Wendy H. Raskind

A p53-dependent mouse spindle checkpoint

Cell cycle checkpoints enhance genetic fidelity by causing arrest at specific stages of the cell cycle when previous events have not been completed. The tumor suppressor p53 has been implicated in a G1 checkpoint. To investigate whether p53 also participates in a mitotic checkpoint, cultured fibroblasts from p53-deficient mouse embryos were exposed to spindle inhibitors. The fibroblasts underwent multiple rounds of DNA synthesis without completing chromosome segregation, thus forming tetraploid and octaploid cells. Deficiency of p53 was also associated with the development of tetraploidy in vivo. These results suggest that murine p53 is a component of a spindle checkpoint that ensures the maintenance of diploidy.

Clonal Development, Stem-Cell Differentiation, and Clinical Remissions in Acute Nonlymphocytic Leukemia

To determine whether acute nonlymphocytic leukemia develops clonally, to study the pattern of differentiation of the involved stem cells, and to determine whether clinical remissions are true remissions, we studied 27 patients with acute nonlymphocytic leukemia who were heterozygous for the X-chromosome-linked glucose-6-phosphate dehydrogenase. In each case, leukemic blast cells manifested only one type of glucose-6-phosphate dehydrogenase, indicating that the malignant process had developed from a single cell. In six elderly patients, circulating erythrocytes, platelets, or both expressed only the glucose-6-phosphate dehydrogenase found in blast cells, indicating that these leukemias had arisen from stem cells with multipotent differentiative expression. In 16 younger adults and children, erythroid cells and platelets were predominantly derived from normal stem cells. In three other cases, the stem cell that gave rise to leukemic blasts apparently also gave rise to erythroid progenitors but not to mature erythrocytes. Heterogeneity was also found during remissions. In 8 of 13 patients, restoration of nonclonal hemopoiesis and repopulation of the marrow by normal stem cells was observed during remission. In the other five patients, marrow stem cells remained partially or completely clonal, even during remission. These data indicate that acute nonlymphocytic leukemia is a heterogeneous disease with respect to differentiation of the stem cells involved by leukemia and the nature of remissions.

Missense Mutations in the Regulatory Domain of PKCγ: A New Mechanism for Dominant Nonepisodic Cerebellar Ataxia

We report a nonepisodic autosomal dominant (AD) spinocerebellar ataxia (SCA) not caused by a nucleotide repeat expansion that is, to our knowledge, the first such SCA. The AD SCAs currently comprise a group of > or =16 genetically distinct neurodegenerative conditions, all characterized by progressive incoordination of gait and limbs and by speech and eye-movement disturbances. Six of the nine SCAs for which the genes are known result from CAG expansions that encode polyglutamine tracts. Noncoding CAG, CTG, and ATTCT expansions are responsible for three other SCAs. Approximately 30% of families with SCA do not have linkage to the known loci. We recently mapped the locus for an AD SCA in a family (AT08) to chromosome 19q13.4-qter. A particularly compelling candidate gene, PRKCG, encodes protein kinase C gamma (PKC gamma), a member of a family of serine/threonine kinases. The entire coding region of PRKCG was sequenced in an affected member of family AT08 and in a group of 39 unrelated patients with ataxia not attributable to trinucleotide expansions. Three different nonconservative missense mutations in highly conserved residues in C1, the cysteine-rich region of the protein, were found in family AT08, another familial case, and a sporadic case. The mutations cosegregated with disease in both families. Structural modeling predicts that two of these amino acid substitutions would severely abrogate the zinc-binding or phorbol ester-binding capabilities of the protein. Immunohistochemical studies on cerebellar tissue from an affected member of family AT08 demonstrated reduced staining for both PKC gamma and ataxin 1 in Purkinje cells, whereas staining for calbindin was preserved. These results strongly support a new mechanism for neuronal cell dysfunction and death in hereditary ataxias and suggest that there may be a common pathway for PKC gamma-related and polyglutamine-related neurodegeneration.

