Clinical phenotype and genetic function analysis of a family with hypomyelinating leukodystrophy-7 caused by POLR3A mutation.

Hypomyelinating leukodystrophy (HLD) is a rare genetic heterogeneous disease that can affect myelin development in the central nervous system. This study aims to analyze the clinical phenotype and genetic function of a family with HLD-7 caused by POLR3A mutation. The proband (IV6) in this family mainly showed progressive cognitive decline, dentin dysplasia, and hypogonadotropic hypogonadism. Her three old brothers (IV1, IV2, and IV4) also had different degrees of ataxia, dystonia, or dysarthria besides the aforementioned manifestations. Their brain magnetic resonance imaging showed bilateral periventricular white matter atrophy, brain atrophy, and corpus callosum atrophy and thinning. The proband and her two living brothers (IV2 and IV4) were detected to carry a homozygous mutation of the POLR3A (NM_007055.4) gene c. 2300G > T (p.Cys767Phe), and her consanguineous married parents (III1 and III2) were p.Cys767Phe heterozygous carriers. In the constructed POLR3A wild-type and p.Cys767Phe mutant cells, it was seen that overexpression of wild-type POLR3A protein significantly enhanced Pol III transcription of 5S rRNA and tRNA Leu-CAA. However, although the mutant POLR3A protein overexpression was increased compared to the wild-type protein overexpression, it did not show the expected further enhancement of Pol III function. On the contrary, Pol III transcription function was frustrated (POLR3A, BC200, and tRNA Leu-CAA expression decreased), and MBP and 18S rRNA expressions were decreased. This study indicates that the POLR3A p.Cys767Phe variant caused increased expression of mutated POLR3A protein and abnormal expression of Pol III transcripts, and the mutant POLR3A protein function was abnormal.

Pedigree Analysis of Nonclassical Cholesteryl Ester Storage Disease with Dominant Inheritance in a LIPA I378T Heterozygous Carrier.

BACKGROUND: Cholesterol ester storage disorder (CESD; OMIM: 278,000) was formerly assumed to be an autosomal recessive allelic genetic condition connected to diminished lysosomal acid lipase (LAL) activity due to LIPA gene abnormalities. CESD is characterized by abnormal liver function and lipid metabolism, and in severe cases, liver failure can occur leading to death. In this study, one Chinese nonclassical CESD pedigree with dominant inheritance was phenotyped and analyzed for the corresponding gene alterations. METHODS: Seven males and eight females from nonclassical CESD pedigree were recruited. Clinical features and LAL activities were documented. Whole genome Next-generation sequencing (NGS) was used to screen candidate genes and mutations, Sanger sequencing confirmed predicted mutations, and qPCR detected LIPA mRNA expression. RESULTS: Eight individuals of the pedigree were speculatively thought to have CESD. LAL activity was discovered to be lowered in four living members of the pedigree, but undetectable in the other four deceased members who died of probable hepatic failure. Three of the four living relatives had abnormal lipid metabolism and all four had liver dysfunctions. By liver biopsy, the proband exhibited diffuse vesicular fatty changes in noticeably enlarged hepatocytes and Kupffer cell hyperplasia. Surprisingly, only a newly discovered heterozygous mutation, c.1133T>C (p. Ile378Thr) on LIPA, was found by gene sequencing in the proband. All living family members who carried the p.I378T variant displayed reduced LAL activity. CONCLUSIONS: Phenotypic analyses indicate that this may be an autosomal dominant nonclassical CESD pedigree with a LIPA gene mutation.

Adult type I Gaucher disease with splenectomy caused by a compound heterozygous GBA1 mutation in a Chinese patient: a case report.

