David Coman

Detection of cryptic pathogenic copy number variations and constitutional loss of heterozygosity using high resolution SNP microarray analysis in 117 patients referred for cytogenetic analysis and impact on clinical practice

Background: Microarray genome analysis is realising its promise for improving detection of genetic abnormalities in individuals with mental retardation and congenital abnormality. Copy number variations (CNVs) are now readily detectable using a variety of platforms and a major challenge is the distinction of pathogenic from ubiquitous, benign polymorphic CNVs. The aim of this study was to investigate replacement of time consuming, locus specific testing for specific microdeletion and microduplication syndromes with microarray analysis, which theoretically should detect all known syndromes with CNV aetiologies as well as new ones. Methods: Genome wide copy number analysis was performed on 117 patients using Affymetrix 250K microarrays. Results: 434 CNVs (195 losses and 239 gains) were found, including 18 pathogenic CNVs and 9 identified as “potentially pathogenic”. Almost all pathogenic CNVs were larger than 500 kb, significantly larger than the median size of all CNVs detected. Segmental regions of loss of heterozygosity larger than 5 Mb were found in 5 patients. Conclusions: Genome microarray analysis has improved diagnostic success in this group of patients. Several examples of recently discovered “new syndromes” were found suggesting they are more common than previously suspected and collectively are likely to be a major cause of mental retardation. The findings have several implications for clinical practice. The study revealed the potential to make genetic diagnoses that were not evident in the clinical presentation, with implications for pretest counselling and the consent process. The importance of contributing novel CNVs to high quality databases for genotype–phenotype analysis and review of guidelines for selection of individuals for microarray analysis is emphasised.

New indications and controversies in arginine therapy

Arginine is an important, versatile and a conditionally essential amino acid. Besides serving as a building block for tissue proteins, arginine plays a critical role in ammonia detoxification, and nitric oxide and creatine production. Arginine supplementation is an essential component for the treatment of urea cycle defects but recently some reservations have been raised with regards to the doses used in the treatment regimens of these disorders. In recent years, arginine supplementation or restriction has been proposed and trialled in several disorders, including vascular diseases and asthma, mitochondrial encephalopathy lactic acidosis and stroke-like episodes (MELAS), glutaric aciduria type I and disorders of creatine metabolism, both production and transportation into the central nervous system. Herein we present new therapeutic indications and controversies surrounding arginine supplementation or deprivation.

De Novo Mutations in SLC1A2 and CACNA1A Are Important Causes of Epileptic Encephalopathies

Epileptic encephalopathies (EEs) are the most clinically important group of severe early-onset epilepsies. Next-generation sequencing has highlighted the crucial contribution of de novo mutations to the genetic architecture of EEs as well as to their underlying genetic heterogeneity. Our previous whole-exome sequencing study of 264 parent-child trios revealed more than 290 candidate genes in which only a single individual had a de novo variant. We sought to identify additional pathogenic variants in a subset (n = 27) of these genes via targeted sequencing in an unsolved cohort of 531 individuals with a diverse range of EEs. We report 17 individuals with pathogenic variants in seven of the 27 genes, defining a genetic etiology in 3.2% of this unsolved cohort. Our results provide definitive evidence that de novo mutations in SLC1A2 and CACNA1A cause specific EEs and expand the compendium of clinically relevant genotypes for GABRB3. We also identified EEs caused by genetic variants in ALG13, DNM1, and GNAO1 and report a mutation in IQSEC2. Notably, recurrent mutations accounted for 7/17 of the pathogenic variants identified. As a result of high-depth coverage, parental mosaicism was identified in two out of 14 cases tested with mutant allelic fractions of 5%-6% in the unaffected parents, carrying significant reproductive counseling implications. These results confirm that dysregulation in diverse cellular neuronal pathways causes EEs, and they will inform the diagnosis and management of individuals with these devastating disorders.

