Elisa Giorgio

A large genomic deletion leads to enhancer adoption by the lamin B1 gene: a second path to autosomal dominant adult-onset demyelinating leukodystrophy (ADLD).

Chromosomal rearrangements with duplication of the lamin B1 (LMNB1) gene underlie autosomal dominant adult-onset demyelinating leukodystrophy (ADLD), a rare neurological disorder in which overexpression of LMNB1 causes progressive central nervous system demyelination. However, we previously reported an ADLD family (ADLD-1-TO) without evidence of duplication or other mutation in LMNB1 despite linkage to the LMNB1 locus and lamin B1 overexpression. By custom array-CGH, we further investigated this family and report here that patients carry a large (∼660 kb) heterozygous deletion that begins 66 kb upstream of the LMNB1 promoter. Lamin B1 overexpression was confirmed in further ADLD-1-TO tissues and in a postmortem brain sample, where lamin B1 was increased in the frontal lobe. Through parallel studies, we investigated both loss of genetic material and chromosomal rearrangement as possible causes of LMNB1 overexpression, and found that ADLD-1-TO plausibly results from an enhancer adoption mechanism. The deletion eliminates a genome topological domain boundary, allowing normally forbidden interactions between at least three forebrain-directed enhancers and the LMNB1 promoter, in line with the observed mainly cerebral localization of lamin B1 overexpression and myelin degeneration. This second route to LMNB1 overexpression and ADLD is a new example of the relevance of regulatory landscape modifications in determining Mendelian phenotypes.

ELOVL5 Mutations Cause Spinocerebellar Ataxia 38

Spinocerebellar ataxias (SCAs) are a heterogeneous group of autosomal-dominant neurodegenerative disorders involving the cerebellum and 23 different genes. We mapped SCA38 to a 56 Mb region on chromosome 6p in a SCA-affected Italian family by whole-genome linkage analysis. Targeted resequencing identified a single missense mutation (c.689G>T [p.Gly230Val]) in ELOVL5. Mutation screening of 456 independent SCA-affected individuals identified the same mutation in two further unrelated Italian families. Haplotyping showed that at least two of the three families shared a common ancestor. One further missense variant (c.214C>G [p.Leu72Val]) was found in a French family. Both missense changes affect conserved amino acids, are predicted to be damaging by multiple bioinformatics tools, and were not identified in ethnically matched controls or within variant databases. ELOVL5 encodes an elongase involved in the synthesis of polyunsaturated fatty acids of the ω3 and ω6 series. Arachidonic acid and docosahexaenoic acid, two final products of the enzyme, were reduced in the serum of affected individuals. Immunohistochemistry on control mice and human brain demonstrated high levels in Purkinje cells. In transfection experiments, subcellular localization of altered ELOVL5 showed a perinuclear distribution with a signal increase in the Golgi compartment, whereas the wild-type showed a widespread signal in the endoplasmic reticulum. SCA38 and SCA34 are examples of SCAs due to mutations in elongase-encoding genes, emphasizing the importance of fatty-acid metabolism in neurological diseases.

Analysis of LMNB1 Duplications in Autosomal Dominant Leukodystrophy Provides Insights into Duplication Mechanisms and Allele-Specific Expression

Autosomal dominant leukodystrophy (ADLD) is an adult onset demyelinating disorder that is caused by duplications of the lamin B1 (LMNB1) gene. However, as only a few cases have been analyzed in detail, the mechanisms underlying LMNB1 duplications are unclear. We report the detailed molecular analysis of the largest collection of ADLD families studied, to date. We have identified the minimal duplicated region necessary for the disease, defined all the duplication junctions at the nucleotide level and identified the first inverted LMNB1 duplication. We have demonstrated that the duplications are not recurrent; patients with identical duplications share the same haplotype, likely inherited from a common founder and that the duplications originated from intrachromosomal events. The duplication junction sequences indicated that nonhomologous end joining or replication‐based mechanisms such fork stalling and template switching or microhomology‐mediated break induced repair are likely to be involved. LMNB1 expression was increased in patients’ fibroblasts both at mRNA and protein levels and the three LMNB1 alleles in ADLD patients show equal expression, suggesting that regulatory regions are maintained within the rearranged segment. These results have allowed us to elucidate duplication mechanisms and provide insights into allele‐specific LMNB1 expression levels.

