A Homozygous Deep Intronic Mutation Alters the Splicing of Nebulin Gene in a Patient With Nemaline Myopathy.

Nemaline myopathy is a rare disorder affecting the muscle sarcomere. Mutations in nebulin gene (NEB) are known to be responsible for about 50% of nemaline myopathy cases. Nebulin is a giant protein which is formed integrally with the sarcomeric thin filament. This complex gene is under extensive alternative splicing giving rise to multiple isoforms. In this study, we report a 6-year-old boy presenting with general muscular weaknesses. Identification of rod-shaped structures in the patient' biopsy raised doubt about the presence of a nemaline myopathy. Next-generation sequencing was used to identify a causative mutation for the patient syndrome. A homozygous deep intronic substitution was found in the intron 144 of the NEB. The variant was predicted by in silico tools to create a new donor splice site. Molecular analysis has shown that the mutation could alter splicing events of the nebulin gene leading to a significant decrease of isoforms level. This change in the expression level of nebulin could give rise to functional consequences in the sarcomere. These results are consistent with the phenotypes observed in the patient. Such a discovery of variants in this gene will allow a better understanding of the involvement of nebulin in neuromuscular diseases and help find new treatments for the nemaline myopathy.

A founder mutation in the PLPBP gene in families from Saguenay-Lac-St-Jean region affected by a pyridoxine-dependent epilepsy.

Pyridoxine-dependent epilepsy (PDE) is a relatively rare subgroup of epileptic disorders. They generally present in infancy as an early onset epileptic encephalopathy or seizures, refractory to standard treatments, with rapid and variable responses to vitamin B6 treatment. Whole exome sequencing of three unrelated families identified homozygous pathogenic mutation c.370_373del, p.Asp124fs in PLPBP gene in five persons. Haplotype analysis showed a single shared profile for the affected persons and their parents, leading to a hypothesis about founder effect of the mutation in Saguenay-Lac-St-Jean region of French Canadians. All affected probands also shared one single mitochondrial haplotype T2b3 and two rare variations in the mitochondrial genome m.801A>G and m.5166A>G suggesting that a single individual female introduced PLPBP mutation c.370_373del, p.Asp124fs in Quebec. The mutation p.Asp124fs causes a severe disease phenotype with delayed myelination and cortical/subcortical brain atrophy. The most noteworthy radiological finding in this Quebec founder mutation is the presence of the temporal cysts that can be used as a marker of the disease. Also, both patients, who are alive, had a history of prenatal supplements taken by their mothers as antiemetic medication with high doses of pyridoxine. In the context of suspected PDE in patients with neonatal refractory seizures, treatment with pyridoxine and/or Pyridoxal-5-phophate has to be started immediately and continued until the results of genetic analysis received. Even with early appropriate treatment, neurological outcome of our patient is still poor.

Whole-exome sequencing identifies homozygous mutation in TTI2 in a child with primary microcephaly: a case report.

BACKGROUND: Primary microcephaly is defined as reduced occipital-frontal circumference noticeable before 36 weeks of gestation. Large amount of insults might lead to microcephaly including infections, hypoxia and genetic mutations. More than 16 genes are described in autosomal recessive primary microcephaly. However, the cause of microcephaly remains unclear in many cases after extensive investigations and genetic screening. CASE PRESENTATION: Here, we described the case of a boy with primary microcephaly who presented to a neurology clinic with short stature, global development delay, dyskinetic movement, strabismus and dysmorphic features. We performed microcephaly investigations and genetic panels. Then, we performed whole-exome sequencing to identify any genetic cause. Microcephaly investigations and genetic panels were negative, but we found a new D317V homozygous mutation in TELOE-2 interacting protein 2 (TTI2) gene by whole-exome sequencing. TTI2 is implicated in DNA damage response and mutation in that gene was previously described in mental retardation, autosomal recessive 39. CONCLUSIONS: We described the first French Canadian case with primary microcephaly and global developmental delay secondary to a new D317V homozygous mutation in TTI2 gene. Our report also highlights the importance of TTI2 protein in brain development.

Identification of a gene signature for different stages of breast cancer development that could be used for early diagnosis and specific therapy.

