NSPlex: an efficient method to analyze non-specific peaks amplified using commercial STR kits.

Commercial short tandem repeat (STR) kits exclusively contain human-specific primers; however, various non-human organisms with high homology to the STR kit's primer sequences can cause cross-reactivity. Owing to the proprietary nature of the primers in STR kits, the origins and sequences of most non-specific peaks (NSPs) remain unclear. Such NSPs can complicate data interpretation between the casework and reference samples; thus, we developed "NSPlex", an efficient method to discover the biological origins of NSPs. We used leftover STR kit amplicons after capillary electrophoresis and performed advanced bioinformatics analyses using next-generation sequencing followed by BLAST nucleotide searches. Using our method, we could successfully identify NSP generated from PCR amplicons of a sample mixture of human DNA and DNA extracted from matcha powder (finely ground powder of green tea leaves and previously known as a potential source of NSP). Our results showed our method is efficient for NSP analysis without the need for the primer information as in commercial STR kits.

Combined use of the Clinical Frailty Scale and laboratory tests in acutely hospitalized older patients.

AIMS: To evaluate the Clinical Frailty Scale (CFS) and a Frailty Index based on laboratory tests (FI-lab) in terms of what each assesses about frailty and to determine the appropriateness of combined use of these two frailty scales. METHODS: This was a prospective observational cohort study in an acute geriatric ward of a university hospital. The FI-lab is the proportion of laboratory parameters that yield abnormal results from a total of 23. The FI-lab and CFS were assessed at admission. Data on activities of daily living (ADL), cognition, geriatric syndromes, and comorbidities were also collected. Main outcomes were in-hospital mortality and 90-day mortality after admission. RESULTS: In total, 378 inpatients (mean age 85.2 ± 5.8 years, 59.3% female) were enrolled. ADL and cognition correlated strongly with the CFS (Spearman's |r|> 0.60) but weakly with the FI-lab (|r|< 0.30). Both the CFS and FI-lab correlated weakly with geriatric syndromes and comorbidities (|r|< 0.40). The correlation between the CFS and FI-lab was also weak (r = 0.28). The CFS and FI-lab were independently associated with in-hospital mortality and 90-day mortality after admission. The Akaike information criterion was lower for models using both the CFS and FI-lab than for models using either tool alone. CONCLUSIONS: The CFS and FI-lab each reflected only some of the aspects of frailty in acutely hospitalized older patients. The model fit was better when the two frailty scales were used together to assess the mortality risk than when either was used alone.

Evidence for Very Recent Positive Selection in Mongolians.

Mongols, the founders of the largest continental empire in history, successfully adapted to the harsh environments of Inner Asia through nomadic pastoralism. Considerable interest exists in ascertaining whether genetic adaptation also contributed to the Mongols' success, and dissecting the genome diversity of present-day populations in Mongolia can help address this question. To this end, we determined the genotypes of nearly 2.4 million single nucleotide polymorphisms (SNPs) of 96 unrelated Mongolian individuals in Ulaanbaatar city, and performed genome-wide scans for population-specific positive selection. We discovered signatures of Mongolian-specific positive selection at the chromosomal region 3p12.1, in which hits in genome-wide association studies were reported for medical and biological traits related to energy metabolism and reproduction. The top SNP, rs117799927, showed a distinctive geographic distribution: the frequency of the derived allele, rs117799927 G, was extremely low among worldwide populations (0.005) but exceptionally high in Mongolians (0.247). Approximate Bayesian computation-based age estimation showed that the rs117799927 G allele emerged or positive selection began to operate 50 generations before the present, near the age of the climate anomaly named Late Antique Little Ice Age. Furthermore, rs117799927 showed significant associations with multiple adiposity-related traits in Mongolians and allelic difference in enhancer activity in cells of adipocyte lineage, suggesting that positive selection at 3p12.1 might be related to adaptation in the energy metabolism system. These findings provide novel evidence for a very recent positive-selection event in Homo sapiens and offer insights into the roles of genes in 3p12.1 in the adaptive evolution of our species.

Influence of AHRR Pro189Ala polymorphism on kidney functions.

