* 162230

NEUROFILAMENT PROTEIN, HEAVY POLYPEPTIDE; NEFH


Alternative titles; symbols

NFH


HGNC Approved Gene Symbol: NEFH

Cytogenetic location: 22q12.2     Genomic coordinates (GRCh38): 22:29,480,218-29,491,390 (from NCBI)


Gene-Phenotype Relationships
Location Phenotype Phenotype
MIM number
Inheritance Phenotype
mapping key
22q12.2 {?Amyotrophic lateral sclerosis, susceptibility to} 105400 AD, AR 3
Charcot-Marie-Tooth disease, axonal, type 2CC 616924 AD 3

TEXT

Description

Three neuron-specific intermediate filaments in mammals, NEFH, NEFM (162250), and NEFL (162280), are the most prominent cytoskeletal components in large myelinated axons (summary by Elder et al., 1998).


Cloning and Expression

In the course of cloning the region between 2 markers, D22S212 and D22S32, that flank the neurofibromin-2 gene (NF2; 607379), Rouleau et al. (1993) identified a gene with a neuronal pattern of expression and a transcript size identical to that of NEFH. Use of NEFH cDNA confirmed the identity.


Gene Structure

Lees et al. (1988) reported that the human heavy neurofilament subunit gene has 3 introns, 2 of which interrupt the protein-coding sequence at identical points to introns in the genes for the 2 smaller neurofilament proteins NFM and NFL.


Mapping

Mattei et al. (1988) used a rat cDNA probe coding for the C-terminal extension of the NEFH gene to assign, by in situ hybridization, the human NEFH gene to 22q12.1-q13.1. The possible implications of the fact that neurologic disorders such as meningioma map to this region were discussed.

There is compelling evidence that the NEFH locus is close to the NF2 locus. For example, Watson et al. (1993) found that the NEFH locus was hemizygous in a deletion that was observed in affected members of a family with NF2 and was estimated to be about 700 kb long. The NF2 locus has been positioned at 22q12.2.

Bucan et al. (1993) mapped the mouse Nefh gene, which they symbolized Nfh, to chromosome 11.


Gene Function

To investigate how disorganized neurofilaments might cause neurodegeneration, Collard et al. (1995) studied axonal transport of newly synthesized proteins in mice that overexpress the human NEFH gene. They observed dramatic defects of axonal transport, not only of neurofilament proteins but also of other proteins, including tubulin and actin. Ultrastructural analysis revealed a paucity of cytoskeletal elements, smooth endoplasmic reticulum, and especially mitochondria in the degenerating axons. Collard et al. (1995) therefore proposed that the neurofilament accumulations observed in these mice cause axonal degeneration by impeding the transport of components required for axonal maintenance, and that a similar mechanism may account for the pathogenesis of ALS in human patients.

Hirokawa and Takeda (1998) reviewed the contributions that gene targeting studies had made to understanding the role of each of the neurofilament component proteins in neurofilament formation and in determination of the axonal caliber.


Molecular Genetics

The tail of the neurofilament heavy subunit is composed of a repeating amino acid motif, usually X-lysine-serine-proline-Y-lysine (XKSPYK), where X is a single amino acid and Y is 1 to 3 amino acids. There are 2 common polymorphic variants of 44 and 45 repeats. The tail probably regulates axonal caliber, with interfilament spacing determined by phosphorylation of the KSP motifs. According to Al-Chalabi et al. (1999), the polymorphic variants had been mislabeled in the published literature as 44 and 43 repeat variants, respectively, and therefore were referred to by them simply as long (L) and short (S) alleles.

Susceptibility to Amyotrophic Lateral Sclerosis

In amyotrophic lateral sclerosis (ALS; 105400), a degenerative disease of motor neurons, depositions of neurofilaments occur in the perikarya and proximal exons. Two lines of evidence suggest that neurofilament accumulation may play a causal pathogenetic role. First, transgenic mice that overexpress neurofilament proteins show motor neuron degeneration (Collard et al., 1995). Second, deletions within the C-terminal KSP repeat region of the neurofilament heavy-subunit gene have been found in some human ALS patients (Figlewicz et al., 1994). Rooke et al. (1996), however, found no variation in the NEFH gene in 117 unrelated cases of familial ALS when the C-terminal KSP repeat region was examined by single-strand conformation analysis of PCR products. Vechio et al. (1996) also found no deletions in the NEFH gene.

