Challenges and Opportunities in Implementing Pharmacogenetic Testing in Clinical Settings.

The clinical implementation of pharmacogenetic biomarkers continues to grow as new genetic variants associated with drug outcomes are discovered and validated. The number of drug labels that contain pharmacogenetic information also continues to expand. Published, peer-reviewed clinical practice guidelines have also been developed to support the implementation of pharmacogenetic tests. Incorporating pharmacogenetic information into health care benefits patients as well as clinicians by improving drug safety and reducing empiricism in drug selection. Barriers to the implementation of pharmacogenetic testing remain. This review explores current pharmacogenetic implementation initiatives with a focus on the challenges of pharmacogenetic implementation and potential opportunities to overcome these challenges.

CIAO1 and MMS19 deficiency: A lethal neurodegenerative phenotype caused by cytosolic Fe-S cluster protein assembly disorders.

PURPOSE: The functionality of many cellular proteins depends on cofactors; yet, they have only been implicated in a minority of Mendelian diseases. Here, we describe the first 2 inherited disorders of the cytosolic iron-sulfur protein assembly system. METHODS: Genetic testing via genome sequencing was applied to identify the underlying disease cause in 3 patients with microcephaly, congenital brain malformations, progressive developmental and neurologic impairments, recurrent infections, and a fatal outcome. Studies in patient-derived skin fibroblasts and zebrafish models were performed to investigate the biochemical and cellular consequences. RESULTS: Metabolic analysis showed elevated uracil and thymine levels in body fluids but no pathogenic variants in DPYD, encoding dihydropyrimidine dehydrogenase. Genome sequencing identified compound heterozygosity in 2 patients for missense variants in CIAO1, encoding cytosolic iron-sulfur assembly component 1, and homozygosity for an in-frame 3-nucleotide deletion in MMS19, encoding the MMS19 homolog, cytosolic iron-sulfur assembly component, in the third patient. Profound alterations in the proteome, metabolome, and lipidome were observed in patient-derived fibroblasts. We confirmed the detrimental effect of deficiencies in CIAO1 and MMS19 in zebrafish models. CONCLUSION: A general failure of cytosolic and nuclear iron-sulfur protein maturation caused pleiotropic effects. The critical function of the cytosolic iron-sulfur protein assembly machinery for antiviral host defense may well explain the recurrent severe infections occurring in our patients.

A luciferase reporter mouse model to optimize in vivo gene editing validated by lipid nanoparticle delivery of adenine base editors.

The rapid development of CRISPR genome editing technology has provided the potential to treat genetic diseases effectively and precisely. However, efficient and safe delivery of genome editors to affected tissues remains a challenge. Here, we developed luminescent ABE (LumA), a luciferase reporter mouse model containing the R387X mutation (c.A1159T) in the luciferase gene located in the Rosa26 locus of the mouse genome. This mutation eliminates luciferase activity but can be restored upon A-to-G correction by SpCas9 adenine base editors (ABEs). The LumA mouse model was validated through intravenous injection of two FDA-approved lipid nanoparticle (LNP) formulations consisting of either MC3 or ALC-0315 ionizable cationic lipids, encapsulated with ABE mRNA and LucR387X-specific guide RNA (gRNA). Whole-body bioluminescence live imaging showed consistent restoration of luminescence lasting up to 4 months in treated mice. Compared with mice carrying the wild-type luciferase gene, the ALC-0315 and MC3 LNP groups showed 83.5% ± 17.5% and 8.4% ± 4.3% restoration of luciferase activity in the liver, respectively, as measured by tissue luciferase assays. These results demonstrated successful development of a luciferase reporter mouse model that can be used to evaluate the efficacy and safety of different genome editors, LNP formulations, and tissue-specific delivery systems for optimizing genome editing therapeutics.

Trauma, Emotional Regulation, and Coping Styles in Individuals with and without Probable Dissociative Disorders in Hong Kong.

