BRCA carriers after risk-reducing bilateral salpingo-oophorectomy: menopausal hormone therapy knowledge gaps, and the impact of physicians' recommendations.

OBJECTIVE: Female carriers of BRCA1/2 gene mutations are at an increased lifetime risk for breast and ovarian cancers. They are recommended to undergo risk-reducing surgery, including bilateral salpingo-oophorectomy (RR-BSO), upon completion of childbearing. RR-BSO surgery decreases morbidity and mortality but results in early menopause. Menopausal hormone therapy (MHT) is under-utilized despite being shown as safe for carriers. We aim to evaluate the factors associated with decision-making regarding MHT use following RR-BSO in healthy BRCA mutation carriers. METHODS: Female carriers aged <50 years who underwent RR-BSO and were followed in a multidisciplinary clinic completed online multiple-choice and free-text questionnaires. RESULTS: A total of 142 women met the inclusion criteria and filled the questionnaire: 83 were MHT users and 59 were non-users. MHT users underwent RR-BSO earlier than non-users (40.82 ± 3.91 vs. 42.88 ± 4.34; p < 0.0001). MHT usage was positively associated with MHT explanation (odds ratio 4.318, 95% confidence interval [CI] [1.341-13.902], p = 0.014), and knowledge regarding the safety of MHT and its effects on general health (odds ratio 2.001, 95% CI [1.443-2.774], p < 0.0001). MHT users and non-users retrospectively evaluated their comprehension of RR-BSO consequences as significantly lower than before surgery (p < 0.001). CONCLUSION: Post-RR-BSO outcomes, including the effects on women's quality of life and its possible mitigation through MHT use, need to be addressed pre surgery by healthcare providers.

Common founder BRCA2 pathogenic variants and breast cancer characteristics in Ethiopian Jews.

PURPOSE: BRCA1/2 founder pathogenic variants (PVs) occur in various populations, but data on the mutational spectrum in Africans are limited. We examined BRCA1/2 PVs in breast cancer patients of Ethiopian Jewish (EJ) origin. METHODS: We retrospectively analyzed BRCA1/2 test results and clinical features of EJ breast cancer patients from seven medical institutions. We obtained heterozygote carrier rates in affected individuals from the laboratories of the largest Israeli HMO (Clalit). Population carrier frequency was determined in EJ controls. RESULTS: We identified three recurrent BRCA2 PVs in 11 EJ breast cancer patients (9 females, 2 males): c.7579delG, c.5159C > A, and c.9693delA. Only c.5159C > A was previously reported in Africans. In women, mean age at diagnosis was 35.7y; 8/9 were diagnosed with advanced disease. All tumors were invasive, 4/9 were triple negative. Only 3/11 carriers had relevant family history. Carrier rate in high-risk breast cancer patients was 11% (3/28; 95%CI [2.3%, 28.2%]). Combined carrier rate among controls was 1.8% (5/280; 95%CI [0.6%, 4.1%]). CONCLUSION: EJs harbor 3 recurrent BRCA2 PVs presenting with relatively severe breast cancer morbidity. Combined with the high BRCA2 carrier rate in the EJ population, these findings merit increasing awareness in this community and suggest that a culturally adapted population screening approach may be warranted.

A role for TENM1 mutations in congenital general anosmia.

Congenital general anosmia (CGA) is a neurological disorder entailing a complete innate inability to sense odors. While the mechanisms underlying vertebrate olfaction have been studied in detail, there are still gaps in our understanding of the molecular genetic basis of innate olfactory disorders. Applying whole-exome sequencing to a family multiply affected with CGA, we identified three members with a rare X-linked missense mutation in the TENM1 (teneurin 1) gene (ENST00000422452:c.C4829T). In Drosophila melanogaster, TENM1 functions in synaptic-partner-matching between axons of olfactory sensory neurons and target projection neurons and is involved in synapse organization in the olfactory system. We used CRISPR-Cas9 system to generate a Tenm1 disrupted mouse model. Tenm1(-/-) and point-mutated Tenm1(A) (/A) adult mice were shown to have an altered ability to locate a buried food pellet. Tenm1(A) (/A) mice also displayed an altered ability to sense aversive odors. Results of our study, that describes a new Tenm1 mouse, agree with the hypothesis that TENM1 has a role in olfaction. However, additional studies should be done in larger CGA cohorts, to provide statistical evidence that loss-of-function mutations in TENM1 can solely cause the disease in our and other CGA cases.

