Prenatal Testing for Variants in Genes Associated with Hereditary Cancer Risk: Laboratory Experience and Considerations.

Prenatal molecular genetic testing for familial variants that cause inherited disorders has been performed for decades and is accepted as standard of care. However, the spectrum of genes considered for prenatal testing is expanding because of genetic testing for hereditary cancer risk (HCR) and inclusion of conditions with associated cancer risk in carrier screening panels. A few of these disorders, such as ataxia telangiectasia and Bloom syndrome, include increased cancer risk as part of the phenotype, already meet professional guidelines for prenatal testing, and may be associated with increased cancer risk in heterozygous carriers. In addition, recent studies implicate heterozygosity for variants in lysosomal storage disease genes in HCR etiology. Currently, there is no specific professional guidance regarding prenatal testing for HCR. To determine the prevalence of such testing, we reviewed 1345 consecutive prenatal specimens received in our laboratory for familial variant-specific testing and identified 65 (4.8%) with a known or likely HCR component, plus 210 (15.6%) for lysosomal storage disease. These specimens were classified into five distinct categories for clarity and to enable evaluation. Our experience assessing prenatal specimens for variants associated with HCR, with or without a constitutional phenotype, provides metrics for and contributes to the points to consider in prenatal testing for HCR.

A cryptic microdeletion del(12)(p11.21p11.23) within an unbalanced translocation t(7;12)(q21.13;q23.1) implicates new candidate loci for intellectual disability and Kallmann syndrome.

In a patient diagnosed with both Kallmann syndrome (KS) and intellectual disability (ID), who carried an apparently balanced translocation t(7;12)(q22;q24)dn, array comparative genomic hybridization (aCGH) disclosed a cryptic heterozygous 4.7 Mb deletion del(12)(p11.21p11.23), unrelated to the translocation breakpoint. This novel discovery prompted us to consider the possibility that the combination of KS and neurological disorder in this patient could be attributed to gene(s) within this specific deletion at 12p11.21-12p11.23, rather than disrupted or dysregulated genes at the translocation breakpoints. To further support this hypothesis, we expanded our study by screening five candidate genes at both breakpoints of the chromosomal translocation in a cohort of 48 KS patients. However, no mutations were found, thus reinforcing our supposition. In order to delve deeper into the characterization of the 12p11.21-12p11.23 region, we enlisted six additional patients with small copy number variations (CNVs) and analyzed eight individuals carrying small CNVs in this region from the DECIPHER database. Our investigation utilized a combination of complementary approaches. Firstly, we conducted a comprehensive phenotypic-genotypic comparison of reported CNV cases. Additionally, we reviewed knockout animal models that exhibit phenotypic similarities to human conditions. Moreover, we analyzed reported variants in candidate genes and explored their association with corresponding phenotypes. Lastly, we examined the interacting genes associated with these phenotypes to gain further insights. As a result, we identified a dozen candidate genes: TSPAN11 as a potential KS candidate gene, TM7SF3, STK38L, ARNTL2, ERGIC2, TMTC1, DENND5B, and ETFBKMT as candidate genes for the neurodevelopmental disorder, and INTS13, REP15, PPFIBP1, and FAR2 as candidate genes for KS with ID. Notably, the high-level expression pattern of these genes in relevant human tissues further supported their candidacy. Based on our findings, we propose that dosage alterations of these candidate genes may contribute to sexual and/or cognitive impairments observed in patients with KS and/or ID. However, the confirmation of their causal roles necessitates further identification of point mutations in these candidate genes through next-generation sequencing.

A microdeletion del(12)(p11.21p11.23) with a cryptic unbalanced translocation t(7;12)(q21.13;q23.1) implicates new candidate loci for intellectual disability and Kallmann syndrome.

