Diane Kissell

Hypomorphic mutations in PRF1, MUNC13-4, and STXBP2 are associated with adult-onset familial HLH

Familial hemophagocytic lymphohistiocytosis (HLH) is a rare primary immunodeficiency disorder characterized by defects in cell-mediated cytotoxicity that results in fever, hepatosplenomegaly, and cytopenias. Familial HLH is well recognized in children but rarely diagnosed in adults. We conducted a retrospective review of genetic and immunologic test results in patients who developed HLH in adulthood. Included in our study were 1531 patients with a clinical diagnosis of HLH; 175 patients were 18 years or older. Missense and splice-site sequence variants in PRF1, MUNC13-4, and STXBP2 were found in 25 (14%) of the adult patients. The A91V-PRF1 genotype was found in 12 of these patients (48%). The preponderance of hypomorphic mutations in familial HLH-causing genes correlates with the later-onset clinical symptoms and the more indolent course in adult patients. We conclude that late-onset familial HLH occurs more commonly than was suspected previously.

Patients with LRBA deficiency show CTLA4 loss and immune dysregulation responsive to abatacept therapy

Trafficking from bedside to bench Typically in translational research, a discovery in cell or molecular biology is later exploited to improve patient care. Occasionally, information flows in the opposite direction. Lo et al. found that patients with an autoimmune disorder caused by deficiency of a protein called LRBA responded dramatically to the drug abatacept (see the Perspective by Sansom). Abatacept contains a segment of a potent inhibitory immune receptor, CTLA4. Experiments prompted by this observation revealed the relationship between the two proteins: LRBA controls the intracellular trafficking and degradation of CTLA4. This information may further improve patient care, because other clinically approved drugs have the desired mechanism of action with potentially fewer side effects. Science, this issue p. 436; see also p. 377 A rare autoimmune disorder is caused by aberrant degradation of a potent inhibitory immune receptor. [Also see Perspective by Sansom] Mutations in the LRBA gene (encoding the lipopolysaccharide-responsive and beige-like anchor protein) cause a syndrome of autoimmunity, lymphoproliferation, and humoral immune deficiency. The biological role of LRBA in immunologic disease is unknown. We found that patients with LRBA deficiency manifested a dramatic and sustained improvement in response to abatacept, a CTLA4 (cytotoxic T lymphocyte antigen-4)–immunoglobulin fusion drug. Clinical responses and homology of LRBA to proteins controlling intracellular trafficking led us to hypothesize that it regulates CTLA4, a potent inhibitory immune receptor. We found that LRBA colocalized with CTLA4 in endosomal vesicles and that LRBA deficiency or knockdown increased CTLA4 turnover, which resulted in reduced levels of CTLA4 protein in FoxP3+ regulatory and activated conventional T cells. In LRBA-deficient cells, inhibition of lysosome degradation with chloroquine prevented CTLA4 loss. These findings elucidate a mechanism for CTLA4 trafficking and control of immune responses and suggest therapies for diseases involving the CTLA4 pathway.

Synergistic defects of different molecules in the cytotoxic pathway lead to clinical familial hemophagocytic lymphohistiocytosis.

Several molecules (LYST, AP3, RAB27A, STX11, STXBP2, MUNC13-4, and PRF1) have been associated with the function of cytotoxic lymphocytes. Biallelic defects in all of these molecules have been associated with familial hemophagocytic lymphohistiocytosis (FHL). We retrospectively reviewed the genetic and immunology test results from 2701 patients with a clinically suspected diagnosis of hemophagocytic lymphohistiocytosis and found 28 patients with single heterozygous mutations in 2 FHL-associated genes. Of these patients, 21 had mutations within PRF1 and a degranulation gene, and 7 were found to have mutations within 2 genes involved in the degranulation pathway. In patients with combination defects involving 2 genes in the degranulation pathway, CD107a degranulation was decreased, comparable to patients with biallelic mutations in one of the genes in the degranulation pathway. This suggests a potential digenic mode of inheritance of FHL as a result of a synergistic function effect within genes involved in cytotoxic lymphocyte degranulation.

