Darrel Waggoner

Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies.

Chromosomal microarray (CMA) is increasingly utilized for genetic testing of individuals with unexplained developmental delay/intellectual disability (DD/ID), autism spectrum disorders (ASD), or multiple congenital anomalies (MCA). Performing CMA and G-banded karyotyping on every patient substantially increases the total cost of genetic testing. The International Standard Cytogenomic Array (ISCA) Consortium held two international workshops and conducted a literature review of 33 studies, including 21,698 patients tested by CMA. We provide an evidence-based summary of clinical cytogenetic testing comparing CMA to G-banded karyotyping with respect to technical advantages and limitations, diagnostic yield for various types of chromosomal aberrations, and issues that affect test interpretation. CMA offers a much higher diagnostic yield (15%-20%) for genetic testing of individuals with unexplained DD/ID, ASD, or MCA than a G-banded karyotype ( approximately 3%, excluding Down syndrome and other recognizable chromosomal syndromes), primarily because of its higher sensitivity for submicroscopic deletions and duplications. Truly balanced rearrangements and low-level mosaicism are generally not detectable by arrays, but these are relatively infrequent causes of abnormal phenotypes in this population (<1%). Available evidence strongly supports the use of CMA in place of G-banded karyotyping as the first-tier cytogenetic diagnostic test for patients with DD/ID, ASD, or MCA. G-banded karyotype analysis should be reserved for patients with obvious chromosomal syndromes (e.g., Down syndrome), a family history of chromosomal rearrangement, or a history of multiple miscarriages.

THE COPPER CHAPERONE CCS DIRECTLY INTERACTS WITH COPPER/ZINC SUPEROXIDE DISMUTASE

Dominantly inherited mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) result in the fatal motor neuron disease familial amyotrophic lateral sclerosis (FALS). These mutations confer a gain-of-function to SOD1 with neuronal degeneration resulting from enhanced free radical generating activity of the copper present in the mutant enzyme. The delivery of copper to SOD1 is mediated through a soluble factor identified as the copper chaperone for SOD1 (CCS). Amino acid sequence alignment of SOD1 and CCS reveals a striking homology with conservation of the amino acids essential for mediating SOD1 homodimerization. Here we demonstrate that CCS and SOD1 directly interact in vitro and in vivo and that this interaction is mediated via the homologous domains in each protein. Importantly, CCS interacts not only with wild-type SOD1 but also with SOD1 containing the common missense mutations resulting in FALS. Our findings therefore reveal a common mechanism whereby different SOD1 FALS mutants may result in neuronal injury and suggest a novel therapeutic approach in patients affected by this fatal disease.

TBX5 genetic testing validates strict clinical criteria for Holt-Oram syndrome

Holt-Oram syndrome (HOS) is an autosomal dominant heart-hand syndrome characterized by congenital heart disease (CHD) and upper limb deformity, and caused by mutations in the TBX5 gene. To date, the sensitivity of TBX5 genetic testing for HOS has been unclear. We now report mutational analyses of a nongenetically selected population of 54 unrelated individuals who were consecutively referred to our center with a clinical diagnosis of HOS. TBX5 mutational analyses were performed in all individuals, and clinical histories and findings were reviewed for each patient without reference to the genotypes. Twenty-six percent of the complete cohort was shown to have mutations of the TBX5 gene. However, among those subjects for whom clinical review demonstrated that their presentations met strict diagnostic criteria for HOS, TBX5 mutations were identified in 74%. No mutations were identified in those subjects who did not meet these criteria. Thus, these studies validate our clinical diagnostic criteria for HOS including an absolute requirement for preaxial radial ray upper limb malformation. Accordingly, TBX5 genotyping has high sensitivity and specificity for HOS if stringent diagnostic criteria are used in assigning the clinical diagnosis.

Clinic-based study of plexiform neurofibromas in neurofibromatosis 1

Individuals with neurofibromatosis 1 (NF1) develop both benign and malignant tumors at an increased frequency. One of the most common benign tumors in NF1 is the plexiform neurofibroma. These tumors cause significant morbidity and mortality on account of their propensity to grow and affect adjacent normal tissues. To determine the clinical profile of plexiform neurofibromas in NF1, we conducted a retrospective review of 68 NF1 patients with plexiform neurofibroma. In our series, 44% of tumors were detected by 5 years of age and most were located in the trunk and extremities. Only two patients developed malignant peripheral nerve sheath tumors in their preexisting plexiform neurofibromas. Lastly, we demonstrate that there were no specific clinical features of NF1 associated with the presence of plexiform neurofibroma. These results underscore the importance of careful serial examinations in the evaluation of patients with NF1.

