Gerardine Quaghebeur

Exome sequencing can detect pathogenic mosaic mutations present at low allele frequencies

The development of next generation sequencing (NGS) has radically transformed the scientific landscape, making it possible to sequence the exome of any given individual in a cost-effective way. The power of this approach has been demonstrated by a number of groups who have identified pathogenic mutations in small pedigrees that have been resistant to traditional genetic mapping. Recently it has become clear that exome sequencing has great potential with respect to sporadic disease and the identification of de novo mutations. This is highlighted by studies reporting whole-exome sequencing of patient–parental trios affected by learning disability, autism and schizophrenia. It is widely anticipated that the introduction of this technique into a clinical setting will revolutionise genetic diagnosis. However, the sensitivity of NGS exome sequencing is currently unclear. Here, we describe the exome sequencing of DNA samples from a patient with double cortex syndrome and her parents, resulting in the detection of a mosaic splicing mutation in LIS1. This variant was found at an allele frequency of just 18%, demonstrating that NGS methods have the capacity to identify pathogenic mosaic mutations present at a low level.

Recessive mutations in SPTBN2 implicate β-III spectrin in both cognitive and motor development

β-III spectrin is present in the brain and is known to be important in the function of the cerebellum. Heterozygous mutations in SPTBN2, the gene encoding β-III spectrin, cause Spinocerebellar Ataxia Type 5 (SCA5), an adult-onset, slowly progressive, autosomal-dominant pure cerebellar ataxia. SCA5 is sometimes known as “Lincoln ataxia,” because the largest known family is descended from relatives of the United States President Abraham Lincoln. Using targeted capture and next-generation sequencing, we identified a homozygous stop codon in SPTBN2 in a consanguineous family in which childhood developmental ataxia co-segregates with cognitive impairment. The cognitive impairment could result from mutations in a second gene, but further analysis using whole-genome sequencing combined with SNP array analysis did not reveal any evidence of other mutations. We also examined a mouse knockout of β-III spectrin in which ataxia and progressive degeneration of cerebellar Purkinje cells has been previously reported and found morphological abnormalities in neurons from prefrontal cortex and deficits in object recognition tasks, consistent with the human cognitive phenotype. These data provide the first evidence that β-III spectrin plays an important role in cortical brain development and cognition, in addition to its function in the cerebellum; and we conclude that cognitive impairment is an integral part of this novel recessive ataxic syndrome, Spectrin-associated Autosomal Recessive Cerebellar Ataxia type 1 (SPARCA1). In addition, the identification of SPARCA1 and normal heterozygous carriers of the stop codon in SPTBN2 provides insights into the mechanism of molecular dominance in SCA5 and demonstrates that the cell-specific repertoire of spectrin subunits underlies a novel group of disorders, the neuronal spectrinopathies, which includes SCA5, SPARCA1, and a form of West syndrome.

Mutations in SNORD118 cause the cerebral microangiopathy leukoencephalopathy with calcifications and cysts

Although ribosomes are ubiquitous and essential for life, recent data indicate that monogenic causes of ribosomal dysfunction can confer a remarkable degree of specificity in terms of human disease phenotype. Box C/D small nucleolar RNAs (snoRNAs) are evolutionarily conserved non-protein-coding RNAs involved in ribosome biogenesis. Here we show that biallelic mutations in the gene SNORD118, encoding the box C/D snoRNA U8, cause the cerebral microangiopathy leukoencephalopathy with calcifications and cysts (LCC), presenting at any age from early childhood to late adulthood. These mutations affect U8 expression, processing and protein binding and thus implicate U8 as essential in cerebral vascular homeostasis.

Dysregulated mitophagy and mitochondrial organization in optic atrophy due to OPA1 mutations

Objective: To investigate mitophagy in 5 patients with severe dominantly inherited optic atrophy (DOA), caused by depletion of OPA1 (a protein that is essential for mitochondrial fusion), compared with healthy controls. Methods: Patients with severe DOA (DOA plus) had peripheral neuropathy, cognitive regression, and epilepsy in addition to loss of vision. We quantified mitophagy in dermal fibroblasts, using 2 high throughput imaging systems, by visualizing colocalization of mitochondrial fragments with engulfing autophagosomes. Results: Fibroblasts from 3 biallelic OPA1(−/−) patients with severe DOA had increased mitochondrial fragmentation and mitochondrial DNA (mtDNA)–depleted cells due to decreased levels of OPA1 protein. Similarly, in siRNA-treated control fibroblasts, profound OPA1 knockdown caused mitochondrial fragmentation, loss of mtDNA, impaired mitochondrial function, and mitochondrial mislocalization. Compared to controls, basal mitophagy (abundance of autophagosomes colocalizing with mitochondria) was increased in (1) biallelic patients, (2) monoallelic patients with DOA plus, and (3) OPA1 siRNA–treated control cultures. Mitophagic flux was also increased. Genetic knockdown of the mitophagy protein ATG7 confirmed this by eliminating differences between patient and control fibroblasts. Conclusions: We demonstrated increased mitophagy and excessive mitochondrial fragmentation in primary human cultures associated with DOA plus due to biallelic OPA1 mutations. We previously found that increased mitophagy (mitochondrial recycling) was associated with visual loss in another mitochondrial optic neuropathy, Leber hereditary optic neuropathy (LHON). Combined with our LHON findings, this implicates excessive mitochondrial fragmentation, dysregulated mitophagy, and impaired response to energetic stress in the pathogenesis of mitochondrial optic neuropathies, potentially linked with mitochondrial mislocalization and mtDNA depletion.

