Paul Renbaum

A dual role for interleukin-1 in hippocampal-dependent memory processes

Ample research demonstrates that pathophysiological levels of the pro-inflammatory cytokine interleukin-1 (IL-1) produces detrimental effects on memory functioning. However, recent evidence suggests that IL-1 may be required for the normal physiological regulation of hippocampal-dependent memory. To substantiate the physiological role of IL-1 in learning and memory we examined the induction of IL-1 gene expression following a learning experience, and the effects of IL-1 signaling blockade, by either genetic or pharmacological manipulations, on memory functioning. We show that IL-1 gene expression is induced in the hippocampus 24h following fear-conditioning in wild type mice, but not in two mouse strains with impaired IL-1 signaling. Moreover, we report that mice with transgenic over-expression of IL-1 receptor antagonist restricted to the CNS (IL-1raTG) display impaired hippocampal-dependent and intact hippocampal-independent memory in the water maze and fear-conditioning paradigms. We further demonstrate that continuous administration of IL-1ra via osmotic minipumps during prenatal development disrupt memory performance in adult mice, suggesting that IL-1 plays a critical role not only in the formation of hippocampal-dependent memory but also in normal hippocampal development. Finally, we tested the dual role of IL-1 in memory by intracerebroventricular (ICV) administration of different doses of IL-1beta and IL-1ra following learning, providing the first systematic evidence that the involvement of IL-1 in hippocampal-dependent memory follows an inverted U-shaped pattern, i.e., a slight increase in brain IL-1 levels can improve memory, whereas any deviation from the physiological range, either by excess elevation in IL-1 levels or by blockade of IL-1 signaling, results in impaired memory.

Mutant Adenosine Deaminase 2 in a Polyarteritis Nodosa Vasculopathy

BACKGROUND Polyarteritis nodosa is a systemic necrotizing vasculitis with a pathogenesis that is poorly understood. We identified six families with multiple cases of systemic and cutaneous polyarteritis nodosa, consistent with autosomal recessive inheritance. In most cases, onset of the disease occurred during childhood. METHODS We carried out exome sequencing in persons from multiply affected families of Georgian Jewish or German ancestry. We performed targeted sequencing in additional family members and in unrelated affected persons, 3 of Georgian Jewish ancestry and 14 of Turkish ancestry. Mutations were assessed by testing their effect on enzymatic activity in serum specimens from patients, analysis of protein structure, expression in mammalian cells, and biophysical analysis of purified protein. RESULTS In all the families, vasculitis was caused by recessive mutations in CECR1, the gene encoding adenosine deaminase 2 (ADA2). All the Georgian Jewish patients were homozygous for a mutation encoding a Gly47Arg substitution, the German patients were compound heterozygous for Arg169Gln and Pro251Leu mutations, and one Turkish patient was compound heterozygous for Gly47Val and Trp264Ser mutations. In the endogamous Georgian Jewish population, the Gly47Arg carrier frequency was 0.102, which is consistent with the high prevalence of disease. The other mutations either were found in only one family member or patient or were extremely rare. ADA2 activity was significantly reduced in serum specimens from patients. Expression in human embryonic kidney 293T cells revealed low amounts of mutant secreted protein. CONCLUSIONS Recessive loss-of-function mutations of ADA2, a growth factor that is the major extracellular adenosine deaminase, can cause polyarteritis nodosa vasculopathy with highly varied clinical expression. (Funded by the Shaare Zedek Medical Center and others.).

A single nucleotide polymorphism in the RAD51 gene modifies cancer risk in BRCA2 but not BRCA1 carriers