Exome sequencing reveals a novel mutation for autosomal recessive non-syndromic mental retardation in the TECR gene on chromosome 19p13

Exome sequencing is a powerful tool for discovery of the Mendelian disease genes. Previously, we reported a novel locus for autosomal recessive non-syndromic mental retardation (NSMR) in a consanguineous family [Nolan, D.K., Chen, P., Das, S., Ober, C. and Waggoner, D. (2008) Fine mapping of a locus for nonsyndromic mental retardation on chromosome 19p13. Am. J. Med. Genet. A, 146A, 1414-1422]. Using linkage and homozygosity mapping, we previously localized the gene to chromosome 19p13. The parents of this sibship were recently included in an exome sequencing project. Using a series of filters, we narrowed the putative causal mutation to a single variant site that segregated with NSMR: the mutation was homozygous in five affected siblings but in none of eight unaffected siblings. This mutation causes a substitution of a leucine for a highly conserved proline at amino acid 182 in TECR (trans-2,3-enoyl-CoA reductase), a synaptic glycoprotein. Our results reveal the value of massively parallel sequencing for identification of novel disease genes that could not be found using traditional approaches and identifies only the seventh causal mutation for autosomal recessive NSMR.

“Molecular rulers” for calibrating phenotypic effects of telomere imbalance

As a result of the increasing use of genome wide telomere screening, it has become evident that a significant proportion of people with idiopathic mental retardation have subtle abnormalities involving the telomeres of human chromosomes. However, during the course of these studies, there have also been telomeric imbalances identified in normal people that are not associated with any apparent phenotype. We have begun to scrutinise cases from both of these groups by determining the extent of the duplication or deletion associated with the imbalance. Five cases were examined where the telomere rearrangement resulted in trisomy for the 16p telomere. The size of the trisomic segment ranged from ∼4-7 Mb and the phenotype included mental and growth retardation, brain malformations, heart defects, cleft palate, pancreatic insufficiency, genitourinary abnormalities, and dysmorphic features. Three cases with telomeric deletions without apparent phenotypic effects were also examined, one from 10q and two from 17p. All three deletions were inherited from a phenotypically normal parent carrying the same deletion, thus without apparent phenotypic effect. The largest deletion among these cases was ∼600 kb on 17p. Similar studies are necessary for all telomeric regions to differentiate between those telomeric rearrangements that are pathogenic and those that are benign variants. Towards this goal, we are developing “molecular rulers” that incorporate multiple clones at each telomere that span the most distal 5 Mb region. While telomere screening has enabled the identification of telomere rearrangements, the use of molecular rulers will allow better phenotype prediction and prognosis related to these findings.

Sarcomere Mutations in Cardiomyopathy with Left Ventricular Hypertrabeculation

Background— Mutations in the genes encoding sarcomere proteins have been associated with both hypertrophic and dilated cardiomyopathy. Recently, mutations in myosin heavy chain (MYH7), cardiac actin (ACTC), and troponin T (TNNT2) were associated with left ventricular noncompaction, a form of cardiomyopathy characterized with hypertrabeculation that may also include reduced function of the left ventricle. Methods and Results— We used clinically available genetic testing on 3 cases referred for evaluation of left ventricular dysfunction and noncompaction of the left ventricle and found that all 3 individuals carried sarcomere mutations. The first patient presented with neonatal heart failure and was referred for left ventricular noncompaction cardiomyopathy. Genetic testing found 2 different mutations in MYBPC3 in trans. The first mutation, 3776delA, Q1259fs, rendered a frame shift at 1259 of cardiac myosin-binding protein C and the second mutation was L1200P. The frameshift mutation was also found in this mother who displayed mild echocardiographic features of cardiomyopathy, with only subtle increase in trabeculation and an absence of hypertrophy. A second pediatric patient presented with heart failure and was found to carry a de novo MYH7 R369Q mutation. The third case was an adult patient with dilated cardiomyopathy referred for ventricular hypertrabeculation. This patient had a family history of congestive heart failure, including pediatric onset cardiomyopathy where 3 individuals in the family were found to have the MYH7 mutation R1250W. Conclusion— Genetic testing should be considered for cardiomyopathy with hypertrabeculation.

