Clinical characteristics of and growth hormone treatment effects on short stature with type 1 insulin-like growth factor receptor (IGF1R) gene alteration.

Short stature with IGF-1 receptor (IGF1R) gene alteration is known as small-for-gestational-age (SGA) short stature with elevated serum IGF1 levels. Its prevalence and clinical characteristics remain unclear. No adapted treatment is available for short stature related to IGF1R gene alteration in Japan, and genetic testing is not yet widely accessible. We investigated short stature with IGF1R gene alterations and analyzed the clinical data of 13 patients using the results of questionnaires issued to the Japanese Society for Pediatric Endocrinology. Four cases were caused by a deletion of chromosome 15q26.3, and eight were caused by heterozygous pathogenic variants in the IGF1R gene. Cases with deletions showed a more severe degree of growth impairment (-4.5 ± 0.43 SD) than those caused by pathological variants (-2.71 ± 0.15 SD) and were accompanied by neurodevelopmental delay. However, cases caused by pathological variants lacked distinctive features. Only three of the 12 cases demonstrated serum IGF1 values exceeding +2 SD, and the other three had values below 0 SD. Four patients did not meet the criteria for SGA at birth. Six patients received GH therapy for SGA short stature and showed improvement in growth rate without any side effects or elevated serum IGF1 levels during treatment. Elevated IGF1 levels (over +2 SD) after GH treatment should be considered a suspicious finding. Owing to the lack of distinctive features, there was a possibility of undiagnosed cases of this condition. Promoting genetic testing and clinical trials on GH administration for this condition is recommended.

A Japanese Boy With Spotted Fever and Overlapping Symptoms of Kawasaki Disease: A Case Report.

Japanese spotted fever (JSF) is a tick-transmitted infection caused by Rickettsia japonica (R. japonica), which is indigenous to Japan. Patients with JSF typically present with fever and spotted erythema on the palms and/or soles, and most of them have site(s) of tick bites. The prognosis is good, but some cases have a fatal course. Kawasaki disease (KD) is a systemic vasculitis with an unknown cause that is characterized by symptoms such as fever, conjunctival injection, oral findings, amorphous rash, rigid edema, and nonsuppurative cervical lymphadenopathy. Although the symptoms of JSF are partially similar to those of KD, case reports of JSF overlapping KD have never been internationally published. Herein, we report a boy with JSF and KD symptoms. A five-year-old boy presented with fever and rash after he had been on a mountain inhabited by R. japonica. On the fifth day, erythema was spotted mainly on his bilateral palms, bilateral cervical lymphadenopathy, rigid edema of his lower feet, and mild conjunctival injection appeared. Intravenous immunoglobulin (IVIG) therapy was performed because these symptoms satisfied five out of the six diagnostic criteria for KD. However, on the sixth day, the fever persisted, and then we readministered IVIG in addition to tosufloxacin and azithromycin since we found a tick-bite eschar, which suggested a complication of JSF. His symptoms resolved soon after this treatment. Coronary artery lesions were never observed. This case indicates that the R. japonica infection overlaps clinically with KD. Tosufloxacin and azithromycin should be considered to avoid the use of minocycline in younger patients with JSF.

MELAS-Derived Neurons Functionally Improve by Mitochondrial Transfer from Highly Purified Mesenchymal Stem Cells (REC).

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episode (MELAS) syndrome, caused by a single base substitution in mitochondrial DNA (m.3243A>G), is one of the most common maternally inherited mitochondrial diseases accompanied by neuronal damage due to defects in the oxidative phosphorylation system. There is no established treatment. Our previous study reported a superior restoration of mitochondrial function and bioenergetics in mitochondria-deficient cells using highly purified mesenchymal stem cells (RECs). However, whether such exogenous mitochondrial donation occurs in mitochondrial disease models and whether it plays a role in the recovery of pathological neuronal functions is unknown. Here, utilizing induced pluripotent stem cells (iPSC), we differentiated neurons with impaired mitochondrial function from patients with MELAS. MELAS neurons and RECs/mesenchymal stem cells (MSCs) were cultured under contact or non-contact conditions. Both RECs and MSCs can donate mitochondria to MELAS neurons, but RECs are more excellent than MSCs for mitochondrial transfer in both systems. In addition, REC-mediated mitochondrial transfer significantly restored mitochondrial function, including mitochondrial membrane potential, ATP/ROS production, intracellular calcium storage, and oxygen consumption rate. Moreover, mitochondrial function was maintained for at least three weeks. Thus, REC-donated exogenous mitochondria might offer a potential therapeutic strategy for treating neurological dysfunction in MELAS.

