Alternating hemiplegia of childhood (AHC) is a rare, severe neurodevelopmental syndrome characterized by recurrent hemiplegic episodes and distinct neurologic manifestations. AHC is usually a sporadic disorder with unknown etiology. Using exome sequencing of seven patients with AHC, and their unaffected parents, we identified de novo nonsynonymous mutations in ATP1A3 in all seven AHC patients. Subsequent sequence analysis of ATP1A3 in 98 additional patients revealed that 78% of AHC cases have a likely causal ATP1A3 mutation, including one inherited mutation in a familial case of AHC. Remarkably, six ATP1A3 mutations explain the majority of patients, including one observed in 36 patients. Unlike ATP1A3 mutations that cause rapid-onset-dystonia-parkinsonism, AHC-causing mutations revealed consistent reductions in ATPase activity without effects on protein expression. This work identifies de novo ATP1A3 mutations as the primary cause of AHC, and offers insight into disease pathophysiology by expanding the spectrum of phenotypes associated with mutations in this gene.
Susceptibility to tuberculosis is historically ascribed to an inadequate immune response that fails to control infecting mycobacteria. In zebrafish, we find that susceptibility to Mycobacterium marinum can result from either inadequate or excessive acute inflammation. Modulation of the leukotriene A4 hydrolase (LTA4H) locus, which controls the balance of pro- and anti-inflammatory eicosanoids, reveals two distinct molecular routes to mycobacterial susceptibility converging on dysregulated TNF levels: inadequate inflammation caused by excess lipoxins and hyperinflammation driven by excess leukotriene B4. We identify therapies that specifically target each of these extremes. In humans, we identify a single nucleotide polymorphism in the LTA4H promoter that regulates its transcriptional activity. In tuberculous meningitis, the polymorphism is associated with inflammatory cell recruitment, patient survival and response to adjunctive anti-inflammatory therapy. Together, our findings suggest that host-directed therapies tailored to patient LTA4H genotypes may counter detrimental effects of either extreme of inflammation.
Falling is a common and morbid condition among elderly persons. Effective strategies to prevent falls have been identified but are underutilized.
Using a nonrandomized design, we compared rates of injuries from falls in a region of Connecticut where clinicians had been exposed to interventions to change clinical practice (intervention region) and in a region where clinicians had not been exposed to such interventions (usual-care region). The interventions encouraged primary care clinicians and staff members involved in home care, outpatient rehabilitation, and senior centers to adopt effective risk assessments and strategies for the prevention of falls (e.g., medication reduction and balance and gait training). The outcomes were rates of serious fall-related injuries (hip and other fractures, head injuries, and joint dislocations) and fall-related use of medical services per 1000 person-years among persons who were 70 years of age or older. The interventions occurred from 2001 to 2004, and the evaluations took place from 2004 to 2006.
Before the interventions, the adjusted rates of serious fall-related injuries (per 1000 person-years) were 31.2 in the usual-care region and 31.9 in the intervention region. During the evaluation period, the adjusted rates were 31.4 and 28.6, respectively (adjusted rate ratio, 0.91; 95% Bayesian credibility interval, 0.88 to 0.94). Between the preintervention period and the evaluation period, the rate of fall-related use of medical services increased from 68.1 to 83.3 per 1000 person-years in the usual-care region and from 70.7 to 74.2 in the intervention region (adjusted rate ratio, 0.89; 95% credibility interval, 0.86 to 0.92). The percentages of clinicians who received intervention visits ranged from 62% (131 of 212 primary care offices) to 100% (26 of 26 home care agencies).
Dissemination of evidence about fall prevention, coupled with interventions to change clinical practice, may reduce fall-related injuries in elderly persons.
To test by genomic analysis whether empty follicle syndrome (EFS) in a family with two affected sisters has a genetic basis.
Whole-exome sequencing in the context of clinical genetics.
Two women (36 and 32 years old at the time of the study) with EFS.
Genetic counseling based on autosomal recessive inheritance.
Main Outcome Measure(s)
Discovery of a mutation in the LH/choriogonadotropin receptor (LHCGR) as the cause of EFS.
