Update of the genotype and phenotype of KMT2D and KDM6A by genetic screening of 100 patients with clinically suspected Kabuki syndrome.

Kabuki syndrome is characterized by a variable degree of intellectual disability, characteristic facial features, and complications in various organs. Many variants have been identified in two causative genes, that is, lysine methyltransferase 2D (KMT2D) and lysine demethylase 6A (KDM6A). In this study, we present the results of genetic screening of 100 patients with a suspected diagnosis of Kabuki syndrome in our center from July 2010 to June 2018. We identified 76 variants (43 novel) in KMT2D and 4 variants (3 novel) in KDM6A as pathogenic or likely pathogenic. Rare variants included a deep splicing variant (c.14000-8C>G) confirmed by RNA sequencing and an 18% mosaicism level for a KMT2D mutation. We also characterized a case with a blended phenotype consisting of Kabuki syndrome, osteogenesis imperfecta, and 16p13.11 microdeletion. We summarized the clinical phenotypes of 44 patients including a patient who developed cervical cancer of unknown origin at 16 years of age. This study presents important details of patients with Kabuki syndrome including rare clinical cases and expands our genetic understanding of this syndrome, which will help clinicians and researchers better manage and understand patients with Kabuki syndrome they may encounter.

Contribution of gene mutations to Silver-Russell syndrome phenotype: multigene sequencing analysis in 92 etiology-unknown patients.

BACKGROUND: Silver-Russell syndrome (SRS) is characterized by growth failure and dysmorphic features. Major (epi)genetic causes of SRS are loss of methylation on chromosome 11p15 (11p15 LOM) and maternal uniparental disomy of chromosome 7 (upd(7)mat). However, IGF2, CDKN1C, HMGA2, and PLAG1 mutations infrequently cause SRS. In addition, other imprinting disturbances, pathogenic copy number variations (PCNVs), and monogenic disorders sometimes lead to SRS phenotype. This study aimed to clarify the frequency and clinical features of the patients with gene mutations among etiology-unknown patients with SRS phenotype. RESULTS: Multigene sequencing was performed in 92 out of 336 patients referred to us for genetic testing for SRS. The clinical features of the patients were evaluated based on the Netchine-Harbison clinical scoring system. None of the patients showed 11p15 LOM, upd(7)mat, abnormal methylation levels for six differentially methylated regions (DMRs), namely, PLAGL1:alt-TSS-DMR on chromosome 6, KCNQ1OT1:TSS-DMR on chromosome 11, MEG3/DLK1:IG-DMR on chromosome 14, MEG3:TSS-DMR on chromosome 14, SNURF:TSS-DMR on chromosome 15, and GNAS A/B:TSS-DMR on chromosome 20, PCNVs, or maternal uniparental disomy of chromosome 16. Using next-generation sequencing and Sanger sequencing, we screened four SRS-causative genes and 406 genes related to growth failure and/or skeletal dysplasia. We identified four pathogenic or likely pathogenic variants in responsible genes for SRS (4.3%: IGF2 in two patients, CDKN1C, and PLAG1), and five pathogenic variants in causative genes for known genetic syndromes presenting with growth failure (5.4%: IGF1R abnormality (IGF1R), SHORT syndrome (PIK3R1), Floating-Harbor syndrome (SRCAP), Pitt-Hopkins syndrome (TCF4), and Noonan syndrome (PTPN11)). Functional analysis indicated the pathogenicity of the CDKN1C variant. The variants we detected in CDKN1C and PLAG1 were the second and third variants leading to SRS, respectively. Our patients with CDKN1C and PLAG1 variants showed similar phenotypes to previously reported patients. Furthermore, our data confirmed IGF1R abnormality, SHORT syndrome, and Floating-Harbor syndrome are differential diagnoses of SRS because of the shared phenotypes among these syndromes and SRS. On the other hand, the patients with pathogenic variants in causative genes for Pitt-Hopkins syndrome and Noonan syndrome were atypical of these syndromes and showed partial clinical features of SRS. CONCLUSIONS: We identified nine patients (9.8%) with pathogenic or likely pathogenic variants out of 92 etiology-unknown patients with SRS phenotype. This study expands the molecular spectrum of SRS phenotype.