Calibration of 6q subtelomere deletions to define genotype/phenotype correlations

Testing for subtelomere abnormalities in patients with idiopathic mental retardation has become a useful diagnostic tool. However, limited data exist regarding genotype/phenotype correlations for specific subtelomere imbalances. We have ascertained five patients with 6q subtelomere deletions either as a result of an isolated deletion or as a result of an unbalanced translocation, and developed a molecular ruler assay utilizing BAC or PAC clones and determined the size of the deleted regions to range from <0.5 to 8 Mb. To establish genotype/phenotype correlations for distal 6q, we compared the clinical features of these patients to previously reported cases of 6q subtelomere and cytogenetically visible deletions and found that they shared multiple abnormalities, suggesting that the causative genes may lie in the region of the smallest 6q subtelomeric deletion, approximately 400 kb from the telomere. However, multiple unique features were present only in patients with cytogenetically visible 6q deletions, indicative that genes involved in the development of these features may lie more proximally on 6q. These initial studies demonstrate the ability to develop genotype/phenotype correlations for subtelomere rearrangements, which will aid in the diagnosis and prognosis of these patients and may help narrow the search for relevant developmental genes.

Recessive TRAPPC11 Mutations Cause a Disease Spectrum of Limb Girdle Muscular Dystrophy and Myopathy with Movement Disorder and Intellectual Disability

Myopathies are a clinically and etiologically heterogeneous group of disorders that can range from limb girdle muscular dystrophy (LGMD) to syndromic forms with associated features including intellectual disability. Here, we report the identification of mutations in transport protein particle complex 11 (TRAPPC11) in three individuals of a consanguineous Syrian family presenting with LGMD and in five individuals of Hutterite descent presenting with myopathy, infantile hyperkinetic movements, ataxia, and intellectual disability. By using a combination of whole-exome or genome sequencing with homozygosity mapping, we identified the homozygous c.2938G>A (p.Gly980Arg) missense mutation within the gryzun domain of TRAPPC11 in the Syrian LGMD family and the homozygous c.1287+5G>A splice-site mutation resulting in a 58 amino acid in-frame deletion (p.Ala372_Ser429del) in the foie gras domain of TRAPPC11 in the Hutterite families. TRAPPC11 encodes a component of the multiprotein TRAPP complex involved in membrane trafficking. We demonstrate that both mutations impair the binding ability of TRAPPC11 to other TRAPP complex components and disrupt the Golgi apparatus architecture. Marker trafficking experiments for the p.Ala372_Ser429del deletion indicated normal ER-to-Golgi trafficking but dramatically delayed exit from the Golgi to the cell surface. Moreover, we observed alterations of the lysosomal membrane glycoproteins lysosome-associated membrane protein 1 (LAMP1) and LAMP2 as a consequence of TRAPPC11 dysfunction supporting a defect in the transport of secretory proteins as the underlying pathomechanism.

Haploinsufficiency of TAB2 causes congenital heart defects in humans

Congenital heart defects (CHDs) are the most common major developmental anomalies and the most frequent cause for perinatal mortality, but their etiology remains often obscure. We identified a locus for CHDs on 6q24-q25. Genotype-phenotype correlations in 12 patients carrying a chromosomal deletion on 6q delineated a critical 850 kb region on 6q25.1 harboring five genes. Bioinformatics prioritization of candidate genes in this locus for a role in CHDs identified the TGF-beta-activated kinase 1/MAP3K7 binding protein 2 gene (TAB2) as the top-ranking candidate gene. A role for this candidate gene in cardiac development was further supported by its conserved expression in the developing human and zebrafish heart. Moreover, a critical, dosage-sensitive role during development was demonstrated by the cardiac defects observed upon titrated knockdown of tab2 expression in zebrafish embryos. To definitively confirm the role of this candidate gene in CHDs, we performed mutation analysis of TAB2 in 402 patients with a CHD, which revealed two evolutionarily conserved missense mutations. Finally, a balanced translocation was identified, cosegregating with familial CHD. Mapping of the breakpoints demonstrated that this translocation disrupts TAB2. Taken together, these data clearly demonstrate a role for TAB2 in human cardiac development.