Heterogenous electrophysiological features in early stage of hereditary transthyretin amyloidosis neuropathy.

INTRODUCTION: Hereditary transthyretin-mediated amyloidosis (ATTRv, v for variant) is a progressive disease caused by mutations in the TTR gene, leading to sensory-motor, axonal and length-dependent neuropathy. However, some patients may show variable electrophysiological pattern. The aim of this study was to evaluate the electrophysiological features of TTR amyloid neuropathy at the time of the first nerve conduction study (NCS) to assess whether there were distinguishing features useful for early diagnosis. METHODS: We retrospectively revised the first electrophysiological findings of ATTRv patients, and we categorized the neuropathy based on nerve conduction slowing, type of involved fibres and distribution pattern of PNS involvement. Cluster analysis was performed to evaluate the prevalence of neuropathy features between the early and late stage of disease, based on disease duration and disability burden assessed by NIS. RESULTS: We recruited 33 patients (27 males) with mean age 63.9 ± 10.8 years, mean disease duration 2.8 ± 2.4 years and mean NIS 47.6 ± 41.8. Overall, the frequency analysis showed that the most common features of ATTRv neuropathy included the categories of axonal, sensory-motor and neuronopathic-like pattern. This electrophysiological pattern of PNS involvement was constant in patients in late stage of disease, whereas ATTRv patients in early stage of disease displayed variable electrophysiological pattern of PNS involvement. DISCUSSION: Our findings demonstrated that ATTRv neuropathy may present at first NCS in a variable way, and it changes over the course of disease. Such heterogeneity makes the suspicion of ATTRv even more challenging at the time of first electrophysiological examination.

Complete agenesis of corpus callosum and unilateral cortical formation anomalies detected on fetal MR imaging: a phenotype strongly associated with the male fetuses.

INTRODUCTION: In a previous study of classifying fetuses with cortical formation abnormalities (CFA) with fetal MR, we noticed a cluster of cases with unilateral CFA and complete agenesis of the corpus callosum (ACC). In this study, we provide a detailed morphological analysis of such fetuses using fetal MR to determine if there are indicators (such as the gender of the fetus) that could be used to delineate a genetic substrate of the phenotype in order to inform future studies. METHODS: We have studied 45 fetuses with the unilateral CFA/ACC phenotype and analysed through an expert consensus panel the location and fine detail of the CFA and the associated findings such as associated anomalies, head size, and sex of the fetus. RESULTS: The frontal lobe was significantly more frequently involved by CFA when compared with other lobes (p < 0.001) but no preference for the left or right hemisphere. CFA most often consisted of excessive/dysmorphic sulcation. The CFA/ACC phenotype was overwhelmingly more frequent in male fetuses (M:F 4.5:1-p < 0.0001). The most frequent associated findings were: ventriculomegaly (16/45 fetuses) and interhemispheric cysts (12/45 cases). CONCLUSIONS: This report highlights the specific phenotype of unilateral CFA/ACC that is much more common in male fetuses. This finding provides a starting point to study possible sex-linked genetic abnormalities that underpin the unilateral CFA/ACC phenotype. KEY POINTS: • We collected fetuses with unilateral cortical formation abnormality and callosal agenesis. • That distinctive neuroimaging phenotype has a strong male gender prevalence (over 80%). • This observation forms the basis of studies about outcomes and genetic substrates.

Cutting-edge applications of fetal MR neuro-imaging in clinical routine: a pictorial essay.

Over time, fetal MR neuro-imaging has undergone continuous improvement; presently, it plays a pivotal role in the diagnosis of an expanding array of complex neurological conditions. Within this pictorial essay, our focus will be exclusively directed towards those cutting-edge clinical applications, which currently yield valuable diagnostic insights on a single case basis. Specifically, the pictorial examples will center on some abnormal entities and their features at an earlier fetal stage.

Onset of Chiari type 1 malformation: insights from a small series of intrauterine MR imaging cases.

