Long-read sequencing identifies a common transposition haplotype predisposing for CLCNKB deletions

doi:10.1186/s13073-023-01215-1

. 2023 Aug 23;15(1):62.

doi: 10.1186/s13073-023-01215-1.

Long-read sequencing identifies a common transposition haplotype predisposing for CLCNKB deletions

Nikolai Tschernoster^{1

2

3}, Florian Erger^{2

3}, Stefan Kohl⁴, Björn Reusch^{2

3}, Andrea Wenzel^{2

3}, Stephen Walsh⁵, Holger Thiele¹, Christian Becker¹, Marek Franitza¹, Malte P Bartram^{3

6}, Martin Kömhoff⁷, Lena Schumacher⁸, Christian Kukat⁸, Tatiana Borodina⁹, Claudia Quedenau⁹, Peter Nürnberg^{1

3}, Markus M Rinschen^{10

11

12}, Jan H Driller¹³, Bjørn P Pedersen¹³, Karl P Schlingmann¹⁴, Bruno Hüttel¹⁵, Detlef Bockenhauer^{5

16}, Bodo Beck^#^{17

18}, Janine Altmüller^#^{19

20

21}

Affiliations

¹ Cologne Center for Genomics (CCG), University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany.
² Institute of Human Genetics, Faculty of Medicine and University Hospital Cologne, University of Cologne, Kerpener Str. 34, 50931, Cologne, Germany.
³ Center for Molecular Medicine Cologne (CMMC), University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany.
⁴ Department of Pediatrics, Cologne Children's Hospital, Cologne, Germany.
⁵ Department of Renal Medicine, UCL, University College London, London, UK.
⁶ Department II of Internal Medicine, University of Cologne, Cologne, Germany.
⁷ Department of Pediatrics, University Marburg, Marburg, Germany.
⁸ FACS & Imaging Core Facility, Max Planck Institute for Biology of Ageing, Cologne, Germany.
⁹ Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Hannoversche Straße 28, 10115, Berlin, Germany.
¹⁰ Department of Biomedicine, Aarhus University, Aarhus, Denmark.
¹¹ Aarhus Institute of Advanced Studies, Aarhus University, Aarhus, Denmark.
¹² Department III of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
¹³ Department of Molecular Biology and Genetics, Aarhus University, Universitetsbyen 81, DK-8000, Aarhus C, Denmark.
¹⁴ Department of General Pediatrics, University Children's Hospital, Münster, Germany.
¹⁵ Max Planck Genome-Centre Cologne, Max Planck Institute for Plant Breeding Research, Cologne, Germany.
¹⁶ Great Ormond Street Hospital for Children, NHS Foundation Trust, London, UK.
¹⁷ Institute of Human Genetics, Faculty of Medicine and University Hospital Cologne, University of Cologne, Kerpener Str. 34, 50931, Cologne, Germany. [email protected].
¹⁸ Center for Molecular Medicine Cologne (CMMC), University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany. [email protected].
¹⁹ Center for Molecular Medicine Cologne (CMMC), University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany. [email protected].
²⁰ Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Hannoversche Straße 28, 10115, Berlin, Germany. [email protected].
²¹ Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Core Facility Genomics, Berlin, Germany. [email protected].

^# Contributed equally.

PMID: 37612755
PMCID: PMC10464140
DOI: 10.1186/s13073-023-01215-1

Long-read sequencing identifies a common transposition haplotype predisposing for CLCNKB deletions

Nikolai Tschernoster et al. Genome Med. 2023.

. 2023 Aug 23;15(1):62.

doi: 10.1186/s13073-023-01215-1.

