Molecular Cytogenetics II: PCR-based diagnosis of chromosomal deletions and microdeletion syndromes

MINI-REVIEW

Molecular Cytogenetics II: PCR-based diagnosis of chromosomal deletions and microdeletion syndromes

Sérgio D.J. Pena

Núcleo de Genética Médica de Minas Gerais (GENE) and Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Av. Afonso Pena, 3111/9, 30130-909 Belo Horizonte, MG. Fax: [phone]. E-mail: [e-mail]

INTRODUCTION

The development of conventional cytogenetic techniques in the 50's resulted in a rapid growth of knowledge of chromosomal syndromes, the most common genetic diseases of man. Following the development of chromosomal banding techniques in the early 70's and after high resolution prometaphase analysis was developed later in that decade, our ability to diagnose small deletions increased and a series of disorders were characterized as microcytogenetic syndromes (de Grouchy, 1982). With the explosion of human molecular genetics in the 80's and 90's we were able to recognize deletions at the submicroscopic level, the so-called microdeletion syndromes. Moreover, it became clear that there is no discontinuity between the genic and the chromosomal level. The same disease, for instance Angelman syndrome, can arise from mutations in the UBE3A gene or be caused by submicroscopic deletions or larger deletions visible at high-resolution cytogenetics.

In the first article in this series (Pena, 1998) we discussed the diagnosis of human trisomies by the use of PCR-based study of hypervariable microsatellites using computer-assisted laser densitometry. We showed that with this approach we could decrease considerably the time necessary to obtain results in amniocentesis and that we could increase significantly our efficiency in diagnosing chromosomal defects in cases of fetal loss. In this second article we wish to describe how PCR-based molecular cytogenetic techniques can be used for the diagnosis of chromosomal deletions and microdeletion syndromes.

PCR-BASED DIAGNOSIS OF TURNER SYNDROME IN FETAL LOSSES

Spontaneous abortions are common events, occurring in approximately 15% of all human pregnancies. Whenever such fetal losses occur, they are associated with psychological trauma that may involve maternal fantasies and feelings of guilt and inadequacy (see, for instance, Neugebauer et al., 1997; Dreger, 1998). We know that chromosomal defects cause 50-60% of all fetal losses in the first trimester of pregnancy (Boué and Boué, 1973; Hassold, 1986). Monosomy X (45,X) corresponds to 20-25% of all cases, triploidy to 15-20% and the several trisomies to approximately 50%. Proving to a couple that their miscarriage was caused by a sporadic chromosomal defect (a "genetic accident") and that continuation of the pregnancy would inevitably culminate in the birth of an abnormal child, significantly helps the couple to cope with the fetal loss. Moreover, establishment of the specific fetal etiology for the miscarriage dispenses with the need to search for a maternal cause of the fetal death. Thus, whenever possible, all miscarriages should be investigated cytogenetically. Unfortunately, conventional cytogenetics depends on the availability of live dividing human cells that are generally only obtained after cell cultures. Culture failures are common because tissues from abortuses and stillborns are often contaminated with bacteria or may have been frozen or fixed in formalin or alcohol. Based on this knowledge we at GENE - Núcleo de Genética Médica developed a multiplex PCR procedure for the study of DNA extracted from tissues of fetal losses that effectively allows the establishment of the fetal sex and the diagnosis of triploidy and trisomies 13, 16, 18 and 21 in 100% of the cases examined (Pena, 1998). To increase the efficiency of this molecular cytogenetics procedure we decided to develop an additional molecular test specific for Turner syndrome, to be applied when the fetal sex was feminine and no abnormalities were seen in the initial multiplex screening test.

As part of an earlier research project, we had already developed a multiplex procedure that permitted the simultaneous amplification of five dinucleotide repeat polymorphisms in a large non-recombining region in the long arm of the X chromosome (Figure 1A; Pereira, R.W. and Pena, S.D.J., unpublished results). Our population studies showed that haplotypic diversity in European, African and Asian populations was larger than 99%. Thus, we expect to see homozygosity at all five loci in less than 1% of the normal population and the presence of a single peak in all the microsatellites in a female miscarried fetus should be taken as strong evidence of loss of heterozygosity due to deletion of a second sexual chromosome (Figure 1A). In a series of non-mosaic Turner syndrome patients ascertained by conventional cytogenetics, this new molecular procedure was proven to have diagnostic sensitivity and specificity of virtually 100% (Pereira, R.W. and Pena, S.D.J., unpublished results). Using the combination of screening for trisomies, triploidy and Turner syndrome, we have been able to diagnose chromosomal abnormalities in dozens of abortuses and stillbirths that could not have been properly assessed by conventional cytogenetics because the samples sent to our clinical laboratory at GENE had been frozen, fixed in formalin or alcohol, or failed to grow in culture due to maceration or bacterial contamination.

