A Peculiar CLL Case with Complex Chromosome 6 Rearrangements and Refinement of All Breakpoints at the Gene Level by Genomic Array: A Case Report.

INTRODUCTION: Chronic lymphocytic leukemia (CLL), the most common leukemia in Western countries, is a mature B-cell chronic lymphoproliferative disorder characterized by the accumulation of neoplastic CD5+ B lymphocytes, functionally incompetent and usually monoclonal in origin, in bone marrow, lymph nodes and blood. Diagnosis occurs predominantly in elderly patients, with a median age reported between 67 and 72 years. CLL has a heterogeneous clinical course, which can vary from indolent to, less frequently, aggressive forms. Early-stage asymptomatic CLL patients do not require immediate therapeutic intervention, but only observation; treatment is necessary for patients with advanced disease or when "active disease" is observed. The most frequent autoimmune cytopenia (AIC) is autoimmune haemolytic anaemia (AHIA). The main mechanisms underlying the appearance of AIC in CLL are not fully elucidated, the predisposition of patients with CLL to suffering autoimmune complications is variable and autoimmune cytopenia can precede, be concurrent, or follow the diagnosis of CLL. CASE PRESENTATION: A 74-year-old man was admitted to the emergency room following the finding of severe macrocytic anaemia during blood tests performed that same day, in particular the patient showed a profound asthenia dating back several months. The anamnesis was silent and the patient was not taking any medications. The blood examination showed an extremely high White Blood Cell count and findings of AIHA in CLL-type mature B-cell lymphoproliferative neoplasia. Genetic investigations: Conventional karyotyping was performed and it obtained a trisomy 8 and an unbalanced translocation between the short arm of chromosome 6 and the long arm of chromosome 11, concurrent with interstitial deletions in chromosomes 6q and 11q that could not be defined in detail. Molecular cytogenetics (FISH) analyses revealed Ataxia Telangiectasia Mutated (ATM) monoallelic deletion (with loss of ATM on derivative chromosome 11) and retained signals for TP53, 13q14 and centromere 12 FISH probes. TP53 and IGHV were not mutated. Array-CGH confirmed trisomy of the entire chromosome 8 and allowed us to resolve in detail the nature of the unbalanced translocation, revealing multiple regions of genomic losses on chromosomes 6 and 11. DISCUSSION: The present case report is an unusual CLL case with complex karyotype and refinement of all breakpoints at the gene level by the genomic array. From a genetic point of view, the case under study presented several peculiarities. CONCLUSIONS: We report the genetic findings of a CLL patient with abrupt disease onset, so far responding properly to treatments despite the presence of distinct genetic adverse traits including ATM deletion, complex karyotype and chromosome 6q chromoanagenesis event. Our report confirms that interphase FISH alone is not able to provide an overview of the whole genomic landscape in selected CLL cases and that additional techniques are required to reach an appropriate cytogenetic stratification of patients.

Mass spectrometry-based assay for the molecular diagnosis of glioma: concomitant detection of chromosome 1p/19q codeletion, and IDH1, IDH2, and TERT mutation status.

The World Health Organization recently revised the diagnosis of glioma, to integrate molecular parameters, including IDH mutations and codeletion (loss of heterozygosity; LOH) of chromosome arms 1p/19q, into the definitions of adult glioma histological subtypes. Mutations in the TERT promoter may also be useful for glioma diagnosis and prognosis. The integration of molecular markers into routine diagnosis requires their rapid and reliable assessment. We propose a MassARRAY (MS)-based test that can identify 1p/19q codeletion using quantitative SNP genotyping and, simultaneously, characterize hotspot mutations in the IDH1, IDH2, and TERT genes in tumor DNA. We determined the reliability of the MS approach testing 50 gliomas and comparing the MS results with those obtained by standard methods, such as short tandem repeat genotyping, array comparative genomic hybridization (array-CGH) and Fluorescence In Situ Hybridization (FISH) for 1p/19q codeletion and Sanger sequencing for hotspots mutations. The results indicate that MS is suitable for the accurate, rapid, and cost-effective evaluation of chromosome deletions combined with hotspot mutation detection. This MS approach could be similarly exploited in evaluation of LOH in other situations of clinical and/or research importance.

Clinical evaluation and molecular screening of a large consecutive series of albino patients.

Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. In this study we recruited 321 albino patients and screened them for the genes known to cause oculocutaneous albinism (OCA1-4 and OCA6) and ocular albinism (OA1). Our purpose was to detect mutations and genetic frequencies of the main causative genes, offering to albino patients an exhaustive diagnostic assessment within a multidisciplinary approach including ophthalmological, dermatological, audiological and genetic evaluations. We report 70 novel mutations and the frequencies of the major causative OCA genes that are as follows: TYR (44%), OCA2 (17%), TYRP1 (1%), SLC45A2 (7%) and SLC24A5 (<0.5%). An additional 5% of patients had GPR143 mutations. In 19% of cases, a second reliable mutation was not detected, whereas 7% of our patients remain still molecularly undiagnosed. This comprehensive study of a consecutive series of OCA/OA1 patients allowed us to perform a clinical evaluation of the different OCA forms.

Screening of PAX6 gene in Italian congenital aniridia patients revealed four novel mutations.

PURPOSE: To uncover underlying mutations in a cohort of Italian patients with aniridia, a rare congenital panocular condition with an incidence ranging from 1:64,000 to 1:100,000. The disease may be found isolated or in association with other syndromes characterized by partial or complete absence of the iris and iris hypoplasia. METHODS: We analyzed the PAX6 gene in 11 patients with aniridia fulfilling the following inclusion criteria: partial or complete absence of the iris and age < 18 years at the time of diagnosis. DNA sequence analysis was integrated with Multiple Ligation Probe Assay (MLPA) analysis. RESULTS: We identified seven PAX6 mutations, including four novel ones. The majority of mutations lie in the DNA-binding domain and all produce a truncated protein. All tested patients did not have WT1 gene deletions thus excluding the WAGR syndrome. We present the clinical findings in the four cases harboring novel mutations. We were unable to identify mutations in four cases with complete aniridia thus indicating that other gene/s could be involved in the disease. CONCLUSIONS: It is important to establish the molecular diagnosis early to avoid repeated and long-term screening for Wilms tumor. Our work further emphasizes that a wide range of ocular phenotypes are associated with loss of function PAX6 mutations. In addition to the possibility of stochastic variations, other genetic variations could play a role as modifier genes, thus giving rise to the observed different ocular phenotypes.