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Here we reported a pedigree that gave birth to two characteristic clinical signs of Kabuki syndrome daughters. They had an intellectual disability with special facial features. Their eyebrows were relatively wide and the rear 1/3 of the eyebrows were light and sparse. Their eyes were long, narrow, valgus and strabismus. Their noses were broad at the root and flat at the tip. They also had skeletal dysplasia, mainly manifested in the short second knuckle of the little fingers of both hands. Genetic studies showed a novel de novo KMT2D variant (c.16343G C; p.R5448P) as a cause of Kabuki syndrome. It was very unlikely that the same de novo mutation occurred in two members of a family. Gonadal mosaicism in one of the parents was suspected. Haplotype construction and clone sequencing were used for mutation source analysis. Finally, we inferred that the haplotype from the mother (Gdel-G-C-T-A) contained the pathogenic mutation. A gonadal mosaicism novel KMT2D mutation was identified in their mother.

Retinitis pigmentosa 77 is caused by mutations of REEP6 (MIM: 609346), which encodes a protein for the development of photoreceptors. Our study was to identify disease-causing variants in three Chinese families using targeted next-generation sequencing (NGS). Multiple lines of computational predictions combined with in vitro cellular experiments were applied to evaluate the pathogenicity of the newly found variants. Three novel variants in REEP6, including one missense variant, c.268G&gt, C, one frameshift variant, c.468delC, and one splicing variant, c.598+1G&gt, C, were found, while c.268G&gt, C was detected in all probands. The three variants were classified as likely pathogenic by the American College of Medical Genetics and Genomics (ACMG). REEP6 variant proteins c.268G&gt, C and c.468delC in cultured cells destabilized the REEP6 protein and induced intracellular inclusions. Our data suggested that REEP6 c.268G&gt, C may be a recurrent causative variant in Chinese autosomal recessive retinitis pigmentosa patients.

To explore the genetic basis for a pedigree affected with hereditary spastic paraplegia type 4 (HSP4).Peripheral venous blood samples were taken from members of the four-generation pedigree and 50 healthy controls for the extraction of genomic DNA. Genes associated with peripheral neuropathy and hereditary spastic paraplegia were captured and subjected to targeted capture and next-generation sequencing. The results were confirmed by Sanger sequencing.DNA sequencing suggested that the proband has carried a heterozygous c.1196CG variant in exon 9 of the SPAST gene, which can cause substitution of serine by threonine at position 399 (p.Ser399Trp) and lead to change in the protein function. The same variant was also detected in other patients from the pedigree but not among unaffected individuals or the 50 healthy controls. Based on the ACMG 2015 guidelines, the variant was predicted to be possibly pathogenic.The c.1196CG variant of the SPAST gene probably underlay the HSP4 in this pedigree.

Congenital hand malformations is rare and characterized by hand deformities. It is highly heterogeneous, both clinically and genetically, which complicates the identification of causative genes and mutations. Recently, targeted next-generation (NGS) sequencing has been successfully used for the detection of heterogeneous diseases, and the use of NGS also has contributed significantly in evaluating the etiology of heterogeneous disease. Here, we employed targeted NGS to screen 248 genes involved in genetic skeletal disorders, including congenital hand malformations. Three pathogenic mutations located in the GJA1, ROR2 and TBX5 genes were detected in three large Chinese families with congenital hand malformations. Two novel mutations were reported, and a known causative mutation was verified in this Chinese population. This is also the first report that the same panel of targeted NGS was employed to perform molecular diagnosis of different subtypes of congenital hand malformations. Our study supported the application of a targeted NGS panel as an effective tool to detect the genetic cause for heterogeneous diseases in clinical diagnosis.

