Molecular diagnosis of neonatal diabetes mellitus using next-generation sequencing of the whole exome

Background

Accurate molecular diagnosis of monogenic non-autoimmune neonatal diabetes mellitus (NDM) is critical for patient care, as patients carrying a mutation in KCNJ11 or ABCC8 can be treated by oral sulfonylurea drugs instead of insulin therapy. This diagnosis is currently based on Sanger sequencing of at least 42 PCR fragments from the KCNJ11, ABCC8, and INS genes. Here, we assessed the feasibility of using the next-generation whole exome sequencing (WES) for the NDM molecular diagnosis.

Methodology/Principal Findings

We carried out WES for a patient presenting with permanent NDM, for whom mutations in KCNJ11, ABCC8 and INS and abnormalities in chromosome 6q24 had been previously excluded. A solution hybridization selection was performed to generate WES in 76 bp paired-end reads, by using two channels of the sequencing instrument. WES quality was assessed using a high-resolution oligonucleotide whole-genome genotyping array. From our WES with high-quality reads, we identified a novel non-synonymous mutation in ABCC8 (c.1455G>C/p.Q485H), despite a previous negative sequencing of this gene. This mutation, confirmed by Sanger sequencing, was not present in 348 controls and in the patient''s mother, father and young brother, all of whom are normoglycemic.

Conclusions/Significance

WES identified a novel de novo ABCC8 mutation in a NDM patient. Compared to the current Sanger protocol, WES is a comprehensive, cost-efficient and rapid method to identify mutations in NDM patients. We suggest WES as a near future tool of choice for further molecular diagnosis of NDM cases, negative for chr6q24, KCNJ11 and INS abnormalities.     

Subtypes of familial hemophagocytic lymphohistiocytosis in Japan based on genetic and functional analyses of cytotoxic T lymphocytes

Background

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare disease of infancy or early childhood. To clarify the incidence and subtypes of FHL in Japan, we performed genetic and functional analyses of cytotoxic T lymphocytes (CTLs) in Japanese patients with FHL.

Design and Methods

Among the Japanese children with hemophagocytic lymphohistiocytosis (HLH) registered at our laboratory, those with more than one of the following findings were eligible for study entry under a diagnosis of FHL: positive for known genetic mutations, a family history of HLH, and impaired CTL-mediated cytotoxicity. Mutations of the newly identified causative gene for FHL5, STXBP2, and the cytotoxicity and degranulation activity of CTLs in FHL patients, were analyzed.

Results

Among 31 FHL patients who satisfied the above criteria, PRF1 mutation was detected in 17 (FHL2) and UNC13D mutation was in 10 (FHL3). In 2 other patients, 3 novel mutations of STXBP2 gene were confirmed (FHL5). Finally, the remaining 2 were classified as having FHL with unknown genetic mutations. In all FHL patients, CTL-mediated cytotoxicity was low or deficient, and degranulation activity was also low or absent except FHL2 patients. In 2 patients with unknown genetic mutations, the cytotoxicity and degranulation activity of CTLs appeared to be deficient in one patient and moderately impaired in the other.

Conclusions

FHL can be diagnosed and classified on the basis of CTL-mediated cytotoxicity, degranulation activity, and genetic analysis. Based on the data obtained from functional analysis of CTLs, other unknown gene(s) responsible for FHL remain to be identified.     

Identification of Two Novel Mutations in the PHEX Gene in Chinese Patients with Hypophosphatemic Rickets/Osteomalacia

Objective

X-linked dominant hypophosphatemia (XLH) is the most prevalent form of inherited rickets/osteomalacia in humans. The aim of this study was to identify PHEX gene mutations and describe the clinical features observed in 6 unrelated Chinese families and 3 sporadic patients with hypophosphatemic rickets/osteomalacia.

Methods

For this study, 45 individuals from 9 unrelated families of Chinese Han ethnicity (including 16 patients and 29 normal phenotype subjects), and 250 healthy donors were recruited. All 22 exons and exon-intron boundaries of the PHEX gene were amplified by polymerase chain reaction (PCR) and directly sequenced.

