Alexander Pearlman

Genome-wide association study to identify single nucleotide polymorphisms (SNPs) associated with the development of erectile dysfunction in African-American men after radiotherapy for prostate cancer.

PURPOSE To identify single nucleotide polymorphisms (SNPs) associated with erectile dysfunction (ED) among African-American prostate cancer patients treated with external beam radiation therapy. METHODS AND MATERIALS A cohort of African-American prostate cancer patients treated with external beam radiation therapy was observed for the development of ED by use of the five-item Sexual Health Inventory for Men (SHIM) questionnaire. Final analysis included 27 cases (post-treatment SHIM score ≤7) and 52 control subjects (post-treatment SHIM score ≥16). A genome-wide association study was performed using approximately 909,000 SNPs genotyped on Affymetrix 6.0 arrays (Affymetrix, Santa Clara, CA). RESULTS We identified SNP rs2268363, located in the follicle-stimulating hormone receptor (FSHR) gene, as significantly associated with ED after correcting for multiple comparisons (unadjusted p = 5.46 × 10(-8), Bonferroni p = 0.028). We identified four additional SNPs that tended toward a significant association with an unadjusted p value < 10(-6). Inference of population substructure showed that cases had a higher proportion of African ancestry than control subjects (77% vs. 60%, p = 0.005). A multivariate logistic regression model that incorporated estimated ancestry and four of the top-ranked SNPs was a more accurate classifier of ED than a model that included only clinical variables. CONCLUSIONS To our knowledge, this is the first genome-wide association study to identify SNPs associated with adverse effects resulting from radiotherapy. It is important to note that the SNP that proved to be significantly associated with ED is located within a gene whose encoded product plays a role in male gonad development and function. Another key finding of this project is that the four SNPs most strongly associated with ED were specific to persons of African ancestry and would therefore not have been identified had a cohort of European ancestry been screened. This study demonstrates the feasibility of a genome-wide approach to investigate genetic predisposition to radiation injury.

Mutations in MAP3K1 Cause 46,XY Disorders of Sex Development and Implicate a Common Signal Transduction Pathway in Human Testis Determination

Investigations of humans with disorders of sex development (DSDs) resulted in the discovery of many of the now-known mammalian sex-determining genes, including SRY, RSPO1, SOX9, NR5A1, WT1, NR0B1, and WNT4. Here, the locus for an autosomal sex-determining gene was mapped via linkage analysis in two families with 46,XY DSD to the long arm of chromosome 5 with a combined, multipoint parametric LOD score of 6.21. A splice-acceptor mutation (c.634-8T>A) in MAP3K1 segregated with the phenotype in the first family and disrupted RNA splicing. Mutations were demonstrated in the second family (p.Gly616Arg) and in two of 11 sporadic cases (p.Leu189Pro, p.Leu189Arg)-18% prevalence in this cohort of sporadic cases. In cultured primary lymphoblastoid cells from family 1 and the two sporadic cases, these mutations altered the phosphorylation of the downstream targets, p38 and ERK1/2, and enhanced binding of RHOA to the MAP3K1 complex. Map3k1 within the syntenic region was expressed in the embryonic mouse gonad prior to, and after, sex determination. Thus, mutations in MAP3K1 that result in 46,XY DSD with partial or complete gonadal dysgenesis implicate this pathway in normal human sex determination.

Breast cancfr–‐influence of growth rate on prognosis and treatment evaluation. A study based on mastectomy scar recurrences

The growth rate of a mammary cancer can be calculated when a recurrence appears in the mastectomy scar. Growth rate can, at times, be approximated from the patients history with reasonable accuracy. Approximately half of breast cancers exhibit rapid growth (tumor doubling time (Td), up to 25 days); one‐third grow at an intermediate rate (Td 26 to 75 days) and 15% grow slowly (Td 76 days or longer). Anatomic (TNM) staging does not define a homogeneous patient group in breast cancer. Within each stage, there are rapid, intermediate, and slow‐growing tumors. The prognosis varies importantly with the growth rate characteristics of the tumor. Survival is the product of the tumor doubling time and the number of tumor doublings through which the patient lives. Slowly growing and intermediate growth rate tumors are associated with a high percentage of 5‐year survivors after mastectomy (between 80 and 100%). Rapidly growing tumors have few 5‐year survivors. When survival after mastectomy is measured in the number of tumor doublings, the differences in survival of the three groups tended to disappear, indicating that in this select group of patients with scar recurrence there was no therapeutic advantage in any of the three groups, despite the differences in the survival times. The appreciable number of patients with tumors exhibiting slow or intermediate growth rates, in any series, suggests that the use of the 5‐year interval is an inadequate measure of therapeutic response in breast cancer and may actually be misleading.