PURPOSE: Morphometric studies on idiopathic Chiari malformation type 1 (CM1) pathogenesis have been mainly based on post-natal neuroimaging. Prenatal clues related to CM1 development are lacking. We present pre- and post-natal imaging time course in idiopathic CM1 and assess fetal skull and brain biometry to establish if clues about CM1 development are present at fetal age. METHODS: Multicenter databases were screened to retrieve intrauterine magnetic resonance (iuMR) of children presenting CM1 features at post-natal scan. Syndromes interfering with skull-brain growth were excluded. Twenty-two morphometric parameters were measured at fetal (average 24.4 weeks; range 21 to 32) and post-natal (average 15.4 months; range 1 to 45) age; matched controls were included. RESULTS: Among 7000 iuMR cases, post-natal scans were available for 925, with postnatal CM1 features reported in seven. None of the fetuses presented CM1 features. Tonsillar descent was clear at a later post-natal scan in all seven cases. Six fetal parameters resulted to be statistically different between CM1 and controls: basal angle (p = 0.006), clivo-supraoccipital angle (p = 0.044), clivus' length (p = 0.043), posterior cranial fossa (PCF) width (p = 0.009), PCF height (p = 0.045), and PCFw/BPDb (p = 0.013). Postnatally, only the clivus' length was significant between CM1 cases and controls. CONCLUSION: Pre- and post-natal CM1 cases did not share striking common features, making qualitative prenatal assessment not predictive; however, our preliminary results support the view that some of the pathogenetic basis of CM1 may be embedded to some extent already in intrauterine life.

A case of severe increase of liver enzymes in a ATTRv patient after one year of inotersen treatment.

BACKGROUND: Inotersen is an antisense oligonucleotide used to treat hereditary transthyretin amyloidosis (ATTRv). The most common drug-related adverse effects (AEs) include thrombocytopenia and glomerulonephritis. Hepatic damage is rare, but liver enzyme monitoring is mandatory. CASE REPORT: A 70-year-old man with ATTRv (Val30Met) treated with inotersen developed a severe increase of transaminases, with normal bilirubin and cholinesterase levels, that forced us to stop therapy. At the same time, other causes of acquired hepatitis were excluded, and the hypothesis of an inotersen-related hepatic toxicity was supported by the normalization of liver enzymes after 40 days from the drug interruption. DISCUSSION: Our case showed that 1-year inotersen treatment can stabilize neurological impairment and even improve quality of life and suggests to carefully monitor liver enzymes in order to avoid an inotersen-related hepatic dysfunction.

Cerebral folate transporter deficiency syndrome in three siblings: Why genetic testing for developmental and epileptic encephalopathies should be performed early and include the FOLR1 gene.

Cerebral folate transporter deficiency syndrome, caused by FOLR-1 mutations is characterized by late infantile onset, severe developmental regression, epilepsy, and leukodystrophy. An extremely low concentration of 5-methyltetrahydrofolate in the cerebrospinal fluid provides a crucial clue to its diagnosis and is a treatment target. Oral or intravenous folinic acid (5-formyltetrahydrofolate) administration improves clinical symptoms and brain magnetic resonance imaging (MRI) findings. We describe three siblings carrying a novel homozygous FOLR1 nonsense mutation, that were referred due to intractable epilepsy and progressive neurological decline. Brain MRI showed hypomyelination and cerebellar atrophy. Folinic acid (oral and intravenous) supplementation, initiated after over 15 years illness, has failed to result in any sizeable clinical or neurophysiological improvement. Cerebral folate transport deficiency bears overlapping clinical features with many severe developmental encephalopathies. It is crucial to recognize FOLR1 signs and establish an early clinical and molecular diagnosis in order to provide timely folinic acid treatment and improve outcome.

Changes in appearance of cortical formation abnormalities in the foetus detected on sequential in utero MR imaging.