Authors

Affiliations

¹ Cologne Center for Genomics (CCG), University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany.
² Institute of Human Genetics, Faculty of Medicine and University Hospital Cologne, University of Cologne, Kerpener Str. 34, 50931, Cologne, Germany.
³ Center for Molecular Medicine Cologne (CMMC), University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany.
⁴ Department of Pediatrics, Cologne Children's Hospital, Cologne, Germany.
⁵ Department of Renal Medicine, UCL, University College London, London, UK.
⁶ Department II of Internal Medicine, University of Cologne, Cologne, Germany.
⁷ Department of Pediatrics, University Marburg, Marburg, Germany.
⁸ FACS & Imaging Core Facility, Max Planck Institute for Biology of Ageing, Cologne, Germany.
⁹ Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Hannoversche Straße 28, 10115, Berlin, Germany.
¹⁰ Department of Biomedicine, Aarhus University, Aarhus, Denmark.
¹¹ Aarhus Institute of Advanced Studies, Aarhus University, Aarhus, Denmark.
¹² Department III of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
¹³ Department of Molecular Biology and Genetics, Aarhus University, Universitetsbyen 81, DK-8000, Aarhus C, Denmark.
¹⁴ Department of General Pediatrics, University Children's Hospital, Münster, Germany.
¹⁵ Max Planck Genome-Centre Cologne, Max Planck Institute for Plant Breeding Research, Cologne, Germany.
¹⁶ Great Ormond Street Hospital for Children, NHS Foundation Trust, London, UK.
¹⁷ Institute of Human Genetics, Faculty of Medicine and University Hospital Cologne, University of Cologne, Kerpener Str. 34, 50931, Cologne, Germany. [email protected].
¹⁸ Center for Molecular Medicine Cologne (CMMC), University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany. [email protected].
¹⁹ Center for Molecular Medicine Cologne (CMMC), University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany. [email protected].
²⁰ Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Hannoversche Straße 28, 10115, Berlin, Germany. [email protected].
²¹ Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Core Facility Genomics, Berlin, Germany. [email protected].

^# Contributed equally.

PMID: 37612755
PMCID: PMC10464140
DOI: 10.1186/s13073-023-01215-1

Abstract

Background: Long-read sequencing is increasingly used to uncover structural variants in the human genome, both functionally neutral and deleterious. Structural variants occur more frequently in regions with a high homology or repetitive segments, and one rearrangement may predispose to additional events. Bartter syndrome type 3 (BS 3) is a monogenic tubulopathy caused by deleterious variants in the chloride channel gene CLCNKB, a high proportion of these being large gene deletions. Multiplex ligation-dependent probe amplification, the current diagnostic gold standard for this type of mutation, will indicate a simple homozygous gene deletion in biallelic deletion carriers. However, since the phenotypic spectrum of BS 3 is broad even among biallelic deletion carriers, we undertook a more detailed analysis of precise breakpoint regions and genomic structure.

Methods: Structural variants in 32 BS 3 patients from 29 families and one BS4b patient with CLCNKB deletions were investigated using long-read and synthetic long-read sequencing, as well as targeted long-read sequencing approaches.

Results: We report a ~3 kb duplication of 3'-UTR CLCNKB material transposed to the corresponding locus of the neighbouring CLCNKA gene, also found on ~50 % of alleles in healthy control individuals. This previously unknown common haplotype is significantly enriched in our cohort of patients with CLCNKB deletions (45 of 51 alleles with haplotype information, 2.2 kb and 3.0 kb transposition taken together, p=9.16×10^-9). Breakpoint coordinates for the CLCNKB deletion were identifiable in 28 patients, with three being compound heterozygous. In total, eight different alleles were found, one of them a complex rearrangement with three breakpoint regions. Two patients had different CLCNKA/CLCNKB hybrid genes encoding a predicted CLCNKA/CLCNKB hybrid protein with likely residual function.

Conclusions: The presence of multiple different deletion alleles in our cohort suggests that large CLCNKB gene deletions originated from many independently recurring genomic events clustered in a few hot spots. The uncovered associated sequence transposition haplotype apparently predisposes to these additional events. The spectrum of CLCNKB deletion alleles is broader than expected and likely still incomplete, but represents an obvious candidate for future genotype/phenotype association studies. We suggest a sensitive and cost-efficient approach, consisting of indirect sequence capture and long-read sequencing, to analyse disease-relevant structural variant hotspots in general.

