A Mild Clinical Phenotype with Myopathic and Hemolytic Forms of Phosphoglycerate Kinase Deficiency (PGK Osaka): A Case Report and Literature Review.

Phosphoglycerate kinase (PGK) deficiency is an X-linked disorder characterized by a combination of hemolytic anemia, myopathy, and brain involvement. We herein report a Japanese man who had several episodes of rhabdomyolysis but was training strenuously to be a professional boxer. Mild hemolytic anemia was noted. The enzymatic activity of PGK was significantly reduced, and a novel missense mutation, p.S62N, was identified in the PGK1 gene. A literature review revealed only one case with a mixed hemolytic and myopathic phenotype like ours. This mild phenotype indicates the complex pathophysiology of PGK deficiency and suggests the benefits of dietary control and exercise.

High-fat diet increases labial groove formation in maxillary incisors and is related to aging in C57BL/6 mice.

OBJECTIVES: The aim of this study was to explore the relationship between the consumption of a high-fat diet and aging-dependent formation of maxillary incisor grooves in C57BL/6 mice, and to identify putative maxillary incisor groove-related genes. METHODS: We fed 2-month-old and 16-month-old C57BL/6 mice on either a chow diet or a high-fat diet for three months and observed changes in maxillary incisor grooves. We examined tissue sections of the maxillary incisors with grooves and carried out transcriptome analysis of the apical tissue fragments of maxillary incisors with/without grooves. RESULTS: Consumption of a high-fat diet for three months resulted in significant increases in both body weight and the number of incisor grooves. Both the number and frequency of incisor grooves increased in an age-dependent manner from 26 to 28 months, during which time an additional groove appeared. There was abnormal differentiation and apoptosis of ameloblasts on the labial surface at the grooves of the maxillary incisors. Transcriptome analysis identified 23 genes as being specific to 24-month-old mice; these included several genes related to apoptosis and cell differentiation. CONCLUSIONS: The study findings indicate that, in C57BL/6 mice, consumption of a high-fat diet increases labial groove formation in maxillary incisors, which is related to aging of the tissue stem cells in the apical root end of the teeth.

A maternal high-fat diet may accelerate adipo-immunologic aging in offspring.

AIM: Maternal obesity and improper nutrition predispose offspring to chronic metabolic diseases. Although the frequency of these diseases increases with aging, the effect of a maternal high-fat diet on aged offspring remains elusive. MAIN METHODS: C57BL/6J female mice were fed a high-fat (HF) diet or a control (CON) diet and then mated. All offspring remained with their birth dam until weaning at 3 weeks. After weaning, the offspring from the HF and CON diet-fed dams were given either the HF diet or CON diet, which resulted in four groups: CON/CON, CON/HF, HF/CON, and HF/HF. All mice were immunized with ovalbumin and then sacrificed at 70 weeks. KEY FINDINGS: The body weights in offspring from dam exposed to a HF diet were significantly higher than those in offspring from dam fed a CON diet in the early stage of life but then became lower in the later stage of life. The serum adiponectin levels were lower in offspring from dam exposed to a HF diet and were correlated with adiposity measured by visceral and subcutaneous fat mass. Non-alcoholic fatty liver disease was much more severe in the livers of offspring from the maternal HF groups. In particular, lobular inflammation and fibrosis were prominent in the HF/HF group. Regarding immunological parameters, senescence-associated T cells were increased, and natural killer T cells were decreased by the effect of both maternal and offspring HF diet. SIGNIFICANCE: We have demonstrated that a maternal high-fat diet may accelerate the adipo-immunologic aging process.

Identification of genetic risk factors of aggressive periodontitis using genomewide association studies in association with those of chronic periodontitis.

To identify the genetic risk factors for aggressive periodontitis (AgP), it is important to understand the progression and pathogenesis of AgP. The purpose of this review was to summarize the genetic risk factors for AgP identified through a case-control genomewide association study (GWAS) and replication study. The initial studies to identify novel AgP risk factors were potentially biased because they relied on previous studies. To overcome this kind of issue, an unbiased GWAS strategy was introduced to identify genetic risk factors for various diseases. Currently, three genes glycosyltransferase 6 domain containing 1 (GLT6D1), defensin α1 and α3 (DEFA1A3), and sialic acid-binding Ig-like lectin 5 (SIGLEC5) that reach the threshold for genomewide significance have been identified as genetic risk factors for AgP through a case-control GWAS.

