Molecular Analysis
Of the 18 patients 88% presented with mutations in the BCKDHB gene, 1 patient had a mutation in the gene BCKDHA, and 1 patient harbored a mutation in DBT. A total of eight mutations were found in the samples, and four of these (50%) were new, previously uncharacterized mutations. The new mutations found were c.[1006 G>A] and c.[718 C>A], in BCKDHB, c.[1013 C>T] in BCKDHA, and c.[1217 G>A] in DBT (Table 1).
This study was able to identify regions with the highest incidence of mutations in exon 6 of BCDKHB. Exons 4, 5, 9 and 10 exhibited 1 mutation in each, as depicted in the schematic representation of BCKDHB in Figure 1.
Table 1 – Pathogenic variants detected in BCKDHA, BCKDHB and DBT genes of Chilean MSUD patients.
Patient
|
Gene
|
Nucleotide
|
Protein Prediction
|
1
|
BCKDHA
|
c.[ 1013 C>T ] §+[ 1013 C>T ] §
|
p.Thr338Ile + Thr338Ile
|
2
|
BCKDHB
|
c[.595_596delAG] +[641 T>A]
|
p. Pro200Stop +Ile214Lys
|
3
|
BCKDHB
|
c.[641 T>A] + [1006 G>A]§
|
p.Ile214Lys + Gly336S
|
4
|
BCKDHB
|
c.[595_596delAG] +[641 T>A]
|
p.Pro200Stop +Ile214Lys
|
5
|
BCKDHB
|
c.[641 T>A] + [641 T>A]
|
p.Ile214KLys+Ile214Lys
|
6
|
DBT
|
c.[1217 G>A]§+ c.[1217 G>A]§
|
p.Gly406Asp+Gly406Asp
|
7
|
BCKDHB
|
c.[595_596delAG]+ [641 T>A]
|
p. Pro200Stop + Ile214Lys
|
8
|
BCKDHB
|
c.[718 C>A]§ + [718 C>A]§
|
p.Pro240Thr+ Pro240Thr
|
9
|
BCKDHB
|
c.[641 T>A] + [641 T>A]
|
p.Ile214KLys+Ile214Lys
|
10
|
BCKDHB
|
c.[392 G>T] +[595_596delAG]
|
p.Gly131Val + Pro200Stop
|
11
|
BCKDHB
|
c.[641 T>A] + [641 T>A]
|
p.Ile214KLys+Ile214Lys
|
12
|
BCKDHB
|
c.[641 T>A] + [641 T>A]
|
p.Ile214KLys+Ile214Lys
|
13
|
BCKDHB
|
c.[392 G>T] + [392 G>T]
|
p.Gly131Val+ Gly131Val
|
14
|
BCKDHB
|
c[.595_596delAG]+ [641 T>A]
|
p. Pro200Stop +Ile214Lys
|
15
|
BCKDHB
|
c.[718 C>A]§ + [718 C>A]§
|
p.Pro240Thr+ Pro240Thr
|
16
|
BCKDHB
|
c[.595_596delAG]+ [641 T>A]
|
p. Pro200Stop +Ile214Lys
|
17
|
BCKDHB
|
c.[1067 C>T] + [1067 C>T]
|
p.Pro356Leu + Pro356Leu
|
18
|
BCKDHB
|
c.[641 T>A] + [641 T>A]
|
p.Ile214KLys+Ile214Lys
|
Figure 1 – Schematic representation of BCKDHB and mutations; the new mutations are presented in bold. black lines with numbers: coding exons; black horizontal lines: introns; untranslated regions (5' and 3' UTR)
Mutation Pathogenicity
Among the mutations already described in the literature, the Ile214Lys mutation of Spanish origin had the highest incidence, totaling 77 % of the patients, followed by mutation Pro200*, also of Spanish origin in 33% (Table 2). Of these mutations, those found in the heterozygous state were p.Gly131Val, p.Pro200Stop, and p.Ile214Lys. Homozygous mutations identified were p.Gly131Val, and p.Ile214lys.
