FMR1 CGG repeat expansion mutation detection and linked haplotype analysis for reliable and accurate preimplantation genetic diagnosis of fragile X syndrome.

Indhu-Shree Rajan-Babu,Samuel S Chong,Mulias Lian,Arnold S.C Tan,Seong Feei Loh,Min Chen,Felicia S.H Cheah,Ethiraj B Prasath

doi:10.1017/erm.2017.10

Abstract

Fragile X mental retardation 1 (FMR1) full-mutation expansion causes fragile X syndrome. Trans-generational fragile X syndrome transmission can be avoided by preimplantation genetic diagnosis (PGD). We describe a robust PGD strategy that can be applied to virtually any couple at risk of transmitting fragile X syndrome. This novel strategy utilises whole-genome amplification, followed by triplet-primed polymerase chain reaction (TP-PCR) for robust detection of expanded FMR1 alleles, in parallel with linked multi-marker haplotype analysis of 13 highly polymorphic microsatellite markers located within 1 Mb of the FMR1 CGG repeat, and the AMELX/Y dimorphism for gender identification. The assay was optimised and validated on single lymphoblasts isolated from fragile X reference cell lines, and applied to a simulated PGD case and a clinical in vitro fertilisation (IVF)-PGD case. In the simulated PGD case, definitive diagnosis of the expected results was achieved for all 'embryos'. In the clinical IVF-PGD case, delivery of a healthy baby girl was achieved after transfer of an expansion-negative blastocyst. FMR1 TP-PCR reliably detects presence of expansion mutations and obviates reliance on informative normal alleles for determining expansion status in female embryos. Together with multi-marker haplotyping and gender determination, misdiagnosis and diagnostic ambiguity due to allele dropout is minimised, and couple-specific assay customisation can be avoided.

Highlights

CGG-triplet repeat hyperexpansions in the 5′ untranslated region of the X-linked fragile X mental retardation 1 (FMR1) gene cause fragile X syndrome (OMIM #300624) via Fragile X mental retardation 1 (FMR1) promoter hypermethylation and sequential loss of the encoded FMR1 protein (Refs [1,2,3])
Unless an allelic size ladder is generated using sequence-verified controls to correct for this mobility shift, repeat-spanning polymerase chain reaction (PCR)-based preimplantation genetic diagnosis (PGD) assays may underestimate CGG repeat size and incorrectly genotype embryos carrying a borderline FMR1 allele in the low-intermediate and low-premutation repeat size ranges
When the paternal and maternal normal alleles are identical in repeat size, this method cannot distinguish homozygous normal from fragile X syndrome-affected female embryos, restricting its application only to couples with informative normal FMR1 alleles (Ref. 27)

Summary

Introduction

CGG-triplet repeat hyperexpansions in the 5′ untranslated region of the X-linked fragile X mental retardation 1 (FMR1) gene cause fragile X syndrome (OMIM #300624) via FMR1 promoter hypermethylation and sequential loss of the encoded FMR1 protein (Refs [1,2,3]). Fragile X syndrome is the most common single-gene heritable form of intellectual disability, and the causative full-mutation (>200 CGGs) has an estimated prevalence rate of ∼1 in 4000 males and 1 in 5000–8000 females (Refs [4, 5]). Affected individuals usually inherit a copy of the mutant FMR1 allele from their mothers, who harbour a full-mutation or an unstable premutation (55−200 CGGs) that is prone to undergo full-mutation expansion during intergenerational transmission. The risk of premutation-to-fullmutation allele transition is largely influenced by the maternal FMR1 allele size, with ∼100% full-mutation inheritance risk reported among offspring of women harbouring ≥100 CGGs (Ref. 6). At-risk couples can avoid fragile X syndrome-affected/ carrier pregnancies by preimplantation genetic diagnosis (PGD) of in vitro fertilisation (IVF)-derived embryos and selective transfer of embryo(s) with normal (5−44 CGGs) FMR1 allele(s) for implantation

Methods

Results

Conclusion