Nemaline myopathies (NMs) are a heterogeneous group of congenital myopathies caused by mutations in different genes associated with the structural and functional proteins of thin muscular filaments. The incidence of NM is estimated to be 1 in 30,000 births, with a carrier frequency of approximately 1/87 (Lehtokari et al., 2014). The spectrum of clinical phenotypes is very wide, ranging from severe neonatal forms to mild disorders in childhood (Sewry et al., 2019; Wang et al., 2020; Wen et al., 2020). NMs commonly show slow or no progression of symptoms, and only 16% of severe neonatal cases present with hypotonia, arthrogryposis, cardiomyopathy, and respiratory failure leading to early infant death (Cassandrini et al., 2017). Generalized weakness is part of the pediatric clinical phenotype of a wide spectrum of neuromuscular disorders and poses clinical diagnostic challenges. Whole-exome sequencing (WES) is now replacing more invasive procedures such as muscle and nerve biopsies and is helping clinicians reach a diagnosis faster (Mubaraki, 2021).

Nebulin is a giant protein of the skeletal sarcomeres that accounts for 4% of the total myofibrillar protein. Homozygous or compound heterozygous pathogenic variants in the nebulin gene (NEB) cause autosomal recessive nemaline myopathy type 2 (NEM2) (OMIM # 256030), which accounts for more than 50% of all NMs (Jin et al., 2014; Malfatti et al., 2014; Piga et al., 2016; Laitila et al., 2020; Laflamme et al., 2021). NEM2 appears in infancy with hypotonia and muscle weakness affecting the face, neck flexors, and proximal limb muscles. The most severe forms of NEM2 lead to death in the first 2 years of life (North and Ryan, 2002).

Nebulin and leiomodin 3, as striated skeletal muscle thin filaments, are stabilized by a kelch-like family member 40 protein detected in striated human fetal muscles, with the highest expression in adult skeletal muscles (Laitila and Wallgren-Pettersson, 2021). This protein maintains sarcomere integrity by modulating the regulatory circuits that control the expression of muscle-specific proteins (Cenik et al., 2015). Sarcomere dysfunction can cause secondary changes in the muscle’s ultrastructure, such as the nemaline rods, that further worsen muscle weaknesses (de Winter and Ottenheijm, 2017). Homozygous or compound heterozygous mutations in the kelch-like family member 40 gene (KLHL40) cause severe autosomal recessive nemaline myopathy type 8 (NEM8) (OMIM # 615348). Deficiencies in KLHL40 gene expression lead to marked reductions in nebulin and leiomodin 3 proteins, structural sarcomere irregularities, and a lethal loss of muscle function (Ravenscroft et al., 2013; Garg et al., 2014). KLHL40 variants are associated with severe forms of NMs that present with fetal akinesia or hypokinesia, contractures, fractures, respiratory failure, swallowing difficulties at birth, and early death in infancy (Kawase et al., 2015).

We report two rare cases of nemaline myopathy with different clinical severity spectrums caused by homozygous variants in NEB and KLHL40, in two consanguineous Arab families (ClinVar accessions SCV003762114--SCV003762117). Clinical and laboratory data were collected from their medical records and analyzed. An extended literature review of previously reported cases with NEB and KLHL40 gene variants was conducted. The research protocol was approved by the appropriate research ethics committees (approval numbers: E016-PI-6/21 and BDF/R&REC/2022-659), and written consent for the publication of both cases, including the use of photographs, was obtained from the parents.

NMs usually present as early-onset proximal muscle weakness, and many cases are suspected prenatally. Prenatal ultrasonographic signs of NMs correlate with a poor prognosis and an increased risk of mortality. Fetal akinesia, polyhydramnios, arthrogryposis, and foot deformities are associated with NEB and KLHL40 gene variants (Lawlor, et al., 2011; Feingold-Zadok et al., 2017; Yeung et al., 2020; Yi et al., 2021). Both patients showed reduced fetal movement, polyhydramnios, neonatal arthrogryposis, and talipes, underlining again the importance of ultrasound imaging for the early detection of NMs. The earlier a rare myopathy is diagnosed, the easier it will be to quickly intervene and manage the condition, identify the recurrence risk, and initiate prevention.

