兽研A型血友病是一种严重的凝血障碍，由X染色体F8基因功能部分或完全丧失引起。这是一种相对常见的凝血障碍，迄今已在至少17个犬种中有报道。然而，目前仅鉴定了五种不同的分子病因。一种分子遗传病因是大片段倒位，该变异在人类中也有描述，并最早在北卡罗来纳大学教堂山分校的一个爱尔兰雪达犬研究群体中被研究。然而，犬中倒位断点连接的确切位置尚未确定。在此，我们描述了一个威玛猎犬家系中一例A型血友病的分子分析，采用长读长测序。我们表明，A型血友病的病因是一个约420kb的倒位，影响F8基因。倒位的断点鉴定位于内含子22中第124,003,000位点，以及氯离子细胞内通道蛋白2（CLIC2）与epsilon-三甲基赖氨酸羟化酶（TMLHE）之间第124,432,668位点（NC_049260.1:g.124003000_124432668inv）。与人类一样，该倒位最可能由内含子22与F8基因下游约420kb区域之间的非等位同源重组引起。通过鉴定倒位断点连接，现在可以在DNA水平检测该病的携带者，从而预防雄性后代中的疾病风险。
引言
A型血友病是一种X连锁疾病[1]，影响内源性凝血途径，由凝血因子VIII（F8）缺乏引起。F8是凝血级联中F9a激活F10所必需的辅因子（F8a）[2]。A型血友病是人类和犬中最常见的遗传性出血性疾病[3–6]。这种X连锁隐性遗传疾病已在人类、猫、犬、马、牛、大鼠和羊中有描述[7–22]。血友病的临床症状包括脐带持续出血、出血时间延长、不同程度的跛行、血肿形成、轻微创伤后或第二次换牙期间过度出血、创伤性损伤、静脉穿刺以及创伤或手术后的内出血[23–25]。
凝血因子VIII由F8基因产生，该基因位于X染色体长臂，主要在肝脏和血管内皮中合成。由于其位于X染色体上，只有雄性患病，尽管在极罕见情况下杂合携带者也可发展为轻度A型血友病。纯合雌性只有在患病雄性与其配对的杂合雌性（也称为传导者）交配时才能繁育产生。
因子VIII在血液中与血管性血友病因子（vWF）结合循环，后者防止其被蛋白水解和激活。在受伤时，血管性血友病因子被切割下来，因子VIII被激活。激活的因子VIII随后作为辅因子，影响激活的因子IX对因子X激活的作用，因此是血液凝血级联中的关键步骤。
根据CDC血友病A突变项目（CHAMP），到2022年人类中已描述了超过4,000种F8基因的致病变异[6]。特别是单核苷酸变异导致约44%的A型血友病病例。在最近的一项研究中，单核苷酸变异约占人类A型血友病的26%[26]。与人类不同，犬中A型血友病的患病率尚不清楚。除了众多病例报告外，只有少数犬A型血友病病例在DNA水平上进行了特征描述。已在爱尔兰雪达犬、杂种犬、红骨猎浣熊犬、拳师犬、德国牧羊犬、哈瓦那犬、硬毛腊肠犬、大丹犬、贵宾混血犬和老英国牧羊犬等中鉴定了A型血友病的致病突变。这些包括两个错义变异（德国牧羊犬、拳师犬）、两个无义变异（德国牧羊犬、短尾犬）、一个插入（罗得西亚脊背犬）、两个缺失（拉布拉多寻回犬、边境牧羊犬）和一个倒位（爱尔兰雪达犬、迷你雪纳瑞犬）[27–37]。
威玛猎犬中尚未报道A型血友病。在本病例中，短读长测序未检测到F8基因编码区的致病变异，因此F8基因中的倒位成为可能病因[32]。在人类中，内含子22倒位（Inv22）至少占所有A型血友病病例的约40%[26,38,39]。在此我们表明，由内含子22中富含GC区域（包含F8A1基因）与F8远端约420kb的同源片段之间的非等位同源重组（NAHR）介导的倒位是威玛猎犬A型血友病的病因。虽然在教堂山实验犬群体中使用纤维FISH分析仅确定了倒位断点的大致位置，但我们在本研究中能够使用长读长测序确定确切的倒位断点连接[32]。该倒位破坏了F8基因。尽管F8的倒位5'部分（外显子1–22）仍可表达，但由于外显子23–26编码区缺失，倒位导致FVIII C1和A2结构域缩短，从而导致凝血因子功能丧失。
方法
伦理声明
魏玛犬的样本为死后获取。所有其他DNA样本均来源于研究所的样本库，并以残余血液样本的形式保存。残余血液样本的使用已获伦理委员会批准，批准号为E5-21（2021-09-27）。本研究符合德国关于适当动物实验的法律和伦理要求。
DNA样本
死亡犬的样本由犬主提供用于分析。我们从母犬（Ne）获取了肝脏、脾脏、肾脏、肌肉和血液样本。患病魏玛犬公犬（Ay）的一颗牙齿、一个毛绒玩具、一个咀嚼玩具以及一个项圈（带有毛发痕迹）可用于DNA提取。
DNA提取
