Differential metabolic secretion between muscular dystrophy mouse-derived spindle cell sarcomas and rhabdomyosarcomas drives tumor type development.

The dystrophin gene (Dmd) is recognized for its significance in Duchenne muscular dystrophy (DMD), a lethal and progressive skeletal muscle disease. Some patients with DMD and model mice with muscular dystrophy (mdx) spontaneously develop various types of tumors, among which rhabdomyosarcoma (RMS) is the most prominent. By contrast, spindle cell sarcoma (SCS) has rarely been reported in patients or mdx mice. In this study, we aimed to use metabolomics to better understand the rarity of SCS development in mdx mice. Gas chromatography-mass spectrometry was used to compare the metabolic profiles of spontaneously developed SCS and RMS tumors from mdx mice, and metabolite supplementation assays and silencing experiments were used to assess the effects of metabolic differences in SCS tumor-derived cells. The levels of 75 metabolites exhibited differences between RMS and SCS, 25 of which were significantly altered. Further characterization revealed downregulation of nonessential amino acids, including alanine, in SCS tumors. Alanine supplementation enhanced the growth, epithelial mesenchymal transition, and invasion of SCS cells. Reduction of intracellular alanine via knockdown of the alanine transporter Slc1a5 reduced the growth of SCS cells. Lower metabolite secretion and reduced proliferation of SCS tumors may explain the lower detection rate of SCS in mdx mice. Targeting of alanine depletion pathways may have potential as a novel treatment strategy.NEW & NOTEWORTHY To the best of our knowledge, SCS has rarely been identified in patients with DMD or mdx mice. We observed that RMS and SCS tumors that spontaneously developed from mdx mice with the same Dmd genetic background exhibited differences in metabolic secretion. We proposed that, in addition to dystrophin deficiency, the levels of secreted metabolites may play a role in the determination of tumor-type development in a Dmd-deficient background.

Longitudinal data of serum creatine kinase levels and motor, pulmonary, and cardiac functions in 337 patients with Duchenne muscular dystrophy.

INTRODUCTION/AIMS: Duchenne muscular dystrophy (DMD) presents with skeletal muscle weakness, followed by cardiorespiratory involvement. The need for longitudinal data regarding DMD that could serve as a control for determining treatment efficacy in clinical trials has increased notably. The present study examined the longitudinal data of Japanese DMD patients collectively and assessed individual patients with pathogenic variants eligible for exon-skipping therapy. METHODS: Patients with DMD who visited Kobe University Hospital between March 1991 and March 2019 were enrolled. Data between the patients' first visit until age 20 years were examined. RESULTS: Three hundred thirty-seven patients were included. Serum creatine kinase levels showed extremely high values until the age of 6 years and a rapid decline from ages 7-12 years. Both the median 10-m run/walk velocity and rise-from-floor velocity peaked at the age of 4 years and declined with age. The values for respiratory function declined from the age of 11 years. The median left ventricular ejection fraction was >60% until the age of 12 years and rapidly declined from ages 13-15 years. Examination of the relationship between pathogenic variants eligible for exon-skipping therapy and longitudinal data revealed no characteristic findings. DISCUSSION: We found that creatine kinase levels and motor, respiratory, and cardiac functions each exhibited various changes over time. These findings provide useful information about the longitudinal data of several outcome measures for patients with DMD not receiving corticosteroids. These data may serve as historical controls in comparing the natural history of DMD patients not on regular steroid use in appropriate clinical trials.

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.

Brothers with Becker muscular dystrophy show discordance in skeletal muscle computed tomography findings: A case report.

Becker muscular dystrophy is caused by DMD mutations and is characterized by progressive muscle atrophy. The wide variations observed in muscle atrophy progression in Becker muscular dystrophy are considered multifactorial, including differences in mutations and environmental factors. In this case, two brothers, aged 2 and 3 years, had the identical DMD mutation, confirming their Becker muscular dystrophy diagnosis. They began using handrails when ascending and descending stairs at the age of 16 due to progressive muscular weakness. Over an 18-year follow-up, the older brother consistently had high serum creatine kinase levels, significantly over median levels. Muscle computed tomography finings revealed that the older brother's gluteus maximus and vastus femoris cross-sectional areas were only half and one-third of the younger brother's, respectively. The mean computed tomography values of gluteus maximus and vastus femoris were significantly lower in the older brother. Our report suggests that muscle atrophy in Becker muscular dystrophy cannot be solely explained by dystrophin mutation or environmental factors.

