Association of rare missense variants in the second intracellular loop of NaV1.7 sodium channels with familial autism.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder often accompanied by intellectual disability, language impairment and medical co-morbidities. The heritability of autism is high and multiple genes have been implicated as causal. However, most of these genes have been identified in de novo cases. To further the understanding of familial autism, we performed whole-exome sequencing on five families in which second- and third-degree relatives were affected. By focusing on novel and protein-altering variants, we identified a small set of candidate genes. Among these, a novel private missense C1143F variant in the second intracellular loop of the voltage-gated sodium channel NaV1.7, encoded by the SCN9A gene, was identified in one family. Through electrophysiological analysis, we show that NaV1.7C1143F exhibits partial loss-of-function effects, resulting in slower recovery from inactivation and decreased excitability in cultured cortical neurons. Furthermore, for the same intracellular loop of NaV1.7, we found an excess of rare variants in a case-control variant-burden study. Functional analysis of one of these variants, M932L/V991L, also demonstrated reduced firing in cortical neurons. However, although this variant is rare in Caucasians, it is frequent in Latino population, suggesting that genetic background can alter its effects on phenotype. Although the involvement of the SCN1A and SCN2A genes encoding NaV1.1 and NaV1.2 channels in de novo ASD has previously been demonstrated, our study indicates the involvement of inherited SCN9A variants and partial loss-of-function of NaV1.7 channels in the etiology of rare familial ASD.

Replication of a rare risk haplotype on 1p36.33 for autism spectrum disorder.

Hundreds of genes have been implicated in autism spectrum disorders (ASDs). In genetically heterogeneous conditions, large families with multiple affected individuals provide strong evidence implicating a rare variant, and replication of the same variant in multiple families is unusual. We previously published linkage analyses and follow-up exome sequencing in seven large families with ASDs, implicating 14 rare exome variants. These included rs200195897, which was transmitted to four affected individuals in one family. We attempted replication of those variants in the MSSNG database. MSSNG is a unique resource for replication of ASD risk loci, containing whole genome sequence (WGS) on thousands of individuals diagnosed with ASDs and family members. For each exome variant, we obtained all carriers and their relatives in MSSNG, using a TDT test to quantify evidence for transmission and association. We replicated the transmission of rs200195897 to four affected individuals in three additional families. rs200195897 was also present in three singleton affected individuals, and no unaffected individuals other than transmitting parents. We identified two additional rare variants (rs566472488 and rs185038034) transmitted with rs200195897 on 1p36.33. Sanger sequencing confirmed the presence of these variants in the original family segregating rs200195897. To our knowledge, this is the first example of a rare haplotype being transmitted with ASD in multiple families. The candidate risk variants include a missense mutation in SAMD11, an intronic variant in NOC2L, and a regulatory region variant close to both genes. NOC2L is a transcription repressor, and several genes involved in transcription regulation have been previously associated with ASDs.

Mutations in the TSGA14 gene in families with autism spectrum disorders.

Linkage to 7q has been the most robust genetic finding in familial autism. A previous scan of multiplex families with autism spectrum disorders found a linkage signal of genome-wide significance at D7S530 on 7q32. We searched a candidate imprinted region at this location for genetic variants in families with positive linkage scores. Using exon resequencing, we identified three rare potentially pathogenic variants in the TSGA14 gene, which encodes a centrosomal protein. Two variants were missense mutations (c.664C>G; p.P206A and c.766T>G; p.C240G) that changed conserved residues in the same protein domain; the third variant (c.192+5G>A) altered splicing, which resulted in a protein with an internal deletion of 16 residues and a G33D substitution. These rare TSGA14 variants are enriched in the affected subjects (6/348 patients versus 2/670 controls, Fisher's exact two tailed P = 0.022). This is the first report of a possible link of a gene with a centrosomal function with familial autism.

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.

Functional characterization of AC5 gain-of-function variants: Impact on the molecular basis of ADCY5-related dyskinesia.