Previous studies showed that dissociation and dissociative disorders (DDs) are prevalent and are associated with considerable individual and social consequences. There are ongoing debates regarding whether dissociation is a response to betrayal trauma across cultures and whether dissociation can be explained by maladaptive coping. Additionally, little is known about the clinical features of individuals with DDs in the Chinese context. This study aimed to investigate the relationship between trauma, emotional regulation, coping, and dissociation. We analyzed baseline data from a randomized controlled trial (N = 101). Participants with dissociative symptoms in Hong Kong completed self-report assessments. Structured interviews were also conducted subsequently. Participants with probable DDs reported more traumatic events (p = .009 to .017) and exhibited significantly higher levels of dysfunctional coping (p < .001) compared to those who reported dissociative symptoms but did not have a DD. Dissociative symptoms were more strongly associated with betrayal trauma than with non-betrayal trauma. Among different emotion regulation and coping strategies, dysfunctional coping was the only significant factor associated with dissociative symptoms (ß = .309, p = .003). Dysfunctional coping was a statistically significant mediator that may explain the relationship between betrayal trauma and dissociative symptoms. Although other mediation paths are also possible and further longitudinal studies are required, our findings highlight the strong link between dysfunctional coping and dissociative symptoms and suggest that coping skills training should be incorporated into interventions for betrayal trauma survivors with dissociative symptoms. Additionally, this study provides evidence for the cross-cultural validity of the betrayal trauma theory. Further studies, however, are required.

COULD FAMILY WELL-BEING MODERATE THE RELATIONSHIP BETWEEN ADVERSE CHILDHOOD EXPERIENCES AND SOMATOFORM DISSOCIATION? A PRELIMINARY INVESTIGATION.

The impacts of adverse childhood experiences (ACEs) have been well documented. One possible consequence of ACEs is dissociation, which is a major feature of post-traumatic psychopathology and is also associated with considerable impairment and health care costs. Although ACEs are known to be associated with both psychoform and somatoform dissociation, much less is known about the mechanisms behind this relationship. Little is known about whether social and interpersonal factors such as family environments would moderate the relationship between ACEs and somatoform dissociation. This paper discusses the importance of having a positive and healthy family environment in trauma recovery. We then report the findings of a preliminary study in which we examined whether the association between ACEs and somatoform dissociation would be moderated by family well-being in a convenience sample of Hong Kong adults (N = 359). The number of ACEs was positively associated with somatoform dissociative symptoms, but this association was moderated by the level of family well-being. The number of ACEs was associated with somatoform dissociation only when the family well-being scores were low. These moderating effects were medium. The findings point to the potential importance of using family education and intervention programs to prevent and treat trauma-related dissociative symptoms, but further investigation is needed.

Identification of Drug Transporter Genomic Variants and Inhibitors That Protect Against Doxorubicin-Induced Cardiotoxicity.

