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Cited by 1Metabolism and Genetic DisordersLysosomal Storage Disorders ResearchGenomics and Rare Diseases

Identification of a Novel Non-Canonical Splice-Site Variant in ABCD1

Open access
Zheng, Feixia, Lin, Zhongdong, Hu, Ying, Shi, Xulai, Zhao, Qianlei, Lin, Zhenlang·2023·Journal of Clinical Medicine
DOI 10.3390/jcm12020473PMC PMC9863105
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Abstract

Cerebral adrenoleukodystrophy (CALD) is a fatal genetic disease characterized by rapid, devastating neurological decline, with a narrow curative treatment window in the early stage. Non-canonical splice-site (NCSS) variants can easily be missed during genomic DNA analyses, and only a few of them in ABCD1 have been explored. Here, we studied a Chinese patient with clinical features similar to those of early-stage CALD but with a negative molecular diagnosis and a sibling who had presumably died of CALD. Trio-based whole-exome sequencing (trio-WES) and RNA sequencing (RNA-Seq) revealed a novel hemizygote NCSS variant c.901-25_901-9 del in ABCD1 intron 1, resulting in a complex splicing pattern. The in vitro minigene assay revealed that the c.901-25_901-9 del construct contained two aberrant transcripts that caused skipping of exon 2 and a small 48-bp deletion on left of the same exon. We identified a novel NCSS variant, that extends the spectrum of the known ABCD1 variants, and demonstrated the pathogenicity of this gene variant. Our findings highlight the importance of combining RNA-Seq and WES techniques for prompt diagnosis of leukodystrophy with NCSS variants.

AI snapshot

Written from the paper’s full text.

TL;DR

This study reports a Chinese boy with early-stage cerebral adrenoleukodystrophy (CALD) and a negative leukodystrophy gene panel, in whom trio-WES plus RNA-Seq identified a novel hemizygous non-canonical splice-site variant in ABCD1, c.901-25_901-9 del. Minigene assays showed the variant causes aberrant splicing with exon 2 skipping and a 48-bp deletion, expanding the ABCD1 variant spectrum and supporting combined WES/RNA-Seq for diagnosis.

Key findings

  • Trio-WES and RNA-Seq identified a novel hemizygous ABCD1 non-canonical splice-site variant, c.901-25_901-9 del; it was classified as a VUS according to ACMG PM2, PP3, and PP4 criteria, and HSF/MaxEnt predicted splicing effects of −51.39% and −390.19%, respectively.
  • RNA-Seq showed exon 2 of ABCD1 was missing. In HEK293 and MCF-7 minigene assays, wild-type produced an expected 570-bp product, while the variant produced two aberrant products: product I (389 bp) skipping entire exon 2 and product II (522 bp) with a 48-bp deletion on the left of exon 2; product I was mainly obtained.
  • The proband was a six-year-old boy with a six-month history of headaches, normal neurological examination, and brain MRI showing mildly enlarged demyelinating lesions in the splenium of the corpus callosum. Plasma C26:0 was 2.39 μM (normally <1.30 μM) and C26:0/C22:0 was 0.040 (normally <0.023); ACTH and corticosteroid levels were normal, and a 55-gene leukodystrophy panel was negative. His deceased elder brother had decline at nine years and died at 14 years.
  • After allogeneic hematopoietic stem cell transplantation three months after first admission, the proband returned to school at 18 months and at 30 months had smooth school performance without major functional disabilities; cerebral demyelination gradually arrested. As of September 2022, only 43 ABCD1 variants in non-canonical splice sites had been reported, and VUSs were the most frequent NCSS variants (65.1%, 28/43).

Methods

  • Single-family clinical and genetic study: the proband and his parents underwent clinical evaluation, plasma VLCFA/ACTH/corticosteroid measurement, brain MRI review, trio-WES on the NovaSeq 6000 platform, Sanger confirmation, and RNA-Seq from blood.
  • RNA-Seq analysis used STAR v.2.6.1e, HTSeq, OUTRIDER v.1.12.0, FRASER v.1.7.0, MaxEnt, and HSF, with visualization in Integrative Genome Viewer.
  • Minigene assay: ABCD1 intron 1 (285 bp), exon 2 (181 bp), and intron 2 (174 bp) were cloned into pcMINI; wild-type and mutant constructs were transfected into HEK293 and MCF-7 cells, and RT-PCR products were analyzed by gel electrophoresis and sequencing.