Results

The PHEX mutations were detected in 6 familial and 3 sporadic hypophosphatemic rickets/osteomalacia. Altogether, 2 novel mutations were detected: 1 missense mutation c.1183G>C in exon 11, resulting in p.Gly395Arg and 1 missense mutation c.1751A>C in exon 17, resulting in p.His584Pro. No mutations were found in the 250 healthy controls.

Conclusions

Our study increases knowledge of the PHEX gene mutation types and clinical phenotypes found in Chinese patients with XLH, which is important for understanding the genetic basis of XLH. The molecular diagnosis of a PHEX genetic mutation is of great importance for confirming the clinical diagnosis of XLH, conducting genetic counseling, and facilitating prenatal intervention, especially in the case of sporadic patients.     

Frequency of KCNC3 DNA variants as causes of spinocerebellar ataxia 13 (SCA13)

Background

Gain-of function or dominant-negative mutations in the voltage-gated potassium channel KCNC3 (Kv3.3) were recently identified as a cause of autosomal dominant spinocerebellar ataxia. Our objective was to describe the frequency of mutations associated with KCNC3 in a large cohort of index patients with sporadic or familial ataxia presenting to three US ataxia clinics at academic medical centers.

Methodology

DNA sequence analysis of the coding region of the KCNC3 gene was performed in 327 index cases with ataxia. Analysis of channel function was performed by expression of DNA variants in Xenopus oocytes.

Principal Findings

Sequence analysis revealed two non-synonymous substitutions in exon 2 and five intronic changes, which were not predicted to alter splicing. We identified another pedigree with the p.Arg423His mutation in the highly conserved S4 domain of this channel. This family had an early-onset of disease and associated seizures in one individual. The second coding change, p.Gly263Asp, subtly altered biophysical properties of the channel, but was unlikely to be disease-associated as it occurred in an individual with an expansion of the CAG repeat in the CACNA1A calcium channel.

Conclusions

Mutations in KCNC3 are a rare cause of spinocerebellar ataxia with a frequency of less than 1%. The p.Arg423His mutation is recurrent in different populations and associated with early onset. In contrast to previous p.Arg423His mutation carriers, we now observed seizures and mild mental retardation in one individual. This study confirms the wide phenotypic spectrum in SCA13.     

Genetic and epigenetic alterations of the NF2 gene in sporadic vestibular schwannomas

Background

Mutations in the neurofibromatosis type 2 (NF2) tumor-suppressor gene have been identified in not only NF2-related tumors but also sporadic vestibular schwannomas (VS). This study investigated the genetic and epigenetic alterations in tumors and blood from 30 Korean patients with sporadic VS and correlated these alterations with tumor behavior.

Methodology/Principal Findings

NF2 gene mutations were detected using PCR and direct DNA sequencing and three highly polymorphic microsatellite DNA markers were used to assess the loss of heterozygosity (LOH) from chromosome 22. Aberrant hypermethylation of the CpG island of the NF2 gene was also analyzed. The tumor size, the clinical growth index, and the proliferative activity assessed using the Ki-67 labeling index were evaluated. We found 18 mutations in 16 cases of 30 schwannomas (53%). The mutations included eight frameshift mutations, seven nonsense mutations, one in-frame deletion, one splicing donor site, and one missense mutation. Nine patients (30%) showed allelic loss. No patient had aberrant hypermethylation of the NF2 gene and correlation between NF2 genetic alterations and tumor behavior was not observed in this study.

Conclusions/Significance

The molecular genetic changes in sporadic VS identified here included mutations and allelic loss, but no aberrant hypermethylation of the NF2 gene was detected. In addition, no clear genotype/phenotype correlation was identified. Therefore, it is likely that other factors contribute to tumor formation and growth.     

Mutation Prevalence of Cerebral Cavernous Malformation Genes in Spanish Patients

Objective

To study the molecular genetic and clinical features of cerebral cavernous malformations (CCM) in a cohort of Spanish patients.

Methods

We analyzed the CCM1, CCM2, and CCM3 genes by MLPA and direct sequencing of exons and intronic boundaries in 94 familial forms and 41 sporadic cases of CCM patients of Spanish extraction. When available, RNA studies were performed seeking for alternative or cryptic splicing.

