Rare Allelic Variants Research Articles

Mucolipin-2 Cation Channel Increases Trafficking Efficiency of Endocytosed Viruses.

ABSTRACTReceptor-mediated endocytosis is a cellular process commonly hijacked by viruses to enter cells. The stages of entry are well described for certain viruses, but the host factors that mediate each step are less well characterized. We previously identified endosomal cation channel mucolipin-2 (MCOLN2) as a host factor that promotes viral infection. Here, we assign a role for MCOLN2 in modulating viral entry. We show that MCOLN2 specifically promotes viral vesicular trafficking and subsequent escape from endosomal compartments. This mechanism requires channel activity, occurs independently of antiviral signaling, and broadly applies to enveloped RNA viruses that require transport to late endosomes for infection, including influenza A virus, yellow fever virus, and Zika virus. We further identify a rare allelic variant of human MCOLN2 that has a loss-of-function phenotype with respect to viral enhancement. These findings establish a mechanistic link between an endosomal cation channel and late stages of viral entry.

Multiple Gene Variants in Hypertrophic Cardiomyopathy in the Era of Next-Generation Sequencing.

Multiple likely pathogenic/pathogenic (LP/P; ≥2) variants in patients with hypertrophic cardiomyopathy were described 10 years ago with a prevalence of 5%. We sought to re-examine the significance of multiple rare variants in patients with hypertrophic cardiomyopathy in the setting of comprehensive and targeted panels. Of 758 hypertrophic cardiomyopathy probands, we included 382 with ≥45 cardiomyopathy genes screened. There were 224 (59%) with ≥1 rare variant (allele frequency ≤0.02%). Variants were analyzed using varying sized gene panels to represent comprehensive or targeted testing. Based on a 45-gene panel, 127 (33%) had a LP/P variant, 139 (36%) had variants of uncertain significance, and 66 (17%) had multiple rare variants. A targeted 8-gene panel yielded 125 (32%) LP/P variants, 52 (14%) variants of uncertain significance, and 14 (4%) had multiple rare variants. No proband had 2 LP/P variants. Including affected family members (total n=412), cluster-adjusted analyses identified a phenotype effect, with younger age (odds ratio, 0.95; 95% confidence interval, 0.92-0.98; P=0.004) and family history of sudden cardiac death (odds ratio, 3.5; 95% confidence interval, 1.3-9.9; P=0.02) significantly more likely in multiple versus single variant patients when considering an 8-gene panel but not larger panels. Those with multiple variants had worse event-free survival from all-cause death, cardiac transplantation, and cardiac arrest (log-rank P=0.008). No proband had multiple LP/P variants in contrast to previous reports. However, multiple rare variants regardless of classification were seen in 4% and contributed to earlier disease onset and cardiac events. Our findings support a cumulative variant hypothesis in hypertrophic cardiomyopathy.

Whole exome sequencing of a consanguineous family identifies the possible modifying effect of a globally rare AK5 allelic variant in celiac disease development among Saudi patients.

Celiac disease (CD), a multi-factorial auto-inflammatory disease of the small intestine, is known to occur in both sporadic and familial forms. Together HLA and Non-HLA genes can explain up to 50% of CD’s heritability. In order to discover the missing heritability due to rare variants, we have exome sequenced a consanguineous Saudi family presenting CD in an autosomal recessive (AR) pattern. We have identified a rare homozygous insertion c.1683_1684insATT, in the conserved coding region of AK5 gene that showed classical AR model segregation in this family. Sequence validation of 200 chromosomes each of sporadic CD cases and controls, revealed that this extremely rare (EXac MAF 0.000008) mutation is highly penetrant among general Saudi populations (MAF is 0.62). Genotype and allelic distribution analysis have indicated that this AK5 (c.1683_1684insATT) mutation is negatively selected among patient groups and positively selected in the control group, in whom it may modify the risk against CD development [p<0.002]. Our observation gains additional support from computational analysis which predicted that Iso561 insertion shifts the existing H-bonds between 400th and 556th amino acid residues lying near the functional domain of adenylate kinase. This shuffling of amino acids and their H-bond interactions is likely to disturb the secondary structure orientation of the polypeptide and induces the gain-of-function in nucleoside phosphate kinase activity of AK5, which may eventually down-regulates the reactivity potential of CD4+ T-cells against gluten antigens. Our study underlines the need to have population-specific genome databases to avoid false leads and to identify true candidate causal genes for the familial form of celiac disease.

