Mutation screening of EXT genes in Chinese patients with multiple osteochondromas

Multiple osteochondromas (MO), a dominantly inherited genetic disorder, is characterized by the presence of multiple osteochondromas in the long bones. EXT1 and EXT2 are the causative genes in most MO patients. We have characterized 9 MO families and 1 sporadic case involving a total of 25 patients. The coding exons of EXT1 and EXT2 were screened in 10 probands affected with MO. In five of the 10 probands novel pathogenic mutations have been identified: two in EXT1 and three in EXT2. Four probands carried recurrent mutations and one proband had no detectable mutation. Our study extends the mutational spectrum in EXT1 and EXT2 and will facilitate the deep understanding of the pathophysiology of the disease.     

Genetic analysis of hereditary multiple exostoses in Tunisian families: a novel frame-shift mutation in the <Emphasis Type="Italic">EXT1</Emphasis> gene

Hereditary multiple exostoses (HME) is an autosomal dominant orthopaedic disorder most frequently caused by mutations in the EXT1 gene. The aim of the present study is to determine the underlying molecular defect of HME in two multigenerational Tunisian families with 21 affected members and to examine the degree of intrafamilial variability. Linkage analysis was performed using three microsatellite markers encompassing the EXT1 locus and mutation screening was carried out by direct sequencing. In family 1, evidence for linkage to EXT1 was obtained on the basis of a maximum LOD score of 4.26 at θ = 0.00 with D8S1694 marker. Sequencing of the EXT1 revealed a heterozygous G > T transversion (c.1019G>T) in exon 2, leading to a missense mutation at the codon 340 (p.Arg340Leu). In family 2 we identified a novel heterozygous 1 bp deletion in the exon 1 (c.529_531delA) leading to a premature codon stop and truncated EXT1 protein expression (p.Lys177LysfsX15). This mutation was associated with the evidence of an intrafamilial clinical variability and considered to be a novel disease-causing mutation in the EXT1 gene. These findings provide additional support for the involvement of EXT1 gene in the HME disease.     

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 Mutation in the 5'-UTR of IFITM5 Creates an In-Frame Start Codon and Causes Autosomal-Dominant Osteogenesis Imperfecta Type V with Hyperplastic Callus

Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous disorder associated with bone fragility and susceptibility to fractures after minimal trauma. OI type V has an autosomal-dominant pattern of inheritance and is not caused by mutations in the type I collagen genes COL1A1 and COL1A2. The most remarkable and pathognomonic feature, observed in ∼65% of affected individuals, is a predisposition to develop hyperplastic callus after fractures or surgical interventions. To identify the molecular cause of OI type V, we performed whole-exome sequencing in a female with OI type V and her unaffected parents and searched for de novo mutations. We found a heterozygous de novo mutation in the 5′-untranslated region of IFITM5 (the gene encoding Interferon induced transmembrane protein 5), 14 bp upstream of the annotated translation initiation codon (c.−14C>T). Subsequently, we identified an identical heterozygous de novo mutation in a second individual with OI type V by Sanger sequencing, thereby confirming that this is the causal mutation for the phenotype. IFITM5 is a protein that is highly enriched in osteoblasts and has a putative function in bone formation and osteoblast maturation. The mutation c.−14C>T introduces an upstream start codon that is in frame with the reference open-reading frame of IFITM5 and is embedded into a stronger Kozak consensus sequence for translation initiation than the annotated start codon. In vitro, eukaryotic cells were able to recognize this start codon, and they used it instead of the reference translation initiation signal. This suggests that five amino acids (Met-Ala-Leu-Glu-Pro) are added to the N terminus and alter IFITM5 function in individuals with the mutation.     

Mutational screening of EXT1 and EXT2 genes in Polish patients with hereditary multiple exostoses

Hereditary multiple exostoses (HME) also known as multiple osteochondromas represent one of the most frequent bone tumor disorder in humans. Its clinical presentation is characterized by the presence of multiple benign cartilage-capped tumors located most commonly in the juxta-epiphyseal portions of long bones. HME are usually inherited in autosomal dominant manner, however de novo mutations can also occur. In most patients, the disease is caused by alterations in the EXT1 and EXT2 genes. In this study we investigated 33 unrelated Polish probands with the clinical and radiological diagnosis of HME by means of Sanger sequencing and MLPA for all coding exons of EXT1 and EXT2. We demonstrated EXT1 and EXT2 heterozygous mutations in 18 (54.6 %) and ten (30.3 %) probands respectively, which represents a total of 28 (84.9 %) index cases. Sequencing allowed for the detection of causative changes in 26 (78.8 %) probands, whereas MLPA showed intragenic deletions in two (6.1 %) further cases (15 mutations represented novel changes). Our paper is the first report on the results of exhaustive mutational screening of both EXT1/EXT2 genes in Polish patients. The proportion of EXT1/EXT2 mutations in our group was similar to other Caucasian cohorts. However, we found that EXT1 lesions in Polish patients cluster in exons 1 and 2 (55.6 % of all EXT1 mutations). This important finding should lead to the optimization of cost-effectiveness rate of HME diagnostic testing. Therefore, the diagnostic algorithm for HME should include EXT1 sequencing (starting with exons 1–2), followed by EXT2 sequencing, and MLPA/qPCR for intragenic copy number changes.     

