Paz Briones

The P28T Mutation in the GALK1 Gene Accounts for Galactokinase Deficiency in Roma (Gypsy) Patients across Europe

Galactokinase deficiency is an inborn error of metabolism that, if untreated, results in the development of cataracts in the first weeks of life. The disorder is rare worldwide, but has a high incidence among the Roma (Gypsies). In 1999, we reported the founder Romani mutation, P28T, identified in affected families from Bulgaria. Subsequent studies have detected the same mutation in Romani patients from different European countries. The screening of 803 unrelated control individuals of Romani ethnicity from Bulgaria, Hungary, and Spain has shown an overall carrier rate of 1:47 and an expected incidence of affected births about 1:10,000. Using disease haplotype analysis, the age of the P28T mutation was estimated at 750 y, preceding the splits of the proto-Roma into the numerous populations resident in Europe today. The findings suggest that the mutation has spread with the early diaspora of the Roma throughout Europe. Superimposed on this old distribution pattern is the new migration wave of the last decade, with large numbers of Roma moving to Western Europe as a result of the economic changes in the East and the wars in former Yugoslavia. The changing demographic pattern of Romani minorities can be expected to lead to a homogenization of the incidence of “private” Romani disorders and founder mutations. The P28T mutation is thus likely to account for a high proportion of galactokinase deficiency cases across Europe. Mutation-based pilot newborn screening programs would provide current incidence figures and help to design long-term prevention of infantile cataracts due to galactokinase deficiency.

Hearing loss in a patient with the myopathic form of mitochondrial DNA depletion syndrome and a novel mutation in the TK2 gene.

Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is a devastating disorder of infancy caused by a significant reduction of the number of copies of mitochondrial DNA in one or more tissues. We report a Spanish patient with the myopathic form of MDS, harboring two mutations in the thymidine kinase 2 gene (TK2): a previously reported deletion (p.K244del) and a novel nucleotide duplication in the exon 2, generating a frameshift and premature stop codon. Sensorineural hearing loss was a predominant symptom in the patient and a novel feature of MDS due to TK2 mutations. The patient survived up to the age of 8.5 y, which confirms that survival above the age of 5 y is not infrequent in patients with MDS due to TK2 deficiency.

Comparison between high performance liquid chromatography and capillary zone electrophoresis for the diagnosis of congenital disorders of glycosylation

Transferrin isoelectric focusing (IEF) is the most widely used method to screen for congenital disorders of glycosylation (CDG). Our aim was to compare high performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE) procedures for serum sialotransferrin analysis. 58 serum samples were processed both by CZE and HPLC: 35 were from paediatric controls, 18 from patients with an altered sialotransferrin IEF pattern and 5 were transferrin variant samples. HPLC analysis was performed with an anion-exchange column with spectrophotometric detection at 470 nm. CZE analysis was done using the commercial CEofix-CDT kit with spectrophotometric detection at 200 nm. Passing-Bablok regression analysis showed good agreement for tri-, tetra- and penta-sialotransferrin by both procedures. But for disialotransferrin, higher values were observed by the HPLC procedure. The HPLC and CZE methods allowed reproducible separation and analysis of single transferrin glycoforms with similar peak patterns. All patients presented values outside the range established in our control population either by HPLC or by CZE, even in patients with moderate forms of CDG that had been difficult to detect by IEF. In conclusion, measurement of sialotransferrin isoforms and interpretation using method-specific reference values may offer some advantages for the diagnosis of CDG as compared with the standard IEF procedure.

Congenital disorder of glycosylation type Ia revealed by hypertransaminasemia and failure to thrive in a young boy with normal neurodevelopment.

