Robert E. Pyatt

Newborn and carrier screening for spinal muscular atrophy

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder caused by mutations in the survival motor neuron (SMN1) gene, affecting approximately 1 in 10,000 live births. The homozygous absence of SMN1 exon 7 has been observed in the majority of patients and is being utilized as a reliable and sensitive SMA diagnostic test. Treatment and prevention of SMA are complementary responses to the challenges presented by SMA. Even though a specific therapy for SMA is not currently available, a newborn screening test may allow the child to be enrolled in a clinical trial before irreversible neuronal loss occurs and enable patients to obtain more proactive treatments. Until an effective treatment is found to cure or arrest the progression of the disease, prevention of new cases through accurate diagnosis and carrier and prenatal diagnosis is of the utmost importance. The goal of population‐based SMA carrier screening is to identify couples at risk for having a child with SMA, thus allowing carriers to make informed reproductive choices. During this study we performed two pilot projects addressing the clinical applicability of testing in the newborn period and carrier screening in the general population. We have demonstrated that an effective technology does exist for newborn screening of SMA. We also provide an estimate of the carrier frequency among individuals who accepted carrier screening, and report on patients knowledge and attitudes toward SMA testing.

Assessment of 2q23.1 microdeletion syndrome implicates MBD5 as a single causal locus of intellectual disability, epilepsy, and autism spectrum disorder

Persons with neurodevelopmental disorders or autism spectrum disorder (ASD) often harbor chromosomal microdeletions, yet the individual genetic contributors within these regions have not been systematically evaluated. We established a consortium of clinical diagnostic and research laboratories to accumulate a large cohort with genetic alterations of chromosomal region 2q23.1 and acquired 65 subjects with microdeletion or translocation. We sequenced translocation breakpoints; aligned microdeletions to determine the critical region; assessed effects on mRNA expression; and examined medical records, photos, and clinical evaluations. We identified a single gene, methyl-CpG-binding domain 5 (MBD5), as the only locus that defined the critical region. Partial or complete deletion of MBD5 was associated with haploinsufficiency of mRNA expression, intellectual disability, epilepsy, and autistic features. Fourteen alterations, including partial deletions of noncoding regions not typically captured or considered pathogenic by current diagnostic screening, disrupted MBD5 alone. Expression profiles and clinical characteristics were largely indistinguishable between MBD5-specific alteration and deletion of the entire 2q23.1 interval. No copy-number alterations of MBD5 were observed in 7878 controls, suggesting MBD5 alterations are highly penetrant. We surveyed MBD5 coding variations among 747 ASD subjects compared to 2043 non-ASD subjects analyzed by whole-exome sequencing and detected an association with a highly conserved methyl-CpG-binding domain missense variant, p.79Gly>Glu (c.236G>A) (p = 0.012). These results suggest that genetic alterations of MBD5 cause features of 2q23.1 microdeletion syndrome and that this epigenetic regulator significantly contributes to ASD risk, warranting further consideration in research and clinical diagnostic screening and highlighting the importance of chromatin remodeling in the etiology of these complex disorders.

Polymorphic variation at the BAT-25 and BAT-26 loci in individuals of African origin. Implications for microsatellite instability testing.

Instability in the repeat size of microsatellite sequences has been described in both hereditary nonpolyposis and sporadic colorectal cancers. Tumors expressing microsatellite instability are identified through the comparison of the repeat sizes at multiple microsatellite loci between tumor and matched normal tissue DNA. The use of a five-marker panel including two mononucleotide repeat microsatellites, BAT-25 and BAT-26, has recently been suggested for the clinical determination of tumor microsatellite instability. The BAT-25 and BAT-26 loci included in this panel have both demonstrated sensitivity to microsatellite instability and normal quasimonomorphic allelic patterns, which has simplified the distinction between normal and unstable alleles. However, in this study, we identified allelic variations in the size of the poly(A) tract at BAT-26 in 12.6% of 103 healthy African-Americans screened. In addition, 18.4% exhibited allelic size variations in the poly(T) tract at BAT-25. Finally, 2.9% showed variant alleles at both BAT-25 and BAT-26 loci. Screening a small population of Nigerians confirmed the polymorphic nature of both loci and the ethnic origin of alleles not identified in other populations studied thus far. Our results dispute the quasimonomorphic nature of both BAT-25 and BAT-26 in all populations and support the need for thorough population studies to define the different allelic profiles and frequencies at microsatellite loci.

