Table 2 Genetic finding in patients with 21OHD-CAH.

From: Genetic diagnosis of CYP21A2-related CAH: adaptive sampling long-read sequencing is an accurate and scalable solution

sample ID

Test or validation

Allele

AS-LRS genotype

Phases by AS-LRSa

Variant class b

Diagnosis Confirmed

Difference to Previous test

Previous test methode

Allele type (Fig. 2)

allele no. d

CAH01

Testing

1:

gene deletion

2

C5

yes

-

hotspot

J

19

2:

gene/pseudogene chimeraf

C5

-

H

8

CAH02

Testing

1:

gene deletion

2

C5

no; carrier

-

hotspot

E

18

2:

-

-

-

A

0

CAH03

Testing

1:

gene deletion

2

C5

yes

-

hotspot

J

19

2:

c.955C>T

C5

-

B

12

CAH04

Testing

1:

c.92C>T

2

C5

yes

-

hotspot

B

17

2:

c.955C>T

C5

-

B

12

CAH05

Testing

1:

c.844G>T

2

C5

yes

-

hotspot

F

3I

2:

c.955C>T

C5

-

B

12

CAH06

Testing

1:

c.293-13C>G

2

C5

yes

-

NR

B

16

2:

gene deletion

C5

-

J

21

CAH07

Testing

1:

gene deletion

2

C5

yes

-

S + M

J

19

2:

c.1451_1452delinsC

C5

-

B

9

CAH08

Testing

1:

gene deletion

2

C5

yes

-

hotspot

E

21

2:

gene deletion

C5

-

J

18

CAH09

Testing

1:

gene deletion

2

C5

yes

-

S + M

J

19

2:

c.1088C>T

C5

-

B

11

CAH10

Testing

1:

c.92C>T

2

C5

yes

-

S + M

B

17

2:

gene deletion

C5

-

J

19

CAH11

Testing

1:

gene deletion

2

C5

yes

-

hotspot

D

1I

2:

c.844G>T

C5

-

F

3I

CAH12

Testing

1:

gene deletion

1

C5

yesc

-

S + M

J

19

2:

gene deletion

C5

-

J

19

CAH13

Testing

1:

c.293-13C>G

2

C5

yes

-

S + M

B

16

2:

c.844G>T

C5

-

F

3I

CAH14

Testing

1:

c.518T>A

2

C5

yes

-

hotspot

B

15

2:

c.518T>A

C5

-

B

15

CAH15

Testing

1:

c.293-13C>G

2

C5

yes

-

SS

B

16

2:

gene/pseudogene chimeraf

C5

-

H

7

CAH16

Testing

1:

gene deletion

2

C5

yes

-

NR

J

19

2:

c.518T>A

C5

-

B

15

CAH17

Testing

1:

gene deletion

2

C5

yes

-

NR

J

19

2:

c.518T>A

C5

-

B

15

CAH18

Validation

1:

c.293-13C>G

2

C5

yes

-

S + M

B

16

2:

c.518T>A

C5

-

B

15

CAH19

Validation

1:

c.293-13C>G

1

C5

yesc

-

S + M

B

16

2:

c.293-13C>G

C5

-

B

16

CAH20

Validation

1:

c.92C>T

2

C5

yes

-

S + M

B

17

2:

c.518T>A

C5

-

B

15

CAH21

Validation

1:

c.293-13C>G

2

C5

yes

-

S + M

B

8

2:

gene deletion

C5

-

I

22

CAH22

Validation

1:

gene deletion

2

C5

no, C5 and C3

-

S + M

J

21

2:

c.1087G>A

C3

-

B

0

CAH23

Validation

1:

c.518T>A

2

C5

yes

-

S + M

B

15

2:

c.923dup

C5

-

B

13

CAH24

Validation

1:

c.293-13C>G

2

C5

yes

-

S + M

B

16

2:

c.293-13C>G

C5

-

B

16

CAH25

Validation

1:

c.92C>T

2

C5

yes

-

S + M

B

17

2:

c.293-13C>G

C5

-

B

16

CAH26

Validation

1:

c.92C>T

2

C5

yes

-

S + M

B

17

2:

c.293-13C>G

C5

-

B

16

CAH27

Validation

1:

c.92C>T

2

C5

yes

-

S + M

F

20

2:

c.(518T>A;844G>T)

C5

-

F

2I

CAH28

Testing

1:

gene/pseudogene chimeraf

2

C5

yes

p.Pro31Leu

hotspot

G

6

2:

gene deletion

C5

p.Pro31Leu

J

19

CAH29

Testing

1:

c.293-13C>G

2

C5

yes

-

hotspot

B

16

2:

gene deletion

C5

c.239-13C>G

J

19

CAH30

Testing

1:

c.188A>T

2

C5

yes

Novel in AS-LRS

hotspot

B

14

2:

gene/pseudogene chimeraf

C5

Deletion

G

5

CAH31

Testing

1:

c.518T>A

2

C5

yes

-

hotspot

B

15

2:

c.1360C>T

C5

Novel in AS-LRS

B

10

CAH32

Testing

1:

gene/pseudogene chimeraf

2

C5

yes

Deletion

S + M

G

4

2:

c.844G>T

C5

-

F

3I

CAH33

Testing

1:

c.293-13C>G

2

C5

yes

-

NR

B

16

2:

duplication of pseudogene, deletion of gene

C5

Deletion

C

4

CAH34

Testing

1:

c.293-13C>G

2

C5

yes

Uncertain

SS

B

16

2:

gene/pseudogene chimeraf

C5

Uncertain

H

7

  1. aNo of alleles possible to phase from AS-LRS data. 1 phase resembles identical alleles.
  2. b Variant classification is based on ACMG guideline [14].
  3. c homozygosity for the same pathogenic allele was detected. As AS-LRS does not have allele dropout, we consider these cases solved as well.
  4. d Numbers correspond to alleles presented graphically in supplementary Fig. S1.
  5. eThe previous test methods were either targeted testing for 8 common C4/C5 variants (hotspot), Sanger sequencing of the entire region (SS), or both Sanger sequencing and MLPA (S + M). In some of the early reports, the laboratory method was not reported (NR), and it was not possible to identify. Historically, the presence of an 8 bp deletion in the PCR product that should represent the gene was reported as 8 bp deletion in CYP21A2, although it may have been difficult to distinguish if this was due to chimeras or gene conversions.
  6. fBy Sanger sequencing of long-range PCR products, gene/pseudogene chimera may be detected as the presence of CYP21A1P-like variants in PCR fragments specific for the CYP21A2 instead of chimeras. The abnormal allele was detected, but the genetic background may be inaccurate. Hence, we use the term gene/pseudogene chimera for variant annotation.
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