Whole genome sequencing analysis reveals post-zygotic mutation variability in monozygotic twins discordant for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a heterogeneous, fatal neurodegenerative disease, characterized by motor neuron loss and in 50% of cases also by cognitive and/or behavioral changes. Mendelian forms of ALS comprise approximately 10-15% of cases. The majority is however considered sporadic, but also with a high contribution of genetic risk factors. To explore the contribution of somatic mutations and/or epigenetic changes to disease risk, we performed whole genome sequencing and methylation analyses using samples from multiple tissues on a cohort of 26 monozygotic twins discordant for ALS, followed by in-depth validation and replication experiments. The results of these analyses implicate several mechanisms in ALS pathophysiology, which include a role for de novo mutations, defects in DNA damage repair and accelerated aging.

De novo mutations in SOD1 are a cause of ALS.

OBJECTIVE: The only identified cause of amyotrophic lateral sclerosis (ALS) are mutations in a number of genes found in familial cases but also in sporadic cases. De novo mutations occurring in a parental gonadal cell, in the zygote or postzygotic during embryonal development can result in an apparently sporadic/isolated case of ALS later in life. We searched for de novo mutations in SOD1 as a cause of ALS. METHODS: We analysed peripheral-blood exome, genome and Sanger sequencing to identify deleterious mutations in SOD1 in 4000 ALS patients from Germany, South Korea and Sweden. Parental kinship was confirmed using highly polymorphic microsatellite markers across the genome. Medical genealogical and clinical data were reviewed and compared with the literature. RESULTS: We identified four sporadic ALS cases with de novo mutations in SOD1. They aggregate in hot-spot codons earlier found mutated in familial cases. Their phenotypes match closely what has earlier been reported in familial cases with pathogenic mutations in SOD1. We also encountered familial cases where de novo mutational events in recent generations may have been involved. CONCLUSIONS: De novo mutations are a cause of sporadic ALS and may also be underpinning smaller families with few affected ALS cases. It was not possible to ascertain if the origin of the de novo mutations was parental germline, zygotic or postzygotic during embryonal development. All ALS patients should be offered genetic counselling and genetic screening, the challenges of variant interpretation do not outweigh the potential benefits including earlier confirmed diagnosis and possible bespoken therapy.

PTBP1 acts as a dominant repressor of the aberrant tissue-specific splicing of ISCU in hereditary myopathy with lactic acidosis.

BACKGROUND: Hereditary myopathy with lactic acidosis (HML) is an autosomal recessive disease caused by an intron mutation in the iron-sulfur cluster assembly (ISCU) gene. The mutation results in aberrant splicing, where part of the intron is retained in the final mRNA transcript, giving rise to a truncated nonfunctional ISCU protein. Using an ISCU mini-gene system, we have previously shown that PTBP1 can act as a repressor of the mis-splicing of ISCU, where overexpression of PTBP1 resulted in a decrease of the incorrect splicing. In this study, we wanted to, in more detail, analyze the role of PTBP1 in the regulation of endogenous ISCU mis-splicing. METHODS: Overexpression and knockdown of PTBP1 was performed in myoblasts from two HML patients and a healthy control. Quantification of ISCU mis-splicing was done by qRTPCR. Biotinylated ISCU RNA, representing wildtype and mutant intron sequence, was used in a pull-down assay with nuclear extracts from myoblasts. Levels of PTBP1 in human cell lines and mice tissues were analyzed by qRTPCR and western blot. RESULTS: PTBP1 overexpression in HML patient myoblasts resulted in a substantial decrease of ISCU mis-splicing while knockdown of PTBP1 resulted in a drastic increase. The effect could be observed in both patient and control myoblasts. We could also show that PTBP1 interacts with both the mutant and wild-type ISCU intron sequence, but with a higher affinity to the mutant sequence. Furthermore, low levels of PTBP1 among examined mouse tissues correlated with high levels of incorrect splicing of ISCU. CONCLUSION: Our results show that PTBP1 acts as a dominant repressor of ISCU mis-splicing. We also show an inverse correlation between the levels of PTBP1 and ISCU mis-splicing, suggesting that the high level of mis-splicing in the skeletal muscle is primarily due to the low levels of PTBP1.

Sequence variations in C9orf72 downstream of the hexanucleotide repeat region and its effect on repeat-primed PCR interpretation: a large multinational screening study.

A large GGGGCC-repeat expansion mutation (HREM) in C9orf72 is the most common known cause of ALS and FTD in European populations. Sequence variations immediately downstream of the HREM region have previously been observed and have been suggested to be one reason for difficulties in interpreting RP-PCR data. Our objective was to determine the properties of these sequence variations with regard to prevalence, the range of variation, and effect on disease prognosis. We screened a multi-national cohort (n = 6981) for the HREM and samples with deviant RP-PCR curves were identified. The deviant samples were subsequently sequenced to determine sequence alteration. Our results show that in the USA and European cohorts (n = 6508) 10.7% carried the HREM and 3% had a sequence variant, while no HREM or sequence variants were observed in the Japanese cohort (n = 473). Sequence variations were more common on HREM alleles; however, certain population specific variants were associated with a non-expanded allele.In conclusion, we identified 38 different sequence variants, most located within the first 50 bp downstream of the HREM region. Furthermore, the presence of an HREM was found to be coupled to a lower age of onset and a shorter disease survival, while sequence variation did not have any correlation with these parameters.