Table 2 – Mutations already described in the literature found in Chilean MSUD patients.
|
Gene
|
Nucleotide
|
Protein Prediction
|
Type
|
Origin
|
Phenotype
|
Reference
|
Frequency in the sample
|
Exon 4
|
BCKDHB
|
c.[392 G>T]
|
p.Gly131Val
|
M
|
NA
|
NA
|
Margutti AV, 2015
|
2/18
|
Exon 5
|
BCKDHB
|
c.[595_596delAG]
|
p.Pro200Stop
|
D
|
Spanish
|
Classic
|
Henneke M, 2003
|
6/18
|
Exon 6
|
BCKDHB
|
c.[641 T>A]
|
p.Ile214Lys
|
M
|
Spanish
|
Classic
|
Rodriguez-Pombo,2006
|
14/18
|
Exon 10
|
BCKDHB
|
c.[1067 C>T]
|
p.Pro356Leu
|
M
|
Portuguese
|
Classic
|
Quental, 2008
|
1/18
|
|
|
|
|
|
|
|
|
|
|
NA: Not Available.
M: Missense.
D: Deletion.
The new mutation Pro240Thr was found in 16% of the samples; it was also located in exon 6 of BCKDHB, and was the most prevalent of the new mutations. After being subjected to in silico analysis, the new mutations were all considered pathogenic (Table 3). Of these new mutations, the ones found in the homozygous state were p.Thr338Ile, p.Gly406Asp and p.Pro240Thr. Those found as heterozygous were p.Pro240Thr and p.Gly336Ser.
Table 3 – New mutations found in Chilean MSUD patients.
Region
|
Gene
|
Nucleotide
|
Protein Prediction
|
Type
|
Pathogenicity
|
Classification
|
Frequency in the sample
|
|
|
|
|
|
Sift®
|
Polyphen-2®
|
Multipred®
|
|
|
Exon 8
|
BCKDHA
|
c.[1013C>T]
|
p.Thr338Ile
|
M
|
Pathogenic
|
Pathogenic
|
Pathogenic
|
Pathogenic
|
1/18
|
Exon 6
|
BCKDHB
|
c.[718C>A]
|
p.Pro240Thr
|
M
|
Pathogenic
|
Pathogenic
|
Pathogenic
|
Pathogenic
|
3/18
|
Exon 9
|
BCKDHB
|
c.[1006G>A]
|
p.Gly336Ser
|
M
|
Pathogenic
|
Pathogenic
|
Pathogenic
|
Pathogenic
|
1/18
|
Exon 10
|
DBT
|
c.[1217G>A]
|
p.Gly406Asp
|
M
|
Pathogenic
|
Pathogenic
|
Pathogenic
|
Pathogenic
|
1/18
|
M: Missense.
Clinical Analisis
Anthropometric Assessment
According to clinical data, patient 6 was born weighing 4 kg, which is characterized as macrosomia, common in newborns of pregnant women who have gestational diabetes, which was the case in this child. Patient number 10 was born with insufficient weight, but her mother had no problems during pregnancy. The mother of child number 14 presented with preeclampsia during pregnancy, and this patient was born with low weight, also a consequence of complications in pregnancy. Seventy two percent (72%) of the children were born with weight and lengths appropriate for their gestational ages, according to the classification of Intergrowth 21st, as seen in Table 4.
Table 4 – Anthropometric data of patients at birth.
Patient
|
Birth Weight (g)
|
Birth Length (cm)
|
Nutritional Diagnostic
|
Pregnancy Complications
|
1
|
3600
|
51
|
AGA
|
no
|
2
|
2825
|
47,5
|
AGA
|
Anemia
|
3
|
2820
|
48
|
AGA
|
no
|
4
|
3290
|
51,5
|
AGA
|
no
|
5
|
2835
|
50
|
AGA
|
no
|
6
|
4000
|
43
|
LGA
|
Gestational Diabetes/ UTI
|
7
|
3240
|
48
|
AGA
|
no
|
8
|
2830
|
49
|
AGA
|
Hepatic Cholestasis
|
9
|
3150
|
50
|
AGA
|
no
|
10
|
2600
|
47
|
SGA
|
no
|
11
|
3900
|
52
|
AGA
|
no
|
12
|
3890
|
51
|
AGA
|
Hydronephros
|
13
|
3050
|
48
|
AGA
|
no
|
14
|
1580
|
42
|
SGA
|
Preeclampsia
|
15
|
3300
|
50
|
AGA
|
no
|
16
|
2940
|
49
|
AGA
|
no
|
17
|
3000
|
50
|
AGA
|
no
|
18
|
2750
|
48
|
AGA
|
no
|
Source: Villar et al. (2014).