Figure 1
- Diagnosis of chromosomal deletions or microdeletions using PCR-based methodology. A, Detection of Turner syndrome (TS) using microsatellites from the X chromosome. Note that the normal control (NC) female is heterozygous for all loci, while the 45,X patient presents total loss of heterozygosity. B, PCR-based screening for infertile males with azoospermia or severe oligospermia. The NC male presents all the Yq11 STSs and also a normal ratio of the areas of the X and Y peaks at AMEL. The 47,XXY patient shows presence of all the Yq11 STSs but the ratio of the areas of the AMEL X and Y peaks is close to 2. The third patient shows a normal ratio of the areas of the AMEL X and Y peaks but the peaks of sY254, sY258 and sY495 are absent, indicating deletion of the DAZ locus. C, Diagnosis of DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCF) by loss of heterozygosity at three dinucleotide microsatellites in the critical 22q11 region. D, Diagnosis of Williams syndrome WS by loss of heterozygosity at three microsatellites in the ELN gene in 7q. In all cases, approximately 20 ng of DNA was subjected to multiplex PCR in which the upper primer of each pair was labeled with Cy5 and purified by HPLC. After PCR, an aliquot of 1 ml was denatured and run on urea-PAGE on an ALF-Express automatic fluorescent DNA sequencer (Pharmacia Biotech, Uppsala, Sweden). The data were analyzed using the Allelelinks software (Pharmacia Biotech).

Approximately half of all clinically recognized post-natal cases of Turner syndrome have an apparently non-mosaic 45,X constitution while the remainder are mosaics with a 45,X line and/or a structurally abnormal X chromosome (Jacobs et al., 1997). The screening test developed by us is based on loss of heterozygosity and thus is not well suited for diagnosis of Turner syndrome after birth, since we could predict a significant number of false negatives, even if we performed quantification of the microsatellite peaks by computer-assisted densitometry (Pena, 1998). However, due to its simplicity and quick results (less than 12 h when necessary), the multiplex X chromosome microsatellite test could be used as a preliminary screening when a rapid diagnosis of monosomy X is needed or desired.

PCR-BASED DIAGNOSIS OF MICRODELETION SYNDROMES

By definition, microdeletions are deletions of sizes at or below the current level of resolution of the light microscope (Shaffer, 1997). In general, routine cytogenetic analysis can resolve 400-500 chromosome bands per haploid karyotype. At this level of resolution, deletions on the order of 5-10 Mb can be visualized. Using high-resolution methods based on cell culture synchronization to obtain prometaphase spreads, resolution can be increased to 650-850 bands, i.e., deletions of roughly 2-5 Mb can be seen. Below this size, deletions can only be seen by molecular methods, using PCR-based diagnosis or FISH (Shaffer, 1997).

Microdeletions produce abnormal phenotypes because they cause haploinsufficiencies (dominant) or absolute deficiencies (recessive). The latter are seen when the only functional copy of the critical chromosome region is deleted, as for instance in microdeletions of the X or the Y chromosomes in males (examples are X-linked Duchenne muscular dystrophy and Y-linked azoospermia) or in microdeletions of autosomal regions subject to imprinting (expressed monoallelically and consequently functionally haploid), as for instance in the Angelman syndrome, caused by maternal deletions in 15q11-15q13 (see below). On the other hand, the disease may be due to haploinsufficiency, i.e., reduced dosage of the relevant gene product. In these cases, for instance the DiGeorge/velocardiofacial syndromes (deletion of 22q11) or the Williams syndrome (deletion of 7q11), although the diseases generally are sporadic in the families, in some cases dominant inheritance from a mildly afflicted parent may be observed. Some genes are extremely dosage sensitive; the best example is the candidate gene PMP22 located at 17p12. When one copy of this gene is deleted the phenotype is a hereditary neuropathy with liability to pressure palsies (HNPP) while gene duplication leads to another neuropathy, Charcot-Marie-Tooth type 1A. The distinction of the two types of microdeletion syndromes is relevant for molecular diagnosis. PCR-based tests for diseases of absolute deficiency may make use of non-polymorphic markers such as sequence tagged sites (STSs) or parent-of-origin imprinted fragments. On the other hand, diseases caused by haploinsufficiency need polymorphic markers for the diagnosis, which is then based on loss of heterozygosity.