Al-Chalabi et al. (1999) presented results strongly suggesting that NEFH motif deletions can be a primary, albeit uncommon, event in amyotrophic lateral sclerosis. They analyzed samples from 2 different populations (UK, 207; Scandinavia, 323) with age-matched controls for each group (UK, 219; Scandinavia, 228) and found 4 novel NEFH tail deletions, each involving a whole motif. These were found in 3 patients with sporadic ALS and in a family with autosomal dominant ALS. In all cases, motif deletions were associated with disease only when paired with the long NEFH allele. All the deletions occurred within a small region of the NEFH tail. Al-Chalabi et al. (1999) proposed a structural organization of the tail and organized reported deletions into logical groups.

In a study of 164 ALS cases and 207 age-matched controls, Tomkins et al. (1998) identified an 84-bp insertion in the NEFH tail. This insertion occurred at nucleotide 2124 and resulted in an extra 4 KSP repeat motifs. By the classification system used by Al-Chalabi et al. (1999), it was a duplication of domains 10 and 11 (2080-2163) with insertion at 2080 (start of domain 10). The normal allele in this case was the S allele, consistent with the hypothesis that length differences or steric effects between NEFH tails may be important. As with the deletions identified by Al-Chalabi et al. (1999), whole domains were involved.

Charcot-Marie-Tooth Disease, Axonal, Type 2CC

In affected members of 2 unrelated families with autosomal dominant axonal Charcot-Marie-Tooth disease type 2CC (CMT2CC; 616924), Rebelo et al. (2016) identified 2 different frameshift mutations in the last exon of the NEFH gene (162230.0002 and 162230.0003). Both mutations resulted in the continued translation of an additional 40 amino acids beyond the stop codon. The mutations were found by whole-exome sequencing and segregated with the disorder in the families. Molecular modeling studies indicated that the frameshift variants with the extra amino acids at the 3-prime end would result in the addition of cryptic amyloidogenic elements (CAE) with a high propensity to form aggregates. Expression of the frameshift mutations into neuronal cells resulted in prominent abnormal perinuclear aggregation of the mutant protein. The transfected cells appeared round and had significantly decreased axon-like projections compared to wildtype, and the aggregates trapped NEFL (162280), kinesin, and other proteins, and thus disrupted the neurofilament network in a toxic gain-of-function manner. Analysis with different truncated constructs indicated that the most distal 22 amyloidogenic amino acids are sufficient and necessary for the formation of aggregates. Rebelo et al. (2016) emphasized that this unusual disease mechanism should be considered during the evaluation of stop-loss variants.


Animal Model

Using homologous recombination, Rao et al. (1998) generated mice lacking the Nefh gene. In peripheral motor and sensory axons, absence of Nefh did not significantly affect the number of neurofilaments or axonal elongation or targeting, but it did affect the efficiency of survival of motor and sensory axons. Because postnatal growth of motor axon caliber continued largely unabated in the absence of Nefh, Rao et al. (1998) concluded that neither interactions mediated by Nefh nor the extensive phosphorylation of it within myelinated axonal segments are essential features of this growth.

Elder et al. (1998) independently created Nefh knockout mice to assess the contribution of NEFH to the development of axon size as well as its effect on the amounts of neurofilaments light and medium (NEFL and NEFM, respectively). In Nefh-null mice, Nefl levels were reduced only slightly, whereas Nefm and tubulin proteins were unchanged. However, the calibers of both large and small diameter myelinated axons were diminished in the Nefh-null mice, and large diameter axons failed to develop in both the central and peripheral nervous systems. Elder et al. (1998) concluded that unlike loss of the NEFL or NEFM subunits, loss of NEFH has only a slight effect on neurofilament number in axons, yet NEFH plays a major role in the development of large diameter axons.

