Genetic testing (Next Generation Sequencing)—A Sherlock Holmes in discovering the truth of ADPKD

Written & infographics by: Dr. Y. Saivani (@DrSaiVani1, @drsaivani.bsky.social)

Consultant, Kurnool Kidney Care, Kurnool, India

“There is nothing more deceptive than an obvious fact.”

― Arthur Conan Doyle, The Boscombe Valley Mystery—a Sherlock Holmes Short Story

Genetic testing underwent a sea of change after the introduction of Next Generation Sequencing (NGS), which unleashed its potential in solving many controversial diagnoses and genetic disorders. However, unraveling its use in autosomal dominant polycystic kidney disease (ADPKD) is complex. Though the disease onset is at birth, the disease culminates in kidney failure typically in the late 5th decade in PKD1 and in the 7th decade in PKD2 (KDIGO Guidelines 2025). ADPKD is a common monogenic genetic disorder but often goes without genetic testing for diagnosis, with many opting for a clinico-radiological diagnosis (with ultrasound and family history). ADPKD is associated with a point population prevalence of 3.96 per 10,000 people, accounting for 5-10% of the ESRD population, and is the 4th leading cause of CKD. Genetic testing in ADPKD is typically saved for special situations and atypical presentations, whereby its clinical role remains most useful as an adjunct in diagnosis as well as providing prognosis and family planning support. 

"To a great mind, nothing is little."

The major genes involved in ADPKD are PKD1 and PKD2, which account for 93%, and other minor genes account for 6–11%. Among the major genes, PKD1 and PKD2 account for  85% and 15%, respectively. The minor genes, in ascending order of severity of renal disease, are  IFT140, GANA B, ALG9, ALG5, DNAJB11, NEK8, and six autosomal dominant polycystic liver disease (ADPLD) genes: ALG8, SEC61B, SEC63, PRKCSH, and LRP5. ADPLD may be present without severe renal disease.

Below is a list of other syndromes that present with kidney cysts and are included in the differential diagnosis for ADPKD.

1. Alport syndrome (COL4A1, COL4A3, COL4A4, COL4A5),

2. Autosomal Dominant Tubulointerstitial Disease (ADTID—MUC1, REN, UMOD, SEC61A1)

3. Tuberous sclerosis (TSC1,2)

4. Von Hippel-Lindau syndrome (VHL)

5. Nephronophthisis (NPHP1-6) 

6. Bardet-Biedl syndrome (BBS1-12) 

7. HNF1B-related disease (HNF1B)

The PKD1 gene is localized on chromosome 16p13.3 with 46 exons. Hence, it is difficult to quantify. PKD1 produces polycystin-1, a large integral protein of unknown function. The PKD2 gene is localized on 4q 21-22, a 15-exon single-copy gene producing polycystin-2, which is a transient receptor potential channel protein. Both polycystin-1 and polycystin-2 proteins regulate cellular functions like proliferation, apoptosis, and fluid secretion. There are many mutations in PKD1 and PKD2 around >1250 and 200, which supports the allelic heterogeneity of the disease. Depending on the type of genes involved, patients are labeled as ADPKD PKD1, ADPKD PKD2, or ADPKD 1FT140 by the KDIGO 2025 guidelines. 

"Life is infinitely stranger than anything that the mind of man could invent.”

- Arthur Conan Doyle, 'A Case Of Identity.'

The phenotypic heterogeneity of ADPKD depends on the type of mutation. Mutations presenting at an earlier age are truncated (PT) (nonsense and frameshift mutations, removing canonical splice sites) and large. Gene alterations presenting at a later age are nontruncated (NT) inframe deletions and insertions (indels) and non-synonymous missense mutations. Occasionally, truncated mutations can present at a later age. The PROPKD score has emerged as a helpful prognostic tool that incorporates these genetic factors and disease risk factors like hypertension, urological complications, and sex, assisting in better prediction of disease outcomes.

Indications for genetic testing:

There are two situations where the genetic testing is clinically indicated and  another group where genetic testing role is evolving.

Clinically Indicated Genetic Testing:

1. No obvious family history with clinical manifestations similar to ADPKD

2. suspected ADPKD with equivocal imaging findings

3. To exclude the diagnosis in a prospective kidney donor in the family, especially if radiological imaging is negative

4. To exclude the disease in prenatal and preimplantation diagnosis 

Preimplantation genetic testing—monogenic disorders (PGT-M)—is a sophisticated method where intracytoplasmic sperm injection (ICSI) is performed on the ovum on day three at the blastocyst stage, whereby 5-10 trophectoderm cells are taken and sent for genetic testing, identifying ADPKD-negative embryos.

In prenatal genetic testing, noninvasive and invasive methods exist.

Noninvasive: Less accurate, performed at 6-7 weeks gestation, and associated with no risks.

Invasive: Methods include chorionic villus sampling, performed at 11-14 weeks gestation, and amniocentesis, performed at 15-16 weeks gestation. Both tests are considered >99% accurate, but they are associated with a risk of miscarriage of <0.5%.

Evolving role of genetic testing in ADPKD

• Risk stratification and initiation of disease-modifying therapies like vaptans

• Delineating the cause of atypical presentations like asymmetric kidneys, syndromic presentations, intrafamilial discordance, and suspicion of somatic mosaicism.

