Identification of compound heterozygous DNAH11 variants in a Han-Chinese family with primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a group of genetically and clinically heterogeneous disorders with motile cilia dysfunction. It is clinically characterized by oto-sino-pulmonary diseases and subfertility, and half of the patients have situs inversus (Kartagener syndrome). To identify the genetic cause in a Han-Chinese pedigree, whole-exome sequencing was conducted in the 37-year-old proband, and then, Sanger sequencing was performed on available family members. Minigene splicing assay was applied to verify the impact of the splice-site variant. Compound heterozygous variants including a splice-site variant (c.1974-1G>C, rs1359107415) and a missense variant (c.7787G>A, p.(Arg2596Gln), rs780492669), in the dynein axonemal heavy chain 11 gene (DNAH11) were identified and confirmed as the disease-associated variants of this lineage. The minigene expression in vitro revealed that the c.1974-1G>C variant could cause skipping over exon 12, predicted to result in a truncated protein. This discovery may enlarge the DNAH11 variant spectrum of PCD, promote accurate genetic counselling and contribute to PCD diagnosis.

Novel and Recurring NOTCH3 Mutations in Two Chinese Patients with CADASIL.

BACKGROUND: Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an autosomal-dominant, inherited, systemic, vascular disorder primarily involving the small arteries. It is characterized by migraine, recurrent ischemic strokes, cognitive decline, and dementia. Mutations in the Notch receptor 3 gene (NOTCH3) and the HtrA serine peptidase 1 gene (HTRA1) are 2 genetic causes for CADASIL. The NOTCH3 gene, located on chromosome 19p13.12, is the most common disease-causing gene in CADASIL. OBJECTIVE: To investigate genetic causes in 2 unrelated Han-Chinese patients with presentations strongly suggestive of CADASIL. METHODS: Exome sequencing was performed on both patients and potential pathogenic mutations were validated by Sanger sequencing. RESULTS: This study reports on 2 unrelated Han-Chinese patients with presentations strongly suggestive of CADASIL, identifying that NOTCH3 mutations were the genetic cause. A common mutation, c.268C>T (p.Arg90Cys), and a novel mutation, c.331G>T (p.Gly111Cys) in the NOTCH3 gene, were detected and confirmed in the patients, respectively, and were predicted to be deleterious based on bioinformation analyses. CONCLUSIONS: We identified 2 NOTCH3 mutations as likely genetic causes for CADASIL in these 2 patients. Our findings broaden the mutational spectrum of the NOTCH3 gene accountable for CADASIL. Clinical manifestations supplemented with molecular genetic analyses are critical for accurate diagnosis, the provision of genetic counseling, and the development of therapies for CADASIL.

Identification of a GNE homozygous mutation in a Han-Chinese family with GNE myopathy.

GNE myopathy is a rare, recessively inherited, early adult-onset myopathy, characterized by distal and proximal muscle degeneration which often spares the quadriceps. It is caused by mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene (GNE). This study aimed to identify the disease-causing mutation in a three-generation Han-Chinese family with members who have been diagnosed with myopathy. A homozygous missense mutation, c.1627G>A (p.V543M) in the GNE gene co-segregates with the myopathy present in this family. A GNE myopathy diagnosis is evidenced by characteristic clinical manifestations, rimmed vacuoles in muscle biopsies and the presence of biallelic GNE mutations. This finding broadens the GNE gene mutation spectrum and extends the GNE myopathy phenotype spectrum.

Identification of a Novel Keratin 9 Missense Mutation in a Chinese Family with Epidermolytic Palmoplantar Keratoderma.

