Ethical considerations of population screening for late-onset genetic disease.

Population-based genetic screening has been a mainstay of public health in the United States for many years. The goal of genetic screening is to identify individuals at increased risk for treatable diseases. The evolution of genetic testing to include multi-disease panels allows for new screening applications which challenge the traditional model of clinical genetics care by the identification of late-onset disorders in an asymptomatic fetus, child, or adult. We present two unique examples of individuals referred to a biochemical genetics clinic due to the detection of late-onset Pompe disease by population-based screening modalities. We review early experiences in counseling and management of pre-symptomatic individuals and highlight some of the primary ethical factors warranting consideration as we enter the era of genomic medicine.

Fabry disease: baseline medical characteristics of a cohort of 1765 males and females in the Fabry Registry.

The Fabry Registry is a global observational research platform established to define outcome data on the natural and treated course of this rare disorder. Participating physicians submit structured longitudinal data to a centralized, confidential database. This report describes the baseline demographic and clinical characteristics of the first 1765 patients (54% males (16% aged < 20 years) and 46% females (13% < 20 years)) enrolled in the Fabry Registry. The median ages at symptom onset and diagnosis were 9 and 23 years (males) and 13 and 32 years (females), respectively, indicating diagnostic delays in both sexes. Frequent presenting symptoms in males included neurological pain (62%), skin signs (31%), gastroenterological symptoms (19%), renal signs (unspecified) (17%), and ophthalmological signs (11%). First symptoms in females included neurological pain (41%), gastroenterological symptoms (13%), ophthalmological (12%), and skin signs (12%). For those patients reporting renal progression, the median age at occurrence was 38 years for both sexes, but onset of cerebrovascular and cardiovascular events was later in females (median 43 and 47 years, respectively) than in males (38 and 41 years, respectively). This paper demonstrates that in spite of the considerable burden of disease in both sexes that begins to manifest in childhood or adolescence, the recognition of the underlying diagnosis is delayed by 14 years in males and 19 years in females. The Fabry Registry provides data that can increase awareness of common symptoms in all age groups, as well as insight into treated and untreated disease course, leading to improved recognition and earlier treatment, and possibly to improved outcomes for affected individuals.

The use of acarbose inhibition in the measurement of acid alpha-glucosidase activity in blood lymphocytes for the diagnosis of Pompe disease.

PURPOSE: Acid alpha-glucosidase is present in various tissues, including blood cells. Historically, enzyme measurement in cultured fibroblasts, or muscle, has been the gold standard to confirm a diagnosis of Pompe disease, due to the possibility of alternate isoenzyme activity masking disease in white cell assays. Enzyme measurement in an isolated lymphocyte population with acarbose, an inhibitor of neutral alpha-glucosidase, has greatly improved the sensitivity and specificity of the test in blood cells allowing for more rapid laboratory testing for Pompe disease. METHODS: An assay for acid alpha-glucosidase was performed with and without inhibitor in lymphocytes from 14 patients with a clinical suspicion of infantile Pompe disease. Concurrent testing was performed in fibroblasts in an independent laboratory. RESULTS: Thirteen of 14 patients demonstrated a clear deficiency in lymphocytes with acarbose inhibition. One patient was indeterminate, although below normal activity, suggesting the need for confirmatory testing. Tissue enzyme activity in all was consistent with infantile Pompe disease, and corroborated enzyme activity seen in lymphocytes. There were no false positives for disease, making the positive predictive value of lymphocyte enzyme testing 100%. CONCLUSION: Enzyme assay using acarbose as an inhibitor, can be performed in isolated lymphocytes for rapid diagnosis of infantile Pompe disease.

Direct profiling of multiple enzyme activities in human cell lysates by affinity chromatography/electrospray ionization mass spectrometry: application to clinical enzymology.

We describe a new method for enzyme analysis using affinity capture followed by electrospray ionization mass spectrometry (ACESIMS) for the quantitative determination of the initial velocities of four heparin-modifying enzymes. These enzymes, when defective in affected children, lead to the lysosomal storage disease known as Sanfilippo syndrome. The method relies on substrates and internal standards conjugated to the molecular handle biotin via a heavy isotope-encodable, mass-adjustable linker. Reaction velocities of the Sanfilippo enzymes in a crude lysate prepared from as little as 2500 human skin fibroblasts can be determined. In addition, the ACESIMS method is widely applicable to the simultaneous analysis of multiple enzymes in a complex biological sample by a single analytical technique and will thus serve as a useful tool in basic and clinical biomedical research.

