Case: K.A./ BIODEF 264, male, Caucasian, 1 day

CasePresentingHPOs:  HP:0008297, HP:0040206, HP:0040210, HP:0001252

HPOText: Plasma Phe level of 290umol/L at 3 days old, 853 umol/L at 6 days old, and 1997 umol/L at 13 days old. Urinary pterins indicated high neopterin/biopterin ratio and increased levels of primapterin. At 1 month old, neopterin and biopterin levels in normal range. At one month old showed mild hypotonia (extermities and trunk), but at 2.5 years Phe diet stopped and patient developed normally.

CasePreviousTesting: n/a

CaseGenotypingMethod: Direct nucleotide-sequence determination of PCR products from blood samples was performed for the coding exons (2-4) of PCBD1.

Variant: NM_000281.3(PCBD1):c.292C>T (p.Gln98Ter)

ClinVar ID: 16799

No-stars, 1 submission from OMIM.

Homozygous, non-LOF.

gnomAD: MAF (Euro-NF)= 2.2291E-4 (29/126606) hets only. No homozygotes recorded

http://gnomad.broadinstitute.org/variant/10-72643730-G-A

AddCaseInfo: The only individual in the family diagnosed with HyperPhe.

VariantEvidence: The mutant Q97X protein was expressed in E.Coli to determine the solubility of the mutant enzyme and the activity. The mQ97X fragment was mostly found in the insoluble fraction of the supernatant. The remaining fraction obtained in the soluble fraction did retain some activity in vitro. Interestingly, when more Q97X was obtained in the soluble fraction (by growing the E.coli at 20 degrees C), the Km (Michaelis constant) was 2-3 times higher than wildtype (text only, no figures to compare).

Patient part of the BIODEF database: PMID:8830181

Case: I.B./ BIODEF 273, male, Ashkenazi Jewish, 3 days

CasePresentingHPOs: HP:0040210, HP:0008297,

HPOText: Plasma Phe levels were 240 umol/L at 3 days old, and 1845 umol/L at 14 days old. A low-Phe diet caused a rapid decline of Phe level to normal within 36 hours. An oral BH4–loading test (10 mg/kg body weight) resulted in a decrease of plasma Phe from 540 μmol/liter to 234 μmol/liter and then to 196 μmol/liter within 4 and 8 h, respectively. At 1 month, pterin and neurotransmitter in the CSF were normal. Starting a 2 month of age the patient was administered a low Phe diet and BH4 for 4 months. After the 4 months, the BH4 was discontinued, and Phe levels are between 120-300 umol/L at 5 years. Patient is developing normally.

CaseNOTHPOs: n/a

CasePreviousTesting: n/a

CaseGenotypingMethod: Direct nucleotide-sequence determination of PCR products from blood samples was performed for the coding exons (2-4) of PCBD1.

Variant: NM_000281.3(PCBD1):c.259G>T (p.Glu87Ter) 

ClinVar ID: 16795

No star, one submission for OMIM

Homozygous

gnomAD: MAF (Ashkenazi Jewish)= 1.183E-3 (12/10146) in heterozygous only. No homozygous reported.

http://gnomad.broadinstitute.org/variant/10-72643763-C-A

AddCaseInfo: The proband's older sister (J.B., 18 . months older) has the same genotype (homozygous for the T79I mutation), but was never noted as having HyperPhe, and was never treated. She has developed "normally." This family is recorded as Family B in the paper.

VariantEvidence: The mutant E86X/E87X protein was expressed in E.Coli to determine the solubility of the mutant enzyme and the activity. The mutant E86X/E87X fragment was mostly found in the insoluble fraction of the supernatant. The remaining fraction obtained in the soluble fraction did retain some enzyme activity in vitro. Interestingly, when more E86X/E87X was obtained in the soluble fraction (by growing the E.coli at 20 degrees C), the Km (Michaelis constant) was similar to wildtype levels (text only, no figures to compare).

Patient part of the BIODEF database: PMID:8830181

Case: J.S./ BIODEF 211, female, Hispanic, 3 days

CasePresentingHPOs: HP:0008297, HP:0001252, HP:0001270

HPOText: Phe levels were 9 umol/L on day 1, 756 ummol/L on day 28. Primapterinuria was diagnosed on day 8. Following oral BH4 administration (2 mg/kg/d), neopterin normalized, and biopterin as well as primapterin increased about eight-fold; the same results were observed when dihydrobiopterin and sepiapterin were administered. At 8.5 years old, on a normal diet, the patient is developing normally with no HyperPhe.

