Lester Weiss

Prenatal identification of a girl with a t(X;4)(p21;q35) translocation: molecular characterisation, paternal origin, and association with muscular dystrophy.

There are 23 females known with Duchenne or Becker muscular dystrophy (DMD or BMD) who have X;autosome translocations that disrupt the X chromosome within band p21. A female with a t(X;4)(p21;q35) translocation was identified prenatally at routine amniocentesis. At birth, she was found to have a raised CK level, consistent with a diagnosis of Duchenne muscular dystrophy. Her cells were fused with mouse RAG cells and the translocated chromosomes were separated from one another and from the normal X chromosome by segregation in the resulting somatic cell hybrids. Southern blot analysis of the hybrids indicated that the translocation occurred on the X chromosome between genomic probes GMGX11 and J-66, both of which lie within the DMD gene. Further localisation with a subfragment of the DMD cDNA clone placed the translocation breakpoint in an intron towards the middle of the gene, confirming that the de novo translocation disrupted the DMD gene. RFLP analysis of the patient, her parents, and the hybrid cell lines showed that the translocation originated in the paternal genome. This brings to six out of six the number of DMD gene translocations of paternal origin, a fact that may be an important clue in future studies of the mechanism by which X;autosome translocations arise.

868 A PRACTICAL METAPHASE MARKER OF THE INACTIVE X CHROMOSOME

The ability to identify the inactivated X chromosome with routine G- or Q-banding would have broad clinical and research applicability. We recently reported that the inactivated X frequently bends or folds in region Xql3Xq21 (Flejter et al, Am J Hum Genet 36:218, 1984). The fold occurs in about 88% of prometaphase, 50% of early metaphase, 30% of midmetaphase, and 10% of late metaphase inactive Xs. In prometaphase, the site of folding includes Xq11.2 and Xq13.3, infrequently extending to Xq21.1. An omega-shaped loop is frequently formed between sub-bands Xq11.2 and Xq13.3. It is paradoxical that the inactive X is the only chromosome identifiable in interphase, yet in metaphase it cannot be distinguished from its active homolog. The specific inactivation-associated fold at region Xq1 resolves that paradox and is a useful marker of the inactive X. 1. The KOP translocation, t(X; 14)(q13; q32), has nearly all of Xq translocated to 14q (Allerdice et al, Am J Med Genet 2:223, 1978). Cell line GM0074 has one normal 14, one Xq-, two der (14) chromosomes, and a Y. One der(14) folded in 10/18 cells scored, confirming translocation of the inactivation center adjacent to 14q distal. 2. Metaphase cells from other primates had a specific fold in the same region as in the human X: at Xq13-Xq21 in 2 gorillas, 1 chimp, 2 pygmy chimps, 1 orang, 1 baboon, 1 rhesus and 1 stump-tail monkey. This is further evidence for evolutionary conservation of the X chromosome. One chimpanzee exhibited the fold at Xq24; we suspect a pericentric inv in this individual. We have not seen a frequent fold in the X of a bat (Tadarida brasiliensis), Chinese hamster (Cricetulus griseus), or rat kangaroo (Potorous tridactylis). 3. Regarding the relationship of X inactivation and intelligence in fra(X)(q28) carriers, we observe that in cells of normal carriers the fold was mostly on the fragile X-positive chromosome, whereas in cells of an affected carrier the fold was mostly on the fragile X-negative chromosome. This is evidence that inactivation of the fragile X chromosome is positively correlated with intelligence in carriers.