IGF2 Mutations.

OBJECTIVE: IGF2 is a paternally expressed growth-promoting gene. Here, we report five cases with IGF2 mutations and review IGF2 mutation-positive patients described in the literature. We also compare clinical features between patients with IGF2 mutations and those with H19/IGF2:IG-DMR epimutations. RESULTS: We recruited five cases with IGF2 mutations: case 1 with a splice site mutation (c.-6-1G>C) leading to skipping of exon 2 and cases 2-5 with different missense mutations (p.(Cys70Tyr), p.(Cys71Arg), p.(Cys33Ser), and p.(Cys45Ser)) affecting cysteine residues involved in the S-S bindings. All the mutations resided on the paternally inherited allele, and the mutation of case 5 was present in a mosaic condition. Clinical assessment revealed Silver-Russell syndrome (SRS) phenotype with Netchine-Harbison scores of ≥5/6 in all the apparently nonmosaic 14 patients with IGF2 mutations (cases 1-4 described in this study and 10 patients reported in the literature). Furthermore, compared with H19/IGF2:IG-DMR epimutations, IGF2 mutations were associated with low frequency of hemihypoplasia, high frequency of feeding difficulty and/or reduced body mass index, and mild degree of relative macrocephaly, together with occasional development of severe limb malformations, high frequency of cardiovascular anomalies and developmental delay, and low serum IGF-II values. CONCLUSIONS: This study indicates that IGF2 mutations constitute a rare but important cause of SRS. Furthermore, while both IGF2 mutations and H19/IGF2:IG-DMR epimutations lead to SRS, a certain degree of phenotypic difference is observed between the two groups, probably due to the different IGF2 expression pattern in target tissues.

Comprehensive analysis of syndromic hearing loss patients in Japan.

More than 400 syndromes associated with hearing loss and other symptoms have been described, corresponding to 30% of cases of hereditary hearing loss. In this study we aimed to clarify the mutation spectrum of syndromic hearing loss patients in Japan by using next-generation sequencing analysis with a multiple syndromic targeted resequencing panel (36 target genes). We analyzed single nucleotide variants, small insertions, deletions and copy number variations in the target genes. We enrolled 140 patients with any of 14 syndromes (BOR syndrome, Waardenburg syndrome, osteogenesis imperfecta, spondyloepiphyseal dysplasia congenita, Stickler syndrome, CHARGE syndrome, Jervell and Lange-Nielsen syndrome, Pendred syndrome, Klippel-Feil syndrome, Alport syndrome, Norrie disease, Treacher-Collins syndrome, Perrault syndrome and auditory neuropathy with optic atrophy) and identified the causative variants in 56% of the patients. This analysis could identify the causative variants in syndromic hearing loss patients in a short time with a high diagnostic rate. In addition, it was useful for the analysis of the cases who only partially fulfilled the diagnostic criteria.

Efficacy and safety of cardioversion with continuous landiolol infusion for atrial tachyarrhythmia in an inflammatory state caused by volvulus in a child with TARP syndrome and postoperative tetralogy of Fallot.

A 2-year-old boy was diagnosed with TARP syndrome and underwent surgery for tetralogy of Fallot. He developed fever and had an acute abdomen. After 12 hours, atrial tachyarrhythmia (300 beats/min [bpm]) occurred. After nine administration of adenosine and two cardioversions, it relapsed promptly. Landiolol (10 µg/kg/min) was administered until the heart rate decreased to 270 bpm, and cardioversion was performed until sinus rhythm was normal. Exploratory laparotomy revealed small bowel volvulus. Systemic inflammation causing an acute abdomen may be associated with atrial tachyarrhythmia in postoperative tetralogy of Fallot. We speculated that landiolol lowered the defibrillation threshold of the atrium.

Safety of lipid emulsion in very low-birthweight infants according to cytokine level.

