Juvenile sudden death in a family with polymorphic ventricular arrhythmias caused by a novel RyR2 gene mutation: evidence of specific morphological substrates

We report on a family with a history of sudden death and effort-induced polymorphic ventricular arrhythmias. The index case was a 17-year-old boy who died suddenly and at postmortem had evidence of fibrofatty replacement in the right ventricular free wall, consistent with arrhythmogenic right ventricular cardiomyopathy, as well as calcium phosphate deposits within the myocytes. A molecular genetics investigation carried out in the paraffin-embedded myocardium of the subject and in blood samples of family members disclosed a missense mutation in exon 3 (230C-->T; A77V) of the cardiac ryanodine receptor type 2 gene. The carriers showed effort-induced polymorphic ventricular tachycardia in the setting of normal resting electrocardiogram and trivial echocardiographic abnormalities, consistent with catecholaminergic polymorphic ventricular tachycardia. The observation of both arrhythmogenic right ventricular cardiomyopathy type 2 and catecholaminergic polymorphic ventricular tachycardia in the same family suggests that the two entities might correspond to different degrees of phenotypic expression of the same disease. This experience underscores the importance of a precise autopsy diagnosis in the case of sudden cardiac death, including molecular genetics, and the mission of pathologists to guide further clinical investigation of family members.     

Cell proliferation and apoptosis during histogenesis of the guinea pig and rabbit cerebellar cortex

Cell proliferation and apoptosis are essential for development of the nervous system. In this study we have investigated the histogenesis of the cerebellar cortex in guinea pig (a precocial species) and rabbit (an altricial species) at different stages of pregnancy and postnatal life. Proliferating cells were identified after labeling with antibodies against the proliferating cell nuclear antigen (PCNA) and/or the Ki-67 antigen. Apoptotic cells were visualized in situ by the TUNEL method and by immunodetection of cleaved caspase 3 and 9. In guinea pigs, both proliferating and apoptotic cells were detected during pre-natal life (E0-E40). Conversely, cell proliferation and apoptosis in rabbits were temporally restricted to early postnatal weeks (P0-P20). In both species cell proliferation was mainly linked to differentiation and migration of the granule cells. In both species, the majority of cells undergoing programmed cell death likely corresponded to granule cells. They were mainly detected in the external granular layer, and were by far more common than previously reported in other locations of the postnatal brain. This study shows that apoptosis is a shared process of cell death during cerebellar development in both altricial and precocial animals, and that there is a direct spatial and temporal correlation between cell proliferation and death in two mammals with different time tables in cerebellar maturation.     

Patient follow-up is a major problem at genetics clinics

Children with genetic diseases must be followed for long periods of time to seek new findings. Other patients require further check-ups and studies to be diagnosed. Some patients never return for medical care after the first consultation, which may have serious consequences. We reviewed 400 medical charts of patients with genetic disease to analyze overall attendance to the genetics clinic, investigate some of the causes of failure to seek medical advice, and determine the differences between those first seen as outpatients or as inpatients. The mean follow-up period was 8.3 months (range 0-79), and the average number of visits was 2.8 (range 1-16). Forty eight percent of the cases first seen as inpatients were evaluated only once and 14% twice; while 22 and 21% of the 300 cases first seen as outpatients attended once and twice, respectively (P = 0.0). Appointment keeping was apparently not affected by the presence or absence of diagnosis. Overall, 97 patients were discharged, 7 died, 55 continued on follow-up, 62 attended other hospital services-but not genetics-and 179 were completely lost to follow-up. Diagnosed patients were counseled more frequently than undiagnosed patients (62 vs. 5%); and 71% of the diagnosed patients first seen as outpatients but only 36% of undiagnosed cases first seen as inpatients were counseled, differences between these two groups were significant (P = 0.005). We conclude that keeping the patient with genetic disease on follow-up is a difficult task. New educational strategies must be planned to improve this worrisome situation.     

Cytotoxic molecule expression and epithelial cell apoptosis in oral and cutaneous lichen planus

We evaluated the expression of T cell-restricted intracellular antigen (Tia-1), granzyme B, and perforin by lymphocytes and the degree of epithelial apoptosis in oral and cutaneous lichen planus (LP) in 51 untreated cases, including 27 oral LP (OLP) and 24 cutaneous LP (CLP) cases. The number of total dermal-positive lymphocytes in OLP and CLP was similar, indicating similar activity of the inflammatory process. Intraepithelial Tia-1-positive, perforin-positive, and granzyme B-positive lymphoid cells were more numerous in OLP than in CLP (P < .05). The epithelial cell apoptotic index (AI) was increased significantly in OLP (P < .05), particularly in erosive-atrophic variants. A linear correlation between AI and the mean +/- SEM number of intraepithelial and dermal perforin+ cells (6.85 +/- 2.44 and 27.48 +/- 10.19, respectively), per 10 high-power fields for OLP and for CLP (1.17 +/- 0.88 and 10.42 +/- 5.74, respectively), was found (intraepithelial, r = 0.50; dermal, r = 0.51; P < .01). These data suggest a pivotal role for perforin in triggering epithelial cell apoptosis. The differences of infiltrating cytotoxic cells and related AI observed in OLP and CLP are in keeping with the clinical behaviors that distinguish these LP variants.     