对所有提供的样本进行了DNA提取。从Ne的组织样本（肝脏、肾脏、脾脏）中仅能提取到严重降解的DNA，不适合进一步分析。血液样本已凝固，因此也不适合DNA提取。从肌肉样本中可提取到足够量的高分子量DNA，用于DNA测序。在Ay的样本中，仅牙齿适合DNA提取。DNA提取使用Blood and Cell Culture DNA Midi Kit（Qiagen）并按照ONT的方案进行修改后完成。为在GridION（ONT）上进行DNA测序，DNA先用g-tube（Covaris）片段化，然后用Qubit（Thermo Fisher Scientific）（Qubit 1x dsDNA Broad Range Kit）测量。必要时用VIVACON 500（Sartorius）浓缩后再用Qubit测量。牙齿的DNA提取使用ChargeSwitch Forensic DNA Purification Kit（Invitrogen）完成。
长读长DNA测序（Nanopore）
DNA测序在GridION（ONT）上进行。测序使用Ligation Sequencing Kit V14（ONT），按照制造商的方案进行。FlowCell FAY11344、FAY11779、FAY34763和FAY34559用于Ne的DNA测序，FlowCell FBA49383用于Ay。GridION上的测序设置为：运行时限72小时，孔扫描频率1.5小时，最小读长1000 bp，读段拆分开启，自适应采样ENRICH REFERENCE，碱基识别HIGH-ACCURACY MODEL 400bps，最小Q值9。以犬X染色体（CFAX）区域NC_049260.1:g.123878920至NC_049260.1:g.124943784作为参考序列（UU_Cfam_GSD_1.0，release 106，NC_049260.1）。该区域（1,064,864 bp）包含GAB3基因的着丝粒部分，并在VAMP7和IL9R基因之间的下游终止。测序运行通过GridION软件MinKNOW（版本23.11.3，ONT）进行控制和监测。每个文件的读段数限制为4000，获得的fastq文件合并为一个文件用于进一步分析。
长读长测序数据的生物信息学分析
使用pycoQC软件检查序列质量（读段数、大小分布、phred等）。合并的fastq文件用Flye组装器（版本2.9.5）[40]进行从头组装。所得重叠群使用Sequencher（版本5.4.6，Gene Codes）和SeqMan NGen（版本18.0.3.2，DNASTAR）比对到犬X染色体的选定区域。此外，将重叠群合并到本地BLAST数据库中，并用F8基因的分离外显子及CFAX区域的其他基因进行搜索。使用犬参考基因组UU_Cfam_GSD_1.0/Dog（canFam4）（NC_049260.1）通过在线平台BLAT将序列数据进行比对。
断点的PCR验证和Sanger测序
引物cF8_contig26283_for（5’-CAGAGCCAGCGACTTCCC-3’）和cF8_contig26283_rev（5’-CAGAGAGACACCGGCCTC-3’）用于验证内含子22的断点。所得PCR产物使用Qsep Bio-Fragment Analyzer（BIOptic Inc.）配合Q-Analyzer软件（版本4.0.0/114）进行分析。扩增产物还通过Sanger测序进行分析，随后在Applied Biosystems 3130xl DNA分析仪上分离。所得序列使用Sequencher软件（版本5.4.6，Gene Codes）或SeqBuilder Pro（版本18.0.3.2，DNASTAR）进行分析。使用兽医研究所样本库中不同品种的多种犬DNA作为阳性和阴性对照。
结果
DNA提取和质量控制
长读长测序通常需要高分子量DNA。这除其他因素外取决于源材料的类型、其分析前来源以及获取和储存方式。在对可用样本进行DNA提取过程中，从Ne的肌肉组织样本中可提取到足够量的高分子量DNA（S1 Fig，泳道1）。相比之下，Ay的毛绒玩具和牙齿样本的DNA提取物已明显降解，大小分布低于3kb（S1 Fig，泳道2、泳道3）。抑制剂对照还显示毛绒玩具DNA样本无法扩增。Ay的其余样本（咀嚼玩具、带毛发痕迹的项圈）无法提取到DNA。
Ne的长读长测序
将来自Ne的纯化并经片段大小选择的DNA测序文库加载到四个FlowCell上。测序运行结束后，首先使用pycoQC对序列数据进行质量检查（S2图）。获得的中位片段长度为1,135–1,548 bp，Phred分数为13.76至16.56（表1）。在所有FlowCell中，中位片段长度均显著短于所选DNA的长度，但所有运行也包含较大比例片段长度> 2,000 bp的reads。
Table 1. Results of long-read sequence data of Ne using pycoQC.
