Miniature integrated micro-spectrometer array for snap shot multispectral sensing.

An array of micro spectrometers for parallel spectral sensing is designed, set up and tested. It utilizes a planar prism grating combination to obtain an almost linear optical system of 6 mm length only. Arranging such micro spectrometers in an array configuration yields 2'000 spectrometers when utilizing a common 4/3" CCD image sensor well adapted to e.g. microscopic image dimensions. The application in microscopic imaging in the 450-900 nm spectral range is demonstrated as proof of concept, which can be adapted to massively parallel sensing in the frame of integrated sensor concepts.

Duplications of the functional CYP21A2 gene are primarily restricted to Q318X alleles: evidence for a founder effect.

CONTEXT: Rare haplotypes with Q318X mutations and duplicated CYP21A2 genes have been reported to occur in different populations to a varying extent. Discrimination between a normal (Q318X mutation on one of the duplicated CYP21A2 genes) and a congenital adrenal hyperplasia (CAH, Q318X mutation without duplicated functional gene) allele is of importance, particularly for prenatal diagnosis and the respective genetic counseling. Although methods to differentiate between such alleles have been published only recently, it remains unclear with which frequency Q318X mutations are associated with duplicated CYP21A2 genes and whether these haplotypes have a common ancestry. SUBJECTS AND METHODS: Human leukocyte antigen (HLA) typing has been performed in 38 unrelated individuals and in 11 family members detected to carry a Q318X mutation in the course of CYP21 genotyping using sequence, multiplex ligation-dependent probe amplification, and Southern blot analyses. RESULTS: The majority (n = 32, 84.2%) of the 38 unrelated individuals carrying the Q318X mutation had the trimodular RCCX haplotype, carrying the Q318X mutation on a duplicated CYP21A2 gene. Twenty-two individuals of these 32 (68.8%) were of the rare HLA-B*50-Cw*06 haplotype, suggesting a common ancestry of this haplotype. In five (13.2%) of the 38 subjects, the Q318X mutation was not associated with a duplicated CYP21A2 gene and thus represents a CAH allele. None of these five patients had the above mentioned HLA haplotype. CONCLUSION: The majority of individuals in whom Q318X mutations are detected carry a duplicated functional CYP21A2 gene and the rare HLA-B*50-Cw*06 haplotype.

Frequency of mitochondrial transfer RNA mutations and deletions in 225 patients presenting with respiratory chain deficiencies.

OBJECTIVE: To evaluate the frequency of pathogenic mtDNA transfer RNA mutations and deletions in biochemically demonstrable respiratory chain (RC) deficiencies in paediatric and adult patients. METHODS: We screened for deletions and sequenced mitochondrial transfer RNA genes in skeletal muscle DNA from 225 index patients with clinical symptoms suggestive of a mitochondrial disorder and with biochemically demonstrable RC deficiency in skeletal muscle. RESULTS: We found pathogenic mitochondrial DNA mutations in 29% of the patients. The detection rate was significantly higher in adults (48%) than in the paediatric group (18%). Only one pathogenic mutation was detected in the neonatal group. In addition, we describe seven novel transfer RNA sequence variations with unknown pathogenic relevance (six homoplasmic and one heteroplasmic) and 13 homoplasmic polymorphisms. One heteroplasmic transfer RNA(Leu(UUR)) A>G mutation at position 3274 is associated with a distinct neurological syndrome. CONCLUSIONS: We provide an estimation of the frequency of mitochondrial transfer RNA mutations and deletions in paediatric and adult patients with respiratory chain deficiencies.

Mitochondrial diseases represent a risk factor for valproate-induced fulminant liver failure.

