The evolving genetic landscape of neuromuscular fetal akinesias

Common genetic neuromuscular disorders that may present as fetal akinesia and that may be missed with massively parallel sequencing approaches

Some forms of fetal akinesia can result from gene defects that may evade detection by short-read sequencing approaches, these include SMA type 0,^22,29,30 and large repeat expansions in DMPK reviewed in Ho et al. ³¹ Given the ever-increasing reliance of diagnostics via massively parallel sequencing, targeted gene panels, exomes or even genomes, it is important to consider the potential for these genes to underly fetal akinesia and to consider whether these genes are included in the sequencing and/or analysis of the ordered test. SMN1 deletions are challenging to detect given the sequence homology with the SMN2 pseudogene and the issues this creates for read alignment and calling of variants. Newer algorithms now exist and can detect SMN1 variants from short-read sequencing data. ³⁰ DMPK expansions, when maternally inherited are particularly prone to further expansion and it is not uncommon for a minimally affected woman to have a severely affected child than can present as fetal akinesia.^4,32 It is also conceivable that some molecularly unsolved fetal akinesias may result from repeat expansions at other loci, that are yet to be identified, and researchers should ensure short tandem repeats (STRs) calling, e.g., via ExpansionHunter de novo, ³² is performed in unsolved cases.

Central nervous system (CNS): Intellectual disability and CNS involvement is found approximately in 25% of AMC cases reviewed by Dieterich et al. ²³ We would like to highlight a couple of examples that may serve as exemplars for overlapping CNS and PNS involvement.

Prenatal onset muscular dystrophies, GMPPB as an exemplar

As with myotonic dystrophy, it is also recognized that variants in typical muscular dystrophy genes can also result in a fetal akinesia/hypokinesia presentation including for example FKTN, FKRP and GMPPB. Variants in GMPPB, encoding a cytoplasmic protein GDP-mannose pyrophosphorylase B, which catalyzes the formation of GDP-mannose required for the O-mannosylation of alpha-dystroglycan (DG), result in a spectrum varying from severe CMD with structural brain abnormalities to limb-girdle muscular dystrophy (LGMD), recurrent rhabdomyolysis without skeletal muscle weakness, asymptomatic hyper-CKemia and congenital myasthenic syndrome due to altered glycosylation of the acetylcholine receptor subunits and other synaptic proteins.^33–35 Although, LGMD is the most common phenotype (60–70%) within this spectrum, prenatal presentation with abnormal intrauterine growth, decreased fetal movements, arthrogryposis, congenital clubfoot deformity, and scoliosis are recognized as part of early onset severe CMD presentations.^33,34 In a recent series from Italy, two of 13 patients with variants in GMPPB presented with arthrogryposis, microcephaly, intellectual disability; one with congenital cataract, and the other with cerebellar polymicrogyria. ³⁵

MAGEL2-associated arthrogryposis

Since 2013, truncating variants, inherited paternally, have been associated with Schaaf-Yang and Chitayat-Hall syndromes, both characterized by AMC.^36,37 Other variable features include intellectual disability, dysmorphic features, hypopituitarism, growth hormone deficiency, intestinal pseudoobstruction, and obesity. MAGEL2 is a key gene associated with Prader-Willi syndrome, a disorder of imprinting, and has been studied extensively; it encodes a ubiquitin ligase enhancer that is required for endosomal protein recycling and is highly expressed in the hypothalamus. ³⁷

Channelopathies

The voltage-gated sodium channel isoforms expressed in either the muscle or the brain generate and conduct action potentials. ³⁸ Dysfunction of these channels leads to either a reduction (loss-of-function) or an increase (gain-of-function) in tissue excitability. Among these channelopathies, recessive SCN4A variants cause fetal akinesia, congenital myopathy and congenital myasthenia, with clinical severity depending on the degree of channel dysfunction. ³⁹ Primarily brain-expressed sodium channel genes, SCN1A, SCN2A, SCN3A and SCN8A have been associated with epilepsy and neurodevelopmental disorders. ³⁸ Recently, patients with antenatal diagnosis of AMC carrying de novo SCN1A variants, who either died in utero or developed neonatal-onset drug-resistant epileptic seizures were identified.^39–41 This unique phenotype is further expanded by movement disorders and malformations of cortical development.^41–43 Delineation of the characteristics of gain-of-function SCN1A-related disease, based on an international cohort of 35 patients, revealed three distinct presentations depending on age at onset and presence of arthrogryposis and movement disorders. ⁴⁴

