Recurring germline mosaicism in a family due to reversion of an inherited derivative chromosome 8 from an 8;21 translocation with interstitial telomeric sequences.

BACKGROUND: Mosaicism for chromosomal structural abnormalities, other than marker or ring chromosomes, is rarely inherited. METHODS: We performed cytogenetics studies and breakpoint analyses on a family with transmission of mosaicism for a derivative chromosome 8 (der(8)), resulting from an unbalanced translocation between the long arms of chromosomes 8 and 21 over three generations. RESULTS: The proband and his maternal half-sister had mosaicism for a der(8) cell line leading to trisomy of the distal 21q, and both had Down syndrome phenotypic features. Mosaicism for a cell line with the der(8) and a normal cell line was also detected in a maternal half-cousin. The der(8) was inherited from the maternal grandmother who had four abnormal cell lines containing the der(8), in addition to a normal cell line. One maternal half-aunt had the der(8) and an isodicentric chromosome 21 (idic(21)). Sequencing studies revealed microhomologies at the junctures of the der(8) and idic(21) in the half-aunt, suggesting a replicative mechanism in the rearrangement formation. Furthermore, interstitial telomeric sequences (ITS) were identified in the juncture between chromosomes 8 and 21 in the der(8). CONCLUSION: Mosaicism in the proband, his half-sister and half-cousin resulting from loss of chromosome 21 material from the der(8) appears to be a postzygotic event due to the genomic instability of ITS and associated with selective growth advantage of normal cells. The reversion of the inherited der(8) to a normal chromosome 8 in this family resembles revertant mosaicism of point mutations. We propose that ITS could mediate recurring revertant mosaicism for some constitutional chromosomal structural abnormalities.

CNVs cause autosomal recessive genetic diseases with or without involvement of SNV/indels.

PURPOSE: Improved resolution of molecular diagnostic technologies enabled detection of smaller sized exonic level copy-number variants (CNVs). The contribution of CNVs to autosomal recessive (AR) conditions may be better recognized using a large clinical cohort. METHODS: We retrospectively investigated the CNVs' contribution to AR conditions in cases subjected to chromosomal microarray analysis (CMA, N = ~70,000) and/or clinical exome sequencing (ES, N = ~12,000) at Baylor Genetics; most had pediatric onset neurodevelopmental disorders. RESULTS: CNVs contributed to biallelic variations in 87 cases, including 81 singletons and three affected sibling pairs. Seventy cases had CNVs affecting both alleles, and 17 had a CNV and a single-nucleotide variant (SNV)/indel in trans. In total, 94.3% of AR-CNVs affected one gene; among these 41.4% were single-exon and 35.0% were multiexon partial-gene events. Sixty-nine percent of homozygous AR-CNVs were embedded in homozygous genomic intervals. Five cases had large deletions unmasking an SNV/indel on the intact allele for a recessive condition, resulting in multiple molecular diagnoses. CONCLUSIONS: AR-CNVs are often smaller in size, transmitted through generations, and underrecognized due to limitations in clinical CNV detection methods. Our findings from a large clinical cohort emphasized integrated CNV and SNV/indel analyses for precise clinical and molecular diagnosis especially in the context of genomic disorders.

Chromosomal microarray analysis on uncultured chorionic villus sampling can be complicated by confined placental mosaicism for aneuploidy and microdeletions.

OBJECTIVE: This study aims to establish the incidence and implications of confined placental mosaicism (CPM) in the context of prenatal chromosomal microarray analysis (CMA). METHODS: We retrospectively reviewed prenatal array data on 1382 consecutive chorionic villus sampling (CVS) specimens spanning the past 6 years, focusing on those for which whole CVS biopsy (both cytotrophoblast and mesenchymal cells) was used for CMA and cultured cells (primarily mesenchyme) was also analyzed or amniotic fluid (AF)/newborn blood was used for confirmation, to determine the frequency of mosaic abnormal findings that were the result of CPM. RESULTS: Out of a total of 1382 consecutive CVS cases, we identified 42 (42/1382 = 3.0%) cases with abnormal array findings suggestive of mosaicism. Among them, 10 cases were unequivocally interpreted as CPM based on a normal AF/newborn blood confirmatory result. In addition, another 10 cases were interpreted as provisional CPM based on normal results on cultured cells. Notably, 40% (8/20) of the cases revealed complex findings, including multiple mosaic aneuploidies, mosaic submicroscopic copy number variation (CNV), and mosaic aneuploidy plus mosaic CNV. CONCLUSION: Abnormal CMA results from CVS specimens should be interpreted with caution when mosaicism is evident or suspected. Furthermore, confirmatory testing on amniotic fluid, which contains cells derived from the fetus, is recommended in these cases.

Positive predictive value estimates for cell-free noninvasive prenatal screening from data of a large referral genetic diagnostic laboratory.

