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.

Translational repression of the McKusick-Kaufman syndrome transcript by unique upstream open reading frames encoding mitochondrial proteins with alternative polyadenylation sites.

BACKGROUND: Upstream open reading frames (uORFs) are commonly found in the 5'-untranslated region (UTR) of many genes and function in translational control. However, little is known about the existence of the proteins encoded by uORFs, and the role of the proteins except translational control. There was no report about uORFs of the McKusick-Kaufman syndrome (MKKS) gene that causes a genetic disorder. METHODS: Northern blotting, 3'-RACE, and bioinformatics were used for determining the length of transcripts and their 3' ends. Luciferase assay and in vitro translation were used for evaluation of translational regulatory activity of uORFs. Immunoblotting and immunocytochemical analyses were used for detection of uORF-derived protein products and their subcellular localization. RESULTS: The MKKS gene generates two types of transcripts: a canonical long transcript that encodes both uORFs and MKKS, and a short transcript that encodes only uORFs by using alternative polyadenylation sites at the 5'-UTR. The simultaneous disruption of the uORF initiation codons increased the translation of the downstream ORF. Furthermore, both protein products from the two longest uORFs were detected in the mitochondrial membrane fraction of HeLa cells. Database searches indicated that such uORFs with active alternative polyadenylation sites at the 5'-UTR are atypical but surely exist in human transcripts. CONCLUSIONS: Multiple uORFs at the 5'-UTR of the MKKS long transcript function as translational repressor for MKKS. Two uORFs are translated in vivo and imported onto the mitochondrial membrane. GENERAL SIGNIFICANCE: Our findings provide unique insights into production of uORF-derived peptides and functions of uORFs.

PGC-1α is a male-specific disease modifier of human and experimental amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating, adult-onset neurodegenerative disorder of the upper and lower motor systems. It leads to paresis, muscle wasting and inevitably to death, typically within 3-5 years. However, disease onset and survival vary considerably ranging in extreme cases from a few months to several decades. The genetic and environmental factors underlying this variability are of great interest as potential therapeutic targets. In ALS, men are affected more often and have an earlier age of onset than women. This gender difference is recapitulated in transgenic rodent models, but no underlying mechanism has been elucidated. Here we report that SNPs in the brain-specific promoter region of the transcriptional co-activator PGC-1α, a master regulator of metabolism, modulate age of onset and survival in two large and independent ALS populations and this occurs in a strictly male-specific manner. In complementary animal studies, we show that deficiency of full-length (FL) Pgc-1α leads to a significantly earlier age of onset and a borderline shortened survival in male, but not in female ALS-transgenic mice. In the animal model, FL Pgc-1α-loss is associated with reduced mRNA levels of the trophic factor Vegf-A in males, but not in females. In summary, we indentify PGC-1α as a novel and clinically relevant disease modifier of human and experimental ALS and report a sex-dependent effect of PGC-1α in this neurodegenerative disorder.

No GGGGCC-hexanucleotide repeat expansion in C9ORF72 in parkinsonism patients in Sweden.

An intronic GGGGCC-hexanucleotide repeat expansion in C9ORF72 was recently identified as a major cause of amyotrophic lateral sclerosis and frontotemporal dementia. Some amyotrophic lateral sclerosis patients have signs of parkinsonism, and many parkinsonism patients develop dementia. In this study we examined if the hexanucleotide repeat expansion was present in parkinsonism patients, to clarify if there could be a relationship between the repeat expansion and disease. We studied the size of the hexanucleotide repeat expansion in a well defined population-based cohort of 135 Parkinson's disease patients and 39 patients with atypical parkinsonism and compared with 645 Swedish control subjects. We found no correlation between Parkinson's disease or atypical parkinsonism and the size of the GGGGCC repeat expansion in C9ORF72. In conclusion, this GGGGCC-repeat expansion in C9ORF72 is not a cause of parkinsonism in the Swedish population.

A novel phosphorylation site mutation in profilin 1 revealed in a large screen of US, Nordic, and German amyotrophic lateral sclerosis/frontotemporal dementia cohorts.