NALCN is a conserved cation channel related to voltage-gated sodium and calcium channels. Loss-of-function variants in NALCN cause an autosomal recessive condition characterized by infantile hypotonia, psychomotor retardation and characteristic facial features. ⁴⁵ On the other hand, autosomal dominant NALCN variants are identified as a cause of congenital DA with pursed facial expression suggesting a hypercontracted phenotype and designated as congenital contractures of the limbs and face with hypotonia and developmental delay (CLIFAHDD). ⁴⁶ Modeling of the later variants indicate that both gain-of-function and loss-of-function changes may underlie the condition. ⁴⁷

Peripheral nervous system (PNS): including motor neuron survival, specification, innervation and myelination

Neurogenic arthrogryposis can represent early-onset neuronopathies, reviewed recently,^48,49 and most frequently arise due to autosomal dominant (usually de novo) variants in BICD2, DYNC1H1 and TRPV4. These genes accounted for diagnoses in 21 of 505 families across two arthrogryposis cohorts.^22,28

ERGIC1 encodes a putative transmembrane protein of the Endoplasmic Reticulum-Golgi Intermediate Compartment (ERGIC), that functions as a tubulovesicular membrane cluster in protein sorting and trafficking. Homozygous missense variants and a homozygous deletion encompassing the promoter and first exon of ERGIC1 are identified as a cause of relatively mild non-syndromic arthrogryposis and neuropathic AMC.^27,50,51 The pathogenic role of ERGIC1 is further validated by mRNA studies revealing complete loss-of-function. ⁵¹

Kinesin motor protein KIF21A, tubulin isotypes, or transcription factors are involved in motor neuron specification or motor nerve development. Falb et al., in a cohort of 23 cases with fetal akinesia, identified biallelic loss-of-function KIF21A variants in five affected fetuses of two unrelated families, and introduced KIF21A as a new factor underlying severe neurogenic FADS with arthrogryposis of multiple joints, pulmonary hypoplasia and facial dysmorphisms. ⁵² This highlights the essential role of kinesins, in various cellular processes including cell-cycle dynamics and progression, ciliogenesis and cilia function, and organization of polar cells during organogenesis. This adds KIF21A, to the list of already known fetal akinesia and other disease-associated kinesin superfamily proteins such as KIF5C and KIF26B.^53,54

Defects in intramuscular innervation are known to cause AMC due to variants in ECEL1/DINE.^55,56 Natera-de Benito et al., identified variants in COL25A1 presenting with a recognizable phenotype characterized by AMC with or without an ocular congenital cranial dysinnervation disorder, belonging to this pathophysiological mechanism. ⁵⁷ COL25A1 encodes a transmembrane-type collagen crucial for intramuscular motor innervation. Proper interaction between muscle-derived collagen XXV and its motor neuron-derived receptors protein tyrosine phosphatases are required for the navigation of motor axons into developing skeletal muscle. ⁵⁸ The first phenotype described due to recessively inherited pathogenic variants in COL25A1 was ocular congenital cranial dysinnervation disorders (CCDDs) in four patients from two families.^59,60 CCDSs, within the spectrum of axonal guidance disorders, result in absent or aberrant innervation of extraocular and/or cranial musculature. Phenotypic spectrum of COL25A1-related disorders is now expanded with a novel contractual phenotype, variable severity of arthrogryposis, ocular CCDD, without CNS involvement and relatively stable/slow progression and respiratory involvement in some of the patients, due to pathogenic homozygous and compound heterozygous variants. ⁵⁷ Collagen XXV is discussed to have a role in regulating the appropriate innervation not only of extraocular muscles, but also of bulbar, axial, and limb muscles in humans.

