Clinical characterization and diagnosis of cystic fibrosis through exome sequencing in Chinese infants with Bartter-syndrome-like hypokalemia alkalosis

Abstract

Cystic fibrosis (CF) is a fatal autosomal-recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. CF is characterized by recurrent pulmonary infection with obstructive pulmonary disease. CF is common in the Caucasian population but is rare in the Chinese population. The symptoms of early-stage CF are often untypical and may sometimes manifest as Bartter syndrome (BS)-like hypokalemic alkalosis. Therefore, the ability of doctors to differentiate CF from BS-like hypokalemic alkalosis in Chinese infants is a great challenge in the timely and accurate diagnosis of CF. In China, sporadic CF has not been diagnosed in children younger than three years of age to date. Three infants, who were initially admitted to our hospital over the period of June 2013 to September 2014 with BS-like hypokalemic alkalosis, were diagnosed with CF through exome sequencing and sweat chloride measurement. The compound heterozygous mutations of the CFTR gene were detected in two infants, and a homozygous missense mutation was found in one infant. Among the six identified mutations, two are novel point mutations (c.1526G>C and c.3062C>T) that are possibly pathogenic. The three infants are the youngest Chinese patients to have been diagnosed with sporadic CF at a very early stage. Follow-up examination showed that all of the cases remained symptom-free after early intervention, indicating the potential benefit of very early diagnosis and timely intervention in children with CF. Our results demonstrate the necessity of distinguishing CF from BS in Chinese infants with hypokalemic alkalosis and the significant diagnostic value of powerful exome sequencing for rare genetic diseases. Furthermore, our findings expand the CFTR mutation spectrum associated with CF.

Keywords

cystic fibrosis / pseudo-Bartter syndrome / hypokalemic alkalosis / CFTR gene / mutations / infants / diagnosis

Introduction

Cystic fibrosis (CF) is the most common autosomal recessive disease among Caucasians and is characterized by high mortality and poor prognosis. CF is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene [¹]. The CF morbidity in live births varies from 1/25 000 in Finland to 1/1800 in Slovakia [²]. However, CF rarely occurs in the Asian population [²]. The CF morbidity in Japan is 1/350 000 [³] but that in China remains to be clarified given that CF is a rare disease in the Chinese population: to date, only approximately 60 Chinese CF cases have been reported [⁴–¹⁶]. Among these cases, approximately 30 cases have been confirmed to have CFTR gene mutations. In China, no infant younger than three years of age has been diagnosed with sporadic CF. CF may lead to abnormalities of the respiratory and digestive systems and the male reproductive system. Moreover, almost all children with CF will suffer from obstructive pulmonary disease with chronic infection. This condition results in progressive pulmonary dysfunction, which is the leading cause of death among CF patients. The uncharacteristic manifestations and the lack of awareness of CF due to its rarity may delay the diagnosis and/or cause the misdiagnosis of CF among Chinese patients, especially in patients with sporadic CF. The early diagnosis of CF in infants and young children less than 3–5 years of age will provide a unique opportunity for early intervention to delay the onset and halt the progression of CF lung disease.

The present paper reports on the cases of three infants who initially presented Bartter syndrome (BS)-like hypokalemic alkalosis and who were ultimately diagnosed with CF through exome sequencing and sweat chloride testing. These infants are the youngest patients in China to have ever been diagnosed with very-early-stage sporadic CF. Follow-up showed that all cases remained symptom-free after early intervention, indicating the potential benefit of the very early diagnosis of CF and timely intervention. The results of this report also demonstrate the necessity of distinguishing CF from BS in Chinese infants with hypokalemic alkalosis and the considerable diagnostic value of powerful exome sequencing for rare genetic diseases.

Materials and methods

Cases and clinical evaluation

Three infants, who were admitted to our hospital over the period of 2013 to 2014, were included in this study. The patients underwent a follow-up survey until September 2016. Written informed consent was obtained from the parents or legal guardians of the recruited patients. This study was approved by the ethics committees of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (China). All blood gas and biochemical tests were performed using an autoanalyzer. Sweat chloride was initially detected with a filter-based method and later confirmed with standard sweat collection and measurement methodology.

