[Identification of a novel variant of NHS gene underlying Nance-Horan syndrome].

OBJECTIVE: To explore the genetic basis for a pedigree affected with Nance-Horan syndrome. METHODS: Clinical manifestation of the patients was analyzed. Genomic DNA was extracted from peripheral blood samples of the pedigree members and 100 unrelated healthy controls. A panel of genes for congenital cataract was subjected to next-generation sequencing (NGS), and candidate variant was verified by Sanger sequencing and bioinformatic analysis based on guidelines of American College of Medical Genetics and Genomics (ACMG). mRNA expression was determined by reverse transcriptase-PCR (RT-PCR). Linkage analysis based on short tandem repeats was carried out to confirm the consanguinity. RESULTS: A small insertional variant c.766dupC (p.Leu256Profs*21) of the NHS gene was identified in the proband and his affected mother, but not among unaffected members and the 100 healthy controls. The variant was unreported in Human Gene Mutation Database (HGMD) and other databases. Based on the ACMG guideline, the variant is predicted to be pathogenic (PVS1+PM2+PM6+PP4). CONCLUSION: The novel variant c.766dupC of the NHS gene probably underlay the X-linked dominant Nance-Horan syndrome in this pedigree.

[Identification of a novel variant of COL4A5 gene in a pedigree affected with Alport syndrome].

OBJECTIVE: To explore the genetic basis for a pedigree affected with Alport syndrome. METHODS: Next generation sequencing and Sanger sequencing was carried out to detect potential variant of the COL4A5 gene among members from the pedigree and 100 unrelated healthy controls. RESULTS: A novel missense c.3293G>T (p.Gly1098Val) variant was found in the COL4A5 gene among 6 affected members but not the unaffected members of the pedigree or the 100 healthy controls. According to the American College of Medical Genetics and Genomics standards and guidelines, the c.3293G>T variant was classified as pathogenic (PP1-strong+PM1+PM2+PP3+PP4). CONCLUSION: By destructing the Gly-X-Y structure of its protein product, the c.3293G>T variant of the COL4A5 gene probably underlies the Alport syndrome in this pedigree. Above finding has enriched the spectrum of COL4A5 variants.

[Identification of a novel splicing variant of IDS gene in a pedigree affected with type II glycosaminoglycan product storage disease].

OBJECTIVE: To analyze variant of IDS gene in a pedigree affected with mucopolysaccharidosis type II (MPS II). METHODS: The proband was subjected to next generation sequencing and Sanger sequencing to identify potential variants. Suspected variant was analyzed by its co-segregation with the disease in the pedigree. Its impact on mRNA splicing was analyzed by using reverse transcription PCR (RT-PCR). RESULTS: A hemizygous IVS1-3T>G variant was found in the IDS gene in the proband. RT-PCR results revealed two abnormal cDNA fragments of 600 bp and 300 bp. The 600 bp fragment had inserted 216 nucleotides at the 3' end of intron 1, while the 300 bp fragment had lost 109 nucleotides at the 5' end of exon 2, which resulted in two truncated proteins comprising 38 and 92 amino acids, respectively, instead of the normal product (550 amino acids). The proband and his mother were respectively hemizygous and heterozygous for the variant. The same variant was not found among 100 normal controls. CONCLUSION: The IVS1-3T>G variant of the IDS gene probably underlies the MPS II in this pedigree by causing reduction or elimination of the IDS protein.

