Diagnosis and high incidence of hyperornithinemia-hyperammonemia-homocitrullinemia (HHH) syndrome in northern Saskatchewan

Abstract

Mutations in the SLC25A15 gene, encoding the human inner mitochondrial membrane ornithine transporter, are thought to be responsible for hyperornithinemia-hyperammonemia-homocitrullinemia (HHH) syndrome, a rare autosomal recessive condition. HHH syndrome has been detected in several small, isolated communities in northern Saskatchewan (SK). To determine the incidence of HHH syndrome in these communities, a PCR method was set up to detect F188Δ, the common French-Canadian mutation. Neonatal blood spots collected from all newborns from the high risk area were genotyped for the F188Δ mutation for seven consecutive years. Using DNA analysis, we estimated that the heterozygote frequency for the mutant allele for HHH syndrome to be about 1 in 19 individuals, predicting one affected child with HHH syndrome for approximately every 1,500 individuals (1 in 1,550 live births; 1 child every 12 years) in this isolated population. The frequency for the mutant allele for HHH syndrome in this isolated community is probably the highest in the world for this rare disorder. We determined that ornithine levels, by tandem mass spectrometry, were not abnormal in newborns with F188Δ mutation, carriers and normals. Ornithine rises to abnormally high levels at some time after birth well past the time that the newborn screening blood spot is collected. The timing or the reasons for the delayed rise of ornithine in affected children with HHH syndrome have not been determined. Newborn screening for HHH Syndrome in this high risk population is only possible by detection of the mutant allele using DNA analysis.

Appendices

Appendix A: Hardy–Weinberg Equation

Hardy-Weinberg equation

$$ {P^2} + 2pq + {q^2} = 1 $$

(1)

Using the total population number of 5,990 and a confirmed diagnosis of HHH in 4 children from the area,Frequency of HHH in the three communities, q ², is:

$$ {q^2} = {4 \mathord{\left/{\vphantom {4 {5990 = 0.00067\left( {{1 \mathord{\left/{\vphantom {1 {0.00067 = 1492.54;\;{\hbox{i}}{\hbox{.e}}{.,}\; \sim {1}\;{\hbox{in}}\;{1500}\;{\hbox{individuals}}}}} \right.} {0.00067 = 1492.54;\;{\hbox{i}}{\hbox{.e}}{.,}\; \sim {1}\;{\hbox{in}}\;{1500}\;{\hbox{individuals}}}}} \right)}}} \right.} {5990 = 0.00067\left( {{1 \mathord{\left/{\vphantom {1 {0.00067 = 1492.54;\;{\hbox{i}}{\hbox{.e}}{.,}\; \sim {1}\;{\hbox{in}}\;{1500}\;{\hbox{individuals}}}}} \right.} {0.00067 = 1492.54;\;{\hbox{i}}{\hbox{.e}}{.,}\; \sim {1}\;{\hbox{in}}\;{1500}\;{\hbox{individuals}}}}} \right)}} $$

Frequency of the mutant allele, q, is:

$$ q = \surd 0.00067 = 0.0259\left( {{1 \mathord{\left/{\vphantom {1 {0.0259 = 38.61;\;{\hbox{i}}{\hbox{.e}}{.,}\;{1}\;{\hbox{in}}\;{38}\;{\hbox{individuals}}}}} \right.} {0.0259 = 38.61;\;{\hbox{i}}{\hbox{.e}}{.,}\;{1}\;{\hbox{in}}\;{38}\;{\hbox{individuals}}}}} \right) $$

Frequency of normal allele, p, is:

$$ p = 1 - q = 1 - 0.0259 = 0.9741\left( {97\% } \right) $$

Frequency of heterozygote, 2pq, is:

$$ 2pq = 2\left( {0.9741 \times 0.0259} \right) = 0.0504\left( {{1 \mathord{\left/{\vphantom {1 {0.0504 = 19.84;\;{\hbox{i}}{\hbox{.e}}{.,}\;{1}\;{\hbox{in}}\;{19}{.8}\;{\hbox{individuals}}}}} \right.} {0.0504 = 19.84;\;{\hbox{i}}{\hbox{.e}}{.,}\;{1}\;{\hbox{in}}\;{19}{.8}\;{\hbox{individuals}}}}} \right) $$

Based on 657 births, we should have found 33 heterozygotes (657/19.8)

The likelihood of two (2) carriers mating to produce an affected off-spring is:

$$ 2pq \times 2pq \times 0.25 = 0.000635\left( {{1 \mathord{\left/{\vphantom {1 {0.00064 = 1562.5;\;{\hbox{i}}{\hbox{.e}}{.,}\; \sim {1}\;{\hbox{in}}\;{1550}\;{\hbox{births}}}}} \right.} {0.00064 = 1562.5;\;{\hbox{i}}{\hbox{.e}}{.,}\; \sim {1}\;{\hbox{in}}\;{1550}\;{\hbox{births}}}}} \right) $$

Based on Table 1 (birth rate of 126 live births/year), one (1) child with HHH syndrome will be born every 12 years (1550 births/126 births/year).

