Test performance and clinical utility of expanded non-invasive prenatal test: Experience on 71,883 unselected routine cases from one single center.

OBJECTIVE: The balance between benefits and risks of discordant outcomes makes the Genome-Wide Non-Invasive Prenatal Test (GW-NIPT) controversial. This study aims to evaluate performance and clinical utility in a wide cohort of unselected clinical cases from a single center when a standardized protocol is applied and integrated with a secondary algorithm for data interpretation. METHOD: In 2 years, over 70,000 pregnant patients underwent GW-NIPT for fetal common trisomies, sex chromosome aneuploidies, rare autosomal aneuploidies, segmental abnormalities (CNVs ≥ 7 Mb) and microdeletions (CNVs < 7 Mb). All samples were uniformly processed with Veriseq NIPT Solution v2 and analyzed using all data metrics along with a home-made algorithm for sequencing data analysis. Results were retrospectively reviewed for clinical outcomes. RESULTS: Among 71,883 eligible cases including twin pregnancies, 1011 (1.4%) received a positive result and 781 were confirmed by invasive prenatal diagnosis. Clinical sensitivity ranged from 99.65% for common trisomy (T21, T18, T13) to 83.33% for microdeletions, while specificity remained high (99.98%) for each class of fetal abnormalities detected. CONCLUSIONS: Integrating a standardized protocol with an internal algorithm allowed discordant results to be reduced, yielding high accuracy. Observed reliability in detecting genome-wide chromosomal conditions reinforced the expanded NIPT utility in clinical practice.

Non-Ceruloplasmin Copper Identifies a Subtype of Alzheimer's Disease (CuAD): Characterization of the Cognitive Profile and Case of a CuAD Patient Carrying an RGS7 Stop-Loss Variant.

Alzheimer's disease (AD) is a type of dementia whose cause is incompletely defined. Copper (Cu) involvement in AD etiology was confirmed by a meta-analysis on about 6000 participants, showing that Cu levels were decreased in AD brain specimens, while Cu and non-bound ceruloplasmin Cu (non-Cp Cu) levels were increased in serum/plasma samples. Non-Cp Cu was advocated as a stratification add-on biomarker of a Cu subtype of AD (CuAD subtype). To further circumstantiate this concept, we evaluated non-Cp Cu reliability in classifying subtypes of AD based on the characterization of the cognitive profile. The stratification of the AD patients into normal AD (non-Cp Cu ≤ 1.6 µmol/L) and CuAD (non-Cp Cu > 1.6 µmol/L) showed a significant difference in executive function outcomes, even though patients did not differ in disease duration and severity. Among the Cu-AD patients, a 76-year-old woman showed significantly abnormal levels in the Cu panel and underwent whole exome sequencing. The CuAD patient was detected with possessing the homozygous (c.1486T > C; p.(Ter496Argext*19) stop-loss variant in the RGS7 gene (MIM*602517), which encodes for Regulator of G Protein Signaling 7. Non-Cp Cu as an add-on test in the AD diagnostic pathway can provide relevant information about the underlying pathological processes in subtypes of AD and suggest specific therapeutic options.

Sudden winter iloprost withdrawal in scleroderma patients during COVID-19 pandemic.

INTRODUCTION: Intravenous iloprost is currently recommended in the treatment of Raynaud's phenomenon (RP) refractory to oral therapy and of digital ulcers (DUs) related to systemic sclerosis (SSc). In real-life practice there is a huge heterogeneity about the Iloprost regimens used. METHODS: A survey was carried out on SSc patients that interrupted Iloprost infusion to compare acral vascular symptoms just before Iloprost withdrawal and just after the missed infusion. Severity, and frequency of RP, new DUs onset or aggravation of those pre-existing were reported. Last available capillaroscopic images were also evaluated. RESULTS: The analysis includes 50 patients. After iloprost withdrawal, 11 patients reported a RP worsening because of enhanced intensity (p = 0.007). Only 8 patients of them also complained of an increased frequency (p = 0.07). None of the patients experienced digital ulcers for the first-time during quarantine. Among the 27 patients with a history of digital ulcers, 9 reported worsening and 7 recurrence of DUs. Overall, 17 patients (34.0 %) complained of a worsening of SSc vascular acral manifestations, namely RP or DUs. Reduced capillary density was associated with RP worsening, in particular, each unit increase of capillary density corresponds to an average 44 % decrease in the odds of RP worsening (OR 0.56, CI 95 % 0.36-0.97, p = 0.037). As for RP worsening, the aggravation of DU was associated with a lower capillary density. CONCLUSIONS: Low capillary density can predict a worsening of both RP and DUs in controlled quarantine conditions within a month after iloprost discontinuation in SSc patients.