Quantification of cellular acid sphingomyelinase and galactocerebroside beta-galactosidase activities by electrospray ionization mass spectrometry.

BACKGROUND: Diagnosis of Niemann-Pick (A and B) and Krabbe diseases is achieved by measurement of the lysosomal enzymes acid sphingomyelinase (ASM) and galactocerebroside beta-galactosidase (GCG), respectively. Conventional assays use radiolabeled or fluorescent substrates and do not allow simultaneous determination of two or more enzymes in the sample. METHODS: We developed a sensitive and specific method to assay ASM and GCG in skin fibroblast homogenates using biotinylated substrate conjugates. The products were purified by bioaffinity capture on streptavidin-agarose beads and, following release, were analyzed by electrospray ionization mass spectrometry. Quantification was achieved using stable-isotope-labeled internal standards that were chemically identical to the products of the enzymatic reactions. RESULTS: The method demonstrated excellent linearity of ASM and GCG enzymatic product formation with the amount of cellular protein and incubation time. The range of ASM activities in fibroblast lysates from six healthy patients was 39-70 nmol. mg(-1). h(-1) compared with 3.7-5.1 nmol. mg(-1). h(-1) in cell lysates from two patients affected with Niemann-Pick A disease. The GCG activities toward the corresponding substrate conjugate were 4.0-6.8 nmol. mg(-1). h(-1) in cell lysates from healthy patients compared with 0.1-0.2 nmol. mg(-1). h(-1) in cell lysates from two patients affected with Krabbe disease. The amounts of substrate conjugates needed per analysis were 15 nmol (14 microg) for both ASM and GCG. CONCLUSIONS: Electrospray mass spectrometry combined with the use of biotinylated substrate conjugates and bioaffinity purification represents a new approach for the diagnosis of lysosomal storage diseases as demonstrated for Niemann-Pick A and Krabbe diseases. No radioactive substrates are used, and the method uses a single instrumental platform to determine both ASM and GCG in one cell sample.

Mutations in the 4-hydroxyphenylpyruvic acid dioxygenase gene are responsible for tyrosinemia type III and hawkinsinuria.

The enzyme 4-hydroxyphenylpyruvic acid dioxygenase (HPD) catalyzes the reaction of 4-hydroxyphenylpyruvic acid to homogentisic acid in the tyrosine catabolism pathway. A deficiency in the catalytic activity of HPD may lead to tyrosinemia type III, an autosomal recessive disorder characterized by elevated levels of blood tyrosine and massive excretion of tyrosine derivatives into urine. It has been postulated that hawkinsinuria, an autosomal dominant disorder characterized by the excretion of 'hawkinsin,' may also be a result of HPD deficiency. Hawkinsin is a sulfur amino acid identified as (2-l-cystein-S-yl, 4-dihydroxycyclohex-5-en-1-yl)acetic acid. Patients with hawkinsinuria excrete this metabolite in their urine throughout their life, although symptoms of metabolic acidosis and tyrosinemia improve in the first year of life. We performed analyses of the HPD gene in a patient with tyrosinemia type III and two unrelated patients with hawkinsinuria. A homozygous missense mutation predicting an Ala to Val change at codon 268 (A268V) in the HPD gene was found in the patient with tyrosinemia type III. A heterozygous missense mutation predicting an Ala to Thr change at codon 33 (A33T) was found in the same HPD gene in the two patients with hawkinsinuria. These findings support the hypothesis that alterations in the structure and activity of HPD are causally related to two different metabolic disorders, tyrosinemia type III and hawkinsinuria.

The Gaucher registry: demographics and disease characteristics of 1698 patients with Gaucher disease.

