Lesch-Nyhan mutation: prenatal detection with amniotic fluid cells.

Cells cultured from the amniotic fluid of a 22-week fetus in a heterozygote for the X-linked Lesch-Nyhan mutation, which results in neurological and developmental disorders, lacked sex chromatin and were unable to incorporate hypoxanthine. The diagnosis of a mutant male was confirmed upon birth of enzyme-deficient, hyperuricemic twin boys whose amniotic membrane cells failed to incorporate hypoxanthine.

CRASH syndrome: clinical spectrum of corpus callosum hypoplasia, retardation, adducted thumbs, spastic paraparesis and hydrocephalus due to mutations in one single gene, L1.

L1 is a neuronal cell adhesion molecule with important functions in the development of the nervous system. The gene encoding L1 is located near the telomere of the long arm of the X chromosome in Xq28. We review here the evidence that several X-linked mental retardation syndromes including X-linked hydrocephalus (HSAS), MASA syndrome, X-linked complicated spastic paraparesis (SP1) and X-linked corpus callosum agenesis (ACC) are all due to mutations in the L1 gene. The inter- and intrafamilial variability in families with an L1 mutation is very wide, and patients with HSAS, MASA, SP1 and ACC can be present within the same family. Therefore, we propose here to refer to this clinical syndrome with the acronym CRASH, for Corpus callosum hypoplasia, Retardation, Adducted thumbs, Spastic paraplegia and Hydrocephalus.

Fragile X syndrome: recognition in young children.

In recent years, a number of articles have appeared in the literature concerning the fragile X syndrome; however, in few cases was the diagnosis of the syndrome in young children discussed. A review of 20 children younger than 7 1/2 years of age who had the fragile X syndrome seen at the Cincinnati Center of Developmental Disorders was undertaken in an attempt to establish guidelines that would aid the practicing physician in determining which children should have a chromosomal analysis. All children were developmentally delayed; 95% had speech delays. Short attention span with hyperactivity, temper tantrums, mouthing of objects persisting at an age beyond when it would be expected, autistic behaviors, and poor gross motor coordination were seen in 50% or more of the children. Mental retardation was present in the family history of 65%, and 90% had a family history of at least one of the following: mental retardation, learning disabilities, or hyperactivity. The most common physical findings were long and/or wide and/or protruding ears, prominent jaw and/or long face, high arched palate, and a flattened nasal bridge. The fragile X syndrome can be recognized by noting key aspects of the behavioral and family histories as well as the physical findings.

Fragile-X syndrome ascertained by the presence of macro-orchidism in a 5-month-old infant.

The fragile-X syndrome, an X-linked form of mental retardation, is estimated to affect one in every 1,000 to 2,000 live-born male infants. Most commonly, fragile-X syndrome has been detected only after patients clearly demonstrate developmental delay, and frequently detection occurs only if the family history is consistent with X-linked mental retardation. Macro-orchidism is a finding commonly associated with the fragile-X syndrome. It has been suggested that the sparsity of reports of macro-orchidism among prepubertal boys with the fragile-X syndrome might be due to lack of careful measurement of the tests rather than to initiation of the enlargement at puberty. A 5-month-old infant with fragile-X syndrome, ascertained through testicular enlargement noted by actual measurement of testicular size as part of his physical examination, is reported.

Decisions following the intrauterine diagnosis of sex chromosome aneuploidy.

This is a report of 327 phone calls to our center concerning the intrauterine diagnosis of sex chromosome abnormalities (SCA). The first author (A.R.) responded to each by counseling either the parents or the referring professional. Sixty-two percent continued the pregnancy. When the parents were counseled directly, the percentage continuing the pregnancy was significantly higher than when the information was transmitted through the professionals. Our results are different from most reports in these situations, and suggest that well-informed couples, counseled by geneticists, are more likely to continue these pregnancies. The possible occurrence of SCA should be considered in preamniocentesis counseling.

Pentasomy X with multiple dislocations.

We describe a pentasomy X (49,XXXXX) patient whose multiple dislocations led to a consideration of the Larsen syndrome. Review of the 11 reported cases of pentasomy X showed that elbow dislocations are known to occur in this syndrome. Our patient is the first to present hypoplasia of the glenoid process with consequent should dislocation. Clinical and radiologic findings of previously reported cases of pentasomy X are reviewed.

