Clinical delineation of the PACS1-related syndrome--Report on 19 patients.

We report on 19 individuals with a recurrent de novo c.607C>T mutation in PACS1. This specific mutation gives rise to a recognizable intellectual disability syndrome. There is a distinctive facial appearance (19/19), characterized by full and arched eyebrows, hypertelorism with downslanting palpebral fissures, long eye lashes, ptosis, low set and simple ears, bulbous nasal tip, wide mouth with downturned corners and a thin upper lip with an unusual "wavy" profile, flat philtrum, and diastema of the teeth. Intellectual disability, ranging from mild to moderate, was present in all. Hypotonia is common in infancy (8/19). Seizures are frequent (12/19) and respond well to anticonvulsive medication. Structural malformations are common, including heart (10/19), brain (12/16), eye (10/19), kidney (3/19), and cryptorchidism (6/12 males). Feeding dysfunction is presenting in infancy with failure to thrive (5/19), gastroesophageal reflux (6/19), and gastrostomy tube placement (4/19). There is persistence of oral motor dysfunction. We provide suggestions for clinical work-up and management and hope that the present study will facilitate clinical recognition of further cases.

Comprehensive next-generation sequence analyses of the entire mitochondrial genome reveal new insights into the molecular diagnosis of mitochondrial DNA disorders.

PURPOSE: The application of massively parallel sequencing technology to the analysis of the mitochondrial genome has demonstrated great improvement in the molecular diagnosis of mitochondrial DNA-related disorders. The objective of this study was to investigate the performance characteristics and to gain new insights into the analysis of the mitochondrial genome. METHODS: The entire mitochondrial genome was analyzed as a single amplicon using a long-range PCR-based enrichment approach coupled with massively parallel sequencing. The interference of the nuclear mitochondrial DNA homologs was distinguished from the actual mitochondrial DNA sequences by comparison with the results obtained from conventional PCR-based Sanger sequencing using multiple pairs of primers. RESULTS: Our results demonstrated the uniform coverage of the entire mitochondrial genome. Massively parallel sequencing of the single amplicon revealed the presence of single-nucleotide polymorphisms and nuclear homologs of mtDNA sequences that cause the erroneous and inaccurate variant calls when PCR/Sanger sequencing approach was used. This single amplicon massively parallel sequencing strategy provides an accurate quantification of mutation heteroplasmy as well as the detection and mapping of mitochondrial DNA deletions. CONCLUSION: The ability to quantitatively and qualitatively evaluate every single base of the entire mitochondrial genome is indispensible to the accurate molecular diagnosis and genetic counseling of mitochondrial DNA-related disorders. This new approach may be considered as first-line testing for comprehensive analysis of the mitochondrial genome.Genet Med 2013:15(5):388-394.

Ubiquilin-1 is a molecular chaperone for the amyloid precursor protein.

Alzheimer disease (AD) is associated with extracellular deposition of proteolytic fragments of amyloid precursor protein (APP). Although mutations in APP and proteases that mediate its processing are known to result in familial, early onset forms of AD, the mechanisms underlying the more common sporadic, yet genetically complex forms of the disease are still unclear. Four single-nucleotide polymorphisms within the ubiquilin-1 gene have been shown to be genetically associated with AD, implicating its gene product in the pathogenesis of late onset AD. However, genetic linkage between ubiquilin-1 and AD has not been confirmed in studies examining different populations. Here we show that regardless of genotype, ubiquilin-1 protein levels are significantly decreased in late onset AD patient brains, suggesting that diminished ubiquilin function may be a common denominator in AD progression. Our interrogation of putative ubiquilin-1 activities based on sequence similarities to proteins involved in cellular quality control showed that ubiquilin-1 can be biochemically defined as a bona fide molecular chaperone and that this activity is capable of preventing the aggregation of amyloid precursor protein both in vitro and in live neurons. Furthermore, we show that reduced activity of ubiquilin-1 results in augmented production of pathogenic amyloid precursor protein fragments as well as increased neuronal death. Our results support the notion that ubiquilin-1 chaperone activity is necessary to regulate the production of APP and its fragments and that diminished ubiquilin-1 levels may contribute to AD pathogenesis.

Diagnostic approaches to apparent homozygosity.

