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Revista chilena de pediatría

versión impresa ISSN 0370-4106

Rev. chil. pediatr. vol.89 no.6 Santiago dic. 2018 


Detection of mutations of the HNF1B gene in children with congenital anomalies of the kidney and urinary tract

M. Nicole Bascur P.1 

M. Luisa Ceballos O.2 

Mauricio Farfán U.2 

Iván Gajardo H.2 

Joaquín López C.2 

1 Guillermo Grant Benavente Hospital, Concepción, Chile.

2 Luis Calvo Mackenna Children's Hospital, Santiago, Chile.



Congenital anomalies of the kidney and urinary tract are caused by genetic alterations mostly unknown. Mutations in the gene that codes for hepatocyte nuclear factor 1B (HNF1B) are the most frequently described monogenic causes. Data are unknown in Chile and Latin America.


To determine the presence of variants of the HNF1B gene in Chilean children with conge nital anomalies of the kidney and/or the urinary tract and their clinical characteristics.

Patients and Method:

Descriptive study with children aged 10 months to 17 years, patients of the Calvo Mackenna Hospital Nephrology Unit, with cystic renal dysplasia, non cystic renal dysplasia/hypoplasia, horses hoe kidney between April and December 2016. HNF1B variants were determined by sequencing of exons 1, 2, 3 and 4 after DNA extraction and amplification. Restriction enzymes were used to define if the variants were homo or heterozygous. Direct family members of index cases were studied with sequencing of the affected exon.


32 patients were included, 43.75% males, median age 11 years. 65.6% of them had non-cystic renal dysplasia, 31.25% cystic renal dysplasia, and 3.15% hor seshoe kidney. In two patients (6.25%) the same heterozygous genetic variant was detected in exon 4, position 1027 (C1027T), not previously described. The study of relatives found the same variant in three out of five individuals, all without congenital nephro-urological anomalies.


We confirmed the presence of a not previously described heterozygous genetic variant of the HNF1B gene. This work initiates the search for this type of mutations in our region which allows us to approach the knowledge of causality, determination of extrarenal involvement, and genetic counseling.

Keywords: Urinary tract; CAKUT; mutations; Congenital anomalies; Kidney; renal dysplasia; renal cysts


Congenital anomalies of the kidney and urinary tract (CAKUT) have an incidence of 1 in 500 live newborns and are currently the most common cause of chronic kidney disease (CKD) in childhood. Mutations in the TCF2 gene, which codes for hepatocyte nuclear factor 1B (HNF1B), are the most common monogenic cause of impaired renal development in CAKUT patients1.

The transcription factor HNF1B regulates the ex pression of genes involved in the early organs development such as the kidney, pancreas, liver, intestine, and lung2. It was originally identified as one of those responsible for type MODY5 diabetes (maturity-onset diabetes of the young), a monogenic disease that is transmitted in an autosomal dominant manner. The observation of the high frequency of renal malformations in these patients made it possible to determine their association with CAKUT( 1 , 2 ).

The TCF2 gene is located in the q12 region of chro mosome 17 and contains nine exons. To date, more than 50 different mutations have been reported, such as missense, nonsense, frame-shift, and splicing. A complete deletion of the gene has been detected in a significant percentage of patients. Out of the reported mutations, most are in exons 1, 2, 3, and 4. Due to the high number of described mutations, the study of the association between this gene and renal pathology is ca rried out mainly through sequencing techniques1.

Renal malformations caused by deletion and/or mutations of the TCF2 gene are very heterogeneous, with uni- or bilateral cystic renal dysplasia as the most frequent finding. Renal aplasia or hypoplasia, and hor seshoe kidney1-3 have also been found.

Extrarenal involvement may include elevated liver enzymes, endocrine, and exocrine pancreatic involvement, genital tract malformations causing infertility, hypomagnesemia, hyperuricemia, and early-onset gout. From a clinical point of view, the presence of extrarenal involvement does not contribute significantly to early diagnosis since it is usually expressed later in life1,2,4.

