Truncating variants of the sterol recognition region of SHH cause hypertelorism phenotype rather than hypotelorism-holoprosencephaly.

Sonic hedgehog signaling molecule (SHH) is a key molecule in the cilia-mediated signaling pathway and a critical morphogen in embryogenesis. The association between loss-of-function variants of SHH and holoprosencephaly is well established. In mice experiments, reduced or increased signaling of SHH have been shown to be associated with narrowing or excessive expansion of the facial midline, respectively. Herein, we report two unrelated patients with de novo truncating variants of SHH presenting with hypertelorism rather than hypotelorism. The first patient was a 13-year-old girl. Her facial features included hypertelorism, strabismus, telecanthus, malocclusion, frontal bossing, and wide widow's peak. She had borderline developmental delay and agenesis of the corpus callosum. She had a nonsense variant of SHH: Chr7(GRCh38):g.155802987C > T, NM_000193.4:c.1302G > A, p.(Trp434*). The second patient was a 25-year-old girl. Her facial features included hypertelorism and wide widow's peak. She had developmental delay and agenesis of the corpus callosum. She had a frameshift variant of SHH: Chr7(GRCh38):g.155803072_155803074delCGGinsT, NM_000193.4:c.1215_1217delCCGinsA, p.(Asp405Glufs*92). The hypertelorism phenotype contrasts sharply with the prototypical hypotelorism-holoprosencephaly phenotype associated with loss-of-function of SHH. We concluded that a subset of truncating variants of SHH could be associated with hypertelorism rather than hypotelorism.

Successful skipping of abnormal pseudoexon by antisense oligonucleotides in vitro for a patient with beta-propeller protein-associated neurodegeneration.

Pathogenic variants in WDR45 on chromosome Xp11 cause neurodegenerative disorder beta-propeller protein-associated neurodegeneration (BPAN). Currently, there is no effective therapy for BPAN. Here we report a 17-year-old female patient with BPAN and show that antisense oligonucleotide (ASO) was effective in vitro. The patient had developmental delay and later showed extrapyramidal signs since the age of 15 years. MRI findings showed iron deposition in the globus pallidus and substantia nigra on T2 MRI. Whole genome sequencing and RNA sequencing revealed generation of pseudoexon due to inclusion of intronic sequences triggered by an intronic variant that is remote from the exon-intron junction: WDR45 (OMIM #300526) chrX(GRCh37):g.48935143G > C, (NM_007075.4:c.235 + 159C > G). We recapitulated the exonization of intron sequences by a mini-gene assay and further sought antisense oligonucleotide that induce pseudoexon skipping using our recently developed, a dual fluorescent splicing reporter system that encodes two fluorescent proteins, mCherry, a transfection marker designed to facilitate evaluation of exon skipping and split eGFP, a splicing reaction marker. The results showed that the 24-base ASO was the strongest inducer of pseudoexon skipping. Our data presented here have provided supportive evidence for in vivo preclinical studies.

Identification of a novel splice-site WWOX variant with paternal uniparental isodisomy in a patient with infantile epileptic encephalopathy.

WOREE syndrome is an early infantile epileptic encephalopathy characterized by drug-resistant seizures and severe psychomotor developmental delays. We report a case of a WWOX splice-site mutation with uniparental isodisomy. A 1-year and 7-month-old girl presented with nystagmus and epileptic seizures from early infancy, with no fixation or pursuit of vision. Physical examination revealed small deformities, such as swelling of both cheeks, folded fingers, rocking feet, and scoliosis. Brain imaging revealed slight hypoplasia of the cerebrum. Electroencephalogram showed focal paroxysmal discharges during the interictal phase of seizures. Vitamin B6 and zonisamide were administered for early infantile epileptic encephalopathy; however, the seizures were not relieved. Despite altering the type and dosage of antiepileptic drugs and ACTH therapy, the seizures were intractable. Whole-exome analysis revealed the homozygosity of WWOX(NM_016373.4):c.516+1G>A. The WWOX mRNA sequencing using peripheral blood RNA confirmed that exon 5 was homozygously deleted. Based on these results, the patient was diagnosed with WOREE syndrome at 5 months. The WWOX variant found in this study is novel and has never been reported before. WOREE syndrome being extremely rare, further case series and analyses of its pathophysiology are warranted.

SALL4 deletion and kidney and cardiac defects associated with VACTERL association.