BACKGROUND: Multiple pharmacogenomic studies have identified the synonymous genomic variant rs7853758 (G > A, L461L) and the intronic variant rs885004 in SLC28A3 (solute carrier family 28 member 3) as statistically associated with a lower incidence of anthracycline-induced cardiotoxicity. However, the true causal variant(s), the cardioprotective mechanism of this locus, the role of SLC28A3 and other solute carrier (SLC) transporters in anthracycline-induced cardiotoxicity, and the suitability of SLC transporters as targets for cardioprotective drugs has not been investigated. METHODS: Six well-phenotyped, doxorubicin-treated pediatric patients from the original association study cohort were recruited again, and human induced pluripotent stem cell-derived cardiomyocytes were generated. Patient-specific doxorubicin-induced cardiotoxicity (DIC) was then characterized using assays of cell viability, activated caspase 3/7, and doxorubicin uptake. The role of SLC28A3 in DIC was then queried using overexpression and knockout of SLC28A3 in isogenic human-induced pluripotent stem cell-derived cardiomyocytes using a CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9). Fine-mapping of the SLC28A3 locus was then completed after SLC28A3 resequencing and an extended in silico haplotype and functional analysis. Genome editing of the potential causal variant was done using cytosine base editor. SLC28A3-AS1 overexpression was done using a lentiviral plasmid-based transduction and was validated using stranded RNA-sequencing after ribosomal RNA depletion. Drug screening was done using the Prestwick Chemical Library (n = 1200), followed by in vivo validation in mice. The effect of desipramine on doxorubicin cytotoxicity was also investigated in 8 cancer cell lines. RESULTS: Here, using the most commonly used anthracycline, doxorubicin, we demonstrate that patient-derived cardiomyocytes recapitulate the cardioprotective effect of the SLC28A3 locus and that SLC28A3 expression influences the severity of DIC. Using Nanopore-based fine-mapping and base editing, we identify a novel cardioprotective single nucleotide polymorphism, rs11140490, in the SLC28A3 locus; its effect is exerted via regulation of an antisense long noncoding RNA (SLC28A3-AS1) that overlaps with SLC28A3. Using high-throughput drug screening in patient-derived cardiomyocytes and whole organism validation in mice, we identify the SLC competitive inhibitor desipramine as protective against DIC. CONCLUSIONS: This work demonstrates the power of the human induced pluripotent stem cell model to take a single nucleotide polymorphism from a statistical association through to drug discovery, providing human cell-tested data for clinical trials to attenuate DIC.

Development of a Dose-Adjusted Polygenic Risk Model for Anthracycline-Induced Cardiotoxicity.

BACKGROUND: Anthracyclines, which are effective chemotherapeutic agents, cause cardiac dysfunction in up to 57% of patients. The cumulative anthracycline dose is a crucial predictor of cardiotoxicity; however, the cumulative dose alone cannot explain all cardiotoxic events. Strongly associated genetic variants in SLC28A3 , UGT1A6 , and RARG contribute to anthracycline-induced cardiotoxicity in pediatric patients and may help identify those most susceptible. This study aimed to examine how these pharmacogenetic effects are modulated by cumulative anthracycline doses in the development of cardiotoxicity. METHODS: A total of 595 anthracycline-treated children were genotyped and cardiotoxicity cases were identified. A dose-stratified analysis was performed to compare the contributions of SLC28A3 rs7853758, UGT1A6 rs17863783, and RARG rs2229774 variants to the development of cardiotoxicity in low-dose (<150 mg/m 2 cumulative dose) and high-dose (>250 mg/m 2 cumulative dose) patient groups. Logistic regression was used to model the relationships between the cumulative anthracycline dose, genetic variants, and cardiotoxicity in the full cohort. RESULTS: At < 150 mg/m 2 cumulative anthracycline dose, the SLC28A3 protective variant did not reach statistical significance [odds ratio (OR) 0.46 (95% confidence interval (CI) 0.10-1.45), P = 0.23], but it was statistically significant at doses >250 mg/m 2 [OR 0.43 (95% CI 0.22-0.78), P = 0.0093]. Conversely, the UGT1A6 and RARG risk variants were either statistically significant or approaching significance at doses <150 mg/m 2 [OR 7.18 (95% CI 1.78-28.4), P = 0.0045 for UGT1A6 and OR 2.76 (95% CI 0.89-7.63), P = 0.057 for RARG ], but not at doses >250 mg/m 2 [OR 2.91 (95% CI 0.80-11.0), P = 0.10; OR 1.56 (95% CI 0.89-2.75), P = 0.12]. CONCLUSIONS: These findings suggest that the SLC28A3 variant imparts more significant protection for patients receiving higher anthracycline doses, whereas the UGT1A6 and RARG risk variants significantly increased the risk of cardiotoxicity at low anthracycline doses.

Systematic Critical Review of Genetic Factors Associated with Cisplatin-induced Ototoxicity: Canadian Pharmacogenomics Network for Drug Safety 2022 Update.