BACKGROUND/AIMS: Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant genodermatosis. It is characterized by diffuse yellow keratoses on the palmoplantar epidermis, with an erythematous border. The keratin 9 gene (KRT9) and less frequently the keratin 1 gene (KRT1) are responsible for EPPK. This study aims to identify and analyse genetic defects responsible for EPPK in a Han Chinese pedigree. METHODS: A four-generation Han Chinese pedigree containing five individuals affected with EPPK was recruited. Exome sequencing, Sanger sequencing, and bioinformatics tools were conducted to identify the mutation in this pedigree. HaCaT cells were transfected with either wild-type or mutated KRT9. Confocal laser immunofluorescence assay, imaging processing, and statistical analysis were performed to evaluate wild-type and mutant KRT9 groups. RESULTS: A novel heterozygous c.1369C>T transition (p.Leu457Phe) in exon 6 of the KRT9 gene was identified in four patients. It co-segregated with the disorder in the family. Functional analysis showed that withdrawal of the filament network from the cell periphery and particle formation were present in about 10% of Leu457Phe-transfected HaCaT cells, while approximately 3% of cells transfected with wild-type KRT9 showed this phenotype. The particles in mutant group were larger than that in wild-type group (P-value < 0.05). CONCLUSION: The variant may be the disease-causing missense mutation and produce dominant negative effects by interrupting keratin network formation. This study indicates the pathogenic role of the KRT9 gene mutation in this pedigree with EPPK, and may be helpful in genetic counseling, prenatal diagnosis and gene-targeted therapies of EPPK.

Compound heterozygous POMT1 mutations in a Chinese family with autosomal recessive muscular dystrophy-dystroglycanopathy C1.

Muscular dystrophy-dystroglycanopathy (MDDG) is a genetically and clinically heterogeneous group of muscular disorders, characterized by congenital muscular dystrophy or later-onset limb-girdle muscular dystrophy accompanied by brain and ocular abnormalities, resulting from aberrant alpha-dystroglycan glycosylation. Exome sequencing and Sanger sequencing were performed on a six-generation consanguineous Han Chinese family, members of which had autosomal recessive MDDG. Compound heterozygous mutations, c.1338+1G>A (p.H415Kfs*3) and c.1457G>C (p.W486S, rs746849558), in the protein O-mannosyltransferase 1 gene (POMT1), were identified as the genetic cause. Patients that exhibited milder MDDG manifested as later-onset progressive proximal pelvic, shoulder girdle and limb muscle weakness, joint contractures, mental retardation and elevated creatine kinase, without structural brain or ocular abnormalities, were further genetically diagnosed as MDDGC1. The POMT1 gene splice-site mutation (c.1338+1G>A) which leads to exon 13 skipping and results in a truncated protein may contribute to a severe phenotype, while the allelic missense mutation (p.W486S) may reduce MDDG severity. These findings may expand phenotype and mutation spectrum of the POMT1 gene. Clinical diagnosis supplemented with molecular screening may result in more accurate diagnoses of, prognoses for, and improved genetic counselling for this disease.

A novel heterozygous COL4A4 missense mutation in a Chinese family with focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is the most common glomerular histological lesion associated with high-grade proteinuria and end-stage renal disease. Histologically, FSGS is characterized by focal segmental sclerosis with foot process effacement. The aim of this study was to identify the disease-causing mutation in a four-generation Chinese family with FSGS. A novel missense mutation, c.1856G>A (p.Gly619Asp), in the collagen type IV alpha-4 gene (COL4A4) was identified in six patients and it co-segregated with the disease in this family. The variant is predicted to be disease-causing and results in collagen IV abnormalities. Our finding broadens mutation spectrum of the COL4A4 gene and extends the phenotypic spectrum of collagen IV nephropathies. Our study suggests that exome sequencing is a cost-effective and efficient approach for identification of disease-causing mutations in phenotypically complex or equivocal disorders. Timely screening for COL4A3/COL4A4 mutations in patients with familial FSGS may help both accurately diagnose and treat these patients.

Novel CLCN7 mutation identified in a Han Chinese family with autosomal dominant osteopetrosis-2.

