Intestinal hypomagnesemia in an Iranian patient with a novel TRPM6 variant: a case report and review of the literature

Abstract

TRPM6 is predominantly expressed in the kidney and colon and encodes a protein containing an ion channel domain and a protein kinase domain. It is crucial for magnesium homeostasis and plays important roles in epithelial magnesium transport and the active magnesium absorption. In this study, we present a 70-day-old Iranian female patient from consanguineous parents with hypomagnesemia and secondary hypocalcemia. She presented with seizures 19 days after birth and refractory watery non-bloody diarrhea. She consequently had failure to thrive. Other features included hypotonia, wide anterior fontanel, ventriculomegaly, and pseudotumor cerebri following administration of nalidixic acid. She had severe hypomagnesemia and hypocalcemia which were treated with magnesium and calcium supplementation. Despite initial unstable response to supplemental magnesium, she eventually improved and the diarrhea discontinued. The patient was discharged by magnesium and calcium therapy. At the last follow-up at age 2.5 years, the patient remained well without any recurrence or complication. Genetic testing by whole-exome sequencing revealed a novel homozygous frameshift insertion–deletion (indel) variant in exon 26 of the TRPM6 gene, c.3693-3699del GCAAGAG ins CTGCTGTTGACATCTGCT, p.L1231Ffs*36. Segregation analysis revealed the TRPM6 heterozygous variant in both parents. Patients with biallelic TRPM6 pathogenic variants typically exhibit hypomagnesemia with secondary hypocalcemia and present with neurologic manifestations including seizures. In some patients, this is also complicated by chronic diarrhea and failure to thrive. Long-term complications are rare and most of the patients show a good prognosis with supplemental magnesium therapy.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13730-023-00785-8.

Introduction

Transient Receptor Potential Melastatin 6 (TRPM6) belongs to TRPM family [1]. TRPMs are Ca²⁺-permeable cation channels localized predominantly on the plasma membrane. The structural machinery of TRPM channels includes intracellular N and C termini, 6 transmembrane segments, and a pore region between segments 5 and 6. The N-terminal domain has a conserved region, and the C-terminal domain contains a TRP motif, a coiled-coil region, and in TRPM6, an alpha protein kinase domain [2].

TRPM6 is predominantly expressed in the kidney and colon and encodes a protein containing an ion channel domain and a protein kinase domain. This channel protein is crucial for magnesium homeostasis and plays an essential role in epithelial magnesium transport and active magnesium absorption in the gut and kidney [3].

Pathogenic variants in TRPM6 are associated with hypomagnesemia with secondary hypocalcemia. Alternatively, spliced transcript variants encoding different isoforms have been noted for this gene. TRPM6 may also be downregulated in the distal convoluted tubule in response to cyclosporine, resulting in magnesium loss [4].

In this article, we report a novel TRPM6 homozygous variant in a 70-day-old female patient with manifestations of hypomagnesemia, secondary hypocalcemia, refractory diarrhea, seizure, failure to thrive and cerebral pseudotumor.

Case report

The index patient (P1) is a 70-day-old female from south Iran born at term to consanguineous parents (Fig. 1a). She experienced episodes of general tonic clonic seizures 19 days after birth which was found to be due to hypocalcemia and hypomagnesemia. The cause of her illness was not diagnosed before admission to our center and she had not received magnesium and calcium supplements. The patient was discharged after stabilization without the need for maintenance treatment. There was no history of miscarriage or early death in the family. She also complained of refractory watery non-bloody diarrhea lasting for about two weeks. She was hospitalized in another center for 10 days and her condition was not improved after taking calcium, magnesium, metronidazole, and nalidixic acid before admission to our center. She had also developed pseudotumor cerebri following administration of nalidixic acid.

Fig. 1.

a Parental consanguinity is shown in patients’ pedigree, b Failure to thrive in patients (photos at 2.5 years), c The change in serum magnesium level during admission til the last follow-up of P1, d Homozygosity is shown in P1 for variant c.3693_3699delGCAAGAGinsCTGCTGTTGACATCTGCT in TRPM6 gene by Sanger sequencing (a). Heterozygosity is shown in his mother (b) and his father (c)

She was breastfed and had recently started infant formula feed. The patient's weight was 3650 g, which was below the normal range for age. There was no evidence of malabsorption; however, she had documented failure to thrive (Z score: − 1.9) evident during her follow-ups (Fig. 1b).

On examination, she had hypotonia with a wide anterior fontanel. The brain ultrasound revealed mild hydrocephaly and increased diameter of the anterior horn of the left (8.5 cm) and right (9 cm) lateral ventricles. The abdominopelvic examination and ultrasound were normal and no urinary tract abnormality or organomegaly was detected.

