Characterization of mutations in the gene doublecortin in patients with double cortex syndrome.

Mutations in the X-linked gene doublecortin, which encodes a protein with no dear structural homologues, are found in pedigrees in which affected females show "double cortex" syndrome (DC; also known as subcortical band heterotopia or laminar heterotopia) and affected males show X-linked lissencephaly. Mutations in doublecortin also cause sporadic DC in females. To determine the incidence of doublecortin mutations in DC, we investigated a cohort of eight pedigrees and 47 sporadic patients with DC for mutations in the doublecortin open reading frame as assessed by single-stranded conformational polymorphism analysis. Mutations were identified in each of the eight DC pedigrees (100%), and in 18 of the 47 sporadic DC patients (38%). Identified mutations were of two types, protein truncation mutations and single amino acid substitution mutations. However, pedigrees with DC displayed almost exclusively single amino acid substitution mutations, suggesting that patients with these mutations may have less of a reproductive disadvantage versus those patients with protein truncation mutations. Single amino acid substitution mutations were tightly clustered in two regions of the open reading frame, suggesting that these two regions are critical for the function of the Doublecortin protein.

Prenatal exposure to disulfiram implicated in the cause of malformations in discordant monozygotic twins.

Female monozygotic (MZ) twins were discordant for congenital structural anomalies: Twin A had a reduction defect of the right forearm; Twin B had a cleft palate. Both infants were small for gestational age. Specific prenatal exposures were identified at different times in the first trimester of pregnancy: crack cocaine, marijuana, disulfiram, heavy ethanol exposure, and cigarettes. The mother's hospitalization in a drug abuse program and incarceration allowed for identification of exposure timing. The cleft palate could have been related to either disulfiram or alcohol exposure; the limb abnormality most likely corresponded to the timing of disulfiram exposure. Discordance of anomalies in these twins may reflect differences in developmental timing, differences in susceptibility to one or more teratogens, or random events occurring within very complex developmental programs, with the thresholds for malformation affected by one or multiple teratogenic compounds.

A comparative study of prenatal diagnosis and perinatal autopsy.

The objective of this study was to assess the accuracy of prenatal ultrasonography and to identify patterns of fetal malformations. All fetal and neonatal autopsies over a 3 year period were compared to prenatal sonographic findings and comparisons were made between the results of the two examinations. We identified 133 fetuses and neonates who had both a complete autopsy and a perinatal autopsy. Approximately 87% of autopsy-demonstrated major abnormalities had been detected by prenatal ultrasonography, with 61% of all malformations detected. Some limitations in accuracy of prenatal diagnosis are unavoidable, but strict attention to a thorough fetal examination should improve accuracy. Autopsy examination remains an important component of the evaluation of perinatal losses, especially if dysmorphology is known or suspected.

Breast and other cancers in families with ataxia-telangiectasia.

Patients who are homozygous for ataxia-telangiectasia have an exceptionally high incidence of cancer. In a group of families expected to have a high proportion of heterozygotes for ataxia-telangiectasia, we tested the hypothesis that such heterozygotes, estimated to make up 0.68 to 7.7 percent of the U.S. white population, also have an excess cancer risk. Retrospective cancer incidence rates in adult blood relatives of patients with ataxia-telangiectasia in 110 white non-Amish families were significantly elevated over the incidence rates in spouse controls (rate ratios, 1.6 for men [P = 0.032]; 2.0 for women [P = 0.013]). For persons who are heterozygous for ataxia-telangiectasia, the relative risk of cancer was estimated to be 2.3 for men (P = 0.014) and 3.1 for women (P = 0.004). Breast cancer in women was the cancer most clearly associated with heterozygosity for ataxia-telangiectasia (rate ratio, 3.0 [P = 0.028]; heterozygote relative risk, 6.8 [P = 0.006]). On the basis of this estimated relative risk of 6.8 and an estimated heterozygote frequency in the general population of 1.4 percent, 8.8 percent of patients with breast cancer in the U.S. white population would be heterozygous for ataxia-telangiectasia. We conclude that heterozygous carriers of the gene for ataxia-telangiectasia have an excess risk of cancer, particularly breast cancer in women.

