Endocrine and behavioural features of Lowe syndrome and their potential molecular mechanisms.

BACKGROUND: Lowe syndrome (LS) is an X linked disease caused by pathogenic variants in the OCRL gene that impacts approximately 1 in 500 000 children. Classic features include congenital cataract, cognitive/behavioural impairment and renal tubulopathy. METHODS: This study is a retrospective review of clinical features reported by family based survey conducted by Lowe Syndrome Association. Frequency of non-ocular clinical feature(s) of LS and their age of onset was summarised. An LS-specific therapy effectiveness scale was used to assess the response to the administered treatment. Expression of OCRL and relevant neuropeptides was measured in postmortem human brain by qPCR. Gene expression in the mouse brain was determined by reanalysis of publicly available bulk and single cell RNA sequencing. RESULTS: A total of 137 individuals (1 female, 89.1% white, median age 14 years (range 0.8-56)) were included in the study. Short stature (height <3rd percentile) was noted in 81% (n=111) individuals, and 15% (n=20) received growth hormone therapy. Undescended testis was reported in 47% (n=64), and median age of onset of puberty was 15 years. Additional features were dental problems (n=77, 56%), bone fractures (n=63, 46%), hypophosphataemia (n=60, 44%), developmental delay and behavioural issues. OCRL is expressed in human and mouse hypothalami, and in hypothalamic cell clusters expressing Ghrh, Sst, Oxt, Pomc and pituitary cells expressing Gh and Prl. CONCLUSIONS: There is a wide spectrum of the clinical phenotype of LS. Some of the features may be partly driven by the loss of function of OCRL in the hypothalamus and the pituitary.

Simple, Fast, and Efficient Method for Derivation of Dermal Fibroblasts From Skin Biopsies.

Primary fibroblasts are a precious resource in the field of translational regenerative medicine. Dermal fibroblasts derived from human subject biopsies are being used as donor tissues for the derivation of patient-specific iPSC lines, which in turn are used for disease modeling, drug screening, tissue engineering, and cell transplantation. We developed a fast and simple protocol to grow dermal fibroblasts from skin biopsies. Using this protocol, we simply and firmly fix the biopsy piece on the surface of a tissue culture-treated plate and allow the fibroblasts to grow. This novel method eliminates any need for enzymatic digestion or mechanical dissociation of the biopsy piece. By using this newly developed protocol, we have successfully established around 100 fibroblast lines characterized by the expression of specific markers [Serpin H1 (Hsp-47), F-actin, and Vimentin]. Finally, we have used many of these fibroblast lines as donor tissues to successfully derive iPSC lines. We have developed a method that is simple, fast, convenient, efficient, and gentle on the cells to derive dermal fibroblasts from human skin biopsies. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Skin biopsy collection and fibroblast derivation Support Protocol 1: Culturing, freezing, and thawing dermal fibroblasts derived from a skin biopsy Support Protocol 2: Characterization of dermal fibroblasts by immunocytochemistry.

Derivation and characterization of the induced pluripotent stem cell line CUIMCi004-A from a patient with a novel frameshift variant in exon 18a of OCRL.

OCRL encodes for an inositol polyphosphate 5-phosphatase, located in the trans-Golgi network, endosomes, endocytic clathrin-coated pits, primary cilia. Mutations in OCRL causes Lowe syndrome (LS), a rare and complex disorder characterized by congenital cataracts, renal tubular dysfunction, and mental retardation. Here we generated an induced pluripotent stem cell (iPSC) line from Peripheral Blood Mononuclear Cell (PBMCs) of a 5-year-old boy with severe obesity carrying a novel pathogenic variant in the brain-expressed isoform of OCRL. The Sendai virus approach was used for reprogramming. The iPSC line CUIMCi004-A may serve as a useful resource to further investigate the tissue-specific function of OCRL.

Efficient Cas9-based Genome Editing Using CRISPR Analysis Webtools in Severe Early-onset-obesity Patient-derived iPSCs.

The CRISPR system is an adaptive defense mechanism used by bacteria and archaea against viruses and plasmids. The discovery of the CRISPR-associated protein Cas9 and its RNA-guided cleavage mechanism marked the beginning of a new era in genomic engineering by enabling the editing of a target region in the genome. Gene-edited cells or mice can be used as models for understanding human diseases. Given its high impact in functional genomic experiments on different model systems, several CRISPR/Cas9 protocols have been generated in the past years. The technique uses a straightforward "cut and stitch" mechanism, but requires an accurate step-by-step design. One of the key points is the use of an efficient programmable guide RNA to increase the rate of success in obtaining gene-specific edited clones. Here, we describe an efficient editing protocol using a ribonucleotide protein (RNP) complex for homology-directed repair (HDR)-based correction of a point mutation in an induced pluripotent stem cell (iPSC) line generated from a 14-year-old patient with severe early-onset obesity carrying a de novo variant of ARNT2. The resulting isogenic iPSC line, named CUIMCi003-A-1, has a normal karyotype, expresses stemness markers, and can be differentiated into progenies from all three germ layers. We provide a detailed workflow for designing a single guide RNA and donor DNA, and for isolating clonal human iPSCs edited with the desired modification. This article also focuses on parameters to consider when selecting reagents for CRISPR/Cas9 gene editing after testing their efficiency with in silico tools. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Design of sgRNAs and PCR primers Basic Protocol 2: Testing the efficiency of sgRNAs Basic Protocol 3: Design of template or donor DNA Basic Protocol 4: Targeted gene editing Basic Protocol 5: Selection of positive clones Basic Protocol 6: Freezing, thawing, and expansion of cells Basic Protocol 7: Characterization of edited cell lines.

