Hereditary spherocytosis (HS) is caused by mutations in genes such as ANK1, EPB42, SLC4A1, SPTA1, or SPTB,¹ leading to altered red blood cell (RBC) membrane proteins, reduced deformability, and decreased RBC lifespan. Dehydrated hereditary stomatocytosis (xerocytosis) is caused by variants in the PIEZO1 gene and, less commonly, KCNN4 variants, affecting RBC hydration and stability.²

The prevalence of HS is generally reported around 1:2000, while dehydrated hereditary stomatocytosis estimates range from 1:10 000 to 1:50 000, though mostly based on sporadic cases or older studies lacking comprehensive diagnostic methods.^{1, 2} Current prevalence estimates may be inaccurate due to underdiagnosis, referral bias, and the lack of systematic population-based testing. A study analyzing complete blood counts of 48 million North American patients suggested that up to 1:8000 individuals could potentially have dehydrated hereditary stomatocytosis based on specific biochemical markers, although definitive confirmation through genetic or specialized testing was not feasible.³ Interestingly, a study identified a gain-of-function PIEZO1 allele in one-third of African Americans, which, in a mouse model, induced a phenotype resembling dehydrated hereditary stomatocytosis and provided significant resistance to malaria.⁴

We tested the hypothesis that both hereditary spherocytosis and dehydrated hereditary stomatocytosis are more frequent in the white general population than previously estimated. For this purpose, we studied 109 039 white individuals of Danish descent from the Copenhagen General Population Study(H-KF 01-144/01) examined between 2003 and 2015.⁵ All individuals had hemoglobin, red cell distribution width (RDW), and MCHC measured, and all individuals had DNA obtained for further genetic analysis. Individuals with biochemical signs of hemolysis were genetically tested for the most frequent causes of hereditary hemolysis. All individuals aged 40–100 years and 25% of inhabitants aged 20–39 years living in a suburban part of the Capital Region of Denmark were invited, of these 43% participated. All participating individuals underwent a physical examination, had blood samples drawn, and completed a questionnaire on lifestyle and health on the day of enrollment. Blood samples for hemoglobin, mean corpuscular volume, and MCHC were drawn at enrollment and analyzed fresh on an ADVIA 120 Hematology System. RDW was calculated as standard deviation of mean corpuscular volume divided by mean corpuscular volume multiplied by 100. As previously proposed by Kaufman et al.³ potential hemolysis was considered in individuals with RBC indices suggesting hemolysis: increased MCHC and high RDW (as a sign of reticulocytosis), or increased MCHC and low hemoglobin. Increased MCHC was defined as MCHC >95th percentile, equal to MCHC >36.3 g/dL for women and MCHC >35.3 g/dL for men. As reticulocyte count was not measured directly, we used RDW as measure of reticulocytosis since high RDW is well correlated with a high reticulocyte count.⁶ High RDW was defined as RDW >95th percentile equal to RDW >14.5% for women and RDW >14.3% for men. Low hemoglobin was defined as hemoglobin ≤10th percentile equal to hemoglobin ≤12.4 g/dL for women and hemoglobin ≤13.5 g/dL for men. DNA from peripheral blood leucocytes was subjected to a broad targeted panel of genes potentially housing variants causing hereditary hemolysis in individuals of Scandinavian descent (Supplementary Methods and Table S1).