OBJECTIVES: We describe 64 foetuses with cortical formation abnormalities (CFA) who had two in utero magnetic resonance (iuMR) exams, paying particular detail to those in which the original classification of CFA category changed between the two studies. The goal was to attempt to quantify the value of third-trimester follow-up studies in CFA foetuses on second-trimester iuMR imaging. METHODS: The 64 foetuses reviewed came from a CFA cohort of 374 foetuses reported in an earlier publication, which detailed a classification for foetal CFA. A consensus panel of senior paediatric neuroradiologists reviewed both studies, described any change in the category of CFA between them, and attempted to predict the possible clinical significance of any differences based on the combined clinical experience of the panel. RESULTS: In 40/64 (62%) foetuses, the CFA description was the same on both studies. In 24/64 (38%) cases, there was a category change which included three foetuses without CFA on first examination, six foetuses where the difference involved change in laterality/symmetry, and in 15 cases the re-classification involved categorical change within the same group. Brain abnormalities other than CFA were present in 30/64 (47%) foetuses on the first study and in 33/64 (52%) on the second. We predicted that prognosis would have changed on the basis of the second study in 8% of cases, all indicating worse prognosis. CONCLUSIONS: We have shown that the extra diagnostic and predicted prognostic yield justifies follow-up studies in the third trimester if a CFA is shown on the second-trimester iuMR imaging. KEY POINTS: • Sixty-four foetuses with cortical formation abnormalities had two iuMR studies, for the vast majority the baseline in the second trimester and the sequential in the third. • In three foetuses, the cortical formation abnormality (CFA) was not visible on the first study. In a further 21 foetuses, the categorical description of the CFA changed between the two studies. Prognosis changed in 8% of the cases following the second iuMR study, and in all cases, the prognosis was worse. • Multiple iuMR studies provide information about the natural history of CFA; the extra diagnostic and predicted prognostic yield justifies follow-up studies.

A Stereoconvergent Tsuji-Trost Reaction in the Synthesis of Cyclohexenyl Nucleosides.

A highly regio- and stereoselective route to d- and l-cyclohexenyl nucleosides has been devised, using the Tsuji-Trost reaction as the key step. Contrarily to the widely accepted mechanism (involving a net retention of configuration), the reaction proceeded in a highly stereoconvergent manner, providing cis nucleosides regardless of the relative configuration of the starting materials. DFT calculations confirmed the experimental data while suggesting the origin of the stereochemical reaction outcome.

Cortical formation abnormalities on foetal MR imaging: a proposed classification system trialled on 356 cases from Italian and UK centres.

OBJECTIVE: To formulate a classification system for foetal cortical formation abnormalities (CFAs) based on in utero magnetic resonance (iuMR) appearances and trial it in 356 cases. METHODS: This retrospective study included all cases of foetal CFA diagnosed between 2000 and 2017 from seven centres in Italy and UK. All of the studies were reviewed by a panel of paediatric neuroradiologists experienced in iuMR with the aid of an algorithm designed to categorise the abnormalities. RESULTS: Consensus expert review confirmed 356 foetuses with CFA and the first level of classification distinguished bilateral CFA (229/356-64%) from unilateral CFA (127/356-36%) cases with sub-classification of the bilateral cases into asymmetric (65/356-18%) and symmetric (164/356-46%) involvement. There was a statistically significant excess of foetuses with small head size, e.g. 17% of the cohort had a bi-parietal diameter < 3rd centile. There was a small but statistically significant excess of males in the cohort. Further categorisation was made on fine anatomical structure. CONCLUSIONS: It is often not possible to classify foetal CFA using the principles and nomenclature used in paediatric neuroradiology. We have created a classification system for foetal CFA based on the analysis of 356 cases and believe that this will assist future research designed to correlate ante-natal and post-natal imaging features and understand the clinical sequelae of CFA described in utero. KEY POINTS: • We describe a morphological classification system of foetal brain cortical formation abnormalities that can be used in clinical practice. • This classification system can be used in future research studies to evaluate the long-term imaging and clinical outcomes of foetal brain cortical formation abnormalities in 17- to 38-week gestational age range. • The practical value of the work is in providing a framework and language to look for imaging clues that may differentiate between different CFA in further studies.

Prenatal magnetic resonance imaging within the 26th week of gestation may predict the fate of isolated upward rotation of the cerebellar vermis: insights from a multicentre study.