A novel missense mutation in LHCGR, p.N400S, was homozygous in sisters with EFS and/or infertility, but not in their unaffected siblings or parents. The mutation was not present in 500 ancestry-matched control subjects. Asparagine at residue 400 is highly conserved and its substitution by serine predicted to alter critical interactions that stabilize LHCGR.
We describe a genetic basis for EFS and provide strong evidence for the existence of genuine EFS in some patients. A mutation impairing the function of LHCGR explains the lack of response of these patients to repeated administration of β-hCG.
Empty follicle syndrome; luteinizing hormone/choriogonadotropin receptor; infertility; next-generation sequencing
Individuals with autism are more likely to carry rare inherited and de novo copy number variants (CNVs). However, further research is needed to establish which CNVs are causal and the mechanisms by which these CNVs influence autism. We examined genomic DNA of children with autism (N=41) and healthy controls (N=367) for rare CNVs using a high-resolution array comparative genomic hybridization platform. We show that individuals with autism are more likely to harbor rare CNVs as small as ∼10 kb, a threshold not previously detectable, and that CNVs in cases disproportionately affect genes involved in transcription, nervous system development, and receptor activity. We also show that a subset of genes that have known or suspected allele-specific or imprinting effects and are within rare-case CNVs may undergo loss of transcript expression. In particular, expression of CNTNAP2 and ZNF214 are decreased in probands compared with their unaffected transmitting parents. Furthermore, expression of PRODH and ARID1B, two genes affected by de novo CNVs, are decreased in probands compared with controls. These results suggest that for some genes affected by CNVs in autism, reduced transcript expression may be a mechanism of pathogenesis during neurodevelopment.
autism; CNV; aCGH; expression; deletion; duplication
Rare copy number variants (CNVs) play a prominent role in the etiology of schizophrenia and other neuropsychiatric disorders1. Substantial risk for schizophrenia is conferred by large (>500 kb) CNVs at several loci, including microdeletions at 1q21.1 2, 3q29 3, 15q13.3 2 and 22q11.2 4 and microduplication at 16p11.2 5. However, these CNVs collectively account for a small fraction (2-4%) of cases, and the relevant genes and neurobiological mechanisms are not well understood. Here we performed a large two-stage genome-wide scan of rare CNVs and report the significant association of copy number gains at chromosome 7q36.3 with schizophrenia (P= 4.0×10-5, OR = 16.14 [3.06, ∞]). Microduplications with variable breakpoints occurred within a 362 kb region and were detected in 29 of 8,290 (0.35%) patients versus two of 7,431 (0.03%) controls in the combined sample (p-value= 5.7×10-7, odds ratio (OR) = 14.1 [3.5, 123.9]). All duplications overlapped or were located within 89 kb upstream of the vasoactive intestinal peptide receptor VIPR2. VIPR2 transcription and cyclic-AMP signaling were significantly increased in cultured lymphocytes from patients with microduplications of 7q36.3. These findings implicate altered VIP signaling in the pathogenesis of schizophrenia and suggest VIPR2 as a potential target for the development of novel antipsychotic drugs.
Inherited mutations in the BRCA2-interacting protein PALB2 are known to be associated with increased risks of breast cancer. In order to evaluate the contribution of PALB2 to familial breast cancer in the United States, we sequenced the coding sequences and flanking regulatory regions of the gene from constitutional genomic DNA of 1144 familial breast cancer patients with wildtype sequences at BRCA1 and BRCA2. Overall, 3.4% (33/972) of patients not selected by ancestry and 0% (0/172) of patients specifically of Ashkenazi Jewish ancestry were heterozygous for a nonsense, frameshift, or frameshift-associated splice mutation in PALB2. Mutations were detected in both male and female breast cancer patients. All mutations were individually rare: the 33 heterozygotes harbored 13 different mutations, 5 previously reported and 8 novel. PALB2 heterozygotes were 4-fold more likely to have a male relative with breast cancer (P=0.0003) and 6-fold more likely to have a relative with pancreatic cancer (P=0.002), and 1.3-fold more likely to have a relative with ovarian cancer (P=0.18). Compared to their female relatives without mutations, increased risk of breast cancer for female PALB2 heterozygotes was 2.3-fold (95%CI [1.5–4.2]) by age 55 and 3.4-fold (95%CI [2.4–5.9]) by age 85. Loss of the wildtype PALB2 allele was observed in laser dissected tumor specimens from heterozygous patients. Given this mutation prevalence and risk, consideration might be given to clinical testing of PALB2 by complete genomic sequencing for familial breast cancer patients with wildtype sequences at BRCA1 and BRCA2.