PHACE(S) Syndrome with Ocular Involvements and No Periocular Hemangioma.

PHACE(S) syndrome is a neurocutaneous disorder with a hallmark finding of an infantile facial hemangioma (IFH) >5 cm. Eye examination of patients with PHACE(S) syndrome with no IFH at periorbital region is reported to be of low yield. We report a unique case of the syndrome with ocular manifestations without periorbital IFH or systemic findings. A 3-week-old female infant with right periauricular IFH >5 cm, extending to the neck and cheek and lower lip IFH was presented. Examination revealed pseudoptosis due to microphthalmia with esotropia and hypertropia. Both corneas were clear with diameters of 5 mm and 10 mm, right eye (RE) and left eye (LE), respectively. There was a posterior polar cataract with a poor view of the fundus RE. Ocular B-scan and magnetic resonance imaging (MRI) findings were suggestive of a dysmorphic globe, vitreous hemorrhage, spherophakia and persistent fetal vasculature RE and normal findings LE. Clinical evaluation, MRI, and MR angiography revealed no other systemic abnormalities. Subsequent follow-up visits revealed progressive clouding of the cornea with neovascularization and the development of phthisis bulbi RE at which point an ocular prosthesis was placed. The IFH was managed with dye laser and with oral propranolol. At 1 year, the patient has remained stable with no development of new local or systemic anomalies, regression of the periauricular and lip IFH, and normal development of the orbital structure RE with an ocular prosthesis in situ. Ocular involvement in patients with PHACE(S) syndrome may be present without periorbital IFH. Regardless of the location of the IFH and the presence or absence of a periocular component, it is recommended that they receive a full initial ophthalmological assessment.

Genetic, electrophysiological, and pathological studies on patients with SCN9A-related pain disorders.

BACKGROUND AND AIMS: Voltage-gated sodium channel Nav1.7, encoded by the SCN9A gene, has been linked to diverse painful peripheral neuropathies, represented by the inherited erythromelalgia (EM) and paroxysmal extreme pain disorder (PEPD). The aim of this study was to determine the genetic etiology of patients experiencing neuropathic pain, and shed light on the underlying pathogenesis. METHODS: We enrolled eight patients presenting with early-onset painful peripheral neuropathies, consisting of six cases exhibiting EM/EM-like disorders and two cases clinically diagnosed with PEPD. We conducted a gene-panel sequencing targeting 18 genes associated with hereditary sensory and/or autonomic neuropathy. We introduced novel SCN9A mutation (F1624S) into a GFP-2A-Nav1.7rNS plasmid, and the constructs were then transiently transfected into HEK293 cells. We characterized both wild-type and F1624S Nav1.7 channels using an automated high-throughput patch-clamp system. RESULTS: From two patients displaying EM-like/EM phenotypes, we identified two SCN9A mutations, I136V and P1308L. Among two patients diagnosed with PEPD, we found two additional mutations in SCN9A, F1624S (novel) and A1632E. Patch-clamp analysis of Nav1.7-F1624S revealed depolarizing shifts in both steady-state fast inactivation (17.4 mV, p < .001) and slow inactivation (5.5 mV, p < .001), but no effect on channel activation was observed. INTERPRETATION: Clinical features observed in our patients broaden the phenotypic spectrum of SCN9A-related pain disorders, and the electrophysiological analysis enriches the understanding of genotype-phenotype association caused by Nav1.7 gain-of-function mutations.

Extracellular Vesicles and Cx43-Gap Junction Channels Are the Main Routes for Mitochondrial Transfer from Ultra-Purified Mesenchymal Stem Cells, RECs.

