Detection of heterozygous SALL1 deletions by quantitative real time PCR proves the contribution of a SALL1 dosage effect in the pathogenesis of Townes-Brocks syndrome

Townes-Brocks syndrome (TBS) is an autosomal dominantly inherited disorder characterized by ear, anal, limb, and renal malformations, and results from mutations in the gene SALL1. All SALL1 mutations previously found in TBS patients create preterminal termination codons. In accordance with the findings of pericentric inversions or balanced translocations, TBS was initially assumed to be caused by SALL1 haploinsufficiency. This assumption was strongly contradicted by a Sall1 mouse knock-out, because neither hetero- nor homozygous knock-out mutants displayed a TBS-like phenotype. A different mouse mutant mimicking the human SALL1 mutations, however, showed a TBS-like phenotype in the heterozygous situation, suggesting a dominant-negative action of the mutations causing TBS. We applied quantitative real time PCR to detect and map SALL1 deletions in 240 patients with the clinical diagnosis of TBS, who were negative for SALL1 mutations. Deletions were found in three families. In the first family, a 75 kb deletion including all SALL1 exons had been inherited by two siblings from their father. A second, sporadic patient carried a de novo 1.9-2.6 Mb deletion including the whole SALL1 gene, and yet another sporadic case was found to carry an intragenic deletion of 3384 bp. In all affected persons, the TBS phenotype is rather mild as compared to the phenotype resulting from point mutations. These results confirm that SALL1 haploinsufficiency is sufficient to cause a mild TBS phenotype but suggest that it is not sufficient to cause the severe, classical form. It therefore seems that there is a different contribution of SALL1 gene function to mouse and human embryonic development.     

Okihiro syndrome is caused by SALL4 mutations

Okihiro syndrome refers to the association of forearm malformations with Duane syndrome of eye retraction. Based on the reported literature experience, clinical diagnosis of the syndrome can be elusive, owing to the variable presentation in families reported. Specifically, there is overlap of clinical features with other conditions, most notably Holt-Oram syndrome, a condition resulting from mutation of the TBX5 locus and Townes-Brocks syndrome, known to be caused by mutations in the SALL1 gene. Arising from our observation of several malformations in Okihiro syndrome patients which are also described in Townes-Brocks syndrome, we postulated that Okihiro syndrome might result from mutation of another member of the human SALL gene family. We have characterized the human SALL4 gene on chromosome 20q13.13-q13.2. Moreover, we have identified literature reports of forelimb malformations in patients with cytogenetically identifiable abnormalities of this region. We here present evidence in 5 of 8 affected families that mutation at this locus results in the Okihiro syndrome phenotype.     

11p14.1 microdeletions associated with ADHD, autism, developmental delay, and obesity

Genomic copy number imbalances are being increasingly identified as an important cause of intellectual disability and behavioral abnormalities. The typical deletion in WAGR syndrome encompasses the PAX6 and WT1 genes, but larger deletions have been associated with neurobehavioral abnormalities and obesity. We identified four patients with overlapping interstitial deletions on 11p14.1 and extending telomeric to the WAGR critical domain. The minimal overlapping critical chromosomal region was 2.3 Mb at 11p14.1. The deletions encompass the BDNF and LIN7C genes that are implicated in the regulation of development and differentiation of neurons and synaptic transmission. All patients with this deletion exhibit variable degrees of developmental delay, behavioral problems, and obesity. Our data show that ADHD, autism, developmental delay, and obesity are highly associated with deletion involving 11p14.1 and provide additional support for a significant role of BDNF in obesity and neurobehavioral problems.     

Alu-related 5q35 microdeletions in Sotos syndrome

Haploinsufficiency of the NSD1 gene due to 5q35 microdeletions or intragenic mutations causes Sotos syndrome (SoS). In 46 of the 49 Japanese deletion cases, common deletion breakpoints were located at two flanking low copy repeats (LCRs), implying that non-allelic homologous recombination (NAHR) between LCRs is the major mechanism for the common deletion. In the other three cases of atypical deletions, the mechanism(s) of deletions remains unanswered. We characterized the atypical microdeletions using fluorescence in situ hybridization (FISH), quantitative real-time polymerase chain reaction (qPCR), and Southern blot hybridization. All the deletion breakpoints in the three cases were successfully determined at the nucleotide level. Two deletions are 1.07 Mb (SoS19) and 1.23 Mb (SoS109) in size, and another consisted of two deletions with sizes of 28 kb and 0.72 Mb, intervened by an intact 29-kb segment (SoS44). All deletions were smaller than a typical 1.9-Mb common deletion. Alu elements were identified in both deletion breakpoints in SoS19, one of deletion breakpoints in SoS109, and both deletion breakpoints of the proximal 28-kb deletion in SoS44. Alu-mediated NAHR is strongly suggested at least in two of atypical deletions. Finally, qPCR is a very useful method to determine deletion breakpoints even in repeat-related regions.     

