Janet Markle

The mutation significance cutoff: gene-level thresholds for variant predictions

Next-generation sequencing (NGS) has made it possible to identify about 20,000 variants in the protein-coding exome of each individual, of which only a few are likely to underlie a genetic disease. Variant-level methods such as PolyPhen-2, SIFT and CADD are useful for obtaining a prediction as to whether a given variant is benign/damaging1–3 or tolerant/intolerant1–3 (we hereafter use the terms benign/deleterious). These methods are commonly interpreted in a binary manner for filtering out benign variants from NGS data, with a single significance cutoff value across all protein-coding genes. PolyPhen-2 and SIFT integrate the fixed cutoff in the software. CADD proposed (but did not recommend for categorical usage) the fixed value of 15 (or another value between 10 and 20). Gene-level methods, such as RVIS, de novo excess and GDI are also useful4–6. Combining fixed gene-level and variant-level cutoffs is also applied in the RVIS hot zone approach4. However, owing to the diversity of medical and population genetic features between human genes and across populations, a uniform cutoff is unlikely to be accurate genome-wide.

The human gene damage index as a gene-level approach to prioritizing exome variants

Significance The protein-coding exome of a patient with a monogenic disease contains about 20,000 variations, of which only one or two are disease causing. When attempting to select disease-causing candidate mutation(s), a challenge is to filter out as many false-positive (FP) variants as possible. In this study, we describe the gene damage index (GDI), a metric for the nonsynonymous mutational load in each protein-coding gene in the general population. We show that the GDI is an efficient gene-level method for filtering out FP variants in genes that are highly damaged in the general population. The protein-coding exome of a patient with a monogenic disease contains about 20,000 variants, only one or two of which are disease causing. We found that 58% of rare variants in the protein-coding exome of the general population are located in only 2% of the genes. Prompted by this observation, we aimed to develop a gene-level approach for predicting whether a given human protein-coding gene is likely to harbor disease-causing mutations. To this end, we derived the gene damage index (GDI): a genome-wide, gene-level metric of the mutational damage that has accumulated in the general population. We found that the GDI was correlated with selective evolutionary pressure, protein complexity, coding sequence length, and the number of paralogs. We compared GDI with the leading gene-level approaches, genic intolerance, and de novo excess, and demonstrated that GDI performed best for the detection of false positives (i.e., removing exome variants in genes irrelevant to disease), whereas genic intolerance and de novo excess performed better for the detection of true positives (i.e., assessing de novo mutations in genes likely to be disease causing). The GDI server, data, and software are freely available to noncommercial users from lab.rockefeller.edu/casanova/GDI.

The mutation significance cutoff: gene-level thresholds for variant predictions [Letter]

Next-generation sequencing (NGS) has made it possible to identify about 20,000 variants in the protein-coding exome of each individual, of which only a few are likely to underlie a genetic disease. Variant-level methods such as PolyPhen-2, SIFT and CADD are useful for obtaining a prediction as to whether a given variant is benign/damaging1–3 or tolerant/intolerant1–3 (we hereafter use the terms benign/deleterious). These methods are commonly interpreted in a binary manner for filtering out benign variants from NGS data, with a single significance cutoff value across all protein-coding genes. PolyPhen-2 and SIFT integrate the fixed cutoff in the software. CADD proposed (but did not recommend for categorical usage) the fixed value of 15 (or another value between 10 and 20). Gene-level methods, such as RVIS, de novo excess and GDI are also useful4–6. Combining fixed gene-level and variant-level cutoffs is also applied in the RVIS hot zone approach4. However, owing to the diversity of medical and population genetic features between human genes and across populations, a uniform cutoff is unlikely to be accurate genome-wide.

Visceral leishmaniasis in two patients with IL‐12p40 and IL‐12Rβ1 deficiencies

Mutations of the IL12B and IL12RB1 genes underlie the development of IL‐12 p40 and IL‐12Rβ1 deficiencies, respectively, both of which cause predisposition to infection with weakly virulent mycobacteria and Salmonella. Infections with other intramacrophagic organisms have only been rarely observed. We identified two patients with visceral leishmaniasis who had autosomal recessive IL‐12 p40 and IL‐12Rβ1 deficiencies, respectively. This finding demonstrates the importance of IFN‐γ immunity in the control of leishmaniasis. We also searched the literature for similar reports in patients with these and other primary immunodeficiencies.

Transduction of Herpesvirus saimiri‐Transformed T Cells with Exogenous Genes of Interest

Human T cells can be transformed and expanded with herpesvirus saimiri (HVS). HVS‐transformed T cells from patients have facilitated the study of a broad range of primary immunodeficiencies (PID) in which T‐cell development or function is altered. However, the utility of HVS‐transformed T cells for genetic studies has been limited by technical challenges in the expression of exogenous genes, including wild‐type or mutant alleles. A novel, gamma retrovirus–based method for the simple and reliable transduction, purification, and study of HVS‐transformed T cells is described.

