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.

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.

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.

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.

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.

The phenotypic and molecular genetic spectrum of Alström syndrome in 44 Turkish kindreds and a literature review of Alström syndrome in Turkey.

Alström syndrome (ALMS) is an autosomal recessive disease characterized by multiple organ involvement, including neurosensory vision and hearing loss, childhood obesity, diabetes mellitus, cardiomyopathy, hypogonadism, and pulmonary, hepatic, renal failure and systemic fibrosis. Alström Syndrome is caused by mutations in ALMS1, and ALMS1 protein is thought to have a role in microtubule organization, intraflagellar transport, endosome recycling and cell cycle regulation. Here, we report extensive phenotypic and genetic analysis of a large cohort of Turkish patients with ALMS. We evaluated 61 Turkish patients, including 11 previously reported, for both clinical spectrum and mutations in ALMS1. To reveal the molecular diagnosis of the patients, different approaches were used in combination, a cohort of patients were screened by the gene array to detect the common mutations in ALMS1 gene, then in patients having any of the common ALMS1 mutations were subjected to direct DNA sequencing or next-generation sequencing for the screening of mutations in all coding regions of the gene. In total, 20 distinct disease-causing nucleotide changes in ALMS1 have been identified, eight of which are novel, thereby increasing the reported ALMS1 mutations by 6% (8/120). Five disease-causing variants were identified in more than one kindred, but most of the alleles were unique to each single patient and identified only once (16/20). So far, 16 mutations identified were specific to the Turkish population, and four have also been reported in other ethnicities. In addition, 49 variants of uncertain pathogenicity were noted, and four of these were very rare and probably or likely deleterious according to in silico mutation prediction analyses. ALMS has a relatively high incidence in Turkey and the present study shows that the ALMS1 mutations are largely heterogeneous; thus, these data from a particular population may provide a unique source for the identification of additional mutations underlying Alström Syndrome and contribute to genotype-phenotype correlation studies.

Detection of biotinidase gene mutations in Turkish patients ascertained by newborn and family screening.

UNLABELLED: The incidence of biotinidase deficiency in Turkey is currently one of the highest in the world. To expand upon the information about the biotinidase gene (BTD) variations in Turkish patients, we conducted a mutation screening in a large series (n = 210) of probands with biotinidase deficiency, using denaturing high-performance liquid chromatography and direct DNA sequencing. The putative effects of novel mutations were predicted by computational program. Twenty-six mutations, including six novels (p.C143F, p.T244I, c.1212-1222del11, c.1320delG, p.V457L, p.G480R) were identified. Nine of the patients were symptomatic at the initial clinical assessment with presentations of seizures, encephalopathy, and lactic acidemia. The most common mutation in this group of symptomatic patients was c.98-104 del7ins3. Among the screened patients, 72 have partial and 134 have profound biotinidase deficiency (BD) of which 106 are homozygous for BTD mutations. The common mutations (p.R157H, p.D444H, c.98-104del7ins3, p.T532M) cumulatively accounted for 72.3% of all the mutant alleles in the Turkish population. CONCLUSION: The identification of common mutations and hot spot regions of the BTD gene in Turkish patients is important for mutation screening in the Turkish population and helps to ascertain carriers, may have impact on genetic counseling and implementing prevention programs.

MCP1 2518 A/G polymorphism affects progression of childhood focal segmental glomerulosclerosis.

Monocyte chemoattractant protein-1 (MCP-1) is a highly specific chemokine for monocytes and plays roles in pathogenesis of various renal diseases. The aim of this study is to investigate the effect of MCP1 2518 A/G polymorphism on the incidence and clinical course of focal segmental glomerulosclerosis (FSGS) in children. MCP1 2518 A/G genotype was identified by PCR-RFLP in 60 biopsy-proven FSGS patients, 76 steroid sensitive nephrotic syndrome (SSNS) patients, and 96 healthy children. MCP-1 levels in urine and serum were measured by ELISA in all patients and the correlations of genotype with MCP-1 levels and clinical outcome were evaluated. The genotype frequencies for MCP1 were similar in all groups. The percentage of patients who develop chronic renal failure was higher in patients with AA allele compared to GA or GG alleles (46% vs. 35% respectively, p < 0.01, Odds ratio: 1.59). Serum MCP-1 levels were similar in all groups, whereas urinary MCP-1 levels of the patients with FSGS (1680 pg/mg creatinine) were significantly higher than that of patients with SSNS (365 pg/mg creatinine, p < 0.05) and healthy controls (348 pg/mg creatinine; p < 0.05). Urinary MCP-1 levels were correlated with the degree of proteinuria in FSGS group (r = 0.529, p = 0.016). Our results suggest that the AA genotype might be a risk factor for the progression of renal disease in FSGS and MCP1 genotyping may help the physicians to predict prognosis in these patients.

Dynamics of the rhomboid-like protein RHBDD2 expression in mouse retina and involvement of its human ortholog in retinitis pigmentosa.