Breast cancer (BC) is a heterogeneous disease where the survival rate of patients decreases with progression of the disease. BC usually has a linear progression, classified into normal/benign, atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC). This study aimed to identify gene signature for each of these subgroups. We performed human transcriptome array analysis on 5 patient samples from each Normal, ADH, IDC and DCIS and 2 replicates of MCF10A cell line representative of each subgroup. We identified SFRP1 and snoRNAs (especially SNORD115 and SNORD114) as the initial regulators of cancer progression, accompanied by significant changes in extracellular matrix organization. Tumor progression to the IDC stage showed upregulation of tumor promoting genes responsible for increased invasion, inflammation, survival in stress environment and metastasis. The gene signatures identified in this study could represent potential biomarkers for each subgroup of breast cancer progression, which could assist in early diagnosis of breast cancer progression as well as treatment interventions. Moreover, these gene signatures could serve in discovery of specific targeted therapies for each subgroup.

Transcriptional signature of lymphoblastoid cell lines of BRCA1, BRCA2 and non-BRCA1/2 high risk breast cancer families.

Approximately 25% of hereditary breast cancer cases are associated with a strong familial history which can be explained by mutations in BRCA1 or BRCA2 and other lower penetrance genes. The remaining high-risk families could be classified as BRCAX (non-BRCA1/2) families. Gene expression involving alternative splicing represents a well-known mechanism regulating the expression of multiple transcripts, which could be involved in cancer development. Thus using RNA-seq methodology, the analysis of transcriptome was undertaken to potentially reveal transcripts implicated in breast cancer susceptibility and development. RNA was extracted from immortalized lymphoblastoid cell lines of 117 women (affected and unaffected) coming from BRCA1, BRCA2 and BRCAX families. Anova analysis revealed a total of 95 transcripts corresponding to 85 different genes differentially expressed (Bonferroni corrected p-value <0.01) between those groups. Hierarchical clustering allowed distinctive subgrouping of BRCA1/2 subgroups from BRCAX individuals. We found 67 transcripts, which could discriminate BRCAX from BRCA1/BRCA2 individuals while 28 transcripts discriminate affected from unaffected BRCAX individuals. To our knowledge, this represents the first study identifying transcripts differentially expressed in lymphoblastoid cell lines from major classes of mutation-related breast cancer subgroups, namely BRCA1, BRCA2 and BRCAX. Moreover, some transcripts could discriminate affected from unaffected BRCAX individuals, which could represent potential therapeutic targets for breast cancer treatment.

Genome-wide methylation analysis of DNMT3B gene isoforms revealed specific methylation profiles in breast cell lines.

AIM: The goal of this study is to characterize the specific methylation profile triggered by DNMT3B protein isoforms expressed at different levels in breast cell lines. MATERIALS & METHODS: Microarray DNA methylation data were analyzed and associated with functional genome annotation data. RESULTS: A large spectrum of DNMT3B3/DNMT3B2 expression ratio values was observed in parental breast cell lines. According to their methylation profiles, hierarchical clustering of untransfected cell lines revealed clustering based on their ER/PR status. Overexpression of DNMT3B3 triggered methylation changes of thousands of CpG sites in breast cells. Based on the trend of methylation changes, the results suggest an antiproliferative action of the DNMT3B3 isoform through a dominant negative effect on its wild-type counterpart DNMT3B2. CONCLUSION: This study revealed specific pathways modulated by DNMT3B isoforms, which could regulate cell proliferation and other biological mechanisms. This illustrates the importance of multiple interactions between isoforms in the complexity of methylation processes.

Analysis of a FANCE Splice Isoform in Regard to DNA Repair.

The FANC-BRCA DNA repair pathway is activated in response to interstrand crosslinks formed in DNA. A homozygous mutation in 1 of the 17 Fanconi anemia (FA) genes results in malfunctions of this pathway and development of FA syndrome. The integrity of this protein network is essential for good maintenance of DNA repair process and genome stability. Following the identification of an alternatively splice isoform of FANCE (Fanconi anemia complementation group E) significantly expressed in breast cancer individuals from high-risk non-BRCA1/2 families, we studied the impact of this FANCE splice isoform (FANCEΔ4) on DNA repair processes. We have demonstrated that FANCEΔ4 mRNA was efficiently translated into a functional protein and expressed in normal and breast cancer cell lines. Following treatment with the crosslinking agent mitomycin C, EUFA130 (FANCE-deficient) cells infected with FANCEΔ4 were blocked into G2/M phase, while cell survival was significantly reduced compared with FANCE-infected EUFA130 cells. In addition, FANCEΔ4 did not allow FANCD2 and FANCI monoubiquitination, which represents a crucial step of the FANC-BRCA functional pathway. As observed for FANCE wild-type protein, localization of FANCEΔ4 protein was confined to the nucleus following mitomycin C treatment. Although FANCEΔ4 protein showed interaction with FANCE, FANCEΔ4 did not support normal function of FANCE protein in this pathway and could have deleterious effects on FANCE protein activity. We have demonstrated that FANCEΔ4 seems to act as a regulator of FANCD2 protein expression level by promoting its degradation. This study highlights the importance of an efficient regulation of alternative splicing expression of FA genes for proper DNA repair.