To investigate the role of the NEFH subunit in neuron function, Zhu et al. (1998) generated mice bearing a targeted disruption of Nefh. The authors found that the lack of Nefh subunits had little effect on axonal calibers and, by electron microscopy, detected no significant changes in the number and packing density of neurofilaments made up of only the Nefl and Nefm subunits. However, they detected an approximately 2.4-fold increase of microtubule density in the large ventral root axons of the Nefh knockout mice. They also observed a corresponding increase in the ratio of assembled tubulin to Nefl protein in insoluble cytoskeletal preparations from the sciatic nerve. Using axonal transport studies, Zhu et al. (1998) detected an increased transport velocity of newly synthesized Nefl and Nefm proteins in motor axons of the Nefh knockout mice. They concluded that the NEFH subunit is a key mediator of iminodipropionitrile-induced axonopathy.

Rebelo et al. (2016) found that expression of pathogenic frameshift variants extending the reading frame of the NEFH gene (162230.0002 and 162230.0003) in zebrafish embryos resulted in significantly shorter axon lengths of motor neurons in a toxic gain-of-function manner. Transfected zebrafish showed decreased amounts of mutant NEFH, likely resulting from rapid degradation of toxic misfolded proteins.


ALLELIC VARIANTS ( 3 Selected Examples):

.0001 AMYOTROPHIC LATERAL SCLEROSIS, SUSCEPTIBILITY TO (1 family)

NEFH, 42-BP DEL, NT1989
  
RCV000015080...

Al-Chalabi et al. (1999) described a Scandinavian pedigree with ALS (105400) in 4 members of 2 generations. The mother of 2 affected individuals in the first of the 2 generations died from a dementing illness with swallowing difficulties, and a brother of an affected female in the second generation had monomelic ALS. A deletion in the NEFH tail involving nucleotides 1989-2030 was found. All affected individuals carried the L allele, and individuals with the S allele were unaffected, suggesting interactions that affect the penetrance of the deletion mutant.


.0002 CHARCOT-MARIE-TOOTH DISEASE, AXONAL, TYPE 2CC

NEFH, 2-BP DEL, 3010GA
  
RCV000210935...

In affected members of a British family (UK1) with autosomal dominant axonal Charcot-Marie-Tooth disease type 2CC (CMT2CC; 616924), Rebelo et al. (2016) identified a heterozygous 2-bp deletion (c.3010_3011delGA) in exon 4 of the NEFH gene, resulting in a frameshift and premature termination (Asp1004GlnfsTer58). The mutation, which was found by whole-exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family. The mutation affects the last coding exon and shifts translation into an alternative reading frame, resulting in continued translation of an additional 40 amino acids beyond the stop codon. The mutation was filtered against the Exome Variant Server database and was not found in 5,200 exomes from additional individuals with a wide range of clinical phenotypes, including other neuropathies. The extra amino acids at the 3-prime end of the protein resulted in the addition of cryptic amyloidogenic elements (CAE) with a high propensity to form toxic aggregates.


.0003 CHARCOT-MARIE-TOOTH DISEASE, AXONAL, TYPE 2CC

NEFH, 4-BP DUP, NT3017
  
RCV000210933

In 4 sibs (family F2) with autosomal dominant axonal Charcot-Marie-Tooth disease type 2CC (CMT2CC; 616924), Rebelo et al. (2016) identified a heterozygous 4-bp duplication (c.3017_3020dup) in exon 4 of the NEFH gene, resulting in a frameshift and premature termination (Pro1008AlafsTer56). The mutation, which was fond by whole-exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family, although DNA from a deceased parent was unavailable. The mutation affects the last coding exon and shifts translation into an alternative reading frame, resulting in continued translation of an additional 40 amino acids beyond the stop codon, similar to the mutation observed in a different family with the disorder (162230.0002). The mutation was filtered against the Exome Variant Server database and was not found in 5,200 exomes from additional individuals with a wide range of clinical phenotypes, including other neuropathies. The extra amino acids at the 3-prime end of the protein resulted in the addition of cryptic amyloidogenic elements (CAE) with a high propensity to form toxic aggregates.