Germline recessive mutations in PI4KA are associated with perisylvian polymicrogyria, cerebellar hypoplasia and arthrogryposis

Polymicrogyria (PMG) is a structural brain abnormality involving the cerebral cortex that results from impaired neuronal migration and although several genes have been implicated, many cases remain unsolved. In this study, exome sequencing in a family where three fetuses had all been diagnosed with PMG and cerebellar hypoplasia allowed us to identify regions of the genome for which both chromosomes were shared identical-by-descent, reducing the search space for causative variants to 8.6% of the genome. In these regions, the only plausibly pathogenic mutations were compound heterozygous variants in PI4KA, which Sanger sequencing confirmed segregated consistent with autosomal recessive inheritance. The paternally transmitted variant predicted a premature stop mutation (c.2386C>T; p.R796X), whereas the maternally transmitted variant predicted a missense substitution (c.5560G>A; p.D1854N) at a conserved residue within the catalytic domain. Functional studies using expressed wild-type or mutant PI4KA enzyme confirmed the importance of p.D1854 for kinase activity. Our results emphasize the importance of phosphoinositide signalling in early brain development.

Subdural effusions and lack of early pontocerebellar hypoplasia in siblings with RARS2 mutations

Mutations in the recently described RARS2 gene encoding for mitochondrial arginyl-transfer RNA synthetase give rise to a disorder characterised by early onset seizures, progressive microcephaly and developmental delay. The disorder was named pontocerebellar hypoplasia type 6 (PCH6) based on the corresponding radiological findings observed in the original cases. We report two siblings with the RARS2 mutation who displayed typical clinical features of PCH6, but who had distinct neuroimaging features. Early scans showed marked supratentorial, rather than infratentorial, atrophy, and the pons remained preserved throughout. One sibling also had bilateral subdural effusions at presentation. The deceleration in head growth pointed to an evolving genetic/metabolic process giving rise to cerebral atrophy and secondary subdural effusions. RARS2 mutations should be considered in infants presenting with seizures, subdural effusions, decelerating head growth and evidence of cerebral atrophy even in the absence of pontocerebellar hypoplasia on imaging.

A de novo frameshift in HNRNPK causing a Kabuki‐like syndrome with nodular heterotopia

Kabuki syndrome is a heterogeneous condition characterized by distinctive facial features, intellectual disability, growth retardation, skeletal abnormalities and a range of organ malformations. Although at least two major causative genes have been identified, these do not explain all cases. Here we describe a patient with a complex Kabuki‐like syndrome that included nodular heterotopia, in whom testing for several single‐gene disorders had proved negative. Exome sequencing uncovered a de novo c.931_932insTT variant in HNRNPK (heterogeneous nuclear ribonucleoprotein K). Although this variant was identified in March 2012, its clinical relevance could only be confirmed following the August 2015 publication of two cases with HNRNPK mutations and an overlapping phenotype that included intellectual disability, distinctive facial dysmorphism and skeletal/connective tissue abnormalities. Whilst we had attempted (unsuccessfully) to identify additional cases through existing collaborators, the two published cases were ‘matched’ using GeneMatcher, a web‐based tool for connecting researchers and clinicians working on identical genes. Our report therefore exemplifies the importance of such online tools in clinical genetics research and the benefits of periodically reviewing cases with variants of unproven significance. Our study also suggests that loss of function variants in HNRNPK should be considered as a molecular basis for patients with Kabuki‐like syndrome.

Neuroimaging findings of babies with microcephaly and presumed congenital Zika virus infection.

Images add to evidence that Zika virus causes microcephaly

SOX2 anophthalmia syndrome in adulthood – a neurodegenerative picture?

