A mutation affecting laminin alpha 5 polymerisation gives rise to a syndromic developmental disorder.

Laminin alpha 5 (LAMA5) is a member of a large family of proteins that trimerise and then polymerise to form a central component of all basement membranes. Consequently, the protein plays an instrumental role in shaping the normal development of the kidney, skin, neural tube, lung and limb, and many other organs and tissues. Pathogenic mutations in some laminins have been shown to cause a range of largely syndromic conditions affecting the competency of the basement membranes to which they contribute. We report the identification of a mutation in the polymerisation domain of LAMA5 in a patient with a complex syndromic disease characterised by defects in kidney, craniofacial and limb development, and by a range of other congenital defects. Using CRISPR-generated mouse models and biochemical assays, we demonstrate the pathogenicity of this variant, showing that the change results in a failure of the polymerisation of α/ß/γ laminin trimers. Comparing these in vivo phenotypes with those apparent upon gene deletion in mice provides insights into the specific functional importance of laminin polymerisation during development and tissue homeostasis.

Genomic testing for suspected monogenic kidney disease in children and adults: A health economic evaluation.

PURPOSE: To assess the relative cost-effectiveness of genomic testing compared with standard non-genomic diagnostic investigations in patients with suspected monogenic kidney disease from an Australian health care system perspective. METHODS: Diagnostic and clinical information was used from a national cohort of 349 participants. Simulation modelling captured diagnostic, health, and economic outcomes during a time horizon from clinical presentation until 3 months post-test results based on the outcome of cost per additional diagnosis and lifetime horizon based on cost per quality-adjusted life-year (QALY) gained. RESULTS: Genomic testing was Australian dollars (AU$) 1600 more costly per patient and led to an additional 27 diagnoses out of a 100 individuals tested, resulting in an incremental cost-effectiveness ratio of AU$5991 per additional diagnosis. Using a lifetime horizon, genomic testing resulted in an additional cost of AU$438 and 0.04 QALYs gained per individual compared with standard diagnostic investigations, corresponding to an incremental cost-effectiveness ratio of AU$10,823 per QALY gained. Sub-group analyses identified that the results were largely driven by the cost-effectiveness in glomerular diseases. CONCLUSION: Based on established or expected thresholds of cost-effectiveness, our evidence suggests that genomic testing is very likely to be cost saving for individuals with suspected glomerular diseases, whereas no evidence of cost-effectiveness was found for non-glomerular diseases.

Mitochondrial DNA 13513G>A Mutation Causing Leber Hereditary Optic Neuropathy Associated With Adult-Onset Renal Failure.

BACKGROUND: Leber hereditary optic neuropathy (LHON) is one of the more common mitochondrial diseases and is rarely associated with mitochondrial renal disease. We report 3 unrelated patients with a background of adult-onset renal failure who presented to us with LHON and were shown to have a heteroplasmic mitochondrial DNA mutation (m.13513G>A). METHODS: Retrospective chart review. RESULTS: All 3 patients had a background of chronic renal failure and presented to us with bilateral optic neuropathy (sequential in 2) and were found to have heteroplasmic m.13513G>A mutations in the MT-ND5 gene. Two of the patients were females (aged 30 and 45 years) with chronic kidney disease from their 20s, attributed to pre-eclampsia, one of whom also had diabetes and sudden bilateral hearing loss. One patient was a male (aged 54 years) with chronic kidney disease from his 20s attributed to IgA nephropathy. His mother had diabetes and apparently sudden bilateral blindness in her 70s. Renal biopsy findings were variable and included interstitial fibrosis, acute tubular necrosis, focal segmental glomerulosclerosis, and IgA/C3 tubular casts on immunofluorescence. Mild improvements in vision followed treatment with either idebenone or a combination supplement including coenzyme Q10, alpha-lipoic acid, and B vitamins. CONCLUSIONS: Our cases expand the clinical syndromes associated with m.13513G>A to include bilateral optic neuropathy and adult-onset renal disease. This highlights that in patients with bilateral, especially sequential, optic neuropathy a broad approach to mitochondrial testing is more useful than a limited LHON panel. Mitochondrial diseases present a diagnostic challenge because of their clinical and genetic variability.

