Novel large deletion involving EVC and EVC2 in Ellis-van Creveld syndrome.

Ellis-van Creveld syndrome is an autosomal recessive skeletal dysplasia that is characterized by thoracic hypoplasia, polydactyly, oral abnormalities, and congenital heart disease. It is caused by pathogenic variants in the EVC or EVC2 genes. We report a case of a newborn with a compound heterozygous variant comprising NM_147127.5: c.1991dup:[p.Lys665Glufs*10] in the EVC2 gene and a novel large deletion involving exon 1 in EVC and exons 1-7 in EVC2.

Duplications in the G3 domain or switch II region in HRAS identified in patients with Costello syndrome.

Costello syndrome (CS) is an autosomal-dominant disorder characterized by distinctive facial features, hypertrophic cardiomyopathy, skeletal abnormalities, intellectual disability, and predisposition to cancers. Germline variants in HRAS have been identified in patients with CS. Intragenic HRAS duplications have been reported in three patients with a milder phenotype of CS. In this study, we identified two known HRAS variants, p.(Glu63_Asp69dup), p.(Glu62_Arg68dup), and one novel HRAS variant, p.(Ile55_Asp57dup), in patients with CS, including a patient with craniosynostosis. These intragenic duplications are located in the G3 domain and the switch II region. Cells expressing cDNA with these three intragenic duplications showed an increase in ELK-1 transactivation. Injection of wild-type or mutant HRAS mRNAs with intragenic duplications in zebrafish embryos showed significant elongation of the yolk at 11 h postfertilization, which was improved by MEK inhibitor treatment, and a variety of developmental abnormalities at 3 days post fertilization was observed. These results indicate that small in-frame duplications affecting the G3 domain and switch II region of HRAS increase the activation of the ERK pathway, resulting in developmental abnormalities in zebrafish or patients with CS.

Functionally confirmed compound heterozygous ADAM17 missense loss-of-function variants cause neonatal inflammatory skin and bowel disease 1.

A disintegrin and metalloprotease 17 (ADAM17) is the major sheddase that processes more than 80 substrates, including tumour necrosis factor-α (TNFα). The homozygous genetic deficiency of ADAM17 causing a complete loss of ADAM17 expression was reported to be linked to neonatal inflammatory skin and bowel disease 1 (NISBD1). Here we report for the first time, a family with NISBD1 caused by functionally confirmed compound heterozygous missense variants of ADAM17, namely c.1699T>C (p.Cys567Arg) and c.1799G>A (p.Cys600Tyr). Both variants were detected in two siblings with clinical features of NISBD1, such as erythroderma with exudate in whole body, recurrent skin infection and sepsis and prolonged diarrhoea. In a cell-based assay using Adam10/17 double-knockout mouse embryonic fibroblasts (Adam10/17-/- mEFs) exogenously expressing each of these mutants, phorbol 12-myristate 13-acetate-stimulated shedding was strongly reduced compared with wild-type ADAM17. Thus, in vitro functional assays demonstrated that both missense variants cause the loss-of-function of ADAM17, resulting in the development of NISBD1. Our study further expands the spectrum of genetic pathology underlying ADAM17 in NISBD1 and establishes functional assay systems for its missense variants.

Molecular diagnosis of an infant with TSC2/PKD1 contiguous gene syndrome.

A 1-month-old Japanese infant with cardiac rhabdomyoma was diagnosed with TSC2/PKD1 contiguous gene syndrome by targeted panel sequencing with subsequent quantitative polymerase chain reaction that revealed gross monoallelic deletion, including parts of two genes: exons 19-42 of TSC2 and exons 2-46 of PKD1. Early molecular diagnosis can help to detect bilateral renal cyst formation and multidisciplinary follow-up of this multisystem disease.

Enhanced angiotensinogen expression in neonates during kidney development.

BACKGROUND: We recently demonstrated that preterm neonates have higher urinary angiotensinogen (AGT) levels than full-term neonates. Here, we tested the hypothesis that enhanced neonatal AGT expression is associated with intrarenal renin-angiotensin system (RAS) status during kidney development. METHODS: We prospectively recruited neonates born at our hospital and healthy children with minor glomerular abnormalities between April 2013 and March 2017. We measured neonatal plasma and urinary AGT levels at birth and 1 year later and assessed renal AGT expression in kidney tissues from neonates and healthy children using immunohistochemical (IHC) analysis. RESULTS: Fifty-four neonates and eight children were enrolled. Although there were no changes in plasma AGT levels, urinary AGT levels were significantly decreased 1 year after birth. Urinary AGT levels at birth were inversely correlated with gestational age, and urinary AGT levels at birth and 1 year later were inversely correlated with estimated glomerular filtration rate 1 year after birth. IHC analysis showed that renal AGT expression in neonates was higher than that in healthy children and inversely correlated with gestational age. CONCLUSIONS: Enhanced AGT expression and urinary AGT excretion may reflect intrarenal RAS activation associated with kidney development in utero.

Analysis of maternal and neonatal factors that influence the nucleated and CD34+ cell yield for cord blood banking.