Table S2 displays the baseline characteristics of the 109 039 individuals in the study, categorized by RBC indices of hemolysis. Among these, 187 individuals had high MCHC and high RDW and 107 had high MCHC and low hemoglobin. Notably, 14 individuals had high MCHC and both low hemoglobin and high RDW (Figure S1). In total, 280 individuals had potential hemolysis, on whom next-generation sequencing was performed. After variant filtering, 48 individuals harbored a variant in genes that can cause hereditary spherocytosis. Of these, 7/48 individuals had definite hereditary spherocytosis (likely pathogenic and pathogenic variants) and 5/48 individuals had probable hereditary spherocytosis (hot variants of uncertain significance [VUS]). Forty-five individuals harbored a variant in genes that can cause dehydrated hereditary stomatocytosis. Of these, 2/45 individuals had definite dehydrated hereditary stomatocytosis (likely pathogenic and pathogenic variants) and 14/45 individuals had probable dehydrated hereditary stomatocytosis (hot VUS). Of the individuals with probable or definite dehydrated hereditary stomatocytosis, 11/16 harbored one of two combinations of variants in PIEZO1, either c.2423G > A/2344G > A or c.2815C > A/c.7374C > G. These variants have previously been reported in cis in the literature and likely constitutes relatively common haplotypes in the European population (Table S2). Among the 19 individuals with probable hereditary spherocytosis or probable dehydrated hereditary stomatocytosis, 2 individuals overlapped (Figure S1). None of the 280 individuals with RBC indices of hemolysis had genetic evidence of other causes of hereditary hemolysis than hereditary spherocytosis or dehydrated hereditary stomatocytosis.

Among the 12 individuals with definite or probable spherocytosis, 3 had been diagnosed with hereditary spherocytosis and 1 had been diagnosed with unspecified anemia. None of the 16 individuals with either definite or probable dehydrated hereditary stomatocytosis had been diagnosed with dehydrated hereditary stomatocytosis, but 2 had been diagnosed with unspecified anemia, 1 had been diagnosed with iron deficiency anemia, and notably 1 had been diagnosed with hereditary spherocytosis (Table 1). None of the 280 sequenced individuals were diagnosed with hereditary lymphedema, which can result from biallelic PIEZO1 loss-of-function variants.

When estimating a conservative prevalence of hereditary spherocytosis in the general population defined as only individuals with variants definitely causing hereditary spherocytosis, the prevalence would be 0.0064% (95% confidence interval [95% CI]: 0.0026%–0.013%) equal to 1:16000. For dehydrated hereditary stomatocytosis, the corresponding conservative prevalence estimate would be 0.0018% (95% CI: 0.00022%–0.0066%) equal to 1:55000 (Figure S2). If combining individuals with either definite hereditary spherocytosis or probable hereditary spherocytosis (the latter based on hot VUS), the prevalence of hereditary spherocytosis would be 0.011% (95% CI: 0.0057%–0.019%) equal to 1:9000. Likewise, when combining individuals with either definite dehydrated hereditary stomatocytosis or probable dehydrated stomatocytosis, the prevalence of dehydrated hereditary stomatocytosis would be 0.015% (95% CI: 0.0084–0.024) equal to 1:7000 (Figure S2). However, it is important to note that including hot VUS in prevalence calculations is speculative and should be interpreted with caution.

Our study is limited by not being able to ascertain with certainty whether some of the individuals sharing potential hemolysis-causing variants were related. We assessed relatedness between carriers of the shared gene variants using a custom implementation of the Somalier relatedness score (https://github.com/brentp/somalier), which we applied to estimate relatedness based on variants with allele frequencies 25%–75% in our overall population (103 variant positions). Due to the limited size of our NGS panel, we cannot calculate a standardized relatedness score, but our custom relatedness rating indicated that relatedness is not likely to be a major determining factor in the individuals we found had shared hemolysis variants. Importantly, all shared variants were classified as hot VUS and thus did not influence the conservative prevalence estimates.

To our knowledge, this is the first study to screen for hereditary spherocytosis and dehydrated hereditary stomatocytosis in the general population using RBC indices and genetic testing in combination.

As only 25% of individuals with genetic evidence of hereditary spherocytosis was correctly diagnosed in our study, previous studies using hospital diagnoses might be inaccurate in assessing prevalence of hereditary spherocytosis in the general population.

Our finding is consistent with Kaufman et al.,³ who analyzed hematological indices from 48 403 254 patients undergoing complete blood counts, predominantly outpatients, with a smaller proportion of hospitalized individuals. This study suggested a prevalence of dehydrated hereditary stomatocytosis of approximately 1:8000 based on specific biochemical indices. However, this study did not have access to DNA on the studied patients, which made genetic confirmation of these biochemical indices impossible.

