smORFer: a modular algorithm to detect small ORFs in prokaryotes.

Emerging evidence places small proteins (≤50 amino acids) more centrally in physiological processes. Yet, their functional identification and the systematic genome annotation of their cognate small open-reading frames (smORFs) remains challenging both experimentally and computationally. Ribosome profiling or Ribo-Seq (that is a deep sequencing of ribosome-protected fragments) enables detecting of actively translated open-reading frames (ORFs) and empirical annotation of coding sequences (CDSs) using the in-register translation pattern that is characteristic for genuinely translating ribosomes. Multiple identifiers of ORFs that use the 3-nt periodicity in Ribo-Seq data sets have been successful in eukaryotic smORF annotation. They have difficulties evaluating prokaryotic genomes due to the unique architecture (e.g. polycistronic messages, overlapping ORFs, leaderless translation, non-canonical initiation etc.). Here, we present a new algorithm, smORFer, which performs with high accuracy in prokaryotic organisms in detecting putative smORFs. The unique feature of smORFer is that it uses an integrated approach and considers structural features of the genetic sequence along with in-frame translation and uses Fourier transform to convert these parameters into a measurable score to faithfully select smORFs. The algorithm is executed in a modular way, and dependent on the data available for a particular organism, different modules can be selected for smORF search.

AUNA2: A Novel Type of Non-Syndromic Slowly Progressive Auditory Synaptopathy/Auditory Neuropathy with Autosomal-Dominant Inheritance.

BACKGROUND: Auditory synaptopathy/neuropathy (AS/AN) is a heterogeneous disorder, which may be caused by environmental factors like postnatal hyperbilirubinemia or by genetic factors. The genetic forms are subdivided into syndromic and non-syndromic types, and show different inheritance patterns with a strong preponderance of autosomal-recessive forms. To date, only a single locus for non-syndromic autosomal-dominant AS/AN (AUNA1) has been reported in a single family, in which a non-coding DIAPH3 mutation was subsequently described as causative. MATERIALS AND METHODS: Here, we report detailed clinical data on a large German AS/AN family with slowly progressive postlingual hearing loss. Affected family members developed their first symptoms in their second decade. Moderate hearing loss in the fourth decade then progressed to profound hearing impairment in older family members. Comprehensive audiological and neurological tests were performed in the affected family members. Genetic testing comprised linkage analyses with polymorphic markers and a genome-wide linkage analysis using the Affymetrix GeneChip® Human Mapping 250K. RESULTS AND CONCLUSION: We identified a large family with autosomal-dominant AS/AN. By means of linkage analyses, the AUNA1 locus was excluded, and putatively linked regions on chromosomal bands 12q24 and 13q34 were identified as likely carrying the second locus for autosomal-dominant AS/AN (AUNA2). AUNA2 is associated with a slowly progressive postlingual hearing loss without any evidence for additional symptoms in other organ systems.

Interactive effects of citalopram and serotonin transporter genotype on neural correlates of response inhibition and attentional orienting.

The brain's serotonergic (5-HT) system has been implicated in controlling impulsive behavior and attentional orienting and linked to impulse control and anxiety related disorders. However, interactions between genotypical variation and responses to serotonergic drugs impede both treatment efficacy and neuroscientific research. We examine behavioral and electrophysiological responses to acute intravenous administration of a selective serotonin reuptake inhibitor (SSRI) while controlling for major genetic differences regarding 5-HT transporter (5-HTT) genotypes. Out of a genotyped sample of healthy Caucasian subjects (n=878) two extreme-groups regarding 5-HTT genotypes were selected (n=32). A homozygous high-expressing group based on tri-allelic 5-HTTLPR and rs25532 (LAC/LAC=LL) was compared to homozygous S allele carriers (SS). Both groups were administered a low dose of citalopram (10mg) intravenously in a double blind crossover fashion and performed a novelty NoGo paradigm while high density EEG was recorded. Interactions between drug and genotype were seen on both behavioral and neurophysiological levels. Reaction slowing following inhibitory events was decreased by the administration of citalopram in the LL but not SS group. This was accompanied by decreases in the amplitude of the inhibitory N2 EEG component and the P3b in the LL group, which was not seen in the SS group. SS subjects showed an increase in P3a amplitudes following SSRI administration to any type of deviant stimulus possibly reflecting increased attentional capture. The acute SSRI response on inhibitory processes and attentional orienting interacts with genotypes regulating 5-HTT gene expression. SS subjects may show increased attentional side effects reflected in increases in P3a amplitudes which could contribute to treatment discontinuation. Inhibitory processes and their neural correlates are affected only in LL subjects. These findings may indicate an underlying mechanism that could relate genotypical differences to altered side effect profiles and drug responses and are compatible with a non-monotonic relationship between 5-HT levels and optimal functioning.

