Methods The authors have investigated 29 patients with clinical suspicion of 5p- syndrome using Chromosomal Microarray (CMA), and have gathered information on previous tests, clinical signs, symptoms, and development of the patients. Results The results showed 23 pure terminal deletions, one interstitial deletion, one deletion followed by a 3 Mb duplication in 5p, three cases of 5p deletion concomitant to duplications larger than 20 Mb in chromosomes 2, 9, and 18, and one 5p deletion with a chromosome Y deletion. CMA showed relevant CNVs not typically associated with 5p- that may have contributed to the final phenotype in these patients. Conclusions The authors have identified three novel rearrangements between chromosomes 5 and 2 (Patient 27), 5 and 18 (Patient 11), and 5 and Y (Patient 22), with breakpoints and overlapped phenotypes that were not previously described. The authors also highlight the need for further molecular investigation using CMA, in different chromosomes beyond chromosome 5 (since those cases did not show only the typical deletion expected for the 5p- syndrome) to explain discordant chromosomal features and overlapped phenotypes to unravel the cause of the syndrome in atypical cases. HIGHLIGHTS The authors The authors have described three novel rearrangements between chromosomes 5 and 2, 5 and 18, and 5 and Y with chromosomal breakpoints and overlapped phenotypes that were not previously described. One of the main atypical features for 5p- syndrome that the authors report was the presence of seizures that was found in the three patients with rearrangements between different chromosomes and in a patient with a deletion followed by duplication in 5p. The authors suggest physicians conduct further molecular investigation in the presence of atypical clinical features for patients with 5p- syndrome suspicion.

Intragenic variants in the SMN1 gene determine the clinical phenotype in 5q spinal muscular atrophy.

OBJECTIVE: The aim of the study was to report the proportion of homozygous and compound heterozygous variants in the survival motor neuron 1 (SMN1) gene in a large population of patients with spinal muscular atrophy (SMA) and to correlate the severity of the disease with the presence of specific intragenic variants in SMN1 and with the SMN2 copy number. METHODS: Four hundred fifty Brazilian patients with SMA were included in a retrospective study, and clinical data were analyzed compared with genetic data; the SMN2 copy number was obtained by multiplex ligation-dependent probe amplification and pathogenic variants in SMN1 by next-generation sequencing. RESULTS: Four hundred two patients (89.3%) presented homozygous exon 7-SMN1 deletion, and 48 (10.7%) were compound heterozygous for the common deletion in one allele and a point mutation in the other allele. Recurrent variants in exons 3 and 6 (c.460C>T, c.770_780dup and c.734_735insC) accounted for almost 80% of compound heterozygous patients. Another recurrent pathogenic variant was c.5C>G at exon 1. Patients with c.770_780dup and c.734_735insC had a clinical phenotype correlated with SMN2 copy number, whereas the variants c.460C>T and c.5C>G determined a milder phenotype independently of the SMN2 copies. CONCLUSIONS: Patients with specific pathogenic variants (c.460C>T and c.5C>G) presented a milder phenotype, and the SMN2 copy number did not correlate with disease severity in this group.

Case of 15q26-qter deletion associated with a Prader-Willi phenotype.

Prader-Willi syndrome (PWS) is one of the common neurogenetic disorders associated with intellectual disability. PWS involves a complex inheritance pattern and is caused by an absence of gene expression on the paternally inherited 15q11.2-q13 region, either due to deletion, maternal uniparental disomy or imprinting defect. The syndrome is characterized principally by severe neonatal hypotonia, a weak suck in infancy that is later followed by hyperphagia and obesity, developmental delay, intellectual disability and short stature. In the case of the chromosome 15q26-qter deletion syndrome or Drayer's syndrome, very few reports have been published. Its characteristics include intrauterine growth restriction, postnatal growth failure, varying degrees of intellectual disability, developmental delay, typical facial appearance and diaphragmatic hernia. The present paper describes a female patient in whom clinical findings were suggestive of PWS and deletion in the 15q26-qter region. Both karyotyping and methylation-specific polymerase chain reaction were shown to be normal. Nevertheless, fluorescence in situ hybridization showed a 15qter deletion that was later mapped by single nucleotide polymorphism (SNP)-array. The deleted genomic region involves the insulin-like growth factor-1 receptor (IGF1R) gene, which is related to short stature, developmental delay and intellectual disability. This case had various clinical characteristics in common with the cases of 15q26-qter deletionand characteristics compatible with PWS.

Gene expression changes associated with trajectories of psychopathology in a longitudinal cohort of children and adolescents.

