FREQMAX provides an alternative approach for determining high-resolution allele frequency thresholds in carrier screening.

As whole-genome data become available for increasing numbers of individuals across diverse populations, the list of genomic variants of unknown significance (VOUS) continues to grow. One powerful tool in VOUS interpretation is determining whether an allele is too common to be considered pathogenic. As genetic and epidemiological parameters vary across disease models, so too does the pathogenic allele frequency threshold for each disease gene. One threshold-setting approach is the maximum credible allele frequency (MCAF) method. However, estimating some of the input values MCAF requires, especially those involving heterogeneity, can present nontrivial statistical challenges. Here, we introduce FREQMAX, our alternative approach for determining allele frequency thresholds in carrier screening. FREQMAX makes efficient use of the data available for well-studied traits and exhibits flexibility for traits where information may be less complete. For cystic fibrosis, more alleles are excluded as benign by FREQMAX than by MCAF. For less-comprehensively characterized traits like ciliary dyskinesia and Smith-Lemli-Opitz syndrome, FREQMAX is able to set the allele frequency threshold without requiring a priori estimates of maximum genetic and allelic contributions. Furthermore, though we describe FREQMAX in the context of carrier screening, its classical population genetics framework also provides context for adaptation to other trait models.

Estimating the relative frequency of leukodystrophies and recommendations for carrier screening in the era of next-generation sequencing.

Leukodystrophies are a heterogeneous group of heritable disorders characterized by abnormal brain white matter signal on magnetic resonance imaging (MRI) and primary involvement of the cellular components of myelin. Previous estimates suggest the incidence of leukodystrophies as a whole to be 1 in 7,000 individuals, however the frequency of specific diagnoses relative to others has not been described. Next generation sequencing approaches offer the opportunity to redefine our understanding of the relative frequency of different leukodystrophies. We assessed the relative frequency of all 30 leukodystrophies (associated with 55 genes) in more than 49,000 exomes. We identified a relatively high frequency of disorders previously thought of as very rare, including Aicardi Goutières Syndrome, TUBB4A-related leukodystrophy, Peroxisomal biogenesis disorders, POLR3-related Leukodystrophy, Vanishing White Matter, and Pelizaeus-Merzbacher Disease. Despite the relative frequency of these conditions, carrier-screening laboratories regularly test only 20 of the 55 leukodystrophy-related genes, and do not test at all, or test only one or a few, genes for some of the higher frequency disorders. Relative frequency of leukodystrophies previously considered very rare suggests these disorders may benefit from expanded carrier screening.

Novel variants in ZNF34 and other brain-expressed transcription factors are shared among early-onset MDD relatives.

There are no known genetic variants with large effects on susceptibility to major depressive disorder (MDD). Although one proposed study approach is to increase sensitivity by increasing sample sizes, another is to focus on families with multiple affected individuals to identify genes with rare or novel variants with strong effects. Choosing the family-based approach, we performed whole-exome analysis on affected individuals (n = 12) across five MDD families, each with at least five affected individuals, early onset, and prepubertal diagnoses. We identified 67 genes where novel deleterious variants were shared among affected relatives. Gene ontology analysis shows that of these 67 genes, 18 encode transcriptional regulators, eight of which are expressed in the human brain, including four KRAB-A box-containing Zn(2+) finger repressors. One of these, ZNF34, has been reported as being associated with bipolar disorder and as differentially expressed in bipolar disorder patients compared to healthy controls. We found a novel variant-encoding a non-conservative P17R substitution in the conserved repressor domain of ZNF34 protein-segregating completely with MDD in all available individuals in the family in which it was discovered. Further analysis showed a common ZNF34 coding indel segregating with MDD in a separate family, possibly indicating the presence of an unobserved, linked, rare variant in that particular family. Our results indicate that genes encoding transcription factors expressed in the brain might be an important group of MDD candidate genes and that rare variants in ZNF34 might contribute to susceptibility to MDD and perhaps other affective disorders.

Pathogenic EFHC1 mutations are tolerated in healthy individuals dependent on reported ancestry.

