20 novel point mutations and one large deletion in EXT1 and EXT2 genes: Report of diagnostic screening in a large Italian cohort of patients affected by hereditary multiple exostosis

Background

Hereditary multiple exostosis represents the most frequent bone tumor disease in humans. It consists of cartilage deformities affecting the juxta-ephyseal region of long bones. Usually benign, exostosis could degenerate in malignant chondrosarcoma form in less than 5% of the cases. Being caused by mutations in the predicted tumor suppressor genes, EXT1 (chr 8q23-q24) and EXT2 (chr 11p11-p12) genes, HMEs are usually inherited with an autosomal dominant pattern, although “de novo” cases are not infrequent.

Aim

Here we present our genetic diagnostic report on the largest Southern Italy cohort of HME patients consisting of 90 subjects recruited over the last 5 years.

Results

Molecular screening performed by direct sequencing of both EXT1 and EXT2 genes, by MLPA and Array CGH analyses led to the identification of 66 mutations (56 different occurrences) and one large EXT2 deletion out of 90 patients (74.4%). The total of 21 mutations (20 different occurrences, 33.3%) and the EXT2 gene deletion were novel. In agreement with literature data, EXT1 gene mutations were scattered along all the protein sequence, while EXT2 lesions fell in the first part of the protein. Conservation, damaging prediction and 3-D modeling, in-silico, analyses, performed on three novel missense variants, confirmed that at least in two cases the novel aminoacidic changes could alter the structure stability causing a strong protein misfolding.

Conclusions

Here we present 20 novel EXT1/EXT2 mutations and one large EXT2 deletion identified in the largest Southern Italy cohort of patients affected by hereditary multiple exostosis.     

McKusick-Kaufman or Bardet-Biedl syndrome? A new borderline case in an Italian nonconsanguineous healthy family

McKusick-Kaufman syndrome (MKS, OMIM #236700) is a rare syndrome inherited in an autosomal recessive pattern with a phenotypic triad comprising hydrometrocolpos (HMC), postaxial polydactyly (PAP), and congenital cardiac disease (CHD). The syndrome is caused by mutations in the MKKS gene mapped onto chromosome 20p12 between D20S162 and D20S894 markers. Mutations in the same gene causes Bardet-Biedl-6 syndrome (BBS-6, OMIM #209900) inherited in an autosomal recessive pattern. BBS-6 comprises retinitis pigmentosa, polydactyly, obesity, mental retardation, renal and genital anomalies. HMC, CHD, and PAP defects can also occur in BBS-6, and there is a significant clinical overlap between MKS and BBS-6 in childhood. We describe a new borderline case of MKS and BBS syndrome and suggest insights for understanding correlation between MKKS gene mutations and clinical phenotype. Here, we report the results of molecular analysis of MKKS in a female proband born in an Italian nonconsanguineous healthy family that presents HMC and PAP. The mutational screening revealed the presence of two different heterozygous missense variants (p.242A>S in exon 3, p.339 I>V in exon 4) in the MKKS gene, and a nucleotide variation in 5'UTR region in exon 2 (-417 A>C).     

Distinct CD4+ CD8+ double-positive T cells in the blood and liver of patients during chronic hepatitis B and C

CD4(+) and CD8(+) T cells, the main effectors of adaptive cellular immune responses, differentiate from immature, non-functional CD4(+)CD8(+) double-positive T (DPT) cells in the thymus. Increased proportions of circulating DPT lymphocytes have been observed during acute viral infections; in chronic viral diseases, the role and repartition of extra-thymic DPT cells remain largely uncharacterized. We performed a phenotypic analysis of DPT cells in blood and liver from patients chronically infected by hepatitis C (HCV) or B (HBV) viruses. The highest percentages of DPT cells, predominantly CD4(high)CD8(low), were observed in patients infected by HCV, while HBV-infected patients mostly displayed CD4(low)CD8(high) and CD4(high)CD8(high) DPT cells. All proportions of DPT cells were higher in liver than in blood with, for each subpopulation referred to above, a correlation between their frequencies in these two compartments. In HCV patients, intra-hepatic DPT cells displayed more heterogeneous activation, differentiation and memory phenotypes than in the blood; most of them expressed CD1a, a marker of T cell development in the thymus. Ex vivo, the inoculation of liver slices with HCV produced in cell culture was accompanied by a disappearance of CD8(high) cells, suggesting a direct effect of the virus on the phenotype of DPT cells in the liver. Our results suggest that, in half of the patients, chronic HCV infection promotes the production of DPT cells, perhaps by their re-induction in the thymus and selection in the liver.     

The expression of the cerato-platanin gene is related to hyphal growth and chlamydospores formation in Ceratocystis platani

Cerato-platanin (CP) is a protein produced by Ceratocystis platani, the causal agent of canker stain disease of plane trees. CP is the first member of the 'cerato-platanin family', and its role as a pathogen-associated molecular pattern (PAMP), inducing defence responses both in host and nonhost plants, is established. However, the primary role of CP and its homologues in the fungal life remains unknown. In the present work, we investigated the regulation of the cp gene during the in vitro growth of C. platani in different conditions and under the effect of potential stress factors. Fungal growth and conidiogenesis were also analysed. Results showed that cp is a single-copy gene whose expression level is strictly associated with hyphal growth and with chlamydospores formation. The analysis of a 1368 bp 5'-flanking region revealed putative motifs that could be involved in the regulation of gene expression in response to stress and developmental cues. Taking into account the localization of CP in the fungal cell wall and the recently published 3D structure of the protein, our results support a role for CP in growth and developmental processes of C. platani.     

