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Variants of uncertain significance (VUS) are commonly found following genomic sequencing, particularly in ethnically diverse populations that are underrepresented in large population databases. Functional characterization of VUS may assist in variant reclassification, however these studies are not readily available and often rely on research funding and good will. We present four individuals from three families at different stages of their diagnostic trajectory with recurrent acute liver failure (RALF) and biallelic NBAS variants, confirmed by either trio analysis or cDNA studies. Functional characterization was undertaken, measuring NBAS and p31 levels by Western blotting, demonstrating reduced NBAS levels in two of three families, and reduced p31 levels in all three families. These results provided functional characterization of the molecular impact of a missense VUS, allowing reclassification of the variant and molecular confirmation of NBAS-associated RALF. Importantly, p31 was decreased in all individuals, including an individual with two missense variants where NBAS protein levels were preserved. These results highlight the importance of access to timely functional studies after identification of putative variants, and the importance of considering a range of assays to validate variants whose pathogenicity is uncertain. We suggest that funding models for genomic sequencing should consider incorporating capabilities for adjunct RNA, protein, biochemical, and other specialized tests to increase the diagnostic yield which will lead to improved medical care, increased equity, and access to molecular diagnoses for all patients.

AIM: To assess the clinical utility of exome sequencing for patients with developmental and epileptic encephalopathies (DEEs). METHOD: Over 2 years, patients with DEEs were recruited for singleton exome sequencing. Parental segregation was performed where indicated. RESULTS: Of the 103 patients recruited (54 males, 49 females; aged 2 weeks-17 years), the genetic aetiology was identified in 36 out of 103 (35%) with management implications in 13 out of 36. Exome sequencing revealed pathogenic or likely pathogenic variants in 30 out of 103 (29%) patients, variants of unknown significance in 39 out of 103 (38%), and 34 out of 103 (33%) were negative on exome analysis. After the description of new genetic diseases, a molecular diagnosis was subsequently made for six patients or through newly available high-density chromosomal microarray testing. INTERPRETATION: We demonstrate the utility of exome sequencing in routine clinical care of children with DEEs. We highlight that molecular diagnosis often leads to changes in management and informs accurate prognostic and reproductive counselling. Our findings reinforce the need for ongoing analysis of genomic data to identify the aetiology in patients in whom the cause is unknown. The implementation of genomic testing in the care of children with DEEs should become routine in clinical practice. WHAT THIS PAPER ADDS: The cause was identified in 35% of patients with developmental and epileptic encephalopathies. KCNQ2, CDKL5, SCN1A, and STXBP1 were the most frequently identified genes. Reanalysis of genomic data found the cause in an additional six patients. Genetic aetiology was identified in 41% of children with seizure onset under 2 years, compared to 18% with older onset. Finding the molecular cause led to management changes in 36% of patients with DEEs.

BACKGROUND: Our primary aim was to evaluate the systematic reanalysis of singleton exome sequencing (ES) data for unsolved cases referred for any indication. A secondary objective was to undertake a literature review of studies examining the reanalysis of genomic data from unsolved cases. METHOD(S): We examined data from 58 unsolved cases referred between June 2016 and March 2017. First reanalysis at 4-13 months after the initial report considered genes newly associated with disease since the original analysis; second reanalysis at 9-18 months considered all disease-associated genes. At 25-34 months we reviewed all cases and the strategies which solved them. RESULT(S): Reanalysis of existing ES data alone at two timepoints did not yield new diagnoses. Over the same timeframe, 10 new diagnoses were obtained (17%) from additional strategies, such as microarray detection of copy number variation, repeat sequencing to improve coverage, and trio sequencing. Twenty-seven peer-reviewed articles were identified on the literature review, with a median new diagnosis rate via reanalysis of 15% and median reanalysis timeframe of 22 months. CONCLUSION(S): Our findings suggest that an interval of greater than 18 months from the original report may be optimal for reanalysis. We also recommend a multi-faceted strategy for cases remaining unsolved after singleton ES.Copyright © 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.

Background Our primary aim was to evaluate the systematic reanalysis of singleton exome sequencing (ES) data for unsolved cases referred for any indication. A secondary objective was to undertake a literature review of studies examining the reanalysis of genomic data from unsolved cases. Methods We examined data from 58 unsolved cases referred between June 2016 and March 2017. First reanalysis at 4–13 months after the initial report considered genes newly associated with disease since the original analysis; second reanalysis at 9–18 months considered all disease‐associated genes. At 25–34 months we reviewed all cases and the strategies which solved them. Results Reanalysis of existing ES data alone at two timepoints did not yield new diagnoses. Over the same timeframe, 10 new diagnoses were obtained (17%) from additional strategies, such as microarray detection of copy number variation, repeat sequencing to improve coverage, and trio sequencing. Twenty‐seven peer‐reviewed articles were identified on the literature review, with a median new diagnosis rate via reanalysis of 15% and median reanalysis timeframe of 22 months. Conclusion Our findings suggest that an interval of greater than 18 months from the original report may be optimal for reanalysis. We also recommend a multi‐faceted strategy for cases remaining unsolved after singleton ES.

