Genotyping Cancer-Associated Genes in Chordoma Identifies Mutations in Oncogenes and Areas of Chromosomal Loss Involving CDKN2A,PTEN, and SMARCB1

The molecular mechanisms underlying chordoma pathogenesis are unknown. We therefore sought to identify novel mutations to better understand chordoma biology and to potentially identify therapeutic targets. Given the relatively high costs of whole genome sequencing, we performed a focused genetic analysis using matrix-assisted laser desorption/ionization-time of flight mass spectrometer (Sequenom iPLEX genotyping). We tested 865 hotspot mutations in 111 oncogenes and selected tumor suppressor genes (OncoMap v. 3.0) of 45 human chordoma tumor samples. Of the analyzed samples, seven were identified with at least one mutation. Six of these were from fresh frozen samples, and one was from a paraffin embedded sample. These observations were validated using an independent platform using homogeneous mass extend MALDI-TOF (Sequenom hME Genotyping). These genetic alterations include: ALK (A877S), CTNNB1 (T41A), NRAS (Q61R), PIK3CA (E545K), PTEN (R130), CDKN2A (R58*), and SMARCB1 (R40*). This study reports on the largest comprehensive mutational analysis of chordomas performed to date. To focus on mutations that have the greatest chance of clinical relevance, we tested only oncogenes and tumor suppressor genes that have been previously implicated in the tumorigenesis of more common malignancies. We identified rare genetic changes that may have functional significance to the underlying biology and potential therapeutics for chordomas. Mutations in CDKN2A and PTEN occurred in areas of chromosomal copy loss. When this data is paired with the studies showing 18 of 21 chordoma samples displaying copy loss at the locus for CDKN2A, 17 of 21 chordoma samples displaying copy loss at PTEN, and 3 of 4 chordoma samples displaying deletion at the SMARCB1 locus, we can infer that a loss of heterozygosity at these three loci may play a significant role in chordoma pathogenesis.     

SCN5A Mutations in Brugada Syndrome Are Associated with Increased Cardiac Dimensions and Reduced Contractility

Background

The cardiac sodium channel (Na_v1.5) controls cardiac excitability. Accordingly, SCN5A mutations that result in loss-of-function of Na_v1.5 are associated with various inherited arrhythmia syndromes that revolve around reduced cardiac excitability, most notably Brugada syndrome (BrS). Experimental studies have indicated that Na_v1.5 interacts with the cytoskeleton and may also be involved in maintaining structural integrity of the heart. We aimed to determine whether clinical evidence may be obtained that Na_v1.5 is involved in maintaining cardiac structural integrity.

Methods

Using cardiac magnetic resonance (CMR) imaging, we compared right ventricular (RV) and left ventricular (LV) dimensions and ejection fractions between 40 BrS patients with SCN5A mutations (SCN5a-mut-positive) and 98 BrS patients without SCN5A mutations (SCN5a-mut-negative). We also studied 18 age/sex-matched healthy volunteers.

Results

SCN5a-mut-positive patients had significantly larger end-diastolic and end-systolic RV and LV volumes, and lower LV ejection fractions, than SCN5a-mut-negative patients or volunteers.

Conclusions

Loss-of-function SCN5A mutations are associated with dilatation and impairment in contractile function of both ventricles that can be detected by CMR analysis.     

Heimler Syndrome Is Caused by Hypomorphic Mutations in the Peroxisome-Biogenesis Genes PEX1 and PEX6

Heimler syndrome (HS) is a rare recessive disorder characterized by sensorineural hearing loss (SNHL), amelogenesis imperfecta, nail abnormalities, and occasional or late-onset retinal pigmentation. We ascertained eight families affected by HS and, by using a whole-exome sequencing approach, identified biallelic mutations in PEX1 or PEX6 in six of them. Loss-of-function mutations in both genes are known causes of a spectrum of autosomal-recessive peroxisome-biogenesis disorders (PBDs), including Zellweger syndrome. PBDs are characterized by leukodystrophy, hypotonia, SNHL, retinopathy, and skeletal, craniofacial, and liver abnormalities. We demonstrate that each HS-affected family has at least one hypomorphic allele that results in extremely mild peroxisomal dysfunction. Although individuals with HS share some subtle clinical features found in PBDs, the diagnosis was not suggested by routine blood and skin fibroblast analyses used to detect PBDs. In conclusion, our findings define HS as a mild PBD, expanding the pleiotropy of mutations in PEX1 and PEX6.     