IRF4 haploinsufficiency in a family with Whipple’s disease

Most humans are exposed to Tropheryma whipplei (Tw). Whipple’s disease (WD) strikes only a small minority of individuals infected with Tw (<0.01%), whereas asymptomatic chronic carriage is more common (<25%). We studied a multiplex kindred, containing four WD patients and five healthy Tw chronic carriers. We hypothesized that WD displays autosomal dominant (AD) inheritance, with age-dependent incomplete penetrance. We identified a single very rare non-synonymous mutation in the four patients: the private R98W variant of IRF4, a transcription factor involved in immunity. The five Tw carriers were younger, and also heterozygous for R98W. We found that R98W was loss-of-function, modified the transcriptome of heterozygous leukocytes following Tw stimulation, and was not dominant-negative. We also found that only six of the other 153 known non-synonymous IRF4 variants were loss-of-function. Finally, we found that IRF4 had evolved under purifying selection. AD IRF4 deficiency can underlie WD by haploinsufficiency, with age-dependent incomplete penetrance.

Disruption of an antimycobacterial circuit between dendritic and helper T cells in human SPPL2a deficiency

Human inborn errors of IFN-γ immunity underlie mycobacterial diseases. We describe patients with Mycobacterium bovis (BCG) disease who are homozygous for loss-of-function mutations of SPPL2A. This gene encodes a transmembrane protease that degrades the N-terminal fragment (NTF) of CD74 (HLA invariant chain) in antigen-presenting cells. The CD74 NTF therefore accumulates in the HLA class II+ myeloid and lymphoid cells of SPPL2a-deficient patients. This toxic fragment selectively depletes IL-12- and IL-23-producing CD1c+ conventional dendritic cells (cDC2s) and their circulating progenitors. Moreover, SPPL2a-deficient memory TH1* cells selectively fail to produce IFN-γ when stimulated with mycobacterial antigens in vitro. Finally, Sppl2a–/– mice lack cDC2s, have CD4+ T cells that produce small amounts of IFN-γ after BCG infection, and are highly susceptible to infection with BCG or Mycobacterium tuberculosis. These findings suggest that inherited SPPL2a deficiency in humans underlies mycobacterial disease by decreasing the numbers of cDC2s and impairing IFN-γ production by mycobacterium-specific memory TH1* cells.Primary immunodeficiency can predispose patients to mycobacterial disease. Casanova and colleagues identify novel human mutations in the enzyme SPPL2A that result in selective accumulation of CD74 in a dendritic cell subset and lead to their death and the failure to mount effective TH1 responses.

The human CIB1–EVER1–EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses

Patients with epidermodysplasia verruciformis (EV) and biallelic null mutations of TMC6 (encoding EVER1) or TMC8 (EVER2) are selectively prone to disseminated skin lesions due to keratinocyte-tropic human &bgr;-papillomaviruses (&bgr;-HPVs), which lack E5 and E8. We describe EV patients homozygous for null mutations of the CIB1 gene encoding calcium- and integrin-binding protein-1 (CIB1). CIB1 is strongly expressed in the skin and cultured keratinocytes of controls but not in those of patients. CIB1 forms a complex with EVER1 and EVER2, and CIB1 proteins are not expressed in EVER1- or EVER2-deficient cells. The known functions of EVER1 and EVER2 in human keratinocytes are not dependent on CIB1, and CIB1 deficiency does not impair keratinocyte adhesion or migration. In keratinocytes, the CIB1 protein interacts with the HPV E5 and E8 proteins encoded by &agr;-HPV16 and &ggr;-HPV4, respectively, suggesting that this protein acts as a restriction factor against HPVs. Collectively, these findings suggest that the disruption of CIB1–EVER1–EVER2-dependent keratinocyte-intrinsic immunity underlies the selective susceptibility to &bgr;-HPVs of EV patients.

Mendelian susceptibility to mycobacterial disease: 2014-2018 update

Mendelian susceptibility to mycobacterial disease (MSMD) is caused by inborn errors of IFN‐γ immunity. Since 1996, disease‐causing mutations have been found in 11 genes, which, through allelic heterogeneity, underlie 21 different genetic disorders. We briefly review here progress in the study of molecular, cellular and clinical aspects of MSMD since the last comprehensive review published in 2014. Highlights include the discoveries of (1) a new genetic etiology, autosomal recessive signal peptide peptidase‐like 2 A deficiency, (2) TYK2‐deficient patients with a clinical phenotype of MSMD, (3) an allelic form of partial recessive IFN‐γR2 deficiency, and (4) two forms of syndromic MSMD: RORγ/RORγT and JAK1 deficiencies. These recent findings illustrate how genetic and immunological studies of MSMD can shed a unique light onto the mechanisms of protective immunity to mycobacteria in humans.