Microdeletion syndromes are commonly seen as de novo mutations, apparently because of a genome-wide predisposition to deletion events. An explanation for this high frequency is that the evolution of the mammalian genome has resulted in the frequent duplication of genes, gene segments and repeat gene clusters. This genome architecture provides the substrate for homologous recombination between nonsynthenic regions of chromosomes and unequal crossovers, resulting in microdeletions and microduplications (Lupski, 1998).

Microdeletion syndromes associated with absolute deficiencies

Prader-Willi syndrome and Angelman syndrome

Prader-Willi syndrome (PWS; MIM # 176270) has a clinical phenotype characterized by neonatal hypotonia and developmental delay, followed by the post-natal development of hyperphagia with subsequent obesity, short stature, hypogonadism and mild to moderate mental retardation. Angelman syndrome (AS; also known as Happy Puppet syndrome, MIM # 105830) is associated with extra-pyramidal symptoms and signs (jerky, unsteady gait with stiff upper arms), hyperactivity, seizures, severe mental retardation with absence of verbal speech and paroxysms of inappropriate laughter. PWS and AS each occur at a frequency of about 1/10,000 to 1/20,000 births (Nicholls et al., 1998). The reason that they are discussed together is that both syndromes are most frequently caused by microdeletions in the chromosomal region 15q11-q13 that is subjected to parental imprinting. PWS and AS are associated with deficiencies of the paternal and maternal chromosome 15, respectively. The deficiencies are caused by microdeletions (~75% of cases of PWS or AS), by uniparental disomies (25% of PWS and 2% of AS) or by imprinting mutations (2% of PWS and 5% of AS). Because of the imprinting, there is differential monoallelic expression of genes in the paternal and maternal chromosome 15 homologues and thus the microdeletions probably result in complete deficiencies of expression of critical genes. The genes have not been identified yet for PWS, which probably arises from multiple deficiencies (contiguous gene syndrome). On the other hand, approximately 20% of patients with the AS do not show chromosomal microdeletions, uniparental disomies or imprinting mutations. Recently, it was shown that these patients have mutations in the gene coding for a ubiquitin-protein ligase (UBE3A) that is involved in the pathway of protein degradation and undergoes brain-specific imprinting (Malzac et al., 1998).

The molecular mechanism for imprinting is differential DNA methylation of the paternal and maternal alleles. This provides the most efficient method for the molecular diagnosis of these syndromes, i.e., methylation analysis. The most adequate region seems to be at a gene coding for a small nuclear RNA (SNRPN), in which the maternal allele is completely methylated and the paternal allele is completely unmethylated.

We have been using a methylation-sensitive PCR assay for the a exon in the 5' region of the SNRPN gene (M-PCR; Kubota et al., 1997). Representative results are shown in Figure 2. This methylation assay detects all deletions, uniparental disomies and imprinting mutations that constitute approximately 98% of classical PWS patients and about 80% of AS patients. As a secondary diagnostic method we have also been using multiplex PCR of three dinucleotide repeats (D15S11, D15S113 and GABRB3) in the Prader-Willi/Angelman critical region (Mutirangura et al., 1993). This test provides independent confirmation of microdeletion and parental isodisomy cases and serves to differentiate patients with parental heterodisomy and imprinting mutations, who do not display loss of heterozygosity.

