Longitudinal changes in global structural brain connectivity and cognitive performance in former hospitalized COVID-19 survivors: an exploratory study.

BACKGROUND: Long-term sequelae of COVID-19 can result in reduced functionality of the central nervous system and substandard quality of life. Gaining insight into the recovery trajectory of admitted COVID-19 patients on their cognitive performance and global structural brain connectivity may allow a better understanding of the diseases' relevance. OBJECTIVES: To assess whole-brain structural connectivity in former non-intensive-care unit (ICU)- and ICU-admitted COVID-19 survivors over 2 months following hospital discharge and correlate structural connectivity measures to cognitive performance. METHODS: Participants underwent Magnetic Resonance Imaging brain scans and a cognitive test battery after hospital discharge to evaluate structural connectivity and cognitive performance. Multilevel models were constructed for each graph measure and cognitive test, assessing the groups' influence, time since discharge, and interactions. Linear regression models estimated whether the graph measurements affected cognitive measures and whether they differed between ICU and non-ICU patients. RESULTS: Six former ICU and six non-ICU patients completed the study. Across the various graph measures, the characteristic path length decreased over time (ß = 0.97, p = 0.006). We detected no group-level effects (ß = 1.07, p = 0.442) nor interaction effects (ß = 1.02, p = 0.220). Cognitive performance improved for both non-ICU and ICU COVID-19 survivors on four out of seven cognitive tests 2 months later (p < 0.05). CONCLUSION: Adverse effects of COVID-19 on brain functioning and structure abate over time. These results should be supported by future research including larger sample sizes, matched control groups of healthy non-infected individuals, and more extended follow-up periods.

Sequencing of the coding exons of the LRP1 and LDLR genes on individual DNA samples reveals novel mutations in both genes.

Five coding polymorphisms in de LRP1 gene, i.e. A217V, A775P, D2080N, D2632E and G4379S were discovered by sequencing its 89 exons in three test-groups of 22 healthy individuals, 29 Alzheimer patients and 18 individuals with different clinical and molecularly uncharacterized lipid metabolism problems. No genetic defect was evident in the LRP1 gene of any of the Alzheimer's disease (AD) patients, further excluding LRP1 as a major genetic problem in AD. Lipoprotein receptor related protein (LRP) A217V (exon 6) was clearly present in all groups as a polymorphism, while D2632E was observed only once in a healthy volunteer. On the other hand, LRP1 alleles A775P, D2080N, and G4379 were encountered only in patients with FH or with undefined problems of lipid metabolism. This finding forced one to also analyze the LDL receptor (LDLR) gene, for which a method was devised to sequence the entire region comprising LDLR exons 2-18. The resulting sequence contig of 33567 nucleotides yielded finally an exact physical map that corrects published and listed LDLR gene maps in many positions. In addition, next to known mutations in LDLR that cause FH, four novel LDLR defects were defined, i.e. del e7-10, exon 9 mutation N407T, a 20 bp insertion in exon 4, and a double mutation C292W/K290R in exon 6. No evidence for pathology connected to the LRP1 'mutations' was obtained by subsequent screening for the five LRP1 variants in larger groups of 110 FH patients and 118 patients with molecularly undefined, clinical problems of cholesterol and/or lipid metabolism. In three individuals with a mutant LDLR gene a variant LRP1 allele was also present, but without direct, obvious clinical compound effects, indicating that the variant LRP1 alleles must, for the present, be considered polymorphisms.

alpha2-macroglobulin- and murinoglobulin-1- deficient mice. A mouse model for acute pancreatitis.

Mice deficient in either or both mouse alpha2-macroglobulin (MAM) and murinoglobulin-1 (MUG1) were generated and proved phenotypically normal under standard conditions. Acute pancreatitis was induced with a diet deficient in choline and methionine, supplemented with ethionine. The mortality was less than 25% in wild-type mice, as opposed to at least 56% in knockout mice, and was highest (70%) in MAM-/- mice, with earliest onset at 2 days. Plasma amylase and lipase levels were increased, but pancreatic tissue appeared histologically variable in individual mice. The clinical symptoms were most severe in MAM-/- mice and, surprisingly, were not aggravated in the double knockout mice, suggesting that the lack of proteinase inhibition capacity was not the major problem. Therefore, we analyzed the expression of 21 different cytokines and polypeptide factors in the pancreas of all experimental groups of mice. Interleukin-1-receptor antagonist mRNA was consistently induced by the diet in the pancreas of MAM-/- mice, and transforming growth factor-beta, tumor necrosis factor-alpha, tumor necrosis factor-beta, beta-lymphotoxin, and interferon-gamma mRNA levels were also increased. The data demonstrate the important role of alpha2-macroglobulin (A2M) in acute pancreatitis as both a proteinase inhibitor and a cytokine carrier. Mice deficient in MAM and/or MUG thus offer new experimental models for defining in vivo the role of the macroglobulins in pancreatitis and in other normal and pathological processes.

