Jennifer Laffin

FMR1 CGG allele size and prevalence ascertained through newborn screening in the United States

BackgroundPopulation screening for FMR1 mutations has been a topic of considerable discussion since the FMR1 gene was identified in 1991. Advances in understanding the molecular basis of fragile X syndrome (FXS) and in genetic testing methods have led to new, less expensive methodology to use for large screening endeavors. A core criterion for newborn screening is an accurate understanding of the public health burden of a disease, considering both disease severity and prevalence rate. This article addresses this need by reporting prevalence rates observed in a pilot newborn screening study for FXS in the US.MethodsBlood spot screening of 14,207 newborns (7,312 males and 6,895 females) was conducted in three birthing hospitals across the United States beginning in November 2008, using a PCR-based approach.ResultsThe prevalence of gray zone alleles was 1:66 females and 1:112 males, while the prevalence of a premutation was 1:209 females and 1:430 males. Differences in prevalence rates were observed among the various ethnic groups; specifically higher frequency for gray zone alleles in males was observed in the White group compared to the Hispanic and African-American groups. One full mutation male was identified (>200 CGG repeats).ConclusionsThe presented pilot study shows that newborn screening in fragile X is technically feasible and provides overall prevalence of the premutation and gray zone alleles in the USA, suggesting that the prevalence of the premutation, particularly in males, is higher than has been previously reported.

Whole-exome sequencing supports genetic heterogeneity in childhood apraxia of speech

BackgroundChildhood apraxia of speech (CAS) is a rare, severe, persistent pediatric motor speech disorder with associated deficits in sensorimotor, cognitive, language, learning and affective processes. Among other neurogenetic origins, CAS is the disorder segregating with a mutation in FOXP2 in a widely studied, multigenerational London family. We report the first whole-exome sequencing (WES) findings from a cohort of 10 unrelated participants, ages 3 to 19 years, with well-characterized CAS.MethodsAs part of a larger study of children and youth with motor speech sound disorders, 32 participants were classified as positive for CAS on the basis of a behavioral classification marker using auditory-perceptual and acoustic methods that quantify the competence, precision and stability of a speaker’s speech, prosody and voice. WES of 10 randomly selected participants was completed using the Illumina Genome Analyzer IIx Sequencing System. Image analysis, base calling, demultiplexing, read mapping, and variant calling were performed using Illumina software. Software developed in-house was used for variant annotation, prioritization and interpretation to identify those variants likely to be deleterious to neurodevelopmental substrates of speech-language development.ResultsAmong potentially deleterious variants, clinically reportable findings of interest occurred on a total of five chromosomes (Chr3, Chr6, Chr7, Chr9 and Chr17), which included six genes either strongly associated with CAS (FOXP1 and CNTNAP2) or associated with disorders with phenotypes overlapping CAS (ATP13A4, CNTNAP1, KIAA0319 and SETX). A total of 8 (80%) of the 10 participants had clinically reportable variants in one or two of the six genes, with variants in ATP13A4, KIAA0319 and CNTNAP2 being the most prevalent.ConclusionsSimilar to the results reported in emerging WES studies of other complex neurodevelopmental disorders, our findings from this first WES study of CAS are interpreted as support for heterogeneous genetic origins of this pediatric motor speech disorder with multiple genes, pathways and complex interactions. We also submit that our findings illustrate the potential use of WES for both gene identification and case-by-case clinical diagnostics in pediatric motor speech disorders.

Childhood Apraxia of Speech (CAS) in two patients with 16p11.2 microdeletion syndrome

We report clinical findings that extend the phenotype of the ∼550 kb 16p11.2 microdeletion syndrome to include a rare, severe, and persistent pediatric speech sound disorder termed Childhood Apraxia of Speech (CAS). CAS is the speech disorder identified in a multigenerational pedigree (‘KE’) in which half of the members have a mutation in FOXP2 that co-segregates with CAS, oromotor apraxia, and low scores on a nonword repetition task. Each of the two patients in the current report completed a 2-h assessment protocol that provided information on their cognitive, language, speech, oral mechanism, motor, and developmental histories and performance. Their histories and standard scores on perceptual and acoustic speech tasks met clinical and research criteria for CAS. Array comparative genomic hybridization analyses identified deletions at chromosome 16p11.2 in each patient. These are the first reported cases with well-characterized CAS in the 16p11.2 syndrome literature and the first report of this microdeletion in CAS genetics research. We discuss implications of findings for issues in both literatures.

