Anna Capalbo

Confirmation of chromosomal microarray as a first-tier clinical diagnostic test for individuals with developmental delay, intellectual disability, autism spectrum disorders and dysmorphic features☆☆☆

BACKGROUND AND OBJECTIVES Submicroscopic chromosomal rearrangements are the most common identifiable causes of intellectual disability and autism spectrum disorders associated with dysmorphic features. Chromosomal microarray (CMA) can detect copy number variants <1 Mb and identifies size and presence of known genes. The aim of this study was to demonstrate the usefulness of CMA, as a first-tier tool in detecting the etiology of unexplained intellectual disability/autism spectrum disorders (ID/ASDs) associated with dysmorphic features in a large cohort of pediatric patients. PATIENTS AND METHODS We studied 349 individuals; 223 males, 126 females, aged 5 months-19 years. Blood samples were analyzed with CMA at a resolution ranging from 1 Mb to 40 Kb. The imbalance was confirmed by FISH or qPCR. We considered copy number variants (CNVs) causative if the variant was responsible for a known syndrome, encompassed gene/s of known function, occurred de novo or, if inherited, the parent was variably affected, and/or the involved gene/s had been reported in association with ID/ASDs in dedicated databases. RESULTS 91 CNVs were detected in 77 (22.06%) patients: 5 (6.49%) of those presenting with borderline cognitive impairment, 54 (70.13%) with a variable degree of DD/ID, and 18/77 (23.38%) with ID of variable degree and ASDs. 16/77 (20.8%) patients had two different rearrangements. Deletions exceeded duplications (58 versus 33); 45.05% (41/91) of the detected CNVs were de novo, 45.05% (41/91) inherited, and 9.9% (9/91) unknown. The CNVs caused the phenotype in 57/77 (74%) patients; 12/57 (21.05%) had ASDs/ID, and 45/57 (78.95%) had DD/ID. CONCLUSIONS Our study provides further evidence of the high diagnostic yield of CMA for genetic testing in children with unexplained ID/ASDs who had dysmorphic features. We confirm the value of CMA as the first-tier tool in the assessment of those conditions in the pediatric setting.

Array-based comparative genomic hybridization in early-stage mycosis fungoides: recurrent deletion of tumor suppressor genes BCL7A, SMAC/DIABLO, and RHOF.

The etiology of mycosis fungoides (MF), the most frequent form of cutaneous T cell lymphoma (CTCL), is poorly understood. No specific genetic aberration has been detected, especially in early‐stage disease, possibly due to the clinical and histological heterogeneity of patient series and to the different sources of malignant cells (skin, blood, or lymph node) included in most studies. Frozen skin biopsies from 16 patients with early‐stage MF were studied using array‐based comparative genomic hybridization. A DNA pool from healthy donors was used as the reference. Results demonstrated recurrent loss of 19, 7p22.1‐p22.3, 7q11.1‐q11.23, 9q34.12, 12q24.31, and 16q22.3‐q23.1, and gain of 8q22.3‐q23.1 and 21q22.12. The 12q24.31 region was recurrently deleted in 7/16 patients. Real‐time PCR investigation for deletion of genes BCL7A, SMAC/DIABLO, and RHOF—three tumor suppressor genes with a putative role in hematological malignancies—demonstrated that they were deleted in 9, 10, and 13 cases, respectively. The identified genomic alterations and individual genes could yield important insights into the early steps of MF pathogenesis.

Syndromic craniosynostosis due to complex chromosome 5 rearrangement and MSX2 gene triplication

Craniosynostosis is a common birth defect (∼1/3,000 births) resulting from chromosome imbalances, gene mutations or unknown causes. We report a 6‐month‐old female with multiple sutural synostosis and prenatal onset growth deficiency, developmental delay, facial dysmorphism, congenital heart defect, and inguinal hernia. An integrated approach of standard cytogenetics, mBAND, locus‐specific FISH, and 75 kb resolution array‐CGH disclosed a complex chromosome 5 rearrangement, resulting in 3 paracentric inversions, 2 between‐arm insertions, and partial duplication of 5q35. An extra copy of the MSX2 gene, which maps within the duplicated segment and is mutated in Boston‐type craniosynostosis, was confirmed by molecular cytogenetic studies. Our study confirms that early fusion of cranial sutures commonly observed in the dup(5q) syndrome is caused by triplication of the MSX2 gene and strongly supports the crucial role of this gene in the development of craniofacial structures.

2q31.2q32.3 Deletion Syndrome : Report of an Adult Patient

A 36‐year‐old patient with a disorder characterized by severe mental retardation, behavioral problems, dysmorphic face, “muscular build,” and hand/foot anomalies, is reported. Following a diagnosis of de novo pericentric inversion of chromosome 8 based on standard cytogenetic analysis, a subsequent 75 kb array‐CGH investigation disclosed a deletion spanning for about 13.7 Mb in the 2q31.2q32.3 region. Whole painting of chromosome 8 established the intrachromosomal nature of the rearrangement and FISH analysis with locus‐specific probes confirmed the deletion on the long arm of chromosome 2. The deleted region, clinical outcome, and medical history in this patient are mainly superimposable to those reported in a published 8‐year‐old boy, suggesting that this genomic segment is prone to rearrangements and its hemizygosity gives rise to a clinically recognizable syndrome. The role of some genes mapping in the deleted region and related with distinct disorders is discussed. Interestingly, deletion of MSTN gene, a negative regulator of muscle growth, was associated in our patient with a “muscular build,” a feature which could be regarded as a handle for clinical recognition of this syndrome.

