Cloning a balanced translocation associated with DiGeorge syndrome and identification of a disrupted candidate gene.

DiGeorge syndrome (DGS), a developmental defect, is characterized by cardiac defects and aplasia or hypoplasia of the thymus and parathyroid glands. DGS has been associated with visible chromosomal abnormalities and microdeletions of 22q11, but only one balanced translocation--ADU/VDU t(2;22)(q14;q11.21). We now report the cloning of this translocation, the identification of a gene disrupted by the rearrangement and the analysis of other transcripts in its vicinity. Transcripts were identified by direct screening of cDNA libraries, exon amplification, cDNA selection and genomic sequence analysis using GRAIL. Disruption of a gene in 22q11.2 by the breakpoint and haploinsufficiency of this locus in deleted DGS patients make it a strong candidate for the major features associated with this disorder.

Association of chromosome 22q11 deletion with isolated anomalies of aortic arch laterality and branching.

OBJECTIVES: The purpose of this study was to determine the frequency of chromosome 22q11 deletions in patients with isolated anomalies of the aortic arch and its branches. BACKGROUND: Chromosome 22q11 deletions are often present in patients with certain forms of congenital cardiovascular disease, including tetralogy of Fallot, truncus arteriosus and interruption of the aortic arch. Among patients with these anomalies, chromosome 22q11 deletion is more common in those with abnormal aortic arch laterality or branching. METHODS: We studied 66 patients with isolated anomalies of the aortic arch and no associated intracardiac defects for deletions within chromosome 22q11, using fluorescence in situ hybridization with the cosmid probe N25 (D22S75). Arch anomalies included: double aortic arch (n = 22); right aortic arch with aberrant left subclavian artery (n = 28); right aortic arch with mirror-image branching and a vascular ring formed by a left-sided ductus from the descending aorta (n = 5); right aortic arch with mirror-image branching and no vascular ring (n = 4); and left aortic arch with aberrant right subclavian artery (n = 7). In addition, four patients had a cervical aortic arch, four had aortic coarctation and six had hypoplasia/atresia of the proximal pulmonary arteries. RESULTS: Chromosome 22q11 deletions were found in 16 patients (24%) across the full spectrum of anomalies studied. Among the morphologic variables analyzed, only hypoplasia/atresia of the proximal pulmonary arteries correlated with the deletion (p = 0.03). Among patients with a double arch, the frequency of chromosome 22q11 deletion was higher in those with an atretic minor arch than it was in those with a patent minor arch (p = 0.02). CONCLUSIONS: Chromosome 22q11 deletion is associated with isolated anomalies of laterality or branching of the aortic arch in 24% of cases in our series. These findings should alert the clinician to consider deletion screening in patients with isolated anomalies of the aortic arch.

Frequency of 22q11 deletions in patients with conotruncal defects.

OBJECTIVES: This study was designed to determine the frequency of 22q11 deletions in a large, prospectively ascertained sample of patients with conotruncal defects and to evaluate the deletion frequency when additional cardiac findings are also considered. BACKGROUND: Chromosome 22q11 deletions are present in the majority of patients with DiGeorge, velocardiofacial and conotruncal anomaly face syndromes in which conotruncal defects are a cardinal feature. Previous studies suggest that a substantial number of patients with congenital heart disease have a 22q11 deletion. METHODS: Two hundred fifty-one patients with conotruncal defects were prospectively enrolled into the study and screened for the presence of a 22q11 deletion. RESULTS: Deletions were found in 50.0% with interrupted aortic arch (IAA), 34.5% of patients with truncus arteriosus (TA), and 15.9% with tetralogy of Fallot (TOF). Two of 6 patients with a posterior malalignment type ventricular septal defect (PMVSD) and only 1 of 20 patients with double outlet right ventricle were found to have a 22q11 deletion. None of the 45 patients with transposition of the great arteries had a deletion. The frequency of 22q11 deletions was higher in patients with anomalies of the pulmonary arteries, aortic arch or its major branches as compared to patients with a normal left aortic arch regardless of intracardiac anatomy. CONCLUSIONS: A substantial proportion of patients with IAA, TA, TOF and PMVSD have a deletion of chromosome 22q11. Deletions are more common in patients with aortic arch or vessel anomalies. These results begin to define guidelines for deletion screening of patients with conotruncal defects.