Lathosterolosis is a rare autosomal recessive disorder of cholesterol biosynthesis. It is caused by defects in the SC5D (sterol C5-desaturase) gene which encodes for the 3-beta-hydroxysteroid-delta-5-desaturase (also called sterol-C5-desaturase or lathosterol dehydrogenase). Only six cases have been described in the literature, but it is possible that a number of patients with milder forms of the condition might have been missed. Lathosterolosis manifests as microcephaly, bilateral cataracts, dysmorphism, limb anomalies, and developmental delay/intellectual disability. Liver involvement is variable and can range from normal liver function tests to portal fibrosis and cirrhosis. Diagnosis is made by demonstration of specific mutations in the SC5D gene and by plasma sterol analysis to confirm elevated lathosterol levels. In this report, we describe a girl with transaminitis in association with developmental delay/intellectual disability, facial dysmorphism, limb anomalies, and bilateral cataracts. Fibroscan showed severe liver fibrosis. Plasma sterol analysis and exome sequencing confirmed the diagnosis of lathosterolosis. Simvastatin treatment resulted in lowering of plasma lathosterol levels, improvement in transaminitis, and liver fibrosis grade, suggesting that children with this condition should be actively treated in order to prevent progression of liver disease.

Background: Individuals with suspected monogenic disease remain unsolved after exome sequencing (ES) for reasons including technical challenges and difficulty in variant interpretation. Aim(s): To evaluate systematic reanalysis of singleton ES data for unsolved cases. Method(s): Data from unsolved cases referred for clinical ES at Victorian Clinical Genetics Services between 01/2016 and 03/2017 was systematically reanalyzed. First reanalysis at 4-13 months after the initial report looked at genes newly associated with disease since the original analysis; second reanalysis at 9-18 months looked at all disease-associated genes. At 25-34 months we reviewed the status of all cases and collated the strategies which solved cases through means other than reanalysis of singleton ES data. Result(s): Fifty-six unsolved cases were referred for syndromic (45%) and non-syndromic (3.5%) neurodevelopmental conditions, multiple congenital anomalies (12.5%), and single system disorders (39%). Reanalysis of existing ES data alone did not yield new diagnoses. Over the same timeframe, nine new diagnoses were obtained (16%): two intragenic deletions not tractable by ES but detected on array (ATAD3A/3B, NIPBL); two missense variants with low coverage in the original singleton ES, detected on trio ES/GS (CHD7; SCN8A); one in-frame deletion detected with a low variant fraction on singleton ES, highlighted as a de novo variant of interest on trio ES (WDR45); and four novel gene discoveries (unpublished). Conclusion(s): All additional diagnoses in this study were derived from strategies other than reanalysis of singleton ES data, illustrating the need to implement a multifaceted strategy for cases remaining unsolved after singleton ES.

Introduction: This is the first report of agranulocytosis with daclizumab therapy in a patient with relapsing and remitting multiple sclerosis (RRMS), who was subsequently diagnosed with CTLA-4 Haploinsufficiency with Autoimmune Infiltration (CHAI). Method(s): Following three months of daclizumab therapy for RRMS, our patient presented to hospital with an exacerbation of lymphocytic enteritis and was diagnosed with combined immunodeficiency with hypogammaglobulinaemia, reduced B cells and switched memory B cells with absent pneumococcal vaccine responses; T-cell lymphopenia with reduced naive T-cells. The patient was investigated for combined immunodeficiency with whole exome sequencing. Six months following commencement of daclizumab therapy, our patient presented to hospital with a one-week history of nausea, vomiting, tachycardia and high fevers (>39degreeC). Further assessment revealed an absolute neutropaenia (0.0 x109/L) and elevated C-reactive protein. No clear infective cause was found despite numerous investigations, including blood, bone marrow, urine and stool cultures. Bone marrow biopsy revealed an agranulocytosis with an apparent maturation block at the myeloblasts stage but no features of haemophagocytosis and Epstein-Barr virus (EBV) in situ hybridization was negative. Neutrophil recovery occurred following daclizumab cessation and the initiation of T cell immunosuppressive therapy with systemic corticosteroids and methotrexate. Result(s): A novel heterozygous missense variant in CTLA4 (c.410C>T; p. Pro137Leu in exon 2 of CTLA4) was identified in our patient, leading to a diagnosis of CHAI. This novel variant affects the CTLA-4 protein at a highly conserved amino acid in an essential functional domain and is classified as 'likely pathogenic'. The proband's brother and mother were also confirmed to carry the mutation. Conclusion(s): This case shows that autoimmune disease may be the presenting feature of primary immunodeficiency and should be appropri