FGFR1OP tagSNP but Not CCR6 Polymorphisms Are Associated with Vogt-Koyanagi-Harada Syndrome in Chinese Han

Background

Polymorphisms of the CC chemokine receptor 6 (CCR6) and FGFR10P tagSNP (locus close to CCR6) at 6q27 have recently been reported to be associated with the susceptibility to several immune-related diseases. This study was designed to determine the association of CCR6 and FGFR10P (tag)SNPs with Vogt-Koyanagi-Harada (VKH) syndrome, an autoimmune disease directed against melanocytes, in two independent Chinese Han populations.

Methodology/Principal Findings

A total of 601 VKH patients and 725 healthy controls from two Chinese Han populations were genotyped by the polymerase chain reaction-restriction fragment length polymorphism method. Hardy-Weinberg equilibrium was tested using the χ² test. Genotype frequencies were estimated by direct counting. Allele and genotype frequencies were compared between patients and controls using the χ² test. The frequency of the A allele of rs2301436 was significantly higher both in Cohort 1 and Cohort 2 as compared with two separate controls (P = 0.044; P = 0.049, respectively). The significance was lost after Bonferroni correction in both cohorts (Pc = 0.516; Pc = 0.392, respectively). The frequency of the A allele was significantly higher in the combined patient group as compared with all controls before and after Bonferroni correction (P = 0.005, Pc = 0.025). The genotype and allele frequencies of rs3093024, rs6902119, rs3093023 and rs968334 were not different between patients with VKH and healthy controls based on analysis either for both cohorts or for the patients and controls in total. Analysis according to extra ocular clinical findings including headache, alopecia and poliosis, vitiligo and tinnitus did not show any association of the five polymorphisms with these parameters.

Conclusion

These results suggest that the rs2301436 tagSNP of FGFR10P is positively associated with susceptibility to VKH syndrome in the tested Chinese Han populations. No association was found for the tested CCR6 SNPs.     

A new case with 10q23 interstitial deletion encompassing both PTEN and BMPR1A narrows the genetic region deleted in juvenile polyposis syndrome

We report on a patient with a contiguous interstitial germline deletion of chromosome 10q23, encompassing BMPR1A and PTEN, with clinical manifestations of juvenile polyposis and minor symptoms of Cowden syndrome (CS) and Bannayan–Riley–Ruvalcaba syndrome (BRRS). The patient presented dysmorphic features as well as developmental delay at the age of 5 months. Multiple polyps along all parts of the colon were diagnosed at the age of 3 years, following an episode of a severe abdominal pain and intestinal bleeding. The high-resolution comparative genomic hybridisation revealed a 3.7-Mb deletion within the 10q23 chromosomal region: 86,329,859–90,035,024. The genotyping with four polymorphic microsatellite markers confirmed a de novo 10q deletion on the allele with a paternal origin, encompassing both PTEN and BMPR1A genes. The karyotype analysis additionally identified a balanced translocation involving chromosomes 5q and 7q, and an inversion at chromosome 2, i.e. 46,XY,t(5;7)(q13.3-q36), inv(2)(p25q34). Although many genetic defects were detected, it is most likely that the 10q23 deletion is primarily the cause for the serious phenotypic manifestations. The current clinical findings and deletion of BMPR1A indicate a diagnosis of severe juvenile polyposis, but the existing macrocephaly and PTEN deletion also point to either CS or BRRS, which cannot be ruled out at the moment because of their clinical manifestation later in life and the de novo character of the deletion. The deletion detected in our patient narrows the genetic region deleted in all reported cases with juvenile polyposis by 0.04 Mb from the telomeric side, mapping it to the region chr10:88.5–90.03Mb (GRCh37/hg19), with an overall length of 1.53 Mb.     

