healthmedicinet

We here report on two siblings with mild MCADD, of whom only one was identified by
newborn screening. The genotypes of patients identified after implementation of NBS
differ from those identified in patients with clinical manifestation 12], 13]. The mutation c.985A??G is the most common mutation in clinically detected patients
and has been found in 80 % of these individuals in homozygosity and in 18 % in heterozygosity
14], 15]. This mutation is considerably less frequent in individuals diagnosed by NBS, while
numerous novel mutations including a second prevalent mutation, c.199 T??C, have
been identified in this group of patients 12], 13]. Individuals carrying these mutations often show significantly lower concentrations
of disease-characteristic acylcarnitines 13], 14]. Our patients were found to be compound heterozygous for the common c.985A??G mutation
and a novel splice site mutation, c.600-18G??A. The c.985A??G mutation (K329E) is
well characterized with respect to its functional consequences. The MCAD protein is
a tetramer of 4 identical subunits. The amino acid change K329E is located in the
C terminal ? domain of the enzyme and compromises tetramer assembly by affecting helix-helix
interactions resulting in degradation of the mutant enzyme 16]. The K329E protein variant has been shown to be thermosensitive 17]. Therefore, febrile episodes may additionally affect protein stability and function
in patients carrying this mutation and may aggravate energy deficiency 18].

The c.600-18G??A mutation has not been described previously. Variant analysis using
a prediction software indicated a splice altering mutation. Indeed, we could demonstrate
the pathogenicity of this mutation by proof of aberrant splicing. We propose an activation
of cryptic splice sites that leads to weakening of the natural splice sites and generates
shortened transcripts. Although missplicing is not complete, the combination of both
mutations in the compound heterozygous state may account for the reduced enzyme activity
and biochemical abnormalities in our patients. The residual enzyme activity measured
with octanoyl-CoA as substrate was 13-24 % of healthy controls. These values are higher
than those usually found in patients with “classical” MCADD, but lower than those
found in heterozygous carriers 19] indicating that the splice site mutation really has a compromising effect on the
MCAD protein. Missplicing is known to be highly tissue and cell type specific as can
be seen from the different splicing patterns in granulocytes and monocytes of our
patients. Though no prediction of the prevalence of missplicing in hepatocytes can
be made from our data, it is conceivable that there is a shift in favour of the aberrant
splice product in this cell type. Furthermore, it has been reported that splicing
processes can be temperature-sensitive. In case that the original splice site is still
functional temperature may affect splice site selection in favour of the aberrant
splice site 20], 21]. Interestingly, we found a lower residual activity during a febrile infection than
at time of physical well-being in patient 2 (Table 1). In the context of MCADD, in which metabolic decompensations are often triggered
by febrile infections, this may be of clinical relevance. Enhanced missplicing during
episodes of increased energy expenditure may aggravate the cellular energy deficiency
and therefore have clinical consequences.

Only one of the two sisters was identified by newborn screening due to a characteristic
acylcarnitine pattern. This is notable as it has been shown that even carriers of
a single c.985A??G mutation may display mildly elevated C8 acylcarnitine concentrations
22], 23]. Lehotay et al. reported significantly higher C8 concentrations of 0.302?±?0.09 ?mol/l
in c.985A??G heterozygotes compared to healthy controls, while the C8 concentration
in our compound heterozygous patient (0.17 ?mol/l) was even below this range. High
C8 levels have been shown to be associated with severe mutations including the common
c.985G??C mutation (in homozygosity), deletions, nonsense or splice site mutations
24]. In a study with 34 MCAD deficient individuals evaluating the correlation between
genotype and biochemical phenotype, patients homozygous for c.985A??G had the highest
levels of neonatal octanoylcarnitine followed by compound heterozygotes of c.985A??G
and other mutations. Patients without c.985A??G and compound heterozygotes for c.985A??G
and c.199 T??C had the lowest levels of C8. According to this, an intermediate elevation
of the C8 acylcarnitine concentration would have been expected in our patient.

Even if the high residual MCAD activity and the mild biochemical expression in our
patients are suggestive of a presumably low clinical penetrance, the potential risk
of metabolic decompensation during severe catabolism cannot be estimated and the clinical
relevance of mild MCADD is difficult to establish. Although the majority of symptomatic
patients present within the first three years of life, several late onset cases have
been reported 25], 26]. Both girls have remained asymptomatic so far, however, they are only 3 and 7 years
old, respectively. Nevertheless, it is of great interest that patient 1 has experienced
many infections and catabolic conditions before diagnosis by family screening not
resulting in any clinical symptoms. The natural course of the disease in individuals
with novel mutations is generally unknown 16]. Many of these children may remain asymptomatic even without detection by NBS. This
is reflected by the much higher detection rate of MCADD by NBS compared to clinically
detected cases in unscreened populations. In contrast, one more recent study on structural
alterations of several mutant variants proposed that novel mutations found in NBS,
including the c.199 T??C mutation, do not bear a lower risk of metabolic decompensation
than that associated with mutations detected in clinically ascertained patients 16], although, to our knowledge, only one individual carrying the c.199 T??C mutation
together with a second mutation has ever been symptomatic during follow-up. The mother
sought emergency room care for the infant two times in the first year for listlessness
associated with gastroenteritis. At age 22 months altered mental status and variable
hypo- and hyperglycemia were reported during hospital admission for hand/foot/mouth
disease 24]. Residual enzyme activities in patients with proposed mild MCADD are in some cases
in the same activity range as in definite heterozygous parents questioning the clinical
relevance of the mutations 10]. So far, there is no consensus to consider certain ACADM genotypes as safe and protective from clinical symptoms 16], 24], however, we are also aware of an overtreatment of many individuals identified by
newborn screening. The same uncertainty is true with respect to the interpretation
of the biochemical phenotype despite a strong relationship between initial C8 levels
and outcome 24].