The proliferative immune response to autologous Epstein-Barr virus transformed lymphoblastoid cells. II. Studies with HLA class II loss variants demonstrate a role for gene products other than DR and DQ.

The Epstein-Barr virus (EBV) transformed lymphoblastoid cell line (LCL-721) and some of its HLA loss mutant derivatives were used to study the immune specificity of the autologous proliferative T cell response to antigens expressed as a result of EBV infection. We have measured secondary and tertiary proliferative responses to well-characterized variants that lack expression of some or all known class II gene products (DR, DQ, and DP). These experiments prove that the region mapping between DR/DQ and glyoxalase I (GLO) of one haplotype controls at least one specific restriction element which is recognized in the autologous response to LCL-721. Furthermore, specific proliferative responses to variants lacking expression of all known class II gene products indicate the recognition of determinants other than DR, DQ, and DP.

Presentation of Candida albicans and purified protein derivative soluble antigens by Epstein-Barr virus-transformed human lymphoblastoid B-cell lines.

Cells other than the macrophage can function as antigen-presenting cells (APCs). These class II-bearing accessory cells include dendritic cells, epidermal Langerhans cells, B cells, murine B-cell tumors, and human Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV-LCL). We investigated the ability of EBV-LCL to present two soluble antigens, Candida albicans and purified protein derivative of tuberculin (PPD). The EBV-LCL derived from B cells of two different individuals can present both antigens to bulk cultures of autologous antigen-primed peripheral blood lymphocytes. The responses of PPD-reactive T-cell clones were weaker to PPD when presented by EBV-LCL than by PBL-APCs, with some clones responding only to PPD presented by PBL-APCs. This suggests that EBV-LCL are not equivalent to PBL monocytes in APC function, and that expression of class II major histocompatibility complex antigen is not sufficient in enabling antigen-presenting capability.

A human T cell clone that mediates the monocyte procoagulant response to specific sensitizing antigen.

A panel of human purified protein derivative of the tubercle bacillus (PPD)-reactive T cell clones was derived by cloning out of soft agar followed by cultivation on inactivated feeder cells in the presence of interleukin-2. 1 of 4 clones tested was able to mediate an increase in monocyte procoagulant activity (PCA) in response to PPD. All four clones had identical surface marker phenotypes (T4+, T8-) and proliferated in response to antigen. The reactive T cell clone possessed no PCA of its own, but upon being presented with PPD was able to instruct monocytes to increase their expression of PCA. Antigen presentation could be performed only by autologous monocytes; allogeneic monocytes from donors unrelated to the donor of the reactive clone could not present antigen to cells of the clone in a way that would initiate the procoagulant response. Cells of the reactive clone did not mediate increased monocyte PCA in response to Candida, even though peripheral blood mononuclear cells from the donor demonstrated increased PCA to both Candida and PPD. Thus, the PCA response to specific antigen can be mediated by a single clone of cells that shows specificity in the recognition of both antigen and antigen presenting cell.

The proliferative immune response to autologous Epstein-Barr virus-transformed lymphoblastoid cells. I. Studies with HLA haplotype loss variants demonstrate a role for MHC-linked genes.

