Molecular and clinical overlap of Angelman and Prader-Willi syndrome phenotypes.

The Prader-Willi (PWS) and Angelman syndromes (AS) share the same apparent cytogenetic and molecular lesions of 15q11-13 and yet exhibit distinct clinical phenotypes. The etiology of PWS or AS appears to depend on the parental origin of the aberrant chromosome 15. Substantial clinical overlap has not been reported between deletion-positive PWS and AS patients. In the present study, we report the clinical, cytogenetic, and molecular findings in three AS patients. The first patient is a mentally retarded woman with a visible deletion of 15q11-13 with typical craniofacial, behavioral, and neurologic changes of AS. This patient is hyperphagic, and she is moderately obese for her height. Her hands and feet are small. These manifestations are more characteristic of PWS and not of AS. The molecular studies showed deletions of maternal origin for five distal PWCR loci. The most proximal locus, D15S18, was not deleted. These findings are identical to those found in our third AS patient who does not have any PWS features. To the best of our knowledge, this is the first report of concurrence of hyperphagia with consequent obesity and the AS phenotype in a patient with a del 15(q11-13) of maternal origin. These clinical findings suggest that overlap in the symptoms of PWS and AS can occur. Our second AS patient presents with atypical molecular findings in that he cannot be classed into any of the three proposed sub-groups of AS patients and may be representative of a fourth sub-group of AS patients.

Detection of molecular rearrangements in Prader-Willi syndrome patients by using genomic probes recognizing four loci within the PWCR.

The Prader-Willi chromosome region (PWCR) in Prader-Willi syndrome patients was analyzed by using genomic DNA probes mapping to 15q11.2-q12. The present report includes analysis of dosage by RFLP and densitometric studies, and analysis of restriction patterns. Twelve Prader-Willi syndrome (PWS) patients were studied: 5 had deletions of 15q11-q13, one had an unbalanced translocation, and 6 were karyotypically normal. Four genomic DNA probes were used: pML34 (D15S9); pTD3-21 (D15S10); IR4-3 (D15S11), a subclone of IR4; and IR10-1 (D15S12), a subclone of IR10 (Donolon et al.: Proc Natl Acad Sci USA 83:4408-4412, 1986). The results presented demonstrate that molecular rearrangements have occurred in 10 of the 12 PWS patients investigated and that the specific rearrangements differ from patient to patient. Patients with apparently similar cytogenetic deletions differ at the molecular level with deletions and/or duplications of various loci. The present study reports molecular alterations within the PWCR in PWS patients reported as cytogenetically normal. However, the 6 karyotypically normal patients are a heterogeneous group with molecular rearrangements ranging from none detected to deletions and/or duplications. These molecular studies suggest that a physical disruption of the PWCR causes the PWS not only in those patients reported to have a cytogenetic aberration but also in those identified as apparently karyotypically normal. The question remains as to whether the PWS patients in whom a molecular abnormality has not been detected have an autosomal recessive form of PWS, a molecular disruption which has not yet been detected, or another mechanism producing an apparently identical phenotype. The order of the 4 loci on chromosome 15 is hypothesized to be cen----D15S9----D15S12----D15S11----D15S10.

Transmission of the fra(X) haplotype from three nonpenetrant brothers to their affected grandsons.

We report on a family showing transmission of the fra(X) gene by 3 nonpenetrant, fra(X) negative, normally intelligent, full and half-brothers to their affected grandsons. The mothers of the affected boys are obligate carriers, fra(X) negative, and of normal intelligence. This family illustrates the "Sherman Paradox" and is compatible with the predictions of the Laird X-inactivation imprinting model. In addition, molecular and/or cytogenetic studies have enabled at-risk relatives to learn more about their carrier fra(X) status and have allowed for more accurate genetic counselling.

Evidence for the role of the maternal immune system in balancing the sex ratio in mice.

Two experiments have been conducted to verify the effect of maternal preimmunization to H-Y antigen on the secondary sex ratio in C57Bl/6 mice. In accord with the theoretical model previously proposed, it was found that extensive immunization to H-Y antigen following splenectomy resulted in a significant increase in sex ratio (males/females) without affecting litter size. Litter size was also not correlated to sex ratio. The data from both experiments suggest that splenectomy alone or restimulation to H-Y antigen (after 30 days) both act to decrease sex ratio. It is concluded that the maternal immune system plays a limited, but significant, role in maintaining the normal sex ratio in mice.

