Mutations in NMNAT1 cause Leber congenital amaurosis and identify a new disease pathway for retinal degeneration

Leber congenital amaurosis (LCA) is a blinding retinal disease that presents within the first year after birth. Using exome sequencing, we identified mutations in the nicotinamide adenine dinucleotide (NAD) synthase gene NMNAT1 encoding nicotinamide mononucleotide adenylyltransferase 1 in eight families with LCA, including the family in which LCA was originally linked to the LCA9 locus. Notably, all individuals with NMNAT1 mutations also have macular colobomas, which are severe degenerative entities of the central retina (fovea) devoid of tissue and photoreceptors. Functional assays of the proteins encoded by the mutant alleles identified in our study showed that the mutations reduce the enzymatic activity of NMNAT1 in NAD biosynthesis and affect protein folding. Of note, recent characterization of the slow Wallerian degeneration (Wld(s)) mouse model, in which prolonged axonal survival after injury is observed, identified NMNAT1 as a neuroprotective protein when ectopically expressed. Our findings identify a new disease mechanism underlying LCA and provide the first link between endogenous NMNAT1 dysfunction and a human nervous system disorder.     

Exome sequencing identifies NMNAT1 mutations as a cause of Leber congenital amaurosis

Leber congenital amaurosis (LCA) is an autosomal recessive retinal dystrophy that manifests with genetic heterogeneity. We sequenced the exome of an individual with LCA and identified nonsense (c.507G>A, p.Trp169*) and missense (c.769G>A, p.Glu257Lys) mutations in NMNAT1, which encodes an enzyme in the nicotinamide adenine dinucleotide (NAD) biosynthesis pathway implicated in protection against axonal degeneration. We also found NMNAT1 mutations in ten other individuals with LCA, all of whom carry the p.Glu257Lys variant.     

Mutations in NMNAT1 cause Leber congenital amaurosis with early-onset severe macular and optic atrophy

In addition to its activity in nicotinamide adenine dinucleotide (NAD(+)) synthesis, the nuclear nicotinamide mononucleotide adenyltransferase NMNAT1 acts as a chaperone that protects against neuronal activity-induced degeneration. Here we report that compound heterozygous and homozygous NMNAT1 mutations cause severe neonatal neurodegeneration of the central retina and early-onset optic atrophy in 22 unrelated individuals. Their clinical presentation is consistent with Leber congenital amaurosis and suggests that the mutations affect neuroprotection of photoreceptor cells.     

Mutations in a new photoreceptor-pineal gene on 17p cause Leber congenital amaurosis

Leber congenital amaurosis (LCA, MIM 204000) accounts for at least 5% of all inherited retinal disease and is the most severe inherited retinopathy with the earliest age of onset. Individuals affected with LCA are diagnosed at birth or in the first few months of life with severely impaired vision or blindness, nystagmus and an abnormal or flat electroretinogram (ERG). Mutations in GUCY2D (ref. 3), RPE65 (ref. 4) and CRX (ref. 5) are known to cause LCA, but one study identified disease-causing GUCY2D mutations in only 8 of 15 families whose LCA locus maps to 17p13.1 (ref. 3), suggesting another LCA locus might be located on 17p13.1. Confirming this prediction, the LCA in one Pakistani family mapped to 17p13.1, between D17S849 and D17S960-a region that excludes GUCY2D. The LCA in this family has been designated LCA4 (ref. 6). We describe here a new photoreceptor/pineal-expressed gene, AIPL1 (encoding aryl-hydrocarbon interacting protein-like 1), that maps within the LCA4 candidate region and whose protein contains three tetratricopeptide (TPR) motifs, consistent with nuclear transport or chaperone activity. A homozygous nonsense mutation at codon 278 is present in all affected members of the original LCA4 family. AIPL1 mutations may cause approximately 20% of recessive LCA, as disease-causing mutations were identified in 3 of 14 LCA families not tested previously for linkage.     

Mutations in DNMT1 cause hereditary sensory neuropathy with dementia and hearing loss

DNA methyltransferase 1 (DNMT1) is crucial for maintenance of methylation, gene regulation and chromatin stability. DNA mismatch repair, cell cycle regulation in post-mitotic neurons and neurogenesis are influenced by DNA methylation. Here we show that mutations in DNMT1 cause both central and peripheral neurodegeneration in one form of hereditary sensory and autonomic neuropathy with dementia and hearing loss. Exome sequencing led to the identification of DNMT1 mutation c.1484A>G (p.Tyr495Cys) in two American kindreds and one Japanese kindred and a triple nucleotide change, c.1470-1472TCC>ATA (p.Asp490Glu-Pro491Tyr), in one European kindred. All mutations are within the targeting-sequence domain of DNMT1. These mutations cause premature degradation of mutant proteins, reduced methyltransferase activity and impaired heterochromatin binding during the G2 cell cycle phase leading to global hypomethylation and site-specific hypermethylation. Our study shows that DNMT1 mutations cause the aberrant methylation implicated in complex pathogenesis. The discovered DNMT1 mutations provide a new framework for the study of neurodegenerative diseases.     

