Carbonic anhydrase polymorphism in a New Jersey population of the white-footed mouse Peromyscus leucopus.

Two electrophoretic forms of erythrocytic carbonic anhydrase were found to be controlled by one autosomal locus with two codominant alleles, CA(f) and CA(8). The gene frequencies for the CA(f) and CA(8) alleles were found to be.15 and.85, respectively, in a sample of 53 mice from Middlesex County, New Jersey. The observed genotypic frequencies indicated that the population was in Hardy-Weinberg equilibrium.

Prenatal diagnosis of the fra(X) syndrome.

The fra(X) syndrome is one of the most common causes of mental retardation, and validation of the reliability and feasibility of making the prenatal diagnosis of this disorder is important for genetic counseling and prevention. We have received a total of 74 amniotic fluid specimens for prenatal diagnosis of fra(X) from worldwide sources. Results were obtained on 68 specimens of which 43 had a documented family history of the fra(X) syndrome. Of the 43 specimens, 23 were male and 4 were prenatally diagnosed as being affected. On the basis of the results, several conclusions follow: 1.) At least 3 different tissue culture methods should be utilized. 2.) At least 150 cells should be scored, preferably 50 from each of 3 different tissue culture methods or 100 from each method if less than 3 methods are used. 3.) While the test appears to be reliable, it should still be considered to be experimental until larger numbers are obtained with completed follow-up of cases.

Prenatal cytogenetic diagnosis of the fragile X chromosome: feasibility and speed of in situ clonal method in amniotic fluid cell tissue culture.

We have had experience with over 300 amniotic fluid specimens for prenatal diagnosis for the fragile X chromosome [fra(X)], and the flask method of tissue culture has been routinely utilized requiring extended tissue culture periods of 3-4 weeks. The use of the in situ clonal method of tissue culture for routine prenatal cytogenetic diagnosis of amniotic fluid cells has shortened tissue culture time and resulted in more rapid reporting; however, it has not been widely employed for fra(X) prenatal diagnosis. The simultaneous use of both methods of tissue culture has resulted in the detection of 2 cytogenetically fra(X) positive cases in amniotic fluid, with more rapid reporting and satisfactory expression of the fra(X) with the in situ clonal method. Thus, the use of the in situ clonal method of tissue culture for fra(X) prenatal diagnosis in amniotic fluid cells is feasible, faster and can serve as a more rapid cytogenetic adjunct to the newer DNA testing methods.

Prenatal cytogenetic diagnosis of the fragile X syndrome in amniotic fluid: calculation of accuracy.

We have completed over 350 prenatal diagnoses for the fragile X [fra(X)] syndrome using amniotic fluid, chorion villus specimen (CVS), fetal blood sampling and molecular methods. A total of 300 amniotic fluid specimens have been received for prenatal diagnosis of the fra(X) syndrome. There was a documented family history of fra(X) in 170/300 amniotic fluid cases, and 23/170 were correctly identified as cytogenetically fra(X) positive (16 male; 7 female). Three males were false-negative, and one female was fra(X) negative but identified as a probable carrier by RFLPs. No fra(X) positive or false-negative results were found in the absence of a fra(X) family history. Because the a priori risk for the fra(X) syndrome for each pregnancy was different and widely variable, the determination of the accuracy of the prenatal diagnosis results requires a consideration of these variables. On this basis, the calculated accuracy of prenatal cytogenetic diagnosis for the fra(X) syndrome is approximately 97%. This accuracy can be improved further with the simultaneous use of molecular methods, especially in view of recent developments.

Prenatal diagnosis of fragile X syndrome: results from parallel molecular and cytogenetic studies.

Since 1985, we have provided coordinated DNA-based and cytogenetic prenatal analysis for couples at risk for offspring afflicted with the fragile X [fra(X)] syndrome. To date, 40 pregnancies have been studied (22 males, 18 females). There were 5 males and 3 females identified to be at high risk by DNA but only 2 males and one female were demonstrated to be cytogenetically expressing the fra(X) prenatally. Of the other 3 males, one was a cytogenetic false negative (i.e. confirmed fra(X)+ at termination of pregnancy). The other 2 remain fra(X)- and are developing normally (undetected recombinants or non-penetrant male carriers). All fetuses at low risk were carried to term and are reported to be normal.

Prenatal diagnosis of the fragile X syndrome: possible end of the experimental phase for amniotic fluid.

