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.

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.

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.

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.

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.

Two Novel HOGA1 Splicing Mutations Identified in a Chinese Patient with Primary Hyperoxaluria Type 3.

BACKGROUND: Twenty-six HOGA1 mutations have been reported in primary hyperoxaluria (PH) type 3 (PH3) patients with c.700 + 5G>T accounting for about 50% of the total alleles. However, PH3 has never been described in Asians. METHODS: A Chinese child with early-onset nephrolithiasis was suspected of having PH. We searched for AGXT, GRHPR and HOGA1 gene mutations in this patient and his parents. All coding regions, including intron-exon boundaries, were analyzed using PCR followed by direct sequence analysis. RESULTS: Two heterozygous mutations not previously described in the literature about HOGA1 were identified (compound heterozygous). One mutation was a successive 2 bp substitution at the last nucleotide of exon 6 and at the first nucleotide of intron 6, respectively (c.834_834 + 1GG>TT), while the other one was a guanine to adenine substitution of the last nucleotide of exon 6 (c.834G>A). Direct sequencing analysis failed to find these mutations in 100 unrelated healthy subjects and the functional role on splicing of both variants found in this study was confirmed by a minigene assay based on the pSPL3 exon trapping vector. In addition, we found a SNP in this family (c.715G>A, p.V239I). There were no mutations detected in AGXT and GRHPR. CONCLUSION: Two novel HOGA1 mutations were identified in association with PH3. This is the first description and investigation on mutant gene analysis of PH3 in an Asian.

Mutation analysis and audiologic assessment in six Chinese children with primary distal renal tubular acidosis.

The objective of this study is to identify ATP6V1B1, ATP6V0A4 and SLC4A1 genes mutations and assess audiologic characteristics in six Chinese children with primary distal renal tubular acidosis from four unrelated families between the ages of 2 and 13 years. Both ATP6V0A4 and ATP6V1B1 genes were preferentially screened in all index cases by direct sequence analysis. If inconclusive then SLC4A1 gene should be analyzed for mutation. Their clinical features, hearing status and inner ear imaging structure were also investigated. Six loss-of-function mutations were identified in six patients. Two novel mutations were identified in either of ATP6V0A4 and ATP6V1B1 genes, respectively. Two probands from different kindreds with mutations in ATP6V1B1 presented early onset profound sensorineural hearing loss (SNHL) and enlarged vestibular aqueduct (EVA). Two from different families carrying ATP6V0A4 mutations manifested early onset moderate mixed HL and moderate SNHL, respectively, the former comorbid with EVA, while the latter not; however, both their elder sisters showed normal hearing and inner ear. These findings expand the spectrum of mutations in the ATP6V0A4 and ATP6V1B1 genes associated with primary dRTA. Our study confirms the association of EVA and mutations in either of these two genes. More studies are necessary to clarify the relationship between dRTA, SNHL, EVA, and gene mutations.

Three exonic variants in the COL4A5 gene alter RNA splicing in a minigene assay.

BACKGROUND: X-linked Alport syndrome (XLAS) is an inherited renal disease caused by rare variants of COL4A5 on chromosome Xq22. Many studies have indicated that single nucleotide variants (SNVs) in exons can disrupt normal splicing process of the pre-mRNA by altering various splicing regulatory signals. The male patients with XLAS have a strong genotype-phenotype correlation. Confirming the effect of variants on splicing can help to predict kidney prognosis. This study aimed to investigate whether single nucleotide substitutions, located within three bases at the 5' end of the exons or internal position of the exons in COL4A5 gene, cause aberrant splicing process. METHODS: We analyzed 401 SNVs previously presumed missense and nonsense variants in COL4A5 gene by bioinformatics programs and identified candidate variants that may affect the splicing of pre-mRNA via minigene assays. RESULTS: Our study indicated three of eight candidate variants induced complete or partial exon skipping. Variants c.2678G>C and c.2918G>A probably disturb classic splice sites leading to corresponding exon skipping. Variant c.3700C>T may disrupt splicing enhancer motifs accompanying with generation of splicing silencer sequences resulting in the skipping of exon 41. CONCLUSION: Our study revealed that two missense variants positioned the first nucleotides of the 5' end of COL4A5 exons and one internal exonic nonsense variant caused aberrant splicing. Importantly, this study emphasized the necessity of assessing the effects of SNVs at the mRNA level.

Four novel variants identified in primary hyperoxaluria and genotypic and phenotypic analysis in 21 Chinese patients.

