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Massively parallel sequencing has markedly improved mendelian diagnostic rates. This study assessed the effects of custom alterations to a diagnostic genomic bioinformatic pipeline in response to clinical need and derived practice recommendations relative to diagnostic rates and efficiency. The Genomic Annotation and Interpretation Application (GAIA) bioinformatics pipeline was designed to detect panel, exome, and genome sample integrity and prioritize gene variants in mendelian disorders. Reanalysis of selected negative cases was performed after improvements to the pipeline. GAIA improvements and their effect on sensitivity are described, including addition of a PubMed search for gene-disease associations not in the Online Mendelian Inheritance of Man database, inclusion of a process for calling low-quality variants (known as QPatch), and gene symbol nomenclature consistency checking. The new pipeline increased the diagnostic rate and reduced staff costs, resulting in a saving of US$844.34 per additional diagnosis. Recommendations for genomic analysis pipeline requirements are summarized. Clinically responsive bioinformatics pipeline improvements increase diagnostic sensitivity and increase cost-effectiveness.

Whole genome sequencing (WGS) improves Mendelian disorder diagnosis over whole exome sequencing (WES); however, additional diagnostic yields and costs remain undefined. We investigated differences between diagnostic and cost outcomes of WGS and WES in a cohort with suspected Mendelian disorders. WGS was performed in 38 WES-negative families derived from a 64 family Mendelian cohort that previously underwent WES. For new WGS diagnoses, contemporary WES reanalysis determined whether variants were diagnosable by original WES or unique to WGS. Diagnostic rates were estimated for WES and WGS to simulate outcomes if both had been applied to the 64 families. Diagnostic costs were calculated for various genomic testing scenarios. WGS diagnosed 34% (13/38) of WES-negative families. However, contemporary WES reanalysis on average 2 years later would have diagnosed 18% (7/38 families) resulting in a WGS-specific diagnostic yield of 19% (6/31 remaining families). In WES-negative families, the incremental cost per additional diagnosis using WGS following WES reanalysis was AU$36,710 (£19,407;US$23,727) and WGS alone was AU$41,916 (£22,159;US$27,093) compared to WES-reanalysis. When we simulated the use of WGS alone as an initial genomic test, the incremental cost for each additional diagnosis was AU$29,708 (£15,705;US$19,201) whereas contemporary WES followed by WGS was AU$36,710 (£19,407;US$23,727) compared to contemporary WES. Our findings confirm that WGS is the optimal genomic test choice for maximal diagnosis in Mendelian disorders. However, accepting a small reduction in diagnostic yield, WES with subsequent reanalysis confers the lowest costs. Whether WES or WGS is utilised will depend on clinical scenario and local resourcing and availability.

Cantú syndrome (CS), characterized by hypertrichosis, distinctive facial features, and complex cardiovascular abnormalities, is caused by pathogenic variants in ABCC9 and KCNJ8 genes. These genes encode gain‐of‐function mutations in the regulatory (SUR2) and pore‐forming (Kir6.1) subunits of KATP channels, respectively, suggesting that channel‐blocking sulfonylureas could be a viable therapy. Here we report a neonate with CS, carrying a heterozygous ABCC9 variant (c.3347G>A, p.Arg1116His), born prematurely at 32 weeks gestation. Initial echocardiogram revealed a large patent ductus arteriosus (PDA), and high pulmonary pressures with enlarged right ventricle. He initially received surfactant and continuous positive airway pressure ventilation and was invasively ventilated for 4 weeks, until PDA ligation. After surgery, he still had ongoing bilevel positive airway pressure (BiPAP) requirement, but was subsequently weaned to nocturnal BiPAP. He was treated for pulmonary hypertension with Sildenafil, but failed to make further clinical improvement. A therapeutic glibenclamide trial was commenced in week 11 (initial dose of 0.05 mg–1 kg–1 day–1 in two divided doses). After 1 week of treatment, he began to tolerate time off BiPAP when awake, and edema improved. Glibenclamide was well tolerated, and the dose was slowly increased to 0.15 mg−1 kg−1day−1 over the next 12 weeks. Mild transient hypoglycemia was observed, but there was no cardiovascular dysfunction. Confirmation of therapeutic benefit will require studies of more CS patients but, based on this limited experience, consideration should be given to glibenclamide as CS therapy, although problems associated with prematurity, and complications of hypoglycemia, might limit outcome in critically ill neonates with CS.