BACKGROUND: Cisplatin is commonly used to treat solid tumors; however, its use can be complicated by drug-induced hearing loss (ie, ototoxicity). The presence of certain genetic variants has been associated with the development/occurrence of cisplatin-induced ototoxicity, suggesting that genetic factors may be able to predict patients who are more likely to develop ototoxicity. The authors aimed to review genetic associations with cisplatin-induced ototoxicity and discuss their clinical relevance. METHODS: An updated systematic review was conducted on behalf of the Canadian Pharmacogenomics Network for Drug Safety, based on the Preferred Reporting Items for Systematic reviews and Meta-Analyses 2020 statement. Pharmacogenomic studies that reported associations between genetic variation and cisplatin-induced ototoxicity were included. The evidence on genetic associations was summarized and evaluated, and knowledge gaps that can be used to inform future pharmacogenomic studies identified. RESULTS: Overall, 40 evaluated reports, considering 47 independent patient populations, captured associations involving 24 genes. Considering GRADE criteria, genetic variants in 2 genes were strongly (ie, odds ratios ≥3) and consistently (ie, replication in ≥3 independent populations) predictive of cisplatin-induced ototoxicity. Specifically, an ACYP2 variant has been associated with ototoxicity in both children and adults, whereas TPMT variants are relevant in children. Encouraging evidence for associations involving several other genes also exists; however, further research is necessary to determine potential clinical relevance. CONCLUSIONS: Genetic variation in ACYP2 and TPMT may be helpful in predicting patients at the highest risk of developing cisplatin-induced ototoxicity. Further research (including replication studies considering diverse pediatric and adult patient populations) is required to determine whether genetic variation in additional genes may help further identify patients most at risk.

Role of Cisplatin Dose Intensity and TPMT Variation in the Development of Hearing Loss in Children.

BACKGROUND: Cisplatin, widely used in the treatment of solid tumors, causes permanent hearing loss in more than 60% of treated children. Previous studies have implicated several clinical factors in the development of ototoxicity, including cumulative cisplatin dose. However, the role of cisplatin dose intensity in the development of hearing loss in children remains unclear. Pharmacogenetic studies have also identified genetic variants in TPMT that increase the risk of cisplatin-induced hearing loss. This study aims to determine whether cisplatin dose intensity contributes to the risk of hearing loss in children and whether genetic variations in TPMT further modifies the risk of cisplatin-induced hearing loss. METHODS: The authors genotyped 371 cisplatin-treated children for the presence of any 3 TPMT -risk variants. Patients were categorized into high-, moderate-, and low-intensity cisplatin dosing groups according to the cisplatin dose administered per unit time. Kaplan-Meier curves were plotted to compare the cumulative incidence of hearing loss between the genotype and dose intensity groups. RESULTS: Patients receiving cisplatin at high dose intensity experienced significantly higher incidences of ototoxicity than those receiving cisplatin at low dose intensity ( P = 9 × 10 -7 ). Further stratification by TPMT genotype revealed that carriers of ≥1 TPMT variants receiving high-intensity cisplatin developed ototoxicity sooner and more often than their wild-type counterparts (93.8% vs. 56.6% at 12 months; P = 5 × 10 -5 ) and noncarriers receiving low-intensity cisplatin (21.2% at 12 months). CONCLUSIONS: Cisplatin dose intensity is strongly associated with ototoxicity development in children, and this risk is further increased by the presence of TPMT -risk alleles.

Noninvasive testing for mycophenolate exposure in children with renal transplant using urinary metabolomics.