Parkin dosage mutations have greater pathogenicity in familial PD than simple sequence mutations

Objective: Mutations in both alleles of parkin have been shown to result in Parkinson disease (PD). However, it is unclear whether haploinsufficiency (presence of a mutation in only 1 of the 2 parkin alleles) increases the risk for PD. Methods: We performed comprehensive dosage and sequence analysis of all 12 exons of parkin in a sample of 520 independent patients with familial PD and 263 controls. We evaluated whether presence of a single parkin mutation, either a sequence (point mutation or small insertion/deletion) or dosage (whole exon deletion or duplication) mutation, was found at increased frequency in cases as compared with controls. We then compared the clinical characteristics of cases with 0, 1, or 2 parkin mutations. Results: We identified 55 independent patients with PD with at least 1 parkin mutation and 9 controls with a single sequence mutation. Cases and controls had a similar frequency of single sequence mutations (3.1% vs 3.4%, p = 0.83); however, the cases had a significantly higher rate of dosage mutations (2.6% vs 0%, p = 0.009). Cases with a single dosage mutation were more likely to have an earlier age at onset (50% with onset at ≤45 years) compared with those with no parkin mutations (10%, p = 0.00002); this was not true for cases with only a single sequence mutation (25% with onset at ≤45 years, p = 0.06). Conclusions: Parkin haploinsufficiency, specifically for a dosage mutation rather than a point mutation or small insertion/deletion, is a risk factor for familial PD and may be associated with earlier age at onset.

Variation in GIGYF2 is not associated with Parkinson disease

Objective: A recent study reported that mutations in a gene on chromosome 2q36-37, GIGYF2, result in Parkinson disease (PD). We have previously reported linkage to this chromosomal region in a sample of multiplex PD families, with the strongest evidence of linkage obtained using the subset of the sample having the strongest family history of disease and meeting the strictest diagnostic criteria. We have tested whether mutations in GIGYF2 may account for the previously observed linkage finding. Methods: We sequenced the GIGYF2 coding region in 96 unrelated patients with PD used in our original study that contributed to the chromosome 2q36-37 linkage signal. Subsequently, we genotyped the entire sample of 566 multiplex PD kindreds as well as 1,447 controls to test whether variants in GIGYF2 are causative or increase susceptibility for PD. Results: We detected three novel variants as well as one of the previously reported seven variants in a total of five multiple PD families; however, there was no consistent evidence that these variants segregated with PD in these families. We also did not find a significant increase in risk for PD among those inheriting variants in GIGYF2 (p = 0.28). Conclusions: We believe that variation in a gene other than GIGYF2 accounts for the previously reported linkage finding on chromosome 2q36-37. GDS = Geriatric Depression Scale; MMSE = Mini-Mental State Examination; NCRAD = National Cell Repository for Alzheimer’s Disease; PD = Parkinson disease; PSG = Parkinson Study Group; UPDRS = Unified Parkinson’s Disease Rating Scale.

Performance Evaluation of the Next-Generation Sequencing Approach for Molecular Diagnosis of Hereditary Hearing Loss

Objective To evaluate the performance of a next-generation sequencing (NGS)–based targeted resequencing genetic test, OtoSeq, to identify the sequence variants in the genes causing sensorineural hearing loss (SNHL). Study Design Retrospective study. Setting Tertiary children’s hospital. Subjects and Methods A total of 8 individuals presenting with prelingual hearing loss were used in this study. The coding and flanking intronic regions of 24 well-studied SNHL genes were enriched using microdroplet polymerase chain reaction and sequenced on an Illumina HiSeq 2000 sequencer. The filtered high-quality sequence reads were mapped to reference sequence, and variants were detected using NextGENe software. Results A total of 1148 sequence variants were detected in 8 samples in 24 genes. Using in-house developed NGS data analysis criteria, we classified 810 (~71%) of these variants as potential true variants that include previously detected pathogenic mutations in 5 patients. To validate our strategy, we Sanger sequenced the target regions of 5 of the 24 genes, accounting for about 29.2% of all target sequence. Our results showed >99.99% concordance between NGS and Sanger sequencing in these 5 genes, resulting in an analytical sensitivity and specificity of 100% and 99.997%, respectively. We were able to successfully detect single base substitutions, small deletions, and insertions of up to 22 nucleotides. Conclusion This study demonstrated that our NGS-based mutation screening strategy is highly sensitive and specific in detecting sequence variants in the SNHL genes. Therefore, we propose that this NGS-based targeted sequencing method would be an alternative to current technologies for identifying the multiple genetic causes of SNHL.