We aimed to identify blood gene expression patterns associated to psychopathological trajectories retrieved from a large community, focusing on the emergence and remission of general psychiatric symptoms. Hundred and three individuals from the Brazilian High-Risk Cohort Study (BHRCS) for mental disorders were classified in four groups according to Child Behavior Checklist (CBCL) total score at the baseline (w0) and after 3 years (w1): low-high (L-H) (N = 27), high-low (H-L) (N = 12), high-high (H-H) (N = 34) and low-low (L-L) groups (N = 30). Blood gene expression profile was measured using Illumina HT-12 Beadchips, and paired analyses comparing w0 and w1 were performed for each group. Results: 98 transcripts were differentially expressed comparing w0 and w1 in the L-H, 33 in the H-L, 177 in the H-H and 273 in the L-L. Of these, 66 transcripts were differentially expressed exclusively in the L-H; and 6 only in the H-L. Cross-Lagged Panel Models analyses revealed that RPRD2 gene expression at w1 might be influenced by the CBCL score at w0. Moreover, COX5B, SEC62, and NDUFA2 were validated with another technique and were also differentially regulated in postmortem brain of subjects with mental disorders, indicating that they might be important not only to specific disorders, but also to general psychopathology and symptoms trajectories. Whereas genes related to metabolic pathways seem to be associated with the emergence of psychiatric symptoms, mitochondrial inner membrane genes might be important over the course of normal development. These results suggest that changes in gene expression can be detected in blood in different psychopathological trajectories.

Gestational Tubal Choriocarcinoma Presenting as a Pregnancy of Unknown Location following Ovarian Induction.

The management of pregnancy of unknown location (PUL) can be a challenging situation, since it can present as several different conditions. Here we describe a rare case of gestational choriocarcinoma arising in the fallopian tube after ovarian induction in an infertile patient. The patient received clomiphene for ovarian induction and had rising levels of human chorionic gonadotropin (hCG) over nine months without sign of pregnancy. After referral to our center, the patient was diagnosed with a paraovarian tumor, which revealed a gestational choriocarcinoma arising in the fallopian tube; the final diagnosis was supported by pathological and cytogenomic analysis. Malignancies, such as gestational trophoblastic disease, should be in the differential diagnosis of PUL; the early recognition of these conditions is key for the proper treatment and favorable outcome.

Deletion of RUNX1 exons 1 and 2 associated with familial platelet disorder with propensity to acute myeloid leukemia.

Familial platelet disorder with propensity to acute myeloid leukemia (FPD/AML) associated with RUNX1 mutations is an autosomal dominant disorder included in the group of the myeloid neoplasms with germ line predisposition. We describe two brothers who were diagnosed with hematological malignancies (one with AML and the other with T-cell lymphoblastic lymphoma). There was a history of leukemia in the paternal family and two of their siblings presented with low platelet counts and no history of significant bleeding. A microdeletion encompassing exons 1-2 of RUNX1 (outside the cluster region of the Runt Homology domain and the transactivation domain) was detected in six family members using array-CGH and MLPA validation. A low platelet count was not present in all deletion carriers and, therefore, it should not be used as an indication for screening in suspected families and family members.

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.

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.

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.

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.

Investigation of Copy Number Variation in Children with Conotruncal Heart Defects / Investigação da Variação no Número de Cópias Gênicas em Crianças com Defeito Cardíaco Conotruncal

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. .

Fundamento: Os defeitos cardíacos congênitos são o grupo de anormalidades estruturais mais prevalentes ao nascimento e uma das principais causas de morbidade e mortalidade infantil. Estudos têm mostrado a contribuição da variação no número de cópias na gênese das malformações cardíacas. Objetivos: Investigar a variação no número de cópias gênicas em crianças com defeito cardíaco conotruncal. Métodos: Multiplex Ligation-dependent Probe Amplification (MLPA) foi realizado em 39 pacientes com defeito cardíaco conotruncal. Avaliação clínica e laboratorial foi realizada em todos os pacientes. Os pais dos probandos que apresentaram alterações também foram investigados. Resultados: Variação no número de cópias foi detectada em 7/39 pacientes: deleção 22q11.2, duplicação 22q11.2, duplicação 15q11.2, duplicação 20p12.2, deleção 19p, duplicação 15q e 8p23.2 com duplicação 10p12.31. As características clínicas foram compatíveis com o relatado na literatura associadas com microdeleção/microduplicação encontrada. Nenhuma dessas alterações foi herdada dos pais. Conclusões: Nossos resultados demonstram que a técnica de MLPA é útil na investigação de microdeleções e microduplicações em defeitos cardíacos congênitos conotruncais. O diagnóstico precoce das variações no número de cópias em pacientes com defeito cardíaco congênito auxilia na prevenção de morbidade e diminuição da mortalidade nesses pacientes. .