UTI: urinary tract infection.
SGA: small for gestational age.
AGA: appropriate for gestational age.
LGA: large for gestational age
Clinical Evaluation
According to the age of onset of symptoms, 95% of the patients in this cohort were considered to have the classic form of the disease, and 5% with intermediate form. The age at diagnosis ranged from 9 days to 7 months. Leucine levels ranged from 440 to 3962 μmol/L at diagnosis (normal range 35 - 217 μmol/L). Neuropsychomotor Developmental Delay (NPMDD) occurred in 13 of the 18 children studied, and 4 had a mild degree, 3 a moderate degree, and 6 had severe DNPMD. Only 2 children did not have NPMDD, and that information was not available for 3. Therefore, in children for whom NPMDD data were available, the majority, 40%, had a serious degree of delayed neuropsychomotor development.
Table 5 – Characterization of Chilean MSUD patients (n = 18) at INTA.
Patient
|
Diagnostic Time (days)
|
Leucine
(μmol/L)
|
NPMDD
|
First Hospitalization
(days)
|
Presentation according to diagnostic time
|
1
|
10
|
741
|
severe
|
10
|
Classic
|
2
|
9
|
750
|
severe
|
10
|
Classic
|
3
|
14
|
3560
|
moderate
|
7
|
Classic
|
4
|
13
|
2638
|
light
|
32
|
Classic
|
5
|
22
|
2600
|
light
|
30
|
Classic
|
6
|
11
|
750
|
NA
|
48
|
Classic
|
7
|
30
|
1640
|
light
|
5
|
Classic
|
8
|
8
|
3962
|
severe
|
2
|
Classic
|
9
|
21
|
2000
|
severe
|
30
|
Classic
|
10
|
9
|
993
|
moderate/severe
|
20
|
Classic
|
11
|
9
|
440
|
severe
|
4
|
Classic
|
12
|
16
|
1027
|
no
|
32
|
Classic
|
13
|
17
|
1090
|
NA
|
36
|
Classic
|
14
|
30
|
1716
|
no
|
10
|
Classic
|
15
|
210
|
1653
|
light
|
90
|
Intermediate
|
16
|
17
|
1467
|
moderate
|
14
|
Classic
|
17
|
17
|
1038
|
severe
|
NA
|
Classic
|
18
|
27
|
2075
|
NA
|
10
|
Classic
|
NA: Not Available.
NPMDD: Neuropsychomotor Developmental Delay
The biochemical tests values at time of diagnosis provided by INTA were quite high. Leucine levels ranged from 440 to 3962 μmol/L (normal range 35-270 μmol/L), valine ranged from 133 to 1464 μmol/L (normal range 51-325 μmol/L), and isoleucine ranged from 38 to 759 μmol/L (normal range 13-135 μmol /L), as shown in Table 6.
Table 6 – Laboratory values at diagnosis of Chilean MSUD patients at INTA.
Pacient
|
Diagnostic time (days)
|
Leucine
(35-270 μmol/L)
|
Valine
(51-325 μmol/L)
|
Isoleucine
(13-135 μmol/L)
|
1
|
10
|
741
|
759
|
402
|
2
|
9
|
750
|
512
|
50
|
3
|
14
|
3560
|
710
|
512
|
4
|
13
|
2638
|
585
|
372
|
5
|
22
|
2600
|
1180
|
730
|
6
|
11
|
750
|
726
|
657
|
7
|
30
|
1640
|
174
|
53
|
8
|
8
|
3962
|
277
|
215
|
9
|
21
|
2000
|
369
|
190
|
10
|
9
|
993
|
838
|
294
|
11
|
9
|
440
|
1464
|
759
|
12
|
16
|
1027
|
117
|
176
|
13
|
17
|
1090
|
480
|
290
|
14
|
30
|
1716
|
215
|
525
|
15
|
210
|
1653
|
1171
|
586
|
16
|
17
|
1467
|
466
|
419
|
17
|
17
|
1038
|
133
|
38
|
18
|
27
|
2075
|
726
|
170
|
According to the signs and symptoms presented by the patients, the most prevalent were axial hypotonia, identified in 83% of the children, followed by NPMDD in 77% of the cases, intellectual deficit in 61%, and food intolerance and urine with characteristic odor in 55%. Strabismus, pyramidal syndrome, and encephalopathy were reported in 44% of patients, seizures in 38%, macrocephaly and need for mechanical ventilation in 27%, gastrostomy and ataxia in 22%, apnea and mucous and skin lesions in 16%, attention deficit disorder and hyperactivity in 14%, extrapyramidal syndrome in 11%, and coma in 5% (table 7).