We have had experience with 260 prenatal diagnosis cases for the fragile X syndrome [fra(X)]; amniotic fluid was received in 230. There was a documented family history of fra(X) in 148 amniotic fluid cases. Our sample includes 91 males. Eleven were correctly identified as fra(X)-positive and 2 were false-negative. Eight of 57 females were fra(X) positive and one was a false-negative. CVS were received in 21 cases with a family history of fra(X), and there were 2 positive results in females and 3 false-negative results in males which were ultimately detected by means of molecular analysis or a subsequent amniotic fluid specimen. RFLPs were utilized in 29 cases (amniotic fluid and CVS); RFLPs identified 2 false-negative cytogenetic results in CVS. Two male fetuses were found to have a high probability of being affected by means of RFLPs, but on the basis of prenatal and postnatal negative fra(X) cytogenetic results and subsequent normal growth and development, they are either unaffected transmitting males or are double recombinants. Three female fetuses were also found to be cytogenetically negative in CVS but had a 90%, 93%, and 99% probability of being affected by RFLPs. On the basis of the data, it can be concluded: 1. Amniotic fluid experience is adequate to eliminate the "experimental" designation providing the limitations are understood and an experienced laboratory is involved. 2. Chorionic villus cells for cytogenetic analysis should still be considered experimental. 3. Negative results with CVS should be confirmed by molecular methods and/or by cytogenetic analysis of another tissue. 4. Multiple approaches can maximize reliability of fra(X) prenatal diagnosis.

Multiple meningiomas in a patient with constitutional ring chromosome 22.

We report on a patient with multiple congenital anomalies and ring chromosome 22 who died at age 16 years of bronchopneumonia. Autopsy documented multiple psammomatous meningiomas of the spinal dura and tentorium. Tumor tissue for cytogenetic analysis was not available. Although abnormalities of chromosome 22 in tumor tissue have been reported, to our knowledge, this is only the third report of a constitutional chromosome 22 abnormality associated with the development of meningiomas. Thus, a constitutional chromosome 22 abnormality may predispose to the development of meningiomas.

Kallmann syndrome associated with complex chromosome rearrangement.

We report on a male with Kallmann syndrome (KS) and an apparently balanced complex chromosome rearrangement (CCR): 46,XY,t(3; 9)(9;12)(q13.2;q21.2p13;q15). This is the first known report of a CCR in the KS and the second reported case of a definitive autosomal chromosome abnormality with KS. Possible relationships between the cytogenetic abnormality and KS are discussed.

Experience with multiple approaches to the prenatal diagnosis of the fragile X syndrome: amniotic fluid, chorionic villi, fetal blood and molecular methods.

We have had experience with 160 prenatal diagnosis cases for the fragile X syndrome [fra(X)] or Martin-Bell Syndrome. In 140, amniotic fluid was utilized; 98 had a documented family history of fra(X). The 94 completed cases included 4 no growth; 56 males of which 7 were fra(X)-positive and 2 false-negative; 38 females of which 5 were fra(X) positive. There was no fra(X) positive result when a family history of mental retardation was not documented as fra(X). Molecular methods (RFLPs) were utilized in 10 amniotic fluid and 5 chorionic villus specimens (CVS). Percutaneous umbilical blood sampling was used in 2 negative cases and 1 fra(X) positive case because of timing, tissue culture failure or confirmation of another method. CVS were received in 13 cases, and RFLPs were utilized in 5 of the CVS cases. There was no positive fra(X) CVS chromosome result in males, 1 positive result in a female, but 2 false negatives were detected by RFLPs. On the basis of the results, it can be concluded that cytogenetic and molecular methods are complementary and best used together and that multiple approaches can enhance the efficiency and reliability of fra(X) prenatal diagnosis.

Deletion of 16q with prolonged survival and unusual radiographic manifestations.

Deletion of 16q is characterized by mental retardation, microcephaly, a characteristic combination of minor facial anomalies, and broad halluces. Various break points have been described. This patient's phenotype is typical of this syndrome, but in addition, unusual radiographic findings were present. This chromosome abnormality is compatible with survival into adulthood. Expression of this phenotype does not appear to be correlated with specific break points.

DNA-based genetic testing in fifty fragile X families.

