Roberta Zuntini

CpG Drives Human Transitional B Cells to Terminal Differentiation and Production of Natural Antibodies

The receptor TLR9, recognizing unmethylated bacterial DNA (CpG), is expressed by B cells and plays a role in the maintenance of serological memory. Little is known about the response of B cells stimulated with CpG alone, without additional cytokines. In this study, we show for the first time the phenotypic modification, changes in gene expression, and functional events downstream to TLR9 stimulation in human B cell subsets. In addition, we demonstrate that upon CpG stimulation, IgM memory B cells differentiate into plasma cells producing IgM Abs directed against the capsular polysaccharides of Streptococcus pneumoniae. This novel finding proves that IgM memory is the B cell compartment responsible for the defense against encapsulated bacteria. We also show that cord blood transitional B cells, corresponding to new bone marrow emigrants, respond to CpG. Upon TLR9 engagement, they de novo express AID and Blimp-1, genes necessary for hypersomatic mutation, class-switch recombination, and plasma cell differentiation and produce Abs with anti-pneumococcal specificity. Transitional B cells, isolated from cord blood, have not been exposed to pneumococcus in vivo. In addition, it is known that Ag binding through the BCR causes apoptotic cell death at this stage of development. Therefore, the ability of transitional B cells to sense bacterial DNA through TLR9 represents a tool to rapidly build up the repertoire of natural Abs necessary for our first-line defense at birth.

Mutations of the Igβ gene cause agammaglobulinemia in man

Agammaglobulinemia is a rare primary immunodeficiency characterized by an early block of B cell development in the bone marrow, resulting in the absence of peripheral B cells and low/absent immunoglobulin serum levels. So far, mutations in Btk, μ heavy chain, surrogate light chain, Igα, and B cell linker have been found in 85–90% of patients with agammaglobulinemia. We report on the first patient with agammaglobulinemia caused by a homozygous nonsense mutation in Igβ, which is a transmembrane protein that associates with Igα as part of the preBCR complex. Transfection experiments using Drosophila melanogaster S2 Schneider cells showed that the mutant Igβ is no longer able to associate with Igα, and that assembly of the BCR complex on the cell surface is abrogated. The essential role of Igβ for human B cell development was further demonstrated by immunofluorescence analysis of the patients bone marrow, which showed a complete block of B cell development at the pro-B to preB transition. These results indicate that mutations in Igβ can cause agammaglobulinemia in man.

An inherited mitochondrial DNA disruptive mutation shifts to homoplasmy in oncocytic tumor cells

A disruptive frameshift mtDNA mutation affecting the ND5 subunit of complex I is present in homoplasmy in a nasopharyngeal oncocytic tumor and inherited as a heteroplasmic germline mutation recurring in two of the patients siblings. Homoplasmic ND5 mutation in the tumor correlates with lack of the ND6 subunit, suggesting complex I disassembly. A few oncocytic areas, expressing ND6 and heteroplasmic for the ND5 mutation, harbor a de novo homoplasmic ND1 mutation. Since shift to homoplasmy of ND1 and ND5 mutations occurs exclusively in tumor cells, we conclude that complex I mutations may have a selective advantage and accompany oncocytic transformation. Hum Mutat 0, 1–6, 2008.

Placing mitochondrial DNA mutations within the progression model of type I endometrial carcinoma.

Mitochondrial DNA (mtDNA) mutations have been described in almost all types of cancer. However, their exact role and timing of occurrence during tumor development and progression are still a matter of debate. A Vogelstein-like model of progression is well established for endometrial carcinoma (EC), however, mtDNA has been scarcely investigated in these tumors despite the fact that mitochondrial biogenesis increase has been shown to be a hallmark of type I EC. Here, we screened a panel of 23 type I EC tissues and matched typical hyperplasia for mutations in mtDNA and in four oncosupressors/oncogenes, namely PTEN, KRAS, CTNNB1 and TP53. Overall, mtDNA mutations were identified in 69% of cases, while mutational events in nuclear genes occurred in 56% of the cases, indicating that mtDNA mutations may precede the genetic instability of these genes canonically involved in progression from hyperplasia to tumor. Protein expression analysis revealed an increase in mitochondrial biogenesis and activation of oxidative stress response mechanisms in tumor tissues, but not in hyperplasia, in correlation with the occurrence of pathogenic mtDNA mutations. Our results point out an involvement of mtDNA mutations in EC progression and explain the increase in mitochondrial biogenesis of type I EC. Last, since mtDNA mutations occur after hyperplasia, their potential role in contributing to genetic instability may be envisioned.