Keywords: Bartter syndrome type 3; CLCNKA; CLCNKB; HiFi-sequencing; Long-read sequencing; Next-generation sequencing; Risk haplotype; Salt-wasting tubulopathy; Structural variant; Target enrichment.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

**Fig. 1**
Clinical findings and pedigree in severely affected family P1. A Pedigree of family P1. B Estimated glomerular filtration rate (eGFR) in all brothers over a 15 years’ timeline. Note that eGFR declines over age in all brothers. C–E Discordant kidney sonograms. C Increased echogenicity, nephrocalcinosis, and reduced cortico-medullary differentiation. D Increased echogenicity, nephrocalcinosis, and multiple large cysts. E Mildly increased echogenicity. F Histologic slide of kidney biopsy showing unspecific focal segmental glomerulosclerosis in P1.1. G–I Destructive gouty arthritis and multiple gout tophi in P1.1

**Fig. 2**
Results of the WGS and linked-read WGS analysis from patient P1.2. A Varbank2 implemented conifer CNV analysis tool showing results from the WGS dataset. Z-scores from patient P1.2 in cyan-coloured bars and also as extrapolated curve. Affected region is indicated by the black horizontal bar. Genes in this region are indicated by purple horizontal bars. The markers (probes throughout the exons) are evenly distributed among the positional X-axis. The black curves represent the control collective, consisting of a fixed set of samples for the given enrichment kit. B Linked-read Loupe browser v.2.1.1 haplotype resolved SV results for patient P1.2 compared to a control sample (matrix view). Genomic region chr1:16.023.378-16.077.660 (hg38) is displayed on y- and x-axis for both alleles, respectively. Number of reads is indicated by green vertical bars. Sequence coverage (barcode overlap) is indicated by the heat map ranging from zero coverage (white) to high coverage (black). C Xdrop indirect sequence capture and ONT long-read sequencing results for patient P1.2 [26]. Gene-specific nucleotides are indicated by red background; breakpoint regions are indicated by grey background. Long stretches of homologous genomic DNA sequence have been shortened, indicated by […]. D Reconstruction of the genomic structural rearrangement of patient P1.2. The three breakpoint regions result in a rearrangement of fragment B and fragment C and the deletion of the genomic DNA in between these fragments. Genomic DNA Fragments were rearranged according to the identified breakpoints. New genomic rearrangement consisting of fragment A-C-B-D. The common sequence transposition haplotype equates to the transposition of fragment C. Genes are indicated in blue with exons as blue vertical bars. Gene orientation is indicated by blue arrows. Colour coding of genomic fragments A (green), B (orange), C (blue), and D (red). Genomic sequence coordinates refer to the hg38 human reference. † Linked-read control carries the *CLCNKA* 3′ UTR sequence transposition haplotype heterozygously

**Fig. 3**
Genomic *CLCNKA/CLCNKB* locus and the common *CLCNKA* 3′ UTR sequence transposition haplotype on chromosome 1p36.13. The genomic environment surrounding the *CLCNKA/CLCNKB* gene locus contains two highly homologous genomic regions (A and A’, sequence homology approx. 80 %). A and A’ are indicated by the yellow and orange boxes, respectively. The homologous region A stretches over 20.157 bp from chr1:16,346,920-16,367,077 (GRCh37/Hg19), region A’ stretches over 20.827 bp from chr1:16,368,759-16,389,585 (GRCh37/Hg19). The exonic sequence homology of *CLCNKA* and *CLCNKB* is 94% [8]. In the alternative transposition haplotype, a 2.2–3-kb large DNA fragment of the *CLCNKA* 3′ UTR (small orange box, arrow head) is replaced by the homologous fragment from the 3′ UTR of *CLCNKB* (small orange box, arrow tail), resulting in the loss of the genomic material downstream of *CLCNKA* and an extra copy of the DNA fragment duplicated from the *CLCNKB* 3′ UTR. Gene length and orientation of *CLCNKA* and *CLCNKB* are indicated by the grey arrows. Exons are indicated as grey bars