The function of aryl hydrocarbon receptor repressor (AHRR) in the kidney is unclear. The present study investigated associations between AHRR Pro189Ala polymorphism and estimated glomerular filtration rates (eGFR), serum creatinine, and hemoglobin levels in 2775 Japanese adults without diabetes. In addition, we examined whether AHRR expression levels in the kidney of control and chronic kidney disease (CKD) rats were changed. Multiple linear regression analyses showed that carriers of the Ala allele had increased eGFR and lower concentrations of serum creatinine and hemoglobin (p < 0.05). Immunohistochemical analysis showed that the expression of AHRR was upregulated in the kidneys of rats with CKD. These findings suggest that AHRR plays distinct roles in kidney functions and hemoglobin values. The effects of the AHRR polymorphism might be intensified in the kidneys of patients with CKD.

Identification of deleterious rare variants in MTTP, PNPLA3, and TM6SF2 in Japanese males and association studies with NAFLD.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a disorder characterized by excessive fat deposits in hepatocytes without excessive alcohol intake. NAFLD is influenced by genetic factors, and the heritability has been estimated at 0.35 to 0.6 by twin studies. We explored rare variants in known NAFLD-associated genes to investigate whether these rare variants are involved in the susceptibility to NAFLD. METHODS: The target genes for re-sequencing were PNPLA3, TM6SF2, and MTTP. All exons of these three genes were amplified from a discovery panel of 950 Japanese males, and the identified rare variants were further tested for genetic association in 3014 individuals from the Japanese general population and for in vitro functional evaluation. RESULTS: Target re-sequencing analysis using next-generation sequencing identified 29 rare variants in 65 Japanese males (6.84%), 12 of which were newly identified base substitutions. A splicing mutation in TM6SF2 that resulted in deletion of 31 amino acids was identified in an NAFLD case. Among eight genotyped rare single-nucleotide polymorphisms (SNPs; minor allele frequency < 0.02), rs143392071 (Tyr220Cys, PNPLA3) significantly increased (odds ratio = 3.52, P = 0.008) and rs756998920 (Val42Ile, MTTP) significantly decreased (odds ratio = 0.03, P = 0.019) the NAFLD risk. Functional assays showed that these two SNPs disrupted protein functions and supported the genetic association. CONCLUSION: Collectively, 1.79% of individuals in our studied population were estimated carriers of rare variants that are potentially associated with NAFLD.

[State of glycemic control in elderly diabetic patients].

AIM: The Japan Diabetes Society and The Japan Geriatric Society made a joint committee and published a new glycemic target in May 2016. Because reports on the state of glycemic control in elderly diabetic patients are insufficient, we investigated the state of glycemic control in this population before the new glycemic target was established. METHODS: We enrolled patients older than 65 years of age who had been prescribed antidiabetic drugs and hospitalized in the geriatric department of Nagoya University Hospital from April 1, 2015, to March 31, 2016. We investigated the participants' HbA1c, prescription of antidiabetic drugs carrying risks of severe hypoglycemia (risk drugs) at hospitalization, cognitive function, basic activities of daily living, and instrumental activities of daily living. RESULTS: A total of 63 patients were enrolled. Thirty-five patients were male, the mean age was 83.1±5.9 years old, and the average HbA1c was 7.6%±1.5%. The numbers of patients assigned to categories I, II, and III were 10, 12, and 41, respectively. For prescription of risk drugs, 6 participants were assigned to category I, 8 to category II, and 22 to category III. Prescription of risk drugs was associated with the HbA1c. Approximately one third of the patients using risk drugs had a lower HbA1c than the target value. CONCLUSIONS: Risk drugs was not significantly associated with the established categories or age. Many of the patients who were prescribed risk drugs had a lower HbA1c than the target values.

ZNF70, a novel ILDR2-interacting protein, contributes to the regulation of HES1 gene expression.

