Double synonymous mutations in exon 9 of the Cullin3 gene restore exon inclusion by abolishing hnRNPs inhibition.

All mutations in exon 9 of the Cullin3 gene associated with pseudohypoaldosteronism type II (PHA II) contribute to exon skipping to different degrees, but the specific molecular mechanism of this aberrant splicing is still unclear. The aims of this study were to investigate the regulatory mechanism underlying two synonymous splicing events, c.1221A > G (p. Glu407Glu) and c.1236G > A (p. Leu412Leu), and to discover a therapeutic strategy for correcting this aberrant splicing by targeting potential regulatory sites. Through a series of RNA pulldown, silver staining, western blotting, small interfering RNA knockdown, in vitro overexpression and single or double site-directed mutagenesis experiments, we first explored the pathogenesis of exon 9 skipping caused by mutations in the CUL3 gene and verified that the main splicing regulators associated with the synonymous c.1221A > G and c.1236G > A mutations were heterogeneous nuclear ribonucleoproteins. In addition, we verified that introducing another synonymous mutation, c.1224A > G (A18G), significantly rescued the abnormal splicing caused by c.1221A > G and c.1236G > A, highlighting the therapeutic potential for the treatment of PHA II.

Functional analysis of the CTNS gene exonic variants predicted to affect splicing.

Cystinosis is a severe, monogenic systemic disease caused by variants in CTNS gene. Currently, there is growing evidence that exonic variants in many diseases can affect pre-mRNA splicing. The impact of CTNS gene exonic variants on splicing regulation may be underestimated due to the lack of routine studies at the RNA level. Here, we analyzed 59 exonic variants in the CTNS gene using bioinformatics tools and identified candidate variants that may induce splicing alterations by minigene assays. We identified six exonic variants that induce splicing alterations by disrupting the ratio of exonic splicing enhancers/exonic splicing silencers (ESEs/ESSs) or by interfering with the recognition of classical splice sites, or both. Our results help in the correct molecular characterization of variants in cystinosis and inform emerging therapies. Furthermore, our work suggests that the combination of in silico and in vitro assays facilitates to assess the effects of DNA variants driving rare genetic diseases on splicing regulation and will enhance the clinical utility of variant functional annotation.

Identification of seven variants in the col4a1 gene that alter RNA splicing by minigene assay.

Type IV collagen is an integral component of basement membranes. Mutations in COL4A1, one of the key genes encoding Type IV collagen, can result in a variety of diseases. It is clear that a significant proportion of mutations that affect splicing can cause disease directly or contribute to the susceptibility or severity of disease. Here, we analyzed exonic mutations and intronic mutations described in the COL4A1 gene using bioinformatics programs and identified candidate mutations that may alter the normal splicing pattern through a minigene system. We identified seven variants that induce splicing alterations by disrupting normal splice sites, creating new ones, or altering splice regulatory elements. These mutations are predicted to impact protein function. Our results help in the correct molecular characterization of variants in COL4A1 and may help develop more personalized treatment options.

A novel heterozygous variant of the SALL1 gene with atypical Townes-Brocks syndrome phenotypes in Chinese family.

Townes-Brocks syndrome (TBS) is an autosomal dominant disorder characterised by the triad of anorectal, thumb, and ear malformations. It may also be accompanied by defects in kidney, heart, eyes, hearing, and feet. TBS has been demonstrated to result from heterozygous variants in the SALL1 gene, which encodes zinc finger protein believed to function as a transcriptional repressor. The clinical characteristics of an atypical TBS phenotype patient from a Chinese family are described, with predominant manifestations including external ear dysplasia, unilateral renal hypoplasia with mild renal dysfunction, and hearing impairment. A novel heterozygous variant c.3060T>A (p.Tyr1020*) in exon 2 of the SALL1 gene was identified in this proband. Pyrosequencing of the complementary DNA of the proband revealed that the variant transcript accounted for 48% of the total transcripts in peripheral leukocytes, indicating that this variant transcript has not undergone nonsense-mediated mRNA decay. This variant c.3060T > A is located at the terminal end of exon 2, proximal to the 3' end of the SALL1 gene, and exerts a relatively minor impact on protein function. We suggest that the atypical TBS phenotype observed in the proband may be attributed to the truncated protein retaining partial SALL1 function.