BACKGROUND: It would be beneficial to be able to predict the cord blood (CB) cell yield from volunteer donors before cell processing. STUDY DESIGN AND METHODS: The maternal and neonatal factors that influence the total nucleated cell (TNC), CD34+ cell, and CFU-GM yields in CB collected for the Chugoku-Shikoku Cord Blood Bank were evaluated. RESULTS: In a univariate analysis, the volume of CB collected was significantly correlated with the TNC, CD34+ cell, and CFU-GM yields (p < 0.001). A longer cord (p < 0.001), larger placenta (p < 0.001), and bigger baby (p < 0.001) were associated with a greater volume of CB. A female baby (p < 0.05) and longer gestational age (p < 0.005) were associated with a higher TNC concentration. A younger maternal age (p < 0.05), larger birth weight (p < 0.001), shorter gestational age (p < 0.001), and shorter time from collection to processing (p < 0.05) were associated with a higher CD34+ cell concentration. A multivariate linear regression analysis was performed to predict the yield and determine first-level selection criteria to start processing when the volume of CB units was on the borderline. However, this formula might not be suitable for actual use. CONCLUSION: Maternal and neonatal factors appeared to affect CB cell yields. These findings might be useful for efficiently collecting more qualified CB units.

Cryopreservation of mobilized blood stem cells at a higher cell concentration without the use of a programmed freezer.

Cryopreservation of peripheral blood stem cells (PBSC) mobilized by chemotherapy combined with or without granulocyte colony-stimulating factor (G-CSF) is an essential part of procedure for anti-cancer strategies. We evaluated whether a higher cell concentration (2 x 10(8)/ml) without the use of a programmed freezer was acceptable for the storage of mobilized PBSC in an autologous setting. Mobilized PBSC were enriched to mononuclear cells (MNC) by Percoll separation and then frozen at cell concentrations of 2-5 x 10(7)/ml (group I, n=20) or 2 x 10(8)/ml (group II, n=44) without the use of a programmed freezer using 5% DMSO, 6% hydroxy ethyl starch, and 4% autologous serum or human albumin. CD34+ cells purified by ISOLEX300 were frozen at 2 x 10(7)/ml (group III, n=22) using the same method. The median recovery rates of CD34+ cells and CFU-GM were, respectively, n.d. (not determined) and 88% in group I, 103 and 64% in group II, and 98 and 53% in group III. There was a statistical significance between the recovery rate of CFU-GM in group III and that in group I ( p=0.02). The median percentage of cell viability after thawing in each group was 89, 87, and 75%, respectively. The median numbers of days after PBSCT to achieve a WBC of >1.0 x 10(9)/l, an absolute neutrophil count of >0.5 x 10(9)/l, and a platelet count of >50 x 10(9)/l were, respectively, 11, 11 and 15 in group I; 12, 12 and 16 in group II; and 12, 12 and 27 in group III. These results suggest that enriched MNC from mobilized PBSC could be frozen at a higher cell concentration (2 x 10(8)/ml) without the use of a programmed freezer, leading to reduction of the toxicities associated with infusion of thawed cells and of costly space required for cell storage.

Effect of granulocyte colony-stimulating factor on bone metabolism during peripheral blood stem cell mobilization.

Granulocyte colony-stimulating factor (G-CSF) has been shown to affect the biochemical markers of bone metabolism, including serum bone alkaline phosphatase (BALP), serum osteocalcin, and urine deoxypyridinoline. To determine the association between bone resorption and formation and the G-CSF-induced mobilization of peripheral blood stem cells (PBSC), we examined these markers during mobilization in 19 healthy donors. The average (+/- SEM) serum BALP level before treatment was 81.6 +/- 17.0 IU/dL, and the level increased significantly to 117.7 +/- 15.8 IU/dL on day 5 of G-CSF administration (P < .0001). The urine deoxypyridinoline level before treatment was 12.3 +/- 2.4 nmol/mmol creatinine, and this level also increased significantly to 19.4 +/- 3.0 nmol/mmol creatinine on day 5 of G-CSF administration (P < .0001). In contrast, the average level of serum osteocalcin significantly decreased from 8.07 +/- 2.88 ng/mL to 1.53 +/- 0.18 ng/mL on day 5 (P = .0353). During G-CSF administration, we also studied the serum levels of various cytokines (IL-1beta, osteoclastogenesis inhibitory factor [OCIF], IL-6, tumor necrosis factor alpha, transforming growth factor beta, interferon-gamma, macrophage colony-stimulating factor) related to bone metabolism. Only the kinetics of OCIF were significantly affected. The serum level of OCIF increased immediately after the start of G-CSF administration and remained high during G-CSF administration. These results demonstrate that high-dose G-CSF affects bone metabolism and that OCIF may play a role in bone metabolism. Consistent with the notion that G-CSF affects bone metabolism, a significant correlation was observed between CD34+ cell yield and the increase in urine deoxypyridinoline but not for the changes in serum BALP and osteocalcin levels. This result suggests that bone resorption is either directly or indirectly related to the mobilization of PBSC by G-CSF.

Successful unrelated cord blood transplantation in an infant with Wiskott-Aldrich syndrome following recurrent cytomegalovirus disease.

We describe successful unrelated cord blood transplantation in a 14-month-old boy with Wiskott-Aldrich syndrome. He had been suffering from recurrent cytomegalovirus (CMV) pneumonia. Ganciclovir was given pretransplantation and posttransplantation, and CMV antigenemia was monitored as a marker of reactivation. The conditioning regimen was cyclophosphamide, busulfan, and antithymocyte globulin. The patient received an HLA 1-locus-mismatched cord blood unit, and the total number of infused nucleated cells was 9.0 x 10(7)/kg. Neutrophil engraftment was achieved on day +20, and a platelet count greater than 50 x 10(9)/L was achieved on day +51. A normal lymphoproliferative response to phytohemagglutinin mitogen was detectable 7 months posttransplantation. Long-term use of ganciclovir prevented CMV reactivation and did not compromise engraftment.