Objective: No studies have examined the association between characteristics of urban areas and future respiratory disease mortality. We examined whether the type of living area during childhood was associated with all-cause and respiratory disease mortality in adulthood.Methods: A total of 81,413 Japanese participants aged 40–79 years old completed a lifestyle questionnaire including the type of childhood living areas. The Cox proportional hazards regression model was used to calculate the multivariable hazard ratios (HRs) with 95% confidence intervals (CIs) of all-cause and respiratory disease mortality.Results: Living in large city areas in childhood was associated with a higher risk of all-cause mortality [HR = 1.05 ], but not with respiratory disease mortality [HR = 1.04 ] compared to rural and remote areas. The excess risk of all-cause and respiratory disease mortality was primarily found in industrial areas among men; the respective multivariable HRs were 1.28  and 1.90 (95% CI: 1.10–3.29).Conclusion: Eliminating childhood health hazards associated with living in industrial areas suggested to reduce the risk of mortality from respiratory diseases in adulthood. According to the 2018 Revision of World Urbanization Prospects, approximately 55% of the global population live in urban areas. This proportion is expected to increase to 68% by 2050 [In the global burden of disease (GBD) study from 2017, the prevalence of respiratory diseases is 7.1%, with chronic obstructive pulmonary disease (COPD) at 3.9% and asthma at 3.6%. About 3.9 million annual deaths and 1,470 disability-adjusted life years (DALYs) per 100,000 are attributable to respiratory diseases worldwide [Air pollution, such as nitrogen dioxide, PM 2.5, or PM 10 is a major risk factor for respiratory disease in industrialized areas . In threFurthermore, childhood SES—including educational level, passive smoking, and malnutrition in the early stages of life—has been associated with childhood respiratory diseases such as asthma and wheezing, and respiratory disease mortality in adulthood –10. SincSince Japan has a history of serious air pollution from the 1900s with remarkable modernization, there is a great need for scientific evidence to research the effect of childhood living environment on lifelong health . TherefoThe Japan Collaborative Cohort Study for Evaluation of Cancer Risk (JACC) study was conducted between 1988 and 1990. The study screened 110,585 participants, including 46,395 men and 64,190 women aged 40–79 years old, from 45 communities across Japan. The methodology of the JACC study has been described elsewhere .In brief, the participants responded to self-administered questionnaires regarding their sociodemographic characteristics, medical history, and lifestyle. From 110,585 participants, we excluded 29,172  in eight areas because the question on the living area during childhood was not included in their questionnaires. Thus, 81,413 participants  were included in the analyses.Before completing the questionnaires, the participants or community representatives were asked for informed consent to participate in this epidemiological study. Informed consent was obtained from each participant in 29 of the 37 study areas . In the remaining eight areas, group consent was obtained from each area leader. The ethics committees approved the investigation protocol of Nagoya University , Osaka University (14285-8), and Hokkaido University (I14-044).We asked participants, “Which area did you live in the longest during the 12 years before you graduated from elementary school?”. In Japan, elementary school graduation is at age 12. In the present study, therefore, childhood was defined as 0–12 years of age. The answers included “large cities with a population of 500,000 or more,” “other cities with a population less than 500,000 (middle city),” “rural and fishing villages,” “remote islands and remote areas,” and “others”. When choosing “large cities with a population of 500,000 or more,” the participants answered one of the following five options: residential, commercial, industrial, mixed city, or other city areas. The mixed city area was defined as the areas with mixed characteristics of residential, commercial, and industrial areas. Areas with characteristics that did not fit into any of these categories were classified as other city areas. Rural and fishing villages, remote islands, and remote areas were combined as rural and remote areas because of the small sample size per area. Finally, we classified their responses into the following seven groups: rural and remote, middle city, residential, commercial, industrial, mixed city, and other city areas.2).We obtained the other information about sociodemographic and lifestyle factors from a self-administered questionnaire at baseline. These included age, sex, adulthood variables of educational level, past medical history such as hypertension and diabetes, height, weight, smoking and alcohol drinking status, exercise and walking habits, mental status, employment status, and eating habits, and childhood passive smoking status. Body mass index was estimated as the body weight (kg) divided by the height squared . Follow-up was continued in 37 areas until the end of 1999 in four areas, 2003 in three areas, 2008 in two areas, and 2009 in the rest of the areas. In this study, the endpoint of death was all-cause  and respiratory diseases (ICD-10 codes: J01-J99). The date of moving from the community was verified through population registration documents. When participants moved out, they were treated as censored cases.Person-years of follow-up were calculated from the date of study enrollment to the date of death, emigration from the community, or the end of follow-up, whichever happened first. The analysis of covariance was used to test for differences in age-adjusted means and the prevalence of baseline characteristics. Hazard ratios (HRs) with 95% confidence intervals (CIs) of all-cause and respiratory disease mortality were calculated using Cox proportional hazards regression models according to the childhood living area category. We confirmed no violation for the proportional hazard assumption in all models. We adjusted for age (continuous), sex (women or men), educational level (≤18 or ≥19 years of age upon completion of education), history of hypertension (yes or no) and diabetes (yes or no), body mass index (sex-specific quintile), smoking status , childhood passive smoking status , alcohol consumption , hours of exercise  and walking , perceived mental stress , employment status (unemployed or employed), frequency of consuming vegetables, fish, fruits, and soybean intakes (quintile). SAS version 9.4  was used for all statistical analyses.The age-adjusted baseline characteristics of the participants are presented according to the type of childhood living areas in During the median follow-up period of 18.8 years, we documented a total of 18,700 deaths  from all causes and 2,367 deaths  from respiratory disease. In this large Japanese cohort study, we found that living in large city areas during childhood was associated with a higher risk of all-cause mortality, but not of respiratory disease mortality, compared to rural and remote areas. On the other hand, the excess risks of all-cause and respiratory disease mortality in adulthood were primarily observed in men who are living in industrial areas among large city areas during childhood. These associations remained statistically significant after adjusting for potential confounders in adulthood. To the best of our knowledge, this is the first study to find a positive association between living in industrial city areas during childhood and mortality from respiratory disease.p = 0.004), 101.4 ml by NO2 (p = 0.005), 79.7 ml by PM2.5 (p = 0.04), and 87.9 ml by elemental carbon (p = 0.007) in eleven air polluted areas, compared to the least polluted area of Los Angeles [Our results were supported by the finding from a previous study on the effect of a child’s environment on the development of respiratory function; the growth deficits of the forced expiratory volume in one second (FEV1) between 10 and 18 years old were 105.8 ml by acid vapor ( Angeles .Childhood SES was associated with educational attainment and adulthood SES, and subsequently, lung disease during adulthood , 14, 15.Air pollution, passive smoking, and malnutrition in childhood can cause impaired lung function or chest illness in childhood, resulting in adult respiratory disease . In the Passive smoking during childhood influences lung function and development and risk for future respiratory diseases in children. Abnormal growth and inflammation of small airways from passive smoking during the postnatal period may account for increased cough, poor lung function, airway resistance, and a higher fraction of exhaled nitric oxide level –25. A coThe strengths of the present study are its prospective study design, sufficient sample size, and long follow-up duration to investigate the association after adjustment for many potential confounders.However, this study had several limitations. First, we obtained information on childhood living areas through self-reports, potentially resulting in false reports. Second, we did not have detailed information, such as the duration of living in industrial areas during childhood, level of air pollution, and childhood SES. Further research is warranted to elucidate the detailed mechanisms of living in large cities and industrial areas during childhood on the risk of respiratory diseases. Finally, since the present results reflect conditions in Japan prior to the 1960’s, caution should be exercised when applying the present results to current situation in urban industrial areas.In conclusion, living in large cities and industrial areas during childhood was positively associated with all-cause mortality. The excess risk of respiratory disease mortality was also observed among men living in industrial areas in childhood after adjusting for potential confounding factors in adulthood. The present study suggests that eliminating childhood health hazards associated with living in industrial areas may reduce the risk of respiratory diseases in adulthood. Further studies are needed to create healthy environments for children to prevent lifelong disease.