Pathogenic Mutations Associated with Legius Syndrome Modify the Spred1 Surface and Are Involved in Direct Binding to the Ras Inactivator Neurofibromin

Neurofibromatosis type I (NF1) and Legius syndrome are rare inherited disorders that share diagnostic symptoms including dermal abnormalities like axillary and inguinal freckling and café au lait spots. In addition, patients suffering from NF1 have a demanding risk for the development of severe tumors of the peripheral and central nervous system among other NF1-specific symptoms. NF1 and Legius syndrome are caused by alterations in the NF1 and SPRED1 genes encoding the Ras inhibitors neurofibromin and Spred1 (sprouty related EVH1 domain-containing protein), respectively. Neurofibromin functions as a Ras-specific GTPase-activating protein (Ras-GAP), and Spred1 enhances Ras inactivation by recruiting neurofibromin from the cytosol to membrane-anchored Ras. In a previous study, we mapped the Spred binding site to the GAP-related domain of neurofibromin (NF1-GAP) and identified the GAPex subdomain as critical for Spred1 binding. Here, we characterize the binding site of these proteins in more detail focusing on a mutant Spred1 variant carrying a pathogenic missense mutation (threonine 102 to arginine). Introduction of this mutation, which locates at the N-terminal EVH1 domain of Spred1, weakens the interaction with neurofibromin by about 3 orders of magnitude without perturbing the protein fold, and the binding site of NF1-GAP on the mutant Spred1(EVH1) variant can be identified by NMR spectroscopy. Taken together, our data provide structural insight into the interaction of Spred1 and neurofibromin and characterize the structural or functional consequence of selected patient-derived mutations associated with Legius syndrome.     

Mutations in FLVCR2 Are Associated with Proliferative Vasculopathy and Hydranencephaly-Hydrocephaly Syndrome (Fowler Syndrome)

Proliferative vasculopathy and hydranencephaly-hydrocephaly syndrome (PVHH), also known as Fowler syndrome, is an autosomal-recessively inherited prenatal lethal disorder characterized by hydranencephaly; brain stem, basal ganglia, and spinal cord diffuse clastic ischemic lesions with calcifications; glomeruloid vasculopathy of the central nervous system and retinal vessels; and a fetal akinesia deformation sequence (FADS) with muscular neurogenic atrophy. To identify the molecular basis for Fowler syndrome, we performed autozygosity mapping studies in three consanguineous families. The results of SNP microarrays and microsatellite marker genotyping demonstrated linkage to chromosome 14q24.3. Direct sequencing of candidate genes within the target interval revealed five different germline mutations in FLVCR2 in five families with Fowler syndrome. FLVCR2 encodes a transmembrane transporter of the major facilitator superfamily (MFS) hypothesized to be involved in regulation of growth, calcium exchange, and homeostasis. This is the first gene to be associated with Fowler syndrome, and this finding provides a basis for further studies to elucidate the pathogenetic mechanisms and phenotypic spectrum of associated disorders.     

Imprinting Status of 11p15 Genes in Beckwith–Wiedemann Syndrome Patients with CDKN1C Mutations

Beckwith–Wiedemann syndrome (BWS) is an imprinting disorder characterized by somatic overgrowth, congenital malformations, and predisposition to childhood tumors. Aberrant expression of multiple imprinted genes, including H19, IGF2, KCNQ1OT1, and CDKN1C, has been observed in BWS patients. It has been estimated that mutations in CDKN1C occur in 12–17% of BWS patients. We have screened 10 autosomal dominant pedigrees and 65 sporadic BWS cases by PCR/heteroduplex analysis and DNA sequencing and have identified four mutations, two of which were associated with biallelic IGF2 expression and normal H19 and KCNQ1OT1 imprinting. One patient demonstrated phenotypic expression of paternally transmitted mutation in this maternally expressed gene, a second proband is the child of one of a pair of monozygotic twin females who carry the mutation de novo, and a third patient exhibited unusual skeletal changes more commonly found in other overgrowth syndromes. When considered with other studies published to date, this work reveals the frequency of CDKN1C mutations in BWS to be only 4.9%. This is the first report of an analysis of the imprinting status of genes in the 11p15 region where CDKN1C mutations were associated with loss of IGF2 imprinting and maintenance of H19 and KCNQ1OT1 imprinting.     

Contiguous gene deletion within chromosome arm 10q is associated with juvenile polyposis of infancy, reflecting cooperation between the BMPR1A and PTEN tumor-suppressor genes

We describe four unrelated children who were referred to two tertiary referral medical genetics units between 1991 and 2005 and who are affected with juvenile polyposis of infancy. We show that these children are heterozygous for a germline deletion encompassing two contiguous genes, PTEN and BMPR1A. We hypothesize that juvenile polyposis of infancy is caused by the deletion of these two genes and that the severity of the disease reflects cooperation between these two tumor-suppressor genes.     

