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Maple Syrup Urine Disease (MSUD) in Association With a Proximal Interstitial 6q13q14.2 Deletion Syndrome

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Poster # 120 2017 ACMG CLINICAL GENETICS MEETING March 21-25
Unusual Association of Maple Syrup Urine Disease with a
Proximal 6q13q14.2 Interstitial Deletion Syndrome
Sansaricq C1 , Perle M. A2 , Kyriakakos A3 , Cox R.P4 , Chuang J.L5 , Chuang D.T5
1 Department of Human Genetics and Genomics, The Mount Sinai School of Medicine, New York, NY
2 Department of Pathology, New York University School of Medicine, New York, NY
3 Department of Pediatrics, The Montefiore Medical Center, New York, NY
4 Department of Medicine, University of Texas Southwestern Medical Center, Dallas, TX
5 Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX


Abstract: An elevated blood leucine level of 916μmol/L was detected by the NY Newborn
Screening Program (NYNSP) and the patient was referred to our center for evaluation and
treatment. The diagnosis was clinically, biochemically and enzymatically confirmed to be a case
of Maple Syrup Urine Disease (MSUD). However, because of associated physical abnormalities
not seen in MSUD a karyotype was obtained that revealed a microscopically visible interstitial
deletion at 6q13-14.2 that encompasses the 6q14.1 site for the MSUD1B component of the
complex enzymatic system responsible for the branched-chain amino acids (BCAA) catabolic
pathway. However, the deletion was demonstrated only on one chromosome raising the
question whether the homolog gene would be affected as well. Parents appeared not to be
carriers for this cytogenetic abnormality detected in their son and in addition enzymatic studies
on their white cells for the branched-chain keto acid dehydrogenase (BCKADH) that when
defective is responsible for MSUD seemed to be within normal limits. The proposed mechanism
for this clinical condition to happen is reviewed. Molecular studies on the patient cells also
disclosed a 2-bp deletion (642delAG) in exon 5 of the homolog E1βgene thus, demonstrating
that the patient is actually a compound heterozygote for this gene. On the one hand this gene is
totally absent due to the chromosomal deletion and on the other hand its homolog is affected by
the 2-bp deletion. In conclusion, our patient showed evidence for two concurrent diagnoses, that
of a 6q deletion syndrome as well as that of MSUD. This is the compound heterozygote state
that allowed expression of this altered E1β recessive gene causing manifestations of MSUD.
This is also the first report of this 2-bp deletion (642delAG) in exon 5 of the E1β gene that
caused MSUD.
Key words: MSUD, MSUDB, 6q deletion, 640delAG in the E1β gene
Introduction: The New York State Newborn Screening Program had identified the case
of a 4 days old infant with an elevated blood leucine level of 916μmol/L. The patient was
immediately referred to our Center for evaluation.
Clinical Report: On arrival to our center, the baby, a 6 days old male, initially appeared
quite sick being lethargic and having intermittent posturing episodes. He was born after a fullterm
and uneventful pregnancy. Delivery was by C-section for repeat and Apgar scores were
within normal limits. Birth weight was 3470 g. Parents were of Italian and Irish extraction and
had no family history of inherited disease. He was on a regular infant formula. Upon physical
examination the baby measured 53 cm in length, and had a head circumference of 35 cm with
dilated scalp veins, a narrow bifrontal diameter, a short and bulbous nose, a fish-like mouth,
micrognathia, a large tongue, with a short frenulum. The ears were prominent, low set with
fleshy lobes. He had narrow palpebral fissures, a short neck with loose skin folds, low hairline,
umbilical and bilateral inguinal hernias and palmar simian creases. A mild hypospadia was also
noted and a strong body odor of maple syrup was felt. An EEG tracing was diffusely abnormal
with bilateral cerebral dysfunction more accentuated on the left side. While the first part of the
history was consistent with a diagnosis of Maple Syrup Urine Disease (MSUD) the remainder
was more suggestive of a chromosomal disorder.
Confirmatory Laboratory Testing: A urine specimen was strongly positive
to a 2-4 DNPH. Plasma branched-chain amino acids (BCAA) by column chromatography
revealed a valine of 1017μmol/L (N 86-190), an isoleucine of 649μmol/L (N 26-91), a leucine of
2900μmol/L (48-100) and an alloisoleucine of 290μmol/L (N 0.00). Enzymatic activity of the
branched-chain keto acid dehydrogenase (BCKADH) was performed on lymphoblastic cell lines
of the parents, the child and his older brother. This enzyme when deficient is the cause of
MSUD. Radioactive CO2 production from ketoisovaleric acid as a substrate was measured in
triplicate for assessment of enzyme activity. Concomitantly a normal control, a known classical
case of MSUD and a PDH assay were conducted to confirm the diagnosis and validation of the
specimens. The results were the following: The normal control had 0.272nmol of evolved
CO2/mg protein versus 3.404 for the PDH assay. The classical MSUD patient had 0 activity
against 2.558 and our proband 0 versus 1.147. Because of absence of residual enzyme activity
a definite diagnosis of classical MSUD was made [Tab 1]. The parents and their older son had
independently been tested for BCKADH activity and were found to be within normal limits.