A novel splicing mutation in the PKD1 gene causes autosomal dominant polycystic kidney disease in a Chinese family: a case report.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic renal disorder in humans, affecting 1 in 400 to 1000 individuals. Mutations PKD1 (which accounts for 85% of ADPKD and produces polycystin-1) and PKD2 (produces polycystin-2) are responsible for this disease. These two polycystins are critical for maintaining normal renal tubular structures during kidney development. CASE PRESENTATION: We performed genetic analysis on a family with ADPKD. DNA samples extracted from ADPKD patient blood were subject to targeted Next generation sequencing for human a panel of renal disease-related genes. A splicing mutation, c.2854-3C > G (also known as IVS11-3C > G), in the PKD1 gene was found in the 3 patients from the family, but was not found in four unaffected relatives and 100 normal control samples. Reverse transcription-PCR (RT-PCR) was performed to analyse the relative mRNA expression in the patient samples. mRNA sequencing showed that 29 bases inserted into the 3'-end of exon 11 in the PKD1 gene lead to a frameshift mutation. CONCLUSIONS: The PKD1 c.2854-3C > G mutation leads to a frameshift mutation during translation of the polycystin-1 protein, which eventually led to ADPKD in the Chinese family.

[Application of droplet digital PCR for non-invasive prenatal diagnosis of single gene disease in two families].

OBJECTIVE: To assess the value of droplet digital PCR (ddPCR) for non-invasive prenatal diagnosis of single gene disease in two families. METHODS: Paternal mutation in cell-free DNA derived from the maternal blood and amniotic fluid DNA was detected by ddPCR. Suspected mutation in the amniotic fluid DNA was verified with Sanger sequencing. RESULTS: The result of ddPCR and Sanger sequencing indicated that the fetuses have carried pathogenic mutations from the paternal side in both families. CONCLUSION: Droplet digital PCR can accurately detect paternal mutation carried by the fetus, and it is sensitive and reliable for analyzing trace samples. This method may be applied for the diagnosis of single gene diseases caused by paternal mutation using peripheral blood sample derived from the mother.

[Novel mutations of XPC gene detected in a family affected with xeroderma pigmentosum group C].

OBJECTIVE: To detect mutations of the XPC (XPC complex subunit, DNA damage recognition and repair factor) gene in a family affected with xeroderma pigmentosum group C (XP-C). METHODS: The patient was subjected to next-generation sequencing and Sanger sequencing. Suspected mutations were validated by Sanger sequencing. Effect of splicing mutation was confirmed by reverse transcription-PCR (RT-PCR). RESULTS: Compound heterozygous mutations of c.2098G to T and c.2034-7_2040del were found in the XPC gene in the proband. Among these, c.2098G to T (p.G700X) is a nonsense mutation resulting in a truncated XPC protein. C.2034-7_2040del involves the -1 position, which may alter the splice donor site of the intron 11 of XPC and result in a truncated XPC protein with loss of amino acids from 940 to 679 positions. The two mutations were not detected among 100 unrelated healthy controls. CONCLUSION: Mutations of c.2098 G to T and c.2034-7_2040del of the XPC gene may lead to abnormal XPC expression and reduction or elimination of normal XPC functions, which may underlie the disease in this family.

[Identification of a novel splicing mutation of PHEX gene in a pedigree affected with X-linked hypophosphatemia].

OBJECTIVE: To identify potential mutation of PHEX gene in two patients from a family affected with X-linked hypophosphatemia (XLH). METHODS: PCR and Sanger sequencing were performed on blood samples from the patients and 100 healthy controls. Reverse transcription-PCR (RT-PCR) was used to determine the mRNA expression in patient samples. RESULTS: A splicing site mutation, IVS21+2T>G, was found in the PHEX gene in both patients but not among the 100 healthy controls. RT-PCR confirmed that exon 21 of the PHEX gene was deleted. CONCLUSION: The novel splicing mutation IVS21+2T>G of the PHEX gene probably underlies the XLH in this pedigree. At the mRNA level, the mutation has led to removal of exon 21 and shift of the open reading frame (p.Val691fsx), resulting in premature termination of protein translation.

[A novel mutation of GLI3 gene underlying synpolydactyly in a family].

