Application of chromosome microarray analysis on short femur length in fetuses

doi:10.3877/cma.j.issn.1673-5250.2023.04.009

Objective

To explore the genetic etiology of fetuses with short femur length (FL) at the genome-wide level by chromosome microarray analysis (CMA) technique.

Methods

Seventy-three pregnant women and their fetuses who had short FL by prenatal ultrasonography and underwent invasive prenatal diagnosis at Guangzhou Women and Children′s Medical Center from January 2019 to December 2020 were enrolled in the study. According to whether they were combined with other obvious structural developmental abnormalities, they were divided into isolated group (n=56) and complex group (n=17). Chromosomal karyotyping and CMA were performed, and results were analyzed by applying CHAS software and related bioinformatics methods. The procedures followed in this study were in accordance with the ethical standards set by the Human Experimentation Committee of Guangzhou Women and Children′s Medical Center, and were approved by this ethical committee (Approval No. [2019] 11600). Written informed consents were obtained form all pregnant women.

Results

① Of these 56 fetuses in isolated group, 6 were combined with ultrasound soft index abnormalities; of these 17 fetuses in complex group, 6 were combined with neurological malformations, 6 with cardiovascular malformations, and 6 with malformations of other skeletal systems. ②The CMA results of 73 fetuses showed that 10 cases contained pathogenic/probably pathogenic copy number variants (CNV), with a total pathogenicity detection rate of 13.7% (10/73), the variants of uncertain significance (VUS) detection rate of 2.7% (2/73), and a benign CNV detection rate of 83.6% (61/73). ③Of the 44 short FL fetuses that underwent both chromosomal karyotyping and CMA testing, 34 were in isolated group and 10 were in complex group. Comparison of the rates of chromosomal karyotype abnormality (10.0%) and abnormal detection by CMA technique (30.0%) in the complex group showed no statistically significant difference (χ²=2.08, P=0.149). ④None of the 73 fetuses were found to contain the known pathogenic locus of FGFR3 gene. Of the 63 CMA-negative fetuses, 7 fetuses were highly suspected of having long-bone dysplasia due to prenatal ultrasonography (long bones of the limbs <3% of the children of the same gestational age), and further whole exome sequencing (WES) testing showed that the COL1A1 mutation (c.2519C>T; p. P840L) was detected in 1 fetus as VUS. ⑤Follow-up showed that 31 (42.5%) fetuses were delivered at term, 15 (48.4%) live births showed a growth rate of long below the 10th percentile of the same-age child in infancy, and other developmental assessments did not show any obvious abnormalities.

Conclusions

CMA has a certain application value in prenatal diagnosis of fetuses with short FL and has a higher detection rate than chromosomal karyotyping. Therefore, it is suggested that CMA should be used as a first-line technique in the prenatal diagnosis of fetuses with short FL on prenatal ultrasound examination, especially when combined with other structural abnormalities.

Key words: Prenatal ultrasound, Fetal short femur length, Structural anomalies, Chromosomal abnormalities, Microdeletion/microduplication

表1 复杂组17例FL偏短并伴其他结构畸形胎儿超声检查及随访结果

胎儿编号	胎龄(周)	与正常值比较	胎儿其他结构异常超声表现	随访结果
1	28	<－2 s(FL)	后颅窝增宽、腹腔积液、心胸比增大、心律不齐	引产终止妊娠
2	26	<－2 s(FL)	心脏室间隔缺损、单脐动脉、羊水过少	引产终止妊娠
3	25	<－2 s(FL)	双足内翻	失访
4	30	<－2 s(FL)	心胸比增大、心包积液、羊水过少	足月顺产，产后诊断为孤立性FL偏短
5	17	<－3 s(四肢长骨)	侧脑室扩张、淋巴管瘤、蛛网膜囊肿、小下颌畸形、心脏室间隔缺损、下肢长骨成角畸形、双足内翻	引产终止妊娠
6	13	<－3 s(四肢长骨)	尺、桡骨发育异常及巨膀胱	失访
7	25	<－3 s(四肢长骨)	胼胝体缺如、小下颌畸形、羊水过多	引产终止妊娠
8	24	<－2 s(FL)	心脏畸形、巨膀胱	引产终止妊娠
9	26	<－2 s(FL)	十二指肠闭锁	引产终止妊娠
10	28	<－2 s(FL)	主动脉缩窄、腹部囊性包块	足月顺产，产后诊断为孤立性FL偏短
11	27	<－2 s(四肢长骨)	小脑发育不良	引产终止妊娠
12	32	<－2 s(FL)	室间隔缺损、双肾及肝回声增强	引产终止妊娠
13	25	<－2 s(FL)	眼距稍宽、颈部皱褶稍厚，鼻骨发育不良、右肾积水	失访
14	28	<－2 s(FL)	侧脑室扩张	足月顺产，产后诊断为孤立性FL偏短
15	25	<－3 s(四肢长骨)	小下颌畸形、双侧脑室扩张、颈后皮肤皱褶增厚、右肾盂扩张、双足内翻	引产终止妊娠
16	34	<－2 s(四肢长骨，FL)	室管膜下囊肿、透明隔腔小、右侧脑室扩张	引产终止妊娠
17	24	<－3 s(四肢长骨)	双足内翻	引产终止妊娠