CasePreviousTesting: n/a

CaseGenotypingMethod: Direct nucleotide-sequence determination of PCR products from blood samples was performed for the coding exons (2-4) of PCBD1.

Variant: NM_000281.3(PCBD1):c.236C>T (p.Thr79Ile)

ClinVar ID: 16797

No stars, 1 submission for OMIM.

Homozygous

gnomAD: n/a

AddCaseInfo: Family history (Family S) of HyperPhe, as the patients older brother, T.S./ BIODEF212, who is 1.5 years older, showed the same biochemical pattern and is now considered normal and normally developing.

At 8.5 years old, on a normal diet, the patient J.S. is developing normally with no HyperPhe.

VariantEvidence: The T78I/T79I variant was retained completely within the insoluble fraction of the E.Coli lysate in which the recombinant proteins were produced. When the soluble fraction was examined for PCBD1 activity, no activity was observed. The authors note that the absence of activity could be due to the absence of the protein in the soluble fraction alone, instead of being indicative of the mutation causing a loss of enzyme activity. Only if the mutant recombinant protein could be retained in the soluble fraction can the true enzyme activity be assessed.

Patient part of the BIODEF database: PMID:8830181

Case: B.E./ BIODEF 272, male, 3 weeks

CasePresentingHPOs: HP:0008297, HP:0001252, HP:0001270

HPOText: Neonatal (3 days old) plasma screen showed Phe levels at 183 umol/L, which is below the normal cutoff. Repeat screen was only performed at 3 weeks old in which the Phe level was shown to be 1600 umol/L. Peak Phe level was at 3.5 weeks and was 2589 umol/L. Child had considerable Phe tolerance ( ~100mg/kg). Initial neopterin-to-biopterin ratio of 15. A BH4-loading test (6 mg/kg) at age 6 wk showed only a slight response (Phe at 0 h, 731 μmol/liter; at 3 h, 612 μmol/liter; and at 6 h, 502 μmol/liter). A week later, at a BH4 dose of 22 mg/kg, there was a definite response (Phe at 0 h, 773 μmol/liter; at 3 h, 517 μmol/liter; and at 6 h, 155 μmol/liter). CSF neurotransmitter concentrations were normal, suggesting a peripheral deficiency. At 4 months of age, the BH4 was discontinued, no low Phe diet, and the Phe levels have remained normal. at 3.5 years, patient developing normally.

CaseNOTHPOs:

CasePreviousTesting: n/a

CaseGenotypingMethod: Direct nucleotide-sequence determination of PCR products from blood samples was performed for the coding exons (2-4) of PCBD1.

Variant: NM_000281.3(PCBD1):c.292C>T (p.Gln98Ter)

ClinVar ID: 16799

No star, one submission for OMIM

Homozygous

gnomAD: MAF (Euro-NF)= 2.2291E-4 (29/126606) hets only. No homozygotes recorded

AddCaseInfo: Family E in the article, and the proband had an older sibling that also carried the homozygous mutation in PCBD1, she was not noted as having increased Phe plasma levels, nor was ever treated with BH4 or low-Phe diet.

VariantEvidence: The mutant Q97X protein was expressed in E.Coli to determine the solubility of the mutant enzyme and the activity. The mQ97X fragment was mostly found in the insoluble fraction of the supernatant. The remaining fraction obtained in the soluble fraction did retain some activity in vitro. Interestingly, when more Q97X was obtained in the soluble fraction (by growing the E.coli at 20 degrees C), the Km (Michaelis constant) was 2-3 times higher than wildtype (text only, no figures to compare).

Patient part of the BIODEF database: PMID:8830181

Case: Male, 5 days onset, Iran

CasePresentingHPOs: HP:0002572, HP:0004923 (Episodic vomiting, Hyperphenylalaninemia)

HPOsFreeText: Brown eyes

CaseNotHPOs: HP:0001249, HP:0032807 (Intellectual disability, Neonatal seizure)

NotHPOsFreeText: N/A

CaseAddInfo: Parents are consanguineous first cousins

CasePreviousTesting: Screened for HPAs by Guthrie test and on special diet

CaseGenotypingMethod: Genomic DNA was extracted from peripheral blood. PAH gene was screened but no mutations were found. BH4-metabolizing genes were screened via STR markers, with PCBD1 having a homozygous pattern. Direct sequencing revealed a homozygous termination codon extension.

