The flowchart showing research steps including variant screening and follow-up study is presented in Figure 2. A total of 8851 secondary variant counts classified as DM or DM? in the HGMD database or P or LP in the ACMG guidelines in 1330 participants were obtained after variant filtering step 1. These secondary variants included 2777 DM variant counts, 5986 DM? variant counts, 28 P variant counts and 537 LP variant counts. The total number of variants was 450, consisting of 186 DM variants, 208 DM? variants, 20 P variants and 203 LP variants (Figure 3a). After variants were filtered in step 2 according to allele frequency, variant type and inheritance mode, there were 998 variant counts including 493 DM variant counts, 422 DM? variant counts, 22 P variant counts and 399 LP variant counts. The total number of variants was 297, with 149 DM variants, 94 DM? variants, 16 P variants and 181 LP variants (Figure 3b).
Characteristics of the included variants
These 297 pass-filter variants in 638 individuals were included in the follow-up study. These variants were in 48 genes among 59 ACMG genes related to secondary findings (Table 1). The top six most frequently observed secondary variants in genes were BRCA2 (86 individuals, 13.5%), APOB (57 individuals, 8.9%), DSP (49 individuals, 7.7%), MYBPC3 (41 individuals, 6.4%), MYH7 (40 individuals, 6.3%) and RYR2 (40 individuals, 6.3%).
We classified these secondary variants into four categories according to gene-associated phenotypes: variants in genes associated with oncogenic diseases, variants in genes associated with cardiogenic diseases, variants in genes associated with connective tissue diseases, variants in genes associated with hypercholesterolemia, and others. The number of individuals carrying secondary variants from maximum to minimum were oncogenic disease (369 individuals, 57.8%), cardiogenetic diseases (322 individuals, 50.5%), hypercholesterolemia (92 individuals, 14.4%), connective tissue diseases (74 individuals, 11.6%) and others (malignant hyperthermia susceptibility, ornithine transcarbamylase deficiency) (41 individuals, 6.4%) (Table 1, Figure 4a). The order is similar to the number of secondary variants identified in each category (Figure 4b). The top three/four most common variants in genes related to different disease categories separately were DSP, MYBPC3, MYH7 and RYR2 (cardiogenic diseases); FBN1, COL3A1, and MYH11 (connective tissue diseases); APOB, LDLR, and PCSK9 (hypercholesterolemia); BRCA2, TSC1, and APC (oncogenic diseases) and RYR1, CACNA1S, and OTC (others) (Table 1).
A comprehensive follow-up study was performed in 638 individuals and their family members. Among these participants, 105 people were lost to follow-up (38 people could not be contacted, 67 people refused to participate in the study), and 532 people finally took part in the follow-up study including family members of 420 participants with hereditary secondary variants and 112 participants with de novo secondary variants. The detailed screening strategies to identify individuals with convincing clinical evidence in the follow-up study is shown in Figure 5. Examplified by follow-up study of participants carrying MPBPC3 variants, strategies including primary medical evaluation and secondary medical evaluation were performed to identify individuals with convincing clinical evidence (Figure 5). Based on the clinical features of different genes associated with secondary findings, 13 families with sufficient clinical evidence were found in the follow-up study while no participants with de novo variants presented convincing clinical manifestation (Table 1).
Sanger sequencing was conducted to validate variants in families with convincing clinical evidence. There was 1 family who refused Sanger sequencing (Table 2). After Sanger sequencing of the 12 families, 10 families with both convincing clinical and genetic evidence were detected (Table 3). 2 variants did not cosegregate with disease phenotypes in 2 families (Table 2). Among these final identified individuals having clinically and genetically confirmed secondary variants, 8 individuals were from the WES 1330 cohort. Therefore, the frequency of the secondary variants with both convincing clinical and genetic evidence in our cohort was 0.6% (8/1330).
Among the 10 families with convincing clinical and genetic evidence, there were 2 families carrying the same COL3A1 variant c.3133G>A. Two families carried two different APOB variants (c.1342G>A, c.35_39del), and 2 families carried two different BRCA1 variants (c.811G>A, c.427G>A). TNNT2 c.422G>A, MSH2 c.2197G>A, MYBPC3 c.2543C>T, and BRCA2 c.1832C>G were each observed in one family separately. In these 9 validated disease-associated variants, 6 variants (TNNT2 c.422G>A, APOB c.1342G>A, MSH2 c.2197G>A, MYBPC3 c.2543C>T, BRCA2 c.1832C>G, BRCA1 c.811G>A) were classified as DM/LP, 2 variants (COL3A1 c.3133G>A, BRCA1 c.427G>A) were classified as DM?/VUS, and 1 variant (APOB c.35_39del) was classified as LP. The gene-associated genotype categories were cardiogenic diseases (TNNT2-related dilated cardiomyopathy, MYBPC3-related hypertrophic cardiomyopathy); connective tissue diseases (COL3A1-related Ehlers-Danlos syndrome-vascular type); hypercholesterolemia (APOB-familial hypercholesterolemia); and oncogenic diseases (BRCA1 or BRCA2-related hereditary breast and ovarian cancer, MSH2-related Lynch syndrome).