Treatment for dehydrated hereditary stomatocytosis differs substantially from that of hereditary spherocytosis.² In dehydrated hereditary stomatocytosis, iron overload is common and may necessitate monitoring and treatment by either iron chelation or phlebotomy. Splenectomy is the cornerstone treatment for individuals with symptomatic hereditary spherocytosis but contraindicated for individuals with dehydrated hereditary stomatocytosis.² Importantly, in our study, one individual had genetic evidence of dehydrated hereditary stomatocytosis but was diagnosed with hereditary spherocytosis at a hospital, potentially leading to mistreatment.

Strengths of our study include the large study population of 109 039 individuals all of whom had RBC indices measured and DNA available for genetic analyses. Our study was conducted using data on individuals from the general population; therefore, findings may be more generalizable compared with studies using either individuals with a pre-existing condition or blood donors.

The prevalence estimates in this study are weakened by only having studied prevalence of hereditary hemolysis according to RBC indices, likely leading to underestimation, as some individuals with hereditary hemolysis might only be detected through other biochemical markers of hemolysis, like high bilirubin, lactate dehydrogenase, or ferritin. Functional validation, for example, by osmotic gradient ektacytometry, was not possible. Additionally, our next-generation sequencing panel is not well-suited for detecting copy number variations or assessing consanguinity, both of which may affect the accuracy of prevalence estimates.

In conclusion, this study estimates the conservative prevalence of hereditary spherocytosis to be at least 1:16 000, potentially rising to 1:9000 when including probable cases based on hot VUS. Similarly, for dehydrated hereditary stomatocytosis, the prevalence is at least 1:55 000 but may increase to 1:7000 when considering hot VUS. While prevalence estimates incorporating hot VUS carry uncertainty, this aligns with previous data by Kaufmann et al.,³ indicating that dehydrated hereditary stomatocytosis may be several-fold more prevalent than previously reported.

遗传性球形红细胞增多症 （HS） 是由 ANK1、EPB42、SLC4A1、SPTA1 或 SPTB1 等基因突变引起的，导致红细胞 （RBC） 膜蛋白改变、变形能力降低和红细胞寿命缩短。脱水遗传性口红细胞增多症（干红细胞增多症）是由 PIEZO1 基因变异引起的，较少见的是 KCNN4 变异，影响红细胞水合作用和稳定性。^{阿拉伯数字}

HS 的患病率通常报告在 1：2000 左右，而脱水遗传性口红细胞增多症的估计范围为 1：10 000 至 1：50 000，尽管主要基于散发病例或缺乏全面诊断方法的较早研究。^1、2由于诊断不足、转诊偏倚和缺乏基于人群的系统性检测，目前的患病率估计可能不准确。一项分析 4800 万北美患者全血细胞计数的研究表明，根据特定的生化标志物，多达 1：8000 的个体可能患有遗传性口形细胞增多脱水，尽管通过基因或专业检测进行明确确认是不可行的。³ 有趣的是，一项研究在三分之一的非裔美国人中发现了功能获得性 PIEZO1 等位基因，在小鼠模型中，它诱导了类似于脱水遗传性口形细胞增多症的表型，并提供了对疟疾的显着抵抗力。⁴

