Early achievement and maintenance of the therapeutic goals using velaglucerase alfa in type 1 Gaucher disease.

INTRODUCTION: Therapeutic goals have been described to monitor achievement, maintenance and continuity of therapeutic response in patients with type 1 Gaucher disease receiving enzyme replacement therapy. AIM: To benchmark the impact of velaglucerase alfa treatment against therapeutic goals for 5 key clinical parameters of type 1 Gaucher disease (anemia, thrombocytopenia, hepatomegaly, splenomegaly and skeletal pathology). METHODS: In an open-label Phase I/II study, twelve adults with symptomatic type 1 Gaucher disease and intact spleens received velaglucerase alfa for 9 months (60 U/kg infusion every other week [EOW]). Eleven patients completed the study and 10 enrolled in a long-term extension. After 1 year, patients who achieved ≥ 2 hematological or organ goals began step-wise dose reduction from 60 to 45 then 30 U/kg EOW. Data for anemia, thrombocytopenia, hepatomegaly, splenomegaly and skeletal pathology at baseline and 4 years are available for 8 patients (3 male, 5 female). The proportion of patients at goal for anemia, thrombocytopenia, hepatomegaly and splenomegaly at baseline was compared with the proportion achieving each goal at 4 years. The proportion achieving the skeletal pathology goal was determined on the basis of Z-score improvement from baseline to 4 years. The proportion of patients who achieved all 5 goals at 4 years was compared with the proportion at goal for all 5 parameters at baseline. RESULTS: At baseline, no patient was at goal for all clinical parameters. After 1 year of treatment, all patients maintained goals present at baseline, and all achieved ≥ 2 goals. All 8 patients began step-wise dose reduction from 60 to 30 U/kg EOW between 15 and 18 months. By year 4 of treatment, all patients met goals for all 5 clinical parameters; therefore 100% achievement was seen for each of the 5 long-term, therapeutic goals. DISCUSSION: In this velaglucerase alfa Phase I/II and extension study, clinically meaningful achievement of each long-term, therapeutic goal was observed for each patient, despite dose reduction after 1 year. This is the first report of a cohort where all patients receiving ERT for type 1 Gaucher disease achieved all 5 of these long-term, therapeutic goals within 4 years of starting treatment and after ≥ 2years dose reduction.

A very good death: measuring quality of dying in end-stage renal disease.

A case is presented of an exceptionally good death after discontinuation of dialysis, and the authors trace the evolution of their attempts at measuring quality of dying in patients with end-stage renal disease. The Dialysis Quality of Dying Apgar is based on the pediatric tool for measuring the condition of newborn babies. Previous research with termination of dialysis has revealed that staff, patients, and families characterize a good death as being pain-free, peaceful, and brief. The quality of dying tool has corresponding domains to which it adds advance care planning and non-pain symptoms. Quantification of patient deaths combined with descriptive narratives can be used to establish benchmarks for the provision of terminal care. Very good deaths need to be recognized and valued as goals for palliative medicine.

A rapid immunoassay predicts BRCA1 and BRCA2 mutations in buccal cells.

We developed a rapid, non-invasive, and inexpensive, assay capable of identifying BRCA1 and BRCA2 (BRCA) mutations in buccal cells. To determine the predictive value of this immunoassay, a double blind study of 13 high risk individuals was conducted by two independent teams. As greater than 90% of BRCA mutations result in protein truncations, a diminished anti-carboxy immunoreactivity relative to anti-amino immunoreactivity was scored as predictive for mutation. Comparison to BRCA DNA analysis was undertaken. The positive and negative predictive values were 90% and 100% respectively (p<0.02), suggesting great promise as an inexpensive and rapid screen for BRCA mutations.

Immunohistochemical analysis of BRCA2 expression in normal human buccal cells.

Previously we demonstrated that protein coded by the BRCA1 gene was expressed in normal human buccal cells. The present study confirmed that BRCA2 protein was similarly expressed in these cells. Messenger RNA for BRCA2 was detected with sequential use of two primer sets. Pooled cell samples from healthy donors reacted strongly with two commercially available antibodies, I17 and C15. Immunoreactivity was present in both cytoplasmic and nuclear compartments. We conclude buccal cells will provide a suitable model for exploration of normal BRCA function.

An antibody assay predictive of BRCA1 mutations in ovarian tumors and normal tissue.

To determine whether immunohistochemistry can identify BRCA1 mutations, immunohistochemical (IH) analysis was undertaken on paraffin sections of paired ovarian cancer and normal tissue using antibodies against both terminal regions of the BRCA1 protein. Ten patients at risk for BRCA1 mutations were studied. The results of BRCA1 mutation analysis and IH were compared. In tumor, IH correctly identified the presence or absence of loss of heterozygosity in all specimens. In all uninvolved specimens, IH correctly identified the presence or absence of a germline mutation. The sensitivity, specificity, positive and negative predictive values were 100% suggesting promise as a rapid and inexpensive screen.