To provide proof of concept by broadening preconception screening beyond targeted testing to inform reproductive risk in consanguineous couples. Consanguineous couples were screened for autosomal recessive and X-linked disorders using the TruSight One panel of 4,813 genes associated with human disease. We recruited 22 couples, of whom 15 elected to have sequencing. We found four couples to be at risk of autosomal recessive disorders, including one with a child affected by Poretti–Boltshauser syndrome (a diagnosis not made prior to the study) and another previously known to carry a β-globin variant. Two couples were found to carry variants unrelated to known family history. These variants were in the genes C5orf42 (associated with Joubert syndrome and orofaciodigital syndrome) and GYS2 (associated with glycogen synthase deficiency). One known variant was not detected—a single exon deletion in FAM20C. We would not expect to identify this variant with the methodology employed. Of the four variants identified, only the β-globin variant would have been found using available commercial preconception screening panels. Preconception screening of consanguineous couples for recessive and X-linked disorders using genomic sequencing is practicable, and is likely to detect many more at-risk couples than any targeted panel could achieve. A couples-based approach greatly reduces the associated analysis and counselling burden.

BACKGROUND Recognition of viral nucleic acids is one of the primary triggers for a type I interferon-mediated antiviral immune response. Inborn errors of type I interferon immunity can be associated with increased inflammation and/or increased susceptibility to viral infections as a result of dysbalanced interferon production. NFX1-type zinc finger-containing 1 (ZNFX1) is an interferon-stimulated double-stranded RNA sensor that restricts the replication of RNA viruses in mice. The role of ZNFX1 in the human immune response is not known. OBJECTIVE We studied 15 patients from 8 families with an autosomal recessive immunodeficiency characterized by severe infections by both RNA and DNA viruses and virally triggered inflammatory episodes with hemophagocytic lymphohistiocytosis-like disease, early-onset seizures, and renal and lung disease. METHODS Whole exome sequencing was performed on 13 patients from 8 families. We investigated the transcriptome, posttranscriptional regulation of interferon-stimulated genes (ISGs) and predisposition to viral infections in primary cells from patients and controls stimulated with synthetic double-stranded nucleic acids. RESULTS Deleterious homozygous and compound heterozygous ZNFX1 variants were identified in all 13 patients. Stimulation of patient-derived primary cells with synthetic double-stranded nucleic acids was associated with a deregulated pattern of expression of ISGs and alterations in the half-life of the mRNA of ISGs and also associated with poorer clearance of viral infections by monocytes. CONCLUSION ZNFX1 is an important regulator of the response to double-stranded nucleic acids stimuli following viral infections. ZNFX1 deficiency predisposes to severe viral infections and a multisystem inflammatory disease.

Massively parallel sequencing has markedly improved mendelian diagnostic rates. This study assessed the effects of custom alterations to a diagnostic genomic bioinformatic pipeline in response to clinical need and derived practice recommendations relative to diagnostic rates and efficiency. The Genomic Annotation and Interpretation Application (GAIA) bioinformatics pipeline was designed to detect panel, exome, and genome sample integrity and prioritize gene variants in mendelian disorders. Reanalysis of selected negative cases was performed after improvements to the pipeline. GAIA improvements and their effect on sensitivity are described, including addition of a PubMed search for gene-disease associations not in the Online Mendelian Inheritance of Man database, inclusion of a process for calling low-quality variants (known as QPatch), and gene symbol nomenclature consistency checking. The new pipeline increased the diagnostic rate and reduced staff costs, resulting in a saving of US$844.34 per additional diagnosis. Recommendations for genomic analysis pipeline requirements are summarized. Clinically responsive bioinformatics pipeline improvements increase diagnostic sensitivity and increase cost-effectiveness.