The human Epstein-Barr virus transformed lymphoblastoid cell line (EBV-LCL) 721 and MHC haplotype loss variants derived from it were utilized to dissect the functional role of MHC genes in the proliferative response of autologous T lymphocytes to EBV-LCL. LCL-721 is heterozygous at all phenotypically defined MHC loci. One type of LCL-721 variant expresses only determinants encoded by the maternal (m) haplotype and the other expresses determinants encoded by the paternal (p) haplotype. Autologous (individual A) primary proliferative responses are strong to each type of haplotype deletant. The strong tertiary responses to the priming haplotype in comparison to the relatively weak responses to the reciprocal haplotype indicate that MHC linked genes encoded by each haplotype are important in the autologous response to EBV-LCL. Similar specific tertiary responses are observed when peripheral blood lymphocytes (PBLs) from the donor's mother are used as responding cells. Allogeneic responses were also studied by priming PBLs from unrelated donors with the haplotype deletants. Quantitative comparisons of the proliferation by primed allogeneic and autologous lymphocytes stimulated by irradiated PBLs from donor A and her mother, and by LCL-721 and its variants, show that some of the tertiary responses involve specific recognition of EBV-LCL while others detect recognition of alloantigens.

Homozygous deletions that simultaneously eliminate expressions of class I and class II antigens of EBV-transformed B-lymphoblastoid cells. I. Reduced proliferative responses of autologous and allogeneic T cells to mutant cells that have decreased expression of class II antigens.

Mutant human B-lymphoblastoid cell lines, 721.174 and 721.180, that have greatly reduced expressions of known class I and class II HLA antigens were produced by two cycles of gamma-ray mutagenesis followed by selection for HLA antigen loss. Residual binding of monoclonal antibodies directed against class II antigens was negligible except for 10% residual binding of SB-binding antibody ILR1. However, deletion of SB was functionally complete as indicated by failure of the mutants to stimulate proliferation of SB-primed lymphocytes. Residual binding of monoclonal antibodies directed against class I "framework" determinants was reduced by 90-95%. However, the binding of monoclonal antibodies directed against beta 2-microglobulin and against the A2 epitope recognized by monoclonal antibody BB 7.2 was about 20% of normal. The identities of the residual class I-like antigens are unknown. The mutants retained full expression of the EBV-induced EBVCS antigen. Mutants 721.174 and 721.180 have lost most, but definitely not all, of their capacity to stimulate primary allogeneic and autologous lymphoproliferative responses. Therefore, the mutants still express antigens other than those that are presently known to stimulate lymphocyte proliferation. By means of studies of the present sort, and in future studies on expression of transferred HLA genes, mutants 721.174 and 721.180 promise to be useful in molecular and functional analysis of MHC-region-encoded gene products and their genetic regulation.

Proliferative responses to a nonspecific factor produced by irradiated stimulating cells can simulate antigen-specific secondary responses in the primed lymphocyte test.

The primed lymphocyte test (PLT) was utilized to investigate the biologic relationship between alloantigens and soluble environmental antigens. Human lymphocytes primed in vitro to the soluble antigens Candida (CAN), Tetanus (TET), or Purified Protein Derivative of Tuberculin (PPD) gave a strong proliferative response when restimulated with the initial soluble antigen in the presence of irradiated peripheral blood lymphocytes (PBL) acting as antigen-presenting cells (APC). Surprisingly, the soluble antigen-primed cells as well as alloantigen-primed cells responded to other soluble antigens in the presence of APC. By testing primed cells with supernatants derived from irradiated lymphocytes plus soluble antigen, it was apparent that the responses observed were in part due to stimulation by a soluble factor produced by the irradiated "APC" in response to the soluble antigen rather than to recognition of widespread cross-reactivity by the primed cells. This "nonspecific" factor production could be diminished by increasing the dose of irradiation to the APC or by using a T lymphocyte depleted- (SRBC-E-) APC population. In addition, certain antigen-reactive T cell clones did not respond to the nonspecific factor to the same degree as the primed bulk cultures. Nevertheless, the recognition of nonspecific stimulation induced by factors produced by irradiated lymphocytes is critical in the interpretation of primed lymphocyte responses to alloantigens or soluble antigens.

Population genetic analyses of insulin dependent diabetes mellitus using HLA allele frequencies.