The NEB gene variant, at exon 80, NM_001271208.1:c.11969A>C (p.(Asp3990Ala)) identified in patient 1 was assessed as a variant of unknown significance that led to the substitution of a strongly conserved nucleotide and amino acid residue. Nebulin has a fundamental role in skeletal muscle and variants affecting highly conserved regions of NEB may be regarded as pathogenic. To our knowledge, this specific variant has not been reported before in the literature or the Genome Aggregation Database (gnomAD) (https://gnomad.broadinstitute.org/); it is also not listed in ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) or Varsome (https://varsome.com/variant/hg38/NEB). It may have a destabilizing effect on the protein, as predicted by Polyphen 2.0, leading to protein dysfunction. This variant is predicted as being deleterious by Combined Annotation Dependent Depletion (CADD), with a scaled score of 28.7 points (https://cadd.gs.washington.edu/snv). Considering the muscular phenotype of polyhydramnios, fetal hypokinesia, arthrogryposis, myopathic facies, neck flexor weakness, a nasal voice, dysarthria, dysphagia, and muscle weakness, we concluded that this homozygous missense NEB gene mutation was a likely candidate for the disease-causing variant seen in patient 1.

Classifying patients early based on their clinical phenotype is challenging, but disease progression during childhood enables the severity of their condition to be clarified (Lehtokari et al., 2014). At the age of 7 years and 10 months, patient 1 had good motor milestones and a slowly progressive, more distal lower limb myopathy corresponding to the classical NEM2 type. Lower-leg MRI showed findings already reported in this NEM2 type (Jungbluth et al., 2004; Gurgel-Giannetti et al., 2022); notably, the myopathy had completely spared the boy’s thigh muscles and predominantly led to atrophy of the left posterior and lateral calf muscles.

Nebulin is also present in the heart and brain, and cardiomyopathy is reported in about 13% of NEM2 patients (Amburgey et al., 2021). Electrocardiography showed bradycardia, but the cardiac ultrasound was reported to be normal for patient 1.

More than 200 NEB pathogenic variants are associated with a wide spectrum of variable phenotypes, from severe to mild forms, with associated learning and intellectual disabilities (variants in exons 27 and 143) (Jin et al., 2014; Lehtokari et al., 2014; Amburgey et al., 2021). No patients have been reported to have variants in exon 80 of the NEB gene. Our patient had a speech delay and mild learning difficulties as a toddler but experienced normal mental development and a slower myopathy progression.

Patient 1 was included in an intensive rehabilitation program and regularly followed up by a multidisciplinary medical team. He has benefited from physiotherapy and speech and behavioral therapies. His good evolution underlines the importance of both genetic diagnosis and early, sustained intervention.

His parents were confirmed carriers of the same NEB variant, and genetic counseling helped them to understand their recurrence risk and to make informed reproductive decisions. They opted for in vitro fertilization (IVF) and preimplantation genetic testing for the NEB gene variant (PGT-M), and their next pregnancy ended at term with two healthy male dizygotic twins. Multiple relatives later requested preconception carrier genetic testing, including for other rare genetic disorders reported in their extended consanguineous family, such as Niemann-Pick disease type 2C (Figure 1).

The KLHL40 gene variant, at exon 3, NM_152393.3:c.1405G>A (p.(Gly469Ser)), identified in our patient 2 led to the substitution of a conserved amino acid residue. This variant was reported before in ClinVar and Varsome as having uncertain significance for NEM8. It was assessed as likely pathological by ACMG, as probably damaging by the in silico prediction tool Polyphen 2.0 (https://genetics.bwh.harvard.edu/pph2/) with a score of 0.996 points (sensitivity: 0.55; specificity: 0.98), as disease-causing by Align GVGD (http://agvgd.hci.utah.edu/) and Mutation Taster (www.mutationtaster.org), and has a deleterious scaled CADD score of 31 points. The same variant was reported as a compound heterozygote with a KLHL40 gene variant c. 1498C>T in two NEM8 patients from Brazil, 17 months and 5 years of age, respectively, with a severe neonatal phenotype but later motor improvement (patients 29 and 30 in the study by Gurgel-Giannetti et al., 2022).

A similar KLHL40 variant appearing at the same position in exon 3, in a conserved functional domain, but with a different substitution, c.1405G>T, p(Gly469Cys), was found in a Japanese family with NEM8 (family 15 in the study by Ravenscroft et al. (2013)). Molecular modeling assessed this substitution as one that most likely destabilized the hydrophobic core of the kelch domain and reduced protein stability. It is highly probable that the KLHL40 gene variant, c.1405G>A, identified in our patient had the same impact and, to the best of our knowledge, this is the first time it has been reported in a family of Arab origin. The KLHL40 gene variant, c.1582G>A, is the common variant reported in Japanese, Kurdish, and Turkish patients with NEM8 (Seferian et al., 2016) but the variant, c.1516A>C, has only been reported in East Asian populations (Yeung et al., 2020). Considering the rarity of these cases, further studies might be needed to identify KLHL40 gene variants linked to NEM8 in the Arab population.