A splice-switching oligonucleotide treatment ameliorates glycogen storage disease type 1a in mice with G6PC c.648G>T.

Glycogen storage disease type 1a (GSD1a) is caused by a congenital deficiency of glucose-6-phosphatase-α (G6Pase-α, encoded by G6PC), which is primarily associated with life-threatening hypoglycemia. Although strict dietary management substantially improves life expectancy, patients still experience intermittent hypoglycemia and develop hepatic complications. Emerging therapies utilizing new modalities such as adeno-associated virus and mRNA with lipid nanoparticles are under development for GSD1a but potentially require complicated glycemic management throughout life. Here, we present an oligonucleotide-based therapy to produce intact G6Pase-α from a pathogenic human variant, G6PC c.648G>T, the most prevalent variant in East Asia causing aberrant splicing of G6PC. DS-4108b, a splice-switching oligonucleotide, was designed to correct this aberrant splicing, especially in liver. We generated a mouse strain with homozygous knockin of this variant that well reflected the pathophysiology of patients with GSD1a. DS-4108b recovered hepatic G6Pase activity through splicing correction and prevented hypoglycemia and various hepatic abnormalities in the mice. Moreover, DS-4108b had long-lasting efficacy of more than 12 weeks in mice that received a single dose and had favorable pharmacokinetics and tolerability in mice and monkeys. These findings together indicate that this oligonucleotide-based therapy could provide a sustainable and curative therapeutic option under easy disease management for GSD1a patients with G6PC c.648G>T.

A novel splice variant of the human MSTN gene encodes a myostatin-specific myostatin inhibitor.

BACKGROUND: Myostatin, encoded by the MSTN gene comprising 3 exons, is a potent negative regulator of skeletal muscle growth. Although a variety of myostatin inhibitors have been invented for increasing muscle mass in muscle wasting diseases, no effective inhibitor is currently available for clinical use. Myostatin isoforms in several animals have been reported to inhibit myostatin, but an isoform has never been identified for the human MSTN gene, a conserved gene among animals. Here, a splice variant of the human MSTN gene was explored. METHODS: Transcripts and proteins were analysed by reverse transcription-PCR amplification and western blotting, respectively. Proteins were expressed from expression plasmid. Myostatin signalling was assayed by the SMAD-responsive luciferase activity. Cell proliferation was assayed by the Cell Counting Kit-8 (CCK-8) assay and cell counting. Cell cycle was analysed by the FastFUCCI system. RESULTS: Reverse transcription-PCR amplification of the full-length MSTN transcript in CRL-2061 rhabdomyosarcoma cells revealed two bands consisting of a thick expected-size product and a thin additional small-size product. Sequencing of the small-size product showed a 963-bp deletion in the 5' end of exon 3, creating exon 3s, which contained unusual splice acceptor TG dinucleotides. The novel variant was identified in other human cell lines, although it was not identified in skeletal muscle. The 251-amino acid isoform encoded by the novel variant (myostatin-b) was identified in CRL-2061 rhabdomyosarcoma cells. Transfection of a myostatin-b expression plasmid into CRL-2061 and myoblast cells inhibited endogenous myostatin signalling (44%, P < 0.001 and 63%, P < 0.001, respectively). Furthermore, myostatin-b inhibited myostatin signalling induced by recombinant myostatin (68.8%, P < 0.001). In remarkable contrast, myostatin-b did not inhibit the myostatin signalling induced by recombinant growth differentiation factor 11 (9.2%, P = 0.70), transforming growth factor ß (+3.1%, P = 0.83) or activin A (+1.1%, P = 0.96). These results indicate the myostatin-specific inhibitory effect of myostatin-b. Notably, the expression of myostatin-b in myoblasts significantly enhanced cell proliferation higher than the mock-transfected cells by the CCK-8 and direct cell counting assays (60%, P < 0.05 and 39%, P < 0.05, respectively). Myostatin-b increased the percentage of S-phase cells significantly higher than that of the mock-transfected cells (53% vs. 80%, P < 0.05). CONCLUSIONS: We cloned a novel human MSTN variant produced by unorthodox splicing. The variant encoded a novel myostatin isoform, myostatin-b, that inhibited myostatin signalling by myostatin-specific manner and enhanced myoblast proliferation by shifting cell cycle. Myostatin-b, which has myostatin-specific inhibitory activity, could be developed as a natural myostatin inhibitor.