The skeletal manifestations of the congenital disorders of glycosylation

The congenital disorders of glycosylation (CDG) are a rapidly expanding disease group with protean presentations. Specific end‐organ involvement leads to significant morbidity and mortality, and the skeletal manifestations are often not appreciated, apart from the common association of osteopaenia with CDG‐Ia. We performed a literature review of all documented skeletal manifestations in reported CDG patients, revealing a diverse range of skeletal phenotypes. We discuss the possible underlying mechanisms of these skeletal manifestations observed in CDG that are important and frequently under‐recognized.

Seizures, ataxia, developmental delay and the general paediatrician : Glucose transporter 1 deficiency syndrome

Aim  Glucose transporter 1 deficiency syndrome (GLUT1‐DS) is an important condition for the general paediatrician’s differential armamentarium. We describe a case series of eight patients in order to raise awareness of this treatable neurometabolic condition. The diagnosis of GLUT1‐DS is suggested by a decreased absolute cerebrospinal fluid (CSF) glucose value (<2.2 mmol/L) or lowered CSF: plasma glucose ratio (<0.4).

A novel splice site mutation in EYA4 causes DFNA10 hearing loss.

Nonsyndromic autosomal dominant sensorineural hearing loss (SNHL) at the DFNA10 locus was described in two families in 2001. Causative mutations that affect the EyaHR domain of the ‘Eyes absent 4’ (EYA4) protein were identified. We report on the clinical and genetic analyses of an Australian family with nonsyndromic SNHL. Screening of the EYA4 gene showed the novel polypyrimidine tract variation ca. 1282‐12T > A that introduces a new 3′ splice acceptor site. This is the first report of a point mutation in EYA4 that is hypothesized to lead to aberrant pre‐mRNA splicing and human disease. The DFNA10 family described is only the fourth to be identified. One individual presented with apparently the same phenotype as other affected members of the family. However, genotyping illustrated that he did not share the DFNA10 disease haplotype. Detailed clinical investigation showed differences in the onset and severity of his hearing loss and thus he is presumed to represent a phenocopy, perhaps resulting from long‐term exposure to loud noise.

Mutations in the nuclear localization sequence of the Aristaless related homeobox; sequestration of mutant ARX with IPO13 disrupts normal subcellular distribution of the transcription factor and retards cell division

BackgroundAristaless related homeobox (ARX) is a paired-type homeobox gene. ARX function is frequently affected by naturally occurring mutations. Nonsense mutations, polyalanine tract expansions and missense mutations in ARX cause a range of intellectual disability and epilepsy phenotypes with or without additional features including hand dystonia, lissencephaly, autism or dysarthria. Severe malformation phenotypes, such as X-linked lissencephaly with ambiguous genitalia (XLAG), are frequently observed in individuals with protein truncating or missense mutations clustered in the highly conserved paired-type homeodomain.ResultsWe have identified two novel point mutations in the R379 residue of the ARX homeodomain; c.1135C>A, p.R379S in a patient with infantile spasms and intellectual disability and c.1136G>T, p.R379L in a patient with XLAG. We investigated these and other missense mutations (R332P, R332H, R332C, T333N: associated with XLAG and Proud syndrome) predicted to affect the nuclear localisation sequences (NLS) flanking either end of the ARX homeodomain. The NLS regions are required for correct nuclear import facilitated by Importin 13 (IPO13). We demonstrate that missense mutations in either the N- or C-terminal NLS regions of the homeodomain cause significant disruption to nuclear localisation of the ARX protein in vitro. Surprisingly, none of these mutations abolished the binding of ARX to IPO13. This was confirmed by co-immunoprecipitation and immmuno fluorescence studies. Instead, tagged and endogenous IPO13 remained bound to the mutant ARX proteins, even in the RanGTP rich nuclear environment. We also identify the microtubule protein TUBA1A as a novel interacting protein for ARX and show cells expressing mutant ARX protein accumulate in mitosis, indicating normal cell division may be disrupted.ConclusionsWe show that the most likely, common pathogenic mechanism of the missense mutations in NLS regions of the ARX homeodomain is inadequate accumulation and distribution of the ARX transcription factor within the nucleus due to sequestration of ARX with IPO13.

