Detection of a variant protein in hair: new diagnostic method in Portuguese type familial amyloid polyneuropathy

Portuguese type familial amyloid polyneuropathy is a hereditary amyloidosis caused by a variant form of transthyretin in which valine is replaced by methionine at position 30.¹,² Transthyretin is produced predominantly in the liver and circulates in the blood as a carrier protein for thyroid hormones and retinol binding protein.¹,² All carriers of this transthyretin variant have large amounts of the variant protein in their blood, so it may be present in the hair just as A and B blood group antigens are. To test this possibility, we examined the immunoreactivity of hair from carriers and control subjects.

Subjects, methods, and results

We tested the immunoreactivity of hair using a monoclonal antibody specific to the methionine variant of transthyretin that does not react with normal transthyretin.³,⁴ We investigated hair from five Japanese patients with familial amyloid polyneuropathy (age range 28-51 years; all black hair), one carrier of the gene for familial amyloid polyneuropathy who was symptom free (61 year old Swedish woman; grey hair), and eight healthy controls (age range 25-48; five were Japanese (black hair), two were Swedish (one blond, one brown hair), and one was Iranian (black hair)). Hair was embedded directly in epoxy resin without being treated, and sections were cut at 3 μm. Sections were immersed in 0.01% hydrogen TRIS buffer (pH 7.4) for 10 minutes to inhibit endogenous peroxidase and then treated with 1% bovine serum albumin to occupy non-specific binding sites. The sections were incubated overnight at room temperature with the monoclonal transthyretin antibody. Immunoreactivity to the antibody was detected by the avidin-biotin complex method (Dakopatts, Glostrup, Denmark). Finally, hairs were stained with True Blue peroxidase substrate (Novakemi, Stockholm, Sweden).

Immunoreactivity was found in the medulla of hairs from the patients with familial amyloid polyneuropathy (figure (right)) and the carrier of the gene for familial amyloid polyneuropathy, but no immunoreactivity was observed in the medulla of hairs from the controls (figure (left)). The reactivity was found even in the tip of the hair, but it was more obvious in the root.

Comment

With this method we were able to detect the presence of a variant form of a protein in hair samples. DNA diagnosis, radioimmunoassay, and high performance liquid chromatography have been used to detect transthyretin variants in blood samples. These methods are, however, time consuming, and obtaining a blood sample may be difficult. In contrast, this immunocytochemical method is simple and samples are easy to obtain. Hair that has fallen out may be tested, which means that the method could be applied in forensic medicine to detect variant proteins other than transthyretin mutations.

Transthyretin has up to 60 known mutations.⁵ Two mutations (in which serine replaces glycine at position 6 and methionine replaces threonine at position 119) are known benign polymorphisms that are present in 10-15% of the population.² Thus, detection of transthyretin variants can be used to identify a particular person. Other variant proteins could also be detected in hair by this method if suitable antibodies were available or could be produced. This may prove an valuable additional tool to DNA diagnostic procedures in identifying particular people because routine DNA analysis is not always specific.

Figure.

Immunoreactivity for variant transthyretin in healthy male volunteer aged 43 (left) and male patient with familial amyloid polyneuropathy aged 36 (right). Arrows indicate medulla of hair