Electrocardiographic R wave amplitude in V6 lead as a predictive marker of cardiac dysfunction in Duchenne muscular dystrophy.

PURPOSE: Duchenne muscular dystrophy (DMD) is an inherited muscular disease characterized by progressive and fatal muscle weakness. Electrocardiographic (ECG) abnormalities, including abnormal R wave amplitudes are frequently observed in DMD. However, clinical implications of abnormal R wave amplitudes remain unclear. Hence, DMD patients were examined for changes in R wave amplitude over time using synthesized 18-lead ECG and the relationship between R wave amplitude and cardiac function. METHODS: The results of 969 ECG examinations of 193 patients with DMD who underwent electrocardiography and echocardiography on the same day were retrospectively reviewed. RESULTS: A negative correlation was observed between R wave amplitude and age. Positive correlations between R wave amplitude and left ventricular ejection fraction were observed in leads V4, V5, V6, syn-V7, syn-V8, and syn-V9, with V6 showing the strongest correlation (r = 0.52). Mean R wave amplitude during cardiac dysfunction was lower than that observed with preserved cardiac function in leads V6 to syn-V9. Patients had preserved R wave amplitude up to three years before the onset of cardiac dysfunction, with a sharp decrease two years before cardiac dysfunction in leads V6 to syn-V9. CONCLUSIONS: In DMD patients, the R wave amplitude decreases with age. The sharp decline in R amplitude two years before cardiac dysfunction indicates that electrophysiological damage to the myocardium of the left ventricle lateral to the posterior wall precedes the finding of cardiac dysfunction. The R amplitude in V6 of the standard 12-lead ECG is a convenient predictive marker of cardiac dysfunction, similar to that of the 18-lead ECG.

All reported non-canonical splice site variants in GLA cause aberrant splicing.

BACKGROUND: Fabry disease is an X-linked lysosomal storage disorder caused by insufficient α-galactosidase A (GLA) activity resulting from variants in the GLA gene, which leads to glycosphingolipid accumulation and life-threatening, multi-organ complications. Approximately 50 variants have been reported that cause splicing abnormalities in GLA. Most were found within canonical splice sites, which are highly conserved GT and AG splice acceptor and donor dinucleotides, whereas one-third were located outside canonical splice sites, making it difficult to interpret their pathogenicity. In this study, we aimed to investigate the genetic pathogenicity of variants located in non-canonical splice sites within the GLA gene. METHODS: 13 variants, including four deep intronic variants, were selected from the Human Gene Variant Database Professional. We performed an in vitro splicing assay to identify splicing abnormalities in the variants. RESULTS: All candidate non-canonical splice site variants in GLA caused aberrant splicing. Additionally, all but one variant was protein-truncating. The four deep intronic variants generated abnormal transcripts, including a cryptic exon, as well as normal transcripts, with the proportion of each differing in a cell-specific manner. CONCLUSIONS: Validation of splicing effects using an in vitro splicing assay is useful for confirming pathogenicity and determining associations with clinical phenotypes.

A case of infantile Barth syndrome with severe heart failure: Importance of splicing variants in the TAZ gene.

Barth syndrome (BTHS) is an X-linked disorder characterized by cardiomyopathy, skeletal myopathy, and 3-methylglutaconic aciduria. The causative pathogenic variants for BTHS are in TAZ, which encodes a putative acyltransferase named tafazzin and is involved in the remodeling of cardiolipin in the inner mitochondrial membranes. Pathogenic variants in TAZ result in mitochondrial structural and functional abnormalities. We report a case of infantile BTHS with severe heart failure, left ventricular noncompaction, and lactic acidosis, having a missense c.640C>T (p.His214Tyr) variant in TAZ, which is considered a pathogenic variant based on the previously reported amino acid substitution at the same site (c.641A>G, p.His214Arg). However, in this previously reported case, heart function was compensated and not entirely similar to the present case. Silico prediction analysis suggested that c.640C>T could alter the TAZ messenger RNA (mRNA) splicing process. TAZ mRNAs in isolated peripheral mononuclear cells from the patient and in vitro splicing analysis using minigenes of TAZ found an 8 bp deletion at the 3' end of exon 8, which resulted in the formation of a termination codon in the coding region of exon 9 (H214Nfs*3). These findings suggest that splicing abnormalities should always be considered in BTHS.

