👤 Simona Petrucci

Clonal mature B-cell lymphoproliferative disorders (B-LPDs) are a heterogeneous group of neoplasia characterized by the proliferation of mature B lymphocytes in the peripheral blood, bone marrow and/or lymphoid tissues. B-LPDs classification into different subtypes and their diagnosis is based on a multiparametric approach. However, accurate diagnosis may be challenging, especially in cases of ambiguous interpretation. Multiparameter flow cytometry (MFC) represents an extensively used technique to detect the presence of different cellular lines in immunology and hematology. MFC results provide an essential contribution to the B-LPDs diagnostic process, even more so considering that panels are constantly integrating novel markers to improve diagnostic accuracy. The aim was to evaluate the contributing role of MFC routinary studies by analyzing the expression and the mean fluorescence intensity (MFI) of CD200, ROR1, and CD43 in various B-LPDs to evaluate their usefulness in the differential diagnosis of these diseases. We retrospectively evaluated 2615 consecutive cases of newly collected samples (mostly from patients with lymphocytosis) analyzed by MFC carried out in the B-LPD diagnostic process referred to the Division of Hematology of the Sapienza University of Rome. We compared the results of CD200, ROR1, and CD43 expression percentage and their MFI between different subtypes of B-LPDs. In chronic lymphocytic leukemia (CLL), CD200, ROR1, and CD43 were always expressed with bright intensity. CLL samples presented high CD200 expression and MFI [CD200%, mean: 100 (range, 24-100); positivity rate: 100%; MFI, median = 125 (range, 10-1200)] statistically higher than mantle cell lymphoma (MCL) (p<0.001), which is usually negative for CD200, and variant hairy cell leukemia (vHCL, according to 2022 ICC) (p<0.001), but comparable with classic HCL (cHCL) (p>0.9). ROR1 resulted expressed in all CLL [ROR1%, mean: 100 (range, 52-100), positivity rate: 100%; MFI, median=50 (range, 10-202)] and MCL cases with comparable MFI (p>0.9). CD43 expression and MFI were significantly higher in CLL [CD43%, mean 99 (range, 59-100); positivity rate: 100%; MFI, median = 130 (range, 41-980)] than in MCL, vHCL, cHCL, and all the others mature B-cell neoplasia (p<0.001). CD200 and CD43 expression and MFI were significantly higher in cHCL compared to vHCL. Among the other mature B-cell neoplasia, CD200 was variably expressed in follicular lymphoma (FL), marginal zone lymphoma (MZL), diffuse large B-cell lymphoma (DLBCL), and lymphoplasmacytic lymphoma (LPL). ROR1 and CD43 presented a very low expression percentage in this latter group, being mostly negative. Persistent polyclonal B-cell lymphocytosis (PPBL) resulted in uniformly positive for CD200 and negative for ROR1 and CD43. Our data suggest that evaluating CD200, ROR1, and CD43 antigens and their intensity of expression, along with commonly used markers in MFC routine panels for B-LPDs, might be extremely useful for prompt diagnostic evaluation in the differential diagnosis of these diseases. Show less

Genetic testing is valuable to confirm molecular diagnosis in nearly 60% of cases suspected of hypertrophic cardiomyopathy (HCM). However, the interpretation of variants, especially those of uncertain significance (VUSs), remains challenging for laboratories and clinicians. In April 2024, the ClinGen Cardiomyopathy Variant Curation Expert Panel (VCEP) adapted the ACMG/AMP criteria for eight of the sarcomeric genes ( Here, two groups of curators reinterpreted variants with the most recent data using the Cardiomyopathy VCEP specifications until a consensus was reached. To streamline the process, we created a semiautomated decision support tool based on these gene-specific rules. The application of the Cardiomyopathy VCEP specifications resulted in the reclassification of 17.4% ( Using gene-specific ACMG/AMP criteria reduces the rate of VUS, increasing diagnostic yield, and informing clinical management in the context of HCM. Nonetheless, ongoing efforts to generate evidence and promote standardization remain essential to improve variant interpretation. Show less

MYBPC3 pathogenic variants are the most common cause of hypertrophic cardiomyopathy (HCM) and are associated with significant phenotypic heterogeneity. Despite their pathogenic potential, MYBPC3 founder variants persist within specific populations. This study investigates the MYBPC3 c.2309-2 A > G splice variant hypothesizing its founder origin in central Italy. The aim was to confirm the presence of a common haplotype, assess its molecular and clinical impact, and compare the phenotype with that of other MYBPC3 founder variants. Among the 5251 HCM patients recruited at eight Italian referral centers, 1108 probands (21.1%) were identified as carriers of pathogenic or likely pathogenic MYBPC3 variants, and among these, 11.6% carried the c.2309-2 A > G variant. Haplotype reconstruction using short tandem repeats and tag-SNPs revealed a unique 5.2 Mb haplotype segregating with the c.2309-2 A > G variant in all carriers. Age estimation suggested that the variant originated approximately 481 years ago, likely in the Lazio region with clustering in Rome. Clinically, carriers exhibited variable expressivity with age-and sex-dependent penetrance. Males showed earlier onset, higher penetrance and greater disease severity compared to females. RNA analysis showed the retention of both introns 23 and 24, and significantly reduced MYBPC3 expression consistent with haploinsufficiency. Comparative analysis with other MYBPC3 founder variants highlighted differences in phenotypic expression, particularly in left ventricular wall thickness and clinical outcomes. This study establishes c.2309-2 A > G as an Italian MYBPC3 founder mutation, enhancing the understanding of HCM genetics and regional founder effects. These findings emphasize the importance of targeted genetic screening and personalized management for MYBPC3 c.2309-2 A > G carriers. Show less

