Biallelic NLRP7 variants in patients with recurrent hydatidiform mole: A review and expert consensus.

Hydatidiform mole (HM) is an abnormal human pregnancy characterized by excessive growth of placental trophoblasts and abnormal early embryonic development. Following a first such abnormal pregnancy, the risk for women of successive molar pregnancies significantly increases. To date variants in seven maternal-effect genes have been shown to cause recurrent HMs (RHM). NLRP7 is the major causative gene for RHM and codes for NOD-like receptor (NLR) family pyrin domain containing 7, which belongs to a family of proteins involved in inflammatory disorders. Since its identification, all NLRP7 variants have been recorded in Infevers, an online registry dedicated to autoinflammatory diseases (https://infevers.umai-montpellier.fr/web/). Here, we reviewed published and unpublished recessive NLRP7 variants associated with RHM, scored their pathogenicity according to the American College of Medical Genetics classification, and recapitulated all functional studies at the level of both the patients and the conceptions. We also provided data on further variant analyses of 32 patients and genotypes of 36 additional molar pregnancies. This comprehensive review integrates published and unpublished data on NLRP7 and aims at guiding geneticists and clinicians in variant interpretation, genetic counseling, and management of patients with this rare condition.

Pregnancy after oocyte donation in a patient with NLRP7 gene mutations and recurrent molar hydatidiform pregnancies.

Molar pregnancies are benign trophoblastic diseases associated with a risk of malignant transformation. If aetiology remains mostly unknown, the risk of recurrent molar pregnancy is around 1.5% after one molar pregnancy and around 25% after 2 molar pregnancies. In the later situation, genetic mutations have been described, increasing hugely this risk. In case of mutations, probability to obtain a normal pregnancy is estimated around 1.8%. We report the case of a Caucasian 30-year-old woman whose previous five spontaneous pregnancies had a negative outcome: a spontaneous miscarriage and then 4 complete hydatidiform moles. Genetic testing revealed that the patient carried two heterozygous mutations in the NLRP7 gene (c.2982-2A > G and Y318CfsX7). According to this, counselling was conducted to advocate for oocyte donation in order to obtain a normal pregnancy. This technique enabled a complication-free, singleton pregnancy that resulted in a healthy term live birth of a 2900 g female. Few months after delivery, the patient presented a new complete hydatidiform mole. Women presented with mutations in the NLRP7, KHDC3L or PADI6 genes are unlikely to obtain normal pregnancies, with a major risk of reproductive failure. In such a context, oocyte donation may be the best option. Only 4 normal pregnancies and deliveries have been published in this situation through this technique to our knowledge.

A single nucleotide polymorphism of IL6-receptor is associated with response to tocilizumab in rheumatoid arthritis patients.

Biological disease-modifying anti-rheumatic drugs (bDMARDs) have changed care of patients with rheumatoid arthritis (RA). However, bDMARDs are costly, can lead to serious infections, and induce a sustained remission in only 30% of RA patients. In this study, we sought to determine if the clinical response to treatment with Tocilizumab (TCZ), an IL-6 inhibitor, varied with genetic background. The efficacy of TCZ was assessed using the European League Against Rheumatism (EULAR) response criteria, measured after 3 months of treatment in two samples of French RA patients (TOCI and ROC studies). Single nucleotide polymorphisms (SNPs) in 21 candidate genes were genotyped using KasPar method (LGC-genomics, UK) and then analyzed to determine their contribution to variation in the response to treatment. One hundred twenty-three patients in the TOCI group (79.8%) and 48 patients in the ROC group (80%) experienced good or moderate EULAR response. The clinical response to treatment was associated with SNP genotype in the gene IL6R, with patients with the homozygous AA-genotype for rs12083537 (IL6R) showing a significantly better response than homozygous or heterozygous patients with the G allele [TOCI: 87.5% of responders for AA genotype vs. 72.2% for AG or GG genotype (p = 0.018); ROC patients: 89.2% of responders for AA genotype vs. 65.2% for AG or GG genotype, p = 0.044]. A meta-analysis combining data from the two cohorts confirmed the lower response rate in patients carrying a copy of the G allele (OR (95% CI) = 0.35 (0.16-0.61), p = 0.001). No association was found with any of the other SNPs tested.