Congenital anomalies of the kidney and urinary tract (CAKUT) can be a part of the VACTERL association, which represents the non-random combination of the following congenital anomalies: vertebral anomalies, anal anomalies, cardiac anomalies, tracheal-esophageal anomalies, kidney anomalies, and limb anomalies. VACTERL association is generally considered to be a non-genetic condition. Exceptions include a patient with a heterozygous nonsense SALL4 variant and anal stenosis, tetralogy of Fallot, sacro-vertebral fusion, and radial and thumb anomalies. SALL4 encodes a transcription factor that plays a critical role in kidney morphogenesis. Here, we report a patient with VACTERL association and a heterozygous 128-kb deletion spanning SALL4 who presented with renal hypoplasia, radial and atrio-septal defects, and patent ductus arteriosus. The present report of SALL4 deletion, in addition to a previously reported patient with VACTERL association phenotype and SALL4 nonsense mutation, further supports the notion that SALL4 haploinsufficiency can lead to VACTERL association.

PDIVAS: Pathogenicity predictor for Deep-Intronic Variants causing Aberrant Splicing.

BACKGROUND: Deep-intronic variants that alter RNA splicing were ineffectively evaluated in the search for the cause of genetic diseases. Determination of such pathogenic variants from a vast number of deep-intronic variants (approximately 1,500,000 variants per individual) represents a technical challenge to researchers. Thus, we developed a Pathogenicity predictor for Deep-Intronic Variants causing Aberrant Splicing (PDIVAS) to easily detect pathogenic deep-intronic variants. RESULTS: PDIVAS was trained on an ensemble machine-learning algorithm to classify pathogenic and benign variants in a curated dataset. The dataset consists of manually curated pathogenic splice-altering variants (SAVs) and commonly observed benign variants within deep introns. Splicing features and a splicing constraint metric were used to maximize the predictive sensitivity and specificity, respectively. PDIVAS showed an average precision of 0.92 and a maximum MCC of 0.88 in classifying these variants, which were the best of the previous predictors. When PDIVAS was applied to genome sequencing analysis on a threshold with 95% sensitivity for reported pathogenic SAVs, an average of 27 pathogenic candidates were extracted per individual. Furthermore, the causative variants in simulated patient genomes were more efficiently prioritized than the previous predictors. CONCLUSION: Incorporating PDIVAS into variant interpretation pipelines will enable efficient detection of disease-causing deep-intronic SAVs and contribute to improving the diagnostic yield. PDIVAS is publicly available at https://github.com/shiro-kur/PDIVAS .

BMP2 is a potential causative gene for isolated dextrocardia situs solitus.

BMP2 (bone morphogenic protein-2) is a member of the TGF-ß superfamily and has essential roles in the development of multiple organs, including osteogenesis. Because of its crucial role in organ and skeletal development, Bmp2 null mice is fetal lethal. The recent report has characterized multiple patients with BMP2 haploinsufficiency, describing individuals with BMP2 sequence variants and deletions associated with short stature without endocrinological abnormalities, a recognizable craniofacial gestalt, skeletal anomalies, and congenital heart disease. However, due to a small number of reported patients with BMP2 haploinsufficiency, the genotype and phenotype correlations are not fully understood. We experienced a family of BMP2 haploinsufficiency with a novel frameshift variant NM_001200.4: c.231dup (p.Tyr78Leufs*38) which was predicted to be "pathogenic" by the American College of Genetics and Genomics (ACGM) criteria. In addition to short stature, impaired hearing ability and minor skeletal deformities, the proband exhibited isolated dextrocardia situs solitus without cardiac anomalies and abnormal locations of other visceral organs. Our study would shed light on the crucial role of BMP2 in determining the cardiac axis, and further studies are needed to assemble more cases to elucidate BMP2 role in human heart development.

Café-au-lait Spots and Cleft Palate: Not a Chance Association.

The recognition of syndromic forms of cleft palate is important for condition-specific management. Here, we report a patient with cleft palate, congenital heart disease, intellectual disability, and café-au-lait spots who had a deletion of chromosome 15q14. The identification of the precise breakpoints using a Nanopore-based long-read sequencer showed that the deletion spanned MEIS2 and SPRED1 loci. Cleft palate and café-au-lait spots can be ascribed to MEIS2 and SPRED1, respectively. Patients with cleft palate and café-au-lait spots should be encouraged to undergo a detailed genomic evaluation, including screening for a 15q14 deletion, to enable appropriate anticipatory medico-surgical management and genetic counseling.