In the initial laboratory evaluation, normal leukocyte count, normal lymphocytes, mild neutropenia, mild anemia, and thrombocytosis were found. The inflammatory markers (erythrocyte sedimentation rate (ESR) and c-reactive protein (CRP)) were normal. Renal function tests (the blood urea nitrogen (BUN) and creatinine (Cr)) were within the normal range for age. Normal levels of serum calcium, phosphorus, and low levels of serum magnesium and serum albumin were detected. The urine and stool analysis and culture were normal (Table 1).

Table 1.

Summary of laboratory findings at first admission of patient 1 (3 days After treatment at 73 days old)

Parameters (unit)	Results	Normal ranges
WBC × 103 (cell/uL)	9.9	6.0–17.5
RBC (× 106/ul)	4.3	2.7–4.5
Hb (gr/dL)	11.4	9.0–14.1
Plt (× 103/ul)	638	150–450
BUN (mg/dL)	8	5–18
Serum Cr (mg/dL)	0.4	0.12–0.8
Serum Na (mEq/L)	138	133–142
Serum K (mEq/L)	5.6	4.0–6.2
Serum P (mg/dL)	6	4.8–7
Serum Ca (mg/dL)	8.9	8.0–10.7
Serum Mg (mg/dL)	0.9	1.6–2.6
CRP (mg/dL)	5	≤ 10
ESR (mm/hr)	2	≤ 10
Urine Random Pro/Cr (mg/mg)	0.48	< 0.2
Urine Random Mg/Cr (mg/mg)	0.18	< 0.2
Urine Random Ca/Cr (mg/mg)	0.98	0.2–0.46
Urine Random P/Cr	1.4	0.85–1.44
Urine Random Na (mEq/L)	59	10–40
Urine Random K (mEq/L)	16	10–40
Urine Random Cl (mEq/L)	48	10–40
PTH (pg/ml)	69	10–55

WBC white blood cell, RBC red blood cell, Hb hemoglobin, Plt platelet, Cr creatinine, BUN blood urea nitrogen, CRP C-reactive protein, ESR estimated sedimentation rate, Pro protein, Mg magnesium, Ca calcium, Na sodium, K potassium, Cl chloride, P phosphorus

She received cefotaxime, azithromycin, phenobarbital, calcium gluconate injection and oral magnesium sulfate supplementation. The patient's initial calcium level was 7 mg/dL and after starting intravenous calcium gluconate 10% in the amount of 1 ml/kg for 48 h, the calcium level became normal.

The serum magnesium level was normalized with this treatment but dropped rapidly after discontinuation in hospital (Fig. 1c).

She also received sodium bicarbonate to correct the metabolic acidosis. Eventually, fever and respiratory distress resolved, diarrhea decreased and the patient became more alert, and her general condition improved. She was discharged and recommended to use Osteocare syrup regularly and manual magnesium sulfate syrup (50%) (0.8 cc daily). A larger amount of magnesium supplement was needed in the case of diarrhea, and in normal conditions, a smaller amount of magnesium was needed. In the last follow-up at age 2 years, she was completely improved and her height, weight, and blood pressure were normal. No other seizure episode or prolonged diarrhea was reported by her parents.

After further investigation, we found that her paternal cousin’s child had documented hypomagnesemia. The cousin (P2) was a 2-year-old female born to consanguineous parents with three healthy siblings. She presented at 2 months of age with seizure, cyanosis, diarrhea, and was found to have hypomagnesemia (serum Mg: 0.8 mg/dL) and hypocalcemia (serum Ca: 5.2 mg/dL). No nephrocalcinosis in renal ultrasound was found and she had normal weight gain. The level of fractional excretion of sodium (FE Na: 0.26%), fractional excretion of magnesium (FE Mg: 6%), and fractional excretion of calcium (FE Ca: 13%) were higher than normal range. She received intra venus (IV) Mg 20% but did not respond. Oral and IV Mg 50% along with calcium gluconate were administered, which normalized serum magnesium and calcium to 1.43 and 9.8 mg/dL, respectively.

To perform genetic analysis in the proband, a whole blood sample was collected in EDTA containing tubes. Genomic DNA was extracted from whole blood using Blood SV-mini kit (GeneAll Biotechnology Co., LTD, South Korea) according to the manufacturer instruction and whole-exome sequencing was performed. Library preparation was performed using Twist Human Core Exome Plus kit (Twist Bioscience, USA) using manufacturer instruction. Sequencing of libraries was done by high-throughput paired-end sequencing using NovaSeq sequencing platform (Illumina Inc., CA, USA).

Analysis of sequencing data was performed using Genome Analysis toolkit (GATK-v3.4.0) and detected variants were annotated. Proper filtering and then interpretation of a short list of variants in terms of pathogenicity was performed based on ACMG (American College of Medical Genetics and Genomics) guideline for variant interpretation [5].