What is MSUD ?
This disease results from the failure of the BCKADH a complex enzymatic system located in the
outer mitochondrial membrane that is responsible for the catabolism of the BCAA leucine,
isoleucine and valine. It is a rare, pan ethnic, autosomal recessive condition with a worldwide
frequency of 1/185000 but only of 1/175 in Mennonite and Amish. The incidence is higher in
Ashkenazi Jews, Druse and Arab populations. BCKADH is made of three different components
called E1, E2 and E3 controlled by a kinase and a phosphatase as regulators for activation and
deactivation. The phosphorylated form is the inactive form of the enzyme. Thus, the enzyme is
activated by dephosphorylation. The E1 component is made of 2 subunits α and β assembled
as a tetramer (α2β2) with thiamine pyrophosphate (TPP). The α subunit is encoded on 19q13.1-
q14.2, has 9 exons and when defective the condition is called MSUD1A. Likewise the β subunit
is on 6q14.1, has 11 exons and if defective is called MSUD1B. E1 works as a decarboxylase
and a dehydrogenase. E2 the second component is a multimer containing 24 dihydrolipoyltransacylase
elements. It is the core of the enzyme and is referred to as DBT. It is encoded on
1p31, has 11 exons and spans 68 KB. There has been about an equal number of cases of
MSUD reported for E1 and E2. The E3 component is located on 7q31-q32 and has 11 exons. It
is a homodimer referred to as DBL, has 14 exons and a span of 20 KB. It acts to regenerate
lipoamide and is a common element for the PDH and the 2-keto-glutaric dehydrogenase
systems. Any alteration in any of the components or the subunits may cause deficiency of the
BCKADH and hence MSUD [Fig 1 A and 1B].
When this system is defective there is accumulation of the BCAA as well as that of their ketoderivatives
in body fluids and tissues causing the clinical manifestations of the disease such as
vomiting, dizziness, intermittent posturing, coma, psychomotor delay, mental retardation, and
death. The patient eliminates in his urine a chemical derived from isoleucine with a strong smell
reminiscent of maple syrup or burnt sugar, hence its name. This substance has been identified
as sotolone.

Another important marker for the disease is presence of alloisoleucine formed from reamination
of and asymmetry of the third carbon of the chemical α-keto-β-methylvaleric acid that
accumulates in body fluids due to the metabolic block. Among the BCAA, leucine is the most
toxic and has the highest plasma concentration. In Europe where maple syrup is not very well
known the disease is sometimes identified as leucinosis or branched-chain keto acid
dehydrogenase deficiency.

Because of the unusual clinical, physical characteristics exhibited by the patient that are not
normally seen in MSUD a karyotype was performed on peripheral blood. G banding analysis
identified a male karyotype with a microscopically visible interstitial micro-deletion on the long
arm (q) of chromosome 6 in all cells studied. High resolution banding assigned the most likely
breakpoints at bands 6q13 and 6q14.2: 46,XY,del(6)(q13q14.2). This deletion involves the loss
of band q14.1 the active site of the gene for E1β that when defective can cause MSUD1B. [Fig
2]. Parental peripheral blood chromosome studies were normal. The finding of this
chromosomal deletion in our patient establishes the diagnosis of a 6q syndrome and explains
his abnormal physical characteristics.
Deletions in the proximal region of chromosome 6q are rare and are associated with variable
phenotypes based on the size and location of a specific deletion. The 6q deletion syndrome
(monosomy 6q) has been reported by a number of authors including Hopkins, RJ et al (1997)
and also Kumar,R et al (1997) who described a specific syndrome with the same area of
chromosomal material lost that has been estimated to be in the range of 3.7 Mb or 37,000,000
bp of DNA. The clinical features including the facial dysmorphism, simian creases, hernias,
hypotonia, psychomotor delay and mental retardation were observed in our case as well. This
would be consistent with the 6q syndrome.

