Ben Anderson

FMR1 premutation carrier frequency in patients undergoing routine population-based carrier screening: insights into the prevalence of fragile X syndrome, fragile X-associated tremor/ataxia syndrome, and fragile X-associated primary ovarian insufficiency in the United States.

Purpose: Fragile X syndrome is caused by expansion and methylation of a CGG tract in the 5′ untranslated region of the FMR1 gene. The estimated frequency of expanded alleles (≥55 repeats) in the United States is 1:257–1:382, but these estimates were not calculated from unbiased populations. We sought to determine the frequency of fragile X syndrome premutation (55–200 repeats) and full mutation (>200 repeats) alleles in nonselected, unbiased populations undergoing routine carrier screening for other diseases.Methods: A previously validated laboratory-developed test using triplet-primed polymerase chain reaction was used to detect premutation and full mutation alleles in an unselected series of 11,759 consecutive cystic fibrosis carrier screening samples and 2011 samples submitted for screening for genetic diseases prevalent among the Ashkenazi Jewish population.Results: Premutations were identified in 48 cystic fibrosis screening samples (1:245) and 15 samples (1:134) from the Ashkenazi Jewish population. Adjusted for the ethnic mix of the US population and self-reported ethnicity in our screening population, the estimated female premutation carrier frequency in the United States was 1:178. The calculated frequency of full mutation alleles was 1:3335 overall, and the calculated premutation frequency in males was 1:400. Based on frequency of larger, ≥70 repeat alleles, and reported penetrance, the calculated fragile X-associated tremor and ataxia syndrome, and fragile X-associated primary ovarian insufficiency frequencies is 1:4848 and 1:3560, respectively.Conclusion: Our calculated fragile X syndrome carrier rate is higher than previous estimates for the US population and warrants further consideration of population-based carrier screening.

Cystic fibrosis screening using the College panel: Platform comparison and lessons learned from the first 20,000 samples

Purpose: To determine the accuracy of two commercially available kits for cystic fibrosis (CF) genotyping and determine allele frequencies for the ACMG/ACOG recommended mutations.Methods: A total of 1,040 consecutive analyses using Roche CF Gold Strips and the ABI CF Genotyper were performed. Subsequently we performed analyses of 20,103 samples.Results: Both kits accurately determined CF genotypes. The I148T mutation was found >100 times more frequently in carrier screening than in CF patients. Asymptomatic patients were identified who are compound heterozygotes for delta F508 and I148T. Four of 13 patients heterozygous for delta F508 and the IVS8-5T polymorphism had some symptoms of CF.Conclusion: Accurate and timely analysis can be performed for the ACMG CF panel. I148T is a low penetrance CF allele.

Novel and recurrent rearrangements in the CFTR gene: clinical and laboratory implications for cystic fibrosis screening

Because standard techniques used to detect mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene do not detect single or multiple exonic rearrangements, the importance of such rearrangements may be underestimated. Using an in-house developed, single-tube, semi-quantitative fluorescent PCR (SQF PCR) assay, we analyzed 36 DNA samples submitted for extensive CFTR sequencing and identified ten samples with rearrangements. Of 36 patients with classic CF, 10 (28%) harbored various deletions in the CFTR gene, accounting for 14% of CF chromosomes. A deletion encompassing the CFTR promoter and exons 1 and 2 was detected in a sample from one proband, and in the maternal DNA as well. In another family, a deletion of the promoter and exon 1 was detected in three siblings. In both of these cases, the families were African American and the 3120+1G>A splice site mutation was also identified. These promoter deletions have not been previously described. In a third case, a deletion of exons 17a, 17b, and 18 was identified in a Caucasian female and the same mutation was detected in the paternal DNA. In the other seven cases, we identified the following deletions: exons 2 and 3 (n=2); exons 4, 5, and 6a; exons 17a and 17b; exons 22 and 23; and exons 22, 23, and 24 (n=2). In our series, the frequency of CFTR rearrangements in classic CF patients, when only one mutation was identified by extensive DNA sequencing, was >60% (10/16). Screening for exon deletions and duplications in the CFTR gene would be beneficial in classic CF cases, especially when only one mutation is identified by standard methodologies.