Oligohydramnios or Anhydramnios and Ultrasonically Normal Renal Echotexture Secondary to Autosomal Recessive Renal Tubular Dysgenesis: An Important Consideration in the Prenatal Setting.

INTRODUCTION: Autosomal recessive renal tubular dysgenesis (ARRTD) is a rare disorder of renal tubular development. ARRTD is a severe condition with high risk of fetal demise and early neonatal death, with only limited case reports of survival over 2 years [Clin Kidney J. 2012 Feb 1;5(1):56-8]. Prenatal diagnosis of ARRTD is challenging, and diagnosis has only previously been confirmed after postnatal or post-mortem investigation. CASE: To the best of our knowledge, we describe the first reported case of utilizing targeted genetic testing on the chorionic villous sample (CVS) to identify a homozygous variant in the angiotensinogen (AGT) gene. DISCUSSION: By substantiating the diagnosis of ARRTD prenatally, we allow timely and appropriate counseling during pregnancy.

Clinical spectrum, prognosis and estimated prevalence of DNAJB11-kidney disease.

Monoallelic mutations of DNAJB11 were recently described in seven pedigrees with atypical clinical presentations of autosomal dominant polycystic kidney disease. DNAJB11 encodes one of the main cofactors of the endoplasmic reticulum chaperon BiP, a heat-shock protein required for efficient protein folding and trafficking. Here we conducted an international collaborative study to better characterize the DNAJB11-associated phenotype. Thirteen different loss-of-function variants were identified in 20 new pedigrees (54 affected individuals) by targeted next-generation sequencing, whole-exome sequencing or whole-genome sequencing. Amongst the 77 patients (27 pedigrees) now in total reported, 32 reached end stage kidney disease (range, 55-89 years, median age 75); without a significant difference between males and females. While a majority of patients presented with non-enlarged polycystic kidneys, renal cysts were inconsistently identified in patients under age 45. Vascular phenotypes, including intracranial aneurysms, dilatation of the thoracic aorta and dissection of a carotid artery were present in four pedigrees. We accessed Genomics England 100,000 genomes project data, and identified pathogenic variants of DNAJB11 in nine of 3934 probands with various kidney and urinary tract disorders. The clinical diagnosis was cystic kidney disease for eight probands and nephrocalcinosis for one proband. No additional pathogenic variants likely explaining the kidney disease were identified. Using the publicly available GnomAD database, DNAJB11 genetic prevalence was calculated at 0.85/10.000 individuals. Thus, establishing a precise diagnosis in atypical cystic or interstitial kidney disease is crucial, with important implications in terms of follow-up, genetic counseling, prognostic evaluation, therapeutic management, and for selection of living kidney donors.

Population data improves variant interpretation in autosomal dominant polycystic kidney disease.

PURPOSE: Autosomal dominant polycystic kidney disease (ADPKD) is a common adult-onset monogenic disorder, with prevalence of 1/1000. Population databases including ExAC have improved pathogenic variant prioritization in many diseases. Due to pseudogene homology of PKD1, the predominant ADPKD disease gene, and the variable disease severity and age of onset, we aimed to investigate the utility of ExAC for variant assessment in ADPKD. METHODS: We assessed coverage and variant quality in the ExAC cohort and combined allele frequency and age data from the ExAC database (n = 60,706) with curated variants from 2000 ADPKD pedigrees (ADPKD Mutation Database). RESULTS: Seventy-six percent of PKD1 and PKD2 were sequenced adequately for variant discovery and variant quality was high in ExAC. In ExAC, we identified 25 truncating and 393 previously reported disease-causing variants in PKD1 and PKD2, 6.9-fold higher than expected. Fifty-four different variants, previously classified as disease-causing, were observed in ≥5 participants in ExAC. CONCLUSION: Our study demonstrates that many previously implicated disease-causing variants are too common, challenging their pathogenicity, or penetrance. The presence of protein-truncating variants in older participants in ExAC demonstrates the complexity of variant classification and highlights need for further study of prevalence and penetrance of this common monogenic disease.