Alu-mediated rearrangement of tumor suppressor genes occurs frequently during carcinogenesis. In breast cancer, this mechanism contributes to loss of the wild-type BRCA1 allele in inherited disease and to loss of heterozygosity in sporadic cancer. To identify genes required for suppression of Alu-mediated recombination we performed a genomewide screen of a collection of 4672 yeast gene deletion mutants using a direct repeat recombination assay. The primary screen and subsequent analysis identified 12 candidate genes including TSA, ELG1, and RRM3, which are known to play a significant role in maintaining genomic stability. Genetic analysis of the corresponding human homologs was performed in sporadic breast tumors and in inherited BRCA1-associated carcinomas. Sequencing of these genes in high risk breast cancer families revealed a potential role for the helicase PIF1 in cancer predisposition. PIF1 variant L319P was identified in three breast cancer families; importantly, this variant, which is predicted to be functionally damaging, was not identified in a large series of controls nor has it been reported in either dbSNP or the 1000 Genomes Project. In Schizosaccharomyces pombe, Pfh1 is required to maintain both mitochondrial and nuclear genomic integrity. Functional studies in yeast of human PIF1 L319P revealed that this variant cannot complement the essential functions of Pfh1 in either the nucleus or mitochondria. Our results provide a global view of nonessential genes involved in suppressing Alu-mediated recombination and implicate variation in PIF1 in breast cancer predisposition.
Nanos family members have been shown to act as translational repressors in the Drosophila and Caenorhabditis elegans germline, but direct evidence is missing for a similar function in vertebrates. Using a tethered function assay, we show that Xenopus Nanos1 is a translational repressor and that association with the RNA is required for this repression. We identified a 14 amino acid region within the N-terminal domain of Nanos1 that is conserved in organisms as diverse as sponge and Human. The region is found in all vertebrates but notably lacking in Drosophila and C. elegans. Deletion and substitution analysis revealed that this conserved region was required for Nanos1 repressive activity. Consistent with this observation, deletion of this region was sufficient to prevent abnormal development that results from ectopic expression of Nanos1 in oocytes. Although Nanos1 can repress capped and polyadenylated RNAs, Nanos1 mediated repression did not require the targeted RNA to have a cap or to be polyadenylated. These results suggest that Nanos1 is capable of repressing translation by several different mechanisms. We found that Nanos1, like Drosophila Nanos, associates with cyclin B1 RNA in vivo indicating that some Nanos targets may be evolutionarily conserved. Nanos1 protein was detected and thus available to repress mRNAs while PGCs were in the endoderm, but was not observed in PGCs after this stage.
Xenopus; Nanos1/Xcat2; germline determination; translational repression
Identification of genes responsible for medically important traits is a major challenge in human genetics. Due to the genetic heterogeneity of hearing loss, targeted DNA capture and massively parallel sequencing are ideal tools to address this challenge. Our subjects for genome analysis are Israeli Jewish and Palestinian Arab families with hearing loss that varies in mode of inheritance and severity.
A custom 1.46 MB design of cRNA oligonucleotides was constructed containing 246 genes responsible for either human or mouse deafness. Paired-end libraries were prepared from 11 probands and bar-coded multiplexed samples were sequenced to high depth of coverage. Rare single base pair and indel variants were identified by filtering sequence reads against polymorphisms in dbSNP132 and the 1000 Genomes Project. We identified deleterious mutations in CDH23, MYO15A, TECTA, TMC1, and WFS1. Critical mutations of the probands co-segregated with hearing loss. Screening of additional families in a relevant population was performed. TMC1 p.S647P proved to be a founder allele, contributing to 34% of genetic hearing loss in the Moroccan Jewish population.