Simple multiplexed PCR-based barcoding of DNA for ultrasensitive mutation detection by next-generation sequencing.

Detection of extremely rare variant alleles within a complex mixture of DNA molecules is becoming increasingly relevant in many areas of clinical and basic research, such as the detection of circulating tumor DNA in the plasma of cancer patients. Barcoding of DNA template molecules early in next-generation sequencing (NGS) library construction provides a way to identify and bioinformatically remove polymerase errors that otherwise make detection of these rare variants very difficult. Several barcoding strategies have been reported, but all require long and complex library preparation protocols. Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing (SiMSen-seq) was developed to generate targeted barcoded libraries with minimal DNA input, flexible target selection and a very simple, short (∼4 h) library construction protocol. The protocol comprises a three-cycle barcoding PCR step followed directly by adaptor PCR to generate the library and then bead purification before sequencing. Thus, SiMSen-seq allows detection of variant alleles at <0.1% frequency with easy customization of library content (from 1 to 40+ PCR amplicons) and a protocol that can be implemented in any molecular biology laboratory. Here, we provide a detailed protocol for assay development and describe software to process the barcoded sequence reads.

Multiple Gene Variants in Hypertrophic Cardiomyopathy in the Era of Next Generation Sequencing

Introduction: Multiple likely pathogenic/pathogenic (LP/P) variants in hypertrophic cardiomyopathy (HCM) were described 10 years ago with a prevalence of 5%. We sought to re-examine the significance of multiple rare variants in the setting of comprehensive and targeted panels. Methods: Of 758 HCM probands, we included 382 seen in a specialised centre with ≥45 cardiomyopathy genes screened. There were 224 (59%) probands with ≥1 rare variant (allele frequency ≤0.02%). Variants were analysed using varying sized panels to represent comprehensive or targeted testing. Results: Based on a 45-gene panel, 127 (33%) had a LP/P variant, 139 (36%) had variants of uncertain significance (VUS) and 66 (17%) probands had multiple rare variants. A targeted 8-gene panel yielded 125 (32%) LP/P variants, 52 (14%) VUS and 14 (4%) probands had multiple rare variants. No proband had 2 LP/P variants. Including affected family members (total n = 412) cluster-adjusted analyses identified a phenotype effect, with younger age (OR 0.95, 95%CI 0.92-0.98, p = 0.004) and family history of sudden cardiac death (OR 3.5, 95%CI 1.3-9.9, p = 0.02) significantly more likely in multiple versus single variant patients when considering an 8-gene panel, but not larger panels. Further, those with multiple variants had worse event-free survival from all cause death, cardiac transplantation and cardiac arrest (log-rank p = 0.008). Conclusion: No proband had multiple LP/P variants, in contrast to previous reports quoting a 5% yield. However, multiple rare variants regardless of classification were seen in 4% and contributed to earlier disease onset and cardiac events. Our findings support a cumulative variant hypothesis in HCM.

Laboratory Diagnosis by Genotyping.

Alpha-1 antitrypsin (AAT) genotyping is useful to confirm the clinical diagnosis of AAT deficiency and determine the specific allelic variant. Genotyping is the reference standard procedure for identifying rare allelic variants and characterizing new variants. It is also useful when there is a discrepancy between the patients' AAT levels and their phenotypes. AAT genotype is determined by an allele-specific genotyping assay for the S, Z, and Mmalton variants and by exome sequencing.

Is CYP2D6 phenotype predictable from CYP2D6 genotype?

