End-Truncated LAMB1 Causes a Hippocampal Memory Defect and a Leukoencephalopathy.

OBJECTIVE: The majority of patients with a familial cerebral small vessel disease (CSVD) referred for molecular screening do not show pathogenic variants in known genes. In this study, we aimed to identify novel CSVD causal genes. METHODS: We performed a gene-based collapsing test of rare protein-truncating variants identified in exome data of 258 unrelated CSVD patients of an ethnically matched control cohort and of 2 publicly available large-scale databases, gnomAD and TOPMed. Western blotting was used to investigate the functional consequences of variants. Clinical and magnetic resonance imaging features of mutated patients were characterized. RESULTS: We showed that LAMB1 truncating variants escaping nonsense-mediated messenger RNA decay are strongly overrepresented in CSVD patients, reaching genome-wide significance (p < 5 × 10-8 ). Using 2 antibodies recognizing the N- and C-terminal parts of LAMB1, we showed that truncated forms of LAMB1 are expressed in the endogenous fibroblasts of patients and trapped in the cytosol. These variants are associated with a novel phenotype characterized by the association of a hippocampal type episodic memory defect and a diffuse vascular leukoencephalopathy. INTERPRETATION: These findings are important for diagnosis and clinical care, to avoid unnecessary and sometimes invasive investigations, and also from a mechanistic point of view to understand the role of extracellular matrix proteins in neuronal homeostasis. ANN NEUROL 2021;90:962-975.

Unsuspected somatic mosaicism for FBN1 gene contributes to Marfan syndrome.

PURPOSE: Individuals with mosaic pathogenic variants in the FBN1 gene are mainly described in the course of familial screening. In the literature, almost all these mosaic individuals are asymptomatic. In this study, we report the experience of our team on more than 5,000 Marfan syndrome (MFS) probands. METHODS: Next-generation sequencing (NGS) capture technology allowed us to identify five cases of MFS probands who harbored a mosaic pathogenic variant in the FBN1 gene. RESULTS: These five sporadic mosaic probands displayed classical features usually seen in Marfan syndrome. Combined with the results of the literature, these rare findings concerned both single-nucleotide variants and copy-number variations. CONCLUSION: This underestimated finding should not be overlooked in the molecular diagnosis of MFS patients and warrants an adaptation of the parameters used in bioinformatics analyses. The five present cases of symptomatic MFS probands harboring a mosaic FBN1 pathogenic variant reinforce the fact that apparently asymptomatic mosaic parents should have a complete clinical examination and a regular cardiovascular follow-up. We advise that individuals with a typical MFS for whom no single-nucleotide pathogenic variant or exon deletion/duplication was identified should be tested by NGS capture panel with an adapted variant calling analysis.

Detailed cell-level analysis of sperm nuclear quality among the different hypo-osmotic swelling test (HOST) classes.

PURPOSE: We studied the quality differences between the different hypo-osmotic swelling test (HOST) classes, as measured by criteria of DNA fragmentation, DNA decondensation, and nuclear architecture. The aim was to find particular HOST classes associated with good-quality metrics, which may be potentially used in ICSI (intra-cytoplasmic sperm injection). METHODS: Ten patients from the Department of Reproductive Medicine at Tenon Hospital (Paris, France) were included. Their semen samples were collected and divided into two fractions: one was incubated in a hypo-osmotic solution as per HOST protocol and sorted by sperm morphology, and a second was incubated without undergoing the HOST protocol to serve as an unsorted baseline. Three parameters were assessed: DNA fragmentation (TUNEL assay), DNA decondensation (chromomycin A3 assay), and nuclear architecture (FISH, with telomeric and whole chromosome painting probes). The different HOST classes were evaluated for these three parameters, and statistical analysis was performed for each class versus the unsorted non-HOST-treated sperm. Results with p<0.05 were considered statistically significant. RESULTS: For each of the parameters evaluated, we found significant differences between HOST-selected spermatozoa and non-selected spermatozoa. Overall, spermatozoa of HOST classes B and B+ exhibited the highest quality based on four metrics (low DNA fragmentation, low DNA decondensation, short inter-telomeric distance, and small chromosome 1 territory area), while spermatozoa of HOST classes A and G exhibited the poorest quality by these metrics. CONCLUSION: In addition to their pathophysiological interest, our results open possibilities of sperm selection prior to ICSI, which may allow for optimization of reproductive outcomes in heretofore unstudied patient populations.

