Genetic Mapping and Exome Sequencing Identify Variants Associated with Five Novel Diseases

The Clinic for Special Children (CSC) has integrated biochemical and molecular methods into a rural pediatric practice serving Old Order Amish and Mennonite (Plain) children. Among the Plain people, we have used single nucleotide polymorphism (SNP) microarrays to genetically map recessive disorders to large autozygous haplotype blocks (mean = 4.4 Mb) that contain many genes (mean = 79). For some, uninformative mapping or large gene lists preclude disease-gene identification by Sanger sequencing. Seven such conditions were selected for exome sequencing at the Broad Institute; all had been previously mapped at the CSC using low density SNP microarrays coupled with autozygosity and linkage analyses. Using between 1 and 5 patient samples per disorder, we identified sequence variants in the known disease-causing genes SLC6A3 and FLVCR1, and present evidence to strongly support the pathogenicity of variants identified in TUBGCP6, BRAT1, SNIP1, CRADD, and HARS. Our results reveal the power of coupling new genotyping technologies to population-specific genetic knowledge and robust clinical data

An X-linked cobalamin disorder caused by mutations in transcriptional coregulator HCFC1

Derivatives of vitamin B12 (cobalamin) are essential cofactors for enzymes required in intermediary metabolism. Defects in cobalamin metabolism lead to disorders characterized by the accumulation of methylmalonic acid and/or homocysteine in blood and urine. The most common inborn error of cobalamin metabolism, combined methylmalonic acidemia and hyperhomocysteinemia, cblC type, is caused by mutations in MMACHC. However, several individuals with presumed cblC based on cellular and biochemical analysis do not have mutations in MMACHC. We used exome sequencing to identify the genetic basis of an X-linked form of combined methylmalonic acidemia and hyperhomocysteinemia, designated cblX. A missense mutation in a global transcriptional coregulator, HCFC1, was identified in the index case. Additional male subjects were ascertained through two international diagnostic laboratories, and 13/17 had one of five distinct missense mutations affecting three highly conserved amino acids within the HCFC1 kelch domain. A common phenotype of severe neurological symptoms including intractable epilepsy and profound neurocognitive impairment, along with variable biochemical manifestations, was observed in all affected subjects compared to individuals with early-onset cblC. The severe reduction in MMACHC mRNA and protein within subject fibroblast lines suggested a role for HCFC1 in transcriptional regulation of MMACHC, which was further supported by the identification of consensus HCFC1 binding sites in MMACHC. Furthermore, siRNA-mediated knockdown of HCFC1 expression resulted in the coordinate downregulation of MMACHC mRNA. This X-linked disorder demonstrates a distinct disease mechanism by which transcriptional dysregulation leads to an inborn error of metabolism with a complex clinical phenotype

Microcephaly and chorioretinopathy due to a homozygous <i>TUBGCP6</i> mutation.

<p>(<b>A</b>) An affected infant has marked microcephaly (>4SD below normal), a receding forehead, diminutive anterior fontanelle, and sutural ridging. She has cognitive delay and visual impairment but is socially engaged. (<b>B</b>) Head circumference and length plots for Mennonite microcephaly patients. (<b>C</b>) Brain magnetic resonance imaging (MRI) shows diffuse pachygyria, normal myelination, and (<b>D</b>) a hypoplastic cerebellar vermis.</p

Corticobasal degeneration in the brain of an infant who died from a homozygous <i>BRAT1</i> mutation.

<p>(<b>A</b>) Throughout frontal, occipital and temporal cortex, there is marked neuronal loss, gliosis with astrocytes (arrowheads) and swollen oligodendroglia. The arrow indicates a perivascular microcalcification (superior frontal gyrus, deep cortex, 10×). (<b>B</b>) The anterior hippocampus is smaller than expected and there is neuronal loss and gliosis in zone CA-1 (Sommer's sector), demarcated from the CA-2 sector by the dotted line (4×). (<b>C</b>) At 60× magnification, the putamen shows a paucity of neurons, abundant Alzheimer Type 2 astrocytes (arrowhead) and scattered microglial nodules (arrow). Heterologous overexpression of N-terminal FLAG-tagged human BRAT1 (<b>D</b>) and hBRAT1 c.638_639insA (<b>E</b>) in mouse IMCD3 cells. Wild-type Brat1 localizes to the nucleus and cytoplasm of mIMCD3 cells. Mutant Brat1 (c.638_639insA) does not localize to the nucleus and instead forms punctate aggregations in the cytoplasm. Similar results were obtained in hARPE-19 cells (data not shown). (<b>F</b>) RT-PCR demonstrating the stability of overexpressed human BRAT1 transcripts (∼2.6 kb) in hARPE-19 cells. A B-actin amplicon (∼450 bp) was used as a loading control on the same gel. (<b>G</b>) Western blot of lysates from human ARPE-19 cells transiently transfected with wt hBRAT1 displaying FLAG-hBRAT1 fusion protein at ∼90 kDa or with hBRAT1 c.638_639insA displaying the truncated FLAG-hBRAT1 mutant fusion protein at ∼44.5 kDa (FLAG-tag and linker = 3.1 kDa). B-actin was labeled as a loading control.</p