Limitations

  • Stated: the authors could not identify the variant or determine VLCFA levels in the extended family members of the mother because they refused genetic and biochemical testing.
  • Stated: the findings are based on a single-family study, and more cases with this variant are needed to validate the conclusions.
  • Evident from the text: the variant remained classified as an ACMG VUS despite functional assays, and no independent replication or additional unrelated cases are reported.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.3390/jcm12020473

Cites

48 of 48 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

25 of 42 cited sources checked · 8 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    The well-established treatments for CALD are useful only as the first signs of abnormalities are noted on MRI, before the onset of any neurological symptoms.

    “However, they are useful only as the first signs of abnormalities are noted on magnetic resonance imaging (MRI) of the brain, before the onset of any neurological symptoms.”
    Quote in the paperSubstantial claim2 of 5 sources checked
    • cites [2] Zhu, 2020, The changing face of adrenoleukodystrophy, Endocr. Rev. source
    • cites [3] Moser, 2018, Newborn screening and emerging therapies for X-linked adrenoleukodystrophy, JAMA Neurol. source
    • cites [4] Pierpont, 2020, Neurocognitive benchmarks following transplant for emerging cerebral adrenoleukodystrophy, Neurology source

      The Discussion (page unavailable) says transplantation at the first appearance of an enhancing MRI lesion appears most protective of functional outcomes and cautions against waiting for detectable neurocognitive abnormalities. It does not say treatment is useful only at that point or becomes useless once neurological symptoms appear. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [5] Pierpont, 2017, Neurocognitive trajectory of boys who received a hematopoietic stem cell transplant at an early stage of childhood cerebral adrenoleukodystrophy, JAMA Neurol. source
    • cites [6] Eichler, 2017, Hematopoietic stem-cell gene therapy for cerebral adrenoleukodystrophy, Engl. J. Med. source

      The introductory passage following the Abstract > Conclusions says transplantation is more likely to be effective early and reports good outcomes when disease is detected by imaging before symptoms. It does not say treatment is useful only at that point: it describes outcomes in symptomatic boys with a high burden of disease as often unfavorable, not invariably so. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
  • Premisecitation

    Other peroxisomal disorders can present with MRI abnormalities resembling X-linked ALD, particularly in patients with late-onset symptoms.

    “In some cases, other peroxisomal disorders, such as Zellweger spectrum disorder [31], acyl-CoA oxidase deficiency [32], and D-bifunctional protein deficiencies [33], have been reported to present with abnormalities that show in an MRI and that resemble X-linked ALD, particularly in patients with late-onset symptoms [34].”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [31] Tran, 2014, Late-onset Zellweger Spectrum Disorder caused by PEX6 mutations mimicking X-Linked adrenoleukodystrophy, Pediatr. Neurol. source
    • cites [32] Suzuki, 2002, Peroxisomal acyl CoA oxidase deficiency, J. Pediatr. source
    • cites [33] Lines, 2014, Peroxisomal D-bifunctional protein deficiency: Three adults diagnosed by whole-exome sequencing, Neurology source

      The Clinical description reports MRI abnormalities, including white-matter lesions, in three siblings with juvenile-onset D-bifunctional protein deficiency. The Mutation identification section notes that white-matter disease had not previously been reported in later-onset patients. The paper does not say these MRI findings resemble X-linked ALD; that comparison is unsupported by this source. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Findingcitation

    The c.901-25_901-9 del variant was classified as a VUS according to the ACMG PM2, PP3, and PP4 criteria.

