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Cited by 10Genomics and Rare DiseasesGenetics and Neurodevelopmental DisordersMuscle Physiology and Disorders

The DOI matches its Crossref record; 32 of the 32 references it cites were found in a registry or an index; none of the 26 sources its claims cite is checked for support yet.

Homozygous WASHC4 variant in two sisters causes a syndromic phenotype defined by dysmorphisms, intellectual disability, profound developmental disorder, and skeletal muscle involvement

Open access
Gangfuß, Andrea, Czech, Artur, Hentschel, Andreas, Münchberg, Ute, Horvath, Rita, Töpf, Ana, O'Heir, Emily, Lochmüller, Hanns, Stehling, Florian, Kiewert, Cordula, Sickmann, Albert, Kuechler, Alma, Kaiser, Frank J., Kölbel, Heike, Christiansen, Jon, Schara‐Schmidt, Ulrike, Roos, Andreas·2022·The Journal of Pathology
DOI 10.1002/path.5812
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Abstract

Recessive variants in WASHC4 are linked to intellectual disability complicated by poor language skills, short stature, and dysmorphic features. The protein encoded by WASHC4 is part of the Wiskott–Aldrich syndrome protein and SCAR homolog family, co‐localizes with actin in cells, and promotes Arp2/3‐dependent actin polymerization in vitro. Functional studies in a zebrafish model suggested that WASHC4 knockdown may also affect skeletal muscles by perturbing protein clearance. However, skeletal muscle involvement has not been reported so far in patients, and precise biochemical studies allowing a deeper understanding of the molecular etiology of the disease are still lacking. Here, we report two siblings with a homozygous WASHC4 variant expanding the clinical spectrum of the disease and provide a phenotypical comparison with cases reported in the literature. Proteomic profiling of fibroblasts of the WASHC4‐deficient patient revealed dysregulation of proteins relevant for the maintenance of the neuromuscular axis. Immunostaining on a muscle biopsy derived from the same patient confirmed dysregulation of proteins relevant for proper muscle function, thus highlighting an affliction of muscle cells upon loss of functional WASHC4. The results of histological and coherent anti‐Stokes Raman scattering microscopic studies support the concept of a functional role of the WASHC4 protein in humans by altering protein processing and clearance. The proteomic analysis confirmed key molecular players in vitro and highlighted, for the first time, the involvement of skeletal muscle in patients. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Overview

AI snapshot

Written from the paper’s full text.

TL;DR

Two consanguineous sisters with a homozygous *WASHC4* c.3041A>G p.(Tyr1014Cys) variant presented with severe intellectual disability, absent speech, dysmorphisms, short stature/microcephaly, and previously unreported skeletal muscle involvement. Proteomic, histological, and CARS/SHG analyses of patient-derived fibroblasts and muscle support *WASHC4* loss affecting protein processing/clearance and neuromuscular proteins.

Key findings

  • The sisters expand the *WASHC4* phenotype: both had profound intellectual disability, absent speech, severe motor delay, hypotonia/areflexia, and dysmorphic features; sibling 1 also had epilepsy, scoliosis, contractures, and recurrent pulmonary infections. Across published cases, abnormal speech development and intellectual disability were present in 12/12 patients and short stature in 10/12.
  • Skeletal muscle involvement was demonstrated for the first time in patients: quadriceps biopsy showed fiber-type disproportion with increased type 2 fibers, subsarcolemmal mitochondrial enrichment, and increased PAS reactivity. CARS microscopy found an average muscle fiber caliber of 22.81 ± 3.92 μm (638 fibers) versus 43.92 ± 23.29 μm in five controls (1457 fibers), indicating atrophy; 581/638 (91%) patient fibers showed subsarcolemmal increased signal at 2921 cm⁻¹ versus 55/1457 (3.77%) control fibers, suggesting protein aggregation.
  • Proteomic profiling of patient fibroblasts quantified 3707 proteins, of which 364 (9.82%) were significantly dysregulated: 174 (4.69%) increased and 191 (5.13%) decreased. GO terms implicated extracellular matrix organization, unfolded protein response, oxidative stress, cholesterol biosynthesis, mitochondrial function, actin filament assembly, endocytosis, lysosome organization, and autophagy.
  • Muscle validation showed increased FYCO1, VCP, p62, ubiquitin, BiP, GRP170, LC3-I to LC3-II conversion, pPERK, and amyloid, plus decreased Rab7 and actin; phalloidin staining indicated decreased actin cytoskeleton in *WASHC4*-diseased muscle fibers.