Mutations in the colony stimulating factor 1 receptor (CSF1R) gene cause hereditary diffuse leukoencephalopathy with spheroids

Hereditary diffuse leukoencephalopathy with spheroids (HDLS) is an autosomal-dominant central nervous system white-matter disease with variable clinical presentations, including personality and behavioral changes, dementia, depression, parkinsonism, seizures and other phenotypes. We combined genome-wide linkage analysis with exome sequencing and identified 14 different mutations affecting the tyrosine kinase domain of the colony stimulating factor 1 receptor (encoded by CSF1R) in 14 families with HDLS. In one kindred, we confirmed the de novo occurrence of the mutation. Follow-up sequencing identified an additional CSF1R mutation in an individual diagnosed with corticobasal syndrome. In vitro, CSF-1 stimulation resulted in rapid autophosphorylation of selected tyrosine residues in the kinase domain of wild-type but not mutant CSF1R, suggesting that HDLS may result from partial loss of CSF1R function. As CSF1R is a crucial mediator of microglial proliferation and differentiation in the brain, our findings suggest an important role for microglial dysfunction in HDLS pathogenesis.     

Positional cloning uncovers mutations in PLCE1 responsible for a nephrotic syndrome variant that may be reversible

Nephrotic syndrome, a malfunction of the kidney glomerular filter, leads to proteinuria, edema and, in steroid-resistant nephrotic syndrome, end-stage kidney disease. Using positional cloning, we identified mutations in the phospholipase C epsilon gene (PLCE1) as causing early-onset nephrotic syndrome with end-stage kidney disease. Kidney histology of affected individuals showed diffuse mesangial sclerosis (DMS). Using immunofluorescence, we found PLCepsilon1 expression in developing and mature glomerular podocytes and showed that DMS represents an arrest of normal glomerular development. We identified IQ motif-containing GTPase-activating protein 1 as a new interaction partner of PLCepsilon1. Two siblings with a missense mutation in an exon encoding the PLCepsilon1 catalytic domain showed histology characteristic of focal segmental glomerulosclerosis. Notably, two other affected individuals responded to therapy, making this the first report of a molecular cause of nephrotic syndrome that may resolve after therapy. These findings, together with the zebrafish model of human nephrotic syndrome generated by plce1 knockdown, open new inroads into pathophysiology and treatment mechanisms of nephrotic syndrome.     

Mutations in the PCNA-binding domain of CDKN1C cause IMAGe syndrome

IMAGe syndrome (intrauterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genital anomalies) is an undergrowth developmental disorder with life-threatening consequences. An identity-by-descent analysis in a family with IMAGe syndrome identified a 17.2-Mb locus on chromosome 11p15 that segregated in the affected family members. Targeted exon array capture of the disease locus, followed by high-throughput genomic sequencing and validation by dideoxy sequencing, identified missense mutations in the imprinted gene CDKN1C (also known as P57KIP2) in two familial and four unrelated patients. A familial analysis showed an imprinted mode of inheritance in which only maternal transmission of the mutation resulted in IMAGe syndrome. CDKN1C inhibits cell-cycle progression, and we found that targeted expression of IMAGe-associated CDKN1C mutations in Drosophila caused severe eye growth defects compared to wild-type CDKN1C, suggesting a gain-of-function mechanism. All IMAGe-associated mutations clustered in the PCNA-binding domain of CDKN1C and resulted in loss of PCNA binding, distinguishing them from the mutations of CDKN1C that cause Beckwith-Wiedemann syndrome, an overgrowth syndrome.     

Mutations in SOX2 cause anophthalmia

A submicroscopic deletion containing SOX2 was identified at the 3q breakpoint in a child with t(3;11)(q26.3;p11.2) associated with bilateral anophthalmia. Subsequent SOX2 mutation analysis identified de novo truncating mutations of SOX2 in 4 of 35 (11%) individuals with anophthalmia. Both eyes were affected in all cases with an identified mutation.     