Nailfold capillaroscopy findings in patients with coronavirus disease 2019: Broadening the spectrum of COVID-19 microvascular involvement.

OBJECTIVE: Increasing evidence points to endothelial dysfunction as a key pathophysiological factor in coronavirus disease-2019 (COVID-19). No specific methods have been identified to predict, detect and quantify the microvascular alterations during COVID-19. Our aim was to assess microvasculature through nailfold videocapillaroscopy (NVC) in COVID-19 patients. METHODS: We performed NVC in patients with a confirmed diagnosis of COVID-19 pneumonia. Elementary alterations were reported for each finger according to a semi-quantitative score. Capillary density, number of enlarged and giant capillaries, number of micro-hemorrhages and micro-thrombosis (NEMO score) were registered. RESULTS: We enrolled 82 patients (mean age 58.8 ± 13.2 years, male 68.3%) of whom 28 during the hospitalization and 54 after recovery and hospital discharge. At NVC examination we found abnormalities classifiable as non-specific pattern in 53 patients (64.6%). Common abnormalities were pericapillary edema (80.5%), enlarged capillaries (61.0%), sludge flow (53.7%), meandering capillaries and reduced capillary density (50.0%). No pictures suggestive of scleroderma pattern have been observed. Acute COVID-19 patients, compared to recovered patients, showed a higher prevalence of hemosiderin deposits as a result of micro-hemorrhages (P = .027) and micro-thrombosis (P < .016), sludge flow (P = .001), and pericapillary edema (P < .001), while recovered patients showed a higher prevalence of enlarged capillaries (P < .001), loss of capillaries (P = .002), meandering capillaries (P < .001), and empty dermal papillae (P = .006). CONCLUSION: COVID-19 patients present microvascular abnormalities at NVC. Currently ill and recovered subjects are characterized by a different distribution of elementary capillaroscopic alterations, resembling acute and post-acute microvascular damage. Further studies are needed to assess the clinical relevance of NVC in COVID-19.

Gut microbiota analysis in systemic sclerosis according to disease characteristics and nutritional status.

OBJECTIVES: Systemic sclerosis (SSc) is a rare multi-organ disorder with a prominent gastrointestinal (GI) involvement. Altered gut microbiota is now considered a pivotal factor associated with the development of immune-mediated and inflammatory diseases. We performed a 16S ribosomal RNA (rRNA) gene-sequencing analysis of fecal microbiota in a cohort of SSc patients and matched healthy controls (HCs), with the aim to obtain some hints about a possible role of dysbiosis in the onset, progression, and severity of the disease. METHODS: We analysed stool samples from 63 SSc patients with different disease duration, phenotype, and nutritional status and from 17 HCs through 16S ribosomal RNA (rRNA) gene-sequencing. RESULTS: Microbial richness was lower for patients with long-standing disease. A similar observation was made for patients with diffuse cutaneous SSc (dsSSc) compared to those with limited variant (lcSSc) and for patients who reported a recent weight loss. Consistent with previous reports, we noted a deviation of the intestinal microbial composition in patients with SSc compared to HCs, with a greater expression of Lactobacillus and Streptococcus and a depletion of Sutterella. Nutritional status, assessed using BMI as a surrogate, appeared to have a marked impact on the gut microbiota, with overweight patients showing lower richness compared both to underweight and normal-BMI patients. CONCLUSIONS: Our findings expand the current knowledge of gut microbiota in SSc and could be useful to identify patients who would most benefit from treatments aimed at restoring the eu-biosis.

NIPAT as Non-Invasive Prenatal Paternity Testing Using a Panel of 861 SNVs.

In 1997, it was discovered that maternal plasma contains Cell-Free Fetal DNA (cffDNA). cffDNA has been investigated as a source of DNA for non-invasive prenatal testing for fetal pathologies, as well as for non-invasive paternity testing. While the advent of Next Generation Sequencing (NGS) led to the routine use of Non-Invasive Prenatal Screening (NIPT or NIPS), few data are available regarding the reliability and reproducibility of Non-Invasive Prenatal Paternity Testing (NIPPT or NIPAT). Here, we present a non-invasive prenatal paternity test (NIPAT) analyzing 861 Single Nucleotide Variants (SNV) from cffDNA through NGS technology. The test, validated on more than 900 meiosis samples, generated log(CPI)(Combined Paternity Index) values for designated fathers ranging from +34 to +85, whereas log(CPI) values calculated for unrelated individuals were below -150. This study suggests that NIPAT can be used with high accuracy in real cases.

Mutation analysis of the FBN1 gene in a cohort of patients with Marfan Syndrome: A 10-year single center experience.