BACKGROUND: Since its debut in 2011, cell-free fetal DNA screening has undergone rapid expansion with respect to both utilization and coverage. However, conclusive data regarding the clinical validity and utility of this screening tool, both for the originally included common autosomal and sex-chromosomal aneuploidies as well as the more recently added chromosomal microdeletion syndromes, have lagged behind. Thus, there is a continued need to educate clinicians and patients about the current benefits and limitations of this screening tool to inform pre- and posttest counseling, pre/perinatal decision making, and medical risk assessment/management. OBJECTIVE: The objective of this study was to determine the positive predictive value and false-positive rates for different chromosomal abnormalities identified by cell-free fetal DNA screening using a large data set of diagnostic testing results on invasive samples submitted to the laboratory for confirmatory studies. STUDY DESIGN: We tested 712 patient samples sent to our laboratory to confirm a cell-free fetal DNA screening result, indicating high risk for a chromosome abnormality. We compiled data from all cases in which the indication for confirmatory testing was a positive cell-free fetal DNA screen, including the common trisomies, sex chromosomal aneuploidies, microdeletion syndromes, and other large genome-wide copy number abnormalities. Testing modalities included fluorescence in situ hybridization, G-banded karyotype, and/or chromosomal microarray analysis performed on chorionic villus samples, amniotic fluid, or postnatally obtained blood samples. Positive predictive values and false-positive rates were calculated from tabulated data. RESULTS: The positive predictive values for trisomy 13, 18, and 21 were consistent with previous reports at 45%, 76%, and 84%, respectively. For the microdeletion syndrome regions, positive predictive values ranged from 0% for detection of Cri-du-Chat syndrome and Prader-Willi/Angelman syndrome to 14% for 1p36 deletion syndrome and 21% for 22q11.2 deletion syndrome. Detection of sex chromosomal aneuploidies had positive predictive values of 26% for monosomy X, 50% for 47,XXX, and 86% for 47,XXY. CONCLUSION: The positive predictive values for detection of common autosomal and sex chromosomal aneuploidies by cell-free fetal DNA screening were comparable with other studies. Identification of microdeletions was associated with lower positive predictive values and higher false-positive rates, likely because of the low prevalence of the individual targeted microdeletion syndromes in the general population. Although the obtained positive predictive values compare favorably with those seen in traditional screening approaches for common aneuploidies, they highlight the importance of educating clinicians and patients on the limitations of cell-free fetal DNA screening tests. Improvement of the cell-free fetal DNA screening technology and continued monitoring of its performance after introduction into clinical practice will be important to fully establish its clinical utility. Nonetheless, our data provide valuable information that may aid result interpretation, patient counseling, and clinical decision making/management.

Capture-based high-coverage NGS: a powerful tool to uncover a wide spectrum of mutation types.

PURPOSE: Next-generation sequencing (NGS) has been widely applied to clinical diagnosis. Target-gene capture followed by deep sequencing provides unbiased enrichment of the target sequences, which not only accurately detects single-nucleotide variations (SNVs) and small insertion/deletions (indels) but also provides the opportunity for the identification of exonic copy-number variants (CNVs) and large genomic rearrangements. METHOD: Capture NGS has the ability to easily detect SNVs and small indels. However, genomic changes involving exonic deletions/duplications and chromosomal rearrangements require more careful analysis of captured NGS data. Misaligned raw sequence reads may be more than just bad data. Some mutations that are difficult to detect are filtered by the preset analytical parameters. "Loose" filtering and alignment conditions were used for thorough analysis of the misaligned NGS reads. Additionally, using an in-house algorithm, NGS coverage depth was thoroughly analyzed to detect CNVs. RESULTS: Using real examples, this report underscores the importance of the accessibility to raw sequence data and manual review of suspicious sequence regions to avoid false-negative results in the clinical application of NGS. Assessment of the NGS raw data generated by the use of loose filtering parameters identified several sequence aberrations, including large indels and genomic rearrangements. Furthermore, NGS coverage depth analysis identified homozygous and heterozygous deletions involving single or multiple exons. CONCLUSION: Our results demonstrate the power of deep NGS in the simultaneous detection of point mutations and intragenic exonic deletion in one comprehensive step.Genet Med 18 5, 513-521.

Impact of heterozygote CFTR mutations in COPD patients with chronic bronchitis.

