Gaia Roversi

Rothmund-Thomson syndrome

Rothmund-Thomson syndrome (RTS) is a genodermatosis presenting with a characteristic facial rash (poikiloderma) associated with short stature, sparse scalp hair, sparse or absent eyelashes and/or eyebrows, juvenile cataracts, skeletal abnormalities, radial ray defects, premature aging and a predisposition to cancer. The prevalence is unknown but around 300 cases have been reported in the literature so far. The diagnostic hallmark is facial erythema, which spreads to the extremities but spares the trunk, and which manifests itself within the first year and then develops into poikiloderma. Two clinical subforms of RTS have been defined: RTSI characterised by poikiloderma, ectodermal dysplasia and juvenile cataracts, and RTSII characterised by poikiloderma, congenital bone defects and an increased risk of osteosarcoma in childhood and skin cancer later in life. The skeletal abnormalities may be overt (frontal bossing, saddle nose and congenital radial ray defects), and/or subtle (visible only by radiographic analysis). Gastrointestinal, respiratory and haematological signs have been reported in a few patients. RTS is transmitted in an autosomal recessive manner and is genetically heterogeneous: RTSII is caused by homozygous or compound heterozygous mutations in the RECQL4 helicase gene (detected in 60-65% of RTS patients), whereas the aetiology in RTSI remains unknown. Diagnosis is based on clinical findings (primarily on the age of onset, spreading and appearance of the poikiloderma) and molecular analysis for RECQL4 mutations. Missense mutations are rare, while frameshift, nonsense mutations and splice-site mutations prevail. A fully informative test requires transcript analysis not to overlook intronic deletions causing missplicing. The diagnosis of RTS should be considered in all patients with osteosarcoma, particularly if associated with skin changes. The differential diagnosis should include other causes of childhood poikiloderma (including dyskeratosis congenita, Kindler syndrome and Poikiloderma with Neutropaenia), other rare genodermatoses with prominent telangiectasias (including Bloom syndrome, Werner syndrome and Ataxia-telangiectasia) and the allelic disorders, RAPADILINO syndrome and Baller-Gerold syndrome, which also share some clinical features. A few mutations recur in all three RECQL4 diseases. Genetic counselling should be provided for RTS patients and their families, together with a recommendation for cancer surveillance for all patients with RTSII. Patients should be managed by a multidisciplinary team and offered long term follow-up. Treatment includes the use of pulsed dye laser photocoagulation to improve the telangiectatic component of the rash, surgical removal of the cataracts and standard treatment for individuals who develop cancer. Although some clinical signs suggest precocious aging, life expectancy is not impaired in RTS patients if they do not develop cancer. Outcomes in patients with osteosarcoma are similar in RTS and non-RTS patients, with a five-year survival rate of 60-70%. The sensitivity of RTS cells to genotoxic agents exploiting cells with a known RECQL4 status is being elucidated and is aimed at optimizing the chemotherapeutic regimen for osteosarcoma.

Targeted next-generation sequencing appoints c16orf57 as clericuzio-type poikiloderma with neutropenia gene.

Next-generation sequencing is a straightforward tool for the identification of disease genes in extended genomic regions. Autozygosity mapping was performed on a five-generation inbred Italian family with three siblings affected with Clericuzio-type poikiloderma with neutropenia (PN [MIM %604173]), a rare autosomal-recessive genodermatosis characterised by poikiloderma, pachyonychia, and chronic neutropenia. The siblings were initially diagnosed as affected with Rothmund-Thomson syndrome (RTS [MIM #268400]), with which PN shows phenotypic overlap. Linkage analysis on all living subjects of the family identified a large 16q region inherited identically by descent (IBD) in all affected family members. Deep sequencing of this 3.4 Mb region previously enriched with array capture revealed a homozygous c.504-2 A>C mismatch in all affected siblings. The mutation destroys the invariant AG acceptor site of intron 4 of the evolutionarily conserved C16orf57 gene. Two distinct deleterious mutations (c.502A>G and c.666_676+1del12) identified in an unrelated PN patient confirmed that the C16orf57 gene is responsible for PN. The function of the predicted C16orf57 gene is unknown, but its product has been shown to be interconnected to RECQL4 protein via SMAD4 proteins. The unravelled clinical and genetic identity of PN allows patients to undergo genetic testing and follow-up.