Expanding the phenotype of CRB2 mutations - A new ciliopathy syndrome?

Recessive CRB2 mutations were recently reported to cause both steroid resistant nephrotic syndrome and prenatal onset ventriculomegaly with kidney disease. We report two Ashkenazi Jewish siblings clinically diagnosed with ciliopathy. Both presented with severe congenital hydrocephalus and mild urinary tract anomalies. One affected sibling also has lung hypoplasia and heart defects. Exome sequencing and further CRB2 analysis revealed that both siblings are compound heterozygotes for CRB2 mutations p.N800K and p.Gly1036Alafs*43, and heterozygous for a deleterious splice variant in the ciliopathy gene TTCB21. CRB2 is a polarity protein which plays a role in ciliogenesis and ciliary function. Biallelic CRB2 mutations in animal models result in phenotypes consistent with ciliopathy. This report expands the phenotype of CRB2 mutations to include lung hypoplasia and uretero-pelvic renal anomalies, and confirms cardiac malformation as a feature. We suggest that CRB2-associated disease is a new ciliopathy syndrome with possible digenic/triallelic inheritance, as observed in other ciliopathies. Clinically, CRB2 should be assessed when ciliopathy is suspected, especially in Ashkenazi Jews, where we found that p.N800K carrier frequency is 1 of 64. Patients harboring CRB2 mutations should be tested for the complete range of ciliopathy manifestations.

Alpha-thalassemia intellectual disability: variable phenotypic expression among males with a recurrent nonsense mutation - c.109C>T (p.R37X).

Alpha-thalassemia intellectual disability, one of the recognizable X-linked disability syndromes, is characterized by short stature, microcephaly, distinctive facies, hypotonic appearance, cardiac and genital anomalies, and marked skewing of X-inactivation in female carriers. With the advent of next generation sequencing, mutations have been identified that result in less severe phenotypes lacking one or more of these phenotypic manifestations. Here we report five unrelated kindreds in which a c.109C>T (p.R37X) mutation segregates with a variable but overall milder phenotype. The distinctive facial appearance of alpha-thalassemia intellectual disability was present in only one of the 18 affected males evaluated beyond the age of puberty, although suggestive facial appearance was present in several during infancy or early childhood. Although the responsible genetic alteration is a nonsense mutation in exon 2 of ATRX, the phenotype appears to be partially rescued by the production of alternative transcripts and/or other molecular mechanisms.

Familial haplotyping and embryo analysis for Preimplantation genetic diagnosis (PGD) using DNA microarrays: a proof of principle study.

PURPOSE: Development of PGD assays for molecular disorders is based on analysis of a familial mutation together with linked polymorphic STR markers; a process which is lengthy and requires the identification of multiple informative markers prior to PGD analysis. On the other hand, whole genome amplification (WGA), in conjunction with microarray platforms, allows the use of a universal assay for the analysis of a very large number of SNP markers at once. The aim of this study was to test high throughput pre-PGD familial haplotyping for in-case blastomere analysis in order to eliminate time-consuming pre-case preparations for each family. METHODS: A PGD cycle was performed for a couple with paternal Charcot Marie Tooth 1A (CMT1A) using a classic multiplex nested PCR approach. Mutant embryos from the case were blindly reanalyzed, as single or multi-cell biopsies, using a multiple displacement amplification-based WGA protocol and microarray SNP analysis. In parallel, relevant genomic DNA samples from the family were also analyzed by SNP microarray. RESULTS: After applying a 'unique informative allele' selection algorithm to the data, this array-based assay reconfirmed the initial diagnosis in all samples. CONCLUSIONS: We describe a PGD method that is both accurate and feasible during the time-frame required for embryo transfer. This strategy greatly reduces the time for pre-case haplotype preparation.