Mitochondria are essential organelles for maintaining intracellular homeostasis. Their dysfunction can directly or indirectly affect cell functioning and is linked to multiple diseases. Donation of exogenous mitochondria is potentially a viable therapeutic strategy. For this, selecting appropriate donors of exogenous mitochondria is critical. We previously demonstrated that ultra-purified bone marrow-derived mesenchymal stem cells (RECs) have better stem cell properties and homogeneity than conventionally cultured bone marrow-derived mesenchymal stem cells. Here, we explored the effect of contact and noncontact systems on three possible mitochondrial transfer mechanisms involving tunneling nanotubes, connexin 43 (Cx43)-mediated gap junction channels (GJCs), and extracellular vesicles (Evs). We show that Evs and Cx43-GJCs provide the main mechanism for mitochondrial transfer from RECs. Through these two critical mitochondrial transfer pathways, RECs could transfer a greater number of mitochondria into mitochondria-deficient (ρ0) cells and could significantly restore mitochondrial functional parameters. Furthermore, we analyzed the effect of exosomes (EXO) on the rate of mitochondrial transfer from RECs and recovery of mitochondrial function. REC-derived EXO appeared to promote mitochondrial transfer and slightly improve the recovery of mtDNA content and oxidative phosphorylation in ρ0 cells. Thus, ultrapure, homogenous, and safe stem cell RECs could provide a potential therapeutic tool for diseases associated with mitochondrial dysfunction.

Highly-purified rapidly expanding clones, RECs, are superior for functional-mitochondrial transfer.

BACKGROUND: Mitochondrial dysfunction caused by mutations in mitochondrial DNA (mtDNA) or nuclear DNA, which codes for mitochondrial components, are known to be associated with various genetic and congenital disorders. These mitochondrial disorders not only impair energy production but also affect mitochondrial functions and have no effective treatment. Mesenchymal stem cells (MSCs) are known to migrate to damaged sites and carry out mitochondrial transfer. MSCs grown using conventional culture methods exhibit heterogeneous cellular characteristics. In contrast, highly purified MSCs, namely the rapidly expanding clones (RECs) isolated by single-cell sorting, display uniform MSCs functionality. Therefore, we examined the differences between RECs and MSCs to assess the efficacy of mitochondrial transfer. METHODS: We established mitochondria-deficient cell lines (ρ0 A549 and ρ0 HeLa cell lines) using ethidium bromide. Mitochondrial transfer from RECs/MSCs to ρ0 cells was confirmed by PCR and flow cytometry analysis. We examined several mitochondrial functions including ATP, reactive oxygen species, mitochondrial membrane potential, and oxygen consumption rate (OCR). The route of mitochondrial transfer was identified using inhibition assays for microtubules/tunneling nanotubes, gap junctions, or microvesicles using transwell assay and molecular inhibitors. RESULTS: Co-culture of ρ0 cells with MSCs or RECs led to restoration of the mtDNA content. RECs transferred more mitochondria to ρ0 cells compared to that by MSCs. The recovery of mitochondrial function, including ATP, OCR, mitochondrial membrane potential, and mitochondrial swelling in ρ0 cells co-cultured with RECs was superior than that in cells co-cultured with MSCs. Inhibition assays for each pathway revealed that RECs were sensitive to endocytosis inhibitor, dynasore. CONCLUSIONS: RECs might serve as a potential therapeutic strategy for diseases linked to mitochondrial dysfunction by donating healthy mitochondria.

The magnitude of CXCR4 signaling regulates resistance to quizartinib in FLT3/ITD+ cells via RUNX1.

CXCR4 antagonists sensitize FLT3/ITD+ AML cells to FLT3 inhibitors; however, CXCR4 signaling can induce apoptosis in AML cells, raising the question of whether CXCR4 signaling exerts divergent effects on FLT3/ITD+ cells. The present study investigated the paradoxical function of CXCR4 in resistance to FLT3 inhibitors. The FLT3 inhibitor quizartinib significantly decreased the number of FLT3/ITD+ Ba/F3 cells, whereas 1 ng/ml CXCL12 showed a significant protective effect against quizartinib. In contrast, CXCL12 over 100 ng/ml significantly decreased FLT3/ITD+ cell viability with concomitant downregulation of Runx1. Moreover, the survival of FLT3/ITD+ Ba/F3 or MOLM13 cells with low surface CXCR4 expression incubated with quizartinib was significantly enhanced by 100 ng/ml CXCL12; however, this protective effect of CXCL12 against quizartinib was barely detected in cells with high surface CXCR4 expression. Although silencing Runx1 downregulated CXCR4 expression, RUNX1 expression levels were significantly higher in CXCR4LOW FLT3/ITD+ Ba/F3 cells incubated with 100 ng/ml CXCL12 than in CXCR4HIGH cells, coincident with an increase in FLT3 phosphorylation. Silencing RUNX1 partially abrogated resistance to quizartinib in CXCR4LOW cells incubated with CXCL12, whereas ectopic RUNX1 significantly restored resistance in CXCR4HIGH cells. These results indicate that CXCR4 signaling of different magnitudes paradoxically regulates resistance to quizartinib in FLT3/ITD+ cells via RUNX1.