Hereditary parkinsonism with dementia is caused by mutations in ATP13A2, encoding a lysosomal type 5 P-type ATPase.

Neurodegenerative disorders such as Parkinson and Alzheimer disease cause motor and cognitive dysfunction and belong to a heterogeneous group of common and disabling disorders. Although the complex molecular pathophysiology of neurodegeneration is largely unknown, major advances have been achieved by elucidating the genetic defects underlying mendelian forms of these diseases. This has led to the discovery of common pathophysiological pathways such as enhanced oxidative stress, protein misfolding and aggregation and dysfunction of the ubiquitin-proteasome system. Here, we describe loss-of-function mutations in a previously uncharacterized, predominantly neuronal P-type ATPase gene, ATP13A2, underlying an autosomal recessive form of early-onset parkinsonism with pyramidal degeneration and dementia (PARK9, Kufor-Rakeb syndrome). Whereas the wild-type protein was located in the lysosome of transiently transfected cells, the unstable truncated mutants were retained in the endoplasmic reticulum and degraded by the proteasome. Our findings link a class of proteins with unknown function and substrate specificity to the protein networks implicated in neurodegeneration and parkinsonism.

Loss-of-function mutations of ILDR1 cause autosomal-recessive hearing impairment DFNB42.

By using homozygosity mapping in a consanguineous Pakistani family, we detected linkage of nonsyndromic hearing loss to a 7.6 Mb region on chromosome 3q13.31-q21.1 within the previously reported DFNB42 locus. Subsequent candidate gene sequencing identified a homozygous nonsense mutation (c.1135G>T [p.Glu379X]) in ILDR1 as the cause of hearing impairment. By analyzing additional consanguineous families with homozygosity at this locus, we detected ILDR1 mutations in the affected individuals of 10 more families from Pakistan and Iran. The identified ILDR1 variants include missense, nonsense, frameshift, and splice-site mutations as well as a start codon mutation in the family that originally defined the DFNB42 locus. ILDR1 encodes the evolutionarily conserved immunoglobulin-like domain containing receptor 1, a putative transmembrane receptor of unknown function. In situ hybridization detected expression of Ildr1, the murine ortholog, early in development in the vestibule and in hair cells and supporting cells of the cochlea. Expression in hair cell- and supporting cell-containing neurosensory organs is conserved in the zebrafish, in which the ildr1 ortholog is prominently expressed in the developing ear and neuromasts of the lateral line. These data identify loss-of-function mutations of ILDR1, a gene with a conserved expression pattern pointing to a conserved function in hearing in vertebrates, as underlying nonsyndromic prelingual sensorineural hearing impairment.

Author Correction: Octa-repeat domain of the mammalian prion protein mRNA forms stable A-helical hairpin structure rather than G-quadruplexes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

New genetic evidence for involvement of the dopamine system in migraine with aura.