The Role of Methylation in Breast Cancer Susceptibility and Treatment.

DNA methylation is a critical mechanism of epigenetic modification involved in gene expression programming, that can promote the development of several cancers, including breast cancer. The methylation of CpG islands by DNA methyltransferases is reversible and has been shown to modify the transcriptional activity of key proliferation genes or transcription factors involved in suppression or promotion of cell growth. Indeed, aberrant methylation found in gene promoters is a hallmark of cancer that could be used as non-intrusive biomarker in body fluids such as blood and plasma for early detection of breast cancer. Many biomarker genes have been evaluated for breast cancer detection. However, in the absence of a unique biomarker having the sufficient specificity and sensitivity, a panel of multiple genes should be used. Treatments targeting aberrant methylation by DNA methyltransferase inhibitors, which trigger re-expression of silenced genes, are now available and allow for better treatment efficiency.

Analysis of ZNF350/ZBRK1 promoter variants and breast cancer susceptibility in non-BRCA1/2 French Canadian breast cancer families.

ZNF350/ZBRK1 is a transcription factor, which associates with BRCA1 to co-repress GADD45A to regulate DNA damage repair, and the expression of ZNF350 is altered in different human carcinomas. In a previous study, we identified ZNF350 genomic variants potentially involved in breast cancer susceptibility in high-risk non-BRCA1/2 breast cancer individuals, which pointed toward a potential association for variants in the 5'-UTR and promoter regions. Therefore, direct sequencing was undertaken and identified 12 promoter variants, whereas haplotype analyses put in evidence four common haplotypes with a frequency>2%. However, based on their frequency observed in breast cancer and unrelated healthy individuals, these are not statistically associated with breast cancer risk. Luciferase promoter assays in two breast cancer cell lines identified two haplotypes (H11 and H12) stimulating significantly the expression of ZNF350 transcript compared with the common haplotype H8. The high expression of the H11 allele was associated with the variant c.-874A. Using MatInspector and Transcription Element Search softwares, in silico analyses predicted that the variant c.-874A created a binding site for the factors c-Myc and myogenin. This study represents the first characterization step of the ZNF350 promoter. Additional studies in larger cohorts and other populations will be needed to further evaluate whether common and/or rare ZNF350 promoter variants and haplotypes could be associated with a modest risk of breast cancer.

Polymorphic variations in the FANCA gene in high-risk non-BRCA1/2 breast cancer individuals from the French Canadian population.

The majority of genes associated with breast cancer susceptibility, including BRCA1 and BRCA2 genes, are involved in DNA repair mechanisms. Moreover, among the genes recently associated with an increased susceptibility to breast cancer, four are Fanconi Anemia (FA) genes: FANCD1/BRCA2, FANCJ/BACH1/BRIP1, FANCN/PALB2 and FANCO/RAD51C. FANCA is implicated in DNA repair and has been shown to interact directly with BRCA1. It has been proposed that the formation of FANCA/G (dependent upon the phosphorylation of FANCA) and FANCB/L sub-complexes altogether with FANCM, represent the initial step for DNA repair activation and subsequent formation of other sub-complexes leading to ubiquitination of FANCD2 and FANCI. As only approximately 25% of inherited breast cancers are attributable to BRCA1/2 mutations, FANCA therefore becomes an attractive candidate for breast cancer susceptibility. We thus analyzed FANCA gene in 97 high-risk French Canadian non-BRCA1/2 breast cancer individuals by direct sequencing as well as in 95 healthy control individuals from the same population. Among a total of 85 sequence variants found in either or both series, 28 are coding variants and 19 of them are missense variations leading to amino acid change. Three of the amino acid changes, namely Thr561Met, Cys625Ser and particularly Ser1088Phe, which has been previously reported to be associated with FA, are predicted to be damaging by the SIFT and PolyPhen softwares. cDNA amplification revealed significant expression of 4 alternative splicing events (insertion of an intronic portion of intron 10, and the skipping of exons 11, 30 and 31). In silico analyzes of relevant genomic variants have been performed in order to identify potential variations involved in the expression of these spliced transcripts. Sequence variants in FANCA could therefore be potential spoilers of the Fanconi-BRCA pathway and as a result, they could in turn have an impact in non-BRCA1/2 breast cancer families.