Clinical and molecular investigation of 37 Japanese patients with multiple acyl-CoA dehydrogenase deficiency: p.Y507D in ETFDH, a common Japanese variant, causes a mortal phenotype.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is an inherited metabolic disease caused by a defect in electron transfer flavoprotein alpha (ETFA), ETF beta (ETFB), or ETF dehydrogenase (ETFDH), and riboflavin metabolism disorders have recently been reported to present as mimicking MADD. MADD is roughly classified into neonatal (type 1 or 2) and later-onset (type 3) forms. To identify clinicogenetic characteristics in Japan, we investigated 37 Japanese patients with MADD diagnosed from 1997 to 2020. The causes of MADD were ETFDH deficiency in 26 patients, ETFA deficiency in four, ETFB deficiency in six, and riboflavin metabolism disorder in one. All 15 patients with the neonatal-onset type died by 2 years of age, while five of 22 patients with the later-onset form died by 3 years of age. Furthermore, 8 of 15 patients with the later-onset form of ETFDH deficiency treated with riboflavin were riboflavin non-responders. p.Y507D in ETFDH was identified as the most common variant (9 of 48 alleles, 18.8%). Of two patients with a homozygous p.Y507D variant, one experienced disease onset and died in the neonatal period, while the other experienced disease onset at two months of age and died at two years old, suggesting that the p.Y507D variant results in fatal outcomes. Our study concluded that more than half of Japanese patients with MADD died by three years old, and more than half of patients with the later-onset form had poor responsiveness to riboflavin, partly due to the unique Japanese p.Y507D variant in ETFDH.

ACTH-independent Cushing's syndrome due to ectopic endocrinologically functional adrenal tissue caused by a GNAS heterozygous mutation: a rare case of McCune-Albright syndrome accompanied by central amenorrhea and hypothyroidism: a case report and literature review.

In a small number of cases, the development of ectopic residual adrenal lesions during embryogenesis causing Cushing's syndrome due to the production of excess cortisol has been reported. A 29-year-old woman was admitted to our hospital for fatigue and recent amenorrhea. Her plasma ACTH was <1.5 pg/mL, and her serum cortisol was 21.4 pg/mL after the 8 mg dexamethasone suppression test, revealing the presence of ACTH-independent Cushing's syndrome; however, her bilateral adrenal glands were atrophied. Abdominal CT revealed a 40-mm round tumor on the right renal hilum and remarkably accumulated 131I-labelled adosterol. CT and bone scintigraphy showed that 99mTc-methylene diphosphonate had accumulated in her dissymmetric skull at the right-frontoparietal region. The tumor on the right renal hilum was laparoscopically removed. Her cortisol levels rapidly decreased to below the normal range, and glucocorticoids were administered to rescue adrenal insufficiency. The resected tumor was yellowish in appearance and 4.5×3.0×2.8 cm in size. Immunohistochemical staining for SF-1, P450scc, CYP17A, CYP21A, and CYP11B1 indicated that this tumor produced cortisol. Exome sequencing analysis revealed that the GNAS heterozygous mutation (c.601C>T, p. Arg201Cys; accession number, NM_000516.5) was found in approximately 20% of the adrenal tumor sample. A mutation of GNAS, encoding the Gsα subunit that mediates GPCR signaling, causes the constitutive activation of adenylyl cyclase, resulting in hypersecretion of hormones regulated by the GPCR. GNAS mutation is one of the major genetic causes of cortisol-producing adrenal tumors independent of ACTH secretion. Considering the combination of GNAS mutation with one of the typical clinical triad characteristics, fibrous dysplasia of bone, we diagnosed this patient with McCune-Albright syndrome accompanied by ACTH-independent Cushing's syndrome caused by an ectopic residual adrenal tumor due to GNAS mutation. This case highlights that GNAS involves a previously unknown pathological mechanism in which inhibition of the natural elimination of remnant tissue leads to ectopic endocrine hypersecretion.

Clinical and molecular features of patients with COL1-related disorders: Implications for the wider spectrum and the risk of vascular complications.