PURPOSE: Sanger sequencing is a mainstay for the identification of gene mutations used in molecular diagnostic laboratories. However, in autosomal recessive disorders, failure of allele amplification can occur for a variety of reasons, leading heterozygous mutations to appear homozygous. We sought to investigate the frequency at which apparently homozygous mutations detected by Sanger sequencing in our laboratory appeared homozygous due to other molecular etiologies. METHODS: A review of 12,406 cases from 40 different genetic tests that were submitted to the Medical Genetics Laboratories at Baylor College of Medicine for Sanger sequence analysis was performed. The molecular status of apparently homozygous cases was further investigated by testing parents using various methods. RESULTS: A total of 291 cases of apparent homozygosity were identified, ranging from 0 to 37% of the total per gene. One-third of the apparently homozygous cases were followed up by parental testing. Parental carrier status was confirmed in 88% of the cases. Of the cases in which parental carrier status could not be confirmed, deletions encompassing point mutations, allele dropout due to single-nucleotide polymorphisms at primer sites, and uniparental isodisomy were observed. CONCLUSION: For individuals with autosomal recessive disorders and apparently homozygous mutations, confirmation by parental testing can rule out other causes of apparent homozygosity, including allele dropout, copy number variations, and uniparental isodisomy.

An integrated approach for classifying mitochondrial DNA variants: one clinical diagnostic laboratory's experience.

PURPOSE: The mitochondrial genome is highly polymorphic. A unique feature of deleterious mitochondrial DNA (mtDNA) mutations is heteroplasmy. Genetic background and variable penetrance also play roles in the pathogenicity for a mtDNA variant. Clinicians are increasingly interested in requesting mtDNA testing. However, interpretation of uncharacterized mtDNA variants is a great challenge. We suggest a stepwise interpretation procedure for clinical service. METHODS: We describe the algorithms used to interpret novel and rare mtDNA variants. mtDNA databases and in silico predictive algorithms are used to evaluate the pathogenic potential of novel and/or rare mtDNA variants. RESULTS: mtDNA variants can be classified into three categories: benign variants, unclassified variants, and deleterious mutations based on database search and in silico prediction. Targeted DNA sequence analysis of matrilineal relatives, heteroplasmy quantification, and functional studies are useful to classify mtDNA variants. CONCLUSION: Clinical significance of a novel or rare variant is critical in the diagnosis of the disease and counseling of the family. Based on the results from clinical, biochemical, and molecular genetic studies of multiple family members, proper interpretation of mtDNA variants is important for clinical laboratories and for patient care.

The UNC-45 chaperone mediates sarcomere assembly through myosin degradation in Caenorhabditis elegans.

Myosin motors are central to diverse cellular processes in eukaryotes. Homologues of the myosin chaperone UNC-45 have been implicated in the assembly and function of myosin-containing structures in organisms from fungi to humans. In muscle, the assembly of sarcomeric myosin is regulated to produce stable, uniform thick filaments. Loss-of-function mutations in Caenorhabditis elegans UNC-45 lead to decreased muscle myosin accumulation and defective thick filament assembly, resulting in paralyzed animals. We report that transgenic worms overexpressing UNC-45 also display defects in myosin assembly, with decreased myosin content and a mild paralysis phenotype. We find that the reduced myosin accumulation is the result of degradation through the ubiquitin/proteasome system. Partial proteasome inhibition is able to restore myosin protein and worm motility to nearly wild-type levels. These findings suggest a mechanism in which UNC-45-related proteins may contribute to the degradation of myosin in conditions such as heart failure and muscle wasting.

Novel variants identified in CKAP2L in two siblings with Filippi syndrome.

Pathogenic variants in CKAP2L have previously been reported in Filippi syndrome (FS), a rare autosomal recessive, craniodigital syndrome characterized by microcephaly, syndactyly, short stature, intellectual disability, and dysmorphic facial features. To date, fewer than 10 patients with pathogenic variants in CKAP2L associated with FS have been reported. All of the previously reported probands have presumed loss-of-function variants (frameshift, canonical splice site, starting methionine), and all but one have been homozygous for a pathogenic variant. Here we describe two brothers who presented with microcephaly, micrognathia, syndactyly, dysmorphic features, and intellectual disability. Whole-exome sequencing of the family identified a missense variant, c.2066G > A;p.(Arg689His), in trans with a frameshift variant, c.1169_1173del;p.(Ile390LysfsTer4), in CKAP2L To our knowledge, these are the first patients with FS to be reported with a missense variant in CKAP2L and only the second family to be reported with two variants in trans.

Duplication within the SEPT9 gene associated with a founder effect in North American families with hereditary neuralgic amyotrophy.

