Clinical Genetic Testing for Fragile X Syndrome by Polymerase Chain Reaction Amplification and Southern Blot Analyses.

Fragile X syndrome (FXS) is characterized by mental retardation and in the vast majority of cases it is caused by expansion of CGG trinucleotide repeats in the 5' untranslated region (or UTR) in the FMR1 gene on the X chromosome. The size and methylation status of CGG repeats are correlated with the clinical phenotype of FMR1-related disorders. The methods used for clinical genetic testing of FXS include polymerase chain reaction (PCR) amplification and Southern blot analyses, which effectively detect alleles with repeats in the normal, intermediate, premutation, and full mutation size ranges, as well as the methylation status of FMR1 promoter region.

Robust identification of mosaic variants in congenital heart disease.

Mosaicism due to somatic mutations can cause multiple diseases including cancer, developmental and overgrowth syndromes, neurodevelopmental disorders, autoinflammatory diseases, and atrial fibrillation. With the increased use of next generation sequencing technology, multiple tools have been developed to identify low-frequency variants, specifically from matched tumor-normal tissues in cancer studies. To investigate whether mosaic variants are implicated in congenital heart disease (CHD), we developed a pipeline using the cancer somatic variant caller MuTect to identify mosaic variants in whole-exome sequencing (WES) data from a cohort of parent/affected child trios (n = 715) and a cohort of healthy individuals (n = 416). This is a novel application of the somatic variant caller designed for cancer to WES trio data. We identified two cases with mosaic KMT2D mutations that are likely pathogenic for CHD, but conclude that, overall, mosaicism detectable in peripheral blood or saliva does not account for a significant portion of CHD etiology.

Evaluation of the Affymetrix CytoScan(®) Dx Assay for developmental delay.

The goal of molecular cytogenetic testing for children presenting with developmental delay (DD) is to identify or exclude genetic abnormalities that are associated with cognitive, behavioral and/or motor symptoms. Until 2010, chromosome analysis was the standard first-line genetic screening test for evaluation of patients with DD when a specific syndrome was not suspected. In 2010, The American College of Medical Genetics and several other groups recommended chromosomal microarray as the first-line test in children with DDs, multiple congenital anomalies and/or autism. This test is able to detect regions of genomic imbalances at a much finer resolution than G-banded karyotyping. Until recently, no chromosomal microarray testing had been approved by the US FDA. This article focuses on the use of the Affymetrix CytoScan(®) Dx Assay (Santa Clara, CA, USA), the first chromosomal microarray to receive FDA approval for the genetic evaluation of individuals with DD.

Phelan-McDermid syndrome: a review of the literature and practice parameters for medical assessment and monitoring.

Autism spectrum disorder (ASD) and intellectual disability (ID) can be caused by mutations in a large number of genes. One example is SHANK3 on the terminal end of chromosome 22q. Loss of one functional copy of SHANK3 results in 22q13 deletion syndrome or Phelan-McDermid syndrome (PMS) and causes a monogenic form of ASD and/or ID with a frequency of 0.5% to 2% of cases. SHANK3 is the critical gene in this syndrome, and its loss results in disruption of synaptic function. With chromosomal microarray analyses now a standard of care in the assessment of ASD and developmental delay, and with the emergence of whole exome and whole genome sequencing in this context, identification of PMS in routine clinical settings will increase significantly. However, PMS remains a rare disorder, and the majority of physicians have never seen a case. While there is agreement about core deficits of PMS, there have been no established parameters to guide evaluation and medical monitoring of the syndrome. Evaluations must include a thorough history and physical and dysmorphology examination. Neurological deficits, including the presence of seizures and structural brain abnormalities should be assessed as well as motor deficits. Endocrine, renal, cardiac, and gastrointestinal problems all require assessment and monitoring in addition to the risk of recurring infections, dental and vision problems, and lymphedema. Finally, all patients should have cognitive, behavioral, and ASD evaluations. The objective of this paper is to address this gap in the literature and establish recommendations to assess the medical, genetic, and neurological features of PMS.

Role of copy number variants in structural birth defects.

BACKGROUND AND OBJECTIVE: Human genomes include copy number variants (CNVs), defined as regions with DNA gains or losses. Pathologic CNVs, which are larger and often occur de novo, are increasingly associated with disease. Given advances in genetic testing, namely microarray-based comparative genomic hybridization and single nucleotide polymorphism arrays, previously unidentified genotypic aberrations can now be correlated with phenotypic anomalies. The objective of this study was to conduct a nonsystematic literature review to document the role of CNVs as they relate to isolated structural anomalies of the craniofacial, respiratory, renal, and cardiac systems. METHODS: All full-length articles in the PubMed database through May 2011 that discussed CNVs and isolated structural defects of the craniofacial, respiratory, renal, and cardiac systems were considered. Search terms queried include CNV, copy number variation, array comparative genomic hybridization, birth defects, craniofacial defects, respiratory defects, renal defects, and congenital heart disease. Reports published in languages other than English and articles regarding CNVs and neurocognitive deficits were not considered. RESULTS: Evidence supports that putatively pathogenic CNVs occur at an increased frequency in patients with isolated structural birth defects and implicate specific regions of the genome. Through CNV detection, advances have been made in identifying genes and specific loci that underlie isolated birth defects. CONCLUSIONS: Although limited studies have been published, the promising evidence reviewed here warrants the continued investigation of CNVs in children with isolated structural birth defects. Patient care and genetic counseling stand to improve through a better understanding of CNVs and their effect on disease phenotype.

Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome.

The generation of reprogrammed induced pluripotent stem cells (iPSCs) from patients with defined genetic disorders holds the promise of increased understanding of the aetiologies of complex diseases and may also facilitate the development of novel therapeutic interventions. We have generated iPSCs from patients with LEOPARD syndrome (an acronym formed from its main features; that is, lentigines, electrocardiographic abnormalities, ocular hypertelorism, pulmonary valve stenosis, abnormal genitalia, retardation of growth and deafness), an autosomal-dominant developmental disorder belonging to a relatively prevalent class of inherited RAS-mitogen-activated protein kinase signalling diseases, which also includes Noonan syndrome, with pleomorphic effects on several tissues and organ systems. The patient-derived cells have a mutation in the PTPN11 gene, which encodes the SHP2 phosphatase. The iPSCs have been extensively characterized and produce multiple differentiated cell lineages. A major disease phenotype in patients with LEOPARD syndrome is hypertrophic cardiomyopathy. We show that in vitro-derived cardiomyocytes from LEOPARD syndrome iPSCs are larger, have a higher degree of sarcomeric organization and preferential localization of NFATC4 in the nucleus when compared with cardiomyocytes derived from human embryonic stem cells or wild-type iPSCs derived from a healthy brother of one of the LEOPARD syndrome patients. These features correlate with a potential hypertrophic state. We also provide molecular insights into signalling pathways that may promote the disease phenotype.