Exploring metabolic alterations in PYCR2 deficiency: Unveiling pathways and clinical presentations of hypomyelinating leukodystrophy 10.

Proline-5-carboxylate reductase 2, encoded by PYCR2 gene, is an enzyme that catalyzes the last step of proline synthesis from pyrroline-5-carboxylate synthetase to proline. PYCR2 gene defect causes hypomyelinating leukodystrophy 10. Up until now, to our knowledge around 38 patients with PYCR2 defect have been reported. Herein, we describe clinical, neuroradiological, biochemical findings, and metabolomic profiling of three new genetically related cases of PYCR2 defects from a large family. Cerebrospinal fluid (CSF) amino acid levels were measured and untargeted metabolomic profiling of plasma and CSF were conducted and evaluated together with the clinical findings in the patients. While plasma and CSF proline levels were found to be totally normal, untargeted metabolomic profiling revealed mild increases of glutamate, alpha-ketoglutarate, and l-glutamate semialdehyde and marked increases of inosine and xanthine. Our findings and all the previous reports suggest that proline auxotrophy is not the central disease mechanism. Untargeted metabolomics point to mild changes in proline pathway and also in purine/pyrimidine pathway.

Turning Trash Into Treasure: A Simple Protocol for Human Mesenchymal Stromal Cell Isolation From Used Bone Marrow Collection Kits.

The therapeutic potential of mesenchymal stromal cells (MSCs) has been extensively investigated in both preclinical and clinical settings. Recent years have witnessed the emergence of numerous isolation protocols and culture techniques, ranging from the selection of subpopulations to preserve stemness to preconditioning strategies aimed at enhancing therapeutic efficacy, tailored to the specific tissue source. In this protocol, we present a straightforward and cost-effective method for isolating human MSCs (hMSCs) from discarded bone marrow collection kits (comprising bag and filter systems) originally intended for removing impurities and unwanted cellular debris from the collected bone marrow aspirate, ensuring the purity of the stem cell population during stem cell transplantation. Utilizing basic laboratory equipment, we demonstrate the isolation of hMSCs, highlighting the expression of specific surface antigens, and multilineage differentiation into adipogenic, osteogenic, and chondrogenic lineages in vitro. This sustainable and resource-efficient approach not only contributes to reducing medical waste but also holds promise for advancing regenerative medicine applications. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Isolation of human mesenchymal stromal cells from bone marrow collection kits Basic Protocol 2: Culture of human mesenchymal stromal cells Basic Protocol 3: Characterization of human mesenchymal stromal cells with flow cytometry analysis Basic Protocol 4: Characterization of human mesenchymal stromal cells with multilineage differentiation under in vitro conditions.

The bridge between cell survival and cell death: reactive oxygen species-mediated cellular stress.

As a requirement of aerobic metabolism, regulation of redox homeostasis is indispensable for the continuity of living homeostasis and life. Since the stability of the redox state is necessary for the maintenance of the biological functions of the cells, the balance between the pro-oxidants, especially ROS and the antioxidant capacity is kept in balance in the cells through antioxidant defense systems. The pleiotropic transcription factor, Nrf2, is the master regulator of the antioxidant defense system. Disruption of redox homeostasis leads to oxidative and reductive stress, bringing about multiple pathophysiological conditions. Oxidative stress characterized by high ROS levels causes oxidative damage to biomolecules and cell death, while reductive stress characterized by low ROS levels disrupt physiological cell functions. The fact that ROS, which were initially attributed as harmful products of aerobic metabolism, at the same time function as signal molecules at non-toxic levels and play a role in the adaptive response called mithormesis points out that ROS have a dose-dependent effect on cell fate determination. See also Figure 1(Fig. 1).

Investigation of androgen receptor gene CAG repeat length polymorphism in pubertal gynecomastia.