Germline mutations and sequence variants of the macrophage scavenger receptor 1 gene are associated with prostate cancer risk

Deletions on human chromosome 8p22-23 in prostate cancer cells and linkage studies in families affected with hereditary prostate cancer (HPC) have implicated this region in the development of prostate cancer. The macrophage scavenger receptor 1 gene (MSR1, also known as SR-A) is located at 8p22 and functions in several processes proposed to be relevant to prostate carcinogenesis. Here we report the results of genetic analyses that indicate that mutations in MSR1 may be associated with risk of prostate cancer. Among families affected with HPC, we identified six rare missense mutations and one nonsense mutation in MSR1. A family-based linkage and association test indicated that these mutations co-segregate with prostate cancer (P = 0.0007). In addition, among men of European descent, MSR1 mutations were detected in 4.4% of individuals affected with non-HPC as compared with 0.8% of unaffected men (P = 0.009). Among African American men, these values were 12.5% and 1.8%, respectively (P = 0.01). These results show that MSR1 may be important in susceptibility to prostate cancer in men of both African American and European descent.     

Mutations of TTN, encoding the giant muscle filament titin, cause familial dilated cardiomyopathy.

Congestive heart failure (CHF) can result from various disease states with inadequate cardiac output. CHF due to dilated cardiomyopathy (DCM) is a familial disease in 20-30% of cases and is associated with mutations in genes encoding cytoskeletal, contractile or inner-nuclear membrane proteins. We show that mutations in the gene encoding giant-muscle filament titin (TTN) cause autosomal dominant DCM linked to chromosome 2q31 (CMD1G; MIM 604145). Titin molecules extend from sarcomeric Z-discs to M-lines, provide an extensible scaffold for the contractile machinery and are crucial for myofibrillar elasticity and integrity. In a large DCM kindred, a segregating 2-bp insertion mutation in TTN exon 326 causes a frameshift, truncating A-band titin. The truncated protein of approximately 2 mD is expressed in skeletal muscle, but western blot studies with epitope-specific anti-titin antibodies suggest that the mutant protein is truncated to a 1.14-mD subfragment by site-specific cleavage. In another large family with DCM linked to CMD1G, a TTN missense mutation (Trp930Arg) is predicted to disrupt a highly conserved hydrophobic core sequence of an immunoglobulin fold located in the Z-disc-I-band transition zone. The identification of TTN mutations in individuals with CMD1G should provide further insights into the pathogenesis of familial forms of CHF and myofibrillar titin turnover.     

TGFB2 mutations cause familial thoracic aortic aneurysms and dissections associated with mild systemic features of Marfan syndrome

A predisposition for thoracic aortic aneurysms leading to acute aortic dissections can be inherited in families in an autosomal dominant manner. Genome-wide linkage analysis of two large unrelated families with thoracic aortic disease followed by whole-exome sequencing of affected relatives identified causative mutations in TGFB2. These mutations-a frameshift mutation in exon 6 and a nonsense mutation in exon 4-segregated with disease with a combined logarithm of odds (LOD) score of 7.7. Sanger sequencing of 276 probands from families with inherited thoracic aortic disease identified 2 additional TGFB2 mutations. TGFB2 encodes transforming growth factor (TGF)-β2, and the mutations are predicted to cause haploinsufficiency for TGFB2; however, aortic tissue from cases paradoxically shows increased TGF-β2 expression and immunostaining. Thus, haploinsufficiency for TGFB2 predisposes to thoracic aortic disease, suggesting that the initial pathway driving disease is decreased cellular TGF-β2 levels leading to a secondary increase in TGF-β2 production in the diseased aorta.     

Mutation of MYH9, encoding non-muscle myosin heavy chain A, in May-Hegglin anomaly

May-Hegglin anomaly (MHA) is an autosomal dominant macrothrombocytopenia of unclear pathogenesis characterized by thrombocytopenia, giant platelets and leukocyte inclusions. Studies have indicated that platelet structure and function are normal, suggesting a defect in megakaryocyte fragmentation. The disorder has been linked to chromosome 22q12-13. Here we screen a candidate gene in this region, encoding non-muscle myosin heavy chain A (MYH9), for mutations in ten families. In each family, we identified one of three sequence variants within either the -helical coiled coil or the tailpiece domain that co-segregated with disease status. The E1841K mutation was found in 5 families and occurs at a conserved site in the rod domain. This mutation was not found in 40 normal individuals. Four families had a nonsense mutation that resulted in truncation of most of the tailpiece. One family had a T1155I mutation present in an affected mother and daughter, but not in the mother's parents, thus representing a new mutation. Among the 30 affected individuals, 21 unaffected individuals and 13 spouses in the 10 families, there was correlation of a variant of MYH9 with the presence of MHA. The identification of MYH9 as the disease gene for MHA establishes the pathogenesis of the disorder, should provide further insight into the processes of normal platelet formation and may facilitate identification of the genetic basis of related disorders.     