Identification of novel genomic markers related to progression to glioblastoma through genomic profiling of 25 primary glioma cell lines.

Identification of genetic copy number changes in glial tumors is of importance in the context of improved/refined diagnostic, prognostic procedures and therapeutic decision-making. In order to detect recurrent genomic copy number changes that might play a role in glioma pathogenesis and/or progression, we characterized 25 primary glioma cell lines including 15 non glioblastoma (non GBM) (I–III WHO grade) and 10 GBM (IV WHO grade), by array comparative genomic hybridization, using a DNA microarray comprising approx. 3500 BACs covering the entire genome with a 1 Mb resolution and additional 800 BACs covering chromosome 19 at tiling path resolution. Combined evaluation by single clone and whole chromosome analysis plus ‘moving average (MA) approach’ enabled us to confirm most of the genetic abnormalities previously identified to be associated with glioma progression, including +1q32, +7, −10, −22q, PTEN and p16 loss, and to disclose new small genomic regions, some correlating with grade malignancy. Grade I–III gliomas exclusively showed losses at 3p26 (53%), 4q13–21 (33%) and 7p15–p21 (26%), whereas only GBMs exhibited 4p16.1 losses (40%). Other recurrent imbalances, such as losses at 4p15, 5q22–q23, 6p23–25, 12p13 and gains at 11p11–q13, were shared by different glioma grades. Three intervals with peak of loss could be further refined for chromosome 10 by our MA approach. Data analysis of full-coverage chromosome 19 highlighted two main regions of copy number gain, never described before in gliomas, at 19p13.11 and 19q13.13–13.2. The well-known 19q13.3 loss of heterozygosity area in gliomas was not frequently affected in our cell lines. Genomic hotspot detection facilitated the identification of small intervals resulting in positional candidate genes such as PRDM2 (1p36.21), LRP1B (2q22.3), ADARB2 (10p15.3), BCCIP (10q26.2) and ING1 (13q34) for losses and ECT2 (3q26.3), MDK, DDB2, IG20 (11p11.2) for gains. These data increase our current knowledge about cryptic genetic changes in gliomas and may facilitate the further identification of novel genetic elements, which may provide us with molecular tools for the improved diagnostics and therapeutic decision-making in these tumors.

The neural progenitor-restricted isoform of the MARK4 gene in 19q13.2 is upregulated in human gliomas and overexpressed in a subset of glioblastoma cell lines

Alterations of 19q13 are frequently observed in glial neoplasms, suggesting that this region harbors at least one gene involved in gliomagenesis. Following our previous studies on structural 19q chromosome rearrangements in gliomas, we have undertaken a detailed FISH analysis of the breakpoints and identified a 19q13.2 intrachromosomal amplification of the MAP/microtubule affinity-regulating kinase 4 (MARK4) gene in three primary glioblastoma cell lines. Recent data suggest that this gene is involved in the Wnt-signaling pathway. We observed that the expression of the alternatively spliced MARK4L isoform is upregulated in both fresh and cultured gliomas and overexpressed in all of the above three glioblastoma cell lines. Interestingly, we also found that MARK4L expression is restricted to undifferentiated neural progenitor cells or proliferating glial precursor cells, whereas its expression is downregulated during glial differentiation. Perturbation of expression using antisense oligonucleotides against MARK4 in glioblastoma cell lines, consistently induced a decreased proliferation of tumor cells. Taken together, these data show that MARK4, which is normally expressed in neural progenitors, is re-expressed in gliomas and may become a key target of intrachromosomal amplification upon 19q rearrangements.