BACKGROUND: Marfan Syndrome (MFS) is a chronic, life-threatening, autosomal dominant connective tissue disorder caused by mutations in the FBN1 gene, coding for fibrillin-1. All organ systems may be affected, but particularly the cardiovascular system, eyes, and skeleton. Mortality generally results from cardiovascular complications, mainly aortic dissection. Currently, the diagnosis of MFS is based on the revised Ghent nosology. Molecular analysis of the FBN1 gene reduces diagnostic uncertainty in patients with suspected MFS or MFS-related disorders (MFS-RD). To date, more than 2700 FBN1 mutations are known. METHODS: Using Next Generation Sequencing (NGS) followed by Multiplex Ligation-dependent Probe Amplification on NGS-negative samples, we screened FBN1 gene on 124 unrelated patients (101 MFS fulfilling revised Ghent criteria, 20 suspected MFS, 3 MFS-RD) enrolled from 2008 to 2018 at the Multidisciplinary Marfan Clinic, Tor Vergata Hospital, Rome. RESULTS: An FBN1 variant was identified in 107/124 (86.3%) patients, including 48 novel variants (46 pathogenic/likely pathogenic, 2 VUS). A pathogenic/likely pathogenic variant was detected in 90/101 (89.1%) MFS patients. Our approach allowed early diagnosis for 10 young patients (age 3-19 years) with suspected MFS. CONCLUSIONS: This study broadens the mutation spectrum of FBN1, providing a full update of the molecular basis of MFS in Italy.

Treatment strategy introducing immunosuppressive drugs with glucocorticoids ab initio or very early in giant cell arteritis: A multicenter retrospective controlled study.

OBJECTIVE: Glucocorticoids (GC) are associated with side effects in giant cell arteritis (GCA). Immunosuppressive therapies (ITs) have given conflicting results in GCA, regarding GC sparing effect. Primary endpoint is to evaluate whether very early introduction of ITs in GCA minimize the rate of GC-induced adverse events, in terms of infections, new onset systemic arterial hypertension, GC-induced diabetes and osteoporotic fractures. METHODS: A multicenter retrospective case-control study included 165 patients. One group included 114 patients who were treated with at least one IT given at diagnosis or within 3 months from the start of GC. A second group included 51 GCA who received only GC or an IT more than 3 months later. RESULTS: The most frequently used ITs were: methotrexate (138 patients), cyclophosphamide (48 patients) and tocilizumab (27 patients). No difference was observed as concerns the follow-up time between groups [48.5 (IQR 26-72) vs 40 (IQR 24-69), p â€‹= â€‹0.3)]. The first group showed a significantly lower incidence of steroid-induced diabetes (8/114, 7% vs 12/51, 23.5%; p â€‹= â€‹0.003) and no differences for the rate of infections (p â€‹= â€‹0.64). The group was also exposed to lower doses of GC at first (p â€‹< â€‹0.0001) and third (p â€‹< â€‹0.0001, rank-sum test) month. Forty-four patients in the first group (38.6%) compared with 34 in the second one (66.7%) experienced at least one relapse (p â€‹= â€‹0.001). CONCLUSION: Very early introduction of IT in GCA lowered the incidence of steroid-induced diabetes, possibly due to the lower doses of GC in the first three months. Relapse rate was even lower.

Multidisciplinary Evaluation of Interstitial Lung Diseases: New Opportunities Linked to Rheumatologist Involvement.

Multidisciplinary team (MDT) discussion is the gold standard in the management of interstitial lung disease (ILD). The rheumatologist is not routinely involved in MDT, even if up to 20% of ILD are related to systemic autoimmune rheumatic diseases (SARD). The study aims to assess the agreement and its variation over time between rheumatologists and pulmonologists in the screening of SARD and between rheumatologists and an MDT extended to rheumatologists (eMDT) in evaluating the progression of SARD. We computed the agreement between the pulmonologist and rheumatologist in the identification of red flags for SARDs of 81 ILD cases and between the rheumatologist alone and eMDT in the confirmation of 70 suspected SARD-ILD progressions. The agreement between rheumatologists and pulmonologists was moderate for the detection of autoimmunity test positivity (κ = 0.475, p < 0.001) and family history of SARD (κ = 0.491, p < 0.001) and fair for the identification of extrapulmonary symptoms (κ = 0.225, p = 0.064) or routine laboratory abnormalities consistent with SARD. The average agreement between the rheumatologist and eMDT in the identification of ILD progression was moderate (κ = 0.436, p < 0.001). The class of agreement improved from the first to the third semester. The average agreement with the rheumatologist ranged from fair to moderate, suggesting that a shared evaluation of SARD-ILD in eMDT could improve the diagnostic work-up and the evaluation of ILD progression.