Exome Sequencing Identifies a Novel Gene,WNK1, for Susceptibility to Pelvic Organ Prolapse (POP)

Pelvic organ prolapse (POP) is a common gynecological disorder; however, the genetic components remain largely unidentified. Exome sequencing has been widely used to identify pathogenic gene mutations of several diseases because of its high chromosomal coverage and accuracy. In this study, we performed whole exome sequencing (WES), for the first time, on 8 peripheral blood DNA samples from representative POP cases. After filtering the sequencing data from the dbSNP database (build 138) and the 1000 Genomes Project, 2 missense variants in WNK1, c.2668G > A (p.G890R) and c.6761C> T (p.P2254L), were identified and further validated via Sanger sequencing. In validation stage, the c.2668G > A (p.G890R) variant and 8 additional variants were detected in 11 out of 161 POP patients. All these variants were absent in 231 healthy controls. Functional experiments showed that fibroblasts from the utero-sacral ligaments of POP with WNK1 mutations exhibited loose and irregular alignment compared with fibroblasts from healthy controls. In sum, our study identified a novel gene, WNK1, for POP susceptibility, expanded the causal mutation spectrums of POP, and provided evidence for the genetic diagnosis and medical management of POP in the future.     

One third of Japanese patients with multiple osteochondromas may have mutations in genes other than EXT1 or EXT2

Multiple osteochondromas (MO; also referred to as hereditary multiple exostoses [HME] in the literature) is an autosomal dominant disorder characterized by benign, cartilage-capped bone tumors that grow from the metaphyses of long bones. Two genes are associated with this disease: EXT1 on 8q24.11-q24.13 and EXT2 on 11p12-p11. Mutations in EXT1 and EXT2 are found in 54-96% of patients with MO and are generally more frequent in EXT1 than in EXT2. We previously studied 43 Japanese families with MO using single-strand conformation polymorphism analysis for EXT1 and EXT2, and reported 23 families (54%) with mutations and 20 families (46%) with no mutations in these genes. Among the families with mutations, 17 families (40%) had mutations in EXT1, and 6 families (14%) had mutations in EXT2. Here we examined the same 43 Japanese families using denaturing high-performance liquid chromatography as an alternative technique. We detected five mutations, three of which are novel, in seven families in addition to the previously described mutations. In summary, we detected mutations in EXT1 or EXT2 in 30 (70%) out of 43 families. Our result suggests the presence of other gene(s) responsible for MO, at least in Japanese patients.     

Hereditary breast cancer in Middle Eastern and North African (MENA) populations: identification of novel, recurrent and founder BRCA1 mutations in the Tunisian population

Germ-line mutations in BRCA1 breast cancer susceptibility gene account for a large proportion of hereditary breast cancer families and show considerable ethnic and geographical variations. The contribution of BRCA1 mutations to hereditary breast cancer has not yet been thoroughly investigated in Middle Eastern and North African populations. In this study, 16 Tunisian high-risk breast cancer families were screened for germline mutations in the entire BRCA1 coding region and exon?Cintron boundaries using direct sequencing. Six families were found to carry BRCA1 mutations with a prevalence of 37.5%. Four different deleterious mutations were detected. Three truncating mutations were previously described: c.798_799delTT (916 delTT), c.3331_3334delCAAG (3450 delCAAG), c.5266dupC (5382 insC) and one splice site mutation which seems to be specific to the Tunisian population: c.212?+?2insG (IVS5?+?2insG). We also identified 15 variants of unknown clinical significance. The c.798_799delTT mutation occurred at an 18% frequency and was shared by three apparently unrelated families. Analyzing five microsatellite markers in and flanking the BRCA1 locus showed a common haplotype associated with this mutation. This suggests that the c.798_799delTT mutation is a Tunisian founder mutation. Our findings indicate that the Tunisian population has a spectrum of prevalent BRCA1 mutations, some of which appear as recurrent and founding mutations.     

