[Perioperative management for fracture in a child with homozygous congenital protein C deficiency].

Congenital protein C (PC) deficiency is one type of hereditary thrombosis. Patients with hereditary thrombosis are at high risk for thrombosis in the perioperative period, but a standard management strategy has not been established. Here we report a case of perioperative management of a fracture in a child with homozygous congenital PC deficiency. The patient was a 3-year-old boy who was diagnosed with congenital PC deficiency at birth. He sustained a traumatic supracondylar fracture of the right humerus and underwent emergency surgery. To prepare for open surgery for fixation of the fracture, warfarin was discontinued, and an activated PC (APC) concentrate was used in combination with vitamin K antagonism. However, warfarin was administered during the scheduled nail extraction because the operation was minimally invasive. No thrombotic or bleeding complications occurred in either operation. In emergency surgery in patients with congenital PC deficiency, the combination of vitamin K and APC concentrate is considered a maintenance option for PC deficiency. Postoperative PT-INR control was difficult in our patient due to the administration of vitamin K and withdrawal of warfarin, and this issue must be addressed in the future. Further case experience is desirable to standardize perioperative management.

Teriparatide-Associated Hypercalcemia Concomitant With Acute Kidney Injury: A Case-Report.

Teriparatide, a recombinant human parathyroid hormone, is an anabolic treatment for osteoporosis with a high risk of fractures. Transient hypercalcemia is an adverse effect of teriparatide and usually resolves within 16h of teriparatide administration owing to its rapid absorption and elimination. Some cases of prolonged hypercalcemia have been reported, but these improved rapidly after teriparatide discontinuation. Here, we describe a rare case of teriparatide-induced hypercalcemia concomitant with acute kidney injury that persisted for four weeks. An 83-year-old woman began taking teriparatide for a vertebral fracture. The patient was immobilized by the fracture. Three weeks later, the patient developed hypercalcemia and acute kidney injury. However, hypercalcemia persisted for four weeks despite the discontinuation of teriparatide and fluid administration. Clinicians should be aware that teriparatide can induce severe hypercalcemia, especially in the setting of immobilization, and that hypercalcemia can persist for more than 3-4 weeks in patients with decreased kidney function.

Detection of hidden intronic DDC variant in aromatic L-amino acid decarboxylase deficiency by adaptive sampling.

Aromatic l-amino acid decarboxylase (AADC) deficiency is an autosomal recessive neurotransmitter disorder caused by pathogenic DOPA decarboxylase (DDC) variants. We previously reported Japanese siblings with AADC deficiency, which was confirmed by the lack of enzyme activity; however, only a heterozygous missense variant was detected. We therefore performed targeted long-read sequencing by adaptive sampling to identify any missing variants. Haplotype phasing and variant calling identified a novel deep intronic variant (c.714+255 C > A), which was predicted to potentially activate the noncanonical splicing acceptor site. Minigene assay revealed that wild-type and c.714+255 C > A alleles had different impacts on splicing. Three transcripts, including the canonical transcript, were detected from the wild-type allele, but only the noncanonical cryptic exon was produced from the variant allele, indicating that c.714+255 C > A was pathogenic. Target long-read sequencing may be used to detect hidden pathogenic variants in unresolved autosomal recessive cases with only one disclosed hit variant.

Novel compound heterozygous ABCA2 variants cause IDPOGSA, a variable phenotypic syndrome with intellectual disability.

The gene for ATP binding cassette subfamily A member 2 (ABCA2) is located at chromosome 9q34.3. Biallelic ABCA2 variants lead to intellectual developmental disorder with poor growth and with or without seizures or ataxia (IDPOGSA). In this study, we identified novel compound heterozygous ABCA2 variants (NM_001606.5:c.[5300-17C>A];[6379C>T]) by whole exome sequencing in a 28-year-old Korean female patient with intellectual disability. These variants included intronic and nonsense variants of paternal and maternal origin, respectively, and are absent from gnomAD. SpliceAI predicted that the intron variant creates a cryptic acceptor site. Reverse transcription-PCR using RNA extracted from a lymphoblastoid cell line of the patient confirmed two aberrant transcripts. Her clinical features are compatible with those of IDPOGSA.

Novel missense variants cause intermediate phenotypes in the phenotypic spectrum of SLC5A6-related disorders.

