Phenotype and Genotype Profile of Children with Primary Distal Renal Tubular Acidosis: A 10-Year Experience from a North Indian Teaching Institute

Abstract

Primary distal renal tubular acidosis (dRTA) or Type 1 RTA in children is caused by a genetic defect (involved genes ATP6V0A4 , ATP6V1B1 , SLC4A1 , FOXI1 , or WDR72 ), which causes tubular transport defects characterized by an inability to appropriately acidify urine with resultant persistent hyperchloremic metabolic acidosis. Retrospective analysis of 28 children (14 males) under the age of 14 years with dRTA seen from 2010 to 2019 was reviewed, and detailed clinic records were analyzed. The clinical features, investigations, and response to treatment were recorded. The median age of the children at presentation was 30 months (range: 9.25–72 months), and the median age at onset of symptoms was 2 months. All the children had growth failure, polyuria, and polydipsia at presentation. Mean serum potassium, pH, bicarbonate, and anion gap at presentation was 2.3 ± 0.5 mmol/L, 7.22 ± 0.09, 13.28 ± 4.37 mmol/L, and 9.3 ± 2.18, respectively. Mean serum potassium, pH, bicarbonate at follow-up was 3.88 ± 0.6 mmol/L, 7.35 ± 0.06, and 20.13 ± 4.17 mmol/L, respectively. The median z-score for the weight for age and height for age at initial presentation was −4.77 (–7.68 to –3.74) and –4.21 (–5.42 to –2.37) and at follow-up was –3.35 (–5.29 to –1.55) and –3.84 (–5.36 to –1.63), respectively. Twenty-two (78.6%) children had medullary nephrocalcinosis. Four children had sensorineural hearing loss. Seven children had genetic testing done, and six had pathogenic or likely pathogenic variants in ATP6V1B1 and ATP6V0A4 gene. Children with dRTA have a guarded prognosis and ATP6V1B1 and ATP6V0A4 mutations are the most common implicated genetic defect in Indian children with distal RTA.

Introduction

Primary distal renal tubular acidosis (dRTA) or Type 1 RTA in children is caused by a genetic defect that causes tubular transport defects characterized by the inability to appropriately acidify urine with resultant persistent normal anion gap hyperchloremic metabolic acidosis, along with hypokalemia, hypercalciuria, and nephrocalcinosis. ¹ It has an early age of onset with clinical manifestations usually in infancy or childhood. Patients often present with growth failure, vomiting, dehydration, polyuria, polydipsia, diarrhea or constipation, urolithiasis, and rickets. ² The α-intercalated cells in the distal renal tubules are unable to properly acidify the urine (urine pH > 5.5) due to the defective secretion of hydrogen ions (H ⁺ ). ³

The aim of the treatment is to correct the biochemical abnormalities as much as possible, and follow up regularly to monitor the growth of the children and the renal functions. In dRTA, adequate alkali in the form of bicarbonate or citrate with a mixture of sodium and potassium salts should be provided to balance the hydrogen ion production, which is usually higher in children. In young infants, the amount of base required ranges from 5 to 8 mmol/kg/day, which decreases as the child grows to 3 to 4 mmol/kg/day ² .

In children, the etiology of primary dRTA is usually an underlying genetic defect while in adults, dRTA is commonly due to underlying secondary disorders like autoimmune disease, infections, or drugs. ⁴ Primary dRTA is a rare genetic disease caused by loss of function mutations in one of these genes ^{5 6 7 8 9} ( ATP6V0A4 , ATP6V1B1 , SLC4A1 , FOXI1 , or WDR72 ) involved in urinary distal acidification, which is transmitted either as an autosomal dominant or recessive trait. ³ Although previous studies on genetic analysis of dRTA patients have been done in different ethnic population, there is limited data on genetic studies in Indian children with dRTA. ^{6 7 8 9} Most of the published literature is on case reports only. ¹⁰ The next generation sequencing technique enables the identification of mutations in dRTA patients and helps distinguish the genotype-phenotype correlation of the disease. ¹¹ A specific genetic diagnosis will help in genetic counseling of the families regarding the nature of the disease. ¹² In general, children with dRTA have a good prognosis if the diagnosis is made early and adequate alkali therapy is continued. Our study was aimed to evaluate the phenotype and genotype profile of children with primary dRTA.