Novel insertion mutation p.Asp610GlyfsX23 in APC gene causes familial adenomatous polyposis in Chinese families

Objective

The aim of the study was to identify the causative gene defects associated with familial adenomatous polyposis (FAP) in two Chinese pedigrees.

Methods

The diagnosis of FAP patients was confirmed by clinical manifestations, family histories, colonoscopy and pathology examinations. Blood samples were collected and genomic DNA was extracted. The mutation analysis of the adenomatous polyposis coli (APC) and human mutY homolog (MUTYH) genes was conducted by direct polymerase chain reaction (PCR) sequencing and multiplex ligation-dependent probe amplification (MLPA).

Results

In pedigree A, the results of direct PCR sequencing revealed a heterozygous insertion mutation at codon 610 in exon 15 of APC gene (c.1828_1829insG), which resulted in frameshift change (p.Asp610GlyfsX23) in all 4 patients, but was absent in the unaffected familial members and controls. In pedigree B, we didn't identify that causative mutations cosegregated with the clinical phenotype in the APC and MUTYH genes.

Conclusions

We identified a novel insertion mutation as the pathogenic gene of FAP in Chinese population, which could enrich the germline mutation spectrum of APC gene, and the prophylactic proctocolectomy for the mutation carrier in family should be considered.     

Ulna/height ratio as clinical parameter separating EXT1 from EXT2 families?

Multiple osteochondromas (MO) is an autosomal-dominant inherited disorder. The two genes responsible (EXT1 and EXT2) have been identified. We investigated 12 MO families for phenotype details and the genetic basis by cosegregation and mutation analysis (seven novel pathogenic mutations [five frameshift, one splice site, and one gross deletion] and one novel missense polymorphism). We found EXT1 to be responsible in seven families (19 affected members) and EXT2 in four families (17 affected members). One family remains undetermined. We found a tendency to a more severe phenotype in EXT1 families. As a novel finding, we could identify a single parameter (ulna/height ratio) that separates EXT1 family from EXT2 family in our series.     

Detection of SRSF2-P95 Mutation by High-Resolution Melting Curve Analysis and Its Effect on Prognosis in Myelodysplastic Syndrome

Hotspot mutations of serine/arginine-rich splicing factor 2 (SRSF2) gene have been identified in a proportion of hematologic malignancies including myelodysplastic syndrome (MDS). The aim of the present study was to develop a new approach to screen SRSF2 mutation and analyze the clinical relevance of SRSF2 mutations in Chinese MDS. A protocol based on high-resolution melting analysis (HRMA) was established to screen SRSF2-P95 mutation in 108 MDS patients and was compared with Sanger sequencing. The clinical relevance of SRSF2 mutations was further evaluated. HRMA identified five (4.6%) cases with SRSF2 mutation, completely validated by Sanger sequencing without false positive or negative results. The sensitivities of HRMA and Sanger sequencing were 10% and 25% for the detection of SRSF2-P95H mutation, respectively, against the background of wild-type DNA. Patients with SRSF2 mutation had shorter overall survival time than those with wild-type SRSF2 in both the whole cohort of cases and those with normal karyotype (P = 0.069 and 0.023, respectively). Multivariate analysis confirmed SRSF2 mutation as an independent risk factor in both patient populations. We established a fast, high-throughput, and inexpensive HRMA-based method to screen SRSF2 mutation, which could be used in clinical diagnostic laboratories. SRSF2 mutations were significantly associated with mortality rate in the MDS affected Chinese.     

FOXC2 Mutations in Familial and Sporadic Spinal Extradural Arachnoid Cyst

Spinal extradural arachnoid cyst (SEDAC) is a cyst in the spinal canal that protrudes into the epidural space from a defect in the dura mater. Most cases are sporadic; however, three familial SEDAC cases have been reported, suggesting genetic etiological factors. All familial cases are associated with lymphedema-distichiasis syndrome (LDS), whose causal gene is FOXC2. However, FOXC2 mutation analysis has been performed in only 1 family, and no mutation analysis has been performed on sporadic (non-familial) SEDACs. We recruited 17 SEDAC subjects consisting of 2 familial and 7 sporadic cases and examined FOXC2 mutations by Sanger sequencing and structural abnormalities by TaqMan copy number assay. We identified 2 novel FOXC2 mutations in 2 familial cases. Incomplete LDS penetrance was noted in both families. Four subjects presented with SEDACs only. Thus, SEDAC caused by the heterozygous FOXC2 loss-of-function mutation should be considered a feature of LDS, although it often manifests as the sole symptom. Seven sporadic SEDAC subjects had no FOXC2 mutations, no symptoms of LDS, and showed differing clinical characteristics from those who had FOXC2 mutations, suggesting that other gene(s) besides FOXC2 are likely to be involved in SEDAC.     