Parkinsonism in spinocerebellar ataxia with axonal neuropathy caused by adult-onset COA7 variants: a case report.

BACKGROUND: Individuals with variants of cytochrome c oxidase assembly factor 7 (COA7), a mitochondrial functional-related gene, exhibit symptoms of spinocerebellar ataxia with axonal neuropathy before the age of 20. However, COA7 variants with parkinsonism or adult-onset type cases have not been described. CASE PRESENTATION: We report the case of a patient who developed cerebellar symptoms and slowly progressive sensory and motor neuropathy in the extremities, similar to Charcot-Marie-Tooth disease, at age 30, followed by parkinsonism at age 58. Exome analysis revealed COA7 missense mutation in homozygotes (NM_023077.2:c.17A > G, NP_075565.2: p.Asp6Gly). Dopamine transporter single-photon emission computed tomography using a 123I-Ioflupane revealed clear hypo-accumulation in the bilateral striatum. However, 123I-metaiodobenzylguanidine myocardial scintigraphy showed normal sympathetic nerve function. Levodopa administration improved parkinsonism in this patient. CONCLUSIONS: COA7 gene variants may have caused parkinsonism in this case because mitochondrial function-related genes, such as parkin and PINK1, are known causative genes in some familial Parkinson's diseases.

Oculofaciocardiodental syndrome caused by a novel BCOR variant.

Oculofaciocardiodental syndrome is caused by variants in the BCL6 corepressor (BCOR) gene. We identified a novel heterozygous frameshift variant, NM_001123385.2(BCOR):c.2326del, that arose de novo in a Japanese girl with characteristic facial features, congenital heart disease, bilateral syndactyly of toes 2 and 3, congenital cataracts, dental abnormalities, and mild intellectual disability. Reports of BCOR variants are rare, and further case accumulation is warranted.

Heterozygous loss-of-function DHX9 variants are associated with neurodevelopmental disorders: Human genetic and experimental evidences.

DExH-box helicases are involved in unwinding of RNA and DNA. Among the 16 DExH-box genes, monoallelic variants of DHX16, DHX30, DHX34, and DHX37 are known to be associated with neurodevelopmental disorders. In particular, DHX30 is well established as a causative gene for neurodevelopmental disorders. Germline variants of DHX9, the closest homolog of DHX30, have not been reported until now as being associated with congenital disorders in humans, except that one de novo heterozygous variant, p.(Arg1052Gln) of the gene was identified during comprehensive screening in a patient with autism; unfortunately, the phenotypic details of this individual are unknown. Herein, we report a patients with a heterozygous de novo missense variant, p.(Gly414Arg) of DHX9 who presented with a short stature, intellectual disability, and ventricular non-compaction cardiomyopathy. The variant was located in the glycine codon of the ATP-binding site, G-C-G-K-T. To assess the pathogenicity of these variants, we generated transgenic Drosophila lines expressing human wild-type and mutant DHX9 proteins: 1) the mutant proteins showed aberrant localization both in the nucleus and the cytoplasm; 2) ectopic expression of wild-type protein in the visual system led to the rough eye phenotype, whereas expression of the mutant proteins had minimal effect; 3) overexpression of the wild-type protein in the retina led to a reduction in axonal numbers, whereas expression of the mutant proteins had a less pronounced effect. Furthermore, in a gene-editing experiment of Dhx9 G416 to R416, corresponding to p.(Gly414Arg) in humans, heterozygous mice showed a reduced body size, reduced emotionality, and cardiac conduction abnormality. In conclusion, we established that heterozygosity for a loss-of-function variant of DHX9 can lead to a new neurodevelopmental disorder.

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.

Rhegmatogenous Retinal Detachment in Musculocontractural Ehlers-Danlos Syndrome Caused by Biallelic Loss-of-Function Variants of Gene for Dermatan Sulfate Epimerase.

