KLHL40 Mutation Associated with Severe Nemaline Myopathy, Fetal Akinesia, and Cleft Palate

Abstract

The congenital myopathies are a heterogeneous group of inherited neuromuscular disorders characterized by early-onset muscular weakness, hypotonia, and developmental delay. The congenital myopathies are further classified into centranuclear myopathies, nemaline myopathies, core myopathies, and congenital fiber-type disproportion based on major pathological features found in muscle biopsies. There is no evidence at present to show that cleft palate is associated with severe nemaline myopathies and fetal akinesia. The results of our study suggest that KLHL40 mutation associated with Cleft palate, nemaline myopathy and fetal akinesia.

Introduction

The congenital myopathies can have variable presentations such as hypotonia, delayed ambulation, and severe cases––fetal akinesia, polyhydramnios, and hydrops. There are several genes causing congenital myopathy. The most common are central core, nemaline, and congenital fiber-type disproportion. Nemaline myopathy is caused by mutations in ten genes Such as LMOD3, NEBULIN, TPM3, ACTA1, TNNT1 and KLHL40 etc. KLHL40 is also known as KBTBD5 (KELCH repeat and BTB/POZ domaincontaining protein 5) and the function of the gene is unknown.

The other symptoms may include contracture swallowing, feeding difficulties, facial weakness and respiratory failure. In this report, we describe cause of fetal presentation with akinesia, polyhydramnios and cleft palate.

The congenital myopathies refer to a clinically and genetically heterogeneous group of inherited skeletal muscle diseases associated with early infantile or childhood onset of motor weakness, developmental delay, and hypotonia, which have a static or slowly progressive course.[1] Pathologically, congenital myopathies have characteristic but not pathognomonic morphological features such as the presence of nuclear centralization, focal myofibrillar disorganization, and protein aggregation.[2] The three main categories of classical congenital myopathies are as follows: (1) centronuclear or myotubular pyopathies that are defined by the presence of internally located myonuclei, (2) core myopathies that have focidevoid of oxidative enzymes in myofibers, and (3) nemaline myopathies that are marked by the presence of electron-dense nemaline bodies or rods within myofibres.[4]

Methods and Results

Clinical evaluation

Mother came to genetic OPD with abnormal sonographic findings for genetic counseling in the antenatal period. The level-II ultrasonography showed polyhydramnios, decreased fetal movements, cleft palate, and skin edema suggesting of hydrops. Keeping a possibility of fetal akinesia deformation sequence, mutation analysis was performed by targeted exome sequencing to cover conditions such as congenital myopathy, Costello syndrome, and Neu–Laxova syndrome.

Genetic analysis and clinical exome sequencing

The parents of the patients gave written signed consent for blood sample collection. Genomic DNA was extracted by standard protocol (Qiagen kit). Clinical exome sequencing (Illumina platform, USA) was performed in patient’s DNA sample and sequencing data were analyzed for sequence variant and identified variant validation was carried out by Sanger sequencing method.

Variant description

A homozygous nonsense variant in exon 1 of the KLHL40 gene (chr3:42727712G>A: depth 53×) that results in a stop codon and premature truncation of the protein at codon 201 (p.Trp201Ter; ENST000003297777) was detected. The observed variation has previously been reported in patient with nemaline myopathy.[5] The Trp201Ter variant has not been reported in the 1000 genome database and has a minor allele frequency of 0.008% and 0.01% in the ExAC database. The in silico prediction of the variant was analysed by likelihood ratio test (LRT) and Mutation tester-2 software (Charite, Berlin).

OMIM phenotype

Nemaline myopathy-8 (OMIM#615348) is caused by homozygous of compound heterozygous mutation in the KLHL40 gene (OMIM*615340). This is a severe autosomal recessive muscle disorder characterized by fetal akinesia or hypokinesia, followed by contractures, fractures, respiratory failure, and swallowing difficulties apparent at birth. Most patients die in infancy. The skeletal muscle biopsy shows numerous small nemaline bodies, frequently with virtually no normal myofibrils.

Discussion

There were two cases with affected fetuses described earlier had presented with severely reduced fetal movement, contractures and also associated with cleft palate. Severe nemaline myopathy has been reported with mutation in LMOD3 and nebulin gene. Mutation in LMOD3 gene (OMIM 616165) was reported family history of cleft lip palate with hypertelorism, hypospadias, and developmental delay. There was also MID1 mutation running in the family.[6] In another case report, a healthy family of first cousin couple had two unaffected children but three boys had died. One died neonatally and two died at the age of 6–8 months. The fourth boy diagnosed with nemaline myopathy had rocker bottom feet, cleft palate, undescended testes, and a weak voice. The patient and mother were homozygous for a deletion in exon 184 in nebulin gene that was detected with biopsy.[7] Mutation in KLHL40 gene identified by whole exome sequencing, homozygous, or compound heterozygous mutation in six nemaline myopathy families but without cleft palate.[5] The different genes implicated in causing nemaline myopathy are listed in Table 1. In our case, we have identified severe myopathy with cleft palate and identified KLHL40 mutation by targeted exome sequencing. An interactome for proteins involved with cleft lip/palate is given in Figure 1.

Table 1.

Associated genes and functions in nemaline myopathies

Gene	Chromosome location	Exons	Function
ACTA1	1q42.13	7	ADP binding, ATP binding, myosin binding, structural constituent of cytoskeleton
TPM2	9p13.3	11	Actin binding, protein heterodimerization activity, structural constituent of cytoskeleton
TPM3	9p13.3	11	Actin filament binding
NEB	2q23.3	183	Actin filament binding, protein binding, structural constituent of muscle
TNNT1	19q13.42	15	Calcium ion binding, calcium-dependent ATPase activity, tropomyosin binding
KLHL40	3p22.1	6	Myogenesis, skeletal-muscle maintenance
CFL2	14q13.1	6	Actin filament binding
KBTBD13	15q22.31	1	Transcription regulation, ion channel tetramarization and gating
LMOD3	3p14.1	4	Actin monomer binding, tropomyosin binding

ADP = adenosine tri-phosphate; ATP = adenosine di-phosphate

Figure 1.

Protein association networking in nemaline myopathies generated through STRING

Congenital myopathies are genetically a group of heterogeneous inherited disease of muscles that manifest clinically in early life, infancy, or characterized by distinctive abnormalities on muscle biopsy. The precise histologic diagnosis of congenital myopathies is sometime difficult to make because of overlapping features.[8] A number of genes have been identified by advanced sequencing methods associated with congenital myopathy and till date there is a large gap and additional genes are yet to be discovered.

Most cases of cleft lip palate are involved with combined effect of environmental and genetic factors during the first week of pregnancy. Cleft lip palate is associated with common syndromes such as CHARGE syndrome, Stickler syndrome, van der Woude syndrome, Pierre Robin sequence, intellectual disabilities, Kabuki syndrome, and DiGeorge syndrome.[9] Recent studies suggest that it could be associated with other anomalies. To the best of our knowledge, this is the first report of nemaline myopathy with KLHL40 mutation associated with cleft palate.

Financial support and sponsorship

This work was supported by the Ministry of Science and Technology, New Delhi, India.

Conflicts of interest

There are no conflicts of interest.