Sci-Thur PM: YIS - 09: Development of a graphite probe calorimeter for absolute clinical dosimetry: Numerical design optimization, prototyping and experimental proof-of-concept.

In this work, the feasibility of absolute dose to water measurements using a small-scale graphite probe calorimeter (GPC) in a clinical environment is established. A numerical design optimization study was conducted by simulating the heat transfer in the GPC resulting from irradiation using a finite element method software package. The choice of device shape, dimensions and materials was made to minimize the heat loss in the sensitive volume of the GPC. The resulting design, which incorporates a novel aerogel-based thermal insulator, was built in-house. Absorbed dose to water measurements were made under standard conditions in a 6 MV 1000 MU/min photon beam and subsequently compared against TG-51 derived values. The average measured dose to water was 95.7 ±1.4 cGy/100 MU, as compared to an expected value of 96.6 cGy/100 MU. The Monte Carlo-calculated graphite to water dose conversion factor was 1.099, while the derived heat loss correction factors varied between 1.005 and 1.013. The most significant sources of uncertainty were the repeatability (type A, 1.4%) and thermistor calibration (type B, 2.1%). The contribution of these factors to the overall uncertainty is expected to decrease significantly upon the implementation of active thermal stabilization provided by a temperature controller and direct electrical calibration, respectively. This work demonstrates the feasibility of using the GPC as a practical clinical absolute photon dosimeter and will serve as the basis for a miniaturized version applicable to small and composite fields.

Sci-Sat AM: Brachy - 03: Feasibility study of the determination of absorbed dose to water using a fricke based system.

By measuring the dose to water directly a metrology standard, independent of air kerma, can be developed to make the basis of HDR brachytherapy dosimetry consistent with current dosimetry methods for external radiation beams. The Fricke dosimeter system, a liquid chemical dosimeter, provides a means of measuring the absorbed dose rate to water directly by measuring the radiation-induced change in absorption of the Fricke solution. In an attempt to measure the absorbed dose to water directly for a 192 Ir HDR brachytherapy source a ring shaped Fricke holder was constructed from PMMA, essentially following the work of Austerlitz et al. (Med. Phys. 2008). Benchmark measurements conducted in a 60 Co beam yielded a standard uncertainty in the absorption reading of 0.16 %, comparable with previous results in the literature. Measurements of the standard uncertainty of the control (unirradiated) solution using the holder yielded 0.2 %, indicating good process control and minimal contamination from the holder itself. However, it was found that the holder sealing method (to allow measurements in a water phantom) significantly contaminated the Fricke solution, resulting in an excessive background reading. Irradiations were therefore conducted in air to determine the feasibility of the procedure. Irradiations with a 17 GBq source gave a standard uncertainty of approximately 0.5 %, indicating that the target uncertainty of 1.5% for the measurement of absorbed dose to water using a Fricke-based primary standard is achievable. This would be comparable with calorimeter-based systems currently being developed.

Information for genetic management of mtDNA disease: sampling pathogenic mtDNA mutants in the human germline and in placenta.

BACKGROUND: Families with a child who died of severe, maternally inherited mitochondrial DNA (mtDNA) disease need information on recurrence risk. Estimating this risk is difficult because of (a) heteroplasmy-the coexistence of mutant and normal mtDNA in the same person-and (b) the so-called mitochondrial bottleneck, whereby the small number of mtDNAs that become the founders for the offspring cause variation in dose of mutant mtDNA. The timing of the bottleneck and of segregation of mtDNA during foetal life determines the management options. Therefore, mtDNA heteroplasmy was studied in oocytes and placenta of women in affected families. RESULTS: One mother of a child dying from Leigh syndrome due to the 9176T-->C mtDNA mutation transmitted various loads of mutant mtDNA to < or =3 of 20 oocytes. This was used to estimate recurrence as < or =5%. She subsequently conceived a healthy son naturally. Analysis of the placenta showed that some segregation also occurred during placental development, with the mutant mtDNA load varying by >10% in a placenta carrying 65% 3243A-->G mutant mtDNA. DISCUSSION: This is the first report of (a) an oocyte analysis for preconception counselling, specifically, refining recurrence risks of rare mutations and (b) a widely different load of a pathogenic mtDNA mutation in multiple oocytes, apparently confined to the germline, in an asymptomatic carrier of an mtDNA disease. This suggests that a major component of the bottleneck occurs during oogenesis, probably early in the foetal life of the mother. The variable mutant load in placenta implies that estimates based on a single sample in prenatal diagnosis of mtDNA disorders have limited accuracy.