FANCM c.5791C>T nonsense mutation (rs144567652) induces exon skipping, affects DNA repair activity and is a familial breast cancer risk factor

Numerous genetic factors that influence breast cancer risk are known. However, approximately two-thirds of the overall familial risk remain unexplained. To determine whether some of the missing heritability is due to rare variants conferring high to moderate risk, we tested for an association between the c.5791C>T nonsense mutation (p.Arg1931*; rs144567652) in exon 22 of FANCM gene and breast cancer. An analysis of genotyping data from 8635 familial breast cancer cases and 6625 controls from different countries yielded an association between the c.5791C>T mutation and breast cancer risk [odds ratio (OR) = 3.93 (95% confidence interval (CI) = 1.28-12.11; P = 0.017)]. Moreover, we performed two meta-analyses of studies from countries with carriers in both cases and controls and of all available data. These analyses showed breast cancer associations with OR = 3.67 (95% CI = 1.04-12.87; P = 0.043) and OR = 3.33 (95% CI = 1.09-13.62; P = 0.032), respectively. Based on information theory-based prediction, we established that the mutation caused an out-of-frame deletion of exon 22, due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. Furthermore, genetic complementation analyses showed that the mutation influenced the DNA repair activity of the FANCM protein. In summary, we provide evidence for the first time showing that the common p.Arg1931* loss-of-function variant in FANCM is a risk factor for familial breast cancer.

RNA processing defects of the helicase gene RECQL4 in a compound heterozygous Rothmund-Thomson patient.

Rothmund–Thomson syndrome (RTS) (OMIM 268400) is an autosomal recessive genodermatosis associated with genomic instability and increased risk of mesenchymal cancers. Mutations in the RECQL4 gene, encoding a protein of the family of Werner (WRN) and Bloom (BLM) helicases, have been identified in a subset of RTS patients. Apart from congenital poikiloderma, the clinical presentation of RTS is widely variable, raising the question of the possible existence of a second locus. Results herein reported on a sporadic Caucasian patient emphasize the concept that mutation analyses at both DNA and RNA level complement the genetic defect suggested by clinical and cytogenetic signs. The patient presented with typical congenital poikiloderma and bone defects and exhibited significant genomic instability in the peripheral blood karyotype. By RECQL4 DNA mutation analysis, he was found to carry a 1473delT (mut 5) on one allele and an AG to AC change at the 3′‐splice site of exon 13 (a variant of mut 4) on the second allele. RT‐PCR analysis of RECQL4 cDNA encompassing the entire helicase domain showed diffuse splicing defects indicating that the loss of a single 3′‐splice signal motif disregulates the correct splice‐site selection and affects the overall RNA processing. The presence of an unstable minisatellite which ends at 3′‐splice site of IVS12 may enhance the mutation at this site. This genomic feature together with a number of short introns in the RECQL4 gene may account for the common missplicing of RECQL4 mRNA. While it is possible that defects of RECQL4 mRNA processing might account for part of the clinical variability observed for this syndrome, only a thorough analysis at both genomic and RNA level may allow a genotype–phenotype correlation in RTS patients, restricting the search of a second RTS locus to the specific patients.

Clericuzio-type poikiloderma with neutropenia syndrome in three sibs with mutations in the C16orf57 gene: delineation of the phenotype

We report on three sibs who have autosomal recessive Clericuzio‐type poikiloderma neutropenia (PN) syndrome. Recently, this consanguineous family was reported and shown to be informative in identifying the C16orf57 gene as the causative gene for this syndrome. Here we present the clinical data in detail. PN is a distinct and recognizable entity belonging to the group of poikiloderma syndromes among which Rothmund–Thomson is perhaps the best described and understood. PN is characterized by cutaneous poikiloderma, hyperkeratotic nails, generalized hyperkeratosis on palms and soles, neutropenia, short stature, and recurrent pulmonary infections. In order to delineate the phenotype of this rare genodermatosis, the clinical presentation together with the molecular investigations in our patients are reported and compared to those from the literature.