Background: Daclizumab is a humanized monoclonal antibody that blocks CD25, the high affinity alpha subunit of the interleukin-2 receptor. Daclizumab therapy targets T regulatory cell and activated effector T cell proliferation to suppress autoimmune disease activity, in inflammatory conditions like relapsing and remitting multiple sclerosis. Here, we present the first report of agranulocytosis with daclizumab therapy in a patient with relapsing and remitting multiple sclerosis. Case presentation: Our patient was a 24-year-old Australian female with a clinical history of atopy, lymphocytic enteritis complicated by B12 deficiency, relapsing and remitting multiple sclerosis, recurrent lower respiratory tract infections, vulval/cervical intraepithelial neoplasia and melanoma. She was commenced on daclizumab therapy after failing several lines of treatment for relapsing and remitting multiple sclerosis. During a hospital admission for lymphocytic enteritis, she was incidentally diagnosed with combined immunodeficiency with hypogammaglobulinaemia and declined proposed regular intravenous immunoglobulin infusions. Following six months of daclizumab therapy, our patient presented to hospital with febrile neutropenia. No clear infective cause was found, despite numerous investigations. However, bone marrow biopsy revealed agranulocytosis with an apparent maturation block at the myeloblasts stage. Neustrophil recovery occurred following cessation of daclizumab and the initiation of T cell immunosuppressive agents including systemic corticosteroids and methotrexate. The patient was further investigated for combined immunodeficiency and whole exome sequencing revealed a novel heterozygous missense variant in cytotoxic T lymphocyte antigen 4 (CTLA4), leading to a diagnosis of CTLA-4 haploinsufficiency with autoimmune infiltration (CHAI). Conclusion(s): This case demonstrates that autoimmune disease may be the presenting feature of primary immunodeficiency and should be appropri

Background: Accurate and timely diagnosis is critical in providing prognostic information and guiding treatment in acutely unwell patients with suspected monogenic disorders. Aim(s): We describe the development and implementation of a rapid genomic diagnosis program in a routine clinical setting. Method(s): Rapid singleton wholeexome sequencing was performed in acutely unwell patients with suspected monogenic disorders from two pediatric tertiary centers. Technical, organizational, and clinician barriers to implementation were addressed through a cycle of continuous audit and improvement. Service performance parameters and the diagnostic and clinical utility of providing rapid WES were assessed. Result(s): During the study period, time to result reduced from 109 days to 13 days (median 18 days), and this was accompanied by changes in referral and test initiation patterns. Of 26 enrolled patients, 14 (54%) received a molecular genetic diagnosis. Clinical management changed in nine diagnosed patients (64%), including the provision of lifesaving treatment, avoidance of invasive biopsies, and palliative care guidance. A result was provided prior to death or discharge from hospital in 16 of 26 cases (62%). The rapid diagnosis of a riboflavin transporter defect in one patient is estimated to have saved 113 days in intensive care (AUD$508,500) compared to providing the result with a standard turnaround time. Discussion(s): The provision of rapid genomic results in acutely unwell patients with suspected monogenic disorders is feasible in the routine clinical setting and has high diagnostic and clinical utility. It challenges traditional clinical and laboratory genetics service models, and requires a whole-of-system approach for successful implementation.

BACKGROUND: Daclizumab is a humanized monoclonal antibody that blocks CD25, the high affinity alpha subunit of the interleukin-2 receptor. Daclizumab therapy targets T regulatory cell and activated effector T cell proliferation to suppress autoimmune disease activity, in inflammatory conditions like relapsing and remitting multiple sclerosis. Here, we present the first report of agranulocytosis with daclizumab therapy in a patient with relapsing and remitting multiple sclerosis. CASE PRESENTATION: Our patient was a 24-year-old Australian female with a clinical history of atopy, lymphocytic enteritis complicated by B12 deficiency, relapsing and remitting multiple sclerosis, recurrent lower respiratory tract infections, vulval/cervical intraepithelial neoplasia and melanoma. She was commenced on daclizumab therapy after failing several lines of treatment for relapsing and remitting multiple sclerosis. During a hospital admission for lymphocytic enteritis, she was incidentally diagnosed with combined immunodeficiency with hypogammaglobulinaemia and declined proposed regular intravenous immunoglobulin infusions. Following six months of daclizumab therapy, our patient presented to hospital with febrile neutropenia. No clear infective cause was found, despite numerous investigations. However, bone marrow biopsy revealed agranulocytosis with an apparent maturation block at the myeloblasts stage. Neustrophil recovery occurred following cessation of daclizumab and the initiation of T cell immunosuppressive agents including systemic corticosteroids and methotrexate. The patient was further investigated for combined immunodeficiency and whole exome sequencing revealed a novel heterozygous missense variant in cytotoxic T lymphocyte antigen 4 (CTLA4), leading to a diagnosis of CTLA-4 haploinsufficiency with autoimmune infiltration (CHAI). CONCLUSION: This case demonstrates that autoimmune disease may be the presenting feature of primary immunodeficiency and should be appropriate