Allelic variants of the follistatin gene in polycystic ovary syndrome.

In an earlier study of 37 candidate genes for polycystic ovary syndrome (PCOS), the strongest evidence for genetic linkage was found with the region of the follistatin gene. We have now carried out studies to detect variation in the follistatin gene and assess its relevance to PCOS. By sequencing the gene in 85 members of 19 families of PCOS patients, we found sequence variants at 17 sites. Of these, 16 sites have variants that are too rare to make a major contribution to susceptibility; the only common variant is a single base pair change in the last exon at a site that is not translated. In our sample of 249 families, the evidence for linkage between PCOS and this variant is weak. We also examined the expression of the follistatin gene; messenger RNA levels in cultured fibroblasts from PCOS and control women did not differ appreciably. We conclude that contributions to the etiology of PCOS from the follistatin gene, if any, are likely to be small.

Kabuki syndrome is not caused by a microdeletion in the DiGeorge/velocardiofacial chromosomal region within 22q 11.2.

Kabuki syndrome (KS) or Niikawa-Kuroki syndrome is a sporadic disorder characterized by postnatal growth retardation, developmental delay, mild to moderate retardation, and a characteristic facial appearance. Cardiovascular defects, clefts of the lip, palate, or both, and musculoskeletal abnormalities occur in about 50% of patients with KS. The cause of this multiple congenital anomaly syndrome is unknown, and investigators have speculated that KS is a contiguous gene-deletion syndrome. Based on the presence of congenital heart defects in patients with KS, it was suggested that this disorder might share a common cause with the 22q11 deletion syndromes. A preliminary study of 2 patients with KS failed to detect a deletion within 22q11. We report the results of fluorescence in situ hybridization with cosmid probes for loci D22S75 (N25) and D22S259 (R32) within the DiGeorge chromosomal region (DGCR) on metaphase spreads from an additional 5 patients, 2 non-Japanese and 3 Japanese, with KS. None of the 5 had deletions at either locus. It is unlikely that KS is caused by a deletion within 22q11.

Evolution of latent hypoparathyroidism in familial 22q11 deletion syndrome.

Latent hypoparathyroidism (LHP), the inability to increase midmolecular parathyroid hormone levels appropriately during a hypocalcemic challenge, was reported previously in an asymptomatic woman with tetralogy of Fallot. This women's fourth child died with DiGeorge anomaly. Seven years later, we restudied the index patient with LHP and evaluated three generations of her family for parathyroid dysfunction, cardiac abnormalities, and del 22(q11). Deletions were found in six relatives, three with conotruncal cardiac defects and three with a structurally normal heart. We found significant transgenerational noncardiac phenotypic variability, including learning difficulties, dysmorphic facial appearance, and psychiatric illness. A spectrum of parathyroid gland dysfunction associated with the del 22(q11) was seen, ranging from hypocalcemic hypoparathyroidism to normocalcemia with abnormally low basal intact parathyroid hormone (iPTH) levels. In addition, LHP found in the index patient 7 years ago had evolved to frank hypocalcemic hypoparathyroidism. In this family, which is the largest family with 22q11 deletions studied to date, parathyroid gland dysfunction evolved over time. We suggest that the calcium parathyroid hormone axis of unrelated patients with del 22(q11) be followed closely for the development of hypocalcemic hypoparathyroidism.

Velo-cardio-facial syndrome and DiGeorge sequence with meningomyelocele and deletions of the 22q11 region.

Approximately 5% of children with neural tube defects (NTDs) have a congenital heart defect and/or cleft lip and palate. The cause of isolated meningomyelocele, congenital heart defects, or cleft lip and palate has been largely thought to be multifactorial. However, chromosomal, teratogenic, and single gene causes of combinations of NTDs with congenital heart defects and/or cleft lip and palate have been reported. We report on 3 patients with meningomyelocele, congenital heart defects, and 22q11 deletions. Two of the children had the clinical diagnosis of velo-cardio-facial syndrome (VCFS); both also have bifid uvula. The third child had DiGeorge sequence (DGS). The association of NTDs with 22q11 deletions has not been reported previously. An accurate diagnosis of the 22q11 deletion is critical as this micro-deletion and its associated clinical problems is transmitted as an autosomal dominant trait due to the inheritance of the deletion-bearing chromosome. We recommend that all children with NTDs and congenital heart defects, with or without cleft palate, have cytogenetic and molecular studies performed to detect 22q11 deletions.

