Demography and clinical characteristics
A total of 24 patients (seven females and seventeen males) from non-related families were finally diagnosed as FA, including one who has already been reported (Case 9) . The median referral age of this cohort was seven years old, and the median age of BMF onset was 4.5 years old (range, 1-15 years old). There were 23 Han Chinese and one Uyghur Chinese, and the geographical distribution spread nationwide though half of the patients came from the south or southwest of China. All patients were referred to our institute because of severe cytopenia except a thirty-year-old boy (Case 11) who was initially diagnosed as MDS for the myeloid dysplasia and increased myeloblasts indicated by BM morphology. Five patients had an indicative family history, among which, two patients had family members died from anemia (Case 4, Case 17), two patients were from consanguineous families (Case 18, Case 24), and one patient was an in vitro fertilize baby whose paternal grandmother died from pancreatic cancer (Table 1).
Fourteen (58.33%) patients were growth-retarded, and 21 (85.7%) patients manifested as congenital malformations. Congenital abnormalities in our cohort included skin pigmentation (13/24), café au lait spots (6/24), spin and limbs deformation (12/24), craniofacial malformations (8/24), genitourinary system malformations (7/24), cardiovascular system defects (2/24), nervous system diseases (2/24), and endocrine system defects (2/24) (Table 1).
Thoroughly evaluation of the hematologic phenotype is crucial to FA patients since BM dysplasia or pathological cytogenetics relate to disease progression and adverse hematopoietic stem cell transplantations (HSCT) outcomes [5,14]. Twenty-three patients’ morphologic test results and 22 patients’ cytogenetics test results before pre-HSCT conditioning regimen and/or chemotherapy were available. BM dysplasia was found in 15/23 (65.22%) patients, including one AML with the myeloblast count of 41% (Case 6) and one myelodysplasia with the blast count of 6% (Case 11). Karyotypes were described according to the International System for Human Cytogenetic Nomenclature 2013 ; at least 20 metaphases were analyzed for each assay. Cytogenetic abnormalities were found in 10/22 (45.45%) patients with clonality found in six patients, and half of the abnormal karyotypes involved chromosome 7 (-7, 7q-, or der(7)t(1;7)) (Table 2). The cytogenetic result of Case 6 who was diagnosed as AML was 46, XX, der(7)t(1;7)(q21;q36) , which was confirmed to be non-constitutional by matched peripheral blood, and the karyotype of patient Case 11 was highly complex (Table 2). All the patients with abnormal karyotypes also manifested as dysplasia on bone marrow smear or had evident blasts, suggesting the initiation of clonal evolution in the hematopoietic system.
Characteristics of mutations
A total of 44 mutations were identified involving seven different FA genes and composed of 16 missense mutations, ten large deletions, eight nonsense mutations, seven frameshift mutations, two splicing mutations, and one deep intron mutation (Figure 1, Table 3). All the large deletions were found within the FANCA gene. 21 (47.73%) mutations identified in our cohort were novel and the majority of mutations were private except FANCA c.367C>T and FANCA c.3627-607_3765+268del, which were shared by two patients separately. (Figure 2, Table 3). We did not find FANCA c.2546delC in our cohort, which accounts for over 30% FANCA mutations in Japanese and Korean patients [24,25].
Among the 24 patients,17 patients carried compound heterozygous mutations, three patients carried homozygous mutations, three patients harbored monoallelic FANCB mutations who were all males, and one patient with a heterozygous FANCE mutation were identified. Biallelic FANCA mutations caused 58.33% (14/24) of the cases, followed by monoallelic FANCB mutations which constituted 12.5% (3/24) of the cases; both FANCD2 and FANCE mutations made up to 8.33% (2/24) of the cases, and FANCC, SLX4, and ERCC4 mutations caused one case each (Table 3). We did not find any case attributed to FANCG mutations, which is the second most prevalent responsible gene in East Asian according to Japanese and Korean studies [24,25]. Despite the limited size of this cohort, we identified three FANCB mutations, making it rank one of the most common causative genes in line with the Japanese study . There were three homozygous mutations, FANCA c.1867C>T, FANCC c.545C>A, and ERCC4 c.257G>A; the latter two mutations were carried by patients both came from consanguineous families, and the FANCC c.545C>A was carried by the only Uyghur patient in our cohort.
Rigorous criteria were adopted in the process of criminal variant identification (Table 3, Table S3). Majority of the patients were assigned with compelling mutations with two exceptions. All mutations were classified as pathogenic or likely pathogenic according to the guideline of ACMG except FANCA c.2611C>G, which was harbored by Case 1. Albeit the ambiguous pathogenicity, it was rare in the general population and was the only possible responsible mutation found in this patient after an exhaustive search for variants in FA-related genes. Hence, we considered it to be the causative mutation. Case 21 carried compound heterozygous FANCE c.1111C>T mutation and FANCE c.1317-237C>G mutation. The c.1111C>T mutation was considered pathogenic, but the c.1317-237C>G mutation is an intron variant and classified as uncertain significance, therefore it was excluded in statistics.
ADLH2 rs671 genotype
14/24 (58.33%) patients in our cohort carried ALDH2-G/A genotype, and the other patients were all ALDH2-G/G genotype. There was no ALDH2-A/A genotype identified (Table 2). The age of BMF onset of ALDH2-G/A patients was significantly younger than that of the ALDH2-G/G patients (p = 0.024, t-test).
Treatment and outcome
Within the 24 patients, continuous medical records of 22 patients can be retrieved except Case 9 and Case 24, who only came to us once and were excluded in this section. All the 22 patients were eligible for HSCT for they were all transfusion-dependent, and HSCT was performed on 18 patients (81.81%). The numbers of patients accepted HSCT from HLA-matched unrelated donors (MUD), HLA-unmatched unrelated donors (UUD), HLA-haploidentical related (sibling or parental) donors (HRD), and HLA-matched related donors (MRD) were four, five, seven, and one, respectively. Another patient accepted HLA-unmatched unrelated cord blood (UUC) HSCT. The other four patients who did not undergo HSCT accepted androgen, cytokine, and/or intermittent transfusion support. All the patients with abnormal karyotype underwent HSCTs. In the HSCT subgroup, 11/18 (61.11%) were ALDH2-G/A genotype. The median follow-up duration was 33.5 months ranged from one month to 84 months. By the end of the study, eight patients (36.36%) have been dead. Seven of them were HSCT-related, mainly severe acute graft-versus-host disease (aGVHD) and/or infections, accounting for 38.89% of the subgroup. One patient who did not receive HSCT died from severe infection (Table 3).