Philippe Burlet

Analysis of mtDNA variant segregation during early human embryonic development: a tool for successful NARP preimplantation diagnosis

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.

Large scale deletions of the 5q13 region are specific to Werdnig-Hoffmann disease.

Spinal muscular atrophy (SMA) is characterised by degeneration of anterior horn cells of the spinal cord and represents the second most common, lethal, autosomal recessive disorder after cystic fibrosis. Based on the criteria of the Internatinal SMA Consortium, childhood SMAs are classified into type I (Werdnig-Hoffmann disease), type II (intermediate form), and type III (Kugelberg-Welander disease). Recently, two genes have been found to be associated with SMA. The survival motor neurone gene (SMN) is an SMA determining gene as it is absent in 98.6% of patients. A second gene, XS2G3, or the highly homologous neuronal apoptosis inhibitory protein gene (NAIP) have been found to be more frequently deleted in type I than in the milder forms (types II and III). We investigated the correlation between the clinical phenotype and the genotype at this loci. A total of 106 patients were classified into type I (44), type II (31), and type III (31) and analysed using SMN, markers C212 and C272, and NAIP mapping upstream and downstream from SMN respectively. The combined analysis of all markers showed a large proportion of type I patients (43%) carried deletions of both SMN and its flanking markers (C212/272) and NAIP exon 5), as compared with none of the patients with type II or III SMA. The presence of large scale deletions involving these loci is specific to Werdnig-Hoffman disease (type I) and allows one to predict the severity of the disease in our series.

Segregation of mtDNA throughout human embryofetal development: m.3243A>G as a model system

Mitochondrial DNA (mtDNA) mutations cause a wide range of serious diseases with high transmission risk and maternal inheritance. Tissue heterogeneity of the heteroplasmy rate (“mutant load”) accounts for the wide phenotypic spectrum observed in carriers. Owing to the absence of therapy, couples at risk to transmit such disorders commonly ask for prenatal (PND) or preimplantation diagnosis (PGD). The lack of data regarding heteroplasmy distribution throughout intrauterine development, however, hampers the implementation of such procedures. We tracked the segregation of the m.3243A>G mutation (MT‐TL1 gene) responsible for the MELAS syndrome in the developing embryo/fetus, using tissues and cells from eight carrier females, their 38 embryos and 12 fetuses. Mutant mtDNA segregation was found to be governed by random genetic drift, during oogenesis and somatic tissue development. The size of the bottleneck operating for m.3243A>G during oogenesis was shown to be individual‐dependent. Comparison with data we achieved for the m.8993T>G mutation (MT‐ATP6 gene), responsible for the NARP/Leigh syndrome, indicates that these mutations differentially influence mtDNA segregation during oogenesis, while their impact is similar in developing somatic tissues. These data have major consequences for PND and PGD procedures in mtDNA inherited disorders. Hum Mutat 32:116–125, 2011.

Mutation dependance of the mitochondrial DNA copy number in the first stages of human embryogenesis

Mitochondrial DNA (mtDNA) content is thought to remain stable over the preimplantation period of human embryogenesis that is, therefore, suggested to be entirely dependent on ooplasm mtDNA capital. We have explored the impact of two disease-causing mutations [m.3243A>G myopathy, encephalopathy, lactic acidosis and stroke-like syndrome (MELAS) and m.8344A>G myoclonic epilepsy associated with ragged-red fibers (MERRF)] on mtDNA amounts in human oocytes and day 4-5 preimplantation embryos. The mtDNA amount was stable in MERRF and control materials, whereas gradually increasing from the germinal vesicle of oogenesis to the blastocyst stage of embryogenesis in MELAS cells, MELAS embryos carrying ∼3-fold higher mtDNA amount than control embryos (P = 0.0003). A correlation between mtDNA copy numbers and mutant loads was observed in MELAS embryos (R(2) = 0.42, P < 0.0013), suggestive of a compensation for the respiratory chain defect resulting from high mutation levels. These results suggest that mtDNA can replicate in early embryos and emphasize the need for sufficient amount of wild-type mtDNA to sustain embryonic development in humans.