New phenotypic diversity associated with the mitochondrial tRNA(SerUCN) gene mutation.

We performed detailed clinical, histopathological, biochemical, in vitro translation and molecular genetic analysis in patients from two unrelated families harbouring the tRNA(SerUCN) 7472C-insertion mutation. Proband 1 developed a progressive neurodegenerative phenotype characterised by myoclonus, epilepsy, cerebellar ataxia and progressive hearing loss. Proband 2 had a comparatively benign phenotype characterised by isolated myopathy with exercise intolerance. Both patients had the 7472C-insertion mutation in identical proportions and they exhibited a similar muscle biochemical and histopathological phenotype. However, proband 2 also had a previously unreported homoplasmic A to C transition at nucleotide position 7472 in the tRNA(SerUCN) gene. This change lengthens further the homopolymeric C run already expanded by the 7472C-insertion. These data extend the phenotypic range associated with the 7472C-insertion to include isolated skeletal myopathy, as well as a MERRF-like phenotype.

No evidence for paternal mtDNA transmission to offspring or extra-embryonic tissues after ICSI.

There is a risk that ICSI may increase the transmission of mtDNA diseases to children born after this technique. Knowledge of the fate and transmission of paternal mitochondrial DNA is important since mutations in mitochondrial DNA have been described in oligozoospermic males. We have used an adaptation of solid phase mini-sequencing to exclude the presence of levels of paternal mtDNA >0.001% in ICSI families. This method is more sensitive than those used in previous studies and is sufficient to detect the likely paternal contribution (approximately 0.1-0.5% from simple calculations of expected dilution during fertilization). Using this method, we were able to detect concentrations as low as 0.001% paternal mtDNA in a maternal mtDNA background. No paternal mtDNA was detected in the embryonic (blood or buccal swabs) tissue of children born after ICSI nor in extra-embryonic tissue (placenta or umbilical cord). In conclusion, we did not detect paternal mtDNA in blood, buccal swabs, placenta or umbilical cord of children born after ICSI. We have found no evidence that ICSI increases the risk of paternal transmission of mtDNA and hence of mtDNA disorders.

Mutations at the mitochondrial DNA polymerase (POLG) locus associated with male infertility.

Human mitochondrial DNA polymerase, encoded by POLG, contains a polyglutamine tract encoded by a CAG microsatellite repeat. Analysis of POLG genotypes in different populations identified an association between absence of the common, ten-repeat allele and male infertility typified by a range of sperm quality defects but excluding azoospermia.

Decrease of 3243 A-->G mtDNA mutation from blood in MELAS syndrome: a longitudinal study.

It is widely held that changes in the distribution of mutant mtDNAs underlie the progressive nature of mtDNA diseases, but there are few data documenting such changes. We compared the levels of 3243 A-->G mutant mtDNA in blood at birth from Guthrie cards and at the time of diagnosis in a blood DNA sample from patients with mitochondrial encephalopathy, lactic acidosis, and strokelike episodes (MELAS) syndrome. Paired blood DNA samples separated by 9-19 years were obtained from six patients with MELAS. Quantification of mutant load, by means of a solid-phase minisequencing technique, demonstrated a decline (range 12%-29%) in the proportion of mutant mtDNA in all cases (P=.0015, paired t-test). These results suggest that mutant mtDNA is slowly selected from rapidly dividing blood cells in MELAS.

Progress in genetic counselling and prenatal diagnosis of maternally inherited mtDNA diseases.