| FlowCell | Fragment length bp (mdn) | Phred score (mdn) |
|---|---|---|
| FAY11344 | 1,152 | 13.76 |
| FAY11779 | 1,135 | 15.73 |
| FAY34559 | 1,548 | 14.87 |
| FAY34763 | 1,482 | 16.56 |
为进一步分析，将所有过滤后的fastq文件合并为一个数据集（Ne_all.fastq；FAY11344：948,684条reads；FAY11779：578,165条reads；FAY34763：2,073,740条reads；FAY34559：964,973条reads；总计4,565,562条reads；21.63GB），并使用flye进行从头组装。共生成127,589个contig，总长度为1,013,464,080 bp（N50 = 10,464），平均覆盖度为5倍（平均覆盖度）。最大的contig长度为161,742 bp。
为进一步分析序列数据，创建了本地BLAST数据库，并使用F8基因的26个外显子以及SMIM9、FUND2C、MTCP1、CMC4、BRCC3、RAB39B、CLIC2和TMLHE基因的选定外显子进行搜索。这些基因位于F8基因的端粒侧，预期应按顺序位于X染色体（CFAX）上。该分析的目的是鉴定数据集中可归属于CFAX上相应区域的单个contig。另一方面，使用Ne_all.fastq数据集与CFAX上的选定区域进行直接比对。该分析应能够组装出尽可能完整且连贯的DNA区域（scaffold）。S1表显示了可从flye数据集中归属于CFAX的contig。
分离得到的contig覆盖了所选区域约47%。对于F8基因，除外显子5–14和20外，所有外显子均可归属，除VBP1外所有端粒侧基因均有代表。还鉴定出另一个长度为16,847 bp的contig_5498，其包含F8基因的外显子21和22。该contig无法实现最佳定位，因为其包含一段约8 kb长的部分，似乎被错误地进行了从头组装。最初，从现有比较中未能鉴定出导致A型血友病的结构变异。然而，必须注意的是，Ne作为雌性犬携带两条X染色体，因此对于结构变异（例如缺失、易位、倒位或类似变异）应为杂合子。此外，所选策略主要选择了与参考基因组相当或相同的正确组装contig。
因此，使用Ne_all.fastq和flye数据集针对CFAX参考基因组区域进行了比对。通过该方法鉴定出contig_26283（S2表）。为验证contig_26283确实为连续的DNA序列，将Ne_all.fastq数据集以contig_26283为参考序列进行比对。如S3图所示，contig_26283由七条重叠reads生成。因此，序列由flye正确组装，contig_26283的DNA片段是Ne基因组的一部分。Contig_26283特别有趣，因为其5'和3'区域分别属于CFAX上F8区域中两个独立的基因组片段，两者相距约420kb（图1）。图1展示了断点扩增子的方向和位置。中心两个颜色相反的箭头指示倒位断点。contig_26283的左侧与F8基因内含子22中X染色体区域NC_049260.1:g.124,002,698至NC_049260.1:g.124,003,000同源，朝向端粒。相反，contig_26283的右侧与跨越位置NC_049260.1:g.124,432,200至NC_049260.1:g.124,432,668的区域同源，该区域位于下游约420kb处，且呈倒置方向，如交叉线所示。同源区域位于CLIC2和TMLHE基因之间（下图）。为清晰起见，该区域所有其他基因以及F8的其余部分均已省略。注意该图未按比例绘制。
[图：contig_26283结构的图形表示。该图展示了contig_26283的组成。Conti]
由于Ay的DNA产量和质量显著较低，仅加载了一个FlowCell（FBA49383）。用于进一步分析的数据集Ay_all.fastq包含1,545,829条reads（N50 = 342），中位片段长度为273 bp，中位Phred评分为14.295。由于所获得的序列数据质量（尤其是片段长度）不足以进行组装，使用Nanofilt软件[41]将Ay_all.fastq数据集逐步缩减为>400、500、600和800 bp的片段，并确定了所得reads的数量。剩余的reads数量分别为303,924条（>400 bp）、130,138条（>500 bp）、71,446条（>600 bp）和33,202条（>800 bp）。由于组装似乎仅对片段长度>800 bp有用，且由于该长度的序列数量较少，预期无法获得染色体区域的足够覆盖度，因此未对Ay的序列数据进行组装。然而，所分离的DNA被认为适合通过直接PCR验证潜在的结构变异。
使用PCR和Sanger测序验证倒位断点
为验证内含子22中的倒位断点，基于contig_26283的序列设计了引物cF8_contig26283_for和cF8_contig26283_rev。需要注意的是，由于F8区域存在广泛的序列同源性，鉴定合适的引物对并非易事。事实证明，无法设计出仅扩增倒位断点周围区域的引物。所用引物在该区域内具有多个结合位点（S3表）。引物cF8_contig26283_for在犬参考基因组内结合八次（下链两次，上链六次），引物cF8_contig26283_rev结合三次（上链一次，下链两次）。然而，基于引物位置理论上可能产生的大多数扩增子过大，或引物位置相距过远，因此可以忽略。尽管如此，在野生型染色体上可产生六个扩增子，由于其大小，可在标准PCR中生成，包括两个与倒位片段大小相当的扩增子（A2、A3）（S4表）。将潜在扩增子（A2、A3）与倒位断点的实验扩增子进行序列比较，揭示了明显的序列差异（S4图）。三个扩增子的比对显示，如果存在倒位，扩增子包含一个22 bp的重复单元，该重复单元在参考序列的潜在扩增子中不存在。因此，始终必须对扩增子进行测序以确定倒位断点的存在。
S5图使用BLAT展示了倒位断点扩增子的具体位置。图的上半部分（A）显示了DNA序列及所用引物在contig_26283内的位置，产生一个772 bp的扩增子。在面板（B）和（C）中显示了与犬参考基因组的比对。扩增子的5´部分比对到CFAX上的NC_049260.1:g.124,002,698至NC_049260.1:g.124,003,000区域，而反向互补的3´部分比对到NC_049260.1:g.124,432,200至NC_049260.1:g.124,432,668区域。