Novel diagnostic features of dysferlinopathies

Reports of dysferlinopathy have suggested a clinically heterogeneous group of patients. We identified specific novel molecular and phenotypic features that help distinguish dysferlinopathies from other forms of limb‐girdle muscular dystrophy (LGMD). A detailed history, physical exam, and protein and mutation analysis of genomic DNA was done for all subjects. Five of 21 confirmed DYSF gene mutations were not previously reported. A distinct “bulge” of the deltoid muscle in combination with other findings was a striking feature in all patients. Six subjects had atypical calf enlargement, and 3 of these exhibited a paradoxical pattern of dysferlin expression: severely reduced by direct immunofluorescence with overexpression on Western blots. Six patients showed amyloid deposits in muscle that extended these findings to new domains of the dysferlin gene, including the C2G domain. Correlative studies showed colocalization of amyloid with deposition of dysferlin. The present data further serve to guide clinicians facing the expensive task of molecular characterization of patients with an LGMD phenotype. Muscle Nerve 42: 14–21, 2010

Identification of a Recurrent Microdeletion at 17q23.1q23.2 Flanked by Segmental Duplications Associated with Heart Defects and Limb Abnormalities

Segmental duplications, which comprise approximately 5%-10% of the human genome, are known to mediate medically relevant deletions, duplications, and inversions through nonallelic homologous recombination (NAHR) and have been suggested to be hot spots in chromosome evolution and human genomic instability. We report seven individuals with microdeletions at 17q23.1q23.2, identified by microarray-based comparative genomic hybridization (aCGH). Six of the seven deletions are approximately 2.2 Mb in size and flanked by large segmental duplications of >98% sequence identity and in the same orientation. One of the deletions is approximately 2.8 Mb in size and is flanked on the distal side by a segmental duplication, whereas the proximal breakpoint falls between segmental duplications. These characteristics suggest that NAHR mediated six out of seven of these rearrangements. These individuals have common features, including mild to moderate developmental delay (particularly speech delay), microcephaly, postnatal growth retardation, heart defects, and hand, foot, and limb abnormalities. Although all individuals had at least mild dysmorphic facial features, there was no characteristic constellation of features that would elicit clinical suspicion of a specific disorder. The identification of common clinical features suggests that microdeletions at 17q23.1q23.2 constitute a novel syndrome. Furthermore, the inclusion in the minimal deletion region of TBX2 and TBX4, transcription factors belonging to a family of genes implicated in a variety of developmental pathways including those of heart and limb, suggests that these genes may play an important role in the phenotype of this emerging syndrome.

Hereditary and somatic DNA mismatch repair gene mutations in sporadic endometrial carcinoma

Editor—Endometrial cancer (EC) is the second most common malignancy in the hereditary non-polyposis colorectal cancer (HNPCC) syndrome.1 In a recent large study, cumulative cancer incidences by the age of 70 in HNPCC mutation carriers were: colorectal 82%, endometrial 60%, gastric 13%, and ovarian 12%.2 Interestingly, in female mutation carriers the incidence of endometrial cancer (60%) exceeded that of colorectal cancer (CRC) (54%), as had been suggested earlier.2 3 Predisposition to HNPCC is the result of germline mutations in the mismatch repair genes.4 Detectable mutations in the two major genes, MLH1 and MSH2 , account for some 3% of all colorectal cancers.5 One might therefore assume that a similar proportion of all endometrial cancer patients would have such mutations; however, in a number of studies addressing this question, extremely few germline mutations have been found. Summarising the studies by Katabuchi et al ,6Kobayashi et al ,7 Lim et al ,8 Gurin et al ,9 and Kowalski et al ,10 only one germline mutation (in MLH1 ) was found in a total of 352 EC patients (0.3%). In these studies, mutations were sought in all patients whose tumours were microsatellite instability (MSI) positive. Recent reports have suggested that MSH6 might account for many endometrial cancers and that families with these mutations show atypical features of HNPCC with endometrial and ovarian cancers outnumbering colorectal cancers.11 12Additionally, MSI, a hallmark of HNPCC, was low in most tumours associated with MSH6 mutations or was preferentially shown by mononucleotide repeats rather than dinucleotide repeats.12-14 Previous studies have reported that 9-25% of sporadic endometrial cancers display microsatellite instability .7 9 15-18 In the majority of cases, this instability arises through hypermethylation of the MLH1 promoter region.9 19-21 This epigenetic change results …

A feasibility study for the newborn screening of spinal muscular atrophy

Purpose: The natural history of spinal muscular atrophy suggests that for maximum effect, therapeutics will need to be administered in the earliest phases of the disease. This will require the adoption of techniques for the genetic analysis of affected individuals at the newborn stage. Our objective was to examine the feasibility surrounding the newborn screening for spinal muscular atrophy.Methods: We investigated the application of real-time polymerase chain reaction technology for newborn screening. A multiplex assay was designed to identify homozygous deletions in SMN1 exon 7 and validated using 266 samples with defined SMN1 and SMN2 copy numbers. Sensitivity and specificity were then evaluated as part of a newborn screening strategy using DNA from 153 blood spots.Results: Real-time technology validation demonstrated correct exclusion of all normal and carrier samples, and identified the homozygous SMN1 exon 7 deletions in all 32 affected samples. In the series of blood spots, all 59 affected samples were correctly identified yielding an analytic sensitivity of 100%; 56 normal and 39 carrier samples were correctly excluded yielding an analytic specificity of 100% for this blood spot series.Conclusion: We demonstrate that effective molecular technology exists and that ethics may soon warrant the newborn screening of spinal muscular atrophy.