A RECOMBINANT CHROMOSOME 9 DERIVED FROM A MATERNAL PARACENTRIC INVERSION: CYTOGENETICS, INTERPHASE NUCLEAR PROJECTIONS, AND IN SITU HYBRIDIZATION USING CENTROMERE AND PARACENUOMERE

The two year old proband has multiple malformations and severe developmental delay. Her karyotype is 46, XY, -9, +rec(9), dup p, inv(9)(q22.1q34.3)mat, with a net duplication of 9pter←q22.1 and deficiency of distal 9q34.3←qter. The rec(9) was derived by two crossovers, one within the inversion loop. The mothers karyotype is 46, XX, inv(9)(q22.1q34.3). Her chromosomes 9 differ in that the #9 with tie inversion has some heterochromatin in the short arm as a normal variant. In the probands rec(9), the variant centromere is now attached to the normal 9q, which indicates that during maternal meiosis there was a crossover between 9ql2 and 9q22. The rec(9) was present and stable in over 300 lymphocyte metaphase cells. Most rec(9)s had one primary constriction at the centromere within the normal 9q segment. This centromere was Cd-positive, and the second centromere was Cd-negative, but 18% of routine Giemsa-stainad cells had two primary constrictions. In the probands fibroblast cells harvested in situ without colcemid, nuclear projections were observed in 10% of interphase cells. Such nuclear projections have been observed whenever a chromosome with one active centromere and one latent centromere is present, suggesting that there was at least some spindle-fiber activity of the latent centrcraere (Am J Hum Genet 26:83, 1974 & Proc Clin Boil Res 26:181, 1978). In situ hybridization with a centromere specific probe (p82H) and a paracentromere specific probe (L6) revealed no differences between the two C-band regions of the rec(9). This suggests that there was no interstitial deletion of heterochromatic or centromeric material. The present case confirms that a stable recombinant chromosome derived fron a paracentric inversion can lead to malformations and suggests that prenatal diagnosis should be made available to families with paracentric inversions.

116 SURGICAL CLOSURE OF PATENT DUCTUS ARTERIOSUS IN CRITICALLY ILL PREMATURE INFANTS

Respiratory distress syndrome(RDS) of prematurity can be complicated by patent ductus arteriosus(PDA) kept open by underlying hypoxia. As RDS clears, a large left to right(L-R) shunt may develop adding pulmonary edema to pre-existing respiratory dysfunction. Since 1978, 27 infants required ductal closure when medical management failed to abolish a significant L-R shunt. The mean birthweight of these infants was 1122 gm with a gestational age of 29 weeks. Most required intubation due to RDS around one hour of life. If the pulmonary edema in these infants could not be controlled medically within 5-7 days, surgical closure was done. The average body weight at surgery was 1034 gm. Two operative deaths (7%) occurred in infants with preexisting renal failure; 3 late deaths (11%) occurred in patients with severe broncho-pulmonary dysplasia(BPD). Postoperative problems were common: BPD-12 cases, hydrocephalus-6, retrolental fibroplasia-5, and necrotizing enterocolitis-2. At mean follow-up of 35 months, 12 were felt to have normal growth and development. Persistent abnormalities were present in the remaining survivors: respiratory dysfunction-5, cerebral palsy-2, deafness-2, and blindness-1. In summary, PDA complicating RDS of prematurity can be closed with low mortality (7%). Although postoperative complications were common, 48% of survivors had normal growth and development at mean follow-up of 35 months.

