Diagnosis of Fanconi Anemia: Mutation Analysis by Multiplex Ligation-Dependent Probe Amplification and PCR-Based Sanger Sequencing

Abstract

<jats:p>Fanconi anemia (FA) is a rare inherited disease characterized by developmental defects, short stature, bone marrow failure, and a high risk of malignancies. FA is heterogeneous: 15 genetic subtypes have been distinguished so far. A clinical diagnosis of FA needs to be confirmed by testing cells for sensitivity to cross-linking agents in a chromosomal breakage test. As a second step, DNA testing can be employed to elucidate the genetic subtype of the patient and to identify the familial mutations. This knowledge allows preimplantation genetic diagnosis (PGD) and enables prenatal DNA testing in future pregnancies. Although simultaneous testing of all FA genes by next generation sequencing will be possible in the near future, this technique will not be available immediately for all laboratories. In addition, in populations with strong founder mutations, a limited test using Sanger sequencing and MLPA will be a cost-effective alternative. We describe a strategy and optimized conditions for the screening of<jats:italic>FANCA, FANCB, FANCC, FANCE, FANCF,</jats:italic>and<jats:italic>FANCG</jats:italic>and present the results obtained in a cohort of 54 patients referred to our diagnostic service since 2008. In addition, the follow up with respect to genetic counseling and carrier screening in the families is discussed.</jats:p>

Screening for mutations in two exons of FANCG gene in Pakistani population

“…This is also the founder mutation in Japanese and Korean populations and is found to have a common ancestor haplotype in East Asia 39 . This mutation is also reported in 2 patients from Iran 29 . Therefore, taking into consideration the prevalence of this mutation in Asian countries and especially in Iran, it was decided to probe into this mutation in our country.…”

“…Sequencing of exon 3 was carried out to screen for the mutation c.307+1G >T, which is the most prevalent mutation in Japanese and Korean population and also found in 2 patients of Iranian ethnicity recently 28,29 . Sequencing analysis of exon 3 revealed no sequence alteration in our cohort of patients.…”

Genetic diagnosis of Fanconi anemia. Literature review

“…Анализ нуклеотидной последовательности для всех известных АФ-генов за-труднен количеством возможных мутаций в каждом, их разнообразием, в том числе в виде крупных инсер-ций или делеций (indel-мутации). Их длина может варьировать от одного до нескольких сотен и даже тысяч нуклеотидных оснований, что подразумевает использование совершенно разных методов молеку-лярно-генетического исследования [60].…”

“…Метод MLPA используют для инициального скрининга делеции в гене FANCA, параллельно ген FANCA полностью сек-венируют. Если пациент мужского пола -исследуют на наличие делеций ген FANCB [60]. Выпущены ком-мерческие наборы для исследования FANCA, FANCD2, PALB, RAD50, RAD51.…”

Genetic Diagnosis of Fanconi Anemia

“…Following a positive chromosome breakage test molecular analysis is performed using either Multiplex Ligation-Dependent Probe Amplification (MLPA) [65], and/or sequencing, in order to identify deletions and/or point mutations in the 16 FANC genes [4,7].…”