Adenylyl cyclases are key points for the integration of stimulatory and inhibitory G protein-coupled receptor (GPCR) signals. Adenylyl cyclase type 5 (AC5) is highly expressed in striatal medium spiny neurons (MSNs), and is known to play an important role in mediating striatal dopaminergic signaling. Dopaminergic signaling from the D1 expressing MSNs of the direct pathway, as well as the D2 expressing MSNs of the indirect pathway both function through the regulation of AC5 activity, controlling the production of the 2nd messenger cAMP, and subsequently the downstream effectors. Here, we used a newly developed cell line that used Crispr-Cas9 to eliminate the predominant adenylyl cyclase isoforms to more accurately characterize a series of AC5 gain-of-function mutations which have been identified in ADCY5-related dyskinesias. Our results demonstrate that these AC5 mutants exhibit enhanced activity to Gαs-mediated stimulation in both cell and membrane-based assays. We further show that the increased cAMP response at the membrane effectively translates into increased downstream gene transcription in a neuronal model. Subsequent analysis of inhibitory pathways show that the AC5 mutants exhibit significantly reduced inhibition following D2 dopamine receptor activation. Finally, we demonstrate that an adenylyl cyclase "P-site" inhibitor, SQ22536 may represent an effective future therapeutic mechanism by preferentially inhibiting the overactive AC5 gain-of-function mutants.

Evidence for the involvement of B lymphoid cells in polycythemia vera and essential thrombocythemia.

Previous studies with the X-chromosome-linked glucose-6-phosphate dehydrogenase (G6PD) as a marker of cellular mosaicism demonstrated that polycythemia vera (PV) and essential thrombocythemia (ET) are clonal disorders of hematopoietic stem cells that can differentiate to erythrocytes, granulocytes, and platelets. To determine if the involved stem cells could also differentiate along the B-lymphoid pathway, we studied one woman with PV and one woman with ET. Of 117 Epstein-Barr virus-transformed B-lymphoblastoid lines expressing a single G6PD derived from the patient with PV, 108 expressed G6PD type A, the type characteristic of the abnormal clone. The ratio of 108:9 was significantly different from the one to one ratio predicted for this patient, which suggested that at least some circulating progenitors for B-lymphoid cell lines differentiate from the stem cell involved by the disease. Results obtained from the patient with ET were similar--104 of the 109 lymphoblastoid lines monotypic for G6PD expression displayed the enzyme type found in the abnormal clone of marrow cells. Therefore, in these patients, PV and ET, like chronic myelogenous leukemia, involve a stem cell pluripotent for the lymphoid as well as the myeloid series.

Pancytopenia as a clonal disorder of a multipotent hematopoietic stem cell.

Hematopoiesis was investigated in a 14-yr-old girl who had a 2-yr history of stable asymptomatic pancytopenia and who was also heterozygous at the structural locus for glucose-6-phosphate dehydrogenase (G-6-PD). There was no morphologic or cytogenetic evidence for preleukemia and no suggestion of Fanconi anemia. In the skin and sheep erythrocytes-rosetted T lymphocytes, the ratio of G-6-PD A/B activities was 1:1. However, only type B activity was found in peripheral blood erythrocytes, granulocytes, and platelets. Most erythroid bursts and all granulocyte/macrophage colonies formed in methylcellulose culture were derived from the abnormal clone. These findings demonstrate that (a) some cases of pancytopenia are stem cell diseases that apparently develop clonally; (b) circulating differentiated cells originate from this clone; (c) despite a hypoproliferative anemia, the in vivo expression of presumably normal (nonclonal) progenitors is suppressed. In this patient, the relationship between clonal dominance and possible malignancy may be assessed prospectively.

Clonal analysis of childhood acute lymphoblastic leukemia with "cytogenetically independent" cell populations.

Acute lymphoblastic leukemia (ALL) is generally regarded as a clonal disease in which a single abnormal progenitor cell gives rise to neoplastic progeny. Five of 463 cases of childhood ALL with adequately banded leukemic cells were found to have two cytogenetically independent cell populations. In addition, two of the four cases tested had more than two rearranged immunoglobulin genes and (or) T cell receptor genes. To investigate the clonality of these unusual leukemias, we examined the neoplastic cells for X-linked markers extrinsic to the disease. Leukemic cells from each of the three patients heterozygous for an X-linked, restriction fragment length polymorphism showed a single active parental allele, suggesting that both apparently independent cell populations developed from a common progenitor. These cases provide evidence that leukemogenesis involves a multistep process of mutation and suggest that karyotypic abnormalities may be a late event of malignant transformation.