OBJECTIVE: Screening for specific coding mutations in the EFHC1 gene has been proposed as a means of assessing susceptibility to juvenile myoclonic epilepsy (JME). To clarify the role of these mutations, especially those reported to be highly penetrant, we sought to measure the frequency of exonic EFHC1 mutations across multiple population samples. METHODS: To find and test variants of large effect, we sequenced all EFHC1 exons in 23 JME and 23 non-JME idiopathic generalized epilepsy (IGE) Hispanic patients, and 60 matched controls. We also genotyped specific EFHC1 variants in IGE cases and controls from multiple ethnic backgrounds, including 17 African American IGE patients, with 24 matched controls, and 92 Caucasian JME patients with 103 matched controls. These variants are reported to be pathogenic, but are also found among unphenotyped individuals in public databases. All subjects were from the New York City metro area and all controls were required to have no family history of seizures. RESULTS: We found the reportedly pathogenic EFHC1 P77T-R221H (rs149055334-rs79761183) JME haplotype in one Hispanic control and in two African American controls. Public databases also show that the EFHC1 P77T-R221H JME haplotype is present in unphenotyped West African ancestry populations, and we show that it can be found at appreciable frequency in healthy individuals with no family history of epilepsy. We also found a novel splice-site mutation in a single Hispanic JME patient, the effect of which is unknown. SIGNIFICANCE: Our findings raise questions about the effect of reportedly pathogenic EFHC1 mutations on JME. One intriguing possibility is that some EFHC1 mutations may be pathogenic only when introduced into specific genetic backgrounds. By focusing on data from multiple populations, including the understudied Hispanic and Black/African American populations, our study highlights that for complex traits like JME, the body of evidence necessary to infer causality is high.

Multiple Myeloma Risk and Outcomes are Associated with Pathogenic Germline Variants in DNA Repair Genes.

First-degree relatives of multiple myeloma (MM) patients are at increased risk for MM, but the contribution of pathogenic germline variants (PGVs) in hereditary cancer genes to MM risk and outcomes is not well characterized. To address this, we analyzed germline exomes in two independent cohorts of 895 and 786 MM patients. PGVs were identified in 8.6% of the discovery cohort and 11.5% of the replication cohort, with a notable presence of high or moderate-penetrance PGVs (PGV-As) in DNA repair genes (3.6% and 4.1%, respectively). PGVs in BRCA1 (OR=3.9, FDR<0.01) and BRCA2 (OR=7.0, FDR<0.001) were significantly enriched in MM patients compared to 134,187 healthy controls. Five of the eight BRCA2 PGV carriers exhibited tumor-specific copy number loss in BRCA2, suggesting somatic loss of heterozygosity. PGV-As were associated with younger age at diagnosis, personal or familial cancer history, and longer progression-free survival after upfront high-dose melphalan and autologous stem cell transplant (p<0.01).

Obtaining accurate p values from a dense SNP linkage scan.

A major concern of resequencing studies is that the pathogenicity of most mutations is difficult to predict. To address this concern, linkage (i.e. co-segregation) analysis is often used to exclude neutral mutations and to better predict pathogenicity among the candidate mutations that remain. However, when linkage disequilibrium (LD) is present in the population but ignored in the analysis, unlinked regions with high LD can inflate the type 1 error and thousands of neutral mutations may be mistakenly included in a follow-up resequencing study, which could dramatically reduce the power to identify causal variants. To illustrate the need for concern, we simulated data on a sparsely spaced panel of single nucleotide polymorphisms (average spacing 1.27 cM) using an LD pattern estimated from real data. In our simulations, we find that the type 1 error of the maximum LOD can be as high as 14%. Therefore, to control the type 1 error of linkage tests we created Haplodrop - a fast and flexible simulation program that generates the haplotypes of founders with LD and then 'drops' these haplotypes with recombination to all non-founders in the pedigree. Haplodrop can be used to control the type 1 error of any linkage test, agrees well with existing software, accommodates arbitrary pedigree structures, and scales easily to the whole genome. Moreover, by correctly excluding mutations that lie in unlinked regions with high LD, Haplodrop should aid significantly in reducing the multiple testing burden of follow-up resequencing studies.

Convergent evolution of chromosomal sex-determining regions in the animal and fungal kingdoms.

Sexual identity is governed by sex chromosomes in plants and animals, and by mating type (MAT) loci in fungi. Comparative analysis of the MAT locus from a species cluster of the human fungal pathogen Cryptococcus revealed sequential evolutionary events that fashioned this large, highly unusual region. We hypothesize that MAT evolved via four main steps, beginning with acquisition of genes into two unlinked sex-determining regions, forming independent gene clusters that then fused via chromosomal translocation. A transitional tripolar intermediate state then converted to a bipolar system via gene conversion or recombination between the linked and unlinked sex-determining regions. MAT was subsequently subjected to intra- and interallelic gene conversion and inversions that suppress recombination. These events resemble those that shaped mammalian sex chromosomes, illustrating convergent evolution in sex-determining structures in the animal and fungal kingdoms.

Designing case-control studies: decisions about the controls.