Gaucher disease (GD) is an autosomal recessive ailment resulting from glucocerebrosidase deficiency caused by a mutation in the GBA1 gene, leading to multi-organ problems in the liver, spleen, and bone marrow. In China, GD is extremely uncommon and has a lower incidence rate than worldwide. In this study, we report the case of an adult male with an enlarged spleen for 13 years who presented with abdominal distension, severe loss of appetite and weight, reduction of the three-line due to hypersplenism, frequent nosebleeds, and bloody stools. Regrettably, the unexpected discovery of splenic pathology suggestive of splenic Gaucher disease was only made after a splenectomy due to a lack of knowledge about rare disorders. Our patient's delayed diagnosis may have been due to the department where he was originally treated, but it highlights the need for multidisciplinary consultation in splenomegaly of unknown etiology. We then investigated the patient's clinical phenotypes and gene mutation features using genetically phenotypical analysis. The analysis of the GBA1 gene sequence indicated that the patient carried a compound heterozygous mutation consisting of two potentially disease-causing mutations: c.907C > A (p. Leu303Ile) and c.1448 T > C (p. Leu483Pro). While previous research has linked the p. Leu483Pro mutation site to neurologic GD phenotypes (GD2 and GD3), the patients in this investigation were identified as having non-neuronopathic GD1. The other mutation, p. Leu303Ile, is a new GD-related mutation not indexed in PubMed that enriches the GBA1 gene mutation spectrum. Biosignature analysis has shown that both mutations alter the protein's three-dimensional structure, which may be a pathogenic mechanism for GD1 in this patient.

Potential regulatory role of the Nrf2/HMGB1/TLR4/NF-κB signaling pathway in lupus nephritis.

OBJECTIVES: Systemic lupus erythematosus is an autoimmune disease that involves multiple organ systems. One of its major complications, lupus nephritis (LN), is associated with a high mortality rate, and children-onset LN have a more severe course and worse prognosis than adults. Oxidative stress and inflammatory responses are involved in LN development and pathogenesis. Thus, this study aimed to explore the role of signaling regulation of the Nrf2/HMGB1/TLR/NF-κB pathway in LN pathogenesis and unravel the expression of TLR4+CXCR4+ plasma cells subset (PCs) in LN. METHODS: C57BL/6 and MRL/lpr mice were divided into four groups: control, model, vector control, and Nrf2 overexpression groups. The vector control and Nrf2 overexpression groups were injected with adenoviral vectors into the kidney in situ. Pathological changes in kidney tissues were observed by hematoxylin-eosin staining. The expression of Nrf2, HMGB1, TLR4, NF-κB, and downstream inflammatory factors in kidney samples was analyzed by quantitative polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. The ratios of TLR4+CXCR4+ PC subsets in the blood and kidneys of mice were determined by flow cytometry. RESULTS: In MRL/lpr mice, Nrf2 was downregulated while HMGB1/TLR4/NF-κB pathway proteins were upregulated. Nrf2 overexpression decreased the expression of HMGB1, TLR4, NF-κB, and its downstream inflammatory cytokines (IL-1ß and TNFα). These cytokines were negatively correlated with an increase in Nrf2 content. PC and TLR4 + CXCR4 + PCs in the blood and kidney samples were significantly increased in MRL/lpr mice; however, they were decreased upon Nrf2 overexpression. CONCLUSION: This study showed severe kidney injury in an LN mouse model and an increased ratio of TLR4 + CXCR4 + PCs. Furthermore, we observed that Nrf2 regulates LN immune response through the Nrf2/HMGB1/TLR4/NF-κB pathway, which can be considered an important target for LN treatment. The clinical value of the findings of our study requires further investigation.

Clinical phenotype and genetic function analysis of a rare family with hereditary leiomyomatosis and renal cell carcinoma complicated with Birt-Hogg-Dubé syndrome.

To date, over 200 families with hereditary leiomyomatosis and renal cell carcinoma (HLRCC) and over 600 families with Birt-Hogg-Dubé (BHD) syndrome have been reported, with low incidence. Here, we describe a patient with suspected rare HLRCC complicated by BHD syndrome. The proband (II1) had characteristic cutaneous leiomyoma-like protrusions on the neck and back, a left renal mass and multiple right renal, liver and bilateral lung cysts. Three family members (I1, II2, II3) had a history of renal cancer and several of the aforementioned clinical features. Two family members (II1, II3) diagnosed with fumarate hydratase (FH)-deficient papillary RCC via pathological biopsy carried two heterozygous variants: FH (NM_000143.3) missense mutation c.1189G>A (p.Gly397Arg) and FLCN (NM_144997.5) frameshift mutation c.1579_1580insA (p.Arg527Glnfs*75). No family member carrying a single variant had renal tumours. In HEK293T cells transfected with mutant vectors, mRNA and protein expression after FLCN p.Arg527Glnfs*75 and FH p.Gly397Arg mutations were significantly lower than those in wild-type (WT) cells. Cell immunofluorescence showed altered protein localisation and reduced protein expression after FLCN p.Arg527Glnfs*75 mutation. The FH WT was uniformly distributed in the cytoplasm, whereas FH protein expression was reduced after the p.Gly397Arg mutation and scattered sporadically with altered cell localisation. Patients with two variants may have a significantly increased penetrance of RCC.

