Patient characteristics
There were 19 patients with FA (16 males and 3 females) from 17 unrelated families evaluated in this study (Fig. 1). The majority of families (12/17, 70%) were from Northern or Central Punjab. Other families were from Southern Punjab, Islamabad, Khyber Pakhtunkhwa, and Azad Kashmir. The median age at FA diagnosis was 7 years (range 4–12) and while all were evaluated for HCT, only 5 patients underwent matched sibling HCT. Eight of the 19 patients were deceased at the time of this study. The median age at death was 8.5 years (range 4–13). Pathogenic variants relevant to FA were identified in 14 families with FANCA being the most common (7/14, 50%). Homozygous variants in FA-associated genes were identified in 12 of the 14 solved families (86%) and 2 probands had compound heterozygous variants. Physical and genetic findings are listed in Tables 1 and 2, respectively.
Table 1
Clinical features of study participants with Fanconi anemia.
Patient ID† | Gender | Age at Diagnosis (Years), Vital Status | Age at and Cause of Death | Skin | Hand | Renal | Other |
1-FA | M | 7, deceased | 9, NR | | | Ectopic kidneys | |
3-FA | M | 5, alive | -- | Café au lait macules | Thumb malformation | | Short stature |
4-FA | M | 7, alive | -- | | | | |
5-FA | M | 11, deceased | 12, NR | | | | Abnormal left leg growth, short stature |
6-FA | M | 9, deceased | 10, hemorrhage NOS | Hyperpigmentation | Polydactyly | | |
7-FA | M | 7, deceased | 8, NR | | | | |
8-FA | M | 4, deceased | 4, brain hemorrhage | | Absent radii and metacarpal bones | | High arched palate, skeletal malformations NOS |
9-FA | F | 10, alive | -- | | | | |
10-FA | M | 5, deceased | 6, hemorrhagic stroke | | Polydactyly | | |
12-FA | M | 11, alive | -- | | | | |
14-FA | M | 12, deceased | 13, NR | | | | |
16-FA | F | 9, alive | -- | | Bilateral thumb malformations | | |
17_01-FA | M | 6, alive | -- | | Thumb malformation | | Immunodeficiency |
17_02-FA | M | 10, alive | -- | | Thumb malformation | | Immunodeficiency |
18-FA | M | 5, alive | -- | | Polydactyly, small right hand | Ectopic kidneys | Short stature |
19-FA | F | 8, unknown | -- | | | | |
20-FA | M | 5, alive | -- | | | | |
21_01-FA | M | 8, deceased | 13, NR | | | | Numerous rosettes with central eosinophilic material on bone marrow biopsy |
21_02-FA | M | 5, alive | -- | | | | Numerous rosettes with central eosinophilic material on bone marrow biopsy |
† All participants had bone marrow failure and were being considered or underwent hematopoietic cell transplantation. Empty boxes indicate where clinical information was not available. |
Table 2
Germline genetic variants identified in study participants with Fanconi anemia.