Table 7 – Signs and symptoms presented by Chilean MSUD patients (n = 18) of INTA.
Signs and Symptoms
|
Prevalence
|
Axial hypotonia
|
83%
|
NPMDD
|
77%
|
Intellectual deficit
|
61%
|
Urine with maple syrup odor
|
55%
|
Food intolerance
|
55%
|
Strabismus
|
44%
|
Encephalopathy
|
44%
|
Pyramidal syndrome
|
44%
|
Seizures
|
38%
|
Mechanical ventilation
|
27%
|
Macrocephaly
|
27%
|
Ataxia
|
22%
|
Gastrostomy
|
22%
|
Apnea
|
16%
|
Mucous and skin lesions
|
16%
|
ADDH
|
14%
|
Extrapyramidal syndrome
|
11%
|
Coma
|
5%
|
NPMDD: Neuropsychomotor Developmental Delay
ADDH: Attention deficit disorder and hyperactivity.
Genotype-Phenotype Correlations
INTA staff ranked the phenotypes of their patients, based on age at diagnosis. Intermittent and sensitive to thiamine phenotypes were not found in this sample. However one patient was classified with an intermediate form of MSUD, with diagnosis done at 90 days of age, and 17 patients with the classical form, with diagnosis made at 9 to 30 days of life (Table 8). The clinical situation of these patients varied, exhibiting NPMDD and poor prognosis, leading in some cases to death. Among patients with the classical phenotype, four died, six had severe NPMDD, two had moderate NPMDD, and two were diagnosed as light NPMDD. In the patient with the intermediate phenotype the NPMDD was light.
Leucine level values were highly variable. Even in patients with classical presentation of the disease, these values ranged from 741 μmol/L to 3962 μmol/L, as shown in Chart 1 and in Table 8.
Table 8 – Genotype-phenotype correlations of Chilean MSUD patients (n = 18).
Patient
|
Birth year
|
First hospitalization (days)
|
Diagnosis Time (days)
|
LEU
(μmol/L)
|
Clinical Situation
|
Phenotype
|
Genotype
|
1
|
2002
|
10
|
10
|
741
|
Severe
|
Classic
|
c.[ 1013 C>T ]§ +[ 1013 C>T ] §
|
2
|
2002
|
10
|
9
|
750
|
Severe
|
Classic
|
c[.595_596delAG] + c.[641T>A]
|
3
|
1995
|
7
|
14
|
3560
|
Moderate
|
Classic
|
c.[641 T>A] + [1006 G>A]§
|
4
|
2006
|
32
|
13
|
2638
|
Light
|
Classic
|
c.[595_596delAG] +c.[641T>A]
|
5
|
2012
|
30
|
22
|
2600
|
Light
|
Classic
|
c.[641 T>A] + [641 T>A]
|
6
|
2001
|
48
|
11
|
750
|
Death
|
Classic
|
c.[1217 G>A]§+ c.[1217 G>A]¶
|
7
|
2004
|
5
|
30
|
1640
|
Death
|
Classic
|
c.[595_596delAG]+c.[641T>A]
|
8
|
2004
|
2
|
8
|
3962
|
Severe
|
Classic
|
c.[718 C>A]§ + [718 C>A]§
|
9
|
2003
|
30
|
21
|
2000
|
Severe
|
Classic
|
c.[641 T>A] + [641 T>A]
|
10
|
1997
|
20
|
9
|
993
|
Moderate/Severe
|
Classic
|
c.[392 G>T] +[595_596delAG]
|
11
|
2006
|
4
|
9
|
440
|
Severe
|
Classic
|
c.[641 T>A] + [641 T>A]
|
12
|
1997
|
32
|
16
|
1027
|
NA
|
Classic
|
c.[641 T>A] + [641 T>A]
|
13
|
1998
|
36
|
17
|
1090
|
Death
|
Classic
|
c.[392 G>T] + [392 G>T]
|
14
|
2003
|
10
|
30
|
1716
|
NA
|
Classic
|
c[.595_596delAG]+ [641T>A]
|
15
|
1999
|
90
|
210
|
1653
|
Light
|
Intermediate
|
c.[718 C>A]§ + [718 C>A]§
|
16
|
1996
|
14
|
17
|
1467
|
Moderate
|
Classic
|
c[.595_596delAG]+ [641 T>A]
|
17
|
2000
|
ND
|
17
|
1038
|
Death
|
Classic
|
c.[1067 C>T] + [1067 C>T]
|
18
|
2002
|
10
|
27
|
2075
|
NA
|
Classic
|
c.[641 T>A] + [641 T>A]
|
NA: Not Available.