BACKGROUND: Despite the common use of mycophenolate in pediatric renal transplantation, lack of effective therapeuic drug monitoring increases uncertainty over optimal drug exposure and risk for adverse reactions. This study aims to develop a novel urine test to estimate MPA exposure based using metabolomics. METHODS: Urine samples obtained on the same day of MPA pharmacokinetic testing from two prospective cohorts of pediatric kidney transplant recipients were assayed for 133 unique metabolites by mass spectrometry. Partial least squares (PLS) discriminate analysis was used to develop a top 10 urinary metabolite classifier that estimates MPA exposure. An independent cohort was used to test pharmacodynamic validity for allograft inflammation (urinary CXCL10 levels) and eGFR ratio (12mo/1mo eGFR) at 1 year. RESULTS: Fifty-two urine samples from separate children (36.5% female, 12.0 ± 5.3 years at transplant) were evaluated at 1.6 ± 2.5 years post-transplant. Using all detected metabolites (n = 90), the classifier exhibited strong association with MPA AUC by principal component regression (r = 0.56, p < .001) and PLS (r = 0.75, p < .001). A practical classifier (top 10 metabolites; r = 0.64, p < .001) retained similar accuracy after cross-validation (LOOCV; r = 0.52, p < .001). When applied to an independent cohort (n = 97 patients, 1053 samples), estimated mean MPA exposure over Year 1 was inversely associated with mean urinary CXCL10:Cr (r = -0.28, 95% CI -0.45, -0.08) and exhibited a trend for association with eGFR ratio (r = 0.35, p = .07), over the same time period. CONCLUSIONS: This urinary metabolite classifier can estimate MPA exposure and correlates with allograft inflammation. Future studies with larger samples are required to validate and evaluate its clinical application.

Acute kidney injury during cisplatin therapy and associations with kidney outcomes 2 to 6 months post-cisplatin in children: a multi-centre, prospective observational study.

BACKGROUND: Few studies describe acute kidney injury (AKI) burden during paediatric cisplatin therapy and post-cisplatin kidney outcomes. We determined risk factors for and rate of (1) AKI during cisplatin therapy, (2) chronic kidney disease (CKD) and hypertension 2-6 months post-cisplatin, and (3) whether AKI is associated with 2-6-month outcomes. METHODS: This prospective cohort study enrolled children (aged < 18 years at cancer diagnosis) treated with cisplatin from twelve Canadian hospitals. AKI during cisplatin therapy (primary exposure) was defined based on Kidney Disease: Improving Global Outcomes (KDIGO) serum creatinine criteria (≥ stage one). Severe electrolyte abnormalities (secondary exposure) included ≥ grade three hypophosphatemia, hypokalemia, or hypomagnesemia (National Cancer Institute Common Terminology Criteria for Adverse Events v4.0). CKD was albuminuria or decreased kidney function for age (KDIGO guidelines). Hypertension was defined based on the 2017 American Academy of Pediatrics guidelines. RESULTS: Of 159 children (median [interquartile range [IQR]] age: 6 [2-12] years), 73/159 (46%) participants developed AKI and 55/159 (35%) experienced severe electrolyte abnormalities during cisplatin therapy. At median [IQR] 90 [76-110] days post-cisplatin, 53/119 (45%) had CKD and 18/128 (14%) developed hypertension. In multivariable analyses, AKI was not associated with 2-6-month CKD or hypertension. Severe electrolyte abnormalities during cisplatin were associated with having 2-6-month CKD or hypertension (adjusted odds ratio (AdjOR) [95% CI]: 2.65 [1.04-6.74]). Having both AKI and severe electrolyte abnormalities was associated with 2-6-month hypertension (AdjOR [95% CI]: 3.64 [1.05-12.62]). CONCLUSIONS: Severe electrolyte abnormalities were associated with kidney outcomes. Cisplatin dose optimization to reduce toxicity and clear post-cisplatin kidney follow-up guidelines are needed. A higher resolution version of the Graphical abstract is available as Supplementary information.

Linkage analysis identifies an isolated strabismus locus at 14q12 overlapping with FOXG1 syndrome region.

