A Noonan-like pediatric patient with a de novo CBL pathogenic variant and an RNF213 polymorphism p.R4810K presenting with cardiopulmonary arrest due to left main coronary artery ostial atresia.

Left main coronary artery ostial atresia (LMCAOA) is an extremely rare condition. Here, we report the case of a 14-year-old boy with Noonan syndrome-like disorder in whom LMCAOA was detected following cardiopulmonary arrest. The patient had been diagnosed with Noonan syndrome-like disorder with a pathogenic splice site variant of CBL c.1228-2 A > G. He suddenly collapsed when he was running. After administering two electric shocks using an automated external defibrillator, the patient's heartbeat resumed. Cardiac catheterization confirmed the diagnosis of LMCAOA. Left main coronary artery angioplasty was performed. The patient was discharged without neurological sequelae. Brain magnetic resonance imaging revealed asymptomatic Moyamoya disease. In addition, RNF213 c.14429 G > A p.R4810K was identified. There are no reports on congenital coronary malformations of compound variations of RNF213 and CBL. In contrast, the RNF213 p.R4810K polymorphism has been established as a risk factor for angina pectoris and myocardial infarction in adults, and several congenital coronary malformations due to genetic abnormalities within the RAS/MAPK signaling pathway have been reported. This report aims to highlight the risk of sudden death in patients with RASopathy and RNF213 p.R4810K polymorphism and emphasize the significance of actively searching for coronary artery morphological abnormalities in these patients.

Hypomethylation of a centromeric block of ICR1 is sufficient to cause Silver-Russell syndrome.

Silver-Russell syndrome (SRS) is a representative imprinting disorder. A major cause is the loss of methylation (LOM) of imprinting control region 1 (ICR1) within the IGF2/H19 domain. ICR1 is a gametic differentially methylated region (DMR) consisting of two repeat blocks, with each block including three CTCF target sites (CTSs). ICR1-LOM on the paternal allele allows CTCF to bind to CTSs, resulting in IGF2 repression on the paternal allele and biallelic expression of H19 We analysed 10 differentially methylated sites (DMSs) (ie, seven CTSs and three somatic DMRs within the IGF2/H19 domain, including two IGF2-DMRs and the H19-promoter) in five SRS patients with ICR1-LOM. Four patients showed consistent hypomethylation at all DMSs; however, one exhibited a peculiar LOM pattern, showing LOM at the centromeric region of the IGF2/H19 domain but normal methylation at the telomeric region. This raised important points: there may be a separate regulation of DNA methylation for the two repeat blocks within ICR1; there is independent control of somatic DMRs under each repeat block; sufficient IGF2 repression to cause SRS phenotypes occurs by LOM only in the centromeric block; and the need for simultaneous methylation analysis of several DMSs in both blocks for a correct molecular diagnosis.

Refractory sacrococcygeal germ cell tumor in Schinzel-Giedion syndrome.

We describe a boy with Schinzel-Giedion syndrome who developed refractory sacrococcygeal germ cell tumor with elements of embryonal carcinoma and immature teratoma. He developed local recurrence soon after tumor resection. The tumor was highly resistant to platinum-based combination chemotherapy, local irradiation, and salvage chemotherapy. Frequent infections resulted in a delay in treatment, although apparent fragility had not been observed clinically. He died from tumor progression at 32 months of age. Intensification of chemotherapy does not seem to be feasible for tumors in patients with Schinzel-Giedion syndrome.

Comprehensive and quantitative multilocus methylation analysis reveals the susceptibility of specific imprinted differentially methylated regions to aberrant methylation in Beckwith-Wiedemann syndrome with epimutations.

