Kyle S. Yau

Mutations in KLHL40 Are a Frequent Cause of Severe Autosomal-Recessive Nemaline Myopathy

Nemaline myopathy (NEM) is a common congenital myopathy. At the very severe end of the NEM clinical spectrum are genetically unresolved cases of autosomal-recessive fetal akinesia sequence. We studied a multinational cohort of 143 severe-NEM-affected families lacking genetic diagnosis. We performed whole-exome sequencing of six families and targeted gene sequencing of additional families. We identified 19 mutations in KLHL40 (kelch-like family member 40) in 28 apparently unrelated NEM kindreds of various ethnicities. Accounting for up to 28% of the tested individuals in the Japanese cohort, KLHL40 mutations were found to be the most common cause of this severe form of NEM. Clinical features of affected individuals were severe and distinctive and included fetal akinesia or hypokinesia and contractures, fractures, respiratory failure, and swallowing difficulties at birth. Molecular modeling suggested that the missense substitutions would destabilize the protein. Protein studies showed that KLHL40 is a striated-muscle-specific protein that is absent in KLHL40-associated NEM skeletal muscle. In zebrafish, klhl40a and klhl40b expression is largely confined to the myotome and skeletal muscle, and knockdown of these isoforms results in disruption of muscle structure and loss of movement. We identified KLHL40 mutations as a frequent cause of severe autosomal-recessive NEM and showed that it plays a key role in muscle development and function. Screening of KLHL40 should be a priority in individuals who are affected by autosomal-recessive NEM and who present with prenatal symptoms and/or contractures and in all Japanese individuals with severe NEM.

Dominant Mutations in KBTBD13, a Member of the BTB/Kelch Family, Cause Nemaline Myopathy with Cores

We identified a member of the BTB/Kelch protein family that is mutated in nemaline myopathy type 6 (NEM6), an autosomal-dominant neuromuscular disorder characterized by the presence of nemaline rods and core lesions in the skeletal myofibers. Analysis of affected families allowed narrowing of the candidate region on chromosome 15q22.31, and mutation screening led to the identification of a previously uncharacterized gene, KBTBD13, coding for a hypothetical protein and containing missense mutations that perfectly cosegregate with nemaline myopathy in the studied families. KBTBD13 contains a BTB/POZ domain and five Kelch repeats and is expressed primarily in skeletal and cardiac muscle. The identified disease-associated mutations, C.742C>A (p.Arg248Ser), c.1170G>C (p.Lys390Asn), and c.1222C>T (p.Arg408Cys), located in conserved domains of Kelch repeats, are predicted to disrupt the molecules beta-propeller blades. Previously identified BTB/POZ/Kelch-domain-containing proteins have been implicated in a broad variety of biological processes, including cytoskeleton modulation, regulation of gene transcription, ubiquitination, and myofibril assembly. The functional role of KBTBD13 in skeletal muscle and the pathogenesis of NEM6 are subjects for further studies.

Identification of KLHL41 Mutations Implicates BTB-Kelch-Mediated Ubiquitination as an Alternate Pathway to Myofibrillar Disruption in Nemaline Myopathy

Nemaline myopathy (NM) is a rare congenital muscle disorder primarily affecting skeletal muscles that results in neonatal death in severe cases as a result of associated respiratory insufficiency. NM is thought to be a disease of sarcomeric thin filaments as six of eight known genes whose mutation can cause NM encode components of that structure, however, recent discoveries of mutations in non-thin filament genes has called this model in question. We performed whole-exome sequencing and have identified recessive small deletions and missense changes in the Kelch-like family member 41 gene (KLHL41) in four individuals from unrelated NM families. Sanger sequencing of 116 unrelated individuals with NM identified compound heterozygous changes in KLHL41 in a fifth family. Mutations in KLHL41 showed a clear phenotype-genotype correlation: Frameshift mutations resulted in severe phenotypes with neonatal death, whereas missense changes resulted in impaired motor function with survival into late childhood and/or early adulthood. Functional studies in zebrafish showed that loss of Klhl41 results in highly diminished motor function and myofibrillar disorganization, with nemaline body formation, the pathological hallmark of NM. These studies expand the genetic heterogeneity of NM and implicate a critical role of BTB-Kelch family members in maintenance of sarcomeric integrity in NM.

SPEG interacts with myotubularin, and its deficiency causes centronuclear myopathy with dilated cardiomyopathy.