Musculocontractural Ehlers-Danlos syndrome, caused by biallelic loss-of-function variants for dermatan sulfate epimerase (mcEDS-DSE), is a rare connective tissue disorder. Eight patients with mcEDS-DSE have been described with ocular complications, including blue sclera, strabismus, high refractive errors, and elevated intraocular pressure. However, a case with rhegmatogenous retinal detachment (RRD) has not been reported. We report our findings in a 24-year-old woman who was diagnosed with mcEDS-DSE in childhood and presented to our clinic with an RRD in the left eye. The RRD extended to the macula and was associated with an atrophic hole. The patient underwent scleral buckling surgery and cryopexy with drainage of subretinal fluid through a sclerotomy under local anesthesia. The sclera did not appear blue but was very thin at the sclerotomy site. The patient developed frequent bradycardia during the surgery. Subretinal or choroidal hemorrhages were not observed intraoperatively; however, a peripapillary hemorrhage was observed one day after operation. The retina was reattached postoperatively, and the peripapillary hemorrhage was absorbed after one month. The peripapillary retinal hemorrhages, thin sclera, and bradycardia were most likely due to the fragility of the eye. The genetic diagnosis of mcEDS-DSE played an important role before and during the surgery by alerting the surgeons to possible surgical complications due to the thin sclera.

Diagnosis of Prader-Willi syndrome and Angelman syndrome by targeted nanopore long-read sequencing.

The CpG island flanking the promoter region of SNRPN on chromosome 15q11.2 contains CpG sites that are completely methylated in the maternally derived allele and unmethylated in the paternally derived allele. Both unmethylated and methylated alleles are observed in normal individuals. Only the methylated allele is observed in patients with Prader-Willi syndrome, whereas only the unmethylated allele is observed in those with Angelman syndrome. Hence, detection of aberrant methylation at the differentially methylated region is fundamental to the molecular diagnosis of Prader-Willi syndrome and Angelman syndromes. Traditionally, bisulfite treatment and methylation-sensitive restriction enzyme treatment or methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) have been used. We here developed a long-read sequencing assay that can distinguish methylated and unmethylated CpG sites at 15q11.2 by the difference in current intensity generated from nanopore reads. We successfully diagnosed 4 Prader-Willi syndrome patients and 3 Angelman syndrome patients by targeting differentially methylated regions. Concurrent copy number analysis, homozygosity analysis, and structural variant analysis also allowed us to precisely delineate the underlying pathogenic mechanisms, including gross deletion, uniparental heterodisomy, uniparental isodisomy, or imprinting defect. Furthermore, we showed allele-specific methylation in imprinting-related differentially methylated regions on chromosomes 6, 7, 11, 14, and 20 in a normal individual together with 4 Prader-Willi patients and 3 Angelman syndrome patients. Hence, presently reported method is likely to be applicable to the diagnosis of imprinting disorders other than Prader-Willi syndrome and Angelman syndrome as well.

Janus kinase inhibitors ameliorate clinical symptoms in patients with STAT3 gain-of-function.

Germline gain-of-function (GOF) variants in the signal transducer and activator of transcription 3 (STAT3) gene is an inborn error of immunity presenting with autoimmunity and lymphoproliferation. Symptoms can vary widely, and no effective treatment has been established. This study investigated the efficacy of Janus kinase (JAK) inhibitors (JAKi) in patients with STAT3-GOF. Four patients were enrolled and their clinical symptoms before and after the initiation of treatment with JAKi were described. A cell stimulation assay was performed using Epstein-Barr virus transformed lymphoid cell lines (EBV-LCLs) that were derived from the patients with STAT3-GOF. The patients presented with various symptoms, and these symptoms mostly improved after the initiation of JAKi treatment. Upon interleukin-6 stimulation, the EBV-LCLs of patients showed enhanced STAT3 phosphorylation compared with those of the EBV-LCLs of healthy controls. In conclusion, four Japanese patients with STAT3-GOF were successfully treated with JAKi. JAKi ameliorated various symptoms and therefore, the use of JAKi could be an effective treatment option for patients with STAT3-GOF.

Clinical report: Chronic liver dysfunction in an individual with an AMOTL1 variant.

AMOTL1 is a member of the Motin protein family and localizes to tight junctions and is involved in cell polarity and paracellular permeability. Pathological variants have been reported in three patients from two separate families in recent years. The clinical spectrum includes cleft lip and palate along with a high incidence of congenital cardiac disease and ear malformations. We report a case of AMOTL1 pathogenic variant in a 11-year-old male patient with nonspecific and chronic liver dysfunction accompanied by persistently elevated liver enzymes since early infancy. Liver biopsy at 8 years of age revealed a mildly dilated central vein and sinusoid with no specific etiology. Liver dysfunction is not a known clinical feature of AMOTL1 malfunction. However, given that the protein is known to be involved in angiogenesis, it may be inferred that abnormalities in this process may lead to liver dysfunction. This is the first report of liver dysfunction identified in a patient with AMOTL1 malfunction, which will shed light on other putative functions of the protein.

