The autosomal CGS discussed in this chapter are presented in order of chromosome number. For each, the chromosomal band that is deleted or duplicated follows the syndrome name. Details of deletion type (terminal or interstitial) are given in each subsection on cytogenetics. Some of these diseases are discussed in separate chapters in this book. Therefore, they will be presented in less detail here and the reader is referred to those chapters.
Monosomy 1p36 [Del(1)(P36)]
The clinical features associated with deletion of the most distal band on the short arm of chromosome 1 were recently delineated.140,141 Patients show a wide range of features including variable degrees of mental retardation, growth delay, seizures and/or abnormal EEGs, hypotonia, developmental delay, early puberty, orofacial clefting or palatal anomalies, enlarged anterior fontanel, dysmorphic features, deafness, and cardiomyopathy.140,141 Monosomy 1p36 is one of the most common microdeletion syndromes, with the incidence of the deletion estimated to be about 1 in 10,000 births.140
The deletion seen in monosomy 1p36 is a terminal deletion of the most distal band on the short arm of chromosome 1. The majority of cases of monosomy 1p36 have been identified through cytogenetic analysis. By molecular analysis and FISH, most deletions are only partial, telomeric deletions of band 1p36 and a few (≈15 percent) are interstitial deletions, retaining the original telomere, at the molecular level.142 For the most part, the size of the deletion and the type of deletion (interstitial or terminal) cannot be distinguished at the light microscope. At the resolution of routine cytogenetic analysis (400 to 550 bands), this deletion is difficult to visualize, accounting for the fact that roughly 50 percent of patients had a “normal” chromosome evaluation prior to the detection of the deletion.140 Three cases of monosomy 1p36 were identified by using telomeric markers to screen 127 patients with unexplained mental retardation;143–145 thus, exemplifying the common occurrence of this deletion and the inherent difficulties in visualizing this region by G-banding. Currently, there are 39 reports of “pure” 1p36 deletions and at least 10 other cases of 1p36 deletions due to derivative chromosomes that contain additional segmental aneusomy from other chromosomes.141 The majority of cases are sporadic. The frequency of parental translocations is about 3 percent.26 This occurrence is smaller than the frequency of familial translocations in other terminal deletions including cri du chat and Miller-Dieker syndromes, in which about 10 percent of cases are due to the malsegregation of a parental translocation.22,27
The deletion sizes have been characterized for 30 patients with monosomy 1p36 using polymorphic microsatellite markers and FISH probes.26 The deletion sizes were found to be quite variable, with the smallest deletions estimated to be less than 5 Mb and the largest deletions encompassing a genetic distance of up to 32 cM.26 The occurrence and severity of the clinical findings correlate with the size of the deletions. Patients with the smallest, most telomeric deletions show mild mental retardation, while those with larger deletions show severe mental retardation, seizures, and deafness. The patients with the largest deletions may also have orofacial clefting.26 The development of this panel of deletion patients and molecular refinement of the deletions will allow for a focused search for genes causing distinct features of the syndrome in specific regions of 1p36. The majority of sporadic deletions (78 percent) occur on the maternally inherited chromosome 1.26 There are no obvious clinical differences between maternally and paternally derived deletions. This maternal bias may reflect the underlying mechanism of deletion in this region that may be more prone to occur during oogenesis.
The majority of patients are identified through good quality cytogenetic studies and FISH using very distal 1p probes. Most patients are deleted for loci p1-79 (D1Z2), D1S243, and CDC2L1 (p58) (MIM 176873). Three patients were identified using telomere screening strategies,143–145 but about 15 percent of patients have interstitial deletions that are missed using these probes.142 Parental chromosomes should be investigated to exclude a familial rearrangement that would carry a significant risk for future pregnancies.
Russell-Silver Syndrome [Dup(7)(P12p13)]
The association of prenatal and postnatal growth retardation with relative sparing of the head, triangular facies and other dysmorphic features, and limb and facial asymmetry was first described by Silver146 and Russell147 independently. Most cases of Russell-Silver syndrome (RSSMIM 180860) are sporadic, but a significant number have been reported to be transmitted in an autosomal dominant fashion (see review148).
A number of chromosomal abnormalities have been associated with the RSS phenotype including deletion of distal 15q,149 deletion of proximal 8q,150 and balanced translocations involving 17q.151,152 No other consistent cytogenetic abnormality was found until recently; two cases of RSS have been described with duplications in the proximal short arm of chromosome 7.153,154 Although the duplicated regions were slightly different, the common region was from 7p12.1 to 7p13.
In 1988, Spence et al.155 described a child who had inherited two copies of chromosome 7 from her mother and failed to inherit a chromosome 7 from her father (maternal uniparental disomy 7). In addition to having cystic fibrosis, due to the inheritance of two copies of a mutant CF gene from her mother, she had postnatal growth retardation. Since that time, several reports of maternal UPD 7 with short stature have been published (see reviews156–158). Specifically, RSS patients have been investigated for UPD and studies have found that about 10 percent of patients have maternal disomy 7.159–162 The critical region for an imprinted locus has been narrowed to the short arm of chromosome 7, based on two cases of partial UPD 7 involving just 7p.163,164 Further refinement, based on the two duplication 7p cases has resulted in a candidate locus.153,154 The growth factor receptor-binding protein 10 (GRB10; MIM 601523) binds both insulin and insulin-like growth-factor receptors. Thus, GRB10 has a growth suppressing function that would be consistent with the phenotype of growth retardation in RSS patients. GRB10 has been shown to be imprinted in humans164a and mutations have been identified in two RSS patients.164b Additionally, the mouse orthologue, Grb10/MegI, shows imprinting.165 Maternal UPD of this region in mouse produces prenatal growth failure, whereas, paternal UPD of this region produces overgrowth,166 making this a good candidate for RSS.
Patients with RSS should be investigated with routine cytogenetic banding methods to exclude a duplication of the short arm of chromosome 7 or other chromosomal abnormalities. Additionally, patients could be investigated for submicroscopic duplications using interphase FISH analysis.153 For those patients showing a normal karyotype, UPD should be investigated with polymorphic markers for the short arm of chromosome 7. This approach would detect segmental UPD, restricted to 7p, as well as holochromosomic UPD 7. In the future, a methylation-specific assay will likely be developed to detect patients with UPD or imprinting mutations.
Williams Syndrome [Del(7)(Q11.23q11.23)]
The clinical features that are now associated with Williams-Beuren syndrome (WBS; MIM 194050) were first independently described by Williams in 1961167 and Beuren in 1962.168 Patients have a distinct facial dysmorphism that includes periorbital fullness, stellate pattern of the irides,169 anteverted nares, long philtrum, and prominent, full lips. Other significant clinical features include infantile hypercalcemia (MIM 143880), mental retardation, gregarious personality, growth deficiency, and considerable cardiac involvement. Cardiovascular anomalies include supravalvular aortic stenosis, peripheral pulmonary artery stenosis, and pulmonic valvular stenosis. Isolated supravalvular aortic stenosis (SVAS; MIM 185500), a congenital narrowing of the ascending aorta, is an autosomal dominant condition that showed linkage to chromosome 7.170 A family segregating isolated SVAS was shown to have a translocation involving chromosome 7 that disrupted the elastin gene.171 Additional mutations of the elastin gene in patients with SVAS provided strong evidence for a critical role for elastin in vascular development.75,76 Given the occurrence of SVAS in WBS, this region was investigated in patients with the disorder. Most cases of WBS are sporadic, although a few families have demonstrated autosomal dominant inheritance.172 WBS occurs in about 1 in 20,000 to 1 in 50,000 live births.173
Chromosomal abnormalities in WBS were investigated, with no consistent deletion or rearrangement detected.174 Given the linkage of SVAS to the long arm of chromosome 7 and the familial t(6;7) associated with SVAS, the elastin gene on chromosome 7 was investigated in patients with WBS and was found to be deleted.46 Subsequently, deletions of the elastin locus have been found using FISH in greater than 90 percent of WBS patients.175 Deletions are not reliably detected by routine cytogenetics, but have been localized to 7q11.23 by high-resolution analysis.176 The deletions are interstitial and occur in a G-negative band, adjacent to the centromere on the long arm of chromosome 7.
