Suspected malnutrition-induced reversible feline skin fragility syndrome in a cat with congenital axial deformities.

A stray cat, an intact female Japanese domestic shorthair cat of unknown age (suspected to be a young adult), was rescued. The cat was lethargic and thin and had marked skin fragility, delayed wound healing without skin hyperextensibility, and hind limb proprioceptive ataxia and paresis. Survey radiography, computed tomography, and magnetic resonance imaging revealed congenital vertebral anomalies, including thoracolumbar transitional vertebrae, scoliosis resulting from a thoracic lateral wedge-shaped vertebra, and a kinked tail, and a dilated spinal cord central canal. Through nutritional support, the cat's general condition normalized, followed by a gradual and complete improvement of skin features. Whole-genome sequencing was completed; however, no pathogenic genetic variant was identified that could have caused this phenotype, including congenital scoliosis. A skin biopsy obtained 7 y after the rescue revealed no remarkable findings on histopathology or transmission electron microscopy. Based on clinical course and microscopic findings, malnutrition-induced reversible feline skin fragility syndrome (FSFS) was suspected, and nutritional support was considered to have improved the skin condition. Key clinical message: This is the second reported case of presumed malnutrition-induced reversible FSFS and was accompanied by long-term follow-up.

Syndrome de fragilité cutanée réversible induit par la malnutrition soupçonné chez un chat avec des difformités axiales congénitales. Un chat errant, une femelle intacte de race japonaise à poil court et d'âge inconnu (suspecté être une jeune adulte), a été secourue. La chatte était léthargique et maigre, et avait une fragilité marquée de la peau, un retard dans la guérison de plaies sans hyperextensibilité de la peau, et une ataxie proprioceptive et parésie des membres postérieurs. Des radiographies, un examen par tomodensitométrie, et de l'imagerie par résonnance magnétique ont révélé des anomalies congénitales des vertèbres, incluant des vertèbres transitionnelles thoraco-lombaires, une scoliose résultant d'une vertèbre thoracique en forme de coin, une queue pliée, et un canal central de la moelle épinière dilaté. Grâce à un soutien nutritionnel, la condition générale du chat s'est stabilisée, suivi d'une amélioration graduelle et complète des caractéristiques de la peau. Le séquençage du génome complet a été effectué; toutefois, aucune variation génétique pathogénique n'a été identifiée qui aurait pu causer ce phénotype, incluant la scoliose congénitale. Une biopsie cutanée obtenue 7 j après le sauvetage n'a révélé aucune trouvaille spéciale à l'histopathologie ou par microscopie électronique à transmission. Basé sur le déroulement clinique et l'examen microscopique, le syndrome de fragilité cutanée réversible félin induit par la malnutrition (FSFS) était suspecté, et le soutien nutritionnel a été considéré comme ayant amélioré la condition cutanée.Message clinique clé :Ce cas est le deuxième cas rapporté de FSFS induit par la malnutrition soupçonné et a fait l'objet d'un suivi à long terme.(Traduit par Dr Serge Messier).

A MinION-based Long-Read Sequencing Application With One-Step PCR for the Genetic Diagnosis of 21-Hydroxylase Deficiency.

CONTEXT: Recently developed long-read sequencing (LRS) technology has been considered an option for CYP21A2 analysis. However, the clinical use of LRS for CYP21A2 analysis is limited. OBJECTIVE: This study's objective is to develop an efficient and low-cost LRS system for CYP21A2 screening. METHODS: A DNA fragment library was prepared in a single polymerase chain reaction (PCR) that covers the entire CYP21A2 gene and all known junctions caused by TNXB gene structural rearrangements, yielding a single 8-kb product of CYP21A2 or CYP21A1P/CYP21A2 chimera. After barcoding, the PCR products were sequenced on a MinION-based platform with Flongle Flow Cell R9.4.1 and R10.4.1. RESULTS: The reference genotypes of 55 patients with 21-hydroxylase deficiency (21OHD) were established using the conventional method with multiplex ligation-dependent probe amplification (MLPA) and nested PCR. LRS using Flongle Flow Cell R9.4.1 yielded consistent results. Additionally, the recently updated LRS "duplex" analysis with Flongle flow cell R10.4.1 was tested to reveal an advantage of accurately sequencing a variant located on the homopolymer region. By introducing a barcode system, the cost was reduced to be comparable to that of conventional analysis. A novel single-nucleotide variation was discovered at the acceptor site of intron 7, c.940-1G > C. We also identified a subtype of the classical chimeric junction CH2, "CH2a," in the region from the latter part of intron 5 to exon 6. CONCLUSION: We successfully established a novel low-cost and highly accurate LRS system for 21OHD genetic analysis. Our study provides insight into the feasibility of LRS for diagnosing 21OHD and other genetic diseases caused by structural rearrangements.