Background: Primary hyperoxaluria (PH) is a rare genetic disorder characterized by excessive accumulation of oxalate in plasma and urine, resulting in various phenotypes due to allelic and clinical heterogeneity. This study aimed to analyze the genotype of 21 Chinese patients with primary hyperoxaluria (PH) and explore their correlations between genotype and phenotype. Methods: Combined with clinical phenotypic and genetic analysis, we identified 21 PH patients from highly suspected Chinese patients. The clinical, biochemical, and genetic data of the 21 patients were subsequently reviewed. Results: We reported 21 cases of PH in China, including 12 cases of PH1, 3 cases of PH2 and 6 cases of PH3, and identified 2 novel variants (c.632T > G and c.823_824del) in AGXT gene and 2 novel variants (c.258_272del and c.866-34_866-8del) in GRHPR gene, respectively. A possible PH3 hotspot variant c.769T > G was identified for the first time. In addition, patients with PH1 showed higher levels of creatinine and lower eGFR than those with PH2 and PH3. In PH1, patients with severe variants in both alleles had significantly higher creatinine and lower eGFR than other patients. Delayed diagnosis still existed in some late-onset patients. Of all cases, 6 had reached to end-stage kidney disease (ESKD) at diagnosis with systemic oxalosis. Five patients were on dialysis and three had undergone kidney or liver transplants. Notably, four patients showed a favorable therapeutic response to vitamin B6, and c.823_824dup and c.145A > C may be identified as potentially vitamin B6-sensitive genotypes. Conclusion: In brief, our study identified 4 novel variants and extended the variant spectrum of PH in the Chinese population. The clinical phenotype was characterized by large heterogeneity, which may be determined by genotype and a variety of other factors. We first reported two variants that may be sensitive to vitamin B6 therapy in Chinese population, providing valuable references for clinical treatment. In addition, early screening and prognosis of PH should be given more attention. We propose to establish a large-scale registration system for rare genetic diseases in China and call for more attention on rare kidney genetic diseases.

Minigene splicing assays reveal new insights into exonic variants of the SLC12A3 gene in Gitelman syndrome.

BACKGROUND: Gitelman syndrome (GS) is a type of salt-losing tubular disease, most of which is caused by SLC12A3 gene variants, and missense variants account for the majority. Recently, the phenomenon of exon skipping, in which variants disrupt normal pre-mRNA splicing, has been related to a variety of diseases. Therefore, we hypothesize that a certain proportion of SLC12A3 variants can result in disease via interfering with the normal splicing process. METHODS: We analyzed 342 previously presumed SLC12A3 missense variants using bioinformatics programs and identified candidate variants that may alter the splicing of pre-mRNA through minigene assays. RESULTS: Our study revealed that, among ten candidate variants, six variants (c.602G>A, c.602G>T, c.1667C>T, c.1925G>A, c.2548G>C, and c.2549G>C) led to complete or incomplete exon skipping by affecting exonic splicing regulatory elements and/or disturbing canonical splice sites. CONCLUSION: It is worth mentioning that this is the largest study on pre-mRNA splicing of SLC12A3 exonic variants. In addition, our study emphasizes the importance of detecting splicing function at the mRNA level in GS and indicates that minigene analysis is a valuable tool for splicing functional assays of variants in vitro.

High-frequency variant p.T60M in NaCl cotransporter and blood pressure variability in Han Chinese.

OBJECTIVE: Frequent studies have confirmed that homozygous or compound heterozygous loss-of-function mutation p.Thr60Met in NaCl cotransporter (NCC) lead to the salt-wasting Gitelman's syndrome (GS) of hypotension. The finding that Thr60 is a key SPAK/OSR1 phosphorylation site on NCC also raises the possible importance of Thr60 in regulating the activity of NCC and blood pressure (BP). However, the association of heterozygous NCC mutation p.Thr60Met and BP has not yet been studied. METHODS: We collected 38 heterozygous mutation p.Thr60Met carriers, respectively, from 14 GS families confirmed by our previous studies and 1,000 unrelated Han Chinese, and matched them pairwise (sex, age ±2 years and BMI ±1) with 38 unrelated healthy controls. BP and biochemistry data were obtained. Student's t test and χ(2) test were used to compare the differences between these study subjects with the heterozygous variant p.T60M and the controls. p < 0.05 was considered statistically significant. RESULTS: p.Thr60Met carriers had markedly lower BP (systolic 110.3 ± 13.5 vs. 119.1 ± 15.0 mm Hg; diastolic 70.2 ± 7.0 vs. 75.4 ± 8.2 mm Hg, p < 0.01) than controls. p.Thr60Met heterozygotes had higher fasting plasma glucose concentration (5.35 ± 0.73 vs. 4.95 ± 0.69 mmol/l, p < 0.01). 14 carriers versus 6 control subjects had impaired fasting glucose (36.8 vs. 15.8%, p < 0.01), and p.Thr60Met carriers had higher 24-hour urinary sodium excretion than controls but not significantly (170.2 ± 35.6 vs. 159.5 ± 39.0 mmol, p = 0.10). CONCLUSIONS: The NCC mutation p.Thr60Met carriers in Han populations have markedly lower BP and slightly higher fasting plasma glucose compared with normal controls.