Variant: NM_000281.4(PCBD1):c.313T>C (p.Ter105Gln)

ClinVar: 428616, https://www.ncbi.nlm.nih.gov/clinvar/variation/428616/

gnomAD: 0.00003301, https://gnomad.broadinstitute.org/variant/10-72643709-A-G

Case: Citron-1/ BIODEF 270, male, 4 days

CasePresentingHPOs: HP:0008297

HyperPhe was noted, with a plasma Phe level of 700uM. Normal neopterin and biopterin levels were observed in the urine, but the presence of primapterin was found.

CaseNOTHPOs:

CasePreviousTesting: n/a

CaseGenotypingMethod: PCR of exons 2-4 of PCBD1 followed by direct sequencing of the products.

Variant 1: NM_000281.3(PCBD1):c.259G>T (p.Glu87Ter)

ClinVar ID 1: 16795

Variant 2: NM_000281.3(PCBD1):c.244T>C (p.Cys82Arg)

ClinVar ID 2: 16796

Compound heterozygous

gnomAD 1: MAF (Ash-Jews)= 1.183E-3, all hets no homozygous.

gnomAD 2: MAF (Euro-NF)= 2.690E-5 all hets, no homozygous noted. http://gnomad.broadinstitute.org/variant/10-72643778-A-G

AddCaseInfo: Parents were tested and found to have normal Phe levels. The father carriers the termination variant, E87X, the mother harbors the C82R, and both parents are heterozygous for their respective PCBD1 variants.

VariantEvidence:  For the Glu87Ter variant, Thony et al., PMID: 9585615 showed that the mutant E86X/E87X protein was expressed in E.Coli to determine the solubility of the mutant enzyme and the activity. The mutant E86X/E87X fragment was mostly found in the insoluble fraction of the supernatant. The remaining fraction obtained in the soluble fraction did retain some enzyme activity in vitro. Interestingly, when more E86X/E87X was obtained in the soluble fraction (by growing the E.coli at 20 degrees C), the Km (Michaelis constant) was similar to wildtype levels (text only, no figures to compare).

For the C82R/C81R variant was analyzed by Ayling et al., 2000 (PMID:10924272) and was found to still be retained in the soluble fraction in bacteria expressing the recombinant protein. Furthermore, enzyme assays show that the soluble C82RPCBD1 variant retained activity, albeit at 40% of the wildtype.

Case: P.1017/ BIODEF 1035, male, Fars, 12.5 years (Dx)

CasePresentingHPOs: HP:0008297

Plasma Phe levels at 12.5 years 1116uM (per BIODEF database). Neopterin level at 1.52U.

CaseNOTHPOs:

CasePreviousTesting: n/a

CaseGenotypingMethod: PCR of D1 and direct sequencing.

Variant: NM_000281.3(PCBD1):c.313T>C (p.Ter105Gln)

ClinVar ID: 428616

Homozygous

gnomAD: MAF (South Asian)= 3.283E-5, hets only, no homozygous. http://gnomad.broadinstitute.org/variant/10-72643709-A-G

AddCaseInfo: The paper notes that the parents of all the individuals were tested. Consanguinity was not noted for parents, but may be likely due to the same variant.

VariantEvidence: No variant evidence was provided in this article. The mutation does change the termination codon and thus extends the protein by 16 amino acids.

Case: BIODEF 306, male, Caucasian, 7 days (per BIODEF website)

CasePresentingHPOs: HP:0008297, HP:0040210

Increased primapterin levels in urine.

CaseNOTHPOs:

CasePreviousTesting: n/a

CaseGenotypingMethod: Assumed PCR of exons 2-4 of PCBD1 followed by direct sequencing. Psuedogenes were not assessed.

Variant: NM_000281.3(PCBD1):c.289G>A (p.Glu97Lys)

ClinVar ID: 91907

Homozygous

gnomAD: MAF (Euro-NF)= 2.320E-5 in hets only, no homozygous. http://gnomad.broadinstitute.org/variant/10-72643733-C-T

AddCaseInfo: Parents noted as consanguineous on the BIODEF website.

VariantEvidence: Expression of recombinant protein in bacteria revealed that the E97K are present in the insoluble fraction compared to wildtype which is found in the soluble. The little amount retained of each in the soluble fraction showed that they retained activity. Dehydratase activity of the patients duodenal mucosa cells showed reduced activity of he E97K (17% activity) mutant compared to wildtype , indicating that this mutation severely reduces the activity of PCBD1.