A family with Duane anomaly and distal limb abnormalities: a further family with the arthrogryposis-ophthalmoplegia syndrome

A two-generation family is reported in which three members have Duane anomaly and distal limb abnormalities. All three affected have photopic electroretinogram responses that are abnormal or at the lower limit of the normal range with normal scotopic responses. Two affected family members also have hearing loss. The likeliest diagnosis is the syndrome listed as "arthrogryposis-ophthalmoplegia syndrome" on the London Dysmorphology Database or as "arthrogryposis with oculomotor limitation and electroretinal abnormalities" or "oculomelic aplasia" in OMIM [MIM 108145]. In view of the similarities with Okihiro syndrome, a search for mutations within the SALL4 gene was undertaken, but none were identified.     

A new 1p36.13-1p36.12 microdeletion syndrome characterized by learning disability,behavioral abnormalities,and ptosis

Two 1p36 contiguous gene deletion syndromes are known so far: the terminal 1p36 deletion syndrome and a 1p36 deletion syndrome with a critical region located more proximal at 1p36.23-1p36.22. We present even more proximally located overlapping deletions from seven individuals, with the smallest region of overlap comprising 1 Mb at 1p36.13-1p36.12 (chr1:19077793-20081292 (GRCh37/hg19)) defining a new contiguous gene deletion syndrome. The characteristic features of this new syndrome are learning disability or mild intellectual disability, speech delay, behavioral abnormalities, and ptosis. The genes UBR4 and CAPZB are considered the most likely candidate genes for the features of this new syndrome.     

Higher frequency of uncommon 1.5-2 Mb deletions found in familial cases of 22q11.2 deletion syndrome

Familial 22q11.2 deletions have been reported as a 6%-28% of the total affected cases of 22q11.2 microdeletion syndrome (del22q11.2). Different deletion genotypes have been described for this disorder, with a predominant 3 Mb deletion present in 90% of the cases, a less common 1.5-2 Mb deletion in 8%, and atypical smaller deletions in 2%. We have studied 15 cases of del22q11.2 from 6 families (two of them three-generation families) that were previously diagnosed through FISH. We have sized the deleted region by allele genotyping of 12-16 polymorphic markers in all cases, and we have found three families affected with the 1.5-2 Mb deletion, two affected with the 3 Mb deletion, and one in which the deletion size could not be determined. This predominance of the smaller 1.5-2 Mb deletions in our familial cases differs from the minor frequency observed in sporadic cases of del22q11.2. This finding suggests that small deletions are more linked to familial inheritance than large ones, possibly due to psychosocial or biological factors associated with differences in the phenotype. Deletion sizing on routine diagnosis may help characterizing the inheritability of 22q11.2 microdeletion syndrome.     

A 5.3Mb deletion in chromosome 18q12.3 as the smallest region of overlap in two patients with expressive speech delay

Interstitial 18q deletions encompassing band 18q12.3 define the del(18)(q12.2q21.1) syndrome. Usual manifestations are mild dysmorphic features, mental retardation, behaviour abnormalities and lack of serious malformation. Seizures have also been found. Recently, more specifically, impairment of expressive language has been reported. We report on two patients with de novo 18q interstitial deletions characterized by oligonucleotide array CGH. The smallest, a 5.3Mb deletion (35.7-40.9Mb) within band q12.3, was found in a 4-year-old girl who suffered mainly from expressive dysphasia. A larger 9.5Mb deletion (34.6-43.9Mb) was observed in a 20-year-old man with a more severe clinical picture including seizures and limited speech. Among the four genes located in the 5.3Mb region, RIT2 (Ras-like without CAAX 2) and SYT4 (synaptotagmin IV), both strongly expressed in the brain, are pointed out as likely candidate genes for language development.     

Townes-Brocks syndrome: twenty novel SALL1 mutations in sporadic and familial cases and refinement of the SALL1 hot spot region

Townes-Brocks syndrome (TBS) is an autosomal dominant malformation syndrome characterized by renal, anal, ear, and thumb anomalies caused by SALL1 mutations. To date, 36 SALL1 mutations have been described in TBS patients. All but three of those, namely p.R276X, p.S372X, and c.1404dupG, have been found only in single families thereby preventing phenotype-genotype correlations. Here we present 20 novel mutations (12 short deletions, five short duplications, three nonsense mutations) in 20 unrelated families. We delineate the phenotypes and report previously unknown ocular manifestations, i.e. congenital cataracts with unilateral microphthalmia. We show that 46 out of the now 56 SALL1 mutations are located between the coding regions for the glutamine-rich domain mediating SALL protein interactions and 65 bp 3' of the coding region for the first double zinc finger domain, narrowing the SALL1 mutational hotspot region to a stretch of 802 bp within exon 2. Of note, only two SALL1 mutations would result in truncated proteins without the glutamine-rich domain, one of which is reported here. The latter is associated with anal, ear, hand, and renal manifestations, indicating that the glutamine-rich domain is not required for typical TBS.     

