[CIS PIDD] [cis-pidd] SCID newborn screening case

Mon Nov 3 09:01:10 EST 2014

Another thought - ZAP70 is not on your targeted sequencing list and might be worth sending as well.

Mike

From: Karin Chen [karin.chen at hsc.utah.edu]
Sent: Friday, October 31, 2014 5:48 PM
To: CIS-PIDD
Subject: [cis-pidd] SCID newborn screening case

Dear All:

We identified a 2 month old with T-B+NK+ SCID detected by TREC newborn screening and would like to know if there are any reasons to delay or NOT move forward with bone marrow transplant (could this child's immune system become 'normal' over time?).

We may be over-thinking this case:

Quick summary:

-TREC # at birth: 0

-currently healthy, in protective isolation, no Omenn syndrome or other autoimmune features, no dysmorphic features

-In recent weeks, CD3+ T cell # increased from 7/uL (5 days old) to 162/uL (6 weeks old) although these are almost all CD4+CD45RO+ T cells.

-lymphocyte mitogen proliferation NORMAL

-research-based T cell proliferation: decreased proliferation to anti-CD3/anti-CD28

-CD4+CD25+CD127dim natural Tregs: 17.2% (of CD4+ population); also with a detectable population of activated T cells (CD25+CD127high CD4+)

-TCR V-beta by flow cytometry with fairly diverse population, no clear oligoclonality

-lymphoma/leukemia flow markers: no monoclonality

-NO maternal chimerism

-IgA and IgM levels are NORMAL (B cell count also normal)

-unclear if thymus is detectable by ultrasound or chest x-ray

-thymic emigrant flow obtained this week CD4+CD45RA+CD31+ --> only 1 cell/uL

-no dysmorphic features to suggest DiGeorge, normal calcium levels'

-Next-gen targeted mutation sequencing of 19 SCID genes only yielded one heterozygous mutation in FOXN1 (c.1418delC, p.Pro473fs) of unclear significance. Child has plenty of hair and no nail defects.  More details of sequencing  and laboratory results BELOW.

The diversity of the CD4+ T cells, somewhat normal function, as well as normal IgM and IgA are giving us second thoughts on the phenotype, but clearly there does not seem to be any thymic output. We plan to perform whole genome sequencing in near future, although this will not delay potential treatment plans.

Thank you for your opinions,

Karin

Karin Chen, MD
Assistant Professor
Department of Pediatrics
Division of Allergy, Immunology & Rheumatology
University of Utah
karin.chen at hsc.utah.edu

Detailed labs below if you are inclined to review:

Lymphocyte subsets 9/9/14 and 9/5/14 (1st week of life), respectively:

CD4, Abs Count [https://help2-lp.co.ihc.com/ResultsReview/images/infoTrans.gif]         1580-4850       cells/uL        3 L     1 L
Absolute CD45RO [https://help2-lp.co.ihc.com/ResultsReview/images/infoTrans.gif]        50-1500         cells/uL        12 L    2 L
Absolute CD45RA [https://help2-lp.co.ihc.com/ResultsReview/images/infoTrans.gif]        200-3400        cells/uL        12 L    5 L
Absolute CD8 [https://help2-lp.co.ihc.com/ResultsReview/images/infoTrans.gif]   680-2470        cells/uL        4 L     1 L
Absolute CD19 [https://help2-lp.co.ihc.com/ResultsReview/images/infoTrans.gif]          430-3300        cells/uL        508     382 L
Absolute Natural Killer Cells [https://help2-lp.co.ihc.com/ResultsReview/images/infoTrans.gif]          80-340  cells/uL        364 H   223
Absolute CD3 [https://help2-lp.co.ihc.com/ResultsReview/images/infoTrans.gif]   2170-6500       cells/uL        11 L    7 L
CD2, Abs Count [https://help2-lp.co.ihc.com/ResultsReview/images/infoTrans.gif]         3800-5300       cells/uL        483 L   227 L
Absolute HLA-DR [https://help2-lp.co.ihc.com/ResultsReview/images/infoTrans.gif]        430-3300        cells/uL        651     396

10/16/14

Lymphocyte Antigen and Mitogen Panel (by tritiated thymidine incorporation) :
Low Lymphocyte responses to Candida
Normal Lymphocyte responses to Tetanus
Normal Lymphocyte responses to PHA
Normal Lymphocyte responses to Con A
Normal Lymphocyte responses to Pokeweed Mitogen

10/20/14
IgG 634 IgM 32 IgA 35 (receiving IVIG)

Lymphocyte subsets (absolute counts) at 6 weeks of age
CD3 162
CD4 150
CD4+CD45RA+ 12
CD4+CD45RO+ 157
CD8+ 19
CD19+ 809
NK cells 200