Whole exome sequencing of the proband revealed a novel homozygous frameshift insertion–deletion (indel) variant in exon 26 of TRPM6 (NM_017662), c.3693-3699delGCAAGAGinsCTGCTGTTGACATCTGCT; p.L1231Ffs*4. This variant has not been previously reported, however, based on ACMG guidelines, this variant was classified as likely pathogenic. The indel would lead to a predicted truncated protein with loss of the C-terminal part of the protein including the alpha-kinase domain. Another TRPM6 variant c.3694del; p.G1232fs*31 at the same region has been reported as pathogenic in a patient with a comparable phenotype [6].

In addition, multiple lines of in silico computational analysis (including Mutation Taster and CADD) support the deleterious effect of this variant on the gene or gene product(s). The variant is absent in population databases (GenomAD, ExAC, 1000G, and our local database). Further analysis by PCR-Sanger sequencing validated homozygous status of detected variant in the patient and showed heterozygosity in both her parents (Fig. 1d). This finding was consistent with autosomal recessive pattern of inheritance.

Discussion

TRPM6 encodes a channel, which allows inward flow of magnesium cations and small amounts of calcium ions (Ca²⁺) to pass through cells [7]. Here, we report a 70-day-old girl with seizures, diarrhea, cerebral pseudotumor, hypomagnesemia, and secondary hypocalcemia in whom we have found a novel homozygous likely pathogenic TRPM6 variant which explains the phenotype.

From a search of the reported cases to date, 101 patients with TRPM6 variants from 76 families have been identified so far (Supplementary Table 1 [8–39]). The first patients with TRPM6 variants were reported in 1997 by Walder et. al [8]. They evaluated three families from two Bedouin Arab tribes, who were presented shortly after birth with a spectrum of neurologic symptoms including restlessness, tremor, neuromuscular hyperexcitability, and seizures. The supplementary treatment with magnesium improved the clinical symptoms in all the patients; however, two patients continued to suffer from refractory seizure and as a result, mental retardation, due to the delay in diagnosis. Genome-wide screening of pooled DNA samples of these patients revealed a region of homozygosity on chromosome 9 between markers D9S1874 and D9S1807. They also suggested an autosomal recessive pattern of inheritance which was later confirmed by another study [9].

The first presentation of the index patient described in the current study was a seizure attack at 19 days of age, which did not recur and no further neurodevelopmental complication was encountered. In the same way, almost all patients described in the literature had variable types of convulsions as the first presentation, namely generalized tonic–clonic, complex partial, myoclonic, and unspecified seizures. The age at the onset of symptoms ranged from 8 days to 6 years.

In 2005, a study was performed on 28 patients from 21 families with a pathogenic variant in TRPM6, in which 12 families were consanguineous. The seizure was the first symptom in 24 patients and tetany was also seen in some patients. Unfortunately, 3 patients experienced permanent neurologic damage due to delayed diagnosis [10]. In the same year, four Polish patients from 3 non-consanguineous families were identified to have pathogenic TRPM6 variants. These patients experienced seizures and convulsions within 2–6 weeks. Using oral magnesium, all patients improved and were alive and well at the time of the study [11].

In 2012, a study was performed on 8 Turkish children from 6 families (5 boys and 3 girls). All patients presented with tetany and convulsions which were caused by hypomagnesemia and secondary hypocalcemia [12].

Five patients with TRPM6 variants from 5 families were studied in 2014. One of them was from a consanguineous family. They presented with seizures which were caused by hypomagnesemia and secondary hypocalcemia. Magnesium therapy was given and all patients are reported to be alive and well [13].

Consistent with this, most of the identified TRPM6 patients had a good prognosis and responded to supplemental magnesium therapy, while a few patients developed neurological disorders or mental impairment. In a study, 5 patients presented with generalized seizures because of hypomagnesemia and secondary hypocalcemia. 4 of them were successfully treated with intravenous magnesium and oral magnesium therapy; however, one died at 8 weeks old due to muscular weakness [14]. Some studies also reported patients who survived the acute phase but progressed into life-long complications, mostly attributed to the delay in the diagnosis [8, 10, 15–18].

In conclusion, we presented a patient with seizures, hypomagnesemia and secondary hypocalcemia in whom we identified a novel variant in TRPM6 (a homozygous frameshift insertion–deletion (indel) variant, c.3693-3699del GCAAGAG ins CTGCTGTTGACATCTGCT, p.L1231Ffs*36). Similar reports of patients, preferably with a comprehensive evaluation of the family members, can help increase knowledge about the phenotypic range of this genetic defect and consequently reduce diagnostic delay and complications.

Supplementary Information

Below is the link to the electronic supplementary material.

Declarations

Conflict of interest

The authors have declared that no conflict of interest exists.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the Ethics Committee of Watson Genetic Laboratory, Tehran, Iran and with the 1964 Helsinki declaration and its later amendments.

Informed consent

Informed consent was obtained from all individual participants included in the study.