Critical mutations were identified in 6 of the 11 original probands and their families, leading to the identification of causative alleles in 20 additional probands and their families. The integration of genomic analysis into early clinical diagnosis of hearing loss will enable prediction of related phenotypes and enhance rehabilitation. Characterization of the proteins encoded by these genes will enable an understanding of the biological mechanisms involved in hearing loss.
Massively parallel sequencing of barcoded DNA samples significantly increases screening efficiency for clinically important genes. Short read aligners are well suited to single nucleotide and indel detection. However, methods for CNV detection from targeted enrichment are lacking. We present a method combining coverage with map information for the identification of deletions and duplications in targeted sequence data.
Sequencing data is first scanned for gains and losses using a comparison of normalized coverage data between samples. CNV calls are confirmed by testing for a signature of sequences that span the CNV breakpoint. With our method, CNVs can be identified regardless of whether breakpoints are within regions targeted for sequencing. For CNVs where at least one breakpoint is within targeted sequence, exact CNV breakpoints can be identified. In a test data set of 96 subjects sequenced across ~1 Mb genomic sequence using multiplexing technology, our method detected mutations as small as 31 bp, predicted quantitative copy count, and had a low false-positive rate.
Application of this method allows for identification of gains and losses in targeted sequence data, providing comprehensive mutation screening when combined with a short read aligner.
In communities with high rates of consanguinity and consequently high prevalence of recessive phenotypes, homozygosity mapping with SNP arrays is an effective approach for gene discovery. In 20 Palestinian kindreds with prelingual nonsyndromic hearing loss, we generated homozygosity profiles reflecting linkage to the phenotype. Family sizes ranged from small nuclear families with two affected children, one unaffected sibling, and parents to multigenerational kindreds with 12 affected relatives. By including unaffected parents and siblings and screening 250 K SNP arrays, even small nuclear families yielded informative profiles. In 14 families, we identified the allele responsible for hearing loss by screening a single candidate gene in the longest homozygous region. Novel alleles included missense, nonsense, and splice site mutations of CDH23, MYO7A, MYO15A, OTOF, PJVK, Pendrin/SLC26A4, TECTA, TMHS, and TMPRSS3, and a large genomic deletion of Otoancorin (OTOA). All point mutations were rare in the Palestinian population (zero carriers in 288 unrelated controls); the carrier frequency of the OTOA genomic deletion was 1%. In six families, we identified five genomic regions likely to harbor novel genes for human hearing loss on chromosomes 1p13.3 (DFNB82), 9p23–p21.2/p13.3–q21.13 (DFNB83), 12q14.3–q21.2 (DFNB84; two families), 14q23.1–q31.1, and 17p12–q11.2 (DFNB85).
hearing; deafness; DFNB; homozygosity mapping; mutation; protein modeling
Exposure to Mycobacterium tuberculosis produces varied early outcomes, ranging from resistance to infection to progressive disease. Here we report results from a forward genetic screen in zebrafish larvae that identify multiple mutant classes with distinct patterns of innate susceptibility to Mycobacterium marinum. A hypersusceptible mutant maps to the lta4h locus encoding leukotriene A4 hydrolase, which catalyzes the final step in the synthesis of leukotriene B4 (LTB4), a potent chemoattractant and proinflammatory eicosanoid. lta4h mutations confer hypersusceptibility independent of LTB4 reduction, by redirecting eicosanoid substrates to anti-inflammatory lipoxins. The resultant anti-inflammatory state permits increased mycobacterial proliferation by limiting production of tumor necrosis factor. In humans, we find that protection from both tuberculosis and multibacillary leprosy is associated with heterozygosity for LTA4H polymorphisms that have previously been correlated with differential LTB4 production. Our results suggest conserved roles for balanced eicosanoid production in vertebrate resistance to mycobacterial infection.