**Fig. 4**
Summary of the *CLCNKB* deletion alleles identified in this study. Breakpoint regions are indicated by coloured vertical lines. Corresponding breakpoint regions are indicated by coloured arrows. Identical breakpoint regions are coloured in the same colour. Two common *CLCNKA* 3′ UTR sequence transpositions identified in this study are indicated by the orange box within the yellow box of *CLCNKA*. Breakpoint region coordinates of the 2.2 kb and 3 kb sized sequence transpositions are listed in Additional file 1: Table S3. Exons are indicated as vertical bars. Orientation and length of *CLCNKA* and *CLCNKB* are indicated by grey arrows. The gene *FAM131C*, located on the negative strand, is indicated by the grey-hatched arrow and is only partially visualized. *FAM131C* exons are indicated by dotted lines. A Deletion allele A is derived from the reference haplotype. Deletion alleles B, C, and D are derived from the smaller 2.2 kb sized sequence transposition haplotype. Deletion alleles E and F are derived from the larger 3 kb sized sequence transposition haplotype. Adjacent breakpoint region coordinates that discriminate the deletion alleles E and F are listed in Additional file 1: Table S3. All patients show the corresponding deletion allele in a homozygous state if not indicated otherwise. B Deletion alleles G and H that result in *CLCNKA*/*CLCNKB* hybrid genes. Since the haplotype-defining genomic sequence is deleted on these alleles, a haplotype determination could not be made. † Patients, compound heterozygous for the deletion alleles B and F; ‡ Heterozygous deletion allele. This patient carries a 5 bp deletion in exon 9 *in trans*

**Fig. 5**
Workup of the ClC-Ka/ClC-Kb hybrid genes in patients P6 and P11. ClC-Ka AA-sequence is indicated in light grey; ClC-Kb AA-sequence is indicated in dark grey; AA changes between wildtype ClC-Ka and ClC-Ka/ClC-Kb hybrid protein are annotated in the corresponding exon. AA changes analysed in detail in Figure 6 are shown in red. A Hybrid gene in patient P11. Breakpoint region in intron 7/8. Genomic sequence at the border between exon 7 and exon 8 divided into triplets and the corresponding AA are shown. Ser218 is the last AA encoded by exon 7 before the breakpoint region. B Hybrid gene in patient P6. Breakpoint region in intron 15/16. Genomic sequence at the border between exon 15 and exon 16 divided into triplets and the corresponding AA are shown. I540 is the last AA encoded by exon 15 before the breakpoint region. AA G541 is encoded by the last two nucleotides of exon 15 and the first nucleotide of exon 16. In the hybrid gene, the first nucleotide in exon 16 encoded by *CLCNKB* has changed from C>T but the corresponding AA G541 remains unchanged in the hybrid protein

**Fig. 6**
Predicted ClC-Ka/ClC-Kb hybrid proteins in patients P6 and P11. A AlphaFold2 prediction of the ClC-Ka1-218b219-687 hybrid homodimer (P11). B AlphaFold2 prediction of the ClC-Ka1-540b541-687 hybrid homodimer (P6). C Cryo-EM structure of the ClC-K homodimer from Bos taurus (PDB ID: 5TQQ, 84,3% seq ID to ClC-Ka and ClC-Kb). D View on the membrane region of ClC-Ka (teal) and ClC-Kb (pastel red) with the amino acid variations shown in sticks shows no significant changes in the ion tunnel between the variants ClC-Ka and ClC-Kb. E Putative adenosine nucleotide binding site in ClC-Ka and ClC-Kb superimposed with an ATP bound in ClC-7 structure (Protein data base: 7jm7). F Interface between the cytosolic CBS domains of the ClC-Ka and ClC-Kb proteins. G Dimerization interface of the CBS domains in the ClC-Ka and ClC-Kb shows distinct differences