Increased Diagnostic Yield of Spastic Paraplegia with or Without Cerebellar Ataxia Through Whole-Genome Sequencing.

Inherited disorders of spasticity or ataxia exist on a spectrum with overlapping causative genes and phenotypes. We investigated the use of whole-genome sequencing (WGS) to detect a genetic cause when considering this spectrum of disorders as a single group. We recruited 18 Korean individuals with spastic paraplegia with or without cerebellar ataxia in whom common causes of hereditary cerebellar ataxia and hereditary spastic paraplegia had been excluded. We performed WGS with analysis for single nucleotide variants, small insertions and deletions, copy number variants (CNVs), structural variants (SVs) and intronic variants. Disease-relevant variants were identified in ABCD1 (n = 3), CAPN1 (n = 2), NIPA1 (n = 1) and PLA2G6 (n = 1) for 7/18 patients (38.9%). A 'reverse phenotyping' approach was used to clarify the diagnosis in individuals with PLA2G6 and ABCD1 variants. One of the ABCD1 disease-relevant variants was detected on analysis for intronic variants. No CNV or SV causes were found. The two males with ABCD1 variants were initiated on monitoring for adrenal dysfunction. This is one of only a few studies to analyse spastic-ataxias as a continuous spectrum using a single approach. The outcome was improved diagnosis of unresolved cases for which common genetic causes had been excluded. This includes the detection of ABCD1 variants which had management implications. Therefore, WGS may be particularly relevant to diagnosing spastic ataxias given the large number of genes associated with this condition and the relatively high diagnostic yield.

Renal genetics in Australia: Kidney medicine in the genomic age.

There have been few new therapies for patients with chronic kidney disease in the last decade. However, the management of patients affected by genetic kidney disease is rapidly evolving. Inherited or genetic kidney disease affects around 10% of adults with end-stage kidney disease and up to 70% of children with early onset kidney disease. Advances in next-generation sequencing have enabled rapid and cost-effective sequencing of large amounts of DNA. Next-generation sequencing-based diagnostic tests now enable identification of a monogenic cause in around 20% of patients with early-onset chronic kidney disease. A definitive diagnosis through genomic testing may negate the need for prolonged diagnostic investigations and surveillance, facilitate reproductive planning and provide accurate counselling for at-risk relatives. Genomics has allowed the better understanding of disease pathogenesis, providing prognostic information and facilitating development of targeted treatments for patients with inherited or genetic kidney disease. Although genomic testing is becoming more readily available, there are many challenges to implementation in clinical practice. Multidisciplinary renal genetics clinics serve as a model of how some of these challenges may be overcome. Such clinics are already well established in most parts of Australia, with more to follow in future. With the rapid pace of new technology and gene discovery, collaboration between expert clinicians, laboratory and research scientists is of increasing importance to maximize benefits to patients and health-care systems.

Nemaline myopathy and distal arthrogryposis associated with an autosomal recessive TNNT3 splice variant.

A male neonate presented with severe weakness, hypotonia, contractures and congenital scoliosis. Skeletal muscle specimens showed marked atrophy and degeneration of fast fibers with striking nemaline rods and hypertrophy of slow fibers that were ultrastructurally normal. A neuromuscular gene panel identified a homozygous essential splice variant in TNNT3 (chr11:1956150G > A, NM_006757.3:c.681+1G > A). TNNT3 encodes skeletal troponin-Tfast and is associated with autosomal dominant distal arthrogryposis. TNNT3 has not previously been associated with nemaline myopathy (NM), a rare congenital myopathy linked to defects in proteins associated with thin filament structure and regulation. cDNA studies confirmed pathogenic consequences of the splice variant, eliciting exon-skipping and intron retention events leading to a frameshift. Western blot showed deficiency of troponin-Tfast protein with secondary loss of troponin-Ifast . We establish a homozygous splice variant in TNNT3 as the likely cause of severe congenital NM with distal arthrogryposis, characterized by specific involvement of Type-2 fibers and deficiency of troponin-Tfast .

