Development of an automated estimation of foot process width using deep learning in kidney biopsies from patients with Fabry, minimal change, and diabetic kidney diseases.

Podocyte injury plays a key role in pathogenesis of many kidney diseases with increased podocyte foot process width (FPW), an important measure of podocyte injury. Unfortunately, there is no consensus on the best way to estimate FPW and unbiased stereology, the current gold standard, is time consuming and not widely available. To address this, we developed an automated FPW estimation technique using deep learning. A U-Net architecture variant model was trained to semantically segment the podocyte-glomerular basement membrane interface and filtration slits. Additionally, we employed a post-processing computer vision approach to accurately estimate FPW. A custom segmentation utility was also created to manually classify these structures on digital electron microscopy (EM) images and to prepare a training dataset. The model was applied to EM images of kidney biopsies from 56 patients with Fabry disease, 15 with type 2 diabetes, 10 with minimal change disease, and 17 normal individuals. The results were compared with unbiased stereology measurements performed by expert technicians unaware of the clinical information. FPW measured by deep learning and by the expert technicians were highly correlated and not statistically different in any of the studied groups. A Bland-Altman plot confirmed interchangeability of the methods. FPW measurement time per biopsy was substantially reduced by deep learning. Thus, we have developed a novel validated deep learning model for FPW measurement on EM images. The model is accessible through a cloud-based application making calculation of this important biomarker more widely accessible for research and clinical applications.

Reconstruction of mandibular defects using synthetic octacalcium phosphate combined with bone matrix gelatin in rat model.

BACKGROUND: Regeneration of bone defects remains a challenge for maxillofacial surgeons. The objective of this study was to assess the osteogenic potential of octacalcium phosphate (OCP) and bone matrix gelatin (BMG) alone and in combination with together in artificially created mandibular bone defects. MATERIALS AND METHODS: In this experimental study Forty-eight male Sprague-Dawley rats (6-8 weeks old) were randomly divided into four groups. Defects were created in the mandible of rats and filled with 10 mg of OCP, BMG, or a combination of both (1/4 ratio). Defects were left unfilled in the control group. To assess bone regeneration and determine the amount of the newly formed bone, specimens were harvested at 7, 14, 21, and 56 days postimplantation. The specimens were processed routinely and studied histologically and histomorphometrically using the light microscope and eyepiece graticule. The amount of newly formed bone was quantitatively measured using histomorphometric methods. Histomorphometric data were analyzed using SPSS software. Mean, standard deviation, mode, and medians were calculated. Tukey HSD test was used to compare the means in all groups. P < 0.05 was considered as statistically significant (i.e., 5% significant level). RESULTS: In the experimental groups, the new bone formation was initiated from the margin of defects during the 7-14 days after implantation. By the end of study, the amount of newly formed bone increased and relatively matured, and almost all of the implanted materials were absorbed. In the control group, slight amount of new bone had been formed at the defect margins (next to the host bone) on day 56. The histomorphometric analysis revealed statistically significant differences in the amount of newly formed bone between the experimental and the control groups (P < 0.001). CONCLUSION: Combination of OCP/BMG may serve as an optimal biomaterial for the treatment of mandibular bone defects.

Inactivating Mutations in Exonuclease and Polymerase Domains in DNA Polymerase Delta Alter Sensitivities to Inhibitors of dNTP Synthesis.

POLD1 encodes the catalytic subunit of DNA polymerase delta (Polδ), the major lagging strand polymerase, which also participates in DNA repair. Mutations affecting the exonuclease domain increase the risk of various cancers, while mutations that change the polymerase active site cause a progeroid syndrome called mandibular hypoplasia, deafness, progeroid features, and lipodystrophy (MDPL) syndrome. We generated a set of catalytic subunit of human telomerase (hTERT)-immortalized human fibroblasts expressing wild-type or mutant POLD1 using the retroviral LXSN vector system. In the resulting cell lines, expression of endogenous POLD1 was suppressed in favor of the recombinant POLD1. The siRNA screening of DNA damage-related genes revealed that fibroblasts expressing D316H and S605del POLD1 were more sensitive to knockdowns of ribonuclease reductase (RNR) components, RRM1 and RRM2 in the presence of hydroxyurea (HU), an RNR inhibitor. On the contrary, SAMHD1 siRNA, which increases the concentration of dNTPs, increased growth of wild type, D316H, and S605del POLD1 fibroblasts. Hypersensitivity to dNTP synthesis inhibition in POLD1 mutant lines was confirmed using gemcitabine. Our finding is consistent with the notion that reduced dNTP concentration negatively affects the cell growth of hTERT fibroblasts expressing exonuclease and polymerase mutant POLD1.