Pediatric acute liver failure: Etiology, outcomes, and the role of serial pediatric end-stage liver disease scores

To describe etiology, short‐term outcomes and prognostic accuracy of serial PELD scores in PALF. Retrospective analysis of children aged ≤16 yr, admitted with PALF under the QLTS, Brisbane, Australia, between 1991 and 2011. PELD‐MELD scores were ascertained at three time points (i) admission (ii), meeting PALF criteria, and (iii) peak value. Fifty‐four children met criteria for PALF, median age 17 months (1 day–15.6 yr) and median weight 10.2 kg (1.9–57 kg). Etiology was known in 69%: 26% metabolic, 15% infective, 13% drug‐induced, 6% autoimmune, and 9% hemophagocytic lymphohistiocytosis. Age <3 months and weight <4.7 kg predicted poor survival in non‐transplanted children. Significant independent predictors of poor outcome (death or LT) were peak bilirubin > 220 μm/L and peak INR > 4. Serial PELD‐MELD scores were higher in the 17 (32%) transplant recipients (mean: [i] 26.8, [ii] 31.8, [iii] 42.6); highest in the 12 (22%) non‐transplanted non‐survivors (mean: [i] 31.6, [ii] 37.2, [iii] 45.7) compared with the 25 (46%) transplant‐free survivors (mean: [i] 25.3, [ii] 26.0, [iii] 30.3). PELD‐MELD thresholds of ≥27 and ≥42 at (ii) meeting PALF criteria and (iii) peak predicted poor outcome (p < 0.001). High peak bilirubin and peak INR predict poor outcome and serial PELD‐MELD is superior to single admission PELD‐MELD score for predicting poor outcome.

Mutations in SH3PXD2B cause Borrone dermato-cardio-skeletal syndrome

Borrone Dermato-Cardio-Skeletal (BDCS) syndrome is a severe progressive autosomal recessive disorder characterized by coarse facies, thick skin, acne conglobata, dysmorphic facies, vertebral abnormalities and mitral valve prolapse. We identified a consanguineous kindred with a child clinically diagnosed with BDCS. Linkage analysis of this family (BDCS1) identified five regions homozygous by descent with a maximum LOD score of 1.75. Linkage analysis of the family that originally defined BDCS (BDCS3) identified an overlapping linkage peak at chromosome 5q35.1. Sequence analysis identified two different homozygous mutations in BDCS1 and BDCS3, affecting the gene encoding the protein SH3 and PX domains 2B (SH3PXD2B), which localizes to 5q35.1. Western blot analysis of patient fibroblasts derived from affected individuals in both families demonstrated complete loss of SH3PXD2B. Homozygosity mapping and sequence analysis in a second published BDCS family (BDCS2) excluded SH3PXD2B. SH3PXD2B is required for the formation of functional podosomes, and loss-of-function mutations in SH3PXD2B have recently been shown to underlie 7 of 13 families with Frank-Ter Haar syndrome (FTHS). FTHS and BDCS share some overlapping clinical features; therefore, our results demonstrate that a proportion of BDCS and FTHS cases are allelic. Mutations in other gene(s) functioning in podosome formation and regulation are likely to underlie the SH3PXD2B-mutation-negative BDSC/FTHS patients.

Primary skeletal dysplasia as a major manifesting feature in an infant with congenital disorder of glycosylation type Ia

D. Coman,* D. Bostock, M. Hunter, P. Kannu, M. Irving, V. Mayne, M. Fietz, J. Jaeken, and R. Savarirayan Genetic Health Services Victoria, Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, Australia Newborn Services, Monash Medical Centre, Melbourne, Australia Department of Radiology, Monash Medical Centre, Melbourne, Australia National Referral Laboratory, Women’s and Children’s Hospital, Adelaide, Australia University Hospital Gasthuisberg, Leuven, Belgium