Three new Alpha1-Antitrypsin deficiency variants help to define a C-Terminal region regulating conformational change and polymerization

Alpha1-antitrypsin (AAT) deficiency is a hereditary disorder associated with reduced AAT plasma levels, predisposing adults to pulmonary emphysema. The most common genetic AAT variants found in patients are the mildly deficient S and the severely deficient Z alleles, but several other pathogenic rare alleles have been reported. While the plasma AAT deficiency is a common trait of the disease, only a few AAT variants, including the prototypic Z AAT and some rare variants, form cytotoxic polymers in the endoplasmic reticulum of hepatocytes and predispose to liver disease. Here we report the identification of three new rare AAT variants associated to reduced plasma levels and characterize their molecular behaviour in cellular models. The variants, called Mpisa (Lys259Ile), Etaurisano (Lys368Glu) and Yorzinuovi (Pro391His), showed reduced secretion compared to control M AAT, and accumulated to different extents in the cells as ordered polymeric structures resembling those formed by the Z variant. Structural analysis of the mutations showed that they may facilitate polymerization both by loosening ‘latch’ interactions constraining the AAT reactive loop and through effects on core packing. In conclusion, the new AAT deficiency variants, besides increasing the risk of lung disease, may predispose to liver disease, particularly if associated with the common Z variant. The new mutations cluster structurally, thus defining a region of the AAT molecule critical for regulating its conformational state

<b><i>Topoisomerase 1</i></b> Promoter Variants and Benefit from Irinotecan in Metastatic Colorectal Cancer Patients

Objective: Topoisomerase 1 (topo-1) is an important target for the treatment of metastatic colorectal cancer (CRC). The aim of the present study was to evaluate the correlation between topo-1 single-nucleotide polymorphisms (SNPs) and clinical outcome in metastatic CRC (mCRC) patients. Methods: With the use of specific software (PROMO 3.0), we performed an in silico analysis of topo-1 promoter SNPs; the rs6072249 and rs34282819 SNPs were included in the study. DNA was extracted from 105 mCRC patients treated with FOLFIRI ± bevacizumab in the first line. SNP genotyping was performed by real-time PCR. Genotypes were correlated with clinical parameters (objective response rate, progression-free survival, and overall survival). Results: No single genotype was significantly associated with clinical variables. The G allelic variant of rs6072249 topo-1 SNP is responsible for GC factor and X-box-binding protein transcription factor binding. The same allelic variant showed a nonsignificant trend toward a shorter progression-free survival (GG, 7.5 months; other genotypes, 9.3 months; HR 1.823, 95% CI 0.8904-3.734; p = 0.1). Conclusion: Further analyses are needed to confirm that the topo-1 SNP rs6072249 and transcription factor interaction could be a part of tools to predict clinical outcome in mCRC patients treated with irinotecan-based regimens

The Importance of N186 in the Alpha-1-Antitrypsin Shutter Region Is Revealed by the Novel Bologna Deficiency Variant

Alpha-1-antitrypsin (AAT) deficiency causes pulmonary disease due to decreased levels of circulating AAT and consequently unbalanced protease activity in the lungs. Deposition of specific AAT variants, such as the common Z AAT, within hepatocytes may also result in liver disease. These deposits are comprised of ordered polymers of AAT formed by an inter-molecular domain swap. The discovery and characterization of rare variants of AAT and other serpins have historically played a crucial role in the dissection of the structural mechanisms leading to AAT polymer formation. Here, we report a severely deficient shutter region variant, Bologna AAT (N186Y), which was identified in five unrelated subjects with different geographical origins. We characterized the new variant by expression in cellular models in comparison with known polymerogenic AAT variants. Bologna AAT showed secretion deficiency and intracellular accumulation as detergent-insoluble polymers. Extracellular polymers were detected in both the culture media of cells expressing Bologna AAT and in the plasma of a patient homozygous for this variant. Structural modelling revealed that the mutation disrupts the hydrogen bonding network in the AAT shutter region. These data support a crucial coordinating role for asparagine 186 and the importance of this network in promoting formation of the native structure

Primary tumor sidedness and benefit from FOLFOXIRI plus bevacizumab as initial therapy for metastatic colorectal cancer. Retrospective analysis of the TRIBE trial by GONO