表1 复杂组17例FL偏短并伴其他结构畸形胎儿超声检查及随访结果

胎儿编号	胎龄(周)	与正常值比较	胎儿其他结构异常超声表现	随访结果
1	28	<－2 s(FL)	后颅窝增宽、腹腔积液、心胸比增大、心律不齐	引产终止妊娠
2	26	<－2 s(FL)	心脏室间隔缺损、单脐动脉、羊水过少	引产终止妊娠
3	25	<－2 s(FL)	双足内翻	失访
4	30	<－2 s(FL)	心胸比增大、心包积液、羊水过少	足月顺产，产后诊断为孤立性FL偏短
5	17	<－3 s(四肢长骨)	侧脑室扩张、淋巴管瘤、蛛网膜囊肿、小下颌畸形、心脏室间隔缺损、下肢长骨成角畸形、双足内翻	引产终止妊娠
6	13	<－3 s(四肢长骨)	尺、桡骨发育异常及巨膀胱	失访
7	25	<－3 s(四肢长骨)	胼胝体缺如、小下颌畸形、羊水过多	引产终止妊娠
8	24	<－2 s(FL)	心脏畸形、巨膀胱	引产终止妊娠
9	26	<－2 s(FL)	十二指肠闭锁	引产终止妊娠
10	28	<－2 s(FL)	主动脉缩窄、腹部囊性包块	足月顺产，产后诊断为孤立性FL偏短
11	27	<－2 s(四肢长骨)	小脑发育不良	引产终止妊娠
12	32	<－2 s(FL)	室间隔缺损、双肾及肝回声增强	引产终止妊娠
13	25	<－2 s(FL)	眼距稍宽、颈部皱褶稍厚，鼻骨发育不良、右肾积水	失访
14	28	<－2 s(FL)	侧脑室扩张	足月顺产，产后诊断为孤立性FL偏短
15	25	<－3 s(四肢长骨)	小下颌畸形、双侧脑室扩张、颈后皮肤皱褶增厚、右肾盂扩张、双足内翻	引产终止妊娠
16	34	<－2 s(四肢长骨，FL)	室管膜下囊肿、透明隔腔小、右侧脑室扩张	引产终止妊娠
17	24	<－3 s(四肢长骨)	双足内翻	引产终止妊娠

表2 CMA检出结果为致病性/可能致病性CNV的10例FL偏短胎儿的相关检查结果及妊娠结局

胎儿编号^a	胎龄	胎儿超声检查结果	CMA结果	染色体核型异常	染色体片段长度	CNV分类	双亲CNV验证	妊娠结局
1	28	<－2 s(FL)	arr(X)×1	p22.33-q28	150.06 Mb	致病性	新发	引产
9	26	<－2 s(FL)	arr(21)×3	q11.2-q22.3	33.08 Mb	致病性	新发	引产
11	27	<－2 s(FL)	arr5p15.33p15.1(113，576-16，550，629)×1	p15.33p15.1	16.44 Mb	致病性	新发	引产
12	32	<－2 s(FL)	arr(21)×3	q11.2-q22.3	33.08 Mb	致病性	新发	引产
15	25	<－3 s(四肢长骨)	arr17q24.3(70，074，147-70，189，241)×1	q24.3	115.00 kb	致病性	新发	引产
17	24	<－3 s(四肢长骨)	arrXp22.33(327，827-737，851)×3	p22.33	410.00 kb	可能致病	新发	引产
18	29	<－2 s(FL)	arr(21)×3	q11.2-q22.3	33.08 Mb	致病性	新发	引产
19	35⁺⁵	<－2 s(FL)	arr(X)×1	p22.33-q28	150.06 Mb	致病性	新发	引产
20	25	<－2 s(FL)	arr(X)×1	p22.33-q28	150.06 Mb	致病性	新发	引产
21	28	<－2 s(FL)	arr12p13.33p11.1(173，786-33，865，197)×3	p13.33p11.1	33.69 Mb	致病性	新发	引产