Osteopetrosis is a heritable bone condition featuring increased bone density due to defective osteoclastic bone resorption. Exome sequencing and Sanger sequencing were conducted in Han Chinese family members, some of whom had typical osteopetrosis, and a novel missense variant c.2350A>T (p.R784W) in the chloride channel 7 gene (CLCN7) was identified. This variant cosegregated with the disorder in the family but was not observed in 800 controls. The data indicate that exome sequencing is a powerful and effective molecular diagnostic tool for detecting mutations in osteopetrosis, which is a genetically and clinically heterogeneous disorder. This discovery broadens the CLCN7 gene mutation spectrum and has important implications for clinical therapeutic regimen decisions, prognosis evaluations, and antenatal diagnoses.

Identification of a novel collagen type IV alpha-4 (COL4A4) mutation in a Chinese family with autosomal dominant Alport syndrome using exome sequencing.

BACKGROUND & OBJECTIVES: Alport syndrome (AS) is an inherited disorder characterized by glomerulonephritis and end-stage renal disease (ESRD). The aim of this study was to identify the gene responsible for the glomerulopathy in a Chinese family with autosomal dominant AS using exome sequencing. METHODS: A 4-generation, 30-member Chinese Han family was enrolled in this study. Exome sequencing was conducted in the proband of the family, and then direct sequencing was performed in family members of the pedigree and 100 normal controls. RESULTS: A novel frameshift mutation, c.3213delA (p.Gly1072GlufsFNx0169), in the collagen type IV alpha-4 gene (COL4A4) was found to be the genetic cause. Neither sensorineural hearing loss nor ocular abnormalities were present in the patients of this family. Other clinical features, such as age of onset, age of ESRD occurring and disease severity, varied among the patients of this family. INTERPRETATION & CONCLUSIONS: A novel frameshift mutation, c.3213delA (p.Gly1072GlufsFNx0169) in the COL4A4 gene, was identified in the Chinese pedigree with autosomal dominant AS. Our findings may provide new insights into the cause and diagnosis of AS and also have implications for genetic counselling.

Advances in the molecular genetics of non-syndromic polydactyly.

Polydactyly is one of the most common inherited limb abnormalities, characterised by supernumerary fingers or toes. It results from disturbances in the normal programme of the anterior-posterior axis of the developing limb, with diverse aetiology and variable inter- and intra-familial clinical features. Polydactyly can occur as an isolated disorder (non-syndromic polydactyly) or as a part of an anomaly syndrome (syndromic polydactyly). On the basis of the anatomic location of the duplicated digits, non-syndromic polydactyly is divided into three kinds, including preaxial polydactyly, axial polydactyly and postaxial polydactyly. Non-syndromic polydactyly frequently exhibits an autosomal dominant inheritance with variable penetrance. To date, in human, at least ten loci and four disease-causing genes, including the GLI3 gene, the ZNF141 gene, the MIPOL1 gene and the PITX1 gene, have been identified. In this paper, we review clinical features of non-syndromic polydactyly and summarise the recent progress in the molecular genetics, including loci and genes that are responsible for the disorder, the signalling pathways that these genetic factors are involved in, as well as animal models of the disorder. These progresses will improve our understanding of the complex disorder and have implications on genetic counselling such as prenatal diagnosis.

Identification of a novel PHEX mutation in a Chinese family with X-linked hypophosphatemic rickets using exome sequencing.

Familial hypophosphatemic rickets (HR), the most common inherited form of rickets, is a group of inherited renal phosphate wasting disorders characterized by growth retardation, rickets with bone deformities, osteomalacia, poor dental development, and hypophosphatemia. The purpose of this study was to identify the genetic defect responsible for familial HR in a four-generation Chinese Han pedigree by exome sequencing and Sanger sequencing. Clinical features include skeletal deformities, teeth abnormalities, hearing impairments and variable serum phosphate level in patients of this family. A novel deletion mutation, c.1553delT (p.F518Sfs*4), was identified in the X-linked phosphate regulating endopeptidase homolog gene (PHEX). The mutation is predicted to result in prematurely truncated and loss-of-function PHEX protein. Our data suggest that exome sequencing is a powerful tool to discover mutation(s) in HR, a disorder with genetic and clinical heterogeneity. The findings may also provide new insights into the cause and diagnosis of HR, and have implications for genetic counseling and clinical management.