Defects in proteins critical to formation and function of the nodes of Ranvier can result in inherited nodopathies that present as fetal akinesia. CNTNAP1 (OMIM 602346) encodes CASPR an essential component of the nodes of Ranvier that are critical to saltatory conduction of action potentials along myelinated axons. Biallelic loss-of-function variants in CNTNAP1 result in fetal akinesia and non-syndromic AMC with reduced motor NCVs and structural defects in myelinated axons of peripheral nerves.^61,62 CNS involvement in the form of cerebral and cerebellar atrophy is also reported. ⁶³ In 2016, the Melki group first described loss-of-function variants in GLDN (OMIM 608603) encoding gliomedin, another key protein of the nodes of Ranvier, as a cause of lethal arthrogryposis (LCCS 11). ⁶⁴ A cell-based assay that examines cell-surface localization of over-expressed tagged wild type and mutant gliomedin is a useful assay for accessing variants of uncertain significance, especially substitutions.^28,64,65 GLDN-related phenotypes also include non-lethal presentations, with survival of patients into infancy, childhood and adolescence with intensive care and chronic respiratory and nutritional support. ⁶⁶

Loss-of-function variants in genes encoding signaling proteins that are critical to myelination of the peripheral nervous system underlie rare forms of AMC, including LGI4, ⁶⁷ ADCY6, ⁶¹ and ADGRG6 (formerly known as GPR126).^68,69 Intrafamilial variability is also described in LGI4-related arthrogryposis. ⁷⁰ Examination of myelination of intramuscular nerve either by immunohistochemistry or electron microscopy can identify defective myelination of the PNS and may aid diagnosis. ⁶⁸

Biallelic loss-of-function variants in the dystonin gene (DST) were recently identified in a patient with arthrogryposis, in whom examination of sciatic nerve showed severe hypomyelination. ⁷¹ Recessive variants in the neuronal isoform of DST have previously been associated with hereditary sensory and autonomic neuropathy (HSN6, MIM: 614653) with onset from the fetal to adult period. Jacob et al. described 17 arthrogryposis patients from 12 families with eight homozygous variants within the largest exon of DST encoding isoform DST-b. These patients presented with arthrogryposis, hypotonia and cardiac involvement, muscle biopsies were myopathic and it was found that DST-b is most abundant in striated muscle. ⁷²

Lukacs et al. identified bi-allelic hypomorphic variants in NMNAT2 in stillborn siblings with FADS, amyoplasia and hydrops. ⁷³ Nicotinamide mononucleotide adenylyltransferases are required for synthesis of NAD ⁺ and are critical for cellular metabolism. Studies in model organisms show NMNAT2 is critical for survival of peripheral nerve axons. ⁶⁵ The phenotypic spectrum associated with biallelic NMNAT2 variants extends to childhood-onset polyneuropathy with neurogenic pain. ⁷³

Mechanotransduction

The first step in a biochemical cascade that translates mechanical inputs into cellular responses, ‘mechanotransduction’, is through the PIEZO2 cation channel.^74–76 Gain-of-function variants in PIEZO2 are known to cause autosomal dominant distal arthrogryposis syndromes; Marden-Walker syndrome (OMIM: 248700), DA type 3 (OMIM: 114300) and type 5 (OMIM: 108145). ⁷⁷ The so called ‘PIEZO2 deficiency’ due to biallelic loss-of-function variants in PIEZO2 are identified as a cause of arthrogryposis syndrome with hypotonia, feeding difficulties, neonatal respiratory insufficiency, delayed motor development, facial dysmorphic features, impaired touch and proprioception, sensory ataxia, absence of deep tendon reflexes, progressive scoliosis, and abnormal skeletal phenotypes.^78–81 Electrophysiological studies demonstrate decreased sensory nerve action potentials. In patients with congenital distal contractures, other than proprioceptive and sensory deficits, involvement of additional biological systems such as urinary dysfunction and diminished perception of urinary urgency, can serve as a clue to diagnose PIEZO2 deficiency syndrome. ⁷⁶