Whole-Exome Sequencing Reveals Mutations in Genes Linked to Hemophagocytic Lymphohistiocytosis and Macrophage Activation Syndrome in Fatal Cases of H1N1 Influenza

BACKGROUND Severe H1N1 influenza can be lethal in otherwise healthy individuals and can have features of reactive hemophagocytic lymphohistiocytosis (HLH). HLH is associated with mutations in lymphocyte cytolytic pathway genes, which have not been previously explored in H1N1 influenza. METHODS Sixteen cases of fatal influenza A(H1N1) infection, 81% with histopathologic hemophagocytosis, were identified and analyzed for clinical and laboratory features of HLH, using modified HLH-2004 and macrophage activation syndrome (MAS) criteria. Fourteen specimens were subject to whole-exome sequencing. Sequence alignment and variant filtering detected HLH gene mutations and potential disease-causing variants. Cytolytic function of the PRF1 p.A91V mutation was tested in lentiviral-transduced NK-92 natural killer (NK) cells. RESULTS Despite several lacking variables, cases of influenza A(H1N1) infection met 44% and 81% of modified HLH-2004 and MAS criteria, respectively. Five subjects (36%) carried one of 3 heterozygous LYST mutations, 2 of whom also possessed the p.A91V PRF1 mutation, which was shown to decrease NK cell cytolytic function. Several patients also carried rare variants in other genes previously observed in MAS. CONCLUSIONS This cohort of fatal influenza A(H1N1) infections confirms the presence of hemophagocytosis and HLH pathology. Moreover, the high percentage of HLH gene mutations suggests they are risk factors for mortality among individuals with influenza A(H1N1) infection.

Maternal Inheritance and Mitochondrial DNA Variants in Familial Parkinson's Disease

BackgroundMitochondrial function is impaired in Parkinsons disease (PD) and may contribute to the pathogenesis of PD, but the causes of mitochondrial impairment in PD are unknown. Mitochondrial dysfunction is recapitulated in cell lines expressing mitochondrial DNA (mtDNA) from PD patients, implicating mtDNA variants or mutations, though the role of mtDNA variants or mutations in PD risk remains unclear. We investigated the potential contribution of mtDNA variants or mutations to the risk of PD.MethodsWe examined the possibility of a maternal inheritance bias as well as the association between mitochondrial haplogroups and maternal inheritance and disease risk in a case-control study of 168 multiplex PD families in which the proband and one parent were diagnosed with PD. 2-tailed Fisher Exact Tests and McNemars tests were used to compare allele frequencies, and a t-test to compare ages of onset.ResultsThe frequency of affected mothers of the proband with PD (83/167, 49.4%) was not significantly different from the frequency of affected females of the proband generation (115/259, 44.4%) (Odds Ratio 1.22; 95%CI 0.83 - 1.81). After correcting for multiple tests, there were no significant differences in the frequencies of mitochondrial haplogroups or of the 10398G complex I gene polymorphism in PD patients compared to controls, and no significant associations with age of onset of PD. Mitochondrial haplogroup and 10398G polymorphism frequencies were similar in probands having an affected father as compared to probands having an affected mother.ConclusionsThese data fail to demonstrate a bias towards maternal inheritance in familial PD. Consistent with this, we find no association of common haplogroup-defining mtDNA variants or for the 10398G variant with the risk of PD. However, these data do not exclude a role for mtDNA variants in other populations, and it remains possible that other inherited mitochondrial DNA variants, or somatic mDNA mutations, contribute to the risk of familial PD.

