CMA of patients’ family members - Clinical application of chromosomal microarray as a first-tie

Patients were counseled with the significance of CMA results of pathogenic CNVs, VOUS and benign CNVs and then parental and sibling testing was offered to aid further interpretation and classification. In 63 family members of 37 patients, CMA showed pathogenic CNVs in five cases (8%, 5/63), VOUS in five cases (8%, 5/63), benign CNVs in 12 cases (19%, 12/63), and no abnormalities in 41 cases (65%, 41/63) (Table 3). Eleven family members showed abnormal phenotypes, which were associated with five pathogenic CNVs (45.5%, 5/11), two VOUS (18.2%, 2/11), and one benign CNVs (9.1%, 1/11), and no abnormalities were detected in three

cases (27.3%, 3/11) (Table 3).

Table 3. The result of CMA in patients’ family members

Abbreviation: CNVs, copy number variations; VOUS, variant of uncertain clinical significance.

All pathogenic CNVs were found in family members with abnormal phenotypes (Table 4). Three pathogenic CNVs were found to be the same or similar in patients’ CMA (Table 2, patient No. 24, 34, and 36). In family group 1 (Table 4), the patient (Table 2, patient No. 24) had a 140 kb deletion at 9p23 (9,387,329-9,528,560). Similarly, the patient’s sibling had a 140 kb deletion at 9p23 (9,387,329-9,525,866), a region that includes PTPRD (protein-tyrosine phosphatase receptor-type delta, OMIM601598) gene, the deletion of which has been reported to be associated with attention deficit hyperactivity disorder and restless leg syndrome.

In family group 2 (Table 4), the patient (Table 2, patient No.34) had a 240 kb deletion at 10q23.31 (89,523,072-89,764,071), and the patient’s mother had one at 10q23.31 (89,541,765-89,764,071), a region that includes PTEN gene, which is associated with BRRS. The patient and mother showed similar clinical phenotypes but the other family members had normal phenotypes and CMA results. In family group 3 (Table 4), not only the

patient (Table 2, patient No.36), but also the mother and sibling had the same clinical features of strabismus nystagmus, and the CMA showed similar results; the patient had a 450 kb duplication at 13q14.11 43,890,050), the sibling had a 450 kb duplication at 13q14.11 (43,438,657-43,890,050), and the mother had a 430 kb duplication at 13q14.11 (43,452,829-43,890,050). As a result of the CMA, the patient (Table 2, patient No. 26) in family group 4 (Table 4) was diagnosed with Leri-Weill syndrome and his younger brother was diagnosed with DiGeorge syndrome (Table 4).

Table 4. Comparison of CMA and chromosomal genetic tests

Abbreviations: CMA, chromosome microarray; FISH, fluorescence in situ hybridization.

C. Chromosome distribution of pathogenic CNVs

The pathogenic CNVs included 17 cases of long contiguous stretches of homozygosity of >5 Mb and one case (Table 2, patient No.22) of uniparental disomy at chromosomes 4 and 14 (Table 2). Genomic alterations occurred on all chromosomes except 19, 20, and Y. The changes were found most commonly (in order of frequency) on chromosomes 2, X, 3, 10, and 21 (Fig.

2).

Fig. 2. Distribution of pathogenic CNVs on chromosomes. Filled blue triangles, gains in patients; empty blue triangles, gains in family members with abnormal phenotypes; filled red triangles, losses in patients; empty red triangles, losses in family members with abnormal phenotypes. Stars show uniparental disomy.

IV. DISCUSSION

ID, DD and MCA are heterogeneous disorders, requiring proper genetic tests for accurate diagnosis, genetic counseling for patients and their families, and additional genetic tests for the family members [2,3,14]. There are many methods for detection of genomic changes [2,3,5]. Conventional G-banded karyotype analysis was the gold standard for detection of chromosomal abnormalities, but its resolution is limited (<5–6 Mb) [5]. Fluorescence in situ hybridization (FISH) was developed in the early 1980s. FISH uses probes labeled with a fluorescent dye to detect specific DNA sequences in chromosomes; FISH is used to detect structural chromosome abnormalities.

Yet, uniparental disomy and germline mutations cannot be detected by FISH and it only detects abnormalities in the targeted regions corresponding to the probes used [5] (Table 5).