BACKGROUND: The aim of this study was to verify whether lipid emulsion treatment aggravates infection and inflammation in very low-birthweight (VLBW) infants. STUDY DESIGN: Very low-birthweight (<1500 g) infants born at <32 weeks gestational age between October 2013 and October 2014 at Dokkyo Medical University Hospital (Mibu, Tochigi, Japan) were treated with or without i.v. nutrition with a lipid emulsion. Infants were excluded who had congenital abnormalities, could not receive i.v. nutrition because of poor general condition, or on physician decision. Lipid emulsion with purified soybean oil was initiated at 0.5 g/kg/day on postnatal day 1. The dose was increased to 1 g/kg/day, and then to 1.5 g/kg/day (maximum dose). Blood tests were performed before (day 1) and after (day 8) initiation of lipid emulsion treatment. Interleukin (IL)-6, IL-8, monocyte chemotactic protein 1 (MCP-1), tumor necrosis factor-α (TNF-α), C-reactive protein (CRP), total bilirubin (T-Bil), direct bilirubin (D-Bil) and insulin were measured. Changes in respiratory condition, amount of oxygen used, and phototherapy duration were investigated. RESULTS: A total of 17 treated and 15 untreated VLBW infants were enrolled. IL-6, IL-8, MCP-1, TNF-α, CRP, T-Bil, D-Bil and insulin on days 1 and 8; respirator or surfactant use; amount of oxygen used; and phototherapy duration were not significantly different between the two groups. CONCLUSIONS: Lipid emulsion treatment did not increase inflammatory cytokine levels or aggravate respiratory disorders. Lipid emulsions, if proven safe, could be used to treat VLBW infants soon after birth, which may prevent extrauterine growth restriction and improve intellectual development prognosis.

Spectrum of mutations and genotype-phenotype analysis in Noonan syndrome patients with RIT1 mutations.

RASopathies are autosomal dominant disorders caused by mutations in more than 10 known genes that regulate the RAS/MAPK pathway. Noonan syndrome (NS) is a RASopathy characterized by a distinctive facial appearance, musculoskeletal abnormalities, and congenital heart defects. We have recently identified mutations in RIT1 in patients with NS. To delineate the clinical manifestations in RIT1 mutation-positive patients, we further performed a RIT1 analysis in RASopathy patients and identified 7 RIT1 mutations, including two novel mutations, p.A77S and p.A77T, in 14 of 186 patients. Perinatal abnormalities, including nuchal translucency, fetal hydrops, pleural effusion, or chylothorax and congenital heart defects, are observed in all RIT1 mutation-positive patients. Luciferase assays in NIH 3T3 cells demonstrated that the newly identified RIT1 mutants, including p.A77S and p.A77T, and the previously identified p.F82V, p.T83P, p.Y89H, and p.M90I, enhanced Elk1 transactivation. Genotype-phenotype correlation analyses of previously reported NS patients harboring RIT1, PTPN11, SOS1, RAF1, and KRAS revealed that hypertrophic cardiomyopathy (56 %) was more frequent in patients harboring a RIT1 mutation than in patients harboring PTPN11 (9 %) and SOS1 mutations (10 %). The rates of hypertrophic cardiomyopathy were similar between patients harboring RIT1 mutations and patients harboring RAF1 mutations (75 %). Short stature (52 %) was less prevalent in patients harboring RIT1 mutations than in patients harboring PTPN11 (71 %) and RAF1 (83 %) mutations. These results delineate the clinical manifestations of RIT1 mutation-positive NS patients: high frequencies of hypertrophic cardiomyopathy, atrial septal defects, and pulmonary stenosis; and lower frequencies of ptosis and short stature.

[Anti RS virus drugs].

Palivizumab is one of the monoclonal antibodies for RS virus(RSV), and has been widely used to preterm infants, infants with bronchopulmonary dysplasia, and infants with congenital heart disease. Palivizumab can reduce admission rate and length of hospital stay due to lower respiratory tract infection by RSV. Palivizumab can also reduce the rate of later recurrent wheezing. Motavizumab, the 2nd generation monoclonal antibody, has 18-fold greater neutralizing capacity to RSV. Clinical trials of motavizumab finished, however, motavizumab has not been granted because of skin complication. In anti RSV drugs, ribavirin administration is not recommended because the effect is unclear. Clinical trials of some new anti RSV drugs and two live attenuated intranasal vaccines are underway.