表2 CMA检出结果为致病性/可能致病性CNV的10例FL偏短胎儿的相关检查结果及妊娠结局

胎儿编号^a	胎龄	胎儿超声检查结果	CMA结果	染色体核型异常	染色体片段长度	CNV分类	双亲CNV验证	妊娠结局
1	28	<－2 s(FL)	arr(X)×1	p22.33-q28	150.06 Mb	致病性	新发	引产
9	26	<－2 s(FL)	arr(21)×3	q11.2-q22.3	33.08 Mb	致病性	新发	引产
11	27	<－2 s(FL)	arr5p15.33p15.1(113，576-16，550，629)×1	p15.33p15.1	16.44 Mb	致病性	新发	引产
12	32	<－2 s(FL)	arr(21)×3	q11.2-q22.3	33.08 Mb	致病性	新发	引产
15	25	<－3 s(四肢长骨)	arr17q24.3(70，074，147-70，189，241)×1	q24.3	115.00 kb	致病性	新发	引产
17	24	<－3 s(四肢长骨)	arrXp22.33(327，827-737，851)×3	p22.33	410.00 kb	可能致病	新发	引产
18	29	<－2 s(FL)	arr(21)×3	q11.2-q22.3	33.08 Mb	致病性	新发	引产
19	35⁺⁵	<－2 s(FL)	arr(X)×1	p22.33-q28	150.06 Mb	致病性	新发	引产
20	25	<－2 s(FL)	arr(X)×1	p22.33-q28	150.06 Mb	致病性	新发	引产
21	28	<－2 s(FL)	arr12p13.33p11.1(173，786-33，865，197)×3	p13.33p11.1	33.69 Mb	致病性	新发	引产