Conventional G-banded karyotype analysis only detects genomic imbalances in ~3% of patients with unexplained DD, ID, ASD, and cogeneital anomalies, whereas CMA detects genomic imbalances in 15–20%

of these patients [3,5,17]. International guidelines (ISCA, ACMG) recommend specific approaches for the use of CMA as a first-tier genetic test in DD, ID, ASD, and cogeneital anomalies [6]. Despite the growing evidence for its diagnostic yield and cost effectiveness, CMA has not yet been implemented as a first-tier diagnostic test in Korea because it is not reimbursed by the Korean National Health Insurance Service.

Park et al. documented 4073 CMA tests with prenatal cases and 1007 postnatal cases, implemented array CGH in 407 patients with ID, MR, or MCA among postnatal cases, and confirmed chromosomal abnormalities of aneuploidy, deletion, and duplication in 34 patients (8.3%) in 2011 [15]. Lee

et al. performed array CGH in 190 Korean DD/ID patients, detected abnormal results in 26 out of 190 (13.7%), and reported that array CGH showed good clinical performance in 2013 [14]. Byeon et al. reported that the conventional G-banded karyotype method detected chromosomal abnormalities in only 9/87 patients, whereas array CGH revealed chromosome imbalances in 28/87 (32.2%) patients with DD, ID, dysmorphic face, or neurologic diseases, including epilepsy, in 2014 [13]. Shin et al.

reported pathogenic CNVs in 15 patients (15.6%) among 96 patients with DD, MR or ASD, and insisted that CMA should be routinely performed for patients with DD, MR, or ASD [4] (Table 6).

Table 5. Comparison of CMA within family group and chromosomal genetic tests

Abbreviation: BRRS, Bannayan Riley Ruvalcaba Syndrome.

Table 6. Comparison of recent chromosomal microarray detection rate in Korea

Abbreviations: ASD, autism spectrum disorder; DD, developmental display;

MR, mental retardation; ID, intellectual disability; MCA, multiple congenital anomalies; CGH, comparative genomic hybridization; CNVs, copy number variations.

In this study, CMA analysis detected pathogenic CNVs in 43.8% (14/32) of cases of congenital anomaly and 25.3% (22/87) cases of neurodevelopmental disorder, which is higher than the average rate in previous reports [3,4,18,19]. The higher detection rate in this study than other reports could be explained by the high proportion of congenital anomaly among the participants. Focusing on neurodevelopmental disorders, the detection rate was 25.3%, which was similar to other reports [4,12,14,20].

Moreover, the type of array, genome coverage, and resolution could affect the detection rate. This study revealed 22 patients with a deletion, 9 patients with duplication, and 5 patients with duplication and deletion among pathogenic CNVs showing similarities with previous studies. Deletion occurs more often than random duplication in the genome and patients with duplications in their genome showed a milder phenotype than those with a deletion [3,18,21].

Pathogenic CNVs were randomly observed in all the chromosomes except 19, 20, and Y. However, duplication was observed mainly in the X chromosome (5/9, 55.6%) which differed from the study of Kaminsky et al.

that reported duplication was common in chromosomes 3, 8, 16, and 17. The small number of participants could explain the difference [2].

This study included patients for whom the CMA results had been disclosed and recommendations had been suggested in genetic counseling.

Then, CMA was performed on 63 family members of 37 patients and the results showed pathogenic CNVs in five members (8%). The comparison of CNVs found in patients and family members indicates the importance of family studies with phenotype examination and pedigree analysis for the accurate interpretation of CNVs found by the CMA test of the proband. The interpretation of VOUS can benefit from information on whether they are

inherited[5] [7]. The definition of a CNV is that at least 1 kb of DNA differs in copy number from a reference genome [6,21,22]. Not all CNVs have the same clinical significance; therefore, they are classified into pathogenic, benign, and VOUS [6,23]. However, with many genetic variants, there remains the possibility that a predicted pathogenic variant is not pathogenic and vice versa. In this study, the screening of one family (Table 4, family group 3) helped to interpret VOUS. The patient (Table 2, patient No. 36), sibling and mother had similar symptoms. CMA showed similar results of a

~450 kb duplication at 13q14.11, involving the genes ESPTI1, DNAJC15, and ENOX1; the patient’s father was normal. The clinical significance of this CNV has not yet been reported, but we interpreted it as pathogenic CNVs this family had due to an ophthalmopathy of abnormal phenotype.

In the US and Europe, CMA is a commonly used clinical diagnostic test with validated clinical utility, and is replacing conventional G-banded karyotype analysis and FISH [5,6]. Although CMA is a powerful diagnostic testing tool, screening using conventional G-banded karyotype analysis is more suitable for the detection of common aneuploidy [8,10]. FISH should be recommended for the diagnosis of well-described syndromes, such as Williams syndrome, and is a more cost-effective diagnostic test than CMA [24,25,26].