A diabetes susceptibility gene, immunoglobulin-like domain containing receptor 2 (Ildr2), encodes a transmembrane protein localized to the endoplasmic reticulum membrane that is closely related to hepatic lipid metabolism. The livers of ob/ob mice in which Ildr2 is transiently overexpressed are relieved of hepatic steatosis. However, the molecular mechanisms through which ILDR2 affects these changes in hepatic lipid metabolism remain unknown. This study aimed to identify ILDR2-interacting proteins to further elucidate the molecular mechanisms underlying the role of ILDR2 in lipid homeostasis. We purified ILDR2-containing protein complexes using tandem affinity purification tagging and identified ZNF70, a member of the Kruppel C2H2-type zinc finger protein family, as a novel ILDR2-interacting protein. We demonstrated that ZNF70 interacts with ZFP64 and activates HES1 transcription by binding to the HES1 promoter. In addition, HES1 gene expression is increased in ILDR2-knockdown HepG2 cells, in which ZNF70 is translocated from the cytoplasm to the nucleus, suggesting that ZNF70 migration to the nucleus after dissociating from the ILDR2-ZNF70 complex activates HES1 transcription. These results support a novel link between ILDR2 and HES1 gene expression and suggest that ILDR2 is involved in a novel pathway in hepatic steatosis.

Replication analysis of genetic association of the NCAN-CILP2 region with plasma lipid levels and non-alcoholic fatty liver disease in Asian and Pacific ethnic groups.

BACKGROUND: The Neurocan-cartilage intermediate layer protein 2 (NCAN-CILP2) region forms a tight linkage disequilibrium (LD) block and is associated with plasma lipid levels and non-alcoholic fatty liver disease (NAFLD) in individuals of European descent but not in the Malay and Japanese ethnic groups. Recent genome-wide resequence studies identified a missense single-nucleotide polymorphism (SNP) (rs58542926) of the transmembrane 6 superfamily member 2 (TM6SF2) gene in the NCAN-CILP2 region related to hepatic triglyceride content. This study aims to analyze the influences of SNPs in this region on NAFLD and plasma lipid levels in the Asian and Pacific ethnic groups and to reveal the reasons behind positive and negative genetic associations dependent on ethnicity. METHODS: Samples and characteristic data were collected from 3,013 Japanese, 119 Palauan, 947 Mongolian, 212 Thai and 401 Chinese people. Hepatic sonography data was obtained from the Japanese individuals. Genotyping data of five SNPs, rs58542926, rs735273, rs1009136, rs1858999, and rs16996148, were used to verify the effect on serum lipid levels by multiple linear regression, and the association with NAFLD in the Japanese population was examined by logistic regression analysis. RESULTS: rs58542926 showed significant association with the plasma triglyceride (TG) level in Japanese (P = 0.0009, effect size = 9.5 (± 3.25) mg/dl/allele) and Thai (P = 0.0008, effect size = 31.6 (± 11.7) mg/dl/allele) study subjects. In Mongolian individuals, there was a significant association of rs58542926 with total cholesterol level (P = 0.0003, 11.7 (± 3.2) mg/dl/allele) but not with TG level. In multiple comparisons in Chinese individuals, rs58542926 was weakly (P = 0.022) associated with TG levels, although the threshold for statistical significance was not reached. In Palauan individuals, there was no significant association with the studied SNPs. rs58542926 also showed significant association with Japanese NAFLD. The minor allele (t) increased NAFLD risk (OR 1.682, 95 % CI 1.289-2.196, p value 0.00013). CONCLUSION: This study confirmed the genetic association of missense SNP of TM6SF2, rs58542926, with plasma lipid levels in multiple East Asian ethnic groups and with NAFLD in Japanese individuals.

Sin3A-associated protein, 18 kDa, a novel binding partner of TRIB1, regulates MTTP expression.

Mammalian tribbles homolog 1 (TRIB1) is a human locus that has been shown to significantly impact plasma lipid levels across several ethnic groups. In addition, the gene has been associated with the occurrence of nonalcoholic fatty liver disease. In the present study, a yeast-two-hybrid system was used to screen for novel molecular targets of TRIB1 binding. Loci corresponding to clones that were positive for TRIB1 binding subsequently were assessed for roles in lipid metabolism in mice using adenoviral constructs to induce knockdown or overexpression. Sin3A-associated protein, 18 kDa (SAP18) was identified as a novel binding partner of TRIB1. Knockdown of the Sap18 in mouse liver decreased plasma lipid levels and increased hepatic lipid levels; SAP18 overexpression showed the opposite effects. Transcriptome analysis of the mouse liver revealed that Sap18 knockdown decreased and SAP18 overexpression increased microsomal TG transfer protein (MTTP) expression levels. Chromatin immunoprecipitation analysis showed that halo-tagged SAP18, halo-tagged TRIB1, and anti-mSin3A antibody enriched precipitates for regulatory sequences of the MTTP gene. Enforced expression of SAP18 enhanced and SAP18 knockdown conversely attenuated the enrichment of MTTP regulatory sequences seen with anti-mSin3A antibody. These studies indicated that SAP18 expression enhanced the recruitment of mSin3A in coordination with TRIB1 to MTTP regulatory elements and increased MTTP expression.