Donohue syndrome (DS), a rare autosomal recessive disease which represents severe insulin resistance, pre- and postnatal growth retardation, hypertrichosis, and dysmorphic features, is caused by mutations in the insulin receptor (INSR) gene. Here, we have reported the clinical, molecular, and biochemical characterizations of a patient with DS. In this article, we have also reported a case with 2 novel INSR mutations and the DS phenotype. Using next-generation sequencing (NGS), we screened 27 known genes involved in inherited maturity-onset diabetes of the young (MODY) and identified compound heterozygous mutations in the INSR gene in the patient with DS, c.62T>G (p.L21R) and c.2563G>T (p.V855F). The positive correlation of these mutations with DS was further validated by Sanger DNA sequencing of his lineal consanguinity, indicating that these pathogenic mutations were inherited maternally and paternally, respectively. Therefore, our finding expanded the number of reported cases of this rare disease and the mutation spectrum of INSR mutation, suggesting that NGS is an accurate, rapid, and cost-effective method for the genetic diagnosis of this rare disease.

To explore the genetic basis for a family with non-syndromic autosomal recessive deafness.The proband and her parents were subjected to physical and audiological examinations. With genomic DNA extracted from peripheral blood samples, next-generation sequencing was carried out using a panel for deafness genes. Suspected mutation was validated by Sanger sequencing and qPCR analysis of her parents.The proband presented bilateral severe sensorineural hearing loss at three days after birth. Her auditory threshold was 110-120 dBnHL but with absence of vestibular and retinal symptoms. Her brother also had deafness but her parents were normal. No abnormality was found upon physical examination of her family members, while audiological examination showed no middle ear or retrocochlear diseases. Next-generation sequencing identified compound heterozygous mutations of the MYO7A gene, including a previously known c.462CA (p. Cys154Ter) and a novel EX43_46 Del, which were respectively derived from her mother and father.The compound heterozygous mutations of the MYO7A gene probably underlie the disease in this family. Our findings has enriched the mutation spectrum for non-syndromic autosomal recessive deafness 2.

To explore the clinical and genetic features of a patient suspected with Juvenile Parkinson's syndrome (JP).Clinical features of the patient were analyzed. Genomic DNA of the patient and his parents was extracted from peripheral blood samples and sequenced by exome capture sequencing. The nature and impact of detected mutations were predicted and validated.The patient displayed typical features including resting tremor, bradykinesia, rigidity, but with excellent response to low dose levodopa. DNA sequencing showed that she has carried compound heterozygous mutations of the Parkin gene, namely c.1381dupC and c.619-1GC, which were respectively inherited from his mother and father. Neither mutation was reported previously. Bioinformatic analysis predicted that both mutations are pathogenic.The patient has JP caused by mutations of the Parkin gene. Exome capture sequencing is an accurate and efficient method for genetic diagnosis of such disease.

Introduction Adams-Oliver syndrome (AOS) is a rare developmental disorder characterized by the combination of aplasia cutis congenita of the scalp vertex and terminal transverse limb defects. DOCK6 (Dedicator of cytokinesis 6) is one of the six identified AOS genes. Methods We performed targeted next-generation sequencing (NGS) of a child with an AOS phenotype. Sanger DNA sequencing further validated her lineal consanguinity. To explore the pathological features of the mutation, a minigene assay was used to investigate the effects of the mutation on splicing. Results Two compound heterozygous DOCK6 mutations (c.4106+2T>C and c.3063 C>G (p.Y1021*)) were identified in this family, and both mutations have not been reported previously. Sanger DNA sequencing indicated that the mutations were inherited maternally and paternally, respectively. The results of the minigene assay showed that the c.4106+2T>C mutation resulted in aberrant splicing and caused a four-nucleotide insertion in the transcript and a premature stop codon. Conclusions Our findings expanded the number of reported cases of this rare disease and the mutation spectrum of DOCK6 mutations, which can serve as the basis for prenatal diagnosis and genetic counseling.

Identification of genes with variants causing non-syndromic hearing loss (NSHL) is challenging due to genetic heterogeneity. The difficulty is compounded by technical limitations that in the past prevented comprehensive gene identification. Recent advances in technology, using targeted capture and next-generation sequencing (NGS), is changing the face of gene identification and making it possible to rapidly and cost-effectively sequence the whole human exome. Here, we characterize a five-generation Chinese family with progressive, postlingual autosomal dominant nonsyndromic hearing loss (ADNSHL). By combining population-specific mutation arrays, targeted deafness genes panel, whole exome sequencing (WES), we identified PDE1C (Phosphodiesterase 1C) c.958G>T (p.A320S) as the disease-associated variant. Structural modeling insights into p.A320S strongly suggest that the sequence alteration will likely affect the substrate-binding pocket of PDE1C. By whole-mount immunofluorescence on postnatal day 3 mouse cochlea, we show its expression in outer (OHC) and inner (IHC) hair cells cytosol co-localizing with Lamp-1 in lysosomes. Furthermore, we provide evidence that the variant alters the PDE1C hydrolytic activity for both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Collectively, our findings indicate that the c.958G>T variant in PDE1C may disrupt the cross talk between cGMP-signaling and cAMP pathways in Ca2+ homeostasis.