Pitt-Rogers-Danks syndrome and Wolf-Hirschhorn syndrome are caused by a deletion in the same region on chromosome 4p 16.3.

Recently, a deletion of chromosome 4pter was found in three patients with Pitt-Rogers-Danks syndrome. We investigated two of these patients, by means of DNA and FISH studies, together with two additional patients with Pitt-Rogers-Danks syndrome, to determine the critical region of the deletion in these patients and to compare this with the critical region in Wolf-Hirschhorn syndrome. All four patients showed terminal deletions of chromosome 4p of different sizes. One of them appeared to have an unbalanced karyotype caused by a cryptic translocation t(4;8) in the mother, resulting in a deletion of chromosome 4pter and a duplication of chromosome 8pter. The localisation of the Wolf-Hirschhorn critical region has been confined to approximately 1 Mb between D4S43 and D4S115. Our study shows that the deletions in four patients with the Pitt-Rogers-Danks syndrome overlap the Wolf-Hirschhorn critical region and extend beyond this in both directions. This study, combined with the fact that our third patient, who was previously described as a Pitt-Rogers-Danks patient, but who now more closely resembles a Wolf-Hirschhorn patient, makes it likely that Pitt-Rogers-Danks and Wolf-Hirschhorn syndromes are different clinical phenotypes resulting from a deletion in the same microscopic region on chromosome 4p16.     

Clinical and molecular delineation of the Greig cephalopolysyndactyly contiguous gene deletion syndrome and its distinction from acrocallosal syndrome

Greig cephalopolysyndactyly syndrome (GCPS) is caused by haploinsufficiency of GLI3 on 7p13. Features of GCPS include polydactyly, macrocephaly, and hypertelorism, and may be associated with cognitive deficits and abnormalities of the corpus callosum. GLI3 mutations in GCPS patients include point, frameshift, translocation, and gross deletion mutations. FISH and STRP analyses were applied to 34 patients with characteristics of GCPS. Deletions were identified in 11 patients and the extent of their deletion was determined. Nine patients with deletions had mental retardation (MR) or developmental delay (DD) and were classified as severe GCPS. These severe GCPS patients have manifestations that overlap with the acrocallosal syndrome (ACLS). The deletion breakpoints were analyzed in six patients whose deletions ranged in size from 151 kb to 10.6 Mb. Junction fragments were found to be distinct with no common sequences flanking the breakpoints. We conclude that patients with GCPS caused by large deletions that include GLI3 are likely to have cognitive deficits, and we hypothesize that this severe GCPS phenotype is caused by deletion of contiguous genes.     

Detection and delineation of an unusual 17p11.2 deletion by array-CGH and refinement of the Smith-Magenis syndrome minimum deletion to approximately 650 kb

Smith-Magenis syndrome (SMS) is a multiple congenital anomaly/mental retardation syndrome and it is characterized by an interstitial deletion of chromosome 17p11.2. SMS patients have a distinct phenotype which is believed to be caused by haploinsufficiency of one or more genes in the associated deleted region. Five non-deletion patients with classical phenotypic features of SMS have been reported with mutations in the retinoic acid induced 1 (RAI1) gene, located within the SMS critical interval. Happloinsufficiency of the RAI1 gene is likely to be the responsible gene for the majority of the SMS features, but other deleted genes in the SMS region may modify the overall phenotype in the patients with 17p11.2 deletions. SMS is usually diagnosed in the clinical genetic setting by FISH analysis using commercially available probes. We detected a submicroscopic deletion in 17p11.2 using array-CGH with a resolution of approximately 1 Mb in a patient with the SMS phenotype, who was not deleted for the commercially available SMS microdeletion FISH probe. Delineation of the deletion was performed using a 32K tiling BAC-array, containing 32,500 BAC clones. The deletion in this patient was size mapped to 2.7 Mb and covered the RAI1 gene. This case enabled the refinement of the SMS minimum deletion to approximately 650 kb containing eight putative genes and one predicted gene. In addition, it demonstrates the importance to investigate deletion of RAI1 in SMS patients.     