Classical Noonan syndrome is not associated with deletions of 22q11.

Deletions of 22q11 cause DiGeorge sequence (DGS), velo-cardio-facial syndrome (VCFS), conotruncal anomaly face syndrome, and some isolated conotruncal heart anomalies. Demonstration of a 22q11 deletion in a patient with manifestations of DGS and Noonan syndrome (NS) has raised the question of whether NS is another of the chromosome 22 microdeletion syndromes. This prompted us to evaluate a cohort of patients with NS for evidence of 22q11 deletions. Five of 6 NS propositi studied in our laboratory with marker N25 (D22S75) did not have a 22q11 deletion. A 2-month-old infant with several findings suggestive of NS did have a 22q11 deletion, suggesting that a small number of 22q11 deletion propositi may present with a NS-like picture. However, most cases of NS must have another cause.

Autosomal dominant "Opitz" GBBB syndrome due to a 22q11.2 deletion.

We report on a family with autosomal dominant paternally inherited "Opitz" GBBB syndrome and an additional case with findings which have been reported in that syndrome. In each case the propositus presented with a vascular ring. Since a vascular ring may be a sign of a 22q11.2 deletion [Zacki et al., 1995], FISH (fluorescence in situ hybridization) studies were performed. These studies demonstrated a 22q11.2 deletion in the 3 affected individuals. Review of Opitz GBBB syndrome and the 22q11.2 microdeletion syndrome demonstrates significant overlap of manifestations including both facial characteristics and structural anomalies. Based on the phenotypic overlap and the presence of a 22q11.2 deletion in our patients with Opitz GBBB syndrome and the presence of a deletion in a patient with lung hypoplasia, absent pulmonary artery, and long segment tracheomalacia, we propose that, in some cases, the Opitz GBBB syndrome may be due to a 22q11.2 deletion. This enlarges the list of "syndromes" associated with the 22q11.2 deletion, which presently includes most patients with DiGeorge, velocardiofacial, and conotruncal anomaly face syndrome.

Unbalanced 15;22 translocation in a patient with manifestations of DiGeorge and velocardiofacial syndrome.

We report on an 8-year-old girl with an unbalanced 15;22 translocation and manifestations of DiGeorge syndrome (DGS), velocardiofacial syndrome (VCFS), and other abnormalities. The main manifestations of our patient were feeding difficulties, respiratory infections, short stature, peculiar face with hypertelorism, prominent nose, abnormal ears, microstomia and crowded teeth, short broad neck and shield chest with pectus deformity and widely spaced nipples with abnormal fat distribution, heart defect, scoliosis, asymmetric limb development, abnormal hands and feet, and hyperchromic skin patches. Cytogenetic studies demonstrated a 45,XX,der(15)t(15;22)(p11.2;q11.2), -22 karyotype. Fluorescence in situ hybridization (FISH) studies confirmed loss of the proximal DiGeorge chromosomal region (DGCR). This case adds to the diversity of clinical abnormalities caused by deletions within 22q11.2.

Preliminary phenotypic map of chromosome 4p16 based on 4p deletions.

We have collected and analyzed clinical information from 11 patients with chromosome 4p deletions or rearrangements characterized by various molecular techniques. Comparing the extent of these patients' deletions with their respective clinical presentations led to the proposal of a preliminary phenotypic map of chromosome 4p. This map consists of regions which, when deleted, are associated with specific clinical manifestations. Nonspecific changes such as mental and growth retardation are not localized, and probably result from the deletion of more than one gene or region. The region associated with most of the facial traits considered typical in Wolf-Hirschhorn syndrome (WHS) patients coincides with the currently proposed WHS critical region (WHSCR), but some anomalies commonly seen in WHS appear to map outside of the WHSCR. The observation of clinodactyly in 2 patients with nonoverlapping deletions allows assignment of these defects to at least 2 separate regions in 4p16. These initial observations and attempts at genotype/phenotype correlation lay the groundwork for identifying the genetic basis of these malformations, a common objective of gene mapping efforts and chromosome deletion studies.

Nasal dimple as part of the 22q11.2 deletion syndrome.

The phenotype of the 22q11.2 microdeletion syndrome is quite variable. We describe 2 patients with a 22q11.2 deletion and a dimpled nasal tip, which, we suggest can be the extreme of the broad or bulbous nose commonly found in the 22q11.2 deletion syndrome, and should not be confused with the more severe nasal abnormalities seen in frontonasal dysplasia.