Eight novel KCNJ1 variants and parathyroid hormone overaction or resistance in 5 probands with Bartter syndrome type 2.

PURPOSE: Bartter syndrome type 2 (BS2) is an autosomal recessive renal tubular disorder, which is caused by the mutations in KCNJ1. This study was designed to analyze and describe the genotype and clinical features of five Chinese probands with BS2. METHODS: Identify KCNJ1 gene variants by the next generation sequencing and evaluate their mutation effects according to 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines. RESULTS: Ten variants including eight novel ones of KCNJ1 gene were found, the most common type was missense variant. The common symptoms and signs from high to low incidence were: polydipsia and polyuria (5/5), one of them (1/5) presented with diabetes insipidus; maternal polyhydramnios and premature delivery (4/5); growth retardation (3/5). Two patients presented with hypochloremic metabolic alkalosis and hypokalemia; whereas the acid-base disturbance was absent in the others. One patient had evident parathyroid hormone (PTH) resistance (hypocalcemia, hyperphosphatemia and markedly elevated PTH levels), three presented with PTH overacting (hypercalcemia, hypophosphatemia and mild elevated PTH levels), and one showed normal blood calcium and phosphorus concentrations with high-normal PTH levels. All patients had nephrocalcinosis and/or hypercalciuria, and one of them complicated with nephrolithiasis. Indomethacin has significant therapeutic effect on the growth retardation, polydipsia and polyuria and treatment was associated with a decrease in urine calcium excretion, normalization of electrolyte disturbance and PTH parameters. CONCLUSIONS: Ten variants of KCNJ1 gene were identified in five Chinese probands. These patients had atypical BS phenotype lacking evident metabolic alkalosis and/or manifesting with PTH overaction/resistance, which reminds clinicians to carefully differentiate BS2 with other parathyroid disorders. This is the first report of BS2 from Chinese populations.

Thirteen novel CLCNKB variants and genotype/phenotype association study in 42 Chinese patients with Bartter syndrome type 3.

PURPOSE: Analyze the genotype of 42 Chinese patients with Bartter syndrome type 3 (BS3) and investigate their correlation between genotype and phenotype. METHODS: Identify CLCNKB gene variants by the next-generation sequencing and the multiplex ligation-dependent probe amplification (MLPA), and then evaluate their mutation effects according to 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines. RESULTS: Thirty-six different variants in CLCNKB gene, including 13 novel ones, were found. The whole gene deletion of CLCNKB gene was the most frequent mutation (40%), and the rate of large deletions is up to 55%. Among 36 variants, six (c.1244T>A, c.1468G>A, c.849_851delCTT, c.359G>T, c.1052G>T, and c.1309G>A) and three (c.228A>C, c.1294_1295TA>CT, and c.1333T>G) variants were classified as "likely pathogenic variants" and "variants with uncertain significance (VUS)," respectively. The other 27 variants were classified as "pathogenic variants". The most common symptoms included: growth retardation (38/42), polydipsia and polyuria (35/42), constipation (31/42), and vomiting (27/42). All patients presented with hypokalemia, hypochloremia, and metabolic alkalosis. The genotype and phenotype association study revealed that who had mutations probably resulting in complete loss of function of both alleles might have severer phenotype. After the treatment that based on indomethacin and potassium chloride, most patients could achieve obvious recovery of growth rate and restoration of hypokalemia. CONCLUSIONS: The present study have found 36 variants of CLCNKB gene, including 13 novel ones, which enrich the human gene mutation database and provide valuable references to diagnosis, treatment, and the genetic counseling of Chinese population.

Eleven novel SLC12A1 variants and an exonic mutation cause exon skipping in Bartter syndrome type I.