Early-onset high myopia (EoHM) is a disease that causes a spherical refraction error of ≥−6 diopters before 10 years of age, with potential multiple ocular complications. In this article, we report a clinical and genetic study of 43 families with EoHM recruited in our center. A complete ophthalmological evaluation was performed, and a sample of peripheral blood was obtained from proband and family members. DNA was analyzed using a customized next-generation sequencing panel that included 419 genes related to ophthalmological disorders with a suspected genetic cause, and genes related to EoHM pathogenesis. We detected pathogenic and likely pathogenic variants in 23.9% of the families and detected variants of unknown significance in 76.1%. Of these, 5.7% were found in genes related to non-syndromic EoHM, 48.6% in genes associated with inherited retinal dystrophies that can include a syndromic phenotype, and 45.7% in genes that are not directly related to EoHM or retinal dystrophy. We found no candidate genes in 23% of the patients, which suggests that further studies are needed. We propose a systematic genetic analysis for patients with EoHM because it helps with follow-up, prognosis and genetic counseling. High myopia (HM), the most challenging type, is defined as a spherical refraction error of −6 spherical diopters (SDs) or more, or an axial length greater than 26 mm. From a physiological point, myopia is a refractive error in which, in a state of relaxed accommodation, light rays parallel to the eye with an origin greater than 6 m away focus anteriorly on the retina after passing through the eye’s refractive system ,2. This The prevalence of myopia and HM worldwide is 30.6% and 2.7%, respectively . The mosThe pathogenesis of myopia relies on genetic, environmental  and microenvironmental  factors. Most children are born hyperopic, and, during the first 2 years after birth, a process known as emmetropization occurs, which is mainly influenced by a change in axial length . From thPhysiopathological factors of the microenvironment such as oxidative stress, inflammation and angiogenesis can also induce EoHM or promote the exacerbation of the disease. Oxidative stress is produced by the metabolism of reactive species of oxygen (ROS). Retinal tissue has the highest oxygen consumption of the body and is directly exposed to natural light. These two factors produce a higher concentration of ROS in the tissue. In addition to this, EoHM patients are known to present an oxidative/antioxidative statue imbalance suggesting that oxidative stress can induce EoHM onset directly . OxidatiZNF644, SCO2, SLC39A5, CCDC111, P4HA2, BSG, CPSF1, NDUFAF7, TNFRSF21, XYLT and DZIP1); four as autosomal recessive  and two linked to chromosome X (ARR3 and OPN1LW). To date, 26 loci have been discovered  [CTNND2, JOANA, CACNA1F and RPGR [PRSS56, BMP3, KCNQ5, LAMA2, TOX, TJP2, RDH5, ZIC2, RASGRF1, GJD2, RBFOX1, SHISA6, FAM150B-ACP1, LINC00340, FBN1, DIS3L-MAP2K1, ARID2-SNAT1 and SLC14A2 [This manuscript focuses on the genetic factors that may be affecting the development of EoHM, in which over 100 genes and 20 chromosomal loci have been identified in association with myopia and the related quantitative traits via linkage analysis, candidate gene analysis, genome-wide association study and next-generation sequencing (NGS). However, only a small number of genes have been identified to predispose individuals to EoHM, suggesting a complex mechanism ,17,18. Eosome X) . Other s SLC14A2 ,23,24.The proper diagnosis of EoHM is essential because it can be the first sign of a syndromic condition. For cases in which an NGS panel is performed with only non-syndromic EoHM-related genes, a syndromic condition, such as Stickler syndrome, might be overlooked. We therefore recommend a broader approach when faced with a child with EoHM. Several ophthalmological diseases, such as childhood glaucoma, retinopathy of prematurity, congenital stationary night blindness and cone-rod dystrophies, are known to be involved in the development of EoHM . It is aThis study is based on the hypothesis of the possible greater involvement of a genetic cause in the development of EoHM when compared with late-onset HM (LoHM). To clarify this, we analyzed 43 families with EoHM to describe not only the pathogenic genes that have already been related to EoHM or related syndromes but also new affected genes that have not been directly related and could be causing this condition.The study results were obtained from an ongoing project for identifying mutations that cause EoHM in a sample of families at a tertiary hospital in Spain. The project also aims to evaluate the implementation of NGS and its relevance as part of the approach for patients with EoHM.A total of 43 patients with EOHM from 43 unrelated families  were recruited based on their phenotype and inclusion criteria  and 0.55 in the left eye (OS). Axial length (AL) was measured with the IOL master 500  with a mean AL of 27.79 mm for the OD and 27.95 mm for the OS. Only 2 patients had an AL shorter than 25.5 mm. The mean spherical refraction was −10.8 SD for the OD and −10.44 SD for the OS, with a spherical equivalent of −11.22 for the OD and −10.44 for the OS .Strabismus was present in 12% of the patients (5/43), with esotropia being the most common type (80%). Twelve percent (5/43) of the patients presented with nystagmus.When we consider only the 37 patients younger than 12 years at the time of the examination, only two (5%) presented with a posterior staphyloma, four (11%) presented with peripapillary atrophy, and 14 (38%) presented with diffuse chorioretinal atrophy. One patient had a colobomatous papilla, and one patient had complete retrolental retinal detachment in one eye . We perfAfter performing an NGS analysis with a specifically designed panel, we found 46 variants in 33 probands in known genes for EoHM . Of thesCOL2A1 and another two had an affected variant in COL11A1, while the rest had an affected variant in TRPM1, CACNA1F, PAX6, OPA1, ARL6, SCO2 and NDP  and in three families   .Considering zygosity, 40 of the variants found were heterozygous (87%), whereas only one variant was homozygous (2%) and five were hemizygous (11%).In this study, we performed a mutational analysis of Spanish patients diagnosed with EoHM implementing a customized NGS panel containing 419 genes related to ophthalmological disorders with a suspected genetic cause. The use of an NGS panel strategy rather than whole exome sequencing as a first approach for the genetic study is in response to the need for a less expensive method that can provide efficient analysis with a low rate of incidental findings.SCO2 and ZNF644. Two patients (from families OFT-00533 and OFT-00554) in our cohort showed previously reported variants in gene SCO2 (OMIM:604272) [SCO2 that have been considered pathogenic or likely pathogenic for high or extreme myopia (>30 diopters). In contrast, Piekutowska-Abramczuk et al. (2016) [SCO2 in the development of EoHM are needed to determine whether the variants reported in the patients are actually the cause of the phenotype or just a risk factor whose further development is affected by the environment. In our cohort, we confirmed that the variants are just a risk factor and not the cause, given that the proband of family OFT-00554 carried the same heterozygous variants as their healthy parents.As previously mentioned, a few genes are related to non-syndromic EoHM, such as :604272) ,37,38,39:604272) . The exa:604272) ,37,40,41ZNF644 (OMIM:614159), a zinc finger transcription factor expressed in the retina and the retinal pigment epithelium. The protein’s biological function has not been identified. As a transcription factor, however, it might regulate genes involved in eye development, triggering a mutant protein able to modify the axial elongation of the eye globe and cause EoHM [Another gene related to non-syndromic autosomal dominant EoHM is 4 OMIM:6159, a zinuse EoHM . Numerouuse EoHM ,40,43,44TRPM1 (OMIM:603576), CACNA1F (OMIM:300110), KCNV2 (OMIM:607604), MERTK (OMIM:604705), RDH5 (OMIM:601617) and ARL6 (OMIM:608845).Inherited retinal dystrophies are sometimes associated with EoHM, which might be the first isolated sign at presentation. Of the 46 variants found in our cohort, ten were found in six genes associated with inherited retinal degeneration  : TRPM1, a gene associated with congenital stationary night blindness (CSNB), a genetically and clinically heterogeneous disease that manifests as non-progressive nyctalopia and with an electronegative full-field electroretinogram. Our cohort had one patient (from family OFT-00074) with heterozygous compound variants in this gene who presented with nystagmus, esotropia and EoHM. The patient underwent an electroretinogram, which showed moderate-severe disease in the peripheral retina, mild-moderate disease in the central retina and an electronegative result for the bilateral scotopic response, which suggests a functional disorder of the bipolar cells. According to Miraldi Utz et al. (2018), patients presenting EoHM, strabismus, nystagmus and a variant in this gene should undergo full-field electroretinography, given that a considerable proportion of these patients might not present nyctalopia at the start and that the procedure could help diagnose CSNB [We reported 4 variants in ose CSNB . In addiCACNA1F, a gene present in the X chromosome and related to Aland Island disease and cone-rod dystrophy. Wutz et al. (2002) mentioned the possibility of hemizygous variants in this gene causing a phenotype of CSNB with EoHM as one of their most common features [Another gene associated with the same phenotype of CSNB is features ,45,46. ORDH5, a gene expressed in the retinal pigmented epithelium and involved in the retinoid cycle, the metabolic pathway that regenerates the visual chromophore following light exposure [One patient from family OFT-00391 had a variant in exposure ,47. The exposure . Althougexposure ,49,50.KCNV2 (OFT-00092), MERTK (OFT-00474) and ARL6 (OFT-00407), genes with a phenotype of retinal cone dystrophy in the case of KCNV2 and retinitis pigmentosa in the other two cases. KCNV2 has EoHM as one of its ocular features. In the case of MERTK and ARL6 where EoHM was not reported, however, this manifestation might be a second consequence of the genetically determined retinal dystrophy.Patients from three families with a phenotype of EoHM and