Hereditary neuralgic amyotrophy (HNA) is an autosomal dominant disorder associated with recurrent episodes of focal neuropathy primarily affecting the brachial plexus. Point mutations in the SEPT9 gene have been previously identified as the molecular basis of HNA in some pedigrees. However in many families, including those from North America demonstrating a genetic founder haplotype, no sequence mutations have been detected. We report an intragenic 38 Kb SEPT9 duplication that is linked to HNA in 12 North American families that share the common founder haplotype. Analysis of the breakpoints showed that the duplication is identical in all pedigrees, and molecular analysis revealed that the duplication includes the 645 bp exon in which previous HNA mutations were found. The SEPT9 transcript variants that span this duplication contain two in-frame repeats of this exon, and immunoblotting demonstrates larger molecular weight SEPT9 protein isoforms. This exon also encodes for a majority of the SEPT9 N-terminal proline rich region suggesting that this region plays a role in the pathogenesis of HNA.

Utilization of targeted array comparative genomic hybridization, MitoMet, in prenatal diagnosis of metabolic disorders.

Metabolic disorders are inborn errors that often present in the neonatal period with a devastating clinical course. If not treated promptly, these diseases can result in severe, irreversible disease or death. Determining the molecular defects in metabolic diseases is important in providing a definitive diagnosis for patient management. Therefore, prenatal diagnosis for families with known mutations causing metabolic disorders is crucial for timely intervention. Here we present three families in which standard Sanger sequencing failed to provide a definitive diagnosis, but the detection of genomic deletions by array comparative genomic hybridization (CGH) specifically targeted to mitochondrial and metabolic disease genes, MitoMet®, was fundamental in providing accurate prenatal diagnosis. In addition, to our knowledge, two deletions are the smallest detected by oligonucleotide array CGH reported for their respective genes, OTC and ARG1. These data highlight the importance of targeted array CGH in patients with suspected metabolic disorders and incomplete or negative sequencing results, as well as its emerging role in prenatal diagnosis.

Atypical presentation of Leigh syndrome associated with a Leber hereditary optic neuropathy primary mitochondrial DNA mutation.

Leber hereditary optic neuropathy (LHON) is caused by point mutations in mitochondrial DNA (mtDNA), and is characterized by bilateral, painless sub-acute visual loss that develops during the second decade of life. Here we report the case of a five year old girl who presented with clinical and neuroradiological findings reminiscent of Leigh syndrome but carried a mtDNA mutation m.11778G>A (p.R340H) in the MTND4 gene usually observed in patients with LHON. This case is unusual for age of onset, gender, associated neurological findings and evolution, further expanding the clinical spectrum associated with primary LHON mtDNA mutations.

UNC-98 links an integrin-associated complex to thick filaments in Caenorhabditis elegans muscle.

Focal adhesions are multiprotein assemblages that link cells to the extracellular matrix. The transmembrane protein, integrin, is a key component of these structures. In vertebrate muscle, focal adhesion-like structures called costameres attach myofibrils at the periphery of muscle cells to the cell membrane. In Caenorhabditis elegans muscle, all the myofibrils are attached to the cell membrane at both dense bodies (Z-disks) and M-lines. Clustered at the base of dense bodies and M-lines, and associated with the cytoplasmic tail of beta-integrin, is a complex of many proteins, including UNC-97 (vertebrate PINCH). Previously, we showed that UNC-97 interacts with UNC-98, a 37-kD protein, containing four C2H2 Zn fingers, that localizes to M-lines. We report that UNC-98 also interacts with the C-terminal portion of a myosin heavy chain. Multiple lines of evidence support a model in which UNC-98 links integrin-associated proteins to myosin in thick filaments at M-lines.

Loss-of-function mutations in Lysyl-tRNA synthetase cause various leukoencephalopathy phenotypes.

OBJECTIVE: To expand the clinical spectrum of lysyl-tRNA synthetase (KARS) gene-related diseases, which so far includes Charcot-Marie-Tooth disease, congenital visual impairment and microcephaly, and nonsyndromic hearing impairment. METHODS: Whole-exome sequencing was performed on index patients from 4 unrelated families with leukoencephalopathy. Candidate pathogenic variants and their cosegregation were confirmed by Sanger sequencing. Effects of mutations on KARS protein function were examined by aminoacylation assays and yeast complementation assays. RESULTS: Common clinical features of the patients in this study included impaired cognitive ability, seizure, hypotonia, ataxia, and abnormal brain imaging, suggesting that the CNS involvement is the main clinical presentation. Six previously unreported and 1 known KARS mutations were identified and cosegregated in these families. Two patients are compound heterozygous for missense mutations, 1 patient is homozygous for a missense mutation, and 1 patient harbored an insertion mutation and a missense mutation. Functional and structural analyses revealed that these mutations impair aminoacylation activity of lysyl-tRNA synthetase, indicating that defective KARS function is responsible for the phenotypes in these individuals. CONCLUSIONS: Our results demonstrate that patients with loss-of-function KARS mutations can manifest CNS disorders, thus broadening the phenotypic spectrum associated with KARS-related disease.