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.

Polar body-based preimplantation genetic diagnosis for N-acetylglutamate synthase deficiency.

OBJECTIVE: We describe a sensitive and highly reliable preimplantation genetic diagnosis (PGD) assay for N-acetylglutamate synthetase (NAGS) deficiency using polar body (PB) analysis in conjunction with multiple markers flanking the gene. This rare autosomal recessive mitochondrial disorder is characterized by hyperammonemia, uncontrollable movements, developmental delay, visual impairment, failure to thrive and vomiting and is caused by mutations in the NAGS gene located on chromosome 17q21.31. METHODS: For a family with an affected child we have developed a multiplex fluorescent PCR protocol that included detection of the specific familial mutation (2729insC) in conjunction with the analysis of five informative polymorphic markers flanking the gene: D17S902, D17S965, D17S1861, D17S791 and D17S1868. Following successful amplification in single-cell fibroblasts, this protocol was used in the couple carriers of NAGS mutation. RESULTS: Of 18 retrieved eggs, 16 were at the M2 stage and 9 fertilized. 12 polar body 1s (PB1) were heterozygotes, 1 homozygote wild-type, 1 total amplification failure, and two showed inconclusive results. Three oocytes that had heterozygote PB1s showed mutant polar body 2 (PB2) indicating a wild-type oocyte. Despite the fact that the specific 2729insC mutation did not amplify in the PGD cycle, analysis of linked markers in PBs was sufficient to ensure an accurate diagnosis in 5 out of 9 oocytes. This cycle resulted in the transfer of 3 embryos originating from oocytes diagnosed as wild-type by PB analysis, with the subsequent birth of healthy twin girls. Postnatal genetic testing revealed that both girls harbored the healthy maternal allele and carried the mutant paternal allele. CONCLUSIONS: Our multiplex-nested PCR protocol based on several linked microsatellite markers offers an efficient and accurate method for PGD for NAGS syndrome even when the mutation is not amplified.

Attitudes of couples identified through screening as carriers of Gaucher disease type 1.

Gaucher disease (GD) type 1 is the most frequent autosomal recessive disorder among Ashkenazi Jews, but because the phenotype is tremendously variable, including it in the 'Ashkenazi Panel' of carrier screening is controversial. As part of a nationwide study conducted in Israel to evaluate the outcomes of carrier screening for GD, we studied the experience of 65/82 (79%) of the couples identified as being at risk for an affected child. We found that pre-test information was regarded as insufficient and improved in post-result counseling. About 70% of the subjects interpreted the genetic counseling as directive, mostly toward prenatal diagnosis (PND) but against pregnancy termination of affected fetuses. We evaluated the various motivations that had led couples to utilize PND. Subjects' attitudes toward pregnancy termination correlated with their specific genotypes, with their perception of the severity of GD and with attending additional medical consultation. Of the 30 interviewed participants who were faced with having an affected fetus, 80% came to terms with their decision to utilize PND, but about half of the few who terminated the pregnancy regret their decision. Despite questionable benefits of screening, most of the participants did not regret having been tested and supported the continuation of this program. We offer explanations for these findings and suggest extensive genetic and medical counseling for any future carrier screening for low penetrance, treatable disease.

Successful polar body-based preimplantation genetic diagnosis for achondroplasia.