讨论
迄今为止，仅报道了一例魏玛犬A型血友病，但遗传病因仍不明确[42]。在本病例中，短读长测序最初未检测到F8基因编码区的任何变异。由于由非等位同源重组引起的F8基因倒位已被描述为人类和实验犬繁育群体中A型血友病的可能病因，我们使用长读长测序来鉴定此类事件[32]。
检测较大的倒位尤其具有挑战性，因为它们通常不会导致偏离或广泛的SNP变异[43,44]。通常，与相应参考序列相比，断点连接处仅存在微小的序列差异[45,46]。在本病例中，情况更为复杂，因为只有母亲拥有足够的DNA用于长读长测序，因此预期倒位为杂合状态[47]。此外，重复区域数量高于平均水平且序列同源性广泛，尤其是在X染色体的远端区域[48]。
尽管目前已有多种生物信息学方法可用于解析此类结构变异，但其检测仍然是一项非平凡的任务[49]。遗憾的是，使用标准程序比对F8区域并未获得确定性结果[50]。因此，我们首先使用flye[40]对长读长序列进行了从头组装。为识别断点连接，我们利用生成的contig构建了本地BLAST数据库，并使用来自潜在断点连接的非重复DNA序列以及F8（外显子22–26）、CLIC2和TMLHE的外显子序列对该数据库进行检索。BLAST检索中DNA序列的初步选择基于以下假设：如同人类一样，本病例中的倒位最可能由F8内含子22中一个富含GC的区域所介导，其中F8A1基因和一段同源片段位于F8远端约420kb处（图2）。在人类中，F8基因的内含子22倒位由内含子22中的同源区域int22h1与CLIC2和TMLHE之间的int22h2/3所引起。int22h1区域（NC_000023.9:g.153,762,285–153,771,797）包含F8A1基因。Int22h2/3位于NC_000023.9:g.154,259,350–154,268,903区域[51–53]。这两个区域对应于犬中导致本文所报道倒位的染色体片段。为识别contig中F8基因的其他部分以及X染色体区域的其他基因，还使用犬参考序列中的其他DNA片段对BLAST数据库进行了检索。随后，将与所选DNA片段具有同源性的Flye contig使用BLAT[54]比对至犬参考基因组（UU_Cfam_GSD_1.0，版本106）。在倒位事件大致位置未知的情况下，所选方法可能过于复杂，但在本病例中，该方法使得待分析的具有潜在断点连接的contig数量迅速减少。
[图：Ne和Ay染色体倒位的图示。该图展示了Ne和A中的倒位过程]
总之，Ay是F8基因内含子22倒位的携带者，该倒位最可能由非等位同源重组以及距F8约420kb处的一段同源片段所引起。该倒位破坏了F8基因，导致FVIII C1和A2结构域缩短。由外显子23–26编码的C1和A2结构域缩短导致凝血因子功能丧失。该倒位遗传自其母亲Ne，后者为该等位基因的携带者。尽管可以预期F8基因的5´区域（外显子1–22）可能仍会表达，但该倒位导致凝血因子FVIII截短，从而引起功能丧失并发展为A型血友病。尽管已有魏玛犬发生A型血友病的报道[42]，但本研究结果首次表明，此前鲜有描述的F8倒位也可作为该犬种A型血友病的遗传病因。
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Haemophilia A is a severe coagulation disorder caused by partial or complete loss of function of the X-chromosomal F8 gene. It is a relatively common coagulation disorder that has been reported in at least 17 dog breeds to date. However, only five distinct molecular causes have been identified so far. One molecular genetic cause is a large inversion, which has also been described in humans and was first studied in a research colony of Irish Setters at the University of North Carolina at Chapel Hill. However, the exact location of the inversion breakpoint junctions in dogs has not been determined. Here, we describe the molecular analysis of a case of haemophilia A in a Weimaraner dog family using long-read sequencing. We show that the cause of haemophilia A is an approximately 420kb inversion affecting the F8 gene. The breakpoints of the inversion were identified in intron 22 at position 124,003,000 and between chloride intracellular channel 2 (CLIC2) and epsilon-trimethyllysine hydroxylase (TMLHE) at position 124,432,668 (NC_049260.1:g.124003000_124432668inv). As in humans, the inversion is most likely caused by non-allelic homologous recombination between intron 22 and a region approximately 420kb downstream of the F8 gene. By identifying the inversion breakpoint junctions, it is now possible to test carriers of the disorder at the DNA level and thus prevent the risk of disease in male offspring.
