Tamim H. Shaikh

Genomic and Genic Deletions of the FOX Gene Cluster on 16q24.1 and Inactivating Mutations of FOXF1 Cause Alveolar Capillary Dysplasia and Other Malformations

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare, neonatally lethal developmental disorder of the lung with defining histologic abnormalities typically associated with multiple congenital anomalies (MCA). Using array CGH analysis, we have identified six overlapping microdeletions encompassing the FOX transcription factor gene cluster in chromosome 16q24.1q24.2 in patients with ACD/MPV and MCA. Subsequently, we have identified four different heterozygous mutations (frameshift, nonsense, and no-stop) in the candidate FOXF1 gene in unrelated patients with sporadic ACD/MPV and MCA. Custom-designed, high-resolution microarray analysis of additional ACD/MPV samples revealed one microdeletion harboring FOXF1 and two distinct microdeletions upstream of FOXF1, implicating a position effect. DNA sequence analysis revealed that in six of nine deletions, both breakpoints occurred in the portions of Alu elements showing eight to 43 base pairs of perfect microhomology, suggesting replication error Microhomology-Mediated Break-Induced Replication (MMBIR)/Fork Stalling and Template Switching (FoSTeS) as a mechanism of their formation. In contrast to the association of point mutations in FOXF1 with bowel malrotation, microdeletions of FOXF1 were associated with hypoplastic left heart syndrome and gastrointestinal atresias, probably due to haploinsufficiency for the neighboring FOXC2 and FOXL1 genes. These differences reveal the phenotypic consequences of gene alterations in cis.

A 22q11.2 deletion that excludes UFD1L and CDC45L in a patient with conotruncal and craniofacial defects.

This work was supported by National Institutes of Health grants DC02027 and HL51533 (to B.S.E, H.M., and E.H.Z.) and HD 26979 (to B.S.E.) from the Mental Retardation and Developmental Disabilities Research Center. S.C.S. is supported by Medical Genetics Research Training grant MH1-T32-GM-08638.

Clustered 11q23 and 22q11 Breakpoints and 3:1 Meiotic Malsegregation in Multiple Unrelated t(11;22) Families

The t(11;22) is the only known recurrent, non-Robertsonian constitutional translocation. We have analyzed t(11;22) balanced-translocation carriers from multiple unrelated families by FISH, to localize the t(11;22) breakpoints on both chromosome 11 and chromosome 22. In 23 unrelated balanced-translocation carriers, the breakpoint was localized within a 400-kb interval between D22S788 (N41) and ZNF74, on 22q11. Also, 13 of these 23 carriers were tested with probes from chromosome 11, and, in each, the breakpoint was localized between D11S1340 and APOA1, on 11q23, to a region </=185 kb. Thus, the breakpoints on both chromosome 11 and chromosome 22 are clustered in multiple unrelated families. Supernumerary-der(22)t(11;22) syndrome can occur in the progeny of balanced-t(11;22) carriers, because of malsegregation of the der(22). There has been speculation regarding the mechanism by which the malsegregation occurs. To elucidate this mechanism, we have analyzed 16 of the t(11;22) families, using short tandem-repeat-polymorphism markers on both chromosome 11 and chromosome 22. In all informative cases the proband received two of three alleles, for markers above the breakpoint on chromosome 22 and below the breakpoint on chromosome 11, from the t(11;22)-carrier parent. These data strongly suggest that 3:1 meiosis I malsegregation in the t(11;22) balanced-translocation-carrier parent is the mechanism in all 16 families. Taken together, these results establish that the majority of t(11;22) translocations occur within the same genomic intervals and that the majority of supernumerary-der(22) offspring result from a 3:1 meiosis I malsegregation in the balanced-translocation carrier.

Identification of familial and de novo microduplications of 22q11.21-q11.23 distal to the 22q11.21 microdeletion syndrome region

Deletions of the 22q11.2 region distal to the 22q11.21 microdeletion syndrome region have recently been described in individuals with mental retardation and congenital anomalies. Because these deletions are mediated by low-copy repeats (LCRs), located distal to the 22q11.21 DiGeorge/velocardiofacial microdeletion region, duplications are predicted to occur with a frequency equal to the deletion. However, few microduplications of this region have been reported. We report the identification of 18 individuals with microduplications of 22q11.21-q11.23. The duplication boundaries for all individuals are within LCRs distal to the DiGeorge/velocardiofacial microdeletion region. Clinical records for nine subjects reveal shared characteristics, but also several examples of contradicting clinical features (e.g. macrocephaly versus microcephaly and upslanting versus downslanting palpebral fissures). Of 12 cases for whom parental DNA samples were available for testing, one is de novo and 11 inherited the microduplication from a parent, three of whom reportedly have learning problems or developmental delay. The variable phenotypes and preponderance of familial cases obfuscate the clinical relevance of the molecular data and emphasize the need for careful parental assessments and clinical correlations.

Tightly Clustered 11q23 and 22q11 Breakpoints Permit PCR-Based Detection of the Recurrent Constitutional t(11;22)

Palindromic AT-rich repeats (PATRRs) on chromosomes 11q23 and 22q11 at the constitutional t(11;22) breakpoint are predicted to induce genomic instability, which mediates the translocation. A PCR-based translocation-detection system for the t(11;22) has been developed with PCR primers flanking the PATRRs of both chromosomes, to examine the involvement of the PATRRs in the recurrent rearrangement. Forty unrelated carriers of the t(11;22) balanced translocation, plus two additional, independent cases with the supernumerary-der(22) syndrome, were analyzed to compare their translocation breakpoints. Similar translocation-specific junction fragments were obtained from both derivative chromosomes in all 40 carriers of the t(11;22) balanced translocation and from the der(22) in both of the offspring with unbalanced supernumerary-der(22) syndrome, suggesting that the breakpoints in all cases localize within these PATRRs and that the translocation is generated by a similar mechanism. This PCR strategy provides a convenient technique for rapid diagnosis of the translocation, indicating its utility for prenatal and preimplantation diagnosis in families including carriers of the balanced translocation.