Gene | Patient ID | Genotype | Variant | Previously reported | ClinVar | gnomAD % MAF‡ All; South Asian ancestry (44) | Consequence of Variant§ (45–47) |
FANCA | 1-FA | Homozygous | c.3788_3790delTCT, p.Phe1263del | (12) | Pathogenic | 0.009929; 0 | In-frame deletion |
3-FA | Compound Heterozygous | g.89871674-89880557del | (14) | NR | | Exons 4–7 Deletion |
g.89861527-89863726del | NR | | Exon 11 Deletion |
4-FA | Homozygous | c.37dupC, p.Gln13Profs*24 | | NR | | Frameshift |
9-FA | Homozygous | g.89856782-89874222del | (15) | NR | | Exons 7–14 Deletion |
17_01-FA and 17_02-FA | Compound Heterozygous | c.2749C > T, p.Arg917* | (14) | Pathogenic | | Stop-gain |
g.89847600-89853759del | (17) | NR | | Exons 15–17 Deletion |
19-FA | Homozygous | c.4070C > A, p.Ala1357Asp | | NR | | Missense, MetaSVM 0.9149, REVEL 0.702, CADD phred 24.1 |
20-FA | Homozygous | c.1541C > A, p.Ala514Asp | | NR | 0.000398; 0.003266 | Missense, MetaSVM 0.876, REVEL 0.851, CADD phred 27.1 |
FANCC | 5-FA | Homozygous | c.1642C > T, p.Arg548* | (16) | Pathogenic | 0.004954; 0.01307 | Stop-gain |
8-FA | Homozygous | c.1642C > T, p.Arg548* |
FANCF | 10-FA | Homozygous | c.785T > G, p.Leu262* | | NR | 0.0007955; 0.003266 | Stop-gain |
FANCG | 18-FA | Homozygous | c.710C > G, p.Ser237* | | NR | | Stop-gain |
21_01-FA and 21_02-FA | Homozygous | c.1471_1473delAAAinsG, p.Lys491Glyfs*9 | (17) | NR | 0.0003976; 0.003266 | Frameshift |
FANCL | 6-FA | Homozygous | c.1092G > A, p.Trp341_Lys364del | (18) | NR | 0.001994; 0.01634 | Exon 13 Skipping |
7-FA | Homozygous | c.1092G > A, p.Trp341_Lys364del |
‡ % MAF in bold indicate these variants were only reported in South Asian populations. Blank cells indicate the variant was not present in gnomAD. |
§ in silico tools were used to predict the pathogenicity of missense variants. |
FANCA
Patient 1-FA presented at seven years of age with aplastic anemia which progressed to severe BMF. He had ectopic kidneys, but no other phenotypic features were reported. WES revealed a homozygous in-frame deletion in exon 38 of FANCA (c.3788_3790delTCT, p.Phe1263del, NC_000016.9:g.89807250_89807252delAGA, rs397507553, ClinVar:41003). Both parents were unaffected carriers and his sibling was wild-type. The c.3788_3790delTCT variant is the most frequently reported FANCA variant and has been observed in multiple populations throughout the world, including FA patients from Pakistan (12, 19, 61), with a particularly high prevalence in Spain and Brazil (12, 13).
Patient 3-FA had an abnormal thumb (Fig. 3A) and café au lait spots noted at birth. He also had short stature and low gonadotrophin hormone levels. FA was diagnosed by chromosome breakage on primary lymphocytes after he presented with neutropenia that progressed to severe BMF at 5 years of age. He underwent successful HLA-matched sibling donor HCT at the age of 6 years. We identified two deletions in FANCA (NC_000016.9:g.89871674_89880557del, and NC_000016.9:89861527_89863726del) affecting exons 4–7 and 11, respectively. These two deletions have been previously reported in Indian FA patient (14). The exon 11 deletion was paternally inherited, while the deletion of exons 4–7 was maternally inherited. Validation by targeted sequencing methods determined that one unaffected sibling did not carry either deletion. Another unaffected sibling was predicted to be a carrier of the exon 4–7 deletion by VS-CNV but there was insufficient DNA for sequencing validation.
Patient 4-FA presented with severe bone marrow failure at the age of 7 years. Hemophagocytosis was reported on his bone marrow biopsy but no other phenotypic information was available. He underwent successful HLA-matched sibling HCT. A homozygous FANCA frameshift variant in exon 1 (c.37dupC, p.Gln13Profs*24, NC_000016.9:g.89882986dupG) was identified by WES. One unaffected sibling was wild-type and the other was a carrier, but parental DNA was not available for analysis.
Patient 9-FA presented with neutropenia which progressed to severe BMF and was diagnosed with FA at 10 years of age. WES revealed and targeted whole gene sequencing validated a large homozygous deletion of exons 7–14 (NC_000016.9:g.89856782_89874222del). Her unaffected sister was a heterozygous carrier. This specific deletion has not been previously reported, but similar large deletions in FANCA have been reported (62).