Identified eleven exon variants in PKD1 and PKD2 genes that altered RNA splicing by minigene assay.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenic multisystem disease caused primarily by mutations in the PKD1 gene or PKD2 gene. There is increasing evidence that some of these variants, which are described as missense, synonymous or nonsense mutations in the literature or databases, may be deleterious by affecting the pre-mRNA splicing process. RESULTS: This study aimed to determine the effect of these PKD1 and PKD2 variants on exon splicing combined with predictive bioinformatics tools and minigene assay. As a result, among the 19 candidate single nucleotide alterations, 11 variants distributed in PKD1 (c.7866C > A, c.7960A > G, c.7979A > T, c.7987C > T, c.11248C > G, c.11251C > T, c.11257C > G, c.11257C > T, c.11346C > T, and c.11393C > G) and PKD2 (c.1480G > T) were identified to result in exon skipping. CONCLUSIONS: We confirmed that 11 variants in the gene of PKD1 and PKD2 affect normal splicing by interfering the recognition of classical splicing sites or by disrupting exon splicing enhancers and generating exon splicing silencers. This is the most comprehensive study to date on pre-mRNA splicing of exonic variants in ADPKD-associated disease-causing genes in consideration of the increasing number of identified variants in PKD1 and PKD2 gene in recent years. These results emphasize the significance of assessing the effect of exon single nucleotide variants in ADPKD at the mRNA level.

Particulate matter of air pollution may increase risk of kidney failure in IgA nephropathy.

IgA nephropathy (IgAN) is characterized by deposition of galactose-deficient IgA1 (Gd-IgA1) in glomerular mesangium associated with mucosal immune disorders. Since environmental pollution has been associated with the progression of chronic kidney disease in the general population, we specifically investigated the influence of exposure to fine particulate matter less than 2.5 µm in diameter (PM2.5) on IgAN progression. Patients with biopsy-proven primary IgAN were recruited from seven Chinese kidney centers. PM2.5 exposure from 1998 to 2016 was derived from satellite aerosol optical depth data and a total of 1,979 patients with IgAN, including 994 males were enrolled. The PM2.5 exposure levels for patients from different provinces varied but, in general, the PM2.5 exposure levels among patients from the north were higher than those among patients from the south. The severity of PM2.5 exposure in different regions was correlated with regional kidney failure burden. In addition, each 10 µg/m3 increase in annual average concentration of PM2.5 exposure before study entry (Hazard Ratio, 1.14; 95% confidence interval, 1.06-1.22) or time-varying PM2.5 exposure after study entry (1.10; 1.01-1.18) were associated with increased kidney failure risk after adjustment for age, gender, estimated glomerular filtration rate, urine protein, uric acid, hemoglobin, mean arterial pressure, Oxford classification, glucocorticoid and renin-angiotensin system blocker therapy. The associations were robust when the time period, risk factors of cardiovascular diseases or city size were further adjusted on the basis of the above model. Thus, our results suggest that PM2.5 is an independent risk factor for kidney failure in patients with IgAN, but these findings will require validation in more diverse populations and other geographic regions.

Two novel variants in a Bardet-Biedl syndrome type 5 patient with severe renal phenotype.

Bardet-Biedl syndrome type 5 (BBS5) has never been reported in Chinese populations. The aim of this study is to report the first BBS5 case in China, explore the phenotype and genotype correlation. The case was male, Han nationality, born with polydactyly and gained weight after birth, accompanied by polydipsia, polyuria and nocturia. He was found to have low vision at the age of 7 years, and having insufficient renal function at the age of 20 years. After hospitalization, he was found to have suffered from atrophy of the whole layer of macular retina, and end stage of kidney disease, presenting with shrinking and cyst-like changes of bilateral kidneys. Whole-exome sequencing was performed among the proband and his parents (Trios), further validated using Sanger sequencing and quantitative polymerase chain reaction. Two novel compound heterozygous variants of BBS5 gene [a missense variant NC_000002.12, NM_152384.3:c.1A>G(p.Met1?) & a large deletion c.(?_-60)_(386 + 1_387-1)del] were detected. BBS is rare, whereas BBS5 is rarer. Herein, we reported a Chinese BBS5 patient with severe renal phenotype and identified two novel BBS5 variants.