A large deletion in the CFTR gene in CBAVD

Purpose: Most cystic fibrosis mutation screening methods do not detect large exon deletions or duplications in the cystic fibrosis transmembrane regulator gene. We looked for such mutations in congenital bilateral absence of the vas deferens patients in whom routine screening assays had identified only one or no cystic fibrosis transmembrane regulator gene mutations.Methods: DNA samples from 48 men with congenital bilateral absence of the vas deferens were tested for exonic deletions and duplications in the cystic fibrosis transmembrane regulator gene using a laboratory-developed semiquantitative fluorescent PCR assay.Results: Semi-quantitative fluorescent PCR identified a large deletion in one (2%) of the 48 patients. This patient, previously characterized as carrying only the IVS8-5T mutation, was found to have a deletion of exons 22-24 of the cystic fibrosis transmembrane regulator gene. In a second patient with the IVS8-5T mutation, we identified a one-base pair insertion in exon 17b that disrupted the reading frame.Conclusions: Analysis of the cystic fibrosis transmembrane regulator gene for exon deletions and duplications should be included for complete study of CBAVD patients, especially those considering assisted reproduction.

CFTR 5T variant has a low penetrance in females that is partially attributable to its haplotype

Purpose: The studys purpose was to understand the molecular basis for different clinical phenotypes of the 5T variant, a tract of 5 thymidines in intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which disrupts processing of CFTR mRNA and reduces synthesis from the corresponding CFTR alleles.Method: We analyzed the polymorphic TG dinucleotide repeat adjacent to the 5T variant in intron 8 and the codon 470 in exon 10. Patients selected for this study were positive for both the 5T variant and the major cystic fibrosis mutation, Delta F508. Almost all Delta F508 mutation alleles occur in a 10TG-9T-470M haplotype. Therefore, it is possible to determine the haplotype of the 5T variant in trans.Results: Of the 74 samples analyzed, 41 (55%) were 11TG-5T-470M, 31 (42%) were 12TG-5T-470V, and 2 (3%) were 13TG-5T-470M. Of the 49 cases for which we had clinical information, 17.6% of females (6/34) and 66.7% of males (10/15) showed symptoms resembling atypical cystic fibrosis. The haplotype with the highest penetrance in females (42% or 5/12) and more than 80% (5/6) in males is 12TG-5T-470V. We also evaluated 12 males affected with congenital bilateral absence of vas deferens and positive for the 5T variant; 10 of 12 had the 12TG-5T-470V haplotype.Conclusion: Overall, the 5T variant has a milder clinical consequence than previously estimated in females. The clinical presentations of the 5T variant are associated with the 5T-12TG-470M haplotype.

Improving the Positive Predictive Value of Non-Invasive Prenatal Screening (NIPS)

We evaluated performance characteristics of a laboratory-developed, non-invasive prenatal screening (NIPS) assay for fetal aneuploidies. This assay employs massively parallel shotgun sequencing with full automation. GC sequencing bias correction and statistical smoothing were performed to enhance discrimination of affected and unaffected pregnancies. Maternal plasma samples from pregnancies with known aneuploidy status were used for assay development, verification, and validation. Assay verification studies using 2,085 known samples (1873 unaffected, 69 trisomy 21, 20 trisomy 18, 17 trisomy 13) demonstrated complete discrimination between autosomal trisomy (Z scores >8) and unaffected (Z scores <4) singleton pregnancies. A validation study using 552 known samples (21 trisomy 21, 10 trisomy 18, 1 trisomy 13) confirmed complete discrimination. Twin pregnancies showed similar results. Follow-up of abnormal results from the first 10,000 clinical samples demonstrated PPVs of 98% (41/42) for trisomy 21, 92% (23/25) for trisomy 18, and 69% (9/13) for trisomy 13. Adjustment for causes of false-positive results identified during clinical testing (eg, maternal duplications) improved PPVs to 100% for trisomy 21 and 96% for trisomy 18. This NIPS test demonstrates excellent discrimination between trisomic and unaffected pregnancies. The PPVs obtained in initial clinical testing are substantially higher than previously reported NIPS methods.