Patients and Methods

Study Population

A retrospective analysis of 28 patients under the age of 14 years with a clinical diagnosis of dRTA, seen from 2010 to 2019 in Renal Children Clinic in the Department of Pediatrics, Post Graduate Institute of Medical Education and Research, Chandigarh, India were included in this study. Informed consent was obtained from all the parents/caregivers of the patients. Ethical approval was obtained from the institutional review board.

Detailed medical records were analyzed retrospectively for demographic variables, clinical features, history of consanguinity, investigations, and response to treatment. The clinical diagnosis of dRTA was based on the presence of growth failure, polyuria and polydipsia at presentation along with normal anion gap, hyperchloremic metabolic acidosis with blood pH below 7.2, bicarbonate below 15 mEq/L, and urine pH of more than 5.5 with normal renal functions. ² Genetic testing was advised to all the patients, but due to financial constraints, only seven families could afford the test. Children with other types of renal tubular acidosis (Type 2 and 4) and secondary (acquired) causes of tubular disorders were excluded. Screening for audiometry and ultrasound of kidney, ureter, and bladder was performed in all the children at presentation. During the study period, the participants had been regularly followed up every 2 months with serum electrolytes and venous blood gas monitoring. Standard care of treatment including oral potassium and bicarbonate supplementation had been ensured in all children.During the study period, out of 5,852 children with renal diseases, 35 children with renal tubular acidosis had been enrolled in our center. Thus, 0.59% of children attending to a tertiary pediatric nephrology service are likely to suffer from RTA, and out of them, 0.47% of children had distal RTA.

The incidence rate was calculated taking into account the number of patients coming to pediatric OPDs, patients enrolled in pediatric renal clinic during the study period, and population in the 1- to 14-year age group of the areas from where patients were referred to our center. Our center caters to all the pediatric nephrology services from the neighboring states as it functions as a referral center. So, the probability of underestimation of the incidence of distal RTA is somewhat low, as most of the detected cases of distal RTA are usually referred to our institute, this being a rare disease requiring super-specialty care. But a significant proportion may be underdiagnosed at the community level, as in a developing country, the knowledge of primary care pediatricians regarding these diseases might not be adequate.

Genetic Testing

Genetic analysis was done by using next generation sequencing. Genomic DNA was extracted from blood, following which a targeted gene capture was performed by using a custom capture kit. For sequencing purposes, Illumina sequencing platform was used, and for the identification of variants, GATK best practices framework and Sentieon (v201808.01) was followed. The minimum coverage depth of 80 to 100× for all the protein-coding regions was ensured. Sentieon aligner was used to align the obtained sequences with the human reference genome (GRCh37/hg19). The relevant mutations based on clinical presentation were identified by using previously published variants in the literature, using a set of disease databases (ClinVar, GWAS, SwissVar, OMIM, and HGMD). Novel variants were also analyzed for pathogenicity in vitro, which were not previously reported. Based on allele frequency in the 1000 genome phase 3, ExAC, EVS, dbSNP147, and 1000 Japanese Genome, etc., the common variants were filtered. Using multiple algorithms such as PolyPhen-2, SIFT, MutationTaster2, mutation assessor, and LRT, the non-synonymous variant effect was calculated. For clinical interpretation, only non-synonymous and splice site variants found in the whole exome panel consisting of a specific set of genes were used. The silent variations which did not result in any change in amino acid in the coding regions were not reported. The variants were classified according to the latest guidelines of the American College of Medical Genetics.

Statistical Analysis

Data were analyzed by using SPSS software version 26.0. Categorical variables were expressed in terms of frequency. Continuous variables were expressed as mean and standard deviation or median with interquartile range. Whether data describing a particular parameter follows a normal distribution or not was assessed by the Kolmogorov–Smirnov normality test. Continuous variables were compared by using paired Student's t -test. For nonparametric variables, the Wilcoxon rank sum test was used as a test of statistical significance. The p -value of less the 0.05 was taken as significant.