BRCA1 and BRCA2 carriers are at increased risk for both breast and ovarian cancer, but estimates of lifetime risk vary widely, suggesting their penetrance is modified by other genetic and/or environmental factors. The BRCA1 and BRCA2 proteins function in DNA repair in conjunction with RAD51. A preliminary report suggested that a single nucleotide polymorphism in the 5′ untranslated region of RAD51 (135C/G) increases breast cancer risk in BRCA1 and BRCA2 carriers. To investigate this effect we studied 257 female Ashkenazi Jewish carriers of one of the common BRCA1 (185delAG, 5382insC) or BRCA2 (6174delT) mutations. Of this group, 164 were affected with breast and/or ovarian cancer and 93 were unaffected. RAD51 genotyping was performed on all subjects. Among BRCA1 carriers, RAD51-135C frequency was similar in healthy and affected women [6.1% (3 of 49) and 9.9% (12 of 121), respectively], and RAD-135C did not influence age of cancer diagnosis [Hazard ratio (HR) = 1.18 for disease in RAD51-135C heterozygotes, not significant]. However, in BRCA2 carriers, RAD51-135C heterozygote frequency in affected women was 17.4% (8 of 46) compared with 4.9% (2 of 41) in unaffected women (P = 0.07). Survival analysis in BRCA2 carriers showed RAD51-135C increased risk of breast and/or ovarian cancer with an HR of 4.0 [95% confidence interval 1.6–9.8, P = 0.003]. This effect was largely due to increased breast cancer risk with an HR of 3.46 (95% confidence interval 1.3–9.2, P = 0.01) for breast cancer in BRCA2 carriers who were RAD51-135C heterozygotes. RAD51 status did not affect ovarian cancer risk. These results show RAD51-135C is a clinically significant modifier of BRCA2 penetrance, specifically in raising breast cancer risk at younger ages.

Population-based screening for breast and ovarian cancer risk due to BRCA1 and BRCA2

Significance Inherited mutations in the tumor suppressor genes BRCA1 and BRCA2 predispose to very high risks of breast and ovarian cancer. For carriers of these mutations, risk-reducing surgery significantly reduces morbidity and mortality. General population screening for BRCA1 and BRCA2 mutations in young adult women could be feasible if accurate estimates of cancer risk for mutation carriers could be obtained. We determined that risks of breast and ovarian cancer for BRCA1 and BRCA2 mutation carriers ascertained from the general population are as high as for mutation carriers ascertained through personal or family history of cancer. General screening of BRCA1 and BRCA2 would identify many carriers who are currently not evaluated and could serve as a model for population screening for genetic predisposition to cancer. In the Ashkenazi Jewish (AJ) population of Israel, 11% of breast cancer and 40% of ovarian cancer are due to three inherited founder mutations in the cancer predisposition genes BRCA1 and BRCA2. For carriers of these mutations, risk-reducing salpingo-oophorectomy significantly reduces morbidity and mortality. Population screening for these mutations among AJ women may be justifiable if accurate estimates of cancer risk for mutation carriers can be obtained. We therefore undertook to determine risks of breast and ovarian cancer for BRCA1 and BRCA2 mutation carriers ascertained irrespective of personal or family history of cancer. Families harboring mutations in BRCA1 or BRCA2 were ascertained by identifying mutation carriers among healthy AJ males recruited from health screening centers and outpatient clinics. Female relatives of the carriers were then enrolled and genotyped. Among the female relatives with BRCA1 or BRCA2 mutations, cumulative risk of developing either breast or ovarian cancer by age 60 and 80, respectively, were 0.60 (± 0.07) and 0.83 (± 0.07) for BRCA1 carriers and 0.33 (± 0.09) and 0.76 (± 0.13) for BRCA2 carriers. Risks were higher in recent vs. earlier birth cohorts (P = 0.006). High cancer risks in BRCA1 or BRCA2 mutation carriers identified through healthy males provide an evidence base for initiating a general screening program in the AJ population. General screening would identify many carriers who are not evaluated by genetic testing based on family history criteria. Such a program could serve as a model to investigate implementation and outcomes of population screening for genetic predisposition to cancer in other populations.

Spinal Muscular Atrophy with Pontocerebellar Hypoplasia Is Caused by a Mutation in the VRK1 Gene

The spinal muscular atrophies (SMAs) are a genetically and clinically heterogeneous group of disorders characterized by degeneration and loss of anterior horn cells in the spinal cord, leading to muscle weakness and atrophy. Spinal muscular atrophy with pontocerebellar hypoplasia (SMA-PCH, also known as pontocerebellar hypoplasia type 1 [PCH1]) is one of the rare infantile SMA variants that include additional clinical manifestations, and its genetic basis is unknown. We used a homozygosity mapping and positional cloning approach in a consanguineous family of Ashkenazi Jewish origin and identified a nonsense mutation in the vaccinia-related kinase 1 gene (VRK1) as a cause of SMA-PCH. VRK1, one of three members of the mammalian VRK family, is a serine/threonine kinase that phosphorylates p53 and CREB and is essential for nuclear envelope formation. Its identification as a gene involved in SMA-PCH implies new roles for the VRK proteins in neuronal development and maintenance and suggests the VRK genes as candidates for related phenotypes.

