Amir Jahic

A Hereditary Spastic Paraplegia Mouse Model Supports a Role of ZFYVE26/SPASTIZIN for the Endolysosomal System

Hereditary spastic paraplegias (HSPs) are characterized by progressive weakness and spasticity of the legs because of the degeneration of cortical motoneuron axons. SPG15 is a recessively inherited HSP variant caused by mutations in the ZFYVE26 gene and is additionally characterized by cerebellar ataxia, mental decline, and progressive thinning of the corpus callosum. ZFYVE26 encodes the FYVE domain-containing protein ZFYVE26/SPASTIZIN, which has been suggested to be associated with the newly discovered adaptor protein 5 (AP5) complex. We show that Zfyve26 is broadly expressed in neurons, associates with intracellular vesicles immunopositive for the early endosomal marker EEA1, and co-fractionates with a component of the AP5 complex. As the function of ZFYVE26 in neurons was largely unknown, we disrupted Zfyve26 in mice. Zfyve26 knockout mice do not show developmental defects but develop late-onset spastic paraplegia with cerebellar ataxia confirming that SPG15 is caused by ZFYVE26 deficiency. The morphological analysis reveals axon degeneration and progressive loss of both cortical motoneurons and Purkinje cells in the cerebellum. Importantly, neuron loss is preceded by accumulation of large intraneuronal deposits of membrane-surrounded material, which co-stains with the lysosomal marker Lamp1. A density gradient analysis of brain lysates shows an increase of Lamp1-positive membrane compartments with higher densities in Zfyve26 knockout mice. Increased levels of lysosomal enzymes in brains of aged knockout mice further support an alteration of the lysosomal compartment upon disruption of Zfyve26. We propose that SPG15 is caused by an endolysosomal membrane trafficking defect, which results in endolysosomal dysfunction. This appears to be particularly relevant in neurons with highly specialized neurites such as cortical motoneurons and Purkinje cells.

Novel Genetic, Clinical, and Pathomechanistic Insights into TFG‐Associated Hereditary Spastic Paraplegia

Hereditary spastic paraplegias (HSPs) are genetically and clinically heterogeneous axonopathies primarily affecting upper motor neurons and, in complex forms, additional neurons. Here, we report two families with distinct recessive mutations in TFG, previously suggested to cause HSP based on findings in a single small family with complex HSP. The first carried a homozygous c.317G>A (p.R106H) variant and presented with pure HSP. The second carried the same homozygous c.316C>T (p.R106C) variant previously reported and displayed a similarly complex phenotype including optic atrophy. Haplotyping and bisulfate sequencing revealed evidence for a c.316C>T founder allele, as well as for a c.316_317 mutation hotspot. Expression of mutant TFG proteins in cultured neurons revealed mitochondrial fragmentation, the extent of which correlated with clinical severity. Our findings confirm the causal nature of bi‐allelic TFG mutations for HSP, broaden the clinical and mutational spectra, and suggest mitochondrial impairment to represent a pathomechanistic link to other neurodegenerative conditions.

The spectrum of KIAA0196 variants, and characterization of a murine knockout: implications for the mutational mechanism in hereditary spastic paraplegia type SPG8