retinal dystrophy had variants in Regarding the association between EoHM and vitreoretinal inherited diseases, Marr et al. (2001), showed an even higher association between HM and systemic and other ophthalmological conditions, taking into account that they did not consider an exclusion criterion the presence of a syndromic phenotype as an exclusion criteria, as we did in our study. Out Of the 112 probands, the authors found that only 8% had non-syndromic HM, 54% had an underlying systemic association, and the remaining 38% remaining had further HM-related ocular problems associated with HM . Other sCOL9A1 (OMIM:120210), COL2A1 (OMIM:120140), COL11A1 (OMIM:120280), CEP290 (OMIM:610142), PCDH15 (OMIM:605514), LRP5 (OMIM:603506), PEX1 (OMIM:602136), CFH (OMIM:134370), OPA1 (OMIM:605290) and NDP (OMIM:300658).Based on these results, the RetNet genes associated with vitreoretinal inherited diseases with syndromic manifestations should be included for patients with EoHM, such as COL2A, COL9A1 and COL11A1), divided into four pathogenic and likely pathogenic variants and three VUS in OFT-00181(whose variant was inherited from the affected mother), OFT-00191 (from the affected father), OFT-00209, OFT-00275, OFT-00453, OFT-00490 and OFT-00590.Fifteen percent of the reported variants in our study were in three collagen genes  are affected, or as autosomal recessive if COL9A1, COL9A2 (OMIM:120260) and COL9A3 (OMIM:120270) are affected [These three genes are considered to cause Stickler’s syndrome, a connective tissue disorder caused by mutations in collagen genes, which can be inherited as an autosomal dominant disorder if affected . This syaffected , orofaciOur study patients were recruited based on the fixed inclusion criteria; therefore, the initial diagnosis was of EoHM, and the finding of the genetic alteration enabled the diagnosis of a possible syndromic disease that had not been previously suspected. A funduscopic examination under sedation was performed on the patients with mutations in one of these three collagen genes, where pathognomonic vitreous alterations for Stickler’s syndrome were found, leading to the assessment of the patients by other specialists. This was the case in our cohort with families OFT-00181 and OFT-00275, in which the genetic diagnosis allowed for a preventive treatment for the existing retinal lesions that could have led to further complications.The importance of an early diagnosis of Stickler’s syndrome relies on closer follow-up by various specialists to evaluate other possible phenotypic disorders, such as facial alterations, cleft palate, elbow hypermobility, femoral head necrosis and valgus knee. Specifically, it allows for a more thorough evaluation by the ophthalmologist to anticipate possible complications and perform preventive treatment to prevent vision loss in these patients. Considering Stickler’s syndrome in patients with HM before 10 years of age is therefore highly relevant, even if it appears to be the only manifestation, given that it could be the presenting symptom ,56.NDP that encodes the norrin protein, a secretory growth factor that regulates retinal angiogenesis [Other syndromes with EoHM included in the ocular phenotype are Marfan syndrome and homocystinuria ,58. Therogenesis . The repogenesis . In thisCEP290  and the other in PCDH15 (a gene responsible for Usher syndrome). Despite the fact that these two genes do not have a phenotype of high myopia, Wan et al. (2018) reported CEP290 and PCDH15 as novel candidate genes of myopia pathogenesis [PCDH15 is included in a novel locus for high-grade myopia [In this study, one patient from family OFT-00177 with EoHM and retinal dystrophy had two VUS, one in ogenesis . Young ee myopia .LRP5 that are related to syndromes without ocular features and exudative vitreoretinopathy. Chen et al. (2018) observed the presence of a synonymous variant in this gene as pathological and the cause of EoHM presented by their patient. The authors suggested that although the variant does not alter the amino acid chain, the altered nucleotide can modify the secondary structure of the protein and can influence protein holding and gene expression [PEX1 is a gene whose variants are related to defects in peroxisome biogenesis. In our cohort, two families (OFT-00223 and OFT-00568) had VUS in this gene. Poll-The et al. (2004) indicated that variants in this gene can produce numerous phenotypes, EoHM being one common feature present in their patients [Families OFT-00178 and OFT-00332 had variants in gene pression . PEX1 ist al. 200 indicatepatients .CFH. Basal laminar drusen is the only ocular phenotype reported with a mutation in CFH, but variants can also produce complement factor H deficiency or hemolytic uremic syndrome. In the last few years, various researchers have tried to link EoHM to changes in CFH. In 2021, García-Gen et al. established the key role of CFH in the development of myopic disease [Another gene of interest is  disease . In our OPA1 and high myopia [OPA1 is known for its relevance in autosomal dominant optic atrophy (ADOA), which is the most common form of hereditary optic neuropathy. In fact, OPA1 mutations are responsible for approximately 90% of cases [OPA1 protein results in abnormal mitochondrial cristae, the release of cytochrome c and apoptosis [OPA1 mutation, and a higher frequency of EoHM [OPA1 challenged the assumption that this mutation induced EoHM. ADOA is known to induce a thinning of the retinal nerve fiber layer and of the ganglion cell layer, inducing a reduction in total macular thickness [Previous studies have proven the relationship between alterations in h myopia ,64. Howepoptosis . In our poptosis . Chen S  of EoHM . Neverthhickness . Li C ethickness .MMP1 (OMIM:120353), MMP9 (OMIM:120361) and MMP10 (OMIM:185260) in families OFT-00223, OFT-00429, OFT-00436 and OFT-00586. In the case of family OFT-00436, the variant was inherited from the affected mother. The relevance of studying this family of proteins relies on their involvement in organizing the tissue, given that the proteins are responsible for the degradation of collagen and other extracellular matrix components. The structural organization and constant remodeling of the sclera is highly dependent on the activity of the fibroblast, which is the major extracellular matrix-producing cell [In contrast to the above, there were a few genes reported in our study that are not clearly considered to cause EoHM or retinal dystrophy but could be related to the onset of EoHM. Given that EoHM is characterized by scleral thinning, we studied another family of proteins: matrix metalloproteinases (MMPs). In this case, we found VUS in the three genes responsible for MMPs: ing cell . Among ting cell . MMP2, ting cell .Given that the increase in myopia in highly myopic patients is almost always associated with an increase in AL, a thinning of the sclera occurs, mainly at the posterior pole. The most relevant consequence is the potential formation of a posterior staphyloma, an area in which the thin sclera becomes ectatic. Even without staphyloma, when the sclera of a highly myopic patient is compared with that of a non-myopic patient, the highly myopic sclera is up to 50% thinner .Due to this constant remodeling and thinning of the sclera, changes in the myopic fundus increase as patients age . When coMMP genes’ expression in the sclera can lead to greater susceptibility to increased axial elongation of the eye [The variation in these  the eye . Accordi the eye . The autMMP2 activation by scleral fibroblasts, which contributes to scleral extracellular matrix degeneration, scleral thinning and possible ocular ectasia [The models used by David et al. (1997) indicated that the mechanical stress on the retina and choroid during eye movements is significantly higher in larger eyes than smaller eyes . Accordi ectasia ,77.MMP1, MMP2, MMP3, MMP8 (OMIM:120355), MMP9, MMP10, MMP11 (OMIM:185261), MMP13 and EOHM [Other studies, however, such as that by Schache et al. (2012) with an Australian cohort and the one by Nakanishi et al. (2010) with a Japanese cohort, found no association between mutations in and EOHM ,78.TIMP2 (OMIM:188825) mRNA expression, a variant of which was present in family OFT-00209. The activity of MMP2 and TIMP2 is correlated to increased MMP2 activity over a critical point, inducing inhibition of TIMP2 activity, thereby favoring collagen degradation [MMP2, TIMP2 and TIMP3 (OMIM:188826) genes were not associated with high myopia in their cohort [MMP activity was also significantly higher in the vitreous samples from the EoHM group than in those of the control group [MMP activity could play a role in the pathogenesis of human high myopia. However, given that studies that have analyzed the relevance of MMP2, TIMP2 and TIMP3 in blood samples have shown no differences between patients with EoHM and controls, further studies are needed to evaluate whether the importance of MMPs and TIMPs relies on changes on a more local level.The development of myopia has been associated with a mean reduction in radation . A studyr cohort . Howeverol group ,80. FromCRYBB3 (OMIM:123630), CRYGC (OMIM:123680), CRYGA (OMIM:123660) and CRYBA1 (OMIM:123610) in four families OFT-00332, OFT-00391, OFT-00493 and OFT-00630, which have not been previously published.Patients with EoHM appear to have more lens changes ,82. The GPR143 (OMIM:300808), which is expressed in the retinal pigment epithelium and is associated with the development of albinism and nystagmus [Another of the EoHM-related genes is ystagmus . We founVDR (OMIM:601769) gene in OFT-00223, which had already been reported as a phenotype of vitamin D resistance but is considered a risk factor for developing EoHM [We found one variant in the ing EoHM ,92,93.LAMA2 (OMIM:156225) is considered to produce muscular dystrophy with very few ocular features. Laminins are structural proteins that are the main components of the extracellular matrix, whose changes in its composition in the sclera are related to an AL alteration. Specifically, laminin alpha 2 is expressed in the sclera and optic nerve, is present in extraocular muscles during development and can act as a guide for retinal ganglion cell growth. Kiefer et al. (2013) and Cheng et al. (2013) reported that this gene can be considered one of the multiple factors that act in the development of myopia [LAMA2. Another gene related to AL abnormalities is ZNRF3 (OMIM: 612062) [ OMIM:1565 is consf myopia ,95. In o 612062) , a proteEPHA2 (OMIM:176946), a gene related to age-related cortical cataracts. This phenotype was reported to have EoHM as an ocular feature [A patient from family OFT-00463 had a variant in  feature . ConsidePAX6 (OMIM:607108), the paired box 6, which is considered a master gene for eye development. The gene’s best-known phenotype is aniridia. According to a meta-analysis by Tang et al. (2014 and 2018), there is a suggestive association between PAX6 and extreme/high myopia but not lower grade myopia, although further studies are necessary for validation [PAX6 plays an important role in controlling eye globe growth, according to Bilbao-Malavé et al. (2020), and has been shown to have a suggestive association with EoHM, as demonstrated in a meta-analysis that included studies mainly performed on Asian populations [A patient from family OFT-00045 had a variant in lidation ,98. PAX6ulations ,100,101.IGF1R, which encodes for IGF1R, a transmembrane receptor tyrosine kinase responsible for mediating proliferation and protection from apoptosis. A study by Wang et al. observed no relationship with mutations in IGF1R or IGF1 in the development of EoHM in a Chinese population [IGFR1 mutations as a cause of EoHM, given that a study performed on chickens by Penha et al. (2011) showed that overminus lens therapy, and therefore hyperopic defocus, influenced the expression of insulin receptor and IGF1R receptor in the retinal pigment epithelium. The shift of the focal plane behind the photoreceptor layer triggers substantially increased eye growth [Lastly, a patient from family OFT-00429 presented a mutation in pulation . Howevere growth ,103.As mentioned above, high myopia may be related to the microenvironment as well as genetics. In 2017, Fedor et al. demonstrated that serum zinc and copper levels in patients with EoHM were lower than those of the control group. As these two factors are antioxidant elements, the existence of an oxidant/antioxidant imbalance in patients with EoHM is suggested . In 2020ZNF644, TIMP2, MMP1, MMP9 and MMP10). Another four genes of the panel, HGF, RBP3, MMP2 and MMP3, have been related to this factor even though the patients did not show any variant in them [Mérida et al. also found an increase in the total nitrite levels, the end product of nitric oxide metabolite, in highly myopic patients. This parameter is considered as one of the origins of oxidative stress damage and reduced antioxidant capacity, but nowadays it has been suggested to play a role as a nitric oxide reservoir that produces nitric oxide in hypoxia conditions. Finally, it is known to act as an eye modulator in developing myopia, acting in ocular growth, intraocular pressure regulation and retinal vascular development. Despite this, it is not easy to understand the results because of the neurodegenerative/neuroprotective role of the nitric oxide. In our panel, we found variants in five genes that have been linked to oxidative stress  affecting 5–10% of these patients. Pathological myopia is the main cause of onset of CNV in patients under 50 years of age . The preFT-00097 . In thosFT-00097 .As previously mentioned, the hypothesis of our study is based on is the potential greater involvement of genetics in the development of EoHM than of LoHM. Zhou et al. (2018) compared the mutation detection rate in a study of LoHM with one of their previous studies, considering the possible involvement of RetNet genes. The authors found a genetic cause in only 7.2% of the patients with LoHM and in 23.8% of those with EoHM , a resulThe combined ophthalmological and genetic approach was performed by the Ophthalmogenetics Multidisciplinary Unit at La Paz University Hospital, Madrid, Spain, according to the tenets of the Declaration of Helsinki and was approved by the ethics committee.Participants: A total of 49 patients from 43 unrelated families aged 4–74 years were recruited for the study, as well as both parents of the proband for those cases in which it was possible. After informing and obtaining written consent from the proband and from the patients’ parents or their guardians, a peripheral venous blood sample was collected to isolate the genomic DNA from leukocytes.The inclusion criteria were as follows: (1) bilateral myopia with a refraction error ≥−6 diopters in at least one eye with onset before 10 years of age; (2) absence of cataracts; (3) absence of corneal disease or other ophthalmological diseases that produce secondary high myopia; and (4) absence of syndromic phenotype. Patients were recruited according to phenotype and inclusion/exclusion criteria and then underwent a genetic analysis.Clinical evaluation: A complete ophthalmological evaluation of the proband was performed, which included best corrected visual acuity, refraction before and after cycloplegia, funduscopic examination, AL measurement, retinography images and OCT.Genetic analysis: Genomic DNA was isolated from leukocytes in peripheral venous blood samples in the preanalytical area of our institute using the commercial Chemagic Magnetic Separation Module I . DNA concentrations were measured by spectrofluorometer quantification using a NanoDrop ND-1000 Spectrophotometer . Paired-end libraries were created using 1 µg of genomic DNA with KAPA HyperPrep Kit  and hybridization with a KAPA HyperCapture Reagent Kit .Custom panel design: The panel was captured using OFT-v3-1 design : HG19 NCBI Build 37.1/GRCh37, the target bases covered 99.5% and the size was 1,245,179 Kb. The mean horizontal coverage was 99.91%, and the mean sequencing depth per nucleotide was 467.Bioinformatic analysis: The first analysis was performed by the Institute of Medical and Molecular Genetics (INGEMM) Clinical Bioinformatics team, who developed an analytical algorithm that identifies point polymorphisms (SNP) and insertions and deletions of small DNA fragments inside the capture regions that are included in the NGS panels. The system comprises a sample pre-processing step, alignment of reads to a reference genome, identification and functional annotation of variants, and variant filtering. All these steps employ open tools widely used in the scientific community, as well as proprietary tools. Furthermore, all phases are designed in a robust manner and include statistical parameters that provide information on the status of the process and the convenience of continuing with the analysis. This system allows for the monitoring of the process and the appropriate quality controls to issue a reliable report on the aforementioned variants. Lastly, the system backs up the raw and processed data, which are stored in a database using encrypted and anonymized records to preserve patient confidentiality.The bioinformatics analysis was performed by the Clinical Bioinformatics Unit of the INGEMM center using the following software tools: trimmomatic-0.36, bowtie2-align version 2.0.6, picard-tools 1.141, samtools version 1.3.1, bedtools v2.26 and GenomeAnalysisTK version 3.3-0. The databases employed were dbNSFP version 3.5, dbSNP v151, ClinVar date 20180930, ExAC-1, SIFT ensembl 66, Polyphen-2 v2.2.2, MutationAssessor, release 3, FATHMM, v2.3, CADD, v1.4 and dbscSNV1.1.Genotype-phenotype correlation: The second analysis consisted of evaluating the pathogenic clinical significance of the variants found in the patients by employing multiple databases. The first and most extended database is VarSome, which contains an algorithm that uses the 2015 ACMG guidelines [idelines , based oFamily studies: When the variant was fully validated in the proband and the family members, the third part of the analysis consisted of studying the phenotypes related to the gene, to determine whether the variant being studying was the causative variant , six had variants in genes related to a syndromic disease that feature retinal dystrophy  and one had a variant in PAX6 that is not included in any of the groups mentioned but was reported to have a suggestive association with EoHM development.Eleven families in our study had pathogenic and likely pathogenic variants, 1one family had a variant in SCO2 and ZNF644) and seven families with variants in genes associated with other retinal dystrophies . These results support the hypothesis previously indicated by other researchers who found 25% and 38% of variants related to this type of ocular disease and who indicated that EoHM could be a secondary consequence of a genetically determined retinal dystrophy. Ten families had variants in genes related to a syndromic disease with retinal dystrophy . Genetic analysis of patients with EoHM is relevant to anticipate possible syndromic manifestations and other ophthalmological conditions, especially in cases when the disease is present as a non-typical phenotype. The detection of variants that indicate other than a simple EoHM allow these patients to be managed by multidisciplinary units and thereby perform more global patient care, preventive treatment in those cases where it is possible and providing closer follow-up to prevent vision loss. Lastly, 14 families had at least one variant in genes such as GPR143, TIMP2, VDR, MMP1, MMP9, MMP10, PAX6, CRYBB3, CRYGC, CRYGA, GRYBA1, IGF1R, EPHA2, ZNRF3 and LAMA2. Although these genes are not clearly considered to cause EoHM or retinal dystrophy, the published information could relate them to the onset of EoHM. Further studies are needed to confirm the hypothesis of the involvement of these genes.Furthermore, 24 families had VUS, two families with variants in genes related to non-syndromic EoHM (The genetic alterations for most patients with EoHM have not yet been defined, with the causative variant found in only a small number of patients. The genetic study of this disease is currently focused on evaluating every single gene suspected of being a candidate of EoHM. We found no candidate genes in 23% of the patients, which suggests that whole exome sequencing and whole genome sequencing studies, animal models and other studies are needed to clarify the disease’s genetic cause.Based on the above, we can state that EoHM is a complex disease based on a combination of genetic, environmental and microenvironmental components. We recommend a systematic genetic analysis of patients with EoHM and of their relatives, given that it promotes better management of family members, helping with follow-up, prognosis and genetic counseling.


