Methylmalonic Aciduria and Homocystinuria Panel

Cobalamin (Cbl or vitamin B12) is an important cofactor in homocysteine metabolism and in branched-chain amino acid and odd-chain fatty acid catabolism. Cbl is necessary for the production of two vital cofactors: Adenosyl-cobalamin (AdoCbl), which is required in the mitochondria for methylmalonyl CoA mutase to convert L-methylmalonyl-CoA to succinyl CoA, and Methyl-cobalamin (MeCbl), which is required in the cytoplasm for methionine synthase to convert homocysteine to methionine. A series of inherited disorders of cobalamin (Cbl) metabolism and transport, designated cblA through cblJ and cblX, as well as transcobalamin II (TC II) deficiency and a few other cblC-like disorders (caused by variants in PRDX1, THAP11, or ZNF143), can lead to elevated levels of methylmalonic acid in the blood and urine and/or hyperhomocysteinemia/homocystinuria. While cblC and the cblC-like disorders, cblD (classic), cblF, cblJ, cblX and TC II deficiencies all present with combined methylmalonic aciduria and homocystinuria, the remaining disorders present with either isolated methylmalonic aciduria or homocystinuria (Gailus et al. 2010. PubMed ID: 20127417; Sloan et al. 1993. PubMed ID: 20301503; Yu et al. 2013. PubMed ID: 24011988; Watkins and Rosenblatt. 2014).

CblC, cblD, cblF and cblJ show clinical variability, but all may be associated with developmental delay, hematological abnormalities, and severe neurological problems such as seizures (Watkins and Rosenblatt. 2014). Patients diagnosed with cblX and the other cblC-like disorders tend to be more severely affected, and may also present with microcephaly, brain abnormalities, facial dysmorphism and other congenital abnormalities (Yu et al. 2013. PubMed ID: 24011988). TC II deficient patients typically present in early infancy with gastrointestinal symptoms and potentially severe hematological deficiencies, and may eventually develop neurological complications if treatment is delayed (Häberle et al. 2009. PubMed ID: 19373259; Ünal et al. 2015. PubMed ID: 25914105). Most affected individuals are diagnosed as infants and children, but a few patients with cblC disorder are not diagnosed until the teenage years or as young adults (Watkins and Rosenblatt 2014). Some patients with increased propionylcarnitine concentrations are identified through newborn screening (Rutsch et al. 2009. PubMed ID: 19136951; Coelho et al. 2012. PubMed ID: 22922874). For further clinical information, please see the individual gene summaries for each of these deficiencies (MMACHC for cblC disease, MMADHC for cblD disease, LMBRD1 for cblF disease, ABCD4 for cblJ disease, HCFC1 for cblX disease and TCN2 for TC II deficiency).

In addition to the disorders detailed above, this sequencing panel also covers the CD320 gene, which has been associated with transient methylmalonic aciduria and/or homocystinuria in a small number of individuals. CD320 deficiency has not been associated with the neurological or hematological symptoms associated with the cblC, cblD, cblF, cblJ, cblX, or cblC-like types of methylmalonic aciduria and homocystinuria. However, individuals carrying pathogenic variants in CD320 may be detected via newborn screening (Quadros et al. 2010. PubMed ID: 20524213; Karth et al. 2012. PubMed ID: 22819238; Watkins and Rosenblatt. 2014).

The disorders included in this panel are very rare in most populations, with a frequency of ~1/50,000 or less, depending on the disorder (Sloan et al. 2018. PubMed ID: 20301503). Obtaining an accurate molecular diagnosis may help in determining the patient’s prognosis, planning for the best treatment and/or management of symptoms, and allowing for reproductive planning.

Combined methylmalonic aciduria and homocystinuria can result from pathogenic variants in at least ten different genes associated with inborn errors of cobalamin transport or metabolism. Nine of the cobalamin disorders covered by this sequencing panel (cblC, cblD, cblF, cblJ, TC II deficiency, transient methylmalonic aciduria and/or homocystinuria due to CD320 deficiency, and the cblC-like disorders caused by PRDX1, THAP11, or ZNF143 variants) are inherited in an autosomal recessive manner. The cblX disorder, caused by HCFC1 pathogenic variants, is inherited in an X-linked recessive manner. Of note, female carriers of HCFC1 pathogenic variants are generally unaffected (Sloan et al. 2018. PubMed ID: 20301503). Pathogenic de novo variants have occasionally been reported in HCFC1. To our knowledge, de novo variants are not a common cause of disease for the remaining genes in this panel.