Neuromuscular junction

Genes encoding subunits of the post-synaptic acetylcholine receptor (AChR) and proteins critical to assembly and clustering of the AChRs and neuromuscular junction (NMJ) formation and development also underlie presentations ranging from FADS to milder Escobar-variant MPS and DA. Biallelic variants in CHRNA1, CHRNB1, ²⁸ CHRND, DOK7, MUSK, RAPSN and UNC50 ⁸² represent ∼11–17% of genetically diagnosed fetal akinesia and arthrogryposis cases.^22,28 These disorders are allelic with congenital myasthenic syndromes, recently reviewed by Ohno et al. ⁸³ Variants in the gene (CHRNG) encoding the fetal (gamma) subunit of the pentameric AChR cause lethal and non-lethal MPS and DA, ⁸⁴ and represents the most frequent causative NMJ gene in arthrogryposis cohorts (12/505 families).^22,28 A nonsense variant in the gene (SLC18A3) encoding the presynaptic vesicular acetylcholine transporter (VAChT) causes FADS. ⁸⁵

In addition to inherited forms of NMJ-associated fetal akinesia, maternal antibodies to fetal AChR isoform (fAChR) can also result in fetal akinesia and arthrogryposis. Fetal AChR antibodies result in a milder myopathic form called ‘fetal acetylcholine receptor inactivation syndrome, (FARIS)’. ⁸⁶ The spectrum of phenotypes, termed ‘fetal acetylcholine receptor antibody-related disorders, (FARAD)’, resulting from antibodies to fAChR, diagnostic and treatment strategies were recently reviewed by Allen et al. ⁸⁷ In this multicenter cohort including 46 patients, contractures and AMC were identified in 31/46 (67.4%). Of interest, diagnosis of myasthenia gravis was not established in 50% of the mothers, and offspring death was due to severe arthrogryposis (7/46, 15.2%) in the antenatal period, or respiratory failure (4/46, 8.7%) during early infancy. Although there are knowledge gaps owing to rarity and under recognition of these conditions, AMC, transient myasthenia gravis of the newborn, and FARIS seem to represent a continuum, and recognition of FARAD is critically important to implement intensive immunomodulatory treatment during pregnancy in myasthenia gravis-affected mothers, and to consider oral salbutamol treatment option for symptomatic offspring survivors.

Skeletal muscle – excitation-contraction coupling

Several key proteins are critical for relaying the signal received at the neuromuscular junction into Ca²⁺ release from the sarcoplasmic reticulum, with sarcoplasmic Ca²⁺ being the key regulator of skeletal muscle contraction. These included the voltage-gated sodium channel within the sarcolemma and T-tubules, encoded by the gene SCN4A, the voltage sensing ryanodine receptor (encoded by RYR1) within the T-tubules and the Ca²⁺ release channel (dihydropyridine receptor, DHPR) within the SR (encoded by CACNA1S). Among these channelopathies, recessive SCN4A variants cause fetal akinesia, congenital myopathy and congenital myasthenia, with clinical severity dependent on the degree of channel dysfunction. ⁸⁸ Biallelic variants in RYR1, frequently loss-of-function variants, are also associated with fetal akinesia, ⁸⁹ whilst dominantly inherited variants are associated with myopathy and susceptibility to malignant hyperthermia. Healthy parents that are carriers of RYR1 variants that cause fetal akinesia may also be at risk of malignant hyperthermia, and this represents an important aspect of genetic counseling.^24,89,90 Dominantly- and recessively inherited CACNA1S variants cause congenital myopathy, with fetal akinesia representing severe manifestation of CACNA1S-myopathy.^28,91,92

