Mutational analysis of DAX1 in patients with hypogonadotropic hypogonadism or pubertal delay.

Although delayed puberty is relatively common and often familial, its molecular and pathophysiologic basis is poorly understood. In contrast, the molecular mechanisms underlying some forms of hypogonadotropic hypogonadism (HH) are clearer, following the description of mutations in the genes KAL, GNRHR, and PROP1. Mutations in another gene, DAX1 (AHC), cause X-linked adrenal hypoplasia congenita and HH. Affected boys usually present with primary adrenal failure in infancy or childhood and HH at the expected time of puberty. DAX1 mutations have also been reported to occur with a wider spectrum of clinical presentations. These cases include female carriers of DAX1 mutations with marked pubertal delay and a male with incomplete HH and mild adrenal insufficiency in adulthood. Given this emerging phenotypic spectrum of clinical presentation in men and women with DAX1 mutations, we hypothesized that DAX1 might be a candidate gene for mutation in patients with idiopathic sporadic or familial HH or constitutional delay of puberty. Direct sequencing of DAX1 was performed in 106 patients, including 85 (80 men and 5 women) with sporadic HH or constitutional delay of puberty and patients from 21 kindreds with familial forms of these disorders. No DAX1 mutations were found in these groups of patients, although silent single nucleotide polymorphisms were identified (T114C, G498A). This study suggests that mutations in DAX1 are unlikely to be a common cause of HH or pubertal delay in the absence of a concomitant history of adrenal insufficiency.

Mutation analysis of the EMX2 gene in Kallmann's syndrome.

OBJECTIVE: To investigate the possibility that a mutation in the human EMX2 gene may be involved in Kallmann's syndrome. DESIGN: In vitro experiment. SETTING: Academic Medical Center. PATIENTS: One hundred and twenty patients with Kallman's syndrome or idiopathic hypogonadotrophic hypogonadism (IHH). INTERVENTION: Peripheral blood leukocytes were used to obtain DNA. MAIN OUTCOMES MEASURES: Single-stranded conformational polymorphism (SSCP) analysis was used to identify possible mutations of the EMX2 gene. RESULTS: One hundred and twenty patients with Kallmann's syndrome or IHH, had no mutations noted in this gene. CONCLUSION: It is unlikely that EMX2 mutations are a clinically significant cause of IHH or Kallman's syndrome.

Prenatal diagnosis of Smith-Lemli-Opitz syndrome in a pregnancy with low maternal serum oestriol and a sex-reversed fetus.

A cytogenetically normal male fetus was subsequently found to have female external genitalia, a cardiac malformation and mid-trimester intra-uterine growth retardation by ultrasound examination. The maternal serum oestriol level was low. The combination of low oestriol and sonographic findings suggested Smith Lemli Opitz syndrome (SLO), which was confirmed by a markedly increased amniotic fluid level of 7-dehydrocholesterol. We review the differential diagnosis of apparent sex reversal in a fetus and low maternal serum oestriol level. To further examine the specificity of low maternal oestriol level as a marker for SLO a follow-up study of 12141 pregnancies screened for Down syndrome using three biochemical markers: alpha-fetoprotein, beta-human chorionic gonadotrophin and oestriol was performed. 26 pregnancies had an oestriol level that was 0.25 MoM or less. SLO was not diagnosed clinically in any of the liveborn children ascertained through a low maternal oestriol level. Nine of the pregnancies ended in spontaneous miscarriage. Although the frequency of SLO in pregnancies with low maternal oestriol levels or sex-reversed fetuses is unknown, the diagnosis of SLO should, nevertheless, be considered in both clinical settings.

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.

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.

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.

Improved sizing of fragile X CCG repeats by nested polymerase chain reaction.

We have developed an improved method for polymerase chain reaction (PCR)-based sizing of the CCG repeat region at the fragile X locus, FMR-1. This method is designed to optimize denaturation and replication of long repeats with high G + C content, which are otherwise refractory to amplification. The method utilizes nested PCR primers to increase sensitivity and specificity. Alkaline denaturation of the genomic template DNA, combined with addition of glycerol and deaza-dGTP, facilitates strand separation. Labeled PCR products are sized on denaturing polyacrylamide gels. For alleles in the normal-to-premutation size range, strong reproducible signals are routinely obtained from small amounts of rapidly prepared DNA. This allows precise determination of the CCG repeat number, providing data related to the expansion potential of the repetitive segment. Detection of large premutations and some full mutations is also enhanced by the improved procedure.

Molecular diagnosis of genetic disease.

DNA-based testing is becoming possible for an increasing number of hereditary diseases as the responsible genes are mapped to individual chromosomes and then isolated and characterized. The strategy for each test depends on the heterogeneity of mutations commonly causing the disease, the distribution of the mutations in the population and the frequency of new mutations. In many cases, interpretation of the test result requires a comparison with relatives known to carry the abnormal gene. Sickle cell anemia is an example of a recessive mutation with a strong ethnic association and little genetic heterogeneity or new mutation. Cystic fibrosis also has a strong ethnic association and a low frequency of new mutation but greater heterogeneity. Fragile X affects all ethnic groups and often appears sporadic, but all cases have the same type of mutation, and a premutation can be found in all affected families. In the near future, the extreme sensitivity of DNA analysis will allow testing for these and similar diseases to be performed in vitro on fertilized embryos before implantation.

Prenatal diagnosis and investigation of a fetus with chondrodysplasia punctata, ichthyosis, and Kallmann syndrome due to an Xp deletion.

We report the prenatal diagnosis of a male fetus with X-linked recessive chondrodysplasia punctata (CDPX), steroid sulphatase (STS) deficiency, X-linked Kallmann syndrome (KAL), and a chromosome deletion at Xp22.31. Biochemical analysis of bone from this case indicates that CDPX is not a defect of vitamin K metabolism. Immunocytochemical study of the brain suggests that KAL is a defect in neuronal migration.

The association of congenital skin disorders with acetylcholinesterase in amniotic fluid.

We describe a fetus with epidermolysis bullosa dystrophica and a fetus with aplasia cutis congenita who were normal by careful ultrasound examination but whose midtrimester amniotic fluids exhibited elevated concentrations of alpha-fetoprotein and presence of acetylcholinesterase. These cases show that serious fetal skin pathology can be a source of amniotic fluid acetylcholinesterase and elevated alpha-fetoprotein concentration and should be considered as part of the differential diagnosis of these amniotic fluid findings.