Introduction
Haemophilia A is an X-linked disorder [1] that affects the intrinsic coagulation pathway and is caused by a deficiency of coagulation factor VIII (F8). F8 is an essential cofactor (F8a) for the activation of F10 by F9a in the coagulation cascade [2]. Haemophilia A is the most common inherited bleeding disorder in humans and dogs [3–6]. The X-linked recessively inherited disorder has been described in humans, cats, dogs, horses, cattle, rats and sheep [7–22]. Clinical signs of haemophilia include persistent bleeding from the umbilical cord, prolonged bleeding time, variable lameness, haematoma formation, excessive bleeding after minor trauma or during second dentition, traumatic injury, venepuncture, and internal bleeding after trauma or surgery [23–25].
Coagulation factor VIII is produced by the F8 gene, which is located on the long arm of the X-chromosome and is mainly synthesised in the liver and vascular endothelia. Due to its location on the X-chromosome, only males are affected, although in very rare cases heterozygous carriers can also develop a mild form of haemophilia A. Homozygous females can only be bred if an affected male is paired with a heterozygous female who are also referred to as conductors.
Factor VIII circulates in the blood bound to von Willebrand factor (vWF), which prevents its proteolysis and activation. In the event of injury, von Willebrand factor is cleaved off and factor VIII is activated. Activated factor VIII then acts as a cofactor, influencing the effect of activated factor IX on the activation of factor X, and is therefore an essential step in the blood coagulation cascade.
According to the CDC Haemophilia A Mutation Project (CHAMP), more than 4,000 causative variants in the F8 gene had been described in humans by 2022 [6]. Single nucleotide variants in particular lead to haemophilia A in around 44% of cases. In a recent study single nucleotide variants account for approximately 26% of haemophilia A in humans [26]. In contrast to humans, the prevalence of haemophilia A in dogs is unknown. Apart from numerous case reports, only a few cases of haemophilia A in dogs have been characterised at the DNA level. Causative mutations for haemophilia A have been identified in Irish Setters, Mongrels, Redbone Coonhounds, Boxers, German Shepherds, Havanese, Wirehaired Dachshunds, Great Danes, Poodle mixes and Old English Shepherds, among others. These include two missense variants (German Shepherd, Boxer), two nonsense variants (German Shepherd, Bobtail), one insertion (Rhodesian Ridgeback), two deletions (Labrador Retriever, Border Collie) and one inversion (Irish Setter, Miniature Schnauzer) [27–37].
Haemophilia A has not yet been reported in Weimaraner dogs. In the present case, no causative variants in the coding region of the F8 gene were detected by short-read sequencing, leaving an inversion in the F8 gene as a possible cause [32]. In humans an intron 22 inversion (Inv22) accounts for at least approximately 40% of all haemophilia A cases [26,38,39]. Here we show that an inversion mediated by non-allelic homologous recombination (NAHR) between a GC-rich region in intron 22, which harbours the F8A1 gene, and a homologous segment approximately 420kb distal to F8 is causative for haemophilia A in the Weimaraner dog. While only a rough position of the inversion breakpoints has been determined in the experimental dog population in Chapel Hill using fibre FISH analysis, we were able to determine the exact inversion breakpoint junctions in the present study using long-read sequencing [32]. The inversion disrupts the F8 gene. Although the inverted 5’ part (exons 1–22) of F8 can still be expressed, the inversion results in a shortening of the FVIII C1 and A2 domains due to the absence of the coding regions of exons 23–26, leading to loss of function of the blood coagulation factor.
Methods
Ethical statement
The samples from the Weimaraner dogs were obtained post mortem. All other DNA samples were sourced from the institute's depository and stored as residual blood samples. The use of residual blood samples was approved by the Ethics Committee under no. E5-21 (2021-09-27). The study complies with German legal and ethical requirements for appropriate animal procedures.
DNA samples
Samples from the deceased dogs were provided by the owners for analysis. We obtained samples of liver, spleen, kidney, muscle, and blood from the mother (Ne). A tooth, a soft toy, a chew toy, and a collar (with traces of hair) from the affected Weimaraner male dog (Ay) were available for DNA extraction.