Identification of a novel GJA3 mutation in congenital nuclear cataract.

PURPOSE: Congenital cataract is a visual impairment that needs correction as early as possible after birth. This study aimed to identify whether genetic defects exist in a Chinese Han pedigree with congenital nuclear cataract. METHODS: A family consisting of six members and three patients with nuclear cataract spanning three generations and 100 unrelated ethnically matched normal subjects were recruited in this study. Exome sequencing was performed in the 24-year-old proband, and Sanger sequencing was then conducted in other family members and 100 normal controls. RESULTS: A novel missense variant, c.428G>A (p.G143E), in the gap junction protein-alpha 3 gene (GJA3) was identified in three patients of the family but unidentified in three family members without lens opacity and 100 normal controls. CONCLUSIONS: A novel missense mutation, c.428G>A (p.G143E), in the GJA3 gene, localized to the cytoplasmic loop, was suggested to be the genetic cause of congenital nuclear cataract, which further expands the gene mutation spectrum. Our findings suggest that exome sequencing is a powerful and cost-effective tool to discover mutation(s) in disorders with high genetic and clinical heterogeneity. Further functional studies in the GJA3 gene mutations may help uncover pathogenic mechanisms of congenital cataract and therefore provide a possible genetic therapy for this disorder.

Identification of a novel COL4A5 mutation in a Chinese family with X-linked Alport syndrome using exome sequencing.

Alport syndrome (AS) is an inherited disorder and clinically characterized by glomerulonephritis and end-stage kidney disease (ESRD). The aim of this study was to identify the gene responsible for glomerulopathy in a 4-generation Chinese pedigree. Exome sequencing was conducted in four patients of the family, and then direct sequencing was performed in other members of the pedigree. A novel missense mutation c.368G>A (p.Gly123Glu) in the collagen type IV alpha-5 gene (COL4A5) was found to be the genetic cause. The p.Gly123Glu mutation occurs prior to Gly-X-Y repeats in the alpha-5 chain of type IV collagen. Neither sensorineural hearing loss nor ocular abnormalities were present in patients of this family. Other clinical features, such as age of onset, age of ESRD, disease severity and complications, varied among patients of this family. Our finding may provide new insights into the cause and diagnosis of AS, and also have implications for genetic counseling.

CADASIL: A NOTCH3-associated cerebral small vessel disease.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common hereditary cerebral small vessel disease (CSVD), pathologically characterized by a non-atherosclerotic and non-amyloid diffuse angiopathy primarily involving small to medium-sized penetrating arteries and leptomeningeal arteries. In 1996, mutation in the notch receptor 3 gene (NOTCH3) was identified as the cause of CADASIL. However, since that time other genetic CSVDs have been described, including the HtrA serine peptidase 1 gene-associated CSVD and the cathepsin A gene-associated CSVD, that clinically mimic the original phenotype. Though NOTCH3-associated CSVD is now a well-recognized hereditary disorder and the number of studies investigating this disease is increasing, the role of NOTCH3 in the pathogenesis of CADASIL remains elusive. AIM OF REVIEW: This review aims to provide insights into the pathogenesis and the diagnosis of hereditary CSVDs, as well as personalized therapy, predictive approach, and targeted prevention. In this review, we summarize the current progress in CADASIL, including the clinical, neuroimaging, pathological, genetic, diagnostic, and therapeutic aspects, as well as differential diagnosis, in which the role of NOTCH3 mutations is highlighted. KEY SCIENTIFIC CONCEPTS OF REVIEW: In this review, CADASIL is revisited as a NOTCH3-associated CSVD along with other hereditary CSVDs.

Identification of a GJA3 mutation in a Chinese family with congenital nuclear cataract using exome sequencing.