Massively parallel sequencing and targeted exomes in familial kidney disease can diagnose underlying genetic disorders.

Inherited kidney disease encompasses a broad range of disorders, with both multiple genes contributing to specific phenotypes and single gene defects having multiple clinical presentations. Advances in sequencing capacity may allow a genetic diagnosis for familial renal disease, by testing the increasing number of known causative genes. However, there has been limited translation of research findings of causative genes into clinical settings. Here, we report the results of a national accredited diagnostic genetic service for familial renal disease. An expert multidisciplinary team developed a targeted exomic sequencing approach with ten curated multigene panels (207 genes) and variant assessment individualized to the patient's phenotype. A genetic diagnosis (pathogenic genetic variant[s]) was identified in 58 of 135 families referred in two years. The genetic diagnosis rate was similar between families with a pediatric versus adult proband (46% vs 40%), although significant differences were found in certain panels such as atypical hemolytic uremic syndrome (88% vs 17%). High diagnostic rates were found for Alport syndrome (22 of 27) and tubular disorders (8 of 10), whereas the monogenic diagnostic rate for congenital anomalies of the kidney and urinary tract was one of 13. Quality reporting was aided by a strong clinical renal and genetic multidisciplinary committee review. Importantly, for a diagnostic service, few variants of uncertain significance were found with this targeted, phenotype-based approach. Thus, use of targeted massively parallel sequencing approaches in inherited kidney disease has a significant capacity to diagnose the underlying genetic disorder across most renal phenotypes.

Diabetic ketoacidosis in an adult with beta-ketothiolase deficiency (BKD) involving a novel ACAT1 variant : first report of established diabetes in BKD and a review of the literature.

BACKGROUND: Diabetes presenting in young adults is often challenging to classify. Diabetic ketoacidosis is typically seen in autoimmune type 1 diabetes mellitus and more rarely in young onset type 2 diabetes mellitus. Beta-ketothiolase deficiency (BKD) is a rare autosomal recessive condition affecting isoleucine catabolism and ketone body metabolism. BKD typically manifests in childhood as recurrent episodes of ketoacidosis, the frequency of which tends to reduce with age. There is a paucity of data with respect to the co-existence of persistent dysglycemia with BKD. CASE PRESENTATION AND LITERATURE REVIEW: We present a novel case of diabetes presenting as diabetic ketoacidosis in a 34-year-old man with BKD, with genetically confirmed compound heterozygosity for variants in ACAT1, including a novel ACAT1 c.481T>C, p.(Tyr161His) variant. Diabetes in people with BKD presents unique diagnostic and management challenges. To further contextualize our findings, we conducted a comprehensive narrative review of the existing literature with respect to dysglycemia in those with BKD, especially in adulthood. There are no existing reports describing diabetes in adults with BKD. Stress hyperglycemia is not uncommon when children with BKD are acutely unwell, with several pediatric case reports describing short-lived hyperglycemia but normal HbA1c measurements during metabolic crises (indicating the absence of persistent hyperglycemia). CONCLUSIONS: This is the first report of diabetic ketoacidosis in an adult with BKD, with an elevated HbA1c consistent with persistent hyperglycemia. This case highlights the importance of checking HbA1c in people with BKD and hyperglycemia in order to uncover potential coexisting diabetes, facilitating timely management and preventing complications. Increased reporting on the longitudinal outcomes of those with rare metabolic disorders is essential for identifying potential associations with conditions like diabetes.