Methods

  • Clinical and genetic study of two affected sisters from consanguineous parents; whole exome sequencing had >80× mean target coverage and filtered for rare (<1%) damaging homozygous variants. The *WASHC4* variant c.3041A>G p.(Tyr1014Cys) was predicted deleterious by SIFT, Polyphen2, and MutationTaster with CADD 29; a *POSTN* variant had CADD 23.4 and was reported benign in ClinVar.
  • Proteomic profiling used data-independent acquisition on patient skin fibroblasts versus control, with GO term analysis restricted to proteins showing >2 or <0.5 fold-change and a significant P value.
  • Muscle analysis used quadriceps biopsy histology, immunofluorescence, immunoblotting, and coherent anti-Stokes Raman scattering/second harmonic generation microscopy; muscle fiber calibers were manually determined from CARS images (638 patient fibers; 1457 fibers from five controls), and 290 spectra were extracted for analysis.

Limitations

  • Stated: the authors note that additional fibroblast cultures and muscle biopsies from *WASHC4* patients or animal models are needed to define the pathological mechanisms as commonly associated with loss of functional *WASHC4*, and that further proteomic studies on different cellular populations and tissues are needed to identify shared or specific pathophysiological cascades.
  • Evident from the text: molecular validation was largely derived from one affected sister’s fibroblasts and muscle biopsy, without independent replication in additional patient tissues.
  • Evident from the text: standardized IQ testing was never possible due to profound intellectual disability, and the literature comparison was limited by missing cranial MRI in 8/12 patients and echocardiography in 9/12 patients.

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

Integrity signals

Registry

Crossref · 10.1002/path.5812

Cites

32 of 32 references verified0 citation errors · 0 not checkable

Statistics

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Verification

Claims the paper makes

0 of 26 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Our proteomic study indicated altered expression of numerous proteins decisive for proper function of the neuromuscular axis.

    “Indeed, our proteomic study indicated altered expression of numerous proteins decisive for proper function of the neuromuscular axis (supplementary material, Table S3).”
    Quote in the paperSubstantial claim
  • Findingown data

    Immunoblotting showed p62 appearing with a band at a lower molecular weight in the muscle protein extract of the patient compared with the control.

    “Remarkably, immunoblotting showed p62 appearing with a band at a lower molecular weight in the muscle protein extract of the patient compared with the control (Figure 4B).”
    Quote in the paperSubstantial claim
  • Findingown data

    An increase of GRP170 was detected in the quadriceps biopsy derived from the same patient.

    “In support of this finding, we detected an increase of its cochaperone GRP170 in the quadriceps biopsy derived from the same patient.”
    Quote in the paperNot a substantial claim
  • Findingown data

    Areas of decreased signal intensity at 2921 cm−1 in the subsarcolemmal region were found in the patient.

    “We also found areas of decreased signal intensity at 2921 cm À1 in the subsarcolemmal region in the patient (Figure 6B1).”
    Quote in the paperNot a substantial claim
  • Findingown data

    Only 55 of 1457 fibers (3.77%) in the control samples showed a comparable finding.

    “Only 55 of 1457 fibers (3.77%) in the control samples showed a comparable finding.”
    Quote in the paperSubstantial claim
  • The quote only partly states this claim. 7
  • Probably not a substantial claim. 27
  • It may state two separate claims as one. 49

  • The quote only partly states this claim.

    Findingown data

    Analysis of the quadriceps muscle biopsy from Sibling 1 revealed no significant changes by H&E staining.

    “Analysis of the quadriceps muscle biopsy revealed no significant changes by H&E staining (Figure 2A).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingown data

    Sibling 2 was able to make her first unaided steps at age 6 years.

    “At the age of 6 years, the patient was able to make her first unaided steps.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Proteomic profiling of WASHC4 patient-derived fibroblasts quantified 3707 proteins, out of which 364 (9.82%) were significantly dysregulated, with 174 (4.69%) significantly increased and 191 (5.13%) significantly decreased.

    “Our approach allowed the quantification of 3707 proteins, out of which 364 (9.82%) were significantly dysregulated (Figure 3A,B).A total of 174 (4.69%) were significantly increased and 191 (5.13%) were significantly decreased (Figure 3B and supplementary material, Figure S1).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    RAB7 and CD63 were decreased in WASHC4 patient-derived fibroblasts and a similar decrease was confirmed in the patient-derived muscle biopsy.