The authors quantified, first, the effect of misclassified controls (i.e., individuals who are affected with the disease under study but who are classified as controls) on the ability of a case-control study to detect an association between a disease and a genetic marker, and second, the effect of leaving misclassified controls in the study, as opposed to removing them (thus decreasing sample size). The authors developed an informativeness measure of a study's ability to identify real differences between cases and controls. They then examined this measure's behavior when there are no misclassified controls, when there are misclassified controls, and when there were misclassified controls but they have been removed from the study. The results show that if, for example, 10% of controls are misclassified, the study's informativeness is reduced to approximately 81% of what it would have been in a sample with no misclassified controls, whereas if these misclassified controls are removed from the study, the informativeness is only reduced to about 90%, despite the reduced sample size. If 25% are misclassified, those figures become approximately 56% and 75%, respectively. Thus, leaving the misclassified controls in the control sample is worse than removing them altogether. Finally, the authors illustrate how insufficient power is not necessarily circumvented by having an unlimited number of controls. The formulas provided by the authors enable investigators to make rational decisions about removing misclassified controls or leaving them in.

A survey of putative anxiety-associated genes in panic disorder patients with and without bladder symptoms.

BACKGROUND: We have previously described a subtype of panic disorder (PD) that we termed 'bladder syndrome', characterized by urological and bladder symptoms (and possibly interstitial cystitis) in the patients and/or their family members and confirmed the validity of this subset in family linkage and association analysis. In this study, we determine (a) whether 20 single-nucleotide polymorphisms (SNPs) reported in the literature can be replicated in a new PD dataset and (b) whether dividing the sample into those with and without the 'bladder syndrome' can help to resolve the genetic heterogeneity within this new sample. METHODS: We selected 20 putative associated SNPs from the literature, taken from studies published since 2004. We tested these SNPs for association in a sample of 351 PD patients and 552 controls, and then divided them into subgroups of 92 patients from bladder families and 259 from nonbladder families. RESULTS: (a) When analyzed in all PD patients, none of the 20 SNPs appeared to be replicated (except for SLC6A4 from our previous study, but in a sample that overlaps substantially with that in our previous report). (b) However, some intriguing findings emerged when we separated bladder from nonbladder families: SLC6A4, reported by us previously, yielded stronger evidence than before (P=0.0018) when examined only in nonbladder families, and in contrast, is not statistically significant in bladder families. Two other markers yielded nominally significant results in bladder families - rs5751876 in ADORA2A (P=0.046) and rs12579350 in TMEM16B (P=0.035) - but were not significant in nonbladder families. (c) Two markers had noticeably lower P-values when we differentiated the women and analyzed them separately - rs12579350 in TMEM16B (P-value decreased from 0.035, as above, to 0.00055) and a different SNP in ADORA2A, rs4822492 (P-value decreases from 0.07 to 0.028). SIGNIFICANCE: Our results indicate that most of the 20 reported associations do not hold up when PD is analyzed as one group. However, our findings provide further evidence that PD with bladder symptoms may be genetically different from PD without bladder. We suggest that it is worth pursuing SLC6A4 in nonbladder PD, and ADORA2A and TMEM16B in bladder PD. Also, the possibility of a male-female difference in PD is worth pursuing. We also briefly discuss issues of replication and multiple tests.

Recapitulation of the sexual cycle of the primary fungal pathogen Cryptococcus neoformans var. gattii: implications for an outbreak on Vancouver Island, Canada.

Cryptococcus neoformans is a human fungal pathogen that exists as three distinct varieties or sibling species: the predominantly opportunistic pathogens C. neoformans var. neoformans (serotype D) and C. neoformans var. grubii (serotype A) and the primary pathogen C. neoformans var. gattii (serotypes B and C). While serotypes A and D are cosmopolitan, serotypes B and C are typically restricted to tropical regions. However, serotype B isolates of C. neoformans var. gattii have recently caused an outbreak on Vancouver Island in Canada, highlighting the threat of this fungus and its capacity to infect immunocompetent individuals. Here we report a large-scale analysis of the mating abilities of serotype B and C isolates from diverse sources and identify unusual strains that mate robustly and are suitable for further genetic analysis. Unlike most isolates, which are of both the a and alpha mating types but are predominantly sterile, the majority of the Vancouver outbreak strains are exclusively of the alpha mating type and the majority are fertile. In an effort to enhance mating of these isolates, we identified and disrupted the CRG1 gene encoding the GTPase-activating protein involved in attenuating pheromone response. crg1 mutations dramatically increased mating efficiency and enabled mating with otherwise sterile isolates. Our studies provide a genetic and molecular foundation for further studies of this primary pathogen and reveal that the Vancouver Island outbreak may be attributable to a recent recombination event.