REFERENCES

  1. Al-Chalabi, A., Andersen, P. M., Nilsson, P., Chioza, B., Andersson, J. L., Russ, C., Shaw, C. E., Powell, J. F., Leigh, P. N. Deletions of the heavy neurofilament subunit tail in amyotrophic lateral sclerosis. Hum. Molec. Genet. 8: 157-164, 1999. [PubMed: 9931323, related citations] [Full Text]

  2. Bucan, M., Gatalica, B., Nolan, P., Chung, A., Leroux, A., Grossman, M. H., Nadeau, J. H., Emanuel, B. S., Budarf, M. Comparative mapping of 9 human chromosome 22q loci in the laboratory mouse. Hum. Molec. Genet. 2: 1245-1252, 1993. [PubMed: 8401507, related citations] [Full Text]

  3. Collard, J.-F., Cote, F., Julien, J.-P. Defective axonal transport in a transgenic mouse model of amyotrophic lateral sclerosis. Nature 375: 61-64, 1995. [PubMed: 7536898, related citations] [Full Text]

  4. Elder, G. A., Friedrich, V. L., Jr., Kang, C., Bosco, P., Gourov, A., Tu, P.-H., Zhang, B., Lee, V. M.-Y., Lazzarini, R. A. Requirement of heavy neurofilament subunit in the development of axons with large calibers. J. Cell Biol. 143: 195-205, 1998. [PubMed: 9763431, images, related citations] [Full Text]

  5. Figlewicz, D. A., Krizus, A., Martinoli, M. G., Meininger, V., Dib, M., Rouleau, G. A., Julien, J.-P. Variants of the heavy neurofilament subunit are associated with the development of amyotrophic lateral sclerosis. Hum. Molec. Genet. 3: 1757-1761, 1994. [PubMed: 7849698, related citations] [Full Text]

  6. Hirokawa, N., Takeda, S. Gene targeting studies begin to reveal the function of neurofilament proteins. J. Cell Biol. 143: 1-4, 1998. Note: Erratum: J. Cell Biol. 143: 1142 only, 1998. [PubMed: 9763415, related citations] [Full Text]

  7. Lees, J. F., Shneidman, P. S., Skuntz, S. F., Carden, M. J., Lazzarini, R. A. The structure and organization of the human heavy neurofilament subunit (NF-H) and the gene encoding it. EMBO J. 7: 1947-1955, 1988. [PubMed: 3138108, related citations] [Full Text]

  8. Mattei, M.-G., Dautigny, A., Pham-Dinh, D., Passage, E., Mattei, J.-F., Jolles, P. The gene encoding the large human neurofilament subunit (NF-H) maps to the q121-q131 region on human chromosome 22. Hum. Genet. 80: 293-295, 1988. [PubMed: 3192217, related citations] [Full Text]

  9. Rao, M. V., Houseweart, M. K., Williamson, T. L., Crawford, T. O., Folmer, J., Cleveland, D. W. Neurofilament-dependent radial growth of motor axons and axonal organization of neurofilaments does not require the neurofilament heavy subunit (NF-H) or its phosphorylation. J. Cell Biol. 143: 171-181, 1998. [PubMed: 9763429, images, related citations] [Full Text]

  10. Rebelo, A. P., Abrams, A. J., Cottenie, E., Horga, A., Gonzalez, M., Bis, D. M., Sanchez-Mejias, A., Pinto, M., Buglo, E., Markel, K., Prince, J., Laura, M., and 10 others. Cryptic amyloidogenic elements in the 3-prime UTRs of neurofilament genes trigger axonal neuropathy. Am. J. Hum. Genet. 98: 597-614, 2016. [PubMed: 27040688, images, related citations] [Full Text]

  11. Rooke, K., Figlewicz, D. A., Han, F., Rouleau, G. A. Analysis of the KSP repeat of the neurofilament heavy subunit in familial amyotrophic lateral sclerosis. Neurology 46: 789-790, 1996. [PubMed: 8618684, related citations] [Full Text]

