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Objectives Individuals at risk for Huntington disease face difficult decisions regarding their reproductive options. Most do not wish to pass on the gene for Huntington disease to their children, but may not be prepared themselves to undergo presymptomatic testing and learn their genetic status. For these reasons, many at-risk individuals with a family history of HD would choose a method of genetic diagnosis that would assure them that they can have children unaffected with HD without revealing their own genetic status (non-disclosing). We have shown that, with a carefully designed and executed programme of non-disclosing preimplantation genetic testing, one can successfully assist at-risk couples to have their own biological children who are free from Huntington disease, without forcing parents to confront knowledge of their own genetic status. Methods Couples where one partner was at 50% risk for Huntington disease underwent in vitro fertilization with preimplantation embryo biopsy and molecular analysis for Huntington disease where appropriate. Results After extensive counselling and informed consent, 10 couples underwent 13 in vitro fertilization and two frozen embryo transfer cycles in a programme for non-disclosing preimplantation genetic diagnosis for Huntington disease. In 11 cycles, embryos determined to be free of Huntington disease were transferred, resulting in five clinical pregnancies. One set of twins and three singleton pregnancies have delivered. One pregnancy resulted in a first-trimester loss. Conclusions The option of non-disclosing preimplantation genetic diagnosis should be reviewed, along with other relevant medical options, when counselling at-risk Huntington disease families. Copyright © 2002 John Wiley & Sons, Ltd.

Purpose: To assess the effectiveness of outpatient treatment of Ovarian Hyperstimulation Syndrome associated with ascites. Methods: Forty-eight patients diagnosed with ovarian hyperstimulation and ascites from 2246 consecutive in vitro fertilization cycles were retrospectively studied. Patients were treated with outpatient transvaginal culdocentesis and rehydration with intravenous crystalloids and albumin every 1–3 days until resolution of symptoms or hospitalization was required. Outcomes measured included incidences of hospitalization, pregnancy outcomes, cycle characteristics, and oocyte donors versus nondonors comparisons. Results: No complications occurred from outpatient treatments, and 91.6% of patients avoided hospitalization. The pregnancy rate in patients undergoing transfer was 84.7%, and the spontaneous loss rate was 16%. Overall, the estradiol on day of hCG was 4331 pg/mL (range 2211–8167), ascites removed was 1910 cm3 (122–4000), and number of outpatient treatments was 3.4 (1–14). Nondonors averaged more outpatient treatments than donors (3.97 vs. 1.85), but similar rates of hospitalization (3/35 vs. 1/13). Conclusions: Outpatient treatment consisting of culdocentesis, intravenous rehydration, and albumin minimized the need for hospitalization in hyperstimulated patients.

Objective: To evaluate the clinical outcome of in vitro fertilization (IVF) treatment cycles from individual oocyte donors who underwent multiple sequential donations. Methods: We reviewed clinical outcome data from sequential anonymous oocyte donation cycles using donors who underwent multiple IVF stimulations. Donors were grouped by the interval between cycles and the cycle number (rank). The primary outcome measure was delivery rate by individual donor per retrieval from the combined derivative fresh and frozen embryo transfers. Results: Duration and amount of gonadotropin therapy and the fertilization rates did not correlate significantly with the interval between cycles or cycle rank. Cumulative delivered pregnancy rates for cycles 1–6 were 51.5%, 54.6%, 50.5%, 51.5%, 51.1%, and 57.6%, respectively. Delivered pregnancy rates did not vary by interval between cycles. Conclusion: Young healthy presumed or proven fertile women can reliably donate oocytes for at least six cycles with the expectation of consistently high pregnancy rates.

Marfan syndrome is an autosomal dominant disorder affecting the skeletal, ocular, and cardiovascular systems. Defects in the gene that encodes fibrillin-1 (FBN1), the main structural component of the elastin-associated microfibrils, are responsible for the disorder. Molecular diagnosis in families with Marfan syndrome can be undertaken by using intragenic FBN1 gene markers to identify and track the disease allele. However, in sporadic cases, which constitute up to 30% of the total, DNA-based diagnosis cannot be performed using linked markers but rather requires the identification of the specific FBN1 gene mutation. Due to the size and complexity of the FBN1 gene, identification of a causative Marfan syndrome mutation is not a trivial undertaking. Herein, we describe a comprehensive approach to the molecular diagnosis of Marfan syndrome that relies on the direct analysis of the FBN1 gene at the cDNA level and detects both coding sequence mutations and those leading to exon-skipping, which are often missed by analysis at the genomic DNA level. The ability to consistently determine the specific FBN1 gene mutation responsible for a particular case of Marfan syndrome allows both prenatal and pre-implantation diagnosis, even in sporadic instances of the disease. © 2001, Wiley-Liss. Inc.

