Silke Appenzeller

Novel genomic techniques open new avenues in the analysis of monogenic disorders

The molecular genetic cause of over 3,000 monogenic disorders is currently unknown. This review discusses how novel genomic techniques like Next‐Generation DNA Sequencing (NGS) and genotyping arrays open new avenues in the elucidation of genetic defects causing monogenic disorders. They will not only speed up disease gene identification but will enable us to systematically tackle previously intractable monogenic disorders. These are mainly disorders not amenable to classic linkage analysis, for example, due to insufficient family size. Most monogenic diseases are caused by exonic mutations or splice‐site mutations changing the amino acid sequence of the affected gene. These mutations can be identified by sequencing of all exons in the human genome (exome sequencing) rendering whole genome sequencing unnecessary in most cases. Genotyping arrays containing 105–2×106 single nucleotide polymorphisms (SNPs) and nonpolymorphic markers allow highly accurate mapping of genomic deletions and duplications not detectable by exome sequencing, which are the second most common cause of monogenic disorders. However, several hundred rare, previously unknown sequence variants affecting the amino acid sequence of the encoded protein are found in the exome of every human individual. Therefore, the main challenge will be the differentiation between the many rare benign variants detected by novel genomic techniques and disease causing mutations. Hum Mutat 32:144–151, 2011.

Polymorphisms in the glial glutamate transporter SLC1A2 are associated with essential tremor

Objective: Sporadic, genetically complex essential tremor (ET) is one of the most common movement disorders and may lead to severe impairment of the quality of life. Despite high heritability, the genetic determinants of ET are largely unknown. We performed the second genome-wide association study (GWAS) for ET to elucidate genetic risk factors of ET. Methods: Using the Affymetrix Genome-Wide SNP Array 6.0 (1000K) we conducted a two-stage GWAS in a total of 990 subjects and 1,537 control subjects from Europe to identify genetic variants associated with ET. Results: We discovered association of an intronic variant of the main glial glutamate transporter (SLC1A2) gene with ET in the first-stage sample (rs3794087, p = 6.95 × 10−5, odds ratio [OR] = 1.46). We verified the association of rs3794087 with ET in a second-stage sample (p = 1.25 × 10−3, OR = 1.38). In the subgroup analysis of patients classified as definite ET, rs3794087 obtained genome-wide significance (p = 3.44 × 10−10, OR = 1.59) in the combined first- and second-stage sample. Genetic fine mapping using nonsynonymous single nucleotide polymorphisms (SNPs) and SNPs in high linkage disequilibrium with rs3794087 did not reveal any SNP with a stronger association with ET than rs3794087. Conclusions: We identified SLC1A2 encoding the major glial high-affinity glutamate reuptake transporter in the brain as a potential ET susceptibility gene. Acute and chronic glutamatergic overexcitation is implied in the pathogenesis of ET. SLC1A2 is therefore a good functional candidate gene for ET.

No evidence for differential methylation of α-synuclein in leukocyte DNA of Parkinson's disease patients†‡§

Alpha-synuclein (a-Syn) is central to the pathogenesis of Parkinson’s disease (PD). Overexpression of a-Syn caused by genomic duplications/triplications is sufficient to cause monogenic PD. Moreover, association studies have identified potentially regulatory polymorphisms in the a-Syn gene (SNCA, Acc. No. NM_000345.3) associated with an increased risk for sporadic PD. These observations demonstrate that the regulation of a-Syn expression is pivotal in the pathogenesis of PD. It has recently been shown that a CpG island in a putative promoter region of intron 1 of SNCA is hypomethylated in brain tissue of PD patients compared with in neurologically healthy controls. Hypomethylation was associated with increased a-Syn expression in cell lines as well as with increased transcriptional activity in a luciferase reporter assay in both studies. In an attempt to identify a biomarker for PD, we analyzed the methylation status of SNCA in bisulfite converted genomic DNA of peripheral white blood cells of 43 patients with idiopathic PD (49% female; average age, 64.8 years; average age at onset, 61 years), 3 patients with monogenic PD because of duplication of the SNCA gene, and 37 neurologically normal controls (50% female; average age, 64.6 years). All patients were diagnosed and recruited in 2 centers that specialize in movement disorders (Kiel and Lübeck). The study was approved by the local ethics committees, and written informed consent was obtained from all participants. The groups with idiopathic PD and controls did not differ significantly regarding sex (P 1⁄4 .36, chi-square test), age (P 1⁄4 1.00, ANOVA), or ethnic origin (all German). White blood cell DNA was prepared after erythrocyte lysis by the same standard salting-out procedure for all patients. Bisulfite conversion, PCR amplification, and pyrosequencing were performed as described previously, except that a PyroMark PCR kit (Qiagen, Hilden, Germany) was used for PCR amplifications. The 2 PCR amplicons (SNCA_a, SNCA_c) allowed for analysis of 2 CpG stretches (Fig. 1). One stretch encompassed CpGs also analyzed in the 2 previous studies on brain tissue by Jowaed et al and Matsumoto et al (Fig. 1 SNCA_c). The second amplicon covered 8 additional CpGs of a CpG island shore in the untranslated first exon of SNCA (Fig. 1, SNCA_a). Figure 1 shows the distribution of the mean CpG methylation per region for each individual in the 3 groups. Comparisons using the Mann– Whitney test did not reveal any significant methylation differences between the groups with idiopathic PD and controls (P > .1), neither using mean methylation per individual across each of the 2 amplicons (Fig. 1) nor comparing single CpG positions (results not shown). The group with monogenic PD caused by duplication of the SNCA gene was too small for a meaningful statistical comparison. Nevertheless, we did not identify any sample with a grossly different methylation pattern in this group (Fig. 1). Taken together, we demonstrate in a sizable group that the analyzed cytosins are not differentially methylated between idiopathic PD patients and neurologically normal controls using DNA derived from peripheral white blood cells. To serve as a biomarker, a laboratory measure needs to be able to discriminate between patients and controls on an individual basis, which is clearly not the case. Peripheral blood leukocytes were chosen because they are easily attainable, which is essential for diagnostic application. It has previously been shown that methylation profiles are often but not always highly correlated between different tissues. However, SNCA has not been specifically investigated in this regard. Methylation differences between blood leukocyte DNA and brain DNA might explain the difference between our results and the ones obtained in brain tissue. A limitation of our study is that not all CpGs analyzed by Jowaed and Matsumoto in brain tissue were amenable to analysis by pyrosequencing because a number of them were located in long poly-T stretches after bisulfite conversion. On the other hand, we analyzed an additional region (SNCA_a), and our pyrosequencing approach has the advantage of allowing fairly accurate quantification of the percentage of methylated versus unmethylated CpGs.