Detailed clinical and genetic evidence of the 9 secondary variants in 10 families are shown as follows along with penetrance calculation of variants in families based on age at onset of gene-associated diseases.
TNNT2 c.422G>A was identified in the mother of the proband with microcephaly (III:2) by trio-WES in family 1. The heterozygous variant was validated by Sanger sequencing in 5 family members who agreed to do the test. Among them, the variant was detected in 4 family members (II:1, II:3, III:2, III:3) and was not found in 1 family member (IV:1) (Figure 6-7). The diagnosis of dilated cardiomyopathy was made by first-line imaging test echocardiography13. The sister of the grandmother of the proband (II:1) was previously diagnosed with dilated cardiomyopathy by echocardiography (Figure 8a). The grandmother of the proband (II:3) often suffered from chest tightness and dizziness, especially after exercise. Echocardiography showed dilated cardiomyopathy (Figure 8b). The mother of the proband (III:2) had occasional chest distress and palpitation, but she did not seek medical advice from the doctors. After we reported the variant to her, she came to the hospital and underwent echocardiography examination. Dilated cardiomyopathy was presented as a result (Figure 8c) (Table 4). As a gene related to child/adult-onset disease, the penetrance of TNNT2 c.422G>A was 3/4 in family 1.
Trio-WES was performed, and APOB c.1342G>A was observed in the mother and the proband who presented with developmental and epileptic encephalopathy (III:3, IV:1). Sanger sequencing revealed the heterozygous variant in 6 family members (II:1, II:2, II:6, III:1, III:3, IV:1) who wanted to undergo the test (Figure 9-10). The diagnosis was made according to the Dutch Criteria for Familial Hypercholesterolemia14. Two family members (I:2, II:1) died of cerebral infarction accompanied by hyperlipidemia at the ages of 71 and 69. The grandfather of the proband had hyperlipidemia with serum total cholesterol (TC) 7.68 mmol/L and low-density lipoprotein cholesterol (LDL-C) 4.97 mmol/L. The younger brother of the grandfather of the proband (II:6) had facial paralysis after cerebral infarction at the age of 50. The TC was 9.22 mmol/L, and LDL-C was 5.04 mmol/L in the recent blood lipid test. The older brother of the mother of the proband (III:1) had hyperlipidemia (TC 7.91 mmol/L, LDL-C 5.17 mmol/L) and had fatty liver detected by abdominal ultrasound examination. The mother of the proband (III:3) also had hyperlipidemia with TC 8.18 mmol/L and LDL-C 5.46 mmol/L (Table 4). As the age at onset of APOB-related familial hyperlipidemia was child/adult, the penetrance of APOB c.1342G>A in family 2 was 5/6.
APOB c.35_39del was identified in the mother of the proband with microcephaly (III:2) after trio-WES. The heterozygous variant was validated in four family members by Sanger sequencing. Among them, three family members carried the variant (II:2, II:3, III:2), and 1 family member (IV:1) did not (Figure 11-12). Familial hypercholesterolemia was diagnosed according to Dutch criteria14. The father of the grandmother of the proband (I:1) was diagnosed with coronary heart disease and hypercholesterolemia 20 years ago. The grandmother (II:2), the brother of grandmother (II:3) and the mother of the proband (III:2) all had hypercholesterolemia with high TC and LDL-C (TC 8.05 mmol/L LDL-C 5.39 mmol/L; TC 7.72 mmol/L LDL-C 4.98 mmol/L; TC 7.99 mmol/L LDL-C 5.51 mmol/L) (Table 4). According to the age at onset of APOB-related familial hyperlipidemia (child/adult), the penetrance of APOB c.35_39del was 3/3 in family 3.
MSH2 c.2197G>A was detected in the father of the proband with microcephaly (III:1) by trio-WES. Three family members (II:1, II:4, III:1) agreed to undergo Sanger sequencing, and all of them were confirmed to carry this heterozygous variant (Figure 13-14). Lynch syndrome was diagnosed by Amsterdam criteria15. The grandmother of the proband died of rectal cancer at the age of 65. The brother of the grandmother (II:4) and father of the proband (III:1) had rectal cancer at the ages of 62 and 41, respectively (Figure 15a-b) (Table 4). The penetrance of adult-onset MSH2-associated Lynch syndrome was 2/3.