The genetics of recurrent hydatidiform moles: new insights and lessons from a comprehensive analysis of 113 patients.

Hydatidiform mole is an aberrant human pregnancy characterized by early embryonic arrest and excessive trophoblastic proliferation. Recurrent hydatidiform moles are defined by the occurrence of at least two hydatidiform moles in the same patient. Fifty to eighty percent of patients with recurrent hydatidiform moles have biallelic pathogenic variants in NLRP7 or KHDC3L. However, in the remaining patients, the genotypic types of the moles are unknown. We characterized 80 new hydatidiform mole tissues, 57 of which were from patients with no mutations in the known genes, and we reviewed the genotypes of a total of 123 molar tissues. We also reviewed mutation analysis in 113 patients with recurrent hydatidiform moles. While all hydatidiform moles from patients with biallelic NLRP7 or KHDC3L mutations are diploid biparental, we demonstrate that those from patients without mutations are highly heterogeneous and only a small minority of them are diploid biparental (8%). The other mechanisms that were found to recur in patients without mutations are diploid androgenetic monospermic (24%) and triploid dispermic (32%); the remaining hydatidiform moles were misdiagnosed as moles due to errors in the analyses and/or their unusual mechanisms. We compared three parameters of genetic susceptibility in patients with and without mutations and show that patients without mutations are mostly from non-familial cases, have fewer reproductive losses, and more live births. Our data demonstrate that patients with recurrent hydatidiform moles and no mutations in the known genes are, in general, different from those with mutations; they have a milder genetic susceptibility and/or a multifactorial etiology underlying their recurrent hydatidiform moles. Categorizing these patients according to the genotypic types of their recurrent hydatidiform moles may facilitate the identification of novel genes for this entity.

Mutations causing familial biparental hydatidiform mole implicate c6orf221 as a possible regulator of genomic imprinting in the human oocyte.

Familial biparental hydatidiform mole (FBHM) is the only known pure maternal-effect recessive inherited disorder in humans. Affected women, although developmentally normal themselves, suffer repeated pregnancy loss because of the development of the conceptus into a complete hydatidiform mole in which extraembryonic trophoblastic tissue develops but the embryo itself suffers early demise. This developmental phenotype results from a genome-wide failure to correctly specify or maintain a maternal epigenotype at imprinted loci. Most cases of FBHM result from mutations of NLRP7, but genetic heterogeneity has been demonstrated. Here, we report biallelic mutations of C6orf221 in three families with FBHM. The previously described biological properties of their respective gene families suggest that NLRP7 and C6orf221 may interact as components of an oocyte complex that is directly or indirectly required for determination of epigenetic status on the oocyte genome.

Identification of a new exon 2-skipped TNFR1 transcript: regulation by three functional polymorphisms of the TNFR-associated periodic syndrome (TRAPS) gene.

BACKGROUND: Mutations in the TNFRSF1A gene encoding the tumour necrosis factor α cell surface receptor, TNFR1, cause TNFR-associated periodic syndrome (TRAPS) and polymorphisms in TNFRSF1A, including rs4149570, rs767455 and rs1800692, are associated with inflammatory diseases. OBJECTIVES: To describe a new exon 2-spliced transcript-TNFR1-d2-and the impact of these three single nucleotide polymorphisms on exon 2 splicing, transcriptional activity of TNFRSF1A and TRAPS phenotype. METHODS: Expression of TNFRSF1A transcripts was performed by reverse-transcription-PCR in a range of human cells and tissues. Exon 2 splicing and transcriptional activity were analysed in HEK293T and SW480 cells by in vitro alternative splicing and luciferase assays, respectively. We constructed haplotypes containing rs4149570, rs767455 and rs1800692 in controls (n=72), patients with TRAPS (n=111) and in TRAPS-like patients (n=450) to compare their distribution and association with clinical features of TRAPS. RESULTS: TNFR1-d2 was expressed in a tissue-specific manner, whereas TNFR1 expression was ubiquitous. Alternative splicing assays showed that the T-A-T haplotype at rs4149570-rs767455-rs1800692 had a significantly higher expression of exon 2-skipping product (p=0.02) compared with the G-G-C haplotype. Transcriptional activity from the T-T haplotype at rs4149570-rs1800692 was increased compared with the G-C haplotype (p=0.03). In patients with TRAPS, rs1800692 T/T homozygotes were excessively rare (p<10(-4)) and TRAPS-like patients with this genotype experienced less fever. CONCLUSIONS: Our study provides a new mechanism of TNFRSF1A regulation whereby three polymorphisms in the promoter, exon 1 and intron 4 have a functional and combined effect on exon 2 splicing, via a coupling mechanism between transcription and splicing. These polymorphisms may affect the phenotype of TRAPS and TRAPS-like patients.