The novel rhomboid-like protein RHBDD2 is distantly related to rhomboid proteins, a group of highly specialized membrane-bound proteases that catalyze regulated intramembrane proteolysis. In retina, RHBDD2 is expressed from embryonic stages to adulthood, and its levels show age-dependent changes. RHBDD2 is distinctly abundant in the perinuclear region of cells, and it localizes to their Golgi. A glycine zipper motif present in one of the transmembrane domains of RHBDD2 is important for its packing into the Golgi membranes. Its deletion causes dislodgment of RHBDD2 from the Golgi. A specific antibody against RHBDD2 recognizes two forms of the protein, one with low (39 kDa; RHBDD2(L)) and the other with high (117 kDa; RHBDD2H) molecular masses in mouse retinal extracts. RHBDD2(L) seems to be ubiquitously expressed in all retinal cells. In contrast, RHBDD2H seems to be present only in the outer segments of cone photoreceptors and may correspond to a homotrimer of RHBDD2(L). This protein consistently co-localizes with S- and M-types of cone opsins. We identified a homozygous mutation in the human RHBDD2 gene, R85H, that co-segregates with disease in affected members of a family with autosomal recessive retinitis pigmentosa. Our findings suggest that the RHBDD2 protein plays important roles in the development and normal function of the retina.

Exome sequencing and cis-regulatory mapping identify mutations in MAK, a gene encoding a regulator of ciliary length, as a cause of retinitis pigmentosa.

A fundamental challenge in analyzing exome-sequence data is distinguishing pathogenic mutations from background polymorphisms. To address this problem in the context of a genetically heterogeneous disease, retinitis pigmentosa (RP), we devised a candidate-gene prioritization strategy called cis-regulatory mapping that utilizes ChIP-seq data for the photoreceptor transcription factor CRX to rank candidate genes. Exome sequencing combined with this approach identified a homozygous nonsense mutation in male germ cell-associated kinase (MAK) in the single affected member of a consanguineous Turkish family with RP. MAK encodes a cilium-associated mitogen-activated protein kinase whose function is conserved from the ciliated alga, Chlamydomonas reinhardtii, to humans. Mutations in MAK orthologs in mice and other model organisms result in abnormally long cilia and, in mice, rapid photoreceptor degeneration. Subsequent sequence analyses of additional individuals with RP identified five probands with missense mutations in MAK. Two of these mutations alter amino acids that are conserved in all known kinases, and an in vitro kinase assay indicates that these mutations result in a loss of kinase activity. Thus, kinase activity appears to be critical for MAK function in humans. This study highlights a previously underappreciated role for CRX as a direct transcriptional regulator of ciliary genes in photoreceptors. In addition, it demonstrates the effectiveness of CRX-based cis-regulatory mapping in prioritizing candidate genes from exome data and suggests that this strategy should be generally applicable to a range of retinal diseases.

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.

Galactosemia in the Turkish population with a high frequency of Q188R mutation and distribution of Duarte-1 and Duarte-2 variations.

Classical galactosemia is an inherited recessive disorder of galactose metabolism caused by deficiency of the enzyme galactose-1-phosphate uridyl transferase (GALT), which is caused by mutations in the GALT gene. In this study, 56 Turkish patients diagnosed with galactosemia were screened for GALT gene mutations using Affymetrix resequencing microarrays. Eleven types of mutations were detected in these patients, including two novel mutations (R258G and G310fsX49) and nine recurrent mutations. We detected six patients who were homozygous for the E340* mutation and for N314D, L218L silent substitutions (Duarte-1 variant) in this study. The haplotype E340*, N314D and L218L has been reported only in Turkish patients, which suggests that the E340* mutation is specific for our population and might be spread by a Turk ancestor. In patients, the Duarte-1 allele was found with a frequency of 10.71%, whereas the Duarte-2 allele was not detected. Duarte-1 and Duarte-2 alleles were found to be present at a frequency of 2.3% and 1.4%, respectively, in the screening of 105 healthy individuals. Considering all detected mutations, it is a very important finding that exons 6 and 10 of the GALT gene account for 79% of all mutant alleles in the Turkish population. The most common mutation is Q188R, with a frequency of 55.35%.

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).

Microarray based mutational analysis of patients with methylmalonic acidemia: identification of 10 novel mutations.

Methylmalonic acidemia is an autosomal recessive metabolic disorder affecting the propionate oxidation pathway in the catabolism of several amino acids, odd-chain fatty acids, and cholesterol. Methylmalonic acidemia is characterized by elevated levels of methylmalonic acid in the blood and urine. Mutations in the MUT gene, encoding methylmalonyl-CoA mutase carries out isomerization of L-methylmalonyl-CoA to succinyl-CoA, cause methylmalonic acidemia. In this study, 30 Turkish patients diagnosed with mut methylmalonic acidemia were screened for mutations using custom designed sequencing microarrays. The study resulted in detection of 22 different mutations, 10 of which were novel: p.Q132*, p.A137G, c.753+1T, p.T387I, p.Q514E, p.P615L, p.D625V, c.1962_1963delTC, p.L674F, and c.2115_2116insA. The most common, p.P615T, was identified in 28.0% of patients. These results suggest that microarray based sequencing is a useful tool for the detection of mutations in MUT in patients with mut methylmalonic acidemia.

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.