Generation of the iPSC line CUIMCi003-A derived from a patient with severe early onset obesity.

Aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) is a basic helix-loop-helix (bHLH/PAS) transcription factor involved in the development of paraventricular nucleus of the hypothalamus (PVH) through the heterodimerization with Single-minded 1 (SIM1) (Michaud et al., 2000). Using a Sendai virus-based approach, the four reprogramming factors OCT3/4, SOX2, KLF4 and C-MYC were delivered into Peripheral Blood Mononuclear Cell (PBMCs) from a 14-year-old girl with early onset obesity carrying a de novo variant (p.P130A) in ARNT2. The resulting iPSC line CUIMCi003-A had a normal karyotype, showed pluripotency and three germ layer differentiation capacity in vitro and was heterozygous for the de novo ARNT2 variant.

Intracellular FMRpolyG-Hsp70 complex in fibroblast cells from a patient affected by fragile X tremor ataxia syndrome.

BACKGROUND: Fragile X-associated tremor/ataxia syndrome is a late-onset neurodegenerative disorder that affects about 40% of carriers of CGG-repeat expansions in the premutation range within the fragile X gene (FMR1). Main clinical features include intention tremor, cerebellar ataxia, and parkinsonism. Recently, great emphasis on the deposition of soluble aggregates produced by a RAN translation process, as main pathogenic mechanism, has been given. These aggregates contain a small protein with a polyglycine stretch on the aminoterminal end named FMRpolyG and, so far, have been isolated and characterized in drosophila and mouse models, in post mortem brain of fragile X-associated tremor/ataxia syndrome patients, in fibroblasts of fragile primary ovarian insufficiency patients, but never in fibroblasts from a fragile X-associated tremor/ataxia living patients. In adult carriers the syndrome is frequently misdiagnosed due to the lack of specific markers. METHODS: We standardized immunocytochemistry, immunoprecipitation and western blot procedures to study and biochemically characterize the FMRpolyG protein in fibroblasts from human skin biopsy. RESULTS: We demonstrate for the first time, in fibroblasts from a patient affected by Fragile X-associated tremor/ataxia syndrome, the presence ex vivo of inclusions consisting of FMRpolyG- Hsp70 soluble aggregates. CONCLUSION: These observations can pave the way to develop a cellular model for studying ex vivo and in vitro the mechanisms involved in the production of FMRpolyG aggregates, their toxicity, and the role of the FMRpolyG-Hsp70 interaction in the pathogenesis of fragile X-associated tremor/ataxia syndrome.

Mutational analysis of TARDBP gene in patients affected by Parkinson's disease from Calabria.

OBJECTIVE: Neurodegenerative diseases are often characterized by the presence of intracellular or extracellular protein aggregates in the central nervous system. Mutations of TARDBP gene have been shown to cause Amyotrophic Lateral Sclerosis and have been reported to present with clinical heterogeneity including parkinsonism. TDP-43 pathology has been observed across a spectrum of neurodegenerative disorders, including Alzheimer's and Parkinson's disease. METHODS: In this study we screened 100 sporadic and 165 familial PD patients and control series (450) for the TARDBP gene. All cases and controls included in this study were born and living in Calabria. RESULTS: The p.N267S heterozygous mutation was detected in one sporadic PD patient. The p.N267S mutation was not found in a control population of 450 healthy individuals and in our 165 familial PD. CONCLUSIONS: Sequencing of the TARDBP gene in our patient cohort identified one sporadic PD carrying the p.N267S mutation. This is the first analysis of TARDBP mutation in sporadic PD patient from South Italy.

DNAJC13 mutation screening in patients with Parkinson's disease from South Italy.

BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disorder, and the most common neurodegenerative form of parkinsonism. Recently, a pathogenic mutation (p.N855S) in DNAJC13 was linked to autosomal dominant Lewy body PD in a Dutch-German-Russian Mennonite multi-incident kindred, and was found in five additional patients. In this study, we performed a comprehensive screening of the DNAJC13 gene in familial PD and sporadic PD to assess the frequency of known and novel rare nonsynonymous variants. METHODS: We screened 563 sporadic and 168 familial PD patients and a control series (n = 1000) for the coding region of DNAJC13. RESULTS: Our sequencing analysis identified two carriers of the c.2708G > A (p.R903K) variant in exon 24 of DNAJC13. One of these carriers is a familial PD. CONCLUSION: The p. R903K variant was not found in 1000 healthy controls and it is localized in a functional domain of the DNAJC13 protein. Further studies are necessary to evaluate the role of DNAJC13 variants in PD.

Analysis of CHCHD2 gene in familial Parkinson's disease from Calabria.

Parkinson's disease (PD) is the most common form of degenerative Parkinsonism with a prevalence of 1% of those older than 65 years. PD is characterized by the combination of slowness of movement (bradykinesia), muscular rigidity, resting tremor, and postural instability. Recently, using a genome-wide linkage analysis and exome sequencing, a group identified a candidate gene (CHCHD2) in a large Japanese family with autosomal dominant Parkinson's disease. The aim of this study was to evaluate the presence of CHCHD2 mutations in a cohort of 165 familial patients with clinically diagnosed PD and 200 control subjects from South Italy. No mutations in CHCHD2 were found in our 165 PD patients. This result suggests that CHCHD2 mutations might not be the common cause of PD in South Italy.

A new PLA2G6 mutation in a family with infantile neuroaxonal dystrophy.

Phospholipase A2-associated neurodegeneration (PLAN), a syndrome of Neurodegeneration with Brain Iron Accumulation (NBIA), is an autosomal recessive disorder caused by mutations in PLA2G6 gene. This gene encodes a calcium-independent group VI phospholipase A2 (iPLA-VI) critical in cell membrane homeostasis. PLAN syndrome encompasses a group of phenotypes with a different age of onset: classic infantile neuroaxonal dystrophy (INAD), atypical neuroaxonal dystrophy of childhood-onset (atypical NAD) and adult-onset PLA2G6-related dystonia-parkinsonism (PARK14). INAD is a severe progressive psychomotor disorder characterized by the presence of axonal spheroids throughout the central and peripheral nervous system. Here we report clinical, genetic and histopathological findings of an INAD consanguineous-family from Senegal. Sanger sequencing analysis revealed a new homozygous PLA2G6-mutation in the proband (c.1483C>T) and the co-segregation of the mutation in this family. Electron microscopy on skin biopsy showed degenerated axons confirming the phenotype. This study contributes to enrich the landscape of PLA2G6-associated INAD mutations and enforce the genotype-phenotype correlation.

C19orf12 gene mutations in patients with neurodegeneration with brain iron accumulation.

A novel subtype of Neurodegeneration with Brain Iron Accumulation (NBIA) recently has been described: mitochondrial membrane protein-associated neurodegeneration (MPAN), caused by mutations of c19orf12 gene. We present phenotypic data and results of screening of C19orf12 in five unrelated NBIA families. Our data led to identify novel pathogenic mutations in C19orf12.

A new SLC20A2 mutation identified in southern Italy family with primary familial brain calcification.

BACKGROUND: Primary familial brain calcification (PFBC) is a rare neurodegenerative disease characterized by bilateral calcifications mostly located in the basal ganglia and in the thalami, cerebellum and subcortical white matter. Clinical manifestations of this disease include a large spectrum of movement disorders and neuropsychiatric disturbances. PFBC is genetically heterogeneous and typically transmitted in an autosomal dominant fashion. Three causative genes have been reported: SLC20A2, PDGFRB and PDGFB. OBJECTIVE: We screened three PFBC Italian families for mutations in the SLC20A2, PDGFRB and PDGFB genes. METHODS: Phenotypic data were obtained by neurologic examination, CT scan and magnetic resonance imaging. Mutation screening of SLC20A2, PDGFRB and PDGFB was performed by sequencing. RESULTS: We identified a new heterozygous deletion c.21_21delG (p.L7Ffs*10) in SLC20A2 gene in one of these families. No mutations were detected in the other two families. CONCLUSIONS: Our data confirm that mutations in SLC20A2 are a major cause of familial idiopathic basal ganglia calcification.

Role of G-protein coupled receptor kinase 5 gene in cognitive impairment in Parkinson's disease.

The objective of our study was to investigate the association of two single nucleotide polymorphisms (SNPs) with genetic risk of dementia. In 212 patients with Parkinson's disease (PD), we investigated two polymorphisms within the G-protein coupled receptor kinase 5 (GRK5) gene (rs2420616, rs4752293) to determine a possible risk factor for dementia. We identified two alleles most significantly present in PD patients with dementia: G and T alleles. We also identified risk haplotypes: GC, and AT. We demonstrated that the SNPs and the related haplotypes could play a central role in predisposing PD patients to cognitive impairment.