Purpose: The purpose was to determine whether the number of embryos available for transfer following IVF in women over age 39 predicted a successful pregnancy outcome. Methods: Retrospective analysis of 455 consecutive IVF cycles in women ≥ years of age. Results: Few cycles were canceled (29/455, 6.4%) or produced no embryos (5/455, 1.1%). Women 40–43 years of age with normal ovarian reserve had a significantly greater delivery rate when ≥4 embryos were available for transfer than when

We report three false negative prenatal diagnostic results, using direct measurement of glycine cleavage enzyme activity in uncultured chorionic villus tissue from 290 pregnancies at risk for non-ketotic hyperglycinaemia (NKH). Testing was done by two centres: Vancouver, Canada and Lyon, France. One false negative result had activity near the lower limit of the normal range but two samples gave completely normal results well within the control range. All three pregnancies continued and the three children were born affected with NKH. Because of the first result, we now counsel that there is a grey zone of uninterpretable activity where affected and normal enzyme values overlap. Because of the other two results we now counsel that there is an approximately 1% chance of a pregnancy with a normal CVS activity resulting in an affected child. The clinical and biochemical findings in the three families are discussed.

The world's first deliveries of normal babies after use of flow cytometric separated human sperm cells (MicroSort®) for preconception gender selection are reported. Offspring were of the desired female gender in 92.9% of the pregnancies. Most of these pregnancies and births were achieved after simple intrauterine insemination.

Maternal uniparental disomy 15 (UPD15), responsible for approximately 25 per cent of Prader-Willi syndrome cases, is usually caused by maternal meiosis I non-disjunction associated with advanced maternal age. These cases may initially be detected as mosaic trisomy 15 during routine prenatal diagnostic studies. In such cases, PCR (polymerase chain reaction) microsatellite analysis of uncultured cells makes prospective prenatal diagnosis for UPD15 possible with results available in 2-4 days. We have performed molecular analyses on a series of seven cases of mosaic trisomy 15 identified in amniotic fluid (AF, n = 3) or chorionic villus samples (CVS, n = 4) from patients initially referred for advanced maternal age or abnormal triple screen. In all cases, the maternal ages were > or = 35 years and maternal meiosis I non-disjunction was documented as the cause of the trisomy in all informative cases (n = 5). Of the three case with mosaic trisomy 15 at amniocentesis, two showed the presence of the trisomy in the fetus. Molecular analysis showed one case with maternal UPD15 in the euploid cell line and one case with biparental inheritance. Both of these families elected to terminate the pregnancies based on the presence of true fetal mosaicism. In the third case, low-level trisomy 15 mosaicism in the amniotic fluid was not confirmed in a follow-up amniotic fluid sample and molecular analysis indicated biparental inheritance in the fetus. For the four trisomy 15 mosaics detected at CVS, molecular analysis was performed on direct amniotic fluid cell lysates for prospective diagnosis of UPD at 14-16 weeks' gestation. Follow-up cytogenetic analysis of the amniotic fluid in all four cases was normal, indicating confined placental mosaicism. Molecular analysis showed one of these four cases to have maternal heterodisomy 15. Based on the likelihood of Prader-Willi syndrome due to maternal UPD15, the couple chose to terminate the pregnancy. The total of two of seven cases of trisomy 15 mosaicism resulting in UPD15 is consistent with the theoretical expectation of one-third and indicates a high risk of UPD in such pregnancies. Therefore, UPD testing should be offered in all cases of mosaic trisomy 15 encountered in CVS or amniocentesis.