Multiple Localization of Endogenous MARK4L Protein in Human Glioma

Background: We have previously shown that the sustained expression of MARK4L transcripts in glioma and neural progenitors (NHNPs) declines after exposure to antisense MARK4L oligonucleotides in glioblastoma cell lines. Array-CGH confirmed the genomic duplication of MARK4L identified by FISH in a glioblastoma cell line. This background together with literature data on the exogenous association of MARK4 with interphase centrosome prompted us to investigate the sub-cellular localization of the endogenous MARK4L protein aiming at achieving insights on its possible role in the pathomechanisms of glioma. Methods: Immunodetection was carried out to validate the specificity of MARK4L antibody in gliomas and NHNPs. Mass spectrometry was applied for MARK4L protein identification in a representative glioblastoma cell line. Combined biochemical fractionation and immunodetection analyses were performed to confirm the sub-cellular localization of MARK4L achieved by immunofluorescence in glioma cell lines. Results: By assigning MARK4L protein within the band immunoprecipitated by the specific antibody we validated our anti-MARK4L antibody. We demonstrated that the endogenous MARK4L: (i) colocalizes with centrosomes at all mitotic stages and resides in centrosome-enriched fractions; (ii) associates with the nucleolus and the midbody and respective fractions, and (iii) co-stains the aberrant centrosome configurations observed in glioma cell lines. Conclusions: The overall data merge on the multiplex entry of MARK4L into the cell cycle and link it to the aberrant centrosomes in glioma cell lines suggesting a possible role of this kinase in the abnormal mitotic processes of human glioma.

SEARCH FOR GENOMIC IMBALANCES IN A COHORT OF 24 CORNELIA DE LANGE PATIENTS NEGATIVE FOR MUTATIONS IN THE NIPBL AND SMC1L1 GENES.

Cornelia de Lange syndrome (CdLS) is a rare, multiple congenital anomaly/mental retardation syndrome characterized by varied clinical signs including facial dysmorphism, pre‐ and post‐natal growth defects, small hands and malformations of the upper limbs. Established genetic causes include mutations in the NIPBL (50–60%), SMC1L1 and SMC3 (5%) genes. To detect chromosomal rearrangements pointing to novel positional candidate CdLS genes, we used array‐CGH to analyze a subgroup of 24 CdLS patients negative for mutations in the NIPBL and SMC1L1 genes. We identified three carriers of DNA copy number alterations, including a de novo 15q26.2‐qter 8‐Mb deletion, and two inherited 13q14.2‐q14.3 1‐Mb deletion and 13q21.32‐q21.33 1.5‐Mb duplication, not reported among copy number variants. The clinical presentation of all three patients matched the diagnostic criteria for CdLS, and the phenotype of the patient with the 15qter deletion is compared to that of both CdLS and 15qter microdeletion patients.

Differential cytogenomics and miRNA signature of the Acute Myeloid Leukaemia Kasumi-1 cell line CD34+38− compartment

The t(8;21) Acute Myeloid Leukaemia (AML) Kasumi-1 cell line with N822K KIT mutation, is a model system for leukemogenesis. As AML initiating cells reside in the CD34(+)CD38(-) fraction, we addressed the refined cytogenomic characterization and miRNA expression of Kasumi-1 cell line and its FACS-sorted subpopulations focussing on this compartment. By conventional cytogenetics, Spectral-Karyotyping and array-CGH the cytogenomic profile of Kasumi-1 cells evidenced only subtle regions differentially represented in CD34(+)CD38(-) cells. Expression profiling by a miRNA platform showed a set of miRNA differentially expressed in paired subpopulations and the signature of miR-584 and miR-182 upregulation in the CD34(+)CD38(-) fraction.