Detection of submicroscopic deletions in band 17p13 in patients with the Miller-Dieker syndrome

The Miller-Dieker syndrome (MDS), a syndrome with lissencephaly, distinctive craniofacial features, growth impairment, and profound developmental failure, has been associated with a deletion of the distal part of chromosome band 17p13. A minority of patients with the syndrome do not have a deletion detectable with current cytogenetic techniques. Using three highly polymorphic DNA probes (pYNZ22, pYNH37.3, and p144D6) we have detected microdeletions in three MDS patients, two of whom had no visible abnormalities of chromosome 17. Loci defined by two of the DNA probes, pYNZ22 and pYNH37.3, were deleted in all three patients. The most distal locus, defined by p144D6, was present in one MDS patient, possibly defining the distal limits of the MDS region in band 17p13.3. None of these loci were absent in one case of lissencephaly without MDS.     

Molecular detection of microscopic and submicroscopic deletions associated with Miller-Dieker syndrome.

Miller-Dieker syndrome (MDS), a disorder manifesting the severe brain malformation lissencephaly ("smooth brain"), is caused, in the majority of cases, by a chromosomal microdeletion of the distal short arm of chromosome 17. Using human chromosome 17-specific DNA probes, we have begun a molecular dissection of the critical region for MDS. To localize cloned DNA sequences to the MDS critical region, a human-rodent somatic cell hybrid panel was constructed which includes hybrids containing the abnormal chromosome 17 from three MDS patients with deletions of various sizes. Three genes (myosin heavy chain 2, tumor antigen p53, and RNA polymerase II) previously mapped to 17p were excluded from the MDS deletion region and therefore are unlikely to play a role in its pathogenesis. In contrast, three highly polymorphic anonymous probes, YNZ22.1 (D17S5), YNH37.3 (D17S28), and 144-D6 (D17S34), were deleted in each of four patients with visible deletions, including one with a ring chromosome 17 that is deleted for a portion of the single telomeric prometaphase subband p13.3. In two MDS patients with normal chromosomes, a combination of somatic cell hybrid, RFLP, and densitometric studies demonstrated deletion for YNZ22.1 and YNH37.3 in the paternally derived 17's of both patients, one of whom is also deleted for 144-D6. The results indicate that MDS can be caused by submicroscopic deletion and raises the possibility that all MDS patients will prove to have deletions at a molecular level. The two probes lie within a critical region of less than 3,000 kb and constitute potential starting points in the isolation of genes implicated in the severe brain maldevelopment in MDS.     

Microdeletions of chromosome 17p13 as a cause of isolated lissencephaly.

Lissencephaly (agyria-pachygyria) is a brain malformation manifested by a smooth cerebral surface, resulting from arrest of neuronal migration at 10-14 wk gestation. Type I, or classical, lissencephaly can occur either in association with the Miller-Dieker syndrome (MDS) or as an isolated finding, termed "isolated lissencephaly sequence" (ILS). About 90% of MDS patients have visible or submicroscopic deletions of 17p13.3. We therefore investigated the possibility that some ILS patients have smaller deletions in this chromosomal region. Forty-five ILS patients with gyral abnormalities ranging from complete agyria to mixed agyria/pachygyria and complete pachygyria were studied. RFLP analysis with five polymorphic loci in 17p13.3 was performed on all patients and their parents. Somatic cell hybrids were constructed on three patients, to confirm a deletion or to determine the boundaries of a deletion. In-situ hybridization using cosmid probes from within a newly defined lissencephaly critical region was performed on 31 patients as a further method of deletion detection. Six submicroscopic deletions were detected (13.3%). Three of the deletions among 45 ILS patients were detected by RFLP analysis, 4 deletions in 31 patients were detected by in situ hybridization, and one deletion was detected only by somatic cell hybrid studies (in situ hybridization was not performed). Overall, in situ hybridization proved to be the most rapid and sensitive method of deletion detection. The centromeric boundary of these deletions overlapped that of MDS patients, while the telomeric boundary for four ILS deletions was proximal to that of MDS and narrows the critical region for a lissencephaly locus.(ABSTRACT TRUNCATED AT 250 WORDS)     

A genetic etiology for DiGeorge syndrome: consistent deletions and microdeletions of 22q11.