Multiple studies have shown that genomic testing has a high diagnostic yield and an impact on clinical management for patients with suspected genetic conditions. Therefore, there has been a push worldwide to apply rapid genomic sequencing in critically ill neonatal and pediatric patients. The goal of this study was to investigate the practicality of applying ultrarapid genomic testing for critically ill neonatal and pediatric patients with suspected monogenic conditions in Australia. The study recruited a total of 108 patients prospectively from March 2018 to February 2019, and data were collected until May 2019. Of the 12 hospitals that acted as collaborating sites, 5 were women's hospitals, 3 women's and children's hospitals, and 4 children's hospitals. Eligible patients included those who were admitted to a neonatal or pediatric intensive care unit (NICU or PICU) and were referred to clinical genetics for a possible monogenic condition. Chromosomal microarray was a requirement before enrollment if there was a suspected chromosomal condition. Chromosomal microarrays were performed concurrently with ultrarapid exome sequencing when the probability of a positive result on microarray was low. Additionally, rapid mitochondrial genome sequencing was performed alongside ultrarapid exome sequencing if a mitochondrial condition was suspected. When possible, ultrarapid exome sequencing was performed in both parents as well as the child (trio). The primary outcome of this study measured the time from the last sample received to the ultrarapid exome sequencing report finalized. Other outcomes measured were the diagnostic yield, change in clinical management after the report was finalized, number of reports returned before hospital discharge, and time from admission to the report being finalized. Of the 108 patients enrolled, the median age was 28 days, with a range of 0 days to 17 years. Overall, 34% were female, 57% were NICU admissions, 33% were PICU admissions, and 9% were from other hospital wards. The mean time it took from the last sample received to the report being issued was 3.3 days (95% confidence interval [CI], 3.2-3.5 days). The mean time from hospital admission to the sequencing report being finalized was 17.5 days (95% CI, 14.6-21.1 days). The mean time from hospital admission to clinical genetics referral was 8.1 days (95% CI, 6.0-10.8 days). Overall, 94% of reports were issued within the goal of 5 calendar days, and 56 molecular diagnoses were made for 55 patients (51%). Among the 62 patients in the NICU, 35 had a clear molecular diagnosis from ultrarapid exome sequencing (56%), whereas in the 36 PICU patients, 17 had a clear molecular diagnosis (47%), and in the 10 patients from other hospital wards, 3 had a clear molecular diagnosis (30%). After the ultrarapid exome sequencing report was finalized, clinical management changed for 48 of the 108 patients enrolled (44%). Overall, the ultrarapid sequencing reports that revealed a clear molecular diagnosis were regarded as “very useful” or “useful” by referring providers in 52 of 55 cases (95%) and “neutral” in 3 of 55 cases (5%). Negative reports were regarded as “very useful” or “useful” in 31 of 53 cases (58%), “neutral” in 20 of 53 cases, and “not useful” in 2 of 53 cases (4%). Altogether, this study demonstrates the utility of ultrarapid exome sequencing in critically ill pediatric patients with a suspected monogenic condition in Australia. Refereed/Peer-reviewed

IMPORTANCE: Widespread adoption of rapid genomic testing in pediatric critical care requires robust clinical and laboratory pathways that provide equitable and consistent service across health care systems.OBJECTIVE: To prospectively evaluate the performance of a multicenter network for ultra-rapid genomic diagnosis in a public health care system.DESIGN, SETTING, AND PARTICIPANTS: Descriptive feasibility study of critically ill pediatric patients with suspected monogenic conditions treated at 12 Australian hospitals between March 2018 and February 2019, with data collected to May 2019. A formal implementation strategy emphasizing communication and feedback, standardized processes, coordination,distributed leadership, and collective learning was used to facilitate adoption.EXPOSURES Ultra-rapid exome sequencing.MAIN OUTCOMES AND MEASURES: The primary outcome was time from sample receipt to ultra-rapid exome sequencing report. The secondary outcomes were the molecular diagnostic yield, the change in clinical management after the ultra-rapid exome sequencing report, the time from hospital admission to the laboratory report, and the proportion of laboratory reports returned prior to death or hospital discharge RESULTS: The study population included 108 patients with a median age of 28 days (range, 0 days to 17 years); 34% were female; and 57% were from neonatal intensive care units, 33% were from pediatric intensive care units, and 9% were from other hospital wards. The meantime from sample receipt to ultra-rapid exome sequencing report was 3.3 days (95% CI,3.2-3.5 days) and the median time was 3 days (range, 2-7 days). The mean time from hospital admission to ultra-rapid exome sequencing report was 17.5 days (95% CI, 14.6-21.1 days) and 93 reports (86%) were issued prior to death or hospital discharge. A molecular diagnosis was established in 55 patients (51%). Eleven diagnoses (20%) resulted from using the following approaches to augment standard exome sequencing analysis: mitochondrial genome sequencing analysis, exome sequencing–based copy number analysis, use of international databases to identify novel gene–disease associations, and additional phenotyping and RNA analysis. In 42 of 55 patients (76%) with a molecular diagnosis and 6 of 53 patients (11%)without a molecular diagnosis, the ultra-rapid exome sequencing result was considered as having influenced clinical management. Targeted treatments were initiated in 12 patients(11%), treatment was redirected toward palliative care in 14 patients (13%), and surveillance for specific complications was initiated in 19 patients (18%).CONCLUSIONS AND RELEVANCE: This study suggests feasibility of ultra-rapid genomic testing in critically ill pediatric patients with suspected monogenic conditions in the Australian public health care system. However, further research is needed to understand the clinical value of such testing, and the generalizability of the findings to other health care settings. Refereed/Peer-reviewed