The discovery of nucleated erythrocytes in maternal circulation provides a potential source for non-invasive prenatal diagnosis. We have evaluated the use of a three-stage procedure to determine the number of cells that are of fetal rather than maternal origin. First, monoclonal antibodies specific for CD45 and CD14 were used in conjunction with a magnetic (MACS) column to deplete unwanted leukocytes from maternal blood. This was followed by a positive MACS enrichment for nucleated erythrocytes, using an anti-CD71 (transferrin receptor) monoclonal antibody. To discriminate between fetal nucleated erythrocytes and those of maternal origin, enriched fractions were simultaneously stained with an anti-fetal haemoglobin (HbF) antibody and hybridized with probes specific for X and Y chromosomes. Samples were then subjected to blind analysis along with negative control samples from non-pregnant volunteers. Using this dual analysis, we were able to determine that less than one nucleated erythrocyte per ml of maternal blood was of fetal origin. Small numbers of these fetal cells were found in 87.5% of pregnancies, ranging from 6 to 35 weeks gestational age. Comparison of HbF and X/Y probe data also suggests that the fetal cells are less suitable for fluorescence in-situ hybridization (FISH) analysis than similar preparations from other sources.

We report the world's first clinical pregnancy resulting from DNA-based enrichment for X-bearing human spermatozoa, for prevention of X-linked hydrocephalus. Sperm separation was followed by embryo biopsy and nested multiplex polymerase chain reaction (PCR) for gender determination. Enriched populations of X- bearing spermatozoa ranging from 80 to 89% pure as determined by fluorescence in-situ hybridization (FISH) resulted in in-vitro fertilization (IVF) rates indistinguishable from normal IVF procedures (65%). In two separate biopsy procedures, 7/9 and 15/16 of the resulting embryos were determined to be female by multiplex PCR. Embryo transfer resulted in a karyotypically normal female fetus. This technique should be widely applicable to gender selection for the prevention of genetic disorders

We report the world's first clinical pregnancy resulting from DNA-based enrichment for X-bearing human spermatozoa, for prevention of X-linked hydrocephalus. Sperm separation was followed by embryo biopsy and nested multiplex polymerase chain reaction (PCR) for gender determination. Enriched populations of X- bearing spermatozoa ranging from 80 to 89% pure as determined by fluorescence in-situ hybridization (FISH) resulted in in-vitro fertilization (IVF) rates indistinguishable from normal IVF procedures (65%). In two separate biopsy procedures, 7/9 and 15/16 of the resulting embryos were determined to be female by multiplex PCR. Embryo transfer resulted in a karyotypically normal female fetus. This technique should be widely applicable to gender selection for the prevention of genetic disorders

Preimplantation genetic diagnosis now represents an alternative reproductive option for parents at high risk of having offspring affected with certain genetic diseases. Progress in the past year has included increasing reliability in embryo sexing by both polymerase chain reaction and fluorescent in situ hybridization techniques; delivery of babies free of specific diseases such as cystic fibrosis, Lesch-Nyhan syndrome, and Tay-Sachs disease; and successful development of molecular techniques for detecting common diseases such as fragile-X syndrome. In addition, sperm separation in combination with preimplantation genetic diagnosis appears to be an exciting advance in yielding more in vitro fertilization female embryos for transfer and subsequent pregnancy in families at risk for X-linked diseases. Accumulated world experience can now be reviewed to provide couples considering preimplantation genetic diagnosis with observed pregnancy rates and accuracy of diagnosis.

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. © 1994 Wiley-Liss, Inc.

Chorionic villus sampling (CVS) with either transcervical catheters or transabdominal needles is a widely-accepted method for prenatal diagnosis. However, there exists a small subset of patients in whom sampling is difficult or impossible with either route because of individual anatomic variations. A new method of chorionic villus biopsy has been developed to circumvent these problems, utilizing transvaginal chorionic needle aspiration guided by an intravaginal ultrasound probe. This technique was performed successfully in 15 patients in whom villi could not be obtained by either of the conventional methods. This method now makes CVS possible in essentially all women regardless of their uterine anatomy or placental placement; it may also prove useful for very early chorionic sampling.

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.

Purpose: To assess the effectiveness of outpatient treatment of Ovarian Hyperstimulation Syndrome associated with ascites. Methods: Forty-eight patients diagnosed with ovarian hyperstimulation and ascites from 2246 consecutive in vitro fertilization cycles were retrospectively studied. Patients were treated with outpatient transvaginal culdocentesis and rehydration with intravenous crystalloids and albumin every 1–3 days until resolution of symptoms or hospitalization was required. Outcomes measured included incidences of hospitalization, pregnancy outcomes, cycle characteristics, and oocyte donors versus nondonors comparisons. Results: No complications occurred from outpatient treatments, and 91.6% of patients avoided hospitalization. The pregnancy rate in patients undergoing transfer was 84.7%, and the spontaneous loss rate was 16%. Overall, the estradiol on day of hCG was 4331 pg/mL (range 2211–8167), ascites removed was 1910 cm3 (122–4000), and number of outpatient treatments was 3.4 (1–14). Nondonors averaged more outpatient treatments than donors (3.97 vs. 1.85), but similar rates of hospitalization (3/35 vs. 1/13). Conclusions: Outpatient treatment consisting of culdocentesis, intravenous rehydration, and albumin minimized the need for hospitalization in hyperstimulated patients.

