Absence of mutations at SERPINI1 gene in a cohort of patients with Cerebral Cavernous Malformations

Cerebral cavernous malformations (CCM) are vascular lesions affecting brain microvessels. While molecular bases of the sporadic condition are not yet well elucidated, familial forms arise following mutations at three different loci KRIT1, CCM2 and PDCD10. However, no germline mutations are detected in a small percentage of families with hereditary history of CCM. In order to detect other possible candidate genes, we performed molecular analysis of SERPINI1 gene in a cohort of patients carrying no mutations in the three CCM loci, aiming to detect mutations likely associated to lesion development. Therefore, we performed molecular analysis of the SERPINI1 gene in a cohort of 18 unrelated patients affected by both familial and sporadic CCM showing no germline causative mutations. Mutational analysis resulted negative and only few single nucleotide polymorphisms were detected. However, the rs11284733 SNP was detected in a high percentage of patients affected by familial form of the disease. This SNP occurs within a noncoding exon retained in an alternative spliced SERPINI1 transcript, suggesting its possible role in gene expression regulation.

Universal alternative splicing of noncoding exons

The human transcriptome is so large, diverse and dynamic that, even after a decade of investigation by RNA sequencing (RNA-Seq), we are yet to resolve its true dimensions. RNA-Seq suffers from an expression-dependent bias that impedes characterization of low-abundance transcripts. We performed targeted single-molecule and short-read RNA-Seq to survey the transcriptional landscape of a single human chromosome (Hsa21) at unprecedented resolution. Our analysis reaches the lower limits of the transcriptome, identifying a fundamental distinction between protein-coding and noncoding gene content: almost every noncoding exon undergoes alternative splicing, producing a seemingly limitless variety of isoforms. Analysis of syntenic regions of the mouse genome shows that few noncoding exons are shared between human and mouse, yet human splicing profiles are recapitulated on Hsa21 in mouse cells, indicative of regulation by a deeply conserved splicing code. We propose that noncoding exons are functionally modular, with alternative splicing generating an enormous repertoire of potential regulatory RNAs and a rich transcriptional reservoir for gene evolution.

Molecular Bases and Phenotypic Determinants of Aromatase Excess Syndrome

Aromatase excess syndrome (AEXS) is a rare autosomal dominant disorder characterized by gynecomastia. This condition is caused by overexpression ofCYP19A1encoding aromatase, and three types of cryptic genomic rearrangement aroundCYP19A1, that is, duplications, deletions, and inversions, have been identified in AEXS. Duplications appear to have causedCYP19A1overexpression because of an increased number of physiological promoters, whereas deletions and inversions would have induced wideCYP19A1expression due to the formation of chimeric genes consisting of a noncoding exon(s) of a neighboring gene andCYP19A1coding exons. Genotype-phenotype analysis implies that phenotypic severity of AEXS is primarily determined by the expression pattern ofCYP19A1and the chimeric genes and by the structural property of the fused exons with a promoter function (i.e., the presence or the absence of a natural translation start codon). These results provide novel information about molecular mechanisms of human genetic disorders and biological function of estrogens.

Identification of a novel A4GALT exon reveals the genetic basis of the P1/P2 histo-blood groups

The A4GALT locus encodes a glycosyltransferase that synthesizes the terminal Galα1-4Gal of the Pk (Gb3/CD77) glycosphingolipid, important in transfusion medicine, obstetrics, and pathogen susceptibility. Critical nucleotide changes in A4GALT not only abolish Pk formation but also another Galα1-4Gal–defined antigen, P1, which belongs to the only blood group system for which the responsible locus remains undefined. Since known A4GALT polymorphisms do not explain the P1−Pk+ phenotype, P2, we set out to elucidate the genetic basis of P1/P2. Despite marked differences (P1 > P2) in A4GALT transcript levels in blood, luciferase experiments showed no difference between P1/P2-related promoter sequences. Investigation of A4GALT mRNA in cultured human bone marrow cells revealed novel transcripts containing only the noncoding exon 1 and a sequence (here termed exon 2a) from intron 1. These 5′-capped transcripts include poly-A tails and 3 polymorphic sites, one of which was P1/P2-specific among > 200 donors and opens a short reading frame in P2 alleles. We exploited these data to devise the first genotyping assays to predict P1 status. P1/P2 genotypes correlated with both transcript levels and P1/Pk expression on red cells. Thus, P1 zygosity partially explains the well-known interindividual variation in P1 strength. Future investigations need to focus on regulatory mechanisms underlying P1 synthesis.

Mutations of the BCL6 proto-oncogene disrupt its negative autoregulation in diffuse large B-cell lymphoma

The BCL6 proto-oncogene encodes a transcriptional repressor whose expression is deregulated by chromosomal translocations in approximately 40% of diffuse large B-cell lymphomas (DLBCLs). The BCL6 regulatory sequences are also targeted by somatic hypermutation in germinal center (GC) B cells and in a fraction of all GC-derived lymphomas. However, the functional consequences of these mutations are unknown. Here we report that a subset of mutations specifically associated with DLBCL causes deregulated BCL6 transcription. These mutations affect 2 adjacent BCL6 binding sites located within the first noncoding exon of the gene, and they prevent BCL6 from binding its own promoter, thereby disrupting its negative autoregulatory circuit. These alterations were found in approximately 16% of DLBCLs devoid of chromosomal translocations involving the BCL6 locus, but they were not found in normal GC B cells. This study establishes a novel mechanism for BCL6 deregulation and reveals a broader involvement of this gene in DLBCL pathogenesis.