Immunohistochemical evaluation of MYC expression in mantle cell lymphoma

To assess the validity and potential clinical utility of evaluating MYC expression by immunohistochemistry (IHC) in mantle cell lymphoma (MCL).

The advantage of using SNP array in clinical testing for hematological malignancies--a comparative study of three genetic testing methods.

Cytogenetic methods, including G-banded chromosome analysis and fluorescence in situ hybridization (FISH) analysis, serve as a critical part of routine clinical testing for hematological malignancies and provide important diagnostic and prognostic information; however, the limitations of cytogenetic methods, including the requirement for actively dividing cells and lower resolution of G-banded chromosome analysis as well as the inability of both G-banded chromosome analysis and FISH to detect copy number neutral loss of heterozygosity (CN-LOH), can result in a failure to detect genomic abnormalities with diagnostic and prognostic significance. Here, we compared the abnormality detection rate of clinically requested testing (i.e., G-banded chromosome analysis and FISH) with high-resolution oligo (i.e., array comparative genomic hybridization (aCGH)) and single-nucleotide polymorphism (SNP)/oligo hybrid (i.e., SNP-CGH) arrays in a series of patients, in an effort to assess the ability of newer technologies to overcome these limitations. This series found the detection rate for SNP-CGH to be 62.5% for myelodysplastic syndrome (MDS) cases and 72.7% for chronic lymphocytic leukemia (CLL) cases, which are significantly higher than the detection rates of aCGH (31.3% for MDS and 54.5% for CLL) and G-banding and/or FISH (43.8% for MDS and 54.5% for CLL). This demonstrates the advantages of combining SNP-CGH with conventional cytogenetics to provide comprehensive clinical information by detecting clonality, large balanced rearrangements, copy number aberrations, and CN-LOH.

Array-based comparative genomic hybridization (aCGH) in the genetic evaluation of stillbirth†‡

This study examined the utility of array‐based comparative genomic hybridization (aCGH) in detecting genetic abnormalities associated with late pregnancy loss. Comparisons were made with classic cytogenetics to test whether aCGH represents a superior methodology for the clinical evaluation of stillbirth. Stillborn infants were selected for aCGH testing from the Wisconsin Stillbirth Service Program (WiSSP) database and tissue bank, based on abnormal clinical findings (presence of at least two abnormalities of two different organs or parts of the body). aCGH analysis was successfully completed in 15 cases which met the clinical criteria and for which sufficient amount of high quality DNA was recovered from archival material. The testing was performed using commercially available 1 Mb BAC arrays. Among 15 tested stillborns, aCGH detected two abnormalities (trisomy 21 and an unbalanced translocation between chromosomes 3 and 10), for an overall detection rate of 13% in stillborns with malformations who had normal or unobtainable cytogenetic results. This preliminary study supports the clinical value of aCGH testing in diagnostic evaluation of stillborns with congenital anomalies.

MECP2 duplication: possible cause of severe phenotype in females.

MECP2 duplication syndrome, originally described in 2005, is an X‐linked neurodevelopmental disorder comprising infantile hypotonia, severe to profound intellectual disability, autism or autistic‐like features, spasticity, along with a variety of additional features that are not always clinically apparent. The syndrome is due to a duplication (or triplication) of the gene methyl CpG binding protein 2 (MECP2). To date, the disorder has been described almost exclusively in males. Female carriers of the duplication are thought to have no or mild phenotypic features. Recently, a phenotype for females began emerging. We describe a family with ∼290 kb duplication of Xq28 region that includes the MECP2 gene where the proposita and affected family members are female. Twin sisters, presumed identical, presented early with developmental delay, and seizures. Evaluation of the proposita at 25 years of age included microarray comparative genomic hybridization (aCGH) which revealed the MECP2 gene duplication. The same duplication was found in the propositas sister, who is more severely affected, and the probands mother who has mild intellectual disability and depression. X‐chromosome inactivation studies showed significant skewing in the mother, but was uninformative in the twin sisters. We propose that the MECP2 duplication caused for the phenotype of the proband and her sister. These findings support evidence for varied severity in some females with MECP2 duplications.

