Dermatologic Manifestations of Homocystinuria Workup

Updated: May 17, 2022
  • Author: Janette Baloghova, MD, PhD; Chief Editor: Dirk M Elston, MD  more...
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Laboratory Studies

The diagnosis is based on the clinical picture and the results of laboratory analysis.

The cyanide nitroprusside reaction in the urine is used as the Brand reaction. In patients with positive screening test results, the diagnosis can be confirmed by analyzing methionine, homocysteine, and cystathionine levels by using paper chromatography, high-performance liquid chromatography (HPLC) with fluorescence detection, high-voltage electrophoresis, and amino acid tests. The reference range methionine level is less than 1 mg/dL (30 µM). Homocysteine levels of up to 0.2 µmol/mL and methionine levels of up to 2 µmol/mL characterize cystathionine synthetase deficiency.

Levels of homocysteine excreted in the urine are more than 200 mg, and the fraction of mixed bisulfite homocysteine and cysteine is established.

In the liver, the enzymatic activity of cystathionine synthase is deficient. This reduced activity can be demonstrated in a liver biopsy specimen.

Cultured fibroblasts derived from healthy skin, as well as from cells in the amniotic fluid, demonstrate cystathionine synthase activity, although the enzyme is not detectable in intact healthy skin. Fibroblasts grown from the skin of patients with homocystinuria are deficient in the enzyme.

Heterozygous patients with homocystinuria have a dominant negative effect. The cblE type of homocystinuria is a rare autosomal recessive disorder, which manifests with megaloblastic anemia. [50]

The most widely used method for newborn screening for homocystinuria is a semiquantitative bacterial inhibition assay for measuring methionine concentration in dried blood spots (DBS).

Because this method has resulted in a number of missed cases due to many factors, in 2004 Febriani developed an HPLC method with fluorescence detection to measure total homocysteine (tHcy) in DBS, which might be useful for newborn screening for homocystinuria. [51] One disk of DBS, 3 mm in diameter, was sonicated in 10 minutes. The extract was reduced with dithioerythritol and was derivatized with 4-aminosulfonyl-7fluoro-2,1,3-benzoxadiazole before injection into HPLC. This method showed good linearity (r = 0.996), precision (coefficient of variation range 2.7-5%), and excellent correlation coefficient between DBS and serum tHcy, both in control (r = 0.932) and patient samples (r = 0.952). By this method, the mean tHcy concentration in DBS of preterm newborns, full-term newborns, and adults was 1.4 ± 1.0, 2.5 ± 1.6, and 4.9 ± 1.5 µmol/L, respectively. The mean tHcy DBS concentrations in two cases of cystathionine beta-synthase (CBS) deficiency and one case of 5,10-methylenetetrahydrofolate reductase deficiency were 22.7 ± 2.88, 29.3 ± 1.90, and 41.3 µmol/L, respectively. This method, which is rapid, user friendly, and reliable, appears applicable to newborn screening of homocystinuria in place of methionine measurement.

Bártl et al developed a rapid screening procedure for simultaneous determination of cystathionine, methionine, and total homocysteine in DBS by liquid chromatography/tandem mass spectrometry. [52]

Serious complications of homocystinuria caused by cystathionine beta-synthase deficiency can be prevented by early intervention. In 2004, Refsum determined the prevalence of 6 specific mutations in 1133 newborn blood samples. [53] These results suggest that homocystinuria is more common than previously reported. Newborn screening for homocystinuria through mutation detection should be further considered.

In the study reported by Yamasaki-Yashiki et al, a new fluorometric microplate assay using a methionine-specific dehydrogenase and the resazurin/diaphorase system was established to determine the L-methionine concentration in an extract from dried blood spots for newborn mass screening for homocystinuria due to cystathionine b-synthase deficiency. [54]

The determination of thiodiglycolic acid levels in urine may help to characterize the metabolic imbalance of substances participating in methionine synthesis, which leads to hyperhomocystinuria. The determination of thiodiglycolic acid levels of the pretreated patient may indicate the degree of success of the treatment. [55]

CBS plays a key role in the intracellular disposal of homocysteine and is the single most common locus of mutations associated with homocystinuria. Sen et al used hydrogen-exchange mass spectrometry to map peptides, whose motions are correlated with transmission of interasteric inhibition and allosteric activation. [56] The mass spectrometric data provide an excellent correlation between kinetically and conformationally distinguishable states of the enzyme. A pathogenic regulatory domain mutant, D444N, is conformationally locked in 1 or 2 states sampled by the wild type of enzyme.

CBS deficiency is usually confirmed by assaying the enzyme activity in cultured skin fibroblasts. Another method of measuring the presence of CBS activity in human plasma or serum is using isotopically labeled substrates and LC-MS/MS instrumentation. Very low catalytic activities of enzymes originating from the liver can be measured in cell-free extracellular fluids. [57]

Smith et al present a study also with sensitive liquid chromatography mass spectrometry (LC–MS) method to assess the CBS activity in different kinds of cell extracts in order to diagnose CBS deficiency at the enzyme level and to evaluate the effects of diet or other manipulations on CBS activity in different cell types. [58] Secondly, it might be used in newborn screening for homocystinuria, since a faster and more sensitive method is required for an accurate diagnosis.

