[CIS PIDD] [cis-pidd] SCID newborn screening case
Keller, Michael
MKeller at childrensnational.org
Mon Nov 3 08:58:25 EST 2014
Hi Karin,
I agree that your mitogen results do not make much sense given the TRECs and T-cell counts. Is there definitely no hypoproteinemia or any reason for lymphatic loss? Even that would be odd for the T-cell counts that you describe, but worth thinking about given the intact IgA/IgM.
I would suggest repeating both the TRECs and the mitogens. While I wouldn't wait on a molecular phenotype, I would iron out inconsistencies before transplantation.
Mike
-------------------------
Michael D. Keller MD
Division of Allergy / Immunology
Children's National Health System
111 Michigan Ave NW, Room 1W-314B
Washington, DC 20010
Clinic: 202.476.3016
Office: 202.476.5843
Fax: 202.476.2280
www.childrensnational.org
________________________________
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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