DiGeorge syndrome (DGS), a developmental field defect of the third and fourth pharyngeal pouches, is characterized by aplasia or hypoplasia of the thymus and parathyroid glands and by conotruncal cardiac malformations. Cytogenetic studies support the presence of a DGS critical region in band 22q11. In the present study, we report the results of clinical, cytogenetic, and molecular studies of 14 patients with DGS. Chromosome analysis, utilizing high-resolution banding techniques, detected interstitial deletions in five probands and was inconclusive for a deletion in three probands. The remaining six patients had normal karyotypes. In contrast, molecular analysis detected DNA deletions in all 14 probands. Two of 10 loci tested, D22S75 and D22S259, are deleted in all 14 patients. A third locus, D22S66, is deleted in the eight DGS probands tested. Physical mapping using somatic cell hybrids places D22S66 between D22S75 and D22S259, suggesting that it should be deleted in the remaining six cases. Parent-of-origin studies were performed in five families. Four probands failed to inherit a maternal allele, and one failed to inherit a paternal allele. On the basis of these families, and of six maternally and five paternally derived unbalanced-translocation DGS probands in the literature, parent of origin or imprinting does not appear to play an important role in the pathogenesis of DGS. Deletion of the same three loci in all 14 DGS probands begins to delineate the region of chromosome 22 critical for DGS and confirms the hypothesis that submicroscopic deletions of 22q11 are etiologic in the vast majority of cases.     

Angelman syndrome: three molecular classes identified with chromosome 15q11q13-specific DNA markers

Angelman syndrome (AS) and Prader-Willi syndrome (PWS) share a cytogenetic deletion of chromosome 15q11q13. To determine the extent of deletion in AS we analyzed the DNA of 19 AS patients, including two sib pairs, with the following chromosome 15q11q13--specific DNA markers: D15S9-D15S13, D15S17, D15S18, and D15S24. Three molecular classes were identified. Class I showed a deletion of D15S9-D15S13 and D15S18; class II showed a deletion of D15S9-D15S13; and in class III, including both sib pairs, no deletion was detected. These molecular classes appear to be identical to those observed in PWS. High-resolution cytogenetic data were available on 16 of the patients, and complete concordance between the presence of a cytogenetic deletion and a molecular deletion was observed. No submicroscopic deletions were detected. DNA samples from the parents of 10 patients with either a class I or a class II deletion were available for study. In seven of the 10 families, RFLPs were informative as to the parental origin of the deletion. In all informative families, the deleted chromosome 15 was observed to be of maternal origin. This finding is in contrast to the paternal origin of the deletions in PWS and is currently the only molecular difference observed between the two syndromes.     

Detection of deletions and cryptic translocations in Miller-Dieker syndrome by in situ hybridization.

Fluorescence in situ hybridization (FISH) using two cosmid probes (41A and P13) from the Miller-Dieker syndrome (MDS) critical region in 17p13.3 was performed in a blinded comparison of three MDS patients with submicroscopic deletions and in four normal relatives used as controls. The controls showed both chromosome 17 homologues labeled in 85%-95% of cells, while each patient showed only one homologue labeled in 75%-80% of cells. Two MDS patients with cryptic translocations were also studied. In one case, a patient and her mother had the same der(17) (p+), but the reciprocal product of the translocation could not be identified in the mother by G-banding (i.e., it was a "half-cryptic" translocation). FISH revealed a 3q;17p translocation. The other case involved a patient with apparently normal karyotype. Because a large molecular deletion was found, a translocation involving two G-negative telomeres (i.e., a "full-cryptic" translocation) was postulated. FISH studies on her father and normal brother showed an 8q;17p translocation. These studies demonstrate that in situ hybridization is an efficient method for deletion detection in Miller-Dieker syndrome. More important, parental studies by FISH on patients demonstrating molecular deletions and a normal karyotype may identify cryptic translocation events, which cannot be detected by other molecular genetic strategies. Similar in situ strategies for deletion detection can be developed for other microdeletion syndromes, such as Prader-Willi/Angelman syndrome or DiGeorge syndrome.     

Deletion at chromosome 16p13.3 as a cause of Rubinstein-Taybi syndrome: clinical aspects.