STAC3 is another key protein involved in excitation-contraction coupling and is thought to play a critical role in the coupling of RYR1 and the DHPR at the muscle triad. A biallelic c.851G > C, p.Trp284Ser variant in STAC3 was first identified to underly Native American myopathy. ⁹³ This and other STAC3 variants have since been identified in other forms of myopathy and in fetal akinesia.^28,94 A founder effect (attributed to a carrier rate of one in 56 of the p.Trp284Ser variant) has recently been described in southern Africa, with 38% of cases presenting with AMC. ⁹⁴ Biallelic, typically loss-of-function variants, in any one of these four proteins can result in a fetal akinesia presentation.^{24,28,90–94} Putative splice-altering variants can be investigated by RNA studies or minigene assays, ²⁸ however variants of unknown significance, in particular missense variants remain a challenge. Assays to investigate functional changes associated with variants in these genes, include electrophysiology and Ca²⁺ assays, however these are time-consuming and require a laboratory with these expertise.^88,89

Skeletal muscle – the sarcomere

Defects within the sarcomere can impair muscle contraction in utero and result in fetal akinesia and arthrogryposis, these include components of the thin and thick filaments and represent a substantial proportion of diagnoses (77/505 families), with autosomal-recessive titinopathy being the single-largest contributor (22/77 diagnoses).^22,28

It is increasingly recognized that biallelic truncating variants in TTN (TTNtv) (Figure 2) can present as fetal akinesia, AMC and recurrent miscarriage.^95–99 Due to extensive alternative splicing of titin within skeletal and cardiac muscle, variants are often annotated in the inferred complete TTN meta-transcript, a theoretical isoform that includes all putative TTN exons. Many of causative variants underlying prenatal-onset disease occur in or alter exons (i.e., extended splice-variants) that are present only in the meta-transcript of TTN, i.e., they are not present in the canonical transcript.^96,97,99 This includes a recurrent c.39974-11T > G splice variant.^96,97,99 Contractures are most evident in upper extremities with elbows being most severely affected. The ‘meta-transcript only’ titinopathy has a recognizable contractural pattern characterized by ulnar abduction and extension of the wrist, persistent myopathy, and striking fibrofatty involvement of hamstrings and calves with relative sparing of the femoral adductors and anterior segment of thighs on muscle imaging. ¹⁰⁰ RNA studies have shown that these meta-transcript exons are more highly expressed in fetal skeletal muscle than adult skeletal muscle.^97,101 In cases with a TTNtv in an exon expressed in cardiac TTN, cardiac involvement can also occur and may also manifest in the carrier parent and siblings.^99–101

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Figure 2.

A 12-month-old infant with arthrogryposis at birth with a myopathic face, nevus glabellar and forehead nevus flammeus, esotropia, and typical posturing of the hands and feet (b, c) with a diagnosis of congenital titinopathy; an 11-month-old infant with KLHL40-related myopathy who presented with arthrogryposis multiplex congenita at birth (d), at the age of 3 months (e), right femur fracture at birth (f), and 11 months (g, h, i), respectively. Note myopathic face, mild ptosis, facial weakness with tented upper lip, clenched hands, external rotation and hyperabduction of the hip with lower extremity contractures (d, e), pectus excavatum deformity, hypotonia on traction and vertical suspension maneuvers, weakness (g, h, i), and muscle biopsy Gomori trichrome staining compatible with nemaline myopathy (j); a 11-year-old case with arthrogryposis multiplex congenita at birth with hyperextension of the neck (k), clenched hand (l), pes equinovarus deformity (m), multiple fractures at birth (n) with axial hypotonic weakness (o, p) and scoliosis, hand posturing and inverted nipple by the age of 11 years (r, s) with axonal neuropathy and neurogenic arthrogryposis.