Profilin 1 is a central regulator of actin dynamics. Mutations in the gene profilin 1 (PFN1) have very recently been shown to be the cause of a subgroup of amyotrophic lateral sclerosis (ALS). Here, we performed a large screen of US, Nordic, and German familial and sporadic ALS and frontotemporal dementia (FTLD) patients for PFN1 mutations to get further insight into the spectrum and pathogenic relevance of this gene for the complete ALS/FTLD continuum. Four hundred twelve familial and 260 sporadic ALS cases and 16 ALS/FTLD cases from Germany, the Nordic countries, and the United States were screened for PFN1 mutations. Phenotypes of patients carrying PFN1 mutations were studied. In a German ALS family we identified the novel heterozygous PFN1 mutation p.Thr109Met, which was absent in controls. This novel mutation abrogates a phosphorylation site in profilin 1. The recently described p.Gln117Gly sequence variant was found in another familial ALS patient from the United States. The ALS patients with mutations in PFN1 displayed spinal onset motor neuron disease without overt cognitive involvement. PFN1 mutations were absent in patients with motor neuron disease and dementia, and in patients with only FTLD. We provide further evidence that PFN1 mutations can cause ALS as a Mendelian dominant trait. Patients carrying PFN1 mutations reported so far represent the "classic" ALS end of the ALS-FTLD spectrum. The novel p.Thr109Met mutation provides additional proof-of-principle that mutant proteins involved in the regulation of cytoskeletal dynamics can cause motor neuron degeneration. Moreover, this new mutation suggests that fine-tuning of actin polymerization by phosphorylation of profilin 1 might be necessary for motor neuron survival.

High-Resolution Melting (HRM) Analysis of the Cu/Zn Superoxide Dismutase (SOD1) Gene in Japanese Sporadic Amyotrophic Lateral Sclerosis (SALS) Patients.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder, and the majority of ALS are sporadic (SALS). Recently, several causative genes for familial ALS (FALS) were identified, but the cause of the SALS is still unknown. This time, we aimed to identify the genetic background of SALS. First, we applied the new sensitive screening methods: high-resolution melting (HRM) analysis. HRM analysis detected 18 out of 19 known SOD1 gene mutations (94.7% sensitivity). Next, we screened SOD1, three novel mutations (C6Y, Q22H, and S134T) were identified in our own 184 SALS cases (1.63% prevalence), and four mutations in another 255 SALS cases (1.56% prevalence) registered from all over Japan. The patients with SOD1 mutations suggested a relatively young onset and limb involvement at onset. The HRM analysis is a sensitive and easy screening method; we will use this method for screening other ALS causative genes and revealing the genetic background of SALS.

Replication analysis of SNPs on 9p21.2 and 19p13.3 with amyotrophic lateral sclerosis in East Asians.

We performed a replication study of the 2 genetic variants, rs2814707 on 9p21.2 and rs12608932 on 19p13.3 that are recently reported to be most significantly associated with sporadic amyotrophic lateral sclerosis (ALS) in Caucasians. Both rs12608932 and rs2814707 showed no evidence of association in Japanese and Chinese (rs12608932, combined p = 0.58, odds ratio [OR] = 1.03, 95% confidence interval [CI] 0.93-1.13; rs2814707, combined p = 0.88, OR = 1.10, 95% CI 0.93-1.30). The association of these loci with susceptibility to sporadic ALS is considered negative in East Asians.

[Novel mutation in X-linked Charcot-Marie-tooth (CMTXI) disease associated with central conduction slowing on brainstem auditory evoked potential (BAEP)].

CMTX1, the second most common type of inherited hereditary motor and sensory neuropathy (HMSN), is associated with mutations of the gene for the gap junction protein connexin 32 (Cx32). In this condition, central conduction velocity is known to be delayed, presumably because mutated Cx32 is expressed in oligodendrocytes. A 45-year-old man presented with a 5-year history of progressive gait disturbance due to leg muscle weakness. The family history revealed that the mother had also progressive gait disturbance in her early 40s, and the younger sister could not walk faster than before at the age of 41. On neurological assessment, the patient exhibited pes cavus, distal muscle atrophy and weakness, and absence of the knee and ankle jerks. Touch sensation was impaired in the both feet. Motor and sensory nerve conduction velocities were reduced to 30-36 m/s with mild temporal dispersion. Sural nerve biopsy revealed diffuse loss of large myelinated fibers with the remaining large and intermediate nerve fibers being frequently surrounded by a thin myelin sheath. Onion bulb formation was only occasional and mild in degree. His hearing acuity was normal on pure-tone audiometry, but BAEP test demonstrated prolonged central conduction time (-I wave 1.8 milliseconds, I-V wave 6.4 milliseconds). The BAEP findings prompted us to choose Cx32 gene to analyze first to find a novel mutation of two (A and T) base pairs deletion at codons 277 and 278 (Met93fs). Thus, the present case indicates that Cx32 gene mutation should be targeted first in case of HMSN with abnormal BAEP.