Affected brothers 17_01-FA and 17_02-FA both presented with abnormal thumbs at birth (Fig. 3B). Small ear canals were also noted in 17_01-FA (Fig. 3C). At the ages of 6 and 10 years, respectively, they presented with severe BMF and immunodeficiency leading to an FA diagnosis. Biallelic variants in FANCA were identified by various NGS methods in both siblings (c.2749C > T, p.Arg917*, NC_000016.9:g.89831327G > A and NC_000016.9:g.89847600-89853759del). WES revealed a maternally-inherited nonsense variant in exon 28 which has been previously identified in an Indian patient with FA and other populations (rs1060501880, ClinVar:408188) (14, 61). A large deletion of exons 15–17 (NC_000016.9:g.89847600-89853759del) was detected by targeted PacBio® long-range sequencing in both affected siblings and has been previously reported in other patients with FA (17). This deletion was not detected in DNA from father’s peripheral blood, but relatedness analyses confirmed paternity with large regions of homozygosity being consistent with offspring from a consanguineous relationship between third-degree relatives. Additionally, analyses of single nucleotide polymorphisms (SNP) in the FANCA locus provided evidence for a possible a genotypic reversion in the paternal hematopoietic stem cells or paternal inheritance as a result of gonadal mosaicism. Both such occurrences have been previously reported in patients with FA (6, 63–65).
Patient 19-FA presented at 8 years of age with neutropenia that progressed to severe BMF by age 9 years. A homozygous FANCA missense variant in exon 41 (c.4070C > A, p.Ala1357Asp, NC_000016.9:g.89805638G > T) was identified by WES. Her unaffected brother is a heterozygous carrier, but parental DNA was not available. FANCA p.Ala1357Asp is not present in gnomAD and is predicted deleterious by in silico tools (MetaSVM score = 0.915, REVEL = 0.702, CADD phred = 24.1).
Patient 20-FA presented with moderate aplastic anemia that progressed to severe BMF by 5 years of age. We identified a homozygous missense variant in FANCA (c.1541C > A, p.Ala514Asp, NC_000016.9:g.89849440G > T, rs1432656621). His unaffected sibling is a heterozygous carrier. Although not previously reported, this missense variant is rare in gnomAD at a MAF of 0.0003977% and is predicted deleterious by in silico tools (MetaSVM score = 0.876, REVEL = 0.851, CADD phred = 27.1).
FANCC
Two unrelated probands, 5-FA and 8-FA, were homozygous for the same FANCC variant (c.1642C > T, p.Arg548*, NC_000009.11:g.97864024G > A). Relatedness analyses determined that these probands were from distinct families. FANCC p.Arg548* (rs104886457, ClinVar:12047) has been previously reported in two FA patients from Pakistan (16).
Patient 5-FA presented with moderate aplastic anemia and progressed to severe BMF at the age of 11 years. He also had short stature and abnormal left leg growth. He had two brothers and one sister who died due to similar complications but without a diagnosis. His two surviving unaffected siblings and parents are all heterozygous carriers.
Patient 8-FA presented with moderate aplastic anemia and progressed to severe BMF by 4 years of age. Skin hyperpigmentation, bone deformities including the absence of metacarpals, thumbs, and radii, and a high arched palate were also reported. He died at the age of 4 years due to a brain hemorrhage before HLA-matched sibling HCT could be performed. One unaffected sibling is a carrier, but parental DNA was not available.
FANCF
Patient 10-FA was homozygous for nonsense variant in FANCF (c.785T > G, p.Leu262*, NC_000011.9:g.22646572A > C, rs368067979). He was diagnosed with FA at 6 years of age when aplastic anemia progressed to severe BMF. He also had polydactyly and died from a hemorrhagic stroke shortly after his FA diagnosis. Parental DNA was not available and the sibling available for testing was not a carrier.
FANCG
Patient 18-FA had an extra digit, a small right hand, short stature, and ectopic kidneys. He was diagnosed with FA at 5 years of age and underwent HCT from his HLA-matched sister for severe BMF and is doing well. A homozygous nonsense variant in exon 6 of FANCG was identified by WES (c.710C > G, p.Ser237*, NC_000009.11:g.35077035G > C). His sibling is wild-type at this locus. The only parent who was available for testing was heterozygous for this loss of function variant.