BackgroundThe hereditary spastic paraplegias (HSPs) are rare neurodegenerative gait disorders which are genetically highly heterogeneous. For each single form, eventual consideration of therapeutic strategies requires an understanding of the mechanism by which mutations confer pathogenicity. SPG8 is a dominantly inherited HSP, and associated with rather early onset and rapid progression. A total of nine mutations in KIAA0196, which encodes the WASH regulatory complex (SHRC) member strumpellin, have been reported in SPG8 patients so far. Based on biochemical and cell biological approaches, they have been suggested to act via loss of function-mediated haploinsufficiency.MethodsWe generated a deletion-based knockout allele for E430025E21Rik, i.e. the murine homologue of KIAA0196. The consequences on mRNA and protein levels were analyzed by qPCR and Western-blotting, respectively. Motor performance was evaluated by the foot-base angle paradigm. Axon outgrowth and relevant organelle compartments were investigated in primary neuron cultures and primary fibroblast cultures, respectively. A homemade multiplex ligation-dependent probe amplification assay enabling identification of large inactivating KIAA0196 deletion alleles was applied to DNA from 240 HSP index patients.ResultsHomozygous but not heterozygous mice showed early embryonic lethality. No transcripts from the knockout allele were detected, and the previously suggested compensation by the wild-type allele upon heterozygosity was disproven. mRNA expression of genes encoding other SHRC members was unaltered, while there was evidence for reduced SHRC abundance at protein level. We did, however, neither observe HSP-related in vivo and ex vivo phenotypes, nor alterations affecting endosomal, lysosomal, or autophagic compartments. KIAA0196 copy number screening excluded large inactivating deletion mutations in HSP patients. The consequences of monoallelic KIAA0196/E430025E21Rik activation thus differ from those observed for dominant HSP genes for which a loss-of-function mechanism is well established.ConclusionsOur data do not support the current view that heterozygous loss of strumpellin/SHRC function leads to haploinsufficiency and, in turn, to HSP. The lethality of homozygous knockout mice, i.e. the effect of complete loss of function, also argues against a dominant negative effect of mutant on wild-type strumpellin in patients. Toxic gain-of-function represents a potential alternative explanation. Confirmation of this therapeutically relevant hypothesis in vivo, however, will require availability of appropriate knockin models.

A novel strumpellin mutation and potential pitfalls in the molecular diagnosis of hereditary spastic paraplegia type SPG8

Hereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous, neurodegenerative movement disorder. A total of eight KIAA0196/strumpellin variants have thus far been associated with SPG8, a rare dominant HSP. We present a novel strumpellin alteration in a small family with clinically pure HSP. We corroborated its causality by comparing it to rare benign variants at several levels, and, along this line, also re-considered previous genetic reports on SPG8. These analyses identified significant challenges in the interpretation of strumpellin alterations, and suggested that at least two of the few families claimed to suffer from SPG8 may have been genetically misdiagnosed.

Brief Report: Novel Genetic, Clinical and Pathomechanistic Insights into TFG-Associated Hereditary Spastic Paraplegia.

Hereditary spastic paraplegias (HSPs) are genetically and clinically heterogeneous axonopathies primarily affecting upper motor neurons and, in complex forms, additional neurons. Here, we report two families with distinct recessive mutations in TFG, previously suggested to cause HSP based on findings in a single small family with complex HSP. The first carried a homozygous c.317G>A (p.R106H) variant and presented with pure HSP. The second carried the same homozygous c.316C>T (p.R106C) variant previously reported and displayed a similarly complex phenotype including optic atrophy. Haplotyping and bisulfate sequencing revealed evidence for a c.316C>T founder allele, as well as for a c.316_317 mutation hotspot. Expression of mutant TFG proteins in cultured neurons revealed mitochondrial fragmentation, the extent of which correlated with clinical severity. Our findings confirm the causal nature of bi‐allelic TFG mutations for HSP, broaden the clinical and mutational spectra, and suggest mitochondrial impairment to represent a pathomechanistic link to other neurodegenerative conditions.

A polymorphic Alu insertion that mediates distinct disease-associated deletions

Large deletions that are associated with insertions of Alu-derived sequence represent a rare, but potentially unique class of alterations. Whether they form by a one-step mechanism or by a primary insertion step followed by an independent secondary deletion step is not clear. We resolved two disease-associated SPAST deletions, which involve distinct exons by long range PCR. Alu-derived sequence was observed between the breakpoints in both cases. The intronic regions that represent the targets of potentially involved Alu retrotransposition events overlapped. Microsatellite- and SNP-based haplotyping indicated that both deletions originated on one and the same founder allele. Our data suggest that the deletions are best explained by two-step insertion–deletion scenarios for which a single Alu retrotransposition event represents the shared primary step. This Alu then mediated one of the deletions by non-homologous end joining and the other by non-allelic homologous recombination. Our findings thus strongly argue for temporal separation of insertion and deletion in Alu insertion-associated deletions. They also suggest that certain Alu integrations confer a general increase in local genomic instability, and that this explains why they are usually not detected during the probably short time that precedes the rearrangements they mediate.