Causative variants in several genes are associated with nemaline myopathy. ¹⁰² Autosomal recessive variants in NEB, the gene encoding nebulin - the giant sarcomeric protein of the thin filament, were initially associated with nemaline myopathy (NEM2), ¹⁰³ however many cases present with in utero onset of muscle weakness resulting in phenotypes of FADS, AMC, and lethal MPS, and/or cystic hygroma and cleft palate.^24,104–107 Of note, variants in KLHL40 (kelch-like family member 40) representing the severe and often lethal end of the nemaline myopathy spectrum (Figure 2) characterized by autosomal recessive FADS were first identified by our group. ¹⁰⁸ A recent systematic review, including 65 patients with KLHL40 variants showed that, antenatal presentation (76%), fetal akinesia or hypokinesia (58%), contractures at birth (76%), and fractures (35%) dominated the clinical picture. Of the 65 patients described, 60% (n = 39) died during the first 4 years of life (intrauterine: n = 12; first year of life: n = 22; before 4 years of age: n = 5). ¹⁰⁹

Variants in genes encoding fetally expressed isoforms of sarcomeric proteins can result in fetal akinesia and arthrogryposis, including those encoding embryonic or perinatal myosin heavy chains, MYH3 and MYH8 respectively. Dominantly inherited or de novo missense variants in MYH3 are well-established to underlie DA, in particular Freeman-Hall Syndrome and Sheldon-Hall Syndrome. ¹¹⁰ More recently, dominant or recessive MYH3 disease has been identified to cause a phenotype including contractures, pterygia and skeletal deformities, and to underlie spondylocarpotarsal synostosis syndrome characterized by intervertebral fusions and fusion of the carpal and tarsal bones.^111–113 Biallelic missense variants in MYH3, both shown to alter splicing and predicted to result in loss of MYH3 expression, and homozygous loss-of-function variants were also identified in cases with a lethal form of spondylocarpotarsal synostosis syndrome. ¹¹⁴

ACTC1 encodes cardiac alpha-actin of the sarcomere in the heart, this gene is also fetally expressed in skeletal muscle. ¹¹⁵ Variants in ACTC1 are typically associated with cardiomyopathies, ¹¹⁶ however in 2023 Chong et al. identified a cohort of patients with DA and congenital heart defects arising from dominantly-inherited or de novo missense variants in ACTC1. ¹¹⁷ Original clinical diagnoses included MPS, Sheldon-Hall or Freeman-Sheldon syndromes. All identified substitutions in ACTC1 in Chong et al. have previously been observed in ACTA1, encoding skeletal muscle alpha-actin, a well-known congenital myopathy gene. De novo or somatic mosaic ACTA1 variants are another well-described cause of fetal akinesia and arthrogryposis, with 5% of all ACTA1 variants associated with ‘fetal abnormalities’ in a recent ACTA1 review. ¹¹⁸ Moreover, identification of a de novo pathogenic variant in ACTA1 and prenatal brain abnormalities including enlarged subarachnoid space and delayed cortical development in FADS suggests a role for skeletal muscle alpha-actin in the CNS. ¹¹⁹

Troponin I (TnI) regulates thin filament activation and muscle contraction, and two isoforms TnI-fast (TNNI2) and TnI-slow (TNNI1), are predominantly expressed in fast- and slow-twitch myofibers, respectively. Genes encoding these isoforms result in congenital myopathies and/or cardiomyopathies. ¹²⁰ Of interest, variants in TNNI2 (OMIM: 191043) are among the known causes of DA, whereas TNNI1 was recently implicated in a single family with dominant proximal arthrogryposis with the caveat that pathogenicity of the reported TnI variant (p.K175*) was not further investigated. ¹²¹ Donkervoort et al., provided detailed characterization of patients with recessive loss-of-function and dominant gain-of-function variants in TNNI1, as the cause of hypo- and hypercontractile muscle phenotypes, respectively. ¹²² Functional and contractility studies support that increased Ca²⁺ sensitivity of force resulting in continuous hypercontractility of the muscle fibers is underlying disease mechanism for presumed gain of function TNNI1 variants in a family with arthrogryposis and elevated serum creatine kinase levels. This variant-specific sarcomeric dysfunction also suggests different targeted therapeutic strategies for recessive and dominant forms of TNNI1-related disease.