Delivering Precision Oncology in a Community Cancer Program: Results From a Prospective Observational Study.

INTRODUCTION: Precision oncology (PO) is a growing treatment approach in the era of next-generation sequencing (NGS) and matched therapies. Effective delivery of PO in the community has not been extensively studied. Our program developed a virtual molecular tumor board (MTB) strategy to help guide PO care. MATERIALS AND METHODS: Over 18 months, eligible adult patients with advanced, incurable solid tumor malignancies were enrolled in a molecular profiling (MP) study using the Foundation Medicine NGS panel. Results were reviewed through a weekly, videoconferenced MTB conducted across our largely rural integrated health system. Recommendations from the MTB were used to identify actionable alterations (AAs). Feasibility of PO care delivery was assessed as the primary outcome. Secondary outcomes included the frequency of AAs, genomic matched treatments, genomic matched clinical trial enrollment, and clinical outcomes. RESULTS: A total of 120 participants with a variety of advanced tumor types were enrolled. Of these, 109 (90.8%) had successful MP. Treatment on the basis of an AA was recommended by the MTB in 58% of patients (63 of 109) who had a successful MP result. For those completing MP, treatments included enrollment in a genomic matched clinical trial (n = 16; 14.6%) and genomic matched treatment with a Food and Drug Administration-approved agent (n = 23; 21.1%). Response and survival data were similar regardless of the matched treatment option chosen. CONCLUSION: A video-conferenced MTB-facilitated NGS testing and treatment delivery system was implemented in our integrated community oncology program. Continued use of this model aims to increase understanding of the impact of PO in this setting.

Mutation in GM2A Leads to a Progressive Chorea-dementia Syndrome.

BACKGROUND: The etiology of many cases of childhood-onset chorea remains undetermined, although advances in genomics are revealing both new disease-associated genes and variant phenotypes associated with known genes. METHODS: We report a Saudi family with a neurodegenerative course dominated by progressive chorea and dementia in whom we performed homozygosity mapping and whole exome sequencing. RESULTS: We identified a homozygous missense mutation in GM2A within a prominent block of homozygosity. This mutation is predicted to impair protein function. DISCUSSION: Although discovered more than two decades ago, to date, only five patients with this rare form of GM2 gangliosidosis have been reported. The phenotype of previously described GM2A patients has been typified by onset in infancy, profound hypotonia and impaired volitional movement, intractable seizures, hyperacusis, and a macular cherry red spot. Our findings expand the phenotypic spectrum of GM2A mutation-positive gangliosidosis to include generalized chorea without macular findings or hyperacusis and highlight how mutations in neurodegenerative disease genes may present in unexpected ways.

A SUCLG1 mutation in a patient with mitochondrial DNA depletion and congenital anomalies.

Defects in two subunits of succinate-CoA ligase encoded by the genes SUCLG1 and SUCLA2 have been identified in mitochondrial DNA (mtDNA) depletion syndromes. Patients generally present with encephalomyopathy and mild methylmalonic acidemia (MMA), however mutations in SUCLG1 normally appear to result in a more severe clinical phenotype. In this report, we describe a patient with fatal infantile lactic acidosis and multiple congenital anomalies (MCAs) including renal and cardiac defects. Molecular studies showed a defective electron transport chain (ETC), mtDNA depletion, and a novel homozygous mutation in the SUCLG1 gene. Although our patient's clinical biochemical phenotype is consistent with a SUCLG1 mutation, it is unclear whether the MCAs observed in our patient are a result of the SUCLG1 mutation or alterations in a second gene. An increasing number of reports have described MCAs associated with mitochondrial disorders and SUCLG1 specifically. Additional studies such as whole exome sequencing will further define whether additional genes are responsible for the observed MCAs.

Orthostatic tremor, progressive external ophthalmoplegia, and Twinkle.