Exome sequencing

A QIAamp DNA Mini Kit (Qiagen, Germany) was used to extract DNA from peripheral blood samples collected from the children and their parents. The presence of genes associated with CF, BS, and chloride-losing diarrhea were tested through exon trapping and next-generation sequencing (sequencing platform: Illumina Pipeline, version 1.3.4). All relevant exons were captured with a microarray chip, and all exons with their flanking 10-bp introns were sequenced with Illumina HiSeq2000. Image analysis and base calling were performed using the Illumina Pipeline. To identify single nucleotide variants (SNVs) and indels, the Burrows Wheeler Aligner Multi-Vision software package was used to align 90-bp clean reads with the human genome reference retrieved from the NCBI database (HG19). SNVs and indels were identified using SOAPsnp software and Samtools, respectively. The average sequencing depth for the target region was more than 449-fold.

Results

Main Bartter-syndrome-like symptoms of the infants with CF

The main clinical characteristics of the infants are summarized in Table 1. All three infants were Han Chinese, were not consanguineous, and had no familial history of similar symptoms. All three infants were initially referred to nephrologists due to failure to thrive, polydipsia, and polyuria, which are typical BS symptoms. Therefore, the blood gas, electrolytes, and renin–angiotensin–aldosterone of the infants were measured, and the results completely corresponded with BS symptoms. Hypokalemic alkalosis was predominant and occurred during the summer season. All three infants had normal blood pressure but presented high blood renin, angiotensin, and aldosterone. The infants had normal total testosterone, adrenocorticotropic hormone, cortisol, dehydroepiandrosterone sulfate, androstenedione, and 17-hydroxyprogesterone levels. The infants were diagnosed with BS with respiratory infection on the basis of the above typical presentations.

Exome sequencing was performed using DNA samples collected from the infants. However, no suspicious mutations were found in genes associated with BS and all other common renal tubulopathies. Conversely, CFTR gene mutations were identified in these infants, and the infants were diagnosed with CF on the basis of genetic analysis.

To confirm the diagnosis of CF, we first measured sweat chloride using a filter-based method and then performed subsequent evaluations using standard sweat collection and measurement. All the analyses showed that the infants had extremely high sweat chloride levels.

CFTR gene mutations

The CFTR gene mutations identified in the three infants and their parents are summarized in Table 2 and Figs. 1–3. Compound heterozygous CFTR gene mutations, including two reported CF-causing mutations [¹⁷,¹⁸] and one novel mutation (c.1526G>C, p.G509A) with unknown pathogenicity, were identified in the first infant. Sorting Intolerant from Tolerant (SIFT) predicted that the novel mutation c.1526G>C is harmless, whereas PolyPhen-2 suggested that the mutation is detrimental. This mutation was absent from 100 healthy Chinese controls and the human 1000 genome database. A homozygous missense mutation (c.2909G>A) of the CFTR gene was identified in the second infant. This mutation, which resulted in amino acid substitution of glycine with aspartic acid (p. G970D) was previously reported in a Chinese patient [¹⁰]. Compound heterozygous mutations of the CFTR gene were identified in the third infant. c.1116+ 1G>A is a splicing mutation that can result in abnormal mRNA splicing and changes in the primary structures of proteins. This mutation was included in the CF mutation database. c.3062C>T is a missense mutation that replaces proline with leucine. The proline residue at this site (p.1021) is highly conserved among many species, and the substitution of proline residues with alanine or serine at this site (p.1021) affects the function of chloride channels [¹⁹]. Therefore, the proline substitution at this site is a CF-causing factor.

Follow-up information

The three infants were followed up separately for 38, 24, and 26 months. The infants received sodium chloride and potassium chloride supplements that were adjusted on the basis of their serum electrolyte concentrations. The blood pH and electrolytes were maintained within the normal range. Infants P-1 and P-3 occasionally had coughs without other complaints. Their sputum cultures were negative.

Discussion

Given its rarity in China, CF is often neglected and is generally undiagnosed unless the patient presents typical CF lesions in the lung and pancreas or has specific familial medical history for CF. Thus, diagnosing CF in Chinese adults, let alone in infants, is very difficult. To the best of our knowledge, the three infants in this study are the youngest sporadic patients to have ever been diagnosed with sporadic CF in China. Moreover, these CF patients were diagnosed in the very early stage of the disease with a predominantly BS-like manifestation but without the obvious involvement of the respiratory system and pancreas. The successful maintenance of the symptom-free state of the infants during follow-up indicates the potential benefit of early diagnosis and timely intervention. The results of this report also demonstrate the significant diagnostic value of powerful exome sequencing for rare genetic diseases and indicate that doctors should be aware that CF, as well as BS, must be considered in infants or very young children with hypokalemic alkalosis.