OBJECTIVES: Androgen receptor gene CAG repeat, AR (CAG)n, polymorphism is thought to have an effect on male reproductive functions and a relationship between long AR (CAG)n and decreased androgenic activity has been shown. Therefore, we hypothesized that in adolescents with long AR CAG repeat the prevalence of pubertal gynecomastia (PG) will be higher and we aimed to investigate the association between AR (CAG)n polymorphism and PG in Turkish adolescents. METHODS: Adolescents with PG between 11 and 19 years of age were enrolled as the study group and healthy individuals without a history of PG, who were at least 14 years of age and Tanner 4 or 5 were enrolled as the control group. The AR (CAG)n length was detected by direct DNA sequencing analysis and reproductive hormones were measured by standardized analyses. RESULTS: The mean AR (CAG)n was 22.3 ± 2.6 (mean ± SD) in the PG group (n=101) and 21.9 ± 3.1 (mean ± SD) in the control group (n=88) (p=0.276). The adolescents with short AR (CAG)n had lower body mass index standard deviation scores (BMI SDS) compared to the adolescents with intermediate and long repeat numbers (p=0.029). CONCLUSIONS: The results of this study showed a lack of direct association between AR (CAG)n and PG. However, the significant relationship between the AR (CAG)n quartiles and BMI SDS suggests that long AR (CAG)n might cause PG indirectly. Further studies are needed to better clarify this relationship.

Clinical and molecular characteristics of carnitineacylcarnitine translocase deficiency with c.270delC and a novel c.408C>A variant.

BACKGROUND: Carnitine-acylcarnitine translocase deficiency (CACTD) is a rare, autosomal recessive, and highly lethal fatty acid oxidation (FAO) disorder caused by defective acylcarnitine transport across the mitochondrial membrane. CACTD is characterized by severe episodes of hypoglycemia and hyperammonemia, seizures, cardiomyopathy, liver dysfunction, severe neurological damage, and muscle weakness. Herein, we described the clinical features, biochemical, and molecular findings of three patients with CACTD, presented with poor feeding, hypoglycemia, liver dysfunctions, and hyperammonemia, but died despite intensive treatment. CASES: All cases had similar signs and symptoms like poor feeding and respiratory failure associated with liver dysfunction. Urinary organic acid profiles in the presence of hypoglycemia and hyperammonemia led us to the possible diagnosis of one of fatty acid ß-oxidation defects. Results of the molecular analyses were compatible with CACTD. In addition to known mutation (c.270delC;p.Phe91Leufs*38) we detected a novel one (c.408C > A;p.Cys136*). CONCLUSIONS: All three cases died despite a very intensive therapy. Based on our experience with these three cases, it can be said that CACTD has a relatively poor prognosis, molecular studies are of most importance in suspected cases for the final diagnosis and such studies might be of help while giving genetic counselling and guidance to parents for future pregnancies.

Homozygous missense VPS16 variant is associated with a novel disease, resembling mucopolysaccharidosis-plus syndrome in two siblings.

Disorders of intracellular trafficking are a group of inherited disorders, which often display multisystem phenotypes. Vacuolar protein sorting (VPS) subunit C, composed of VPS11, VPS18, VPS16, and VPS33A proteins, is involved in tethering of endosomes, lysosomes, and autophagosomes. Our group and others have previously described patients with a specific homozygous missense VPS33A variant, exhibiting a storage disease phenotype resembling mucopolysaccharidosis (MPS), termed "MPS-plus syndrome." Here, we report two siblings from a consanguineous Turkish-Arabic family, who have overlapping features of MPS and intracellular trafficking disorders, including short stature, coarse facies, developmental delay, peripheral neuropathy, splenomegaly, spondylar dysplasia, congenital neutropenia, and high-normal glycosaminoglycan excretion. Whole exome sequencing and familial segregation analyses led to the homozygous NM_022575.3:c.540G>T; p.Trp180Cys variant in VPS16 in both siblings. Multiple bioinformatic methods supported the pathogenicity of this variant. Different monoallelic null VPS16 variants and a homozygous missense VPS16 variant had been previously associated with dystonia. A biallelic intronic, probably splice-altering variant in VPS16, causing an MPS-plus syndrome-like disease has been very recently reported in two individuals. The siblings presented herein display no dystonia, but have features of a multisystem storage disorder, representing a novel MPS-plus syndrome-like disease, associated for the first time with VPS16 missense variants.

Characterization of human bone marrow niches with metabolome and transcriptome profiling.

Bone marrow (BM) niches are special microenvironments that work in harmony with each other for the regulation and maintenance of hematopoiesis. Niche investigations have thus far been limited to various model organisms and animal studies; therefore, little is known about different niches in healthy humans. In this study, a special harvesting method for the collection of BM from two different anatomical regions in the iliac crest of humans was used to investigate the presence of different niches in BM. Additionally, metabolomic and transcriptomic profiles were compiled using comparative 'omics' technologies, and the main cellular pathways and corresponding transcripts and metabolites were identified. As a result, we found that the energy metabolism between the regions was different. This study provides basic broad data for regenerative medicine in terms of the design of the appropriate microenvironment for in vitro hematopoietic niche modeling, and identifies the normal reference values that can be compared in hematological disease.