The role of TRIB1 in lipid metabolism; from genetics to pathways.

The plasma concentration of lipids is a heritable risk factor for the development of atherosclerosis and related coronary artery diseases (CAD). Mammalian tribbles homologue 1 (TRIB1) is a human locus, the downstream linkage disequilibrium (LD) block of which affects plasma low-density lipoprotein (LDL)-associated cholesterol, triglyceride (TG) levels and CAD across multiple ethnic groups. In addition, association of TRIB1 with non-alcoholic fatty liver disease (NAFLD) has also been shown. A regulatory sequence that enhances TRIB1 promoter activity was identified in the LD block and the minor allele of a single nt polymorphism (SNP, rs6982502) in this regulatory sequence reduces the activity of the TRIB1 promoter. The minor allele of rs6982502 is a risk allele for increasing plasma lipid levels and NAFLD. Trib1 deficiency increases plasma cholesterol and TGs in mice and overexpression of TRIB1 in mouse liver reduces these factors. Expression of rate-limiting lipogenic enzymes is increased in Trib1-knockout mouse liver and decreased with overexpression. Recently, carbohydrate-responsive element-binding protein (ChREBP) emerged as a novel binding partner of TRIB1. Furthermore, novel binding partner, Sin3A (Swi-independent 3A)-associated protein, 18 kDa, was identified, which activates microsomal TG transfer protein (MTTP) expression by binding with MTTP regulatory elements in co-ordination with mSin3A and TRIB1. Very recently, a small molecular compound that up-regulates TRIB1 expression in HepG2 cells has been discovered. Further exploration of the binding partners of TRIB1 and their involvement in lipid metabolism may aid discovery of novel pharmacological targets for the management of dyslipidaemia and steatosis.

Common variants of GIP are associated with visceral fat accumulation in Japanese adults.

Animal studies have demonstrated that glucose-dependent insulinotropic polypeptide (GIP) and GIP receptor (GIPR) contribute to the etiology of obesity. In humans, genomewide association studies have identified single nucleotide polymorphisms (SNPs) in the GIPR gene that are strongly associated with body mass index (BMI); however, it is not clear whether genetic variations in the GIP gene are involved in the development of obesity. In the current study, we assessed the impact of GIP SNPs on obesity-related traits in Japanese adults. Six tag SNPs were tested for associations with obesity-related traits in 3,013 individuals. Multiple linear regression analyses showed that rs9904288, located at the 3'-end of GIP, was significantly associated with visceral fat area (VFA). Moreover, rs1390154 and rs4794008 showed significant associations with plasma triglyceride levels and hemoglobin A1c levels, respectively. Among the significant SNPs, rs9904288 and rs1390154 were independently linked with SNPs in active enhancers of the duodenum mucosa, the main GIP-secreting tissue. The haplotypes of these two SNPs exhibited stronger associations with VFA. Numbers of VFA-increasing alleles of rs9904288 and BMI-increasing alleles of previously identified GIPR SNPs showed a strong additive effect on VFA, waist circumference, and BMI in the subject population. These novel results support the notion that the GIP-GIPR axis plays a role in the etiology of central obesity in humans, which is characterized by the accumulation of visceral fat.

Unilateral gluteal myositis as a unique presentation in mesenteric Kikuchi-Fujimoto disease.