Genetic mapping of seven Plain disorders.

<p>The results of autozygosity mapping using Affymetrix GeneChip 10 K or 50 K SNP microarrays are plotted for each disorder. The x-axis depicts chromosomal location on autosomes. Yellow peaks represent the number of contiguous homozygous SNPs shared by affected individuals and the purple peaks depict location scores. (<b>A</b>) Autozygosity mapping of two affected individuals identified a single, large block of homozygosity on chromosome 6 (yellow peak). Genotyping of 6 unaffected siblings excluded this homozygous block, but identified 12 genomic regions greater than 5 Mb in size (red peaks) that were consistent with linkage in the family. (<b>B</b>) List of genomic regions consistent with linkage in the single nuclear family with infantile parkinsonism-dystonia syndrome. Panels <b>C–H</b> provide mapping plots for the other 6 disorders. For two disorders (<b>C,D</b>), 50 K microarrays were used after 10 K microarrays failed to unequivocally localize the disease gene. The other four disorders (<b>E–H</b>) were mapped with 10 K microarrays.</p

Overexpression of mouse HARS in mIMCD3 cells.

<p>(<b>A</b>) Wild-type N-terminal FLAG-HARS localizes to the cytoplasm. (<b>B</b>) Mutant N-terminal FLAG-HARS (p.Tyr454Ser) localizes to the cytoplasm in a manner that is indistinguishable from the wild-type localization shown in <b>A</b>. Transfected and non-transfected cells were labeled with anti-FLAG M2 monoclonal antibody and AlexaFluor 488-conjugated anti-mouse IgG₁ (green fluorescence). (<b>C</b>) Reaction velocity vs. human tRNA^His concentration for histidine aminoacylation of tRNA^His by wild-type murine HARS (HARS) and p.Tyr454Ser (Y454S) HARS. Scale bar = 10 µm in <b>A</b> and <b>B</b>.</p

Pathogenic Variant Summary.

<p>Pathogenic Variant Summary.</p

Genetic Mapping Summary.

<p>Genetic Mapping Summary.</p

Symptomatic epilepsy and skull dysplasia due to a homozygous <i>SNIP1</i> mutation.

<p>(<b>A</b>) Two affected brothers presented with severe psychomotor delay, intractable seizures, bulbous nose, wide mouth and tongue, broad jaw with protuberant angles, short hands, short tapered fingers, and broad thumbs. (<b>B,C</b>) Brain MRI (B, axial T2; C, coronal T1) MRI showed enlarged ventricles, a thin corpus callosum, hypomyelination, and an irregular, undulating skull surface. (<b>D</b>) Mouse FLAG-SNIP1 (wt) fusion protein, when transiently overexpressed in mIMCD3 cells, localizes to the nucleus in a punctate pattern consistent with transcriptional complexes. (<b>E</b>) Mouse FLAG-SNIP1 (p.Glu353Gly) localizes to the nucleus, but with a more aggregated distribution. (<b>F</b>) <i>Top</i> – Reverse-transcriptase PCR from three wild-type mSNIP1-transfected samples and four c.1058A>G mSNIP1-transfected samples. mSNIP1 amplicon – 400 bp. mGAPDH (loading control) – 1037 bp amplicon. <i>Bottom</i> – Western blot of lysates from mIMCD3 cells transiently transfected with wt (lanes wt_1&2) or c.1058A>G (lanes p.Glu353Gly_1&2) mSNIP1 displaying the FLAG-mSNIP fusion protein at ∼48 kDa. The 140-kDa non-specific band was used as a loading control. Data shown are two out of four replicate sets of transfections.</p