Congenital cataract, a clinically and genetically heterogeneous lens disorder is defined as any opacity of the lens presented from birth and is responsible for approximately 10% of worldwide childhood poor vision or blindness. To identify the genetic defect responsible for congenital nuclear cataract in a four-generation Chinese Han family, exome and direct Sanger sequencings were conducted and a missense variant c.139G>A (p.D47N) in the gap junction protein-alpha 3 gene (GJA3) was identified. The variant co-segregated with patients of the family and was not observed in unaffected family members or normal controls. The above findings indicated that the variant was a pathogenic mutation. The mutation p.D47N was found in the first extracellular loop (El) domain of GJA3 protein. Our data suggest that exome sequencing is a powerful tool to discover mutation(s) in cataract, a disorder with high genetic heterogeneity. Our findings may also provide new insights into the cause and diagnosis of congenital nuclear cataract and have implications for genetic counseling.

Auricular fistula: a review of its clinical manifestations, genetics, and treatments.

Auricular fistula is a common congenital auricular malformation, characterized as a small opening in the skin and a subcutaneous cyst. It can be classified in different ways according to positions of pits and directions of fistula tracts. The term preauricular fistula and variant type of preauricular fistula (postauricular fistula) are used. Auricular fistula prevalence varies in countries and populations, and its actual prevalence is presently unknown. The most accepted and widely cited theory of auricular fistula etiopathogenesis is an incorrect or incomplete fusion of six auricular hillocks that are mesenchymal proliferations. Auricular fistula can occur either sporadically or genetically. The pattern in inherited cases is thought to be incomplete autosomal dominant, with variable expressions, reduced penetrance, and inapparent gender differences. Auricular fistula has several forms and is reported as being a component of many syndromes. In the field of genetics, currently, there is no related review to comprehensively summarize the genetic basis of auricular fistula and related disorders. This article provides a comprehensive review of auricular fistula, especially congenital preauricular fistula, which accounts for the majority of auricular fistula, by summarizing the clinical manifestations, histological and embryological development, genetics, examinations, and treatments, as well as syndromes with auricular fistula.

The role of the PLA2G6 gene in neurodegenerative diseases.

PLA2G6-associated neurodegeneration (PLAN) represents a continuum of clinically and genetically heterogeneous neurodegenerative disorders with overlapping features. Usually, it encompasses three autosomal recessive diseases, including infantile neuroaxonal dystrophy or neurodegeneration with brain iron accumulation (NBIA) 2A, atypical neuronal dystrophy with childhood-onset or NBIA2B, and adult-onset dystonia-parkinsonism form named PARK14, and possibly a certain subtype of hereditary spastic paraplegia. PLAN is caused by variants in the phospholipase A2 group VI gene (PLA2G6), which encodes an enzyme involved in membrane homeostasis, signal transduction, mitochondrial dysfunction, and α-synuclein aggregation. In this review, we discuss PLA2G6 gene structure and protein, functional findings, genetic deficiency models, various PLAN disease phenotypes, and study strategies in the future. Our primary aim is to provide an overview of genotype-phenotype correlations of PLAN subtypes and speculate on the role of PLA2G6 in potential mechanisms underlying these conditions.

Novel compound heterozygous variants in the USH2A gene associated with autosomal recessive retinitis pigmentosa without hearing loss.

Background: Retinitis pigmentosa (RP) is a group of progressive inherited retinal dystrophies characterized by the primary degeneration of rod photoreceptors and the subsequent loss of cone photoreceptors because of cell death. It is caused by different mechanisms, including inflammation, apoptosis, necroptosis, pyroptosis, and autophagy. Variants in the usherin gene (USH2A) have been reported in autosomal recessive RP with or without hearing loss. In the present study, we aimed to identify causative variants in a Han-Chinese pedigree with autosomal recessive RP. Methods: A six-member, three-generation Han-Chinese family with autosomal recessive RP was recruited. A full clinical examination, whole exome sequencing, and Sanger sequencing, as well as co-segregation analysis were performed. Results: Three heterozygous variants in the USH2A gene, c.3304C>T (p.Q1102*), c.4745T>C (p.L1582P), and c.14740G>A (p.E4914K), were identified in the proband, which were inherited from parents and transmitted to the daughters. Bioinformatics analysis supported the pathogenicity of the c.3304C>T (p.Q1102*) and c.4745T>C (p.L1582P) variants. Conclusions: Novel compound heterozygous variants in the USH2A gene, c.3304C>T (p.Q1102*) and c.4745T>C (p.L1582P), were identified as the genetic causes of autosomal recessive RP. The findings may enhance the current knowledge of the pathogenesis of USH2A-associated phenotypes, expand the spectrum of the USH2A gene variants, and contribute to improved genetic counseling, prenatal diagnosis, and disease management.

