Highly accurate quantification of allelic gene expression for population and disease genetics.

Analysis of allele-specific gene expression (ASE) is a powerful approach for studying gene regulation, particularly when sample sizes are small, such as for rare diseases, or when studying the effects of rare genetic variation. However, detection of ASE events relies on accurate alignment of RNA sequencing reads, where challenges still remain, particularly for reads containing genetic variants or those that align to many different genomic locations. We have developed the Personalised ASE Caller (PAC), a tool that combines multiple steps to improve the quantification of allelic reads, including personalized (i.e., diploid) read alignment with improved allocation of multimapping reads. Using simulated RNA sequencing data, we show that PAC outperforms standard alignment approaches for ASE detection, reducing the number of sites with incorrect biases (>10%) by ∼80% and increasing the number of sites that can be reliably quantified by ∼3%. Applying PAC to real RNA sequencing data from 670 whole-blood samples, we show that genetic regulatory signatures inferred from ASE data more closely match those from population-based methods that are less prone to alignment biases. Finally, we use PAC to characterize cell type-specific ASE events that would be missed by standard alignment approaches, and in doing so identify disease relevant genes that may modulate their effects through the regulation of gene expression. PAC can be applied to the vast quantity of existing RNA sequencing data sets to better understand a wide array of fundamental biological and disease processes.

Multiple marker abundance profiling: combining selected reaction monitoring and data-dependent acquisition for rapid estimation of organelle abundance in subcellular samples.

Measuring changes in protein or organelle abundance in the cell is an essential, but challenging aspect of cell biology. Frequently-used methods for determining organelle abundance typically rely on detection of a very few marker proteins, so are unsatisfactory. In silico estimates of protein abundances from publicly available protein spectra can provide useful standard abundance values but contain only data from tissue proteomes, and are not coupled to organelle localization data. A new protein abundance score, the normalized protein abundance scale (NPAS), expands on the number of scored proteins and the scoring accuracy of lower-abundance proteins in Arabidopsis. NPAS was combined with subcellular protein localization data, facilitating quantitative estimations of organelle abundance during routine experimental procedures. A suite of targeted proteomics markers for subcellular compartment markers was developed, enabling independent verification of in silico estimates for relative organelle abundance. Estimation of relative organelle abundance was found to be reproducible and consistent over a range of tissues and growth conditions. In silico abundance estimations and localization data have been combined into an online tool, multiple marker abundance profiling, available in the SUBA4 toolbox (http://suba.live).

Late onset spinal motor neuronopathy is caused by mutation in CHCHD10.

OBJECTIVE: A study was undertaken to identify the responsible gene defect underlying late onset spinal motor neuronopathy (LOSMoN/SMAJ; Online Mendelian Inheritance in Man #615048), an autosomal dominant disease mapped to chromosome 22q11.2. METHODS: The previous genetic linkage approach by microsatellite haplotyping was continued in new families. A whole genome sequencing was performed to find all possibly pathogenic mutations in the linked area. The detected variations were verified by Sanger sequencing. RESULTS: Six new SMAJ families were identified based on the unique founder haplotype. A critical recombination in 1 family restricted the linked area to 727kb between markers SHGC-106816 and D22S345. In whole genome sequencing a previously unknown mutation c.197G>T p.G66V in CHCHD10 was identified. The mutation was shown to segregate with the disease in 55 patients from 17 families. INTERPRETATION: Mutation c.197G>T p.G66V in CHCHD10 is the cause of the lower motor neuron syndrome LOSMoN/SMAJ. During the preparation of this article other mutations were reported to cause frontotemporal dementia-amyotrophic lateral sclerosis syndrome, indicating that the CHCHD10 gene is largely important for the motor and cognitive neuronal systems.

Serum Creatine, Not Neurofilament Light, Is Elevated in CHCHD10-Linked Spinal Muscular Atrophy.