SLC5A6 encodes the sodium-dependent multivitamin transporter, a transmembrane protein that uptakes biotin, pantothenic acid, and lipoic acid. Biallelic SLC5A6 variants cause sodium-dependent multivitamin transporter deficiency (SMVTD) and childhood-onset biotin-responsive peripheral motor neuropathy (COMNB), which both respond well to replacement therapy with the above three nutrients. SMVTD usually presents with various symptoms in multiple organs, such as gastrointestinal hemorrhage, brain atrophy, and global developmental delay, at birth or in infancy. Without nutrient replacement therapy, SMVTD can be lethal in early childhood. COMNB is clinically milder and has a later onset than SMVTD, at approximately 10 years of age. COMNB symptoms are mostly limited to peripheral motor neuropathy. Here we report three patients from one Japanese family harboring novel compound heterozygous missense variants in SLC5A6, namely NM_021095.4:c.[221C>T];[642G>C] p.[(Ser74Phe)];[(Gln214His)]. Both variants were predicted to be deleterious through multiple lines of evidence, including amino acid conservation, in silico predictions of pathogenicity, and protein structure considerations. Drosophila analysis also showed c.221C>T to be pathogenic. All three patients had congenital brain cysts on neonatal cranial imaging, but no other morphological abnormalities. They also had a mild motor developmental delay that almost completely resolved despite no treatment. In terms of severity, their phenotypes were intermediate between SMVTD and COMNB. From these findings we propose a new SLC5A6-related disorder, spontaneously remitting developmental delay with brain cysts (SRDDBC) whose phenotypic severity is between that of SMVTD and COMNB. Further clinical and genetic evidence is needed to support our suggestion.

Exome-wide benchmark of difficult-to-sequence regions using short-read next-generation DNA sequencing.

Next-generation DNA sequencing (NGS) in short-read mode has recently been used for genetic testing in various clinical settings. NGS data accuracy is crucial in clinical settings, and several reports regarding quality control of NGS data, primarily focusing on establishing NGS sequence read accuracy, have been published thus far. Variant calling is another critical source of NGS errors that remains unexplored at the single-nucleotide level despite its established significance. In this study, we used a machine-learning-based method to establish an exome-wide benchmark of difficult-to-sequence regions at the nucleotide-residue resolution using 10 genome sequence features based on real-world NGS data accumulated in The Genome Aggregation Database (gnomAD) of the human reference genome sequence (GRCh38/hg38). The newly acquired metric, designated the 'UNMET score,' along with additional lines of structural information from the human genome, allowed us to assess the sequencing challenges within the exonic region of interest using conventional short-read NGS. Thus, the UNMET score could provide a basis for addressing potential sequential errors in protein-coding exons of the human reference genome sequence GRCh38/hg38 in clinical sequencing.

A heterozygous germline deletion within USP8 causes severe neurodevelopmental delay with multiorgan abnormalities.

Ubiquitin-specific protease 8 (USP8) is a deubiquitinating enzyme involved in deubiquitinating the enhanced epidermal growth factor receptor for escape from degradation. Somatic variants at a hotspot in USP8 are a cause of Cushing's disease, and a de novo germline USP8 variant at this hotspot has been described only once previously, in a girl with Cushing's disease and developmental delay. In this study, we investigated an exome-negative patient with severe developmental delay, dysmorphic features, and multiorgan dysfunction by long-read sequencing, and identified a 22-kb de novo germline deletion within USP8 (chr15:50469966-50491995 [GRCh38]). The deletion involved the variant hotspot, one rhodanese domain, and two SH3 binding motifs, and was presumed to be generated through nonallelic homologous recombination through Alu elements. Thus, the patient may have perturbation of the endosomal sorting system and mitochondrial autophagy through the USP8 defect. This is the second reported case of a germline variant in USP8.

Complete SAMD12 repeat expansion sequencing in a four-generation BAFME1 family with anticipation.

Benign adult familial myoclonic epilepsy type 1 (BAFME1) is an autosomal dominant, adult-onset neurological disease caused by SAMD12 repeat expansion. In BAFME1, anticipation, such as the earlier onset of tremor and/or seizures in the next generation, was reported. This could be explained by intergenerational repeat instability, leading to larger expansions in successive generations. We report a four-generation BAFME1-affected family with anticipation. Using Nanopore long-read sequencing, detailed information regarding the sizes, configurations, and compositions of the expanded SAMD12 repeats across generations was obtained. Unexpectedly, a grandmother-mother-daughter triad showed similar repeat structures but with slight repeat expansions, despite quite variable age of onset of seizures (range: 52-14 years old), implying a complex relationship between the SAMD12 repeat expansion sequence and anticipation. This study suggests that different factor(s) from repeat expansion could modify the anticipation in BAFME1.