OBJECTIVE: To detect mutation of GLI3 gene in a family affected with autosomal dominant synpolydactyly. METHODS: Genomic DNA was extracted from peripheral blood samples from members of the family and 100 unrelated healthy controls. Potential mutation was screened by next-generation sequencing and confirmed by Sanger sequencing. RESULTS: A heterozygous frameshift mutation c.480dupC was identified in the GLI3 gene among all patients from the family. The same mutation was not found in unaffected family members and the 100 healthy controls. CONCLUSION: The c.480dupC of the GLI3 gene probably underlies the synpolydactyly in this family.

[A novel pathogenic mutation of CRYGD gene in a congenital cataract family].

OBJECTIVE: To detect the disease-causing mutation in a pedigree affected with autosomal dominant congenital cataract. METHODS: Genomic DNA was extracted and purified from peripheral blood samples from members of the pedigree and 100 healthy controls. Coding regions of 18 candidate genes were screened with PCR and Sanger sequencing. Identified mutations were verified among 100 healthy individuals to exclude single nucleotide polymorphisms. RESULTS: A heterozygous nonsense mutation c.471G>A of the CRYGD gene, which resulted in p.Trp157Term, was identified in all three patients. The same mutation was not found in the two normal individuals from the family and 100 healthy controls. The nonsense mutation was predicted to be "disease causing" by Mutation t@sting program. CONCLUSION: The nonsense mutation c.471G>A of the CRYGD gene probably underlies the congenital cataract in the pedigree.

[Application of droplet digital PCR technology for genetic testing and prenatal diagnosis of spinal muscular atrophy].

OBJECTIVE: To explore the clinical application of droplet digital PCR (ddPCR) for genetic testing and prenatal diagnosis of spinal muscular atrophy (SMA) with deletion of SMN1 gene exon 7. METHODS: A total of 138 clinical samples, including 121 peripheral blood, 13 amniotic fluid, 2 umbilical cord blood and 2 chorionic villi from 56 SMA families, were tested by both ddPCR and multiplex ligation-dependent probe amplification (MLPA). Results of the two approaches were analyzed with commercial software QuantaSoft (ddPCR) and Coffalyser (MLPA), respectively. RESULTS: Among the 138 cases, 25 had two copies, 84 had one copy, and 29 had null copy of exon 7 of the SMN1 gene. The results of ddPCR and MLPA were completely consistent. CONCLUSION: As a rapid, precise and economically efficient method, ddPCR will provide a new choice for genetic testing of SMA.

[Identification of a novel splicing mutation of PKD1 gene in a pedigree affected with autosomal dominant polycystic kidney disease].

OBJECTIVE: To identify potential mutations of PKD1 gene in a family affected with autosomal dominant polycystic kidney disease (ADPKD). METHODS: The coding regions of the PKD1 gene were subjected to PCR and Sanger sequencing. Reverse transcription-PCR (RT-PCR) was used to determine the relative mRNA expression in the patient. RESULTS: A splicing site mutation, c.8791+1_8791+5delGTGCG (IVS23+1_+5delGTGCG), was detected in the PKD1 gene in all 5 patients from the pedigree but not in 6 phenotypically normal relatives and 40 healthy controls. Sequencing of RNA has confirmed that there were 8 bases inserted in the 3' end of exon 23 of the PKD1 gene. CONCLUSION: The novel c.8791+1_8791+5delGTGCG mutation has created a new splice site and led to a frameshift, which probably underlies the ADPKD in the family. Above finding has enriched the mutation spectrum of the PKD1 gene.

Flexible Strain Sensors Based on an Interlayer Synergistic Effect of Nanomaterials for Continuous and Noninvasive Blood Pressure Monitoring.