We tested the hypothesis that de novo copy number variation (CNV) is associated with autism spectrum disorders (ASDs). We performed comparative genomic hybridization (CGH) on the genomic DNA of patients and unaffected subjects to detect copy number variants not present in their respective parents. Candidate genomic regions were validated by higher-resolution CGH, paternity testing, cytogenetics, fluorescence in situ hybridization, and microsatellite genotyping. Confirmed de novo CNVs were significantly associated with autism (P = 0.0005). Such CNVs were identified in 12 out of 118 (10%) of patients with sporadic autism, in 2 out of 77 (3%) of patients with an affected first-degree relative, and in 2 out of 196 (1%) of controls. Most de novo CNVs were smaller than microscopic resolution. Affected genomic regions were highly heterogeneous and included mutations of single genes. These findings establish de novo germline mutation as a more significant risk factor for ASD than previously recognized.
A 60-year-old man was readmitted 1 year after bioprosthetic aortic valve replacement for recurrent endocarditis. Transthoracic 2-dimensional color Doppler revealed a novel finding of a left-to-right shunt from the left ventricular outflow tract to the right atrium immediately superior to the septal leaflet of the tricuspid valve consistent with an acquired Gerbode defect. Real-time 3-dimensional echocardiography was used to accurately delineate the course of the shunt. To avoid overestimating right ventricular systolic pressure by mistaking such a shunt for an eccentric jet of tricuspid regurgitation, it is important to accurately differentiate the two. Real-time 3-dimensional echocardiography now provides rapid, detailed 3-dimensional appreciation of the origin and course of such shunts with easy facility of orienting views to the flows of interest by cropping. Such information can help design optimal surgical or catheter-based therapy.
Aortic valve replacement; Gerbode defect; Three-dimensional echocardiography
It is general practice to correct cardiac chamber size for body size by the process of scaling or normalization. Normalization is most commonly performed using simple linear or isometric correction; however, there is increasing evidence that this approach may be flawed. Likewise, there is little agreement concerning the appropriate scaling variable (measure of body size) for normalization. Therefore, we aimed to establish the optimal method for correcting the differences in body size in a large population of echocardiographically normal paediatric subjects.
Methods and results
We compared the relative ability of standard size variables including height (HT), body weight (BW), body mass index (BMI), and body surface area (BSA), in both isometric and allometric models, to remove the effect of body size in 4109 consecutive echocardiographically normal subjects <18 years of age, using the left atrial dimension (LAD) as a reference standard. Simple linear normalization resulted in significant residual correlations (r = −0.57 to −0.92) of the indexed value with the body size variable, the correlations with weight (WT) and BSA actually increasing. In contrast, correction by the optimal allometric exponent (AE) removed the effects of the indexed variable (residual correlations −0.01 to 0.01), with BW and BSA best removing the effects of all the measures of body size.
Conventional linear correction for body size is inaccurate in children and paradoxically increases the relationship of the indexed parameter with WT and BSA. Conversely, correction using the optimal AE removes the effect of that variable, with WT best correction for all measures of body size.
Scaling; Allometric; Echocardiography; Linear; Classification
c-Myc-deficient mice fail to develop normal vascular networks and c-Myc-deficient embryonic stem cells fail to provoke a tumor angiogenic response when injected into immune compromised mice. However, the molecular underpinnings of these defects are poorly understood. To assess whether c-Myc indeed contributes to embryonic vasculogenesis we evaluated c-Myc function in Xenopus laevis embryogenesis. Here we report that Xc-Myc is required for the normal assembly of endothelial cells into patent vessels during both angiogenesis and lymphangiogenesis. Accordingly, the specific knockdown of Xc-Myc provokes massive embryonic edema and hemorrhage. Conversely, Xc-Myc overexpression triggers the formation of ectopic vascular beds in embryos. c-Myc is required for normal expression of Slug/Snail2 and Twist, and either XSlug/Snail2 or XTwist could compensate for defects manifest by Xc-Myc knockdown. Importantly, knockdown of Xc-Myc, XSlug/Snail2, or XTwist within the lateral plate mesoderm, but not the neural crest, provoked embryonic edema and hemorrhage. Collectively, these findings support a model in which c-Myc, Twist, and Slug/Snail2 function in a regulatory circuit within lateral plate mesoderm that directs normal vessel formation in both the vascular and lymphatic systems.