Figure 2
- Methylation-sensitive PCR assay for the a exon in the 5' region of the SNRPN gene (M-PCR). In this gene the maternal allele is completely methylated and the paternal allele is completely unmethylated. One microgram of purified DNA was treated with sodium bisulfite and submitted to PCR with primers specific for the paternal and maternal alleles as described by Kubota et al. (1997). PCR can be multiplex, i.e., primers for both paternal and maternal alleles are mixed (lane 1) or each allele can be amplified independently (lanes 2 and 3). The PCR products are visualized after electrophoresis in 6% polyacrylamide gels and staining with silver salts and include a maternal 174-bp band and a paternal 100-bp band (arrows) as well as a small primer dimer (PD) band produced in the PCR for the paternal allele (arrow). In patients with the Prader-Willi syndrome (PW) a normal maternal band is seen (lane 4) but no paternal band is present (lane 5). In patients with the Angelman syndrome (AS) a maternal band is not seen (lane 6) but a normal paternal band is present (lane 7). NC, Normal control.

PCR-based molecular diagnosis of Y chromosome microdeletions

Male sterility affects 7-10% of all men and, in turn, 10% of these (~1% of all men) have azoospermia or severe oligospermia. Barring obstructive causes, the most common etiology is genetic: 10-15% of all patients present sexual chromosomal aberrations (Klinefelter syndrome - 47,XXY, or 46,XX males) and another 10-20% present microdeletions of the long arm of the Y chromosome (Wieacker and Jakubiczka, 1997). The microdeletion occurs in a region of the Y chromosome (Yq11.23) called AZF ("azoospermia factor"), where several genes involved in spermatogenesis are concentrated. Clinical studies based on studies of STSs (sequence tagged sites) in azoospermic or severely oligospermic individuals have shown that microdeletions cluster in three separate regions that have been called AZFa, AZFb e AZFc (Vogt et al., 1996). A gene isolated from AZFb, RBM (RNA-binding motif; formerly YRRM, MIM # 400006) turned out to belong to a multigene family that is widely distributed on the Y chromosome. At least two of the RBM (RBM1 and RBM2) genes lie in AZFb. Approximately 8% of the men with seriously impaired spermatogenesis have microdeletion in the STS intervals corresponding to these two genes. Another gene, DAZ (deleted in azoospermia, MIM # 400003), has been identified in AZFc. Like RBM, DAZ is also part of a multigene family. Deletions of DAZ are associated with variable testicular defects ranging from complete absence of germ cells (Sertoli-cell-only syndrome) to various spermatogenesis arrests. Deletions of the most centromeric portion of Yq11 (AZFa) are seen in azoospermic patients, but specific transcripts have yet to be defined in this region.

It thus seems clear that genes in several regions of the long arm of the human Y chromosome play a crucial role in spermatogenesis. At GENE the detection of microdeletions of the Y chromosome is performed by sequential multiplex PCR reactions using STSs located in all the three critical spermatogenesis regions in the long arm of the Y chromosome, with emphasis on DAZ (Figure 1B). The STSs are multiplexed with amelogenin, thus also permitting the simultaneous diagnosis of Klinefelter syndrome by computer-assisted laser densitometry (Figure 1B; Pena, 1998).

What is the significance of diagnosing AZF microdeletions? First, microdeletions do not prevent fertilization by intra-cytoplasmic sperm injections (ICSI). The problem is that sperm from affected males also carry the Y microdeletion and there is a significant (although surprisingly not absolute) risk that male offspring would inherit the microdeletion (McLaren, 1998). Thus, from the point of view of etiological diagnosis, prognosis and genetic counseling, the ascertainment of AZF microdeletions is a worthwhile procedure that should be pursued, together with the molecular screening for Klinefelter syndrome in all azoospermic and severely oligospermic patients.

Duchenne muscular dystrophy

The Duchenne and Becker muscular dystrophies (DMD and BMD) are caused by molecular defects in the dystrophin gene in Xp21. Approximately 65% of dystrophin gene mutations are microdeletions (Beggs, 1994). In 1988 Chamberlain et al. were the first to develop a deletion screening of the dystrophin locus using multiplex DNA amplification. Modern protocols (Beggs, 1994) allow screening of 26 different dystrophin gene exons using only three complementary multiplex PCR assays. At least one of these exons is missing in >95% of the deletion cases identified with the more cumbersome Southern blotting techniques using dystrophin cDNA probes. This excellent sensitivity of the PCR-based diagnosis has made it the method of choice for the diagnosis of these muscular dystrophies in boys, replacing the much more expensive and invasive muscle biopsy.