  12. Rouleau, G. A., Merel, P., Lutchman, M., Sanson, M., Zucman, J., Marineau, C., Hoang-Xuan, K., Demczuk, S., Desmaze, C., Plougastel, B., Pulst, S. M., Lenoir, G., Bijlsma, E., Fashold, R., Dumanski, J., de Jong, P., Parry, D., Eldrige, R., Aurias, A., Delattre, O., Thomas, G. Alteration in a new gene encoding a putative membrane-organizing protein causes neuro-fibromatosis type 2. Nature 363: 515-521, 1993. [PubMed: 8379998, related citations] [Full Text]

  13. Tomkins, J., Usher, P., Slade, J. Y., Ince, P. G., Curtis, A., Bushby, K., Shaw, P. J. Novel insertion in the KSP region of the neurofilament heavy gene in amyotrophic lateral sclerosis. Neuroreport 9: 3967-3970, 1998. [PubMed: 9875737, related citations] [Full Text]

  14. Vechio, J. D., Bruijn, L. I., Xu, Z., Brown, R. H., Jr., Cleveland, D. W. Sequence variants in human neurofilament proteins: absence of linkage to familial amyotrophic lateral sclerosis. Ann. Neurol. 40: 603-610, 1996. [PubMed: 8871580, related citations] [Full Text]

  15. Watson, C. J., Gaunt, L., Evans, G., Patel, K., Harris, R., Strachan, T. A disease-associated germline deletion maps the type 2 neurofibromatosis (NF2) gene between the Ewing sarcoma region and the leukaemia inhibitory factor locus. Hum. Molec. Genet. 2: 701-704, 1993. [PubMed: 8102569, related citations] [Full Text]

  16. Zhu, Q., Lindenbaum, M., Levavasseur, F., Jacomy, H., Julien, J.-P. Disruption of the NF-H gene increases axonal microtubule content and velocity of neurofilament transport: relief of axonopathy resulting from the toxin beta,beta-prime-iminodipropionitrile. J. Cell Biol. 143: 183-193, 1998. [PubMed: 9763430, images, related citations] [Full Text]


Cassandra L. Kniffin - updated : 4/27/2016
Dawn Watkins-Chow - updated : 11/6/2002
Victor A. McKusick - updated : 3/9/1999
Orest Hurko - updated : 5/8/1996
Creation Date:
Victor A. McKusick : 5/12/1988
carol : 06/24/2016
carol : 4/28/2016
ckniffin : 4/27/2016
carol : 7/11/2014
terry : 3/14/2013
alopez : 7/8/2010
carol : 11/7/2002
tkritzer : 11/6/2002
tkritzer : 11/6/2002
alopez : 6/14/2000
alopez : 6/14/2000
terry : 6/13/2000
carol : 6/23/1999
terry : 6/9/1999
carol : 3/23/1999
terry : 3/9/1999
mark : 5/8/1996
terry : 5/3/1996
terry : 7/6/1995
mark : 6/9/1995
carol : 9/20/1993
carol : 7/7/1993
carol : 6/21/1993
carol : 6/18/1993

* 162230

NEUROFILAMENT PROTEIN, HEAVY POLYPEPTIDE; NEFH


Alternative titles; symbols

NFH


HGNC Approved Gene Symbol: NEFH

Cytogenetic location: 22q12.2     Genomic coordinates (GRCh38): 22:29,480,218-29,491,390 (from NCBI)


Gene-Phenotype Relationships

Location Phenotype Phenotype
MIM number
Inheritance Phenotype
mapping key
22q12.2 {?Amyotrophic lateral sclerosis, susceptibility to} 105400 Autosomal dominant; Autosomal recessive 3
Charcot-Marie-Tooth disease, axonal, type 2CC 616924 Autosomal dominant 3

TEXT

Description

Three neuron-specific intermediate filaments in mammals, NEFH, NEFM (162250), and NEFL (162280), are the most prominent cytoskeletal components in large myelinated axons (summary by Elder et al., 1998).


