A gene pathogenicity tool "GenePy" identifies missed biallelic diagnoses in the 100,000 Genomes Project.

PURPOSE: The 100,000 Genomes Project diagnosed a quarter of affected participants, but 26% of diagnoses were not on the applied gene panel(s); with many being de novo variants. Assessing biallelic variants without a gene panel is more challenging. METHODS: We sought to identify missed biallelic diagnoses using GenePy, which incorporates allele frequency, zygosity, and a user-defined deleterious metric, generating an aggregate GenePy score per gene, per participant. We calculated GenePy scores for 2862 recessive disease genes in 78,216 100,000 Genomes Project participants. For each gene, we ranked participant GenePy scores and scrutinized affected participants without a diagnosis, whose scores ranked among the top 5 for each gene. In cases which participant phenotypes overlapped with the disease gene of interest, we extracted rare variants and applied phase, ClinVar, and ACMG classification. RESULTS: 3184 affected individuals without a molecular diagnosis had a top-5-ranked GenePy score and 682 of 3184 (21%) had phenotypes overlapping with a top-ranking gene. In 122 of 669 (18%) phenotype-matched cases (excluding 13 withdrawn participants), we identified a putative missed diagnosis (2.2% of all undiagnosed participants). A further 334 of 669 (50%) cases have a possible missed diagnosis but require functional validation. CONCLUSION: Applying GenePy at scale has identified 456 potential diagnoses, demonstrating the value of novel diagnostic strategies.

When is an SNP not an SNP?

Genomic duplications are important sources of structural change and gene innovation. In humans, the most recent and highly identical sequences (>90% homology, >1 kb long) are known as segmental duplications (SDs). Single-nucleotide variants or single-nucleotide polymorphisms within SDs have not been systematically assessed due to limitations around mapping short-read sequencing data. Single-nucleotide variant rs62486260 was flagged in a study of familial renal stone disease but it was unclear whether it was real or an artifact resulting from the presence of a SD. We describe in silico and wet-lab approaches to investigate this, using segment-specific long-PCR assays, followed by short PCR for Sanger sequencing. Our conclusion was that rs62486260 is an artifact. Our approach can be generalized to deal with other such situations.

The method described includes a two-step procedure for determining whether an apparent single-nucleotide polymorphism may be an artifact resulting from the presence of a duplicated genomic region/pseudogene. Step one involves identifying sequence differences between the two duplicated regions and designing a long PCR assay to specifically amplify each region separately. Step 2 involves amplifying a short PCR product which flanks the single-nucleotide polymorphism of interest, from the long products generated in step 1.

A Role for Genetic Modifiers in Tubulointerstitial Kidney Diseases.

With the increased availability of genomic sequencing technologies, the molecular bases for kidney diseases such as nephronophthisis and mitochondrially inherited and autosomal-dominant tubulointerstitial kidney diseases (ADTKD) has become increasingly apparent. These tubulointerstitial kidney diseases (TKD) are monogenic diseases of the tubulointerstitium and result in interstitial fibrosis and tubular atrophy (IF/TA). However, monogenic inheritance alone does not adequately explain the highly variable onset of kidney failure and extra-renal manifestations. Phenotypes vary considerably between individuals harbouring the same pathogenic variant in the same putative monogenic gene, even within families sharing common environmental factors. While the extreme end of the disease spectrum may have dramatic syndromic manifestations typically diagnosed in childhood, many patients present a more subtle phenotype with little to differentiate them from many other common forms of non-proteinuric chronic kidney disease (CKD). This review summarises the expanding repertoire of genes underpinning TKD and their known phenotypic manifestations. Furthermore, we collate the growing evidence for a role of modifier genes and discuss the extent to which these data bridge the historical gap between apparently rare monogenic TKD and polygenic non-proteinuric CKD (excluding polycystic kidney disease).

Hemizygous loss of function mutations in CLCN5 causing end-stage kidney disease without Dent disease phenotype.

Dent disease type 1 is suspected in the presence of a complete phenotype of low molecular weight (LMW) proteinuria, hypercalciuria and at least one of the following: nephrocalcinosis, nephrolithiasis, haematuria, hypophosphatemia or chronic kidney disease (CKD). We present two brothers who presented with CKD alone. In the absence of typical clinical features, further assessment of LMW proteinuria and hypercalciuria was not undertaken. Whole-genome sequencing revealed hemizygous loss of function mutations in chloride voltage-gated channel 5 (CLCN5) consistent with Dent disease. Dent disease should, therefore, be considered in patients with an incomplete phenotype, including unexplained CKD alone.

A genotype-to-phenotype approach suggests under-reporting of single nucleotide variants in nephrocystin-1 (NPHP1) related disease (UK 100,000 Genomes Project).