我们检验了遗传性球形红细胞增多症和脱水遗传性口红细胞增多症在白人普通人群中比以前估计的更频繁的假设。为此，我们研究了 109 039 年至 2003 年间检查的哥本哈根一般人群研究 （H-KF 01-144/01） 中的丹麦血统白人个体。⁵ 所有个体都测量了血红蛋白、红细胞分布宽度 （RDW） 和 MCHC，并且所有个体都获得了 DNA 以进行进一步的遗传分析。对具有溶血生化体征的个体进行遗传性溶血最常见原因的基因检测。所有 40-100 岁的个人和 25% 的 20-39 岁居住在丹麦首都地区郊区的居民都被邀请，其中 43% 参加了。所有参与的人在入组当天都接受了身体检查，抽取了血样，并完成了一份关于生活方式和健康的问卷。在入组时抽取血样检测血红蛋白、平均红细胞体积和 MCHC，并在 ADVIA 120 血液学系统上进行新鲜分析。RDW 计算为平均红细胞体积除以平均红细胞体积的标准差乘以 100。正如 Kaufman 等人之前提出^的那样3，在红细胞指数提示溶血的个体中考虑了潜在的溶血：MCHC 升高和高 RDW（作为网织红细胞增多症的标志），或 MCHC 升高和低血红蛋白。MCHC 增加定义为 MCHC >95th percentile，等于女性 MCHC >36.3 g/dL 和男性 MCHC >35.3 g/dL。由于网织红细胞计数没有直接测量，我们使用 RDW 作为网织红细胞增多症的测量，因为高 RDW 与高网织红细胞计数密切相关。⁶ 高 RDW 定义为 RDW >95th percentile 等于女性的 RDW >14.5% 和男性的 RDW >14.3%。低血红蛋白定义为血红蛋白≤第 10 个百分位数等于女性血红蛋白 ≤12.4 g/dL，男性血红蛋白 ≤13.5 g/dL。来自外周血白细胞的 DNA 受到广泛的靶向基因组的影响，这些基因可能包含导致斯堪的纳维亚血统个体遗传性溶血的变异（补充方法和表 S1）。

表 S2 显示了研究中 109 039 例个体的基线特征，按溶血的 RBC 指数分类。其中，187 例 MCHC 高、RDW 高，107 例 MCHC 高、血红蛋白低。值得注意的是，14 例个体具有高 MCHC 以及低血红蛋白和高 RDW （图 S1）。总共有 280 人患有潜在的溶血，对他们进行了下一代测序。经过变异过滤，48 个个体的基因中携带可导致遗传性球形红细胞增多症的变异。其中，7/48 个体有明确的遗传性球形红细胞增多症（可能是致病性和致病性变异），5/48 个体可能患有遗传性球形红细胞增多症（意义不确定的热变异 [VUS]）。45 个个体携带可导致脱水遗传性口红细胞增多症的基因变异。其中，2/45 个体患有明确的脱水遗传性口红细胞增多症（可能是致病性和致病性变异），14/45 个体可能患有脱水遗传性口红细胞增多症（热 VUS）。在可能或明确患有脱水遗传性口红细胞增多症的个体中，11/16 携带 PIEZO1 中两种变异组合之一，c.2423G > A/2344G > A 或 c.2815C > A/c.7374C > G。这些变异以前在文献中以顺式形式报道过，并且可能构成欧洲人群中相对常见的单倍型 （表 S2）。在 19 名可能患有遗传性球形红细胞增多症或可能脱水的遗传性口红细胞增多症的个体中，有 2 名个体重叠（图 S1）。280 例具有溶血红细胞指数的个体中没有除遗传性球形红细胞增多症或脱水遗传性口红细胞增多症外其他原因遗传性溶血的遗传证据。

在 12 例明确或可能的球形红细胞增多症个体中，3 例被诊断为遗传性球形红细胞增多症，1 例被诊断为未指明的贫血。16 名明确或可能患有脱水遗传性口红细胞增多症的个体均未被诊断为脱水遗传性口红细胞增多症，但 2 名被诊断为未指明的贫血，1 名被诊断为缺铁性贫血，值得注意的是 1 名被诊断为遗传性球形红细胞增多症（表1）。280 名测序个体均未被诊断出患有遗传性淋巴水肿，这可能是由双等位基因 PIEZO1 功能丧失变异引起的。

