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Clinical and genetic analysis in a family with familial renal glucosuria: Identification of an N101K mutation in the sodium-glucose cotransporter 2 encoded by a solute carrier family 5 member 2 gene.

Abstract We report the identification of a mutation in the solute carrier family 5 member 2 (SLC5A2) gene, which encodes sodium–glucose cotransporter 2, in a family with familial renal glucosuria. The proband was a 26‐year‐old Japanese man referred to the diabetes division with repeated glucosuria without hyperglycemia. His mother, uncle and grandfather also had a history of glucosuria. A heterozygous missense mutation (c.303T>A:p.N101K) in SLC5A2 was identified in the patient and his mother, but not in 200 chromosomes from 100 healthy and unrelated individuals, or in 3,408 Japanese individuals in the Tohoku Medical Megabank. Furthermore, bioinformatics software predicted that this lesion would be pathogenic. We infer that the mutation led to clinically relevant sodium–glucose cotransporter 2 dysfunction. The patient showed no symptoms of hypoglycemia, but continuous glucose monitoring confirmed asymptomatic hypoglycemia.


INTRODUCTION
Familial renal glucosuria (FRG) is a rare renal tubular disorder characterized by isolated persistent glucosuria without hyperglycemia 1 . Mutations in the solute carrier family 5 member 2 (SLC5A2) gene, which encodes sodium-glucose cotransporter 2 (SGLT2), are responsible for the vast majority of FRG cases 2,3 . Most individuals with heterozygous mutations have mild glucosuria; those with homozygous or compound heterozygous mutations usually have severe glucosuria. In pedigrees with mild glucosuria, FRG is often inherited as an autosomal dominant trait 4 ; pedigrees with severe glucosuria suggest autosomal recessive inheritance 5 . However, recent studies have shown that inheritance of FRG might best be described as a codominant trait with variable penetrance 2 .
SGLT2 inhibitors have been proposed as a novel approach for the treatment of diabetes; such inhibitors are a new category of glucose-lowering drugs with insulin-independent action. Thus, FRG patients might provide an ideal model for evaluating the long-term safety of these agents.
FRG has been suggested to have a prevalence of 0.007% in Japanese schoolchildren 6 . However, the genetic basis of FRG in Japan is largely unknown. In the present study, clinical and genetic findings are described in a family with FRG associated with a SLC5A2 gene mutation.

Participants
The pedigree of a family with FRG is shown in Figure 1. The proband was a 26-year-old Japanese man who was referred to the diabetes division because of repeated glucosuria. The patient was first noted to have glucosuria without hyperglycemia and any other symptoms at the age of 3 years. His growth rate and intellectual development were normal.

Genetic analysis
After obtaining informed consent from the patient and his parents, blood samples were collected. Genomic DNA was extracted from peripheral white blood cells using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). We sequenced 14 coding exons in SLC5A2 together with their flanking intronic regions (Appendix S1; Table S1).

Pathogenicity prediction
The functional consequences of mutations were predicted using three online tools: Polyphen 2 (http://genetics.bwh.harvard.edu/ pph2/), SIFT (http://sift.jcvi.org) and MutationTaster (http:// www.mutationtaster.org). These in silico tools predict the effect of an amino acid substitution on protein function based on the degree of the conservation of the amino acid residues and the changes of protein structure [7][8][9] .

Continuous glucose monitoring
To evaluate the possible pathogenic effect of the SLC5A2 mutation on glycemic excursion, daily glucose profiles and low-glucose events were monitored for 14 consecutive days with a continuous glucose monitoring (CGM) system (FreeStyle Libre; Abbott Japan Co., Ltd., Chiba, Japan).

Ethical considerations
Ethical approval for this study was obtained from the ethics committee of Koseiren Tsurumi Hospital (approval No. 19-006), and the study conforms to the provisions of the Declaration of Helsinki.

RESULTS
Clinical characteristics of the participants are shown in Table 1. Laboratory tests, such as blood glucose and fasting counter-regulatory hormones, detected no abnormalities, except for glucosuria. The proband's glucose tolerance was normal during a 3-h, 75-g oral glucose tolerance test.
A T-to-A transition at complementary deoxyribonucleic acid (cDNA) position 303 of SLC5A2 (c.303T>A) was identified in the patient and his mother; this mutation corresponded to an asparagine-to-lysine substitution at position 101 of the SGLT2 protein (N101K) ( Figure 2). The proband and his mother were heterozygous for the mutation. The mutation was not detected in the proband's father, nor in 200 chromosomes derived from 100 healthy, unrelated individuals.
Online analysis using Polyphen 2, SIFT and MutationTaster suggested that the N101K mutation would be deleterious and could be associated with FRG ( Figure S1).
Although the proband did not complain of symptoms of hypoglycemia, two weeks of CGM showed both nocturnal and diurnal asymptomatic hypoglycemia (<70 mg/dL) for a mean of 236 min/day ( Figure 3).