Han family with essential tremor caused by the P421L variant of the TENM4 gene in China.

BACKGROUND: Essential tremor (ET) is an autosomal dominant inheritance disorder. Mutations in fusion sarcoma (FUS), mitochondrial serine peptidase 2 (HTRA2), teneurin transmembrane protein 4 (TENM4), sortilin1 (SORT1), SCN11A, and notch2N-terminal-like (NOTCH2NLC) genes are associated with familial ET. METHODS: A proband with ET was tested using whole-exome sequencing and repeat-primed polymerase chain reaction. Subsequently, the family members were screened for the suspected mutation, and the results were verified using Sanger sequencing. The relationship between pedigree and phenotype was also analyzed, and structural and functional changes in the variants were predicted using bioinformatics analysis. RESULTS: In a family with ET, the proband (III4) and the proband's father (II1), grandfather (I1), uncle (II2), and cousin (III5) all presented with involuntary tremors of both upper limbs. The responsible mutation was identified as TENM4 c.1262C > T (p.P421L), which showed genetic co-segregation in the family survey. AlphaFold predicted a change in the spatial position of TENM4 after the P421L mutation, which may have affected its stability. AlphaFold also predicted P421L to be a deleterious variation, which would lead to lower degrees of freedom of the TENM4 protein, thereby affecting the protein's structure and stability. According to the bioinformatics analysis, TENM4 (p.P421L) may reduce the signal reaching the nucleus by affecting the expression of TENM4 messenger RNA (mRNA), thereby impairing the normal oligodendrocyte differentiation process and leading to impaired myelination. CONCLUSION: This study revealed that the TENM4 (p.P421L) pathogenic missense variation was responsible for ET in the proband.

Function of PHEX mutations p.Glu145* and p.Trp749Arg in families with X-linked hypophosphatemic rickets by the negative regulation mechanism on FGF23 promoter transcription.

X-linked hypophosphatemic rickets (XLH) is characterized by increased circulating fibroblast growth factor 23 (FGF23) concentration caused by PHEX (NM_000444.5) mutations. Renal tubular resorption of phosphate is impaired, resulting in rickets and impaired bone mineralization. By phenotypic-genetic linkage analysis, two PHEX pathogenic mutations were found in two XLH families: c.433 G > T, p.Glu145* in exon 4 and c.2245 T > C, p.Trp749Arg in exon 22. Immunofluorescence showed that the localization of p.Glu145* and p.Trp749Arg mutant and secretory PHEX (secPHEX) changed, with decreased expression. In a HEK293T cell model co-transfected with PHEX, secPHEX, and FGF23, wild-type PHEX, secPHEX, and FGF23 proteins were distributed in the cell membrane or endoplasmic reticulum, while the mutant was located in the nuclear membrane and cytoplasm. qPCR of p.Glu145* revealed decreased PHEX and secPHEX mRNA expression in cells, with no difference in mRNA expression of p.Trp749Arg. Both mutations decreased intracellular PHEX endopeptidase activity. Western blot analysis showed decrease in mutant and secPHEX protein expression and no FGF23 protein expression in single-transfected PHEX and secPHEX cells. In cells co-transfected with FGF23, PHEX and secPHEX mutation promoted FGF23 expression. Dual-luciferase reporter gene was used to detect the effect of PHEX on FGF23 promoter. The dual-luciferase reporter gene showed that after PHEX overexpression, the activity of mutant firefly luciferase was significantly higher than that of wild type. The regulatory mechanism between PHEX and FGF23 is still unclear, but we found that PHEX is a direct transcriptional inhibitor of FGF23 and affects the expression of FGF23. This study verified the pathogenicity of the two variants and revealed the possible regulatory mechanism between PHEX and FGF23.