Whole transcriptome profiling reveals the RNA content of motor axons

Most RNAs within polarized cells such as neurons are sorted subcellularly in a coordinated manner. Despite advances in the development of methods for profiling polyadenylated RNAs from small amounts of input RNA, techniques for profiling coding and non-coding RNAs simultaneously are not well established. Here, we optimized a transcriptome profiling method based on double-random priming and applied it to serially diluted total RNA down to 10 pg. Read counts of expressed genes were robustly correlated between replicates, indicating that the method is both reproducible and scalable. Our transcriptome profiling method detected both coding and long non-coding RNAs sized >300 bases. Compared to total RNAseq using a conventional approach our protocol detected 70% more genes due to reduced capture of ribosomal RNAs. We used our method to analyze the RNA composition of compartmentalized motoneurons. The somatodendritic compartment was enriched for transcripts with post-synaptic functions as well as for certain nuclear non-coding RNAs such as 7SK. In axons, transcripts related to translation were enriched including the cytoplasmic non-coding RNA 7SL. Our profiling method can be applied to a wide range of investigations including perturbations of subcellular transcriptomes in neurodegenerative diseases and investigations of microdissected tissue samples such as anatomically defined fiber tracts.

Landscape of somatic mutations in sporadic GH-secreting pituitary adenomas

CONTEXT Alterations in the cAMP signaling pathway are common in hormonally active endocrine tumors. Somatic mutations at GNAS are causative in 30-40% of GH-secreting adenomas. Recently, mutations affecting the USP8 and PRKACA gene have been reported in ACTH-secreting pituitary adenomas and cortisol-secreting adrenocortical adenomas respectively. However, the pathogenesis of many GH-secreting adenomas remains unclear. AIM Comprehensive genetic characterization of sporadic GH-secreting adenomas and identification of new driver mutations. DESIGN Screening for somatic mutations was performed in 67 GH-secreting adenomas by targeted sequencing for GNAS, PRKACA, and USP8 mutations (n=31) and next-generation exome sequencing (n=36). RESULTS By targeted sequencing, known activating mutations in GNAS were detected in five cases (16.1%), while no somatic mutations were observed in both PRKACA and USP8. Whole-exome sequencing identified 132 protein-altering somatic mutations in 31/36 tumors with a median of three mutations per sample (range: 1-13). The only recurrent mutations have been observed in GNAS (31.4% of cases). However, seven genes involved in cAMP signaling pathway were affected in 14 of 36 samples and eight samples harbored variants in genes involved in the calcium signaling or metabolism. At the enrichment analysis, several altered genes resulted to be associated with developmental processes. No significant correlation between genetic alterations and the clinical data was observed. CONCLUSION This study provides a comprehensive analysis of somatic mutations in a large series of GH-secreting adenomas. No novel recurrent genetic alterations have been observed, but the data suggest that beside cAMP pathway, calcium signaling might be involved in the pathogenesis of these tumors.