    “This novel variant was classified as a VUS according to the ACMG PM2, PP3, and PP4 criteria [6], and confirmed using Sanger sequencing (Figure 2B).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [6] Eichler, 2017, Hematopoietic stem-cell gene therapy for cerebral adrenoleukodystrophy, Engl. J. Med. source

      The Methods section on clinical, imaging, and laboratory assessments describes PCR analysis of vector integration sites, not classification of the c.901-25_901-9 del variant. Searches for the variant and for ACMG, VUS, and Sanger sequencing found no passage addressing the claimed classification. (LLM, conf=0.74)

      Experimental — accuracy not yet measured
  • Premisecitation

    False-positive VLCFA results have been reported in approximately 2.5% of the healthy population, hemolyzed samples, and patients with a ketogenic diet.

    “Although VLCFAs are regarded as the most important biomarkers for the majority of peroxisomal disorders, false-positive results have been reported in approximately 2.5% of the healthy population, hemolyzed samples, and patients with a ketogenic diet [35,36].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [35] Theda, 1993, Increased very long chain fatty acids in patients on a ketogenic diet: A cause of diagnostic confusion, J. Pediatr. source
    • cites [36] Engelen, 2012, X-linked adrenoleukodystrophy (X-ALD): Clinical presentation and guidelines for diagnosis, follow-up and management, Orphanet. J. Rare Dis. source

      The Differential diagnosis > Increased VLCFA section says hemolysis and dietary causes, including a ketogenic diet, can produce false-positive plasma VLCFA results. It does not report a false-positive rate of approximately 2.5% in healthy people. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
  • Interpretationreasoning

    Prompt diagnosis can improve the prognosis of CALD in boys.

    “Thus, prompt diagnosis can improve the prognosis of CALD in boys [2,3,4,5,6].”
    Quote in the paperSubstantial claim2 of 5 sources checked
    • cites [2] Zhu, 2020, The changing face of adrenoleukodystrophy, Endocr. Rev. source
    • cites [3] Moser, 2018, Newborn screening and emerging therapies for X-linked adrenoleukodystrophy, JAMA Neurol. source
    • cites [4] Pierpont, 2020, Neurocognitive benchmarks following transplant for emerging cerebral adrenoleukodystrophy, Neurology source
    • cites [5] Pierpont, 2017, Neurocognitive trajectory of boys who received a hematopoietic stem cell transplant at an early stage of childhood cerebral adrenoleukodystrophy, JAMA Neurol. source
    • cites [6] Eichler, 2017, Hematopoietic stem-cell gene therapy for cerebral adrenoleukodystrophy, Engl. J. Med. source
  • Classified as probably not a substantial claim. 21
  • May combine two claims. 15
  • The quote only partly states this claim. 1

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The percentage of leukodystrophy cases without a specific diagnosis has been estimated to be 20-30%.

    “Nevertheless, the percentage of leukodystrophy cases without a specific diagnosis has been estimated to be 20–30% [7,39,40].”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [7] Vanderver, 2016, Whole exome sequencing in patients with white matter abnormalities, Ann. Neurol. source

      The Introduction estimates that nearly half of patients presenting with a possible leukodystrophy remain unresolved, rather than 20–30%. The Methods (Cohort description) also report 90 persistently unsolved cases among 191 identified cases, approximately 47%. (LLM, conf=0.87)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [39] Verdura, 2022, Diagnosis of genetic white matter disorders by singleton whole-exome and genome sequencing using interactome-driven prioritization, Neurology

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [40] Kevelam, 2016, Update on leukodystrophies: A historical perspective and adapted definition, Neuropediatrics source

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    The most likely disease-causing variants were prioritized according to the American College of Medical Genetics and Genomics classification guidelines and the clinical phenotype of the patients.

    “The most likely disease-causing variants were prioritized according to the American College of Medical Genetics and Genomics (ACMG) classification guidelines [20] and the clinical phenotype of the patients.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [20] Richards, 2015, Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology, Genet Med. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    VLCFAs are regarded as the most important biomarkers for the majority of peroxisomal disorders.

