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ObjectiveTo identify a consistent pattern of brain MRI imaging in primary complex I deficiency. Complex I deficiency, a major cause of respiratory chain dysfunction, accounts for various clinical presentations, including Leigh syndrome. Human complex I comprises seven core subunits encoded by mitochondrial DNA (mtDNA) and 38 core subunits encoded by nuclear DNA (nDNA). Moreover, its assembly requires six known and many unknown assembly factors. To date, no correlation between genotypes and brain MRI phenotypes has been found in complex I deficiencies.Design and subjectsThe brain MRIs of 30 patients carrying known mutation(s) in genes involved in complex I were retrospectively collected and compared with the brain MRIs of 11 patients carrying known mutations in genes involved in the pyruvate dehydrogenase (PDH) complex as well as 10 patients with MT-TL1 mutations.ResultsAll complex I deficient patients showed bilateral brainstem lesions (30/30) and 77% (23/30) showed anomalies of the putamen. Supratentorial stroke-like lesions were only observed in complex I deficient patients carrying mtDNA mutations (8/19) and necrotising leucoencephalopathy in patients with nDNA mutations (4/5). Conversely, the isolated stroke-like images observed in patients with MT-TL1 mutations, or the corpus callosum malformations observed in PDH deficient patients, were never observed in complex I deficient patients.ConclusionA common pattern of brain MRI imaging was identified with abnormal signal intensities in brainstem and subtentorial nuclei with lactate peak as a clue of complex I deficiency. Combining clinico-biochemical data with brain imaging may therefore help orient genetic studies in complex I deficiency.

Background: Diseases arising from mitochondrial DNA (mtDNA) mutations are usually serious pleiotropic disorders with maternal inheritance. Owing to the high recurrence risk in the progeny of carrier females, “at-risk” couples often ask for prenatal diagnosis. However, reliability of such practices remains under debate. Preimplantation diagnosis (PGD), a theoretical alternative to conventional prenatal diagnosis, requires that the mutant load measured in a single cell from an eight cell embryo accurately reflects the overall heteroplasmy of the whole embryo, but this is not known to be the case. Objective: To investigate the segregation of an mtDNA length polymorphism in blastomeres of 15 control embryos from four unrelated couples, the NARP mutation in blastomeres of three embryos from a carrier of this mutation. Results: Variability of the mtDNA polymorphism heteroplasmy among blastomeres from each embryo was limited, ranging from zero to 19%, with a mean of 7%. PGD for the neurogenic ataxia retinitis pigmentosa (NARP) mtDNA mutation (8993T→G) was therefore carried out in the carrier mother of an affected child. One of three embryos was shown to carry 100% of mutant mtDNA species while the remaining two were mutation-free. These two embryos were transferred, resulting in a singleton pregnancy with delivery of a healthy child. Conclusions: This PGD, the first reported for a mtDNA mutation, illustrates the skewed meiotic segregation of the NARP mtDNA mutation in early human development. However, discrepancies between the segregation patterns of the NARP mutation and the HV2 polymorphism indicate that a particular mtDNA nucleotide variant might differentially influenced the mtDNA segregation, precluding any assumption on feasibility of PGD for other mtDNA mutations.

Background: Mitochondrial DNA (mtDNA) mutations cause a wide range of serious genetic diseases with maternal inheritance. Because of the high transmission risk and the absence of therapy in these disorders, at-risk couples often ask for prenatal diagnosis (PND). However, because heteroplasmy load (coexistence of mutant and wild-type mtDNA) may vary among tissues and with time, the possibility that a single fetal sample may not reflect the whole neonate impedes prenatal diagnosis of mtDNA diseases. Methods: We performed 13 prenatal diagnoses for the NARP (neurogenic weakness, ataxia, retinitis pigmentosa) m.8993T→G mtDNA mutation (p.Leu156Arg) in the ATP synthase subunit 6 gene. Analyses were performed on chorionic villous (CVS) and/or amniocyte samples carried out at various stages of pregnancy, using a method enabling quantification of low DNA amounts. Results: Maternal mutant loads ranged from 0 to 75% in blood and had no predictive value for the fetus status, except for women with no detectable mutant DNA, whose fetuses were consistently mutation-free. In 8/13 PND, mutant load was <30%. These children are healthy at 2–7 years of age. In 5/13 PND, mutant load ranged from 65 to 100%, and parents preferred to terminate the pregnancies (15–22 weeks of gestation). Single-cell analysis of 20 trophoblastic cells and 21 amniocytes isolated from two affected fetuses found an average mutant load close to the overall CVS and amniocyte mutant load, despite striking intercellular variation. The m.8993T→G mutant loads, assessed in 7, 17, 11, and 5 different tissues from 4 terminations, respectively, were identical in all tissues from a given individual (mean (SD) 78 (1.2)%, 91 (0.7)%, 74 (2)%, and 63 (1.6)% for the 4 fetuses, respectively). Conclusions: Our results indicate that the placental/amniotic mutant loads do reflect the NARP mutant mtDNA load in the whole fetus, even when the sample amount is small, and suggest that heteroplasmy level remains stable during pregnancy, at least after 10 weeks of gestation. Although these data establish the feasibility of PND for this mutation, assessing more precisely the correlation between mutant load and disease severity should further help in interpreting PND results.