In an apparently balanced translocation t(7;12)(q22;q24)dn exhibiting both Kallmann syndrome (KS) and intellectual disability (ID), we detected a cryptic heterozygous 4.7 Mb del(12)(p11.21p11.23) unrelated to the translocation breakpoint. This new finding raised the possibility that KS combined with neurological disorder in this patient could be caused by gene(s) within this deletion at 12p11.21-12p11.23 instead of disrupted or dysregulated genes at the genomic breakpoints. Screening of five candidate genes at both breakpoints in 48 KS patients we recruited found no mutation, corroborating our supposition. To substantiate this hypothesis further, we recruited six additional subjects with small CNVs and analyzed eight individuals carrying small CNVs in this region from DECIPHER to dissect 12p11.21-12p11.23. We used multiple complementary approaches including a phenotypic-genotypic comparison of reported cases, a review of knockout animal models recapitulating the human phenotypes, and analyses of reported variants in the interacting genes with corresponding phenotypes. The results identified one potential KS candidate gene ( TSPAN11 ), seven candidate genes for the neurodevelopmental disorder ( TM7SF3 , STK38L , ARNTL2 , ERGIC2 , TMTC1 , DENND5B , and ETFBKMT ), and four candidate genes for KS with ID ( INTS13 , REP15 , PPFIBP1 , and FAR2 ). The high-level expression pattern in the relevant human tissues further suggested the candidacy of these genes. We propose that the dosage alterations of the candidate genes may contribute to sexual and/or cognitive impairment in patients with KS and/or ID. Further identification of point mutations through next generation sequencing will be necessary to confirm their causal roles.

Incidental molecular diagnoses and heterozygous risk alleles in a carrier screening cohort.

PURPOSE: Expanded pan-ethnic carrier screening is an effective tool for the management of reproductive risk. However, growth in the number of conditions screened, in combination with increasingly more comprehensive test methodologies, can lead to the detection of genetic findings that may affect the health of the tested individual. The objective of this study was to investigate the frequency of pathogenic genotypes in a presumed healthy carrier screening cohort to facilitate broader discussions regarding disclosure of genetic information from carrier screening. METHODS: A retrospective analysis of 73,755 targeted carrier screens was performed to identify individuals with pathogenic genotypes and heterozygous risk alleles. RESULTS: In this study, we identified 79 individuals (0.11%) with pathogenic genotypes associated with moderate to profound autosomal recessive or X-linked conditions. In addition, 10 cases had chromosome X dosage abnormalities suggestive of a sex chromosome abnormality. Heterozygote risk alleles represented the majority of ancillary findings in this cohort, including 280 female carriers of FMR1 premutation alleles, 15 heterozygous females with pathogenic DMD variants, and 174 heterozygotes with pathogenic variants in genes that may confer increased risk for somatic malignancies in the heterozygous state. CONCLUSION: These data suggest that nearly 1% of individuals undergoing carrier screening will have a finding that may require clinical evaluation or surveillance.

Screening for autosomal recessive and X-linked conditions during pregnancy and preconception: a practice resource of the American College of Medical Genetics and Genomics (ACMG).

Carrier screening began 50 years ago with screening for conditions that have a high prevalence in defined racial/ethnic groups (e.g., Tay-Sachs disease in the Ashkenazi Jewish population; sickle cell disease in Black individuals). Cystic fibrosis was the first medical condition for which panethnic screening was recommended, followed by spinal muscular atrophy. Next-generation sequencing allows low cost and high throughput identification of sequence variants across many genes simultaneously. Since the phrase "expanded carrier screening" is nonspecific, there is a need to define carrier screening processes in a way that will allow equitable opportunity for patients to learn their reproductive risks using next-generation sequencing technology. An improved understanding of this risk allows patients to make informed reproductive decisions. Reproductive decision making is the established metric for clinical utility of population-based carrier screening. Furthermore, standardization of the screening approach will facilitate testing consistency. This practice resource reviews the current status of carrier screening, provides answers to some of the emerging questions, and recommends a consistent and equitable approach for offering carrier screening to all individuals during pregnancy or preconception.