3q29 Microdeletion: a mental retardation disorder unassociated with a recognizable phenotype in two mother-daughter pairs.

The 3q29 microdeletion syndrome (del 3q29) is a novel genomic disorder identified after the introduction of microarray‐based technology. The phenotype of the reported patients is variable, including mental retardation and subtle facial anomalies. We report on two mother–daughter pairs, heterozygous for 3q29, and review clinical features of all known affected individuals. Del 3q29 syndrome is associated with nonspecific clinical features, including mild‐to‐moderate developmental delay, microcephaly, and mild facial dysmorphisms such as short philtrum and high nasal bridge. Facial anomalies were nonoverlapping and nondistinct, also within each mother–daughter pair. Parental transmission of del 3q29 could be more frequent than previously considered. Malformations are rare, occurring only in single subjects. The phenotypic diversity of affected patients and the lack of distinct dysmorphisms suggest that this disorder cannot be recognized on clinical ground alone. Del 3q29 should be searched in subjects with unexplained mild/moderate mental retardation, microcephaly, and minor nonspecific facial anomalies.

Deletion 2p15-16.1 syndrome: case report and review.

We report on a 9‐year‐old female patient with facial anomalies and developmental delay, heterozygous for three de novo rearrangements: a paracentric inversion of chromosome 7, an apparently balanced translocation between chromosome 1 and 7, involving the same inverted chromosome 7, detected by standard cytogenetic analysis [46,XX, der(7) inv(7)(q21.1q32.1)t(1;7)(q23q32.1)]; and a 2p16.1 deletion, spanning about 3.5 Mb of genomic DNA, shown by SNP‐array analysis [arr 2p16.1 (56,706,666–60,234,485)x1 dn]. Clinical features and cytogenetic imbalance in our patient were similar to those reported in five published cases, suggesting that this genomic region is prone to recombination and its hemizygosity results in a distinct although variable spectrum of clinical manifestations.

De-novo 2.15 Mb terminal Xq duplication involving MECP2 but not L1CAM gene in a male patient with mental retardation.

Distal Xq disomy in males results in characteristic phenotypes that typically include mental retardation, microcephaly, prominent hypotonia and hypogonadism. The 8-year-old male patient reported here presented with mental retardation, prominent ears, abnormally wide and unstable gait and flat occiput. He did not have microcephaly or hypogonadism. Subtelomeric multi-fluorescence in-situ hybridization analysis identified a duplicated terminal portion of chromosome Xq/Yq located distally on Yp. Further analysis of the duplicated region using additional FISH probes, specific for the distal Xq and Yp chromosomal regions, and array comparative genomic hybridization analysis using the 244 K oligo-array of Agilent, showed that it spans approximately 2.15 Mb of the terminal Xq region and includes MECP2 but not L1CAM gene. This is the smallest well-characterized terminal Xq duplication reported to date. Genes proximal to MECP2 that are not duplicated in our patient are likely responsible for additional clinical manifestations including characteristic facial dysmorphic features, microcephaly, hypogonadism and more severe hypotonia, as noted in patients with larger distal Xq duplications. Our patients features are similar to previously reported MECP2 gene duplication cases, thus suggesting minor or no contribution of duplicated genes distal of MECP2 to the reported phenotype.

HDR (Hypoparathyroidism, Deafness, Renal dysplasia) syndrome associated to GATA3 gene duplication