Persistent clonal areas and clonal expansion in Barrett's esophagus.

Three patients with Barrett's esophagus who had cytogenetic abnormalities detected in their metaplastic epithelium developed high-grade dysplasia or adenocarcinoma during prospective surveillance over a period of 1.5 to 6 years. In the 3 cases, cytogenetic abnormalities that were associated with the most advanced histological lesions were present in samples obtained 11, 25, and 48 months prior to the diagnosis of high-grade dysplasia or carcinoma. In a fourth patient, marker chromosomes found in a Barrett's adenocarcinoma were also present in an esophageal region spatially removed from the tumor. In all four patients, clonal cytogenetic abnormalities were present in samples obtained at widespread locations in the Barrett's segment. These observations suggest that in some patients with Barrett's esophagus clonal proliferations arise in regions of benign histology and spread to involve large areas of Barrett's mucosa. These clones persisted when the disease progressed to high-grade dysplasia or adenocarcinoma.

17p allelic deletions and p53 protein overexpression in Barrett's adenocarcinoma.

Barrett's esophagus is a condition in which the stratified squamous epithelium of the esophagus is replaced by metaplastic columnar epithelium that predisposes to the development of esophageal adenocarcinoma. Allelic deletions of 17p and alterations of p53 including elevated p53 protein levels have been observed in many different tumors. To investigate the presence of 17p allelic deletions and p53 protein overexpression in Barrett's adenocarcinomas, we have combined the use of restriction fragment length polymorphism analysis, multiparameter flow cytometry, and DNA content cell sorting. The combined use of these methodologies permits the purification of aneuploid tumor cells for restriction fragment length polymorphism analysis of 17p allelic deletions and the evaluation of p53 protein expression by multiparameter flow cytometry in the same aneuploid tumor cell populations. We analyzed 15 aneuploid populations and one tetraploid populations from 13 Barrett's adenocarcinomas for 17p allelic deletions and p53 protein overexpression to determine whether both of these alterations are involved in carcinogenesis in Barrett's esophagus. Twelve of 13 tumors (92%) had 17p allelic deletions, and 8 of 13 tumors (62%) had p53 protein overexpression. Eight of the 12 tumors (67%) with 17p allelic deletions also had p53 protein overexpression. These data indicate that both 17p allelic deletions and p53 protein overexpression are frequently involved in carcinogenesis in Barrett's esophagus.

Frequent loss of heterozygosity at the retinoblastoma locus in human esophageal cancers.

Abnormalities in the retinoblastoma tumor suppressor gene (Rb) have been observed in a large number of human cancers. Loss of heterozygosity is a common mode of allelic inactivation of Rb and other tumor suppressor genes. We investigated DNA from 61 primary human esophageal tumors for loss of heterozygosity at the Rb locus using a polymerase chain reaction-based restriction fragment length polymorphism assay. Of informative cases, we found loss of heterozygosity in 14 of 26 (54%) squamous cell carcinomas and 5 of 14 (36%) adenocarcinomas. These data support the hypothesis that Rb inactivation is involved in the pathogenesis and/or progression of esophageal cancer.

Clonal development of myeloproliferative disorders: clues to hematopoietic differentiation and multistep pathogenesis of cancer.