Detection of allele frequencies of common c. 511C>T and c.625G>A variants in the ACADS gene in the Turkish population.

Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is a rare inborn error of mitochondrial fatty acid oxidation and protein misfolding disorder. Our aim was to detect the number of Turkish patients diagnosed with SCADD in the literature and to determine the allele frequencies of two common variants (c.511C > T and c.625G > A) in the Turkish population. Five Turkish patients with SCADD were reported in the literature from four unrelated families. We also investigated allele frequencies of common variants of c.511C > T and c.625G > A, which confer susceptibility to SCADD, which were found to be 1.7% and 20.2%, respectively. Both of these susceptibility variants were found to be high in the Turkish population as they are worldwide.

Autoinflammation in addition to combined immunodeficiency: SLC29A3 gene defect.

INTRODUCTION: H Syndrome is an autosomal recessive (AR) disease caused by defects in SLCA29A3 gene. This gene encodes the equilibrative nucleoside transporter, the protein which is highly expressed in spleen, lymph node and bone marrow. Autoinflammation and autoimmunity accompanies H Syndrome (HS). AIM: The aim was to further elucidate the mechanisms of disease by molecular studies in a patient with SLC29A3 gene defect. PATIENT AND METHODS: Mitochondrial dysfunction, lysosomal integrity, cytokine response in response to stimulation with different pattern recognition receptor ligands, and circulating cell-free mitochondrial-DNA(ccf-mtDNA) level in plasma were analyzed compared to controls to understand the cellular triggers of autoinflammation. RNA sequencing (RS) analyses were also performed in monocytes before/after culture with lipopolysaccharide. RESULTS: Patient had progressive destructive arthropathy in addition to clinical findings due to combined immunodeficiency. Pure red cell aplasia (PRCA), vitiligo, diabetes, multiple autoantibody positivity, lymphopenia, increased acute phase reactants were present. Recent thymic emigrants (RTE), naïve T cells were decreased, effector memory CD4 + T cells, nonclassical inflammatory monocytes were increased. Patient's peripheral blood mononuclear cells secreted more IL-1ß and IL-6, showed lysosomal disruption and significant mitochondrial dysfunction compared to healthy controls. Plasma ccf-mtDNA level was significantly elevated compared to age-matched controls (p < 0.05). RNA sequencing studies revealed decreased expression of NLR Family Caspase Recrument-Domain Containing 4(NLRC4), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4(PFKFB4), serine dehydratase(SDS), heparan sulfate(Glucosamine) 3-O-sulfotransferase 1(HS3ST1), neutral cholesterol ester hydrolase 1 (NCEH1), and interleukin-8 (IL-8) in patient's monocytes compared to controls. Longstanding PRCA, which is possibly autoimmune, resolved after initiating monthly intravenous immunoglobulins (IVIG) and low dose steroids to the patient. CONCLUSION: Although autoinflammation and autoimmunity are reported in HS, by functional analyses we here show in the present patient that over-active inflammasome pathway in HS might be related with mitochondrial and lysosomal dysfunction. Increased plasma ccf-mtDNA may be used as a biomarker of inflammasomopathy in HS. HS should be included in the classification of primary immunodeficiency diseases.

Presentation of 14 alkaptonuria patients from Turkey.