Genetic Analysis and Functional Study of a Pedigree With Bruck Syndrome Caused by PLOD2 Variant.

Background: Bruck syndrome (BS) is a rare autosomal recessive inherited osteogenesis imperfecta disease characterized by increased bone fragility and joint contracture. The pathogenic gene of type I BS is FKBPl0, whereas that of type II BS is PLOD2. No significant difference has been found in the clinical phenotype between the two types of BS. In this study, we performed genetic analysis of a BS pedigree caused by PLOD2 variant and studied the corresponding cellular function. Methods: Serum biochemistry, parathyroid hormone (PTH), 25-hydroxyvitamin D [25-(OH) D], osteocalcin, and 24-h urinary calcium levels of a family member with BS was assessed. The genes of the proband were analyzed by second-generation sequencing and exon capture techniques. Sanger sequencing was also performed for the suspected responsible variant of the family member. Wild- and variant-type lentivirus plasmids were constructed by gene cloning and transfected into HEK293T cells. Cell function was verified by real-time quantitative polymerase chain reaction, western blotting, and immunofluorescence detection. Results: In this pedigree, the proband was found to have a homozygous variant c.1856G > A (p.Arg619His) in exon 17 of PLOD2 (NM_182943.3). His consanguineous parents and sisters were p.Arg619His heterozygous carriers. The mRNA expression of PLOD2 in the constructed p.Arg619His variant cells was significantly upregulated, while the expression of PLOD2 and collagen I protein in the cell lysate was significantly downregulated. Immunofluorescence revealed that the wild-type PLOD2 was mainly located in the cytoplasm, and the expression of the PLOD2 protein after c.1856G > A variant was significantly downregulated, with almost no expression, aligning with the western blot results. The serum sodium, potassium, calcium, phosphorus, magnesium, alkaline phosphatase, PTH, 25-(OH) D, osteocalcin, and 24 h urinary calcium levels of the proband, his parents, and sisters were normal. Conclusion: Through gene and cell function analyses, PLOD2 Arg619His missense variant was preliminarily confirmed to cause BS by reducing protein expression.

Analysis of a Family with Brugada Syndrome and Sudden Cardiac Death Caused by a Novel Mutation of SCN5A.

Background: Brugada syndrome is a hereditary cardiac disease associated with mutations in ion channel genes. The clinical features include ventricular fibrillation, syncope, and sudden cardiac death. A family with Brugada syndrome with sudden cardiac death was analyzed to locate the associated mutation in the SCN5A gene. Methods and Results: Three generations of a Han Chinese family with Brugada syndrome were recruited in the study; their clinical phenotype data were collected and DNA samples extracted from the peripheral blood. Next-generation sequencing was carried out in the proband, and candidate genes and mutations were screened using the full exon capture technique. The family members who participated in the survey were tested for possible mutations using Sanger sequencing. Six family members were diagnosed with Brugada syndrome, including four asymptomatic patients. A newly discovered heterozygous mutation in the proband was located in exon 25 of SCN5A (NM_000335.5) at c.4313dup(p.Trp1439ValfsTer32). Among the surviving family members, only those with a Brugada wave on their electrocardiogram carried the c.4313dup(p.Trp1439ValfsTer32) variant. Bioinformatics prediction revealed that the frameshift of the c.4313dup (p.Trp1439ValfsTer32) mutant led to a coding change of 32 amino acids, followed by a stop codon, resulting in a truncated protein product. Conclusion: The newly discovered mutation site c.4313dup(p.Trp1439ValfsTer32) in exon 25 of SCN5A may be the molecular genetic basis of the family with Brugada syndrome.

Clinical and Genetic Analysis of a Family With Sitosterolemia Caused by a Novel ATP-Binding Cassette Subfamily G Member 5 Compound Heterozygous Mutation.