OBJECTIVES: We investigated whether prenatal magnetic resonance imaging (MRI) within 26 weeks of gestation (GW) may predict the fate of isolated upward rotation of the cerebellar vermis (URCV). METHODS: This retrospective multicentre observational study included foetuses diagnosed with isolated URCV in prenatal MRI performed within 26 GW. Isolated URCV was defined by a brainstem-vermis angle (BVA) ≥ 12° in the MR midline sagittal view without abnormalities of the supratentorial structures, brainstem, or cerebellum hemispheres. The assessments included the BVA, clival-supraoccipital angle, transverse diameter of the posterior cranial fossa, tentorial angle, width of the cisterna magna (WCM), ventricular width, vermian diameters, hypointense stripes, and cerebellar tail sign. Late prenatal or postnatal MRI was used as a reference standard to assess the final vermian fate (rotated/de-rotated). RESULTS: Forty-five foetuses (mean GW at prenatal MRI = 21.5 ± 1.4 weeks) were included. In the reference standard, the vermis was de-rotated in 26 cases (57.7%). At least two of the following criteria were used to predict the persistence of URCV at imaging follow-up: BVA ≥ 23°, WCM ≥ 9 mm, and the cerebellar tail sign. The results were a sensitivity of 84.21% (95% CI, 60.4-96.6%), specificity of 80.8% (95% CI, 60.6-93.4%), positive predictive value of 76% (95% CI, 58.7-87.8%), and negative predictive value of 87.5% (95% CI, 70.9-95.2%). CONCLUSIONS: MRI within 26 GW on foetuses diagnosed with isolated URCV may predict delayed cerebellar vermis de-rotation, which is associated with good neurodevelopmental outcome in most cases. KEY POINTS: • Foetal MRI is a valuable tool in predicting the fate of isolated upward-rotated cerebellar vermis. • A wider angle between the brainstem and vermis is associated with higher risk of persistence of vermian rotation. • The presence of ≥ 2 factors among a brainstem-to-vermis angle ≥ 23°, width of the cisterna magna ≥ 9 mm, and the presence of the "cerebellar tail sign" has a sensitivity of 84.21% (95% CI, 60.4-96.6%) and specificity of 80.8% (95% CI, 60.6-93.4%) in predicting the persistence of the vermian rotation at imaging follow-up.

High resolution post-mortem MRI of non-fixed in situ foetal brain in the second trimester of gestation: Normal foetal brain development.

PURPOSE: To describe normal foetal brain development with high resolution post-mortem MRI (PMMRI) of non-fixed foetal brains. METHODS: We retrospectively collected PMMRIs of foetuses without intracranial abnormalities and chromosomal aberrations studied after a termination of pregnancy due to extracranial abnormalities or after a spontaneous intrauterine death. PMMRIs were performed on a 3-T scanner without any fixation and without removing the brain from the skull. All PMMRIs were evaluated in consensus by two neuroradiologists. RESULTS: Our analysis included ten PMMRIs (median gestational age (GA): 21 weeks; range: 17-28 weeks). At 19 and 20 weeks of GA, the corticospinal tracts are recognisable in the medulla oblongata, becoming less visible from 21 weeks. Prior to 20 weeks the posterior limb of the internal capsule (PLIC) is more hypointense than surrounding deep grey nuclei; starting from 21 weeks the PLIC becomes isointense, and is hyperintense at 28 weeks. From 19-22 weeks, the cerebral hemispheres show transient layers: marginal zone, cortical plate, subplate, and intermediate, subventricular and germinal zones. CONCLUSION: PMMRI of non-fixed in situ foetal brains preserves the natural tissue contrast and skull integrity. We assessed foetal brain development in a small cohort of foetuses, focusing on 19-22 weeks of gestation. KEY POINTS: • Post-mortem magnetic resonance imaging (PMMRI) of non-fixed head is feasible. • PMMRI of unfixed in situ foetal brains preserves the natural tissue contrast. • PMMRI provide a good depiction of the normal foetal brain development. • PMMRI of unfixed in situ foetal brains preserves the skull integrity. • PMMRI pattern of foetal brain development at early gestational age is described.

A Semisynthetic Approach to New Immunoadjuvant Candidates: Site-Selective Chemical Manipulation of Escherichia coli Monophosphoryl Lipid†A.

A semisynthetic approach to novel lipid A derivatives from Escherichia coli (E. coli) lipid A is reported. This methodology stands as an alternative to common approaches based exclusively on either total synthesis or extraction from bacterial sources. It relies upon the purification of the lipid A fraction from fed-batch fermentation of E. coli, followed by its structural modification through tailored, site-selective chemical reactions. In particular, modification of the lipid pattern and functionalization of the phosphate group as well as of the sole primary hydroxyl group were accomplished, highlighting the unusual reactivity of the molecule. Preliminary investigations of the immunostimulating activity of the new semisynthetic lipid A derivatives show that some of them stand out as promising, new immunoadjuvant candidates.