Predictive Clinical and Biological Criteria for Gene Panel Positivity in Suspected Inherited Autoinflammatory Diseases: Insights from a Case-Control Study.

In order to assess the clinical and biological criteria that predict gene panel positivity in patients with a suspected inherited genetic autoinflammatory disease, we conducted a case-control study. These new selection criteria could replace the national multidisciplinary staff approval before performing genetic testing that has been required since 2019. The study involved 119 positive gene panels matched by panel sizes to 119 randomly selected negative gene panels. The patients were referred to our laboratory for genetic testing between June 2012, and March 2023. The clinical and biological criteria were extracted from a prospectively filled database. We focused our evaluation on accuracy and the positive predictive value. Neonatal symptom onset and deafness had the highest accuracies among all criteria associated with the positivity panel, with 92.9% (88.6; 96.0) and 92.6% (88.5; 95.6), respectively. However, it is important to note that the associated Positive Predictive Values (PPVs) cannot exceed 50%. Despite finding a statistical association between clinical and biological criteria and panel positivity, the predictive values of these criteria were not sufficient to recommend Next-Generation Sequencing (NGS) gene panel testing without the national multidisciplinary staff evaluation.

NLRP7 in the spectrum of reproductive wastage: rare non-synonymous variants confer genetic susceptibility to recurrent reproductive wastage.

BACKGROUND: NLRP7 mutations are responsible for recurrent molar pregnancies and associated reproductive wastage. To investigate the role of NLRP7 in sporadic moles and other forms of reproductive wastage, the authors sequenced this gene in a cohort of 135 patients with at least one hydatidiform mole or three spontaneous abortions; 115 of these were new patients. METHODS/RESULTS: All mutations were reviewed and their number, nature and locations correlated with the reproductive outcomes of the patients and histopathology of their products of conception. The presence of NLRP7 mutations was demonstrated in two patients with recurrent spontaneous abortions, and some rare non-synonymous variants (NSVs), present in the general population, were found to be associated with recurrent reproductive wastage. These rare NSVs were shown to be associated with lower secretion of interleukin 1ß and tumour necrosis factor and therefore to have functional consequences similar to those seen in cells from patients with NLRP7 mutations. The authors also attempted to elucidate the cause of stillbirths observed in 13% of the patients with NLRP7 mutations by examining available placentas of the stillborn babies and live births from patients with mutations or rare NSVs. A number of severe to mild placental abnormalities were found, all of which are known risk factors for perinatal morbidity. CONCLUSIONS: The authors recommend close follow-up of patients with NLRP7 mutations and rare NSVs to prevent the death of the rare or reduced number of babies that reach term.

Screening for NLRP7 mutations in familial and sporadic recurrent hydatidiform moles: report of 2 Tunisian families.

A familial or sporadic recurrent hydatidiform mole is a rare autosomal recessive condition that has been associated with biallelic mutations in the nucleotide-binding, leucine-rich repeat, pyrin domain 7 (NLRP7) gene (19q13.42). Cases from different ethnic origins have been reported earlier. Here we report the first Tunisian patients: 2 sisters with homozygous NLRP7 mutations (p.E570X) and 1 sporadic case with no mutation in NLRP7. Our results extend the number of familial recurrent reproductive wastages due to mutations in NLRP7. We suggest that mutations screening of NLRP7 could be proposed more systematically in women with recurrent pathologic pregnancy outcomes of unknown origin. The rare cases with a typical clinical picture, which were not related to NLRP7 mutation as in our sporadic case, should be investigated more to identify the causative gene.

Mevalonate Kinase-Associated Diseases: Hunting for Phenotype-Genotype Correlation.