The Influence of LPS-Induced Maternal Inflammation on Postnatal Collagen-Induced Arthritis.

Maternal health and nutritional status influence offspring health and the diseases that may develop in them. The effects of maternal inflammation on offspring from the perspective of the inflammatory response and immune changes are not fully understood. We hypothesized that maternal inflammation modulates immune and metabolic functions, affecting the pathophysiology of inflammatory diseases in offspring. This study investigated whether maternal inflammation affects the onset of collagen-induced arthritis (CIA), a murine model of human rheumatoid arthritis. Female DBA/1J mice received a single intraperitoneal injection of lipopolysaccharide (LPS) 5 days before conception. Male offspring of LPS-treated dams were placed in the maternal LPS group (MLG). To induce CIA, type II collagen (CII) was emulsified with Freund's complete adjuvant and injected twice into each mouse, at 13 and 16 weeks. The offspring were sacrificed at 26 weeks to analyze immunological and metabolic parameters. The degree of joint swelling at an early stage of CIA was lower in the MLG than in the control group. From histological analysis, the severity of joint destruction (severity of arthritis score) and CII-specific IgG titer were significantly lower in the MLG. However, at 26 weeks, serum interleukin (IL)-6 levels, an index of CIA disease activity, were significantly higher in the MLG. Moreover, serum leptin levels were lower in the MLG, and a negative correlation between leptin and serum IL-6 was observed. In conclusion, maternal inflammation does not merely suppress inflammation; it may delay CIA in offspring. The analysis of inflammatory cytokines and leptin concentrations at 26 weeks suggests that the pathophysiology of arthritis was worsening. This study also suggests that maternal inflammation modulates postnatal inflammatory response patterns in offspring.

Analysis of an ADTKD family with a novel frameshift mutation in MUC1 reveals characteristic features of mutant MUC1 protein.

BACKGROUND: Medullary cystic kidney disease Type 1 is an autosomal dominant tubulointerstitial kidney disease (ADTKD). Recently, mucin 1 (MUC1) was identified as a causal gene of medullary cystic kidney disease (ADTKD-MUC1). However, the MUC1 mutation was found to be a single cytosine insertion in a single copy of the GC-rich variable number of tandem repeats (VNTRs), which are very difficult to analyze by next-generation sequencing. To date, other mutations have not been detected in ADTKD-MUC1, and the mutant MUC1 protein has not been analyzed because of the difficulty of genetically modifying the VNTR sequence. METHODS: We conducted whole-exome analyses of an ADTKD family by next-generation sequencing. We also performed histopathological analyses of a renal biopsy from a pedigree family member. We constructed a mutant protein expression vector based on the patient genome sequence and characterized the nature of the mutant protein. RESULTS: We found a novel frameshift mutation before the VNTR in the MUC1 gene. The resulting mutant MUC1 protein had a very similar amino acid sequence and predicted 3D structure to the previously reported mutant protein. Notably, the recombinant mutant MUC1 protein was trapped in the cytoplasm and appeared to self-aggregate. The patient native mutant protein was also found in urine exosomes. CONCLUSIONS: This novel frameshift mutation in the MUC1 gene and consequent mutant protein may contribute to the future discovery of the pathophysiology of ADTKD-MUC1. The mutant MUC1 protein in urine exosomes may be used for non-DNA-related diagnosis.

Effects of in vitro heat shock on immune cells in diet-induced obese mice.

Obesity has been associated with impaired immune responses and inflammation. The mechanisms underlying these immune disturbances in obesity are not yet clarified. This study investigated the effects of in vitro heat shock (HS) on immune cells from the point of view of thymocyte apoptosis and T-cell mitogen-stimulated splenocyte cytokine production as well as the heat shock protein 70 (HSP70) protein levels in diet-induced obese mice to explore a possible association between the disturbance of T cell immunity and HS response in obesity. Obese mice had increased apoptotic and necrotic thymocytes populations and increased splenocyte cytokine production of both proinflammatory and anti-inflammatory cytokines compared with lean mice. The in vitro HS at 42°C decreased the rate of live cells in thymocytes, and the degree of the decrease was larger in obese mice compared with lean mice. The in vitro HS increased the intracellular and extracellular HSP70 protein levels in thymocytes and splenocytes, while the effects of obesity on the HSP70 protein levels were not obvious. The in vitro HS prior to T cell mitogen stimulation decreased IFN-γ and IL-10 production by mitogen-stimulated splenocytes. This change in cytokine production due to HS was not affected by obesity. The obvious alteration of the HSP70 protein levels and association between cytokine production and the HS response in obesity were not found in this obesity model; however, our results indicate an association between the viability of thymocytes and an altered HS response in obesity and provide evidence that the increase in thymocyte apoptosis and acceleration of thymus involution in obesity could be, in part, due to the alteration of the HS response.