Congenital disorders of glycosylation (CDG), formerly called carbohydrate deficient glycoprotein syndromes, are a group of inherited metabolic errors resulting in defective synthesis or processing of N-glycoproteins or, in some cases, of O-glycans. Hypoglycosylation of proteins leads to variable, generally severe, problems in affected individuals. Since CDG was first described in 1980 (1) several subtypes have been identified. Following recent recommendations (2), congenital disorders of N-glycosylation are divided into two groups. CDG type I comprises defects in the assembly of N-glycans in cytosol and endoplasmic reticulum, and CDG type II refers to defects in the processing of the protein-bound glycans in the endoplasmic reticulum or in the Golgi compartment. The most frequent subtype is CDG Ia, which originates from a phosphomannomutase 2 deficiency caused by a mutation of the PMM gene locus on chromosome 16p13 (3,4). We report the case of a 6-year-old boy who presented at 2 years of age with abnormal serum transaminases and failure to thrive as the main signs of a type Ia CDG syndrome. Neurologic signs were detected only on careful examination of the patient, as he showed only slight motor clumsiness and his neurodevelopment quotient (Brunet-Lézine) was normal.

Severe encephalopathy associated to pyruvate dehydrogenase mutations and unbalanced coenzyme Q10 content

Coenzyme Q10 (CoQ10) deficiency is associated to a variety of clinical phenotypes including neuromuscular and nephrotic disorders. We report two unrelated boys presenting encephalopathy, ataxia, and lactic acidosis, who died with necrotic lesions in different areas of brain. Levels of CoQ10 and complex II+III activity were increased in both skeletal muscle and fibroblasts, but it was a consequence of higher mitochondria mass measured as citrate synthase. In fibroblasts, oxygen consumption was also increased, whereas steady state ATP levels were decreased. Antioxidant enzymes such as NQO1 and MnSOD and mitochondrial marker VDAC were overexpressed. Mitochondria recycling markers Fis1 and mitofusin, and mtDNA regulatory Tfam were reduced. Exome sequencing showed mutations in PDHA1 in the first patient and in PDHB in the second. These genes encode subunits of pyruvate dehydrogenase complex (PDH) that could explain the compensatory increase of CoQ10 and a defect of mitochondrial homeostasis. These two cases describe, for the first time, a mitochondrial disease caused by PDH defects associated with unbalanced of both CoQ10 content and mitochondria homeostasis, which severely affects the brain. Both CoQ10 and mitochondria homeostasis appears as new markers for PDH associated mitochondrial disorders.

Expanding the clinical phenotypes of MT-ATP6 mutations

Mitochondrial DNA mutations at MT-ATP6 gene are relatively common in individuals suffering from striatal necrosis syndromes. These patients usually do not show apparent histochemical and/or biochemical signs of oxidative phosphorylation dysfunction. Because of this, MT-ATP6 is not typically analyzed in many other mitochondrial disorders that have not been previously associated to mutations in this gene. To correct this bias, we have performed a screening of the MT-ATP6 gene in a large collection of patients suspected of suffering different mitochondrial DNA (mtDNA) disorders. In three cases, biochemical, molecular-genetics and other analyses in patient tissues and cybrids were also carried out. We found three new pathologic mutations. Two of them in patients showing phenotypes that have not been commonly associated to mutations in the MT-ATP6 gene. These results remark the importance of sequencing the MT-ATP6 gene in patients with striatal necrosis syndromes, but also within other mitochondrial pathologies. This gene should be sequenced at least in all those patients suspected of suffering an mtDNA disorder disclosing normal results for histochemical and biochemical analyses of respiratory chain.

Role of creatine as biomarker of mitochondrial diseases

Recent investigations have suggested creatine (Cr) as an additional biomarker of mitochondrial diseases. With the aim of corroborating previous findings, we have studied plasma Cr in a cohort of 33 patients with different mitochondrial diseases. Cr was clearly increased in 9 out of 33 patients. Therefore, positive patients represent only 28% of the total number, suggesting that Cr is not a sensitive biomarker of mitochondrial diseases although it does present an acceptable specificity (83%). High plasma Cr, together with other biomarkers, might be useful to reinforce the diagnosis of mitochondrial diseases.

Peripheral neuropathy with ataxia in childhood as a result of the G8363A mutation in mitochondrial DNA.