In November 1996, word reached the University of Washington that Philip Fialkow and his wife, Helen, had died while trekking in Nepal. Over a 30-year period, Dr Fialkow and his colleagues used the cellular mosaicism resulting from X-chromosome inactivation in females as a marker system to investigate the clonal development of human hematopoietic disorders. This review discusses the impact that these studies have had on our understanding of hematopoietic stem cell relationships and the pathogenesis of human neoplasia in general. To appreciate the special role played by studies on clonality, it is necessary to consider how little was known about the origin of leukemias and myeloproliferative disorders and the limited techniques available for their study in the early to mid 1960s. Dr Fialkow and his coworkers were the first to show that myeloproliferative disorders and acute myelogenous leukemias (AML) are clonal diseases at the time of diagnosis and to elucidate the level of differentiation manifested by the originating cell type. Although the myelodysplastic disorders were found to involve a pluripotent stem cell, heterogeneity was found in the level of stem cell involvement in AML. Evidence was obtained to support a multistep pathogenesis of these diseases as well as a clonal but cytogenetically normal stage in some cases of Ph-positive chronic myelogenous leukemia, AML, acute lymphoblastic leukemia and myelodysplasia.

The effect of long-term marrow culture on the origin of colony-forming cells in acute myeloblastic leukemia: studies of two patients heterozygous for glucose-6-phosphate dehydrogenase.

Long-term marrow cultures (LTMCs) provide a selective growth advantage for cytogenetically normal cells in patients with acute and chronic myeloid leukemias. In the present study, LTMCs were established from two patients with newly diagnosed acute myeloid leukemia (AML) who were heterozygous for the X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD). Initially only leukemic clusters grew from cells plated in semisolid medium, but after 1 or more weeks in LTMC, morphologically normal granulocyte-macrophage colonies were detected. Nonetheless, in one of the patients, more than 80% of these colonies expressed the G6PD type observed in the leukemic blast cells, indicating a probable neoplastic derivation for many of them. In the second patient, colonies cultured during the first 3 weeks of the LTMC were predominantly derived from clonal progenitors, whereas after week 4 the colonies were derived from normal stem cells. Colonies derived from clonal or normal stem cells were not morphologically distinguishable. These data support the conclusion that LTMC has a selective anti-leukemic effect on marrow cells from some patients. However, normalization of colony growth is by itself not a sufficient criterion for determination of whether committed progenitor cells from patients with AML are derived from normal or leukemic stem cells.

Further evidence for the existence of a clonal Ph-negative stage in some cases of Ph-positive chronic myelocytic leukemia.

The Ph chromosome abnormality is involved in the pathogenesis of almost all patients with chronic myelocytic leukemia (CML). Previous studies on the B-lymphoid cell lineage in two patients with Ph-positive CML suggest that there may also be a clonal Ph-negative stage in CML and that the Ph-positive stage arises by subclonal expansion. To determine whether this is a frequent or a rare occurrence, 14 additional glucose-6-phosphate dehydrogenase (G6PD)-heterozygous patients with CML were studied. In five of these patients there was a statistically significant excess of Ph-negative B-lymphoid cell lines expressing the same G6PD type expressed in the corresponding CML clone. In no case was an excess of B-lymphoid lines expressing the opposite G6PD type recovered. These data provide further evidence that in some patients the Ph chromosome arises in a pluripotent stem cell from a pre-existing Ph-negative clone that enjoys a growth advantage.

Clonal remission in childhood acute myeloid leukemia is an infrequent event.

Acute myeloid leukemia (AML) is a heterogeneous group of diseases that differ in pattern of both remission and lineage involvement. The observation that hematopoiesis remains clonal in some patients with AML in complete clinical remission suggests that the acute phase may develop from a clinically unrecognized preleukemic clone. To investigate the characteristics and significance of clonal remissions in childhood AML, we used X-chromosome-linked polymorphisms to study granulocytes obtained from pediatric female patients in complete clinical remission. Remission granulocytes from only one of 17 evaluable patients were clonally derived, suggesting that clonal remission is an infrequent event in childhood AML.

Host origin of marrow stromal cells obtained from marrow transplant recipients and transformed in vitro by simian virus-40.

We evaluated the karyotypes of marrow-derived stromal cell lines established by simian virus-40 (SV-40) transformation of long-term cultures from four men who received marrow transplants from women donors. In all cases a normal female karyotype was found in marrow cells. In contrast, a Y chromosome was identified in metaphase cells from the stromal lines, suggesting that following successful marrow transplantation, cells in the marrow microenvironment susceptible to SV-40 transformation remain host in origin.

