Clinical utility of targeted next-generation sequencing panel in routine diagnosis of hereditary hemolytic anemia: A national reference laboratory experience.

INTRODUCTION: Hereditary hemolytic anemias (HHA) comprise a heterogeneous group of disorders resulting from defective red blood cell (RBC) cytoskeleton, RBC enzyme deficiencies, and hemoglobin (Hb) synthesis disorders such as thalassemia or sideroblastic anemia. MATERIALS AND METHODS: Our hemolytic anemia diagnostic next-generation sequencing (NGS) panel includes 28 genes encoding RBC cytoskeletal proteins, membrane transporter, RBC enzymes, and certain bilirubin metabolism genes. The panel covers the complete coding region of these genes, splice junctions, and, wherever appropriate, deep intronic or regulatory regions are also included. Four hundred fifty-six patients with unexplained hemolytic anemia were evaluated using our NGS panel between 2015 and 2019. RESULTS: We identified pathogenic/likely pathogenic variants in 111/456 (24%) patients that were responsible for the disease phenotype (e.g., moderate to severe hemolytic anemia and hyperbilirubinemia). Approximately 40% of the mutations were novel. As expected, 45/456 (10%) patients were homozygous for the promoter polymorphism in the UGT1A1 gene, A(TA)7 TAA (UGT1A1*28). 8/45 homozygous UGT1A1*28 cases were associated with additional pathogenic mutations causing hemolytic anemia, likely exacerbating hyperbilirubinemia. The most common mutated genes were membrane cytoskeleton genes SPTA1, and SPTB, followed by PKLR. Complex interactions between SPTA1 low expression alleles, alpha-LELY and alpha-LEPRA alleles, and intragenic SPTA1 variants were associated with hereditary pyropoikilocytosis and autosomal recessive hereditary spherocytosis in 23/111 patients. CONCLUSIONS: Our results demonstrate that hemolytic anemia is underscored by complex molecular interactions of previously known and novel mutations in RBC cytoskeleton/enzyme genes, and therefore, NGS should be considered in all patients with clinically unexplained hemolytic anemia and in neonates with hyperbilirubinemia. Moreover, low expression alleles alpha-LELY and alpha-LEPRA should be included in all targeted HHA panels.

A pathway-based association analysis identified FMNL1-MAP3K14 as susceptibility genes for leprosy.

The nuclear transcription factor-κB (NF-κB) plays a pivotal role in controlling both innate and adaptive immunity and regulates the expressions of many immunological mediators. Abundant evidences have showed the importance of NF-κB pathway in the host immune responses against Mycobacterium leprae in the development of leprosy. However, no particular association study between leprosy and NF-κB pathway-related gene polymorphisms was reported. Here, we performed a large-scale and two-stage candidate association study to investigate the association between 94 NF-κB pathway-related genes and leprosy. Our results showed that rs58744688 was significantly associated with leprosy (P = 7.57 × 10-7 , OR = 1.12) by combining the previous genomewide association data sets and four independent validation sample series, consisting of a total of 4631 leprosy cases and 6413 healthy controls. This founding implicated that MAP3K14 and FMNL1 were susceptibility genes for leprosy, which suggested the involvement of macrophage targeting and NF-κB pathway in the development of leprosy.

CUBN and NEBL common variants in the chromosome 10p13 linkage region are associated with multibacillary leprosy in Vietnam.

Leprosy is caused by infection with Mycobacterium leprae and is classified clinically into paucibacillary (PB) or multibacillary (MB) subtypes based on the number of skin lesions and the bacillary index detected in skin smears. We previously identified a major PB susceptibility locus on chromosome region 10p13 in Vietnamese families by linkage analysis. In the current study, we conducted high-density association mapping of the 9.5 Mb linkage peak on chromosome region 10p13 covering 39 genes. Using leprosy per se and leprosy subtypes as phenotypes, we employed 294 nuclear families (303 leprosy cases, 63 % MB, 37 % PB) as a discovery sample and 192 nuclear families (192 cases, 55 % MB, 45 % PB) as a replication sample. Replicated significant association signals were revealed in the genes for cubilin (CUBN) and nebulette (NEBL). In the combined sample, the C allele (frequency 0.26) at CUBN SNP rs10904831 showed association [p = 1 × 10(-5); OR 0.52 (0.38-0.7)] with MB leprosy only. Likewise, allele T (frequency 0.42) at NEBL SNP rs11012461 showed association [p = 4.2 × 10(-5); OR 2.51 (1.6-4)] with MB leprosy only. These associations remained valid for the CUBN signal when taking into account the effective number of tests performed (type I error significance threshold = 2.4 × 10(-5)). We used the results of our analyses to propose a new model for the genetic control of polarization of clinical leprosy.

The structure, function, and regulation of Mycobacterium FtsZ.

