Genetic backgrounds and genotype-phenotype relationships in anthropometric parameters of 116 Japanese individuals with Noonan syndrome.

Noonan syndrome (NS) is caused by pathogenic variants in genes encoding components of the RAS/MAPK pathway and presents with a number of symptoms, including characteristic facial features, congenital heart diseases, and short stature. Advances in genetic analyses have contributed to the identification of pathogenic genes in NS as well as genotype-phenotype relationships; however, updated evidence for the detection rate of pathogenic genes with the inclusion of newly identified genes is lacking in Japan. Accordingly, we examined the genetic background of 116 individuals clinically diagnosed with NS and the frequency of short stature. We also investigated genotype-phenotype relationships in the context of body mass index (BMI). Genetic testing revealed the responsible variants in 100 individuals (86%), where PTPN11 variants were the most prevalent (43%) and followed by SOS1 (12%) and RIT1 (9%). The frequency of short stature was the lowest in subjects possessing RIT1 variants. No genotype-phenotype relationships in BMI were observed among the genotypes. In conclusion, this study provides evidence for the detection rate of pathogenic genes and genotype-phenotype relationships in Japanese patients with NS, which will be of clinical importance for accelerating our understanding of the genetic backgrounds of Japanese patients with NS.

Multiplex Real-Time PCR-Based Newborn Screening for Severe Primary Immunodeficiency and Spinal Muscular Atrophy in Osaka, Japan: Our Results after 3 Years.

In newborn screening (NBS), it is important to consider the availability of multiplex assays or other tests that can be integrated into existing systems when attempting to implement NBS for new target diseases. Recent developments in innovative testing technology have made it possible to simultaneously screen for severe primary immunodeficiency (PID) and spinal muscular atrophy (SMA) using quantitative real-time polymerase chain reaction (qPCR) assays. We describe our experience of optional NBS for severe PID and SMA in Osaka, Japan. A multiplex TaqMan qPCR assay was used for the optional NBS program. The assay was able to quantify the levels of T-cell receptor excision circles and kappa-deleting recombination excision circles, which is useful for severe combined immunodeficiency and B-cell deficiency screening, and can simultaneously detect the homozygous deletion of SMN1 exon 7, which is useful for NBS for SMA. In total, 105,419 newborns were eligible for the optional NBS program between 1 August 2020 and 31 August 2023. A case each of X-linked agammaglobulinemia and SMA were diagnosed through the optional NBS and treated at early stages (before symptoms appeared). Our results show how multiplex PCR-based NBS can benefit large-scale NBS implementation projects for new target diseases.

Haploinsufficiency of NKX2-1 is likely to contribute to developmental delay involving 14q13 microdeletions.

Nucleotide variations or deletions in the NK2 homeobox 1 gene (NKX2-1), located at 14q13.3, lead to symptoms associated with the brain, lungs, and thyroid, and the combination of these phenotypes is clinically recognized as the brain-lung-thyroid syndrome. Many types of nucleotide variants of NKX2-1 have been identified, and phenotypic variability has been reported. Chromosomal deletions involving NKX2-1 have also been reported; however, phenotypic differences between patients with nucleotide variants of NKX2-1 and patients with chromosomal deletions involving NKX2-1 have not been well established. Recently, we identified seven patients with 14q13 microdeletions involving the NKX2-1. Most patients exhibited developmental delay. This inquiry arises regarding the potential existence of haploinsufficiency effects beyond those attributed to NKX2-1 within the 14q13 microdeletion. However, a literature review has shown that developmental delay is not rare in patients with nucleotide alterations in NKX2-1. Rather, motor function impairment may have affected the total developmental assessment, and the haploinsufficiency of genes contiguous to NKX2-1 is unlikely to contribute to developmental delay.

Craniosynostosis in molecularly diagnosed Kabuki syndrome: Prevalence and clinical implications.

Kabuki syndrome (KS) is characterized by growth impairment, psychomotor delay, congenital heart disease, and distinctive facial features. KMT2D and KDM6A have been identified as the causative genes of KS. Craniosynostosis (CS) has been reported in individuals with KS; however, its prevalence and clinical implications remain unclear. In this retrospective study, we investigated the occurrence of CS in individuals with genetically diagnosed KS and examined its clinical significance. Among 42 individuals with genetically diagnosed KS, 21 (50%) exhibited CS, with 10 individuals requiring cranioplasty. No significant differences were observed based on sex, causative gene, and molecular consequence among individuals with KS who exhibited CS. Both individuals who underwent evaluation with three-dimensional computed tomography (3DCT) and those who required surgery tended to exhibit cranial dysmorphology. Notably, in several individuals, CS was diagnosed before KS, suggesting that CS could be one of the clinical features by which clinicians can diagnose KS. This study highlights that CS is one of the noteworthy complications in KS, emphasizing the importance of monitoring cranial deformities in the health management of individuals with KS. The findings suggest that in individuals where CS is a concern, conducting 3DCT evaluations for CS and digital impressions are crucial.