Results

Demographic and Clinical Features

A total of 28 children (14 males, 50%) with a clinical diagnosis of dRTA were found to be under treatment and followed up during the study period. The median age of the children at presentation was 30 months (interquartile range [IQR]: 9.25–72), and the median age at onset of symptoms was 2 months (range: 1–9). All the children had growth failure, polyuria, and polydipsia at presentation. Acute presentation with loose stools and vomiting was present in eight (28.5%) children, and one child had hypokalemic paralysis. Mean serum potassium, pH, bicarbonate, and anion gap at presentation was 2.3 ± 0.5 mmol/L, 7.22 ± 0.09, 13.28 ± 4.37 mmol/L, and 9.3 ± 2.18, respectively. Mean serum potassium, pH, bicarbonate at follow-up was 3.88 ± 0.6 mmol/L ( p  < 0.001), 7.35 ± 0.06 ( p  < 0.0001), and 20.13 ± 4.17 mmol/L ( p  < 0.0001), respectively. The mean blood urea and serum creatinine at presentation was 25.5 ± 12.3 and 0.32 ± 0.13 mg/dL, respectively and on follow-up was 26.8 ± 10.2 ( p  = 0.12) and 0.33 ± 0.14 mg/dL ( p  < 0.001), respectively. The median weight and height at initial presentation was 6.85 (4.46–11.9) kg and 71.75 (57–94.35) cm and at follow-up was 12.3 (7.9–18.9) kg and 92.96 (66.75–106.25) cm, respectively. The median z-score for weight for age and height for age at initial presentation was −4.77 (−7.68 to −3.74) and −4.21 (−5.42 to −2.37) and at follow up was −3.35 (−5.29 to −1.55; p  < 0.0001) and −3.88 (−5.36 to −1.63) ( p  = 0.45), respectively. Twenty-two (78.6%) children had medullary nephrocalcinosis, and hypercalciuria was seen in the majority of the cases (82.1%). Rickets was seen in 18 (64.2%) children. The clinical and demographic profiles of children with dRTA have been shown in Table 1 and Fig. 1 . All children received potassium supplements and alkali therapy. Three children had parental consanguinity. Four children had sensorineural hearing loss (SNHL). Out of the four children with SNHL, one child underwent cochlear implantation and is doing well. At the last follow-up (median follow-up duration: 18.5 months, range: 10.5–24.05 months), 22 (78.5%) children had normal serum potassium and 19 (67.8%) had growth failure. The frequency of various key biochemical and anthropometric variables at presentation and on follow-up has been described in Table 2 .

Table 1. Clinical and demographic profile of children with distal renal tubular acidosis.

Variables at presentation	n  = 28
Age of onset (mo) (median and IQR)	2 (1–9)
Age at presentation (mo) (median and IQR)	30 (9.25–72)
Follow up duration (mo) (median and IQR)	18.5 (10.5–24.05)
Male: Female	14:14
Parental consanguinity	3 (10.7%)
Growth failure
At presentation	28 (100%)
At follow-up	22 (78.5%)
Polyuria
At presentation	28 (100%)
At follow-up	11 (39.3%)
Polydipsia
At presentation	28 (100%)
At follow-up	12 (42.8%)
Hearing loss	4 (14.3%)
Medullary nephrocalcinosis	22 (78.6)
Rickets
At presentation	18 (64.2%)
At follow-up	6 (21.4%)
Loose stools
At presentation	8 (28.5%)
At follow-up	1 (3.5%)

Fig. 1.

Clinical characteristics of children with primary distal renal tubular acidosis at the time of presentation and at last follow-up.

Table 2. Laboratory findings of children with distal renal tubular acidosis at presentation.