A customized scaffolds approach for the detection and phasing of complex variants by next-generation sequencing.

Next-generation sequencing (NGS) is widely used in genetic testing for the highly sensitive detection of single nucleotide changes and small insertions or deletions. However, detection and phasing of structural variants, especially in repetitive or homologous regions, can be problematic due to uneven read coverage or genome reference bias, resulting in false calls. To circumvent this challenge, a computational approach utilizing customized scaffolds as supplementary reference sequences for read alignment was developed, and its effectiveness demonstrated with two CBS gene variants: NM_000071.2:c.833T>C and NM_000071.2:c.[833T>C; 844_845ins68]. Variant c.833T>C is a known causative mutation for homocystinuria, but is not pathogenic when in cis with the insertion, c.844_845ins68, because of alternative splicing. Using simulated reads, the custom scaffolds method resolved all possible combinations with 100% accuracy and, based on > 60,000 clinical specimens, exceeded the performance of current approaches that only align reads to GRCh37/hg19 for the detection of c.833T>C alone or in cis with c.844_845ins68. Furthermore, analysis of two 1000 Genomes Project trios revealed that the c.[833T>C; 844_845ins68] complex variant had previously been undetected in these datasets, likely due to the alignment method used. This approach can be configured for existing workflows to detect other challenging and potentially underrepresented variants, thereby augmenting accurate variant calling in clinical NGS testing.

Comparison of pan-ethnic and ethnic-based carrier screening panels for individuals of Ashkenazi Jewish descent.

The intent of carrier screening is to identify individuals at risk for having a child with a genetic disorder. American College of Medical Genetics and Genomics (ACMG) guidelines currently recommend that individuals of Ashkenazi Jewish (AJ) descent be screened for carrier status for nine disorders. However, a joint statement from five professional organizations acknowledges benefits of expanded carrier screening and this is becoming common practice. To better understand the impact of expanded carrier screening for the AJ population, we performed a retrospective analysis comparing detection rates for AJ individuals screened by two targeted panels: a pan-ethnic panel comprising 87 disorders and an AJ panel comprising an 18-disorder subset of the pan-ethnic panel. We also extrapolated the detection rates for the 18 AJ disorders from the pan-ethnic panel data and for the nine ACMG-recommended disorders using data from both panels. We found that with the pan-ethnic panel 431/1150 (37.5%) individuals were carriers of at least one disorder, compared to 319/1248 (25.6%) individuals with the AJ panel. If the pan-ethnic panel cohort were tested in the AJ panel or for the nine ACMG-recommended disorders, the detection rates would have been 280/1150 (24.3%) and 207/1150 (18.0%) respectively. Therefore, the pan-ethnic expanded carrier screening panel of 87 disorders increased the carrier detection rate in AJ individuals by approximately 50% and 100%, respectively, compared with a panel of 18 disorders considered relevant to the AJ population and the ACMG-recommended disorders. Twenty disorders accounted for the difference in carrier detection rates between the pan-ethnic and AJ panels. Of these, three were among the 10 most commonly identified disorders. Our findings reinforce published data that targeted AJ panels are less effective than a pan-ethnic panel in carrier detection among AJ individuals and provide metrics to address the impact of expanded carrier screening in this population.

Translocations disrupting PHF21A in the Potocki-Shaffer-syndrome region are associated with intellectual disability and craniofacial anomalies.