FtsZ is a widely distributed major cytoskeletal protein involved in the archaea and bacteria cell division. It is the most critical component in the division machinery and similar to tubulin in structure and function. Four major roles of FtsZ have been characterized: cell elongation, GTPase, cell division, and bacterial cytoskeleton. FtsZ subunits can be assembled into protofilaments. Mycobacteria consist of a large family of medical and environmental important bacteria, such as M. leprae, M. tuberculosis, the pathogen of leprosy, and tuberculosis. Structure, function, and regulation of mycobacteria FtsZ are summarized here, together with the implication of FtsZ as potential novel drug target for anti-tuberculosis therapeutics.

Evaluation of the combination of BP180-NC16a enzyme-linked immunosorbent assay and BP230 enzyme-linked immunosorbent assay in the diagnosis of bullous pemphigoid.

BACKGROUND: Bullous pemphigoid (BP) is an acquired autoimmune subepidermal blistering disease characterized by circulating IgG autoantibodies directed against BP180 and BP230 hemidesmosomal proteins. Previous studies have demonstrated that antibodies against the NC16a domain of BP180 mediate BP pathogenesis, while antibodies against BP230 enhance the inflammatory response. Recently, commercial BP180-NC16a enzyme-linked immunosorbent assay (ELISA) and BP230 ELISA kits were developed to detect anti-BP180 and anti-BP230 autoantibodies in human BP sera. AIMS: To evaluate the efficacy of BP180-NC16a ELISA and BP230 ELISA in the initial diagnosis of BP. METHODS: Sera from 62 BP patients and 62 control subjects were tested by BP180-NC16a ELISA and BP230 ELISA and compared with findings from indirect immunofluorescence (IIF) and immunoblotting (IB) to determine the sensitivity and specificity of these assays. RESULTS: The sensitivities of BP180-NC16a ELISA and BP230 ELISA were 87.1% (54/62) and 56.5% (35/62), respectively, and the specificities of both were 100% (62/62). Using both ELISAs for diagnosis increased the sensitivity to 95.2% (59/62) and was statistically comparable with IB sensitivity. CONCLUSIONS: ELISA is a convenient, effective, and reliable method for serodiagnosis of BP, and combined use of BP180-NC16a ELISA and BP230 ELISA can increase the sensitivity of this diagnostic approach.

In vitro polymerization of Mycobacterium leprae FtsZ OR Mycobacterium tuberculosis FtsZ is revived or abolished, respectively, by reciprocal mutation of a single residue.

A single residue that dramatically influences polymerization of principal cell division protein FtsZ of Mycobacterium leprae (MlFtsZ) and Mycobacterium tuberculosis (MtFtsZ) has been identified. Soluble, recombinant MlFtsZ did not show polymerization in vitro, in contrast to MtFtsZ, which polymerised. Mutation of the lone non-conserved residue T172 in the N-terminal domain of MlFtsZ to A172, as it exists in MtFtsZ, showed dramatic polymerization of MlFtsZ-T172A in vitro. Reciprocal mutation of A172 in MtFtsZ to T172, as it exists in MlFtsZ, abolished polymerization of MtFtsZ-A172T in vitro. While T172A mutation enhanced weak GTPase activity of MlFtsZ, reciprocal A172T mutation marginally reduced GTPase activity of MtFtsZ in vitro. These observations demonstrate that the residue at position 172 plays critical role in the polymerization of MlFtsZ and MtFtsZ. A possible evolutionary correlation between the presence of polymerization-adversive or polymerization-favouring residue at position 172 in FtsZ and generation time of the respective bacterium are discussed.

Molecular basis for the peripheral nerve predilection of Mycobacterium leprae.

Mycobacterium leprae, the causative organism of leprosy, has a unique predilection for Schwann cells, the glial cells of the peripheral nervous system. M. leprae invasion of Schwann cells leads to the neurological damage that underlies the sensory motor loss and subsequent deformity and disability associated with this disease. Recent studies have begun to elucidate the early events of M. leprae infection of Schwann cells on a molecular level, and the host and bacterial factors that determine the neural predilection of this bacterium. These advances have now provided novel insights into the mechanisms of bacterial interactions with host cells.

How does Mycobacterium leprae target the peripheral nervous system?

Mycobacterium leprae has the capacity to invade the peripheral nervous system and cause neuropathy. The molecular mechanisms responsible have remained unknown until recently. Identification of the endoneurial laminin-2 isoform and its receptor alpha-dystroglycan as neural targets of M. leprae has not only opened up a new area of scientific inquiry into the pathogenesis of neurological damage in leprosy, but has also revealed unexpected biological properties of these important host molecules.

Dystroglycan inside and out.