Studies on the prevalence of mutations in the TCF2 gene in CAKUT carrier patients have been carried out in Europe, the United States, and Japan5-7, however, data are unknown in Latin America. The objective of this work is to determine the presence of variants of the gene coding for HNF1B (TCF2) in Chilean children with congenital anomalies of the kidney and/or urinary tract and their phenotypic characteristics.

Patients and Methods


Prospective, observational, descriptive study with patients of the Luis Calvo Mackenna Hospital Nephrology Unit for nine months, between April and Decem ber 2016. The presence of at least one of the following phenotypes was considered as inclusion criteria: uni- or bilateral cystic renal dysplasia, uni- or bilateral re nal dysplasia or hypoplasia, and horseshoe kidney. The ultrasound evaluation was performed by pediatric ra diologists at the Luis Calvo Mackenna Hospital. From the ultrasound point of view, renal dysplasia was defi ned as the finding of alteration of the corticomedullary differentiation and/or diffuse hyperechogenicity of the renal parenchyma; hypoplasia due to a renal length less than 2 SD for age8. Patients with obstructive uropathies and/or moderate to severe vesicoureteral reflux (grade III to V) that could generate secondary renal damage were excluded, and those with clinical and/or molecu lar evidence of other genetic anomalies that explained the malformations they presented in the nephrourinary tract, such as, for example, autosomal dominant or re cessive polycystic kidney disease.

Molecular and ultrasound studies were carried out on the first-degree relatives of the index cases.

Detection of TCF2 gene variants

Genomic DNA was obtained from peripheral blood using the MagNa Pure Compact kit (Roche) according to the manufacturer’s protocol. The first four exons of the TCF2 gene were amplified through Polymerase Chain Reaction (PCR), specific primers were used for exons 1, 2, 3, and 4 (Promega) previously described in the literature9. The purity of the obtained amplicon was evaluated using a 2% agarose gel. The PCR product was sequenced through the Sanger technique, using the services of Macrogen (Korea) with 100% sequencing coverage. Bioinformatic analysis of the sequences was performed with Sequencher 5.4.5 (Gene Codes Corp.). As a reference, the NM_00458.2 sequence of the TCF2 gene was used.

To determine if the found variant affected one or both alleles (homo or heterozygous), the Polymerase Chain Reaction-Restriction Fragment Length Poly morphism (PCR-RFLP) technique was used. Exon 4, which presented the variant under study, was amplified by PCR, and the amplified product was digested with the Earl enzyme (Promega), capable of recognizing the DNA segment that incorporates the nucleotide variant position. The enzymatic digestion products were analy zed through Electrophoresis in polyacrylamide gels.

Biochemical Study

All patients included in the study were evaluated for plasma biochemical parameters, including: Creatinine (mg/dl), urea nitrogen (mg/dl), blood glucose (mg/dl), GPT-GOT transaminases (UI/L), uric acid (mg/dl), and magnesium (mEq/L). The samples were processed in the Luis Calvo Mackenna Hospital Central Laboratory. The calculation of the glomerular filtration rate was perfor med using the Schwartz Formula10.

A molecular and renal ultrasound study was carried out on the first-degree relatives of the index cases.

Ethical Aspects

The study was approved by the Human Research Ethics Committee of the School of Medicine, Univer sity of Chile, and the Luis Calvo Mackenna Hospital Management. Informed Consent was obtained from the responsible adult in all cases and Assent in patients over 12 years of age.


32 patients were included, 18 female (56.25%), the median age was 11 years, ranging from ten months to 17 years. 21 out of the 32 had non-cystic renal dysplasia (65.6%); from this group, 76.2% had bilateral involvement. Ten patients (31.25% of the sample) presented cystic dysplasia, bilateral in three of them. One patient was admitted to the study with a horseshoe kidney diagnosis (Figure 1).