In order to systematically test the hypothesis that genetic variation in the dopamine system contributes to the susceptibility to migraine with aura (MA), we performed a comprehensive genetic association study of altogether ten genes from the dopaminergic system in a large German migraine with aura case-control sample. Based on the genotyping results of 53 variants across the ten genes in 270 MA cases and 272 controls, three genes-DBH, DRD2 and SLC6A3-were chosen to proceed to additional genotyping of 380 MA cases and 378 controls. Four of the 26 genotyped polymorphisms in these three genes displayed nominally significant allelic P-values in the sample of 650 MA patients and 650 controls. Three of these SNPs [rs2097629 in DBH (uncorrected allelic P value = 0.0012, OR = 0.77), rs7131056 in DRD2 (uncorrected allelic P value = 0.0018, OR = 1.28) and rs40184 in SLC6A3 (uncorrected allelic P value = 0.0082, OR = 0.81)] remained significant after gene-wide correction for multiple testing by permutation analysis. Further consideration of imputed genotype data from 2,937 British control individuals did not affirm the association with DRD2, but supported the associations with DBH and SLC6A3. Our data provide new evidence for an involvement of components of the dopaminergic system-in particular the dopamine-beta hydroxylase and dopamine transporter genes-to the pathogenesis of migraine with aura.

Replication study of the insulin receptor gene in migraine with aura.

We performed the first replication study for the reported association of the insulin receptor gene (INSR) with migraine with aura (MA). Two of 35 SNPs (rs1052371 and rs2860174) reached borderline significance (best uncorrected allelic p value of 0.052 for rs2860174) in stage 1 of our study (270 MA patients, 280 controls). As rs2860174 was 1 of the 5 SNPs with prior evidence of association, we also genotyped this SNP in our stage 2 sample (679 MA patients, 368 controls), and it was nonsignificant (allelic p value 0.478). The combined analysis of our samples showed just a nonsignificant trend for rs2860174 (p=0.1). However, the joint analysis of our study and the initial study reporting an association-including 1278 Caucasian MA patients and 1337 Caucasian controls altogether-displayed a significant allelic p value of 0.005. In conclusion, further association studies for rs2860174 with even larger numbers of individuals are required to exclude or confirm definitely a small effect of this SNP on migraine susceptibility.

Octa-repeat domain of the mammalian prion protein mRNA forms stable A-helical hairpin structure rather than G-quadruplexes.

Misfolding and aggregation of prion protein (PrP) causes neurodegenerative diseases like Creutzfeldt-Jakob disease (CJD) and scrapie. Besides the consensus that spontaneous conversion of normal cellular PrPC into misfolded and aggregating PrPSc is the central event in prion disease, an alternative hypothesis suggests the generation of pathological PrPSc by rare translational frameshifting events in the octa-repeat domain of the PrP mRNA. Ribosomal frameshifting most commonly relies on a slippery site and an adjacent stable RNA structure to stall translating ribosome. Hence, it is crucial to unravel the secondary structure of the octa-repeat domain of PrP mRNA. Each of the five octa-repeats contains a motif (GGCGGUGGUGGCUGGG) which alone in vitro forms a G-quadruplex. Since the propensity of mRNA to form secondary structure depends on the sequence context, we set to determine the structure of the complete octa-repeat region. We assessed the structure of full-length octa-repeat domain of PrP mRNA using dynamic light scattering (DLS), small angle X-ray scattering (SAXS), circular dichroism (CD) spectroscopy and selective 2'-hydroxyl acylation analysis by primer extension (SHAPE). Our data show that the PrP octa-repeat mRNA forms stable A-helical hairpins with no evidence of G-quadruplex structure even in the presence of G-quadruplex stabilizing agents.

A novel HSF4 gene mutation (p.R405X) causing autosomal recessive congenital cataracts in a large consanguineous family from Pakistan.