A comparison between constitutive and inducible transgenic expression of the PhRIP I gene for broad-spectrum resistance against phytopathogens in potato

Objectives

To engineer broad spectrum resistance in potato using different expression strategies.

Results

The previously identified Ribosome-Inactivating Protein from Phytolacca heterotepala was expressed in potato under a constitutive or a wound-inducible promoter. Leaves and tubers of the plants constitutively expressing the transgene were resistant to Botrytis cinerea and Rhizoctonia solani, respectively. The wound-inducible promoter was useful in driving the expression upon wounding and fungal damage, and conferred resistance to B. cinerea. The observed differences between the expression strategies are discussed considering the benefits and features offered by the two systems.

Conclusions

Evidence is provided of the possible impact of promoter sequences to engineer BSR in plants, highlighting that the selection of a suitable expression strategy has to balance specific needs and target species.

     

Inappropriate use of clinical practices in Canada: a systematic review

Background:Inappropriate health care leads to negative patient experiences, poor health outcomes and inefficient use of resources. We aimed to conduct a systematic review of inappropriately used clinical practices in Canada.Methods:We searched multiple bibliometric databases and grey literature to identify inappropriately used clinical practices in Canada between 2007 and 2021. Two team members independently screened citations, extracted data and assessed methodological quality. Findings were synthesized in 2 categories: diagnostics and therapeutics. We reported ranges of proportions of inappropriate use for all practices. Medians and interquartile ranges (IQRs), based on the percentage of patients not receiving recommended practices (underuse) or receiving practices not recommended (overuse), were calculated. All statistics are at the study summary level.Results:We included 174 studies, representing 228 clinical practices and 28 900 762 patients. The median proportion of inappropriate care, as assessed in the studies, was 30.0% (IQR 12.0%–56.6%). Underuse (median 43.9%, IQR 23.8%–66.3%) was more frequent than overuse (median 13.6%, IQR 3.2%–30.7%). The most frequently investigated diagnostics were glycated hemoglobin (underused, range 18.0%–85.7%, n = 9) and thyroid-stimulating hormone (overused, range 3.0%–35.1%, n = 5). The most frequently investigated therapeutics were statin medications (underused, range 18.5%–71.0%, n = 6) and potentially inappropriate medications (overused, range 13.5%–97.3%, n = 9).Interpretation:We have provided a summary of inappropriately used clinical practices in Canadian health care systems. Our findings can be used to support health care professionals and quality agencies to improve patient care and safety in Canada.

As health care systems struggle with sustainability, there is increased recognition that a substantial percentage of the health care received is inappropriate.¹ Inappropriate health care occurs when effective clinical practices are underused, ineffective clinical practices are overused or other practices are misused. It can lead to negative patient experiences,² poor health outcomes^3,4 and inefficient use of scarce health care resources.⁵ In response, there is widespread professional and policy interest in reducing inappropriate health care in Canada and abroad. For example, in 2014, Choosing Wisely Canada,⁶ a physician-led campaign in partnership with the Canadian Medical Association, was established. This initiative encourages conversations between clinicians and patients about low-value or overused care in efforts to reduce inappropriate care. Choosing Wisely Canada is endorsed across Canada by all provincial and territorial medical associations (https://choosingwiselycanada.org/about/).Although reducing inappropriate health care is a high priority for health care professionals, agencies and governments in Canada, designing effective initiatives for quality improvement has been a difficult goal to achieve without knowledge of which clinical practices are inappropriately used. This is further challenged because Canada does not have a mandatory and comprehensive national tracking system for quality. The Canadian Institute for Health Information (CIHI) houses multiple Canadian health databases, but it does not collect information on all clinical practices. Therefore, a systematic synthesis is necessary to provide an overview of inappropriate health care in Canada.⁷ Summaries of inappropriately used clinical practices exist for several countries: United States,^8,9 United Kingdom¹⁰ and Australia.¹¹ Each of these syntheses found high levels (50% on average) of inappropriately used practices and laid the foundation for several quality improvement initiatives in these countries. We aimed to conduct a systematic review to estimate the nature and amount of inappropriately used clinical practices in Canada.     

Trichoderma spp. alleviate phytotoxicity in lettuce plants (Lactuca sativa L.) irrigated with arsenic-contaminated water

The influence of two strains of Trichoderma (T. harzianum strain T22 and T. atroviride strain P1) on the growth of lettuce plants (Lactuca sativa L.) irrigated with As-contaminated water, and their effect on the uptake and accumulation of the contaminant in the plant roots and leaves, were studied. Accumulation of this non-essential element occurred mainly into the root system and reduced both biomass development and net photosynthesis rate (while altering the plant P status). Plant growth-promoting fungi (PGPF) of both Trichoderma species alleviated, at least in part, the phytotoxicity of As, essentially by decreasing its accumulation in the tissues and enhancing plant growth, P status and net photosynthesis rate. Our results indicate that inoculation of lettuce with selected Trichoderma strains may be helpful, beside the classical biocontrol application, in alleviating abiotic stresses such as that caused by irrigation with As-contaminated water, and in reducing the concentration of this metalloid in the edible part of the plant.