Abnormalities in type I procollagen genes (COL1A1 and COL1A2) are responsible for hereditary connective tissue disorders including osteogenesis imperfecta (OI), specific types of Ehlers-Danlos syndrome (EDS), and COL1-related overlapping disorder (C1ROD). C1ROD is a recently proposed disorder characterized by predominant EDS symptoms of joint and skin laxity and mild OI symptoms of bone fragility and blue sclera. Patients with C1ROD do not carry specific variants for COL1-related EDS, including classical, vascular, cardiac-valvular, and arthrochalasia types. We describe clinical and molecular findings of 23 Japanese patients with pathogenic or likely pathogenic variants of COL1A1 or COL1A2, who had either OI-like or EDS-like phenotypes. The final diagnoses were OI in 17 patients, classical EDS in one, and C1ROD in five. The OI group predominantly experienced recurrent bone fractures, and the EDS group primarily showed joint hypermobility and skin hyperextensibility, though various clinical and molecular overlaps between OI, COL1-related EDS, and C1ROD as well as intrafamilial phenotypic variabilities were present. Notably, life-threatening vascular complications (vascular dissections, arterial aneurysms, subarachnoidal hemorrhages) occurred in seven patients (41% of those aged >20 years) with OI or C1ROD. Careful lifelong surveillance and intervention regarding bone and vascular fragility could be required.

A Simple Flow Injection Analysis-Tandem Mass Spectrometry Method to Reduce False Positives of C5-Acylcarnitines Due to Pivaloylcarnitine Using Reference Ions.

Flow injection analysis−tandem mass spectrometry (FIA-TMS) has been applied in a first-tier test of newborn screening (NBS). Although isovalerylcarnitine (i-C5), which is a diagnostic indicator of isovaleric acidemia (IVA), is isobaric with pivaloylcarnitine (p-C5), 2-methylbutyrylcarnitine, and n-valerylcarnitine, these isomers cannot be distinguished by the FIA-TMS. There are many reports of false positives derived from p-C5 due to the use of pivalate-conjugated antibiotics. In this study, we developed a new FIA-TMS method to distinguish i-C5 and p-C5. We found that the intensity ratio of product ions for i-C5 and p-C5 was different in a certain range even under the same analytical conditions. The product ions with the most distinct differences in ionic intensity between the isomers and the collision energies that produce them were determined to be m/z 246.2 > 187.1 and −15 V, respectively. In addition to the quantification ion, a reference ion was defined, and the similarity of the i-C5 and p-C5 reference ion ratios (i-C5 score and p-C5 score, respectively) were used to estimate which isomer (i-C5 and p-C5) was responsible for elevated C5 acylcarnitine in dried blood spots (DBSs). As a result of analyses of 11 DBS samples derived from pivalate-conjugated antibiotics and four DBS samples from IVA patients using our method, it was found that our method was able to correctly determine the type of C5-acylcarnitine (i-C5 or p-C5) in the DBS samples. Implementation of this new FIA-TMS method into the current NBS protocol will allow for a reduction in false positives in IVA.

Preemptive hematopoietic cell transplantation for asymptomatic patients with X-linked lymphoproliferative syndrome type 1.

Few reports have examined whether prophylactic allogeneic hematopoietic cell transplantation (HCT) for X-linked lymphoproliferative syndrome type 1 (XLP1) improves the prognosis. We compared the prognosis of symptomatic probands and affected siblings in the same family. Twenty-two cases (10 probands and 12 affected siblings) in Japan, the United Kingdom, and the United States were analyzed. The overall survival (OS) rate at 5 years after diagnosis was 70.0% in probands and 91.7% in affected siblings (p = 0.0789). The prognosis of patients who developed symptoms of XLP1 before HCT and those who did not was also compared. The 5-year probability of OS from the time of diagnosis in asymptomatic patients (100%) was significantly better than that in symptomatic patients (66.7%). These results suggested that early HCT as soon as the diagnosis is made improves the prognosis in asymptomatic XLP1 patients.

