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Author: Prashant Sharma, Undiagnosed Diseases Program, National Human Genome Research Institute, National Institutes of Health
Competing interests: Prashant Sharma is an author of the article discussed in this blog.
Image Caption: Image of wild-type (left) and littermate Hephl1 knockout (right) mice, with an insert showing hair abnormalities in a patient with biallelic HEPHL1 variants.
Image credit: Sharma et al.
Scientific inquiry is an exploration of the unknown, a first-ever journey to an unspecified destination. Of course, that’s what makes research so exciting and rewarding. The National Institutes of Health Undiagnosed Diseases Program  brings together clinical and research experts across multiple specialties to evaluate individuals with rare and undiagnosed diseases using advanced technologies.
In the featured article  of the May issue of PLOS Genetics, we were able to connect the finding of anomalous hair in a young boy to a particular gene (HEPHL1), and we confirmed this connection by showing that mice lacking this gene grow the curly, kinked whiskers shown in the image above. The loss of HEPHL1 function observed in the boy and in mice leads to abnormalities in the structure of hair, strongly suggesting that the function of the multi-copper oxidase enzyme encoded by this gene is important for proper hair development and growth.
Copper serves as a catalytic cofactor in many different metalloenzymes, and perturbation of the management of copper in the body leads to a wide spectrum of impairments, causing several genetics diseases. One of the best known of these is Menkes kinky hair syndrome, which is characterized by the presence of a peculiar “kinky hair” trait and degeneration of the brain, bones and connective tissue. Menkes is an X-linked disease caused by disruptive mutations in the ATP7A gene, which encodes a protein that uses ATP to pump copper across cellular membranes. How the copper deficiency in Menkes disease patients leads to hair abnormalities is not completely understood, but it has been suggested that the low activity of the enzyme sulfhydryl oxidase (which depends on copper for its activity) results in fewer of the disulfide bridges that provide structural strength and elasticity in hair .
Through the NIH Undiagnosed Diseases program, we evaluated a patient who presented clinically with abnormal hair (pili torti and trichorrhexis nodosa) and cognitive dysfunction. The hair abnormalities observed in our patient resemble those found in Menkes disease, but sequence analysis of the ATP7A gene and relevant biochemical testing showed that ATP7A wasn’t involved in causing our patient’s clinical features. To identify other variants that might be contributing, we sequenced all of the protein-coding regions of the patient’s DNA, along with those of his parents, and identified two deleterious mutations, one from each parent (making him a so-called “compound heterozygote”), in the HEPHL1 gene, located on chromosome 11.
HEPHL1 belongs to a family of proteins known as Multi-Copper Oxidases (MCOs). The main function of HEPHL1’s two closest relatives, ceruloplasmin and hephaestin, is to catalyze the oxidation of ferrous iron [Fe (II)] to the more easily mobilizable ferric form [Fe (III)]. The resulting Fe (III) can then bind to a circulating protein called transferrin which distributes it throughout the body. One unique property of MCOs is the presence of three copper-binding sites that can house six copper atoms. Mutations that alter the structure of these sites are likely to affect their ability to bind copper, and this in turn may alter the iron-oxidizing activity (ferroxidase activty) of the enzyme.
Our study demonstrates that each mutation found in the patient led to complete loss of iron-oxidizing activity, likely due to loss of copper from the HEPHL1 enzyme, and defective glycosylation (attachment of sugars). As a consequence, we found unexpectedly high levels of iron in skin cells taken from the patient, presumably due to inadequate oxidation of Fe (II) to Fe (III) and reduced export of iron from the cells.
To help us explore the physiological consequences of the loss of HEPHL1 activity more thoroughly, we used a gene targeting approach to make mice with a disrupted Hephl1 gene. Interestingly, as shown in the above image, all mice that had a complete deletion of both copies of their Hephl1 gene (homozygotes) had short, curled whiskers (vibrissae) throughout their life. The curly whisker in Hephl1 knockout mice resemble the pili torti found in our patient with two different HEPHL1 mutations. Mice with one copy of the normal Hephl1 gene (so-called heterozygotes, like the patient’s parents) didn’t have curly whiskers, showing that complete loss of HEPHL1 function is needed to affect the structure of the hair.
How HEPHL1 mechanistically regulates hair growth and development will require further in-depth analysis, and the curly whiskers from Hephl1 knockout mice could help these investigations. Our preliminary results showed that HEPHL1 regulates the activity of the enzyme lysyl oxidase, which needs copper for its enzymatic activity. This raises the intriguing possibility that, in addition to playing an iron-related role, HEPHL1 could also serve as a copper oxidase for other cuproenzymes, whose activity may be required to provide structural strength to hair shafts. A recent genome-wide transcriptome analysis also identified Hephl1 as a signature gene of the transient amplifying cells (TACs) of the mouse hair follicles, where it is much more highly expressed than its fellow MCOs ceruloplasmin and hephaestin . This suggests that HEPHL1’s ferroxidase activity could play a more specific role in the hair follicle, where it can’t be compensated by another related MCO. In conclusion, our study identified HEPHL1 as a novel gene responsible for hair abnormalities and highlights the importance of exploring the role of HEPHL1 — and its interconnections with other key regulators — in developing new therapeutic strategies to treat hair disorders.
- Gahl WA, Mulvihill JJ, Toro C, Markello TC, Wise AL, Ramoni RB, et al. (2016) The NIH Undiagnosed Diseases Program and Network: Applications to modern medicine. Mol Genet Metab. 2016;117(4):393-400. Epub 2016/02/06. doi: 10.1016/j.ymgme.2016.01.007. PMID: 26846157; PMCID: PMCPMC5560125.
- Sharma P, Reichert M, Lu Y, Markello TC, Adams DR, Steinbach PJ, et al. (2019) Biallelic HEPHL1 variants impair ferroxidase activity and cause an abnormal hair phenotype. PLoS Genet. 2019;15(5):e1008143. Epub 2019/05/28. doi: 10.1371/journal.pgen.1008143. PMID: 31125343; PMCID: PMCPMC6534290.
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