Auditory Neuropathy Spectrum Disorder Progressing with Motor and Sensory Neuropathy Caused by an ATP1A1 Variant.

We encountered a 27-year-old Japanese woman with sensorineural deafness progressing to motor and sensory neuropathy. At 16 years old, she had developed weakness in her lower extremities and hearing impairment, which gradually deteriorated. At 22 years old, combined audiological, electrophysiological, and radiological examination results were consistent with auditory neuropathy spectrum disorder (ANSD). Genetic analyses identified a previously reported missense variant in the ATP1A1 gene (NM_000701.8:c.1799C>G, p.Pro600Arg). Although sensorineural deafness has been reported as a clinical manifestation of ATP1A1-related disorders, our case suggested that ANSD may underlie the pathogenesis of deafness in ATP1A1-related disorders. This case report broadens the genotype-phenotype spectrum of ATP1A1-related disorders.

Efficient detection of somatic UBA1 variants and clinical scoring system predicting patients with variants in VEXAS syndrome.

OBJECTIVES: To efficiently detect somatic UBA1 variants and establish a clinical scoring system predicting patients with pathogenic variants in VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. METHODS: Eighty-nine Japanese patients with clinically suspected VEXAS syndrome were recruited [81 males and 8 females; median onset age (IQR) 69.3 years (62.1-77.6)]. Peptide nucleic acid-clamping PCR (PNA-PCR), regular PCR targeting exon 3 clustering UBA1 variants, and subsequent Sanger sequencing were conducted for variant screening. Partitioning digital PCR (pdPCR) or targeted amplicon deep sequencing (TAS) was also performed to evaluate the variant allele frequency (VAF). We developed our clinical scoring system to predict UBA1 variant-positive and ­negative patients and assessed the diagnostic value of our system using receiver operating characteristic (ROC) curve analysis. RESULTS: Forty patients with reported pathogenic UBA1 variants (40/89, 44.9%) were identified, including a case having a variant with VAF of 1.7%, using a highly sensitive method. Our clinical scoring system considering >50 years of age, cutaneous lesions, lung involvement, chondritis, and macrocytic anaemia efficiently predicted patients with UBA1 variants (the area under the curve for the scoring total was 0.908). CONCLUSIONS: Genetic screening with the combination of regular PCR and PNA-PCR detected somatic UBA1 variants with high sensitivity and specificity. Our scoring system could efficiently predict patients with UBA1 variants.

Long-term remission of VEXAS syndrome achieved by a single course of CHOP therapy: A case report.

We herein describe the case of a 52-year-old male patient who presented with fever, arthritis, and neutrophilic dermatosis in 2013 and subsequently experienced macrophage activation syndrome treated with high-dose glucocorticoid therapy. Due to the persistent symptoms refractory to several immunomodulatory and immunosuppressive (IS) drug therapies with dapsone, methotrexate, tacrolimus, infliximab (IFX), and tocilizumab (TCZ), he received prednisolone (PSL) ≥20 mg/day to suppress disease activity. In 2017, Epstein-Barr virus (EBV)-associated haemophagocytic lymphohistiocytosis (HLH) was diagnosed and initially treated with immunochemotherapy consisting of dexamethasone, cyclosporine (CyA), and etoposide (ET). Because of the suboptimal response to the initial therapy, cytoreduction therapy consisting of CHOP (combination chemotherapy consisting of cyclophosphamide, doxorubicin, vincristine, and PSL) was administered. This regimen improved the EBV-associated HLH. Later, the patient's condition stabilised with methylprednisolone 1 mg/day and CyA 100 mg/day. In 2022, ubiquitylation-initiating E1 enzyme (UBA1) variant analysis using Sanger sequencing of peripheral blood leukocytes detected a previously reported somatic variant (NM_003334.3: c.118-1G>C), confirming the diagnosis of vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome. The clinical course in the present case suggested the possibility that CHOP could be a potential treatment option for VEXAS syndrome, in the pathophysiology of which the expansion of clones with UBA1 variant seems to play a pivotal role.

Biallelic structural variations within FGF12 detected by long-read sequencing in epilepsy.