Broad Phenotypic Heterogeneity due to a Novel SCN1A Mutation in a Family With Genetic Epilepsy With Febrile Seizures Plus

Genetic (generalized) epilepsy with febrile seizures plus is a familial epilepsy syndrome with marked phenotypic heterogeneity ranging from simple febrile seizure to severe phenotypes. Here we report on a large Israeli family with genetic (generalized) epilepsy with febrile seizures plus and 14 affected individuals. A novel SCN1A missense mutation in exon 21 (p.K1372E) was identified in all affected individuals and 3 unaffected carriers. The proband had Dravet syndrome, whereas febrile seizure plus phenotypes were present in all other affected family members. Simple febrile seizures were not observed. Phenotypes were found at both extremes of the genetic (generalized) epilepsy with febrile seizures plus spectrum and distribution of phenotypes suggested modifying familial, possibly genetic factors. We suggest that families with extreme phenotype distributions can represent prime candidates for the identification of genetic or environmental modifiers.

Febrile infection-related epilepsy syndrome (FIRES) is not caused by SCN1A, POLG, PCDH19 mutations or rare copy number variations

Aim  Febrile infection‐related epilepsy syndrome (FIRES) is an enigmatic seizure disorder in childhood with an innocuous febrile infection triggering severe and intractable multifocal epilepsy, mostly with status epilepticus. FIRES shares several phenotypic features with epilepsies seen in patients with protocadherin 19 (PCDH19), sodium channel protein type 1 subunit alpha (SCN1A), and DNA polymerase subunit gamma‐1 (POLG) mutations. The aim of the study was the mutation analysis of these prime candidate genes in a cohort of patients with FIRES. Additionally, given that rare copy number variations (CNVs) have recently been established as important risk factors for epilepsies, we performed a genome‐wide CNV analysis.

Autosomal-dominant striatal degeneration is caused by a mutation in the phosphodiesterase 8B gene.

Autosomal-dominant striatal degeneration (ADSD) is an autosomal-dominant movement disorder affecting the striatal part of the basal ganglia. ADSD is characterized by bradykinesia, dysarthria, and muscle rigidity. These symptoms resemble idiopathic Parkinson disease, but tremor is not present. Using genetic linkage analysis, we have mapped the causative genetic defect to a 3.25 megabase candidate region on chromosome 5q13.3-q14.1. A maximum LOD score of 4.1 (Theta = 0) was obtained at marker D5S1962. Here we show that ADSD is caused by a complex frameshift mutation (c.94G>C+c.95delT) in the phosphodiesterase 8B (PDE8B) gene, which results in a loss of enzymatic phosphodiesterase activity. We found that PDE8B is highly expressed in the brain, especially in the putamen, which is affected by ADSD. PDE8B degrades cyclic AMP, a second messenger implied in dopamine signaling. Dopamine is one of the main neurotransmitters involved in movement control and is deficient in Parkinson disease. We believe that the functional analysis of PDE8B will help to further elucidate the pathomechanism of ADSD as well as contribute to a better understanding of movement disorders.

Genetic landscape of sporadic unilateral adrenocortical adenomas without PRKACA p.Leu206Arg mutation

CONTEXT Adrenocortical adenomas (ACAs) are among the most frequent human neoplasias. Genetic alterations affecting the cAMP/protein kinase A signaling pathway are common in cortisol-producing ACAs, whereas activating mutations in the gene encoding β-catenin (CTNNB1) have been reported in a subset of both benign and malignant adrenocortical tumors. However, the molecular pathogenesis of most ACAs is still largely unclear. OBJECTIVE The aim of the study was to define the genetic landscape of sporadic unilateral ACAs. DESIGN AND SETTING Next-generation whole-exome sequencing was performed on fresh-frozen tumor samples and corresponding normal tissue samples. PATIENTS Ninety-nine patients with ACAs (74 cortisol-producing and 25 endocrine inactive) negative for p.Leu206Arg PRKACA mutation. MAIN OUTCOME MEASURES Identification of known and/or new genetic alterations potentially involved in adrenocortical tumorigenesis and autonomous hormone secretion, genotype-phenotype correlation. RESULTS A total of 706 somatic protein-altering mutations were detected in 88 of 99 tumors (median, six per tumor). We identified several mutations in genes of the cAMP/protein kinase A pathway, including three novel mutations in PRKACA, associated with female sex and Cushings syndrome. We also found genetic alterations in different genes involved in the Wnt/β-catenin pathway, associated with larger tumors and endocrine inactivity, and notably, in many genes of the Ca(2+)-signaling pathway. Finally, by comparison of our genetic data with those available in the literature, we describe a comprehensive genetic landscape of unilateral ACAs. CONCLUSIONS This study provides the largest sequencing effort on ACAs to date. We thereby identified somatic alterations affecting known and novel pathways potentially involved in adrenal tumorigenesis.

MRI Features of Pontine Autosomal Dominant Microangiopathy and Leukoencephalopathy (PADMAL)

Cerebral small vessel disease is a topic of growing interest for both the scientific community and the aging society. We report the magnetic resonance imaging (MRI) characteristics of a recently found autosomal dominantly inherited microangiopathy.