Breakpoints in complex chromosomal rearrangements correspond to transposase-accessible regions of DNA from mature sperm.

Constitutional complex chromosomal rearrangements (CCRs) are rare cytogenetic aberrations arising in the germline via an unknown mechanism. Here we analyzed the breakpoint junctions of microscopically three-way or more complex translocations using comprehensive genomic and epigenomic analyses. All of these translocation junctions showed submicroscopic genomic complexity reminiscent of chromothripsis. The breakpoints were clustered within small genomic domains with junctions showing microhomology or microinsertions. Notably, all of the de novo cases were of paternal origin. The breakpoint distributions corresponded specifically to the ATAC-seq (assay for transposase-accessible chromatin with sequencing) read data peak of mature sperm and not to other chromatin markers or tissues. We propose that DNA breaks in CCRs may develop in an accessible region of densely packaged chromatin during post-meiotic spermiogenesis.

Newborn screening for spinal muscular atrophy in Osaka -challenges in a Japanese pilot study.

OBJECTIVE: This study aimed to establish an optional newborn screening program for spinal muscular atrophy (SMA-NBS) in Osaka. METHODS: A multiplex TaqMan real-time quantitative polymerase chain reaction assay was used to screen for SMA. Dried blood spot samples obtained for the optional NBS program for severe combined immunodeficiency, which covers about 50% of the newborns in Osaka, were used. To obtain informed consent, participating obstetricians provided information about the optional NBS program to all parents by giving leaflets to prospective parents and uploading the information onto the internet. We prepared a workflow so that babies that were diagnosed with SMA through the NBS could be treated immediately. RESULTS: From 1 February 2021 to 30 September 2021, 22,951 newborns were screened for SMA. All of them tested negative for survival motor neuron (SMN)1 deletion, and there were no false-positives. Based on these results, an SMA-NBS program was established in Osaka and included in the optional NBS programs run in Osaka from 1 October 2021. A positive baby was found by screening, diagnosed with SMA (the baby possessed 3 copies of the SMN2 gene and was pre-symptomatic), and treated immediately. CONCLUSION: The workflow of the Osaka SMA-NBS program was confirmed to be useful for babies with SMA.

Interstitial deletions in the proximal regions of 6q: 12 original cases and a literature review.

Interstitial microdeletions in the proximal region of the long arm of chromosome 6 are rare. Herein we have reported 12 patients with developmental delays associated with interstitial microdeletions in 6q ranging from q12 to q22. The microdeletions were detected by chromosomal microarray testing. To confirm the clinical significance of these deletions, genotype-phenotype correlation analysis was performed using genetic and predicted loss-of-function data. SIM1 was recognized as the gene responsible for developmental delay, particularly in Prader-Willi syndrome-like phenotypes. Other genes possibly related to developmental delay were ZNF292, PHIP, KCNQ5, and NUS1. To further establish the correlation between the genotype and phenotype, more patient information is required.

The novel and recurrent variants in exon 31 of CREBBP in Japanese patients with Menke-Hennekam syndrome.

Menke-Hennekam syndrome-1 (MKHK1) is a congenital disorder caused by the heterozygous variants in exon 30 or 31 of CREBBP (CREB binding protein) gene mapped on 16p13.3. It is characterized by psychomotor delay, variable impairment of intellectual disability (ID), feeding difficulty, autistic behavior, hearing impairment, short stature, microcephaly, and facial dysmorphisms. The CREBBP loss-of-function variants cause Rubinstein-Taybi syndrome-1 (RSTS1). The function of CREBBP leading to MKHK1 has not been clarified so far, and the phenotype of MKHK1 significantly differs from that of RSTS1. We examined six patients with de novo pathogenic variants affecting the last exon of CREBBP, and they shared the clinical features of MKHK1. This study revealed that one frameshift and three nonsense variants of CREBBP cause MKHK1, and inferred that the nonsense variants of the last exon could further help in the elucidation of the etiology of MKHK1.