Strabismus is a common condition, affecting 1%-4% of individuals. Isolated strabismus has been studied in families with Mendelian inheritance patterns. Despite the identification of multiple loci via linkage analyses, no specific genes have been identified from these studies. The current study is based on a seven-generation family with isolated strabismus inherited in an autosomal dominant manner. A total of 13 individuals from a common ancestor have been included for linkage analysis. Among these, nine are affected and four are unaffected. A single linkage signal has been identified at an 8.5 Mb region of chromosome 14q12 with a multipoint LOD (logarithm of the odds) score of 4.69. Disruption of this locus is known to cause FOXG1 syndrome (or congenital Rett syndrome; OMIM #613454 and *164874), in which 84% of affected individuals present with strabismus. With the incorporation of next-generation sequencing and in-depth bioinformatic analyses, a 4 bp non-coding deletion was prioritised as the top candidate for the observed strabismus phenotype. The deletion is predicted to disrupt regulation of FOXG1, which encodes a transcription factor of the Forkhead family. Suggestive of an autoregulation effect, the disrupted sequence matches the consensus FOXG1 and Forkhead family transcription factor binding site and has been observed in previous ChIP-seq studies to be bound by Foxg1 in early mouse brain development. Future study of this specific deletion may shed light on the regulation of FOXG1 expression and may enhance our understanding of the mechanisms contributing to strabismus and FOXG1 syndrome.

The Acceptability and Potential Benefits of a Web-Based Psychoeducation Program for People with Pathological Dissociation: A Pilot Evaluation Study.

Pathological dissociation is a common trauma-related condition and is associated with considerable health and social care needs. People with pathological dissociation typically require psychosocial interventions, but there are many challenges in providing such interventions for this vulnerable population. An important question that remains unexplored is whether web-based interventions are acceptable and beneficial for people with pathological dissociation. This paper reports the findings of a pilot evaluation study of a web-based psychoeducation program for people with pathological dissociation. A total of 80 participants were included. The preliminary findings revealed that: most participants were satisfied with the web-based program (e.g., most participants agreed that the program helped them understand [94.1%] and manage [66.7%] their mental health conditions and remain hopeful for recovery [78.4%]); and participants had improvements in symptom management (large effect; partial eta squared = .143) and self-esteem (medium-to-large effect; partial eta squared = .111) after the program. However, no improvements in clinical symptoms were observed on measures of post-traumatic or dissociative symptoms. The dropout rate at posttest was 36.3%. This is one of the first studies that evaluated web-based interventions for people with pathological dissociation. Although the web-based program is well-accepted by the participants and might be associated with some potential benefits, the limitations of this pilot study should be acknowledged. Implications are discussed in terms of the directions for future developments of the program and the needs for further evaluation.

The False Memory Debate A Reply to Otgaar.

The author replies to a critique of his paper about false memories in an effort to clarify issues on which there is disagreement. A key point made is that the validity of dissociation as a phenomenon, that is, as reported symptoms, does not depend on theories about the mechanisms of dissociation. There are no proven mechanisms for any mental health symptoms or DSM-5 diagnoses including anxiety, depression, psychosis and substance use disorders.

Gene expression profiles complement the analysis of genomic modifiers of the clinical onset of Huntington disease.

Huntington disease (HD) is a neurodegenerative disorder that is caused by a CAG repeat expansion in HTT. The length of this repeat, however, only explains a proportion of the variability in age of onset in patients. Genome-wide association studies have identified modifiers that contribute toward a proportion of the observed variance. By incorporating tissue-specific transcriptomic information with these results, additional modifiers can be identified. We performed a transcriptome-wide association study assessing heritable differences in genetically determined expression in diverse tissues, with genome-wide data from over 4000 patients. Functional validation of prioritized genes was undertaken in isogenic HD stem cells and patient brains. Enrichment analyses were performed with biologically relevant gene sets to identify the core pathways. HD-associated gene coexpression modules were assessed for associations with neurological phenotypes in an independent cohort and to guide drug repurposing analyses. Transcriptomic analyses identified genes that were associated with age of HD onset and displayed colocalization with gene expression signals in brain tissue (FAN1, GPR161, PMS2, SUMF2), with supporting evidence from functional experiments. This included genes involved in DNA repair, as well as novel-candidate modifier genes that have been associated with other neurological conditions. Further, cortical coexpression modules were also associated with cognitive decline and HD-related traits in a longitudinal cohort. In summary, the combination of population-scale gene expression information with HD patient genomic data identified novel modifier genes for the disorder. Further, these analyses expanded the pathways potentially involved in modifying HD onset and prioritized candidate therapeutics for future study.

