Alexandre Bolze

Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis

Whole-exome sequencing reveals activating STAT1 mutations in some patients with autosomal dominant chronic mucocutaneous candidiasis disease.

Whole-genome sequencing is more powerful than whole-exome sequencing for detecting exome variants

Significance Whole-exome sequencing (WES) is gradually being optimized to identify mutations in increasing proportions of the protein-coding exome, but whole-genome sequencing (WGS) is becoming an attractive alternative. WGS is currently more expensive than WES, but its cost should decrease more rapidly than that of WES. We compared WES and WGS on six unrelated individuals. The distribution of quality parameters for single-nucleotide variants (SNVs) and insertions/deletions (indels) was more uniform for WGS than for WES. The vast majority of SNVs and indels were identified by both techniques, but an estimated 650 high-quality coding SNVs (∼3% of coding variants) were detected by WGS and missed by WES. WGS is therefore slightly more efficient than WES for detecting mutations in the targeted exome. We compared whole-exome sequencing (WES) and whole-genome sequencing (WGS) in six unrelated individuals. In the regions targeted by WES capture (81.5% of the consensus coding genome), the mean numbers of single-nucleotide variants (SNVs) and small insertions/deletions (indels) detected per sample were 84,192 and 13,325, respectively, for WES, and 84,968 and 12,702, respectively, for WGS. For both SNVs and indels, the distributions of coverage depth, genotype quality, and minor read ratio were more uniform for WGS than for WES. After filtering, a mean of 74,398 (95.3%) high-quality (HQ) SNVs and 9,033 (70.6%) HQ indels were called by both platforms. A mean of 105 coding HQ SNVs and 32 indels was identified exclusively by WES whereas 692 HQ SNVs and 105 indels were identified exclusively by WGS. We Sanger-sequenced a random selection of these exclusive variants. For SNVs, the proportion of false-positive variants was higher for WES (78%) than for WGS (17%). The estimated mean number of real coding SNVs (656 variants, ∼3% of all coding HQ SNVs) identified by WGS and missed by WES was greater than the number of SNVs identified by WES and missed by WGS (26 variants). For indels, the proportions of false-positive variants were similar for WES (44%) and WGS (46%). Finally, WES was not reliable for the detection of copy-number variations, almost all of which extended beyond the targeted regions. Although currently more expensive, WGS is more powerful than WES for detecting potential disease-causing mutations within WES regions, particularly those due to SNVs.

Whole-Exome-Sequencing-Based Discovery of Human FADD Deficiency

Germline mutations in FASL and FAS impair Fas-dependent apoptosis and cause recessively or dominantly inherited autoimmune lymphoproliferative syndrome (ALPS). Patients with ALPS typically present with no other clinical phenotype. We investigated a large, consanguineous, multiplex kindred in which biological features of ALPS were found in the context of severe bacterial and viral disease, recurrent hepatopathy and encephalopathy, and cardiac malformations. By a combination of genome-wide linkage and whole-exome sequencing, we identified a homozygous missense mutation in FADD, encoding the Fas-associated death domain protein (FADD), in the patients. This FADD mutation decreases steady-state protein levels and impairs Fas-dependent apoptosis in vitro, accounting for biological ALPS phenotypes in vivo. It also impairs Fas-independent signaling pathways. The observed bacterial infections result partly from functional hyposplenism, and viral infections result from impaired interferon immunity. We describe here a complex clinical disorder, its genetic basis, and some of the key mechanisms underlying its pathogenesis. Our findings highlight the key role of FADD in Fas-dependent and Fas-independent signaling pathways in humans.

Ribosomal Protein SA Haploinsufficiency in Humans with Isolated Congenital Asplenia

Spleen Knockout Explained Isolated congenital asplenia (ICA) is a rare disorder where patients are born without a spleen and are at increased risk of bacterial infection but have no other developmental abnormalities. Through sequence analysis of familial and sporadic cases, Bolze et al. (p. 976, published online 11 April) found that ICA patients carry mutations in the gene encoding ribosomal protein SA and as a result express about half the normal amount of this protein. The mechanism by which reduced expression of a housekeeping protein causes an organ-specific defect remains unclear. A rare human disorder, characterized by the absence of a spleen at birth, is associated with mutations in a ribosomal protein. Isolated congenital asplenia (ICA) is characterized by the absence of a spleen at birth in individuals with no other developmental defects. The patients are prone to life-threatening bacterial infections. The unbiased analysis of exomes revealed heterozygous mutations in RPSA in 18 patients from eight kindreds, corresponding to more than half the patients and over one-third of the kindreds studied. The clinical penetrance in these kindreds is complete. Expression studies indicated that the mutations carried by the patients—a nonsense mutation, a frameshift duplication, and five different missense mutations—cause autosomal dominant ICA by haploinsufficiency. RPSA encodes ribosomal protein SA, a component of the small subunit of the ribosome. This discovery establishes an essential role for RPSA in human spleen development.

Inherited IL-17RC deficiency in patients with chronic mucocutaneous candidiasis

Autosomal-recessive IL-17RA, IL-17RC, and ACT1 deficiencies and autosomal-dominant IL-17F deficiency in humans underlie susceptibility to chronic mucocutaneous candidiasis.

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.