Background Alkaptonuria (OMIM: 203500) is an inborn error of metabolism due to homogentisate 1,2-dioxygenase homogentisic acid 1,2 dioxygenase (HGD) enzyme deficiency. Due to the enzyme deficiency, homogentisic acid cannot be converted to maleylacetoacetate and it accumulates in body fluids. Increased homogentisic acid is converted to benzoquinones, the resulting benzoquinones are converted to melanin-like pigments, and these pigments are deposited in collagen - this process is called ochronosis. In patients with alkaptonuria, the urine is darkened, which is misinterpreted as hematuria, the incidences of renal stones, arthritis and cardiac valve calcification are increased, and spontaneous tendon ruptures, prostatitis and prostate stones can be encountered. The present study aimed to evaluate the HGD gene mutations in 14 patients with alkaptonuria. Methods Fourteen patients diagnosed with alkaptonuria and followed up from 1990 to 2014 were retrospectively evaluated. Their demographic, clinical and treatment-related data were retrieved from hospital files. For mutation analysis, genomic DNAs of the patients were isolated from their peripheral blood samples. Variations in the HGD gene were scanned on the HGD-mutation database (http://hgddatabase.cvtisr.sk). Results Among 14 patients, the female/male ratio was 1/1 and the median age was 9 years (range, 6-59 years). All patients were symptomatic at their first visit and the most common symptom was dark urine (71%) followed by arthralgia. Independent of the urinary homogentisic acid concentrations, patients with the presenting symptom of arthralgia were elder. Nine different mutations including p.Ser59AlafsX52, p.Gly161Arg, p.Asn219Ser, p.Gly251Asp, p.Pro274Leu, p.Arg330Ser, p.Gly372Ala, c.656_657insAATCAA and a novel mutation of p.Val316Ile were detected. All of the pediatric-age patients (n = 13) were treated with ascorbic acid at a dose of 250-1000 mg/day. Conclusions Nine different HGD gene mutations with a novel one, p.Val316Ile, were detected. The most common mutation was p.Ser59AlafsX52 for the HGD gene followed by p.Gly161Arg and p.asn219Ser, which can be considered specific to the Turkish population.

Complement Factor I Gene Polymorphism in a Turkish Age-Related Macular Degeneration Population.

OBJECTIVE: Evaluation of Complement Factor I (CFI) rs10033900 and rs2285714 polymorphism frequencies in patients with age-related macular degeneration (AMD) and healthy controls in a Turkish population. METHODS: A total of 111 eyes of 111 AMD patients and 96 eyes of 96 healthy controls, only one eye of individuals, were included in the study; however, 2 patients' and 4 controls' samples were excluded as analyses could not be performed for rs10033900 polymorphism. The AMD patients and control group (>50 years) lacked corneal, lenticular, vitreal opacity. However, these patients did not have any retinal diseases apart from AMD. Venous blood samples of patients were collected. Central macular thickness, subfoveal choroidal thickness (SCT), presence of reticular drusen, epiretinal membrane, and pigment epithelial detachment were investigated using Spectral-Domain Optical Coherence Tomography, and the largest diameter of atrophic areas measured. Drusen properties were documented from fundus photographs. The lesion width was calculated by using fundus fluorescein angiography. RESULTS: There was no difference between patient and control groups and polymorphism distributions. The frequency of the CT allele was higher in patients with dry-type AMD with retinal pigment epithelial abnormality (p = 0.041). SCT was significantly thinner in TT allele carriers with rs2285714 polymorphism (p = 0.030). No significant relationship was found between the other parameters and polymorphism distributions. Con-clusion: CFI rs10033900 and rs2285714 polymorphisms in a Turkish population were not associated with AMD.

Exon 2 deletion represents a common mutation in Turkish patients with fructose-1,6-bisphosphatase deficiency.

Fructose-1,6-bisphosphatase (FBPase) deficiency is an autosomal recessive inborn error of gluconeogenesis. We aimed to investigate clinical and biochemical findings and molecular genetic data in ten Turkish patients with fructose-1,6-bisphosphatase deficiency. Ten Turkish patients who were diagnosed with fructose-1,6-biphosphatase deficiency in a single center from 2013 to 2019 were included in this study. Their clinical and laboratory data were collected retrospectively. All patients were hospitalised in intensive care unit mostly after catabolic stress conditions such as infections, starvation and rarely fructose consumption. Prognosis was good after correct diagnosis and treatment. Molecular analyses of FBP1 gene revealed a homozygous exon 2 deletion in eight patients, a novel homozygous c.910_911dupTT mutation in one patient and a homozygous IVS5 + 1G > A splicing mutation in one patient. Exon 2 deletion (previously termed exon 1) was found to be the most common mutation in Turkish fructose-1,6-biphosphatase deficiency patients.

Five novel ALMS1 gene mutations in six patients with Alström syndrome.

BACKGROUND: Alström syndrome is a rare autosomal recessive inherited disorder caused by mutations in the ALMS1 gene. METHODS: We describe the clinical and five novel mutational screening findings in six patients with Alström syndrome from five families in a single center with distinct clinical presentations of this condition. RESULTS: Five novel mutations in ALMS1 in exon 8 and intron 17 were identified, one of them was a compound heterozygous: c.2259_2260insT, p.Glu754*; c.2035C>T p.Arg679*; c.2259_2260insT, p.Glu754*; c.5969C>G, p.Ser1990*; c.6541C>T, p. Gln2181*/c.11666-2A>G, splicing. One patient had gallstones, this association, to our knowledge, has not been reported in Alström syndrome previously. CONCLUSIONS: Early diagnosis of Alström syndrome is often difficult in children and adolescents, because many of the clinical features develop over time. Early diagnosis can initiate an effective managemen of this condition, and it will help to reduce future damage.