Centronuclear myopathies (CNMs) are characterized by muscle weakness and increased numbers of central nuclei within myofibers. X-linked myotubular myopathy, the most common severe form of CNM, is caused by mutations in MTM1, encoding myotubularin (MTM1), a lipid phosphatase. To increase our understanding of MTM1 function, we conducted a yeast two-hybrid screen to identify MTM1-interacting proteins. Striated muscle preferentially expressed protein kinase (SPEG), the product of SPEG complex locus (SPEG), was identified as an MTM1-interacting protein, confirmed by immunoprecipitation and immunofluorescence studies. SPEG knockout has been previously associated with severe dilated cardiomyopathy in a mouse model. Using whole-exome sequencing, we identified three unrelated CNM-affected probands, including two with documented dilated cardiomyopathy, carrying homozygous or compound-heterozygous SPEG mutations. SPEG was markedly reduced or absent in two individuals whose muscle was available for immunofluorescence and immunoblot studies. Examination of muscle samples from Speg-knockout mice revealed an increased frequency of central nuclei, as seen in human subjects. SPEG localizes in a double line, flanking desmin over the Z lines, and is apparently in alignment with the terminal cisternae of the sarcoplasmic reticulum. Examination of human and murine MTM1-deficient muscles revealed similar abnormalities in staining patterns for both desmin and SPEG. Our results suggest that mutations in SPEG, encoding SPEG, cause a CNM phenotype as a result of its interaction with MTM1. SPEG is present in cardiac muscle, where it plays a critical role; therefore, individuals with SPEG mutations additionally present with dilated cardiomyopathy.

Whole exome sequencing in foetal akinesia expands the genotype-phenotype spectrum of GBE1 glycogen storage disease mutations

The clinically and genetically heterogenous foetal akinesias have low rates of genetic diagnosis. Exome sequencing of two siblings with phenotypic lethal multiple pterygium syndrome identified compound heterozygozity for a known splice site mutation (c.691+2T>C) and a novel missense mutation (c.956A>G; p.His319Arg) in glycogen branching enzyme 1 (GBE1). GBE1 mutations cause glycogen storage disease IV (GSD IV), including a severe foetal akinesia sub-phenotype. Re-investigating the muscle pathology identified storage material, consistent with GSD IV, which was confirmed biochemically. This study highlights the power of exome sequencing in genetically heterogeneous diseases and adds multiple pterygium syndrome to the phenotypic spectrum of GBE1 mutation.

Congenital Titinopathy: Comprehensive characterization and pathogenic insights: Congenital Titinopathy

Comprehensive clinical characterization of congenital titinopathy to facilitate diagnosis and management of this important emerging disorder.

G.P.18

Two problems for molecular diagnosis of neurogenetic disorders are high levels of genetic heterogeneity and involvement of large genes, e.g. titin. These problems result in many patients not being diagnosed by current Sanger-sequencing based diagnostics. To offset these problems we have developed a TargetSeq (Life Technologies) capture panel consisting of 335 disease genes with mutations detectable by next generation sequencing (NGS). The genes were 276 neurogenetic and 59 cardiomyopathy genes, targeted together because of clinical overlap. The 335 panel genes were captured from pools of 16 bar-coded patient DNA samples and sequenced 16 patients at a time using Ion Proton™ (Life Technologies) sequencing. Variant analysis was by either custom Annovar or Cartagenia (Cartagenia, Inc.) based pipelines. Testing the panel identified 88% of the known small-scale mutations in 28 positive controls. Known mutations deliberately chosen in regions not sequenced well by NGS were not detected. Using the FishingCNV package, we identified 90% of CNV controls, including 100% of CMT1A/HNPP duplication/deletion controls. Although one single exon CNV was detected, not all small CNVs were detected. We have screened >300 molecularly undiagnosed probands, identifying mutations in 59 disease genes, many of which were not previously analysed by the diagnostic laboratory due to Sanger sequencing costs. Using the targeted panel is not ethically different to traditional sequential Sanger sequencing diagnostics. The panel needs updating regularly with new disease gene discoveries. We now receive samples from all round Australasia for analysis of the 335-gene panel, which is cheaper than Sanger.