We discovered biallelic intragenic structural variations (SVs) in FGF12 by applying long-read whole genome sequencing to an exome-negative patient with developmental and epileptic encephalopathy (DEE). We also found another DEE patient carrying a biallelic (homozygous) single-nucleotide variant (SNV) in FGF12 that was detected by exome sequencing. FGF12 heterozygous recurrent missense variants with gain-of-function or heterozygous entire duplication of FGF12 are known causes of epilepsy, but biallelic SNVs/SVs have never been described. FGF12 encodes intracellular proteins interacting with the C-terminal domain of the alpha subunit of voltage-gated sodium channels 1.2, 1.5, and 1.6, promoting excitability by delaying fast inactivation of the channels. To validate the molecular pathomechanisms of these biallelic FGF12 SVs/SNV, highly sensitive gene expression analyses using lymphoblastoid cells from the patient with biallelic SVs, structural considerations, and Drosophila in vivo functional analysis of the SNV were performed, confirming loss-of-function. Our study highlights the importance of small SVs in Mendelian disorders, which may be overlooked by exome sequencing but can be detected efficiently by long-read whole genome sequencing, providing new insights into the pathomechanisms of human diseases.

A missense variant at the RAC1-PAK1 binding site of RAC1 inactivates downstream signaling in VACTERL association.

RAC1 at 7p22.1 encodes a RAC family small GTPase that regulates actin cytoskeleton organization and intracellular signaling pathways. Pathogenic RAC1 variants result in developmental delay and multiple anomalies. Here, exome sequencing identified a rare de novo RAC1 variant [NM_018890.4:c.118T > C p.(Tyr40His)] in a male patient. Fetal ultrasonography indicated the patient to have multiple anomalies, including persistent left superior vena cava, total anomalous pulmonary venous return, esophageal atresia, scoliosis, and right-hand polydactyly. After birth, craniofacial dysmorphism and esophagobronchial fistula were confirmed and VACTERL association was suspected. One day after birth, the patient died of respiratory failure caused by tracheal aplasia type III. The molecular mechanisms of pathogenic RAC1 variants remain largely unclear; therefore, we biochemically examined the pathophysiological significance of RAC1-p.Tyr40His by focusing on the best characterized downstream effector of RAC1, PAK1, which activates Hedgehog signaling. RAC1-p.Tyr40His interacted minimally with PAK1, and did not enable PAK1 activation. Variants in the RAC1 Switch II region consistently activate downstream signals, whereas the p.Tyr40His variant at the RAC1-PAK1 binding site and adjacent to the Switch I region may deactivate the signals. It is important to accumulate data from individuals with different RAC1 variants to gain a full understanding of their varied clinical presentations.

Genome-wide identification of tandem repeats associated with splicing variation across 49 tissues in humans.

Tandem repeats (TRs) are one of the largest sources of polymorphism, and their length is associated with gene regulation. Although previous studies reported several tandem repeats regulating gene splicing in cis (spl-TRs), no large-scale study has been conducted. In this study, we established a genome-wide catalog of 9537 spl-TRs with a total of 58,290 significant TR-splicing associations across 49 tissues (false discovery rate 5%) by using Genotype-Tissue expression (GTex) Project data. Regression models explaining splicing variation by using spl-TRs and other flanking variants suggest that at least some of the spl-TRs directly modulate splicing. In our catalog, two spl-TRs are known loci for repeat expansion diseases, spinocerebellar ataxia 6 (SCA6) and 12 (SCA12). Splicing alterations by these spl-TRs were compatible with those observed in SCA6 and SCA12. Thus, our comprehensive spl-TR catalog may help elucidate the pathomechanism of genetic diseases.

Long-read sequencing revealing intragenic deletions in exome-negative spastic paraplegias.

Hereditary spastic paraplegias (HSPs) are a heterogeneous group of neurodegenerative disorders characterized by progressive spasticity and weakness in the lower extremities. To date, a total of 88 types of SPG are known. To diagnose HSP, multiple technologies, including microarray, direct sequencing, multiplex ligation-dependent probe amplification, and short-read next-generation sequencing, are often chosen based on the frequency of HSP subtypes. Exome sequencing (ES) is commonly used. We used ES to analyze ten cases of HSP from eight families. We identified pathogenic variants in three cases (from three different families); however, we were unable to determine the cause of the other seven cases using ES. We therefore applied long-read sequencing to the seven undetermined HSP cases (from five families). We detected intragenic deletions within the SPAST gene in four families, and a deletion within PSEN1 in the remaining family. The size of the deletion ranged from 4.7 to 12.5 kb and involved 1-7 exons. All deletions were entirely included in one long read. We retrospectively performed an ES-based copy number variation analysis focusing on pathogenic deletions, but were not able to accurately detect these deletions. This study demonstrated the efficiency of long-read sequencing in detecting intragenic pathogenic deletions in ES-negative HSP patients.

Ocular Manifestations of Peters Plus-Like Syndrome in 8q21.11 Microdeletion Syndrome.

