Severe Hemolytic Anemia and Metabolic Acidosis at Birth with Glutathione Synthetase Deficiency and Progressive Neurological Symptoms on Follow-Up.

BACKGROUND Glutathione synthetase deficiency (GSD) is a rare autosomal recessive disorder caused by glutathione synthetase (GSS) gene variants that occur in 1 in 1 million individuals. The severe form of GSD is characterized by hemolytic anemia, metabolic acidosis with 5-oxoprolinuria, progressive neurological symptoms, and recurrent bacterial infections. This case report presents a male Japanese infant with severe hemolytic anemia and metabolic acidosis at birth caused by GSD, who developed progressive neurological symptoms on follow-up. CASE REPORT A Japanese male term infant developed severe hemolytic anemia and metabolic acidosis in the early neonatal period. We suspected GSD based on his symptoms and a high 5-oxoproline urine concentration. We began correcting his metabolic acidosis and administering vitamins C and E supplements. The patient required blood transfusion twice during the acute phase for hemolytic anemia. After age 1 month, he maintained good control of metabolic acidosis and hemolytic anemia. A definitive diagnosis of GSD was made based on high concentrations of 5-oxoproline in urine, low concentrations of glutathione and GSS activity in erythrocytes, and genetic testing. Several episodes of febrile convulsions were started at age 11 months, but none occurred after 2 years. At the last follow-up at age 25 months, metabolic acidosis and hemolytic anemia were well controlled, but he had mild neurodevelopmental delay. CONCLUSIONS This case report shows that GSD can present with severe hemolytic anemia and metabolic acidosis at birth, and manifest with subsequent neurological impairment despite early diagnosis and treatment. Therefore, a careful long-term follow-up that includes neurological evaluation is essential for patients with GSD.

Dravet syndrome and hemorrhagic shock and encephalopathy syndrome associated with an intronic deletion of SCN1A.

OBJECTIVE: Hemorrhagic shock and encephalopathy syndrome (HSES) is a serious condition that requires intensive care and is associated with a high mortality rate. However, its pathogenesis remains unclear. In the present study, a genetic analysis was performed to determine the genetic background of patients with clinically suspected Dravet syndrome (DS) who developed HSES. METHODS: Whole exome sequencing was performed, followed by minigene analysis of the intron variant detected by whole exome sequencing to confirm its effect on splicing. RESULTS: Whole exome sequencing revealed a novel 21-bp deletion in intron 3 of SCN1A NM_001165963.4 (NC_000002.11:g.166073675_166073695del). This deletion was not found in the patient's parents and was proven to be de novo. Minigene analysis revealed an aberrant mRNA lacking 40 and 106 bp from the 5' end of exon 4 of SCN1A. Therefore, we diagnosed this case as DS due to the deletion in intron 3 of SCN1A. CONCLUSIONS: We report a case of DS with HSES caused by a 21-bp deletion in the intron of SCN1A that was confirmed by minigene analysis. The present case met Levin's criteria for HSES and the splicing analysis of SCN1A is an important finding. This study has important implications for understanding HSES pathogenesis.

Antisense oligonucleotide induced pseudoexon skipping and restoration of functional protein for Fukuyama muscular dystrophy caused by a deep-intronic variant.

Fukuyama congenital muscular dystrophy (FCMD) is an autosomal recessive disorder caused by fukutin (FKTN) gene mutations. FCMD is the second most common form of childhood muscular dystrophy in Japan, and the most patients possess a homozygous retrotransposal SINE-VNTR-Alu insertion in the 3'-untranslated region of FKTN. A deep-intronic variant (DIV) was previously identified as the second most prevalent loss-of-function mutation in Japanese patients with FCMD. The DIV creates a new splicing donor site in intron 5 that causes aberrant splicing and the formation of a 64-base pair pseudoexon in the mature mRNA, resulting in a truncated nonfunctional protein. Patients with FCMD carrying the DIV present a more severe symptoms, and currently, there is no radical therapy available for this disorder. In the present study, we describe in vitro evaluation of antisense oligonucleotide mediated skipping of pseudoexon inclusion and restoration of functional FKTN protein. A total of 16 19-26-mer antisense oligonucleotide sequences were designed with a 2'-O-methyl backbone and were screened in patient-derived fibroblasts, lymphoblast cells and minigene splice assays. One antisense oligonucleotide targeting the exonic splice enhancer region significantly induced pseudoexon skipping and increased the expression of normal mRNA. It also rescued FKTN protein production in lymphoblast cells and restored functional O-mannosyl glycosylation of alpha-dystroglycan in patient-derived myotubes. Based on our results, antisense oligonucleotide-based splicing correction should be investigated further as a potential treatment for patients with FCMD carrying the DIV.One Sentence Summary Antisense oligonucleotide treatment restored normal FKTN protein production and functional O-mannosyl glycosylation of alpha-dystroglycan via pseudoexon skipping in patient-derived cells carrying the compound heterozygous deep-intronic variant of Fukuyama muscular dystrophy.