MR imaging appearance of laryngeal atresia (congenital high airway obstruction syndrome): unique course in a fetus.

Congenital high airway obstruction syndrome (CHAOS) is a rare life-threatening syndrome. Most cases are diagnosed prenatally by US. We report a fetus with this syndrome that showed a unique course revealed on MRI. Ultrasonography at 22 weeks demonstrated that the fetus had ascites and bilaterally enlarged hyperechoic lungs. Congenital infection, congenital cystic adenomatoid malformation or CHAOS was suspected. Subsequent MRI performed at 24 weeks demonstrated bilaterally enlarged high-signal lungs, dilated bronchi, massive ascites, subcutaneous oedema and polyhydramnios. MRI confirmed the diagnosis of CHAOS. A second MRI at 35 weeks showed that the bilateral lung enlargement, ascites, oedema and polyhydramnios had resolved, but that the appearance of the airway was unchanged. The infant was delivered by caesarean section at 38 weeks of gestation and immediate tracheostomy was performed. This spontaneous regression was explained by a tracheo-oesophageal fistula that may have decreased the intrathoracic pressure.

Elevated white blood cell count as a risk factor of coronary artery disease: inconsistency between forms of the disease.

White blood cells (WBC) destabilize coronary artery plaques and an elevated WBC count is a risk factor of coronary artery disease (CAD). Nevertheless, the differences between the forms of CAD in the relationship with WBC count remain to be elucidated. To study these differences, we reviewed the health-checkup records from 1994 to 1999 for 6021 Japanese post office workers without any cardiovascular abnormalities. Baseline WBC counts of patients with acute coronary syndrome (ACS) were significantly higher than those of subjects free from coronary artery discase (mean +/- SD = 9210 +/- 2703/microL vs 6205 +/- 1635/microL, P<0.001), while the patients with stable angina pectoris (sAP) (6233 +/- 1528/microL) were similar to subjects without coronary artery disease in baseline WBC counts. Hypertension at the baseline was related to sAP (relative risk [95% CI] = 61.78 [17.29 to 78.66]) but not to ACS. Conversely, hypercholesterolemia and cigarette smoking were risk factors for ACS (relative risk [95% CI] = 11.48 [2.39 to 18.03] and 10.04 [3.00 to 12.12], respectively) but not for sAP. Multivariate logistic regression analysis found only WBC count (1000/microL) discriminated between ACS and sAP (P=0.038, odds ratio 2.049 [1.042-4.016]). We conclude that an elevated WBC count may be a risk factor of ACS but not sAP, and this finding is consistent with previous reports demonstrating the effects of WBCs in the destabilization of coronary artery plaques.

Ultrastructural and cytochemical characterization of human cord blood cells.

As part of a study to identify the characteristics of cord blood cells, we examined their morphological features by electron microscopy. Additionally, we cultured CD34-positive cells derived from cord blood and from bone marrow to perform morphological observations, as well as cytochemical examinations following the peroxidase reaction. Compared with normal peripheral blood cells, cord blood cells frequently showed immature morphology and a unique ultrastructure, such as nuclear pockets in neutrophils, several crystalloids in a single eosinophilic granule, and deformed nuclei in lymphocytes. In contrast to bone marrow cells, cord blood cells yielded a large number of cells of immature myelo-monocytic lineages in cell culture, and demonstrated a weaker peroxidase reaction. We identified that cord blood cells were different from normal peripheral blood cells and bone marrow cells, confirming the functional differences that were previously assumed.

Prenatal diagnosis of hypochondrogenesis using fetal MRI: a case report.

We describe the successful prenatal diagnosis of hypochondrogenesis by MRI. Fetal MR findings were the presence of a conspicuous cartilaginous structure in the basioccipital region, ill-defined ossification of the cervical vertebral bodies, hypoplastic thorax, retarded ossification of the pubic bones, and broad, short long bones. In contrast, fetal US revealed only the presence of short long bones. MRI accurately delineated the axial skeleton in this case and is an effective clinical tool for diagnosing skeletal dysplasias in utero.