IMPORTANCE: Orthostatic tremor (OT) is a high-frequency (13-18 Hz) leg tremor occurring in standing position. Orthostatic tremor has an unknown pathophysiologic mechanism. It is thought to be sporadic but siblings with OT from 3 unrelated families were reported. No mutations have been reported in OT. We describe a patient with OT carrying a C10orf2 TWINKLE mutation to highlight the possible association of OT with mitochondrial dysfunction and mutations in the mitochondrial replicative helicase Twinkle. OBSERVATIONS: A man in his late 60s had ptosis and tremor on standing for 30 years, followed by development of progressive external ophthalmoplegia. Polygraphic recordings revealed an orthostatic synchronic tremor with 17.5-Hz frequency. Electromyography/nerve conduction studies showed evidence for a mild myopathy and associated mild axonal sensorimotor peripheral neuropathy. Muscle biopsy revealed ragged red fibers; mild cerebral atrophy was evident by magnetic resonance imaging. Molecular analysis revealed a novel heterozygous missense mutation at an evolutionarily conserved residue of the C10orf2 TWINKLE gene. CONCLUSIONS AND RELEVANCE: Although the incidental association of OT and C10orf2 TWINKLE mutation is possible, the simultaneous onset of OT and eyelid ptosis at a much younger age than usually observed for OT raises the possibility of mitochondrial dysfunction and loss of mitochondrial DNA integrity in the pathogenesis of OT.

Sequence analysis of the whole mitochondrial genome and nuclear genes causing mitochondrial disorders.

The diagnosis of mitochondrial disorders has increased considerably over the past few years. However, the genetics are complex, as the causative mutations can be in either the mitochondrial or the nuclear genome. Identification of the molecular defects in the causative genes is the key to a definitive diagnosis of the disease. Here, we describe PCR-based sequence analysis of the entire mitochondrial genome and a group of nuclear genes known to cause mitochondrial disorders.

Alternative splicing of sept9a and sept9b in zebrafish produces multiple mRNA transcripts expressed throughout development.

BACKGROUND: Septins are involved in a number of cellular processes including cytokinesis and organization of the cytoskeleton. Alterations in human septin-9 (SEPT9) levels have been linked to multiple cancers, whereas mutations in SEPT9 cause the episodic neuropathy, hereditary neuralgic amyotrophy (HNA). Despite its important function in human health, the in vivo role of SEPT9 is unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we utilize zebrafish to study the role of SEPT9 in early development. We show that zebrafish possess two genes, sept9a and sept9b that, like humans, express multiple transcripts. Knockdown or overexpression of sept9a transcripts results in specific developmental alterations including circulation defects and aberrant epidermal development. CONCLUSIONS/SIGNIFICANCE: Our work demonstrates that sept9 plays an important role in zebrafish development, and establishes zebrafish as a valuable model organism for the study of SEPT9.

Requirement of inositol 1,4,5-trisphosphate receptors for tumor-mediated lymphocyte apoptosis.

Tumor cells strategically down-regulate Fas receptor expression to evade immune attack and up-regulate expression of Fas ligand to promote apoptosis of infiltrating T lymphocytes. Many pathways leading to apoptotic cell death require calcium release from inositol 1,4,5-trisphosphate receptors (IP3Rs). Here, we show that Fas-dependent killing of Jurkat T lymphoma cells by SW620 colon cancer cells requires calcium release from IP3R. General suppression of IP3R signaling significantly reduced SW620-mediated Jurkat cell apoptosis. Significantly, a specific inhibitor of apoptotic calcium release from IP3R strongly blocked lymphocyte apoptosis. Thus, selective pharmacological targeting of apoptotic calcium release from IP3R may enhance tumor cell immunogenicity.

The UCS family of myosin chaperones.

The canonical UCS (UNC-45/Cro1/She4p) protein, Caenorhabditis elegans UNC-45, was one of the earliest molecules to be shown genetically to be necessary for sarcomere assembly. Genetic analyses of homologues in several fungal species indicate that the conserved UCS domain functionally interacts with conventional type II and unconventional type V myosins. In C. elegans and other invertebrate species, UNC-45 and its orthologues interact with both sarcomeric and non-sarcomeric myosins whereas, in vertebrates, there are two UNC-45 isoforms: a general cell (GC) and a striated muscle (SM) isoform. Although the mechanism of action of UCS proteins is unknown, recent biochemical studies suggest that they may act as molecular chaperones that facilitate the folding and/or maturation of myosin.