Since LC–MS methods use stabile isotope dilution, they usually offers the highest precision of the available techniques. This intra-assay variation is higher than a previously published the LC–MS method (1.4%) [57] ; however, those activities were measured in plasma and expressed as nmol/h X L. The CBS activity in cell extracts in this study is expressed per mg protein, which makes the rather inaccurate spectrophotometric protein determination (intra-assay CV 6%) the dominant factor invariation. In addition, the overall analysis time was reduced from 2 days to only 1 day for LC–MS methods. Another benefit of a LC–MS method is the possibility of up-scaling it to 96-wells plates, which enables high-throughput analyses. [58]  

For second-tier testing in newborn screening, LC–MS methods offer the possibility of measurement of CBS activity in plasma or cells exhibiting low CBS activity, like peripheral blood mononuclear cells. This would considerably lower diagnosis time and costs since cell culture would not be required. [58]

It is known that methionine in individuals affected by homocystinuria may not elevate until 7 days after birth. It is better to detect homocystinuria in newborns by quantification of the total homocysteine present. A quick and easy 96-well-plate method that requires a run time of 3 minutes per sample was developed. The method involves liquid chromatography tandem mass spectrometry and a deuterium-labeled homocystine internal standard. Authors anticipate the implementation of this method as a second-tier test to improve the screening algorithm for homocystinuria. [59]

Scherer et al showed that acute hyperhomocysteinemia (in vivo study) significantly reduced cholinesterase activity in the serum of rats of all ages tested. [60] They also observed that 500 μM homocysteine added to the incubation medium (in vitro study) significantly inhibited cholinesterase activity both in serum of rats and humans. These findings seem to reinforce the proposed associations of cholinesterase activity with hyperhomocysteinemia.

Patients with homocystinuria due to remethylation defects have an isolated brain choline deficiency, probably secondary to depletion of labile methyl groups produced by the transmethylation pathway. [61] Although biochemical studies suggest mild peripheral creatine deficiency, brain creatine is in the reference range, indicating a possible compartmentation phenomenon. Paradoxical increase of S-adenosylmethionine suggests that secondary inhibition of methylases contributes to the transmethylation defect in these conditions.

CBS carriers tend to have a higher total homocysteine level in the presence of folate deficiency than noncarriers. [24]

Sosvorová et al developed and evaluated a novelized gas chromatography method with flame ionization detection (GC-FID) tailored for the assessment of small changes in homocysteine concentrations in cerebrospinal fluid (CSF) during lumbar drainage in patients diagnosed with hydrocephalus. Lumbar drainage led to a decrease in homocysteine concentration, and decreasing levels corresponded to amelioration of the clinical state. Determination of CSF homocysteine in patients with confirmed or suspected hydrocephalus may serve as an independent marker for deciding on their further treatment strategy. [62]


Other Tests

Testing for heterozygosity may be valuable. The results can be used to guide the use of preventative measures such as reduced methionine intake and pyridoxine supplementation. Such testing is especially helpful in families of patients with homocystinuria.

Electroencephalographic abnormalities may be reflected as increased intracerebral pressure.

Cellular integrity may be affected in patients with high homocysteine levels, thus indicating that phase angle could be a valuable indicator of prognosis in classic homocystinuria. Phase angle is a measurement derived from bioelectrical impedance analysis that reflects cell membrane integrity and intracellular and extracellular water distribution. It is an independent predictor of mortality in several pathological conditions, such as cancer, amyotrophic lateral sclerosis, HIV infection, and kidney disease. [63]

In families with deceased probands, genetic diagnosis can be made by testing the parental MMACHC genes. If both of the parents have a MMACHC mutation at a heterozygous level, the proband’s genotype can be deduced. Zong et al report a study with 10 pedigrees, with the probands having clinically and biochemically confirmed combined methymalonic aciduria and homocystinuria. Nine variations in MMACHC were identified. Chorionic villi samples were collected in the first trimester of pregnancy for prenatal genetic diagnosis for three families. One fetus was found to be affected by cblC deficiency with compound heterozygous mutations of MMACHC, one fetus was determined to be a mutation carrier, while the third fetus had a normal genotype. By transabdominal chorionic villi sampling and DNA sequencing, genetic prenatal diagnose is performed and proved to be accurate and convenient. [64]

Hayıroglu et al present a case of 19-year-old boy. On electrocardiography, there was ST segment elevation in aVR derivation and ST segment depression in all other derivations. Transthoracic echocardiography revealed global hypokinesia. Merely plaques were detected ın coronary angiography. [65]