PURPOSE: Expression of imprinted genes is regulated by DNA methylation of differentially methylated regions (DMRs). Beckwith-Wiedemann syndrome is an imprinting disorder caused by epimutations of DMRs at 11p15.5. To date, multiple methylation defects have been reported in Beckwith-Wiedemann syndrome patients with epimutations; however, limited numbers of DMRs have been analyzed. The susceptibility of DMRs to aberrant methylation, alteration of gene expression due to aberrant methylation, and causative factors for multiple methylation defects remain undetermined. METHODS: Comprehensive methylation analysis with two quantitative methods, matrix-assisted laser desorption/ionization mass spectrometry and bisulfite pyrosequencing, was conducted across 29 DMRs in 54 Beckwith-Wiedemann syndrome patients with epimutations. Allelic expressions of three genes with aberrant methylation were analyzed. All DMRs with aberrant methylation were sequenced. RESULTS: Thirty-four percent of KvDMR1-loss of methylation patients and 30% of H19DMR-gain of methylation patients showed multiple methylation defects. Maternally methylated DMRs were susceptible to aberrant hypomethylation in KvDMR1-loss of methylation patients. Biallelic expression of the genes was associated with aberrant methylation. Cis-acting pathological variations were not found in any aberrantly methylated DMR. CONCLUSION: Maternally methylated DMRs may be vulnerable to DNA demethylation during the preimplantation stage, when hypomethylation of KvDMR1 occurs, and aberrant methylation of DMRs affects imprinted gene expression. Cis-acting variations of the DMRs are not involved in the multiple methylation defects.

MLL2 and KDM6A mutations in patients with Kabuki syndrome.

Kabuki syndrome is a congenital anomaly syndrome characterized by developmental delay, intellectual disability, specific facial features including long palpebral fissures and ectropion of the lateral third of the lower eyelids, prominent digit pads, and skeletal and visceral abnormalities. Mutations in MLL2 and KDM6A cause Kabuki syndrome. We screened 81 individuals with Kabuki syndrome for mutations in these genes by conventional methods (n = 58) and/or targeted resequencing (n = 45) or whole exome sequencing (n = 5). We identified a mutation in MLL2 or KDM6A in 50 (61.7%) and 5 (6.2%) cases, respectively. Thirty-five MLL2 mutations and two KDM6A mutations were novel. Non-protein truncating-type MLL2 mutations were mainly located around functional domains, while truncating-type mutations were scattered through the entire coding region. The facial features of patients in the MLL2 truncating-type mutation group were typical based on those of the 10 originally reported patients with Kabuki syndrome; those of the other groups were less typical. High arched eyebrows, short fifth finger, and hypotonia in infancy were more frequent in the MLL2 mutation group than in the KDM6A mutation group. Short stature and postnatal growth retardation were observed in all individuals with KDM6A mutations, but in only half of the group with MLL2 mutations.

Precise definition of the breakpoints of an apparently balanced translocation between chromosome 3q26 and chromosome 7q36: Role of KMT2C disruption.

When a de novo balanced reciprocal translocation is identified in the patient, the cause of phenotype of the patient can be explained by detecting the breakpoints of the genes. Here, we report a 3-year-old patient with developmental delay, autism spectrum disorder, and distinctive facial features who had an apparently balanced translocation between chromosome 3q26 and chromosome 7q36. Nanopore long-read sequencing revealed that balanced translocation disrupted the KMT2C gene, the haploinsufficiency of which leads to Kleefstra syndrome 2 characterized by delayed psychomotor development, variable intellectual disability and mild dysmorphism. Nanopore long-read sequencing was shown to be useful in elucidating the exact genetic etiology of patients with nonspecific clinical findings.

Axenfeld-Rieger anomaly and Axenfeld-Rieger syndrome: clinical, molecular-cytogenetic, and DNA array analyses of three patients with chromosomal defects at 6p25.

Clinical phenotypes of and genetic aberrations in three unrelated Japanese patients with Axenfeld-Rieger anomalies and various accompanying malformations of systemic organs are described. GTG-banded chromosome analysis showed terminal deletions of the short arm of chromosome 6 in two patients and an inversion, inv(6)(p25q14), in the other. FISH and DNA array analyses revealed that the two patients with deletions had 5.0-5.7 Mb and 6.6 Mb 6p terminal deletions, respectively, and FOXC1 was apparently deleted in both patients. In the other patient, the inversion breakpoint at 6p25 was estimated to be in or very close to the FOXC1 locus, but DNA array analysis did not reveal a deletion around the breakpoint. Common extraocular findings in these patients included broad forehead, brachycephaly, hypertelorism, downslanting palpebral fissures, small anteverted nose, and cardiac defects. Two patients also exhibited autistic characteristics. The two patients with deletions exhibited poor muscle tone and developmental delays. Most of these extraocular findings were similar to those found in previous patients with FOXC1 mutations and distinct from those found in patients with PITX2 mutations, who frequently develop umbilical and dental anomalies. We suggest that the psychomotor retardation is a clinical manifestation associated with a deletion of multiple contiguous genes in the 6p terminus and that this phenomenon is similar to the 6p25 deletion syndrome. Understanding the relationship between genetic lesions and the spectrum of extraocular findings in patients with Axenfeld-Rieger anomalies may lead to better clinical management.