Proline incorporation by cultured skin fibroblasts from patients with duchenne muscular dystrophy.

The incorporation of proline by cultured skin fibroblasts derived from normal individuals and patients with Duchenne muscular dystrophy (DMD) was analyzed by SDS-gel electrophoresis combined with a double-labeling procedure [Pediatr. Res., 12: 887 (1978)]. DMD fibroblasts showed increased proline incorporation into protein of approximately 130 000 daltons which could be degraded partially by collagenase. This difference was observed only at 7 days after seeding, and may be due to slight differences in growth rate as comparison of cells harvested at 2 and 7 days indicated that increased proline incorporation into high molecular weight protein was associated with day 7 cells, whether normal or DMD. During 14 days in culture normal and DMD strains did not differ in protein content, doubling time or incorporation of [3H]leucine and [14C]proline into cellular protein, although the ratio of [14C]proline to [3H]leucine incorporated was greater for DMD fibroblasts at 7 days. Incorporation of [14C]proline and [3H]leucine into extracellular proteins was greater in DMD fibroblast cultures. These subtle differences support the hypothesis that the DMD gene is expressed in fibroblasts.

Long-range restriction mapping and linkage analysis of the Prader-Willi chromosome region (PWCR).

In an attempt to elucidate the relationship between genetic alterations at chromosomal bands 15q11.2-12 and the Prader-Willi syndrome (PWS), we have constructed a long-range restriction map of this region using a combination of pulsed-field gel techniques and the infrequently cutting restriction enzymes NotI, MluI, SalI, SfiI, NruI, SacII, and BssHII. Four previously reported probes mapping to 15q11.2-12 and known to be deleted in PWS patients were used to construct the physical map of this region. The loci recognized by these four probes have been localized to a 2600-kb partial SalI restriction fragment and a 3200-kb partial EcoRI restriction fragment. Linkage studies were performed on nine families to estimate the recombination rates between these loci. The calculated lod scores did not indicate significant linkage between any of the four loci. The contrast between the physical distance and the observed recombination frequency suggests that these four loci are located in a recombinational "hot spot."

Assignment of the sorbitol dehydrogenase locus to human chromosome 15 pter leads to q21.

The locus for sorbitol dehydrogenase (SORD, E.C. 1.1.1.14) has been shown to segregate with hexosaminidase A and mannose phosphate isomerase in a series of human-Chinese hamster somatic cell hybrids. Cytogenetic analysis supports the assignment to chromosome 15 and further defines the gene locus to the region 15pter leads to q21.

Immunogenetics of sex determination of the polled goat.

Using the protein A rosette technique, it was found that the gene for polledness (P) in goats is associated with the presence of H-Y antigen on the cell surface of cultured fibroblasts. Two XX intersex goats (P/P) were found to be H-Y+, and one heterozygous (P/+) normal XX female was also found to express H-Y antigen at a low level. The expression of H-Y antigen by intersex goats was found to be lower than that of normal XY males. The statistical analysis suggests that animals of the same genotype and sex might differ in the density of H-Y antigen on their cell surface, which might explain the variability in primary sex determination among intersex goats. The association between the gene for sex reversal and H-Y antigen is discussed in relation to the location of the H-Y gene(s).

Prenatal diagnosis in Canada--1990: a review.

This paper presents the results of a detailed study of the genetic prenatal diagnostic services available in Canada in 1990. All 22 genetic centres offering prenatal diagnostic services as well as the 64 laboratories processing samples were surveyed. Data were collected from each to determine what testing was being done, how many women were being tested, for what conditions, and with what outcomes. Also, data on the 35 formal outreach sites were collected. The statistics presented are for the 1990 calendar year. This survey was conducted for the Canadian Royal Commission on New Reproductive Technologies.

The human complement after trypsin pretreatment as compared to the Paris standard.