    “Although VLCFAs are regarded as the most important biomarkers for the majority of peroxisomal disorders, false-positive results have been reported in approximately 2.5% of the healthy population, hemolyzed samples, and patients with a ketogenic diet [35,36].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [35] Theda, 1993, Increased very long chain fatty acids in patients on a ketogenic diet: A cause of diagnostic confusion, J. Pediatr. source
    • cites [36] Engelen, 2012, X-linked adrenoleukodystrophy (X-ALD): Clinical presentation and guidelines for diagnosis, follow-up and management, Orphanet. J. Rare Dis. source

      The Biochemical and molecular diagnosis section says elevated plasma VLCFA confirms X-ALD in a male with suggestive symptoms. The Differential diagnosis > Increased VLCFA section also links elevated VLCFA to several other peroxisomal disorders. Neither section establishes that VLCFA are the most important biomarkers for the majority of peroxisomal disorders. Page numbers are not available in our copy. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    NGS genetic testing, including gene panel testing, WES, or WGS, is the most successful approach for diagnosing rare diseases with the highest diagnostic yield.

    “NGS genetic testing, including gene panel testing, WES, or whole genome sequencing (WGS), is the most successful approach for diagnosing rare diseases with the highest diagnostic yield [7,37].”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [7] Vanderver, 2016, Whole exome sequencing in patients with white matter abnormalities, Ann. Neurol. source

      The Results and Discussion support WES as a useful diagnostic approach for patients with unresolved white matter abnormalities: it yielded clinical diagnoses in 42% of the study cohort and increased diagnoses beyond standard approaches. The Discussion explicitly says further research is needed to compare WES, WGS, and targeted panels, so the paper does not establish that NGS is the most successful approach or has the highest yield across rare diseases. Page numbers are not provided in our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [37] Lionel, 2018, Improved diagnostic yield compared with targeted gene sequencing panels suggests a role for whole-genome sequencing as a first-tier genetic test, Anesthesia Analg. source

      The Introduction says WES has increased diagnostic yield, and the Results report that WGS diagnosed 41% of this pediatric cohort versus 24% with conventional testing. The paper supports a diagnostic advantage for WGS, but not the broader claim that NGS testing—including gene panels, WES, and WGS collectively—is the highest-yield approach for rare diseases generally. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    NCSS variants are considered VUSs unless their effect on splicing has been validated.

    “According to the ACMG standards and guidelines, NCSS variants are considered VUSs unless their effect on splicing has been validated [20].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [20] Richards, 2015, Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology, Genet Med. source

      The BP7 Synonymous variants section (p. 17) recommends uncertain-significance classification when a synonymous variant has a suspected splicing effect, pending functional evaluation or other evidence. The Proposed Criteria section (p. 10) also says variants default to uncertain significance when pathogenic or benign criteria are unmet. Neither passage establishes that all noncanonical splice-site (NCSS) variants must remain VUSs unless their splicing effect is validated; the guideline expressly allows other evidence to inform classification. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Trio-WES has substantially increased the number of known leukodystrophies and improved diagnoses.

    “Trio-WES has substantially increased the number of known leukodystrophies and improved diagnoses [7,8,39].”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [7] Vanderver, 2016, Whole exome sequencing in patients with white matter abnormalities, Ann. Neurol. source

      The Abstract reports that trio-or-larger WES yielded clinical diagnoses in 42% of 71 previously unresolved patients, and the Discussion estimates an approximately 20% diagnostic increase when WES is added to standard approaches. The paper does not show that WES substantially increased the number of known leukodystrophies; its Results describe diagnoses involving genes already associated with those disorders. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [8] Schiffmann, 2019, Diagnosis, prognosis, and treatment of leukodystrophies, Lancet Neurol. source
    • cites [39] Verdura, 2022, Diagnosis of genetic white matter disorders by singleton whole-exome and genome sequencing using interactome-driven prioritization, Neurology

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Genetic screening using next-generation sequencing is the most successful approach with the highest diagnostic yield in cases of underlying genetic disorders, such as CALD, and provides the fastest results.