Skeletal muscle – intermediate filament

Variants in FLNC, the gene encoding filamin C, which is an actin binding protein involved in sarcomere stability and maintenance, are usually associated with cardio- and/or skeletal myopathies, including myofibrillar myopathies and nemaline myopathy. A recurrent de novo FLNC variant has been more recently associated with DA with cardiomyopathy.^28,123

Other genetic causes of fetal akinesia

The first human phenotype attributable to loss-of-function of the myogenic factor MyoD is perinatally lethal FADS in a single consanguineous family with affected sibships. ¹²⁴ The MyoD family of transcriptional regulators is among the earliest described example of a developmental fate-controlling transcription factors and includes two primary-muscle lineage determining factors, MyoD and Myf-5.^124,125 To date, there is a handful of patients reported with pathogenic MYOD1 variants within a spectrum from severe lethal FADS to milder phenotypes. Main clinical features are craniofacial anomalies, respiratory insufficiency, diaphragmatic anomalies, hypotonia, arthrogryposis and renal anomalies. Bilateral vocal cord paralysis in an infant with hand and finger anomalies, inspiratory stridor, hypotonia and respiratory insufficiency is defined as a part of the clinical spectrum, ¹²⁶ and phenotype is further expanded with a 38-year-old pregnant woman who presented at 30 weeks’ gestation with respiratory distress and preeclampsia, who had a previous history of slowly-progressive dyspnea, generalized weakness and previous surgery for ptosis and prognathia. ¹²⁷

Glycogenosis type IV (GSD IV, OMIM: #232500) or Andersen's disease is a rare autosomal recessive metabolic disorder due to pathogenic variants in GBE1, leading to glycogen branching enzyme deficiency and an excessive deposition of structurally abnormal, amylopectin-like glycogen in affected tissues (liver, skeletal muscle, heart, nervous system, etc.). Historically, GSD type IV was classified into six clinical subtypes depending on the hepatic and/or neuromuscular presentation, age of onset and severity.^128,129 Severe neuromuscular presentations were categorized under fatal perinatal (hydrops fetalis, joint contractures, perinatal death) or congenital/neonatal subtype (severe hypotonia, muscle atrophy, need for mechanical ventilation, and death in the neonatal period or early infancy). Therefore, GBE1 is included in various diagnostic gene panels such as congenital myopathy, arthrogryposis and fetal akinesia. Recently, a phenotypic characterization score is proposed to evaluate neuromuscular, hepatic and cardiac involvement, thus reflecting the multidimensional clinical continuum. ¹³⁰ Lefevre et al., described characteristics of 10 cases from eight families with the severe neuromuscular form of GSD IV in comparison with the literature, including a total of 51 cases (perinatal: n = 16; congenital: n = 35). ¹³¹ Main antenatal features are defined as FADS or arthrogryposis/joint contractures often associated with muscle atrophy, decreased fetal movement, cystic hygroma, and/or hydrops fetalis, whereas presentation at birth was severe with hypotonia and/or muscle atrophy, need for mechanical ventilation, cardiomyopathy, retrognathia and arthrogryposis, resulting in stillbirth or death within the 1^st month of life. ¹³² The authors highlight the importance of performing a pathological examination of both the fetus and placenta, including unexplained early miscarriages, and considering prenatal exome to facilitate diagnosis. ¹³¹ Of note, out of three cases with the neuromuscular forms of GSD IV in the absence of characteristic polyglucosan bodies on muscle biopsy, which highlights the importance of including GBE1 in the molecular diagnostic work-up even the absence of hallmark pathology. ¹³²

Tissue-specific extracellular matrix (ECM) dysregulation

Recently, Boschann et al., identified biallelic ADAMTS15 pathogenic variants in five affected individuals from four unrelated families with DA, characterized by congenital flexion contractures of the interphalangeal joints and hypoplastic or absent palmar creases. ¹³³ Phenotype also includes knee, Achilles tendon, and toe contractures, spinal stiffness, scoliosis, and orthodontic abnormalities. The ADAMTS/L superfamily comprises several metalloproteases and related glycoproteins (ADAMTS-like proteins), which are implicated in microfibril assembly ECM regulating pathways. ¹³¹ Combined with the RNA data, the authors hypothesize that ADAMTS15 plays a critical role in perimuscular connective tissue and tendon development. ¹³⁴