Association between coding variability in the LRP gene and the risk of late-onset Alzheimer's disease.

We have sequenced the entire (89 exons) open reading frame of the LRP gene in 12 cases of Alzheimer's disease (AD) from Northern France. We have found no novel changes but confirm the occurrence of a polymorphism in exon 6 of the gene (A216V). This polymorphism is rare (2.8% of controls) and is in linkage equilibrium with previously reported polymorphisms. The V216 allele is negatively associated with the disease in a large case-controlled series. These data suggest that the LRP receptor may be involved in the pathobiology of AD, but the association that we report here cannot explain the previously reported genetic data implicating the LRP gene in AD. If the LRP gene is a major site of genetic variability leading to AD, there must be other biologically relevant variability in promoter or other regulatory elements of this large gene.

Analysis of the human LRPAP1 gene coding for the lipoprotein receptor-associated protein: identification of 22 polymorphisms and one mutation.

The lipoprotein receptor-associated protein (RAP) is considered a chaperone protein for the lipoprotein receptor-related protein (LRP) and for the other members of the LDL receptor family. Genetic analysis is anticipated to help in delineating groups or individuals with potential defects or problems in this regard. A combined amplification/sequencing strategy was developed to analyze the human LRPAP1 gene for polymorphisms and mutations. The LRPAP1 gene was amplified from genomic DNA in four long-range PCR amplicons, 2.4 to 7.6 kb in size. Three amplicons were finally used as templates with 14 sequencing primers to obtain the sequence of the eight exons and large portions of adjacent introns from individual DNA. This strategy, applied to sequence the LRPAP1 gene of 14 unrelated, normal individuals revealed, in total, 23 distinct mutations and polymorphisms, mostly intronic substitutions and deletions. In this small group 1 expressed mutation was encountered on one allele in 2 unrelated individuals: a G to A transition results in the replacement of valine by methionine in exon 7 at position 311 of the human RAP precursor protein.

Strategy to sequence the 89 exons of the human LRP1 gene coding for the lipoprotein receptor related protein: identification of one expressed mutation among 48 polymorphisms.

The human lipoprotein receptor related protein (LRP) binds and internalizes a diverse set of ligands, making LRP the most multifunctional endocytic receptor known. This is possible due to the large size, i.e., 600 kDa, of the receptor protein containing three clusters of putative ligand binding domains, each structurally comparable to the classical LDL receptor. Based on previous structural analysis of the human LRP1 gene (Van Leuven et al., 1994, Genomics, 24: 78-89), a strategy was developed to sequence the 89 exons of the LRP1 gene, including partial intron sequences. The gene was amplified from individual genomic DNA by long-range PCR, in 14 amplicons sized between 0.4 and 11 kb that were used as templates for 110 sequencing primers. In total, 48 mutations and intronic polymorphisms were identified. Two previously reported polymorphisms, i.e., in the promoter region and in exon 3, were precisely defined by sequencing. The first expressed mutation, i.e., an alanine to valine transition at position 217 of the LRP precursor protein, was detected on one allele in 2 of 33 individuals. Although the strategy is still subject to refinement, this approach is reported to allow others to analyze genetic differences in the human LRP1 gene, with particular reference to the recently reported association with late-onset Alzheimer disease.

Molecular cloning and characterization of the human interleukin-11 receptor alpha-chain gene, IL11RA, located on chromosome 9p13.

The human gene coding for the interleukin-11 receptor (IL11RA) was cloned and its structure analyzed. The gene is composed of 13 exons comprising nearly 10 kb of DNA that was completely sequenced. The intron-exon boundaries were determined based on the mouse Etl2 and interleukin-11 receptor cDNAs that were recently cloned. The protein sequence predicted by the human gene was over 83% identical with its murine counterpart, with very strict conservation of functionally important domains and signatures. Fluorescence in situ hybridization showed the gene to be located on human chromosome 9p13, syntenic with the mouse etl2 gene on chromosome 4. The coding exons of the Interleukin-11 gene were sequenced in a patient with the cartilage-hair hypoplasia syndrome, which has been linked to a gene on chromosome 9, but no functional mutations were detected.

