Differences in DNA methylation status explain phenotypic variability in patients with 5p- syndrome.

Cri Du Chat syndrome, or 5p- syndrome, is characterized by a terminal or interstitial deletion on the short arm of chromosome 5 that causes variable clinical manifestations, including high-pitched cry in newborns, delayed growth, and global development. Different cytogenomic rearrangements, family history, and environmental factors may hinder the genotype-phenotype association. Thus, the phenotypic variability of this syndrome may not be limited only to variations in gene structure, such as deletions and duplications. It is possible that other mechanisms related to the activation or inactivation of promoters and/or exons of actively transcribed genes, such as DNA methylation are involved. Therefore, we studied the genome-wide methylation status profile of peripheral blood samples from fifteen patients with Cri du Chat Syndrome and nine control samples through the array method to look for Differentially Methylated Regions. We found that Differentially Methylated Regions outside the 5p region are mainly associated with regulating gene transcription, splicing, and chromatin remodeling. Most biological pathways are related to transcription, histone and chromatin binding, spliceosome and ribosomal complex, and RNA processing. Our results suggest that changes in the 5p region can cause an imbalance in other chromosomal regions capable of affecting gene modulation and thus explain the phenotypic differences in patients with 5p-.

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.

Gene expression profile suggesting immunological dysregulation in two Brazilian Bloom's syndrome cases.

BACKGROUND: Bloom syndrome (BS) is a rare autosomal recessive chromosome instability disorder. The main clinical manifestations are growth deficiency, telangiectasic facial erythema, immunodeficiency, and increased risk to develop neoplasias at early age. Cytogenetic test for sister chromatid exchanges (SCEs) is used as a diagnostic marker for BS. In addition, most patients also present mutations in the BLM gene, related to defects in the DNA repair mechanism. However, the molecular mechanism behind the pathogenicity of BS is still not completely understood. METHODS: We describe two patients confirmed with BS by SCE and molecular analysis. Also, we performed the gene expression profile by the RNA-seq methodology in mRNA transcripts for differential gene expression analysis using as a biological condition for comparison BS versus health controls. RESULTS: We detected 216 differentially expressed genes related to immunological pathways such as positive regulation and activation of B cells, immune effector process and absence of difference of DNA repair genes expression. In addition; we also observed differentially expressed genes associated with apoptosis control, such as BCL2L1, CASP7, CDKN1A, E2F2, ITPR, CD274, TNFAIP6, TNFRSF25, TNFRSF13C, and TNFRSF17. CONCLUSION: Our results suggest that the combination of altered expression of genes involved in signaling pathways of immune response and apoptosis control may contribute directly to the main characteristics observed in BS, such as recurrent infections, growth failure, and high risk of cancer. Transcriptome studies of other instability syndromes could allow a more accurate analysis of the relevant gene interactions associated with the destabilization of the genome. This is a first description of the profile of differential gene expression related to immunological aspects detected in patients with BS by RNA-seq.

Breakpoint delineation in 5p- patients leads to new insights about microcephaly and the typical high-pitched cry.

BACKGROUND: Cri du chat syndrome (CdCS) is a rare syndrome caused by a partial or complete deletion of the short arm of chromosome 5 (5p-). The main clinical features include a high-pitched cry, facial asymmetry, microcephaly, round face at birth, epicanthal folds, hypotonia, delayed growth and development. METHODS: We studied 14 Brazilian patients with CdCS using genomic array in order to better define the 5p breakpoints and recognize copy number variations (CNVs) that contribute to clinical manifestations associated with the syndrome. RESULTS: Array confirmed terminal deletions in 13 patients and an interstitial deletion in one patient. It was also possible to map the breakpoints and associate a genomic region of 4.7 Mb to the development of head circumference and cat-like cry. We also found other CNVs concomitant to the 5p deletion including a 9p duplication, a 17q deletion, and a 22q deletion in three different patients. CONCLUSION: With advancements of molecular cytogenomic methods in the last two decades, it was possible to evidence cryptic alterations and improve the genotype-phenotype correlation. In this work, we describe a new genomic region associated with microcephaly and cat-like cry and highlight the importance of precise delineation of 5p deletion breakpoints and detection of other CNVs in CdCS patients to improve genotype-phenotype correlation to perform a complete clinical and molecular diagnosis.

Mosaic Trisomy 12 Associated with Overgrowth Detected in Fibroblast Cell Lines.

Mosaic trisomy 12 is a rare anomaly, and only 9 cases of live births with this condition have been reported in the literature. The clinical phenotype is variable, including neuropsychomotor developmental delay, congenital heart disease, microcephaly, cutaneous spots, facial asymmetry, prominent ears, hypotonia, retinopathy, and sensorineural hearing loss. A 2-year-old female presented with neuropsychomotor developmental delay, prominent forehead, dolichocephaly, patchy skin pigmentation, and unexpected overgrowth at birth. Cytogenetic analysis of her peripheral blood showed normal results, suggesting the presence of a chromosomal alteration in other tissues. Further studies using G-banding and FISH performed on fibroblasts from both hyper- and hypopigmented regions identified a 47,XX,+12/46,XX karyotype. To the best of our knowledge, no patients with mosaic trisomy 12 associated with overgrowth have been reported to date. Congenital overgrowth and neonatal overgrowth have been frequently linked to Pallister-Killian syndrome (PKS; OMIM 601803). This case suggests the possibility of an association of genes present in the 12p region with fetal overgrowth, considering that chromosomal duplications could lead to an increase in the production of aberrant transcripts and disturbing gene dosage effects. This case highlights the importance of cytogenetic analysis in different tissues to provide relevant information to the specific genotype/phenotype correlation.