1',5'-Anhydro-L-ribo-hexitol Adenine Nucleic Acids (α-L-HNA-A): Synthesis and Chiral Selection Properties in the Mirror Image World.

The synthesis and a preliminary investigation of the base pairing properties of (6' → 4')-linked 1',5'-anhydro-L-ribo-hexitol nucleic acids (α-L-HNA) have herein been reported through the study of a model oligoadenylate system in the mirror image world. Despite its considerable preorganization due to the rigidity of the "all equatorial" pyranyl sugar backbone, α-L-HNA represents a versatile informational biopolymer, in view of its capability to cross-communicate with natural and unnatural complements in both enantiomeric forms. This seems the result of an inherent flexibility of the oligonucleotide system, as witnessed by the singular formation of iso- and heterochiral associations composed of regular, enantiomorphic helical structures. The peculiar properties of α-L-HNA (and most generally of the α-HNA system) provide new elements in our understanding of the structural prerequisites ruling the stereoselectivity of the hybridization processes of nucleic acids.

Oligonucleotides containing a ribo-configured cyclohexanyl nucleoside: probing the role of sugar conformation in base pairing selectivity.

The synthesis and a preliminary evaluation of the pairing properties of ribo-cyclohexanyl nucleic acids (r-CNA) is herein reported. Incorporation of a single r-CNA nucleotide into natural duplexes did not enhance their stability, while a very high pairing selectivity for RNA was found. As deduced by comparative analysis of Tm and NMR data, a relationship between pairing selectivity and conformational preferences of the "sugar" moiety of r-CNA (and more generally of six-membered nucleic acids) was suggested.

A combined fermentative-chemical approach for the scalable production of pure E. coli monophosphoryl lipid A.

Lipid A is the lipophilic region of lipopolysaccharides and lipooligosaccharides, the major components of the outer leaflet of most part of Gram-negative bacteria. Some lipid As are very promising immunoadjuvants. They are obtained by extraction from bacterial cells or through total chemical synthesis. A novel, semisynthetic approach to lipid As is ongoing in our laboratories, relying upon the chemical modification of a natural lipid A scaffold for the fast obtainment of several other lipid As and derivatives thereof. The first requisite for this strategy is to have this scaffold available in large quantities through a scalable process. Here, we present an optimized fed-batch fermentation procedure for the gram-scale production of lipid A from Escherichia coli K4 and a suitable phenol-free protocol for its purification. A study for regioselective de-O-phosphorylation reaction was then performed to afford pure monophosphoryl lipid A with an attenuated endotoxic activity, as evaluated by cytokine production in human monocytic cell line THP-1 in vitro. The reported method for the large-scale obtainment of monophoshoryl lipid A from the fed-batch fermentation broth of a recombinant strain of E. coli may permit the access to novel semisynthetic lipid A immunoadjuvant candidates.

Sulfur-assisted domino access to bicyclic dihydrofurans: case study and early synthetic applications.

A DDQ-mediated domino reaction (up to six steps in a single process) has been developed to selectively provide substituted dihydrofurans from a common starting material containing a cyclic bis-thioenol ether. Study of the reaction mechanism highlighted a role played by the sulfur-containing moiety in influencing reaction rate and stereoselectivity.

Machine Learning for Early Diagnosis of ATTRv Amyloidosis in Non-Endemic Areas: A Multicenter Study from Italy.