After WES sequencing of trios, the proband with microcephaly (IV:3) and mother (III:5) of family 1 carried the secondary heterozygous variant COL3A1 c.3133G>A. Sanger sequencing was applied to the family members who agreed to perform the test. Five people carried the variant (III:2, III:3, III:5, IV:2, IV:3), and 2 people (III:1, IV:1) did not (Figure 16-17). The diagnosis for Ehlers-Danlos syndrome-vascular type was made by clinical features and COL3A1 mutations16,17. The brother of the proband’s grandfather (II:1) reported a history of inguinal hernia. The grandfather of the proband (II:2) had an acute onset severe headache at the age of 47, and subarachnoid hemorrhage was detected by brain tomography angiography (CT) (Figure 18). Brain computed tomography angiography (CTA) was performed, and an aneurysm of the internal carotid system was identified (Figure 19a). Surgical interventions were conducted. The younger sister of the mother (III:3) had headache of unknown reason at the age of 28. Due to the cerebral aneurysm hemorrhage of her father, brain CTA was performed, and an internal carotid system aneurysm was found (Figure 19b). After being informed that he carried the same variant as his family members, the asymptomatic brother of the mother (III:2) was suggested to undergo brain CTA, and an aneurysm of the internal carotid system was also identified (Figure 19c). The mother and sister of the proband showed dermatopathological features of Ehlers-Danlos syndrome-vascular type. The mother of the proband (III:5) had a brown-black plaque above the left ankle manifesting atrophic skin and ulcers (Figure 20a) and ecchymosis on the left side of the knee (Figure 20b). The sister of the proband (IV:2) presented with scar hyperplasia surrounded by hypopigmented pityriasis versicolor-like lesions over the knee (Figure 20c) (Table 4). The penetrance was 4/5 based on child/adult age at onset of COL3A1-related Ehlers-Danlos syndrome-vascular type.
The WES of trios in family 2 identified the same heterozygous variant COL3A1 c.3133G>A in the proband with developmental delay (IV:1) and her mother (III:2). Sanger sequencing validated the variant in 4 family members. The diagnosis of Ehlers-Danlos syndrome-vascular type was made as stated in family 5. Three family members (II:5, III:2, IV:1) carried the variant, and 1 family member (III:3) did not (Figure 21-22). The younger brother of the grandfather of the proband died of acute myocardial infarction at the age of 22 (II:4). The grandfather (II:5) and mother (III:2) of the proband had a history of gastric perforation (Table 4). The penetrance was 2/3 based on child/adult age at onset of COL3A1-related Ehlers-Danlos syndrome-vascular type.
Trio-WES results showed that the father of the proband with microcephaly (IV:1) carried the heterozygous MYBPC3 c.2543C>T in family 7. Three family members of family 7 underwent Sanger sequencing. Two family members were verified to carry the variant (III:1, IV:1), and 1 family member (V:1) was not (Figure 23-24). The European Society of Cardiology (ESC) on the diagnosis and management of hypertrophic cardiomyopathy was used as a diagnostic criterion18. Four family members died suddenly. Among them, two family members (I:2, II:3) had sudden death before 40 with unknown cause. Two family members (II:1 and III:3) died of cardiac arrest at the ages of 45 and 38, respectively. The grandfather of the proband (III:1) was admitted to the hospital due to shortness of breath and chest tightness, and echocardiography detected features of hypertrophic cardiomyopathy (Figure 25a). The asymptomatic father of the proband (IV:1) was advised to undergo echocardiography after she was informed that she was a variant carrier. Echocardiography also showed evidence of hypertrophic cardiomyopathy (Figure 25b) (Table 4). The penetrance of MYBPC3 c.2543C>T in family 7 was 2/2 (child/adult onset).
BRCA2 c.1832C>G was identified by trio-WES in the mother of the proband who presented with developmental delay (III:2). The heterozygous variant was validated by Sanger sequencing in 5 family members, including 4 carriers (II:2, II:3, III:2, III:3) and 1 noncarrier (II:4) (Figure 26-27). One of the family members died of breast cancer at the age of 55. Pathological examination proved that two members had breast cancer in the left breast (II:3, III:2) (Figure 28a-b) (Table 4). The penetrance of the variant was 2/4 in total and 2/3 in females in family 8 according to adult-onset BRCA2-associated breast cancer.
BRCA1 c.811G>A was detected in the proband with microcephaly by WES. Sanger sequencing was performed on 4 family members who agreed to the test. Three family members (I:2, II:1, II:2) were carriers of the variant, and 1 family member (II:4) was a noncarrier (Figure 29-30). Breast cancers were diagnosed histologically in two family members (I:2, II:2) (Figure 31a-b) (Table 4). Due to the adult onset of BRCA1-related breast cancer, the penetrance was 2/3 in total and 2/3 in females in family 9.
BRCA1 c.427G>A was identified in the mother (III:3) and the proband who presented with developmental and epileptic encephalopathy (IV:1) by trio-WES. Sanger sequencing validated the variant in four family members. Among them, 3 family members (II:2, III:1, III:3) carried the variant, and 1 family member (II:3) did not (Figure 32-33). Pathological findings proved that the three family members had breast cancer in the right breast (Figure 34a-b) (Table 4). The penetrance of the variant was 3/3 according to adult-onset BRCA1-related breast cancer.