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.

Lysinuric protein intolerance exhibiting renal tubular acidosis/Fanconi syndrome in a Japanese woman.

Lysinuric protein intolerance (LPI), caused by pathogenic variants of SLC7A7, is characterized by protein aversion, failure to thrive, hyperammonemia, and hepatomegaly. Recent studies have reported that LPI can cause multiple organ dysfunctions, including kidney disease, autoimmune deficiency, pulmonary alveolar proteinosis, and osteoporosis. We report the case of a 47-year-old Japanese woman who was initially diagnosed with renal tubular acidosis (RTA), Fanconi syndrome, and rickets. At the age of 3 years, she demonstrated a failure to thrive. Urinary amino acid analysis revealed elevated lysine and arginine levels, which were masked by pan-amino aciduria. She was subsequently diagnosed with rickets at 5 years of age and RTA/Fanconi syndrome at 15 years of age. She was continuously treated with supplementation of vitamin D3, phosphate, and bicarbonate. A renal biopsy at 18 years of age demonstrated diffuse proximal and distal tubular damage with endocytosis-lysosome pathway abnormalities. Distinctive symptoms of LPI, such as protein aversion and postprandial hyperammonemia were not observed throughout the patient's clinical course. The patient underwent a panel-based comprehensive genetic testing and was diagnosed with LPI. As the complications of LPI involve many organs, patients lacking distinctive symptoms may develop various diseases, including RTA/Fanconi syndrome. Our case indicates that proximal and distal tubular damages are notable findings in patients with LPI. The possibility of LPI should be carefully considered in the management of RTA/Fanconi syndrome and/or incomprehensible pathological tubular damage, even in the absence of distinctive symptoms; furthermore, a comprehensive genetic analysis is useful for diagnosing LPI.

Clinical features and morphology of collagen fibrils in patients with vascular Ehlers-Danlos based on electron microscopy.

Background: Vascular-type Ehlers-Danlos syndrome (vEDS) is caused by collagen III deficit resulting from heterogeneous mutations in COL3A1, which occasionally causes sudden death due to arterial/visceral rupture. However, it is difficult to conduct basic research on the pathophysiology of vEDS. Moreover, the number of patients with vEDS is small, limiting the number of available samples. Furthermore, the symptoms of vEDS may vary among family members, even if they share the same mutation. Accordingly, many aspects of the pathology of vEDS remain unknown. Therefore, we investigated the structural abnormalities in collagen fibrils and endoplasmic reticulum (ER) stress in skin samples using electron microscopy as well as their relationship with clinical symptoms in 30 patients with vEDS (vEDS group) and 48 patients without vEDS (disease-negative control group). Methods: Differences between the two groups were evaluated in terms of the sizes of collagen fibrils using coefficient of variation (COV). Results: COV was found to be significantly higher in the vEDS group than in the disease-negative control group, indicating irregularity in the size of collagen fibrils. However, in the vEDS group, some patients had low COV and seldom experienced serious complications and ER stress. Conclusion: ER stress might affect collagen fibril-composing proteins. Moreover, as this stress varies among people based on environmental factors and aging, it may be the underlying cause of varying vEDS symptoms.

Clinical and molecular delineation of classical-like Ehlers-Danlos syndrome through a comprehensive next-generation sequencing-based screening system.