Very large deletions of Xp21 may also delete other contiguous genes, such as those causing chronic granulomatous disease, McLeod syndrome, retinitis pigmentosa, mental retardation, glycerol kinase and adrenal hypoplasia congenita, sometimes resulting in very complex phenotypes (Ballabio, 1991; Shaffer, 1997). Obviously, any one of these deficiencies can also occur as an individual microdeletion disease.

Microdeletion syndromes caused by haploinsufficiency

CATCH-22 syndrome (cardiac abnormality/abnormal facies, T cell deficit due to thymic hypoplasia, cleft palate, hypocalcemia due to hypoparathyroidism resulting from 22q11 deletion)

Several malformation syndromes are associated with heterozygous microdeletions within 22q11.2 (del22q11), including the DiGeorge syndrome (MIM # 18840, DGS) and the velocardiofacial syndrome (also called Shprintzen syndrome, MIM # 19243, VCFS). The two syndromes are quite common, with an estimated prevalence of 1/4000 live births each. VCFS is characterized by cleft palate or velopharyngeal insufficiency, typical facial dysmorphology, learning disabilities and conotruncal congenital heart defects. A common recently discovered feature of the VCFS is the development of important psychiatric disturbances in adolescence. The DGS patients share many of the features seen in VCFS but display most prominently hypocalcemia due to hypoparathyreoidism and immune deficiencies due to congenital aplasia or hypoplasia of the thymus. The presence of the same chromosomal deletion in both DGS and VCFS and their phenotypic overlap led Wilson et al. (1993) to propose CATCH22 (cardiac Abnormality/abnormal facies, T cell deficit due to thymic hypoplasia, cleft palate, hypocalcemia due to hypoparathyroidism resulting from 22q11 deletion) as a collective acronym. Deletions of 22q11 are seen in 15-20% of patients with conotruncal congenital heart disease (i.e., malformations of the left ventricular ejection tract and aortic arch) including tetralogy of Fallot (Webber et al., 1996), in 8% of children with cleft palate and in 38% of patients with idiopathic velopharyngeal insufficiency (Zori et al., 1998). In addition, del22q11 are frequent in cases of idiopathic learning disability (Murphy et al., 1998) and can be seen in 1-2% of all schizophrenic individuals (Yan et al., 1998).

In both syndromes the deleted region is estimated to span the same region of about 2-3 Mb. No reason is known for the phenotypic variability, and although several genes have been identified in this region, the role of any of them in the pathogenesis of VCFS/DGS remains to be defined. It is believed that a developmental field defect involving neural cell migration or differentiation in the pharyngeal arches and that haploinsufficiency of a gene disrupts proper development of these systems, leading to multiple organ and tissue abnormalities (Saint-Jore et al., 1998). Deletions of 22q11 are seen in ~80-90% of patients with VCFS/DGS; remaining cases may be associated with abnormalities of human chromosome 10 (Schuffenhauer et al., 1998).

At GENE we adopted a screening test for VCFS/DGS using multiplex PCR of three dinucleotide repeats (D22S264, D22S941 and D22S944) in the critical 22q11 region (Bonnet et al., 1997; Figure 1C). These primers detect 95% of cases of the CATCH-22 syndrome, whose presence is indicated by loss of heterozygosity at all three loci. Chance homozygosity for all three microsatellites is expected in only 1% of the Brazilian population. Positive screening results should ideally be confirmed by studying the parents with the same markers.