Most deletions in patients with WBS are of a consistent size of approximately 2 Mb.177 The region is flanked by low-copy, region-specific repeats178 that likely contribute to the deletion process through homologous recombination and unequal crossing over (see “Molecular Mechanisms Leading to Contiguous Gene Syndromes” below).179,180 The deletion region contains at least 15 known genes.176 The only gene that has been definitively shown to cause clinical features in WBS is the elastin locus. Functional monosomy for elastin results in the SVAS and is presumed to be responsible for other features in WBS including renal artery stenosis, hypertension, hoarse voice, small genitalia, premature aging of the skin, and perhaps some of the facial characteristics. However, individuals with isolated SVAS due to mutations or small deletions in the elastin gene do not have the facial features, hypercalcemia, or mental retardation. Based on these observations, WBS is considered a true CGS, with other genes likely contributing to the overall phenotype.175 Although most patients have consistent-sized deletions, a few patients have been described with larger deletions.181 These patients have more severe mental retardation and many have seizures.181
The deletion in 7q11.23 cannot be reliably detected using routine chromosome analysis. FISH is the only reliable means for detecting the deletions. Greater than 90 percent of patients demonstrate a deletion by FISH using a probe containing the elastin locus.175
Langer-Giedion Syndrome [Del(8)(Q24.1q24.1)]
The association of sparse hair, a characteristic facial appearance including a pear-shaped nose with bulbous tip and tented alae nasi, and cone-shaped epiphyses was first described by Giedion in 1966,182 and termed the trichorhinophalangeal syndrome type I (TRPS I; MIM 190350). Affected individuals are usually intellectually normal. In 1969, several groups independently reported patients with features of TRPS I, who, in addition, exhibited multiple cartilaginous exostoses and mental retardation, a combination that has come to be known as the trichorhinophalangeal syndrome type II (TRPS II) or Langer-Giedion syndrome (LGS) (MIM 150230).183 Other clinical features of LGS may include microcephaly, large, protruding ears, elongated upper lip, micrognathia, lax skin, and short stature.183,184 Although mental retardation was originally considered a constant feature, intelligence is extremely variable. Approximately 29 percent of patients show normal or borderline intelligence, 17 percent have mild mental retardation, 40 percent are moderately retarded, and 14 percent are profoundly retarded.184
TRPS I is inherited as an autosomal dominant disorder. Families with multiple cartilaginous exostoses (ME; MIM 13370) identical with that seen in LGS have been observed with autosomal dominant transmission. LGS is generally a sporadic condition with the notable exception of a set of twins and one case of an affected father and daughter.184
In 1980, Buhler et al.185 and Pfeiffer186 reported cases of LGS with de novo interstitial deletions in the long arm of chromosome 8. Subsequently, this was confirmed and the specific location of the deletion determined to be band 8q24.1.184,187 Approximately 75 percent of LGS patients have cytogenetically detectable deletions in this region by conventional banding or high-resolution chromosome analysis.44
These observations led to the hypothesis that LGS was a CGS caused by the deletion of several genes in 8q24.1, including the TRPS1 gene, the multiple exostoses gene, and a locus or loci causing mental retardation.1 The finding of a small number of LGS patients with normal intelligence and visible or submicroscopic deletions suggests that the mental retardation locus is not located between the TRPS I and multiple exostoses loci.44 Mapping of TRPS I to 8q24.1 was initially confirmed by the identification of two patients with visible deletions who had TRPS I but without exostoses or mental retardation.188,189 A locus for multiple exostoses in 8q24.1 was initially supported by the observation of a patient with a balanced translocation and breakpoint in 8q24.1.190
Linkage analysis of families with the autosomal dominant condition hereditary multiple exostoses has demonstrated genetic heterogeneity, with an estimated 70 percent of families showing linkage to 8q24.1.191 Molecular studies to define the smallest region of overlap among LGS deletions has been reported by two groups,44,192 and is estimated at less than 2 Mb. In addition to 12 visible deletions, submicroscopic deletions were detected by quantitative Southern blot analysis in two patients with apparently balanced translocations and two patients with apparently normal karyotypes.
The gene for the multiple exostosis I locus has been cloned (EXT1; MIM 133700).193 Patients with isolated multiple exostoses have shown loss of function mutations in EXT1.194 These patients do not display other features of LGS, lending support to the hypothesis that functional monosomy (haploinsufficiency) causes the multiple exostosis phenotype in LGS and that LGS is a CGS with other genes causing the features associated with TRPS I and the mental retardation. Recently, the gene for the isolated TRPS I phenotype was cloned.195 The gene contains a putative GATA-binding zinc finger and two potential nuclear localization signals. Some patients were found to have submicroscopic deletions containing this gene, while about 70 percent of patients with the TRPS I phenotype had mutations within the gene. In addition to the identification of the EXT1 locus in the LGS critical region, the fact that TRPS1 is deleted in some patients with TRPS I and all patients with LGS; that TRPS1 is disrupted in two patients with balanced chromosomal abnormalities and TRPS I; and that TRPS1 contains inactivating mutations in patients with TRPS I, makes LGS a true CGS.