DNA extraction
DNA was extracted from all samples provided. Only severely degraded DNA could be extracted from the tissue samples (liver, kidney, spleen) of Ne, which was not suitable for further analysis. The blood sample had already coagulated and was therefore also unsuitable for DNA extraction. Sufficient DNA with a high molecular weight could be extracted from the muscle sample, which was used for DNA sequencing. Of the samples from Ay, only the tooth was suitable for DNA extraction. DNA extraction was performed using the Blood and Cell Culture DNA Midi Kit (Qiagen) with modifications according to a protocol from ONT. For DNA sequencing on the GridION (ONT), DNA was fragmented with a g-tube (Covaris) and then measured with the Qubit (Thermo Fisher Scientific) (Qubit 1x dsDNA Broad Range Kit). If necessary, the concentration was increased with VIVACON 500 (Sartorius) and then measured again with the Qubit. DNA was extracted from the tooth using the ChargeSwitch Forensic DNA Purification Kit (Invitrogen).
Long-read DNA sequencing (Nanopore)
DNA sequencing was performed on a GridION (ONT). The Ligation Sequencing Kit V14 (ONT) was used for sequencing and performed according to the manufacturer's protocol. FlowCells FAY11344, FAY11779, FAY34763 and FAY34559 were used for sequencing the DNA of Ne and FlowCell FBA49383 was used for Ay. The settings for sequencing on the GridION were: run limit 72h, pore scan freq. 1.5h, minimum read length 1000 bp, read splitting ON, adaptive sampling ENRICH REFERENCE, basecalling HIGH-ACCURACY MODEL 400bps, Min Q score 9. The region NC_049260.1:g.123878920 to NC_049260.1:g.124943784 of the canine X chromosome (CFAX) was used as reference sequence (UU_Cfam_GSD_1.0, release 106, NC_049260.1). This region (1,064,864 bp) contains the centromeric part of the GAB3 gene and ends downstream between the VAMP7 and IL9R genes. The sequencing run was controlled and monitored with the GridION software MinKNOW (version 23.11.3, ONT). The number of reads per file was limited to 4000 and the fastq files obtained were merged into one file for further analysis.
Bioinformatic analysis of the long-read sequencing data
The quality of the sequences (number of reads, size distribution, phred, etc.) was checked with the pycoQC software. The merged fastq files were assembled de novo with the Flye assembler (version 2.9.5) [40]. The resulting contigs were aligned to the selected region of the canine X chromosome using Sequencher (version 5.4.6, Gene Codes) and SeqMan NGen (version 18.0.3.2, DNASTAR). In addition, the contigs were merged into a local BLAST database and searched with the isolated exons of the F8 gene and other genes of the CFAX region. The canine reference genome UU_Cfam_GSD_1.0/Dog (canFam4) (NC_049260.1) was used to align the sequence data with the online platform BLAT.
PCR validation and Sanger sequencing of breakpoints
The primers cF8_contig26283_for (5’-CAGAGCCAGCGACTTCCC-3’) and cF8_contig26283_rev (5’-CAGAGAGACACCGGCCTC-3’) were used to validate the breakpoint of intron 22. The obtained PCR products were analysed with a Qsep Bio-Fragment Analyzer (BIOptic Inc.) using Q-Analyzer software (version 4.0.0/114). Amplicons were also analysed by Sanger sequencing and subsequent separation on an Applied Biosystems 3130xl DNA analyser. The sequences obtained were analysed using Sequencher software (version 5.4.6, Gene Codes) or SeqBuilder Pro (version 18.0.3.2, DNASTAR). Various dog DNAs of different breeds from the repository of the Veterinary Institute were used as positive and negative controls.
Results
DNA extraction and quality control
High-molecular-weight DNA is generally required for long-read sequencing. This depends, among other things, on the type of source material, its pre-analytical origin, and how it is obtained and stored. During DNA extraction from the samples available, sufficient quantities of high-molecular-weight DNA could be extracted from the muscle tissue sample of Ne (S1 Fig, lane 1). In contrast, the DNA isolates from the soft toy and tooth sample of Ay were already significantly degraded and showed a size distribution below 3kb (S1 Fig, lane 2, lane 3). An inhibitor control also showed that the soft toy DNA sample could not be amplified. No DNA could be extracted from the remaining samples of Ay (chew toy, collar with hair traces).
Long-read sequencing of Ne
The sequencing libraries of purified and size-selected DNA from Ne were loaded onto four FlowCells. After the runs, the sequence data were first checked for quality using pycoQC (S2 Fig). The median fragment lengths obtained ranged from 1,135–1,548 bp and the Phred scores ranged from 13.76 to 16.56 (Table 1). The median fragment lengths were significantly shorter than the length of the selected DNA in all FlowCells, but all runs also contained larger proportions of reads with fragment lengths > 2,000 bp.