FA2H-related disorders: a novel c.270+3A>T splice-site mutation leads to a complex neurodegenerative phenotype

Homozygous mutations in the gene for fatty acid 2‐hydroxylase (FA2H) have been associated in humans with three neurodegenerative disorders: complicated spastic paraplegia (SPG35), leukodystrophy with spastic paraparesis and dystonia, and neurodegeneration with brain iron accumulation. Here, we describe a novel homozygous c.270+3A>T mutation in an Italian consanguineous family. In two affected brothers (age at molecular diagnosis 22y and 15y; age at last follow‐up 24y and 17y), altered FA2H function led to a severe phenotype, with clinical features overlapping those of the three FA2H‐associated disorders. Both patients showed childhood onset progressive spastic paraparesis, mild pyramidal and cerebellar upper limb signs, severe cognitive impairment, white‐matter disease, and cerebellar, brainstem, and spinal cord atrophy. However, absence of dystonia, drowsiness episodes, and a subtle globus pallidus involvement suggested that FA2H mutations result in a clinical spectrum, rather than causing distinct disorders. Although clinical heterogeneity is apparent, larger numbers of patients are needed to establish more accurate genotype–phenotype correlations.

Identification of different Ikaros cDNA transcripts in Philadelphia-positive adult acute lymphoblastic leukemia by a high-throughput capillary electrophoresis sizing method

Ikaros is a member of the Kruppel family of zinc finger DNA-binding proteins. The findings of this study demonstrated that both aberrant splicing and genomic deletion leading to different non-DNA-binding transcripts are common features of Philadelphia chromosome-positive acute lymphoblastic leukemia. Background Ikaros is the prototypic member of a Kruppel-like zinc finger transcription factor subfamily that is required for normal hematopoietic cell differentiation and proliferation, particularly in the lymphoid lineages. Alternative splicing can generate multiple Ikaros isoforms that lack different numbers of exons and have different functions. Shorter isoforms, which lack the amino-terminal domain that mediates sequence-specific DNA binding, exert a dominant negative effect and inhibit the ability of longer heterodimer partners to bind DNA. Design and Methods In this study, we developed a high-throughput capillary electrophoresis sizing method to detect and quantify different Ikaros cDNA transcripts. Results We demonstrated that Philadelphia chromosome-positive acute lymphoblastic leukemia cells expressed high levels of the non-DNA-binding isoform Ik6 that was generated following IKZF1 genomic deletions (19/46 patients, 41%). Furthermore, a recurring 60 bp insertion immediately upstream of exon 5, at the exon 3/exon 5 junction, was frequently detected in the Ik2 and Ik4 isoforms. This insertion occurred either alone or together with an in-frame ten amino acid deletion that was due to a 30 bp loss at the end of exon 7. Both the alterations are due to the selection of alternative cryptic splice sites and have been suggested to cause impaired DNA-binding activity. Non-DNA-binding isoforms were localized in the cytoplasm whereas the DNA-binding isoforms were localized in the nucleus. Conclusions Our findings demonstrate that both aberrant splicing and genomic deletion leading to different non-DNA-binding Ikaros cDNA transcripts are common features of Philadelphia chromosome-positive acute lymphoblastic leukemia.