Myc; Slug/Snail2; Twist; Vasculogenesis; Lymphangiogenesis; Xenopus
The aim of this study was to determine the genomic structure of the deletions on chromosome 17 in ovarian carcinomas from women with inherited BRCA1 mutations. Normal and tumor DNA from 14 ovarian tumors associated with inherited BRCA1 mutations were extracted and tested for loss of heterozygosity (LOH) at microsatellite markers along chromosome 17. Finer mapping using more microsatellite markers and single nucleotide polymorphisms (SNPs) helped further define the LOH margins. The genomic repeated elements within the LOH breakpoint regions were identified using the University of California Santa Cruz Genome Database and the frequencies were compared to regions of equal GC percentages across the genome. Of the 14 ovarian tumors, 12 showed LOH of the entire chromosome 17. The other two tumors lost the distal end of the 17q arm. The breakpoints of these two tumors occurred in regions with significantly high frequencies of SINE repeating elements, specifically Alu elements. Ovarian tumors of high grade and stage have large regions of LOH along chromosome 17, with most tumors showing loss of the entire chromosome. In those tumors with retention of part of chromosome 17, LOH margins suggest that a high Alu content may have a role in the deletions.
We studied a consanguineous family (Family A) from the island of Newfoundland with an autosomal recessive form of prelingual, profound, nonsyndromic sensorineural hearing loss. A genome-wide scan mapped the deafness trait to 10q21-22 (max LOD score of 4.0; D10S196) and fine mapping revealed a 16 Mb ancestral haplotype in deaf relatives. The PCDH15 gene was mapped within the critical region and was an interesting candidate because truncating mutations cause Usher syndrome type IF (USH1F) and two missense mutations have been previously associated with isolated deafness (DFNB23). Sequencing of the PCDH15 gene revealed 33 sequencing variants. Three of these variants were homozygous exclusively in deaf siblings but only one of them was not seen in ethnically matched controls. This novel c.1583 T>A transversion predicts an amino-acid substitution of a valine with an aspartic acid at codon 528 (V528D). Like the two DFNB23 mutations, the V528D mutation in Family A occurs in a highly conserved extracellular cadherin (EC) domain of PCDH15 and is predicted to be more deleterious than the previously identified DFNB23 missense mutations (R134G and G262D). Physical assessment, vestibular and visual function testing in deaf adults ruled out syndromic deafness because of Usher syndrome. This study validates the DFNB23 designation and supports the hypothesis that missense mutations in conserved motifs of PCDH15 cause nonsyndromic hearing loss. This emerging genotype–phenotype correlation in USH1F is similar to that in several other USH1 genes and cautions against a prognosis of a dual sensory loss in deaf children found to be homozygous for hypomorphic mutations at the USH1F locus.
PCDH15; isolated deafness; usher syndrome type IF; hypomorphic alleles
Duplications in Xq28 involving MECP2 have been described in patients with severe mental retardation, infantile hypotonia, progressive spasticity, and recurrent infections. However, it is not yet clear to what extent these and accompanying symptoms may vary. In addition, the frequency of Xq28 duplications including MECP2 has yet to be determined in patients with unexplained X-linked mental retardation and (fe)males with severe encephalopathy. In this study, we used multiplex ligation-dependent probe amplification to screen Xq28 including MECP2 for deletions and duplications in these patient cohorts. In the group of 283 patients with X-linked mental retardation, we identified three Xq28 duplications including MECP2, which suggests that approximately 1% of unexplained X-linked mental retardation may be caused by MECP2 duplications. In addition, we found three additional MECP2 duplications in 134 male patients with mental retardation and severe, mostly progressive, neurological symptoms, indicating that the mutation frequency could be as high as 2% in this group of patients. In 329 female patients, no Xq28 duplications were detected. In total, we assessed 13 male patients with a MECP2 duplication from six unrelated families. Moderate to severe mental retardation and childhood hypotonia was noted in all patients. The majority of the patients also presented with absent speech, seizures, and progressive spasticity as well as ataxia or an ataxic gait and cerebral atrophy, two previously unreported symptoms. We propose to implement DNA copy number testing for MECP2 in the current diagnostic testing in all males with moderate to severe mental retardation accompanied by (progressive) neurological symptoms.
MECP2; Xq28; XLMR; encephalopathy; duplications