Identification of a 55-bp deletion in the glucocerebrosidase gene in Gaucher disease: phenotypic presentation and implications for mutation detection assays.

A 55-bp deletion in exon 9 of the glucocerebrosidase gene was identified in a 28-year-old male affected with Gaucher disease. The diagnosis was established during an evaluation for mild pancytopenia and was confirmed by bone marrow histology and biochemical studies. The patient is of German ancestry. Initial DNA testing indicated homozygosity for the N370S mutation. However, subsequent testing of the patient's parents suggested that the patient and his mother carried a null allele by our assay for N370S. Further molecular studies identified a 55-bp deletion in exon 9 of the glucocerebrosidase gene (g.6767_6822del55). This deletion has been previously reported in a patient with severe Gaucher disease (1), and is present in the glucocerebrosidase pseudogene. In the previously reported case, initial DNA testing also suggested the genotype N370S/N370S, but further mutation studies were undertaken because clinical severity was greater than expected for that genotype. In contrast, our patient has an unusually mild clinical course. Thus, clinical severity cannot be reliably used to determine when to test for the presence of the 55-bp deletion. While the 55-bp deletion is not reported to be common, its actual frequency may be underestimated since it eludes detection by many standard clinical assays for Gaucher disease. This report points out the need to consider this deletion mutation which may cause erroneous interpretation of results in existing assays for the common mutations N370S and L444P. Furthermore, the importance of recommending parental analysis for individuals who test homozygous for autosomal mutations is highlighted.

Screening semen donors for hereditary diseases. The Fairfax cryobank experience.

OBJECTIVE: To study the carrier frequency of hereditary diseases in potential semen donors with no family history of a genetic disease. STUDY DESIGN: Carrier screening was performed on potential semen donors for chromosomal abnormalities, cystic fibrosis, alpha-1-antitrypsin deficiency, hemoglobinopathies, Tay-Sachs disease, Gaucher disease, Canavan disease, and hereditary breast and ovarian cancer (the BRCA1 185delAG mutation). The screening regimen used for each donor was dictated by his ethnic background. RESULTS: Among 361 individuals screened for chromosomal abnormalities, 1 carried an inversion, and 4 were possible mosaics. Fifteen of 407 potential donors carried cystic fibrosis, 18 of 209 carried alpha-1-antitrypsin deficiency, and 2 of 74 carried a hemoglobinopathy. No carriers of Tay-Sachs disease (56 screened), Gaucher disease (32 screened), Canavan disease (22 screened) or the BRCA1 185delAG mutation (22 screened) were found. CONCLUSION: Screening semen donors for a number of genetic diseases that are passed silently from generation to generation is warranted since family history alone cannot identify them.

Prenatal determination of genotypes Kell and Cellano in at-risk pregnancies.

OBJECTIVE: To evaluate the accuracy of a DNA-based testing methodology in determining the KEL1 and KEL2 (Kell and Cellano) genotype of fetuses at risk for Kell or Cellano hemolytic disease. STUDY DESIGN: DNA was extracted from chorionic villus samples (CVS) or amniotic fluid (AF) cells, a portion of the Kell gene was amplified, the amplified product was cut with a restriction enzyme that recognizes the KEL1 nucleotide substitution, and the digested product was run on a polyacrylamide gel to separate the fragments. This analysis was routinely run on uncultured cells to provide rapid results. Testing of parental DNA was performed in conjunction with fetal analysis to ensure that their alleles were detectable with this DNA test. RESULTS: We determined the fetal KEL1 and KEL2 genotype in 1 CVS and 65 AF specimens. Forty-eight of them were determined to be KEL2, 17 were KEL1/2, and 1 was KEL1. Among the fetuses born to date, follow-up information was available on 14 of them, 11 KEL2 and 3 KEL1/2. In all 14 there was complete correlation between the DNA analysis and the serotype or clinical course. CONCLUSION: Determination of the fetal KEL1 and KEL2 genotype using this DNA-based method provides accurate and timely information that can aid the prenatal care of women sensitized to these Kell antigens.

A fragile X mosaic male with a cryptic full mutation detected in epithelium but not in blood.

Individuals with developmental delay who are found to have only fragile X premutations present an interpretive dilemma. The presence of the premutation could be an unrelated coincidence, or it could be a sign of mosaicism involving a full mutation in other tissues. To investigate three cases of this type, buccal epithelium was collected on cytology brushes for Southern blot analysis. In one notable case, the blood specimen of a boy with developmental delay was found to have a premutation of 0.1 extra kb, which was shown by PCR to be an allele of 60 +/- 3 repeats. There was no trace of a full mutation. Mosaicism was investigated as an explanation for his developmental delay, although the condition was confounded by prematurity and other factors. The cheek epithelium DNA was found to contain the premutation, plus a methylated full mutation with expansions of 0.9 and 1.5 extra kb. The three populations were nearly equal in frequency but the 1.5 kb expansion was the most prominent. Regardless of whether this patient has clinical signs of fragile X syndrome, he illustrates that there can be gross tissue-specific differences in molecular sub-populations in mosaic individuals. Because brain and epithelium are more closely related embryonically than are brain and blood, cryptic full mutations in affected individuals may be evident in epithelial cells while being absent or difficult to detect in blood. This phenomenon may explain some atypical cases of the fragile X phenotype associated with premutations or near-normal DNA findings.