    “Genetic screening using next-generation sequencing (NGS) is the most successful approach with the highest diagnostic yield in cases of underlying genetic disorders, such as CALD, and provides the fastest results [7,8].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [7] Vanderver, 2016, Whole exome sequencing in patients with white matter abnormalities, Ann. Neurol. source

      The Discussion (page unavailable) reports that whole-exome sequencing would increase diagnoses by about 20% in a cohort with genetic white matter disorders. It does not establish that NGS has the highest diagnostic yield or provides the fastest results, and says comparative effectiveness needs further study. The cited passages do not establish the claim specifically for CALD. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [8] Schiffmann, 2019, Diagnosis, prognosis, and treatment of leukodystrophies, Lancet Neurol. source

  • Classified as probably not a substantial claim.

    Premisecitation

    ALD is caused by ABCD1 mutations.

    “ALD is caused by ABCD1 mutations [1].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [1] Mallack, E., Gao, K., Engelen, M., and Kemp, S. (2022). Structure and function of the ABCD1 variant database: 20 years, 940 pathogenic variants, and 3400 cases of adrenoleukodystrophy. Cells, 11. source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    ALD is the most common peroxisomal disorder and has multiple phenotypes without an established genotype-phenotype relationship.

    “ALD is the most common peroxisomal disorder and has multiple phenotypes without an established genotype–phenotype relationship [1].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [1] Mallack, E., Gao, K., Engelen, M., and Kemp, S. (2022). Structure and function of the ABCD1 variant database: 20 years, 940 pathogenic variants, and 3400 cases of adrenoleukodystrophy. Cells, 11. source

      The Introduction describes ALD as having a striking clinical spectrum with no established genotype–phenotype relationship; the Abstract names adrenal insufficiency, myelopathy, and leukodystrophy. Our copy gives ALD’s estimated birth prevalence but does not establish that it is the most common peroxisomal disorder. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    For patients in whom WES is uninformative, RNA-Seq of blood has been reported to show diagnostic utility in rare diseases by generating a 7.5% diagnostic rate and 16.7% improved candidate gene resolution.

    “For patients in whom WES is uninformative, RNA-Seq of blood has been reported to show diagnostic utility in rare diseases by generating a 7.5% diagnostic rate and 16.7% improved candidate gene resolution [45].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [45] Smail, 2019, Identification of rare-disease genes using blood transcriptome sequencing and large control cohorts, Nat. Med. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The majority of noncoding variants are involved in the production of aberrant transcripts through exon skipping, cryptic site use, or intron retention.

    “The majority of noncoding variants are involved in the production of aberrant transcripts through exon skipping, cryptic site use, or intron retention [44].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [44] Mucaki, 2020, Expression changes confirm genomic variants predicted to result in allele-specific, alternative mRNA splicing, Front. Genet. source

      The Introduction says the majority of mutations in a set of validated splice-variant predictions were associated with exon skipping, cryptic site use, or intron retention. It does not extend that finding to the majority of all noncoding variants, and it classifies some resulting isoforms as alternative rather than aberrant. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    NCSS variants are less straightforward to interpret because they exhibit considerable sequence variation and can easily be missed during genomic DNA analyses.

    “Non-CSS (NCSS) variants are less straightforward to interpret because they exhibit considerable sequence variation and can easily be missed during genomic DNA analyses [43].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [43] Anna, 2018, Splicing mutations in human genetic disorders: Examples, detection, and confirmation, J. Appl. Genet. source

      The Summary (page not available) says splicing mutations outside canonical sites can be missed during genomic DNA analysis. The section on branch points and polypyrimidine tracts (page not available) says their degenerate consensus sequences make variants hard to interpret, but it establishes that sequence-variation point for those motifs rather than for NCSS variants as a whole. (LLM, conf=0.78)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Previous studies have reported that RNA-Seq helps interpret VUSs identified through WES/WGS when linked to an aberrant transcript event, and detects variants missed by current standard diagnostic approaches in rare diseases.