Genetic polymorphism of cytochrome P450s results in clinically significant modifications in patients' drug metabolizing capacities. CYP2D6 has a crucial role in the elimination of several clinically important drugs (antiarrhythmics, beta-adrenergic blockers, psychopharmacons and analgesics); however, the prediction of the phenotypic appearance of CYP2D6 is a challenge. Since single nucleotide polymorphisms and gene copy number variations (gene deletion and multiplication) frequently occur in CYP2D6 gene, CYP2D6 activity particularly depends on the genetic factors.Microsomal CYP2D6 activities (dextromethorphan O-demethylation) and CYP2D6 genotypes for the most frequent allelic variants (CYP2D6*3, *4, *5, *6, *10, *41 and duplication) were determined in 128 human liver samples derived from Hungarian organ donors. Substantial inter-individual variations were observed in CYP2D6 metabolic activities that were successfully predicted from the CYP2D6 genotypes in 67% of the donors. The underestimation of CYP2D6 phenotypes in 12.5% of the donors was assumed to be originated from the overlapping ranges of CYP2D6 activity among similar diplotypes or from the presence of −1584C>G in the promoter region evoking increased transcription of the wild-type CYP2D6 allele. In an appreciable number of donors (20.3%), the genotype-based CYP2D6 phenotype prediction was overestimated because of the rare CYP2D6 allelic variants which were not included in our genotyping platform or some external factors that could alter CYP2D6 activity (medication with CYP2D6 substrate/inhibitor) and hepatic function (Augmentin therapy, chronic alcohol consumption).In conclusion, CYP2D6 genotyping for the most frequent allelic variants was able to reliably predict CYP2D6 phenotypes in most donors; however, the external factors modifying the phenotypic appearance of CYP2D6 had to be taken into account. Personalized medication strategy should include monitoring of CYP2D6 genotype in a more comprehensive manner and should take external factors into consideration for an appropriate prediction of CYP2D6 metabolizing capacity.

NKp30 isoforms and NKp46 transcripts in metastatic melanoma patients: Unique NKp30 pattern in rare melanoma patients with favorable evolution

ABSTRACTGiven the NK cell-based immunosurveillance of melanoma, we investigated the prognostic value of NKp46 transcript and NKp30 isoform (NKp30A, NKp30B and NKp30C) profiling in blood of 187 melanoma patients including 13 long survivors (LS), metastatic patients that have controlled the disease. Compared to healthy volunteers (HV), patients had reduced amounts of transcripts of the three NKp30 isoforms (NKp30 A, B and C) but similar ratios between NKp30 isoforms (ΔAB, ΔAC, ΔBC). Stratification of patients according to disease stage showed higher NKp30C and lower NKp46 transcripts in stage IV patients. Furthermore, patients with previous history of conventional chemotherapy displayed reduced NKp30A transcripts. The expression levels of NKp30 isoforms failed to predict survival from sampling of patients, while NKp46 expression predicted melanoma outcome. LS patients displayed elevated NKp30A levels, accordingly high ΔAB and ΔBC ratios, and a unique pattern of rare allelic variants of NKp30 SNPs. Moreover, NK cells from LS displayed correlated NKp30/NKp46 membrane expression, high spontaneous and NKp30- or NKp46-triggered degranulation. These data outline the impact of NKp30 and NKp46 transcripts on melanoma evolution and identify unique genetic features of NKp30 associated with higher NK activation in rare LS melanoma patients that control a metastatic disease.

A rare missense mutation in CHRNA4 associates with smoking behavior and its consequences.

Using Icelandic whole-genome sequence data and an imputation approach we searched for rare sequence variants in CHRNA4 and tested them for association with nicotine dependence. We show that carriers of a rare missense variant (allele frequency=0.24%) within CHRNA4, encoding an R336C substitution, have greater risk of nicotine addiction than non-carriers as assessed by the Fagerstrom Test for Nicotine Dependence (P=1.2 × 10−4). The variant also confers risk of several serious smoking-related diseases previously shown to be associated with the D398N substitution in CHRNA5. We observed odds ratios (ORs) of 1.7–2.3 for lung cancer (LC; P=4.0 × 10−4), chronic obstructive pulmonary disease (COPD; P=9.3 × 10−4), peripheral artery disease (PAD; P=0.090) and abdominal aortic aneurysms (AAAs; P=0.12), and the variant associates strongly with the early-onset forms of LC (OR=4.49, P=2.2 × 10−4), COPD (OR=3.22, P=2.9 × 10−4), PAD (OR=3.47, P=9.2 × 10−3) and AAA (OR=6.44, P=6.3 × 10−3). Joint analysis of the four smoking-related diseases reveals significant association (P=6.8 × 10−5), particularly for early-onset cases (P=2.1 × 10−7). Our results are in agreement with functional studies showing that the human α4β2 isoform of the channel containing R336C has less sensitivity for its agonists than the wild-type form following nicotine incubation.

Variants of CARD14 gene and psoriasis vulgaris in southern Chinese cohort.