Relative Burden of Large CNVs on a Range of Neurodevelopmental Phenotypes

While numerous studies have implicated copy number variants (CNVs) in a range of neurological phenotypes, the impact relative to disease severity has been difficult to ascertain due to small sample sizes, lack of phenotypic details, and heterogeneity in platforms used for discovery. Using a customized microarray enriched for genomic hotspots, we assayed for large CNVs among 1,227 individuals with various neurological deficits including dyslexia (376), sporadic autism (350), and intellectual disability (ID) (501), as well as 337 controls. We show that the frequency of large CNVs (>1 Mbp) is significantly greater for ID–associated phenotypes compared to autism (p = 9.58×10−11, odds ratio = 4.59), dyslexia (p = 3.81×10−18, odds ratio = 14.45), or controls (p = 2.75×10−17, odds ratio = 13.71). There is a striking difference in the frequency of rare CNVs (>50 kbp) in autism (10%, p = 2.4×10−6, odds ratio = 6) or ID (16%, p = 3.55×10−12, odds ratio = 10) compared to dyslexia (2%) with essentially no difference in large CNV burden among dyslexia patients compared to controls. Rare CNVs were more likely to arise de novo (64%) in ID when compared to autism (40%) or dyslexia (0%). We observed a significantly increased large CNV burden in individuals with ID and multiple congenital anomalies (MCA) compared to ID alone (p = 0.001, odds ratio = 2.54). Our data suggest that large CNV burden positively correlates with the severity of childhood disability: ID with MCA being most severely affected and dyslexics being indistinguishable from controls. When autism without ID was considered separately, the increase in CNV burden was modest compared to controls (p = 0.07, odds ratio = 2.33).

Temperature-sensitive mutations in the III–IV cytoplasmic loop region of the skeletal muscle sodium channel gene in paramyotonia congenita

Paramyotonia congenita (PMC), a dominant disorder featuring cold-induced myotonia (muscle stiffness), has recently been genetically linked to a candidate gene, the skeletal muscle sodium channel gene SCN4A. We have now established that SCN4A is the disease gene in PMC by identifying two different single-base coding sequence alterations in PMC families. Both mutations affect highly conserved residues in the III-IV cytoplasmic loop, a portion of the sodium channel thought to pivot in response to membrane depolarization, thereby blocking and inactivating the channel. Abnormal function of this cytoplasmic loop therefore appears to produce the Na+ current abnormality and the unique temperature-sensitive clinical phenotype in this disorder.

Writing problems in developmental dyslexia ; Under-recognized and under-treated

The International Dyslexia Association defines dyslexia as unexpected problems of neurobiological origin in accuracy and rate of oral reading of single real words, single pseudowords, or text or of written spelling. However, prior research has focused more on the reading than the spelling problems of students with dyslexia. A test battery was administered to 122 children who met inclusion criteria for dyslexia and qualified their families for participation in a family genetics study that has been ongoing for over a decade. Their parents completed the same test battery. Although a past structural equation modeling study of typically developing children identified a significant path from handwriting to composition quality, the current structural equation modeling study identified a significant path from spelling to composition for children and their parents with dyslexia. Grapho-motor planning did not contribute uniquely to their composition, showing that writing is not just a motor skill. Students with dyslexia do have a problem in automatic letter writing and naming, which was related to impaired inhibition and verbal fluency, and may explain their spelling problems. Results are discussed in reference to the importance of providing explicit instruction in the phonological, orthographic, and morphological processes of spelling and in composition to students with dyslexia and not only offering accommodation for their writing problems.