    “Previous studies have reported that RNA-Seq helps interpret VUSs identified through WES/WGS when linked to an aberrant transcript event, and detects variants missed by current standard diagnostic approaches in rare diseases [42,45,46].”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [42] Kremer, 2017, Genetic diagnosis of Mendelian disorders via RNA sequencing, Nat. Commun. source
    • cites [45] Smail, 2019, Identification of rare-disease genes using blood transcriptome sequencing and large control cohorts, Nat. Med. source

      The introductory paragraph and summary (section labeled NOTE; no page given) report that blood RNA-seq aided diagnosis after uninformative WES, identifying causal genes and variants in 7.5% of cases. The paper also describes combining exome or genome variants with RNA-seq splicing outliers to narrow candidates. Our copy does not establish that the variants interpreted in those analyses were specifically classified as VUSs, so that part of the claim is more specific than the evidence. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [46] Cummings, 2017, Improving genetic diagnosis in Mendelian disease with transcriptome sequencing, Sci. Transl. Med. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Approximately 50-75% of patients with Mendelian disorders do not receive a genetic diagnosis based on WES that could identify disease-causing variants in noncoding regions.

    “Approximately 50–75% of patients with Mendelian disorders do not receive a genetic diagnosis based on WES that could identify disease-causing variants in noncoding regions [41,42].”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [41] Wortmann, 2015, Whole exome sequencing of suspected mitochondrial patients in clinical practice, J. Inherit. Metab. Dis. source

      The Discussion reports that WES diagnosed 42 of 109 patients suspected of having a mitochondrial disorder (39%), leaving approximately 61% without a genetic diagnosis. That supports a figure within the claimed range, but only for this specific cohort; the paper does not establish a 50–75% rate across Mendelian disorders or attribute undiagnosed cases to disease-causing variants in noncoding regions. Page numbers were not provided in our copy. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [42] Kremer, 2017, Genetic diagnosis of Mendelian disorders via RNA sequencing, Nat. Commun. source

  • May combine two claims.

    Findingown data

    The proband's deceased elder brother had progressive neurological deterioration and died at age 14.

    “The deceased elder brother of the proband showed a decline in school performance at nine years of age, followed by progressive deterioration in behavior, hearing, vision, and motor function. He died at the age of 14 years.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Until the last visit, 30 months after treatment, the boy had achieved smooth school performance without major functional disabilities.

    “Until the last visit, 30 months after treatment, the boy had achieved smooth school performance without major functional disabilities.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    The proband received allogeneic hematopoietic stem cell transplantation three months after first admission, without major adverse events.

    “After CALD diagnosis, the proband received allogeneic hematopoietic stem cell transplantation three months after the first admission, without major adverse events.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    RNA-Seq analysis revealed a complex splicing pattern for ABCD1, with only a small number of ABCD1 transcripts and exon 2 missing.

    “The RNA-Seq analysis revealed a complex splicing pattern for ABCD1 (Figure 2A). Only a small number of ABCD1 transcripts were identified. Examination of mRNA transcripts revealed that exon 2 of ABCD1 was missing.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Trio-WES identified five variants (ABCD1, CPT2, AP4B1, NDUFS1, and ENTPD1) associated with white matter abnormalities, leukodystrophy, or very long chain fatty acid accumulation and segregating according to the predicted genetic model.

    “Five variants, ABCD1, CPT2, AP4B1, NDUFS1, and ENTPD1 (Table S2), identified using trio-WES were found to be associated with “white matter abnormalities, leukodystrophy, or very long chain fatty acid accumulation” and segregated according to the predicted genetic model (X-linked recessive, autosomal recessive, and compound heterozygote).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PCR product I [exon A-exon B] was 389 bp and skipped the entire exon 2.

    “PCR product I [exon A–exon B] was 389 bp and skipped the entire exon 2, whereas product II [exon A–△exon 2 (133 bp)–exon B] was 522 bp with a 48-bp deletion on the left of exon 2.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The proband's mother carried heterozygous variants of ABCD1 and was asymptomatic.

    “The proband’s mother, who carried heterozygous variants of ABCD1, was asymptomatic.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingreasoning

    The c.901-25_901-9 del variant is a maternal variant following an X-linked recessive model of inheritance.