Mevalonate kinase-associated diseases (MKAD) are caused by pathogenic mutations in the mevalonate kinase gene (MVK) and encompass several phenotypically different rare and hereditary autoinflammatory conditions. The most serious is a recessive systemic metabolic disease called mevalonic aciduria, and the most recently recognized is disseminated superficial actinic porokeratosis, a dominant disease limited to the skin. To evaluate a possible correlation between genotypes and (1) the different MKAD clinical subtypes or (2) the occurrence of severe manifestations, data were reviewed for all patients with MVK variants described in the literature (N = 346), as well as those referred to our center (N = 51). The genotypes including p.(Val377Ile) (homozygous or compound heterozygous) were more frequent in mild systemic forms but were also sometimes encountered with severe disease. We confirmed that amyloidosis was more prevalent in patients compound heterozygous for p.(Ile268Thr) and p.(Val377Ile) than in others and revealed new associations. Patients homozygous for p.(Leu264Phe), p.(Ala334Thr) or compound heterozygous for p.(His20Pro) and p.(Ala334Thr) had increased risk of severe neurological or ocular symptoms. All patients homozygous for p.(Leu264Phe) had a cataract. The variants associated with porokeratosis were relatively specific and more frequently caused a frameshift than in patients with other clinical forms (26% vs. 6%). We provide practical recommendations focusing on phenotype-genotype correlation in MKAD that could be helpful for prophylactic management.

Phenotypic Associations of PSTPIP1 Sequence Variants in PSTPIP1-Associated Autoinflammatory Diseases.

Pathogenic variants in the PSTPIP1 gene cause pyogenic sterile arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome. They were also identified in a broad spectrum of phenotypes. As their interpretation is sometimes challenging, we discuss the genotype-phenotype association in PSTPIP1-associated autoinflammatory diseases (PAIDs) in light of a recent consensus classification of variant pathogenicity. Only 7 of 39 (18%) of the PSTPIP1 variants found in all reported cases and our national reference center (161 patients [114 probands]) were pathogenic. They were clearly associated with PAPA and PSTPIP1-associated myeloid-related proteinemia inflammatory syndrome (PAMI), reflecting a variable clinical expression of PAIDs.

TNFR1-d2 carrying the p.(Thr79Met) pathogenic variant is a potential novel actor of TNFα/TNFR1 signalling regulation in the pathophysiology of TRAPS.

Binding of tumour necrosis factor α (TNFα) to its receptor (TNFR1) is critical for both survival and death cellular pathways. TNFα/TNFR1 signalling is complex and tightly regulated at different levels to control cell fate decisions. Previously, we identified TNFR1-d2, an exon 2-spliced transcript of TNFRSF1A gene encoding TNFR1, whose splicing may be modulated by polymorphisms associated with inflammatory disorders. Here, we investigated the impact of TNFRSF1A variants involved in TNFR-associated periodic syndrome (TRAPS) on TNFR1-d2 protein expression and activity. We found that TNFR1-d2 could be translated by using an internal translation initiation codon and a de novo internal ribosome entry site (IRES), which resulted in a putative TNFR1 isoform lacking its N-terminal region. The kinetic of assembly of TNFR1-d2 clusters at the cell surface was reduced as compared with full-length TNFR1. Although co-localized with the full-length TNFR1, TNFR1-d2 neither activated nuclear factor (NF)-κB signalling, nor interfered with TNFR1-induced NF-κB activation. Translation of TNFR1-d2 carrying the severe p.(Thr79Met) pathogenic variant (also known as T50M) was initiated at the mutated codon, resulting in an elongated extracellular domain, increased speed to form preassembled clusters in absence of TNFα, and constitutive NF-κB activation. Overall, TNFR1-d2 might reflect the complexity of the TNFR1 signalling pathways and could be involved in TRAPS pathophysiology of patients carrying the p.(Thr79Met) disease-causing variant.

Positive Impact of Expert Reference Center Validation on Performance of Next-Generation Sequencing for Genetic Diagnosis of Autoinflammatory Diseases.