Employing a systematic approach to biobanking and analyzing clinical and genetic data for advancing COVID-19 research

Within the GEN-COVID Multicenter Study, biospecimens from more than 1000 SARS-CoV-2 positive individuals have thus far been collected in the GEN-COVID Biobank (GCB). Sample types include whole blood, plasma, serum, leukocytes, and DNA. The GCB links samples to detailed clinical data available in the GEN-COVID Patient Registry (GCPR). It includes hospitalized patients (74.25%), broken down into intubated, treated by CPAP-biPAP, treated with O₂ supplementation, and without respiratory support (9.5%, 18.4%, 31.55% and 14.8, respectively); and non-hospitalized subjects (25.75%), either pauci- or asymptomatic. More than 150 clinical patient-level data fields have been collected and binarized for further statistics according to the organs/systems primarily affected by COVID-19: heart, liver, pancreas, kidney, chemosensors, innate or adaptive immunity, and clotting system. Hierarchical clustering analysis identified five main clinical categories: (1) severe multisystemic failure with either thromboembolic or pancreatic variant; (2) cytokine storm type, either severe with liver involvement or moderate; (3) moderate heart type, either with or without liver damage; (4) moderate multisystemic involvement, either with or without liver damage; (5) mild, either with or without hyposmia. GCB and GCPR are further linked to the GCGDR, which includes data from whole-exome sequencing and high-density SNP genotyping. The data are available for sharing through the Network for Italian Genomes, found within the COVID-19 dedicated section. The study objective is to systematize this comprehensive data collection and begin identifying multi-organ involvement in COVID-19, defining genetic parameters for infection susceptibility within the population, and mapping genetically COVID-19 severity and clinical complexity among patients.Subject terms: Genetics research, Viral infection     

Antidepressant response and well-being in pre-, peri- and postmenopausal women with major depressive disorder treated with fluoxetine

BACKGROUND: We assessed the impact of menopausal status on treatment response and well-being in a cohort of outpatient women with major depressive disorder (DSM-III-R criteria), who received treatment with fluoxetine (20 mg/day for 8 weeks). METHODS: Menopausal status was defined based on age, presence of menstrual irregularity or amenorrhea and vasomotor symptoms. Remission and response of depression were defined as a 17-item Hamilton Depression Rating Scale (HAM-D-17) score or=50%, respectively. Well-being was assessed by self-rating with the Symptom Questionnaire. Remitters were followed up for 28 additional weeks. RESULTS: No differences in rates of response and remission as well as in levels of well-being were observed among pre- (n = 121), peri- (n = 28) and postmenopausal (n = 35) women at the endpoint of the acute phase, even after adjustment for baseline depression severity. Residual symptoms, however, were significantly more common in postmenopausal women, except for the continuation phase endpoint. Differences in residual symptoms during the acute phase subsided after adjustment for baseline depression severity. CONCLUSIONS: Overall, menopausal status did not significantly affect the response to fluoxetine treatment and the degree of posttreatment well-being among major depressive disorder patients.     

NK-active cytokines IL-2, IL-12, and IL-15 selectively modulate specific protein kinase C (PKC) isoforms in primary human NK cells.

Natural killer (NK) cell function is largely modulated by growth factors and cytokines. In particular, interleukin (IL)-2, IL-12, and IL-15 have major effects on the proliferative and cytotoxic activities of NK cells against tumor and virus-infected cells. It is thought that the members of the protein kinase C (PKC) family of serine/threonine kinases play an important role in mediating the pleiotropic effects of cytokines on their target cells. We have investigated the downstream effects generated in purified human NK cells by IL-2, IL-12, and IL-15 on PKCalpha and PKCepsilon--a canonical and a novel isoform of PKC, respectively. By means of Western blotting, PKC activity assays, and immunofluorescence performed on highly purified preparations of primary human NK cells, we demonstrate that: 1) the three cytokines have similar effects on PKCalpha and PKCepsilon activities; 2) whereas PKCepsilon activity is induced by cytokine stimulation, PKCalpha activity is inhibited; and 3) both the induction of PKCepsilon and the inhibition of PKCalpha functional activity are relatively early events in NK cells, while longer cytokine stimulations do not generate significant variations in enzyme activity, suggesting that the activation of both the canonical and novel isoforms of PKC are events required in the early phases of cytokine-induced NK cell stimulation.     

