Laura Bernardini

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.

Recurrent microdeletion at 17q12 as a cause of Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome: two case reports

BackgroundMayer-Rokitansky-Kuster-Hauser syndrome (MRKH) consists of congenital aplasia of the uterus and the upper part of vagina due to anomalous development of Müllerian ducts, either isolated or associated with other congenital malformations, including renal, skeletal, hearing and heart defects. This disorder has an incidence of approximately 1 in 4500 newborn girls and the aetiology is poorly understood.Methods and Resultswe report on two patients affected by MRKH syndrome in which array-CGH analysis disclosed an identical deletion spanning 1.5 Mb of genomic DNA at chromosome 17q12. One patient was affected by complete absence of uterus and vagina, with bilaterally normal ovaries, while the other displayed agenesis of the upper part of vagina, right unicornuate uterus, non cavitating rudimentary left horn and bilaterally multicystic kidneys. The deletion encompassed two candidate genes, TCF2 and LHX1. Mutational screening of these genes in a selected group of 20 MRKH females without 17q12 deletion was negative.ConclusionDeletion 17q12 is a rare albeit recurrent anomaly mediated by segmental duplications, previously reported in subjects with developmental kidney abnormalities and diabetes. The present two patients expand the clinical spectrum associated with this imbalance and suggest that this region is a candidate locus for a subset of MRKH syndrome individuals, with or without renal defects.

High-resolution SNP arrays in mental retardation diagnostics: how much do we gain?

We used Affymetrix 6.0 GeneChip SNP arrays to characterize copy number variations (CNVs) in a cohort of 70 patients previously characterized on lower-density oligonucleotide arrays affected by idiopathic mental retardation and dysmorphic features. The SNP array platform includes ∼900 000 SNP probes and 900 000 non-SNP oligonucleotide probes at an average distance of 0.7 Kb, which facilitates coverage of the whole genome, including coding and noncoding regions. The high density of probes is critical for detecting small CNVs, but it can lead to data interpretation problems. To reduce the number of false positives, parameters were set to consider only imbalances >75 Kb encompassing at least 80 probe sets. The higher resolution of the SNP array platform confirmed the increased ability to detect small CNVs, although more than 80% of these CNVs overlapped to copy number ‘neutral’ polymorphism regions and 4.4% of them did not contain known genes. In our cohort of 70 patients, of the 51 previously evaluated as ‘normal’ on the Agilent 44K array, the SNP array platform disclosed six additional CNV changes, including three in three patients, which may be pathogenic. This suggests that about 6% of individuals classified as ‘normal’ using the lower-density oligonucleotide array could be found to be affected by a genomic disorder when evaluated with the higher-density microarray platforms.

Deletions of NF1 gene and exons detected by multiplex ligation-dependent probe amplification

To estimate the contribution of single and multi-exon NF1 gene copy-number changes to the NF1 mutation spectrum, we analysed a series of 201 Italian patients with neurofibromatosis type 1 (NF1). Of these, 138 had previously been found, using denaturing high-performance liquid chromatography or protein truncation test, to be heterozygous for intragenic NF1 point mutations/deletions/insertions, and were excluded from this analysis. The remaining 63 patients were analysed using multiplex ligation-dependent probe amplification (MLPA), which allows detection of deletions or duplications encompassing ⩾1 NF1 exons, as well as entire gene deletions. MLPA results were validated using real-time quantitative PCR (qPCR) or fluorescent in situ hybridisation. MLPA screening followed by real-time qPCR detected a total of 23 deletions. Of these deletions, six were single exon, eight were multi-exon, and nine were of the entire NF1 gene. In our series, deletions encompassing ⩾1 NF1 exons accounted for ∼7% (14/201) of the NF1 gene mutation spectrum, suggesting that screening for these should now be systematically included in genetic testing of patients with NF1.

Spectrum of epilepsy in terminal 1p36 deletion syndrome

Purpose: Previous reports have summarized the seizures types occurring in 1p36 deletion syndrome. To better define the spectrum of epilepsy, we studied 91 patients (median age 7.8 years) with confirmed 1p36 deletion.

Mutations in ZBTB20 cause Primrose syndrome

Primrose syndrome and 3q13.31 microdeletion syndrome are clinically related disorders characterized by tall stature, macrocephaly, intellectual disability, disturbed behavior and unusual facial features, with diabetes, deafness, progressive muscle wasting and ectopic calcifications specifically occurring in the former. We report that missense mutations in ZBTB20, residing within the 3q13.31 microdeletion syndrome critical region, underlie Primrose syndrome. This finding establishes a genetic link between these disorders and delineates the impact of ZBTB20 dysregulation on development, growth and metabolism.

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.

Nablus mask‐like facial syndrome is caused by a microdeletion of 8q detected by array‐based comparative genomic hybridization

In 2000, Teebi reported on a 4‐year‐old boy with a distinctive pattern of malformation, which he termed the “Nablus mask‐like facial syndrome” (OMIM# 608156). Characterization of this syndrome has been difficult because of the paucity of patients described in the medical literature and its unknown etiology and pathogenesis. We present two patients with Nablus mask‐like facial syndrome who both display a microdeletion in the 8q21‐8q22 region detected by array‐based comparative genomic hybridization. Patient 1, a boy, has a distinct facial appearance characterized by severe blepharophimosis, tight‐appearing glistening facial skin, sparse and unruly hair, a flat and broad nose, and distinctive ears that are triangular in shape with prominent antihelices. He also demonstrates camptodactyly, contractures, unusual dentition, cryptorchidism, mild developmental delay, and a happy demeanor. Patient 2, a girl with a strikingly similar phenotype, was previously described in a report by Salpietro et al. 2003 . She has distinctive ears, dental anomalies, and developmental delay. The etiology of her pattern of malformation was not identified at that time. Although high‐resolution chromosome and subtelomeric FISH analyses were normal, array‐based comparative genomic hybridization revealed an approximately 4 Mb deletion involving the 8q21.3‐8q22.1 region in both patients. This region encompasses a number of genes that may contribute to this unique phenotype. These results demonstrate a chromosomal microdeletion as the etiology of Nablus mask‐like facial syndrome and emphasize the diagnostic utility of array‐based comparative genomic hybridization in the evaluation of multiple malformation syndromes of previously unrecognized causation.