Congenital Dyserythropoietic Anemia Type IV with KLF1 E325K
Mutation –
a New Case with Dysmorphic Male Genitalia .
Gita Massey MD1, Alden Chesney MD2,
Yaddanapudi Ravindranath MBBS3
1Children’s Hospital of Richmond, VCU Health System
Division of Pediatric Hematology, Oncology and Cellular Therapeutics,
Richmond VA
2 Virginia Commonwealth University Medical Center
Division of Clinical Pathology, Richmond VA
3Wayne State University School of Medicine
Division of Pediatric Hematology Oncology, Detroit, MI
Corresponding Author:
Gita V Massey, MD
CHoR – Children’s Pavilion
Pediatric Hematology-Oncology
1000 Easr Broad St 1st Floor
Richmond, VA 23219
E-mail:
gita.massey@vcuhealth.org
Phone: 804-828-9605
FAX: 804-628-5848
Word Count: Abstract 76; Main Text 1155
Number of Tables and Figures: 2
Running Title: Congenital Dyserythropoeitic Anemia Type IV (CDA-IV)
Key Words: hemolytic anemia, hydrops fetalis, gonadal dysmorphism, KLF1
mutation
Abbreviations: CDA-IV - Congenital Dyserythropoeitic Anemia Type IV
LSD – lyposomal storage disorder
AML – anti-Mullerian hormone
Abstract:
Congenital Dyserythropoietic Anemia Type IV (CDA-IV) is still an
emerging new disease with approximately 10 cases reported over the past
three decades. CDA-IV is known to be caused by a specific mutation in
exon 3 of KLF1, an erythroid transcription factor KLF1 with substitution
of glutamic acid with lysine at residue 325 (KLF1 E325K). Because of the
rarity of this disorder the presenting features are incompletely
defined; especially the non-erythroid comorbidities. Here we report a
new case, a male child, presenting with fetal hydrops and dysmorphic
external genitalia.
Introduction:
Congenital Dyserythropoietic Anemia type IV (CDA-IV) is an exceedingly
rare disease, with only approximately 10 cases reported over the past 3
decades1-10. It is defined by a specific genetic
mutation in exon 3 of KLF1 (chr19p13.2), an erythroid transcription
factor with substitution of glutamic acid with lysine at residue 325
(KLF1 E325K). The hematologic presentation is variable, but is defined
by ineffective erythropoiesis leading to anemia. There are also
non-erythroid comorbidities which remain poorly
defined11,12. We report here a new case of a male
child presenting with fetal hydrops and dysmorphic external genitalia.
Case Description:
KC is a firstborn male infant to a Caucasian mother and African-American
father. Pre-natal ultrasound at 30 weeks noted cardiomegaly and repeat
ultrasound at 35 weeks suggested hydrops fetalis leading to emergency
Caesarean section. Born at an outside hospital, the infant was found to
have severe anemia, thrombocytopenia, coagulopathy, hepatosplenomegaly
and cardiomyopathy compatible with hydrops fetalis. The left testicle
was not palpable and he was thought to have a micropenis. Initial
laboratory findings are shown in Table 1.
He required a 1 month neonatal intensive care admission during which he
underwent an exchange transfusion, multiple transfusions of packed red
blood cells, platelets, and fresh frozen plasma. A bone marrow aspirate
was thought to be non-diagnostic. A liposomal storage disorder (LSD) was
suspected, but enzyme testing for N-acetyl-alpha-glucosaminidase as well
as molecular testing for LSD were negative. Newborn metabolic/genetic
disease screening was reported as negative.
KC first presented to our institution at 5 months of age. At that time
hepatosplenomegaly had resolved, but he still required intermittent
transfusions of packed red blood cells. He was noted to have a
micropenis, undescended testicle, scrotal tethering and excess
suprapubic fat pad. He had been recently transfused, however in the
following 4 weeks, hemoglobin dropped to 7.3 g/dl with 7.3%
reticulocytes, MCV 89.0 fl, platelets 222 10e9/L, WBC 10.0 10e9/L with
16% nRBC, 46.5% neutrophils, 38.7% lymphocytes, 13.4% monocytes,
0.7% eosinophils and 0.7% basophils. Smear revealed mild myeloid left
shift beyond band stage, macrocytic anemia with polychromasia, moderate
anisopoikilocytosis with atypical erythrocytes including, spherocytes,
ovalocytes and occasional fragmented red cells. Frequent nRBCs were
identified (Figure 1a).