The frequencies of very long-chain acyl-CoA dehydrogenase deficiency genetic variants in Japan have changed since the implementation of expanded newborn screening.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency has been a target of expanded newborn screening (ENBS) using tandem mass spectrometry in Japan. Since the implementation of ENBS, a number of novel ACADVL variants responsible for VLCAD deficiency have been identified. In this study, genotypic differences in Japanese patients with VLCAD deficiency were investigated before and after ENBS. The ACADVL variants in 61 subjects identified through ENBS (ENBS group) and in 40 patients who subsequently developed clinical symptoms without undergoing ENBS (pre-ENBS group) were compared. Subjects in the ENBS group underwent genetic testing and/or VLCAD enzyme activity measurements. Patients in the pre-ENBS group were stratified into three clinical phenotypes and underwent genetic testing. This study revealed that the variants p.K264E, p.K382Q and c.996dupT were found in both groups, but their frequencies were lower in the ENBS group (5.2%, 3.1% and 4.2%, respectively) than in the pre-ENBS group (16.5%, 12.7% and 10.1%, respectively). In addition, p.C607S, p.T409M, p.M478I, p.G289R, p.C237R, p.T260M, and p.R229* were exclusively identified in the ENBS group. Among these variants, p.C607S exhibited the highest frequency (18.8%). The patients who were heterozygous for p.C607S demonstrated 7-42% of control enzyme activity. p.C607S is suspected to be unique to Japanese individuals. According to a comparison of enzyme activity, patients with the p.C607S variant may exhibit higher enzyme activity than those with the p.A416T, p.A180T, p.R450H, and p.K264E variants, which are responsible for the myopathic form of the disease. The VLCAD deficiency genotypes have changed since the initiation of ENBS in Japan.

NUDT15 is a key genetic factor for prediction of hematotoxicity in pediatric patients who received a standard low dosage regimen of 6-mercaptopurine.

6-Mercaptopurine (6-MP) is commonly used for treatment of acute lymphoblastic leukemia (ALL). The incidence of hematotoxicity caused by this drug is quite high in Asians even using a standard low dosage regimen. The present study was aimed to elucidate the impact of thiopurine S-methyltransferase (TPMT), a nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15), inosine triphosphatase (ITPA) and ATP Binding Cassette Subfamily C Member 4 (ABCC4) polymorphisms on hematotoxicity in pediatric patients who received a standard low starting dose of 6-MP. One hundred and sixty-nine pediatric patients were enrolled and their genotypes were determined. Patients who carried NUDT15∗3 and NUDT15∗2 genotypes were at a 10-15 fold higher risk of severe neutropenia than those of the wild-type during the early months of the maintenance phase. Risk of neutropenia was not significantly increased in patients with other NUDT15 variants as well as in patients with TPMT, ITPA or ABCC4 variants. These results suggest that NUDT15 polymorphisms particularly, NUDT15∗3 and NUDT15∗2, play major roles in 6-MP-induced severe hematotoxicity even when using a standard low dosage of 6-MP and genotyping of these variants is necessary in order to obtain precise tolerance doses and avoid severe hematotoxicity in pediatric patients.

Prevalence of common aneuploidy in twin pregnancies.

The incidence of chromosomal abnormalities in twin pregnancies is not well-studied. In this retrospective study, we investigated the frequency of chromosomal abnormalities in twin pregnancies and compared the incidence of chromosomal abnormalities in dichorionic diamniotic (DD) and monochorionic diamniotic (MD) twins. We used data from 57 clinical facilities across Japan. Twin pregnancies of more than 12 weeks of gestation managed between January 2016 and December 2018 were included in the study. A total of 2899 and 1908 cases of DD and MD twins, respectively, were reported, and the incidence of chromosomal abnormalities in one or both fetuses was 0.9% (25/2899) and 0.2% (4/1908) in each group (p = 0.004). In this study, the most common chromosomal abnormality was trisomy 21 (51.7% [15/29]), followed by trisomy 18 (13.8% [4/29]) and trisomy 13 (6.9% [2/29]). The incidence of trisomy 21 in MD twins was lower than that in DD twins (0.05% vs. 0.5%, p = 0.007). Trisomy 21 was less common in MD twins, even when compared with the expected incidence in singletons (0.05% vs. 0.3%, RR 0.15 [95% CI 0.04-0.68]). The risk of chromosomal abnormality decreases in twin pregnancies, especially in MD twins.

Heterozygous missense variant of the proteasome subunit ß-type 9 causes neonatal-onset autoinflammation and immunodeficiency.