Purpose : The craniosynostoses are characterized by premature fusion of one or more cranial sutures. The relative contribution of previously reported genes to craniosynostosis in large cohorts is unclear. Here we report on the use of a massively parallel sequencing panel in individuals with craniosynostosis without a prior molecular diagnosis. Methods : A 20-gene panel was designed based on the genes’ association with craniosynostosis, and clinically validated through retrospective testing of an Australian and New Zealand cohort of 233 individuals with craniosynostosis in whom previous testing had not identified a causative variant within FGFR1-3 hot-spot regions or the TWIST1 gene. An additional 76 individuals were tested prospectively. Results : Pathogenic or likely pathogenic variants in non-FGFR genes were identified in 43 individuals, with diagnostic yields of 14% and 15% in retrospective and prospective cohorts, respectively. Variants were identified most frequently in TCF12 (N = 22) and EFNB1 (N = 8), typically in individuals with nonsyndromic coronal craniosynostosis or TWIST1-negative clinically suspected Saethre–Chotzen syndrome. Clinically significant variants were also identified in ALX4, EFNA4, ERF, and FGF10. Conclusion : These findings support the clinical utility of a massively parallel sequencing panel for craniosynostosis. TCF12 and EFNB1 should be included in genetic testing for nonsyndromic coronal craniosynostosis or clinically suspected Saethre–Chotzen syndrome.

Craniosynostosis is one of the most common craniofacial disorders encountered in clinical genetics practice, with an overall incidence of 1 in 2,500. Between 30% and 70% of syndromic craniosynostoses are caused by mutations in hotspots in the fibroblast growth factor receptor (FGFR) genes or in the TWIST1 gene with the difference in detection rates likely to be related to different study populations within craniofacial centers. Here we present results from molecular testing of an Australia and New Zealand cohort of 630 individuals with a diagnosis of craniosynostosis. Data were obtained by Sanger sequencing of FGFR1, FGFR2, and FGFR3 hotspot exons and the TWIST1 gene, as well as copy number detection of TWIST1. Of the 630 probands, there were 231 who had one of 80 distinct mutations (36%). Among the 80 mutations, 17 novel sequence variants were detected in three of the four genes screened. In addition to the proband cohort there were 96 individuals who underwent predictive or prenatal testing as part of family studies. Dysmorphic features consistent with the known FGFR1-3/TWIST1-associated syndromes were predictive for mutation detection. We also show a statistically significant association between splice site mutations in FGFR2 and a clinical diagnosis of Pfeiffer syndrome, more severe clinical phenotypes associated with FGFR2 exon 10 versus exon 8 mutations, and more frequent surgical procedures in the presence of a pathogenic mutation. Targeting gene hot spot areas for mutation analysis is a useful strategy to maximize the success of molecular diagnosis for individuals with craniosynostosis.

Contains fulltext : 79485.pdf (Publisher’s version ) (Closed access) Saethre-Chotzen syndrome (SCS) is a rare autosomal dominant syndrome involving craniosynostosis, craniofacial abnormalities, and syndactyly. A recent Scandinavian study reported an increased risk of breast cancer in individuals with a clinical diagnosis of SCS. Because of the potential importance of this finding, we organized a multicenter study enrolling people with TWIST1 mutation confirmed SCS to determine if an increased risk of cancer is present. This study did not identify any cases of breast or ovarian cancer in a cohort of equivalent power to that reported previously. These results provide clinical reassurance that at present there is no evidence for breast cancer screening above standard practice for individuals with SCS.