DISCUSSION
In healthy individuals, the kidneys filter approximately 180 g of glucose per day; nearly 100% of the glucose is reabsorbed in the proximal convoluted tubule through SGLTs to maintain glucose homeostasis 10 . SGLT2 is responsible for the active transport of glucose across the brush border membrane, and is expressed almost exclusively in the kidney, accounting for the bulk of glucose reabsorption 10 . Mutations in the SLC5A2 gene, which encodes SGLT2, have been reported to be responsible for FRG 2,3 .
In the present study, a heterozygous missense mutation (c.303T>A:p.N101K) in SLC5A2 was identified in a Japanese family with FRG. This mutation was not detected in 200 chromosomes from 100 healthy and unrelated individuals, or in 3,408 Japanese individuals in the Tohoku Medical Megabank, which is the largest genome database based on wholegenome sequences of the Japanese general population (https:// jmorp.megabank.tohoku.ac.jp/201905/variants/by-gene/SLC5A2). In the Genome Aggregation Database derived from 123,136 exome sequences and 15,496 whole-genome sequences from unrelated individuals sequenced as part of various diseasespecific and population genetic studies, only one example of this variant (rs1306939757) has been reported, corresponding to a frequency of 0.0000319 (http://gnomad.broadinstitute.org/), indicating the rarity of this variant. The N101 amino acid residue was found to be highly conserved across SGLT2 homologs in multiple species and among human SGLT subtypes ( Figure S2). In humans, only SGLT3, which is encoded by the SLC5A4 gene, harbors a replacement of the asparagine residue (by threonine). However, SGLT3 is not a cotransporter, but rather a glucosensor expressed in cholinergic neurons at the neuromuscular junction 11 , suggesting that the N101 amino acid residue in those SLC5 members that co-transport glucose might play a critical role in the symporter function. Furthermore, all three of the bioinformatics programs consulted here predicted that the N101K mutation would be pathogenic ( Figure S1). Further work will be required to prove that the N101K mutation actually results in a loss of function, thereby increasing renal glucosuria.
Given the clinical manifestations shown by the patient, we infer that the mutation leads to clinically relevant SGLT2 dysfunction.
Notably, in the present study, the heterozygous patient had severe glucosuria (urine glucose: 43.0 g/1.73 m 2 /24 h), whereas his mother (also heterozygous for the same mutation) did not (urine glucose: 0.2 g/1.73 m 2 /24 h). This result indicates that the inheritance of renal glucosuria is best described as codominant, and its variable penetrance might relate to the compensatory ability of the wild-type allele or environment factors might be associated with the glucose reabsorption 2 . Notably, although Sglt2-/mice excrete much of the filtered glucose, urinary glucose concentrations do not appreciably differ between Sglt2+/and wildtype mice, indicating that one intact Sglt2 allele is sufficient to achieve normal renal glucose reabsorption 12 .
To our knowledge, this pedigree represents the first case in which CGM has been carried out in a patient with FRG. Renal glucosuria is thought to be a "non-disease"; the great majority of affected individuals do not have any complaints, and only rarely have been reported to show a propensity to hypoglycemia 2,13 . However, CGM of this proband suggested the presence of asymptomatic hypoglycemia (as judged by frequency and severity; Figure 3) in an otherwise asymptomatic Fasting plasma glucagon levels were measured using the sandwich enzyme-linked immuno sorbent assay (Mercodia, Uppsala, Sweden). BMI, body mass index; eGFR, estimated glomerular filtration rate; HOMA-R, homeostasis model assessment for insulin resistance; IRI, immunoreactive insulin.   FRG patient. Because of their experience with extended hypoglycemic periods since birth, patients with FRG might be accustomed to hypoglycemia. The present study had two limitations. First, hypoglycemia was detected with CGM, but not by laboratory test or by selfmonitoring of blood glucose. Conflicting data about the accuracy of CGM at low-glucose levels have been reported 14,15 . Second, details of clinical phenotypes were studied only in a proband in the present study. Urinary glucose excretion in FRG varies widely among patients, ranging from 0.6 to 202 g/ 1.73 m 2 /24 h 2,16 . The present patient excreted large amounts of urinary glucose, despite being heterozygous for the mutation. Although FRG has been considered to be a "non-disease," the condition sometimes might be accompanied by asymptomatic hypoglycemia on an everyday basis, as shown in the present case. Further studies on CGM in patients with FRG will be required to define the pathological characteristics of this disease.

DISCLOSURE
The authors declare no conflict of interest.

SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the end of the article. Figure S1 | Pathogenicity prediction. Figure S2 | Multiple amino acid sequence alignments. Table S1 | Primers used for solute carrier family 5 member 2 gene polymerase chain reaction and sequencing analysis. Appendix S1 | Mutation analysis.