BACKGROUND: Hereditary transthyretin amyloidosis with polyneuropathy (ATTRv) is an adult-onset multisystemic disease, affecting the peripheral nerves, heart, gastrointestinal tract, eyes, and kidneys. Nowadays, several treatment options are available; thus, avoiding misdiagnosis is crucial to starting therapy in early disease stages. However, clinical diagnosis may be difficult, as the disease may present with unspecific symptoms and signs. We hypothesize that the diagnostic process may benefit from the use of machine learning (ML). METHODS: 397 patients referring to neuromuscular clinics in 4 centers from the south of Italy with neuropathy and at least 1 more red flag, as well as undergoing genetic testing for ATTRv, were considered. Then, only probands were considered for analysis. Hence, a cohort of 184 patients, 93 with positive and 91 (age- and sex-matched) with negative genetics, was considered for the classification task. The XGBoost (XGB) algorithm was trained to classify positive and negative TTR mutation patients. The SHAP method was used as an explainable artificial intelligence algorithm to interpret the model findings. RESULTS: diabetes, gender, unexplained weight loss, cardiomyopathy, bilateral carpal tunnel syndrome (CTS), ocular symptoms, autonomic symptoms, ataxia, renal dysfunction, lumbar canal stenosis, and history of autoimmunity were used for the model training. The XGB model showed an accuracy of 0.707 ± 0.101, a sensitivity of 0.712 ± 0.147, a specificity of 0.704 ± 0.150, and an AUC-ROC of 0.752 ± 0.107. Using the SHAP explanation, it was confirmed that unexplained weight loss, gastrointestinal symptoms, and cardiomyopathy showed a significant association with the genetic diagnosis of ATTRv, while bilateral CTS, diabetes, autoimmunity, and ocular and renal involvement were associated with a negative genetic test. CONCLUSIONS: Our data show that ML might potentially be a useful instrument to identify patients with neuropathy that should undergo genetic testing for ATTRv. Unexplained weight loss and cardiomyopathy are relevant red flags in ATTRv in the south of Italy. Further studies are needed to confirm these findings.

Synthesis and evaluation of folate-based chlorambucil delivery systems for tumor-targeted chemotherapy.

The development of tumor-targeting drug delivery systems, able to selectively transport cytotoxic agents into the tumor site by exploiting subtle morphological and physiological differences between healthy and malignant cells, currently stands as one of the most attractive anticancer strategies used to overcome the selectivity problems of conventional chemotherapy. Owing to frequent overexpression of folate receptors (FRs) on the surface of malignant cells, conjugation of cytotoxic agents to folic acid (FA) via suitable linkers have demonstrated to enhance selective drug delivery to the tumor site. Herein, the chemical synthesis and biological evaluation of two novel folate-conjugates bearing the anticancer agent chlorambucil (CLB) tethered to either an aminoether (4,7,10-trioxa-1,13-tridecanediamine) or a pseudo-ß-dipeptide (ß-Ala-ED-ß-Ala) linker is reported. The two drug delivery systems have been prepared in high overall yields (54% and 34%) through straightforward and versatile synthetic routes. Evaluation of cell specificity was examined using three leukemic cell lines, undifferentiated U937 (not overexpressing FRs, FR(-)), TPA-differentiated U937 (overexpressing FRs, FR(+)), and TK6 (FR(+)) cells. Both conjugates exhibited high specificity only to FR(+) cells (particularly TK6), demonstrating comparable antitumor activity to CLB in its free form. These data confirm the reliability of folate-based drug delivery systems for targeted antitumor therapy; likewise, they lay the foundations for the development of other folate-conjugates with antitumor potential.

Quantitative Sensory Testing in Late-Onset ATTRv Presymptomatic Subjects: A Single Center Experience.

Backgrounds Hereditary transthyretin amyloidosis (ATTRv) presymptomatic subjects undergo multidisciplinary evaluation to detect, as early as possible, a subclinical involvement of multisystem disease. Quantitative sensory testing (QST) that investigates and discriminates the function of C, Aδ and Aß fibers is included as an instrumental test to monitor nerve fiber function. The purpose of this study was to evaluate the role of QST in the context of the multidisciplinary evaluation in late onset carriers. Methods Four-teen presymptomatic (namely carriers) were enrolled. Subjects underwent thermal [cold and warm detection threshold (CDT, WDT), cold and heat pain (CP and HP)] and tactile QST in four body sites: foot dorsum, distal lateral leg, distal thigh, hand dorsum. Results Overall, presymptomatic subject showed a significant difference in all thermal QST findings compared to the control group. All subjects had at least one altered thermal QST finding; the sites more frequently altered were foot and leg, whilst the thermal modalities which were more frequently abnormal were CDT, WDT and CP. Conclusions Our study highlights the importance of performing thermal QST in subjects carrying TTR mutation, given the high frequency of abnormal findings. Notably, performing both innocuous and painful stimulation in foot and/or leg increases the chance of detecting nerve fiber dysfunction. Moreover, the investigation of the hand may provide useful information in monitoring disease progression before the Predicted Age of Disease Onset (PADO).