We report on 3 siblings (2 females and 1 male) with chronic progressive external ophthalmoplegia (CPEO), compatible with inherited mitochondrial cytopathy. The younger of the two sisters died at the age of 37 due to progressive respiratory failure. The older one presented with a status epilepticus at the age of 39 and was treated with valproate. Five months after the start of treatment, she developed fulminant liver failure and died. The brother has suffered from CPEO since early childhood but has had so far no other symptoms of a mitochondrial disease. A muscle biopsy from the younger sister revealed ragged-red fibers and decreased activities of complex I and IV of the respiratory chain but no pathogenic mutations in the mitochondrial tRNA genes or in several locations in the coding region of the mitochondrial genome. In the older sister's liver (obtained post-mortem), mitochondrial DNA was fragmented and could not be investigated. The clinical presentation and the biochemical findings suggest that all 3 siblings suffered from a mitochondrial cytopathy. Since mitochondrial cytopathies and valproate-induced fulminant liver failure are both rare events, an association between them is likely. Mitochondrial diseases should therefore be considered as a risk factor for valproate-induced liver failure and be excluded before treatment with valproate.

Microvesicular steatosis, hemosiderosis and rapid development of liver cirrhosis in a patient with Pearson's syndrome.

BACKGROUND/AIMS: Pearson's marrow-pancreas syndrome consists of refractory sideroblastic anemia with vacuolization of marrow precursors and exocrine pancreas dysfunction. Patients with this disease usually have large deletions of the mitochondrial genome. We report a patient with Pearson's syndrome who had predominantly hepatic manifestations such as microvesicular steatosis, hemosiderosis and rapidly developing cirrhosis. METHODS: Analysis of the mitochondrial and nuclear genomes, determination of enzyme activities and of the hepatic iron content were performed using standard techniques of molecular biology and biochemistry. RESULTS: The patient had typical ringed sideroblasts in a bone marrow smear and a 7436-bp deletion of the mitochondrial genome in all tissues investigated, compatible with Pearson's syndrome. He died within 3 months after birth due to liver failure. Histopathological analysis of the liver revealed complete cirrhosis with signs of chronic cholestasis, microvesicular steatosis and massive hemosiderosis. In addition, the patient was heterozygous for the C282Y and H63D mutations of the hemochromatosis gene. CONCLUSIONS: Pearson's syndrome should be added to the list of neonatal diseases which can cause microvesicular steatosis, hepatic accumulation of iron and liver cirrhosis.

A systematic mutation screen of 10 nuclear and 25 mitochondrial candidate genes in 21 patients with cytochrome c oxidase (COX) deficiency shows tRNA(Ser)(UCN) mutations in a subgroup with syndromal encephalopathy.

COX deficiency is believed to be the most common defect in neonates and infants with mitochondrial diseases. To explore the causes of this group of disorders, we examined 25 mitochondrial genes (three COX subunit genes and 22 tRNA genes) and 10 nuclear COX subunit genes for disease associated mutations using PCR-SSCP and direct sequencing of polymorphic SSCP fragments. DNA from one patient with severe COX deficiency and with consanguineous parents was entirely sequenced. The patient population consisted of 21 unrelated index patients with mitochondrial disorders and predominant (n=7) or isolated (n=14) COX deficiency. We detected two distinct tRNA(Ser)(UCN) mutations, which have been recently described in single kindreds, in a subgroup of four patients with COX deficiency, deafness, myoclonic epilepsy, ataxia, and mental retardation. Besides a number of nucleotide variants, a single novel missense mutation, which may contribute to the disease phenotype, was found in the mitochondrial encoded COX 1 gene (G6480A). Mutations in nuclear encoded COX subunit genes were not detected in this study.

Progressive myoclonus epilepsy and mitochondrial myopathy associated with mutations in the tRNA(Ser(UCN)) gene.

We report seven unrelated families with mitochondrial tRNA(Ser(UCN)) gene mutations at three different loci. A novel G7497A mutation is found in two families, both of which present with progressive myopathy, ragged-red fibers, lactic acidosis, and deficiency of respiratory chain complexes I and IV. This mutation presumably affects the tertiary tRNA(Ser(UCN)) dihydrouridine interaction. Mutations 7472 insC and T7512C, found in three and two families, respectively, are associated with myoclonus epilepsy, deafness, ataxia, cognitive impairment, and complex IV deficiency. No ragged-red fibers or ultrastructural abnormalities are seen. It is interesting that 6 of our 7 index patients are apparently homoplasmic, indicating a minor pathogenetic power of the tRNA(Ser(UCN)) mutations.