Spontaneous conversion to estrogen receptor expression by the human breast epithelial cell line, MCF-10A.

The human breast epithelial cell line, MCF-10A, derived from tissue from a woman undergoing a cutaneous mastectomy for fibrocystic breast disease, is negative for estrogen receptor expression, has undergone minimal genetic changes, retains many of the characteristics of normal breast epithelium and fails to exhibit growth in nude mice. When transfected with a functional copy of the estrogen receptor, both ER and MDM2 expression are negatively regulated by the presence of increasing concentrations of estradiol, as previously reported. We obtained the MCF-10A cell line from the American Type Culture Collection and confirmed that it was negative for ER expression. After approximately 20 passages under differing growth conditions, one subline was determined to be positive for ER expression. Growth of this ER-positive subline in phenol red-free media supplemented with charcoal-dextran stripped serum in the presence of nanomolar concentrations of estradiol failed to modulate ER and MDM2 expression, and induced expression of both pS2 and cathepsin D. Simultaneously with these observations, we observed that this subline, unlike the parent MCF-10A line, overexpressed P53 protein with a nuclear localization. Intermediate levels of the P53-inducible protein p21 WAF1/Cip1 were also detected in the ER-positive subline whereas levels of this protein in the parent subline were barely detectable, as measured by immunohistochemical methods. We conclude from these studies that ER expression and P53 alteration may constitute early steps in progression of malignant potential for breast cancer development.

Impact of genetic counseling on primary and preventive care in obstetrics and gynecology.

OBJECTIVE: To evaluate the utility of the prenatal three-generation pedigree in assessment of the obstetric patient's primary medical risks. STUDY DESIGN: In a case series, 250 charts of patients referred for amniocentesis on the basis of advanced maternal age were reviewed for a significant genetic risk of a primary care disorder. RESULTS: A total of 40 patients (16%) were at significantly increased risk for a primary care disorder. Thirty-eight patients (15.2%) were at increased risk for medical conditions for which early screening, detection and/or intervention are established. CONCLUSION: For the advanced maternal age population, formal genetic risk assessment performed prior to amniocentesis can be beneficial in primary care risk assessment.

Prenatal diagnosis and clinical features of an individual with tetrasomy 18p and trisomy 18q mosaicism.

Prenatal diagnosis and clinical follow up of a patient with mosaicism for anomalies of chromosome 18 are reported. The fetus appeared on ultrasound to have multiple anomalies, including clubbed feet, abnormal hand positioning, edema of the scalp, cleft palate, and polyhydramnios. The karyotype on amniocytes was 47,XY,+i(18p). Postnatally, the peripheral blood karyotype was 46,XY,+i(18q), whereas the skin fibroblast karyotype was 47,XY,+i(18p). The infant had many features consistent with those previously described in cases of tetrasomy 18p and some that were consistent with trisomy 18q.

Immunohistochemical analysis of BRCA1 expression in normal human buccal cells.

While tumor suppressor properties of BRCA1 are well documented, less is understood concerning the normal function of this protein in healthy adult tissues. We demonstrate here that BRCA1 protein is expressed in human buccal cells. Expression of BRCA1 mRNA transcripts in buccal cells was confirmed by PCR of reverse transcribed RNA. Three BRCA1 antibodies, D20, MS110 and MS13, reacted positively with cytospin deposited buccal cells, demonstrating apparent cytoplasmic and nuclear distribution of BRCA1 protein. Double antibody immunofluorescent, binding of D20 and MS110 was differentially distributed. We conclude that buccal cells provide a cell source for exploration of BRCA1 protein function in normal human adults.

The usefulness of a prenatal genetic questionnaire in genetic risk assessment.

OBJECTIVE: To evaluate a prenatal questionnaire as a genetic screen and as an aid in pre-amniocentesis genetic risk assessment. METHODS: In a retrospective cohort study, charts were reviewed for 158 consecutive women of advanced maternal age referred for genetic counseling. Genetic risks identified by use of a questionnaire completed by 79 consecutive patients were compared with those risks identified by the referring physician, those identified during subsequent three-generation pedigree analysis, and to genetic risks identified by pedigree evaluation of 79 consecutive individuals who underwent genetic counseling without the aid of a questionnaire (controls). RESULTS: Sixteen (20%) of the questionnaires revealed a previously unidentified genetic risk. The sensitivity and specificity of the questionnaire were determined to be 40.0 and 97.4%, respectively. Pedigree analysis alone (control group) identified significantly more at-risk pedigrees than did the questionnaire alone (34 versus 20%, P < .05), but identified significantly fewer at-risk pedigrees than obtained from the study group patients who completed a questionnaire and pedigree evaluation (34 versus 50.6%, P < .05). Of all 158 patients, 15.2% (n = 24) underwent additional testing on the basis of genetic risk assessment. There was no difference between the study and control groups in additional evaluations performed (P = 1.0). CONCLUSION: A three-generation pedigree is superior to a questionnaire in genetic risk assessment. The questionnaire was not sufficiently sensitive to serve independently as an adequate genetic screen or risk assessment tool and did not influence subsequent fetal evaluation. Assessment of the sensitivity and specificity of prenatal genetic questionnaires should be undertaken before their routine clinical use.