    “Along this line, two further proteins involved in autophagy, RAB7 and CD63, were decreased in WASHC4 patient-derived fibroblasts and a similar decrease was confirmed in the patient-derived muscle biopsy (Figure 4B and supplementary material, Figure S1).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingcitation

    Ropers et al did not report developmental delay, deviating head circumference, behavioral abnormalities, or dysmorphic features, whereas all patients described thereafter showed these symptoms, including clear dysmorphic features.

    “Ropers et al did not report developmental delay, deviating head circumference, behavioral abnormalities, or dysmorphic features [10], whereas all patients described thereafter showed these symptoms, including clear dysmorphic features.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10]

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

  • It may state two separate claims as one.

    Findingown data

    GO term-based analysis revealed that proteins increased in patient-derived cells affect extracellular matrix organization and cell adhesion, protein folding and IRE1-mediated unfolded protein response, regulation of beta-amyloid clearance, positive regulation of smooth muscle cell proliferation, response to oxidative stress, and lipoprotein catabolic processes.

    “Our GO term-based analysis focusing on biological processes and cellular compartments revealed that proteins increased in the patient-derived cells are among others affecting extracellular matrix (ECM) organization and cell adhesion, protein folding and IRE1-mediated unfolded protein response, regulation of beta-amyloid clearance, positive regulation of (smooth) muscle cell proliferation, response”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Clinical comparison of the two WASHC4 patients described here with those previously reported in the literature revealed that the most frequent common symptoms include poor language skills/language delay (12/12) and ID (12/12), followed by short stature (10/12).

    “A clinical comparison of the two WASHC4 patients described here with those previously reported in the literature was performed.This revealed that the most frequent common symptoms include poor language skills/ language delay (12/12) and ID (12/12), followed by short stature (10/12).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingnone given

    These areas tend to have a roundish shape.

    “These areas tend to have a roundish shape.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingown data

    Sibling 2 was able to walk a few steps independently.

    “She was able to walk a few steps independently.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Galectin-3 was decreased in WASHC4 patient-derived fibroblasts and the decrease was confirmed in the patient-derived quadriceps biopsy.

    “Galectin-3 coordinates the mobilization of core autophagy regulators in response to damaged endomembranes and was identified as decreased in WASHC4 patient-derived fibroblasts; confirmational immunological studies on the patient-derived quadriceps biopsy confirmed a decrease of galectin-3 (Figure 4B and supplementary material, Figure S1).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingown data

    Sibling 2 received hearing aids at age 12 months due to pathological BERA results.

    “At the age of 12 months, she was equipped with hearing aids due to pathological BERA results.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Sibling 1 showed delayed general development: only sounding; no sitting and reduced head control at 14 months; standing with support at 20 months.

    “General development was delayed (only sounding; no sitting and reduced head control at 14 months; standing with support at 20 months).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingown data

    Sibling 2 did not gain weight adequately despite regular feeding via PEG tube.

    “Despite regular feeding via the PEG tube, she did not gain weight adequately.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Immunostaining on a muscle biopsy derived from the same patient confirmed dysregulation of proteins relevant for proper muscle function, thus highlighting an affliction of muscle cells upon loss of functional WASHC4.

    “Immunostaining on a muscle biopsy derived from the same patient confirmed dysregulation of proteins relevant for proper muscle function, thus highlighting an affliction of muscle cells upon loss of functional WASHC4.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Findingown data

    X-ray of Sibling 2's left hand at age 6 years showed significantly retarded bone age, due to a low (not measurable) anti-Müllerian hormone (AMH) level; ovarian insufficiency was suspected.

    “At the age of 6 years, X-ray of her left hand showed a significantly retarded bone age, due to a low (not measurable) anti-Müllerian hormone (AMH) level; ovarian insufficiency was suspected and further controls were recommended.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    The POSTN variant c.2396A>C; p.(Phe799-Ser) has a CADD score of 23.4, is predicted deleterious by two of three tools, and is reported benign in ClinVar.

    “The WASHC4 variant is predicted to be deleterious by three in silico tools (SIFT, Polyphen2, and MutationTaster) and has a CADD score of 29, whereas the POSTN variant has a lower CADD score of 23.4, is predicted to be deleterious by two of the three tools tested, and has been reported as benign in ClinVar.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    The patient's average muscle fiber caliber was 22.81 ± 3.92 μm (638 fibers), compared with 43.92 ± 23.29 μm in five healthy individuals (1457 fibers), indicating atrophic muscle fibers.