Heterogeneity in clinical presentation is not merely explained by the type of mutation but also the complexity of genes.

Scenarios impacting clinical phenotype:

1.  Severe and early onset of the disease—an infant or neonate presenting with the enlarged cystic kidneys reaching ESRD by teenage

   1. Due to contiguous deletion of genes PKD1 and TSC2 resulting in ADPKD by teenage

   2. Biallelic gene mutations—in one gene PKDPT (protein truncating mutation), and in another gene, PKDNT (non-truncating mutation) abnormality resulting in ADPKD at early age 

   3. Digenic—Along with PKD1, presence of another mutation like another syndromic gene or PKD2

   4. Presence of bilineal disease—marked discordance in family  If one of the parents has ADPKD PKDPT and another has ADPKD PKDNT, and if the children are more than 75% affected, we should suspect this.

2. Marked intrafamilial variability—marked variance in familial manifestation of the disease suggests

   1. Presence of other risk factors like diabetes and hypertension.

   2. Presence of digenic disease

   3. Presence of genetic mosaicism

   4. Uniparental isodisomy results in gene abnormality during meiosis, resulting in genetic differences in family members. 

3. No apparent family history

   1. May be sporadic with an absence of parental records

   2. Presence of somatic mosaicism—the presence of two lineages of cells in the same individual, depending on the amount of mutated cell lineages. Clinically, somatic mosaicism presents as de novo, unilateral, mild, asymmetric involvement

4. Atypical renal imaging: ADPKD usually presents with the symmetric distribution of multiple cysts. However, around 16% present with asymmetric cysts. Among them, 40% may not have a family history. Asymmetric cysts with mild renal insufficiency suggest PKD1NT and PKD2NT mutations. The presence of severe renal insufficiency may suggest involvement of genes like DNAJB1 or thin basement membrane disease COL4A3.

5. Syndromic presentation: The syndromic presentation will provide clues for the diagnosis

6. ARPKD—presence of hepatic fibrosis

7. TSC—presence of hamartomas in target organs

8. ADTKD - presence of gout and hyperuricemia - UMOD involvement

9. ADTKD - HNF1B - presence of Maturity Onset of diabetes of the young (MODY) and genitourinary malformation.

PKD screening in the era of the Next Generation Sequence (NGS) (multiple parallel generation system) assay:

“Education never ends; it is a series of lessons, with the greatest for the last.”

― Sir Arthur Conan Doyle, His Last Bow

Rapid evolution of molecular diagnostics from laborious Sanger analysis (which can be used even now if the genomic mutation of the family member is known) to the high-throughput simultaneous screening of multiple disease genes (NGS). ADPKD PKD1 (46 exons) genetic analysis is very complex in view of the large gene size and rich guanosine and cytosine content (GC). It contains two parts: PKD1 (1-33) and PKD1 (34-46). PKD1(1-33) analysis is very difficult due to duplication and the presence of six pseudogenes (having more mutations than normal genes), giving false positive results. Rosetti et al. first reported a protocol that employed five long-range PCRs to generate PKD1-specific amplicons from the duplicated region, followed by 65 nested PCRs to screen the entire gene, which is used as the gold standard for research purposes. PKD1 (34 - 46) exon is a single-copy gene, and screening is easy, similar to PKD2 and other minor genes.

Targeted gene assay: Customized panel of genes are measured 

a. DNA capture technique: DNA capture methods enrich genomic DNA fragments from the target regions (i.e., multiple pre-selected genes with a combined size up to 50 Mb) hybridized to custom-designed oligonucleotide probes of 55-120 base pairs. In general, these methods are more time- and cost-efficient for target sequence enrichment than PCR-based methods and may detect large gene rearrangements

b. Long-range locus-rich PCR technique—The use of locus-specific PCR-based enrichment is laborious but provides a robust approach against “contamination” with pseudogene sequences. It also enables more versatility for PCR primer design, higher target sequence enrichment in GC-rich regions, and minimal off-target sequence capture. As a result, there is a low likelihood for ambiguous sequence mapping and false positive results with relatively straightforward data analysis. However, “allelic bias or drop-out” and difficulty in detecting large gene rearrangements are the drawbacks of these methods.

Whole Exome Sequencing: Whole protein-coding genes are measured

Whole Genome Sequencing: the entire genome is measured; similar to DNA capture, it has a high false positive rate due to pseudogenes and poorer measurement at GC-rich genes of PKD1. It has the advantage of diagnosing other large gene deletions and other syndromic genes.

Conclusion: Recent advancements in NGS have helped with the diagnosis of ADPKD in atypical presentations like asymmetric kidneys, severe and early disease, radiologic dilemmas, negative family history, diagnosis of exclusion in prospective kidney donors from the same family, and prenatal and preimplantation genetic diagnosis. The type of mutation can be used for prognostication and to initiate precise and personalized disease-modifying therapy. Guidelines do not advise presymptomatic genetic testing in children, as there are no specific disease-modifying therapies.

Mentors

Dr Sophia Ambruso

Dr Elba Medina

Dr Edgar Lerma

Dr. Brian Rifkin

Dr Kajaree Giri