A composite G-banding diagram after trypsin pretreatment of metaphase chromosomes from 5 different individuals (2 males and 3 females) is presented and compared with the Paris diagram. The patterns obtained by the present technique were very similar to those previously reported. It was found that the darkly staining bands were much more consistent in appearance than the lightly staining bands and that there was little individual variation.

A cytogenetic survey of 14,069 newborn infants. I. Incidence of chromosome abnormalities.

Data from a chromosome examination of 14,069 consecutive newborn infants is presented. Successful karyotypes were obtained on 13,939 babies using short-term blood cultures and conventional staining methods. Of those, 13,645 babies had normal chromosomes; 64 (0.46%) had a major chromosome abnormality; and 230 (1.65%) had a marker chromosome; giving a total of 294 (2.11%) babies with a major chromosome abnormality or distinctive marker chromosomes. Six male babies with sex chromosome abnormalities had a 47,XXY and four a 47,XYY karyotype, and three were mixoploids. Five female babies had a 47,XXX karytotype and two were mixoploids. There were three babies with ambiguous external genitalia, all with normal karyotypes. Fourteen babies had 21-trisomy; there were three 18-trisomics and one 13-trisomic. The mother of one 18-trisomy baby had a balanced (18;21) translocation. Twenty-four infants had a balanced chromosome rearrangement. Eleven of these were reciprocal and thirteen were Robertsonian translocations. One baby had an unbalanced derivative chromosome resulting from an 18;11 insertion. Two infants with additional unidentified fragments were detected. Two hundred and thirty babies (1:60) carying distinctive chromosome variants were detected. The commonest variant was the Yq+ among males (0.89%). Other common variants involved the short arms of the D and G groups (0.32% and 0.57%, respectively) 16q+ (0.09%), and 1q+ (0.04%). The results of the present study when combined with five other comparable studies, thus comprising a total of 46,150 newborn infants, indicates that the frequency of major chromosome abnormalities is between 1:150 and 1:200 live-born babies. This represents a small proportion of all conceptuses with chromosome abnormalities, which has been estimated as being approximately 1:20. It is thus clear that chromosome abnormalities form a major part of the genetic load carried by the human population. The development of chromosome banding techniques already has increased, and with further increase, the complexities of human cytogenetics and may reveal many additional rearrangements undetectable by conventional methods.

Localization of human gene loci using spontaneous chromosome rearrangements in human-Chinese hamster somatic cell hybrids.

Analysis of human-Chinese hamster somatic cell hybrids with spontaneously derived chromosome structural changes has provided data for the regional and subregional localization of gene loci which have previously been assigned to human chromosomes 2, 12, and X. Correlation of the expression of human gene loci with the human chromosome complements present in somatic cell hybrids indicates that the cytoplasmic malate dehydrogenase (MDH1) locus is in the 2p23yields2pter region, and red cell acid phosphatase (AcP1) is at or adjacent to 2p23. The cytoplasmic isocitrate dehydrogenase (IDH1) locus is at or adjacent to 2q11, peptidase B (Pep B) is at or adjacent to 12q21, lactate dehydrogenase B (LDH B) is in the 12q21yiedls12pter region, glucose-6-phosphate dehydrogenase (G6PD) is in the Xq24yieldsXqter region, and the gene loci for phosphoglycerate kinase (PGK), alpha-galactosidase (alpha-gal), and hypoxanthine guanine phosphoribosyltransferase (GPRT) are in the Xp21yieldsXq24 region.

Regional localization of loci for human PGM and 6PGD on human chromosome one by use of hybrids of Chinese hamster-human somatic cells.

The human gene loci phosphoglucomutase(1) (PGM(1), EC 2.7.5.1) and 6-phosphogluconate dehydrogenase (6PGD, EC 1.1.1.43), which are located on human chromosome one, have been assigned to a specific region of the short arm of that chromosome, by use of a hybrid cell line derived from a Chinese hamster cell line deficient in hypoxanthine phosphoribosyl transferase and a strain of human diploid fibroblasts. Cytogenetic analysis of a hybrid clone maintained for about 50 generations in vitro revealed two populations of cells, the first containing a human chromosome one with a break point at band 1p33, such that about 25% of the short arm of this chromosome was deleted. The second cell population contained a normal chromosome one. Biochemical analysis of subclones derived by cloning this mixed population revealed two phenotypic classes, one of which expressed all three chromosome-one markers, PGM(1), 6PGD, and peptidase C (Pep C), while the other expressed only Pep C. Cytogenetic analysis showed that the subclones expressing all three markers carried the normal human chromosome one, while those expressing only Pep C carried the deleted chromosome. These data indicate that the human gene loci PGM(1) and 6PGD are located on the short arm of chromosome one distal to the break point, while Pep C lies elsewhere on the chromosome.