Dravet syndrome and hemorrhagic shock and encephalopathy syndrome associated with an intronic deletion of SCN1A.

OBJECTIVE: Hemorrhagic shock and encephalopathy syndrome (HSES) is a serious condition that requires intensive care and is associated with a high mortality rate. However, its pathogenesis remains unclear. In the present study, a genetic analysis was performed to determine the genetic background of patients with clinically suspected Dravet syndrome (DS) who developed HSES. METHODS: Whole exome sequencing was performed, followed by minigene analysis of the intron variant detected by whole exome sequencing to confirm its effect on splicing. RESULTS: Whole exome sequencing revealed a novel 21-bp deletion in intron 3 of SCN1A NM_001165963.4 (NC_000002.11:g.166073675_166073695del). This deletion was not found in the patient's parents and was proven to be de novo. Minigene analysis revealed an aberrant mRNA lacking 40 and 106 bp from the 5' end of exon 4 of SCN1A. Therefore, we diagnosed this case as DS due to the deletion in intron 3 of SCN1A. CONCLUSIONS: We report a case of DS with HSES caused by a 21-bp deletion in the intron of SCN1A that was confirmed by minigene analysis. The present case met Levin's criteria for HSES and the splicing analysis of SCN1A is an important finding. This study has important implications for understanding HSES pathogenesis.

Detecting pathogenic deep intronic variants in Gitelman syndrome.

Gitelman syndrome (GS) is a rare, autosomal recessive, salt-losing tubulopathy caused by loss of function in the SLC12A3 gene (NM_000339.2), which encodes the natrium chloride cotransporter. The detection of homozygous or compound heterozygous SLC12A3 variants is expected in GS, but 18%-40% of patients with clinical GS carry only one mutant allele. Previous reports identified some pathogenic deep intronic variants in SLC12A3. Here, we report the screening of SLC12A3 deep intronic variants in 13 patients with suspected GS carrying one mutated SLC12A3 allele. Variant screening used the HaloPlex Target Enrichment System Kit capturing whole introns and the promotor region of SLC12A3, followed by SureCall variant analysis. Rare intronic variants (<1% frequency) were identified, and pathogenicity evaluated by the minigene system. Deep intronic variant screening detected seven rare SLC12A3 variants from six patients. Only one variant showed pathogenicity in the minigene system (c.602-16G>A, intron 4) through activation of a cryptic acceptor site. No variants were detected in the promotor region. Deep intronic screening identified only one pathogenic variant in patients with suspected GS carrying monoallelic SLC12A3 variants. Our results suggest that deep intronic variants partially explain the cause of monoallelic variants in patients with GS.

Evaluation of Suspected Autosomal Alport Syndrome Synonymous Variants.

Background: Alport syndrome is an inherited disorder characterized by progressive renal disease, variable sensorineural hearing loss, and ocular abnormalities. Although many pathogenic variants in COL4A3 and COL4A4 have been identified in patients with autosomal Alport syndrome, synonymous mutations in these genes have rarely been identified. Methods: We conducted in silico splicing analysis using Human Splicing Finder (HSF) and Alamut to predict splicing domain strength and disruption of the sites. Furthermore, we performed in vitro splicing assays using minigene constructs and mRNA analysis of patient samples to determine the pathogenicity of four synonymous variants detected in four patients with suspected autosomal dominant Alport syndrome (COL4A3 [c.693G>A (p.Val231=)] and COL4A4 [c.1353C>T (p.Gly451=), c.735G>A (p.Pro245=), and c.870G>A (p.Lys290=)]). Results: Both in vivo and in vitro splicing assays showed exon skipping in two out of the four synonymous variants identified (c.735G>A and c.870G>A in COL4A4). Prediction analysis of wild-type and mutated COL4A4 sequences using HSF and Alamut suggested these two variants may lead to the loss of binding sites for several splicing factors, e.g., in acceptor sites and exonic splicing enhancers. The other two variants did not induce aberrant splicing. Conclusions: This study highlights the pitfalls of classifying the functional consequences of variants by a simple approach. Certain synonymous variants, although they do not alter the amino acid sequence of the encoded protein, can dramatically affect pre-mRNA splicing, as shown in two of our patients. Our findings indicate that transcript analysis should be carried out to evaluate synonymous variants detected in patients with autosomal dominant Alport syndrome.