WHEN ARE ANEUPLOID CELLS CLINICALLY SIGNIFICANT|[quest]|

Some workers suggest a causal relationship between multiple miscarriages or offspring with trisomy 21 and low frequency hyperdiploidy in the parents. Others consider low frequency aneuploidy of no clinical significance. We compared the frequency of aneuploid cells in five groups of subjects, karyotyped from 1978-83: 79 parents of trisomy 21 patients, 164 other 1st and 2nd degree relatives of Downs patients, 702 subjects with multiple miscarriage, 341 phenotypically and karyotypically normal control parents (e.g., parent of a dysmorphic child or member of a translocation family), and 1,165 others (e.g., chromosomally normal dysmorphic and retarded individual, etc.). In all, 47,595 cells were analyzed from 2,451 subjects. A cell was called hyperdiploid only if the extra chromosome(s) was recognizable and structurally normal.We found significant age and sex effects, but no other differences among the five groups of patients. Autosomal hypodiploidy (3.8% of cells) had no between group differences, but 45,X cells were age and sex associated: adult females .33%, adult males .17%, younger females .10%, and younger males .16%. Autosomal hyperdiploidy (.11% of cells) had no group, sex, or age differences. The frequency of X chromosome hyperdiploid cells was age and sex associated: adult females .26%, younger females .00%, adult males .04%, and younger males .00%. Females had a marked increase of X aneuploidy with age:age under 23 had .09% X0 and .00% +X cells,age 23 to 34 had .31% X0 and .21% +X cells, andage 34 to 50 had .64% X0 and .60% +X cells.In summary, the frequency of aneuploid cells was greater in females than males and was positively correlated with advancing age. Such cells were not more frequent in couples with multiple miscarriages or offspring with trisomy 21.

PRENATAL DIAGNOSIS OF A GIRL WITH MUSCULAR DYSTROPHY CAUSED BY DE NOVO t|[lpar]|X;4|[rpar]| |[lpar]|p21;q35|[rpar]|

In May, 1983 we obtained amniotic fluid for chromosome studies from a couple who were both 31 years old. The indication was parental anxiety because the mother worked closely with mentally retarded adults. The amniotic fluid cell karyotype was female with a balanced translocation: 46,X,t(X;4)(p21;q35) with the normal X preferentially inactivated. Alpha fetoprotein and detailed ultrasound examinations were normal. Chromosome studies of the parents were normal, so the translocation apparently arose as a new mutation.Two special circumstances complicated the counseling. First, a de novo rearrangement ascertained by amniocentesis carries roughly a 5% increased risk of birth defects over the general 3-5% population incidence of birth defects, but the available risk estimates do not appear to include the risk of mental retardation without malformation, since follow-up has been sporadic of such cases identified prospectively. Second, in a balanced X/autosome translocation the structurally normal X is genetically inactivated, which allows expression of any abnormal genes on the translocation X. There are at least 13 other girls with Duchenne muscular dystrophy who carry a de novo X/autosome translocation with a breakpoint at Xp21. In the absence of a family history of muscular dystrophy, these findings suggest a point mutation due to the break at Xp21.We advised the parents that the fetus was at some increased risk (of uncertain magnitude) for having malformations, retardation, or muscular dystrophy. The couple continued the pregnancy. At age 3 months, the infant has a normal appearance, developmental milestones and neurologic exam. However, at 24 hours of age her serum CPK was 21,450 IU/L. Subsequent values were 1,260 and 3,100 at 1 and 3 months, respectively. Such neonatal and infancy levels of CPK are strongly suggestive of Duchennes muscular dystrophy.

Mesodermal Induction Defect as a Possible Cause of Ear Malformations

Deafness due to inner ear anomalies is rarely associated with malformations of the auricles. We describe two brothers with profound congenital sensorineural deafness, abnormal vestibular function, normal ossicles, and delayed motor development. Since the external and inner ear originate from distinctly separate structures, the embryogenesis of this malformation association is less clear than in the more common association of external and middle anomalies, where the latter two structures are derived from the first and second branchial arches. The combination of auricular and inner ear anomalies, with sparing of the middle ear structures, can be explained on the assumption that mesodermal induction is responsible for normal differentiation of both the otocyst and of the branchial arch ectoderm. A recessive mutant gene may lead to a deficiency of a mesodermal inducer substance of a target tissue receptor site. A similar mechanism may be involved in other multiple malformation syndromes, whereby a mutant gene acting during a specific period of organogenesis causes disruption of the normal induction-competence relationship.