In order to try to detect heterogeneity within insulin dependent diabetes mellitus (IDDM) and to distinguish a mode of inheritance of IDDM, population genetic analyses were performed using HLA allele frequencies. HLA-A and -B typing performed on 231 IDDM individuals and 268 controls from the southeastern U.S. showed significant increases with IDDM in A2, B8, B15 and B18, and significant decreases in Aw23, B7, B14 and B17. The combination of HLA-B8/B15 showed a greatly increased risk (RR = 25.5). Between the 120 IDDM individuals and 123 controls HLA-DR typed, HLA-DR3 and -DR4 were significantly increased among the IDDM group and DR2 and DR7 were decreased. The risk for DR3/4 was 29.2. It appeared that the B15 association was secondary to the DR4, but the B8/DR3 association showed no difference. Using the method of Curie-Cohen, no significant increases in risk were found for the B8/B15 or DR3/DR4 heterozygotes when compared to the respective homozygotes. Using the method of Thomson and Bodmer, the dominant mode of inheritance was excluded for DR4 only. There was a significant increase in B15 and DR4 in those with onset before age 20. No significant differences were found among the DR phenotypes with respect to season.

Evidence for genetic admixture as a determinant in the occurrence of insulin-dependent diabetes mellitus in U.S. blacks.

In recent years, it has been proposed that genetic admixture may have played a role in the increased frequency of insulin-dependent diabetes mellitus (IDDM) in young U.S. blacks relative to African blacks. In support of this proposal, the similar associations of specific markers of the major histocompatibility complex (MHC) with IDDM in U.S. blacks with respect to U.S. whites have been cited. To determine whether racial admixture was a factor in the increased prevalence, we did three analyses of admixture. In the first we used nine genetic markers (ABO, Rh, Fy, Hp, Gc, Pl, OR, Tfr, and Gm) and determined that there was significantly greater than zero genetic contribution from whites in our sample of U.S. black IDDM patients (9.6 +/- 2.3%, P less than 0.01) when a sample of U.S. blacks without IDDM was used as one "parental" population. In the next two analyses, we estimated the amounts of genetic contribution from whites in the U.S. blacks with and without IDDM using reported gene frequencies for West African blacks for four genetic markers (ABO, Rh, Fy, and Hp). The estimate of admixture (21.4 +/- 2.8%) for the black IDDM sample was greater than that for the U.S. black controls (17.9 +/- 2.3%), although the difference was not significant. Our estimate of genetic contribution from whites, 21.4% for black IDDM patients, supports the assumptions of 20% admixture which MacDonald and Rotter and Hodge used to test their respective models for the inheritance of IDDM. These results support the hypothesis that admixture with the white population is, in part, responsible for the increase in prevalence of IDDM seen in U.S. blacks.

HLA associations with insulin-dependent diabetes mellitus in a sample of the American Black population.

It has been of considerable interest to determine if the HLA associations with insulin-dependent diabetes mellitus (IDDM) in blacks are the same as in whites in the United States. Seventy-nine black IDDM patients who were under the age of 40 at onset 283 black controls were HLA typed for A, B and C specificities. This is the largest sample of black IDDM patients yet reported. Analysis of HLA antigen frequencies for these samples has revealed an increased frequency of HLA B8 (p less than 0.01), B3 (p less than 0.02) and B15(p less than 0.006), and a decreased frequency of B14 (p less than 0.01). The estimate of relative risks for B8 and B15 was 2.6 and 4.4, respectively. Comparison of a subsample of the black patients (n = 61) and controls (N = 137) revealed increased frequencies of DR3 (p less than 0.0004), DR4 (p less than 0.0002), and DR7 (p less than 0.03) in the IDDM patients. Both HLA DR2 and DR5 were decreased in the diabetic patients (p less than 0.0001 and p less than 0.0002, respectively). All p values were uncorrected. The associations of HLA antigens with IDDM in our black American sample are essentially the same as those found in white IDDM patients. This suggests that the occurrence of IDDM in American blacks may be due to admixture of white genes.