Peripheral neuropathy has been identified in children with mitochondrial encephalomyopathies but not as a main clinical landmark. Here we report the clinical, electrophysiologic, biochemical, and genetic findings in a family who harbors the G8363A mutation in the tRNALys gene of mitochondrial DNA. Affected individuals presented with peripheral neuropathy and ataxia as the main clinical sign. Additional involvement included muscle weakness and multiple lipomatosis. Other common clinical characteristics associated with the G8363A mutation, such as cardiomyopathy and myoclonus epilepsy, were not observed. These findings suggest that a mitochondrial disease should be considered in the differential diagnosis of children with here-doataxic syndrome and peripheral neuropathy of unknown origin.

Association between coenzyme Q10 and glucose transporter (GLUT1) deficiency

BackgroundIt has been demonstrated that glucose transporter (GLUT1) deficiency in a mouse model causes a diminished cerebral lipid synthesis. This deficient lipid biosynthesis could contribute to secondary CoQ deficiency. We report here, for the first time an association between GLUT1 and coenzyme Q10 deficiency in a pediatric patient.Case presentationWe report a 15 year-old girl with truncal ataxia, nystagmus, dysarthria and myoclonic epilepsy as the main clinical features. Blood lactate and alanine values were increased, and coenzyme Q10 was deficient both in muscle and fibroblasts. Coenzyme Q10 supplementation was initiated, improving ataxia and nystagmus. Since dysarthria and myoclonic epilepsy persisted, a lumbar puncture was performed at 12 years of age disclosing diminished cerebrospinal glucose concentrations. Diagnosis of GLUT1 deficiency was confirmed by the presence of a de novo heterozygous variant (c.18+2T>G) in the SLC2A1 gene. No mutations were found in coenzyme Q10 biosynthesis related genes. A ketogenic diet was initiated with an excellent clinical outcome. Functional studies in fibroblasts supported the potential pathogenicity of coenzyme Q10 deficiency in GLUT1 mutant cells when compared with controls.ConclusionOur results suggest that coenzyme Q10 deficiency might be a new factor in the pathogenesis of G1D, although this deficiency needs to be confirmed in a larger group of G1D patients as well as in animal models. Although ketogenic diet seems to correct the clinical consequences of CoQ deficiency, adjuvant treatment with CoQ could be trialled in this condition if our findings are confirmed in further G1D patients.

X-inactivation of HSD17B10 revealed by cDNA analysis in two female patients with 17β-hydroxysteroid dehydrogenase 10 deficiency.

17β-Hydroxysteroid dehydrogenase 10 (HSD10) is a mitochondrial enzyme involved in the degradation pathway of isoleucine and branched-chain fatty acids. The gene encoding HSD10, HSD17B10, has been reported as one of the few genes that escapes X-inactivation. We previously studied two female patients with HSD10 deficiency, one of them was severely affected and the other presented a mild phenotype. To elucidate as to why these two carriers were so differently affected, cDNA analyses were performed. The HSD17B10 cDNA of eight control cell lines, two hemizygous patients and two carriers was obtained from cultured fibroblasts, amplified by PCR and sequenced by standard methods. All HSD17B10 cDNAs were quantified by real-time PCR. In the fibroblasts of the female patient who presented with the severe phenotype, only the mutant allele was identified in the cDNA sequence, which was further confirmed by relative quantification (RQ) of HSD17B10 cDNA. This is in agreement with an unfavourable X-inactivation. The other female patient, with slight clinical affectation, showed the presence of both mutant and wild-type alleles in the cDNA sequence, which was confirmed by RQ of HSD17B10 cDNA in fibroblasts. This is in line with normal X-inactivation and the expression of both alleles in different cells (functional mosaicism). RQ results of HSD17B10 cDNA did not differ significantly between male and female controls, which indicate that the genetic doses of mRNA of HSD17B10 was the same in both sexes. In conclusion, these results suggest that the HSD17B10 gene does not escape X-inactivation as has been reported previously.