The preliminary results of an international collaborative study examining premature menopause in fragile X carriers are presented. A total of 760 women from fragile X families was surveyed about their fragile X carrier status and their menstrual and reproductive histories. Among the subjects, 395 carried a premutation, 128 carried a full mutation, and 237 were noncarriers. Sixty-three (16%) of the premutation carriers had experienced menopause prior to the age of 40 compared with none of the full mutation carriers and one (0.4%) of the controls. Based on these preliminary data, there is a significant association between fragile X premutation carrier status and premature menopause.

Preimplantation genetic testing (PGT) on embryos from couples at risk for Huntington disease can achieve disease prevention in offspring without disclosure of parental genotype. This strategy may also be applicable to other extremely deleterious dominant traits.

Using fluorescent in-situ hybridization (FISH) we have evaluated, on a blind basis, the efficiency of flow cytometry to separate human X- and Y-chromosome bearing spermatozoa. Our data demonstrate that human spermatozoa can be sorted to a purity of 80-90% for X spermatozoa and of 60-70% for Y spermatozoa. Our results using triple FISH fully agree with the sorting treatment used in each case and corroborate the efficiency of the flow sorting technique for sperm sex selection. In these limited samples (200-500 sperm/donor), the frequencies of disomic or diploid spermatozoa were not increased when comparing the sorted samples with unselected samples or with our control series.

Marfan syndrome (MFS) is an autosomal dominant disease that affects the skeletal, ocular and cardiovascular systems. Defects in the gene that codes for fibrillin (FBN-1) are responsible for MFS. Here we report the world's first use of preimplantation genetic testing (PGT) to achieve a clinical pregnancy and live birth of a baby free of a Marfan mutation. One or two blastomeres from each embryo were tested for a CA repeat within the FBN-1 gene. The prospective mother is homozygous for the CA repeat (2/2) and has two normal copies of the FBN-1 gene, while the prospective father is heterozygous for the CA repeat (1/2), and is affected with the Marfan syndrome. In the father's family, allele 2 segregates with the mutated FBN-1 gene. For PGT, any embryo diagnosed as heterozygous for the CA repeat (1/2) would be presumed to have inherited normal FBN-1 genes from the father and the mother and be unaffected. One in-vitro fertilization (IVF) cycle yielded 12 embryos for preimplantation testing; six of the embryos were heterozygous for the CA repeat (1/2) and presumed to be free of the Marfan mutation. Five of the six embryos were subsequently transferred into the uterus. The fetus was tested by chorionic villus sampling and found to be free of the Marfan mutation by the same linkage analysis, had a normal fetal echocardiogram, and was normal at birth.

Four cases having mosaicism for a small marker or ring [45,X/46,X,+mar or 45,X/46,X,+r] chromosome were ascertained following cytogenetic studies requested because of minor anomalies (cases 1, 3, and 4) and/or short stature (cases 2 and 4). While all 4 cases had traits typical of Ullrich-Turner syndrome (UTS), cases 1, 3, and 4 had manifestations not usually present in UTS, including unusual facial appearance, mental retardation/developmental delay (MR/DD) (cases 3 and 4), and syndactylies (case 1). The facial appearances of cases 1 and 3 were similar yet distinct from that of case 4. Using fluorescence in situ hybridization (FISH), each of the markers in these 4 cases was identified as having been derived from an X chromosome. The level of mosaicism for the mar/r(X) cell line in these cases varied from 70% (case 1) to 16% (case 4) but was not apparently correlated with the presence of MR/DD. Replication studies demonstrated a probable early replication pattern for the mar/r(X) in cases 1, 3, and 4, while the marker in case 2 was apparently late replicating. To date, 41 individuals having mosaicism for a small mar/r(X) chromosome have been described. Interestingly, most of the 14 individuals having a presumedly active mar/r(X) demonstrated clinical findings atypical of UTS, including abnormal facial changes (11) and MR/DD (13). MR was noted most frequently in those cases having at least 50% mosaicism for the marker or ring. In contrast, atypical UTS facial appearance or MR/DD was not noted in 14 of the 16 cases with UTS who carried a probable late replicating marker or ring. In conclusion, although the phenotype of 45,X/46,X,mar/r(X) individuals appears to be influenced by the genetic content and degree of mosaicism for the mar/r(X), the most significant factor associated with MR/DD appears to be the activity status of the mar/r(X) chromosome. Thus, our 4 cases provide further support for the hypothesis that a lack of inactivation of a small mar/r(X) chromosome may be a factor leading to the MR and other phenotypic abnormalities seen in this subset of individuals having atypical UTS.