Atypical splicing variants in PKD1 explain most undiagnosed typical familial ADPKD.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic cause of kidney failure and is primarily associated with PKD1 or PKD2. Approximately 10% of patients remain undiagnosed after standard genetic testing. We aimed to utilise short and long-read genome sequencing and RNA studies to investigate undiagnosed families. Patients with typical ADPKD phenotype and undiagnosed after genetic diagnostics were recruited. Probands underwent short-read genome sequencing, PKD1 and PKD2 coding and non-coding analyses and then genome-wide analysis. Targeted RNA studies investigated variants suspected to impact splicing. Those undiagnosed then underwent Oxford Nanopore Technologies long-read genome sequencing. From over 172 probands, 9 met inclusion criteria and consented. A genetic diagnosis was made in 8 of 9 (89%) families undiagnosed on prior genetic testing. Six had variants impacting splicing, five in non-coding regions of PKD1. Short-read genome sequencing identified novel branchpoint, AG-exclusion zone and missense variants generating cryptic splice sites and a deletion causing critical intron shortening. Long-read sequencing confirmed the diagnosis in one family. Most undiagnosed families with typical ADPKD have splice-impacting variants in PKD1. We describe a pragmatic method for diagnostic laboratories to assess PKD1 and PKD2 non-coding regions and validate suspected splicing variants through targeted RNA studies.

Introme accurately predicts the impact of coding and noncoding variants on gene splicing, with clinical applications.

Predicting the impact of coding and noncoding variants on splicing is challenging, particularly in non-canonical splice sites, leading to missed diagnoses in patients. Existing splice prediction tools are complementary but knowing which to use for each splicing context remains difficult. Here, we describe Introme, which uses machine learning to integrate predictions from several splice detection tools, additional splicing rules, and gene architecture features to comprehensively evaluate the likelihood of a variant impacting splicing. Through extensive benchmarking across 21,000 splice-altering variants, Introme outperformed all tools (auPRC: 0.98) for the detection of clinically significant splice variants. Introme is available at https://github.com/CCICB/introme .

Participant Choice towards Receiving Potential Additional Findings in an Australian Nephrology Research Genomics Study.

The choices of participants in nephrology research genomics studies about receiving additional findings (AFs) are unclear as are participant factors that might influence those choices. Methods: Participant choices and factors potentially impacting decisions about AFs were examined in an Australian study applying research genomic testing following uninformative diagnostic genetic testing for suspected monogenic kidney disease. Results: 93% of participants (195/210) chose to receive potential AFs. There were no statistically significant differences between those consenting to receive AFs or not in terms of gender (p = 0.97), median age (p = 0.56), being personally affected by the inherited kidney disease of interest (p = 0.38), or by the inheritance pattern (p = 0.12-0.19). Participants were more likely to choose not to receive AFs if the family proband presented in adulthood (p = 0.01), if there was family history of another genetic disorder (p = 0.01), and where the consent process was undertaken by an adult nephrologist (p = 0.01). Conclusion: The majority of participants in this nephrology research genomics study chose to receive potential AFs. Younger age of the family proband, family history of an alternate genetic disorder, and consenting by some multidisciplinary team members might impact upon participant choices.

The HIDDEN Protocol: An Australian Prospective Cohort Study to Determine the Utility of Whole Genome Sequencing in Kidney Failure of Unknown Aetiology.