Identification of two novel variants of BCS1L gene in a patient with classical GRACILE syndrome.

BCS1L pathogenic variants cause widely different clinical phenotypes. Disease phenotypes can be as mild as Björnstad syndrome, characterized by pili torti (abnormal flat twisted hair shafts) and sensorineural hearing loss, or as severe as GRACILE syndrome, characterized by growth restriction, aminoaciduria, cholestasis, iron overload, lactic acidosis and early death. BCS1L pathogenic variants are also linked to an undefined complex III deficiency, a heterogeneous condition generally involving renal and hepatic pathologies, hypotonia, and developmental delays. So far, all patients with GRACILE syndrome carry a homozygous p.Ser78Gly variant in BCS1L gene by reviewing articles. A 24-day-old boy presented with typical clinical phenotype of GRACILE syndrome. The Whole Exome Sequencing confirmed that the patient had a missense variant (c.245C > T, p.Ser82Leu) and a small deletion (c.231_232delCA, p. Ser78Cysfs*9) in BCS1L gene inherited from his father and mother separately, he died at 5 months of age. We reported a patient with GRACILE syndrome and identified two novel variants in BCS1L gene. Our study expands the mutational spectrum of BCS1L gene associated with GRACILE syndrome and will be beneficial for genetic diagnosis.

Role of abnormal energy metabolism in the progression of chronic kidney disease and drug intervention.

Chronic kidney disease (CKD) is a severe clinical syndrome with significant socioeconomic impact worldwide. Orderly energy metabolism is essential for normal kidney function and energy metabolism disorders are increasingly recognized as an important player in CKD. Energy metabolism disorders are characterized by ATP deficits and reactive oxygen species increase. Oxygen and mitochondria are essential for ATP production, hypoxia and mitochondrial dysfunction both affect the energy production process. Renin-angiotensin and adenine signaling pathway also play important regulatory roles in energy metabolism. In addition, disturbance of energy metabolism is a key factor in the development of hereditary nephropathy such as autosomal dominant polycystic kidney disease. Currently, drugs with clinically clear renal function protection, such as Angiotensin II Type 1 receptor blockers and fenofibrate, have been proven to improve energy metabolism disorders. The sodium-glucose co-transporter inhibitors 2 that can mediate glucose metabolism disorders not only delay the progress of diabetic nephropathy, but also have significant protective effects in non-diabetic nephropathy. Hypoxia-inducible factor enhances ATP production to the kidney by improving renal oxygen supply and increasing glycolysis, and the mitochondria targeted peptides (SS-31) plays a protective role by stabilizing the mitochondrial inner membrane. Moreover, several drugs are being studied and are predicted to have potential renal protective properties. We propose that the regulation of energy metabolism represents a promising strategy to delay the progression of CKD.

Identification of seven exonic variants in the SLC4A1, ATP6V1B1, and ATP6V0A4 genes that alter RNA splicing by minigene assay.

Primary distal renal tubular acidosis (dRTA) is a rare tubular disease associated with variants in SLC4A1, ATP6V0A4, ATP6V1B1, FOXⅠ1, or WDR72 genes. Currently, there is growing evidence that all types of exonic variants can alter splicing regulatory elements, affecting the precursor messenger RNA (pre-mRNA) splicing process. This study was to determine the consequences of variants associated with dRTA on pre-mRNA splicing combined with predictive bioinformatics tools and minigene assay. As a result, among the 15 candidate variants, 7 variants distributed in SLC4A1 (c.1765C>T, p.Arg589Cys), ATP6V1B1 (c.368G>T, p.Gly123Val; c.370C>T, p.Arg124Trp; c.484G>T, p.Glu162* and c.1102G>A, p.Glu368Lys) and ATP6V0A4 genes (c.322C>T, p.Gln108* and c.1572G>A, p.Pro524Pro) were identified to result in complete or incomplete exon skipping by either disruption of exonic splicing enhancers (ESEs) and generation of exonic splicing silencers, or interference with the recognition of the classic splicing site, or both. To our knowledge, this is the first study on pre-mRNA splicing of exonic variants in the dRTA-related genes. These results highlight the importance of assessing the effects of exonic variants at the mRNA level and suggest that minigene analysis is an effective tool for evaluating the effects of splicing on variants in vitro.