De Novo Truncating Mutations in AHDC1 in Individuals with Syndromic Expressive Language Delay,Hypotonia, and Sleep Apnea

Clinical whole-exome sequencing (WES) for identification of mutations leading to Mendelian disease has been offered to the medical community since 2011. Clinically undiagnosed neurological disorders are the most frequent basis for test referral, and currently, approximately 25% of such cases are diagnosed at the molecular level. To date, there are approximately 4,000 “known” disease-associated loci, and many are associated with striking dysmorphic features, making genotype-phenotype correlations relatively straightforward. A significant fraction of cases, however, lack characteristic dysmorphism or clinical pathognomonic traits and are dependent upon molecular tests for definitive diagnoses. Further, many molecular diagnoses are guided by recent gene-disease association discoveries. Hence, there is a critical interplay between clinical testing and research leading to gene-disease association discovery. Here, we describe four probands, all of whom presented with hypotonia, intellectual disability, global developmental delay, and mildly dysmorphic facial features. Three of the four also had sleep apnea. Each was a simplex case without a remarkable family history. Using WES, we identified AHDC1 de novo truncating mutations that most likely cause this genetic syndrome.     

Identification of SCN1A and PCDH19 Mutations in Chinese Children with Dravet Syndrome

Background

Dravet syndrome is a severe form of epilepsy. Majority of patients have a mutation in SCN1A gene, which encodes a voltage-gated sodium channel. A recent study has demonstrated that 16% of SCN1A-negative patients have a mutation in PCDH19, the gene encoding protocadherin-19. Mutations in other genes account for only a very small proportion of families. TSPYL4 is a novel candidate gene within the locus 6q16.3-q22.31 identified by linkage study.

Objective

The present study examined the mutations in epileptic Chinese children with emphasis on Dravet syndrome.

Methods

A hundred children with severe epilepsy were divided into Dravet syndrome and non-Dravet syndrome groups and screened for SCN1A mutations by direct sequencing. SCN1A-negative Dravet syndrome patients and patients with phenotypes resembling Dravet syndrome were checked for PCDH19 and TSPYL4 mutations.

Results

Eighteen patients (9 males, 9 females) were diagnosed to have Dravet syndrome. Among them, 83% (15/18) had SCN1A mutations including truncating (7), splice site (2) and missense mutations (6). The truncating/splice site mutations were associated with moderate to severe degree of intellectual disability (p<0.05). During the progression of disease, 73% (11/15) had features fitting into the diagnostic criteria of autism spectrum disorder and 53% (8/15) had history of vaccination-induced seizures. A novel PCDH19 p.D377N mutation was identified in one SCN1A-negative female patient with Dravet syndrome and a known PCDH19 p.N340S mutation in a female non-Dravet syndrome patient. The former also inherited a TSPYL4 p.G60R variant.

Conclusion

A high percentage of SCN1A mutations was identified in our Chinese cohort of Dravet syndrome patients but none in the rest of patients. We demonstrated that truncating/splice site mutations were linked to moderate to severe intellectual disability in these patients. A de novo PCDH19 missense mutation together with an inherited TSPYL4 missense variant were identified in a patient with Dravet syndrome.     

Genotyping of Novel SNPs in BMPR1B,BMP15, and GDF9 Genes for Association with Prolificacy in Seven Indian Goat Breeds

Goats form the backbone of rural livelihood and financial security systems in India but their population is showing decreasing trend. Improvement of reproductive traits such as prolificacy offers a solution to stabilize the decreasing goat population and to meet the nutritional needs of growing human population. In the present study, six novel SNPs in three candidate genes for prolificacy (BMPR1B, BMP15, and GDF9) were genotyped in seven breeds of Indian goats to evaluate their association with litter size. Tetra primer ARMS-PCR and PCR-RFLP based protocols were developed for genotyping six novel SNPs, namely, T(-242)C in BMPR1B; G735A and C808G in BMP15; and C818T, A959C, and G1189A in GDF9 gene. The effect of breed was highly significant (p ≤ 0.01) on litter size but the effect of genotype was nonsignificant. The effect of parity on litter size was also significant in the prolific Black Bengal breed. The litter size differences observed between breeds are attributed to breed differences. Novel mutations observed at different loci in GDF9, BMP15, and BMPR1B genes do not contribute to the reproductive capability of the investigated breeds. Further studies with more number of breeds and animals exploring association of these novel SNPs with reproductive traits may be fruitful.