This paper reports eight cases of non-mosaic, rare, and typically lethal trisomies diagnosed in chorionic villi and not confirmed by amniocentesis. Four cases were 47,XX, + 16; two cases were 47,XX, +2; one was 47,XX, + 12; and one was 47,XY, +7. There have been no known complications in any of these gestations. These eight cases were found in a series of approximately 12 000 samples processed in our laboratory (0.07 per cent). We conclude that (1) rare non-mosaic trisomy not reflecting the fetal condition is an occasional source of diagnostic ambiguity in chorionic villus sampling; and (2) when encountered, a follow-up amniocentesis should be recommended to the patient to confirm or rule out the abnormality. We propose the term ‘confined placental abnormality’ to describe non-mosaic trisomies and other related abnormalities found only in chorionic tissue.

Chorionic villus sampling (CVS) in the first trimester of pregnancy provides a safe and effective method for the early prenatal diagnosis of cytogenetic abnormalities in multiple gestations. In this multicentre study involving 126 twin and 2 triplet gestations primarily at risk because of advanced maternal age, the overall success rate of obtaining an adequate villus sample from each fetus was 99.2 per cent. For women of advanced maternal age, the rate of combined losses of chromosomally normal fetuses due to spontaneous abortion, stillbirths, and neonatal deaths was 5.0 per cent, compared with a 4.0 per cent total loss rate following CVS in singleton pregnancies derived from the same population (Rhoads et al., 1989). There was a 100 per cent success rate in obtaining a cytogenetic analysis; a cytogenetic abnormality was present in five of the multiple gestations (3.9 per cent) and involved seven fetuses (2.7 per cent). There were no diagnostic errors and no cases of normal cytogenetic diagnosis followed by the birth of a cytogenetically abnormal newborn. Based on cases of XX/XY admixture, cell contamination derived either from maternal decidua or the other twin occurred in 6 of 256 samples (2.3 per cent), giving an overall estimate of the frequency of cell contamination of 4.6 per cent; these cases did not present a diagnostic problem. However, there were two cases (0.8 per cent) in which the fetal sex was incorrect, due either to complete maternal cell contamination or to the possibility that in error one twin was sampled twice.

Purpose: To assess the effectiveness of outpatient treatment of Ovarian Hyperstimulation Syndrome associated with ascites. Methods: Forty-eight patients diagnosed with ovarian hyperstimulation and ascites from 2246 consecutive in vitro fertilization cycles were retrospectively studied. Patients were treated with outpatient transvaginal culdocentesis and rehydration with intravenous crystalloids and albumin every 1–3 days until resolution of symptoms or hospitalization was required. Outcomes measured included incidences of hospitalization, pregnancy outcomes, cycle characteristics, and oocyte donors versus nondonors comparisons. Results: No complications occurred from outpatient treatments, and 91.6% of patients avoided hospitalization. The pregnancy rate in patients undergoing transfer was 84.7%, and the spontaneous loss rate was 16%. Overall, the estradiol on day of hCG was 4331 pg/mL (range 2211–8167), ascites removed was 1910 cm3 (122–4000), and number of outpatient treatments was 3.4 (1–14). Nondonors averaged more outpatient treatments than donors (3.97 vs. 1.85), but similar rates of hospitalization (3/35 vs. 1/13). Conclusions: Outpatient treatment consisting of culdocentesis, intravenous rehydration, and albumin minimized the need for hospitalization in hyperstimulated patients.

We report an 11-month-old infant with cri du chat syndrome and cataracts. The chromosomal abnormality was transmitted via a balanced 5/11 translocation from a phenotypically normal mother. The child underwent bilateral cataract extraction and was fitted with extended wear contact lenses. Congenital cataracts may be associated with cri du chat syndrome.