Spectrum of MLL2 (ALR) mutations in 110 cases of Kabuki syndrome.

Kabuki syndrome is a rare, multiple malformation disorder characterized by a distinctive facial appearance, cardiac anomalies, skeletal abnormalities, and mild to moderate intellectual disability. Simplex cases make up the vast majority of the reported cases with Kabuki syndrome, but parent-to-child transmission in more than a half-dozen instances indicates that it is an autosomal dominant disorder. We recently reported that Kabuki syndrome is caused by mutations in MLL2, a gene that encodes a Trithorax-group histone methyltransferase, a protein important in the epigenetic control of active chromatin states. Here, we report on the screening of 110 families with Kabuki syndrome. MLL2 mutations were found in 81/110 (74%) of families. In simplex cases for which DNA was available from both parents, 25 mutations were confirmed to be de novo, while a transmitted MLL2 mutation was found in two of three familial cases. The majority of variants found to cause Kabuki syndrome were novel nonsense or frameshift mutations that are predicted to result in haploinsufficiency. The clinical characteristics of MLL2 mutation-positive cases did not differ significantly from MLL2 mutation-negative cases with the exception that renal anomalies were more common in MLL2 mutation-positive cases. These results are important for understanding the phenotypic consequences of MLL2 mutations for individuals and their families as well as for providing a basis for the identification of additional genes for Kabuki syndrome.

Molecular karyotyping in 17 patients and mutation screening in 41 patients with Kabuki syndrome.

The Kabuki syndrome (KS, OMIM 147920), also known as the Niikawa-Kuroki syndrome, is a multiple congenital anomaly/mental retardation syndrome characterized by a distinct facial appearance. The cause of KS has been unidentified, even by whole-genome scan with array comparative genomic hybridization (CGH). In recent years, high-resolution oligonucleotide array technologies have enabled us to detect fine copy number alterations. In 17 patients with KS, molecular karyotyping was carried out with GeneChip 250K NspI array (Affymetrix) and Copy Number Analyser for GeneChip (CNAG). It showed seven copy number alterations, three deleted regions and four duplicated regions among the patients, with the exception of registered copy number variants (CNVs). Among the seven loci, only the region of 9q21.11-q21.12 (approximately 1.27 Mb) involved coding genes, namely, transient receptor potential cation channel, subfamily M, member 3 (TRPM3), Kruppel-like factor 9 (KLF9), structural maintenance of chromosomes protein 5 (SMC5) and MAM domain containing 2 (MAMDC2). Mutation screening for the genes detected 10 base substitutions consisting of seven single-nucleotide polymorphisms (SNPs) and three silent mutations in 41 patients with KS. Our study could not show the causative genes for KS, but the locus of 9q21.11-q21.12, in association with a cleft palate, may contribute to the manifestation of KS in the patient. As various platforms on oligonucleotide arrays have been developed, higher resolution platforms will need to be applied to search tiny genomic rearrangements in patients with KS.

Comprehensive clinical and molecular studies in split-hand/foot malformation: identification of two plausible candidate genes (LRP6 and UBA2).

