Scanning for telomeric deletions and duplications and uniparental disomy using genetic markers in 120 children with malformations.

We screened 120 children with sporadic multiple congenital anomalies and either growth or mental retardation for uniparental disomy (UPD) or subtelomeric deletions. The screening used short tandem repeat polymorphisms (STRP) from the subtelomeric regions of 41 chromosome arms. Uninformative marker results were reanalyzed by using the next available marker on that chromosome arm. In total, approximately 25,000 genotypes were generated and analyzed for this study. Subtelomeric deletions of 1 Mb in size were excluded for 27 of 40 chromosome arms. Among the 120 subjects none was found to have UPD, but five subjects (4%, 95% confidence interval 1-9%) were found to have a deletion or duplication of one or more chromosome arms. We conclude that UPD is not a frequent cause of undiagnosed multiple congenital anomaly syndrome. In addition, we determined that 9p and 7q harbor chromosome length variations in the normal population. We conclude that subtelomeric marker analysis is effective for the detection of subtelomeric duplications and deletions, although it is labor intensive. Given a detection rate that is similar to prior studies and the large workload imposed by STRPs, we conclude that STRPs are an effective, but impractical, approach to the determination of segmental aneusomy given current technology.

Linkage disequilibrium at the Angelman syndrome gene UBE3A in autism families.

Autistic disorder is a neurodevelopmental disorder with a complex genetic etiology. Observations of maternal duplications affecting chromosome 15q11-q13 in patients with autism and evidence for linkage and linkage disequilibrium to markers in this region in chromosomally normal autism families indicate the existence of a susceptibility locus. We have screened the families of the Collaborative Linkage Study of Autism for several markers spanning a candidate region covering approximately 2 Mb and including the Angelman syndrome gene (UBE3A) and a cluster of gamma-aminobutyric acid (GABA(A)) receptor subunit genes (GABRB3, GABRA5, and GABRG3). We found significant evidence for linkage disequilibrium at marker D15S122, located at the 5' end of UBE3A. This is the first report, to our knowledge, of linkage disequilibrium at UBE3A in autism families. Characterization of null alleles detected at D15S822 in the course of genetic studies of this region showed a small (approximately 5-kb) genomic deletion, which was present at somewhat higher frequencies in autism families than in controls.

Microsatellite analysis reveals a high incidence of maternal cell contamination in 46,XX products of conception consisting of villi or a combination of villi and membranous material.

OBJECTIVE: With the use of microsatellite analysis, we sought to determine the incidence of maternal cell contamination in 46,XX products of conception consisting of villi or a combination of villi and membranous material. STUDY DESIGN: Deoxyribonucleic acid from cultured fibroblasts of 46,XX products of conception specimens and a corresponding maternal blood sample were obtained from 31 women. Maternal and fetal genotypes for several highly polymorphic microsatellite markers were compared. RESULTS: Maternal cell contamination was present in 26 (89.7%) of the 29 products of conception specimens from which conclusive results were obtained. The contamination appeared to completely obscure the fetal material in 24 of these specimens. CONCLUSIONS: A significant proportion of 46,XX karyotypes from products of conception represents maternal cell contamination. When maternal cells rather than fetal cells are karyotyped, no information is gained regarding the chromosome constitution of the abortus, and genetic counseling regarding recurrence risks for future pregnancies may be inaccurate. Thus laboratories should exercise caution when reporting normal female karyotypes on products of conception and should consider using microsatellite analysis to determine whether 46,XX results are truly representative of the fetal karyotype.

Prenatal diagnosis of mosaicism for triploidy and trisomy 13.

Mosaicism for trisomy 13 and triploidy was detected by amniocentesis performed at 18 weeks' gestation because of fetal anomalies. Pregnancy continued and a live-born male was delivered vaginally at 37 weeks. The infant had features common to both trisomy 13 and triploidy: intrauterine growth retardation (IUGR), small abnormal ears, cleft palate, and a small jaw. In addition, he had complete cutaneous syndactyly of fingers 3 and 4 and partial syndactyly of the toes, as seen in triploidy. Mixoploidy for trisomy 13 and triploidy was confirmed postnatally in blood, skin, and placenta. Examination of chromosome heteromorphisms and DNA markers suggested the presence of two maternal contributions in the triploid cell line. In addition, the extra chromosome 13 in the trisomic cell line was derived from the mother.

Lack of association of the (AAAT)6 allele of the GXAlu tetranucleotide repeat in intron 27b of the NF1 gene with autism.