Many patients with LGS show visible deletions at 8q24.1 using routine cytogenetics. FISH using probes containing the TRPS1 gene aid in the detection of submicroscopic deletions in TRPS I patients who do not have multiple exostoses,195 while FISH probes containing the EXT1 locus are helpful in identifying patients with the full LGS clinical spectrum.196
Potocki-Shaffer Syndrome [Del(11)(P11.2p12)]
In 1993, Shaffer et al., reported a family that was segregating an insertional translocation between chromosomes 11 and 13 that resulted in deletion of 11p11.2 in some family members. These individuals had skull defects (wormian bones and biparietal foramina), brachycephaly, developmental delay, mental retardation, and genital anomalies.197 One family member had multiple exostoses but his sister, who also had the deletion, did not have this trait, so it was concluded that the multiple exostoses were an incidental finding in the brother. Subsequently, additional patients were described with del(11)(p11.2p12) and were all found to have multiple exostoses.198,199 Radiographic reexamination of the sister from the original family revealed multiple exostoses.198 The full spectrum of the Potocki-Shaffer syndrome (PSS; MIM 601224) has been delineated to include mental retardation, biparietal foramina, minor facial dysmorphism, multiple cartilaginous exostoses and micropenis in males.198 In 1990, Lorenz et al. reported a patient with an unusual presentation of acrocephalosyndactyly.200 Shaffer et al. proposed that although this patient had a normal karyotype, many of the same features were found in the family reported with del(11)(p11.2p12). Upon reexamination of the karyotype, the case reported by Lorenz et al. was found to have a deletion of 11p11.2.199
The interstitial deletion associated with PSS can be visualized by routine cytogenetic analysis. However, given that the deleted band is the first G-negative band adjacent to the centromere, the deletion may be overlooked.197–199 In fact, the majority of patients reported to date had at least one “normal” karyotype before the deletion was identified.197–199 One submicroscopic deletion, detectable by polymorphic microsatellite analysis, has been reported.199 FISH analysis using a probe containing the multiple exostosis 2 gene (EXT2) has been used to confirm a known 11p11.2 deletion.201
The extent of the deletion has been delineated and appears relatively large (≈20 cM distance).198,199 Both maternally and paternally derived deletions have been observed.198,199 The deletion is presumed to include at least three loci contributing to this syndrome; one for multiple exostoses, one for biparietal foramina, and one for mental retardation. This assumption was based on families who segregate autosomal dominant isolated multiple exostoses that are linked to chromosome 11,202 one family with a smaller deletion in 11p11.2 with multiple exostoses and biparietal foramina but no mental retardation,199 and large families segregating parietal foramina as an isolated autosomal dominant condition (PFM1; MIM 168500). The multiple exostosis 2 locus was cloned (EXT2; MIM 133701)203,204 and mutations in isolated multiple exostoses patients have been identified.204
Most patients with PSS will have visible deletions using routine cytogenetic methods. However, given the proximity of this G-negative band to the centromere of chromosome 11 and the fact that the deletion in most patients with PSS was missed initially, FISH using a probe containing the EXT2 gene should be used in patients with mental retardation and multiple exostoses who do not fit the criteria for Langer-Giedion syndrome.201
WAGR Syndrome [Del(11)(P12p14)]
The term WAGR syndrome defines the association of Wilms tumor, aniridia, genitourinary dysplasia, and mental retardation (WAGR).205,206 The clinical features, and the genetics of Wilms tumor, or nephroblastoma (MIM 194070), are described in Chap. 38. Aniridia type 2 (AN2; MIM 106210) is a bilateral developmental disorder of the eye, characterized by the complete or partial aplasia or dysplasia of the iris and optic nerve hypoplasia, leading to vision impairment. In some patients, the phenotype can be progressive, owing to cataract, corneal opacification, lens dislocation, and glaucoma.207 There is genetic evidence that aniridia is the human equivalent of the murine small eye mutation.208
Genitourinary abnormalities observed in patients with WAGR syndrome include ambiguous genitalia (pseudohermaphroditism), undescended testes, hypospadias, fused kidneys, urinary tract anomalies, and gonadoblastoma.11,209–212 The association of these features with a complex nephropathy and with Wilms tumor is also known as the Denys-Drash syndrome.213 This raises the possibility that the latter clinical entity may be part of the WAGR syndrome.214 Additional features observed in patients with WAGR syndrome include variable mental retardation, growth retardation, and hemihypertrophy.11,215,216
The identification of interstitial deletions involving the 11p13 region in patients with WAGR syndrome suggested a common genetic etiology for its various components.11,217 It was postulated that the complex phenotype observed in patients with WAGR syndrome was the manifestation of a CGS due to a deletion of adjacent genes on 11p13.11,217 Since then, many patients carrying various types of rearrangements of 11p13, and showing features of the WAGR syndrome, have been identified, lending support to this hypothesis.216–221
Molecular characterization of patients carrying 11p13 abnormalities has permitted the assignment of loci involved in specific phenotypic manifestations of the WAGR syndrome.222–228 Distinction between the Wilms tumor and aniridia loci was possible through the study of patients with familial 11p13 translocations showing isolated aniridia without Wilms tumor.223,229,230 Furthermore, independent evidence for the presence of an aniridia gene on 11p13 came from linkage studies in families with isolated autosomal dominant aniridia.228,231
Several detailed physical maps of 11p13, spanning regions containing deletion and translocation breakpoints from patients with WAGR syndrome, have been constructed.208,232,233 Overlap cloning of the 11p13 region led to the isolation of the Wilms tumor gene (WT1), which encodes a zinc finger-containing transcription factor.234,235 In Wilms tumor, constitutional heterozygous deletions or mutations involving the Wilms tumor gene are followed by loss of heterozygosity through a somatic-cell event. The genetic mechanism involved in the pathogenesis of Wilms tumor is, therefore, a good example of Knudson's two-hit hypothesis.236
There is convincing evidence that the Wilms tumor gene is also involved in the genitourinary abnormalities seen in patients with WAGR syndrome. Point mutations in the Wilms tumor gene were found in patients with Denys-Drash syndrome. These mutations must have a dominant-negative effect based on the evidence that they are present only in one allele of the Wilms tumor gene and that the genitourinary abnormalities found in Denys-Drash patients are far more severe than those observed in WAGR deletions. The molecular mechanisms underlying the dominant-negative nature of these mutations have not yet been identified.237
Following the identification of the Wilms tumor gene, “chromosome walking” and “jumping” in 11p13 led to the positional cloning of another disease gene from this region, the aniridia gene.238 The PAX6 gene was found to be disrupted in aniridia-associated chromosomal aberrations and to be expressed in all tissues affected in aniridia patients.238 The identification of point mutations in PAX6 in patients with sporadic aniridia239 and of mutations in the murine Pax6 orthologue in mice with the small-eye phenotype240 provided definitive evidence for the involvement of this gene in aniridia. However, the possibility still exists that more than one aniridia locus might be present in 11p13.239 Unlike humans, mice with deletions spanning both the mouse orthologues of the human Wilms tumor and the PAX6 genes, do not have nephroblastoma.208
Most deletions resulting in WAGR are large, encompassing 11p13 that can be seen using routine banding methods. For those patients showing partial phenotypes, FISH using probes containing WT1 or PAX6 may help identify submicroscopic deletions.241
Beckwith-Wiedemann Syndrome [Dup(11)(P15.5p15.5)]
Beckwith-Wiedemann syndrome (BWS; MIM 130650) consists of multiple growth abnormalities, including macroglossia, omphalocele, visceromegaly, and gigantism. Importantly, there is an increased predisposition (5 to 20 percent) to several childhood malignancies including Wilms tumor, adrenocortical carcinoma, hepatoblastoma, and rhabdomyosarcoma.4 Additional discussions of clinical features and the genetics of BWS are found in Chaps. 15, 18, and 38.
Eighty-five percent of BWS cases are sporadic, but a number of familial cases have been reported with or without associated chromosomal aberrations. It is now generally accepted that BWS can be transmitted as a Mendelian trait, influenced by imprinting and that the molecular bases of the disorder is heterogeneous with variable expression (see Chap. 18).