Potocki-Shaffer syndrome (PSS) is a contiguous gene disorder due to the interstitial deletion of band p11.2 of chromosome 11 and is characterized by multiple exostoses, parietal foramina, intellectual disability (ID), and craniofacial anomalies (CFAs). Despite the identification of individual genes responsible for multiple exostoses and parietal foramina in PSS, the identity of the gene(s) associated with the ID and CFA phenotypes has remained elusive. Through characterization of independent subjects with balanced translocations and supportive comparative deletion mapping of PSS subjects, we have uncovered evidence that the ID and CFA phenotypes are both caused by haploinsufficiency of a single gene, PHF21A, at 11p11.2. PHF21A encodes a plant homeodomain finger protein whose murine and zebrafish orthologs are both expressed in a manner consistent with a function in neurofacial and craniofacial development, and suppression of the latter led to both craniofacial abnormalities and neuronal apoptosis. Along with lysine-specific demethylase 1 (LSD1), PHF21A, also known as BHC80, is a component of the BRAF-histone deacetylase complex that represses target-gene transcription. In lymphoblastoid cell lines from two translocation subjects in whom PHF21A was directly disrupted by the respective breakpoints, we observed derepression of the neuronal gene SCN3A and reduced LSD1 occupancy at the SCN3A promoter, supporting a direct functional consequence of PHF21A haploinsufficiency on transcriptional regulation. Our finding that disruption of PHF21A by translocations in the PSS region is associated with ID adds to the growing list of ID-associated genes that emphasize the critical role of transcriptional regulation and chromatin remodeling in normal brain development and cognitive function.

Reporting of diagnostic cytogenetic results.

This appendix, developed by the staff at the Clinical Cytogenetics Laboratory at the Brigham and Women's Hospital, includes a comprehensive list of current "macros" or standardized statements used to facilitate reporting of cytogenetic results. These are provided as a reference for other laboratories. The statements are organized under the general categories of constitutional or acquired abnormalities and subdivided into analysis type (GTG-banding or FISH). Multi-specimen usage macros are included that can be applied to two or more specimen types.

Reporting of diagnostic cytogenetic results.

This appendix, developed by the staff at the Clinical Cytogenetics Laboratory at the Brigham and Women's Hospital, includes a comprehensive list of current "macros" or standardized statements used to facilitate reporting of cytogenetic results. These are provided as a reference for other laboratories. The statements are organized under the general categories of constitutional or acquired abnormalities and subdivided into analysis type (GTG-banding or FISH). Multi-specimen usage macros are included that can be applied to two or more specimen types.

Characterization of apparently balanced chromosomal rearrangements from the developmental genome anatomy project.

Apparently balanced chromosomal rearrangements in individuals with major congenital anomalies represent natural experiments of gene disruption and dysregulation. These individuals can be studied to identify novel genes critical in human development and to annotate further the function of known genes. Identification and characterization of these genes is the goal of the Developmental Genome Anatomy Project (DGAP). DGAP is a multidisciplinary effort that leverages the recent advances resulting from the Human Genome Project to increase our understanding of birth defects and the process of human development. Clinically significant phenotypes of individuals enrolled in DGAP are varied and, in most cases, involve multiple organ systems. Study of these individuals' chromosomal rearrangements has resulted in the mapping of 77 breakpoints from 40 chromosomal rearrangements by FISH with BACs and fosmids, array CGH, Southern-blot hybridization, MLPA, RT-PCR, and suppression PCR. Eighteen chromosomal breakpoints have been cloned and sequenced. Unsuspected genomic imbalances and cryptic rearrangements were detected, but less frequently than has been reported previously. Chromosomal rearrangements, both balanced and unbalanced, in individuals with multiple congenital anomalies continue to be a valuable resource for gene discovery and annotation.

Maternally inherited duplication of chromosome 7, dup(7)(p11.2p12), associated with mild cognitive deficit without features of Silver-Russell syndrome.