Inducing maternal inflammation promotes leptin production in offspring but does not improve allergic symptoms in a mouse model of allergic rhinitis.

AIMS: The intrauterine environment is considered to affect immunological development in fetus, leading to an increased risk of developing allergy. In particular, maternal lipopolysaccharides (LPS) administration might regulate the development of allergic disease in offspring. Several studies have shown that being obese relates to a higher prevalence of allergic diseases compared to normal weight. The present study explored the effects of inducing maternal inflammation with LPS before pregnancy on body weight, physical composition including body fat, adipokine production, and pathology of allergic rhinitis in offspring. MAIN METHODS: Female mice received a single intraperitoneal injection of LPS (2 µg/g BW). After 5 days of LPS administration, female mice were mated with males, and experimental allergic rhinitis was induced in female offspring. Immunization and nasal challenge with ovalbumin (OVA) were performed at 7 and 8 weeks of age. Allergic rhinitis-like symptoms, OVA-specific IgE and adipokines in sera, body weight, fat pad weight, and cytokine production by splenocytes in these 9-week-old offspring. KEY FINDINGS: Maternal LPS administration results in a significant increase in body weight, visceral fat accumulation, and serum leptin concentration, and the dominance of Th1 in Th balance. Nevertheless, there was no statistical difference in OVA-specific IgE titer and allergic-like symptoms between the groups. SIGNIFICANCE: In conclusion, maternal LPS promoted leptin production and altered Th balance in mice offspring, but not improved allergic symptoms in a mouse model of allergic rhinitis. It might suggest that inflammation during pregnancy plays a role in the adipose tissue function which could diversely influence allergic inflammation in offspring.

Mutation in VPS33A affects metabolism of glycosaminoglycans: a new type of mucopolysaccharidosis with severe systemic symptoms.

Mucopolysaccharidoses (MPS) are a group of genetic deficiencies of lysosomal enzymes that catabolize glycosaminoglycans (GAG). Here we describe a novel MPS-like disease caused by a specific mutation in the VPS33A gene. We identified several Yakut patients showing typical manifestations of MPS: coarse facial features, skeletal abnormalities, hepatosplenomegaly, respiratory problems, mental retardation, and excess secretion of urinary GAG. However, these patients could not be diagnosed enzymatically as MPS. They showed extremely high levels of plasma heparan sulphate (HS, one of GAG); 60 times the normal reference range and 6 times that of MPS patients. Additionally, most patients developed heart, kidney, and hematopoietic disorders, which are not typical symptoms for conventional MPS, leading to a fatal outcome between 1 and 2-years old. Using whole exome and Sanger sequencing, we identified homozygous c.1492C > T (p.Arg498Trp) mutations in the VPS33A gene of 13 patients. VPS33A is involved in endocytic and autophagic pathways, but the identified mutation did not affect either of these pathways. Lysosomal over-acidification and HS accumulation were detected in patient-derived and VPS33A-depleted cells, suggesting a novel role of this gene in lysosomal functions. We hence propose a new type of MPS that is not caused by an enzymatic deficiency.

A Putative Association of a Single Nucleotide Polymorphism in GPR126 with Aggressive Periodontitis in a Japanese Population.