An Antisense Oligonucleotide against a Splicing Enhancer Sequence within Exon 1 of the MSTN Gene Inhibits Pre-mRNA Maturation to Act as a Novel Myostatin Inhibitor.

Antisense oligonucleotides (ASOs) are agents that modulate gene function. ASO-mediated out-of-frame exon skipping has been employed to suppress gene function. Myostatin, encoded by the MSTN gene, is a potent negative regulator of skeletal muscle growth. ASOs that induce skipping of out-of-frame exon 2 of the MSTN gene have been studied for their use in increasing muscle mass. However, no ASOs are currently available for clinical use. We hypothesized that ASOs against the splicing enhancer sequence within exon 1 of the MSTN gene would inhibit maturation of pre-mRNA, thereby suppressing gene function. To explore this hypothesis, ASOs against sequences of exon 1 of the MSTN gene were screened for their ability to reduce mature MSTN mRNA levels. One screened ASO, named KMM001, decreased MSTN mRNA levels in a dose-dependent manner and reciprocally increased MSTN pre-mRNA levels. Accordingly, KMM001 decreased myostatin protein levels. KMM001 inhibited SMAD-mediated myostatin signaling in rhabdomyosarcoma cells. Remarkably, it did not decrease GDF11 mRNA levels, indicating myostatin-specific inhibition. As expected, KMM001 enhanced the proliferation of human myoblasts. We conclude that KMM001 is a novel myostatin inhibitor that inhibits pre-mRNA maturation. KMM001 has great promise for clinical applications and should be examined for its ability to treat various muscle-wasting conditions.

Human Dystrophin Dp71ab Enhances the Proliferation of Myoblasts Across Species But Not Human Nonmyoblast Cells.

Dystrophin Dp71 is an isoform produced from the Dp71 promoter in intron 62 of the DMD gene, mutations in which cause Duchenne muscular dystrophy. Dp71 is involved in various cellular processes and comprises more than 10 isoforms produced by alternative splicing. Dp71ab, in which both exons 71 and 78 are deleted, has a hydrophobic C-terminus that is hydrophilic in Dp71. Therefore, Dp71ab is believed to have different roles from Dp71. Previously, we reported that Dp71ab enhanced the proliferation of human myoblasts. Here, we further characterized Dp71ab, focusing on the activation of cell proliferation. Dp71ab increased the proliferation of immortalized human myoblasts in a dose-dependent manner. In contrast, Dp71 suppressed proliferation in a dose-dependent manner. Consistent with these opposite effects, eGFP-tagged Dp71ab and mCherry-tagged Dp71 showed different cellular distributions, with Dp71ab mostly in the nucleus. Notably, human Dp71ab enhanced the proliferation of rat and mouse myoblasts. Despite these findings, human Dp71ab did not enhance the proliferation of human nonmyoblast cells, including rhabdomyosarcoma cells. We concluded that Dp71ab is a myoblast-specific proliferation enhancer. In further studies, Dp71ab will be employed for the expansion of myoblasts in clinical settings.

A sandwich ELISA kit reveals marked elevation of titin N-terminal fragment levels in the urine of mdx mice.

The mdx mouse is a model of Duchenne muscular dystrophy (DMD), a fatal progressive muscle wasting disease caused by dystrophin deficiency, and is used most widely in preclinical studies. Mice with dystrophin deficiency, however, show milder muscle strength phenotypes than humans. In human, the introduction of a sandwich enzyme-linked immunosorbent assay (ELISA) kit revealed a more than 700-fold increase in titin N-terminal fragment levels in the urine of pediatric patients with DMD. Notably, the urinary titin level declines with aging, reflecting progression of muscle wasting. In mouse, development of a highly sensitive ELISA kit has been awaited. Here, a sandwich ELISA kit to measure titin N-terminal fragment levels in mouse urine was developed. The developed kit showed good linearity, recovery, and repeatability in measuring recombinant or natural mouse titin N-terminal fragment levels. The titin N-terminal fragment concentration in the urine of mdx mice was more than 500-fold higher than that of normal mice. Urinary titin was further analyzed by extending the collection of urine samples to both young (3-11 weeks old) and aged (56-58 weeks old) mdx mice. The concentration in the young group was significantly higher than that in the aged group. It was concluded that muscle protein breakdown is active and persistent in mdx mice even though the muscle phenotype is mild. Our results provide an opportunity to develop DMD treatments that aim to alleviate muscle protein breakdown by monitoring urinary titin levels.