Clinical phenotype of hereditary spastic paraplegia due to KIF1C gene mutations across life span.

Hereditary spastic paraplegias (HSPs) are a group of genetic disorders resulting in pyramidal tract impairment, predominantly in lower limbs. KIF1C gene has recently been identified as one of the genetic causes of HSP and associated with pure or complicated HSP. We present three patients with complicated HSP from two unrelated families, who had early onset progressive cerebellar signs and developed pyramidal tract signs during follow-up. Whole exome sequencing in these patients followed by segregation analysis identified novel truncating KIF1C mutations (c.463C> T; p.R155∗ and c.2478delA; p.Ala828Argfs∗13). Neuroimaging findings showed cerebral and upper cervical spinal atrophy, bilateral symmetrical pyramidal tract involvement, and focal cerebral white matter lesions. Patients with KIF1C mutations may present with cerebellar signs and pyramidal findings may emerge later, therefore complicated HSP should be considered in the differential diagnosis of unidentified cases with cerebellar dysfunction.

A New Chapter for Mesenchymal Stem Cells: Decellularized Extracellular Matrices.

From orthopedic to neurological disorders, stem cells are used as platforms to understand disease mechanisms and considered as novel and promising treatment options, especially when the valid therapeutic approaches are unavailable or ineffective. There are different stem cell types in the literature, however the spindle-shaped, colony forming and multilineage-differentiating cells, also known as mesenchymal stem cells (MSC) are very popular, as MSC can be isolated from different tissues with minimal ethical concerns and without tumor formations, which make them easily accessible and widely used in vitro and in vivo studies. In the literature, MSC have been shown to have therapeutic effects and orchestrate the healing process via their mobilization, migration, differentiation capacities, immunomodulation properties and/or secretion of bioactive factors. Nowadays, MSC derived extracellular matrices (ECM), which are part of the secreted/produced bioactive molecules from MSC; draw attention of researchers due to their key roles in cell biology. Several groups have isolated ECM from in vitro cultured MSC using different methods of decellularization techniques for tissue-engineering approaches. According to current knowledge, decellularized ECM (dECM) influence growth, adhesion, differentiation, migration, apoptosis, proliferation, and phenotype of cells, covering almost all cellular events. In this comprehensive review we focused on MSC and the isolation methods and effects of MSC derived dECM (MSC-dECM).

A patient with mitochondrial disorder due to a novel mutation in MRPS22.

MRPS22 gene defect is a very rare newly discovered mitochondrial disorder. We report a 4-month-old severely affected male infant with MRPS22 mutation. Whole exome sequencing revealed a novel homozygous splicing mutation c.339 + 5 G > A in MRPS22 gene. He has mild dysmorphism, hypotonia, developmental delay but not hypertrophic cardiomyopathy and tubulopathy which differ from other majority of reported patients. Therefore, hypertrophic cardiomyopathy and tubulopathy may not be considered as constant features of MRPS22. With this case report, we also present first symmetrical bilateral brainstem and medial thalamic lesions, and cerebellar and cerebral atrophy on a brain MR imaging follow-up of ten months.

Discovery of biomarkers in rare diseases: innovative approaches by predictive and personalized medicine.

There are more than 8000 rare diseases (RDs) that affect >5 % of the world's population. Many of the RDs have no effective treatment and lack of knowledge creates delayed diagnosis making management difficult. The emerging concept of the personalized medicine allows for early screening, diagnosis, and individualized treatment of human diseases. In this context, the discovery of biomarkers in RDs will be of prime importance to enable timely prevention and effective treatment. Since 80 % of RDs are of genetic origin, identification of new genes and causative mutations become valuable biomarkers. Furthermore, dynamic markers such as expressed genes, metabolites, and proteins are also very important to follow prognosis and response the therapy. Recent advances in omics technologies and their use in combination can define pathophysiological pathways that can be drug targets. Biomarker discovery and their use in diagnosis in RDs is a major pillar in RD research.