Table 1. Results of long-read sequence data of Ne using pycoQC.
| FlowCell | Fragment length bp (mdn) | Phred score (mdn) |
|---|---|---|
| FAY11344 | 1,152 | 13.76 |
| FAY11779 | 1,135 | 15.73 |
| FAY34559 | 1,548 | 14.87 |
| FAY34763 | 1,482 | 16.56 |
For further analysis, all filtered fastq files were merged into one dataset (Ne_all.fastq; FAY11344: 948,684 reads; FAY11779: 578,165 reads; FAY34763: 2,073,740 reads; FAY34559: 964,973 reads; total 4,565,562 reads; 21.63GB) and assembled de novo with flye. A total of 127,589 contigs with a total length of 1,013,464,080 bp (N50 = 10,464) and an average 5-fold coverage (mean coverage) were generated. The largest contig had a length of 161,742 bp.
For further analysis of the sequence data, a local BLAST database was created and searched with the 26 exons of the F8 gene and selected exons of the SMIM9, FUND2C, MTCP1, CMC4, BRCC3, RAB39B, CLIC2, and TMLHE genes. These genes are located telomeric to the F8 gene and should be located on the X-chromosome (CFAX) in the expected order. The aim of this analysis was to identify individual contigs in the dataset that could be assigned to the corresponding sections on CFAX. On the other hand, the Ne_all.fastq dataset was used to perform a direct alignment with the selected region on CFAX. This analysis should enable the assembly of a DNA region (scaffold) that is as complete and coherent as possible. S1 Table shows the contigs that could be assigned from the flye dataset to CFAX.
The isolated contigs covered approximately 47% of the selected region. For the F8 gene, all exons except exons 5–14 and 20 could be assigned, and all telomeric genes except VBP1 were represented. Another contig_5498 with a length of 16,847 bp was also identified, which contained exons 21 and 22 of the F8 gene. This contig could not be optimally positioned because it contained an approximately 8 kb long part that appeared to have been incorrectly de novo assembled. Initially, no structural variants causative of haemophilia A could be identified from the available comparisons. However, it must be noted that Ne, as a female dog, carries two X chromosomes and would therefore be heterozygous for a structural variant (e.g., deletion, translocation, inversion, or similar). In addition, the chosen strategy mainly selected correctly assembled contigs that were comparable or identical to the reference genome.
Therefore, an alignment against the CFAX reference genome region was performed using the Ne_all.fastq and flye data sets. With this approach contig_26283 was identified (S2 Table). To verify that contig_26283 was indeed a continuous DNA sequence, the Ne_all.fastq dataset was aligned with contig_26283 as a reference sequence. As shown in S3 Fig, contig_26283 is generated by seven overlapping reads. Hence the sequences were assembled correctly by flye and the DNA fragment of contig_26283 is part of the genome of Ne. Contig_26283 was particularly interesting because the 5’ and 3’ regions belong to two separate genome segments in the F8 region on CFAX, which are about 420kb apart (Fig 1). Fig 1 illustrates the orientations and locations of the breakpoint amplicon. The two opposing-coloured arrows in the centre indicate the inversion breakpoint. The left side of contig_26283 is homologous to the X-chromosomal region NC_049260.1:g.124,002,698 to NC_049260.1:g.124,003,000 in intron 22 of the F8 gene, oriented towards the telomere. In contrast, the right-hand side of contig_26283 is homologous to a region spanning positions NC_049260.1:g.124,432,200 to NC_049260.1:g.124,432,668, approximately 420kb downstream, and in an inverted orientation, as indicated by the crossed lines. The homologous region is located between the genes for CLIC2 and TMLHE (lower panel). For clarity, all other genes in the region, as well as the remaining parts of the F8, have been omitted. Note that the diagram is not to scale.
[图：Graphical representation of the structure of contig_26283. The figure illustrates the composition of contig_26283. Conti]
Due to the significantly lower yield and quality of the DNA of Ay, only one FlowCell was loaded (FBA49383). The dataset Ay_all.fastq used for further analysis contained 1,545,829 reads (N50 = 342) with a median fragment length of 273 bp and a median Phred score = 14.295. Since the quality (especially fragment lengths) of the sequence data obtained was insufficient for assembly, the Ay_all.fastq dataset was gradually reduced to fragments of > 400, 500, 600, and 800 bp using Nanofilt software [41], and the number of resulting reads was determined. The remaining number of reads were 303,924 (>400 bp), 130,138 (>500 bp), 71,446 (>600 bp), and 33,202 (>800 bp). Since assembly only appeared to be useful for fragment lengths >800 bp and, due to the small number of sequences with this length, sufficient coverage of the chromosomal region was not to be expected and therefore the sequence data from Ay were not assembled. However, the isolated DNA was considered suitable for validation of potential structural variants by direct PCR.