Periodontitis is an inflammatory disease causing loss of tooth-supporting periodontal tissue. Disease susceptibility to the rapidly progressive form of periodontitis, aggressive periodontitis (AgP), appears to be influenced by genetic risk factors. To identify these in a Japanese population, we performed whole exome sequencing of 41 unrelated generalized or localized AgP patients. We found that AgP is putatively associated with single nucleotide polymorphism (SNP) rs536714306 in the G-protein coupled receptor 126 gene, GPR126 [c.3086 G>A (p.Arg1029Gln)]. Since GPR126 activates the cAMP/PKA signaling pathway, we performed cAMP ELISA analysis of cAMP concentrations, and found that rs536714306 impaired the signal transactivation of GPR126. Moreover, transfection of human periodontal ligament (HPDL) cells with wild-type or mutant GPR126 containing rs536714306 showed that wild-type GPR126 significantly increased the mRNA expression of bone sialoprotein, osteopontin, and Runx2 genes, while mutant GPR126 had no effect on the expression of these calcification-related genes. The increase in expression of these genes was through the GPR126-induced increase of bone morphogenic protein-2, inhibitor of DNA binding (ID) 2, and ID4 expression. These data indicate that GPR126 might be important in maintaining the homeostasis of periodontal ligament tissues through regulating the cytodifferentiation of HPDL cells. The GPR126 SNP rs536714306 negatively influences this homeostasis, leading to the development of AgP, suggesting that it is a candidate genetic risk factor for AgP in the Japanese population.

HDR: a statistical two-step approach successfully identifies disease genes in autosomal recessive families.

In the search for sequence variants underlying disease, commonly applied filtering steps usually result in a number of candidate variants that cannot further be narrowed down. In autosomal recessive families, disease usually occurs only in one generation so that genetic linkage analysis is unlikely to help. Because homozygous recessive mutations tend to be inherited together with flanking homozygous variants, we developed a statistical method to detect pathogenic variants in autosomal recessive families: We look for differences in patterns of homozygosity around candidate variants between patients and control individuals and expect that such differences are greater for pathogenic variants than random candidate variants. In six autosomal recessive mitochondrial disease families, in which pathogenic homozygous variants have already been identified, our approach succeeded in prioritizing pathogenic mutations. Our method is applicable to single patients from recessive families with at least a few dozen control individuals from the same population; it is easy to use and is highly effective for detecting causative mutations in autosomal recessive families.

Beyond Homozygosity Mapping: Family-Control analysis based on Hamming distance for prioritizing variants in exome sequencing.

A major challenge in current exome sequencing in autosomal recessive (AR) families is the lack of an effective method to prioritize single-nucleotide variants (SNVs). AR families are generally too small for linkage analysis, and length of homozygous regions is unreliable for identification of causative variants. Various common filtering steps usually result in a list of candidate variants that cannot be narrowed down further or ranked. To prioritize shortlisted SNVs we consider each homozygous candidate variant together with a set of SNVs flanking it. We compare the resulting array of genotypes between an affected family member and a number of control individuals and argue that, in a family, differences between family member and controls should be larger for a pathogenic variant and SNVs flanking it than for a random variant. We assess differences between arrays in two individuals by the Hamming distance and develop a suitable test statistic, which is expected to be large for a causative variant and flanking SNVs. We prioritize candidate variants based on this statistic and applied our approach to six patients with known pathogenic variants and found these to be in the top 2 to 10 percentiles of ranks.

Leveling the Playing Field in Homozygosity Mapping Using Map Distances.

Studies of linkage disequilibrium (LD) and its variation in the genome are of central importance for understanding evolutionary history, population structure, and selective sweeps. Extreme forms of the latter may result in runs of homozygosity (ROH). In human gene mapping, long ROHs are the basis for homozygosity mapping (HM) with length measured in terms of Mb (106 base pairs physical distance). LD varies greatly over the human genome so that long ROHs tend to occur preferentially in regions of high LD and ROHs of the same length in different regions are not strictly comparable. Thus, in human gene mapping, LD appears as a confounder that needs to be taken into account in the interpretation of ROHs. The effect of varying LD can be mitigated by working on a scale of centimorgans (cM, genetic distance) instead of Mb. We demonstrate this effect for HapMap 3 data on chromosome 19 and show examples with different ROH lengths depending on whether physical or genetic lengths are used. These results suggest that HM should preferably be done on genetic rather than physical distances.

Early detection of vulnerable atherosclerotic plaque for risk reduction of acute aortic rupture and thromboemboli and atheroemboli using non-obstructive angioscopy.