The predictive value of dermatoglyphic anomalies in the diagnosis of fra(X)-positive Martin-Bell syndrome (MBS)

In a representative group of 160 institutionalized mentally retarded males without Down syndrome, prospective dermatoglyphic-cytogenetic studies were performed in order to assess the utility of the dermatoglyphic index system of Rodewald [1986] for an efficient ascertainment of patients with Martin-Bell syndrome (MBS). A negative (abnormal) score was found in 32 men (20 +/- 3%), 14 of whom (predictive value: 44 +/- 9%) were fra(X)-positive. This prevalence of 14/160 = 9 +/- 2% patients with fra(X)-positive MBS indicates that in our study most, if not all, MBS patients have been detected by the simple pre-screening of dermatoglyphics. In the MBS patients, there was no correlation between the dermatoglyphic scores and percentage of fra(X)-positive cells.

A dermatoglyphic study of a group of Sicilian children with fragile-X syndrome.

In a dermatoglyphic study of 14 fra(X) boys (compared with a control group of 191 normal schoolboys), we observed the following statistically significant (p less than 0.01) differences: 1) lower frequency of ulnar loops on the fingertips, particularly on the 2nd and 3rd fingers, with a corresponding increase of whorls; 2) transverse course of main line A; 3) increased frequency of abnormal palmar creases. The log score index of Simpson et al [1984] identified 71.4% of our patients and that of Rodewald et al [1986] 64.2%. The different values of these indexes can probably be attributed to ethnic differences. We think that by combining the results of dermatoglyphic analysis from several centers a more discriminatory log score index can be obtained.

Prenatal diagnosis of the fragile X--the Australasian experience.

Identification of increasing numbers of females heterozygous for fragile X linked mental retardation together with improved genetic counselling is creating a growing demand for prenatal diagnosis of fra(X). However, present cytogenetic techniques are somewhat unreliable and our collaborative approach has endeavoured to improve quality of cell culture systems and the sensitivity of fra(X) detection. Since 1985 we have exchanged cell cultures between our laboratories for verification of diagnostic results and comparison of induction techniques and have benefitted from larger numbers of cells scored for fra(X). Our 50 cases represent almost all of such studies undertaken in Australasia (Australia and New Zealand). Ten cases were unequivocally fra(X) positive; there was discrepancy between laboratories in 4 cases and one false-negative case. We propose a protocol to enhance fra(X) detection and conclude that, provided care is exercised, couples at risk of a fra(X) pregnancy can benefit from prenatal cytogenetic diagnosis.

Esophageal dysmotility in brothers with an FG-like syndrome.

We present 4 brothers with developmental delay, minor anomalies, and symptoms due to gastrointestinal dysmotility. There was some resemblance with FG syndrome, although none of the brothers had sufficient findings to make this diagnosis. The index case presented with at age 1 month with screaming episodes, mild gastro-esophageal reflux (GER), and severe constipation. Esophageal manometry studies were consistent with the diagnosis of "nutcracker esophagus." Symptomatic and manometric improvement followed treatment with oral calcium channel blockers. Two older and less severely affected brothers had similar manometric findings but did not require treatment. A fourth brother with symptoms in infancy now has normal esophageal manometry findings. These boys in all likelihood have an X-linked syndrome with manifestations of FG syndrome, in which treatment with calcium channel blockers, produces clinical and manometric improvement. The FG syndrome is an X-linked syndrome of multiple congenital anomalies/mental retardation with facultative manifestations of gastrointestinal dysmotility, including gastro-esophageal reflux, severe feeding difficulties, and constipation. Esophageal dysmotility, in particular "nutcracker esophagus," should be suspected in infants with the FG syndrome and screaming attacks.

Validity of cytogenetic analyses from trophoblast tissue throughout gestation.

Cytogenetic findings in the Münster Chorionic Villi Sampling (CVS) program are presented after 1,184 first trimester transcervical samplings and 131 second and third trimester placentacenteses. In the first trimester series the abnormality rate is low (2.4%) in patients with only an age-dependent aneuploidy risk. In this group terminations were performed in only 1.6% because of aneuploidy. True mosaicism was found more frequently after CVS, and the risk of maternal cell contamination seems higher as compared to amniocentesis. There are no obvious differences in the overall rate of diagnostic errors after both procedures, when metaphases after direct preparation and chorionic cell cultures are analysed and doubtful findings such as mosaicism are adequately followed up by amniocentesis. The cytogenetic techniques also offer a very rapid approach to karyotyping in the second and third trimester. We found a high rate of aneuploidy (15%) when placentacentesis was performed after sonographic diagnosis of fetal abnormalities. We conclude that cytogenetic analysis from trophoblast tissue is an accurate diagnostic tool applicable from first to third trimester of pregnancy.