Comprehensive genetic analysis using next-generation sequencing for the diagnosis of nephronophthisis-related ciliopathies in the Japanese population.

Nephronophthisis is an autosomal-recessive kidney disease that is caused by abnormalities in primary cilia. Nephronophthisis-related ciliopathies (NPHP-RCs) are a common cause of end-stage kidney disease (ESKD) in children and adolescents. NPHP-RCs are often accompanied by extrarenal manifestations, including intellectual disability, retinitis pigmentosa, or polydactyly. Although more than 100 causative genes have been identified, its diagnosis is difficult because the clinical features of each mutation often overlap. From September 2010 to August 2021, we performed genetic analysis, including next-generation sequencing (NGS), in 574 probands with kidney dysfunction and retrospectively studied cases genetically diagnosed with NPHP-RCs. RESULTS: We detected mutations related to NPHP-RCs in 93 patients from 83 families. Members of 60 families were diagnosed using NGS, and the mutations and the corresponding number of families are as follows: NPHP1 (24), NPHP3 (10), OFD1 (7), WDR35 (5), SDCCAG8 (4), BBS10 (3), TMEM67 (3), WDR19 (3), BBS1 (2), BBS2 (2), IFT122 (2), IFT140 (2), IQCB1 (2), MKKS (2), SCLT1 (2), TTC21B (2), ALMS1 (1), ANKS6 (1), BBS4 (1), BBS12 (1), CC2D2A (1), DYNC2H1 (1), IFT172 (1), and MAPKBP1 (1). A total of 39 cases (41.9%) progressed to ESKD at the time of genetic analysis, whereas 58 cases (62.3%) showed extrarenal manifestations, the most common being developmental delay, intellectual disability, and autism spectrum disorder in 44 patients. Comprehensive genetic analysis using NGS is useful for diagnosing patients with NPHP-RCs.

Last Nucleotide Substitutions of COL4A5 Exons Cause Aberrant Splicing.

INTRODUCTION: COL4A5 is a causative gene of X-linked Alport syndrome (XLAS). Male patients with XLAS with nonsense variants have the most severe phenotypes of early onset end-stage kidney disease (ESKD); those with splicing variants have middle phenotypes and those with missense variants have the mildest phenotypes. Therefore, genotyping for male patients with XLAS can be used to predict kidney prognosis. Single-base substitutions at the last nucleotide position in each exon are known to affect splicing patterns and could be splicing variants. Nevertheless, in XLAS, these variants are generally considered to be missense variants, without conducting a transcript analysis, which underestimates some patients as having mild phenotypes. This study aimed to investigate whether single-base substitutions at the last nucleotide position of COL4A5 exons cause aberrant splicing. METHODS: In total, 20 variants were found in the Human Gene Mutation Database (n = 14) and our cohort (n = 6). We performed functional splicing assays using a hybrid minigene analysis and in vivo transcript analyses of patients' samples when available. Then, we investigated genotype-phenotype correlations for patients with splicing variants detected in this study by comparing data from our previous studies. RESULTS: Among the 20 variants, 17 (85%) caused aberrant splicing. Male patients with splicing variants had more severe phenotypes when compared with those with missense variants. Findings from the in vivo analyses for 3 variants were identical to those from the minigene assay. CONCLUSION: Our study revealed that most single-base substitutions at the last nucleotide position of COL4A5 exons result in splicing variants, rather than missense variants, thereby leading to more severe phenotypes.

Mutation spectrum analysis of DMD gene in Indonesian Duchenne and Becker muscular dystrophy patients.

Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the DMD gene. The full mutation spectrum of the DMD gene in Indonesian patients is currently unknown. Mutation-specific therapies are currently being developed, such as exon skipping or stop codon read-through therapy. This study was conducted with the aim of identifying the mutation spectrum of the DMD gene in Indonesia to guide future development and application of feasible therapeutic strategies. Methods: This study is a cross sectional study that enrolled 43 male patients with a clinical suspicion of DMD or BMD. Multiplex ligation-dependent probe amplification (MLPA) reaction was performed to screen for the common mutations in the DMD gene. Results: Out of 43 subjects, deletions accounted for 69.77% (n=30) cases, while duplications were found in 11.63% (n=5) cases. One novel duplication spanning exons 2 to 62 was identified. Deletion mutations clustered around the distal (66.67%) and proximal (26.67%) hot spot regions of the DMD gene while duplication mutations were observed solely at the proximal region. Two false positive cases of single exon deletion detected through MLPA were attributed to sequence mutations affecting primer ligation sites, confirming the need to validate all single exon deletions when using this screening method. Analysis of available maternal DNA samples showed that the rate of de novo mutations (48.15%) appears higher than expected in this population. Out of 31 patients who were classified as DMD based on clinical and genotype characterizations, 60.47% (n=26) of cases were suitable for exon skipping therapy. Conclusion: This is the first comprehensive study showing the feasibility of implementing the MLPA method for routine screening of DMD patients in Indonesia. This is also the first study showing the potential applicability of exon skipping therapy in the majority of DMD cases in the country.

Clinical features of autosomal recessive polycystic kidney disease in the Japanese population and analysis of splicing in PKHD1 gene for determination of phenotypes.

BACKGROUND: Autosomal recessive polycystic kidney disease (ARPKD) is caused by mutations in the PKHD1 gene. The clinical spectrum is often more variable than previously considered. We aimed to analyze the clinical features of genetically diagnosed ARPKD in the Japanese population. METHODS: We conducted a genetic analysis of patients with clinically diagnosed or suspected ARPKD in Japan. Moreover, we performed a minigene assay to elucidate the mechanisms that could affect phenotypes. RESULTS: PKHD1 pathogenic variants were identified in 32 patients (0-46 years). Approximately one-third of the patients showed prenatal anomalies, and five patients died within one year after birth. Other manifestations were detected as follows: chronic kidney disease stages 1-2 in 15/26 (57.7%), Caroli disease in 9/32 (28.1%), hepatic fibrosis in 7/32 (21.9%), systemic hypertension in 13/27 (48.1%), and congenital hypothyroidism in 3 patients. There have been reported that truncating mutations in both alleles led to severe phenotypes with perinatal demise. However, one patient without a missense mutation survived the neonatal period. In the minigene assay, c.2713C > T (p.Gln905Ter) and c.6808 + 1G > A expressed a transcript that skipped exon 25 (123 bp) and exon 41 (126 bp), resulting in an in-frame mutation, which might have contributed to the milder phenotype. Missense mutations in cases of neonatal demise did not show splicing abnormalities. CONCLUSION: Clinical manifestations ranged from cases of neonatal demise to those diagnosed in adulthood. The minigene assay results indicate the importance of functional analysis, and call into question the fundamental belief that at least one non-truncating mutation is necessary for perinatal survival.

Assessment of catabolic state in infants with the use of urinary titin N-fragment.

BACKGROUND: Urinary titin N-fragment levels have been used to assess the catabolic state, and we used this biomarker to evaluate the catabolic state of infants. METHODS: We retrospectively measured urinary titin N-fragment levels of urinary samples. The primary outcome was its changes according to postmenstrual age. The secondary outcomes included differences between gestational age, longitudinal change after birth, influence on growth, and relationship with blood tests. RESULTS: This study included 219 patients with 414 measurements. Urinary titin N-fragment exponentially declined with postmenstrual age. These values were 12.5 (7.1-19.6), 8.1 (5.1-13.0), 12.8 (6.0-21.3), 26.4 (16.4-52.0), and 81.9 (63.3-106.4) pmol/mg creatinine in full, late, moderate, very, and extremely preterm infants, respectively (p < 0.01). After birth, urinary levels of titin N-fragment exponentially declined, and the maximum level within a week was associated with the time to return to birth weight in preterm infants (ρ = 0.39, p < 0.01). This was correlated with creatine kinase in full-term infants (ρ = 0.58, p < 0.01) and with blood urea nitrogen in preterm infants (ρ = 0.50, p < 0.01). CONCLUSIONS: The catabolic state was increased during the early course of the postmenstrual age and early preterm infants. IMPACT: Catabolic state in infants, especially in preterm infants, was expected to be increased, but no study has clearly verified this. In this retrospective study of 219 patients with 414 urinary titin measurements, the catabolic state was exponentially elevated during the early postmenstrual age. The use of the urinary titin N-fragment clarified catabolic state was prominently increased in very and extremely preterm infants.