Sitosterolemia (OMIM ##210250), also known as phytosterolemia, is a rare autosomal recessive disorder caused by mutations in the ATP-binding cassette subfamily G member 5 (ABCG5) or member 8 (ABCG8) genes. This leads to abnormal functions of the transporter sterolin-1 protein encoded by ABCG5 and sterolin-2 protein encoded by ABCG8, respectively, which can hinder the formation of stable ABCG5/G8 heterodimers, decreasing its ability to transport sterols. As a result, phytosterols in tissue or plasma are significantly increased, leading to early onset atherosclerosis-related diseases and xanthelasma of tendons and skin. In this study, whole exome sequencing was performed on a Chinese Han proband with sitosterolemia to capture the target gene and screen for suspected pathogenic mutations. Sanger sequencing of the family members was performed to verify the relationship between family genetics and phenotypes. The structural and functional changes in the transporter sterolin-1 protein after the responsible mutation were predicted using bioinformatics analysis. A novel compound heterozygous mutation in the ABCG5 gene (NM_022436) was identified in a proband with sitosterolemia, one of which was inherited from the father: c.296T >G (p.M99R), and one from the mother: c.-76 C >T. SIFT, Polyphen2, and Mutation Taster software predicted that p.M99R may be the responsible variant and a novel variant. RNAFold software predicts that c.-76 C >T may affect the transcriptional information or the binding of RNA binding proteins by regulating the structure of RNA, and ultimately affect gene transcription or RNA stability and translation. Swiss model software predicts that the amino acid sequence around p.M99R is highly conserved, and p.M99R leads to instability of the tertiary structure of the ABCG5/ABCG8 heterodimer. GPS 5.0 predicted that M99R affects the phosphorylation of nearby amino acid sequences, and DUET and VarSite software predicted that M99R affects the stability of sterolin-1 and cause disease. The p.M99R and c.-76 C >T mutations led to the formation of unstable heterodimers, which disturbed sterol absorption and excretion in vivo. The compound heterozygous variants c.296 T >G (p.m99r) and C.-76 C >T on exon 3 of ABCG5 in this family may be the molecular genetic basis of sitosterolemia.

A novel compound heterozygous variant linked to hematuria in a family with hereditary factor VII deficiency.

BACKGROUND: Hereditary factor VII deficiency (FVIID) is a rare congenital autosomal recessive bleeding disorder. In clinical manifestations, its onset is caused by variant of the F7 gene (NM_019616) with strong heterogeneity. We identified a family with hematuria caused by a novel F7 compound heterozygous variant and investigated the FVIID-dependent mechanism impacted by these variants. METHODS: Coagulation factors in the proband were functionally verified. We located pathogenic variants in relevant genes using next-generation sequencing after target enrichment and verified them by Sanger sequencing. We examined the coagulation activity and secretion pattern of recombinant FVII variants expressed in cells and observed their location and stability by immunofluorescence. RESULTS: We found a missense variant c.1207G>A (p.Gly403Ser) and a frameshift variant c.154_155del (p.Arg53fs) in the F7 gene of the proband. FVII activity tests showed that the variants significantly decreased its presence in the cell culture supernatant. Moreover, the R53fs mutant lacked the FVII functional domain and had no detectable activity. Immunofluorescence indicated that the p.Gly403Ser variant was distributed to the cell membrane and cytoplasm, whereas the FVII R53fs variant was not detected. Deficient FVII protein function and severe coagulation disorder are the likely causes of hematuria and other bleeding symptoms in the proband. CONCLUSIONS: The newly discovered F7 gene variants enrich the spectrum of hereditary FVII deficiency and provide a new foundation for the diagnosis and treatment of this type of coagulation disorder.

Different phenotypes of neurological diseases, including alternating hemiplegia of childhood and rapid-onset dystonia-parkinsonism, caused by de novo ATP1A3 mutation in a family.

