Diagnosis and management of RDs until recently relied on the assessment of the primary physician along with biochemical, radiological, and pathological tests. The overlapping spectrum of manifestations was the major reason for the limitations in the exact clinical diagnosis of renal diseases [Mansilla et al., 2019]. Molecular diagnosis of inherited kidney diseases remains a challenge due to their expanding phenotypic spectra, as well as, the constantly growing list of disease-causing genes [Bullich., 2018]. According to Jha et al end-stage renal disease (ESRD), is an increasing public health issue with an estimated prevalence of 8-16% worldwide [Jha., 2013]. According to Mele et al [Mele et al., 2011] ESRD necessitates dialysis or transplantation for survival and is a cause of morbidity and mortality. Hence, knowing the underlying genetic cause of RD will help in planning novel therapies and strategies for treatment /management and prevention.
The rapid changes in the methodology of genetic testing, especially NGS have become important to identify causative disease genes, which will facilitate the use of molecular diagnosis and reduce the need for invasive diagnostic procedures, such as renal biopsy [Bullich et al., 2018]. In our present study, twenty cases were evaluated by NGS-based panel tests, and phenotype-associated gene variants were identified in nineteen cases. This high diagnostic yield of 95% was achieved because the cases were selected for exome testing after genetic counseling which included pedigree analysis and syndrome identification based on clinical, radiological, and family details.
The patients included in the present study were clinically categorized into two groups: Polycystic Kidney Disease (PKD) and Syndromic cases with renal involvement.
POLYCYSTIC KIDNEY DISEASE (PKD)
PKD is an inherited disorder in which clusters of non-cancerous water-like fluid-filled sacs develop that interfere with the ability of the kidney to filter waste. The most common form of PKD is Autosomal dominant PKD (ADPKD), which is the adult onset and Autosomal recessive PKD (ARPKD) is the neonatal form of PKD. ADPKD is caused by mutations in PKD1 and PKD2 genes that code for two different proteins called Polycystin 1 and Polycystin 2 respectively. They work together for the normal functioning of the kidney. ARPKD phenotype is variable and is caused by mutations in Polycystic Kidney and Hepatic Disease 1 (PKHD1) gene that codes for fibrocystin protein, a cliliary protein spanning kidney cell membrane and has also been associated with PKD [Harris and Torres., 2009]. Damaging sequence variations in these genes lead to dysfunction of proteins resulting in numerous progressively enlarging cysts in the kidneys, leading to structural damage and loss of function progressively causing end-stage renal disease. From our study PKD cases had a very high molecular diagnostic rate with five cases with PKD1, two cases with PKD2, and one case with PKHD1 variants. This is similar to what was reported by Groopman who demonstrated maximum variants from PKD1 and PKD2 genes from samples of kidney diseases[Groopman et al., 2019].
According to the American College of Medical Genetics and Genomics (ACMG) guidelines, 17/19 (89%) of the cases from the current study were reported with a pathogenic or likely pathogenic variant. In a study by Mansilla et al., 2019, NGS based genetic testing on patients with various renal phenotypes had identified 43% of variants to be pathogenic and likely pathogenic. In the present study two VUS were identified: (i) Case 1.2, which was a fetus, with PKD2 exon 6 c.1505 A>G p.Tyr502Cys variant. Segregation analysis of parents revealed that the father had the same variant and he was referred to radiology and nephrology surveillance prior to prenatal evaluation. (ii) Case 1.7, a 46 year old female with no family history of PKD was identified with PKD1 exon 18 c.7409C>T p.Pro2470Leu.She was counselled for targeted segregation analysis in the family to identify if this was a denovo variant.
SYNDROMIC CASES WITH RENAL INVOLVEMENT
There ar several syndromes in which abnormalities in the renal system are observed. Eleven syndromic cases after genetic counselling were offered NGS testing which was able to provide a diagnosis to all the cases.
Three cases clinically diagnosed with progressive loss of kidney function were identified with pathogenic variants in COL4 genes associated with Alport syndrome (AS), two of them (2.1 & 2.2) had homozygous COL4A3 pathogenic variants indicating autosomal recessive inheritance and one case (2.3) had a X linked heterozygous COL4A5 variant associated with X linked dominant inheritance (Table2). AS is a hereditary disease caused by mutations in collagen type IV alpha chain genes (COL4A3/COL4A4/COL4A5, OMIM: 12007, 120131, 303630). The progressive damage to the basement membrane with mutated form of type 4 collagen leads to microscopic hematuria, sensorineural deafness, anterior lenticonus, and progressive kidney dysfunction leading to end-stage renal disease [Kashtan and Michael., 1996]. Girimaji reported an autosomal recessive pathogenic novel variant c.1891G>A in exon 26 of COL4A3 gene leading to end stage renal disease from an Indian patient born to a consanguineous couple [Girimaji et al., 2020].