If a CMA result is normal, it does not mean that the genome is normal.

Balanced translocation or inversions and low-level mosaic cases could be missed by CMA [27,28]. Therefore, a whole-chromosome analysis technique such as conventional G-banded karyotype analysis may be considered for patients with normal CMA results.

CMA has a limitation of interpretation with VOUS. The interpretation of VOUS can be helped by further testing of family study and exome

sequencing. Family study can inform whether it is inherited from a parent or it occurred de novo in the patient.

In summary, this experience demonstrates that CMA is a powerful cytogenetic test and chromosome aberration detection application for patients with congenital anomalies, DD, ID and ASD that enable the rapid diagnosis. I suggest that the CMA test should be covered by insurance and performed widely in clinical practices, which would help diagnose DD, ID, ASD and congenital anomaly patients accurately and provide the patients and their families with proper genetic counseling.

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-국문초록-

국내 발달지연과 정신지체 환자에 대한 우선적 검사로서 염색체 마이크로에레이의 임상적 적용에 대한 연구

이 형 남

아주대학교 대학원 의학과/의학전공 지도교수 김현주, 정선용

배경: 염색체 마이크로어레이 검사(CMA)는 염색체 전체를 고해상도로 빠르고 정확하게 복제수 변이(CNV)를 조사할 수 있는 세포유전학적 진단 검사법이다. 국제적 가이드라인은 발달 장애, 지적 장애, 자폐 범주성 장애, 선천성 기형 환자들에게 우선적 임상 진단 검사로서 적합한 검사법으로 추천하고 있다. 하지만, 국내에서는 건강 보험법상 임상진단 검사로 사용할 수 없어 CMA 검사가 일반적으로 사용되고 있지는 못한 실정이다. 본 연구에서는 CMA 검사가 발달 장애, 지적

장애, 자폐 범주성 장애, 선천성 기형 등의 환자에게 우선적 진단 검사로서의 적 유용성과 한계점에 대해 연구하고자 하였다.

방법: 2012 년 12 월부터 2016 년 7 월 동안 염색체 이상이 의심되었으나 염색체 검사에서는 정상 핵형으로 관찰된 환자 119 명을 대상으로 하였다. 이중 87 명은 신경발달장애(발달지연 70 명, 지적장애 16 명, 자폐증 1 명)이고, 32 명은 발달지연이나 지적장애 증상을 가지고 있는 선천성 기형 환자였다. 환자 중 37 명은 가족 검사가 가능한 총 63 명의 환자가족들에 대해 CMA 검사를 실시하였으며, 검사결과를 바탕으로 유전상담을 시행하였다.

결과: 환자 119 명의 CMA 분석 결과 pathogenic CNV 36 명(30.3%), variants of uncertain clinical significance(VOUS) 11 명(7.6%), benign CNV 24 명(17.6%), 정상 53 명 (44.5%)으로 확인되었다. 환자 37 명의 가족들 63 명에 대한 CMA 결과는 pathogenic CNV 5 명 (8%), VOUS 5 명(8%), benign CNV 12 명(19%), 정상 41 명(65%)으로 분석되었다. 환자 가족 중 11 명은 비정상적 표현형을 보였으며 이들의 CMA 분석결과 pathogenic CNV 5 명(45.5%), VOUS 2 명(18.2%), benign CNV 1 명(9.1%), 정상 3 명(27%)로 pathogenic CNV 발견 빈도가 높았다. 환자 총 119 명중 pathogenic CNV 가 36 명으로

30.3%의 높은 빈도로 확인 되었다. 환자군에 따라 신경발달장애 환자 87 명중 22 명으로 25.3%로 확인되나 선천성 기형 환자 32 명중 14 명에서 확인되어 43.8%의 높은 비율을 확인할 수 있었다.

결론: 본 연구에서 신경발달장애 및 선천성 기형 환자에 대한 CMA 검사결과 30.3%에서 pathogenic CNVs 가 발견되었다. 비록 VOUS 결과의 해석적 한계점 등이 분명히 있지만 CMA 검사가 발달 장애, 지적 장애, 자폐 범주성 장애, 선천성 기형 등의 환자를 대상으로 한 우선적 염색체이상 검사법으로서 매우 유용하다는 것을 시사한다.

핵심어: 염색체 마이크로어레이, 복제수변이, 신경발달장애, 선천성 기형, 발달지연, 지적장애

문서에서 Clinical application of chromosomal microarray as a first-tier chromosome aberration test for patients with developmental delay and intellectual disability in Korea (페이지 20-0)