Appendix B: test for deviation from Hardy–Weinberg equilibrium

The Hardy-Weinberg expectation (Exp) of each genotype is given by the sum of the probability of each genotype multiplied by the total. Thus, in our study, using the results from Table 2 (Results of F188Δ genotype analysis), we obtain the following:

$$ \begin{array}{*{20}{c}} {{\hbox{Exp}}\;\left( {{\hbox{wild}}\;{\hbox{genotype}}} \right) = {{\hbox{p}}^2}{\hbox{n = }}{{\left( {0.9741} \right)}^2} \times 657 = 0.9489 \times 657 = 623.43} \\{{\hbox{Exp}}\;\left( {{\hbox{carrier}}\;{\hbox{genotype}}} \right) = {\hbox{2pqn = 2}} \times \left( {0.9741 \times 0.0259 \times 657} \right) = 2 \times 16.58 = 33.16} \\{{\hbox{Exp}}\;\left( {{\hbox{mutant}}\;{\hbox{genotype}}} \right){ = }{{\hbox{q}}^2}{\hbox{n = }}{{\left( {0.0259} \right)}^2} \times 657 = 0.44} \\\end{array} $$

Pearson’s chi-square (X ²) test, at 1 degree of freedom (number of genotype−number of alleles = 3 − 2 = 1) and 5% significance level is given by the following:

$$ \begin{array}{*{20}{c}} {{X^2}} & { = {{{{\left[ {{\hbox{number}}\;{\hbox{of}}\;{\hbox{wild}}\;{\hbox{genotype}}\;{ - }\;{\hbox{Exp}}\left( {{\hbox{wild}}\;{\hbox{genotype}}} \right)} \right]}^2}} \mathord{\left/{\vphantom {{{{\left[ {{\hbox{number}}\;{\hbox{of}}\;{\hbox{wild}}\;{\hbox{genotype}}\;{ - }\;{\hbox{Exp}}\left( {{\hbox{wild}}\;{\hbox{genotype}}} \right)} \right]}^2}} {{\hbox{Exp}}\left( {{\hbox{wild}}\;{\hbox{genotype}}} \right)}}} \right.} {{\hbox{Exp}}\left( {{\hbox{wild}}\;{\hbox{genotype}}} \right)}}} \\{} & + \\{} & {{{{{\left[ {{\hbox{number}}\;{\hbox{of}}\;{\hbox{carriers}}\;{ - }\;{\hbox{Exp}}\left( {{\hbox{carrier}}\;{\hbox{genotype}}} \right)} \right]}^2}} \mathord{\left/{\vphantom {{{{\left[ {{\hbox{number}}\;{\hbox{of}}\;{\hbox{carriers}}\;{ - }\;{\hbox{Exp}}\left( {{\hbox{carrier}}\;{\hbox{genotype}}} \right)} \right]}^2}} {{\hbox{Exp}}\left( {{\hbox{carrier}}\;{\hbox{genotype}}} \right)}}} \right.} {{\hbox{Exp}}\left( {{\hbox{carrier}}\;{\hbox{genotype}}} \right)}}} \\{} & + \\{} & {{{{{\left[ {{\hbox{number}}\;{\hbox{of}}\;{\hbox{mutants}}\;{ - }\;{\hbox{Exp}}\left( {{\hbox{mutant}}\;{\hbox{genotype}}} \right)} \right]}^2}} \mathord{\left/{\vphantom {{{{\left[ {{\hbox{number}}\;{\hbox{of}}\;{\hbox{mutants}}\;{ - }\;{\hbox{Exp}}\left( {{\hbox{mutant}}\;{\hbox{genotype}}} \right)} \right]}^2}} {{\hbox{Exp}}\left( {{\hbox{mutant}}\;{\hbox{genotype}}} \right)}}} \right.} {{\hbox{Exp}}\left( {{\hbox{mutant}}\;{\hbox{genotype}}} \right)}}} \\\end{array} $$

Thus, for our study group,

$$ \begin{array}{*{20}{c}} {{X^2}} & { = {{{{\left[ {628 - 623.43} \right]}^2}} \mathord{\left/{\vphantom {{{{\left[ {628 - 623.43} \right]}^2}} {{{623.43 + {{\left[ {25 - 33.16} \right]}^2}} \mathord{\left/{\vphantom {{623.43 + {{\left[ {25 - 33.16} \right]}^2}} {{{33.16 + {{\left[ {4 - 0.44} \right]}^2}} \mathord{\left/{\vphantom {{33.16 + {{\left[ {4 - 0.44} \right]}^2}} {0.44}}} \right.} {0.44}}}}} \right.} {{{33.16 + {{\left[ {4 - 0.44} \right]}^2}} \mathord{\left/{\vphantom {{33.16 + {{\left[ {4 - 0.44} \right]}^2}} {0.44}}} \right.} {0.44}}}}}}} \right.} {{{623.43 + {{\left[ {25 - 33.16} \right]}^2}} \mathord{\left/{\vphantom {{623.43 + {{\left[ {25 - 33.16} \right]}^2}} {{{33.16 + {{\left[ {4 - 0.44} \right]}^2}} \mathord{\left/{\vphantom {{33.16 + {{\left[ {4 - 0.44} \right]}^2}} {0.44}}} \right.} {0.44}}}}} \right.} {{{33.16 + {{\left[ {4 - 0.44} \right]}^2}} \mathord{\left/{\vphantom {{33.16 + {{\left[ {4 - 0.44} \right]}^2}} {0.44}}} \right.} {0.44}}}}}}} \\{} & { = \left( {{{20.88} \mathord{\left/{\vphantom {{20.88} {623.43}}} \right.} {623.43}}} \right) + \left( {{{66.56} \mathord{\left/{\vphantom {{66.56} {33.16}}} \right.} {33.16}}} \right) + \left( {{{12.67} \mathord{\left/{\vphantom {{12.67} {0.44}}} \right.} {0.44}}} \right)} \\{} & { = 0.033 + 2.01 + 28.79} \\{} & { = 30.83} \\\end{array} $$

The 5% significant level for 1 degree of freedom is 3.84 (from X ² table). Since the calculated X ² value (30.83) is greater than this value (30.83 vs 3.84), the null hypothesis (the population is in Hardy-Weinberg equilibrium) is rejected.