Split-hand/foot malformation (SHFM) is a clinically and genetically heterogeneous condition. We sequentially performed screening of the previously identified Japanese founder 17p13.3 duplication/triplication involving BHLHA9, array comparative genomic hybridization, and whole exome sequencing (WES) in newly recruited 41 Japanese families with non-syndromic and syndromic SHFM. We also carried out WES in seven families with nonsyndromic and syndromic SHFM in which underlying genetic causes including pathogenic copy-number variants (CNVs) remained undetected in our previous studies of 56 families. Consequently, we identified not only known pathogenic CNVs (17p13.3 duplications/triplications [n = 21], 2q31 deletion [n = 1], and 10q24 duplications [n = 3]) and rare variants in known causative genes (TP63 [n = 3], DLX5 [n = 1], IGF2 [n = 1], WNT10B [n = 3], WNT10B/PORCN [n = 1], and PORCN [n = 1]), but also a de novo 19q13.11 deletion disrupting UBA2 (n = 1) and variants that probably affect function in LRP6 (n = 1) and UBA2 (n = 1). Thus, together with our previous data based on testing of 56 families, molecular studies for a total of 97 families with SHFM revealed underlying genetic causes in 75 families, and clinical studies for the 75 families indicated a certain degree of correlation between genetic causes and phenotypes. The results imply that SHFM primarily occurs as a genetic disorder with genotype-phenotype correlations. Furthermore, the results together with previous data such as the development of SHFM in Lrp6 knockout mice, the presence of SHFM in two subjects with 19q13 deletions involving UBA2, and strong mouse Uba2 expression in the developing limb buds, imply that LRP6 and UBA2 represent plausible candidate genes for SHFM.

A case of 45,X/47,XXX mosaic Turner syndrome with limb length discrepancy.

Patients with Turner syndrome (TS) frequently show short stature and skeletal deformities, such as kyphosis and scoliosis. However, to the best of our knowledge, limb length discrepancy (LLD) has not yet been reported in patients with TS. The case of a 12-yr-old girl with 45,X/47,XXX mosaic TS showing LLD is herein presented. She was on GH therapy for short stature and was noted to have scoliosis in the standing position at a regular examination; however, the scoliosis became less evident in the supine position, which is indicative of LLD. The length of the left leg was 5.0 cm shorter than that of the right leg when measured. She was referred to orthopedics and underwent right distal femoral and right proximal tibial staple epiphysiodesis to shorten the abnormally long limb at 10 yr 6 mo of age. One year after the operation, the LLD decreased from 5.0 to 1.5 cm. During this period, GH was continued. LLD is a rare complication in TS, but when patients with TS show scoliosis in the standing position, re-evaluation for scoliosis in the supine position should be performed and the lengths of both legs should be measured.

Miller-Dieker Syndrome with unbalanced translocation 45, X, psu dic(17;Y)(p13;p11.32) detected by fluorescence in situ hybridization and G-banding analysis using high resolution banding technique.

Lissencephaly is one of the central nervous system anomalies of Miller-Dieker Syndrome (MDS). Fetuses with lissencephaly have an abnormal smooth brain with fewer folds and grooves that will be detected by ultrasounds or fetal magnetic resonance imaging (MRI) after 30 weeks of gestation. We report a fetus with lissencephaly diagnosed as Miller-Dieker Syndrome postnatally. G banded chromosome analysis revealed 45,X,psu dic(17;Y)(p13;p11.32).ish dic (17;Y)(LIS1-,RARA+, SRY+, DYZ3+) by G-banding analysis using high resolution banding technique. Fetal delayed cortical development will be the findings to perform further investigations including fluorescence in situ hybridization analysis for MDS, a 17p13.3 microdeletion syndrome, pre/postnatally. This will be the first case of MDS with unbalanced translocation between deleted short arm of chromosome 17 and Y chromosome.

Genetic and prenatal findings in two Japanese patients with Schinzel-Giedion syndrome.

We report two Japanese patients with Schinzel-Giedion syndrome. When polyhydramnios is observed, additional fetal findings such as overlapping fingers, hydrocephalus, hydronephrosis, and very characteristic facial appearance comprising high, prominent forehead, hypertelorism, and depressed nasal root may suggest Schinzel-Giedion syndrome.

Japanese founder duplications/triplications involving BHLHA9 are associated with split-hand/foot malformation with or without long bone deficiency and Gollop-Wolfgang complex.