Primary cutaneous large B-cell lymphoma of the leg: histogenetic analysis of a controversial clinicopathologic entity

This study analyzes the pathologic and molecular features of 5 cases of primary cutaneous large B-cell lymphoma of the leg (PCLBCL-leg), recently included in the European Organization for Research and Treatment of Cancer (EORTC) classification of primary cutaneous lymphoma. PCLBCL-leg accounts for 5% to 10% of all primary cutaneous B-cell lymphoma (PCBCL), usually affects elderly patients and carries a worse prognosis than other forms of PCBCL. It has been proposed that the malignant cells of PCLBCL-leg originate from germinal center (GC)-related cells, but their effective normal counterpart is unclear, and the rationale behind the inclusion of this lymphoma as a separate entity is based on its prognosis rather than on its proved histogenesis. All of our cases of PCLBCL-leg morphologically resembled diffuse large B-cell lymphoma (DLBCL), but to better define their histogenesis, we also analyzed various phenotypic and genotypic markers, including mutations of the Ig and of BCL-6 genes, as well as expression of the bcl-6, MUM1, and CD138/syndecan-1 proteins. Immunohistochemically, all of our cases stained for the L-26/CD20cy and CD79a antigens and expressed the bcl-2, bcl-6, and MUM-1 proteins but were negative for both the CD10/CALLA and CD138 antigens. With respect to molecular analysis, the lymphoma population of all PCLBCL-leg carried hypermutation of Ig genes, and all but 1 case also harbored mutations of the BCL-6 gene. Our results indicate that PCLBCL-leg are similar both under the morphofunctional and molecular profiles to most DLBCL of other sites. Thus, caution seems justified before definitely considering PCLBCL of the leg as a distinct entity.     

A patient with novel MBOAT7 variant: The cerebellar atrophy is progressive and displays a peculiar neurometabolic profile

Mutations in the MBOAT7 gene have been described in 43 patients, belonging to 18 families, showing nonspecific clinical features (intellectual disability [ID], seizures, microcephaly or macrocephaly, and mild to moderate cerebellar atrophy) that make the clinical diagnosis difficult. Here we report the first Italian patient, a 22.5‐year‐old female, one of the oldest reported, born to apparently consanguineous parents. She shows severe ID, macrocephaly, seizures, aggressive outbursts, hyperphagia. We also documented progressive atrophy of the cerebellar vermis, that appeared not before the age of 7. The whole‐exome sequencing of the trio identified a novel homozygous variant c.1057_1058delGCinsCA (p.Ala353His) in the MBOAT7 gene. The variant is considered to be likely pathogenic, since it is absent from population database and it lies in a highly conserved amino acid residue. This disorder has a neurometabolic pathogenesis, implicating a phospholipid remodeling abnormalities. A brain hydrogen‐magnetic resonance spectroscopy (H‐MRS) examination in our patient disclosed a peculiar neurometabolic profile in the cerebellar hemispheric region. This new finding could address the clinical suspicion of MBOAT7‐related disorder, among the wide range of genetic conditions associated with ID and cerebellar atrophy. Moreover, the documented progression of cerebellar atrophy and the worsening of the disease only after some years open to the possibility of a therapeutic window after birth.     

Highly immunogenic and protective recombinant vaccine candidate expressed in transgenic plants

Vaccine development has been hampered by difficulties in developing new and safe adjuvants, so alternative technologies that offer new avenues forward are urgently needed. The goal of this study was to express a monoclonal recombinant immune complex in a transgenic plant. A recombinant protein consisting of a tetanus toxin C fragment-specific monoclonal antibody fused with the tetanus toxin C fragment was designed and expressed. Immune complex formation occurred between individual fusion proteins to form immune complex-like aggregates that bound C1q and FcgammaRIIa receptor and could be targeted to antigen-presenting cells. Unlike antigen alone, the recombinant immune fusion complexes were highly immunogenic in mice and did not require coadministration of an adjuvant (when injected subcutaneously). Indeed, these complexes elicited antibody titers that were more than 10,000 times higher than those observed in animals immunized with the antigen alone. Furthermore, animals immunized with only 1 mug of recombinant immune complex without adjuvant were fully protected against lethal challenge. This the first report on the use of a genetic fusion between antigen and antibody to ensure an optimal expression ratio between the two moieties and to obtain fully functional recombinant immune complexes as a new vaccine model.