An increased number of TRAP positive cells (tartrate resistant acid phosphatase) was observed in BT and BTP. We conclude that PBMT positively influenced the repair of bone defects filled with B and T.In this preclinical protocol, an adjunct method is used in an attempt to overcome the limitations of conventional therapeutic approaches applied to bone repair of large bone defects filled with scaffolds. Thus, we evaluate the effects of photobiomodulation therapy (PBMT) on the bone repair process on defects filled with demineralized bovine bone (B) and fibrin sealant (T). The groups were BC (blood clot), BT (B + T), BCP (BC + PBMT), and BTP (B + T + PBMT). Microtomographically, BC and BCP presented a hypodense cavity with hyperdense regions adjacent to the border of the wound, with a slight increase at 42 days. BT and BTP presented discrete hyperdensing areas at the border and around the B particles. Quantitatively, BCP and BTP  showed higher mean bone density volume in relation to BC and BT . Histologically, BC and BCP presented deposition of immature bone at the periphery and at 42 days new bone tissue became lamellar with organized total collagen fibers. BT and BTP showed inflammatory infiltrate along the particles, but at 42 days, it was resolved, mainly in BTP. In the birefringence analysis, BT and BTP, the percentage of red birefringence increased , but green birefringence was similar in relation to 14 days . The number of osteocytes in the neoformed bone matrix proportionally reduced in all evaluated groups. Immunostaining of bone morphogenetic protein (BMP—2/4), osteocalcin (OCN), and vascular endothelial growth factor (VEGF) were higher in BCP and BTP when compared to the BC and BT groups ( Bone tissue presents good regenerative capacity, and, on several occasions, it has the property of restoring its original bone structure both in architecture and in physiology ,2. HowevIn addition, in cases of large bone defects in which the regenerative physiological capacities are exceeded, reconstructive surgery procedures with bone grafting as an important treatment technique are required ,8. AmongGiven these limitations, there is an increase in the search for mechanically and biologically adequate bone substitutes capable of repairing critical-size bone defects by a new tissue with morphofunctional characteristics similar to the original ,12. WithOften, bone substitutes are incorporated into three-dimensional scaffolds, such as fibrin sealants, to form a graft mixture moldable to the receptor bed, preventing the dispersion of particles and providing structural stability, favoring the adhesion of bioactive molecules and cells, and providing conditions for proliferation and cell differentiation . Among tIn recent years, innovative modalities, such as low-intensity pulsed ultrasound, photobiomodulation therapy (PBMT) with low-level laser, and hyperbaric oxygen therapy, or their combination, are being evaluated in an attempt to improve metabolism and accelerate the bone repair process ,20,21,22Despite the previous existence of publications on the use of three-dimensional scaffolds in regenerative medicine and the growing interest in adjuvant methods that seek to overcome the limitations of conventional therapeutic approaches for bone repair, few studies have investigated the effect of PBMT associated with biomaterials on the process of bone regeneration.Therefore, we aim to evaluate the effects of PBMT in the repair of critical bone defects in rat calvaria filled with bone substitute and fibrin sealant, by means of qualitative and quantitative microtomographic, histomorphological studies using Masson’s trichrome and Picrosirius red staining, and immunohistochemical analysis with BMP—2/4, OCN, VEGF and TRAP immunomarkers.2/g; and compressive strength, 35 MPa.We used the inorganic matrix Bio-Oss™ , from bovine bone cortical material with a calcium-phosphorus ratio and a structure very close to human hydroxyapatite , with the following physicochemical characteristics: granules of cancellous bone (0.25–1 mm); demineralized bovine bone (B) matrix–xenograft; sterilization, γ-irradiation; pores sizes, macropores (300–1500 µm) and micropores ; amount, 2.0 g vial; purification process, multi-phase ; intracrystalline spaces, 3–26 nm; porosity, 70–75% of the total volume; internal surface area, 100 mThe fibrin sealant Tisseel (T) has part of its constituents derived from human plasma. Tisseel Lyo™ is the trademark manufactured by Baxter Healthcare Ltd., Norfolk, United Kingdom, with Ministry of Health Registration ANVISA No. 1.0683.0182. It is available in four glass vials containing: component 1 containing Tisseel Lyo powder —freeze-dried, with 91 mg/mL human fibrinogen; component 2 containing powdered thrombin—freeze-dried, with 500 IU/mL human thrombin; component 3 containing aprotinin—diluent solution of component 1 containing 3000 UIC/mL synthetic aprotinin; component 4 containing calcium chloride solution—diluent for component 2 containing 40 μmol/mL of calcium chloride. Tisseel Lyo™ contains 0.6–5 IU/mL human coagulation factor XIII, which is isolated from plasma together with human fibrinogen.In preparation and reconstitution of the fibrin sealant, all vials were initially preheated in a water bath for approximately 3 min at a temperature of 33–37 °C, controlled by a mercury thermometer . The aprotinin solution was then transferred to the flask containing the freeze-dried Tisseel Lyo™ powder and the calcium chloride solution into the vial containing freeze-dried thrombin, shaking rapidly, but avoiding foaming. The flasks with the solutions of sealant protein and thrombin solution were then returned to the water bath for one minute to be used. The solutions were maintained at this temperature throughout the surgical procedure until the moment of use within 4 h after reconstitution A.Rattus norvegicus), adult male, healthy, and weighing around 400 g.Thirty-six Wistar rats were obtained from the bioterium of the Ribeirão Preto , following the inclusion criteria: Wistar rats  in an air-conditioned environment, period dark–light 12/12 h, humidity 60% ± 10, air exhaustion, lighting 150 lux/1 m floor, maximum noise 70 dB (decibel), and temperature 22 °C ± 2 °C.The animals were received at 42 days of age and, during the entire experimental period, they were housed in cages containing four animals each . The cages had feeders and drinkers “ad libitum” . This preclinical study was performed according to the ARRIVE  guidelines  and expen = 16  and BT, n = 20  and that these groups were further subdivided into BC, n = 8 , BCP, n = 8 , BT, n = 10 , and BTP, n = 10 , according to the treatment provided  and Ketamine 50 mg/kg . A semi-lunar incision was made to expose the cortical bone extensively and thus to avoid damages to the periosteum and adjacent tissues by the trephine.TM  to avoid osteonecrosis, in the center of parietal bones, including the sagittal suture . . 3 of castituted A4. The aAfter the grafts had been repositioned, the periosteum and overlying tissues were repositioned to reduce the risk of invagination into the bone defect .For post-surgical recovery, the animals were exposed to incandescent light and received a single oral dose of paracetamol 200 mg/kg  dissolved in water available in the cages.TM IBRAMED, Amparo, Brazil)  laser (Laserpulse Brazil) C was use Brazil)  21,28].,28.TM IBTM—Commerce of Materials for Laboratories LTDA, Campinas, Brazil) for 48 h and subsequently sent for microtomographic examination.The animals were randomly divided to be euthanized after 14 and 42 days of the surgical procedure. They received an excessive dose of xylazine/ketamine, following the guidelines of the National Council for the Control of Animal Experimentation (CONCEA/Brazil). In the BC and BCP groups, 4 animals/period; in BT and BTP, 5 animals/period. The skin-coated skulls were collected and fixed in 10% phosphate-buffered formalin . The images were captured with 0.73° at each pace, 13.76 µm voxel, and further reconstructed using NReconTM v.1.6.8.0, SkyScan, 2011 . Ring artefact and beam hardening corrections were applied in reconstruction. Afterward, the reconstructed images were realigned using DataViewerTM 1.4.4.0 software .After fixation in formalin solution, the specimens were subjected to X-ray beam scan in the computed microtomographic machine SkyScan 1174v2 , based on the average diameter and minimum height (8 × 0.8 mm) obtained between the samples, to quantify the structures. Subsequently, the binarization of the images  was performed with the upper and lower limits  for neoformed bone 130–72 and biomaterial 130–255, and then the volume of each phase. Afterward, the bone volume (BV), percent bone volume , and bone mineral density (BMD) were measured for each dataset and the remaining particle percentages were taken by subtracting the bone volume (BV/TV) of the bigger amount of bone particles from the bone volume (BV/TV) of total amount of bone particles (The region of interest was chosen as a sleeve around new-formed bone (NFB) and bone particles individually with 0.5 mm of thickness in the plan coronal, using the CTAnarticles .TM III  and 0.44% sodium hydroxide  for a period of 7 weeks. Specimens were submitted to standard histological procedures for embedding in HistosecTM .Subsequently, the pieces were washed in tap water for 24 h and placed in 10% ethylenediaminetetraacetic acid (EDTA) solution containing 4.13% TitriplexTM, Tokyo, Japan). Subsequently, the sections were stained by Masson’s trichrome, hematoxylin, and eosin , picrosirius red solution and against Harris hematoxylin stain , and immunostaining for markers of bone formation and resorption .Semi-serial sections with a 5 μm thick coronal plane were used to reach the center of the 8 mm defect using the scale free size tool available in DP controller software  at approximate magnifications of 10 and 40×. In sections stained with Masson’s trichrome, the evaluation consisted of description of the healing events such as granulation tissue, bone neoformation and remodeling, inflammation, and the interaction between the biomaterial bone graft and newly formed bone.The histologic sections were analyzed by light microscopy  bone, which stains green. This technique is also used for connective tissue, staining its collagen fibers in green, highlighting them from other tissue elements.TM, Microsystems, Wetzlar, Germany) connected to confocal laser microscope and capture system . Three histological fields (10× magnification) were captured corresponding fully to the defect. All remaining bone present in the histological fields and removed from the images were then selected to avoid counting these fibers in Adobe Photoshop CS6 software.Picrosirius red staining was used to determine the quality and quantity of a new organic matrix in the studied periods of 14 and 42 days of repair. Images were obtained from the defect using higher-resolution Leica DFC 310FX camera  and the total area, the biomaterial area of the particles and the area of connective tissue plus newly formed bone, was determined with dashed lines, yielding values in pixels2. The birefringence analysis of the collagen content of bone healing defects is shown in The Images were transferred to AxioVision Rel. 4.8 Ink analysis software  area analysis of each fiber type per color was assessed. Woven bone was recognized by its random, unorganized fibrillar pattern, usually with polarization colors ranging from red-orange (poorly organized woven bone) and lamellar bone (bright green/yellow), depending on fiber width.TM MicroImaging GmbH, Jena, Deutschland). The areas of bone neoformation and medullary channels were manually surrounded with dashed lines to determine the total matrix area in pixels2 (px2 = 0.528 mm2). Then, three horizontal lines were drawn from each total matrix area to count the osteocyte nuclei in each one. The collected data were analyzed using Microsoft Excel 2016 and GraphPad PrismTM Software, Inc, with significance level set at p < 0.05  at 95 °C for 20 min. Then, endogenous peroxidase and nonspecific sites were blocked using 3% hydrogen peroxide for 1 h and bovine serum albumin  for 12 h, respectively.Standard deparaffinization, rehydration, and antigen-recovery procedures were used. Briefly, xylene and ethanol were used to deparaffinize and rehydrate the tissues and, for antigen recovery, the tissues were incubated in a sodium citrate buffer in a pressurized chamber , anti-BMP 2/4 , and anti-OCN  e anti-TRAP . For signal amplification, the sections were incubated