Classical-like Ehlers-Danlos syndrome (clEDS) is an autosomal recessive disorder caused by complete absence of tenascin-X resulting from biallelic variation in TNXB. Thus far, 50 patients from 43 families with biallelic TNXB variants have been identified. Accurate detection of TNXB variants is challenging because of the presence of the pseudogene TNXA, which can undergo non-allelic homologous recombination. Therefore, we designed a genetic screening system that is performed using similar operations to other next-generation sequencing (NGS) panel analyses and can be applied to accurately detect TNXB variants and the recombination of TNXA-derived sequences into TNXB. Using this system, we identified biallelic TNXB variants in nine unrelated clEDS patients. TNXA-derived variations were found in >75% of the current cohort, comparable to previous reports. The current cohort generally exhibited similar clinical features to patients in previous reports, but had a higher frequency of gastrointestinal complications (e.g., perforation, diverticulitis, gastrointestinal bleeding, intestinal obstruction, rectal/anal prolapse, and gallstones). This report is the first to apply an NGS-based screening for TNXB variants and represents the third largest cohort of clEDS, highlighting the importance of increasing awareness of the risk of gastrointestinal complications.

Homozygous splice site variant affecting the first von Willebrand factor A domain of COL12A1 in a patient with myopathic Ehlers-Danlos syndrome.

Myopathic Ehlers-Danlos syndrome (mEDS) is a subtype of EDS that is caused by abnormalities in COL12A1. Up-to-date, 24 patients from 15 families with mEDS have been reported, with 14 families showing inheritance in an autosomal dominant manner and one family in an autosomal recessive manner. We encountered an additional patient with autosomal recessive mEDS. The patient is a 47-year-old Japanese man, born to consanguineous parents with no related features of mEDS. After birth, he presented with hypotonia, weak spontaneous movements, scoliosis, and torticollis. He had soft palms but no skin hyperextensibility or fragility. Progressive scoliosis, undescended testes, and muscular torticollis required surgery. During adulthood, he worked normally and had no physical concerns. Clinical exome analysis revealed a novel homozygous variant in COL12A1 (NM_004370.6:c.395-1G > A) at the splice acceptor site of exon 6, leading to in-frame skipping of exon 6. The patient was diagnosed with mEDS. The milder manifestations in the current patient compared with previously reported patients with mEDS might be related to the site of the variant. The variant is located in the genomic region encoding the first von Willebrand factor A domain, which affects only the long isoform of collagen XII, in contrast to the variants in previously reported mEDS patients that affected both the long and short isoforms. Further studies are needed to delineate comprehensive genotype-phenotype correlation of the disorder.

A patient with pleuroparenchymal fibroelastosis carrying a novel fibrillin-2 gene variant.

Pleuroparenchymal fibroelastosis is a recently recognized clinical entity characterized by interstitial pneumonia with proliferating elastin in the upper lung regions. Pleuroparenchymal fibroelastosis is categorized as idiopathic or reported depending on the coexistent initiating factors; however, congenital contractural arachnodactyly, which is caused by abnormal production of elastin based on a mutation in the fibrillin-2 gene, is rarely reported with lung lesion resembling pleuroparenchymal fibroelastosis. We present a case of pleuroparenchymal fibroelastosis in a patient with a novel mutation in the fibrillin-2 gene, which encodes the prenatal fibrillin-2 protein as a scaffold for elastin.

Cancer gene analysis of liquid-based cytology specimens using next-generation sequencing: A technical report of bimodal DNA- and RNA-based panel application.

BACKGROUND: As liquid-based cytology (LBC) specimens harbor high-quality DNA, genomic analysis using LBC specimens is beneficial for integrative diagnosis. This study aimed to clarify the feasibility of LBC specimens for a bimodal application of DNA- and RNA-based next-generation sequencing (NGS) panels. METHODS: LBC specimens were prepared from cultured human cancer HEC59 cells using commercially available fixatives (Cellprep, CytoRich Red, and SurePath solutions), and were subjected to NGS for a feasibility study. Clinical LBC specimens of thyroid and salivary gland tumors were prepared using CytoRich Red solution. After DNA and RNA extraction, NGS analyses were performed in a single run using combined DNA- and RNA-based custom-made cancer panels for the detection of gene mutations and fusions. RESULTS: High-quality DNA and RNA were obtained, and the expected gene mutations and fusions were detected in HEC59 cells using all types of LBC fixatives. Most available clinical cases (18 out of 20) exhibited pathogenic gene mutations (15 cases) and fusion genes (3 cases) using the bimodal DNA- and RNA-based panels. Overall, 18 cases (90%) showed oncogenic mutations or fusion genes of diagnostic values. CONCLUSION: Simultaneous application of bimodal DNA- and RNA-based gene panels was useful in NGS analysis using residual LBC specimens for integrative diagnosis. Residual LBC specimens for genomic analysis, including fusion gene analysis, are particularly useful for obtaining genomic information before surgical resection.