Although most BWS patients are cytogenetically normal,205 a number of cases have been observed with abnormalities involving the most distal band on 11p. At least 15 cases have been reported with unbalanced translocations or other abnormalities that produce partial trisomy for 11p15.5.53 In all cases in which it has been determined, the origin of the duplicated segment is paternal. In contrast, six cases have been reported with apparently balanced translocations or inversions involving 11p15, and in all cases the abnormality was inherited from the mother.53
In familial cases, linkage analysis has confirmed the localization of familial BWS to 11p15.5.242,243 The most significant finding in sporadic BWS has been the identification of partial paternal uniparental disomy for the distal short arm of chromosome 11 in 20 percent of patients.244–246 Different from the holochromosomic UPD for chromosome 15 observed in PWS and AS, the UPD in BWS is usually confined to the 11p15 region. All cases analyzed showed isodisomy (homozygosity) for markers in 11p15, but normal biparental inheritance elsewhere on chromosome 11. This indicates that the UPD is a somatic event rather than a meiotic nondisjunction event, resulting in the segmental isodisomy. Evidence for somatic mosaicism for UPD has been observed at the single-cell level,247 confirming that the UPD arises after fertilization. The molecular genetics of BWS are quite complex, involving genomic imprinting and perhaps several genes. A detailed discussion is presented in Chap. 18.
Currently the diagnosis of BWS is based on clinical findings. About 20 percent of cases have partial, paternal isodisomy of distal 11p, but the molecular analysis is complicated by the presence of mosaicism. A small percentage of patients demonstrate biallelic expression of IGF2, which is normally expressed from only the paternally inherited chromosome 11,248 and a small number of patients have mutations in the p57KIP2 gene.249 For the majority of the remaining patients, loss of imprinting (LOI) for the gene LIT1 (long QT intronic transcript 1) appears to be the most common molecular basis causing BWS250 .
Prader-Willi Syndrome [Del(15)(Q11.2q13)]
Prader-Willi syndrome (PWS; MIM 176270) was first described in 1956251 and is a relatively common cause of genetic obesity and mental retardation, with a frequency of approximately 1 in 10,000 to 20,000 births. Severe hypotonia and poor suck during the neonatal period usually requiring gavage feeding characterize this syndrome. Males usually have undescended testes and females may have hypoplastic labia. By 2 to 3 years of age, patients develop hyperphagia leading to obesity unless caloric intake is strictly regulated. A characteristic facial appearance is present including a narrow bitemporal diameter, almond-shaped eyes, upslanting palpebral fissures, and strabismus. Other physical features include short stature, small hands and feet, and fair hair and skin color (hypopigmentation) relative to other family members. The hypopigmentation is a clinical finding suggesting a deletion instead of UPD as the underlying mechanism, because this likely reflects haploinsufficiency of the P locus. All individuals with PWS have some degree of cognitive dysfunction, with a wide range of formal IQ scores. Significant behavioral abnormalities are present, including stubbornness, temper tantrums, and poor peer interactions. Excellent reviews of the clinical features and diagnostic criteria for PWS are available.252–254
Autosomal recessive and autosomal dominant modes of inheritance had been ascribed to PWS before the finding of a chromosome deletion in most patients.255 In the great majority of cases, the condition is sporadic. Familial cases of PWS are infrequent, but at least seven cases have been identified with small deletions causing imprinting mutations.256,257 At least one familial case due to a balanced chromosomal rearrangement is known.258 Based on the very rare occurrence of familial PWS with normal karyotype, Cassidy259 estimated the recurrence risk for PWS as less than 1 in 1000 and may reflect gonadal mosaicism for trisomy 15.
Hawkey and Smithies12 first suggested a relationship between chromosome 15 and PWS in 1976. This was based on their patient with a 15;15 Robertsonian translocation and a review of the literature showing several similar abnormalities. Subsequent high-resolution cytogenetic investigation revealed a small, interstitial deletion of proximal 15q in more than half of all PWS patients.260,261 Using cytogenetic polymorphisms, Butler and coworkers made the interesting observation that the de novo interstitial deletions in PWS were paternal in origin.262,263 It is now recognized that about 70 percent of PWS patients have interstitial deletions in 15q12. The deletion size is about 4 Mb.264 Given the proximity of this band to the centromere and the variable block of long arm heterochromatin, cytogenetic analysis by G-banding is not always reliable.265 FISH analysis using a probe contained within the deletion region provides the most reliable means for detecting deletions in the cytogenetics laboratory. Patients with balanced Robertsonian translocations are now recognized to likely have UPD,156,266 rather than submicroscopic deletions and should be further investigated by DNA polymorphisms or methylation analysis (see “Diagnostics” below).
Several molecular mechanisms can result in PWS: microdeletions of paternal origin in 70 percent of cases; maternal, holochromosomic UPD in about 28 percent of cases; and imprinting mutations in a few (≈2 percent) cases. Most patients have consistent-sized deletions of ≈4 Mb. The deletion is flanked by ∼ 400 kb genomic regions with high sequence identity, which may promote homologous recombination events leading to deletion.266a, 419 Study of PWS patients with atypical deletions or unusual translocations have allowed refinement of the critical region. The PWS region is probably less than 1 Mb and is bounded by D15S13 on the centromeric side,267 and D15S10 on the telomeric side.268 This region has been separated from the AS critical region, also mapping to 15q12, by a familial case of AS in which a submicroscopic deletion involving the region from D15S10 to GABRB3 (β3-subunit of the GABA receptor).269 When the deletion was inherited from a female, children were affected with AS. When the deletion was inherited from a male, there was no phenotypic abnormality. This important case provided strong evidence that PWS and AS were caused by separate loci with AS distal to PWS. The gene causing AS is now known (see “Angelman Syndrome” below).
Patients are considered to have imprinting mutations if they have biparental inheritance of the PWS region, but a maternal methylation pattern on both chromosomes. Methylation is most reliable if analyzed at the CpG island of the 5′ end of SNRPN .270 About half of the patients with imprinting mutations have small deletions very near the major promoter for SNRPN.256,271 These small deletions are consistently of familial origin while patients with imprinting mutations without identifiable deletions are consistently sporadic.272
Within the PWS critical region, a number of paternally expressed genes have been identified: SNRPN (large and small open reading frame), IPW (no open reading frame), ZNF127, NDN (necdin), and one or two necdin-like genes referred to as NDNL1 or MAGEL2.256,273,274 In addition, there is evidence for at least 13 paternally expressed transcripts in the PWS region.275 Any of these transcripts may be causally involved in producing the PWS phenotype. The gene for small nuclear ribonucleoprotein-associated peptide N (SNRPN) has been debated to be the PWS gene. As reviewed in Mann and Bartolomei,273 mice with UPD have been generated that show early postnatal lethality that may be analogous to the hypotonia and poor feeding seen in PWS. Mouse models have also been generated that deleted Snrpn and the region containing the imprinting center and these also result in postnatal lethality. Mice that have just an intragenic deletion of Snrpn have no lethality or obvious phenotypic features indicating that, at least in mice, mutation involving more than just Snrpn is necessary to produce the PWS phenotype. There are conflicting reports as to whether inactivating mutations in mouse necdin cause lethality.276,277
Deletion of the P gene is likely responsible for the hypopigmentation present in the majority of PWS deletion patients.278,279 Because paternal and maternal deletions of the P gene produce similar phenotypic consequences, this represents a gene dosage effect without imprinting. The P locus has also been implicated in type II oculocutaneous albinism (OCA2; MIM 203200) by linkage to this region of chromosome 15,280 and by identification of recessive mutations.279 One case of PWS who also had OCA2 was found to have a paternal deletion on one chromosome 15 and a mutation in the P locus on the maternal homologue.47
Although deletions of chromosome 15 account for the majority of PWS cases, about 30 percent of PWS do not show deletions by cytogenetic or molecular methods. Uniparental disomy (UPD) is now known to account for the majority of nondeletion PWS. UPD, the inheritance of both chromosome homologues from a single parent, was first proposed by Engel in 1980,281 and first observed in humans by Spence and coworkers in 1988,155 in a patient with cystic fibrosis and growth retardation who inherited both chromosome 7 homologues from her mother. Since then, UPD has been found for a number of human chromosomes and may be a significant cause of mental retardation and other developmental abnormalities.282
Several groups have shown that maternal UPD for chromosome 15 is a common cause of PWS, accounting for approximately 28 percent of cases.267,283 Maternal UPD leads to paternal deficiency of chromosome 15 and absence of expression of any genes expressed exclusively from the paternal homologue. Interestingly, maternal UPD cases are associated with advanced maternal age, whereas deletion cases of PWS are not.267,283 Most UPD cases demonstrate maternal heterodisomy (the inheritance of two different maternal homologues) (Fig. 65-8), suggesting a meiosis I nondisjunction event.267,283,284 Cases of trisomy 15 mosaicism have been observed in chorionic villus cells during prenatal diagnosis for advanced maternal age, but only normal karyotypes were observed at follow-up amniocentesis studies.285–287 At birth, clinical features of PWS led to molecular investigations and the discovery of maternal UPD. These data suggest a model for the origin of UPD in PWS as shown in Fig. 65-8. Advanced maternal age produces an increased risk for maternal nondisjunction and trisomy 15 conception. Because trisomy 15 is lethal, loss of one chromosome 15 postzygotically would “rescue” this lethal condition. However, if the paternal chromosome 15 is lost (presumed to occur in one-third of cases), maternal UPD 15 results in PWS. Additional discussion on PWS can be found in Chap. 15.