To detect potential mutation in a Chinese pedigree affected with familial exudative vitreoretinopathy (FEVR).Clinical data of the pedigree was collected. Coding regions of candidate genes were amplified by PCR and subjected to next generation sequencing (NGS). Suspected mutations were verified by Sanger sequencing and segregation analysis.Two novel heterozygous mutations (c.1695dupC and c.552-563del) were respectively detected in the LRP5 and ZNF408 genes in the proband. Both mutations were inherited from the affected mother. By Sanger sequencing, the c.552-563del mutation was also detected among unaffected members, while the c.1695dupC mutation was only detected in affected members from the pedigree and was not recorded by the HGMD, NCBI, or 1000 genome database. Upon prenatal diagnosis, the fetus was found to carry the same mutations.Combined NGS and Sanger sequencing not only can reduce the time required for diagnosis but also enable accurate prenatal diagnosis for FEVR.

We determined whether a child with 17α-hydroxylase/17, 20-lyase deficiency possessed the sex-determining region (SRY) gene, and examined the mutations present in the CYP17A1 gene that led to 17α-hydroxylase/17, 20-lyase deficiency. In the child, karyotype analysis was performed and polymerase chain reaction analysis and electrophoretic techniques were used to identify the SRY gene. A total of 50 normal individuals were included as a control group. Polymerase chain reaction and DNA sequencing were used to identify CYP17A1 gene mutations in all samples. The karyotype of the child was 46, XY, which was inconsistent with her social sex, SRY was positive, and a compound heterozygous mutation p. Thr101Ilefs*2 in exon 2 and p. Thr306Ale in exon 5 were identified in the CYP17A1 gene. These mutations were inherited from her parents. In the 20 normal individuals, these mutations were not identified. In the child, sex reversal may have been caused by CYP17A1 mutations. The compound heterozygous mutation of p. Thr101Ilefs*2 and p. Thr306Ale is a new gene mutation of 17α-hydroxylase/17, 20-lyase deficiency.

Waardenburg syndrome (WS) is an auditory-pigmentary disorder with varying combinations of sensorineural hearing loss and abnormal pigmentation. The present study aimed to investigate the underlying molecular pathology and provide a method of prenatal diagnosis of WS in Chinese families. A total of 11 patients with WS from five unrelated Chinese families were enrolled. A thorough clinical examination was performed on all participants. Furthermore, patients with WS underwent screening for mutations in the following genes: Paired box 3 (PAX3), melanogenesis associated transcription factor (MITF), SRY-box 10, snail family transcriptional repressor 2 and endothelin receptor type B using polymerase chain reaction sequencing. Array-based comparative genomic hybridization was used for specific patients whose sequence results were normal. Following identification of the genotype of the probands and their parents, prenatal genetic diagnosis was performed for family 01 and 05. According to the diagnostic criteria for WS, five cases were diagnosed as WS1, while the other six cases were WS2. Genetic analysis revealed three mutations, including a nonsense mutation PAX3 c.583C>T in family 01, a splice-site mutation MITF c.909G>A in family 03 and an in-frame deletion MITF c.649_651delGAA in family 05. To the best of the authors' knowledge the mutations (c.583C>T in PAX3 and c.909G>A in MITF) were reported for the first time in Chinese people. Mutations in the gene of interest were not identified in family 02 and 04. The prenatal genetic testing of the two fetuses was carried out and demonstrated that the two babies were normal. The results of the present study expanded the range of known genetic mutations in China. Identification of genetic mutations in these families provided an efficient way to understand the causes of WS and improved genetic counseling.