Variables	At presentation	At last follow-up	p -Value
Blood pH	7.22 ± 0.09	7.35 ± 0.06	<0.0001
Serum bicarbonate (mmol/L)	13.28 ± 4.37	20.13 ± 4.17	<0.0001
Anion gap	9.3 ± 2.18	4.78 ± 1.16	<0.0001
Serum sodium (mmol/L)	141.3 ± 5.65	141.7 ± 3.3	0.67
Serum potassium (mmol/L)	2.3 ± 0.5	3.88 ± 0.6	<0.001
Serum chloride (mEq/L)	118 ± 10	103 ± 5.1	<0.001
Blood urea (mg/dL)	25.5 ± 12.3	26.8 ± 10.2	0.12
Serum creatinine (mg/dL)	0.32 ± 0.13	0.33 ± 0.14	<0.001
Urine pH	6.87 ± 0.63	7.18 ± 0.61	0.005
Height (z-score) mean ∗ Height (z-score) median (IQR)	−4.55 ± 3.57 −4.21 (3.87)	−3.84 ± 2.87 −3.88 (4.05)	0.45
Weight (z-score) mean ∗ Weight (z-score) median (IQR)	−7.1 ± 7.95 −4.77 (4.18)	−4.31 ± 4.64 −3.35 (4.04)	<0.0001

Abbreviation: IQR, interquartile range.

^∗p -Value was calculated by using Wilcoxon's sign rank-sum test.

Genetic Analysis

Seven children with a clinical diagnosis of dRTA had genetic testing done and six children had pathogenic mutations. History of consanguinity was present in three children. Genetic testing for ATP6V0A4 , ATP6V1B1 , SLC4A1 , FOXI1 , or WDR72 was done in all the seven children. Among seven children, one child had novel mutations (in exon 2 and 6) in the ATP6V1B1 gene ( Table 3 ). Out of six children, nonsense, missense, and 5′ splice site mutation were found in two children each. 5′ splice site mutations were detected only in the ATP6V0A4 gene. No pathogenic mutation was detected in one child.

Table 3. Genetic analysis of children with distal renal tubular acidosis.

S No.	Gene	Location	Variant	Type of mutation	Inheritance	Novel/ Reported	Classification according to ACMG
1	ATP6V0A4	Intron 16	c.1691 + 1G > A	5′ splice site	AR	Reported 20	Pathogenic
2	ATP6V1B1	Exon 1	c.91C > T	Nonsense	AR	Reported 22	Pathogenic
3	ATP6V1B1	Exon 2 Exon 6	c.123C > G c.541C > T	Nonsense Nonsense	AR	Novel	Likely pathogenic
4	ATP6V1B1	Exon 10	c.1037C > G	Missense	AR	Reported 6	Likely pathogenic
5	ATP6V0A4	Exon 14	c.1346G > A	Missense	AR	Reported 21	Likely pathogenic
6	ATP6V0A4	Intron 16	c.1691 + 1G > A	5′ splice site	AR	Reported 20	Pathogenic

Abbreviations: AR, autosomal recessive; ACMG, American College of Medical Genetics and Genomics.

Clinical Features with ATP6V0A4 and ATP6V1B1 Mutations

All children with ATP6V0A4 and ATP6V1B1 mutations had an early age of disease onset (mean age: 1 vs. 1.5 months). Children with ATP6V0A4 mutations had severe acidosis at the initial presentation when compared with those with ATP6V1B1 mutations (mean pH and HCO ₃ levels were 6.95, 7.21, and 5.1 and 11.6 mmol/L, respectively). Serum potassium levels at the time of presentation was lower in children with ATP6V0A4 mutations than those with ATP6V1B1 mutations (mean serum K+ was 2 and 2.63 mmol/L, respectively). The clinical features such as growth failure, polyuria, polydipsia, and medullary nephrocalcinosis were similar in both ATP6V0A4 and ATP6V1B1 mutations. Hearing loss was seen in children with ATP6V1B1 mutations, and it was not seen in children with ATP6V0A4 mutations.