The importance of genetic counseling before amniocentesis.

OBJECTIVE: To determine the adequacy of genetic risk assessment among primary care providers and to evaluate the efficacy of genetic counseling before "routine" genetic amniocentesis. STUDY DESIGN: A retrospective cohort study was undertaken. Charts of 275 consecutive patients referred for genetic counseling and amniocentesis on the basis of advanced maternal age (AMA) were compared with charts of 103 consecutive patients referred for an abnormal maternal serum alpha-fetoprotein (MSAFP) finding. Pedigree information obtained during counseling of these patients was compared with the family histories charted by the referring physician. RESULTS: In 35.6% of pedigrees evaluated, a significant genetic risk was discovered during genetic consultation that had not been noted by the referring physician. Furthermore, 9.8% of AMA patients and 10.7% of patients with abnormal MSAFP results underwent additional genetic testing or screening on the basis of genetic counseling. Additional genetic testing of 0.8% of amniotic fluid specimens was done on the basis of the genetic risk assessment elicited during counseling. Although a significant difference in increased genetic risk was observed between the AMA and abnormal MSAFP groups (AMA 30.8% positive, MSAFP 48.5% positive; relative risk 0.81, confidence limit 0.70 to 0.93), no significant difference was observed between the two groups with regard to patient interventions (relative risk 0.97, confidence limit 0.79 to 1.21) or amniotic fluid testing (p = 0.57, not significant). CONCLUSIONS: The data support the importance of genetic counseling before amniocentesis. Furthermore, the findings support the relevancy and usefulness of genetic counseling in more accurately ascertaining genetic risk and in maximizing the benefits of genetic evaluation of patients seemingly at low risk for other genetic diseases.

Differences in clinical expression of hypertrophic cardiomyopathy associated with two distinct mutations in the beta-myosin heavy chain gene. A 908Leu----Val mutation and a 403Arg----Gln mutation.

BACKGROUND: The disease gene for hypertrophic cardiomyopathy (HCM) has been identified as the beta-myosin heavy chain (beta-MHC) gene in some HCM families. We describe extensive clinical evaluations in two kindreds with two distinct point mutations in the beta-MHC gene. METHODS AND RESULTS: We used single-strand confirmation polymorphism (SSCP) gel analysis of polymerase chain reaction-amplified products capturing each of the 40 beta-MHC gene exons to identify distinct missense mutations in two HCM kindreds. Clinical, ECG, and echocardiographic studies were performed in the two kindreds: kindred 2755 with amino acid 908Leu----Val mutation and kindred 2002 with amino acid 403Arg----Gln mutation. The morphological appearances of HCM were similar in these two kindreds. However, the two kindreds differed with respect to disease penetrance, age of onset of disease, and incidence of premature sudden death. Twelve of 31 adults (greater than or equal to 17 years) with the disease gene in kindred 2755 did not have left ventricular hypertrophy (LVH), and only five of these had ECG abnormalities. Thus, the disease penetrance in adults with this mutation was only 61%. None of 11 children aged less than 16 years had LVH. The 908 mutation was associated with a low incidence of cardiac events: Only two sudden deaths and one syncope occurred in 46 individuals with the mutant allele. In contrast, LVH was present in all 11 adults in kindred 2002 with the 403 mutation (100% disease penetrance). In addition, three of four affected children were symptomatic and had clinical evidence of HCM. The disease in this kindred was severe and resulted in six premature sudden deaths. Seven additional patients had syncope or presyncope. CONCLUSIONS: In some kindreds, the HCM disease gene is more prevalent than indicated by echocardiography and ECG. Some point mutations may be associated with a more malignant prognosis. Preclinical identification of children with mutations associated with a high incidence of sudden death and syncope provides the opportunity to evaluate efficacy of early therapeutic interventions.

Blood exposure in single versus double gloving during pelvic surgery.

In light of continued reports suggesting the inadequacy of surgical gloves as sterile barriers, as well as an increasing number of reports describing intraoperative cross infections, a prospective study was performed comparing the presence of visible blood on the hands of surgeons wearing single or double gloves during 45 consecutive major obstetric and gynecologic operations. Single-gloved hands revealed the presence of visible blood in 38% of cases (n = 42) whereas visible blood was noted in only 2% of double-gloved hands (n = 48) (p less than 0.001). These results demonstrate that the sterile barrier between surgeon and patient was compromised intraoperatively and that particles the size of red blood cells were able to cross this barrier. In addition, these data suggest single gloving may be less than optimal in maintaining a sterile barrier, as well as strongly suggesting that double gloving can improve the integrity of the patient-surgeon sterile barrier during pelvic surgery.