    “From the CARS measurements of the patient's muscle biopsy, an average muscle fiber caliber of 22.81 Æ 3.92 μm (638 fibers) was determined.From comparison with the average muscle fiber caliber of five healthy individuals (43.92 Æ 23.29 μm; 1457 fibers), the patient displays atrophic muscle fibers.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Patients 8-12 had developmental delay and severely affected speech development in combination with borderline (patient 8), mild (patients 9 and 10), and severe ID (patients 11 and 12).

    “Patients 8-12 had a developmental delay and severely affected speech development in combination with borderline (patient 8), mild (patients 9 and 10), and severe ID (patients 11 and 12).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Sibling 2 showed dysmorphic features including enlarged and coarsened nasal tip with deviation to the right, small eyes with moderate ptosis, and low-set ears.

    “She showed clearly visible dysmorphic features such as an enlarged and coarsened nasal tip with deviation to the right side, small eyes with moderate ptosis and low-set ears (Figure 1H,I), a global developmental delay with suspicion of severe intellectual disability (ID), and stereotypic movements in combination with absent speech.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Findingown data

    Hirsutism was present in patient 10 and patient 11 but not in patient 12.

    “Hirsutism was present in patient 10 and patient 11 (but not in her sister, patient 12).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Sibling 1 showed dysmorphic features including enlarged and coarsened nasal tip with deviation to the left, small upper lip vermilion, small eyes with moderate ptosis and mild strabismus, high arched eyebrows with discreet synophrys, low-set ears with enlarged ear lobes, prominent forehead with low-set hairline, and hirsutism.

    “She showed significant dysmorphic features such as an enlarged and coarsened nasal tip with deviation to the left side, a small upper lip vermilion, small eyes with moderate ptosis and mild strabismus, high arched eyebrows with discreet synophrys, as well as low-set ears with enlarged ear lobes and a prominent forehead with low-set hairline and hirsutism (Figure 1B,C,F,G).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    The WASHC4 variant is predicted to be deleterious by three in silico tools (SIFT, Polyphen2, and MutationTaster) and has a CADD score of 29.

    “The WASHC4 variant is predicted to be deleterious by three in silico tools (SIFT, Polyphen2, and MutationTaster) and has a CADD score of 29, whereas the POSTN variant has a lower CADD score of 23.4, is predicted to be deleterious by two of the three tools tested, and has been reported as benign in ClinVar.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingown data

    Epilepsy was only mentioned in patient 11.

    “Epilepsy was only mentioned in patient 11.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Sibling 1 developed epilepsy with generalized tonic-clonic seizures at age 4.5 years and became seizure-free on oxcarbazepine.

    “At age 4.5 years, the patient developed epilepsy with generalized tonic-clonic seizures and received oxcarbazepine (seizure-free on therapy).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Sibling 1 had global developmental delay with suspicion of severe ID, stereotypic movements, autoaggression, and absent speech at age 9 years.

    “At the age of 9 years, she presented with a global developmental delay with suspicion of severe ID, stereotypic movements, and autoaggression in combination with absent speech.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Molecular karyotyping of Sibling 1 showed no relevant dosage aberrations but revealed five large homozygous areas encompassing about 98 Mb, in line with parental consanguinity.

    “Molecular karyotyping (Affymetrix Genome-Wide SNP_6.0Array) showed no relevant dosage aberrations but revealed five large homozygous areas, encompassing about 98 Mb, which is in line with the known parental consanguinity.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Sibling 2 had short stature (height 104 cm, -3.79z), dystrophy (weight 17 kg, -2.56z), and microcephaly (head circumference 49 cm, -2.20z) at age 7 years.

    “At the age of 7 years, she presented with short stature (height 104 cm, À3.79z), dystrophy (weight 17 kg, À2.56z), and microcephaly (head circumference 49 cm, À2.20z) (z-scores [18]).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Sibling 1 had short stature (height 121 cm, -2.62z) and microcephaly (head circumference 49 cm, -2.95z) but normal weight (26 kg, -1.15z) at age 9 years.

    “Short stature [height 121 cm (À2.62z)] and microcephaly (head circumference 49 cm (À2.95z)] but normal weight [26 kg (À1.15z)] (z-scores [18]) were found.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    The two patients described in this study showed a very significant motor delay and failure to develop speech in combination with a profound ID.