High genetic carrier frequency of Wilson's disease in France: discrepancies with clinical prevalence.

BACKGROUND: Wilson's disease (WD) is a rare autosomal recessive metabolic disease caused by ATP7B gene mutations tat cause excessively high copper levels, particularly in the liver and brain. The WD phenotype varies in terms of its clinical presentation and intensity. Diagnosing this metabolic disorder is important as a lifelong treatment, based on the use of copper chelating agents or zinc salts, is more effective if it's started early. Worldwide prevalence of WD is variable, with an average of 1/30,000. In France, a recent study based on French health insurance data estimated the clinical prevalence of the disease to be around 3/200,000. METHODS: To estimate the genetic prevalence of WD in France, we analysed the ATP7B gene by Next Generation Sequencing from a large French cohort of indiscriminate subjects. RESULTS: We observed a high heterozygous carrier frequency of ATP7B in France. Among the 697 subjects studied, 18 variants classified as pathogenic or probably pathogenic were found at heterozygous level in 22 subjects (22 alleles/1394 alleles), yielding a prevalence of 0.032 or 1/31 subjects. CONCLUSIONS: This considerable and unexplained discrepancy between the heterozygous carrier frequency and the clinical prevalence of WD may be explained by the clinical variability, the incomplete penetrance and the existence of modifiers genes. It suggests that the molecular analysis of ATP7B should be interpreted with caution, always alongside copper assays (ceruloplasmin, relative exchangeable copper, 24 h-urinary copper excretion) with particular respect to exome sequencing.

An AluYa5 Insertion in the 3'UTR of COL4A1 and Cerebral Small Vessel Disease.

Importance: Cerebral small vessel diseases (CSVDs) account for one-fifth of stroke cases. Numerous familial cases remain unresolved after routine screening of known CSVD genes. Objective: To identify novel genes and mechanisms associated with familial CSVD. Design, Setting, and Participants: This 2-stage study involved linkage analysis and a case-control study; linkage analysis and whole exome and genome sequencing were used to identify candidate gene variants in 2 large families with CSVD (9 patients with CSVD). Then, a case-control analysis was conducted on 246 unrelated probands, including probands from these 2 families and 244 additional probands. All probands (clinical onset

Extension of the Clinicoradiologic Spectrum of Newly Described End-Truncating LAMB1 Variations.

Objectives: To refine the clinical spectrum of a very recently identified phenotype associated with LAMB1 end-truncating pathogenic variations. Methods: Detailed clinical, neuropsychological, and MRI investigation of 6 patients from 2 unrelated families segregating end-truncating LAMB1 variations. Results: All patients harbored a LAMB1 end-truncating pathogenic variation. The specific association of a hippocampal type episodic memory dysfunction and a diffuse leukoencephalopathy was observed in all 4 patients aged older than 50 years, slightly worsening over time in 2 patients with several years of follow-up. Additional unspecific neurologic symptoms are reported, such as episodes of numbness, language troubles, or faintness in these 4 patients and the 2 younger ones. Discussion: The association of an extensive leukoencephalopathy with an episodic memory dysfunction of the hippocampal type is strongly suggestive of a LAMB1 end-truncating variation in adults older than 50 years. Early cognitive complaints and imaging abnormalities might exist decades before. Additional transient manifestations can be observed, and this association should lead to LAMB1 screening to avoid unnecessary invasive investigations.

Scaling of petiole anatomies, mechanics and vasculatures with leaf size in the widespread Neotropical pioneer tree species Cecropia obtusa Trécul (Urticaceae).

