The polycystic kidney disease autosomal dominant (ADPKD), international codes classified 753.12, 753.13 (ICD-9) and Q61.2, Q61.3 (ICD-10), is the most common hereditary nephropathy causing kidney failure and need for treatment renal replacement therapy (RRT). There are renal cysts that grow progressively replacing the entire normal renal parenchyma and this is accompanied by fibrosis and inflammation of the interstitium.1 It is a monogenic and/or bigenic disease2 that can affect other organs besides the kidney and cause high blood pressure, urinary tract infections, lumbar or abdominal pain, hematuria, nephrolithiasis and kidney failure, as well as cysts of the liver, pancreas and epididymis, choroidal and arachnoid plexus, brain aneurysms, heart abnormalities and abdominal hernias, among others.3 The intensity of these manifestations is variable even between individuals of the same family.4

The prevalence of the disease is not well determined since there are not specific official records, although recent studies indicate that the prevalence is between 4.76–9.14 cases/10,000 births.5 Our registry, made up of 298 families and 1,164 patients identified in the Granada healthcare setting, identifies between 16–31 new cases/year (during the 2000−2018p eriod), with an average incidence of 2.34 cases/105 people/year,6 consistent with data reported in the study Else - Kroener - Fresenius-ADPKD in southwestern Germany.7

Most cases are due to variants in the PKD1 gene (16p13.3), and to a lesser extent in the PKD2 (4q22.1), GANAB (11q12.3) genes8 and DNAJB11 (3q27.3), with few cases of combined involvement of the PKD1 and PKD2 genes.9 These genes encode the proteins polycystin 1 (PKD1), polycystin 2 (PKD2) and the α subunit of glucosidase II (GANAB), which are very ubiquitous in the body, although they are mainly part of the primary cilium.10 Its alteration will cause a decrease in intracellular calcium with an increase in cAMP, alterations in cell proliferation and polarity, an increase in fluid secretion and an increase in the extracellular matrix, all of which are implicated in the cystogenesis, but the exact mechanism is still unknown.10,11

Our aim is to demonstrate that the new variant c.7292T> A identified in the PKD1 gene is responsible for ADPKD in 4 families from the Alpujarra de Granada, apparently not related by a genealogical tree, as well as to identify the existence of a possible founder effect.

Medical histories, genealogical trees and ultrasounds have been obtained in members of the 4 families affected by PKRAD from the geographical region of the Alpujarra of Granada, that has a registered population of 25,000 people: one family is from Órgiva, with 5,570 inhabitants and the other three from Pórtugos, with 403 inhabitants.12

Genetic studies were performed by massive sequencing (NGS) in people with a diagnosis of ADPKD.13 The variant c.7292T> A (p.Leu2431Gln) was identified in heterozygosity in the PKD1 gene, not described in the databases or in the literature. Additionally the in silico analysis was contradictory: the PoliPhen2, SIFT and Mutation Taster programs indicate a deleterious effect while Align GVGD indicated a benign effect. With this situation it was necessary to plan the segregation study: genetic analyzes were performed in affected relatives (complete sequencing of the PKD1 gene or study of known point mutation, depending on each case) and in healthy relatives (study of known point mutation). In each family, affected and healthy individuals were studied. All participants were informed and signed the consent to participate in the study; in the case of minors parents authorization was requested.

Genetic studies were carried out at the Institute of Genomic Medicine (IMEGEN) using the NextGeneDx ® massive sequencing panel (NGS), which includes the PKD1, PKD2 and GANAB genes, following the following methodology:

• 1

  Genomic DNA extraction from the lymphocytes of the blood sample.

• 2

  Library preparation using the Nextera XT kit (Ilumina).

• 3

  Sequencing of the libraries (2 × 150) in the MiSeq sequencer (Ilumina).

• 4

  Bioinformatic analysis of the sequences obtained.

• 5

  Confirmation of variants by Sanger sequencing.

Pathogenicity of the variants discovered was evaluated using population databases such as the Human Gene Mutation Database (HGMD), Clinvar, GenomeAD, LOVD and, the one that offers the most specific information, the ADPKD Mutation Database, which identifies 2,601 changes in the structure of the PKD1 gene, between silent, pathogenic and of uncertain or indeterminate clinical significance.14

For point mutations in patients with an already known mutation, Sanger sequencing was performed by the Imegen laboratory.