NEM8 is characterized by severe and distinctive fetal and neonatal features, and by an early death in the first months of life (Ravenscroft et al., 2013), with rare cases surviving into adolescence (Seferian et al., 2016). As previously reported (Kawase et al., 2015; Yi et al., 2021), our patient 2, also had fetal hypokinesia and polyhydramnios, a severe phenotype at birth with respiratory failure, long bone fractures, arthrogryposis, and hypotonia. Despite the early interventions, she passed away at the age of 10 weeks. The severity of patient 2’s clinical phenotype in association with the abnormal myofibrils and rod bodies identified by the muscular biopsy, supported the molecular findings and certified the diagnosis of a rare severe case of NEM8. When accepted by the family, a muscle biopsy can clarify the gene variant's impact on the structure and functionality of muscle fibers (Wang et al., 2020). The homozygous KLHL40 gene variant, c.1405G>A, may explain the intrauterine fetal death and the other neonatal fetal death previously recorded in this family.

A heterozygous missense variant was identified in exon 31 of the CREBBP gene, NM_004380.2:c.5600G>A (p.(Arg1867Gln)), in patient 1. This variant is classified as pathogenic by the American College of Medical Genetics and Genomics (ACMG), with a score of 17 points (https://varsome.com/variant/hg38/rs1131691326) and has a deleterious scaled CADD score of 28.9 points. Variants in exons 30 and 31 of the CREBBP gene are associated with Menke-Hennekam syndrome 1 (https://www.omim.org/entry/618332) (Menke et al., 2016; Menke et al., 2018). Two patients with craniofacial dysmorphism, intellectual disability, and the same CREBBP variant c.5600G>A, were previously reported, including a six-year-old girl [patient 8 in the study by Menke et al. (2016)] and a 57-year-old man [patient C16 in the study by Menke et al. (2018)]. Both patients had other genomic changes (del6p12.3 and dup9q34.3, respectively) with unclear influences on their phenotype. Some of their facial dysmorphic features were assessed as possibly associated with this CREBBP gene variant, including a depressed nasal bridge, a short nose, anteverted nares, a short columella, a long philtrum, and decreased elbow extension, which were present in our patient but were also reported previously in the NEM2 phenotype. Patient 1 had a speech delay but not a developmental delay. Segregation analysis studies were conducted on the parents and other relatives (their siblings and cousins). The patient’s father has the same CREBBP variant, but none of the signs reported in the literature; therefore, it remains uncertain whether this is a case of non-penetrance and whether this variant has any influence on our patient’s phenotype.

Patient 2 was reported to also be homozygous for a missense variant of uncertain significance in the FAT4 gene, c.13178A>T, (p.(Asp4393Val)), which was not reported in gnomAD but has been predicted as damaging by the in silico tool, Mutation Taster. Pathogenic variants in the FAT4 gene are associated with autosomal recessive Van Maldergem syndrome type 2, and Hennekam lymphangiectasia-lymphedema syndrome type 2. Hypotonia, respiratory difficulties, and talipes are also features of Van Maldergem syndrome. A typically periventricular nodular heterotopia linked to this syndrome could not be ruled out; therefore, the impact of this variant on our patient’s phenotype, if any, is uncertain.

The professional care and guidance received led to an excellent collaboration with the families of both patients. Genetic counseling facilitated an understanding of complex genetic pathologies, clarifying the impact of consanguinity and the importance of prevention. The counseling helped both families to cope with this rare disease diagnosis and to make informed decisions going forward, regarding adequate management and future offspring. Getting another healthy pregnancy with the help of IVF and PGT-M for the NEB gene variant brought a lot of joy to the first family, and both families have become strong advocates of the importance of preconception genetic counseling in the context of consanguinity and a history of affected children.

Homozygous variants of the NEB2 gene, c.11969A>C, and the KLHL40 gene, c.1405G>A, were correlated with the phenotypes of the two patients presented here. This study enriches our knowledge about the phenotypic spectrum of nemaline myopathy caused by variants in NEB and KLHL40 and highlights the importance of detailed prenatal, neonatal, and infancy assessments of muscular weakness associated with complex systemic features. Variants of uncertain significance in genes associated with nemaline myopathies may also contribute to the phenotype. Early and multidisciplinary intervention can improve the outcome, especially in patients with mild forms of the disease. WES is essential for clarifying complex clinical phenotypes encountered in patients from consanguineous families. Targeted carrier screening of extended family members facilitates accurate genetic counseling and enables genetic prevention.