Kikuchi-Fujimoto disease (KFD) is a self-limiting disease, characterised by fever and cervical lymphadenopathy. Lymphadenopathy without cervical lymph node involvement is rare and may mimic lymphoma. Although KFD can be associated with extranodal involvement, muscle involvement has not been reported. Herein, we report a novel case of unilateral gluteal myositis associated with mesenteric KFD in a patient who presented with persistent fever and right hip pain. Radiological imaging revealed an inflammatory lesion on the right gluteal muscle and multiple enlarged abdominal lymph nodes. No cervical lymphadenopathy was observed. A mesenteric lymph node biopsy was performed, and the histopathological findings led to a diagnosis of KFD. By day 29, the patient's body temperature gradually returned to normal without any therapeutic intervention. Follow-up radiological imaging showed resolution of the gluteal lesion and a significant decrease in abdominal lymph node size. Considering the clinical course, the unilateral myositis may have developed as an extranodal involvement of KFD. Even if the clinical findings appear unrelated to those of KFD, a differential diagnosis that includes KFD should be considered in patients with unknown origin of fever.

Relationship between cognitive function and phase angle measured with a bioelectrical impedance system.

PURPOSE: The purpose of this study was to investigate the relationship between cognitive function and phase angle (PhA), an indicator of muscle quality. METHODS: This cross-sectional study enrolled outpatients who visited a memory clinic at the Nagoya University hospital from January 2016 to June 2022. We enrolled 153 participants with body composition measurements. Inclusion criteria were a Mini-Mental State Examination score of 20-30 and a clinical diagnosis of Alzheimer's dementia (AD) or amnesic mild cognitive impairment (aMCI). The background characteristics of the participants were compared according to AD and aMCI. Next, linear regression analysis was performed with PhA as the objective variable. In addition, logistic regression analysis was performed for AD diagnosis. RESULTS: PhA was lower in the AD group (P = 0.009). In linear regression analysis, PhA consistently decreased with worsening ADAS score. In logistic regression analysis, high PhA was associated with absence of AD. Gender-specific analyses showed these associations existed only in men. CONCLUSIONS: Our study of patients with AD and aMCI found that PhA decreased with worsening of cognitive function. Compared with aMCI, AD was associated with significantly lower PhA. Our results strengthen the limited evidence in the literature showing that low muscle quality is associated with poor cognitive function.

Frailty Index Based on Common Laboratory Tests for Patients Starting Home-Based Medical Care.

OBJECTIVES: To determine whether a Frailty Index based on laboratory tests (FI-lab) is associated with clinical outcomes independently of a standard nonlaboratory Frailty Index (FI-clinical) in older patients starting home-based medical care. DESIGN: Secondary analysis of data from a multicenter prospective cohort study. SETTING AND PARTICIPANTS: Patients aged ≥65 years who were starting home-based medical care services provided by doctors and nurses at Nagoya, Japan. METHODS: We calculated FI-lab (proportion of abnormal results out of 25 commonly tested laboratory parameters) and FI-clinical using 42 items based on data obtained at enrollment. The primary outcome was mortality within 2 years after starting home-based medical care. A sensitivity analysis was also conducted with 1-year mortality as the outcome. Other outcomes included hospitalization and nursing home admission within 2 years. RESULTS: In total, 188 patients (mean age 79.9 ± 10.2 years, 57.5% male) were included. The median FI-lab was 0.40 [interquartile range (IQR) 0.29-0.50] and the median FI-clinical was 0.32 (IQR 0.24-0.43). Sixty-nine patients (36.7%) died within 2 years of starting home-based medical care. A Cox proportional hazards regression analysis including age, sex, FI-lab, and FI-clinical as independent variables revealed that FI-lab was associated with 2-year mortality independently of FI-clinical [FI-lab per 0.1 unit, odds ratio (OR) 1.49, 95% CI 1.25-1.77; FI-clinical per 0.1 unit, OR 1.13, 95% CI 0.90-1.41]. The sensitivity analysis showed similar results for 1-year mortality. Neither FI-lab nor FI-clinical was associated with hospitalization or nursing home admission within 2 years. CONCLUSIONS AND IMPLICATIONS: FI-lab was associated with 2-year mortality in patients starting home-based medical care, independently of FI-clinical, and may be useful for risk assessment in this population. Studies with larger sample sizes are needed.

Association of marital relationship with quality of life among older adults with mild cognitive impairment and mild dementia.

The marital relationship is associated with the quality of life among those with cognitive impairment, but sarcopenia status seems to play an important role in the association.