Although the leaf economic spectrum has deepened our understanding of leaf trait variability, little is known about how leaf traits scale with leaf area. This uncertainty has resulted in the assumption that leaf traits should vary by keeping the same pace of variation with increases in leaf area across the leaf size range. We evaluated the scaling of morphological, tissue-surface and vascular traits with overall leaf area, and the functional significance of such scaling. We examined 1,271 leaves for morphological traits, and 124 leaves for anatomical and hydraulic traits, from 38 trees of Cecropia obtusa Trécul (Urticaceae) in French Guiana. Cecropia is a Neotropical genus of pioneer trees that can exhibit large laminas (0.4 m2 for C. obtusa), with leaf size ranging by two orders of magnitude. We measured (i) tissue fractions within petioles and their second moment of area, (ii) theoretical xylem hydraulic efficiency of petioles and (iii) the extent of leaf vessel widening within the hydraulic path. We found that different scaling of morphological trait variability allows for optimisation of lamina display among larger leaves, especially the positive allometric relationship between lamina area and petiole cross-sectional area. Increasing the fraction of pith is a key factor that increases the geometrical effect of supportive tissues on mechanical rigidity and thereby increases carbon-use efficiency. We found that increasing xylem hydraulic efficiency with vessel size results in lower leaf lamina area: xylem ratios, which also results in potential carbon savings for large leaves. We found that the vessel widening is consistent with hydraulic optimisation models. Leaf size variability modifies scaling of leaf traits in this large-leaved species.

A novel deep intronic variant in ATP7B in five unrelated families affected by Wilson disease.

BACKGROUND: Wilson disease is an autosomal recessive metabolic disorder resulting from accumulation of excess copper especially in the liver and brain. This disease is mainly characterized by hepatic disorders and less frequently by neuro-psychiatric disturbances. This recessive disease is due to mutation in ATP7B, which codes for an ATPase involved in copper-transport across the plasma membrane. Molecular diagnosis of WD is positive in approximately 98% of cases. Also, in few cases, WD patients present a single deleterious mutation (heterozygous) or no mutation after sanger and NGS standard sequencing analysis of ATP7B. Therefore, in these problematic WD cases, we hypothesized that deleterious mutations reside in intronic regions of ATP7B. METHODS: Complete ATP7B gene was sequenced by Next Generation Sequencing including its promoter. RESULTS: Five unrelated families with Wilson disease shared the same novel, deep intronic NG_008806.1 (ATP7B_v001):c.2866-1521G>A variant in ATP7B. Analysis of RNA transcripts from primary fibroblasts of one patient confirmed the deleterious impact of the intronic variant on splicing and its likely pathologic effect in this compound heterozygote. CONCLUSION: This discovery of a novel intronic mutation in ATP7B has improved the molecular diagnosis of WD in the French patient cohort to greater than 98%. Thus, we recommend complete sequencing of ATP7B gene, including introns, as a molecular diagnostic approach in cases of clinically confirmed WD which lack pathogenic exon or promoter variants in one or both alleles.

Association of ABI3 and PLCG2 missense variants with disease risk and neuropathology in Lewy body disease and progressive supranuclear palsy.