BACKGROUND: Hereditary cataracts are most frequently inherited as autosomal dominant traits, but can also be inherited in an autosomal recessive or X-linked fashion. To date, 12 loci for autosomal recessive cataracts have been mapped including a locus on chromosome 16q22 containing the disease-causing gene HSF4 (Genbank accession number NM_001040667). Here, we describe a family from Pakistan with the first nonsense mutation in HSF4 thus expanding the mutational spectrum of this heat shock transcription factor gene. METHODS: A large consanguineous Pakistani family with autosomal recessive cataracts was collected from Quetta. Genetic linkage analysis was performed for the common known autosomal recessive cataracts loci and linkage to a locus containing HSF4 (OMIM 602438) was found. All exons and adjacent splice sites of the heat shock transcription factor 4 gene (HSF4) were sequenced. A mutation-specific restriction enzyme digest (HphI) was performed for all family members and unrelated controls. RESULTS: The disease phenotype perfectly co-segregated with markers flanking the known cataract gene HSF4, whereas other autosomal recessive loci were excluded. A maximum two-point LOD score with a Zmax=5.6 at theta=0 was obtained for D16S421. Direct sequencing of HSF4 revealed the nucleotide exchange c.1213C>T in this family predicting an arginine to stop codon exchange (p.R405X). CONCLUSION: We identified the first nonsense mutation (p.R405X) in exon 11 of HSF4 in a large consanguineous Pakistani family with autosomal recessive cataract.

Genetic association studies of the chromosome 15 GABA-A receptor cluster in migraine with aura.

Recently, a novel susceptibility locus for migraine with aura (MA) on chromosome 15q containing three GABA-A receptor subunits has been identified by linkage analysis in several large pedigrees. To further study the role of this locus in MA etiology we genotyped 56 SNPs capturing the known common haplotype variations of these three candidate genes in a sample comprising 270 MA patients and 273 matched controls. In a single marker analysis, four SNPs displayed nominally significant (P < 0.05) association with MA. However, after permutation-based correction for the number of tests performed, the P-values of these SNPs were non-significant. Furthermore, a replication study of two of these SNPs in a second independent sample of 379 MA patients and 379 controls did not result in a significant finding. We also compared haplotype estimates based on case-control genotypes. Again we could not demonstrate a significant association with the phenotype after correction for multiple testing. In summary, we found no convincing evidence for an involvement of common SNPs at the GABA-A receptor cluster on 15q11-q12 in the pathophysiology of MA.

Dynamic m6A methylation facilitates mRNA triaging to stress granules.

Reversible post-transcriptional modifications on messenger RNA emerge as prevalent phenomena in RNA metabolism. The most abundant among them is N6-methyladenosine (m6A) which is pivotal for RNA metabolism and function; its role in stress response remains elusive. We have discovered that in response to oxidative stress, transcripts are additionally m6A modified in their 5' vicinity. Distinct from that of the translationally active mRNAs, this methylation pattern provides a selective mechanism for triaging mRNAs from the translatable pool to stress-induced stress granules. These stress-induced newly methylated sites are selectively recognized by the YTH domain family 3 (YTHDF3) "reader" protein, thereby revealing a new role for YTHDF3 in shaping the selectivity of stress response. Our findings describe a previously unappreciated function for RNA m6A modification in oxidative-stress response and expand the breadth of physiological roles of m6A.

Dysfunction of the MDM2/p53 axis is linked to premature aging.

The tumor suppressor p53, a master regulator of the cellular response to stress, is tightly regulated by the E3 ubiquitin ligase MDM2 via an autoregulatory feedback loop. In addition to its well-established role in tumorigenesis, p53 has also been associated with aging in mice. Several mouse models with aberrantly increased p53 activity display signs of premature aging. However, the relationship between dysfunction of the MDM2/p53 axis and human aging remains elusive. Here, we have identified an antiterminating homozygous germline mutation in MDM2 in a patient affected by a segmental progeroid syndrome. We show that this mutation abrogates MDM2 activity, thereby resulting in enhanced levels and stability of p53. Analysis of the patient's primary cells, genome-edited cells, and in vitro and in vivo analyses confirmed the MDM2 mutation's aberrant regulation of p53 activity. Functional data from a zebrafish model further demonstrated that mutant Mdm2 was unable to rescue a p53-induced apoptotic phenotype. Altogether, our findings indicate that mutant MDM2 is a likely driver of the observed segmental form of progeria.