Genotypic and phenotypic analysis in 51 Chinese patients with primary distal renal tubular acidosis.

The aim of this study was to analyze the genetic variants of 51 Chinese patients with distal renal tubular acidosis (dRTA) and explore the correlation between their genotype and phenotype. Eight variants of SLC4A1, 19 variants of ATP6V0A4, and 16 variants of ATP6V1B1 have been identified, and of which 14 were novel ones. Eleven patients with autosomal dominant dRTA, and four patients with autosomal recessive dRTA were caused by genetic defects in SLC4A1; 18 and nine patients with recessive dRTA were resulted by defects in ATP6V0A4 and ATP6V1B1 respectively; no causal gene was identified in seven patients. Mutation frequency of SLC4A1 in Chinese populations was more common than Europeans. The incidence of deafness in ATP6V0A4 and ATP6V1B1 groups was 16.7% and 54.5%, respectively. The frequency of CKD in adults, children and infants was 100%, 51%, and 3%, separately. Our study will further expand the mutation spectrum of primary dRTA and provide valuable references to genetic counseling of Chinese populations.

Novel gain-of-function mutation of TRPC6 Q134P contributes to late onset focal segmental glomerulosclerosis in a Chinese pedigree.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS, OMIM®#603 965) is an overriding cause that leads to end-stage renal disease (ESRD). As a member of TRP superfamily, mutations of TRPC6 gene are closely linked to FSGS. By now, 20 missense mutations have been reported, among them, nine gain-of-function (GOF), and five loss-of-function (LOF) mutations have been recognized according to the effect on TRPC6 channel activity. Systematic investigations of functional mutations will provide valuable evidences for understanding the pathophysiology of TRPC6 involved in FSGS. The aim of this study is to investigate the pathogenicity of a novel TRPC6 mutation p.Q134P in FSGS. METHODS: High-throughput sequencing was performed to analyse 436 genes which are associated with hereditary kidney diseases in a Chinese pedigree. Then we constructed TRPC6 expression plasmids of wide type and variant. Immunofluorescence, cell-surface biotinylation assays and electrophysiology were used to analyse the localization, cell surface expression, and calcium transport activity of TRPC6. RESULTS: A novel variant c.401A>C (p.Q134P) in exon 2 of TRPC6 gene was found. There was no significant difference between the expression levels of p.Q134P mutant and WT TRPC6 protein in the whole cell lysate and cell-surface fraction. Q134P mutant-bearing TRPC6 elicited much higher Ca+ current amplitude than WT. CONCLUSION: We identified a novel GOF mutation p.Q134P of TRPC6 which contributed to late-onset FSGS. Our study expands the mutational spectrum of TRPC6 associated with FSGS and furtherly supports the hypothesis of calcium dose-response dependency that a moderate increased calcium influx elicited a mild FSGS phenotype.

From Bartter's syndrome to renal tubular acidosis in a patient with Hashimoto's thyroiditis: A case report
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Hashimoto's thyroiditis (HT) is an autoimmune disease that can cause the dysfunction of glands. Moreover, autoimmune disease is an under-recognized cause of several types of tubular dysfunction such as renal tubular acidosis (RTA), Gitelman's syndrome (GS), and Bartter's syndrome (BS). However, the potential mechanism of acquired BS and RTA associated with autoimmune diseases remains unclear. A 55-year-old female patient presented with numbness in both lower extremities for 6 months. She had a 2-year history of HT. Laboratory findings showed hypokalemia, metabolic alkalosis, and elevated plasma aldosterone concentration and renin activity. Urinary analysis revealed renal wasting of potassium, sodium, and chloride. Therefore, she was diagnosed as having HT and secondary BS and was treated with oral methylprednisolone, potassium chloride, and spironolactone. Two weeks later, her serum potassium levels restored to normal. After that, however, the patient was lost to follow-up. Two years later, she was re-hospitalized for progressive muscle weakness and quadriplegia. Laboratory results demonstrated severe hypokalemia, hyperchloremic metabolic acidosis, and inappropriate alkaline urine. Thus, distal RTA (dRTA) was confirmed, and she was prescribed potassium citrate and prednisone, resulting in the correction of hypokalemia and acid-base disturbance within 2 weeks after discharge. We report an HT patient who initially developed hypokalemic metabolic alkalosis and then developed metabolic acidosis within a few years. To our knowledge, this is the first report of two completely opposite acid-base disturbances associated with renal tubular diseases occurring consecutively in the same patient with HT. Our case may provide a valuable clue for exploring the mechanism of renal involvement in autoimmune diseases.

