Daniela Zantomio

Predicting the diagnosis of autism spectrum disorder using gene pathway analysis

Autism spectrum disorder (ASD) depends on a clinical interview with no biomarkers to aid diagnosis. The current investigation interrogated single-nucleotide polymorphisms (SNPs) of individuals with ASD from the Autism Genetic Resource Exchange (AGRE) database. SNPs were mapped to Kyoto Encyclopedia of Genes and Genomes (KEGG)-derived pathways to identify affected cellular processes and develop a diagnostic test. This test was then applied to two independent samples from the Simons Foundation Autism Research Initiative (SFARI) and Wellcome Trust 1958 normal birth cohort (WTBC) for validation. Using AGRE SNP data from a Central European (CEU) cohort, we created a genetic diagnostic classifier consisting of 237 SNPs in 146 genes that correctly predicted ASD diagnosis in 85.6% of CEU cases. This classifier also predicted 84.3% of cases in an ethnically related Tuscan cohort; however, prediction was less accurate (56.4%) in a genetically dissimilar Han Chinese cohort (HAN). Eight SNPs in three genes (KCNMB4, GNAO1, GRM5) had the largest effect in the classifier with some acting as vulnerability SNPs, whereas others were protective. Prediction accuracy diminished as the number of SNPs analyzed in the model was decreased. Our diagnostic classifier correctly predicted ASD diagnosis with an accuracy of 71.7% in CEU individuals from the SFARI (ASD) and WTBC (controls) validation data sets. In conclusion, we have developed an accurate diagnostic test for a genetically homogeneous group to aid in early detection of ASD. While SNPs differ across ethnic groups, our pathway approach identified cellular processes common to ASD across ethnicities. Our results have wide implications for detection, intervention and prevention of ASD.

Convergent evidence for mGluR5 in synaptic and neuroinflammatory pathways implicated in ASD

The pathogenesis of Autism Spectrum Disorder (ASD), a serious neurodevelopmental disorder, is poorly understood. We review evidence for alterations in glutamatergic signalling in the aetiology of ASD, with a focus on the metabotropic glutamate receptor-5 (mGluR5). mGluR5 signalling is important for synapse formation, neuroplasticity and long term potentiation as well as neuroprotection and has been shown to have a regulatory role in neuroinflammation. Evidence for neuroinflammation in ASD is supported by increase in pro-inflammatory cytokines in the blood and cerebrospinal fluid (CSF) and increased number and activation of microglia in postmortem dorsolateral prefrontal cortex (DLPFC). mGlur5 signalling has also been shown to downregulate microglial activation. Therefore, we focus on mGluR5 as a potential unifying explanation for synapse alteration and neuroinflammation seen in ASD. Data from mGluR5 knockout mouse models, and syndromic and non syndromic forms of ASD are discussed in relation to how alterations in mGluR5 are associated with ASD symptoms. This review supports altered mGluR5 functioning as a convergent point in ASD pathogenesis and indicates more research is warranted into mGluR5 as a potential therapeutic target.

Decreased expression of mGluR5 within the dorsolateral prefrontal cortex in autism and increased microglial number in mGluR5 knockout mice: Pathophysiological and neurobehavioral implications

Metabotropic glutamate receptor 5 (mGluR5) and microglial abnormalities have been implicated in autism spectrum disorder (ASD). However, controversy exists as to whether the receptor is down or upregulated in functioning in ASD. In addition, whilst activation of mGluR5 has been shown to attenuate microglial activation, its role in maintaining microglial homeostasis during development has not been investigated. We utilised published microarray data from the dorsolateral prefrontal cortex (DLPFC) of control (n=30) and ASD (n=27) individuals to carry out regression analysis to assess gene expression of mGluR5 downstream signalling elements. We then conducted a post-mortem brain stereological investigation of the DLPFC, to estimate the proportion of mGluR5-positive neurons and glia. Finally, we carried out stereological investigation into numbers of microglia in mGluR5 knockout mice, relative to wildtype littermates, together with assessment of changes in microglial somal size, as an indicator of activation status. We found that gene expression of mGluR5 was significantly decreased in ASD versus controls (p=0.018) as well as downstream elements SHANK3 (p=0.005) and PLCB1 (p=0.009) but that the pro-inflammatory marker NOS2 was increased (p=0.047). Intensity of staining of mGluR5-positive neurons was also significantly decreased in ASD versus controls (p=0.016). Microglial density was significantly increased in mGluR5 knockout animals versus wildtype controls (p=0.011). Our findings provide evidence for decreased expression of mGluR5 and its signalling components representing a key pathophysiological hallmark in ASD with implications for the regulation of microglial number and activation during development. This is important in the context of microglia being considered to play key roles in synaptic pruning during development, with preservation of appropriate connectivity relevant for normal brain functioning.

Gelatinous transformation of the bone marrow as a late morphological change in imatinib mesylate treated chronic myeloid leukaemia

from the effect of IgE-type myeloma. An in vitro finding from a human IgE myeloma derived cell line may shed some light on another possible mechanism. After the myeloma cell line had been cultivated for more than 12 years, the investigators found newly expressed IgA2 accompanied by a lower rate of IgE production. Similar to our finding, they failed to demonstrate a recombinatory isotype switch from the e to the a2 locus at the DNA level. The authors hypothesised that after longterm cultivation, a regulator which normally regulates isotype expression through RNA splicing or termination was altered, causing the a2 expression. Thus, it is possible that chemotherapy treatment on our patient might have exerted selection pressure on plasma cells in causing differential splicing of RNA via an altered regulator. Proper identification of rare myeloma variants like IgD or IgE requires laboratory scientists to be alerted to unmatched light chain bands in routine serum immunofixation and take appropriate action to retest using IgD and IgE-type heavy chain antisera.