Polysymptomatic complaints and Briquet's syndrome in polycystic ovary disease.

To test the hypothesis that there is an association between polycystic ovary disease and Briquet's syndrome, the authors administered a health questionnaire to infertile women with polycystic ovary disease, infertile women with tubal disease, and normal women. The patients with polycystic ovary disease endorsed significantly more physical and psychological complaints than either control group. Structured interviews revealed that five of the 39 (13%) met diagnostic criteria for definite or probable Briquet's syndrome. This study gives support to an association between polysymptomatic complaints, Briquet's syndrome, and polycystic ovary disease.

Familial spastic paraparesis: evaluation of locus heterogeneity, anticipation, and haplotype mapping of the SPG4 locus on the short arm of chromosome 2.

Familial spastic paraparesis (SPG) is a clinically and genetically heterogeneous group of disorders. At least three loci have been implicated in autosomal dominant pure SPG and mutations in either of two loci may cause the X-linked form. Although the penetrance is high for all forms by age 60, there is wide variation in clinical characteristics, including age of onset. Two-point and multi-point linkage analyses in nine families provided supportive evidence that the most common form of SPG is linked to chromosome 2 (SPG4). Haplotype analysis localized the critical region to a 6 cM interval between D2S392 and D2S367. By haplotype analysis, the disease in at least one family does not appear to be linked to any of the presently known SPG loci, suggesting that there is at least one additional SPG gene. Evaluation at ages of onset in 11 families gave suggestive evidence for anticipation with mean age of onset in parents (41.3 years) being older than mean age of onset in children (26.9 years; P < 0.005).

Correlation between cytogenetic and molecular findings in human chronic myelogenous leukemia lines EM-2 and EM-3.

Few established cell lines derived from patients with chronic myelogenous leukemia have been reported. Cytogenetic examinations of two independently derived Philadelphia chromosome (Ph)-positive cell lines from a patient with chronic myelogenous leukemia were performed serially from their initiation in 1982 to the present. Subcultures of each of these lines maintained separately in two laboratories for over 2 years were compared for degree of divergence. The modal chromosome number declined substantially within the first few months and slowly thereafter. Despite hyperploidy, these lines have remained remarkably stable cytogenetically. For each line, the modal chromosome number is hypotetraploid, multiple copies of Ph are present and no normal chromosome #9 remains. Only a few marker chromosomes have arisen. Despite the multiple copies of Ph, a single bcr restriction pattern was seen, suggesting duplication of a single Ph, rather than independent translocation events. These lines should be very useful for in vitro studies of chronic myelogenous leukemia.

Inactivation of p53 and the development of tetraploidy in the elastase-SV40 T antigen transgenic mouse pancreas.

Human pancreatic cancer develops in association with an acquired genomic instability, but the events that lead to instability are difficult to investigate because they occur sporadically and unpredictably. The elastase-SV40 T antigen transgenic mouse model of pancreatic adenocarcinoma reproducibly proceeds through a diploid --> tetraploid --> multiple aneuploid sequence of genetic abnormalities. We investigated the relationship between inactivation of p53 and development of tetraploidy in this model. Because T antigen inactivates p53 by forming a stable complex with it, we used multiparameter flow cytometry to assess p53 expression in pancreatic samples of transgenic and control mice between 8 and 24 days of age. On day 18, a cell cycle-specific inactivation of p53 developed between diploid G, and S phase and was associated with the appearance of a cycling tetraploid cell population that had p53 protein overexpression in both G1- and S-phase cells. Cytogenetic analysis of pancreatic samples confirmed the development of a tetraploid cell population. Inactivation of p53 in diploid cells of the transgenic pancreas is followed by the development of a tetraploid cell population. We have shown previously that this tetraploid intermediate is predisposed to progression to aneuploidy because it has abnormal mitotic poles. Therefore, our results suggest that inactivation of p53 by T antigen leads to formation of a tetraploid cell intermediate that is predisposed to chromosome segregation abnormalities and the development of multiple aneuploid cell populations.