Figure 1 Characterization of the sample according to type of Nephrourolo- gical Malformation. 

Regarding renal function, 12 patients had CKD in stage 1, six patients in stage 2, and 14 patients in stage 5. Out of the latter group, two patients were on peritoneal dialysis and 12 patients were transplanted (Figure 2).

Figure 2 Characterization of the sample according to the Chronic Kidney Disease Stage. 

The blood glucose, transaminases, magnesium, and uric acid levels of the 32 patients were within normal ranges.

The same heterozygous variant (Figure 3) was found in two of the 32 studied patients, corresponding to 6.25% of the sample. This variant is located in exon 4, at position 1027, determines a change in the nitrogenous base (cytosine-to-thymine - C1027T), which re sults in a change in the amino acid encoded at position 343 of proline-to-serine.

One of the patients affected by this variant is a 17-year-old adolescent with left cystic renal dysplasia and normal renal function. The other index case is an 8-year-old male patient with bilateral non-cystic renal dysplasia who is currently transplanted (Table 1). These patients have no known blood relationship.

The presence of this variant was studied in the family of one of the index cases since the other patient has adoptive parents and the history of his/her biologi cal parents is unknown. The same heterozygous variant (C1027T) was found in three out of the five studied family members (one of the parents and two of his/ her three siblings), which shows that it is not a de novo variant (Figure 3). These studied relatives are asymptomatic, with normal renal and urinary tract ultrasound study.

Table 1 Características generales de los casos índices. 

Figure 3 Polyacrylamide gel electrophoresis showing products of the exon 4 digestion with restriction enzyme. Exon 4, previously amplified through PCR, is digested using Earl restriction endonuclease, which recognizes the DNA segment that incorporates the variant. Eigth samples were analyzed, the first 3 on the left are from control patients and the 5 on the right correspond to the 2 index cases and their 3 relatives variant carriers. It can be observed that in the controls the enzyme does not fragment the DNA, and in the carriers of the variant one of the alleles is fragmented and the other remains intact, which allows us to show that it is a heterozygous variant. The fragmentation by the enzyme originate smaller pieces of DNA that migrate in the gel with greater speed and can be observed. 


This is the first time that a study has been carried out in Chile, which tries to find an association bet ween CAKUT and variants of the gene that encodes for HNF1B.

We analyzed 32 non-consanguineous patients with CAKUT phenotype and identified two of them with the same heterozygous variant in exon 4 of the gene coding for HNF1B. This variant is not previously described in the literature, therefore its pathogenic role is unknown, although finding an identical alteration in both patients could suggest it. In order to determine its pathogenici ty, an in silico analysis (computer simulation) was used with three available software: Polyphen-2, SIFT, and Mutation Taster. The first two predict a benign and to lerable effect; the third indicates that this variant could be the cause of illness.

The first index case is a 17-year-old adolescent with history of physiological pregnancy, without prenatal ultrasound abnormalities, born by vaginal delivery at 38 weeks, birth weight 2,850 g. She was admitted in the Polyclinic of Nephrology in February 2012, at 13 years of age, due to the ultrasound finding of a left kidney with signs of cystic dysplasia during a study of ima ges due to low-back pain. A DMSA renal scintigraphy showed left kidney scintigraphic exclusion. Within her examination, it highlights blood creatinine 0.68 mg/dl and normal urine test. The patient evolves asymptoma tic and normotensive. In April 2015, she is referred to adult medicine for further follow-up. The second index case, a 10-year-old male, has history of irregularly controlled pregnancy, with no known maternal pathology or abnormal ultrasound findings. Born by vaginal delivery at 35 weeks, birth weight 2,020 g. He was admit ted to the Polyclinic of Nephrology in November 2010 referred from another pediatric center, with diagnoses of bilateral renal dysplasia and end-stage chronic renal disease in peritoneal dialysis, to start a pre-transplant study. In his history, the situation of social vulnerability due to family abandonment stands out. He receives a kidney transplant from a donor who died in February 2014, at the age of five. The patient is currently five years post-transplant, under adoptive family care, with an estimated 70 ml/min/1,73m2 filtration rate.