Course and Follow-up
The patient underwent dialysis and quickly recovered but remained very delayed. The hypotonia
persisted. He turned over at 12 months, walked at 2 years and spoke at 5. At 4 years and 9
months his IQ was 47. He had a few batches of decompensation but was able to maintain plasma
leucine levels between 305 to 916 mol/L. He had delayed ossification. At 11 months his bone age
was that of a 3 months old but had an unusual opacification on the base of the second and third
metacarpal bones, femoral head dislocation and left hydronephrosis. At 15 years of age he was on
the 25th percentile for height and the 90th for weight. After this age the family moved out of state
and we lost contact with them. We learned that he had died around age 33 of an acute
complication of his illness.

In our case two concurrent diagnoses of MSUD and of the 6q deletion syndrome were well
established. MSUD is an autosomal recessive condition and the 6q deletion syndrome a dominant
one. Can these conditions be linked together? The chromosomal deletion was established as ”de
novo” since the parents did not have any structural defects in their DNA therefore they could not
have transmitted this to their offspring. This deletion involves the loss of an E1 gene part of the
BCKADH responsible for the BCAA catabolic pathway. However, this would not fully explain the
occurrence of MSUD in the patient who would only be heterozygote for this condition assuming
the presence of a normal gene on the homologous chromosome. As long as this gene remained
unaffected, the patient should not suffer from any clinical abnormality. Presumably, in order for a
patient to be affected one would assume some alteration or abnormality in the homologous gene.
Actually, this is the case here. Indeed, molecular studies on the homolog E1 gene revealed a 2-
bp (AG) deletion in the exon 5 of the gene (642delAG) that lead to a frameshift followed by a stop
codon resulting in total loss of BCKADH activity Fig 3A and 3B. This situation did create a
compound heterozygote as, on the one hand, due to the chromosome deletion, we have a total
absence of the E1 gene the site of which is at 6q14.1 while, on the other hand the homolog gene
is affected by the 2-bp deletion (AG) that allowed for expression of the MSUD. It is likely that this
latter recessive gene mutation probably originated from one of the unidentified carrier parents who
presumably had a near normal BCKADH activity that remained unrecognized as such.
In fact this is why this case is unusual and even unique because the calculated risk of association
of these two different mutations is infinitely small being at least in the range of 0.000007. This
gene mutation is actually a “novel” one and is being first reported here in addition to the 7 known
that can cause MSUD1B.

They are: 52insG frameshift after glycine (-33)
92del11 frameshift after glycine (-21)
IVS5+1 G>T skipping of exon 5 and 6
954delT frameshift after serine 268

Our patient showed both, evidence for the unusual association of two concurrent diagnoses, that of
MSUD and that of the 6q deletion syndrome. This is also the first report of a “de novo” mutation
(642delAG) in exon 5 of the E1 gene causing MSUD. This unusual combination of two different
deletions caused a compound heterozygote on chromosome 6q that allowed expression of a
recessive mutated gene
Bibliography (abridged)
Chuang DT, and Shih VE: Maple Syrup Urine Disease. (Branched-Chain Ketoaciduria in: Scriver,
Beaudet , Valle, Sly, Childs, Kinzler, Vogelstein. The Metabolic and Molecular Bases of Inherited
Disease, 8th edition 2001 McGraw-Hill, Inc. p 1971-2005
Hopkin RJ, Schorry E, Bofinger M, Milatovich A, Stern HJ, Jayne C, and Sa HM. : New insights into
the phenotypes of 6q deletion (1997) AM J Med Genet 70:377-386
Kumar R, Riordan D, Dawson AJ, and Chudley AE. Proximal Interstitial 6q Deletion. (1997) Am J Med
Genet 71:353-356.
Edelmann L, Wasserstein MP, Kornreich R, Sansaricq C, Snyderman SE, Diaz GA. Maple Syrup
Urine Disease: Identification and frequency determination of a novel founder mutation in the
Ashkenazi Jewish population (2001) Am J Hum Genet 69:863-868

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