Hereditary spastic paraplegia: Advances in genetic research

Hereditary spastic paraplegia (HSP) is a diverse group of inherited disorders characterized by progressive lower-extremity spasticity and weakness. Insight into the genetic basis of these disorders is expanding rapidly. Uncomplicated autosomal dominant, autosomal recessive, and X-linked HSP are genetically heterogeneous: different genes cause clinically indistinguishable disorders. A locus for autosomal recessive HSP is on chromosome 8q. Loci for autosomal dominant HSP have been identified on chromosomes 2p, 14q, and 15q. One locus (Xq22) has been identified for X-linked, uncomplicated HSP and shown to be due to a proteolipoprotein gene mutation in one family. The existence of HSP families for whom these loci are excluded indicates the existence of additional, as yet unidentified HSP loci. There is marked clinical similarity among HSP families linked to each of these loci, suggesting that gene products from HSP loci may participate in a common biochemical cascade, which, if disturbed, results in axonal degeneration that is maximal at the ends of the longest CNS axons. Identifying the single gene defects that cause HSPs distal axonopathy may provide insight into factors responsible for development and maintenance of axonal integrity. We review clinical, genetic, and pathologic features of HSP and present differential diagnosis and diagnostic criteria of this important group of disorders. We discuss polymorphic microsatellite markers useful for genetic linkage analysis and genetic counseling in HSP. NEUROLOGY 1996;46: 1507-1514

Mutations in the EXT1 and EXT2 Genes in Hereditary Multiple Exostoses

Hereditary multiple exostoses (EXT; MIM 133700) is an autosomal dominant bone disorder characterized by the presence of multiple benign cartilage-capped tumors (exostoses). Besides suffering complications caused by the pressure of these exostoses on the surrounding tissues, EXT patients are at an increased risk for malignant chondrosarcoma, which may develop from an exostosis. EXT is genetically heterogeneous, and three loci have been identified so far: EXT1, on chromosome 8q23-q24; EXT2, on 11p11-p12; and EXT3, on the short arm of chromosome 19. The EXT1 and EXT2 genes were cloned recently, and they were shown to be homologous. We have now analyzed the EXT1 and EXT2 genes, in 26 EXT families originating from nine countries, to identify the underlying disease-causing mutation. Of the 26 families, 10 families had an EXT1 mutation, and 10 had an EXT2 mutation. Twelve of these mutations have never been described before. In addition, we have reviewed all EXT1 and EXT2 mutations reported so far, to determine the nature, frequency, and distribution of mutations that cause EXT. From this analysis, we conclude that mutations in either the EXT1 or the EXT2 gene are responsible for the majority of EXT cases. Most of the mutations in EXT1 and EXT2 cause premature termination of the EXT proteins, whereas missense mutations are rare. The development is thus mainly due to loss of function of the EXT genes, consistent with the hypothesis that the EXT genes have a tumor- suppressor function.

Actionable exomic incidental findings in 6503 participants: challenges of variant classification

Recommendations for laboratories to report incidental findings from genomic tests have stimulated interest in such results. In order to investigate the criteria and processes for assigning the pathogenicity of specific variants and to estimate the frequency of such incidental findings in patients of European and African ancestry, we classified potentially actionable pathogenic single-nucleotide variants (SNVs) in all 4300 European- and 2203 African-ancestry participants sequenced by the NHLBI Exome Sequencing Project (ESP). We considered 112 gene-disease pairs selected by an expert panel as associated with medically actionable genetic disorders that may be undiagnosed in adults. The resulting classifications were compared to classifications from other clinical and research genetic testing laboratories, as well as with in silico pathogenicity scores. Among European-ancestry participants, 30 of 4300 (0.7%) had a pathogenic SNV and six (0.1%) had a disruptive variant that was expected to be pathogenic, whereas 52 (1.2%) had likely pathogenic SNVs. For African-ancestry participants, six of 2203 (0.3%) had a pathogenic SNV and six (0.3%) had an expected pathogenic disruptive variant, whereas 13 (0.6%) had likely pathogenic SNVs. Genomic Evolutionary Rate Profiling mammalian conservation score and the Combined Annotation Dependent Depletion summary score of conservation, substitution, regulation, and other evidence were compared across pathogenicity assignments and appear to have utility in variant classification. This work provides a refined estimate of the burden of adult onset, medically actionable incidental findings expected from exome sequencing, highlights challenges in variant classification, and demonstrates the need for a better curated variant interpretation knowledge base.

Expression of the gene defect in X-linked agammaglobulinemia.

Although X-linked agammaglobulinemia was one of the first immunodeficiencies described,1 the genetic defect responsible for this disorder has not yet been identified. X-linked agammaglobulinemia is...