Preimplantation genetic diagnosis for autosomal recessive polycystic kidney disease.

Autosomal recessive polycystic kidney disease (ARPKD) is one of the most common hereditary renal cystic diseases, and is caused by mutations in the PKHD1 gene. Due to the poor prognosis, there is a strong demand for prenatal diagnosis. Preimplantation genetic diagnosis (PGD) represents an alternative because it avoids the physical and emotional trauma of a pregnancy termination in the case of an affected fetus. A standardized single-cell diagnostic procedure was developed, based on haplotype analysis, enabling PGD to be offered to couples at risk of transmitting ARPKD. Six linked markers within (D6S1714 and D6S243), or in close proximity to (D6S272, D6S436, KIAA0057, D6S1662) the PKHD1 gene were tested by multiplex nested-polymerase chain reaction (PCR), using a Qiagen multiplex PCR kit. PCR analyses were carried out on 50 single lymphocytes. The amplification rate was excellent (100%), with an allele drop-out (ADO) rate ranging from 0 to 8%. Five PGD cycles were performed and 23 embryos were biopsied and analysed using this test. Transferable embryos were obtained in 4 cycles, resulting in two pregnancies and the birth of a healthy boy. This standardized diagnostic procedure allowed the detection of recombination, contamination, and ADO events, providing high assay accuracy with wide applicability.

The loss of the snoRNP chaperone Nopp140 from Cajal bodies of patient fibroblasts correlates with the severity of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a common autosomal recessive neurodegenerative disease caused by reduced survival motor neuron (SMN) levels. The assembly machinery containing SMN is implicated in the biogenesis of the spliceosomal small nuclear ribonucleoproteins (snRNPs). SMN is present in both the cytoplasm and nucleus, where it transiently accumulates in subnuclear domains named Cajal bodies (CBs) and functions in the maturation of snRNPs and small nucleolar (sno)RNPs. The impact of lowering SMN levels on the composition of CBs in SMA cells is still not completely understood. Here, we analyse the CB composition in immortalized and primary fibroblasts from SMA patients. We show that the U snRNA export factors PHAX and chromosome region maintenance 1 and the box C/D snoRNP core protein fibrillarin concentrate in CBs from SMA cells, whereas the box H/ACA core proteins GAR1 and NAP57/dyskerin show reduced CB localization. Remarkably, the functional deficiency in SMA cells is associated with decreased localization of the snoRNP chaperone Nopp140 in CBs that correlates with disease severity. Indeed, RNA interference knockdown experiments in control fibroblasts demonstrate that SMN is required for accumulation of Nopp140 in CBs. Conversely, overexpression of SMN in SMA cells restores the CB localization of Nopp140, whereas SMN mutants found in SMA patients are defective in promoting the association of Nopp140 with CBs. Taken together, we demonstrate that only a subset of CB functions (as indicated by the association of representative factors) are impaired in SMA cells and, importantly, we identify the decrease of Nopp140 localization in CBs as a phenotypic marker for SMA.

Data from Artificial Models of Mitochondrial DNA Disorders Are Not Always Applicable to Humans

Mitochondrial DNA (mtDNA) mutations, a major cause of maternally inherited human diseases, are commonly characterized by the coexistence of mutant and wild-type mtDNA molecules within a cell (called “heteroplasmy” or “mutation load”). Usually, the higher the mutation load, the more severe the disease. Because of the high risk of recurrence in siblings and the absence of treatment options, couples at risk of transmitting mtDNA mutations often ask for prenatal diagnosis (PND) or preimplantation genetic diagnosis (PGD).