Case report: further delineation of AEBP1-related Ehlers-Danlos Syndrome (classical-like EDS type 2) in an additional patient and comprehensive clinical and molecular review of the literature.

The Ehlers-Danlos Syndromes (EDS), a group of hereditary connective tissue disorders, were classified into 13 subtypes in the 2017 International Classification. Recently, a new subtype of EDS called classical-like EDS type 2 (clEDS2), which is caused by biallelic variants in the adipocyte enhancer binding protein 1 (AEBP1) gene, was identified. We describe the 11th patient (9th family) with clEDS2, who was complicated by a critical vascular event (superior mesenteric artery aneurysm and rupture). A next-generation sequencing panel-based analysis revealed compound heterozygous variants in AEBP1: NM_001129.5:c.[2296G>T]; [2383dup], p.[(Glu766*)]; [(Glu795Glyfs*3)]. Light microscopic analyses showed increased interfibrillar spaces in the reticular dermis, a disorganized arrangement of collagen fibers, and decreased collagen content. An electron microscopic analysis showed the presence of collagen fibrils with irregular contours (flower-like appearance) and small collagen fibrils. A biochemical analysis showed reduced secretion of type I and type III procollagen. Clinical and molecular features of the current patient and all previously reported patients were reviewed comprehensively. Manifestations noted in most cases (>80%) included skin features (hyperextensibility, atrophic scars, easy bruising, excessive skin/skin folding, delayed wound healing, translucency, piezogenic papules), skeletal features (generalized joint hypermobility, dislocations/subluxations, pes planus), dental abnormalities, and neuromuscular abnormalities. Critical complications, each occurring in a single case, included superior mesenteric artery multiple aneurysm and rupture, aortic root dilation requiring surgery, and bowel rupture. Most AEBP1 variants were predicted or experimentally confirmed to lead to nonsense-mediated mRNA decay, whereas one variant resulted in a protein that was retained intracellularly and not secreted. Clinical, molecular, pathological, and biochemical features of the current patient, as well as a review of all previously reported patients, suggest the importance of the aortic carboxypeptidase-like protein encoded by AEBP1 in collagen fibrillogenesis.

Pathophysiological Investigation of Skeletal Deformities of Musculocontractural Ehlers-Danlos Syndrome Using Induced Pluripotent Stem Cells.

Musculocontractural Ehlers-Danlos syndrome caused by mutations in the carbohydrate sulfotransferase 14 gene (mcEDS-CHST14) is a heritable connective tissue disorder characterized by multiple congenital malformations and progressive connective tissue fragility-related manifestations in the cutaneous, skeletal, cardiovascular, visceral, and ocular systems. Progressive skeletal deformities are among the most frequent and serious complications affecting the quality of life and activities of daily living in patients. After establishing induced pluripotent stem cells (iPSCs) from cultured skin fibroblasts of three patients with mcEDS-CHST14, we generated a patient iPSC-based human osteogenesis model and performed an in vitro assessment of the phenotype and pathophysiology of skeletal deformities. Patient-derived iPSCs presented with remarkable downregulation of osteogenic-specific gene expression, less alizarin red staining, and reduced calcium deposition compared with wild-type iPSCs at each stage of osteogenic differentiation, including osteoprogenitor cells, osteoblasts, and osteocytes. These findings indicated that osteogenesis was impaired in mcEDS-CHST14 iPSCs. Moreover, the decrease in decorin (DCN) expression and increase in collagen (COL12A1) expression in patient-derived iPSCs elucidated the contribution of CHST14 dysfunction to skeletal deformities in mcEDS-CHST14. In conclusion, this disease-in-a-dish model provides new insight into the pathophysiology of EDS and may have the potential for personalized gene or drug therapy.