In the accompanying paper, a chromosomal localization of the Rubinstein-Taybi syndrome by cytogenetic investigations with fluorescence in situ hybridization techniques at chromosome 16p13.3 is described. We investigated 19 of these patients and their parents (a) to ascertain the parental origin of the chromosome with the deletion in families where such a deletion was detected, (b) to disclose whether uniparental disomy plays a role in etiology, and (c) to compare clinical features in patients with a deletion to those in individuals in whom deletions were not detectable. Molecular studies showed a copy of chromosome 16 from each parent in all 19 patients. Uniparental disomy was also excluded for five other chromosome arms known to be imprinted in mice. None of the probes used for determining the origin of the deleted chromosome proved to be informative. The clinical features were essentially the same in patients with and without visible deletion, with a possible exception for the incidence of microcephaly, angulation of thumbs and halluces, and partial duplication of the halluces. A small deletion at 16p13.3 may be found in some patients with Rubinstein-Taybi syndrome. Cytogenetically undetectable deletions, point mutations, mosaicism, heterogeneity, or phenocopy by a nongenetic cause are the most probable explanations for the absence of cytogenetic or molecular abnormalities in other patients with Rubinstein-Taybi syndrome.     

Human chromosome 17 NotI linking clones and their use in long-range restriction mapping of the Miller-Dieker chromosome region (MDCR) in 17p13.3

A NotI linking library constructed from flow-sorted human chromosome 17 material was screened to aid in construction of a long-range restriction map of the Miller-Dieker chromosome region (MDCR) in 17p13.3. A total of 66 clones were mapped to one of eight regions of chromosome 17 using a somatic cell hybrid panel, and 44/66 (67%) of these clones cross-hybridized to rodent DNA on Southern blots. Of these, 24 clones were tested and all mapped to mouse chromosome 11, the homolog of human chromosome 17. Four linking clones mapped to 17p13.3 and were used for pulsed-field gel electrophoresis studies along with six other anonymous probes previously mapped to this region. Clone L132 was found to be deleted in all Miller-Dieker patients tested (n = 15) and therefore lies within the critical region for this disorder. It detects two NotI fragments (180 and 320 kb), one of which (320 kb) was shared by YNZ22 and YNH37, two probes previously shown to be co-deleted in all patients with the Miller-Dieker syndrome (MDS). These results indicate that all MDS patients share a minimum deletion region of greater than 370 kb. Two other NotI clones, L53 and L125, mapped telomeric to the MDS critical region and share a 600-kb MluI fragment with each other and with YNZ22/YNH37. This provides a 930-kb MluI map that encompasses the distal boundary of the MDS critical region but does not include the proximal boundary. A total of over 2 Mbp is represented in the MluI fragments by probes in subband p13.3, a cytogenetic region estimated to be 3-4 Mbp.     

Molecular cytogenetic aberrations in Tunisian patients with multiple myeloma identified by cIg-FISH in fixed bone marrow cells

Cytogenetic studies in multiple myeloma (MM) are hampered by the hypo-proliferative nature of plasma cells. In order to circumvent this problem, we have used a combination of immunolabeling of cytoplasmic Ig light chains (λ or κ) and FISH (cIg-FISH), which allowed a comprehensive detection of the most common and/or recurrent molecular cytogenetic aberrations on fixed bone marrow cells of 70 Tunisian patients. Translocations involving the chromosome 14q32 region were observed in 32 cases (45.7%), including 18 cases with a t(11;14), 8 cases with a t(4;14), and 2 cases with a t(14;16). Deletions of the 13q14 region (D13S319/RB1) were detected in 18.6%, and deletions of the 17p13 region (TP53) in 5.7% of the cases, respectively. Of all patients with a D13S319/RB1 deletion, 61.5% also carried a 14q32 translocation, whereas TP53 deletions were associated with a t(11;14) in 2 cases (50%) and a D13S319 deletion in 1 case (25%). Our results suggest that there is a correlation between the presence of 14q32 translocations and chromosome 13q14 deletions in MM patients and that cIg-FISH is more sensitive as compared to conventional karyotyping in detecting molecular cytogenetic abnormalities in this disease.     