Hypokalemic alkalosis is rare in infants. The most common causes of this symptom are BS and pseudo-BS. BS is caused by dysfunctional electrolyte reabsorption arising from ion channel mutations in the renal tubules. BS occurs in early infancy, and its typical clinical manifestations are growth retardation, hypokalemic alkalosis, diarrhea or vomiting, and other symptoms. The patients’ blood renin–angiotensin–aldosterone system (RAAS) levels increase, whereas their blood pressure remains normal [²⁰]. Pseudo-BS presents with hypokalemic alkalosis caused by salt loss for various reasons, including the use of diuretics, pyloric stenosis, chronic diarrhea, and periodic vomiting. Patients with pseudo-BS have normal blood RAAS levels.

All the infants in our study presented growth retardation, hypokalemic alkalosis, and normal blood pressure. The three infants were admitted to the nephrology division with an initial diagnosis of BS. Iatrogenic factors, such as the use of diuretics, were absent. Infants P-1 and P-3 did not experience persistent diarrhea or vomiting. However, infant P-2 had occasional diarrhea prior to being diagnosed with hypokalemic alkalosis. The three infants have histories of coughing and bronchitis. Their fasting blood glucose levels were normal, and their urine sugar was negative. Thus, we excluded other causes of secondary hypokalemic alkalosis.

Patients with CF manifest hypokalemic alkalosis, especially after hotshot [²¹]. Scurati-Manzoni et al. reviewed [²²] 262 cases of CF reported worldwide over the period of 1951 to 2013. All of the cases manifested electrolyte disturbance. Iatrogenic factors (e.g., long-term vomiting or diarrhea and pancreatic disease) were excluded from the main causes of electrolyte disturbance. Of the clinical manifestations, hypochloremia accounted for 98.2% (219/223), hyponatremia for 94.5% (206/218), hypokalemia for 81% (161/199), and metabolic alkalosis for 91.7% (188/205). The average sodium, potassium, chloride, and bicarbonate levels of the patients were approximately 125, 2.8, 71, and 37 mmol/L, respectively. No Chinese children were included in the review. In addition, no reports on Chinese children with CF and presenting electrolyte disturbances have been available to date.

The three infants included in our study initially presented hypokalemic alkalosis. Their average serum sodium, potassium, chloride, and bicarbonate levels were 109.9, 2.35, 63.7, and 36.4 mmol/L, respectively. Our patients showed more severe hyponatremia and hypochloremia than those reported in the literature [²²]. Their 24 h urine potassium, sodium, and chlorine levels were in the normal range. The patients did not exhibit severe electrolyte leakage from the kidneys although their sweat chloride concentrations drastically increased. At the same time, the parents of all three children complained that the infants had polydipsia. These findings suggested that the infants’ electrolyte losses are mainly due to sweat loss. Increased perspiration upon exposure to high summer temperatures may lead to severe manifestations in those infants and are an important cause of hypokalemic alkalosis. This result is consistent with that reported in the literature.

In this study, the three infants underwent RAAS testing early in the morning in a supine position. The results showed that their RAAS levels considerably increased as a likely result of hypovolemia in the infants.

Alkalosis is usually caused by bicarbonate resorption and the abnormal secretion of hydrogen ions and potassium ions arising from renal tubular lesions; these functions are mainly adjusted by the chloride ion-bicarbonate exchanger located in the intercalary cells of the collecting tubes [²³]. In infants with CF, hypokalemic alkalosis results from high chloride loss through sweating. Chloride depletion enables the renal tubules to generate more HCO₃⁻, causing potassium ions to shift from the extracellular to the intracellular space. Blood volume contraction induces secondary hyperaldosteronism and hypokalemia-metabolic alkalosis, which are both responsible for hypokalemic alkalosis. Additionally, in infants, extracellular fluid accounts for a large proportion of the total body fluid, and infants usually have a weak body fluid-regulatory function. Anorexia, diarrhea, vomiting, and other symptoms lead to further decreases in the amounts of extracellular fluid. The resultant increase in antidiuretic hormones results in hyponatremia. The increase in aldosterone aggravates hypokalemia. Therefore, hypokalemic alkalosis often occurs in infants with CF and is an important manifestation of CF. According to one study [²²], infants with CF who suffer from electrolyte disturbances are all younger than 2.5 years of age. Among these infants, 60% are not initially diagnosed with CF, and some are diagnosed with BS. In our study, the infants were younger than the infants included in other reports, with an average age of only 6.3 months. The infants showed severe hypokalemic alkalosis and were initially diagnosed with BS with mild pulmonary manifestations.