Assessment of the upper limb muscles in patients with Fukuyama muscular dystrophy: Noninvasive assessment using visual ultrasound muscle analysis and shear wave elastography.

Fukuyama-type congenital muscular dystrophy (FCMD) is severe, childhood-onset muscular dystrophy. Recently, our group has discovered a potential treatment using antisense oligonucleotides. Therefore, an effective, reliable, and objective method of assessing muscle is needed. Ultrasound is a minimally invasive tool that can be applied without radiation exposure or pain. Evaluating tissue stiffness by shear wave elastography (SWE) has especially recently attracted attention. Here, we aimed to evaluate SWE value of the upper limb muscles: biceps brachii, triceps brachii, brachioradialis, abductor pollicis brevis, and abductor finger muscle in patients with FCMD. Upper extremity function was evaluated by visual muscle ultrasound analysis (VMUA) and SWE in 13 patients with FCMD and 20 healthy controls. The motor function evaluation tool was used to evaluate motor function, and the correlation with the dynamics of the SWE was determined. VMUA scaled using the Heckmatt scale was higher in patients with FCMD. SWE was also significantly higher and stiffer in the biceps brachii and brachioradialis in patients with FCMD. Furthermore, the severity of FCMD symptoms was correlated with muscle stiffness. We conclude that VMUA and SWE can be useful tools for monitoring muscle atrophy and upper limb function in patients with FCMD.

A case of a parthenogenetic 46,XX/46,XY chimera presenting ambiguous genitalia.

Sex-chromosome discordant chimerism (XX/XY chimerism) is a rare chromosomal disorder in humans. We report a boy with ambiguous genitalia and hypospadias, showing 46,XY[26]/46,XX[4] in peripheral blood cells. To clarify the mechanism of how this chimerism took place, we carried out whole-genome genotyping using a SNP array and microsatellite analysis. The B-allele frequency of the SNP array showed a mixture of three and five allele combinations, which excluded mosaicism but not chimerism, and suggested the fusion of two embryos or a shared parental haplotype between the two parental cells. All microsatellite markers showed a single maternal allele. From these results, we concluded that this XX/XY chimera is composed of two different paternal alleles and a single duplicated maternal genome. This XX/XY chimera likely arose from a diploid maternal cell that was formed via endoduplication of the maternal genome just before fertilization, being fertilized with both X and Y sperm.

Human CD134 (OX40) expressed on T cells plays a key role for human herpesvirus 6B replication after allogeneic hematopoietic stem cell transplantation.