Comprehensive genetic screening for vascular Ehlers-Danlos syndrome through an amplification-based next-generation sequencing system.

Vascular Ehlers-Danlos syndrome (vEDS) is a hereditary connective tissue disorder (HCTD) characterized by arterial dissection/aneurysm/rupture, sigmoid colon rupture, or uterine rupture. Diagnosis is confirmed by detecting heterozygous variants in COL3A1. This is the largest Asian case series and the first to apply an amplification-based next-generation sequencing through custom panels of causative genes for HCTDs, including a specific method of evaluating copy number variations. Among 429 patients with suspected HCTDs analyzed, 101 were suspected to have vEDS, and 33 of them (32.4%) were found to have COL3A1 variants. Two patients with a clinical diagnosis of Loeys-Dietz syndrome and/or familial thoracic aortic aneurysm and dissection were also found to have COL3A1 variants. Twenty cases (57.1%) had missense variants leading to glycine (Gly) substitutions in the triple helical domain, one (2.9%) had a missense variant leading to non-Gly substitution in this domain, eight (22.9%) had splice site alterations, three (8.6%) had nonsense variants, two (5.7%) had in-frame deletions, and one (2.9%) had a multi-exon deletion, including two deceased patients analyzed with formalin-fixed and paraffin-embedded samples. This is a clinically useful system to detect a wide spectrum of variants from various types of samples.

Diffuse-Type Tenosynovial Giant Cell Tumor Arising in the Temporomandibular Joint Extending to the External Auditory Canal: A Case Report and Literature Review.

A 74-year-old Japanese woman with a 1-year history of right preauricular pain and a 2-month history of bleeding from the right ear was admitted to our department. Tumor was observed in the anterior wall in the right external auditory canal. Bony swelling of the right preauricular area was palpated. Computed tomography revealed an ill-defined, osteogenic tumor around the mandibular condyle with a destructive bony lesion involving the temporal bone. Magnetic resonance imaging revealed a 2.0 × 1.5 × 1.3-cm solid tumor around the mandibular condyle, exhibiting a low-intensity signal on T1-weighted imaging and an isointense central area surrounded by low-signal intensity on T2-weighted imaging. Histological examination of biopsy specimens revealed diffuse-type tenosynovial giant cell tumor (D-TGCT). After the feeding arteries for the tumor were embolized, the patient underwent surgery with combined temporal craniotomy and mandibular condylectomy. The soft and cystic tumor with calcification located in the extradural space was totally resected along with the mandibular condyle. No facial paralysis or recurrence was evident as of 6 months postoperatively. To date, only 23 cases of D-TGCT arising in the temporomandibular joint (TMJ) with ear involvement have been reported since 2011. We report successful resection of a rare case of D-TGCT arising in the TMJ.

Severe Cerebral Small Vessel Disease Caused by the Uniallelic p.A252T Variant of HTRA1.

Objective: To investigate the clinical effect of a heterozygous missense variant of HTRA1 on cerebral small vessel disease (CSVD) in a large Japanese family with a p.A252T variant. Methods: We performed clinical, laboratory, radiologic, and genetic evaluations of members of a previously reported family with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). Results: Two family members were previously reported patients with CARASIL. Among 6 uniallelic p.A252T carriers, 2 had neurologic symptoms with brain MRI abnormalities, 2 showed CSVD on the MRI only, and the other 2 were unaffected. Clinical phenotypes of 2 heterozygous patients were comparable with those of patients with CARASIL, whereas the other 3 heterozygous patients had developed milder and later-onset CSVD. One heterozygous carrier was asymptomatic. Discussion: Previous studies have suggested that uniallelic p.A252T causes disease. However, our study revealed that patients with uniallelic p.A252T can have severe and young-onset CSVD. The clinical manifestations of uniallelic variant carriers were highly variable, even within the same family. Male and atherosclerotic risk factors were considered to be additional factors in the severity of neurologic symptoms in uniallelic p.A252T carriers, suggesting that strict control of vascular risk factors can prevent vascular events in uniallelic HTRA1 carriers.

Case report: Mild phenotype of a patient with vascular Ehlers-Danlos syndrome and COL3A1 duplication mutation without alteration in the [Gly-X-Y] repeat sequence.