当估计遗传性球形红细胞增多症在一般人群中的保守患病率时，定义为仅具有明确导致遗传性球形红细胞增多症的变异的个体，患病率为 0.0064%（95% 置信区间 [95% CI]：0.0026%–0.013%），等于 1：16000。对于脱水遗传性口红细胞增多症，相应的保守患病率估计值为 0.0018%（95% CI：0.00022%–0.0066%），等于 1：55000（图 S2）。如果将具有明确遗传性球形红细胞增多症或可能的遗传性球形红细胞增多症（后者基于热 VUS）的个体合并，则遗传性球形红细胞增多症的患病率将为 0.011%（95% CI：0.0057%–0.019%），等于 1：9000。同样，当合并患有明确脱水遗传性口红细胞增多症或可能患有脱水口红细胞症的个体时，脱水遗传性口红细胞增多症的患病率为 0.015%（95% CI：0.0084–0.024），等于 1：7000（图 S2）。然而，需要注意的是，在患病率计算中包括热 VUS 是推测性的，应谨慎解释。

我们的研究受到限制，因为无法确定一些共享潜在溶血导致变异的个体是否相关。我们使用 Somalier 相关性评分 （https://github.com/brentp/somalier） 的定制实施来评估共享基因变异携带者之间的相关性，我们将其应用于根据我们总体中等位基因频率为 25%-75% 的变异（103 个变异位置）来估计相关性。由于我们的 NGS 检测组合的规模有限，我们无法计算标准化的相关性评分，但我们的自定义相关性评级表明，相关性不太可能成为我们发现的具有共享溶血变异的个体的主要决定因素。重要的是，所有共享变体都被归类为热 VUS，因此不会影响保守的患病率估计。

据我们所知，这是第一项使用 RBC 指数和基因检测相结合在普通人群中筛查遗传性球形红细胞增多症和脱水遗传性口红细胞增多症的研究。

由于在我们的研究中只有 25% 的具有遗传性球形红细胞增多症遗传证据的个体被正确诊断，因此以前使用医院诊断的研究在评估普通人群中遗传性球形红细胞增多症的患病率可能不准确。

我们的发现与 Kaufman 等人3 分析了 48 403 254 名接受全血细胞计数的患者的血液学指标，主要是门诊患者，住院患者比例较小。这项研究表明，根据特定的生化指标，脱水遗传性口红细胞增多症的患病率约为 1：8000。然而，这项研究无法获得所研究患者的 DNA，这使得这些生化指标的基因确认变得不可能。

脱水遗传性口红细胞增多症的治疗与遗传性球形红细胞增多症的治疗有很大不同。² 在脱水遗传性口红细胞增多症中，铁超负荷很常见，可能需要通过铁螯合或静脉切开术进行监测和治疗。脾切除术是有症状的遗传性球形红细胞增多症患者的基础治疗，但禁用于脱水遗传性口红细胞增多症患者。² 重要的是，在我们的研究中，一个人有脱水遗传性口红细胞增多症的遗传证据，但在医院被诊断出患有遗传性球形红细胞增多症，可能导致误诊。

我们研究的优势包括 109 039 人的庞大研究人群，他们都测量了红细胞指数并提供了可用于遗传分析的 DNA。我们的研究是使用来自普通人群的个体数据进行的;因此，与使用已有疾病的个体或献血者的研究相比，研究结果可能更具普遍性。

本研究中的患病率估计值因仅根据 RBC 指数研究遗传性溶血的患病率而被削弱，这可能导致低估，因为一些遗传性溶血个体可能只能通过其他溶血生化标志物检测到，如高胆红素、乳酸脱氢酶或铁蛋白。例如，通过渗透梯度 ektacytometry 进行功能验证是不可能的。此外，我们的下一代测序检测组合不太适合检测拷贝数变异或评估血缘关系，这两者都可能影响患病率估计的准确性。

总之，本研究估计遗传性球形红细胞增多症的保守患病率至少为 1：16 000，当包括基于热 VUS 的可能病例时，可能会上升到 1：9000。同样，对于脱水遗传性口红细胞增多症，患病率至少为 1：55 000，但在考虑热 VUS 时可能会增加到 1：7000。虽然包含热 VUS 的患病率估计存在不确定性，但这与 Kaufmann 等人之前的数据一致，³ 表明脱水遗传性口形细胞增多症的患病率可能比以前报道的要高出几倍。