with biotinylated secondary anti-mouse/rabbit IgG antibody generated for 2 h  and subsequently treated with streptavidin conjugated to horseradish peroxidase—HRP  for 1 h. The development was carried out using the 3,3′-diaminobenzidine tetrachlorhydrate  as a chromogen. Counterstaining with Harris hematoxylin was performed for the samples used to detect VEGF, BMP—2/4, and OCN, and counterstaining did not use counterstaining in the samples used to detect TRAP, to assist in the counting of immunostained cells. As negative control, the specimens were submitted to the same procedures, eliminating the use of the primary antibody.Histological sections were divided into four batches and each batch was incubated with one of the following primary antibodies: anti-VEGF  with attached digital camera , connected to the microcomputer, at 40× and 100× magnification of the original augmentation, over the entire length of the bone defect.This evaluation was executed with the aid of an optical microscope . The extent of staining was recorded by a trained and blinded examiner for the experimental groups. For VEGF, BMP—2/4 and OCN, a semi-quantitative analysis was performed using three histological sections of each animal.The scores for quantification were as follows ,32: scorTM, San Diego, CA, USA). First, the data obtained were subjected to a normality test (Kolmogorov–Smirnov) and homogeneity of variance (Bartlett’s test). The data homogeneously distributed  were subsequently subjected to parametric analysis: one-way ANOVA followed by the Tukey test to compare different treatments (BC vs. BCP vs. BT vs. BTP) for each experimental period, and a “t” test to compare the influence of time (14 or 42 days) for each treatment.All tests were performed using GraphPad Prism version 5.00 for Windows (GraphPad Softwarep < 0.05).The percentage data obtained for the micro-CT and histology were submitted to Pearson’s correlation test. For nonparametric data , Kruskal–Wallis followed by Dunn’s post-test and the Mann–Whitney test were used because of the non-normal distribution of the data. The tests were performed with a 5% statistical significance level .The descriptive analysis of the microtomographic images is shown in In the 14-day period, a hypodense cavity was observed in both the BC and BCP groups, which corresponded to the bone fragment removed, laterally delimited by the hyperdense bone border and by hyperdense regions related to the centripetal growth of the newly formed bone tissue. In the groups BT and BTP, every area of the defect was filled by hyperdense xenograft particles and correctly positioned, without extravasation.At 42 days, evaluating all extension of the defect in the BC and BCP groups, an increase was observed in the hyperdensity of the area near the edges of the defect and some islets, with a more organized structure mainly in the BCP group. In the same period, the BT and BTP groups presented the defects still completely filled with the xenograft particles, with a discrete appearance of fine bone trabeculae on the edge of the defect and overlapping the dura mater.TM Company, Kontich, Belgium) are presented in 3) was 212% greater than that of the defect in groups BC and BCP (mean 36.37 mm3) and did not change at 42 days.The morphometric data of the 3D microtomographic images of volume and percentage obtained in the CTAn program (Skyscan3) occupied 35.89% of the TV, with no differences between groups and periods.Regarding the material volume (MV), only in the BT and BTP groups the biomaterial particles (MV of 40 mm3) and increased to 15.69% (BV = 5.68 mm3) at 42 days. Meanwhile, in the groups treated with biomaterial, they were similar between groups and periods, occupying 15.97% (BV = 17.84 mm3).In the analysis of bone volume (BV) in all experimental groups, bone formation was discreet and occurred near the edge of the defect and on the surface of the dura mater. In the groups filled with clot (BC and BCP), at 14 days the BV/TV averaged 8.11% (BV = 2.98 mm3) of the defect, decreasing to 84.51% (StV = 30.25 mm3) at the end of the experiment because of the increase in bone formation. In the BT and BTP groups, due to the presence of biomaterial particles, the StV/TV was only 48.13% (StV = 54.65 mm3), with no differences between groups and periods.Hypodense images (black) referred to connective tissue, StV, as seen in histological sections. At 14 days, in the BC and BCP groups, the soft tissue occupied 91.89% , but without significant difference in relation to the other BCP, BT, and BTP  A1,A2,C3.At this period, the percentage of green birefringence fibers was higher in the BTP group (average 3.59%) compared to the other experimental groups, with significant difference from the BC group (mean 1.23%), but without statistical difference in relation to BCP and BT  A1,A2,C2.Regarding the percentage of fibers with yellow birefringence, there was no significant difference in all groups analyzed A1,A2,C4.At 42 days, red-orange birefringence gradually decreased in the BC and BCP groups , but without significant difference, while green birefringence increased proportionally  B1,B2,C3.2). The comparative evaluation between the experimental groups, at 14 and 42 days, did not reveal a statistically significant difference . In contrast, in evaluating the influence of time within each group, the same pattern was observed with a significant decrease in osteocyte density values in BC, BCP, BT, and BTP at 42 compared to 14 days . Additionally, the same immunostaining profile was found at 42 days. Throughout the experimental period, an influence of time was noted on the increase in the number of cells with positive staining for TRAP activity only in the BTP group   statistically significant compared to BCP, BT, and BTP  .Laser photobiomodulation therapy directly interferes with tissue healing, increasing local circulation, cell proliferation, and collagen synthesis ,34,35. HAnimal calvaria is an anatomical region widely used in experimental models of critical-size bone defects because of morphological and embryonic similarity to the craniofacial region ,37,38. IIn the search to aid the repair of defects, GaAIAs laser photobiomodulation therapy has been indicated as an adjunct to the surgical technique because it is able to reach deeper tissues because of malabsorption by water and skin pigments ,41. In tThe infrared wavelength (830 nm) in continuous mode was chosen because of its lower loss, which can reach up to 37% of its intensity after a depth of 2 mm . With thIn a preliminary study by our group of researchers, previously published, with qualitative microtomography (2D), histomorphological, and histomorphometric evaluations of new bone tissue on slides stained with hematoxylin and eosin (HE), it was possible to conclude that the support system (biocomplex), formed by fibrin sealant and xenograft, associated with the proposed PBMT protocol, created a positive microenvironment in the bone repair process .In addition, the scientific literature reports numerous intrinsic characteristics of these materials directly correlated with the mechanism of action in the bone regeneration process, suggesting, when associated, the formation of a tissue construction with synergistic effects ,53,54,55From this, we decided to deepen with the realization of this new study with new analyses, to corroborate or not these findings through new evaluations. 3D microtomographic quantification was performed to provide a detailed view of bone growth from end to end of the defect . A new hIn the two-dimensional reconstructions obtained by μ-CT, the BC and BCP groups at 14 days presented a hypodense cavity corresponding to the space filled by granulation tissue and osteoid matrix, confirmed in the histological sections. In the 42-day period, there was an increase in the hyperdensity of these neoformed areas, related to mineralized bony trabeculae, limited to the borders of the wound, with some islets more evident in the biostimulated BCP animals. These results are in agreement with the findings of Wang et al. , which sIn the BT and BTP groups, all animals had xenograft particles at the insertion site for up to 42 days, without significant variation in the percentage of the total volume of the biomaterial. This finding can be explained by studies by Galindo-Moreno et al. , which rThrough the coronal and transaxial sections, it was possible to observe the correct positioning of the graft in the surgical area, without extravasation of the particles. This is due to the agglutinating action of the fibrin sealant on the graft particles, which ensured a proper fixation. The same findings were observed by Chen et al. , Brown, Histologically, at 14 days, the BC and BCP experimental groups had a large amount of loose connective tissue and bone formation limited to the periphery of the defect. These results corroborate studies that affirmed that new bone formation begins at the defect margins, overlapping the dura mater, probably stimulated by growth factors such as morphogenetic proteins, released in this region because of vascular rupture resulting from the surgical procedure and the underlying presence of periosteum, which is a source of mesenchymal stem cells .At study completion, new bone formation remained on the wound margin, with thicker, compact bone trabeculae, and complete closure of the defect occurred by fibrous connective tissue. This cellular behavior agrees with studies that reported as a critical-size bone defect, since the lack of growth and nutrition factors at the center of the surgical area limits osteoblastic differentiation, facilitating the growth of fibroblasts ,70. In tAt 42 days, this inflammatory infiltrate showed to be in the resolution phase, being more evident in the group treated with laser photobiomodulation, BTP, a result also observed by Kazancioglu et al.  and de ORegarding the material volume of the demineralized bovine bone (B), in the BT group there was a slight decrease in the means when compared to BTP . This may be related to the direct action of laser photobiomodulation in the activation of nuclear factor-κB ligand (RANKL), influencing the maturation and differentiation process of osteoclasts responsible for the degradation of particles ,79. At 4When we compare the quantitative data of the previous study , carriedRegarding the histochemical analysis of collagen fibers by picrosirius red staining, all groups initially analyzed demonstrated the formation of thin and disorganized type III collagen fibers with a gradual increase in birefringence throughout the periods . At 42 dIn the immunohistochemical results, BMP—2/4, OCN, and VEGF immunostaining were significantly higher in BCP and BTP when compared to the BC and BT groups. Such data are indicative of the positive effect of laser biostimulation from the initial stages of bone matrix synthesis (BMP—2/4) to the final stages of bone mineralization and renewal (OCN) . FurtherIn the groups treated with biomaterial, a significantly higher number of TRAP-positive cells was observed, possibly because of the reduction in the rate of resorption by osteoclasts during repair. Conflicting data are presented in these groups when combined with the greater amount of new bone, suggesting that accelerated resorption of graft granules before 30 days is not beneficial to bone formation, which was confirmed by the percentage of new bone tissue formed lower in the groups treated with clot at the end of the experiment .The number of osteocytes in the newly formed bone matrix proportionally reduced in all groups evaluated, following the physiological evolution of the newly formed bone tissue (woven to lamellar bone), which suggests a similar bone maturity for the treatments . In summWe can consider as a limitation of this study the absence of comparative tests with autogenous graft, considered the gold standard, but which would increase the number of animals and is already included in many studies published in the scientific world.This study aimed to evaluate the effects of photobiomodulation in the repair of critical bone defects in rat calvaria filled with bone substitute and derived from human blood fibrin sealant. In view of the results obtained, it was concluded that the association of two scaffolds, bone substitute and fibrin sealant, to the proposed protocol for photobiomodulation therapy, with the use of a low-level laser, improved the formation of new bone during the restoration of bone gaps, suggesting it is a promising strategy, mainly for the treatment of large defects .