Histomorphometric Analysis of Newly-formed Bone Using Octacalcium Phosphate and Bone Matrix Gelatin in Rat Tibial Defects.

BACKGROUND: Repair of bone defects is challenging for reconstructive and orthopedic surgeons. In this study, we aimed to histomorphometrically assess new bone formation in tibial bone defects filled with octacalcium phosphate (OCP), bone matrix gelatin (BMG), and a combination of both. METHODS: A total of 96 male Sprague Dawley rats aged 6-8 weeks weighing 120-150 g were randomly allocated into three experimental (OCP, BMG, and OCP/BMG) and one control group (n=24 in each group). The defects in experimental groups were filled with OCP (6 mg), BMG (6 mg), or a combination of OCP and BMG (6 mg, 2:1 ratio). No material was used to fill the defects in the control group and the defect was only covered with Surgicel. Samples were taken on days 7, 14, 21, and 56 after the surgery. The sections were stained with hematoxylin-eosin (H&E) and assessed using light microscopy. RESULTS: In our experimental groups, bone formation was started from the margins of the defect towards the center with an increasing rate during the study period. Moreover, the formed bone was more mature. Bone formation in our control group was only limited to the margins of the defect. The newly formed bone mass was significantly higher in the experimental groups (P=0.001). CONCLUSION: OCP, BMG, and OCP/BMG compound enhanced osteoinduction in long bones.

CTC1 mutations in a Brazilian family with progeroid features and recurrent bone fractures.

BACKGROUND: Cerebroretinal microangiopathy with calcifications and cysts (CRMCC) is an autosomal recessive disorder caused by pathogenic variants of the conserved telomere maintenance component 1 (CTC1) gene. The CTC1 forms the telomeric capping complex, CST, which functions in telomere homeostasis and replication. METHODS: A Brazilian pedigree and an Australian pedigree were referred to the International Registry of Werner Syndrome (Seattle, WA, USA), with clinical features of accelerated aging and recurrent bone fractures. Whole exome sequencing was performed to identify the genetic causes. RESULTS: Whole exome sequencing of the Brazilian pedigree revealed compound heterozygous pathogenic variants in CTC1: a missense mutation (c.2959C>T, p.Arg987Trp) and a novel stop codon change (c.322C>T, p.Arg108*). The Australian patient carried two novel heterozygous CTC1 variants, c.2916G>T, p.Val972Gly and c.2926G>T, p.Val976Phe within the same allele. Both heterozygous variants were inherited from the unaffected father, excluding the diagnosis of CRMCC in this pedigree. Cell biological studies demonstrated accumulation of double strand break foci in lymphoblastoid cell lines derived from the patients. Increased DSB foci were extended to non-telomeric regions of the genome, in agreement with previous biochemical studies showing a preferential binding of CTC1 protein to GC-rich sequences. CONCLUSION: CTC1 pathogenic variants can present with unusual manifestations of progeria accompanied with recurrent bone fractures. Further studies are needed to elucidate the disease mechanism leading to the clinical presentation with intra-familial variations of CRMCC.

Biallelic WRN Mutations in Newly Identified Japanese Werner Syndrome Patients.

Werner syndrome (WS) is a rare autosomal recessive disorder characterized by systemic accelerated aging. It is caused by pathogenic variants of the WRN gene that encodes a nuclear helicase. In this report, we describe 4 newly identified WS cases among those referred to the Japanese Werner Consortium, Chiba University, Japan. All 4 cases were compound heterozygotes of the Japanese founder mutation, c.3139-1G>C, and a novel null pathogenic variant, c.1587G>A, c.2448+1G>A, or c.3233+1G>T, or an amino acid substitution variant, c.1720G>A, p.Gly574Arg. These 3 null pathogenic variants were not previously described. The p. Gly574Arg was previously reported in a European patient, and the identification of the second p. Gly574Arg case, with classical WS features, further confirmed the pathogenic nature of this variant. For the case with c.3233+1G>T, we determined the phase of 2 disease-causing mutations and demonstrated that they are on different chromosomes. This assay would be particularly important for those cases with ambiguous clinical diagnosis.

Uncommon cause of cirrhosis-A case of Werner syndrome with a novel WRN mutation.

Werner syndrome is a rare progeroid syndrome caused by the WRN gene mutation. It is characterized by a general appearance of premature aging, diabetes mellitus, and atherosclerosis, and an increased risk of malignancies. We report a patient who presented with hematemesis due to cirrhosis of liver and was subsequently diagnosed with Werner syndrome. Further genetic analysis showed a novel mutation in the WRN gene which has not previously been reported. Werner syndrome should be considered for the cases of liver cirrhosis when accompanied by the features of accelerated aging.