A novel mouse model for pyridoxine-dependent epilepsy due to antiquitin deficiency.

Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive disease caused by mutations in the ALDH7A1 gene leading to blockade of the lysine catabolism pathway. PDE is characterized by recurrent seizures that are resistant to conventional anticonvulsant treatment but are well-controlled by pyridoxine (PN). Most PDE patients also suffer from neurodevelopmental deficits despite adequate seizure control with PN. To investigate potential pathophysiological mechanisms associated with ALDH7A1 deficiency, we generated a transgenic mouse strain with constitutive genetic ablation of Aldh7a1. We undertook extensive biochemical characterization of Aldh7a1-KO mice consuming a low lysine/high PN diet. Results showed that KO mice accumulated high concentrations of upstream lysine metabolites including ∆1-piperideine-6-carboxylic acid (P6C), α-aminoadipic semialdehyde (α-AASA) and pipecolic acid both in brain and liver tissues, similar to the biochemical picture in ALDH7A1-deficient patients. We also observed preliminary evidence of a widely deranged amino acid profile and increased levels of methionine sulfoxide, an oxidative stress biomarker, in the brains of KO mice, suggesting that increased oxidative stress may be a novel pathobiochemical mechanism in ALDH7A1 deficiency. KO mice lacked epileptic seizures when fed a low lysine/high PN diet. Switching mice to a high lysine/low PN diet led to vigorous seizures and a quick death in KO mice. Treatment with PN controlled seizures and improved survival of high-lysine/low PN fed KO mice. This study expands the spectrum of biochemical abnormalities that may be associated with ALDH7A1 deficiency and provides a proof-of-concept for the utility of the model to study PDE pathophysiology and to test new therapeutics.

The cumulative incidence of cisplatin-induced hearing loss in young children is higher and develops at an early stage during therapy compared with older children based on 2052 audiological assessments.

BACKGROUND: Ototoxicity is a common adverse event of cisplatin treatment. The authors investigated the development of cisplatin-induced hearing loss (CIHL) over time in children with cancer by age and examined the influence of other clinical characteristics on the course of CIHL. METHODS: Data from Canadian patients with childhood cancer were retrospectively reviewed. Hearing loss was graded according to International Society of Pediatric Oncology criteria. The Kaplan-Meier method was applied to estimate the cumulative incidence of CIHL for the total cohort and according to age. Cox regression models were used to explore the effects of independent variables on CIHL development up to 3 years after the start of therapy. RESULTS: In total, 368 patients with 2052 audiological assessments were included. Three years after initiating therapy, the cumulative incidence of CIHL was highest in patients aged ≤5 years (75%; 95% confidence interval [CI], 66%-84%), with a rapid increase observed to 27% (95% CI, 21%-35%) at 3 months and to 61% (95% CI, 53%-69%) at 1 year, compared with patients aged >5 years (48%; 95% CI, 37%-62%; P < .001). The total cumulative dose of cisplatin at 3 months (per 100 mg/m2 increase: hazard ratio [HR], 1.20; 95% CI, 1.01-1.41) vincristine (HR, 2.87; 95% CI, 1.89-4.36) and the total duration of concomitantly administered antibiotics (>30 days: HR, 1.85; 95% CI, 1.17-2.95) further influenced CIHL development over time. CONCLUSIONS: In young children, the cumulative incidence of CIHL is higher compared with that in older children and develops early during therapy. The course of CIHL is further influenced by the total cumulative dose of cisplatin and other ototoxic (co-)medication. These results highlight the need for audiological monitoring at each cisplatin cycle.

Length of Uninterrupted CAG, Independent of Polyglutamine Size, Results in Increased Somatic Instability, Hastening Onset of Huntington Disease.