V-beta TCR repetoire:
Number Of Markers 27 markers
TCR V-Beta 1 3.64 % 1.90-4.67
TCR V-Beta 2 3.06 % L 4.16-8.91
TCR V-Beta 3 1.59 % 0.64-8.22
TCR V-Beta 4 1.32 % 1.08-3.76
TCR V-Beta 5.1 2.91 % L 3.77-7.39
TCR V-Beta 5.2 1.48 % 0.87-2.27
TCR V-Beta 5.3 0.97 % 0.73-4.57
TCR V-Beta 7.1 1.18 % L 1.28-4.33
TCR V-Beta 7.2 1.57 % 0.02-3.12
TCR V-Beta 8 4.49 % 2.78-8.56
TCR V-Beta 9 0.88 % L 2.62-4.15
TCR V-Beta 11 1.49 % 0.81-1.50
TCR V-Beta 12 4.21 % H 1.08-1.93
TCR V-Beta 13.1 4.79 % 2.62-9.42
TCR V-Beta 13.2 1.72 % 1.31-6.55
TCR V-Beta 13.6 1.92 % 1.29-2.50
TCR V-Beta 14 2.88 % 2.75-4.45
TCR V-Beta 16 7.06 % H 0.47-2.91
TCR V-Beta 17 2.35 % L 3.45-7.88
TCR V-Beta 18 1.31 % 0.55-1.81
TCR V-Beta 20 11.82 % H 1.19-4.60
TCR V-Beta 21.3 3.28 % H 1.69-2.56
TCR V-Beta 22 2.72 % 2.46-4.62
TCR V-Beta 23 1.00 % 0.50-1.47
Interpretation See Note
Flow cytometric analysis of the peripheral blood specimen
indicates that 26% of the leukocytes are lymphocytes, and 14% of
the lymphocytes are T-cells. Analysis of T-cell antigen receptor
variable beta chain (TCR-VB) expression by the total T-cells
(percentages shown), in conjunction with CD3, CD4, and CD7, show
TCR-VB expression levels close to or within the normal range.
When analyzed separately, the individual CD4 positive T-cell
subsets (CD3+, CD4+, CD7+, 55% of T-cells), and (CD3+, CD4+,
CD7-, 30% of T-cells) also show TCR-VB expression levels close
to or within the normal range. CD4 negative T-cells are too few
in number to accurately assess TCR-VB expression. The viability
of the specimen is 96
IMPRESSION:
No evidence of a monoclonal T-cell population using TCR-VB
analysis in conjunction with analysis of CD3, CD4, and CD7.
These results cannot completely rule out the possibility of a
small T-cell clone being present.

10/22/14
Chimerism studies: No maternal engraftment detected.

10/27/14
Leuk/Lymph Phenotype, Impression See Note
Number Of Markers 22 markers
Leuk/Lymph Phenotype, % CD2 31 %
Leuk/Lymph Phenotype, % CD3 15 %
Leuk/Lymph Phenotype, % CD4 13 %
Leuk/Lymph Phenotype, % CD5 47 %
Leuk/Lymph Phenotype, % CD7 37 %
Leuk/Lymph Phenotype, % CD8 1 %
Leuk/Lymph Phenotype, % Gamma-Delta 1 %
Leuk/Lymph Phenotype, % CD1 1 %
Leuk/Lymph Phenotype, % CD16 30 %
Leuk/Lymph Phenotype, % CD56 18 %
Leuk/Lymph Phenotype, % CD57 5 %
Leuk/Lymph Phenotype, % CD10 14 %
Leuk/Lymph Phenotype, % CD19 52 %
Leuk/Lymph Phenotype, % CD20 52 %
Leuk/Lymph Phenotype, % Kappa 30 %
Leuk/Lymph Phenotype, % Lambda 22 %
Leuk/Lymph Phenotype, % CD34 See Note %
Leuk/Lymph Phenotype, % CD11b 5 %
Leuk/Lymph Phenotype, % CD13 6 %
Leuk/Lymph Phenotype, % CD33 5 %
Leuk/Lymph Phenotype, % CD64 4 %
Leuk/Lymph Phenotype, % CD45 See Note %

PERIPHERAL BLOOD, FLOW CYTOMETRIC IMPRESSION:
Phenotypically normal myeloid cells, with slightly increased
eosinophils (approximately 13% of leukocytes) and phenotypically
normal T-cells with an increased CD4:CD8 ratio of 13.0, without
flow cytometric evidence of monoclonality, acute leukemia, or
lymphoproliferative disorder.
COMMENT:
The significance of the increased CD4 positive T cells is
uncertain but likely reflects a reactive T-cell population,
since they do not show phenotypic abnormalities. Please
correlate the results from this study morphologically and
clinically for proper interpretation
ANALYSIS:
Flow cytometric analysis of the peripheral blood specimen
indicates that 51% of the CD45 positive leukocytes are
lymphocytes, 4% are monocytes, and 46% are myeloid cells. 15% of
the lymphocytes (percentages shown) are T-cells, 27% are NK
cells and 52% are B-cells. The T-cells appear phenotypically
normal with an increased CD4:CD8 ratio of 13.0. The NK cells
appear phenotypically normal with expression of CD2, CD7, CD16,
CD56, partial CD57 and partial CD8. The B-cells express partial
CD5, partial CD10, CD19, and CD20, and are polytypic, with a
kappa:lambda ratio of 1.4. The myeloid cells include
approximately 35% eosinophils (CD10 negative, CD16 negative) but
otherwise demonstrate phenotypically normal expression patterns
for the antigens evaluated, and actual percentages should be
determined morphologically or using other laboratory methods.
The remaining myeloid cells demonstrate phenotypically normal
expression patterns for the markers evaluated, including CD11b,
CD10, CD13, CD16, CD33, and CD64 and include 0.36% CD34 positive
myeloblasts. The viability of the specimen is 97%.