1209 MESODERMAL INDUCTION DEFECT AS A POSSIBLE CAUSE OF EAR MALFORMATIONS

Deafness due to inner ear anomalies is rarely associated with malformation of the auricles. Two brothers born of consanguineous parents, with profound congenital sensorineural deafness, malformed auricles, abnormal vestibular function and delayed motor development, had low-set, cupped, pointed auricles with folded helices, narrow external auditory canals and normal tympanic membranes. Caloric stimulation failed to elicit nystagmus. Temporal bone laminography revealed bilateral absence of the cochlea and normal ossicles. Since the external and inner ear originate from distinctly separate structures, the embryogenesis of this malformation association is less clear than the more common association of external and middle ear anomalies. In the latter, the first and second branchial arches are the common derivative of both structures. The association of auricular and inner ear anomalies, with sparing of the middle ear, can be explained on the assumption that mesodermal induction is responsible for differentiation of both otocyst and branchial arch ectoderm. A recessive mutant gene may be responsible for the production of a deficient mesodermal inductor substance or a deficient cell surface receptor of the target tissue, rendering it unresponsive. A similar mechanism may be involved in other multiple malformation syndromes, whereby a mutant gene acting during a specific period of organogenesis causes disruption of the normal induction competence relationship.

568 PATTERN AND ASYNCHRONY OF LATE X REPLICATION IN CASES OF MULTIPLE X

The replication patterns of human chromosomes can be visualised using the RBG technique. Five to six hours prior to termination of lymphocyte culture, BudR is added to a concentration of 75 mcg/ml. Prepared slides are exposed to 33258 Hoechst and sunlight concurrently, and subsequently stained with Giemsa. The BudR-incorporated (late replicating) regions are stained pale, and the resultant pattern is similar to R-banding. We have been examining the fine details of X chromosome replication in subjects with structural or numerical X abnormalities. In early metaphase cells of children with multiple X chromosomes the late-replicating Xs do not replicate synchronously. The degree of asynchrony varies from cell to cell. However, the late-replicating X chromosomes follow a single sequence of replication. Xp22, p11, and q13 replicate first (98%), followed by q26 (95%), q24 and q28 (80%), and q22 (60%). (Percentage indicates the probability of replication prior to addition of BudR). The late replicating major bands are q12 (10%), q23 and q27 (5%), q25 (2%), q21 and p21 (0%). Late replicating sub-bands are seen in p22 (60% of Xs), p11 (30% of Xs), and q13 (10% of Xs). Very early metaphase chromosomes are still under examination and are likely to reveal other late-replicating sub-bands. Exceptions to the above sequence of replication include late replication of Xq13 (4% of Xs) and fusion of q22, q24, or q26 possibly due to early replication of q23 or q25 (7% of Xs).

THYROID ABNORMALITIES IN TWENTY CHILDREN WITH TURNER SYNDROME

Hashimoto thyroiditis, Addison disease, and diabetes mellitus occurs with a greater than normal frequency among patients with Turner syndrome. Most studies demonstrating this have been of patients over 20 years old. Therefore, 20 pediatric patients with Turner syndrome were recalled to obtain subjective and objective data reflecting thyroid function. Thyroxine (T4) and thyroid-stimulating hormone (TSH) were measured in serum samples by radioimmunoassay, and the serum antithyroid antibody titer (Thab) obtained by the tanned red cell agglutination method. Hypothyroidism was diagnosed in the presence of a decreased T4 and an elevated TSH. Hashimoto thyroiditis was presumed when a hypothyroid patient had an elevated Thab.Ten patients with Turner syndrome had structural abnormalities of the X chromosome. Three were hypothyroid, four had elevated serum Thab titers, and one had a goiter and low T4 without other evidence of thyroid dysfunction. Ten patients with Turner syndrome did not have structural abnormality of the X chromosome. None of these were hypothyroid, although five were considered to be at risk by virtue of an elevated serum Thab. One of these had a goiter. The Thab titer was elevated in nine of 15 patients over 10 years old, but in only one of five under 10 years. The three hypothyroid patients were over 10 years of age.Screening of children with Turner syndrome for evidence of thyroid dysfunction is clearly indicated.