Down syndrome and recent demographic trends in Manitoba.

Two hundred and thirty-three children born in Manitoba with Down syndrome between 1965 and 1974 were ascertained and the maternal ages obtained. Mean maternal age was found to have declined in this period both for all livebirths and to a greater extent for Down syndrome children. Though the proportion of mothers of Down syndrome infants with a maternal age of less than 35 years remained the same, the proportion of mothers aged 35 to 39 years increased and the age specific incidence of Down syndrome became significantly greater for women in this age group. Reduction in the proportion of Down syndrome births to women over 40 years and the increased incidence of Down syndrome in children of women aged 35 to 39 years has important consequences for the planning of amniocentesis programmes.

The effect of gamma irradiation of human cells and somatic cell hybridization on sister chromatid exchange frequencies in the hamster chromosomes of hamster-human hybrid lines.

Sister chromatid exchange (SCE) frequency was studied in human/hamster hybrid lines in which the human cells had been exposed to gamma irradiation at doses of 0, 10, 20, and 40 grays. A higher SCE frequency was found in the hamster chromosomes in the hybrid lines than was found in the hamster parental cell line CHW1103. There was also a dose-dependent increase of SCE frequency in the unirradiated hamster chromosomes in the hybrid lines in which the human parental cells had been irradiated.

Human chromosomes control growth of human-Chinese hamster somatic cell hybrids.

Several independent human-Chinese hamster hybrid cell lines have been monitored for growth rate and chromosome composition. The rate of growth, as indicated by doubling time during log phase, was positively correlated with the number of human chromosomes, and human chromosomes 3 and 21 were present in stable hybrid cell lines more frequently than expected.

Localization of the processed gene for human ceruloplasmin to chromosome region 8q21.13----q23.1 by in situ hybridization.

A processed gene for human ceruloplasmin has been localized to 8q21.13---q23.1 by in situ hybridization. This result confirms the assignment of this locus to chromosome 8 by Southern blot analysis using human x hamster somatic-cell hybrids and localizes the gene to the long arm of chromosome 8.

Somatic recombination rather than uniparental disomy suggested as another mechanism by which genetic imprinting may play a role in the etiology of Prader-Willi syndrome.

Six Prader-Willi syndrome (PWS) patients with normal karyotypes and their parents were analyzed to determine the nature of the molecular aberrations present in the proximal region of 15q and to determine the parental origin of the aberrant chromosome 15. In addition, the likelihood that uniparental disomy plays a significant role in the etiology of PWS patients with normal karyotypes was studied. Restriction fragment length polymorphisms (RFLPs) recognized by seven probes [pML34 (D15S9), pTD3-21, pCGS0.9, pCGS1.1 (D15S10), IR4.3 (D15S11), IR10.1 (DS15S12), p189-1 (D15S13), IR39 (D15S18), and CMW-1 (D15S24)] mapping to the Prader-Willi chromosome region (PWCR) and an additional two probes [pMS1-14 (D15S1); the cDNA of neuromedin B] mapping elsewhere on chromosome 15 were analyzed in the six PWS patients and their parents. Copy number of each locus within the PWCR was determined by densitometry. Molecular rearrangements of the proximal region of 15q were observed in all of the six probands and the origin of the aberrant chromosome 15 when determined was consistently paternal in origin. While data obtained from our six patients does not support the mechanism of disomy, results obtained from three of the six patients show more complex rearrangements hypothesized to have resulted from somatic recombination. These rearrangements have resulted in acquired homozygosity and the lack of a paternal allele at various loci within the PWCR. The presence of only a maternal contribution at certain loci as the result of somatic recombination may be another mechanism by which genetic imprinting plays a role in the presentation of the PWS phenotype.