Identification of two novel ALS2 mutations in infantile-onset ascending hereditary spastic paraplegia.

Biallelic mutations of ALS2 cause a clinical spectrum of overlapping autosomal recessive neurodegenerative disorders: infantile-onset ascending hereditary spastic paralysis (IAHSP), juvenile primary lateral sclerosis (JPLS), and juvenile amyotrophic lateral sclerosis (ALS2). We report on eleven individuals affected with IAHSP from two consanguineous Pakistani families. A combination of linkage analysis with homozygosity mapping and targeted sequencing identified two novel ALS2 mutations, a c.194T > C (p.Phe65Ser) missense substitution located in the first RCC-like domain of ALS2/alsin and a c.2998delA (p.Ile1000*) nonsense mutation. This study of extended families including a total of eleven affected individuals suggests that a given ALS2 mutation may lead to a phenotype with remarkable intrafamilial clinical homogeneity.

Rare missense variants in ATP1A2 in families with clustering of common forms of migraine.

Migraine is a recurrent neurovascular disease. Its two most common forms-migraine without aura (MO) and migraine with aura (MA)-both show familial clustering and a complex pattern of inheritance. Familial hemiplegic migraine (FHM) is a rare monogenic subform caused by mutations in the calcium channel gene CACNA1A or the Na(+)/K(+)-ATPase gene ATP1A2. An involvement of FHM genes in the pathogenesis of common forms of migraine is not proven. We therefore systematically screened ATP1A2 in families with several members affected by MA and/or MO. We identified two novel missense alterations [c.520G>A (p.E174 K) and c.1544G>A (p.C515Y)] in two out of 45 families, which were not found in 520 control chromosomes. Functional studies of these variants in Xenopus oocytes by two-electrode voltage clamp measurements and radiochemical determination of ATPase activity showed that C515Y leads to a complete loss of function comparable with the effect of FHM-mutations whereas for E174 K no functional alteration could be found in the in vitro assays. In conclusion we propose that rare variants in ATP1A2 are involved in the susceptibility to common forms of migraine, because of 1) the absence of alterations in controls, 2) the particular pattern of segregation in both families, 3) the high conservation of mutated residues in Na(+)/K(+)-ATPases, 4) the functional effect of C515Y, and 5) the involvement of ATP1A2 in a monogenic form of migraine.

De novo FUS mutations are the most frequent genetic cause in early-onset German ALS patients.

In amyotrophic lateral sclerosis (ALS) patients with known genetic cause, mutations in chromosome 9 open reading frame 72 (C9orf72) and superoxide dismutase 1 (SOD1) account for most familial and late-onset sporadic cases, whereas mutations in fused in sarcoma (FUS) can be identified in just around 5% of familial and 1% of overall sporadic cases. There are only few reports on de novo FUS mutations in juvenile ALS patients. To date, no systematic evaluation on the frequency of de novo FUS mutations in early-onset ALS patients has been conducted. Here, we screened a cohort of 14 early-onset sporadic ALS patients (onset age <35 years) to determine the frequency of mutations in C9orf72, SOD1, and FUS in this defined patient cohort. All patients were recruited prospectively by a single center in a period of 38 months. No mutations were detected in SOD1 or C9orf72; however, we identified 6 individuals (43%) carrying a heterozygous FUS mutation including 1 mutation that has not been described earlier (c.1504delG [p.Asp502Thrfs*27]). Genetic testing of parents was possible in 5 families and revealed that the mutations in these patients arose de novo. Three of the 6 identified patients presented with initial bulbar symptoms. Our study identifies FUS mutations as the most frequent genetic cause in early-onset ALS. Genetic testing of FUS thus seems indicated in sporadic early-onset ALS patients especially if showing predominant bulbar symptoms and an aggressive disease course.

Identification of a novel mutation in the coding region of the grey-lethal gene OSTM1 in human malignant infantile osteopetrosis.