图1 广州市妇女儿童医疗中心产前诊断中心总结的胎儿FL偏短的遗传学诊断流程图注：FL为股骨长度，WES为全外显子组测序，CMA为染色体微阵列分析

[1]	Speer PD, Canavan T, Simhan HN, et al. Prenatal midtrimester fetal long bone measurements and the prediction of small-for-gestational-age fetuses at term [J]. Am J Perinatol, 2014, 31(3): 231-236. DOI: 10.1055/s-0033-1345260.
[2]	Beke A, Papp C, Tóth-Pál E, et al. Trisomies and other chromosome abnormalities detected after positive sonographic findings [J]. J Reprod Med, 2005, 50(9): 675-691.
[3]	Rao R, Platt LD. Ultrasound screening: status of markers and efficacy of screening for structural abnormalities [J]. Semin Perinatol, 2016, 40(1): 67-78. DOI: 10.1053/j.semperi.2015.11.009.
[4]	Kaijomaa M, Ulander VM, Ryynanen M, et al. Risk of adverse outcomes in euploid pregnancies with isolated short fetal femur and humerus on second-trimester sonography [J]. J Ultrasound Med, 2016, 35(12): 2675-2680. DOI: 10.7863/ultra.16.01086.
[5]	Weisz B, David AL, Chitty L, et al. Association of isolated short femur in the mid-trimester fetus with perinatal outcome [J]. Ultrasound Obstet Gynecol, 2008, 31(5): 512-516. DOI: 10.1002/uog.5349.
[6]	Aviram A, Bardin R, Wiznitzer A, et al. Midtrimester isolated short femur length as a predictor of adverse pregnancy outcome [J]. Fetal Diagn Ther, 2015, 38(3): 205-211. DOI: 10.1159/000375446.
[7]	Mathiesen JM, Aksglaede L, Skibsted L, et al. Outcome of fetuses with short femur length detected at second-trimester anomaly scan: a national survey [J]. Ultrasound Obstet Gynecol, 2014, 44(2): 160-165. DOI: 10.1002/uog.13286.
[8]	Agathokleous M, Chaveeva P, Poon LC, et al. Meta-analysis of second-trimester markers for trisomy 21 [J]. Ultrasound Obstet Gynecol, 2013, 41(3): 247-261. DOI: 10.1002/uog.12364.
[9]	Chandler N, Best S, Hayward J, et al. Rapid prenatal diagnosis using targeted exome sequencing: a cohort study to assess feasibility and potential impact on prenatal counseling and pregnancy management [J]. Genet Med, 2018, 20(11): 1430-1437. DOI: 10.1038/gim.2018.30.
[10]	Liu J, Huang L, He Z, et al. Clinical value of genetic analysis in prenatal diagnosis of short femur [J]. Mol Genet Genomic Med, 2019, 7(11): e978. DOI: 10.1002/mgg3.978.
[11]	Bardin R, Hadar E, Haizler-Cohen L, et al. Cytogenetic analysis in fetuses with late onset abnormal sonographic findings [J]. J Perinat Med, 2018, 46(9): 975-982. DOI: 10.1515/jpm-2017-0071.
[12]	Riggs ER, Andersen EF, Cherry AM, et al. Technical standards for the interpretation and reporting of constitutional copy-number variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen) [J]. Genet Med, 2020, 22(2): 245-257. DOI: 10.1038/s41436-019-0686-8.
[13]	Mohr-Sasson A, Toussia-Cohen S, Shapira M, et al. Long-term follow-up on fetuses with isolated sonographic finding of short long bones: a cohort study [J]. Arch Gynecol Obstet, 2020, 301(2): 459-463. DOI: 10.1007/s00404-019-05421-4.
[14]	American College of Obstetricians and Gynecologists Committee on Genetics. Committee Opinion No. 581: the use of chromosomal microarray analysis in prenatal diagnosis [J]. Obstet Gynecol, 2013, 122(6): 1374-1377. DOI: 10.1097/01.AOG.0000438962.16108.d1.
[15]	Bardin R, Hadar E, Haizler-Cohen L, et al. Cytogenetic analysis in fetuses with late onset abnormal sonographic findings [J]. J Perinat Med, 2018, 46(9): 975-982. DOI: 10.1515/jpm-2017-0071.
[16]	Tzadikevitch K, Singer A, Maya I, et al. Chromosomal microarray should be performed for cases of fetal short long bones detected prenatally [J]. Arch Gynecol Obstet, 2021, 303(1): 85-92. DOI: 10.1007/s00404-020-05729-6.
[17]	Bethune M. Literature review and suggested protocol for managing ultrasound soft markers for Down syndrome: thickened nuchal fold, echogenic bowel, shortened femur, shortened humerus, pyelectasis and absent or hypoplastic nasal bone [J]. Australas Radiol, 2007, 51(3): 218-225. DOI: 10.1111/j.1440-1673.2007.01713.x.
[18]	Richards S, Aziz N, Bale S, et al, Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology [J]. Genet Med, 2015, 17(5): 405-424. DOI: 10.1038/gim.2015.30.
[19]	Papp C, Beke A, Mezei G, et al. Prenatal diagnosis of Turner syndrome: report on 69 cases [J]. J Ultrasound Med, 2006, 25(6): 711-717; quiz 718-720. DOI: 10.7863/jum.2006.25.6.711.
[20]	Klopocki E, Hennig BP, Dathe K, et al. Deletion and point mutations of PTHLH cause brachydactyly type E [J]. Am J Hum Genet, 2010, 86(3): 434-439. DOI: 10.1016/j.ajhg.2010.01.023.
[21]	Maass PG, Wirth J, Aydin A, et al. A cis-regulatory site downregulates PTHLH in translocation t(8；12)(q13；p11.2) and leads to brachydactyly type E [J]. Hum Mol Genet, 2010, 19(5): 848-860. DOI: 10.1093/hmg/ddp553.
[22]	Cox JJ, Willatt L, Homfray T, et al. A SOX9 duplication and familial 46，XX developmental testicular disorder [J]. N Engl J Med, 2011, 364(1): 91-93. DOI: 10.1056/NEJMc1010311.
[23]	Matsushita M, Kitoh H, Kaneko H, et al. A novel SOX9 H169Q mutation in a family with overlapping phenotype of mild campomelic dysplasia and small patella syndrome [J]. Am J Med Genet A, 2013, 161A(10): 2528-2534. DOI: 10.1002/ajmg.a.36134.
[24]	Smyk M, Obersztyn E, Nowakowska B, et al. Recurrent SOX9 deletion campomelic dysplasia due to somatic mosaicism in the father [J]. Am J Med Genet A, 2007, 143A(8): 866-870. DOI: 10.1002/ajmg.a.31631.
[25]	Barca-Tierno V, Aza-Carmona M, Barroso E, et al. Identification of a Gypsy SHOX mutation (p.A170P) in Léri-Weill dyschondrosteosis and Langer mesomelic dysplasia [J]. Eur J Hum Genet, 2011, 19(12): 1218-1225. DOI: 10.1038/ejhg.2011.128.
[26]	Binder G, Ranke MB, Martin DD. Auxology is a valuable instrument for the clinical diagnosis of SHOX haploinsufficiency in school-age children with unexplained short stature [J]. J Clin Endocrinol Metab, 2003，88(10): 4891-4896. DOI: 10.1210/jc.2003-030136.
[27]	Benito-Sanz S, Royo JL, Barroso E, et al. Identification of the first recurrent PAR1 deletion in Léri-Weill dyschondrosteosis and idiopathic short stature reveals the presence of a novel SHOX enhancer [J]. J Med Genet, 2012, 49(7): 442-450. DOI: 10.1136/jmedgenet-2011-100678.