BACKGROUND:Recent mutation analysis identified several missense mutations in CARD14 in psoriasis.OBJECTIVES:We performed the genomic sequence analysis on CARD14 in southern Chinese Han Cantonese with Psoriasis Vulgaris (PsV) to reveal more causative missense mutations.METHODS:A total of 131 patients with PsV and 207 matched controls were included. We conducted sequence analysis of all the exon and exon-intron boundaries of CARD14 in the group of PsV patients and subsequent case control analysis of potential sequence variants of significance.RESULTS:We found five rare mutations and four of them are annotated or reported. Only the variant (c.1291C>G) has not been reported and annotated, but the variant was also found in controls. No significant difference was detected among all rare variant allele frequencies of patients and controls.CONCLUSION:None of the new definite variants were pathogenic. The other pathogenic mutations for PsV are still elusive in our cohort.

Moroccan study of HLA (-A, -B, -C, -DR, -DQ) polymorphism in 647 unrelated controls: Updating data

The scope of this study is to investigate the HLA (Human Leukocyte Antigen) distribution and polymorphism in a large sample of healthy Moroccans in order to extend and update the available data. 647 unrelated Moroccan controls originating from diverse regions of the country were typed using microlymphocytotoxicity for HLA-A and -B, sequence-specific-primer amplification for -C, -DR, and -DQ and Luminex HD for specific -DR. The most frequent allele groups detected were HLA-A2 (19.2%), -B44 (12.4%), -C*07 (24.4%), -DRB1*03 (16.9%), -DRB1*04 (18.4%), -DQB1*02 (28.7%) and -DQB1*03 (27.8%). The most predominant specific alleles found for DRB1 were: *03:01, *04:02, *04:05, *07:01, *11:01, *13:02 and *15:01. Rare allelic variants were detected, for the first time in Moroccan population, at the DRB1*03 (*03:52, *03:54, *03:56), DRB1*07 (*07:07, *07:11, *07:16) and DRB1*11 (*11:70) locus. The most frequent haplotypes were: A2-B44, A30-B18, A2-C*16, A30-C*06, B14-C*08, B58-C*07, B45-C*06, DRB1*03-DQB1*02, DRB1*04-DQB1*03, DRB1*07-DQB1*02 and DRB1*15-DQB1*06. Comparison of genetic distances and haplotypes with other populations shows that the Moroccans are genetically closer to North Africans and Europeans than to sub-Saharan Africans. Our results reflect the high degree of HLA polymorphism in the Moroccan population and provide a useful baseline of healthy Moroccan controls for disease association and anthropological studies.

HLA DQB1*06:02 Negative Narcolepsy with Hypocretin/Orexin Deficiency

To identify rare allelic variants and HLA alleles in narcolepsy patients with hypocretin (orexin, HCRT) deficiency but lacking DQB1*06:02. China (Peking University People's Hospital), Czech Republic (Charles University), Denmark (Golstrup Hospital), Italy (University of Bologna), Korea (Catholic University), and USA (Stanford University). CSF hypocretin-1, DQB1*06:02, clinical and polysomnographic data were collected in narcolepsy patients (552 with and 144 without cataplexy) from 6 sites. Numbers of cases with and without DQB1*06:02 and low CSF hypocretin-1 were compiled. HLA class I (A, B, C), class II (DRBs, DQA1, DQB1, DPA1, and DPB1), and whole exome sequencing were conducted in 9 DQB1*06:02 negative cases with low CSF hypocretin-1. Sanger sequencing of selected exons in DNMT1, HCRT, and MOG was performed to exclude mutations in known narcolepsy-associated genes. Classic narcolepsy markers DQB1*06:02 and low CSF hypocretin-1 were found in 87.4% of cases with cataplexy, and in 20.0% without cataplexy. Nine cases (all with cataplexy) were DQB1*06:02 negative with low CSF hypocretin-1, constituting 1.7% [0.8%-3.4%] of all cases with cataplexy and 1.8% [0.8%-3.4%] of cases with low CSF hypocretin independent of cataplexy across sites. Five HLA negative subjects had severe cataplexy, often occurring without clear triggers. Subjects had diverse ethnic backgrounds and HLA alleles at all loci, suggesting no single secondary HLA association. The rare subtype DPB1*0901, and homologous DPB1*10:01 subtype, were present in 5 subjects, suggesting a secondary association with HLA-DP. Preprohypocretin sequencing revealed no mutations beyond one previously reported in a very early onset case. No new MOG or DNMT1 mutations were found, nor were suspicious or private variants in novel genes identified through exome sequencing. Hypocretin, MOG, or DNMT1 mutations are exceptional findings in DQB1*06:02 negative cases with hypocretin deficiency. A secondary HLA-DP association may be present in these cases. These represent particularly difficult diagnostic challenges.