Novel variants in aromatic L-amino acid decarboxylase deficiency: Case report of sisters with mild phenotype.

BACKGROUND: Aromatic L-amino acid decarboxylase (AADC) deficiency, caused by a pathogenic variant in the dopa decarboxylase (DDC) gene, is a rare neurometabolic disorder in which catecholamine and serotonin are not synthesized. From a large number of reports, it has been recognized that most affected patients show severe developmental delay in a bedridden state and are unable to speak. On the other hand, patients with a mild phenotype with AADC deficiency have been reported, but they number only a few cases. Therefore, the variation of phenotypes of the disease appears to be broad, and it may be challenging to diagnose an atypical phenotype as AADC deficiency. CASE REPORT: We report novel compound heterozygous variants in DDC (c.202G > A and c.254C > T) in two sisters, whose main complaint was mild developmental delay, by whole-exome sequencing (WES). Additionally, we describe their clinical features and provide an image that shows the variants located at different sites responsible for the catalysis of AADC in a three-dimensional structure. The patients were prescribed a Monoamine oxidase (MAO) inhibitor after diagnosis. INTERPRETATION: Our cases indicate that a comprehensive genomic approach helps to diagnose AADC deficiency with atypical features, and underscore the significance of understanding the variations of this disorder for diagnosis and appropriate treatment.

Protocol for isolating young adult parvalbumin interneurons from the mouse brain for extraction of high-quality RNA.

Dysfunction in the parvalbumin (PV) subclass of GABAergic interneurons is implicated in several neurodevelopmental disorders that evolve in severity with postnatal developmental stages. Understanding the molecular underpinnings of the postnatal changes in the function of PV interneurons has been limited by the difficulty in the isolation of pure adult PV interneurons and high-quality RNA. Here, we describe our protocol for the isolation of pure young adult PV interneurons and preparation of high-quality RNA from these cells. For complete details on the use and execution of this protocol, please refer to Joseph et al. (2021).

Blended phenotype of combination of HERC2 and AP3B2 deficiency and Angelman syndrome caused by paternal isodisomy of chromosome 15.

Angelman syndrome is a neurodevelopmental disorder characterized by intellectual disability (ID), a distinctive gait pattern, abnormal behaviors, severe impairment in language development, and characteristic facial features. Most cases are caused by the absence of a maternal contribution to the imprinted region on chromosome 15q11-q13. Here, we present the first reported case of a 3-year-old boy with an atypical phenotype of Angelman syndrome due to uniparental isodisomy with two recessive homozygous pathogenic variants: in HERC2 and AP3B2. Known phenotypes related to HERC2 and AP3B2 include ID and early infantile epileptic encephalopathy, respectively. The patient had severe global developmental delay and profound ID and showed a happy demeanor, stereotypic laughter, and hand-flapping movements, but also irritability. Craniofacial dysmorphic features, including brachycephaly, strabismus, wide ala nasi, short philtrum, wide open mouth, and slight hypopigmentation were seen. Progressive microcephaly was noted. Magnetic resonance imaging of the brain showed delayed myelination and cerebral atrophy. Trio whole exome sequencing and CGH-SNP array analysis revealed paternal uniparental isodisomy of chromosome 15 and two coexisting recessive diseases resulting from homozygous HERC2 and AP3B2 pathogenic variants. The pathogenic variant in HERC2 was inherited from his heterozygous-carrier father, and the variant in AP3B2 was de novo. We suppose that these unusual features were the combination of the effect of three concomitant disorders.

Clinical spectrum of individuals with de novo EBF3 variants or deletions.

Hypotonia, ataxia and delayed development syndrome (HADDS) (MIM#617330) is a neurodevelopmental disorder caused by heterozygous pathogenic variants in EBF3 (MIM; 607,407), which is located on chromosome 10q26, and was first reported in 2017. To date, missense, nonsense and frameshift variants have been reported as causes of HADDS, and EBF3 pathogenic variants have been predicted to result in nonsense-mediated mRNA decay and haploinsufficiency. It was also reported that total deletion of EBF3 associated with a 10q26.3 microdeletion also causes HADDS symptoms, supporting the concept that HADDS results from haploinsufficiency of EBF3. Here, we report eight unrelated individuals with heterozygous pathogenic variants of EBF3 or haploinsufficiency of EBF3 due to 10q26 deletion, who exhibit clinical findings including craniofacial features of HADDS. In a detailed examination of clinical manifestations in this study, revealed that neurogenic bladder was diagnosed in infancy (the median 6.5 months), was more frequent than previously reported, and required cystostomy in all but one case. For psychomotor delay, it was also found that their motor/skills values were significantly lower than their cognition/adaptation values (p = 0.0016; paired t-test). Therefore, that HADDS is a recognizable syndrome that shares its characteristic facial features, and that neurogenic bladder diagnosed in infancy and psychomotor delay with marked delay in motor/skills are noteworthy findings in the diagnosis and management of individuals with HADDS.