Mitochondrial DNA is almost entirely maternally inherited. Thousands of copies of mitochondrial DNA are present in every nucleated cell and in most normal individuals these are virtually identical (homoplasmy). Mitochondrial DNA diseases may be caused by mutations in either mitochondrial (Nature 1988;331:717-719) or nuclear genes (Nature 1989;339(6222):309-311; Br J Hosp Med 1996;55:712-716) and hence give rise to maternal or autosomal patterns of inheritance. Antenatal diagnosis of mitochondrial diseases based on chorionic villus sampling is available for Mendelian disorders and the syndromes caused by mutations at bp 8993 (associated with both Leigh's syndrome or neurogenic weakness ataxia and retinitis pigmentosa). However, prenatal diagnosis of many other maternally inherited mitochondrial DNA diseases is less reliable because it is not possible to predict the way in which heteroplasmic mitochondrial DNA mutations segregate within tissues with confidence. This review focuses on the substantial progress that has been made recently, and on the applicability of prenatal diagnosis to genetic counselling in this field.

Evidence from human oocytes for a genetic bottleneck in an mtDNA disease.

We have examined oocytes from a patient with Kearn-Sayre syndrome caused by mtDNA rearrangements. In mtDNA diseases, mutant and wild-type mtDNA frequently coexist in affected individuals (the condition of heteroplasmy). The proportion of mutant mtDNA transmitted from mother to offspring is variable because of a genetic bottleneck, and the "dose" of mutant mtDNA received influences the severity of the phenotype. The feasibility of prenatal diagnosis is critically dependent on the nature and timing of this bottleneck. Significant levels of rearranged mtDNA were detectable in the majority of the patient's oocytes, by use of multiplex PCR, with wide variation, in the levels of mutant and wild-type molecules, between individual oocytes. We also used length variation in a homopolymeric C tract, which is often heteroplasmic in normal controls, to identify founder subpopulations of mtDNAs in this patient's oocytes. We present direct evidence that the number of segregating units (n) is three to five orders of magnitude less than the number of mitochondria in the human female oocyte. In some cases, the best estimate of n may correspond to a single mitochondrion, if it is assumed that intergenerational transmission of mtDNA can be treated as a single sampling event. The bottleneck appears to contribute a major component of the variable transmission from mother to oocyte, in this patient and in a control. That this bottleneck had occurred by the time that oocytes were mature advances the prospects for prenatal diagnosis of mtDNA diseases.

A common mitochondrial DNA variant is associated with insulin resistance in adult life.

Mitochondrial DNA is maternally inherited. Mitochondrial DNA mutations could contribute to the excess of maternal over paternal inheritance of non-insulin-dependent diabetes mellitus (NIDDM). We therefore investigated the relationship between this variant, insulin resistance and other risk factors in a cohort which had been well characterised with respect to diabetes. Blood DNA was screened from 251 men born in Hertfordshire 1920-1930 in whom an earlier cohort study had shown that glucose tolerance was inversely related to birthweight. The 16189 variant (T--> C transition) in the first hypervariable region of mitochondrial DNA was detected using the polymerase chain reaction and restriction digestion. DNA analysis showed that 28 of the 251 men (11%) had the 16189 variant. The prevalence of the 16189 variant increased progressively with fasting insulin concentration (p < 0.01). The association was independent of age and body mass index and was present after exclusion of the patients with NIDDM or impaired glucose tolerance. We found that insulin resistance in adult life was associated with the 16189 variant. This study provides the first evidence that a frequent mitochondrial variant may contribute to the phenotype in patients with a common multifactorial disorder.

Homopolymeric tract heteroplasmy in mtDNA from tissues and single oocytes: support for a genetic bottleneck.