    “The two patients A Gangfuß et al described in this study showed a very significant motor delay and failure to develop speech in combination with a profound ID (not possible to be tested with a standardized test).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Sibling 2 had severe global developmental delay with absent speech and dysmorphic stigmata (enlarged and coarsened nasal tip) at age 3.5 years.

    “At 3.5 years, she presented with a severe global developmental delay with absent speech and dysmorphic stigmata (enlarged and coarsened nasal tip).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Sibling 1 had areflexia and prominent generalized muscle hypotonia leading to right convex scoliosis, contractures of the elbows, knees, and ankles, and was only able to walk a few steps with support with an unstable broad-based gait.

    “Clinical examination revealed areflexia and prominent generalized muscle hypotonia leading to right convex scoliosis (Figure 1D,E,F); contractures of the elbows, knees, and ankles; and she was only able to walk a few steps with support, with an unstable broad-based gait pattern.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Based on the report of further patients and our study, in total 27 different features were described; the majority of these features were present in only some of the patients, some features even only in single patients.

    “Based on the report of further patients and our study, in total 27 different features were described.The majority of these features were present in only some of the patientssome features even only in single patients.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Collagen VI was increased in fibroblasts and confirmed increased within the ECM in the patient-derived quadriceps biopsy.

    “Proteomic profiling revealed an increase of collagen VI in fibroblasts and confirmational studies on the patient-derived quadriceps biopsy showed an increase of collagen VI within the ECM (supplementary material, Figure S1).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Tenascin was increased in WASHC4-deficient patient-derived fibroblasts and the sarcoplasmic increase was confirmed in quadriceps muscle fibers derived from the same patient.

    “The ECM protein tenascin, implicated in the guidance of migrating neurons, synaptic plasticity, and neuronal regeneration, was also identified to be increased in WASHC4-deficient patient-derived fibroblasts and the sarcoplasmic increase could be confirmed in quadriceps muscle fibers derived from the same patient (Figure 4B and supplementary material, Figure S1).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Ropers et al did not mention developmental delay, deviating head circumference, or dysmorphic features, whereas all patients described thereafter had clear dysmorphic features.

    “Moreover, the authors did not mention a developmental delay (including age of walking), deviating head circumference or dysmorphic features, whereas all patients described thereafter had clear dysmorphic features, for example a prominent and wide nasal tip (patients 8, 11, and 12), a prominent forehead and synophrys (patients 8, 9, and 10) or dysmorphic ears (patients 8 and 10).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Dystrophy due to feeding difficulties was prominent in patients 11 and 12 but was not described by the other authors.

    “Although dystrophy due to feeding difficulties was prominent in patients 11 and 12, this was not described by the other authors.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Patient 8 developed macrocephaly, while patients 10 and 11 showed microcephaly.

    “While patient 8 developed macrocephaly, patients 10 and 11 showed microcephaly.”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data

    Brain MRI of Sibling 1 revealed agenesis of the pituitary gland.

    “brain magnetic resonance imaging (MRI) revealed agenesis of the pituitary gland.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Histological findings showed a reduction in succinate dehydrogenase (SDH) and an increase of type 2 fibers, and the calculation of muscle fiber diameter indicated atrophy.

    “Moreover, an affliction of the skeletal muscles is indicated by our histological findings showing a reduction in succinate dehydrogenase (SDH) and an increase of type 2 fibers as well as by the calculation of muscle fiber diameter indicating atrophy.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingcitation

    Behavioral abnormalities were not mentioned by Ropers et al but were described in four out of the remaining five patients.

    “Behavioral abnormalities were not mentioned by Ropers et al [10] but were described in four out of the remaining five patients.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10]

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

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Findingown data

    Cranial MRI was performed in only 4 out of 12 patients, and echocardiography in only 3 out of 12.

    “Cranial MRI was performed in only 4 out of the 12 patients, and echocardiography in only 3 out of 12.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Findingown data

    The parents are healthy, consanguineous (cousins), of Middle Eastern descent (United Arab Emirates).

    “The parents are healthy, consanguineous (cousins I ), of Middle Eastern descent (United Arab Emirates).”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data

    Proteomic findings suggested a decrease of the actin cytoskeleton in WASHC4 patient-derived fibroblasts.