Results

A total of 26 pathogenic mutations, 22 of which predict truncated proteins, were identified in 29 familial forms and in three sporadic cases. The repertoire includes six novel non-sense and frameshift mutations in CCM1 and CCM3. We also found four missense mutations, one of them located at the third NPXY motif of CCM1 and another one that leads to cryptic splicing of CCM1 exon 6. We found four genomic deletions with the loss of the whole CCM2 gene in one patient and a partial loss of CCM1and CCM2 genes in three other patients. Four families had mutations in CCM3. The results include a high frequency of intronic variants, although most of them localize out of consensus splicing sequences. The main symptoms associated to clinical debut consisted of cerebral haemorrhage, migraines and epileptic seizures. The rare co-occurrence of CCM with Noonan and Chiari syndromes and delayed menarche is reported.

Conclusions

Analysis of CCM genes by sequencing and MLPA has detected mutations in almost 35% of a Spanish cohort (36% of familial cases and 10% of sporadic patients). The results include 13 new mutations of CCM genes and the main clinical symptoms that deserves consideration in molecular diagnosis and genetic counselling of cerebral cavernous malformations.     

MET Genetic Abnormalities Unreliable for Patient Selection for Therapeutic Intervention in Oropharyngeal Squamous Cell Carcinoma

Background

Identification of MET genetic alteration, mutation, or amplification in oropharyngeal squamous cell carcinoma (OPSCC) could lead to development of MET selective kinase inhibitors. The aim of this study was to assess the frequency and prognostic value of MET gene mutation, amplification, and protein expression in primary OPSCC.

Methods

A retrospective chart review was conducted of patients treated for single primary OPSCC between January 2007 and December 2009. Pre-treatment OPSCC tissue samples were analyzed for MET mutations, gene amplification, and overexpression using Sanger sequencing, FISH analysis, and immunohistochemistry respectively. Univariate and multivariate analyses were used to analyze correlations between molecular abnormalities and patient survival.

Results

143 patients were included in this study. Six cases (4%) were identified that had a genetic variation, but previously described mutations such as p.Tyr1235Asp (Y1235D) or p.Tyr1230Cys (Y1230C) were not detected. There were 15 high polysomy cases, and only 3 cases met the criteria for true MET amplification, with ≥10% amplified cells per case. Immunohistochemistry evaluation showed 43% of cases were c-MET negative and in 57% c-MET was observed at the tumor cell level. Multivariate analysis showed no significant association between MET mutation, amplification, or expression and survival.

Conclusions

Our study shows a low frequency of MET mutations and amplification in this cohort of OPSCC. There was no significant correlation between MET mutations, amplification, or expression and patient survival. These results suggest that patient selection based on these MET genetic abnormalities may not be a reliable strategy for therapeutic intervention in OPSCC.     

In silico characterization of a novel pathogenic deletion mutation identified in XPA gene in a Pakistani family with severe xeroderma pigmentosum

Background

Xeroderma Pigmentosum (XP) is a rare skin disorder characterized by skin hypersensitivity to sunlight and abnormal pigmentation. The aim of this study was to investigate the genetic cause of a severe XP phenotype in a consanguineous Pakistani family and in silico characterization of any identified disease-associated mutation.

Results

The XP complementation group was assigned by genotyping of family for known XP loci. Genotyping data mapped the family to complementation group A locus, involving XPA gene. Mutation analysis of the candidate XP gene by DNA sequencing revealed a novel deletion mutation (c.654del A) in exon 5 of XPA gene. The c.654del A, causes frameshift, which pre-maturely terminates protein and result into a truncated product of 222 amino acid (aa) residues instead of 273 (p.Lys218AsnfsX5). In silico tools were applied to study the likelihood of changes in structural motifs and thus interaction of mutated protein with binding partners. In silico analysis of mutant protein sequence, predicted to affect the aa residue which attains coiled coil structure. The coiled coil structure has an important role in key cellular interactions, especially with DNA damage-binding protein 2 (DDB2), which has important role in DDB-mediated nucleotide excision repair (NER) system.

Conclusions

Our findings support the fact of genetic and clinical heterogeneity in XP. The study also predicts the critical role of DDB2 binding region of XPA protein in NER pathway and opens an avenue for further research to study the functional role of the mutated protein domain.     

A Novel PRPF31 Mutation in a Large Chinese Family with Autosomal Dominant Retinitis Pigmentosa and Macular Degeneration

Purpose

This study was intended to identify the disease causing genes in a large Chinese family with autosomal dominant retinitis pigmentosa and macular degeneration.