Clinical, cytogenetic, and molecular characterization of seven patients with deletions of chromosome 22q13.3.

We have studied seven patients who have chromosome 22q13.3 deletions as revealed by high-resolution cytogenetic analysis. Clinical evaluation of the patients revealed a common phenotype that includes generalized developmental delay, normal or accelerated growth, hypotonia, severe delays in expressive speech, and mild facial dysmorphic features. Dosage analysis using a series of genetically mapped probes showed that the proximal breakpoints of the deletions varied over approximately 13.8 cM, between loci D22S92 and D22S94. The most distally mapped locus, arylsulfatase A (ARSA), was deleted in all seven patients. Therefore, the smallest region of overlap (critical region) extends between locus D22S94 and a region distal to ARSA, a distance of > 25.5 cM.     

Localization of the Miller-Dieker critical region is proximal to locus D17S34 (p144D6) in 17p13.3

A child with normal growth and development and the abnormal karyotype 46,XY,17ps, was analyzed using molecular probes localized to 17p13. The results indicated the presence of two copies of the probes YNZ22.1 (D17S5) and YNH37.3 (D17S28), previously shown to be deleted in all Miller-Dieker (MDS) patients studied. However, the patient was hemizygous for probe p144D6 (D17S34), which is absent in approximately 75% of the MDS patients. As the patient is active at 9 months of age, with no clinical signs of MDS, the results confirm that the absence of locus D17S34 does not lead to the phenotypic expression of MDS. Furthermore, this deletion should assist in defining the distal limits of this contiguous gene syndrome.     

Molecular analyses of 17p11.2 deletions in 62 Smith-Magenis syndrome patients.

Smith-Magenis syndrome (SMS) is a clinically recognizable, multiple congenital anomalies/mental retardation syndrome caused by an interstitial deletion involving band p11.2 of chromosome 17. Toward the molecular definition of the interval defining this microdeletion syndrome, 62 unrelated SMS patients in conjunction with 70 available unaffected parents were molecularly analyzed with respect to the presence or absence of 14 loci in the proximal region of the short arm of chromosome 17. A multifaceted approach was used to determine deletion status at the various loci that combined (i) FISH analysis, (ii)PCR and Southern analysis of somatic cell hybrids retaining the deleted chromosome 17 from selected patients, and (iii) genotype determination of patients for whom a parent(s) was available at four microsatellite marker loci and at four loci with associated RFLPs. The relative order of two novel anonymous markers and a new microsatellite marker was determined in 17p11.2. The results confirmed that the proximal deletion breakpoint in the majority of SMS patients is located between markers D17S58 (EW301) and D17S446 (FG1) within the 17p11.1-17p11.2 region. The common distal breakpoint was mapped between markers cCI17-638, which lies distal to D17S71, and cCI17-498, which lies proximal to the Charcot Marie-Tooth disease type 1A locus. The locus D17S258 was found to be deleted in all 62 patients, and probes from this region can be used for diagnosis of the SMS deletion by FISH. Ten patients demonstrated molecularly distinct deletions; of these, two patients had smaller deletions and will enable the definition of the critical interval for SMS.     

Molecular analysis of 24 Alagille syndrome families identifies a single submicroscopic deletion and further localizes the Alagille region within 20p12.

Alagille syndrome (AGS) is a clinically defined disorder characterized by cholestatic liver disease with bile duct paucity, peculiar facies, structural heart defects, vertebral anomalies, and ocular abnormalities. Multiple patients with various cytogenetic abnormalities involving 20p12 have been identified, allowing the assignment of AGS to this region. The presence of interstitial deletions of varying size led to the hypothesis that AGS is a contiguous gene deletion syndrome. This molecular analysis of cytogenetically normal AGS patients was performed in order to test this hypothesis and to refine the localization of the known AGS region. Investigation of inheritance of simple tandem repeat polymorphism alleles in 67 members of 24 cytogenetically normal Alagille families led to the identification of a single submicroscopic deletion. The deletion included loci D20S61, D20S41, D20S186, and D20S188 and presumably intervening uninformative loci D20S189 and D20S27. The six deleted loci are contained in a single YAC of 1.9 Mb. The additional finding of multiple unrelated probands who are heterozygous at each locus demonstrates that microdeletions at known loci within the AGS region are rare in cytogenetically normal patients with this disorder. This suggests that the majority of cases of AGS may be the result of a single gene defect rather than a contiguous gene deletion syndrome.     