BACKGROUND: The Gaucher Registry, the largest database of patients with Gaucher disease (GD) worldwide, was initiated to better delineate the progressive nature of the disorder and determine optimal therapy. This report describes the demographic and clinical characteristics of 1698 patients with GD before they received enzyme replacement therapy. METHODS: Physicians worldwide who treat patients with GD were invited to submit prospective and retrospective data for an ongoing registry, using standardized data collection forms, for central processing and review. RESULTS: Most patients were from the United States (45%) and Israel (17%), but patients are from 38 countries. Most (94%) had type 1 GD, fewer than 1% had type 2, and 5% had type 3. Mutant allele frequency data, available for 45% of patients, showed the most common alleles to be N370S (53%), L444P (18%), 84GG (7%), and IVS2+1 (2%). Twenty-five percent of L444P homozygotes (13 of 52 patients) had type 1 GD phenotype. Mean age at diagnosis in patients with the N370S/N370S genotype was 27.2 years (SD, 19.7 years); in L444P/L444P patients, 2. 3 years (SD, 3.2 years). Histories of bone pain and radiological bone disease were reported by 63% and 94% of patients, respectively; both were more likely in asplenic patients than in patients with spleens. Mean spleen and liver volumes were 19.8 and 2.0 multiples of normal, respectively. Anemia and thrombocytopenia were present in 64% and 56%, respectively. Thrombocytopenia was present in 13% of asplenic patients. CONCLUSIONS: The Gaucher Registry permits a comprehensive understanding of the clinical spectrum of GD because of the uniquely large sample size. The Registry will be useful in evaluating the effects of specific therapies in GD and the possible influences of environment, ethnicity, and genotype on the natural history of the disorder.

Severe type II Gaucher disease with ichthyosis, arthrogryposis and neuronal apoptosis: molecular and pathological analyses.

Severe infantile Gaucher disease associated with ichthyosis and neonatal death is a rare subgroup of Type II Gaucher disease. This group of infants has little, if any, detectable beta-glucocerebrosidase activity, and prior genetic analyses have been limited in detecting the mutations responsible for this phenotype. We document an Hispanic infant succumbing with arthrogryposis and collodion membrane covering the skin who had no detectable beta-glucocerebrosidase activity in tissue samples and who was homozygous for a rare recombinant allele, RecNciI. Microscopic evaluation demonstrated accumulation of Gaucher cells in visceral organs and extensive loss of neurons in the anterior horns, brainstem, and cortex of the nervous system. The apoptosis of neuronal cells from the anterior horns and brainstem are a reasonable explanation for the arthrogryposis and neonatal death, respectively.

Rabies in two privately owned domestic rabbits.

Two privately owned domestic rabbits (Oryctolagus cuniculus) in Maryland were found to be infected with the raccoon variant of the rabies virus in 1998. Both rabbits had an acute onset of anorexia and paralysis or paresis of the left forelimb; 1 also developed head tremors and a head tilt. One of the rabbits became ill 25 days after being attacked by a raccoon (Procyon lotor) and was euthanatized 3 days after onset of illness. The other rabbit, which was housed in an outdoor hutch, died 4 days after onset of clinical signs; the source of infection in that rabbit remains unknown. Currently, there is not a rabies vaccine approved for use in rabbits; thus, the only way to prevent the infection in rabbits is to prevent exposure. Veterinarians in rabies-enzootic areas should be familiar with the clinical signs of rabies in rabbits and should caution rabbit owners about the need to protect their pets from contact with wildlife.

Gaucher disease: recommendations on diagnosis, evaluation, and monitoring.