Large cryptic genomic rearrangements with apparently normal karyotypes detected by array-CGH

BackgroundConventional karyotyping (550 bands resolution) is able to identify chromosomal aberrations >5-10 Mb, which represent a known cause of intellectual disability/developmental delay (ID/DD) and/or multiple congenital anomalies (MCA). Array-Comparative Genomic Hybridization (array-CGH) has increased the diagnostic yield of 15-20%.ResultsIn a cohort of 700 ID/DD cases with or without MCA, including 15 prenatal diagnoses, we identified a subgroup of seven patients with a normal karyotype and a large complex rearrangement detected by array-CGH (at least 6, and up to 18 Mb). FISH analysis could be performed on six cases and showed that rearrangements were translocation derivatives, indistinguishable from a normal karyotype as they involved a similar band pattern and size. Five were inherited from a parent with a balanced translocation, whereas two were apparently de novo. Genes spanning the rearrangements could be associated with some phenotypic features in three cases (case 3: DOCK8; case 4: GATA3, AKR1C4; case 6: AS/PWS deletion, CHRNA7), and in two, likely disease genes were present (case 5: NR2F2, TP63, IGF1R; case 7: CDON). Three of our cases were prenatal diagnoses with an apparently normal karyotype.ConclusionsLarge complex rearrangements of up to 18 Mb, involving chromosomal regions with similar size and band appearance may be overlooked by conventional karyotyping. Array-CGH allows a precise chromosomal diagnosis and recurrence risk definition, further confirming this analysis as a first tier approach to clarify molecular bases of ID/DD and/or MCA. In prenatal tests, array-CGH is confirmed as an important tool to avoid false negative results due to karyotype intrinsic limit of detection.

Messenger RNA processing is altered in autosomal dominant leukodystrophy

Adult-onset autosomal dominant leukodystrophy (ADLD) is a slowly progressive neurological disorder characterized by autonomic dysfunction, followed by cerebellar and pyramidal features. ADLD is caused by duplication of the lamin B1 gene (LMNB1), which leads to its increased expression. The molecular pathways involved in the disease are still poorly understood. Hence, we analyzed global gene expression in fibroblasts and whole blood of LMNB1 duplication carriers and used Gene Set Enrichment Analysis to explore their gene signatures. We found that LMNB1 duplication is associated with dysregulation of genes involved in the immune system, neuronal and skeletal development. Genes with an altered transcriptional profile clustered in specific genomic regions. Among the dysregulated genes, we further studied the role of RAVER2, which we found to be overexpressed at mRNA and protein level. RAVER2 encodes a putative trans regulator of the splicing repressor polypyrimidine tract binding protein (PTB) and is likely implicated in alternative splicing regulation. Functional studies demonstrated an abnormal splicing pattern of several PTB-target genes and of the myelin protein gene PLP1, previously demonstrated to be involved in ADLD. Mutant mice with different lamin B1 expression levels confirmed that Raver2 expression is dependent on lamin B1 in neural tissue and determines an altered splicing pattern of PTB-target genes and Plp1. Overall our results demonstrate that deregulation of lamin B1 expression induces modified splicing of several genes, likely driven by raver-2 overexpression, and suggest that an alteration of mRNA processing could be a pathogenic mechanism in ADLD.

Novel mutation of SLC20A2 in an Italian patient presenting with migraine

Dear Sirs, Idiopathic basal ganglia calcifications (IBGC), also known as Fahr’s disease, are rare neurological diseases characterized by symmetric calcium deposits in the basal ganglia and other brain regions. Clinically, IBGC patients show high phenotypic heterogeneity, both in the neuradiological findings and in clinical manifestations. Recently, PDGFRB, PDGFB and SLC20A2 have been identified as causative genes for IBGC [1]. We report the case of an Italian patient with IBGC associated with a novel mutation in the SLC20A2 gene, who presented with episodic migraine. In September 2013, a 48-year-old woman presented to our outpatient clinic with a 29 years history of headache. The recurrent attacks were characterized by pulsating pain of moderate intensity in frontotemporal location, associated with severe nausea and photo/phonophobia, lasting up to 72 h, with 6–8 episodes a month. The reported symptoms fulfilled ICHD-III beta version criteria for episodic migraine without aura (code 1.1) [2]. The medical history of the patient was unremarkable. A positive family history for migraine (mother and maternal aunt) and psychosis (another maternal aunt) was reported. Neurological examination showed hyperreflexia and slight neck rigidity, while no cerebellar signs were identified. A computed tomography scan showed severe calcifications at the bilateral globus pallidus, caudate nuclei, putamen, and dentate nuclei (Fig. 1a). Laboratory tests were normal (including serum 25-hydroxyvitamin D and calcium concentrations) and excluded any parathyroid dysfunction. Neuropsychological screening showed a mild impairment in verbal fluency and mild attention deficit. The remaining neuropsychological tests had normal scores, also in those for visuospatial functions. STAIx-1 and STAIx-2 tests showed high level of anxiety. The patient received genetic counselling and on the basis of the neuroradiological findings with calcifications both in basal ganglia and cerebellum, SLC20A2 was sequenced. We analyzed the candidate gene by direct genomic sequencing of the coding exons, performed on an ABI Prism 3130 XL platform. We identified a novel frameshift mutation p.Val507Glufs*2 in the isoform 1 (c.1520_1521delTG, exon 8, NM_006749) (Fig. 1b, c). This genetic change was predicted to change an aminoacid and insert a stop codon, likely leading to a degradation of the mutated messenger RNA due to nonsense-mediated decay [3]. No other relative was available for segregation analysis. SCL20A2 encodes the type III sodium-dependent phosphate transporter 2, broadly expressed and with high levels in brain. SLC20A2 gene mutations have been reported in China, Brazil, Japan, and Spain [3–12], and all are predicted E. Rubino (&) L. Pinessi Neurology I, Department of Neuroscience ‘‘Rita Levi Montalcini’’, University of Torino, Via Cherasco 15, 10126 Torino, Italy e-mail: elisa.rubino@unito.it

Possible influence of a non-synonymous polymorphism located in the NGF precursor on susceptibility to late-onset Alzheimer's disease and mild cognitive impairment.