Molecular fragile X screening in normal populations.

In December, 1993, we initiated a pilot project in which DNA fragile X (fraX) testing was offered during routine prenatal or genetic counseling to all pregnant women seen at the Genetics & IVF Institute, most of whom were referred for the indication of advanced maternal age. A brochure on fragile X syndrome was sent to each patient prior to her appointment and was reviewed by a counselor or physician during the counseling session. As of June 1995, 3,345 patients were offered testing; 474 women with no identified family history of mental retardation or learning disability and 214 women with a positive family history accepted the test on a self-pay basis. The second population screened was 271 potential donors in our anonymous egg donor program. DNA from blood was tested by Southern blot using EcoRI/EagI and StB12.3. If an expansion was detected, CGG repeat number was determined by PCR-based analysis. Among the 474 patients with unremarkable family histories, three fraX carriers were identified (repeat sizes = 60+), whereas none were found in the 214 patients with a positive family history. Among the potential egg donors, two high borderline patients were identified (repeat sizes = between 50 and 59). Our ongoing study indicates that screening of pregnant or preconceptual populations for fraX carrier status using DNA testing is accepted by many patients and is an important addition to current medical practice.

DNA-based prenatal determination of the RhEe genotype.

BACKGROUND: Maternal antibodies to RhE may cause severe hemolytic disease. Based on recent RhD and RhCE sequence information, we have developed a DNA-based testing methodology to determine the RhEe genotype of fetuses at risk for RhE hemolytic disease from amniotic fluid (AF) or chorionic villus samples. CASE: RhEe testing was undertaken in a fetus at risk for RhE hemolytic disease. Maternal serum anti-E titers had risen between 12-15 weeks' gestation. Optical density (OD450) AF readings also rose slightly between 22-24 weeks' gestation. Both maternal serum titers and AF bilirubin measurements provided early indications that the fetus might have the RhE antigen. Using amniotic cells obtained at the first amniocentesis, DNA was extracted and analyzed for the RhE gene sequence. The use of two primer pairs from distinct sites in the RhCE gene, plus analysis of parental DNA, greatly minimized the possibility of false results. The fetus was determined to be Rhe/Rhe by molecular analysis. The DNA result was confirmed by serologic typing at birth. CONCLUSION: DNA-based RhEe genotyping of at-risk fetuses provides accurate and timely information that is useful in the management of RhE-sensitized pregnancies.

Recent advances in reproductive genetic technologies.

New possibilities for the diagnosis and treatment of reproductive and genetic disorders are becoming available as a result of a series of recent technical advances. Intracytoplasmic sperm injection (ICSI) allows treatment of numerous infertile men whose sperm cannot penetrate the egg to initiate fertilization. Molecular genetic testing provides clients of reproductive age with additional information that permits prevention of genetic diseases such as fragile X syndrome, the leading cause of inherited mental retardation. Preimplantation genetic testing (PGT) offers couples who carry genetic disorders the prospect of having children with a greatly decreased risk of initiating a pregnancy involving an affected individual. Flow-cytometric sperm separation offers a new, effective approach for prevention of X-linked genetic disorders. Two major causes of recurrent pregnancy loss (RPL) involve recurrent trisomies and immunological disorders. Of the latter, 70% of studied populations of patients can attain live births with simple treatment protocols. Maternal serum assays involving multiple markers reduce both false positives and false negatives in detection of trisomies. Despite these advances in research, many safe and effective methods of diagnosis and treatment remain under-utilized in the clinical arena.

Molecular analysis of the RhD genotype in fetuses at risk for RhD hemolytic disease.

The objective of this study was to evaluate the accuracy of a DNA-based testing methodology in determining the RhD genotypes of fetuses at risk for RhD hemolytic disease. We designed a multiplex polymerase chain reaction-based test based on recent RhD and RhCE sequence information. To improve the accuracy of the results, two different portions of the RhD gene were examined. Deoxyribonucleic acid was extracted from fetal specimens, portions of the RhD gene were amplified by the polymerase chain reaction, and the amplified product was run on a polyacrylamide gel to look for the presence or absence of the RhD gene. We tested 67 amniotic fluid and two chorionic villus specimens to determine the fetal RhD genotype in pregnancies at risk for RhD hemolytic disease. Forty-seven of the 69 specimens were determined to be Rh-positive, and 22 were Rh-negative. Fifty of the 69 fetal specimens--31 Rh-positive and 19 Rh-negative--were serotyped at birth. In all 50, there was complete correlation between the DNA analysis and the serotyping results. RhD gene analysis is a rapid and reliable method that provides an accurate fetal genotype to aid in the prenatal care of RhD-alloimmunized women.