Methods

A genome scan analysis was conducted in this family for disease gene preliminary mapping. Snapshot analysis of selected SNPs for two-point LOD score analysis for candidate gene filter. Candidate gene PRPF31 whole exons'' sequencing was executed to identify mutations.

Results

A novel nonsense mutation caused by an insertion was found in PRPF31 gene. All the 19 RP patients in 1085 family are carrying this heterozygous nonsense mutation. The nonsense mutation is in PRPF31 gene exon9 at chr19:54629961-54629961, inserting nucleotide “A” that generates the coding protein frame shift from p.307 and early termination at p.322 in the snoRNA binding domain (NOP domain).

Conclusion

This report is the first to associate PRPF31 gene''s nonsense mutation and adRP and JMD. Our findings revealed that PRPF31 can lead to different clinical phenotypes in the same family, resulting either in adRP or syndrome of adRP and JMD. We believe our identification of the novel “A” insertion mutation in exon9 at chr19:54629961-54629961 in PRPF31 can provide further genetic evidence for clinical test for adRP and JMD.     

Whole Exome Analysis Identifies Frequent CNGA1 Mutations in Japanese Population with Autosomal Recessive Retinitis Pigmentosa

Objective

The purpose of this study was to investigate frequent disease-causing gene mutations in autosomal recessive retinitis pigmentosa (arRP) in the Japanese population.

Methods

In total, 99 Japanese patients with non-syndromic and unrelated arRP or sporadic RP (spRP) were recruited in this study and ophthalmic examinations were conducted for the diagnosis of RP. Among these patients, whole exome sequencing analysis of 30 RP patients and direct sequencing screening of all CNGA1 exons of the other 69 RP patients were performed.

Results

Whole exome sequencing of 30 arRP/spRP patients identified disease-causing gene mutations of CNGA1 (four patients), EYS (three patients) and SAG (one patient) in eight patients and potential disease-causing gene variants of USH2A (two patients), EYS (one patient), TULP1 (one patient) and C2orf71 (one patient) in five patients. Screening of an additional 69 arRP/spRP patients for the CNGA1 gene mutation revealed one patient with a homozygous mutation.

Conclusions

This is the first identification of CNGA1 mutations in arRP Japanese patients. The frequency of CNGA1 gene mutation was 5.1% (5/99 patients). CNGA1 mutations are one of the most frequent arRP-causing mutations in Japanese patients.     

Copy Number Variation Analysis in Familial BRCA1/2-Negative Finnish Breast and Ovarian Cancer

Background

Inherited factors predisposing individuals to breast and ovarian cancer are largely unidentified in a majority of families with hereditary breast and ovarian cancer (HBOC). We aimed to identify germline copy number variations (CNVs) contributing to HBOC susceptibility in the Finnish population.

Methods

A cohort of 84 HBOC individuals (negative for BRCA1/2-founder mutations and pre-screened for the most common breast cancer genes) and 36 healthy controls were analysed with a genome-wide SNP array. CNV-affecting genes were further studied by Gene Ontology term enrichment, pathway analyses, and database searches to reveal genes with potential for breast and ovarian cancer predisposition. CNVs that were considered to be important were validated and genotyped in 20 additional HBOC individuals (6 CNVs) and in additional healthy controls (5 CNVs) by qPCR.

Results

An intronic deletion in the EPHA3 receptor tyrosine kinase was enriched in HBOC individuals (12 of 101, 11.9%) compared with controls (27 of 432, 6.3%) (OR = 1.96; P = 0.055). EPHA3 was identified in several enriched molecular functions including receptor activity. Both a novel intronic deletion in the CSMD1 tumor suppressor gene and a homozygous intergenic deletion at 5q15 were identified in 1 of 101 (1.0%) HBOC individuals but were very rare (1 of 436, 0.2% and 1 of 899, 0.1%, respectively) in healthy controls suggesting that these variants confer disease susceptibility.

Conclusion

This study reveals new information regarding the germline CNVs that likely contribute to HBOC susceptibility in Finland. This information may be used to facilitate the genetic counselling of HBOC individuals but the preliminary results warrant additional studies of a larger study group.     