OBJECTIVE: To characterize serum biomarkers in mitochondrial CHCHD10-linked spinal muscular atrophy Jokela (SMAJ) type for disease monitoring and for the understanding of pathogenic mechanisms. METHODS: We collected serum samples from a cohort of 49 patients with SMAJ, all carriers of the heterozygous c.197G>T p.G66V variant in CHCHD10. As controls, we used age- and sex-matched serum samples obtained from Helsinki Biobank. Creatine kinase and creatinine were measured by standard methods. Neurofilament light (NfL) and glial fibrillary acidic protein (GFAP) were measured with single molecule array (Simoa), fibroblast growth factor 21 (FGF-21), and growth differentiation factor 15 (GDF-15) with an enzyme-linked immunosorbent assay. For non-targeted plasma metabolite profiling, samples were analyzed with liquid chromatography high-resolution mass spectrometry. Disease severity was evaluated retrospectively by calculating a symptom-based score. RESULTS: Axon degeneration marker, NfL, was unexpectedly not altered in the serum of patients with SMAJ, whereas astrocytic activation marker, GFAP, was slightly decreased. Creatine kinase was elevated in most patients, particularly men. We identified six metabolites that were significantly altered in serum of patients with SMAJ in comparison to controls: increased creatine and pyruvate, and decreased creatinine, taurine, N-acetyl-carnosine, and succinate. Creatine correlated with disease severity. Altered pyruvate and succinate indicated a metabolic response to mitochondrial dysfunction; however, lactate or mitochondrial myopathy markers FGF-21 or GDF-15 was not changed. CONCLUSIONS: Biomarkers of muscle mass and damage are altered in SMAJ serum, indicating a role for skeletal muscle in disease pathogenesis in addition to neurogenic damage. Despite the minimal mitochondrial pathology in skeletal muscle, signs of a metabolic shift can be detected.

Human disease caused by loss of fast IIa myosin heavy chain due to recessive MYH2 mutations.

Striated muscle myosin heavy chain is a molecular motor protein that converts chemical energy into mechanical force. It is a major determinant of the physiological properties of each of the three muscle fibre types that make up the skeletal muscles. Heterozygous dominant missense mutations in myosin heavy chain genes cause various types of cardiomyopathy and skeletal myopathy, but the effects of myosin heavy chain null mutations in humans have not previously been reported. We have identified the first patients lacking fast type 2A muscle fibres, caused by total absence of fast myosin heavy chain IIa protein due to truncating mutations of the corresponding gene MYH2. Five adult patients, two males and three females, from three unrelated families in UK and Finland were clinically assessed and muscle biopsy was performed in one patient from each family. MYH2 was sequenced and the expression of the corresponding transcripts and protein was analysed in muscle tissue. The patients had early-onset symptoms characterized by mild generalized muscle weakness, extraocular muscle involvement and relatively favourable prognosis. Muscle biopsy revealed myopathic changes including variability of fibre size, internalized nuclei, and increased interstitial connective and adipose tissue. No muscle fibres expressing type IIa myosin heavy chain were identified and the MYH2 transcripts were markedly reduced. All patients were compound heterozygous for truncating mutations in MYH2. The parents were unaffected, consistent with recessive mutations. Our findings show that null mutations in the fast myosin heavy chain IIa gene cause early onset myopathy and demonstrate that this isoform is necessary for normal muscle development and function. The relatively mild phenotype is interesting in relation to the more severe phenotypes generally seen in relation to recessive null mutations in sarcomeric proteins.

A novel COL6A2 mutation causing late-onset limb-girdle muscular dystrophy.

Limb-girdle muscular dystrophies (LGMD) are genetic disorders characterized by weakness of predominantly proximal limb and trunk muscles due to progressive loss of muscle tissue. Collagen VI-related muscular dystrophies usually display more generalized muscle involvement combined with contractures and/or hyperlaxity of distal finger joints. LGMD-like phenotype of collagenopathy has only rarely been described and as reported is usually of childhood onset. We identified a Finnish family with COL6A2-related LGMD with autosomal dominant inheritance and very late onset at 40-60 years of age. Since the mutation was previously unreported, the pathognomonic findings on muscle MRI were the decisive clue for the correct diagnosis.

Adipokines and inflammatory markers in elderly subjects with high risk of type 2 diabetes and cardiovascular disease.