Five patients with novel overlapping interstitial deletions in 8q22.2q22.3

High‐resolution microarray technology has facilitated the detection of submicroscopic chromosome aberrations and characterization of new microdeletion syndromes. We present clinical and molecular data of five patients with previously undescribed overlapping interstitial deletions involving 8q22.2q22.3. All deletions differ in size and breakpoints. Patients 1–4 carry deletions between 5.25 and 6.44 Mb in size, resulting in a minimal deletion overlap of 3.87 Mb (from 100.69 to 104.56 Mb; hg18) comprising at least 25 genes. These patients share similar facial dysmorphisms with blepharophimosis, telecanthus, epicanthus, flat malar region, thin upper lip vermillion, down‐turned corners of the mouth, and a poor facial movement/little facial expression. They have a moderate to severe developmental delay (4/4), absent speech (3/4), microcephaly (3/4), a history of seizures (3/4), postnatal short stature (2/4), and a diaphragmatic or hiatal hernia (2/4). Patient 5 was diagnosed with a smaller deletion of about 1.92 Mb (containing nine genes) localized within the deletion overlap of the other four patients. Patient 5 shows a different facial phenotype and a less severe mental retardation. In Patients 1–4, COH1 is involved in the deletion (in total or in part), but none of them showed clinical features of Cohen syndrome. In two patients (Patients 2 and 4), ZFPM2 (also called FOG2, a candidate gene for congenital diaphragmatic hernias) was partly deleted. We suggest that patients with a microdeletion of 8q22.2q22.3 may represent a clinically recognizable condition characterized particularly by the facial phenotype and developmental delay. More patients have to be evaluated to establish a phenotype–genotype correlation. © 2011 Wiley‐Liss, Inc.     

Array comparative genomic hybridization in patients with congenital diaphragmatic hernia: mapping of four CDH-critical regions and sequencing of candidate genes at 15q26.1-15q26.2

Congenital diaphragmatic hernia (CDH) is a common birth defect with a high mortality and morbidity. There have been few studies that have assessed copy number changes in CDH. We present array comparative genomic hybridization data for 29 CDH patients to identify and map chromosome aberrations in this disease. Three patients with 15q26.1-15q26.2 deletions had heterogeneous breakpoints that overlapped with the critical 4 Mb region previously delineated for CDH, confirming 15q26.1-15q26.2 as a critical region for CDH. The three other most compelling CDH-critical regions for genomic deletions based on these data and a literature review are located at chromosomes 8p23.1, 4p16.3-4pter, and 1q41-1q42.1. Based on these recurrent deletions at 15q26.1-15q26.2, we hypothesized that loss-of-function mutations in a gene or genes from this region could cause CDH and sequenced six candidate genes from this region in more than 100 patients with CDH. For three of these genes (CHD2, ARRDC4, and RGMA), we identified missense changes and that were not identified in normal controls; however, none of these alterations appeared unambiguously causal with CDH. These data suggest that CDH caused by chromosome deletions at 15q26.2 may arise because of a contiguous gene deletion syndrome or may have a multifactorial etiology. In addition, there is evidence for substantial genetic heterogeneity in CDH and diaphragmatic hernias can be non-penetrant in patients who have deletions involving CDH-critical regions.     

Identification of novel deletions of 15q11q13 in Angelman syndrome by array-CGH: molecular characterization and genotype-phenotype correlations

Angelman syndrome (AS) is a neurodevelopmental disorder characterized by mental retardation, absent speech, ataxia, and a happy disposition. Deletions of the 15q11q13 region are found in approximately 70% of AS patients. The deletions are sub-classified into class I and class II based on their sizes of approximately 6.8 and approximately 6.0, respectively, with two different proximal breakpoints and a common distal breakpoint. Utilizing a chromosome 15-specific comparative genomic hybridization genomic microarray (array-CGH), we have identified, determined the deletion sizes, and mapped the breakpoints in a cohort of 44 cases, to relate those breakpoints to the genomic architecture and derive more precise genotype-phenotype correlations. Interestingly four patients of the 44 studied (9.1%) had novel and unusually large deletions, and are reported here. This is the first report of very large deletions of 15q11q13 resulting in AS; the largest deletion being >10.6 Mb. These novel deletions involve three different distal breakpoints, two of which have been earlier shown to be involved in the generation of isodicentric 15q chromosomes (idic15). Additionally, precise determination of the deletion breakpoints reveals the presence of directly oriented low-copy repeats (LCRs) flanking the recurrent and novel breakpoints. The LCRs are adequate in size, orientation, and homology to enable abnormal recombination events leading to deletions and duplications. This genomic organization provides evidence for a common mechanism for the generation of both common and rare deletion types. Larger deletions result in a loss of several genes outside the common Angelman syndrome-Prader-Willi syndrome (AS-PWS) critical interval, and a more severe phenotype.