Rachel Beeri

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.

Functional variant of KLOTHO: a breast cancer risk modifier among BRCA1 mutation carriers of Ashkenazi origin.

Klotho is a transmembrane protein that can be shed and act as a circulating hormone and is a putative tumor suppressor in breast cancer. A functional variant of KLOTHO (KL-VS) contains two amino acid substitutions F352V and C370S and shows reduced activity. Germ-line mutations in BRCA1 and BRCA2 substantially increase lifetime risk of breast and ovarian cancers. Yet, penetrance of deleterious BRCA1 and BRCA2 mutations is incomplete even among carriers of identical mutations. We examined the association between KL-VS and cancer risk among 1115 Ashkenazi Jewish women: 236 non-carriers, 631 BRCA1 (185delAG, 5382insC) carriers and 248 BRCA2 (6174delT) carriers. Among BRCA1 carriers, heterozygosity for the KL-VS allele was associated with increased breast and ovarian cancer risk (hazard ratio 1.40, 95% confidence intervals 1.08–1.83, P=0.01) and younger age at breast cancer diagnosis (median age 48 vs 43 P=0.04). KLOTHO and BRCA2 are located on 13q12, and we identified linkage disequilibrium between KL-VS and BRCA2 6174delT mutation. Studies in breast cancer cells showed reduced growth inhibitory activity and reduced secretion of klotho F352V compared with wild-type klotho. These data suggest KL-VS as a breast and ovarian cancer risk modifier among BRCA1 mutation carriers. If validated in additional cohorts, the presence of KL-VS may serve as a predictor of cancer risk among BRCA1 mutation carriers.

X-inactivation in Fabry disease ☆

BACKGROUND Fabry disease is one of three X-linked lysosomal disorders. Because of X-chromosome inactivation (XCI), wherein there is (random) transcriptional silencing of one of the X-chromosomes in each female cell, females are mosaic for the expression of (some) X-linked genes. Thus, based on penetrance and expression, some females heterozygous for Fabry disease are symptomatic but not to the same degree as hemizygous males. The purpose of this study was to ascertain whether skewed X-inactivation favoring the mutant α-galactosidase A allele exists in our cohort of female heterozygotes of Fabry disease. METHOD All patients were evaluated by physical examination and ascribed disease-specific severity sub-scores for each of the four categories (cardiac, renal, neurological, general) and a total score using the Mainz Severity Score Index (MSSI). Blood samples were drawn for enzymatic activity of α-galactosidase A and for DNA extraction for analysis for α-galactosidase A mutations. XCI ratios were determined from peripheral blood leukocyte samples. The X-chromosome inactivation ratio was determined in each heterozygote. RESULTS Of 77 samples, only 18.2% were highly skewed (80/20). Only 14.3% of samples with nonsense mutations were highly skewed. There were no correlations between the XCI ratios and age, enzymatic activity of α-galactosidase A, MSSI sub-scores or total score, or with the clinical signs of cardiac involvement, neuropathic pain, or proteinuria. CONCLUSION These findings are comparable with others in Fabry disease, i.e., essentially the same as seen in normal non-elderly female population, raising the question of the mechanism underlying symptomatic phenotypic expression in heterozygous females with Fabry disease.

Female Sex Bias in Human Embryonic Stem Cell Lines

The factors limiting the rather inefficient derivation of human embryonic stem cells (HESCs) are not fully understood. The aim of this study was to analyze the sex ratio in our 42 preimplantation genetic diagnosis (PGD)-HESC lines, in an attempt to verify its affect on the establishment of HESC lines. The ratio between male and female PGD-derived cell lines was compared. We found a significant increase in female cell lines (76%). This finding was further confirmed by a meta-analysis for combining the results of all PGD-derived HESC lines published to date (148) and all normal karyotyped HESC lines derived from spare in vitro fertilization embryos worldwide (397). Further, gender determination of embryos demonstrated that this difference originates from the actual derivation process rather than from unequal representation of male and female embryos. It can therefore be concluded that the clear-cut tendency for female preponderance is attributed to suboptimal culture conditions rather than from a true gender imbalance in embryos used for derivation of HESC lines. We propose a mechanism in which aberrant X chromosome inactivation and/or overexpression of critical metabolic X-linked genes might explain this sex dimorphism.

