Population-Wide Duchenne Muscular Dystrophy Carrier Detection by CK and Molecular Testing

Abstract

Carrier screening of Duchenne muscular dystrophy (DMD) has not been widely evaluated. To identify definite DMD female carriers prior to or in early pregnancy, we studied a large population of reproductive age females and provided informed reproductive options to DMD carriers. 37268 females were recruited from the Hangzhou Family Planning Publicity and Technology Guidance Station/Hangzhou Health Service Center for Children and Women, Hangzhou, China, between October 10, 2017, and December 16, 2018. CK activity was measured with follow-up serum DMD genetic testing in subjects with hyperCKemia, defined as CK > 200 U/L. The calculated upper reference limit (97.5^th percentile) of serum creatine kinase (CK) for females aged 20-50 years in this study was near the reference limit recommended by the manufacturer (200 U/L), above which was defined as hyperCKemia. 427 females (1.2%) harbored initially elevated CK, among which 281 females (response rate of 65.8%) accepted CK retesting. DMD genetic testing was conducted on 62 subjects with sustained serum CK > 200 U/L and 16 females with a family history of DMD. Finally, 6 subjects were confirmed to be DMD definite carriers. The estimated DMD female carrier rate in this study was 1 : 4088 (adjusting for response rate), an underestimated rate, since only 50% to 70% of DMD female carriers manifest elevated serum CK, and carriers in this study may have been missed due to lack of follow-up or inability to detect all DMD pathogenic variants by current genetic testing.

Figure 1

Screening workup of asymptomatic creatine kinase elevation in females.

Figure 2

Results of screening program for DMD carriers in Hangzhou city, Zhejiang province, China, during October 10, 2017, to December 16, 2018.

Figure 3

CK characteristics of the study population by age. Bars indicate 2.5^th and 97.5^th percentile.

Figure 4

Pedigrees (left) and the results of DMD gene testing (right). (a) Pedigree of the family of carrier 1 (III2). The carrier status is indicated by a circle with a dot, sloid symbols (affected), clear symbols (unaffected). The result of MLPA analysis on the right shows exon 44del in DMD gene (the horizontal axis represents exon number of DMD, and the vertical axis shows the relative peak area ratios compared to the mean of control samples). (b) Pedigree of the family of carrier 2 (I2). The carrier status is indicated by a circle with a dot, sloid symbols (affected), clear symbols (unaffected). The result of MLPA on the right shows exon 43del in DMD gene. (c) Pedigree of the family of carrier 3 (I2). The carrier status is indicated by a circle with a dot, sloid symbols (affected), clear symbols (unaffected). The result of MLPA on the right shows exon 52-54del in DMD gene. (d) A: pedigree of the family of carrier 4 (I2). The carrier status is indicated by a circle with a dot, abortion (a dot), clear symbols (unaffected). PCR-based Sanger sequencing on the right validates the results of next generation sequencing: the arrow represents the carrier (I2) with 1 heterozygous insertion variant c.10364 dup T of exon 73 (upper right) as well as the same but hemizygous variant in the aborted embryo (lower right). B: the amino acid sequences of the wild type and mutant were subjected to DNAMAN, revealing that the variant produced a truncated dystrophin. (e) Pedigree of the family of carrier 5 (I2). The carrier status is indicated by a circle with a dot, clear symbols (unaffected). The result of MLPA on the right shows exon 52_60dup in DMD gene. (f) A: pedigree of the family of carrier 6 (I2). The carrier status is indicated by circle with a dot, clear symbols (unaffected). PCR-based Sanger sequencing on the right validates the results of next generation sequencing: the arrow represents the carrier (I2) with 1 heterozygous missense variant c.7555G>A of exon 52. (b) Multiple species alignment analysis showed the high evolution conservation of amino acid sequence at the missense site.