Huntington disease (HD) is caused by a CAG repeat expansion in the huntingtin (HTT) gene. Although the length of this repeat is inversely correlated with age of onset (AOO), it does not fully explain the variability in AOO. We assessed the sequence downstream of the CAG repeat in HTT [reference: (CAG)n-CAA-CAG], since variants within this region have been previously described, but no study of AOO has been performed. These analyses identified a variant that results in complete loss of interrupting (LOI) adenine nucleotides in this region [(CAG)n-CAG-CAG]. Analysis of multiple HD pedigrees showed that this LOI variant is associated with dramatically earlier AOO (average of 25 years) despite the same polyglutamine length as in individuals with the interrupting penultimate CAA codon. This LOI allele is particularly frequent in persons with reduced penetrance alleles who manifest with HD and increases the likelihood of presenting clinically with HD with a CAG of 36-39 repeats. Further, we show that the LOI variant is associated with increased somatic repeat instability, highlighting this as a significant driver of this effect. These findings indicate that the number of uninterrupted CAG repeats, which is lengthened by the LOI, is the most significant contributor to AOO of HD and is more significant than polyglutamine length, which is not altered in these individuals. In addition, we identified another variant in this region, where the CAA-CAG sequence is duplicated, which was associated with later AOO. Identification of these cis-acting modifiers have potentially important implications for genetic counselling in HD-affected families.

Bi-allelic GOT2 Mutations Cause a Treatable Malate-Aspartate Shuttle-Related Encephalopathy.

Early-infantile encephalopathies with epilepsy are devastating conditions mandating an accurate diagnosis to guide proper management. Whole-exome sequencing was used to investigate the disease etiology in four children from independent families with intellectual disability and epilepsy, revealing bi-allelic GOT2 mutations. In-depth metabolic studies in individual 1 showed low plasma serine, hypercitrullinemia, hyperlactatemia, and hyperammonemia. The epilepsy was serine and pyridoxine responsive. Functional consequences of observed mutations were tested by measuring enzyme activity and by cell and animal models. Zebrafish and mouse models were used to validate brain developmental and functional defects and to test therapeutic strategies. GOT2 encodes the mitochondrial glutamate oxaloacetate transaminase. GOT2 enzyme activity was deficient in fibroblasts with bi-allelic mutations. GOT2, a member of the malate-aspartate shuttle, plays an essential role in the intracellular NAD(H) redox balance. De novo serine biosynthesis was impaired in fibroblasts with GOT2 mutations and GOT2-knockout HEK293 cells. Correcting the highly oxidized cytosolic NAD-redox state by pyruvate supplementation restored serine biosynthesis in GOT2-deficient cells. Knockdown of got2a in zebrafish resulted in a brain developmental defect associated with seizure-like electroencephalography spikes, which could be rescued by supplying pyridoxine in embryo water. Both pyridoxine and serine synergistically rescued embryonic developmental defects in zebrafish got2a morphants. The two treated individuals reacted favorably to their treatment. Our data provide a mechanistic basis for the biochemical abnormalities in GOT2 deficiency that may also hold for other MAS defects.

Glutaminase Deficiency Caused by Short Tandem Repeat Expansion in GLS.

We report an inborn error of metabolism caused by an expansion of a GCA-repeat tract in the 5' untranslated region of the gene encoding glutaminase (GLS) that was identified through detailed clinical and biochemical phenotyping, combined with whole-genome sequencing. The expansion was observed in three unrelated patients who presented with an early-onset delay in overall development, progressive ataxia, and elevated levels of glutamine. In addition to ataxia, one patient also showed cerebellar atrophy. The expansion was associated with a relative deficiency of GLS messenger RNA transcribed from the expanded allele, which probably resulted from repeat-mediated chromatin changes upstream of the GLS repeat. Our discovery underscores the importance of careful examination of regions of the genome that are typically excluded from or poorly captured by exome sequencing.