Preimplantation genetic diagnosis (PGD)--prevention of the birth of children affected with endocrine diseases.

Abstract Objective: To develop a reliable and accurate preimplantation genetic diagnosis (PGD) method in six families with endocrine diseases: persistent hyperinsulinemic hypoglycemia of infancy (PHHI), congenital adrenal hyperplasia (CAH) salt-wasting form, Sanjat-Sakati syndrome and multiple endocrine neoplasia 2A (MEN 2A). Methods: For each disease a battery of at least four informative markers surrounding the tested gene were identified and for each family a protocol of multiplex fluorescent markers was developed and performed on single cells. Results: PGD for PHHI was performed in three families. In family 1 two healthy children were born from different cycles, in family 2 three healthy children were born from two cycles, and in family 3 a healthy boy was born. For CAH in one family a healthy girl was born. One PGD cycle for Sanjat-Sakati resulted in a clinical pregnancy that was terminated due to high nuccal translucency (46X0). For one family with MEN 2A disease, the eighth PGD cycle resulted in birth of healthy twins. In all children genetic confirmation of the healthy status was performed. Conclusions: PGD is an effective method for preventing birth of affected children with endocrine disorders. Increasing the awareness of clinicians to the availability of these methods is most important.

Population screening for BRCA1/BRCA2 founder mutations in Ashkenazi Jews: proactive recruitment compared with self-referral.

Purpose:Population screening of three common BRCA1/BRCA2 mutations in Ashkenazi Jews (AJ) apparently fulfills screening criteria. We compared streamlined BRCA screening via self-referral with proactive recruitment in medical settings.Methods:Unaffected AJ, age ≥25 years without known familial mutations, were either self-referred or recruiter-enrolled. Before testing, participants received written information and self-reported family history (FH). After testing, both non-carriers with significant FH and carriers received in-person genetic counseling. Psychosocial questionnaires were self-administered 1 week and 6 months after enrollment.Results:Of 1,771 participants, 58% were recruiter-enrolled and 42% were self-referred. Screening uptake was 67%. Recruited enrollees were older (mean age 54 vs. 48, P < 0.001) and had less suggestive FH (23 vs. 33%, P < 0.001). Of 32 (1.8%) carriers identified, 40% had no significant FH. Post-test counseling compliance was 100% for carriers and 89% for non-carrier women with FH. All groups expressed high satisfaction (>90%). At 6 months, carriers had significantly increased distress and anxiety, greater knowledge, and similar satisfaction; 90% of participants would recommend general AJ BRCA screening.Conclusion:Streamlined BRCA screening results in high uptake, very high satisfaction, and no excess psychosocial harm. Proactive recruitment captured older women less selected for FH. Further research is necessary to target younger women and assess other populations.Genet Med advance online publication 08 December 2016

An intronic haplotype in α galactosidase A is associated with reduced mRNA expression in males with cryptogenic stroke.

Persons with unexplained early-onset stroke have been targeted for screening surveys for Fabry disease, the most common of the three X-linked lysosomal disorders, because Fabry patients with stroke are more likely to have the life-threatening progressive cardiac and renal manifestations and would therefore most benefit from early diagnosis and intervention with enzyme replacement therapy (ERT). Among 175 Israeli patients with unexplained cryptogenic stroke screened for mutations in the Fabry α galactosidase A (GLA) gene, sequencing identified six with 2-4 GLA intronic variants, one of whose father and three sisters had the same variants. Two variants, c.640-16A>G (g.10115A>G) in intron 4 and c.1000-22C>T (g.10956C>T) in intron 6, were common to all patients. However, three males with a common four variant intronic haplotype had low residual enzyme activity and ~50% reduced mRNA expression. Transcript splice-site defects were not identified in any of the index cases and X-chromosome inactivation was not highly skewed in the six females. These data do not suggest that GLA intronic variants, per se, are pathogenic. Nonetheless, it is clear that a certain intronic haplotype in males with cryptogenic stroke is associated with reduced GLA expression and function.

Prevention of lysosomal storage diseases and derivation of mutant stem cell lines by preimplantation genetic diagnosis.