BACKGROUND: CD134 (OX40), which is a cellular receptor for human herpesvirus-6B (HHV-6B) and expresses on activated T cells, may play a key role for HHV-6B replication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). OBJECTIVES: Therefore, we examined the CD134 expression on T cells and HHV-6B replication after allo-HSCT, and analyzed the correlation between them. STUDY DESIGN: Twenty-three patients after allo-HSCT were enrolled. The percentages of CD134-positive cells within the CD4+ and CD8+ cell populations were measured by flow cytometry, and the viral copy number of HHV-6B was simultaneously quantified by real-time PCR. The correlation between CD134 and HHV-6B viral load was then statistically analyzed. RESULTS: HHV-6B reactivation occurred in 11 of 23 patients (47.8%). CD134 expression was seen on T cells and was coincident with the time of peak viral load. The percentage of CD134-positive cells decreased significantly when HHV-6B DNA disappeared (p = .005 in CD4+ T cells, p = .02 in CD8+ T cells). In the 4 patients who underwent umbilical cord blood transplantation (UCBT), the viral load varied with the percentage of CD134-positive cells. In the comparison between the HHV-6B reactivation group and non-reactivation group, maximum percentages of CD134-positive cells among CD4+ T cells in reactivation group were significantly higher than those in non-reactivation group (p = .04). CONCLUSIONS: This is the first study to show that a correlation of CD134 expression on T cells with HHV-6B replication after allo-HSCT, especially in UCBT. The results possibly indicate that CD134 on T cells plays a key role for HHV-6B replication after allo-HSCT.

Corrigendum: Novel missense mutation in DLL4 in a Japanese sporadic case of Adams-Oliver syndrome.

This corrects the article DOI: 10.1038/jhg.2017.48.

Novel missense mutation in DLL4 in a Japanese sporadic case of Adams-Oliver syndrome.

Adams-Oliver syndrome (AOS, OMIM; 100300) is a rare genetic disease characterized by aplasia cutis congenita, terminal transverse limb defects and cutis marmorata with vascular anomalies such as congenital heart defects. The etiology of this syndrome has remained largely unknown but defective Notch signaling during vascular formation has been suggested. Here we describe a sporadic Japanese newborn case with clinically diagnosed AOS. Trio whole-exome sequencing identified a de novo, novel, heterozygous missense mutation in the Delta-like 4 ligand gene (DLL4 c.572G>A, p.Arg191His) in the patient. DLL4 functions as a requisite ligand for NOTCH1 receptor, which is essential for vascular formation. Amino acid substitution of Arg191 to His was predicted by molecular models to interfere with direct binding between DLL4 and NOTCH1. DLL4 has recently been identified as a causative gene of an autosomal dominant type of AOS with milder symptoms. The case described here showed gradual recovery from skull defects after birth and no psychomotor developmental delay has been observed. This is the second report of an AOS case with DLL4 mutation, and the phenotypic characteristics between the two cases are compared and discussed.

Comprehensive analysis of serum cytokines/chemokines in febrile children with primary human herpes virus-6B infection.

Cytokines and chemokines induced by primary human herpes virus (HHV)-6B infection may play a critical role in the clinical manifestations of infection. In this study, we analyzed 40 cytokines/chemokines in febrile children with primary HHV-6B infection. Blood samples from 233 febrile and 36 afebrile patients 0-3 years of age were used for this study. In febrile patients, primary HHV-6B infection was determined by detection of HHV-6B DNA without anti-HHV-6 immunoglobulin G in the blood (HHV-6B group). Infection by other pathogens was assumed when HHV-6B DNA was not detected in the blood (non-HHV-6B group). Of the 233 febrile patients, 30 patients (13%) were diagnosed with primary HHV-6B infection. To analyze serum cytokines/chemokines, patients were randomly chosen from the HHV-6B (n = 25) and non-HHV-6B groups (n = 8). Sera from 25 afebrile patients were used as a control. When comparing the levels of 40 cytokines/chemokines between the HHV-6B and control groups, we found that four chemokines (chemokine [C-X-C motif] ligand [CXCL] 11, CXCL10, CXCL16, and chemokine [C-C motif] ligand [CCL] 2) were significantly upregulated in the HHV-6B group compared with those in the control. Of these, only CXCL11 levels were significantly higher in the HHV-6B group than in the non-HHV-6B group. Because the induction of CCL2 was already reported in an early study, we found, for the first time, the induction of three new chemokines, i.e., CXCL11, CXCL10, and CXCL16 in patients with primary HHV-6B infection. Importantly, we demonstrated that serum CXCL11 levels increased specifically in patients with HHV-6B infection.

Transcutaneous bilirubin monitoring predicts unexplained late-onset hemolysis in a very low birthweight infant.

