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.

The Ashkenazi Jewish carrier screening panel: evolution, status quo, and disparities.

OBJECTIVE: Although prenatal/preconception carrier screening recommendations for individuals of Ashkenazi Jewish descent (AJ) were published by American College of Medical Genetics and Genomics (2008) and American College of Obstetrics and Gynecology (2009), scientific advances have led to widely varied screening panels. Mutation carrier frequencies are sometimes based on small, homogeneous AJ populations. This study sought to update the state of AJ screening for the obstetrician by assessing laboratory screening panel compositions as well as assessing literature and laboratory carrier frequencies for common AJ mutations. METHODS: A literature review (1991-2013) was performed for AJ disease carrier frequencies. AJ screening data from six screening laboratories were collected. AJ panel composition was compared across 16 commercial and academic laboratories. RESULTS: Overall literature and laboratory carrier frequencies of AJ mutations were similar, although the Walker-Warburg syndrome laboratory carrier frequency was almost twice that in the literature. Laboratory AJ disease panel composition varied widely, from 8 to 25 diseases. CONCLUSIONS: Current AJ panels vary widely by laboratory, resulting in disparate levels of screening. Consideration of an updated professional standard for prenatal/preconception AJ screening based on carrier frequency rates, level of disease burden, availability of screening, and cost of technology may be useful in providing equitable and appropriate care for those planning a pregnancy.

Cystic fibrosis carrier testing in an ethnically diverse US population.

BACKGROUND: The incidence of cystic fibrosis (CF) and the frequency of specific disease-causing mutations vary among populations. Affected individuals experience a range of serious clinical consequences, notably lung and pancreatic disease, which are only partially dependent on genotype. METHODS: An allele-specific primer-extension reaction, liquid-phase hybridization to a bead array, and subsequent fluorescence detection were used in testing for carriers of 98 CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] mutations among 364 890 referred individuals with no family history of CF. RESULTS: One in 38 individuals carried one of the 98 CFTR mutations included in this panel. Of the 87 different mutations detected, 18 were limited to a single ethnic group. African American, Hispanic, and Asian individuals accounted for 33% of the individuals tested. The mutation frequency distribution of Caucasians was significantly different from that of each of these ethnic groups (P < 1 × 10⁻¹°). CONCLUSIONS: Carrier testing using a broad mutation panel detects differences in the distribution of mutations among ethnic groups in the US.

A Role for Chromosomal Microarray Testing in the Workup of Male Infertility.

Genetic analysis is a critical component in the male infertility workup. For male infertility due to oligospermia/azoospermia, standard guidelines recommend karyotype and Y-chromosome microdeletion analyses. A karyotype is used to identify structural and numerical chromosome abnormalities, whereas Y-chromosome microdeletions are commonly evaluated by multiplex PCR analysis because of their submicroscopic size. Because these assays often require different Vacutainer tubes to be sent to different laboratories, ordering is prone to errors. In addition, this workflow limits the ability for sequential testing and a comprehensive test result. A potential solution includes performing Y-microdeletion and numerical chromosome analysis-the most common genetic causes of oligospermia/azoospermia-by chromosomal microarray (CMA) and reflexing to karyotype as both assays are often offered in the cytogenetics laboratory. Such analyses can be performed using one sodium heparin Vacutainer tube sample. To determine the effectiveness of CMA for the detection of clinically significant Y-chromosome microdeletions, 21 cases with known Y microdeletions were tested by CytoScan HD platform. CMA studies identified all known Y-chromosome microdeletions, and in 11 cases (52%) identified additional clinically important cytogenetic anomalies, including six cases of 46, XX males, one case of isodicentric Y, two cases of a dicentric Y, and three cases of terminal Yq deletions. These findings demonstrate that this testing strategy would simplify ordering and allow for an integrated interpretation of test results.

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.

High allelic heterogeneity between Afro-Brazilians and Euro-Brazilians impacts cystic fibrosis genetic testing.

Cystic fibrosis (CF) is an autosomal recessive disease caused by at least 1,000 different mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). To determine the frequency of 70 common worldwide CFTR mutations in 155 Euro-Brazilian CF patients and in 38 Afro-Brazilian CF patients, we used direct PCR amplification of DNA from a total of 386 chromosomes from CF patients born in three different states of Brazil. The results show that screening for seventy mutations accounts for 81% of the CF alleles in Euro-Brazilians, but only 21% in the Afro-Brazilian group. We found 21 different mutations in Euro-Brazilians and only 7 mutations in Afro-Brazilians. The frequency of mutations and the number of different mutations detected in Euro-Brazilians are different from Northern European and North American populations, but similar to Southern European populations; in Afro-Brazilians, the mix of CF-mutations is different from those reported in Afro-American CF patients. We also found significant differences in detection rates between Euro-Brazilian (75%) and Afro-Brazilian CF patients (21%) living in the same state, Minas Gerais. These results, therefore, have implications for the use of DNA-based tests for risk assessment in heterogeneous populations like the Brazilians. Further studies are needed to identify the remaining CF mutations in the different populations and regions of Brazil.

Simultaneous detection of multiple point mutations using allele-specific oligonucleotides.

This unit describes high-throughput mutation analysis using hybridization with pooled allele-specific oligonucleotide (ASO) probes. The approach can be used to screen one gene for many allelic mutations or to screen several loci for several allelic mutations each. Because tetramethyl ammonium chloride (TMAC) is added to the hybridization solution, the melting temperature of each oligonucleotide is independent of G-C content and oligonucleotides of the same length can be hybridized simultaneously. The pooled probes will give a positive hybridization signal from any PCR-amplified DNA sample containing a sequence complementary to any of the ASOs in the pool of oligonucleotide sequences. If many PCR-amplified samples are spotted onto a single membrane, multiple individuals can then be screened simultaneously for many mutant sequences. This multiple ASO hybridization technique is appropriate only for circumstances when hybridization with any one of the pooled probes is expected to be uncommon.

The I148T CFTR allele occurs on multiple haplotypes: a complex allele is associated with cystic fibrosis.

PURPOSE: To determine whether intragenic changes modulate the cystic fibrosis (CF) phenotype in individuals who are positive for the I148T allele. METHODS: The genes from individuals who carried at least one copy of the I148T allele were analyzed for additional changes that may be acting as genetic modifiers. RESULTS: Seven of eight individuals with a known or suspected diagnosis of CF who carried I148T in combination with a severe CF mutation also carried 3199del6. Eight apparently healthy adult individuals who were compound heterozygous for I148T and a severe CF mutation or homozygous for I148T did not carry the deletion ( = 0.0014). The I148T allele occurs on at least three haplotypes: an IVS-8 9T background, a 7T background, or a 9T + 3199del6 background. The 3199del6 allele was not identified in 386 non-CF chromosomes. CONCLUSIONS: It is concluded that I148T occurs on at least three haplotypes and the complex allele I148T + 9T + 3199del6 is associated with a classic CF phenotype.