While mtDNA polymorphisms at single base positions are common, the overwhelming majority of the mitochondrial genomes within a single individual are usually identical. When there is a point-mutation difference between a mother and her offspring, there may be a complete switching of mtDNA type within a single generation. It is generally assumed that there is a genetic bottleneck whereby a single or small number of founder mtDNA(s) populate the organism, but it is not known at which stages the restriction/amplification of mtDNA subtype(s) occur, and this uncertainty impedes antenatal diagnosis for mtDNA disorders. Length polymorphisms in homopolymeric tracts have been demonstrated in the large noncoding region of mtDNA. We have developed a new method, T-PCR (trimmed PCR), to quantitate heteroplasmy for two of these tracts (D310 and D16189). D310 variation is sufficient to indicate clonal origins of tissues and single oocytes. Tissues from normal individuals often possessed more than one length variant (heteroplasmy). However, there was no difference in the pattern of the length variants between somatic tissues in any control individual when bulk samples were taken. Oocytes from normal women undergoing in vitro fertilization were frequently heteroplasmic for length variants, and in two cases the modal length of the D310 tract differed in individual oocytes from the same woman. These data suggest that a restriction/amplification event, which we attribute to clonal expansion of founder mtDNA(s), has occurred by the time oocytes are mature, although further segregation may occur at a later stage. In contrast to controls, the length distribution of the D310 tract varied between tissues in a patient with heteroplasmic mtDNA rearrangements, suggesting that these mutants influence segregation. These findings have important implications for the genetic counselling of patients with pathogenic mtDNA mutations.

Prospects for DNA-based prenatal diagnosis of mitochondrial disorders.

Mitochondria have their own DNA which is maternally inherited. Mitochondrial DNA (mtDNA) diseases are extremely variable because of the genetics of mtDNA and the unique pathogenesis of these disorders. This makes predicting the prognosis and the transmission of mtDNA disorders difficult. While mtDNA polymorphisms at a single base position are common, the overwhelming majority of the mitochondrial genomes within a single human individual are usually identical. When there is a point mutation difference between a mother and her offspring, there may be a complete switching of mtDNA type within a single generation. It is generally assumed that there is a genetic bottleneck whereby a single or small number of founder mtDNA(s) populate the organism, but it is not known at which stages the restriction/amplification of mtDNA subtype(s) occur, and this uncertainty impedes antenatal diagnosis for mtDNA disorders. Autosomally inherited disorders of mitochondrial function may be caused by mutations in genes for the components of the respiratory chain and for the machinery of mitochondrial biogenesis, which are nuclear-encoded. Accurate diagnosis of these disorders is important as prenatal diagnosis is available in a minority of cases.

Do sequence variants in the major non-coding region of the mitochondrial genome influence mitochondrial mutations associated with disease?

Several different mutations in human mitochondrial DNA (mtDNA) have been associated with disease, but their origins and the basis of the wide phenotypic variability remain to be elucidated. We initially investigated three patients with heteroplasmic disease associated mutations of mtDNA for the presence of cis mutations in the major non-coding region that might influence their origins or pathology. A T --> C transition at nt 16 189 previously identified in one patient with the 3243 G:C mutation was associated with heteroplasmic length variation. Identical length variation was found in patient-derived cybrid lines containing 0-97.5% 3243 G:C. Similarly, heteroplasmic length variation was demonstrated in 2/6 other probands with both the 3243 mutation and the 16,189 polymorphism. The distribution of length variants in probands and in asymptomatic family members was identical in all cases. Thus length variation appears to be independent of the level of 3243 mutant mtDNA and hence probably arose within both 3243 G:C and 3243 A:T mtDNAs. We suggest that the 16,189 polymorphism reflects a predisposition to the formation or fixation of several different mutations in mitochondrial tRNA-LeuUUR.

Intracellular heteroplasmy for disease-associated point mutations in mtDNA: implications for disease expression and evidence for mitotic segregation of heteroplasmic units of mtDNA.