Sudden onset of nephrotic syndrome in an asymptomatic Fabry patient: a case report.

BACKGROUND: Fabry disease (FD) is an X-linked lysosomal storage disorder caused by the mutation of the GLA gene, encoding the α-galactosidase, which is responsible for the catabolism of neutral glycosphingolipids. Microalbuminuria or low-grade proteinuria, and continuously progressive renal failure are common manifestations in FD males. However, sudden onset of nephrotic syndrome in FD, is rarely reported. CASE REPORT: A 32-year-old Chinese man was admitted to our hospital because of sudden onset of generalized edema due to nephrotic syndrome. He denied hypohidrosis, nocturia, and any history of episodic hand or foot pain. A few scattered angiokeratoma can be found on the low back skin on examination. Except for the similar locating pattern of angiokeratoma, no evident abnormality was found in the laboratory work up and physical examination of his younger brother. The patient was diagnosed with FD companying with minimal change disease by renal biopsy. Genetic analysis on our patient and his sibling revealed a nonsense GLA gene variant (c.707G > A, p.Trp236*), which has been previously reported in FD. Immunotherapy alone (steroids and tacrolimus), but without enzyme replacement therapy, much improved the massive proteinuria. Follow up to date, his 24-h urine protein is stable at about 0.5 g, and renal function keeps normal. CONCLUSION: Sudden onset of nephrotic syndrome, although rare, may occur in FD, even as the primary renal manifestation, but this usually suggests additional renal disease. Immunosuppressive treatment should be considered in such FD patient companying with nephrotic syndrome.

Cast nephropathy associated with monoclonal immunoglobulin M-secreting mucosa-associated lymphoid tissue B-cell lymphoma
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The kidney is among the various anatomical sites involved in mucosa-associated lymphoid tissue (MALT) lymphoma. A variety of renal pathological types, including membranous glomerulopathy, membranoproliferative glomerulonephritis, crescentic IgA nephropathy, minimal change disease, and cryoglobulinemic glomerulopathy, have been reported in MALT lymphoma patients. However, cast nephropathy is extremely rare in MALT lymphoma. Herein, we describe the case of a patient with a history of MALT lymphoma of the stomach and small intestine 7 years before presenting with acute kidney failure 1 year after chemotherapy cessation. Monoclonal IgM-λ was detected in the serum, and kidney biopsy showed λ light chain deposition-based cast nephropathy. In addition, MALT recurrence was discovered in the stomach rather than intestinal tissue by gastrointestinal endoscope, and no lymphoplasmacytic infiltration was found in bone marrow. After 1 year of chemotherapy, renal function was partially restored, and the level of serum λ chain, serum IgM, and 24-hour urine protein all decreased. Our case illustrates that MALT lymphoma is prone to recurrence and grows slowly, moreover, with the characteristic of monoclonal immunoglobulin production and kidney infiltration.

The urate-lowering efficacy and safety of febuxostat versus allopurinol in Chinese patients with asymptomatic hyperuricemia and with chronic kidney disease stages 3-5.