JAK2 unmutated polycythaemia is frequent but under-investigated in original publications. In this retrospective cohort study, we investigated the clinical and laboratory data, underlying causes, management and outcomes of JAK2 unmutated polycythaemia patients. (2) Methods: The hospital database was searched to identify JAK2 unmutated patients fulfilling WHO 2016 Hb/Hct criteria for PV  between 2008 and 2019. Clinical and laboratory data were collected and analysed. (3) Results: From 727,731 screened patients, 294 (0.04%) were included, the median follow-up time was 47 months. Epo and P50 showed no clear pattern in differentiating causes of polycythaemia. In 30%, the cause remained idiopathic, despite extensive work-up. Sleep apnoea was the primary cause, also in patients under 30. Around 20% had received treatment at any time, half of whom had ongoing treatment at the end of follow-up. During follow-up, 17.2% developed a thromboembolic event, of which 8.5% were venous and 8.8% arterial. The mortality was around 3%. (4) Conclusions: Testing for Epo and P50 did not significantly facilitate identification of underlying causes. The frequency of sleep apnoea stresses the need to investigate this condition. Idiopathic forms are common. A diagnostic flowchart based on our data is proposed here. NGS testing should be considered in young patients with persisting polycythaemia, irrespective of Epo and P50 levels. Polycythaemia is defined by an increase in haemoglobin (Hb) concentration, haematocrit (Hct) or red blood cell (RBC) count above the reference range adjusted to age, sex and living altitude. JAK2 unmutated polycythaemia is frequent in clinical practice and is usually classified into polycythaemia vera (PV), secondary and hereditary polycythaemia. Nowadays, with the discovery of JAK2 driver mutations, PV no longer represents a diagnostic difficulty. A more serious challenge is JAK2 unmutated polycythaemia, which encompasses a heterogenous group of entities.According to the revised World Health Organization (WHO) classification of myeloid neoplasms from 2016, there are clear diagnostic criteria for PV, including haemoglobin (Hb) > 16.5 g/dL in men and Hb > 16 g/dL in women, or haematocrit (Hct) > 49% in men and Hct > 48% in women, or red blood cell (RBC) mass > 25% above mean normal predicted value [Hereditary causes of JAK2 unmutated polycythaemia should be investigated, especially in cases of long-term polycythaemia, in young patients and if family history is positive. According to the literature , testingIn clinical practice, therapy guidelines for PV are frequently applied for the management of JAK2 unmutated polycythaemia, which may result in overtreatment of these benign conditions.There is a myriad of publications outlining the diagnostic work-up of JAK2 unmutated polycythaemia. However, since the discovery of JAK2 V617F in 2005, the vast majority of them are reviews based on expert suggestions, not on original research work. There are only singular studies that systematically analysed causes, diagnostic steps and outcomes, including thrombotic events in patients with JAK2 unmutated polycythaemia ,5.Therefore, in this retrospective cohort study, we aimed to investigate the clinical and laboratory data of underlying causes, as well as the real-world management and outcomes, of patients with JAK2 unmutated polycythaemia at a Swiss tertiary reference centre.For this study, we assumed that in every patient in whom no clinical underlying cause was found to explain polycythaemia, JAK2 V617F mutation should have been investigated. As a result, we focused on those patients whose laboratory confirmed polycythaemia and where JAK2 V617F mutation was tested, the result was available, and it was negative. Using this stepwise identification process , patientThe hospital database (Microsoft SQL Server Management Studio) was searched to identify polycythaemia patients in the study period between 1 October 2008 and 31 July 2019. This was performed using different strategies: for inpatients, we used the ICD-10 coding system (D45 for polycythaemia vera or D75.1 for secondary polycythaemia); for outpatients, key words were used . All patients ≥ 15 years of age were included in the search. All identified patients were individually evaluated to confirm if they met the Hb/Hct WHO 2016 criteria for PV.All laboratory data was filtered and collected automatically using our hospital’s laboratory system . If more than one measurement was available for full blood count (FBC), Epo or P50, the nearest measurement to identification of JAK2 V617f mutation was chosen for analysis. Laboratory results not automatically retrievable, such as molecular reports, were manually acquired from the clinical records. The clinical data of patients was manually collected by a clinical record review. Alive/dead status and date of death, if applicable, was determined from the hospital or state insurance database.The follow-up period was defined as the time between first documented polycythaemia, according to WHO 2016 criteria, and the last available Hb/Hct values. The data collection cut-off was 31 July 2019. The availability of a JAK2 V617F result was considered crucial to definitively rule out the diagnosis of PV. Patients were considered lost to follow-up if no second Hb/Hct measurement was performed. To evaluate patients’ outcomes, the occurrence of new venous or arterial thrombosis and death were included. Data regarding thromboembolic events were retrieved from clinical records and only documented (not suspected) events were taken into account. Young patients were defined as being under 30 years of age.The causes of polycythaemia were divided into 5 groups: (1) relative (spurious); (2) reactive, oxygen-driven, consisting of all cases where low cellular oxygen seems to play a role; (3) reactive Epo or hormonal-driven, with cases in which an endogenous or exogenous substance seems to play a role; (4) congenital, including those with proven molecular abnormality, congenital hemoglobinopathy and also Down’s Syndrome, known to be associated with polycythaemia; (5) patients with an undetermined (idiopathic) cause . If we iWe classified cases as relative polycythaemia only if normal RBC mass was confirmed on nuclear measurement of the ratio between erythrocyte volume and plasma volume (EV/PV). RCM and plasma volume were obtained using chromium-labelled RBC and albumin, respectively. Dehydration, Gaisböck syndrome and capillary leak syndrome were considered as causes only if confirmed by a clinician. Sleep apnoea was classified as a cause only if it was confirmed by a polysomnography. Altitude was classified as a cause only if the patient lived above 1500 metres over the sea level. Smoking was classified as a cause only if the patient was an active smoker at the time of diagnosis of polycythaemia, irrespective of pack-years smoked. Respiratory disease, such as chronic obstructive pulmonary disease (COPD) and pneumopathy, were considered as causes only if a diagnosis was confirmed by a pneumonologist. Cardiac diseases were considered as causes only when there was a right–left shunt resulting in a cyanotic heart disease confirmed by a cardiologist. Increased Epo was considered as a cause if it was above the reference range used at our centre 4.30–29.0 mU/mL). Noncancer kidney diseases were only classified as causes when there seemed to be a clear renal arterial problem, for instance one small kidney or atherosclerotic stenosis vessels, or in cases of hydronephrosis or real cystic diseases. Androgen therapy was considered as a cause only if a patient was being treated with androgens when polycythaemia was diagnosed. As far as neoplasia related to polycythaemia is concerned, we only included cancers which have been published to be related with polycythaemia, and therefore the following cancers were included in this group: renal ,7,8; adr.0 mU/mL.2 test. A value of p < 0.05 was considered significant in both tests. For the follow-up analysis, 13 patients who did not have a repeated Hb/Hct measurement were excluded. The missing laboratory data were excluded from analysis.The data collection and analysis were conducted in Excel Statistics 2019, IBM SPSS Statistics for Windows (version 27), and R-statistics (version 4.1.2). A non-normal distribution of all variables was assumed, and median and range values were reported. The Kruskal–Wallis test was used to compare medians of three or more groups. The frequencies of categorical variables were compared using Pearson’s χThe search was conducted across available medical reports of 727,731 patients, 239,516 inpatients and 488,215 outpatients with any record fitting our criteria.We identified 4391 patients whose medical records reported polycythaemia (or a similar keyword). Of these, 282 occurrences were duplicates (both inpatient and outpatient) and therefore their records were merged. The remaining 4109 patients that represent 0.56% of all screening patients were examined for Hb/Hct WHO 2016 criteria for PV. In this process, we confirmed 1483 patients  fulfilling the Hb/Hct criteria. From 1483 patients, 391 were tested for JAK2 V617 mutation with 297 (76%) of them being JAK2 V617F negative and 94 (24%) positive. Thus, these 94 JAK2 V617F positive MPN patients were excluded from our analysis. Additionally, three patients were excluded due to the presence of another clonal mutation (CALR mutation (N = 2); JAK2 exon 12 (N = 1)). In total, 294 patients , were classified as having JAK2 unmutated polycythaemia, and they represent the final cohort of this study. Their laboratory and clinical data, as well as the management and outcome, were evaluated and reported here. The flowchart of patient selection is presented in In 205 of 294 patients with JAK2 unmutated polycythaemia (70% of our study cohort), an underlying physiopathological mechanism to explain polycythaemia was found. Among those, 14 (5%) patients were diagnosed with relative (spurious), 139 (47%) with reactive oxygen-driven, 40 (14%) with reactive Epo- or hormone-driven and 12 (4%) with congenital polycythaemia. In 89 (30%) patients, the cause was idiopathic. Causes were subclassified, as presented in The causes of JAK2 unmutated polycythaemia according to the frequency of their occurrence are also presented in Among young patients , determined cause  was more frequent than unexplained polycythaemia . Moreover, in this group, the most frequently determined causes of polycythaemia were sleep apnoea  and smoking . In 17 young patients with persistent polycythaemia at the end of follow-up, the cause was unexplained. We are aiming to investigate this population for congenital causes of polycythaemia as well as myeloproliferative and myelodysplastic diseases.2 all p-values > 0.10).There was a great over-representation of males  in the cohort, but the ratio of males to females in each of the five subgroups  was not p = 0.007). Specifically, patients have higher median age in all groups other than congenital polycythaemia, and idiopathic polycythaemia .In contrast, the median age at diagnosis of polycythaemia was statistically different according to the pathophysiological underlying attributed mechanisms of polycythaemia , though in 13/294 (4%) patients, follow-up was not available. In the last available blood results, the majority of patients 159/281 (57%) still fulfilled Hb/Hct WHO 2016 criteria for PV, indicating persistent polycythaemia. In 135/281 (48%) patients, polycythaemia resolved during