Searching for a needle in the haystack: Comparing six methods to evaluate heteroplasmy in difficult sequence context

Mitochondrial DNA (mtDNA) mutations have been involved in disease, aging and cancer and furthermore exploited for evolutionary and forensic investigation. When investigating mtDNA mutations the peculiar aspects of mitochondrial genetics, such as heteroplasmy and threshold effect, require suitable approaches which must be sensitive enough to detect low-level heteroplasmy and, precise enough to quantify the exact mutational load. In order to establish the optimal approach for the evaluation of heteroplasmy, six methods were experimentally compared for their capacity to reveal and quantify mtDNA variants. Drawbacks and advantages of cloning, Fluorescent PCR (F-PCR), denaturing High Performance Liquid Chromatography (dHPLC), quantitative Real-Time PCR (qRTPCR), High Resolution Melting (HRM) and 454 pyrosequencing were determined. In particular, detection and quantification of a mutation in a difficult sequence context were investigated, through analysis of an insertion in a homopolymeric stretch (m.3571insC).

Autosomal recessive hereditary spastic paraplegia with thin corpus callosum: a novel mutation in the SPG11 gene and further evidence for genetic heterogeneity

Background and purpose:  Autosomal Recessive Hereditary Spastic Paraplegia with Thin Corpus Callosum (AR‐HSPTCC) is a clinically and genetically heterogeneous complicated form of spastic paraplegia. Two AR‐HSPTCC loci have been assigned to chromosome 15q13‐15 (SPG11) and chromosome 8p12‐p11.21 respectively. Mutations in the SPG11 gene, encoding the spatacsin protein, have been found in the majority of SPG11 families. In this study, involvement of the SPG11 or 8p12‐p11.21 loci was investigated in five Italian families, of which four consanguineous.

Where Birt–Hogg–Dubé meets Cowden Syndrome: mirrored genetic defects in two cases of syndromic oncocytic tumours

Birt–Hogg–Dubè (BHD) is an autosomal dominant syndrome characterised by skin fibrofolliculomas, lung cysts, spontaneous pneumothorax and renal cancer. The association of benign cutaneous lesions and increased cancer risk is also a feature of Cowden Syndrome (CS), an autosomal dominant disease caused by PTEN mutations. BHD and CS patients may develop oncocytomas, rare neoplasias that are phenotypically characterised by a prominent mitochondrial hyperplasia. We here describe the genetic analysis of a parotid and a thyroid oncocytoma, developed by a BHD and a CS patient, respectively. The BHD lesion was shown to maintain the wild-type allele of FLCN, while losing one PTEN allele. On the other hand, a double heterozygosity for the same two genes was found to be the only detectable tumorigenic hit in the CS oncocytoma. Both conditions occurred in a context of high chromosomal stability, as highlighted by comparative genomic hybridisation analysis. We conclude that, similarly to PTEN, FLCN may not always follow the classical Two Hits model of tumorigenesis and may hence belong to a class of non-canonical tumour suppressor genes. We hence introduce a role of PTEN/FLCN double heterozygosity in syndromic oncocytic tumorigenesis, suggesting this to be an alternative determinant to pathogenic mitochondrial DNA mutations, which are instead the genetic hallmark of sporadic oncocytic tumours.

Two distinct thyroid tumours in a patient with Cowden syndrome carrying both a 10q23 and a mitochondrial DNA germline deletion

Background Cowden syndrome (CS) is an autosomal dominant disorder characterised by macrocephaly, specific mucocutaneous features and predisposition to benign and malignant tumours. Detectable mutations in the PTEN gene account for 80–85% of cases. Methods/Results Here, the authors report a patient with macrocephaly and typical CS mucocutaneous features who developed dysplastic cerebellar gangliocytoma and two synchronous thyroid cancers of papillary and oncocytic type, in whom a germline 500-Kb deletion on chromosome 10q23 including PTEN was detected. Molecular characterisation of thyroid cancer led to the identification of the oncogenic BRAFV600E mutation in the papillary carcinoma. BRAFV600E has been proposed to cause cancer only in the presence of a tumour-suppressor mutation, which, in this case, could be the PTEN deletion. In the oncocytic carcinoma, a large deletion in the mitochondrial-DNA-encoded MTND1 was found, associated with respiratory complex I disassembly, which was subsequently shown to be a constitutional, de novo genetic lesion. Conclusions This is the first reported case of a patient with CS carrying constitutional deletions in both the nuclear and the mitochondrial genome that might help elucidate some aspects of CS pathogenesis.