Model for the mechanism of uniparental disomy (UPD) in Prader-Willi and Angelman syndromes. Maternal chromosome 15 homologues are shown in white, with 2 different centromeres white or black, and paternal 15 homologues black. A maternal meiosis I nondisjunction event produces disomic and nullisomic eggs. Fertilization of the disomic egg by a nullisomic sperm (gamete complementation) produces maternal heterodisomy, as does fertilization by a normal sperm with subsequent loss of the paternal homologue. Fertilization of the nullisomic egg by a normal sperm produces monosomy 15, a lethal condition. Subsequent duplication of the single paternal homologue would “rescue” this lethal event and produce AS with complete isodisomy (homozygosity for all markers). (From Mutirangura et al.284 Used by permission of Human Molecular Genetics.)
A joint American Society of Human Genetics/American College of Medical Genetics committee has made diagnostic testing recommendations.288 The recommendations for patients suspected to have PWS are to perform chromosome analysis to exclude gross structural rearrangements of chromosome 15 or other chromosomal rearrangements and FISH to detect deletions of 15q12 (see Fig. 65-3A). Those patients who have had a prior normal cytogenetic study should be studied by an assay that detects methylation differences within loci of the PWS region. This analysis can detect abnormalities within the region, but cannot distinguish between deletions, UPD, or imprinting mutations. Those patients who show an abnormal methylation pattern should have FISH to confirm a deletion or DNA analysis with polymorphic markers to identify UPD. Abnormal methylation with two methylated copies of the CpG island at SNRPN with evidence for biparental inheritance of chromosome 15 defines an imprinting mutation. Analysis can be performed by Southern blotting270 or with PCR after bisulfite treatment of the DNA.289,290 Neither the FISH nor the methylation analysis requires parental blood samples to be analyzed. However, analysis of polymorphisms within the PWS region requires a comparison of parental alleles to the child's alleles. The clinical observation of trisomy rescue suggests that it would be prudent to test for UPD in all instances of normal karyotype at amniocentesis following the identification of trisomy 15 in CVS.
Angelman Syndrome [Del(15)(Q11.2q13)]
Angelman syndrome (AS; MIM 105830) is associated with severe mental retardation, seizures, and absence of speech development. These patients also have an ataxic gait, inappropriate bouts of laughter, and characteristic facies including a wide mouth, protruding tongue, prominent jaw, and thin upper lip.291–294 AS patients may also show hypopigmentation, apparently due to deletion and haploinsufficiency of the P locus. This feature is the only apparent clinical overlap with PWS other than mental retardation.
AS is sporadic in the majority of cases; however, a number of familial cases have been reported. Because familial cases usually involve affected sibs with normal parents, AS was sometimes erroneously thought to be an autosomal recessive disease.295 Subsequent to the finding of chromosome 15 abnormalities in the majority of AS cases, linkage analysis in several familial AS cases demonstrated linkage to chromosome 15, consistent with autosomal dominant transmission of an imprinted locus.296–298 AS occurs in about 1 in 15,000 births.
Deletions of chromosome 15 were first described in AS patients in 1987299 and have subsequently been shown to be present in the majority of patients.300 Although it was proposed initially that the PWS and AS cytogenetic deletions were of somewhat different size and location in proximal 15q,301 it has been demonstrated by molecular analysis that the breakpoints of the deletions are the same in the two disorders,302,303 and the mechanism of deletion involving flanking repeats appears the same.266a, 419 The difference in phenotype is due solely to the differential parental origin of the deletion—exclusively paternal origin of deletions in PWS262,263,267,304,305 and exclusively maternal origin of deletion in AS.269,300,306,307 As in PWS, the deletion of 15q12 in AS can be difficult to visualize in some patients due to its proximity to the centromere and pericentromeric heterochromatin.265 FISH, therefore, provides a more reliable means of detecting these deletions.
Considerable progress has been made in delineating the molecular mechanisms that can result in AS. In AS, paternal disomy has been observed in 3 to 5 percent of cases, but not as frequently as maternal disomy in PWS.284,308–310 This lack of a maternal homologue is consistent with absence of expression of a gene or genes expressed exclusively from the maternal homologue and has the same effect as maternal deletion of this region. Also different from PWS, paternal disomy in AS more often displays isodisomy (homozygosity) for the entire chromosome (Fig. 65-8).284,308,309 This is perhaps best explained by a monosomy 15 conception, followed by a postzygotic event that duplicates the single paternal homologue (Fig. 65-8). Because in this case monosomy 15 arises as a result of a maternal nondisjunction event, this is the reciprocal product of the same event producing maternal UPD for chromosome 15. Paternal UPD arising by maternal nondisjunction would be predicted to be associated with advanced maternal age. Too few cases are available for statistical analysis, but in the small number of reported cases, the maternal age is elevated.284
Among the remaining patients who do not demonstrate deletions or UPD, 4 to 6 percent have mutations in the UBE3A gene, 3 percent have imprinting mutations by methylation analysis, and 10 to 14 percent have no identifiable molecular defect.311 The UBE3A gene encodes the E6-AP ubiquitin-protein ligase. Point mutations, mostly resulting in truncated proteins,77,78 when inherited on the maternal chromosome, result in functional nullisomy (the paternal copy is silenced in parts of the brain in a tissue-specific manner312) and AS.313,314 More extensive mutation studies of patients without the ≈4 Mb deletion, UPD, or imprinting mutation identified loss of function mutations in 75 to 80 percent of families with more than one affected case and in 14 to 23 percent of isolated cases.315,316 Very large pedigrees with multiple affected individuals can occur with point mutations in UBE3A,297,315 and with imprinting mutations caused by small deletions.271 As for PWS, imprinting mutations occur usually on an inherited basis involving small deletions in the imprinting center or sporadically with no deletion identified.272,317 The smallest region of overlap for imprinting center deletions causing AS is found ≈5 kb centromeric to the deletions causing PWS and is narrowed to 880 bp.318,319 The variability of the AS phenotype may be due to genes that modify the basic phenotype caused by maternal deficiency of UBE3A. Certainly, this is the case for the light pigmentation seen in AS deletion patients, due to haploinsufficiency of the P protein, but epilepsy may be more severe in deletion patients.311 Thus, although maternal deficiency for UBE3A causes a relatively complete AS phenotype, deletion patients may still be considered as having a CGS.