Cloning and Expression

In the course of cloning the region between 2 markers, D22S212 and D22S32, that flank the neurofibromin-2 gene (NF2; 607379), Rouleau et al. (1993) identified a gene with a neuronal pattern of expression and a transcript size identical to that of NEFH. Use of NEFH cDNA confirmed the identity.


Gene Structure

Lees et al. (1988) reported that the human heavy neurofilament subunit gene has 3 introns, 2 of which interrupt the protein-coding sequence at identical points to introns in the genes for the 2 smaller neurofilament proteins NFM and NFL.


Mapping

Mattei et al. (1988) used a rat cDNA probe coding for the C-terminal extension of the NEFH gene to assign, by in situ hybridization, the human NEFH gene to 22q12.1-q13.1. The possible implications of the fact that neurologic disorders such as meningioma map to this region were discussed.

There is compelling evidence that the NEFH locus is close to the NF2 locus. For example, Watson et al. (1993) found that the NEFH locus was hemizygous in a deletion that was observed in affected members of a family with NF2 and was estimated to be about 700 kb long. The NF2 locus has been positioned at 22q12.2.

Bucan et al. (1993) mapped the mouse Nefh gene, which they symbolized Nfh, to chromosome 11.


Gene Function

To investigate how disorganized neurofilaments might cause neurodegeneration, Collard et al. (1995) studied axonal transport of newly synthesized proteins in mice that overexpress the human NEFH gene. They observed dramatic defects of axonal transport, not only of neurofilament proteins but also of other proteins, including tubulin and actin. Ultrastructural analysis revealed a paucity of cytoskeletal elements, smooth endoplasmic reticulum, and especially mitochondria in the degenerating axons. Collard et al. (1995) therefore proposed that the neurofilament accumulations observed in these mice cause axonal degeneration by impeding the transport of components required for axonal maintenance, and that a similar mechanism may account for the pathogenesis of ALS in human patients.

Hirokawa and Takeda (1998) reviewed the contributions that gene targeting studies had made to understanding the role of each of the neurofilament component proteins in neurofilament formation and in determination of the axonal caliber.


Molecular Genetics

The tail of the neurofilament heavy subunit is composed of a repeating amino acid motif, usually X-lysine-serine-proline-Y-lysine (XKSPYK), where X is a single amino acid and Y is 1 to 3 amino acids. There are 2 common polymorphic variants of 44 and 45 repeats. The tail probably regulates axonal caliber, with interfilament spacing determined by phosphorylation of the KSP motifs. According to Al-Chalabi et al. (1999), the polymorphic variants had been mislabeled in the published literature as 44 and 43 repeat variants, respectively, and therefore were referred to by them simply as long (L) and short (S) alleles.

Susceptibility to Amyotrophic Lateral Sclerosis

In amyotrophic lateral sclerosis (ALS; 105400), a degenerative disease of motor neurons, depositions of neurofilaments occur in the perikarya and proximal exons. Two lines of evidence suggest that neurofilament accumulation may play a causal pathogenetic role. First, transgenic mice that overexpress neurofilament proteins show motor neuron degeneration (Collard et al., 1995). Second, deletions within the C-terminal KSP repeat region of the neurofilament heavy-subunit gene have been found in some human ALS patients (Figlewicz et al., 1994). Rooke et al. (1996), however, found no variation in the NEFH gene in 117 unrelated cases of familial ALS when the C-terminal KSP repeat region was examined by single-strand conformation analysis of PCR products. Vechio et al. (1996) also found no deletions in the NEFH gene.

Al-Chalabi et al. (1999) presented results strongly suggesting that NEFH motif deletions can be a primary, albeit uncommon, event in amyotrophic lateral sclerosis. They analyzed samples from 2 different populations (UK, 207; Scandinavia, 323) with age-matched controls for each group (UK, 219; Scandinavia, 228) and found 4 novel NEFH tail deletions, each involving a whole motif. These were found in 3 patients with sporadic ALS and in a family with autosomal dominant ALS. In all cases, motif deletions were associated with disease only when paired with the long NEFH allele. All the deletions occurred within a small region of the NEFH tail. Al-Chalabi et al. (1999) proposed a structural organization of the tail and organized reported deletions into logical groups.