Proband 21_01-FA and his brother 21_02-FA were diagnosed with FA at the ages of 8 and 5 years, respectively. 21_01-FA had pancytopenia that progressed rapidly following his diagnosis with FA and he died due to a brain hemorrhage. Currently, 21_02-FA does not have cytopenias. The bone marrow of both FA-affected brothers was reported to have numerous rosettes with central eosinophilic material surrounded by small cells seen in a background of fibrosis. The affected brothers have a homozygous frameshift variant in exon 11 of FANCG (c.1471_1473delAAAinsG, p.Lys491Glyfs*9, NC_000009.11:g.35075283_35075285delTTTinsC, rs1018027137). One of their unaffected siblings was heterozygous for this variant. This variant has been previously reported in a heterozygous patient with FA (17).
FANCL
Unrelated probands 6-FA and 7-FA were both homozygous for a recently identified FANCL South Asian founder variant. Donovan et al. established this single nucleotide variation (NC_000002.11:g.58387243C > T) induces aberrant mRNA splicing to skip exon 13 (c.1021_1092del, p.Trp341_Lys364del, rs577063114), resulting in a 24 amino acid deletion from the RING domain of FANCL (18). The genomAD MAF of this variant is 0.001994% in all populations and 0.01634% in South Asian populations.
A pairwise comparison between cases 6-FA and 7-FA was performed to assess potential relationships. A genotype comparison on approximately 7300 common SNPs between the probands and their siblings showed no indication of relatedness between families 6-FA and 7-FA. Parental sequencing data was not available.
Patient 6-FA had an extra thumb and areas of skin hyperpigmentation. He presented with aplastic anemia that progressed to severe BMF at the age of 9 years. Although he was treated with androgens while awaiting an HLA sibling matched HCT, he died at 10 years of age due to an unspecified hemorrhage. Patient 7-FA was diagnosed with FA after presenting with severe BMF at the age of 7 years old. He died shortly after his diagnosis at the age of 8 years due to an unreported cause.
Gene Unknown Families
Rare heterozygous variants in FA pathway genes identified in probands 12-FA, 14-FA, and 16-FA are reported in Supplementary Table 2. These individuals had chromosome breakage testing consistent with FA. Proband 12-FA had no dysmorphology but was diagnosed with FA at the age of 11 years after presenting with BMF and underwent a successful HLA-matched sibling HCT. No rare deleterious variants were identified in the 22 FA-associated genes.
FA was diagnosed in proband 14-FA at 12 year of age and he died 1 year after diagnosis due to an unreported cause. 14-FA was a heterozygous carrier for a variant of uncertain significance (VUS) in FANCN and a likely benign FANCO variant.
Bilateral thumb malformations were noted at birth in proband 16-FA. She was diagnosed with aplastic anemia at age 9. She underwent a successful HLA-matched sibling HCT. Heterozygous VUS were present in FANCA, FANCD2, FANCI, and FANCP. The FANCP variant (c.2209C > T, p.Arg737Cys, NC_000016.9:g.3642818G > A, rs140706384) may be deleterious as it has a REVEL score of 0.449 and CADD score of 26.2, but the MetaSVM score was predicted as tolerated; additional functional studies are required to determine potential pathogenicity. There were no other deleterious variants or large CNV events detected in FANCP, so this patient remains gene unknown.
Variants in patients with FA from South Asia and the Middle East
Figure 2A is a heat map showing the distribution of studies published from South Asia and the Middle East on the genetic etiology of FA. Reports from Turkey, India, and Pakistan were the most common, followed by Iran and Saudi Arabia. Figure 2B describes the variants reported to cause FA in multiple populations in South Asia and the Middle East. The majority of genetic studies of patients with FA from South Asia and the Middle East focused on targeted sequencing efforts for FANCA (10 out of 29 studies).
The majority of reported variants occurred in FANCA and were private to their respective populations. All large deletions, SNPs, and small insertion/deletion variants reported in patients with FA in these regions can be found in Supplementary Tables 3, 4, and 5, respectively. The only large deletions reported were in FANCA, similar to our findings and consistent with others. We also identified the recently identified FANCL founder mutation (c.1092G > A, p.Trp341_Lys364del) in 2 families from Pakistan (18).