Four patients and one fetus were seen with homozygous pathogenic/likely pathogenic variants in genes associated with Bardet Biedl syndrome. Of the five cases, one case (2.4) had a homozygous pathogenic variant in BBS9 gene, and the remaining had variants in BBS10 gene (Table2). Segregation analysis in parents was performed in case 2.7 and 2.8, showing that both parents were carriers for the reported variant and this information could be used for subsequent prenatal diagnosis.
BBS is an autosomal recessive disorder was identified is a ciliopathy disorder, affecting vision, intelligence, genitalia renal function and also show polydactyly, however, variable expression and heterogeneity of features is seen (Priya et al ., 2016). Although globally BBS1 is considered to be the most common type, however, an earlier report from India by Chandrasekhar et al (2018) also showed BBS10 to be most common in India. A founder mutation, C91fs*95 was also found in a fetal sample identified to have polycystic kidneys in prenatal ultrasound of a non-consanguineous couple with a history of previous terminated pregnancy with the similar renal features. Segregation analysis of the couple revealed them to be carriers. This founder mutation has also been reported in other studies from India, Chandrasekhar et al (2018).
A child experiencing dehydration, nausea, poor weight gain, rickets with kidney stones was identified with a homozygous likely pathogenic variant in the ATP6V1B1 gene, which impairs the function of the V-ATPase proton pump resulting in the loss of control on pH of the fluid in the inner ear leading to sensorineural hearing loss and loss of control on acidity of the blood leading to demineralization of bones and other consequences of metabolic acidosis, which Is characterized by hyperchloremic metabolic acidosis, hypokalemia, hypercalciuria, hypocitraturia, nephrocalcinosis, and conserved glomerular filtration rate. [Escobar et al., 2016]. Sethi reported a pathogenic variant in exon 1 of ATP6V1B1 in an Indian family with distal RTA and sensorineural deafness [Sethi., et al 2009].
The Segregation analysis in parents of case 2.9 revealed them to be carriers. The couple was counseled that the recurrence risk is 25% for all subsequent pregnancies and were explained about using this variant for prenatal diagnosis.
Two cases were identified with X linked gene variants. One was X-linked hypophosphataemia (XLH) and the other was Hemizygous Nephrogenic Diabetes incipidus (Table 2): i) Mutations in Phosphate-regulating Endopeptidase Homolog X- linked (PHEX) gene cause hypophosphataemia, impaired synthesis of active vitamin D (1,25 (OH)2 vitamin D), rickets, mineralization defect of bones, teeth and disproportionate short stature . PHEX gene encodes a cell-surface-bound protein-cleavage enzyme that is predominantly expressed in osteoblasts, osteocytes and teeth causes renal phosphate wasting. The family in the present study had multiple members effected (figure 1) showed a pathogenic, heterozygous variant in exon 15 of PHEX gene. The degree of severity among the affected members of the family harboring the above mentioned variation was different, suggestive of X linked dominant inheritance with variable expressivity. ii) Nephrogenic Diabetes inipidus (NDI) causes water imbalance due to the inability of the kidney to concentrate urine from the blood stream in response to antidiuretic hormone vasopressin (AVP) leading to large volume of un-concentrated urine (case 2.11) (Ma et al., 2020). 90% of the NDI cases are due to sequence changes in AVPR2 gene present on the X chromosome, which in X-linked recessive inheritance affects males mainly and the rest 10% are due to sequence changes in the AQP2 gene variants which result in both autosomal recessive and autosomal dominant inheritance (Fujiwara and Bichet, 2005). In the case included in the study nonsense deletion at 553 position results in premature truncation of the protein at codon 185 leading to NDI. Tayfur et al., 2018 reported a case of NDI in a six year boy with a hemizygous variant in exon 2 of AVPR2 gene resulting in the incorporation of Tyrosine in place of Histidine that ultimately leads to inappropriate protein folding.
In our study 11/50 showed to have a syndromic condition after first round of genetic counselling. All eleven cases had a molecular diagnosis after testing, giving us a very high diagnostic yield. Five of the cases which showed the features of Bardet Biedl syndrome (BBS) were confirmed by molecular diagnosis of BBS with four of them having a pathogenic variant in BBS10 gene and one in BBS9 gene. Three of the cases were diagnosed to have Alport syndrome, of which two had variants in COL4A3 causing autosomal dominant inheritance and one had COL4A5 variant that cause X linked dominant Alport syndrome. All three variants were in exonic region, two were frameshift mutations responsible for protein termination post Arginine truncation. The third variant was a missense variant altering the protein in codon 1412.
According to Liebau and Benzing investigations of rare genetic disorders can serve as a starting point for the development of novel therapeutic strategies in RDs that are inherited [Liebau and Benzing, 2011]. NGS based genetic testing if applied at an early stage of the diagnostic process, reduces the unnecessary financial burden involved in repeated hospital visits and laboratory tests especially in a country like India, where the healthcare is not covered by the governmental schemes for majority of the population.