DiGeorge anomaly with renal agenesis in infants of mothers with diabetes.

We report on 2 infants with the DiGeorge anomaly born to diabetic mothers treated with insulin. Both infants had unilateral renal agenesis. One of the mothers has manifestations suggestive of velo-cardio-facial syndrome (VCFS). Cytogenetic studies on both patients and the mother with apparent VCFS were normal. Molecular studies utilizing probes from the DiGeorge critical region did not demonstrate a 22q11 microdeletion in either patient or the mother with apparent VCFS. We conclude that maternal diabetes is a pathogenetic factor in the DiGeorge anomaly, and infants of diabetic mothers who have this anomaly should also be screened for renal agenesis.

Deletions and microdeletions of 22q11.2 in velo-cardio-facial syndrome.

Velo-cardio-facial syndrome (VCFS), an autosomal dominant disorder, is characterized by cleft palate, cardiac defects, learning disabilities and a typical facial appearance. Less frequently, VCFS patients have manifestations of the DiGeorge complex (DGC) including hypocalcemia, hypoplastic or absent lymphoid tissue and T-cell deficiency suggesting that these 2 conditions share a common pathogenesis. Here, we report the results of cytogenetic and molecular studies of 15 VCFS patients. High-resolution banding techniques detected an interstitial deletion of 22q11.21-q11.23 in 3 patients. The remaining 12 patients had apparently normal chromosomes. Molecular analysis with probes from the DiGeorge Chromosome Region (DGCR) within 22q11 detected DNA deletions in 14 of 15 patients. In 2 families, deletions were detected in the affected parent as well as the propositus suggesting that the autosomal dominant transmission of VCFS is due to segregation of a deletion. Deletions of the same loci previously shown to be deleted in patients with DGC explains the overlapping phenotype of VCFS and the DGC and supports the hypothesis that the cause of these two disorders is the same.

Skeletal anomalies and deformities in patients with deletions of 22q11.

Skeletal anomalies in patients with a 22q11.2 deletion are reported infrequently. We report the skeletal findings in 108 patients with a 22q11.2 deletion, of whom 37 (36%) had a skeletal anomaly. Twenty-two patients (20%) had anomalies of the limbs, 7 of the upper limb, including preaxial or postaxial polydactyly. An anomaly of the lower limb was found in 16 patients, including postaxial polydactyly, clubfoot, severely overfolded toes, and 2-3 toe cutaneous syndactyly. Chest films of 63 patients were examined; 30% of them had abnormal findings, most commonly supernumerary ribs (17%) or a "butterfly" vertebral body (11%). Hypoplastic vertebrae, hemivertebrae, and vertebral coronal clefts were also noted. Thus, skeletal anomalies are not uncommon in patients with a 22q11.2 deletion and may occur more frequently than recognized previously.

Cognitive and behavior profile of preschool children with chromosome 22q11.2 deletion.

A microscopic deletion of chromosome 22q11.2 has been identified in most patients with the DiGeorge, velocardiofacial syndrome, conotruncal anomaly face syndrome, and in some patients with isolated conotruncal cardiac anomalies. This study presents the neurodevelopmental outcome, including cognitive development, language development, speech, neuromuscular development, and behavioral characteristics of 40 preschool children (ages 13 to 63 months) who have been diagnosed with the 22q11.2 deletion. The impact of cardiac disease, cardiac surgery, and the palatal anomalies on this population was also studied. In the preschool years, children with a 22q11.2 deletion are most commonly found to be developmentally delayed, have mild hypotonia, and language and speech delays. The more significantly delayed children are at high risk to be subsequently diagnosed with mild or moderate mental retardation. The global delays and the variations in intelligence found are directly associated with the 22q11.2 deletion and are not explained by physical anomalies such as palatal defects or cardiac defects, or therapeutic interventions such as cardiac surgery. Our findings demonstrate that there is a pattern of significant speech disorders within this population. All of the children had late onset of verbal speech. Behavioral outcomes included both inhibition and attention disorders. Early intervention services are strongly recommended beginning in infancy to address the delays in gross motor skills, speech and language, and global developmental delays.

Cloning and expression of primate Daxx cDNAs and mapping of the human gene to chromosome 6p21.3 in the MHC region.