Neonatal Hyperbilirubinemia in Glucose-6-Phosphate Dehydrogenase-deficient Heterozygotes

Objectives. We assessed the incidence of hyperbilirubinemia, defined as serum total bilirubin ≥15 mg/dL (256 μmol/L), in a cohort of Sephardic Jewish female neonates at risk for glucose-6-phosphate dehydrogenase (G-6-PD) deficiency with especial emphasis on the heterozygotes. We studied the roles of hemolysis by blood carboxyhemoglobin (COHb) determinations and of the variant promoter of the gene for the bilirubin-conjugating enzyme uridine 5′-diphosphate glucuronosyltransferase 1 (UGT1A1) seen in Gilberts syndrome in the pathogenesis of the hyperbilirubinemia. Methods. Consecutively born, healthy, term, female neonates were screened for G-6-PD deficiency and observed clinically with serum bilirubin evaluations as indicated for hyperbilirubinemia. On day 3, blood was sampled for COHb, total hemoglobin (tHb), and a mandatory serum bilirubin determination. COHb, determined by gas chromatography, was expressed as percentage of tHb and corrected for inspired carbon monoxide (COHbc). DNA was analyzed for the G-6-PD Mediterranean563T mutation and for the variant UGT1A1 gene. Results. The cohort included 54 G-6-PD-deficient heterozygotes, 19 deficient homozygotes, and 112 normal homozygotes. More heterozygotes (12/54, 22%; relative risk: 2.26; 95% CI: 1.07–4.80) and deficient homozygotes (5/19, 26.3%; relative risk: 2.68; 95% CI: 1.05–6.90) developed hyperbilirubinemia, than did normal homozygotes (11/112, 9.8%). Third-day serum bilirubin values that were obtained from 144 neonates were significantly higher in both heterozygotes (11.2 ± 3.7 mg/dL [192 ± 64 μmol/L]) and G-6-PD-deficient homozygotes (12.0 ± 3.0 mg/dL [206 ± 52 μmol/L]) than in the G-6-PD normal homozygotes (9.4 ± 3.4 mg/dL [160 ± 58 μmol/L). In contrast, COHbc values were higher only in G-6-PD-deficient homozygotes (0.74% ± 0.14%) and not in heterozygotes (0.69% ± 0.19%, not statistically significant), compared with control values (0.63% ± 0.19%). High COHbc values were not a prerequisite for the development of hyperbilirubinemia in any of the G-6-PD genotypes. A greater incidence of hyperbilirubinemia was found among the G-6-PD-deficient heterozygotes, who also had the variant UGT1A1 gene, in both heterozygous (6/20, 30%) and homozygous (4/8, 50%) forms, than was found in their counterparts with the normal UGT1A1 gene (2/26, 7.7%). This effect was not seen in the G-6-PD normal homozygote group. A color reduction screening test for G-6-PD deficiency identified only 20.4% (11/54) of the heterozygotes. Conclusions. We showed that G-6-PD-deficient heterozygotes, categorically defined by DNA analysis, are at increased risk for neonatal hyperbilirubinemia. The screening test that was used was unable to detect most heterozygotes. Increased bilirubin production was not crucial to the development of hyperbilirubinemia, but presence of the variant UGT1A1 gene did confer increased risk. bilirubin, carbon monoxide, carboxyhemoglobin, females, gas chromatography, Gilberts syndrome, glucose-6-phosphate dehydrogenase deficiency, hemolysis, hyperbilirubinemia, neonates, polymerase chain reaction, Sephardic Jews, screening test, uridine 5′-diphosphate glucuronosyltransferase.

Prevalence of glucocerebrosidase mutations in the Israeli Ashkenazi Jewish population.

Gaucher disease is the most prevalent inherited disease among Ashkenazi Jews. It is very heterogeneous due to a large number of mutations within the glucocerebrosidase gene, whose impaired activity is the cause for this disease. Aiming at determining Gaucher carrier frequency among the Ashkenazi Jewish population in Israel, 1,208 individuals were molecularly diagnosed for six mutations known to occur among Ashkenazi Jewish Gaucher patients, using the newly developed Pronto™ Gaucher kit. The following mutations were tested: N370S, 84GG, IVS2+1, D409H, L444P, and V394L. Molecular testing of these mutations also allows identification of the recTL allele. The results indicated that Gaucher carrier frequency is 1:17 within the tested population. The prevalence of N370S carriers is 1:17.5. This implies that ˜1:1225 Ashkenazi Jews will be homozygous for the N370S mutation. Actually, in our study of 1,208 individuals one was found to be homozygous for the N370S mutation. The actual number of known Ashkenazi Jewish Gaucher patients with this genotype is much lower than that expected according to the frequency of the N370S mutation, suggesting a low penetrance of this mutation. Results of loading experiments in cells homozygous for the N370S mutation, as well as cells homozygous for the L444P and the D409H mutations, exemplified this phenomenon. Hum Mutat 12:240–244, 1998.