Diagnostic testing recommendations were made by a joint American Society of Human Genetics/American College of Medical Genetics committee prior to the identification of the AS gene.288 The recommendations for patients suspected to have AS are similar to that for PWS. The first step is to perform chromosome analysis to exclude gross structural rearrangements of chromosome 15 or other chromosomes and FISH to detect deletions of 15q12. Those patients who have had a prior normal cytogenetic study should be studied by methylation analysis. Because this assay cannot distinguish between deletions, UPD, or imprinting mutations, those patients who show an abnormal methylation pattern should have FISH to confirm a deletion or DNA analysis with polymorphic markers to identify UPD. Those patients who do not carry a deletion or show an abnormal methylation pattern should be investigated for mutations in the UBE3A gene. Unfortunately, a variety of mutations have been identified such that individual mutations must be identified by sequencing UBE3A.315,316
Rubinstein-Taybi Syndrome [Del(16)(P13.3)]
Rubinstein-Taybi syndrome (RTS; MIM 180849) is a well-defined, complex disorder comprised of facial abnormalities (microcephaly, broad nasal bridge, prominent or beaked nose, downward-slanting palpebral fissures, micrognathia, and ear abnormalities), broad thumbs and toes, and mental retardation. The prevalence at birth has been estimated to be 1 in 125,000.320 Although autosomal dominant transmission has been occasionally observed, most cases are sporadic. Although Rubinstein-Taybi syndrome was postulated to be a chromosomal syndrome321 and was listed by Schmickel1 as an excellent candidate for a CGS, the identification of mutations in a single gene (see below) has redefined the etiology of this disorder.
Three unrelated cases of RTS have been identified involving balanced translocations between chromosome 16 and chromosomes 2, 7, or 20.322 In each case, the breakpoint in chromosome 16 occurred in 16p13.3, suggesting that this was the site of the Rubinstein-Taybi syndrome gene(s). Following this clue, Breuning et al.322 studied 24 patients with Rubinstein-Taybi syndrome by FISH analysis using cosmid probes in this region of chromosome 16. They identified submicroscopic deletions in 6 of 24 (25 percent) of patients, confirming that 16p13.3 is the location of Rubinstein-Taybi gene(s) and that microdeletions account for a significant percentage of cases. There were no apparent clinical differences between patients with deletions and those without.323 Subsequent FISH studies have shown that a smaller proportion of patients (≈10 percent) have deletions and the remaining patients are likely to have mutations in the CBP gene.324,325
Using RTS translocation breakpoints and microdeletion patients, the RTS critical region was narrowed to 15 kb.326 The microdeletions ranged in size from 130 kb to greater than 650 kb. The CREB binding protein (CREBBP or CBP; MIM 600140), a nuclear protein that is a coactivator in cyclic-AMP-regulated gene expression, was shown to be disrupted in RTS patients with translocations.326 Sequence analysis of CBP in nondeletion, nontranslocation patients showed a variety of mutations.326 Because the clinical findings among deletion RTS patients and patients with CBP mutations did not differ, RTS is now considered a condition caused by a single gene with pleiotropic effects. The mutations in CBP are all dominant loss-of-function mutations resulting in functional monosomy.
A minority of RTS patients show gross structural cytogenetic anomalies. Approximately 10 percent of patients will have submicroscopic deletions by FISH.324,325 Unfortunately, there is not one FISH probe that detects most deletions. Using a panel of 5 cosmid probes spanning the CBP gene, Blough et al.324 found a variety of deletions in RTS patients including deletion of the 5′ end, but not the 3′ end of the gene; deletion extending through both the 5′ and 3′ ends of the gene; deletion of the 3′ end, but not the 5′ end; and an interstitial deletion in the 3′ end of the gene. The majority of patients are presumed to have mutations in the CBP gene that are best identified through DNA sequencing or a protein truncation assay.326
Smith-Magenis Syndrome [Del(17)(P11.2p11.2)]
In 1986 two groups described a total of 15 patients with an interstitial deletion of 17p11.2 who had very similar phenotypes, including brachycephaly with a broad face and nasal bridge, flat midface, brachydactyly, and mental retardation associated with hyperactivity, and often self-destructive behavior.327,328 This deletion syndrome has been termed the Smith-Magenis syndrome (SMS; MIM 182290) and is a relatively common recurring cytogenetic deletion. A series of 32 cases confirmed the clinical features described above, and added the common features of speech delay and hoarse, deep voice.67 In addition, a high percentage of patients have ophthalmologic findings, otolaryngologic abnormalities (including hearing loss), visceral anomalies involving the heart and kidneys, and a significant sleep disturbance.329 The frequency of Smith-Magenis syndrome has been estimated to be 1 in 25,000,67 which would make this deletion syndrome more common than cri du chat syndrome (with a frequency of 1 in 50,000),27 and about as common as Prader-Willi syndrome (at 1 in 10,000 to 20,000).
To date, all patients with Smith-Magenis syndrome have visible interstitial deletions of band 17p11.2 detectable by good quality routine cytogenetic analysis.67 As seen in the Potocki-Shaffer syndrome, the deletion of this very proximal G-negative band on 17p may be overlooked in some cases. A FISH probe was developed containing the human homologue of the Drosophila melanogaster Flightless-1 gene (FLI1), which was found to map within the SMS critical region.330 This probe has been deleted in 100 percent of patients tested (L.G. Shaffer and J.R. Lupski, unpublished data). FISH was helpful in reevaluation of two cases suspected to have mosaic deletion who were found to be nonmosaic by FISH, but were shown to have deletions smaller than the common deletion.331,332
The deletions in SMS are of consistent size of about 5 Mb.67–70 The deletion is flanked by low-copy repeat-gene clusters that are likely involved in unequal crossing over events that result in the deletion58 (see “Molecular Mechanisms Leading to Contiguous Gene Syndromes” below). The reciprocal duplication product has been seen in patients with a milder phenotype that is unrelated to SMS.43 SMS has been hypothesized to be a CGS 67 and although a number of genes have been directly identified in the deletion region, none have been implicated in the syndrome phenotype.333
The interstitial deletion in 17p11.2 can be visualized using routine banding methods. Currently, FISH can be used to confirm a deletion or exclude a submicroscopic deletion (see Fig. 65-3J). A multiprobe cocktail containing a probe for the PWS/AS region on 15q12, the WBS region on 7q11.23, the DGS/VCFS region on 22q11.2, and the SMS region, identified a 17p11.2 deletion in a patient with developmental delay, referred for cytogenetic analysis.334 Additionally, interphase FISH has been used to identify duplications of this same genomic region [dup(17)(p11.2p11.2)].43
Duplication 17p11.2 [Dup(17)(P11.2p11.2)]
The clinical findings in seven patients with dup(17)(p11.2p11.2) are relatively mild.43 The patients were ascertained for developmental delay and chromosome analysis revealed an apparently increased band size for the 17p11.2 region. Interphase FISH using the FLT1 probe from the SMS critical region demonstrated duplications.43 The phenotype of dup(17)(p11.2p11.2) consists of generally normal-appearing facies for most patients, short stature, mild to borderline mental retardation and behavioral difficulties. Dental abnormalities such as malocclusion and crowded teeth were found in six of seven patients. Notably, no major organ developmental abnormalities were observed, which is in sharp contrast to what is found in patients with SMS who are deleted for the same genomic region.