A Drosophila Genetic Resource of Mutants to Study Mechanisms Underlying Human Genetic Diseases

Invertebrate model systems are powerful tools for studying human disease owing to their genetic tractability and ease of screening. We conducted a mosaic genetic screen of lethal mutations on the Drosophila X chromosome to identify genes required for the development, function, and maintenance of the nervous system. We identified 165 genes, most of whose function has not been studied in vivo. In parallel, we investigated rare variant alleles in 1,929 human exomes from families with unsolved Mendelian disease. Genes that are essential in flies and have multiple human homologs were found to be likely to be associated with human diseases. Merging the human data sets with the fly genes allowed us to identify disease-associated mutations in six families and to provide insights into microcephaly associated with brain dysgenesis. This bidirectional synergism between fly genetics and human genomics facilitates the functional annotation of evolutionarily conserved genes involved in human health.

Family-based tests applied to extended pedigrees identify rare variants related to hypertension.

The application of family-based tests to whole-genome sequenced data provides a new window on the role of rare variant alleles in the etiology of disease. By applying family-based tests to these data, we can now identify rare variants associated with disease. Approaches for common variants, by contrast, require large sample sizes for power, and are powerless when faced with rare variants. When we tested Yip et al's 2011 family-based association tests for rare variants on pedigrees from the Genetic Analysis Workshop 18, we found that weighted collapsing methods generally have more power than unweighted methods, but are more prone to type I errors. We then evaluated a sliding window modification of the weighted family-based association tests for rare variants method. Although this modification inflates the rate of false positives, it significantly increases the power of family-based association tests for rare variants to identify causal rare variants.

Age-related macular degeneration: genetics and biology coming together.

Genetic and genomic studies have enhanced our understanding of complex neurodegenerative diseases that exert a devastating impact on individuals and society. One such disease, age-related macular degeneration (AMD), is a major cause of progressive and debilitating visual impairment. Since the pioneering discovery in 2005 of complement factor H (CFH) as a major AMD susceptibility gene, extensive investigations have confirmed 19 additional genetic risk loci, and more are anticipated. In addition to common variants identified by now-conventional genome-wide association studies, targeted genomic sequencing and exome-chip analyses are uncovering rare variant alleles of high impact. Here, we provide a critical review of the ongoing genetic studies and of common and rare risk variants at a total of 20 susceptibility loci, which together explain 40-60% of the disease heritability but provide limited power for diagnostic testing of disease risk. Identification of these susceptibility loci has begun to untangle the complex biological pathways underlying AMD pathophysiology, pointing to new testable paradigms for treatment.

Detection of new genetic profiles and allelic variants in improperly classified grapevine accessions

Thirty-seven grapevine accessions, collected in Central Italy, were characterized by morphological and genetic analysis, according to guidelines developed by European Union programs of grapevine research and standardization. Traditional denominations of some sampled varieties were revealed to be incorrect; moreover, 10 synonymies and 12 homonymies were recognized. Ampelographic and ampelometric measurements of leaf characters were performed. These data generated a phenotypic similarity matrix and a relative diagram showing morphological differences between specimens. Many samples presented different morphology even in the presence of the same genotype, probably as a result of various environmental pressures. Grapevines were typed by 12 microsatellite loci and then compared with the CRA-VIT genetic resource database. Twenty-five SSR profiles were clearly identified as well-known cultivars, while nine genotypes did not find a direct correspondence: these samples could represent putative new autochthonous Latial Vitis vinifera cultivars or hybrid varieties. The genetic approach also detected three new (169 and 173 in VVMD27 locus; 179 in ISV2 locus) and seven rare allelic variants. Plant sample classification by oral history, morphological observations, and molecular results were compared and discussed. Scions of samples were planted in the Botanic Garden of the University of Rome "Tor Vergata", to preserve grapevine biodiversity and to protect possible new autochthonous varieties.

Frequency of rare allelic variation in candidate genes among individuals with low and high urinary calcium excretion.

