Year: 2023
Type: editorial
Abstract: In 1963, two sisters from Northern Ireland with mental deficiency and seizures were discovered to have homocystine in their urine (1). In addition to the mental deficiency and seizures, these sisters had dislocated ocular lens (ectopia lentis), malar flush, genu valgum, and pes cavus, all features now known to be characteristic of classic homocystinuria (HCU) (2). This discovery was of particular significance because HCU was the first amino acid metabolic disorder discovered that closely resembled phenylketonuria (PKU), the prototypical metabolic disorder that had been described almost 30 years earlier as the first biochemical cause of mental deficiency. Although other amino acid disorders were described before HCU (1), they either presented clinically in the newborn or had much less clinical import, whereas those with HCU, like PKU, are normal until several months of age when irreversible developmental delay is apparent and subsequently mental deficiency appears. HCU differs from PKU, however, in that the clinical complications in HCU include somatic features, whereas in PKU the effect is exclusively mental deficiency. In HCU these somatic features resemble Marfan syndrome (3). Importantly, HCU, like PKU, can be controlled by limiting an essential protein amino acid in the diet. In HCU this amino acid is methionine, which leads to the synthesis of homocysteine; in PKU the amino acid is phenylalanine. Thus, in both disorders, preventing the clinical complications requires presymptomatic initiation of the diet to reduce the increased concentrations of methionine and homocysteine in HCU and of phenylalanine in PKU, and this requires presymptomatic diagnosis, i.e., detection in the newborn. Consequently, HCU was one of the first disorders to be added to newborn screening (NBS) following the success of PKU screening (4). Unfortunately, NBS screening HCU has only been partially successful because of false-negative results. It is probable that less than 50% of those with HCU are detected by NBS. The reason NBS “misses” such a high percentage of those with HCU is that identification requires elevated methionine, a secondary metabolite, rather than elevated homocysteine, the primary metabolite. As a secondary metabolite, the concentration of methionine may be normal or perhaps only very slightly elevated and below the cutoff level on the first or second day of life when the blood specimen for NBS is usually collected. Consequently, many of those with HCU are not correctly diagnosed and treated until they are adolescents or young adults and by then have developed irreversible complications such as mental deficiency, ectopia lentis, osteoporosis, thromboembolism such as stroke, or any combination of these problems. Often they have been misdiagnosed since childhood as Marfan syndrome due to their skeletal appearance and/or ectopia lentis and have not benefitted from the diet that could have prevented these abnormalities (5). Attempts to increase the sensitivity of methionine for identifying HCU in the newborn, such as lowering the methionine cutoff (6) or adding a methionine/phenylalanine ratio (7) to aid interpretation of the NBS result, have only marginally reduced the false-negative rate. However, homocysteine as the primary metabolite in HCU would be expected to be elevated in the newborn blood specimen and would be far more effective in screening for HCU (5). So why is methionine rather than homocysteine measured in NBS? This is because methionine is one of the amino acids that is routinely measured in NBS (8), whereas the method for measuring homocysteine has required a separate and specific test that includes a reducing agent to free homocysteine from its disulfide and protein-bound forms (9). This assay is performed in a few screening programs but only as a second-tier test on newborn blood specimens that showed elevated methionine in the first-tier screen to further evaluate the possibility of HCU, not as a first-tier test (10). This could change. In this issue of Clinical Chemistry, Pickens and his associates at the Centers for Disease Control and Prevention describe a method for measuring homocysteine in the newborn specimen that is designed for primary first-tier NBS (11). Using a combination of tris(2-carboxyethyl) phosphine reducing agent to free homocysteine from its disulfide and protein-bound forms and N-ethylmaleimide to derivatize homocysteine, they measured the analytes by flow injection tandem mass spectrometry, the tandem mass spectrometry system used in NBS. Thus, homocysteine could be added to the other analytes routinely measured in routine first-tier NBS. They also show that this method clearly distinguished newborns with HCU from those with increased methionine due to other causes such as total parental nutrition. However, as recognized by the authors, the method is not yet ready for routine NBS. Performance of some biomarkers was affected, most notably in the case of acylcarnitines that utilized surrogate internal standards C5:1, C14:1, C3DC + C5OH, and ornithine. Performance of other markers such as guanidinoacetate appeared to be affected by suspected matrix effects and ion suppression in relation to their high QC samples. In addition, the parallel reaction monitoring spectrum of N-ethylmaleimide (m/z 126) indicated the presence of a product m/z 80 ion, which could interfere with valine (m/z 126 >80 transition) measurements. They plan to investigate methods for reducing ion suppression, including the removal of tris(2-carboxyethyl) phosphine from the specimen extract, methods to reduce the additional sample preparation overhead to facilitate routine NBS, adding an internal standard to remove the need for the surrogate internal standards used for quantifying analytes in the described method, and investigating alternative internal standards for those suspected of having differential ionization or interferences. Should these further investigations be successful, this method could greatly increase the identification of HCU in routine NBS and allow for early presymptomatic treatment, thereby substantially reducing the number of individuals tragically impaired by HCU because of delayed diagnosis and treatment. In addition, it could also allow newborn identification of cobalamin disorders in which elevated homocysteine is a key feature. These disorders are due to defects in the formation or transport of vitamin B12 coenzymes, and many are not currently included in NBS (12). Thus, including homocysteine in routine NBS would represent a major advance in the NBS process. HCU, classic homocystinuria; PKU, phenylketonuria; NBS, newborn screening. The corresponding author takes full responsibility that all authors on this publication have met the following required criteria of eligibility for authorship: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Nobody who qualifies for authorship has been omitted from the list. Harvey Levy (Conceptualization-Lead, Data curation-Lead, Methodology-Equal, Writing—original draft-Lead, Writing—review & editing-Lead) and Inderneel Sahai (Methodology-Supporting, Validation-Equal, Writing—review & editing-Supporting) Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: None declared. None declared. None declared. None declared. H.L. Levy, PI of Boston site for the Travere Pegbatinase clinical trial (Travere contracts with the Boston Children’s Hospital). None declared. None declared.
Source: Clinical Chemistry
Authors: Harvey L Levy, Inderneel Sahai
Language: English
Subfield: Clinical Biochemistry
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W4360599437