Achondroplasia, the most common form of dwarfism, is a candidate for preimplantation genetic diagnosis (PGD) because a single mutation accounts for almost all cases. Multiplex fluorescent assay including the common G380R mutation in the FGFR3 gene and eight close polymorphic markers was developed. First and second polar bodies (PB) were used for PGD analysis. An affected woman was treated with routine long-protocol ovarian stimulation and puncture. In the first PGD cycle, out of four fertilized oocytes, PB analysis revealed two mutant oocytes, one with total amplification failure of the maternal allele and one with inconclusive results. In the second PGD cycle, 14 oocytes were retrieved following a higher FSH dose and by performing oocyte retrieval and by placing the patient in the anti-Trendelenburg position using abdominal pressure to allow all follicles to be drained. Following PB analysis, two embryos containing the wild-type FGFR3 allele were transferred. This led to an uncomplicated pregnancy and delivery by Caesarean section at week 38 of a healthy boy, carrying the FGFR3 wild-type maternal allele. In conclusion, oocyte retrieval, while difficult in patients with achondroplasia, can be successfully performed. PB analysis is a reliable and sensitive method for PGD for maternal achondroplasia.

Advantages of multiple markers and polar body analysis in preimplantation genetic diagnosis for Alagille disease.

OBJECTIVE: The development of a preimplantation genetic diagnosis (PGD) protocol for Alagille syndrome (AGS), a rare autosomal dominant disorder with hepatic, cardiac and ophthalmologic involvement. METHODS: We developed a polar body (PB)-based multiplex fluorescent PCR reaction for a female affected with AGS. The protocol included analysis of the Jagged 1 (JAG1) familial mutation and five closely linked highly polymorphic markers (D20S162, D20S901, D20S894, and D20S186). RESULTS: In two cycles of PGD 9 of ten embryos were accurately diagnosed by assessment of first and second PBs, one embryo required additional blastomere biopsy. CONCLUSIONS: This protocol takes advantage of the larger window of opportunity for transfer and the increased accuracy of diagnosis afforded by the combination of PB biopsy and multiple marker analysis. Two cycles resulted in the transfer of two and three mutation-free embryos and a subsequent pregnancy as measured by the rising hCG levels.

Cancer risks among BRCA1 and BRCA2 mutation carriers.

BRCA1 and BRCA2 mutations increase breast and ovarian cancer risks substantially enough to warrant risk reduction surgery, despite variable risk estimates. Underlying this variability are methodological issues, and also complex genetic and nongenetic effects. Although many modifying factors are unidentified, known factors can already be incorporated in individualised risk prediction.

Single-sperm analysis for haplotype construction of de-novo paternal mutations: application to PGD for neurofibromatosis type 1.

BACKGROUND: Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder caused by mutations in the neurofibromin gene. Approximately, 50% of cases are caused by de-novo mutations. Even when the NF1 mutation is known, accuracy of PGD is highly enhanced by simultaneous analysis of linked markers. In a childless couple referred to PGD, the male carried a de-novo mutation, precluding the possibility of typing relatives to establish the mutation-associated haplotype. We developed a single-sperm haplotype analysis strategy to establish the haplotype linked to the NF1 mutation. METHODS: Spermatozoa from freshly ejaculated semen were used as a substrate for multiplex PCR on single sperm. RESULTS: In addition to the NF1 mutation, six informative polymorphic markers flanking the NF1 gene (D17S1294, D17S1849, D17S841, D17S975, NF1TG2 and NF1AC5) were linked to individual alleles in single sperm from the affected male. CONCLUSIONS: Single-sperm analysis established the haplotypes of both mutant and wild-type NF1 alleles and enabled the implementation of a PGD protocol using polymorphic marker analysis. This method is generally applicable to PGD for any disease in which the haplotype of paternal mutations cannot be determined by typing relatives.

[Combination of heterozygote blood coagulation factor V Leiden carriage with erythrocytosis as a cause of the hip deep vein thrombosis]. / Sochetanie geterozigotnogo nositel'stva leidenskoi mutatsii faktora svertyvaniia krovi s éritrotsitozom kak prichina tromboza glubokikh ven bedra.