SCID Panel Specimen         Whole Blood

SCID Panel Interpretation   See Note

Reason for referral: Abnormal newborn screening (TREC) and

severe T cell deficiency

Result: One pathogenic mutation was detected in the FOXN1 gene

Pathogenic Mutation

Gene: FOXN1 (NM_003593)

Nucleic Acid Change: c.1418delC; Heterozygous

Amino Acid Alteration: p.Pro473fs

Interpretation:

One pathogenic mutation, c.1418delC, p.Pro473fs, was detected in

the FOXN1 gene by massively parallel sequencing and confirmed by

Sanger sequencing. The p.Pro473fs mutation has not been reported

in the scientific literature, gene specific mutation databases,

nor has it been previously identified in our laboratory.

However, it is predicted to alter the proteins amino acid

sequence beginning at codon 473, leading to a premature stop

codon and a truncated or absent protein.

Pathogenic mutations in FOXN1 are causative for autosomal

recessive T-cell immunodeficiency, congenital alopecia and nail

dystrophy (MIM:601705). Thus, this patient is at least a

carrier, and may be affected if a second pathogenic FOXN1

mutation is present on the opposite chromosome but could not be

detected by this assay (eg, deep intronic mutations, or

regulatory region mutations). Of note, heterozygous FOXN1

p.Arg255Ter carriers that belonged to one extended pedigree

were all reported to exhibit nail dystrophy (see reference

Auricchio et al.)

No pathogenic mutations were detected by massively parallel

sequencing in the other 18 genes included in this panel. No

large exonic deletions or duplications were identified in any of

the 19 genes analyzed by CGH array

Recommendations:

Medical screening and management should rely on clinical

findings and family history. Genetic consultation is

recommended. At-risk family members should be offered testing

for the identified mutation (Familial Mutation, Targeted

Sequencing; ARUP test code 2001961)

References:

Auricchio, L et al: Nail dystrophy associated with a

heterozygous mutation of the nude/SCID human FOXN1 (WHN) gene.

Arch Dermatol 2005; 141:647-648

This result has been reviewed and approved by Karl V.

Voelkerding, M.D.

Background Information for Severe Combined Immunodeficiency

(SCID) Panel, Sequencing and Deletion/Duplication, 19 genes

Characteristics of SCID: Absence or very low number of T-cells

and no or very low T-cell function as measured by flow cytometry

and response to mitogens. Lack of B-cells or failure of B-cells

to generate antibodies. Clinical symptoms associated with SCID

include severe infections, chronic diarrhea, candidiasis,

failure to thrive, sepsis and meningitis with onset usually

between three to six months of age

Incidence: Combined estimated incidence of SCID is 1 in 50,000

births, and may be more prevalent in cultures that commonly

practice consanguineous marriage or there is a founder mutation.

Inheritance: X-linked for IL2RG, autosomal recessive for all

other genes tested.

Penetrance: Unknown

Cause: Pathogenic mutations in ADA, AK2/ADK2, CD247/CD3Z, CD3D,

CD3E, CORO1A, DCLRE1C/ARTEMIS, FOXN1, IL2RG, IL7R, JAK3, LIG4,

NHEJ1, PNP, PRKDC, PTPRC/CD45, RAG1, RAG2, RMRP and other

unknown genes

Clinical Sensitivity: Approximately 90 percent

Methodology: Targeted capture of all coding exons and

exon-intron boundaries followed by massively parallel

sequencing, and deletion/duplication analysis by tiled

comparative genomic hybridization (CGH) array of the following

genes: ADA, AK2/ADK2, CD247/CD3Z, CD3D, CD3E, CORO1A,

DCLRE1C/ARTEMIS, FOXN1, IL2RG, IL7R, JAK3, LIG4, NHEJ1, PNP,

PRKDC, PTPRC/CD45, RAG1, RAG2, and RMRP. Sanger sequencing is

performed for bases with insufficient coverage and to confirm

all reported variants. The Hg 19 Human Genome build is used for

data analysis

Analytical Sensitivity: 95 percent for sequencing and 100

percent for CGH analysis

Analytical Specificity: greater than 99 percent for sequencing

and 99.5 percent for CGH analysis

Limitations: Regulatory region mutations, deep intronic

mutations, breakpoints of large deletion/duplications,

translocations and deletions/duplications of exon 1 in ADA, exon

11 in CORO1A, exons 4, 6, and 8 in DCLRE1C, and exons 3, 6, and

9 in JAK3 gene are not assayed. Small deletions or insertions

may not be detected. Diagnostic errors can occur due to rare

sequence variations.

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