Autosomal recessive malignant infantile osteopetrosis (ARO) is characterized by severe osteosclerosis, pathologic fractures, hepatosplenomegaly, and pancytopenia. The pathophysiological basis is inadequate bone resorption due to osteoclast dysfunction. In the majority of cases, mutations in either of two human genes cause this fatal disorder: TCIRG1, encoding a subunit of the osteoclast H(+)-ATPase, and the voltage-gated chloride channel gene CLCN7. We excluded both genes in a small inbred family with malignant infantile osteopetrosis and undertook linkage analysis of several candidate loci that are involved in murine osteopetrosis. A region spanning more than 20 cM between the markers D6S1717 and D6S1608 on chromosome 6q21 was found to be homozygous in the affected child. This locus is syntenic to the genomic region harboring the gene for the osteopetrotic mutant mouse grey-lethal (gl). Recently, mutations in a novel gene of unknown function were described in the grey-lethal mouse and in one human patient. Mutation screening of the grey-lethal gene (OSTM1), revealed a homozygous 2-bp deletion in exon 2 (c.415_416delAG) in the affected child. No mutations could be found in six independent ARO patients who had tested negative for mutations in TCIRG1 and CLCN7. In summary, we describe the identification of a novel mutation in the coding sequence of the human grey-lethal gene, which is the second OSTM1 mutation found in human ARO, confirming the involvement of this gene in the pathogenesis of this severe bone disease.

A homozygous splice site mutation in TRAPPC9 causes intellectual disability and microcephaly.

Autosomal recessive intellectual disability is believed to be particularly prevalent in highly consanguineous populations and genetic isolates and may account for a quarter of all non-syndromic cases. Mutations in more than 50 genes have been reported to be involved in autosomal recessive intellectual disability, including TRAPPC9 (MIM 611966), mutations of which have been identified in six families from different geographical origins. We performed a clinical and molecular genetic study of a consanguineous Pakistani family segregating intellectual disability and microcephaly. SNP-array-based homozygosity mapping revealed suggestive linkage to four genomic regions including one on chromosome 8 that contained TRAPPC9. We detected a homozygous TRAPPC9 splice donor site mutation (c.1024+1G>T) that cosegregated with intellectual disability in the family and led to skipping of exon 3 and exons 3 and 4 in blood-derived patient RNA. We have thus identified a novel splice site mutation leading to exon skipping and premature termination of TRAPPC9 translation. These data further suggest that TRAPPC9 mutations -unlike mutations in the vast majority of the known intellectual disability-associated genes- constitute a more frequent cause of autosomal-recessive cognitive deficits, especially when microcephaly is also present.

Genome-wide association study of migraine implicates a common susceptibility variant on 8q22.1.

Migraine is a common episodic neurological disorder, typically presenting with recurrent attacks of severe headache and autonomic dysfunction. Apart from rare monogenic subtypes, no genetic or molecular markers for migraine have been convincingly established. We identified the minor allele of rs1835740 on chromosome 8q22.1 to be associated with migraine (P = 5.38 × 10⁻9, odds ratio = 1.23, 95% CI 1.150-1.324) in a genome-wide association study of 2,731 migraine cases ascertained from three European headache clinics and 10,747 population-matched controls. The association was replicated in 3,202 cases and 40,062 controls for an overall meta-analysis P value of 1.69 × 10⁻¹¹ (odds ratio = 1.18, 95% CI 1.127-1.244). rs1835740 is located between MTDH (astrocyte elevated gene 1, also known as AEG-1) and PGCP (encoding plasma glutamate carboxypeptidase). In an expression quantitative trait study in lymphoblastoid cell lines, transcript levels of the MTDH were found to have a significant correlation to rs1835740 (P = 3.96 × 10⁻5, permuted threshold for genome-wide significance 7.7 × 10⁻5. To our knowledge, our data establish rs1835740 as the first genetic risk factor for migraine.