Genetic marker polymorphisms on chromosome 8q24 and prostate cancer in the Dutch population: DG8S737 may not be the causative variant.

Prostate cancer is the most commonly diagnosed cancer in men in Europe and Northern America. Genome-wide association studies (GWAS) have detected an association with markers on chromosome 8q24. Allele -8 of microsatellite DG8S737 with 22 repeats and allele A of the single-nucleotide polymorphism (SNP) rs1447295 have been found to be significantly associated with prostate cancer. As GWAS are subjected to type 1 error, confirmation studies are required to validate the results. Here, we analysed the same markers in 277 cases and 282 controls from the Netherlands using a nested case–control study. Incident prostate cancer cases and controls selected were identified in the population of the Netherlands Cohort Study. We also investigated clinical features of the disease by stratifying by tumour stage. We did not replicate the association with the SNP rs1447295-A allele (P=0.10), although the effect estimate was in the same direction as previous studies (odds ratio (OR), 1.38). Interestingly a statistically significant decreased risk was observed for DG8S737 allele -8 (OR, 0.62; P=0.03). The apparent protective effect of the DG8S737 -8 allele observed in this study contrasts with the Amundadottir study. This suggests that DG8S737 and rs1447295 might be tightly linked markers flanking the actual causative variant and that there may be potentially more than one high-risk haplotype present in the Caucasian population. This short report highlights the importance of validation, although further confirmation is still needed.

Integrative Genomics Identifies Molecular Alterations that Challenge the Linear Model of Melanoma Progression

Superficial spreading melanoma (SSM) and nodular melanoma (NM) are believed to represent sequential phases of linear progression from radial to vertical growth. Several lines of clinical, pathologic, and epidemiologic evidence suggest, however, that SSM and NM might be the result of independent pathways of tumor development. We utilized an integrative genomic approach that combines single nucleotide polymorphism array (6.0; Affymetrix) with gene expression array (U133A 2.0; Affymetrix) to examine molecular differences between SSM and NM. Pathway analysis of the most differentially expressed genes between SSM and NM (N = 114) revealed significant differences related to metabolic processes. We identified 8 genes (DIS3, FGFR1OP, G3BP2, GALNT7, MTAP, SEC23IP, USO1, and ZNF668) in which NM/SSM-specific copy number alterations correlated with differential gene expression (P < 0.05; Spearmans rank). SSM-specific genomic deletions in G3BP2, MTAP, and SEC23IP were independently verified in two external data sets. Forced overexpression of metabolism-related gene MTAP (methylthioadenosine phosphorylase) in SSM resulted in reduced cell growth. The differential expression of another metabolic-related gene, aldehyde dehydrogenase 7A1 (ALDH7A1), was validated at the protein level by using tissue microarrays of human melanoma. In addition, we show that the decreased ALDH7A1 expression in SSM may be the result of epigenetic modifications. Our data reveal recurrent genomic deletions in SSM not present in NM, which challenge the linear model of melanoma progression. Furthermore, our data suggest a role for altered regulation of metabolism-related genes as a possible cause of the different clinical behavior of SSM and NM.