The TSC2 c.2742+5G>A variant causes variable splicing changes and clinical manifestations in a family with tuberous sclerosis complex.

Background: Tuberous sclerosis complex (TSC) is a genetic, variably expressed, multisystem disease characterized by benign tumors. It is caused by pathogenic variants of the TSC complex subunit 1 gene (TSC1) and the TSC complex subunit 2 gene (TSC2). Genetic testing allows for early diagnosis, genetic counseling, and improved outcomes, but it did not identify a pathogenic variant in up to 25% of all TSC patients. This study aimed to identify the disease-causing variant in a Han-Chinese family with TSC. Methods: A six-member, three-generation Han-Chinese family with TSC and three unrelated healthy women were recruited. A comprehensive medical examination, a 3-year follow-up, whole exome sequencing, Sanger sequencing, and segregation analysis were performed in the family. The splicing analysis results obtained from six in silico tools, minigene assay, and patients' lymphocyte messenger RNA were compared, and quantitative reverse transcription PCR was used to confirm the pathogenicity of the variant. Results: Two affected family members had variable clinical manifestations including a rare bilateral cerebellar ataxia symptom. The 3-year follow-up results suggest the effects of a combined treatment of anti-epilepsy drugs and sirolimus for TSC-related epilepsy and cognitive deficits. Whole exome sequencing, Sanger sequencing, segregation analysis, splicing analysis, and quantitative reverse transcription PCR identified the TSC2 gene c.2742+5G>A variant as the genetic cause. This variant inactivated the donor splice site, a cryptic non-canonical splice site was used for different splicing changes in two affected subjects, and the resulting mutant messenger RNA may be degraded by nonsense-mediated decay. The defects of in silico tools and minigene assay in predicting cryptic splice sites were suggested. Conclusions: This study identified a TSC2 c.2742+5G>A variant as the genetic cause of a Han-Chinese family with TSC and first confirmed its pathogenicity. These findings expand the phenotypic and genetic spectrum of TSC and may contribute to its diagnosis and treatment, as well as a better understanding of the splicing mechanism.

Identification of novel compound heterozygous variants in the DNAH1 gene of a Chinese family with left-right asymmetry disorder.

Most internal organs in humans and other vertebrates exhibit striking left-right asymmetry in position and structure. Variation of normal organ positioning results in left-right asymmetry disorders and presents as internal organ reversal or randomization. Up to date, at least 82 genes have been identified as the causative genetic factors of left-right asymmetry disorders. This study sought to discover potential pathogenic variants responsible for left-right asymmetry disorder present in a Han-Chinese family using whole exome sequencing combined with Sanger sequencing. Novel compound heterozygous variants, c.5690A>G (p.Asn1897Ser) and c.7759G>A (p.Val2587Met), in the dynein axonemal heavy chain 1 gene (DNAH1), were found in the proband and absent in unaffected family members. Conservation analysis has shown that the variants affect evolutionarily conserved residues, which may impact the tertiary structure of the DNAH1 protein. The novel compound heterozygous variants may potentially bear responsibility for left-right asymmetry disorder, which results from a perturbation of left-right axis coordination at the earliest embryonic development stages. This study broadens the variant spectrum of left-right asymmetry disorders and may be helpful for genetic counseling and healthcare management for the diagnosed individual, and promotes a greater understanding of the pathophysiology.