AIM: To reject one of the variants of inherited thrombophylia in a 64-year-old patient with deep thrombosis of leg veins and high hemoglobin and red cell levels. MATERIAL AND METHODS: The study was made of antithrombin III and protein C, protein S levels; resistance to activated protein C; molecular structure of DNA coding factor 5; methylenetetrahydrofolate reductase. RESULTS: The patient was diagnosed to have heterozygote factor V Leiden mutation. The replacement of arginine by glutamine in position 506 of factor V molecule raises the risk of thrombosis. This risk was aggravated by high hemoglobin, red cells, hematocrit, low volume of circulating plasma, smoking. The patient had normal levels of leukocytes and platelets, normal spleen size, slightly lowered level of erythropoietin. CONCLUSION: The presence of thrombosis in patients with erythremia or erythrocytosis rejects one of the thrombophilia forms.

Differing pathogenesis of perinatal bilirubinemia in glucose-6-phosphate dehydrogenase-deficient versus-normal neonates.

The objective was to compare the contribution to perinatal bilirubinemia of hemolysis and UDP-glucuronosyltransferase (UGT) gene promoter polymorphism, seen in Gilbert's syndrome, between glucose-6-phosphate dehydrogenase (G-6-PD)-deficient and -normal neonates. Serum total bilirubin (STB) values from 52 G-6-PD-deficient and 166 G-6-PD-normal term, male neonates, sampled within 3 h of delivery (first sample) and on d 3 (second sample), were analyzed in relation to blood carboxyhemoglobin corrected for inspired CO (COHbc), an accurate index of hemolysis, and UGT promoter genotype. COHbc values (% total Hb) were greater in G-6-PD-deficient neonates than controls: first sample 1.00 +/- 0.25% versus 0.84 +/- 0.24%, p < 0.0001; second sample 0.83 +/- 0.20% versus 0.76 +/- 0.19%, p = 0.002. First sample COHbc and STB values did not correlate in either the G-6-PD-deficient or control groups, whereas second sample COHbc values correlated significantly with corresponding STB values in the control population only (r = 0.28, p = 0.0007). At second sampling, there was a higher allele frequency of the variant UGT promoter among those with STB values > or =75th percentile than those <75th among the G-6-PD-deficient neonates (0.60 versus 0.33, respectively, p = 0.025), but not controls (0.31 versus 0.40, respectively, p = 0.24). Among those infants with at least one variant UGT promoter allele, STB values were higher in the G-6-PD-deficient neonates than controls at second sampling only (181 +/- 56 microM versus 149 +/- 46 microM, respectively, p = 0.03). Both within and between the G-6-PD-deficient and control groups, our data demonstrate changing and differing contributions of hemolysis and UGT promoter polymorphism to bilirubinemia during the first 3 d of life.

Neonatal diabetes mellitus, enteropathy, thrombocytopenia, and endocrinopathy: Further evidence for an X-linked lethal syndrome.

We describe an unusual family with a fatal genetic syndrome of neonatal diabetes mellitus (DM), enteropathy, endocrinopathy, and severe infections with variable thrombocytopenia. All affected individuals are male; X-linked inheritance is likely. The most common clinical features are neonatal DM, inanition, and enteropathy; a variety of other autoimmune phenomena are less frequent. Clinical variability within and among families is common, including lack of one or more cardinal features. The syndrome is usually fatal, but survival is sometimes possible with immunosuppressive therapy. Clinical variability and frequent new mutations may contribute to poor recognition and underreporting of similar cases.

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.

X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy.

To determine whether human X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome (IPEX; MIM 304930) is the genetic equivalent of the scurfy (sf) mouse, we sequenced the human ortholog (FOXP3) of the gene mutated in scurfy mice (Foxp3), in IPEX patients. We found four non-polymorphic mutations. Each mutation affects the forkhead/winged-helix domain of the scurfin protein, indicating that the mutations may disrupt critical DNA interactions.