The literature describes cases of patients with in herited or de novo variants in HNF1B in about 50% of cases, respectively1. We studied the family of one of the index cases, finding that three family members carried the same heterozygous variant of exon 4. The very different phenotypes of the two index patients and the absence of renal malformations in the relatives carrying the variant reaffirms the lack of genotype-phenotype correlation, which is already described in the literatu re and suggests an inheritance with incomplete penetrance6. Therefore, the pathogenic role of this genetic variant is uncertain. An indirect way of approaching this could be to determine its relative absence in a sig nificant sample of healthy population, which could be done in a cost-effective way through PCR-RFLP.

The variants frequency of the gene coding for HNF1B in our study was 6.25%, which is consistent with results described in other series, ranging from 5 to 31%1. In 2006, Ulinski11 published a descriptive study of 80 French patients carrying CAKUT, showing TCF2 alterations in 31% of them, either mutation or comple te deletion of the gene. Thomas12, in 2011 published a US series of patients carrying renal aplasia and/or hypoplasia, finding 5% of patients with alterations of the gene coding for HNF1B. Different publications have shown that the variants frequency of this gene is higher in patients with CAKUT who present uni- or bilateral renal cysts, reaching 50% of the studied children in this group11,13.

No alterations were observed in uric acid or plasma magnesium levels, nor transaminases or blood glucose elevation. This can be explained because much of the extrarenal manifestations of the HNF1B variants manifest late in life.

We cannot fail to mention the limitations of our study. In the first place, due to cost reasons, only the variants of exons 1, 2, 3 and 4 were analyzed, with a total of nine. In addition, no search was made for gene deletions, which would have required other technique types. This explains the relatively low frequency of variants in the TCF2 gene found in our series. On the other hand, the fact that none of our patients have presented abnormal basal blood glucose does not allow us to rule out diabetes or prediabetic states since there was no glucose tolerance test and/or blood insulin measu rement. In this study, no imaging tests were performed to rule out anatomical abnormalities of the pancreas or internal genitalia, which may be extrarenal manifesta tions of HNF1B variants. Finally, we cannot rule out in our patients abnormalities in other involved genes in renal development, which have also been associated with CAKUT: Ret, GDNF, Pax2, Six2, UMOD, BMP4, among others14.


This work is the first study carried out in Chile that looks for gene variants coding for HNF1B, whose alterations are recognized as the most frequent monogenic cause of CAKUT, and has allowed us to recognize a new variant whose pathogenic value we do not know.

The genotypic characterization of this group of patients is far from being a reality in clinical practice, however, in selected groups could be very useful for ge netic counseling, prevention, and search for extrarenal manifestations.

Scientific research that allows us to know the ge notypic characteristics of patients with nephrourinary malformations provides a basis for a thorough unders tanding of the origin of these diseases and is the starting point for future studies.

Ethical Responsibilities

Human Beings and animals protection: Disclosure the authors state that the procedures were followed according to the Declaration of Helsinki and the World Medical Association regarding human experimentation developed for the medical community.

Data confidentiality: The authors state that they have followed the protocols of their Center and Local regulations on the publication of patient data.

Rights to privacy and informed consent: The authors have obtained the informed consent of the patients and/or subjects referred to in the article. This do cument is in the possession of the correspondence author.

Conflicts of Interest: Authors declare no conflict of interest regarding the present study.

Financial Disclosure: Luis Calvo Mackenna Hospital Research Award, No vember 2015 version. SAVAL 2015 Research Projects Award.

Aknowledgments: To Dr. Francisco Cano Schuffeneger, for review of the manuscript.


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Received: June 18, 2018; Accepted: November 12, 2018

Correspondence: Nicole Bascur. E-mail:

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