Hypertrophic cardiomyopathy (HCM) is a genetic cardiac muscle disease characterized by clinical and genetic heterogeneity. Genetic testing can reveal the presence of disease-causing variants in genes encoding sarcomere proteins. However, it yields inconclusive or negative results in 40-60% of HCM cases, owing to, among other causes, technical limitations such as the inability to detect pathogenic intronic variants. Therefore, we aimed to increase the diagnostic yield of molecular analysis for HCM by improving the Show less

The role of genetic testing over the clinical and functional variables, including data from the cardiopulmonary exercise test (CPET), in the hypertrophic cardiomyopathy (HCM) risk stratification remains unclear. A retrospective genotype-phenotype correlation was performed to analyze possible differences between patients with and without likely pathogenic/pathogenic (LP/P) variants. A total of 371 HCM patients were screened at least for the main sarcomeric genes Show less

Hereditary multiple osteochondromas (HMO) is a rare autosomal dominant skeletal disorder, caused by heterozygous variants in either EXT1 or EXT2, which encode proteins involved in the biogenesis of heparan sulphate. Pathogenesis and genotype-phenotype correlations remain poorly understood. We studied 114 HMO families (158 affected individuals) with causative EXT1 or EXT2 variants identified by Sanger sequencing, or multiplex ligation-dependent probe amplification and qPCR. Eighty-seven disease-causative variants (55 novel and 32 known) were identified including frameshift (42%), nonsense (32%), missense (11%), splicing (10%) variants and genomic rearrangements (5%). Informative clinical features were available for 42 EXT1 and 27 EXT2 subjects. Osteochondromas were more frequent in EXT1 as compared to EXT2 patients. Anatomical distribution of lesions showed significant differences based on causative gene. Microscopy analysis for selected EXT1 and EXT2 variants verified that EXT1 and EXT2 mutants failed to co-localize each other and loss Golgi localization by surrounding the nucleus and/or assuming a diffuse intracellular distribution. In a cell viability study, cells expressing EXT1 and EXT2 mutants proliferated more slowly than cells expressing wild-type proteins. This confirms the physiological relevance of EXT1 and EXT2 Golgi co-localization and the key role of these proteins in the cell cycle. Taken together, our data expand genotype-phenotype correlations, offer further insights in the pathogenesis of HMO and open the path to future therapies. Show less

Blepharospasm (BSP) is a type of focal dystonia characterized by involuntary orbicularis oculi spasms that are usually bilateral, synchronous, and symmetrical. Despite strong evidence for genetic contributions to BSP, progress in the field has been constrained by small cohorts, incomplete penetrance, and late age of onset. Although several genetic etiologies for dystonia have been identified through whole-exome sequencing (WES), none of these are characteristically associated with BSP as a singular or predominant manifestation. We performed WES on 31 subjects from 21 independent pedigrees with BSP. The strongest candidate sequence variants derived from in silico analyses were confirmed with bidirectional Sanger sequencing and subjected to cosegregation analysis. Cosegregating deleterious variants (GRCH37/hg19) in CACNA1A (NM₀₀₁₁₂₇₂₂₂.1: c.7261₇₂₆₂delinsGT, p.Pro2421Val), REEP4 (NM₀₂₅₂₃₂.3: c.109C>T, p.Arg37Trp), TOR2A (NM₁₃₀₄₅₉.3: c.568C>T, p.Arg190Cys), and ATP2A3 (NM₀₀₅₁₇₃.3: c.1966C>T, p.Arg656Cys) were identified in four independent multigenerational pedigrees. Deleterious variants in HS1BP3 (NM₀₂₂₄₆₀.3: c.94C>A, p.Gly32Cys) and GNA14 (NM₀₀₄₂₉₇.3: c.989₉₉₀del, p.Thr330ArgfsTer67) were identified in a father and son with segmental cranio-cervical dystonia first manifest as BSP. Deleterious variants in DNAH17, TRPV4, CAPN11, VPS13C, UNC13B, SPTBN4, MYOD1, and MRPL15 were found in two or more independent pedigrees. To our knowledge, none of these genes have previously been associated with isolated BSP, although other CACNA1A mutations have been associated with both positive and negative motor disorders including ataxia, episodic ataxia, hemiplegic migraine, and dystonia. Our WES datasets provide a platform for future studies of BSP genetics which will demand careful consideration of incomplete penetrance, pleiotropy, population stratification, and oligogenic inheritance patterns. Show less

Serum proteome investigations have raised an incredible interest in the research of novel molecular biomarker, nevertheless few of the proposed evidences have been translated to the clinical practice. One of the limiting factors has been the lack of generally accepted guidelines for clinical proteomics studies and the lack of a robust analytical and pre-analytical ground for the proposed classification models. Pre-analytical issues may results in a deep impact for biomarker discovery campaign. In this study we present a systematic evaluation of sample storage and sampling conditions for clinical proteomics investigations. We have developed and validated a linear MALDI-TOF-MS protein profiling method to explore the low protein molecular weight region (5-20 kDa) of serum samples. Data normalization and processing was performed using optimise peak detection routine (LIMPIC) able to describe each group under investigation. Data were acquired either from healthy volunteers and from multiple sclerosis patients in order to highlight ex vivo protein profile alteration related to different physio-pathological conditions. Our data showed critical conditions for serum protein profiles depending on storage times and temperatures: 23 degrees C, 4 degrees C, -20 degrees C and -80 degrees C. We demonstrated that upon a -20 degrees C short term storage, characteristic degradation profiles are associated with different clinical groups. Protein signals were further identified after preparative HPLC separation by peptide sequencing on a nanoLC-Q-TOF TANDEM mass spectrometer. Apolipoprotein A-IV and complement C3 protein fragments, transthyretin and the oxidized isoforms in different apolipoprotein species represent the major molecular features of such a degradation pattern. Show less