BACKGROUND: In term infants, transcutaneous bilirubin (TcB) monitoring can be used to predict hemolytic hyperbilirubinemia. However, it is not clear whether the technique can also be used to predict unexplained late-onset hemolysis in very low birthweight (VLBW) infants. CASE PRESENTATION: The case was an infant with a birthweight of 1154 g who developed unexplained late-onset hemolysis at 8 days of age. The hyperbilirubinemia rapidly worsened, and therefore both phototherapy and exchange transfusion were performed. TcB levels were measured using the JM-105 jaundice meter and found to have increased by >3 mg/dL since before the onset, demonstrating for the first time that the device clearly detects changes in hemolytic rate. CONCLUSIONS: Although TcB levels did not correspond directly with total serum bilirubin levels in VLBW infants, the two values exhibited parallel changes in this case. Therefore, serial TcB monitoring may be useful in the early prediction of unexplained late-onset hemolysis in VLBW infants.

Prevalence of small for gestational age (SGA) and short stature in children born SGA who qualify for growth hormone treatment at 3 years of age: Population-based study.

BACKGROUND: To treat children born small for gestational age (SGA) with severe short stature, treatment with growth hormone (GH) has been approved in the USA, Europe, and Japan, but no population-based studies have reported their prevalence. The aims of this study were to investigate the prevalence of SGA and short stature in children born SGA who qualify for GH treatment at 3 years of age in a Japanese population. METHODS: A population-based study was conducted in Kobe, Japan with 27 228 infants who were born between 2006 and 2008 and followed until 3 years of age. Prevalence of birthweight (BW) or birth length (BL) ≤ -2.0 standard deviation scores (SDS) for gestational age (GA; definition of SGA) was calculated. Short children born SGA who qualify for GH treatment at 3 years of age were estimated using the following criteria: BW and BL below the 10th percentile for GA, BW or BL ≤ -2.0 SDS for GA, and 2.5 SDS below the mean height for age. RESULTS: The prevalence of SGA was 3.5%. The estimated prevalence of short stature in children born SGA who met the criteria for GH treatment was 0.06%. The prevalence in infants born <34 weeks (0.39%) was significantly higher than that in infants born 34-41 weeks GA (0.05%, P = 0.02). CONCLUSIONS: The prevalence of SGA and short stature in children born SGA who qualify for GH treatment is approximately 1 of 30 infants and 1 of 1800 children, respectively. The risk is increased when children are born <34 weeks GA.

Extremely preterm infants small for gestational age are at risk for motor impairment at 3 years corrected age.

BACKGROUND: Few studies have targeted psychomotor development and associated perinatal risk factors in Japanese very low birth weight (VLBW) infants who are severely small for gestational age (SGA). DESIGN/SUBJECTS: A single-center study was conducted in 104 Japanese VLBW infants who were born preterm, due to maternal, umbilical cord, or placental abnormalities, between 2000 and 2007. Psychomotor development as a developmental quotient (DQ) was assessed using the Kyoto Scale of Psychological Development at 3 years corrected age. Severely SGA was defined as birth weight or length below -2 standard deviation values of the mean values at the same gestation. VLBW infants were divided into 2 subgroups based on gestational age at birth: ⩾28 weeks (n=64) and <28 weeks (n=40). DQs of infants with severe SGA were compared with those of infants who were appropriate for gestational age (AGA). Factors associated with developmental disabilities in VLBW infants with severe SGA (n=23) were determined. RESULTS: In the group born at ⩾28 weeks gestation, infants with severe SGA had normal DQ values and did not significantly differ from those with AGA. However, in the group born at <28 weeks gestation, severe SGA infants had significantly lower postural-motor DQ values than AGA infants. Gestational age <28 weeks was an independent factor for low postural-motor DQ, regardless of the cause of severe SGA or pregnancy termination. CONCLUSIONS: Extremely preterm newborns with severe SGA are at risk of motor developmental disability at age 3 years.

Incidence of short stature at 3†years of age in late preterm infants: a population-based study.