Inflammation plays a significant role in pathogenesis of diabetes and atherosclerosis. Increased adiposity with an upregulation of cytokines in prediabetes has been associated with vascular inflammation and considered a leading causal factor for type 2 diabetes (T2D). Information on adipokines and inflammatory markers in prediabetes, defined by hemoglobin A1C (HbA1c) 5.7-6.4% in addition to impaired fasting glucose (IFG) and impaired glucose tolerance (IGT), are sparse. We conducted a population-based cross-sectional study (part of a follow-up study) of inhabitants of Oulu, Finland, born in 1935. Inflammatory markers and traditional risk markers of 367 subjects were measured. The glucose status was determined by an oral glucose tolerance test (OGTT) and HbA1c. Inflammatory markers and glycemic levels were analysed using analysis of covariance (ANCOVA). Of the participants, 193 were normoglycemic, 82 had prediabetes and 40 T2D. Inflammatory cytokines were significantly higher in subjects with prediabetes as compared to normoglycemic subjects: IL-4 (14.9 vs 5.9 pg/ml, p = 0.041), IP-10 (251 vs 209 pg/ml, p = 0.05), TNF-α (10.4 vs 6.9 pg/ml, p = 0.027), RANTES (43.3 vs 33.1 pg/ml, p = 0.009), CD40L (3708 vs 1671 pg/ml, p = 0.010) and VEGF (269 vs 174 pg/ml, p = 0.013). These inflammatory cytokines remained significant even after adjustment for waist circumference. The differences in inflammatory markers in prediabetic and T2D subjects were not statistically significant. Prediabetes was associated with low-grade inflammation with increased inflammatory cytokine levels, while the levels in prediabetic subjects were comparable to those in T2D subjects. The associations were independent of visceral adiposity.

SNCA mutation p.Ala53Glu is derived from a common founder in the Finnish population.

Mutations in SNCA are rare causes of familial Parkinson's disease (PD). We have previously described a novel p.Ala53Glu mutation in 2 Finnish families. To assess this mutation's frequency among Finnish PD patients, we screened 110 PD patients (mean age-of-onset 60 years) from Western Finland by Sanger sequencing of the third coding exon of SNCA. In addition, a sample of 47 PD subjects (mean age-of-onset 53 years) originating from Southern and Eastern Finland were studied using next-generation sequencing covering SNCA. Only one new individual with the p.Ala53Glu mutation was identified, confirming that this mutation is a rare cause of PD in the Finnish population. To search for a possible common origin of the p.Ala53Glu mutation, haplotype analysis was conducted in 2 families and in a patient from a third family (6 affected subjects) using both STR markers and a genome-wide SNP array. The results show that patients with the p.Ala53Glu mutation share a haplotype spanning a minimum of 5.7 Mb suggesting a common founder.

Late-onset spinal motor neuronopathy - a common form of dominant SMA.

We previously described two Finnish families with a new autosomal dominant late-onset spinal motor neuronopathy that was mapped to chromosome 22q11.2-q13.2. In the current screening study of 43 lower motor neuron disease patients from Finland and Sweden, we identified 26 new late-onset spinal motor neuronopathy patients sharing the founder haplotype. In addition to the main symptoms and signs: painful cramps, fasciculations, areflexia and slowly evolving muscle weakness, new features such as mild bulbar findings, were identified. The disease is relatively benign in terms of life expectancy and rate of disability progression, and it is therefore noteworthy that three patients were initially misdiagnosed with ALS. Significant recombinants in this new patient cohort restricted the disease locus by 90% to 1.8Mb. Late-onset spinal motor neuronopathy seems not to be very rare, at least not in Finland, with 38 patients identified in a preliminary ascertainment.

Autosomal dominant late-onset spinal motor neuronopathy is linked to a new locus on chromosome 22q11.2-q13.2.

Spinal muscular atrophies (SMAs) are hereditary disorders characterized by degeneration of lower motor neurons. Different SMA types are clinically and genetically heterogeneous and many of them show significant phenotypic overlap. We recently described the clinical phenotype of a new disease in two Finnish families with a unique autosomal dominant late-onset lower motor neuronopathy. The studied families did not show linkage to any known locus of hereditary motor neuron disease and thus seemed to represent a new disease entity. For this study, we recruited two more family members and performed a more thorough genome-wide scan. We obtained significant linkage on chromosome 22q, maximum LOD score being 3.43 at marker D22S315. The linked area is defined by flanking markers D22S686 and D22S276, comprising 18.9 Mb. The region harbours 402 genes, none of which is previously known to be associated with SMAs. This study confirms that the disease in these two families is a genetically distinct entity and also provides evidence for a founder mutation segregating in both pedigrees.