Missense variants ABI3_rs616338-T and PLCG2_rs72824905-G were previously associated with elevated or reduced risk of Alzheimer's disease (AD), respectively. Despite reports of associations with other neurodegenerative diseases, there are few studies of these variants in purely neuropathologically diagnosed cohorts. Further, the effect of these mutations on neurodegenerative disease pathologies is unknown. In this study, we tested the effects of ABI3_rs616338-T and PLCG2_rs72824905-G on disease risk in autopsy cohorts comprised of 973 patients diagnosed neuropathologically with Lewy body disease (LBD-NP) and 1040 with progressive supranuclear palsy (PSP), compared to 3351 controls. LBD-NP patients were further categorized as high, intermediate and low likelihood of clinical dementia with Lewy bodies (DLB-CL) based on DLB Consortium criteria. We also tested for association with both Braak neurofibrillary tau tangle (nTotal = 2008, nPSP = 1037, nLBD-NP = 971) and Thal phase amyloid plaque scores (nTotal = 1786, nPSP = 1018, nLBD-NP = 768). Additionally, 841 PSP patients had quantitative tau neuropathology measures that were assessed for genetic associations. There was no statistically significant association with disease risk for either LBD-NP or PSP in our study. LBD intermediate category disease risk was significantly associated with ABI3_rs616338-T (OR = 2.65, 95% CI 1.46-4.83, p = 0.001). PLCG2_rs72824905-G was associated with lower Braak stage (ß = - 0.822, 95% CI - 1.439 to - 0.204, p = 0.009). This effect was more pronounced in the PSP (ß = - 0.995, 95% CI - 1.773 to - 0.218, p = 0.012) than LBD-NP patients (ß = - 0.292, 95% CI - 1.283 to 0.698, p = 0.563). PLCG2_rs72824905-G also showed association with reduced quantitative tau pathology for each lesion type and overall tau burden in PSP (ß = - 0.638, 95% CI - 1.139 to - 0.136, p = 0.013). These findings support a role for PLCG2_rs72824905-G in suppressing tau neuropathology. ABI3_rs616338-T may influence disease risk specifically in the LBD-NP intermediate category comprised of patients with diffuse neocortical or limbic LB, concurrently with moderate or high AD neuropathology, respectively. Our study provides a potential mechanism of action for the missense PLCG2 variant and suggests a differential disease risk effect for ABI3 in a distinct LBD-NP neuropathologic category.

In vitro tests and modelization of bicarbonate kinetics and mass transfers during online hemodiafiltration.

This paper proposes an in vitro hemodiafiltration (HDF) model in which the patient is represented by a 2 L bag of fresh heparinized bovine blood circulated by a 4008H monitor through a 0.6 m2 hemodialyzer to investigate kinetics of bicarbonate (HCO,) during online post-dilution HDF. Five tests were carried out, with three ultrafiltration rates, zero (HD test), 30 and 50 ml/min. Blood gases, pH, HCO3, hematocrit and electrolytes were measured with an ABL 77 (Radiometer) blood gas analyzer, and HCO3- was simultaneously measured with a biochemistry analyzer. The variation over time of plasma HCO3- concentrations was also calculated using mass conservation and the model of Legallais et al (JMS 168, 2000, 3-15). Agreement between theoretical and measured concentrations was good during the first 25 minutes of each test, corresponding to the time necessary to dialyze the blood. In hemodialysis (HD), there was an HCO3- mass transfer into blood through the membrane due to diffusion which vanished after 40 minutes, while in HDF tests, blood lost HCO3 due to ultrafiltration after 10 minutes. With reinjection, the net HCO3- mass flow rate to the "patient" decayed, from 1.8 mmol/min at t = 0 to zero at the end of the test (t = 60 min), and was higher in HD than in HDF "Patient" dialysance, taking into account reinjection, was positive in all tests, and decayed from about 110 ml/ min to 40 m/min at the end of dialysis. These data confirmed that online HDF automatically corrects acidosis without creating alkalosis when HCO3- dialysate concentration is around 30 mmol/L.

Various Genital and Reproductive Phenotypes in 46,XX/46,XY Chimeras.

Tetragametic chimeras are due to the fusion of 2 different zygotes after fertilization. When occurring between embryos of different chromosomal sex, the phenotype ranges from fertile individuals to infertile patients and even to patients with variations in sex development. Here, we report 3 new cases of XX/XY chimeras, one in a young boy carrying an abnormal gonad which turned out to be an ovary and 2 in phenotypically normal infertile men, one of whom had been diagnosed previously as a XX-SRY negative male. These cases highlight the importance of combining several cytogenetic and molecular techniques on different tissues for a proper diagnosis and an appropriate prognosis.

Body fluid volumes measurements by impedance: A review of bioimpedance spectroscopy (BIS) and bioimpedance analysis (BIA) methods.