[1]	Houmei Han, Xuan Sheng, Dequan Liu, Yang Gao, Hong Yin. Value of ultrasound in prenatal diagnosis of fetal annular pancreas[J]. Chinese Journal of Medical Ultrasound (Electronic Edition), 2023, 20(01): 46-50.
[2]	Yan Ding, Huaxuan Wen, Mengyu Zhang, Siqi Chen, Xin Wen, Guiyan Peng, Qing Zeng, Dandan Luo, Yimei Liao, Yue Qin, Meiling Liang, Shengli Li. Prenatal ultrasound study of fetal cerebellar fissure[J]. Chinese Journal of Medical Ultrasound (Electronic Edition), 2023, 20(01): 14-22.
[3]	Shujie Zhang, Fan Jiang, Ping Luo, Lili Gu, Yuwei Gao, Nan. Zhou. Preliminary study on parameters of the choroid plexus of fetal lateral ventricle during 11 to 13⁺⁶ weeks of gestation and analysis of abnormal cases[J]. Chinese Journal of Medical Ultrasound (Electronic Edition), 2022, 19(09): 888-893.
[4]	Juan Zhang, Qingqing Wu, Li Wang, Tiejuan Zhang, Jijing Han. Value of prenatal ultrasound in differential diagnosis of mature and immature fetal sacrococcygeal teratoma[J]. Chinese Journal of Medical Ultrasound (Electronic Edition), 2022, 19(08): 796-800.
[5]	Xinmei Zhang, Huawei Zhao, Yu Xia, Yixiu Zhang, Qing Dai, Yuxin Jiang. Prenatal ultrasound characteristics and prognosis of fetal ovarian cysts[J]. Chinese Journal of Medical Ultrasound (Electronic Edition), 2021, 18(05): 444-450.
[6]	Wei Xiong, Shengli Li, Qingxiu Lin, Meifang Zhang. Pregnancy outcome and prognosis of fetal lateral ventriculomegaly[J]. Chinese Journal of Medical Ultrasound (Electronic Edition), 2021, 18(03): 290-295.
[7]	Dandan Luo, Huaxuan Wen, Guiyan Peng, Yi Lin, Yimei Liao, Meiling Liang, Yue Qin, Qing Zeng, Jing Dang, Shengli Li. Value of smart fetus technique in prenatal ultrasound examination during the second and third trimesters[J]. Chinese Journal of Medical Ultrasound (Electronic Edition), 2020, 17(11): 1061-1069.
[8]	Feifei Wang, Shiqiao Tan, Xiaodong Wang, Haiyan Yu. Ductus venosus blood flow spectrum of ultrasonic testing in perinatal period[J]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2017, 13(02): 131-134.
[9]	Hong Su, Ming Chen, Hongfeng Wang, Na Zhang, Xinhua Wu, Qi Ma, Qingxin Shen, Chengcheng Hu, Menghua Chen. Prenatal ultrasonographic diagnosis of fetal congenitally corrected transposition of the great arteries[J]. Chinese Journal of Clinicians(Electronic Edition), 2018, 12(11): 619-625.
[10]	Qiang Zhao. Research progress of genetic factors in congenital heart diseases[J]. Chinese Journal of Clinical Laboratory Management(Electronic Edition), 2017, 05(02): 74-77.

Viewed

Full text

Abstract

Please choose a citation manager

Content to export