During the past 4 years (1985-1989), we have analyzed 171 cases in 50 fragile X [fra(X)] families by DNA linkage methods. Most (140 cases; 81%) were for carrier detection, both female (98 cases; 57%) and male (41 cases; 24%). Women who were obligate carriers of the fra(X) mutation accounted for an additional 6 "prior-to-pregnancy" cases. Four pregnancies have subsequently occurred with 3 having been successfully monitored (one male, 2 females). One pregnancy miscarried early prior to testing. Prenatal diagnoses (26 cases; 15%) accounted for the remainder of cases (15 males, 11 females). These will be discussed in the companion paper by Shapiro et al. (Am J Med Genet, 1991). A diagnosis in the cytogenetically uninformative carrier cases was reached in greater than 75% of analyses with a panel of 5 probes: 3 proximal (F9, DXS105, DXS98) and 2 distal (F8, DSX52). Five additional probes, 3 proximal (DXS10, DSX51, DSX102) and 2 distal (DSX15, DXS33), were used in cases that were resistant to analysis with the standard panel. In 60% of cases, flanking markers were identified (proximal and distal). Given this panel, only 5% of cases did not have any informative markers identified. Thus, molecular methods can provide a useful adjunct to cytogenetic analysis in most situations. An unusual association between the rare allele (A1) of DXS10 with the X chromosome carrying the fra(X) mutation was observed. This occurred in both male and female carriers in the uppermost generation tested. The basis for this association is uncertain at the present time.

Cytogenetic diagnosis of the fragile X syndrome: efficiency, utilization, and trends.

In order to assess the impact of the increasing awareness of the fra(X) syndrome and a broader approach to fra(X) testing, we analyzed our laboratory experience for 1980-1988. In 1981-1986, there was an average of 80 cases/year (62 male; 18 female). The 103 (74 male; 29 female) cases in 1987 represent a 45% increase over the prior 3 years; this sustained in 1988 with 106 cases. The fra(X) positive yield decreased from a high of 49% in 1980 to an average of 20% (range 15-24%) in 1981-1984, 10% (range 9-11%), in 1985-1987 and 7% in 1988. The positive rate for males and females was nearly identical in both time periods. The positive yield for mentally retarded individuals with a family history of mental retardation dropped from an average of 20% for 1981-1984 and 33% for 1985-87 to 13% for 1988; however, the positive fra(X) rate for mentally retarded individuals decreased from an average of 23% in 1981-1984 to 9% in 1985-1987 and 7% in 1988. The decreasing fra(X) yield and increasing case load are directly attributable to the relaxation of criteria for referral and testing related to the referral of all mentally retarded patients, and to the perceived malpractice liability for not doing a "complete" evaluation. Although the burden for cytogenetic laboratories is considerable, the yield of positive fra(X) cases is still worthwhile, and may be maximized by the use of improved screening criteria.

The use of early simultaneous percutaneous umbilical blood sampling (PUBS) and amniocentesis for prenatal fragile X chromosome diagnosis.

Early simultaneous percutaneous umbilical blood sampling (PUBS) and amniocentesis for prenatal diagnosis were undertaken for the first time in a 17-week gestation fetus at risk for the fragile X [fra (X)] syndrome. Metaphase spreads from 300 fetal lymphocytes were examined within 5 days following PUBS, while approximately 5 weeks were required for the analysis of 148 amniocytes. The chromosomes were interpreted as normal (46,XX) and the fetus as fragile X-negative at the time of prenatal diagnosis. This was cytogenetically confirmed after delivery of a healthy term female infant. Our results suggest that early PUBS may become a useful adjunct to amniocentesis because of shorter culture time and earlier diagnosis.

Asymmetry of methylation with FMR-1 full mutation in two 45,X/46,XX mosaic females associated with normal intellect.

The full FMR-1 mutation is known to cause the fragile X syndrome [Fra(X)], but variable expression in females, including normal to deficient intellect, may be related to random X-inactivation (lyonization). We have evaluated 2 mosaic 45,X/46,XX females who are cytogenetically fra(X) positive, have an FMR-1 full mutation, and are of normal intellect. There were 50% fra(X) chromosomes in the 45,X cells of one of the females; this has not been reported previously. In both patients, there was a strong asymmetry of FMR-1 methylation with the normal allele being totally or 90% unmethylated and the mutant allele being similarly methylated. Thus, the apparent selective inactivation of the full mutant FMR-1 allele appears to have resulted in limited expression with normal intellect. The presence of the fra(X) chromosome in 45,X cells is unique; however, there may be no relationship to the asymmetric inactivation of the mutant allele which could be due to chance or a mechanism yet to be delineated.