Apparent ‘JAK2‐negative’ polycythaemia vera due to compound mutations in exon 14

A somatic activating mutation in exon 14 of JAK2 involving the pseudokinase domain (c.1849G>T; p.V617F), is found in ~95% of patients with polycythaemia vera (PV), and is a major diagnostic criterion in the 2008 World Health Organization (WHO) classification (Swerdlow et al, 2008) and its proposed forthcoming revision (Barbui et al, 2015). A further 2–3% of PV patients have mutations in exon 12 of JAK2, so that very few PV patients are ‘JAK2-negative’, lacking a somatic mutation in that gene. As there are many alternative causes of absolute or relative erythrocytosis the diagnosis sometimes remains in doubt when no JAK2 mutation is identified. Here we report a case of WHO-defined PV with no JAK2 mutation detected on routine testing using a single nucleotide primer extension assay for JAK2 V617F and direct sequencing of exon 12. Targeted next generation sequencing (NGS) of the entire coding region of JAK2 revealed a novel mutation in exon 14 (c.1849_1853GTCTG>TTTCT; p.V617F/ C618L). This mutation resulted in failure of the JAK2 V617F mutation-specific primer to bind to the target, causing a false negative result with amplification only of the wild type allele. Direct sequencing of exon 14 in 7 additional cases of ‘JAK2negative’ polycythaemia without a conclusive diagnosis, identified one other compound mutation, demonstrating the diagnostic utility of broader sequencing of JAK2 in such cases. The index case (Patient 1) was a 58-year-old male who presented with itching for 2 years and recent onset night sweats. Red cell indices are shown in Table I. The bone marrow (BM) biopsy showed panmyelosis (Fig 1A) with grade 3/4 reticulin fibrosis and iron deficiency. The karyotype was normal, but single nucleotide polymorphism (SNP)-array analysis demonstrated a 23 6 MB block of uniparental disomy (UPD) at 9p24.3–p21.3, involving the JAK2 gene. Targeted NGS identified the JAK2 V617F mutation in 40% of 4362 reads, always with C618L in cis (data not shown), and confirmed by direct sequencing (Fig 1A). Subsequently we tested 7 additional patients (summarized in Table I) who were referred by their treating clinicians with an elevated haemoglobin concentration, subnormal or lownormal serum erythropoietin (EPO) levels (<29 lower limit of normal) and no JAK2 mutation in routine diagnostic testing (V617F and exon 12). Direct sequencing of JAK2 exon 14 in genomic DNA extracted from peripheral blood leucocytes or BM aspirates revealed a compound mutation (c.1849G>T;1852T>C; p.V617F/C618R) in one additional patient (Patient 2). The other six patients had normal results. Patient 2 was a 76-year-old male with asymptomatic polycythaemia. The BM showed panmyelosis (Table I, Fig 1B) with iron deficiency and grade 2/4 reticulin fibrosis. The karyotype and SNP-array analysis were normal. Erythroid burst-forming units from Patients 1 and 2 were grown in the absence of EPO and the individual colonies sequenced. In Patient 1, the majority of colonies were homozygous for the compound mutation, suggesting that UPD was a secondary event, as is usual in classical PV (Wang et al, 2014). No V617F/C618R homozygous colonies were identified in Patient 2, consistent with the absence of UPD by SNP-array. Only ~20% of PV patients lack a substantial UPD clone (Godfrey et al, 2012). JAK2 C618R has been reported as a sole mutation in a suspected myeloproliferative neoplasm (Ma et al, 2009), which could be consistent

Severe intravenous immunoglobulin-induced hemolysis with pigment nephropathy managed with red cell exchange.

We report on the use of red cell exchange in a case of severe intravenous immune globulin induced hemolysis and pigment nephropathy. Renal impairment and hemoglobinuria were not ameliorated by supportive measures including hydration. Partial red cell exchange with group O blood reduced hemoglobinuria and appeared to stabilize renal function. This is the first report on the use of red cell exchange in this clinical setting. J. Clin. Apheresis 31:464–466, 2016.

Response to Belgard et al.

We thank the Editor for the opportunity to respond to the letter from Belgard et al.1 In their letter, these authors consider that the issue of ethnic population stratification may have negatively impacted the findings in our original manuscript.2 We agree that population stratification is an important issue that needs to be accounted for in such analyses.

Response to Robinson et al.

Recently, Robinson et al.1 published results suggesting that our genetic classifier for autism spectrum disorder (ASD) has no translational value, as they did not replicate our accuracy in their independent cohort. They state that our classifier was confounded by a combination of issues, particularly population stratification as well as call rates between platforms.

Ask the expert (transfusion session): severe intravascular haemolysis following intravenous immunoglobulin (IVIg) administration and use of red cell exchange

We describe the first case of severe intravascular haemolysis secondary to IVIg treated with partial red cell exchange with Group O blood. A 62-year-old male was administered high dose IVIg (1.4g/kg in three divided doses) for a new diagnosis of chronic inflammatory demyelinating polyneuropathy. Within 24 hours of completing his IVIG infusion, he developed severe intravascular haemolysis, complicated by haemoglobuinuria and acute renal failure. Confirmatory tests included haemolysis screen, elution of anti-A and anti-B antibodies off the patient’s red cells and absence of other causes for haemolysis. The case illustrates patient risk factors for haemolytic IVIG reactions including: high IVIg dose >100g, non-O blood group (patient being AB RhD+) and discusses secretor status. Preventive measures including IVIg product processing are discussed. Although management of this condition is largely supportive, there may be a need to transfuse and group specific blood should be avoided. This patient received a single red cell exchange transfusion with Group O blood, with rapid resolution of renal impairment.