INTRODUCTION: Bartter syndrome type I (BS1) has been rarely reported in large groups. On the other hand, the phenomenon of exon skipping, in which exonic mutations result in abnormal splicing, has been reported to be associated with various diseases. Specifically, mutations that result in the disruption of exonic splicing enhancers (ESEs) and/or the creation of exonic splicing silencers (ESSs) can promote exon skipping. However, the aberrant exon skipping caused by an exonic variant in such splicing regulatory elements (SREs) sequences has never been reported in the causal gene of SLC12A1 in BS1. METHODS: We analyze the variants in nine Chinese families with BS1, including eight with antenatal BS (aBS) and one presenting as classical BS (cBS), by next-generation sequencing. Then we used bioinformatics programs to analyze all these variants found in this study and identify candidate mutations that may induce exon skipping. Furthermore, the effects of identified variants were classified according to the 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines. RESULTS: Fifteen different variants of SLC12A1 gene were identified, including 11 novel ones. Two of the nine probands were homozygotes, the rest seven ones were compound heterozygotes. One candidate variant (c.1435C>G), not only significantly reduced ESEs scores but also markedly increased ESSs scores, were further investigated by mini-gene splicing assay, and found this single-nucleotide substitution causes abnormal splicing in vitro (exclusion of exon 11). Finally, among 15 variants, 9, 3, and 3 were classified as "pathogenic variants", "likely pathogenic variants", "variants with uncertain significance", respectively. CONCLUSION: These data would enrich the human gene mutation database (HGMD) and would provide valuable references to the genetic counseling and diagnosis of BS1 for Chinese population. Additionally, our results suggest that aberrant exon skipping is one previously unrecognized mechanism by which an exonic variant in SLC12A1 can lead to BS1.

A novel SLC12A3 splicing mutation skipping of two exons and preliminary screening for alternative splice variants in human kidney.

BACKGROUND: Gitelman's syndrome is a mild autosomal recessive disorder caused by inactivating mutations of SLC12A3. However, severe phenotype may be associated with compound heterozygous nonfunctional variants such as frameshift and splicing mutations. Because most multi-exon genes are alternatively spliced as shown by recent studies, SLC12A3, with 26 exons, is likely to be alternatively spliced as well. METHODS: A case of Gitelman's syndrome with both physical and mental retardation was investigated by genetic analysis. In addition, the alternative splice variants of SLC12A3 were screened by RT-PCR. RESULTS: A novel intron 7 and exon 8 boundary mutation (a successive 13-nucleotide transition: intron 7 as -1 G>A plus exon 8 +1 to +12 delCGGACATTTTTGinsCCGAAAATTTT) was identified in this patient besides a missense mutation Thr60Met. Further cDNA analysis revealed the novel mutation led to skipping of exons 7 and 8. Furthermore, we found an aberrant splice product skipping of exon 7 and identified two high-abundance alternative splice transcripts. CONCLUSION: This is the first report of a splice mutation of SLC12A3 with multiple-exon skipping in Gitelman's syndrome. This study provides further evidence for the severe phenotype of Gitelman's syndrome and its association with underlying mutations. Additionally, we demonstrated that the pre-mRNA of SLC12A3 was complex spliced.

A novel mutation in exon 9 of Cullin 3 gene contributes to aberrant splicing in pseudohypoaldosteronism type II.

Pseudohypoaldosteronism type II (PHAII) is a rare renal tubular disease that is inherited in an autosomal dominant manner. Mutations in four genes (WNK1,WNK4,CUL3, and KLHL3) have been identified to be responsible for this disease. Cullin 3 (CUL3) and KLHL3 are subunits of Cullin-RING E3 ubiquitin ligase complexes, and the serine-threonine kinases WNK1 and WNK4 are substrates of this ubiquitin ligase. For CUL3, all mutations associated with PHAII exclusively lead to exon 9 skipping. In this study, we identified a Chinese PHAII kindred caused by a novel synonymous mutation (c.1221A > G p.Glu407Glu) in CUL3, and explored its effects on exon 9 abnormal splicing through an in vitro splicing assay and study of the patients' RNA. We obtained evidence that this synonymous mutation leads to complete exon 9 skipping, and in silico bioinformatics analysis demonstrated that the CUL3 c.1221A > G mutation might decrease the ratio of exonic splicing enhancers and silencers. This is the first report of PHAII in Chinese patients with a novel CUL3 mutation. Our findings add a novel pathogenic splicing variant to the CUL3 mutational spectrum and provide reference for further research on mechanisms of splicing modulation and development of potential therapeutic reagents for PHAII.