Implementing Homocysteine into First-Tier Mass Spectrometry Tests in Newborn Screening for Detection of Homocystinurias in New York State
Ethical Legal Social Issues (ELSI) Public Health and Policy
-
Primary Categories:
- Public Health Genetics
-
Secondary Categories:
- Public Health Genetics
Introduction:
Homocystinurias (HCU) are a group of inherited disorders of homocysteine (Hcy) metabolism associated with high blood level of total Hcy (tHcy). The most common causes of HCU include reduced activity of the enzyme cystathionine beta-synthase (CBS) and defective remethylation to methionine due to cobalamin (vitamin B12) or folate deficiencies. The New York State (NYS) Newborn Screening (NBS) Program introduced mass spectrometry (MS) to screen for HCU via measurement of methionine (Met) levels in 2002 and made continuous efforts to optimize the screening algorithm, aiming to identify HCU in pre-symptomatic newborns. However, screening based on Met levels alone exhibits a considerable number of false-negative and -positive results as Met can be low in the remethylation disorders, high/normal in CBS deficiency, and high in newborns with nutritional disparities and/or hepatic dysfunction. Implementing the detection of tHcy in first-tier MS assay would improve the sensitivity and specificity in the detection of HCU in NBS.
Methods:
3.2-mm punches from dried blood spot (DBS) specimens including residual DBS with known HCU were extracted with methanol containing internal standards. The internal standard contained known concentrations of stable isotope analogs including d8-homocystine. Extracted samples were reduced by tris(2-carboxyethyl)phosphine (TCEP) and dried. Dried samples were treated by N-ethylmaleimide (NEM) for selective thiol derivatization followed by butyl ester derivatization. Samples were analyzed by tandem MS/MS using multiple reaction monitoring (MRM). Hcy-NEM and d4-Hcy-NEM were tuned and optimized to determine the best MRM transitions and MS parameters.
Results:
The linearity of the method for each analyte, including tHcy was determined by analyzing blood samples fortified with five known concentrations of the analytes. All analytes, including Hcy wrere linear across a broad dynamic concentration range. With an average tHcy population level of 0.8 µmol/L, the concentrations of tHcy in NBS DBS specimens from two babies with CBS deficiency were 7.1 µmol/L and 6.7 µmol/L, respectively. In one specimen from a patient with cobalamin C disease, the tHcy concentration was 16.3 µmol/L while a specimen from a patient with cobalamin J disease showed a tHcy concentration of 0.6 µmol/L.
Conclusion:
This preliminary study demonstrates the potential of quantifying tHcy during first-tier screening to better detect HCU. Multiplexing Hcy into existing first-tier screening MS screen workflows may reduce false-negative and -positive rates and minimize the impact of adding a new test. Future work is needed to validate the method, establish reference ranges, and analyze the data to define algorithms.
Homocystinurias (HCU) are a group of inherited disorders of homocysteine (Hcy) metabolism associated with high blood level of total Hcy (tHcy). The most common causes of HCU include reduced activity of the enzyme cystathionine beta-synthase (CBS) and defective remethylation to methionine due to cobalamin (vitamin B12) or folate deficiencies. The New York State (NYS) Newborn Screening (NBS) Program introduced mass spectrometry (MS) to screen for HCU via measurement of methionine (Met) levels in 2002 and made continuous efforts to optimize the screening algorithm, aiming to identify HCU in pre-symptomatic newborns. However, screening based on Met levels alone exhibits a considerable number of false-negative and -positive results as Met can be low in the remethylation disorders, high/normal in CBS deficiency, and high in newborns with nutritional disparities and/or hepatic dysfunction. Implementing the detection of tHcy in first-tier MS assay would improve the sensitivity and specificity in the detection of HCU in NBS.
Methods:
3.2-mm punches from dried blood spot (DBS) specimens including residual DBS with known HCU were extracted with methanol containing internal standards. The internal standard contained known concentrations of stable isotope analogs including d8-homocystine. Extracted samples were reduced by tris(2-carboxyethyl)phosphine (TCEP) and dried. Dried samples were treated by N-ethylmaleimide (NEM) for selective thiol derivatization followed by butyl ester derivatization. Samples were analyzed by tandem MS/MS using multiple reaction monitoring (MRM). Hcy-NEM and d4-Hcy-NEM were tuned and optimized to determine the best MRM transitions and MS parameters.
Results:
The linearity of the method for each analyte, including tHcy was determined by analyzing blood samples fortified with five known concentrations of the analytes. All analytes, including Hcy wrere linear across a broad dynamic concentration range. With an average tHcy population level of 0.8 µmol/L, the concentrations of tHcy in NBS DBS specimens from two babies with CBS deficiency were 7.1 µmol/L and 6.7 µmol/L, respectively. In one specimen from a patient with cobalamin C disease, the tHcy concentration was 16.3 µmol/L while a specimen from a patient with cobalamin J disease showed a tHcy concentration of 0.6 µmol/L.
Conclusion:
This preliminary study demonstrates the potential of quantifying tHcy during first-tier screening to better detect HCU. Multiplexing Hcy into existing first-tier screening MS screen workflows may reduce false-negative and -positive rates and minimize the impact of adding a new test. Future work is needed to validate the method, establish reference ranges, and analyze the data to define algorithms.