Preimplantation genetic diagnosis (PGD) allows birth of unaffected children for couples at risk for a genetic disorder. We present the strategy and outcome of PGD for four lysosomal storage disorders (LSD): Tay-Sachs disease (TSD), Gaucher disease (GD), Fabry disease (FD), and Hunter syndrome (HS), and subsequent development of stem cell lines. For each disease, we developed a family-specific fluorescent multiplex single-cell PCR protocol that included the familial mutation and informative markers surrounding the mutation. Embryo biopsy and PGD analysis were performed on either oocytes (polar bodies one and two) or on single blastomeres from a six-cell embryo. We treated twenty families carrying mutations in these lysosomal storage disorders, including 3 couples requiring simultaneous analysis for two disorders (TSD/GD, TSD/balanced Robertsonian translocation 45XYder(21;14), and HS/oculocutaneus albinism). These analyses led to an overall pregnancy rate/embryo transfer of 38% and the birth of 20 unaffected children from 17 families. We have found that PGD for lysosomal disorders is a safe and effective method to prevent birth of affected children. In addition, by using mutant embryos for the derivation of stem cell lines, we have successfully established GD and HS hESC lines for use as valuable models in LSD research.

PGD for germline mosaicism.

The aim of this study was to develop and perform a preimplantation genetic diagnosis (PGD) assay discriminating between wild-type and mutant alleles in two families with germline mosaicism. Family 1 had two children affected with severe myoclonic epilepsy (SCNA1A del exons 1-22). Family 2 had two children with tuberous sclerosis (TSC2 C1327T) and two healthy children. Neither mutation was detected in genomic DNA derived from the parents in either family. Informative microsatellite markers flanking SCNA1A and TSC2 along with the identified mutations were used to construct haplotypes. For tuberous sclerosis, single spermatozoa were analysed using a multiplex assay that included six informative markers and the TSC2 mutation. In family 1, deletion in the maternal allele was detected in the affected child. In family 2, both affected children and one healthy child shared the same paternal allele. To confirm mutant paternal transmission, single spermatozoa were analysed for the mutation along with six markers. Of 44 single spermatozoa, four showed the mutant T allele, allowing linkage between the mutation and the genetic markers. Both families delivered healthy children following IVF/PGD. In conclusion, germline mosaicism complicates allele assignment when constructing haplotypes for PGD. Sperm analysis is a useful tool for verifying allelic linkage.

Rac1 Polymorphisms and Thiopurine Efficacy in Children With Inflammatory Bowel Disease.

Objectives: Thiopurines are effective for maintenance of remission in inflammatory bowel disease (IBD) in only about half of patients. Predictors of response may assist in selecting the most appropriate patients for thiopurine therapy. Thiopurines inhibit Rac1, a GTPase that exerts an antiapoptotic effect on T-lymphocytes. A genetic association was recently demonstrated between a Rac1 single nucleotide polymorphism (SNP) and poorer response to thiopurines in adult patients with Crohn disease. We aimed to determine whether Rac1 SNPs are associated with response to thiopurines in children with IBD. Methods: Children with IBD treated with thiopurines were prospectively followed for 1 year and were genotyped for 3 Rac1 SNPs previously found to be relevant to IBD: rs10951982, rs4720672, and rs34932801. The rate of sustained steroid-free remission (SSFR) without treatment escalation by 12 months was compared between wild types (WTs) and heterozygotes. Results: A total of 59 patients were studied (63% boys, 80% having Crohn disease, mean age 13 ± 4.1). Nineteen of the 41 WT (46%) and 9 of the 15 (60%) heterozygotes for rs10951982 were in SSFR (P = 0.55). Similarly, 21 of the 45 (47%) WT and 8 of the 12 (67%) heterozygotes for rs4720672 were in remission (P = 0.33). Finally, 21 of the 45 (47%) WT and 3 of the 5 (60%) heterozygotes for rs34932801 were in remission (P = 0.66). All of the 3 comparisons remained nonsignificant in a sensitivity analysis of only the patients with Crohn disease. Conclusions: We did not find an association between 3 Rac1 SNPs and thiopurine effectiveness by 12 months in a prospective study of children with IBD. Other predictors of response should be sought to optimize patient selection for thiopurine therapy.