BACKGROUND: Timely diagnosis and continued monitoring of patients with type I Gaucher disease is critical because skeletal involvement can permanently disable patients and visceral organ involvement can lead to abdominal pain and secondary hematologic and biochemical complications. OBJECTIVE: To seek clinical consensus for minimum recommendations for effective diagnosis and monitoring of patients with type I Gaucher disease. PARTICIPANTS, EVIDENCE, AND CONSENSUS PROCESS: Contributing authors collaborated in quarterly meetings over a 2-year period to synthesize recommendations from peer-reviewed publications and their own medical experiences. These physicians care for most patients with Gaucher disease in the United States and serve as the US Regional Coordinators for the International Collaborative Gaucher Group Registry, the world's largest database for this disorder. CONCLUSIONS: The definitive method of diagnosis is enzyme assay of beta-glucocerebrosidase activity. Schedules differ for monitoring complications of type I Gaucher disease, depending on symptoms and whether enzyme replacement therapy is used. Hematologic and biochemical involvement should be assessed by complete blood cell count, including platelets, acid phosphatase, and liver enzymes, at baseline and every 12 months in untreated patients and every 3 months and at enzyme replacement therapy changes in treated patients. Visceral involvement should be assessed at diagnosis using magnetic resonance imaging or computed tomographic scans. Skeletal involvement should be assessed at diagnosis using T1- and T2-weighted magnetic resonance imaging of the entire femora and plain radiography of the femora, spine, and symptomatic sites. Follow-up skeletal and visceral assessments are recommended every 12 to 24 months in untreated patients, and every 12 months and at enzyme replacement therapy changes in treated patients.

Non-insulin dependent diabetes mellitus in African-American youths of Arkansas.

Non-insulin dependent diabetes mellitus (NIDDM) occurs more frequently in certain adult populations, including African-Americans. Recently an increase in the incidence of NIDDM has been observed among African-American youths in Arkansas. Clinical presentations among these youths vary from asymptomatic to severe diabetic ketoacidosis. From a chart review, data were examined to determine which physical, biochemical, and autoimmune characteristics were most helpful in appropriate classification of NIDDM vs insulin dependent diabetes mellitus (IDDM). It is apparent that several diagnostic and treatment issues need to be addressed to improve the management of African-American youths with NIDDM.

Characteristics of youth-onset noninsulin-dependent diabetes mellitus and insulin-dependent diabetes mellitus at diagnosis.

OBJECTIVE: To describe the characteristics of youth-onset noninsulin-dependent diabetes mellitus (NIDDM) at diagnosis and compare them with youths with insulin-dependent diabetes mellitus (IDDM) when matched for age, sex, and geographic region of residence. STUDY DESIGN: Medical records of youths referred for evaluation of diabetes to a pediatric tertiary care center from 1988 to 1995 were reviewed to identify youths diagnosed with NIDDM. Patients selected for study met National Diabetes Data Group criteria for type of diabetes. RESULTS: Fifty patients with NIDDM were reviewed and compared with similar IDDM patients. The NIDDM female:male ratio was 1.6:1 and 74% were African-American. Only 18% of the IDDM patients were African-American. The mean body mass index +/- standard error at diagnosis of NIDDM patients was 35 +/- 1.1 kg/m in contrast to IDDM, 20 +/- .8 kg/m. Ninety-six percent of NIDDM and 24% of IDDM youths had a body mass index >/=85th percentile. More then 30% of NIDDM youths presented with hypertension. Diabetic ketoacidosis was present in >25% of NIDDM patients. Acanthosis nigricans was documented in 86% of NIDDM and 0% of IDDM patients. CONCLUSIONS: In Arkansas, youths with NIDDM are characterized by significant obesity in contrast to youths with IDDM. Physical characteristics such as obesity, acanthosis nigricans, and hypertension on examination of any youth with new-onset diabetes should raise suspicion of NIDDM.

The identification of a (CGG)6AGG insertion within the CGG repeat of the FMR1 gene in Asians.

We have evaluated the structure of the CGG repeat within the FMR1 gene of an Asian population and found the most common size of the repeat to be 29 and 30 with a minor population of 36 repeats. We have isolated and sequenced DNA containing the 36 repeats and found the basis sequence to be (CGG)9AGG(CGG)9AGG-(CGG)6AGG(CGG)9; with a (CGG)6)AGG insertion, designated as 9A9A6A9. Of 144 Asian chromosomes, 11 (8%) had sequences with this insertion. Six different variations of the basic sequence were observed in the population: 9A9A6A2A9, 9A9A6A11, 9A9A16, 9A9A15, 8A9A6A6A9, and 11A6A6A9. All but one of the chromosomes with the insertion had the haplotype of DXS548/ FRAXAC1: 194/D suggesting that the sequences with the 6A insertion arose from a single ancestral allele. We have not observed the insertion in the FMR1 gene of Caucasians or Native Americans. The (CGG)6AGG insertion may be unique to Asians.