G.P.35

TTN encodes titin, the largest human protein. In striated muscle two titin molecules align “head to head” to span the full length of the sarcomere, providing a scaffold for sarcomere organisation, a sensing and signalling hub, and both passive and active modulation of muscle contraction. Dominant mutations in TTN are established causes of cardiomyopathy, tibial muscular dystrophy, and hereditary myopathy with early respiratory failure. More recently, several individuals with two truncating mutations have been described with increased internalised nuclei (CNM) with or without multi-minicores (MMC). Using next generation sequencing, we identified 14 individuals from 11 families with compound heterozygous or homozygous truncating TTN mutations. Presentation was in utero or during infancy in all cases. Weakness of truncal and respiratory muscles was often prominent, and early-onset scoliosis and respiratory failure were common complications. Three affected individuals had dilated cardiomyopathy (DCM) or left ventricular dysfunction, and one carrier parent developed DCM in later life. Two individuals had congenital aortic abnormalities (coarctation and stenosis). Many affected individuals had distinctive clinical features which are unusual for congenital myopathies, including congenital or early-onset hand and foot deformities, congenital scoliosis, spinal rigidity, cleft palate, distal hypermobility and short stature. The most common histological abnormality was a mixed CNM-MMC pattern. Novel histological patterns included typical congenital fibre-type disproportion (CFTD) and CNM-MMC with caps and nemaline rods. The presence of palatal clefts, facial dysmorphology, and cardiac malformations in several individuals with TTN mutations suggests that titin plays a role in foetal development. Mutations in TTN should be considered in any individual with predominant truncal and respiratory weakness, and CNM, MMC, CFTD, caps, rods, or any combination of these, on muscle biopsy.

Clinical Application of Circulating Tumor Cells and Circulating Tumor DNA in Uveal Melanoma

Purpose To evaluate the feasibility of using circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) for the management of uveal melanoma (UM). Patients and Methods Low-coverage whole-genome sequencing was used to determine somatic chromosomal copy number alterations (SCNAs) in primary UM tumors, ctDNA, and whole-genome amplified CTCs. CTCs were immunocaptured using an antimelanoma-associated chondroitin sulfate antibody conjugated to magnetic beads and immunostained for melanoma antigen recognised by T cells 1 (MART1)/glycoprotein 100 (gp100)/S100 calcium-binding protein β (S100β). ctDNA was quantified using droplet digital polymerase chain reaction assay for mutations in the GNAQ, GNA11, PLCβ4, and CYSLTR2 genes. Results SCNA analysis of CTCs and ctDNA isolated from a patient with metastatic UM showed good concordance with the enucleated primary tumor. In a cohort of 30 patients with primary UM, CTCs were detected in 58% of patients (one to 37 CTCs per 8 mL of blood), whereas only 26% of patients had detectable ctDNA (1.6 to 29 copies/mL). The presence of CTCs or ctDNA was not associated with tumor size or other prognostic markers. However, the frequent detection of CTCs in patients with early-stage UM supports a model in which CTCs can be used to derive tumor-specific SCNA relevant for prognosis. Monitoring of ctDNA after treatment of the primary tumor allowed detection of metastatic disease earlier than 18F-labeled fluorodeoxyglucose positron emission tomography in two patients. Conclusion The presence of CTCs in localized UM can be used to ascertain prognostic SCNA, whereas ctDNA can be used to monitor patients for early signs of metastatic disease. This study paves the way for the analysis of CTCs and ctDNA as a liquid biopsy that will assist with treatment decisions in patients with UM.

G.P.273

Mutations in the gene ( KLHL40 ), encoding Kelch-like family member 40, were recently identified in patients presenting with severe autosomal recessive nemaline myopathy (NEM8). Using exome sequencing or targeted re-sequencing of 276 known neuromuscular disease genes through a TargetSeq (Life Technologies) capture panel, we have identified two additional cases with KLHL40 mutations. Both were from consanguineous kindreds. Case 1 was diagnosed with severe SMA and congenital fractures. We identified a homozygous nonsense mutation in KLHL40 (c.46C>T, p. Gln16*). Muscle biopsy identified variation in fibre size and miliary nemaline bodies by electron microscopy seen as very fine scattered granules upon Gomori trichrome staining. Case 2 presented with severe nemaline myopathy, the biopsy showing numerous tiny nemaline bodies. We identified a homozygous missense mutation in KLHL40 (c.931C>A, p. Arg311Ser). Neither of the variants was present in 1000genomes or EVS databases. These data further suggest that muscle biopsy may be useful to inform genetic testing and that KLHL40 should be considered in patients presenting with severe miliary nemaline myopathy. Nemaline myopathy should also be considered in the differential diagnosis of severe SMA, especially in patients presenting with congenital fractures and contractures. The normal biological function of KLHL40 is largely unknown and therefore the basis of the severe muscle disease resulting from mutations of KLHL40 is also unknown. In investigations of control and diseased mouse and patient muscle, we show that KLHL40 is more abundant in the mouse soleus and diaphragm (muscles that comprise oxidative type I and IIA myofibres) than in the EDL, gastrocnemius and quadriceps muscles (type II glycolytic predominant). In addition we show that KLHL40 is increased in dystrophic mouse ( mdx ) and regenerating patient muscle (including DMD), suggesting that it may serve as a novel marker of skeletal muscle regeneration.