De novo non-synonymous DPYSL2 (CRMP2) variants in two patients with intellectual disabilities and documentation of functional relevance through zebrafish rescue and cellular transfection experiments.

Collapsin response mediator protein 2 (Crmp2) is an evolutionarily well-conserved tubulin-binding cytosolic protein that plays critical roles in the formation of neural circuitry in model organisms including zebrafish and rodents. No clinical evidence that CRMP2 variants are responsible for monogenic neurogenic disorders in humans presently exists. Here, we describe two patients with de novo non-synonymous variants (S14R and R565C) of CRMP2 and intellectual disability associated with hypoplasia of the corpus callosum. We further performed various functional assays of CRMP2 variants using zebrafish and zebrafish Crmp2 (abbreviated as z-CRMP2 hereafter) and an antisense morpholino oligonucleotide [AMO]-based experimental system in which crmp2-morphant zebrafish exhibit the ectopic positioning of caudal primary (CaP) motor neurons. Whereas the co-injection of wild-type z-CRMP2 mRNA suppressed the ectopic positioning of CaP motor neurons in Crmp2-morphant zebrafish, the co-injection of R566C or S15R, z-CRMP2, which corresponds to R565C and S14R of human CRMP2, failed to rescue the ectopic positioning. Transfection experiments of zebrafish or rat Crmp2 using plasmid vectors in HeLa cells, with or without a proteasome inhibitor, demonstrated that the expression levels of mutant Crmp2 protein encoded by R565C and S14R CRMP2 variants were decreased, presumably because of increased degradation by proteasomes. When we compared CRMP2-tubulin interactions using co-immunoprecipitation and cellular localization studies, the R565C and S14R mutations weakened the interactions. These results collectively suggest that the CRMP2 variants detected in the present study consistently led to the loss-of-function of CRMP2 protein and support the notion that pathogenic variants in CRMP2 can cause intellectual disabilities in humans.

De novo non-synonymous CTR9 variants are associated with motor delay and macrocephaly: human genetic and zebrafish experimental evidence.

CTR9 is one of five genes that form the PAF1 complex, which binds to RNA polymerase II and plays critical roles in transcriptional elongation and transcription-coupled histone modifications including histones H3K4me3 and H3K36me3. In this study, de novo CTR9 non-synonymous variants (p.(Glu15Asp) and p.(Pro25Arg)) were detected in two unrelated patients with macrocephaly, motor delay, and intellectual disability. A pull-down assay showed that the mutant CTR9 proteins had stronger affinities to the PAF1 protein than the wild-type protein. Functional analyses using zebrafish showed that the knockout of the ctr9 gene caused motor defects and enlargement of the telencephalon, which is homologous to the mammalian cerebrum. The rescue experiment, in which the human CTR9 mutants were introduced into ctr9-knockout zebrafish, failed to maintain the swimming posture of the ctr9-knockout fish, suggesting that the human CTR9 mutant proteins do not function normally in vivo. In addition, the overexpression of human CTR9 mutant mRNA caused telencephalon enlargement in zebrafish larvae, suggesting that the human CTR9 mutant proteins interfered with normal endogenous CTR9 function. We concluded that the two missense variants in CTR9 (p.(Glu15Asp) and p.(Pro25Arg)) cause a new syndrome involving macrocephaly, motor delay and intellectual disability through the loss of the normal function of CTR9 and the inhibition of the normal intrinsic CTR9 function of the contralateral allele.

Precocious puberty in a case of Simpson-Golabi-Behmel syndrome with a de novo 240-kb deletion including GPC3.

Here, we report a Japanese patient with Simpson-Golabi-Behmel syndrome involving a de novo 240-kb deletion including a part of GPC3. The patient showed pre- and postnatal macrosomia associated with coarse face, macrocephaly, supernumerary nipples, and cryptorchidism and characteristically presented with precocious puberty, mostly evaluated as advanced pubertal age of 15 years at the chronological age of 11.5 years.