Impaired proteasome activity due to genetic variants of certain subunits might lead to proteasome-associated autoinflammatory syndromes (PRAAS). Here we report a de novo heterozygous missense variant of the PSMB9 proteasome subunit gene in two unrelated Japanese infants resulting in amino acid substitution of the glycine (G) by aspartic acid (D) at position 156 of the encoded protein ß1i. In addition to PRAAS-like manifestations, these individuals suffer from pulmonary hypertension and immunodeficiency, which are distinct from typical PRAAS symptoms. The missense variant results in impaired immunoproteasome maturation and activity, yet ubiquitin accumulation is hardly detectable in the patients. A mouse model of the heterozygous human genetic variant (Psmb9G156D/+) recapitulates the proteasome defects and the immunodeficiency phenotype of patients. Structurally, PSMB9 G156D interferes with the ß-ring-ßring interaction of the wild type protein that is necessary for 20S proteasome formation. We propose the term, proteasome-associated autoinflammatory syndrome with immunodeficiency (PRAAS-ID), to indicate a separate category of autoinflammatory diseases, similar to, but distinct from PRAAS, that describes the patients in this study.

Diagnosis of Mucopolysaccharidoses and Mucolipidosis by Assaying Multiplex Enzymes and Glycosaminoglycans.

Mucopolysaccharidoses (MPS) and mucolipidosis (ML II/III) are a group of lysosomal storage disorders (LSDs) that occur due to a dysfunction of the lysosomal hydrolases responsible for the catabolism of glycosaminoglycans (GAGs). However, ML is caused by a deficiency of the enzyme uridine-diphosphate N-acetylglucosamine:lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase, EC2.7.8.17), which tags lysosomal enzymes with a mannose 6-phosphate (M6P) marker for transport to the lysosome. A timely diagnosis of MPS and ML can lead to appropriate therapeutic options for patients. To improve the accuracy of diagnosis for MPS and ML in a high-risk population, we propose a combination method based on known biomarkers, enzyme activities, and specific GAGs. We measured five lysosomal enzymes (α-L-iduronidase (MPS I), iduronate-2-sulfatase (MPS II), α-N-acetylglucosaminidase (MPS IIIB), N-acetylglucosamine-6-sulfatase (MPS IVA), and N-acetylglucosamine-4-sulfatase (MPS VI)) and five GAGs (two kinds of heparan sulfate (HS), dermatan sulfate (DS), and two kinds of keratan sulfate (KS)) in dried blood samples (DBS) to diagnose suspected MPS patients by five-plex enzyme and simultaneous five GAGs assays. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) for both assays. These combined assays were tested for 43 patients with suspected MPS and 103 normal control subjects. We diagnosed two MPS I, thirteen MPS II, one MPS IIIB, three MPS IVA, two MPS VI, and six ML patients with this combined method, where enzymes, GAGs, and clinical manifestations were compatible. The remaining 16 patients were not diagnosed with MPS or ML. The five-plex enzyme assay successfully identified MPS patients from controls. Patients with MPS I, MPS II, and MPS IIIB had significantly elevated HS and DS levels in DBS. Compared to age-matched controls, patients with ML and MPS had significantly elevated mono-sulfated KS and di-sulfated KS levels. The results indicated that the combination method could distinguish these affected patients with MPS or ML from healthy controls. Overall, this study has shown that this combined method is effective and can be implemented in larger populations, including newborn screening.

The feasibility of Gazefinder under 12 months of age infants.

Eye-tracking to evaluate gaze patterns has developed as an assessment tool for children with autism spectrum disorder (ASD). Gazefinder is one of Eye-tracking devices and few studies have investigated whether it can measure the gaze data of infants under 12 months of age. We conducted a prospective cross-sectional study from April 2019 to March 2020 in a periodic health checkup in Ohchi County, Shimane, Japan. Participants included infants between 4 and 11 months of age who were not suspected the presence of developmental problems. Ninety-three participants' datapoints were analyzed. The mean age was 6.5 months and mean developmental quotient was 88%. The mean fixation time percentage of all sequences was 81.0% (standard deviation; 4.4), and there was no significant difference in each age group. Infants in all groups showed a significantly higher predilection for eyes than for mouths. There was a positive association of age with human gaze and a negative association with geometric gaze. Moreover, we confirmed that joint attention skills were enhanced in accordance with their growth process. The eye-tracking data were almost corresponding to previous studies' data of infant with typical development and Gazefinder could be applied to infants starting at 4 months of age.