    “Prompted by our proteomic findings suggesting a decrease of the actin cytoskeleton, phalloidin staining (visualizing the actin cytoskeleton) was performed in the WASHC4 patientderived quadriceps biopsy, which revealed less intensive immunoreactivity suggesting a decrease of actin cytoskeleton in WASHC4-diseased muscle fibers (supplementary material, Figure S1).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Sibling 1 had frequent febrile pulmonary infections necessitating antibiotic treatment, and CT showed signs of primary lung disease (thickened walls and ecstatic bronchi as well as milk glass opacity).

    “Due to frequent febrile pulmonary infections necessitating antibiotic treatment, additional diagnostic interventions (chest X-ray, polysomnography, anti-body investigations) were performed at the age of 4.5 years but did not identify an underlying cause.Computed tomography (CT) showed signs for a primary lung disease (thickened walls and ecstatic bronchi as well as milk glass opacity).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Findingown data

    Sibling 1 had abnormal brainstem-evoked response audiometry (BERA) and received hearing aids at age 5 years.

    “Due to abnormal brainstemevoked response audiometry (BERA), hearing aids were implemented at the age of 5 years but were not well tolerated.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Sibling 1 had deterioration of motor skills during pulmonary infections and needed a long recovery time.

    “During pulmonary infections, she presented with deterioration of motor skills and needed a long recovery time.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    p62 staining in the WASHC4 patient showed slightly lower signal intensities but simultaneously a higher percentage of points with maximum signal intensity along with sarcoplasmic clustering.

    “For p62 staining, the WASHC4 patient showed slightly lower signal intensities but simultaneously a higher percentage of points with maximum signal intensity along with sarcoplasmic clustering, supporting the concept of protein aggregation (Figure 5B).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Sibling 2 had generalized hypotonia and areflexia.

    “Generalized hypotonia and areflexia were present.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Age of walking was slightly delayed in patients 8, 9, and 10 but severely retarded in patients 11 and 12, with patient 11 still not able to walk unassisted at age 9 years.

    “Age of walking was slightly delayed in patients 8, 9, and 10 but was severely retarded in patients 11 and 12, with patient 11 still not being able to walk unassisted at the age of 9 years.”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data

    Sibling 1 had persisting feeding difficulties requiring insertion of a percutaneous endoscopic gastrostomy tube (PEG).

    “Persisting feeding difficulties required insertion of a percutaneous endoscopic gastrostomy tube (PEG).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. The quote only partly states this claim.

    Findingown data

    Bronchoscopy of Sibling 1 showed signs of tracheomalacia.

    “Bronchoscopy showed signs of tracheomalacia.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductnone given

    Other CH3 substances exist in cells from which signals are detected.

    “In addition to protein aggregates, other ═CH 3 substances exist in cells from which signals are detected.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductnone given

    For Gene Ontology term analysis, only proteins identified as >2 or <0.5 fold-change with a significant P value were included.

    “For Gene Ontology (GO) term analysis, only those proteins identified as >2 or <0.5 fold-change with a significant P value were included.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Mutations in tenascin have been linked to sensorineural hearing loss, a clinical finding in our patients.

    “Interestingly, mutations in tenascin have been linked to sensorineural hearing loss [23], a clinical finding in our patients.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [23]

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

  • Probably not a substantial claim.

    Study design and conductown data

    Likely pathogenic variants were identified applying standard filtering criteria: minor allele frequency less than 1% and high to moderate variant effect predictor.

    “Likely pathogenic variants were identified applying standard filtering criteria: minor allele frequency less than 1% and high to moderate variant effect predictor.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim.

    Premisecitation

    E-CARS allows the imaging of small objects, which are not detected in F-CARS.

    “Since it is known that E-CARS allows the imaging of small objects, which are not detected in F-CARS [31], these positions may indicate areas with very small objects present and may potentially represent the accumulation of proteins.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [31]

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

  • It may state two separate claims as one.

    Interpretationcitation

    This molecular observation is in keeping with the described role of the WASHC4 protein in the proper function of the endoplasmic/sarcoplasmic reticulum, which might affect protein processing and clearance.

    “In addition, this molecular observation is in keeping with the described role of the WASHC4 protein in the proper function of the endoplasmic/sarcoplasmic reticulum [20] which might affect protein processing and clearance.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [20]

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

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductown data

    A total of 290 spectra were used for the spectra presented in the Results section: A: 59; B: 58; Ref: 38; Controls: 135.