This paper reviews various bioimpedance methods permitting to measure non-invasively, extracellular, intracellular and total body water (TBW) and compares BIA methods based on empirical equations of the wrist-ankle resistance or impedance at 50 kHz, height and weight with BIS methods which rely on an electrical model of tissues and resistances measured at zero and infinite frequencies. In order to compare these methods, impedance measurements were made with a multifrequency Xitron 4200 impedance meter on 57 healthy subjects which had undergone simultaneously a Dual X-ray absorptiometry examination (DXA), in order to estimate their TBW from their fat-free-mass. Extracellular (ECW) and TBW volumes were calculated for these subjects using the original BIS method and modifications of Matthie[Matthie JR. Second generation mixture theory equation for estimating intracellular water using bioimpedance spectroscopy. J Appl Physiol 2005;99:780-1], Jaffrin et al. [Jaffrin MY, Fenech M, Moreno MV, Kieffer R. Total body water measurement by a modification of the bioimpédance spectroscopy method. Med Bio Eng Comput 2006;44:873-82], Moissl et al. [Moissl UM, Wabel P, Chamney PW, Bosaeus I, Levin NW, et al. Body fluid volume determination via body composition spectroscopy in health and disease. Physiol Meas 2006;27:921-33] and their TBW resistivities were compared and discussed. ECW volumes were calculated by BIA methods of Sergi et al. [Sergi G, Bussolotto M, Perini P, Calliari I, et al. Accuracy of bioelectrical bioimpedance analysis for the assessment of extracellular space in healthy subjects and in fluid retention states. Ann Nutr Metab 1994;38(3):158-65] and Hannan et al. [Hannan WJ, Cowen SJ, Fearon KC, Plester CE, Falconer JS, Richardson RA. Evaluation of multi-frequency bio-impedance analysis for the assessment of extracellular and total body water in surgical patients. Clin Sci 1994;86:479-85] and TBW volumes by BIA methods of Kushner and Schoeller [Kushner RF, Schoeller DA. Estimation of total body water by bioelectrical impedance analysis. Am J Clin Nutr 1986;44(3):417-24], Lukaski et al. [Lukaski HC, Bolonchuk WW. Estimation of body fluid volumes using tetrapolar bioelectrical impedance measurements. Aviat Space Environ Med 1988;59:1163-9], Hannan et al. [Hannan WJ, Cowen SJ, Fearon KC, Plester CE, Falconer JS, Richardson RA. Evaluation of multi-frequency bio-impedance analysis for the assessment of extracellular and total body water in surgical patients. Clinical Science 1994;86:479-85], Deurenberg et al. [Deurenberg P, van der Koy K, Leenen R, Westrate JA, Seidell JC. Sex and age specific prediction formulas for estimating body composition from bioelectric impedance: a cross validation study. Int J Obesity 1991;15:17-25] These volumes were compared against those given by BIS method and, in the case of TBW, with those by DXA. For ECW, a good agreement was found between various BIS methods and that of Sergi while Hannan's values were higher. Both Matthie's and Moissl's methods gave mean TBW resistivities and volumes lower than those of Jaffrin's and DXA methods. Kushner et al. method gave values of TBW not significantly different from those of Jaffrin et al. and DXA, as Hannan's method in men, but Lukaski and Deurenberg methods led to an underestimation.

A comparison of bicarbonate kinetics and acid-base status in high flux hemodialysis and on-line post-dilution hemodiafiltration.