BACKGROUND: The spectrum of neurological diseases related to ATP1A3 gene mutations is highly heterogeneous and exhibits different phenotypes. Phenotype overlaps, including alternating hemiplegia of childhood (AHC), early infantile epileptic encephalopathy, and rapid-onset dystonia-parkinsonism (RDP), can also occur at extremely low incidences. Currently, over 90 types of pathogenic mutations have been identified in ATP1A3. PATIENTS AND METHODS: The family of a 2-year-11-month-old proband with AHC was recruited for this clinical investigation. The proband was screened for candidate mutation gene sites using next-generation sequencing and target-region capture technology. Sanger sequencing was used to identify carriers among family members. RESULTS: The mother of the proband with AHC was diagnosed with dystonia (later diagnosed as RDP). The biochemical and immune indices of the proband and the mother were not abnormal. Moreover, brain imaging of the proband revealed no significant abnormalities. However, the electroencephalogram of the mother was mildly abnormal, with no spike wave discharge. Brain MRI revealed slight cerebellar atrophy. Electromyography revealed neurogenic damage, with a decrease in the conduction velocity of the left ulnar and radial nerves. Based on the sequencing data, both the proband and her mother carried c.823G > C p. (Ala275Pro) heterozygotes; other family members were not identified as carriers. With a PolyPhen-2 score of 0.997 and SIFT score of 0.001, this mutation can be considered damaging. CONCLUSION: Family genotype-phenotype correlation analysis revealed that the phenotype and gene mutation were co-segregated, suggesting that it may be a pathogenic mutation.

A thrombophilia family with protein S deficiency due to protein translation disorders caused by a Leu607Ser heterozygous mutation in PROS1.

BACKGROUND: Protein S deficiency (PSD) is an autosomal dominant hereditary disease. In 1984, familial PSD was reported to be prone to recurrent thrombosis. Follow-up studies have shown that heterozygous protein S (PROS1) mutations increase the risk of thrombosis. More than 300 PROS1 mutations have been identified; among them, only a small number of mutations have been reported its possible mechanism to reduce plasma protein S (PS) levels. However, whether PROS1 mutations affect protein structure and why it can induce PSD remains unknown. METHODS: The clinical phenotypes of the members of a family with thrombosis were collected. Their PS activity was measured using the coagulation method, whereas their protein C and antithrombin III activities were measured using methods such as the chromogenic substrate method. The proband and her parents were screened for the responsible mutation using second-generation whole exon sequencing, and the members of the family were verified for suspected mutations using Sanger sequencing. Mutant and wild type plasmids were constructed and transfected into HEK293T cells to detect the mRNA and protein expression of PROS1. RESULTS: In this family, the proband with venous thrombosis of both lower extremities, the proband's mother with pulmonary embolism and venous thrombosis of both lower extremities, and the proband's younger brother had significantly lower PS activity and carried a PROS1 c. 1820 T > C:p.Leu607Ser heterozygous mutation (NM_000313.3). However, no such mutations were found in family members with normal PS activity. The PS expression in the cell lysate and supernatant of the Leu607Ser mutant cells decreased, while mRNA expression increased. Immunofluorescence localization showed that there was no significant difference in protein localization before and after mutation. CONCLUSIONS: The analysis of family phenotype, gene association, and cell function tests suggest that the PROS1 Leu607Ser heterozygous mutation may be a pathogenic mutation. Serine substitution causes structural instability of the entire protein. These data indicate that impaired PS translation and synthesis or possible secretion impairment is the main pathogenesis of this family with hereditary PSD and thrombophilia.

Review and Analysis of Two Gitelman Syndrome Pedigrees Complicated with Proteinuria or Hashimoto's Thyroiditis Caused by Compound Heterozygous SLC12A3 Mutations.

Gitelman syndrome (GS) is an autosomal recessive inherited salt-losing renal tubular disease, which is caused by a pathogenic mutation of SLC12A3 encoding thiazide-sensitive Na-Cl cotransporter, which leads to disturbance of sodium and chlorine reabsorption in renal distal convoluted tubules, resulting in phenotypes such as hypovolemia, renin angiotensin aldosterone system (RAAS) activation, hypokalemia, and metabolic alkalosis. In this study, two GS families with proteinuria or Hashimoto's thyroiditis were analyzed for genetic-phenotypic association. Sanger sequencing revealed that two probands carried SLC12A3 compound heterozygous mutations, and proband A carried two pathogenic mutations: missense mutation Arg83Gln, splicing mutation, or frameshift mutation NC_000016.10:g.56872655_56872667 (gcggacatttttg>accgaaaatttt) in exon 8. Proband B carries two missense mutations: novel Asp839Val and Arg904Gln. Both probands manifested hypokalemia, hypomagnesemia, hypocalcinuria, metabolic alkalosis, and RAAS activation; in addition, the proband A exhibited decreased urinary chloride, phosphorus, and increased magnesium ions excretion, complicated with Hashimoto's Thyroiditis, while the proband B exhibited enhanced urine sodium excretion and proteinuria. The older sister of proband B with GS also had Hashimoto's thyroiditis. Electron microscopy revealed swelling and vacuolar degeneration of glomerular epithelial cells, diffuse proliferation of mesangial cells and matrix, accompanied by a small amount of low-density electron-dense deposition, and segmental fusion of epithelial cell foot processes in proband B. Light microscopy showed mild mesangial hyperplasia in the focal segment of the glomerulus, hyperplasia, and hypertrophy of juxtaglomerular apparatus cells, mild renal tubulointerstitial lesions, and one glomerular sclerosis. So, long-term hypokalemia of GS can cause kidney damage and may also be susceptible to thyroid disease.