Monogenic autoinflammatory diseases (AIDs) are caused by variants in genes that regulate innate immunity. The current diagnostic performance of targeted next-generation sequencing (NGS) for AIDs is low. We assessed whether pre-analytic advice from expert clinicians could help improve NGS performance from our 4 years of experience with the sequencing of a panel of 55 AIDs genes. The study included all patients who underwent routine NGS testing between September 2014 and January 2019 at the laboratory of autoinflammatory diseases (Montpellier, France). Before March 2018, all medical requests for testing were accepted. After this time, we required validation by a reference center before NGS: the positive advice could be obtained after a face-to-face consultation with the patient or presentation of the patient's case at a multidisciplinary staff meeting. Targeted NGS resulted in an overall 7% genetic confirmation, which is consistent with recent reports. The diagnostic performance before and after implementation of the new pre-requisite increased from 6% to 10% (p = 0.021). Our study demonstrated, for the first time, the beneficial effect of a two-step strategy (clinical expert advice, then genetic testing) for AIDs diagnosis and stressed the possible usefulness of the strategy in anticipation of the development of pan-genomic analyses in routine settings.

Association of TRAF1-C5 with risk of uveitis in juvenile idiopathic arthritis.

OBJECTIVES: Numerous single nucleotide polymorphisms (SNPs) have been associated with JIA, but few of these studies were replicated. We conducted a candidate-gene approach study to assess if some SNPs could be related to clinical subtypes or other clinical features of the juvenile idiopathic arthritis (JIA) such as the risk of uveitis, ANA positivity and the age at onset. METHODS: SNPs in six genes were analysed: PTPN22, STAT4, TRAF1-C5, TGFbeta, TNFAIP3, and C12orf30. We retrospectively included 104 JIA patients, fulfilling the ILAR classification criteria. Association between SNPs and JIA clinical subtypes, the presence of ANA, risk of uveitis and age at onset was evaluated using a Chi2 test or a Fischer test. RESULTS: No associations between different clinical subtypes as well as presence of ANA and the 6 SNPs were found. However, the AA genotype of TRAF1-C5 appeared associated in a subgroup of patients with uveitis in oligoarticular and polyarticular forms [OR 3.77 (95% CI: 1.1067, 12.8527); P=0.066]. Furthermore, AA TRAF1-C5 was significantly more frequent in antinuclear antibodies (ANA) positive patients presenting uveitis, compared to patients without uveitis and without ANA (P<0.05). CONCLUSION: TRAF1-C5 genotype could identify JIA patients with a risk to develop uveitis especially in oligo and polyarticular forms and among ANA-positive children. Given the moderate size of our cohort, this association has to be confirmed in other studies.

Clinical dose effect and functional consequences of R92Q in two families presenting with a TRAPS/PFAPA-like phenotype.

BACKGROUND: TNF receptor-associated syndrome (TRAPS) is a dominantly inherited autoinflammatory condition caused by mutations in the TNFRSF1A gene. The mechanism underlying the variable expressivity of the common variant R92Q (rs4149584; c.362G>A; p.Arg121Gln) is unclear and is of critical importance for patient care and genetic counseling. This study evaluated the impact of the number of R92Q mutations in two unique unrelated families. METHODS: Two patients with undefined but clear autoinflammatory symptoms were referred for genetic diagnosis. Blood samples were collected from the available family members to screen autoinflammatory genes and assess key steps of the TNFR1-mediated signaling pathway using flow cytometry and ex vivo culture. RESULTS: R92Q homozygosity was demonstrated for the two probands. In family 1, the segregation analysis revealed TRAPS-like symptoms in all carriers, with a more severe presentation in the proband, whereas in family 2, the heterozygous parents were totally asymptomatic, suggesting recessive transmission. Functional studies revealed a nonclassical pathogenesis of TRAPS in the two probands and suggested a compensatory mechanism without clear dose effect. CONCLUSION: We observed for the first time a possible clinical dose effect of R92Q. This work highlights the importance of familial studies to reconcile the contradictory reports published on the pathogenicity of this variant.

NLRP7 mutation analysis in sporadic hydatidiform moles in Tunisian patients: NLRP7 and sporadic mole.