Introduction: Myopathy, encephalopathy, lactic acidosis, and stroke-like (MELAS) syndrome, a maternally inherited disorder that is among the most common mitochondrial DNA (mtDNA) diseases, is usually associated with the m.3242A>G mutation of the mitochondrial tRNAleu gene. Very few data are available with respect to prenatal diagnosis of this serious disease. The rate of mutant versus wild-type mtDNA (heteroplasmy) in fetal DNA is indeed considered to be a poor indicator of postnatal outcome. Materials and methods: Taking advantage of a novel semi-quantitative polymerase chain reaction test for m.3243A>G mutant load assessment, we carried out nine prenatal diagnoses in five unrelated women, using two different fetal tissues (chorionic villi v amniocytes) sampled at two or three different stages of pregnancy. Results: Two of the five women, although not carrying m.3243A>G in blood or extra-blood tissues, were, however, considered at risk for transmission of the mutation, as they were closely related to MELAS-affected individuals. The absence of 3243A>G in the blood of first degree relatives was associated with no mutated mtDNA in the cardiovascular system (CVS) or amniocytes, and their three children are healthy, with a follow-up of 3 months–3 years. Among the six fetuses from the three carrier women, three were shown to be homoplasmic (0% mutant load), the remaining three being heteroplasmic, with a mutant load ranging from 23% to 63%. The fetal mutant load was fairly stable at two or three different stages of pregnancy in CVS and amniocytes. Although pregnancy was terminated in the case of the fetus with a 63% mutant load, all other children are healthy with a follow-up of 3 months–6 years. Conclusion: These data suggest that a prenatal diagnosis for MELAS syndrome might be helpful for at-risk families.

Hirschsprung disease (HSCR), the most common hereditary cause of intestinal obstruction, shows considerable variation and complex inheritance. Coding sequence mutations in RET , GDNF , EDNRB , EDN3 and SOX10 lead to long-segment (L-HSCR) and syndromic HSCR but fail to explain the transmission of the much more common short-segment form (S-HSCR). We conducted a genome scan in families with S-HSCR and identified susceptibility loci at 3p21, 10q11 and 19q12 that seem to be necessary and sufficient to explain recurrence risk and population incidence. The gene at 10q11 is probably RET , supporting its crucial role in all forms of HSCR; however, coding sequence mutations are present in only 40% of linked families, suggesting the importance of noncoding variation. Here we show oligogenic inheritance of S-HSCR, the 3p21 and 19q12 loci as RET -dependent modifiers, and a parent-of-origin effect at RET . This study demonstrates by a complete genetic dissection why the inheritance pattern of S-HSCR is nonmendelian.

Preimplantation genetic diagnosis (PGD) first consisted of the selection of female embryos for patients at risk of transmitting X-linked recessive diseases. Advances in molecular biology now allow the specific diagnosis of almost any Mendelian disease. For families with an identified X-linked recessive disease-causing mutation, non-specific diagnosis by sex identification can be considered as a sub-standard method, since it involves the unnecessary disposal of healthy male embryos and reduces success rate by diminishing the pool of embryos eligible for transfer. The most telomeric part of the X-chromosome long arm is a highly gene-rich region encompassing disease genes such as haemophilia A, X-linked adrenoleukodystrophy, X-linked hydrocephalus and incontinentia pigmenti. We developed five single-cell triplex amplification protocols with microsatellite markers DXS1073, DXS9901 (BGN), G6PD, DXS1108, DXS8087 and F8C-IVS13 located in this Xq terminal region. These tests allow the diagnosis of all diseases previously mentioned providing that the genetic material allowing the identification of the morbid allele can be obtained. The choice of the microsatellite set to use depends on the localisation of the gene responsible for the diagnosed pathology and on the informativity of the markers in particular families. Single-cell amplification efficiency was assessed on single lymphocytes. Amplification rate of the different markers ranged from 89-97% with an allele drop out rate of 2-19%. So far PGD has been carried out for three carrier females at risk of transmitting X-linked adrenoleukodystrophy, X-linked hydrocephalus and hemophilia A. The latter one is now pregnant.

Complex I deficiency, the most common cause of mitochondrial disorders, accounts for a variety of clinical symptoms and its genetic heterogeneity makes identification of the disease genes particularly tedious. Indeed, most of the 43 complex I subunits are encoded by nuclear genes, only seven of them being mitochondrially encoded. In order to offer urgent prenatal diagnosis, we have studied an inbred/multiplex family with complex I deficiency by using microsatellite DNA markers flanking the putative disease loci. Microsatellite DNA markers have allowed us to exclude the NDUFS7, NDUFS8, NDUFV1 and NDUFS1 genes and to find homozygosity at the NDUFS4 locus. Direct sequencing has led to identification of a homozygous splice acceptor site mutation in intron 1 of the NDUFS4 gene (IVS1nt -1, G-->A); this was not found in chorion villi of the ongoing pregnancy. We suggest that genotyping microsatellite DNA markers at putative disease loci in inbred/multiplex families helps to identify the disease-causing mutation. More generally, we suggest giving consideration to a more systematic microsatellite analysis of putative disease loci for identification of disease genes in inbred/multiplex families affected with genetically heterogeneous conditions.