A novel mitochondrial tRNA(Phe) mutation inhibiting anticodon stem formation associated with a muscle disease.

We have identified a novel mitochondrial (mt) DNA mutation in the tRNA(Phe)-gene in a patient with an isolated mitochondrial myopathy. This T to C transition at position 618 disrupts a strictly conserved base pair within the anticodon stem of tRNA(Phe). Computer analysis showed that the affected base pair is essential for anticodon stem formation of tRNA(Phe). The mutant mtDNA was heteroplasmic in skeletal muscle (95% mutant) and peripheral blood cells (20% mutant) from the patient but was undetectable in blood cells from his healthy sister. The patient presented with ragged red fibers and reduced activities of complex I and complex III in skeletal muscle. The T618C mutation described here is the second found in this region. Both mutations affect the same base pair of the tRNA(Phe) anticodon stem substantiating the pathogenic nature of both mutations.

Detection and characterization of mitochondrial DNA rearrangements in Pearson and Kearns-Sayre syndromes by long PCR.

We used a strategy based on long PCR (polymerase chain reaction) for detection and characterization of mitochondrial DNA (mtDNA) rearrangements in two patients with clinical signs suggesting Pearson syndrome and Kearns-Sayre syndrome (KSS), respectively, and one patient with myopathic symptoms of unidentified origin. Mitochondrial DNA rearrangements were detected by amplification of the complete mitochondrial genome (16.6 kb) using long PCR with primers located in essential regions of the mitochondrial genome and quantified by three-primer PCR. Long PCR with deletion-specific primers was used for identification and quantitative estimation of the different forms of rearranged molecules, such as deletions and duplications. We detected significant amounts of a common 7.4-kb deletion flanked by a 12-bp direct repeat in all tissues tested from the patient with Pearson syndrome. In skeletal muscle from the patient with clinical signs of KSS we found significant amounts of a novel 3.7-kb rearrangement flanked by a 4-bp inverted repeat that was present in the form of deletions as well as duplications. In the patient suffering from myopathic symptoms of unidentified origin we did not detect rearranged mtDNA in blood but found low levels of two rearranged mtDNA populations in skeletal muscle, a previously described 7-kb deletion flanked by a 7-bp direct repeat and a novel 6.6-kb deletion with no repeat. These two populations, however, were unlikely to be the cause of the myopathic symptoms as they were present at low levels (10-40 ppm). Using a strategy based on screening with long PCR we were able to detect and characterize high as well as low levels of mtDNA rearrangements in three patients.

Combined 3-methylglutaconic and 3-hydroxy-3-methylglutaric aciduria with endocardial fibroelastosis and dilatative cardiomyopathy in male and female siblings with partial deficiency of complex II/III in fibroblasts.

We report on 2 children, brother and sister, who presented with cardiomyopathy and muscular hypotonia at the age of B months. They both excreted significant amounts of 3-hydroxy-3-methylglutaric acid (3-HMG) and 3-methylglutaconic acid (3-MGC) but no 3-methylglutaric acid (3-MG). Enzyme analysis in fibroblasts revealed normal activities of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase and of 3-methylglutaconyl hydratase and other enzymes of 3-HMG metabolism. Loading tests with leucine did not affect the excretion of 3-HMG and 3-MGC. The girl died as a result of her cardiomyopathy, while the boy recovered and was treated with cardiac supportive therapy. He showed a steady improvement during his clinical course with biochemical normalization of the urinary excretion of 3-HMG, concomitant with marked improvement in the hypertrophic cardiomyopathy. In cultured fibroblasts from both patients a reduced activity of complex II/III of the respiratory chain was measured which may be the cause of this new type of 3-HMG uria. Analysis of mitochondrial DNA heart muscle, liver and fibroblast culture of the patient did not reveal any major mitochondrial DNA rearrangements (deletion, duplication) or any point mutation that had been described in association with mitochondrial cardiomyopathy.