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Bax. J Transl Genet Genom 2020;4:1-16 I http://dx.doi.org/10.20517/jtgg.2020.08 Page 5

cardiac arrest and supraventricular tachycardia, mitral valve prolapse and systolic heart murmurs,
cardiomyopathy and endocarditis [2,16,22,47,61] .

Laboratory investigations
Although confirming a diagnosis of MNGIE is usually very straightforward, diagnostic testing is not
routinely performed by the majority of chemical pathology laboratories. A genetic confirmation of MNGIE
is mandatory. Testing for MNGIE is accomplished by the measurement of plasma and urine thymidine
and 2’-deoxyuridine concentrations, the measurement of thymidine phosphorylase activity and TYMP
sequencing.

Thymidine and 2’-deoxyuridine measurements are performed using high-performance liquid
chromatography with ultraviolet spectrophotometric (HPLC-UV) or tandem mass spectrometric
detection [62-64] . In contrast to healthy unaffected individuals who have undetectable plasma levels of these
metabolites (< 0.05 µmol/L), patients with MNGIE have markedly elevated plasma concentrations of
thymidine (> 3 µmol/L) and 2’-deoxyuridine (> 5 µmol/L) [12,13] . The urinary excretion of thymidine and
2’-deoxyuridine is also increased in patients but attention should be paid to the possibility of metabolite
[11]
catabolism by contaminating bacteria . Preservatives used for chemical urinalysis specimens, such
tartaric acid, boric acid, perchloric acid or thymol, should be included in collections that are not analysed
immediately.

Thymidine phosphorylase activity is measured either spectrophotometrically or by HPLC-UV by the
endpoint determination of the thymine formed after incubation of buffy coat homogenates in the presence
[65]
of an excess of the enzyme’s substrate, thymidine . The assay of enzyme activity is generally required
to complement the measurement of plasma metabolite concentrations, or following the identification of
novel variants of the TYMP gene, or when clinics do not have access to sequencing of TYMP. Thymidine
phosphorylase activity is severely reduced in the leukocytes of patients with MNGIE, showing either no
activity or activities less than 10% (0-46 nmol thymidine formed/hour/mg protein) of healthy unaffected
controls (253-1000 nmol thymidine formed/hour/mg protein) [11,13] . Heterozygous carriers of TYMP
mutations have approximately 35% of residual thymidine phosphorylase activity and have undetectable
[66]
concentrations of plasma deoxyribonucleosides .
The benchmark for the diagnosis of MNGIE is the identification of homozygous or compound
heterozygous allelic pathogenic TYMP variants, which are detected by Sanger or next generation gene
sequencing, mitochondrial disease gene panels or whole exosome sequencing. The Human Gene Mutation
Database (HGMD Professional 2019.4, accessed January 2020) reports 97 different mutations which
have been mapped to exonic or intronic regions, with some identified as benign and other as pathogenic
[67]
variants . These mutations include: 59 missense/nonsense, 14 splice site mutations, 13 small deletions,
7 small insertions, 2 small indels, 1 gross deletion and 1 gross insertion . These mutations have been
[68]
mapped to either exonic or intronic regions, with some identified as benign and the others as pathogenic
variants. In the event of a non-identified mutation or variant of uncertain significance being detected,
metabolite and thymidine phosphorylase activity testing should be performed to confirm or exclude a
[10]
diagnosis of MNGIE .
Few patients have presented with endocrine and other metabolic dysfunctions, including diabetes,
elevations in amylase, glucose intolerance and hypergonadotropic hypogonadism but these are non-specific
and largely do not contribute to the diagnosis of MNGIE [16,21,69] .

Histological and biochemical studies on skeletal muscle biopsies may reveal abnormalities of a
mitochondrial myopathy, including ragged-red fibres, cytochrome c oxidase-deficient fibres, ultra-