A Hereditary Enteropathy Caused by Mutations in the SLCO2A1 Gene,Encoding a Prostaglandin Transporter

Previously, we proposed a rare autosomal recessive inherited enteropathy characterized by persistent blood and protein loss from the small intestine as chronic nonspecific multiple ulcers of the small intestine (CNSU). By whole-exome sequencing in five Japanese patients with CNSU and one unaffected individual, we found four candidate mutations in the SLCO2A1 gene, encoding a prostaglandin transporter. The pathogenicity of the mutations was supported by segregation analysis and genotyping data in controls. By Sanger sequencing of the coding regions, 11 of 12 other CNSU patients and 2 of 603 patients with a diagnosis of Crohn’s disease were found to have homozygous or compound heterozygous SLCO2A1 mutations. In total, we identified recessive SLCO2A1 mutations located at seven sites. Using RT-PCR, we demonstrated that the identified splice-site mutations altered the RNA splicing, and introduced a premature stop codon. Tracer prostaglandin E2 uptake analysis showed that the mutant SLCO2A1 protein for each mutation exhibited impaired prostaglandin transport. Immunohistochemistry and immunofluorescence analyses revealed that SLCO2A1 protein was expressed on the cellular membrane of vascular endothelial cells in the small intestinal mucosa in control subjects, but was not detected in affected individuals. These findings indicate that loss-of-function mutations in the SLCO2A1 gene encoding a prostaglandin transporter cause the hereditary enteropathy CNSU. We suggest a more appropriate nomenclature of “chronic enteropathy associated with SLCO2A1 gene” (CEAS).     

Genetic diagnosis of autosomal dominant polycystic kidney disease by targeted capture and next-generation sequencing: Utility and limitations

Mutation-based molecular diagnostics of autosomal dominant polycystic kidney disease (ADPKD) is complicated by genetic and allelic heterogeneity, large multi-exon genes, and duplication sequences of PKD1. Recently, targeted resequencing by pooling long-range polymerase chain reaction (LR-PCR) amplicons has been used in the identification of mutations in ADPKD. Despite its high sensitivity, specificity and accuracy, LR-PCR is still complicated. We performed whole-exome sequencing on two unrelated typical Chinese ADPKD probands and evaluated the effectiveness of this approach compared with Sanger sequencing. Meanwhile, we performed targeted gene and next-generation sequencing (targeted DNA-HiSeq) on 8 individuals (1 patient from one family, 5 patients and 2 normal individuals from another family). Both whole-exome sequencing and targeted DNA-HiSeq confirmed c.11364delC (p.H3788QfsX37) within the unduplicated region of PKD1 in one proband; in the other family, targeted DNA-HiSeq identified a small insertion, c.401_402insG (p.V134VfsX79), in PKD2. These methods do not overcome the screening complexity of homology. However, the true positives of variants confirmed by targeted gene and next-generation sequencing were 69.4%, 50% and 100% without a false positive in the whole coding region and the duplicated and unduplicated regions, which indicated that the screening accuracy of PKD1 and PKD2 can be largely improved by using a greater sequencing depth and elaborate design of the capture probe.     

Identification of a Novel Mutation in the COL2A1 Gene in a Chinese Family with Spondyloepiphyseal Dysplasia Congenita

Spondyloepiphyseal dysplasia congenita (SEDC) is an autosomal dominant chondrodysplasia characterized by disproportionate short-trunk dwarfism, skeletal and vertebral deformities. Exome sequencing and Sanger sequencing were performed in a Chinese Han family with typical SEDC, and a novel mutation, c.620G>A (p.Gly207Glu), in the collagen type II alpha-1 gene (COL2A1) was identified. The mutation may impair protein stability, and lead to dysfunction of type II collagen. Family-based study suggested that the mutation is a de novo mutation. Our study extends the mutation spectrum of SEDC and confirms genotype-phenotype relationship between mutations at glycine in the triple helix of the alpha-1(II) chains of the COL2A1 and clinical findings of SEDC, which may be helpful in the genetic counseling of patients with SEDC.     