Williams syndrome

The Williams syndrome (WS; also called Williams-Beuren syndrome; MIM # 194050) is a disorder that in full-blown form includes supravalvular aortic stenosis, multiple peripheral pulmonary arterial stenoses, elfin face, mental and statural deficiency, characteristic dental malformation, and infantile hypercalcemia. The frequency of Williams syndrome has been estimated to be about 1 in 10,000. The syndrome is caused by haploinsufficiency of genes at 7q11.23 (Ewart et al., 1993). A common WS deletion region has been delineated and the size of the deletion is estimated to be more than 1 Mb (Perez Jurado et al., 1996) and includes many genes, including the elastin gene (ELN) and LIM-kinase 1 (LIMK1). Haploinsufficiency of the former is thought to cause the vascular defects of WS while hemizygosity for the latter may be associated with impaired visual-spatial constructive cognition (Meng et al., 1998). Thus, WS emerges as a contiguous gene deletion syndrome and haploinsufficiency of individual genes is apparently responsible for distinct phenotypes. Other transcripts in the critical region include the replication factor C subunit (RFC2), the human frizzled homologue, the syntaxin 1A gene (STX1A) and a gene belonging to the immunophilin FK-506 binding protein gene family (FKBP6). The latter is deleted in 40/40 WS patients tested and may be associated with the hypercalcemia and growth delay seen in the syndrome (Meng et al., 1998). In a series of 235 patients, Lowery et al. (1995) identified molecular cytogenetic deletions by FISH in 96% of patients with classic WS. Likewise, Perez Jurado et al. (1996) used 13 polymorphisms and determined that 94% of patients had a deletion of the ELN locus. Wu et al. (1998) defined the minimal critical deletion region on 7q in 63 WS patients, using 10 microsatellite markers and 5 fluorescence in situ hybridization probes flanking the ELN gene. These studies showed deletions of consistent size. In all informative cases deleted at ELN, the deletion extended from D7S489U to D7S1870.

At GENE we developed a screening test for the WS syndrome using multiplex PCR of three microsatellites (D7S1870, Eln and Elni1) in the ELN locus (Figure 1D). The presence of WS is indicated by loss of heterozygosity at all three loci. Chance homozygosity for all three microsatellites is expected in less than 1% of the Brazilian population. Positive screening results should ideally be confirmed by studying the parents with the same markers.

Other microdeletion syndromes

We have chosen to describe in some detail only the syndromes for which we have first-hand molecular diagnostic experience. Space does not allow us to discuss many other important microdeletion syndromes, such as the Wilms/Aniridia syndrome associated with microdeletions in 11p13, the Miller-Dieker syndrome (lissencephaly) on 17p13.3, Langer-Giedion syndrome on 8q24.1 and Smith-Magenis syndrome on 17p11.2. We plan to establish routine PCR-based tests for these in the near future using testing strategies very similar to the ones described in this mini-review. For further information and literature on these other microdeletion syndromes we recommend the review by Shaffer (1997).

CONCLUSIONS

In this brief review we have shown our experience in the molecular diagnosis of chromosomal deletions and microdeletion syndromes using a simple and straightforward PCR-based approach. It might be questioned why not use the more established techniques based on chromosomal or interphase fluorescent in situ hybridization (FISH) that have been commonly applied for diagnosis in these cases (Adinolfi and Crolla, 1994; Shaffer, 1997). We have previously indicated that conventional cytogenetics has the inconveniences of depending on the availability of live dividing human cells, of being very time consuming, even in the best equipped laboratories, and of depending on technical expertise that can only be developed after prolonged training and extensive experience (Pena, 1998). FISH on chromosome spreads certainly increases significantly the level of resolution for diagnosis of microdeletions, but suffers from basically these same drawbacks, with considerable aggravation of the technical complexity. On the other hand, it is true that interphase in situ hybridization dispenses with the need for live dividing cells, but it is still slow, labor intensive and needs considerable expertise, besides depending on the availability of large cloned probes. A recent study of interphase FISH in 315 cases of prenatal diagnosis conducted at a leading laboratory (D'Alton et al., 1997) showed that almost 20% of the samples were uninformative or unreportable and that the mean time for FISH diagnosis was 2.8 days. In contrast, PCR-based studies only depend on easily available primers, can always provide results in less than 24 h if needed, are technically simple and highly automatable and thus can be offered to the public as highly useful screening tests at considerably lower prices than FISH.

ACKNOWLEDGMENTS

The experiments with the original results described in this mini-review were performed at GENE - Núcleo de Genética Médica, by Rosane Sturzeneker, Helena B.B.L. Martins-da-Costa, Elen E.R.F. Carvalho, Márcia C.B.N. Campos and Riva P. Oliveira. I am grateful to Rinaldo W. Pereira and Andreia Vercesi who helped in technical development.

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(Received December 1, 1998)

Publication Dates

• Publication in this collection
  01 Mar 1999
• Date of issue
  Dec 1998

History

• Received
  12 Mar 1997