OBJECTIVES: This study aimed to investigate the incidence of short stature at 3 years of age in a Japanese cohort of late preterm infants who were born at 34-36 weeks' gestational age (GA). We compared these late preterm infants with term infants (37-41 weeks' GA), and evaluated the effect of birth weight on the incidence of short stature. METHODS: A longitudinal population-based study of 26 970 neonates who were born between 34 weeks' and 41 weeks' GA in 2006-2008 was conducted in Kobe, Japan. Of these neonates, 1414 were late preterm and 25 556 were term infants. The late preterm infants were then divided into three subgroups based on birth weight as determined by Japanese neonatal anthropometric charts for GA at birth: large-for-GA (n=140), appropriate-for-GA (AGA, n=1083), and small-for-GA (SGA, n=191). The incidence of short stature at 3 years of age was calculated in the late preterm group and compared with that in the term group, and between the AGA and SGA groups with late preterm birth. RESULTS: The incidence of short stature in the late preterm group was 2.9%, which was significantly higher than that in the term group (1.4%). Late preterm SGA infants developed short stature with a significantly higher (9.4%) incidence than that of late preterm AGA infants (2.1%). CONCLUSIONS: The incidence of short stature in 3-year-old children who were late preterm infants has a 2-fold higher risk than that in term infants. The risk of developing short stature is increased 4.5-fold if they are SGA.

Standard values for the urine HVA/VMA ratio in neonates as a screen for Menkes disease.

BACKGROUND: Menkes disease is a lethal disorder associated with copper metabolism. Although early treatment with copper-histidine injections can improve outcomes, early diagnosis is difficult because the clinical features of Menkes disease are subtle or do not manifest in affected neonates. Previous report stated that the low activity of dopamine ß-hydroxylase, a copper-dependent enzyme, leads to increases in the urine homovanillic acid/vanillylmandelic acid (HVA/VMA) ratios in patients with Menkes disease, and indicated that a urine HVA/VMA ratio cut-off value of >4 is useful in screening for Menkes disease. METHODS: We examined the standard values of the urine HVA/VMA ratio in unaffected neonates and assessed its use as a screening parameter for Menkes disease among neonates. In total, 112 neonates, aged between 1 and 6 days, were enrolled in the study and were classified into 2 groups based on their urine HVA/VMA ratios: high (>4) and low (⩽ 4). RESULTS: Multivariate logistic analysis revealed that mechanical ventilation was an independent risk factor for a high urine HVA/VMA ratio (odds ratio: 21.94; 95% confidence interval: 2.82-247.03; p=0.004). The mean urine HVA/VMA ratio was 2.47 ± 0.67 among 92 neonates who did not receive mechanical ventilation. CONCLUSION: This study established standard values for the urine HVA/VMA ratio in newborn babies that could be useful in screening for Menkes disease among neonates.

Association of a vascular endothelial growth factor polymorphism with the development of bronchopulmonary dysplasia in Japanese premature newborns.

Our objective was to correlate vascular endothelial growth factor (VEGF) genetic polymorphisms with the risk of bronchopulmonary dysplasia (BPD) development in premature newborns. Fifty-five newborns with BPD (BPD: median gestational age [GA]: 27 weeks, birthweight [BW]: 786 g) and 42 newborns without BPD (non-BPD: median GA: 29 weeks, BW: 1,165 g), who were born at <32 weeks gestational age and were admitted to Kobe University Hospital, were included. BPD was defined as oxygen dependency at 36 weeks postmenstrual age. Genomic DNA was extracted from the umbilical cord, cord blood, or buccal mucosa. Six VEGF genotypes (-1498T > C, -1154G > A, -634C > G, -7C > T, 936C > T, and 1612G > A) were determined by DNA sequencing. Clinical characteristics, and allele and genotype frequencies of VEGF in the BPD and non-BPD groups were analyzed. G allele frequencies in -634C > G of the BPD group were significantly higher than in the non-BPD group (66.4% vs. 50%, P = 0.02). -634C > G genotype distributions differed significantly between the BPD and non-BPD groups (BPD: CC 7%/CG 53%/GG 40%; non-BPD: CC 24%/CG 52%/GG 24%; P = 0.04). Multivariate logistic regression showed that duration of ventilation, VEGF-634G > C G alleles, and male gender were independent risk factors for BPD. In conclusion, polymorphism VEGF -634C > G may influence the risk of BPD.