Autosomal recessive whole gene deletions of nephrocystin-1 (NPHP1) result in abnormal structure and function of the primary cilia. These deletions can result in a tubulointerstitial kidney disease known as nephronophthisis and retinal (Senior-Løken syndrome) and neurological (Joubert syndrome) diseases. Nephronophthisis is a common cause of end-stage kidney disease (ESKD) in children and up to 1% of adult onset ESKD. Single nucleotide variants (SNVs) and small insertions and deletions (Indels) have been less well characterised. We used a gene pathogenicity scoring system (GenePy) and a genotype-to-phenotype approach on individuals recruited to the UK Genomics England (GEL) 100,000 Genomes Project (100kGP) (n = 78,050). This approach identified all participants with NPHP1-related diseases reported by NHS Genomics Medical Centres and an additional eight participants. Extreme NPHP1 gene scores, often underpinned by clear recessive inheritance, were observed in patients from diverse recruitment categories, including cancer, suggesting the possibility of a more widespread disease than previously appreciated. In total, ten participants had homozygous CNV deletions with eight homozygous or compound heterozygous with SNVs. Our data also reveals strong in-silico evidence that approximately 44% of NPHP1 related disease may be due to SNVs with AlphaFold structural modelling evidence for a significant impact on protein structure. This study suggests historical under-reporting of SNVS in NPHP1 related diseases compared with CNVs.

A gene pathogenicity tool 'GenePy' identifies missed biallelic diagnoses in the 100,000 Genomes Project.

The 100,000 Genomes Project (100KGP) diagnosed a quarter of recruited affected participants, but 26% of diagnoses were in genes not on the chosen gene panel(s); with many being de novo variants of high impact. However, assessing biallelic variants without a gene panel is challenging, due to the number of variants requiring scrutiny. We sought to identify potential missed biallelic diagnoses independent of the gene panel applied using GenePy - a whole gene pathogenicity metric. GenePy scores all variants called in a given individual, incorporating allele frequency, zygosity, and a user-defined deleterious metric (CADD v1.6 applied herein). GenePy then combines all variant scores for individual genes, generating an aggregate score per gene, per participant. We calculated GenePy scores for 2862 recessive disease genes in 78,216 individuals in 100KGP. For each gene, we ranked participant GenePy scores for that gene, and scrutinised affected individuals without a diagnosis whose scores ranked amongst the top-5 for each gene. We assessed these participants' phenotypes for overlap with the disease gene associated phenotype for which they were highly ranked. Where phenotypes overlapped, we extracted rare variants in the gene of interest and applied phase, ClinVar and ACMG classification looking for putative causal biallelic variants. 3184 affected individuals without a molecular diagnosis had a top-5 ranked GenePy gene score and 682/3184 (21%) had phenotypes overlapping with one of the top-ranking genes. After removing 13 withdrawn participants, in 122/669 (18%) of the phenotype-matched cases, we identified a putative missed diagnosis in a top-ranked gene supported by phasing, ClinVar and ACMG classification. A further 334/669 (50%) of cases have a possible missed diagnosis but require functional validation. Applying GenePy at scale has identified potential diagnoses for 456/3183 (14%) of undiagnosed participants who had a top-5 ranked GenePy score in a recessive disease gene, whilst adding only 1.2 additional variants (per individual) for assessment.

Exome sequencing identifies a disease variant of the mitochondrial ATP-Mg/Pi carrier SLC25A25 in two families with kidney stones.

BACKGROUND: Calcium kidney stones are common and recurrences are often not preventable by available empiric remedies. Their etiology is multifactorial and polygenic, and an increasing number of genes are implicated. Their identification will enable improved management. METHODS: DNA from three stone-formers in a Southampton family (UK) and two from an Italian family were analyzed independently by whole exome sequencing and selected variants were genotyped across all available members of both pedigrees. A disease variant of SLC25A25 (OMIM 608745), encoding the mitochondrial ATP-Mg/Pi carrier 3 (APC3) was identified, and analyzed structurally and functionally with respect to its calcium-regulated transport activity. RESULTS: All five patients had a heterozygous dominant SLC25A25 variant (rs140777921; GRCh37.p13: chr 9 130868670 G>C; p.Gln349His; Reference Sequence NM_001006641.3). Non-stone formers also carried the variant indicating incomplete penetrance. Modeling suggests that the variant lacks a conserved polar interaction, which may cause structural instability. Calcium-regulated ATP transport was reduced to ~20% of the wild type, showing a large reduction in function. CONCLUSION: The transporter is important in regulating mitochondrial ATP production. This rare variant may increase urine lithogenicity through impaired provision of ATP for solute transport processes in the kidney, and/or for purinergic signaling. Variants found in other genes may compound this abnormality.