Discussion

This study presents the details of 28 patients with a clinical diagnosis of dRTA and mutation analysis in seven patients. The diagnosis of dRTA is usually made by clinical features and biochemical findings. ³ The median age of onset of symptoms was 2 months (range: 1–9) with growth failure, polyuria, and polydipsia at presentation. Alkali therapy is the mainstay of treatment for children with dRTA. All children received potassium supplements and alkali therapy for hypokalemia and metabolic acidosis. Children with early age of onset have severe metabolic acidosis in comparison to those with older age of onset and adults. The need for an increased dose of bicarbonate in younger patients in our study was consistent with other studies. ^{3 6 13} In the presence of acidosis, there is increased in urinary calcium excretion which can lead to increased risk of nephrolithiasis/nephrocalcinosis, which usually decreases with the administration of alkali therapy. ^{14 15} In our cohort, hypercalciuria was seen in the majority of the cases (82.1%) with nephrocalcinosis in 78.6% of the cases.

In children with long-standing metabolic acidosis, failure to thrive with features of rickets is a common clinical presentation secondary to bone mineralization. However, the improvement in growth is noted in children if adequate treatment with alkali is started. ¹³ In our cohort, 78.5% had growth failure on follow-up. The weight for age ( p  < 0.0001) was significantly affected on follow-up in comparison to height for age ( p  = 0.45). This can be explained by the shorter duration of follow-up (18.5 months, range: 10.5–24.05 months) as well as malnutrition due to inadequate calorie intake secondary to polydipsia in our patients. The majority of the children had improvement in polyuria and polydipsia on follow-up.

Out of the seven patients who underwent molecular testing, one patient did not show any mutation. Six patients had a pathogenic mutation diagnosis. SLC4A1 mutation was not seen in our cohort. Various studies on the genotype-phenotype correlation in patients with dRTA have been published in literature. ^{1 7 8 9 16 17 18 19} In a study by Palazzo et al, the genotype-phenotype correlation was done in 89 patients with dRTA, where an underlying genetic cause was detected in 71.9% of the cases. They found that mutation in the ATP6V0A4 gene was as common as the mutation in the ATP6V1B1 gene in patients with the recessive disease and the clinical features such as sensorineural hearing loss in the sporadic cases was not a specific indicator for the underlying causal gene. ¹⁹ In the current study, the majority had proven mutation in the ATP6V1B1 and ATP6V0A4 genes. All patients had an autosomal recessive form of dRTA. In our cohort, we found that one child had novel mutations (exon 2 and 6) in the ATP6V1B1 gene and had sensorineural hearing loss. In patients with dRTA, SNHL is often seen to be associated. In a study by Besouw et al, all six children with ATP6V1B1 gene mutation had hearing loss (HL) and only one child with ATP6V0A4 gene had HL. ⁶ The early onset of HL has been noted to be diagnosed before 10 years of age in those with ATP6V1B1 mutation when compared with patients with ATP6V0A4 mutation, where the onset of HL is usually diagnosed beyond the second decade of life. ^{6 20 21 22} All four children with SNHL in our study also had an early onset SNHL before 6 months of age, among which three had ATP6V1B1 gene mutation and in the other child, mutation analysis was not done, which emphasizes the importance of regular hearing assessment in children with dRTA even in the absence of clinical signs and symptoms of deafness. One of the children with ATP6V1B1 gene mutation with SNHL underwent cochlear implantation and is currently doing well postsurgery. Based on these findings, complete genetic testing should be done in patients with dRTA, irrespective of their clinical phenotype.

Our study had certain limitations. We could not perform genetic analysis in all the children with dRTA, and hence, we could not establish genotype-phenotype correlation analysis. We did not assess the calorie intake which could have an impact on the growth parameters. Although our data depict a 10-year experience, the follow-up duration was less as most of the patients were lost to follow up. We did not screen any family members; however, there was no family history of RTA.

Conclusion

ATP6V1B1 and ATP6V0A4 mutations are the most commonly implicated genetic defects in Indian children with distal RTA. Sensorineural hearing loss was exclusively present in children with a pathogenic variant in the ATP6V1B1 gene. There was a delay in the diagnosis noted from the disease onset to the presentation at the hospital.

Funding Statement

Funding None.