Mutations in MAP3K1 tilt the balance from SOX9/FGF9 to WNT/β-catenin signaling

In-frame missense and splicing mutations (resulting in a 2 amino acid insertion or a 34 amino acid deletion) dispersed through the MAP3K1 gene tilt the balance from the male to female sex-determining pathway, resulting in 46,XY disorder of sex development. These MAP3K1 mutations mediate this balance by enhancing WNT/β-catenin/FOXL2 expression and β-catenin activity and by reducing SOX9/FGF9/FGFR2/SRY expression. These effects are mediated at multiple levels involving MAP3K1 interaction with protein co-factors and phosphorylation of downstream targets. In transformed B-lymphoblastoid cell lines and NT2/D1 cells transfected with wild-type or mutant MAP3K1 cDNAs under control of the constitutive CMV promoter, these mutations increased binding of RHOA, MAP3K4, FRAT1 and AXIN1 and increased phosphorylation of p38 and ERK1/2. Overexpressing RHOA or reducing expression of MAP3K4 in NT2/D1 cells produced phenocopies of the MAP3K1 mutations. Using siRNA knockdown of RHOA or overexpressing MAP3K4 in NT2/D1 cells produced anti-phenocopies. Interestingly, the effects of the MAP3K1 mutations were rescued by co-transfection with wild-type MAP3K4. Although MAP3K1 is not usually required for testis determination, mutations in this gene can disrupt normal development through the gains of function demonstrated in this study.

North African Jewish and non-Jewish populations form distinctive, orthogonal clusters

North African Jews constitute the second largest Jewish Diaspora group. However, their relatedness to each other; to European, Middle Eastern, and other Jewish Diaspora groups; and to their former North African non-Jewish neighbors has not been well defined. Here, genome-wide analysis of five North African Jewish groups (Moroccan, Algerian, Tunisian, Djerban, and Libyan) and comparison with other Jewish and non-Jewish groups demonstrated distinctive North African Jewish population clusters with proximity to other Jewish populations and variable degrees of Middle Eastern, European, and North African admixture. Two major subgroups were identified by principal component, neighbor joining tree, and identity-by-descent analysis—Moroccan/Algerian and Djerban/Libyan—that varied in their degree of European admixture. These populations showed a high degree of endogamy and were part of a larger Ashkenazi and Sephardic Jewish group. By principal component analysis, these North African groups were orthogonal to contemporary populations from North and South Morocco, Western Sahara, Tunisia, Libya, and Egypt. Thus, this study is compatible with the history of North African Jews—founding during Classical Antiquity with proselytism of local populations, followed by genetic isolation with the rise of Christianity and then Islam, and admixture following the emigration of Sephardic Jews during the Inquisition.

De Novo 12;17 translocation upstream of sox9 resulting in 46,xx testicular disorder of sex development

Individuals with rare cytogenetic variants have contributed to our understanding of the genetics of sex development and its disorders. Here, we report on a child with a de novo 12;17 translocation, 46,XX,t(12;17)(q14.3;q24.3) chromosome complement, resulting in SRY‐negative 46,XX testicular disorder of sex development (46,XX DSD without campomelic dysplasia). The chromosome 12 breakpoint was mapped via array comparative genomic hybridization (aCGH) of a hybrid somatic cell line to 64.2–64.6 Mb (from the p arm telomere). The chromosome 17 breakpoint was mapped to 66.4–67.1 Mb, that is, upstream of SOX9. The location of the chromosome 17 breakpoint was refined by fluorescence in situ hybridization (FISH) at ≥776 kb upstream of SOX9. Thus, the 12;17 translocation removed part of the SOX9 cis‐regulatory region and replaced it with a regulatory element from pseudogene LOC204010 or the next gene, Deynar, of chromosome 12, potentially causing up‐regulation of the testis‐determining SOX9 gene during gonadal development and the phenotype of 46,XX testicular DSD.

“Seminoma with trophocarcinoma”. A clinical variant of seminoma

Seminoma occasionally contains foci of trophocarcinoma (embryonal carcinoma), or else has histologic features of aggressive growth. The resultant variants are classified into: anaplastic seminoma, borderline seminoma, and seminoma with trophocarcinoma. The latter variant is characterized by specific histologic features and, compared with pure seminoma, by more aggressive clinical behavior, larger required tumor lethal radiation dose, and lower survival rate. The intimate association of primordial trophoblastic structures with seminoma is of histogenetic interest.

The Adjuvant Effect of Lucanthone (Miracil D) in Clinical Radiation Therapy

Clinical trials were undertaken to determine whether lucanthone (miracil D) affects radiation-induced regression in measurable pulmonary metastases and advanced squamous-cell oral and pharyngeal tumors. The time required for 50% tumor regression was decreased by approximately 50% in those patients who received lucanthone in addition to irradiation. These results indicate that lucanthone has a definite adjuvant effect when used together with irradiation.