A novel allele of the GXAlu tetranucleotide repeat in intron 27b of the neurofibromatosis 1 (NF1) gene has recently been reported to be present in 4.7% of autistic patients but not in controls. We have found the novel GXAlu allele absent in 204 patients from the South Carolina Autism Project and 200 controls. The autism population studied includes a significant number of patients with hypotonia, stereotyped behaviors, or postural, gait, and motor abnormalities similar to those seen in the patients previously reported to possess the novel GXAlu allele. This suggests that the novel (AAAT)6 GXAlu allele is not associated with autism.

Usefulness and limitations of FISH to characterize partially cryptic complex chromosome rearrangements.

Interpretation of a complex chromosome rearrangement (CCR) using only G-band analysis is difficult and potentially inaccurate. We present two patients with de novo, partially cryptic, CCRs that illustrate both the value and limitations of using fluorescence in situ hybridization (FISH) whole chromosome paint probes to characterize these types of rearrangements. In a patient referred because of features of Townes-Brocks syndrome, G-band analysis revealed an unbalanced CCR involving 3 chromosomes (2,11 and 16) and at least 4 breakpoints. A more complex rearrangement involving two cryptic insertions and at least 6 breakpoints, however, was detected using whole chromosome paint probes specific for the 3 chromosomes involved in the rearrangement. In this case, FISH studies were essential for accurate characterization of this patient's rearrangement. In a second patient, G-band analysis revealed that a 12-year-old male with obesity, small genitalia, attention deficit disorder, learning disabilities, and behavior problems, carried a CCR involving 4 chromosomes (3, 5, 10 and 13) with 6 breakpoints. This rearrangement seemed unbalanced, with missing terminal 3p26. 2-pter material. Our G-band interpretation of this karyotype was confirmed by FISH using whole chromosome paint probes specific for the involved chromosomes. Although no evidence of the "missing" 3pter material was observed using a chromosome 3 paint, FISH analysis using a chromosome 3p unique telomere probe identified telomeric 3p material on the distal long arm of the derivative 10 chromosome. This case illustrates the limited value of painting probes to detect small rearrangements, especially those involving terminal chromosome regions.

The HOPA gene dodecamer duplication is not a significant etiological factor in autism.

A recent study has suggested that a dodecamer duplication in the HOPA gene in Xq13 may occur in a significant portion of male patients with autism. We have determined the incidence of this duplication in 202 patients from the South Carolina Autism Study. The incidence of the duplication was not significantly different between patients and controls. Three of the female patients inherited the duplication from nonautistic fathers. In addition, there was no systematic skewing of X inactivation in the female patients with the duplication, or in nonautistic mothers and sisters with the duplication. These findings suggest that the dodecamer duplication in the HOPA gene does not play a significant role in the etiology of autism.

Evidence that a dodecamer duplication in the gene HOPA in Xq13 is not associated with mental retardation.

A recent study suggested that a dodecamer duplication in exon 42 of the HOPA gene in Xq13 may be a significant factor in the etiology of X-linked mental retardation. In an effort to investigate this possibility, we determined the incidence of the dodecamer duplication in cohorts of non-fragile X males with mental retardation from three countries, cohorts of fragile X males from two countries, 43 probands from families with X-linked mental retardation and control cohorts from three countries. The duplication was found in 3.6-4.0% of male patients from two non-fragile X groups (Italy and South Carolina), in 1.2% from another non-fragile X group (South Africa), but in no male patients from families with X-linked mental retardation (South Carolina). The dodecamer duplication was also found in several white males with fragile X syndrome from France (5%) and South Africa (22.2%). Additionally, the duplication was found in 1.5% of South Carolinian newborn males, 2.5% South Carolinian male college students, 5% Italian male controls and 4.5% of the white South African controls. None of the black South African non-fragile X individuals with mental retardation, the fragile X or the control samples tested carried the duplication, suggesting that the duplication is rare in the black South African population. The incidence of the duplication was not significantly different between any of the groups in the study. Therefore, results of our studies in four different populations do not corroborate the findings of the previous study, and indicate that the HOPA dodecamer duplication does not convey an increased susceptibility to mental retardation.

DNA methylation analysis with respect to prenatal diagnosis of the Angelman and Prader-Willi syndromes and imprinting.