OBJECTIVES: To compare bicarbonate kinetics and acid base status in HD and HDF for the same patient; and to investigate the effect of patient physiologic parameters on these kinetics. METHODS: In order to monitor HCO3- kinetics during dialysis, acid-base parameters, pH, blood gases partial pressures, and HCO3- concentrations were recorded during 3 regular dialysis (HD) and 3 on-line post-dilution HDF sessions performed on 12 patients, using same dialysis fluid with a 38 mmol/l HCO3- concentration. HCO3- mass transfers through the hemodialyzers membranes and into the patients were continuously calculated during the sessions from HCO3- concentrations, together with HCO3-dialysance. The"apparent" HCO3-gain was calculated by integrating over time the instantaneous mass transfer from dialyzer and re-infusion fluid to the patient. A second method consisted in calculating the patient apparent bicarbonate space (ABS) and HCO3- mass (ABS times plasma concentration) at beginning and end of session. RESULTS: No significant differences were observed between acid base parameters at the end of HD and HDF sessions. In contrast to urea clearances, HCO3- dialysances decayed with time during sessions from 110 to 140 ml/min to about 40 ml/min after one hour. The net HCO3- gain was taken as the difference between final and initial HCO3-masses. This net gain was in average 63% of apparent gain in HD and 74% in HDF. CONCLUSIONS: Uremic acidosis was well corrected without risk of alkalosis. An unexpected result was the continuous decay of bicarbonate dialysance both in HD and HDF during runs.

Extracellular volume measurements using bioimpedance spectroscopy-Hanai method and wrist-ankle resistance at 50 kHz.

A method for extrapolating the extracellular water (ECW) resistance from wrist-ankle resistance at 50 kHz (R (50)) is proposed in this paper, in order to enable 50 kHz impedancemeters to use the BIS-Hanai equation for determination of ECW. Values of R (50) and the ECW resistance extrapolated at zero frequency R (e) were measured in a first group of 57 healthy volunteers, using a Xitron 4200 multifrequency impedancemeter and mean values (b) of the ratio R (50)/R (e) in men and women were used to determine individual values of R (e50), the ECW resistance extrapolated from R (50), which were substituted to R (e) in the BIS-Hanai equation. For validation, the method was compared against ECW measured with the Xitron (V (ex)) in a second group of 31 healthy volunteers, using values of b of first group. Values of R (e50) in this second group were found to be not significantly different from corresponding values of R (e) with p-values of Student test of 0.346 for men and 0.300 for women. ECW volumes (V (e50)) calculated from R (e50) were also found not significantly different from those of the Xitron with Student paired test p values of 0.277 in men and 0.393 in women. Our method gave a better agreement with V (ex) than two bioimpedance analysis methods from the literature, especially in women. It was also tested on a 50 kHz single frequency impedancemeter (BodyExplorer, Juwell Medical) on a third group of 21 subjects and gave ECW volumes not significantly from those of the Xitron with p = 0.531 for men and 0.096 for women.

Measurements of body composition in limbs and trunk using a eight contact electrodes impedancemeter.

Regional body composition measurements may be achieved in a single operation with impedancemeters equipped with four plantar and four hand electrodes. By measuring sequentially the resistances of five current lines connecting the hands and feet and solving a system of five linear equations, it is possible to calculate the resistances of each limb and the trunk. The impedancemeter used in this study was a prototype with four plantar electrodes and four additional contact electrodes for the hands. Its electronic hardware was identical to that of a Tefal commercial foot-to-foot impedancemeter (FFI). The Tefal FFI was used for measuring weight, whole body fat-free mass (FFM) and fat tissue mass (FM). Impedance and DXA measurements were taken sequentially on a 1st cohort of 170 healthy adults, aged from 19 to 75 years, to obtain equations relating appendicular FFM measured by DXA to their resistances, subject weight and height. For appendicular FM, correlations of the body FM measured by the FFI, age and BMI were used. Trunk FFM was obtained by subtracting appendicular FFM from FFM of trunk+limbs obtained by the same method as that for appendicular FFM. For an independent validation, these equations were tested on a 2nd cohort of 87 subjects (18-74 years) who underwent the same impedance and DXA protocol. Comparison of FFM and FM by impedance and by DXA in the limbs and the trunk using paired Student's t-tests, showed that they were not significantly different both in the 1st and validation cohorts. Mean FFM differences between impedance and DXA were -0.018+/-0.48 kg for right arms and -0.039+/-0.85kg for right legs of men in validation cohort. This work confirms that eight contact electrodes bioimpedance can measure appendicular and trunk FFM and FM in good agreement with DXA, at least in a healthy population.