Prenatal diagnosis in known fragile X carriers.

Prenatal diagnosis for fragile X syndrome was performed in 34 pregnancies of 33 known carriers, on 22 chorionic villus samples (CVS), and 15 amniocentesis samples. Fetal and maternal DNA were analyzed by the EagI/EcoRI Southern blot of Rousseau et al. [1991: N Engl J Med 325:1673-1681], with detection of full mutations ensured by a second loading with brief electrophoresis. As a supplemental assay for full mutations, cytogenetic induction was performed in 20 cases. Positive cytogenetic results were helpful in confirming full mutations in CVS cases where the fetal DNA was intermediate in appearance, between a large premutation and a small full mutation. Of 8 mothers with full mutations, the fetal results were 5 full, 2 normal, and 1 premutation (whose mother was a full/pre compound heterozygote). Of 26 mothers with premutations, the fetal results were 5 full, 13 normal, 7 premutation, and 1 uninterpretable (maternal contamination). Maternal premutations were sized in kb by Southern blot and in CGG repeat number by PCR; the predicted correlation between maternal length and penetrance was seen. Follow-up studies include 3 full mutations and 2 premutations confirmed by DNA analysis at birth. Maternal contamination of CVS samples was encountered in 3 of 22 cases, illustrating the value of EagI in detecting maternal (lyonized) chromosomes.

Molecular confirmation of alpha 1-antitrypsin genotypes in newborn dried blood specimens.

Deficiency of alpha 1-antitrypsin (alpha 1AT), a common hereditary disorder of Caucasians, is associated with an increased risk for early-onset chronic obstructive pulmonary disease and childhood liver dysfunction. The two most common deficiency variants, PiS and PiS, are both single base-pair substitutions causing amino acid modifications, although neither mutation creates or destroys a naturally occurring restriction site. Dried blood specimens (DBS) submitted to the New York State Department of Health for mandated newborn screening tests were tested for alpha 1AT activity using a fluorometric elastase inhibition assay. A second DBS from specimens determined to be alpha 1AT deficient was phenotyped on an agarose isoelectric focusing gel. Genotypic confirmation was performed by amplifying, directly from a DBS, the regions of the DNA containing the S and Z mutation. The Z mutation was analyzed with a modified primer designed to create an artificial restriction site in the normal allele. TaqI digestion produces two bands, a 157- and a 22-bp fragment. The single base substitution in PiS individuals eliminates this TaqI restriction site, thus showing the same 179-bp fragment before and after digestion. A primer mismatch placed close to the S mutation creates a restriction site in the normal allele, producing a 100-bp product after TaqI digestion. The restriction site is abolished in individuals that carry the S mutation, with a 121-bp product observed before and after digestion. Of 11,081 specimens screened, 3 PiS neonates, all Caucasian, were detected by these methodologies for an estimated incidence of 1:2019 in the Caucasian or 1:3694 in the general population in New York State.(ABSTRACT TRUNCATED AT 250 WORDS)

Neonatal screening for cystic fibrosis: addition of molecular diagnostics to increase specificity.

Newborn screening for cystic fibrosis (CF) has been carried out on approximately 106,000 neonates in western Pennsylvania since 1987 using the immunoreactive trypsinogen (IRT) assay on dried filter paper blood specimens (DBS). Molecular analysis utilizing a duplicate DBS from the same sample was implemented in November 1989 for newborns having elevated IRT levels. DNA is amplified directly from the DBS and the amplified products are tested for the delta F508 deletion and several common exon 11 mutations. Substituting dUTP for dTTP in the PCR reaction and an initial treatment with uracil N-glycosylase (UNG) virtually eliminates PCR carryover contamination. The number of confirmed cases of CF is 20, giving an estimated incidence of 1:5287 in the western Pennsylvania population. Eight of the CF patients are homozygous and 12 are compound heterozygotes for the delta F508 deletion and a second mutation. Two of the compound heterozygotes carry the G551D mutation and one has the R553X mutation. Twenty-one additional neonates that are heterozygous for the delta F508 mutation are normal carriers for CF. In approximately 55% of the cases, molecular analysis of the CF gene confirmed the diagnosis of CF prior to sweat testing. The incorporation of molecular analysis into our CF screening program increases the specificity of the screening strategy and has the potential to decrease the false positive and sweat test referral rate, reduce parental anxiety, and bring CF infants to the attention of physicians more rapidly.

Psychosocial characteristics of institutionalized adolescents: resilient or at risk?

The primary purpose of this investigation was to provide a psychosocial description of 187 institutionalized adolescents (ages 11-17). The findings suggest that these adolescents may not be as psychologically dysfunctional as is commonly surmised, considering their accumulated life events, family problems, and current displacement. Although the literature is replete with studies that describe characteristics purported to typify at-risk adolescents, there is no substantive demographic base concerning institutionalized adolescents. The authors acknowledge the limitations inherent in their sample and, consequently, offer their findings as a "place to start," and encourage others to follow this line of research.