The continuous, noninvasive monitoring of human blood pressure (BP) through the accurate detection of pulse waves has extremely stringent requirements on the sensitivity and stability of flexible strain sensors. In this study, a new ultrasensitive flexible strain sensor based on the interlayer synergistic effect was fabricated through drop-casting and drying silver nanowires and graphene films on polydimethylsiloxane substrates and was further successfully applied for continuous monitoring of BP. This strain sensor exhibited ultrahigh sensitivity with a maximum gauge factor of 34357.2 (∼700% sensitivity enhancement over other major sensors), satisfactory response time (∼85 ms), wide strange range (12%), and excellent stability. An interlayer fracture mechanism was proposed to elucidate the working principle of the strain sensor. The real-time BP values can be obtained by analyzing the relationship between the BP and the pulse transit time. To verify our strain sensor for real-time BP monitoring, our strain sensor was compared with a conventional electrocardiogram-photoplethysmograph method and a commercial cuff-based device and showed similar measurement results to BP values from both methods, with only minor differences of 0.693, 0.073, and 0.566 mmHg in the systolic BP, diastolic BP, and mean arterial pressure, respectively. Furthermore, the reliability of the strain sensors was validated by testing 20 human subjects for more than 50 min. This ultrasensitive strain sensor provides a new pathway for continuous and noninvasive BP monitoring.

OGM and WES identifies translocation breakpoints in PKD1 gene in an polycystic kidney patient and healthy baby delivered using PGT.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common autosomal dominant genetic diseases. Whole exome sequencing (WES) is a routine tool for diagnostic confirmation of genetic diseases, and it is usually performed to confirm the clinical diagnosis in ADPKD. Reciprocal translocation is the most common chromosomal structural abnormalities and most of its carriers have normal phenotypes until they are encountered infertility problems in adulthood. However, for the polycystic kidney disease caused by abnormal chromosome structure, WES is difficult to achieve the purpose of gene diagnosis. METHODS: ADPKD-related genes were detected by WES; Chromosomal karyotyping and Optical Genome Mapping (OGM) were used to detect structural variant; The genomic break-point locations and the abnormal splicing were detected by reverse transcription-PCR and Sanger sequencing; The karyomapping gene chip and Next-Generation Sequencing (NGS) were performed to screen aneuploidy and to distinguish the non-carrier embryos from the carrier embryos. RESULTS: No pathogenic variant was found after the first round of WES analysis. Karyotyping data showed 46, XX, t (16; 17) (p13.3; q21.3). With the help of OGM, the translocation breakpoint on chromosome 16 was located within the PKD1 gene. With re-analysis of WES raw data, the breakpoint of translocation was verified to be located at the c.10618 + 3 of PKD1 gene. Based on this molecular diagnosis, a non-carrier embryo was selected out from three blastocysts. With preimplantation genetic testing (PGT) after in vitro fertilization (IVF), it was then transferred into uterus. With confirmation by prenatal and postnatal testing, the pedigree delivered a healthy baby. CONCLUSION: We identified a case of ADPKD caused by balanced translocation and assisted the patient to have a healthy child. When the phenotype was closely related with a monogenic disease and the WES analysis was negative, chromosomal structural analysis would be recommended for further genetic diagnosis. Based on the precision diagnosis, preventing the recurrence of hereditary diseases in offspring would be reachable.

Apolipoprotein E epsilon 2 allele and low serum cholesterol as risk factors for gastric cancer in a Chinese Han population.

Apolipoprotein E (apoE) mediates lipid metabolism both in peripheral and in the brain. The human APOE gene has three polymorphic alleles that influence the risk for various types of cancer and neurodegenerative diseases. A potential association between APOE allele and the risk for gastric cancer has been implicated, but the specific allele involved and potential associations with the subtype and the grade of cancer malignancy need further clarification. We screened the APOE genotype in 550 gastric cancer patients and 550 non-cancer control individuals and found that the presence of the APOE ε2 and lower serum total cholesterol are associated with an increased risk for gastric cancer (all P ≤ 0.0005). Interestingly, APOE ε2 is also correlated with increased risk for both intestinal and diffuse histotypes but not with TN classification or stage in gastric cancer patients, suggesting that APOE polymorphic alleles are associated with the risk of development but unlikely the progression of gastric cancer. Since ε2 carriers have lower levels of serum total cholesterol than non-ε2 carriers, our findings suggest that the increased risk for gastric cancer by APOE ε2 allele might be mediated through lowered serum total cholesterol levels.