Extensive size variability of the GGGGCC expansion in C9orf72 in both neuronal and non-neuronal tissues in 18 patients with ALS or FTD.

A GGGGCC-repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) among Caucasians. However, little is known about the variability of the GGGGCC expansion in different tissues and whether this correlates with the observed phenotype. Here, we used Southern blotting to estimate the size of hexanucleotide expansions in C9orf72 in neural and non-neural tissues from 18 autopsied ALS and FTD patients with repeat expansion in blood. Digitalization of the Southern blot images allowed comparison of repeat number, smear distribution and expansion band intensity between tissues and between patients. We found marked intra-individual variation of repeat number between tissues, whereas there was less variation within each tissue group. In two patients, the size variation between tissues was extreme, with repeat numbers below 100 in all studied non-neural tissues, whereas expansions in neural tissues were 20-40 times greater and in the same size range observed in neural tissues of the other 16 patients. The expansion pattern in different tissues could not distinguish between diagnostic groups and no correlation was found between expansion size in frontal lobe and occurrence of cognitive impairment. In ALS patients, a less number of repeats in the cerebellum and parietal lobe correlated with earlier age of onset and a larger number of repeats in the parietal lobe correlated with a more rapid progression. In 43 other individuals without repeat expansion in blood, we find that repeat sizes up to 15 are stable, as no size variation between blood, brain and spinal cord was found.

A blinded international study on the reliability of genetic testing for GGGGCC-repeat expansions in C9orf72 reveals marked differences in results among 14 laboratories.

BACKGROUND: The GGGGCC-repeat expansion in C9orf72 is the most frequent mutation found in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Most of the studies on C9orf72 have relied on repeat-primed PCR (RP-PCR) methods for detection of the expansions. To investigate the inherent limitations of this technique, we compared methods and results of 14 laboratories. METHODS: The 14 laboratories genotyped DNA from 78 individuals (diagnosed with ALS or FTD) in a blinded fashion. Eleven laboratories used a combination of amplicon-length analysis and RP-PCR, whereas three laboratories used RP-PCR alone; Southern blotting techniques were used as a reference. RESULTS: Using PCR-based techniques, 5 of the 14 laboratories got results in full accordance with the Southern blotting results. Only 50 of the 78 DNA samples got the same genotype result in all 14 laboratories. There was a high degree of false positive and false negative results, and at least one sample could not be genotyped at all in 9 of the 14 laboratories. The mean sensitivity of a combination of amplicon-length analysis and RP-PCR was 95.0% (73.9-100%), and the mean specificity was 98.0% (87.5-100%). Overall, a sensitivity and specificity of more than 95% was observed in only seven laboratories. CONCLUSIONS: Because of the wide range seen in genotyping results, we recommend using a combination of amplicon-length analysis and RP-PCR as a minimum in a research setting. We propose that Southern blotting techniques should be the gold standard, and be made obligatory in a clinical diagnostic setting.

The defective splicing caused by the ISCU intron mutation in patients with myopathy with lactic acidosis is repressed by PTBP1 but can be derepressed by IGF2BP1.

Hereditary myopathy with lactic acidosis (HML) is caused by an intron mutation in the iron-sulfur cluster assembly gene ISCU, which leads to the activation of cryptic splice sites and the retention of part of intron 4. This incorrect splicing is more pronounced in muscle than in other tissues, resulting in a muscle-specific phenotype. In this study, we identified five nuclear factors that interact with the sequence harboring the mutation and analyzed their effect on the splicing of the ISCU gene. The identification revealed three splicing factors, SFRS14, RBM39, and PTBP1, and two additional RNA binding factors, matrin 3 (MATR3) and IGF2BP1. IGF2BP1 showed a preference for the mutant sequence, whereas the other factors showed similar affinity for both sequences. PTBP1 was found to repress the defective splicing of ISCU, resulting in a drastic loss of mutant transcripts. In contrast, IGF2BP1 and RBM39 shifted the splicing ratio toward the incorrect splice form.

Tissue-specific splicing of ISCU results in a skeletal muscle phenotype in myopathy with lactic acidosis, while complete loss of ISCU results in early embryonic death in mice.

Hereditary myopathy with lactic acidosis (HML) is caused by an intron mutation in the iron-sulphur cluster assembly gene (ISCU) leading to incorporation of intron sequence into the mRNA. This results in a deficiency of Fe-S cluster proteins, affecting the TCA cycle and the respiratory chain. The proteins involved in the Fe-S machinery are evolutionary conserved and shown to be fundamental in all organisms examined. ISCU is expressed at high levels in numerous tissues in mammals, including high metabolic tissues like the heart, suggesting that a drastic mutation in the ISCU gene would be damaging to all energy-demanding organs. In spite of this, the symptoms in patients with HML are restricted to skeletal muscle, and it has been proposed that splicing events may contribute to the muscle specificity. In this study we confirm that a striking difference in the splicing pattern of mutant ISCU exists between different tissues. The highest level of incorrectly spliced ISCU mRNA was found in skeletal muscle, while the normal splice form predominated in patient heart. The splicing differences were also reflected at a functional level, where loss of Fe-S cluster carrying enzymes and accumulation of iron were present in muscle, but absent in other tissues. We also show that complete loss of ISCU in mice results in early embryonic death. The mice data confirm a fundamental role for ISCU in mammals and further support tissue-specific splicing as the major mechanism limiting the phenotype to skeletal muscle in HML.