We recorded sociodemographic, clinical and other variables that could be related to the impact of the mutation on the kidney and the patient by doing survival studies. Geno-Pro programs were used to make the genealogical trees and the statistical package SPSS 15.0. Data are expressed in terms of mean ± SD, range, median, and percentage, as appropriate. Kidney and patient survival studies were performed with the Kaplan-Meier test, considering significance if p < 0.05.

Sociodemographic, clinical and genetic characteristics of the studied population are summarized in Table 1. Due to the accessibility conditions, genetic study was performed in 20 of the 66 patients diagnosed of ADPKD by ultrasound criteria by Ravine and Pei.13 In 8 patients the genetic study was performed by mass sequencing and in 12 by Sanger sequencing to the study of known point mutations. Genetic analysis revealed the presence of the variant c.7292T> A (p.Leu2431Gln) in all affected patients. Likewise, 8 relatives without clinical and ultrasound evidence of the disease were studied and the variant was not identified in any of the 8 relatives.

In these 4 families, a 62.9% of affected were women. Diagnosis was made at 29.3 ± 15.82 years of age (age range 1–59), in 64.8% after delivery of a first child. The presence of a family history and hematuria were the two main causes that led to the diagnosis. In affected relatives, hepatic cysts were identified in 59.3%, a history of hemorrhagic stroke in 3.3%, and cysts in the epididymis or ovary in 13%. The age of initiation of renal replacement therapy (RRT) was 55.8 ± 7.62 years (age range 44–67); death occurred at 63.1 ± 9.32 years (range 48–76 years), the most frequent causes were: unknown cause, cardiovascular and renal failure frequently. The median renal survival was 58.5 ± 0.77 years and the patient's median survival was 67.2 ± 3.54 years, without significant differences based on sex. Of the deceased patients, 52.2% required RRT and 94.4% had some degree of renal failure (IR).

Our variant, not identified in the general or specific human gene variant registries, is located in exon 18 at chromosomal position chr16: 2156596 where an adenine to thymine transversion substitution occurs that predicts a missense mutation (missense) with the modification of leucine (L) by glutamine (Q) at codon 2431 (c.7292T> A, L2431Q) (Table 2).

This change is located in the extramembranous region, REJ domain of polycystin 1 (PKD1). The domain in PKD1 is almost 1,000 amino acids in length between exons 15 and 27. It is likely composed of multiple structural domains. There are six fully conserved cysteine residues that can form disulfide bridges.15 The function of this domain is not well established, although it has been related to the regulation of calcium ion flux.16 Some authors suggest that it is an essential region for the biological activity of polycystin-1.17 The REJ domain is located in the vicinity of the GPS domain (G-protein coupled proteolytic site), which is essential for a normal structure and function of the kidney. Some studies have shown that the GPS domain function requires the integrity of the REJ region. In the Mayo Clinic PKD database there are described around 65 missense variants classified as causes of disease in this region.15 Other missense variants classified as probably pathogenic are described in this same region very close to the protein sequence, such as c.7300C> T, p.Arg2434Trp (R2434W),18 c.7301G> A, p.Arg2434Gln (R2434Q)19 and p. Leu2433Arg (L2433R).20

Some in silico predictors classify this variants as deleterious and others as benign. This is not registered in the ExAC, ESP5400 and 1000 Genomes databases which indicates its low frequency in the population. This contradiction of predictors causes the variant to be classified as indeterminate. The segregation study is compelling: the variant is seen in 20 affected people and in none of the 8 healthy individuals studied. That is, the variant set aside with the disease. To classify a variant, it is recommended to follow the criteria of the American College of Medical Genetics and Genomics.21 In 2015, this consensus guide was written trying to actualize, as far as possible, criteria to decide on the classification of variants. It proposes a work system through a series of weighted criteria that allows a score to be made to decide on the greater or lesser probability of pathogenicity of the finding, with criteria in favor and criteria against:

Our work indicates 1) that the variant c.7292T> A in the PKD1 gene is pathogenic and therefore must be classified as such and 2) that its distribution in the Alpujarra region of Granada suggests a founder effect. In situations like this, segregation studies are necessary to characterize pathogenic variants not yet described and facilitate reproductive options such as TGP.

This work has been funded by the José Luis Castaño-SEQC Foundation, 2018-2019 post-residency scholarship.

The authors declare that they have no conflict of interests.