    “A total of 290 spectra were used for the spectra presented in the Results section: A: 59; B: 58; Ref: 38; Controls: 135.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductown data

    The proteomic profiling data have been deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD024222.

    “The proteomic profiling data have been deposited at the ProteomeXchange Consortium via the PRIDE [19] partner repository with the dataset identifier PXD024222.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [19]

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

  • Probably not a substantial claim.

    Study design and conductcitation

    Proteomic profiling on human skin fibroblasts was performed using a data-independent acquisition (DIA) approach with control and WASHC4-patient fibroblast samples processed and analyzed in parallel.

    “To elucidate cellular pathophysiological processes upon loss of functional WASHC4, proteomic profiling on human skin fibroblasts was performed utilizing a dataindependent acquisition (DIA) approach as described previously [15], whereby control and WASHC4-patient fibroblast samples were processed and analyzed in parallel.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [15]

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

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductown data

    Whole exome sequencing was performed with a mean target coverage of >80x.

    “Whole exome sequencing (WES) was performed by the Genomics Platform at the Broad Institute of MIT and Harvard (Cambridge, MA, USA).Libraries were created with an Illumina (San Diego, CA, USA) exome capture (38 Mb target) and sequenced with a mean target coverage of >80x.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    SHG signal indicates highly organized substances, for example, collagen or ordered protein aggregates.

    “An SHG signal indicates highly organized substances, for example, collagen [25] or ordered protein aggregates [26].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [25]

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

    • cites [26]

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

  • It may state two separate claims as one.

    Study design and conductreasoning

    Aggregations of certain proteins (e.g., FYCO1, p62, or BiP) may be visible by fluorescence staining but do not alter the total protein concentration enough to be visible in the CARS image.

    “Thus, aggregations of certain proteins, e.g.FYCO1, p62, or BiP, may be visible by fluorescence staining but do not alter the total protein concentration enough to be visible in the CARS image.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Interpretationown data

    Taken together, these findings highlight that skeletal muscles are affected, defined by profound protein dysregulations and impaired protein processing and clearance.

    “Taken together, these findings highlight that skeletal muscles also are affecteddefined by profound protein dysregulations and impaired protein processing and clearance.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Study design and conductown data

    A total of 638 fibers were analyzed for the patient, and 1457 fibers were analyzed from five control samples.

    “A total of 638 fibers were analyzed for the patient.As a reference, 1457 fibers were analyzed from five control samples.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    The protein encoded by WASHC4 is part of the Wiskott-Aldrich syndrome protein and SCAR homolog family, co-localizes with actin in cells, and promotes Arp2/3-dependent actin polymerization in vitro.

    “The protein encoded by WASHC4 is part of the Wiskott-Aldrich syndrome protein and SCAR homolog family, co-localizes with actin in cells, and promotes Arp2/3-dependent actin polymerization in vitro.”
    • Also stated in: § Introduction · plain
      “Human WASH proteins are part of the WASP family, co-localize with actin in cells, and promote Arp2/3-dependent actin polymerization in vitro [6].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [6]

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

  • Probably not a substantial claim. It may state two separate claims as one.

    Premisecitation

    The signals at 2868 and 2847 cm−1 result from CH2 vibrations and are mainly associated with lipids.

    “The signals at 2868 and 2847 cm À1 result from ═CH 2 vibrations and are mainly associated with lipids [29,30].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [29]

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

    • cites [30]

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

  • It may state two separate claims as one.

    Interpretationreasoning

    The observed subsarcolemmal increased signal at 2921 cm−1 indicates increased protein content at these positions, pointing towards protein aggregation.

    “Given that this signal is characteristic for proteins [27,28], this microscopic finding indicates an increased protein content at these positions, thus pointing towards protein aggregation.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductown data

    Differentiation between certain proteins using CARS was not performed in this study.

    “In addition, the differentiation between certain proteins using CARS has not been followed here.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Study design and conductnone given

    Parental informed consent including permission to publish data and photographs was obtained for both patients, and the ethics committee of University Medicine Essen granted ethical approval (19-9011-BO).

    “Parental informed consent including permission to publish data and photographs was obtained for both patients, and the ethics committee of University Medicine Essen granted ethical approval (19-9011-BO).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one. The quote only partly states this claim.

    Interpretationown data

    The clinical report of the patients presented in this study expands the phenotypical spectrum of WASHC4 patients by adding ptosis, muscle weakness, scoliosis, and contractures in combination with fiber-type disproportion and protein dysregulation as well as recurrent pulmonary infections, epilepsy, and various endocrinological features to the list of clinical features.