CpG demethylation enhances alpha-synuclein expression and affects the pathogenesis of Parkinson's disease

Background

Alpha-synuclein (SNCA) gene expression is an important factor in the pathogenesis of Parkinson''s disease (PD). Gene multiplication can cause inherited PD, and promoter polymorphisms that increase SNCA expression are associated with sporadic PD. CpG methylation in the promoter region may also influence SNCA expression.

Methodology/Principal Findings

By using cultured cells, we identified a region of the SNCA CpG island in which the methylation status altered along with increased SNCA expression. Postmortem brain analysis revealed regional non-specific methylation differences in this CpG region in the anterior cingulate and putamen among controls and PD; however, in the substantia nigra of PD, methylation was significantly decreased.

Conclusions/Significance

This CpG region may function as an intronic regulatory element for SNCA gene. Our findings suggest that a novel epigenetic regulatory mechanism controlling SNCA expression influences PD pathogenesis.     

Mutations in the gene DNAJC5 cause autosomal dominant Kufs disease in a proportion of cases: study of the Parry family and 8 other families

Background

The Neuronal Ceroid Lipofuscinoses (NCL) comprise at least nine progressive neurodegenerative genetic disorders. Kufs disease, an adult-onset form of NCL may be recessively or dominantly inherited. Our study aimed to identify genetic mutations associated with autosomal dominant Kufs disease (ADKD).

Methodology and Principal Findings

We have studied the family first reported with this phenotype in the 1970s, the Parry family. The proband had progressive psychiatric manifestations, seizures and cognitive decline starting in her mid 20 s. Similarly affected relatives were observed in seven generations. Several of the affected individuals had post-mortem neuropathological brain study confirmatory for NCL disease. We conducted whole exome sequencing of three affected family members and identified a pLeu116del mutation in the gene DNAJC5, which segregated with the disease phenotype. An additional eight unrelated affected individuals with documented autosomal dominant or sporadic inheritance were studied. All had diagnostic confirmation with neuropathological studies of brain tissue. Among them we identified an additional individual with a p.Leu115Arg mutation in DNAJC5. In addition, a pAsn477Ser change in the neighboring gene PRPF6, a gene previously found to be associated with retinitis pigmentosa, segregated with the ADKD phenotype. Interestingly, two individuals of the Parry family did report visual impairment.

Conclusions

Our study confirmed the recently reported association of DNAJC5 mutations with ADKD in two out of nine well-defined families. Sequence changes in PRPF6 have not been identified in other unrelated cases. The association of vision impairment with the expected PRPF6 dysfunction remains possible but would need further clinical studies in order to confirm the co-segregation of the visual impairment with this sequence change.     

Genotype-phenotype correlation in Japanese patients with familial Mediterranean fever: differences in genotype and clinical features between Japanese and Mediterranean populations

Introduction

Familial Mediterranean fever (FMF) is a hereditary autoinflammatory disease characterized by recurrent self-limiting fever and serositis that mainly affects Mediterranean populations. Many patients with FMF have been reported in Japan due to increasing recognition of this condition and the availability of genetic analysis for the gene responsible, MEFV. The present study was performed to elucidate the clinical characteristics of Japanese FMF patients and to examine the precise genotype-phenotype correlation in a large cohort of Japanese FMF patients.

Methods

We analyzed the MEFV genotypes and clinical manifestations in 116 patients clinically diagnosed as having FMF and with at least one mutation.

Results

The most frequent mutation in Japanese patients was E148Q (40.2%), followed by M694I (21.0%), L110P (18.8%), P369S (5.4%), and R408Q (5.4%). In contrast, common mutations seen in Mediterranean patients, such as M694V, V726A, and M680I, were not detected in this population. The clinical features with M694I were associated with more severe clinical course compared to those seen with E148Q. P369S/R408Q showed variable phenotypes with regard to both clinical manifestations and severity. Patients with M694I showed a very favorable response to colchicine therapy, while those with P369S and R408Q did not.

Conclusions

Clinical features and efficacy of treatment in Japanese FMF patients vary widely according to the specific MEFV gene mutation, and therefore genetic analysis should be performed for diagnosis in cases of Japanese FMF.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0439-7) contains supplementary material, which is available to authorized users.     