NPMDD: Neuropsychomotor Developmental Delay.
In an attempt to establish correlations between genotype and phenotype, it was observed that among the mutations found in the homozygous state, the c. [1013 C>T] or p.Thr338Ile (new), c. [1217 G>A ] or p.Gly406Asp (new), c. [392 G>T] or p.Gly131Val, c. [1067 C>T] or p.Pro357Leu, and c. [641 T>A] or p.Ile214Lys expressed the classic form in 100% of the patients. The new mutation c. [718 C>A ] or p.Pro240Thr, when homozygous, was associated with the intermediate form of the disease in 50% of cases. All heterozygous mutations were found in patients with classic phenotypes (Chart 1).
Chart 1 – Representation of leucine values in μmol/L at diagnosis in patients classified with the classic phenotype, according to age at diagnosis.
Also, a representative chart of the correlation between leucine values and diagnostic age was drawn. Leucine values ranged very heterogeneously according to age at MSUD diagnosis; hence, it was not possible to identify any correlations among them (Chart 2).
Chart 2 –Correlation between leucine values in μmol/L and age at diagnosis in days, with a p value of 0.989.
Once the p.Ile214Lys mutation was identified in 11 of the 18 samples, it was possible to combine clinical information regarding these patients in an attempt to establish genotype/phenotype correlations. The following were assessed by Fisher`s exact test: correlation between the p.Ile214Lys mutation and the degree of NPMDD, the mutation and the classic and intermediate phenotypes, the allele type (hetero- and homozygous), and the severity of NPMDD, and the allele type and the classic and intermediate phenotypes. None of the correlations were associated with a probability value of significance smaller than 5%. Thus we cannot state that there is any correlation between these variables in this sampling (Table 9).
Table 9 – Associations between patients with the p.Ile214Lys mutation and the type of allele.
Mutation
|
NPMDD
|
Total
|
P-Value
|
|
Light/Moderate
|
Severe/Death
|
|
|
Others
|
1
|
6
|
7
|
0.282
|
Ile214Lys
|
4
|
4
|
8
|
|
|
|
|
|
|
Mutation
|
Phenotype
|
Total
|
P-Value
|
|
Intermediate
|
Classic
|
|
|
Others
|
1
|
6
|
7
|
0.245
|
Ile214Lys
|
0
|
11
|
11
|
|
|
|
|
|
|
Allele
|
NPMDD
|
Total
|
P-Value
|
|
Light/Moderate
|
Severe/Death
|
|
|
Heterozygous
|
3
|
2
|
5
|
0.394
|
Homozygous
|
1
|
2
|
3
|
|
|
|
|
|
|
Allele
|
Phenotype
|
Total
|
P-Value
|
|
Intermediate
|
Classic
|
|
|
Heterozygous
|
0
|
6
|
6
|
0.245
|
Homozygous
|
0
|
5
|
5
|
|
p-value<0,05
NPMDD: Neuropsychomotor Developmental Delay
Others: mutations identified in this study other than I214K.