Lack of X inactivation: loss of one X inactivation center in a case with mos45,X,-21, +der(21)t(X;21) (p21.3;p11.2)/46,X,t(X;21) (p21.3;p11.2).

We present a girl with a mos45,X,-21, +der(21)t(X;21) (p21.3;p11.2)/46,X,t(X;21) (p21.3;p11.2) chromosome constitution. The ratio of these cells was 59/26 in phytohemagglutinin (PHA)-stimulated lymphocytes. The 45,X,der(21)t(Xp-;21p+) cells lacked an X inactivation center located at Xq13 on the derivative X chromosome; in these cells, the whole normal X chromosome and the distal part of Xp translocated onto the derivative chromosome 21 were early replicating. She had moderate mental retardation and other findings different from those that occur in the Ullrich-Turner syndrome. Her phenotype may be due to the functional excess of the distal part of Xp on the derivative 21 in 45,X,der(21)t(Xp-;21p+) cells; thus, this might be another type of the "lack of X-inactivation" syndrome.

The prenatal detection of the fragile X chromosome: review of recent experience.

The fragile X chromosome has been identified in specimens from 17 male and 10 female fetuses in 11 laboratories throughout the world, obtained from at least 79 fetuses at increased risk for the fra(X) syndrome. Of these, 19 were confirmed, 6 were pending, 1 was negative and 1 could not be confirmed. Twenty-five of the 79 cases were studied in our laboratory (Institute for Basic Research [IBR]) and resulted in fra(X) demonstration in specimens from 3 male and 5 female fetuses. All 3 males and 2 of the 5 females have been confirmed. When amniocytes from the two confirmed female fetuses were exposed to FUdR after culturing in Chang medium, fra(X) frequencies were virtually negative indicating that Chang medium should not be used in fragile X studies at least when FUdR is used to induce fragility. Finally, amniocytes from a fra(X) male fetus studied in 3 different laboratories exhibited strikingly different frequencies. To date, we have experienced no false-positives or negatives, but the latter case was controversial. It is recommended that laboratories undertaking fra(X) prenatal detection use a combination of at least two different proven induction systems as well as complementary DNA marker studies to prevent false negative diagnosis.

Second trimester prenatal diagnosis of the fragile X.

The fra(X) chromosome was detected in 5 samples of amniotic fluid cells in a series of 23 pregnancies at risk. The prenatal results were confirmed in 2 male abortuses, one with a relatively high and one with a very low frequency of expression in both amniocytes and fetal tissue. In a third male fetus with low expression in amniocytes, the fra(X) was not detected in the fetal tissues tested. In another male with low expression in amniocytes the fra(X) was not detected after birth. In one female with a low expression in amniocytes, a very high frequency (28%) was detected in cord blood after birth. Low expression of the fra(X) was found in a 4-year-old normally developed girl, where the prenatal results had been negative. In 4 males and 4 females the negative prenatal diagnoses were confirmed after birth. This study indicates that prenatal diagnosis of the fragile X after amniocentesis may be complicated, either due to technical problems related to the use of amniotic fluid cells, or due to genetic heterogeneity, or both. Part of this heterogeneity could be due to the existence of normal male transmitters. Also, it seems that the frequency of expression in amniocytes from female carriers can not be used for the prediction of the frequency in blood after birth.

Characterization of new PCR based markers for mapping and diagnosis: AC dinucleotide repeat markers at the DXS237 (GMGX9) and DXS102 (cX38.1) loci.

Genomic insert DNAs from 45 probes representing 113.4 kb of the X chromosome were screened for AC dinucleotide repeat sequence. Two new AC repeat sequences were identified with length polymorphism based on variation in repeat copy number. One at DXS237 exhibits 44% heterozygosity and is potentially useful for rapid diagnosis and mapping of X-linked disorders in Xp22.3. The other, at DXS102 in Xq26, has 71% heterozygosity. This marker will improve accuracy of diagnoses by linkage for families with Börjeson-Forssman-Lehmann syndrome. Review of the literature has identified 31 PCR based markers on the X chromosome, with minimum heterozygosity of 50%, applicable to the mapping and diagnosis of X-linked disorders.