Molecular Analysis and Phenotypic Study in 14 Chinese Families with Bietti Crystalline Dystrophy

Purpose

To investigate the clinical features and cytochrome P450 family 4 subfamily V polypeptide 2 (CYP4V2) gene mutations in 14 Chinese families with Bietti crystalline dystrophy (BCD).

Methods

Seventeen patients from 14 unrelated Chinese families with BCD were recruited for complete clinical ophthalmic examination and genetic study. The 11 exons of CYP4V2 were amplified from genomic DNA of all patients and their family members by polymerase chain reaction (PCR) and then sequenced. Exons of TIMP3 were also sequenced in BCD patient associated with choroidal neovascularization (CNV). One hundred and seventy unrelated healthy Chinese subjects were screened for mutations in CYP4V2.

Results

All 17 patients with BCD had mutations in CYP4V2; one of these mutations was novel (c.219T>A, p.F73L) and four other mutations had been reported. The p.F73L mutation was a commonly detected mutation in our study (seven out of 34 alleles), either in the homozygous state or in the heterozygous state. Among the patients, considerable phenotypic variability was detected, both within and between families. Screening of TIMP3 did not find any mutation in the BCD patient associated with CNV.

Conclusion

The novel CYP4V2 c.219T>A (p.F73L) mutation may be another recurrent mutation in Chinese patients with BCD. Our study expands the mutation spectrum of CYP4V2 and characterizes novel genotype–phenotype associations in Chinese patients with BCD.     

Autosomal Recessive Transmission of MYBPC3 Mutation Results in Malignant Phenotype of Hypertrophic Cardiomyopathy

Background

Hypertrophic cardiomyopathy (HCM) due to mutations in genes encoding sarcomere proteins is most commonly inherited as an autosomal dominant trait. Since nearly 50% of HCM cases occur in the absence of a family history, a recessive inheritance pattern may be involved.

Methods

A pedigree was identified with suspected autosomal recessive transmission of HCM. Twenty-six HCM-related genes were comprehensively screened for mutations in the proband with targeted second generation sequencing, and the identified mutation was confirmed with bi-directional Sanger sequencing in all family members and 376 healthy controls.

Results

A novel missense mutation (c.1469G>T, p.Gly490Val) in exon 17 of MYBPC3 was identified. Two siblings with HCM were homozygous for this mutation, whereas other family members were either heterozygous or wild type. Clinical evaluation showed that both homozygotes manifested a typical HCM presentation, but none of others, including 5 adult heterozygous mutation carriers up to 71 years of age, had any clinical evidence of HCM.

Conclusions

Our data identified a MYBPC3 mutation in HCM, which appeared autosomal recessively inherited in this family. The absence of a family history of clinical HCM may be due to not only a de novo mutation, but also recessive mutations that failed to produce a clinical phenotype in heterozygous family members. Therefore, consideration of recessive mutations leading to HCM is essential for risk stratification and genetic counseling.     

Differential Gene Repertoire in Mycobacterium ulcerans Identifies Candidate Genes for Patho-Adaptation

Background

Based on large genomic sequence polymorphisms, several haplotypes belonging to two major lineages of the human pathogen Mycobacterium ulcerans could be distinguished among patient isolates from various geographic origins. However, the biological relevance of insertional/deletional diversity is not understood.

Methodology

Using comparative genomics, we have investigated the genes located in regions of difference recently identified by DNA microarray based hybridisation analysis. The analysed regions of difference comprise ∼7% of the entire M. ulcerans genome.

Principal Findings

Several different mechanisms leading to loss of functional genes were identified, ranging from pseudogenization, caused by frame shift mutations or mobile genetic element interspersing, to large sequence polymorphisms. Four hot spot regions for genetic instability were unveiled. Altogether, 229 coding sequences were found to be differentially inactivated, constituting a repertoire of coding sequence variation in the rather monomorphic M. ulcerans.

Conclusions/Significance

The differential gene inactivation patterns associated with the M. ulcerans haplotypes identified candidate genes that may confer enhanced adaptation upon ablation of expression. A number of gene conversions confined to the classical lineage may contribute to particular virulence of this group comprising isolates from Africa and Australia. Identification of this spectrum of anti-virulence gene candidates expands our understanding of the pathogenicity and ecology of the emerging infectious disease Buruli ulcer.