Dual Sensory Impairment Predicts an Increased Risk of Postdischarge Falls in Older Patients.

OBJECTIVES: The purpose of this study was to determine the associations of vision impairment, hearing impairment, and comorbid vision and hearing impairment [ie, dual sensory impairment (DSI)] on admission to hospital with falls within 3 months of discharge in older patients. DESIGN: This prospective multicenter study included patients admitted to and discharged from geriatric wards at 3 university hospitals and 1 national medical center in Japan between October 2019 and July 2023. SETTING AND PARTICIPANTS: Of 1848 individuals enrolled during the study period, 1141 were excluded, leaving 707 for inclusion in the analysis. METHODS: Participants' background factors were compared in terms of whether they had a fall during the 3 months postdischarge. Logistic regression analysis was then performed using the presence or absence of falls after discharge as the objective variable. Three models were created using vision impairment, hearing impairment, and DSI as covariates. Other covariates included physical function, cognitive function, and depression. In addition, logistic regression analysis was performed with falls during hospitalization as the objective variable. RESULTS: DSI was significantly more common in the falls group (P = .004). Logistic regression analysis showed that the risk of falls after discharge was higher in patients with DSI (odds ratio 3.432, P = .006) than in those with vision or hearing impairment alone. When adjusted for physical function, cognitive function, depression, and discharge location, DSI was significantly associated with an increased risk of falls after discharge (odds ratio 3.107, P = .021). The association between DSI and falls during hospitalization did not reach statistical significance, but a trend was observed. CONCLUSIONS AND IMPLICATIONS: This study is the first to show an association between DSI and falls after discharge. Simple interventions for patients with DSI may be effective in preventing falls, and we suggest that they be actively implemented early during hospitalization.

Objective physical function declines in the absence of subjective physical complaints among patients with amnestic mild cognitive impairments and mild alzheimer's disease.

PURPOSE: To examine the extent to which patients with amnestic mild cognitive impairment (aMCI) or Alzheimer's disease (AD) perceive their own physical decline. METHODS: This study included 4450 outpatients (1008 normal cognition [NC], 1605 aMCI, and 1837 mild AD) who attended an initial visit to a memory clinic between July 2010 and June 2021. Their physical function was assessed by the Timed Up and Go test, one-leg standing test, and grip strength. For physical complaints, data were obtained on reports of fear of falling and dizziness or staggering. Logistic regression analysis was performed to compare the patients' physical function and complaints for each stage of NC, aMCI, and mild AD. RESULTS: Objective physical function declined from aMCI and the mild AD stage, but subjective physical complaints decreased by 20-50% in aMCI and 40-60% in mild AD compared with the NC group. CONCLUSION: As objective physical functional declined from the aMCI stage onward, subjective physical complaints decreased. This suggests a need for objective assessment of physical function in aMCI and mild AD patients even when they have no physical complaints in the clinical setting.

Unfavorable switching of skewed X chromosome inactivation leads to Menkes disease in a female infant.

Menkes disease is an X-linked disorder of copper metabolism caused by mutations in the ATP7A gene, and female carriers are usually asymptomatic. We describe a 7-month-old female patient with severe intellectual disability, epilepsy, and low levels of serum copper and ceruloplasmin. While heterozygous deletion of exons 16 and 17 of the ATP7A gene was detected in the proband, her mother, and her grandmother, only the proband suffered from Menkes disease clinically. Intriguingly, X chromosome inactivation (XCI) analysis demonstrated that the grandmother and the mother showed skewing of XCI toward the allele with the ATP7A deletion and that the proband had extremely skewed XCI toward the normal allele, resulting in exclusive expression of the pathogenic ATP7A mRNA transcripts. Expression bias analysis and recombination mapping of the X chromosome by the combination of whole genome and RNA sequencing demonstrated that meiotic recombination occurred at Xp21-p22 and Xq26-q28. Assuming that a genetic factor on the X chromosome enhanced or suppressed XCI of its allele, the factor must be on either of the two distal regions derived from her grandfather. Although we were unable to fully uncover the molecular mechanism, we concluded that unfavorable switching of skewed XCI caused Menkes disease in the proband.