Contains fulltext : 71384.pdf (Publisher’s version ) (Closed access) We present prenatal and postnatal features of a patient with severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN). Mutation analysis confirmed the clinical diagnosis by detecting the FGFR3 Lys650Met mutation. This case, one of only six with molecular analysis reported in the literature, confirms the severe morbidity and adds to the reports with early mortality associated with SADDAN. Clinical-radiological characteristics of all reported cases of SADDAN are reviewed and discussed.

Recent methodological advances have improved the detection rate for dystrophin mutations, but there are no published studies that have measured the clinical utility of these protocols for carrier detection compared with conventional carrier testing protocols that use pedigree, serum creatine kinase levels and linkage analysis.The clinical utility of a combined mutation detection protocol was measured. It involved quantitative PCR procedures followed by DNA sequence analysis for the identification of dystrophin mutation carriers in 2101 women at risk of being carriers from 348 mutation-known Duchenne or Becker muscular dystrophy pedigrees.The combined mutation detection protocol identified a mutation in 96% and 82% of index cases of Duchenne muscular dystrophy and Becker muscular dystrophy, respectively. An additional 692 (33%) potential carriers were correctly classified by the combined mutation detection protocol compared with pedigree, serum creatine kinase levels and linkage analysis. Significantly lower mutation carrier rates were identified in the mothers of isolated cases with deletion mutations than predicted from theoretical considerations, but these findings were not confirmed for duplication and DNA sequence mutations.There are significant clinical benefits to be gained from a combined mutation detection protocol for carrier detection. It is recommended that mutation-specific carrier frequencies for the different classes of dystrophin mutations should be taken into account in genetic counselling practice.

Contains fulltext : 71488.pdf (Publisher’s version ) (Closed access)

Pathogenic mutations in FGFR2 and TWIST genes are detected in the majority of individuals with Crouzon, Pfeiffer, Apert, and Saethre-Chotzen syndromes. In contrast, mutations have been identified rarely in cases of nonsyndromic, single suture craniosynostosis. Recently, two studies confirming somatic mosaicism with local expression of an FGFR mutation have been reported. This study investigates whether somatic mosaicism could account for nonsyndromic, single suture craniosynostosis. Eight individuals with single suture craniosynostosis who were negative for known mutations in FGFR1-3 and TWIST after screening in their leucocyte DNA were tested for the presence of pathogenic mutations in suture cell-derived DNA. Five had sagittal synostosis, two had metopic synostosis, and the other unicoronal synostosis. Osteoprogenitor cells from surgically excised fusing sutures and an adjacent open suture were cultured. DNA from the cultured cells grown to passage 3 was then examined for underlying FGFR and TWIST mutations. No mutations within the exons of the FGFR or TWIST genes studied were identified in any suture cells. This study found no evidence to support the notion that mosaicism for FGFR or TWIST mutations, normally associated with syndromal forms of craniosynostosis, occur in single suture craniosynostosis. Thus, any underlying genetic defects must occur in regions outside those normally implicated in syndromal craniosynostosis, or this disorder could arise as a consequence of some other epigenetic modification.

We present a case of Beare-Stevenson syndrome with a broad range of phenotypic features including craniosynostosis, cutis gyrata, choanal stenosis, bifid scrotum with perineal hypospadias and a caudal appendage. The paternal age at the time of conception was 62 years consistent with a paternal age effect. Mutation analysis was undertaken and demonstrated the FGFR2 Y375C mutation. This case, one of only nine with molecular analysis, confirms the significant morbidity associated with this syndrome.

The AmplideX FMR1 assay is a commercially produced PCR-based method, capable of detecting expanded and full mutation alleles in fragile X syndrome. The aims of this project were to firstly determine the suitability of this assay for routine diagnostic use and, if appropriate, to implement the test within an Australian genetic laboratory setting. A validation study was undertaken on a total of 91 clinical peripheral blood samples with reference to the ‘gold standard’, fragile X PCR with or without reflex Southern blotting. The assay was found to have both a sensitivity and specificity of 100%. In addition, key parameters including accuracy, precision, robustness and measurement uncertainty were assessed and found to be of an acceptable standard. The assay was subsequently subjected to a NATA accreditation review and incorporated into the standard laboratory workflow. A post-implementation assessment was performed to assess several factors including turn-around-times, cost of test provision and the maintenance of testing quality. This project represents the largest validation study of the Ampli-deX assay within an Australian diagnostic genetic laboratory. The resultant implementation within the NSW reference laboratory may herald a change in the current Australasian paradigm for fragile X syndrome testing.