PURPOSE: The aim of this study was to report a case of Peters plus-like syndrome, which revealed to have an 8q21.11 microdeletion by copy number variation analysis using exome data. METHODS: A 6-month-old Japanese boy presented with bilateral corneal opacity since birth. The right eye maintained central corneal transparency with slightly inferior nasal and superior peripheral corneal opacities. The entire cornea was opacified in the left eye, particularly in the superior quadrants with vascularization, suggesting Peters anomaly. Identification of intraocular structures in the left eye was difficult; however, hypoplasia of the circumferential anterior iris stroma appeared bilaterally present, and no abnormalities were present in the posterior segment on funduscopic examination of the right eye and ultrasonography in the left eye. He had several facial malformations in addition to corneal opacity, but no other external abnormalities. General examination, including biochemical tests of blood and urine, physiological and imaging tests including abdominal echo, auditory brain stem response, brain computed tomography, and magnetic resonance imaging, showed no abnormalities. However, the patient showed intellectual disability and delayed motor development. RESULTS: Although his karyotype was normal, copy number variation analysis using exome data and subsequent quantitative polymerase chain reaction identified a de novo 4.6-Mb deletion at 8q21.11q21.13; thus, the patient was diagnosed with 8q21.11 microdeletion syndrome. CONCLUSIONS: We identified a de novo 4.6-Mb deletion at 8q21.11q21.13 in a patient with ophthalmic anterior segment dysgenesis and systemic complications, clinically diagnosed as Peters plus-like syndrome. Clinically, the 8q21.11 microdeletion syndrome shows a phenotype similar to that of Peters plus syndrome, and a genetic diagnosis is required.

Clinical and genetic features of Japanese cases of MDS associated with VEXAS syndrome.

VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a new disease entity with autoinflammatory disorders (AID) driven by somatic variants in UBA1 that frequently co-exists with myelodysplastic syndromes (MDS). Clinicopathological and molecular features of Japanese cases with VEXAS-associated MDS remain elusive. We previously reported high prevalence of UBA1 variants in Japanese patients with relapsing polychondritis, in which 5 cases co-occurred with MDS. Here, we report clinicopathological and variant profiles of these 5 cases and 2 additional cases of MDS associated with VEXAS syndrome. Clinical characteristics of these cases included high prevalence of macrocytic anemia with marked cytoplasmic vacuoles in myeloid/erythroid precursors and low bone marrow (BM) blast percentages. All cases were classified as low or very low risk by the revised international prognostic scoring system (IPSS-R). Notably, 4 out of 7 cases showed significant improvement of anemia by treatment with prednisolone (PSL) or cyclosporin A (CsA), suggesting that an underlying inflammatory milieu induced by VEXAS syndrome may aggravate macrocytic anemia in VEXAS-associated MDS. Targeted deep sequencing of blood samples suggested that MDS associated with VEXAS syndrome tends to involve a smaller number of genes and lower risk genetic lesions than classical MDS.

Bi-allelic SNAPC4 variants dysregulate global alternative splicing and lead to neuroregression and progressive spastic paraparesis.

The vast majority of human genes encode multiple isoforms through alternative splicing, and the temporal and spatial regulation of those isoforms is critical for organismal development and function. The spliceosome, which regulates and executes splicing reactions, is primarily composed of small nuclear ribonucleoproteins (snRNPs) that consist of small nuclear RNAs (snRNAs) and protein subunits. snRNA gene transcription is initiated by the snRNA-activating protein complex (SNAPc). Here, we report ten individuals, from eight families, with bi-allelic, deleterious SNAPC4 variants. SNAPC4 encoded one of the five SNAPc subunits that is critical for DNA binding. Most affected individuals presented with delayed motor development and developmental regression after the first year of life, followed by progressive spasticity that led to gait alterations, paraparesis, and oromotor dysfunction. Most individuals had cerebral, cerebellar, or basal ganglia volume loss by brain MRI. In the available cells from affected individuals, SNAPC4 abundance was decreased compared to unaffected controls, suggesting that the bi-allelic variants affect SNAPC4 accumulation. The depletion of SNAPC4 levels in HeLa cell lines via genomic editing led to decreased snRNA expression and global dysregulation of alternative splicing. Analysis of available fibroblasts from affected individuals showed decreased snRNA expression and global dysregulation of alternative splicing compared to unaffected cells. Altogether, these data suggest that these bi-allelic SNAPC4 variants result in loss of function and underlie the neuroregression and progressive spasticity in these affected individuals.