Copy-Number Variation of the Glucose Transporter Gene SLC2A3 and Congenital Heart Defects in the 22q11.2 Deletion Syndrome

The 22q11.2 deletion syndrome (22q11DS; velocardiofacial/DiGeorge syndrome; VCFS/DGS) is the most common microdeletion syndrome and the phenotypic presentation is highly variable. Approximately 65% of individuals with 22q11DS have a congenital heart defect (CHD), mostly of the conotruncal type, and/or an aortic arch defect. The etiology of this phenotypic variability is not currently known. We hypothesized that copy-number variants (CNVs) outside the 22q11.2 deleted region might increase the risk of being born with a CHD in this sensitized population. Genotyping with Affymetrix SNP Array 6.0 was performed on two groups of subjects with 22q11DS separated by time of ascertainment and processing. CNV analysis was completed on a total of 949 subjects (cohort 1, n = 562; cohort 2, n = 387), 603 with CHDs (cohort 1, n = 363; cohort 2, n = 240) and 346 with normal cardiac anatomy (cohort 1, n = 199; cohort 2, n = 147). Our analysis revealed that a duplication of SLC2A3 was the most frequent CNV identified in the first cohort. It was present in 18 subjects with CHDs and 1 subject without (p = 3.12 × 10(-3), two-tailed Fishers exact test). In the second cohort, the SLC2A3 duplication was also significantly enriched in subjects with CHDs (p = 3.30 × 10(-2), two-tailed Fishers exact test). The SLC2A3 duplication was the most frequent CNV detected and the only significant finding in our combined analysis (p = 2.68 × 10(-4), two-tailed Fishers exact test), indicating that the SLC2A3 duplication might serve as a genetic modifier of CHDs and/or aortic arch anomalies in individuals with 22q11DS.

Characterization of CDC45L: a gene in the 22q11.2 deletion region expressed during murine and human development

Division of Human Genetics and Molecular Biology, The Children’s Hospital of Philadelphia, 1002 Abramson Research Center, 34th Street and Civic Center Blvd., Philadelphia, Pennsylvania 19104, USA Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, USA Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA

A patient with mosaic partial trisomy 18 resulting from dicentric chromosome breakage.

We present a patient with minor dysmorphic features and a mosaic karyotype with two different abnormal cell lines, both involving abnormalities of chromosome 18. Twenty percent of cells studied (4/20) had 46 chromosomes with a large derivative pseudoisodicentric chromosome 18. This chromosome was deleted for 18pter and duplicated for part of proximal 18p (18p11.2 based on fluorescence in situ hybridization (FISH) studies and all of 18q. The two copies of portions of chromosome 18 were fused in an inverted fashion (duplicated for 18qter‐>18p11.3). The smaller der(18) was present in 80% of cells studied (16/20) and had a normal q‐arm, while the p‐arm was missing the subtelomere region but had duplication of a part of 18p. FISH studies showed that the larger derivative 18 contained the 18q subtelomere at each end, but the 18p subtelomere was absent, consistent with fusion of two regions within 18p resulting in deletion of the subtelomeric regions. The smaller der(18) was also missing the 18p subtelomere (with normal 18q as expected). Further testing with BAC clones mapping within 18p11.2 showed that these sequences were duplicated and inverted in both of the der(18)s. These findings lead us to hypothesize that the smaller der(18) was derived from the larger, dicentric 18 following anaphase bridge formation, with breakage distal to the duplicated segment.

The 22q11 low copy repeats are characterized by unprecedented size and structure variability

Low copy repeats (LCRs) are recognized as a significant source of genomic instability, driving genome variability and evolution. The chromosome 22 LCRs (LCR22s) are amongst the most complex regions in the genome and their structure remains unresolved. These LCR22s mediate non-allelic homologous recombination (NAHR) leading to the 22q11 deletion syndrome (22q11DS), causing the most frequent genomic disorder. Using fiber FISH optical mapping, we have de novo assembled the LCR22s in 33 cell lines. We observed a high level of variation in LCR22 structures, including 26 different haplotypes of LCR22A with alleles ranging from 250 Kb to over 2,000 Kb. An additional four haplotypes were detected using Bionano mapping. Further, Bionano maps generated from 154 individuals from different populations suggested significantly different LCR22 haplotype frequencies between populations. Furthermore, haplotype analysis in nine 22q11DS patients resulted in the localization of the NAHR site to a 160 Kb paralog between LCR22A and –D in seven patients and to a 31 Kb region in two individuals with a rearrangement between LCR22A and –B.. This 31 Kb region contains a palindromic AT-rich repeat known to be a driver of chromosomal rearrangements. Our study highlights an unprecedented level of polymorphism in the structure of LCR22s, which are likely still evolving. We present the most comprehensive map of LCR22 variation to date, paving the way towards investigating the role of LCR variation as a driver of 22q11 rearrangements and the phenotypic variability in 22q11DS patients as well as in the general population.