The duplication is difficult to see by routine G-banding and is likely underascertained. By G-banding, the duplication appears as a broader 17p11.2 band. Those patients that have been identified, have been confirmed using FISH on interphase nuclei.43
Duplication of 17p11.2 represents the apparent reciprocal recombination of the common deletion found in del(17)(p11.2p11.2) patients with Smith-Magenis syndrome (see “Molecular Mechanisms Leading to Contiguous Gene Syndromes” below). By interphase, multicolor FISH analysis, all duplications were determined to be direct (tandem) in nature.43
The diagnosis of dup(17)(p11.2p11.2) is made by the astute observation of a broader region of the light-staining 17p11.2 band. Confirmation should be made by interphase FISH diagnosis using probes that have been shown to be deleted in the Smith-Magenis syndrome deletion (see Fig. 65-3I and 65-3K).
Miller-Dieker Syndrome [Del(17)(P13.3)]
Miller-Dieker syndrome (MDS) (MIM 247200) is a multiple malformation syndrome characterized by type I lissencephaly and a characteristic facial appearance.335–337 Lissencephaly is a severe brain malformation whereby the typical convolutions (gyri and sulci) are absent and the brain surface is smooth.337–339 The specific sequelae of lissencephaly include bitemporal hollowing, a small jaw, and neurologic abnormalities such as severe to profound mental retardation. In addition to the brain abnormalities, a characteristic facial appearance is present in MDS, which includes prominent forehead, bitemporal hollowing, a short nose with upturned nares, protuberant upper lip, thin vermilion border of the upper lip, and small jaw. Lissencephaly can also be observed as an isolated finding with relatively normal facial appearance, which is referred to as isolated lissencephaly sequence (ILS).337,339,340 The brain malformation may be as severe as in MDS or less severe, with mixed agyria and pachygyria or pachygyria alone. Patients may have subtle facial changes such as bitemporal hollowing and small jaw, but other birth defects are rare. All have profound mental retardation; about half achieve no developmental skills, while the remainder are able to show some social responses, roll over, and make some semipurposeful movements of their hands.
MDS was initially considered an autosomal recessive disorder based on the occurrence of several families with multiple affected sibs.335 Subsequently, it was shown that most of the known familial cases were due to a parental chromosomal rearrangement.341 Although most cases of MDS and ILS are now known to be de novo deletions or mutations of a chromosome 17 gene, an X-linked form of lissencephaly is well documented and the gene has been identified.342,343
A chromosomal etiology for MDS was first recognized by the identification of a ring chromosome 17 in a patient with deletion of a small region at distal 17p and distal 17q.14 Subsequent to this initial report, high-resolution chromosome analysis was performed on a series of MDS patients and identified visible deletions in approximately half.45 Most abnormalities are de novo deletions, but several familial cases have been due to parental balanced translocations and inversions. High-resolution chromosome analysis in 58 ILS patients has been normal with the exception of one de novo balanced translocation between chromosome 2 and the X chromosome [46,XX,t(X;2)(q22;p25)] in a female with classic lissencephaly.337,340,344 This translocation has subsequently been shown to disrupt the doublecortin (DCX; MIM 300121) gene on the X chromosome.342,343
A variety of molecular techniques have been used to detect submicroscopic deletions in MDS and ILS patients.345,346 FISH analysis for detection of submicroscopic deletions in this region has proved to be the most sensitive and efficient method for diagnosis and for mapping the boundaries of patient deletions.22 With this method, visible or submicroscopic deletions are detected in over 90 percent of MDS patients and in 40 percent of patients with ILS.22,337,344,347 Importantly, analysis of parents of children in whom a deletion is observed by FISH has revealed the presence of “cryptic” translocations or inversions that were not detectable by high-resolution chromosome analysis.22 Because a parental rearrangement produces a substantial recurrence risk for unbalanced offspring, it is imperative that parental studies be performed by FISH following the identification of a deletion in a child.
Definition of the smallest region of overlap among a collection of patient deletions revealed a 350-kb “critical region” in MDS and ILS patients,344 and a gene has been identified in the critical region.348 Subsequently, it was determined that the 5′ end was chimeric and derived from more distal 17p. The correct 5′ end of the gene was then determined.349 The gene symbol for lissencephaly 1 gene (LIS1) gene encodes a subunit of the brain platelet-activating factor acetylhydrolase (PAFAH). All truncation mutations and lissencephaly causing point mutations or intragenic deletions result in a reduction in the amount of correctly folded LISI protein.350 LIS1 is a microtubule-associated phosphoprotein.351 Mutations found in ILS patients supports that LIS1 is responsible for the brain malformation found in MDS and ILS, but that the additional clinical features found in MDS are likely caused by an additional gene(s).347,349,352 These findings support that MDS is still likely to be a CGS. A knockout for the LIS1 gene in the mouse has confirmed haploinsufficiency as the underlying molecular mechanism for lissencephaly because heterozygotes have the neuronal migration defects similar to the human brain anomaly.353
Although some patients have visible deletions detectable by routine cytogenetic methods, FISH is the most reliable method for detecting the deletions in MDS and ILS. Probes containing the LIS1 gene detect deletions in over 90 percent of MDS patients and 40 percent of ILS patients.347 Parental FISH analyses should be conducted to detect parental translocations. Parents who carry translocations involving 17p13.3 are at substantial risk for having unbalanced offspring.354 ILS patients not found to have deletions are likely to have point mutations in LIS1 or DCX.352,355
Alagille Syndrome [Del(20)(P11.23p11.23)]
Alagille syndrome (AGS; MIM 118450) was first described in 1969,356 and is characterized by chronic cholestasis due to paucity of intrahepatic bile ducts, typical facial appearance (including broad forehead, deep-set eyes, mild hypertelorism, bulbous tip of the nose, and small pointed chin), peripheral pulmonic stenosis, vertebral anomalies (“butterfly” vertebrae), and posterior embryotoxon of the eye. Many cases are sporadic, but autosomal dominant inheritance with reduced penetrance and variable expressivity is suggested by family studies.357,358 AGS occurs in about 1 in 70,000 births and is the second most common cause of infantile intrahepatic cholestasis.359
An interstitial deletion in proximal 20p was first reported in 1986 by Byrne et al.360 in a patient who was small for gestational age and had features of AGS. At least 15 cases have subsequently been reported with deletions in chromosome 20 involving bands p11.23-p12.2 in association with AGS, confirming this localization and causing its inclusion in the list of CGS disorders.361,362 However, a prospective study of an additional 14 patients with Alagille syndrome failed to identify any deletions in 20p by high-resolution cytogenetic analysis or limited FISH analysis with a single cosmid probe from this region of chromosome 20.363 Molecular identification of mutations in the Jagged 1 (JAG1) gene in patients with Alagille syndrome led to the investigation of deletions in patients with AGS. Using a FISH probe containing JAG1, 6 percent of AGS patients demonstrated a deletion.357
The use of linkage analysis and two AGS families with balanced translocations involving 20p helped narrow the AGS critical region.364 The region was further narrowed and the candidate gene, Jagged 1, was identified.364,365 JAG1 encodes a ligand for the transmembrane Notch 1 receptor. Mutations have been identified in 75 percent of patients.357 A small percentage of patients (6 percent) have microdeletions that include the JAG1 locus, while the vast majority (69 percent) have a variety of mutations in JAG1.357 The majority of mutations lead to a nonfunctional protein, supporting a gene dosage, haploinsufficiency model in this disease. The finding of JAG1 mutations in patients with the entire AGS clinical spectrum and in patients with isolated heart defects,366 is consistent with a single gene defect with variable expressivity and pleiotropic effects.
