Clinical and bi-genomic DNA findings of patients suspected to have mitochondrial diseases.

Background: Mitochondrial diseases are the most common group of inherited metabolic disorders, causing difficulties in definite diagnosis due to clinical and genetic heterogeneity. Clinical components are predominantly associated with pathogenic variants shown in nuclear or mitochondrial genomes that affect vital respiratory chain function. The development of high-throughput sequencing technologies has accelerated the elucidation of the genetic etiology of many genetic diseases that previously remained undiagnosed. Methods: Thirty affected patients from 24 unrelated families with clinical, radiological, biochemical, and histopathological evaluations considered for mitochondrial diseases were investigated. DNA isolated from the peripheral blood samples of probands was sequenced for nuclear exome and mitochondrial DNA (mtDNA) analyses. MtDNA sequencing was also performed from the muscle biopsy material in one patient. For segregation, Sanger sequencing is performed for pathogenic alterations in five other affected family members and healthy parents. Results: Exome sequencing revealed 14 different pathogenic variants in nine genes encoding mitochondrial function peptides (AARS2, EARS2, ECHS1, FBXL4, MICOS13, NDUFAF6, OXCT1, POLG, and TK2) in 12 patients from nine families and four variants in genes encoding important for muscle structure (CAPN3, DYSF, and TCAP) in six patients from four families. Three probands carried pathogenic mtDNA variations in two genes (MT-ATP6 and MT-TL1). Nine variants in five genes are reported for the first time with disease association: (AARS2: c.277C>T/p.(R93*), c.845C>G/p.(S282C); EARS2: c.319C>T/p.(R107C), c.1283delC/p.(P428Lfs*); ECHS1: c.161G>A/p.(R54His); c.202G>A/p.(E68Lys); NDUFAF6: c.479delA/p.(N162Ifs*27); and OXCT1: c.1370C>T/p.(T457I), c.1173-139G>T/p.(?). Conclusion: Bi-genomic DNA sequencing clarified genetic etiology in 67% (16/24) of the families. Diagnostic utility by mtDNA sequencing in 13% (3/24) and exome sequencing in 54% (13/24) of the families prioritized searching for nuclear genome pathologies for the first-tier test. Weakness and muscle wasting observed in 17% (4/24) of the families underlined that limb-girdle muscular dystrophy, similar to mitochondrial myopathy, is an essential point for differential diagnosis. The correct diagnosis is crucial for comprehensive genetic counseling of families. Also, it contributes to making treatment-helpful referrals, such as ensuring early access to medication for patients with mutations in the TK2 gene.

Clinical pattern, mutations and in vitro residual activity in 33 patients with severe 5, 10 methylenetetrahydrofolate reductase (MTHFR) deficiency.

BACKGROUND: Severe methylenetetrahydrofolate reductase (MTHFR) deficiency is a rare inborn defect disturbing the remethylation of homocysteine to methionine (<200 reported cases). This retrospective study evaluates clinical, biochemical genetic and in vitro enzymatic data in a cohort of 33 patients. METHODS: Clinical, biochemical and treatment data was obtained from physicians by using a questionnaire. MTHFR activity was measured in primary fibroblasts; genomic DNA was extracted from cultured fibroblasts. RESULTS: Thirty-three patients (mean age at follow-up 11.4 years; four deceased; median age at first presentation 5 weeks; 17 females) were included. Patients with very low (<1.5%) mean control values of enzyme activity (n = 14) presented earlier and with a pattern of feeding problems, encephalopathy, muscular hypotonia, neurocognitive impairment, apnoea, hydrocephalus, microcephaly and epilepsy. Patients with higher (>1.7-34.8%) residual enzyme activity had mainly psychiatric symptoms, mental retardation, myelopathy, ataxia and spasticity. Treatment with various combinations of betaine, methionine, folate and cobalamin improved the biochemical and clinical phenotype. During the disease course, patients with very low enzyme activity showed a progression of feeding problems, neurological symptoms, mental retardation, and psychiatric disease while in patients with higher residual enzyme activity, myelopathy, ataxia and spasticity increased. All other symptoms remained stable or improved in both groups upon treatment as did brain imaging in some cases. No clear genotype-phenotype correlation was obvious. DISCUSSION: MTHFR deficiency is a severe disease primarily affecting the central nervous system. Age at presentation and clinical pattern are correlated with residual enzyme activity. Treatment alleviates biochemical abnormalities and clinical symptoms partially.