Postnatal serum concentrations of endogenous free fatty acids in newborns admitted to the neonatal intensive care unit: effects on unbound bilirubin.

BACKGROUND: Few studies have reported the characterization of postnatal serum concentrations of endogenous free fatty acids (FFAs) in high-risk newborns and their effects on unbound bilirubin (UB). METHODS: Serum concentrations of FFA, albumin (Alb), UB and total bilirubin (TB) were measured in 713 samples obtained within 5 days after birth from 439 newborns without intravenous lipid supplementation admitted to the neonatal intensive care unit (NICU). Serum FFA was reported as the day-specific percentile-based curve. Serum FFA and FFA/Alb ratios were compared in term and preterm patients. To assess the impact of FFA on UB, daily changes in FFA/Alb and UB/TB ratios were compared in term patients without receiving phototherapy or any drugs, and linear regression analysis was performed between FFA/Alb ratio and serum UB concentration or UB/TB ratio using 140 sera with hyperbilirubinemia of term and preterm patients. RESULTS: A percentile-based curve showed that serum FFA peaked at 1 day of age and progressively decreased. Serum FFA and the FFA/Alb ratio were significantly higher in term than in preterm patients at birth and 1 and 3 days of age. FFA/Alb ratio significantly changed over 5 days after birth, but UB/TB ratio remained constant. FFA/Alb ratio did not correlate with serum UB concentration or UB/TB ratio in sera with hyperbilirubinemia. CONCLUSIONS: We assessed postnatal concentrations of serum FFA in a large number of high-risk newborns admitted to the NICU. The concentration of endogenous FFAs in newborns admitted to the NICU was not rising until it influenced UB.

Dried umbilical cord is a potential material for retrospective diagnosis of intrauterine enterovirus infection.

OBJECTIVE: To determine whether dried umbilical cords (UCs) are useful for retrospective diagnosis of intrauterine enterovirus (EV) infection. METHODS: Dried UCs in two patients with neonatal EV sepsis and 10 neonates without infectious signs were enrolled. Viral RNA was extracted from their dried UCs, and nested reverse transcription polymerase chain reaction (RT-PCR) was performed. RESULTS: Infection routes estimated by the clinical course were intrauterine infection in Case 1 and post-natal horizontal infection in Case 2. EV-RNA was detected from dried UC in Case 1, but not in Case 2 and 10 neonates. CONCLUSIONS: This report showed the potential use of dried UCs for retrospective diagnosis of intrauterine EV infection.

Quantitative evaluation of ventricular dilatation using computed tomography in infants with congenital cytomegalovirus infection.

BACKGROUND: Infants with congenital cytomegalovirus infection (CCMVI) may develop brain abnormalities such as ventricular dilatation, which may potentially associate with sensorineural hearing loss. There is currently no recognized method for quantitative evaluation of ventricle size in infants with CCMVI. Our objectives were to establish a method for quantitative evaluation of ventricle size using computed tomography (CT) in infants with CCMVI, and determine a cut-off value associated with abnormal auditory brainstem response (ABR) early in life. DESIGN/SUBJECTS: This study enrolled 19 infants with CCMVI and 21 non-infected newborn infants as a control group. Infants with CCMVI were divided into two subgroups according to ABR at the time of initial examination: normal ABR (11 infants) or abnormal ABR (8 infants). Ventricle size was assessed by calculating Evans' index (EI) and lateral ventricle width/hemispheric width (LVW/HW) ratio on brain CT images, and was compared among groups. A cut-off ventricle size associated with abnormal ABR was determined. RESULTS: EI and LVW/HW ratio were significantly higher in the CCMVI with abnormal ABR group than the control and CCMVI with normal ABR groups. Cut-off values of 0.26 for EI and 0.28 for LVW/HW ratio had a sensitivity of 100% and 100%, respectively, and a specificity of 73% and 91%, respectively, for association with abnormal ABR. CONCLUSIONS: We established a method for quantitative evaluation of ventricle size using EI and LVW/HW ratio on brain CT images in infants with CCMVI. LVW/HW ratio had a more association with abnormal ABR in the early postnatal period than EI.