BRCA1 germline mutations in women with uterine serous papillary carcinoma

Objective To determine the possible effects and incidence of BRCA1 and BRCA2 germline mutations in uterine serous papillary carcinoma. Methods We screened DNA from 12 women with uterine serous papillary carcinoma for BRCA1 and BRCA2 germline mutations common in the Jewish population (BRCA1–185delAG and 5382insC, BRCA2–6174delT). In women with germline mutations, tumor DNA was screened for loss of heterozygosity at the appropriate loci. Results Nine women were of Jewish Ashkenazi origin and three were non-Ashkenazi. Two of nine Ashkenazi women were carriers of germline mutations: one 185delAG mutation and one 5382insC mutation. Five women had histories of breast carcinoma before diagnosis of uterine serous papillary carcinoma. Family histories of seven women had at least one first-degree relative with malignant disease. Of those, four had at least one first-degree relative with breast, ovarian, or colon carcinoma. Both carriers had strong family histories of breast-ovarian carcinoma. Loss of heterozygosity analysis found loss of the wild-type BRCA1 allele in the primary uterine tumors. Conclusion BRCA1 germline mutations were observed in two of nine of the women in this series. The loss of heterozygosity in the tumor tissue of the carriers, coupled with the high frequency of family and patient histories of breast or ovarian malignancies, suggest that uterine serous papillary carcinoma might be a manifestation of familial breast-ovarian cancer.

Single Nucleotide Polymorphisms That Increase Expression of the Guanosine Triphosphatase RAC1 Are Associated With Ulcerative Colitis

BACKGROUND & AIMS RAC1 is a guanosine triphosphatase that has an evolutionarily conserved role in coordinating immune defenses, from plants to mammals. Chronic inflammatory bowel diseases are associated with dysregulation of immune defenses. We studied the role of RAC1 in inflammatory bowel diseases using human genetic and functional studies and animal models of colitis. METHODS We used a candidate gene approach to HapMap-Tag single nucleotide polymorphisms in a discovery cohort; findings were confirmed in 2 additional cohorts. RAC1 messenger RNA expression was examined from peripheral blood cells of patients. Colitis was induced in mice with conditional disruption of Rac1 in phagocytes by administration of dextran sulfate sodium. RESULTS We observed a genetic association between RAC1 with ulcerative colitis in a discovery cohort, 2 independent replication cohorts, and in combined analysis for the single nucleotide polymorphisms rs10951982 (P(combined UC) = 3.3 × 10(-8), odds ratio = 1.43 [95% confidence interval: 1.26-1.63]) and rs4720672 (P(combined UC) = 4.7 × 10(-6), odds ratio = 1.36 [95% confidence interval: 1.19-1.58]). Patients with inflammatory bowel disease who had the rs10951982 risk allele had increased expression of RAC1 compared to those without this allele. Conditional disruption of Rac1 in macrophage and neutrophils of mice protected against dextran sulfate sodium-induced colitis. CONCLUSIONS Human studies and knockout mice demonstrated a role for the guanosine triphosphatase RAC1 in the development of ulcerative colitis; increased expression of RAC1 was associated with susceptibility to colitis.

FMR1 Epigenetic Silencing Commonly Occurs in Undifferentiated Fragile X-Affected Embryonic Stem Cells

Summary Fragile X syndrome (FXS) is the most common heritable form of cognitive impairment. It results from epigenetic silencing of the X-linked FMR1 gene by a CGG expansion in its 5′-untranslated region. Taking advantage of a large set of FXS-affected human embryonic stem cell (HESC) lines and isogenic subclones derived from them, we show that FMR1 hypermethylation commonly occurs in the undifferentiated state (six of nine lines, ranging from 24% to 65%). In addition, we demonstrate that hypermethylation is tightly linked with FMR1 transcriptional inactivation in undifferentiated cells, coincides with loss of H3K4me2 and gain of H3K9me3, and is unrelated to CTCF binding. Taken together, these results demonstrate that FMR1 epigenetic gene silencing takes place in FXS HESCs and clearly highlights the importance of examining multiple cell lines when investigating FXS and most likely other epigenetically regulated diseases.