A role for protein phosphatase PP1γ in SMN complex formation and subnuclear localization to Cajal bodies

Summary The spinal muscular atrophy (SMA) gene product SMN forms with gem-associated protein 2–8 (Gemin2–8) and unrip (also known as STRAP) the ubiquitous survival motor neuron (SMN) complex, which is required for the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs), their nuclear import and their localization to subnuclear domain Cajal bodies (CBs). The concentration of the SMN complex and snRNPs in CBs is reduced upon SMN deficiency in SMA cells. Subcellular localization of the SMN complex is regulated in a phosphorylation-dependent manner and the precise mechanisms remain poorly understood. Using co-immunoprecipitation in HeLa cell extracts and in vitro protein binding assays, we show here that the SMN complex and its component Gemin8 interact directly with protein phosphatase PP1&ggr;. Overexpression of Gemin8 in cells increases the number of CBs and results in targeting of PP1&ggr; to CBs. Moreover, depletion of PP1&ggr; by RNA interference enhances the localization of the SMN complex and snRNPs to CBs. Consequently, the interaction between SMN and Gemin8 increases in cytoplasmic and nuclear extracts of PP1&ggr;-depleted cells. Two-dimensional protein gel electrophoresis revealed that SMN is hyperphosphorylated in nuclear extracts of PP1&ggr;-depleted cells and expression of PP1&ggr; restores these isoforms. Notably, SMN deficiency in SMA leads to the aberrant subcellular localization of Gemin8 and PP1&ggr; in the atrophic skeletal muscles, suggesting that the function of PP1&ggr; is likely to be affected in disease. Our findings reveal a role of PP1&ggr; in the formation of the SMN complex and the maintenance of CB integrity. Finally, we propose Gemin8 interaction with PP1&ggr; as a target for therapeutic intervention in SMA.

Successful pre-implantation genetic diagnosis for Hirschsprung disease

To the Editor : Hirschsprung disease (HSCR, OMIM142623) is the consequence of a lack of neurons in the myenteric and submucosal plexuses along a variable length of the intestine. In isolated (non-syndromic) HSCR, the recurrence risk in the sibship of the index case varies from 1% to 33%, taking into account (i) the gender and the length of the aganglionic segment of the index case and (ii) the gender of the sib (1, 2). HSCR is genetically heterogeneous, mutations being identified in 11 genes thus far (3). The major susceptibility gene is RET (OMIM164761), which encodes a receptor tyrosine kinase (4). RET mutations are identified in about 50% of familial cases, offering to these families the possibility to perform prenatal diagnosis (PND) or pre-implantation genetic diagnosis (PGD). Such procedures remain however controversial, due to the incomplete sex-dependent penetrance of RET mutations (50% and 70% in females and males, respectively) (2, 5), and because this disorder is curable by surgical procedures. A couple was referred to our PGD center because the male partner and his two children suffered from severe HSCR caused by a RET exon 4 mutation (c.751insGC). Despite surgery undertook in the neonatal period, the son suffered from constipation requiring daily laxatives or enemas, and the 6-year-old daughter still complained of soiling. This couple experienced PND and terminated the pregnancy of a carrier fetus. The PGD test was developed on 50 heterozygous lymphocytes by coamplification of the c.751insGC RET mutation with a polymorphic marker located in intron 5 of the RET gene, using multiplex nested polymerase chain reaction (PCR) (Table 1) (6). Amplification failure and allele drop-out rates were respectively of 5% and 8% for the RET mutation, and 4% and 2% for the intron 5 marker. No contamination was detected. The couple underwent one treatment cycle, 17 oocytes were sampled, of whom 13 fertilized (76%), resulting in four embryos at day 3. Conclusive results were obtained for three of the four embryos (Fig. 1). All embryos were diagnosed to be unaffected, and two were transferred on day 5, resulting in a twin pregnancy, and the birth of two healthy boys. Pre-implantation diagnosis was confirmed on cord blood DNA. The use of PGD for other purpose than serious heritable disorders raises ethical and legal discussions (7, 8). French Law links the practice of PGD to PND, stipulating that PGD must be carried out inside a multidisciplinary network licensed for PND and supervised by the French Biomedicine Agency. Access to these procedures is indeed limited to couple being at high risk of giving birth to a child affected with a serious genetic disease, incurable at the time of the diagnosis. In this respect, PGD for HSCR has been considered as questionable. First, surgery can cure the disease. Surgical therapies are however associated with an unsatisfactory long-term prognosis for many (9, 10).