There was no known family history of a hemolytic disorder. Diagnostic
work-up revealed Hemoglobin electrophoresis of 68.2% Hemoglobin A,
2.1% Hemoglobin A2 and 29.7% Hemoglobin F. Quantitative measurements
of glucose 6-phosphatase and pyruvate kinase enzyme activities were
normal, as was RBC Band 3 protein reduction for hereditary
spherocytosis. Because of the frequent transfusions, and thus possible
inaccuracy of the previous tests, Clinical Exome Sequence Analysis was
performed through GeneDx (https://www.genedx.com; Gaithersburg,
PA; USA). KP was found to be heterozygous for a pathogenic variant in
the KLF1 gene (c.973 G>A; p.E325K). This was a de novo
mutation and established the diagnosis of congenital dyserythropoeitic
anemia (CDA) type 4. He remains transfusion dependent and on iron
chelation therapy and has developed hemolytic facies and splenomegaly.
Discussion:
Clinical diagnosis of KLF1 E325K associated Congenital Dyserythropoietic
anemia – (CDA -IV) remains complex and CDA-IV as a cause of non-immune
fetal hydrops is under appreciated. This and the Detroit case underscore
this point.4,13 Fetal hydrops was suspected in both
cases. Blood group incompatibility was excluded by clinical testing. The
child described here was noted to have severe anemia with high MCV - a
smear review was not available but the extraordinarily high white cell
count suggested the presence of a leukoerythroblastic picture. Post
transfusion blood examination shows certain hall marks of the aberrant
erythroid maturation and block in the fetal to beta globin transition by
KLF1 E325K- ie persistent high fetal hemoglobin, lack of CD44 surface
expression red cells (Figure 1b) and the presence of increased nRBC,
some with binuclearity. The latter are indicative of the well identified
enucleation defect associated with the KLF1 E325K
mutation.3,14 Documentation of other features such as
the presence of embryonic hemoglobin require isoelectric focusing or
complex proteomics. Search for Colton a-/b- caused by aquaporin1 (AQP1)
deficiency and In(Lu) blood group ( Lutheran blood group/ BCAM
deficiency caused by lack of CD44 on red cell membrane) are likewise
complex and are not readily available. However, CD44 surface marker
testing by flowcytometry is feasible and could be incorporated in to the
testing of non-immune hydrops cases with erythroblastosis fetalis as it
discriminates between native and transfused red
cells.3,15
Non-hematologic comorbidities in CDA-4 have not received sufficient
focus. The hemolytic anemia appears to be more severe in males and in
addition two thirds of affected males showed genital dysmorphology
including complete sex reversal in the Detroit
case.3,15 Two had no genital dysplasia
described.5,7 Our present case, a 46 XY male has
micropenis, undescended left testis with scrotal tethering. An immediate
alternative hormonal cause is not evident. Retrospective evaluation of
clinical exome sequencing did not identify a pathogenic germline
mutation nor insertions/deletions in genes reported to cause disorders
of sex differentiation (with the exception of CYP21A2 which has poor
coverage by exome sequencing). In fact, the only pathogenic mutation
identified on clinical exome sequencing was the E325K mutation in KLF1,
a transcription factor regulating erythroid differentiation and
maturation. Endocrine evaluation at 37 months of age in the present
child showed the following laboratory values- total testosterone:
<2.5 ng/dl (<2.5-10); androstenedione: <10
ng/dl (0-22); dihydroepiandrostenedione <20ng/dl (0-67);
17-hydroxyprogesterone: 11ng/dl (0-90); FSH 0.6mlU/ml (0.9-12.0);
anti-Mullerian hormone (AMH): >960ng/ml - (Males at birth
32.7-262.9); inhibin B: 306.8 pg/ml (42-268). Elevated levels of AMH
have not been previously reported but suggest an adaptive response to an
as yet unidentified early gonadal dysgenesis event.
Identification and reporting of additional cases of CDA-IV particularly
those in 46 XY males will be helpful in fully delineating the
non-erythroid off-target effects of the KLF1 E325K mutant transcription
factor. Progress toward this goal has also been made in the recent
development of a human cellular model of CDA-IV which may allow for
future analysis of the disordered biological processes associated with
this rare molecular defect.16
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