Structure of the gene (LRP1) coding for the human alpha 2-macroglobulin receptor lipoprotein receptor-related protein.

The alpha 2-macroglobulin receptor or lipoprotein receptor-related protein (A2MR/LRP) is an amazingly large and multifunctional receptor. The active receptor protein is derived from a 600-kDa precursor, encoded by a 15-kb mRNA, cloned and sequenced in human, mouse, and chicken. We report here the cloning of the entire human gene (LRP1) coding for A2MR/LRP. The gene covered about 92 kb and a total of 89 exons were identified, varying in size from 65 bases (exon 86) to 925 bases (exon 89). The introns varied from 82 bases (intron 53) to about 8 kb (intron 6). In the introns, 3 complete and 4 partial Alu sequences were identified. In intron 44 a complex repetitive sequence posed a cloning problem since it was not retrieved from any genomic library screened. Interexon PCR from exon 43 to 45 yielded a fragment of 2.5 kb. Attempts to subclone this fragment yielded inserts ranging between 0.8 and 1.6 kb. Sequencing of 3 subclones with different-size inserts revealed a complex repetitive element with a different size in each subclone. In the mouse LRP gene this intron was much smaller, and no repetitive sequence was observed. In 18 unrelated individuals no difference in size was observed when analyzed by interexon PCR.

Genomic cloning of the mouse LDL receptor related protein/alpha 2-macroglobulin receptor gene.

The LDL receptor-related protein (LRP) or alpha 2-macroglobulin receptor (A2mr) is encoded by a 15-kb mRNA in mouse and human. Probes encompassing different regions of the mouse cDNA were used to isolate clones from a cosmid library of mouse strain 129. Four overlapping cosmids were used for restriction mapping and Southern blot analysis. This map and hybridization data obtained with oligonucleotide probes from the 5' and 3' ends of the Lrp cDNA demonstrated that the mouse gene is approximately 85 kb in size. The Lrp promoter region was sequenced and reveals strong evolutionary conservation of putative regulatory elements between mouse and human. The present study will facilitate detailed elucidation of the function of LRP in vivo.

Molecular cloning of the mouse gene coding for alpha 2-macroglobulin and targeting of the gene in embryonic stem cells.

We have cloned the mouse gene coding for alpha 2-macroglobulin in overlapping lambda clones and have analyzed its structure. The gene contains 36 exons, coding for the 4.8-kb cDNA that we cloned previously. Including putative control elements in the 5' flanking region, the gene covers about 45 kb. A region of 3.8 kb, stretching from 835 bases upstream of the cDNA start site to exon 4, including all intervening sequences, was sequenced completely. The analysis demonstrated that the putative promoter region of the mouse A2M gene differed considerably from the known promoter sequences of the human A2M gene and of the rat acute-phase A2M gene. Comparison of the exon-intron structure of all known genes of the A2M family confirmed that the rat acute phase A2M gene is more closely related to the human gene than to the mouse A2M gene. To generate mice with the A2M gene inactivated, an insertion type of construct containing 7.5 kb of genomic DNA of the mouse strain 129/J, encompassing exons 16 to 19, was synthesized. A hygromycin marker gene was embedded in intron 17. After electroporation, 198 hygromycin-resistant ES cell lines were isolated and analyzed by Southern blotting. Five ES cell lines were obtained with one allele of the mouse A2M gene targeted by this insertion construct, demonstrating that the position and the characteristics of the vector served the intended goal.

Molecular cloning and sequencing of the murine alpha-2-macroglobulin receptor cDNA.

We have molecularly cloned and sequenced the mouse alpha-2-macroglobulin receptor cDNA. The cDNA contained 14849 bases with one large open reading frame of 4545 codons which is one more than in the corresponding human cDNA. Comparison of the predicted mouse and human receptor proteins revealed the very conserved nature of this receptor with an overall amino acid identity of more than 97%. A dramatic example of this is the presence of 331 cysteine residues predicted in the mouse protein, of which 327 are positionally conserved relative to human.