Murine Daxx, a protein that binds to Fas and enhances Fas-mediated apoptosis through a signal transduction pathway involving the Jun amino-terminal kinase, was recently described. Here we report the cloning of human and monkey Daxx cDNAs, the widespread expression of Daxx mRNA in human tissues, and the mapping of the human Daxx gene to 6p21.3 in the major histocompatibility complex (MHC) region. The location of the Daxx gene, which is implicated in the pathway for deletion of autoreactive lymphocytes, in the MHC region may shed light on the genetic basis of autoimmune diseases.

High pressure 2H nuclear magnetic resonance study of the gel phases of dipalmitoylphosphatidylcholine.

The 2H-NMR lineshapes of dipalmitoylphosphatidylcholine perdeuterated in the acyl chains were studied in a 15% dispersion in water as a function of pressure from 1 bar to 5 kbar over the temperature range from 7 degrees C to 75 degrees C. Increasing pressure in the gel state had the same effect as lowering the temperature: the lineshape gradually changed from a motionally averaged to a rigid lattice type spectrum with much of the intensity in the shoulders at +/- 63 kHz. At very high pressures and low temperatures (7 degrees C, 2.5 kbar; 25 degrees C, 5 kbar) even the methyl portion of the spectrum became a rigid lattice type spectrum at +/- 21 kHz. In addition to the liquid crystalline phase, five gel phases were detected. Using different techniques to determine the phase transitions, a general pressure-temperature phase diagram was constructed.

PCR assay for screening patients at risk for 22q11.2 deletion.

Deletions of 22q11.2 have been detected in the majority of patients with DiGeorge, velocardiofacial, and conotruncal anomaly face syndromes by either cytogenetic analysis, fluorescence in situ hybridization (FISH), or Southern blot hybridization. However, these techniques may not be the most efficient or cost-effective means of screening large numbers of "at-risk" patients. Therefore, we developed a PCR assay to assess a patient's likelihood of having a 22q11.2 deletion based on homozygosity at consecutive markers in the DiGeorge chromosomal region. The sensitivity and specificity of PCR screening were evaluated in a cohort of cardiac patients. We conclude that a PCR-based assay is a reliable and efficient means of identifying which patients are at greatest risk for a 22q11.2 deletion and should have FISH studies to confirm their deletion status.

Recognition of tubo-ovarian abscess in adolescents with pelvic inflammatory disease.

PURPOSE: Ultrasonography of the pelvis is commonly used to diagnose tubo-ovarian abscess (TOA) in patients with pelvic inflammatory disease (PID). Our objective was to determine whether the clinical features of PID differ in adolescents with and without TOA. METHODS: A retrospective design was used to derive and validate a clinical model differentiating adolescents with PID who did and did not have TOA. The study population consisted of hospitalized adolescents with a discharge diagnosis of PID. Of the 208 patients discharged from January 1, 1990, to July 31, 1993, 87 (42%) met published criteria for PID and comprised the derivation set. Of the 63 patients from August 1, 1993, to June 24, 1994, 30 (48%) met criteria and comprised the validation set. All patients had pelvic ultrasonography performed during hospitalization. The ultrasonography records were reviewed retrospectively for TOA, ovarian and uterine size, clarity of tissue planes, and endometrial or cul-de-sac fluid. Medical records were reviewed for sociodemographic characteristics, medical and sexual history, physical examination, laboratory results, and hospital course. RESULTS: TOA was present in 17% of the derivation set and 20% of the validation set. A six-variable model developed on the derivation set performed best in differentiating the TOA and non-TOA groups: last menstrual period > 18 days prior to admission (60% and 17%), previous PID (53% and 22%), palpable adnexal mass (13% and 3%), white blood cell count > or = 10,500/microliters (33% and 64%), erythrocyte sedimentation rate > 15 mm/h (33% and 64%), and heart rate > 90/min (40% and 78%). In the derivation and validation sets, the model correctly identified 78 and 83% of the TOA groups and 88 and 77% of the non-TOA groups. The area under the receiver operating characteristic curve of the model was 0.92 in the derivation set and 0.87 in the validation set. CONCLUSIONS: We conclude that clinical characteristics help identify adolescents with acute PID who have TOA. These patients may have fewer signs of acute illness than those without TOA and may develop symptoms later in the menstrual cycle.