Our study investigated the association of rare allelic variants with extremes of 24-hour urinary calcium excretion because higher urinary calcium excretion is a dominant risk factor for calcium-based kidney stone formation. We resequenced 40 candidate genes potentially related to urinary calcium excretion in individuals from the Nurses' Health Studies I & II and the Health Professionals Follow-up Study. A total of 960 participants were selected based on availability of 24-hour urine collection data and level of urinary calcium excretion (low vs. high). We utilized DNA sample pooling, droplet-based target gene enrichment, multiplexing, and high-throughput sequencing. Approximately 64% of samples (n = 615) showed both successful target enrichment and sequencing data with >20-fold deep coverage. A total of 259 novel allelic variants were identified. None of the rare gene variants (allele frequencies <2%) were found with increased frequency in the low vs. high urinary calcium groups; most of these variants were only observed in single individuals. Unadjusted analysis of variants with allele frequencies ≥2% suggested an association of the Claudin14 SNP rs113831133 with lower urinary calcium excretion (6/520 versus 29/710 haplotypes, P value = 0.003). Our data, together with previous human and animal studies, suggest a possible role for Claudin14 in urinary calcium excretion. Genetic validation studies in larger sample sets will be necessary to confirm our findings for rs113831133. In the tested set of candidate genes, rare allelic variants do not appear to contribute significantly to differences in urinary calcium excretion between individuals.

Genetics of Childhood-onset Schizophrenia

Schizophrenia is a heritable disorder. The genetic architecture of schizophrenia is complex and heterogeneous. This article discusses genetic studies of childhood-onset schizophrenia (COS) and compares findings in familial aggregation, common allele, and rare allele studies of COS with those for adult-onset schizophrenia (AOS). COS seems to be a rare variant of AOS with greater familial aggregation of schizophrenia spectrum disorders and higher occurrence of rare allelic variants. The usefulness of genetic screening for diagnosis and individualized treatment is limited; however, identifying common pathways through which multiple genes adversely affect neural systems offers great promise toward developing novel pharmacologic interventions.

Human platelet‐specific antigen frequencies in Indonesian population

Alloantibodies against human platelet antigens (HPAs) are responsible for the development of alloimmune thrombocytopenia including platelet transfusion refractoriness (PTR) and neonatal alloimmune thrombocytopenia (NAIT). Therefore, transfusion of HPA-compatible platelets is of importance for the management of these diseases. Determination of the allele frequency of the major HPA systems for Indonesian blood donors and the development of the first HPA-typed donor registry in Indonesia. DNA derived from 500 Indonesian healthy blood donors was genotyped for HPA-1 to HPA-6 and HPA-15 alleles by the use of polymerase chain reaction sequence-specific primer method. The gene frequencies of the rare allelic variants HPA-1b, -2b, -3b, -4b, -5b, -6b and -15b were 0·023, 0·060, 0·493, 0·052, 0·032, 0·044 and 0·049, respectively. However, donors homozygous for the HPA-1b, -2b and -6b were not found in this cohort, indicating that the risks of alloimmunisation caused by incompatibility of these three HPA systems are extremely low. In contrast, alloimmunisation against HPA-3, -4, -5 and -15 systems is anticipated. The development of an HPA-genotyped registry for donors homozygous for HPA-1b, -2b and -6b is desired for the optimum management of PTR patients and children with NAIT.

M89V Sialic Acid Acetyl Esterase (SIAE) and All Other Non-Synonymous Common Variants of This Gene Are Catalytically Normal

Catalytically defective rare variants of Sialic acid Acetyl Esterase (SIAE) have previously been linked to autoimmunity. Studies presented here confirm that the M89V SIAE protein and all other products of common variant alleles of SIAE are catalytically normal. Although overexpressing transfected non-lymphoid cells secrete small amounts of SIAE that can associate with the cell surface, normal human lymphocytes do not exhibit cell surface SIAE, supporting genetic evidence in mice that indicates that this protein functions in a lymphocyte intrinsic manner. Analyses of the plasma proteome also indicate that SIAE is not secreted in vivo. A re-analysis exclusively of catalytically defective rare variant alleles of SIAE in subjects in which this gene was completely sequenced confirmed an association of SIAE with autoimmunity. A subset of catalytically defective rare variant SIAE alleles has previously been typed in a large genotyping study comparing a diverse group of disease subjects and controls; our re-analysis of this data shows that catalytically defective alleles are enriched in disease subjects. These data suggest that SIAE may be associated with autoimmunity and that further study of catalytically defective rare variant SIAE alleles in terms of autoimmune disease susceptibility is strongly warranted.