To the Editor: GATA3haploinsufficiencydue to loss-of-function mutations or deletions is the only known pathogenic mechanism causing HDR (Hypoparathyroidism, sensorineural Deafness, Renal dysplasia) triad (OMIM #146255) (1, 2). Mutations in regulatory sequences and genetic heterogeneity could explain the small subset of HDR syndrome patients negative to GATA3 screening (1–3). We report on a girl presenting HDR triad associated with psychomotor delay, facial dysmorphisms, bilateral cleft lip and palate, heart defect and abnormal fingers and toes, resulting from a complex rearrangement of 10p15p14 region. She was the firstborn of healthy non-consanguineous parents. Pregnancy was complicated by growth retardation. A prenatal ultrasound scan at 20 weeks detected unilateral renal hypoplasia. Amniocentesis disclosed a 46,XX karyotype. She was born by cesarean section at 36 weeks of gestation. Birth weight was 2490 g (3rd centile), length 49 cm (50th centile), and head circumference 32.5 cm (,3rd centile). Apgar scores were 9 and 10 at 1 and 5 min. Mild hypocalcemia (7.5 mg/dl; normal range: 8.8–10.8 mg/dl) was diagnosed on the third day of life. Clinical examination disclosed high-arched and sparse eyebrows, synophrys, hypertelorism, upslanting palpebral fissures, bilateral cleft lip and palate (Fig. 1a), tapering fingers, andmalposed toeswith cutaneous syndactyly of second and third toes. Cerebral ultrasound revealed bilateral subependymal hemorrhage and asymmetric lateral ventricles; echocardiography showed Tetralogy of Fallot; renal ultrasound confirmed left kidney hypodysplasia with hyperechogenic parenchyma and cortical microcysts and right pyelic ectasia; cystoureterography demonstrated second-grade left vesicoureteral reflux; acoustic otoemission testing disclosed bilateral deafness. At 14 months, herweightwas 11.9 kg (90th centile), height 81 cm (90th–97th centile) and head circumference 45.5 cm (25th centile). Developmental milestones were mildly delayed. Subtelomeric regions fluorescence in situ hybridization (FISH) analysis (ToTelVysion Kit; Abbott Molecular, Abbott Park, IL) disclosed 10pter de novo deletion, which was further characterized by array-comparative genomic hybridization (44K Chip; Agilent Technologies, Waldbronn, Germany). This analysis confirmed 10p15.3p15.1 deletionspanningabout6.5 Mb, fromA_14_P134493 (138,206 bp) toA_14_P107348 (6,561,124 bp) probes, and detected 10p15.1p14 duplication spanning 1.9 Mb, from A_14_P107636 (6,639,966 bp) to A_14_P124746 (8,457,497 bp), based on May 2004 release mapping data (hg17; http://www. ensembl.org) (Fig. 1b). Both were de novo imbalances not listed among copy number polymorphisms (http://projects.tcag.ca/variation/). Break points were confirmed by dual-color FISH, using BAC clones (32K Library; BACPAC Resources, Oakland, CA), and real-time polymerase chain reaction (PCR) assays (Fig. 2). The rearrangement likely originated by the breakage–fusion–bridge step (4), but no evident genomic architectural feature possibly underlying this mechanism mapped nearby the break points. The duplicated segment included GATA3 and 1.5 Mb upstream and 0.3 Mb downstream of this gene (Fig. 2b). Real-time PCR assay confirmed a twofold increase in theGATA3 copynumber comparedwithparents’ and threehealthycontrols’DNAs (datanot shown). Complementary DNA direct sequencing did not disclose any alteration in GATA3 sequence. The present observation suggests that both GATA3 deletion and duplication could lead to a similar phenotype. This mechanism has been demonstrated for other transcription factors involved in developmental control processes, in agreement with the balance hypothesis’ (5–12). Indeed, several studies in mouse have shown a direct relationship between organogenesis and GATA3 expression levels with a temporal and

Microduplications in 22q11.2 and 8q22.1 associated with mild mental retardation and generalized overgrowth

The 22q11.2 microduplication is a genomic disorder, characterized from a variable phenotype ranging from different defects to normality. The most common microduplication of 22q11.2 is 3 Mb in size, but there are also cases reported with atypical duplications between 0.8 Mb and 6Mb. Here, we describe a case of a child with macrocephaly, overgrowth with advanced bone age, attention deficits, evidence of mild mental retardation and dysmorphic features. An array-CGH analysis detected a 252 Kb duplication at the 22q11.2 region inherited from mother and 142 Kb duplication at 8q22.1 region inherited from father. Both parents show mild dysmorphic features. The duplicated genes in chromosomes 22q and 8q are TOP3B and PGCP, respectively. We describe for the first time a patient carrying the smaller atypical 22q11.2 duplication who also presents with mild mental retardation and generalized overgrowth. This patient has an additional duplication in 8q22.1 which may act as a genomic modifier of its clinical phenotype.

Dandy-Walker malformation and Wisconsin syndrome: novel cases add further insight into the genotype-phenotype correlations of 3q23q25 deletions

BackgroundThe Dandy-Walker malformation (DWM) is one of the commonest congenital cerebellar defects, and can be associated with multiple congenital anomalies and chromosomal syndromes. The occurrence of overlapping 3q deletions including the ZIC1 and ZIC4 genes in few patients, along with data from mouse models, have implicated both genes in the pathogenesis of DWM.Methods and resultsUsing a SNP-array approach, we recently identified three novel patients carrying heterozygous 3q deletions encompassing ZIC1 and ZIC4. Magnetic resonance imaging showed that only two had a typical DWM, while the third did not present any defect of the DWM spectrum. SNP-array analysis in further eleven children diagnosed with DWM failed to identify deletions of ZIC1-ZIC4. The clinical phenotype of the three 3q deleted patients included multiple congenital anomalies and peculiar facial appearance, related to the localization and extension of each deletion. In particular, phenotypes resulted from the variable combination of three recognizable patterns: DWM (with incomplete penetrance); blepharophimosis, ptosis, and epicanthus inversus syndrome; and Wisconsin syndrome (WS), recently mapped to 3q.ConclusionsOur data indicate that the 3q deletion is a rare defect associated with DWM, and suggest that the hemizygosity of ZIC1-ZIC4 genes is neither necessary nor sufficient per se to cause this condition. Furthermore, based on a detailed comparison of clinical features and molecular data from 3q deleted patients, we propose clinical diagnostic criteria and refine the critical region for WS.