Maternal Chromosome Xp Deletion Identified by Prenatal Cell-free DNA Screening

Newborn girls are affected by classic Turner syndrome at a rate of approximately one out of 2500 to 3000 live births. The most common form of Turner syndrome is due to monosomy X (45, X), with some individuals exhibiting mosaicism for the 45, X cell line (45, X/46, XX). Perhaps the rarest form of Turner syndrome is due to a partial loss of X chromosome material. Typical clinical signs of Turner syndrome include but are not limited to unexplained growth failure, delayed puberty, infertility, and amenorrhea. As the infertility features of Turner syndrome are often caused by ovarian dysgenesis and failure, only 10% of women with Turner syndrome can experience spontaneous pregnancies. Those who do achieve pregnancy are at high risk of cardiovascular (e.g., aortic) complications, miscarriage, and stillbirth. However, variable expressivity is observed in women with variant Turner syndrome, which can lead to underdiagnosis. We describe the ability of an established cellfree DNA (cfDNA)-based prenatal screening assay to detect a maternal chromosome Xp deletion causing variant Turner syndrome in a patient with spontaneous pregnancy. A peripheral blood specimen from a 39-year-old pregnant woman at 11 weeks’ gestation was submitted to Quest Diagnostics for prenatal cfDNA screening, with the clinical indication of advancedmaternal age. At the time of submission, the patient did not report any known personal history of chromosome abnormality. She presented with a height of 5 ft, a history of hypothyroidism, and various mental health concerns. Her medical history also included a previous elective abortion and infertility secondary to increased folliclestimulating hormone levels. She had no documented indication of an abnormal maternal serum screen, abnormal ultrasound finding(s), or clinically significant family history. The current pregnancy was achieved via intrauterine insemination from sperm donation. Prenatal cfDNA screening was conducted using massively parallel shotgun sequencing to detect fetal aneuploidy of chromosomes 21, 18, and 13, as well as the sex chromosomes. The cfDNA sequencing data showed a markedly lower level of cfDNA sequencing products at chromosome Xp by means of noticing a copy number ratio of approximately 0.5 (normalized bin count) of typical female X chromosome content (Figure 1). At a fetal fraction of 9.73%, the level of cfDNA products was lower than expected for a fetal Xp deletion in an unaffected mother. Therefore, the Xp deletion was suspected to be maternal in origin. The cfDNA screen was negative for trisomies 21, 18, and 13, and fetal sex was determined to be female based upon absence of Y chromosome material. As there was a confounding effect of the suspected maternal deletion, fetal sex chromosome aneuploidy status could not be assessed. The result report included a recommendation to offer maternal chromosomal microarray (CMA) analysis [Affymetrix CytoScan® HD SNP-array platform and Chromosome Analysis Suite (ChAS; version 3.0, Affymetrix)] to confirm and characterize the deletion. Follow-up CMA analysis of the mother’s DNA confirmed a maternal 39.5-Mb deletion of Xp22.33p11.4, consistent with the prenatal cfDNA screening finding and a diagnosis of variant Turner syndrome (46, XXp-) (Figure 1). After discussing these results with the laboratory genetic counselor, the ordering provider offered the patient invasive diagnostic testing to confirm this deletion in the fetus. There was explanation that the patient’s short stature and infertility secondary to increased follicle-stimulating hormone suggested a mild clinical phenotype in comparison with classic Turner syndrome. These features are consistent with the patient’s molecular diagnosis of variant Turner syndrome due to a chromosome Xp deletion. After consideration of the provided information, the patient declined invasive diagnostic testing in favor of postnatal evaluation to confirm the diagnosis. If the fetus inherits the deletion, early intervention with growth and sex hormone therapies can mitigate short stature and aid development of secondary sexual characteristics. Increasing the use of prenatal cfDNA screening in the general pregnancy population may identify a higher prevalence of