It has been recently reported that patients compound heterozygous for a pathogenic variant in MMACHC and a variant that disrupts the intron 5 splice acceptor site of the PRDX1 gene (which is adjacent to the MMACHC gene) present with cblC type methylmalonic aciduria and homocystinuria. Disruption of the splice site at the junction of intron 5 and exon 6 of PRDX1 was shown to lead to PRDX1 exon 6 skipping, transcription of antisense MMACHC RNA, and resultant hypermethylation of the MMACHC promoter and exon 1. This lead to silencing of MMACHC gene expression (Guéant et al. 2018. PubMed ID: 29302025).

See individual gene summaries for additional information about molecular biology of the gene products and spectra of pathogenic variants.

Although the overall sensitivity of this test panel is not precisely known, most pathogenic variants reported for the genes in this panel are of the type which can be detected using standard PCR and automated sequencing methods.

In one large study examining 118 patients with confirmed cblC disease, 227 pathogenic variants were identified on 236 alleles, for an overall sensitivity of ~96% (Lerner-Ellis et al. 2009. PubMed ID: 19370762). Regarding MMADHC pathogenic variant detection, two studies which examined a total of 10 patients with cblD defect found MMADHC pathogenic variants in either a compound heterozygous or homozygous state in all affected individuals (Coelho et al. 2008. PubMed ID: 18385497; Miousse et al. 2009. PubMed ID: 19058814). Similarly, in the largest published study of patients with cblF deficiency, all 12 affected patients were found to have LMBRD1 pathogenic variants in either a compound heterozygous or homozygous state (Rutsch et al. 2009. PubMed ID: 19136951). Regarding HCFC1 pathogenic variant detection, Yu and colleagues (2013. PubMed ID: 24011988) reported pathogenic variants in 13 out of 17 male patients who were suspected to have or cellularly diagnosed with cblC type methylmalonic aciduria and homocystinuria but did not harbor pathogenic variants in the MMACHC gene. Finally, based on collective totals of transcobalamin II deficient patients reported in the literature, the clinical sensitivity of TCN2 sequencing is estimated to be ~73% (29 pathogenic TCN2 alleles out of 40 total alleles, with only one patient counted if multiple family members were affected) (Li et al. 1994a. PubMed ID: 7849710; Li et al. 1994b. PubMed ID: 7980584; Namour et al. 2003. PubMed ID: 14632784; Prasad et al. 2008. PubMed ID: 18956254 ; Häberle et al. 2009. PubMed ID: 19373259; Ratschmann et al. 2009. PubMed ID: 19581117; Nissen et al. 2010. PubMed ID: 20607612; Schiff et al. 2010. PubMed ID: 20352340; Ünal et al. 2015. PubMed ID: 25914105; Pupavac et al. 2016. PubMed ID: 26827111).

For cblJ disorder, CD320 deficiency, cblC type methylmalonic acidemia due to variants in PRDX1, and combined methylmalonic acidemia and homocystinuria due to variants in THAP11 or ZNF143, clinical sensitivity cannot be estimated because only a small number of patients with ABCD4, CD320, PRDX1, THAP11, or ZNF143 pathogenic variants have been reported.

This test is performed using Next-Gen sequencing with additional Sanger sequencing as necessary.

This panel provides 100% coverage of all coding exons of the genes plus 10 bases of flanking noncoding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. We define coverage as ≥20X NGS reads or Sanger sequencing. PGnome panels typically provide slightly increased coverage over the PGxome equivalent. PGnome sequencing panels have the added benefit of additional analysis and reporting of deep intronic regions (where applicable).

Dependent on the sequencing backbone selected for this testing, discounted reflex testing to any other similar backbone-based test is available (i.e., PGxome panel to whole PGxome; PGnome panel to whole PGnome).