Ami Haviv

A-to-I RNA editing occurs at over a hundred million genomic sites, located in a majority of human genes

RNA molecules transmit the information encoded in the genome and generally reflect its content. Adenosine-to-inosine (A-to-I) RNA editing by ADAR proteins converts a genomically encoded adenosine into inosine. It is known that most RNA editing in human takes place in the primate-specific Alu sequences, but the extent of this phenomenon and its effect on transcriptome diversity are not yet clear. Here, we analyzed large-scale RNA-seq data and detected ∼1.6 million editing sites. As detection sensitivity increases with sequencing coverage, we performed ultradeep sequencing of selected Alu sequences and showed that the scope of editing is much larger than anticipated. We found that virtually all adenosines within Alu repeats that form double-stranded RNA undergo A-to-I editing, although most sites exhibit editing at only low levels (<1%). Moreover, using high coverage sequencing, we observed editing of transcripts resulting from residual antisense expression, doubling the number of edited sites in the human genome. Based on bioinformatic analyses and deep targeted sequencing, we estimate that there are over 100 million human Alu RNA editing sites, located in the majority of human genes. These findings set the stage for exploring how this primate-specific massive diversification of the transcriptome is utilized.

Biallelic SZT2 Mutations Cause Infantile Encephalopathy with Epilepsy and Dysmorphic Corpus Callosum

Epileptic encephalopathies are genetically heterogeneous severe disorders in which epileptic activity contributes to neurological deterioration. We studied two unrelated children presenting with a distinctive early-onset epileptic encephalopathy characterized by refractory epilepsy and absent developmental milestones, as well as thick and short corpus callosum and persistent cavum septum pellucidum on brain MRI. Using whole-exome sequencing, we identified biallelic mutations in seizure threshold 2 (SZT2) in both affected children. The causative mutations include a homozygous nonsense mutation and a nonsense mutation together with an exonic splice-site mutation in a compound-heterozygous state. The latter mutation leads to exon skipping and premature termination of translation, as shown by RT-PCR in blood RNA of the affected boy. Thus, all three mutations are predicted to result in nonsense-mediated mRNA decay and/or premature protein truncation and thereby loss of SZT2 function. Although the molecular role of the peroxisomal protein SZT2 in neuronal excitability and brain development remains to be defined, Szt2 has been shown to influence seizure threshold and epileptogenesis in mice, consistent with our findings in humans. We conclude that mutations in SZT2 cause a severe type of autosomal-recessive infantile encephalopathy with intractable seizures and distinct neuroradiological anomalies.

Expansion of the spectrum of TUBB4A-related disorders: a new phenotype associated with a novel mutation in the TUBB4A gene

Mutations in the TUBB4A gene have been identified so far in two neurodegenerative disorders with extremely different clinical features and course: whispering dysphonia, also known as dystonia type 4 (DYT4), and hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC). We describe a patient with slowly progressive spastic paraparesis, segmental dystonia, intellectual disability, behavioral problems, and evidence of permanent, incomplete myelination associated with progressive cerebellar atrophy. Whole exome sequencing revealed a novel E410K de novo heterozygous mutation in the TUBB4A gene. The clinical and radiological picture of our patient is different from the classic phenotype; thus, it expands the phenotypic variation of TUBB4A-gene-related disorders.

Microcephaly Thin Corpus Callosum Intellectual Disability Syndrome Caused by Mutated TAF2

BACKGROUND The combination of microcephaly, pyramidal signs, abnormal corpus callosum, and intellectual disability presents a diagnostic challenge. We describe an autosomal recessive disorder characterized by microcephaly, pyramidal signs, thin corpus callosum, and intellectual disability. METHODS We previously mapped the locus for this disorder to 8q23.2-q24.12; the candidate region included 22 genes. We performed Sanger sequencing of 10 candidate genes; to ensure other genes in the candidate region do not harbor mutations, we sequenced the exome of one affected individual. RESULTS We identified two homozygous missense changes, p.Thr186Arg and p.Pro416His in TAF2, which encodes a multisubunit cofactor for TFIID-dependent RNA polymerase II-mediated transcription, in all affected individuals. CONCLUSIONS We propose that the disorder is caused by the more conserved mutation p.Thr186Arg, with the second sequence change identified, p.Pro416His, possibly further negatively affecting the function of the protein. However, it is unclear which of the two changes, or maybe both, represents the causative mutation. A single missense mutation in TAF2 in a family with microcephaly and intellectual disability was described in a large-scale study reporting on the identification of 50 novel genes. We suggest that a mutation in TAF2 can cause this syndrome.

Reply to: The many faces of TUBB4A mutations

Dear Sirs, We have read with interest the letter by Lohmann and Klein regarding our paper Expansion of the spectrum of TUBB4Arelated disorders: a new phenotype associated with a novel mutation in the TUBB4A gene. Lohmann and Klein expand our knowledge regarding the first family described with DYT4 dystonia in 1985 and agree with us that the more recently described phenotype of hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) is a different and much more severe phenotype. We described a novel mutation (E410K) in the TUBB4A gene associated with a new phenotype, different from DYT4 dystonia and H-ABC. Lohmann and Klein suggested that the described clinicoradiological phenotype was in keeping with the H-ABC spectrum. However, as described in our original paper, our patient demonstrates only mild and slowly progressive leg spasticity with retained ambulation in contrast to the typical presentation of H-ABC, which shows an earlier and faster progression resulting in a disabling motor disorder. The brain MRI findings are also different, demonstrating only a regional and mild myelin deficit compared to diffuse hypomylination in H-ABC and no atrophy of the basal ganglia. Lohmann and Klein inquired regarding the frequency of the E410K variant in ethnically matched controls. This variant is not mentioned in the Exome variant server as well as in the dbSNP databases. No information is available regarding the frequency in Moroccan Jewish population. Lohmann and Klein mention the fact that there were 29 de novo variants in the patient’s DNA,whichmay have contributed to the phenotype. We have analyzed these genes and found that none of them have been described in associationwith neurologic symptoms except for IQSEC2. Mutations in this gene have been associated with nonsyndromic intellectual disability and epilepsy, but the variant found in our patient (A1311T) is predicted to be benign according to SIFT, Polyphen2, and mutation taster. We greatly appreciate the clarification by Lohmann and Klein regarding the confusion in the literature and OMIM nomenclature of the beta tubulin genes. They indicate that the article by Breuss et al. does not deal with mutations in TUBB4A but rather in TUBB5. The table they have added is extremely helpful. We have requested publication of an ERRATUM regarding our paper stating that the paper by Breuss et al. should be omitted from the discussion, table, and reference list, since it does not describe another presentation of TUBB4A Lubov Blumkin and Ayelet Halevy contributed equally to this work. L. Blumkin : T. Lerman-Sagie Pediatric Neurology Unit, Wolfson Medical Center, Holon, Israel