Dystroglycan connects the extracellular matrix and cytoskeleton. Key findings in the past year indicate that dystroglycan interacts with a wider repertoire of extracellular ligands than originally appreciated, that dystroglycan plays a critical role in organizing extracellular matrix molecules on the cell surface and in basement membranes, and that at least two human pathogens utilize dystroglycan to gain access to host cells. Together, these advances begin to help elucidate important biological roles for dystroglycan in development and disease.

Role of alpha-dystroglycan as a Schwann cell receptor for Mycobacterium leprae.

alpha-Dystroglycan (alpha-DG) is a component of the dystroglycan complex, which is involved in early development and morphogenesis and in the pathogenesis of muscular dystrophies. Here, alpha-DG was shown to serve as a Schwann cell receptor for Mycobacterium leprae, the causative organism of leprosy. Mycobacterium leprae specifically bound to alpha-DG only in the presence of the G domain of the alpha2 chain of laminin-2. Native alpha-DG competitively inhibited the laminin-2-mediated M. leprae binding to primary Schwann cells. Thus, M. leprae may use linkage between the extracellular matrix and cytoskeleton through laminin-2 and alpha-DG for its interaction with Schwann cells.

Dystroglycan in development and disease.

Our understanding of the structure and function of dystroglycan, a cell surface laminin/agrin receptor, has increased dramatically over the past two years. Structural studies, analysis of its binding partners, and targeted gene disruption have all contributed to the elucidation of the biological role of dystroglycan in development and disease. It is now apparent that dystroglycan plays a critical role in the pathogenesis of several muscular dystrophies and serves as a receptor for a human pathogen as well as being involved in early development, organ morphogenesis, and synaptogenesis.

Immunolocalization of cell-wall-deficient forms of Mycobacterium tuberculosis complex in sarcoidosis and in sinus histiocytosis of lymph nodes draining carcinoma.

In sarcoidosis, pleomorphic chromogens (PCs) occur as multivariate pigmented elements within sinusoids of lymph nodes (sinusoidal phase) and as tiny "round bodies" detectable in granulomas (generalized phase). The sinusoidal phase occurs in other conditions as well and characteristically contains yeastlike bodies also known as H-W bodies. To elucidate the antigenic profile of all variant forms, 28 cases of sarcoidosis (series A) and 14 cases of malignancy associated sinus histiocytosis (series B) were studied immunohistochemically with panels of various antibodies, including antimycobacterial MAbs specific for M tuberculosis complex (TB68, TB71), for M. leprae (MMP-I-3C3) and for cross-reactive mycobacterial antigens (F24-2-3 and F116-5, the latter recognizing superoxide dismutase). Results for series A indicate that: 1) PCs are cell-wall-deficient (CWD) mycobacterial forms belonging to M. tuberculosis complex (over 95%); 2) both phases are antigenically identical parts of the L-cycle; 3) "round bodies" of the "infective" phase have an endolysosomal evolution; 4)uncommon vacuolated forms represent a labile spheroplast stage; 5) the yeastlike bodies are specialized sinusoidal large bodies of unknown function. Results for series B show that in roughly two thirds of cases the pigmented forms are also CWD mycobacteria, have the same immunophenotype as sarcoid PCs in 35.7% of cases, have a much higher incidence of labile vacuolated forms and, finally, that malignancy associated "pseudosarcoid" granulomas do not differ antigenically from genuine sarcoid granulomas. Unlike conventional mycobacteria, PCs do not express cytoskeletal proteins consistently. Their general reactivity for HBcAg raises the possibility of phage interactions being responsible for the L-cycle since it may reflect shared epitopes between unrelated virus entities.

Polyspecificity of human monoclonal antibodies reactive with Mycobacterium leprae, mitochondria, ssDNA, cytoskeletal proteins, and the acetylcholine receptor.

The origin of autoantibodies against ubiquitous autoantigens (e.g., single-stranded (SS) DNA, cytoskeletal proteins, mitochondria) is obscure. Patients with lepromatous leprosy have many such autoantibodies in their serum. In order to study the polyspecificities of human autoantibodies expressed during infection with Mycobacterium leprae we prepared human monoclonal antibodies derived from the fusion of peripheral blood lymphocytes of a patient with lepromatous leprosy to the human lymphoblastoid line GM 4672. Hybridomas were tested for binding to a DNAse-treated sonicate of M. leprae and a panel of autoantigens. Of the primary (uncloned) cultures, 14% bound ssDNA, 35% bound M. leprae, 11% bound both M. leprae and ssDNA, and 16% bound to mitochondria. Several also bound to the acetylcholine receptor of Torpedo marmorata. Monoclonal antibodies derived from separate primary cultures revealed similar cross-reactions between several autoantigens and M. leprae. In addition, one antibody was identified which bound to mitochondria and the acetylcholine receptor, and which was recognized by an anti-idiotypic antibody which bears the "internal image" of the acetylcholine receptor. These results suggest that antigenic mimicry may play a role in eliciting autoantibody expression from the immune repertoire.