MLPA-based evidence for sequence gain: pitfalls in confirmation and necessity for exclusion of false positives.

Multiplex ligation-dependent probe amplification (MLPA) has become a standard method for identifying copy number mutations in diagnostic and research settings. The occurrence of false-positive deletion findings and the underlying causes are well recognized, whereas false-positive duplication/amplification findings have not been appreciated so far. We here present three pertinent cases which were only identified on extended, nonstandard secondary analyses. We also offer and experimentally validate a potential explanation. Our findings imply that MLPA data indicating gain of genomic sequence require validation on an independent sample or by an independent method.

Maternal mosaicism for IDUA deletion clarifies recurrence risk in MPS I

Mucopolysaccharidosis I (MPS I) is a rare autosomal recessive multisystem lysosomal storage disorder. It is caused by biallelic loss-of-function variants in IDUA, encoding alpha-l iduronidase. Here, we describe an individual affected by MPS I due to a paternally inherited deletion of IDUA exons 1 and 2, c.(?_-88)_(299+1_300-1)del and a whole-gene deletion of IDUA (?_-88?)_(*136?)del secondary to maternal somatic mosaicism. We define a previously unreported mutational mechanism for this disorder.

A nonstop variant in REEP1 causes peripheral neuropathy by unmasking a 3′UTR-encoded, aggregation-inducing motif

Single‐nucleotide variants that abolish the stop codon (“nonstop” alterations) are a unique type of substitution in genomic DNA. Whether they confer instability of the mutant mRNA or result in expression of a C‐terminally extended protein depends on the absence or presence of a downstream in‐frame stop codon, respectively. Of the predicted protein extensions, only few have been functionally characterized. In a family with autosomal dominant Charcot‐Marie‐Tooth disease type 2, that is, an axonopathy affecting sensory neurons as well as lower motor neurons, we identified a heterozygous nonstop variant in REEP1. Mutations in this gene have classically been associated with the upper motor neuron disorder hereditary spastic paraplegia (HSP). We show that the C‐terminal extension resulting from the nonstop variant triggers self‐aggregation of REEP1 and of several reporters. Our findings support the recently proposed concept of 3′UTR‐encoded “cryptic amyloidogenic elements.” Together with a previous report on an aggregation‐prone REEP1 deletion variant in distal hereditary motor neuropathy, they also suggest that toxic gain of REEP1 function, rather than loss‐of‐function as relevant for HSP, specifically affects lower motor neurons. A search for similar correlations between genotype, phenotype, and effect of mutant protein may help to explain the wide clinical spectra also in other genetically determined disorders.

Development and validation of a TTR-specific copy number screening tool, and application to potentially relevant patient cohorts

TTR amyloidosis (ATTR) is a fatal condition caused by extracellular deposits of misfolded transthyretin. Patients often present with cardiac disease, but manifestations may also involve other organs including the peripheral nervous system. ATTR is considered familial when heterozygous mutations in the TTR gene are present (ATTRmutant or ATTRm), or acquired when no TTR aberrations are detected (ATTRwildtype or ATTRwt). We hypothesized that TTR copy number variants (CNVs), which would escape the standard diagnostic approaches, contribute to ATTR-related phenotypes, and developed a multiplex ligation-dependent probe amplification-based (MLPA-based), TTR-specific copy number screening tool. High inter-sample and intra-sample homogeneity of MLPA signals and the expected drop in signal intensity for restriction digest-based positive controls validated this tool. Subsequent application to 13 patients diagnosed with ATTRwt, and to 93 patients presenting with late onset and presumably inherited polyneuropathy did not identify TTR CNVs. We discuss insufficient sensitivity of the assay as well as non-existence and non-pathogenicity of TTR CNVs as potentially underlying our negative finding, but suggest size and composition of our cohorts as more likely explanations. Our CNV-screening tool will be made available to initiatives interested in screening additional and potentially more appropriate patient samples.