Very few AGS patients have visible cytogenetic abnormalities. Even though only 6 percent of patients are deleted by FISH, cytogenetic and FISH analyses rapidly identify abnormalities of 20p. Unfortunately, for the majority of patients not demonstrating a cytogenetic abnormality, more laborious mutation analysis of JAG1 is needed. To date, only 2 of the 35 mutations identified occurred at the same location in the gene.357 Therefore, direct sequencing or other methods to detect coding region alterations are the only available options to identify mutations in the majority of AGS patients.
Digeorge Syndrome/Velocardiofacial Syndrome [Del(22)(Q11.2q11.2)]
In 1965, DiGeorge367 described a patient with hypoparathyroidism and cellular immune deficiency similar to other patients he had seen with absent parathyroids and thymus at autopsy; the condition has become known as DiGeorge syndrome (DGS; MIM 188400). Since then a number of patients have been reported with absent or hypoplastic thymus and absent or hypoplastic parathyroid, frequently in combination with conotruncal-type cardiac malformations (interrupted aortic arch or truncus arteriosus).368 In fact, congenital heart disease is the most frequent presenting feature in these patients and has been the major contributor to the high morbidity in the first weeks of life. Dysmorphic facial features, including hypertelorism, cleft palate, bifid uvula, and low-set ears, are frequently observed.368 Most of the patients who survive infancy are mildly to moderately retarded. The incidence of DGS/VCFS has been estimated at 1 in 4000,369 making it the most common, recognized, microdeletion syndrome.
The velocardiofacial syndrome (VCFS), or Shprintzen syndrome (MIM 192430), was initially described as a separate disorder, unrelated to DiGeorge syndrome. It is characterized by cleft palate, cardiac anomalies, and typical facies, including prominent nose, broad nasal root, narrow palpebral fissures, and retrognathia.370 It is probably the most common syndrome associated with clefting, with approximately 8 percent of all patients presenting with cleft palate but without cleft lip (see review370). Cardiac abnormalities are found in 85 percent of patients, the most common being ventricular septal defect with or without a right aortic arch. Virtually all patients have some degree of learning disability, with microcephaly and mental retardation present in about 40 percent of cases. Other behavioral and psychiatric problems have also been observed. Significant overlap in phenotypic features exists between DGS and VCFS. Stevens et al.371 first proposed that VCFS represents the inherited form of DGS. Retrospective review of the literature has led several authors to conclude that many published cases of DGS actually represent VCFS.370
The etiology of DGS is heterogeneous, having been associated with several different chromosome abnormalities as well as teratogenic causes including retinoic acid embryopathy and the fetal alcohol syndrome.372,373 Most cases of DGS are sporadic, but a number of familial cases have been described with apparent autosomal dominant transmission. As discussed above, these familial cases of DiGeorge probably represent the whole clinical spectrum associated with VCFS and deletions of proximal 22q. Additionally, in some families with DGS or VCFS, there are individuals with congenital heart defects but minimal dysmorphism. Because of this, Wilson et al.24 performed molecular studies on a series of nine cases of familial congenital heart disease. Submicroscopic deletions in 22q11 were identified in five of the nine families. In four cases, the parent who had the deletion had a relatively mild heart anomaly, while in the fifth case, the father had a deletion and was unaffected. Interestingly, the five families with deletions exhibited heterogeneity in the nature of the cardiac anomaly between individuals in the family, while the four families without deletion all had unaffected parents and identical defects in the affected children. These observations provide additional evidence that deletions of 22q11 can have a wide spectrum of clinical expression, even within families.
DGS has been reported in association with a number of different chromosome abnormalities, most frequently deletions of proximal 22q and less frequently deletions of 10p13.374 The specific association with deletion of the proximal portion of the long arm of chromosome 22 was first proposed by de la Chapelle et al. in 1981,13 based on the observation of an unbalanced translocation producing partial monosomy 22 and a review of previously published monosomy 22 cases. High-resolution cytogenetic studies have identified subtle visible deletions of band 22q11.2 in approximately one-third of cases.375–377 In VCFS, visible deletions of 22q11.2 have been detected by high-resolution cytogenetic studies in only a small number of cases.378 Submicroscopic deletions have been identified in the majority of DGS and VCFS patients using Southern blot or FISH analysis.378–380
Extensive progress has been made in the molecular characterization of the 22q11.2 CGS. Initial studies used RFLP and dosage analysis of Southern blots to demonstrate the presence of submicroscopic deletions in 22q11 in DGS patients. Combined data from two groups showed that although only about one-third of DGS patients showed cytogenetically detectable deletions, almost 100 percent of patients could be shown to have deletions by molecular methods.375,376 The smallest region of overlap among these deletions included three common loci that represent a distance of at least 750 kb.376 There was no apparent correlation between the size of deletion and severity of phenotype, even between patients with visible cytogenetic deletions as compared with those with submicroscopic deletions. FISH analysis with a cosmid probe from the DGS critical region has been demonstrated as an efficient method to detect submicroscopic deletions.379,380 As described previously for Miller-Dieker syndrome, FISH has become the method of choice for diagnostic studies in DGS.
Molecular studies in VCFS have produced results essentially identical to those in DGS. From two studies, 26 of 27 VCFS patients were demonstrated to have submicroscopic deletions of probes in 22q11 by RFLP or quantitative Southern blot analysis.378,381,382 With the currently available DNA probes, the deleted region for DGS and VCFS cannot be distinguished.
The deletion in 22q11.2 has been delineated. The common deletion region is 3 Mb in size and is flanked by low-copy repeats.383 The breakpoints in 22q11.2 deletions associated with the majority of DGS and VCFS patients are identical.384 Some patients show an alternate breakpoint resulting in smaller (1.5 Mb) deletions.383,384
A mouse model has been developed through an engineered heterozygous deletion spanning the region orthologous to human 22q11.2. The mice show defects consistent with DiGeorge syndrome including cardiovascular anomalies.385 These mutants are important in understanding of the cellular mechanisms leading to developmental abnormalities of the fourth pharyngeal arch arteries.
The interstitial deletion in proximal 22q cannot be reliably detected by routine G-banding. FISH using probes within the common deletion region have shown that greater than 90 percent of patients are deleted.378–380 Patients not demonstrating a deletion by FISH for probes in the critical region should have a chromosome analysis to exclude gross structural rearrangements of chromosome 22 or other chromosomes. Some patients with deletions of 10p13-p14 show features of DGS, and FISH with probes from that region has been helpful in identifying these patients.374 In cases of deletion and given the wide clinical expression, parental chromosomes should be examined using FISH to exclude a familial deletion.