The mortality rate due to rupture of aortic dissection and aortic aneurysm is approximately 90%. Acute aortic rupture can be fatal prior to hospitalization and has proven difficult to diagnose correctly or predict. The in-hospital mortality rate of ruptured aortic aneurysm ranges from 53 to 66%. Emergency surgical and endovascular treatments are the only options for ruptured aortic dissection and aortic aneurysm. No method of systematic early detection or inspection of vessel injury is available at the prevention stage. Regardless of the improvement in many imaging modalities, aortic diameter has remained a major criterion for recommending surgery in diagnosed patients. Previous reports have suggested a relationship between vulnerable plaque and atherosclerotic aortic aneurysm. Non-obstructive angioscopy is a new method for evaluating intimal injury over the whole aorta. It has been used to identify many advanced atherosclerotic plaques that were missed on traditional imaging modalities before aneurysm formation. Non-obstructive angioscopy has shown that atherosclerosis of the aorta begins before that of the coronary artery, which had been noted on autopsy "in vivo". Strong or repetitive aortic injuries might cause sudden aortic disruption. Aortic atheroma is also a risk factor of stroke and perivascular embolism. Detecting aortic vulnerable atherosclerotic plaque on non-obstructive angioscopy may not only clarify the pathogenesis of acute aortic rupture and "aortogenic" thromboemboli and atheroemboli but also play a role in the pre-emptive medicine.

Quantitative analysis of coronary vessels with optimized intracoronary CT number.

BACKGROUND: Variability in intracoronary computed tomography (CT) number may influence vessel quantification. We confirmed the feasibility of a novel method for measuring vessel diameter and area using coronary CT angiography (CCTA) with an optimized intracoronary CT number, 350 HU. METHODS: We performed intravascular ultrasound (IVUS) imaging in 52 patients with significant stenosis detected by coronary CT angiography targeting 350 HU using a CT number-controlling system. We measured 0-to-0 HU distances in the cross-sectional coronary images of 32 patients. We analyzed the ratio of 0-to-0 HU distances in CT images to media-to-media distances in IVUS images (C:I ratio). The area of ≥0 HU for 103 representative points in the remaining 20 patients was compared to the area of the traced external elastic membrane (EEM) in IVUS images. RESULTS: There was a strong correlation between 0-to-0 HU distance in CT images and media-to-media diameter in IVUS images (r = 0.97, p<0.001). The C:I ratio was 1.1. EEM area was estimated by dividing the area of ≥0 HU by the square of C:I. There was also a strong correlation between the estimated EEM area and the EEM area in IVUS images (r = 0.95, p<0.001). CONCLUSIONS: Media-to-media diameter and EEM area can be estimated by CCTA targeting the optimized intracoronary CT number when blood vessel borders are defined at 0 HU.

CT number-controlling system for reproducibility of intracoronary CT number on follow-up coronary CT angiography.

BACKGROUND: Coronary computed tomography angiography (CCTA) may be useful for noninvasive follow-up; however, evaluation of coronary stenosis and CT number of plaque may be inaccurate under different vessel enhancement of contrast media. We examined the reproducibility of the CT number of repeat CCTA using our original CT number-controlling system (CTN-CS), which selects contrast level by a multiple regression equation using body surface area and peak CT number and peak time on timing bolus and during CCTA. METHODS AND RESULTS: One hundred seventy-two patients who underwent serial CCTA were prospectively and randomly assigned to 3 groups. In the first group, Group A, the amount of contrast for the second CCTA was determined by CTN-CS to match the intracoronary CT number of the first CCTA. In Group B, each patient received the same amount of intravenous contrast in both CCTA examinations. In Group C, 0.7 mL/mg body weight (BW) of contrast medium (350 mgI/mL) was used for baseline and follow-up CCTAs. The regression of repeated CCTAs was the best in Group A (r=0.85, p<0.001) vs. Group B (r=0.52, p<0.001), and Group C (r=0.61, p<0.001). The absolute difference between intracoronary CT numbers of the second and first CCTA was the lowest in Group A (24.8 ± 21.8HU), followed by Group B (37.6 ± 26.2 HU; p<0.05) and Group C (46.5 ± 34.4HU; p<0.001). CONCLUSIONS: Using CTN-CS, the difference of intracoronary CT numbers of the second and first CCTA was the smallest when compared to CCTAs using the same contrast volumes or constant volumes per body weight.