BACKGROUND: While the dose of allopurinol is limited in patients with chronic kidney disease (CKD), information is lacking concerning the efficacy, safety, and maintenance dose of febuxostat in Chinese patients with hyperuricemia and with CKD stages 3-5. METHODS: A single center, prospective cohort study was conducted in patients with CKD stages 3-5 and with hyperuricemia who had not yet begun to undergo renal replacement therapy. We enrolled 208 patients who were newly treated with febuxostat (n = 112) or allopurinol (n = 96) in this study. The efficacy of febuxostat was determined by the proportion of patients with serum uric acid (sUA) < 360 µmol/L at the end of the study and changes of renal function. RESULTS: The target of sUA < 360 µmol/L was reached by 96.4% of participants in the febuxostat group and 37.5% in the allopurinol group at 6 months. The eGFR in the febuxostat group showed an increase from 28.45 to 30.65 mL/min/1.73 m2 at 6 months, while in the allopurinol group, the eGFR decreased from 28.06 to 24.39 mL/min/1.73 m2. Linear regression analysis showed that the reduction in sUA was significantly associated with an increase in eGFR and decrease in proteinuria. We found that 83.0% of the patients could remain with sUA < 360 µmol/L at a maintenance dose of febuxostat 20 mg/day. CONCLUSION: Febuxostat had superior urate-lowering efficacy to that of allopurinol in Chinese Han patients with hyperuricemia with CKD stages 3-5, and the reduction in sUA levels was associated with a slower progression of renal function.

Identification of a novel TSC2 c.3610G > A, p.G1204R mutation contribute to aberrant splicing in a patient with classical tuberous sclerosis complex: a case report.

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by hamartomas in any organ systems. Mutations in the TSC1 or TSC2 gene lead to the dysfunction of hamartin or tuberin proteins, which cause tuberous sclerosis complex. CASE PRESENTATION: We describe the clinical characteristics of patients from a Chinese family with tuberous sclerosis complex and analyze the functional consequences of their causal genetic mutations. A novel heterozygous mutation (c.3610G > A) at the last nucleotide of exon 29 in TSC2 was identified. On the protein level, this variant was presumed to be a missense mutation (p.Gly1204Arg). However, the splicing assay revealed that this mutation also leads to the whole TSC2 exon 29 skipping, besides the wild-type transcript. The mutated transcript results in an in-frame deletion of 71 amino acids (p.Gly1133_Thr1203del) and its ratio with the normal splice product is of about 44:56. CONCLUSIONS: The novel c.3610G > A TSC2 mutation was identified in association with tuberous sclerosis complex. And it was proven to code both for a missense-carrying transcript (56%), and for an isoform lacking exon 29 (44%).

Novel compound heterozygous ATP6V1B1 mutations in a Chinese child patient with primary distal renal tubular acidosis: a case report.

BACKGROUND: Distal renal tubular acidosis (dRTA) is a heterogeneous disorder characterized by normal anion gap metabolic acidosis. Autosomal recessive dRTA is usually caused by mutations occurring in ATP6V1B1 and ATP6V0A4 genes,encoding subunits B1 and a4 of apical H+-ATPase, respectively. The heterogeneous clinical manifestations of dRTA have been described in different ethnic groups harboring distinct mutations. Most of the reported cases are from Europe and Africa. At present, the prevalence of primary dRTA is still poorly elucidated in Chinese population. CASE PRESENTATION: A 2-year and six-month-old female patient was hospitalized because of recurrent hypokalemia, hyperchloremic metabolic acidosis and growth retardation. Laboratory investigations presented a normal anion gap hyperchloremic metabolic acidosis, hypokalemia, and inappropriate alkaline urine. Renal ultrasound indicated bilateral nephrocalcinosis. Bilateral sensorineural hearing loss (SNHL) was confirmed with moderately severe (45 dB) on the left ear and severe (80 dB) on the right ear, which was accompanied with enlarged vestibular aqueduct (EVA) on both sides. According to these findings, a diagnosis of dRTA was made. To identify the pathogenic gene mutation, all coding regions of ATP6V1B1 and ATP6V0A4 gene, including intron-exon boundaries, were analyzed using PCR followed by direct sequence analysis. The splicing variants were verified in peripheral blood leucocytes of the patient by RT-PCR. As a result, two novel heterozygous mutations in ATP6V1B1 were identified in the child. One mutation was a successive 2-nucleotide deletion in exon 2(c.133-134delTG), which caused a marked nonsense mediated mRNA decay. The other was a guanine to adenine substitution of the first nucleotide of intron 8(c.785 + 1 G > A), which led to the exclusion of exon 8. After treatment with sodium citrate, potassium citrateand citric acid, metabolic acidosis and hypokalemia were corrected, but her hearing decreased gradually during the 2 years and had to accept the use of bilateral hearing aids. CONCLUSIONS: We described two novel dRTA associated mutations in ATP6V1B1 identified in a Chinese child patient accompanying with SNHL and EVA. Our study will help to expand the understanding of this rare disease in Chinese population.