Loss-of-function mutations in PTPN11 cause metachondromatosis, but not Ollier disease or Maffucci syndrome

Metachondromatosis (MC) is a rare, autosomal dominant, incompletely penetrant combined exostosis and enchondromatosis tumor syndrome. MC is clinically distinct from other multiple exostosis or multiple enchondromatosis syndromes and is unlinked to EXT1 and EXT2, the genes responsible for autosomal dominant multiple osteochondromas (MO). To identify a gene for MC, we performed linkage analysis with high-density SNP arrays in a single family, used a targeted array to capture exons and promoter sequences from the linked interval in 16 participants from 11 MC families, and sequenced the captured DNA using high-throughput parallel sequencing technologies. DNA capture and parallel sequencing identified heterozygous putative loss-of-function mutations in PTPN11 in 4 of the 11 families. Sanger sequence analysis of PTPN11 coding regions in a total of 17 MC families identified mutations in 10 of them (5 frameshift, 2 nonsense, and 3 splice-site mutations). Copy number analysis of sequencing reads from a second targeted capture that included the entire PTPN11 gene identified an additional family with a 15 kb deletion spanning exon 7 of PTPN11. Microdissected MC lesions from two patients with PTPN11 mutations demonstrated loss-of-heterozygosity for the wild-type allele. We next sequenced PTPN11 in DNA samples from 54 patients with the multiple enchondromatosis disorders Ollier disease or Maffucci syndrome, but found no coding sequence PTPN11 mutations. We conclude that heterozygous loss-of-function mutations in PTPN11 are a frequent cause of MC, that lesions in patients with MC appear to arise following a "second hit," that MC may be locus heterogeneous since 1 familial and 5 sporadically occurring cases lacked obvious disease-causing PTPN11 mutations, and that PTPN11 mutations are not a common cause of Ollier disease or Maffucci syndrome.     

20 novel point mutations and one large deletion in EXT1 and EXT2 genes: Report of diagnostic screening in a large Italian cohort of patients affected by hereditary multiple exostosis

Background

Hereditary multiple exostosis represents the most frequent bone tumor disease in humans. It consists of cartilage deformities affecting the juxta-ephyseal region of long bones. Usually benign, exostosis could degenerate in malignant chondrosarcoma form in less than 5% of the cases. Being caused by mutations in the predicted tumor suppressor genes, EXT1 (chr 8q23-q24) and EXT2 (chr 11p11-p12) genes, HMEs are usually inherited with an autosomal dominant pattern, although “de novo” cases are not infrequent.

Aim

Here we present our genetic diagnostic report on the largest Southern Italy cohort of HME patients consisting of 90 subjects recruited over the last 5 years.

Results

Molecular screening performed by direct sequencing of both EXT1 and EXT2 genes, by MLPA and Array CGH analyses led to the identification of 66 mutations (56 different occurrences) and one large EXT2 deletion out of 90 patients (74.4%). The total of 21 mutations (20 different occurrences, 33.3%) and the EXT2 gene deletion were novel. In agreement with literature data, EXT1 gene mutations were scattered along all the protein sequence, while EXT2 lesions fell in the first part of the protein. Conservation, damaging prediction and 3-D modeling, in-silico, analyses, performed on three novel missense variants, confirmed that at least in two cases the novel aminoacidic changes could alter the structure stability causing a strong protein misfolding.

Conclusions

Here we present 20 novel EXT1/EXT2 mutations and one large EXT2 deletion identified in the largest Southern Italy cohort of patients affected by hereditary multiple exostosis.     

Whole Exome Sequencing Identifies Recessive PKHD1 Mutations in a Chinese Twin Family with Caroli Disease

Background

Mutations in PKHD1 cause autosomal recessive Caroli disease, which is a rare congenital disorder involving cystic dilatation of the intrahepatic bile ducts. However, the mutational spectrum of PKHD1 and the phenotype-genotype correlations have not yet been fully established.

Methods

Whole exome sequencing (WES) was performed on one twin sample with Caroli disease from a Chinese family from Shandong province. Routine Sanger sequencing was used to validate the WES and to carry out segregation studies. We also described the PKHD1 mutation associated with the genotype-phenotype of this twin.

Results

A combination of WES and Sanger sequencing revealed the genetic defect to be a novel compound heterozygous genotype in PKHD1, including the missense mutation c.2507 T>C, predicted to cause a valine to alanine substitution at codon 836 (c.2507T>C, p.Val836Ala), and the nonsense mutation c.2341C>T, which is predicted to result in an arginine to stop codon at codon 781 (c.2341C>T, p.Arg781*). This compound heterozygous genotype co-segregates with the Caroli disease-affected pedigree members, but is absent in 200 normal chromosomes.

Conclusions

Our findings indicate exome sequencing can be useful in the diagnosis of Caroli disease patients and associate a compound heterozygous genotype in PKHD1 with Caroli disease, which further increases our understanding of the mutation spectrum of PKHD1 in association with Caroli disease.