the follow-up period: in 13/135 with treatment and in 118/135 without any intervention (4 patients had unknown status). At the time of the last follow-up, 53 patients had persistent, idiopathic polycythaemia  and 17 of these were younger than 30 years of age and were exclusively male. There were no clinical or laboratory data on thrombophilia available.For the diagnostic work-up, in 153/294 (52%) patients, the Epo level was tested, in 240/294 (82%), the P50 was tested, 54/294 (18%) were evaluated with a bone marrow examination, 62/294 (21%) with a stem cell culture, 48/294 (16%) with an EV/PV and in 98/294 (33%), for JAK2 exon 12 mutation. Epo was suppressed in 15/153 of Epo-tested patients (10%). Exactly 113 were tested for both Epo and P50. Epo level was normal or increased and P50 was <24 mmHg in 20/113 of those tested (18%), whereas the Epo level was normal or increased and P50 was ≥24 mmH in 93/113 (82%). Of the 196 patients in whom JAK2 exon 12 was not tested, PV was excluded with bone marrow investigation in 22, whereas 95 had persistent polycythaemia at the end of follow-up, but only 3 had suppressed Epo. In the 3/4 of patients with suppressed Epo and idiopathic cause, JAK2 exon 12 was tested, and it was negative. Specific conditions according to Epo and P50 levels are listed in 2, p < 0.001). The median time between the JAK2 V617F negative report and involvement of a haematologist was 25 days (range 0 to +7306), and 29 patients were seen by a haematologist before the diagnosis of JAK2 unmutated polycythaemia due to other issues.In 227 cases (77%), a haematologist was involved in the diagnostic work-up and/or management of polycythaemia independent of the underlying cause. The only exception to this was observed in patients in which reactive Epo or hormone-driven erythrocytosis was diagnosed, where the majority  were not seen by a haematologist . Among these patients, some received more than one type of treatment. The majority were treated with phlebotomy only, followed by phlebotomy with ASS and ASS only. Few patients were treated with combination of three types of therapy. At the end of follow-up, 22/61 (36% of ever treated) had ongoing therapy . The most frequent venous thromboembolic event was pulmonary embolisms  and deep venous thrombosis , followed by splanchnic , catheter associated , ophthalmic , cerebral  and cardiac with one right atrial thrombus (3%). The most frequent arterial thrombotic event was stroke , followed by myocardial infarction  and other arterial peripheral thromboembolisms . Mostly, patients suffered from a single event . However, some suffered from two  or more events . Among eight patients with cancer, two had one venous thromboembolic event. There was no significant gender difference in the prevalence of venous and arterial thromboembolism: venous thromboembolic events in males  and females  ; arterial thromboembolism in males  and females  (Chi-square according Pearson’s p = 0.45). Furthermore, we analysed the occurrence of thromboembolism according to the age of patients by dividing them into three age groups: 0–30 (N = 54), 31–50 (N = 105) and >50 years old (N = 122). There were no significant differences between the three age groups for both venous and arterial thromboembolism: venous thromboembolism: age 0–30: 3/54 (6%); age 31–50: 10/105 (10%); age >51: 12/122 ; arterial thromboembolism: age 0–30: 3/54 (6%); age 31–50: 8/105 (8%); age >51: 15/122 . During follow-up, a number of thromboembolic events were observed. In total, 33 venous in 25/294 (8.5%) patients and 32 arterial in 26/294 (8.8%) were identified . No pati2p = 0.26).Venous thromboembolism occurred before the diagnosis of polycythaemia in some patients . Likewise, there was no significant difference in prevalence of arterial thromboembolism between the five subgroups . In the idiopathic group, there were 7/33 (21%) venous and 7/32 (27%) arterial thrombotic events. We have not observed any thromboembolism in three patients with mutations causing congenital polycythaemia identified by our in-house Next Generation Sequencing (NGS) panel for polycythaemia [Further, there were no significant differences in occurrence of venous thromboembolisms when considering the underlying physiopathological mechanisms between the 1–5 subgroups  with unknown alive/dead status were excluded. At the end of follow-up, the majority were alive . Among the eight who died, the majority were in the reactive oxygen-driven  and some in the Epo/hormone-driven erythrocytosis  group; however, the exact causes of their deaths are unknown.In our cohort of patients, we have not observed any MPN during the follow-up.This study, as one of the few since the introduction of JAK2 V617F testing in 2005 , systemaThese real-world data, extracted from clinical practice patients’ files, showed a prevalence of polycythaemia fulfilling Hb/Hct criteria for PV in 0.2% of all screened patients. Furthermore, the prevalence of JAK2 unmutated polycythaemia was higher  than the prevalence of JAK2 mutated polycythaemia . The prevalence of PV in our cohort is lower than previously reported by others, at 44–57 per 100,000 .We confirmed the diagnostic challenge of JAK2 unmutated polycythaemia. Indeed, though a cause was identified in nearly two thirds of patients, in the remaining third, despite extensive work-up, the cause remained unexplained. Interestingly, sleep apnoea was the single most frequent identifiable cause in any age group. The likelihood of sleep apnoea as a cause of reactive polycythaemia is probably underestimated, particularly in younger patients, and our data show the need to emphasise it.We have also observed that no systematic sequential diagnostic work-up of JAK2 unmutated polycythaemia was followed. It seems sensible to recommend that once PV, as well as obvious secondary causes of polycythaemia, are excluded , the next step should be to investigate for sleep apnoea, irrespective of patients’ age, including a referral for respiratory evaluation and measurement of carboxyhaemoglobin (COHb).For the interpretation of results, we applied diagnostic algorithms proposed by others ,3 to ourWe have noticed that JAK2 exon 12 mutation was investigated in only one third of our cohort. Indeed, JAK2 exon 12 should be explored systematically, particularly if Epo is suppressed . A possiImaging can make a clear contribution while investigating underlying causes of polycythaemia, such as benign kidney pathology , adrenal disorders (Cushing’s syndrome), as well as Epo-producing tumours . However, the most appropriate time to suggest imaging in the work-up algorithm is difficult to define. Probably, clinical judgment is the best strategy for not missing a relevant diagnosis. The measurement of the RBC mass by EV/PV examination, either confirming or ruling out absolute erythrocytosis, is a diagnostic step that in some patients will be essential for a better diagnosis. In recent years, the inclusion of NGS has made an enormous contribution in the diagnosis of congenital forms of polycythaemia ,22. ThisWe found that in almost half of patients, polycythaemia was no longer present at the end of follow-up, suggesting that in many patients there may be transient triggers, or that appropriate interventions such as smoking cessation, use of devices to improve night-time breathing, weight loss and/or discontinuing androgen treatments might have contributed to treat polycythaemia.In this cohort, a haematologist was involved in the diagnostic work-up and/or management of polycythaemia independent to the underlying cause in 77% of patients. This high percentage may be related to the characteristics of patients themselves, in whom JAK2 testing was considered necessary, but it may also be related to the characteristics of our tertiary care centre that offers specific haematology care. Therefore, the data presented here might not be representative of the management of such patients in primary or secondary care centres. An exception was, however, observed in patients with hormone-driven polycythaemia, since 53% of them were not seen by a haematologist.Treatment recommendations of secondary polycythaemia are different from those of PV . We can Historically, the management of non-PV erythrocytosis has been conflicted by unfounded concerns regarding the risk of thrombosis, arising from a poor characterization of congenital erythrocytosis forms, except for Chuvash polycythaemia, known for its thrombotic tendency . In our In contrast to what is observed in PV, we did not observe any transformation of polycythaemia into any other disease. The overall mortality rate was around 3% within a median follow-up period of 47 months. We do not have data regarding causes of deaths, and we do not know whether this is different when compared to a matched general population. However, the observed mortality is clearly lower than for PV, reported as 10% during a large observational study .The strength of this study is the enormous number of patients screened, the confirmation of polycythaemia with Hb/Hct results, the availability of a negative JAK2 test in the patients investigated and access to clinical data of all of them, which makes the cohort very consistent. This study has, however, a number of limitations, mainly related to its retrospective nature and low patient number in some subgroups. In the screened population with confirmed polycythaemia by Hb/Hct laboratory values, JAK2 mutation testing was not performed in 74% of all patients, therefore these were excluded from the analysis. We assume that possibly there was another clinical cause found to explain polycythaemia, however we cannot exclude that, if JAK2 had been tested in all of them, the prevalence or the repartition of JAK2 unmutated polycythaemia would have been different. We also cannot rule out that some patients may have been misdiagnosed as having another chronic myeloid neoplasm. Furthermore, the diagnostic procedure, as well as the decision to treat an individual patient, was made at the discretion of a leading physician. Therefore, there are missing data, since not all diagnostic tests were performed, and in some patients the reason to treat was not always comprehensible. Moreover, no data regarding therapy and risk factors of thromboembolic events, such as cardiovascular diseases, laboratory data including presence of thrombophilia, especially antiphospholipid antibodies or classification of events as provoked or unprovoked, was available. Moreover, FBC results were not consistently available at the time of thromboembolic events in all patients. Likewise, although the deaths could be identified, thanks to the reports in the insurance database, their causes were not available.In conclusion, this study shows that once PV is ruled out as a cause of polycythaemia by genetic testing, the search for causes of unmutated JAK2 polycythaemia is complex and, in one third of the patients, it may not be found. This study also illustrates that for the work-up of patients with polycythaemia, an orderly diagnostic approach is needed with a major awareness of different possible underlying causes. We find that some diagnostic algorithms are helpful in the resulting interpretation of many but not in all cases. We suggest, therefore, particularly in young patients, to consider common causes such as sleep apnoea, which should not be underestimated, and in some cases the investigation of hereditary causes, using new diagnostic methods such as NGS, should be considered.