See this image and copyright information in PMC

References

1. Bartter FC, Pronove P, Gill JR JR, Maccardle RC. Hyperplasia of the juxtaglomerular complex with hyperaldosteronism and hypokalemic alkalosis. A new syndrome. Am J Med. 1962;33:811–28. doi: 10.1016/0002-9343(62)90214-0. - DOI - PubMed
1. Kleta R, Bockenhauer D. Salt-losing tubulopathies in children: what's new, what's controversial? J Am Soc Nephrol. 2018;29:727–39. doi: 10.1681/ASN.2017060600. - DOI - PMC - PubMed
1. Rodríguez-Soriano J. Bartter and related syndromes: the puzzle is almost solved. Pediatr Nephrol. 1998;12:315–27. doi: 10.1007/s004670050461. - DOI - PubMed
1. Gitelman HJ, Graham JB, Welt LG. A new familial disorder characterized by hypokalemia and hypomagnesemia. Trans Assoc Am Physicians. 1966;79:221–35. - PubMed
1. Kondo A, Nagano C, Ishiko S, Omori T, Aoto Y, Rossanti R, et al. Examination of the predicted prevalence of Gitelman syndrome by ethnicity based on genome databases. Sci Rep. 2021;11:16099. doi: 10.1038/s41598-021-95521-6. - DOI - PMC - PubMed

Publication types

Actions

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions

Substances

Actions
Actions

Supplementary concepts

Actions

LinkOut - more resources

Full Text Sources

[1] Bartter FC, Pronove P, Gill JR JR, Maccardle RC. Hyperplasia of the juxtaglomerular complex with hyperaldosteronism and hypokalemic alkalosis. A new syndrome. Am J Med. 1962;33:811–28. doi: 10.1016/0002-9343(62)90214-0. - DOI - PubMed

[2] Bartter FC, Pronove P, Gill JR JR, Maccardle RC. Hyperplasia of the juxtaglomerular complex with hyperaldosteronism and hypokalemic alkalosis. A new syndrome. Am J Med. 1962;33:811–28. doi: 10.1016/0002-9343(62)90214-0. - DOI - PubMed

[3] Kleta R, Bockenhauer D. Salt-losing tubulopathies in children: what's new, what's controversial? J Am Soc Nephrol. 2018;29:727–39. doi: 10.1681/ASN.2017060600. - DOI - PMC - PubMed

[4] Kleta R, Bockenhauer D. Salt-losing tubulopathies in children: what's new, what's controversial? J Am Soc Nephrol. 2018;29:727–39. doi: 10.1681/ASN.2017060600. - DOI - PMC - PubMed

[5] Rodríguez-Soriano J. Bartter and related syndromes: the puzzle is almost solved. Pediatr Nephrol. 1998;12:315–27. doi: 10.1007/s004670050461. - DOI - PubMed

[6] Rodríguez-Soriano J. Bartter and related syndromes: the puzzle is almost solved. Pediatr Nephrol. 1998;12:315–27. doi: 10.1007/s004670050461. - DOI - PubMed

[7] Gitelman HJ, Graham JB, Welt LG. A new familial disorder characterized by hypokalemia and hypomagnesemia. Trans Assoc Am Physicians. 1966;79:221–35. - PubMed

[8] Gitelman HJ, Graham JB, Welt LG. A new familial disorder characterized by hypokalemia and hypomagnesemia. Trans Assoc Am Physicians. 1966;79:221–35. - PubMed

[9] Kondo A, Nagano C, Ishiko S, Omori T, Aoto Y, Rossanti R, et al. Examination of the predicted prevalence of Gitelman syndrome by ethnicity based on genome databases. Sci Rep. 2021;11:16099. doi: 10.1038/s41598-021-95521-6. - DOI - PMC - PubMed

[10] Kondo A, Nagano C, Ishiko S, Omori T, Aoto Y, Rossanti R, et al. Examination of the predicted prevalence of Gitelman syndrome by ethnicity based on genome databases. Sci Rep. 2021;11:16099. doi: 10.1038/s41598-021-95521-6. - DOI - PMC - PubMed

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

Long-read sequencing identifies a common transposition haplotype predisposing for CLCNKB deletions

Affiliations

Long-read sequencing identifies a common transposition haplotype predisposing for CLCNKB deletions

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

Similar articles

References

Publication types

MeSH terms

Substances

Supplementary concepts

LinkOut - more resources

Full Text Sources

Abstract

Conflict of interest statement

Figures

Similar articles

References

Publication types

MeSH terms

Substances

Supplementary concepts

Related information

LinkOut - more resources

Full Text Sources