To the Editor : Heterozygous, loss of function mutations in the high mobility group (HMG) gene, SOX2 , encoding the sex-determining region Y-box 2 (MIM 184429) protein, lead to severe developmental eye and brain malformations, and some anophthalmos-esophagealgenital syndrome cases (1). However, the fate of these cases into adulthood is unknown. We report an adult with SOX2 anophthalmia syndrome who is exhibiting features suggesting neurodegeneration. A 35-year-old male with bilateral anophthalmia was referred for genetic diagnosis. He was the third son born at term to unrelated parents (mother 25 years, father 32 years), weighing 6lb 9oz. Post-delivery, he developed peripheral cyanosis and was diagnosed with bilateral anophthalmia on admission to intensive care. In early childhood, he suffered recurrent infections and poor feeding with persistent choking episodes. He had delayed motor development, sitting at 17 months, walking at 3.5–4 years. He developed an atypical seizure disorder, treated with valproate. He had a duplex kidney and ureteric reflux, with recurrent infections. His speech and language development was delayed: at 3 years, he spoke ∼30 words, losing some by 4 years. By 10 years his language, motor and social development had not significantly progressed. Puberty occurred normally. His brother had mild sensorineural deafness; there was no other relevant family history. Over recent years, his condition deteriorated. He lost speech and experienced increasing difficulties with walking and swallowing, premature graying, occasional drop attacks and cyanotic episodes of unknown aetiology. His electrocardiography and echocardiography were normal. His electroencephalography at 31 years showed some frontotemporal sharp and slow waves, but not diagnostic of epilepsy. His kidney function was reduced on imaging with Tc-99m-mercaptoacetyltriglycine (MAG 3) scan (right 33%; left 67%). His examination showed bilateral anophthalmia, short palpebral apertures, underdeveloped orbital bones, slightly square earlobes and underdeveloped helices (Fig. 1a,b). His head circumference was 56 cm (25–50%). He had spastic diplegia, and fatigued easily. His magnetic resonance imaging scan at 31 years showed prominent cerebrospinal fluid spaces and ventricles, consistent with global loss of brain volume (Fig. 1c–e). Since there are no other scans for comparison, the timeframe for the change is unknown. There were non-specific scattered areas of increased T2 signal intensity within predominantly frontal subcortical white matter. These are of unknown clinical significance and do not resemble heterotopia. The pituitary fossa was present and anterior pituitary tissue was identifiable. Hippocampal volume was reduced bilaterally. Molecular analysis of the SOX2 gene revealed a de novo mutation c. 181C>T p.Gln61X in the HMG DNA-binding domain predicted to lead to haploinsufficiency of SOX2. This mutation has been reported in another case (2). Our patient had typical SOX2 syndrome with bilateral anophthalmia, seizure disorder, spastic diplegia, pervasive neurodevelopmental disorder, renal anomalies and swallowing difficulties (1). Brain malformations are frequent in SOX2 cases, mainly hypothalamopituitary anomalies, corpus callosal agenesis or hippocampal malformations, occasionally, hydrocephalus and deafness (2). Our patient’s white matter lesions have not been reported before. It is unclear if they relate to his neurological decline. Although most SOX2 cases exhibit severe phenotypes with major eye anomalies, phenotypic variability is emerging; some individuals even have normal or minimally affected eyes (3). Contributing factors include genetic background, mutation type, local and stochastic factors. Since SOX2 belongs to a close family of SOX genes, there may be genetic redundancy within the group. Sox2 paralogues of the Sox B1 subgroup, Sox1 or Sox3, can rescue Sox2 deficiency in animal models. The mutation type may be important: a fourgeneration family with varying ocular phenotypes has been reported with a SOX2 missense mutation within the partner factor-binding domain, not involving the HMG domain (3). Although the overwhelming majority of SOX2 cases are de novo and severe, phenotypic variability and occasional mosaicism suggest caution when advising family members about recurrence risk. Here, it would be sensible to screen our patient’s brother, whose only symptom is deafness. There is evidence supporting a generalized role for Sox2 in both neurogenesis and neuropreservation from mouse models of central nervous system (CNS) development (4). Compound heterozygous mouse embryos with markedly reduced Sox2 expression display decreased hippocampal and subventricular neurogenesis analogous to hippocampal and periventricular abnormalities seen in SOX2 cases. SOX2

Activation of an exonic splice-donor site in exon 30 of CDK5RAP2 in a patient with severe microcephaly and pigmentary abnormalities.

This report constitutes the first report of a cryptic exonic splice‐donor site in CDK5RAP2, highlights the importance of evaluating novel splice mutations, and suggests that the phenotypic range associated with CDK5RAP2 mutations may include skin pigmentary abnormalities.