Early identification of genetic kidney disease allows personalised management, clarification of risk for relatives, and guidance for family planning. Genetic disease is underdiagnosed, and recognition of genetic disease is particularly challenging in patients with kidney failure without distinguishing diagnostic features. To address this challenge, the primary aim of this study is to determine the proportion of genetic diagnoses amongst patients with kidney failure of unknown aetiology, using whole genome sequencing (WGS). A cohort of up to 100 Australian patients with kidney failure of unknown aetiology, with onset <50 years old and approved by a panel of study investigators will be recruited via 18 centres nationally. Clinically accredited WGS will be undertaken with analysis targeted to a priority list of ∼388 genes associated with genetic kidney disease. The primary outcome will be the proportion of patients who receive a molecular diagnosis (diagnostic rate) via WGS compared with usual -care (no further diagnostic investigation). Participant surveys will be undertaken at consent, after test result return and 1 year subsequently. Where there is no or an uncertain diagnosis, future research genomics will be considered to identify candidate genes and new pathogenic variants in known genes. All results will be relayed to participants via the recruiting clinician and/or kidney genetics clinic. The study is ethically approved (HREC/16/MH/251) with local site governance approvals in place. The future results of this study will be disseminated and inform practical understanding of the potential monogenic contribution to kidney failure of unknown aetiology. These findings are anticipated to impact clinical practice and healthcare policy. Study Registration: [https://dora.health.qld.gov.au], identifier [HREC/16/MH/251].

Use of Whole-Genome Sequencing for Mitochondrial Disease Diagnosis.

BACKGROUND AND OBJECTIVES: Mitochondrial diseases (MDs) are the commonest group of heritable metabolic disorders. Phenotypic diversity can make molecular diagnosis challenging, and causative genetic variants may reside in either mitochondrial or nuclear DNA. A single comprehensive genetic diagnostic test would be highly useful and transform the field. We applied whole-genome sequencing (WGS) to evaluate the variant detection rate and diagnostic capacity of this technology with a view to simplifying and improving the MD diagnostic pathway. METHODS: Adult patients presenting to a specialist MD clinic in Sydney, Australia, were recruited to the study if they satisfied clinical MD (Nijmegen) criteria. WGS was performed on blood DNA, followed by clinical genetic analysis for known pathogenic MD-associated variants and MD mimics. RESULTS: Of the 242 consecutive patients recruited, 62 participants had "definite," 108 had "probable," and 72 had "possible" MD classification by the Nijmegen criteria. Disease-causing variants were identified for 130 participants, regardless of the location of the causative genetic variants, giving an overall diagnostic rate of 53.7% (130 of 242). Identification of causative genetic variants informed precise treatment, restored reproductive confidence, and optimized clinical management of MD. DISCUSSION: Comprehensive bigenomic sequencing accurately detects causative genetic variants in affected MD patients, simplifying diagnosis, enabling early treatment, and informing the risk of genetic transmission.

Attitudes and Practices of Australian Nephrologists Toward Implementation of Clinical Genomics.

INTRODUCTION: Genomic testing is becoming widely available as a diagnostic tool, although widespread implementation is not yet established in nephrology. METHODS: An anonymous electronic survey was administered to investigate experience and confidence with genomic tests, perceived clinical utility of genomic services, preferences for service delivery models, and readiness for implementation among nephrologists. Questions were guided by a comprehensive literature review and published tools, including a validated theoretical framework for implementation of genomic medicine: Consolidated Framework for Implementation Research (CFIR). RESULTS: Responses were received from 224 clinicians, of which 172 were eligible for analysis. Most clinicians (132 [76%]) had referred at least one patient to a genetics clinic. Despite most clinicians (136 [85%]) indicating that they believed genetic testing would be useful, only 39 (23%) indicated they felt confident to use results of genomic testing, with pediatric clinicians feeling more confident compared with adult clinicians (12 of 20 [60%] vs. 27 of 149 [18%]), P < 0.01, Fisher exact). A multidisciplinary renal genetics clinic was the preferred model among clinicians surveyed (98 of 172 [57%]). A key implementation barrier highlighted related to the hospital or organizational culture and/or environment. Specific barriers noted in quantitative and qualitative responses included inadequate staffing, learning resources, and funding. CONCLUSIONS: Our findings suggest support for genomic testing among nephrologists, with a strong preference for a multidisciplinary model (involving a nephrologist, clinical geneticist, and genetic counselor). Broad-ranging interventions are urgently required to shift the current culture and ensure successful implementation of genomics in nephrology, including reducing knowledge gaps, increased funding and resources, disease-specific guidelines, and streamlining of testing processes.