Background: Vascular-type Ehlers-Danlos syndrome (vEDS) is an autosomal dominant inherited disorder caused by a deficit in collagen III as a result of heterogeneous mutations in the α1 type III collagen gene (COL3A1). Patients with vEDS often experience the first major complications in their early 20s and >80% have at least one complication by their 40s, reducing their average life expectancy to 48 years. Most commonly, vEDS variants are heterozygous missense substitutions of a base-pair encoding a glycine (Gly) residue of the [Gly-X-Y] repeat of the COL3A1 protein. When a peptide chain derived from a mutant allele is present in the procollagen triple helical structure, the helical structure cannot be maintained. Therefore, typically, the mutated collagen peptide induces a dominant negative effect on procollagen production. We reported the case of a patient with vEDS and a unique novel duplication mutation without alteration in the [Gly-X-Y] triplet repeat sequence. Case presentation: A 58-year-old man developed a sudden disorder of consciousness and abdominal pain and was consequently taken to a nearby hospital, where an intra-abdominal aneurysm was found, in addition to mild small joint hypermobility and acrogeria. There has been no history of spontaneous pneumothorax, dislocation, or subcutaneous hematoma. The analysis of genomic DNA from a blood sample identified a likely pathogenic in-frame duplication mutation in the COL3A1 gene coding region. Interestingly, this mutation is not expected to alter the [Gly-X-Y] triplet repeat sequence. We verified the mutation's pathogenicity by performing an analysis of synthetic procollagen from cultured skin fibroblasts, electron microscopy, and mRNA expression analysis of unfolded protein response sensors for endoplasmic reticulum (ER) stress. Conclusion: Although the clinical findings of the case were mild, when compared to typical vEDS, decreased α1 collagen III levels and morphological abnormalities of the collagenous bundles were observed in the patient samples when compared with the normal control samples. Our evidence supports the conclusion that this variant is pathogenic. However, unlike the common vEDS, ER stress was not observed, and the mild phenotype presentation was suggested to be due to the unique mutation, allowing the triple helical structure to be maintained to a certain extent.

Clinical and molecular features of patients with COL1-related disorders: Implications for the wider spectrum and the risk of vascular complications.

Abnormalities in type I procollagen genes (COL1A1 and COL1A2) are responsible for hereditary connective tissue disorders including osteogenesis imperfecta (OI), specific types of Ehlers-Danlos syndrome (EDS), and COL1-related overlapping disorder (C1ROD). C1ROD is a recently proposed disorder characterized by predominant EDS symptoms of joint and skin laxity and mild OI symptoms of bone fragility and blue sclera. Patients with C1ROD do not carry specific variants for COL1-related EDS, including classical, vascular, cardiac-valvular, and arthrochalasia types. We describe clinical and molecular findings of 23 Japanese patients with pathogenic or likely pathogenic variants of COL1A1 or COL1A2, who had either OI-like or EDS-like phenotypes. The final diagnoses were OI in 17 patients, classical EDS in one, and C1ROD in five. The OI group predominantly experienced recurrent bone fractures, and the EDS group primarily showed joint hypermobility and skin hyperextensibility, though various clinical and molecular overlaps between OI, COL1-related EDS, and C1ROD as well as intrafamilial phenotypic variabilities were present. Notably, life-threatening vascular complications (vascular dissections, arterial aneurysms, subarachnoidal hemorrhages) occurred in seven patients (41% of those aged >20 years) with OI or C1ROD. Careful lifelong surveillance and intervention regarding bone and vascular fragility could be required.

Clinical and pathophysiological delineation of musculocontractural Ehlers-Danlos syndrome caused by dermatan sulfate epimerase deficiency (mcEDS-DSE): A detailed and comprehensive glycobiological and pathological investigation in a novel patient.