All three infants in our study were able to maintain serum electrolytes within the normal range after taking oral electrolytes and even remained relapse-free from pseudoBS during the 2–3 years of follow-up. Their lung lesions did not develop, and their pancreases were normal. These findings imply the benefits of early diagnosis and intervention in these infants.

Through exome sequencing, we successfully excluded mutations in the genes associated with BS and all other common renal tubulopathies and instead identified CFTR gene mutations in these infants. Six variants were found in different sites without hotspots. F508del is the most frequent mutation of CFTR in Caucasians but is rare among the Chinese [⁴,¹⁶]. Previous studies have reported four CF-causing variants. Among the two novel mutations, c.1526G>C is heterozygous for 1526G→C in exon 11. This mutation causes missense changes in G509A and coexists with two other CF-causing mutations in other alleles. This mutation is absent from the genomes of 100 healthy Chinese controls and from the human 1000 genome database. Moreover, PolyPhen-2 predicted that the mutation is harmful. Thus, c.1526G>C might be important in the development of CF in infants. The second novel mutation c.3062C>T results in the substitution of a highly conserved proline with leucine. Thus, this mutation is possibly pathogenic. A comparison of gene mutations failed to show a relationship between the infants’ serum electrolyte disturbances and specific CFTR gene mutations. BS-like manifestations in CF infants develop as salt loss through sweat and are aggravated during summer. Accordingly, all our cases presented with hypokalemic alkalosis during summer.

CF is a rare disease in China. The uncharacteristic manifestations of and failure to recognize hypokalemic alkalosis may delay diagnosis and cause the misdiagnosis of CF among Chinese patients. These factors may explain why the reported CF incidence among the Chinese population is considerably lower than that among Asian immigrants in Canada (1/9200) [²⁴] and Great Britain (1/10 000) [²⁵]. The infant and preschool (2–5 years) years provide a unique window of opportunity for postponing or even delaying the onset of CF lung disease. Chinese pediatricians must provide considerable attention to CF, especially in children with hypokalemic alkalosis.

Hypokalemic alkalosis is a common diagnostic indicator of BS. Patients with hypokalemic alkalosis are often admitted to the pediatric nephrology department. Sweat chloride is an important diagnostic criterion for CF [²⁶]. Consequently, patients with suspected CF, especially infants, must undergo sweat chloride concentration testing. If necessary, gene analysis can be used to detect CFTR mutations. Next-generation sequencing is a powerful, high throughput, time-efficient, and inexpensive technique for the identification of gene mutations. The prediction of the pathogenicity of several novel gene variants, however, presents challenges to clinicians. Nevertheless, in addition to the functional verification of new gene variants through experiments, some practical and convenient strategies are available to predict the pathogenicity of novel mutations in suspected genes.

Macroduct sweat collection and analysis are currently accepted diagnostic methods for CF. Given the low CF incidence in China, few Chinese hospitals and medical laboratories use this test. We initially used a simple filter paper method to quantify the sweat electrolytes of suspected CF patients. This method has been previously applied to other children in our department. All children, including patients with true BS and healthy infants, showed chloride concentrations of less than 45 mmol/L. We also found that the chloride concentrations of the parents of infants with CFs was less than 45 mmol/L. Considering the increasing number of patients with CF in China and the importance of measurement methodologies for sweat chloride, we recommend importing and applying the standard Macroduct collection system and Sweat-Chek conductivity analyzer [²⁷].

In summary, we are the first to report on the diagnosis of CF among Chinese infants whose initial clinical manifestations were BS-like hypokalemic alkalosis with mild respiratory manifestations. The infants must be monitored for the development of lesions in the respiratory system. Our study indicates the necessity of distinguishing CF from BS in Chinese infants with hypokalemic alkalosis, demonstrates the diagnostic value of powerful exome sequencing in rare genetic diseases, and expands the CFTR mutation spectrum in CF.

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