Application of fluorescent in situ hybridization with X and Y chromosome specific probes to buccal smear analysis.

Conventional X- and Y-chromatin and fluorescent in situ hybridization (FISH) analysis based on X- and Y-chromosome specific probes were conducted from buccal smear, on 15 normal males, 15 normal females, and 9 cases suspected of sex chromosome anomalies. The proportion of X- and Y-chromatin in normal females and males was 12% +/- 3% and 51.5% +/- 4.9%, respectively, by the conventional X- and Y-chromatin procedure. The CEP-X/Y analysis by FISH for the same specimens provided a proportion of 98.8% +/- 0.7% cells with XX signals in the normal females and 99.8% +/- 0.4% cells with XY signals in the normal males. The FISH method was superior to the conventional procedure in nine cases suspected of sex chromosome anomalies, including one case of mosaicism. The results of CEP-X/Y will sometimes be false; it will not detect structural anomalies of sex chromosomes, and it is not intended to detect low level mosaicism. However, the test is useful for rapid screening of sex chromosome aneuploidy at a fraction of the cost for chromosome analysis. The FISH test is also appropriate to detect tissue specific sex chromosome mosaicism, especially if it is relatively high. This FISH test is best used as an adjunct to chromosome analysis whenever possible.

Parents' adaptation to early diagnosis of sex chromosome anomalies.

In 56 structured psychiatric interviews parents were asked to describe their experience as participants in the Denver prospective study of children with sex chromosome anomalies in order to assess its impact on attitudes toward the identified child and on family relationships. It was found that most achieved satisfactory understanding of the diagnosis with minimal disturbance, preferred early disclosure, denied its influence on parent-child and parent-parent relationships, and were reasonably comfortable in sharing diagnostic information with the child. Environmental and cultural factors did not correlate with the responses obtained. Emphasis directed toward obstacles in the adaptive process permitted evaluation of reported parental anxieties arising from faulty or delayed communication of the diagnosis, a child's adjustment to problems of growth and development, and, for parents of children with 45,X and 47,XXY chromosome constitutions, anxiety regarding anticipated difficulty in sexual maturation and fertility. The assessment interviews afforded additional opportunity for clinical discussion and counseling with parents on issues of concern to them.

Partial fra(X) phenotype with megalotestes in fra (X)-negative patients with acquired lesions of the central nervous system.

We describe 3 men with acquired lesions of the central nervous system (one male with tumor of the third ventricle, two males with peri- or postnatal accidents). In all three patients macroorchidism was associated with facial characteristics similar to those found in fra(X) males, but they were fra(X)-negative. The finding of megalotestes associated with a partial fra(X) phenotype in the present patients and, more particularly, the documentation of a hypothalamic tumor in the first patient, suggests that a specific hypothalamic lesion in fra(X) males is responsible for some of their manifestations.

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.

Differential expression of fragile site Xq27 in cultured fibroblasts from hemizygotes and heterozygotes and its implications for prenatal diagnosis.

Expression of the fragile site Xq27 (fraXq27) was studied in metaphases derived from fibroblasts of 8 hemizygotes and 2 heterozygotes, cultured in 2 different media containing reduced concentrations of folic acid. The proportion of fra(X) (q27)-positive metaphases ranged from 1.0 to 14%, which is considerably lower than in lymphocyte cultures from the same individual, but differed with respect to the culture medium used. Four hemizygotes showed a higher proportion of fra(X)-positive cells in medium 199 than in methotrexate-exposed cultures. No fra(X)-negative cultures were observed when the folic acid inhibitor methotrexate was added to medium 199. Thus, in all 10 individuals carrying the fra(X), the fragile site could be detected in fibroblast cultures. We conclude that the expression of fra(X) (q27) in cultured fibroblasts should be studied using at least 2 types of culture conditions, because familial, i.e. genetic differences may influence the expression of this fragile site in fibroblasts. The implications for prenatal diagnosis are discussed on the basis of investigations in 15 pregnancies at risk for the trait.