OBJECTIVE: To identify a consistent pattern of brain MRI imaging in primary complex I deficiency. Complex I deficiency, a major cause of respiratory chain dysfunction, accounts for various clinical presentations, including Leigh syndrome. Human complex I comprises seven core subunits encoded by mitochondrial DNA (mtDNA) and 38 core subunits encoded by nuclear DNA (nDNA). Moreover, its assembly requires six known and many unknown assembly factors. To date, no correlation between genotypes and brain MRI phenotypes has been found in complex I deficiencies. DESIGN AND SUBJECTS: The brain MRIs of 30 patients carrying known mutation(s) in genes involved in complex I were retrospectively collected and compared with the brain MRIs of 11 patients carrying known mutations in genes involved in the pyruvate dehydrogenase (PDH) complex as well as 10 patients with MT-TL1 mutations. RESULTS: All complex I deficient patients showed bilateral brainstem lesions (30/30) and 77% (23/30) showed anomalies of the putamen. Supratentorial stroke-like lesions were only observed in complex I deficient patients carrying mtDNA mutations (8/19) and necrotising leucoencephalopathy in patients with nDNA mutations (4/5). Conversely, the isolated stroke-like images observed in patients with MT-TL1 mutations, or the corpus callosum malformations observed in PDH deficient patients, were never observed in complex I deficient patients. CONCLUSION: A common pattern of brain MRI imaging was identified with abnormal signal intensities in brainstem and subtentorial nuclei with lactate peak as a clue of complex I deficiency. Combining clinico-biochemical data with brain imaging may therefore help orient genetic studies in complex I deficiency.

Fetal DNA and cells present in maternal blood have previously been used for non-invasive prenatal diagnosis. However, some fetal cells can persist in maternal blood after a previous pregnancy. Fetal rhesus status and sex determination have been performed by using amplification by real-time polymerase chain reaction (PCR) of fetal DNA sequences present in maternal circulation; no false-positive results related to persistent fetal DNA from a previous pregnancy have been reported. This idea has recently been challenged. An SRY real-time PCR assay was performed on the serum of 67 pregnant women carrying a female fetus but having previously given birth to at least one boy and on the serum of 30 healthy non-pregnant women with a past male pregnancy. In all cases, serum was negative for the SRY gene. These data suggest that fetal DNA from a previous pregnancy cannot be detected in maternal serum, even by using a highly sensitive technique. Therefore, non-invasive prenatal diagnosis by fetal sex determination for women at risk of producing children with X-linked disorders, and fetal RHD genotyping is reliable and secure as previously demonstrated.

BACKGROUND: Mitochondrial DNA (mtDNA) mutations cause a wide range of serious genetic diseases with maternal inheritance. Because of the high transmission risk and the absence of therapy in these disorders, at-risk couples often ask for prenatal diagnosis (PND). However, because heteroplasmy load (coexistence of mutant and wild-type mtDNA) may vary among tissues and with time, the possibility that a single fetal sample may not reflect the whole neonate impedes prenatal diagnosis of mtDNA diseases. METHODS: We performed 13 prenatal diagnoses for the NARP (neurogenic weakness, ataxia, retinitis pigmentosa) m.8993T arrow right G mtDNA mutation (p.Leu156Arg) in the ATP synthase subunit 6 gene. Analyses were performed on chorionic villous (CVS) and/or amniocyte samples carried out at various stages of pregnancy, using a method enabling quantification of low DNA amounts. RESULTS: Maternal mutant loads ranged from 0 to 75% in blood and had no predictive value for the fetus status, except for women with no detectable mutant DNA, whose fetuses were consistently mutation-free. In 8/13 PND, mutant load was <30%. These children are healthy at 2-7 years of age. In 5/13 PND, mutant load ranged from 65 to 100%, and parents preferred to terminate the pregnancies (15-22 weeks of gestation). Single-cell analysis of 20 trophoblastic cells and 21 amniocytes isolated from two affected fetuses found an average mutant load close to the overall CVS and amniocyte mutant load, despite striking intercellular variation. The m.8993T arrow right G mutant loads, assessed in 7, 17, 11, and 5 different tissues from 4 terminations, respectively, were identical in all tissues from a given individual (mean (SD) 78 (1.2)%, 91 (0.7)%, 74 (2)%, and 63 (1.6)% for the 4 fetuses, respectively). CONCLUSIONS: Our results indicate that the placental/amniotic mutant loads do reflect the NARP mutant mtDNA load in the whole fetus, even when the sample amount is small, and suggest that heteroplasmy level remains stable during pregnancy, at least after 10 weeks of gestation. Although these data establish the feasibility of PND for this mutation, assessing more precisely the correlation between mutant load and disease severity should further help in interpreting PND results.