In a study of 164 ALS cases and 207 age-matched controls, Tomkins et al. (1998) identified an 84-bp insertion in the NEFH tail. This insertion occurred at nucleotide 2124 and resulted in an extra 4 KSP repeat motifs. By the classification system used by Al-Chalabi et al. (1999), it was a duplication of domains 10 and 11 (2080-2163) with insertion at 2080 (start of domain 10). The normal allele in this case was the S allele, consistent with the hypothesis that length differences or steric effects between NEFH tails may be important. As with the deletions identified by Al-Chalabi et al. (1999), whole domains were involved.

Charcot-Marie-Tooth Disease, Axonal, Type 2CC

In affected members of 2 unrelated families with autosomal dominant axonal Charcot-Marie-Tooth disease type 2CC (CMT2CC; 616924), Rebelo et al. (2016) identified 2 different frameshift mutations in the last exon of the NEFH gene (162230.0002 and 162230.0003). Both mutations resulted in the continued translation of an additional 40 amino acids beyond the stop codon. The mutations were found by whole-exome sequencing and segregated with the disorder in the families. Molecular modeling studies indicated that the frameshift variants with the extra amino acids at the 3-prime end would result in the addition of cryptic amyloidogenic elements (CAE) with a high propensity to form aggregates. Expression of the frameshift mutations into neuronal cells resulted in prominent abnormal perinuclear aggregation of the mutant protein. The transfected cells appeared round and had significantly decreased axon-like projections compared to wildtype, and the aggregates trapped NEFL (162280), kinesin, and other proteins, and thus disrupted the neurofilament network in a toxic gain-of-function manner. Analysis with different truncated constructs indicated that the most distal 22 amyloidogenic amino acids are sufficient and necessary for the formation of aggregates. Rebelo et al. (2016) emphasized that this unusual disease mechanism should be considered during the evaluation of stop-loss variants.


Animal Model

Using homologous recombination, Rao et al. (1998) generated mice lacking the Nefh gene. In peripheral motor and sensory axons, absence of Nefh did not significantly affect the number of neurofilaments or axonal elongation or targeting, but it did affect the efficiency of survival of motor and sensory axons. Because postnatal growth of motor axon caliber continued largely unabated in the absence of Nefh, Rao et al. (1998) concluded that neither interactions mediated by Nefh nor the extensive phosphorylation of it within myelinated axonal segments are essential features of this growth.

Elder et al. (1998) independently created Nefh knockout mice to assess the contribution of NEFH to the development of axon size as well as its effect on the amounts of neurofilaments light and medium (NEFL and NEFM, respectively). In Nefh-null mice, Nefl levels were reduced only slightly, whereas Nefm and tubulin proteins were unchanged. However, the calibers of both large and small diameter myelinated axons were diminished in the Nefh-null mice, and large diameter axons failed to develop in both the central and peripheral nervous systems. Elder et al. (1998) concluded that unlike loss of the NEFL or NEFM subunits, loss of NEFH has only a slight effect on neurofilament number in axons, yet NEFH plays a major role in the development of large diameter axons.

To investigate the role of the NEFH subunit in neuron function, Zhu et al. (1998) generated mice bearing a targeted disruption of Nefh. The authors found that the lack of Nefh subunits had little effect on axonal calibers and, by electron microscopy, detected no significant changes in the number and packing density of neurofilaments made up of only the Nefl and Nefm subunits. However, they detected an approximately 2.4-fold increase of microtubule density in the large ventral root axons of the Nefh knockout mice. They also observed a corresponding increase in the ratio of assembled tubulin to Nefl protein in insoluble cytoskeletal preparations from the sciatic nerve. Using axonal transport studies, Zhu et al. (1998) detected an increased transport velocity of newly synthesized Nefl and Nefm proteins in motor axons of the Nefh knockout mice. They concluded that the NEFH subunit is a key mediator of iminodipropionitrile-induced axonopathy.