CONTEXT: Hydatidiform mole, an aberrant human pregnancy, is commonly a nonrecurrent disease. Recently, a rare autosomal recessive form of familial and/or recurrent molar pregnancies was associated with mutations in the NLRP7 gene. OBJECTIVE: To investigate whether NLRP7 mutations exist in Tunisian women with sporadic hydatidiform moles. DESIGN: Genomic DNA from 38 unrelated Tunisian patients with sporadic hydatidiform moles were screened by sequencing all NLRP7 exons. A high-resolution melting curve analysis was performed on 170 DNA controls to analyze new sequence variants. RESULTS: More than 13% of these patients were heterozygous for NLRP7 mutations. We found 2 novel missense mutations in the heterozygous state, c.544G>A (p.Val182Met) in 1 patient and c.1480G>A (p.Ala494Thr) in 2 patients, and 2 already reported mutations, c.1532A>G (p.Lys511Arg) and c.2156C>T (p.Ala719Val), in 2 patients. None of these mutations were identified in 170 controls except for 1 woman who was heterozygous for p.Val182Met. CONCLUSION: As homozygous NLRP7 mutations are associated with recurrent hydatidiform mole or conception loss, the heterozygous state could represent a risk factor for nonrecurrent mole.

TGF beta1 polymorphisms are candidate predictors of the clinical response to rituximab in rheumatoid arthritis.

OBJECTIVE: To evaluate the association between several candidate single-nucleotide polymorphisms (SNPs) and responsiveness to rituximab in patients with rheumatoid arthritis (RA). METHODS: Sixty-three RA patients were included. Nine genes (13 SNPs) were subsequently analyzed, including those coding for cytokines involved in synovitis (IL10, LTA, TGFß1, TNF-α, TNF receptor II) and genes associated with RA susceptibility (-C5 TRAF1, STAT4, TNFAIP3 and PTPN22). RESULTS: Forty-four patients were defined as responders and 19 as nonresponders. TGFß1 Codon 10 and TGFß1 Codon 25 SNPs were both associated with clinical response (probability to respond to treatment with the Codon 10C/T genotype: OR = 1.6; P = 0.002, and with the Codon 25 G/C genotype: OR = 1.6; P = 0.025). The probability to be a responder when the TGFß Codon10 C/T and TGFß Codon 25 G/C genotypes were co-inherited, doubled (OR = 2.6; P = 0.008). CONCLUSION: The TGFß1 SNPs are associated with a good response to rituximab therapy and as such could be useful genetic biomarkers in predicting therapy outcome.

A comparison of restriction fragment length polymorphism, tetra primer amplification refractory mutation system PCR and unlabeled probe melting analysis for LTA+252 C>T SNP genotyping.

BACKGROUND: From the wide range of methods currently available for genotyping, we wished to identify a quick, reliable and affordable approach for routine use in our laboratory for LTA+252 C>T SNP screening. METHODS: We set up and compared three genotyping methods for SNP detection: restriction fragment length polymorphism (RFLP), tetra primer amplification refractory mutation system PCR (TPAP) and unlabeled probe melting analysis (UPMA). The SNP model used was LTA+252 C>T, a cytokine gene polymorphism that has been associated with response to treatment in rheumatoid arthritis. The study was performed using 46 samples from healthy Caucasian volunteers. RESULTS: Allele and genotype distribution was similar to that previously described in the same population. All three genotyping methods showed good reproducibility and are suitable for a medium scale throughput molecular platform. UPMA was the most cost effective, reliable and safe method since it required the shortest technician time, could be performed in a single closed tube and involved automatic data analysis. CONCLUSION: This work is the first to compare these three genotyping techniques and provides evidence for UPMA being the method of choice for LTA+252 C>T SNP genotyping.

The spectrum of NLRP7 mutations in French patients with recurrent hydatidiform mole.

OBJECTIVES: The NLRP7 gene (19q13.42) is associated with recurrent and/or familial hydatidiform moles. Several mutations, histopathological types and reproductive outcomes have been described. We studied our recurrent hydatidiform mole cases recorded since 1999 in order to identify links between clinic, histology and genetics. STUDY: We present here the gestational history and the spectrum of NLRP7 mutations in our French series. DESIGN: We performed a retrospective study from clinical forms received for genetic diagnosis. Cases declaration was based on a voluntary initiative coming from French practitioners, subjected to patients' agreement. RESULTS: Among 12 recurrent hydatidiform moles investigated, we identified 3 cases of confirmed homozygous NLRP7 mutation and 3 cases of heterozygous NLRP7 mutation. One patient bore a novel mutation p.Leu880Ser in a homozygous state. CONCLUSIONS: We here identified a new homozygous NLRP7 mutation. Unfortunately, no modern therapeutic option has proven effective to obtain evolutive pregnancies. Then, fundamental and clinical researches seem to be necessary. Moreover, collecting RHM cases is essential.