Population genetics of the D4S139, D10S28, D17S74 and D17S79 VNTR loci among Asian, black, Caucasian, Hispanic and Native American populations from Seattle.

We studied four variable number of tandem repeat (VNTR) loci (D4S139, D10S28, D17S74, and D17S79) in five ethnic populations from the Seattle metropolitan area. DNA samples purified from randomly chosen individuals were digested with Hae III or Hinf I and probed with pH30, for D4S139; TBQ7 for D10S28; pCMM86 for D17S74 and pAC256 for D17S79. The allele frequencies, expected Hardy-Weinberg values, observed heterozygosities and genetic distances among the populations were obtained for all these loci. D4S139 restriction fragment lengths (RFLs) varied in size from 1.4 to 22 kilobase pairs (kbp). The observed heterozygosities (H) varied from 84% in Native American populations to 94% among Blacks. D10S28 RFLs varied in size from 650 base pairs (bp) to 10.1 kbp. H varied from 90% in Native Americans to 96% in Caucasians and Hispanics. D17S74 RFLs varied in size from 782 bp to 9.3 kbp. H varied from 87% in Asians to 92% among Blacks. D17S79 RFLs varied in size from 400 bp to 3 kbp. H varied from 87% in Hispanics to 95% in the Black population. The frequencies of genotypes of the loci conformed to Hardy-Weinberg equilibrium with the exception of the D17S79 in Hispanics and Native Americans. The genetic distances between the populations were also determined.

Reliability and validity of breast cancer screening belief scales in African American women.

The purpose of this study was to describe the psychometric development of culturally sensitive scales to measure beliefs related to mammography and breast self-examination screening. Health Belief Model constructs of susceptibility, benefits, barriers, and self-efficacy were refined from previously existing instruments to reflect cultural sensitivity. A total of 329 African American women 45 to 64 years of age were included in the sample. Construct validity was tested using confirmatory factor analysis and testing of theoretical hypotheses. Cronbach alpha reliability coefficients ranged from .65 to .90, and test-retest reliability ranged from .40 to .68.

Lactose metabolism and time to pregnancy.

OBJECTIVE: To investigate whether, in the absence of galactosemia, relatively high intestinal lactase activity or low activity of an enzyme involved in galactose catabolism reduces fertility, as it does in the presence of galactosemia. DESIGN: Retrospective cohort study. SETTING: Healthy women selected from the community. PATIENTS: Fifty-three married women. INTERVENTION: Urinary galactose after an oral lactose challenge (a measure of intestinal lactase activity), erythrocyte galactose-1-phosphate uridyltransferase (transferase) activity, and transferase polymorphisms by isoelectric focusing. MAIN OUTCOME MEASURE: Pregnancy rate (number of pregnancies divided by number of months at risk) in the 12 months after stopping use of birth control to become pregnant. RESULTS: Relatively high urinary galactose was not related to a decreased rate of pregnancy during the first 12 months (> or = 24.6 compared with < or = 14.3 mg: relative risk [RR] = 1.9; 95% confidence interval [CI] = 0.86 to 4.0). Relatively high transferase activity was not related to an increased rate of pregnancy (> or = 19.5 compared with < or = 17.2 mumol/h per g hemoglobin: RR = 1.1; 95% CI = 0.56 to 2.4). Low-activity transferase polymorphisms were not related to a decreased rate (RR = 1.2; 95% CI = 0.58 to 2.5). CONCLUSION: Our study does not support the hypothesis that the biologic variation in galactose metabolism that exists in the general population influences infertility.

Bone marrow transplantation in Hunter syndrome.

Hunter syndrome (mucopolysaccharidosis II) is a rare X-linked disorder of mucopolysaccharide metabolism that typically progresses to severe mental retardation and death by 18 years of age. A child with Hunter syndrome received an allogeneic bone marrow transplantation from an unaffected human leukocyte antigen-identical sibling at the age of 29 months without complications. Despite full and sustained engraftment now at 70 months after transplantation, the patient's neurocognitive abilities have continued to deteriorate. In this case, replacement of defective marrow-derived macrophages by bone marrow transplantation was not effective in preventing the neurologic progression of the disease in a child with the severe phenotype of Hunter syndrome.