Baijayanta Maiti

Proteomic identification of FHL1 as the protein mutated in human reducing body myopathy

Reducing body myopathy (RBM) is a rare disorder causing progressive muscular weakness characterized by aggresome-like inclusions in the myofibrils. Identification of genes responsible for RBM by traditional genetic approaches has been impossible due to the frequently sporadic occurrence in affected patients and small family sizes. As an alternative approach to gene identification, we used laser microdissection of intracytoplasmic inclusions identified in patient muscle biopsies, followed by nanoflow liquid chromatography-tandem mass spectrometry and proteomic analysis. The most prominent component of the inclusions was the Xq26.3-encoded four and a half LIM domain 1 (FHL1) protein, expressed predominantly in skeletal but also in cardiac muscle. Mutational analysis identified 4 FHL1 mutations in 2 sporadic unrelated females and in 2 families with severely affected boys and less-affected mothers. Transfection of kidney COS-7 and skeletal muscle C2C12 cells with mutant FHL1 induced the formation of aggresome-like inclusions that incorporated both mutant and wild-type FHL1 and trapped other proteins in a dominant-negative manner. Thus, a novel laser microdissection/proteomics approach has helped identify both inherited and de novo mutations in FHL1, thereby defining a new X-linked protein aggregation disorder of muscle.

Clinical, histological and genetic characterization of reducing body myopathy caused by mutations in FHL1

We recently identified the X-chromosomal four and a half LIM domain gene FHL1 as the causative gene for reducing body myopathy, a disorder characterized by progressive weakness and intracytoplasmic aggregates in muscle that exert reducing activity on menadione nitro-blue-tetrazolium (NBT). The mutations detected in FHL1 affected highly conserved zinc coordinating residues within the second LIM domain and lead to the formation of aggregates when transfected into cells. Our aim was to define the clinical and morphological phenotype of this myopathy and to assess the mutational spectrum of FHL1 mutations in reducing body myopathy in a larger cohort of patients. Patients were ascertained via the detection of reducing bodies in muscle biopsy sections stained with menadione-NBT followed by clinical, histological, ultrastructural and molecular genetic analysis. A total of 11 patients from nine families were included in this study, including seven sporadic patients with early childhood onset disease and four familial cases with later onset. Weakness in all patients was progressive, sometimes rapidly so. Respiratory failure was common and scoliosis and spinal rigidity were significant in some of the patients. Analysis of muscle biopsies confirmed the presence of aggregates of FHL1 positive material in all biopsies. In two patients in whom sequential biopsies were available the aggregate load in muscle sections appeared to increase over time. Ultrastructural analysis revealed that cytoplasmic bodies were regularly seen in conjunction with the reducing bodies. The mutations detected were exclusive to the second LIM domain of FHL1 and were found in both sporadic as well as familial cases of reducing body myopathy. Six of the nine mutations affected the crucial zinc coordinating residue histidine 123. All mutations in this residue were de novo and were associated with a severe clinical course, in particular in one male patient (H123Q). Mutations in the zinc coordinating residue cysteine 153 were associated with a milder phenotype and were seen in the familial cases in which the boys were still more severely affected compared to their mothers. We expect the mild end of the spectrum to significantly expand in the future. On the severe end of the spectrum we define reducing body myopathy as a progressive disease with early, but not necessarily congenital onset, distinguishing this condition from the classic essentially non-progressive congenital myopathies.

A mutation in the SEPN1 selenocysteine redefinition element (SRE) reduces selenocysteine incorporation and leads to SEPN1‐related myopathy

Mutations in SEPN1 result in a spectrum of early‐onset muscle disorders referred to as SEPN1‐related myopathy. The SEPN1 gene encodes selenoprotein N (SelN), which contains the amino acid selenocysteine (Sec). Incorporation of Sec occurs due to redefinition of a UGA codon during translation. Efficient insertion requires a Sec insertion sequence (SECIS) in the 3′UTR and, for at least a subset of selenoprotein genes, a Sec redefinition element (SRE) located adjacent to the UGA codon. We report the effect of three novel and one previously reported point mutation in the SelN SRE element on Sec insertion efficiency. Notably, the previously reported mutation c.1397G>A (p.R466Q), which weakens the secondary structure of the SRE element, reduces Sec insertion efficiency and SelN RNA levels. Muscle from patients with this mutation have negligible levels of SelN protein. This data highlights the importance of the SRE element during SelN expression and illustrates a novel molecular mechanism by which point mutations may lead to SEPN1‐related myopathy. Hum Mutat 0, 1–6, 2008.

DMD exon 1 truncating point mutations: amelioration of phenotype by alternative translation initiation in exon 6

Mutations in the DMD gene result in two common phenotypes associated with progressive muscle weakness: the more severe Duchenne muscular dystrophy (DMD) and the milder Becker muscular dystrophy (BMD). We have previously identified a nonsense mutation (c.9G>A; p.Trp3X) within the first exon of the DMD gene, encoding the unique N‐terminus of the 427‐kDa muscle isoform of the dystrophin protein. Although this mutation would be expected to result in severe disease, the clinical phenotype is very mild BMD, with ambulation preserved into the seventh decade. We identify the molecular mechanism responsible for the amelioration of disease severity to be initiation of translation at two proximate AUG codons within exon 6. Analysis of large mutational data sets suggests that this may be a general mechanism of phenotypic rescue for point mutations within at least the first two exons of the DMD gene. Our results directly demonstrate, for the first time, the use of alternate translational initiation codons within the DMD gene, and suggest that dystrophin protein lacking amino acids encoded by the first five exons retains significant function. Hum Mutat 0:1–8, 2009.

Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice

Most mutations that truncate the reading frame of the DMD gene cause loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity has been shown to result from alternative translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. Here we demonstrate that this isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid inducible. We confirmed IRES activity by both peptide sequencing and ribosome profiling in muscle from individuals with minimal symptoms despite the presence of truncating mutations. We generated a truncated reading frame upstream of the IRES by exon skipping, which led to synthesis of a functional N-truncated isoform in both human subject–derived cell lines and in a new DMD mouse model, where expression of the truncated isoform protected muscle from contraction-induced injury and corrected muscle force to the same level as that observed in control mice. These results support a potential therapeutic approach for patients with mutations within the 5′ exons of DMD.Most mutations that truncate the reading frame of the DMD gene cause loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity can result from alternate translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. This novel isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid-inducible. IRES activity is confirmed in patient muscle by both peptide sequencing and ribosome profiling. Generation of a truncated reading frame upstream of the IRES by exon skipping leads to synthesis of a functional N-truncated isoform in both patient-derived cell lines and in a new DMD mouse model, where expression protects muscle from contraction-induced injury and corrects muscle force to the same level as control mice. These results support a novel therapeutic approach for patients with mutations within the 5’ exons of DMD.

The ZZ Domain of Dystrophin in DMD: Making Sense of Missense Mutations

Duchenne muscular dystrophy (DMD) is associated with the loss of dystrophin, which plays an important role in myofiber integrity via interactions with β‐dystroglycan and other members of the transmembrane dystrophin‐associated protein complex. The ZZ domain, a cysteine‐rich zinc‐finger domain near the dystrophin C‐terminus, is implicated in forming a stable interaction between dystrophin and β‐dystroglycan, but the mechanism of pathogenesis of ZZ missense mutations has remained unclear because not all such mutations have been shown to alter β‐dystroglycan binding in previous experimental systems. We engineered three ZZ mutations (p.Cys3313Phe, p.Asp3335His, and p.Cys3340Tyr) into a short construct similar to the Dp71 dystrophin isoform for in vitro and in vivo studies and delineated their effect on protein expression, folding properties, and binding partners. Our results demonstrate two distinct pathogenic mechanisms for ZZ missense mutations. The cysteine mutations result in diminished or absent subsarcolemmal expression because of protein instability, likely due to misfolding. In contrast, the aspartic acid mutation disrupts binding with β‐dystroglycan despite an almost normal expression at the membrane, confirming a role for the ZZ domain in β‐dystroglycan binding but surprisingly demonstrating that such binding is not required for subsarcolemmal localization of dystrophin, even in the absence of actin binding domains.

PET Imaging in Movement Disorders

Positron emission tomography (PET) has revealed key insights into the pathophysiology of movement disorders. This paper will focus on how PET investigations of pathophysiology are particularly relevant to Parkinson disease, a neurodegenerative condition usually starting later in life marked by a varying combination of motor and nonmotor deficits. Various molecular imaging modalities help to determine what changes in brain herald the onset of pathology; can these changes be used to identify presymptomatic individuals who may be appropriate for to-be-developed treatments that may forestall onset of symptoms or slow disease progression; can PET act as a biomarker of disease progression; can molecular imaging help enrich homogenous cohorts for clinical studies; and what other pathophysiologic mechanisms relate to nonmotor manifestations. PET methods include measurements of regional cerebral glucose metabolism and blood flow, selected receptors, specific neurotransmitter systems, postsynaptic signal transducers, and abnormal protein deposition. We will review each of these methodologies and how they are relevant to important clinical issues pertaining to Parkinson disease.

Corrigendum: Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice (Nature Medicine (2014))

Nat Med. 20, 992–1000 (2014); doi:10.1038/nm.3628; corrected 25 August 2014; corrected after print 13 March 2015 In the version of this article initially published, three participants of the study were not included as co-authors. Also, one of the individuals mentioned in the Acknowledgments section of the report was incorrectly included and thus has been removed at their request, and the name of another individual mentioned in the Acknowledgments was originally misspelled (“Fabbri” should have been “Fabris”).

Erratum: Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice: (Nature Medicine (2014) 20 (992-1000) DOI:10.1038/nm.3628)

In the version of this article initially published online, the third sentence of the Abstract read “Gene expression analysis identified higher expression of JAK-STAT signaling targets in 3-week-old relative to 18-month-old mice,” when it should have read “Gene expression analysis identified higher expression of JAK-STAT signaling targets in 18-month-old relative to 3-week-old mice.” The error has been corrected for the print, PDF and HTML versions of this article.

Atypical CSF findings in West Nile neuroinvasive disease: A diagnostic and therapeutic conundrum.

West Nile virus (WNV) infections are typically asymptomatic. Less than 1% present with meningitis, encephalitis, myelitis, or acute flaccid paralysis, collectively coined West Nile neuroinvasive disease (WNND).1 In 2012, the most severe outbreak since 2003, there were 5,387 cases reported, of which 2,734 (51%) were WNND, resulting in 243 deaths.2