Right-sided metastatic colorectal cancer (mCRC) patients have poor prognosis and achieve limited benefit from first-line doublets plus a targeted agent. In this unplanned analysis of the TRIBE study, we investigated the prognostic and predictive impact of primary tumor sidedness in mCRC patients and the differential impact of the intensification of the chemotherapy in subgroups defined according to both primary tumor sidedness and RAS and BRAF mutational status

The molecular species responsible for α₁‐antitrypsin deficiency are suppressed by a small molecule chaperone

The formation of ordered Z (Glu342Lys) α1‐antitrypsin polymers in hepatocytes is central to liver disease in α1‐antitrypsin deficiency. In vitro experiments have identified an intermediate conformational state (M*) that precedes polymer formation but this has yet to be identified in vivo. Moreover, the mechanism of polymer formation and their fate in cells have been incompletely characterised. We have used cell models of disease in conjunction with conformation‐selective monoclonal antibodies and a small molecule inhibitor of polymerization to define the dynamics of polymer formation, accumulation and secretion. Pulse‐chase experiments demonstrate that Z α1‐antitrypsin accumulates as short chain polymers that partition with soluble cellular components and are partially secreted by cells. These precede the formation of larger, insoluble polymers with a longer half‐life (10.9 +/‐ 1.7 h and 20.9 +/ 7.4 h for soluble and insoluble polymers respectively). The M* intermediate (or a byproduct thereof) was identified in the cells by a conformation‐specific monoclonal antibody. This was completely abrogated by treatment with the small molecule which also blocked the formation of intracellular polymers. These data allow us to conclude that: the M* conformation is central to polymerization of Z α1‐antitrypsin in vivo; preventing its accumulation represents a tractable approach for pharmacological treatment of this condition; polymers are partially secreted; and polymers exist as two distinct populations in cells whose different dynamics have likely consequences for the aetiology of the disease

The molecular species responsible for α1-antitrypsin deficiency are suppressed by a small molecule chaperone

The formation of ordered Z (Glu342Lys) α1-antitrypsin polymers in hepatocytes is central to liver disease in α1-antitrypsin deficiency. In&nbsp;vitro experiments have identified an intermediate conformational state (M*) that precedes polymer formation, but this has yet to be identified in&nbsp;vivo. Moreover, the mechanism of polymer formation and their fate in cells have been incompletely characterised. We have used cell models of disease in conjunction with conformation-selective monoclonal antibodies and a small molecule inhibitor of polymerisation to define the dynamics of polymer formation, accumulation and secretion. Pulse-chase experiments demonstrate that Z α1-antitrypsin accumulates as short-chain polymers that partition with soluble cellular components and are partially secreted by cells. These precede the formation of larger, insoluble polymers with a longer half-life (10.9&nbsp;±&nbsp;1.7&nbsp;h and 20.9&nbsp;±&nbsp;7.4&nbsp;h for soluble and insoluble polymers, respectively). The M* intermediate (or a by-product thereof) was identified in the cells by a conformation-specific monoclonal antibody. This was completely abrogated by treatment with the small molecule, which also blocked the formation of intracellular polymers. These data allow us to conclude that the M* conformation is central to polymerisation of Z α1-antitrypsin in&nbsp;vivo; preventing its accumulation represents a tractable approach for pharmacological treatment of this condition; polymers are partially secreted; and polymers exist as two distinct populations in cells whose different dynamics have likely consequences for the aetiology of the disease

Mini-invasive approach to preneoplastic and neoplastic endometrial lesions. Comparative study among histological, cytological and immunohistochemical diagnosis