We report on a familial duplication in the short arm of chromosome 7, dup(7)(p11.2p12), present in three generations. The duplication was identified by GTG-banding and fluorescence in situ hybridization (FISH) with a whole chromosome 7 DNA painting probe that verified that the duplicated material originated from chromosome 7. The multicolor banding (mBAND) was used to refine the breakpoint assignment. The duplication identified in the proband was also present in her son and mother. All three carriers have mild cognitive deficiencies. Interstitial duplications of the short arm of chromosome 7, although relatively uncommon, have been described in association with a variety of clinical features, including mental retardation of varying severity. Duplication of the p11.2p13 region on chromosome 7 was reported in association with Silver-Russell syndrome (SRS), and an overlapping dup(7)(p11.2p14.1)dn was described in an individual with autistic disorder. Furthermore, a potentially overlapping maternally transmitted inverted duplication, dup(7)(p13p12.2), was reported in patients with cognitive delay. These observations and the phenotype of our duplication carriers suggest that partial trisomy of the proximal 7p region causes cognitive deficiency. The maternal origin of the duplication is of special interest in light of genomic imprinting and implication of the 7p11-p13 region in the SRS etiology. Locus-specific FISH targeting a growth factor receptor binding protein 10 (GRB10), the strong candidate for SRS residing at 7p12.2, showed that it is not duplicated in our patients. Our study helps refine the SRS critical region on 7p and extends our understanding of the clinical manifestations associated with 7p duplications.

Disruption of diacylglycerol kinase delta (DGKD) associated with seizures in humans and mice.

We report a female patient with a de novo balanced translocation, 46,X,t(X;2)(p11.2;q37)dn, who exhibits seizures, capillary abnormality, developmental delay, infantile hypotonia, and obesity. The 2q37 breakpoint observed in association with the seizure phenotype is of particular interest, because it lies near loci implicated in epilepsy in humans and mice. Fluorescence in situ hybridization mapping of the translocation breakpoints showed that no known genes are disrupted at Xp11.2, whereas diacylglycerol kinase delta (DGKD) is disrupted at 2q37. Expression studies in Drosophila and mouse suggest that DGKD is involved in central nervous system development and function. Electroencephalographic assessment of Dgkd mutant mice revealed abnormal epileptic discharges and electrographic seizures in three of six homozygotes. These findings implicate DGKD disruption by the t(X;2)(p11.2;q37)dn in the observed phenotype and support a more general role for DGKD in the etiology of seizures.

Micronuclei with multiple copies of the X chromosome: do chromosomes replicate in micronuclei?

The formation of a micronucleus due to chromosome lagging is a well known mechanism of chromosomal loss. However, the post-mitotic fate of the micronucleus and the chromosomal DNA within it is poorly understood. We observed micronuclei (MN) that had multiple copies of the X chromosome (ranging from 4 to 10) when analyzing cultured human lymphocytes using fluorescence in situ hybridization (FISH). A possible mechanism for this observation is that the chromosome(s) or chromatid(s) contained within the micronuclei successfully completed one or more cycles of replication after their expulsion from the primary nucleus.

Human chromosomes with shorter telomeres and large heterochromatin regions have a higher frequency of acquired somatic cell aneuploidy.

Both telomere shortening and increases in aneuploidy frequencies have been associated with aging. To test if these chromosomal attributes are correlated, chromosome-specific telomere lengths and aneuploidy frequencies were estimated and compared. Aneuploidy frequencies were determined for 10 autosomes (1, 3, 5, 8, 9, 10, 13, 16, 17, 21) and the X chromosome in lymphocytes, and for chromosomes 17 and X in buccal mucosa cells. Overall, chromosomal loss was seen more often than gain in lymphocytes, with the highest loss rates being observed for chromosomes X (3.03%), 17 (2.00%), and the autosomes having large blocks of heterochromatin (1 [1.93%]; 16 [1.53%]; and 9 [1.05%]). The frequencies of loss were significantly lower in the buccal mucosa cells compared to lymphocytes for chromosomes 17 (P = 0.006) and X (P = 0.003). However, the chromosome 17 trisomy frequencies did not vary between tissues. Using a semi-quantitative FISH assay to estimate chromosome-specific telomere length, a significant negative correlation (r = -0.379; P = 0.007) was seen for chromosomal aneuploidy and telomere length, with chromosomes having higher loss rates being noted to have shorter telomeres. Collectively, these studies show that acquired, spontaneous chromosomal loss is associated with multiple factors including the amount of heterochromatin, the chromosome's telomere length, and tissue-specific factors.