BACKGROUND: Cigarette smoking causes Chronic Obstructive Pulmonary Disease (COPD), the 3rd leading cause of death in the U.S. CFTR ion transport dysfunction has been implicated in COPD pathogenesis, and is associated with chronic bronchitis. However, susceptibility to smoke induced lung injury is variable and the underlying genetic contributors remain unclear. We hypothesized that presence of CFTR mutation heterozygosity may alter susceptibility to cigarette smoke induced CFTR dysfunction. Consequently, COPD patients with chronic bronchitis may have a higher rate of CFTR mutations compared to the general population. METHODS: Primary human bronchial epithelial cells derived from F508del CFTR heterozygotes and mice with (CFTR+/-) and without (CFTR+/+) CFTR heterozygosity were exposed to whole cigarette smoke (WCS); CFTR-dependent ion transport was assessed by Ussing chamber electrophysiology and nasal potential difference measurements, respectively. Caucasians with COPD and chronic bronchitis, age 40 to 80 with FEV1/FVC < 0.70 and FEV1 < 60% predicted, were selected for genetic analysis from participants in the NIH COPD Clinical Research Network's Azithromycin for Prevention of Exacerbations of COPD in comparison to 32,900 Caucasian women who underwent prenatal genetic testing. Genetic analysis involved an allele-specific genotyping of 89 CFTR mutations. RESULTS: Exposure to WCS caused a pronounced reduction in CFTR activity in both CFTR (+/+) cells and F508del CFTR (+/-) cells; however, neither the degree of decrement (44.7% wild-type vs. 53.5% F508del heterozygous, P = NS) nor the residual CFTR activity were altered by CFTR heterozygosity. Similarly, WCS caused a marked reduction in CFTR activity measured by NPD in both wild type and CFTR heterozygous mice, but the severity of decrement (91.1% wild type vs. 47.7% CF heterozygous, P = NS) and the residual activity were not significantly affected by CFTR genetic status. Five of 127 (3.9%) COPD patients with chronic bronchitis were heterozygous for CFTR mutations which was not significantly different from controls (4.5%) (P = NS). CONCLUSIONS: The magnitude of WCS induced reductions in CFTR activity was not affected by the presence of CFTR mutation heterozygosity. CFTR mutations do not increase the risk of COPD with chronic bronchitis. CFTR dysfunction due to smoking is primarily an acquired phenomenon and is not affected by the presence of congenital CFTR mutations.

Complex rearrangements in patients with duplications of MECP2 can occur by fork stalling and template switching.

Duplication at the Xq28 band including the MECP2 gene is one of the most common genomic rearrangements identified in neurodevelopmentally delayed males. Such duplications are non-recurrent and can be generated by a non-homologous end joining (NHEJ) mechanism. We investigated the potential mechanisms for MECP2 duplication and examined whether genomic architectural features may play a role in their origin using a custom designed 4-Mb tiling-path oligonucleotide array CGH assay. Each of the 30 patients analyzed showed a unique duplication varying in size from approximately 250 kb to approximately 2.6 Mb. Interestingly, in 77% of these non-recurrent duplications, the distal breakpoints grouped within a 215 kb genomic interval, located 47 kb telomeric to the MECP2 gene. The genomic architecture of this region contains both direct and inverted low-copy repeat (LCR) sequences; this same region undergoes polymorphic structural variation in the general population. Array CGH revealed complex rearrangements in eight patients; in six patients the duplication contained an embedded triplicated segment, and in the other two, stretches of non-duplicated sequences occurred within the duplicated region. Breakpoint junction sequencing was achieved in four duplications and identified an inversion in one patient, demonstrating further complexity. We propose that the presence of LCRs in the vicinity of the MECP2 gene may generate an unstable DNA structure that can induce DNA strand lesions, such as a collapsed fork, and facilitate a Fork Stalling and Template Switching event producing the complex rearrangements involving MECP2.

De novo variants in H3-3A and H3-3B are associated with neurodevelopmental delay, dysmorphic features, and structural brain abnormalities.

The histone H3 variant H3.3, encoded by two genes H3-3A and H3-3B, can replace canonical isoforms H3.1 and H3.2. H3.3 is important in chromatin compaction, early embryonic development, and lineage commitment. The role of H3.3 in somatic cancers has been studied extensively, but its association with a congenital disorder has emerged just recently. Here we report eleven de novo missense variants and one de novo stop-loss variant in H3-3A (n = 6) and H3-3B (n = 6) from Baylor Genetics exome cohort (n = 11) and Matchmaker Exchange (n = 1), of which detailed phenotyping was conducted for 10 individuals (H3-3A = 4 and H3-3B = 6) that showed major phenotypes including global developmental delay, short stature, failure to thrive, dysmorphic facial features, structural brain abnormalities, hypotonia, and visual impairment. Three variant constructs (p.R129H, p.M121I, and p.I52N) showed significant decrease in protein expression, while one variant (p.R41C) accumulated at greater levels than wild-type control. One H3.3 variant construct (p.R129H) was found to have stronger interaction with the chaperone death domain-associated protein 6.

Comprehensive 5-year study of cytogenetic aberrations in 668 infertile men.