Rebelo et al. (2016) found that expression of pathogenic frameshift variants extending the reading frame of the NEFH gene (162230.0002 and 162230.0003) in zebrafish embryos resulted in significantly shorter axon lengths of motor neurons in a toxic gain-of-function manner. Transfected zebrafish showed decreased amounts of mutant NEFH, likely resulting from rapid degradation of toxic misfolded proteins.


ALLELIC VARIANTS 3 Selected Examples):

.0001   AMYOTROPHIC LATERAL SCLEROSIS, SUSCEPTIBILITY TO (1 family)

NEFH, 42-BP DEL, NT1989
SNP: rs606231212, ClinVar: RCV000015080, RCV000057189

Al-Chalabi et al. (1999) described a Scandinavian pedigree with ALS (105400) in 4 members of 2 generations. The mother of 2 affected individuals in the first of the 2 generations died from a dementing illness with swallowing difficulties, and a brother of an affected female in the second generation had monomelic ALS. A deletion in the NEFH tail involving nucleotides 1989-2030 was found. All affected individuals carried the L allele, and individuals with the S allele were unaffected, suggesting interactions that affect the penetrance of the deletion mutant.


.0002   CHARCOT-MARIE-TOOTH DISEASE, AXONAL, TYPE 2CC

NEFH, 2-BP DEL, 3010GA
SNP: rs876657411, ClinVar: RCV000210935, RCV001853394

In affected members of a British family (UK1) with autosomal dominant axonal Charcot-Marie-Tooth disease type 2CC (CMT2CC; 616924), Rebelo et al. (2016) identified a heterozygous 2-bp deletion (c.3010_3011delGA) in exon 4 of the NEFH gene, resulting in a frameshift and premature termination (Asp1004GlnfsTer58). The mutation, which was found by whole-exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family. The mutation affects the last coding exon and shifts translation into an alternative reading frame, resulting in continued translation of an additional 40 amino acids beyond the stop codon. The mutation was filtered against the Exome Variant Server database and was not found in 5,200 exomes from additional individuals with a wide range of clinical phenotypes, including other neuropathies. The extra amino acids at the 3-prime end of the protein resulted in the addition of cryptic amyloidogenic elements (CAE) with a high propensity to form toxic aggregates.


.0003   CHARCOT-MARIE-TOOTH DISEASE, AXONAL, TYPE 2CC

NEFH, 4-BP DUP, NT3017
SNP: rs876657412, ClinVar: RCV000210933

In 4 sibs (family F2) with autosomal dominant axonal Charcot-Marie-Tooth disease type 2CC (CMT2CC; 616924), Rebelo et al. (2016) identified a heterozygous 4-bp duplication (c.3017_3020dup) in exon 4 of the NEFH gene, resulting in a frameshift and premature termination (Pro1008AlafsTer56). The mutation, which was fond by whole-exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family, although DNA from a deceased parent was unavailable. The mutation affects the last coding exon and shifts translation into an alternative reading frame, resulting in continued translation of an additional 40 amino acids beyond the stop codon, similar to the mutation observed in a different family with the disorder (162230.0002). The mutation was filtered against the Exome Variant Server database and was not found in 5,200 exomes from additional individuals with a wide range of clinical phenotypes, including other neuropathies. The extra amino acids at the 3-prime end of the protein resulted in the addition of cryptic amyloidogenic elements (CAE) with a high propensity to form toxic aggregates.


REFERENCES

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Contributors:
Cassandra L. Kniffin - updated : 4/27/2016
Dawn Watkins-Chow - updated : 11/6/2002
Victor A. McKusick - updated : 3/9/1999
Orest Hurko - updated : 5/8/1996

Creation Date:
Victor A. McKusick : 5/12/1988

Edit History:
carol : 06/24/2016
carol : 4/28/2016
ckniffin : 4/27/2016
carol : 7/11/2014
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carol : 6/23/1999
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carol : 9/20/1993
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