Objective: To compare the accuracy of cytology plus immunoistochemistry vs histology in the preoperative diagnosis of endometrial malignancy. Methods: We prospectively analyzed 142 women with a proliferative endometrial lesion undergoing operative hysteroscopy (ISC): at the time of ISC, the fluid used for saline contrast sonohysterography (SCSH) was collected for cytological analysis and compared to histology. In 9 women a markers board (Notch-1+ER-\u3b1+PR-\u3b2) expression was analyzed semiquantitatively in term of presence and intensity, on both glandular and stromal samples. Results: Table 1 shows the comparison between cytological and histological diagnosis. ISC histological results Benign Lesions n=134 Malignant Lesions n=8 Endometrial Polyps n=124 Hypertrophy n=3 Typical hyperplasia n=7 Atypical hyperplasia n=4 Cancer n=4 CTM - 0 0 0 0 0 CTM + 0 0 0 0 3 SCSH cytological results Atypia - 116 3 6 1 0 Atypia + 2 0 0 3 3 Inadequate (5%) 5 (4 cervical cells) (1 scant sample) 0 1 (1 cervical cells) 0 1 (hypocellulated) Cytological sampling was inadequate in 7 cases (5%). The K value between cytology and histology was 98.4% for benign and 85.7% for malignant lesions. Notch-1 revealed a changing expression pattern: absent in benign lesions, focal and marked in atypical hyperplasia and widespread and marked in cancers. Moreover Notch-1 expression was mild and focal in originally cyto-hystologycal benign lesions which turned into atypical hyperplasia during follow up. In cancer cases, ER-\u3b1 and PR-\u3b2 were widespread and markedly expressed either in the glandular or stromal layer. Conclusions: Cytological analysis could be used as a screening test, at least for women at high surgical risk. Notch-1+ER-\u3b1+PR-\u3b2 expression could be predictive for the risk of endometrial malignancy even at an earlier stadium than hyperplasia and could be used to identify the glandular or stromal origin of cancer thus helping in identifying women at increased risk of malignancy

Both ghrelin deletion and unacylated ghrelin overexpression preserve muscles in aging mice

Sarcopenia, the decline in muscle mass and functionality during aging, might arise from age-associated endocrine dysfunction. Ghrelin is a hormone circulating in both acylated (AG) and unacylated (UnAG) forms with antiatrophic activity on skeletal muscle. Here, we show that not only lifelong overexpression of UnAG (Tg) in mice, but also the deletion of ghrelin gene (Ghrl KO) attenuated the age-associated muscle atrophy and functionality decline, as well as systemic inflammation. Yet, the aging of Tg and Ghrl KO mice occurs with different dynamics: while old Tg mice seem to preserve the characteristics of young animals, Ghrl KO mice features deteriorate with aging. However, young Ghrl KO mice show more favorable traits compared to WT animals that result, on the whole, in better performances in aged Ghrl KO animals. Treatment with pharmacological doses of UnAG improved muscle performance in old mice without modifying the feeding behavior, body weight, and adipose tissue mass. The antiatrophic effect on muscle mass did not correlate with modifications of protein catabolism. However, UnAG treatment induced a strong shift towards oxidative metabolism in muscle. Altogether, these data confirmed and expanded some of the previously reported findings and advocate for the design of UnAG analogs to treat sarcopenia

Prevalence and Determinants of Liver Disease in Relatives of Italian Patients With Advanced MASLD

Background &amp; Aims: Metabolic dysfunction associated steatotic liver disease (MASLD) has a strong genetic component. The aim of this study was to examine noninvasively the prevalence of MASLD and of advanced fibrosis in relatives of patients with advanced MASLD and the risk factors for liver involvement, with a focus on the contribution of common genetic risk variants. Methods: We prospectively enrolled 98 consecutive probands with advanced fibrosis and/or hepatocellular carcinoma caused by MASLD and 160 nontwin first-degree relatives noninvasively screened for MASLD and advanced fibrosis at 4 Italian centers. We evaluated common genetic determinants and polygenic risk scores of liver disease. Results: Among relatives, prevalence of MASLD was 56.8% overall, whereas advanced fibrosis was observed in 14.4%. At multivariable analysis in relatives, MASLD was associated with body mass index (odds ratio [OR], 1.31 [1.18–1.46]) and tended to be associated with diabetes (OR, 5.21 [0.97–28.10]), alcohol intake (OR, 1.32 [0.98–1.78]), and with female sex (OR, 0.54 [0.23–1.15]), whereas advanced fibrosis was associated with diabetes (OR, 3.13 [1.16–8.45]) and nearly with body mass index (OR, 1.09 [1.00–1.19]). Despite that the PNPLA3 risk variant was enriched in probands (P =.003) and overtransmitted to relatives with MASLD (P =.045), evaluation of genetic risk variants and polygenic risk scores was not useful to guide noninvasive screening of advanced fibrosis in relatives. Conclusions: We confirmed that about 1 in 7 relatives of patients with advanced MASLD has advanced fibrosis, supporting clinical recommendations to perform family screening in this setting. Genetic risk variants contributed to liver disease within families but did not meaningfully improve fibrosis risk stratification