We investigated fatty alcohol metabolism in eight patients with Sjögren-Larsson syndrome, and in nine obligate heterozygotes. Fatty alcohol: nicotinamide-adenine dinucleotide oxidoreductase (FAO) activity was deficient in cultured skin fibroblasts (mean 18% of normal, n = 8) and peripheral blood leukocytes (mean 22% of normal, n = 3) from patients with Sjögren-Larsson syndrome. The palmitoyl coenzyme A-inhibitable component of FAO activity was decreased to 10% and 15% of normal in fibroblasts and leukocytes, respectively, of patients with Sjögren-Larsson syndrome. Most affected patients accumulated long-chain fatty alcohol in plasma, with a greater relative accumulation of octadecanol (mean threefold greater than normal) than hexadecanol (mean twofold greater than normal). Erythrocyte lipid alkyl ether linkages derived from hexadecanol were slightly increased in three of four patients. Fibroblasts and leukocytes from heterozygotes with Sjögren-Larsson syndrome showed mean FAO activities that were intermediate between those seen in homozygotes and in normal control subjects. The heterozygotes had normal fatty alcohol concentrations in plasma. These studies demonstrate FAO deficiency in patients with Sjögren-Larsson syndrome, and suggest that accumulation of fatty alcohol or its metabolic products may be important in the pathogenesis of this disorder.

We describe a large consanguineous German-Acadian ("Cajun") family from a rural area in Louisiana in which 11 persons in two generations had the Maroteaux-Lamy syndrome. The mutant arylsulfatase B enzyme in this family was similar to the mutant enzyme in previously studied families in its cross-reactivity with specific antibodies to the enzyme, but it differed in both its electrophoretic mobility and its residual enzymatic activity. These findings indicate that a different mutational event leading to Maroteaux-Lamy syndrome occurred in this family.

CONTEXT—Chromosomal abnormalities that involve the proximal region of chromosome 15q occur relatively frequently in the human population. However, interstitial triplications involving one 15 homologue are very rare with three cases reported to date. OBJECTIVE—To provide a detailed molecular characterisation of four additional patients with interstitial triplications of chromosome 15q11-q14. DESIGN—Molecular analyses were performed using DNA markers and probes specific for the 15q11-q14 region. SETTING—Molecular cytogenetics laboratory at the University of Chicago. SUBJECTS—Four patients with mild to severe mental retardation and features of Prader-Willi syndrome (PWS) or Angelman syndrome (AS) were referred for molecular cytogenetic analysis following identification of a suspected duplication/triplication of chromosome 15q11-q14 by routine cytogenetic analysis. MAIN OUTCOME MEASURES—Fluorescence in situ hybridisation (FISH) was performed to determine the type of chromosomal abnormality present, the extent of the abnormal region, and the orientation of the extra chromosomal segments. Molecular polymorphism analysis was performed to determine the parental origin of the abnormality. Methylation and northern blot analyses of the SNRPN gene were performed to determine the effect of extra copies of the SNRPN gene on its methylation pattern and expression. RESULTS—Fluorescence in situ hybridisation (FISH) using probes within and flanking the Prader-Willi/Angelman syndrome critical region indicated that all patients carried an intrachromosomal triplication of proximal 15q11-q14 in one of the two chromosome 15 homologues (trip(15)). In all patients the orientation of the triplicated segments was normal-inverted-normal, suggesting that a common mechanism of rearrangement may have been involved. Microsatellite analysis showed the parental origin of the trip(15) to be maternal in three cases and paternal in one case. The paternal triplication patient had features similar to PWS, one maternal triplication patient had features similar to AS, and the other two maternal triplication patients had non-specific findings including hypotonia and mental retardation. Methylation analysis at exon 1 of the SNRPN locus showed increased dosage of either the paternal or maternal bands in the paternal or maternal triplication patients, respectively, suggesting that the methylation pattern shows a dose dependent increase that correlates with the parental origin of the triplication. In addition, the expression of SNRPN was analysed by northern blotting and expression levels were consistent with dosage and parental origin of the triplication. CONCLUSIONS—These four additional cases of trip(15) will provide additional information towards understanding the phenotypic effects of this abnormality and aid in understanding the mechanism of formation of other chromosome 15 rearrangements. Keywords: chromosome 15 triplication; Prader-Willi syndrome; Angelman syndrome; autism