The Angelman (AS) and Prader-Willi syndromes (PWS) are clinically distinct neurobehavioural syndromes resulting from loss of maternal (AS) or paternal contributions (PWS) of imprinted genes within the chromosomal 15q11-q13 region. The molecular diagnosis of both syndromes can be made by a variety of techniques, including DNA methylation, DNA polymorphism and molecular cytogenetic analyses. DNA methylation analysis at three major loci (ZNF127, PW71 and 5' SNRPN) has been successfully used for the postnatal diagnosis of AS and PWS. Methylation analysis, in contrast to other techniques, can reliably be used to diagnose all three major molecular classes (deletion, uniparental disomy and imprinting mutation) of PWS, and three of the four major classes of AS. In this study we demonstrate that methylation analysis can also be successfully used in prenatal diagnosis, by examining specimens obtained from amniocentesis and chorionic villus sampling. Correct prenatal diagnoses were obtained in 24 out of 24 samples using the 5' SNRPN locus; 4 out of 15 using the ZNF127 locus; and 10 out of 18 using the PW71 locus. Therefore, our data indicate that although the DNA methylation imprints of ZNF127 and 5' SNRPN arise in the germline and are present in brain, only 5' SNRPN maintains the imprint in tissues suitable for the prenatal diagnosis of AS and PWS.

Diffusion-weighted magnetic resonance imaging in boys with neural cell adhesion molecule L1 mutations and congenital hydrocephalus.

The phenotype of severe congenital hydrocephalus secondary to neural cell adhesion molecule L1 (L1CAM) gene mutations includes the distinct finding of brainstem corticospinal tract hypoplasia. Using diffusion-weighted imaging (DWI), we failed to demonstrate anisotropy in the corticospinal tracts of the basis pontis in 4 affected boys with L1CAM mutations. The DWI findings correlated with the neuropathological findings in a fifth patient. DWI may be a useful technique to screen for boys with L1CAM mutations.

Severe haemophilia A in a female: a compound heterozygote with nonrandom X-inactivation.

We report a case of severe haemophilia A (<1% factor VIII level) in a female resulting from an interesting and improbable combination of events. The patient inherited a factor VIII intron 22 inversion from her carrier mother, as well as a second factor VIII inversion involving intron 22 that arose de novo on her paternally derived X chromosome. In addition, the patient's paternally derived X chromosome had been preferentially inactivated in 95+% of her somatic cells. The patient's mother, who was clinically unaffected, carried an intron 22 inversion as well and also showed nonrandom X-inactivation. The patient's mother had a brother with severe haemophilia A. It is therefore likely that the mother's inversion was on her maternally derived X chromosome. Since she was unaffected, it is likely that her inversion-bearing X was the one that was preferentially inactivated.

Chromosome duplications and deletions and their mechanisms of origin.

Duplications and deletions of the same gene loci or chromosome regions are known to produce different clinical manifestations and are significant factors in human morbidity and mortality. Extensive cytogenetic and molecular cytogenetic studies with cosmid and YAC probes in two patients with unique mosaicism for reciprocal duplication-deletion allowed us to further understand the origin of these abnormalities. The first patient's mosaic karyotype was 46,XX, inv dup(11) (q23q13)/46,XX,del(11)(q13q23). The second patient had a 46,XY,dup(7)(p11.2p13)/46,XY,del(7)(p11.2p13)/46,XY karyotype. Fluorescence in situ hybridization studies on the first patient placed the two breakpoints near the folate-sensitive fragile sites FRA11A and FRA11B. The presence of repeated sequences responsible for these fragile sites may have been involved in the patient's duplication-deletion. Our investigation leads us to conclude that, in addition to known mechanisms (such as unequal crossovers between homologs, unequal sister chromatid exchanges, excision of intrachromatid loops, and meiotic recombination within a single chromatid), duplication-deletion can also arise by the formation of an overlying loop followed by an uneven crossover at the level of the DNA strand.

Townes-Brocks syndrome.

Townes-Brocks syndrome (TBS) is an autosomal dominant disorder with multiple malformations and variable expression. Major findings include external ear anomalies, hearing loss, preaxial polydactyly and triphalangeal thumbs, imperforate anus, and renal malformations. Most patients with Townes-Brocks syndrome have normal intelligence, although mental retardation has been noted in a few.

The site of a missense mutation in the extracellular Ig or FN domains of L1CAM influences infant mortality and the severity of X linked hydrocephalus.