    “Along this line, the clinical report of the patients presented in this study expands the phenotypical spectrum of WASHC4 patients by adding ptosis (as seen in patients with neuromuscular junction defects), muscle weakness, scoliosis, and contractures in combination with fibertype disproportion (as seen, for example, in congenital myopathies) and protein dysregulation as well as recurrent pulmonary”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationown data

    The proteomic analysis confirmed key molecular players in vitro and highlighted, for the first time, the involvement of skeletal muscle in patients.

    “The proteomic analysis confirmed key molecular players in vitro and highlighted, for the first time, the involvement of skeletal muscle in patients.”
    Quote in the paperSubstantial claim
72 of 72

  • The paper relies on this without support.

    Collagen is a component of the ECM.

    “Since collagen is a component of the ECM, this points toward an increase in the ECM in the area around the muscle fibers.”
1 of 1

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Figure caption reverses the reported galectin-3 change

    Major

    The results report decreased galectin-3 in patient muscle, but the Figure 4 caption reports an increase.

  • Figure caption reverses the reported tenascin change

    Minor

    The results say tenascin was increased in patient muscle fibers, whereas the Figure 4 caption lists it as decreased.

  • Conflicting totals for distinct clinical features

    Minor

    The results say the patient comparison revealed 43 different features, whereas the discussion says 27 different features were described in total.

3 of 3

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Metadata

Authors
Gangfuß, A., Czech, A., Hentschel, A., Münchberg, U., Horvath, R., Töpf, A., O'Heir, E., Lochmüller, H., Stehling, F., Kiewert, C., Sickmann, A., Kuechler, A., Kaiser, F. J., Kölbel, H., Christiansen, J., Schara‐Schmidt, U., & Roos, A.
Affiliations
  • University of Duisburg-Essen (DE)
  • Leibniz Institute for Analytical Sciences - ISAS (DE)
  • Department of Clinical Neurosciences, John Van Geest Centre for Brain Repair, School of Clinical Medicine University of Cambridge Cambridge UK
  • Newcastle upon Tyne Hospitals NHS Foundation Trust (GB)
  • Center for Mendelian Genomics, Program in Medical and Population Genetics Broad Institute of MIT and Harvard Cambridge MA USA
  • University of Ottawa (CA)
  • Essen University Hospital (DE)
  • Department of Pediatric Neurology, Centre for Neuromuscular Disorders, Centre for Translational Neuro‐ and Behavioral Sciences University Duisburg‐Essen Essen Germany
Corresponding author
Gangfuß, Andrea
ORCID
Venue
The Journal of Pathology
Publisher
Wiley
Published
2021-11-18
Volume
256
Issue
1
Pages
93-107
ISSN
  • 0022-3417 (ISSN-L)
  • 1096-9896
DOI
10.1002/path.5812
OpenAlex
W3202170491
Cited by
10
References
32
Citations by year
2026: 2 · 2025: 2 · 2024: 1 · 2023: 1 · 2022: 4
FWCI
0.72
Type
journal article
Language
en
Open access
Licence
Locations
  • The Journal of Pathology (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Universitätsbibliographie, Universität Duisburg-Essen (submittedVersion)
  • Repositori digital de la UPF (Universitat Pompeu Fabra) (submittedVersion, cc-by-nc-nd)
  • Apollo (University of Cambridge) (submittedVersion, cc-by-nc-nd)
  • Apollo
Funders
  • Canada Foundation for Innovation (CFI‐JELF 38412)
  • Canadian Institutes of Health Research (FDN‐167281)
  • European Regional Development Fund
  • European Research Council (309548)
  • French Muscular Dystrophy Association (21466)
  • University of Kentucky (MR/N025431/1)
  • National Human Genome Research Institute (R01 HG009141, UM1 HG008900)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomics and Rare Diseases
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetics and Neurodevelopmental Disorders
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Muscle Physiology and Disorders
Keywords
homozygous variant, intellectual disability, developmental disorders, skeletal muscle involvement, short stature, Wiskott-Aldrich syndrome protein, protein clearance, zebrafish model, coherent anti-Stokes Raman scattering microscopy, siblings
MeSH terms
Exome Sequencing, Child, Developmental Disabilities, Humans, Intellectual Disability, Mutation, Pedigree, Phenotype, Muscle, Skeletal, Siblings, Proteomics

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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.

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