Case: Patient 4/ BIODEF 263, female, 1 week

CasePresentingHPOs: HP:0008297, HP:0040210, HP:0040206

At 1 week old plasma showed Phe levels at 258 umol/L. Repeat screening at 9 days old showed Phe levels at 900 umol/L. Amino acid quantitation of plasma showd a Phe level of 1200 umol/L. Urine analysis showed low BH4, high neopterin, low biopterin, and the presence of primapterin. A diet of low protein and phenylalanine were begun, and continue with 100 mg/kg of phenylalanine per day. Patient is 6 years old and developing normally.

CaseNOTHPOs:

CasePreviousTesting: n/a 

CaseGenotypingMethod: PCR of exons 2-4 of PCBD1 followed by direct sequencing.

Variant: NM_000281.3(PCBD1):c.292C>T (p.Gln98Ter)

ClinVar ID: 16799

Homozygous

gnomAD: n/a

AddCaseInfo: No information about the family.

VariantEvidence: Expression of recombinant protein in bacteria revealed that both the Q98X variants are present in the insoluble fraction compared to wildtype which is found in the soluble. the little amount retained of each in the soluble fraction showed that they retained activity.

Case: Patient 3/ BIODEF 216, male, Morrocan, 1 month

CasePresentingHPOs: HP:0008297, HP:0040210, HP:0040206

HyperPhe presented a 1 month old with plasma levels of 1850 umol/L. A low Phe diet was initiated for 8 months. Urinary pterin profile showed high neopterin, low biopterin, and elevated primapterin. The diet was ceased at 2 years old, and the child has normal Phe levels (110 umol/L).

CaseNOTHPOs:

CasePreviousTesting: n/a

CaseGenotypingMethod: PCR of exons 2-4 of PCBD1 followed by direct sequencing.

Variant: NM_000281.3(PCBD1):c.289G>A (p.Glu97Lys)

ClinVar ID: 91907

Homozygous

gnomAD: MAF (Euro-NF)= 2.320E-5 in hets only, no homozygous. http://gnomad.broadinstitute.org/variant/10-72643733-C-T

AddCaseInfo: Parents are consanguineous. Older brother (by 17 years) presented with HyperPhe in the neonatal period, but no information on whether he was a carrier of the mutation.

VariantEvidence: Expression of recombinant protein in bacteria revealed that the E97K are present in the insoluble fraction compared to wildtype which is found in the soluble. the little amount retained of each in the soluble fraction showed that they retained activity. Per Ayling et al., 2000 (PMID:10924272), another patient (BIODEF 306) that was homozygous for the same E97K mutation, showed reduced activity of PCBD1 from duodenal mucosa cells compared to wildtype (17% activity), indicating that this mutation severely reduces the activity of PCBD1.

Case: Patient 2/ BIODEF 319, female, Caucasian, 10 days

CasePresentingHPOs: HP:0008297, HP:0040210, HP:0040206

HPOtext: Conceived by IVF. HyperPhe detected by neonatal screen, 240umol/L. Plasma Phe levels at 10 days olf was 585 umol.L. BH4 loading test normalized Phe levels. Urine levels showed high neopterin and low biopterin. PCD levels in patient's blood sample were normal. Urine reevalutation showed increased primapterin, again indicating PCD deficiency. cofactor replacement therapy was started, which improved growth trajectories on a normal diet. At 2 years old is still on diet and developing normally.

CaseNOTHPOs:

CasePreviousTesting: n/a

CaseGenotypingMethod: PCR of exons 2-4 of PCBD1 followed by direct sequencing.

Variant 1: NM_000281.3(PCBD1):c.289G>A (p.Glu97Lys)

ClinVar ID 1: 91907

Variant 2: NM_000281.3(PCBD1):c.292C>T (p.Gln98Ter)

ClinVar ID 2: 16799

compound heterozygous

gnomAD variant 1: MAF (Euro-NF)= 2.320E-5 in hets only, no homozygous. http://gnomad.broadinstitute.org/variant/10-72643733-C-T

gnomAD variant 2 n/a

AddCaseInfo: Father carries E97K variant, mother carrier of the Q98X variant, neither are symptomatic.

VariantEvidence: Expression of recombinant protein in bacteria revealed that both the E97K and Q98X variants are present in the insoluble fraction compared to wildtype which is found in the soluble. the little amount retained of each in the soluble fraction showed that they retained activity.