Allelotype of human thyroid tumors: loss of chromosome 11q13 sequences in follicular neoplasms.

Two classes of genes are the targets of mutations involved in human tumorigenesis: oncogenes, the activation of which leads to growth stimulation, and tumor suppressor genes, which become tumorigenic through loss of function, often through allelic deletion. To obtain evidence for a role for tumor suppressor genes in thyroid tumorigenesis, we examined DNA from 80 thyroid neoplasms for loss of heterozygosity in multiple chromosomal loci using 19 polymorphic genomic probes. None of the informative thyroid tumors studied had allelic loss detected with probes for chromosome 2q (D2S44), 3p (D3F15S2, D3S32), 3q (D3S46), 4p (D4S125), 6p (D6S40), 8q (D8S39), 9q (D9S7), 12p (D12S14), 13q (D13S52), 17p (D17S30), or 18q (D18S10). One of eight of the follicular adenomas had a 10q deletion detected with marker D10S15, and one of 26 had a 10q deletion detected with D10S25. One of two of the follicular carcinomas had an 11p deletion in the H-ras locus. The most significant findings were on chromosome 11q13, the site containing the putative gene predisposing to multiple endocrine neoplasia type I. Four of 27 follicular adenomas had loss of heterozygosity for probes in this region. Allelic deletions were detected with the following probes: D11S149, PYGM, D11S146, and INT2. None of 13 informative papillary carcinomas and none of two follicular carcinomas had loss of heterozygosity detectable with these 11q13 markers. Allelic loss is a relatively infrequent event in human thyroid tumors. Deletions of chromosome 11q13 are present in about 14% of follicular, but not papillary, neoplasms.(ABSTRACT TRUNCATED AT 250 WORDS)     

D8S7 is consistently deleted in inverted duplications of the short arm of chromosome 8 (inv dup 8p)

Ten patients with inverted duplication of 8p (inv dup 8p) were studied with cytogenetic, biochemical and molecular techniques. The duplication for the region 8p12-p22 was always associated with a deletion of the locus D8S7 (mapped in 8p23.1) as demonstrated with the probe pSW50 by both in situ hybridization and Southern blot. Restriction fragment length polymorphisms detected by probes pSW50 (1 case) and by pG2LPL35 (locus LPL) (two cases) were informative as to a maternal origin of the anomaly. The activity of glutathione reductase, whose gene maps in the duplicated region at 8p21.1, was increased in all patients. The recognizable phenotype of inv dup 8p includes neonatal hypotonia, prominent forehead, large mouth with everted lower lip, abnormally shaped large ears, brain malformations and severe mental retardation. Our findings indicate that the chromosome rearrangement is homogeneous at least for the presence of the deletion and support the hypothesis of a common mechanism of origin.     

The 13q- syndrome: the molecular definition of a critical deletion region in band 13q32.

Patients with interstitial deletions of the long arm of chromosome 13 may have widely varying phenotypes. From cytogenetic analysis, we have postulated that there is a discrete region in 13q32 where deletion leads to a syndrome of severe malformations, including digital and brain anomalies. To test this hypothesis at the molecular level, we have studied the deletions in 17 patients; 5 had severe malformations, while the remaining 12 had only minor malformations. Our results indicate that the deletions in the severely affected patients all involve an overlapping region in q32, while the deletions in the mildly affected patients include some, but not all, of this overlapping region. Our findings are consistent with the hypothesis that the severely malformed 13q- phenotype results from the deletion of a critical region in 13q32. This region is presently defined as lying between D13S136 and D13S147 and is on the order of 1 Mb in size.     

Chromosome 1p36 deletions: the clinical phenotype and molecular characterization of a common newly delineated syndrome.