Musculocontractural Ehlers-Danlos syndrome caused by dermatan sulfate epimerase deficiency (mcEDS-DSE) is a rare connective tissue disorder. This is the first report describing the detailed and comprehensive clinical and pathophysiological features of mcEDS-DSE. The patient, with a novel homozygous nonsense variant (NM_013352.4:c.2601C>A:p.(Tyr867*)), exhibited mild skin hyperextensibility without fragility and small joint hypermobility, but developed recurrent large subcutaneous hematomas. Dermatan sulfate (DS) moieties on chondroitin sulfate/DS proteoglycans were significantly decreased, but remained present, in skin fibroblasts. Electron microscopy examination of skin specimens, including cupromeronic blue-staining to visualize glycosaminoglycan (GAG) chains, revealed coexistence of normally assembled collagen fibrils with attached curved GAG chains and dispersed collagen fibrils with linear GAG chains from attached collagen fibrils across interfibrillar spaces to adjacent fibrils. Residual activity of DS-epi1, encoded by DSE, and/or compensation by DS-epi2, a minor homolog of DS-epi1, may contribute to the mild skin involvement through this "mosaic" pattern of collagen fibril assembly.

Genome sequencing and RNA sequencing of urinary cells reveal an intronic FBN1 variant causing aberrant splicing.

Exome sequencing and panel testing have improved diagnostic yield in genetic analysis by comprehensively detecting pathogenic variants in exonic regions. However, it is important to identify non-exonic pathogenic variants to further improve diagnostic yield. Here, we present a female proband and her father who is diagnosed with Marfan syndrome, a systemic connective tissue disorder caused by pathogenic variants in FBN1. There are also two affected individuals in the siblings of the father, indicating the genetic basis in this family. However, panel testing performed by two institutions reported no causal variants. To further explore the genetic basis of the family, we performed genome sequencing of the proband and RNA sequencing of urinary cells derived from urine samples of the proband and her father because FBN1 is strongly expressed in urinary cells though it is poorly expressed in peripheral blood mononuclear cells. Genome sequencing identified a rare intronic variant (c.5789-15G>A) in intron 47 of FBN1 (NM_000138.4), which was transmitted from her father. RNA sequencing revealed allelic imbalance (monoallelic expression) of FBN1, retention of intron 47, and fewer aberrant transcripts utilizing new acceptor sites within exon 48, which were confirmed by RT-PCR. These results highlighted urinary cells as clinically accessible tissues for RNA sequencing if disease-causing genes are not sufficiently expressed in the blood, and the usefulness of multi-omics analysis for molecular diagnosis of genetic disorders.

Clinical and molecular features of 66 patients with musculocontractural Ehlers-Danlos syndrome caused by pathogenic variants in CHST14 (mcEDS-CHST14).

BACKGROUND: Musculocontractural Ehlers-Danlos syndrome is caused by biallelic loss-of-function variants in CHST14 (mcEDS-CHST14) or DSE (mcEDS-DSE). Although 48 patients in 33 families with mcEDS-CHST14 have been reported, the spectrum of pathogenic variants, accurate prevalence of various manifestations and detailed natural history have not been systematically investigated. METHODS: We collected detailed and comprehensive clinical and molecular information regarding previously reported and newly identified patients with mcEDS-CHST14 through international collaborations. RESULTS: Sixty-six patients in 48 families (33 males/females; 0-59 years), including 18 newly reported patients, were evaluated. Japanese was the predominant ethnicity (27 families), associated with three recurrent variants. No apparent genotype-phenotype correlation was noted. Specific craniofacial (large fontanelle with delayed closure, downslanting palpebral fissures and hypertelorism), skeletal (characteristic finger morphologies, joint hypermobility, multiple congenital contractures, progressive talipes deformities and recurrent joint dislocation), cutaneous (hyperextensibility, fine/acrogeria-like/wrinkling palmar creases and bruisability) and ocular (refractive errors) features were observed in most patients (>90%). Large subcutaneous haematomas, constipation, cryptorchidism, hypotonia and motor developmental delay were also common (>80%). Median ages at the initial episode of dislocation or large subcutaneous haematoma were both 6 years. Nine patients died; their median age was 12 years. Several features, including joint and skin characteristics (hypermobility/extensibility and fragility), were significantly more frequent in patients with mcEDS-CHST14 than in eight reported patients with mcEDS-DSE. CONCLUSION: This first international collaborative study of mcEDS-CHST14 demonstrated that the subtype represents a multisystem disorder with unique set of clinical phenotypes consisting of multiple malformations and progressive fragility-related manifestations; these require lifelong, multidisciplinary healthcare approaches.