Comprehensive Genetic Analysis Reveals Complexity of Monogenic Urinary Stone Disease

Abstract

Introduction: Because of phenotypic overlap between monogenic urinary stone diseases (USD), gene-specific analyses can result in missed diagnoses. We used targeted next generation sequencing (tNGS), including known and candidate monogenic USD genes, to analyze suspected primary hyperoxaluria (PH) or Dent disease (DD) patients genetically unresolved (negative; N) after Sanger analysis of the known genes. Cohorts consisted of 285 PH (PHN) and 59 DD (DDN) families.

Methods: Variants were assessed using disease-specific and population databases plus variant assessment tools and categorized using the American College of Medical Genetics (ACMG) guidelines. Prior Sanger analysis identified 47 novel PH or DD gene pathogenic variants.

Results: Screening by tNGS revealed pathogenic variants in 14 known monogenic USD genes, accounting for 45 families (13.1%), 27 biallelic and 18 monoallelic, including 1 family with a copy number variant (CNV). Recurrent genes included the following: SLC34A3 (n = 13), CLDN16 (n = 8), CYP24A1 (n = 4), SLC34A1 (n = 3), SLC4A1 (n = 3), APRT (n = 2), CLDN19 (n = 2), HNF4A1 (n = 2), and KCNJ1 (n = 2), whereas ATP6V1B1, CASR, and SLC12A1 and missed CNVs in the PH genes AGXT and GRHPR accounted for 1 pedigree each. Of the 48 defined pathogenic variants, 27.1% were truncating and 39.6% were novel. Most patients were diagnosed before 18 years of age (76.1%), and 70.3% of biallelic patients were homozygous, mainly from consanguineous families.

Conclusion: Overall, in patients suspected of DD or PH, 23.9% and 7.3% of cases, respectively, were caused by pathogenic variants in other genes. This study shows the value of a tNGS screening approach to increase the diagnosis of monogenic USD, which can optimize therapies and facilitate enrollment in clinical trials.

Keywords: Dent disease; kidney stones; molecular genetics; monogenic; primary hyperoxaluria.

Graphical abstract

Figure 1

Flow chart showing the design of the study for the suspected primary hyperoxaluria (PH) and Dent disease (DD) popuations. (a) The composition of the Sanger-resolved populations and number of PH-negative (PHN) and DD-negative (DDN) patients screened with the targeted next generation sequencing (tNGS) panel are shown. (b) Mutated genes detected from the tNGS of the PHN (left) and DDN (right) populations. (c) An overall summary of the associated genes in the resolved biallelic (left) and monoallelic (right) families.

Figure 2

Examples of genetic results from 5 families. (a) DDN has 3 SLC34A3 variants: c.413C>T (p.Ser138Phe); c.1576_1578del (p.Leu527del); and c.448+1G>A (p.Lys149?). Analysis of data from other families (not shown) and published data indicated that c.413C>T (p.Ser138Phe) and c.1576_1578del (p.Leu527del) are likley on the same allele. Analysis of the tNGS reads showed that c.413C>T (p.Ser138Phe) and c.448+1G>A (p.Lys149?) are on different alleles (left), with the Sanger sequence shown (right), and so this patient has a biallelic genotype. (b) Patient DDN41 also has 3 SLC34A3 variants: c.1453C>T (p.Arg485Cys); c.1454G>A (p.Arg485His); and c.1585A>T (p.Ile529Phe). The conservation of p.Arg485 is shown in multisequence alignment (left), with the phase data from the targeted next generation sequencing (tNGS) reads showing that c.1453C>T (p.Arg485Cys) and c.1585A>T (p.Ile529Phe) are on the same allele and c.1454G>A (p.Arg485His) is on the other allele. (c) Patient DDN39 has 2 SLC34A3 variants, an intronic deletion of 30 bp within IVS5, c.560+23_561-42del (p.Arg187?), plus the missense variant c.1058G>T (p.Arg353Leu). The deletion shown in next generation sequencing (NGS) reads (left) and Sanger sequence (right) leaves a very small intron (65 bp) that may not be excised efficiently. (d) In pedigree PHN2 (left), 3 siblings have end-stage kidney disease (ESKD), and in 2 (where samples were available; PHN2-1 and PHN2-2) the atypical splicing variant c.81-3C>G (p.Asp28?) to APRT was detected in homozygosity, shown by NGS (center) and Sanger sequence (right). This novel variant in IVS1 is predicted to eliminate the splice acceptor site. (e) In PHN20 a CNV deletion was detected with the genes ABCG2 and SPP1 (chr 4q) using the 90-gene panel. Follow-up microarray analysis detected a 18Mb deletion (left) containing 72 genes (right).

Figure 3

Renal imaging of primary hyperoxaluria−negative (PHN) and Dent disease−negative (DDN) cohort depicting the spectrum of renal phenotypes. (a) Abdominal computed tomography (CT) without (w/o) contrast of DDN6 with biallelic SLC34A3 pathogenic variants causing HHRH showing diffuse severe medullary nephrocalcinosis (NC). (b) CT of DDN36 with Bartter syndrome type 2 due to KCNJ1 pathogenic variants showing mild NC plus stones. (c) CT of DDN51 with a monoallelic CYP24A1 pathogenic variant showing tiny calyceal tip stones.