Genotype-Directed Dosing Leads to Optimized Voriconazole Levels in Pediatric Patients Receiving Hematopoietic Stem Cell Transplantation

Invasive fungal infections are a significant cause of morbidity and mortality in recipients of hematopoietic stem cell transplantation (HSCT), warranting antifungal prophylaxis as a standard of care in these patients. Voriconazole is commonly used in this setting because of its broad-spectrum activity and available dosage forms. There is wide well-known inter- and intrapatient variability in voriconazole concentrations, in part because concentrations are affected by common CYP2C19 polymorphisms. In 2 successive studies we have optimized voriconazole dosing to achieve target voriconazole serum concentrations using a genotype-specific dosing algorithm for antifungal prophylaxis in the post-HSCT period. In our pilot study all patients undergoing HSCT who received voriconazole antifungal prophylaxis were prospectively followed. Voriconazole concentrations were monitored weekly and doses adjusted until concentrations reached between 1 and 5.5 μg/L. The most common CYP2C19 polymorphisms were determined and correlated with voriconazole dose and time required to reach the target concentration range. In the subsequent study patients receiving voriconazole prophylaxis were dosed based on their CYP2C19 genotype and followed prospectively. In the pilot study 25 patients received voriconazole as antifungal prophylaxis for a median of 49 days (range, 15 to 196 days). The median time to reach the target concentration was 34 days for extensive metabolizers and 11 days for poor metabolizers. Three patients were genotyped as intermediate metabolizers; they reached the target concentration in a median of 56 days. Similarly, 2 patients who were genotyped as ultrarapid metabolizers reached the target range in 18 and 25 days. The time and dose required to reach the adequate concentration showed a trend toward correlation with individual CYP2C19 genotype, although voriconazole concentrations showed large interpatient variability in wild-type patients (extensive metabolizers). In our follow-up study, 20 patients received voriconazole prophylaxis prospectively dosed based on their CYP2C19 genotype. The median times to reach the target concentration using genotype-guided dosing were 9, 6.5, and 4 days for ultrarapid, extensive, and intermediate metabolizers, respectively. Overall, the median time to reach the target concentration with genotype-guided dosing was 6.5 days compared with a median time of 29 days when all patients were started on the same dose regardless of CYP2C19 genotype (P < .001). Our data show that traditional voriconazole dosing does not lead to timely achievement of target levels for fungal prophylaxis. However, a genotype-directed dosing algorithm allows patients to reach the voriconazole target range significantly sooner, providing better prophylaxis against fungal infections in the immediate post-transplant period.

Genetic Analysis through OtoSeq of Pakistani Families Segregating Prelingual Hearing Loss

Objective To identify the genetic cause of prelingual sensorineural hearing loss in Pakistani families using a next-generation sequencing (NGS)–based mutation screening test named OtoSeq. Study Design Prospective study. Setting Research laboratory. Subjects and Methods We used 3 fluorescently labeled short tandem repeat (STR) markers for each of the known autosomal recessive nonsyndromic (DFNB) and Usher syndrome (USH) locus to perform a linkage analysis of 243 multigenerational Pakistani families segregating prelingual hearing loss. After genotyping, we focused on 34 families with potential linkage to MYO7A, CDH23, and SLC26A4. We screened affected individuals from a subset of these families using the OtoSeq platform to identify underlying genetic variants. Sanger sequencing was performed to confirm and study the segregation of mutations in other family members. For novel mutations, normal hearing individuals from ethnically matched backgrounds were also tested. Results Hearing loss was found to co-segregate with locus-specific STR markers for MYO7A in 32 families, CDH23 in 1 family, and SLC26A4 in 1 family. Using the OtoSeq platform, a microdroplet PCR-based enrichment followed by NGS, we identified mutations in 28 of the 34 families including 11 novel mutations. Sanger sequencing of these mutations showed 100% concordance with NGS data and co-segregation of the mutant alleles with the hearing loss phenotype in the respective families. Conclusion Using NGS-based platforms like OtoSeq in families segregating hearing loss will contribute to the identification of common and population-specific mutations, early diagnosis, genetic counseling, and molecular epidemiology.