Sarilumab use in severe SARS-CoV-2 pneumonia.

BACKGROUND: Interleukin-6 signal blockade showed preliminary beneficial effects in treating inflammatory response against SARS-CoV-2 leading to severe respiratory distress. Herein we describe the outcomes of off-label intravenous use of Sarilumab in severe SARS-CoV-2-related pneumonia. METHODS: 53 patients with SARS-CoV-2 severe pneumonia received intravenous Sarilumab; pulmonary function improvement or Intensive Care Unit (ICU) admission rate in medical wards, live discharge rate in ICU treated patients and safety profile were recorded. Sarilumab 400 mg was administered intravenously on day 1, with eventual additional infusion based on clinical judgement, and patients were followed for at least 14 days, unless previously discharged or dead. FINDINGS: Of the 53 SARS-CoV-2pos patients receiving Sarilumab, 39(73·6%) were treated in medical wards [66·7% with a single infusion; median PaO2/FiO2:146(IQR:120-212)] while 14(26·4%) in ICU [92·6% with a second infusion; median PaO2/FiO2: 112(IQR:100-141.5)].Within the medical wards, 7(17·9%) required ICU admission, 4 of whom were re-admitted to the ward within 5-8 days. At 19 days median follow-up, 89·7% of medical inpatients significantly improved (46·1% after 24 h, 61·5% after 3 days), 70·6% were discharged from the hospital and 85·7% no longer needed oxygen therapy. Within patients receiving Sarilumab in ICU, 64·2% were discharged from ICU to the ward and 35·8% were still alive at the last follow-up. Overall mortality rate was 5·7%. INTERPRETATION: IL-6R inhibition appears to be a potential treatment strategy for severe SARS-CoV-2 pneumonia and intravenous Sarilumab seems a promising treatment approach showing, in the short term, an important clinical outcome and good safety.

Genotypic Categorization of Loeys-Dietz Syndrome Based on 24 Novel Families and Literature Data.

Loeys-Dietz syndrome (LDS) is a connective tissue disorder first described in 2005 featuring aortic/arterial aneurysms, dissections, and tortuosity associated with craniofacial, osteoarticular, musculoskeletal, and cutaneous manifestations. Heterozygous mutations in 6 genes (TGFBR1/2, TGFB2/3, SMAD2/3), encoding components of the TGF-ß pathway, cause LDS. Such genetic heterogeneity mirrors broad phenotypic variability with significant differences, especially in terms of the age of onset, penetrance, and severity of life-threatening vascular manifestations and multiorgan involvement, indicating the need to obtain genotype-to-phenotype correlations for personalized management and counseling. Herein, we report on a cohort of 34 LDS patients from 24 families all receiving a molecular diagnosis. Fifteen variants were novel, affecting the TGFBR1 (6), TGFBR2 (6), SMAD3 (2), and TGFB2 (1) genes. Clinical features were scored for each distinct gene and matched with literature data to strengthen genotype-phenotype correlations such as more severe vascular manifestations in TGFBR1/2-related LDS. Additional features included spontaneous pneumothorax in SMAD3-related LDS and cervical spine instability in TGFB2-related LDS. Our study broadens the clinical and molecular spectrum of LDS and indicates that a phenotypic continuum emerges as more patients are described, although genotype-phenotype correlations may still contribute to clinical management.

Rhabdoid tumor predisposition syndrome caused by SMARCB1 constitutional deletion: prenatal detection of new case of recurrence in siblings due to gonadal mosaicism.

Rhabdoid tumors are aggressive malignancies that show loss-of-function mutations of SMARCB1 gene, a member of the SWI/SNF chromatin-remodeling complex controlling gene transcription. One-third of patients affected by rhabdoid tumor harbor a germ-line mutation of SMARCB1 defining a rhabdoid tumor predisposition syndrome. The occurrence of a second somatic mutation determines the development of neoplasia in a two-hit model. Most germ-line mutations occur de novo, and few cases of recurrence in a sibship have been described. Here we report on a new Italian family with recurrence of SMARCB1 germ-line deletion in two siblings due to gonadal mosaicism. The deletion was identified in the 9-month-old proband with malignant rhabdoid tumor of the right kidney and disseminated metastases. Testing of both parents confirmed the de novo origin of the mutation, but recurrence was then detected prenatally in a new pregnancy. This is the sixth family with malignant rhabdoid tumor predisposition syndrome with the recurrence of the same germ-line SMARCB1 mutation in the sibship but not in healthy parents, suggesting that gonadal mosaicism is a less rare event than supposed. The clinical outcome in our patient confirms previous data of poorer outcome in patients with rhabdoid tumor predisposition syndrome.