The complex network of neurotrophic factors is supposed to play a role in neurodegeneration, but the effect of variations in their coding genes on susceptibility to sporadic Alzheimers disease is not established. The mature form of nerve growth factor (NGF) derives from a precursor, proNGF, which was recently discovered to exert crucial functions in brain. We designed a case-control association study to test the hypothesis as to whether polymorphisms located in the proNGF genomic region influence the liability to Alzheimers disease and its prodromal form, mild cognitive impairment. Three independent case-control samples, with individuals aged >60 years, were collected in Italian Alzheimer Units. One polymorphism located in the proNGF region, rs6330, demonstrated a minor allele frequency >5% and was used in the association study. The minor allele of rs6330 was more frequent in patients from the three sample series as compared to respective normal controls. Multivariate logistic regression showed a significant association under the dominant model in one cohort (OR 1.83, 95% CI 1.00-3.54) and in the pooled case-control sample (OR 1.47, 95% CI 1.03-2.08). These findings further suggest that proNGF may play a role in Alzheimer-type neurodegeneration and that genetic variations in the NGF locus may influence the occurrence of sporadic, late-onset Alzheimers disease.

Severe fluoropyrimidine-related toxicity: clinical implications of DPYD analysis and UH2/U ratio evaluation

The fluoropyrimidines are commonly used in chemotherapeutic cancer medicine, but many patients still experience severe adverse side effects from these drugs. We observed a severe toxicity in a 50-year-old woman treated with capecitabine and docetaxel for a metastatic breast cancer. Since dihydropyrimidine dehydrogenase (DPD) is the main candidate for pharmacogenetic studies on 5-FU toxicity, the entire coding sequence and exon-flanking intronic regions of the DPYD gene were sequenced in the patient. None of the previously described deleterious variants were detected. Also, the haplotype-based analysis failed to reveal DPYD variations associated with 5-FU toxicity. We also evaluated the UH2/U ratio in plasma as an index of 5-FU pharmacokinetics. The UH2/U value did not demonstrate low DPD activity in the patient. We discuss the advantages and limitations of this approach, particularly concerning the clinical applications of 5-FU pharmacogenetics in the family setting.

Whole exome sequencing is necessary to clarify ID/DD cases with de novo copy number variants of uncertain significance: Two proof-of-concept examples

Whole exome sequencing (WES) is a powerful tool to identify clinically undefined forms of intellectual disability/developmental delay (ID/DD), especially in consanguineous families. Here we report the genetic definition of two sporadic cases, with syndromic ID/DD for whom array—Comparative Genomic Hybridization (aCGH) identified a de novo copy number variant (CNV) of uncertain significance. The phenotypes included microcephaly with brachycephaly and a distinctive facies in one proband, and hypotonia in the legs and mild ataxia in the other. WES allowed identification of a functionally relevant homozygous variant affecting a known disease gene for rare syndromic ID/DD in each proband, that is, c.1423C>T (p.Arg377*) in the Trafficking Protein Particle Complex 9 (TRAPPC9), and c.154T>C (p.Cys52Arg) in the Very Low Density Lipoprotein Receptor (VLDLR). Four mutations affecting TRAPPC9 have been previously reported, and the present finding further depicts this syndromic form of ID, which includes microcephaly with brachycephaly, corpus callosum hypoplasia, facial dysmorphism, and overweight. VLDLR‐associated cerebellar hypoplasia (VLDLR‐CH) is characterized by non‐progressive congenital ataxia and moderate‐to‐profound intellectual disability. The c.154T>C (p.Cys52Arg) mutation was associated with a very mild form of ataxia, mild intellectual disability, and cerebellar hypoplasia without cortical gyri simplification. In conclusion, we report two novel cases with rare causes of autosomal recessive ID, which document how interpreting de novo array‐CGH variants represents a challenge in consanguineous families; as such, clinical WES should be considered in diagnostic testing.

Copy number variants analysis in a cohort of isolated and syndromic developmental delay/intellectual disability reveals novel genomic disorders, position effects and candidate disease genes.

Array‐comparative genomic hybridization (array‐CGH) is a widely used technique to detect copy number variants (CNVs) associated with developmental delay/intellectual disability (DD/ID).