Mutations in RDH12 encoding a photoreceptor cell retinol dehydrogenase cause childhood-onset severe retinal dystrophy

We identified three consanguineous Austrian kindreds with 15 members affected by autosomal recessive childhood-onset severe retinal dystrophy, a genetically heterogeneous group of disorders characterized by degeneration of the photoreceptor cells. A whole-genome scan by microarray analysis of single-nucleotide polymorphisms (ref. 2) identified a founder haplotype and defined a critical interval of 1.53 cM on chromosome 14q23.3-q24.1 that contains the gene associated with this form of retinal dystrophy. RDH12 maps in this region and encodes a retinol dehydrogenase proposed to function in the visual cycle. A homozygous 677A-->G transition (resulting in Y226C) in RDH12 was present in all affected family members studied, as well as in two Austrian individuals with sporadic retinal dystrophy. We identified additional mutations in RDH12 in 3 of 89 non-Austrian individuals with retinal dystrophy: a 5-nucleotide deletion (806delCCCTG) and the transition 565C-->T (resulting in Q189X), each in the homozygous state, and 146C-->T (resulting in T49M) and 184C-->T (resulting in R62X) in compound heterozygosity. When expressed in COS-7 cells, Cys226 and Met49 variants had diminished and aberrant activity, respectively, in interconverting isomers of retinol and retinal. The severe visual impairment of individuals with mutations in RDH12 is in marked contrast to the mild visual deficiency in individuals with fundus albipunctatus caused by mutations in RDH5, encoding another retinal dehydrogenase. Our studies show that RDH12 is associated with retinal dystrophy and encodes an enzyme with a unique, nonredundant role in the photoreceptor cells.     

Somatic histone H3 alterations in pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas

To identify somatic mutations in pediatric diffuse intrinsic pontine glioma (DIPG), we performed whole-genome sequencing of DNA from seven DIPGs and matched germline tissue and targeted sequencing of an additional 43 DIPGs and 36 non-brainstem pediatric glioblastomas (non-BS-PGs). We found that 78% of DIPGs and 22% of non-BS-PGs contained a mutation in H3F3A, encoding histone H3.3, or in the related HIST1H3B, encoding histone H3.1, that caused a p.Lys27Met amino acid substitution in each protein. An additional 14% of non-BS-PGs had somatic mutations in H3F3A causing a p.Gly34Arg alteration.     

Exome sequencing identifies truncating mutations in PRRT2 that cause paroxysmal kinesigenic dyskinesia

Paroxysmal kinesigenic dyskinesia is the most common type of paroxysmal movement disorder and is often misdiagnosed clinically as epilepsy. Using whole-exome sequencing followed by Sanger sequencing, we identified three truncating mutations within PRRT2 (NM_145239.2) in eight Han Chinese families with histories of paroxysmal kinesigenic dyskinesia: c.514_517delTCTG (p.Ser172Argfs*3) in one family, c.649dupC (p.Arg217Profs*8) in six families and c.972delA (p.Val325Serfs*12) in one family. These truncating mutations co-segregated exactly with the disease in these families and were not observed in 1,000 control subjects of matched ancestry. PRRT2 is a newly discovered gene consisting of four exons encoding the proline-rich transmembrane protein 2, which encompasses 340 amino acids and contains two predicted transmembrane domains. PRRT2 is highly expressed in the developing nervous system, and a truncating mutation alters the subcellular localization of the PRRT2 protein. The function of PRRT2 and its role in paroxysmal kinesigenic dyskinesia should be further investigated.     

Mutations in PCSK9 cause autosomal dominant hypercholesterolemia

Autosomal dominant hypercholesterolemia (ADH; OMIM144400), a risk factor for coronary heart disease, is characterized by an increase in low-density lipoprotein cholesterol levels that is associated with mutations in the genes LDLR (encoding low-density lipoprotein receptor) or APOB (encoding apolipoprotein B). We mapped a third locus associated with ADH, HCHOLA3 at 1p32, and now report two mutations in the gene PCSK9 (encoding proprotein convertase subtilisin/kexin type 9) that cause ADH. PCSK9 encodes NARC-1 (neural apoptosis regulated convertase), a newly identified human subtilase that is highly expressed in the liver and contributes to cholesterol homeostasis.