Familial hypomagnesaemia, Hypercalciuria and Nephrocalcinosis associated with a novel mutation of the highly conserved leucine residue 116 of Claudin 16 in a Chinese patient with a delayed diagnosis: a case report.

BACKGROUND: Sixty mutations of claudin 16 coding gene have been reported in familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) patients. Recent investigations revealed that a highly conserved glycine-leucine-tryptophan (115G-L-W117) motif in the first extracellular segment (ESC1) of claudin 16 might be essential for stabilization of the appropriately folded ECS1 structure and conservation of normal claudin 16 function. However, neither missense nor nonsense mutation has ever been described in this motif. Our study aimed at identifying mutations in a Chinese patient with FHHNC and exploring the association between genotype and phenotype. CASE PRESENTATION: A 33-year-old female presented with 4 years history of recurrent acute pyelonephritis without other notable past medical history. Her healthy parents, who aged 56 and 53 respectively, were second cousins, and her only sibling died from renal failure without definite cause at age 25. Renal ultrasound imaging demonstrated atrophic kidneys and bilateral nephrocalcinosis. The laboratory workup revealed impaired renal function (Stage CKD IV), hypocalcemia and mild hypomagnesemia, accompanied with marked renal loss of magnesium and hypercalciuria. During the follow-up, treatment with calcitriol and calcium but not with magnesium was difficult to achieve normal serum calcium levels, whereas her serum magnesium concentration fluctuated within normal ranges. In the end, the patient unavoidably reached ESRD at 36 years old. The clinical features and family history suggested the diagnosis of FHHNC. To make a definite diagnosis, we use whole-exome sequencing to identify the disease-causing mutations and Sanger sequencing to confirm the mutation co-segregation in the family. As a result, a novel homozygous mutation (c.346C > G, p.Leu116Val) in 115G-L-W117 motif of claudin 16 was identified. Her parents, grandmother and one of her cousins carried heterozygous p.Leu116Val, whereas 200 unrelated controls did not carry this mutation. CONCLUSIONS: We described a delayed diagnosis patient with FHHNC in the Chinese population and identified a novel missense mutation in the highly conserved 115G-L-W117 motif of claudin 16 for the first time. According to the reported data and the information deduced from 3D modeling, we speculate that this mutation probably reserve partial residual function which might be related to the slight phenotype of the patient.

Genotype/Phenotype Analysis in 67 Chinese Patients with Gitelman's Syndrome.

BACKGROUND: Gitelman's syndrome (GS) is an autosomal recessive renal tubular disorder, which is caused by the mutations in SLC12A3. This study was designed to analyze the characteristics of the genotype and phenotype, and follow-up in the largest group of Chinese patients with GS. METHODS: Sixty-seven patients with GS underwent SLCl2A3 analysis, and their clinical characteristics and biochemical findings as well as follow-up were reviewed, aiming to achieve a better description of GS. Additionally, the association of genotype and phenotype was explored. RESULTS: Forty-one different mutations were identified within these 67 GS patients, including 11 novel mutations and 5 recurrent ones. Typical hypocalciuria and hypomagnesemia were not found in 6 (9%) and 8 (11.9%) patients, respectively. Male patients and those harboring severe mutations in both alleles had significant higher urinary fractional excretion (FE) of potassium, magnesium and chlorine. In addition, there were 2 patients who had chronic kidney disease (estimated glomerular filtration rate <60 ml/min/1.73 m2) and 32 patients with abnormal glucose metabolism. CONCLUSIONS: We identified 41 mutations related to GS, containing 11 novel variants and 5 high-frequency ones, which should facilitate earlier and more accurate diagnosis of GS. FE of electrolytes in urine may be more sensitive in the phenotype evaluation and differential diagnosis than corresponding serum electrolytes. Hypokalemia and hypomagnesemia in GS were difficult to correct; however, spironolactone might be helpful for hypokalemia to some degree. Compared with normal people, patients with GS were at higher risk of developing type 2 diabetes.