Validation of the inversion breakpoint using PCR and Sanger sequencing
To validate the inversion breakpoint in intron 22, primers cF8_contig26283_for and cF8_contig26283_rev were developed based on the sequence of contig_26283. It should be noted that identifying a suitable primer pair was not straightforward due to the extensive sequence homologies in the F8 region. It proved impossible to develop primers that amplified only the region around the inversion breakpoint. The primers used had multiple binding sites within the region (S3 Table). Primer cF8_contig26283_for binds eight times (twice on the lower strand, six times on the upper strand), and primer cF8_contig26283_rev binds three times (once on the upper strand, twice on the lower strand) within the canine reference genome. However, most of the theoretically possible amplicons based on the primer positions would be too large, or the primer positions were too far apart and could therefore be disregarded. Nevertheless, on the wild-type chromosome six amplicons can be produced which, due to their size, can be generated in a standard PCR, including two amplicons (A2, A3) that were comparable in size to the inversion fragment (S4 Table). Sequence comparison of the potential amplicons (A2, A3) with the experimental amplicon of the inversion breakpoint revealed a clear sequence difference (S4 Fig). Alignment of the three amplicons showed that if the inversion is present, the amplicon contains a 22 bp repeat unit that does not occur in the potential amplicons of the reference sequence. Sequencing of the amplicon is therefore always mandatory to determine the presence of the inversion breakpoint.
S5 Fig illustrates the specific location of the inversion breakpoint amplicon using BLAT. The upper part of the figure (A) shows the DNA sequence with the positions of the primers used within contig_26283, generating a 772 bp amplicon. In panel (B) and (C) the alignments with the canine reference genome are shown. The 5´ part of the amplicon aligned to region NC_049260.1:g.124,002,698 to NC_049260.1:g.124,003,000 on CFAX whereas the reverse complement 3´ part aligned to region NC_049260.1:g.124,432,200 to NC_049260.1:g.124,432,668.
Discussion
To date, only one case of haemophilia A in Weimaraner dogs has been reported, but the genetic cause remains unclear [42]. In the present case, short-read sequencing did not initially detect any variants in the coding regions of the F8 gene. As an inversion in the F8 gene caused by non-allelic homologous recombination has been described as a possible cause of haemophilia A in both humans and an experimental dog breeding population, we used long-read sequencing to identify such an event [32].
Detecting larger inversions is particularly challenging, as they do not usually result in deviating or extensive SNP variants [43,44]. Often, there are only minor sequence differences at the breakpoint junctions compared to the corresponding reference sequence [45,46]. In the present case, the situation was complicated by the fact that only the mother had sufficient DNA available for long-read sequencing and, consequently, heterozygosity for the inversion was to be expected [47]. In addition, there is an above-average number of repetitive regions and extensive sequence homologies, especially in the distal region of the X chromosome [48].
Although various bioinformatic approaches now exist for elucidating such structural variants, detection remains a non-trivial task [49]. Unfortunately, the use of standard programmes for aligning the F8 region did not yield conclusive results [50]. We therefore initially assembled the long-reads de novo using flye [40]. To identify the breakpoint junctions, we created a local BLAST database with the generated contigs and searched it using non-repetitive DNA sequences from the potential breakpoint junctions, as well as exonic sequences of F8 (exons 22–26), CLIC2, and TMLHE. The initial selection of DNA sequences for the BLAST search was based on the assumption that, as in humans, the inversion in the present case was most likely mediated by a GC-rich region in intron 22 of F8, where the F8A1 gene and a homologous segment are located approximately 420kb distal to F8 (Fig 2). In humans, the intron 22 inversion in the F8 gene is caused by the homologous regions int22h1 in intron 22 and int22h2/3 between CLIC2 and TMLHE. The int22h1 region (NC_000023.9:g.153,762,285–153,771,797) contains the F8A1 gene. Int22h2/3 is located in the region NC_000023. 9:g.154,259,350–154,268,903 [51–53]. Both regions correspond to the chromosomal segments in dogs responsible for the inversion reported here. To identify additional parts of the F8 gene and other genes of the X-chromosomal region within the contigs, the BLAST database was also searched using other DNA fragments from the canine reference sequence. Flye contigs with homologies to the selected DNA fragments were then aligned against the dog reference genome (UU_Cfam_GSD_1.0, release 106) using BLAT [54]. The chosen approach may be too complex in cases where the approximate location of the inversion event is unknown, but in the present case it led to a rapid reduction in the number of contigs to be analysed with the potential breakpoint junction.
[图：Graphical representation of chromosomal inversion in Ne and Ay. The figure illustrates the inversion process in Ne and A]
In summary Ay was carrier of an inversion in intron 22 of the F8 gene most likely caused by non-allelic homologous recombination and a homologous segment approximately 420kb distal from F8. The inversion disrupts the F8 gene, resulting in a shortening of the FVIII C1 and A2 domains. The shortening of the C1 and A2 domains, encoded by exons 23–26, leads to loss of function of the coagulation factor. The inversion was inherited from its mother Ne, who was a carrier of the allele. Although it can be expected that the 5´ region of the F8 gene (exons 1–22) might still be expressed, the inversion causes truncation of coagulation factor FVIII, resulting in loss of function and the development of haemophilia A. Although the occurrence of haemophilia A in Weimaraner dogs has been reported [42], the present findings show for the first time that the previously rarely described inversion in F8 can also be the genetic cause of haemophilia A in this dog breed.
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