Reciprocal deletion and duplication at 2q23.1 indicates a role for MBD5 in autism spectrum disorder

Copy number variations associated with abnormal gene dosage have an important role in the genetic etiology of many neurodevelopmental disorders, including intellectual disability (ID) and autism. We hypothesize that the chromosome 2q23.1 region encompassing MBD5 is a dosage-dependent region, wherein deletion or duplication results in altered gene dosage. We previously established the 2q23.1 microdeletion syndrome and report herein 23 individuals with 2q23.1 duplications, thus establishing a complementary duplication syndrome. The observed phenotype includes ID, language impairments, infantile hypotonia and gross motor delay, behavioral problems, autistic features, dysmorphic facial features (pinnae anomalies, arched eyebrows, prominent nose, small chin, thin upper lip), and minor digital anomalies (fifth finger clinodactyly and large broad first toe). The microduplication size varies among all cases and ranges from 68 kb to 53.7 Mb, encompassing a region that includes MBD5, an important factor in methylation patterning and epigenetic regulation. We previously reported that haploinsufficiency of MBD5 is the primary causal factor in 2q23.1 microdeletion syndrome and that mutations in MBD5 are associated with autism. In this study, we demonstrate that MBD5 is the only gene in common among all duplication cases and that overexpression of MBD5 is likely responsible for the core clinical features present in 2q23.1 microduplication syndrome. Phenotypic analyses suggest that 2q23.1 duplication results in a slightly less severe phenotype than the reciprocal deletion. The features associated with a deletion, mutation or duplication of MBD5 and the gene expression changes observed support MBD5 as a dosage-sensitive gene critical for normal development.

Quality assurance for Duchenne and Becker muscular dystrophy genetic testing: development of a genomic DNA reference material panel.

Duchenne and Becker muscular dystrophies (DMD/BMD) are allelic X-linked recessive disorders that affect approximately 1 in 3500 and 1 in 20,000 male individuals, respectively. Approximately 65% of patients with DMD have deletions, 7% to 10% have duplications, and 25% to 30% have point mutations in one or more of the 79 exons of the dystrophin gene. Most clinical genetics laboratories test for deletions, and some use technologies that can detect smaller mutations and duplications. Reference and quality control materials for DMD/BMD diagnostic and carrier genetic testing are not commercially available. To help address this need, the Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the genetic testing and the DMD/BMD patient communities and the Coriell Cell Repositories, have characterized new and existing cell lines to create a comprehensive DMD/BMD reference material panel. Samples from 31 Coriell DMD cell lines from male probands and female carriers were analyzed using the Affymetrix SNP Array 6.0 and Multiplex Ligation-Dependent Probe Amplification (MRC-Holland BV, Amsterdam, the Netherlands), a multiplex PCR assay, and DNA sequence analysis. Identified were 16 cell lines with deletions, 9 with duplications, and 4 with point mutations distributed throughout the dystrophin gene. There were no discordant results within assay limitations. These samples are publicly available from Coriell Institute for Medical Research (Camden, NJ) and can be used for quality assurance, proficiency testing, test development, and research, and should help improve the accuracy of DMD testing.

Variability in pathogenicity prediction programs: impact on clinical diagnostics.

Current practice by clinical diagnostic laboratories is to utilize online prediction programs to help determine the significance of novel variants in a given gene sequence. However, these programs vary widely in their methods and ability to correctly predict the pathogenicity of a given sequence change. The performance of 17 publicly available pathogenicity prediction programs was assayed using a dataset consisting of 122 credibly pathogenic and benign variants in genes associated with the RASopathy family of disorders and limb‐girdle muscular dystrophy. Performance metrics were compared between the programs to determine the most accurate program for loss‐of‐function and gain‐of‐function mechanisms. No one program correctly predicted the pathogenicity of all variants analyzed. A major hindrance to the analysis was the lack of output from a significant portion of the programs. The best performer was MutPred, which had a weighted accuracy of 82.6% in the full dataset. Surprisingly, combining the results of the top three programs did not increase the ability to predict pathogenicity over the top performer alone. As the increasing number of sequence changes in larger datasets will require interpretation, the current study demonstrates that extreme caution must be taken when reporting pathogenicity based on statistical online protein prediction programs in the absence of functional studies.