Monitoring tyrosinaemia type I: Blood spot test for nitisinone (NTBC).

BACKGROUND: Quantification of nitisinone, 2-(nitro-4-trifluoromethylbenzoyl)1,3-cyclohexanedione (NTBC) has been repeatedly described. Nevertheless monitoring of NTBC has not yet become part of routine therapy surveillance in tyrosinaemia type I (OMIM 276700). We developed a blood spot test to facilitate collection and transport of samples. Furthermore, the test material can be used for determination of other parameters like tyrosine and succinylacetone. METHOD: For quantification of NTBC in blood spots filter paper discs of 3.2mm diameter were extracted with 150 µL methanol containing mesotrione as internal standard (IS). Analysis was done by UPLC-MS/MS on a Xevo mass spectrometer (ESI+), (MRM). Parent ions were 330.05 for NTBC and 340.05 for IS, daughter ions were m/z 217.95 and m/z 125.95 for NTBC, and m/z 227.95 and m/z 103.95 for IS. RESULTS: The calibration curve for NTBC in blood spots was linear from 0.1 µmol/L to 100 µmol/L. Recovery exceeded 73.1%, CV intraday and interday were below 9.6%. Instrumental run time was 2.5 min. Sensitivity of the method was 0.1 µmol/L. NTBC concentrations in plasma were higher than in blood spots by a factor of 1.56 ± 0.13. CONCLUSION: As demonstrated in patients with tyrosinaemia type I quantification of NTBC by UPLC-MS/MS in blood spots is feasible and gives valuable information for monitoring NTBC treatment.

Biotinidase deficiency: novel mutations and their biochemical and clinical correlates.

Biotinidase deficiency is a defect in the recycling of the vitamin biotin. Biotin supplementation can markedly improve the neurological and cutaneous symptoms of affected children and prevent symptoms in children identified by newborn screening or treated since birth. We have determined thirteen novel mutations in children with the disorder. Two nonsense mutations, eight single missense mutations, three allelic double missense mutations, and two are polymorphisms were identified in the biotinidase gene (BTD). One of the missense mutations, c.734G>A (p. C245Y), is the first to be reported that alters the cysteine in the putative location crucial for ester formation and binding of the biotinyl-moiety in the active site of the enzyme. These mutations add to the growing list of mutations that are helping to delineate structure/function relationships of the enzyme.

Highest heterogeneity for cystic fibrosis: 36 mutations account for 75% of all CF chromosomes in Turkish patients.

We analyzed the CFTR locus in 83 Turkish cystic fibrosis patients to identify mutations, haplotypes, and the carrier frequency in the population. We detected 36 different mutations in 125 (75%) of the total 166 CF chromosomes. Seven novel mutations were identified: four missense (K68E, Q493P, E608G, and V1147I), two splice-site (406 -3T > C and 3849 +5G > A), and one deletion (CFTRdele17b,18). The data showed that the Turkish population has the highest genetic heterogeneity at the CFTR locus reported so far. The results of this thorough molecular analysis at the CFTR locus of a population not of European descent shows that CF is not uncommon in all such populations. The large number of mutations present, as well as the high heterogeneity in haplotypes associated with the mutations suggests that most of the mutations have persisted for a long time in the population. Consistently, the carrier frequency is assessed to be high, indicating that the disease in the population is ancient.