Congenital Asplenia in Mice and Humans with Mutations in a Pbx/Nkx2-5/p15 Module

The molecular determinants of spleen organogenesis and the etiology of isolated congenital asplenia (ICA), a life-threatening human condition, are unknown. We previously reported that Pbx1 deficiency causes organ growth defects including asplenia. Here, we show that mice with splenic mesenchyme-specific Pbx1 inactivation exhibit hyposplenia. Moreover, the loss of Pbx causes downregulation of Nkx2-5 and derepression of p15Ink4b in spleen mesenchymal progenitors, perturbing the cell cycle. Removal of p15Ink4b in Pbx1 spleen-specific mutants partially rescues spleen growth. By whole-exome sequencing of a multiplex kindred with ICA, we identify a heterozygous missense mutation (P236H) in NKX2-5 showing reduced transactivation in vitro. This study establishes that a Pbx/Nkx2-5/p15 regulatory module is essential for spleen development.

Isolated Congenital Asplenia: A French Nationwide Retrospective Survey of 20 Cases

OBJECTIVE To better describe the natural history, mode of inheritance, and the epidemiological and clinical features of isolated congenital asplenia, a rare and poorly understood primary immunodeficiency. STUDY DESIGN A French national retrospective survey was conducted in hospital pediatric departments. A definitive diagnosis of ICA was based on the presence of Howell-Jolly bodies, a lack of detectable spleen, and no detectable cardiovascular malformation. RESULTS The study included 20 patients (12 males and 8 females) from 10 kindreds neither related to each other nor consanguineous. The diagnosis of ICA was certain in 13 cases (65%) and probable in 7 cases (35%). Ten index cases led to diagnosis of 10 additional cases in relatives. Five cases were sporadic and 15 were familial, suggesting autosomal dominant inheritance. Median age was 12 months at first infection (range, 2-516 months), 11 months at diagnosis of asplenia (range, 0-510 months), and 9.9 years at last follow-up (range, 0.7-52 years). Fifteen patients sustained 18 episodes of invasive bacterial infection, caused mainly by Streptococcus pneumoniae (61%). Outcomes were poor, with 9 patients (45%) dying from fulminant infection. CONCLUSIONS ICA is more common than was previously thought, with an autosomal dominant inheritance in at least some kindreds. Relatives of cases of ICA should be evaluated for ICA, as should children and young adults with invasive infection.

Exome and genome sequencing for inborn errors of immunity

The advent of next-generation sequencing (NGS) in 2010 has transformed medicine, particularly the growing field of inborn errors of immunity. NGS has facilitated the discovery of novel disease-causing genes and the genetic diagnosis of patients with monogenic inborn errors of immunity. Whole-exome sequencing (WES) is presently the most cost-effective approach for research and diagnostics, although whole-genome sequencing offers several advantages. The scientific or diagnostic challenge consists in selecting 1 or 2 candidate variants among thousands of NGS calls. Variant- and gene-level computational methods, as well as immunologic hypotheses, can help narrow down this genome-wide search. The key to success is a well-informed genetic hypothesis on 3 key aspects: mode of inheritance, clinical penetrance, and genetic heterogeneity of the condition. This determines the search strategy and selection criteria for candidate alleles. Subsequent functional validation of the disease-causing effect of the candidate variant is critical. Even the most up-to-date dry lab cannot clinch this validation without a seasoned wet lab. The multifariousness of variations entails an experimental rigor even greater than traditional Sanger sequencing-based approaches in order not to assign a condition to an irrelevant variant. Finding the needle in the haystack takes patience, prudence, and discernment.

A Mild Form of SLC29A3 Disorder: A Frameshift Deletion Leads to the Paradoxical Translation of an Otherwise Noncoding mRNA Splice Variant

We investigated two siblings with granulomatous histiocytosis prominent in the nasal area, mimicking rhinoscleroma and Rosai-Dorfman syndrome. Genome-wide linkage analysis and whole-exome sequencing identified a homozygous frameshift deletion in SLC29A3, which encodes human equilibrative nucleoside transporter-3 (hENT3). Germline mutations in SLC29A3 have been reported in rare patients with a wide range of overlapping clinical features and inherited disorders including H syndrome, pigmented hypertrichosis with insulin-dependent diabetes, and Faisalabad histiocytosis. With the exception of insulin-dependent diabetes and mild finger and toe contractures in one sibling, the two patients with nasal granulomatous histiocytosis studied here displayed none of the many SLC29A3-associated phenotypes. This mild clinical phenotype probably results from a remarkable genetic mechanism. The SLC29A3 frameshift deletion prevents the expression of the normally coding transcripts. It instead leads to the translation, expression, and function of an otherwise noncoding, out-of-frame mRNA splice variant lacking exon 3 that is eliminated by nonsense-mediated mRNA decay (NMD) in healthy individuals. The mutated isoform differs from the wild-type hENT3 by the modification of 20 residues in exon 2 and the removal of another 28 amino acids in exon 3, which include the second transmembrane domain. As a result, this new isoform displays some functional activity. This mechanism probably accounts for the narrow and mild clinical phenotype of the patients. This study highlights the ‘rescue’ role played by a normally noncoding mRNA splice variant of SLC29A3, uncovering a new mechanism by which frameshift mutations can be hypomorphic.