    “The mutant gene ABCD1 (RefSeq NM_000033.4: c.901-25_901-9 del) is strongly correlated with clinical measures and is a maternal variant following an X-linked recessive model of inheritance.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    The leukodystrophy multi-gene panel test was the first approach in this case.

    “Therefore, the leukodystrophy multi-gene panel test was the first approach in this case.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductcitation

    The study was approved by the Research Ethics Board of the Second Affiliated Hospital of Wenzhou Medical University and conducted in accordance with the Declaration of Helsinki.

    “The study was approved by the Research Ethics Board of the Second Affiliated Hospital of Wenzhou Medical University (2021-K-127-02) and conducted in accordance with the Declaration of Helsinki [11].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [11] World Medical Association (2013). World Medical Association Declaration of Helsinki: Ethical principles for medical research involving human subjects. JAMA, 310, 2191–2194. source

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Study design and conductnone given

    Written consent was obtained from the patient and his legal guardians.

    “Written consent was obtained from the patient and his legal guardians, where necessary.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisereasoning

    VUSs are the most frequent NCSS variants of ABCD1, at 65.1% (28/43).

    “To date, VUSs are the most frequent NCSS variants of ABCD1 (65.1%, 28/43).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    As of September 2022, only 43 ABCD1 variants located in non-canonical splice sites have been reported in the ABCD1 Variant Database.

    “As of September 2022, only 43 ABCD1 variants located in NCSSs have been reported in the ABCD1 Variant Database (https://adrenoleukodystrophy.info/, accessed on 1 September 2022).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Interpretationreasoning

    A likely explanation is that the variant may disrupt ABCD1 splicing, thereby causing a premature termination of transcription and degradation of defective transcripts.

    “A likely explanation is that the variant may disrupt ABCD1 splicing, thereby causing a premature termination of transcription and degradation of defective transcripts.”
    Quote in the paperNot a substantial claim
28 of 28

  • The authors state this as an assumption.

    The deceased elder brother had CALD.

    “a sibling who had presumably died of CALD”
    “He was suspected of having had a frontal form of CALD.”

  • The paper relies on this without support.

    The minigene assay in HEK293 and MCF-7 cells recapitulates endogenous ABCD1 splicing.

    “To determine whether the proband variant NM_000033.4: c.901-25_901-9 del disrupted the splicing of ABCD1, the sequences of intron 1 (285 bp), exon 2 (181 bp), and intron 2 (174 bp) were amplified and expressed as minigenes (exon A–intron A–multiple cloning site–intron B–exon B) in HEK293 and MCF-7 cells.”

  • The paper relies on this without support.

    RNA-Seq of blood reflects the splicing pattern of ABCD1 in the affected central nervous system.

    “Furthermore, we analyzed their brain MRI scans and the results of genetic tests, and performed trio-WES of the proband’s and his parents’ samples and RNA-Seq of the proband’s samples.”
3 of 3

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Authors
Zheng, F., Lin, Z., Hu, Y., Shi, X., Zhao, Q., & Lin, Z.
Affiliations
  • Wenzhou Medical University (CN)
  • Department of Pediatrics, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325027, China
ORCID
Zhao, Qianlei
Venue
Journal of Clinical Medicine
Publisher
MDPI AG
Published
2023-01-06
Volume
12
Issue
2
Pages
473
ISSN
2077-0383 (ISSN-L)
DOI
10.3390/jcm12020473
PMC
PMC9863105
OpenAlex
W4313648614
Semantic Scholar
7c15d3aa8031e63414c1cdc5d13569eca1cc5ec0
Cited by
1
References
48
Citations by year
2023: 1
FWCI
0.15
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Clinical Medicine (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
Funders
the Wenzhou Science and Technology Bureau of Zhejiang Province (Y20210002)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Clinical Biochemistry › Metabolism and Genetic Disorders
  • Health Sciences › Medicine › Physiology › Lysosomal Storage Disorders Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomics and Rare Diseases
Keywords
cerebral adrenoleukodystrophy, ABCD1, exon skipping, hemizygous mutation, variant pathogenicity, minigene assay, Chinese patients

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-20.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-20.

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