Background Hypophosphatemic rickets, including familial hypophosphatemic vitamin D-resistant rickets, which commonly manifests in childhood, is generally hereditary. X-linked dominant hypophosphatemic rickets (XLH, MIM307800), caused by inactivating mutations in the PHEX gene, is the most common form. This study aimed to identify the gene mutations responsible for three cases of XLH and its clinical phenotype. Methods We conducted a genetic diagnosis and clinical phenotypic linkage analysis of three pedigrees with XLH. Three probands finally diagnosed as XLH were analyzed by next-generation sequencing (NGS). Sanger sequencing was used for mutation scanning in other family members. Results For the three patients with XLH, the age of onset ranged from 1.5 to 2 years and their heights were less than three standard deviations (SDs) below the median. The patients exhibited curved deformities in both lower limbs, hypophosphatemia, elevated serum FGF23 levels and elevated levels of blood alkaline phosphatase, with normal levels of blood parathyroid hormone (PTH) and calcium. X-ray analysis of the limbs and chest revealed characteristic rickets signs. Three candidate pathogenic mutations were identified in PHEX (NM_000444.5): c.433G>T (p.Glu145*, p.E145*) in exon 4, c.1735G>A (p.Gly579Arg, p.G579R) (rs875989883) in exon 17 and c.2245T>C (p.Trp749Arg, p.W749R) in exon 22. The nonsense mutation (p.E145*) in PHEX is novel and is predicted to cause a truncation of the encoded protein, resulting in loss of function. Conclusions The novel nonsense mutation (p.E145*) in PHEX is possibly involved in inherited XLH.

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.

In the present study whole-exome sequencing using the Complete Genomics platform was employed to scan a proband from a split-hand/split-foot malformation (SHFM) 4 family. The missense mutation c.728G>A (p.Arg243Gln) in the TP63 gene was revealed to be associated with SHFM. Sanger sequencing confirmed the sequences of the proband and his father. The father was diagnosed with SHFM and harbored a CGG-to-CAG mutation in exon 5, which produced a R243Q substitution in the zinc binding site and dimerization site of TP63. The R243Q mutation was predicted to be pathogenic by PolyPhen-2. The proband, who was diagnosed with four digit SHFM, exhibited a more severe phenotype. X-ray analysis returned the following results: Absence of third phalange bilaterally and third metacarpus of the left hand; absence of the second toes bilaterally and partial third toes; and partial fusion of the second, third and metatarsal bones of the right side with deformity of the second metatarsal of the right side. Osteochondroma was present in the fourth proximal radial metacarpal of the left hand and the basal and proximal parts of the second metatarsal of the right side. The proband's father had five digits in both feet. These results indicate that the R243Q mutation produces a novel phenotype named SHFM4. The present study revealed that the R243Q mutation in the TP63 gene produced a novel phenotype named SHFM4, thereby demonstrating the mutational overlap between ectrodactyly-ectodermal dysplasia-cleft syndrome and SHFM4.

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.

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.

BackgroundsGrowth hormone deficiency (GHD) is a major cause of congenital short stature. GHD patients have significantly decreased serum leptin levels, which are regulated by gene polymorphism of leptin and leptin receptor. This study thus investigated the relationship between gene polymorphism and susceptibility to GHD.Material/MethodsA case-control study was performed using 180 GHD children in addition to 160 healthy controls. After the extraction of whole genomic DNA, the genotypes of leptin and leptin receptor gene loci were analyzed by sequencing for single-nucleotide polymorphism.ResultsThe frequency distribution of all alleles identified in leptin gene (loci rs7799039) and leptin receptor gene (loci rs1137100 and rs1137101) fit Hardy-Weinberg equilibrium. There was a significant difference in allele frequency at loci rs7799039 or rs1137101, as individuals with heterozygous GA allele had lower (rs7799039) or higher (rs1137101) GHD risk. No significant difference in allele frequency was discovered at loci rs1137100 (p>0.05), which was unrelated to GHD susceptibility.ConclusionsGene polymorphism of leptin (loci rs7799039) and leptin receptor (loci rs1137101) are correlated with GHD susceptibility.

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 DCM-associated mutations predominates its onset. Therefore, the present study investigated the pathogenesis of DCM via pedigree analysis and genetic diagnosis by massive whole-exome screening, and targeted exon capture. To study the familial gene-phenotype association, the exon and splice sites of 325 hereditary disease-associated genes in the proband with familial dilated cardiomyopathy (FDC), including 61 cardiac disease-associated genes, such as the lamins A/C (LMNA), were analyzed by ultra-high 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 voltage-gated 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 PolyPhen-2 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.

BackgroundBruck 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.MethodsSerum 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.ResultsIn this pedigree, the proband was found to have a homozygous variant c.1856G &gt; 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 &gt; 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.ConclusionThrough gene and cell function analyses, PLOD2 Arg619His missense variant was preliminarily confirmed to cause BS by reducing protein expression.