Interphase cytofission maintains genomic integrity of human cells after failed cytokinesis

In cell division, cytokinesis is tightly coupled with mitosis to maintain genomic integrity. Failed cytokinesis in humans can result in tetraploid cells that can become aneuploid and promote cancer. However, the likelihood of aneuploidy and cancer after a failed cytokinesis event is unknown. Here we evaluated cell fate after failed cytokinesis. We interrupted cytokinesis by brief chemical treatments in cell populations of human epithelial lines. Surprisingly, up to 50% of the resulting binucleate cells generated colonies. In RPE1 cells, 90% of colonies obtained from binucleate founders had a karyotype that matched the parental cell type. Time-lapse videomicroscopy demonstrated that binucleate cells are delayed in the first growth phase of the cell cycle (G1) and undergo interphase cellular fission (cytofission) that distributes nuclei into separate daughters. The fission is not compatible with delayed cytokinesis because events occur in the absence of polymerized microtubules and without canonical components of the cytokinetic machinery. However, the cytofission can be interrupted by inhibiting function of actin or myosin II. Fission events occur in both two- and three-dimensional culture. Our data demonstrate that cytofission can preserve genomic integrity after failed cytokinesis. Thus, traction-mediated cytofission, originally observed in Dictyostelium, is relevant to human biology—where it seems to be an evolutionarily conserved mechanism that can preserve genomic integrity.

DNA Methylation Assay for X-Chromosome Inactivation in Female Human iPS Cells

Remarkable interest in the epigenetic status of human induced pluripotent stem (iPS) cells inspired numerous studies of their X-inactivation patterns. However, both the presence and the absence of X-inactivation have been described to date in undifferentiated iPS cells. The reasons for the discordant results between different studies are unclear, and further X-inactivation testing is warranted for all female human iPS cell lines. Some of the inconsistency in the current data most likely results from the use of different X-inactivation assays by different authors. We provide a detailed protocol for a simple, reliable and affordable X-inactivation assay based on promoter methylation and CAG-repeat polymorphism in the human androgen receptor (AR) gene at Xq11.2. This assay is commonly used in clinical genetic laboratories and we propose that it could be ideal for routine assessment and monitoring of the X-inactivation status in female human iPS cell lines.

Clinical and Molecular Characterization of Overlapping Interstitial Xp21-p22 Duplications in Two Unrelated Individuals

Development and implementation of high‐density DNA arrays demonstrated the important role of copy number changes on the X chromosome in the etiology of developmental delay and mental retardation (MR). We describe two unrelated patients with developmental delay due to similar interstitial duplications at Xp21‐p22. The first patient is a 6‐month‐old male with multiple affected family members including many females. The second patient is a 5‐year‐old adopted female. In both patients, chromosome analysis and array comparative genomic hybridization (aCGH) showed duplications of overlapping regions at Xp21‐p22. The duplicated segments contain numerous genes associated with MR, including AP1S2, NHS, CDKL5, RPS6KA3, SMS, and ARX. Except for developmental delay, there is little phenotypic overlap between the male and the female patient. Additionally, the female patient and affected female relatives of the male patient have variable severities of cognitive impairment, likely due to different X‐inactivation patterns and effects of other, nonduplicated genes important for normal development. These cases illustrate that increased gene dosage of X‐linked MR genes lead to cognitive impairment. Precise delineation of chromosome rearrangements by aCGH and identification of genes within duplicated segments helped in establishing genotype–phenotype correlations for each of our patients, in comparing them to each other, as well as with previously reported cases of Xp21‐p22 duplications. However, we show that even with detailed molecular characterization, phenotype prediction remains challenging in patients with structural abnormalities of the X chromosome.