The karyotype of Philadelphia chromosome-negative, bcr rearrangement-positive chronic myeloid leukemia.

Philadelphia (Ph) chromosome negative chronic myeloid leukemia (CML) can be distinguished from clinically similar disorders on the basis of the presence of rearrangement of the breakpoint cluster region (bcr) of chromosome 22. We have identified six patients with Ph-negative CML, each with bcr rearrangement. Apparently normal karyotypes were observed in two cases, and a third contained a rearrangement that did not appear to involve chromosomes 9 or 22. The other three cases had translocations involving chromosome band 9q34 but no case contained the common derivative chromosome 9pter----9q34::22q11----22qter. One case appeared to contain either a deletion of an unrearranged bcr locus in approximately 50% of cells or duplication of rearranged bcr, both 5' and 3' of the chromosome 22 breakpoint. Considerable complexity exists in the types of genetic changes that can juxtapose bcr and the c-abl oncogene in CML. Based on the molecular and cytogenetic analyses of these and other cases described in the literature, we conclude that most cases of true Ph-negative CML arise from submicroscopic genetic exchanges rather than masking of simple t(9;22)(q34;q11) translocations by secondary rearrangements.

Establishment of new human prostatic cancer cell line (JCA-1).

The establishment of a new human prostatic cancer cell line is described. This cell line was derived from a poorly to moderately differentiated prostatic adenocarcinoma. It has been maintained in tissue culture for fourteen months and has been passed fifty-two times. This cell line has an ability to form colonies in soft agar suspension cultures, and also is transplantable to nude mice. Tumors grown in nude mice revealed a poorly differentiated adenocarcinoma with positive PSA staining. Acid phosphatase activity was detected in freeze-thawed cells by enzymatic assay. A karyotype analysis demonstrated aneuploidy with a model chromosomal number of 69 and six marker chromosomes.

Disomic balanced reciprocal translocation.

The previously unreported and unique finding of a complete disomy of an apparently balanced reciprocal translocation is described. The parents are second cousins once removed and each parent contributed the same balanced reciprocal translocation chromosome. Although the complete disomy involves balanced translocation chromosomes from unaffected parents, it is possible that a hemizygous state of some genes may be present on each translocation chromosome, which in a disomic homozygous state could result in an abnormal phenotype, as manifested by infantile seizures in this patient.

Molecular markers of fragile X: clinical aspects of carrier detection and prenatal diagnosis.

In our families, a determination of carrier or affected status was made in more than 75% of cases using a standard panel of five marker systems: three proximal (F9, DXS105, DXS98) and two distal (F8, DXS52). Five additional systems, three proximal (DXS10, DXS51, DXS102) and two distal (DXS15, DXS33), were used in cases resistant to analysis with the standard panel. In 60% of cases, flanking markers were identified (proximal and distal). Utilizing the complete panel, only 5% of cases did not have any informative markers identified. In order to facilitate the appropriate application of molecular methods, several simple rules should be followed by the genetic service provider when dealing with fra(X) families: 1. Cytogenetic prescreening of females may be helpful. Only negative or ambiguous fra(X) expression levels justify the labor-intensive DNA-based family studies. 2. At present, DNA-based studies are the only way to ascertain male carrier status. 3. Every effort should be made to perform DNA-based studies under maternal phase-known conditions. 4. Information should be collected and pedigrees prepared for both sets of maternal grandparents. 5. Fathers of female consultands should be directly DNA-typed to rule out non-paternity. 6. Genetic counseling should be conservative, i.e. the "worst" scenario presented to these families, in order to avoid underestimating the risk of transmission to the next generation.

Prader-Willi syndrome and Robertsonian translocations involving chromosome 15.

A case of Prader-Willi syndrome is presented in which high resolution chromosome analysis revealed not only a familial Robertsonian translocation [t(13q15q)], but also a del(15) (q11.2q13) of the chromosome 15 not involved in the translocation. While there have been numerous reports of Robertsonian translocations involving chromosome 15 in patients with Prader-Willi syndrome, in this case, the Robertsonian translocation was shown to be unrelated to the clinical findings.