Deletions of the distal short arm of chromosome 1 (1p36) represent a common, newly delineated deletion syndrome, characterized by moderate to severe psychomotor retardation, seizures, growth delay, and dysmorphic features. Previous cytogenetic underascertainment of this chromosomal deletion has made it difficult to characterize the clinical and molecular aspects of the syndrome. Recent advances in cytogenetic technology, particularly FISH, have greatly improved the ability to identify 1p36 deletions and have allowed a clearer definition of the clinical phenotype and molecular characteristics of this syndrome. We have identified 14 patients with chromosome 1p36 deletions and have assessed the frequency of each phenotypic feature and clinical manifestation in the 13 patients with pure 1p36 deletions. The physical extent and parental origin of each deletion were determined by use of FISH probes on cytogenetic preparations and by analysis of polymorphic DNA markers in the patients and their available parents. Clinical examinations revealed that the most common features and medical problems in patients with this deletion syndrome include large anterior fontanelle (100%), motor delay/hypotonia (92%), moderate to severe mental retardation (92%), growth delay (85%), pointed chin (80%), eye/vision problems (75%), seizures (72%), flat nasal bridge (65%), clinodactyly and/or short fifth finger(s) (64%), low-set ear(s) (59%), ear asymmetry (57%), hearing deficits (56%), abusive behavior (56%), thickened ear helices (53%), and deep-set eyes (50%). FISH and DNA polymorphism analysis showed that there is no uniform region of deletion but, rather, a spectrum of different deletion sizes with a common minimal region of deletion overlap.     

Chromosome 17p13.3 deletion syndrome: aCGH characterization,prenatal findings and diagnosis,and literature review

We report a molecular cytogenetic characterization of 17p13.3 deletion syndrome by array comparative genomic hybridization (aCGH), fluorescence in situ hybridization (FISH) and quantitative polymerase chain reaction (qPCR) in a fetus with lissencephaly, corpus callosum dysgenesis, ventriculomegaly, microcephaly, intrauterine growth restriction (IUGR), polyhydramnios and single umbilical artery. aCGH analysis revealed a 3.17-Mb deletion at 17p13.3, or arr [hg19] 17p13.3 (0–3,165,530)×1. The qPCR assays revealed a maternal origin of the deletion. Metaphase FISH analysis detected the absence of the LIS1 probe signal on the aberrant chromosome 17. The karyotype was 46,XX,del(17)(p13.3). We review the literature of chromosome 17p13.3 deletion syndrome with prenatal findings and diagnosis, and suggest that prenatal ultrasound detection of central nervous system anomalies such as lissencephaly, corpus callosum dysgenesis/agenesis, ventriculomegaly and microcephaly associated with IUGR, polyhydramnios, congenital heart defects, abdominal wall defects and renal abnormalities should include a differential diagnosis of chromosome 17p13.3 deletion syndrome.     

Somatic cell hybrids, sequence-tagged sites, simple repeat polymorphisms, and yeast artificial chromosomes for physical and genetic mapping of proximal 17p.

Somatic cell hybrids retaining the deleted chromosome 17 from 15 unrelated Smith-Magenis syndrome (SMS) [del(17)(p11.2p11.2)] patients were obtained by fusion of patient lymphoblasts with thymidine kinase-deficient rodent cell lines. Seventeen sequence-tagged sites (STSs) were developed from anonymous markers and cloned genes mapping to the short arm of chromosome 17. The STSs were used to determine the deletion status of these loci in these and four previously described human chromosome 17-retaining hybrids. Ten STSs were used to identify 28 yeast artificial chromosomes (YACs) from the St. Louis human genomic YAC library. Four of the 17 STSs identified simple repeat polymorphisms. The order and location of deletion breakpoints were confirmed and refined, and the regional assignment of several probes and cloned genes were determined. The cytogenetic band locations and relative order of six markers on 17p were established by fluorescence in situ hybridization mapping to metaphase chromosomes. The latter data confirmed and supplemented the somatic cell hybrid results. Most of the hybrids derived from [del(17)(p11.2p11.2)] patients demonstrated a similar pattern of deletion for the marker loci and were deleted for D17S446, D17S258, D17S29, D17S71, and D17S445. However, one of them demonstrated a unique pattern of deletion. This patient is deleted for several markers known to recognize a large DNA duplication associated with Charcot-Marie-Tooth (CMT) disease type 1A. These data suggest that the proximal junction of the CMT1A duplication is close to the distal breakpoint in [del(17)(p-11.2p11.2)] patients.