Postnatal Arx transcriptional activity regulates functional properties of PV interneurons.

The transcription factor Aristaless-related X-linked gene (Arx) is a monogenic factor in early onset epileptic encephalopathies (EOEEs) and a fundamental regulator of early stages of brain development. However, Arx expression persists in mature GABAergic neurons with an unknown role. To address this issue, we generated a conditional knockout (CKO) mouse in which postnatal Arx was ablated in parvalbumin interneurons (PVIs). Electroencephalogram (EEG) recordings in CKO mice revealed an increase in theta oscillations and the occurrence of occasional seizures. Behavioral analysis uncovered an increase in anxiety. Genome-wide sequencing of fluorescence activated cell sorted (FACS) PVIs revealed that Arx impinged on network excitability via genes primarily associated with synaptic and extracellular matrix pathways. Whole-cell recordings revealed prominent hypoexcitability of various intrinsic and synaptic properties. These results revealed important roles for postnatal Arx expression in PVIs in the control of neural circuits and that dysfunction in those roles alone can cause EOEE-like network abnormalities.

Emergence of dorsal-ventral polarity in ESC-derived retinal tissue.

We previously demonstrated that mouse embryonic stem cell (mESC)-derived retinal epithelium self-forms an optic cup-like structure. In the developing retina, the dorsal and ventral sides differ in terms of local gene expression and morphological features. This aspect has not yet been shown in vitro Here, we demonstrate that mESC-derived retinal tissue spontaneously acquires polarity reminiscent of the dorsal-ventral (D-V) patterning of the embryonic retina. Tbx5 and Vax2 were expressed in a mutually exclusive manner, as seen in vivo Three-dimensional morphometric analysis showed that the in vitro-formed optic cup often contains cleft structures resembling the embryonic optic fissure. To elucidate the mechanisms underlying the spontaneous D-V polarization of mESC-derived retina, we examined the effects of patterning factors, and found that endogenous BMP signaling plays a predominant role in the dorsal specification. Further analysis revealed that canonical Wnt signaling, which was spontaneously activated at the proximal region, acts upstream of BMP signaling for dorsal specification. These observations suggest that D-V polarity could be established within the self-formed retinal neuroepithelium by intrinsic mechanisms involving the spatiotemporal regulation of canonical Wnt and BMP signals.

p75 neurotrophin receptor signaling in the nervous system.

The neurotrophin receptor p75(NTR) has long been known as a receptor for neurotrophins that promote survival and differentiation. Consistent with the role of neurotrophins, p75(NTR) is expressed during the developmental stages of the nervous system. However, p75(NTR) is re-expressed in various pathological conditions in the adult. We now know that p75(NTR) has the ability to elicit bi-directional signals, that result in the inhibition as well as the promotion of the neurite outgrowth. p75(NTR) is a key receptor for myelin-derived inhibitory cues that contribute to the lack of regeneration of the central nervous system.

Promotion of axon regeneration by myelin-associated glycoprotein and Nogo through divergent signals downstream of Gi/G.

Several myelin-derived proteins have been identified as components of the CNS myelin that prevents axonal regeneration in the adult vertebrate CNS. Activation of RhoA has been shown to be an essential part of the signaling mechanism of these proteins. Here we report an additional signal, which determines whether these proteins promote or inhibit axon outgrowth. Myelin-associated glycoprotein (MAG) and Nogo trigger the intracellular elevation of Ca2+ as well as the activation of PKC, presumably mediated by G(i)/G. Neurite outgrowth inhibition and growth cone collapse by MAG or Nogo can be converted to neurite extension and growth cone spreading by inhibiting conventional PKC, but not by inhibiting inositol 1,4,5-triphosphate (IP3). Conversely, neurite growth of immature neurons promoted by MAG is abolished by inhibiting IP3. Activation of RhoA is independent of PKC. Thus, a balance between PKC and IP3 is important for bidirectional regulation of axon regeneration by the myelin-derived proteins.