Studies in vitro have shown that a respiratory-deficient phenotype is expressed by cells when the proportion of mtDNA with a disease-associated mutation exceeds a threshold level, but analysis of tissues from patients with mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) have failed to show a consistent relationship between the degree of heteroplasmy and biochemical expression of the defect. One possible explanation for this phenomenon is that there is variation of heteroplasmy between individual cells that is not adequately reflected by the mean heteroplasmy for a tissue. We have confirmed this by study of fibroblast clones from subjects heteroplasmic for the MELAS 3243 (A-->G) mtDNA mutation. Similar observations were made with fibroblast clones derived from two subjects heteroplasmic for the 11778 (G-->A) mtDNA mutation of Leber's hereditary optic neuropathy. For the MELAS 3243 mutation, the distribution of mutant mtDNA between different cells was not randomly distributed about the mean, suggesting that selection against cells with high proportions of mutant mtDNA had occurred. To explore the way in which heteroplasmic mtDNA segregates in mitosis we followed the distribution of heteroplasmy between clones over approximately 15 generations. There was either no change or a decrease in the variance of intercellular heteroplasmy for the MELAS 3243 mutation, which is most consistent with segregation of heteroplasmic units of multiple mtDNA molecules in mitosis. After mitochondria from one of the MELAS 3243 fibroblast cultures were transferred to a mitochondrial DNA-free (rho0) cell line derived from osteosarcoma cells by cytoplast fusion, the mean level and intercellular distribution of heteroplasmy was unchanged. We interpret this as evidence that somatic segregation (rather than nuclear background or cell differentiation state) is the primary determinant of the level of heteroplasmy.

Duplications of mitochondrial DNA in Kearns-Sayre syndrome.

mtDNA duplications were detectable in 10 of 10 patients with mtDNA deletions and Kearns-Sayre syndrome (KSS) and in none of 8 patients with chronic progressive external ophthalmoplegia (CPEO). Thus, duplications of mtDNA seem to be a distinctive feature of KSS, including patients where Pearson's syndrome is the first manifestation. Diabetes mellitus was identified in 4 of 7 patients with high or moderate levels of mtDNA duplications. The balance of mtDNA rearrangements may be central to the pathogenesis of this unique group of disorders.

Pyruvate dehydrogenase deficiency. Clinical presentation and molecular genetic characterization of five new patients.

Fibroblast cultures from five patients with early onset severe encephalopathy and lactic acidosis were studied for evidence of pyruvate dehydrogenase (PDH) deficiency. Three males had significantly reduced activity (0.29-0.45 nmol/mg protein/min versus normal controls 0.7-1.1 nmol/mg protein/min); two females had PDH activity within the normal range. However, as the majority of cases of PDH deficiency result from defects in the X-linked E1 alpha subunit and both females had biased patterns of X-inactivation (making it impossible to rule out the possibility that they were heterozygous for an E1 alpha gene defect) molecular genetic studies were performed. cDNA from the male patients was sequenced and mis-sense mutations found: Y243N (T-->A) in exon 7, D315A (G-->A) in exon 10 and R378H (G-->A) in exon 11. Single-strand conformation polymorphism analysis of amplified genomic DNA fragments and sequencing revealed a mis-sense mutation M282L (A-->C) in one female and a frameshift mutation caused by insertion of T (R288ins) in the other. Adding to recent descriptions of new mutations, this report emphasizes the allelic heterogeneity of the condition. The identification of mutations in females with a suggestive clinical phenotype, even when peripheral fibroblasts do not show deficient PDH activity, illustrates the importance of molecular analysis of this disease.

Neurodevelopmental abnormalities and lactic acidosis in a girl with a 20-bp deletion in the X-linked pyruvate dehydrogenase E1 alpha subunit gene.

We describe a girl with developmental abnormalities of the CNS and a lactic acidosis whose cultured fibroblasts showed a profound deficiency of pyruvate dehydrogenase complex (PDHC) activity (patient = 0.14 nmol/mg protein per minute, controls = 0.7 to 1.1 nmol/mg protein per minute). Immunocytochemistry demonstrated the fibroblast culture to be mosaic, with 14% of cells expressing the PDHC E1 alpha subunit protein in normal amounts and the remaining 86% having no detectable immunoreactive activity. Direct sequencing of cDNA for the X-linked PDHC E1 alpha subunit established that the patient was heterozygous for a 20-bp deletion beginning in the codon for Ser300 of the derived amino acid sequence. The pattern of methylation at the DXS255 locus suggested predominant expression of the X chromosome carrying the mutant allele in the fibroblast culture. There was a good correlation between the residual PDHC activity, the proportion of cells with immunoreactive E1 alpha protein, and the X chromosome inactivation ratio, demonstrating the importance of X-inactivation for expression of this X-linked neurometabolic disease in females.