PURPOSE: The causes of male infertility are heterogeneous but more than 50% of cases have a genetic basis. Specific genetic defects have been identified in less than 20% of infertile males and, thus, most causes remain to be elucidated. The most common cytogenetic defects associated with nonobstructive azoospermia are numerical and structural chromosome abnormalities, including Klinefelter syndrome (47,XXY) and Y chromosome microdeletions. To refine the incidence and nature of chromosomal aberrations in males with infertility we reviewed cytogenetic results in 668 infertile men with oligozoospermia and azoospermia. MATERIALS AND METHODS: High resolution Giemsa banding chromosome analysis and/or fluorescence in situ hybridization were done in 668 infertile males referred for routine cytogenetic analysis between January 2004 and March 2009. RESULTS: The overall incidence of chromosomal abnormalities was about 8.2%. Of the 55 patients with abnormal cytogenetic findings sex chromosome aneuploidies were observed in 29 (53%), including Klinefelter syndrome in 27 (49%). Structural chromosome abnormalities involving autosomes (29%) and sex chromosomes (18%) were detected in 26 infertile men. Abnormal cytogenetic findings were observed in 35 of 264 patients (13.3%) with azoospermia and 19 of 365 (5.2%) with oligozoospermia. CONCLUSIONS: Structural chromosomal defects and low level sex chromosome mosaicism are common in oligozoospermia cases. Extensive cytogenetic assessment and fluorescence in situ hybridization may improve the detection rate in males with oligozoospermia. These findings highlight the need for efficient genetic testing in infertile men so that couples may make informed decisions on assisted reproductive technologies to achieve parenthood.

Publisher Correction: Non-invasive prenatal sequencing for multiple Mendelian monogenic disorders using circulating cell-free fetal DNA.

In the version of this article originally published, some cases that were presented in Fig. 3 should have been underlined but were not. The appropriate cases have now been underlined. The error has been corrected in the print, PDF and HTML versions of the article.

Non-invasive prenatal sequencing for multiple Mendelian monogenic disorders using circulating cell-free fetal DNA.

Current non-invasive prenatal screening is targeted toward the detection of chromosomal abnormalities in the fetus1,2. However, screening for many dominant monogenic disorders associated with de novo mutations is not available, despite their relatively high incidence3. Here we report on the development and validation of, and early clinical experience with, a new approach for non-invasive prenatal sequencing for a panel of causative genes for frequent dominant monogenic diseases. Cell-free DNA (cfDNA) extracted from maternal plasma was barcoded, enriched, and then analyzed by next-generation sequencing (NGS) for targeted regions. Low-level fetal variants were identified by a statistical analysis adjusted for NGS read count and fetal fraction. Pathogenic or likely pathogenic variants were confirmed by a secondary amplicon-based test on cfDNA. Clinical tests were performed on 422 pregnancies with or without abnormal ultrasound findings or family history. Follow-up studies on cases with available outcome results confirmed 20 true-positive, 127 true-negative, zero false-positive, and zero-false negative results. The initial clinical study demonstrated that this non-invasive test can provide valuable molecular information for the detection of a wide spectrum of dominant monogenic diseases, complementing current screening for aneuploidies or carrier screening for recessive disorders.

Mosaicism for r(X) and der(X)del(X)(p11.23)dup(X)(p11.21p11.22) provides insight into the possible mechanism of rearrangement.

We report a patient with a unique and complex cytogenetic abnormality involving mosaicism for a small ring X and deleted Xp derivative chromosome with tandem duplication at the break point. The patient presented with failure to thrive, muscular hypotonia, and minor facial anatomic anomalies, all concerning for Turner syndrome. Brain MRI revealed mild thinning of the corpus callosum, an apparent decrease in ventricular white matter volume, and an asymmetric myelination pattern. Array comparative genome hybridization analysis revealed mosaicism for the X chromosome, deletion of the short arm of an X chromosome, and a duplication of chromosome region Xp11.21-p11.22. G-banded chromosome and FISH analyses revealed three abnormal cell lines: 46,X,der(X)del(X)(p11.23)dup(X)(p11.21p11.22)/46,X,r(X)(q11.1q13.1)/45,X. The small ring X chromosome was estimated to be 5.2 Mb in size and encompassed the centromere and Xq pericentromeric region. X chromosome inactivation (XCI) studies demonstrated a skewed pattern suggesting that the ring X remained active, likely contributing to the observed clinical features of brain dysmyelination. We hypothesize that a prezygotic asymmetric crossing over within a loop formed during meiosis in an X chromosome with a paracentric inversion resulted in an intermediate dicentric chromosome. An uneven breakage of the dicentric chromosome in the early postzygotic period might have resulted in the formation of one cell line with the X chromosome carrying a terminal deletion and pericentromeric duplication of the short arm and the second cell line with the X chromosome carrying a complete deletion of Xp. The cell line carrying the deletion of Xp could have then stabilized through self-circularization and formation of the ring X chromosome.