The L1 cell adhesion molecule (L1CAM) plays an important role in axon growth, fasciculation, and neural migration. Mutations in the L1CAM gene produce a phenotype characterised by X linked hydrocephalus, mental retardation, spastic paraplegia, adducted thumbs, and agenesis of the corpus callosum. We have conducted a detailed analysis of the phenotypic effects of missense mutations in the extracellular portion of L1CAM, following a study that differentiated between "key" amino acid residues critical for maintaining the conformation of the extracellular immunoglobulin type C-like (Ig) or fibronectin type III-like (FN) domains and surface residues of less certain significance. We have analysed the data from 71 published cases and seven patients whose mutations were detected in our laboratory to determine if the site of a missense mutation in the Ig or FN domains correlated with the severity of hydrocephalus, presence of adducted thumbs, or survival past infancy. Mutations affecting the key residues in either type of domain were more likely to produce a phenotype with severe hydrocephalus, adducted thumbs, and lifespan less than one year than were mutations affecting surface residues. In addition, mutations affecting the FN domains were more likely than those affecting Ig domains to produce a phenotype with severe hydrocephalus, with less certain effects on adducted thumbs and lifespan. Mutations in key residues of the FN domains were particularly deleterious to infant survival. These data provide information that may be useful in predicting some aspects of the phenotypic effects of certain L1CAM mutations.

Maternal meiosis I non-disjunction of chromosome 15: dependence of the maternal age effect on level of recombination.

Non-disjoined chromosomes 15 from 115 cases of uniparental disomy (ascertained through Prader-Willi syndrome) and 13 cases of trisomy of maternal origin were densely typed for microsatellite loci spanning chromosome 15q. Of these 128 cases a total of 97 meiosis I (MI) errors, 19 meiosis II (MII) errors and 12 mitotic errors were identified. The genetic length of a map created from the MI errors was 101 cM, as compared with a maternal length of 137 cM based on CEPH controls. No significant differences were detected in the distribution of recombination events along the chromosome arm and a reduction was seen for most of the chromosome 15 intervals examined. It was estimated that 21% of tetrads leading to MI non-disjunction were achiasmate, which may account for most or all of the reduction in recombination noted. The mean age of mothers of cases involving MI errors which showed no transitions from heterodisomy to isodisomy was significantly lower (32.7) than cases showing one or more observable transitions (36.3) (P < 0.003, t -test). However, even among chiasmate pairs the highest mean maternal age was seen for multiple exchange tetrads. Chromosome-specific differences in maternal age effects may be related to the normal distribution of exchanges (and their individual susceptibilities) for each chromosome. However, they may also reflect the presence of multiple factors which act to ensure normal segregation, each affected by maternal age in a different way and varying in importance for each chromosome.

Constitutional del(19)(q12q13.1) in a three-year-old girl with severe phenotypic abnormalities affecting multiple organ systems.

We present the clinical, cytogenetic, and molecular studies on a constitutional deletion of 19q ascertained prenatally due to decreased fetal activity and IUGR. Chromosome analysis by GTG banding on amniocytes suggested a del(19)(q13.1q13.3), but the analysis of microsatellites by PCR demonstrated that the deletion involved the distal segment of q12 and the proximal segment of q13.1 (15 cM). The severely affected female infant born at 38 weeks has clinical findings that may be related to haploinsufficiency of specific genes within 19q12.1-->q13.1 that control important processes of normal development and cell function.

The mechanisms involved in formation of deletions and duplications of 15q11-q13.

Haplotype analysis was undertaken in 20 cases of 15q11-q13 deletion associated with Prader-Willi syndrome (PWS) or Angelman syndrome (AS) to determine if these deletions arose through unequal meiotic crossing over between homologous chromosomes. Of these, six cases of PWS and three of AS were informative for markers on both sides of the deletion. For four of six cases of paternal 15q11-q13 deletion (PWS), markers on both sides of the deletion breakpoints were inferred to be of the same grandparental origin, implying an intrachromosomal origin of the deletion. Although the remaining two PWS cases showed evidence of crossing over between markers flanking the deletion, this was not more frequent than expected by chance given the genetic distance between proximal and distal markers. It is therefore possible that all PWS deletions were intrachromosomal in origin with the deletion event occurring after normal meiosis I recombination. Alternatively, both sister chromatid and homologous chromosome unequal exchange during meiosis may contribute to these deletions. In contrast, all three cases of maternal 15q11-q13 deletion (AS) were associated with crossing over between flanking markers, which suggests significantly more recombination than expected by chance (p = 0.002). Therefore, there appears to be more than one mechanism which may lead to PWS/AS deletions or the resolution of recombination intermediates may differ depending on the parental origin of the deletion. Furthermore, 13 of 15 cases of 15q11-q13 duplication, triplication, or inversion duplication had a distal duplication breakpoint which differed from the common distal deletion breakpoint. The presence of at least four distal breakpoint sites in duplications indicates that the mechanisms of rearrangement may be complex and multiple repeat sequences may be involved.