Screening and function discussion of a hereditary renal tubular acidosis family pathogenic gene.

Hereditary distal renal tubular acidosis (dRTA) is a rare disease of H+ excretion defect of α-intercalated cells in renal collecting duct, caused by decreased V-ATPase function due to mutations in the ATP6V1B1 or ATP6V0A4 genes. In the present study, a genetic family with 5 members of the complete dRTA phenotype were found with distal tubule H+ secretion disorder, hypokalemia, osteoporosis, and kidney stones. A variant NM_020632.2:c.1631C > T (p.Ser544Leu) in exon 16 on an ATP6V0A4 gene associated with dRTA was detected by next generation sequencing target region capture technique and verified by Sanger sequencing, which suggested that except for one of the patients who did not receive the test, the other four patients all carried the p.S544L heterozygote. In transfected HEK293T cells, cells carrying p.S544L-mut showed early weaker ATPase activity and a slower Phi recovery rate after rapid acidification. By immunofluorescence localization, it was observed that the expression level of p.S544L-mut on the cell membrane increased and the distribution was uneven. Co-immunoprecipitation showed the a4 subunit of ATP6V0A4/p.S544L-mut could not bind to the B1 subunit, which might affect the correct assembly of V-ATPase. The present study of dRTA family suggests that the p.S544L variant may be inherited in a dominant manner.

Genetic mutation of familial dilated cardiomyopathy based on next­generation semiconductor sequencing.

Dilated cardiomyopathy (DCM) is a complex myocardial disease of multifactorial etiologies, including enlarged cardiac chambers and contractile dysfunction. It has been suggested that the inheritance of DCM­associated mutations predominates its onset. Therefore, the present study investigated the pathogenesis of DCM via pedigree analysis and genetic diagnosis by massive whole­exome screening, and targeted exon capture. To study the familial gene­phenotype association, the exon and splice sites of 325 hereditary disease­associated genes in the proband with familial dilated cardiomyopathy (FDC), including 61 cardiac disease­associated genes, such as the lamins A/C (LMNA), were analyzed by ultra­high multiplex polymerase chain reaction and the Ion AmpliSeq™ Inherited Disease Panel. The present study also conducted Sanger DNA Sequencing for family members with global minor allele frequencies <1% to verify potential pathogenic mutation sites. A total of three rare missense mutations were detected, including heterozygous c.244G>A in LMNA, c.546C>G in potassium voltage­gated channel subfamily KQT (KCNQ4) and c.1276G>A in EYA transcriptional coactivator and phosphatase 1 (EYA1), indicating a glutamic acid to lysine substitution at amino acid 82 (p.E82K) in LMNA, a p.F182L in KCNQ4 (a mutation associated with pathogenic deafness) and p.G426S in EYA1 (associated with Branchiootorenal syndrome 1 and Branchiootic syndrome 1 pathogenesis). In the present study, a carrier with slight hearing impairment was detected in the family analyzed; however, no patients with deafness or branchiootorenal syndrome were observed. LMNA p.E82K revealed SIFT and PolyPhen­2 scores of 0 and 1, respectively. In the second generation, 3 patients with DCM underwent permanent pacemaker implantation due to sick sinus syndrome, atrioventricular block and unstable cardiac electrophysiology. The present study suggested that LMNA p.E82K may contribute to the pathogenesis of FDC and concomitant atrioventricular block. At present, only three families with DCM resulting from similar mutations have been reported. The present study demonstrated the strong pathogenic effects of LMNA p.E82K on DCM.