BACKGROUND: Limb malformations are rare disorders with high genetic heterogeneity. Although multiple genes/loci have been identified in limb malformations, underlying genetic factors still remain to be determined in most patients. METHODS: This study consisted of 51 Japanese families with split-hand/foot malformation (SHFM), SHFM with long bone deficiency (SHFLD) usually affecting the tibia, or Gollop-Wolfgang complex (GWC) characterized by SHFM and femoral bifurcation. Genetic studies included genomewide array comparative genomic hybridization and exome sequencing, together with standard molecular analyses. RESULTS: We identified duplications/triplications of a 210,050 bp segment containing BHLHA9 in 29 SHFM patients, 11 SHFLD patients, two GWC patients, and 22 clinically normal relatives from 27 of the 51 families examined, as well as in 2 of 1,000 Japanese controls. Families with SHFLD- and/or GWC-positive patients were more frequent in triplications than in duplications. The fusion point was identical in all the duplications/triplications and was associated with a 4 bp microhomology. There was no sequence homology around the two breakpoints, whereas rearrangement-associated motifs were abundant around one breakpoint. The rs3951819-D17S1174 haplotype patterns were variable on the duplicated/triplicated segments. No discernible genetic alteration specific to patients was detected within or around BHLHA9, in the known causative SHFM genes, or in the exome. CONCLUSIONS: These results indicate that BHLHA9 overdosage constitutes the most frequent susceptibility factor, with a dosage effect, for a range of limb malformations at least in Japan. Notably, this is the first study revealing the underlying genetic factor for the development of GWC, and demonstrating the presence of triplications involving BHLHA9. It is inferred that a Japanese founder duplication was generated through a replication-based mechanism and underwent subsequent triplication and haplotype modification through recombination-based mechanisms, and that the duplications/triplications with various haplotypes were widely spread in Japan primarily via clinically normal carriers and identified via manifesting patients. Furthermore, genotype-phenotype analyses of patients reported in this study and the previous studies imply that clinical variability is ascribed to multiple factors including the size of duplications/triplications as a critical factor.

Heterozygous mutations in cyclic AMP phosphodiesterase-4D (PDE4D) and protein kinase A (PKA) provide new insights into the molecular pathology of acrodysostosis.

Acrodysostosis without hormone resistance is a rare skeletal disorder characterized by brachydactyly, nasal hypoplasia, mental retardation and occasionally developmental delay. Recently, loss-of-function mutations in the gene encoding cAMP-hydrolyzing phosphodiesterase-4D (PDE4D) have been reported to cause this rare condition but the pathomechanism has not been fully elucidated. To understand the pathogenetic mechanism of PDE4D mutations, we conducted 3D modeling studies to predict changes in the binding efficacy of cAMP to the catalytic pocket in PDE4D mutants. Our results indicated diminished enzyme activity in the two mutants we analyzed (Gly673Asp and Ile678Thr; based on PDE4D4 residue numbering). Ectopic expression of PDE4D mutants in HEK293 cells demonstrated this reduction in activity, which was identified by increased cAMP levels. However, the cells from an acrodysostosis patient showed low cAMP accumulation, which resulted in a decrease in the phosphorylated cAMP Response Element-Binding Protein (pCREB)/CREB ratio. The reason for this discrepancy was due to a compensatory increase in expression levels of PDE4A and PDE4B isoforms, which accounted for the paradoxical decrease in cAMP levels in the patient cells expressing mutant isoforms with a lowered PDE4D activity. Skeletal radiographs of 10-week-old knockout (KO) rats showed that the distal part of the forelimb was shorter than in wild-type (WT) rats and that all the metacarpals and phalanges were also shorter in KO, as the name acrodysostosis implies. Like the G-protein α-stimulatory subunit and PRKAR1A, PDE4D critically regulates the cAMP signal transduction pathway and influences bone formation in a way that activity-compromising PDE4D mutations can result in skeletal dysplasia. We propose that specific inhibitory PDE4D mutations can lead to the molecular pathology of acrodysostosis without hormone resistance but that the pathological phenotype may well be dependent on an over-compensatory induction of other PDE4 isoforms that can be expected to be targeted to different signaling complexes and exert distinct effects on compartmentalized cAMP signaling.